..jvr;ni The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924104225911 "6^P TrTF— ' . — yL^^^f^ORNELL UNIVERSITY. THE GIFT OF ROSWELL p. FL-GWER * FOR THE USE OF THE- N. Y *STATE VETERINARY CGLLEQE. 1897 ^!^^"*Saiii 3 <>X WORKS OF J. A. MANDEL PUBLISHED BY JOHN WILEY & SONS. A Text-book of Physiological Chemistry. By Olof Hammarsten, Professor of Medical and Physiological Chemistry in the University of Upsala. Au1;horized translation, from the second Swedish edition and from the author's eplarged and revised German edition, by John A. Mandel, Assistant to the Chair of Chemistry, etc., in the Bellevue Hospital Medical College and in the Col- lege of the City of New York. 8vo, cloth, $400. Handboolc for Bio-Chemical Laboratory. i2mo, cloth, Si. 50. A TEXT-BOOK OS PHYSIOLOGICAL CHEMISTRY. BY OLOF HAMMAESTEN, Professor of Medical and Physiological Chemistry in thi i . nn K ' University of Upsala. I-* LlDHM ^ut^^ori^tir Cranslalion FROM THE AUTHOR'S ENLARGED AND REVISED THIRD GERMAN EDITION JOHN A. MANDEL, Professor of Inorganic Chemistry and Physics, and Adjunct Professor of Physiological Chemistry in the University and Bellevue Hospital Medical College. SECOND EDITION. FIRST THOUSAND. t , < If, NEW YORK: JOHN WILEY & SONS. London • CHAPMAN & HALL, Limited. 1898. Copyriglit, BY JOHN A. MANDEL. t>\ollO^ I 896 ^f .>^-^^^ «^V . ^■'". ..-; ,. ROBERT DRUMMOND, PRINTER, NEW YORK. PEEFACE TO THE SECOND GERMAN EDITION. After the appearance of the first Swedish edition of this text- book I was asked by several colaborers abroad to provide a German translation, which was at that time impossible for several reasons. But I found it very difficult to decline a similar proposal which I received from many colleagues after the second edition appeared. I yielded, therefore, to their expressed wishes; bat I found after a time that it was impossible to obtain a translator in this special province of science, notwithstanding the unwearied exertions of my publisher. Nothing remained for me but to undertake the transla- tion myself; hence I ask the reader's indulgence for possible idiomatic or orthographic errors. Specialists will at once perceive that the book before them is not a complete or detailed text-book. My intention was merely to sup- ply students and physicians with a condensed and as far as possible objective representation of the principal results of physiologico- chemical research and also with the principal features of physio- logico-chemical methods of work. It seems to me that I have followed a common, practical, even if not strictly correct usage in allowing space in this book to the more important pathologico- chemical facts, although I have given the book the title Text-book of Physiological Chemistry. The arrangement of subject-matter, which deviates considerably from that generally followed in text-books, was caused by the manner in which physiological chepiistry is studied in Sweden. Here physiologico- and pathologico-chemical laboratory practice is obligatory on all students of medicine. In the arrangement of such practical work I continually kept in view that it should not consist of isolated, purely chemical or analytico-chemical problems, bat that always, as far as possible, it should go hand in hand with the study of the different chapters of chemical physiology. IV PREFACE. The stady of physiologico-chemical processes withia the animal body miisfc precede the study of its component parts, its fluids and tissues; and this latter study, according to my experience, will then only inspire true interest if the study of the physiological signifi- cance of those component parts be closely pursued in connection with that of the transformations which take place in these fluids and tissues. In Tiew of this arrangement of subject-matter, and in order to render my book of greater interest and utility to those who do not wish to take cognizance of its analytico-chemical part, I hare dis- tinguished the latter by different setting of the type. With the exception of urinary analysis, which practically is of particular importance and which has been treated somewhat elaborately, this part in general depicts only the main points in the methods of preparation and of analytical methods. The instructor who su- perintends the. laboratory practice and who chooses the problems for work has ample opportunity to give the beginner the necessary advanced directions, and for the more experienced student, as well as for the specialist, the excellent works of Hoppb-Setlbe, Neubauee-Huppbbt, and others render more explicit directions superfluous. Olof Hammaesten. Upsala, October, 1890. TRANSLATOE'S PREFACE TO THE FIRST AMERICAN EDITION. Knowing the demands of the medical student and practising physician for a more extended knowledge of physiological chemis- try, and at the same time knowing the lack of literature on this subject in the English language, I have been led to make a transla- tion of this most admirable work. The subject of physiological chemistry is being more and more advanced in this country, until it will soon become an obligatory study in our medical schools, and the enlargement of the literature on the subject will greatly help its progress. It will be seen at a glance that the work is well suited as a laboratory book, for it contains the best methods for the prepara- tion, detection, and quantitative estimation of most of the sub- stances found in the organism and its excretions and secretions. At the author's request I have made no additions or changes what- soever in the manuscript, and it may seem that some of the methods described, especially those on urinary analysis, are too lengthy and troublesome for the practising physician; however, the quick or clinical methods are well described in smaller handbooks on the subject. In the work of translation I have adhered as closely as possible to the author's enlarged Qerman edition and also the orig- inal Swedish edition, and therefore the literary errors will perhaps be pardoned. I must here express my appreciation to Mon. A. Boukgougnon, who has kindly gone carefully over the manuscript and read the proof-sheets. J. A. Mandbl. New Yobk, October, 1893. PREFACE TO THE THIRD GERMAN EDITION. The prcEent edition, which difEers from the second in the arrangement of matter, contains three new chapters. The wonder- f al development of our knowledge of the chemistry of the carbo- hydrates in recent times has made it necessary to introduce a special chapter on this subject; and as the two chief groups of organic foods, the protein substances and the carbohydrates, are treated of in special chapters, the third group, the fats, likewise has a chap- ter devoted to it. It also appears appropriate to treat the rather extensive subject of the chemistry of respiration in a special chapter and not, as heretofore, in connection with the blood. Another deviation from the earlier editions is that the present edition is supplied with the references to the literature, in pursuance of the request made on many sides. This edition is also thoroughly revised and enlarged according to the advancement of the science; still it was naturally impossible to incorporate into the text the various papers appearing or accessible to me during the printing of this edition. Olof Hammaesten. Upbala, April, 1895. TRANSLATOR'S PREFACE TO THE SECOND AMERICAN EDITION. As the subject of physiological chemistry has been rather generally introduced into the curriculum of our medical schools, and as the first American edition was one of the few authoritative works on this important subject, I was led to prepare a second American edition from the third, revised, German edition. At the request of the author no changes or additions have been made with the exception of the incorporation of the author's Addenda into the text. J. A. Mandel. New Yoke, October, 1898. vii CONTENTS. CHAPTER I. PAGE iNTRODDCTION 1 CHAPTER II. Protein Substances 17 CHAPTER III. Caebohydbates. 59 CHAPTER IV. ANIMAIiFATS 8t CHAPTER V. The Animai, Cell 88 CHAPTER VI. The Blood Ill CHAPTER VII. Chyle, Lymph, Transudations and Exudations 180 CHAPTER VIII. The Liter 206 CHAPTER IX. Digestion 2S1 CHAPTER X. Tissues op the Connective Substance 342 CHAPTER XI. Muscle 360 iz X CONTENTS. CHAPTER XII. PAQE Beain and Nekves 390 CHAPTER XHI. OjtGANs OP Generation 403 CHAPTER XIV. Milk 420 CHAPTER XV. The Ubine 445 CHAPTER XVI. The Skin and its Secretions 573 CHAPTER XVII. Chemistry of Respiration 583 CHAPTER XVIII. Metabolism '607 PHYSIOLOGICAL CHEMISTRY. CHAPTEE I. INTRODUCTION. It follows from the law of the conservation of force and matter that living beings, plants and animals, can neither prodcice new matter nor new force. They are only called upon to appropriate and assimilate already existing material and to transform it into new forms of force. Out of a few relatively simple combinations, especially carbon- dioxide and water, together with ammonium compounds or nitrates, and a few mineral substances, which serve as its food, the plant- builds up the extremely complicated constituents of its organism,, proteids, carbohydrates, fats, resins, organic acids, etc. The chemical work which is performed in the plant must therefore, in the majority of cases, consist in syntheses; but besides these, processes of reduction take place to a great extent. The vis viva of the sunlight induces the green parts of the plant to split ofF oxygen from the carbon dioxide and water, and therefore the chief coustituents of the plant contain less oxygen than the material serving as food. The vis viva of the sun, which produces this- splitting, is not lost; it is, only transformed into another form of force— into the potential energy or chemical tension of the fre& oxygen on the one side, and the combinations less oxygenated, pro- duced by the synthesis, on the other side. These conditions are not the same in animals. They are dependent either directly, as the herbivora, or indirectly, as the carnivora, upon plant-life, from which they derive the three chief groups of organic nutritive matter — proteids, carbohydrates, and 2 INTRODUCTION. fats. These bodies, of which the protein substances and fat form the chief mass of the animal body, undergo within the animal organism a splitting and oxidation, and yield as final products •exactly the above-mentioned chief components of the nutrition of plants, namely, carbon dioxide, water, and ammonia derivatives, which are rich in oxygen and have feeble potential energy. The chemical tension, which is partly combined with the free oxygen and partly stored up in the above-mentioned more complex chem- ical compounds, is transformed into vis viva, heat, and mechanical work. While in the plant reduction processes and syntheses, which are active in the conversion of living force into potential energy or chemical tension, are the prevailing forces, we find in the animal body the reverse of this, namely, splitting and oxidation processes, which convert chemical tension into living force {vis viva). This difference between animals and plants must not be over- rated, nor must we consider that there exists a sharp boundary-line between the two. This is not the case. There are not only lower plants, free from chlorophyll, which in regard to chemical processes represent intermediate steps between higher plants and animals, but the difference existing between the higher plants and animals is more of a quantitative than a qualitative kind. Plants require oxygen as peremptorily as do animals. Like the animal, the plant also, in the dark and by means of those parts which are free from chlorophyll, takes up oxygen and eliminates carbon dioxide, while in the light the oxidation processes going on in the green parts are overshadowed or hidden beneath the more intense reduction proc- esses. Like the animal the fermentive fungi transform chemical tension into living energy and heat; and even in a few of the higher plants — as the aroidem when bearing fruit — a considerable develop- ment of heat has been observed. The reverse is found in the animal organism, for, besides oxidation and splitting, reduction processes and syntheses also take place. The contrast which seem- ingly exists between aaimals and plants consists merely in that in the animal organism the processes of oxidation and splitting are prevalent, while in the plant those of reduction and synthesis have thus far been observed. WoHLER ' in 1834: furnished the first example of stxthetical PROCESSES within the animal organism. He showed that when ' Berzelius, Lehib. d. Chemie, iibersetzt von Waliler, Bd. 4, Dresden, 1831. S. 376, Anm. ANIMAL OXIDATIONS. 3 benzoic acid is introdaced into the stomach it reappears as hippuric acid in the urine, after it combines with glyoocoll (amido-acetic acid). Since the. discovery of this synthesis, which may be ex- pressed by the following equation, <:!.H..COOH-f-NH,.CH,.COOH=NH(O.H..GO).OH,.COOH+H,0, Benzoic acid Glycocoll Hippuric acid and which is- ordinarily considered as a type of an entire series of syntheses occurring in the body where water is eliminated, the number of kaown syntheses in the animal kingdom has increased considerably. Many of these syntheses have also been artificially produced outside of the organism, and numerous examples of animal syntheses of which the course is absolutely clear will be found in the following pages. Besides these well-studied syntheses, there occur in the animal body also similar processes unquestionably of the greatest importance to animal life, but of which we know nothing with positiveness. We enumerate as examples of this kind of synthesis the reformation of the red-blood pigment (the haemo- globin), the formation of the different proteids from the peptones, the formation of fat from carbohydrates, and others. The chemical processes in the animal body we have mentioned a,bove as consisting chiefly of oxidation and splitting processes. The oxygen of inhaled air, as also that of the blood, is now called neutral, molecular oxygen, and the old assumption that ozone occurs in the organism has now been discarded for several reasons. There are but few substances which can be oxidized within the animal organism by the neutral oxygen; while, on the contrary, proteids and fat, which form the chief part of the organic constit- uents of the animal body, are almost iadifferent to neutral oxygen. The question arises, how then is the oxidation of these and other bodies possible in the animal organism ? Formerly the view was generally accepted that animal oxida- TiON took place in. the fluids, while to-day we are of the opinion, derived from the investigations of Pflugeb and his pupils,' that it is connected with the form-elements and the tissues. The question how this oxidation in the form-elements proceeds and how it is induced cannot be answered with certainty. 'PflUger, Pflilger's Archiv, Bdd. 6 and 10; Pinkler, ibid, Bdd. 10 and U ; Oertman, ibid., Bdd. 14 and 15 ; Hoppe-Seyler, ibid., Bd. 7. * INTRODUCTION. The cause of the animal oxidation is considered, by Peldgek and several other investigators, to be dependent upon the special contitution of the protoplasmic proteids. This investigator call& the proteids outside of the organism, and also those which circulate in the blood and fluids, " non-living proteids " as compared to thos& which are converted by the activity of the living cell into living protoplasm,' which he calls " living proteids." It is now also con- sidered that this "living proteid " differs from the " non-liviug^ proteid " by a greater mobility of the atoms within the molecule, and it may be characterized by a greater inclination towards intra- molecular changes of position of these atoms. The reason for these greater intramolecular movements Pfluger ^ ascribes to the presence of cyanogen, Loew ' to the presence of aldehydic groups, and Latham ° attributes it to the presence of a chain of cyanalcohols in the proteid molecule. Pflugek considers these differences between ordinary proteids and living protoplasmic proteids as the cause for the oxidation processes in the animal organism. These processes show certain similarity to the oxidation of phosphorus in an atmosphere contain- ing oxygen. In this process the phosphorus is not only itself oxidized, but, as it splits the oxygen molecules and sets free oxygen atoms (active oxygen), it may cause at the same time an indirect or Secondary oxidizing action upon other bodies present. In an analogous way the living protoplasmic proteid, which is not, like dead proteid, indifferent to molecular oxygen, may cause a splitting of the oxygen molecule, thus becoming itself oxidized, and at the same time setting oxygen atoms free, which may cause a secondary oxidation of other less oxidizable substances. Active oxygen may also be produced, according to 0. Nasse,* by a hydroxylization of the constituents of the protoplasm with the splitting off of molecules of water. If benzaldehyde is shaken with water and air an oxidation of the benzaldehyde into benzoic acid takes place, while oxidizable substances present at the same time may also be oxidized. The simultaneous presence of potassium iodide and starch or tincture of gnaiacum causes a blue coloration because the hydroxyl (OH) takes the place of the hydrogen in the ' Pfliiger's Archiv, Bd. 10. 2 Loew and Bokorny, Pfliiger's Archiv, Bd. 35, and Loew, ibid., Bd. 30. ; » British Medical Journal, 1886. * Rostocker Zeitung, 1891, No. 534. ANIMAL OXIDATIONS. o aldehyde group and these two hydrogen atoms, one derived from the aldehyde and the other from the splitting of the water, have a splitting action on the molecular oxygen. Nassb and Eosin^g ' have found that certain varieties of proteid have the property of'' feeing hydroxylized in the presence of water, and a series of oxida- tions in the animal body may, according to Nasse, be accounted for by the oxygen atoms set iiee in the hydroxylization similar to that of benzaldehyde. Another very widely diffused view exists in regard to the origin of the activity of the oxygen, namely, that by the decomposition processes in the tissues reducing substances are formed which split the oxygen molecule, uniting with one oxygen atom and setting the other free. The formation of reducing substances during fermentation and putrefaction is generally kuown. The butyric fermentation of Brilcke, Wiener Sitzungsbericht, Bd. 43. 1861. 12 INTRODUCTION. peroxide is, according to Alex. Schmidt," not dependent upon the enzyme, but is caused by the contamination of the enzyme with constituents from the protoplasm. This coincides with the obser- vations of Jacobson " on emulsin, pancreas enzyme, and diastase that the catalytic property may be destroyed by proper means with- out diminishing the specific enzymotic action. The continued heating of their solutions above + 80° C. generally destroys most of the enzymes. In the dry state, however, certain enzymes may be heated to 100° or indeed to 150°-160° C. without losing their power. The enzymes are precipitated from their solutions by alcohol. We have no characteristic reactions for the enzymes in general, and each enzyme is characterized by its specific action and by the conditions under which it operates. Bat it must be stated that, however the difEerent enzymes may vary in action, they all seem to have this in common, that by their presence an impulse is gipen to split more complicated combinations into simpler ones, whereby the atoms arrange themselves from an unstable equilibrium into a more stable one, chemical tension is transformed into living force, and new products are formed with lower heat of combustion than the original substance. The presence of water seems to be a necessary factor in the perfection of such decompositions, and the chemical process seems to consist in the taking up of the elements of water. The action of the enzymes may be markedly influenced by external conditions. The reaction of the liquid is of special im- portance. Certain enzymes act only in acid, others, and the majority, on the contrary act only in neutral or alkaline liquids. Certain of them act in very faintly acid as well as in neutral or alkaline solutions, but best at a specific reaction. The temperature exercises also a very important infiuence. In general the activity of enzymes increases to a certain limit with the temperature. This limit is not always the same, but is dependent upon the quantity of enzyme.' The products of the enzymotic processes exercise a retarding influence. Additions of various kinds may have a re- tarding and others an accelerating action. Fekmi and Peebtossi * have studied the action of various iafla- ' Al. Schmidt, Zar Blutlehre. Leipzig, 1892. ^ ZeitscUr. f. physiol, Chemie, Bd. 16, S. 340. 'Tammann, Zeitschr. f. physiol. Chem., Bd. 16, S. 271. «Zeitscbr. f. Hygiene, Bd. 18. ENZYMES AND PTOMAINES. 13 eiices on the enzymes. Starting with the assumption that when the free ions are set free by the action of enzymes the electrical conductivity of the water must be raised, 0. Nasse ' experimented with soluble starch, partly boiled and partly unboiled, and diastase, and determined the resistance according to Kohleausch's method and observed a considerable increase in the conductivity of the active diastase solutions. The animal enzymes are divided into several groups. The most studied of these are the hydrolytic enzymes found in the digestive canal. The three most important groups are the amylolytic or diastatic, the proteolytic or those converting proteids into soluble modifications, and the steatolytic or fat-splitting enzymes. The coagulating enzymes form a peculiar group. The mode of action of these enzymes, amongst which we reckon chymosin (rennin) or casein-coagulating, and fibrin ferment or blood-coagulating, is still less known than the others. The manner in which these enzymes work is still obscure, but their action may be considered, in several respects, as very closely related to the so-called catalytic or contact action. As above stated, the enzymes are of great importance for the chemical processes going on in the digestive tract, but we have to add that the results of their action are greatly complicated by processes of putrefaction which take place in the intestine at the same time, and which are caused by micro-organisms. Micro- organisms therefore exercise a certain influence on the physiological processes of the animal body. These organisms, when they enter the animal fluids and tissues and develop and increase, are of the greatest pathological importance, and modern bacteriology in rela- tion to the doctrine of infectious diseases, founded by Pasteur and Koch, gives eflBcient testimony to these facts. Putrefaction caused within the animal fluids and tissues by lower organisms may produce, among others, combinations of a basic nature. Such bodies were first found by Selmi in human cadavers, and called by him cadaver alkaloids or ptomaines. These ptomaines, which have been isolated from cadavers and some from putrefying proteid mixtures, have been closely studied by Selmi," ' Rostocker Zeitung, 1894. ' Sulle ptomaiue od alcaloidi cadaverici e loro importanza in tossicologia. Bologna, 1878. Ber, d, deutsch. chem. Oesellsch., Bd. 11. 14 INTBODUCTION. Bkieger,' and GtAUTIER," and are considered as products of chem- ical processes caused by putrefaction microbes. The first ptomaine to be analyzed was colUdin, C,,H,,N, obtained by Nencki/ on the putrefaction of gelatin. Since then many ptomaines have been anlayzed by Gautiee, and especially by Bkieger. Certain of the ptomaines originate undoubtedly from lecithin and other so-called extractives of the tissues, but the majority seem to be derived from the protein substances by decomposition. Some ptomaines, although all belong to the aliphatic series, contain oxygen and others are free from oxygen. The majority of the true ptomaines belong to the latter group. Most of the ptomaines isolated by Briegbk are diamines or compounds derived from the same. Amongst the diamines we have two, cadqverin or pentamethylendiamin, CjH,,N„ and putrescin or tetramethylendi- amin, C,H,jN,, which are of special interest because they have been found in the intestinal tract and urine in certain pathological con- ditions, namely, cholera'' and cystinuria.' Some of the ptomaines are exceedingly poissonons, while others are not. The poisonous ones are called toxines, according to the suggestion of Briegbr. The formation of such toxines in the decompositions caused by putrefactive microbes makes it probable that the lower organisms acting in infections diseases also produce poisonous substances which may cause by their action the symptoms or complications of the disease. Brieger, who has become prominent by his study of this subject, has been able to isolate from typhus cultures a sub- stance called typliotoxin which has a poisonous action on animals; and he has also prepared another substance, tetanin' from the amputated arm of a patient with tetanus, animals inoculated with which die exhibiting symptoms of developed tetanus. As above stated, the chemical processes in animals and plants do not stand in opposition to each other; they offer differences ' Ueber Ptomaine, Parts 1, 2, and 3. Berlin, 1885-1886. • Traite de cUiinie appliquee ^ ia physiologie. Tome 2, 1873. Compt. rendus. Tome 94. ^ Ueber die Zersetzung der Gelatine, etc. Bern, 1876. * Brieger, Berlin, klin. Wochenschr., 1887. 'Baumann and Udransky, Zeitschr. f. pbysiol. Chem., Bdd. 13 and 15; Brieger and Stadthagen, Berlin, klin . Wochenschr, , 1889. •Biieger. Arch. f. pathoi. Anat., Bdd, 113 and 115. Also Sitzungsber. d. Berl. Akad. d. W., 1889, and Berl. klin. Wochenschr,, 1888. LBUCOMAINES. 15 indeed, bat still they are of the same kind from a qualitative stand- point. Pflugek says that there exists a blood-relationship between all living cells of the animal and vegetable kingdoms, and that they originate from the same root; and if the organisms consisting of one cell can decompose protein substances in such a manner as to produce poisonous substances, why should not the animal body, which is only a collection of cells, be able to produce under physiological conditions similar poisonous substances ? It has been known for a long time that the animal body possesses this ability to a great extent, and as well-known evidence of this ability we may mention various nitrogenized extractives and poisoaous constituents of the secretions of certain animals. Those substances of basic nature which are incessantly and regularly produced as products of the decomposition of the protein substances in the living organism, and which therefore are to be considered as products of the physi- ological exchange of material, have been called hucomaines by Gautier ' in contradistinction to the ptomaines and toxines pro- duced by micro-organisms. These bodies, to which belong several well-known animal extractives, were isolated by Gautier from animal tissues such as the muscles. The hitherto known leuco- maines, of which a few are poisonous in small amounts, belong to the cholin, the uric acid, and the creatinin group. The leucomaines are considered as being of certain importance as causes of disease. It has been contended that when these bodies accumulate on account of an incomplete excretion or oxidation in the system, an auto-intoxication may be produced (Bouchard"). The toxines and the poisonous leucomaines are, however, neither the only nor the most active poison produced by the plant or animal cell. Later investigations have shown that certain plants as well as animals can produce proteids which are exceedingly poisonous. Such poisonous proteids have, for example, been isolated from the jequirity and castor beans, as also from the venom of snakes, spiders, and other animals. The toxic proteids produced by pathogenic micro-organisms are of special interest. Proteids have been isolated from the cultures of various pathogenic microbes 'Bull. Boc. chim., 43, and A. Gautier, Sur les alcaloldes deTivSa de la de- itmction bactSrienne ou phjsiologique des tissus animaux. Paris, 1886. ' Bouchard, Lemons sur les auto-intoxications dans les maladies. Paris, 1887. 16 INTRODUCTION. within the last few years (Beiegee and Frankel') which are exceedingly poisonous, and which reproduce the symptoms of the infection more exactly than the toxine. These proteids have been called toxalbumms by Beiegee and Feankel. It is of great interest that we know also of proteid bodies some of which, like the so-called dlexmes in the blood serum, have a germicidal, or bactericidal action, while others make the animal body immune against infection with a certain microbe or protect the body against the poison produced by the microbe. The great importance of these observations is apparent, but as it is not within the range of this book we will not further discuss the subject. The nature of these remarkable proteids wiU be given somewhat in detail in the following chapter. > Berl. klin. Wochenschi., 1890. CHAPTER II. THE PROTEIN SUBSTANCES. The chief mass of the organic constituents of animal tissues consists of amorphous, nitrogenized, very complex bodies of high molecular weight. These bodies, which are either proteids in a special sense or bodies nearly related thereto, take first rank among the organic constituents of the animal body on account of their great abundance. For this reason they are classed together in a special group which has received the name protein group (from Ttpcorevo, I am the first, or take the first place). The bodies belonging to these several groups are called protein substances, although in a few cases the proteid bodies in a special sense are designated by the same name. The several protein substances contain carbon, hydrogen, ni- trogen, and oxygen. The majority contain also sulphur, a few phosphorus, and a few also iron. Copper has been found in some few cases. On heating the protein substances they gradually decompose, producing inflammable gases, ammoniacal compounds, carbon dioxide, water, nitrogenized bases, as well as many other bodies, and at the same time they emit a strong odor of burnt horn or wool. More highly heated they leave a porous, shining mass of carbon, and when this is thoroughly burnt an ash is obtained con- sisting chiefly of calcium and magnesium phosphates. The ques- tion whether the mineral bodies left by burning exist as impurities or whether they are constituents of the protein molecule has not been decided. It is at present impossible to decide on a classification of the' protein substances based upon their properties, reactions, and con- stitution, as well as upon their solubilities and precipitations, corresponding to the demands of science. The best classification is perhaps the following systematic summary of the better known 17 18 THE PROTEIN SUB8TANGES. aad studied animal protein substances, due chiefly to Hoppe- Setlbr and Drechsel." I. Simple Froteids or Albuminous Bodies. Seralbumin, Albumins \ Ovalbumin, Lactalbumin. Serglobulin, Fibrinogen, Globulins \y'''';. Muscuhn, Crystallin, ' Vitellins {?). ( Casein, ' ' ' \ Ovovitellin if), and others. f Acid albuminate, \ Alkali albuminate. Albumoses and Peptones. , i •. -., ^ . ■. ( Filrin, Coagulated Froteids i Nucleo-albumius . . Albuminates Proteids coagulated by heat, and others. II. Compound Froteids. Hsemoglobins. Glyeoproteids A Mucins and Mucinoids, Hyalogens, Ichthulin, Helicoproteid. „ , , . , ( Nucleoliiston, Nucleoproteids \ ^ . i -, j j.v ^ ( Gytoglobm, and others. III. Albumoids or Albuminoids. Xeratin. Elastin. Collagen. Reticulin. (Amyloid.) (Fibroin, Sericin, Cornein, Spongin, Conchiolin, Byssas, and others.) •See "EiweisskBrper," Ladenburg's Handworterbuch der Chemie, Bd. 3, S. 5.34-589. SIMPLE PROTEIDS. 19 To this summary must be added that we often find in the investigations of animal fluids and tissues protein substances which do not coincide with the above scheme, or do so only with difficulty. At the same time it must be remarked that bodies will be found which seem to rank between the different groups, hence it is very -difficult to sharply divide these groups. I. Simple Proteids or Albuminous Bodies. The simple proteids are never-failing constituents of the animal and vegetable organisms. They are especially found in the animal body, where they form the solid constituents of the muscles, glands, and the blood serum, and they are so generally distributed that there are only a few animal secretions and excretions, such as the tears, perspiration, and perhaps urine, in which they are entirely absent or only occur as traces. All albuminous bodies contain carbon, hydrogen, nitrogen, oxygen, and sulphur;' a few contain also phosphorus. Iron is generally found in traces in their ash, and it seems to be a regular constituent of a certain group of the albuminous bodies, namely, the nncleo-albumins. The composition of the different albuminous bodies varies a little, but the variations are within relatively close Jimits. For the better studied animal proteids the following com- position of the ash-free substance has been given : 50.6 —54.5 per cent. H . . . . . . . 6.5 — 7.3 N 15.0 —17.6 S 0.3 — 2.3 P 0.42— 0.85 21.50 — 33.50 A part of the nitrogen of the proteid molecule is loosely com- Tiined and splits off easily as ammonia by the action of alkalies -(Nasse'). Sulphur shows the same property in nearly all albumi- ' An exception is found in the myeoprotein of putrefaction bacteria and the anthraxprotein of the anthrax bacillus, which are sulphur-free proteids. See Nencki and SchafEer, Journ. f. prakt. Chem., Bd. 20 (N. P.), and Nencki, Ber. d. deutsch. chem. Gesellech. , Bd. 17. ' Pflilger's Archiv, Bd. 6. 20 THE PROTEIN SUBSTANCES. nous bodies (Pleitmann,' Dakilewsky," Kkugee'). A part of the sulphur separates as potassium or sodium sulphide on boiling with caustic potash or soda, and may be detected by lead acetate. What remains can only be detected after fusing with nitre and sodium carbonate and testing for sulphates. The proteid molecule therefore contains at least 3 atoms of sulphur. The molecular weight of the proteids has not been determined with accuracy up to the present time, therefore it is impossible to give them formulae. The molecular weight of ovalbumin as determined by Sabanejew and Alexandrow' is about 14. .300. For the alkali albuminate, in whose formation from native albumins a part of the nitrogen and^ the loosely bound sulphnr is split off, Lieberkuhn has given the formula C„H,.,N.,SO,,. The constitution of the proteid bodies, notwithstanding numer- ous investigations, is still unknown. By heating proteids with barium hydrate and water in sealed tubes at 150°-300° C. for several days, Schutzestberger ' obtained a number of products among which were ammonia, carbon dioxide, oxalic acid, acetic acid, and, as chief product, a mixture of amido-acids. This mix- ture contained, besides a little tyrosin and a few other bodies, chiefly acids of the series C„H5„+iN0, {leucines) and C„Hj„_iNOj {leucemes). The leucines and leuceines are formed from more complicated substances, with the general formula C,„Hj„NjO,, by hydrolytic splitting. These substances are called glucoproteins by Schutzenbbrger on account of their sweet taste. The sulphur of the proteids yields sulphites. The three bodies, carbon dioxide, oxalic acid, and ammonia, are formed in the same relative propor- tion as in the decomposition of urea and oxamid; therefore ScHtJT- zenbeeger suggests that perhaps albumin may be considered as a very complex ureid or oxamid. Suoh a conclusion cannot be derived from the above decomposition prt)cesses for several reasons, and the attempts to prepare urea directly by oxidation have also given negative results. On fusing proteids with caustic alkali, ammonia, mercaptan, and other volatile products are generated; also leucin, from which ' Annal. der Chem und Pharm., Bd. 66. 'Zeitsclir. f. physiol. Chem., Bd. 7. 'Pfliiger's Archiv, Bd. 43. *See Maly's Jahresber., Bd. 21, S.' 11. ' Annal. de Chim. et Phys. (5), 16, and Bull. soc. chim , 23 and 24. SIMPLE PROTEIDS. 21 volatile fatty acids, such as acetic acid, valerianic acid, and alao butyric acid, are formed; and tyrosin, from which phenol, indol, and skatol are produced. On boiling with mineral acids, or still better by boiling with hydrochloric acid and tin chloride (Hlasi- ■\VETZ and Habermann '), the proteids yield amido-acids, such as leucin, aspartic acid, glutamic acid, and tyrosin (and from vegetable albumin Schulze and Barbieri" obtained a-phenyl- amidopropionic acid), also sulphuretted hydrogen, ammonia, and nitrogenized bases (Drechsel'). As an essential difference between the action of acids and alkalies (barium hydrate) on albumins, Drechsel suggests that by the action of acids carbon dioxide, oxalic and acetic acids are not prodaced. Amongst the bases obtained by Drechsel from casein and by his pupils E. Fischer and M. Siegfried * from other proteids and gelatine on boiling with hydrochloric acid and tin chloride, we have one having the formula O^H^K^O, or GjE^NjO + H,0, which seems to be homologous to creatin or creatinin and called lysatin or lysatinin by Drechsel. On boiling lysatinin with baryta- water it yields urea amongst other cleavage products, and it is therefore possible to prepare urea artificially from albumin, without oxidation, by the liydrolysis of this base. Another substance, called lysm, has the formula C,H,^N,Oj. From its formula we find that it is homologous with ornithm, C^H^N^O, (Jaffe), which it resembles in certain respects (see Chapter XV). Lysin, which is probably diamidocaproic acid, and lysatinin have been shown by Drechsel and Hedin to be produced in the tryptic digestion of fibrin. Drechsel ' also found diamidoacetic acid amongst the oleayage products of casein. Proteids are decomposed by the action of proteolytic enzymes in the presence of water. First proteid bodies of lower molecular v^eight are formed — albumoses and peptones — and then on further decomposition amido-acids such as leucin, tyrosin, and aspartic acid. Both lysin and lysatinin may be produced on far-reaching ' Annal. d. Chem. u. Pharm., Bdd. 1.59 and 169. 'Ber. d deutsch. chem. Gesellscli., Bd. 16. ' Sitzungsber. d. matli.-phys. Klasse der k. saclis. Gesellscli. d. Wissen- schaften. 1889. * Drechsel gives a complete review of his own and his pupils Fischer, Siegfried and Hedin's investigations on this subject in Du Bois-Reymond's Archiv, 1891 : "Der Abbau der EiweissstofEe." 'Ber. d. k. sSchs, Gesellsch. d. Wissensch., 1893. 22 THE PROTEIN SUBSTANCES. decomposition (in tryptic digestion). On the extensive decomposi- tion a chromogen may also be formed, which gives a violet color with chlorine- or bromine-water. This chromogen, which is formed in all far-reaching decompositions of proteids where leucin and tyrosin are formed, is called proteinochromogen by Stadelmank ' and tryptophan by Nbumeistek." Kencki ' considers this chromo- gen as the mother substance of various animal pigments. Nencki * has found on the addition of bromine to the digestive fluid contain-^ ing proteinochromogen that at least two different bodies containing different quantities of bromine are produced. Both bodies show,, although not obtained quite pure, a close relationship to- certain animal pigments in regard to elementary composition. One stands- close to hsematoporphyrin, or bilirabin, and the other to the animal melanins. A great many substances are produced in the putrefaction of proteids. First the same bodies as are formed in the decomposition by means of proteolytic enzymes are produced, and then a farther decomposition occurs with the formation of a large number of bodies belonging to both the alipathic and aromatic series. Belong- ing to the first series we have ammonium salts of volatile fattj acids, such as caproic, valerianic, and butyric acids, also carbon dioxide, methane, hydrogen, sulphuretted hydrogen, methyl- mercaptau,' and others. The ptomaines also belong to these products and are probably formed by very different chemical processes or even syntheses. B. Salkowski" divides the putrefactive products of the aro- matic series into three groups: (a) the phenol group, to which tyrosin, the aromatic oxy-acids, phenol, and cresol belong; (5) the phenyl group, including phenylacetic acid and phenylpropionic acid; and lastly (c) the indol group, which includes indol, skatol, and skatolcarbonic acid. These various aromatic products are formed during the putrefaction with access of air. Nencki and Bovet' obtained only p. -oxyphenylpropionic acid, phenylpropionic acid, and skatolacetic acid on the putrefaction of proteids by ' Zeitschr. f . Biologie, Bd. 26. ^Ibid., S. 339. ' Schweizerische Wochenschr. f. Pharmacie, 1891. * Ber. d. deutsch. cliepi. Gesellsoh, , Bd. 38. 'See Nencki and Sieber : Monatshefte f. Chem., Bd. 10. «Zeitschr. f. physiol. Chem., Bd. 13, S. 315. 'Monatshefte f. Chem., Bd, 10. CLEAVAGE PRODUCTS OF PORTEIDS. 23 anaerobic schizomycetes in the absence of oxygen. These three acids are produced by the action of nascent hydrogen on the corre- sponding amido-aeid, namely, tyrosin, phenylamidopropionic acid, and skatolamidoacetic acid, and these three last-mentioned amido- acids exist, according to Nencki, preformed in the proteid mole- cule. On the putrefaction of proteids, as well as their decomposition by means of acids or alkalies and also by certain enzymes, among other products amido-acids are produced, and these have a certain significance for the probable formation of the proteids. It is more than likely that in the synthesis of proteids in the plant from the ammonia or the nitric acid of the soil, amido-acids or acid amids, among which asparagin plays an important r61e, are produced ; and from these the albuminous bodies are derived by the influence of glucose or other non-nitrogenized combinations. Since Grimaux ' was able to prepare by synthetical means from amido-acids bodies which in certain regards were similar to pro- tein substances, so later Schutzenbeegek," by heating a mixture of leucines and lenceines with urea and phosphoric anhydride, obtained a substance which was so similar to peptone in its behavior with several reagents that it was called pseudopeptone. The synthetical preparation of protein-like substances by Lilienfeld and WoLKOWicz " in Kossel's laboratory is of great importance. The experiments started from the observation of Cuetius and GoEBEL- that amidoacetic acid ethylester readily splits with the PO "N^TT PTT separation of a base whose formula is probably NH Schmiedeberg, Mitth. aus d. zool. Stat, zu Neapel, Bd, 8, 1883. • Hilger, Pflliger's Archiv, Bd. 3. •Krukenberg, Zeitschr. f. Biologie, Bd. 23. "Zeitschr. f. physiol. Chem., Bd. 15. » Pflftger's Archiv, Bd. 36. 48 THE PROTEIN SUBSTANCES. the glands of the snail Helix pomatia. It has the following composition : C 46.99 ; H 6.78 ; N 6.08 ; S 0.62 ; P 0.47^. It is converted into a gummy, Isevorotatory carbohydrate, called animal sinistrin, by the action of alkalies . On boiling with an acid it yields a dextrorotatory, reducible substance. Nueleoproteids. With this name we designate those compound proteids which yield true nucleins (see Chapter V) on pepsin diges- tion and those which yield, besides proteids, xanthin bases or so-called nuclein bases on boiling with dilate mineral acids. The nucleoproteids seem to be widely diffused in the animal body. They occur chiefly in the cell nuclei, but they also of tea occur in the protoplasm. They may also pass into the animal fluids on the destruction of the cells, hence nucleoproteids have also been found in blood serum. They may be considered as combinations of a proteid nucleus with a side chain, which Kossel ' calls the pkostetic group. This side chain, which contains the phosphorus, yields on the decomposition of certain nucleoproteids, such as from the yeast cell," or from the pancreas,' besides nuclein bases also reducing substances, which form crystalline combinations with phenyl- hydrazin. It is still an open question as to the formation of reducing substances from other nucleoproteids. This prostetic group may be split off as nucleic acid (see Chapter V) by the action of alkalies. The nucleoproteids seem to be dissimilar according to the kind of nucleic acid split off because they yield differing relative amounts of the various xanthin bases. The nucleoproteids are acids whose alkali compounds are solu- ble in water and which coagulate on heating (this is true at least for all genuine nucleoproteids investigated up to the present time). They may be precipitated from their alkali compounds by acetic acid, and the precipitate is more or less soluble in an excess of the acid. A confusion may occur here with nucleoalbumins and also with mucin substances. This confusion can be avoided by warming the body for some time on the water-bath with dilute sulphuric acid, and on cooling filtering and saturating the filtrate with ammonia and testing for xanthin bodies by an ammoniacal solution of silver nitrate. Any precipitate formed is examined more closely by the methods as given in Chapter V. I Verb. d. physiol. Gesellsch. zu Berlin, 1893-93, No. 1. ' A, Kossel, Du Bois-Eeymond's Archiv, Physiol Abth., 1891. ' O. Hammarsten, Zeitschr. f, physiol. Chem., Bd. 19. KERATINS. 49 The properties of the various nncleoproteids are given more in detail in the various chapters which follow. III. Albumoids or Albuminoids. Under this name we collect into a special group all those protein bodies which cannot be placed in either of the other two groups, although they differ essentially among themselves and from a chemical standpoint do not show any radical difference from the true proteid bodies. The most important and abundant of the bodies belonging to this group are important constituents of the animal skeletoQ or the cutaneous structure. They occur as a rule in an insolable state in the organism, and they are distinguished in most cases by a pronounced resistance to reagents which dissolve proteids or to chemical reagents in general. The Keratin Group. , Keratin is the chief constituent of the horny structure, of the epidermis, of hair, wool, of the nails, hoofs, horns, feathers, of tortoise-shell, etc., etc. Keratin is also found as neurokeratin (Kuhne') in the brain and nerves. The shell- membrane of the hen's egg seems also to consist of keratin. It seems that there exist more than one keratin, and these form a special group of bodies. This fact, together with the difficulty in isolating the keratin from the tissues in a pure condition without a partial decomposition, is sufficient explanation for the variation in the elementary composition given below. As examples the analyses of a few tissues rich in keratin and of keratins are given as follows: c H N S Human hair... 50.65 6.36 17.14 5.00 20.85 (V. Labr)" Nail 51.00 6.94 17.51 a.8o 21.75 (Mdldbr)s Neurokeratin. . 56.11-58.46 7.26-8.02 11.46-14.32 1.63-2.24 (KUHNH:)> Horn (average). 50.86 6.94 3.30 (HORDACZEWSKl)* Tortoise-shell... 54.89 6.56 i6!77 2.22 19'56 (Mulder)^ Shell-membrane 49.78 6.64 16.43 4.25 22.90 (Lindvall)"* Mohr' has determined the quantity of sulphar in various keratin substances. The percentage varies from 2.6 to 5.3. Sul- phur is at least in part in loose combination, and it is partly ' Kilhne and Ewald, Verb d. naturhistor.-med. Vereins zu Heidelberg (N. F.), Bd. 1 ; also Kiiline and Chittenden, Zeitschr. f. Biologie, Bd. 26. ' Annal. d. Chem, u. Pharm., Bd. 45. = Versuch einer allgem. physiol Chem. Braunschweig, 1844-51. * See Drechsel in Ladenburg's Handw8rterbuch d. Chem., Bd. 3. ' See Maly's Jahresbericht, 1881. « Zeitschr. f, physiol. Chem., Bd. 20. 60 TBS PMOTEm SUBSTANCES. removed by the action of alkalies (as sulphides), or indeed in part by boiling with water. Combs of lead after long usage become black, and this is due to the action of the sulphur of the hair. On heating keratin with water in sealed tubes at a temperature of 150° to 200" C. it dissolves, with the elimination of sulphuretted hydrogeu, forming a non-gelatinizing liquid which contains albu- Jnose (called keratinose by Keukenbeeg ') and peptone (f). Kera- tin is dissolved by alkalies, especially on heating, forming, besides ■alkali sulphides, albumoses and peptones (?). The decomposition products of keratins are moreover the same as the true proteids. On boiling with acids we obtain besides leucin ■and tyrosin, which occurs in relatively great amounts (1-5^), asparaginic acid' and glutamic acid,' ammonia, and sulphuretted" hydrogen. Hedin* has obtained a little lysin and considerable lysatinin from horn shavings. Besides these he obtained a sulphur compound whose hydrochloric-acid combination had the composi- tion C.jHjjN^O^SCl,, and another body which is perhaps identical with serin. There is no doubt that the keratins are derived from the proteids. Dkechsel ^ is also of the opinion that in the keratin a part of the oxygen of the proteids is exchanged for sulphur, and a part of the leucin, or any other amido-acid, is exchanged for tyrosin. Keratin and proteids give the same decomposition products, wibh the exception that the former gives proportionally a greater quantity of tyrosin (1-5^). Among the sulphurized cleavage products of keratin Emmerlins ' found cystin, and Suter ' thio- lactic acid. Suter could not detect either cystin or cystein. Among the cleavage products obtained by the action of hydrochloric acid and tin chloride Hedin " obtained a base which is probably identical with the base arginin, 0,H,,lSr,03, isolated by Schulze -and Stbigee " from lupin and malt acrospire. ' Untersueh. iiber d. chem. Bau d. EiweisskSrper. Sitzuugsber. d. Jenaischen Gesellsch. f. Med. u. Naturwissensch. , 1886. 2 Kreusler, Journ. f. prakt. Chem., Bd. 107. ' Horbaczewski, Sitzungsber. d. k. k. Wien. Akad. d. Wiasensch., Bd. 80. < Kgl. fysiogr. Sallsk. i Lund handlingar, Bd. 4 ; also Maly'a Jahiesber., -Bd. 23. ' Drechsel in Ladenburg's HandwSrterbuch d. Chem., Bd. 3. « Chemiker-Zeitung, No. 80, 1894. •> Zeitschr. f. physiol. Chem., Bd. 30. « lUd., Bd. 80. ' Ibid., Bd. 11, S. 43. ELA8TIN. 51 Bodies occur in the animal kingdom which form intermediate bodies between coagulated albumin and keratin. C. Th. Mobster ' has detected such a body in the tracheal cartilage, which forms a ■net-like basement membrane. This substance appears to be related to keratin on account of its solubilities and on the quantity of the sulphur (which turns lead black) it contains, while according to its solubility in gastric juice it must stand close to the proteids. Another substance, more similar to keratin, forms the horny layer in the gizzard of birds. According to J. Hedebtius ° this substance is insoluble in gastric or pancreatic juice and acts qnite similar to keratin. It contains only 1% sulphur, and yields on decomposition only very little tyrosin besides considerable leucin. Keratin is amorphous or takes the form of the tissues from which it was prepared. On heating it decomposes and generates an odor of burnt horn. It is insoluble in water, alcohol, or ether. On heating with water to 150°-200° C. it dissolves. It also dis- solves gradually in caustic alkalies, especially on heating. It is not dissolved by artificial gastric juice or by trypsin solutions. Keratin gives the xanthoproteic reaction, as well as the reaction with Millon's reagent, even though they are not always typical. In the preparation of keratin a finely divided horny structure is treated first with boiling water, then consecutively with diluted acid, pepsin-hydrochloric acid, and alkaline trypsin solution, and, lastly, with water, alcohol, and ether. Elastin occurs in the connective tissue of higher animals, some- times in such large quantities that it forms a special tissue. It occurs most abundantly in the cervical ligament (ligamentum nuchas). Elastin is generally considered as a sulphur-free substance. According to the investigations of Chittenden' and Haet,° it is a question whether or not elastin does not contain sulphur, which is removed by the action of the alkali in its preparation. H. Schwakz' has been able to prepare an elastin containing sulphur from the aorta by another method, and this sulphur can be removed by the action of alkalies, without changing the properties of the elastin. Elastin is hence perhaps a protein substance containing sulphur which exists only loosely combined. The most trustworthy analyses ' Maly's Jahresber., Bd. 18. = Skandinav. Arct. f. Ttysiol., Bd. 3. ' iieitschr. f. Biologie, Bd. 35. * Zeitschr. f. physiol. Chem., Bd. 18. 52 THE PROTEIN SUBSTANCES. of elastin from the cervical ligament (Nos. 1 and 2) and from the aorta (No. 3) have given the following results : c H N s O 1. 2. 3. 54.33 54 34 53.95 6.99 7.27 7.03 16.75 16.70 16.67 0.38 21.94 (HORBACZEWSKI)' 21.79 (Chittenden and Hast)'' (H. SCHWABZ)» The splitting products of elastin are the same as for the true proteids with the difEerence that glycocoU but no aspartic and glutamic acids are obtained.* Tyrosin is only obtained in small quantities. Schwaez was able to detect lysatinin in the decom- position products, but not lysin positively. On putrefaction ' no indol or phenol is obtained, but Schwaez, on the contrary, obtained indol, skatol, benzol, and phenols, but no methylmercaptan, on fusing aorta-elastin with caustic potash. On heating with water in closed vessels, on boiling with dilute acids, or by tfee action of proteolytic enzymes, the elastin dissolves and splits into two chief products, called by Hoebaczewski hemielastin and elastinpeptone. According to Chittenden and Haet, these products correspond to two albumoses designated by them protoelastose and deutero- elastose. The first is soluble in cold water and separates on heat- ing, and its solution is precipitated by mineral acid as well as by acetic acid and potassium ferrocyanide. The watery solution of the other does not become cloudy on heating, and is not precipi- tated by the above-mentioned reagents. Pure dry elastin is a yellowish- white powder; in the moist state it appears like yellowish- white threads or membranes. It is insolu- ble in water, alcohol, or ether, and shows a resistance against the action of chemical reagents. It is not dissolved by strong caustic alkalies at the ordinary temperature, and only slowly at the boiling temperature. It is very slowly attacked by cold concentrated sulphuric acid, and it is relatively easily dissolved on warming with strong nitric acid. Blastins of differing origins act differently with cold concentrated hydrochloric acid; for instance, elastin from the aorta dissolves readily therein, while elastin from the ligamentnm nuchse, at least from old animals, dissolves with difficulty. Elastin ' Zeitschr. f. physiol. Chem., Bd. 6. 2 Zeitsclir. f. Biologie, Bd. 25. 3 Zeitschr. f. pkysiol. Chem., Bd. 18. * See Drechsel in Ladenburg's HandwSrterbuch d. Chem., Bd. 3, and Hor- baczewski , Monatshefte f . Chem. , Bd. 6. * WSlchli, Journ. f. prakt. Chem., Bd. 17. COLLAGEN. 53 is more readily dissolved by warm concentrated hydrochloric acid. It responds to the xanthoproteic reaction and with Millon's reagent. On account of its great resistance to chemical reagents, elastin may be prepared (best from the ligamentum nuch») in the follow- ing way: First boil with water, then with 1% caustic potash, then again with water, and lastly with acetic acid. The residue is treated with cold 5^ hydrochloric acid for twenty-four hours, care- fully washed with water, boiled again with water, and then treated with alcohol and ether. ScHWATZ first incompletely digested the tissues with pepsin, washed first with soda solution and then with water, and boiled lastly with water until the elastic substance was dissolved away. The dried and powdered substance is again digested with gastric juice and treated as above, and then boiled with water until the contaminating reticulin-like substance is completely removed. Collagen, or gelatine-forming substance, occurs very extensively in the animal kingdom. The flesh of cephalopods is claimed to contain collagen.' Collagen is the chief constituent of the fibrils of the connective tissue and (as ossein) of the organic substances of the bony structure. It also occurs in the cartilaginous tissues as chief constituent, but it is here mixed with other substances, pro- ducing what was formerly called chondrigen. Collagen from different tissues has not quite the same composition, and probably there are several varieties of collagen. By continuously boiling with water (more easily in the presence of a little acid) collagen is converted into gelatine. Hofmeister' found that gelatine, on being heated to 130° C, is again trans- formed into collagen ; and this last may be considered as the anhy- dride of gelatine. Collagen and gelatine have about the same composition : C H N S-l-0 Collagen 50.75 6.47 17.86 34.93 (Hofmeistbr) » Gelatine (from hartshorn). 49.31 6.55 18.37 35.77 (Mtjldbk)* Gelatine (from bones) 50.00 6.50 17.50 36.00 (Fbemy)« Purified Gelatine 50.14 6.69 18.13 .... (Paal)' The gelatine contains about 0.6^ sulphur, which probably ' Hoppe-Seyler, Physiol. Chem. Berlin, 1877-81. S. 97. «Zeitschr. f. physiol. Chem., Bd. 2. 'Annal. d. Chem. u. Pharm., Bd. 45. *Jahresber. d. Chem., 1854. 'Ber. d. deutsch. chem. Gesellsch., Bd. 35, S. 1308. 54 THE PBOTEIN SUBSTANCES. belongs to the gelatine and does not exist there as an imparity from the proteids. The decomposition products of collagen are the same as those of gelatine. Gelatine under similar conditions as the proteids yields amido-acids, such as leucin, aspartic and glutamic acids, but no tyrosin, which is especially important. It yields, on the contrary, large quantities of glycocoU, to which the name gelatine sugar is given on account of its sweet taste. Lysin and lysatinin have also been obtained from gelatine by Dkechsbl and E. Fischer.' On putrefaction gelatine yields neither tyrosin, indol nor skatol,' in which it differs from the proteids. Still the aromatic group is not absent in gelatine, and it acts like the oxidized proteid, the oxyprotsulphonic acid, yielding benzoic acid (Malt'). Collagen is insoluble in water, salt solutions, dilute acids, and alkalies, but it swells up in dilute acids. By continuous boiling with water it is converted into gelatine. It is dissolved by the gastric juice and also by the pancreatic juice (trypsin solution) when it has previously been treated with acid or heated with water above + 70° C* By the action of ferrous sulphate, corrosive sub- limate, or tannic acid, collagen shrinks greatly. Collagen treated by these bodies does not putrefy, and bannic acid is therefore of great importance in the preparation of leather. Gelatine or glutin is colorless, amorphous, 'and transparent in thin layers. It swells in cold water without dissolving. It dissolves in warm water, forming a sticky liquid, which solidifies on cooling when sufficiently concentrated. The quantity of ash contained in gelatine is of the greatest importance in the gelatinization of gela- tine solutions, as shown by 0. Nasse and A. Keugbk,' namely, a diminished quantity of ash diminishes the gelatinization power. Gelatine solutions are not precipitated on boiling, neither by mineral acids, acetic acid, alum, lead acetate, nor mineral salts in general. A gelatine solution acidified with acetic acid may be pre- cipitated by potassium ferrocyanide on carefully adding the reagent, ' See Drechsel, Der Abbau der Eiweissk8rper. Du Bois-Reymoiid's Archiv, 1891. ' See literature on the cleavage products of gelatine : Drechsel in Laden- burg's HandwOrterbuch, Bd. 3. «Monat§Uefte f. Chem., Bd. 10. ^ Kilhne and Ewald, Verh. d. naturhist. med. Vereins in Heidelberg, 1877^ Bd. 1. 'See Maly's Jahresber., Bd. 19, S. 29. GELATINE. 55 but on the addition of too much potasBium ferrocyanide the liquid remains clear. Gelatine solntions are precipitated by tannic acid in the presence of salt; by acetic acid and common salt in sub- stance; mercuric chloride in the presence of HCl and NaCl; meta- phosphoric acid, phosphomolybdic acid in the presence of acid ; and lastly by alcohol, especially when neutral salts are present. Gela- tine solntions do not difiase. Gelatine gives the biuret reaction, but not Adamkibwicz's. It gives Millon's reaction and the xantho- proteic acid reaction so faintly that it probably occurs from an impurity consisting of proteids. By continuous boiling with water glntin is converted into a non-gelatinizing modification called /J-glutin by Nassb. According to Nasse and Keugee the specific rotatory power is hereby reduced from — 167°. 5 to about — 136°. On long-continued boiling with water, especially in the presence of dilute acids, also in the gastric or tryptic digestion, the gelatine is transformed into gelatine albu- moses, so-called gelatoses and gelatine peptones, which diffuse more or less readily. According to Hofmeistbe ' two new substances, semiglutin and Jiemicollin, are formed. The former is insoluble in alcohol of 70- 80^ and is precipitated by platinum chloride. The latter, which is not precipitated by platinum chloride, is soluble in alcohol. CHiTTEiirDEif and Sollbt " have obtained in the peptic and tryptic digestion aproto- and a deuterogelatose, besides some true peptone. The elementary compositiou of the gelatoses does not essentially differ from 'that of the gelatine. Paal ' has prepared gelatine peptone hydrochlorides from gelatine by the action of dilute hydro- chloric acid. Some of these salts are soluble in ethyl and methyl alcohol, and others insoluble therein. The peptones obtained from these salts contain less carbon and more hydrogen than the glutin from which they originated, showing that hydration has taken, place. The molecular weight of the gelatine peptone as determined by PiAL by Eaoult's method was 200 to 353, while that for gelatine was 878 to 960. Collagen may be obtained from bones by extracting them with hydrochloric acid (which dissolves the earthy phosphates) and then carefully removing the acid with water. It may be obtained from 'Zeitschr. f. physiol. Chem., Bd. 2. 'Journ. of physiol., Vol. 13. 'Ber. d. deutsch. chem. (Jesellach,, Bd. 35. 56 THE PROTEIN SUBSTANCES. tendons by extracting with lime-water or dilute alkali, (which dis- solve the proteids and mucin) and then thoroughly washing with water. Gelatine is obtained by boiling collagen with water. The finest commercial gelatine always contains a little proteid, which may be removed by allowing the finely divided gelatine to swell up in water and thoroughly extracting with large quantities of fresh water. Then dissolve in warm water and precipitate with alcohol. Chondrin or cartilage gelatine is only a mixture of glutin witli the specific constituents of the cartilage and their transformation products. Reticulin. The reticular tissues of the lymphatic glands con- tain a variety of fibres which have also been found by Mall ' in the spleen, intestinal mucosa, liver, kidneys, and lungs. These fibres consist of a special substance, reticulin, investigated by Siegfried.' Eeticulin has the following composition: C 52.88; H 6.97; N 15.63; S 1.88; P 0.34; ash 2.37. The phosphorus occurs in organic combination. It yields no tyrosin on splitting with hydro- chloric acid. It yields, on tlie contrary, sulphuretted hydrogen, ammonia, lysin, lysatinin, and amido-valerianic acid. On con- tinuous boiling with water, or more readily with dilute alkalies, reticulin is converted into a body which is precipitated by acetic acid, and at the same time phosphorus is split off. Eeticulin is insoluble in water, alcohol, ether, lime-water, sodium carbonate, and dilute mineral acids. It is dissolved, after several weeks, on standing with caustic soda at the ordinary tem- perature. Pepsin hydrochloric acid or trypsin do not dissolve it. Reticulin responds to the biuret, xanthoproteic, and Adamkiewicz's reactions, but not with Millon^'s reagent. It may be prepared as follows, according to Siegfkied : Digest intestinal mucosa with trypsin and alkali. Wash the residue, extract with ether, and digest again with trypsin and then treat with alcohol and ether. On careful boiling with water the collagen present either as contamination or as a combination with reticulin is removed. The thoroughly dried residue consists of reticulin. Skeletins are a number of nitrogenized substances which form the skeletal tissue of various classes of invertebrates so designated by Krukenbbkg.' These substances are chitin, spongin, con- chiolin, cornein, and fibroin (silk). Of these chitin does not belong Abhandl. d. math.-phys. Klasse d. kgl. sachs. Qesellsch. d. Wiss., 1891. ' Ueber die chemlschen Eigenschaften des reticulirten Gewebes. Inaugural dissertation. Leipzig, 1893. ' Grundziige einer vergl. Physiol, d. thier. Gertistsubst. Heidelberg, 1885. SEELETINS. 57 to the protein substances, and fibroin (silk) is hardly to be classed as a skeletin. On,ly those so-called skeletins will be given that acbually belong to the protein group. Spongin forms the chief mass of the ordinary sponge. It gives no gelatine on boiling with acids, but yields leucin and glycocoll and no tyrosin. Zalo- COSTAS' claims to have found tyrosin and also butalanin and glycalanin (CtHuNjO,). Conchiolin is found in the shells of mussels and snails and also in the egg-shells of these animals. It yields leucin but no tyrosin. The Byssus contains a substance, closely related to conchiolin, which is soluble with difficulty. Cornein forms the axial system of the Antipathes and Gorgonia. It gives leucin and a crystallizable substance, cornierystaUin (Kbtikenberq). Fibroin and Serioin are the two chief constituents of raw silk. By the action of superheated water the sericin dissolves and gelatinizes on cooling (silk gelatine), while the more difficultly soluble fibroin remains undissolved in the shape of the original fibre. On boiling with acid the fibroin yields alanin (Weyl''), glycocoll, and a great deal (5-8^) of tyrosin. Fibroin is dissolved in cold concentrated hydrochloric acid with the expulsion of 1% nitrogen as ammonia, and it is converted into another, nearly related substance called terieoin (Wbtl). Sericin yields no glycocoll, but leucin and a crystallizable substance called serin (amidoethylenlactic acid). The composition of the above-mentioned bodies is as follows : C H N S Conchiolin (from snail-eggs) 50.93 6.88 17.86 0.31 34.34 (Krukbnbebg )' Spongin 46.50 6.30 16.20 " 48.75 6.85 16.40 . Cornein 48.96 5.90 16.81 . Fibroin 48.33 6.37 18.31 . " 48.30 6.50 19.30 . Sericin.... 44.33 6.18 18.30 . 37.50 (Croockewitt)* (POSSBLT)' 38 33 (Krukknbebg)« 27.19 (Cramer)' 36.00 (Vl6N0N)8 30.30 (Cramer) Amyloid, so called by Viechow, is a protein substance appear- ing under pathological conditions in the internal organs, such as the spleen, liver, and kidneys, as infiltrations; and in serous mem- branes as granules with concentric layers. It probably also occurs as a constituent of certain prostate calculi. Amyloid has not been obtained pure, therefore its composition cannot be given with cer- tainty. Friedeeich and Kekule ' found 53.6; H 7.0; N 15.0; and S + 24.4^. Kuhnb and Kudxbff'" found 1.3^ sulphur. Amyloid is not related to the carbohydrates in the ordinary sense, and on boiling with acids it gives neither glucose nor any other ' Compt. rend., Tome 107. ' Ber. d. deutsch. chem. Gesellsch., Bd. 21. » Ibid., Bd. 18, and Zeitschr. f Biologie, Bd. 22. * Annal. d. Chem. u. Pharm., Bd. 48. ' Ibid., Bd. 45. • Ber. d. deutsch. chem. Gesellsch., Bd. 17. ' Journ. f . prakt. Chem. , Bd. 96. « Compt. rend., Tome 115. ' Virchow's Archlv, Bd. 16. •» Ibid., Bd. 33. 58 TEE PROTEIN SUBSTANCES. reducing sabstance. On the contrary, it yields lencin and tyrosine According to Kkaweow,' amyloid yields a residue similar to chitin on boiling with strong caustic alkali. It is insoluble in -water, alcohol, ether, dilute hydrochloric acid, and acetic acid. It is dissolved in concentrated hydrochloric acid or caustic alkali, and is converted into acid or alkali albuminates depending upon the agents employed. According to Kostjurik," amyloid is dissolved by the gastric juice, which is the reverse of older theories. A. Tscheemak ' found that the amyloid from the liver and spleen was readily soluble in alkalies, less soluble in organic acids and mineral acid, as well as by peptic or tryptic digestion or by heating in sealed tubes with water. First soluble, unchanged amyloid is formed, which is then transformed into albu- minates, albumoses, and peptones. All these products give the same color reactions as the mother-substance. Tscheemak con- siders amyloid as a coagulated proteid. Amyloid gives the xantho- proteic reaction and the reactions of Millon and Adamkiewicz. Its most important property is its behavior with certain coloring matters. It is colored reddish brown or a dingy violet by iodine ; a violet or blue by iodine and sulphuric acid ; red by methylaniline iodide, especially on the addition of acetic acid ; and red by aniline green. Amyloid is prepared by extracting the tissue with cold and then boiling water, afterwards with alcohol and ether. After boiling with alcohol containing hydrochloric acid and digesting with gastric juice, that which is insoluble is considered as amyloid. As the amyloid may be dissolved by the gastric juice (KosTJUKiiir), the utility of this method seems doubtful. ' Centralbl. f. d. med. Wissensch, 1892. ' Wien. med. Jahrbilcher, 1886. Cit. from Maly's Jahresber., Bd. 16, S. 32. 3 Zeitsclir. f. physiol. Chem., Bd. 20. CHAPTEE III. THE CARBOHYDRATES. We designate witli this name bodies which occur especially abundant in the plant kingdom. As the protein bodies form the chief portion of the solids in animal tissues, so the carbohydrates form the chief portion of the dry substance of the plant structure. They occur in the animal kingdom only in proportionately small quantities either free or in combinations with more complex mole- cules, forming compound proteids. Carbohydrates are of extraor- dinarily great importance as food for both man and animals. The carbohydrates contain carbon, hydrogen, and oxygen. The last two elements occur in the same proportion as they do in water, namely, 2 : 1, and this is the reason why the name carbohydrates has been given to them. This name is not quite pertinent, if strictly considered; because even though we have bodies, such as acetic acid and lactic, which are not carbohydrates and still have their oxygen and hydrogen in the relationship to form water, never- theless we also have sugars (rhamnose, C,H,jOJ which have these two elements in another proportion. Heretofore it was thought possible to characterize as carbohydrates those bodies which con- tained 6 atoms of carbon, or a multiple, in the molecule, but this is not considered valid at the present time. We have true carbohy- drates containing less than 6 and also those containing 7, 8, and 9 carbon atoms in the molecule. The carbohydrates have no proper- ties or characteristics in general which difEerentiate them from other bodies; on the contrary, the various carbohydrates are in many cases very different in their external properties. Under these circumstances it is very difficult to give a positive definition of carbohydrates. From a chemical standpoint we can say that all carbohydrates are aldehyde or ketone derivatives of hexatomic alcohols. The 59 60 THE CARBOITYDEATJES. simplest carbohydrates, the simple sugars or monosaccharides, are either aldehyde or ketone derivatives of these alcohols, and the more complex carbohydrates seem to be derived from these by the forma- tion of anhydrides. It is a fact that the more complex carbo- hydrates yield two or even more molecules of the simple sugars when made to undergo hydrolytic splitting. The carbohydrates are generally divided into three chief groups, namely, monosaccharides, disaccharides, axid. polysaccharides. Our knowledge of the carbohydrates and their structural rela- tionships have been very much extended by the pioneering investi- gations of KiLiANi ' and especially those of E. Fischek.'' As the carbohydrates occur chiefly in the plant kingdom it is naturally not the place here to give a complete discussion of the numerous carbohydrates known up to the present time. According to the plan of this work it is only possible to give a short review of those carbohydrates which occur in the animal kingdom or are of special importance as food for man and animals. Monosaccharides. All varieties of sugars, the monosaccharides as well as disaccha- rides, are characterized by the termination " ose," to which a root' is added signifying their origin or other relations. According to the number of carbon atoms contained in the molecule the mono- saccharides are divided into tioses, tetroses, pentoses, hexoses, heptoses, and so on. All monosaccharides are either aldehydes or ketones of hex- atomic alcohols. The first are termed aldoses and the other ketoses. Ordinary glucose is an aldose, while ordinary fruit-sugar (fructose) is a ketose. The difference may be shown by the structural formula of these two varieties of sugar : Glucose = OH,(OH).CH(OH).CH{OH).OH(OH).CH(OH).CHO; Fructose = CH,(OH).CH(OH).CH(OH).CH(OH).CO.CH,(OH). A difference is also observed on oxidation. The aldoses can be converted into oxyacids having the same quantity of carbon, while ' Ber. d. deutsch. chem. Gesellsch., Bdd. 18, 19, and 20. 'See E. Fischer's lecture: " Synthesen in der Zuckergruppe, " Ber. d. deutsch. chem. Gesellscb., Bd. 23, S. 3114. An excellent work on Carbohydrates is ToUen's "Kurzes Handbuch der Kohlehydrate, " Breslau, 1888, which gives a complete review of the literature , MONOSACCHARIDES 61 the ketoses yield acids having less carbon. On mild oxidation the aldoses yield monobasic oxyacids and dibasic acids on more energetic oxidation. Thus ordinary glucose yields gluconic acid in the first case and saccharic acid in the second. Gluconic acid = CH,(OH).[CH(OH)],.COOH; Saccharic acid = COOH.[CH(OH)]..COOH. The monobasic oxyacids are of the greatest importance in the artificial formation of the monosaccharides. These acids, as lac- tones, can be converted into their respective aldehydes (correspond- ing to the sugars) by the action of nascent hydrogen. On the other hand they may be transformed into stereo-isomeric acids on heating with chinolin, pyridin, etc., and the stereo-isomeric sugars may be obtained from these by reduction. Numerous isomers occur among the monosaccharides, and espe- cially in the hexose group. In certain cases, as for instance in glucose and fructose, we are dealing with a different constitution (aldoses and ketoses), but in most cases we have stereo-isomerism due to the presence of asymmetric carbon atoms. The monosaccharides are converted into the corresponding alcohols by nascent hydrogen. Thus ababinosb, which is a pentose, CuH,„0„ is transformed into the pentatomic alcohol, AEABiT, CjH^Oj. The three hexoses, glucose, pructose, and GALACTOSE, C,H|jOj, are transformed into the corresponding three hexatomic alcohols, sorbite, mannite, and dulcite, C,H,,0,. Inversely, the corresponding sugars may be prepared from their alcohols by careful oxidation. Similar to the ordinary aldehydes and ketones the sugars may be made to take up hydrocyanic acid. Cyanhydrines are thus formed. These addition products are of special interest in that they make the artificial preparation possible of sugars rich in ci.rbon from sugars poor in carbon. As example, if we start from glucose we obtain glucocyanhydriu on the addition of hydrocyanic acid: CH,(0H).rCH(0H)]4.C0H + HCN = CHj(OH). rCH(0H)l4.CH(0H).CN. On the saponification of glucocyanhydriu the cor- responding oxyacid is formed: CH,(0H).[CH{0H)]4.CH(0H).CN -f- 3H,0 = CHj(OH).[CH(OH)]..CH(OH).COOH + NH,. By the action of nascent hydrogen on the lactone of this acid we obtain glucoheptose, CtHuOt. The monosaccharides give the corresponding oximes with hydro- xylamin; thus glucose yields glucosoxime, CHj(OH).[CH(OH)],. CH : JN'.OH. These combinations are of importance on account of 62 TEE CABB0HTDRATE8. the fact, as found by Wohl,' that they are the starting-point in the building up of varieties of sugars, namely, the preparation of sugars poor in carbon from those rich in carbon. The monosaccharides are strong reducing bodies, similar to the aldehydes. They reduce metallic silver from ammoniacal silver solutions, and also several metallic oxides, such as copper, bismuth, and mercury oxides, on warming their alkaline solutions, ^his property is of the greatest importance in their detection and quan- titative estimation. The behavior of the sugars to phenylhydrazin acetate is of special importance. Their watery solutions first yield htdka- zoNES with phenylhydrazin acetate, and then osazonbs on lengthy warming in the water-bath. The reaction takes place as follows: (a) CHj(OH),[CH(OH)]a.CH(OH).CHO + HjN.NH.C.H. = CH,(OH).[CH(OH)]a.CH(OH)CH:N.NH.C,H. + H,0. Phenylglucoshydrazon , (h) CHj(0H)[CH(0H)]5.CH(0H).CH : N.NH.CHs + HjN.NH.C.H. = CHa(0H).[CH(0H)]3.C : CH ■ N.NH.CaH. N.NH.OeHs + H,0 -I- H,. Phenylglucosazon. The hydrogen is not evolved, but acts on a second molecule of phenylhy- drazon and splits it into anilin and ammonia : H,N.NH.C.H5 + H, = HjN.C.Hi + NH3. The osazones are yellow crystalline combinations, which differ from each other in melting-point, solubility, and optical properties and hence have received great importance in the characterization of certain sugars. They have also become of extraordinarily great importance in the study of the carbohydrates for other reasons. Thus they are very good means of precipitating sugars from solu- tion in which they occur mixed with other bodies, and they are of the greatest importance in the artificial preparation of sugars. On splitting, by the short action of gentle heat and fuming hydrochloric acid, the osazones yield phenylhydrazin hydrochloride and so-called osones, bodies which are ketoaldehydea : CH,(CiH).[CH(OH)],.C.CH:]Sr.NH.C.H. N.NH.C.H, +2H,0 + 2HC1 = 20.H..NH.NH,.HC1 -f CH,(OH).[CH(OH)],.CO.CHO. Ospne. ' Ber. d. deutsch. chem. Gesellsph., Bd. 26, S. 730. MONOSACOHAlilDES. 63 The ketoses are obtained from the osones by redaction with zinc dnst and acetic acid : CH,(OH).[CH(OH)],CO.CHO + 2H = CH,(OH).[CH(OH)]..OO.CH,(OH). If we start with an aldose, we do not get the same sugar back again, but an isomere ketose, and in this way we can convert glucose into fructose. We can also pass from the osazones to the corresponding sugars (ketoses) in other ways, namely, by direct reduction of the osazones with acetic acid 'and zinc dust. The corresponding osamin is first formed, and then on treating with nitrous acid a ketose is obtained : €H,(OH).[CH(OH)],.C.CH:N.NH.C.H. N.NH.C.H. + H,0 + 4H = Phenylgluoosazon. €H,(OH).[CH(OH)]..CO.CH,(NH,)+C.H..NH.NH,+C.H..NH, Isoglucosamin. and ■CH,(OH).[CH(OH)],.CO.CH,(NH,) + HNO, = CH,(OH).[CH(OH)]..CO.CH,(OH) + N, + H,0. Fructose. From what has been stated we see that there are various ways ■of preparing sugars artificially. They may be prepared (1) by the careful oxidation of the related alcohols ; (3) reduction of the corre- -sponding monobasic oxyacids; (3) splitting of the osazone with hydrochloric acid and a reduction of the osone; (4) direct reduction -of the osazone and treating the osamin with nitrous acid; (5) syntheses from conibinations poor in carbon (see syntheses of the hexoses). The monosaccharides are colorless and odorless bodies, neutral in reaction, with a sweet taste, readily soluble in water, generally solu- ble with difficulty in absolute alcohol, and insoluble in ether, and some of which crystallize well in the pure state. They are optically active, some Isevorotatory and others dextrorotatory; but there are also optically inactive modifications (racemic), which are formed from two optically opposed components. We designate the optical activity of the carbohydrates with the letter 1- for laevogyrate, d- for dextrogyrate, and i- for inactive. These are only partly useful. Thus dextrorotatory glucose is 64 THE CARBOHYDRATES. designated d-glucose, Isevorotatory 1-glucose, and the inactive i-glucose. Emil Pischek has used these signs in another sense. He designates by these signs the homogeneousness of the various kinds of sugars instead of their optical activity. For example, he does not designate the laevorotatory fructose, 1-fructose, but d-fructose, showing its close relation to dextrorotatory d-glucose. This designation is generally accepted, and the above-mentioned signs only show the optical properties in a few cases. Specific rotation means the rotation in degrees produced by 1 gm. substance dissolved in 1 cc. liquid placed in a tube 1 d. cm. long. The reading is ordi- narily made at -\- 20° C. and witb a bomogeneous sodium light. The sp. rota- tion with this light is represented by a(D), and is expressed by the following formula ; a(D) = ± — r, in which a represents the reading of degrees, 1 the length of the tube In decimetres, and p the weight of substance in 1 cc. of the liquid. Inversely the per cent P of substance can be calculated, when the specific rotation is known, by the formula P = — —, in which s represents the known specific rotation. A freshly prepared sugar solution often shows another rotation form, when it is allowed to stand for some time. If the rotation gradually diminishes, this is called birotation, while a gradual increase in the rotation is called half-rotation. The birotation and half-rotation may be immediately abolished by the addition of very little ammonia (1 p. m.). C. Schultze and Tollins." Many monosaccharides, but not all, ferment with yeast, and it has been shown that only those varieties of sugar containing 3, 6, or 9 atoms of carbon in the molecule are fermentable with yeast. Still amongst the hexoses we find exceptions, namely, a few artifi- cially prepared hexoses do not ferment with yeast. Various kinds of schizomycetes cause a different fermentation, such as lactic and butyric acid fermentation and mucilaginous fermentation. The simple varieties of sugar occur in part in nature as such already formed, which is the case with both of the very important sugars, grape-sugar and fructose. They also occur in great abund- ance in nature as more complex carbohydrates (di- and polysaccha- rides) ; also as ester combinations with different substances, as so-called glucosides. Among the groups of monosaccharides known at the present time, those containing less than five and more than six carbon atoms in the molecule have no great importance in zoo-chemistry, although they are of high scientific interest. Of the other two groups the hexoses are of the greatest importance, because in the ' Annal. d. Chem. u. Pharm., Bd. 371. PENTOSES. 65 past only those carbohydrates with six carbon atoms were considered as true carbohydrates. As the pentoses have been the subject of zoo-chemical investigations of late, they will also be given in short. Pentoses (C.H,„OJ. As a rule the pentoses do not occur as such in nature, but are formed in the hydrolytic splitting of several complex carbohydrates, the so-called pentosanes, especially on boiling gums with dilute mineral acids. They exist very widely distributed in the plant kingdom, and are especially of great importance in the building up of certain plant constituents. They have only thus far been found in exceptional cases in animals. Salkowski and Jasteowitz ' have found a pentose in the urine of those addicted to the morphine habit. A pentose has been found by the author" amongst the cleavage products of a nucleoproteid from the pancreas. The pentoses seem to be of importance as food for herbivorous animals. Salkowski ' and Ckemer ' have shown that the pentoses xylose, arabinose, and rhamnose are absorbed by rabbits and hens, and that these animals utilize the pentoses, and even form glycogen therefrom. The pentoses seem to be absorbed by human beings, but the views in regard to their assimilation are somewhat disputed.' The pentoses are non-fermentable, reducible aldoses. On heat- ing with sulphuric or hydrochloric acids they yield furfurol, but no levulinic acid. The furfurol passing over on distilling with hydro- chloric acid may not only be used in the detection (with aniline acetate paper which is colored red with furfurol), but also in the quantitative estimation of pentoses (or pentosanes). On warniing with hydrochloric acid containing phloroglucin a beautiful red solution is the result, and this solution gives a sharply-defined absorption band on the right of the sodium line. The most im- portant pentoses are akabinose and xylose. Arabinose (dextro-rotatory arabinose, pectin sugar) is obtained on boiling gum arable or cherry-gum with 3,"^ sulphuric acid. It crystallizes, has a sweet taste, melts at about 160°, and is strongly 1 Centralbl. f. d. med. Wissenscli., 1893, S. 337 and 593. ^ Zeitscbr. f. physiol. Chem., Bd. 19. « Centralbl. f. d. med. Wissensch., 1892, S. 387 and 593. < Zeitscbr. f. Biologie, Bd. 29. ' See Ebstein, Virchow's Arch. , Bd. 129, and Cramer, Zeitscbr. f. Biologie, Bd. 29. ■66 THE CABBOHTDRATES. dextro-rotatory. Its osazon melts at 157-158° C. The artificially prepared laevogyrate as well as the optically inactive arabinose are known. Xylose (wood sugar). This body is obtained with the previous stereo-isomeric pentose on boiling wood gums with dilute acids. It crystallizes, is feebly dextrogyrate, and gives an osazon, which melts at about 160° C. Amongst the pentoses we have ribose, obtained on the reduction of the lac- tone of ribonic acid, which is produced from arobonic acid. Shamnose, which used to be called isodulcite, is a methylpentose, CeHuOn , and is obtained from JifEerent glucosides (quercitin, xanthorhamnin, etc. ). Hexoses (C.H,.,0.). The most important and best-known simple sugars belong to ■ this group, and the remaining bodies considered as carbohydrates ((with the exception of arabinose and inosite) are anhydrides of this group. Certain hexoses, such as dextrose and fructose, occur in nature already formed, while others are produced by the hydrolytic splitting of other more complicated carbohydrates or glucosides. Others, such as manuose or galactose, are formed by the hydrolytic cleavage of natural products; while some, on the contrary, such as gulose, talose, and others, are obtained only by artificial means. All hexoses, as also their anhydrides, yield levulinic acid, CjHjO,, besides formic acid and humiis substances, on boiling with dilute mineral acids. Some of the hexoses are fermentable with yeast, while the artificially prepared hexoses do not, or at least only with great difficulty and incompletely. Some hexoses are aldoses, while others are ketoses. Belonging -to the first group we have mannose, glucose, gulose, galactose, and TALOSE, and to the other fructose, and possibly also sorbi- KOSE. We differentiate also between the d, 1, and i modifica- tions, for instance, d-, 1-, and i-glucose; hence the number of isomers is very great. The most important syntheses of the carbohydrates have been made by E. Fischer and his pupils chiefly within the members of the hexose group. A short summary of the syntheses of hexoses is given below. The first artificial preparation of glucose was made by Btjtlekow.' On treating trioxymethylen, a polymer of formaldehyde, with lime-water he ' Ann. d. Chem. u. Pharm., Bd. 120 , Compt. rend., Tome 53. DEXTROSE. 67 obtained a faintly sweetish syrup called methylenUan. Loew ' later obtained about the same product on the condensation of formaldehyde in the presence of bases, and he called this product formose. E. Fischer * has shown that this formose syrup consists of a mixture of a nonfermentable sugar, formoae, and a fermentable sugar, a-acrose. This last-mentioned hexose is the starting-point for further syntheses. The name a-aorose has been given to these bodies because they are obtained from acrolein bromide by the action of bases (Fischeh). They are also obtained admixed with ft-acrose on the oxidation of glycerin with bromine in the presence of sodium carbonate, and treating the resulting mixture of glycerin, aldehyde, and dioxyaceton, CHs(OH).CH(OH).CHO and CHj(OH).CO.CHj(OH) with alkalies. A condensation talies place with the formation of hexoses. a-acrose may be isolated from the above mixture and obtained pure by first converting it into its osazon and then retransforming this into the sugar, a-acrose is identical with i-fructose. With yeast one-half, the liEvogyrate d-fructose ferments, while the dextrogyrate 1-fructose remains. The i- and 1-fructose may be prepared in this way. On the reduction of a-acrose we obtain a acrit, which is identical with i-mannite. On oxidation of i-mannite we obtain i-mannose, from which only 1-mannose remains on fermentation. On further oxidation of i-mannose it yields i-mannonic acid. The two active mannonic acids may be separated from each other by the fractional crystallization of their strychnin or morphin salts. The two corresponding mannoses may be obtained from these two acids, d- and 1-mannonic acids, by reduction. d-fructose is obtained from d-mannose by the method given on page 63, using the osazon as an intermediate step. The d- and 1-mannnnic acids are partly converted into d- and 1-gluconic acid on heating with chinolin, and d- or 1-glucose is obtained on the reduction of these acids. 1-glucose is best prepared from 1-arabinose by means of the cyanhydrin, reaction, using 1-gluconic acid as the intermediate step. The combination of 1- and d-gluconic acid, forming i-gluconic acid, yields i-glucose on reduction. The artificial preparation of sugars by means of condensation of formal- dehyde has received special interest because, according to Baetbr's assimila- tion hypothesis of plants, formaldehyde is first formed by the reduction of carbon dioxide, and the sugars are produced by the condensation of this formal- dehyde. BOKORNT ' has shown, by special experiments on algse Spirogyra, that formaldehyde sodium sulphite was split by the living algse cells. The formal- dehyde set free is immediately condensed to carbohydrate and precipitated as starch. Among the hexoses known at the present time only dextrose, fructose, and galactose are really of physiological chemical interest ; therefore the other hexoses will only be incidentally mentioned. Dextrose (d. -glucose), gltcosb, geape-sugar, and diabetic SUGAK, occurs abundantly in the grape, and also, often accompanied with levnlose (d. -fructose), in honey, sweet fruits, seeds, roots, etc. It occurs in the intestinal tract during digestion, also in small quantities in the blood and lymph, and as traces in other animal fluids and tissues. It only occurs as traces in urine under normal 1 Journ. f. prakt. Chem., Bd. 33, and Ber. d. deutsch. chem, Gesell., Bdd. 20, 21, 32. 'Ibid., Bd. 21. 'Biplog. Centralbl., Bd. 12, S. 331 and 481. 68 TEE CARBOHTJDRATES. conditions, while in diabetes the quantity is very large. It is formed in the hydrolytic cleavage of starch, dextrin, and other compound carbohydrates, as also in the splitting of glucosides. Properties of Dextrose. Dextrose crystallizes sometimes with 1 mol. water of crystallization in warty masses or small leaves or plates, and sometimes when free from water in needles. The sugar containing water of crystallization melts even below 100° C. and loses its water of crystallization at 110° C. The anhydrous sugar melts at 146° C, and is converted into glucosan, O^H^Oj, at 170° C. with the elimination of water. On strongly heating it is converted into caramel and then decomposed. Grape-sugar is readily soluble in water. This solution, which is not as sweet as a cane-sugar solution of the same strength, is dextrogyrate and shows strong birotation. The specific rotation is somewhat dependent upon concentration of the solution, but the specific rotation of a watery solution of 1-15^ anhydrous dextrose at +20° C. may be considered as + 53°.6. Dextrose dissolves sparingly in cold, but more freely in boiling, alcohol. 100 parts alcohol -of sp.gr. 0.837 dissolves 1.95 parts anhydrous glucose at + 17°.5 C. and 37.7 parts at the boiling temperature (Anthon '). Glucose is insoluble in ether. If an alcoholic caustic-alkali solution is added to an alcoholic solution of glucose, an amorphous precipi- tate of insoluble alkali compound is formed. On warming this compound it decomposes easily with the formation of a yellow or brownish color, which is the basis of the following reaction. Mooee's Test. If a glucose solution is treated with about J of its volume of caustic potash or soda and warmed, the solution becomes first yellow, then orange, yellowish brown, and lastly dark brown. It has at the same time a faint odor of caramel, and this odor is more pronounced on acidification. Glucose forms many crystallizable combinations with NaCl, of which the easiest to obtain is (CjH^OJj.NaCl -f H,0, which forms large colorless six-sided double pyramids or rhomboids with 13.40j^ NaCl. Glucose in neutral or very faintly acid (by an organic acid) solution passes into alcoholic fermentation with beer-yeast, CjH = 2C,H,.OH + 3CO,. The most favorable temperature for this fermentation is 34° C. according to Jodblauer." Besides the • Cited from Tollens' Handbuch. « Hoppe-Seyler's Handbuch, 6. Auf . , 1893, S. 63. REACTIONS FOR DEXTROSE. 69 alcohol and carbon dioxide there are formed, especially at higher temperatures, small quantities of homologous alcohols (amyl-alco- hol), glycerin, and succinic acid. In the presence of acid milk or cheese the grape-sugar passes, especially in the presence of a base such as ZnO or CaCOj, into lactic-acid fermentation. The lactic acid may then further pass into butyric-acid fermentation : 3C,H,0, = C.H.O, + 2C0, + 4H. Grape-sugar reduces several metallic oxides, such as copper oxide, bismuth oxide, mercuric oxide, in alkaline solutions, and the most important reactions for sugar are based on this fact. Trommer's test is based on the property that glucose possesses of reducing copper-hydrated oxide in alkaline solution into sub- oxide. Treat the glucose solution with about |— J vol. caustic soda and then carefully add a dilute copper-sulphate solution. The copper-hydrated oxide is thereby dissolved, forming a beautiful blue solution, and the addition of copper sulphate is continued until a very small amount of hydrate remains undissolved in the liquid. This is now warmed and a yellow hydra'ted suboxide or red suboxide separates even below the boiling-point. If too little copper salt has been added, the test will be yellowish brown in color as in Moore's test; but if an excess of copper-salt has been added, the excess of hydrate is converted on boiling into a dark-brown hydrate which interferes with the test. To prevent these difficulties the so-called Fehling's solution may be employed. This reagent is obtained by mixing before use equal volumes of an alkaline solution of Eochelle salts and a copper-sulphate solution (see Quantitative Estimation of Sugar in the Urine in regard to concentration). This solution is not reduced or noticeably changed by boiling. The tartrate holds the excess of copper-hydrate oxide in solution, and an excess of the leagent does not interfere in the performance of the test. In the presence of sugar this solution is reduced. BoTTGBR-ALMEif's test is based on the property glucose possesses of reducing bismuth oxide in alkaline solution. The reagent best adapted for this purpose is obtained, according to Ntlai^dbr's ' modification of Almen's original test, by dissolving 4 grms. Eochelle salt in 100 parts 10^ caustic-soda solution and adding 2 grms. bismuth subnitrate and digesting on the water-bath until as much of the bismuth salt is dissolved as possible. If a glucose ' Zeitschr. f. physiol, Chem., Bd. 8. 70 TEE CARBOHYDBATES. solution is treated with about ^ vol., or with a larger quantity of the solution when large quantities of sugar are present, and boiled for a few minutes, the solution becomes first yellow, then yellowish brown, and lastly nearly black, and after a 'time a black deposit of bismuth (?) settles. On heating with phbntlhydeazin acetate a dextrose solution gives a precipitate consisting of fine yellow crystalline needles which are nearly insoluble in water but soluble in boiling alcohol, and which separate again on treating the alcoholic solution with water. The crystalline precipitate consists of phenylglucosazone. This compound melts when pure at 204-205° C. Glucose is not precipitated by a lead-acetate solution, but is almost completely precipitated by an ammoniacal basic lead-acetate solution. On warming the precipitate becomes flesh-color or rose- red (Kubner's reaction'). If a watery solution of grape-sugar is treated with benzotl- CHLOEiDE and an excess of caustic soda, and shaken until the odor of benzoylchloride has disappeared, a precipitate of benzoic-acid ester of glucose will be produced which is insoluble in water or alkali (Baumann'). If -J-l c.c. of a dilute watery solution of glucose is treated with a few drops of a 15^ alcoholic solution of a-napMJiol, the liquid is colored a beautiful violet on the addition of 1-2 c.c. concentrated sulphuric acid (Molisch"). This reaction depends on the forma- tion of furfurol from the sugar by the action of the sulphuric acid. Diazobbnzol-SUIjPHONIC acid gives with a dextrose solution made alkaline with a fixed alkali a red color, after 10-15 minutes gradually changing to violet. Orthonitrophbnyl-pbopiolic acid yields indigo when boiled with a small quantity of sugar and sodium carbonate, and this is converted into indigo-white by an excess of sugar. An alkaline solution of grape-sugar is colored deep red on being warmed with a dilute solution of picric acid. A more complete description as to the performance of these several tests will be given in detail in a subsequent chapter (on the urine) . Dextrose is prepared pure by the following simple method of SoxHLET and Tolleks, being a modification of Schwaz's* method : ' Zeitschr. f. Biologie, Bd. 30. ' Ber. d. deutsch. chem. Gesellsch. , Bd. 19 ; also Kueny, Zeitschr. f . physioU Chem., Bd. 14. ' Monatshefte f. Chem., Bd. 7, and Centralbl. f. d. med. Wissensch., 1887„ S. 34 and 49. * Tollens' Handbuch der Kohlehydrate, S. 39. D- FRUCTOSE. 71 Treat 12 litres alcohol with 480 c.c. faming hydrochloric acid and warm to 45-50° C. ; gradually add 4 kilos powdered cane-sugar^ and allow to cool after heating for 2 hours, when all the sugar will have dissolved and been inverted. To incite crystallization, some crystals of anhydrous dextrose are added, and after several days the crystals are sucked dry by the air-pump, washed with dilute alcohol to remove hydrochloric acid and crystallized from alcohol or methyl alcohol. According to Toller's it is best to dissolve the sugar in one. half its weight of water on the water-bath and then add double this volume of 90-95j^ alcohol. In detecting dextrose in animal fluids or extracts of tissues we may make use of the above-mentioned reduction-tests, the optica! determination, the fermentation, aud phenylhydrazin tests. For the quantitative estimation the reader is referred to the chapter on nrine. Those liquids containing proteids must first have these removed by coagulation with heat and addition of acetic acid, or by precipitation with alcohol or metallic salts, before testing for dextrose. In regard to the diflBcalties of operating with blood and serous fluids we refer the student to the works of Schenk/ KoHMANS",' Abblbs,' and Sebgbn".* The galosoB are stereo-isomers of dextrose and may be prepared artificially, d-gulose is obtained on the reduction of d-gulonio acid, which is derived on the reduction of glycuronic acid (see chapter on urine). Mannoses. — d-maanose, also called aeminose, is obtained with d-fructose, ore the careful oxidation of d-niannite. It is also obtained on the hydrolysis of natural carbohydrates, such as salep slime, and reserve cellulose (especially from the shavings from the ivory-nut). It is dextrorotatory, readily ferments with beer-yeast, gives a hydrazon not readily soluble in water, and an osazon. which is identical with that from d-glucose. d-fructose, also called levulose, feuit-sugar, occurs, as above stated, mixed with dextrose extensively distributed in the plant kingdom and also in honey. It is formed in the hydrolytic cleavage of cane-sugar and other carbohydrates, but it is readily obtained by the hydrolytic splitting of innlin. In extraordinary cases of diabetes mellitus we find fructose in the urine. This sugar has won special dietetic importance in diabetes on account of its being readily assimilated. Pructose crystallizes with difficulty in needles partly anhydrous and partly containing water. It is readily soluble in water, but nearly insoluble in cold absolute alcohol, though rather readily in boiling alcohol. Its watery solution is laevogyrate, but the state- ' PflUger's Archiv, Bdd. 46 and 47. 2 Centralbl. f . Physiol. , Bd. 4. * Zeitschr. f. physiol Chem., Bd. 15. * Centralbl. f. Physiol., Bdd. 4 and 8. i'-i- J LIE CARllOHYDRATES. ments ia regard to the specific rotation are quite variable. Fructose ferments with yeast, and gives the same reduction tests as dextrose and also the same osazone. It gives a combination with calcium which is less soluble than the corresponding dextrose combination. Fructose, as above stated, is best obtained by the hydrolytic splitting of inulin, by warming with faintly acidulated water. Sorbinose (sorbin) is obtained from the juice of the berry of the mountain ash under certain conditions. It is crystalline and is Isevogyrate, and is con- verted into sorbit by reduction , hence it seems to be a ketose which is stereo- isomeric.with fructose. Galactose (not to be mistaken for lactose or milk-sugar) is obtained on the hydrolytic cleavage of milk-sugar and by hydrolysis of other carbohydrates, especially varieties of gums and slime bodies. It is also obtained on heating cerebrin, a nitrogenized glucoside prepared from the brain, with dilute mineral acids. It crystallizes in needles or leaves, which melt at 168° 0. It is somewhat less soluble than dextrose in water. It is dextrogyrate, and shows multirotation. It ferments with yeast (although not as rapidly as dextrose) ; still the statements on this subject are contra- dictory. G-alactose reduces Fehling's solution to a less extent than dextrose, and 10 c.c. of this solution are reduced, according to SoxHLET, by 0.0511 gm. galactose in 1^ solution. Its phenylosazon melts at 193° C. On oxidation it first yields galactonic acid and then mucic acid. Both 1- and i-galactose have been artificially prepared. Talose is a sugar which is artificially prepared by the reduction of talonic acid. Talonic acid is obtained from d-galactonic acid by heating it witli chino- lin or pyridin to 140-150° C. Disaccharides, Some of the varieties of sugar belonging to this group occur ready formed in nature Thus we have cane-sugar and milk-sugar. Some, on the contrary, such as maltose and isomaltose, are produced by the partial hydrolytic cleavage of complicated carbohydrates. Isomaltose is besides this also obtained from glucose by reversion (see below). The disaccharides or hexobioses are to be considered as anhy- drides, derived from two monosaccharides with the exit of 1 mol. water. Corresponding to this, their general formula is Cj^H^^O,,. On hydrolytic cleavage, on the addition of water, they yield two CANE sua AH. 73 molecules of hexoses, and indeed either two molecules of the same hexose or two different hexoses. Thus: Cane-sugar -|- H,0 = glucose + fructose; Maltose + H,0 = glucose + glucose; Milk-sugar + H,0 = glucose -|- galactose. The fructose turns the polarized ray more to the left than the glucose does to the right ; hence the mixture of hexoses obtained on the cleavage of cane-sugar has an opposite rotation to the cane-sugar itself. On this account the mixture is called invert sugak, and the hydrolytic splitting is designated as inversion. This term inversion is not only used for the splitting of cane-sugar, but is also used for the hydrolytic cleavage of compound sugars into monosaccharides. The reverse reaction, whereby monosaccharides are condensed into complicated carbohydrates, is called reversion. We subdivide the disaccharides into two groups. One, to which cane-sugar belongs, where the members have not the property of reducing certain metallic oxides and of reacting with pheny^hy- drazin. The other group, on the contrary, to which the two maltoses and milk-sugar belong, the members act like monosac- charides in regard to the reduction tests, and yield osazones with phenylhydrazin. The members of this last group have the char- acter of aldehyde-alcohols; hence they are given the following formula : 0— CH, CH,(OH).[CH(OH)],.CH< i ^0— CH. [CH(OH)],CHO. Cane-sugar or Sacchakose occurs extensively distributed in the plant kingdom. It occurs to greatest extent in the stalk of the sugar-millet and sugar-cane, the roots of the sugar-beet, the trunk of certain varieties of palms and maples, in carrots, etc. Cane-sugar is of extraordinarily great importance as a food and condiment. Cane-sngar forms large, colorless monoclinic crystals. On heat- ing it melts in the neighborhood of 160° C, and on heating stronger it turns brown, forming so-called caramel. It dissolves very readily in water, and according to Scheibler ' 100 parts saturated sugar solution contains 67 parts sugar at 20° C. It dissolves with diffi- ' See ToUens' Handbuoh der Kohlehydrate, S. 121. Ti THE CARBOHTDBATES. culty in strong alcohol. Cane-sugar is strongly dextrorotatory. The specific rotation is only slightly modified by concentration, but is markedly changed by the presence of other inactive substances. The specific rotation is (a)D = + 66°. 5. Cane-sugar acts indifferently towards Mooke's test and to the ordinary reduction tests, and it does not react with phenylhydrazin. It does not ferment directly, but ferments after inversion, which can be brought about by an enzym, invertin, contained in the yeast. An inversion of cane-sugar also takes place in the intestinal canal. Concentrated snlphnric acid blackens cane-sugar very quickly even at the ordinary temperature, and anhydrous oxalic acid acts the same on warming on the water-bath. Various products are obtained on the oxidation of cane-sugar, dependent upon the variety of oxidizing material and also upon the intensity of the action. Saccharic acid and oxalic acid are the most im- portant products. The reader is referred to complete text-books on chemistry for the preparation and quantitative estimation of cane-sugar. Maltose (malt-sugae) is formed in the hydrolytic cleavage of starch by malt diastase,. saliva, and pancreatic juice. It is obtained from glycogen under the same conditions (see Chapter VIII). Maltose is also produced transitorily in the action of sulphuric acid on starch. Maltose forms the fermentable sugar of the potato or grain mash, and also of the beerwort. Maltose crystallizes with 1 mol. water of crystallization in fine white needles. It- is readily soluble in water, rather easily in alcohol, but insoluble in ether. Its solutions are dextrorotatory, and show birotation. The specific rotation is {a)D = + 137°. Maltose fements readily and completely with yeast, and acts like dextrose in regard to the reduction tests. It yields phenylmaltosa- zone on w^arming with phenylhydrazin for 1^ hours. This phenyl- maltosazone melts at 306° C. Maltose differs from dextrose chiefly in the following: It does not dissolve as readily in alcohol, has a stronger dextrorotatory power, has a feebler reducing action on Feeling's solution. 10 c.c. Pehling's solution is, according to SoxHLET,' reduced by 77.8 milligrams anhydrous maltose in approximately 1^ solution. Isomaltose. This variety of sugar is produced, as has been ' Cit. from Tollens' Handbuch, S, 153. STARCH. 75 shown by Fischer,' besides dextrin-like products, by the action of faming hydrochloric acid on glucose. It is also formed, besides ordinary maltose, in the action of diastase on starcli paste. It is also produced, with maltose, by the action of saliva or pancreatic juice (KuLZ and Vogel ') or blood-serum (Kohmann ') on starch. It also occurs in beer and in technical starch-sugar. Isomaltose dissolves very readily in water, has a pronounced sweetish taste, ferments but slowly. It is dextrorotatory, and has very nearly the same power of rotation as maltose. Isomaltose is characterized by its osazone. This forms fine yellow needles, which begin to form drops at 140° C. and melt at 150-153° C. It is rather easily soluble in hot water. Milk-sugar (lactose). As this sugar occurs exclusively in the animal world, in the milk of human beings and animals, it will be treated of in a following chapter (on milk). Trehalose is a hexobiose found in fungi. Melebiose is a saccharose obtained with d-fructose in the partial hydrolytic cleavage of raffinose (a hexotriose) occurring in beetroot molasses. Melebiose splits into galactose and glucose. Polysaccharides. If we exclude the hexotrioses and the few remaining sugar-like polysaccharides, this group includes a great number of very complex carbohydrates, which occur only in the amorphous condition or not as crystals in the ordinary sense. Contrary to the bodies belonging to the other groups, these have no sweet taste. Some are soluble in water, while others swell up therein, especially in warm water, and finally are neither dissolved nor visibly changed. Polysaccharides are ultimately converted into monosaccharides by hydrolytic cleavage. The polysaccharides (not sugar-like) are ordinarily divided into the following chief groups: starch group, gum and vegetable-muci- lage group, and cellulose group. Starch Group (C,H,„OJx. Starch, Ajiylum. (C,H, OJx. This substance occurs in the plant kingdom very extensively distributed in the difierent parts of ' Ber. d. deutscli. ohem. Gesellsch., Bd. 23, S. 3687. = Zeitscur. f. Biologic, Bd. 31, ' Centralbl f , d. med. Wissenscb., 1893, S. 849. 1Q THE CARBOHYDRATBS. the plant, especially as reserve food in the seeds, roots, tubers, and trunk. . Starch is a white, odorless, and tasteless powder, consisting of small grains, which have a stratified structure and different shape and size in different plants. According to the ordinary opinion the starch-grains consist of two different substances, starch GKANULOSB and STARCH CELLULOSE, of which the first only goes into solution on treatment with diastatic enzymes. Starch is considered insoluble in cold water. The grains swell np in warm water and burst, yielding a paste. Starch is insoluble in alcohol and ether. On heating starch with water alone, or heating with glycerin to 190° C, or on treating the starch-grains with 6 parts dilute hydrochloric acid of sp. gr. 1.06 at ordinary temperature for 6 to 8 weeks,' it is converted into soluble starch (amylo DEXTRIN", amidulin). Soluble starch is also formed as an intermediate step in the conversion of starch into dextrose by dilute acids or diastatic enzymes. Starch-granules swell up and form a pasty mass in caustic potash or soda. This mass gives neither Moore's nor Trommer's test. Starch-paste does not ferment with yeast. The most characteristic test for starch is the blue coloration produced by iodine in the presence of hydroiodic acid or alkali iodides." This blue coloration disappears on the addition of alcohol or alkalies, and also on warming, but reappears again on cooling. On boiling with dilute acids starch is converted into glucose. In the conversion by means of diastatic enzymes we have as a rule, besides dextrin, maltose, and isomaltose, only very little glucose. We are considerably in the dark as to the kind and number of intermediate products produced in this process (see dextrin). Starch may be detected by means of the microscope and by the iodine reaction. Starch is quantitatively estimated, according to Sachsse's method,' by converting it into sugar by hydrochloric acid and then determining the sugar by the ordinary methods. Inulin, (CjH,„Oj)x + H,0, occurs in the underground parts of many compositse, especially in the roots of the inula helenium, the tubers of the dahlia, the varieties of helianthus, etc. It is ordi- narily obtained from the tubers of the dahlia. ' See Tollens' Handbuch. S. 187. ' Mylius, Ber. d. deutscb. chem. Gesellsch., Bd. 30, S. 683, and Zeitsclir. f. physiol. Chem., Bd. 11. » Tollens' Handbuch, S. 184. GUMS AND VEGETABLE MUCILAGEH. 77 Inulin. forms a white powder, similar to starch, consisting of sphseroid crystals, which are readily soluble in warm water without forming a paste. It separates slowly on cooling, but more rapidly on freezing. Its solutions are laevogyrate and are precipitated by alcohol, and are only colored yellow with iodine. Inulin is con- verted into the laevogyrate monosaccharide fructose, on boiling with dilute sulphuric acid. Diastatic enzymes have no or very slight action on. inulin.' Lichenin (moss-staech) occurs in many lichens, namely, in Iceland moss. It is not soluble in cold water, but swells up into a jelly. It is soluble in bot water, forming a jelly on allowing the concentrated solution to cool. It is colored yellow by iodine, and yields glucose on boiling witb dilute acids. Liclienin is not changed by diastatic enzymes such as ptyalin or amylopsin (NlLSON*). Glycogen. This carbohydrate, which stands to a certain extent between starch and dextrin, is principally found in the animal kingdom, hence it will be treated in a subsequent chapter (on the liver). The Gums and Vegetable Mucilages (C,H,„OJx. These bodies may be divided into two chief groups, according to their origin and occurrence, namely, the dextrin group and the vegetable gums or mucilages. The dextrines stand in close relation- ship to the starches and are formed therefrom as intermediate products in the action of acids and diastatic enzymes. The vari- ous kinds of vegetable gums and vegetable mucilages occur, on the contrary, as natural products in the plant kingdom, and some may be separated from certain plants as amorphous, transparent masses and others may be extracted from certain parts of the plant, such as the wood and seeds, by proper solvents. The dextrines yield as final products only hexoses, and indeed only dextrose on complete hydrolysis. The vegetable gums and the mucilages yield, on the contrary, not only hexoses, but also an abundance of pentoses (gum arable and wood-gum), d-galactose occurs often amongst the hexoses, and as differentiation from the dextrines they yield mucic acid on oxidation with nitric acid. The dextrines, as well as the ordinary varieties of gums and mucilages, are precipitated by alcohol. Basic lead acetate precipitates the gums and mucilages, but not the dextrins. ' ToUens' Handbuch, S. 303. ' Upsala Lakaref, forh., Bd. 28. 78 THE CARB0HTDEATE8. Dextrin (British gum) is produced on heating starch to 200- 210° C, or by heating starch, which has previously been moistened with water containing a little nitric acid, to 100-110° C. Dex- trins are also produced by the action of dilute acids and diastatic enzymes on starch. We are not quite clear in regard to the steps taking place in the above processes, but the ordinary views are as follows : Soluble starch is the first product, from which a dextrin, erythrodextrin, which is colored red by iodine, and sugar are formed by hydrolytic splitting. On further splitting of this erythrodextrin more sugar and a dextrin, achroodextrin, which is not colored by iodine, is formed. From this achroodextrin after successive splittings we have sugar and dextrins of lower molecular weights formed, until finally we have sugar and a dextrin, malto- dextrin, which refuses to split further, as final products. The views are rather contradictory in regard to the number of dextrins which occur as intermediate steps. The sugar formed is isomaltose, from which maltose and very little dextrose are produced. Another view is that first several dextrins are formed consecutively in the successive splitting with hydration, and then finally the sugar is formed by the splitting of the last dextrin. ' The various dextrins have not as yet been separated from each other, nor isolated as chemical individuals ; hence the characteristic properties and reactions can only be given for the dextrins in general. The dextrins appear as an amorphous, white or yellowish-white powder which is readily soluble in water. Their concentrated solutions are viscid and sticky, similar to gum solutions. The dextrins are dextrogyrate, the specific rotation of maltodextrin being («)!) = + 174°. 5. They are insoluble or nearly so in alcohol, and insoluble in ether. "Watery solutions of dextrins are not precipitated by basic lead acetate. Dextrins dissolve copper oyy hydrate in alkaline liquids, forming a beautiful blue solution. The question whether or not perfectly pure dextrin reduces Fbhling's solution is undecided. According to Brucke ' a non- reducible dextrin may be obtained by warming a solution of achroo- dextrin with an excess of alkaline copper solution and then ' In regard to the new theories see Lintner and DilU, Ber. d. deutsch. chem. Gesellsch , Bd. 26, S. 2533, and Scheiblerand Mittelmeier, ibid., Bd. 33, S. 3060, and Bd, 26, S. 3930. ' Vorlesungen uber Physiologie. Wien, 1874. S. 381. CELLULOSE. Y9 precipitating with alcohol. According to Scueihler and Mittel- meier' the dextrin obtained by the action of acid is a polysaccharide •of an aldehydic nature, hence it acts as a reducing agent. The dextrins are not directly fermentable. The behavior of the various dextrins to iodine has been given above, but it must be remarked that, according to Musculus and Meter," erythrodextrin is only a mixture of achroodextrin with a little soluble starch. The vegetable gums are soluble in water, forming solutions which are viscid but may be filtered. We designate, on the contrary, as vegetable mucilages those varieties of gum which do not or only partly dissolve in water, and which swell up therein to a greater or less extent. The natural varieties of gum and mucilage to which several generally known and important substances, such as gum arable, wood-gum, cherry-gum, salep and quince mucilage, and probably also the little-studied pectin substances, belong will not be treated of in detail, because of their unimportance from a zoo- physiological standpoint. The Cellulose Group (C,H,„OJx. Cellulose is that carbohydrate, or perhaps more correctly mix- ture of carbohydrates, which forms the chief constituent of the walls of the plant-cells. This is true for at least the walls of the young oells, while in the walls of the older cells the cellulose is extensively incrusted with a substance called lignin. The true celluloses are characterized by their great insolubility. They are insoluble in cold or hot water, alcohol, ether, dilute acids, and alkalies. We have only one specific solvent for cellulose, and that is an ammoniacal solution of copper oxide called Schweitzer's reagent. The cellulose may be precipitated from this solvent by the addition of acids, and obtained as an amorphous powder after washing with water. Cellulose is converted into a substance, so-called amyloid, which gives a blue coloration with iodine by the action of concen- trated sulphuric acid. By the action of strong nitric acid or a mixture of nitric acid and concentrated sulphuric-acid celluloses is converted into nitric-acid esters or nitro-cellulose, which are highly explosive and have found great practical use. » Ber. d. deutsch. chem. Gesellsch., Bd. 23, S. 3060, and Bd. 36, S. 3930. ' Zeitschr. f. physiol. Chem., Bd. 4, S. 451. 80 THE CARBOHYDRATES. The ordinary celluloses when treated at the ordinary tempera- ture with strong sulphuric acid and then boiled for some time after diluting with water is converted into dextrose. Other varieties of cellulose have a different behavior, namely, we have a cellulose which yields mannose on the preceding treatment. This substance, called mannoso-cellulose by E. Schulze," occurs in the coffee-bean, as well as in the cocoanut and sesame cake, and is not to be con- sidered as belonging to the hemicellulose group. Hemicelluloses are, according to E. Schulze, those constituents of the cell-wall related to cellulose which differ from the ordinary cellulose by dissolving on heating with strongly diluted mineral acids, such as 1.25^ sulphuric acid, with a splitting into monosac- charides. The sugars produced hereby are of different kinds. The hemicellulose from the yellow lupin yields galactose and arabinose, from the rye and wheat bran arabinose and xylose, and from the ivory-nut — called reserve cellulose by Reiss " — mannose. The cellulose, at least in part, undergoes decomposition in the intestinal tract of man and animals. A closer discussion of the nutritive value of cellulose will be given in a future chapter (on digestion). The great importance of the carbohydrates in the animal economy and to animal metabolism will also be given in following chapters. 'Zeitsclir. f. physiol. Chem., Bd. 16. 'Ber. d. deutsch. chem. Gesellsch., Bd. 23. CHAPTEE rV. THE ANIMAL FATS. The fats form the third chief group of the organic foods of man and animals. They occur very widely distributed in the animal and plant kingdoms. Pat occurs in all organs and tissues of the animal organism, though the quantity may be so variable that a tabular exhibit of the amount of fab in different organs is of little interest. The marrow contains the largest quantity, having over 960 p. m. The three most important deposits of fat in the animal organism are the intermuscular connective tissue, the fatty tissue in the abdominal cavity, and the subcutaneous connective tissues. Amongst the plants the seeds and fruit, and in certain instances also the roots, are rich in fat. The fats consist nearly entirely of so-called neutral fats with only very small quantities of fatty acids. The neutral fats are esters of the triatomic alcohol, glycerin, with monobasic fatty acids. These esters are triglycerides, that is, the three hydrogen atoms of the hydroxyl of the glycerin are replaced by the fatty-acid radicals, and their general formula is therefore OjH^.Oj.E,. The animal fats consist chiefly of esters of the three fatty acids, stearic, palmitic, and oleic acids. In the plant kingdom triglycerides of other fatty acids, such as laaric acid, linoleic acid, erucic acid, etc., sometimes occur abundantly. The animal fats are of the greatest interest and consist of a mixture of varying quantities of teisteaein, teipalmitin, and TRIOLEIN, having an average elementary composition of C 76.5, H 12.0, and 11.5 per cent. Fats from different species of animals, and even from different parts of the same animal, have an essentially different consistency, depending upon the relative amounts of the different fats. In solid fats— as tallow — tristearin and tripalmitin are in excess, while the 81 82 THE ANIMAL PATS. less solid fats are characterized by a greater abundance of tripal- mitin and triolein. This last-mentioned fat is found in greater quantities proportionally in cold-blooded animals, and this accounts ior the fat of these animals remaining fluid at temperatures at which the fat of warm-blooded animals solidifies. Human fat from different organs and tissues contains, in round numbers, 670-800 J), m. triolein. The melting-point of different fats depends upon the composition of the mixtures, and it not only varies for fat from different tissues of the same animal, but also for the fat from the same tissues in various kinds of animals. Neutral fats are colorless or yellowish and, when perfectly pure, odorless and tasteless. They are lighter than water, on which they float when in a molten condition. They are insoluble in water, dissolve in boiling alcohol, but separate on cooling, — often in crystals. They are easily soluble in ether, benzol, and chloroform. The fluid neutral fats give an emulsion when shaken with a solu- tion of gam or albumin. With water alone they give an emulsion only after vigorous and prolonged shaking, but the emulsion is not persistent. The presence of some soap causes a very fine and per- manent emulsion to form easily. Fat produces spots on paper which do not disappear; it is not volatile; it boils at about 300° 0. w^ith partial decomposition, and burns with a luminous and smoky flame. The fatty acids have most of the above-mentioned proper- ties in common with the neutral fats, but differ from them in being soluble in alcohol-ether, in having an acid reaction, and by not giving the acrolein test. The neutral fats generate a strong irritat- ing vapor of acrolein, due to the decomposition of glycerine, C3Hj(0H)3 — 2HjO — CjHjO, when heated alone, or more easily when heated with potassium bisulphate or with other substances removing water. The neutral fats may be split by the addition of the constituents of water according to the following equation: C3H^(0E)3 -\- SH^O = C3H^(0H)3 + SHOE. This splitting may be produced by the pancreatic enzyme or by superheated steam. "We most frequently decompose the neutral fats by boiling them with caustic alkali not too concentrated, or, still better (in zoochemical researches), with an alcoholic potash solution. By this procedure, which is called sapouification, the alkali salts of the fatty acids (soaps) are formed. If the saponification is made with lead oxide, then lead-plaster, .lead-salt of the fatty acids, is produced. We do not only call the STEARIN AND PALMITIN. 83 splitting of neutral fats by alkalies saponification, but also the splitting of neutral fats into fatty acifls and glycerin in general. On keeping fats for a long time in contact with air they undergo a change, becoming yellow in color, acid in reaction, and develop an unpleasant odor and taste. It becomes rancid, and in this change a part of the fat is split into fatty acids and glycerin, and then an oxidation of the free fatty acids takes place, producing volatile bodies of an unpleasant odor. The rancidity is not due, as shown by Gaffky and Eitsert,' to the presence of microbes. According to these investigators the change is due to the combined action of air and light. In certain animal fats, as in milk-fat, small quantities of triglycerides of lower fatty acids, such as butyric, caproic acids, etc., occur. The same is observed in fat from certain animals, although little studied. Still these are of minor importance as compared to the three most important fats of the animal body, namely, tristearin, tripalmitin, and triolein. Stearin, or tristearin, C,II^(C,,Hj^O,)„ occurs especially in the solid varieties of tallow, but also in the vegeta,ble fats. Stearic acid, C„H„0„ is found in the free state in decomposed pus, in the expectorations in gangrene of the lungs, and in cheesy tuberculous masses. It occurs as lime-soap in excrements and adipocere, and in this last product also as an ammonia soap. It perhaps exists as sodium soap in the blood, transudations, and pus. Stearin is the hardest and most insoluble of the three ordinary neutral fats. It is nearly insoluble in cold alcohol and soluble with great difficulty in cold ether (225 parts). It separates from warm alcohol on cooling as rectangular, less frequently as rhombical plates. The statements in regard to the melting-point are some- what varied. Pure stearin, according to Heintz," melts between + 55° and 71°. 5. The stearin from the fatty tissues (not pure) melts at + 63° C. Stearic acid crystallizes (on cooling from boiling alcohol) in large, shining, long-rhombical scales or plates. It is less soluble than the other fatty acids and melts at 69.3° C. Its barium salt contains 19.49^ barium. Palmitin, tripalmitin, C3H.(C„H.,0,)3. Of the two solid varieties of fats, palmitin is the one which occurs in predominant ' Naturwissenschaftl. Wocbensclir. , 1890. 'Annal. il, Chem, u. Pharm . Bd. 92, S, 300. 84: TEE ANIMAL FATS. quantities in human fat (Langek).' Palmitin is present in all animal fats and in several kinds of vegetable fats. A mixture of stearin and palmitin was formerly called MAEGAKiiir. Palmitic acid, C^Hj^O^. As to occarreuce, about the same remarks apply as to stearic acid. The mixture of these two acids has been called margaric acid, and this mixture occurs — often as very long, thin, crystalline plates — in old pus, in expectorations from gangrene of the lungs, etc. Palmitin crystallizes, on cooling from a warm saturated solution in ether or alcohol, in starry rosettes of fine needles. The mix- ture of palmitin and stearin, called margarin, crystallizes, on cool- ing from a solution, as balls or round masses which consist of short or long, thin plates or needles which often appear like blades of grass. Palmitin, like stearin, has a variable melting and solidifying point, depending upon the way it has been previously treated. The melting-point is often given as + 63°. According to other statements" it melts at 50°. 5 0., solidifies on fdrther heat and melts again at 66°. 50 C. Palmitic acid crystallizes from an alcoholic solution in tufts of fine needles. It melts at + 63° C. ; still the admixture with stearic acid, as Hbiktz has shown, essentially changes the melting and solidifying points according to the relative amounts of the two acids. Palmitic is somewhat more soluble in cold alcohol than stearic acid; but they have about the same solubility in boiling alcohol, ether, chloroform, and benzol. Olein, TEiOLBiN, C,H^(C,jH330j3, is present in all animal fats and in greater quantities in plant fats. It is a solvent for stearin and palmitin. Oleic acid, elaic acid, C.^Hj^O,, occurs probably as soaps in the intestinal canal during digestion and in the chyle. Olein is, at ordinary temperatures, a nearly colorless oil of a specific gravity of 0.914, without odor or marked taste. It solidifies ' in crystalline needles at — 5° C. It becomes rancid quickly if exposed to the air. It dissolves with difficulty in cold alcohol, but more easily in warm alcohol oi- in ether. It is converted into its isomer, blaidust, by nitrous acid. Oleic acid forms at ordinary temperature a colorless, tasteless, and odorless oily liquid which solidifies in crystals at about + 4° C, which then melt again at -f li" 0. On being heated it yields, ' Monatshef te t, Chem. , Bd. 2. 'K. Benedikl, Analyse der Fette. Berlin, 1886. S. 39. DETECTION OF FATS. 85 besides volatile fatty acids, sebacic acid, C,„Ii,jO„ which crystal- lizes in shining plates and melts at -f 127° C. Oleic acid is con- verted by nitrons acid into its isomer, elaidic acid, which is a solid, melting at -f 45° C. Oleic acid is insoluble in water, but dissolves in alcohol, ether, and chloroform. With concentrated sulphuric acid and some cane-sugar it gives a beautiful red or reddish-violet liquid whose color is similar to that produced in PETTEifKOFER's test for bilc-acids. If the watery solution of the alkali combinations of oleic acid is precipitated with lead acetate, a white, tough, sticky mass of lead oleate is obtained which is not soluble in water and only slightly in alcohol, but is soluble in ether {differing from the lead-salts of the •other two fatty acids). An acid related to oleic acid, dobglic acid, which is solid at 0° C, liquid at -{- 16°, and soluble in alcohol, is found in the blubber of the Balmna rostrata. Kuebatopf ' has demonstrated the presence of linoleic acid in the fat of the silurus, sturgeon, seal, and certain other animals. To detect the presence of fat in an animal fluid or tissue the fat must first be extracted with ether. After the evaporation of the •ether the residue is tested for fat and the acrolein test must not be neglected. If this test gives positive results, then neutral fats are present; if the results are negative, then only fatty acids are present. If the above residue after evaporation gives the acrolein test, then a small portion is dissolved in alcohol-ether free from acid and which has been colored bluish violet by tincture of alkanet. If the color becomes red, a mixture of neutral fat and fatty acids is present. In this case the fat is treated in the warmth with a soda solution and evaporated on the water-bath, constantly stirring until all the water is removed. The fatty acids hereby combine with the alkali, forming soaps, while the neutral fats are not saponi- fied under these conditions. If this mixture of soaps and neutral fats is treated with -water and then shaken with pure ether, the neutral fats are dissolved, while the soaps remain in the watery solution. The fatty acids may be separated from this solution by the addition of a mineral acid which sets the acid free. The neutral fats separated from the soaps by mean of ether are often contaminated with cholesterin, which must be separated in quantitative determinations by saponification with alcoholic caustic potash. The cholesterin is not attacked by the caustic alkali, while the neutral fats are saponified. After the evaporation of the alcohol the residue is dissolved in water and shaken with ether, which dissolves the cholesterin. The fatty acids are separated from the watery solution of the soaps by the addition of a mineral acid. If a mixture of soaps, neutral fats, and fatty acids is originally ' Maly's Jahresber., Bd, 32. 86 THE ANIMAL FATS. present, it is treated first with water, then agitated with ether fre& from alcohol, which dissolves the fat and fatty acids, while the soaps remain in the solution, with the exception of a very small amount which is dissolved by the ether. To detect and to separate the different varieties of neutral fats from each other it is best first to saponify them with alcoholic potash, or still better with sodium alcoholate, according to Kossel, Obekmullee, and Keijger.' After the evaporation of the alcohol they are dissolved in water and precipitated with sugar of lead. The lead oleate is then separated from the other two lead-salts by repeated extraction with ether. The residue insoluble in ether is decomposed on the water-bath with an excess of soda solution, evaporated to dryness, finely pulverized, and extracted with boiling alcohol. The alcoholic solution is then fractionally precipitated by barium acetate or barium chloride. In one fraction the amount of barium is determined, and in the other the melting-point of the fatty acid set free by a mineral acid. The fatty acids occurring originally in the animal tissues or fluids as free acids or as soaps are converted into barium salts and investigated as above. The fats are poor in oxygen but rich in carbon and hydrogen. They therefore represent a large amount of chemical potential energy, and they correspondingly yield large quantities of heat on combustion. They *ake first rank amongst the foods in this regard and are therefore of very great importance in animal life. "We will speak more in detail of this significance, also of fat formation and the behavior of the fats in the body, in the following chapters. The LBCiTHiKS, which stand in close relationship to the fats, will be treated of in a subsequent chapter. The following bodies append themselves to the ordinary animal fats. Spermaceti. In the living spermaceti or white whale there is found in a large cavity in the skull an oily liquid called spermaceti, which on cooling after death separates into a solid crystalline part, ordinarily called spermaceti, and into a liquid, spermacbti-oil. This last is separated by pressure. Sper- maceti is also found in other whales and in certain species of dolphin. The puriiied, solid spermaceti, which is called cetin, is a mixture of esters of fatty acids. The chief constituent is the cetyl-palmitic ester mixed with small quantities of compound ethers of lauric, myrisitic, and stearic acids with radicals of the alcohols, lethal, CuHjj.OH, mbthal, C14HJ9.OH, and stbthai,, CibHst.-OH. Cetin is a snow-white mass shining like mother-of-pearl, crystallizing in plates, brittle, fatty to the touch, and which has a varying melting-point of -j- 30° to 50° C, depending upon its purity. Cetin is insoluble in water, but dissolves easily in cold ether or volatile and fatty oils. It dissolves in boiling alcohol, but crystallizes on cooling. It is saponified with diflBculty by a solu- tion of caustic potash in water, but with an alcoholic solution it saponifies, readily and the above-mentioned alcohols are set free. 'Zeitschr. f. physiol. Chem., Bdd. 14, 15, and 16. ETIIAL 87 Ethal, or cetyl alcohol, CieHas.OH, which also occurs in the coccygeal gland of ducks and geese (Dss Jonoe>) and in smaller quantities in beeswax, forms white, transparent, odorless, and tasteless crystals which are insoluble in water but dissolve easily in alcohol and ether. Ethal melts at 49. 5° ( '. Spermaceti-oil yields on saponification valerianic acid, small amounts of solid fatty acids, and physetolbic acid. This acid forms colorless and odor- less, needle-shaped crystals which easily dissolve in alcohol and ether and melt at + 34° C. Beeswax may be treated here as concluding the subject of fats. It contains three chief constituents: 1 Cbeotic acid, Cj7H640a, which occurs as cetyl ether in Chinese wax and as free acid in ordinary wax. It dissolves in boiling alcohol and separates as crystals on cooling. The cooled alcoholic extract of wax contains (3) ceeolbin, which is probably a mixture of several bodies, and (3) MYKisiN, which forms the chief constituent of that part of wax which is insoluble in warm or cold alcohol. Myrisin consists chiefly of pabnitic-acid ether of melissyl (myricyl) alcohol, CsoHai.OH. This alcohol is a silky, shining, crystalline body melting at -|- 85° C. ' Zeitschr. f. physiol. Chem., Bd. 3. ' CHAPTER V. THE ANIMAL CELL. The cell is the unit of the manifold, variable forms of the organism ; it forms the simplest physiological apparatus, and as such is the seat of chemical processes. It is generally admitted that all chemical processes of importance do not take place in the animal fluids, but transpire in the cells, which may be considered as the chemical laboratory of the organism. It is also principally the cells which, through their greater or less activity, regulate or govern the range of the chemical processes and also the intensity of the total exchange of material. It is natural that the chemical investigation of the animal cell «hould in most cases coincide with the study of those tissues of which it forms the chief constituent. Only in a few cases can the cells be directly, by relatively simple manipulations, isolated in a rather pure state from the tissues, as, for example, in the investi- gation of pus or of tissue very rich in cells. But even in these cases the chemical investigation may not lead to any positive results in regard to the constituents of the uninjured living cells. By the process of chemical transformation new substances may be formed on the death of the cell, and at the same time physiological con- stituents of the cell may be destroyed or transported into the snr- Tounding menstruum and therefore escape investigation. For this and other reasons we possess only a very limited knowledge of the constituents and the composition of the cell, especially of the living one. While young cells of different origin in the early period of their existence may show a certain similarity in regard to form and chemical composition, they may, on further development, not only take the most varied forms, but may also ofEer from a chemical standpoint the greatest diversity. As a description of the constit- PSOTOPLASM. 89 ueats and composition of the different cells occurriDg in the animal organism is nearly equivalent to a demonstration of the chemical properties of most animal tissues, and as this exposition will be found in their respectiye chapters, we will here only discuss the chemical constituents of the young cells or the cells in general. In the study of these constituents we are confronted with another difficulty, namely, we must difEerentiate by chemical research between those constituents which are essentially necessary for the life of the cells and those which are casual, i.e., stored up as reserve ma;terial or as metabolic products. In this connection we have only been able, thus far, to learn of certain substances which seem to occur in every developing cell. Such bodies, called PRIMARY by KossEL," are, besides water and certain mineral con- stituents, proteids, nucleoproteids or nuclein, lecithins, glycogen (?), and cholesterin. Those bodies which do not occur in every developing cell are called secondary. Amongst these we liave fat, glycogen (?), pigments, etc. It must not be forgotten that it is still possible that other primary cell constituents may exist, but unknown to us, and we also do not known whether all the primary constituents of the cell are necessary or essential for the life and functions of the same. We do not know, for example, whether the ever-present cholesterin is an excretory product of the meta- bolism within the cell or whether it is necessary for the life and development of the same. Another important question is the division of the various cell constituents between the two morphological components of the cell, namely, the protoplasm and the nucleus. This is very difficult to decide for many of the constituents, nevertheless it is appropriate to difEerentiate between the protoplasm and the nucleus. The Frotoplasm of the developing cell consists during life of a semi-solid mass, contractile under certain conditions and readily changeable, which is rich in water and whose chief portion consists of protein substances. If the cell be deprived of the physiological conditions of life, or if exposed to destructive exterior influences, such as the action of high temperatures, of chemical agents, or indeed of distilled water, the protoplasm dies. The albuminous bodies which it contains coagulate at least partially, and other chemical changes are found to take place. The alkaline reaction ' Verhandl. der physiol. Geaellsch. zu Berlin, 1890-91, Nos. 5 and 6. 90 THE ANIMAL CELL. of the living cell may be converted into an acid by the appearance of paralactic acid, and the carbohydrate, glycogen, which habitually occurs in the young generative cell may after its death be quickly changed and consumed. The question as to the structure of the protoplasm has been answered in various ways. According to the ordinary view the body of the cell, the cytoplasm, contains a network, the spongio- PLASM, in the meshes of which is a more homogeneous, structureless substance, hyaloplasm. It has also been admitted that the spongioplasm consists of a special substance, plastin, which will be described later, and that the hyaloplasm consists chiefly of proteid. Besides this the protoplasm contains granules of various kinds which behave differently with dyes and sometimes vacuoles contain- ing fluid. The proteids of the protoplasm consist, according to the general view, chiefly of globulins. Albumins have also been found besides the globulins. There is no doubt at present that the albumins occur in the cells only as traces, or at least only in trifling quanti- ties. The presence of globulins can hardly be disputed, although certain cell constituents described as globulins have been shown on closer investigation to be nucleoalbumins or nucleoproteids. This is true for the so-called /S-globulin isolated from the lymphatic glands by Halliburton. On the contrary, according to this investigator, the so-called a-cell globulin, coagulating at 47-50° C, and occurring in all cells, is a true globulin; ' In opposition to the view that the chief mass of the anima.1 cell consists of true proteids, the author ' expressed the opinion several years ago, that the chief mass of the protein substances of the cells does not consist of proteids in the ordinary sense, but con- sists of more complex phosphorized bodies, and that the globulins and albumins are to be considered as nutritive material for the cells or as destructive products in the chemical transformation of the protoplasm. This view has received substantial support by inves- tigations within the last few years. Alex. Schmidt ' has come to the view, by investigations on various kinds of cells, that they contain only very little proteid, and that the chief mass consists of ' See Halliburton, On tlie Chemical Physiology of the Animal Cell 1893 No. 1, King's College Physiol. Laboratory. 2 Pflilger's Arch., Bd. 36, S. 449. 3 Alex. Schmidt, Zur Blutlehre, Leipzig. 1892. PROTEIN SUBSTANCES OF THE CELLS. 91 very complex protein sabstances. Lilibnfeld has also fouad on a quantitative analysis of leucocytes from the thymus gland only 1.76^ proteid (in the dried substance), in the ordinary sense. The protein substances of the cells consist chiefly of compound vroteids, and these are divided between the glycoproteid and the nucleo-proteid groups. It is impossible at present to state the extent of nucleoalbumins in the cells because thus far in most cases no exact difference has been made between them and the nucleo- proteids. Hoppe-Seylbr ' calls vitellin a regular constituent of all protoplasm. This body used to be considered as a globulin, but later researches have shown that the so-called vitelline bodies may be of various kinds. Certain vitellins seem to be nucleoalbumins, and it is therefore very probable that cells habitually contain The nucleoproteids take a very prominent place among the com- pound proteids of the cell. The various substances isolated by different investigators from animal cells, such as tissue fibrinogen (WooLDRiDGE ") , cytogloUn &nd. preglohultn (Alex. Schmidt'), or nucleoMston (Kossel and Lilienfeld''), belong to this group. The cell constituent which swells up to a sticky mass with common salt solution and called Kovida's hyaline substance, also belongs to this group. The above-mentioned different protein substances have only been simply designated as constituents of the cells. The next question is which of these belong to the protoplasm and which to the nucleus. At present we can give no positive answer to this question. According to Kossel and Lilienfeld,' the cell nucleus of the leucocytes contains a nucleoproteid, besides nucleins, as chief constituent, and sometimes perhaps also nucleic acid (see below), while the body of the cells contains chiefly pure proteids besides other substances, and only a little nucleoalbumin, containing a very small quantity of phosphorus. This view coincides well with the observations of Lilienfeld on the behavior of the protoplasm and cell nucleus on one side as compared with the proteids and nnclein 1 Physiol. Chem., 1877-1881, S. 76. ' Die Gerinnung des Blutes. Leipzig, 1891. * Zur Blutlelire. 4 Lilienfeld, Zeitschr. f. physiol. Chem., Bd. 18. ' Ueher die Wahlverwandschaft der Zellelemente zu gewissen Parbstoffen. Verhandl. d. physiol. Qesellsch. zu Berlin, No. 11, 1893. 92 THE ANIMAL CELL. substances with certain coloring matters; bat it seems to be inconsistent with the quantibative composition of the leucocytes as found by Liliebtfeld. If we admit, according to Kossel and LiLiBNFELD, that the nucleoproteid, called by them nucleohiston, belongs only to the nucleus of the leucocytes of the thymus gland, then 77.45 parts of the 79.21 parts of proteins in 100 parts of the dried substance belongs to the nucleus and only 1.76 parts to the protoplasm. As the lymphocytes of the thymus gland of the calf contain only one nucleus, in which the mass of the nucleus sur- passes that of the cytoplasm, it is natural that the relative propor- tion of the various protein substances in these cells cannot be taken as a standard for the composition of other cells richer in cytoplasm. Complete investigations in regard to the distribution of protein substances in the protoplasm and nucleus of other cells have not been made. If we consider for the present that the cells rich in protoplasm contain, as a rule, only very little true proteid, we are hardly wrong in considering it probable that the protoplasm con- tains chiefly nacleoalhumins and compound proteids besides traces of albumin and a little globulin. These compound proteids are in certain cases glycoproteids,. but otherwise nucleoproteids which differ from the nucleoproteids of the nucleus in being pooi-er in phosphorus, besides containing a great deal of proteid and only less of the prostetic group, and hence have no specially pronounced acid character. The nucleoproteids of the nucleus are on the contrary, as shown by LiLiENFELD and Kossel, rich in phosphorus and of a strongly acid character. These nucleoproteids will be treated of in speaking of the nncleins of the nucleus. In cases in which the protoplasm is surrounded by an outer, condensed layer or a cell membrane, this envelope seems to consist of albumoid substances. -In a few cases these substances seem to be closely related to elastin; in other cases, on the contrary, they seem rather to belong to the keratin group. The chemical processes by which these albumoid substances are formed from the albuminous bodies or compound proteids of the protoplasm are unknown. Among the non-proteid substances of the cell we must first men- tion lecithin, which exists as a positive constituent of the proto- plasm. It is difiBcult to say whether it also exists in the nucleus. LECITHIN. 93 Lecithin. This body is, accordiilg to the investigations of Stbeckek,' Hundeshagen/ and Gilson,' an ether-like combina- tion of glycerophosphoric acid substituted by two fatty acid radicals, with a base, cholin. Therefore there may be different lecithins according to the fatty acid contained in the lecithin molecule. One of these — distearyllecttliin — ^has been closely studied by Hoppe- Seylbr and Diaconow :* C..H.0NPO. = HO.(CH,),N.C,H,.0(OH)PO.O.C'H. : (C,.H,.OJ,. In agreement with this, if lecithin be boiled with baryta-water it yields fatty acids, glycerophosphoric acid, and cholin. It is only slowly decomposed by dilute acids. Besides small quantities of glycerophosphoric acid (perhaps also distearylglycerophosphoric acid) we have large. quantities of free phosphoric acid split off. Glycerophosphoric acid (H0),P0.0.C,H,(0II), is a bibasic acid, which probably only occurs in the animal fluids and tissues as splitting product of lecithin. The cholin, which is identical with the bases sinkalin (in mustard-seed) and aman^iti^st (in agaricus mnscarius), has the formula HO.N(CH3)3.C,H,.(DH, and is therefore considered as trimethylethoxylium hydrate. Cholin, on the contrary, is not identical with the base, neurin", prepared by Liebreich as a decomposition product from the brain, which is considered as trimethylvinylium hydrate, HO.N(CHj)3.CjH3. The combination of cholin with hydrochloric acid gives with platinum chloride a crystalline double combination which is easily soluble in water, insoluble in alcohol and ether, and which crystallizes in six- sided orange-colored plates. This combination is used in detecting this base. Lecithin occurs, as Hoppe-Sbtlbr ' has especially shown, widely diffused in the vegetable and animal kingdoms. According to this investigator, it occurs also in many cases in loose combina- tion with other bodies, such as albuminous bodies, hEemoglobin, and others. Lecithin, according to Hoppe-Setler, is found in nearly all animal and vegetable cells thus far studied, and also in nearly all animal fluids. It is specially abundant in the brain, nerves, ' Annal. d. Chem. u. Pharm. , Bd. 148. « Journ. f. praJit. Chem., Bd. 28. ^Zeitschr. f. physiol. Chem., Bd. 18. * Hoppe-Seyler's Med. cbem. Untersuch., S. 221 and 405. 'Physiol. Chem., 1877-1881, p. 57. 94 THE ANIMAL CELL. fish-eggs, yolk of the egg, eldfctrical organs of the Torpedo electricus, semen and pns, and also in the muscles and blood-corpuscles, blood- plasma, lymph, milk, and bile, as well as in other animal juices and liquids. Lecithin is also found in pathological tissues or liquids. Lecithin may be obtained in grains or warty masses composed of small crystalline plates by strongly cooling its solution in strong alcohol. In the dry state it has a waxy appearance, is plastic and soluble in alcohol, especially on heating (to 40-50° C.) ; it is less soluble in ether. It is dissolved also by chloroform, carbon disul- phide, benzol, and fatty oils. It swells in water to a pasty mass which shows under the microscope slimy,, oily drops and threads, so-called myelin forms (see Chapter XII). On warming this swollen mass or the concentrated alcoholic solution, decomposition takes place with the production of a brown color. On allowing the solu- tion or the swollen mass to stand, decomposition takes place and the reaction becomes acid. In putrefaction lecithin yields glycero- phosphoric acid and cholin; the latter further decomposes with the formation of methylamiu, ammonia, carbon dioxide, and marsh-gas (Hasebeoek '). If dry lecithin be heated it decomposes, takes fire and burns, leaving a phosphorized coke. On fusing with caustic alkali and saltpetre it yields alkali phosphates. Lecithin is easily carried down during the precipitation of other compounds such as the proteid bodies, and may thereforf-. very greatly change the solubilities of the latter. Lecithin combines with acids and bases. The combination with hydrochloric acid gives with platinum chloride a double salt which is insoluble in alcohol, soluble in ether, and which contains 10.2^ platinum. It may be prepared tolerably pure from the yolk of the hen's egg by the following methods, as suggested by Hoppe-Seylee and DiACONOW." The yolk, deprived of albumin, is extracted with cold ether until all the yellow color is removed. Then the residue is extracted with alcohol at 50-60° C. After the evaporation of the alcoholic extract at 50-60° C, the sirupy matter is treated with ether and the insoluble residue dissolved in as little alcohol as possible. Oh cooling this filtered alcoholic solution to — 5° to — 10° C. the lecithin gradually separates in small granules. The ether, however, contains considerable of the lecithin. The ether is 'Zeitschr, f. physiol. Chem., Bd. 12. 'HoppeSeyler's Med.-cUem, Untersuch. PREPARATION OF LECITHIN. 95 distilled ofE and the residue dissolved in chloroform and the lecithin precipitated from this solution by means of aceton (AltmaNN '). According to Gilsok, a new portion of lecithin may be obtained from the ether used in extracting the yolk by dissolving the residue after the evaporation of the ether in petroleum ether and then shaking this solution with alcohol.^ The petroleum ether takes the fat, while the lecithin remains dissolved in the alcohol and may be obtained therefrom rather easily by using the proper precautions. The detection and the quantitative estimation of lecithin in animal fluids or tissues is based on the solubility of the lecithin (at 50-60° 0.) in alcohol-ether, by which the phosphoric acid or glycerophosphoric acid salts which may be present at the same time are not dissolved. The alcohol-ether extract is evaporated, the residue dried and fused with soda and saltpetre. Phosphoric acid is formed from the lecithin, and it can be used in the detection and quantitative estimation. The distearyllecithin yields 8.798^ PjOj- This method is, however, not exactly correct, for it is possible that other phosphorized organic combinations, such as jecorin (see Chapter Vni) and protagon (Chapter XII) may have passed into the alcohol-ether extract. The residue of the evaporated alcohol- ether extract may be boiled for an hour with baryta-water, filtered, the excess of barium precipitated with CO,, and filtered while hot. The filtrate is concentrated to a sirupy consistency, extracted with absolute alcohol, and the filtrate precipitated with an alcoholic solution of platinum chloride. The precipitate after filtration may be dissolved in water and allowed to crystallize over sulphuric acid. Protagons, which are found in the leucocytes and pus cells, are also to be considered as a constituent of protoplasm. These phos- phorized bodies occur principally in the brain and nerves and hence will be described in a following chapter. Glycogen, discovered by Cl. Bbenaed and Hensin, is found in developing animal cells and especially in developed embryonic tissues. According to Hoppb-Setlee it seems to be a never-failing constituent of the cells, which show amoeboidal movement, and he found this carbohydrate in the leucocytes, but not in the developed motionless pus-corpuscles. Salomok and afterwards others have, however, found glycogen in pus." From the relationship which seems to exist between glyco- gen and muscular work (see Chapter XI), it is presumable that a consumption of glycogen takes place in the movement of animal pro- toplasm. On the other hand, the extensive occurrence of glycogen in embryonic tissues, as also its occurrence in pathological tumors 'Cited from Hoppe-Seyler's Handbuch, etc., 6. Aufl., S. 84. ' In regard to the literature on glycogen see Chap. VIII. 96 THE ANIMAL CELL. and in abundant cell-formation, speaks for the importance of this tody in the formation and development of the cell. In adult animals glycogen occurs in the muscles and certain other organs, but principally in the liver; therefore it will be com- pletely described in connection with this organ (Chapter VIII). Glycogen has been directly detected as a constituent of the proto- plasm of various cells. Another body, or perhaps more correctly a group of bodies which occur widely distributed in the animal and vegetable king- doms, and which occur regularly in the cells, are the cholesterins. The best-known representative of this group is ordinary cholestenn,' which is the chief constituent of certain biliary calculi and exists in abundant quantities in the brain and nerves. It is hardly admissi- ble that this body is of direct importance for the life and develop- ment of the cell. ■ It must be considered that the cholesterin, as accepted by Hoppe-Seyler,' is a cleavage product appearing in the cell during the processes of life. According to Hoppe-Sbylbr the same is true for the fats, which do not occur constantly in the cells and have nothing to do in the ordinary processes of life. There is no doubt that cholesterin exists as a constituent of the protoplasm, but its existence in the nucleus is questionable. The cell nucleus has a rather complex structure. It consists in part of a mitoplasm, which consists of fibriles which form a net- work, and another part, which is less solid and homogeneous, called the hyaloplasm. The mitoplasm differs from the hyaloplasm in a stronger affinity for many dyes. On account of this behavior the first is called the chromatic substance or chromatin, and the other the achromatic substance or achromatin. The hyaloplasm of the nucleus is considered as a mixture of proteids. The mitoplasm seems to contain the more specific con- stituent of the nucleus, namely, the nuclein substances. Besides this it is alleged to also contain another substance, plastm. This last is less soluble than the nuclein substances and does not have the property, like them, of fixing dyes. The chief constituents of the cell nucleus are the nuclems, the nucleoproteids, and in a few cases nucleic acid. Nucleins. By the name nuclein Hoppe-Setleb and Mie- SCHEE^ designated the chief constituent of the nucleus of the ' See Chap. VIII. = Physiol. Chem., S. 81. ' Hoppe-Seyler, Med.-chem. Untersuch. , S. 453. NUCLEIN8 AND P8EUD0NUCLEIN8. 97 pus cell first isolated by them. Since it has been shown by- repeated research that similar bodies occar extensively in the animal and plant kingdoms, especially in organs rich in cells, we have for some time designated as nucleins a number of phosphorized bodies which are in part derived as cleavage products from the nucleo- albumins and in part form the chief constituent of the cell nucleus. According to IIoppe-Setlee, these bodies may be divided into three groups. The first, to which belongs the nuclein of yeast, pus, nucleated red blood-corpuscles, and probably of the cell nucleus in general, yield as splitting products, on boiling with acids, pro- teid bodies, xanthin bases, and phosphoric acid. To the second group, which yield as splitting products proteid and phosphoric acid, belongs the nuclein of the yolk of the egg and casein — in other words, the nucleo-albumins in general; and to the third group, which gives as splitting products only phosphoric acid and xanthin bases, belongs only the nuclein of the sperm of thj salmon. Those nuclein substances which do not yield nuclein bases on splitting — such, for instance, as nuclein from casein and vitellin — are to be separated from the others. Kossel ' has suggested the name paranuclein for these nuclein substances. As the paranucleins amongst themselves are very different and have only an apparent similarity to the true nucleins, the author " has proposed the name pseudonucleins for them. The nuclein of spermatozoa, which does not yield any proteid on splitting, shows a great similarity to the substance obtained by Altmann from the nucleins of Hoppe-Seylee's first group by the action of alkalies. This substance was called nucleic acid by Altmakn' and Kossel,* and hence this nuclein will be called nucleic acid in the future. The nuclein of the first group is, according to Kossel, true nuclein or simply nuclein. This nuclein, which gives phosphoric acid as well as proteid and xanthin bases on splitting with acids, is considered by Kossel as a combination between proteid and nucleic acid. Pseudonucleins or Pabanucleins. These bodies are obtained ' Du Bois-Reymond's Arch., 1891. 'Zeitschr. f. physiol. Cliem., Bd. 19. »Du Bois-Keymond's Arch., 1889. *Iiid., 1891. 98 THE ANIMAL GBLL. as an insoluble residue on the digestion of nucleoalbumins or phosphoglycoproteids with pepsin hydrochloric acid. Attention is •called to the fact that the pseudonuclein may be dissolved by the presence of too much acid or by a too energetic peptic digestion. Pseudonucleins contain phosphorus, which, as shown by Liebek- MANN,' is split off as metaphosphoric acid by mineral acids. The pseudonucleins are very dissimilar. One group of these, whose most important representative is the long-known pseudonuclein from casein, yields no reducing substance on boiling with mineral acids, while the other group, to which the pseudonuclein from ichthulin belongs, does yield such a substance. The pseudonucleins are amorphous bodies insoluble in water, .tilcohol, and ether, bat readily soluble in dilute alkalies. They are not soluble in very dilute acids, and may be precipitated from their isolation in dilute alkalies by adding acid. They give the proteid reactions very strongly. In preparing a pseudonuclein, dissolve the mother-substance in liydrochloric acid of 1-2 p. m., filter if necessary, and add pepsin solution, and allow to stand at the bodily temperature for about 24 hours. The precipitate is filtered off, washed with water, and purified by alternately dissolving in very faintly alkaline water and xeprecipitating with acid. Hucleins or True Nucleiks. These bodies are obtained as an insoluble or difiicultly soluble residue on the digestion of nucleo- proteids with pepsin hydrochloric acid. They are rich in phos- phorus, about 5^ and above, and according to LiEBEEMAiirN' metaphosphoric may also be split oil from the true nncleins (yeast nuclein). The nncleins are decomposed into proteid and nucleic acid by caustic alkali, and as different nucleic acids exist, so there also exist different nncleins. Certain nncleins, such as yeast nuclein and that isolated by the author ' from the pancreas and mammary gland, give a reducing carbohydrate on boiling with dilate acids, while other nucleins, like that from the thymus gland does not. All nucleins yield xantMn iases or nuclein bases so called by Kossel, on boiling with dilute acids. The nucleins contain iron to a considerable extent. They act like rather strong acids. ' Ber. d. deutsch. chem. Gesellsch., Bd. 21, and Centralbl. f. d. med Wissensch., Bd. 37. 'Pfliiger's Arcli., Bd. 47. ^Zeitschr. f. pliysiol Chem., Bd. 19. NUCLEIC ACIDS. 99 The nucleins are colorless, amorphous, insoluble, or only slightly soluble in water. They are insoluble i:i alcohol and ether. They are more or less readily dissolved by dilute alkalies. Pepsin hydro- chloric acid or dilute mineral acids do not dissolve them, or only to a slight extent. The nucleins give the biuret test and Millox's reaction. They show a great affinity for many dyes, especially the basic ones, and take these up with avidity from watery or alcoholic solutions. On burning they yield an acid coke containing meta- phosphoric acid and which is very difficult to consume. On fusion with saltpetre and soda/the nucleins yield alkali phosphates. According to Liebermann ' the nucleins are combinations of pro- teids with metaphosphoric mixed with xanthin bases. To prepare nucleins from cells or tissues, first remove the chief mass of proteids by artificial digestion with pepsin hydrochloric acid, lixiviate the residue with very dilute ammonia, filter, and precipitate with hydrochloric acid. The precipitate is further digested with gastric juice, washed and purified by alternately dis- solving in very faintly alkaline water, and reprecipitating with an acid, washing with water, and treating with alcohol-ether. A nuclein may be prepared more simply by the digestion of a nucleo- proteid. In the detection of nucleins we make use of the above- described method and testing for phosphorus in the product after fusing with saltpetre and soda. Naturally the phosphates, lecithins (and jecorin) must first be removed by treatment with acid, alcohol, and ether, respectively. We must specially call attention to the fact, as shown by Liebeemann," of the very great difficulty in removing lecithin by means of alcohol-ether. No exact methods are known for the quantitative estimation of nucleins in organs or tissues. Nucleic Acids. Kossel differentiates between the various nucleic acids by the decomposition products. All yield nuclein bases as cleavage products, but the nucleic acid from bull sperma ■ tozoa yields chiefly xanthin, while that from the calf's thymus yields only adenin. According to Kossel ' it is probable that there exist four nucleic acids, one for each nuclein base, namely, an adenylic, a guanylic acid, etc. The nucleic acids thus far investigated, with the exception of adenylic acid from the calf's thymus, were only mixtures of several nucleic acids. Another circumstance which makes the acceptance of many nucleic acids necessary is that certain. "Centralbl, f. d. med. Wiasensch., Bd. 27. ''PflUger's Arch., Bd. 54. »Ber. d. deutsch. chem. Qesellsch., Bd. 26. S. 2753. 100 TSE ANIMAL CELL. of the nucleic acids, such as those from yeast, pancreas, and the mammary glands, give reducing carbohydrates or carbohydrate groups, while the others, such as the nucleic acid from the calf's thymus, salmon, and carp sperm, do not. In the far-reaching splitting of adenylic acid with sulphuric acid Kossel and Neu- MANsr ' obtained levulinic acid. A general formula for the nucleic acids cannot be given, and the composition of the different nucleic acids analyzed is naturally very different. The nucleic acids do not contain any sulphur, but do contain nitrogen and phosphorus in the relation of 3 : 1, according to Kossel." The quantity of phosphorus is large. In the nucleic acid, with the formula C^.H^IST^P^O,,, obtained by Mibschee'' from salmon sperm, the quantity of phosphorus was over 9^. KossBL* assumes tliat the nucleic acid contains a nucleus which consists of phosphorus atoms combined similar to polymetaphosphoric acids. According- to Liebermann' the nucleic acids contain metaphosphoric acid, probably the mono-acid, and he has also, as above stated, split off metaphosphoric acid from nuclein. Other acids rich in phosphorus are formed by the action of alkali or boiling water on nucleic acids. From adenylic acid and later from other nucleic acids KossBL and Neumann ' have prepared an acid called by them ihyminie acid, which on boiling with sulphuric acid yields a crystalline substance, thymin, having the formula CBHaNjOj.' From the thymin they obtained a new cleavage product, a base called cytodn, with the probable formula C„H3„N,«0, + 5HjO. The nucleic acids are amorphous, white, and of a strongly acid reaction. They are readily soluble in ammoniacal or alkaline water. They are not precipitated from these solutions by an excess of acetic acid, but are precipitated by a slight excess of hydrochloric acid, especially in the presence of alcohol. They are insoluble in alcohol and ether. The nucleic acids give precipitates with pro- teids which have been considei-ed as nucleins. The question whether these precipitates are real nucleins has not been settled. Nucleic acid may be best prepared, according to Altmank " from yeast. Each 1000 c.c. of yeast is treated with 3250 c.c. dilute ' Sitzungsber. d. Berl. Akad. d. Wissensch. , Bd. 18, 1894. « Du Bois-Eeymond's Arch., 1893. ^L. c. *L. c. ; see also Ceutralbl. 1 d. med. Wissensch., 1893, S. 497. = Piliiger's Arch., Bd. 47, and Centralbl. f. d. med. Wissensch., 1893 S 465 and 737. 'Ber. d. deutsch. chem. Gesellsch., Bd. 36, and Sitzungsber. derBerl. Akad 1. c. 'Du Bois-Reymond's Arch., 1894, Physiol. Abth. ^Ibid., 1889, Physiol. Abth., S. 524. NUCLE0HI8T0N. 101 -caustic soda of about 3^ for five minutes at the temperature of the room. The chief portion of the sodium hydrate is then neutralized with hydrochloric acid, and then acetic acid added in excess. The liquid separated from the precipitated proteids is acidified with hydrochloric acid until it contains 3-5 p. m. HCl, and then mixed ■with an equal volume of alcohol of the same acidity. Impure nucleic acid separates out and may be purified by dissolving in ammoniacal water and repeatedly treating, as above, with acetic acid, hydrochloric acid, and alcohol. Plastin. — On tlie solution of the nucleins from cell nuclei of certain plants in dilute soda solution a residue is obtained which is characterized by its great insolubility. This substance, of which the spongioplasm of the body of the cell and the nucleus granules are alleged to be composed, is considered as a nuclein modification of great insolubility, although its nature is not known. Nucleoproteids with relatively high percentage of phosphorus and of a markedly acid character occur in cell nuclei. Like the nucleins they are also combinations of proteid with nucleic acid. They are, however, richer in proteid than the nucleins, and differ from them in that their neutral solutions decompose with the splitting off of coagulated proteid on boiling, and also in that they yield nucleins on their peptic digestion. Among the nucleoproteids the most carefully studied is nucleohiston. Nucleohiston is the name given by Kossel and Lilienfeld ' to the nucleoproteid isolated by them from the calf's thymus. Its composition is: C 48.46; H 7.00; N 16.86; P. 3.03S; S 0.701; 33.95^. On heating its solution it splits into coagulated proteid. On peptic digestion it yields nuclein. On treating with hydro- chloric acid of 0.8j^ it splits into nuclein and a proteid substance soluble in hydrochloric acid, and which differs from other proteids in being insoluble in an excess of ammonia. Kossel has called this substance histon. Nucleohiston is precipitated from a neutral solution by means •of acetic acid, and is not redissolved by an excess of acetic acid. The neutral solution is precipitated by alcohol, but not on saturating with MgSO,. Nucleohiston is easily dissolved in dilute alkalies or alkali carbonates. It is soluble in glacial acetic acid, hydrochloric and sulphuric acids. The relationship of the nucleins and histon to the coagulation of the blood will be spoken of in Chapter VI. Nucleohiston is prepared by precipitating the filtered watery extract of the gland, free from cellular elements, with acetic acid, and purifying by repeated solution in water slightly alkaline with ' Zeitschr. f. physiol. Chem., Bd. 18. 102 THE ANIMAL CELL. soda and precipitating with acetic acid. Finally it is washed with water containing acetic acid and then with alcohol, then extracted with cold and hot absolute alcohol and lastly with ether. The compound proteids ' described by other investigators under the names tissue fibrinogen and cell fibrinogen are to be considered as impure nucleohiston or bodies very closely related thereto. The cytoglobin and preglobulin described by Alex. Schmidt ' as important cell constituents also belong to the same group as the nucleohiston. Cytoglobin is to be considered as the alkali combi- nation of preglobulin. The residue remaining on the complete exhaustion of the cells with alcohol, water, and common-salt solution is called eytin by Alex. Schmidt. The relationship of these bodies to the coagulation of blood will be spoken of in Chapter VI. Among the decomposition products of nuclein substances the xanthin bases are of especially great interest. Xanthin Bases. With this name we designate a group of bodies consisting of carbon, hydrogen, nitrogen, and in most cases also of oxygen, which, by their composition, show a relationship not only among themselTes, but also with uric acid. These bodies are xanthin, hypoxanthin, episarhin, guanin, adenin, heteroxanthin, paraxanthin, and carnin. The bodies theobeomin and theo- PHTLLIN (both dimethylxanthin) and caffeih" (trimethylxanthin) occurring in the vegetable kingdom also belong to this group. The composition of these bodies occurring in the animal body is as follows : Uric acid CsHiNjOs Xanthin CsHiNiOj Heteroxanthin (methylxanthini^iju. C»HbN,Oj Paraxanthin (dimethylxanthiir): . . .... , . . CHbNiOj Guanin , . OeHjNjO Hypoxanthin ,. . . CbH^NiO Adenin .... CsH^N^ Episarkin. , , , CiHeNsO (?) Carnin .^^ CHsN.Os After SALOMOiir ' had shown the occurrence of xanthin bases in young cells the importance of the xanthin bases as decomposition products of cell nuclei and of nucleins was shown by the pioneering researches of Kossel, who discovered adenin and theophyllin. In those tissues in which, as in the glands, the cells have kept their original state the xanthin bases are not found free, but in combi- nation with other atomic groups (nucleins). In such tissue, on the contrary, as in muscles, which are poor in cell nuclei, the xanthin bases are found in the free state. As the xanthin bases, as sug- gested by Kossel, stand in close relationship to the cell nucleus it 1 See p. 91. s Zur Blutlehre. " Sitzungsber. d. Bot. Vereins der Provinz Brandenburg, 1880. XANTUIX BASES 10? is easy to understand why the quantity of these bodies is so greatly increased when large quantities of nucleated cells appear in such places as were before relatively poorly endowed. As an example of this we have in leucaemia blood extremely rich in leucocytes. In such blood Kossel' found 1.04 p. m. xanthin bases, against only traces in the normal blood. That the xanthin bases are also inter- mediate steps in the formation of urea or uric acid in the animal organism, is probable, and will be shown later (see Chapter XV). Only a few of the xanthin bases have been found in the urine' or in the muscles. Only four xanthin bases — xanthin, guanin, hypoxanthin, and adenin, — have been obtained, thus far, as cleav- age products of nucleins. In regard to the other xanthin bases we refer the reader to their respective chapters. Only the above four bodies, the real nuclein bases, will be treated of at this time. Of these four bodies the xanthin and gnanin form one special group, and hypoxanthin and adenin another. By the action of nitrous acid guanin is converted into xanthin and adenin into hypoxanthin. C.H.N.O.NH + HNO, = O.H.N.O, + N, + H,0 ; Guanin. Xanthin. C.H,N,.NH + HNO, = C,H.N,0 + N, + H,0. Adenin. Hypoxanthin. By putrefaction guanin is converted into xanthin and adenin into hypoxanthin. On cleavage with hydrochloric acid all four of the bodies are converted into ammonia, glycoeoll, carbon dioxide, and formic acid. Uric acid yields, under the same conditions, ammonia and carbon dioxide, and also glycoeoll. On oxidation with hydrochloric acid and potassium chlorate xanthin, bromadenin, and bromhypoxanthin yield alloxan and urea; guanin yields guanidin, parabanic acid (an oxidation product of alloxan), and carbon dioxide. Uric acid in acid solution is oxidized into urea, alloxan and then further into parabanic acid. The close relationship of these bases to each other and to uric acid is apparent. Xanthin has been prepared synthetically by Q-autier " by heating hydrocyanic acid with water and acetic acid. The nnclein bases form crystalline salts with mineral acids, which ire decomposed by water with the exception of the adenin ' Zeitschr. f. physiol. Chem., Bd. 7, S. 22. «Compt. rend., Tome 98, p. 1523. 104 THE ANIMAL CELL. salts. They are easily dissolyed by alkalies, while with ammonia their action is somewhat different. They are all precipitated from acid solution by phosphotungstic acid, also they separate as a silver combination on the addition of ammonia and ammoniacal silver- nitrate solution. These precipitates are soluble in boiling nitric acid of 1.1 sp. gr. AH xanthin bases with the exception of cafEein and theobromin are precipitated by Fehling's solution (see Chap. XV) in the presence of a reducing substance such as hydroxylamin (Dkbchsbl and Balke '). Copper sulphate and sodium bisulphite may also be used to advantage in their precipitation (Kkugee '). This behavior of the xanthin bases is made use of in their precipita- tion and preparation. Xanthin, C.H,N,0, = ^J'^h.O^ N^ >^^ (^- Fischer =), is found in the muscles, liver, spleen, pancreas, kidneys, testicles, carp-sperm, thymus, and brain. It occurs in small quantities as a physiological constituent of urine, and it has been found rarely as a urinary sediment or calculus. It was first observed in such a stone by Makcet. Xanthin is found in larger amounts in a few varieties of guano (Jar vis guano). Xanthin is amorphous, or forms granular masses of crystals. It is very slightly soluble in water, in 14,151-14,600 parts at -f 16° C, and in 1300-1500 parts at 100° 0. (Almen'). It is in- soluble in alcohol or ether, but is dissolved by alkalies or acids. With hydrochloric acid it gives a crystalline, diificultly soluble com- bination. With very little caustic . soda it gives a readily crystal- lizable combination, which is easily dissolved by an excess of alkali. Xanthin dissolved in ammonia gives with silver nitrate an insolu- ble, gelatinous precipitate of xanthin silver. This precipitate is dissolved by nitric acid, and by this means an easily soluble crystal- line double combination is formed. A watery xanthin solution is precipitated on boiling with copper acetate. At ordinary tempera- tures xanthin is precipitated by mercuric chloride and by ammoni- acal basic lead acetate. It is not precipitated with basic lead acetate alone. When evaporated to. dryness in a porcelain dish with nitric acid 'Zur Kenutniss der XantWnkSrper. Inaug. Diss. Leipzig, 1893. *Zeitschr. f. physiol. Chem., Bd. 18. 'Annal. d. Chem., Bd. 215. . i ••Journ. f. prakt. Chem., Bd. 96. GUANIN. 105 xauthin gives a yellow residue, which turns, on the addition of caustic soda, first red, and, after heating, purple-red. If we add some chloride of lime to some caustic soda in a porcelain dish and add the xanthin to this mixture, at first a dark green and then quickly a brownish halo forms around the xanthin grains and then disappears (Hoppe-Seylek). If xanthin be warmed in a small vessel on the water-bath with chlorine-water and a trace of nitric acid and evaporated to dryness, when the residue is exposed under a bell-jar to the vapors of ammonia a red or purple-violet color is produced (Weidel's reaction). n ■ n TTxr r^ NH.CH : C.NH ^^ ^ . . Guanm, C.H,N,0 = ^^ c.nH.C • N>*-'^- Guamn is found in organs rich in cells, such as the liver, spleen, pancreas, testicles, and in salmon-sperm. It is further found in the muscles (in very small amounts), in the scales and in the air-bladder of certain fishes as iridescent crystals of guanin lime ; in the retina epithelium of fishes, in guano, and in the excrement of spiders it is found as chief constituent. It also occurs in human and pig urine. Under pathological conditions it has been found in leucEemic blood, and in the muscles, ligaments, and articulations of pigs with guanin gout. Guanin is a colorless, ordinarily amorphous powder which may be obtained as small crystals by allowing its solution in concentrated ammonia to spontaneously evaporate. It is nearly insoluble in water, alcohol, and ether. It is easily dissolved by mineral acids and alkalies, but it dissolves with great difficulty in ammonia. Ac- cording to WuLFF ' 100 c.c. of cold ammonia solution containing 1, 3, and 5^ NH, dissolve 9, 15, and 19 milligrammes guanin re- spectively. The solubility is relatively increased in hot ammonia solution. The hydrochloric-acid salt readily crystallizes, and this has been recommended by Kossel " in the microscopical detection of guanin on account of its behavior to polarized light. Very dilute guanin solutions are precipitated by both picric acid and metaphosphoric acid. These precipitates may be used in the quan- titative estimation of guanin. The silver combination dissolves with difficulty in boiling nitric acid, and on cooling the double combina- tion crystallizes out readily. Guanin acts like xanthin in the ■Zeitschr. f. physiol. Chem., Bd. 17, S. 505. ' Ueber die chem. Zusammensetzung der Zelle. Verhandl. der physiol. Gesellsoh. zu Berlin. 1890-1891. Nos. 5 and 6. 100 THE ANIMAL CELL. nitric-acid test, but gives with alkalies on heating a more bluish- violet color. A warm solution of gnanin hydrochloride gives with a cold saturated solution of picric acid a yellow precipitate consist- ing of silky needles (Capkanica '). With a concentrated solution of potassium bichromate a gnanin solution gives a crystalline, orange-red precipitate, and with a concentrated solution of potas- sium ferricyanide a yellowish-brown, crystalline precipitate (Ca- pbanica). The composition of these and other guanin combinations have been studied by Kossel and "Wtjlfe." oTTAT^ NH.CH: C.NH^ „„ Hypoxanthin or Saekin, O^H^N.O = i,„ . -^ n, . fx > 00 or ^' ^ ■ /,■ ^^ >C0 (Keugek"). This body is found in the same CH.NH.C ! N ^ ' ■ tissues as xanthin. It is especially abundant in the sperm of the salmon and carp. Hypoxanthin occurs also in the marrow and in very small quantities in normal urine, and, as it seems, also in milk. It is found in rather considerable quantities in the blood and urine in leucaemia. Hypoxanthin forms very small colorless crystalline needles. It dissolves in 300 parts cold and 78 parts boiling water. It is nearly insoluble in alcohol, bat is dissolved by acids and alkalies. The combination with hydrochloric acid is crystalline, but is more soluble than the corresponding xanthin combination. This combination is easily soluble in dilute alkalies and ammonia. The silver combina- tion dissolves with difficulty in boiling nitric acid. On cooling a mixture of two hypoxanthin silver, nitrate combinations not having a constant composition separates out. On treating this mixture with ammonia and excess of silver nitrate in the warmth, a hypo- xanthin silver combination is formed, which when dried at 120° C. has a constant composition, 2(CjH,Ag,]Sr,0)HjO, and which is used in the quantitative estimation of hypoxanthin. Hypoxanthin picrate is soluble with difficulty, but if a boiling-hot solution of the same is treated with a neutral or only faintly acid solution of silver nitrate the hypoxanthin is nearly quantitatively precipitated as the compound C,H,AgN,0.C,H,{N0J30H. Hypoxanthin does not form any combination with metaphosphoric acid. When treated ' ZeitscliT. f. physiol. Ohem. , Bd. 4, S. 333. ^Ibid.. Bd. 17, S 468. 'lUd., Bd. 18, S. 459. ADENIN. 107 like xanthin, with nitric acid, it yields a nearly colorless residue which on warming with alkali does not turn red. Hypoxanthin does not give Weidel's reaction. After the action of hydrochloric acid and zinc a hypoxanthin solution becomes first ruby-red and then brownish red in color on the addition of an excess of alkali (KOSSEL '). Adenin," C,H.N. = CH : N.C : N >^ ^^-^^ °'' 1^ OTT * P 'N'TT y:,' „ ■ /,' ,, >C (NH), Kkugee,' was first found by Kossel in CH.NH.C -.^ ^ ' . •' the pancreas. It occurs in all nucleated cells, but in greatest quantities in the sperm of the carp and in the thymus. Adenin has also been found in lencaemic urine (StadthagEjST ■"). It may be obtained in large quantities from tea-leaves. Adenin crystallizes with 3 mol. water of crystallization in long needles which become opaque gradually in the air, but much more rapidly when warmed. If the crystals are warmed slowly with a quantity of water insufii- cient for solution, they become suddenly cloudy at 53° C, a charac- teristic reaction for adenin. It dissolves in 1088 parts cold water, but is easily soluble in warm. It is insoluble in ether, but some- what soluble in hot alcohol. Adenin is easily soluble in acids and alkalies. It is more easily soluble in ammonia solution than guanin, but less soluble than hypoxanthin. The silver combination of adeuin is difficultly soluble in warm nitric acid, and deposits on cool- ing as a crystalline mixture of adenin silver nitrates. With picric acid adenin forms a compound, CjHjN5.0|,Hj(N0,)30H, which is very insoluble and which separates more readily than the hypoxan- thin picrate and which can be used in the quantitative estimation of adenin. We also have an adenin mercury picrate. Adenin gives a precipitate with metaphosphoric acid, if the solution is not too dilute, which dissolves in an excess of the acid. Adenin hydro- chloride gives with gold chloride a double combination which con- sists in part of leaf -shaped aggregations and in part of cubical or prismatic crystals, often with rounded corners. This compound is used in the microscopic detection of adenin. With the nitric-acid test and with Weidel's reaction adenin acts in the same way as 'Zeitschr. f. physiol. Chem., Bd. 12. 5 See Kossel, Md., Bdd. 10 and 13. *md., Bd. 18, S. 459. ■"Virchow's Arcli., Bd. 109. 108 THE AmMAL CELL. hypoxanthin. The same is true for its behavior to hydrochloric acid, and zinc and subsequent addition of alkali. The principle for the preparation, detection, and the quantita- tive estimation of the four above-described xanthin bodies in organs and tissues is, according to Kossel and his pupils, as follows: The finely divided organ or tissue is boiled for three or four hours with sulphuric acid of about 5 p. m. The filtered liquid is freed from proteid by basic lead acetate, and the new filtrate is treated with sulphuretted hydrogen to remove the lead, again filtered, concen- trated, and, after adding an excess of ammonia, precipitated with ammoniacal silver nitrate. The silver combination (with the addi- tion of some urea to prevent nitrification) is dissolved in not too large a quantity of boiling nitric acid of sp. gr. 1.1, and this solu- tion filtered boiling hot. On cooling the xanthin silver remains in the solution, while the double combination of guanin, hypoxanthin, and adenin crystallizes out. The xanthin silver may be precipi- tated from the filtrate by the addition of ammonia, and the xanthin set free by means of sulphuretted hydrogen. The three above- mentioned silver nitrate combinations are decomposed in water with ammonium sulphide and heat; the silver sulphide is filtered, the filtrate concentrated, saturated with ammonia, and digested on the water-bath. The guanin remains undissolved, while the other two bases pass into solution. A part of the guanin is still retained by the silver sulphide, and may be liberated by boiling it with dilute hydrochloric acid and then saturating the filtrate with ammonia. When the above filtrate, containing the adenin and hypoxanthin, which has been, if necessary, freed from ammonia by evaporation, is allowed to cool, the adenin separates, while the hypoxanthin remains in solution. According to Balke ' we can to advantage precipitate the xanthin bases with copper sulphate and hydroxyla- min as above mentioned and then further separate the bodies. The prominent points in the above method are made use of in the quantitative estimation of xanthin bases. The xanthin is weighed as xanthin silver. The three silver nitrate combinations are transformed into the corresponding silver eombination by the addition of ammonia with silver nitrate and then this acted on, after thorough washing, by ammonium sulphide. Guanin is weighed as such. The ammoniacal filtrate containing the adenin and hypoxanthin, ?ind which must not be mixed with the hydrochloric- acid extract of the silver sulphide, is neutralized and treated with a cold concentrated solution of sodium picrate until the solution is pronouncedly i yellow. The adenin picrate is filtered off imme- diately, washed on the filter with water, dried at above 100° C, and weighed. The filtrate containing the hypoxanthin is gradually treated, while boiling hot, with silver nitrate, and when cold treated with silver nitrate to see whether precipitation has been complete. ' Zur Kenntnisse der Xanthihkorper. Inaug. Diss. Leipzig, 1893. MINERAL CONSTITUENTS OP THE CELL 109 The hypoxanthin picrate is washed, dried at 100° C, and weighed. In regard to the composition of these compounds see pages 106 and 107. This method of separating adenin and hypoxanthin presup- poses that the liqaid does not contain any hydrochloric acid. The above method of separation with ammonia does not give exact results on account of the not inconsiderable solubility of guanin in warm ammonia. According to Kossel and Wulff ' the guanin may therefore be precipitated from sufficiently dilute solu- tions by an excess of metaphosphoric acid and the nitrogen deter- mined in the washed precipitate by Kjeldahl's method. The adenin and hypoxanthin may be precipitated from the filtrate by ammoniacal silver nitrate. The silver compound is decomposed with very dilute hydrochloric acid and the adenin separated from the hypoxanthin according to the suggestion of Bkuhns." Mineral bodies are never-failing constituents of the cell. These mineral bodies are potassium, sodium, calcium, magnesium, iron, phosphoric acid, and chlorine. In regard to the alkalies we find in general in the animal organism that the sodium combinations are more abundant in the fluids, and the potassium combinations in the form-constituents and in the protoplasm. Corresponding to this the cell contains potassium, chiefly as phosphate, while the sodium and chlorine combinations occur less abundantly. According to the ordinary views the potassium combinations, especially the potassium phosphate, are of the greatest importance for the life and development of the cell, even though we do not knovr the nature of the importance. In regard to the phosphoric acid there seems to be no doubt that its importance lies chiefly in that it takes part in the forma- tion of nucleins and thereby indirectly makes possible the processes of growth and division, which are dependent upon the cell nucleus. LoEW s has shown, by means of cultivation experiments on algae Spirogyra, that only on the supplying of phosphates (in his experi- ments potassium phosphate) was the nutrition of the cell nucleus made possible, and thereby the growth and division of the cells. The cells of the Spirogyra can be kept alive and indeed produce starch and proteids for some time without a supply of phosphates, but its growth and propagation sufEers. Phosphoric acid is also without doubt of importance in the formation of the lecithins. Iron seems to occur especially in the nucleus, because the ' Zeitschr. f. physiol. Chem., Bd. 17. Ubid.. Bd. 14, S. 559. » Biologisches Centralblatt, Bd. 11, 1891, S. S69. 110 THE ANIMAL CELL. nucleins are Ferj rich therein. The regular occurrence of earthy phosphates in all cells and tissues, as also the difficulty or rather the impossibility of separating these bodies from the protein bodies without modifying them, leads to the supposition that these mineral bodies are of unknown but nevertheless great importance for the life of the cell, as well as the chemical processes going on within them. CHAPTER VI. THE BLOOD. The blood is to be considered from a certain standpoint as a fluid tissue, and it consists of a transparent liquid, tbe Mood-plasma, in which a vast number of solid particles, the red and white blood- corpuscles (and the blood-plates) are suspended. Outside of the organism the blood, as is well known, coagulates more or less quickly ; but this coagulation is accomplished generally in a few minutes after leaving the body. All varieties of blood do not coagulate with the same degree of rapidity. Some coagulate more quickly, others more slowly. Among the varieties of blood thus far investigated the blood of the horse coagulates most slowly. 'The coagulation may be more or less retarded by quickly cooling ; and if we allow equine blood to flow directly from the vein into a glass cylinder which is not too wide and which has been cooled, and let it stand at 0° C, the blood may be kept fluid for several days. An upper, amber-yellow layer of plasma gradually separates from a lower, red layer composed of blood-corpuscles with only a little plasma. Between these we observe a whitish-gray layer, which consists of white blood-corpuscles. The plasma thus obtained and filtered is a clear amber-yellow alkaline liquid which remains fluid for some time when kept at 0° C, but soon coagulates at the ordinary temperature. The coagulation of the blood may be prevented in other ways. After the injection of peptone or, more correctly, albumose solu- tions into the blood (in the living dog), the blood does not coagulate on leaving the veins (Fam-o," Sohmidt-Mulheim '). The plasma obtained from such blood by means of centrifugal force is called > Du Bois-Reymond's Archiv, 1881, S. 277. 'iWa., 1880. Ill 113 THE BLOOD. "peptone-plasma.'''' The coagulation of the blood of warm-blooded animals is prevented by the injection of an effusion of the moath of the officinal leech into the blood-current (Hayceaft'). If the blood-circulation of a dog is cut off from the li ver and intestine and the blood allowed to flow only through the head and the viscera of the thoracic cavity, the coagulation property . of the blood is destroyed (Pawlow, Bohr"). The statement as to the non- coagulability of the blood after the excision of the liver and abnominal cavity could not be confirmed by Contejean/ If we allow the blood to flow directly, while we stir it, into a neutral salt solution — best a saturated magnesium-sulphate solution (1 vol. salt solution and .3 vols, blood) — we obtain a mixture of blood and salt which remains uncoagulated for several days. The blood-corpuscles which, because of their adhesiveness and elasticity, would otherwise pass easily through the pores of the filter-paper are made solid and stiff by the salt, so that they may be easily filtered. The plasma thus obtained, which does not coagulate spontaneously, is called ' ' salt-plasma. ' ' An especially good method of preventing coagulation of blood consists in drawing the blood into a dilute solution of potassium oxalate, so that the mixture contains 0.1^ oxalate (Aethus and Pages '). The soluble calcium salts of the blood are precipitated by the oxalate, and hence the blood loses its coagulability. On coagulation there separates in the previously fluid blood an insoluble or a very difficultly soluble albuminous substance, fibrin. When this separation takes place without stirring, the blood coagu- lates to a solid mass which, when carefully severed from the sides of the vessel, contracts, and a clear, generally yellow-colored liquid, the ilood-serum, exudes. The solid coagulum which encloses the blood-corpuscles is called the blood-clot (placenta sanguinis). If the blood is beaten during coagulation, the fibrin separates in elastic threads or fibrous masses, and the defihrinated blood which separates is sometimes called cruor," and consists of blood-corpuscles and ' Proc. physiol. Soc, 1884, p. 13, and Arch. f. exp. Pathol, und Pharm. 1884, Bd. 18. ^Centralbl. f. Physiol., 1888, No. 11. 'Arch, de Physiol., Ser. 5, Tome 7. *IUd., Tome 2, 1890, and Compt. rend., 1891, Tome 112, No. 4. 5 The name cruor is used in different senses. We sometimes understand thereby only the blood when coagulated to a red solid mass, in other cases the blood-clot after the separation of the serum, and lastly the sediment consisting BLOOD PLASMA. 113 blood-serum. Deflbrinated blood consists of blood-corpnsoles and serum, while uncoagulated blood consists of blood-corpuscles and blood-plasma. The essential chemical difference between blood- serum, and blood-plasma is that the blood-serum does not contain the mother-substance of fibrin, the fibrinogen, which exists in the blood-plasma, and the serum is proportionally richer in another body, the fibrin ferment (see page 116). I. Blood-plasma and Blood-serum. The Blood-plasma. In the coagulation of the blood a chemical transformation takes place in the plasma. A part of the proteids separates as insoluble fibrin. The albuminous bodies of the plasma must therefore be first described. They are, as far as we know at present, fibrinogen., serglolulin, and seralbumin. Fibrinogen occurs in blood-plasma, chyle, lymph, and in certain transudations and exudations.' It has the general properties of the globulins, but differs from other globulins as follows: In a moist condition it forms white flakes which are soluble in dilute common- salt solutions, and which easily conglomerate into tough, elastic masses or lumps. The solution in NaCl of 5-10^ coagulates on. heating to + 53° to 55° C, and the faintly alkaline or nearly neutral weak salt solution coagulates at + 56° C, or at exactly the same temperature at which the blood-plasma coagulates. Fibrin- ogen solutions are precipitated by an equal volume of a saturated common-salt soluion, and are completely precipitated by adding an excess of NaCl in substance (thus differing from serglobulin) . It differs from myosin of the muscles, which coagulates at about the same temperature, and from other albuminous bodies, in the property of being converted into fibrin under certain conditions. Fibrinogen has a strong decomposing action on hydrogen peroxide." It is quickly made insoluble by precipitation with water or with of red blood-corpuscles whicli is obtained from defibrinated blood by means of centrifugal force or by letting it stand. ' The question as to the occurrence of other fibrinogens (Wooldkidqe) will be spoken of in connection with the complete discussion of the coagulation of the blood. (See further on.) ' In regard to fibrinogen the reader is referred to the author's investigations. Pflilger's Archiv, Bdd. 19 and 32. 114 THE BLOOD. dilate acids. Its specific rotation is ar{D) = — 52.5° according to MiTTELBACH.' Fibrinogen may be easily separated from the salt-plasma by pre- cipitation with an equal volume of a saturated NaOl solution. For further purification the precipitate is pressed, redissolved in an 8^ salt solution, the filtrate precipitated by a saturated-salt solution as above,' and after precipitating in this way three times the pre- cipitate at last obtained is pressed between filter-paper and finely divided in water. The fibrinogen dissolves with the aid of the .small amount of NaOl contained in itself, and the solution may be made salt-free by dialysis with very faintly alkaline water. From vtransudations we ordinarily obtain a fibrinogen which is strongly contaminated with lecithin and which can hardly be purified with- out decomposing. The method for the detection and quantitative estimation of fibrinogen in a liquid is based on its property of yielding fibrin on the addition of a little blood, of serum, or of fibrin ferment. The fibrinogen stands in close relation to its transformation- product, the fibrin. Fibrin is the name of that proteid body which separates on the so-called spontaneous coagulation of blood, lymph, and transuda- tions, as also in the coagulation of a fibrinogen solution after the addition of serum or fibrin ferment (see below). If the blood is beaten during coagulation, the fibrin separates in elastic, fibrous masses. The fibrin of the blood-clot may be beaten to small, less elastic, and not particularly fibrous lumps. The typical, fibrous, and elastic white fibrin, after washing, stands in regard to its solubility close to the coagulated proteids. It is insoluble in water, alcohol, or ether. It expands in hydrochloric acid of 1 p. m., as also in caustic potash or soda of 1 p. m., to a gelatinous mass, which dissolves at the ordinary temperature only after several days, but at the temperature of the body it dissolves more readily but still slowly. Fibrin expands in a 5-10^ solution of common salt or saltpetre, but only dissolves very slowly at ordi- nary temperature, while at 40° C. it dissolves more readily. At present we cannot positively state what action the presence of micro-organisms or contaminating enzymes have on this solution. According to Arthus and Hubek," and also lately to Dareste,' there is no doubt of the solubility of fibrin in neutral salt solutions iZeitschr. f. physiol. Chem., Bd. 19. 'Arch, de Physiol., Ser. 5, Tome 5. 'Ibid., Tome 7. FIBRIN. 115 -without the action of micro-organisms. According to GREEJf ' two globulins are formed in this solution of fibrin. Fibrin decomposes hydrogen peroxide, but this property is destroyed by heating or by the action of alcohol. What has been said of the solubility' of fibrin relates only to the typical fibrin obtained from the arterial blood of mammals or man hy whipping and washing first with water and with common-salt solution, and then with water again. The blood of various kinds of animals yields fibrin with somewhat different properties, and according to Febmi' pig-fibrin dissolves much more readily in hydrochloric acid of 5 p. m. than ox-fibrin. Fibrins of varying purity or originating from blood from different parts of the body have unlike solubilities. The fibrin obtained by beating the blood and purified as above described is always contaminated by enclosed blood-corpuscles or remains thereof, and also by lymphoid cells. It can only be obtained pure from filtered plasma or filtered transudations. For the pure preparation, as well as for the quantitative estimation of fibrin, the spontaneously coagulating liquid is at once, or the non- spontaneously coagulating liquid only after the addition of blood- serum or fibrin ferment, thoroughly beaten with a whale-bone, and the separated coagulum is washed first in water, and then with a 5^ ' common-salt solution, and again with water, and lastly extracted with alcohol and ether. If the fibrin is allowed to stand in contact with the blood from which it was formed for some time, it partly dissolves (fibrinolysis — Dastre'). This fibrinolysis must be pre- vented in the quantitative estimation of fibrin (Dastke). A pure fibrinogen solution may be kept at the ordinary tem- perature until putrefaction begins without showing a trace of fibrin coagulation. But if to this solution we add a water-washed fibrin- clot or a little blood-serum, it immediately coagulates and may yield perfectly typical fibrin. The transformation of the fibrinogen into fibrin requires the presence of another body contained in the blood-clot and in the serum. This body, whose importance in the coagulation of fibrin was first observed by Buchanan,* was later rediscovered by Alexander Schmidt' and designated "_/?Jrm- ' Journal of Physiol., Vol. 8, p. 513. 'Zeitscbr. f. Biologie, Bd. 28, S. 229. « Archives de Physiol. (5), Tome 5, No. 3, and Tome 6, No. 4, p. 670. * London Med. Gazette, 1845, p. 617. Cit. by Qamgee, Journal of Physiol., 1879. ' Pflilger's Archiv, Bd. 6, S. 413. 116 THE BLOOB. ferment.'''' The nature of this enzymotic body has not been ascer- tained. Although many investigators, especially English, consider fibrin-ferment as a globulin, still more recent experiments of Pekblhaeing,' Weight,' and Lilienfeld' show that it is a nucleoalbumin or perhaps a nncleoproteid. Fibrin ferment, which is now called thrombin by Ales. Schmidt,* is produced, according to Pekelhaeikg, by the action of soluble calcium salts on a pre- formed zymogen existing in the non-coagulated plasma. Schmidt admits of the presence of such a mother-substance of the fibrin ferment in the blood and calls it prothrombin. The zymogen as well as the fibrin ferment is less soluble in an excess of acetic acid than the globulins, and yields a nuclein or a pseudonuolein on peptic digestion. Thrombin corresponds to other enzymes in that the very smallest amount of it produces an action and its solution becomes inactive on heating. It is most active at about 40° 0. Th& zymogen, according to Pekelhaking, is destroyed at about + 65° C, while the ferment is destroyed at about the same or a little higher temperature, 70-75° 0. The isolation of the fibrin-ferment has been tried in several ways. Ordinarily it may be prepared by the following method pro- posed by Alex. Schmidt'*: Precipitate the serum or defibrinated blood with 15-30 vols, of alcohol and allow it to stand a few • months. The precipitate is then filtered and dried over sulphuric acid. The ferment may be extracted from the dried powder by means of water. A globulin-free thrombin solution may be prepared as follows, according to the authoe": The globulins are separated from ox-serum by completely saturating with magnesium sulphate, filter- ing and diluting the filtrate with water, and then adding very dilute caustic-soda solution, with constant stirring until a rather abundant, flocky precipitate of Mg(OH)j is obtained. This precipitate, which contains a great deal of the ferment, is washed, pressed, dissolved in water with the aid of acetic acid until neutral, and then freed irom salts by means of dialysis. ' Verliandel. d. kon. Akad. d. Wetensch. te Amsterdam, Deel 1, No. 3, 1892. ' Proc. of Roy. Irish Acad. (3), Vol. 3, and Lecture on Tissue- or Cell-fibrin- ogen, Lancet, 1893; also on Wooldridge's Method, etc., British. Med. Journal Sept., 1891. 'Du Bois-Reymond's Archiv, 1892, and Ueber Leukocyten und Blutgerin- nung, Verhandl. d. physiol, Gesellsch. zu Berlin, 1892. ^Zur Blutlehre. Leipzig, 1892. 'PflUger's Archiv, Bd. 6. ^Ibid. Bd. 18, S. 89. OOAOULATION. 117 Thrombin can be precipitated from this solution, according to Pekelhaeing,' by the proper addition of acetic acid. According to this investigator, it is best to dialyze the above filtrate saturated with MgSO, and then precipitate with acetic acid. He has been able to obtain thrombin directly from the blood-seram by diluting with water and adding acetic acid until the serglobulin, which first precipitates, is at least in great part redissolved. The thrombin is purified by repeated solution in alkaline water and reprecipitating with acetic acid. If a fibrinogen solution containing salt, as above prepared, is treated with a solution of " fibriu-ferment, " it coagulates at the ■ordinary temperature more or less quickly and yields a typical fibrin. Besides the fibrin ferment the presence of neutral salts is necessary, for without them Alex. Schmidt '' has shown the coagulation of fibrin does not take place. The presence of soluble calcium salts is likewise an essential condition for the formation of fibrin (Abthtjs and Pages, Pekelharing), and the fibrin separated always con- tains calcium. The quantity of fibrin obtained on coagulation is always smaller than the amount of fibrinogen from which the fibrin is derived, and we always find a small amount of protein substance in the solution. It is therefore not improbable that the coagulation of fibrin, in accordance with the views of Dekis, is a splitting process in which the soluble fibrinogen is split into an insoluble albuminous body, the fibrin, which forms the chief mass, and a soluble protein substance which is only formed in small amounts. We find a globulin-like substance which coagulates at about + 64° C. in blood-serum as well as in the serum from coagulated fibrinogen solutions. This substance is called fibrin- fflobulin by the AUTHOE.' The question whether this substance exists in the fibrinogen solution as contamination or is formed as a splitting product has not been positively decided. ' The lime-salts, as above stated, are a necessary factor in the coagulation. According to Pekelhaeistg,* they act as follows: The fibrin-ferment or thrombin is a calcium combination of the zymogen, the prothrombin. In coagulation the calcium is trans- ferred to the fibrinogen by the thrombin, forming insoluble fibrin ■containing calcium. The thrombin is hereby reconverted into 'L. c. ♦Pflilger's Aichiv, Bd. 11, S. 291-304 ; also Bd. 13, S. 103. 'Ibid., Bd. 33. * Verhandel. d. kon. Akad. d. WettenscU. te Amsterdam, Deei 1 , No. 3, 1893. 118 THE BLOOD. prothrombin, which takes up more calciam, being converted into thrombin again, which then gives up its calcium to a new portion of fibrinogen, and so on. The process has great similarity to the formation of ether from alcohol by sulphuric acid. LiLiENFELD ' has described his experiments and views in an extensive memoir. According to him the fibrinogen may be split by acetic acid, and also by the nuclein substances of the leucocytes (these also act in alkaline solution), into a proteid body, which is- precipitated readily, tJirombosin, and an albnmose-like substance, which gives the biuret reaction and which retards coagulation. Thrombosin passes into fibrin in the presence of soluble calcium salts, without further addition inasmuch as fibrin is nothing but the calcium combination of thrombosin. The above cleavage of fibrinogen into thrombosin and a soluble proteid substance may also take place in the absence of calcium salts, and these are only neces- sary for the separation of the calcium combination of thrombosin, i.e., fibrin. Fibrin-ferment, which is a globulin according to LiLiENFELD, is not a precursor but a product of the coagulation. The coagulation process is considered by Lilienfeld and most, investigators as a cleavage of the fibrinogen, and the essential difference between his theory and the others consists in that the coagulation exciter is not the fibrin-ferment bnt a nncleoproteid which is the lenconuclein derived from the nucleohiston by cleavage. Halliburton and Brodib" have raised an objection to the statement of Pekelhaking as to the identity of fibrin-ferment with a nncleoproteid or its calcium combination occurring in the blood- plasma. Pekelhaeing^ has repudiated this in a recent article. He has shown, in opposition to the views of Hallibubto:n" and Liliestfeld, that the fibrin-ferment yields nuclein in careful pepsin digestion, hence it must be a nncleoproteid. In a work which appeared after the death of Alex. Schmidt' he has given his position on the work of other investigators in this field, but as this extensive work is chiefly of a critical nature we cannot discuss it. According to Dogiel and Holzmaitn ' the fibrin coagulation ' Zeitschr. f. physiol. Chem., Bd. SO. 2 Journal of Physiol , Vol. 17. sCentralbl. f. Physiol., 1895, Heft 3. "■ Weitere Beitrage zur Blutlehre. Wiesbaden, 1895. ' Compt. rend. d. congrSs internat. des sciences medicales a CopenhaKue 1884, Tome 1, p. 135. ' 8ER0L OB ULIN. 1 1 » consists in an oxidation of fibrinogen. The relationship of oxygen to the coagulation is indeed not clear, and that it has a certain influ- ence on the coagulation cannot be denied; still, as coagulation may take place in the absence of free oxygen, the above view does not seem to be based on sufficient fact. Although the processes of coagulation are still not clear, never- theless they consist essentially in the conversion of the fibrinogen of the plasma into fibrin. The coagulation of the blood is a much more complicated process than the coagulation of a fibrinogen solu- tion, inasmuch as the first involves other important questions, as, for instance, the reason for the blood remaining fluid in the body, the origin of the fibrin-ferment, and the importance of the form- elements in the coagulation. A .fuller discussion of the various hypotheses and theories concerning the coagulation of the blood must therefore be given later. Serglobulin, also called paraglolulin (KttHNE'), fibrinoplastia substance (Alex. Schmidt"), serum-casein (Panum^), occurs in the plasma, serum, lymph, transudations and exudations, in the white and red corpuscles, and probably in many animal tissues and form-elements, though in small quantities. It is also found in the urine in many diseases. Serglobulin is without doubt not an individual substance, but consists of a mixture of two or more protein bodies which cannot be completely and positively separated from each other. Under these circumstances the statements in regard to the properties of the serglobnlins is naturally somewhat uncertain. According to our present knowledge it has the following properties:* Serglobulin has the general properties of the globulins. In a moist condition it forms a snow-white flaky mass neither tough nor elastic. The essential difEerences between serglobulin and fibrinogen are the following: Serglobulin solutions are only incompletely pre- cipitated by adding NaCl to saturation, and not precipitated at all by an equal volume of a saturated common-salt solution. The coagulation temperature is, with 5-10^ NaCl in solution, -f 75° C. It is completely precipitated by MgSO, in substance added to sat- uration, as also by an equal volume of a saturated solution of ' Lebrbucb d. physiol. Cbem. Leipzig, 1866-68. 'Arch. f. Anat. u. Physiol., 1861, S. 545, and 1862, S. 438. ' Virchow's Archiv, Bd. 4. *See Hammarsten, Ueber Paraglobulin, Pflilger's Arohiv, Bdd. 17 and 18. 120 THE BLOOD. ammonium snlphate. The specific rotatory power, according td Fbedbeicq," for serglobulin (from ox-blood) solutions containing salt is a(D) = - 47.8°. ' According to K. Mokner' serglobulin yields a reducing sub- stance on boiling with, a dilute acid. The question whether the substance we have heretofore called serglobulin is a glycoproteid or whether it is a mixture of globulin with a glycoproteid has not been positiTely decided up to the present time. Serglobulin may be easily separated as a fine flocculent precipi- tate from blood-serum by neutralizing or making faintly acid with acetic acid and then diluting with 10-20 vols, of water. For further parification this precipitate is dissolved in dilute common- salt solution, or in water by the aid of the smallest possible amount of alkali, and then reprecipitated'by diluting with water or by the addition of a little acetic acid. The serglobulin may also be separated from the serum by means of magnesium or ammonium sulphate; in these cases it is difficult to completely remove the salt by dialysis. The serglobulin from blood-serum is always contami- nated by lecithin and so-called fibrin-ferment. A serglobulin free from fibrin-ferment may be prepared from ferment-free transu- dations, as sometimes from hydrocele fluids, and this shows that serglobulin and fibrin-ferment are different bodies. For the detec- tion and the quantitative estimation of serglobulin we may use the precipitation by magnesium sulphate added to saturation (author '), or by an equal volume of a saturated neutral ammonium sulphate solution (Hofmbister and Kauder and Pohl*). In the quantitative estimation the precipitate is collected on a weighed filter, washed with the salt solution employed, dried with the filter at about 115° C, then washed with boiling-hot water, so as to completely remove the salt, extracted with alcohol and ether, dried, weighed and burnt to determine the ash. Seralbumin is found in large quantities in blood-serum, blood- plasma, lymph, transudations, and exudations. Probably it also occurs in other animal liquids and tissues. The proteids which pass into the urine under pathological conditions consists largely of seralbumin. In the dry state seralbumin forms a transparent,^ gummy, brittle, hygroscopic mass, or a white powder which may be heated to 100° C. without decomposing. Its solution in water gives the > Bull. Acad. Roy. de Belg. (2), Tome 50. •Centralbl. f. Physiol., 1893, No. 30. 'Pfliiger'sArchiv, Bd. 17, S. 447. 'Arch. f. exp. Path. a. Pharm., Bd. 30, S. 411 and 426. ' SBRALBUMIN. 121 ordinary reactions for albumins; the specific rotatory power for seralbumin free from paraglobulin, obtained, from human transuda- tions, is, according to Stark,' a(D) = — 62.6° to — 64.6°. The coagulation temperature of a seralbumin solution is -(- 70° to -|- 75° C, according to most authorities, but this varies to a great extent with a varying concentration and amount of salt (Staek). A 1-2^ seralbumin solution may, in the presence of very little NaCl, coagulate at + 50° C. or below; in the presence of b% NaCl it coagulates at + 75° to + 90° C. By the careful addition of acid the coagulation temperature maybe lowered; by the addition of alkali it may be raised. In blood-serum from certain animals and in human transudations Halliburton " found the coagulation to take place on heating to the following temperatures: + 70° to 73° C. ; 77° to 78° C. ; and 82° to 85° C. He therefore considers the seralbumin as a mixture of three albumins, a, /?, and y, which coagulate at the three points mentioned. In cold-blooded animals he found only the a-albumin. Gurbee ^ has prepared crystallized proteid from blood-serum of the horse, which seems to correspond to three different seralbumins. Seralbumin differs from the albumin of the white of the hen's egg in the following particulars: it is more laevogyrate; the precipi- tate formed by hydrochloric acid easily dissolves in an excess of the acid; is rendered less insoluble by alcohol; and lastly it acts dif- ferently inside of the organism. If egg-albumin is introduced into the blood system it passes into the urine, while seralbumin does not. A solution of seralbumin positively free from mineral bodies has never yet been prepared. A solution as poor as possible in salts does not coagulate either on boiling or on the addition of alcohol. After the addition of a little common salt it coagulates in both cases. In preparing seralbumin, first remove the globulins according to JoHAN-ssoN-,* by saturating with magnesium sulphate at about + 30° C, and filtering at the same temperature. The cooled filtrate is separated from the crystallized salt and is treated with acetic acid so that it contains about 1^. The precipitate formed is filtered, pressed, dissolved in water with the addition of alkali to neutral reaction, and the solution freed from salt by dialysis. The ' Maly's Jahresbericlit, Bd. 11. ' Journal of Physiol. . Vols. 5 and 7. • Sitzungsber. d. Wilrzb. phys. med. Qesellsch., 1894. *Zeltschr, f. physiol. Ctom., Bd. 9, S. 317. 122 THE BLOOD. seralbumin may also be separated from the filtrate saturated with magnesium sulphate by adding sodium sulphate to saturation at about + 40° C. (Stakk'). The pressed precipitate is also in this case dissolved in water and the solution freed from salt by dialysis. The albumin may be obtained in a solid form from the dialyzed solution either by evaporating the solution to dryness at gentle heat or by precipitating with alcohol, which must be removed quickly. In the detection and quantitative estimation of seralbumin, the filtrate from the globulins which have been removed by magnesium sulphate is heated to boiling, after the addition of a little acetic acid if necessary. -The simplest way is to consider the difference between the total proteids and the globulins as seralbumin. Summary of the elementary composition of the above mentioned and described albuminous bodies : C H N S Fi brinogeu 52. 93 6. 90 16. 66 1 . 35 23. 26 (Hammabstbn) Fibrin 53.68 6.83 16.91 1.10 32.48 Fibrin-globulin 52.70 6.98 16.06 Serglobulin 52.71 7.01 15.85 1.11 33.33 Seralbumin (1) 53.06 6.85 16.04 1.80 32.26 Seralbumin (2) 52.35 6.65 15.88 3.35 33.97 The seralbumin (3) came from a human exudation, and the other bodies from horse s blood. The fibrin was prepared from a filtered common-salt plasma. The Blood-serum. As above stated, the blood-serum is the clear liquid which is pressed out by the contraction of the blood-clot. It differs chiefly from the plasma in the absence of fibrinogen and an abundance of fibrin-ferment. Considered qualitatively the blood-serum contains the same chief constituents as the blood-plasma. Blood-serum is a sticky liquid which is more alkaline than the plasma. The specific gravity in man is 1.027 to 1.032, average 1.028. The color is strongly or faintly yellow; in human blood- serum it is pale yellow with a shade towards green, and in horses it is often amber-yellow. The serum is ordinarily clear ; after a meal it may be opalescent, cloudy, or milky white, according to the amount of fat contained in the food. Besides the above-mentioned bodies, the following constituents are found in the blood-plasma or blood-serum : Fat occurs from 1-7 p. m. in fasting animals. After partaking 'Maly's Jahresber., Bd. 11. GLUCOSE AND OLYOOLYSIS. 123 of food the amount is increased to a great extent. We also find soaps (Hoppe-Sbtlee'), cholesierin, and lecithin. Glucose seems to be a physiological constituent of the plasma. According to the investigations of Abeles, Ewald, Kulz, T. Mekiitg,^ and Seegen," the sugar found in the plasma is glucose. Otto* found in the plasma, besides glucose, another reducing, non-fermentable substance. The amount of glucose in the blood is about 1-1.6 p. m. Otto found in human blood 1.18 p. m. glucose and 0.29 p. m. of the other reducing substance. According to Jacobsen,' this is soluble in ether and is closely related to jecorin. The amount of glucose in the blood seems to be almost indepeadent of the food ; nevertheless after feeding with large quantities of glucose and dextrin Bleile' observed a signifi- cant increase of glucose. If the amount is more than 3 p. m., according to Cl. Beknaed,' the glucose passes into the urine, pro- ducing glycosuria. The different amounts of glucose in the blood from different vessels and under various conditions will be fully discussed later. The glycogen found in the blood does not seem to come from the plasma, but from the leucocytes. Bernakd ' has shown that the quantity of sugar in the blood diminishes more or less rapidly on leaving the veins. Lepiitb," associated with Baeral, has specially studied this decrease in the quantity of sugar and calls it glycolysis. Lepine and Baeral as well as Arthus '° have shown that this glycolysis takes place in the complete absence of micro-organisms." It seems to be due to a ' Zeitschr. f . physiol. Chem. , Bd. 8. ' Du Bois-Reymond's Archiv, 1877, S. 379. This article contains numerous references. " Pflilger's Archiv, Bd. 40. *lbid., Bd. 35. Contains a good review of the literature of sugar in the blood. sCentralbl. f. Physiol., 1893, Part 13, ' Du Bois-Reymond s Archiv, 1879, S. 67-69. ' Lefons sur le diabfite. Paris, 1877. 8 Ibid. "Lyon medical, Tome 63 and 63 , Compt. rendus, Tome 110, 113, and 113 ; Lepine, Le ferment glycolytique et la pathogenie du diabfite (Paris, 1891), and Revue analytique et critique des travaux, etc., in Arch, de med. exper. (Paris, 1893). '» Arch, de Physiol. , July 1891 and April 1892. " A critical review of the various methods of removing proteids from the blood in sugar estimations is given by Seegeu, Centralbl. f. Physiol., 1893, Heft 17. 124 THE BLOOD. soluble enzyme whose activity is destroyed by heating to + 54° C. This enzyme is derived, according to the above investigators, from the leucocytes and is, according to Lbpinb, delivered from the pancreas to the blood. According to Lbpin-b' the pancreas con- tains a zymogen of the glycolytic enzyme occurring in the blood. This zymogen, which is converted into the enzyme by the action of sulphuric acid of 2 p. m. at 38° C, is nothing but the diastatic enzyme. Eohmann and Spitzbe' and Spitzbk,' who have shown the occurrence of a glycolysis under the influence of not only the blood but also various tissues, consider this, as first shown by Kkaus,' a process of oxidation. This oxidation is brought about by the oxygen of the oxidation ferment occurring in the form- elements. According to Aethus and Colbnbrandek ' this gly- colysis is only a post-mortem process and not a vital one. Blood-plasma contains an enzyme which converts starch and glycogen into sugar (Eohmann " and Bial'). This enzyme also occurs in the lymph, but not in the form-elements of the blood. The sugar found in the serum by enzyme action is partly maltose or isomaltose and partly dextrose. These various sugars are pro- duced in differing quantities in the various phases of the enzymotic processes. This is accounted for, by recent researches of Rohmann and C. Hamburgbe,' by the presence of two differing enzymes in the blood. One of these enzymes is diastase, which converts starch and glycogen into maltose. The other differs from invertin and, according to Eohmann, is probably an enzyme, identical with glucase, occurring in the plant kingdom, and which has the prop- erty of splitting maltose into dextrose. Among the bodies which are found in the blood, and without doubt met with in smaller or greater amounts in the plasma, are to be mentioned urea, uric acid (found in human blood by Abelbs ') , > Compt. rend., Tome 130. ''BeT. d. deutsch. chem. Gesellsch., Bd. 38. » Pfluger's Arohiv, Bd. 60. ■•Zeitsclir. f. klin. Med., Bd. 31. • ' Maly's Jahresber., Bd. 23. « Ber. d. deutsoli. oliem. Gesellsch., Bd. 35, and Pfluger's Arch., Bd. 53. ' Ueber das diastatische Ferment des Lymph- und Blutserums. Inaug. Diss. Breslau, 1893. Contains also the older literature. See also Pfliiger'3 ATch.,Bdd. 53, 54, and 55. ' Ber. d. deutsch. chem. Gesellsch., Bd. 27, and Pfluger's Arch. j Bd. 60. ' Wien. med. Jahrbiicher, 1887. EXTRA CTIVES. 12 rt ceratin, carbamic acid, paralactic acid, and hippuric acid. The quaatity of urea depends npon the natrifcive condition of the animal. During starvation Schondoefe ' found a minimum of 0.348 p. m., and in the highest stages of urea formation a maximum of 1.529 p. m. Under pathological conditions the following bodies have been found: xanthm bases, leucm, tyrosin, and biliary con- stituents. The coloring matters of the blood-serum are very little known. In equine blood-serum biliary coloring matters, bilirubin, besides other coloring matters, often occur. The yellow coloring matter of the serum seems to belong to the group of lui&ms, which are often called lipochromes or fat-coloring matters. From ox-serum Krukenberg' was able to isolate with amyl alcohol a so-called lipochrome whose solution shows two absorption-bands, of which one encloses the line F and the other lies between F and G. The mineral bodies in serum and plasma are qualitatively, but not quantitatively, the same. A part of the calcium, magnesium, and phosphoric acid is removed on the coagulation of the fibrin. By means of dialysis, the presence of sodium chloride, which forms the chief mass or 60-70^ of the total mineral bodies, also lime- salts, sodium carbonate, besides traces of sulphuric and phosphoric acids and potassium, may be directly shown in the serum. Traces of silicic acid, fluorine, copper, iron, manganese, and ammonia are claimed to have been found in the serum. As in most animal fluids, the chlorine and sodium are in the blood-serum in excess of the phosphoric acid and potassium (the occurrence of which in the serum is even doubted). The acids found in the ash are not snflEL- cient to saturate the bases found, a condition which shows that a part of the bases is combined with organic substances, perhaps proteids. The gases of the blood-serum, which consist chiefly of carbon dioxide with only a little nitrogen and oxygen, will be described when treating of the gases of the blood. Because of the difficulty of obtaining. plasma only a few analyses have been made. As an example the results of the analyses of the blood-plasma of the horse will be given below. The analysis No. 1 waa made by Hoppe-Seylbe.' No. 2 is the average of the results ' Pflttger's Archiv, Bd. 54. » Sitzber. d. Jen. Gesellsch. f. Med., 1886. » Cit. from V. Gorup-Besanez's LehrbucU der physiol. Chem., 4. Aufl., p. 346. 126 THE BLOOD. of three analyses made by the author.' The figures are given in 1000 parts of the plasma. No. 1. No. 2. Water 908.4 917.6 Solids 91.6 82.4 Total proteids 77.6 69.5 Fibrin 10.1 6.5 Globulin 38.4 Seralbumin 24 . 6 Fat 1.2^ Extractive substances 4.01 ioq Soluble salts 6.4 ( ' Insoluble salts l-7j As an example of the composition of blood-seram with special regard to the relationship of the different proteids to each other, the following analyses are given. The results are in 1000 parts. Serum from SoUds. Total Albumin- ous Bodies, Ser- globulin. Ser- albumin, Lecithin, Fat, Salts, etc. Ser- globulin Ser- albumin. Authority. Man . . . Horse . . Oi Dog.,.. Hen.,.. Frog... Eel . . . 92.07 85.97 89.65 54.00 76.20 72.57 74.99 58.20 39.49 25.40 67.30 31.04 45.65 41.69 20.50 7.84 21.80 52.80 45.16 26.92 33.30 37,70 31.65 3.60 14.50 15.88 13.40 14.66 14.51 1 1.5 1 0.591 1 Hammaestbk ' 0,843 1 1,8 1 4.03 1 0,165 1 0.275 Salveoli" Hammarsteh Halliburton* According to Halliburton, the amount of the albumins in comparison with the globulins in cold-blooded animals is not only proportionally smaller, but the total amount of albuminous bodies is smaller than in the warm-blooded animals. By a comparative investigation of serum and plasma from the same individual, we find more serglobnlin in the one than in ' Pfluger's Archiv, Bd, 17. « Ibid. » Du Bois-Reymond's Archiv, 1881, S. 275. * Journ. of Physiol., Vol. 7, pp, 319-321. MINERAL CONSTITUENTS. 127 the other. The reason for this may lie partly in the fact that in the coagnlation of fibrin from the fibrinogen some fibrin-globulin is formed which in the quantitative estimation is precipitated with the serglobulin, and partly because the white corpuscles yield serglobu- lin in the fibrin coagulation (Alex. Schmidt). The quantity of mineral bodies in the serum has been deter- mined by many investigators. The conclusions drawn from the analyses is that there exists a rather close correspondence between human and animal blood- serum, and it is therefore sufficient to give here the analysis of C. Schmidt' of (1) human blood, and of (3) pig- and (3) ox-blood by BuNGB." As in the calcination of lecithin and proteids incor- rect results are obtained for the phosphoric and sulphuric acids, these results will not be given below. All figures correspond to 1000 parts of serum. 1 2 ^ KjO 0.387-0.401 0.273 0.254 Na,0 4.290-4.290 4.272 4.351 CI 3.565-3.659 3.611 3.717 CaO 0.155-0.155 0.136 0.126 MgO 0.101 0.038 0.045 FejOs O.OIJ 0.011 The amount of NaCl is about 6 p. m., and it is remarkable that this amount of NaOl remains almost constant, so that with food containing an excess of NaCl it is quickly eliminated by the urine, and with food poor In chlorides the amount in the blood first decreases, but increases after taking chlorides from the tissues. The secretion of chlorides by the urine is thereby diminished. The amount of phosphoric acid, calculated as Na,HPO„ in the serum freed from lecithin has been determined as 0.03-0.09 p. m. by Sertoli' and Mkoczkowski' in different varieties of serum. The small amount of iron sometimes found in the serum probably originates from a contamination with the blood-coloring matters. ' Cit. Hoppe-Seyler's Physiol. Chem., 1881, S. 439. ' Zeitschr. f . Biologie, Bd. 12, S. 206-208. • Hoppe-Seyler's Med. chem. Untersuch., S. 350. « Centralbl. f. d. med. Wissensoh., 1878, No. 20. 12S . THE BLOOD. II. The Form-elements of the Blooti. The Red Blood-corpuscles. The blood-corpnscles are round, biconcave disks withonfc mem- brane and nucleus in man and mammalia (with the exception of the llama, the camel, and their congeners). In the latter animals, as also in birds, amphibia, and fishes (with the exception of the cyclostoma), the corpuscles have in general a nucleus, are biconvex and more or less elliptical. The size varies in different animals. In man they have an average diameter of 7 to 8 /^ (/^ = 0.001 mm.) and a maximum thickness of 1.9//. The volume of a single red corpuscle of a horse amounts to 0.00000003858 c.mm. and of a pig 0.0000000435 c.mm. (Wendblstadt and L. BleibtkSu'). The weight of a red corpuscle of a horse is, according to the same investigators, 0.00000004307 mg. Their specific gravity is 1.088 to 1.105. They are heavier than the blood-plasma or serum, and therefore sink in these liquids. In the discharged blood they may lie sometimes with their flat surfaces together, forming a cylinder like a roll of coin. The reason for this is unknown, but as it may be observed in defibrinated blood it seems probable that the forma- tion of fibrin has nothing to do with it. Seen with the microscope, each blood-corpuscle has a pale yellow color, and only in moderately thick layers is the color somewhat reddish. The number of red blood-corpnscles is different in the blood of various animals. In the blood of man there are generally 5 million red corpuscles in 1 c.mm., and in woman 4 to 4.5 million. On diluting the blood with water and alternately freezing and thawing it, as also on shaking it with ether, or by the action of chloroform or bile, a remarkable change takes place. The blood- coloring matters, which are hardly free in the blood-corpnscles, but rather, according to the view of Hoppe-Seylbe, are combined with some other substance, perhaps lecithin, are by this means set free from these combinations and pass into solution, while the remainder of each blood-corpuscle forms a swollen mass. By the action of carbon dioxide, by the careful addition of acids, acid salts, tincture of iodine, or certain other bodies, this residue, rich in albumin, condenses and in many cases the form of the blood-corpuscles may ' Pflilger's Archiv, Bd. 52. STROMA. 129 Tae again obtained. This residue has been called the stroma of the red blood-corpuscles. To isolate the stromata of the blood-corpnscles they are washed first by diluting the blood with 10-30 vols, of a 1-2^ common-salt solution and then separating the mixture by centrifugal force or by allowing it to stand at a low temperature. This is repeated a few times until the blood-corpuscles are freed from serum. These purified blood-corpuscles are, according to Wooldbidge, mixed with 5-6 vols, of water and then a little ether is added until com- plete solution is obtained. The leucocytes gradually settle to the bottom, a movement which may be accelerated by centrifugal force, and the liquid which separates therefrom is very carefully treated with a 1^ solution of KHSO, until it is about as dense as the original blood. The separated stromata are collected on a filter and quickly washed. WoOLDRiDGE ' found as constituents of the stroma lecithin, cholesterin, nucleoalbumin, and a globulin which, according to Halliburton, is probably a nucleoproteid which he calls cell- glolulin. No nuclein substances or seralbumin or albumoses could. ■ be detected by Halliburton and Friend.' The nucleated red blood-corpnscles of the bird contain, according to Plosz and Hoppb-Setler,' nuclein and an albuminous body which swells to a slimy mass in a 10^ common-salt solution, and which seems to be closely related to the hyaline substance {hyaline substance of Eovida) occurring in the lymph-cells. The red blood-corpuscles> without any nucleus are, as a rule, very poor in proteid but are rich in hasmoglobin; the nucleated corpuscles are richer in proteid and poorer in haemoglobin. A gelatinous, fibrin-like proteid body may be obtained from the red blood-corpuscles under certain circumstances. This fibrin-like mass has been observed on freezing and then thawing the sediment of the blood-corpuscles, or on discharging the spark from a large Leyden jar through the blood, or on dissolving the blood-corpnscles of one kind of animal in the serum of another (Landois, stroma- fibrin'). In none of these cases has it been shown that we have to deal with a fibrin formation at the expense of the stroma. It seems ' Du B;)is-Reymond's Archiv, 1881, S. 387. ' Journal of Physiol., Vol. 10. ' Hoppe-Seyler's Med. chem. Untersuch. , S. 510. * Ceutralbl. f. d. med. WissensoU., 1874, S. 421. 130 THE BLOOD. only to have been shown that the red blood-corpuscles of frog's blood contain fibrinogen (Alex. Schmidt and Semmbr'). The mineral bodies of the red corpuscles are chiefly potassium, phosphoric acid, and chlorine; in the red corpuscles of man, the dog, and the ox sodium has also been found. The most important constituent of the blood -corpuscles from a physiological standpoint is the red coloring matter. Blood-coloring Matters. According to Hoppe-Seyler' the coloring matter of the red blood-corpuscles is not in a free state, but combined with some otner substance. The crystalline coloring matter, the haemoglobin or oxyhsemoglobin, which may be isolated from the blood, is con- sidered, according to Hoppe-Setler, as a cleavage prod act of this combination, and it acts in many ways unlike the questionable com- bination itself. This combination is insoluble in water and uncrys- tallizable. It strongly decomposes hydrogen peroxide without beiog oxidized itself; it shows a greater resistance to certain chemical reagents (as potassium ferricyanide) than the free coloring matter, and lastly it gives ofE its loosely combined oxygen much more easily in vacuum than the free pigment. To distinguish between the cleavage products, the haemoglobin and the oxyhemoglobin, Hoppe-Setler ' calls the combination of the blood-coloring matter of the venous blood-corpuscles pMebin, and that of the arterial ' arterin. Since the above-mentioned combination of the blood- coloring matters with other bodies, for example (if they really do exist) with lecithin, have not been closely studied, the following statements will only apply to the free pigment, the haemoglobin. The color of the blood depends in part on hcemoglobm or pseudo- TiCBmoglolin (see below), and in part on a molecular combination of this with oxygen, the oxyhmnoglolin. We find in blood after asphyxiation almost exclusively heemoglobin and pseudo-haemo- globin, in arterial blood disproportionately large amounts of oxy- haemoglobin, and in venous blood a mixture of both. Blood-color- ing matters are found also in striated as well as in certain smooth muscles, and lastly in solution in different invertebrates. The quantity of haemoglobin in human blood may indeed be somewhat ' Alex. Schmidt in Pfliiger's Arcliiv, Bd. 11, S. 530-559. ' Zeitschr. f. physiol. Cliem., Bd. 13. » Ibid., S. 495. COMPOSITION OF HMMOQLOBIN. 131 Tariable under different circumstances, bnt amounts averaging about 14^ or 8.5 grammes have been determined for each kilo of the weight of the body. Haemoglobin belongs to the group of compound proteids, and yields as cleavage products, besides very small amounts of volatile fatty acids and other bodies, chiefly proteid (96^) and a coloring matter, hcBmochromogen (4^), containing iron, which in the pres- ence of oxygen is easily oxidized into hwmatin. The hsemoglobin prepared from different kinds of blood has not exactly the same composition, which seems to indicate the presence of different haemoglobins. The analyses of different investigators of the hsemoglobin from the same kind of blood do not always agree with one another, which probably depends upon the somewhat various methods of preparation. The following analyses are given as examples of the constitution of different haemoglobins : Haemoglobin from the Dog.. Horse 53.85 54.57 54.87 51 15 Ox 54.66 Pig 54 17 " 54.71 Guinea-pig 54.13 Squirrel . . 54.09 Goose 54.36 Hen 53.47 7.33 7.33 6.97 6 76 7.35 7.38 7 38 7.36 7.39 7.10 7.19 N 16.17 16.38 17.81 17.94 17.70 16.38 17.43 16.78 16.09 16.31 16.45 Pe P=0. S 0.390 0.430 21 84 (Hoppe-Seyler>) 0..568 0.336 30.93 (Jaquet^) 0.650 0.470 19.73 (Ko8SBL») 0.890 0.835 38.43 (Zinoffskt^) 0.447 0.400 19.543 (Hupners) 0.660 0.430 31.360 (Otto«) 0.479 0.399 19 603 (Hufner) 580 0.480 30.680 (Hoppb-Setlbr) 0.400 0.590 31.440 0,540 0.430 30.690 0.77 0.857 0.335 33.500 0.197 (Jaquet) The question whether the amount of phosphorus in the haemo- globin from birds exists as a contamination or as a constituent has not been decided. According to Inoko' the haemoglobin from goose's blood consists of a combination between nucleic acid and haemoglobin. In the haemoglobin from the horse (Zixoffsky), the pig, and the ox (Hufkbr) we have 1 atom of iron to 2 atoms of sulphur, while in the hsemoglobin from the dog (Jaquet) the relation is 1 to 3. Prom the data of the elementary analysis, as • Med. chem. Untersuch. , S. 370. « Zeitschr. f. pliysiol. Chem., Bd. 14, S. 296. » Ibid., Bd. 3, S. 150. • Ibid., Bd. 10. » Beitr. z, Physiol., Pestsohr. f. C. Ludwig, 1887, S. 74-81. • Zeitschr. f. physiol. Chem., Bd. 7, S. 61. ' Ibid., Bd. 18. 132 THE BLOOD. also from the amount of loosely combined oxygen, HuFifBE ' has calculated the molecular weight of dog-hffimoglobin as 14,139 and the formula C,„H,„„]Sr,„FeS,0,,,. 'The molecular weight is there- fore very high. The haemoglobin from various kinds of blood not only shows a diverse constitution, but also a different solubility and crystalline form, and a varying quantity of water of crystallization ; hence we infer that there are several kinds of hsemoglobin. Bohr" is a very zealous advocate of this supposition. He has been able to obtain haemoglobin from dog and horse blood, by fractional crystallization, which had different power of combining with oxygen and containing different quantities of iron. Hoppe- Setlek ° had already prepared two different forms of haemoglobin crystals from horse's blood, and Bohr concludes from a collection of these observations that the ordinary haemoglobin consists of a mixture of different haemoglobins. In opposition to this state- ment Huener' has shown that only one haemoglobin exists in ox blood, and that this is probably true for the blood of many other animals. Oxyhsemoglobiu, which has also been called h^matoglobulist or H^MATOCETSTALLIN, is a molccular combination of haemoglobin and oxygen. For each molecule of haemoglobin 1 molecule of oxygen exists; and the amount of loosely combined oxygen which is united to 1 grm. hemoglobin (of the dog) has been determined by Huener' as 1.34 c.c. (calculated at 0° C. and 760 mm. mer- cury). According to Bohb' the facts are different. He differentiates between four different oxyliaimoglobins, according to the quantity of oxygen which they absorb, namely, or-, [i-, y~, and ^-oxyhaemoglobin, all having the same absorp- tion-spectrum and 1 gm. combining with respectively 0'4, 0-8, 1-7, and 2-7 cc. oxygen at the temperature of the room and with an oxygen pressure of 150 mm. mercury. The ;K-oxyhaBmoglobin is the ordinary one obtained by the customary method of preparation. Bohb designates as a-oxyhsemoglobin the crystalline powder obtained by drying ;r-oxyhEemoglobin in the air. On dissolving ct-oxyhaemoglobin in water it is converted into /S-oxyhsemoglobin without decomposition, and the quantity of iron is increased. On keeping a solution of Journ. f. prakt. Chem., Bd. 33. ' "Sur les combinaisons de Themoglobine avec I'oxygfine." Extrait du Bulletin de I'Academie Royale Danoise des sciences, 1890 ; also Centralbl f Physiol., 1890, S. 249. ^ Zeitschr. f. physiol. Chem. , Bd. 2. * Du Bois-Reymond's Archiv, 1894. OXTH^MOQLOBm. 133 y-oxyhsBmoglobin in a sealed tube it is transformed into (S-oxyhsemoglobin, altliough the circamstances of this change are not known. According to HUfner ' these are nothing but a mixture of genuine and partly decomposed haemoglobins. The ability of haemoglobin to take np oxygen seems to be a function of the iron it contains, and when this is calculated as about 0.33-0.40j^, then 1 atom of iron in the haemoglobin corre- sponds to about 3 atoms = 1 molecule of oxygen. The combination of haemoglobin with oxygen is, as has been stated, loose and dis- sociatable, and the quantity of oxygen taken up by a haemoglobin solution depends upon the pressure of the oxygen at that tempera- ture. If this latter be decreased by means of a vacuum, especially on gently heating or by passing some indifferent gas through the solution, all of the oxygen may be expelled from an oxyhaemoglobin solution so that only haemoglobin remains. The reverse of this is true of a haemoglobin solution which by its remarkable attraction Jor oxygen may be converted into oxyhaemoglobin. Oxyhaemoglobin is generally considered as a weak acid. Oxyhaemoglobin has been obtained in crystals from several varieties of blood. These crystals are blood-red, transparent, silky, and may be 2-3 mm. long. The oxyhaemoglobin from squirrel's blood crystallizes in six-sided plates of the hexagonal system ; the other varieties of blood yield needles, prisms, tetrahedra, or plates which belong to the rhombic system. The quantity of water of crystallization varies between 3-lOj^ for the different oxyhfemo- ^lobins. When completely dried at a low temperature over sul- phuric acid the crystals may be heated to 110-115° 0. without decomposing. At higher temperatures, somewhat above 160° C, they decompose, giving an odor of burnt horn, and leave, after complete combustion, an ash consisting of oxide of iron. The oxyhaemoglobin crystals from difficultly crystallizable kinds of blood, for example from such as ox's, human, and pig's blood, are easily soluble in water. The oxyhaemoglobin from easily crystal- lizable blood, as from that of the horse, dog, squirrel, and guinea- pig, are soluble with difficulty in the order above given. The oxyhaemoglobin dissolves more easily in a very dilute solution of alkali carbonate than in pure water, and this solution may be kept. The presence of a little too much alkali causes the oxyhaemoglobin to quickly decompose. Thp crystals are insoluble without decolor- ' Du Bois-Reymond's Archiv, 1894. 134 THE BLOOD. ization in absolute alcohol. According to Nescki,' it is hereby converted into an isomeric or polymeric modification, called by him parahcemogloMn. Oxyhsemoglobin is insoluble in ether, chloroform, benzol, and carbon disulphide. A solution of oxyhsemoglobin in -water is precipitated by many metallic salts, but is not precipitated by sugar of lead or basic lead acetate. On heating the watery solution it decomposes at 60° to 70° C, and it splits off proteid and hsematin. It is also decom- posed by acids, alkalies, and many metallic salts. It gives the ordinary reactions for proteids with the ordinary proteid reagents, which first decompose the oxyhsemoglobin with the splitting off of proteid. The oxyhsemoglobin may, when it is gradually oxidized, act as an " ozone exciter" by the decomposition of neutral oxygen, con- verting it into active oxygen (Pflugee ") . It may also have another relation to ozone, since it has the property of an " ozone trans- mitter" in that it causes the reaction of certain reagents (turpen- tine) containing ozone upon ozone reagents such as tincture of guaiacum. A sufficiently dilute solution of oxyhsemoglobin or arterial blood sliows a spectrum with two absorption-bands between the Fkaun- HOFEB lines D and E. The one band, a, which is narrower but darker and sharper, lies on the line D; the other, broader, less defined and less dark band, /?, lies at E. These bands can be detected in a layer of 1 cm. thick of a 0.1 p. m. solution of oxyhsemoglobin. In a still weaker dilution the band /? first dis- appears. By increased concentration of the solution the two bands become broader, the space between them smaller or entirely obliter- ated, and at the same time the blue and violet part of the spectrum is darkened. The oxyhsemoglobin may be differentiated from other coloring matters having a similar absorption-spectrum by its behavior towards reducing substances. (See below.) A great many methods have been proposed for the preparation of oxyhsemoglobin crystals, but in their chief features they all agree with the following method as suggested by Hoppe-Setlee ' : The washed blood-corpuscles (best those from the dog or the horse) are stirred with 2 vols, water and then shaken with ether. After ' Nenchi and Sieber, Ber. d. deutsch. cliem. Gtesellsch., Bd. 18. » Pfluger's Archiv, Bd. 10, S. 353. 3 Med. cbem. Untersuot., S. 181. HJSMOOLOBIN. 135 decanting the ether and allowing the ether which is retained by the hlood solution to evaporate in an open dish in the air, cool the filtered blood solution to 0° C, add while stirring J vol. of alcohol also cooled, and allow to stand a few days at — ^° to — 10° C. Tlie crystals which separate may be repeatedly recrystallized by dissolving in water of about 35° C, cooling and adding cooled alcohol as above. Lastly, they are washed with cooled water con- taining alcohol {\ vol. alcohol) and dried in vacuum at 0° C. or a lower temperature. According to Gschbidlen's ' investigations, oxyhsemoglobin crystals may be obtained from difficultly crystalliz- able varieties of blood by allowing the blood first to putrefy slightly in sealed tubes. After shaking with air by which the blood is again arterialized, proceed as above. For the preparation of oxy haemoglobin crystals in small quanti- ties from blood easily crystallized, it is often sufficient to stir a drop of blood with a little water on a microscope slide and allow the mixture to evaporate so that the drop is surrounded by a dried ring. After covering with a thin glass, the crystals gradually appear radiating from the ring. These crystals are formed in a surer manner if the blood is first mixed with some water in a test-tube and shaken with ether and a drop of the lower deep-colored liquid treated as above on the slide. Hsemoglobin, also called eeducbd h^moslobin or purple CEUORiN (Stokes ■'), occurs only in very small quantities in arterial blood, in larger quantities in venous blood, and is nearly the only blood-coloring matter after asphyxiation. Hsemoglobin is much more soluble than the oxyhsemoglobin, and it can therefore only be obtained as crystals with difficulty. These crystals are as a rule isomorphous to the corresponding oxyhaemoglobin crystals, but are darker, having a shade towards blue or purple, and are decidedly more pleochromatic. Its solu- tions in water are darker and more violet or purplish than solutions of oxyhsemoglobin of the same concentration. They absorb the blue and the violet rays of the spectrum in a less marked degree, but strongly absorb the rays lying between C and D. In proper dilution the solution shows a spectrum with one broad, not sharply defined band between D and E. This band does not lie in the middle between D and E, but is towards the red end of the spectrum, a little over the line D. A hsemoglobin solution actively absorbs oxygen from the air and is converted into an oxyhsemo- globin solution. ' Pfliiger's Archiv, Bd 16. ' Philosophical Magazine, Vol. 28, No. 190, Nov. 1864. 136 THE BLOOD. A solution of oxy haemoglobin may be easily concerted into a solution having the spectrum of haemoglobin by means of a yacuum, by passing an indifferent gas through it, or by the addition of a reducing substance, as, for example, an ammoniacal ferro-tartarte solution (Stokes' reduction-liquid). If an oxyheemoglobin solution or arterial blood is kept in a sealed tube, we observe a gradual consumption of oxygen and a reduction of the oxyhemoglobin into hsemoglobin. If the solution has a proper concentration, a crystal- lization of hsemoglobin may occur in the tube at lower temperatures (Hufnee'). Fseudohaemoglobin. Ludwig and Siegekied ° have observed that blood which has been reduced by hyposulphites so completely that the oxyhaemoglobin spectrum disappears and only the hsemo- globin spectrum is seen yields large amounts of oxygen when exposed to a vacuum. Blood which has been reduced by the passage of a stream of hydrogen through it until the oxyhaemo- globin spectrum disappears acts in the same manner. Hence a loose combination of haemoglobin and oxygen exists which gives the haemoglobin spectrum, and this combination is called pseudo- haemoglobin by Ludwig and Siegekied. Pseudohaemoglobin, whose presence has been detected in asphyxiation blood from dogs, is considered by the author as an intermediate step between haemoglobin and oxyhaemoglobin, on the reduction of the latter. Methaemoglobin. This name has been given to a coloring matter which is easily obtained from oxyhaemoglobin as a trans- formation product and which has been correspondingly found in transudations and cystic fluids containing blood, in urine, in haematuria or haemoglobinuria, also in urine and blood on poisoning with potassium chlorate, amy] nitrite or alkali nitrite, and many other bodies. Metheemoglobin does not contain any oxygen in molecular or dissociable combination, but still the oxygen seems to be of impor- tance in the formation of methaemoglobin, because it is formed from oxyhaemoglobin in the absence of oxygen or oxidizing agents, and not from haemoglobin. If arterial blood be sealed up in a tube, it, gradually consumes its oxygen and becomes venous, and by this absorption of oxygen a little methaemoglobin is formed. The same occurs on the addition of a small quantity of acid to the blood. ' Zeitschr. f. physiol. Chem., Bd. 4, S. 382. ' ' Du Bois-Reymond's Archiv, 1890 ; also see Ivo Novi, Pfluger's Archiv, Bd. 56. METRJEM00L(jB1N. 137 / By the sj^ontaueous decomposition of blood some methaemoglobin is formed, and by the action of ozone, potassium permanganate, potassium ferricyanide, chlorates, nitrites, nitrobenzol, pyrogallol, pyrocatechin, acetanilid, and certain other bodies on the blood an abundant formation of methsemoglobin takes place. According to the investigations of HCfnee, li^ULZ, and Otto' methsemoglobin contains just as much oxygen as oxy haemoglobin, but it is more strongly combined. Jadeeholm ' and Saarbach ' claim that a methsemoglobin solution is first converted into an oxyhsemoglobin and then into a haemoglobin solution by reducing substances, while Hoppe-Seylek and Akaki ' claim that it is con- verted directly into a hsemoglobin solution. Methaemoglobin has the same constitution as oxyhaemoglobin (HuFNEE and Otto). It was first shown by them that it crystal- lizes in brownish-red needles, prisms, or six-sided plates. It dis- solves easily in water; the solution has a brown color and becomes a beautiful red on the addition of alkali. The solution of the pure substance is not precipitated by basic lead acetate alone, but by basic lead acetate and ammonia. The absorption-spectrum of a watery or acidified solution of methsemoglobin is, according to Jadeeholm and Beetin-Sans,° very similar to that of haematin in acid solution, but is easily distinguished from the latter since, on the addition of a little alkali and a reducing substance, the former passes over to the spectrum of reduced hsemoglobin, while a hffimatin solution under the same conditions gives the spectrum of an alkaline haemochromogen solution (see below). Methsemoglobin in alkaline solution shows two absorption-bands which are like the two oxyhaemoglobin bands, but they differ from these in that the band yS is stronger than a. By the side of the band a and united with it by a shadow lies a third, fainter band between C and D, near to D. According to other investigators, Araki and Dix- TEiCH,' a neutral or faintly acid methaemoglobin solution shows only one characteristic band a between C and Z>, and the second ' Zeitschr. f. physiol. Chem., Bd. 7. ' Nord. raed. Arkiv, Bd. 16, and Zeitschr. f. Biologie, Bd. 16. » Pflilger's Archiv, Bd. 38. * Zeitschr. f physiol. Chem., Bd. 14. » Compt. rend , Tome 106. * Arch. f. exp. Path. u. Pharm., Bd. 29. Important references on methse- moglobin are given by Otto, PflQger's Archiv, Bd. 31. 138 THE BLOOD. band between D and E is only due to contamination with oxyhsemoglobin. Crystallized methsemoglobin may be easily obtained by treating- a concentrated solution of oxyhsemoglobin with a sufficient quantity of concentrated potassium ferricyanide solution to give the mixture a porter-brown -color. After cooling to 0° C. add i vol. cooled alcohol and allow the mixture to stand a few days in the cold. The crystals may be easily purified by recrystallizing from water by the addition of alcohol. Carbon Monoxide Hsemoglobin ' is the molecular combination between 1 mol. haemoglobin and 1 mol. CO. This combination is stronger than the oxygen combination of haemoglobin. The oxygen is for this reason easily driven ofE by carbon monoxide, and this explains the poisonous action of carbon monoxide, which kills by the expulsion of the oxygen of the blood. Hufkek" has deter- mined the dissociation constant of carbon-monoxide hsemoglobin and finds it equal to 0.074 for a solution containing on an average 11 gm. in 100 c.c. at a temperature of 32.7° C. The dissociation constant of carbon monoxide hemoglobin is hence aboat 33 times smaller than that of oxyheemoglobin under nearly the same condi- tions (jff'for oxy haemoglobin = 2.44). Carbon monoxide haemoglobin is formed by saturating blood or a haemoglobin solution with carbon monoxide, and may be obtained as crystals by the same means as oxyhaemoglobin. These crystals are isomorphous to the oxyhaemoglobiu crystals, but are less soluble and more stable, and their bluish-red color , is more marked. For the detection of carbon-monoxide haemoglobin its absorption spec- trum is of the greatest importance. This spectrum shows two- bands which are very similar to those of oxyhaemoglobin, but they occur more towards the violet part of the spectrum. These bands do not change noticeably on the addition of reducing substances; this constitutes an important difference between carbon monoxide and oxyhaemoglobin. If the blood contains oxyhemoglobin and carbon-monoxide haemoglobin at the same time, we obtain on the addition of a reducing substance (ammoniacal ferrotartrate solation) a mixed spectrum originating from the haemoglobin and carbon- monoxide haemoglobiu. ' In reference to carbon monoxide haemoglobin see especially Hoppe-Seyler, Med. cbem. Untersuch., S. 201; Centralbl. f. d. med. -Wissenscb., 1864 and 1865; Zeitschr. f. physiol. Chem., Bdd. 1 and 13. * Du Bois-Beymond's Archiv, Physiol. Abth., 1895. CARBON-DIOXIDE HEMOGLOBIN. 13& A great many reactions have been suggested for the detection of carbon-monoxide haemoglobin in medico-legal cases. A simple and at the same time a good one is Hoppe-Seyler's soda test. The blood is treated with doable its volume of caastio-soda solution of 1.3 sp. gr., by vchich ordinary blood is converted into a dingy brownish mass, which when spread out on porcelain is brown with a shade of green. Carbon-monoxide blood gives under the same conditions a red mass, which if spread out on porcelain shows a beautifal red color. Several modifications of this test have been proposed. Carbon monoxide metheemoglobin has been prepared by Weil and v. Anebp ' by the action of potassium permanganate on carbon monoxide haemoglobin, but this is contradicted by Bbrtin-Sans and Moitessibr.'' Sulphur methsemoglobiu is the name given by Hoppe-Seyler ' to that coloring matter which is formed by the action of sulphuretted hydrogen on oxyhaemoglobin. The solution has a greenish-red, dirty color and shows an absorption band in the red. This coloring matter is claimed to be the greenish color seen on the surface of putre- fying flesh. Carbon-dioxide Haemoglobin, CarhohcemogloUn. Haemoglobin, according to Bohr* and Torup,' also forms a molecular combina- tion with carbon dioxide whose spectrnm is similar to that of haemoglobin. According to Bohr there are three different carbo- haemoglobins, namely, a-, ft-, and /-carbohEemoglobi n, in which 1 gm. combines with respectively 1.5, '5, and 6 c.c. CO, (measured at 0° C. and 760 mm.) at + 18° C. and a pressure of 60 mm. mercury. If a haemoglobin solation is shaken with a mixture of oxygen and carbon dioxide, the haemoglobin combines loosely with the oxygen as well as carbon dioxide, independently of each other, just as if each gas existed alone (Bohr). He considers that the two gases are combined with different parts of the haemoglobin, namely, the oxygen with the pigment nucleus and the carbon dioxide with the proteid component. According to Torup the hasmoglobin mast therefore be partly decomposed by the carbon dioxide setting free some proteid. ' Du Bois-Reymond's Archiv, 1880. 2 Compt. rend.. Tome 113. 2 Med. chem. Untersuch., S. 151; also see Araki, Zeitschr. f. physiol. Chem., Bd. 14. * "Etudes sur les combinaisons du sang avec I'acide carbonique." Extrait du Bull, de I'Acad. Danoise, 1890, also Centralbl. f, Physiol., Bd. 4, 1890. s Maly's Jahresber., Bd. 17, S. 115. 140 THE BLOOD. Nitric-oxide Haemoglobin ' is also a crystalline molecnlar com- binatioa which is eren stronger than the carbon-monoxide haemo- globin. Its solution shows two absorption-bands which are paler and less sharp than the carbon-monoxide hsemoglobin bands, and they do not disappear on the addition of redncing bodies. Hsemoglobin also forms a molecular combination with acetylene. Methse- moglobin solutions become of a beautiful red color by the action of hydrocyanic acid, and, according to Robert, '^ cyanmethmmoglobin is probably formed, _ Its spectrum is very similar to that of hsemoglobin, but it is not converted into oxyhsemoglobin on shaking with air. Decomposition products of the blood-coloring matters. By its decomposition haemoglobin yields, as above stated, a proteid, which has been called glohm, and aferruginotis^j^mew^ as chief prodncts. If the decomposition takes place in the absence of oxygen, a color- ing matter is obtained which is called by Hoppb-Sbtlbk hcemo- chromogen, by other investigators (Stokes) reduced hmmatin. In the presence of oxygen, heemochromogen is quickly oxidized to haematin, and we therefore obtain in this case hcematin as a colored decomposition product. As haemochromogen is easily converted by oxygen into hsematin, so this latter may be reconverted into haemo- chromogen by reducing substances. Hsemochromogen was discovered by Hoppe-Setlee.' He has also been able to obtain this coloring matter as crystals. H^mo- chromogen is, according to Hoppe-Setler, the colored atomic group of hsemoglobin and its combination with gases, and this atomic group is combined with proteids in the coloring matter. The characteristic absorption of light depends on the hfemochromo- gen, and it is also this atomic group which binds in the oxyhaemo- globin 1 mol. oxygen and in the carbon-monoxide haemoglobin 1 mol. carbon monoxide with 1 atom iron. Hoppe-Setler has observed a combination between haemochromogen and carbon mon- oxide, and this combination shows the spectral appearance of carbon monoxide haemoglobin. An alkaline haemochromogen solution has a beautiful red color. It shows two absorption-bands, iirst described by Stokes, of which the one is darker and lies between D and JE, and the other, broader ' See Herrmann and Reichert in Du Bois-Keymond's Archiv, 1865, and Hoppe-Seyler, Med. chem. Untersuch., S. S04. ' Ueber Cyanmethiemoglobiu und den Nachweis der Blausaure. Stutt- gart, 1891. ' Zeitschr. f. physiol. Chem., Bd. 12. H.EMATm. 141 but not so dark, covers the lines E and b. In acid solution hsemo- chroinogen shows four bands, which, according to Jaderholm,' depend on a mixture of hsemochromogen and hsematoporphy/ia (see below), this last formed by a partial decomposition resulting from the action of the acid. Hsemochromogen may be obtained as crystals by the action of caustic soda on haemoglobin at 100° C. iu the absence of oxygen (Hoppe-Setlee). By the decomposition of hasmoglobin by acids (of course in the absence of air) we obtain hsemochromogen con- taminated with a little hsematoporphyrin. An alkaline haemo- chromogen solution is easily obtained by the action of a reducing substance (Stokes' reduction liquid) on an alkaline haematin solu- tion. Haematin, also called Oxyhsematin, is sometimes found in old transudations. It is formed by the action of gastric or pancreatic juices on oxyhaemoglobin, and is therefore also found in the faeces after hemorrhage iu the intestinal canal, and also after a meat diet and food rich in blood. It is stated that haematin may occur in urine after poisoning with arseninretted hydrogen. As shown above, the haematin is formed by the decomposition of oxyhemo- globin, or at least of hsemoglobin, in the presence of oxygen. Bertiit-Sans and Moitessier" have prepared an intermediate body between oxyhsmoglobia and hsemochromogen. This reduced haematin shows one band whose middle lies over D. The constitution of haematin may, according to Hoppe-Set- LER,' be expressed by the formula Cj.Hj.lSr^FeO,. According to Nencki and Sieber it has the formula CjjH^N.PeO,, and they claim that haematin is a hydrate of a body not yet isolated, haemin, C„H3„N,Fe03. Haematin is amorphous, dark brown or bluish black. It may be heated to 180° C. without decomposition; on burning it leaves a residue consisting of iron oxide. It is insoluble in water, dilute acids, alcohol, ether, and chloroform, but it dissolves slightly in warm glacial acetic acid. Haematin dissolves in acidified alcohol or ether. It easily dissolves in alkalies, even when very dilute. The alkaline solutions are dichroitic ; in thick layers they appear red by transmitted light, and in thin layers greenish. The alkaline solu- ' Nord. med. Arkiv, Bd. 16. ' Compt. rend., Tome 116. •Med. chem. Uutersuob., S, 535. 142 THE BLOOD. tions are precipitated by lime- and baryta- water, as also by solutions of neutral salts of the alkaline earths. The acid solutions are always brown. An acid hsematin solution absorbs the red part of the spectrum less and the violet part more. The solution shows a rather sharply defined band between C and D whose position may change with the variety of acid used as a solvent.. Between D and F &, second, much broader, less sharply defined band occurs which by proper dilution of the liquid is converted into two bands. The one between 5 and F, lying near F, is darker and broader, the other, between D and E, lying near ^, is lighter and narrower. Also by proper dilution a fourth very faint band is observed between D and E lying near D. Heematin may thus in acid solution show four absorption-bands; ordinarily one sees distinctly only the bands between Cand D and the broad, dark band — or the two bands — between D and F. In alkaline solution the heematin shows a broad absorption-band, which lies in greatest part between and Z), but reaches a little over the line D towards the right in the space between B and E. Hsemin, Hjemin Crystals, or TEiCHMAifN''s Crystals. Heb- min, according to Hoppe-Seylee, is a combination between hse- matin and hydrochloric acid, having the formula Cj.Hjj^N^PeO^.HCl. Nencki and Siebee designate as hsemin, on the contrary (see page 141), a body not yet isolated, of the formula Cj^Hj^N^FeO,, which may be considered as an anhydride of hsematin or CH..]^ PeO — H,0. The haemin crystals are, according to the latest views, a combination of this substance, hsemin, and HCl, according to the formula Cj.H^^lSr.PeOj.HCl. The analyses of the hydrochloride and hydrobromide of hsematin by Hufner and Kustee ' lead to the same formula. According to Nbncki and Sibbbb the hsemin crystals are a double combina- tion with the solvent, amyl alcohol or acetic acid, which is used in their preparation; while Hoppe-Sbtler claims that the solvent is only held mechani- cally by the crystals. The formula of the hsemin crystals prepared by means of amyl alcohol is, according to Nbnck.i and Sibbbb, (C3,H3oN4Fe03.HC])4.C5H,jO. Haemin crystals form in large masses a bluish-black powder, but are so small that they can only be seen by the microscope. They consist of dark -brown or nearly brownish-black, long, rhombic, or spool-like crystals, isolated, or grouped as crosses, rosettes, or starry ' Ber. d. deutsch. chem. Gesellsch., Bd. 37, S. 573. H^-tlMIN. 143 forms. They are insoluble in water, dilute acids at the normal temperature, alcohol, ether, and chloroform. They are slightly dissolved by glacial acetic acid and warmth. They dissolve in acidified alcohol, as also in dilute caustic or carbonated alkalies; and in the last case they form, besides alkali chlorides, soluble hsematin alkali, from which the haematin may be precipitated by an acid. The preparation of hsemin crystals is always the starting-point for the preparation of hsematin. According to Hoppb-Seylbe,' shake the blood-corpuscles which have been washed with common- salt solution with water and ether, then filter the solution of blood-coloring matters, concentrate strongly, mix with 10-20 vols, glacial acetic acid, and heat for 1-2 hours on the water- bath. After diluting with several volumes of water, allow the liquid to stand a few days. The crystals which separate are then washed with water, boiled with acetic acid, and then washed again with water, alcohol, and ether. Nencki and Siebee coagulate the sediment of the blood-corpuscles by alkali, allow the coagulum to dry incompletely in the air, rub it fine, and then boil it with amyl alcohol after the addition of a little hydrochloric acid. The crystals which separate from the filtrate after cooling are washed with water, alcohol, and ether. If hsemin crystals be dissolved in dilute caustic alkali, hsematin may be precipitated from the solution by the addition of acid ; and from this hsematin pure hsemin crystals may be prepared by heating with glacial acetic acid and a little common salt. In preparing hsemin crystals in small amounts proceed in the following manner: The blood is dried after the addition of a small quantity of common salt, or the dried blood may be rubbed with a trace of common salt. The dry powder is placed on a microscope- slide, moistened with glacial acetic acid, and then covered with the cover-glass. Add, by means of a glass rod, more glacial acetic acid by applying the drop at the edge of the cover-glass, until the space between the slide and the cover-glass is full. Now warm over a very small fiame, with the precaution that the acetic acid does not boil and pass with the powder from under the cover-glass. If no crystals appear. after the first warming and cooling, warm again, and if necessary add some more acetic acid. After cooling, if the experiment has been properly performed, a number of dark-brown or nearly black hsemin crystals of varying forms will be seen. Hsematin is dissolved by concentrated sulphuric acid in the presence of air, forming a purple-red liquid. The iron is here split off and the new coloring matter, called hcsmatoporphyrin by Hoppe- ' Med. chem. Untersuoh., S. 379. 114 THE BLOOD. Seyler,' is iron-free. The hsematin yields with concentrated sul- phuric acid, in the absence of air, a second iron-free coloring matter called hmmatoUn (Hoppe-Setlbr). Haematoporphyrin may also be prepared by the action of glacial acetic acid saturated with hydrobromic acid on hsemin crystals (Nbncki and Siebee"). Hsematoporphyrin, 0,,H„N,0,. This pigment, according to Mac MuifN",' occurs as a physiological coloring matter in certain animals. It has been repeatedly observed in the last few years in human urine especially after the use of sulphonal (see Chapter XV on the urine). This coloring matter is, according to Nbncki and SiEBER, an isomer of the bUe-pigment bilirubin, and its formation from hsematin can be expressed by the following equation : C„H„N,0,Pe + 2H,0 - Fe = aC^H.^N^O,. A pigment closely allied to the urinary pigment urobilin has been obtained by the action of reducing substances on haemotoporphyrin (Hoppe-Setler,' Kencki and Sieber,' Le Nobel," Mac Muifiir '). On the administration of haemotoporphyrin to rabbits, Nbstcki and KoTSCHT ' observed that a part was reduced to a substance similar to urobilin. The combinations of haemotoporphyrin with Na and with HCl have been obtained as crystals by Nencki and Sieber. The acid alcoholic solutions have a beautiful purple color, which becomes violet-bine on the addition of large quantities of acid. The alkaline solution has a beautiful red color, especially when not too much alkali is present. Haematoporphyrin prepared by various methods- may differ somewhat in solubility and in color of solution, but their characteristic absorption-spectra are essentially the same. An alcoholic solution of haematoporphyrin, acidulated with hydrochloric or sulphuric acid, shows two absorption-bands, of which one is fainter and narrower and lies between C and D, near D. The other is much darker, sharper and broader and lies ' Med. chem. Untersuch., S. 528. ^ Monatshefte f. Chem., Bd. 9. ' Journ. of Physiol., Vol. 7. ■* Med. chem. Untersuch., S. 533. 5 Monatshefte f. Chem., Bd. 9. ' Pflliger's Archiv, Bd. 40. ■" Proc. Roy. See, 1880, No. 208 ; Journ. of Physiol., Vol. 10. 8 Monatshefte f. Chem., Bd. 10. E^MATOIDIN. 145 in the middle between D and E. An absorption extends from these bands towards the red, terminating with a dark edge, which may be considered as a third band between the other two. A dilate alkali solution shows four bands, namely, a band between G and D ; a second, broader, surrounding D and with its broadest part between D and E; a third, between D and E nearly at E; and lastly a fourth, broad and dark band between b and F, On the addition of an alkaline zinc-chloride solution the spectrum changes more or less rapidly,' and finally a spectrum is obtained with only two bands, of which one surrounds D and the other lies between D and E. Hsematoidin, thus called by Viechow, is a coloring matter which crystallizes in orange-colored rhombic plates, and which occurs in old blood extravasations, and whose origin from the blood- coloring matters seems to be established (Langhans, Cokdua, Quincke, and others '). A solution of hsematoidin shows no absorption-bands, but only a strong absorption of the violet to the green (Ewald '). According to most observers, hsematoidin is identical with the bile-coloring matter bilirubin. It is not identical with the crystallizable lutein from the corpora lutea of the ovaries of the cow (Piccolo and Liebbn,' Kuhne and Ewald). In the detection of the above-described blood-coloring matters the spectroscope is the only entirely trustworthy means of investi- gation. If it is only necessary to detect blood in general and not to determine definitely whether the coloring matter is hsemoglobiu, methsemoglobin, or hsematin, then the preparation of hsemin crys- tals is an absolute positive proof. The reader is referred to more extended text-books for exacter methods for the detection of blood in chemico-legal cases, and it is perhaps sufficient to give here the chief points of the investigation. If spots on clothes, linen, wood, etc., are to be tested for the presence of blood, it is best, when possible, to scratch or shave off as much as possible, rub with common salt, and from this prepare the haemin crystals. On obtaining positive results the presence of blood is not to be doubted. If you do not obtain sufficient material by the above means, then soak the spot with a few drops of water in a watch-crystal. If a colored solution is thus obtained, then ' Hammarsten, Skan. Arcli. f. Physiol., Bd. 3. ' A compreliensive review of the literature pertaining to hsematoidin may be found in Stadelmann; Der Icterus, etc. Stuttgart, 1891. Pages 3 and 45. 3 Zeitschr. f. Biologie, Bd. 22, S. 475. * Cit. from Gorup-Besanez: Lehrbuch d. physiol. Chem., 4. Aufl., 1878. 146 THE BLOOD. remove the fibres, wood-shavings, and the like as far as possible, and allow the solution to dry in the watch-glass. The dried residue may be partly used for the spectroscope test directly, and part may be employed in the preparation of the hsemin crystals. It also serves to detect hsemochromogen in alkaline solution after previous treatment with alkali and the addition of reducing substances. If a colorless solution is obtained after soaking with water, or the spots are on rusty iron, then digest with a little dilute alkali (5 p. m.). In the presence of blood the solution gives, after neutralization with hydrochloric acid and drying, a residue which may give the hsemin crystals with glacial acetic acid. Another part of the alkaline solution shows, after the addition of Stokes' reduc- tion liquid, the absorption-bands of haemochromogen in alkaline solution. \ The methods proposed for the quantitative estimation of the blood -coloring matters are partly chemical and partly physical. Among the chemical methods to be mentioned is the ashing of the blood and the determination of the amount of iron contained therein, from which the amount of haemoglobin may be calculated. Another method consists in first saturating the blood completely with oxygen. Now pump out thoroughly this oxygen, and calculate from the amount of oxygen the amount of hsemoglobin liresent (GRfiHANT ' and Quinqtjadd'). None of these methods is reliable. The physical methods consist either in a colorimetric or a spec- troscopic investigation. The principle of Hoppe-Setlee's coloHmetric method is that a measured quantity of blood is diluted with an exactly measured quantity of water until the diluted blood solution has the same color as a pure oxyhaemoglobin solution of a known strength. The amount of coloring matter present in the undiluted blood may be easily calculated from the degree of dilution. In the colorimetric testing we use a glass vessel with parallel sides containing a layer of liquid 1 cm. thick (heematinometer of Hoppe-Seyler). The method is good, and the inconvenience that the normal solution of oxyhaemoglobin does not keep for any length of time without decomposing may be prevented by preserving the solution in sealed tubes. The oxyhaemoglobin is gradually reduced to a hsemoglobin solution which may be kept for years, and when required for use it is converted into an oxyhsetnoglobin solution by aerating. Accord- '.ng to an improved method by Hoppe Seylee,' it is much better to use a solution of carbon-monoxide hsemoglobin, as normal solution. The blood solution in this case is saturated with carbon monoxide ' Compt. rend., Tome 75. « Ibid., Tome 76. 'Zeitschr. f. physiol. Chem., Bd. 16, and Lehrbuch d. physlol. u. pathol. chein. Analyse, 6. Aufl., 189a SPaCTBOPHOTOMETEIC ESTIMATION. 147 and the two solutions compared in a specially constructed colori- metric double pipette (see original article). The replacing of the oxy haemoglobin solution by a solution of picrocarmin, as suggested by certain investigators, is to be rejected according to Hoppe- Setlee. The quantitative estimation of the blood -coloring matters by means of the spectroscope may be done in different ways, but at the present time the spectrophotometnc method is chiefly used, and this seems to be the most reliable. This method ' is based on the fact that the extinction coeiScient of a colored liquid for a certain region of the spectrum is directly proportional to the concentration, so that O : E = C^ : E^, when G and C, represent the different concentra- tions and E and E the corresponding coefficient of extinction. G From the equation ^ = -^ it follows that for one and the same Ml Ji, ^ coloring matter this relation, which is called the absorption ratio, must be constant. If the absorption ratio is represented by A, the determined extinction coefficient by E, and the concentration (the amount of coloring matter in grams in 1 c.c.) by C, then G= A. E. Different apparatus have been constructed (Viekoedt and HuFNER ") for the determination of the extinction coefficient which is equal to the negative logarithm of those rays of light which remain after the passage of the light through a layer 1 cm. thick of an absorbing liquid. In regard to these apparatus the reader is referred to other text-books. As control the extinction coefficients are determined in two different regions «f the spectrum, namely, DZ2E—Do4:E and D6SE—DSiE. The coustanfs or the absorption ratio for these two regions of the spectrum are designated by HiJPNER by A and A'. Before the determination the blood must be diluted with water, and if the proportion of dilution of the blood be represented by V, then the concentration or the amount of coloring matter in 100 parts of the undiluted blood is C= 100 .V. A . E and C = 100 . r. A' E'. The absorption ratio or the constants in the two above-mentioned regions of the spectrum have been determined for oxy haemoglobin, haemoglobin, carbon- monoxide haemoglobin, and methsemoglobin. The figures for the above coloring matters obtained from canine blood are as follows ; Oxyheemoglobin Ao = 0,001330 and A'o = 0.001000 Hemoglobin A- = 0.001091 " .4'r = 0.001351 Carbon monoxide haemoglobin Ac = 0.001130 " A'„ = OOOlOOO MethEemoglobin .^to= 0.003696 " ^'m= 0.002798 The quantity of each coloring matter may be determined in a mixture of two blood coloring matters by this method, which is of special importance in ' See Vierordt, Die Anwendung des Spektralapparates zu Photometrie, etc. (Tubingen, 1873), and Hafner, Zeitschr. f. physiol. Chem., Bd. 3; v. NoorJen, Ibid., Bd. 4; Otto, Ibid., Bd. 7 ; and Pflllger's Archiv, Bdd. 31 and 36. 'L. c. 148 THE BLOOD. the determination of the quantity of oxyhaemoglobin and hsBmoglobin present in blood at the same time. If we represent by E and E' the extinction coeffi- cients of the mixture in the above-mentioned regions of the spectrum, by ^o and A'o and Ar and A'r the constants for oxyhaemoglobin and reduced haemoglobin, and by V the degree of dilution of the blood, then the percentage of oxyhsemo- globin JSo and of (reduced) haemoglobin Hr is and A oAr — Jlo-^ r Among the many apparatus constructed for clinical purposes for the quantitative estimation of heemoglobin the haemometer of Fleischl ' is to be preferred. The determination by this apparatus is made by comparing the color of the blood diluted with water with the color of a wedge-shaped movable prism of red glass. If the blood shows the same color as the glass prism, then the amount of haemoglobin in the blood may be directly read from the scale. The amount of haemoglobin is expressed as percentage of the physiologi- cal amount of haemoglobin. Many other coloring matters are found besides the often-occurring haemo- globin in the blood of invertebra. In a few arachnidae, Crustacea, gasteropodae, and cephalopodae a body analogous to haemoglobin containing copper, hcBmo- eyanin, has been found by Frbdericq.^ By the taking up of loosely bound oxygen this body is converted into blue oxyhmmocyanin, and by the escape of tfie oxygen becomes colorless again. A coloring matter called chlorocruorin by Lankestbr' is found in certain chaetopodse. Hmmeryth'Hn,* so called by Kictr- KBNBBEG but first observed by Schwalbb, is a red coloring matter from certain gephyrea. Besides haemocyanin we find in the blood of certain Crustacea the red coloring matter tetronerythrin (Hallibtjhton*), which is also widely spread in the animal kingdom. Eehinochrom, so named by Mac Munn,* is a brown coloring matter occurring in the perivisceral fluid of a variety of echino- derms. The quantitative constitution of the red blood-corpuscles is diffi- cult to determine, and we have hardly any suificiently trustworthy analyses of them. The amount of water varies in different varieties of blood between 570-630 p. m., with a corresponding amount, 430-370 p. m., of solids. The chief mass, about ■^, of the dried substance consists of haemoglobin (in human find canine blood). ' See V. Jaksch, Klinische Diagnostik, 4. Aufl., p. 18. « Extrait des Bulletins de I'Acad. Roy. de Belgique (2), Tome 46, 1878. 3 Journ. of Anat. and Physiol., 1868, p. 114, and 1870, p. 119. ■> See Physiol. Studien, Reihe 1, Abth. 3. Heidelberg, 1880. ' Journal of Physiol., Vol. 6. 5 Quart. Journ. Microsc. Science, 1885. HsBraoglobin. Albumin. Lecithin. Cholesterin. 868-943 122-51 7.3-3.5 3.5 . 865 126 5.9 3.6 . 627 364 4.6 4.8 . 467 525 LEUC00TTE8. 149 According to the analyses of Hoppe-Setlee ' and his pupils, the red corpuscles contain in 1000 parts of the dried substance: Human blood . . « . Dog " . . Goose " . . Snake " . . Of special interest is the varying proportion of the hajmoglobin to the proteid in the nucleated and in the non-nucleated blood- corpuscles. These last are much richer in haemoglobin and poorer in proteid than the others. According to M. and L. Blbibtreu and Wendelstadt' the amount of nitrogen in the red corpuscles seems to be constant in certain animals, such as the horse and the pig. The quantity of proteid (inclusive of haemoglobin) in the moist corpuscles of the horse was 468.5 and in the pig 443.6 p. m. as calculated by the above experiments from the quantity of nitrogen. The amount of mineral bodies, as far as they have been deter- mined, in the moist corpuscles is 4.8-8.9 p. m. The chief mass consists of potassium, phosphoric acid, and chlorine. The blood- corpuscles of ox-blood contain, according to Bungb, more sodium and chlorine than phosphoric acid and potassium. The blood- corpuscles of the pig and horse contain no sodium (Bunge '). Human-blood corpuscles contain, according to Wanaoh,' about five times as much potassium as sodium, on an average 3.99 p. m. potassium and 0.75 p. m. sodium. The White Blood-corpuscles and the Blood-plates. The White Blood-corpuscles, also called Leucocytes or Lymphoid Cells, which occur in the blood in varying forms and sizes, form in a state of rest spherical lamps of a sticky, highly refractive power, capable of motion, non-membranous protoplasm, which show 1-4 nuclei on the addition of water or acetic acid. In human and mammalian blood they are larger than the red blood- corpuscles. They have also a lower specific gravity than the red ' Med. chem. Untersuch., S. 390 and 393. « Pflilger's Archlv, Bdd. 51 and 52. » Zeitschr. f. Blologie, Bd. 12, S. 306, 207. * Maly's Jahresber., Bd. 18, S. 88. 150 TEE BLOOD. corpuscles, move in the circulating blood nearer to the walls of the- vessel, and have also a slower motion. The nnmbet of white blood-corpuscles varies not only in the different blood-vessels, but also under different physiological condi- tions. As an average we have only 1 white corpuscle for 350^500 red corpuscles. According to the investigations of Alex. Schmidt ' and his pupils, the leucocytes are destroyed in great part on the discharge of the blood before and during coagulation, so that dis- charged blood is much poorer in leucocytes than the circulating blood. The correctness of this statement has been denied by other investigators. From a histological standpoint we generally discriminate between the different kinds of colorless blood-corpuscles; chemically consid- ered, however, there is no known essential difference between them. With regard to their importance in the coagulation of fibrin Alex. Schmidt and his pupils distinguish between the leucocytes which are destroyed by the coagulation and those which are not. The^ last mentioned give with alkalies or common-salt solutions a slimy mass; the first do not show such behavior. The protoplasm of the leucocytes has during life amoeboid movements which partly make possible the wandering of the cells and partly the taking up of smaller grains or foreign bodies within the same. On these grounds the occurrence of myosin in them has been admitted even without any special proof thereof. Alex. Schmidt ' claims to have found serglobulin in equine-blood leuco- cytes which had been washed with ice-cold water. There are also certain leucocytes as above stated which yield a slimy mass when treated with alkalies or NaCl solutions, which seem to be identical with the so-called hyaline substance of Eovida found in the pus- cells. On digesting the leucocytes with water a solution of a proteid body is obtained which can be precipitated by acetic acid and which is not soluble in an excess of the acid and forms the chief mass of the leucocytes. This substance, which is undoubt- edly related to coagulation, has been described under different names (see Chapter V), and consists, chiefly at least, of nucleo- histon. Glycogen has been found in the leucocytes by Hoppe-Setlbb,' ' Pflilger's Archiv, Bd. 11. » L.C. ' Physiol. Chem. Berlin, 1878-1881. S. 82. BLOOD-PLATES. 151 Salomon,' Gabeitschewsky," and other investigators. The glycogen found, by Huppert/ Czernt,' and others in the blood probably originated from the leucocytes. The constituents of the leucocytes are the same as the constituents of the cell as described in Chapter V. The blood-plates (Bizzozbeo's), haematoblasts (Hatem), whose nature and physiological importance have been much questioned, are pale, colorless, gummy disks, round or more oval in shape and generally with a diameter two or three times smaller than the red blood-corpuscles. Certain investigators claim that the blood-plates occur preformed in the circulating blood, while others on the con- trary deny this. According to Lowit ' the blood-plates are formed from the leucocytes with the elimination of globulin substance, hence they are also called globulin-plates. According to MosB2f these globulin-plates are not identical with the true blood-plates, and these first are derived very likely from the latter. The blood- plates separate into two substances by the action of difEerent reagents, namely, one which is homogeneous and non-refractive, while the other is highly refractive and granular. Blood-plates readily stick together and attach themselves to foreign bodies. According to the important researches of Kossel and Lilien- FELD ' the blood-plates consist of a chemical combination between proteid and nuclein, and hence they are called nuclein-plates by LiLiENFBLD. According to this investigator they are derivatives of the cell nucleus, a view which is in accord with Hlata's state- ments. It seems certain that the blood- plates stand in a certain, relationship to the coagulation of blood, and according to Lilien- FELD the fibrin coagulation is indeed a function of the cell nucleus. The importance of these formations to blood coagulation will be referred to later. ' Deutsch. med. Wochenschr., 1877, Nos. 8 and 35. ' Arch. f. exp. Path, und Pharm.. Bd. 28. » Centralbl. f. Physiol., 1893, Part 14. • Arch. f. exp. Path, uad Pharm., Bd. 31. ' In regard to the literature nf the blood-plates, see Lilienfeld, Du Bois- Reymond's Archiv, 1893, and R Mosen, ibid., 1893. 'L.c; also Lilienfeld, " Leukocyten und Blutgerinnuug," V^rtandJ. d. physipl. 3esellsch. zu Berlin, 1893. 152 THE BLOOD. III. The Blood as a Mixture of Plasma and Blood- corpuscles. The blood in itself is a thick, sticky, lighter or darker red opaque liquid having a salty taste and a faint odor difEering in different kinds of animals. On the addition of sulphuric acid to the blood the odor is more pronounced. In adult human beings the specific gravity ranges between 1.045 and 1.075. It has an average of 1.058 for grown men and a little less for women. According to Scheekenziss ' the foetal blood has a lower specific gravity than the blood of grown persons. Lloyd Jones ' found that the specific gravity is highest at birth and lowest in children when about two years old and in pregnant women. The determi- nations of Llotd Jones, Hammbrschlag,' and others show that the variation of the specific gravity, dependent upon age and sex, corresponds to the variation in the quantity of haemoglobin. The determination of the specific gravity is most accurately done by means of the pyknometer. For clinical purposes where only small amounts are available it is best to proceed with the method as suggested by Hammbeschlag. Prepare a mixture of chloroform and benzol of about 1.050 sp. gr. and add a drop of the blood to this mixture. If the drop rises to the surface then add benzol, and if it sinks add chloroform. Continue this until the drop of blood suspends itself midway and then determine the specific gravity of the mixture by means of an areometer. The reaction of the blood is alkaline. The amount of alkali, calculated as Na^CO,, is in the dog about 2 (Zuntz*), in rabbits about 2.5 (Lassae'), and in man 3.38-3.90 p. m. (v. Jaksoh'). The alkaline reaction diminishes outside of the body, and indeed the more quickly the greater the original alkalinity of the blood. This depends on the formation of acid in the blood, in which the red blood-corpuscles seem to take part in some way or another. After excessive muscular activity the alkalinity is diminished on • Cit. from Maly's Jahresber., Bd. 18, S. 85. ' Journ. of Physiol., Vol. 8. ' Wien. klin. WoclienscliT. , 1890, and Zeitschr. f. klin. Med., Bd. 30. • CentralW. f. d. med. Wissensch.,Bd. 5, S. 531 and 801. ' Pfliiger's Archiv, Bd. 9. • Zeitschr. f. klin. Med., Bd. 13, S. 350. COAGULATION OF THE BLOOD. 153 account of the formation of acid in the muscles (Peipee,' Cohn- stein'), and it is also decreased after the continuous use of acids (Lassak, Frbudberg"). The color of the blood is red — light scarlet-red in the arteries and dark bluish-red in the reins. Blood free from oxygen is dichroitic, dark red by reflected light, and green by transmitted light. The blood-coloring matters occur in the blood-corpuscles. For this reason blood is opaque in thin layers and acts as a " deck- farbe." If the haemoglobin is removed from the stroma and dissolved by the blood-liquid, by any of the above-mentioned means the blood becomes transparent and acts then like a " lake color." Less light is now reflected from its interior, and this laky blood is therefore darker in thicker layers. On the addi- tion of salt solutions to the blood-corpuscles they shrink and more light is reflected and the color appears lighter. A great abundance of red corpuscles makes the blood darker, while by diluting with serum or by a greater abundance of white corpuscles the blood becomes lighter in appearance. The diilerent colors of arterial and of venous blood depend on the varying quantity of gas contained in these two varieties of blood or, better, on the difEerent amounts of oxyhaemoglobin and haemoglobin they contain. The most striking property of blood consists in its coagulating within a shorter or longer time, but as a rule very shortly after leaving the vein. DifEerent kinds of blood coagulate with varying rapidity; in human blood the first marked sign of coagulation is seen in 2-3 minutes, and within 7-8 minutes the blood is thoroughly converted into a gelatinous mass. If the blood is allowed to coagulate slowly, the red corpuscles have time to settle more or less before the coagulation, and the blood-clot then shows an upper, yellowish-gray or reddish-gray layer consisting of fibrin enclosing chiefly colorless corpuscles. This layer has been called crusta inflammatoria or phlogistica, because it has been especially observed in inflammatory processes, and is considered one of the characteristics of them. This crusta or " huffy coat " is not char- acteristic of any special disease, and it occurs chiefly when the blood coagulates slowly or when the blood-corpuscles settle more quickly ' Virchow's Arch., Bd. 116. ' Ibid., Bd. 130, which has also references to the works of Minkowski, Zuntz, and Geppert. ' Virchow's Arch., Bd. 125. 154 THE BLOOD. than usual. A buffy coat is of tea observed in the slow-coagulating equine blood. The blood from the capillaries is not supposed to have the power of coagulating. Coagulation is retarded by cooling, by diminishing the oxygen and increasing the amount of carbon dioxide, which is the reason that venous blood and to a much higher degree blood after asphyxia- tion coagulates more slowly than arterial blood. The coagulation may be retarded or prevented by the addition of acids, alkalies, or ammonia, even in small quantities; by concentrated solutions of neutral alkali salts and alkaline earths, alkali oxalates and fluorides ; also by egg-albumin, solutions of sugar or gum, glycerin, or much water; also by receiving the blood in oil. Coagulation may be prevented by the injection of an albumose solution or by an infusion of the leech into the circulating blood, but this in- fusion of the leech acts in the same way on blood just expelled. According to Dastee ' the coagulation of the blood of a dog may be gradually prevented by a series of bleedings and re-injection of the defibrinated blood. The reason for this non-coagulation lies in the lack of fibrinogen. The coagulation may be facilitated by rais- ing the temperature ; by contact with foreign bodies, to which the blood adheres; by stirring or beating it; by admission of air; by diluting with very small amounts of water; by the addition of platinum-black or finely powdered carbon ; by the addition of laky blood, which does not act by the presence of dissolved blood-coloring matters, but by the stromata of the blood-corpuscles (WooL- dkidge"), and also by the addition of the leucocytes from the lymphatic glands, or a watery saline extract of the lymphatic glands, testicles, or thymus. The active constituent of such a watery extract is the nucleoproteid called tissue fibrinogen or nucleo- histon. An important question to answer is why the blood remains fluid in the circulation while it quickly coagulates when it leaves the cir- culation. "When the blood leaves the vein it comes under new, abnormal conditions. It cools off, comes in contact with the air, its motion stops, and it is deprived of the influence of the living walls of the vessels. That the cooling is not the reason of the coagulation is proved by the fact that cooling is a good means of retarding ' Compt. rend d. soc. biol.. Tome 45, and Arch, de physiol., Ser. 5, Tome 5. « Die Qerinnung des Blutes (published by M. V. Frey, Leipzig, 1891). COAGULATION OF THE BLOOD. 155 coagulation. That the contact with air is not essential is shown bj the fact that when blood is collected over mercury, so that it cannot absorb or expel any gas, it likewise coagulates. That the cessation of the motion does not cause the coagulation follows, since blood collected over mercury coagulates whether it is shaken or not, and further from the fact that motion, such as beating the blood, facilitates the coagulation. The reason why blood coagulates on leaving the body is therefore to be sought for in the influence which the walls of the living and entire blood-vessels exert upon it. These views are derived from the observations of many investigators. From the observations of Hewson,' Lister," and Phedericq' it is known that when a vein full of blood is ligatured at the two ends and removed from the body, the blood may remain fluid for a long time. Bruckb* allowed the heart removed from a tortoise to beat at 0° C, and found that the blood remained nncoagulated for some days. The blood from another heart qnickly coagulated when collected over mercury. In a dead heart, as also in a dead blood-vessel, the blood soon coagulates, and also when the walls of the vessel are changed by pathological processes. What then is the influence which the walls of the vessels exert on the liquidity of the circulating blood ? Freund " has found that the blood remains fluid when collected by means of a greased canula under oil or in a vessel smeared with vaseline. If the blood collected in a greased vessel be beaten with a glass rod previously oiled, it does not coagulate, bat it quickly coagulates on beatingit with an unoiled glass rod or when it is poured into a vessel not greased. The non-coagulability of blood collected under oil has been confirmed later by Hatcraft and Carlier." Freund found on further investigating that the evaporation of the upper layers of blood or their contamination with small quantities of dust causes a coagulation even in a vessel treated with vaseline. Accord- ing to Freund, it is this adhesion between the blood or between its form-elements and a foreign substance — and the diseased walls of ' Hewson's works, ed. by Gulliver, London, 1876. » Proc. Roy. Soc, Vol. 12. » Becherches sur la constitution du plasma sangnin, Gand, 1878. * Vircliow's Archiv, Bd. 13. 'Wlen. med. Jahrb., 1886. • Journal of Anat. and Physiol., Vol. 32. 156 THE BLOOB. the vessel also act as such — that gives the impulse towards coagula- tion, while the lack of adhesion prevents the blood from coagulat- ing. This adhesion of the form-elements of the blood to certain foreign substances seems to induce changes which apparently stand in a certain relatioQsbip to the coagulation of the blood. The views in regard to these changes are very contradictory. According to Alex. Schmidt ' and the Doepat school, an abun- dant destruction of" the leucocytes takes place in coagulation, and important constituents for the coagulation of the fibrin pass into the plasma. According to Lowit' and other experimenters the essential is not a destruction of the leucocytes, but an elimination of coastituents from the cells into the plasma. This process is called plasmoschisis by Lowit. According to BizzozOKO ' and others, the leucocytes are not the startiag-point in the fibrin formation, but rather the blood-plates. This view is in good accord with the recent investigations of LiLiENFELD and MosEN." ■ According to Liliekeeld the blood- plates are considered as derived from the cell nucleus and according to this author the fibrin coagulation is a function of the cell nucleus. This view is contradicted by Geiesbach" because, as he claims, the nucleus cannot take part in the coagulation, but that in the first place a part of the cell body is destroyed by plasmoschises, and this even while the nucleus remains still intact. Wooldeidgb' takes a very peculiar position in regard to this question, namely, he considers the form-elements as only of second- ary importance in coagulation. As found by him, a peptone- plasma, which has been freed from all form-constituents by means of centrifugal force, yields abundant fibrin when it is not separated ' Pfliiger's Archiv, Bd, 11. The works of Alex. Schmidt are found in Arch, f. Anat. und Physiol., 1861, 1863; Pfluger's Arch., Bdd. 6, 9, 11, 13. See especially Alex. Schmidt, Zur Blutlehre (Leipzig, 1893), which also gives the work of his pupils. ' Wien. Sitzungsber., Bdd. 89 and 90, and Prager med. Wochenschr., 1889. Referred to in Centralhl. f. d. med. Wissensch., Bd. 28, S.265. = Virchow's Arch., Bd. 90; Centralhl. f. d. med. Wissensch., 1882, S. 17, 161, 353, .563; ibid., 1883; Virchow's Festschrift, 1891. "L-c. ^ Pfluger's Archiv, Bd. 50, and Centralhl. f. d. med. Wissensch., 1892, S. 497. «L.c. SCHMIDTS THEORY OF COAGULATION. 157 from a substance which precipitates on cooling. This sabstance, which WooLDRiDGE has called A-fibrinogen, seems to be identical with Lowit's globulin-plates, and it consists in all probability of a nucleoproteid, which is perhaps identical with prothrombin as isolated by Pekelharing.' As this nucleoproteid originates, acording to the unanimous view of several investigators, from the form-elements of the blood, either the blood-plates or leucocytes, Wooldkidge's experiments do not seem to contradict the generally accepted view that the form-elements of the blood are of the greatest importance in the coagulation of the same. The views are greatly divided in regard to those bodies which are eliminated from the form-elements of the blood before and during coagulation. According to Alex. Schmidt' the leucocytes, like all cells, contain two chief groups of constituents, one of which accelerates coagulation, while the other retards or hinders it. The first may be extracted from the cells by alcohol, while the other cannot be extracted. Blood-plasma contains only traces of thrombin, accord- ing to Schmidt, bat does contain its antecedent, prothrombin. The bodies which accelerate coagulation are neither thrombin nor pro- thrombin, but they act in this wise in that they split off thrombin from the prothrombin. On this account they are called zymoplastic substances by Alex. Schmidt. The nature of these bodies is unknown, and according to Lilienfbld^ KH^PO, is found amongst them, and Schmidt has given no notice of their behavior to the lime-salts, which have been found to have zymoplastic activity by other investigators. The constituents of the cells which hinder coagulation and which are insoluble in alcohol-ether are compound proteids and have been called cytogloMn and preglolulin by Schmidt. The retarding action of these bodies may be suppressed by l!he addition of zymoplastic substances, and the yield of fibrin on coagulation in this case is much greater than in the absence of the compound proteid-retarding coagulation. Tin's last supplies the material from which the fibrin is produced. The process is, accord- ing to Schmidt, as follows: The preglobnlin first splits, yielding serglobulin, then from this the fibrinogen is derived, and from this ' Ueber das Fibrinferment. Verhandl. d. kon. Akad. van. Wetensch. te Amsterdam, Deel 1, No. 3, 1892. ' Zur Blutlebre. » Weitere BeitrSge zur Kenntniss der Blutgerinnung. Berlin, 1893. 158 THE BLOOD. latter the fibrin is produced. The object of the thrombin is two- fold. The thrombin first splits the fibrinogen from the paraglobulin and then converts the fibrinogen into fibrin. Alex. Schmidt is now agreed with most investigators that fibrin is produced by an enzymotic transformation of the fibrinogen, and the influence of the serglobulin, as observed by him on the quantity of fibrin formed, he explains now by the assumption that the fibrinogen is produced by the splitting of the serglobulin. According to Schmidt the retarding action of the cells is prominent during life, while the accelerating action is especially pronounced outside of the body or by coming in contact with foreign bodies. The parenchymous masses of the organs and tissues, through which the blood flows in the capillaries, are those cell masses which serve to keep the blood fluid during life (Alex. Schmidt). LiLiENFELD " has given further proofs as to the occurrence in the form-elements of the blood of bodies which accelerate or retard the coagulation. According to this author the nature of these bodies is very markedly different from Schmidt's idea. While, according to Schmidt, the coagulation accelerators are bodies solu- ble in alcohol, and the proteids exhausted with alcohol only act retardingly on coagulation, Lilibnfeld states that the substance which acts acceleratingly and retardingly on coagulation consists of a nucleoproteid, namely, nucleohiston. Nncleohiston readily splits into leuconuclein and histon, the first of which acts as a coagulation excitant, while the other, introduced into the blood- vascular system, either intravascular or extravascular, robs the blood of its property of coagulating. Introduced into the circulatory system the nucleo- histon splits into its two components. It therefore causes extensive coagulation on one side and makes the remainder of the blood uncoagulable on the other. Liliestfeld'' is of the view that fibrinogen does not exist dis- solved in the plasma of the circulating blood. It passes into the plasma on the disintegration of the leucocytes and originates from the substance of cell nuclei of the leucocytes. Nucleohiston may be directly transformed into fibrin. Lilieneeld's theory at the ' See Lilienfeld : Ueber Leukocyten und Blutgerinnuug. Verhandl. d. physiol. Gesellscli. zu Berlin, No. 11, 1893; Deber den fl.issigen Zustand des Blutes, etc., Und., No, 16, 1893; and Weitere Beitea^e zur Kentnisse der Blut- gerinnung, ibid., July, 1893. » Zeitschr. f. physlol. Gbem., Bd. 20. LILmNFELli'S THEORY OF COAGULATION. 159 present time is that on leaving the veins the leucocytes of the blood are destroyed or the nuclein substance passes into the plasma. This naclein substance splits the fibrinogen into thrombosin and a sub- stance which gives the biuret reaction. The thrombosin combines with the soluble calcium salts, forming fibrin. The leuconuclein is therefore the real coagulation exciter (not the fibrin ferment); the histon split from the nucleohiston has, on the contrary, a retarding action on coagulation. As the blood-plates contain nuclein, they as well as the leucocytes take an active part in the fibrin coagula- tion. Brucke showed long ago that fibrin left an ash containing cal- cium phosphate. The fact that calcium salts may facilitate or even cause a coagulation in liquids poor in ferment has been known for several years through the researches of the author,' Green,' KisGER, and Sainsbury." The necessity of the lime-salts for coagulation was first shown positively by the important investiga- tions of Arthus and Pages.' In regard to the manner in which the lime-salts act we have only lately been able to come to a result. Freund ' has given the following explanation for the action of lime-salts: The alkali phosphates pass from the form-elements into the plasma, which is richer in lime-salts and forms calcium phos- phate. If the quantity of calcium phosphate in the plasma or other coagulable liquid is so great that it cannot be kept completely in solution, then, according to Freund, the separation of the excess is the cause of a part of the proteids becoming insoluble, that is, a cause for coagulation. Weighty objections can be made against this view, and it is also confuted by Latschenberger ' and Strauch.' According to Pekelharing" the process is as follows: The prothrombin is converted into thrombin by the action of the soluble lime-salts and fluids otherwise capable of coagulation, which contains only prothrombin, but no thrombin can therefore be brought to 1 Nova Acta reg. Soc. Sclent, Upsala, Ser. Ill, Vol. 10, 1879. ' Journ. of Physiol., Vol. 8. * Ibid., Vols. 11 and 2. * M. Arthus, Eecherches sur la Coagulation du sang., Paris, 1890; Arthus €t Pag6s; Nouvelle Theorie, etc., Arch, de Physiol. (5), Tome 2, 1890. * Wien. med. Jahrb., 1888, S. 359. « Ibid., 1888, 8. 479, and Wien. med. Wochenschr., 1889. ' Dissertation. Dorpat, 1889. Ref. Maly's Jahresber., Bd. 19. » Virchow's Festschrift, Bd. 1, 1891. 160 TEE BLOOD. coagalation by the addition of soluble liine-salts. Thrombin, according to Pekblhaeing, is a lime combination of prothrombin, and the process of coagalation consists in that the thrombin carries the lime to the fibrinogen, which is converted into the insoluble combination of fibrin and lime. The thrombin is hereby recon- verted into prothrombin, which again takes up lime to be trans- formed into thrombin, which gives up its lime to a new portion of fibrinogen, converting it into fibrin ; and so on. This explanation of the process is only a hypothesis, but the formation of thrombin from a mother-substance by the action of soluble lime-salts is, on the contrary, a positively proven fact. It is a question whether the prothrombin exists in the plasma of the circulating blood or whether it is a body eliminated from the form-elements before coagulation. Alex. Schmidt claims that the circnlatiug plasma contains prothrombin, but Pekelhaeing dis- claims this. Blood-plasma obtained by means of leech infusion does not coagulate on the addition of lime-salts, but does on the addition of a prothrombin solution. The form-elements, especially the blood-plates, are particularly well preserved by such plasma; and according to Pekelhaeing it is probable that the circulating plasma does not contain any mentionable amounts of prothrombin, and that this body emerges from the form-elements, perhaps the blood-plates, before coagulation. . In opposition to the view of Alex. Schmidt, who considers the fibrin coagulation as an enzymotic process, Wooldridge ' is of the opinion that the fibrin ferment is not the cause of the coagulation, but is a product of the chemical processes taking place during coagulation. Wooldeidge claims, on the contrary, that lecithin and protein substances containing lecithin are of the greatest im- portance in the coagulation. This product is obtained by cooling the peptone-plasma which has been centrifugated, and the substance which separates has been called by Wooldridge ^-fibrinogen. The plasma, according to Wooldeidge, contains in itself all quali- ties necessary to produce a coagulation, and the form-elements are only of a secondary importance. Peptone-plasma which has been centrifugated and which is entirely free from form-elements, but contains the ^-fibrinogen, coagulates on diluting with water, by the passage of carbon dioxide through the liquid, or after the addi- 1 The summary of the observations of Wooldridge are found in the pre- viously cited publication, "Die Qerinnung des Blutes " (M. v, Frey, 1891). INTRAVASCULAB COAGULATION. 161 tion of a little acetic acid, and the fibrin ferment is thereby formed. WOOLDRIDGE designates as C-flbrinogen the ordinary fibrinogen isolated by the method suggested on page 113. This fibrinogen occurs indeed in transudations, but it only occurs in the peptone- plasma in very small quantities. A third fibrinogen occurs in the greatest amounts in the peptone-plasma, and this is the mother- substance of the C-fibrinogen, and called 5-fibrinogen by Wool- DRiDGE. The .B-flbrinogen is converted into fibrin by lecithin and leucocytes from the lymphatic glands, but not by fibrin ferment or blood-serum. After the previous action of serum or fibrin ferment the 5-fibrinogen yields fibrin on diluting with water. The one most essential for the fibrin coagulation is, according to Wool- DRiDGE, a reciprocal action between A- and 5-fibrinogen. An exchange of lecithin from the ^.-fibrinogen to the .6-fibrinogen takes place. Halliburton ' has opposed weighty arguments to this theory, It is also difiicult to find in Wooldeidge's discussion conclusive proofs for the above views, and the experiments by which they are supported are interpreted with difiiculty. On account of the very complicated condition of the question of coagulation at the present time, it is impossible to draw any definite conclusions from the observations of Wooldridge. Intravascular coagulation. It has been shown by Alex. Schmidt and his students, as also by Wooldridge, Wright,' and others, that an intravascular coagulation may be brought about by the intravenous injection into the circulating blood of a large quantity of a thrombin solution, as also by the injection of leuco- cytes or tissue fibrinogen (impure nucleohiston). In rabbits this coagulation may extend through the entire vascular system, while in dogs it is ordinarily confined to the portal system. The blood in the other parts of the vascular system has generally a decreased coagulat ability. If too little of the above-mentioned bodies be injected, then we only observe a marked retarding tendency in the coagulation of the blood. According to Wooldridge we can generally maintain that after a short stage of accelerated coagula- > Journal of Physiol.. Vol. 9. 2 A study of the intravascular coagulation, etc.. Proceed, of the Roy. Irish Acad, (3), Vol. 2 ; see also Wright : Lecture on tissue or cellfibrinogen , The Lancet ; 1892 ; and Wooldridge's Method of producing immunity, etc. , Brit. Med. Journal, Sept. 1891. 162 THE BLOOD. bility, which may lead to a total or partial intravascular coagalation, a second stage of a diminished or even arrested coagulability of the blood follows. The first stage is designated as the positive and the other the negative phase of coagulation. These statements have been confirmed by several investigators. There is no doubt that the positive phase is brought about by an abundant introduction of thrombin, or by a rapid and abundant formation of the same. According to Alex. Schmidt, the zymo- plastic substances soluble in alcohol are active in these processes, while according to the investigations of Pekelhabin^G this action is caused more likely by the leuconucleins, split off from the nucleohiston. According to Wooldkidgb, his tissue fibrinogen does not produce any intravascular coagulation if it is freed from contaminating bodies by means of alcohol. This corresponds with the statements of Alex. Schmidt, but still further investigations are necessary. In regard to the origin of the negative phase, attention has been called to histon, which has a retarding action on coagulation, and which is split off from the nucleohiston. According to Wright and Pekelharing, the retarding substances are albumoses, which are formed in the decomposition of the nucleoproteids. Albumoses have been detected by these investigators in the blood of animals during this phase, and also in the urine after intravenous injection of tissue fibrinogen. According to Pekelhaeing, the albumoses act by combining with the calcium of the blood, and in this wise preventing coagulation. Grosjeazs" ' has found that blood which has regained its property of coagulation 34 hours after an albumose injection will not have its coagulation prevented by a fresh injec- tion of albumose, hence it is immune against albumose injection. He also infers from these experiments that the albumose, to have a preventing action at all, must first undergo a change in the organism. This has been further studied by Contejean," who finds that under the influence of injected albumose a special sub- stance is secreted in the animal body which prevents coagulation. This seems to be brought about by means of the liver and intestine. A dog may be made immune against the preventive action of albumose by previously injecting a small quantity of " peptonized blood" into the vessels. The body hereby loses its property of ' Travaux du laboratoire de L. Frederiq. Tome 4. Ligge, 1893. ' Arch, de Physiol., Ser. 5, Tome 7. QUANTITATIVE COMPOSITION OF THE BLOOD. 163 3)roducing substances which prevent coagulation under the influence of injected albumoses. Weight gives as reason why the intravascular coagulation of the blood of a dog is ordinarily confined to the portal system, in the fact that it contains larger quantities of carbon dioxide. An increased quantity of carbon dioxide in the blood favors the appear- ance of the positive phase, and an intravascular coagulation may be produced in dogs, who are asphyxiated by clamping the trachea, by injecting tissue fibrinogen (impure nucleohiston). The gases of the blood will be treated of in Chapter XVII (on respiration). IV. The Quantitative Composition of the Blood. The quantitative analysis of blood cannot be of value for the blood as an entirety. We must ascertain on one side the relation- ship of the plasma and blood-corpuscles to each other, and on the other side the constitution of each of these two chief constituents. The difficulties which stand in the way of such a task, especially in regard to the living, nou-coagulated blood, have not been removed. Since the constitution of the blood may difEer not only in different vascular regions, but also in the same region under different cir- oumstances, which renders also a number of blood analyses neces- sary, it can hardly appear remarkable that our knowledge of the constitution of the blood is still relatively limited. The relative volume of blood-corpuscles and serum in defibri- nated blood may be determined, according to L. and M. Blbib- TEEU,' by various methods if the defibrinated blood is mixed with different proportions of NaCl solutions of 6 p. m. (1 vol. salt solution to 1 vol. blood), the blood-corpuscles allowed to settle to the bottom or facilitated by centrifugal force, and the clear super- natant mixture of serum and common-salt solution siphoned off. The methods are as follows : 1. Determine the quantity of nitrogen in at least two different portions of the mixture of serum and salt solution by means of Kjeldahl's method and calculate the quantity of proteid corre- sponding thereto by multiplying with 6.25, and the relative volume of blood X and also the volume of the structural elements {I — x) is found by the following equation : («■ - «Ja; = p, - p,. ' Pflllger's Arcliiv, Bd. 51. 164 THE BLOOD. In this equation (for mixtures 1 and 2), J, or 5, represents the volume of blood in the^ mixture, s, or s, the volume of salt solution, and fij or e, the quantity of proteid in a certain volume of each mixture. 2. Determine the specific gravity of the blood-serum, the salt solution and at least one of the mixtures of serum and salt solution by means of a pyknometer. The relative volume of serum x is, found in this by the following equations: s S-E X = 'in this equation s and h represent the volumes of salt solution and blood mixed. S represents the specific gravity of the obtained serum and salt solution obtained on allowing the blood-corpuscles to settle, S„ the sp. gr. of the serum, and K that of the salt solu- tion. For horses' blood, two other, shorter methods may be made use of (see the original article). Hambubger ' raises special objections to the above methods, but according to Bleibtebu they are of no practical importance aa long as the blood is not diluted with more than an equal volume of the salt solution." ETKMAiir" and Hbdin"* have raised important objections to Bleibtreu's method. They have shown by different methods that the red corpuscles are not changed in volume only in such salt solutions which are isotonic with the plasma or serum. (In regard to the osmotic pressure of the blood-corpuscles and the isotonic relationship of salt solutions and serum, see Hambttegee '.) Such a solution is not one containing 6 p. m. NaCl, for human, ox, or horse's blood, but rather one containing about 9 p. m. KaCl (Lackschewitz '). The blood-corpuscles swell up in a solution of 6 p. m. NaOl, and therefore an abundant exchange of constituents takes place between them and the salt solution: hence Bleibtebu's method is incorrect. Hedin, as before him Bieenacki,'' could not obtain corresponding results of the volume of corpuscles calculated from the nitrogen determined. The question arises whether this method is available if we use an isotonic salt solution. According to Hedin this is not true, as he has found that the red blood- corpuscles take up considerable quantities of plasma proteid, even ' Centralbi; f. Physiol., Bd. 7, S. 161. ' See M. Bleibtreu, Pflilger's Archiv, Bd. 55. 5 Pfluger's Arch., Bd. 60. * Ibid., and Skand. Arch. f. Physiol., Bd. 5. 5 Virohow's Arch. , Bd. 140, S. 505. • Pfluger's Arch., Bd. 59. ' Zeitschr. f. physiol. Chem., Bd. 19. QUANTITATIVE BLOOD ANALYSIS. 165 in isotonic common-salt solution, without changing their volume. This statement is disputed by M. Bleibteeu," and the analyses made by using an isotonic salt solution, although not numerous, have led to very good results. For clinical purposes the relative volume of corpuscles in the blood may be determined by the use of a small centrifuge called hmmatocrit, constructed by Blix and described and tested by Hedix.' a measured quantity of blood is mixed with a known volume (best an equal volume) of a fluid which prevents coagala- tion. This mixture is iutroduced into a tube and then centrif iiged. Hedin uses Mullek's solution as a diluting fluid and Daland ' a 3.5^ solution of potassium bichromate. After complete centrifu- gation the layer of blood-corpuscles is read ofE on the graduated tube, and the volume of blood-corpuscles calculated in 100 vols, of the blood therefrom. By means of comparative counts Hedin and Daland have found that an approximately constant relation exists between the volume of the layer of blood-corpuscles and the number ■of red corpuscles under physiological conditions, so that the number of corpuscles may be calculated from the volume. Daland has shown that such a calculation gives approximate results also in ■disease, when the size of the blood-corpuscles does not essentially deviate from the normal. In certain diseases, such as pernicious anaemia, this method gives such inaccurate results that it cannot be used. The uselessness of this method for the exact estimation of the volume of blood-corpuscles has been demonstrated* by L. Bleib- TRBU. ' Eykmak as well as Hediit repudiate the objections made by Bleibtbeu against the haematocrit method; but they also show that Muller's solution as well as the 2.5^ potassium bichromate solution causes the blood-corpuscles to swell up, and hence lead to incorrect results. According to Hedin, in working with the haematocrit dilute the blood, which is kept fluid by a 1 p. m. oxalate solution, with an equal volume of a solution containing 9 p. m. NaCl. Under these conditions the determination of the volume of blood-corpuscles by the haematocrit method is very serviceable. This method is not available for the exact determina- tion of the volume of corpuscles, because the sediment of blood- corpuscles to all appearances does not consist only of blood-cor- puscles, but also some plasma. If we know the relationship between the volume of corpuscles and blood liquid we can also estimate the relative weights by determining the specific gravity of the blood and serum. In direct determinations of the proportion by weight we proceed in the fol- lowing way : ' Pflager's Arch., Bd. 60. » Skandinav. Arch. f. Physiol., Bd. 2, S. 134 and 361. » Fortschritte d. Med., Bd. 9, 1891. < Biernacki, Zeitscbr. f. physiol. Chem., Bd. 19. ' Berl. klin. Wochenschr. , 1893, No. 30. 166 THE BLOOD. If any substance is found in the blood which belongs exclusively to the plasma and does not occar in the blood-corpuscles, then the amount of plasma containfed in the blood may be calculated if we determine the amount of this substance in 100 parts of the plasma, or serum, respectiyely, on one side and in 100 parts of the blood on the other. If we represent the amount of this substance in the plasma by p and in the blood by h, then the amount of x in the . , , . • 100 ■ * plasma from 100 parts of blood is a; = . Such a substance, which occurs only in the plasma, is fibrin according to Hoppe-Setlee,' sodium according to Bunge" (in certain kinds of blood), and sugar according to Otto.' The experimenters just named have tried to determine the amount of the plasma and blood-corpuscles, respectively, in different kinds of blood, starting from the above-mentioned substances. Another method, suggested by Hoppe-Setlek,' is to determine the total amount of hffimoglobin and proteids in a portion of blood, and on the other hand the amount of hemoglobin and proteids in the blood-corpuscles (from an equal portion of the same blood), which have been sufficiently washed with common-salt solution by centrifugal force. The figures obtained as a difference between these two determinations correspond to the amount of proteids- which was contained' in the serum of the first portion of blood. If we now determine the proteids in a special portion of serum of the same blood, then the amount of serum in the blood is easily deter- mined. The usefulness of this method has been confirmed by BuifGE by the control experiments with the sodium determinations. If the amount of serum and blood-corpuscles in the blood is known,, and we then determine the amount of the different blood-constit- uents in the blood-serum on one side and of the total blood on the other, the distribution of these different blood-constituents in the two chief components of the blood, plasma, and blood-corpuscles may be ascertained. According to the just-mentioned procedure,, the following analyses of pig's blood and ox's blood have been made' by BuNGE. The analyses of human blood have been made by C. Schmidt' according to another method, which perhaps have given rather too high results for the weight of the blood-corpuscles.. AH figures represent parts in 1000 parts of blood. ' Handb. d. pliysiol. und pathol. chem. Analyse, 6. Aufl., S. 417. 2 Zeitschr. f. Biologie, Bd. 12. 3 Pfluger's Arcliiv, Bd. 35, S. 480-482. * See Handb. d. physiol. und pathol. chem. Analyse, 6. Aufl. ' Cited and partly recalculated from v. Gorup-Besanez, Lehrb. der physiol.- Chem., 4. Aufl., S. 345. QUANTITATIVE COMPOSITION DIP THE BLOOD. 167 Water Solids Hffimo^lobiu and } Proteid f • • • Remaining or^. bodies, Inorganic bodies K,0 Ha,0 CaO MgO Fe,0, CI P.O. Pig's Blood. Blood- cor- puscles 276.100 160.700 6.800 3.900 2.421 0.069 0.657 0.903 Serum 663.2 617.900 45.300 38.100 2.800 4.300 0.164 2.406 •0.072 0.021 0.006 2.034 0.106 Ox's Blood. Blood- cor- puscles 318.7 191.200 127.500 2.400 1.500 0.838 0.667 0.006 0.521 0.224 Serum 681.3 622.200 59.100 49.900 3.800 6.400 0.173 2.964 0.070 0.031 0.007 2.633 0.181 Human Blood. Man's Blood- cor- puscles 613.02 349.690 163.380 3.740 1.586 0.241 0.898 0.61i5 Serum 486.96 439.020 47.960 43.820 4.140 0.163 1.661 1.722 O.OTl Woman's. Blood- cor- puscles 396.24 272.660 123.680 130.130 3.560 1.412 0.648 0.363 0.643 Serum 603.78 651.999 51.770 46.700 6.070 0.200 1.916 Hoppb-Seylek, Sachaejin,' and Otto" found 584.9-693.5 p. m. plasma and 415.1-306.6 p. m. blood-corpuscles in horse's blood. BuNGE " found, on the contrary, in an analysis 468.5 p. m. serum and 531.5 p. m. blood-corpuscles— more blood-corpuscles, therefore, than serum. For human blood Arronet' has found 478.8 p. m. blood-corpuscles and 521.3 p. m. serum (in defibri- nated blood) as an average of nine deter minatio us. Schneideb ' found 349.6 and 650.4 p. m. respectively in women. , The relationship between blood-corpuscles and plasma varies; in the blood of men it is aboub 50^ of the weight of the blood, while in women it is somewhat more. The quantity of plasma in animals is often greater, and in certain cases it may indeed be two thirds of the weight of the blood. The relationship between the corpuscular elements and the plasma may undergo marked fluctuation. L. and M. Bleibtreu found in 10 experiments with defibrinated horse- blood that the relative volume of form-elements varied between 261.4 and 409.5 p. m. The relative volume of blood liquid to the corpuscular elements varies according to the manner in which the blood is drawn from the animal. L. and M. Bleibtreu" have found that the blood from a killed animal is regularly richer in ' Hoppe-Seyler's Physiol. Chem., 1877-1881, S. 447. 5 Pflilger's Archiv, Bd. 35. M. 0. * Maly's Jaliresber., Bd. 17, S. 139. > Centralld. f. Physiol., Bd. 5, S. 362. «L. c. 168 THE BLOOD. corpuscles than blood taken from the veins. Water occurs in the greatest amount in the plasma or serum, which latter ordinarily contains at least ^ water, while the blood-corpuscles contains only a little more than ^ or about | water. Iron probably occurs only in the blood-corpuscles. Chlorine and sodium prevail in the plasma, while potassium and phosphoric acid prevail in the blood- corpuscles. In a few varieties of blood (pig's and horse's blood) the sodium is found exclusively in the plasma or serum, the potas- sium prevailing in the blood-corpuscles (Bunge '■). In dog's and ox's blood the blood-corpuscles are, however, richer in sodium than in potassium (Bunge). In man the potassium exists in large quantities in the blood-corpuscles and only in very small quantities in the plasma (C. Schmidt,'-' "Wanach"). The alkaline earths occur chiefly in the plasma. Manganese has also been found in the blood, as well as traces of lithium, copper, lead, and silver. The blood as a whole contains in ordinary cases 770-820 p. m. water, with 180-330 p. m. solids; of these 173-330 p. m. are organic and 6-10 p. m. inorganic. The organic consist, deducting 6-13 p. m. extractive bodies, of proteids and haemoglobin. The amount of this last-mentioned body in human blood is about 130-150 p. m. The quantity of hemoglobin in dog's blood is about the same; and BuNGE found 114 p. m. haemoglobin in pig-blood and 89.4 p. m. in ox-blood. The amount of sugar in the blood is on an average 1-1.5 p. m. The quantity of urea, which varies between 0.3 and 1.5 p. m., is greater after partaking of food than during fasting (Geehan't and QuiNQUAUD,* Schondoefe'). The quantity of uric acid may be 0.1 p. m. in bird's blood (v. Scheoedee'). Lactic acid was first found in human blood by Salomoii and then by Gaglio, Bee- iiiSTEEBLAU and Ieisawa.' The quantity of lactic acid may vary considerably. Beelineeblau found 0.71 p. m. as maximum. ' L. c. "L. c. ^ Maly's Jahresber., Bd. 18, S. 88. * Journal de I'anatomie et de la physiol., Tome 30, and Compt. rend.. Tome 98. ' Pfliiger's Arch., Bd. 54. « Ludwig's Festschrift, 1887, p. 89. ' Zeitschr. f. physiol. Chem., Bd. 17, which also gives the older literature. . BLOOD IN DIFFERENT VASCULAR REGIONS. 169 The Composition of the Blood in Different Vascular Regions and under Different Physiological Conditions. Arterial and Venous Blood. The most striking difierence between these two kinds of blood is the variation in color caused by their containing different amounts of gas and different amounts of oxyhaemoglobin and haemoglobin. The arterial blood is light red; the venous blood is dark red, dichroitic, greenish by transmitted light through thin layers. The arterial coagulates more quickly than the venous blood. The latter, on account of the transudation which takes place in the capillaries, is somewhat poorer in water but richer in blood-corpuscles and heemoglobin than the arterial blood, but this is denied by modern investigators. According to Keugee ' and his pupils the quantity of dry residue and liaBmo- globin in blood from the carotid artery and from the jugular vein (in cats) are the same. Rohmann and Muhsam ' could not detect any diflerence in the quantity of fat in arterial and venous blood. Blood from the Portal Vein and the Hepatic Vein. The blood of the hepatic vein is poorer in ordinary red blood-corpuscles but richer in white and so-called young red blood-corpuscles. A few investigators have concluded from this that a formation of red blood-corpuscles takes place in the liver, while others claim that a destruction takes place. In consequence of the small quantities of bile and lymph found relatively to the large quantity of blood circulating through the liver in a given time, we can hardly expect to detect a positive difEerence in the composition between the blood of the portal and hepatic veins by chemical analysis. The statements in regard to such a diflerence are in fact contradictory. For example, Deos- DOFJ ' has found more haemoglobin in the hepatic than in the portal vein, while Otto* found less. Keugee " finds that the quantity of hsemoglobin, as well as the solids, in the blood from the vessels pass- ing to and from the liver is different, but a constant relationship cannot be determined. The disputed question as to the varying ' Zeitschr. f. Biologie, Bd. 26. , ' PflQger's Archiv, Bd. 46. ' Zeitschr. f. physiol. Cliem., Bd. 1. « Christiania Videnskabs. Selskabs Forhandlinger, 1886, No. 11. See Maly's Jahresber., Bd. 17, S. 134. ' Zeitsclir. f. Biologie, Bd. 36. 170 TBE BLOOD. quantities of sugar in the portal and hepatic veins will be discussed in a following chapter (see Chapter VIII, on the formation of sugar in the liver). After a meal rich in carbohydrates the blood of the portal vein not only becomes richer in dextrose, but may contain also dextrin and other carbohydrates (t. Meeing,' Otto"). The amount of urea in the blood from the hepatic vein is greater than in other blood (Gkehant and Quistquaud ') . Blood of the Splenic Vein is decidedly richer in leucocytes than the blood from the splenic artery. The red blood-corpuscles of the blood from the splenic vein are smaller than the ordinary, less flat- tened, and show a greater resistance to water. The blood from the splenic vein is also claimed to be richer in water, fibrin, and albumin than the ordinary venous blood (Bbclaed''). According to V. MiDDENDOKFF,^ it is richer in hoBmoglobin than arterial blood. Keugbk * and his pupils have found that the blood from the vena lienalis is generally richer in haemoglobin and solids than arterial blood; still the contrary is often found. The blood from the spleni'c vein coagulates slowly. The Blood from the Veins of the Glands. The blood circulates with greater rapidity through a gland during activity (secretion) than when at rest, and the outflowing venous blood has therefore during activity a lighter red color and a greater amount of oxygen. Because of the secretion the venous blood also becomes somewhat poorer in water and richer in solids. The blood from the Muscular Veins Shows an opposite behavior, for during activity it is darker and more venous in its properties because of the increased absorption of oxygen by the muscles and still greater production of carbon dioxide than when at rest. Menstrual Blood has, according to an old statement, not the power of coagulating. This statement is nevertheless false, and the apparent uncoagulability depends in part on the womb and the vagina retaining the blood-clot, so that only fluid cruor is at times eliminated, and in part on a contamination with vaginal mucus w^hich disturbs the coagulation. > Du Bois-Reymond's Archiv, 1887, S. 412 and 431. ^ See note 4, page 169. 2 Journal d. I'anatomie et de la physiol., Tome 20, and Compt. rend., Tome 98. * Arch, generale de medecine. Tome 18. s Cit. from Centralbl. f. Physiol., Bd. 3, S. 753. « Zeitscbr. f. Biologie, Bd. 26. BLOOD AT DIFFERENT PEEI0D8 OF LIFE. iTl Tlie Blood of the Two Sexes. Woman's blood coagulates some- what more quickly, has a lower specific gravity, a greater amount of water, and a smaller quantity of solids than the blood of man. The amount of blood-corpuscles and haemoglobin is somewhat smaller in woman's blood. The amount of haemoglobin is, accord- ing to Otto, 146 p. m. for man's blood and 133 p. m. for woman's. During pregnancy Nasse ' has observed a decrease in the specific gravity, with an increase in the amount of water until the end of the eighth month. Prom then the specific gravity increases, and at delivery it is normal again. The amount of* fibrin is some- what increased (Bbcqueeel and Eodiee,' Nasse). The number of blood-corpuscles seems to decrease. In regard to the amount of hsemoglobin the statements are somewhat contradictory. Cohn- stein ' found the number of red corpuscles diminished in the blood of pregnant sheep as compared to non-pregnant, but the red cor- puscles were larger, and the quantity of hsemoglobin in the blood was greater in the first case. The Blood at Different Periods of Life. Foetal blood is strik- ingly poorer in blood- corpuscles and hsemoglobin than the blood of the adult. The foetal blood at the moment of birth has, according to ScHEREENziss,* a lower specific gravity, a markedly lower amount of hsemoglobin, and a little less fibrin, but a greater amount of mineral bodies, especially proportionally more sodium (but less potassium) than the blood of adults. A few hours after birth the blood of the child has the same or greater quantity of hemoglobin than the blood of the mother (Cohnsteist, Zutttz,' Otto '). The quantity of hsemoglobin and blood-corpuscles quickly increases after birth; still they do not both increase at the same rate, as the amount of haemoglobin increases much faster. Two or three days after birth the hajmoglobin reaches a maximum (20-31^), which is greater than at any other period of life. This is the cause of the great abundance of solids in the blood of new-born infants as observed by several investigators. The quantity of haemoglobin and blood-corpuscles sinks gradually from this first maximum to a > Maly's Jahresber., Bd. 7, S. 129. 2 Traite de cliiiuie patliol. Paris, 1854. P. 59. • Pflilger's Archiv, Bd. 34, S. 333. • Maly's Jahresber., Bd. 18. ' Pflilger's Arch. , Bd. 34, S. 178. • Maly's Jahresber., Bdd. 15 and 17. 172 THE BLOOD. minimum of about 11^ hsemoglobin, which minimum appears in human, beings between the fourth and eighth years. The quantity of hsemoglobin then increases again until about the twentieth year, when a second maximum of 13> 7-15^ is reached. The haemoglobin remains at this point only towards the forty-fifth year, and then gradually and slowly decreases (Leichtenstben,' Otto '). Accord- ing to older statements, the blood at old age is poorer in blood- corpuscles and albuminous bodies but richer in water and salts. The Influence of Food on the Blood. In complete starvation no decrease in the amount of solid blood constituents is found to take place (Panum ' and others). The amount of hasmoglobin is a little increased (Stjbbotin,' Otto), and also the number of red blood- corpuscles increases (Worm Mullbe,' Buntzbis''), which probably depends on the fact that the blood-corpuscles are not so quickly transformed as the serum. As after-effect the inanition causes an anaemic condition. After a rich meal the relative number of blood-corpuscles, especially after secretion of digestive juices or absorption of nutri- tive liquids, may be increased or diminished (Buntzen, Lbiohtbn'- STEEn). The number of colorless blood-corpuscles may be increased to such an extent, after a diet rich in proteids, that a true digestion leucocytosis appears (Hobmbistbe and Pohl'). After a diet rich in fat the plasma becomes, even after a short time, more or less milky-white, like an emulsion. The constitution of the food acts essentially on the amount of heemoglobin in the blood. The blood of herbivora is generally poorer in haemoglobin than that from carnivora, and Subbotin' has observed in dogs after a partial feed- ing with food rich in carbohydrates that the amount of haemoglobin sank from the physiological average of 137.5 p. m. to 103.2-93.7 p. m. According to LEiCHTEirsTEEif a gradual increase in the amount of haemoglobin is found to take place in the blood of human beings on enriching the food, and according to the same investigator • Untersuch. liber den HSmoglobingehalt des Blutes im gesunden und kranken Zustande. Leipzig, 1878. ' Maly's Jahresber., Bd. 17. ' Vircbow's Arcb., Bd. 39. * Zeitschr. f. Biologie, Bd. 7. ' Transfusion und Pletbora. Cbristiania, 1875. 6 Om ErniBringens og Blodtabets Indflydelse pi, Blodet. KjSbenbavu, 1879. See also Maly's Jabresber., Bd. 9. ' Arcb. f . exp. Patb. und Pharm. , Bd. 35. BLOOD UNDER ABNORMAL CONDITIONS. 173 the blood of lean persons is generally somewhat richer la hffimo- globin than blood from fat ones of the same age. The addition of iron salts to the food greatly influences the number of blood- corpuscles and especially the amount of hasmoglobin they contain. The action of the iron salts is obscure. According to Buif ge ' they probably combine with the sulphuretted hydrogen of the intestinal canal and thereby prerent the iron, associated in the food as protein combination, from being eliminated as iron sul- phide. The Composition of the Blood under Abnormal Conditions may be changed either by the appearance of a foreign substance or by the quantities of any one or more of the blood constituents being abnormally increased or diminished. Changes of this last kind occur frequently. An increase in the number of red corpuscles, a true " plethora POLTCTTH^MIRA," takes place after transfusion of blood of the same species of animal. According to the observations of Panum " and Worm Muller,' the blood-liquid is quickly eliminated and transformed in this case, — the water being eliminated principally by the kidneys, and the albumin burned into urea, etc., — while the blood - corpuscles are preserved longer and cause a " polt- OTTH^MiA. ' ' A relative increase in the number of red corpuscles is found after abundant transudations from the blood, as in cholera and heart-failure, with considerable accumulation. A decrease in the number of red corpuscles occurs in ansmia from different causes. Very excessive hemorrhage causes an acute anEsmia or more correctly oligaemia. Even during the hemorrhage the remaining blood becomes richer in water by diminished secretion and excretion, as also by an abundant absorption of parenchymous fluid somewhat poorer in proteids and strikingly poorer in red blood-corpuscles. The oligaemia passes soon into a hydraemia. The amount of proteid then gradually increases again; but the re-formation of the red blood-corpuscles is slower, and after the hydrsemia follows also an oligocythsemia. After a little time the number of blood-corpuscles rises to normal ; but the re-formation of haemoglobin does not keep pace with the re-formation of the cor- puscles, and a chlorotic condition may appear. A considerable ■ Zeitschr. f. Physiol. Cliem., Bd. 9. » Vircbow's Arch., Bd. 29. ' Transfusion und Plethora. Christiania, 1875. 174 TEE BLOOD. decrease in the number of red corpuscles occurs also in chronic anjemia and chlorosis; still in such cases an essential decrease in the amount of haemoglobin occurs without an essential decrease in the number of blood-corpuscles. The decrease in the amount of hffimoglobin is more characteristic of chlorosis than a decrease in the number of red corpuscles. A very considerable decrease in the number of red corpuscles (300,000-400,000 in 1 c.mm.) and diminution in the amount of heemoglobin (i-tV) occurs in pernicious anaemia (Hatem, Laachb'). On the contrary, the individual red corpuscles are larger and richer in haemoglobin than they ordinarily are, and the number stands in an inverse relationship to the amount of haemo- globin (Hatem). Besides this the red corpuscles often, but not always, show in pernicious anaemia remarkable and extraordinary irregularities of form and size, which QuiisrcKE ' has termed poiki- locytosis. The Composition of the Red Corpuscles. Irrespective of the changes in the amount of haemoglobin, as just mentioned, the com- position of the blood-corpuscles may be changed in other ways. By abundant transudations, as in cholera, the blood-corpuscles may give up water, potassium, and phosphoric acid to the concentrated plasma and become correspondingly richer in organic substances (C. Schmidt"). By a few other transudation processes, as in dysentery and dropsy with albuminuria, a considerable amount of proteid passes from the blood; the plasma becomes richer in water, and the blood-corpuscles take up water and so become poorer in organic substance (0. Schmidt). The number of leucocytes may, as above mentioned, increase considerably under physiological conditions, such as after a meal rich in proteids (physiological leucocytosis). Under pathological conditions a hyperleucocytosis may occur, and according to Vie- CHOW ' this occurs in all pathological processes in which the lym- phatic glands take part. Leucocytosis occurs prominently in leucaemia, which is characterized by the very great abundance of leucocytes in the blood. The number of leucocytes is not only absolutely increased in this disease, but also in proportion to the ' Die Anaemie. Christiania, 1883. ^ Deutsch. Arcli. 1 klin. Med., Bdd. 20 and 35. ' Cit. from Hoppe-Seyler's Physiol. Cliem., 1877-1881. * Virohow's Gesammelte Abhandl. zur wissensch. Med. Bd. 3. QUANTITY OB' WATER, PROTEIDS, FAT, ETC. 175 number of red blood-corpuscles, which is considerably diminished in leucaemia. The blood from a leucasmic patient has a lower specific gravity than the ordinary (1.035-1.040) and a lighter color, as if it were mixed with pus. The reaction is alkaline, but after death is often acid, probably due to a decomposition of the con- siderably increased lecithin. In leucsmic blood, volatile fatty acids, lactic acid, glycero-phosphoric acid, large amounts of xanthin bodies (Salomon,' Kossel"), and the so-called Charcot's crystals (see Chapter XIII) have been found. The quantity of water in the blood is increased in general dropsy, with or without kidney disease, in different forms of anaemia, in scurvy, and in febrile diseases. The amount of water is diminished in abundant transudations, by powerful laxatives, in diarrhoea, and especially in cholera. The amount of proteids in the blood may be relatively increased (htpbealbuminosis) in cholera and after the action of laxatives. A decrease in the amount of proteids (HYPALBUiiiNOsis) occurs after direct loss of proteids from the blood, as in hemorrhage, albuminuria, dysentery, copious formation of pus, etc., etc. The amount of fibrin is increased (htpeeinosis) in inflammatory dis- eases, pneumonia, acute muscular rheumatism, and erysipelas, in which the blood yields a " ceusta phlogistica " because it coagu- lates more slowly. The statements in regard to the occurrence of a hyperinosis in scurvy and hydraemia seems to require further con- firmation. A decrease in the amount of fibrin (hypinosis) has not been observed with certainty in any disease. The amount of fat in the Hood (lip^mia) increases, irrespective of the increase after a diet rich in fat, in drunkards, in corpulent individuals, after fracture of the bones, and also in diabetes. In the last-mentioned case the increase in fat depends, according to Hoppe-Setlbe,' upon defective digestion. An increase in the amount of fat in the blood has also been observed in diseases of the liver, Bright's disease, tuberculosis, malaria, and cholera. T. Jaksch * has observed volatile fatty acids in the blood (lipa- ciDiEMiA) in febrile diseases and sometimes in diabetes. The amount of salts in the blood is increased in dropsy, dysen- ' Arch. f. Anat., Physiol, und wissenscli. Med., 1876, > Zeitachr. f. physiol. Chem., Bd. 7, S. 32. » Physiol. Chem., 1877-1881, S. 433. * Zeitschr. f. kiln. Med., Bd. 11. 176 THE BLOOD. tery, and in cholera immediately after the first violent attack, but diminishes later after the attack in cholera, in scurvy, and in inflammatory diseases. The decrease of alkali salts, especially common salt, is only trifling, but in pneumonia the salt disappears almost entirely from the urine. A decrease in the alkalinity of the blood has been observed in many cases, as in fevers, uraemia, carbon- monoxide poisoning, diseases of the liver, leucaemia, pernicious anaemia, and diabetes. The quantity of glucose is increased in diabetes (mellitaemia). Hoppe-Setlbr ' found in one case 9 p. m. glucose in the blood. According to Claude Beknaed,' when the quantity of glucose in the blood amounts to 3 p. m. it passes into the urine. The quan- tity of urea is augmented in fevers, also in increased metabolism of proteids. A further increase in the amount of urea in the blood occurs in retarded micturition, as in cholera as well as in cholera infantum (K. Mobnee ') , and in affections of the kidneys and the urinary passages. After a ligature of the ureters or after extirpation of the kidneys of animals an accumulation of urea takes place in the blood. In uraemia, ammonia may occur in the blood, which origi- nates from a decomposition of the urea. Uric acid is foand increased in the blood in gout (Gaerod,* Salomon '); oxalic acid was also found in the blood in the same disease by Gaeeod. According to v. Jaksch fevers alone do not lead to uricacidcemia. Uric acid occurs in relatively large quantities, up to 0.08 p. m., in affections of the kidneys, anaemia, and especially such conditions which lead to the symptoms of dyspnoea. J^uclein bases occur sometimes in very small quantities (v. Jaksch). Among the foreign bodies which are found in the blood the following must be mentioned here: biliaet acids and biliaet PIGMENTS (which latter may occur under physiological conditions in a few varieties of blood) in iceterus; leucin and tyeosin in acute atrophy of the liver; acbton" specially in fevers (v. Jaksch '). In melanaemia, especially after conbinuons malarial fever, black, less often light brown or yellowish, grains of pigment occur in the blood, which, according to the generally received opinion, come > Physiol. Cbem., S. 430. ' Le9ons sur le diabete. » See Maly's Jahresber. , Bd. 17, S. 453. * Med. Surg. Transactions, Vols. 31 and 87. ' Zeitschr. f. physiol. Chem., Bd. a. « Ueber Acetonurie und Diaceturie. Berlin, 1885. QUANTITY OF BLOOD. 177 ■from the spleen. After poisoning with potassinm chlorate, methse- moglobin is observed in human and in canine blood (Mabchand ' and Cahn"); but, on the contrary, no formation of methsemo- globin takes place in the blood of rabbits (Stokvis' and Kim- MTSBE*). A formation of methffimoglobin may be caused at the expense of the haemoglobin by the inhalation of amyl nitrite, as also by the action of a number of other medicinal bodies (Hatem,' DiTTBiCH," and others). The quantity of Mood is indeed somewhat variable in different species of animals and in different conditions of the body; in general we consider the entire quantity of blood in adults as about iV-tV of the weight of the body, and in new-born infants about ^. Fat individuals are relatively poorer in blood than lean ones. During inanition the quantity of blood decreases less quickly than the weight of the body (Panum '), and it may therefore be also pro- portionally greater in starving individuals than in well-fed ones. By careful bleeding the quantity of blood may be considerably diminished without any dangerous symptoms. The loss of blood to \ of the normal quantity has as sequence no durable sinking of the blood-pressure in the arteries; while the smaller arteries accom- modate themselves to the small quantities of blood by contracting (WoEJi MtJLLER*). A loss of blood to \ oi the quantity reduces the blood-pressure considerably, and a loss of ^ of the blood in adults is dangerous to life. The faster the bleeding the more dangerous it is. New-born infants are very sensitive to loss of blood, and likewise fat, old, and weak persons cannot stand much loss of blood. Women can stand loss of blood better than men. The quantity of blood may be considerably increased by the injection of blood from the same species of animal (Panum," Landois," WoBM MuLLER,' PoNFicK "). According to Woem 1 Virchow's Archiv, Bd. 77, and Arch. f. exp. Path. u. Pharm., Bd. 22. ' Arch. f. exp. Path. u. Pharm., Bd. 24. ^Ibid., Bd. 21. •• Maly's Jahresber., Bd. 14, S. 243. ' Comp. rend.. Tome 102. « Arch. f. exp. Path. u. Pharm., Bd. 29. ■" Virchow's Arch., Bd. 29. ' Transfusion und Plethora. Christiania, 1875. ' Nord. med. Ark., Bd. 7; Virchow's Arch., Bd. 63. '» Centralbl. f. d. med. Wissensch., 1875, and Die Transfusion des Blutes. Leipzig, 1875. " Virchow's Arch., Bd. 62. 178 THE BLOOD. MtJLLER the normal quantity of blood may indeed be increased to 83^ without producing any abnormal conditions or lasting high blood-pressure. An increase of the quantity of blood to 150^ may be directly dangerous to life (Worm Mullbk). If the quantity of blood of an animal is increased by transfusion with blood of the same kind of animal, an abundant formation of lymph takes place. The water in excess is eliminated by the urine ; and as the proteid ■of the blood-serum is quickly decomposed, while the red blood- corpuscles are destroyed much more slowly (Tschikjew," Foestek,' Panum,' Worm Mullee'), a polycythaemia is gradually produced. If blood of another kind is transfused, then under certain con- ditions, according to the quantity of blood introduced, more or less menacing symptoms appear. These appear, for instance, when the blood-corpuscles of the receiver are dissolved easily by the serum of the introduced blood, as, for example, the blood-corpuscles of rabbits on transfusion with a difEerent kind of blood, or the reverse, when the blood-corpuscles of the transfused blood are dissolved by the blood of the receiver; for instance, when the blood of a dog is transfused with rabbit's or lamb's blood, or the blood of a man with lamb's blood (Landois*). Before dissolving, the blood-corpuscles may unite in tough agglomerated heaps, which clog up the smaller vessels (Landois). On the other hand, the stromata of the dis- solved blood-corpuscles may also give rise to an extensive intra- vascular coagulation, causing death. The transfusion should therefore when possible be made with the blood of the same kind of animal, and for the resuscitating action of the blood it is immaterial whether or not it contains the fibrin or the mother-substance of the same. The action of trans- fused blood depends on its blood-corpuscles, and therefore defibri- nated blood acts just like non-deflbrinated (PAifUM,* Lakdois"). The property of blood-serum of a certain species of animals of dissolving or destroying the blood-corpuscles of another has been called the globulicidal action of the serum. According to Dabbmbekg,' Bdchner,' and others, this ' Arbeiten aus der physiol. Anstalt zu Leipzig, 1874, S. 292. « Zeitschr. f. Biologie, Bd. 11. ' Virchow's Archiv, Bd. 39. «L. c. 'L. c. « Sem. medic, 1891, No. 51. Cit. from Maly's Jahresber., Bd. 22. ' Arch. f. Hygiene, Bd. 10 ; Miinchener med. Wocheuschr. , 1892, No. 8, and Berl. klin. Wochenschr,, 1892, No. 19. QUANTITY OF BLOOD. 179 property stands in certain relationship to its bactericidal or so-called micro- iieidal action, and these two actions, which have much in common, may be retarded by heating the blood-serum to 55-65° C. The microbicidal action is in part connected with the presence of certain protein bodies acting like enzymes, called alexins, and in part to certain mineral bodies such as sodium chloride and alkali. Somewhat similar conditions are also necessary for the globulicidal action. Makaqliano ' has found that tlie blood-serum in many diseases, such as pneumonia, malaria, typhus, leucmaeia, cancerous cachexia, etc., has a destructive action on the red blood-corpuscles. He found the quantity of sodium chloride diminished in such serum, and the globulicidal action was prevented by the addition of NaCl sufficient to make the serum normal in salt. The quantity of blood in the difEerent organs depends essentially on the activity of the same. During work the exchange of material in an organ is more active than when at rest, and the increased metabolism is connected with a more abundant flow of blood. Although the total quantity of blood in the body remains constant, the distribution of the blood in the various organs may be difEerent at difEerent times. As a rule, the quantity of blood in an organ can be an approximate measure of the more or less active meta- bolism going on in the same, and from this point of view the dis- tribution of the blood in the difEerent organs and groups of organs is of interest. According to Eanke," to whom we are especially indebted for our knowledge of the relationship of the activity of the organs to the quantity of blood contained therein, of the total quantity of blood (in the rabbit) about ^ comes to the muscles in rest, i to the heart and the large blood-vessels, ^ to the liver, and i to the other organs. • Berl. kiln. Wochenschr., 1893, No. 31. » Die Blutvertheilung und der Thatigkeitswechsel der Organs. Leipzig, 1871. CHAPTEE VII. CHYLE, LYMPH, TRANSUDATIONS AND EXUDATIONS. I. Chyle and Lymph. The lymph is the mediator in the exchange of constituents between the blood and tissues. The bodies necessary for the nutri- tion of the tissue pass from the blood into the lymph, and the tissues deliver water, salts, and products of metabolism into th& lymph. The lymph therefore originates partly from the blood and partly from the tissues. From a purely theoretical standpoint we^ can, according to Hbidenhain, differentiate between blood-lymph and tissue-lymph according to origin. It is impossible at the present time to completely separate what one or the other source delivers; but, thanks to the pioneering investigations of Hbiden- HAIN", we have means of exciting a copious flow from one or the other sources of lymph. The action of these means, Heidenhain's lymphagogues, will be closely studied later. According to older views the lymph was only considered as a, filtrate from the blood-fluid. Since the investigations of Heidbn- HAiN ' and Hambukger ' this view cannot be maintained. Accord- ing to these investigators the lymph is considered under physiological conditions in part as a product of the active, secretory property of the cells of the blood-capillaries. In chemical respect the lymph is the same as plasma and con- tains qualitatively the same bodies as this; The most essential difference is of a quantitative nature and consists in that the lymph is poorer in proteids. No essential chemical difference has been found between the lymph and the chyle of starving animals. ' Pfluger's Arch., Bd. 49. 2 Zeitschr. f. Biologie, Bd. 27, S. 259, and Bd. 30, S. 143; see Ziegler's Beitr. z. pathol. Anat., etc., Bd. 14, S. 443. 180 PROTEIDa OF OETLE AND LTMPH. 181 After the assimilation of fatty food the chyle differs from the lymph ia its wealth of minutely divided fat-globules, which give it a milky appearance; hence the old name " milk-juice." Chyle and lymph, like the plasma, contain seralbumin, serglo- bulin, fibrinogen, and fibrin-ferment. The two last-mentioned bodies occur only in very small amounts; therefore the chyle and lymph coagulate slowly (but spontaneously) and yield but little fibrin. Like other liquids poor in fibrin-ferment, chyle and lymph do not at once coagulate completely, but repeated coagulations take place. The extractive bodies seem to be the same as in plasma. Glucose is found in about the same quantity as in the blood-serum, hut in larger quantities than in the blood; this depends on the fact that the blood-corpuscles contain no glucose. According to EoHMANK and Bial ' lymph contains a diastatic enzyme similar to that in blood-plasma, and Lepine ' has found that the chyle of a ■digesting dog has great glycolytic activity. Dastkb ' has studied the glycolytic activity of horse's and cow's lymph, and he finds that it is retarded by the presence of 2 p. m. potassium oxalate. He could also detect glycogen in the cow-lymph which existed in the plasma but not in the form-elements. The amount of urea has been determined by "Wurtz* as 0.12-0.38 p. m. The mineral bodies appear to be the same as in plasma. As form-elements leucocytes and red blood-corpuscles are common to both chyle and lymph. When it has not left the villi of the intestine chyle contains very few leucocytes, but in the vessels on the peritoneal side of the intestine it is richer in leucocytes. The greatest quantity of leucocytes is found in the chyle between the great mesenteric gland and the cisterna chyli. The chyle is poorer in leucocytes in the thoracic duct, probably because a mixing takes place here with lymph that is poorer iu form-constituents from other parts of the body. Bed blood-corpuscles occur in the chyle and lymph in very small quantities. In these liquids, which seem to be free from oxygen, the blood-corpuscles are darker-colored, and only after they iiave come in contact with the air do they have the light-red color ' Pflllger's Aroliiv, Bdd. 53, 53, and 55. ' Compt. rend.. Tome 110. » Arch. d. Physiol. , Ser. 5, Tome 7. * Compt. rend., Tome 49. 182 CHTLE, LTMPE, TRANSUDATIONS AND EXUDATIONS. of oxyhsemoglobin and give the surface of the fibrin-clot a beaatif nl light-red appearance. It has been suggested that this red color originates from the transition forms between red and white blood- corpuscles, in which blood-coloring matters are first formed by the action of the oxygen. The chyle of starving animals has the appearance of lymph. After partaking of fat or food rich in fat it is milky, and this is partly due to the presence of large fat-globules, as in milk, or partly, and indeed chiefly, the finely divided fat. The nature of the fats occurring in the chyle depends on the variety of fat in the food. The disproportionally greater part consists of neutral fats, and even after feeding with abundant amounts of free fatty acids. Munk' found in the chyle chiefly neutral fats with a small quantity of fatty acids or soaps. The gases of the chyle have not been studied, and it seems that the gases of an entirely normal human lymph have not thus far been investigated. The gases from dog-lymph contain only traces of oxygen and consist of 37.4-53.1^ CO, and l.Q^ N (author ") calcu- lated at 0° C. and 760 mm. mercury. The chief mass of the carbon dioxide of the lymph seems to be firmly chemically com- bined. Comparative analyses of blood and lymph have shown that the lymph contains more carbon dioxide than arterial, but less than venous, blood. The tension of the carbon dioxide of lymph is, according to Pflugek and Strassbueg,' smaller than in venous, but greater than in arterial, blood. The quantitative composition of the chyle must naturally be very variable. The analyses thus far made refer only to that mixture of chyle and lymph which is obtained from the thoracic duct. The specific gravity varies between 1.007 and 1.043. As example of the composition of human chyle we will here give two analyses. The first is by Owbk-Eebs,* of the chyle of an executed person, and the second by Hoppe-Setlee,' of the chyle in a case of rupture of the thoracic duct. In the latter case the fibrin had previously separated. The results are in 1000 parts. > Vircliow's Arch., Bdd. 80 and 133. » Die Gase der Hundelymphe. Arbeit, aus d. physiol. Anstalt zu Leipzig, 1871. 3 Pfliiger's Arch., Bd. 6, p. 85. ■> Cit. from Hoppe-Seyler, Physiol. Chem., S. 595. 6 aid. , S. 597. COMPOSITION OF CHYLE. 183 No. 1. No. 3. Water 904.8 940.73 water Solids 95.3 59.28 solids Fibrin traces Albumin 70.8 36.67 albumin Fat 9.3 7.33 fat 3.85 soaps f 0.83 lecithin Remaining organic bodies 10.8 ^Jl S^^L^tractives Salts 4.4 \l . 58 water extractives 80 soluble salts 35 insoluble salts The quantity of fat is very variable and may be considerably increased by partaking food rich in fats. J. Munk and A. Eosen- STEiN ' have investigated the lymph or chyle obtained from a lymph fistula at the end of the upper third of the leg of a girl 18 years old and weighing 60 kg., and the highest quantity of fat in the chylous lymph was 47 p. m. after partaking of fat. In the starvation lymph from the same patient they found only 0.6-3.6 p. m. fat. The quantity of soaps was always small, and on partaking of 41 gm. fat the quantity of soaps was only about -^ of the neutral fats. A great many analyses of chyle from animals have been made, and they chiefly show the fact that the chyle is a liquid with a very changeable composition which stands closely related to blood-plasma, but with the chief difierence that it contains more fat and less solids. The reader is referred to special works for these analyses, as, for example, to v. Goeup-Besanez's " Lehrbuch der physiolo- gischen Chemie," 4th edition. The composition of the lymph is also very changeable, and its specific gravity shows about the same variation as the chyle. In the following analyses, 1 and 3, made by Gubleb and Quetenite,' are the results obtained from lymph from the upper part of the thigh of a woman aged 39; and 3, made by v. Schebee,' is an analysis of lymph from the sac-like dilated lymphatic vessels of the sper- matic cord. No. 4 was made by 0. Schmidt,' the data being obtained from lymph from the neck of a colt. The results are in parts per 1000. ' Virchow's Arch., Bd. 133. « Cit. from Hoppe-Seyler's Physiol. Chem., S. 591. » lUd., S. 591. See Hamburger, Zeitschr. f. Biologie, Bd. 37, S. 359, and Bd. 30, S. 143. Also Hambarger, Hydrops von mikrobiellem Ursprung, in Beitr. zur path. Anat. und zur allg. Pathol., Bd. 14, S. 443. ' Journal of Physiol., Vols. 16 and 17. » Journ. of Physiol., Vol. 18. * Pflilger's Arch., Bd. 59. ' Virchow's Arch., Bd. 185, and Pflilger's Arch., Bd. 59. 188 CHYLE, L7MPR, TRANSUDATIONS AND EXUDATIONS. tion, but a pathological and toxic one which increases the perme- ability of the capillary walls. The lymphagognes of the second series act, according to Star- ling, first by osmosis, causing an abundant flow of water into the blood and thereby increasing the pressure in the capillaries, produc- ing a stronger filtration. The more abundant current of lymph in the thoracic duct is caused in this case by a greater pressure in the abdominal capillaries. II. Transudations and Exudations. The serous membranes are normally kept moistened by liquids ■whose quantity is only sufiicient in a few instances, as in the pericardial cavity and the subarachnoidal space, for a complete chemical analysis to be made of them. Under diseased conditions an abundant transudation may take place from the blood into the serous cavities, into the subcutaneous tissues, or under the epider- mis ; and in this way pathological transudations are formed. Such true transudations, which are similar to lymph, are generally poor in form-elements and leucocytes, and yield only very little or almost no fibrin, while the inflammatory transudations, the so-called exudations, are generally rich in leucocytes and yield proportionally more fibrin. As a rule, the richer a transudation is in leucocytes the closer it stands to pus, while when it has a diminished quantity of leucocytes it is more nearly like real transudations or lymph. It is ordinarily accepted that filtration is of the greatest import- ance in the formation of transudations and exudations. The facts coincide with this view, namely, that all these fiuids contain the salts and extractive bodies occurring in the blood-plasma in about the same quantity as the blood-plasma, while the amount of proteids is habitually smaller. "While the diflerent fluids belonging to this group have about the same quantities of salts and extractive bodies, they differ from each other chiefly in containing differing quantities of proteid and form-elements, as well as varying quantities of trans- formation and decomposition products of these latter — changed blood-corloring matters, cholesterin, etc., etc. It must be apparent that the circulation and pressure conditions must have an essential influence on the quantity and composition of the transudations, but their action has been little studied. An increase in the vein-pressure causes, according to SEiiTATOE,' an ' Vircliow's Arch., Bd. 111. TRANSUDATIONS AND EXUDATIONS. 189 increase in the quantity of transudation and the quantity of proteid contained, while the amount of salts does not markedly change. Nothing positive is known in regard to the variations in the quantity of proteid by simple arterial hyperemia. The process, as suggested by Cohnheim," of the changed perme- ability of the capillary walls in disease is a second important factor in the formation of transudations. The circumstance that the greatest quantity of proteid occurs in transudations in inflammatory processes, to which is also due the abundant quantity of form- elements in such transudations, has been explained by this hypothe- sis. The greater quantity of proteid in the transudations in formative irritation is in great part explained by the large amount of destroyed form-elements. The interesting observation made by PAiJKtJLL," that in such cases in which an inflammatory irritation has taken place the fluid contains nucleoalbumin (or nucleo- proteids?), while these substances do not occur in transudations in the absence of inflammatory processes, can be explained by the presence of form-elements. As the secretory importance of the capillary endothelium has been made probable by the investigations of HBiDEiirHAiN and Hambuegek, it is a priori to be expected that an abnormal increased secretory activity of the endothelium is a third cause of transuda- tions. Certain observations of Hambuegee in a case of dropsy,' in which the transudation was probably produced by the lymph- exciting action of a metabolic product formed by a bacterium, speak for the correctness of this assumption. Hamburgee therefore considers the irritation of the endothelium of the capillaries by means of a special substance exciting lymph-flow and formed in disease as a third cause of the transudations. The question whether this substance acts secretory in HEiDEifHAiN^'s sense or increases the permeability in Staeling's sense must be proved. That the conditions of the blood-capillaries in the different vascular regions have an effect on the quantity of proteid has been partly explained by the varying secretory activity of the capillary endothelium (C. Schmidt'). For example, the amount of proteid in the peeicaedial, pleueal, and peritostbal fluids is con- ' Colinlieim, Vorlesungen ilber allg. Path., 3. Aufl., Part 1. " TJpsala Lakarefs. Pdrhandl., Bd. 37, and Maly's Jahresber., Bd. 33. • See Ziegler's Beitrage, Bd. 14. * Cit. from Hoppe-Seyler's Physiol. Chem,, p. 607. 190 CH7LE, LYMPH, TRANSUDATIONS AND EXUDATIONS. siderably greater than in those fluids which are found in the sub- arachnoidal SPACE, in the suBCUTA]>rEOUS tissues, or in the AQUEOUS HUMOE, which are poor in proteid. The condition of the blood also greatly affects the transudations, for in hydrsemia the amount of proteid in the transudation is very small. With the increase of the age of a transudation, of a hydrocele fluid for instance, the quantity of proteid is increased, probably by resorp- tion of water, and indeed exceptional cases may occur in which the amount of proteid, without any previous hemorrhage, is even greater than in the blood-serum. The proteids of transudations are chiefly seralbumin, serglobulin, and a little fibrinogen. The non-inflammatory transudations do not as a rule coagulate spontaneously, or very slowly. On the addition of blood or blood-serum they coagulate. Inflammatory exudations coagulate spontaneously. Paijkull ' has shown that these often contain nucleoalbumin. Mucoid substances, which were first observed by the AUTHOK " in a few cases of ascitic fluid, without complication with ovarial tumors, seem, according to Paijkull, to be regular constituents of transudations. The rela- tionship between globulin and seralbumin varies very much in different cases, but, as Hoffmann ' and Pigeaud ' have shown, the variation is in each case the same as the blood-serum of the indi- vidual. The speciflc gravity runs rather parallel with the quantity of proteid. The varying specific gravity has been suggested as a means of differentiation between transudations and exudations by Ebuss," as the first often show a specific gravity below 1015-1010, while the others have a specific gravity of 1018 or above. This rule holds good in many but not in all cases. The gases of the transudations consist of carbon dioxide besides small amounts of nitrogen and traces of oxygen. The tension of the carbon dioxide is greater in the transudations than in the blood. On mixing with pus the amount of carbon dioxide is decreased. The extractives are, as above stated, the same as in the blood- plasma; but sometimes extractive bodies occur, such as allantoin in 'L.c. ' Zeitschr. f . pliysiol. chem., Bd. 15. " Arch. f. exp. Path. u. Pharm., Bd. 16. * See Maly's Jaliresber., Bd. 16. ' Deutsch. Arcli. f. klin. Med., Bd. 28. PERICARDIAL FLUID. 191 dropsical fluids (Moscatelli '), which have not beea detected in the blood. Urea seems to occar in very variable amonnts. Glucose, or at least a fermentable substance which reduces copper oxide in alkaline liquids, occurs in most transudations. Succinic acid has been found in a few cases in hydrocele fluids, while in other cases it is entirely absent. Leucin and tyrosin have been found in trans- udations from diseased livers and in pus-like transudations which have undergone decomposition. Among other extractives found in transudations we must mention uric acid, allantoin, xanthtn, creaiin, inosit, ani j)yrocaiechin. As above stated, irrespective of the varying number of form- elements contained in the different transudations, the quantity of proteid is the most characteristic chemical distinction in the com- position of the various transudations; therefore a quantitative analysis is only of importance in so far as it considers the quantity ■of proteid. On this account the following quantitative composition is referred to the chief weight, the quantity of proteid. Pericardial Fluid. The quantity of this fluid is also, under certain physiological conditions, so large that a sufficient quantity for chemical investigation was obtained from a person who had been executed. This fluid is lemon-yellow in color, somewhat sticky, and yields more fibrin than other transudations. The amount of solids, according to the analyses performed by v. GrOBUP-BESANEZ," Wachsmuth," and Hoppb-Setler,* is 37.6-44.9 p. m., and the amount of proteid is 32.8-24.7 p. m. The analysis made by the AUTHOR of a fresh pericardial fluid from a young man who had been executed yielded ^the following results, calculated in 1000 parts by weight: Water 960.85 Solids 39. 15 (Fibrin 0.31 Proteids 28.60 ■< Globulin. .. . 5.95 (Albumin 32.34 Solublesalts S.eoJNaCl 7,38 Insoluble salts 0.15 Extractive bodies 2.00 > Zeitschr. f. physiol. Chem., Bd. 13. ' V. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., S. 401. • Virohow's Arch., Bd. 7. * Physiol. Chem., S. 605. 192 CHYLE, LTMPM, TRANSUDATIONS AND EXUDATIONS. Fkiend ' has found nearly the same composition for a pericar- dial fluid from a horse, with the exception that this liquid was relatively richer in globulin. The ordinary statement that pericar- dial fluids are richer in fibrinogen than other transudations is hardly based on sufficient proof. In a case of chylopericardium, which was probably due to the rupture of a chylus vessel or caused by a capillary exudation of chyle because of stoppage, Hasebeoek " found in 1000 parts of the analyzed fluid 103.61 parts solids, 73.79 albuminous bodies, 10.77 fat, 3.34 cholesterin, 1.77 lecithin, and 9.34 salts. The pleural fluid occurs under physiological conditions in such small quantities that a chemical analysis of the same cannot be made. Under pathological conditions this fluid may show very variable properties. In a few cases it is nearly serous, in others again sero-fibrinous, and in others similar to pus. There is a corre- sponding variation in the specific gravity and the properties in general. If a pus-like exudation is kept closed for a long time in the pleural cavity, a more or less complete maceration and solution of the pus-corpuscles is found to take place. The ejected^ yellowish- brown or greenish fluid may then be as rich in solids as the blood- serum; and an abundant flocculent precipitate of a nucleoalbumin (the pyin of early writers) may be obtained on the addition of acetic acid. This precipitate is soluble with difficulty in an excess of acetic acid. Numerous analyses, by many investigators,' of the quantitative composition of pleural fluids under pathological conditions are at hand. From these analyses we learn that in hydrothorax the specific gravity is lower and the quantity of proteid less than in pleuritis. In the first case the specific gravity is generally less than 1015, and the quantity of proteid 10-30 p. m. In acute pleuritis the specific gravity is generally higher than 1020, and the quantity of proteid 30-66 p. m. The quantity of fibrinogen, which in hydrothorax is about 0.1 p. m., may amount to more than 1 p. m. in pleuritis. In pleurisy with an abundant gathering of pus the specific gravity may rise even to 1030, according to the observations 1 Halliburton : Text-book of Chem. Physiol., etc. London, 1891. P. 347. ' Zeitschr. f. physiol. cbem., Bd. 12. ' See the works of Mehu, Runeberg, F. Hoffmann, Reuss, Neuenkirchen, all of which are cited in Bernheim's paper in Virchow's Arch., Bd. 131, S. 274. See also Paijkull, 1. c, and Halliburton's Text-book, p. 346. PEBITONMAL FLUID. 193 of the AUTHOR. The quantity of solids is often 60-70 p. m., and may be even more than 90-100 p. m. (authoe). Mucoid sub- stances have also been detected in pleural fluids by Paijktjll.. Cases of chylous pleurisy are also known ; in such a case Mehu " found 17.93 p. m. fat and cholesteria in the fluid. The quantity of peritoneal fluid is very small under physiological conditions. The investigations refer only to the fluid under diseased conditions {dropsical or ascitic fluid). The color, trans- parency, and consistency of these may vary greatly. In cachectic conditions or a hydrsemic condition of the blood the fluid- has little color, is milky, opalescent, watery, does not coagu- late spontaneously, has a very low specific gravity, 1005-1010-1015, and is nearly free from form-elements. The ascitic fluid in portal stagnation, or generally in venous stagnation, has a low specific gravity and ordinarily less than 20- p. m. proteid, although in certain cases the quantity of proteid may rise to 35 p. m. In carcinomatous peritonitis it may have a cloudy, dirty-gray appearance, due to its richness in form-elements of various kinds. The specific gravity is then higher, the qaantity of solids greater, and it often coagulates spontaneously. In inflamma- tory processes it is straw- or lemon-yellow in color, somewhat cloudy or reddish, due to leucocytes and red blood-corpuscles, and from great richness in leucocytes it may appear more like pus. Ifc coagulates spontaneously, and may be relatively richer in solids. Ifc contains regularly 30 p. m. or more proteid (although exceptions with less proteid occur), and may have a specific gravity of l.OSO' or above. By rupture of a chylous vessel the dropsical fiuid may be rich in very finely emulsified fat (chylous ascites). In such cases 3.86-10.30 p. m. fat has beea found in the dropsical fluid (GuiNOCHET,' Hay'), or even 17-43 p. m. fat has been found by Minkowsky. By admixture of this fluid with the fluid from an ovarian cyst it may sometimes contain pseudomucin (see Chapter XIII). We also have cases in which the ascitical fluid contains mucoids which may be precipitated by alcohol after removal of the proteids by coagulation at boiling temperature. Such substances, which yield a reducible substance on boiling with acids, have been ' Arch. gen. de med., 1886, Tome 2. Cit from Maly's Jaliresber., Bd. IS. ' See Straus, Arch, de physiol, Tome 18. Cit. from Maly's Jahrfesber., Bd. 17. . 2 See Maly's Jahresber., Bd. 16, S. 475. 194 CHYLE, LYMPH, TRANSUDATIONS AND EXUDATIONS. found by the author in tuberculous peritonitis and in cirrhosis hepatis syphilitica in men. According to the investigations of Paijkull' these substances seem to occur often and perhaps habitually in the ascitic fluids. As the quantity of proteid in ascitic fluids is dependent upon the same circumstances as in other transudations and exudations, it is sufiQcient to give the following example of the composition, taken from Beestheim's ° treatise. The results are expressed in 1000 parts of the fluid: Max. Min. Mean, Cirrhosis of the liver 34.5 5.6 9.69 — 2t. 06 Brighfs disease 16.11 10.10 5.6 10.36 Tuberculous and idiopatbic peritonitis 55.8 18.72 30.7 — 37.95 Carcinomatous peritonitis 54.20 27.00 35. 1 — 58.96 Urea bas also been found in ascitical iluids, sometimes only as traces, somb- times in larger quantities (4 p. m. in albuminuria), also uric acid, allantoin in cirrhosis of tbe liver (Moscatelli'), xanthin, creatin, cliolesterin, and glucose. Hydrocele and Spermatocele Fluids. These fluids differ from each other in various ways. The hydrocele fluids are generally colored light or darker yellow, sometimes brownish with a shade of green. They have a relatively higher specific gravity, 1.016-1.026, with a variable but generally higher amount of solids, an average of 60 p. m. They sometimes coagulate spontaneously, sometimes only after the addition of fibrin-ferment or blood. They contain leucocytes as chief form-elements. Sometimes they contain smaller or larger amounts of cholesterin crystals. The spermatocele fiuids, on the contrary, are as a rule colorless, thin, cloudy like water mixed with milk. They sometimes have an acid reaction. They have a lower specific gravity, 1.006-1.010, a lower amount of solids — an average of about 13 p. m., — and do not coagulate either spontaneously or after the addition of blood. They are, as a rule, poor in proteid and contain spermatozoa, cell-detritus, and fat-globules as form-constituents. To show the unequal com- position of these two kinds of fluids we will give the average results (calculated in parts per 1000 parts of the fluid) of 17 analyses of hydrocele fluids and 4 of spermatocele fluids made by the author :* 'L. c. ' L. c. As it was impossible to derive mean figures from those given by Bernheim, the author has given above the maximum and minimum of the averages given by him. 3L. c. * Upsala Lakaref. F5rh., Bd. 14, and Maly's Jahresber., Bd. 8, S. 347. CEBEBBO-aPINAL FLUID. 195 Hydrocele, Spermatocele. Water 938.85 986.33 Solids 61.15 13.17 Fibrin 0.59 Globulin 13.25 0.59 Seralbumin 35.94 1.83 Ether extractive bodies 4.03 ' Soluble salts 8.60 J- 10.76 Insoluble salts 0.( il In the hydrocele fluid traces of urea and a reducing substance have been found, and in a few cases also succinic add and inoait. A hydrocele fluid may, according to Dbvellaed,' sometimes contain paralbumin or metalbumin (?). Cases of chylous hydrocele are also known. Cerebro-spinal Fluid. This fluid has heretofore been considered as a secretion and not a transudation. But as we now consider not ■only the lymph as part secretion, but also the transudations, such a difference between this fluid and the others cannot be maintained. The cerebro-spinal fluid is thin, water-clear, of low speciflc gravity, 1007-1008. The spina bifida fluid is very poor in solids, 8-10 p, m., with only 0.19-1.6 p. m. proteid. The fluid of chronic lydrocephalus is somewhat richer in solids (13-19 p. m.) and pro- teids. According to Hallibueton' the proteid of the cerebro- spinal fluid is a mixture of globulin and albumoses; occasionally some peptone occurs, and more rarely, in special cases, seralbumin appears. An optically inactive, non-fermentable, reducing sub- stance, seeminglj pyrocatechm (Hallibueto]^), has been observed in this fluid. The older statement that the cerebro-spinal fluid differs from the other transudations in a greater wealth of potassium salts has not been confirmed by recent investigations of YvoN " and Hallibueton. According to Cavazzani * the cerebro-spinal fluid is more alkaline and richer in solids in the morning than in the evening. Aqueous Humor. This fluid is clear, alkaline, and has a speciflc gravity of 1.003-1.009. The amount of solids is on an average 13 p. m., and the amount of proteids only 0.8-1.3 p. m. The proteid consists of seralbumin and globulin and very little fibrinogen. According to GEUEKHAGBif,'' it contains paralactic acid, another dextrogyrate substance, and a reducing body which is not similar ' Bull. soc. chim., Tome 49, p. 617. ' Halliburton's Text-book, pp. 355-361. » Journ. de Pharm. et de Chim. (4 Ser.), Tome 26. * Maly's Jahresber., Bd. 23, S. 346. ' Pflfiger's Arch., Bd. 43. 196 CETLE, LTMPH, TRANSUDATIONS AND EXUDATIONS. to glucose or dextrin. Pautz ' found urea and sugar in the aqueoas humor of oxen. Blister-fluid. The content of blisters caused by burns, and of vesicator blisters and the blisters of the pemphigus chronicus, is generally a fluid rich in solids and proteids (40-65 p. m.). This is especially true of the contents of vesicatory blisters, which also con- tain a substance that reduces copper oxide. The fluid of the pemphigus is slimy and alkaline in reaction. The fluid of subcutaneous oedema. This is, as a rule, very poor in solids, purely serous, does not contain fibrinogen, and has a specific gravity of 1.005-1.010. The quantity of proteids is in most cases lower than 10 p. m., — according to Hoffmann 1-8 p. m., — and in serious affections of the kidneys, generally with amyloid degeneration, less than 1 p. m. has been shown (Hoff- mann'). The oedema fluid also habitually contains urea, 1-3' p. m., and also a reducing substance. The FLUID OF THE TAPBWOBM cyst is related to the transudations. It is thin and colorless, and has a specific gravity of 1.005-1.015. The quantity of solids is l4r-30 p. m. The chemical constituents are glucose (3.5 p. m.), inoait, traces of urea, creatin, succinic acid, and salts (8.3-9.7 p. m.). Proteids are only found in traces, and then only after an inflammatory irritation. In the last-mentioned case 7 p. m. proteids have been found in the fluid. The Synovial Fluid and Fluid in Synovial Cavities around Joints, etc. The synovia is hardly a transudation, but it is often treated as an appendix to the transudations. The synovia is an alkaline, sticky, fibrousj yellowish finid which is cloudy, from the presence of cell-nuclei and remains of destroyed cells, but is sometimes clear. It contains also, besides proteids and salts, a substance similar to mucin in, physical properties. The nature of these niucin-like constituents of physiological synovial fiuids has not been determined. The author ' has found a mucin- like substance in pathological synovial fluid, but it was not true mucin. It acts like a nucleoalbumin or a nucleoproteid, and gave no reducing substance when boiled with acid. Salkowsei * also found a mncin-like substance in a pathological synovial fluid, which was neither mucin nor nucleoalbumin. He called the sub stance " synovin.'''' ' Zeitschr. f. Biologie, Bd. 31. « Deutsch. Arch. f. klin. Med., Bd. 44. 3 Upsala Lakaref. POrhandl., Bd. 17. * Virchow's Arch.. Bd. 131. PUS. 197 The composition of synovia is not constant, but varies in rest and in motion. In the last-mentioned case the quantity of fluid is less, but the amount of the mucin-like body, proteids, and of the extractive bodies is greater, while the quantity of salts is diminished. This may be seen from the following analyses by Fberichs." The figures represent parts per 1000. I. Synovia from II. Synovia (rem a Stall-fed Ox. a Field-fed Ox. •Water 969,9 943.5 Solids 30.1 51.5 Mucin-like body . 3.4 5.6 Proteids and extractives 15.7 35.1 Fat 0.6 o!7 Salts 11.3 9.9 The synovia of new-born babes corresponds to that of resting animals. The fluid of the bursas mucosae, as also the fluid in the synovial cavities around joints, etc., is similar to synovia from a ■qualitative standpoint. III. Pus. Pus is a yellowish-gray or yellowish-green, creamy mass of a faint odor and an unsavory, sweetish taste. It consists of a fluid, the pus-serum, in which solid particles, the pus-cells, swim. The number of these cells varies so considerably that the pus may at one time be thin and at another time so thick that it scarcely contains a drop of serum. The specific gravity, therefore, may also greatly vary, namely, between 1.020 and 1.040, but ordinarily it is 1.031- 1.033. The reaction of fresh pus is generally alkaline, but it may become neutral or acid from a decomposition in which fatty acids, glycero-phosphoric acid, and also lactic acid are formed. It may become strongly alkaline when putrefaction occurs'with the forma- tion of ammonia. In the chemical investigation of pus the pus-serum and the pus- corpuscles must be studied separately. Pus-serum. Pus does not coagulate spontaneously nor after the addition of defibrinated blood. The fluid in which the pus- ■corpuscles are suspended is not to be compared with the plasma, but rather with the serum. The pus-serum is pale yellow, yellowish green, or brownish yellow, and has an alkaline reaction. It con- ' Wagner's HandwSrterbuoli, Bd. 3, Abth. 1. S. 463. 198 CHTLE, LYMPH, TRANSUDATIONS AND EXUDATIONS. tains, for- the most part, the same constituents as the blood-sernmj but sometimes besides these — when, for instance, the pus has remained in the body for a long time — it contains a nucleoalbumin or nncleoproteid which is precipitated by acetic acid and soluble with great difiacnlty in an excess of the acid {pyin of the older authors). This nucleoalbumin seems to be formed from the hyaline Bubstance of the pus-cells by maceration. The pus-serum contains, moreover, at least in many cases, no fibrin-ferment. According to the analyses of Hoppb-Setlbe," the pus-serum contains in 1000' parts: I. II. Water 913.7 905.65 Solids 86 3 9435 Proteids 63.33 77.21 Lecithin 1.50 0.56 Fat 0.26 0.39 Cholesterin 0.53 0.87 Alcohol extractives . • 1.53 0.73 Water extractives 11.58 6.93 Inorganic salts 7.73 7.77 The ash of pus-serum has the following composition, calculated to 1000 parts of the serum : I. II. NaCl.. .* 5.33 5.39 Na^SOi 0.40 0.31 NajHPO, 0.98 0.46 Na^COs 0.49 1.13 CasCPOi),... 0.49 0.31 Mg3(P04)2 0.19 0.13 POa (in excess) .05 The pus-corpuscles are generally thought to consist in great part- of emigrated white blood-corpuscles (emigration hypothesis), and their chemical properties have therefore been given above. We consider the molecular grains, fat-globules, and red blood-corpus- cles rather as casual form-elements. The pus-cells may be separated from the serum by centrifugal force, or by decantation directly or after dilution with a solution of sodium sulphate in water (1 vol. saturated sodium-sulphate solution and 9 vols, water), and then washed by this same solution in the same manner as the blood-corpuscles. The chief constituents of the pus-corpuscles are albuminous bodies of which the largest proportion seems to be a nncleoproteid which is insoluble in water and which expands into a tough, slimy ' Med. chem. Untersuch., S. 490. PUS. 199 mass when treated with a 10,"^ common-salt solution. This proteid substance, which is soluble in alkali but quickly changed thereby, is called Eoyidas's hyaline substance, and the property of the pus of being converted into a slime-like mass by a solution of common salt depends on this substance. Besides this substance we find in the pus-cells also an albuminous body which coagulates at 48-49° 0., as well as serglobulin (?), seralbumin, a substance similar to coagu- lated albumin (Miescher),' and lastly peptone (Hofmeistek)." We also find in the protoplasm of the pus-cells, besides the pro- teids, lecithin, cholesterin, xanthin bodies, fat, and soaps. Hoppe- Setlee has found cerebrin, a decomposition product of a protagon- like substance, in pus (see Chapter XII). Kossbl and Feettag " have isolated from pus two substances, pyosin and pyogemn, which belong to the cerebrin group (see Chapter XII). Hoppe-Sbylee * claims that glycogen appears only in the living, contractile white blood-cells and not in the dead pus-corpuscles. Salomon '' has nevertheless found glycogen in pus. The cell-nucleus contains nuclein and nucleoproteids. The mineral constituents of the pus-corpuscles are potassium, sodium, calcium, magnesium, and iron. A part of the alkalies is found as chlorides, and the remainder, as well as the other bases, exists as phosphates. The quantitative composition of the pus-cells from the analyses of Hoppe-Setlee is as follows, in parts per 1000 of the dried substance : I. II. Proteids 137.62) Nuclein 342,57U85.85 673.69 Insoluble bodies 305.66) Lecithin I i^qqq 75.64 Fat \ ^^^-^^ 75.00 Cholesterin 74.00 73.83 Cerebrin 51.99 ) Extractive bodies 44.33 j MINEKAL SUBSTANCES IN 1000 PARTS OP THE DRIED SUBSTANCE. NaCl 4.35 Caa(PO,)a 2.05 Mg,(PO.), 1.13 FePO, 1.06 PO, 9.16 Na 0.68 K traces (?) ' Hoppe-Seyler's Med. chem. Untersuch., S. 441. = Zeitschr. f. physiol. Chem. , Bd. 4. 'Ibid., Bd. 17, S. 453. * Physiol. Chem., S. 790. 5 Deutsch. med. Wochenschr., 1877, No. 8. 200 CHYLE, LYMPH, TRANSUDATIONS AND EXUDATIONS. MlBSCHBE has obtained other results for the alkali combinations, namely : potassium phosphate 13, sodium phosphate 6.1, earthy phosphate and iron phosphate 4.3, sodium chloride 1.4, and phosphoric acid combined with organic substances 3.14-2.03 p. m. In pus from congested abscesses which have stagnated for some time we find peptone, leucin, and ty rosin, free fatty acids, and mlatile fatty acids, such as formic acid, butyric acid, valerianic acid. We also sometimes find cliondrin (?) and glutin (?), urea, glucose (in diabetes), tile-pigments and Ule-acids (in catarrhal icterus). As more specific but not constant constituents of the pus we must mention the following: pyin, which seems to be a nucleo- albumin or nucleoproteid precipitable by acetic acid, and also pyinic acid and chlorrhodinic acid, which have been so little studied that they cannot be more fully treated here. In many cases a blue, more rarely a green, color has been observed in the pus. This depends on the presence of a variety of vibrios (Lucke) from which Fordos ' and Luckb " have isolated a crystallizable coloring matter partly blue and partly yellow, pyocy- anin and pyoxanthose. Appendix. Lymphatic Glands, Spleen, etc. The Lymphatic Glands. The cells of the lymphatic glands are found to contain the protein substances occurring generally in cells (Chapter V, p. 90-91). Albumoses and peptones may also occur as products of a post-mortem decomposition. Besides the other ordinary tissue-constituents, such as collagen, reticulin, elastin, and nuclein, we find in the lymphatic glands also cholesterin, fat, glycogen, sarcolactic acid, xanthin bodies, and leucin. In the inguinal glands of an old woman Oidtmann* found 714.32 p. m. water, 384.5 p. m. organic and 1.16 p. m. inorganic substances. The Spleen. The pulp of the spleen cannot be freed from blood. The mass which is separated from the spleen capsule and the structural tissue by pressure and which ordinarily serves as material for chemical investigations is therefore a mixture of blood and spleen constituents. For this reason the albuminous bodies of ' Compt. rend., Tome 51 and 56. ' Arch. f. klin. Chirurg., Bd. 3. • V. Qorup-Besanez, Lehrbuch, 4, Aufl., S. 732. THE SPLEEN. 201 the spleen are little known. As characteristic constituents we have albuminates containing iron, and especially a protein substance which does not coagulate on boiling, and which Is precipitated by acetic acid and yields an ash containing much phosphoric acid and iron oxide.' The pulp of the spleen, when fresh, has an alkaline reaction, but quickly turns acids, due partly to the formation of free paralactic acid and partly perhaps to glycero-phosphoric acid. Besides these two acids there have been found in the spleen also volatile fatty adds, as formic, acetic, and butyric acids, as well as succinic acid, neutral fats, cholesterin, traces of leucin, inosit (in ox-spleen), .HcylUt, a body related to inosit (in the spleen of plagiostoma), glycogen (in dog-spleen), uric acid, xanthin bodies, and iecorin (Baldi'). Among the constituents of the spleen the deposit rich in iron, which consists of ferruginous granules or conglomerate masses of them, and closely studied by ISTasse, is of special interest. These iron grains produced by the transformation of the red corpuscles, and which also occur in old thrombi, are chiefly produced when stagnant blood-corpuscles are not dissolved, and they may be formed either extracellular or intracellular when the blood-corpuscles are taken up by the colorless cells. This deposit does not occur to the same extent in the spleen of all animals. It is found especially abundant in the spleen of the horse. Nasse'ou analyzing the grains (from the spleen of a horse) obtained 840-630 p. m. organic and 160-370 p. m. inorganic subsbances. These last consisted of 566-726 p. m. Fe,03, 205-388 p. m. P,0„, and 67 p. m. earths. The organic substances consisted chiefly of proteids (660-800 p. m.), nuclein, 52 p. m. (maximum), a yellow coloring matter, extractive bodies, fat, cholesterin, and lecithin. In regard to the mitieral constituents it is to be observed that the amount of iron in adults is strikingly large, and further that the amount of sodium and phosphoric acid is smaller than that of potassium and chlorine. The amount of iron in new-born and young animals is small (Lapicque,* Keuger, and Pernou"), in ' V. Gorup-Besanez, Lehrbuch, 4. Aufl., S. 717. * Du Bois-Eeymond's Arch., 1887, Suppl. » Maly's Jahresber., Bd. 19, S. 315. *lUd., 20, S. 368 » Zeitschr. f. Bio]ogie, Bd. 27. 202 CHTLE, LYMPH, TRANSUDATIONS AND EXUDATIONS. adults more appreciable, and in old animals sometimes very con- siderable. Nasse ' found nearly 50 p. m. iron in the dried pulp of the spleen of an old horse. The quantitative analyses of the human spleen by Oidtmank * give the following results: In men he found 750-694 p. m. water and 350-306 p. m. solids. In that of a woman he found 774.8 p. m. water and 225.2 p. m. solids. The quantity of inorganic bodies was in men 4.9-7.4 p. m., and in women 9.5 p. m. In regard to the pathological processes going on in the spleen we must specially recall the abundant re-formation of leucocytes in lencEemia and the appearance of amyloid substance (see page 57). The physiological functions of the spleen are little known with the exception of its importance in the formation of leucocytes. Some consider the spleen as an organ for the dissolution of the red blood-corpuscles, and the occurrence of the above-mentioned deposit rich in iron seems to confirm this view. Other investigators regard the spleen as a blood-forming organ. Several investigators claim the occurrence of nucleated preliminary steps in the formation of red corpuscles in the spleen or of younger red corpuscles in the blood of the splenic vein. The spleen has also been claimed to play an important part in digestion. The organ is known to enlarge after a meal, and this enlargement is thought by Schiff ° and Hekzen * to be connected with the filling of the pancreas with enzymes. According to the above-mentioned investigators, after the extirpation of the spleen the pancreas does not produce any enzyme which digests proteids, but Heidenhetm ' and Ewald " have not been able to confirm this fact. According to later investigations of Heezen,' an enzyme which digests proteids is produced in the spleen during its enlarge- ment. An increase in the quantity of uric acid eliminated has been observed by many investigators (see Chapter XV) in lineal leu- caemia, while the reverse has been observed under the influence of ' Cit. from Hoppe-Seyler's Physiol. Chera., S. 730. ' Cit. from v. Gorup-Besanez, Lehrbuch, 4. Aufl., S. 719. = Arch. f. Heillmnde, Bd. 3, Schweiz. Zeitschr. f. wiss. Med., 1862. * Pfliiger's Arch , Bd. 30, S. 295 and 308. ' L. Hermann's Handb. d. Physiol. , Bd. 5, S. 206. « Verhandl. d. physiol. Ges. in Berlin, 1878. 'Maly's Jahresber., Bd. 18, S. 193. THE THTMUa. 203 qninin in large doses, which produces an enlargement of the spleen. We have here a rather positive proof that there is a close relation- ship between the spleen and the formation of uric acid. This relationship has lately been studied by Hokbaczewski.' He has shown that when the spleen pulp and blood of calves is allowed to act on each other, under certain conditions and temperature, in the presence of air, large quantities of uric acid are formed. Under other conditions he obtained from the spleen pulp only xanthin bases with uo or very little uric acid. Hoebaczbwski has also shown that the uric acid originates from the nucleins of the spleen, which yield uric acid and xanthin bases according to the experi- mental conditions. The spleen has the same property as the liver of retaining foreign bodies, metals and metalloids. The Thymus. Besides proteids and substances belonging to the connective group, we find small quantities of fat, leucin, succinic acid, lactic acid, and glucose. The large quantity of xanthin bodies, chiefly adenin, is remarkable — 1.79 p. m. in the fresh gland, or 19.19 p. m. in the dried substance (KossfeL and Schikblbr"). LiLiEXFELD ' has found inosit and protagon in the cells of the thymus. The quantitative composition of the lymphocytes of the thymus of a calf is, according to Lilibjtfeld's * analysis, as fol- lows. The results are given in 1000 parts of the dried substance. Proteids 17.6 Leuconuclein 687.8 Histon ,86.7 Lecitlim 15.\ Fat 40.2 Cholesterin 44 Glycogen °-0 The dried substance of the leucocytes amounted to an average of 114.9 p. ra. Potassium and phosphoric acid are prominent mineral constituents. Lilienpeld found KH.PO, amongst the bodies soluble in alcohol. Oidtmaitn ' found 807.06 p. m. water, 193.74 p. m. organic and 0.3 p. m. inorganic substances in the gland of a child two weeks old. ' Monatshefte f. Chem., 1889, and Wien. Sitzungsber. 1891, Math. Naturw. Klasse, Abthl. 3. ' Zeitscbr. f . physiol. Chem. , Bd. 13. » IMd., Bd. 18, S. 473. 4L. c. • Cit. from v. Gorup-Besanez, Lehrbuch, 4. Aufl,, S. 733. 204 GHTLE, LYMPH, TRANSUDATIONS AND EXUDATIONS. The Thyroid Gland. The chemical constitueats of this gland are little known. Bubnow' has obtained a protein substance called by him " thyreoproteine,'" by extracting the gland with common-salt solution or by very dilute caustic potash. This body has about the same amount of nitrogen, but smaller amounts of carbon and hydrogen than, the proteids in general. The fluid found in the vesicle sometimes contains a mucin-lihe substance which is precipitated by an excess of acetic acid. Gotjklat ' could not find any mucin but only a nucleoalbumin in the thyroid gland of oxen. Besides these, other substances have been found in the extract of the glands,, such as leucin, xanthin, Jiypoxanthin, lactic and succinic acids. OiDTMANiS' ' found in the thyroid gland of an old woman 832.4 p. m. water, 176.7 p. m. organic and 0.9 p. m. inorganic substances. He found 772.1 p. m. water, 223.4 p. m. organic and 4.5 p. m. inorganic substances in an infant two weeks old. In " STKUMA CYSTICA " Hoppb-Setlee found hardly any pro- teid in the smaller glandular vessels, but an excess of mucin, while in the larger he found a great deal of proteid, 70-80 p. m.* Gholesterin is regularly 'found in such cysts, sometimes in such large quantities that the entire contents form a thick mass of cholesterin plates. Crystals of calcium oxalate also occur frequently. The contents of the struma cysts are sometimes of a brown color due to decomposed coloring matter, methcemogloMn (and hgematin?). Bile- coloring matters have also been found in such cysts. (In regard to the paralbumins and colloids which have been found in struma cysts and colloid degeneration, see Chapter XIII.) Little is known in regard to the functions of the thyroid gland. From a chemical standpoint the view is worth suggesting tbat the so-called myxoedema, which is a slimy infiltration or abundant extuberance of the connective tissue of the subcutaneous cell-tissue especially of the head and throat (besides other disturbances) stands in connection with the failing of the activity of the thyroid gland. HoRSLET and Halliburton' found in monkeys, but not in pigs, that the amount of mucin in the tissue was increased after extirpat- ing the thyroid gland. ' Zeitsohr. f. physiol. Chem., Bd. 8. ' Journal of Physiol. , Vol. 16. » Cit. from v. Gorup-Besanez, Lehrbuch, S. 733. « Hoppe-Sevler, Physiol. Chem., S. 731. « Brit. Med, Journ., 1885 ; also Maly's Jahresber., Bd. 18, S. 334. THE SUPRARENAL CAPSULE. 205 We have no explanation as to the action of the gland in these cases. In consideration of the very favorable therapeutical results which have been obtained in many cases of myxoedema by the injection of a watery or glycerin extract of the gland or the admin- istration of the gland of sheep, it seems probable that myxoedema is caused by an intoxication produced by metabolic products, which are otherwise destroyed or made harmless by the gland. The Suprarenal Capsule. — Besides proteids, substances of the connective tissue, and salts, we find in the suprarenal capsule inosit, palmitin, lecithin, neurin, and glycero-phosphoric acid, which last gives the poisonous properties of the watery extract of the gland (Maeino-Zuco and Guarnieei '), and some leucin, which is probably a decomposition product. The statement that benzoic acid, Tiippuric acid, and biliary acids occur in this gland could not be confirmed by Stadelmakn.' In the medulla there have been found one or more chromogens which are converted into a red pigment by the action of air, light, warmth, haloid or metallic salts (VuLPiAN, Kbijkenbbeg^). Pyrocatechin also probably occurs therein. Because of the amount of chromogen contained in the suprarenal body, a connection is claimed between the abnormal deposition of pigment in the skin, which is characteristic of Addi- son's disease, and the diseased changes which often occur in the suprarenal body. Nothing positive is known as to the functions of the suprarenal capsule. The extirpation of the suprarenal capsule of a dog is always a fatal operation (Langlois). Death is hastened by the injection of blood from an animal killed by this operation, while the blood from a healthy animal has no action. Perhaps we have here also to deal with an intoxication produced by metabolic products, which are made harmless or destroyed by the suprarenal capsules under normal conditions. The investigations of Abelous and Langlois and others seem to confirm this view 1 Maly's Jahresber., Bd. 18, S. 231. » Zeitschr. f. physiol. Chem., Bd. 18. * Vircliow's Arcli. , Bd. 101. CHAPTER VIII. THE LIVER. The liver, which is the largest organ of the body, stands in close relationship to the blood-forming organs. The importance of this organ in the physiological composition of the blood is evident from the fact that the blood coming from the digestive tract, laden ivith absorbed bodies, must circulate through the liver before it is driven by the heart through the different organs aad tissues.. It has been proved, at least for the carbohydrates, that an assimilation of the absorbed nutritive bodies which are brought to the liver by the blood of the portal vein takes place in this organ. The occurrence of synthetical processes in the liver has been positively proved by special observations. It is possible that in the liver certain am- monia combinations are converted into urea or uric acid (in birds), while certain products of putrefaction in the intestine, such as phenol, may be converted by synthesis into ethereal sulphuric acids by the liver (Pflugeb and Kochs '). The liver has also the prop- erty of removing and retaining heterogeneous bodies from the blood, and this is not only true of metallic salts, which are often retained by this organ, but also, as Schiff, Lautenbbrgee, Jacques, Hegee, and Rogee" have shown, the alkaloids are retained and are probably partjally decomposed in the liver. Toxins are also retained by ,the liver and hence this organ has a protective action against poisons. Even though the liver is of assimilatory importance and purifies the blood coming from the digestive tract, it is at the same time a secretory organ which eliminates a specific secretion, the bile, in the 1 Pfliiger's Arch. , Bd. 30 and Bd. 23, S. 169. « Roger, Action du foie sur les poisons (Paris, 1887) ; Bouchard. Lefons sur les autointoxications dans les Maiadies (Paris, 1887); and E. Kotliar in Arch, des sciences biologique de St. PetersbouTg, Tome 3, No. 4, p. 587. 306 THM LIVER 8UBBTANCE. 20-7 production of which the red blood-corpuscles are destroyed, or at least one of their constituents, the haBmoglobin. It is generally admitted that the liver acts contrariwise during foatal life, at that time forming the red blood-corpuscles. There is no doubt that the chemical operations going on in this organ are manifold and must be of the greatest importance for the organism; but unfortunately we know very little about the kind and extent of these processes. Among them are two principal ones which will be fully treated in this chapter, after we have first described the constituents and the chemical composition of the liver. One of these processes seems to be of an assimilatory nature and refer to the formation of glycogen, while the other refers to the production and secretion of the bile. The reaction of the liver-cell is alkaline during life, but becomes acid after death. This change is probably due to the formation of lactic acid, causing a coagulation of the proteids of the protoplasm of the cell. A positive difference between the albu- minous bodies of the dead and the living, non-coagulated proto- plasm has not been observed. The proteids of the liver were first carefully investigated by Plosz.' He found in the watery extract of the liver an albuminous substance which coagulates at -|- 45° C, also a globulin which coagulates at + 75° C, a nucleoalbumin which coagulates at H- 70° C, and lastly a proteid body which is nearly related to coagulated albumins and which is insoluble in dilute acids or alkalies at the ordinary temperature, but dissolves on the applica- tion of heat, being converted into an albuminate. HALLiBUETOiir ' has found two globulins in the liver-cells, one of which coagulates at 68-70° C, and the other at 45-50° C. He also found, besides traces of albumin, a nucleoalbumin (nucleoproteid) which contained 1.45^ phosphorus and a coagulation-point of 60° C. The liver- cells contain, besides these proteids, a large quantity of difficultly soluble protein bodies (see Plosz). St. Zaleski ' has found in the liver a proteid containing iron, in which the iron is more or less strongly combined. It is unknown what relation this bears to the above-mentioned proteids. The fat of the liver occurs partly as very small globules and > Pflliger's Arch., Bd. 7. ' Journal of Physiol., Vol. 13, Sappl. 1893. » Zeitschr. f. physiol. Chem., Bd. 10, S. 486. 208 THE LIVER. partly, especially in nursing children and sucking animals, as also after food rich in fat, as rather large fat-drops. This infiltration of fat, which may be made so abundant by proper food that it appears similar in the highest degree to a pathological fatty liver, begins in the periphery of the acini and extends towards the centre. If the amount of fat in the liver is increased by an infiltration, the water decreases correspondingly, while the quantity of the other solids remains little changed. In fatty degeneration this is differ- ent. In this process the fat is formed from the protoplasm of the cell, and the quantity of the other solids is therefore diminished while the amount of water is only slightly changed. To illustrate this, we give below the results from a normal liver, and also the results obtained by Peels ' in fatty degeneration and fatty infiltra- tion. The results are in 1000 parts. Water. Fat. Remaining Solids. Normal liver 770 20-35 307-195 Fatty degeneration 816 87 97 Fatty infiltration 616-621 195-340 184-145 Among the extractive substances besides glycogen, which will be treated of later, we find rather large quantities of xanthin lases. Kosse-l" found in 1000 parts of the dried substance 1.97 p. m. guanin, 1.34 p. m. hypoxanthin, and 1.21 p. m. xanthin. Adenin is also contained in the liver. In addition there have been found urea and uric acid (especially in birds), and indeed in larger quan- tities than in the blood, paralactic acid, leucin, jecorin, and cystin. In pathological cases inosit and tyrosin have been detepted. The occurrence of bile-coloring matters in the liver-cell under normal conditions is doubtful; but in retention of the bile the cells may absorb the coloring matter and become colored thereby. Jecorin was first found by Drechsbl' in the liver of a horse, and later by Baldi* in the liver and spleen of other animals, in the muscles and blood of the horse, and in the human brain. It contains sulphur and phosphorus, but its constitution is not positively knovpn. Jecorin dissolves in ether, but is precip- itated from this solution by alcohol. It reduces copper oxide, and it solidifies after boiling with alkalies to a gelatinous mass. It may lead to errors in the investigations of organs or tissues, for it can easily be mistaken for lecithin on account of its solubilities and because it contains phosphorus. The mineral bodies of the liver consist of phosphoric acid, potassium, sodium, alkaline earths, and chlorine. The potassium > Centralbl. f. d. med. "Wissensch., Bd. 11, S. 801. = Zeitschr. f . physiol. Chem., Bd. 8, S. 408. ' Ber. d. sSchs. Ges. d. Wissensch., 1886, S. 44. * Du Bois-Reymond's Arch., Physiol. Abth., 1887. Suppl. S. 100. IRON IN TEE LIVER-CELLS. 209 is in excess of ths sodium. Iron is a regular constituent of the liver, but in very variable amounts, 0.3-11.8 p. m. calculated for the dried substance of the liver of different animals (St. Zaleski '). Bunge' has found 0.01-0.355 p. m. iron in the blood-free liver of young cats and dogs. This was calculated on the liver substance freshly washed with a 1^ NaCl solution. Calculated on 10 kilos bodily weight, the iron in the livers amounted to 3.4-80.1 mgm. The richness of the liver of new-born animals in iron is of special interest; a condition which follows from the analyses of St. Zaleski, but especially studied by Krugbr, Meyer, and Pernou.' In oxen and cows they found 0.246-0.376 p. m. iron (calculated on the dry substance), and in the cow fcetus about ten times as much. The liver-cells of a calf a week old contain about seven times as much iron as the full-grown animal; the quantity sinks in the first four weeks of life, when it about reaches the same amount as in the grown animal. Lapicque ' has also found that in rabbits the quantity of iron in the liver steadily diminishes from the eighth day to three months after birth, namely, from 10 to 0.4 p. m., calculated on the dry substance. " The foetal liver-cells bring an abundance of iron into the world to be used up, within a certain time, for a purpose not well known." A part of the iron exists as phosphate, and the greater part in combination with the protein bodies (St. Zaleski). P. KRUGER'has determined the quantity of calcium in the liver-cells of oxen in various stages of development, and has found that the average quantity was only 0.71 p. m. of the dried substance in full-grown oxen and 1.23 p. m. in calves. In the fcetus of the cow it is lower than in calves, but it shows two maxima during foetal life, one in the first to the fifth month, and the other in the tenth month, of pregnancy. At these times the liver-cells contain about 45^ more calcium than in full-grown oxen. During pregnancy the iron and calcium are antagonistic; namely, an increase in the quantity of calcium causes a diminution in the iron, and an increase in the iron causes a decrease in the calcium. Kkugbb found 23.8 p. m. sulphur, 12.8 p. m. phosphorus, and 0.55 p. m. iron in the liver-cells of adult persons and 35.6 p. m. ' L. c, S. 464-479. ^ Zeitsclir. f. physiol. Chem., Bd. 17, S. 78. ' Zeitschr. f. Biologie, Bd. 37, S. 439. * Maly's Jahresber., Bd. 30, S. 368. ' Zeitschr. f. Biologie, Bd. 31. 210 TEE LIVER. snlpliur, 15.4 p. m. phosphorus, and 3.14 p. m. iron in those of new-born infants. Copper seems to be a physiological constituent. Foreign metals, such as lead, zinc, and others (also iron), are easily taken up and retained for a long time by the liver. y. BiBKA ' found in the liver of a young man who had suddenly died, 763 p. m. water and 238 p. m. solids, consisting of 35 p. m'. fat, 152 p. m. proteid and gela bin-forming substance, and 61 p. m. extractive substances. Glycogen and its Formation. Glycogen was discovered by Been aed and Hbnsen ' independ- ently of each other. It is a carbohydrate closely related to the starches or dextrins with the general formula C^H^Oj, perhaps 6(CjH,„0,) + HjO (KuLZ and Boekteagee '). The largest quan- tities are found in the liver of full-grown animals, and smaller quantities in the muscles (Beenaed, Nasse *). It is found in very small quantities in nearly all tissues of the animal body. Its occur- rence in lymphoid cells, blood, and pus has been mentioned in . previous chapter, and it seems to be a regular constituent of all cells capable of development. Glycogen was first shown to exist in embryonic tissues by Beenard and Kuhne,' and it seems on the whole to be a constituent of such tissues in which a rapid cell- formation and cell -development is taking place. It is also present in rapidly forming pathological swellings (Hoppe-Sbyleb °). Cer- tain animals, as certain muscles, are very rich in glycogen (Bizio '). Glycogen also occurs in the plant kingdom, especially in many fungi. The quantity of glycogen in the liver, as also in the muscles, depends essentially upon the food. In starvation it disappears nearly completely after a short time, but more rapidly in small than in large animals. According to the old views it disappears earlier from the muscles than from the liver. According to the later ' See V. Gorup-Besanez, Lehrbuch, 4. Aufl., S. 711. = CI. Bernard, Comp. rend., Tome 44, p. 578; and Hensen, Virchow's'Arcli Bd. 11, S. 395. 3 Pfluger's Arch., Bd. 24, S. 19. * Ibid., Bd. 2, S. 97. ' See Kilhne, Lehrb. d. physiol. Chem., 1868, S. 307. « Pflilger'a Arch., Bd. 7, S. 409. ^ Comp. rend.. Tome 62, p. 675. QLTCOOEN. 211 determinations of Aldehoff ' on hens, pigeons, rabbits, cats, and horses, which have been confirmed by Kulz and Heegenhahk ' and others, the muscle glycogen has a greater resistance to destruc- tion than liver glycogen. After partaking of food especially abun- dant in carbohydrates, the liver becomes rich again in glycogen, the greatest increment occurring 14 to 16 hours after eating (Kulz '). Hekgenhahn found on experiments with hens that the appearance of the maximum of glycogen in the liver was also dependent upon the quantity of carbohydrates partaken of. The maximum of liver glycogen was reached on supplying 10 gms. cane-sugar in 13 hours, and after 30 gms. in 20 hours. The maximum of muscle glycogen is reached after 20-34 hours, independently of the quantity of cane- sugar supplied. The quantity of liver glycogen may amount to 130-160 p. m. after partaking of large quantities of carbohydrates. Ordinarily it is considerably less, namely, 13-30 to 40 p. m. The quantity of glycogen of the, liver (and also the muscles) is also dependent upon rest and activity, because during activity the quantity diminishes. Kulz * has shown that by hard work the quantity of glycogen in the liver (of dogs) is reduced to a minimum in a few hours. The muscle glycogen does not diminish to the same extent as the liver glycogen. Kulz was able to completely ■consume the liver as well as the muscle glycogen of a rabbit in 3-5 hours by qualified strychnin poisoning. Glycogen forms an amorphous, white, tasteless, and inodorous powder. It, gives an opalescent solution with water Vhich, when allowed to evaporate in the water-bath, forms a pellicle over the surface that disappears again on cooling. The solution is dextro- gyrate, (or) D = + 196''.63 (Huppbkt'). The specific rotatory power is given somewhat difEerently by various investigators. A solution of glycogen, especially on the addition of NaOl, is colored wine-red by iodine. It may hold copper oxy hydrate in solution in alkaline liquids, but does not reduce it. A solution of glycogen in water is not precipitated by potassium-mercuric iodide and ' Zeitschr. f. Biologie, Bd. 35, S. 137. Contains a summary of the literature. ' Ibid., Bd. 37, S. 314. * PflUger's Arch., Bd. 34, S. 1-114. This important article contains numer- ous data in regard to the literature of the glycogen question. * PflUger's Arch., Bd. 34, and " BeitrSge zur Kenntniss des Glykogens." C. Ludwig's Festschrift Marburg, 1891. « Zeitschr. f. physiol. Chem., Bd. 18, S. 137. 212 THE LIVER. hydrochloric acid, but is precipitated by alcohol (on the addition of NaCl when necessary) or ammoniacal lead acetate. It gives a- white granular precipitate of benzoyl glycogen with benzoyl chlo- ride and canstic soda. Glycogen is not decomposed on prolonged boiling with dilute caustic potash, but it seems to be changed slightly (ViNTSCHGAU and Dietl'). By diastatic enzymes glyco- gen is converted into maltose or dextrose, depending upon the nature of the enzyme. It is transformed into dextrose by diluta mineral acids. The preparation of pure glycogen (simplest from the liver) ia generally performed by the method suggested by Beucke, of which the main points are the following: Immediately after the death of the animal the liver is thrown into boiling water, then finely divided and boiled several times with fresh water. The filtered extract is- now sufficiently concentrated, allowed to cool, and the proteida- removed by alternately adding potassium -mercuric iodide and hydrochloric acid. The glycogen is precipitated from the filtered liquid by the addition of alcohol until the liquid contains 60 vols, per cent. The glycogen is first washed on the filter with QO^ and then with 95^ alcohol, then treated with ether and dried over sul- phuric acid. It is always contaminated with mineral substances. To be able to extract the glycogen from the liver or especially from muscles and other tissues completely, which is essential in a quan- titative estimation, these parts must first be boiled ;for a few hours with a dilute solution of caustic potash, say 4 gms. KOH to 100 gms. liver and 400 c.c. water (Kulz). Proteid-free glycogen may be prepared according to the method suggested by Huizinga,' in which the liver tissue is extracted with a mixture of equal volumes of a saturated mercuric-chloride solution and Bsbaoh's reagent (10 gm. picric acid and 30 gms. citric acid in a liter). The glycogen is precipitated by alcohol and treated with alcohol and ether. The quantitative estimation is best performed according to the described method of Bbucke-Kulz.= It is to be observed that it is necessary to heat the liver for 2-3 hours and muscle 4-8 hours with caustic-potash solution. This liquid must not be concentrated too far, and must not contain more than 2^ caustic potash. It is neutralized by hydrochloric acid and precipitated by the alternate addition of potassium-mercuric iodide and hydrochloric acid. The precipitate must be removed from the filter at least four times, sus- pended in water with the addition of a few drops HOI and potas- sium-mercuric iodide, and refiltered so that all the glycogen is ' Pfliiger's Arch., Bd. 13, S. 353. ' Pfluger's Arch., Bd. 61. ' See R. Kulz, Zeitschr. f. Biologie, Bd. 32, S. 161. PREPARATION OF OLYCOGEN. 213 obtained in the filtrates. These are then precipitated with double their volume of alcohol, filtered after 13 hours, the precipitate dissolved in a little warm water, treated on cooling with HCl and potassium-mercuric iodide, filtered, and the filtrate again precipi- tated with alcohol. Filter and carefully wash the contents of the filter with alcohol and ether, dry, weigh, and incinerate to deter- mine the quantity of ash present. It sometimes happens that the liquid, after complete precipita- tion of the proteids with HCI and potassium-mercuric iodide, is cloudy and does not filter clear. In this, case add 2-3^ vols. 95^ alcohol according to Pflugek's' suggestion. After the liquid becomes clear and the precipitate has settled it can be filtered. The precipitate is dissolved in a 2^ caustic-potash solution and again precipitated by hydrochloric acid and potassium-mercuric iodide. Then proceed as above described. The new method as suggested by Fbankel," in which the glycogen is extracted from the tissues by a 3-4^ water solution of trichloracetic acid, seems not to be reliable, according to Wbidbn- BAUM.' Numerous investigators have endeavored to determine the origin of glycogen in the animal body. It is positively established by the unanimous observations of many investigators * that the varieties of sugars and their anhydrides, dextrins and starches, have the prop- erty of increasing the quantity of glycogen in the body. The state- ments are somewhat disputed in regard to the action of the pentoses. Ckemee' found that various pentoses such as rhaminose, xylose, and arabinose have a positive influence on the glycogen formation in rabbits and hens, and Salkowski ' obtained the same result on feeding rabbits and a hen on arabinose. Fbentzbl ' found, on the contrary, no glycogen formation on feeding xylose to a rabbit which •had previously been made glycogen-free by strychnin poisoning. The hexoses, and the carbohydrates derived therefrom, do not all possess the ability of forming or accumulating glycogen to the same extent. Thus 0. Voit ' and his pupils have shown that ' Pflilger's Arch,, Bdd. 53 and 55. « Ibid., Bdd. 52 and 55. '/Wrf., Bdd. 54 and 55. * In reference to the literature on this subject see E. Killz, Pflilger's Arch., 3d. 24, and Ladwig-Festschrift, 1891 ; WolfEberg, Zeitschr. f. Biologie, Bd. 12, and C. Voit, ibid. , Bd. 28, S. 245. ' Zeitschr. f. Biologie, Bd. 29. » Centralbl. f. d. med. Wissensch., 1893, No. 11. ■" Pflager's Arch., Bd. 56. « Zeitschr. £. Biologie, Bd. 28. 214 THE LIVER. dextrose has a more powerful action than cane-sugar, while milk- sugar acts disproportionately less (in rabbits and hens) than dex- trose, leevulose, cane-sugar, and maltose. The following substances when introduced into the body also increase the quantity of glycogen in the liver : glycerin, gelatin, arlutin, and also, according to the investigations of Kulz,' erytJirit, quercit, dulcit, mannit, inosit, allyl and crotyl alcohols,' gly cur onic anhydride, saccharic acid, mucic acid, sodium tartrate, saccharin, isosaccharin, and urea. Ammonium carbonate, glycocoll, and asparagin may also, according to RoHMANiir,'' cause an increase in the amount of glycogen in the liver. According to Nebblthau ' other ammonium salts and cer- tain amides, also certain narcotics, hypnotics, and antipyretics, produce an increase in the glycogen of the liver. This action of the antipyretics (especially antipyrin) had been shown by Lepine. and POETEEBT." The fats, notwithstanding the above-mentioned action of glycer- in, have no action on the quantity of glycogen in the liver, accord- ing to the statements of most investigators. The views in regard to the action of proteids have been very contradictory in the past. It is undoubtedly settled from many observations that the proteids also increase the liver glycogen. Amongst these observations we must include certain feeding experiments with boiled beef (NAUnrxsr) or blood fibrin (v. Mering), and especially the very careful experiments made by E. Kulz * on hens with pure proteids such as casein, seralbumin, and ovalbumin. Woleebbeg ' has also shown that a more abundant accumulation of glycogen takes place after feeding with proteids and carbohydrates in proper proper tions- than with carbohydrate food with only a little proteid. MiUKA ' has made experiments to demonstrate the role of the innlin as a glycogen-former in starving rabbits. In certain cases the quantity of glycogen was increased, in others, on the coatrary, not afEected. The inconstancy of the results of these tests may be dependent upon the fact that the inulin introduced was only partly ' E. Kulz, Ludwig's Festschrift, 1891. °- Pfluger's Arch., Bd. 39. 5 Zeitschr. f . Biologie, Bd. 28, S. 138. " Comp. rend., Tome 106, p. 1033. ' Cit. Ludwig's Festschrift. The complete literature in regard to the gly- cogen formation from proteids will he found here. 8 Zeitschr. f. Biologie., Bd. 16, S. 266. ^ Ibid.. Bd. 32. FORMATIOX OF OLYGOGEN. 215 or only slowly transformed into laevulose, and hence the absorbed sngar could not always cause an accumulation of the glycogen. MiUEA also mentions the results of the older experiments and also gives the older literature. If we raise the question as to the action of the various bodies in the accumulation of glycogen in the liver we must call to mind that a reformation of glycogen takes place in this organ, and also a con- sumption of the same.' An accumulation of glycogen may be caused by an increased formation of glycogen, but also by a dimin- ished consumption, or by both. We do not known how all the above-mentioned various bodies act in this regard. Certain of them probably have a retarding action on the transformation of glycogen in the liver, while others perhaps are more combustible and in this way protect the glycogen. Some probably excite the liver-cells to a more active glycogen formation, while others yield material from which the glycogen is formed and are glycogen-forniers in the true sense of the word. TIjb knowledge of these last-mentioned bodies is of the greatest importance in the question as to the origin of glycogen in the animal body, and the chief interest attaches itself to the question, to what extent are the two chief groups of food, the proteids and carbohydrates, glycogen- f ormers ? The great importance of the carbohydrates in the formation of glycogen has given rise to the opinion that the glycogen in the liver is produced from other carbohydrates (glucose) by a synthesis in which water separates with the formation of an anhydride (Luchsixger and others). This theory {anhydride theory) has found opponents because it neither explains the formation of glycogen from such bodies as proteids, carbohydrates, gelatin, and others, nor the cir- cumstance that the glycogen is always the same independent of the properties of the carbohydrate introduced, whether it is dextro- or Iffivo-gyrate. It is therefore the opinion of many investigators that all glycogen is formed from proteid, and that this splits into two parts, one containing nitrogen and the other free from nitrogen : the latter is the glycogen. According to these views, the carbo- hydrates act only in that they spare the proteid and the glycogen produced therefrom {sparing theory of Weiss, Wolffbekg, and others"). ' See Wolffberg, 1. c. ' See Wolffberg, 1. c, in regard to these two theories. 216 THE LIVER. In opposition to this view E. Voit,* by feeding experiments with rice, which is poor in nitrogen, and 0. VoiT ' and his pupils, by tests with dextrose, Isevalose, maltose, and cane-sugar, have shown that the quantity of glycogen stored up in the body, after partaking of large amounts of carbohydrates, is sometimes so large that it cannot be covered by the proteids decomposed during the same time. In these cases we must admit of glycogen formation from sugars. The investigations of C. VoiT show that dextrose directly or Isvulose either directly or after previous conversion into dextrose passes into glycogen in the liver. Maltose and cane-sugar must first probably be transformed into dextrose or invert-sugar in the intestinal tract. Milk-SLigar and galactose seem, according to Kausoh and SociK," although contrary to the observations of Voit, to form glycogen directly if the absorption in the intestine is sufficiently abundant. There is no doubt that feeding with pure proteids leads to an accumulation of glycogen, and at the present time we must admit that glycogen can be formed from proteids as well as from carbo- hydrates. The manner in which glycogen is formed from proteids is not known. The view held by certain investigators that carbohydrates split ofE directly from the genuine proteids has not sufficient basis, and therefore the glycogen formation is often explained according to Pflugbe,* by a synthesis from the proteids after a complex cleavage. Like the carbohydrates in general, so has glycogen without any doubt a great importance in the formation of heat and development of energy in the animal body. The possibility of the formation of fat from glycogen must not be denied. Glycogen is generally con- sidered as accumulated reserve food in the liver and formed in the liver-cells. Where does the glycogen existing in the other organs, such as the muscles, originate ? Is the glycogen of the muscles formed on the spot or is it transmitted to the muscles by the blood ? These questions cannot yet be answered with positiveness, and the investigations on this subject by diilerent experimenters' have given contradictory results. The later experiments of KtJLZ,' in ' Zeitsclir. f. Biologie, Bd. 35, S. 543. ^Ibid., Bd. 38. ' Arcli. f. exp. Path. u. Pharm., Bd. 31. ^ Pfliiger's Arch., Bd. 42. * See Minkowski and Lave?, Arch. f. exp. Path. u. Pharm., Bd. 33. * Zeitsohr. f. Biologie, Bd. 27. FORMATION OF SUGAR IN THE LIVER. 2l7 which he studied the glycogen formation by passing blood contain- ing cane-sugar through the muscle, has led to no conclusive results. If we consider that the blood and lymph contain a diastatic enzyme which transforms glycogen into dextrose, and also that the glycogen regularly occurs in the form-elements and is not dissolved in the fluids, it seems probable that the glycogen is not transmitted by the blood to the organs in solution, but perhaps more likely, if the leucocytes do not act as carriers, is formed on the spot from the dextrose. The glycogen formation seems to be a general function of the cells. In adults the liver, which is very rich in cells, has the property, on account of its anatomical position, of transforming large quantities of dextrose into glycogen. The question now arises whether there is any foundation for the statement that the liver glycogen is transformed into dextrose. As first shown by Bernard and repeated by many investigators, the glycogen in a dead liver is gradually changed into dextrose, and this sugar formation is caused, as Bernard supposed and Arthus and Htjber ' proved, by a diastatic enzyme. This post-mortem sugar formation led Bernard to the assumption of the formation of sugar from glycogen in the liver during life. Bernard ' sug- gested the following arguments for this theory: The liver always contains some sugar under physiological conditions, and the blood from the hepatic vein is always somewhat richer in sugar than the blood from the portal vein. The correctness of either or both of these statements has been disputed by many investigators. Payt, EiTTER, ScHiFF, EuLENBBRG, LussANA, Abelbs, and others deny the occurrence of dextrose in the liver during life, and also the greater amount of dextrose in the blood from the hepatic vein is disclaimed by them and certain other investigators. A few inves- tigators claim that a greater amount of sugar may occur in the hepatic vein under certain circumstances, and they consider in these cases that it is caused by the operation. The doctrine as to the physiological formation of sugar in the liver has obtained an energetic advocate in Sebgen." He main- tains, after numerous experiments, that the liver regularly contains ' Arch, de phvsiol., (5) Tome 4. » In regard to" the literature on sugar formation in the liver see Bernard, Lefons sur le diabfete. Paris, 1877. Seegen, Die Zuckerbildung im Thierkorper. Berlin, 1890. M. Bial, Pflilger's Arch., Bd. 55, S. 434. 2 See Seegen, Die Zuckerbildung im ThierkOrper. Berlin, 1890. 218 THE LIVES. considerable amounts of sugar. He has observed an increase of 3j^ in the quantity of dextrose in the liver of a dog kept alive by pass- ing arterial blood through the organ, and lastly he has also found in a very great number of experiments on dogs that the blood from the hepatic vein always contains more — even double as much — sugar than the blood from the portal vein. Although Seegek energetically espouses the doctrine of Bbb- HAED as to the vital sugar formation in the liver, still it deviates essentially from Beenakd in that he claims the dextrose is not derived from the glycogen. According to Seegen the sugar is formed from peptones and fat. The observations on which he bases this view seem hardly to be correct," according to the control experiments made by many investigators. The statement of Lepine " as to the occurrence of an enzyme in the blood which has the property of transforming peptone into sugar could not be sub- stantiated by BiAL. The circumstance that the blood-sugar rapidly sinks to ^^ of its original quantity, or even disappears when the liver is cut out of the circulation, speaks for a vital formation of sugar in the liver (Seegen, Bock, and HoFFMAN^isr''). In geese whose livers were removed from the circulation Minkowski' found no sugar in the blood after a few hours. We will also learn shortly of certain poisons and operative changes which may cause an abundant elimi- nation of sugar, but only when the liver contains glycogen. If we recall the fact shown by Eohmann and Bial ' that the lymph as well as the blood contains a diastatic enzyme, then several reasons speak for the view of Beenakd that the post-mortem formation of sugar from the glycogen in the liver is a continuation of the vital process. Although it is unanimous that the post-mortem sugar formation is produced by a diastatic enzyme, still several investiga- tors, such as Dastee and NoEL-PATOiir,' are of the view that sugar formation is not caused in life by an enzyme, but by a vital process of the cell protoplasm. ' A compilation of these control experiments may be found in Bial, Pflu- ger's Arcli. , Bd. 55. ^ Compt. rend., Tome 115 and 116. 3 See Seegen, 1. c, pp. 183-184. * Arch. f. exp. Path. u.'Pharm., Bd. 21. ' See pp. 124 and 181, this hook. « See Noel-Paton, On Hepatic Glycogenesi^. Phil. Trans, of the Roy. Soc. London, vol. 185, B. 1894. <C08URIA. 219 The relationship of the sugar eliminated in the urine under certain conditions, such as in diabetes mellitus, certain intoxications, lesions of the nervous system, etc., to the glycogen of the liver il also an important question. It does not enter into the plan and scope of this book to enter into detail into the various views in regard to glycosuria and diabetes. The appearance of dextrose in the urine is a symptom which may have essentially different causes, depending upon dif- ferent circumstances. Only a few of the most important points will be mentioned. The blood contains always about an average of 1.5 p. m., while the urine at most contains only traces. When the quantity of sugar in the blood rises to 3 p. m. or above, then sugar passes into the urine. The kidneys have the property to a certain extent of preventing the passage of blood-sugar into the urine; and it follows from this that an elimination of sugar in the urine may be caused partly by a reduction or suppression of this above-mentioned activity and partly also by an abnormal increase of the quantity of sugar in the blood. The first seems, according to v. Mbeistg and Minkowski, to be the case in phlorhizin ' diabetes, v. MEEiife has found that a strong glycosuria appears in man and animals on the administration of the glucoside phlorhizin, and that the quantity of sugar in the blood is not increased, but somewhat diminished. In this form of diabetes we have, according to Minkowski, abnormal processes in the kidneys. According to Levbne ' phlorhizin diabetes is not produced by an increased elimination of sugar by the kidneys, but more likely an increased formation of sugar in these organs. He found generally more sugar in the venous blood of the kidneys than in the arterial blood, and he also found considerably more sugar after injection of phlorhizin than under normal conditions. He agrees with the observations of other investigators such as Peaus- NiTZ, Cebmeb, and Rittbe, that in phlorhizin diabetes the sugar is formed from the protein substances. All other forms of glycu- ' In regard to the literature on phlorhizin diabetes see : v. Mering, Zeitschr. f. kiln. Med., Bdd. 14 and 16; Minkowski, Berl. klin. Wochenschr., 1893, No. 5, and Arch. f. exp. Path. u. Pharm., Bd. 31; Moritz and Prausnitz, ZeitBchr. f. Biologie., Bdd. 27 and 29; Kdlz and Wright, ibid.,.BA. 27, S. 181; Cremerand Ritter, ibid., Bdd. 28 and 39. » Journal of Physiol., Vol. 17. 220 THE LIVER. soria or diabetes depend, on the contrary, as far as known, to an increased quantity of sugar in the blood, namely, a hyperglucmmia. A hyperglncaemia may be caused in various ways. It may be caused, for example, by the introduction of more sugar than the body can destroy. The property of the animal body to assimilate the different varieties of sugar has naturally a limit. If too much sugar is intro- duced into the intestinal tract at one time, so that the so-called assimilation limit (see Chapter XIX on absorption) is overreached, then the excess of absorbed sugar passes into the urine. This form of glycosuria is called alimentary glycosuria,^ and it is caused by the passage of more sugar into the blood than the liver and other organs can destroy. As the liver cannot transform all the sugar into glycogen which comes to it in alimentary glycosuria, it is possible that a glycosuria may be brought about by the activity of the liver to transform sugar into glycogen being changed or reduced by disease. It is difficult to state in how far such a glycosuria occurs, but according to Seegekt the lighter forms of diabetes are produced in this way. We difEerentiate between light and severe forms of diabetes. In the first the urine only contains sugar when carbohydrates are taken as food, while in the other case the urine contains sugar even with food entirely free from carbohydrates. According to the view of Sbegen," in light forms of diabetes the liver is incapable of transforming all the carbohydrates introduced into glycogen, or to utilize this in a proper way, and the activity of the liver-cells is also reduced or changed in these cases. This view is nevertheless hardly based on sufficient proof. A hyperglucsemia, which passes into a glycosuria, may also be brought about by an excessive formation of sugar from the glycogen within the animal body. The so-called piqure, and also probably those glycosurias which occur after other lesions of the nervous system, belong to the above group of glycosurias. The glycosuria produced on poisoning with carbon monoxide, curare, strychnin, morphin, etc., also belong to this group. That the glycosuria produced in these cases is due to an increased transformation of the glycogen follows from the fact that no glycosuria appears, under the above-mentioned circum- ' See Moritz, Arcb. f. klin. Med., Bd. 46, 1890. ' Die Zuckerbildune, etc. Lecture 15. DIABETES. 221 stances, when the liver has been previoasly made free from glycogen by starvation or other means.' A hyperglucffimia with glycosuria may also be caused by a de- creased activity of the animal body to consume or destroy the sugar. In this case the sugar must accumulate in the blood, and the forma- tion of diabetes mellitus is now generally explained by this process. The inability of diabetics to destroy or consume the sugar does not seem to be connected with any decrease in the oxidation energy of the cells, as both varieties of sugar, dextrose and Itevulose, both of which can be oxidized with the same readiness, act differently in the body of diabetics. Laevulose is, according to Kulz ' and other investigators, contrary to dextrose, utilized to a great extent in the organism, and may even cause a deposit of glycogen in the liver in animals with pancreas-diabetes (Mi2srK0WSKi'). In this diabetes the ability of the cells to utilize the dextrose is diminished, and this diminution of ability seems to be in some way dependent upon the pancreas. The investigations of Minkowski, v. Meeing, Uome- Nicis, and later by other investigators' have shown that a true diabetes of a severe kind is caused by the total extirpation of the pancreas of many animals, especially dogs. As in man in severe forms of diabetes, so also in dogs with pancreas-diabetes an abundant elimination of sugar takes place even on the complete exclusion of carbohydrates in the food, and the formation of sugar in these cases is derived from the protein substances. It seems in man with diabetes that the ability of the sugar destruction is never quite arrested ; in dogs with pancreas-diabetes Miitkowski and v. Mee- ing, as also Hedon,' have been able, in a few cases, to detect that the total quantity of sugar introduced with the food passed into the urine. ' See Bock, Pfltiger's Arch., Bd. 5; Bock and Hoffmann, Expt. Studien Uber Diabetes (Berlin, 1874). CI. Bernard, Lefons sur le diabSte (Paris); T. Araki, Zeitscbr. f. pbysiol. Chem., Bd. 15, S. 351. ' Beitrage zur Patb. und Tber. des Diabetes mellitus Marburg, 1874. ' Arcb. f. exp. Patb. u. Pbarm., Bd. 31. * See Minkowski, TJntesucbrungen iiber Diabetes mellitus nacb Exstirpa- tion des Pankreas (Leipzig, 1893), and the chapter on diabetes in v. Noorden's Lehrbuch der Path, des StofEwechsels (Berlin, 1893), which contains a very copious index of the literature. In regard to diabetes see also CI. Bernard, Lemons sur le diabfete (Paris), and Seegen, Die Zuckerbildung im ThierkOrper (Berlin, 1890). ' Arch, de Physiol., (5) Tome 5. 222 THE LIVER. Artificial pancreas-diabetes may also in other respects present exactly the same picture as diabetes in man; and as in the past we have always looked to the liver for the cause of diabetes, our atten- tion is now more and more called to the pancreas. We do not know in what respect the pancreas stands to diabetes. We will refer to this question again in a subsequent chapter (Chapter IX). The Bile and its Formation. By the establishment of a biliary fistula, an operation which was first performed by Schwann ' in 1844 and which has been improved lately by Dastee," it is possible to study the secretion of the bile. This secretion is continuous, but with varying intensity. It takes place under a very low pressure ; therefore an apparently unimpor- tant hindrance in the outflow of the bile, namely, a stoppage of mucus in the exit ot the secretion of large quantities of viscous bile, may cause stagnation and absorption of the bile by means of the 'ymphatic vessels (absorption icterus). The quantity of bile secreted in the 34 hours in dogs can be exactly determined. The quantity secreted by different animals varies, and the limits ure 3.9-36.4 gm. bile per kilo of weight in the 34 hours.' The statements aS to the extent of bile secretion in man are few and not to be depended on. Eanke ' found (using a method which is not free from criticism) a secretion of 14 gm. bile with 0.44 gm. solids per kilo in 34 hours. Noel-Paton ' observed a 61-year-old woman with biliary fistula for 33 days, and found an average of 638 cc. with 8.378 gm. solids in 34 hours. MAto-RoBSOK,' whose observations on a woman 43 years old with biliary fistula extended over 15 months, found an average of 863 cc. for the 34 hours' bile secretion. The axjthoe ' found 650 cc. as the maximum quantity 1 Arch, f. Anat. und Physiol., 1844. = Arch, de Physiol., Tome 33. ' In regard to the quantity of bile secreted in animals see Heidenhain, Die Ctallenahsonderung' m Hermanns Handbuch der Physiol. , Bd. 5, and Stadel- mann, Ber Icterus und seine verschiedenen Formen (Stuttgart, 1891). * Die Blutvertheilung und der Thatigkeitswechsel der Organe. Leipzig, 1871. 5 Rep. Lab. Roy. Coll. Phys. Edinb., Vol. 3. » Proc. Roy. Soc, Vol. 47 ' Nova Acta Reg. Soc. Scient. Upsala, Ser. 3, Vol 16, 1893. BILE. 223 in a man and 950 cc. in a woman. Such determinations are of doubtful value, because in most cases it follows from the composition of the collected bile that we are not dealing with a secretion of normal liver-bile. The quantity of bile secreted is, however, as specially shown by Stadelmann,' subject to such great variation even under physio- logical conditions that the study of these circumstances which influ- ence the secretion is very difficult and uncertain. The contradic- tory statements by different investigators may probably be explained by this fact. In starvation the secretion diminishes. According to Luk- JANOW" and Albertoni," under these conditions the absolute quantity of solids decreases, while the relative quantity increases. After partaking of food the secretion increases again. The state- ments are contradictory in regard to the time necessary after par- taking of food before the secretion reaches its maximum. After a careful examination and compilation of all the existing statements Heideithain * has come to the conclusion that in dogs the curve of rapidity of secretion shows two maxima, the first at the 3d to 5th hour, and the second at the 13th to 15th hour, after partaking of food. According to the older statements, the proteids, of all the differ- ent foods, cause the greatest secretion of bile, while the carbohy- drates diminish, or at least excite much less than the proteids. It is nevertheless positive that an increase in the bile secretion takes place after a continuous over-abundant meat diet. We are by no means agreed as to the action of the fats. While many older investigators have not observed any increase, but rather the reverse, in the secretion of bile after feeding with fats, the newer experi- ments made by Eosenberg ''show that the fats have a more power- ful exciting action on the secretion of bile than the other foods, and that olive-oil is a strong cholagogue. This statement seems, according to the investigations of Majj-delstamm,' not to be suffi- ciently proven. ' Der Icterus. » Zeitschir. f. physiol. Chem., Bd. 16. " Recherches sur la secretion biliaire. Turin, 1893. * Hermann's Handb., Bd. 5, and Stadelmann, Der Icterus. « PflUger's Arch., Bd. 46. • Ueber den Einfluss einiger Arzneimittel auf Sekretion und Zusammensetz- ung der Galle. Dissert. Dorpat, 1890. In regard to the action of various foods 224 TEE LIVER. The question whether there exist special medicinal bodies, so-called cholagogues, which have a specific exciting action on the secretion of bile has been answered in very different ways. Many, especially the older investigators, have observed an increase in the bile secretion after the use of certain therapeutic agents such as calomel, rhubarb, jalap, turpentine, olive-oil, sodium salicylate, etc., while others, especially the later investigators, have arrived at quite opposite results. Prom all appearances this contradiction is due to the great irregularity of the normal secretion, which may be readily mistaken in tests with therapeutic agents. Schiff's' view, that the bile absorbed from the intestinal canal increases the secretion of bile and hence acts as a cholagogue, seems to be a positively proven fact by the investigations of several experi- menters." The bile is a mixture of the secretion of the liver-cells and the so-called mucus which is secreted by the glands of the biliary passages and by the mucous membrane of the gall-bladder. The secretion of the liver, which is generally poorer in solids than the bile from the gall-bladder, is thin and clear, while the bile collected in the gall-bladder is more ropy and viscous on account of the absorption of water and the admixture of " mucus, " and cloudy because of the admixture of cells, pigments, and the like. The specific gravity of the bile from the gall-bladder varies considerably, being-in man between I.OIQ and 1.040. Its reaction is alkaline. The color changes in different animals r golden yellow, yellowish brown, olive-brown, brownish green, grass-green, or bluish green. Bile obtained from an executed person immediately after death is golden yellow or yellow with a shade of brown. Still cases occur in which ' fresh human bile has a green color. The ordinary post-mortem bile has a variable color. The bile of certain animals has a peculiar odor; as example, oV-bile has an odor of musk, especially on warm- ing. The taste of bile is also different in different animals. Human as well as ox bile has a bitter taste with a sweetish after-taste. The on the secretion of bile see Heidenhain in Hermann's Handbucli, etc., and Stadelmann, Der Icterus. 1 Pflilger's Arch., Bd. 3. ' See Stadelmann, Der Icterus, and the dissertations of his pupils, namely, Winteler, Expt.-Beitrage zur Frage des Kreislaufes der Qallefluaug Diss. Dorpat, 1852), and Cfertner, Expt. BeltrSge zur Physiol, und Path, der Qallen- sekretion" (Inaug, Diss. Jurjew, 1893). BILE SALTS. 225 l)ile of the pig and rabbit has an intense persistent hitter taste. Ou heating bile to boiling it does not coagalate. It contains (in the ox) only traces of true mucin, and its ropy properties depend, it seems, chiefly on the presence of a nucleoalbumin similar to mucin (Paijkull '). The adthoi} " has, on the contrary, found trae mucin in human bile. The specific constituents of the bile are bile-acids combined with alkalies, bile-pigments, and besides small quantities. of lecithin, cholesterin, soaps, tieutral fats, urea, and mineral sub- stances, chiefly sodium chloride, calcium, and magnesium phos- phate, and iron. Traces of copper also occur. Bile Salts. The thus-far best studied bile-acids may be divided into two groups, the glycocholic and taurochoUc acid groups. As found by the authoe," a third group of bile-acids occur in the shark and probably also in other animals. They are rich in sulphur, and like the ethereal sulphuric acids they split off sulphuric acid on- boiling with hydrochloric acid. All glycocholic acids contain. nitrogen, but are free from sulphur and can be split with the addi- tion of water into glycocoll (amido-acetic acid) and a nitrogen-free acid, cholalic acid. All taurocholic acids contain nitrogen and sulphur and are split, with the addition of water, into tanrin (amido-ethylsulphonic acid) and cholalic acid. The reason of the existence of different glycocholic and taurocholic acids depends on- the fact that there are several cholalic acids. The different bile-acids occur in the bile as alkali salts, generally in oombinatioQ with sodium, but in sea-fishes as potassium salts. In the bile of certain animals we find almost solely glycocholic acid, in others only taurocholic acid, and in other animals a mixture of both (see below). All alkali salts of the biliary acids are soluble in water and alcohol, but insoluble in ether. Their solution in alcohol is there- fore precipitated by ether, and this precipitate, with the proper care in manipulation, gives, for nearly all kinds of bile thus far investigated, rosettes or balls of fine needles or 4-6-sided prisms (Plattner's crystallized bile). Fresh human bile also crystallizes readily. The bile-acids and their salts are optically active and dextrorotatory. The former are dissolved by concentrated sul- phuric acid at the ordinary temperature, forming a reddish-yellow ' Zeitschr. f. pbysiol. Chem., Bd. 13. ' Nova Acta reg. soc. sclent. Upsala, Ser. 3, Vol. 16. « Investigation not published. 326 THE LIVER. liquid which has a beantifal green fluorescence. On carefully -warming with concentrated sulphuric acid and a little cane-sugar, the bile-acids give a beautiful cherry-red or reddish-violet liquid. Pbttenkofek's reaction for bile-acids is based on this behavior. Pettbnkoffbe's test for bile-acids is performed as follows : A small quantity of bile in substance is dissolved in a small porcelain dish in concentrated sulphuric acid and warmed, or some of the liquid containing the bile-acids is mixed with concentrated sul- phuric acid, taking special care in both cases that the temperature does not rise higher than 60-70° 0. Then a 10^ solution of cane- sugar is added, drop by drop, continually stirring with a glass rod. 'The presence of bile is indicated by the production of a beautiful red liquid, whose color does not disappear at the ordinary tempera- ture, but becomes more bluish violet in the course of a day. This red liquid shows a spectrum with two absorption-bands, the one at F and the other between D and E, near E. This extremely delicate test fails^ however, when the solution is heated too high or if an improper quantity — generally too much — of the sugar is added. In the last-mentioned case the sugar easily carbonizes and the test becomes brown or dark brown. The reac- tion readily fails if the sulphuric acid contains sulphurous acid or the lower oxides of nitrogen. Many other substances, such as pro- teids, oleic acid, amyl alcohol, morphin, and others, give a similar reaction, and therefore in doubtful cases the spectroscopic examina- tion of the red solution must not be forgotten. Pettenkofee's test for the bile-acids depends essentially on the fact that furfurol is formed from the sugar by the sulphuric acid, and this body can therefore be substituted for the sugar in this test (Mtlius). According to Mtlius ' and v. Udranszkt ' a 1 p. m. solution of furfurol should be used. Dissolve the bile, which must first be purified by animal charcoal, in alcohol. To each c. c. of alcoholic solution of bile i-u a test-tube add 1 drop of the furfurol solution and 1 c. c. cone, sulphuric acid, and cool when necessary so that the test does not become too warm. This reaction, when performed as described, will detect ^-^—^ milligram cholalic acid (t. Udranszkt). Other modifications of Pettebtkoeek's test have been proposed. Glycocholie Acid. The constitution of that glycocholic acid, 1 Zeitschr. f. physiol. Chem., Bd. 11, S. 492. »iMd.,Bd. 12, S. 370. BILM AGIDS. 227 occurring in human and ox bile, which has been most studied is represented by the formula C,„H„]SrO,. Glycooholic acid is absent or nearly so in the bile of carnivora. On boiling with acids or alkalies this acid, which is analogous to hippuric acid, is converted into cholalic acid and glycocoll. Glycooholic acid crystallizes in fine, colorless needles or prisms. It is soluble with difSculty in water (in about 300 parts cold and 120 parts boiling water), and is easily precipitated from its alkali- salt solution by the addition of dilate mineral acids. It is readily soluble in strong alcohol, but with great difficulty in ether. The solutions have a bitter but at the same time sweetish taste. The salts of the alkalies and alkaline earths are soluble in alcohol and water. The salts of the heavy metals are mostly insoluble or soluble with difficulty in water. The solution of the alkali salts in water is precipitated by sugar of lead, copper-oxide and ferric salts, and silver nitrate. The preparation of pure glycooholic acid may be performed in several ways. We may precipitate the bile, which has been freed from mucus by means of alcohol and the alcohol removed by 'evap- oration, by a solution of lead acetate. The precipitate is then decomposed by a soda solution and heat, evaporated to dryness, and the residue extracted with alcohol, which dissolves the alkali glyco- cholate. The alcohol is distilled from the filtered solution and the residue dissolved in water; this solution is now decolorized by animal charcoal, and the glycooholic acid precipitated from the solution by the addition of a dilute mineral acid. The acid may be obtained in crystals either from boiling water, on cooling, or from strong alcohol by the addition of ether. The reader is referred to more exhaustive works for other methods of preparation. Hyo-glyoooholic Acid, CaTHiaNOs, is the crystalline glycocholic acid obtained from the bile of the pig.. It is very insoluble in water. The alkali salts, ^jchose solutions have an intense bitter taste without any sweetish after-taste, are pre- cipitated by CaClj, BiiCla, and MgCU, and may be salted out like a soap by NajSO« when added in sufficient quantity. Besides this acid there occurs in the bile of the pig still another glycocholic acid (JoLDsr'). The glycocholate in the bile of the rodent is also precipitated by the above- mentioned salts, but cannot, like the corresponding salt in the human or ox bile, be precipitated on saturating with a neutral salt (NaaS04). Guano bile- acid possibly belongs to the glycocholic-acid group, and is found in Peruvian .guano but has not been thoroughly studied. Taurocholic Acid. This acid, which is found in the bile of man, carnivora, oxen and a few other herbivora, such as sheep and ' Zeitschr, f. physiol. Chem., Bdd. 13 and 13. 228 THE LIVER. goats, has the constitution C^H^NSO,. On boiling with acids and alkalies it splits into cholalic acid and taurin. Tanrocholic acid may be obtained, though only with difiBcnlty, in fine needles which deliquesce in the air (Pabkb '). It is very soluble in water, and can hold the difficultly soluble glycocholic acid in solution. This is the reason why a mixture of glycocholate with a, sufficient quantity of taurocholate, which often occurs in ox-bile, is not precipitated by a dilute acid. Taurocholic acid is readily soluble in alcohol, but insoluble in ether. Its solutions have^ a bitter-sweet ' taste. Its salts are, as a rule, readily soluble in water, and the solutions of the alkali salts are not precipitated by copper sulphate, silver nitrate, or sugar of lead. Basic lead acetate gives, on the contrary, a precipitate which is soluble in boiling alcohol. Taurocholic acid is best prepared from decolorized, crystallized dog-bile, which contains only taurocholate. The solution of this, bile is precipitated by basic lead acetate and ammonia, and the washed precipitate dissolved in boiling alcohol. The filtrate is now treated with H,S, and this filtrate is evaporated at a gentle heat to a small volume, and treated with an excess of water-free ether. The acid sometimes partially crystallizes. Cheno-taarocholic Acid. This is the most essential acid of goose-bile and has the formula C2sH49NSOe. This acid, though little studied, is amorphous and soluble in water and alcohol. As repeatedly mentioned above, the two bile-acids split on boiling with acids or alkalies into non-nitrogenous cholalic acid and glycocoll or taurin. Therefore we will now describe the products, of this cleavage. Cholalic Acid. The ordinary cholalic acid obtained as a decom- position product of human and ox bile, which occurs regularly in the contents of the intestine and in the urine in icterus, has, accord- • ing to Steeckbe " and nearly all recent investigators, the constitu- tion C,,H„Oj. According to Mtlius,' cholalic acid is a monobasio alcohol-acid with a secondary and two primary alcohol groups. Its (CHOH formula may therefore be written C H , •< (CH^OH) . On oxida- (COOH ' Hoppe-Seyler, Med. chem. TJntersuch., S. 160. 2 The important investigations of Strecker on the bile-acids may be found in. Ann. d. Chem. u. Pharm., Bdd. 65, 67, and 70. s Ber. d. deutsch. chem. Gesellsoh., Bd. 19, pp. 369-379 and 2000-3009. CHOLALia ACID. 229 iion it first yields dehydrocholalic acid (authok)', and then hilianic ■acid (Cleve)." The formulae of these acids (when we take C,, for the cholalic acid) are G^^,fi^ aad C„H„Oj. On reduction' (in putrefaction) cholalic acid may yield desoxy cholalic acid, C^H^^O, (Mtlius).' Cholalic acid crystallizes partly with oae molecule of water, in rhombic plates or prisms, and partly in larger rhombic tetrahedra or octahedra with 1 mol. of alcohol of crystallization (Mtlius). These crystals become quickly opaque and porcelain-white in the air. They are quite insoluble in water (in 4000 parts cold and 750 parts boiling), rather soluble in alcohol, but soluble with difiSculty in ether. The amorphous cholalic acid is less insoluble. The solu- tions have a bitter-sweetish taste. The crystals lose their alcohol of crystallization only after a lengthy heating to 100-120° 0. The acid free from water and alcohol melts at + 195° C. The alkali salts are readily soluble in water, 'but when treated with a concentrated caustic or carbonated alkali solution may be separated as an oily mass which becomes crystalline on cooling. The alkali salts are not readily soluble in alcohol, and on the evap- oration of the alcohol they may crystallize. The specific rotatory power of the sodium salt is (ar)D = + 31°. 4. The watery solution of the alkali salts, when not too dilute, is precipitated immediately or after some time by sugar of lead or by barium chloride. The barium salt crystallizes in fine, silky needles, and it is rather insolu- ble in cold, but somewhat easily soluble in warm water. The barium salt, as well as the lead salt which is insoluble in water, is soluble in warm alcohol. Cholalic acid is best prepared from ox-bile by the following method as suggested by Mtlius: ' Boil the bile for 24 hours with 5 parts its weight of a 30^ caustic-soda solution, replacing the water lost by evaporation. Now saturate the liquid with CO^ and evap- orate nearly to dryness. The residue is extracted with 96^ alcohol, and this alcoholic. extract diluted with water until it contains at the most 20^ alcohol, and completely precipitated with a BaCl, solu- tion. The precipitate, which contains besides fatty acids also the choleio acid, is filtered and the cholalic acid precipitated from the filtrate by hydrochloric acid. After the cholalic acid has gradually crystallized out it is repeatedly recrystallized from alcohol or methyl alcohol. ' Ber. d. deutsch, chem. Gesellsch., Bd. 14, S. 71. « Bull. Soc. Chim., Tome 35. » L. c. * Zeitselir. f. physiol. Chem.. Bd. 12. 230 TBE LIVER. Choleio Acid is another cholalic acid with the formula 0„H„0,.. (Lassak-Cohn ') named by Latschinoff.' This acid, which oc- curs in varying but always small quantities in ox-bile, is probably identical with desoxycholallc acid. Oholeic acid first yields dehy- drocJioleic acid, C^Hj^O,, and then cholanic acid, C^H^Oj, on oxidation. Oholeic acid may be obtained from the above-mentioned barium precipitate by first converting the barium salts into sodium salts by sodium carbonate and then fractionally precipitating the fatty acids- by barium acetate and separating the choleic acid from the filtrate by hydrochloric acid and recrystallizing several times from glacial acetic acid. Fellic Acid, 0„H,„0„ is a cholalic acid, so called by Schotten,' and which he obtained from human bile, along with the ordinary acid. This acid is crystalline, is insoluble in water, and yields barium and magnesium salts which are very insoluble. It does not- give Pettbnkofek's reaction easily and gives a more reddish-blue color. The conjugate acids of human bile have not been investigated. To all appearance human bile contains under different circum- stances various conjugate bile-acids. In some cases the bile-salts of human bile are precipitated by BaCl,, and in others not. Accord- ing to the latest statements of Lassae-Cohit ' three cholalic acids may be prepared from human bile, namely, ordinary cholalic acid, choleic acid, and fellic acid. The hyo-glycocholic and cheno-taurocholic acids as well as the glycocholic acid of the bile of rodents yield corresponding cholalic acids. On boiling with acids, on putrefaction in the intestine, or on heating, cholalic acids lose water and are converted into an anhydride, the so-called dyslysin. The dyslysin, d^Jl^fi,, corre- sponding to ordinary cholalic acid, and which occurs in fseces, is amorphous, insoluble in water and alkalies. Choloidic acid, C^HjjO,, is called the first anhydride or an intermediate product in the formation of dyslysin. On boiling dyslysin with caustic alkali it is recomverted into the corresponding cholalic acid, > Zeitscbr. f. pliysiol. Chem., Bd. 17, S. 606. ' Ber. d. deutsch. chem. Gesellsch., Bdd. 18 and 30. ^ Ibid., Bi. 11, S. 268. ■• Ber. d. deutsch. chem. Gesellsch., Bd. 37, S. 1339. GLTCOCOLL AND TAURIN. 231 GlycocoU, C,H,NO„ or amido-acetic acid, NH^.CH^.COOH, also called glycin, or sagar of gelatin, has been found in the muscles of pecten irradians, but it is of special interest as a decom- position product of certain protein substances — ^gelatin and spongin — as also of hippuric acid or glycocholic acid on splitting them by boiling with acids. GlycocoU forms colorless, often large, hard rhombic crystals or four-sided prisms. The crystals taste sweet and dissolve easily in cold (4.3 parts) water. They are insoluble in alcohol and ether; in warm spirits of wine they dissolve, but with difficulty. GlycocoU combines with acids and bases. Under the last-mentioned combina- tions we must mention those with copper and silver. GlycocoU dissolves copper hydroxide in alkaline liquids, but does not reduce it at the boiling temperature. A boiling-hot solution of glycocoll dissolves freshly precipitated copper hydroxide, forming a blue liquid from which dark-blue needles crystallize on cooling, if the liquid is sufficiently concentrated. The combination of glycocoll with HCl is soluble in water and alcohol. Glycocoll is best prepared from hippuric acid by boiling it 10-lX hours with 4 parts of dilute sulphuric acid, 1 : 6. After cooling separate the benzoic acid, concentrate the filtrate, remove the remainder of the benzoic acid by shaking with ether, remove the sulphuric acid by BaCOj, and evaporate the filtrate to crystalliza- tion. In the preparation and quantitative estimation of glycocoll from gelatin we can proceed according to Gonneemann's ' modi- fication of Ch. Fischeb's" method. The gelatin is decomposed by sulphuric acid, the sulphuric aoid removed by lead carbonate, the glycocoll transformed into hippuric acid by benzoyl chloride and caustic soda. This solution is acidified with sulphuric acid, extracted with acetic ether, and the syrupy residue of acetic ether dissolved in chloroform containing benzol. The precipitated hip- puric acid after 24 hours is collected on a filter and first washed with chloroform containing benzol and then with pure chloroform. Taurin, C,H,NS03, or amido-ethylsulphonic acid, NH 0,11^. SOjOH. This body is well known as a splitting product of tauro- cholic acid, and may occur in small quantities in the contents of the intestine. It has also been found in the lungs and kidneys of oxen and in the blood and muscles of cold-blooded animals. Taurin crystallizes in colorless, often in large, shining, 4-6-sided prisms. It dissolves in 15-16 parts of water at ordinary tempera- ' Pflliger's Arcli., Bd. 59. ' Zeitsclir. f. pbysiol. Chem., Bd 19. 232 TEE LIVER. tares, but rattier more easily in warm water. It is insoluble in absolute alcohol and ether; in cold spirits of wine it dissolves slightly, but more when warm. Taurin yields acetic and sulphurous acids, but no alkali sulphides, on boiling with strong caustic alkali. The amount of sulphur can be determined as sulphuric acid after fusing with saltpetre and soda. Taurin combines with metallic oxides. The combination with mercuric oxide is white, insoluble, and is formed when a solution of taurin is boiled with freshly pre- cipitated mercuric oxide (J. Lang'). This combination may be used in detecting the presence of taurin. Taurin is not precipitated by metallic salts. The preparation of taurin from bile is very simple. The bile is boiled a few hours with hydrochloric acid. The filtrate from the dyslysin and choloidic acid is concentrated well in the water-bath, and filtered so as to remove the common salt and other substances which have separated. Then evaporate to dryness, and treat the residue with strong alcohol, which dissolves the hydrochlorate of glycocoll, while the taurin remains. (The alcoholic solution of hydrochlorate of glycocoll may be used in the preparation of glycocoll by evaporating the alcohol and dissolving the residue in water, decomposing the solution with lead hydroxide, filtering, and freeing the solution from lead by H,S, and strongly concentrating this filtrate. The crystals which separate are dissolved and decolor- ized by animal charcoal, and the solution evaporated to crystalliza- tion. ) The above-obtained residue containing the taurin is dissolved in as little water as possible, filtered warm, and treated with an excess of alcohol. The crystalline precipitate which immediately forms is filtered as soon as possible, and the taurin now separates, on cooling, in very long needles or prisms. These crystals may be purified by reerystallization from a little warm water. Though the taurin shows no positive reactions, it is cliiefly identified by its crystalline form, by its solubility in water and insolubility in alcohol, by its combination with mercuric oxide, by its non-precipitability by metallic salts, and above all by its con- taining sulphur. The DBTBGTioiir of Bile-acids in Animal Fluids. To obtain the bile-acids pure so that Pbttenkofbr's test can be applied to them, the proteid and fat must first be removed. The proteid is removed by making the liquid first neutral and then add- ing a great excess of alcohol, so that the mixture contains at least 85 vols, per cent of water-free alcohol. Now filter, extract the pre- cipitated proteid with fresh alcohol, unite all filtrates, distil the alcohol, and evaporate to dryness. The residue is completely exhausted with strong alcohol, filtered, and the alcohol entirely > See Maly's Jahresber., Bd. 6, S. 73. BILIRUBIN. 233 evaporated from the filtrate. The new residue is dissolved in water, and filtered if necessary, and the solution precipitated by basic lead acetate and ammonia. The washed precipitate is dis- solved in boiling alcohol, filtered while warm, and a few drops of soda solution added. Then evaporate to dryness, extract the residue with absolute alcohol, filter, and add an excess of ether. The precipitate now formed may be used for Pettenkofee's test. It is not necessary to wait for a crystallization; but one must not consider the crystals which form in the liquid as being positively crystallized bile. It is also possible for needles of alkali acetate to be formed. For the detection of bile-acids in urine see Chapter XV. Bile-pigments. • The bile-coloring matters known thus far are relatively numerous, and in all probability there are still more. Most of the known bile-pigments are not found in the normal bile, but occur either in post-mortem bile or, principally, in the bile concrements. The pigments which occur under physiological con- ditions are the reddish-yellow bilirubin, the green biliverdin, and sometimes there is also observed in fresh human bile a pigment closely allied to hydrobilirubin. The pigments found in gall-stones are (besides the bilirubin and biliverdin) bilifuscin, bihprasi?i, bilihumin, bilicyanin (and choletelin ?). Besides these, others have been observed in human and animal bile. The two above-men- tioned physiological pigments, bilirubin and biliverdin, are those which serve to give the golden-yellow or orange-yellow or sometimes greenish color to the bile, or when, as is most frequently the case in ox-bile, the two pigments are present in the bile at the same time, producing the difEerent shades between reddish brown and green. Bilirubin. This pigment, according to the common accepta- tion, has the formula 0„H„N,0, (Malt') and is designated by the names cholbpyeehin, biliph^in, bilifulvin, and n.iJMA- TOiDiN. It occurs chiefly in the gall-stones as bih'rubin-calcium. It occurs in the liver-bile of all vertebrates and in the bladder-bile especially in man and carnivora; sometimes, however, the latter when fasting or in a starving condition may have a green bile. It occurs also in the contents of the small intestine, in blood-serum of the horse, in old blood extravasations (as haematoidin), and in the urine and the yellow-colored tissue in icterus. Bilirubin is derived in all probability from haematin, which it closely resembles. It ' Wien. Sitzungsber., Bdd. 57 and 70. 234 THE LIVER. is converted into hydrobiliruUn, Cj^H^N^O, (Malt ') by hydrogen in a nascent state. It is claimed by several investigators to be identical with the urinary pigment urobilin, as well as with sterco- hilin (Masius and Vanlaib"), which is found in the contents of the intestine. There is no doubt that a great similarity exists between these pigments, but their identity is emphatically, denied by MacMunn.' On oxidation bilirubin yields biliverdin and other coloring matters (see below). Bilirubin is partly amorphous and partly crystalline. The amorphous bilirubin is a reddish-yellow powder of nearly the same color as amorphous antimony sulphide; the crystalline bilirubin has nearly the same color as crystallized chromic acid. The crystals, which can easily be obtained by allowing a solution of bilirubin in chloroform to spontaneously evaporate, are reddish-yellow, rhombic plates, whose obtuse angles are often rounded. Bilirubin is insoluble in water, slightly soluble in ether, some- what more soluble in alcohol, easily soluble in chloroform, especially in the warmth, and less soluble in benzol, carbon disulphide, amyl alcohol, fatty oils, and glycerin. Its solutions show no absorption- bands, but only a continuous absorption from the red to the violet end of the spectrum, and they have, even on diluting greatly, (1 : 600000) in a layer 1.5 c.cm. thick a decided yellow color. If a dilute solution of bilirubin in water is treated with an excess of ammonia and then with a zinc-chloride solution, the liquid is first colored deep orange and then gradually olive-brown and then green. This solution first gives a darkening of the violet and blue part of the spectrum and then the bands of alkaline cholecyanin (see below), or at least the bands of the pigment in the red between and D close to C. This is a good reaction for bilirubin. The combina- tions of bilirubin with alkalies are insoluble in chloroform, and bilirubin may be separated from its solution in chloroform by shak- .ing with dilute caustic alkali (differing from lutein). Solutions of bilirubin-alkali in water are precipitated by the soluble salts of the alkaline earths and also by metallic salts. If an alkaline solution of bilirubin be allowed to stand in con- tact with the air, it gradually absorbs oxygen and green biliverdin is formed. Biliverdin is also formed from bilirubin by oxidation. ' Ann. d. Chera., Bd. 163. * Centralbl. f. d. med. Wissensch., 1871, 8. 369. » Journal of Physiol., Vol. 10, p. 71. REACTIONS FOR BILE PIGMENTS. 235 nndor other conditionB. A green coloring matter similar in appear- ance is formed by the action of other reagents such as CI, Br, and I. In these cases it does not seem to be biliverdin, but a sub- stitution product of bilirubin (Thudichum,' Maly'), which is obtained. Gmelin's Reaction for Bile-pigments. If we carefully pour under a solution of bilirabin-alkali in water nitric acid containing some nitrous acid, we obtain a series of colored layers at the junc- ture of the two liquids, in the following order from above down- wards: green, blue, violet, red, and reddish yellow. This color reaction, Gmelin's test, is very delicate and serves to detect the presence of one part bilirubin in 80,000 parts liquid. The green ring must never be absent; and also the reddish violet must be present at the same time, otherwise the reaction may be confused with that for lutein, which gives a blue or greenish ring. The nitric acid must not contain too much nitrous acid, for then the reaction takes place too quickly and it does not become typical. Alcohol must not be present in the liquid, because, as is well known, it gives a play of colors, in green or blue, with the acid. Huppeet's Reaction. If a solution of bilirubin-alkali is treated with mijk of lime or with calcium chloride and ammonia, a precipi- tate is produced consisting of bilirubin-calcinm. If this moist pre- cipitate, which has been washed with water, is placed in a test-tube and the tube half filled with alcohol which has been acidified with sulphuric acid, and heated to boiling for some time, the liquid becomes emerald-green or bluish green in color. This reaction is a good and easily performed test for bile-pigments. In regard to the modifications of Gmelin's test and certain other reactions for bile-pigments, see Chapter XV (Urine). That the characteristic play of colors in Gmelin's test is the result of an oxidation is generally admitted. The first oxidation step is the green biliverdin. Then follows a blue coloring matter which Heinsids and Campbell " call bilicyanin and Stokvis ' calls cholecyanin, and which shows a characteristic absorption-spectrum. The neutral solutions of this coloring matter are, according to Stokvis, bluish green or steel-blue with a beautiful blue fluores- ' Journal of the Chem Soc, (3) Vol. 13. » Wien. Sitzungaber., Bd. 72. « Pflilger's Arcli. , Bd. 4, S. 529. * Centralbl. f. d. med, Wissensch., 1873, S. 786. 236 THE LIVEB. cence. The alkaline solutions are green and have no marked fluorescence. The neutral and alkaline solutions show three absorp- tion-bands, one sharp and dark in the red between and D, nearer to C; a second, less defined, covering D; and a third, forming only a faint shadow, in the green, exactly in the middle, between D and E. The strongly acid solutions are violet-blue and show two bands, described by Jaffe, between the lines G and E, separated from each other by a narrow space near D. The next oxidation step after these blue coloring matters is a red pigment, and lastly a yellowish-brown pigment, called choletelin by Malt,' which in neutral alcoholic solutions does not give any absorption spectrum, but in acid solution gives a band between h and F. Bilirubin is best prepared from gall-stones of oxen, these con- cretions being very rich in bi]irubin.-calcium. The finely powdered concrement is first exhausted with ether and then with boiling water, so as to remove the cholesterin and bile-acids. The powder is then treated with hydrochloric acid, which sets free the pigment. Wash thoroughly with water and alcohol, dry, and extract re- peatedly with boiling chloroform. After distilling the chloroform from the solution, treat the powdered residue with absolute alcohol to remove the bilifuscin; dissolve the remaining bilirubin in a little chloroform; precipitate it from this solution by alcohol, and do this several times if necessary. The bilirubin is finally dissolved in boiling chloroform and allowed to crystallize on cooling. The quantitative estimation of bilirubin may be made by the spectro- photometrical method, according to the steps suggested for the blood-coloring matters. Biliverdin, C,„H,jNjO,. This body, which is formed by the oxidation of bilirubin, occurs in the bile of many animals, in vomited matter, in the placenta of the bitch (?), in the shells of birds' eggs, in the urine in icterus, and sometimes in gall-stones, although in very small quantities. Biliverdin is amorphous, or at least it has not been obtained in well-defined crystals. It is insoluble in water, ether, and chloro- form (this is true at least for the artificially prepared biliverdin, while the green pigment of ox-bile is soluble in chloroform, accord- ing to MacMunn"), but is soluble in alcohol or glacial acetic acid, showing a beautiful green color. It is dissolved by alkalies, giving 1 Wien. Sitzungsber., Bd. 59. See also Jaffe, Centralbl. f. d. med. Wis- sensch., 1868, and Heinsius and Campbell, Pfluger's Arch., Bd. 4. 2 Journal of Physiol., Vol. 6. BILIVERDIN. 237 a brownish-green color, and this solution is precipitated by acids, as well as by calcium, barium, and lead salts. Biliverdin gives Huppekt's and Gmelin's reactions, commencing with the blue color. lb is converted into hydrobilirubin by nascent hydrogen. On allowing the green bile to stand, also by the action of ammonium sulphide, the biliverdin may be reduced to bilirubin (Hatcraft and ScoFiBLD '). Biliverdin is most simply prepared by allowing a thin layer of an alkaline solution of bilirubin to stand exposed to the air in a dish until the color is brownish green. The solution is then precipitated by hydrochloric acid, the precipitate washed with water until no HCl reaction is obtained, then dissolved in alcohol and the pigment again separated by the addition of water. Any bilirubin present may be removed by means of chloroform. Bilifuscin, so named by Stadbleu', is an amorphous brown pigment, soluble in alcohol and alkalies, nearly insoluble in water and ether, and soluble with great difficulty in chloroform (when bilirubin is not present at the same time). When pure bilifuscin does not give Gmblin's reaction. It is found in post-mortem bile and gall-stones. Bilipraain is a green pigment prepared by Stadeler from gall-stones, which perhaps is only a mixture of biliverdin and bilirubin. Bilihumin is the name given by Stadeler to that brownish amorphous residue which is left after extracting gall-stones with chloroform, alcohol, and ether. It does not give Gmblin's test. Bilicyanin is also found in human gallstones (Heinsius and Campbell). CliololuBmatin, so called by MacMunn," is a pigment often occurring in sheep- and ox-bile and character- ized by four absorption-bands, and which is formed from haematin by the action of sodium amalgam. In the dried condition obtained by the evapo- ration of the chloroform Solution it is green, and in alcoholic solution olive- brown. Gjielin's and Huppbet's reactions are generally used to detect the presence of bile-pigments in animal fluids or tissues. The first, as a rule, can be performed directly, and the presence of proteid does not interfere with it, but, on the contrary, it brings out the play of colors more strikingly. If blood -coloring matters are present at the same time, the bile-coloring matters are first precipi- tated by the addition of sodium phosphate and milk of lime. This precipitate containing the bile-pigments may be used directly in Huppert's reaction, or may be treated with water and some hydro- chloric acid, and then shaken with chloroform free from alcohol, and this chloroform solution used in testing for the bile-pigments. ' Centralbl. f. Physiol., 1889, S. 222, and Zeitschr. f. physiol. Chem , Bd. 14. ' Vierteljahrschr. d. naturf. Gesellsch. in Zvirich, Bd. 8, cited from Hoppe- Seyler, Physiol, u. path. chem. Analyse, 6. Aufi., S. 325. » Journal of Physiol., Vol. 6. 238 THB LIVER. Bilirubin is detected in blood, according to Hbdenius,' by precipi- tating the proteins by alcohol, filtering and acidifying the filtrate with hydrochloric or sulphuric acid, and boiling. The liquid becomes of a greenish color. Serum and serous fluids may be boiled directly with a little acid after the addition of alcohol. Besides the bile-acids and bile-pigments we also have in the bile cholesterin, lecithin, palmitin, stearin, olein, and soaps of the corre- sponding/a^^^ acids. Lassae-Cohn " has also found myristic acid in ox-bile. In some animals the bile contains a diastatic enzyme. Cholin and glycero-pliosphoric acid, when they are present, may be considered as decomposition products of lecithin. Urea occurs, though only as traces, as a physiological constituent of human, ox, and dog bile. Urea occurs in the bile of the shark and ray in such large quantities that it forms one of the chief constituents of the bile.° The mineral constituents of the bile are, besides the alkalies, to which the bile acids are united, sodium and potassium chloride, calcium and magnesium phosphate, and iron — 0.04-0.115 p. m. in human bile, chiefly combined with phosphoric acid (YouifG'). Traces of copper are habitually present, and traces of zinc are often found. Sulphates are entirely absent or only occur in very small amounts. The quantity of iron in the bile varies very much. According to K"ovi ' it is dependent upon the kind of food, and in dogs it is lowest with a bread diet and highest with a meat diet. According to Dastee ' this is not the case. The quantity of iron in the bile varies even though a constant diet is kept up, and the variation is dependent upon the formation and destruction of blood. The question as to the extent of elimination by the bile of the iron introduced into the body has received various answers! There is no doubt that the liver has the property of collecting and retaining iron and also other metals from the blood. Certain investigators, such as No VI and Kun^kbl,' are of the opinion that the introduced and transitorily retained iron in the liver is eliminated by the bile, ' TJpsala Lakaref. PQrh., Bd. 29. ' Zeitschr. f. pliysiol. Chem., Bd. 17. ' Investigation not published by tbe author. * Journal of Auat. and Physiol., Vol. 5, p. 158. " See Maly's Jahresber., Bd. 30, S. 873. « Arch, de Physiol., (5) Tome 3. ' Pfluger's Arch., Bd. 14. COMPOSITION OF THE BILE. 239 ■while others, such as Hamburger," Gottlieb,' and Anselm," deny any sach elimination of iron by the bile. Quantitative Composition of the Bile. Complete analyses of human bile have been made by Hoppe-Seyler and his pupils. The bile was removed as fresh as possible from the gall-bladder of cadavers whose livers showed no remarkable change. The following figares of Socoloff* are the average of six analyses, and those of . Hoppe-Seylee ' of five analyses. The relationship between the glycocholate and taurocholate was found by fusing the precipitate, consisting of biliary alkalies obtained by ether from the alcoholic extract, with saltpetre and soda. On determining the amount of sulphur in the fused mass the tauroeholic acid can be calculated from this. 100 parts BaSO, correspond to 230.86 parts tauroeholic acid. The figures are parts per 1000. Trifanowski." Socoloff. Hoppb-Seylbr. I. n. Mucin ,24.8 13.0) ^^ ^^ 12.9 Remaining bodies insol. in alcohol. 4.5 14.6 j 1.4 Taurocholate 7.5 19.2 15.67 8.7 Glycocholate 21.0 4.4 49.04 30.3 Soaps 8.1 16.3 14.60 13.9 Cholesterin 2.5 3.3 3.5 Lecithin ) c o 0,2 5.3 Fat j ^■'' 3.6 7.3 Ferric phosphate .. . ... 0.166 Older and less complete analyses of human bile have been made by Feeeichs and v. GtOeup-Besakez. The bile analyzed by them was fro!n perfectly healthy persons who had been executed or accidentally killed. The two analyses of Peerichs are, respec- tively, of (I) an 18-year-old and (II) a 33-year-old male. The analyses of v. Goeup-Besanez are of (I) a man of 49 and (II) a woman of 39. The results are, as usual, in parts per 1000. Fbeeiichs.' v. Gobdp-Besanez." I. II. I. n. Water 860.0 859.2 822.7 898.1 Solids 140.0 140.8 177.3 101.9 Biliary salts 72.2 91.4 107.9 56.5 Mucus and pigments 26.6 29 8 22.1 14.5 Cholesterin 1.6 2.6) 473 g^ 9 Fat 3.2 9.2) Inorganic substances 6.5 7.7 10.8 6.2 ' Zeitschr. f. physiol. Chem., Bdd. 2 and 4. ^Ibid., Bd. 15. ' TJeber die Eisenausscheidung der Qalle, Inaug. Diss. Dorpat, 1891. *;PflUger's Arch., Bd. 12. » Physiol. Chem., S. 301. « Pflilger's Arch., Bd. 9. ' Cit. from Hoppe-Seyler's Physiol. Chem., S. 299. »Ibid. 240 TEE LIVEB. Human liver-bile is poorer in solids thian the bladder-bile. In several cases it only contained 12-18 p, m. solids, bat the bile in these cases is hardly to be considered as normal. Jacobsen' found 23.4-22.8 p. m. solids in a specimen of bile. The authoe,* who had occasion to analyze the iiver-bile in seven cases of biliary fistula, has repeatedly found 25-28 p. m. solids. In a case of a corpulent woman the quantity of solids in the liver-bile varied between 30.10-38.6 p. m. in ten days. Human bile sometimes, but not always, contains sulphur in an ethereal sulphuric-acid combination. The quantity of such sulphur may even amount to l~k of the total sulphur. Human bile is Iiabitually richer in glycocholic than in taurocholic acid. In six cases of liver-bile analyzed by the author the relationship of taurocholic to glycocholic acid varied between 1 : 2.07 and 1 : 14.36. The bile analyzed by Jacobsen contained no tauro- cholic acid. As example of the composition of human liver-bile we give the following results of three analyses made by the author. ° The results are calculated in parts per 1000. Solids 35.200 35.360 35.400 Water ,974 800 964.740 974.600 Miicin and pigments .5.290 4.290 5.150 Bile-salts 9.310 18.240 9.040 Taurocholate 3.034 2.079 3.180 Glycocholate 6.276 16.161 6.860 Fatty acids from soaps 1.230 1.360 1.010 Cliolesterin 0.630 1.600 1.500 Lecithin I ^990 0-574 0.650 Pat S 0.956 0.610 Soluble salts 8.070 6.760 7.250 Insoluble salts 0.250 0.490 0.310 Baginsky and Sommerfbld^ have found true mucin, mixed with some nuoleoalbumin, in the bladder-bile of children. The bile contained on an average 896.5 p. m. water; 103.5 p. m. solids; 30 p. m. mucin; 9.1 p. m. mineral substances; 25.2 p. m. bile-salts, of which 16.3 p. m. were glycocholate and 8.9 p. m. taurocholate; 3.4 p. m. cholesterin; 6.7 p. m. fat, and 2.8 p. m. leucin. Amongst the mineral constituents the chlorine and sodium occur to the greatest extent. The relationship between potassium ' Ber. d. deutscli. chem. Gesellscli., Bd. 6. ' Nova Acta Reg. Soc. Sclent. Upsala, Bd. 16. »L. c. * Verhandl. d. physiol. Gesellsch. zu Berlin, 1894-95, Kos. 13, 14, 15. COMPOSITION OF TEE BILE. 241 and sodium varies considerably in different biles. Sulphuric acid and phosphoric acid only occur in very small quantities. The quantity of iron in the liver-bile in three cases investigated by the AUTHOR was 0.018-0.044 p. m., calculated on the fresh bile. The quantity of pigment in human bile is, according to Noel- Patois',' 0.4-1.3 p. m. for a case of biliary fistula. The method used in determining the pigments in this case was not quite trust- worthy. More exact results obtained by spectro-photometric methods are on record for dogs' bile. According to Stadelmann * dogs' bile contains on an average 0.6-0.7 p. m. bilirubin. At the most, only 7 milligrams pigment are secreted per kilo of body ia the 24 hours. In animals the relative proportion of the two acids varies very- much. It has been found, on determining the amount of sulphur, that, so far as the experiments have gone, taurocholic acid is the prevailing acid in carnivorous mammalia, birds, snakes, and fishes. Among the herbivora sheep and goats have a predominance of taurocholic acid in the bile. Ox-bile sometimes contains tauro- cholic acid in excess, in other cases glycocholic acid predominates, and in a few cases the latter occurs almost alone. The bile of the- rabbit, hare, and kangaroo contains, like the bile of the pig, almost exclusively glycocholic acid. A distinct influence on the relative amounts of the two bile-acids by different foods has not beeiL detected. Ritter ' claims to have found a decrease in the quantity of taurocholic acid in calves when they pass from the milk to the plant diet. In the above-mentioned calculation of the taurocholic acid from the quantity of sulphur in the bile-salts it must be remarked that no exact conclusion can be drawn from this calculation as long as we have not investigated whether other kinds of bile contain sul- phur in combinations other than taurocholic acid, as in human and shark bile. The gases of the bile consist of a large quantity of carbon diox- ide, which increases with the amount of alkalies, only traces of oxygen, and a very small quantity of nitrogen. Little is known in regard to the properties of the bile in disease. The quan- tity of urea is found to be considerably increased in uraemia. Leucin and tyrosin are observed in acute yellow atrophy of the liver and in typhus. Traces • Rep. Lab. Roy. Soc. Coll. Phys. Edinb., Vol. 3. ' Der Icterus, etc. Stuttgart, 1891. = Cit, from Maly's Jahresber., Bd. 6, S. 195. 242 THE LIVER. of albumin (without regard to nucleoalbumin) liave several times been found in the human bile. The so-called pigmentary aelioUa, or the secretion of a^ bile containing bile-acids but no bile-pigments, has also been repeatedly noticed. In all such cases observed by Rittbk ' he found a fatty degeneration of the liver-cells, in return for which, even in excessive fat infiltration, a normal bile containing pigments was secreted. The secretion of a bile nearly free from bile-acids has been observed by Hoppb-Setlbr' in amyloid degeneration of the liver. In animals, dogs, and especially rabbits it has been observed that the blood-pigments pass into the bile in poisoning and in other cases, causing a destruction of the blood-corpuscles, as also after intravenous hsemoglobin injec- tion (Wbbthbimkr and Meter,* Filbhnb,* Stern '). Chemical Formation of the Bile. The first question to be answered is the following: Do the specific constituents of the bile, the bile-acids and bile-pigments, originate in the liver; and if this is the case, do they come from this organ only, or are they also formed elsewhere ? The investigations of the blood, and especially the comparative investigations of the blood of the portal and hepatic veins under normal conditions, have not given any answer to this question. To decide this, therefore, it is necessary to extirpate the liver of animals or isolate it from the circulation. If the bile constituents are not formed in the liver, or at least not alone in this organ, but only eliminated from the blood, then, after the extirpation or removal of the liver from the circulation, an accumulation of the bile constituents is to be expected in the blood and tissues. If the bile constituents, on the contrary, are formed exclusively in the liver, then the above operation naturally would give no such resalt. If the choledochus duct is tied, then the bile constituents will be collected in the blood or tissues whether they are formed in the liver or elsewhere. From these principles Kobnek ° has tried to demonstrate by experiments on frogs that the bile-acids are produced exclusively in the liver. While he was unable to detect any bile-acids in the blood and tissues of these animals after extirpation of the liver, still he was able to discover them on tying the choledochus duct. The investigations of Ludwig and Fibischl ' show that in the dog the ' Compt. rend.. Tome 74, and Journ. de I'auat. et de la physiol,, 1873. ' Physiol. Chem., S. 317. • Compt. rend.. Tome 108. * Virchow's Arch., Bd. 131. ^ IMd., Bd. 123. 8 See Heidenhain, Physiologie der Absonderungsvorgange in Hermann's ilandbuch, Bd. 5. ' Arbeiten aus der physiol. Anstalt zu Leipzig, Jahrgang 9. FORMATION OF BILE ACIDS. 243 bile-acids originate in the liver alone. After tying the choledochus dact they observed that the bile constituents were absorbed by the lymphatic vessels and passed into the blood through the thoracic dnct. Bile-acids could be detected in the bl'ood after such an operation, while they could not be detected in the normal blood. But when the choledochus and thoracic ducts were both tied at the same time, then not the least trace of bile-acids could be detected in the blood, while if they are also formed in other organs and tissues they should have been present. Other ways have been tried to demonstrate the formation of bile-acid in the liver-cells. Alex. Schmidt and Kallmbtbr ' have shown that the isolated liver-cells, which have been washed with a physiological NaCl solution, have the property, in the presence of hEemoglobin and glycogen, of increasing the quantity twofold, of snbstances soluble in alcohol but insoluble in ether. This tends to show the formation of bile alkalies. From older statements of Cloez and Vulpiast as well as Vie- CHOW the bile-acids also occur in the suprarenal capsule. These statements have not been confirmed by later investigations of Stadelmann and Beiek." At the present time we have no ground for supposing that the bile-acids are formed elsewhere than in the liver. It has been indubitably proved that the bile-pigments may be formed in other organs besides the liver, for, as is generally admitted, the coloring matter haematoidin, which occurs in old blood extravasations, is identical with the bile-pigment bilirubin (see page 145). Latschenbebger ' has also observed in horses, under pathological conditions, a formation of bile-pigments from the blood-coloring matters in the tissues. Also the occurrence of bile-pigments in the placenta seems to depend on their formation in that organ, while the occurrence of small quantities of bile-pig- ments in the blood-serum of certain animals probably depends on an absorption of the same. Although the bile-pigments may be formed in other organs besides the liver, still it is of first importance to know what bearing this organ has on the elimination and formation of bile-pigments. ' Kallmeyer, Ueber die Entstehung der Qallensauren, etc." Inaug. Diss. Dorpat, 1889. ' Zeitschr. f. physiol. Chem., Bd. 18. This contains the older literature. » Maly's Jahresber., Bd. 16, S. 301, and Monatshefte f. Chem., Bd. 9. 244 THE LIVER. In this regard it must be recalled that the liver is an excretory- organ for the bile-pigments circnlating in the blood. Tabcha- NOFi' ' has observed, in a dog with biliary fistula, that intravenous injection of bilirubin causes a very considerable increase in the bile- pigments eliminated. This statement has been confirmed lately by the investigations of Vossius." ' ISTumerous experiments have been made to decide the question whether the bile-pigments are only eliminated by the liver or whether they are also formed therein. By experimenting on pigeons STEEi*r ' was able to detect bile-pigments in the blood-serum five hours after tying the biliary passages alone, while after tying all the vessels of the liver and also the biliary passages no bile- pigments could be detected either in the blood or the tissues of the animal, which was killed 10-12 hours after the operation. Min- kowski and !N"ATr]srT]sr * have also found that poisoning with arseniuretted hydrogen produces a liberal formation of bile-pigments and the secretion, after a short time, of a urine rich in biliverdin in previously healthy geese. In geese with extirpated livers this does not occur. No such experiments can be carried out on mammalia, as they do not live long enough after the operation ; still there is no doubt that this organ is the chief seat of the formation of bile-pigments under physiological conditions. In regard to the materials from which the bile-acids are pro- duced, it may be said with certainty that the two components, glycocoll and taurin, which are both nitrogenized, are formed from the protein bodies. In regard to the origin of the non-nitrogenized cholalic acid, which was formerly considered as originating from the fats, we know nothing positively. The blood-coloring matters are considered as the mother-sub- stance of the bile-pigments. If the identity of hsematoidin and bilirubin was settled beyond a doubt, then this view might be con- sidered as proved. Independently, however, of this identity, which is not admitted by all investigators, the view that the bile-pigments are derived from the blood-coloring matters has strong arguments in its favor. It has been shown by several experimenters that a ' Pflilger's Arch. , Bd. 9. ' Cit. from Stadelmann, Der Icterus, etc. » Arch. f. exp. Path. u. Pharm., Bd. 19. * Ibid., Bd. 21 FORMATION OF BILE PIGMENTS. 245 yellow or yellowish-red pigment can be formed from the blood- coloring matters, which gives Gmelin's test, and which, though it may not form a complete bile-pigment, is at least a step in its formation (Latschenbeegeb '). A farther proof of the formation of the bile-pigments from the blood -coloring matters consists in the fact that haematin yields urobilin, which is identical with hydro- bilirabin, on redaction (Hoppb-Setlek and others). Other inves- tigators (Nenoki and Siebee and Le Nobel") claim that the substance thus obtained is not true urobilin, but, all things consid- ered, it seems to be so very nearly related that this relationship can be considered as a proof of the formation of bilirubin from blood- pigments. Farther, hsematoporphyrin (see page 144) and bilirubin are isomers, according to Nencki and Siebee, and nearly allied. The formation of bilirubin from the blood-coloring matters is shown, according to the observations of several investigators j° by the ajjpearance of free hsemoglobin in the plasma — produced by the destruction of the red corpuscles by widely differing influences (see "below) or by the injection of haemoglobin solution — causing an increased formation of bile-pigments. The amount of pigments in the bile is not only considerably increased, but the bile-pigments may even pass into the urine under certain circumstances (icterus). After the injection of hsemoglobin solution into a dog either subcu- taneously or in the peritoneal cavity, Stadelmank and GoEO- DECKi ' observed in the secretion of pigments by the bile an increase of 61^ which lasted for more than twenty-four hours. If, then, iron-free bilirubin is derived froLx the haematin con- taining iron, then iron must be split off. This process may be represented by the following formula, according to Nenoki and Siebee,* C„H„N,0,Pe + 2H,0 - Fe = 3C,.H„N,0„ though in reality it is probably more complicated. The question in what form or combination the iron is split ofE is of special interest, and also whether it is eliminated by the bile. This latter does not seem to be the case. In 100 parts of bilirubin which are eliminated by the bile there are only 1.4-1.6 parts iron, according ' L. c. ' See Chapter VI on the blood, p. 144. ' See Stadelmann, Der Icterus, etc. ■• Arch. f. exp. Path. u. Pharm., Bd. 24. 246 THE LIVER. to Kttkkel ' ; while 100 parts lisematin contain about 9 parts iron. Minkowski and Basekin' have also found that the abundant formation of bile-pigments occurring in poisoning by arseniuretted hydrogen does not increase the quantity of iron in the bile. The quantity apparently does not correspond with that in the decom- posed blood-coloring matters. On the contrary, it seems as if the iron, at least for a. time, is re-tained by the liver as a pigment rich in iron. Such a pigment containing iron, which was formed by the decomposition of haemo- globin,, was observed by Nauktk and Minkowski ' in the livers of birds, in arseniuretted hydrogen icterus. Latschenbeegbr * claims that a yellow or yellowish-red pigment, " choleglolin,^'' is. derived from the blood-coloring matters, and acts as a step in the formation of the bile-pigments; and besides this he mentions another body consisting of dark grains and containing iron, which he designates as melanin. Neumann ' has observed in blood extravasations and thrombi, besides hsematoidin, a pigment con- taining iron, for which he has proposed the name hmmatosiderin. What relationship does the formation of bile-acids bear to the formation of bile-pigments ? Are these two chief constituents of the bile derived simultaneously from the same material, and can we detect a certain connection between the formation of bilirubin and bile-acids in the liver ? The investigations of Stadblmann ° teach us that this is not the case. With increased formation of bile- pigments the bile-acids decrease and the supply of hEsmoglobin to the liver acts in strongly increasing the formation of bilirubin, but simultaneously strongly decreases the production of bile-acids. According to Stadelmann the formation of bile-pigments and bile- acids is due to a special activity of the cells. An absorption of bile from the liver by the lymphatic vessels and the passage of the bile constituents into the blood aad urine occurs in retarded discharge of the bile, and usually in different forms of hepatogenic icterus. But bile-pigments may also pass into the nrine under other circumstances, especially in animals where a ' Pfluger's Arch., Bd. 14, S. 353. *ArcL. f. exp. Path. u. Pharm., Bd. 28. »L. c. * Ibid. ' Virchow's Arch., Bd. 111. • Der Icterus, etc. BILE C0NCREMENT8. 247 solution or destruction of the red blood-corpuscles takes place through injection of water or a solution of biliary salts, through poisoning by ether, chloroform, arseninretted hydrogen, phosphorus, or toluylendiamin ; and in other cases. This occurs also in man in grave infections diseases. We have therefore a second form of icterus, in which the blood-coloring matters are transformed into bile-pigments elsewhere than in the liver, namely, in the blood — a hcBmatogenic or anhepatogenic icterus. The occurrence of a hsema- togenic icterus has been made very probable by the investigations of Minkowski and Nausttn, Afanassiew, SiLBERMAiirN, and especially STAnELMANN. ' This statement has been proven in cer- tain of the above-mentioned cases, as after poisoning with phos- phorus, toluylendiamin, and arseninretted hydrogen, by direct experiment. The icterus is also in these cases heptogenic ; it depends upon an absorption of bile-pigments from the liver, and this absorption seems to originate in the different cases in somewhat different ways. Thus the bile may be viscous and cause a stowing of the bile by counteracting the low secretion pressure. In other cases the fine biliary passages may be compressed by an abnormal swelling of the liver-cells, or a catarrh of the bile-passages may occur causing a stowage of the bile (Stadelmann). The other forms of so-called haematogenic icterus are now explained in an analogous way. Bile Concretions. The concrements which occur in the gall-bladder vary consider- ably in size, form, and number, and are of three kinds, depending upon the kind and nature of the bodies forming their chief mass. One group of gall-stones contains lime-pigment as chief constituent, the other cholesterin, and the third calcium carbonate and phos- phate. The concrements of the last-mentioned group occur very seldom in man. The so-called cholesterin stones are those which occur most frequently in man, while the lime-pigment stones are not found very often in man, but often in oxen. The pigment-stones are generally not large in man, but in oxen and pigs they are sometimes found the size of a walnut or even larger. In most cases they consist chiefly of bilirubin-calcium with ' The literature belonging to this subject is found in Stadelmann, Der Icterus etc. Stuttgart, 1891. 248 THE LIVER. little or no biliverdin. Sometimes also small black or greenish black, metallic-looking stones are found, which consist chiefly of bilifascin along with biliverdin. Iron and copper seem to be regular constituents of pigment-stones. Manganese and zinc have also been found a few cases. The pigment-stones are generally heavier than water. The cholesterin-stones, whose size, form, color, and structure may vary greatly, are often lighter than water. The fractured surface is radiated, crystalline, and frequently shows crystalline, concentric layers. The cleavage fracture is waxy in appearance, and the fractured surface when rubbed by the nail also becomes like -wax. By rubbing against each other in the gall-bladder they often become faceted or take other remarkable shapes. Their surface is sometimes nearly white and waxlike, but generally their color is variable. They are sometimes smooth, in other cases they are rough or uneven. The quantity of cholesterin in the stones varies from 642-981 p. m. (Eittee'). The cholesterin-stones also some- times contain variable amounts of lime-pigments which give them a very changeable appearance. Cholesterin, C,,H„0, or, according to Obekmullee, C„H„0. •Cholesterin is generally considered as a monatomic alcohol of the formula OjjH„.OH. According to the investigations of Obee- MULLEE," who has analyzed several cholesterin compounds, it seems , that the formula is rather Cj,H^jOH. It yields a colored hydro- carbon, cholesterilin, with concentrated sulphuric acid, and this hydrocarbon is claimed by Wetl ' to be closely related to the terpene group. Cholesterin is also claimed to be closely allied to cholalic acid. Cholesterin occurs in small amounts in nearly all animal fluids and juices. It occurs only rarely in the urine, and then in very small quantities. It is also found in the different tissues and organs — especially abundant in the brain and the nervous system, — further in the yolk of the egg, in semen, and in wool-fat (together with isocholesterin). It appears also in the contents of the intes- tine, in excrements, and in the meconium. It occurs pathologi- cally especially in gall-stones, as well as in atheromatous cysts, in pus, in tuberculous masses, old transudations, cystic fluids, sputum, ' Journal de I'anat. et de la physiol. , 1873. ' Dn Bols-Reymond's Arch., 18S9, and Zeitschr. f. physiol. Chem., Bd. 15 •Ibid., 1896,8.182. 0H0LE8TERIN. 249 and tnmors. Several kinds of cholesterin seem to occur in the plant world. Cholesterin which crystallizes from warm alcohol on cooling, and that which is present in old transudations, contains 1 mol. of water of crystallization, melts at 145° C, and forms colorless, trans- parent plates whose sides and angles frequently appear broken and whose acute angle is often 76° 30' or 87° 30'. In large quantities it appears as a mass of white plates which shine like mother-of-pearl and have a greasy feel. Cholesterin is insoluble in water, dilute acids and alkalies. It is neither dissolved nor changed by boiling caustic alkali. It is easily soluble in boiling alcohol, and crystallizes on cooling. It dis- solves readily in ether, chloroform, and benzol, and also in the volatile or fatty oils. It is dissolved to a slight extent by alkali salts of the bile-acids. Among the many combinations of cholesterin studied by Obee- MULLER ' the propionic ester, C,IIj.CO.O.C„H„, is of special interest. This is used in the detection of cholesterin. For the detection of cholesterin we make use of its reaction with concen- trated sulphuric acid, which, as above stated, gives a colored hydro- carbon with this acid. If a mixture of five parts sulphuric acid and one part water acts on a cholesterin crystal, this crystal will show colored rings, first a bright carmine-red and then violet. This fact is made use of in the microscopic detection of cholesterin. Another test, and one very good for the microscopical detection of cholesterin, consists in treating the crystals first with the above dilute acid and then with some iodine solution. The crystals will be gradually colored violet, bluish green, and a beautiful blue. Salkowski's ' Reaction. — The cholesterin is dissolved in chloro- form and then treated with an equal volume of concentrated sul- phuric acid. The cholesterin solution becomes first bluish red, then gradually more violet-red, while the sulphuric acid appears dark red with a greenish fluorescence. If the chloroform solution is poured into a porcelain dish it becomes violet, then green, and finally yellow. Liebeemakk-Bukchard's ' Reaction. — Dissolve the cholesterin 'L. c. « Pflilger's Arch., Bd. 6. ' C. Liebermann, Ber. d. deutsoli. chem. Gesellsch., Bd. 18, S. 1805. H. Burohard, Beitrftge zur Kenntniss der Cholesterine . Rostock, 1889. 250 THE LIVEB. in about % c.c. chloroform and add first 10 drops acetic anhy- dride and then concentrated salphurie acid drop by drop. The mixture will first be beautiful red, then blue, and finally, if not too much cholesterin or sulphuric acid is present, a permanent green. In the presence of very little cholesterin the green color may appear immediately. Pare, dry cholesterin when fused in a test-tube over a low flame with 3 to 3 drops propionic anhydride yields a mass which on cooling is first violet, then blue, green, orange, carmine red, and finally copper-red. It is best to re-fuse the mass on a glass rod and then to observe the rod on cooling, holding it against a dark background (Obbrmullbk).' Schipf's Reaction. If a little cholesterin is placed in a porcelain dish with the addition of a few drops of a mixture of two to three vols. cone, hydrochloric acid or sulphuric acid and one vol. of a medium solution of ferric chloride, and carefully evaporated to dryness over a small flame, a reddish-violet residue is first obtained and then a bluish violet. If a small quantity of cholesterin is evaporated to dryness with a drop of concentrated nitric acid, we obtain a yellow spot which becomes deep orange- red with ammonia or caustic soda (not a characteristic reaction). iBOCholesterin. This body, so called by Schtjlzb," is isomeric with the ordinary cholesterin and occurs in wool-fat, and is therefore found in abundant quantities in so-called lanolin. It does not give Salkowski's reaction. It melts at 138-138°. 5. We make use of the so-called cholesterin-stones in the prepara- tion of cholesterin. The powder is first boiled with water and thei.'. repeatedly boiled with alcohol. The cholesterin which on cooling separates from the warm filtered solution is boiled with a solution of caustic potash in alcohol bo as to saponify any fat. After thi , evaporation of the alcohol we extract the cholesterin from the residue with ether, by which the soaps are not dissolved, filter, evaporate the ether, and purify the cholesterin by recrystallization from alcohol-ether. The cholesterin may be extracted from tissues and fluids by first extracting with ether and then purifying as above. It is detected and determined quantitatively in tissue, etc., by this same method. It is ordinarily easily detected in transudations and pathological formations by means of the microscope. ' L. c. 2 Ber. d. deutsch. chem. Qesellch., Bd. 6; Journal f. prakt. Chem., N. F. Bd. 25, S. 458; and Zeitschr. f. physiol. Chem., Bd. 14, S. 532. See also B. Schulze and J. Barbieri, Journal f. prakt. Chem. , N. P. Bd. 25, S. 159. CHAPTEE IX. DIGESTION. The purpose of the digestion is to separate those constituents of the food which serve as the nutriment of the body from those which are useless, and to separate each in such a form that it may be taken up by the blood from the alimentary canal and employed for the various purposes in the organism. This demands not only mechanical but also chemical action. The first action, which is essentially dependent upon the physical properties of the food, con- sists in a tearing, cutting, crashing, or grinding of the food, and serves chiefly to convert the nutritive bodies into a soluble and easily absorbed form, or in the splitting of the same into simpler combinations for use in the animal synthesis. The solution of the nutritive bodies may take place in certain cases by the aid of water alone, but in most cases a chemical metamorphosis or splitting is necessary, and is effected by means of the acid or alkaline fluids secreted by the glands. The study of the processes of digestion from a chemical standpoint must therefore begin with the digestive fluids, their qualitative and quantitative composition, as well as their action on the nutriments and foods. I. The Salivary Grlands and the Saliva. The salivary glands are partly albuminous glands (as the parotid in man and mammalia and the submaxillary in rabbits), partly mucous glands (as some of the small glands in the buccal cavity and the sublingual and submaxillary glands of many animals), and partly mixed glands (as the submaxillary gland in man). The alveoli of the albumin-glands contain cells which are rich in albumin, but contain no mucin. The alveoli of the mucin-glands contain cells rich in mucinogen or mucin but poor in albumin. 251 2.52 DiaESTION. Cells rich in proteid also occur in the submaxillary and suHingual glands between the mucous cells and the membrana propria, which in a few cases takes the form of a crescent (lunula, according to Glanuzzi), and in other cases the cells rich in mucin are sur- rounded as by a ring, and sometimes certain alveoli may be com- pletely filled. By continuous secretion the mncin-cells seem to give up all their mucin (Ewald, Stohe), so that only albumin-cells are to be seen (Heidenhaik'). During rest the mucin is re-formed. According to the analyses of Oidtman N ' the salivary glands of a dog contain 790 p. m. water, 300 p. m. organic and 10 p. m. inorganic solids. Among the solids we find mucin, proteids, amongst which nucleoalbumin or nucleoproteid, nuclein, diastatic enzyme and its zymogen,^ besides extractive bodies, leucin, xanthin bases, and mineral substances. The saliva is a mixture of the secretion of the above-mentioned groups of glands; therefore it is proper that we first study each of the different secretions by itself, and then the mixed saliva. The submaxillary saliva in man may be easily collected by intro- ducing a canula through the papillary opening into Wharton's duct. The submaxillary saliva has not always the same composition or properties; this depends essentially upon the conditions under which the secretion takes place. That is to say, the secretion is partly dependent on the cerebral, partly on the sympathetic, ner- vous system. In consequence of this dependence the two distinct varieties of submaxillary secretion are distinguished as chorda- and sympathetic saliva. A third kind of saliva, the so-called paralytic saliva, is secreted after poisoning with curara or after the severing of the glandular nerves. The difference between chorda- and sympathetic saliva (in dogs) consists chiefly in their quantitative constitution, namely, the less abundant sympathetic saliva is more viscous and richer in solids, especially in mucin, than the more abundant chorda-saliva. The specific gravity of the chorda-saliva of the dog is 1.0039-1.0056 and ' In regard to these conditions see text-books on histology and the article "Die Absonderungsvorgange " by Heidenhaiu in Hermann's Handbuch der Physiologie, Bd. 5, S. 57. ' Cit. from Gorup Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., S. 732. The figures there given amount to 1010 parts instead of 1000 parts. ' See especially Warren, Centralbl. f. Physiol., Bd. 8, S. 311. SALIVA. 253 contains from 12-14 p. m. solids (Eckhakd"). The sympathetic has a specific gravity of 1.0075-1.018, with 16-28 p. m. solids. The gases of the chorda-saliva Lave been investigated by Pflugek." He found 0.5-0.8^ oxygen, 0.9-1^ nitrogen, and 64.73-85.13^ carbon dioxide — all results calculated at 0° C. and 760 mm. pres- sure. The greater part of the carbon dioxide was chemically com- bined. The two kinds of submaxillary secretion just named have not thus far been separately studied in man. The secretion may be excited by a moral emotion, by mastication, and by irritating the mucous membrane of the mouth, especially with acid-tasting substances. The submaxillary saliva in man is ordi- narily clear, rather thin, a little ropy, and froths easily. Its reac- tion is alkaline. The specific gravity is 1.002-1.003, and it contains 3.6-4.5 p. m. solids.' We find as organic constituents mucin, traces of proteid and diastatic enzyme, which is absent in several species of animals. The inorganic bodies are alkali chlorides, sodium and magnesium phosphates, besides bicarbonates of the alkalies and calcium. Oehl* finds 0.036 p. m. potassium sulpho- cyanide in this saliva. The Sutlingaal Saliva. — The secretion of this saliva is also influenced by the cerebral and the sympathetic nervous system. The chorda-saliva, which is secreted only to a small extent, con- tains numerous salivary corpuscles, but is otherwise transparent and very ropy. Its reaction is alkaline and contains, according to Heidenhain,' 27.5 p. m. solids (in dogs). The sublingual secretion in man has been investigated by Oehl.' It was clear, mucilaginous, more alkaline than the sub- maxillary saliva, and contained mucin, diastatic enzyme, and potas- sium sulphocyanide. Buccal mucus can only be obtained pure from animals by the method of Bidder and Schmidt, which consists in tying the exit to all the large salivary glands and cutting off their secretion from the mouth. The quantity of liquid secreted under these circum- ' Cit. from Kllhne, Lebrb. d. physiol. Chem., S. 7. » Pfliiger's Arch. Bd. 1. 2 See Maly, Chemie der VerdauungssSfte und der Verdauung in Her- mann's Handb., Bd. 5, Th. 3, S. 18. * Canstatfs Jabresbericht d. Med., 1865, 1, S. 130. * Studien d. physiol. Instituts zu Breslau, Heft 4. •L. c. 254 DIQESTION. stances (in dogs) was so very small that the investigators named were able to collect only 2 grms. buccal mucus in the course of twenty-four hours. It is a thick, ropy, sticky liquid containing mucin; it is rich in form-elements, above all in flat epithelium- cells, mucous cells, and salivary corpuscles. The quantity of solids in the buccal mucus of the dog is, according to Bidder and Schmidt,' 9.98 p. m. Parotid Saliva. The secretion of this saliva is also partly dependent on the cerebral nervous system (n. glossopharyngeus) and partly on the sympathetic. The secretion may be excited by mental emotions and by irritation of the glandular nerves, either directly (in animals) or reflexly, by mechanical or chemical irrita- tion of the mucous membrane of the mouth. Among the chemical irritants the acids take first place, while alkalies and pungent sub- stances have little action. Sweet-tasting bodies, such as honey, are said to have no effect. Mastication has great influence in the secre- tion of parotid saliva, which is especially marked in certain herbivora. Human parotid saliva may be collected by the introduction of a canula into Stekson's duct. This saliva is thin, less alkaline than the submaxillary saliva (the first drops are sometimes neutral or acid), without special odor or taste. It contains a little albumin but no mucin, which is to be expected from the construction of the gland. It also contains a diastatic enzyme, which, however, is absent in many animals. The quantity of solids varies between 5 and 16 p. m. The specific gravity is 1.003-1.012. Potassium snlphocyanide seems to be present, though it is not a constant con- stituent. KiJLz" found 1.4=6^ oxygen, 3.2^ nitrogen, and in all 66.7^ carbon dioxide in human parotid saliva. . The quantity of firmly combined carbon dioxide was 62^. The mixed buccal saliva in man is a colorless, faintly opalescent, slightly ropy, easily frothing liquid without special odor or taste. It is made turbid by epithelium-cells, mucous and salivary corpus- cles, and often by food residues. Like the submaxillary and parotid saliva, on exposure to the air it becomes covered with an incrusta- tion consisting of calcium carbonate and a small quantity of an organic substance, or it gradually becomes cloudy. Its reaction is ' Die Verdauungssafte tind der StofEwechsel (Mitau and Leipzig, 1852), S. 5. « Zeitschr. f . Biologie, Bd. 23. PTTALIN. 255 alkaline, but occasionally also acid. According to Stickek,' fresh saliva may be acid a few hours after a meal. Two or three hours after breakfast and four to five hours after dinner the maximum of acidity occurs, and it may also be faintly acid from midnight to morning. The specific gravity varies between 1.003 and 1.008, and the quantity of solids between 5 and 10 p. m. The solids, irre- spective of the form-constituents mentioned, consist of albumin, mucin, ptyalin, and mineral bodies. It is also claimed that urea is a normal constituent of the saliva. The mineral bodies are alkali chlorides, bicarbonates of the alkalies and calcium, phosphates, and traces of sulphates and sulphocyanides. Salphocyanides, which, although not constant, occur in the saliva of man and certain animals, may be easily detected by first acidifying the saliva with hydrochloric acid and treating with a very dilute solution of ferric chloride. To make the test more conclusive it is best, as control, to take an equal quantity of acidified water and then add ferric chloride. Another, simpler method, proposftd by GscHEiDLBN," consists in putting in a drop or two of the saliva on filter-paper which has previously been -dipped in an amber- colored solution of ferric chloride containing hydrochloric acid, and then dried. Each drop of saliva containing sulphocyanide will give a reddish spot. If the quantity of sulphocyanide is so small that it cannot be detected directly, concentrate the saliva after the addition of a little alkali, acidify strongly with hydrochloric acid, and shake repeatedly with ether, evaporating the latter after the addition of water containing alkali over a gentle heat; then test the remaining liquid. Ptyalin, or salivary diastase, is the amylolytic enzyme of the saliva. This enzyme is found in human saliva, but not in that of all animals. It occurs not only in adults, but also in new-born infants. Zweifel' claims that the ptyalin in new-born infants occurs only in the parotid gland, but not in the submaxillary. In the latter it appears only two months after birth. According to H. Goldschmidt* the saliva (parotid saliva) of the horse does not contain ptyalin, but a zymogen of the same, while in other animals and man the ptyalin is formed from the zymogen during secretion. In horses the zymogen is transformed into ' Deutsch. med. Zeitung, 1889. Cit. from Centralbl. f, Physiol., Bd. 3, S. 237. ' Maly's Jahresber., Bd. 4, S. 91. ' Untersuchungen aber den Verdauungsapparat der Neugeborenen. Ber- IJn, 1874. * Zeitschr. f. physiol. Chem., Bd. 10. 256 DIGESTION. ptyalia daring mastication, and the bacteria seem to give the impulse to this change. During precipitation with alcohol the zymogen is changed into ptyalin. Ptyalin has not been isolated in a pare form up to the present time. It can be obtained purest by Cohnhbim's ' method, -which consists in carrying the enzyme down mechanically with a calcium- phosphate precipitate and washing the precipitate with water, which dissolves the ptyalin, and from which it can be obtained by precipi- tating with alcohol. For the study or demonstration of the action of ptyalin we may use a watery or glycerin extract of the salivary glands, or simply the saliva itself. Ptyalin, like other enzymes, is characterized by its action. This consists in converting starch into dextrin and sugar. In regard to the process going on in this conversion we are not quite clear. In general it may be described as follows: In the first stages soluble starch or amidulin is formed. From this amidulin, erythrodextrin and sugar are produced by hydrolytic cleavage. The erythrodextrin then splits into a-achroodextrin and sugar. From this achroodextrin by splitting /S-achroodextrin and sugar are formed, and finally this /J-achroodextrin splits into sugar and y- achroodextrin. According to a few investigators the number ot dextrins formed, as intermediate steps is difEerent. It is only within a very short time that we have been made clear as to the kind of sugar produced in this process. For a long time it was considered that dextrose was the sugar formed from starch and glycogen, but SEEGEisr " and 0. Kassb ' have shown that this is not true. MuscuLUS and v. Meeiitg ' have shown that the sugar formed by the action of saliva, amylopsin, and diastase upon starch and glycogen is in greatest part maltose. This has been substantiated by Beowst and Heron.' Lately E. Kulz and J. Vogel° have demonstrated that in the saccharification of starch and glycogen isomaltose, maltose, and some dextrose are formed, the varying quantities depending upon the amount of ferment and length of 1 Virchow's Arch. , Bd. 28. = Centralbl. f. d. med. Wissenscli., 1876, S. 851, and PflUger's Arch Bd. 19. * Pfliiger's Arch., Bd. 14. * Zeitschr. f. physiol Chem., Bd. 3. ' Liebig's Annalen, Bdd. 199 and 304. « Zeitschr. f. Biologie, Bd. 31. ACTION OF PTTALIN. 257 time of digeBtion. As, according to Tebb,' the salivary glands, as well as the pancreas, contain an inverting enzyme, it is still un- decided whether the formation of dextrose is due to the diastatic enzyme or to the invertin alone. According to Eohmakn and Hamburger '' the saliva contains diastase and glucase. The same is true for the pancreatic juice and intestinal juice. In relation to the blood-serum all tLese secretions are relatively poor in glucase, and this is especially true for saliva. Ptyalin is not identical with malt diastase. It is most active at about + 40° C, while, according to Chittendbk and MARTiif,' LiNTNER, and Eckhard,* malt diastase is most active at + 50° to 55° C. The action of ptyalin in various reactions has been the subject of numerous investigations.' Naturally the alkaline saliva is very active, but it is not as active as when neutral. It may be still more active under circumstances in faintly acid reaction, and according to Chittendeit and Smith it acts better when enough hydrochloric acid is added to saturate the proteids present than when only simply- neutralized. When the acid combined proteid exceeds a certain amount, then the diastatic action is diminished. The addition of alkali to the saliva decreases its diastatic action; on neutralizing the alkali with acid or carbon dioxide the retarding or preventive action of the alkali is arrested. According to Schibrbbck carbon dioxide has an accelerating action in neutral liquids, while Ebstbiit claims that it has as a rule a retarding action. Organic as well as inor- ganic acids, when adde^ in sufficient quantity, may stop the dias- tatic action entirely. The degree of acidity necessary in this case is not always the same for a certain acid, but is dependent upon the quantity of ferment. The same degree of acidity in the presence of large amounts of ferment has a weaker action than in the pres- ■ Journal of Physiol., Vol. 15. ' Ber. d. deutsch. cliem. Gesellsch., Bd. 27, and PflUger's Arch., Bd. 60. • Studies from the Laborat. of Physiol. Chem. of Tale College, Vol. 1, 1885. * Journ. f. prakt. Chem., N. F. Bd. 41. 5 See Hammarsten, Maly's Jahresber., Bd. 1 ; Chittenden and Griswold, ibid., Bd. 11 ; Langley, Journal of Physiol., Vol. 3 ; Nylen, Maly's Jahresber., Bd. 13, S. 341 ; Chittenden and Ely, ibid., S. 343 ; Langley and Eves, Jour- nal of Physiol., Vol. 4 ; Chittenden and Smith, Yale College Studies, Vol. 1, 1885, p. 1 ; John, Centralbl. f. 'kim. Med., Bd. 12 ; Schlesinger, Virchow'a Arch , Bd. 135 ; Shierbeck, Skand. Arch. f. Physiol., Bd. 3; Ebstein and C. Schulze, Virchow's Arch., Bd. 134. ■358 DIGESTION. ence of smaller quantities. Hydrochloric acid is of special physio- logical interest in this regard, namely, it prevents the formation of sugar even in very small amounts (0.03 p. m.). Hydrochloric acid has not only the property of preventing the formation of sugar, but, as shown by Langlet, Ntlen, and others, may entirely destroy the enzyme. This is important in regard to the physiological sig- nificance of the saliva. That boiled starch (paste) is quickly, and unboiled starch only slowly, converted into sugar is also of in- terest. Various kinds of unboiled starch are converted with difEerent degrees of rapidity. The rapidity with which ptyalin acts increases, at least under conditions otherwise favorable, with the amount of enzyme and with an increasing temperature to a little above +40° 0. Foreign sub- stances, such as metallic salts,' have different effects. Certain salts even in small quantities completely arrest the action ; for example, HgCl, accomplishes this result by the presence of only 0.05 p. m. Other salts, such as magnesium sulphate, in small quantities (0.35 p. m.) accelerate, and in larger quantities (5 p. m.) check the action. The presence of peptone has an accelerating action on the sugar formation (Ohittbndek and Smith and others). The accumulation of the products of the amylolytic decomposition also checks the action of the saliva. This has been shown by special experiments made by Sh. Lea.' He made parallel experiments with digestions in test-tubes and in dialyzers, and found on the removal of the products of the amylolytic decomposition by dialysis that the formation of sugar took place quicker, but also that consid- erably more maltose and less dextrin was formed. To show the action of saliva or ptyalin on starch the three ordinary tests for dextrose may be used, namely, Moore's or Tkommer's test or the bismuth test (see Chapter XV). It is also necessary, as a control, to first test the starch-paste and the saliva for the presence of dextrose. The steps formed in the transforma- -tion of starch into amidulin, erythrodextrin, and achroodextrin may be shown by testing with iodine. The quantitative composition of the mixed saliva must vary con- siderably, not only because of individual differences, but also hecause under varying conditions there may be an unequal division 1 See O. Nasse, Pflilger's Arch., Bd. 11, and Chittenden and Painter, Tale U^ollege Studies, Vol. 1, 1885, p. 52. ' Journ. of Physiol., Bd. 11. COMPOSITION OF THE SALIVA. 259 of the secretion from the differeut glands. We give below a few analyses of human saliva as example of its composition. The results are in parts per 1000. Water Solids Mucus and epithelium Soluble organic substances . (Ptyalin of early investigators), Sulphocyanides Salts n g S S 5 ' » g g H 1 S 1 ^ B ^ to 1 g IS a 1^ 992.9 995.16 994.1 988.3 994.7 7.1 4.84 5.9 11.7 5.8 3.5-8.4 in filtered saliva. 1.4 1.62 3.13 3.8 1.34 1.42 8.27 0.06 0.10 0.064 to , 0.09 1.9 1.82 2.19 1.03 994.2 5.8 2.3 1.4 0.04 3.2 Hammbkbacheb found in 1000 parts of the ash from human saliva; potash 457.2, soda 95.9, iron oxide 50.11, magnesia 1.55, sulphuric anhydride (SOs) 68.8, phosphoric anhydride (PjOb) 188.48, and chlorine 183.53. The quantity of saliva secreted during 34 hours cannot be exactly determined, but has been calculated by Bidder and Schmidt" to be 1400-1500 grms. The most abundant secretion occurs daring meal-times. According to the calculations and determinations of Tuczbk ' in man, 1 grm. of gland yields 13 grms. secretion in the course of one hour during mastication. These figures correspond fairly well with those representing the average secretion from 1 grm. of gland in animals, namely, 14.2 grms. in the horse and 8 grms. in oxen. The quantity of secretion per hour may be 8 to 14 times greater than the entire mass of glands, and there is probably no gland in the entire body, as far as we know at present — the kidneys not excepted — whose ability of secretion under physiological conditions equals that of the salivary glands. A remarkably abundant secretion of saliva is induced by pilocarpin, while atropin, on the contrary, prevents it. Though an abundant secretion of saliva is produced, as a rule, ' Zeitschr. f. physiol. Chem., Bd. 5. The other analyses are cited from Maly, Chemie der VerdauungssSfte, Hermann's Handbuch d. Physiol., Bd. 5. Th. 2, S. 14. 'L. c, S. 13. ' Zeitschr. f. Biologie, Bd. 13. 260 DIGESTION. by an increased supply of blood, still it is not a simple filtration process, as seen from the following circnnistances. The secretion- pressure is greater than the blood-pressure in the carotid, and in poisoning by atropin, which paralyzes the secretory nerves, an increased supply of blood is produced by irritation of the chorda, but no secretion. The salivary glands have moreover a specific property of eliminating certain substances, such as potassium salts (Salkowski),' iodine, and bromine combinations, but not others, such as iron combinations. It is also noticeable that the saliva is richer in solids when it is eliminated quickly by gradually increased irritation, and in larger quantities than when the secretion is slower and less abundant (Heidenhain)." The amount of salts increases also to a certain degree by an increasing rapidity of elimination (Hbidenhain, Weethee,' Langlet and Pletchbk,' Kovi'). The chemical changes taking place during secretion are un- known, but it is probable that, like the secretion processes in general, the secretion of saliva is closely connected with the pro- cesses in the cells. The chemical processes going on in these cells during secretion are still unknown. Heidbnhaii^ claims that the" mucin cells of the submaxillary gland are destroyed during secretion, and in the period of rest the mucin or mucinogen reappears in these cells. Ewald' claims that they only discharge their mucin. These observations still do not throw any light upon the chemical processes going on. The Physiological Importance of the Saliva. The quantity of water in the saliva renders possible the effects of certain bodies on the organs of taste, and it also serves as a solvent for a part of the nutritive substances. The importance of the saliva in mastication is especially marked in herbivora, and there is no question of its importance in facilitating the act of swallowing. The power of converting starch into sugar does not belong to the saliva of all animals, and even when it possesses this property the intensity varies in diflerent animals. In man, whose saliva forms sugar rapidly, a formation of sugar from (boiled) starch undoubtedly takes place in the mouth, but how far this action goes on after the morsel ' Vircliow's Arch., Bd. 53. « Pflilger's Arch., Bd. 17. 3 Ibid., Bd. 38. * Proo. Roy. Soc, Vol. 45, and especially Philos. Trans., Vol. 180, ' Du Bois-Eeymond's Arch., 1888. « See Heidenhain in Hermann's Haudb., Bd. 5, Th. 1, S. 64, etc. OLANDS OF TES STO.VAOH. 261 has entered the stomach depends upon the rapidity with which the acid gastric juice mixes with the swallowed food, and also upon the relative amounts of the gastric juice and food in the stomach. The large quantity of water which is swallowed with the saliva must be absorbed and pass into the blood, and it must go through an intermediate circulation in the organism. Thus the organism possesses in the saliva an active medium by which a constant stream, conveying the dissolved and finely divided bodies, passes into the blood from the intestinal canal during digestion. Salivary Conorements. The so-called tartar is yellow, gray, yellowish gray, brown or black, and has a stratified structure. It may contain more thaji 200 p. m. organic substances, which consist of mucin, epithelium, and lbpto- THKIX-CHAINS. The chief pait of the inorganic constituents consists of calcium carbonate and phosphate. The salivary calculi may vary in size from that of a small grain to that of a pea or still larger (a salivary calculus has been found weighing 18.6 grms.), and it contains a variable quantity of organic sub- stances, 50-380 p. m., which remain on extracting the calculus with hydro- •chloric acid. The chief inorganic constituent is calcium carbonate. II. The Glands of the Mucous Membrane of the Stomach, and the Gastric Juice. Since of old, the glands of the mucous coat of the stomach have been divided into two distinct kinds. Those which occur in the greatest abundance and which have the greatest size in the fundus are called fundus glands, also rennin or pepsin glands. Those which occur only in the neighborhood of the pylorus have received the name of pyloric glands, sometimes also, though incorrectly, called mucous glands. The mucous coating of the stomach is covered throughout with a layer of columnar epithelium which is generally considered as consisting of goblet cells that produce mucus by a metamorphosis of the protoplasm. The fundus glands contain two kinds of cells : adelomorphic or chief cells, and delomoephic or parietal cells, the latter formerly called rennin or pepsin cells. Both kinds consist of protoplasm rich in proteids; but their relationship to coloring matters seems to show that the albuminous bodies of both are not identical. The nucleus must consist chiefly of nuclein. Besides the above-mentioned constituents the fundus glands contain as more specific constituents two zymogens, which are the mother-substances of the pepsin and the rennin, besides a small quantity of fat and cholesterin. The pyloric glands contain cells which are generally considered 262 DIQESTION. as related to the above-mentioned chief cells of the fundus glands. As these glands were formerly thought to contain a larger quantity of mucin, they were also called mucous glands. According to Hbidenhaist, independent of the columnar epithelium of the. excretory ducts, they take no part worthy of mention in the forma- tion of mucus, which, according to his views, is effected by the epithelium covering the mucous membrane. The pyloric glands also seem to contain the zymogens referred to above. Alkali chlorides, alkali phosphates, and calcium phosphates are found in the mucous coating of the stomach. Liebbrmann' has obtained an acid-reacting residue on digesting the mucosa of the stomach with pepsin hydrochloric acid, which strangely contained no nuclein, but only a proteid containing lecithin, called lecithalbumin. To this lecithalbumin he ascribes a great importance in the secretion of hydrochloric acid (see below). The Gastric Juice. The observations of Helm" and Beau- mont ' on persons with gastric fistula led to the suggestion that, gastric fistulas be made on animals, and this operation was first per- formed by Bassow * in 1843 on a dog. Veesteuil " performed the same on a man in 1876 with successful results. These fistulas in animals afford an excellent means of studying the secretion of gastric juice and also the stomachic digestion. In a fasting condition the mucous coat is often nearly dry; sometimes, especially in certain herbivora, it is covered with a layer of viscid so-called mucus. If food is introduced into the stomach, or if the mucous membrane is irritated in some way, then a secre- tion of a thin, acid fluid, the real gastric juice, takes place. The secretion may be produced by mechanical or thermal irritation (introduction of cold water or pieces of ice into the stomach), or by chemical irritants. Among the latter we include alcohol and ether, which when in too great concentration do not produce a physio- logical secretion, but a transudation of a neutral or faintly alkaline fluid containing albumin. To this class of irritants belong carbon ' Pfluger's Arch., Bd. 50. 3 Helm, Zwei Krankengeschichten. Wien, 1803. Cit. from Hermann's Handbuch, Bd. 5, Th. 3, S. 39. 2 "The Physiology of Digestion," 1833. * Bull, de la soc. des natur. de Moscou, Tome 16. Cited from Maly in Hermann's Handbuch, Bd. 5, S. 38. 5 See Ch. Richet, Du sue gastrique chez I'homme et les animaux Paris 1878, p. 158. GASTRIC JUICE. y(j;3 dioxide and hydrochloric acid; the last especially increases the secretion of pepsin (Jaworsky'), spices, meat extracts, neutral salts, such as NaCl (which acts like alcohol in too great concentra- tion), and alkali carbonates. The alkali carbonates are supposed by certain investigators to first neutralize the acid and then produce a continuous secretion of acid gastric juice. The statements in regard to the action of diSerent bodies on the secretion of gastric juice are still rather uncertain and often contradictory. The secretion of gastric juice is reflexly stimulated from the mouth. After the introduction of water into the stomach a rela- tively scanty and not less constant flow of secretion takes place ; while on the contrary if digestible food is introduced a more abun- dant and continuous secretion is observed (Schifp," Heidenhain'). But in these cases the secretion does not take place immediately,, but only after the absorption of the soluble bodies has commenced. This fact justifies the usual custom of commencing a meal with fluid nutritives, snch as soup. The beautiful experiments made by Pawlow and Schoumow-Simanowsky ' have shown that the secretion of gastric juice is stimulated reflexly from the mouth, and also that this reflex is discontinued on cutting through the vagi, and that the secretion in the stomachic glands is caused by the central nervous system through special secretory nerve-flbres, analo- gous to the secretion of saliva and pancreatic juice. The Qualitative and Quantitative Composition of the Gastric Juice. The gastric juice, which can hardly be obtained pure and free from residues of the food or from mucus and saliva, is a clear, or only very faintly cloudy, and in man nearly colorless fluid of an insipid, acid taste and strong acid reaction. It contains, as form- elements, glandular cells or their nuclei, mucus-corpuscles, and more or less changed columnar epithelium. The acid reaction of the gastric juice depends on the presence of free acid, which, as we have learned from the investigations of C. Schmidt,' Richet," and others, consists, when the gastric juice is pure and free from particles of food, chiefly or nearly so of hydro- ' Deutsch. med. Wocliensclir., 1887. = Lepons sur la pbysiol. de la digestion, Tome 2, 1867. ' Pflilger's Arch., Bd. 19. / * Du Bois-Reyinond's Arch., 1895. = Bidder and Schmidt, Die Verdauuugssafte, etc., S. 44. " L. c. 264 DIGESTION. chloric acid. Ooktejean ' has regularly found traces of lactic acid ill the pure gastric juice of fasting dogs. After partaking of food, especially after a meal rich in carbohydrates, lactic acid occurs abundantly, and sometimes acetic and butyric acids. The quantity of free hydrochloric acid in the gastric Juice of sheep is about 1.3 p. m., and in dogs, according to the ordinary statements, about 2-3 p. m. ScHOUMOw-SiMANOWSKT ^ has observed a considerably higher degree of acidity in perfectly pure and fresh gastric juice of a dog, namely, 4,6-5.8 p. m. Eiasantsew ' states that the gastric juice of the cat is very similar to that of the dog and has about the same degree of acidity, 4.11-5.84 p. m., and an average of 5.20 p. m. EiCHET * found as average for 80 determinations of human gastric juice 1.7 p. m. free hydrochloric acid, with a variation between 0.5 and 3 p. m. According to Szabo,' Bwald,° and others, the human gastric juice contains usually about 2-3 p. m. HCl. EiCHET has shown that the acid gastric juice acts in many respects different from free hydrochloric acid of the same concen- tration, and he concludes from this that the hydrochloric acid is not free, but combined with organic substances (lencin). Contejean is of the same opinion, and has found that gastric juice dissolves •cobalt hydrocarbonate with more difficulty and slower than a hydrochloric acid of the same concentration. Perfectly fresh gastric jaice seems to contain a little coagulable proteid, but contains peptone and albumoses on standing for some time. Among the organic bodies a little mucin is found and two enzymes, pepsin and rennin, especially in man. The sulphocyanic acid found by Kelliistg ' in the contents of the stomach is consid- ered by Nencki and Schoumow-Simanowsky " as a normal con- stituent of pure saliva-free gastric juice of dogs. The specific gravity of gastric juice is low, 1.001-1.010. It is therefore correspondingly poor in solids. As examples of the ' Contrib, a I'etude de la physiol. de restomac. Thesis. Paris, 1892. Maly's Jahresber., Bd 23, S. 393. ' Arch. f. exp. Path. u. Pharm., Bd. 33. ' Arch, des Sciences biol. de St. Petersbourg, Tome 3. «L. c. ' Zeitschr. f. physiol. Chem., Bd. 1. * C. A. Ewald, Klinik der Verdauungskrankheiten, 1890. ' Zeitschr. f. physiol Chem., Bd. 18. * Arch. f. exp. Path. u. Pharm., Bd, 34, and Ber. d. deutsch. Chem. Gesellsch. , Bd. 38. PEP8IN. 265 compositioa of different kinds of gastric juice the analyses of C. Schmidt ' are here given. It must be remarked that the human gastric juice analyzed was diluted by saliva and water and should therefore not be considered as normal. The figures are parts per 1000. Water Solids Organic substance NaCl CaClj KCl NH4CI Free hydrocUoric acid (HCl). Ca,(PO,)j Mg,(P04)j FePO. Human Gas- tric Juice mixed with Saliva. 994.40 5.60 3.19 1.46 0.06 0.55 0.30 0.13 Gastric • Juice from Dog free from Saliva. 973.0 27.0 17.1 3.5 0.6 1.1 0.5 3.1 1.7 0.3 0.1 Gastric Juice from Dog contain- ing Saliva. 971.3 28.8 17.3 3.1 1.7 1.1 0.5 2.3 3.3 0.3 0.1 Gastric Juice of Sheep. 986.15 13.85 4.05 4.36 0.11 1.53 0.47 1.33 1.18 0.57 0.33 The other physiologically important constituents of gastric juice are pepsin and rennin. Pepsin. This enzyme is found, with the exception of certain fishes, in all vertebrates thus far investigated. Pepsin occurs in adults and in new-born infants. This condi- tion is different in new-born animals. While in a few herbivora, such as the rabbit, pepsin occurs in the mucous coat before birth, this enzyme is entirely absent at the birth of those carnivora which have thus far been examined, such as the dog and cat. In various invertebrates a ferment has also been found wMcb has a prote- olytic action in acid solutions. It has been shown that this enzyme, neverthe- less, is not in all animals identical with ordinary pepsin. Darwin has fur- ther found that certain plants which feed upon insects secrete an acid juice which dissolves proteid, but it is still doubtful whether these plants contain any pepsin, v. Gorup-Bbsanbz ' has isolated from vetch-seed an enzyme which acts like pepsin, but whose identity with pepsin is doubtful. Pepsin is as difiicult to isolate in a pure condition as other enzymes.' The purest pepsin was that prepared by Bbucke and ' Cit. from v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., S. 494. » Ber. d. deutsch. chem. Qesellsch., Bdd. 7 and 8. » Schoumow-Simanowsky, Arch. f. exp. Path. u. Pharm., Bd. 33, has ob served that the pure, fresh gastric juice of a dog deposits a protein substance containing chlorine, on cooling, and this he considers as pure pepsin. This substance is, however, precipitated by certain proteid reagents which even do 266 DIGESTION. SuKDBERG ; this gave negative results with most reagents for pro- teids. Pepsin, therefore, does not seem to be a true albuminoua substance. It is, at least in the impure condition, solable in water and glycerin. It is precipitated by alcohol, but only slowly- destroyed. It is quickly destroyed by heating its watery solution to boiling. According to Bieknacki ' pepsin in neutral solutions is destroyed by heating to + 55° C. In the presence of 3 p. m. HCl a temperature of 55° C. is without action; the pepsin is destroyed by heating to 65° C. for five minutes. On adding peptone and certain salts the pepsin may be heated to 70° C. with- out decomposing. In the dry state it can, on the contrary, be heated to over 100° 0. without losing its physiological action. The only property which is characteristic of pepsin is that it dissolves proteid bodies in acid, but not in neutral or alkaline, solutions with the formation of albnmoses and peptones. The methods for the preparation of relatively pure pepsin depend, as a rule, upon its property of being thrown down with finely divided precipitates of other bodies, such as calcium phos- phate or cholesterin. The rather complicated methods of Bkucke'' and Sundbekg' are based upon this property. A relatively pure pepsin solution intended for digestion tests and of efEective action may be prepared by the following method as suggested by Malt.* The mucous membrane (of the pig's stomach) is treated with water containing phosphoric acid, and the filtrate precipitated by lime- water ; the precipitate, which contains the pepsin, is then dissolved in water by the addition of hydrochloric acid, and the salts removed by dialysis, by which means the pepsin which does not diffus^T remains in the dialyzer. A pepsin solution somewhat impure but rich in pepsin, and which can be kept for years, may be obtained if, as suggested by v. Wittichs/ we extract the finely divided mucous membrane with glycerin, or better with glycerin which contains 1 p. m. HCl. To each part by weight of the mucous coat add 10-20 parts glycerin. This is filtered after 8-14 days. The pepsin (together with much albumin) may be precipitated by alcohol from this extract. If this extract is to be used directly for digestion tests, then to 100 c. c. of water which has been acidified with 1-4 p. m. HCl add 2-3 c. c. of the extract. not precipitate very powerful commercial pepsin, and tlierefore it cannot be a pure enzyme. ' Zeitschr. f. Biologie, Bd. 38. ' Wien. Sitzungsber. , Bd. 43. ' Zeitscbr. f. pbysiol. Cbem., Bd. 9. * Pfliiger's Arcb., Bd. 9. » Ibid., Bd. 2. ACTION OF PEPSIN. <2,Q'J For digestion tests an infusion of the mucous membrane of the stomach may be used directly in many cases. The mucous coat is carefully washed with water (if a pig's stomach is used) and finely cut; if a calf's stomach is employed, only the outer layer of the mucous coat is scraped off with a watch-glass or the back of a knife. The pieces of mucous membrane or the slimy masses obtained by scraping are rubbed with pure quartz-sand, treated with acidified water, and allowed to stand for 24 hours in a cool place and then filtered. In the preparation of artificial gastric juice that part only of the mucous coat richest in pepsin is used ; the pyloric part is of little value. A strong, impure infusion may generally be obtained from the pig's stomach, while a relatively pure and powerful infusion is obtained from the stomach of birds (hens). The stomachs of fish (pike) also yield a tolerably pure and active infusion. An active and rather pure artificial gastric juice may be prepared by scraping the inner layers of a calf's stomach from which the pyloric end has been removed. For a medium-sized calf's stomach 1000 c. c. of acidified water must be used. The degree of acidity required in the infusion depends upon the use to which the gastric juice is to be put. If it is to be employed in the digestion of fibrin, an acidity of 1 p. m. HCl must be selected, while, on the contrary, if it is to be used for the digestion of hard-boiled-egg albumin, an acidity of 2-3 p. m. HCl is prefer- able. This last-mentioned degree of acidity is generally the better, because the infusion is preserved thereby, and at all events it is so rich in pepsin that it may be diluted with water until it has an acidity of 1 p. m. HCl without losing any of its solvent action on unboiled fibrin. The preparation of acid infusions is nowadays unnecessary on account of the ability of getting various pepsin preparations in commerce which have a remarkable activity. Such a pepsin preparation can be purified when necessary by following the method suggested by Kuhke.' Precipitate the pepsin together with the albumoses by ammonium sulphate, press the precipitate and dis- solve in dilute hydrochloric acid, and let it undergo auto-digestion. On repeating this again and then i-emoving the salts by dialysis we obtain an extraordinarily active pepsin, but which is still less pure than when obtained by the methods of Bbdcke and Sundbbkg. Hie A ction of Pepsin on Proteids. Pepsin is inactive in neutral or alkaline reactions, but in acid liquids it dissolves coagulated albuminous bodies. The proteid always swells and becomes trans- parent before it dissolves. Unboiled fibrin swells up in a solution containing 1 p. m. HCl, forming a gelatinous mass, and does not dissolve at ordinary temperature within a couple of days. Upon ' Zeitsohr. f. Biologie, Bd. 23, S. 428. 268 DIQESTION. the addition of a little pepsin, however, this swollen mass dissolves quickly at an ordinary temperature. Hard-boiled-egg albumin, cut in thin pieces with sharp edges, is not perceptibly changed by dilute acid (3-4 p. m. HCl) at the temperature of the body in the course of several hours. But the simultaneous presence of pepsin causes the edges to become clear and transparent, blunt and swollen, and the albumin gradually dissolves. From what has been said above in regard to pepsin, it follows that proteids may be employed as a means of detecting pepsin in liquids. Fibrin may be employed as well as hard-boiled-egg albu- min, which latter is used in the form of slices with sharp edges. As the fibrin is easily digested at the normal temperature, while the pepsin test with egg-albumin requires the temperature of the body, and as the test with fibrin is somewhat more delicate, it is often preferi'ed to that with egg-albumin. When we speak of the ^'pepsin te^" without further explanation, we ordinarily under- stand it as the test with fibrin. This test nevertheless requires care. The fibrin used should be ox fibrin and not pig fibrin, which last is dissolved too readily with dilute acid alone. The unboiled fibrin may be dissolved by acid alone without pepsin, but this generally requires more time. In testing with unboiled fibrin at normal temperature, it is advisable to make a control test with another portion of the same fibrin with acid alone. Since at the temperature of the body unboiled fibrin is easier dissolved by acid alone, it is best always to work with boiled fibrin. As pepsin has not, thus far, been prepared in a positively pure condition, it is impossible to determine the absolute quantity of pepsin in a liquid. It is only possible to compare the relative amounts of pepsin in two or more liquids, which may be done in several ways. As the best of these we give the following method as suggest by Brucke. If two pepsin solutions A and B are to be compared with each other rela- tively to the amounts of pepsin they contain, they must first be brought to the proper degree of acidity, about 1 p. m. HCl, care being taken that one is not more diluted than the other. Then prepare a large number of specimens of each solution by diluting with hydrochloric acid of 1 p. m. HCl, so that they contain respectively J, \, |, y\, ^^, and so on, the amount of pepsin in the original liquid being 1. If the original quantity of pepsin in the two liquids is designated by p and p', we then have the two series of liquids : A B Ip Ip' iP iP' iP W JisP tW ^jP IsP ACTION OF PEPSIN. 269 TUen a small piece of boiled-egg albumin, obtained by cutting thin slices ■with a cork-cutter, is placed in each test, or a small flake of fibrin is added Of course care must be taken to add the same-sized slice of egg-albumin or flake of fibrin. Now observe and note exactly the time when each test of the two (Series begins to digest and when it ends, and it will be found that certain tests of one series make about the same progress as certain tests of the other series. It may be inferred from this that they contain about the same quan- tity of pepsin. As example, it is found in one series of tests that the digestive rapidity of the tests phP^-P-h^^ about the same as the tests p' i, p' i, p'l; therefore we conclude that the liquid A is about four times as rich in pepsin as the liquid B. Another method as suggested by Mkttb ' gives more exact results according to the investigations of Samojloff. « Draw up liquid white of egg in a glass tube of about 1 to 2 mm. diameter and coagulate the albumin in the tube by heating, cut the ends of the tube off sharply, add two tubes to each test-tube with a few cc. of acid pepsin solution, allow to digest at the bodily tempera- ture, and after a certain time measure the lineal extent of the digested layer of albumin in the various tests. From the rapidity of digestion expressed from the extent of the digested layer, we can calculate the relative quantity of pepsin, according to the rule first found by Schutz,* that the energy of diges- tion of different pepsin solutions is the square root of the quantity of pepsin it contains. This rule, however, only applies to sufficiently dilute pepsin solu- tions. The rapidity of the pepsin digestion depends on several circum- stances. Thus different acids are unequal in their action; hydro- chloric acid shows a more powerful action than any other, whether an organic or an inorganic acid. The degree of acidity is also of the greatest importance. With hydrochloric acid the degree of acidity is not the same for different proteid bodies. For fibrin it is 0.8-1 p. m., for myosin, casein, and vegetable albumin about 1 p. m., for hard-boiled-egg albumin, on the contrary, about 3.5 p. m. The rapidity of the digestion increases, at least to a certain point, with the quantity of pepsin present, unless the pepsin added is contaminated by a large quantity of products of digestion, which may prevent its action. The accumulation of products of digestion has a retarding action on digestion, although, according to Chit- tendek: and AMEHMAiir,' the removal of the digestion products by means of dialysis does not essentially change the relationship be- tween the albumoses and true peptones. Pepsin acts slower at low temperatures than it does at higher. It is even active in the neighborhood of 0°C., but digestion takes place very slowly at this temperature. With increasing temperature the rapidity of diges- tion also increases until about 40° C, when the maximum is reached. ' Cited from SamoilofE. See foot-note 8. ' Arch, des Sciences biol. de St. PStersbourg, Tome 2, S. 699. 3 Zeitschr. f. physiol. Chem., Bd. 9, S. 577. * Journal of Physiol., 1893. 270 DIGESTION. According to the investigations of Platjm' it is probable that the relationship between albumoses and peptones remains the same, irrespective of whether the digestion took place at a low or high temperature as long as the digestion is continuous for some time. If the swelling up of the proteid is prevented, as by the addition of neutral salts, such as NaCl in sufficient amounts, or by the addition of bile to the acid liquid, digestion can be prevented to a greater or less extent. Foreign bodies of different kinds produce different ac- tions, in which naturally the variable quantities in which they are added are of the greatest importance. Salicylic acid and carbolic acid hinder the digestion, while arsenious acid promotes it (Chit- TENDBisr), and hydrocyanic acid is relatively indifferent. Alcohol in large quantities (10^ and above) disturbs the digestion, while small quantities act indifferently. Metallic salts in very small quan- tities may indeed sometimes accelerate digestion, but otherwise they tend to retard it. The action of metallic salts in different cases can be explained in different ways, but they often seem to form with proteJds insoluble or difficultly-soluble combinations. The alkaloids may also retard the pepsin digestion (CHiTTEiirDEN and Allbh').' A very large number of observations have been made in regard to the action of foreign substances on artificial pepsin digestion, but as these observations have not given any direct result in regard to the action of these same substances on natural digestion, we will not here further discuss them. Tlie Products of the Digestion of Proteids hy Means of Pepsin and Acid. In the digestion of nucleoproteids or nucleo-albumins an insoluble residue of nuclein or pseudo-nuclein always remains. With experiments on casesin Salkowski' has shown that the para- nuclein first split off which contains according to Willdenow,* phosphorus in organic combination, may be dissolved by continuous digestion. Some orthophosphoric acid is hereby split off, but an organic phosphorized acid is also formed. Fibrin also yields an insoluble residue, which consists, at least in great part, of nuclein, derived from the form-elements enclosed ' Zeitschr. f. Biologie, Bd. 28. » Tale College Studies, Vol. 1, p. 76. See also Chittenden and Stewart, iMd., Vol. 3, p. 60. ' Centralbl. f. d. med. Wissensch., 1893, S. 385 and 467. See also Sontag, ibid., S. 419, and Moraczewski, Zeitschr. f. physiol, Chem., Bd. 30. •* " Zur Kenntuiss der peptischen Verdauung des Kaseins." Inaug Diss Bern., 1893. PRODUCTS OF THE ACTION OF PEPSIN. 21 1 in the blood-clot. This residue which remains in the digestion of certain albuminous bodies is called dyspeptone by Meissnbr. ' If . the solution is filtered after a finished digestion and neutralized, it gives in different cases a more or less abundant precipitate of acid albuminate, or a mixture of albuminates called parapeptone by Meissnek. After filtering this precipitate and concentrating the filtrate again, some proteid often separates in the warmth. If this precipitate be filtered, the filtrate now contains alhumoses SLndi pep- tones in the ordinary sense, while the so-called true peptone of KuHKE may sometimes be entirely absent, and in general is obtained in quantity worth mentioning only after a more continu- ous and intensive digestion. The relationship between the albu- moses and peptones in the ordinary sense changes very much in different cases and in the digestion of various albuminous bodies. For instance, a larger quantity of primary albumoses is obtained from fibrin than from hard-boiled egg albumin or from the pro- teids of meat. In the digestion of unboiled fibrin an intermediate product may be obtained in the earlier stages of the digestion — a globulin which coagulates at + 55° C. (Hasebeoek''). For information in regard to the different albumoses and peptones which are formed in pepsin digestion, the reader is referred to pre- vious pages (33-39). Action of Pepsin Hydrochloric Acid on other Bodies. The gela- tin-forming substance of the connective tissue, of the cartilage and of the bones, from which last the acid only dissolves the inorganic substances, is converted into gelatin by digesting with gastric juice. The gelatin is further changed so that it loses its property of gelatinizing and is converted into a so-called gelatin peptone (see page 55). True mucin (from the submaxillary) is dissolved by the gastric juice and yields a substance similar to peptone and a reducing substance similar to that obtained by boiling with a mineral acid. Elastin is dissolved more slowly and yields the above-mentioned substances (page 52). Keratin and the epidermis formation are insoluble. Nuclein is not dissolved and the cell- nuclei are therefore insoluble in gastric juice. The animal cell- membrane is, as a rule, more easily dissolved the nearer it stands to elastin, and it dissolves with greater difficulty the more closely it is ' The works of Meissner on pepsin digestion are found in Zeitschr. f. rat. Med., Bdd. 7, 8, 10, 12, and 14. ' Zeitschr. f. physiol. Chem., Bd. 11. 272 DIGESTION. related to keratin. The membrane of the plant-cell is not dissolved. OxyhwmogloUin is changed into haematin and acid albuminate, the latter undergoing further digestion. It is for this reason that blood is changed into a dark-brown mass in the stomach. The gastric juice does not act on fat, but, on the contrary, on fatty tissue, dissolving the cell-membrane, setting the fat free. Gastric juice has no action on starch or the simple varieties of sugar. The statements in regard to the ability of gastric juice to invert cane- sugar are very contradictory. At least, this action of the gastric juice is not constant, and, according to Voit, ' if it is present at all, it is probably due to the action of the acid. Pepsin alone, as above stated, has no action on proteids, and an acid of the intensity of the gastric juice can only very slowly, if at all, dissolve coagulated albumin at the temperature of the body. Pepsin and acid together not only act more quickly, but qualita- tively they act otherwise than the acid alone. If liquid proteid is digested with hydrochloric acid of 2 p.m., it is converted into acid albuminates ; but if pepsin is previously added to the acid, the formation of syntonin takes place essentially slower under the same conditions (Meissnbe). From this it is inferred that a part of the hydrochloric acid is combined with the pepsin, and we have here a proof of the existence of a paired acid, called by C. Schmidt pepsin hydrochloric acid. It has been further suggested that this hypothetical acid is possibly decom- posed in digestion into free pepsin and free hydrochloric acid, which in statu naseendi dissolves proteids to a certain degree. The pepsin set free reunites with a new portion of acid, forming pepsin hydrochloric acid, and in contact with proteids is further decomposed as above described. It is hardly necessary to mention that this statement is only an unproved hypothesis. Renniu or chtmosin is the second enzyme of the gastric juice. It occurs in human gastric under physiological conditions, but may be absent under special pathological conditions, such as carcinoma, atrophy of the mucous membrane, and certain chronic catarrhs (Boas, Johnson, Klempbkbb).' It is habitually found in the neutral, watery infusion of the fourth stomach of the calf and sheep, especially in an infusion of the fundus part. In other mammals and in birds it is seldom found, and in fishes hardly ever in the neutral infusion. ' Zeitschr. f. Biologie, Bd. 28, also contains the literature. ' A good review of the literature may be found in Szydlowski, Beitrag zur Kenntuiss des Labenzym nach Beobachtungen an Sauglingen, Jahrb. f. Kin- derheilkunde, N. F., Bd. 34. RENNIN. ' 273 In these cases a rennin-forming substance, & rennin zymogen, occurs which is converted into rennin by the action of an acid. Eennin is just as difficult to prepare in a pure state as the other enzymes. , The purest rennin enzyme thus far obtained did not give the ordinary proteid reactions. On heating its solutions it is destroyed, and indeed more easily in acid than in neutral solutions. If an active and strong infusion of a mucous coat in water containing 3 p. m. HCl is heated to 37-40° C. for 48 hours, the rennin is destroyed, while the pepsin remains. A pepsin solu- tion free from rennin can be obtained in this way. Eennin is characterized by its physiological action, which consists in coagu- lating milk or a casein solution containing lime, if neutral or very faintly alkaline. Eennin may be carried down by other precipitates like other enzymes, and thus may be obtained relatively pure. It may also be obtained, contaminated with a great deal of proteids, by extract- ing the mucous coat of the stomach with glycerin. A comparatively pure solution of rennin may be obtained in the following way. An infusion of- the mucous coat of the stomach in hydrochloric acid is prepared and then neutralized, after which it is repeatedly shaken with new quantities of magnesium carbonate until the pepsin is precipitated. The filtrate, which should, act strongly on milk, is precipitated by basic lead acetate, the precipi- tate decomposed with very dilute sulphuric acid, the acid liquid: filtered and treated with a solution of stearin soap. The rennin is carried down by the fatty acids set free, and when these last are placed in water and removed by shaking with ether, the rennin remains in the watery solution. A fasting animal may secrete a strongly-acid gastric juice. The acid of the gastric juice then cannot be derived from the foods, but must originate in the mucous coat. As the pyloric glands, which contain no parietal cells, secrete an alkaline secretion according to Hbidenhain ' and Klemensibwicz, ' while the fundus glands, which contain these cells, yield an acid secretion, it is generally assumed with IIeidenhain' that the parietal cells are of special importance in the secretion of free hydrochloric acid — a statement which other observations tend to confirm. The later investigations of Fbankel' and Coktejean' seem to ' Pflilger's Arch., Bdd. 18 and 19. See also Hermann's Handbuch, Bd. 5, Th. 1, " AbBonderungsvorgange." ' Wien. Sitzungsber, Bd. 71. » Pflilger's Arch., Bdd. 48 and 50. ■• Contribution &, I'ltude de la physiol. de I'estomac, Th6se, Paris, 1893. Also Maly's Jahresber., Bd. 33, S. 393. 274 ' DiaESTION. contradict this statement. They claim that the chief cells as well as the parietal cells take part in the formation of acid. That the hydrochloric acid must originate from the chlorides of the blood is evident, and Kahn ' has given a direct proof for this. He found in dogs that after a sufficiently long common-salt starva- tion that the stomach secreted a gastric juice containing pepsin but no free hydrochloric acid. On the administration of soluble chlo- rides a gastric juice containing hydrochloric acid was immediately secreted. We do not know how the secretion of free hydrochloric .acid originates. Whereas it used to be considered that the chlorides were decomposed by an electrolysis or by organic acids produced in the mucosa, we now rather generally accept the process as suggested by Malt. Malt ' has called attention to the fact that, on account of the presence of a large quantity of free carbon dioxide in the blood and the avidity of the same, there must be present among the numerous combinations of acids and bases which exist in the serum traces of free hydrochloric acid in addition to acid salts. As these traces of hydrochloric acid are removed from the blood by means of rapid diffusion by the glands, the mass-action of the carbon dioxide must set free new traces of hydrochloric acid in the blood. In this way may. be explained the secretion in the blood of large quantities of hydrochloric acid from the chlorides, but the proof that the hydrochloric acid set free passes into the gastric juice simply by diffusion, is missing. Similar processes in other animal glands render it probable that here, as in other cases of secretion, we have to deal with a yet unexplained specific secretory action of the glandular cells. L Liebermann^ has lately proposed a new theory for the secretion of hydro- chloric acid. According to him lecithalbumin occurs in the glandular cells, and this combines readily with alkalies. The more active metabolism in the glands during work leads to an abundant formation of carbon dioxide, and this carbon dioxide by its mass-action sets hydrochloric acid free from the chlorides. The hydrochloric acid passes into the secretion by diffusion, while the alkalies com- bine with the lecithalbumin. In regard to details of this theory we must refer the reader to the original article. Nencki and Schoumow-Simanowsky ' have confirmed, on dogs, theo bservations of KuLz, ' namely, that the introduction of ' Zeitschr. f. physiol. Chem., Bd. 10. = lUd., 1. ' Pfluger's Arch., Bd. 50. •• Arch, des Sciences biol. de St. Petersboug, Tome 3. ' Zeitschr. f . Blologie, Bd. 23. PROPEPSIN. 215 alkali bromides or iodides causes a replacement of the hydrochoric acid of tlie gastric juice by HBr or to a smaller extent by HI. On the determination of the quantity of chlorine in the Tarious tissues and fluids under normal conditions and after the adminis- tration of NciBr they have shown that bromine can also replace the chlorine in the organism. After an abundant meal, when the store of pepsin in the stom- ach is completely exhausted, Schiff claims that certain bodies, especially dextrin, have the property of causing a supply of pepsin in the mucous membrane. This "charge theory," though experi- mentally proved by several investigators, has nevertheless not yet been confirmed. On the contrary, the statement of Schiff' that a substance forming pepsin, a "pepsinogen" or ^'propepsin," occurs in the ventricle has been proved. Langlet' has shown positively the existence of such a substance in the mucous coat. This sub- stance, propepsin, shows a comparatively strong resistance to dilute alkalies (a soda solution of 5 p. m.), which easily destroy pepsin (Langlet). Pepsin, on the other hand, withstands better than propepsin the action of carbon dioxide, which quickly destroys the latter. The occurrence of a rennin zymogen in the mucous coat has been mentioned above. The question in which cells the two zymogens, especially the propepsin, are produced has been extensively discussed for several years. Formerly it was the general opinion that the parietal cells were pepsin cells, but since the investigations of HEiDENHAiKr and his pupils, Langlbt and others, the formation of pepsin has been shifted to the chief cells. Peankel and Contejean have lately presented objections to the views of HBiDBKHAiiir that certains cells produce the zymogens and others only the acid. The Pyloric Secretion. That part of the pyloric end of the dog's stomach which contains no fundus glands was dissected by Klemensibwicz, one end being sewed together in the shape of a blind sack and the other sewed into the stomach. From the fistula thus created he was able to obtain the pyloric secretion of a living animal. This secretion is alkaline, viscous, jelly-like, rich in mucin, of a specific gravity of 1.009-1.010, and containing 16.5-20.5 p. m. solids. It has no effect on fat, but acts, though very slowly, on starch, converting it mto sugar, and contains • Le9ons sur la physiol. de la digestion, 1867, Tome 3. ' Langley and Edkins, Journ. of Physiol. , Vol. 7. 276 DIGESTION. ordinarily pepsin, which sometimes occurs in considerable amounts-. This has been observed by Hbidbn-hain in permanent pyloric fistula. Contejean' has investigated the pyloric secretion in other ways, and finds that it contains both acid and pepsin. The alkaline reaction of the secretions investigated by HeidenhaiN" and Klemeistsiewicz is due, according to Contejean, to an abnor- mal secretion caused by the operation, because the stomach readily yields an alkaline juice instead of an acid one under abnormal conditions. Akekman" has found, in accordance with Heiden- HAiN and KLEMEifSiEWicz, that the pyloric secretion of a dog was alkaline. He could never detect free acid, but always pepsin and rennin. The secretion of the juice of the stomach is dependent to a great extent upon the excitement acting on the mucous coat of the stomach, and it follows from this that the quantity of secretion under different conditions must vary considerably. The statements of the quantity of gastric juice secreted in a certain time are there- fore so unreliable that they need not be taken into account. The Chyme and the Digestion in the Stomach. By means of the mechanical irritation of the mucous coat of the stomach, as well as by the chemical irritation caused by the food and saliva, an abundant secretion of gastric juice occurs. The food is thereby freely mixed with liquid and is gradually converted into a pulpy mass, called the chyme. This mass is acid in reaction, and, with the exception of the interior of large pieces of meat or other solid foods, the chyme is acid throughout. The transformation products of the digestion of proteids and carbohydrates can be detected in the chyme; likewise more or less changed undigested residues of swallowed food, which indeed form the chief mass of the chyme. In the chyme morsels of meat more or less changed are found which, when unboiled meat is partaken of, may be much swollen and slippery; Muscle and caetilage are also often swollen and slippery, while pieces of bone sometimes show a rough and uneven surface after the digestion has continued for some time, which de- pends upon the fact that the gelatinous substances of the bone are attacked more quickly by the gastric juice than the earthy parts. Milk coagulates in the stomach by the combined action of the ren- nin and the acid, but in certain cases by the action of the acid ■ L. c. » Skand. Arch. f. Physiol., Bd. 5. DIGESTION IN THE STOMACH. 277 alone. From the relative quantities of the swallowed milk to the other food either large and solid lumps of cheese are formed or smaller lumps or grains which are divided in the pulpy mass. Cow's milk regularly yields large, solid masses or lumps; human milk gives, on the contrary, a fine, loose coagulum or a fine precip- itate which is immediately dissolved in part by the acid liquid. The milk-sugar may pass into lactic-acid fermentation, and this, accord- ing to RiCHET, is the reason why the acid reaction of the contents of the stomach is greater at the end of the digestion of a meal con- sisting mainly of milk. Bread, especially when not too fresh, is converted rather easily into a pulpy mass in the stomach. Other vegetable foods, such as POTATOES, may, if not sufficiently masticated, often be found in the contents of the stomach, very little changed, several hours after a meal. Staech is not converted into sugar by the gastric juice, but in the first phases of the digestion, before a large quantity of hydro- ■chloric acid has accumulated, it seems that the action of the saliva ■continues, and therefore the presence of dextrin and sugar can be detected in the contents of the stomach. Besides this the carbohy- drates in the stomach may in part undergo a lactic-acid fermenta- tion, caused by the micro-organisms present. According to the investigations of Ellenbeegek and Hoff- meistee' on horses and pigs, after a meal rich in amylaceous bodies in the first phase of the digestion, an amtioltsis takes place with the formation of lactic acid; then gastric juice containing hydro- chloric acid is secreted, when a second phase in which peotbolysis takes place. As a rule, the formation of lactic acid decreases as the .secretion of hydrochloric acid increases. Ewald and Boas'' claim that a similar condition also exists in human beings. They claim that there is in the first stage of digestion a predominance of lactic acid in the stomach, in the second a simultaneous occurrence of lactic and hydrochloric acids, and in the third stage almost exclu- sively hydrochloric acids. Kjaeegaaed' has lately formed the same conclusions from his investigations on children and robust persons. In nersons with altered blood-vessels due to senility the ' Maly's Jahresber., Bdd. 15 and 16. ' Virchow's Arch., Bd. 101. ' Om. Ventrikelfordbjelsen hos sunde Mennesker, Kjobenhavn, 1888. See Maly's Jahresber., Bd. 19. 278 DIGESTION. contents of the stomacli show chiefly the presence of lactic acid. Such persons digest large amounts of carbohydrates, while the- digestion of albuminous bodies is decreased. From recent investi- gations, making use of his new method of estimating lactic acid. Boas ' considers that after partaking of carbohydrates lactic acid does not occur in the stomach under normal conditions nor during the continual lack of hydrochloric acid. Lactic acid, on the con- trary, is regularly found in carcinoma. The FATS which are not fluid at the ordinary temperature melt in the stomach at the temperature of the body and become fluid. In the same way the fat of the fatty tissues is set free in the stomach by the gastric juice which digests the cell-membrane. The gastric juice itself seems to have no action on fats." The soluble salts of the food naturally are found dissolved in the liquids of the contents of the stomach; but the insoluble salts may also be dis- solved by the acid of the gastric juice. Since the hydrochloric acid of the gastric juice prevents the contents of the stomach from fermenting with the generation of gas, those gases which occur in the stomach probably depend, at least in great measure, upon the swallowed air and saliva, and upon those gases generated in the intestine and returned thraugh the pyloric valve. Planek' found in the stomach-gases of a dog 66-68^ N, aS-SS/'^ CO,, and only a small quantity, 0.8-6.1^ of oxygen. Schibebeck * has shown that a part of the carbon di- oxide is formed by the mucous membrane of the stomach. The tension of the carbon dioxide in the stomach corresponds, accord- ing to him, to 30-40 mm. Hg in the fasting condition. It increases after partaking food, independently of the kind of food, and may rise to 130-140 mm. Hg during digestion. The curve of the carbon- dioxide tension in the stomach is the same as the curve of acidity in the different phases of digestion, and Schibebeck has also found that the carbon-dioxide tension is considerably increased by pilo- carpin, but diminished by nicotin. According to him, the carbon dioxide of the stomach is a product of the activity of the secretory cells. ' Berlin, klin. Wochenschr., 1895. ' See Contejean, " Sur la digestion gastriquedelagraisse," Arch., de Physi- ologie, (5) Bd. 6. 3 Wien. Sitzungsber., Bd. 42, 1860, * Skan. Arch. f. Physiol., Bdd. 3 and 5. DIGESTION IN THE STOMACH. 273 According as the food is finely or coarsely divided it passes sooner or later througli the pylorus into the intestine. From Busch's' observations on a human intestinal fistula, it required generally 15-30 minutes after eating for undigested food, such as pieces of meat, to pass into the upper part of the small intestine. In a case of duodenal fistula in a human being observed by KUHNE," he saw, ten minutes after eating, uncurdled but still coagulable milk and small pieces of meat pass out of the fistula. The time in which the stomach unburdens itself of its contents depends, however, upon the rapidity with which the quantity of hydrochloric acid increases, for it seems to act as a sort of irritant and causes the opening of the pylorus. Many other conditions- also come into play, namely, the activity of the gastric juice, the quantity and character of the food, etc., etc., and therefore the time required to empty the stomach must be variable. Kichet' observed in a case of stomachic fistula that in man the quantity of food which is in the stomach the first three hours is not essentially changed, but that in the course of a quarter of an hour nearly all is driven out, so that only a small residue remains. Kuhste * has made about the same observations on dogs and human beings. He found, indeed, in dogs that in the first nour small quantities of meat passed into the intestine every ten minutes ; but he also observed that in dogs, on an average, about five hours after eating, in man somewhat earlier, a free emptying into the intestine takes place. According to other investigators, the emptying of the human stomach does not take place suddenly, but gradually, Beaumont '' found in his extensive observations on the Canadian hunter, St. Maetin, that the stomach, as a rule, is emptied 1^-5^ hours after a meal, depending upon the character of the food. The time in which different foods leave the stomach depends also upon their digestibility. In regard to the unequal digestibil- ity in the stomach of foods rich in proteids, which really form the object of the action of the gastric juice, a distinction must be made between the rapidity with which the proteids are converted into albnmoses and peptones and the rapidity with which the food is ' Vircliow's Arch., Bd. 14. ' Lelirbuch d. physiol. Chem., S. 53. ^ ' L. c. ' ^Ibid. 280 DIGESTION. converted into chyme, or at least so prepared that it may easily pass into the intestine. This distinction is especially important from a practical standpoint. When a proper food is to be decided upon in cases of diminished stomachic digestion, it is important to select such foods as, independent of the diificulty or ease with which their proteid is peptonized, leave the stomach easily and quickly, and which require as little action as possible on the part of this organ. Prom this point of view those foods, as a rule, are most digestible which are fluid from the start or may be easily liquefied in the stomach ; but these foods are not always the most digestible in the sense that their proteid is most easily peptonized. As an example, hard-boiled white of egg is more easily peptonized than fluid white of egg at a degree of acidity of 1-3 p. m. HCl ;' nevertheless we consider, and justly, that an unboiled or soft-boiled egg is easier to digest than a hard-boiled one. Likewise uncooked meat, when it is not chopped very fine, is not more quickly but more slowly peptonized by the gastric juice than the cooked, but if it is divided sufficiently fine it is often more quickly, peptonized than the cooked. The greater or less facility with which the different albuminous foods are peptonized by the gastric juice has been comparatively little studied, and as the conditions in the stomach are more com- plicated, results obtained with artificial gastric juice are often of no value for the practising physician and should in any case be used only with the greatest caution. Under these circumstances we cannot enter more deeply into this subject, but the reader is referred to text-books on dietetics and the study of foods. As our knowledge of the digestibility of the different foods in the stomach is slight and dubious, so also our knowledge of the action of other bodies, such as alcoholic drinks, bitter principles, spices, etc, on the natural digestion is very uncertain and imper- fect. The difficulties which stand in the way of this kind of in- vestigation are very great, and therefore the results obtained thus far are often ambiguous or conflict with each other. For example, certain investigators have observed that small quantities of alcohol or alcoholic drinks do not prevent but rather facilitate digestion ; others observe only a disturbing action; while other investigators believe to have found that the alcohol first acts somewhat as a dis- ' Wawrinsky, TJpsala Lakarefs. Fdrli., Bd. 8; see also Maly's Jahresber., Bd. 3. THE aroMAcn as a diqestive organ. 281 turbing agent, but afterwards, when it is absorbed, it produces an abundant secretion of gastric juice, and thereby facilitates digestion (Gluzinski," Chittenden"). The digestion of sundry foods is not dependent on one organ alone, but divided among several. For this reason it is to be expected that the various digestive organs can act for one another to a certain point, and that therefore the work of the stomach could be taken up more or less by the intestine. This in fact is the case. Thus the stomach of a dog has been almost completely extirpated (Czbrny," Caevallo, and Panchon '), and also that part necessary in the digestive process has been eliminated by plugging the pyloric opening (Ludavig and Ogata '), and in both cases it was possible to keep the animal alive, well fed, and strong. In these cases it is evident that the digestive work of the stomach was taken up by the intestine. That the stomach nevertheless, during normal conditions, bears an essential part of the process of digestion may be inferred from the fact that the products of pro- teolysis can generally be detected in the contents of the human stomach even shortly after a meal. By tests on dogs that had been given meat-powder, Cahn ° found large quantities of peptone in the stomach, and this progressed to the same extent as the diges- tion, although absorption took place, as shown by Sohmidt- MULHEIM. ' It is, however, quite generally assumed that no peptonization of the proteids worth mentioning occurs in the stomach, and that the albuminous foods are only prepared in the stomach for the real digestive processes in the intestine. That the stomach serves in the first place as a storeroom follows from its shape, and this func- tion is of special value in certain new-born animals, for instance in dogs and cats. In these animals the secretion of the stomach con- tains only hydrochloric acid but no pepsin, and the casein of the milk is converted by the acid alone into solid lumps or a solid coagulum which fills the stomach. Small portions of this coagulum 1 Deutsch. Arcli. f. klin. Med., Bd. 39. » Centralbl. f. d. med. Miss., 1889, S. 435. ' Czerny, Beitrage zui- operativen Cliirurgie. Stuttgart, 1878. Cited from Bunge, Lelirbucb der physiol u. path. Chem. , p. 150. * Arch, de physiol., (5) Tome 7, p. 106. ' Du Bois-Reymond's Arch, 1883. • Zeitschr. f. klin. Med., Bd. 12. ■> Du Bois-Reymond's Arch., 1879, S. 39. 282 DIGESTION. pass in to the intestine only little by little, and an overburdening of the intestine is thus prevented. In other animals, such as the snake and certain fishes, which swallow their food entire, it is certain that the major part of the process of digestion takes place in the stomach. The importance of the stomach in digestion cannot at once be decided. It varies for different animals, and it may vary in the same animal, depending upon the division of the food, the rapidity with which the peptonization takes place, the more or less rapid increase in the amount of hydrochloric acid, and so on. It is a well-known fact that the contents of the stomach may be kept without decomposing for some time by means of hydrochloric acid, while, on the contrary, when the acid is neutralized a fer- mentation commences by which lactic acid and other organic acids are formed. The hydrochloric aeid of the gastric juice has unques- tionably an anti-fermentive' action, and also, like dilute mineral acids, an antiseptic action. This action is of importance, as many disease micro-organisms may be destroyed by the gastric juice. The comma bacillus of cholera is killed by the normal acid gastric juice, while if it is introduced into the stomach after an injection of a soda solution it may remain active. Also varieties of pyogenic streptococcus and the staphylococcus pyog. aureus are killed by the acid gastric juice. Still the gastric juice does not act on all micro- organisms, and especially in the state of spores they can withstand its action. As an example, the tubercle-virus is not destroyed by the gastric juice, and the spores of the anthrax bacteria are not always destroyed by the hydrochloric acid of the gastric juice.' Because of this action ° the chief importance of the gastric juice is now considered to be its antiseptic action. In opposition to this view Cabvallo and Pachon have shown in a dog with extirpated stomach that putrefying meat could be partaken of without disturb- ing the digestion. After death, if the stomach still contains food, digestion goes on of itself not only in the stomach, but also in the neighboring organs,, during the slow cooling of the body. This leads to, the question, why does the stomach not digest itself during life? Ever since ' See Kiihne's Lehrbuch, p. 57; Bunge, Lehrbuch, pp. 142 and 153; F. Cobn, Zeitscbr. f. pbysiol. Chem., Bd. 14; Hirsclifeld, Pfliiger's Arcb., Bd. 47. ^ See Falk, Vircbow's Arcb., Bd. 93; E. Prank, Deutscb. med. Wocb- enscbr., 1884, No. 34; R. Kocb, ibid., 1884, No. 45. ^ Bunge, 1. 0. ABNORMAL BTOMACEIO DIGESTION. 283 Pavt ' has shown that after tying the smaller blood-vessels of the stomach of dogs the corresponding part of the mucous membrance was digested, efforts have been made to find the cause in the neutralization of the acid of the gastric juice by the alkali of the blood. That the reason for the non-digestion during life is to be sought for in the normal circulation of the blood cannot be contra- dicted ; but it is more probably found in the fact that the living mucous coat nourished by the alkaline blood shows quite different absorption, diffusion, and filtration properties than the dead mucous coat. This last was shown long ago by Eanke." Under pathological conditions irregularities in the secretion as well as in the absorption and in the mechanical work of the stomach may occur. Pepsin is almost always present, but the absence of the rennin, as above stated, may occur in many cases (Boas, JoHNSOK, Klempeebr'). In regard to the acid, it should be mentioned that sometimes this secretion may be increased so that an abnormally acid gastric juice is secreted, and sometimes may be decreased so that little or hardly any hydrochloric acid is secreted. A hypersecretion of acid gastric juice sometimes occurs. In the secretion of too little hydrochloric acid the same conditions appear as after the neutralization of the acid contents of the stomach out- side of the organism. Fermentation processes now appear in which, besides lactic acid, there appear also volatile fatty acids, such as butyric and acetic acids, etc., and gases like hydrogen. These fermentation products are therefore often found in the stomach in cases of chronic catarrh of the stomach, which may give rise to belching, pyrosis, and other symptoms. Among the foreign substances found in the contents of the stomach we have ueba, or ammonium carbonate derived therefrom in uraemia; blood, which generally forms a dark-brown mass through the presence of hasmatin, due to the action of the gastric juice; bile, which, especially during vomiting, easily finds its way through the pylorus into the stomach, but whose presence seems to be without importance. If it is desired to test the gastric juice or the contents of the stomach tor pepsin, fibrin may be employed. If this is thoroughly ' PWlos. Transactions, Vol. 153, Part 1, and Guy's Hospital Reports, Vol. 13. 2 See Ranke, Grundzilge der Pliyaiol., 3. Aufl.. 1875, S. 111-120. » See foot-note, p. 372. -284 DIGESTION. ■washed immediately after beating the blood, well pressed a^d placed ia glycerin, it may be kept in serviceable condition an indefinitely long time. The gastric juice or the matter contained in the stomach — the latter, if necessary, having been previously diluted with 1 p. m. hydrochloric acid — ^is filtered and tested with fibrin at ordinary temperature. (It must not be forgotten that a control test must be made with acid alone and another portion of the same fibrin.) If the fibrin is not noticeably digested within one or two hours, no pepsin is present, or at most there are only slight traces. In testing for rennin the liquid must be first carefully neutral- ized. To 10 c. c. unboiled amphoteric (not acid) reacting cow's milk add 1-2 c. c. of the filtered neutralized liquid; but care must be taken not to add too much of the liquid from the stomach, for the coagulation may be retarded or prevented by diluting the milk. In the presence of rennin the milk should coagulate to a solid mass at the temperature of the body in the course of 10-20 minutes without changing its reaction. If the milk is diluted too much by the addition of the liquid of the stomach, only coarse flakes are obtained and no solid coagulum. Addition of lime-salts is to be avoided, as they in great excess may produce a partial coagulation even in the absence of rennin. In many cases it is especially important to determine the degree of acidity of the gastric juice. This may be done by the ordinary titration methods. Pheuol phthalein must not be used as an indi- cator, for we get too high results in the presence of large quantities of proteids. Good results may be obtained, on the contrary, by using very delicate litmus-paper. As the acid reaction of the con- tents of the stomach may be caused simultaneously by several acids, still the degree of acidity is here, as in other cases, expressed in only one acid, e.g., HCl. Generally the acidity is expressed by the If number of c. c. of — caustic soda which is required to neutralize the several acids in IDO c. c. of the liquid of the stomach. An acidity of 43^ means that 100 c. c. of the liquid of the stomach N required 43 c. c. of — caustic soda to neutralize it. The acid reaction may be partly due to free acid, partly to acid salts (monophosphates), and partly to both. According to Leo ' we can test for acid phosphates by calcium carbonate, which is not neutralized therewith, while the free acids are. If the gastric con- tent has a neutral reaction after shaking with calcium carbonate and the carbon dioxide is driven out by a current of air, then it contains only free acid; if it has an acid reaction, then acid phos- phates are present; and if it is less acid than before, it contains ' Centralbl. f. d. med. Wissensch., 1889, S. 481, and Diagnostik der Krank- heiten der Verdauungsorgane (Berlin, 1890) ; also Pfliiger's Arch., Bd. 48, S. 614. DETECTION OF ACID IN THE STOMACH. 285 both free acid and acid phosphate. This method can also be applied in the estimation of free acid (see below). It is also important to be able to ascertain the nature of the acid or acids occurring in the contents of the stomach. For this pur- pose, and especially for the detection of free hydrochloric acid, a great number of color reactions have been proposed, which are all based upon the fact that the coloring substance gives a character- istic color with very small quantities of hydrochloric acid, while lactic acid and the other organic acids do not give these colorations, or only in a certain concentration, which can hardly exist in the contents of the stomach. These reagents are a mixture of ferbio ACETATE and POTASSIUM SULPHOCTANIDB Solution (Mohe's reagent has been modified by several investigators), iiETHYLANiLiif-TiOLET, TROP^OLiN^ 00, Congo red, malachite-geebn, phlobogluoin- VANiLLiK, BENzoptJEPUBiK 6 B, and others. As reagents for free lactic acid IJFFELMANif suggests a strongly diluted, amethyst-blue solution of EEEEic CHLOEiDB and caebolic acid or a strongly diluted, nearly colorless solution of feeeic chloeide. These give a yellow with lactic acid, but not with hydrochloric acid or with volatile fatty acids. Instead of the untrustworthy lactic-acid reac- tion of Uffelmann, Boas ' makes use, in the detection and estima- tion of lactic acid, of the property of lactic acid of being oxidized into aldehyde and formic acid on careful oxidation with sulphuric acid and manganese dioxide. The aldehyde is detected by its forming iodoform with an alkaline iodine solution or by its forming aldehyde mercury with Nesslbe's reagent. The quantitative N estimation consists in the formation of iodoform with — iodine solution and caustic potash, adding an excess of hydrochloric acid and titrating with a -r-x sodium arsenite solution and retitrating with iodine solution, after the addition of starch-paste, until a blue coloration is obtained. The value of these reagents in testing for free hydrochloric acid or lactic acid is still disputed." Among the reagents for free hydro- chloric acid, Mohe's test (even though not very delicate), GuNz- bueg's test with phloroglucin-vanillin, and the test with tropseolin 00, performed in moderate heat as suggested by Boas, seem to be the most valuable. If these tests give positive results, then the presence of hydrochloric acid may be considered as proved. A negative result does not eliminate the presence of hydrochloric acid, as the delicacy of these reactions has a limit, and also the simul- taneous presence of proteid, peptones, and other bodies influences ' Deutsch. med. Wochensclir., 1893, and Milnchener med. Wocliensclir., 1893. ' In regard to the extensive literature on this question we refer to v. Jaksch, Klinische Diaguostik innerer Krankheiten, 4. Aufl., 1896, Section 5. 286 DIGESTION. ^he reactions more or less. The reactions for lactic acid may also give negative results in the presence of comparatively large quanti- ties of hydrochloric acid in the liquid to be tested. -Sngar, sulpho- cyanides, and other bodies may act with these reagents similarly to lactic acid. In order to be able to correctly judge of the value of the differ- ent reagents for free hydrochloric acid, it- is naturally of greatest importance to be clear in regard to what we mean by free hydro- chloric acid. It is a well-known fact that hydrochloric acid com- bines with proteids, and a considerable part of the hydrochloric acid may therefore exist in the contents of the stomach, after a meal rich in proteids, in combination with proteids. This hydrochloric acid combined with proteids, as well as that which is combined with amido-acids, cannot be considered as free, and it is for this reason that certain investigators consider such methods as those of Leo and Sjoqvist, which will be described below, as of little value. However, it must be remarked that, according to the unanimous experience of many investigators, the hydrochloric acid combined with proteids and also that combined with amido-acids (Saleowski and KuMAGAWA ') is physiologically active. Those reactions (color reactions) which only respond to actually free hydrochloric acid do not show the physiologically active hydrochloric acid. The sugges- tion of determining the " physiologically active " hydrochloric acid instead of the " free " seems to be correct in principle; and as the conceptions of free and physiologically active hydrochloric acid do not cover one another we must always be clear, if we want to determine the actually free or the physiologically active hydrochloric acid, before we judge of the value of a certain reaction. As the above-mentioned reactions for hydrochloric acid and organic acids are not sufficient in exact investigations, still- they may serve in many cases for clinical purposes, and it will suffice to refer the reader to other text-books, and especially to "Klinische Diagnostik innerer Krankheiten,^^ by R. v. Jaksch, 4:th edition, 1896, for the performance and the relative value of these tests. Among the many methods suggested for the quantitative esti- mation of hydrochloric acid not combined with inorganic bases, the two following are the most trustworthy. The method of K. Morneb and Sjoqvist depends on the fol- lowing principle: "When the gastric juice is evaporated to dryness with barium carbonate and then calcined the organic acids burn up and give insoluble barium carbonate, while the hydrochloric acid forms soluble barium chloride. Prom the quantity of this the original amount of hydrochloric acid can be calculated. 10 c. c. of the filtered contents of the stomach is mixed in a small platinum or silver dish with a knife-point of barium carbonate free from chlorides, and evaporated to dryness. The residue is burnt and ' Vircliow's Arch., Bd. 138. B8TIMATI0N OF ACID IN THE STOMACH. 287 allowed to glow for a few minutes. The cooled carbon is gently rubbed with water and completely extracted with boiling water, and the filtrate (about 60 c. c.) treated with an equal volume of alcohol and S-i c. c. sodium acetate solution (10^ acetic acid and 10^ acetate). The amount of barium in the filtrate is determined by titration with a solution of potassium bichromate, in which the alcohol facilitates the precipitation of the barium chromate, while the acetate prevents in part the precipitation of the calciuum car- bonate and in part the setting free of hydrochloric acid. The potassium-bichromate solution should contain about 8.5 grms. potassium bichromate to the litre. Its titre must exactly corre- spond with an — barium-chloride solution, "and the procedure is the same as in the titration of the BaCl^ solution obtained from the contents of the stomach. A paper moisteaed with tetramethylpara- phenylendiamin is used as indicator; this is colored blue by a "bichromate in acetic-acid solution. In titrating we add chromate solution as long as the barium chromate precipitated does not apparently increase, then test with the indicator-paper after each addition until it gives a decided blue coloration within one minute, and stop adding chromate solution. As the titre of the chromate solution has been determined by an — BaCl, solution, it is easy to calculate the quantity of HCl in 10 c. c. of the gastric juice corre- sponding to the number of c. c. of the chromate solution used. If tbe total acidity is determined in a second portion of the gastric juice, then the quantity of lactic acid or other organic acids repre- sented as HCl may be calculated. v. Jaksoh suggests precipi- tating the barium with sulphuric acid and weighing the sulphate instead of titrating. Sjoqvist ' has modified his method of deter- mining hydrochloric acid by precipitating the solution of BaCl, by ammonium chromate in the presence of acetic acid. This precipi- tate is dissolved in water by the aid of a little HCl, then titrated with a potassium-iodide solution and hydrochloric acid, and now titrated with sodium hyposulphite. The reactions take place as follows: 4HC1 + 2BaC0, = 2BaCl, + 2H,0 + 3C0,; 2Ba01, + ^(NH.),CrO, = 2BaCrO. + 4NH,C1; aBaCrO, -f- 16HC1 -j- 6KI = 2BaCl, + Cr,Cl, + 8H,0 + 6KC1 -f 31,; and 31, + 6Na,S,0, = 6NaI + 3Na,S,0,. Each c.c. of the hyposulphite corresponds to 3 mgm. HCl. Other modifications of this method have been pro- posed by Salkowski and Fawitzki," Boas,' and Bourgbt.* This method of Moknek-Sjoqvist gives, according to Leo ' and Koss- 1 Skan. Arch. f. Physiol., Bd. 5. ' Virchow's Arch., Bd. 133. » Centralbl. f. klin. Med., Bd. 13. < Schmidt's Jahrbucher, 1891, Bd. 229 (Reference). «L. c. 288 DIGESTION. LEE,' in the presence of phosphates, too low valnes, but it is other- wise very good. Lbo's Method." 10 c. c. of the filtered gastric Juice is treated with about 5 c. c. calcium-chloride solution, and the total acidity N determined by — caustic-soda solution, using litmus as the indi- cator. Then shake 15 c. c. of the same gastric juice with pure, finely powdered calcium carbonate, filter through a dry filter, remove the carbon dioxide from the filtrate by means of a current of air, measure off exactly 10 c. c. of the liquid and treat with 5 c. c. of the calcium-chloride solution, and add litmus and titrate again. The difference 'between the two titrations shows the acidity due to free acid. Any fatty acids present may be shaken out from another portion by ether and the acidity determined on the spon- taneous evaporation of the ether. By determining the electrical resistance Sjoqvist has been able to determine the amount of actually free acid and that combined with alkali in a mixture of hydrochloric acid and alkali monophos- phate. He finds that the quantity of hydrochloric acid found by Mornek-Sjoqvist's method in such mixtures corresponds very closely to the quantity actually present. He upholds his method in opposition to Leo's method, which, according to him, does not give accurate results for free acid. Other methods have been proposed by Oahn and v. Meking, HoFFMANK, Winter and Hatem, and Beaun. According to Kosslek ' the three last-mentioned methods are not quite service- able. In testing for volatile fatty acids the contents of the stomach should not be directly distilled, as volatile fatty acids may be formed by the decomposition of other bodies, such as proteid and haemoglobin. The neutralized contents of the stomach are there- fore precipitated with alcohol at ordinary temperature, filtered quickly, pressed, and repeatedly extracted with alcohol. The alco- holic extracts are made faintly alkaline by soda, and the alcohol distilled. The residue is now acidified by sulphuric or phosphoric acid and distilled. The distillate is neutralized by soda and evap- orated to dryness on the water-bath. The residue is extracted with absolute alcohol, filtered, the alcohol distilled off, and this residue dissolved in a little water. This solution may be directly tested for acetic acid with sulphuric acid and alcohol or with ferric chloride. J'ormic acid may be tested for by silver nitrate, which quickly gives a black precipitate; and butyric acid is detected by the odor after the addition of an acid. In regard to the methods for more fully ' Zeitschr. f. pliysiol. Chem., Bd. 17. 'L. c. * L. c. ; see also Mizerski and L. Nencki, Arch, des sciences biologiques, St. Petersbourg, Tome 1. INTESTINAL JUICE. 289 investigating the different volatile fatty acids, the reader is referred to other text-books. III. The Glands of the Mucous Memhrane of th6 Intestine and their Secretions. The Secretion of Brunner's Glands. These glands are partly considered as small pancreas glands and partly as mucons or salivary glands. Their importance in various animals is different. Accord- ing to Grutznee,' in dogs they are closely related to the pyloric glands and contain pepsin. The statements in regard to the occur- rence of a diastatic enzyme are contradictory, and the difBcnlty of collecting the secretion from these glands free from contamination makes these assumptions somewhat unreliable. The Secretion of Lieberkuhn's Glands. The secretion of these glands has been studied by the aid of a fistula in the intestine according to the method of Thirx" and Vblla.' Very little if any secretion takes place in fasting animals (dog) when the mucons membrane is not irritated. The secretion begins in the first hour after partaking of food, but the maximum varies with the quantity and character of the food.* Mechanical, chemical, or electrical irritation excites a secretion or increases that already began (Thirt). Laxatives do not increase the secretion, while pilocarpin produces a very abundant one (Masloff ' and Vella). The quantity of this secretion in the course of 24 hours has not been exactly determined. In the upper part of the small intestine of the dog this secretion is scanty, slimy, and gelatinous; in the lower part it is more fiuid, wibh gelatinous lumps or fiakes (Eohmaitk '). Intestinal juice has a strong alkaline reaction, generates carbon dioxide on the addition of an acid, and contains (in dogs) nearly a constant quantity of NaCl and Na^COj, 4.8-5 and 4-5 p. m. respectively (G-ttmilewski,' Kohmann). It contains proteid (Thirt found 8.01 p. m.), the quantity decreasing with the duration of the elimination. The ' Pflilger's Arch., Bd. 12. « Wien. Sitzungsber., Bd. 50. ' Moleschott's Untersuch., Bd. 13. * See Heidenhain in Hermann's Handbuoh, Bd. 5, Th. 1, S. 170. " Cited from Heidenhain, iUd., S. 171. • Pfiilger's Arch., Bd. 41. ■> Ibid., Bd. 39. 290 DIGESTION. quantity of solids varies. In dogs the quantity of solids is 12.3- 24.1 p. m., and in sheep 46-47 p. m. The specific gravity of the intestinal juice of the dog, according to the observations of Thiry, is 1.010-1.0107. The action of the intestinal juice has been studied by many investigators, but the statements concerning it are at variance. According to certain experimenters it has the power of converting starch into sugar, but others claim that it has not the property. However, it seems generally accepted, as shown by Paschutih',' Beown and Hbeon," Bastianelli,° and others, that the intestinal juice or an infusion of the mucous membrane has an inserting .action' on cane-sugar or maltose. This has been further confirmed by MiUBA, Pawtz and Vogel." Lactose does not seem to be inverted by the intestinal juice in the absence of micro-organisms. ' The action on carbohydrates takes place more quickly and to a greater extent in the upper part of the intestine, and correspond- ingly the absorption of starch and sugar occurs more quickly in the tipper part than in the lower section of the intestine (Lannois and Lepiste," Eohmawn). Intestinal juice does not split neutral fats, but it has the prop- erty, like other alkaline fluids, of emulsifying the fats. In regard to its action on albuminous bodies most investigators agree that the intestinal juice has no action on boiled proteid or meat, while it dissolves fibrm according to Thiet. Albumoses are not converted into peptones (Wenz,' Bastianblli). Contrary to other investi- gators, ScHiFi' * claims that the juice from a successful fistula opera- tion digests not only coagulated proteid and lumps of casein, but also unboiled and boiled meat. The lack of action on proteids which was observed by other investigators Schife attributes to the abnormal juice with which they experimented. Schiff also obtained from an operation not entirely successful a juice whose ' Centralbl. f. d. med. Wissensch., 1870, S. 561. "^ Annal. d. Chem. u. Pbarm., Bd. 204. ' Moleschott's Untersuoh. zur Naturlehre, Bd. 14. This contains all the ■older literature. * Zeitschr. f . Biologie, Bd. 33. ' Voit and Lusk, Zeitschr. f. Biologie, Bd. 28. « Arch, de Physiol. (3) Tome 1. ■" Zeitschr. f. Biologie, Bd. 32. ' Centralbl. f. d. med. Wissensch., 1868, S. 357. THIS PANOBEAS. 291 action oa proteid and meat was no greater than that studied by Thiry and other investigators. Hainan intestinal juice in a case of anus prmternaturalis has been investigated by Demakt.' This juice showed itself entirely inactive on albuminous bodies, even on fibrin and on fats. It only had a very faint action on boiled starch. Tubbt and Manning " have investigated human intestinal juice. The specific gravity was on an average 1.0069. The reaction was alkaline, and an abundant development of carbon dioxide took place on adding acid. Proteids were not digested ; starch was first saccharified very slowly, while cane-sugar and maltose were inverted by the juice. Fats were both emulsified and saponified. These experiments on the action of the intestinal juice on food introduced into the intestine in cases of isolated loop of the intestine in animals, and in human intestine in cases of anus prceternaturalis, have not given any positive results, because of the putrefaction processes going on in the intestine. The secretion of the glands in the large intestine seems to con- sist chiefiy of mucus. Fistulas have also been introduced into these parts of the intestine, which are chiefiy if not entirely to be consid- ered as absorption organs. The investigations on the action of this secretion on nutritive bodies have not as yet yielded any positive Jesuits. IV. Pancreas and Pancreatic Juice. In invertebrates, which have no pepsin digestion and which also have no formation of bile, the pancreas, or at least an analogous organ, seems to be the essential digestion gland. On the contrary, an anatomically characteristic pancreas is absent in certain verte- brates and in certain fishes. Those functions which should be per- formed by this organ seem to be performed in these animals by the liver, which may be rightly called hepatopanckeas. In man and in most vertebrates the formation of bile and of certain secretions containing enzymes important for digestion is divided between the two organs, the liver and the pancreas. The pancreas gland is similar in certain respects to the parotid gland. The secreting elements of the former consist of nucleated ' Virehow's Arch., Bd. 75. « Guy's Hosp. Report, Vol. 48, p. 377 ; also Centralbl. f. d. med. Wissensch., 1893, S. 945. 2y2 DIGESTION. cells whose basis forms a mass ricli in proteids, which expand ia water and in which two distinct zones exist. The outer zone is more homogeneous, the inner cloudy due to a quantity of granules. The nucleus lies about midway between the two zones, but this position may change with the varying relative size of the two zones. According to Hbidenhaii^^,' the inner part of the cells diminishes in size during the first stages of digestion, in which the secretion is active, while at the same time the outer zone enlarges owing to the absorption of new material. In a later stage, when the secretion has decreased and the absorption of the nutritive bodies has taken place, the inner zone enlarges at the expense of the outer, the sub- stance of the latter having been converted into that of the former. Under physiological conditions the glandular cells are undergoing a constant change, at one time consuming from the inner part and at another time growing from the outer part. The inner granular zone is converted into the secretion, and the outer, more homo- geneous zone, which contains the repairing material, is then con- verted into the granular substance. Besides considerable quantities of proteids, globulin, nucleo- proteid (see Chapter II), and albumin, we find in this glaad several enzymes, or, more correctly, zymogens, which will be described later. "We also find in this gland nuclein, leucin (butalanin), tyrosin (not in the perfectly fresh gland), xanthin, 1-8 p. m., hypoxanthm, 3-4 p. m., guanin, 2-7.5 p. m. (all figures are calcu- lated for the dried substance, Kossbl ') , adenin, inosit, lachc acid, volatile fatty acids, fats, and mineral substances. According to the investigations of Oidtmann,' the human pancreas contains 745.3 p. m. water, 245.7 p. m. organic and 9.5 p. m. inorganic sub- stances. The purpose of the pancreas is to produce very important enzymes for digestion; but besides this it also has another very important function. As already stated in a preceding chapter, it is of the greatest importance in metabolism, namely, for the trans- formation of dextrose in the animal body. In this regard it is well known that in dogs and certain other animals (but not in pigeons and geese) , the extirpation of the gland causes a marked diabetes, ' Pfliiger's Arch., Bd. 10. ^ Zeitsclir. f . physlol. Chem. , Bd. 8. ' v Gorup-Besanez, Lehrbuch, 4. Aufl., S. 733. PANCREAS AND DIABETES. 293 at least in most cases. We do not know how this diabetes is brought about. According to the brothers Cavazzaki/ the pancreas diabetes is not caused by a decreased combustion of the normal quantity of sugar formed, but by an abnormal increase in the formation of sugar in the liver, and the extirpation of the pancreas acts, according to them, by causing a lesion of the plexus coeliacus. They have found that irritation of this plexus produced an increased production of sugar in the liver, and they claim that the extirpation of the pancreas induces a degenerative irritation of the plexus, which is similar to the paralytic secretion in the salivary glands. In opposition to this view the investigations of Minkowski, Hedon, Lancebeaux, Thiroloix," and others have been presented, namely, tliat on sub- ■cutaneoasly transplanting a portion of the pancreas the function of the pancreas in transforming or producing sugar is not disturbed. After the removal of the intra-abdominal portion of the gland the animal in this case did not acquire diabetes. If the subcutaneously enveloped portion of pancreas is further removed, then an elimina- tion of sugar of great intensity takes place. Chauveau and Kaufmann ' are of the opinion that after the extirpation of the pancreas an abnormal increase in the formation of sugar in the liver takes place. The pancreas, according to them, regulates the formation of sugar in the liver by means of two nerve- centres, a retarding and an irritating centre. The pancreas irritates the retarding centre and retards the irritating centre of the liver, and it has a double action on retarding the sugar production. The extirpation of the pancreas removes the irritation of the retarding centres, the activity of the irritating centres is thereby raised, and in consequence a strong hyperglycaemia takes place. In considera- tion of the above-mentioned action of transplanted pieces of pan- creas, we must accept in these cases that the irritating action on the questionable centres under normal conditions is exercised by some other unknown internal secretory products of the gland. The ordinary view in regard to the origin of diabetes is, how- ever, as above (Chapter VIII) stated, that we have not to do with an increased production of sugar, but more likely a decreased trans- • See Centralbl. f. Physiol., Bd. 7, S 217 * See Minkowski, Arch. f. erp. Path. u. Pharm., Bd. 31. •Mem. Soc. Biol., 1893, p. 29. Cited from Centralbl. f. Physiol., Bd. 7, S. 317. 294 DIQESTION. formation of the sugar in the animal body. We must also admit that the pancreas has the ability, in some way or other, of regulat- ing the consumption of sugar; but we do not know how it acts. Lepinb ' has made an experiment to explain this action. Accord- ing to him a glycolysis regularly takes place in the blood (see Chapter VI), and the enzyme active in this change is secreted from the pancreas to the blood. On the extirpation of the pancreas naturally this function of the gland is removed, hence a hypergly- caBmia is produced. Important exceptions have been made against- this hypothesis by several investigators,'' and the action of the pancreas in the elimination of sugar still stands unexplained. According to Chauveau and Kaufmann ' a formation of sugar takes place in the liver, partly from the glycogen and partly from other bodies — carbohydrates, proteids, and fats — which on the^ destruction of tissues, the histolysis, are taken up by the blood and carried to the liver, where they are transformed into sugar. The pancreas has a preventive action on the sugar production of the liver, as also on the histolysis. This is caused by means of au unknown product of the inner secretion, which product passes into the blood. All three factors, the sugar production in the liver as well as the ianer secretion of the pancreas and the histolysis, are, according to Kaufmanit, influenced in a double way by the nervous system, namely, partly exciting and partly retarding. The exciting action on the liver and on histolysis has simultaneously a preventive action on the internal secretion of the pancreas, and this therefore causes an increased formation of sugar in a threefold way. The pre- ventive action on the liver and histolysis causes a simultaneous exci- tation of the internal secretion of the pancreas, and the formation of sugar under these conditions is reduced for three reasons. Mae- cusE ' has found that in frogs, in which Aldehoff has shown that diabetes may be produced- on the extirpation of the pancreas, no diabetes appears on as perfect extirpation of the liver as possible. Pancreatic Juice. This secretion may be obtained by adjusting & fistula in the excretory duct, according to the methods suggested by Bernard," Ludwig," and Heidenhaiit. ' If the operation is- ' See foot-note No! 9, p 123, Chapter VI. » See Minkowski, Arch. f. exp. Path. u. Pharm., Bd. 31, S. 174. ' Arch, de Physiol., Ser. 5, Tome 7. ■• Da Bois-Reymond's Arch., 1894. ' LeQons de Pbysiologie, Tome 2, p. 190. « See Bernstein, Arbeiten a. d physiol. Anstalt zu Leipzig, 1869, S. 1. ' Pfluger's Arch., Bd. 10, S. 604. PANCREATIO JTJIOE. 295 performed with sufficient rapidity and dexterity on an animal which has been well fed a few hours before, there is obtained from the fistula, as a rule, immediately after the operation {temporary fistula) a secretion rich in solids, viscid, very active, and which may be considered as normal pancreatic juice. Ordinarily, however, the gland becomes diseased in a few hours or days after the operation, and the secretion which then flows out of the fistula {permanent fistula) is more liquid, deficient in solids, and in certain other respects different from the secretion obtained immediately after the operation. Still a permanent fistula may also sometimes yield a normal secretion for a long time (Heidenhaik), while the tem- porary fistula in careless operations may give no secretion or only an abnormal juice. In herbivora, such as rabbits, whose digestion is uninterrupted, the secretion of the pancreatic juice is continuous. In carnivora it seems, on the contrary, to be intermittent and dependent on the digestion. During starvation the secretion almost stops, bat com- mences again after partaking of food. Food seems to act in a twofold manner. First, it may, with the more abundant flow of blood during the digestion, which is seen by the red color of the gland, convey a larger quantity of nutritive material to the gland, and thereby cause the secretion of a juice rich in solid nutritive bodies. In another way the food may also, by the irritation which it produces on the mucous coab of the stomach and the duodenum, cause an increased secretion. That the food indeed acts in these two ways follows from the fact that other substances, such as ether, may reflexily act on the mucous membrane of the stomach or intestine, causing a secretion of pancreatic juice, but in starvation a thin fluid is secreted, and after partaking of food a thick fluid is produced. According to the observations of Bbrnstbist, HEiDBif- HAiJT, and others, the secretion increases rapidly after eating, and it reaches its maximum in the course of the first three hours. From this time the secretion diminishes, but may again increase from the Sth-^th hour, when generally large quantities of food pass from the stomach to the intestine. Then it again decreases continuously from the 9th-llth hour, and stops entirely at the 15th-16th hour. In regard to the action of various bodies on the secretion Becker ' has found that the introduction of 1-3 gm. sodium chloride or bicarbonate diminishes the quantity of juice secreted by dogs and ' Arch, des Sciences biol. de St. Petersbourg, Tome 2, No, 3, p. 433. 296 digestion: decreases the proteolytic action of the same, while the introduction of distilled water or, still more, carbonated water increases the secretion. Pilocarpin, according to Gottlieb,' increases the secre- tion in rabbits. Accoiding to the same investigator the introduc- tion of irritants such as mustard-oil, acids, and alkalies into the duodenum causes reflexly an increased secretion. The statements as to the amount of pancreatic juice secreted in the course of 24 hours are variable and not trustworthy. It seems positively proved that the permanent fistula yields a considerably larger quantity of secretion than the temporary. While Kefeb- STEiN and Hallwachs, and Schmidt and Keogbe, find that the quantity of juice secreted from the first is 45-100 grms. per kilo during 34 hours, Bidder and Schmidt and Bidder and Skee- BiTZKT claim that the quantity from the temporary fistula is 3.5-5 grms. per kilo in the same time." In regard to the constituents and composition of the pancreatic juice, a distinction must be made between the secretion of a tem- porary and of a permanent fistula. The secretion flowing from the former is in dogs a clear, colorless, nearly sirupy, odorless fluid of an alkaline reaction which is very rich in proteid, and sometimes containing so large a quantity that it coagulates like white of egg when heated. Besides proteids the juice contains also three enzymes — one diastatic, one fat-splitting, and one which dissolves proteids. The last-mentioned has been called trypsin by Kuhiste. Besides the above-mentioned bodies the pancreatic juice habitually contains small quantities of leucin, fat, and soaps. As mineral con- stituents it contains chiefly alkali chlorides, also alkali carbonates, and some phosphoric acid, lime, magnesia, and iron. The secretion from the permanent flstula always contains less solids, especially proteid and enzymes, than that from a temporary fistula. A long time after the operation it is more fluid, more strongly alkaline, and the property which the juice from the tem- porary fistula has of dissolving proteids is often absent, or the secre- tion shows it in only a slight degree. As an example of the difEerent composition of the juice from a temporary and from a permanent fistula we give below the analysis of C. Schmidt.' The figures represent parts per 1000. ' Arch. f. exp. Path. u. Pharm., Bd. 33. ' Cited from Kiihne's Lehrbuch., S. 114. ' Cited from Maly, Chemie der Verdauungssafte in Hermann's Handbach, Bd. 5, Theil 3, S. 189. AMTLOPSm. 297 Juice from a Temporary Juice from a Permanent Fistula. Fistula. Water 900.8 884.4 976'8. 979.9 984.6 Solids 99.3 115.6 23.3 20.1 15.4 ■Organic substance 90.4 16.4 13.4 9 2 Ash 8.8 6.8 7.5 6^1 The mineral constituents of the secretion from the temporary fistula con- sisted chiefly of NaCl, 7.4 p. m. In the pancreatic juice of rabbits 11-36 p. m. solids have been found, and in that from sheep 14.3-36.9 p. m. In the pancreatic juice of the horse 9-15.5 p. m. solids have been found ; in that of the pigeon, 13-14 p. m. The human pancreatic juice has been analyzed by Hertkr ' in a case of stoppage of the exit of the juice by the pressure of a cancer. This juice, which could hardly be considiered as normal, was clear, alkaline, without odor, and contained the three enzymes. It contained peptone, but no other proteid. The quantity of solids was 24.1 p. m. Of these 6.4 p. m. were soluble in alco- hol. It contained 11.5 p. m. peptone (and enzymes) and 6.2 p. m. mineral substances. Zawadsky ' has analyzed the pancreatic juice of a young woman with a fistula, and found 864.05 p. m. water, 132.51 p. m. organic and 3.44 p. m. in- organic substances. The quantity of protein bodies was 92.05 p. m. Among the constituents of the pancreatic juice, the three enzymes are the most important. Amylopsin or pancreatic diastase, which according to Koro- wiisr' and Zweifel* is not found in new-born infants and, does not appear until more than one month after birtli, seems, although not identical with ptyalin, to be nearly related to it. Amylopsin acts very energetically upon boiled starch, especially at -|- 37° to 40° C, and forms, similar to the action of saliva, besides dextrin, chiefly isomaltose and maltose, with only very little dextrose (Musoulus and V. Meeing,' Kulz and Vogel"), The dextrose is probably formed by the action of the invertin ' existing in the gland and juice. If the natural pancreatic jaice is not to be obtained, then the gland, best after it has been exposed a certain time (34 hours) to the air, may be treated with water or glycerin. ' This infusion or the glycerin extract diluted with water (when a glycerin has been used which has no reducing action) may be tested directly with starch-paste. It is safer, however, to first precipitate the enzyme ' Zeitschr. f . physiol. Chem. , Bd. 4. 2 Centralbl. f. Physiol., Bd. 5, 1891, S. 179. ' See Maly's Jahresber. , Bd. 3. * Untersuchungen ilber den Verdauungsapparat der Neugeborenen. Berlin, 1874. ' Zeitschr. f. physiol. Chem., Bd. 2. ' See Tebb, Journal of Physiol., Vol. 15, and Abelous, C. R. Soc. de biol., 1891. 298 DIGESTION. from the glycerin extract by alcohol, and wash with this liquid, dry the precipitate over sulphuric acid, and extract with water. The enzyme is dissolved by the water. The detection of sagar may be made in the same manner as in the saliva. Steapsin or Fat-splitting Enzyme. The action of the pancreatie jaice on fats is twofold. First, the neutral fats are split into fatty acids and glycerin, which is an enzymotic process; and secondly, it has also the property of emulsifying the fats. The action of the pancreatic juice in splitting the fats may be shown in the following way. Shake olive-oil with caustic soda and ether, siphon ofE the ether and filter if necessary, then s'hake the ether repeatedly with water and evaporate at a gentle heat. In this way we obtain a residue of fat free from fatty acids which is neutral, and which dissolves in acid-free alcohol and is not colored red by alkanet tincture. If such fat is mixed with perfectly fresh alkaline pancreatic juice or with a freshly prepared infusion of the fresh gland and treated with a little alkali or with a faintly alkaline glycerin extract of the fresh gland (9 parts glycerin and 1 part 1^ soda solution for each gramme of the gland), and some litmus tincture added and the mixture warmed to + 37° 0. , the alkaline reaction will gradually disappear and an acid one take its place. This acid reaction depends upon the conversion of the neutral fats by the enzyme into glycerin and free fatty acids. The splitting of the neutral fats may also be shown more exactly by the foUowiag method. The mixture of neutral fats (absolutely free from fatty acids) and pancreatic juice or pancreas infusion is digested at the temperature of the body and treated with some soda and repeatedly shaken with fresh quantities of ether until all the unsplit neutral fats are removed. Then it is made acid with sul- phuric acid, after which shake tlie acid liquid with ether, evaporate the ether, and test the residue for fatty acids. Another simple process for the demonstration of the fat-splitting action of the pancreas glands is the following (Ol. Bebnaed) : A small portion of the perfectly fresh, finely divided gland substance is first soaked in alcohol (of 90^). Then the alcohol is removed as far as possible by pressing between blotting-paper, after which the pieces of gland are covered with an ethereal solution of neutral butter-fat (which may be obtained by shaking milk with caustic soda and ether). After the evaporation of the ether the pieces of gland covered with butter-fat are pressed between two watch-glasses and then gently heated to 37° to 40° C. in this position. After a certain time a marked odor of butyric acid appears. The action of the pancreatic juice in splitting fats is a process analogous to that of saponification, the neutral fats being decom- posed, by the addition of the elements of water, into fatty acids and TRYPSIN. 299 glycerin according to the following formula: C.H^.Oj.K, (neutral fat) + 3H,0 = C.H..O..H. (glycerin) + 3(H.0.R) (fatty acid). This depends upon a hydrolytic splitting, which was first positively proved by Beenaed ' and Beethelot." The pancreas-enzyme also decomposes other esters just as it does the neutral fats (Nbncki,' Baas'). The pancreas-enzyme which decomposes fats has been less studied than the other pancreas-enzymes, and it has indeed been questioned whether or not the decomposition of the neutral fats in the intestine may not be effected through lower organisms. According to the investigations of Nencki, it seems that the pan- creas actually contains an enzyme which decomposes fats. This enzyme, which is still little known, appears to be very sensitive to acids, and it is often absent in acid glands not perfectly fresh. If a watery infusion of the gland prepared cold be treated with cal- cined magnesia, then the enzyme in question will, according to Danilewski,' be retained by the magnesia precipitate. The fatty acids which are split off by the action of the pan- creatic juice combine in the intestine with the alkalies, forming soaps which have a strong emulsifying action on the fats, and thus the pancreatic juice becomes of great importance in the emulsifica- tion and the absorption of the fats. Trypsin. The action of the pancreatic jaice in digesting pro- teids was first observed by Beenaed, but first proved by Corvi- SART.' It depends upon a special euzyme called by Kuhnb trypsin. Strictly speaking, this enzyme does not occur in the gland itself. In the gland, more probably, a zymogen occurs from which the enzyme is split off or formed during secretion, also by the action of water, acids, alcohol, and other substances. According to Albee- TOis'i,' this zymogen is found in the gland in the last third of the intra-uterine life. The purest trypsin thus far prepared, isolated by Kuhne,' is soluble in water, but insoluble in alcohol or glycerin. The less pure ' Ann. de chim. et physique (3 ser.), Tome 35. 2 Jahresber. d. Cliem., 1855, S. 733. 5 Arcb. f. exp. Path. u. Pharm., Bd. 20. * Zeitscbr. f. physiol. Chem., Bd. 14, S. 416. ' Virchow's Arch. , Bd. 25. • Gaz. hebdomadaire, 1857, Nos. 15, 16, 19. Cited from Bunge, Lehrbucb. S. 174. ' See Maly's Jahresber., Bd. 8, S. 254. 8 Verb. d. naturh.-med. Vereins zu Heidelberg, (N. F.) Bd. 1, Heft 8. 300 DIGESTION. enzyme, on the coutrary, is soluble in glycerin. If the solution of the enzyme in water is heated to the boiling-point with the addition of a little acid, it decomposes into coagalated proteid and peptone (Kuhne). According to the investigations of Bieenacki ' trypsin in 0.25-0.5^ soda solution is destroyed in 5 minutes by heating to 50° C. It is destroyed by heating its neutral solution to 45° C. The presence of albumoses or certain ammonium salts in alkaline trypsin solutions hare a protective action to a certain extent. Trypsin is destroyed by gastric juice. Like other enzymes, trypsin is characterized by its physiological action. This action consist in dissolving proteids and especially fibrin in alkaline, neutral, or even faintly acid solutions with readiness. The preparation of pure trypsin has been tried by various experimenters. The purest seems to have been prepared according to the rather complicated method of Kuhnb." In studying the action of trypsin a less pure preparation may often answer, and various methods of preparing such have been proposed, but we cannot describe all of them. For the production of a glycerin extract (Heidenhain ') the gland should be rubbed with glass powder or pure quartz-sand, this mass carefully mixed wibh acetic acid of 1% (1 c. c. to each grm. of gland), then for each part of the gland-mass add 10 parts of glycerin, and filter after about three days. By precipitating the glycerin extract with alcohol and redis- solving the precipitate in water, we obtain a solution which has a powerful digestive action. A watery infusion of the gland may be made only after it has been exposed to the air for 24 hours, and 5-10 parts of water for each part by weight of the gland should be used. According to Kuhne* the impure trypsin is allowed to undergo autodigestion in a 0.2^ soda solution and in the presence of thymol. After the conversion of the albumoses into peptones the trypsin may be precipitated by ammonium sulphate. An active but impure infusion may be obtained by digesting the finely divided gland for a few days with water containing 5-10 c. c. chloroform per liter (Salkowski '). A very active trypsin may be prepared by extracting the finely divided gland of oxen, free from water and blood, with water con- taining 0.01-0.05^ NH3. The filtered extract gives a precipitate with acetic acid which has great digestive powers and which can be further purified. (Not published investigations of the authoe.) ' Zeitschr. f. Biologie, Bd. 28. ♦ Verli. d. naturli.-med. Vereins zu Heidelberg, (N. F.) Bd. 1, Heft 3. ' Pflilger's Arch., Bd. 10. *Centralbl. f. d. med. Wissenscli. , 1886, S. 629. » Deiitscli. med. Wochenschr. , 1888, No. 16. ACTION OF TRYPSIN. 301 The action of trypsin on proteids is best demonstrated by the use of fibrin. Very considerable quantities of this albuminoas body are dissolved by a small amount of trypsin at 37-40° C. It is always necessary to make a control test with fibrin alone, with or without the additioa of alkali. Fibrin is dissolved by trypsin with- out any putrefaction; the liquid has a pleasant odor somewhat like bouillon. To completely exclude putrefaction a little thymol, chloroform, or ether should be added to the liquid. Trypsin diges- tion differs essentially from pepsin digestion in that the first takes place in neutral or alkaline reaction and not, as is necessary for pepsin digestion, in an acidity of 1-2 p. m. HCl, and further by the fact that the proteids dissolve in trypsin digestion without previously swelling up. Many circumstances exert a marked influence on the rapidity of the trypsin digestion. "With an increase in the quantity of enzyme present the digestion is hastened at least to a certain point, and the same is also true of an increase in temperature at least to about + 40° C, at which temperature the proteid is very rapidly dissolved by the trypsin. The reaction is also of the greatest importance. Trypsin acts energetically in neutral, or still better in alkaline, solu- tions, and best in an alkalinity of 3-4 p. m. NajCOj. Free mineral acids, even in very small quantities, completely prevent the diges- tion. If the acid is not actually free, but combined with albumi- nous bodies, then the digestion may take place quickly when the acid combination is not in too great excess (Chittenden and Cummins '). Organic acids act less disturbingly, and in the pres- ence of 0.2 p. m. lactic acid and the simultaneous presence of bile and common salt the digestion may indeed proceed more quickly than in a faintly altaline liquid (Lindbergbr'). Carbon dioxide, according to Schierbeck,' has a retarding action in acid solutions, but it accelerates the tryptic digestion in faintly alkaline liquids. Foreign bodies, such as borax and potassium cyanide, may promote tryptic digestion, while other bodies, such as salts of mercury, iron, and others (Chittenden and Cummins), or salicylic acid in large quantities, may have a preventive action. The nature of the pro- teids is also of importance. Unboiled fibrin is, relatively to most ' Studies from the Physiol. Chem. Laboratory of Tale College, New Haven, 1885, Vol. I, p. 100. 2 See Maly's Jahresber., Bd. 13, S. 380. ■ Skan. Arch. f. Physiol., Bd. 3. 802 DIGESTION. other albuminous bodies, dissolved so yery quickly that the diges- tion test with raw fibrin gives an incorrect idea of the power of trypsin to dissolve coagulated albuminous bodies in general. An accumulation of products of digestion tends to hinder the trypsin digestion. Tlie Products of the Trypsin Digestion. In the digestion of nnboiled fibrin a globulin which coagulates at + 55-60° 0. may be obtained as an intermediate product (Hbeemann '). Moreover from fibrin, as well as from other albuminous bodies, emanate alhumoses and peptones, leucin, tyrosin, and aspartic acid, a little lysin, lysatinin (Hedin"), and ammonia (Hieschlee ") , and also the so-called protein chromogen* or tryptophan,'' a substance whose nature is not known, but which gives a reddish-violet product, so- called proteinochrom, with chlorine or bromine. When putrefaction has not been entirely prevented numerous other bodies appear which will be spokeu of later in connection with the putrefaction process going on ia the intestine. In the trypsin digestion, in contrast to the pepsin digestion, pure peptones, not precipitated by ammonium sulphate, are relatively easily and quickly formed. The peptone, according to Kuhne, consists entirely of antipeptone, and the above- mentioned decomposition products, leucin and the others, are formed by the decomposition of the hemipeptone. We will now consider the decomposition products, leucin and tyrosin, formed in the trypsin digestion of proteids. Leucin, C^Hj^NO^, or amido-caproic acid, more recently called a-amido-isobutylacetic acid, (OHJ,CH.CH,.CH(NH,). COOH.° Leucin is f6rmed not only in the trypsin digestion of proteids, but also from the protein substances by their decomposi- tion on boiling with diluted acids or alkalies, by fusing with alkali hydrates, and by putrefaction. Because of the ease with which leucin and tyrosin are formed in the decomposition of protein sub- stances, it is difficult to positively decide whether these bodies when found in the tissues are constituents of the living body or are only to be considered as decomposition products formed after death. ' Zeitsclir. f, physiol. Chem., Bd. 11. ' See Drecltsel, Du Bois-Reymond's Arch., 1891. ' Zeitschr. f. physiol. Chem., Bd. 10, S. 302. * Stadelmann, Zeiischr. f. Biologie, Bd. 26. ' Neumeister, ibid., Bd. 26, S. 329. « See Schulze and Likiernik, Zeitschr. f. physiol. Chem., Bd. 17, and Gmelin, ibid., Bd. 18. LEUCIN. 303 Leucin has been found iu the pancreas and its secretion, in the spleen, thymus, and lymph-glands, in the thyroid gland, in the salivary glands, ia the kidneys, brain, and liver (but mostly in dis- ease). It also occurs in the wool of sheep, in dirt from the skin (inactive epidermis) and between the toes, and its decomposition products have the disagreeable odor of the perspiration of the feet. It is found pathologically in atheromatous cysts, ichthyosis scales, pas, blood, and urine (in diseases of the liver). Leucin also occurs in the vegetable kingdom. Leucin has been prepared synthetically by Hufnee ' from isovaleraldehyde-ammonia and hydrocyanic acid. This leucin is optically inactive. Inactive leucin may also be prepared, as shown by E. ScHTJLZE, Baebieri and Bosshaed," by the cleavage of pro- teids with baryta at 160° C. or on heating ordinary leucin with baryta-water to the same temperature. The Isevorotatory modi- fication may be formed from the inactive leucin by the action of penicillum glaucum. The leucin obtained in the pancreatic digestion of proteids as well as in their cleavage with hydrochloric acid, seems always to be the dextrorotatory variety." CoHsr' has, however, obtained a leucin difEering from the ordinary leucin in the tryptic digestion of fibrin. Hufnee has prepared an isomer of leucin from monobromcaproic acid and ammonia. It is a question whether there exist natural leucins corresponding to normal caproic acid. On oxidation the leucins yield the corresponding oxyacids (leucinio acids). The leucins are decomposed on heating, evolving carbon dioxide, ammonia, and amylamin. On heating with alkalies, as also iu putrefaction, it yields valerianic acid and ammonia. Leucin crystallizes when pure in shining, white, very thin plates, usually forming round knobs or balls, either appearing like hyalin or alternating light or dark concentric layers which consist of radial groups of crystals. Leucin as obtained from the animal fluids and tissues is very easily soluble in water and rather easily in alcohol. Pure leucin is soluble with difficulty; according to certain statements it dissolves in about 39 parts of water at ordinary tem- peratures or little higher, and according to others in 46 parts. This ' Journ. f. prakt. Chem., N. F., Bd. 1. « Zeitsohr. f . physiol. Chem. , Bdd. 9 and 10. » In regard to contradictory statements see Hoppe-Seyler's Handbuch, 6. Aufl., p. 134. * Zeitsohr. f. physiol. Chem., Bd. 20. 304 DI0B8TI0N. difference may be due, according to Gmelin,' to the fact that the optically active leucin may be variable mixta res of the dextro- and lEBVorotatory modifications. The inactive leucin is the most insolu- ble. The specific rotation of the ordinary leucin, dissolved in hydrochloric acid, is (a)D = +17.5. Leucin is readily soluble in alkalies and acids. On slowly heating to 170° 0. it melts and sublimes in white, woolly flakes which are similar to sublimed zinc oxide. A marked odor of amylamin is generated at the same time. The solution of leucin in water is not, as a rule, precipitated by metallic salts. The boiling-hot solution may, however, be precipi- tated by a boiling-hot solution of copper acetate. If the solution of leucin is boiled with sugar of lead and then ammonia be added to the cooled solution, shining crystalline leaves of leucin-lead oxide separate. When boiled with leucin, copper oxyhydrate is dissolved without reduction. Leucin is recognized by the appearance of the balls or knobs under the microscope, by its action when heated (sublimation test), and by Schbeek's test. This last consists in the lencin yielding a colorless residue when carefully evaporated with nitric acid on platinum-foil, and this residue when warmed with a few drops of caustic soda gives a color varying from a pale yellow to brown (depending on the purity of the lencin), and on further concentrat- ing over the flame it agglomerates into an oily drop which rolls about on the foil. Tyrosin, C^H^NOj, or jb-oxtphbntl-amidopeopionic acid, HO.C,H,.C,H,(NH,).COOH, is derived from most protein sub- stances (not gelatin) under the same conditions as leucin, which it habitually accompanies. It is especially found with leucin in large quantities in old cheese [Tvpos), from which it derives its name. Tyrosin has not with certainty been found in perfectly fresh organs, with the exception, perhaps, of the spleen and pancreas of cattle. It occurs in the intestine in the digestion of albuminous substances, and it has about the same physiological and pathological importance as leucin. Tyrosin was prepared by Belenmbyer and Lipp" from p- amido-phenylalanin by the action of nitrons acid. On fusing with caustic alkali it yields p-oxybenzoic acid, acetic acid, and ammonia. ' Zeitschr. f. physiol. Chem., Bd. 18. ' Ber. d. deutsch. chem. Gesellsch. zu Berlin, Bd. 15, S. 1544. T7R0SIN. 305 By pntrefactioa it may yield p-hydrocoumaric acid, oxypheayl- acetio acid, and p-cresol. Tyrosin in a very impure state may be in the form of balls similar to leucin. The purified tyrosin, on the contrary, appears as colorless, silky, fine needles which are often grouped into tufts. or balls. It is soluble with difficulty in water, being dissolved by 3454 parts water at + 20° 0. and 154 parts boiling water, separat- ing, however, as tufts of needles on cooling* It dissolves more easily in the presence of alkalies, ammonia, or a mineral acid. It is difficultly soluble in acetic acid. Crystals of tyrosin separate from an ammoniacal solution on the spontaneous evaporation of the ammonia. The solution of the tyrosin obtained from protein sub- stances by the action of acids has always a faint Isevorotatory power. Tyrosin prepared synthetically or by decomposition of proteids by baryta is optically inactive.' Tyrosin is not soluble in alcohol or ether. It is identified by its crystalline form and by the following reactions : Pieia's Test. Tyrosinis dissolved in concentrated sulphuric acid by the aid of heat, by which tyrosin-sulphuric acid is formed ; i t is allowed to cool, diluted with water, neutralized by BaCOj, and filtered. On the addition of a solution of ferric chloride the filtrate gives a beautiful violet color. This reaction is disturbed by the presence of free mineral acids and by the addition of too much; ferric chloride. Hofmann's Test. If some water is poured on a small quantity of tyrosin in a test-tube and a few drops of Millon's reagent added and then the mixture boiled for some time, the liquid becomes a beautiful red and then yields a red precipitate. Mercuric nitrate may first be added, then, after this has boiled, nitric acid contaia- ing some nitrous acid. Schereb's Test. If tyrosin is carefully evaporated to dryness with nitric acid on platinum-foil, a beautiful yellow residue (nitro- tyrosin nitrate) is obtained, which gives a deep reddish-yellow color with caustic soda. This test is not characteristic, as other bodies give a similar reaction. Leucin and tyrosin may be prepared in large quantities by boil- ing albuminous bodies or albuminoids with dilate mineral acids. Ordinarily we boil hoof -shavings (2 parts) with dilute sulphuric acid ' See Mauthner, Wien. Sitzungsber., Bd. 85, and E. Schulze, Zeitschr. f. physiol Chem., Bd. 9. 306 DIGESTION. (5 parts concentrated acid and 13 parts water) for 24 hoars. After boiling the solution it is diluted with water and neutralized while still warm with milk of lime and then filtered. The calcium sul- phate is repeatedly boiled with water, and the several filtrates are united and concentrated. The lime is precipitated from the coa- centrated liqnid by oxalic acid and the precipitate filtered ofE, repeatedly boiled with water, all filtrates united and evaporated to crystallization. "What first crystallizes consists chiefly of tyrosin with only a little Igucin. By concentration a new crystallization may be produced in the mother-liquor, which consists of leucin with some tyrosin. To separate leucin and tyrosin from each other their different solubilities in water may be taken advantage of in preparing them on a large scale, but surer and better results are obtained by the following method of Hlasiwbtz and HABEBMAisrif.' The crystalline mass is boiled with a large quantity of water and enough ammonia to dissolve it. To this boiling-hot solution enough basic lead acetate is added until the precipitate formed is nearly white; now filter, heat the light yellow filtrate to boiling, neutralize with sulphuric acid, and filter while boiling hot. After cooling, nearly all the tyrosin is precipitated, while the leucin remains in the solution. The tyrosin may be purified by recrystallizing from boil- ing water or from ammoniacal water. The above-mentioned mother-liqnor rich in leucin is treated with H,S, the filtrate con- centrated and boiled with an excess of freshly precipitated cojfper oxyhydrate. A part of the leucin is precipitated, and the residue remains in the solution and partly crystallizes as a cuprous com- pound on cooling. The copper is removed from the precipitate and solution by means of H,S, the filtrate decolorized when necessary with animal charcoal, strongly concentrated and allowed to crystal- lize. The leucin obtained from the precipitate is quite pure, while that from the solution is somewhat contaminated. If one is working with small quantities, the crystals, which con- sist of a mixture of the two bodies, may be dissolved in water and this solution precipitated with basic lead acetate. The filtrate is treated with H,S, the new filtrate evaporated to dryness, and the residue treated with warm alcohol which dissolves the leucin but not the tyrosin. The remaining tyrosin is purified by recrystalliza- tion from ammoniacal alcohol. Leucin may be purified by recrys- tallization from boiling alcohol or by precipitating it as leucin lead oxide, treating the precipitate suspended in water with H,S and evaporating the filtered solution to crystallization. To detect the presence of leucin and tyrosin in animal fluids or iissues the albumin must first be removed by coagulation with the addition of acetic acid and then precipitated by basic lead acetate. The filtrate is treated with H^S, this filtrate evaporated to a sirup or to dryness, and the two bodies in the residue are separated from -each other by boiling alcohol and then purified as above stated. ' Annal. d. Chem. u. Pharm., Bd. 169, S. 160. ASPARTIC ACID. 307 Aspartic Acid, C,II,N'0„ or amido-succiitio acid, C,H,(]SrHJ. (COOH)j. This acid is obtained in the trypsin digestion of fibrin and gelatin. It may also be obtained by the decomposition of albuminous bodies or albuminoids with acids (see Chapter II). It has also been found in beet-root molasses, and lastly it is very widely difEused in the vegetable kingdom as the amid aspaeaginb (amido- succinic-acid amid), which seems to be of the greatest importance in the development and formation of the albuminous bodies, Aspartic acid dissolves in 256 parts water at + 10° 0. and in 18.6 parts boiling water, and crystallizes on cooling as rhombic prisms. The acid prepared from protein substances is optically active, and is dextrogyrate in a solution strongly acid with nitric acid, and Isevogyrate in a watery solution. It forms with copper oxide a crystalline combination which is soluble in boiling-hot water and nearly insoluble in cold water, and which may be used in the preparation of the pure acid from a mixture with other bodies. In regard to methods of preparation see Hlasiwbtz and Habbk- MANN ' and E. Schulzb." The action of trypsin on other bodies has not been thoroughly studied. An enzyme has been found in the pancreas of the pig and certain herbivora, which is not identical with trypsin and which causes the coagulation of neutral or alkaline milk (Kuhne and EoBBETs'). Gelatin is dissolved by the pancreatic juice and is •converted into gelatin-peptone. According to Kuhne and Ewald * neither glycocoU nor leucin is formed. The gelatin-forming sub- stance of the connective tissues is not directly dissolved by trypsin, but only after it has been treated with acids or soaked in water at + 70° C. By the action of trypsin on hyalin cartilage the cells dissolve, leaving the nucleus. The basis is softened and shows an indis- tinctly constructed network of collagenous substance (Kuhnb and Ewald). The elastic substance, the structureless membrane, and the membrane of the fat-cells are also dissolved. Parenchymatous ■organs, such as the liver and the muscles, are dissolved all but the nucleus, connective tissue, fat-corpusoles, and the remainder of 'L. c. » Zeitschr. f. physiol. Chem., Bd. 9. ■ See Maly's Jahresber., Bd. 9, S. 224 ; also Sidney Edkins, Journal of Physiology, Vol. 13, which contains all the literature. * Verh. d. naturh.-med. Vereins zu Heidelberg, (N. F.) Bd. 1. 308 DIGESTION. the nervous tissue. If the muscles are boiled, then the connec- tive tissue is also dissolved. Mucin and certain nucleins are dis- solved and split by trypsin solutions. The digestibility of casein pseudonuclein in trypsin solutions has been shown recently by Sebelein." Popoff' had previously shown the same for the nuclein from the thymus. Gumlich' and Weintraud* hav& shown that the nucleins are only partly utilized in the intestine. Trypsin seems to be without action on chitin and horny sub- stance. OxyJicemoglohin is decomposed by trypsin with the splitting off of haamatin. Hmmoglolin, on the contrary, when the access of oxygen is completely prevented, is not decomposed by trypsin (Hoppb-Setlbr ^) . Trypsin does not act on fats or carbohydrates. It has already been brought out above that trypsin does not exist ready formed in the gland, but more likely, as Heidenhain ° has shown, the gland contains a corresponding zymogen. The maximum quantity of such zymogen in the gland occurs 14-16-18 hours after an abundant meal, and the minimum 6-10 hours after. Tfiis zymogen is not converted by glycerin into trypsin, but is easily changed by water and acids. A soda solution of 1-1.5^, on the contrary, prevents almost entirely the conversion of the zymogen. If we allow the gland to lie in the air it gradually becomes acid, and this leads to the formation of an enzyme in which the oxygen seems to be active, as is usual in the conversion of the zymogen into trypsin. It is very probable also that the two other enzymes are formed from corresponding zymogens, and this has been shown by Liveesidge' to be plausible in the case of the diastatic enzyme. After a plentiful meal Heidenhain found in dogs in the first stages of digestion, when the secretion of pancreatic juice was most active, that the glandular cells became smaller owing to the con- sumption of the inner granular zone, while the outer zone at the same time took up new material and became larger. In these stages the quantity of zymogen is smallest. At a later period, 13-30 hours after a meal, the inner zone is re-formed from the outer, and the larger this ione is the larger the quantity of zymogen in the gland ' Zeitsclir. f. pliysiol. Chem., Bd. 30. 'i^iU, Bd. 18. 2 Ibid., Bd. 18. ■• Verb an dl. d. physiol. Gesellsch. zu Berlin, 1895. <• Pliysiol. Chem., S. 367. « Pfluger's Arcli. , Bd. 10. ' Journal of Physiol., Vol. 8. VHEMWAL PROCESSES IN THE INTESTINE. 309 seems to be. The zymogen consequently belongs to tbe inner zone, and the secretion consists therefore, at least in part, in a destruction or dissolution of this zone whereby the substance of the gland itself is changed into the secretion (Heidenhain). This view, however, is in opposition to that of Lewaschew,' who observed that in animals which have starved and whose pancreas are nearly free from zymogen, the inner granular zone is just as much developed as under normal conditions and containing abundant quantities of zymogen. We are still . completely in the dark regarding the nature of the chemical processes which take place in the conversion of the zymogen into the enzyme. V. The Cliemical Processes in the Intestine. The action which belongs to each digestive secretion may be essentially changed by mixing with other digestive fluids ; and since the digestive fluids which flow into the intestine are mixed with still another fluid, the bile, it will be readily understood that the combined action of all these fluids in the intestine makes the chemi- cal processes going on therein very complicated. As the acid of the gastric juice acts destructively on ptyalin, this enzyme has no further diastatic action, even after the acid of the gastric juice has been neutralized in the intestine. The bile has, at least in certain animals, a faint diastatic action which in itself can hardly be of any great importance, but which shows that the bile has not a preventive but rather a beneficial influence on the energetic diastatic action of the pancreatic juice. Maetih" and Williams" have observed a beneficial action of the bile on the diastatic action of the pancreas infusion. To this may be added that the organized ferments which occur habitually in the intestine and sometimes in the food have partly a diastatic action and partly produces a lactic-acid and butyric-acid fermentation. The maltose which is formed from the starch seems to be converted into glucose in tlie intestine. Cane-sugar is inverted in the intestine, but, according to the observations of Voit and Ldsk," milk-sugar is not inverted in the intestine of rabbits. Cellulose, especially the finer and more tender, is undoubtedly partly dissolved in the intestine ; > Pfliiger's Arch., Bd. 37. ' Proceed, of Roy. Soc, Vols. 45 and 48. 1 Zeitschr. f. Biologie, Bd. 28, S. 375. 310 DIGESTION. the products formed hereby are not jery well known. It has been shown by Tappbnier that cellulose may undergo fermentation, caused by the action of micro-organisms with the production of marsh-gas, acetic acid, and butyric acid ; still we do not know to what extent the cellulose is destroyed in this way." Bile possesses the power of dissolving fats in so slight a degree that it is scarcely worthy of mention. It is, however, without doubt of greater importance that the bile, as Nencki" and Each- FOED ° have shown, facilitates the fat-splitting action of the pan- creatic juice. This splitting of the fats into fatty acids and glycerin is an important factor in the absorption of the fats. The fatty acids combine with the alkalies of the intestinal and pancreatic juices, producing soaps which are partly absorbed as such and partly exer- cise a powerful action on the absorption of the fats. There is no doubt that the chief part of the fats in the food is absorbed as a fine emulsion, and the soaps are of great importance in the forma- tion of this emulsion. If to a soda solution of about 2 p. m. Na,COj we add pure, perfectly neutral olive-oil in not too large quantity, we obtain, after vigorous shaking, a transient emulsion. If, on the contrary, we add to the same quantity of soda solution an equal amount of com- mercial olive-oil (which always contains free fatty acids), we need only turn the vessel over for the two liquids to mix and immediately we have a very finely divided and permanent emulsion making the- liquid appear like milk. The free fatty acids of the always some- what rancid commercial oil combine with the alkali to form soaps which act" to emulsify the fats (Brucke,* Gad '). This emulsifying action of the fatty acids split off by the pancreatic juice is undoubtedly assisted by the habitual occurrence of free fatty acids in the food,, and also by the splitting ofE of fatty acids from the neutral fats by the putrefaction in the intestine. These fatty acids must combine with the alkalies in the intestine and form soaps. ' On the digestion of cellulose see Henneberg and Stohmann, Zeitscbr. f. Biologie, Bd. 31, S. 613 ; v. Knieriem, ibid., S. 67; Hofmeister, Arch. f. wiss. u. prakt. Thierheilkunde, Bd. 11; Weiske, Zeitschr. f. Biologie, Bd. 32, S. 373 ; Tappeiner, ibid., Bdd. 30 and 34; and Mallfivre, Pflixger's Arch.^ Bd. 49. •2 Arch. f. exp. Path. u. Pharm., Bd. 30. ' Journal of Physiol., Vol. 12. « Wien. Sitzungsber., Bd. 61, Abth. 2. . * Du Bois-Reyuiond's Arch. , 1878, CHEMICAL PROCESSES IN THE INTESTINE. 311 This emulsification of fats by means of the action of the pan- creatic juice or by soaps formed in other ways can only take place in an alkaline solution. In the contents of the intestine, as long as they are acid, such an emulsion can hardly occur. On the con- trary, it undoubtedly occurs at the point where the fat comes in contact with an alkaline secretion under a mucous membrane, or in general where it meets with sufficient alkali to form an emulsion. In the acid contents of the intestine of dogs, which had been kept on food rich in fat, Ludwig and Cash ' observed no emulsion. After tying the two pancreas excretory ducts they found a remark- ably fine emulsion in the chylous vessels, though the fat in the contents of the intestine was not emulsified. In this case it is possible that the free fatty acid which is hardly ever absent in the fat of the food, and which may be produced also by putrefaction in the intestine, forms soaps with the alkali of the mucous coat of the intestine and produces the emulsion iu the chylous vessels. It must not be forgotten that, according to many observations, an emulsion of the fats may be produced by means of proteid, independently of the reaction. In this regard reference should be made to the statement of Kuhne ' that the pancreatic juice from a permanent fistula which is poor in proteid has the emulsification property to a less degree than the juice from a temporary fistula which is rich in proteid. Kuhne has also shown that this emulsification property is not to be ascribed to the alkali, as faintly acid juices also have this property. Claude Beenabd found long ago in his experiments on rabbits, in which, animals the choledochus duct was inosculated to the small intestine above the pancreas passages, that when their food contained a large proportion of fat the chylous vessels of the intestine above the pancreas passages were transparent, but below the same they were milky-white, and from this concluded that the bile alone, without the pancreatic juice, does not emulsify fats. Dastee ' tried the reverse experiment in dogs, namely, tying the choledochus duct aud producing a biliary fistula, through which the bile would flow into the intestine below the month of the pancreatic passages. .• "When the animals were killed after a meal rich in fat, the chylous vessels were first milky-white below the opening of the ' Du Bois-Reymond's Arch., 1880. » Lehrbuch d. physiol. Chem., 1868, S. 123. » Arch, de pliysiol., (5) Tome 2, p. 315. 312 DIGESTION. biliary fistula. Dastke draws the following conclasion from this: that combined action of the bile and the pancreatic juice is neces- sary for the absorption of the fats — a deduction which coincides with the above-mentioned observations of Nencki and Eachford. The importance of the bile and the pancreatic juice for the absorp- tion of fats will be discussed in detail later (see Absorption) . Bile completely prevents pepsin digestion in artificial digestion, and it may also retard the swelling up of the proteids. The passage of bile into the stomach during digestion, on the contrary, seems according to several investigators, especially Oddi ' and Dastee," to have no retarding action on stomachic digestion. Bile has no solvent actioa on proteids in neutral or alkaline reaction, but still it may have an influence on proteid digestion in the intestine. The acid contents of the stomach, containing an abundauce of proteids, give with the bile a precipitate of proteids and bile-acids. This precipitate carries a part of the pepsin with it, and for this reason, and also on account of the partial or complete neutralization of the acid of the gastric juice by the alkali of the bile and the pancreatic juice, the pepsin digestion cannot proceed further in the intestine. On the contrary, the bile does not disturb the digestion of proteids by the pancreatic juice in the intestine. The action of these digestive secretions, as above stated, is not disturbed by the bile, especially not by the faintly acid reaction due to organic acids which are habitually found in the upper parts of the intestine. In a dog killed while digestion is going on, the faintly acid, bile- containing mattier of the intestine shows regularly a strong digestive action on proteids. The precipitate formed on the meeting of the acid contents of the stomach with the bile easily redissolves in an excess of bile and also in the NaCl formed in the neutralization of the hydrochloric acid of the gastric juice. This may take place even under faintly acid reaction. Since in man the excretory ducts of the bile and the - pancreatic juice open near one another, in consequence of which the acid contents of the stomach are probably immediately in great part neutralized by the bile as soon as it enters, it is doubtful whether a precipitation of proteids by the bile occurs in the^ntestine. Besides the previously mentioned processes caused by enzymes, there are others of a different nature going on in the intestine, ' Centralbl. f. Physiol., 1887, S. 313. »L. c. CHEMICAL PR0CE8ISE8 IN THE INTESTINE. 313 namely, the fermentation and pntrefaetion processes caused by micro-organisms. These are less intense in the upper parts of the intestine, but increase in intensity towards the lower part of the same, and decrease in the large intestine because of the absorption of water. Fermentation but not putrefaction processes occnr in the small intestine as long as the contents are strongly acid. Macfadyek, M. Nbkcki, and N. Siebbk ' have investigated a case of human anus praeternaturalis, in which the fistula occurred at the lower end of the ileum, and they were able to investigate the con- tents of the intestine after it had been exposed to the action of the mucous membrane of the entire small intestine. The mass was yellow or yellowish brown, due to bilirubin, had an acid reaction which, calculated as acetic acid, amounted to 1 p. m. The con- tents were nearly odorless, having an empyreumatic odor recalling that of volatile fatty acids, and only seldom had a putrid odor re- calling that of indol. The essential acid present was acetic acid, accompanied with fermentation lactic acid and paralactic acid, volatile fatty acids, succinic acid, and bile acids. Ooagulable pro- teids, peptone, mucin, dextrin, dextrose, and alcohol were present. Leucin and tyrosin could not be detected. According to the above-mentioned investigators, the proteids are only to a very slight extent, if at all, decomposed by the microbes in the small intestine of man. The microbes present in the small intestine preferably decompose the carbohydrates, forming ethyl alcohol and the above-mentioned organic acids. Free hydrochloric acid does not occur in the small intestine, and it is the organic acids that prevent the putrefaction of the proteids in the intestine and also reduce the decomposition of the carbohydrates. Farther investigations of Jakowskt ' lead to the same result, namely, that in man the putrefaction of the proteids does not take place in the small but in the large intestine. This putre- faction of the proteids is not the same as the pancreatic digestion, and these two processes are essentially different because of the products they yield. In the pancreatic digestion of proteids there are formed, as far as we know at present, besides albumoses and peptones, lysin, lysatinin, proteinchromogen, amido-acids, and am- monia. In the putrefaction of the proteids we have, indeed, the same products formed at the beginning, but the decomposition ' ArcK. f. exp. Path. u. Pharm., Bd. 28, S. 311. ' Arch, des sciences biol. de St. Petersbourg, Tome 1, 1893. • 314 DIGESTION. proceeds considerably further and a number of products are developed which have become known through the labors of numer- ous investigators, Nencki, BAUMANiT, Bbiegbk, H. and E. Sal- KOWSKI, and their pupils. The products which are formed in the putrefaction of proteids are (in addition to albumoses, peptones^ amido-acids, and ammonia) indol, skatol, paracresol, phenol^ phenyl-propionic acid, and phenyl-acetic acid, also paraoxyphenyl- acetic acid and Tiydroparacumaric acid (besides paracresol, pro- duced in the putrefaction of tyrosin), volatile fatty acids, carton dioxide, hydrogen, marsh-gas, methylmercaptan, and sulphuretted hydrogen. In the putrefaction of gelatin neither tyrosin nor indol is formed, while gycocoll is produced. Among these products of decomposition a few are of special interest because of their behavior within the organism and because after their absorption they pass into the urine. A few, such as the oxyacids, pass unchanged into the urine. Others, such as phenol, are directly transformed into ethereal sulphuric acids by synthesis, and are eliminated as such by the urine ; on the . contrary, others, such as indol and skatol, are only converted into ethereal sulphuric acids after oxidation (for details see Chapter XV). The quantity of these bodies in the urine varies also with the extent of the putre- factive processes in the intestine; at least this is true for the ethereal sulphuric acids. Their quantity increases in the urine with a stronger putrefaction, and the reverse takes place, as Baumann ' has shown by experiments on dogs, when the intestine has been disinfected by calomel, namely, they then disappear from the urine. Among the above-mentioned putrefactive products in the intes- tine the two following, indol and skatol. must be carefully discussed. OH ' / X Indol, C,H,]Sr = C,H^ OH, and Skatol, or methtl- \ / NH O.OH, INDOL, 0,H,N = C,H, OH, are two bodies which stand \ / NH > Zeitschr. f. physiol. Chem.. Bd. 10. INBOL AND SKATOL. 315 in close relationship to the indigo substances, and which are formed from the albuminous bodies by their putrefaction, or by fusion -with caustic alkali. Hence they occur habitually in the human intes- tinal canal and, after oxidation into indoxyl and skatoxyl respec- tively, pass, at least partly, into the urine as the corresponding ethereal sulphuric acids and also as glycuronic acids. These two bodies have been prepared synthetically in many ways. Both may be obtained from indigo by reducing it with tin and hydrochloric acid and heating this reduction product with zinc- dust (Baeyer'). Indol may be formed from skatol by passing it through a red-hot tube. Indol suspended in water is in part oxidized into indigo-blue by ozone (Nencki'). Indol and skatol crystallize in shining leaves, and their melting- points are + 53° and 95° C. respectively. Indol has a peculiar excrementitious odor, while skatol has an intense fetid odor (skatol obtained from indigo should be odorless). Both bodies are easily volatilized by steam, skatol more easily than indol. They may both be removed from the watery distillate by ether. Skatol is the more insoluble of the two in boiling water. Both are easily soluble in alcohol, and give with picric acid a combination consisting of red crystalline needles. If a mixture of the two picrates be distilled with ammonia, they both pass over without decomposition ; while if they are distilled with caustic soda, the indol but not the skatol is decomposed. The watery solution of indol gives with fuming nitric acid a red liquid, and then a red precipitate of nitroso-indol nitrate (Nbncki "). It is better to first add two or three drops of nitric acid, and then a 2j^ solution of potassium nitrite, drop by drop (Salkowski'). Skatol does not give this reaction. An alcoholic solution of indol treated with hydrochloric acid colors a pine chip cherry-red. Skatol does not give this reaction. Skatol dissolves in concentrated hydrochloric acid with a violet coloration. On warming skatol with sulphuric acid a beautiful purple-red coloration is obtained (Ciamician- and Magnanini '). For the detection of indol and skatol in, and their preparation from, excrement and putrefying mixtures, the main points of the • Annal. d. Chem. u. Pharm., Bd. 140, and Supplbd. 7, S. 56 ; also Ber. d. deutscU. chem. Gesellscli., Bdd. 1 and 3. ' Ber. d. deutsch. chem. Gesellsch., Bd. 8, S. 737. 8 Ibid., Bd. 8, S. 722 and 1517. ' Zeitschr. f. physiol. chem., Bd. 8, S. 447. ' Ber. d. deutsch. chem. Gesellsch., Bd. 21, S. 1928. 316 DIGESTION. usual method are as follows: The mixture is distilled after acidify- ing with acetic acid; the distillate is then treated with alkali (to combiae with any phenol which may be present) and again distilled. From this second distillate the two bodies, after the addition of hydrochloric acid, are precipitated by picric acid. The picrate pre- cipitate is then distilled with ammonia. The two bodies are obtained from the distillate by repeated shaking with ether and evaporation of the several ethereal extracts. The residue, contain- ing indol and skatol, is dissolved in a very small quantity of absolute alcohol and treated with 8-10 vols, of water. Skatol is precipi- tated, but not the indol. The further treatment necessary for their separation and purification will be found in other works. The gases which are produced by the decomposition processes are mixed in the intestinal tract with the atmospheric air swallowed with the saliva, and as the gas generated by different foods varies, so the mixture of gases after various foods should have a dissimilar composition. This is found to be true. Oxygen is only found in very faint traces in the intestine; this may be accounted for in part by the formation of reducing substances in the fermentation processes which combine with the oxygen, and partly, perhaps chiefly, to a diffusion of the oxygen through the tissues of the walls of the intestine. To show that these processes take place mainly in the stomach the reader is referred to page 378, on the composition of the gases of the stomach. Nitrogen is habitually found in the intestine, and it is probably due chiefly to the swallowed air, or perhaps in part, as Bttkge ' claims, to a diffusion of nitrogen from the tissues of the intestinal walls to the intestine. The carbon dioxide originates partly from the contents of the stomach, partly from the putrefaction of the proteids, partly from the lactic-acid and butyric-acid fermentation of carbohydrates, and partly from the setting free of carbon dioxide from the alkali carbonates of the pancreatic and intestinal juices by their neutralization through the hydrochloric acid of the. gastric juice and by organic acids formed in the fermentation. Hydrogen occurs in largest quantities after a milk diet and in smallest quantities after a purely meat diet. This gas seems to be formed chiefly from the butyric-acid fermentation of carbohydrates, although it may occur in large quantities in the putrefaction of proteids under certain circumstances. There is no doubt that the methyhnercaptan and sulphuretted hydrogen which occurs normally in the intestine originates from the proteids. The ' Lehrbucli d. pbysiol. u. path. Chem., 1. Aufl., S. 368. PUTREFACTIVE PROCESSES IN THE INTESTINE. 317 marsh-gas undoubtedly originates in the putrefaction of proteids. As proof of this Kugb ' found 36.45^ marsh-gas in the human intestine after a meat diet. He found a still greater quantity of this gas after a diet consisting of leguminous plants ; this coincides with the observation that marsh-gas may be produced by a fermea- tation of carbohydrates, but especially of cellulose (Tappeinek"). Such an origin of marsh-gas, especially in herbivora, is to be expected. A small part of the marsh-gas and carbon dioxide may also depend on the decomposition of lecithin (Hasebeobk ') . Putrefaction in the intestine not only depends upon the com- position of the food, but also upon the albuminous secretions and the bile. Among the constituents of bile which are changed or decomposed we have not only the pigments — the bilirubin yields hydrobilirabin and a brown pigment — but also the bile-acids, especially taurocholic acid. Glycocholic acid is more stable, and a part is found unchanged in the excrement of certain animals, while taurocholic acid is so completely decomposed that it is entirely absent in the faeces. In the foetus, in whose intestinal tract no putrefaction processes occur, we find, on the contrary, undecom- posed bile-acids and bile-pigments in the contents of the intestine. The reduction of bilirubin into hydrobilirubin does not, according to Macfadten, Neucki, and Sieber,' take place in man in the small but in the large intestine. That the secretions rich in proteids are of importance in putre- faction in the intestine follows from the fact that putrefaction may also continue during complete fasting. From the observations of MiJLLEE ' on Cetti it was foand that the elimination of indican during starvation rapidly decreased and after the third day of starvation it had entirely disappeared, while the phenol elimination, which at first decreased so that it was nearly minimum, increased again from the fifth day of starvation and on the eighth or ninth day it was three to seven times as much as in man under ordinary circumstances. In dogs, on the contrary, the elimination of indican during starvation is considerable, but the phenol elimination is minimum. Among the secretions which undergo putrefaction in • Wien. Sitzungsber. , Bd. 44. 'L. c. ' Zeitschr. f. physiol Chem., Bd, 12. ■" Arcli. f. exp. Path. u. Pliarm., Bd. 38. » Berlin, klin. Wochenschr., 1887, No. 24. 318 DIGESTION. the intestine, the pancreatic juice, which putrefies most readily, takes first place. Pisenti ' found, in his experiments on dogs, that the elimination of indican by the urine greatly diminished after tying the pancreatic ducts, but that it increased again when the animal was given pancreas peptones or pancreatic juice. From the foregoing facts we conclude that the products formed by the putrefaction in the intestine are in part the same as those formed in digestion. The putrefaction may Lie oi benefit to the organism so far as the formation of such products as albumoses, peptones, and perhaps also certain amido-acids is concerned. On the contrary, the formation of further splitting products is to be considered as a loss of valuable material, and it is therefore im- portant that putrefaction in the intestine is kept within certain limits. If an animal is killed while digestion in the intestine is going on, the contents of the small intestine give out a peculiar but not putrescent odor. Also the odor of the contents of the large intestine is far less offensive than a putrefying pancreas infusion or a putrefying mixture rich in proteid. From this we may conclude that putrefaction in the intestine is ordinarily not nearly as intense as outside of the organism. It seems thus to be provided, under physiological conditions, that putrefaction shall not proceed too far, and the factors which here come under consideration are probably of different kinds. Absorption is undoubtedly one of the most important of them, and it has been proved by actual observation that the putrefaction increases, as a rule, as the absorption is checked and fiuid masses accumulate in the intestine. The character of the food also has an unmistakable influence, and it seems as if a large quantity of carbo- hydrates in the food acts against putrefaction (Hirschleb"). It has been shown by P5h:l, Bieenacki, Eovighi, Wiisttbe- ifiTZ, and SoHMiTZ " that milk and kephir have a specially strong preventive action on putrefaction. , This action, according to ScHMiTZ, is not due to the casein, but chiefly to the lactose and also in part to the lactic acid. A specially strong preventive action on putrefaction has been ' See Maly's Jahresber., Bd. 17, S. 377. ' Zeitschr. f. physiol. Chem. , Bd. 10, S. 306. 'Ibid., 17, S. 401, which, gives references to the older literature, and Bd. 19. See also Salkowski, Centralbl., f. d. med. Wiss., 1893, S. 467. PUTREFACTIVE PROCESSES IN THE INTESTINE. 319 ascribed for a, long time to the bile. This anti-putrid action is not due to neutral or faintly alkaline bile, whicii itself easily putrefies, but to the free bile-acids, especially taurocholic acid (Maly and Emich,' Lindbergee ") . There is no question that the free bile- acids have a strong preventive action on putrefaction outside of the organism, and it is therefore difficult to deny such an action in the intestine. Notwithstanding this the anti-putrid action of the bile in the intestine is contradicted by certain investigators (Voit,° EoHlIAlirK'*). Biliary fistulse have been established so as to study the import- ance of the bile in digestion (Schwank," Blondlot,' Bidder and Schmidt,' and others). As a result it has been observed that with fatty foods an imperfect absorption of fat regularly takes place, and the excrements contain, therefore, an excess of fat and have a light- gray or pale color. The extent of deviation from the normal after the operation is essentially dependent upon the character of the food. If an animal is fed on meat and fat, then the quantity of food must be considerably increased after the operation, otherwise the animal will become very thin, and indeed die with symptoms of starvation. In these cases the excrements have the odor of carrion, and this was considered a proof of the action of the bile in checking putrefaction. The emaciation and the increased want of food depend, naturally, upon the imperfect absorption of the fats, whose high calorific value is reduced and must be replaced by the taking up of larger quantities of other nutritive bodies. If the quantity of proteids and fats be increased, then this last, which can be only very incompletely absorbed, accumalates in the intestine. This accumulation of the fats in the intestine only renders the action of the digestive juices on proteids more difficult, and these last increase the amount of putrefaction. This explains the appearance of fetid faeces, whose pale color is not due to a lack of bile-pigments, but to a surplus of fat (Rohmann, Voit). If the animal is, on the contrary, fed on meat and carbohydrates, it may remain quite normal, and the leading o£E of the bile does not cause any increased ' Monatsheft f. Chem., Bd. 4. » Maly's Jahresber., Bd. 14, S. 334. » Beitr. z. Biologie. Jubilaumaschrift. Stuttgart, 1882. * Pflttger's Arch., Bd. 29. ' Miller's Arch. f. Anat. u. Physiol., 1844. ' Essai sar les fonctions du foie et de ses annexes. Paris, 1846. ^ Die YerdauungssSfte und der StofCwechsel, S. 98. 320 DIGESriON. putrefaction. The carbohydrates may be uninterruptedly absorbed in such large quantities that they replace the fat of the food, and this is the reason why the animal on such a diet does not become emaciated. As with this diet the putrefaction in the intestine is no greater than under normal conditions even though the bile is absent, it would seem that the bile in the intestine exercises no pre- ventive action on putrefaction. We must remember, however, that the presence of free acids counteracts putrefaction, and further that the carbohydrates yield free acids by acid fermentation within the intestine. It is there- fore conceivable that to the carbohydrates, which, according to HiKSCHLER, are capable of checking putrefaction without entering into an acid fermentation, the antiseptic action of the bile is due. It cannot be denied that the bile under ordinary conditions, with a mixed diet deficient in carbohydrates, has a preventive action on the putrefaction in the intestine. Limboukg ' has shown that it acts in an antiseptic sense, so that the destrnction of the proteids, giving rise to simpler products less valuable, or perhaps even injurious, in the organism, is checked. Although the question how the putrefactive processes in the intestine under physiological conditions are kept within certain limits cannot be answered positively, still it may be asserted that the acid reaction of the upper parts of the intestine and the absorp- tion of water in the lower parts are important factors. That the acid reaction in the intestine has a preventive influence on the putrefactive processes follows from the existing relation between the degree of acidity of the gastric Juice and the putrefac- tion in the intestine. After the investigations and observations of Kast, Stadelmajstn, Wasbutzki, Bibrnacki, and Mestee had proven that an increased putrefaction in the intestine occurred when the quantity of hydrochloric acid in the gastric Juice was diminished or deficient, Schmitz ' has lately shown in man that on the administration of hydrochloric acid, producing a hyperacidity of the gastric Juice, the putrefaction in the intestine may be checked. Excrements. It is evident that the residue which remains after completed digestion and absorption in the intestine must be differ- ent, both qualitatively and quantitatively, according to the variety ' Zeitschr. f. physio]. Chem., Bd. 13. ' Ibid., Bd. 19, S. 401, wliich includes all the pertinent literature. EXCREMENT. 321 and quantity of the food. In man the quantity of excrement from a mixed diet is 120-150 grms., with 30-37 grms. solids, per 24 hours, while the quantity from a vegetable diet, according to Voit,,'^ was 333 grms., with 75 grms. solids. With a strictly meat diet the excrements are scanty, pitch-like, and colored nearly black by hsBmatin and iron sulphide. The scanty excrements in starvation have a similar appearance. A large quantity of coarse bread yields a great amount of light-colored excrement. If there is a large pro- portion of fat, it takes a lighter, clayey appearance. The decom- position products of the bile-pigments seem to play only a small part in the normal color of the faeces. The constituents of the faeces are of different kinds. We find in the excrements digestible or absorbable constituents of the food, such as muscle-fibres, connective tissues, lumps of casein, grains of starch, and fat which have not had sufficient time to be completely digested or absorbed in the intestinal tract. In addition the excre- ments contain indigestible bodies, such as remains of plants, keratin substances, nuclein, and others; also form-elements originating; from the mucous coat and the glands; constituents of the different secretions, such as mucin, cholalic acid, dyslysin, and cholesterin; mineral bodies of the food and the secretions; and, lastly, products of putrefaction or of the digestion, such as skatol, indol, volatile fatty acids, lime, and magnesia soaps. Occasionally, also, parasites of different kinds occur; and lastly, the excrements contain micro- organisms, fungi of different kinds, sometimes in such large quantities that the chief mass of the excrements seems to consist of micro-organisms (v. Jaksch"). That the mucous membrane of the intestine by its secretion and by the abundant quantity of detached epithelium contributes essentially to the formation of excrement follows from the observa- tions first made by L. Hermann," who separated a loop of intestine, washed it clean and united the two ends, forming a ring, and restored the continuity of the remainder of the intestine. He found in a few days a mass resembling f feces which he called " ring faeces." 1 Zeitschr. f. Biologie, Bd. 25, S. 264. » Klinische Diagnostik, 3 Aufl. S. 302. » Pflilger's Arch., Bd. 46. See also Ehrenthal, ibid., Bd. 48; Bernstein, ibid., Bd. 53; Klecki, Centralbl. f. Physiol., 1898, S. 736, and F. Volt, Zeitsolir. . f. Biologie, Bd. 29. 322 DIGESTION. The reaction of the excrements is very changeable. It is often acid in the inner part, while the outer layers in contact with the macous coat have an alkaline reaction. In nursing infants it is habitually acid (Wegscheidek '). The odor is perhaps chiefly due to skatol, which was first found in the excrements by Bkiegee, and so named by him. Indol and other substances also take part in the production of odor. The color is ordinarily light or dark brown, and depends above all upon the nature of the food. Medicinal bodies may give the fseces an abnormal color. The excrements are colored black by iron and bismuth, yellow by rhubarb, and green by calomel. This last-mentioned color was formerly accounted for by the formation of a little mercury sulphide, but now it is said that calomel checks the putrefaction and the decomposition of the bile-pigments, so that a part of the bile-pigments pass into the f feces as biliverdin. According to Lesage '' a green color of the excrements in children is caused partly by biliverdin and partly by a pigment produced from a bacillus. In the yolk-yellow or green- ish-yellow excrements of nursing infants we can detect bilirubin. iNeither bilirubin nor biliverdin seems to exist in the excrements of mature persons under normal conditions. On the contrary, we find STEECOBiLiifr (Masius and Vanlaie), which, according to certain investigators, is identical with hydrobilirubin (Malt), which is obtained from bilirubin by a reduction process, and urobilin (Jaffe) — a view contested by MacMunk." Bilirubin may occur i n pathological cases in the fasces of mature persons. It has been observed in a crystallized state (as hsematoidin) in the fseces of children as well as of grown persons (Uffelmann," v. Jaksch °). The absence of bile (acholic faeces) causes the excrements to have, as above stated, a gray color, due to large quantities of fat; this may, however, be partly attributed to the absence of bile-pig- ments. In these cases a large quantity of crystals has been observed (Geehakdt, v. Jaksch) which consist chiefly of magnesia soaps (OESTEELEif) or sodium soaps (Stadelmaitn'). Hemorrhage in the upper parts of the digestive tract yields, when it is not very abundant, a dark-brown excrement, due to haematin. ' See Maly's Jaliresber., Bd. 6, S. 482. = Ibid., Bd. 18, S. 336. » See Chapter VIII, on the bile, p. 234. * Deutsch. Arch. f. klin. Med., Bd. 24. ' Klinische Diagnostik, 4. Aufl., S. 373. • In regard to fat crystals in the faeces see v. Jaksch, 1. c, p. 374. MECONIUM. 323 ExCKBTiN, SO named by Mabcet,' is a crystalline body occurring in human excrement, but which, according to Hoppk-Sbyler, is perhaps only impure •cholesterin. Excrbtolic acid is the name given by Marcet to an oily body with an excrementitious odor. In consideration of the very variable composition of excrements their quantitative analyses are of little value and therefore will be ■omitted. Meconium is a dark brownish-green, pitchy, mostly acid mass •without any strong odor. It contains greenish-colored epithelium cells, cell-detritus, numerous fat-globules, and cholesterin plates. The amount of water and solids is respectively 720-800 and 280-200 p. m. Among the solids we find mucin, bile-pigments and bile- acids, cholesterin, fats, soaps, calcium and magnesium phosphates. Sugar and lactic acid, albuminous bodies (?) and peptones, also leucin and tyrosin and the other products of putrefaction occurring in the intestine, are absent. Meconium may contain undecomposed tanrocholic acid, bilirubin and biliverdin, but it does not contain any hydrobilirubin, which is considered as proof of the non-exist- ence of putrefactive processes in the digestive tract of the fcetus. In medico-legal cases it is sometimes necessary to decide whether ispots on linen or other substances are caused by meconium. In such cases we have the following conditions : The spot caused by meconium has a brownish-green color and can be easily separated from the material because, on account of the ropy property of the meconium, it is difiBcult to wet through. When moistened with water it does not develop any special odor, but on warming with dilute sulphuric acid it has a somewhat fetid odor. It forms with water a slimy, greenish-yellow liquid containing brown flakes. The solution gives with an excess of acetic acid an insoluble precipitate of mucin; on boiling it does not coagulate. The filtered, watery extract gives GtMELIN's, but still better Huppert's, reaction for bile-pigments. The liquid precipitated by an excess of milk of lime gives a nearly colorless filtrate, which after concentration gives PBTTBiirKOFEE's reaction. The contents of the intestine under abnormal conditions are perhaps less the subject of chemical analysis than of an inspection or microscopical investigation. On this account the question as to the properties of the contents of the intestine in different diseases •cannot be thoroughly treated here. The question as to the difEerent processes which, so far as they are dependent on secretion and absorption, cause an abnormal consistency, a thinning of the excre- ' Annal. de chim. et de phys., Tome 59 324 DIGESTION. menfcs, possesses a certain interest. Such excrements may in part be produced by arrested absorption of liquid from the intestine for some reason or other, and in part caused by an increased secretion or a transudation of liquids into the intestine. A diminished absorption (of water) may be caused by a more active movement of the intestine, which causes their contents ta pass quickly, and in this way the action of laxatives is often ex- plained. A diminished absorption may also be due to a decreased activity of the absorbing cells. In absorption, which is generally accepted to-day, the cells of the mucous coat take an active part, and anything which acts disturbingly on the protoplasm of these cells must also exercise an influence on the absorption. This con- dition with regard to the action of laxatives has been especially noted by Hoppe-Setlee. ' According to him, it is also probable that such laxatives, of which only traces are required for absorp- tion, by a direct action on the intestinal epithelium — whether the absorption is made more difficult, or a transudation made possible, or whether the action of these two is simultaneous — cause watery evacuations. According to Eohmakn,^ concentrated salt solutions act by a decreased absorption activity. A thin evacuation may be produced by an increased elimination of fluid into the intestine, and there are many investigators wha consider it positively proved that a transudation of liquid into the intestine is cansed by the action of saline laxatives. The character of the intestinal epithelium is undoubtedly an important factor in the production of such a transudation, and when this is caused by the saline laxatives it probably is produced by action on the epithelium. We must admit with Hoppb-Setler and other investigators that the most important regulator of the flow of liquid through the intestinal mucous membrane is the intes- tinal epithelium. It is the epithelium which renders possible the stream of fluid contrary to the laws of osmosis, and which under normal conditions prevents a transudation into the intestine. Bodies which affect the epithelium may therefore cause a transuda- tion, and this is found to be especially abundant after ejection of the intestinal epithelium. The most striking example of this is observed in Asiatic cholera, in, which the epithelium is largely expelled and an extraordinarily abundant transudation takes place. ' Physiol. Chem., S. 359 and 361. = Pfluger's Arch., Bd. 41. INTESriNAL CONOBBMENTB. 325 Appendix. Intestinal Concrements. Calculi occur very seldom in. human intestine or in the intestine «f carnivora, but they are quite common in herbivora. Foreign bodies or undigested residues of food may, when for some reason or other they are retained in the intestine for some time, become incrusted with salts, especially ammonium -magnesium phosphate or magnesium phosphate, and these salts form usually the chief con- stituent of the concrements. In man they are sometimes oval or Tound, yellow, yellowish gray, or brownish gray, of variable size, consisting of concentric layers and containing chiefly ammonium- magnesium phosphate, calcium phosphate, besides a small quantity of fat or pigment. The nucleus ordinarily consists of some foreign body, such as the stone of a fruit, a fragment of bone, or something similar. In those countries where bread made from oafc-bran is an important food, we often find in the large intestine ^balls similar to the so-called hair-balls (see below). Such calculi contain calcium and magnesium phosphate (about 70^), oat-bran (15-18^), soaps and fat (about lOjg). Concretions which contain very much (about 14:%) fat seldom, occur and those consisting of fibrin clots, sinews, or pieces of meat incrusted with phosphates are also rare. Intestinal calculi often occur in animals, especially in horses fed on bran. These calculi, which attain a very large size, are hard and heavy (as much as 8 kilos) and consist in great iJart of concen- tric layers of ammonium-magnesium phosphate. Another variety of concrements which occurs in horses and cattle consists of gray- colored, often very large, but relatively light stones which contain plant residues and earthy phosphates. Stones of a third variety are sometimes cylindrical, sometimes spherical, smooth, shining, brownish on the surface, consisting of matted hairs and plant-fibres, and termed hair-dalls. The so-called " ^gagkopila," which probably originate from the antilopus eupicapea, belong to this group, and are generally considered as nothing else than the hair- balls of cattle. The so-called oriental hezoar-stone belongs also to the intestinal concrements, and probably originates from the intestinal tract of the CAPEA JEGASEUS and antilope doecas. We may have two varieties of bezoar-stones. One is olive-green, faintly shining. 326, DIGESTION. formed of concentric layers. On heating it melts with the develop- ment of an aromatic odor. It contains as chief constituent litho- FELLic acid, OjjHj.O,, which is related to cholalic acids, and besides this a bile-acid, lithobilic acid. The others are nearly blackish brown or dark green, very glossy, consisting of concentric layers, and do not melt on heating. They contain as chief constituent ELLAGic ACID, a derivative of tannic acid, of the formula C,,H,Oj> which gives a deep blue color with an alcoholic solution of ferric chloride. This last-mentioned bezoar-stone originates, to all appearances, from the food of the animal. Ambergris is generally considered an intestinal concrement of the sperm- whale. Its chief constituent is ambbain, which is a non-nitrogenous sub- stance perhaps related to cholesterin. Ambrain is insoluble in water and is. not changed by boiling alkalies. It dissolves in alcohol, ether, and oils. VI. Absorption. The problem of digestion consists in part in separating the= valuable constituents of the food from the useless constituents and to dissolve or transform these first into forms which are necessary in the processes of absorption. In discussing the absorption processes we must treat of the form into which the different foods, are transformed before absorption, of the manner in which this is accomplished, and, lastly, of the forces which act in these processes. Peptone is the final product of the digestion of albuminous bodies. Now as peptone is a very soluble and a relatively easily diffusible modification of proteids, it is not difficult to admit the deduction that proteids must be changed into peptone in order that- it may be readily absorbed. Certain observations of FuifKES ' on animals confirm this view. He found in an untied intestinal knot of a living animal that the peptone (in the old sense of the word) was absorbed consid^rably faster than other proteids. There is also no doubt that a part of the proteids is invariably absorbed from the intestinal canal as peptones, or more correctly perhaps as albumoses and peptones. But it has been positively settled by the investiga- tions of Bkucke," Bacjee and Voit," Eichhoest,* Czekett and Latschenbeegbb,' that non-peptonized proteids, casein, myosin^ ' SeeKiihne's Lehrb. d. physiol. Chem., S. 145. ' Wien. Sitzungsber. , Bdd. 37 and 59. ' Zeitschr f. Biologie, Bd. 5. * Pflilger's Arch., Bd. 4, ' Virchow's Arch., Bd. 59. ABSORPTION OF PB0TEID8. 327 and alkali albaminates are absorbed from the intestine — a matter which is of practical importance especially with regard to the nutritive clysters. If the proteids can be absorbed partly as snch and partly as peptone or albamoses, then the question arises, how much more can it be absorbed in one form than in the other ? This question cannot be decisively answered. Several investig£^- tions have been made on this subject, but it is hardly possible to draw any positive conclusion from them. In feeding experiments on pigs Elleubeegbb and Hofmeistek ' found that meat was only slowly digested and the quantity of albumoses and peptones in the intestinal canal was always very small. Ewald and Gumlich ' have obtained the same results in regard to the quantity of peptone in normal human stomachs after partaking of meat. Although the albumoses and peptones are rather readily (perhaps more readily than other proteids) absorbed, still it is clear that no positive con- clusion can be drawn as to the abundance of peptone-formation from the small quantities of albumoses or peptones found in a certain portion of the intestine. The investigations of Schmidt- MuLHBiM ' of the contents of the stomach and intestine of dogs who were killed at various times after a meal of boiled meat show that the quantity of peptone in the intestinal canal is considerably larger than the quantity of simply dissolved proteids, and this seems to indicate that in these cases the greatest part of the proteids is absorbed as peptones (or albumoses). In what way are the albumoses and peptones absorbed, and how are they conveyed to the tissues? Ludwig and Schmidt-Mul- HEiM * tied the jugular and humeral arteries and lymphatic vessels of both sides of a dog, completely cutting off the chyle from the blood circulation, as shown later on dissection. They found that the absorption from the intestine hereby was not impaired, and it fol- lows from this that the proteids do not reach the blood through the lymphatic vessels, but through the walls of the intestinal epithelium. The observations of Munk and Rosekstein " on a patient with a lymphatic fistula have "led to the same conception. They observed that the quantity of proteid in the chyle did not materially increase ' Du Bois-Reymond's Arch., 1890. » Berl. klin. Wochenschr. , 1890, No. 44. • Du Bois-Reymond's Arcli., 1879. * Ibid., 1877, S. 549. « Virchow's Arch., Bd. 133. 328 niOESTION. after a meal rich in proteids. K either albumoses nor peptones are found in the chyle after a meal rich in proteids. As the peptones (albnmoses incladed) do not pass in to the lymph, it is to be expected that peptones may be found in the blood during or after digestion. This is not the case. Schmidt- Mulheim ' and Hofmeistee ' only found traces of peptone in the serum or blood, and according to JSTeumbistbr " not even traces exist in the blood. What becomes of the peptone absorbed from the intestine ? If peptone is introduced into the circulating blood it is quickly eliminated from the blood by means of the urine (Plosz and Oyebgyai,' Hofmeistee," Schmidt-Mulheim'). The same takes place on the subcutaneous injection of peptone. Iformal urine does not contain any peptone, and the absence of this body in the blood after digestion cannot be explained by the statement that an elimi- nation of this peptone takes place through the kidneys. As the peptone introduced in the blood is quickly eliminated through the kidneys, while that formed in the intestine does not pass into the nrine, we can perhaps consider that this peptone is retained normally by the liver and is consumed, and only that peptone which finds its way into the circulating blood by evasion from this organ passes into the urine. This supposition, however, is untenable. IfEUMEiSTEE ' has investigated the portal blood of rabbits in whose stomachs large quantities of albumoses and peptones had been introduced, and found therein only traces of the body in question. He has also shown that when we supply the liver of a dog with the portal-blood peptone (ampho-peptone), this is not retained by the liver, but is eliminated with the urine. Shoee ° has arrived at similar results in regard to the importance of the liver, and has also shown that the spleen cannot transform peptone. Peptone seems to pass neither into the blood nor the chylous vessels, and the following observation of Ltjdwig and Salvioli" bears out this > Du Bois-Reymond's Arcli., 1880. ^ Zeitschr. f . pliysiol. Chem, , Bdd. 5 and 6. ^ Zeitschr. f. Biologie, Bd. 34, S. 272. * Pfluger's Arch., Bd. 10. ' Zeitschr. f. ptysiol. Chem., Bd. 5. « Da Bois-Reymond's Arch., 1880. ■■ See Neumeister, Sitzungsber. d. phys.-med. Gesellsch, zu Wlirzburg, 1889, and Zeitschr. f . Biologie, Bd. 24. ' Journal of Physiol., Vol. 11. ' Du Bois-Reymond's Arch. , 1880, Supplement. TRAN8F0nMATI0N OF ALBUMOSEB AND PEPTONES. 329 assumption. These iavestigators introduced a peptone solution into a double-ligatured, isolated piece of the small intestine, which was kept alive by passing defibrinated blood through it and observed that the peptone disappeared from the intestine, but that the blood passing through did not contain any peptone. All observations indicate that the albumoses and peptones are transformed in some way in the intestine or intestinal wall. Certain investigators, such as v. Ott,' Nadine Popoff,'' and Julia Bkinck ° are of the opinion that the albumoses and peptones of gastric digestion are transformed into seralbumin before they pass into the walls of the digestive tract. This transformation is brought about by means of the epithelium cells, as also by the living activity of a fungus called by Julia Bkinck micrococcus resti- tuens. No positive proofs have been presented for this view. The view that the transformation of the albumoses and peptones takes place after they have been taken up by the mucous membrane lias better foundation. The above-mentioned experiments of LuDWiG and Salvioli confirm this, as do also the observations of HoFMEisTEE ' — according to whom the walls of the stomach and the intestine are the only parts of the body in which peptones occur constantly during digestion — that peptone (at the temperature of the body) after a time disappeared from the excised but apparently still living mucous coat of the stomach. Peptone seems to undergo a change in the mucosa of the digestive canal. If, then, peptone already disappears in the mucous coat, or at least in the walls of the digestive tract, the question naturally arises, what becomes of the peptone in the mucous membrane ? The experiments of Malt,' Plosz and Gtbbgyai,' Adamkibwicz,' ZuJTTZ,' and Pollitzek" have established that the albumoses and peptones may be substituted for proteid in the food, and may also be converted into ordinary proteid. "We must then assume that ' Du Bois-Reymond's Arch., 1883, ' Zeitscbr. f. Biologie, Bd. 35. » Ibid. , Bd. 35, S. 453. * Zeitschr. f . physiol. Chem. , Bd. 6. ' Pfluger's Arob., Bd. 9. •L.c. ' Die Natur und der NSbrwertb des Peptons. Berlin, 1877. " Pfltlger's Arch., Bd. 87, S. 313. « Ibid., Bd. 37, S. 801. 330 DIOESTION. peptone is already converted into proteid in the mucous membrane of the digestive canal. According to Hofmeistee ' a considerable increase of leucocytes, occurs in the adenoid tissues during digestion, an observation which is in close accord with that of Pohl," who found that in dogs after an albuminous diet the venous blood of the intestine contains more leucocytes than the arterial blood. According to Hofmeistee, leucocytes play an important part in the absorption and assimilation of the peptones. They may take up the peptones and be the means, of transporting them to the blood, and secondly by their growth, regeneration, and increase may stand in close relation to the trans- formation and assimilation of the peptones. Hbidenhaust,' who- considers that the transformation of peptone into proteid in the mucous membrane is positively settled, does not attribute so great an importance to these last in the absorption of the peptones as Hofmeistee, chiefly on the ground of comparative estimation of the quantity of absorbed peptones and leucocytes. He considers it most probable that the reconversion of the peptones into proteid takes place in the epithelium layers. This view is further corrobo- rated by the investigations of Shoee.' The extent of the proteid absorption is dependent essentially upon the kind of food introduced, since as a rule the protein sub- stances from an animal source are much more completely absorbed than from a vegetable source. As proof of this we give the follow- ing observations: In his experiments on the utilization of certain foods in the intestinal canal of man Eubnee ° found with an exclu- sive animal diet on partaking of an average of 738-884 grms. fried meat or 948 grms. eggs per day that the nitrogen deficit with the excrement was only 3.5-3.8^ of the total introduced nitrogen. With exclusive milk diet the results were somewhat unfavorable, since after partaking of 4100 grms. milk the nitrogen deficit rose indeed to 13^. The conditions are quite different with vegetable food, as shown by .the experiments of Metee,' Eubnek,' Hultgeen 1 Arch. f. exp. Path. u. Pharm., Bdd. 19, 20, and 22. 'Ibid., Bd. 25. » Pailger's Arch., Bd. 43. *L. c. ' Zeitschr. f. Biologie, Bd. 15. » lUd., Bd. 7. ■'Ibid., Bd. 15. ABSORPTION OF PROTEIDS. 331 and Landergeen,' who made experiments with various kinds of rye bread and found that the loss of nitrogen through the faeces amounted to 22-48^. Experiments with other vegetable foods, and also the investigations of Schusteb," Cramer/ Meineet,* Mori,'' and others on the utilization of foods with mixed diets, have led to- similar results. All through we see that the loss of nitrogen by the excrement increases with an abundant amount of vegetable food in the diet. The reason for this is manifold. The often large quantity of cellulose present in vegetable foods impedes the absorption of pro- teids. The stronger irritation produced by the vegetable food itself or by the organic acids formed in the fermentation in the intestinal canal causes a stronger peristalsis which drives the con- tents of the intestine quicker than otherwise along the intestinal canal. Another and most important reason is the fact that a part of the vegetable protein substances seems to be indigestible. In speaking of the functions of the stomach we stated that after the removal or excision of this organ an abundant digestion and absorption of proteids may take place. It is therefore of interest to learn how the digestion and absorption of proteids go on after the extirpation of the second proteid-digesting organ, the pancreas. In this regard Minkowski and Abelmann ° found after the total extirpation of the pancreas of dogs thab the utilization of the pro- teids was on an average 44^, and after partial extirpation 54j^. Sandmetek ' focind in dogs after the extirpation of f or ^ of the gland,, and leaving pieces not in connection with the intestine, that the utilization of the proteids amounted to 62-70^. All three investigators found that on the addition of raw ox-pancreas to the- food the utilization of the proteids was essentially increased, and on the addition of sufficient finely chopped pancreas Sakdmeter observed even a proteid absorption which did not difEer much from that of a normal dog. It seems that the destructive action of the- ' Nord. mr-d. Arkiv., Bd. 21, No. 8. ' See Voit, TJntersucli. der Kost, etc., S. 142. ' Zeitschr. f. physiol. Chem., Bd. 6. * Ueber Massenernahrung. Berlin, 1885. ' Kellner and Mori, Zeitschr. f . Biologie, Bd. 25. • Ueber die Ausnutzung der NahrungsstofEe nach Pankreasexstirpation, etc. Inaug. Diss. Dorpat, 1890. Cited from Maly's Jahresber., Bd. 30. Zeitschr. f. Biologie, Bd. 31. 332 ■ BIOESTION. gastric juice on the trypsin does not assert itself under these circumstances, or only to a slight extent. The carbohydrates are, it seems, chiefly absorbed as monosac- charides. Glucose, IseTulose, and galactose are probably absorbed as such. The two disaccharides, cane-sugar and maltose, ordinarily undergo an inversion in the intestinal tract and are converted into glucose and Isevulose. Lactose, according to Voit and Lusk," is not inverted and is absorbed as such except what undergoes lactic- acid fermentation. The polysaccharides are also finally converted into monosaccharides, although in certain cases an absorption of dextrin may take place. According to the observations of Otto ' and V. Mering" the portal blood contains besides dextrose a dextrin-like carbohydrate after a carbohydrate diet. A part of the carbohydrates is destroyed by fermentation in the intestine, with the formation of lactic and acetic acids. The different varieties of sugars are absorbed with varying degrees of rapidity, bat as a general thing they are absorbed very quickly. With experiments on dogs Albeetoni * found on intro- ducing 100 grms. of the sugar that during the iirst hour 60 gfms. dextrose were absorbed, maltose and cane-sugar 70-80, and lactose ouly 30-40 grms. He finds that lactose is relatively more readily absorbed from dilute solutions than from concentrated ones. On the introduction of starch even in very considerable quanti- ties into the intestinal tract no dextrose passes into the urine, which probably depends in this case upon the absorption and assimilation and the slow sacchariflcation taking place at the same pace. If, on the contrary; large quantities are introduced at one time, then an elimination of sugar by the urine takes place, and this elimination of sugar is called alimentary glycosuria. In these cases the assimi- lation of the sugar and the absorption do not take place at the same pace, hence the liver and the remaining organs do not have the necessary time to fix and utilize the sugar. This glycosuria may also in part be due to the fact that the introduction of considerable quantities of sugar forces the sugar in absorption not only in the ordinary way through the blood-vessels to the liver (see below), but ' Zeitschr. f . Biologie, Bd. 38. = Cliristiania Videusk. Selskabs Porli., 1886, No. 11, and Maly's Jahresber., Bd. 17. ' Du Bois-Reymond's Arch., 1877. * Manifire de se comporter des sacres, etc. Arch. ital. de Biol., Tome 15. ABSORPTION OF CARBOBTDBATES. 333 also in parb by passing into the blood circulation through the lymphatic vessels, evading the liver. That quantity of sugar to which we must raise the sugar par- taken of to produce an alimentary glycosuria gives, according to HoFMEiSTEE,' the assimilation limit for that same sugar. This limit is difiEerent for various kinds of sugar; and it also varies for the same sugar not only in different animals, but also for different members of the same kind, as also for the same individual under different circumstances. In general we can say that in regard to the ordinary varieties of sugar, such as dextrose, Isevulose, cane-sugar, maltose, and lactose, the assimilation limit is highest for dextrose and lowest for lactose. We must admit that with an overabundant quantity of sugars in the intestinal tract the disaccharides do not have sufficient time for their complete inversion; hence it is not remarkable that disaccharides have been found in the urine in cases of alimentary glycosuria.^ From the investigations of Ludwig andv. Meeing' and others we learn in regard to the way in which the sugars pass into the blood-stream, namely, that they as well as bodies soluble in water do not ordinarily pass over into the chylous vessels in measurable quantities, but are in greatest part taken up by the blood in the capillaries of the villi and in this way pass into the mass of the blood. These investigations have been confirmed by observations of I. MuNK and Eosbis^stein * on human beings. The reason why the sugar and other soluble bodies do not pass over into the chylous vessels in appreciable quantity is, according to Heidenhain,' to be found in the anatomical conditions, in the arrangement of the capillaries close under the layer of epithelium. Ordinarily these capillaries find the necessary time for the taking up of the water and the solids dissolved in it. But when a large quantity of liquid, such as a sugar solution, is introduced into the intestine at once, this is not possible, and in these cases a part of the dissolved bodies passes into the chylous vessels and the thoracic duct (Ginsbeeg" and Eohmann'). ' Arch. f. exp. Path. u. Pharm., Bdd. 35 and 36. ' For the literature in regard to the passage of various kinds of sugars into the urine see C. Voit, Ueber die Glykogenbildung, Zeitschr. f . Biologie, Bd. 38. " Du Bois-Eeymond's Arch., 1877. 'L. c. ' Pflilger's Arch., Bd. 43, Suppl. • Ibid., Bd. 44. 'iSiU, Bd. 41. 334 DIGESTION. The introduction of larger quantities of sugar into the intestine at one time can readily cause a disturbance with diarrhceal evacua- tions of the intestine. If the carbohydrate is introduced in the form of starch, then very large quantities may be absorbed without causing any disturbance and the absorption may be very com- plete. EuBNEE ' found the following: On partaking 508-670 grms. carbohydrate as wheat bread per day the part not absorbed amounted to only 0.8-3.6^. For peas, where 357-588 grms. were eaten, the loss was 3.6-75^, and for potatoes (718 grms.) 7.6^. CoiS'STANTiKiDi " found on partaking 367-380 grms. carbohydrates, •chiefly as potatoes, a loss of only 0.4-0.7^. In the experiments of EuBKEK," as also of Hultqeen and LAWDEEGKEif,* with rye bread the utilization of carbohydrates was less complete, although the loss in a few cases rose even to 10.4-10.9^. It at least follows from the •experiments made thus far that man can absorb more than 500 grms. carbohydrates per diem without difBculty. We generally consider the pancreas as the most important ■organ in the digestion and absorption of amylaceous bodies, and it is a question how these bodies are absorbed after the extirpation of the pancreas. Minkowski and Abelmanx " found that in dogs after total extirpation of the pancreas only 57-71^ of the amylaceous Ijodies were absorbed. In the experiments of the brothers Cavaz- ZAifNi " only 47^^ of the starch introduced was used by the animal with the pancreas removed. Emulsiflcation seems to be of the greatest importance in the absorption of fats. The fats may be absorbed in part as soaps, but the quantity absorbed in this form is very small as compared to that which is absorbed as an emulsion. The emulsion is undoubtedly the most important form in which fats are absorbed, and the neutral Jats as well as the free fatty acids, when they occur in large quan- tities in the intestine, form an emulsion. The fatty acids are not absorbed as such or as soaps. The investigations of I. Munk,' and later confirmed by others,' have shown that the fatty acids undergo ' L. c. and Zeitschr. f . Biologie, Bd. 19. » Zeitschr. f. Biologie, Bd. 23. » Ibid., Bd. 15. « Nord. med. Arkiv., Bd. 21. ' L. c. See Maly's Jaliresber.,Bd. 20. « Centralbl. f. Physiol., Bd. 7. ' Virohow's Arch., Bd. 80. 8 See V. Walther, Du Bois-Reymond's Arch., 1890, and Minkowski, Arch. 1 exp. Path. u. Pharm., Bd. 21, S. 373. ABSORPTION OF FATS. 335 in great part a synthesis into neutral fats in the walls of the intes- tine or, according to Walthee,' in the intestine, and carried as euch by the stream of chyle into the blood. Through nnmeroas investigations we also know that among all the nutritive bodies the fats are the only substances that under ■ordinary conditions pass into the blood through the lymphatic ^vessels and the thoracic duct. It does not follow from this that all or the greater part of the fat takes this course, and according to the experiments of V. Walthbr and 0. Frank ' the reverse is true, namely, only a very small part of the fat, or at least of the fatty acids partaken of, passes into the chylous vessels. On feeding dogs with fatty acids Walthee found that in the course of several hours only very few grammes of fat were carried away with the lymph current, although the intestine had absorbed 40-60 grms. fat. Frank has reached a similar conclusion, and indeed found that by the excision of the thoracic duct an absorption of fatty acids took place to a considerable extent. These observations do not seem to be applicable to the absorption of neutral fats or of the absorption in man under normal conditions. Munk and Eosenstein in their investigations on a girl with lymph fistula found 60^ of the fat partaken of in the chyle, and of the total quantity of fat in the chyle only 4-5$^ existed as soaps. On feeding with a foreign fatty acid, such as erncic acid, they found 37^ of the introduced body as neutral fat in the chyle. The completeness with which fats are absorbed depends, under normal conditions, essentially upon the kind of fat. In this regard we know, especially from the investigations of Munk ° and Aen- SCHiNK,' that the varieties of fat with high melting-points, such as mutton tallow and especially stearin, are not so completely absorbed as the fats with low melting-points, such as hog- and goose-fat, olive- oil, etc. The kind of fat also has an influence upon the rapidity of absorption, as Munk and Eosenstein found that solid mutton- fat was absorbed more slowly than fluid lipanin. The extent of absorption in the intestinal tract is under physiological con- ditions very considerable. In a case of a dog investigated by VoiT ' he found that out of 350 grms. of fat (butter) partaken, 346 ' Walther, 1. c. * Du Bois-Keymond's Arch., 1893. • Virchow's Arch., Bdd. 80 and 85. * Zeitschr. f . Biologie, Bd. 26. • Ibid., Bd. 9. 33 b DIGESTION. grms. were absorbed in the intestinal canal, and according to the investigations of Etjbwer ' the human intestine can absorb over 300 grms. fat per diem. The fats are, according to Rctbnbk, much more completely absorbed when free, in the form of butter or lard, than when enclosed in the cell-membranes, as in bacon. The bile as well as the pancreas is of the greatest importance in the absorption of fats. Through numerous observations of many investigators, such as Bidder and Schmidt,^ Voit,' Eohmajs^ij^,* Fk. Mullek,'' I. MuNK,° and others, it has been shown that the exclusion of the bile from the intestinal tract diminishes the absorption of fat to such an extent that only \ to about -J of the quantity of fat ordi- narily absorbed undergoes absorption. In icterus with entire exclusion of the bile a considerable decrease in the absorption of fat is noticed. As under normal conditions, so also in the absence of bile in the intestine the more readily melting parts of the fats are more completely absorbed than those which have a high melting- point. I. MuNK found in his experiments with lard and mutton tallow on dogs that the absorption of the high melting tallow was. reduced twice as much as the lard on the exclusion of the bile from the intestine. We also learn from the investigations of Rohmann' and I. MuKK that in the absence of bile the relationship between fatty acids and neutral fats is changed, namely, about 80-90^ of the fat existing in the faeces consists of fatty acid, while under normal conditions the faeces contain 1 part neutral fat to about 3-2^ parts free fatty acids. We cannot positively state how this relatively increased quantity of fatty acids in the fat of the faeces is produced on the exclusion of the bile from the intestine. According to the investigations of MuifK it does not in the least depend upon the fact that the fatty acids are less readily absorbed than the neutral fats, for Just the reverse is the case. There is no doubt that the bile is of great importance in the absorption of fats. Still there is also no doubt that rather consider- able quantities of fat may be absorbed from the intestine in the ' Zeitsohr. f. Biologie, Bd. 15. ' Die Verdauungssafte und der StofEweclisel, S. 223. ' Beitrage z. Biologie, Jubilaumsschrift fiir v. BischofE. Stuttgart, 1883. *PflilgeT's Arch., Bd. 29. ' Sitzungsber. d. physik. -med. Gesellscli. zu Wurzburg, 1885. • Virohow's Arch., Bd. 122. ABSORPTION OF FATS. 337 absence of bile. What relation does the pancreas bear to this ques- tion ? According to Beenaed the presence of pancreatic juice in the intestine is necessary in the absorption of fats. This view has found support in the investigations of Minkowski and Abelmann ' ou the absorption of fats after the extirpation of the pancreas in dogs. These investigators found that the fat introduced in the food was not absorbed at all after the complete extirpation of the pancreas. Milk was an exception, and a greater or smaller part (38-53^) of its fat was absorbed. It is diflBcult to state anything positive about the significance of these observations, since there are other investigations which have led to diflerent results. Sandmetee " found in hia experiments on dogs that the utilization of the non-emulsified fat was very variable. Sometimes no fat was absorbed, while at other times in the same animal 30 or even 78^ of the administered fat was absorbed. In a series of experiments administering emulsified fat in the form of milk about 42j^ was absorbed . Teichmann ° has also found that after ligaturing the pancreatic duct in rabbits the absorption of fat was not noticeably disturbed, and Fe. Mullee ' had occasion to observe in a patient with pancreatic fistula that in human beings a considerable absorption of fat may take place in the intestine with- out pancreatic juice. The question as to the importance of the pancreatic juice in the' absorption of fats is still somewhat disputed. It is certain at least that the pancreatic juice is of very great importance for the absorption of fats, and it is also certain that the absorption of fats is most considerable in the simultaneous presence of bile and pancreatic juice in the intestine. We can give no explanation for this last fact. The common acceptance is that to form an emulsion of the fats a previous splitting is necessary, and this is; produced by the pancreatic juice, accelerated by the bile. Many doubts have been raised against this statement, and to what has been said already (page 310) we must add the following: In the experi- ments of Minkowski and Abelmann the masses of fat eliminated ' Ueber die Ausnutzung der NahrungsstofEe nach Pankreasexstirpation, etcs. Inaug. Diss. Dorpat, 1890. » Zeitschr. f Biologie, Bd. 31. ' Mikroskop. Beitr. z. Lehre von der Pettresorption. Diss. Breslau, 1891. Cited from Neuraeister, I/slirb. d. physiol. Chem. Jena, 1897. S. 336. ■* Cited from Neumeister, Lehrb. d. physiol. Chem. Jena, 1897. S. 387. 338 DIGESTION. by the fseces were in great part split even in the absence of the pancreas, and according to the investigations of Hedok and Wille ' an abundant splitting of the fats may take place in the intestine even in the absence of the bile as well as of the pancreatic Jaice. The extent of action of microbes and other unknown factors in -this splitting has not yet been determined. Prom these experiments we cannot draw any positive conclusion as to the importance of the splitting of the fat for emulsiflcation under normal conditions, because on the exclusion of the pancreatic juice from the intestine the secretion of alkali carbonates, which are important in the emulsiflcation of the fats as well as for the normal processes in the intestine, sufEers essentially in quantity. In the experiments of Min^kowski and Abblmann" the ethereal extract of the fat masses of the fseces consisted of 80,'^ fatty acids, ■which were chiefly free and only combined with alkali to a slight extent. V. Haelbt ' has made experiments on the absorption of fats (milk) in dogs with extirpated pancreas. The passage of the fats from the stomach to the intestine in these dogs was retarded, and Haklet found not only as much fat in the intestinal tract as Tvas introduced, but also a little which was derived from the secre- •itions and excretions of the intestine. This experiment gave entirely different results from Abelmakst's experiment, and Haelet ex- plains this by the fact that in ABBLMAiir2sr's experiment the action of intestinal bacteria was not excluded or reduced to a minimum, as in his. The fact that milk is the only form in which fat can be absorbed in dogs in the absence of pancreatic juice (Minkowski) may, according to him, be explained in the fact that this fat emulsion is permanent in acid as well as in neutral or alkaline reaction. Prom -these observations, and from the conflrming observations of Sakd- metee that a considerable absorption of other fats may take place in dogs with extirpated pancreas when with the fat food we add finely chopped ox-pancreas, Minkowski suggests that the proteids are of the greatest importance in the emulsiflcation of fats. This view is in accordance with the older statements of Bernaed and KtJHifB,^ but has not been the subject of thorough research. > See Maly's Jahresber., Bd. 22, S. 38. » Journal of Physiol., Vol. 18. ' See page 311. ABBOBPTION IN GENERAL. 339 The soluble salts are also absorbed with the water. The proteids and peptone which can dissolve a considerable quantity of salts, such as earthy phosphates which are otherwise insoluble in alkaline water, are of great importance in the absorption of such salts. Water, according to the experiments of v. Meeing,' as also of Olet and Rondeau," on dogs is not absorbed to any appreciable amount in the stomach. Alcohol, on the contrary, is absorbed to a great extent in the stomach. The extent of the absorption of .dissolved bodies seems to be dependent upon the concentration of the solution, and according to Beandl ' the difference between the absorption in the stomach and intestine consists in that the absorp- tion in the first organ takes place better in greater concentration and in the second in less concentration. Thus, for example, a solu- tion of cane- or grape-sngar is most completely absorbed by the intestine in a concentration of 0.5^. With increasing concentration the absorption diminishes, and in a concentration of 5^ a disturb- ance takes place. In the stomach an appreciable absorption first occurs with a concentration of 5%, and then increases to about 20^. The presence of other bodies which cause an irritation on the mucous membrane seems to be valuable for absorption, and accord- ing to Beakdl chloral hydrate, sugar, and potassium-iodide solu- tions are better absorbed in the presence of alcohol than from pure •watery solution. The soluble constituents of the digestive secretions may, like other dissolved bodies, be absorbed, as is demonstrated by the passage of peptone into urine; the enzymes may also be absorbed. The occurrence of urobilin in urine attests the absorption of the bile-constituents under physiological conditions notwithstanding the question as to the occurrence of very small traces of bile-acids in the urine is disputed. The absorption of bile-acids by the intestine seems to be positively proven by other observations. Tappeinee * introduced a solution of bile-salts of a known concentration into an intestinal knot, and after a time investigated the contents. He found that in the jejunum and the ileum, but not in the duodenum, an absorption of bile-acids took place, and further that of the two ' Centralbl. f. Physiol., Bd. 7, S. 533. » C. E. Soc. de Biol., 1898. • Zeitschr. f. Biologie, Bd. 39. This containB all the older literature relat- ing to this question. < Wien. Sitzungsber., Bd. 77. 340 DIGESTION. bile-acids only the glycocholic acid was absorbed in the jejnnnm. Further, Schiff ' long ago expressed the opinion that bile under- goes an intermediate circulation, in such wise that it is absorbed from the intestine, then carried to the liver by the blood, and lastly eliminated from the blood by this organ. Although this view has met with some opposition, still its correctness seems to be established by the researches of various investigators, and more recently by Peevost and Biket," as also and specially by Stadelmann and his pupils.' After the introduction of foreign bile into the intestine of an animal the foreign bile-acids appear again in the secreted bile. Little is known concerning the forces taking part in absorption. Osmosis and filtration were formerly considered as the most impor- tant factors. But as in regard to the peptones, whose formation in the digestion was considered as taking place especially in the interest of a facilitated osmosis and filtration, but whose conditions have been found quite different and much more complicated, so in the absorption theory there is a still greater contrast between former and present views, the latter inclining to the theory that absorption is a process connected with the vital properties of the cells (Hoppe- Setler'). Investigations in this direction have been made by Heidenhain ' and his pupils Eohmann", and Gttmilewsky ' ; and these investigations have shown that the cells take an active part in the absorption, and that this action is independent of the processed caused by an unequal difEnsibility of the different bodies. For example, in a solution which contains equal quantities of grape- sugar and sodium sulphate the sugar will be almost completely absorbed in a certain time, while the salt, which has the greater diffnsibility, still remains in considerable amounts in the intestine. According to the latest investigations of Heidenhain' ' on the absorption of blood-serum and common-salt solutions from the intestine of dogs, no doubt can now exist that the cells have a special physiological force besides which under certain circumstances osmosis may operate, but under other circumstances an absorption ' Pfluger's Arcb., Bd. 3. « Compt. rend., Tome 106. ' See reference, page 224. * Physiol. Chem., S. 348. ' Pfluger's Arch., Bdd. 43 and 56. *iJ8d., Bd. 41. •< Ibid , Bd. 39. <> Ibid., Bd. 56. ABSORPTION IN GENKRAL. 341 may take place with the entire exclusion of osmosis. It is also known that certain pigments are absorbed and others not, and the oells seem to have the property of discriminating between the different substances. The absorption of dissolved bodies seems to be connected with a specific activity of the living cell, the living protoplasm. In the absorption of bodies not dissolved, of the emulsified fats, iorces take part which are not known. That the bile performs the most important part in the absorption of fats is very generally admitted, but how the bile acts in this process is not yet deter- mined. V. WiSTiNGHAUSEN ' has found that fat rises higher in a capillary tube moistened with bile than when moistened with water, and further that fluid fat filters more easily through a dead membrane dipped in bile than when dipped in water. From these observations, whose correctness has lately been disputed by Gad and Gropek,' the inference has been drawn that bile facilitates the capillary attraction and thereby accelerates the absorption of the fats. The epithelium layer of the intestinal mucous membrane ■cannot be compared with a dead membrane soaked in water, and it is therefore doubtful if the above-mentioned action of bile can have any influence on the absorption of fats in the intestine. That the -absorption of fats is caused by the lymphoid migratory cells (ZAWARTKiifr,' ScHAEER*) is disputed by Gruenhaqbn- " and Hbideuhaik.' According to them, the fat takes its way chiefly through the epithelium cells. How these last act is, like the nature of their action in absorption, enveloped in darkness. ' See the translation of Wistinghausen's dissertation by Steiner in Du Bois- Beymond's Arch., 1873. ' Du Bois-Reymond's Arch., 1889. » Pfluger's Arch., Bd. 31. * Ibid. , Bd. 33. 'Arch. f. mikroskop. Anat., Bd. 29. « Pflilger's Arch,, Bd. 43. CHAPTER X. TISSUES OF THE CONNECTIVE SUBSTANCE. I. The Connective Tissues. The form-elements of the typical connective tissues are cells of various kinds, of a not very well known chemical composition, and gelatin-yielding fibrils, which like the cells are imbedded in an interstitial or intracellular substance. The fibrils consist of collagen. The interstitial substance contains chiefly mucin besides serglohulin and seralbumin., which occur in the parenchymatous fluid (Lobbisch'). The connective tissue also often contains fibres or formations consisting of elastin, sometimes in such great quantities that the connective tissue is transformed into elastic tissue. According to Mall " a third variety of fibres, the reticular fibres, also occur, and these according to Siegfried consist of eetiouliit. If finely divided tendons are extracted in cold water, the albuminous bodies soluble in the nutritive fluid in addition to a little mucin are dissolved. If the residue is extracted with half- saturated lime-water, then the mucin is dissolved (Rollett,' Loebisch) and may be precipitated from the filtered extract by saturating with acetic acid. The digested residue contains the fibrils of the connective tissue together with the cells and the elastic substance. The fibrils of the connective tissue are elastic and swell slightly in water, somewhat more in dilute alkalies or in acetic acid. On the other haad, they shrink by the action of certain metallic salts, such as ferrous sulphate or mercuric chloride, and tannic acid, which forms an insoluble combination with the collagen. Among ' Zeitschr. f. physiol. Chem., Bd. 10. » Kgl. Sachs. Gesellscb. d. Wissensoh., 1891, Bd. 17, Math.-phys. Klasse. » Wien. Sitzungsber., Bd. 39. 342 CONNECTIVE TISSUE AND CARTILAGE. 343 these combinations, which prevent putrefaction of the collagen, that with tannic acid has been found of the greatest technical importance in the preparation of leather. In regard to tendon mucin see page 45, and in regard to collagen, gelatin, elastin, and reticulin, pages 51-56. The tissues described under the names mucous or gelatinous tissues are characterized more by their physical than their chemical properties and have been but little studied. So much, however, is known, that the mucous or gelatinous tissues contain, at least im certain cases, as in the acalephse, no mucin. The umbilical cord is the most accessible material for the inves- tigation of the chemical constituents of the gelatinous tissues. The mucin occurring therein has been described on page 45. C. Th. MoRNEE ' has found a mucoid in the vitreous humor which contains 12.27^ nitrogen and 1.19$^ sulphur. Young connective tissue is richer in mucin than old. Halli- BUETON ' found an average of 7.66 p. m. mucin in the skin of very young children and only 3.85 p. m. in the skin of adults; In so-called myxoedema, in which a reformation of the connective tissue of the skin takes place, the quantity of mucin is also increased. II. Cartilage. Cartilaginous tissue consists of cells and an originally hyaline matrix, which, however, may become changed in such wise that there appears in it a network of elastic fibres or connective-tissue fibrils. Those cells that offer great resistance to the action of alkalies and acids have not been carefully studied. According to former views, the matrix was considered as consisting of a body analogous to collagen, so-called chondrigen, which under similar conditions- passes, like collagen, into a corresponding gelatin called chondritx- or cartilage-gelatin. The recent investigations of Moeoohowetz ^ and others, but especially those of C. Th. Moenee,* have shown that the matrix of the cartilage consists of a mixture of collagen with other bodies. • Zeitschr. f. physiol. Chem., Bd. 18, S. 250. ' Mucin in Myxoedema. Further Analyses. Kings College. Collected Papers No. 1, 1893. ' Verliandl. d. naturhist.-med. Vereins zu Heidelberg, Bd. 1, Heft 5. * Skand. Arch. f. Physiol., Bd. 1. 341 TISSUES OF THE CONNECTIVE SUBSTANCE. The tracheal, thyroideal, cricoidal, and arytenoidal cartilages of fall-grown cattle contain, according to Mokitee, four constituents in the matrix, namely, chondromucoid, chondroitin-sulphuric acid, collagen, and an albuminoid. Chondromucoid. This body, according to Moenee, has the composition C 47.30, H 6.43, N 12.58, S 3.43, 31.38^. Sulphur is in part loosely combined and may be split off by the action of alkalies, and a part separates as sulphuric acid -when boiled with hydrochloric acid. Chondromucoid is decomposed by dilute alkalies and yields alkali albuminate, peptone substances, chondroitin- sulphuric acid, alkali sulphides, and some alkali sulphates. On iboiling with apids it yields acid albuminate, peptone substances, PflUger's Arch., Bd. 24. » IMd., Bd. 35, S. 45. ' lUd., Bd. 43. 376 MUSCLE. nervous system seems also to have a similar inflaence on the post- mortem acidification of the muscles (Geoss "). Hermann and his pupils " consider the rigor mortis as a final slowly proceeding mascle- contraction identical with the ordinary contraction. Gotschlich ' has indeed made the statement that rest, activity, and rigor of the muscles are identical processes in principle. This cannof at present he positively proven from a chemical standpoint. When the muscle passes into rigor mortis it becomes shorter and thicker, harder and non-transparent, less ductile. The acid part of the amphoteric reaction becomes stronger, which is explained by most investigators by a formation of lactic acid. There is hardly any doubt that this increase in acidity may at least in part be due to a transformation of a part of the diphosphate into monophosphate by the lactic acid. The statements in regard to the occurrence also of free lactic acid or not in the rigor mortis muscle are contradic- tory.* The chemical processes which take place in rigor of the muscles, besides the formation of acid, are the following : By the coagulation of the plasma a myosin-clot is produced which is the cause of the hardening and of the diminished transparency of the muscle. The appearance of this clot may be hastened by the simultaneous occurrence of lactic acid. Carbon dioxide is also formed, which does not seem to be a direct oxidation product, but a product of the cleavage processes. Hermann ^ claims that carbon ■dioxide is produced in the removed muscle, even in the absence of oxygen, when it passes into rigor mortis. As many investigators admit of an increased formation of lactic acid on the appearance of rigor mortis, the question arises, from what constituents of the muscle is this acid derived ? The most probable explanation is that the lactic acid is produced from the glycogen,, as certain investigators, such as Nassb* and Weethee,' have observed a decrease in the quantity of glycogen in rigor of the 1 Centralbl. f. Physiol., Bd. 2, S. 91. '' See Bierfreand, 1. c. ' Pfltlger's Arch., Bd. 56. * It is impossible to enter into details of the disputed statements as to the reaction of the muscles, etc. We will only refer to the works of ROhmann, Pfluger'B Arch., Bdd. 50 and 55, and Hefter, Arch. f. exp. Path. u. Pharm., Bd. 31. ' Untersuchungen iiber den StofEwechsel der Muskeln, etc. Berlin, 1867. • Beitr. z. Physiol, der Kontraktil. Substanz, Pfltlger's Arch., Bd. 3. •" Pfliiger's Arch., Bd. 46. METABOLISM IN THE MUSCLE. 377 muscle. On the other side, Bohm' has observed cases in which no consumption of glycogen took place in rigor of the, muscle, and he has also found that the quantity of lacbic acid prodaced is not pro- portional to the quantity of glycogen. It is therefore possible that the consumption of glycogen and the formation of lactic acid in the muscles are two processes independent of each other, and, as above stated in regard to the formation of paralactic acid, the lactic acid of the muscle may be considered as a decomposition product of proteid. , The origin of the carbon dioxide is also not to be sought for in the decomposition of the glycogen or dextrose. Ppluser and Stintzing " have found that in the muscle a substance occurs which evolves large quantities of carbon dioxide on boiling with water, and it is probably this substance which is decomposed with the formation of carbon dioxide in tetanus as well as in rigor. TissOT " has observed a true respiration in removed muscle, which is independent of the putrefactive processes, and by which oxygen is absorbed and carbon dioxide eliminated, this being contrary to Hermann's assertion. The carbon dioxide eliminated originates from two sources. A part is preformed in the muscle and is only physically evolved carbon dioxide, and another part is formed in the removed muscle. After the muscles have been rigid for some time they relax again and the muscles become softer. This is in part produced by the strong acid dissolving the myosin clot and in part, and in all prob- ability mainly, upon the commencement of putrefaction. Metabolism in the Inactive and Active Muscles. It is admitted by a number of prominent investigators, Pflugbe and Colasanti,' ZuNTZ and Eohkig,' and others, that the exchange of material in the muscles is regulated by the nervous system. When at rest, when there is no mechanical exertion, we have a condition which ZuNTZ and Koheig have designated '■'■chemical tonus.'''' This tonus seems to be a reflex tonus, for it may be reduced by discon- tinuing the connection between the muscles and the central organ of the nervous system by cutting through the spinal cord or the ' Pflllger'3 Arch.. Bdd. 23 and 46. 'Ibid., Bd. 18. 2 Arch, de Physiol., Ser. 5, Tome 7. ■* See tlie works of Pflilger and his pupils in Pflilger's Arch., Bdd. 4, 12, 14, 16, 18. ^IHd., Bd. 4, 8. 57 ; also Zuntz., ibid., Bd. 12, S. 532. 378 MUSCLE. mnscle-nerves, or by paralyzing the same by means of cnrara poison. It may also be reduced or checked by adjusting the temperature between the skin and the surrounding medium; or it may be increased by the reverse, by irritating the nerves of the skin by cooling. The possibility of reducing the chemical tonus of the muscles by any of the aboye-mentioned means, but especially by the action of curara, offers an important means of deciding the extent and kind of chemical processes going on in the muscles when at rest. In comparative chemical investigation of the processes in the active and the inactive muscles several methods of procedure have been adopted. The removed homologous, active and inactive muscles have been compared, also the arterial and venous muscle- blood in rest and activity, and lastly the total exchange of material, the receipts and expenditures of the organism, have been investi- gated under these two conditions. By investigations according to these several methods it has been found that the active muscle takes up oxygen from the blood and returns to it carbon dioxide, and also that the quantity of oxygen taken up is greater than the oxygen contained in the carbon dioxide eliminated at the same time. The muscle, therefore, holds in some form of combination a part of the oxygen taken up while at rest. During activity the exchange of material in the muscle, and therewith the exchange of gas, is increased. The animal organism takes up considerably more oxygen in activity than when at rest, and eliminates also considerably more carbon dioxide. The quan- tity of oxygen which leaves the body as carbon dioxide during activity is considerably larger than the quantity of oxygen taken up at the same time ; and the venous muscle-blood is poorer in oxygen and richer in carbon dioxide during activity than during rest. The exchange of gases in the muscles during activity is the reverse of that at rest, for the active muscle gives up a quantity of carbon dioxide which does not correspond to the quantity of oxygen taken up, but is considerably greater. It follows from this that in muscular activity not only does oxidation take place, but also split- ting processes occur. This follows also from the fact that removed blood-free muscles when placed in an atmosphere devoid of oxygen can labor for some time and also yield carbon dioxide (Hermann '). During muscular inactivity, in the ordinary sense, a consump- tion of glycogen takes place. This is inferred from the observations >L. c. OONSUMPTION OF OL7C0GEN IN THE MUSOLE. 379 of several investigators that the quantity of glycogen is increased and its corresponding consumption reduced in those muscles whose chemical tonus is reduced either by cutting through the nerve or for other reasons (Bekbtaed,' Chastdblon'," Way," and others). In activity this consumption of glycogen is increased, and it has been positively proved by the researches of several investigators (Nassb,* Weiss," Kulz,' Maecose,' Manche,' Mokat and Dufoue') that the quantity of glycogen in the muscles in activity decreases quickly and freely. By investigating with the muscles in situ, especially on the levator labii superioris of a horse, Chauteat: and KAUEMANiir '° have not only confirmed the above facts in regard to the exchange of gas during rest and activity, but they also found that the muscles remove sugar from the blood, and indeed considerably more during activity than when at rest. They found (calculating the amount found in 1 gramme of muscle per minute to 1 kilo per hour) that 1 kilo of muscle removes 2.186 grms. sugar from the blood per hour during rest, while it removes 8.416 grms. per hour in activity. Strong objections to the conclusions drawn from these experi- ments have been made by SEEGSiii'"; although these experiments may not be quite conclusive, still it cannot be denied that an increased consumption of sugar takes place during activity. Other investigators, such as Quinqtjaud," Moeat and Dueoub, have observed a consumption of the sugar derived from the blood during work, and finally iu this connection we must recall that Sebgen " and still earlier CHAuvEAtr have come to a similar conclusion by special investigations. According to SBEGEisr the blood-sugar is on the whole the source of heat and work. SsEGBif " has determined ' Compt. rend., Tome 48, p. 673. ' Pflttger's Arch. , Bd. 13. > Arch. f. exp. Path. u. Pharm., Bd. 34. * Pflilger's Arch., Bd. 2. ' Wien. Sitzungsber., Bd. 64, Abth. 1. « See Kttlz in Ludwig's Festschrift. Marburg, 1891. ' PflUger's Arch., Bd. 39. « Zeitschr. f . Biologie, Bd. 25. 9 Arch, de Physiol. (5) Bd. 4. '» Oompt. rend., Tome 103, 104, and 105. " Centralbl. f. Physiol., Bd. 8, S. 417. " Maly's Jahresber., Bd. 16, S. 321. " Die Zuckerbildung im ThierkOrper (Berlin, 1890), and Pfltiger's Arch., Bd. 50. '^ Centralbl. f. Physiol., Bd. 8, and DuBois-Eeymond's Arch., 1895. 380 MUBGLB. the quantity of sugar in the arterial and venous blood of the muscle during rest and when directly or indirectly irritated, but obtained no constant results. He found, on the contrary, generally a very considerable consumption of the glycogen in the active muscles. Sbegen calculates that in his experiments, with the assumption that the glycogen was completely oxidized, the glycogen in great- est part served as heat-former and only to a small extent, in most cases 5-10^ of its store of energy, as mechanical work. The entire quantity of glycogen in the animal body is, according to Sebgbn, only suflBcient to supply a small fraction of the mechanical work of the body, and the most important source of mechanical work and of heat lies, according to him, in the blood-sugar. The amphoteric reaction of the inactive muscles is changed during activity to an acid reaction (DuBois-Ebtmond and others), and the acid reaction increases to a certain point with the work. The quickly contracting pale muscles produce, according to {tLBISS,' more acid during activity than the more slowly contract- ing red muscles. The acid reaction appearing during activity was formerly considered due to the formation of lactic acid, a view which has been contradicted by Astaschewskt,' Pplugek and Waeken,' who found less lactic acid in the tetanized muscle than when at rest. Moitaki * also found a decrease in the quantity of lactic acid during activity, and according to Hefter ' the quantity of lactic acid in the muscle is diminished in tetanus produced by poison. Contrary to these investigations Marcuse " and Werxhee ' have been able to prove the f oririation of lactic acid during activity ; still the statements are very contradictory. Other observations speak for a formation of lactic acid during activity. Thus Spiro " found an increase in the quantity of lactic acid in the blood during work. CoLASAifTi and Moscatelh' found small quantities of lactic acid in human urine after strenuous marches, and Weether observed abundance of lactic acid in the urine of ■PflUger's Arch., Bd. 41. ^ Zeitschr. f. physiol. Chem., Bd. 4. » Pfluger's Arch., Bd. 24. « Maly's Jaliresber. , Bd. 19, S. 303. » Arch. f. exp. Path. u. Pharm., Bd. 31. •L. c. ' Pfluger's Arch., Bd. 46. * Zeitschr. f. physiol. Chem., Bd. 1. 9 Maly's Jahresber., Bd. 17, S. 212. REACTION OF THB MUSCLE. 881 frogs after tetanization. According to Hoppe-Seylek,' on the contrary, in agreement with his view in regard to the formation of lactic acid, a formation of lactic acid does not take place regularly during work, but only when insufficient oxygen is supplied. Zillb- SEN " has also found that on artificially cutting off the oxygen from the muscles during life more lactic acid was formed than under normal conditions. It is evident that the experiments with the muscles in situ, in other words with muscles through which blood is passing, cannot yield any conclusion to the above question, as the lactic acid formed during work may perhaps be removed by the blood. The following objections can be made against those experiments in which lactic acid has been found after moderate work in the blood or the urine, as also especially against the experiments with re- moved active muscles, namely, that in these cases the supply of oxygen to the muscles was not sufficient, and that the lactic acid formed thereby is not, corresponding to the views of Hoppe-Seyleb, a perfectly normal process. The question as to the formation of lactic acid in the active muscle under perfectly physiological con- ditions is still an open one. According to Wetl and Zeitlee," the active muscle contains more phosphoric acid (in part formed by the decomposition of lecithin) than the inactive muscle. As in the dead muscle, so in the active muscle, the somewhat stronger acid reaction is in part due to a greater quantity of monophosphate. The amount of proteids in the removed muscles is, according to the older investigators, decreased by work. The correctness of this statement is, however, disputed by other investigators. Also the older statements in regard to the nitrogenous extractive bodies of the muscle in rest and in activity are uncertain. According to the recent researches of Monari,* the total quantity of creatin and creatinin is increased by work ; and indeed the amount of creatinin is especially augmented by an excess of muscular activity. The creatinin is formed essentially from tte creatin. In excessive activity Moitaei also found xantho-creatinin in the muscle, and the quantity was one tenth of that of the creatinin. The quantity ' L. c. and Zeitschr. f. physiol Chem., Bd. 19, S. 476. ' Zeit8chr. f. physiol. Chem., Bd. 15. » Zeitschr. f. physiol. Chem., Bd. 6, S. 557. ■> Maly's Jahresber., Bd. 19, S. 396. 382 MUaCLB. of xanthin bodies is, according to Monari, decreased under the influence of work. It seems to have been positively shown that the active muscle contains a smaller quantity of bodies soluble in water and a larger quantity of bodies soluble in alcohol than the resting muscle (Hblmholtz).' An attempt has been made to solve the question relative to the behavior of the nitrogenized constituents of the muscle at rest and during activity by determining the total quantity of nitrogen eliminated under these different conditions of the body. While formerly it was held with Liebig that the elimination of nitrogen by the urine was increased by muscular work, the researches of several experimenters,' especially those of Voit" on dogs and Pettenkofbe and Voit" on men, have led to quite different results. They have shown, as has also lately been confirmed by other investigators, especially Hikschfeld,* that during work no increase or only a very insignificant increase in the elimination of nitrogen takes place. We should not omit to mention the fact that a series of experiments has been made showing a significant increase in the metabolism of proteids during or after work. We have as example the observations of PLiisrT ' and Pavy ' on a pedestrian, v. Wolff, v. Funke, Keeuzhage and Kellkee ' on a horse, and lately those of Aegutii^skt ' and Keummachee " on , themselves, which show an undoubted increase in the elimination of nitrogen during or after work. The elimination of nitrogen is mainly dependent upon causes which will be spoken of later (Chapter XVIII), such as the quantity and composition of the food, the condition of the adipose tissue, the action of work on the respiratory mechanism, etc., etc., all of which can hardly have received sufficient consideration in the last- ' Arch. f. Anat. u. Physiol., 1845. ' Untersuchungen uber den Einfluss des Kochsalzes, des Eafiees und der Muskelbewegungen auf den StofEwechsel (Mttnohen, 1860), and Zeitschr. f. Biologie, Bd. 3. » Zeitschr. f . Biologie, Bd. 2. * Virchow's Arcli., Bd. 121. ' Journal of Anat. and Physiol., Vols. 11 and 12. « The Lancet, 1876 and 1877. ' Cited by Voit in Hermann's Handbuch, Bd. 6, S. 197. » Pflttger's Arch., Bd. 46. 9iMd.,Bd. 47. MLIMINATION OF NITROGEN. 383 mentioned experiments.' The strong proof which the very careful experiments of VoiT, of Pettenkofek and Voit, and of Hiesch- FELD furnish in support of this theory is hardly affected by these investigations, though we must admit that this question is still some- what unsettled. Even if we consider the question that muscular work does not cause any increase in the elimination of nitrogen as quite positively proved, still we do not exclude the possibility of an increased metabolism of proteids in the muscle. It is possible on account of the functional exchange action of the organs, of which Eanke ' has made a special study, that an increased metabolism of proteid in the muscles may be compensated by a simultaneous decreased metabolism of proteid in other organs. But however this may be, the modern view is, notwithstanding, that the metabolism of proteid in the muscle is not increased by activity. The quantity of metabolic products containing sulphur may also be a measure of the extent of the metabolism of proteids, and this quantity may be determined by estimating the suphur in the urine. An increase in the elimination of sulphur after work has been observed for a long time by En'GELMAN'N, ° and also by Flint and Pavt. As sulphuric acid and also non-oxidized sul- phur are eliminated by the urine, it is necessary to determine the total sulphur eliminated during work and after work. Beck and Benedikt ' have made investigations of this kind, and they find that the elimination of sulphur is increased by work and diminished after work, which speaks for an increased proteid metabolism during work. I. Munk ' by observations on resting and working persons has given further proof that the elimination of nitrogen and sulphur (also phosphoric acid and potash) runs parallel with the metabolism of proteid. The increased elimination of sulphur was not in the neutral sulphur, but nearly entirely in the oxidized sulphur. The investigations on the amount of fat in removed muscles during activity and at rest have not led to any definite results. The metabolic experiments of VoiT on a starving dog, and those of ' See Voit in Hermann's Handbucb, Bd. 6, Kap. 3, Absolin., 9; I. Munk, Du Bois-Reymond's Arch., 1890; and Hirschfeld, 1. c. ' Die Blutvertheiltmg und der Thatigkeitsweohsel der Organe. Leipzig, 1871. ' Du Bois-Heymond's Arch. , 1871. * PflUger's Arch., Bd. 54. ' Verhandl. d. physiol. Gesellsch. zu Berlin, 1894r-95. 384 MUSCLE. Pbttenkofer and Voit on a man, offer strong proofs to show that an increased decomposition of the fat takes place during activity. If the results of the investigations thus far made of the chemical processes going on in the active and inactive muscle were collected together, we would find the following characteristics for the active muscle. The active muscle takes up more oxygen and gives off more carhon dioxide than the inactive muscle ; still the elimination of carbon dioxide is increased considerably more than the absorption CO, of oxygen. The respiratory quotient, -7^' is found to be regularly raised during work ; still this rise, which will be explained in detail in a following chapter on metabolism, can hardily be conditioned on the kind of processes going on in the muscle during activity with a sufficient supply of oxygen. In work a consumption of carbohy- drates, glycogen, and sugar takes place. A consumption of sugar seems only to have been shown in muscle with blood circulation, while a consumption of glycogen also has been observed in removed muscle. The acid reaction of the muscle becomes greater with work. In regard to the extent of a re-formation of lactic acid opinion is divided. Eespecting the behavior of fats in removed muscles nothing is known with certainty, though an increase in the con- sumption of fat in the organism has been observed in certain cases during activity. An increase in the nitrogenous extractive bodies of the creatin group seems also to occur. In regard to the proteid bodies the views are contradictory ; but an increased elimination of nitrogen as a direct consequence of muscular exertion has thus far not been positively proved. In close connection with the above-mentioned facts we have the question as to the origin of muscular activity so far as it has its origin in chemical processes. In the past the generally accepted opinion was that of Libbig, that the source of muscular action con- sisted of a metabolism of the proteid bodies; to-day another gener- ally accepted view prevails. Fick and "Wislicenus ' climbed the Faulhorn and calculated the amount of mechanical force expended in the attempt. "With this they compared the mechanical equiva- lent transformed in the same time from the proteids, calculated from the nitrogen eliminated with the urine, and found that the work really performed was not by any means compensated by the ' Vierteljahrschr. d. Zurich, naturf. Gesellscli., Bd. 10. Cited from Cen- tralbl. f. d. med. Wiss., 1866, S. 309. SOURCE OF MUSCULAR FORCE. 385 consumption of proteid. It was therefore proved by this that pro- teids alone cannot be the source of muscular actiyity, and that this depends in great measure on the metabolism of non-nitrogenous substances. Many other observations have led to the same result, especially the experiments of VoiT, of Pettenkofer and Voit, and of other investigators, whose experiments show that while the elim- ination of nitrogen remains unchanged, the elimination of carbon dioxide during work is very considerably increased. It is also generally considered as positively proved that muscular work is produced, at least the greatest part, by the metabolism of non- nitrogenous substances. Nevertheless we are not warranted in the statement that muscular activity is produced entirely at the cost of the non-nitrogenous substances, and that the proteid bodies are without importance as a source of force. The recent investigations of Pfluger ' are of great interest in this connection. He fed a bulldog for more than 7 months with meat which alone did not contain sufficient fat and carbohydrates for the production of heart activity, and then let him work very- hard for periods of 14, 35, and 41 days. The positive results ob- tained by these series of experiments was that " complete muscular activity may be effected to the greatest extent in the absence of fat and carbohydrates," and the ability of proteids to serve as a source of muscular energy cannot be denied. Among the non-nitrogenous bodies we must accord to carbohy- drates, glycogen, and sugar the first place as sources of force. That the fats are also to be considered as a source for force is very probable, and the researches of Voit ' on starving and working dogs give support to this theory. The view, as accepted by several in- vestigators, that all three chief groups of organic food or muscle constituents may serve as source of force seems to be true. A few investigators are of the opinion, as formulated by Bunge," that the muscles first consume the supply of non-nitrogenous nutritive bodies, and that the proteids are only secondarily attacked. Pflugee is, on the contrary, of the opposite opinion. According to him no muscular work takes place without a decomposition of proteid, and the living cell substance prefers always the proteid and rejects the fat and sugar, contenting itself with these only when proteids are absent. ' Pflilger's Arch., Bd. 50. ' Ueber den Einfluss des Kochsalzes, etc., 1. c. ' Lehrbuch d. physiol. u. pathol. Chem., 1. Aufl., S. 345. 386 MU8CI/E. ZuNTz/ in collaboration with Fkbktzel and Loeb, has made 'experiments in dogs from which he concludes, that at least in these experiments (part in staryation and part with such an abundant food that a deposition of nitrogen took place even after hard work) the animals preferred the non-nitrogenous bodies which were offered as food to defray the work done. ZuKfTZ has also shown that the foods may supply work approximately in proportion as they con- sume oxygen and according to their heat of combustion. Quantitative Composition of the Muscle. A large number of analyses have been made of the flesh of various animals for purely practical purposes, in order to determine the nutritive value of different varieties of meat; but we have no exact scientific analyses with sufl&cient regard to the quantity of different albuminous bodies and the remaining muscle-constituents, or these analyses are in- complete or of little value. To give the reader some idea of the variable composition of muscle-substance we give the following summary, chiefly obtained from K. B. Hofman"n's ' book. The figures are parts per 1000. Muscles of Muscles of Muscles of Cold-blooded Mammalia. Birds. Animals. Solids 217-355 237-283 300 Water 745-783 717-773 800 •Organic bodies 208-345 217-263 180-190 Inorganic bodies 9-10 10-19 10-20 Myosin 35-106 29.8-111 29.7-87 Stroma substance (Danilewsky) 78-161 88.0-184 70.0-121 Alkali albuminate 29-30 — — Creatin 2 3.4 2.3 Xanthin bases 0.4-0.7 0.7-0.3 — Inosi nic acid (barium salt) 0.1 0.1-0.8 — Protic acid ... — — 7.0 Taurin 0.7 (horse) — 1.1 Innsit 0.03 — _ 'Glycogen 4-5 — 3-5 Lactic acid 0.4-0.7 — — Phosphoric acid 3.4-4 8 Potash 3.0-4.0 Soda 0.4 Lime 2 Magnesia.... 4 Sodium chloride 0.04-0.1 Iron oxide 0.03-0.1 In this table, which has little value because of the variation in the composition of the muscles, we have no results as to the ' Du Bois-Reymond's Arch,, 1894. « Lehrbuch d. Zoochem. Wien, 1876, S. 104. COMPOSITION OF TUB MU80LE. 387 estimates of fat. Owing to the variable quantity of fat in meat it is hardly possible to quote a positive average for this body. After most careful efforts to remove the fat from the muscles without chemical means, it has been found that a variable amount of inter- muscular fat, which does not really belong to the muscular tissue, always remains. The smallest quantity of fat in the muscles from lean oxen is, according to Geouvek, 6.1 p. m., and according to Peteesen 7.6 p. m. This last observer also found regularly a smaller amount of fat, 7.6-8.6 p. m., in the fore quarter of oxen, and a greater amount, 30.1-34.6 p. m., in the hind quarter of the animal. A low amount of fat has also been found in the muscles of wild animals. B. Konig and Faewick found 10.7 p. m. fat in the muscles of the extremities of the hare, and 14.3 p. m. in the muscles of the partridge. The muscles of pigs and fattened ani- mals are, wjien all the adherent fat is removed, very rich in fat, amounting to 40-90 p. m. The muscles of certain fishes also con- tain a large amount of fat. According to Almen, the flesh of the salmon, mackerel, and eel contains respectively 100, 164, and 339 p. m. fat.' The quantity of vtatee in the muscle is liable to considerable variation. The amount of fat has a special influence on the quan- tity of water, and we find, as a rule, that the flesh which is deficient in water is correspondingly rich in fat. The quantity of water does not depend alone upon the amount of fat, but upon many other circumstances, among which we must mention the age of the ani- mal. In young animals the organs in general, and therefore also the muscles, are poorer in solids and richer in water. In man the amount of water decreases until mature age, but increases again towards old age. Work and rest also influence the amount of water, for the active muscle contains more water than tlie inactive. The uninterruptedly active heart should therefore be the muscle richest in water. That the amount of water may vary independently of the amount of fat is strikingly shown by comparing the muscles of dif- ferent species of animals. In cold-blooded animals the muscles generally have a greater amount of water, in birds a lower. The comparison of the flesh of cattle and flsh shows very strikingly the different amounts of water (independent of the amount of fat) in ' In regard to the literature and complete statements on the composition of flesh of various animals, see KiSnig, Chemie der menschlichen Nahrungs- und Oenussmittel, 3. Aufl. 388 MUSCLE. the flesh of different animals. According to the analysis of Almeit,' the muscles of lean oxen contain 15 p. m. fat and 767 p. m. water; the flesh of the pike contains only 1.5 fat and 839 p. m. water. For certain purposes, as, for example, in experiments on meta- bolism, it is important to know the elementary composition of flesh. In regard to the quantity of nitrogen we generally accept Voit's ' figure, namely, 3.4^, as an average for fresh lean meat. According to Nowak ° and Huppeet * this quantity may vary about 0.6^, and in more exact investigations it is therefore neces- sary to specially determine the nitrogen. According to Salkow- SEi,' of the total nitrogen of beef 77.4j^ was insoluble proteids, 10.08^ soluble proteids, and l%.b%fo other soluble bodies. Complete elementary analyses of flesh have recently been made with great care by Akgutinsky.' The average for ox-flesh dried in vacuo and free from fat and with the glycogen deducted was as follows : 49.6; ' H 6.9; N 15.3; + S 23.0; and ash 5.2^. The relationship of the carbon to nitrogen, which Akgutinskt calls the " flesh quoti- ent," is on an average 3.24: 1. Non-striated Muscles. The smooth muscles have a neutral or alkaline reaction (Du- Bois-Rbtmond)' when at rest. During activity they are acid, which is inferred from the observations of Bbknsteik," who found that the nearly continually contracting sphincter muscle of the Anodonta is acid during life. The smooth muscles may also, ac- cording to HBiDBNHAi} Cited from KQhne's Lehrbucb, S. 333. ♦ IMd. CHAPTER XII. BRAIN AiSfD NERVES. On account of the difficulty of making a mechanical separation and isolation of the different tissue-elements of the nervous central organ and the nerves, we must resort to a few microchemical re- actions, chiefly to qualitative and quantitative investigations of th& different parts of the brain, in order to study the different chemical composition of the cells and the nerve-tubes. The chemical investi- gation of this part is accompanied with the greatest difficulty; and although our knowledge of the chemical composition of the brain and nerves has been somewhat extended by the investigations of modern times, still we must admit that this chapter is as yet one of the most obscure and complicated in physiological chemistry. Proteids of different kinds have been shown to be chemical con- stituents of the brain and nerves. A part of these are insoluble in water and dilute neutral-salt solutions, and part are soluble therein.. Among the latter we find albumin and globulin. Nucleoalbumin, which is often considered as an alkali albuminate, also occurs.. Hallibueton ' found two globulins in the brain, one of which coagulates at 47-50° 0. and the other at 70° C. He found in the gray matter a nucleoalbumin which coagulated at 55-60° C. and contained 0.5^ phosphorus. It seems unquestionable that the albuminous bodies belong chiefly to the gray substance of the brain and to the axis-cylinders. The same remarks apply to nuclein, which v. Jacksch " found in large quantities in the gray substance. Neurokeratin see page 49), which was first detected by KuHNB, and which partly forms the neuroglia, and which as a ' On the Chemical Physiology of the Animal Cell. King's College, London, Physiological Laboratory. Collected Papers, No. 1, 1893. » Pfluger's Arch., Bd. 13. 390 THE BRAIN. 391 double sheath envelops the outside of the nerve medulla under Schwann's sheath and the inner axis-cylinders, chiefly occurs in the white substance (Kuhne and Chittenden,' Baumstaek).' The phosphorized substance protagon must be considered as one of the chief constituents, perhaps the only constituent (Baum- staek), of the white substance. This last-mentioned substance, if we keep for the present to the most carefully studied protagon — because there are perhaps several different protagons — yields as decomposition products lecithin, fatty acids, and a nitrogenous substance, cerebrin ; this last probably does not occur preformed in the brain, but is more likely a product of transformation. That lecithin also is pre-existent in the brain and nerves can hardly be doubted. The investigations thus far made have not shown decidedly whether it is more abundant in the gray or the white substance. Fatty acids and neutral fats may be prepared from the brain and nerves; but as these may be readily derived from a decom- position of lecithin and protagon, which exist in the fatty tissue between the nerve-tubes, it is difl&cult to decide what part the fatty acids and neutral fats play as constituents of the real nerve-substance. Gholesterin is also found in the brain and nerves, a part free and a part in chemical combination of which we know nothing about (Baumstaek). Cholesterin seems to occur in greater abundance in the white substance. Besides these substances the nerve tissue, especially the white substance, contains doubtless a number of other constituents not well known, and among which are several containing phosphorus. Thudichum asserted that he had isolated a number of phosphorized substances from the brain which he divided into three principal groups : Tcepalines, myelines, and leciihines. But- thus far this assertion Has not been confirmed by other in- vestigators. By allowing water to act on the contents of the medulla, round or oblong double-contoured drops or fibres, not unlike double-con- toured nerves, are formed. This remarkable formation, which can also be seen in the medulla of the dead nerve, has been called " my eline forms," ani they were formerly considered as produced from a special body, "myeline." Myeline forms may, however, b& obtained from other bodies, such as impure protagon, lecithin, fat, and impure cholesterin, and they depend on a decomposition of the > Zeitschr. f. Biologie, Bd. 36. « Zeitschr. f. physiol. Chem., Bd. 9. 39-2 BRAIN AND NERVES. constituents of the medulla. According to Gad and Heymans ' myeline is lecithin in a free condition or in loose chemical com- bination. The extractive bodies seem to be almost the same as in the mus- cles. We find creatin, which may, however, be absent (Baum- staek), xanthin bases, inosit, lactic acid (also fermentation lactic acid), uric acid,jecorin (according to BALDi,°in the human brain), and neuridin, C^Hj^N,, discovered by Bbibgbb' and which is most interesting because of its appearance in the putrefaction of animal tissues or in cultures of the typhoid bacillus. Under patho- logical conditions leucin and urea have been found in the brain. Urea is also a physiological constituent of the brain of cartilaginous fishes. Of the above-mentioned constituents of the nerve-substance protagon and its decomposition products, the cerebrins or cerebro- sides, must be specially described. Protagoii. This body, which was discovered by Liebkbich, is a nitrogenized and phosphorized substance whose elementary com- position, according to Gamgbb and Blakkbnhobn^,* is 66.39, H 10.69, N 2.39, and P 1.068 per cent. Baumstabk ' and Ruppbl ' obtained the same figures, while Libbebich ' found an average of 2.80^ N" and 1.23^ P. Kossbl and Feettag,' who obtained still higher figures for the nitrogen, namely, 3.25$^, and somewhat lower figures for the phosphorus, 0. 97^, found some sulphur, an average of 0.51^, regularly in the protagon. Euppel also found some sul- phur, but in such small quantity that he considered it as a contami- nation. On boiling with baryta- water protagon yields the decompo- sition products of lecithin, namely, fatty acids, glycerophosphoric dcid, and cholin (neurin ?), and besides this also cerebrin. Kossel and Peettag found that protagon not only yielded cerebrin in its decomposition, but two and perhaps indeed three cerebrosides (see below), namely cbebbbin, kebasiu (homocerebrin), and eitcepha- Liif. Because of this behavior, and also because of the varying ' Da Bois-Eeymond's Arch., 1890. ' Ibid., 1887, Supplbd. ' Brieger, Ueber Ptomaine. Berlin, 1885 and 1886. * Zeitschr. f. physiol. Chem., Bd. 3. 'Ibid.. Bd. 9. « Zeitschr. f . Biologie, Bd. 31. ' Annal. d. Chem. u. Pharra., Bd. 184. 8 Zeitschr. f. physiol. Chem., Bd. 17. PBOTAGON. 393 elementary composition although the greatest care was taken in the preparation, Fkbttag considers it very probable that there are more than one protagon. On boiling with dilute mineral acids, protagon yields among other substances a reducing carbohydrate. On oxidation with nitric acid protagon yields higher fatty acids. Protagon appears, when dry, as a loose white powder. It dis- solves in alcohol* of 85 vols, per cent at -\- 45° C, but separates on cooling as a snow-white, flaky precipitate, consisting of balls or groups of fine crystalline needles. It decomposes on heating even below 100° C. It is hardly soluble in cold alcohol or ether, but dissolves on warming. It swells in little water, decomposes partly. With more water it swells to a gelatinous or pasty mass, which with much water yields an opalescent liquid. On fusing with saltpetre and soda, alkali phosphates are obtained. Protagon is prepared in the following way : An ox-brain as fresh as possible, with the blood and membranes carefully removed, is ground fine and then extracted for several hours with alcohol of 85 vols, per cent at + 45° C, filtered at the same temperature, and the residue extracted with warm alcohol until the filtrate does not yield a precipitate at 0° C. The several alcoholic extracts are cooled to 0° C. and the precipitates united and completely extracted with cold ether, which dissolves the cholesterin and lecithin-like bodies. The residue is now strongly pressed between filter-paper and allowed to dry over sulphuric acid or phosphoric anhydride. It is now pulverized, digested with alcohol at + 45° C, filtered and slowly cooled to 0° C. The crystals which separate may be purified when necessary by recrystallization. The same steps are taken when we wish to detect the presence of protagon. On decomposing protagon or the protagons by the gentle action of alkalies we obtain as cleavage products, as above stated, one or more bodies, which Thudichum' has embraced under the name cerebrosides. The cerebrosides are nitrogenous substances free from phosphorus, which yield a reducing variety of sugar (galactose) on boiling with dilute mineral acids. On fusing with potash or by oxidation with nitric acid they yield higher fatty acids, palmitic or stearic acids. The cerebrosides isolated from the brain are cerebri n, kerasin, and encephalin. The bodies isolated by Kossel and Fret- tag from pus, pyosin and pyogenin, also belong to the cerebrosides. ' Thudichum, Grundziige der anatomischen und kliaischen Chemie, Berlin, 1886. 394 BRAIN AND NERVSS. Cerebrin. Under this name W. Mullee ' first described a nitrog- enous substance, free from phosphorus, which he obtained by ex- tracting a brain-mass, which had been previously boiled with baryta- water, with boiling alcohol. Following a method essentially the same, but differing somewhat, Geoghegan' prepared from the brain a cerebrin with the same properties as MtLiEB's, but con- taining less nitrogen. According to Pakcus ' the cerebrin isolated by Geoghegan as well as by MtJliee consists of a mixture of three bodies, "cerebrin," " homocerebrin, " and " encephalin." KossEL and Fkeytag' isolated two cerebrosides from protagon which were identical with the cerebrin and homocerebrin of Pae- cus. According to these investigators the two bodies phrenosin and kerasin as described by Thudichum seem to be identical with cerebrin and homocerebrin. Cerebrin, according to Parous, has the following composition : C 69.08, H 11.47, N 2.13, 17.23^ which corresponds with the analyses made by Kossel and Feeytag. No formula has been given to this body. In the dry state it forms a pure white, odorless, and tasteless powder. On heating it melts, decomposes gradually, smells like burnt fat, and burns with a luminous flame. It is insoluble in water, dilute alkalies, or baryta-water. It is also insoluble in cold alcohol and in cold or hot ether. On the contrary, it is soluble in boiling alcohol and separates as a flaky precipitate on cooling, and this is found to consist of a mass of balls or grains on micro- scopical examination. Cerebrin forms an insoluble compound with baryta which decomposes by the action of carbon dioxide. Cere- brin dissolves in concentrated sulphuric acid, and on warming the solution it becomes blood-red. The variety of sugar split off on boiling with mineral acids — the so-called brain-sugar — is, according to Thieefeldee," galactose. Kerasin (according to Thudichum) or homocerebrin (accord- ing to Paecus) has the following composition: C 70.06, H 11.60, N 2.23, and 16.11^. Encephalin has the composition C 68.40, H 11.60, N 3.09, and 16.91^. Both bodies remain in the mother liquor after the impure cerebrin has precipitated from the ' Annal. d. Chem. u. Pharm., Bd. 105. ' Zeitsclir. f. physiol. Chem., Bd. 3. 3 Ueber einige neue Gehirnstoffe, Inaug.-Diss., Leipzig, 1881. «L. c. ' Zeitschr. f. physiol. Chem., Bd. 14. CBRBBBINS AND NEURIDIN. 395 warm alcohol. These bodies have the tendency of separating as gelatinous masses. Kerasin is homologous to cerebrin, but dissolves more easily in warm alcohol and also in warm ether. It may be obtained as extremely fine needles. Encephalin is, according to Paecus, a transformation product of cerebrin. In perfectly pure state it crystallizes in small lamellae. It swells into a pasty mass in warm water. Like cerebrin and kerasin, it yields a reducing sub- stance (probably galactose) on boiling with dilute acid. The cerebrins are generally prepared according to Mullee's method. The brain is first stirred with baryta-water until it ap- pears like thin milk and then it is boiled. The insoluble parts are removed, pressed, and repeatedly boiled with alcohol, which is fil- tered while boiling hot. The impure cerebrin which separates on cooling is freed from cholesterin and fat by means of ether, and then purified by repeated solution in warm alcohol. According to Paeoits this repeated solution in alcohol is continued until no gelatinous separation of homocerebrin or encephalin takes place. According to Geoghegan's method the brain is first extracted with cold alcohol and ether and then boiled with alcohol. The pre- cipitate which separates on the cooling of the alcoholic filtrate is treated with ether and then boiled with baryta-water. The insolu- ble residue is purified by repeated solution in boiling alcohol. The cerebrin may also be obtained from other organs by employ- ing the above methods. The quantitative estimation, when such is desired, may be performed in the same way. KossEL and Feettag prepare cerebrin from protagon by sa- ponifying it in a solution in methyl alcohol with a hot solution of caustic baryta in methyl alcohol. The precipitate is filtered off and decomposed in water by carbon dioxide, and the cerebrin or cerebroside extracted from the insoluble residue by hot alcohol. Neuridin, CsHnNj, is a non-poisonous diamin discovered by Brieger, and which was obtained by him in the putrefaction of meat and gelatine, and from cultures of the typhoid bacillus. It also occurs under physiological conditions in the brain, and as traces in the yolk of the egg. Neuridin dissolves in water, and yields on boiling with alkalies a mixture of dimethylamin and trimethylamin. It dissolves with difficulty in amyl-alco- hol. It is insoluble in ether or absolute alcohol. In the free state neuridin has a peculiar odor, suggesting semen. With hydrochloric acid it gives a combination crystallizing in long needles. With platinic chloride or gold chloride it gives crystallizable double combinations which are valuable in its preparation and detection. The so-called corpusctila amtlacba, which occur on the upper surface of the brain and in the pituitary gland, are colored more or less pure violet by iodine and more blue by sulphuric acid and iodine. They consist, perhaps, of the same substance as certain prostatic calculi, but they have not been closely investigated. 396 BRAIN AND NEBVE8. Quantitative Composition of the Brain. The quantity of water is greater in the gray than in the white substance, and greater in new-born or young individuals than in grown ones. The brain of the foetus contains 879-926 p. m. water. According to the obser- Tations of Weisbach ' the amount of water in the several parts of the brain (and in the medulla) varies at different ages. The fol- lowing figures are in 1000 parts — A for men and B for women : 20-30 Years. 30-50 Years. 50-70 Years. 70 -94 Years. A. 7. A. F. A. 5? A. W. the brain 695.6 683.9 683.1 703.1 701.9 689.6 726.1 722 Gray ditto 833.6 826.3 836.1 830.6 838.0 838.4 847.8 839.5 «yri 784.7 792.0 795 9 772.9 796. 1 796.9 802.3 801.7 Cerebellum 788.3 794.9 778.7 789.0 787.9 784.5 803.4 797.9 Pons varoli 784.6 740.3 725.5 722.0 730.1 714.0 727.4 '724.4 Medulla oblongata. 744.3 740.7 733.5 739.8 733.4 730.6 736.3 733.7 Quantitative analyses of the brain have also been made by Peteowsky " on an ox-brain and by Baumstaek ' on the brain of a borse. In the analysis of Petkowsky the protagon has not been considered, and all organic, phosphorized substances were calcu- lated as lecithin. On these grounds these analyses are not of much value from a certain standpoint. In Baumstaek's analyses the ^ray and the white substance could not be sufficiently separated, and these analyses, on this account, show partly an excess of white and partly an excess of gray substance; nearly one half of the ■organic bodies, chiefly consisting of bodies soluble in ether, could not be exactly analyzed. Neither of these analyses gives sufficient explanation of the quantitative composition of the brain. The analyses made up to the present time give, as above stated, an unequal division of the organic constituents in the gray and white substance. In the analyses of Peteowsky the quantity of proteids and gelatin-forming substances in the gray matter was somewhat more than one half, and in the white about one quarter, of the solid organic substances. The quantity of cholesterin in the white was about one half, and in the gray substance about one fifth, of the solid bodies. A greater quantity of soluble salts and extractive bodies was found in the gray substance than in the white (Baumstaek). The following analyses of Baumstaek give the most important known constituents of the brain calculated in 1000 1 Cited from K. B. Hofmann's Lehrb. d. Zoocliemie (Wien, 1876), S. 121. > Pfluger's Arch,, Bd. 7. ' Zeitschr. f. physiol. Chem., Bd. 9. COMPOSITION OF THE BBAIN. 397 parts of the fresh, moist brain. A represents chiefly the white, and B chiefly the gray, substance. A. B. Water 695.35 769.97 Solids 304.65 230.0S Protagon 25.11 10.80 Insoluble proteld and connective tissue 50. 03 60. 7& Cholesterin, free 18.19 6.30 combined 26.96 17.51 Nuclein 2.94 1.9» Neurokeratin 18.93 10.45 Mineral bodies 5.33 5.63 The remainder of the solids probably consists chiefly of lecithin and other phosphorized bodies. Of the total amount of phosphorus 15-20 p. m. belongs to the nuclein, 50-60 p. m. to the protagon, 150-160 p. m. to the ash, and 770 p. m. to the lecithin and the other phosphorized organic substances. The quantity of neurokeratin in the nerves and in the different parts of the brain has been carefully determined by Kuhnb and Chittenden." They found 3.16 p. m. in the plexus brachialis, 3.12 p. m. in the edge of the cerebellum, 22.434 p. m. in the white substance of the cerebrum, 25.72-29.02 p. m. in the white sub- stance of the corpus callosum, and 3.27 p. m. in the gray substance of the edge of the cerebrum (when free as possible from white sub- stance). The white is very considerably richer in neurokeratin than the peripheric nerves or the gray substance. According to Geifeiths' neurochitin replaces neurokeratin in insects and Crustacea, the quantity of the first being 10.6-12 p. m. The quantity of mineral constituents in the brain amounts to 2.95-7.08 p. m. according to Geoghbgan.' He found in 1000 parts of the fresh, moist brain 0.43-1.32 01, 0.956-2.016 P0„ 0.244-0.796 CO,, 0.102-0.220 SO., 0.01-0.098 Fe,(PO,)„ 0.005- 0.022 Ca, 0.016-0.072 Mg, 0.58-1.778 K, 0.450-1.114 Na. The gray substance yields an alkaline ash, the white an acid ash. Appendix. The Tissue and Fluids of the Eye. The retina contains in all 865-899.9 p. m. water, 57.1-84.5 p. m. proteid bodies — myosin, albumin, and mucin (?), 9.5-28.9 p. m. lecithin, and 8.2-11.2 p. m. salts (Hoppe-Sbylee and " Zeitsclir. f. Biologie, Bd. 26. ' Compt. rend., Tome 115. ' Zeitsclir. f. physiol. Chem., Bd. 3. 398 BBAIN AND NERVES. €ahn '). The mineral bodies consist of 423 p. m. Na,HPO, and 353 p. m. ISTaCl. Those bodies which form the different segments of the rods and cones have not been closely studied, and the greatest interest is therefore connected with the coloring matters of the retina. Visual purple, also called rhodopsin, erythropsin, or visual BED, is the pigment of the rods. Boll " observed in 1876 that the layer of rods in the retina during life had a purplish-red color which was bleached by the action of light. Kuhnb ' showed later that this red color might remain for a long time after the death of t)ie animal if the eye was protected from daylight or investigated by a sodium light.. Under these conditions it was also possible to isolate and closely study this substance. Visual red (Boll) or visual purple (Kuhkb) has become known mainly by the investigations of KiJHKE. The pigment occurs chiefly in the rods and only in their outer parts. In animals whose retina has no rods the visual purple is absent, and is also neces- sarily absent in the macula lutea. In a variety of bat {rhinolopJius . hipposideros), in hens, pigeons, and new-born rabbits, no visual purple has been found in the rods. A solution of visual purple in water which contains 2-5j^ crystallized bile, which is the best solvent for it, is purple-red in color, quite clear, and not fluorescent. On evaporating this solu- tion in vacuo we obtain a residue similar to ammonium carminate which contains violet or black grains. If the above solution is dialyzed with water, the bile diffuses and the visual purple separates as a violet mass. Under all circumstances, even when still in the retina, the visual purple is quickly bleached by direct sunlight, and with diffused light with a rapidity corresponding to the in- tensity of the light. It passes from red and orange to yeHow. Eed light bleaches the visual purple slowly ; the ultra-red light does not bleach it at all. A solution of visual purple shows no special absorption-bands, but only a general absorption which extends from the red side, beginning at D, to the line G. The strongest absorption is found at E. Visual purple when heated to"52°-53° C. is destroyed after several ' Zeitschr. f. Physiol. Chem., Bd. 5. 2 Monatssohr. d. Berl. Akad., 13 Nov. 1876. 2 The investigations of Kllhne and his pupils Ewald and Ayres on the vis- ual purple will be found in Untersuchungen aus dem physiol. Institut der Universitat Heidelberg, Bdd. 1 und 3. VISUAL PURPLE. 399 hours, and almost instantly when heated to + 76° C. It is also destroyed by alkalies, acids, alcohol, ether, and chloroform. On the contrary, it. resists the action of ammonia or alum solution. As the visual purple is easily destroyed by light, it must there- fore also be regenerated during life. Kuhne has also found that the retina of the eye of the frog becomes bleached when exposed for a long time to strong sunlight, and that its color gradually returns when the animal is placed in the dark. This regeneration of the visual purple is a function of the living cells in the layer of the pigment-epithelium of the retina. This may be inferred from the fact that a detached piece of the retina which has been bleached by light may have its visual purple restored if the detached piece of the retina be carefully laid on the chorioidea having layers of the pigment-epithelium attached. The regeneration has, it seems, nothing to do with the dark pigment, the melanin or fuscin, in the epithelium-cells. A partial regeneration seems, according to KtJHNE, to be possible ' in the completely removed retina. On ac- count of this property of the visual purple of being bleached by light during life we may, as KuHifE has shown, under special con- ditions and by observing special precautions, obtain after death by the action of intense light or more continuous light the picture of bright objects, such as windows and the like — so-called optograms. The physiological importance of visual purple is unknown. It follows that the visual purple is not essential to sight, since it is absent in certain animals and also in the cones. Visual purple must always be prepared exclusively in a sodium light. It is extracted from the net membrane by means of a watery solution of crystallized bile. The filtered solution is evaporated in vacuo or dialyzed until the visual purple is separated. To prepare a visual-purple solution, perfectly free from haemo- globin, KtJHKE' suggests to precipitate it from it solution in bile by Mg SO, in substance, or to treat the retina, which has been previously hardened by alum and then lixiviated with water and 10$^ NaCl solution, with bile. The pigments of the cones. In the inner segments of the cones of birds, reptiles, and fishes a small f at-globnle of varying color is found. Kuhnb ' Las isolated from this fat a green, a yellow, and a red pigment called respectively cldorophan, xanthophan, and rJwdophan. ' Zeitschr. f. Biologie, Bd. 33. ' Kahne, Die nichtbestandigen Farben der Netzhaut. Untersuch. aus dem physiol. Institut Heidelberg, Bd. 1, S. 341. 400 BRAIN AND NERVES. The dark pigment of the epithelium-cells of the net membrane, which was formerly called melanin, but since named fuacin by Kuhnb and Mat, ' dis- solves in concentrated caustic alkalies or concentrated sulphuric acid on warm- ing, but, like melanins in general (see Chapter XVI), has been little studied. The pigment occurring in the pigment-cells of the chorioldea seems to be iden- tical with the f uscin of the retina. The vitreous humor is often considered as a variety of gelatinous tissue. The membrane consists, according to C. Moeitee/ of a gelatine-forming substance. The fluid contains a little proteid and a mucoid, hyalomucoid, which was first shown by Moknee, and which is not precipitated by acetic acid. This contains 12.27^ N and 1.19^ S. Among the extractives we find a little urea accord- ing to Picaed" 5 p.m., according to EinLMAifisr * 0.64 p. m. Pautz ' found besides some urea also paralactic acid, and, in con- firmation of the statements of Chabbas, Jesnee, and Kuhn, also glucose in the vitreous humor of oxen. The reaction of the vitreous humor is alkaline, and the quantity of solids amounts to about lip. m. The quantity of mineral bodies is about 9 p. m., and the albuminous bodies 0.7 p. m. In regard to the aqueous humor see page 195. The crystalline lens. That substance which forms the capsule of the lens ; has been recently investiga,ted by C. Moenbe. It belongs, according to him, to a special group of protein, which are called tnembranins. The membranin bodies are insoluble at the ordinary temperature in water, salt solutions, dilute acids and alkalies, and, like the mucins, yield a reducing substance on boiling with dilute mineral acids. They contain sulphur, which blackens lead. The membranins are colored a very beautiful red by MiL- LOJf's reagent, but give no characteristic reaction with concen- trated hydrochloric acid or Adamkiewicz's reagent. They are dissolved with great difficulty by pepsin-hydrochloric acid or trypsin solution. They are dissolved by dilute acids and alkalies in the warmth. Membranin of the capsule of the lens contains 14.10^ N and 0.83^ S, and is a little less soluble than that from Des- CEMETS membrane. The chief mass of the solids of the crystalline lens consists of • Kiihne, ibid., Bd. 3, S. 324. "Zeitschr. f. physiol. Chem., Bd. 18. ' Gamgee's Physiol. Chem., p. 454. * Maly's Jahresber., Bd. 6, S. 3:9. 'Zeitschr. f. Biologie, Bd. 31. PEOTEIDS OF THE OBTBTALLINE LENS. 401 proteids, whose nature has been investigated by C. MonuEii. ' Some of these proteids are insoluble in dilute salt solution, and others soluble therein. Tlie Insoluble Proteid. The lens-fibres consist of a proteid sub- stance which is insoluble in water and salt solution to which MoKNBR has given the name albumoid. It dissolves readily in very dilute acids or alkalies. Its solution in caustic potash of 0. 1^ is very similar to an alkali-albuminate solution, but coagulates at about 50° C. on nearly complete neutralization and addition of 8^ NaCl. Albumoid has the following composition: C 53.12, H 6.8, N 16.62, and S 0.79$^. The lens-fibres themselves contain 16.61^ N and 0.77^ S. The inner parts of the lens are considerably richer in albumoid than the outer. The quantity of albumoid in the entire lens amounts on an average to about 48^ of the total weight of proteids of the lens. Tlie Soluble Proteid consists, exclusive of a very small quantity of ALBUMIN, of two globulins, a- and /S-OKXSTALLiiir. These two. globulins differ from each other in this manner: fc-crystallin con- tainsl6.68$^ N and 0.56^ S; /?-crystallin, on the contrary, 17.04$^ N and 1.27j^ S. The first coagulates at about 72° C. and the other at- 63° C. Besides this, /S-crystallin is precipitated from salt-free solution with greater diflSculty by acetic acid or carbon dioxide. These globulins are not precipitated by an excess of NaCl at either the ordinary temperature or 30° C. Magnesium or sodium sul- phate in substance precipitates both globulins, on the contrary, at 30° C. These two globulins are not equally divided in the mass of the lens. The quantity of a-crystallin diminishes in the lens from without inwards, /?-crystallin, on the contrary, from within out- wards. A Bbchamp ' distinguisbes the two following albuminous bodies in the watery extract of the crystalline lens : fhoAxaymaae, which coagulates at -f 55° C, and contains a diastatic enzyme, and has a specific rotatory power of (a)j = — 41°, and the cryatalbumin. with a specific rotatory power of (rt)j = _ 80°. 3. From the residue of the lens, which was insoluble in water, Bbchamp extracted, by means of hydrochloric acid, an albuminous body having a specific rotatory power of (a)j = — 80°. 3 which he called crysialjibrin. The lens does not seem to contain any proteid bodies which coagulate spontaneously like fibrinogen. That cloudiness which appears after death depends, according to Kunhe, ' upon the un- ' Zeitschr. f. physiol. Chem., Bd. 18. This contains also the pertinent lit- erature. 'Compt. rend., Bd. 90. ' Lehrbuch d. physiol. Chem., S. 405. 402 BBAIN AND NERVES. equal changing of the concentration of the contents of the lens- tubes. This change is produced by the altered ratio of diffusion. A cloudiness of the lens may also be produced in life by a rapid removal of water, as, for example, when a frog is plunged into a salt or sugar solution (Kukde '). The appearance of cloudiness in diabetes has been attributed by some to the removal of water. The views on this subject are, however, contradictory. The average results of four analyses made by Laptsohinsky " of the lens of oxen are here given, calculated in parts per lOOQ : Proteids 349.3 Lecithin 3.3 Cholesterin 3.3 Fat 3.9 Soluble salts 5.3 Insoluble salts 3.3 In cataract the amount of proteid is diminished and the amount of cholesterin increased. The quantity of the different proteids in the fresh moist lens of oxen is as follows, according to Morkek': Albumoid (lens-fibres) 170 p. m. , /J-crystallin 110 " ar-crystallin 68 *' Albumin 3 " The corneal tissue has been previously treated of (page 348).. The sclerotic has not been closely investigated, and the choroid coat is chiefly of interest because of the coloring matter, melanin, it con- tains (see Chap. XVI). Tears consist of a water-clear, alkaline fluid of a saltish taste. According to the analyses of Lekch * they contain 983 p. m. water, 18 p. m. solids, with 5 p. m. albumin and 13 p. m. N"aCl. The Fluids of the Inner Ear. The perilymph and endolymph are alkaline fluids which, besides salts, contain — in the same amounts as in transudations — ^traces of albumin, and in certain animals (codfish) also mucin. The quantity ot mucin is greater in the perilymph than in the endolymph. Otoliths contain 745-795 p. m. inorganic substance, which con- sists chiefly of crystallized calcium carbonate. The organic sub- stance is very like mucin. ' Cited from Ktlline, 1. c. « Pflilger's Arch., Bd. 13. »L. c. ■• Cited from Gorup-Besanez, Lehrb. d. physiol Chem., 4. Aufl., S. 401. CHAPTEE XIII. ORGANS OP GENERATION. (a) Male Generative Secretions. The testis have been little investigated chemically. We find in the testis of animals proteid bodies of different kinds, seralbu- min, alkali albuminate (?), and an albuminous body related to EoviDAs' hyaline substance, also leucin, tyrosin, creatin, xanthin bases, cholesterin, lecithin, inosit, and/ai. In regard to the occur- rence of glycogen the statements are somewhat contradictory. Daeeste' found in the testis of birds starch-like granules, which were colored blue with difficulty by iodine. The semen as ejected is a white or whitish-yellow, viscous, sticky fluid of a milky appearance, with whitish, non -transparent lumps. The milky appearance is due to spermatozoa. Semen is heavier than water, contains albumin, has a neutral or faintly alkaline reaction and a peculiar specific odor. Soon after ejection semen becomes gelatinous, as if it were coagulated, but afterwards becomes more fiuid. When diluted with water white flakes or shreds separate (Hbnle's fibrin). According to the analyses of Vauquelin' human semen contains 900 p. m. water and 100 p. m. solids, with 60 p. m. organic and 40 p. m. inorganic sub- stance, of which 30 p. m. is calcium phosphate. Among the albuminous bodies Posner' claims th&t propeptone occurs even in the absence of the spermatozoa. The semen in the vas deferens differs chiefly from the ejected semen in that it is without the peculiar odor. This last depends on the admixture with the secretion of the prostate. This secretion, ' Compt. rend. , Tome 74. ' Cited from Lehmann's Lehrb. d. physiol. Chem. (Leipzig, 1853), Bd. 2, 8. 303 ' Berlin, klin. Wochenschr. , 1888, No. 21, and Centralbl. f. d. med. Wis- sensch., 1890, S. 497. 403 404 ORQANB OF GENERATION. according to Iveksbn'," has a milky appearance and ordinarily an alkaline reaction, very rarely a neutral one, contains small amounts of proteids and mineral bodies, especially NaCl. Besides these it contains a crystalline combination of phosphoric acid with a base, CjHjN. This combination has been called Bottcher's spermine crystals, and it is claimed that the specific odor of the semen is due to a partial decomposition of these crystals. The crystals which appear on slowly evaporating the semen, and which are also observed in anatomical preparations kept in alcohol and in desiccated egg-albumin, are identical, according to SoHEEiKBE, with Chaecot's Crystals found in the blood, and in the lymphatic glands in leucsemia. They are, according to SoHEEiNEE,' a combination of phosphoric acid with a base, spe'F- min, CjHjN, which he discovered. Spermin. The views in regard to the nature of this base are not unanimoils. According to the investigations of Ladenbttrg and Abel ' it is not improba- ble that spermin is identical with ethylenimin, but this identity is disputed by Majeut and A. Schmidt,^ andalso by Poehl.' The compound of spermin with phosphoric acid — Bbttcher's spermine crystals — is insoluble in alcohol, ether, and chloroform, soluble with difficulty in cold water but more readily in hot water, and easily soluble in dilute acids o» alkalies, also alkali carbonates and ammonia. The base is precipitated by tannic acid, mercuric chloride, gold chloride, platinic chloride, potassium-bismuthic iodide, and phospho- tungstic acid. Spermin has a tonic action, and according to Poehl ' it has a marked action on the oxidation processes of the animal body. The spermatozoa show a great resistance to chemical reagents in general. They do not dissolve completely in concentrated sul- phuric acid, nitric acid, acetic acid, nor in boiling-hot soda solu- tions. They are soluble in a boiling-hot causticrpotash solution. They resist putrefaction, and after drying they may be obtained again in their original form by moistening them with a 1% com- riion-salt solution. By careful heating and burning to an ash the shape of the spermatozoa may be seen in the ash. The quantity of ash is about 50 p. m. and consists mainly (f ) of potassium phos- phate. The spermatozoa show well-known movements, but the cause of this is not known. This movement may continue for a very long time, as under some conditions it may be observed for several days » Nord. med. Ark., Bd. 6; also Maly's Jahresber., Bd. 4, S. 358. • Annal. d. Chem. u. Pharm., Bd. 194. • Ber. d. deutsch. chem. Gesellsch., Bd. 21. « ma., Bd. 24. 6 Compt. rend., Tome 115. • Berlin, klin. Wochenschr., 1893, No. 36. SPERMATOZOA. 405 in the body after death, and in the secretion of the uterus longer than a week. Acid liquids stop these movements immediately; they are also destroyed by strong alkalies, especially ammoniacal liquids, also by distilled water, alcohol, ether, etc. The movements con- tinue for a longer time in faintly alkaline liquids, especially in alkaline animal secretions, and also in properly diluted neufral salt- -solutions. According to the investigations of Mieschek,' there are lecithin and nuclein, but no cerebrin, in the speematozoa of bulls. The head of the spermatozoa contains nuclein, which forms probably the outer part of the head; albumin, which forms the contents of the head; and lastly a substance rich in sulphur which has not T)een studied. The tail dissolves in gastric juice after continuous digestion, and seems to consist of proteids or allied bodies which show a variable resistance towards pepsin-hydrochloric acid. The SPEEMATOZOA of the Ehiite salmon show, according to MiESCHEB, a great resistance. With caustic-potash and soda solu- tions they give a cloudy, gelatinous mass which is precipitated as shreds by acids; but these shreds do not dissolve in an excess of the acid. They are strongly attacked by a 10-15^ solution of NaCl or NaNO, , and the semen is converted by such a solution into a stiff .gelatin. The head is attacked, but not the tail or the middle part. This last-mentioned part, like the tail, contains albumin, which is dissolved by hydrochloric acid of 1 p. m., but not in NaCl. MiESCHEE also found lecithin, fat, cholesterin, guanin, and sarhm in relatively large amounts in the salmon-semen. The organic constituent occurring in the largest amount in the salmon-semen is, according to Meischee, a combination of nuclein with the base protamin, which is soluble in water but insoluble in alcohol or ether. According to Kossel, the nuclein of the spermatozoa is nucleic acid (see Chapter V), and a combination of nucleic acid and protamin is supposed to exist therein. Protamin, This base is, like its salts, hardly possible to obtain in perfectly characteristic crystals. The platinum double salt has the following composi- tion, according to Piccard'' : PtCl, -\- 2(HCl.C8HiaN,50j). The compounds with hydrochloric or nitric acid dissolve readily in water and with diflSculty in alcohol. They are insoluble in ether. The base is precipitated by silver nitrate, potassium-mercuric iodide, potassium ferricyanide, and phospho- molybdic acid. ' Verb. d. naturf. Qesellsch. in Basel, Bd. 6; also Maly's Jahresber., Bd. 4, S. 337. » Maly's Jahresber., Bd. 4, S. 355. 406 ORGAJ!^S OF OENERATION. According to Miesohee, the spermatozoa of salmon contain 487 p. m. nuclein, 268 p. m. protamin, 103 p. m. proteid bodies, 75 p. m. lecithin, 23 p. m. cholesterin, and 45 p. m. fat. PiC0ABl> found 60-80 p. m. guanin and sarkin in ripe semen. Kossbl and SCHINDLER ' found no guanin, but xanthin and large amounts of adenin and hypoxanthin, in the semen of the carp. Inoko," who investigated the semen of bulls, boars, and salmon,, found the four ordinary nuclein bases in all. The xanthin bases occurred habitually in greater quantity than the sarkin bases, and the relationship between the two was very variable. Sp rmatin is a name which has been given to a constituent similar to alkali albuminate, but it has not been closely studied. prostatic concrements are of two kinds. One is very small, generally oval in shape with concentric layers. In young but not in older persons they ar& colored blue by iodine (Iversen).' The other kind is larger, sometimes the size of the head of a pin, and consisting chiefly of calcium phosphate (about 700 p. m.) with only a very small amount, about 160 p. m., organic substance.. (b) Female Generative Organs. The stroma of the ovaries are of little interest from a physio- logico-chemical standpoint, and the most important constituent of the ovaries, the GraaflBan /oZHcfos with the ovum, have thus far not been the subject of a careful chemical investigation. The fluid in the follicles (of the cow) do not contain, as has been stated, the peculiar bodies, paralbumin or metalbumin, which are found in certain pathological ovarial fluids, but seems to be a serous liquid. The corpora lutea are colored yellow by an amorphous pigment called lutein. Besides this, another coloring-matter sometimes occurs which is not soluble in alkali ; it is crystalline, but not iden- tical with bilirubin or hsematoidin ; but it may be identified as a lutein by its spectroscopic behavior (Piccolo and LiEBEif, Kuhne and Ewald).' The cysts often occurring in the ovaries are of special patholog- ical interest, and these may have essentially different contents, de- pending upon their variety and origin. The serous cysts (Hydeops eolliculoeum Geaaeii), which are formed by a dilation of the Graafian follicles, contain a serous. ' Zeitschr. f. physlol, Chem., Bd. »i6i(?., Bd. 18. 'L. c. * See Chapter VI, p. 145. SEROUS AND PROLIFEROUS CYSTS. -iUT liquid which has & specific gravity of 1.005-1.022. A specific gravity of 1.020 is less frequent. Generally the specific gravity is lower, 1.005-1.014, with 10-40 p. m. solids. As far as is known, the contents of these cysts do not essentially differ from other serous liquids. The proliferous cysts (myxoid cysts, colloid cysts), which are developed from Pfluger's epithelium-tuhes, may have a con- tents of a very variable composition. We sometimes find in small cysts a semi-solid, transparent, or somewhat cloudy or opalescent mass which appears like solidified glue or quivering jelly, and which has been called colloid because of its physical properties. In other cases the cysts contain a thick, tough mass which can be drawn out into long threads, and as this mass in the different cysts is more or less diluted with serous liquids their contents may have a variable consistency. In other cases the small cysts may also contain a thin, watery fluid. The color of the contents is also variable. In certain cases they are bluish white, opalescent, and in others yellow, yellowish brown, or yellowish with a shade of green. They are often colored more or less chocolate- brown or red-brown, due to the decomposed blood- coloring matters. The reaction is alkaline or nearly neutral. The specific gravity, which may vary considerably, is generally 1.015-1.030, but may in few cases be 1.005-1.010 or 1.050-1.055. The amount of solids is very variable. In rare cases they amount to only 10-20 p. m. ; ordinarily they vary between 50-70-100 p. m. In a few cases 150-200 p. m. solids have been found. As form-elements we find red and white blood-corpuscles, gran- ular cells, partly fat-degenerated epithelium and partly large so- called Glugb's corpuscles, fine granular masses, epithelium-cells, cliolesterin crystals, and colloid corpuscles — ^large, circular, highly refractive formations. Though the contents of the proliferous cyst may have a variable composition, still it may be characterized in typical cases by its slimy or ropy consistency; by its grayish-yellow, chocolate-brown, sometimes whitish-gray color; and by its relatively high specific gravity, 1.015-1.025. Such a liquid does not ordinarily show a spontaneous fibrin-coagulation. We consider colloid, metalbumin, and paralbumin as character- istic constituents of these cysts. Colloid. This name does not designate any particular chemical 408 ORGANS OF GENERATION. substance, but is given to the contents of tumors with certain phys- ical properties similar to gelatin jelly. Colloid is found as a diseased product in several organs. Colloid is a gelatinous mass, insoluble in water and acetic acid; it is dissolved by alkalies and gives a liquid which is not precipitated by acetic acid or by acetic acid and potassium ferrocyanide. Ac- cording to Pfannbnstiel ' such a colloid is designated /J-pseu- ■domucin. Sometimes a colloid is found which, when treated with a very •dilute alkali, gives a solution similar to a mucin solution. On T)oiling with acids colloid gives a reducing substance. It is related to mucin, and it is considered by certain investigators as a trans- formed mucin. A colloid found by Wurtz " in the lungs contains C 48.09, H 7.47, N 7.00, and 37.44 ^. Colloids of different origin seem to have an unequal composition. Metalhumin. This name Scheebk ' gave to a protein substance found by him in an ovarial fluid. The metalbumin was considered by ScHBKER to be an albuminous body, but it belongs to the mucin jgroup, and it is for this reason c&lled pseudomucin by the author.'' Fseudomucin. This body, which, like mucin, gives a reducing substance when boiled with acids, is a mucoid of the following composition: C 49.75, H 6.98, IST 10.38, S 1.25, 31.74^ (author). With water pseudomucin gives a slimy, ropy solution, and it is this isubstance which gives the fluid contents of the ovarial cysts their typical ropy property. Its solutions do not coagulate on boiling, ibut only become milky-opalescent. Unlike mucin solutions, pseu- domucin solutions are not precipitated by acetic a.cid. With alcohol they give a coarse flocculent or thready precipitate which is soluble even after having been kept under water or alcohol for a long time. MiTJUKOFB ' has isolated and investigated a colloid from an ovarial cyst. It had the following composition: C 51.76, H 7.76, N. 10.7, S 1.09, and 28.69 ^, and differed from mucin and pseudomucin » Arch. f. Gynak., Bd. 38, ' See Lebert, Beitr. zur Kenntnniss des Gallertkrebses, Virchow's Arch., Bd. 4. »Verh. d. phySik.Tmed. Gesellsch. in WUrzburg, Bd. 3, and Sitzungsber. der physik.-med. Gesellsch. in Wurzburg ftlr 1864-1865; Wllrzburg med. Zeitschr., Bd. 7. i * Zeitschr. f. physiol. Chem., Bd. 6. ' Ueber das paramucin. Inaug.-Diss., Berlin, 1895. PSEUDOMUOm. 409 by reducing Feeling's solution before boiling with acid. He calls it paramucin. Paralbumin is another substance discovered by Schekee," and which occurs in ovarial liquids and also in ascites fluids with the simultaneous presence of ovarial cysts and rupture of the same. It is therefore only a mixture of pseudomucin with variable amounts of proteid, and the reactions of paralbumin are correspondingly variable. The detection of metalbumin and paralbumin is naturally con- nected with the detection of pseudomucin. A typical ovarial fluid containing pseudomucin is, as a rule, suflBciently characterized by its physical properties, and a special chemical investigation is only necessary in cases where a serous fluid contains very small amounts of pseudomucin. We proceed in the following way: The proteid is removed by heating to boiling with the addition of acetic acid ; the filtrate is strongly concentrated and precipitated by alcohol. The precipitate is carefully washed with alcohol, and then dissolved in water. A part of this solution is digested with saliva at the temperature of the body and then tested for glucose (derived from glycogen or dextrin). If glycogen is present, it will be converted into glucose by the saliva; precipitate again with alcohol and then proceed as in the absence of glycogen. In this last-mentioned case, first add acetic acid to the solution of the alcohol precipitate in water so as to precipitate any existing mucin. The precipitate produced is filtered, the filtrate treated with 2^ HCl, and warmed on the water-bath until the liquid is deep brown in color. In the presence of pseudomucin this solution gives Tkommbk's test. The other protein bodies which have been found in cystic fluids are ser globulin and seralbumin, peptone (?), mucin, and mucin- peptone (?). Fibrin only occurs in exceptional cases. The quantity of mineral bodies on an average amounts to about 10 p. m. The amount of extractive bodies (cholesterin and urea) and fat is ordi- narily 2-4 p. m. The remaining solids, which constitute the chief mass, are albuminous bodies and pseudomucin. The intraligamentary, papillary cysts contain a yellow, yellowish-green, or brownish-green liquid which contains either no pseudo-mucin or very little. The speciflc gravity is generally rather high, 1.032-1.036, with 90-100 p. m. solids. The principal con- stituents are the albuminous bodies of blood-serum. The rare tubo-ovarial cysts contain as a rule a watery, serous fluid containing no pseudomucin. ' L. c. 410 ORGANS OF OENEBATION. The parovarial cysts or the cysts of the ligamekta lata may attain a considerable size. In general, and when quite typical, the contents are watery, mostly very pale yellow-colored, water-clear or only slightly opalescent liquids. The specific gravity is low, 1.002- 1.009; and the solids only amount to 10-20 p. m. Pseudomucin does not occur as a typical constituent; proteid is sometimes absent, and when it does occur the quantity is very small. The principal part of the solids consists of salts and extractive bodies. In exceptional cases the fluid may be rich in proteid and may show a higher specific gravity. In regard to the quantitative composition of the fluid from ovarial cysts we refer the reader to the work of Oeeum. ' The Egg. The small ova of man and mammals cannot, for evident reasons, be the subject of a searching chemical investigation. Up to the present time the eggs of birds, amphibians, and fishes have been investigated, but above all the hen's egg. We will here occupy ourselves with the constituents of this last. The yolk of the hen's egg. In the so-called white yolk, which forms the germ with a process reaching to the centre of the yolk {latebra), and also a layer found between the yolk and yolk-mem- brane, we find proteid, nuclein, lecithin, and potassium (Liebbe- mann)\ The occurrence of glycogen is doubtful. The yolk- membrane consists of an albumoid similar in certain respects to keratin (Liebermann). The principal part of the yolk — the nutritive yolk or yellow — is a viscous, non-transparent, pale-yellow or orange-yellow alkaline emulsion of a mild taste. The yolk contains vitellin, lecithin, cholesterin, fat, coloring matters, ^tia.ees of neuridin (Bkiegek),' glucose in very small quantities, and mineral bodies. The occur- rence of cerebrin and of granules similar to starch ^Dareste) * has not been positively proved. Ovovitellin. This body is generally considered as a globulin, but it resembles a nucleoalbumin more. The question as to what ' Kemiske Studier over Ovariecystevcedsker, etc. Koebenhavn, 1884. See also Maly's Jahresber., Bd. 14, S. 459. ' Pfluger's Arch., Bd. 43. » Ueber Ptomaine. Berlin, 1885. < Compt. rend., Tome 73. OVOVITELLIN. 411 relationship other protein substances which, like the aleuron-grains of certain seeds and the so-called " dotterpldttchen " of the eggs of certain fishes and amphibians, are related to ovovitellin, bear to this substance, is a question which requires further investigation. The ovovitellin which has been prepared from the yolk of eggs is not a pure albuminous body, but always contains lecithin. Hoppe-Seyleb found 25^ lecithin in vitellin and also some pseudo- nuclein. The lecithin may be removed by boiling alcohol, but the vitellin is changed thereby, and it is therefore probable that the leci- thin is chemically united with the vitellin (Hoppe-Setlbe).' BuNGE ' prepared a pseudonuclein by digesting the yolk with gas- tric juice, and this pseudonuclein, according to him, is of great importance in the formation of the blood, and on these grounds he called it hcematogen. This hsmatogen — ^whose composition is as follows: C 42.11, H 6.08, N 14.73, S 0.55, P 5.19, Fe 0.29, and 31.05j^ — seems to be a decomposition product of vitellin. Vitellin is similar to the globulins in that it is insoluble in water, but on the contrary soluble in dilute neutral-salt solutions (although the solution is not quite transparent). It is also soluble in hydrochloric acid of 1 p. m. and in very dilute solutions of alka- lies or alkali carbonates. It is precipitated from its salt solution by diluting with water, and when allowed to stand some time in contact with water the vitellin is gradually changed, forming a sub- stance more like the albuminates. The coagulation temperature for the solution containing salt (NaCl) lies between + 70° and 76° C. or, when heated very rapidly, at about + 80° 0. Vitellin differs from the globulins in yielding pseudonuclein by pepsin digestion. It is not always or only in part precipitated by NaCl in substance. The chief points in the preparation of ovovitellin are as follows : The yolk is thoroughly agitated with ether; the residue is dissolved in a 10^ common-salt solution, filtered, and the vitellin precipitated by adding an abundance of water. The vitellin is now purified by repeatedly redissolving in dilute common-salt solutions and pre- cipitating by water. Ichthulin, which occurs in the eggs of the carp and other fishes, is, according to KosSBL and Walter, ' an amorphous modification of the crystalline body ieMhidin, which occurs in the eggs of the carp. Ichthulin is precipitated on diluting with water. It used to be considered as a vitellin. According to Walteb it yields a pseudonuclein on peptic digestion, and this pseudonuclein ' Med. chem. Untersuch., S. 216. ' Zeitschr. f. physiol. Chem., Bd. 9. »/M£?..Bd. 15. 412 ORGANS OF GENERATION. gives a reducing carboliydrate on boiling with, sulphuric acid. Iclitbulin has the following composition : C 53.42 ; H 7.63 ; N 15.63 ; 32.19; S 0.41 ; P 0.43. It also contains iron. The yolk also contains, besides vitellin, alhali-alhuminute and albumin. The fat of the yolk of the egg is, according to Libbekmann",' a mixture of a solid and a liquid fat. The solid fat consists chiefly of tripalmitin with some stearin. On the saponification of the egg-oil LiEBBEMANN obtained 40^ oleic acid, 38.04^ palmitic acid, and 15.31j^ stearic acid. The fat of the yolk of the egg contains less carbon than other fats, which may depend on the presence of mono- and diglyeerides or on a quantity of fatty acid deficient in carbon {LlEBEEMAKSr). Lutein. Yellow or orange-red amorphous coloring matters occur in the yellow of the egg and in seTeral other places in the animal organism ; for instance, in the blood-serum and serous fluids, iatty tissues, milk-fat, corpora lutea, and in the fat-globules of the retina. These coloring matters, which also occur in the vegetable kingdom (Thudichtjm'), have been called luteines or lipochromes. The luteines, which among themselves show somewhat different properties, are all soluble in alcohol, ether, and chloroform. They differ from the bile-pigment, bilirubin, in that they are not separated from their solution in chloroform by water containing alkali, and also in that they do not give the characteristic play of colors with nitric acid containing a little nitrous acid, but give a transient blue color, and lastly they give an absorption-spectrum of ■ordinarily two bands, of which one covers the line F and the other lies between the lines F and G. The luteines withstand the action of alkalies so that they are not changed when we remove the fats present by means of saponification. Lutein has not been prepared pure. Malt ' has found two pigments free from iron in the eggs of a water-spider (maja squinado), one a red, mtelloru- bin, and the other a yellow pigment, vitellolutein. Both of these pigments are colored blue by nitric acid containing nitrous acid, and beautifully green by concentrated sulphuric acid. The absorption-bands, especially of the vitello- lutein, correspond very nearly with those of ovolutein. The mineral bodies of the yolk of the egg consist, according to POLECK,' of 51.2-65.7 parts soda, 89.3-80.5 potash, 123.1-133.8 'L. c. » Centralbl. f. d. med. Wissensch., 1869, No. 1. » Monatshefte f . Chem. , Bd. 2. * Cited from QorupBesanez, Lehrbuch d. physiol. Chem., 4. Aufl., S. 740. WHITE OF THE EGG. 41^ lime, 20.7-21.1 magnesia, 14.5-11.90 iron oxide, 638.1-667.0 phosphoric acid, and 5.5-14.0 parts silicic acid in 1000 parts of the ash. We find phosphoric acid and lime the most abundant, and then potash, which is somewhat greater in quantity than the soda. These results are not, however, quite correct, first, because no dissolved phosphate occurs in the yolk (Liebekmann), and secondly, in burning, phosphoric and sulphuric acids are produced and these drive away the chlorine, which is not accounted for in the preceding analyses. The yolk of the hen's egg weighs about 12-18 grms. The quantity of water and solids amounts, according to Pabkes,' to 471.9 p. m. and 528.1 p. m. respectively. Among the solids he found 156.3 p. m. proteid, 3.53 p. m. soluble and 6.12 p. m. in- soluble salts. The quantity of fat, according to Paekes, is 228.4 p. m., the lecithin, calculated from the amount of phosphorus in the organic substance in the alcohol-ether extract, was 107.2 p. m., and tlie cholesterin 17.5 p.m. The white of the egg is a faint-yellowish albuminous fiuid en- closed in a framework of thin membranes; and this fluid is in itself very liquid, but seems viscous because of the presence of these fine membranes. That substance which forms the membranes, and of which the chalaza consists, seems to be a body nearly related to horn substances (Liebermakn'). The white of the egg has a specific gravity of 1.045 and always has an alkaline reaction. It contains 850-880 p. m. water, 100-130 p. m. proteid bodies, and 7 p. m. salts. Among the extractive bodies Lehmann found a fermentable variety of sugar which amounted to 5 p. m. or, according to Meissijek, 80 p. m. of the solids.' Besides these, we find in the white of the egg traces of fats, soaps, lecithin, and cholesterin. • The wWte of the egg during incubation becomes transparent on boiling and acts in many respects like alkali-albuminate. This albumin Tabchanobf* called " tataibumin." The albuminous bodies of the white of the egg belong partly to the globulin and partly to the albumin group. Besides these, the white of the egg contains a mucoid substance. ' Hoppe-Seyler, Med. chem. Untersuch. , Heft 2, S. 309 'L. c. • Cited from Gorup-Besanez, Lehrbuch, 4. Aufl., S. 739. * PflUger's Arch., Bdd. 31, 33, and 39. 414 ORGANS OF GENERATION. The ovglolulin is, according to Dillnek/ closely related to s,erglobulin. On diluting the white of the egg with water it partly separates. It is also precipitated by magnesium sulphate. The quantity of globulins in the white of the egg is on an average 6.67 p. m., or about 67 p. m. of the total proteids. According to COBIN and Bbeaed/ we have two globulins in the white of the egg, one coagulating at + 57.6° C, and the other at + 67° C. Ovalbumin, or the albumin of the white of the egg. Ovalbumin was first obtained in a crystalline form by Hofmeister', by allow- ing its solution in a half -saturated ammonium-sulphate solution to evaporate very slowly. This crystalline ovalbumin is later further studied by Gabeiel,* Bojtdztnski and Zoja,* and the two last- mentioned investigators were able, by fractional crystallization, to show that ovalbumin was probably a mixture of several albumins of about the same elementary composition but with somewhat differ- ent coagulation-temperature, solubility, and specific rotation. In the main these results are in accord with the views of many other investigators, such as G-autiee,' Bechamp,' Coein^ and Beeaed,' on the occurrence of several albumins, but in details they do not agree very well. According to Gatjtiek and Bechamp ovalbumin is a mixture of two albumins with the coagulation-temperature of 60-63° and 71-74° 0. respectively, while according to OoEiifr and Beeaed it is a mixture of three albumins with the coagulation- temperature of 67, 73, and 82° C, respectively. According to BosTDZYNSKi and Zoja the portion which dissolves with difficulty coagulates at 64.5°, while the readily soluble portion coagulates at 55.5-56° C. The elementary composition of ovalbumin has not been positively established. Bondztnski and Zoja found 52.07- 52.44, H 6.95-7.26, N. 15.11-15.58, and S 1.61-1.70^ for four different fractions, which agree well with the results of the author, namely, 53.25, H 6.90, N 15.25, S 1.67-1.93^. Hofmeistee,' > Upsala Lakarefs. F5rh., Bd. 20; also Maly's Jahresber., Bd. 15, S. 31. * Travaux du laboratolre de I'Universite de Li§ge, Tome 3; also Maly's Jahresber., Bd. 18, S. 13. » Zeitschr. f. pliyslol. Chem., Bdd. 14 and 16. * lUd., Bd. 15. 5 Ibid., Bd. 19. ' Bull, de la soc. chim., Tome 14. ' lUd., Tome 31. « L. c. ' Zeitschr. f. physiol. Chem., Bd. 16. VALB UMIN AND VOMUCOID. 415 on the contrary, found higher figures, 53.28^, for the carbon and lower, 15.0 and 1.09^, for the nitrogen and sulphur respectively. The specific rotation was determined by Starke ' as a (D)= — 38°. BoNDZYNSKi and Zoja found 25.8-36.3°, 29.16°, 34.18°, and 43.54° for various fractions. Ovalbumin has the properties of the albumins in general, but differs from seralbumin in the following : Its specific rotation is lower. It is quickly rendered insoluble by alcohol. It is precipitated by a suflBcient quantity of hydrochloric acid, but dissolves with greater diflSiculty than seralbumin in an excess of the acid. Ovalbumin in solution, when introduced into the blood-circulation, passes into the urine, which is not the case with seralbumin. Ovalbumin, or, more correctly, the mixture of albumins, may be obtained, according to Starke, by precipitating the globulins by MgSO, at 20° 0. and saturating the filtrate with Na,SO, at the same temperature. The ovalbumin which separates is filtered, pressed, dissolved in water, and freed from salts by dialysis. The dialyzed solution is then evaporated in a vacuum or at 40°— 50° C. If precipitated with alcohol, albumin becomes quickly insoluble. To prepare crystallized ovalbumin mix the white of egg, previously beaten and separated from the foam, with an equal vol- ume of a saturated solution of ammonium sulphate, filter off the globulin, and allow the filtrate to evaporate slowly in not too thin layers at the temperature of the room. The mass, which separates after a time, is dissolved in water, treated with ammonium sulphate solution until a cloudiness commences, and then allowed to stand. After repeated recrystallizations the mass is treated either with alcohol, which makes the crystals insoluble, or they are dissolved in water and purified by dialysis. The albumin does not crystallize from this solution on spontaneous evaporation. Ovomucoid. ,This substance, first observed by Nbumeister" and considered by him as pseudo-peptone and then later studied by Salkowski," is, according to C. Th. MoRi^rER,' a mucoid with 13.65^ nitrogen and 3.20^ sulphur. On boiling with dilute min- eral acids it yields a reducing substance. Ovomucoid exists to a great extent in hens' eggs, the solids of which, in round numbers, contain lOf^. A solution of ovomucoid is not precipitated by mineral acids ' Upsala Lakarefs P6rh., Bd. 16; also Maly's Jahresber., Bd. 11, S. 17. ' Zur Physiologie der Eiweissresorptlon, etc. Zeitschr. f. Biologie., Bd., 27. ' Centrabl. f . d. med. Wissonsch. 1893. ' Zeitschr. f. physiol. Chem. Bd. 18. 416 OSQAJf^S OF OENEBATION. nor by organic acids, with the exception of phosphotungstic acid and tannic acid. It is not precipitated by metallic salts, but basic lead acetate and ammonia give a precipitate. Ovomucoid is pre- cipitated by alcohol, but sodium chloride, sodium sulphate, and magnesium sulphate give no precipitates either at the ordinary temperature nor when added to saturation at 30° C. Its solutions are not precipitated by an equal volume of a saturated solution of ammonium sulphate, but are precipitated on adding more salt thereto. The substance is not precipitated on boiling, but the part which has become insoluble in cold water and then dried is precipitated when dissolved in boiling water. Ovomucoid may be prepared by removing all the proteids by boiling with the addition of acetic acid, and then concentrating the filtrate and precipitating with alcohol. The substance is puri- fied by repeated solution in water and precipitating with alcohol. The mineral bodies of the white of the egg have been analyzed by POLEOK and Webbe.' They found in 1000 parts of the ash: 276.6-284.5 grms. potash, 235.6-329.3 soda, 17.4-29 lime, 16-31.7 magnesia, 4.4-5.5 iron oxide, 238.4-285.6 chlorine, 31.6-48.3 phosphoric acid (P,OJ, 13.2-26.3 sulphuric acid, 2.8-20.4 silicic acid, and 96.7-116 grms. carbon dioxide. Traces of fluorine have also been found (Nickles"). The ash of the white of the egg con- tains, as compared with the yolk, a greater amount of chlorine and alkalies, and a smaller amount of lime, phosphoric acid, and iron. The Shell-membrane and the Egg-shell. The shell-membrane consists, as above stated (page 49), of a keratin substance. The shell contains very little organic substance, 36-65 p. m. The chief mass, more than 900 p. m., consists of calcium carbonate; besides this there are very small amounts of magnesium carbonate and earthy phosphates. The difEerent coloring of birds' eggs depends upon several different coloring matters. Among these we find a red or reddish-brown pigment called " ooro- Mn" hj SoRBY,' which is peAaps identical with hsematoporphyrin. The green or blue coloring matter, Sorby's oocyan, seems, according to Ltbber- MANN,* and Kruebneehg,' to be partly biliverdin and partly a blue deriva- tive of the tile-pigments. ' Cited from Hoppe Seyler's Physiol. Chem., S. 778. ' Conaip. rend.. Tome 43. " Cited frcim Krukenberg, Verh. d. phys. -ch^u. Q-esellBch. in WUrzburg, Bd. 17. * Ber. d. deutsch. chem. Qesellsoh., Bd. 11. 'L. c. SBELL MEMBRANE AND THE EGG-SHELL. 417 The eggs of birds have a space at their blunt end filled with gas; this gas contains on an average 18.9-19.9 per cent oxygen (Hufnee). ' The weight of a hen's egg varies between 40-60 grammes and may weigh sometimes 70 grms. The shell and shell-membrane together, when carefully cleaned, but still in the moist state, weigh 5-8 grms. The yolk weighs 13-18 and the white 23-34, or about double. The white of the egg of cartilaginous and bony fishes contains only traces of true albumin, and the cover of the frog's egg consists, according to Giacosa,' of mucin. The crystalline formations (yolk-spherules or dotierpldttehen) which have been observed in the egg of the tortoise, frog, ray, shark, and other fishes, and which are described by Valenciennes and Fkemt ' under the names emydin, ichthin, iehthidin, and ichthulin, seem, as above stated in con- nection with ichthulin, to consist chiefly of phosphoglycoproteids. The egg- of the river-crab and the lobster contain the same pigment as the shell of the animal. This pigment, called cyanocrystallin, becomse red on I oiling in water. In fosil eggs (of aptenodttes, pelecands, and hall^us) in old guano deposits a yellowish-white, silky, laminated combination has been found which is called guanovidtt, (NH.jjSO, -f- SKjSO, + 3KHS0, + 4HjO, and which is easily soluble in water, but is insoluble in alcohol and ether. Those eggs which develop outside of the mother-organism must; contain all the elements necessary for the young animals. One- finds, therefore, in the yolk and white of the egg an abundant quantity of albuminous bodies of different kinds, and especially a phosphorized proteid in the yolk. "Further, we also find lecithin in the yolk, which seems habitually to occur in the developing cell. The occurrence of glycogen is doubtful, and the carbohydrates are perhaps represented by a very small amount of glucose and ovomu- coids. On the contrary, the egg contains a large proportion of fat, which doubtless is an important source of nutrition and res- piration for the embryo. The cholesterin and the lutein can hardly have a direct influence on the development of the embryo. The egg also seems to contain the ^mineral bodies necessary for the development of the young animal. The lack of phosphoric acid is compensated by an abundant amount of phosphorized organic sub- stance, and the nucleoalbumin containing iron, from which the hffmatogen (see page 411) is formed, is doubtless, as Bunge claims, of great importance in the formation of the haemoglobin containing ' Du Bois-Reymond's Arch., 1893. ' Zeitschr. f. physiol. Chem., Bd. 7. » Cited from Hoppe-Seyler's Physiol. Chem., S. 77. 418 ORGANS OF GENERATION. iron. The silicic acid necessary for the develoiiment of the feath- ers is also found in the egg. During the period of incubation the egg loses weight, chiefly due to loss of water. The quantity of solids, especially the fat and the proteids, diminishes and the egg gives off not only carbon dioxide, but also, as LiBBEKMANiir ' has shown, nitrogen or a nitrog- enous substance. The loss is compensated by the absorption of oxygen, and it is found that during incubation a respiratory exchange of gas takes place. While the quantity of dry substance in the egg during this period always decreases, the quantity of mineral bodies, proteid, and fat always increases in the embryo. The increase in the amount of fat in the embryo depends, accord- ing to LiEBBEMANN, in great part upon a taking up of the nutri- tive yolk in the abdominal cavity. The weight of the shell and the quantity of lime-salts contained therein remains unchanged during incubation. The yolk and white together contain the necessary quantity of lime for development. The most complete and careful chemical investigation on the development of the embryo of the hen has been made by Liebee- MANN. From his researches we may quote the following : In the earlier stages of the development, tissues very rich in water are formed, but on the continuation of the development the quantity of water decreases. The absolute quantity of bodies soluble in water increases with the development, while their relative quantities, as compared to the other solids, continually decreases. The quantity of bodies soluble in alcohol quickly increases. A specially impor- tant increase is noticed in the fat, whose quantity is not very great even on the fourteenth day, but after that it becomes considerable. The quantity of albuminous bodies and albuminoids insoluble in water grows continually and regularly in such a way that their absolute quantity increases while their relative quantity remains nearly unchanged. Libbermann found no gelatin in the embryo of the hen. The embryo does not contain any gelatin-forming substance until the tenth day, and from the fourteenth day on it contains a body which when boiled with water gives a substance similar to chondrin. A body similar to mucin occurs in the enibryo when about six days old, but then disappears. The quan- tity of haemoglobin shows a continual increase compared to the -weight of the body. LiEBEEMAiir]!r found that the relationship of ' Pfluger's Arch., Bd. 43. AMNIOTIC FLUID. 419 the hfemoglobin to the bodily weight was 1 : 728 on the eleventh day and 1:421 on the twenty-first day. The tissue of the placenta has not thus far been the subject of detailed chem- ical investigation. In the edges of the placenta of bitches and of cats a crya- tallizable orange-colored pigment (bilirubin ?) has been found, and also a green amorphous pigment, Meckel's Juematochlorin, which is considered as biliverdin by Ktti.' Preyer^ questions the identity of these pigments with biliverdin. From the cotyledons of the placenta in ruminants a white or faint rose-col- ored creamy fluid, the uterine milk, can be obtained by pressure. It is alkaline in reaction, but becomes acid quickly. Its specific gravity is 1.033-1.040. It contains as form-elements fat-globules, small granules, and epithelium-cells. We have found 81 3-120.9 p. m. solids, 61.2-105.6 p. m. proteid, about 10 p. m. fat. and 3.7-8 3 p. m. ash in the uterine milk. The fluid occurring in the so-called geape-mole (mola racemosa) has a low specific gravity, 1 009-1.012, and contains 19.4-36.3 p. m. solids with 9-10 p. m. protein bodies and 6-7 p. m. ash. The amniotic fluid is in women thin, whitish, or pale yellow; sometimes it is somewhat yellowish brown and cloudy. White flakes separate. The form-elements are mucus-corpuscles, epithe- lium-cells, fat-drops, and lanugo hair. The odor is stale, the reaction neutral or faintly alkaline. The specific gravity is 1.002- 1.028. The amniotic fluid contains the constituents of ordinary transu- dations. The amount of solids at birth is hardly 20 p. m. In the earlier stages of pregnancy the fluid contains more solids, especially proteids. Among the albuminous bodies, Wetl ' found one sub- stance similar to vitellin, and with great probability also ser- albumin, besides small quantities of mucin. Glucose is regularly found in the amniotic fluid of cows, but not in human beings. On the contrary, the human amniotic fluid contains some urea and allantoin. The quantity of these may be increased in hydramnion (Peochownick,* HAEifACK),' which depends on an increased secre- tion by the kidneys and skin of the foetus. Creatin and lactates are questionable constituents of the amniotic fluid. The quantity of urea in the amniotic fluid is, according to Peochownick, 0. 16 p. m. In the fluid in hydramnion, PEOOHOWincK and Haenack found respectively 0.34 and 0.48 p. m. urea. The chief mass of the solids consists of salts. The quantity of chloridel (NaCl) is 5.7-6.6 p. m. • Maly's Jahresber., Bd. 3, S. 887. » Die Blutkrystalle (Jena, 1871), S. 189 ; Du Bois-Reymond's and Reicheit's Arch., 1876. » lUd. * Arch. f. Gynak., Bd. 11; also Maly's Jahresber., Bd. 7, S. 155. » Berlin klin. Wochenschr., 1888, No. 41. CHAPTEE Xrv. MILK. The chemical constituents of the mammary glands have been little studied. The protoplasm of the cells is rich in proteid, which consists in great part of casein or a substance nearly related. If all the milk is removed from the mammary gland by thorough washing, the cells still contain a large quantity of proteids which swell up to a slimy, ropy, or fibrous mass when very dilute alkali (1-3 p. m. KOH) is added. These proteids consist mainly of nucleoproteid, which is gradually changed by the action of the alkali. This nucleoproteid gives a reducing substance on boiling with dilute acids. If the mammary gland is boiled with water, the protoplasm of the cell is decomposed and a nucleo- proteid passes into solution, which may be precipitated by the addition of acetic acid, and which is characterized by its greater insolubility in acetic acid, compared with casein. This nucleo- proteid, which may well be considered as a protoplasm-nucleo- proteid changed by heat, also gives on boiling with dilute mineral acids a reducing substance whose nature is not known. The relation this nucleoproteid bears to lactose or the mother-substance of the same has not been determined. According to Beet," the secreting glands contain a body which on boiling with dilute mineral acids yields a reducing substance. Such "a substance, which acts as a step towards the formation of lactose, has also been observed by Thierfeldek.' Fat seems to be a never-failing con- stituent of the cell, at least in the secreting gland, and this fat may be observed in the protoplasm as large or small globules similar to milk-globules. The extractive bodies of the mammary glands have ' Compt.. rend., Tome 98. ' Pfiuger's Arch., Bd. 33, and Maly's Jahresber., Bd. 13, S. 156. 420 cows MILK. 421 teen little investigated, but among them we find considerable amounts of xanthin bases. As human milk and milk of animals are essentially of the same constitution, it seems best to speak first of the one most thoroughly investigated, namely, cow's milk, and then of the essential proper- ties of the remaining important varieties of milk. Cow's Milk. Cow's milk forms, as all milks do, an emulsion which consists of very finely divided fat suspended in a solution consisting chiefly of proteid bodies, milk-sugar, and salts. Milk is non- transparent, white, whitish yellow, or in thin layers somewhat bluish white, of a faint, insipid odor and mild, faintly sweetish taste. . The specific gravity is 1.028 to 1.0345 at + 15° C. The reaction of perfectly fresh milk is generally amphoteric. The extent of the acid and alkaline part of this amphoteric reac- tion has been determined by different investigators, especially Thornbe,' Sebelin," and Coueastt.' The results are different on using different indicators, and also the milk from various animals, as well as at different times djiring the lactation period, differs somewhat. The first and last portions of the same milking have a different reaction. Cotjeant has determined the alkaline N part by — sulphuric acid, using blue lacmoid as indicator and the N acid part by :rp: caustic soda, using phenolphthalein as indicator. He found, as average for the first and last portions of the milking of twenty cows, that 100 cc. milk had the same alkaline reactiori N ior blue lacmoid as 41 cc. — caustic soda, and the same acid reac- N tion for phenolphthalein as 19.5 cc. r^ sulphuric acid. Milk gradually changes when exposed to the air, and its reac- tion becomes more and more acid. This depends on a gradual transformation of the milk-sugar into lactic acid, caused by micro- organisms. ' Cbem. Ztg., Bd. 16, S. 1469. «/6i(i.,Bd. 16, S. 597. ' Ueber die Reaktion der Kuh- und Frauenmilcli, etc. luaug.-Diss. Bonn, 1891; also Pflttger's Arcli., Bd. 50. 422 MILK. Entirely fresh amphoteric milk does not coagulate on boiling, hut forms a skin consisting of coagulated casein and lime-salts, which rapidly re-forms after being removed. Even after passing a current of carbon dioxide through the fresh milk it does not coagulate on boiling. In proportion as the formation of lactic acid advances this behavior changes, and soon a stage is reached when the milk, which has previously had carbon dioxide passed through it, coagulates on boiling. At a second stage it coagulates alone on heating; then it coagulates by passing carbon dioxide alone without boiling; and lastly, when the formation of lactic acid is sufficient, it coagulates spontaneously at the ordinary temperature, forming a solid mass. It may also happen, especially in the warmth, that the casein-clot contracts and a yellowish or yellowish-green acid liquid (acid whey) separates. If the drawn is sterilized by heating and contact with micro- organisms prevented, the formation of lactic acid may be entirely stopped. The formation of acid may also be prevented, at least for some time, by many antiseptics, such as salicylic acid (1 : 5000), thymol, boracic acid, and other bodies. If freshly drawn amphoteric milk is treated with rennet, it coagulates quickly, especisflly at the temperature of the body, to a solid mass (curd) from which a yellowish fluid (sweet whey) i& gradually pressed out. This coagulation occurs without any change in the reaction of the milk, and therefore it is distinct from the acid coagulation. Milk sometimes undergoes a peculiar kind of coagulation, being converted into a thick, ropy, slimy mass (thick milk). This conversion depends, accord- ing to Schmidt- Mulheim,' upon a peculiar change in which the milk-sugar is made to undergo a slimy transformation. This transformation is caused by a special organized ferment. In cow's milk we find as form-elements a few colostrum cor- puscles (see Colostrum) and a few pale nucleated cells. The number of these form-elements is very small compared with the immense amount of the most essential form-constituents, the milk- globules. The Milk-globules. These consist of extremely small drops of fat whose number is, according to Woll," 1.03-5.75 million in '■ Pfluger's Arch., Bd. 27. ' On the Conditions influencing the Number and Size of Fat-globules ia Cow's Milk. Wisconsin Expt. Station, Vol. 6, 1892. MILK-OLOBULES. 123 1 c.mm., and whose diameter is 0.0024-0.0046 mm. and 0.0037 mm. as average for animals of different races. It is unquestionable that the milk-globules contain fat, and we consider it as positive that all the milk-fat exists in them. Another and disputed ques- tion is whether the milk-globules consist entirely of fat or whether they also contain proteid. According to the observations of Asoherson,' drops of fat, when dropped in an alkaline proteid solution, are covered with a fine albuminous coat, a so-called haptogen-memhrane. As milk on shaking with ether does not give up its fat, or only very slowly, in the presence of a great excess of ether, and as this takes place very readily after the addition of acids or alkalies, which dissolve proteids, it was formerly thought that the fat-glohtiles of the milk were en- veloped in a proteid coat. A true membrane has not been detected'; and since, when no means of dissolving the proteid is resorted to — for example, when the milk is precipitated by carbon dioxide after the addition of very little acetic acid, or when it is coagulated by rennet — the fat can be very easily extracted by ether, the theory of a special albuminous membrane for the fat-globules has been gener- ally abandoned. The observations of Quincke ' on the behavior of the fat-globules in an emulsion prepared with gum have led, at the present time, to the conclusion that each fat-globule in the milk is surrounded by a stratum of casein solution by means of molecular attraction, and this prevents the globules from uniting with each other. Everything that changes the physical property of the casein in the milk or precipitates it must necessarily help the solution of the fat in ether, and it is in this way that the alkalies, acids, and rennet work. If we accept this view, which requires further proof, we must not overlook the fact that the fat-globules remain unchanged when the milk under agitation is coagulated with rennet. In this case we find an immense number of un- changed milk-globules in the whey, and if we wish to admit of a stratum of proteids around the fat-globules proceeding from the molecular attraction , we must not consider that it is entirely due to casein, but to proteid in general. If the fat-globules are filtered ofE and washed on a filter, we always obtain (Radbnhausen and Danilewskt) ' after their treatment with ether a residue consisting of proteid. Prom this behavior the deduction has been made that the fat-globules, even though they have no real membrane, consist, neverthe- less, of fat and proteid. The extreme difficulty of completely removing the albuminous bodies of the milk by washing the fat on the filter renders it neces- sary to exercise great caution in drawing a conclusion. The question as to the ' Arch. f. Anat. u. Physiol.. 1840. » Pflllger's Arch., Bd. 19. ' Forschungen auf dem Gebeite der Viehhaltung (Bremen, 1880), Heft 9. 424 MILK. composition of the milk-globules, and especially as to the possible amount of proteid, cannot be decided at present. The milk-fat has a rather variable specific gravity, which ac- cording to BoHK ' is 0.949-0.996 at + 15° 0. The milk-fat, which is obtained under the name of butter, consists in great part of the neutral fats palndtin, olein, and stearin. Besides these it contains, as triglycerides, myristic acid, small quantities of butyric acid and caproic acid, traces of caprylic acid, capric acids, lauric acid, and arachidic acids. Butter which has been exposed to the action of sunlight contains also formic acid (Dtjclaux). Milk-fat also con- tains a small quantity of lecithin and cholesterin, also a yellow col- oring matter. The quantity of volatile fatty acids in butter is, ac- cording to DuCLATJX," on an average about 70 p. m., of which 37-51 p. m. is butyric acid and 20-33 p. m. is caproic acid. The non-volatile fat consists of ^-^ to -j^ olein, and the remainder of a mixture of palmitin and stearin. According to other investigators milk-fat has a different composition. KOBFOED* found in butter from Jutland besides oleic acid two other acids not belonging to the series CnHjnOa, having the formulae CiBHaB04 and (probably) 'CaBHsiOfi. In 100 parts fatty acids he found 66 parts acids of the series C„HanOa, name- ly, 2 stearic acid, 38 palmitic acid, 23 myristic acid, 8 lauric acid, 1.5 butyric iicid, 3 caproic acid, 8 capric acid, and 0.5 caprylic acid. According to Wanklyn * butter does not contain any palmitic acid. It contains instead an acid called by him oMepal/milio acid, with the formula (CieHsoOa)^ and not be- longing to the oleic acid series. The relative quantities of the different fatty jaclds do not seem to be constant, and they differ at various times during lac- tation. The quantity of volatile fatty acids in butter-fat is of great practical im- portance in the methods for detecting the presence of foreign fats in butter. This detection is performed generally according to Rbichbbt's process based on Hbhnbk and Angbll's method. The fat is saponified with alcoholic pot- ash and the alcohol evaporated. The soaps are dissolved in water, and then distilled with an excess of phosphoric acid. The quantity of volatile fatty acids in the distillate is determined by titration with decinormal alkali. With butter of proper composition 2.5 grms. should yield a distillate requiring 14-13 c.c. for neutralization, and at least not less than 13 c.c. of the decinormal alkali. In proportion as the butter contains a greater quantity of foreign fats the quantity of alkali required becomes smaller. We cannot here describe in detail the different modifications of this process as well as the newer methods. The milk-plasma, or that fluid in which the fat-globules are sus- pended, contains at least three different albuminous bodies, casein, lactoglobulin, and lactalhumin, and two carbohydrates, of which only ' Studier over Maelk. KjSbenhavn, 1880, and Maly's Jahresber., Bd. 10, S. 183. ' Compt. rend., Tome 104. ' Bull, de I'Acad. Roy. Danoise, 1891 . ■• Chem. News, Vol. 63. CASEIN. 425 one, the milk-sugar, is of great importance. The milk-plasma also contains extractive bodies, traces of urea, creatin, creatinin, Jiypo- xanthin (?), lecithin, cholesterin, citric acid (Soxhlet and Hen- kel),' and lastly also mineral bodies and gases. Casein. This protein substance, which thus far has been de- tected positively only in milk, belongs to the nucleoalbumins, and differs from the albuminates by its containing phosporus and by its behavior with the rennet enzyme. Casein from cow's milk has the following composition: C 53.0, H 7.0, N 15.7, S 0.8, P 0.85, and 22. 65$^. Its specific rotation is, according to Hoppe-Seylbr," somewhat variable; in neutral solution it is a (D) = — 80°. The question whether the casein from different varieties of milk is iden- tical or whether there are several different caseins has not been positively determined. Casein when dry appears like a fine white powder which, after heating to 100° C. or somewhat above, shows the properties and solubilities of freshly precipitated, still-moist casein. Casein is only slightly soluble in water or in neutral-salt solutions. According to Aethus ' it is rather easily soluble in a 1^ solution of sodium fiuo- ride, ammonium, or potassium oxalate. It acts like a rather strong acid, dissolves readily in water on the addition of very little alkali, forming a neutral or acid liquid, and lastly it .dissolves in water in the presence of calcium carbonate, from which it expels the carbon dioxide. If casein is dissolved in lime-water and this solution care- fully treated with very dilute phosphoric acid until it is neutral in reaction, the casein appears to remain in solution, but is probably only swollen as in milk, and the liquid contains at the same time a large quantity of calcium phosphate without any precipitate or any visible suspended particles. The casein solutions containing lime are opalescent and have on warming the appearance of milk deficient in fat. Therefore it is not impossible that the white color of the milk is due partly to the casein and calcium phosphate. Soldner has prepared two calcium combinations of casein with 1.55 and 2. 36j^ CaO, and these combinations are designated di- and tricalcium casein by Coueant. ' ' Cited from F. SOldner, Die Salze der Milch, etc. Landwirthsch. Ver- suchsstation, Bd. 35. Separatabzug, S. 18. ' Handb. d. physioI. u. patliol. chem. Analyse, 6. Aufl., S. 359. ' Theses presentees ft la faculte des sciences de Paris, 1893. «L. c. 426 MILK. Casein solutions do not coagulate on boiling, but are covered, like milk, with a skin. They are precipitated by very little acid, but the presence of neutral salts retards the precipitation. A casein solution containing salt or ordinary milk requires, therefore, more acid for precipitation than a salt-free solution of casein of the same concentration. The precipitated casein dissolves very easily again in a small excess of th% acid, but less easily in an excess of acetic acid. The acid solutions are precipitated by mineral acids in excess. Casein is precipitated from neutral solutions or from milk by common salt or magnesium sulphate in substance without chang- ing its properties. Metallic salts, such as copper sulphate, com- pletely precipitate the casein from neutral solutions. The property which is the most characteristic of casein is that it coagulates with rennet in the presence of a sufficiently great amount of lime-salts. In solutions free from lime-salts the casein does not coagulate with rennet; but it is changed so that the solu- tion (even if the enzyme is destroyed by heating) yields a coagulated mass, having the properties of curd, if lime-salts are added. The rennet enzyme, rennin, has therefore an action on casein even in the absence of lime-salts, and these last are only necessary for the coagulation or the separation of the curd. This fact, which was first proved by the afthor,' has lately been confirmed by Aethus and Pages." Peters ' claims to have found that paracasein, when dissolved in lime-water, may be repeatedly coagulated by rennet., According to Peters rennet also coagulates alkali albuminate, as also vegetable proteid bodies precipitated by acids (wheat and peas) when dissolved in lime-water. Several enzymes existing in the planet kingdom also have the same action as rennet. The curd formed on the coagulation of milk contains large quantities of calcium phosphate. According to Soxhlet and SoLDNER,* the soluble lime-salts are only of essential importance in coagulation, while the calcium phosphate is without importance. According to Coukant' the calcium casein on coagulation may carry down with it, if the solution contains dicalcium phosphate, a ' Maly's Jabresber., Bdd. 2 and 4; also Hammarsten, Zur Kenntniss des Kaseins und der Wirkung des Labfermentes. Nova Acta Reg. Soc. Scient, Upsala, 1877. Festscbrift. ' Arcb. de Physiol. (5), Tome 3, and Mem. Soc. bid.. Tome 43. ' Unters. liber das Lab und die Lab&hnlichen Fermente. Rostock, 1894. ■■L. c. = L. c. PROPERTIES OF CASEIN. ^11 part of this as tricalcium phosphate, leaving monocalcinm phosphate in the solution. The chemical processes which take place in the rennet coagalation have not been thoronghly investigated; still several observations seem to show that Tjasein splits partly into a difficultly soluble body, 'paracasein or curd, whose composition closely resembles that of casein and which forms the chief product, and partly into an easily soluble substance, similar to albumose, wAey-^ro^eirf, which is deficient in carbon and nitrogen (50.3^ C and 13.2^ N, Kostnbk") and which is produced in very small quantities. Paracasein" is not further changed by the rennet enzyme, and it has not the same property of holding calcium phos- phate in solution as casein has. In the digestion of casein with pepsin hydrochloric acid pseudo- nnclein is split off. The quantity of psendonuclein split off is, according to Moeaczewski,' very considerable, from 1.29 to 21.10j^ of the digested casein. Salkowski and Hahk * and Siebblien ^ have also found with Moeaczbwski that the quantity of pseudo- nuclein split off in the peptic digestion of casein is very variable. Sebblien as well as WiLLDBisrow and Mokaczewski could not bring all the psendonuclein in solution by contLnnons digestion. The quantity of phosphorus in the psendonuclein also varies between 0.88 and 6.86^, and of the casein phosphorus varying quantities, 6 to 60^, were obtained in the psendonuclein. All the phosphorus of the casein was never obtained as psendonuclein, and Moea- OZEWSKI draws the conclusion from his investigations that the pseu- donuclein from the beginning does not contain all the phosphorus of the casein. Casein may be prepared in the following way: The milk ia diluted with 4 vols, water and the mixture treated with acetic acid to 0.75 to 1 p. m. Casein thus obtained is purified by repeated solution in water with the aid of the smallest quantity of alkali possible, by filtrating and reprecipitating with acetic acid, and ' See Maly's Jaliresber., Bd. 11, S. 14. ' It has been recently proposed to designate the ordinary casein as caseino- gen, and the curd as casein. Although such a proposition is theoretically cor- rect, it leads in practice to confusion. On this account the author calls the card paracasein, according to Sohulze and ESse (Landwirthsch. Versuohsstat., Bd. 31). ' Zeitschr. f. physiol. Chem., Bd. 20. * Pfluger's Arch., Bd. 50. ' Zeitschr. f. physiol. Chem., Bd. 20. 428 MILK. thoroughly washing with water. Most of the milk-fat is retained by the filter on the first filtration, and the casein contaminated with traces of fat is purified by treating with alcohol and etlier. Ladoglolulin was obtained by Sebelien ' from cow's milk by saturating it with NaCl in substance (which precipitated the casein), and saturating the filtrate with magnesium sulphate. As far as it has been investigated it had the properties of serglobulin, with which it is perhaps identical. Lactalbumin was first prepared in a pnie state from milk by Sebeliek." Its compositiou is, according to Sbbelien, C 53.19, H 7.18, N 15.77, S 1.73, 23.13^. Lactalbumin has the proper- ties of the albumins. It coagulates, according to the concentration and the amount of salt in solution, at + 72° to 84° C. It is similar to seralbumin, but differs from it in having a considerably lower specific rotatory power: a (D) = — 37°. The principle of the preparation of lactalbumin is the same as for the preparation of seralbumin from serum. The casein and the globulin are removed by MgSO, in substance and the filtrate treated as previously stated (page 122). The occurrence of other albuminous bodies, such as albumoses and peptones, in milk has not been positively proved. These bodies are easily produced as laboration products from the other proteids of the milk. Such a laboration product is Millon'b and Comaille's lactoprotein, which is a mixture of a little casein with changed albumin, and albumose,* which is formed by the chemical operations. Milk-sugar, Lactose, 0„II„0„ + H,0. This sugar with the absorption of water can be split into two glucoses, dextrose and galactose. It yields mucic acid by the action of dilute nitric acid, besides other organic acids. Levulinic acid is formed, besides formic acid and humin substances, by the stronger action of acids. By the action of alkalies amongst other products we find lactic acid and pyrocatechin. Milk-sugar occurs, as a rule, only in milk, but it has also been found in the urine of pregnant women on stagnation of milk. According to the statements of Pappel and Eichmond ' the milk of the Egyptian buffalo does not contain milk-sugar, but a sugar which they call tewfikose. ' Zeitschr. f. physiol. Chem., Bd. 9. « L. c. ' See Hammarsten, Ueber das Laktoproteln. Nord. med. Arkiv., Bd. 8, No. 10; also Maly's Jahresber., Bd. 6, S. 13. ' * Journ. Chem, Soc, London, 1894, p. 754. MILK-SVOAB. 429* Milk-sugar occurs ordinarily as colorless rhombic crystals ■with 1 mol. of water of crystallization, which is driven off by slowly heating to 100° C, but more easily at 130-140° C. At 170° to 180° C. it is converted into a brown amorphous mass, lactocaramel, C,H,„0,. Milk-sugar dissolves in 6 parts cold and in 2.5 parts boiling water; it has a faint sweetish taste. It does not dissolve in ether or absolute alcohol. lbs solutions are dextrogyrate. The rotatory power, which on heating the solution to 100° C. becomes constant, is a (D) — -f- 52.5°. Milk-sugar combines with bases; the alkali combinations are insoluble in klcohol. Milk-sugar is not fermentable with pure yeast. It undergoes, on the contrary, alcoholic fermentation by the action of certain schizomycetes, and lactic acid is produced thereby. The prep- aration of milk- wine, " kumyss" from mare's milk and " kephir " from cow's milk is based upon this fact. Micro-organisms produce a lactic-acid fermentation in lactose, and this explains the ordinary souring of milk. Lactose responds to the reactions of grape-sugar, such as Moore's or Tkommer's, and the bismuth test, which will all be described in Chapter XV on the urine. It also reduces mercuric oxide in alkaline solutions. After warming with phenylhydrazin acetate it gives on cooling a yellow crystalline precipitate of phenyl- lactosazon, C„II„N^O,. It differs from cane-sugar by giving posi- tive reactions with Mooee's or Tkommee's and the bismuth test, and also that it does not darken when heated with anhydrous oxalic acid to 100° C. It differs from grape-sugar and maltose by its solubility and crystalline form ; but especially by its not fermenting with yeast and by yielding mncic acid with nitric acid. For the preparation of milk-sugar we make use of the by-product in the preparation of cheese, the sweet whey. The proteid is removed by coagulation with heat and the filtrate evaporated to a syrup. The crystals which separate after a certain time are recrys- tallized from water after decolorizing with animal charcoal. A pure preparation may be obtained from the commercial milk-sugar by repeated recrystallization. The quantitative estimation of milk- sugar may in part be performed by the polaristrobometer and partly by means of titration with Feeling's solution. 10 c. c. of Feeling's solution corresponds to 0.067 grm. milk-sugar in 0.5-1.5^ solution and boiling for 6 minutes (in regard to Feeling's solution and the titration of sugar, see Chapter XV). KiTTHAUSBN ' has found another carbohydrate in milk which is soluble in ' Jourii. f. prakt. Chem., N. F., Bd. 15. 430 MILK. water, non-crystallizable, whicli has a faint reducing action, and which yields on boiling with an acid a body having a greater reducing power. Landwbhk ' considers this as animal gum, and B^champ ^ as dextrin. According to J. Hbbz ' granules occur in milk, which act like starch with iodine and which are perhaps animal starch. The mineral bodies of milk will be treated in connection with its quantitative composition. The methods for the quantitative analysis of milk are very numerous, and as they cannot all be treated of here, we will giv^e the chief points of a few of the most trustworthy and most fre- quently employed methods. In determining the solids a carefully weighed quantity of milk is mixed with an equal weight of heated quartz sand, fine glass powder, or asbestos. The evaporation is first done on the water- bath and finished in a current of Carbon dioxide or hydrogen not above 100° 0. The mineral bodies are determined by ashing the milk, using ithe precautions mentioned in the text-books. The results obtained for the phosphoric acid are incorrect on account of the burning of phosphorized bodies, such as casein and lecithin. We must there- fore, according to Soldnee,' subtract 35^ from the total phosphoric acid found in the milk. The quantity of sulphate in the ash also depends on the burning of the proteids. In the determination of the total amount of proteids we make use of EiTTHAUSEif's * method, namely, precipitate the milk with copper sulphate. This method gives incorrect results because the copper hydroxide does not give up all its water of hydration on drying the precipitate, but only after ashing the same. The results for the proteids are therefore somewhat too high. I. Munk ' has modified this process in this wise, that he precipitates all the pro- teids by means of copper oxyhydrate at boiling heat and determines the nitrogen in the precipitate by means of Kjbldahl's method. This modification gives exacter results. The method of Puls ' and Stenbeeg ' consists in first diluting the neutralized milk with some water and then treating with alcohol until the mixture contains 70-85 vols, per cent alcohol. The pre- cipitate is collected on a filter, washed with warm 70^ alcohol, extracted with ether, dried, weighed, burnt, and the residue reweighed. The traces of proteid which remain in the filtrate and ' Pfliiger's Arch., Bd. 39 and 40. » Bull. soc. chim. (Ser. 3), Tome 6. » Chem. Ztg.,Bd. 16, S. 1594. * Landwirtbsch. Versuchsstat., Bd. 35. 5 Journal f. prakt. Chem., N. F., Bd. 15. • Virchow's Arch , Bd. 134. ' Pfliiger's Arch. , Bd. 13. ' Nord. med. Arkiv., Bd. 9; also Maly's Jahresber., Bd. 7, S. 169. ANALYSIS OF MILK. 4S1 wash-liquor are precipitated by tannic acid. 63^ of the tannic acid precipitate is considered as proteid, and this must be added to the proteid found directly. This method gives exact and good results, but is more complicated. According to Sebelien's ' method, 3-5 grms. of milk are diluted with an equal volume of water, a little common-salt solution added, and precipitated with an excess of tannic acid. The pre- cipitate is washed with cold water, and then the quantity of nitrogen determined by Kjeldahl's method. The total nitrogen found when multiplied by 6.37 (casein and lactalbumin contain both 16.7^ nitrogen) gives the total quantity of albuminous bodies. This method, which is readily performed, gives very good results. I. MuNK used this method in the analysis of woman's milk. In this case the quantity of nitrogen found must be multiplied by 6.34. According to Munk's analyses nearly ^ of the total nitrogen of cow's milk and -^ of woman's milk is derived from the extractives. To determi ne the casein and albumins separately we may make use of the method first suggested by Hoppe-Setlee and Tolmax- SCHEFF,' in which the casein is precipitated by magnesium sulphate. According to SEBBLiEiir,°the milk is diluted with its own volume of a saturated magnesium-sulphate solution, then saturated with the salt in substance, the precipitate filtered and washed with a saturated magnesium-sulphate solution. The nitrogen is deter- mined in the precipitate by KjbldahJv's method, and the quantity of casein determined by multiplying the result by 6.37. The quantity of lactalbumin may be calculated as the difference between the casein and the total proteids found. The lactalbumin may also be precipitated by tannic acid from the filtrate containing MgSO^ from the casein precipitate, diluted with water, and the nitrogen determined by Kjeldahl's method and the result multiplied by 6.87. The quantity of globulins in milk cannot be exactly determined. A minimum result can be obtained by first precipitating the casein completely by NaCl in substance, and then precipitating the globu- lins in the filtrate by magnesium sulphate (Sebelibbt). The casein may also be precipitated from the diluted milk by acetic acid and the globulin precipitated after neutralization by means of MgSO,. In these cases we obtain somewhat high results, because of the presence of traces of casein which remain behind. The/a^ is gravimetrically determined by thoroughly extracting the dried milk with ether, evaporating the ether from the extract, and weighing the residue. The fat may be determined by aerometric means by adding alkali to the milk, shaking with ether, and deter- mining the specific gravity of the fat solution by means of Soxhlbt's apparatus. In determining the amount of fat in a large number of ' Zeitschr. f. physiol. Chem., Bd. 13. ' Hoppe-Seyler, Med. chem. Untersuch., Heft 2. »L. c. 432 MILK. samples the lactocrit of De Laval may be used -with success. The milk is first mixed with an equal volume of a mixture of glacial acetic acid and concentrated sulphuric acid, warmed 7-8 minutes on the water-bath, the mixture placed in graduated tubes, and these in the centrifugal machine at + 50° C. The height of the layer of fat gives its quantity. The numerous and very exact analyses of NiLSON ' have shown that with milks containing small quantities of fat, below l.Sj^, the older corrections are unnecessary, and that this method gives excellent results if we use lactic acid treated with 5^ hydrochloric acid instead of the above mixture of glacial acetic acid and sulphuric acid. In determining the milk-sugar first the proteids are removed. For this purpose we precipitate either with alcohol, which must be evaporated from the filtrate, or by diluting with water, and remov- ing the casein by the addition of a little acid, and the lactalbumin by coagulation at boiling heat. The sugar is determined by titration with Fehling's or Knapp's solution (see Chap. XV) . The prin- ciple of titration is the same as for the titration of sugar in urine : 10 c. c. of Fbhliitg's solution corresponds to 0.0676 grm. milk- sugar; 10 c. c. of Knapp's solution corresponds to 0.0311-0.0310 grm. milk-sugar, when the saccharine liquid contains about i-1^ sugar. In regard to the modus operandi of the titration we must refer the reader to more complete works and to Chapter XV. Instead of the volumetric determinations the following steps may be taken: A measured quantity of the milk-sugar solution is treated with an excess of Fbhling's solution, boiled, the copper suboxide filtered and reduced in a current of hydrogen, and the metallic copper weighed. Soxhlet " has given a table which sim- plifies the calculations in such cases. The sugar may also be determined by the polariscope, and with ease, because the filtrates containing milk-sugar are generally color- less. The determination is quickly performed, but does not give exact results. The quantitative composition of cow's milk is naturally very variable. The average obtained by Konig ' is as follows in 1000 parts : Water. Solids. Casein. Albumin. Pats. Sugar. Salts 871.7 128.3 30.3 5.3 36.9 48.8 7.1 35.5 The quantity of mineral bodies in 1000 parts of cow's milk is, according to the analyses of Soldkeb,' as follows: K,0 1.72, Na,0 ' Maly's Jahresber., Bd. 21, S. 142. ' Journal f. prakt. Chem., 1880. ' Chemie der menschichen Nahrungs- uud Genussmittel, 3. Aufl. *L. c. COLOSTRUM. 433 0.51, CaO 1.98, MgO 0.30, P,0. 1.82 (after correction for the pseudonuclein), CI 0.98 grins. Bunge" foand 0.0035 grm. Fe,0,. According to Soldnee, the K, Na, and CI are found in the same quantities in whole milk as in milk-serum. Of the total phosphoric acid 36-56j^ is not simply dissolved and also 53-73^ of the lime. A part of this lime is combined with the caseiu; the remainder is found united with the phosphoric acid as a mixture of di- and tri- calcium pohsphate, which is kept dissolved or suspended by the casein. The ba^es are in excess of the mineral acids in the milk- serum. The excess of the first is combined with organic acids, which correspond to 3.6 p. m. citric acid (Soldnek"). The gases of the milk consist chiefly of CO,, besides a little N and traces of 0. Pfluger" found 10 vols, per cent CO, and 0.6 vol. per cent N, calculated at 0° C. and 760 mm. pressure. The variation in the composition of cow's milk depends on, several circumstances. The colostrum, or the milk which is secreted before calving and in the first few days after, is yellowish, sometimes alkaline, but often acid, of higher specific gravity, 1.046-1.080, and richer in solids than ordinary milk. The colostrum contains, besides fat- globules, an abundance of colostrum-corpuscles — nucleated granular cells 0.005-0.035 mm. in diameter with abundant fat-granules and fat-globules. The fat of colostrum has a somewhat higher melting- point and is poorer in volatile fatty acids than the fat from ordinary milk (NiLSOK*)' The quantity of cholesteria and lecithin is generally greater. The most apparent difference between it and ordinary milk is that colostrum coagulates on heating to boiling because of the absolute and relatively greater quantities of globulin and albumin it contains. The quantity of the first of these two albuminous bodies may indeed amount to several per cent (Sebe- LiEiT '). The composition of colostrum is very variable. KoNiG ' gives as average the following figures in 1000 parts: i/Vater. Solids. Casein. Albamin and Globulin. Fat. Sugar. Salts. 746.7 253.3 40.4 136.0 35.9 26.7 15.6 The constitution of milk is changed during lactation, and it ' Zeitschr. f. Biologie, Bd. 10. 2 L. c. ' Pfliiger's Arch., Bd. 2. * Maly's Jahresber., Bd. 17, S. 169. ^ Ibid., Bd. 18. S. 102. • L. I-. 434 MILK. becomes richer in casein but poorer in fat and milk-sugar. Tlie evening milk is richer in fat than the morning milk (Alex. Mullbe and EisE2srsTUCK; Nilson and others'). The breed of the animal also has a great influence on the milk. The- influence food exercises upon the composition of milk will be discussed in connection with the chemistry of the milk secretion. In the following we give the average composition of skimmed milk and dertain other preparations of milk : Water. Proteids. Fat. Sugar. Lactic Acid. Salts. Skimmed milk. 906.6 31.1 7.4 47.5 7.4 Cream 655.1 36.1 267.5 35.2 6.1 Buttermilk.... 902.7 40.6 9.3 37.3 3.4 6.7 Whey... 933.4 8.5 2.3 47.0 3.3 6.5 Ktjmyss and kkphik are obtained, as above stated, by the alcoholic and lactie-acid fermentation of the milk-sugar, the first from mare's milk and the last from cow's milk. Large quantities of carbon dioxide are formed thereby, and also the albuminous bodies of the milk are partly convened into albumoses and peptones, which increases the digestibility. The quantity of lactic acid in these preparations may be about 10-20 p. m. The quantity of alcohol varies from 10 to 35 p. ui. Milk from other Animals. Ooat's milk has a more yellowish color and another, more specific, odor than cow's milk. The coagulation obtained by acid or rennet is more solid and is harder than that from cow's milk. Shbbp'b milk is similar to goat's milk, but has a higher specific gravity and contains a greater amount of solids. Mabe's milk is alkaline and contains a casein which is not precipitated by acids in lumps or solid masses, but, like the casein from woman's milk, in fine flakes. This casein is only incompletely precipitated by rennet, and it is very similar also in other respects to the casein of human milk. According to Beil, * the casein from mare's and cow's milk is the same, and the different behavior of the two varieties of milk is due to different amounts of salts and to a differ- ent relation between the casein and the albumin. The milk of the ass is sim- ilar to human milk. The milk of carnivora, the bitch and cat, are acid in reaction and very rich in solids. The composition of the milk of these animals varies very much with the composition o f the food. To illustrate the composition of the milk of other animals the following figures, the compilation of KoNiG, will be given. As the milk of each variety of animals may have a variable composition, these figures may only be con- sidered as examples of the composition of mii of different kinds. Milk of the Water. Solids. Proteids. Fat. Sugar. Salts. Dog 754.4 24r).6 99.1 95.7 ' 31.9 7.3 Cat 816.3 183.7 90.8 83.3 49.1 5 8 Gnat 869 1 130.9 36.9 40.9 44.5 8.6 Sheep 833.0 165.0 57.4 61.4 39 6 6 6 Cow 871.7 128.3 35.5 36,9 48 8 71 Horse 900 6 99.4 18 9 10 9 66 5 31 Ass 900.0 100.0 21.0 13.0 63.0 3 Pig. .....833.7 167.3 60.9 64 4 40 4 10 6 Elephant.. 678.5 331.5 30.9 195.7 88.4 6.5 Dolphin'.. 468.7 513.3 437.5 4.6 ' See KOnig, 1. c, Bd. 1, S. 313, and Nilson, 1. c. « Studien ilber die Eiweissstoffie des Kumys und Kefir. St. Petersburg, 1886. (Ricker.) 5 Frankland, Chem. News, 1890, vol. 61. HUMAN MILK. 435 Haman Hilk. Woman's milk is amphoteric in reaction. According to Coueant' its reaction is relatively stronger alkaline than cow's milk, bat has nevertheless a lower absolute reaction for alkalinity as well as acidity. Coueant found between the tenth day and four- teenth month after confinement rather constant results. The alka- linity as well as the acidity were a little lower than in childbed. 100 c. c. of the milk had the same average alkalinity as 10.8 c. c. ttt; caustic soda and the same acidity as 3.6 c. c. — r acid. The 10 •' 10 relationship between the alkalinity and acidity was for woman's milk as 3 : 1, and in cow's milk as 3.1 : 1. Human milk also contains fewer fat-globules than cow's milk, but they are larger in size. The specific gravity of woman's milk varies between 1026 and 1036, generally between 1028 and 1034. According to Monti " the specific gravity of the milk from healthy, robust women is 1030-1035. The specific gravity is highest in well-fed and lowest in poorly fed women. The fat of woman's milk has been investigated by Ruppel.' It forms a yellowish, white mass, similar to ordinary butter, having a specific gravity of 0.966 at + 15° C. It melts at 34.0° and solidifies at 20.2° C. The following fatty acids can be obtained from the fat, namely, butyric, caproic, capric, myristic, palmitic, stearic, and oleic acids. The fat from woman's milk is, according to RUPPEL, relatively poor in volatile fatty acids. Laves ' found only traces of butyric acid in the fat from woman's milk. The melting- point of the fat was 30-31° and of the free fatty acids 37-39° C. The non-volatile fatty acids consist of one-half oleic acid, while among the solid fatty acids myristic ap-d palmitic acids are found to a greater extent than stearic acid. The essential qualitative difference between woman's and cow's milk seems to lie in the proteids or in the more accurately deter- mined casein. A number of older and younger investigators' claim ' Ueber die Beaktion der Kuh- und Frauenmilcli, etc. Inaug. Diss. Bonn, 1891; also Pflttger's Arch., Bd. 50. » Arch. f. Kinderheilkunde, Bd. 13. • Zeitsch. f. Biologie, Bd. 31. * Zeitschr. f. physiol. Chem., Bd. 19. ' See Biedert, Untersuchungen tiber die cbemischen Untersohiede dar 436 MILK. that the casein from woman's milk has other properties than that from cow's milk. The essential differences are the following : The casein from woman's milk is precipibated with greater difficulty with acids or salts; it does not coagulate regalarly in the milk after the addition of rennet; it may be precipitated by gastric juice, but dissolves completely and easily in an excess of the same; the casein precipitate produced by an acid is more easily soluble in an excess of the acid; and lastly, the clot formed from the casein of woman's milk does not appear in such large and coarse masses as the casein from cow's milk, but is more loose and flocculent. This last-men- tioned fact is of great importance, since it explains the generally admitted easy digestibility of the casein from woman's milk. The question as to whether the above-mentioned differences depend on a decided difference in the two caseins or only on an unequal relationship between the casein and the salts in the two varieties of milk, or upon other circumstances, has been recently investigated. According to Szontagh ' the casein from human milk does not yield any pseudonuclein on pepsin digestion and hence it cannot be a nucleoalbumin. Wroblbwski " has recently arrived at the same results and also found that the two caseins had a different composi- tion. He found the following for the composition of casein from woman's milk: C 52.34, H 7.33, N 14.97, P 0.68, S 1.117, 33.66^. Woman's milk also contains lactalbumin besides the casein and a protein substance which is very rich in sulphur (4.7^) and relatively poor in carbon (We6blb{vski). The statements as to the occurrence of albumoses and peptones are disputed as in many other cases. No positive proof as to the occurrence of albu- moses and peptones in fresh milk has been given. The quantitative composition of woman's milk is, even after those differences are eliminated which depend on the imperfect analytical methods employed, variable to such an extent that it is impossible to give any average results. Eliminating certain of the older, incorrect analyses, we here give only examples from the average results of a few modern investigators, taken from a very large number of analyses (Pfeiffeb). The following figures are parts per 1000 : Menschen- und Kuhmilch. Stuttgart, 1884. Langgaard, Vircliow's Arch., Bd. 65. Makris, Studieu fiber die EiweisskOrper der Fraueu- und Kuhmilch. Inaug. Diss. Strassburg, 1876. ' Maly's Jahresber., Bd. 22, S. 168. ' Beitrage zur Kenntniss des Frauenkasei'ns. Inaug. -Diss. Bern, 1894. C0MP08ITI0N OF MUMAN MILK. 437 «76.0 «91.0 872.4 «93.0 890.6 877.9 Solids. 124.0 109.0 127.6 108.0 109.4 122.1 Froteids. 22.10 23 60 17.91) 19 00 16.13 17.24 25.30 Fat. 38.10 25.60 33.00 43.20 32.38 29.15 38.90 Clioles- teriu. 0.32 Sugar. 60.90 55.60 53.90 59.70 57.94 59.92 55.40 Salts. 2.90 4.20 2.80 1.65 2.09 2.50 BlBL' t01.mat8chefp ' Geuber ' Christenn * |gigy^?-''J?(PFBI.EER» Mbndes de Leon ' Although the composition of woman's milk is very variable, and uotwithstauding that in a few cases higher resalts (about 40 p. m.) have been obtained, by later analyses, for proteid bodies, still it seems that woman's milk in general contains less proteid s and more sugar than cow's milk. The quantity of casein is not only abso- lutely but also relatively smaller in proportion to the quantity of albumin in woman's than in cow's milk. According to Schbibe ' the quantity of citric acid is smaller in woman's milk than in cow's milk. A further difference between woman's and cow's milk is that the first is richer in lecithin but poorer in mineral bodies, especially CaO and P,0, (it contains only \ and i, respectively, of the corre- .sponding quantity\of these mineral bodies in cow's milk). In regard to the quantity of mineral bodies in woman's milk the analyses of Bungb ° are most reliable. He analyzed the milk of a woman, fourteen days after delivery, whose diet contained very little common salt for four days previous to the analysis (A), and again three days later after a daily addition of 30 grms. NaCl to the food (B). Bukge found the following figures in 1000 parts of the milk: A B K,0 0.780 0.703 Na,0 0.332 0.2.57 CaO 0.328 343 MifO 0.064 0.065 Fe,Oa 0.004 0.006 P,0, 0.473 0.469 CI 0.438 0.445 ' Maly's Jahresber., Bd. 4, S. 168. ' Hoppe-Seyler, Med. chem. Untersuch., Heft 2. • Ball, de la soc. chim., Tome 23. < Maly's Jahresber.. Bd. 7, S. 171. ' Jahrb. f. Kinderheilkunde. Bd. 20; also Maly's Jahresber., Bd. 13. ' Ueber die Zusammensetzung der Frauenmilch. Inaug. Diss, der Univ. Heidelberg. 1881; also Maly's Jahresber., Bd. 12. ' Landwirthsch. Versuchsstat. , Bd. 39. • Zeilschr. f. Biologie, Bd. 10. 438 MILK. The relationship of the two bodies, potassium and sodinm, tO' each other may, according to Bukgb, vary considerably (1.3-4.4 equivalents potash to 1 of soda). By the addition of salb to the food the quantity of sodium and chlorine in the milk increases, while the quantity of potassium decreases. The gases of woman's, milk have been investigated by Ktjlz.' He found 1.07-1.44 c. c. oxygen, 2.35-2.87 c. c. carbon dioxide, and 3.37-3.81 c. c. nitrogen in 100 c. c. milk. The proper treatment of cow's milk by diluting with water and by certain additions in order to render it a proper substitute for woman's milk in the nourishment of babes cannot be determined before the difference in the albuminous bodies of these two kinds of milk has been completely studied. The colostram has a higher specific gravity, 1.040-1.060, a greater quantity of coagnlable proteids, and a deeper yellow color than ordinary woman's milk. Even a few days after delivery the color becomes less yellow, the quantity of albumin vless, and the number of colostrum-corpuscles diminishes. Clemm ' has analyzed the colostrum at different periods before and after delivery, and the following are his results in parts per 1000: Four Weeks before Delivery. Seventeen Days before Delivery. Nine Days before Delivery. Tweuty- lour Hours after Delivery.' Two Days 1 2 Delivery. Water Solids 943.2 54.8 853.0 148.0 851.7 148.3 858.5 141.5 843: 157.0 867.9 132 1 21 8 • 28.8 7.1 17. S 4.4 69.0 41.3 39.5 4.4 74.8 30.2 43.7 4.5 80.7 23.5 3S.4 5.4 "5.1' Fat 48 6 Milk-sugar Salts 61.0 The total quantity of proteids seems to decrease with the dura- tion of lactation. Pfeifpek' found the average figures for the total proteids for the two first days, the first week, the second week, the second month, and the seventh month to be 86.04, 34.42, 22.88, 18,43, and 15.21 p. m., respectively. Simon* claims that the amount of casein is smaller in the first stages of lactation and ' Zeitschr. f. Biologie, Bd. 32. ' Cited from Hoppe-Seyler's Physiol. Chem., p. 734. »L. c. * Die Frauenmilch. Berlin, 1838. HUMAN C0L08TBUM. 439 then increases considerably; but according to Pfeiffek just the reverse takes place. The amount of fat shows no regular and con- stant variation daring lactation. According to Vernois and Becquebel' the quantity of milk-sagar decreases in the first months, but increases in the eighth to the tenth month. Accord- ing to Pfeiffee the quantity of sugar increases regularly from the delivery to the third to fourth month, and then it is somewhat variable. The two mammary glands of the same woman may yield somewhat different milk, as shown by Sodbdat ' and later by Brunner.' Also the different por- tions of milk from the same milking may have different compositions. The first portions are always poorer in fat. According to l'Hbritibr,* Vbbnois, and Becquerel the milk of blonds contains less casein than that of brunettes, a difference which Tolmatscheff ' conld not substantiate. Women of weak constitutions yield a milk ricbpr in solids, especially in casein, than women with strong constitutions (V. and B.). According to Vernois and Becquerel, the age of the woman has an effect on the composition of the milk, so that we fiml a greater quantity of proteids and fat in women 15-20 years old and a smaller quantity of sugar. The small- est quantity of proteids and the greatest quantity of sugar are found at 20 or from 25-30 years of age. According to V. and B., the milk with the first-bom is richer in water — with a proportionate diminution of the quantity of casein, sugar, and fat — than after several deliveries. The influence of menstruation seems to slightly diminish the milk-sugar and to considerably increase the fat and casein (V. and B.). Witch's Milk is the secretion of the mammary glands of new-born, children of both sexes immediately after birth. This secretion has from a qualitative standpoint the same constitution as milk, but may show important differences' and variations from a quantitative point of view. Schlossberger and Hatjff,' Otjbler and Qdevenne,' and v. Genser' have made analyses of this milk and give the following results : 10.5-28 p. m. proteids, 8.2-14.6 p. m. fat, and 9-60 p. m. sugar. As milk is the only form of nourishment during a certain period of the life of man and mammals, it must contain all the nutritioua bodies necessary for life. This fact is shown by the milk-contain- ing representatives of the three chief groups of organic nutritive substances, proteids, carbohydrates, and fat; and all milk seems to contain also some lecithin. The mineral bodies in milk must also occur in proper proportion, and on this point the observations of ' Compt. rend., Tome 36, and Vernois et Becquerel, Du lait chez la femme dans I'etat de sante, etc. Paris, 1853. ' Compt. rend., Tome 71. » Pflttger's Arch., Bd. 7. * Traite de chim. pathol. Paris, 1842. Cited from Hoppe-Seyler's Physiol. Chem.. p. 738. ' Hoppe-Seyler, Med. chem. Untersuch., 8. 278. • Annal. de Chem. u. Pharm., Bd 96. ' Gaz. mfid. de Paris, 1856 p. 15. e Jahrb. f. Kinderheilkunde, N. F., Bd. 9, 8. 60. 440 MILK. BuNGE on dogs are of special interest. He found that the mineral bodies of the milk occur in about the same relative proportion as they do in the body of the sucking animal. Btjnge ' found in 1000 parts of the ash the following results (A represents resulbs from the new-born dog and B the milk from the bitch) : A B KjO 114 2 149.8 Na,0 106.4 88.0 CaO 395.2 372.4 MgO 18.2 15.4 Fe,0 7.3 1.3 PaOs 394.3 342.2 CI 83.5 169.0 BuNGE explains the fact that the milk-ash is richer in potash and poorer in soda than the new-born animal by saying that in the growing animal the ash of the muscles rich in potash relatively increases and the cartilage rich in soda relatively decreases. Bungb seeks to explain the high amount of chlorine in the milk-ash also teleologically by the statement that the chlorides not only serve to build up the tissues, but are indispensable in the secretions of the kidneys. In regard to the amount of iron we find an unexpected condition, the ash of the new-born animal containing six times as much as the milk-ash. This condition BuifGE explains by the fact founded on his and Zalesky's experimeuts, that the quantity of iron in the total organism is highest at birth. The new-born Minimal has therefore a supply of iron for the growth of its organs even at its birth. The influence of the food on the composition of the milk is of interest from many points of view and has been the subject of many investigations. Prom these investigations we learn that in human beings as well as in animals an insufiicient diet decreases the quantity of milk and the quantity of solids in the same, while abundant food increases both. From the observations of Decaisne ' on nursing women during the siege of Paris in 1871, the quantity of casein, fat, sugar, and salts, but especially the fat, was found to decrease with insuflBcient food, while the quantity of lactalbumin was found to be somewhat increased. Food rich in proteids increases the quantity of milk, and also the solids contained, espe- cially the fat. The quantity of sugar in woman's milk is found by certain investigators to be increased after food rich in proteids, 'Zeitschr. f. physiol. Chem., Bd. 18. ' Gaz. med. de Paris, 1871, S. 317; cited from Hoppe-Seyler, 1. c, S. 739. CUEMISTRY OF THE MILK 8E0BETI0N. 441 while others claim it is diminished. Food rich in fat may (ia sheep) cause an increase in the quantity of fat in the milk. An increase in the quantity of fat in cow's milk because of an addition of fat to the fodder has only been observed after a previous insuffi- cient diet, but not after a sufficient and rich diet. After feeding with palm-oil cake a one-sided increase in the fat of cow's milk was observed. The presence of large quantities of carbohydrates in the food seems to cause no constant, direct action on the quantity of the milk-constituents.' In carnivora, as shown by SstJBOTiN," the secretion of milk-sugar proceeds uninterruptedly on a diet consisting exclusively of lean meat. Watery food gives a milk containing an excess of water of little value. In the milk from cows which were fed on distillers' grains Commaille' found 906.5 p. m. water, 26.4 p. m. casein, 4.3 p. m. albumin, 18'.2 p. m. fat, and .33.8 p. m. sugar. Such milk has a peculiar sour, sharp after-taste. Chemistry of the Milk-secretion. That the actually dissolved constituents occurring in milk pass into the secretion, not alone by filtration or diffusion, but more likely are secreted by a specific secretory activity of the glandular elements, is shown by the fact that milk-sugar, which is not found in the blood, is to all appear- ances formed in the glands themselves. A further proof lies in the fact that the lactalbumin is not identical with seralbumin; and lastly, as Buuge * has shown, the mineral bodies secreted by the milk are in quite different proportions from those in the blood- serum. Little is known in regard to the formation and secretion of the specific constituents of milk. The older theory, that the casein was produced from the lactalbumin by the action of an enzyme, is incorrect and originated probably from mistaking an alkali-albumi- nate for casein. Better founded is the statement that the casein originates from the protoplasm of the gland-cells, which seem to ' In regard to the literature on the action of various foods on woman's milk, see Zalesky, " Ueber die Einwirkung der Nahrung auf die Zusammensetzung und Nahrhaftigkeit der Frauenmilch," Berlin, klin. Wochenschr., 1888, which also contains the literature on the importance of the food on the composition of other varieties of milk. In regard to the extensive literature on the influence of various foods on the milk production of animals, see KSnig, Chem. d. menschl. Nahrungs- und Genussraittel, 3. Aufl., Bd. 1, S. 298. 'Centralbl. f. d. med. Wissensoh., 1866, S. 337. » Cited from KSnig, Bd. 3, S. 235. * Lehrbuchd. physiol. und pathol. Chem., 1. Aufl., S. 98. 442 MILK. consist of casein or a substance related to it. The previously men- tioned (page 430) nucleoproteid of the gland-cells appears to be re- lated to casein, and it may possibly form its mother-substance. There does not seem to be any doubt that the protoplasm of the cells takes part in the secretion in such a manner that it becomes itself a con- stituent of the secretion. According to Heidenhaist,' the alveoles dontain a simple layer of cells, which, in the inactive gland, are' flat, polybedrous, and with single nucleus, while in the active gland they often have several nuclei, are rich in proteid, and are high and cylindrical in form. In the inner part of the cell turned towards the cavity of the acinus, single fat-granules are formed during the secretion which are broken off with the edge of the cells. The broken-off or destroyed cell-substance in the secretion dissolves in the milk, filling the lumen of the acinus, while the cells take up nutrition by their outer parts, and grow, and replace the inner parts used in the secretion. This reminds us of the action of the pancreas-cells in the secretion of the pancreatic juice. The colos- trum-corpuscles are not, according to Heidenhain, degenerated fat-cells, but are contractile elements originating from the epithe- lium, which take up finely divided fat and thereby obtain their quantity of fat-globules. That the milk-fat is produced by a formation of fat in the protoplasm, and that the fat-globules are set free by their destruc- tion, is a generally admitted opinion which, however, does not exclude the possibility that the fat is in part taken up by the glands from the blood and eliminated with its secretion. A formation of fat from carbohydrates in the animal organism is at the present day considered as positively proved, and it is also possible that the milk- glauds also produce fats from the carbohydrates brought to them by the blood. It is a well-known fact that an animal gives off for a long time, daily, considerably more fat in the milk than it receives as food, and this proves that at least a part of the fat secreted by the milk is produced from proteids or carbohydrates, or perhaps from both. The question as to how far this fat is produced directly in the milk-glands, or from other organs and tissues, and brought to the gland by means of the blood, cannot be decided. The origin of the milk-sugar is not known. Muntz ' calls attention to the fact that a number of very widely diffused bodies ' Hermann's Handbuch d. Physiol., Bd. 5, Thl. 1, S. 380. » Compt. rend., Tome 102. GHEMISTRT OF THE MILK BEGMETION. 443. in the vegetable kingdom — vegetable mucilage, gums, pectin bodiea — yfeld galactose as products of decomposition, and he believes, theJlfore, that the milk-sugar may be formed in herbivora by a synthesis from dextrose and galactose. This origin of milk-sugar does not answer for carnivora, as they produce milk-sugar when fed on food consisting entirely of lean meat. The observations of BEnr and Thierfeldee ' that a mother-substance of the milk-sugar, a saccharogen, occurs in the glands cannot, as the nature of this mother-substance is still unknown, give further explanation as to the formation of milk-sugar. The question whether the above- mentioned (page 420) proteid, which yields a reducing substance when boiled with dilute acids, has anything to do with the forma- tion of milk-sugar cannot be answered until further thorough inves- tigations have been made on this subject. The passage of foreign substances into the milk stands in close connection with the chemical processes of the milk-secretion. It is a well-known fact that milk acquires a foreign taste from the food of the animal, which is in itself a proof that foreign bodies pass into the milk. This fact becomes of special importance in reference to such injurious substances as may be introduced into the organism of the nursing child by means of the milk. Among these substances may be mentioned opium and mor- phine, which after large doses pass into the milk and act on the child. Alcohol may also pass into the milk, but not probably in such quantities as to have any direct action on the nursing child. "^ Alcohol is claimed to have been detected in the milk after feeding cows with brewer's grains. Among inorganic bodies, iodine, arsenic, bismuth, antimony,, zinc, lead, mercury, and iron have been found in milk. In icterus neither bile-acids nor bile-rpigments pass into the milk. Under diseased conditions no constant change has been found in woman's milk. In isolated cases Sohlossbekgbk/ Jolt and Filhol* have observed indeed a markedly abnormal composition, but no positive conclusion can be de- rived therefrom. The changes in cow's milk in disease have been little studied. In tubercu- losis of the udder Stobch ' found tubercule bacilli in the milk, and he also 'L. c. ' Klingemann, Virchow'a Arch., Bd. 186. • Annal. d. Chem. u. Pharm. , Bd. 96. « Cited from v. Gorup-Besanez, Lehrb., 4. Aufl., S. 438. • See Bang, Om Tuberkulose i Koens Yver og om tuberkulOs Mttlk. Nord.. med. Arkiv., Bd. 16; also Maly's Jahresber., Bd. 14, S. 170. 444 MILK. found that the milk became more and more diluted during the disease with a serous liquid similar to blood-serum, so that the glands finally, instead of yielding milk, only gave blood-serum or a serous fluid. HussoN ' founjMthe milk from cows sick with murrain contained more proteids but considafebly less fat and (in diflicult cases) less sugar than normal milk. '■-:^ The milk may be blue or red in color, due to the development of micro- organisms. The formation of concrements in the exit-passages of the cow's udder ar« •often observed. They consist chiefly of calcium carbonate, or of carbonate and phosphate with only a small amount of organic substances. ' Compt. rend.. Tome 73. CHAPTEE XV. THE URINE. The urine is the most important excretion of the animal organ- ism; it is the means of eliminating the nitrogenous metabolic products, also the water and the soluble mineral substances ; and in many cases it furnishes important data relative to the metabolism, quantitatively by its variation, and qualitatively by the appearance of foreign bodies in the excretion. Also in many cases we are able from the chemical or morphological constituents which the urine abstracts from the kidneys, ureters, bladder, and urethra to judge of the condition of these organs; and lastly, urinary analysis affords an excellent means of deciding the question how certain medicines or other foreign substances introduced into the organism are absorbed and chemically changed. Urinary analysis has furnished very important particulars especially relative to the last-mentioned question in regard to the nature of the chemical processes taking place within the organism, and it is therefore not only an important aid in diagnosis to the physician, but it is also of the greatest im- portance to the toxicologist and the physiological chemist. In studying the secretions and excretions the relationship must be sought between the chemical structure of the secreting organ and the chemical composition of its secreted products. Investigations with respect to the kidneys and the urine have led to very few . results from this standpoint. Although the anatomical relation of the kidneys has been carefully studied, their chemical composition has not been the subject of thorough analytical research. In cases in which a chemical investigation of the kidneys has been under- taken, it has only been in general on the organ as such, and not on the different anatomical parts. An enumeration of the chemical constituents of the kidneys known at the present time can, there- fore, only have a secondary value. 445 446 THE UBINE. In the kidneys we find albnmiaons bodies of different kinds. According to Hallibukton ' the kidneys do not contain any albumin, but only globulin and nucleoalbumin. The globulin coagulates at about 52° C. and the nucleoalbumin at 63° 0. The quantity of phosphorus in the latter is 0.37^. According to LiEBEBMANN " the kidueys contain leciihalbumin, and he ascribes to this body a special importance in the secretion of acid urines, namely, he claims that the lecithalbumin, which acts like an acid, decomposes in part the alkali-salts of the blood-plasma in the cells, -combining with the alkalies. Besides the above protein substances and the albuminoids of the connective-substance group, the kidneys contain a bodi/ similar to mucin. The question as to whether pure mucin really exists in the kidneys has not been decided. The body similar to mucin, which is a nucleoalbumin, and which gives no reducing substance when boiled with acids (Lonnbeeg"), belongs chiefly to the papillse, while the cortical substance is richer in a non-mucin-like nucleoalbumin. Fat occurs only in very small amounts in the cells of the tortuous urinary passages. Among the extractive bodies of the kidneys we find xanthin bodies, also urea, uric acid (traces), glycogen, leucin, inosit, taurin, and cystin (in ox-kidneys). The quantitative analyses of the kidneys thus far made possess little interest. Oidtmakk* found 810.94 p. m. water, 179.16 p. m. organic and 0.99 p. m. inorganic substance in the kidney of an old Tvoman. The fluid collected under pathological conditions, as in hydronephrosis, is thin with a variable but generally low specific gravity. Usually it is stravr- yellow or paler in color, and sometimes colorless. Most frequently it is clear, or only faintly cloudy from vehite blood-corpuscles and epithelium-cells ; in a few cases it is so rich in form-elements that it appears like pus. Proteids occur generally only in small amounts; sometimes it is entirely absent, and in a few rare cases the amount is nearly as large as in the blood-serum. Urea occurs sometimes in considerable amounts when tbe parenchyma of the kidneys is only in part atrophied; in complete atrophy the urea may be entirely absent. I. Physical Properties of Urine. Consistency, transparency, odor, and taste of urine. Urine is under physiological conditions a thin liquid and gives, when shaken with air, a froth which quickly subsides. Human urine or urine ' Journal of Physiol.. Vol. 13, Suppl. « Pfluger's Arch., Bdd. 50 nnd ,54. » See Maly's Jabresber., Bd. 20. * Cited from v. Gorup-Besanez, Lehrb., 4. Aufl., S. 733. PHYSICAL PBOPBETIEB OF URINE. 447 from carnivora, which is habitually acid, appears clear and trans- parent, often faintly fluorescent, immediately after voiding. When allowed to stand for a little while human urine shows a light cloud {nubecula) which consists of the so-called " mucus " and generally also contains a few epithelium-cells, mucus-corpuscles, and urate- grannles. The presence of a larger quantity of urates renders the urine cloudy, and a clay-yellow, yellowish-brown, rose-colored, or often brick-red precipitate (sedimentum lateritium) settles on cool- ing because of the greater insolubility of the urates at the ordinary temperature than at the temperature of the body. This cloudiness disappears on gently warming. In new-born infants the cloudiness of the urine during the first 4-5 days is due to epithelium, mucus- corpuscles, uric acid, and urates. The urine of herbivora, which is habitually neutral or alkaline in reaction, is very cloudy on account of the carbonates of the alkaline earths present. Human urine may sometimes be alkaline under physiological conditions. In this case it is made cloudy by the earthy phosphates, and this cloudiness does not disappear on warming, differing in this respect from the ^dimentum lateritium. Urine has a salty and faintly bitter taste produced by sodium chloride and urea. The odor of urine is peculiarly aromatic; the bodies which produce this odor are unknown. The color of urine is normally pale yellow when the specific gravity is 1.020. The color otherwise depends on the concentration of the urine and varies from pale straw-yellow, when the urine con- tains small amounts of solids, to a dark reddish yellow or reddish brown in stronger concentration. As a rule the intensity of the color corresponds to the concentration, but under pathological con- ditions exceptions occur, and such an exception is found in diabetic urine, which contains a large amount of solids and has a high specific gravity and a pale yellow color. The reaction of urine depends essentially upon the composition of the food. The camivora void an acid, the herbivora a neutral or alkaline, urine. If a carnivora is put on a vegetable diet, its urine may become less acid or neutral, while the reverse occurs when an lierbivora is starved, that is, when it lives upon its own flesh, as then the urine voided is acid. The urine of a healthy man on a mixed diet has an acid reac- tion, and the sum of the acid equivalents is greater than the sum of the base equivalents. This depends on the fact that in the physi- 448 THE URINE. ological combustion of neutral substances (proteids and others) "within the organism acids are produced, chiefly sulphuric acid, but also phosphoric and organic acids, such as hippuric, uric, and oxalic acid, also aromatic oxyacids and others. From this it follows that the acid reaction is not due to one acid alone. We do not know to what extent any one acid takes part in the acid reaction; but as the sum of the base equivalents is greater than, or at least the same as, the sum of the inorganic acid equivalents, the acid reaction mast be due in greatest part to organic acids or acid salts. It is generally considered that the acid reaction of human urine is caused by double-acid alkali-phosphate (monophosphate). The quantity of acid-reacting bodies or combinations eliminated by the urine in 34 hours, when calculated as oxalic acid or hydrochloric acid, is respectively 2-4 and 1.15-2.3 grms. The composition of the food is not the only influence which afEects the degree of acidity of human urine. For example, after taking food, at the beginning of digestion, when a larger amount of gastric juice containing hydrochloric acid is secreted, the urine may be neutral or even alkaline. The statements of various inves- tigators are rather contradictory in regard to the time of the appear- ance of the maximum and minimum of the acidity, which may in part be explained by the different individuality and different condi- tions of life of the persons investigated. It has not infrequently been observed that perfectly healthy persons in the morning void a neutral or alkaline urine which is cloudy from earthy phosphates. The effect of muscular activity on the acidity of urine has not been positively determined. According to Hoffmann ' and Ringstedt" muscular work raises the degree of acidity, but Aducco " claims that it decreases it. Abundant perspiration reduces the acidity (Hoffmann). In man and carnivora it seems that the degree of acidity of the urine cannot be increased above a certain point, even though mineral acids or organic acids which are burnt up with difficulty are taken in large quantities. When the supply of carbonates of the fixed alkalies stored up in the organism for this purpose is not sufficient to combine with the excess of acid, then ammonia is split ' Zar Semiologie des Earns. Inaug.-Diss. Berlin, 1884. See Maly's Jah- resber., Bd. 14, S. 313. « See Maly's Jahresber., Bd. 30, S. 196. ■ Ibid., Bd. 17, S. 179. DETERMINATION OF THE ACIDITY. 449 from the proteids or their decomposition products, and the excess of acid combines therewith, forming ammonium salts which pass into the urine. In herbivora this splitting of ammonia and forma- tion of ammonia salts does not seem to take place, and the herbivora therefore soon die when acids are given. Nevertheless the degree of acidity of human urine may be easily diminished so that the reaction is neutral or alkaline. This occurs after the taking of carbonates of the fixed alkalies or of such salts of vegetable acids — tartaric-acid, citric-acid, and malic-acid salts — as are easily burnt into carbonates in the organism. Under pathological conditions, as in the absorption of alkaline transudations, the urine may become alkaline (Quincke ') . The degree of acidity cannot be determined by the ordinary acidimetric process, since the urine contains di-hydrogen phosphate, MH,PO„ besides hydrogen di-phosphate, M,HPOj. In the titration . the di-hydrogen phosphate is changed gradually into M,HPO„ and we obtain at a certain point a mixture of the two phosphates in variable proportions, which mixture is not neutral but amphoteric. Since it is generally admitted that the acid reaction of urine is due to the di-hydrogen phosphate, it is therefore best to express the degree of acidity by the amount of di-hydrogen phosphate present. If we wish to calculate the degree of acidity of the urine as di-hydrogen phosphate or, still more simply, as phosphoric anhy- dride, P,Oj, contained in this salt, the titration is performed accord- ing to the method of Malt and Hoffmann,' which is as follows: The urine (100-200 c. c.) is treated with an exactly measured quantity of \ normal caustic-soda solution, which is more than suffi- cient to convert all the phosphate into basic phosphate, or, in other words, enough to make the urine strongly alkaline. Then an approximate f normal BaCl, solution (142.8 grms. BaCl^aH^O in a litre) is added until no further precipitate is formed. By this means all the phosphoric acid is precipitated from the urine. Filter through a dry filter, measure a quantity corresponding to 50 or 100' c. c. of the original urine from the filtrate, and titrate with J normal sulphuric acid until a neutral reaction is obtained, using litmus-paper as an indicator. If the amount found by this titration be subtracted from the original amount of caustic soda added to this; volume of uriue, the difference is the amount of caustic soda neces- sary to convert the existing di-hydrogen and hydrogen di -phosphates, into normal phosplr.ite. If we designate this by a, and the quantity of total P,0, in milligrammes in the same quantity of urine, as > Zeitsclir. f. klin. Med., Bd. 7, Suppl., 1884. ' Maly, Zeitschr. f. anal. CUem., Bd. 15, andF. Hoffmann, Arch. d. Heil- knnde, Bd. 17. 450 THE UBI2fE. ■determined by a method which will be described later, by g, then we obtain the quantity of P^O^ in milligrammes in the di-hydrogen ' phosphate s by the following formula: s = 17.75a — g. If, for example, in a case in which the conversion of both phos- phates into normal phosphate in 100 c. c. of the urine required 20. 0. c. caustic soda, while the total quantity of P^O^ in 100 c. o. urine was 375 milligrammes, then s = 17.75 X 20 - 375 = 80 milli- grammes. The quantity of P.,0, as simple acid phosphate was therefore 195 milligrammes. This method, according to Libblein,' gives too high figures for the di-hydrogen phosphate. The quantity of alkali used is too great because of the formation of basic barium phosphate. Lieb- JjEVSS recommends the following method as suggested by Fkbund.' Pirst determine the total quantity of phosphoric acid in the urine by means of titration with uranium solution and then precipitate the phosphoric acid existing as simple acid salts in another portion iby barium chloride, and determine the phosphoric acid remaining in & portion of the filtrate as monophosphate by titration with uranium solution. According to Libblein 10 c. c. of a normal bariam chloride solution (133 grm. BaCl„3H,0 in 1 litre) are used to precipitate each 100 milligrammes total phosphoric acid existing as simple acid salts, the filtrate made up to 100 c. c. and the phosphoric acid determined in 50 c. c. thereof. In the precipitation of the urine with BaCl^ about 3^ of the phosphoric acid existing as simple acid salts remains in solution as double acid salt, and hence a correspond- ing correction must be made. As one third of the phosphoric acid is combined with fixed bases as double acid salts, Libblein is of the opinion that in calculating the acidity of a urine only two thirds of this phosphoric acid is to be ascribed thereto. Frbund and Tobppbr^ have lately suggested a method for the determination of the acidity as well as the alkalinity of the urine by means of titration with — caustic soda, or — hydrochloric acid, using phe-. nolphthalein, sodium alizarin sulphonate, or a solutinn of Poiribb's blue as indicators. Lieblbik's ^ investigations do not speak in favor of this jnethod, A urine with an alkaline reaction caused by fixed alkalies has a very different diagnostic value from one whose alkaline reaction is caused by the presence of ammonium carbonate. In the latter case we have to deal with a decomposition of the urea of the urine by the action of micro-organisms. ' Zeitsehr. f. physiol. Ciiem,, Bd. 20. 5 Centralbl. f. d. med. Wissensch., 1892, S. 689. 8 Zeitsehr. f. physiol. Chem., Bd. 19, S. 84. * L. c. SPECIFIC GBAVITT OF URINE. 451 If we wish to determine whether the alkaline reaction of the urine is due to ammonia or fixed alkalies, we dip a piece of red litmns-paper into the urine and allow it to dry exposed to the air or to a gentle heat. If the alkaline reaction is due to ammonia, the paper becomes red again; but if it is caused by fixed alkalies, it remains blue. The specific gravity of urine, which is dependent upon the relationship existing between the quantity of water secreted and the solid urinary constituents, especially the urea and sodium chloride, may vary considerably, but is generally 1.017-1.030. After drink- ing large quantities of water it may fall to 1.002, while after profuse perspiration or after drinking very little water it may rise to 1.035- 1.040. In new-born infants the specific gravity is low, 1.007-1.005. The determination of the specific gravity is an important means of learning the average amount of solids eliminated from the organism with the urine, and on this account the determinatiou becomes of true value only when at the same time the quantity of urine voided in a given time is determined. The different portions of urine voided in the course of the 24 hours are collected, mixed together, the total quantity measured, and then the specific gravity taken. The determination of the specific gravity is most accurately obtained with the pyknometer. Eor ordinary cases the specific gravity may be determined with sufficieut accuracy by means of areometers. The areometers found in the trade, or urinometers, are graduated from 1.000 to 1.040; for exact observations it is better to use two urinometers, one graduated from 1.000 to 1.020, and the other from 1.020 to 1.040. A special urinometer is that of Heller, which is graduated according to Baume's. scale, from to 8. Each degree corresponds to 7 degrees of the ordinary urinometer, and as the zero-point of Hellee's urinometer corre- sponds to the figure 1000, then the 1, 1.5, 2, 2.5, 3, etc., degrees of Heller's urinometer correspond to 1.007, 1.0105, 1.014, 1.0176, 1.024, etc., of the ordinary specific gravity. To determine the specific gravity of urine, if necessary filter the urine, or if it contains a urate sediment, first dissolve it by gentle heat, then pour the clear urine into a dry cylinder, avoiding the formation of froth. Air-babbles or froth, when present, must be removed with a glass rod or filter-paper. The cylinder, which must be about | full, must be wide enough to allow the urinometer to swim freely in the liquid without touching the sides. The oyliuder and urinometer should both be dry or previously washed with the urine. On reading, the eye is brought on a level with the lower meniscus — which occurs when the surface of the liquid and the lower limb of the meniscus coincide; the reading is then made from. 452 THE URINE. the point where this curved line cuts the scale of the urinometer. If the eye is not in the same horizontal plane with the convex line of the meniscus, but is too high or too low, the surface of the liquid assumes the shape of an ellipse, and the reading in this position is incorrect. Before reading press the urinometer gently down into the liquid and then allow it to rise, and wait until it is at rest. If the quantity of urine at disposal is not sufficient to fill the cylinder to the proper height it may be diluted, according to cir- cumstances, with an equal volume or several volumes of water. This does not give quite accurate results, and with small quantities of urine it is best to determine the specific gravity by means of the pyknometer. Each urinometer is graduated for a certain temperature, which is marked on the instrument, or at least on the best. If the nriue is not at the proper temperature, the following corrections must be made: For every three degrees above the normal temperature one unit of the last order is added to the reading, and for every three degrees below the normal temperature one unit (as above) is sub- tracted from the specific gravity observed. For example, when a urinometer graduated for -f- 15° 0. shows a specific gravity of 1.017 at + 24° C, then the specific gravity at + 15° C. = 1.017 + 0.003 = 1.020. II. Organic Physiological Constituents of the Urine. t Urea, Ur, which is ordinarily considered as carbamid, C0(NH5)j, may be synthetically prepared in several different ways, namely, from carbonyl-chloride, or carbonic-acid ethyl-ether and ammonia, 0001, + 2NH3 = 00(NHJ, + 2HC1, or (C,H,),.0,.CO + 2NH. = 2(0,II,.0H) + 00(NHJ,; by the metameric decomposition of ammonium -cyanate, CO: KNH, = 00(]SrHJ, (Wohlee, 1828); and in many other ways. It is also formed by the decomposition or oxidation of certain bodies found in the animal organism, such as creatin and uric acid. Urea is found most abundant in the urine of carnivora and man, but in smaller quantities in that of herbivora. The quantity in human urine is ordinarily 20-30 p. m. It has also been found in small quantities in the urine of certain birds and amphibians. Urea occurs in the perspiration in small quantities, and as traces in the blood and in most of the animal fluids. It also occurs in rather large quantities in the blood, liver, muscle (v. SchroeSee ') and bile" of sharks. Urea is also found in certain tissues and organs of ' Zeitscbr. f. physiol. Chem., Bd. 14. ' Investigations not published 1 y tbe author. UBBA. 453 mammals, especially in the liver and spleen, and in smaller quanti- ties in the muscles. Under pathological conditions, as in obstructed ■excretion, urea may appear to a considerable extent in the animal fluids and tissues. The quantity of urea which is voided in 34 hours on a mixed diet is in a grown man about 30 grms., for women somewhat less. Children void absolutely less, but relative to their body-weight the excretion is larger than in grown persons. The physiological sig- nificance of urea lies in the fact that this body forms in man and •carnivora, from a quantitative standpoint, the most important ni- trogenous final product of the metabolism of proteid bodies. On this account the elimination of urea varies to a great extent with the amount of proteid transformed, and above all with the quantity •of absorbable proteids in the food taken. The elimination of urea is greatest after an exclusive meat diet, and lowest, indeed less than during starvation, after the consumption of non-nitrogenous bodies, for these diminish the metabolism of the proteids of the body. If the consumption of the proteids of the body is increased, then the elimination of urea is correspondingly increased. This is found to a rather high degree in certain diseases with fever: also in other cases of increased elimination of nitrogen, such as after poisoning with arsenic, antimony, and phosphorus, by a diminished supply of oxygen — as in severe and conliinuous dyspnoea, poisoning with carbon monoxide, hemorrhage, etc. — it used to be considered that it was due to an increased elimination of urea because no exact difference was made between the quantity of urea and the total quantity of nitrogen in the urine. Eecent researches have com- pletely demonstrated the untrustworthiness of these observations. Since Pflugek and Bohlan'D ' have shown that 16j^ of the total nitrogen of the urine exists under physiological conditions as other •combinations, not urea, attention has been called to the relative relationship of the different nitrogenous constituents of the urine to each other, and it has been found, under pathological conditions, that this relationship may vary very considerably, especially in regard to the urea. We have numerous estimations by different investigators, such as Bohland," E. Schultze,' Camekeb,* ' PflUger'3 Arcli., Bdd. 38 and 43. « Ibid., Bd. 43. > Ibid.. Bd. 45. « Zeitsolir. f. Biologie, Bdd. 34, 37, and 38. 454: THE URINE. VoGES,' MoKNER and Sjoqtist,' Gumlich,^ and others, on the relationship of the different nitrogenoas constituents t,o eacJi other in normal urine of adults. Sjoqtist ' has made similar estimations on new-bora babes from 1-7 days old. Prom all these analyses we obtain the following figures, A for adults and B for new-born babes.: Of the total nitrogen, we have: A B Urea 84-91^ 73-76 Ammonia 3-5 7.8-9.6 Uric acid 1-3 3.0-8.5 Remaining nitrogenous substances (extractives) 7-13 7.3-14.7 The different relationship between uric acid, ammonia, and urea nitrogen in children and adults is remarkable, since the nrine of children is considerably richer in uric acid and ammonia and con- siderably poorer in urea than the lirine of adults. In disease tli& proportion of the nitrogenous substances may be markedly changed and a decrease in the quantity of urea and an increase in the quan- tity of ammonia have been observed in certain diseases of the liver. This will be treated of in detail in connection with the formation of urea in the liver. It is natural that there is a diminished forma- tion of urea in diminished administration of proteids or diminished consumption of proteids. In diseases of the kidneys which disturb or destroy the integrity of the epithelium of the tortuous urinary passage the elimination of urea is considerably diminished. Formation of urea in the organism. The experiments to pro- duce urea directly from proteids by oxidation have not led to any positive results. On the contrary Drbchsel, as mentioned in Chapter II, has obtained lysin and lysatin as products of the hydrolytic cleavage of proteids and obtained urea from the lysatin. by the action of alkalies. According to Deechsel and Hedin' (see Chap. II, p. 31, and Chap. IX, p. 302) these two bodies are pro- duced by the hydrolytic cleavage of proteids by trypsin, and it is also possible that a part of the urea may be formed by a hydrolytic cleavage of proteids with these two bodies as intermediate steps. Creatin and creatinin, which are homologues of lysatin, are products of the destruction of proteid in the animal body and alsa ' Ueber die Mischung der sticlsstoilhaltigen Bestandtheile im Ham, etc. Inaug.-Diss. Berlin, 1893. Cited from Maly's Jahresber., Bd, 33. ' Slcand. Arch. f. Physiol., Bd. 3. See also Sj5qvist, Nord. med. Arkiv.» 1893, No. 36. * Zeitscbr. f . pbysiol Chem., Bd. 17. * Nord. med. Arljlv., 1894, No. 10. FORMATION OF UREA. 455 yield nrea by the action of alkalies, hence they may be steps in the formation of urea in the body. In the decomposition of proteid bodies we ordinarily obtain, as mentioned in Chapter II, amido-acids of various kinds, hence we consider the amido-acids as intermediate steps in the formation of urea from proteids. It has been shown that leucin and glycocoU (ScHULTZEN and Nencki,' Salkowski") and aspartfc acid (v. Knibkiem') may be in part transformed into urea within the organism. The nature of the chemical processes by which these transformations are effected is not positively known. Schmiede- BEEG claims that the nitrogenous combinations in which the nitrogen exists in the group NII,-CH, are decomposed in the organism with the formation of ammonia, and also that the am- monium carbonate is then converted by a synthesis into urea. The correctness of the last statement has been recently confirmed by. many investigators. Thus the researches of v. Kutieeiem,* Sal- KOWSKI,' FbDEK," I. MUNK,' GOBAKTDA,' SCHMIEDEBEKG and Fb.Walter," and Hallerwokden,'" on the behavior of ammonium salts in the animal body and the elimination of the ammonia under various conditions, have shown that the ammonium salts with strong acids act differently in the organism of carnivora and herbivora, while ammonium carbonate or such salts which are burnt into car- bonate in the organism are transformed into urea by carnivora as well as herbivora. The researches of v. Scheodee " have given an explanation as to the organ in which urea is formed. By passing blood which had been treated with ammonium carbonate or am- monium formate through a dog's liver he found a very considerable formation of urea, and these observations have been confirmed by the very careful observations of Salomon"." The formation of 'Zeitschr. f. Biologie, Bd. 8. ^ Zeitschr. f. pliysiol. Chem., Bd. 4. ' Zeitschr. f. Biologie, Bd. 10. *Ibid., Bd. 10. ' Zeitschr. f. physiol, Chem., Bd. 1. • Zeitschr. f. Biologie, Bd. 13. ' Zeitschr. f. physiol. Chem,, Bd. 3. « Arch. f. Path. u. Pharm., Bd. 13. 'Ibid., Bd. 7. ^0 Ibid., Bd. 10. " Ibid., Bd. 15. " Virchow's Arch., Bd. 97. 456 THE URINE. urea from ammoniam carbonate is to be considered as a synthesis with the expulsion of water. The formation of urea from amido-acids has been explained in other ways. Schultzen and Nencki' have expressed the view- that the amido-acids yield carbamic acid in the animal body, which tlien is transformed into urea. This view has later received farther support by more important observations. Drechsel " has shown that the amido-acids yield carbamic acids by oxidation in alkaline flaid outside of the organism, and he obtained urea from ammonium carbamate by passing an alternate electric current through its solu- tion, namely, by alternate oxidation and reduction. Deechsel has also been able to detect small quantities of carbamates in blood, and later in conjunction with Abel ' he detected carbamic acid in alka- line horse's urine. Deechsel therefore accepts the formation of nrea from ammoniam carbamate, and according to him the alternat- ing oxidation and reduction take place in the following way: H^N.O.OO.NH, + =H,N.O.CO.NH, + H,0 and H,]Sr.O.CO.NH, -f- H, = H^N.CO.NH, + H,0. Urea. Abel and Muiehead * have later observed an abundant elimina- tion of carbamic acid in human and dog's urine after the adminis- tration of large quantities of milk of lime, and finally the regular appearance of this acid in normal acid human and dog's urine has been made very probable by M. Nes^cki and HAHisr.' These last- mentioned investigators have also given very important support to the theory of the formation of urea from ammonium carbamate by , observations on dogs with Bck's fistula. In this case the portal vein is directly connected with the inferior vena cava, and a com- munication is thus established so that the blood of the portal vein flows directly into the vena cava, without passing through the liver. Nencki and Hahn observed violent symptoms of poisoning in dogs ' Zeitschr. f. Biologie,, Bd. 8. ' Ber. d. sachs. Gesellsch. d. Wissenscli, , 1875. See also Journ. f . prakt. Chem. (N. F.), Bdd. 13, 16, and 32. ' Da Bois-Reymoud's Arch., 1891, S. 336. * Arch. f. exp. Path. u. Pharm., Bd. 31. ' Hahn, Massen, Nencki et Pawlow, La fistula d'Eck de la veine cave in- ferieur et de la veiue porte, etc. Arch, des sciences biol. de St. Petersbourg, Tome 1, No. 4, 1893; also Arch. f. exp. Path. u. Pharm., Bd, 33, S, 161. FORMATION OF URBA. 457 after this operation, and. these symptoms were quite identical with those obtained on introducing carbamate into the blood. These symptoms also appear after the introduction of carbamate into the stomach, while the introduction of carbamate into the stomach of a normal dog had no action. As these observers also found that the urine of the dog on which the operation was made was richer in carbamate than that of the normal dog, they conclude that the symptoms were due to the non-transformation of the ammonium carbamate into urea in the liver, and they consider the ammonium carbamate as the substance from which the urea is derived in the liver of mammals. The view as to the formation of urea from ammoniam carbamate does not contradict the above statement as to the transformation of carbonates into urea, since we can imagine that the carbonate is first converted into carbamate with the expulsion of a molecule of water, and that this then is transformed into urea with the expulsion of a second molecule of water. Besides the above-mentioned theories as to the formation of urea we have others, which will not be given because the only theory which has thus far been positively demonstrated is the formation of urea from ammonium compounds in the liver. The question in which organ urea is formed has been the subject of numerous investigations. From the researches of numerous investigators, Pebvost and Dumas, Meissnek, Voit, Gbehant, GscHBiDLBN', Salkowski, and V. Schroder,' it has been found that the extirpation of the kidneys causes a considerable increase in the quantity of urea in the blood, and that the kidneys therefore, if they produce urea at all, are not the only organs which can pro- duce it. By experiments performed on the removed kidneys, which were analogous to the above-mentioned experiments on the removed liver, T. Schroder has shown that neither the kidneys nor the muscles nor the remaining tissues of the lower extremities of the dog have the property of forming urea from ammonium carbonate. The liver is the only organ where the formation of urea from ammonium compounds has been proved with certainty, and the question arises as to the importance of these compounds to the urea synthesis in the liver. Is all or the chief mass of the urea formed from ammonium compounds in the liver ? ' Arch, f exp. Path. u. Pharm., Bdd. 15 and 19. In regard to the above- cited researches and the older literature on this subject we refer the reader to V. Schroder, and also Voit, Zeitschr. f . Biologie, Bd. 4. 458 THE URINE. No satisfactory answer can be given at present to this question. If urea is formed from ammonium combinations in the lirer, then ■we can expect a diminished or reduced formation of urea and a corresponding increase in the elimiuation of ammonia in extirpa- tion of the liver. The normal relationship between ammonia and urea in the urine must in these cases be essentially changed. In order to demonstrate this, experiments have been made on animals, and the urine in men with liver disease has been ex- amined. The extirpa.tion and atrophy experiments on animals made by different methods by JSTbncki and Hahn,' Slosse," and Liebleik ' have shown that a rather marked increase of ammonia and a diminished elimination of urea take place after the operation, but also that there are cases in which, irrespective of the pronounced atrophy, an abundant formation of urea takes place and no appre- ciable if any change in the proportion of ammonia to the total nitrogen and urea is observed. The observations on human beings with diseases of the liver lead to similar results. In this regard the numerous investigations of Hallerworden,* Stadelmanist," Frankel," Pawitzki,' Morjstee and Sjoqvist," Gumlich," v. Nooeden,'" Weintraud," Munzer," and WiNTERBERG " and others on the urine in cirrhosis of the liver, acute yellow atrophy of the liver, and phosphorus poisoning, are available. We learn from these investigations that in certain cases the proportion of the nitrogenous substances may be so changed that urea is only 50-60^ of the total nitrogen, while in other cases, on the contrary, even in very extensive atrophy of the liver-cells, the formation of urea is not diminished, neither is the proportion ' L. c. » Du Bois-Reymond's Arch., 1890. 3 Arch. f. exp. Path. u. Pharm., Bd. 32. " Arch. f. exp. Path. u. Pharm,, Bd. 13. = Deatsch. Arch. f. klin. Med., Bd. 33. « Berlin klin. Wochenschr., Jahrg. 1878 and 1892. ' Deutsch. Arch. f. klin. Med., Bd. 45. 8 Skand. Arch. f. Physiol., Bd. 2; see also SjSqvist, Nord. med. Arkiv., Jahrg. 1893, No. 36. ' Zeitschr. f. physiol. Chem., Bd. 17. '» Lehrb. d. Pathol, des StofEwechsels, S. 387. " Arch. f. exp. Path. u. Pharm., Bd. 31. " Deutsch. Arch. f. klin. Med., Bd. 53. " Milnzer and Winterberg, Arch. f. exp. Path. u. Pharm., Bd. 83. PBOPERTIES AND REACTIONS OF UREA. 459 between the total nitrogen, urea, and ammonia essentially changed. Even in the cases in which the formation of urea was relatively diminished and the elimination of ammonia considerably increased we must not without further investigation assume a reduced abil- ity of the organism to produce urea. An increased elimination of ammonia may, as shown by Munzek in the case of acute phosphorus poisoning, be dependent upon the formation of abnormally large quantities of acids, caused by abnormal metabolism, and these acids require a greater quantity of ammonia for their neutralization. For the present we are not justified in the statement that the liver is the only organ in which urea is formed, and continued investigation only can yield further information as to the extent and importance of the formation of urea from ammonia compounds in the liver. Properties and Reactions of Urea. Urea crystallizes in needles or in long, colorless, four-sided, often hollow, anhydrous rhombic prisms. It has a neutral reaction and produces a cooling sensation on the tongue like saltpetre. It melts at 130-133° C, but decom- poses already at about 100° 0. At ordinary temperatures it dis- solves in equal weight of water and in five parts alcohol ; it requires one part boiling alcohol for solution; it is insoluble in alcohol-free ether and also in chloroform. If urea in substance is heated in a test-tube, it melts, decomposes, gives ofi ammonia, and leaves finally a non-transparent white residue which, among other sub- stances, contains also cyanuric acid and biuret, which dissolves in water, giving a beautiful reddish-violet liquid with copper sulphate and alkali (biuret reaction). On heating with baryta-water or caustic alkali, also in the so-called alkaline fermentation of urine caused by micro-organisms, urea splits into carbon dioxide and ammonia with the addition of water. The same decomposition products are produced when urea is heated with concentrated sul- phuric acid. An alkaline solution of sodium hypobromite decom- poses urea into nitrogen, carbon dioxide, and water according to the equation CON,H. + 3NaOBr = 3NaBr + CO, + 2H,0 + N,. With a concentrated solution of furfurol and hydrochloric acid urea in substance gives a coloration passing from yellow, green, blue to violet and then beautiful purple-violet after a few minutes 460 THE URINE. (Schiff's ' reaction). According to Huppert" the test is best per- formed by taking 2 c. c. of a concentrated furfurol solution, 4-6 drops concentrated hydrochloric acid, and adding to this mixture, ■which must not be red, a small crystal of urea. A deep violet coloration appears in a few minutes. Urea forms crystalline combinations with many acids. Among these the one with nitric acid and the one with oxalic acid are the most important. TJeea Nitrate, C0(NH,),.HN0,. On crystallizing quickly this combination forms thin rhombic or six-sided overlapping tiles, colorless plates, whose point has an angle of 83°. When crystal- lizing slowly, larger and thicker rhombic pillars or plates are obtained. This combination is rather easily soluble in pure water, but is considerably less soluble in water containing nitric acid ; it may be obtained by treating a concentrated solution of urea with an excess of strong nitric acid free from nitrous acid. On heat- ing this combination it volatilizes without leaving a residue. This compound may be employed with advantage in detecting smiill amounts "f urea. A drop of the concentrated solution is placed on a micro- scope-slide and the cover-glass placed upon it; a drop of uitric acid is then placed on the side of the cover-glass and allowed to flow under. The forma tion of crystals begins where the solution and the nitric acid meet. Alkali nitrates may crystallize very similarly to urea nitrate when they are contaui- inated with other bodies; therefore, in testing for urea, the crystals must be identified as urea nitrate by heatiuLf and by other means. Urea Oxalate, 3.C0(]SrHJj.H,C,0,. This compound is more sparingly soluble in water than the nitric-acid compound. It is obtained in rhombic or six-sided prisms or plates on adding a saturated oxalic-acid solution to a concentrated solution of urea. Urea also forms combinations with mercuric nitrate in variable proportions. If a very faintly acid mercuric-nitrate solution is added to a two-per-cent solution of urea and the mixture carefully neutralized, a combination is obtained of a constant composition which contains for every 10 parts of urea 72 parts mercuric oxide. This compound serves as the basis of Libbig's titration method. Urea combines also with salts, forming mostly crystallizable com- binations, as, for instance, with sodium chloride, with the chlorides of the heavy metals, etc. An alkaline but not a neutral solution of urea is precipitated with mercuric chloride. The method of preparing urea from urine is chiefly as follows: Concentrate the urine, which has been faintly acidified with sul- ' Ber. d. deutsch. chem. Gesellsch., Bd. 10. 2 Huppert-Neubauer, Analyse des Harnes, 10. Aufl., S. 296. ESTIMATION OF UUEA. 461 phuric acid, at a low temperature, add an excess of nitric acid, at the same time keeping tiie mixture cool, press the precipitate well, decompose it in water with freshly precipitated barium carbonate, dry on the water-bath, extract the residue with strong alcohol, decolorize when necessary with animal charcoal, and filter while warm. The urea which crystallizes on cooling is purified by recrystallization from warm alcohol. A further quantity of urea may be obtained from the mother-liquor by concentration. The urea is purified from contaminating mineral bodies by redissolving in alcohol-ether. If it is only necessary to detect the presence of urea in urine, it is sufficient to concentrate a little of the urine on a watch-glass and after cooling treat with an excess of nitric acid. In this way we obtain crystals of urea nitrate. Quantitative Estimation of Urea in urine. The methods sug- gested for this purpose are those of Liebig by titration, of Hbintz and Eagsky, also that of Kjeldahl, by which the total nitrogen is determined, and those of Bunsbn and Knop-Hufnbe and Mokner-Sjoqvist, where urea is intended to be determined as such. Among these methods, that of Liebig, which is perhaps the one most frequently employed by physicians, and that of Moener- Sjoqtist will here be carefully explained. In regard to the others, whose chief points only will be spoken of here, the student is referred to other text-books. Liebig's method is based upon the fact that a dilute solution of merouric nitrate under proper conditions precipitates all the urea, forming a compound of constant composition. As indicator, a soda solution or a thin paste of sodium bicarbonate is used. An excess of mercuric nitrate produces herewith a yellow or yellowish-brown combination, while the combination of urea and mercury is white. Peluger' has given full particulars of this method; therefore we «will describe Pflugee's modification of Liebig's method. As phosphoric acid is also precipitated by the mercuric-nitrate solution, this must be removed from the urine by the addition of a baryta solution before titration. Pfluger also suggested that the acidity produced by the mercury solution be neutralized during titration by the addition of a soda solution. The liquids necessary for the titration are the following: 1. Mercuric Nitrate Solution. This solution is calculated for a 2j^ urea solution, and 20 c. c. of the first should correspond to 10 0. c. of the latter. Bach c. c. of the mercury solution corresponds to 0.01 grm. urea. As a small excess of HgO is necessary in the urine to make the final reaction (with alkali carbonate or bicar- bonate) appear, each c. c. of the mercury solution must contain ' Pfliiger, and PflUger and Bohland, in PflUger's Arch., Bdd. 21, 36, 37, and 40. 462 TEE UBINB. 0.0773 instead of 0.0720 grm. HgO. The mercury solation con- tains therefore 77.3 grms. HgO in one litre. The solution may be prepared from J)ure mercury or mercuric oxide by dis- solving in nitric acid, Tlie soliiiion, freed as completely as possible from an excess of acid, is diluted by the careful addition of water, stirring meanwhile, until it has a specific gravity of 1.10 or a little higher at -|- 20° C. The solu- tion is standardized with a 2% solution of pure urea which has been dried over sulphur. c acid, and the operation conducted as will be described later. If the solution is too concentrated, it is corrected by the careful addition of the neces- sary amount of water, avoiding precipitation of basic salt, and titrating again. The solution is correct if 19.8 c. c. of it added at once to 10 c. c. of the urea solution and the necessary quantity of normal soda solution (11-18 c. c. or more) to u arly c impletely neutralize the liquid, gives the final reaction when exactly 20 c. c. of the mercury solution have been employed. 3. Baryta Solution. This consists of 1 vol. barium-nitrate and 3 vols, bari am- hydrate solation, bobh saturated at the ordinary temperature. ^ 3. Normal Soda Solution. This solution contains 53 grms. pure anhydrous sodium carbonate in 1 litre of water. According to Pfluger a solution having a specific gravity of 1.053 is sufficient. The amoant of this soda solution necessary to completely neutralize the acid set free during the titration is determined by titrating with a pure 3^ urea solation. To facilitate operations a table can be made showing the qaantity of soda solution necessary when from 10 to 35 c. c. of the mercury solation is used. Before the titration the following mast be considered. The chlorides of the urine interfere with the titration in that a part of the mercuric nitrate is transformed into mercuric chloride, which does not precipitate the urea. The chlorides of the urine are there- fore removed by a silver-nitrate solution, which also removes any bromine or iodine combinations which may exist in the urine. If the urine contains proteid in noticeable amounts, it must be removed by coagulation and the addition of acetic acid, but care must be taken that the concentration and the volume of the urine is not changed during these operations. If the urine contains ammonium carbonate in notable quantities, caused by alkaline fermentation, this titration method cannot be applied. The same is true of urine containing leucin, tyrosin, or medicinal preparations precipitated by mercuric nitrate. In cases where the urine is free from proteid or sugar and not specially poor in chlorides, the qaantity of urea, and also the approximate quantity of mercuric nitrate necessary for the titration, may be learned from the specific gravity. A specific gravity of 1.010 corresponds to about 10 p. m., a specific gravity of 1.015 generally somewhat less than 15 p. m., and a specific gravity of 1.015-1.030 about 15-30 p. m. urea. With a specific gravity higher than 1.030 the urine generally contains more than 30 p. m. of urea, and above this point the amount of urea increases much more rapidly than the specific gravity, so that with a specific ESTIMATION OF UREA. • 463 gravity of 1.030 it contains over 40 p. m. urea. Fever urines with a specific gravity above 1.030 sometimes contain 30-40 p. m. urea, or even more. Pkepakation for the Titeation. If a large amount of urea is suspected from a higii specific gravity, the urine must first be diluted with a carefully measured quantity of water, so that the amount of urea is reduced below 30 p. m. In a special portion of the same urine the amount of chlorides is determined by one of the methods which will be given later, and the number of c. c. of silver-nitrate solution necessary is noted. Then a larger quantity of urine, say 100 c. c, is mixed with one half or, if this is not sufficient to precipitate all the sulphuric and phosphoric acids, with an equal volume of the baryta solution; it is then allowed to stand a little while, and the precipitate is flkered through a dried filter. From the filtrate containing the urine diluted with water a proper quantity, corresponding to about 60 c. c. of the original urine, is measured, and exactly neutralized with nitric acid added from a burette, so that the exact quantity employed is known. The neutralized mixture of urine and baryta is treated with the proper quantity of silver-nitrate solution necessary to completely precipitate the chlorides, which was ascertained by a previous determination. The mixture containing a known volume of urine is now filtered through a dried filter into a flask, and from the filtrate an amount is measured corresponding to 10 c. c. of the original urine. Execution of the Titeation. Nearly the total quantity of mercuric-nitrate solution to be used, and which is known from the specific gravity of the urine, is added at once, and immediately afterwards the quantity of soda solution necessary, as indicated by the table. If the mixture becomes yellowish in color, then too much mercury solution has been added and another determination must be made. If the test remains white, and if a drop taken out and placed on a glass plate with a dark background and stirred with a drop of a thin paste of sodium bicarbonate does not give a yellow color, the addition of mercury solution is continued by adding | and then -^ c. c, and testing after each addition in the following way : A drop of the mixture is placed on a glass plate with a dark background beside a small drop of the bicarbonate paste. If the color after stirring the two drops together is still white after a few seconds, then more mercury solution must be added; if, on the contrary, it is yellowish, then — if not too much mercury solution has been added by inattention — the result to ^ c. c. has been found. By this approximate determination, which is sufficient in many cases, we have fixed the minimum amount of mercury solution necessary to add to the quantity of urine in question, and we now proceed to the final determination. A second quantity of the filtrate, corresponding to 10 c. c. of the original urine, is filtered, and the same quaiitity of mercury solution added at one time as was found necessary to produce the 464 . THE URINE. final reaction, and immediately after the corresponding amount of soda solution, which must not indicate the end of the reaction. Then add the mercury solution in ^ c. c. without neutralizing with soda, until a drop taken out and mixed with the soda solution gives a yellow coloration. If this final reaction is obtained after the addition of 0.1-0.3 c. c, then the titration may he considered as finished. If, on the contrary, a larger quantity is necessary, the addition of the mercury solution must be continued until a final reaction is obtained with simple carbonate, and the titration repeated again, adding the quantity of mercury solution used in the jirevious test at one time, and also adding the corresponding amount of soda solution. If we obtain the end reaction by the addition of i'„ c. c, we may consider the titration as finished. If in each titration a quantity of filtrate containing urine and baryta corresponding to 10 c. c. of the original urine is used, then the calculations are very simple, since 1 c. c. of mercuric-nitrate solution corresponds to 0.01 grm. of urea. As the mercury solution is made for a %% urea solution, the filtrate of urine and baryta being generally deficient in urea (if the quantity of urea is above 3^^, it is easy to avoid any mistake by dilating the urine at the beginning of the operation), a mistake occurs here which can be corrected in the following way, according to Pflugee: To the measured volume of the filtrate from the urine (the filtrate with baryta after neutraliza- tion with nitric acid, precipitation with silver nitrate and filtration) the quantity of normal soda solution employed is added, and from this sum the volume of mercury solution used is subtracted. The remainder is then multiplied by 0.08, and the product subtracted from the number of c. c. of mercury solution used. For example, if the filtrate (urine and baryta -|- nitric acid -j- silver nitrate) measured 35.8 c. c, and the number of c. o. of soda solution used in the titration 13.8 c. c, and the mercury solution 30.5 c. c, we have then 30.5 - {(39.6 - 20.5) X 0.08} = 20.5 - 1.53 = 18.97, and the corrected quantity of mercury solution is therefore 18.97 c. c. If the measured c. c. of the filtrate (in this case 35.8 c. c.) corresponds to 10 c. c. of the original urine, then the amount of urea is 18.97 X 0.01 = 0.1897 = 18.97 p. m. urea. Besides the urea other nitrogenous constituents of the urine are precipitated by the mercury solution. In the titration we really do not obtain the quantity of urea, but, as Pelugee has shown, the total quantity of nitrogen in the urine expressed as urea. As urea contains 46.67 p. c. IST, the total quantity of nitrogen in the urine may be calculated from the quantity of urea found. The results obtained by Libbiq-Pplugee's titration method for the total nitrogen, Pflugee has shown, correspond well with the ESTIMATION OF UREA. 465 results obtained by Kjbldahl's ' method, which was first (1860) used by Almen ' for urea deteriniQatious, and modified by Pfluger and BoHLAND.' This method consists in heating the urine a few hours with an excess of concentrated or fuming sulphuric acid (5 c. c. urine and 40 c. c. sulphuric acid) until all the nitrogen has been converted into ammonia, and after the addition of an excess of caustic soda the ammonia is distilled into — sulphuric acid and the amount of ammonia determined by titration. Bxjnsbn's ' Ukea Detbemination-. The principle of this method consists in heating the urine or urea solution in a sealed glass tube to a high temperature with an alkaline barium-chloride solution. The urea splits into carbon dioxide and ammonia, which may be determined separately. This method has been very care- fully tested by Pflugee and his pupils Bohland and Blbibtbeu," and essentially improved. They fonnd that very accurate results can be obtained by this me'thod if the other nitrogenous constituents of the urine are first precipitated by a mixture of hydrochloric acid and phospho-tungstic acid, and then the filtrate made faintly alkaline with milk of lime, and lastly heated with alkaline barium- chloride solution in a sealed tube. The carbon dioxide and the ammonia can be determined (by distilling with magnesia and receiv- ing the distillate in — acid and titrating). In the last case a cor- rection must be made (according to Schlosin&'s method) for the ammonia pre-existing in the urine. Pflugek and Bleibteeu have essentially changed this method in the following way: They pre- cipitate the other nitrogenous urinary constituents with hydro- chloric acid and phospho-tungstic acid, make the filtrate faintly alkaline with milk of lime, determine the pre-existing ammonia iu a part of this filtrate according to Schlosing's method (observing certain precautions), and then placing the other part of the filtrate (about 15 c. c.) in a large flask which contains 10 grms. crystallized phosphoric acid, heat to 230-260° C. for about three hoars. All the urea is decomposed, and the ammonia split off combines with ' Zeitschr. f. anal. Cliem., Bd. 32; also Wilfartb, Chem. Centralbl., 1885, and Argutinsky, Pflilger's Arcli., Bd. 46. ' Aug. Almen, Om urinafsOndriug och Uraemle. Dissert. XJpsala, 1860. ' Pflilger's Areh., Bdd. 35, 36, and 44. * Anal. d. Cbem. u. Phariu. , Bd. 65. s Pflilger's Arcli., Bdd. 38, 43, and 44. 466 THE URINE. the phosphoric acid. After cooling, an excess of caustic soda is added and the ammonia distilled into a titrated acid, which must then be retitrated. After subtracting the quantity of pre-existing ammonia very accurate results are obtained for the ammonia orig- inating from the nrea (and perhaps from an unknown ureid present in the urine). KiiTOP-HtiFKBE's METHOD ' is based on the fact that urea by the action of sodium hypobromite splits into water, carbon dioxide (which dissolves in the alkali), and nitrogen, whose volume is measured (see page 459). This method is less accurate than the preceding ones, and therefore in scientific work it is discarded. It is of value to the physician and for practical purposes because of the ease and rapidity with which it may be performed, even though it may not give very accurate results. For practical purposes a series of difEerent apparatus have been constructed to facilitate the use of this method." Among these Bsbach's ureomeier deserves to be especially mentioned. In regard to the Veagents necessary for the determination of urea, and also for instructions in the use of this instrument, we must refer the reader to the directions accompany- ing the apparatus. For pure urea solations Esbaoh's apparatus gives qaite exact results. The determination of urea in urine by this method always gives results somewhat too low, and as a rule a result is obtained which on an average is about 0.1^ lower than that obtained with Liebig's titration method. Moenek-Sjoqvist Mbthow." According to this method the nitrogenous constituents of the urine, with the exception of the urea and ammonia, are first precipitated by alcohol-ether after the addition of a solution of barium chloride and barium hydrate and then the urea determined in the concentrated filtrate, after driving off the ammonia, by Kjeldahl's nitrogen estimation. Tbe procedure is as follows: Mix 5 c. c. of the urine in a flask with 5 c. c. saturated Bad, solution, in which 5fc barium hydrate is dissolved. Then add 100 c. c. of a mixture of two parts 97^ alcohol and 1 part ether and allow this to stand in the closed flask overnight. The precipitate is filtered off and washed with alcohol- ether. The alcohol and ether is removed from the filtrate by dis- ' Knop, Zeitsclir. f. analyt. Chem., Bd. 9; HUfuer, Jour. f. prakt. Chem. (N P.), Bd. 3. See also Huppert-Neubauer, 10. Aufl. ^ S '6 Huppert-Neubauer. 3 Skand. Arch. f. Physiol., Bd. 3. OREATININ. , 467 tillation at about 65° C. (not above 60° C). When the liquid is reduced to about 25 c. c. a little water and calcined magnesia are added and the evaporation continued until the vapors are no longer alkaline in reaction, which generally is found before it is concen- trated to 15-10 c. c. This concentrated liquid is transferred into a proper flask by the aid of a little water, treated with a few drops of concentrated sulphuric acid and further concentrated on the water- bath. Now 20 c. c. pure concentrated sulphuric acid are added and the process carried out according to Kjbldahl. According to BoDTKER ' the addition of magnesia is unnecessary, and it is best to avoid it entirely as it easily leads to a small loss of urea. This exact method is to be recommended. Carbamic Acid, HjN.COOH. This acid is not known in the free state, but only as salts. Ammonium carbamate is produced by the action of dry ammo- nia on dry carbon dioxide. Carbamic acid is also produced by the action of potassium permanganate on proteid and several other nitrogenous organic bodies. We have already spoken of the occurrence of carbamic acid in human and animal urines in connection with the formation of urea. The calcium salt, which is soluble in water and ammonia but insoluble in alcohol, is most im- portant in the detection of this acid. The solution of the calcium salt in water becomes cloudy on standing, but much quicker on boiling, and calcium car- bonate separates. Carbamic acid ethylester (urethan), as shown by Jafpe,' may pass, by the mutual action of alcohol and urea, into the alcoholic extract of the urine when working with large quantities of urine. Creatinin, C,H,N,0, or NH : C^^-prr \ /,tt > is generally con- sidered as the anhydride of creatin (see page 366) foand in the muscles. It occurs in human urine and in that of certain mam- malia. It has also been found in ox-blood, milk, though in very small amounts, and in the flesh of certain fishes. According to Johnson " a creatinin occurs in fresh ox-flesh which differs from that occurring in urine and from which the creatin of the muscles is formed by bacterial action. The quantity of creatinin in human urine is for a grown man, voiding a normal quantity of urine in the 24 hours, 0.6-1.3 grms. (Neubauee *), or on an average 1 grm. The quautity is dependent on the food, and decreases in starvation. Sucklings do not gen- erally eliminate any creatinin, and it only appears in the urine whea ' Zeitaehr. f. physiol. Chem., Bd. 17. » Ibid., Bd. 14. » Proc. Koy. Soc, Vol. 50. Cited from Maly'a Jahresber., Bd. 22. * Huppert-Neubauer, Harnanalyse, 10. Aufl., S. 387. 468 . THE URINE. the milk is replaced by other food. The quantity of creatinin in urine varies as a rule with the quantity of urea, although it is increased more by flesh (because the flesh contains creatin) than by proteid. Grocco ' and Moitessibe ' claim that the elimination of creatinin is increased by muscular activity. The behavior of creatinin in disease is little known. By increased metabolism the amount is increased, while by decreased exchange of material, as in ansemia and cachexia, it is diminished. Creatinin crystallizes in colorless, shining monoclinic prisms- which differ from creatin crystals in not becoming white with loss of v/ater when heated to 100° C. It dissolves in 11.5 parts cold water, but more easily in warm water. It requires nearly 100 parts- cold absolute alcohol for solution,' but it is more soluble in warm alcohol. It is nearly insoluble in ether. In alkaline solution creatinin is converted into creatin very easily on warming. Creatinin gives an easily soluble crystalline combination with hydrochloric acid. A solution of creatinin acidified with mineral acids gives crystalline precipitates with phospho-tungstic or phospho-molybdic acids even in very dilute solutions (1 : 10,000) (Keener,^ Hofmeister'). It is precipitated, like urea, by mercuric-nitrate solution. Among the compounds of creatinin, that with zinc chloride, creatinin zinc-chloride, (C,H,NjO),ZnClj, is of special interest. This combination is obtained when a sufficiently concentrated solution of creatinin in alcohol is treated with a con- centrated, faintly acid solution of zinc chloride. Free mineral acids dissolve the combination, hence they must not be present; this, however, may be prevented, when they are present, by an addition of sodium acetate. In the impure state, as ordinarily obtained from urine, creatinin zinc chloride forms a sandy, yellowish powder which under the microscope appears as fine needles forming concen- tric groups, mostly complete rosettes or yellow balls or tufts, or grouped as brushes. On slowly crystallizing, or when very pure, more sharply defined prismatic crystals are obtained. This com- bination is sparingly soluble in water, Creatinin acts as a reducing agent. Mercuric oxide is reduced ' See Maly's Jahresber., Bd. 16, S. 199. = Compt. rend. soc. biol., Tome 43. Cited from Maly's Jahresber., Bd. 21. ' This statement is taken from Huppert-Neubauer's book. Hoppe-Seyler's Handb., 6- Aufl., S. 144, gives other figures. •• Pflilger's Arch., Bd. 2, S. 220. ' Zeitschr. f. physiol. Chem. , Bd. 5. PBOPEBTIES AND REACTIONS OF OBEATININ. 469 to metallic mercury, and oxalic acid and methylguanidin (methyl- uramin) are formed. Creatinin also reduces copper hydroxide in alkaline solution, forming a colorless soluble combination, and only after continuous boiling with an excess of copper salt is free sub- oxide of copper formed. Creatinin interferes with Teommer's test for sugar, partly because it has a reducing action and partly by retaining the copper suboxide in solution. The combination with Ibid., Bd. 10. « Ann. d. Chem. u. Pharm., Bd. 119. 470 THE URINE. with CaGl, solation until all the phosphoric acid is precipitated; it is filtered and washed with water, the filtrate and the wash-water nuited, and evaporated to a syrup after acidifying with acetic acid. This syrup is mixed while hot with 60 c. c. of 95-97^ alcohol. This mixture is transferred to a beaker, and the residue in the evaporating-dish is completely and carefully removed and added. The liquid is allowed to stand covered for at least eight hours in the cold. Then it is filtered through a small filter, the precipitate washed with alcohol, the filtrate evaporated if necessary until the volume is 50-60 c. c, then allowed to cool and ^ c. c. of an acid- free zinc-chloride solution of a specific gravity ofyl.20 is added; it. is stirred, and the covered beaker is left standing in a cool place for two or three days. The precipitate is collected on a small dried and weighed filter, using the filtrate to wash the crystals from the beaker. After allowing the crystals to completely drain ofE, they are washed with a little alcohol until the filtrate gives no reaction for chlorine, and dried at 100° 0. 100 parts creatinin zinc-chloride contain 63.44 parts creatinin. As the precipitate is never qnita pure, the quantity of zinc must be carefully determined, in exact experiments, by evaporating with nitric acid, heating, washing the oxide of zinc with water (to remove any NaCl), drying, heating, and weighing. 22.4 parts zinc oxide correspond to 100 parts- creatinin zinc chloride. The preparation of creatinin zinc chloride on a large scale from urine is done in the same way. The creatinin is obtained from the creatinin zinc chloride by boiling with lead hydroxide, filtering, decolorizing the filtrate with animal charcoal, evaporating, treating the residue with strong alcohol (which leaves the creatin undis- solved), evaporating to crystallization, redissolving in water, and recrystallizing. In regard to the modifications of Neubaube's method for the quantitative estimation of creatinin the reader is referred to Sal- KOWSKi.' Kolisch" has given a new method for estimating creatinin in urine which consists in precipitating the creatinin from the alcoholic extract by an alcoholic solution of mercuric chloride acidified with acetic acid. The nitrogen is exactly determined in the carefully washed precipitate by Kjbldahl's method. Kolisch uses the following solution as precipi tant : 30 parts mercuric chlo- ride, 1 part sodium acetate, 3 drops glacial acetic acid, and 125 parts, absolute alcohol. Xanthocreatinin, CtHioN.O. This body, which was first prepared from meat extract by Gautibk,' has been found by Monabi* in dog's urine after ' Zeitschr. f. physiol Chem., Bdd. 10 and 14. ' Centralbl. f. innere Medizin, 1895. ^ Bull, de I'Acad. de med. (2), Tome 15, and Bull, de la soc. chim., Toma 48. « See Maly's Jahresber., Bd. 17, S. 182. URIC ACID. 471 the injection of creatiniii into the abdominal cavity, and in human urine after several hours of exhausting marches. According to Colasanti" it occurs to a relatively greater extent in lion's urine. Stadtha&en' considers the xanthocreatinin, isolated from human urine after strenuous muscular activity, as impure creatinin. Xanthocreatinin forms sulphur-yellow thin plates, similar to cholesterin, which have a Ijitter taste. It dissolves in cold water and in alcohol, and gives a crystalline combination with hydrochloric acid and a double compound with gold and platinum chloride. It gives a combination with zinc chloride, which crystallizes in fine needles. Xanthocreatinin has a poisonous action. Uric Acid, Ur, C.H.N.O,. The structural formula of this acid, /NH.C.NHX according to Medicus, is C0<[ C.NH/'^^' ^^^ ^^"^ ^"^^ \HN.CO may therefore he considered, from its constitution as a derivative of acrylic acid, as acrylic acid diureid. Uric acid has been synthetically prepared by Horbaczewski ' in several ways. On fusing urea and glycocoU, uric acid is formed according to the formula 3C0N,H, + C,H.NO, = C^H.N^O, + 3H,0 + 3NH3, and in this reaction hydantoin and biuret are formed as intermediate products. On melting methylhydantoin with urea or methylhydantoin with biuret or with allophanic-acid amyl-ester Hokbaczewski obtained methyl-uric acid. He also obtained uric acid on heating triphlor-lactic acid, or still better trichlor-lactic acid-amid, with an excess of nrea. If we eliminate from the reaction the numerous by-products (cyanuric acid, carbon dioxide, etc.), then this process may be expressed by the formula. C,C1.H,0,N + aCON.H. = O.H.N.O. + H,0 + NH.Cl -f 2HC1. On strongly heating uric acid it decomposes with the formation of UKBA, HTDKOCTAKIC ACID, OTANURIC ACID, and AMMONIA. On heating with concentrated hydrochloric acid in sealed tubes to 170° C. it splits into gltcocoll, carbon dioxide, and ammonia. By the action of oxidizing agents a splitting and oxidation takes place, and either monoureid or diureid is produced. By oxidation with lead peroxide, carbon dioxide, oxalic acid, urea, and ALLANTOiN, which last is glyoxyldinreid, are produced (see below). ' Arch. ital. de Biologie, Tome 15. ' Zeitschr. f. klin. Med. , Bd. 15. ' Monatshefte f. Chem., Bdd. 6 and 8. See also Behrend and Boosen, Her. d. deutsch. chem. Oesellsch., Bd. 21, S. 999. 472 THE URINE. By oxidatioa with nitric acid in the cold ukea and a monoureid, the mesoxalyl urea or ALLOXAif, are obtained, O.H.N.Oj + + H,0 = C^H^N^O, + (NHJ,CO. On warming with nitric acid, .alloxan yields carbon dioxide, and oxalyl nrea or pakabakic acid, CjHjNjO,. By the addition of water the parabanic acid passes into ■OXALUKic ACID, CjH^NjO,, traces of which are found in the nrine and which easily split into oxalic acid and urea. Uric acid occurs most abundantly in the nrine of birds and of scaly amphibians, in which animals the greater part of the nitrogen of the urine appears in this form. Uric acid occurs frequently in the nrine of carnivorous mammalia, bat is sometimes absent; in nrine of herbivora it is habitually present, though only as traces; in human nrine it occurs in greater but still small and variable amounts. Traces of uric acid are also found in several organs and tissues, as in the spleen, lungs, heart, pancreas, liver (especially in Mrds), and in the brain. It habitually occurs in the blood of birds ((Meissnbk'). Traces have been found in human blood under ^normal conditions (Abbles"). Under pathological conditions it occurs to an increased extent in the blood in pneumonia (v. Jaksch '), but also in leucamia and arthritis. Uric acid also ■occurs in large quantities in "chalk-stones," certain urinary •calculi, and in guano. It has also been detected in the urine of insects and certain snails. The amount of uric acid eliminated with the human urine is subject to considerable variation, but amounts on an average to 0.7 grm. during 24 hours on a mixed diet. The relationship of the uric acid to the urea on a mixed diet is on an average 1 : 50-1 : 70.' In new-born infants and in the first days of life the elimination of nric acid is increased (Makes °), and the relation between the uric acid and urea is about 1 : 13-14. Sjoqvist ° found the relationship in new-born infants to be 1 : 6.42-17.1. ' Zeitsclir. f. rat. med. (3), Bd. 31. Cited from Hoppe-Seyler's Physiol. Chem., S. 482. = Wien. med. JahrbilcUer, 1887. Cited from Maly's Jaliresber. , Bd. 17. ' TJeber die klin. Bedeutung des Vorkommens der HarnsSure, etc. Prager Festschrift. Berlin, 1896. S. 79. ^ A very good tabular summary of the variation in the elimination of uric acid and the relationship of total nitrogen to uric-acid nitrogen is found in v. Noorden's Lehrbuch der Pathologie des StofEwechsels, S. 54. ' See Centralbl. f. d. med. Wissensch., 1888, S. 2. « Nord. med. Arkiv., 1894, No. 10. URIC ACID. 473 In regard to the action of food we know from the observations of Eanke,' Mares," and Cambeee" that the elimination of uric acid is diminished in starvation, and that it quickly increases on partaking food, especially proteid food. MareS found the mini- mum about 13 hours after the last meal, and a strong increase about •i-5 hours after meat diet. This increase after a meal rich in pro- teid IIOEBACZEWSKi ' explains by the digestion leucocytoeis (see below) which habitually appears. It is quite generally accepted that the quantity of uric acid eliminated with vegetable food is smaller than with a meat diet, in which case the quantity may rise to 2 grms. or over per 24 hours.' The statements in regard to the influence of other circumstances, as also of different bodies, on the elimination of uric acid are rather contradictory. This is in part due to the fact that the older inves- tigators nsed an inaccurate method (HEiiirTz's method), and also, as shown especially by Mares and Salkowski,' that the extent of uric-acid elimination is dependent in the first place upon the indi- viduality. According to Sohondorff ' the drinking of water, con- trary to older statements, does not have any effect on the elimination of uric acid. According to Clar ' and Haig ' alkalies increase the elimination of uric acid, while according to Salkowski they diminish, and according to Hermastn '° they have no influence on the elimination. Hoebaczewski and Kanera" found an increased elimination of uric acid after the administration of glycerin, while no increase was observed after partaking sodium acrylate (Horbac- CEWSKi"). Certain medicines, such as quinin and atropin, diminish, while others, such as pilocarpin, increase, the elimination of uric acid. According to Horbaczewski'' and his pupils the first cause ' J. Eauke, Beobachtungen und Versuche uber die Ausscheidung der Harn- saure, etc. MUnchen, 1858. 'L. c. ' Zeitsehr. f. Biologie, Bd. 26. * Wien. Sitzungsber., Bd. 100, Abth. 3, 1891. ' In regard to the action of various diets the reader is referred to the above- cited authors, and especially to A. Hermann, Arch. f. klin. Med., Bd. 43. « Virchow's Arch., Bd. 117. ' PflUger's Arch., Bd. 46. « Centralbl. f. d. med. Wissensoh., 1888, No. 25. ' Journal of Physiol., Vol. 8. '» Arch. f. klin. Med., Bd. 43. " Wien. Sitzungsber., Bd. 97. '« Monatshefte f. Chem., Bd. 10. "Wien. Sitzungsber., Bd. 100. 474 THE VmNE. a diminution of the number of leucocytes in the blood, while the last cause an increase in the number. Little is known in regard to the elimination of uric acid in dis- ease. The uric acid introduced into the organism of a dog is in great part, as shown by Fbekichs and Wohlee,' converted into urea, aud as urea is also formed by the action of oxidizing agents on uric acid outside of the body, uric acid has been often considered as a step towards the formation of urea in the organism. Such a view is not, however, well founded, and the statement that in diseases with an incomplete supply of oxygen and diminished oxida- tion an increased formation of uric acid is produced has not been proved. With regard to the pathological relations we really only know two conditions in which the elimination of uric acid is increased, namely, in fever aad leucaemia. In fevers the uric acid eliminated is increased after the crisis, but it is undecided whether the quantity is increased at the height of the fever as compared to the normal." In leucaemia the elimination is increased absolutely as well as relatively to the urea (EAiirKE,' Salkowski,* Fleischee and Penzoldt,' Stadthagen," Sticker,' Bohland and Schuez," and others), and the relationship between the uric acid and urea (total nitrogen recalculated as urea) may be even 1 : 9, while under normal conditions, according to difEerent investigators, it is 1 : 40 to 66 to 100. The elimination of uric acid may be diminished in gout shortly before and during the attack. Formation of Uric Acid in the organism. The formation of uric acid in birds is increased by the administration of ammonia- salts (v. Schroder'). Urea acts in the same way (Meter and Jafee "), while in the organism of mammalia uric acid is more or less completely converted into urea. Minkowski " observed in geese with extirpated livers a very significant decrease in the elimination of uric acid, while the elimination of ammonia was ' Annal. d. Chem. u. Pliarm., Bd. 65. ' See V. Noorden, Lebrbucli d. Pathol, des Stoffwechsels, S. 311 and 218. 3 Schmidt's Jahrb., 1859. ■• Virchow's Arch. , Bd. 50. 5 Arch. f. klin. Med., Bd. 36. • Virchow's Arch., Bd. 109. ' Zeitschr. f. klin. Med., Bd. 14. » Pflilger's Arch., Bd. 47. ' Zeitschr. f. physiol. Chem., Bd. 2. '» Ber. d. deutsch. Chem. Gesellsch., Bd. 10 " Arch. f. exp. Path. u. Pharm., Bd. 31. FORMATION OF URIC ACID. 475, increased to a corresponding degree. Tliis indicates a participation, of ammonia in the formation of uric acid in the organism of birds j and as Minkowski has also found after the extirpation of the liver that considerable amounts of lactic acid occur in the urine, it is- probable that the uric acid in birds is produced in the liver, perhaps from lactic acid and ammonia by synthesis. Amido-acids — ^leucin, glycocoll, and aspartic acid — increase the elimination of uric acid in birds (v. Kniekiem '), but whether the amido-acids are first decomposed with the splitting off of ammonia is still unknown. We have no basis for the statement as to the formation of uric acid from ammonium salts in the human and mammalian liver. T. Mach" has shown that a small part of the uric acid in birds- originates from hypoxanthin, and a similar origin for the uric acid of mammalia is also very probable (Minkowski). The xanthin bases, as stated in Chapter V, originate from the nncleins, and Hokbaczewski ' gives the same origin for uric acid. According to this investigator uric acid is not derived from the naclein with the xanthin bases as intermediate steps, but uric acid or xanthin bases originate rather from the same mother-substance, the nuclein substances, according to circum- stances. ' Uric acid is formed when a cleavage precedes an oxidation, and xanthin bases, on the contrary, by cleavage without oxidation. Several circumstances speak for this origin of uric acid in the organism. Hokbaczbwski has prepared uric acid from tissues rich in nuclein, such as the spleen-pulp, and from spleen nuclein by slight putrefaction, subsequent oxidation with blood, and then cleavage by boiling. If the oxidation was neglected, he obtained an eqaivalent quantity of xanthin bases. The nuclein prepared from the spleen-pulp when introduced into the animal body causes an increase in the elimination of uric acid, which Horbaczbwski con- siders is not due to a direct transformation of the nuclei n. According to him it may be due indirectly to tlie leacocytosis pro- duced by the nuclein. According to Hoebaczbwski the nric acid originates chiefly from the naclein of the destroyed leucocytes, and the greater the number of leucocytes in the blood the greater is the destruction of the same, and hence the elimination of uric acii • Zeitschr. f. Biologie, Bd. 13. ' Aroli. f. exp. Path. u. Pharm., Bd. 24. ' Wlen Sitzungsber., Bd. 100. 476 THE URINE. is correspondingly increased. Observations on the elimination of uric acid stand in good accord with this theory. Thus, for example, new-born children eliminate more uric acid than adults because of the leucocytosis going on. The increase in the elimination of uric acid after food rich in proteid is explained by the leacocytosis, as also the abundant formation of uric acid, after animal as compared with yegetable food. Leucaemia, in which the elimination of uric acid is greatly increased, is characterized by an abnormally great number of leucocytes in the blood. Such medicaments, which increase the number of lencocytes, also increase in general ' the elimination of uric acid. _ It seems positively proven that a certain relationship exists hetween the elimination of uric acid and the quantity of leucocytes in the blood, and Horbaczewski's view that the uric acid is a product of the destruction of the leacocytes is very acceptable. The positive proof that uric acid actually originates in the destruc- tion of the leucocytes and not in some other way, in their reforma- tion or as a metabolic product, has, as stated by Mares," not been given. We cannot say anything positive in regard to the organ or organs in which uric acid is formed. After the extirpation of the kidneys of snakes (Zaleskt °) and birds (v. Schroder *) an accumulation of uric acid in the blood and tissues has been observed. This shows that the kidneys of these animals are not the only organ producing uric acid, and any direct proof of the formation of this acid in the kidneys has not to the present time been demonstrated. A direct ,relationship between the spleen and the formation of uric acid, also in man, has been sought by several investigators. According to the investigations of HoRBACZEWSKi this relationship seems to be of an indirect kind, as it probably stands in close connection with the importance of the spleen to the formation of the leucocytes. If uric acid is derived in man and mammals chiefly from nuclein, then we must look for its formation where a destruction of tissues containing nuclein takes place, although, according to Horbaczewski, it originates in the ' Horbaczewski, 1. c. ' Wien Sitzungsber. , Bd. 101, Abth. 3, and " Zur Theorie der Harnsaare- bildung im Saugethierorganismus. " Prag, 1892. 3 Cited from Hermann's Handb. , Bd. 5, Till. 1, S. 305. ^ Du Bois-Reymoud's Arch., 1880, Suppl. Bd., and Ludwig's Festschrift, 1887. PROPERTIES AND REACTIONS OF URIC ACID. 477 first place ia the destrnction of the leucocytes. We have no posi- tive basis for the statement that uric acid is formed in the liver of man and mammals, but the formation of uric acid in the liver of birds is shown to be highly probable by the researches of Min- kowski. Properties and Reactions of Uric Acid. Pure uric acid is a white, odorless, and tasteless powder consisting of very small rhom- bical prisms or plates. Impure uric acid is easily obtained as some- what larger, colored crystals. In quick crystallization, small, apparently colorless, thin, four- sided rhombic prisms are formed, which can only be seen by the aid of the microscope, and these sometimes appear as spools because of the rounding of their obtuse angles. The plates are sometimes six- sided, irregularly developed; in other cases they are rectangnlar with partly straight and partly jagged sides; and in other cases they show still more irregular forms, the so-called dumb-bells, etc. In slow crystallization, as when the urine deposits a sediment or when treated with acid, large, always colored crystals separate. Examined with the microscope these crystals appear always yellow or yellowish brown in color. The most ordinary form is the whetstone shape formed by the rounding off of the obtuse angles of the rhombic plate. The whetstones are generally connected together, two or more crossing each other. Besides these forms, rosettes of prismatic crystals, irregular crosses, brown-colored rough masses of destroyed needles and prisms occur, also other forms. Uric acid is insoluble in alcohol and ether; it is rather easily soluble in boiling glycerin, very difficultly soluble in cold water (14,000-15,000 parts), and difficultly soluble in boiling water (in 1800-1900 parts). It is soluble in a warm solution of sodium diphosphate, and in the presence of an excess of uric acid mono- phosphate and acid urate are produced. Sodium phosphate is con- sidered as a solvent for the uric acid in the urine. According to EiJDEL' urea is an important solvent. 1000 c. c. of a 2^ urea solution can hold on an average 0.529 grm. uric acid in solution, and as the daily quantity of urine is 1500-2000 c. c, and this con- tains 2^ urea, it is possible for the urea alone to hold nearly all of the uric acid eliminated in solution. Piperazin (diethylendiamin), C,H,„1^„ is also a good solvent for uric acid. Uric acid dissolves ' Arcb. f. exp. Path. u. Pharm., Bd. 30. 478 THE URINE. without decomposing ia concentrated sulphuric acid. It is com- pletely precipitated from the urine by picric acid (Jaffb '). Uric acid is dibasic and correspondingly forms two series of salts, neutral and acid. According to Bence Jones' hyperacid salts, QUADEiuEATES, with the general formula (MHU + H,U) also •occur. Of the alkali urates the neutral potassium and lithium salts dis- solve most easily, and the ammonium salt dissolves with difficulty. The acid-alkali urates are very insoluble, and separate as a sediment {sedimentum lateriiium) from concentrated urine on cooling. The salts with alkaline earths are very insoluble. If a little uric acid in substance is treated on a porcelain dish with a few drops of nitric acid, the uric acid dissolves on warming with a strong development of gas, and after thoroughly drying on the water-bath a beautiful red residue is obtained, which turns a purple-red (ammonium purpurate or murexide) on the addition of a little ammonia. If, instead of the ammonia, we add a little caustic soda (after cooling), the color becomes more blue or bluish violet. This color disappears quickly on warming, differing from certain xanthin bodies. This reaction is called the murexide test. If uric acid is converted into alloxan by the careful action of nitric acid and the excess of acid carefully expelled on treating this with a few drops concentrated sulphuric acid and commercial benzol (containing thiophen), a beautiful blue coloration is obtained {Denigbs' ^ reaction). Uric acid does not reduce an alkaline solution of bismuth, but •does, on the contrary, an alkaline copper-hydroxide solution. In the presence of only a little copper salt we obtain a white precipitate consisting of copper urate. In the presence of more copper salt red suboxide separates. The method for the volumetric estimation of uric acid as suggested by Arthacd and Butte,' as well as the method suggested by KRtJGEE and Wules,' is based on the insolu- bility of copper urate. ' Zeitscbr. f, pliysiol. Chem., Bd. 10. » Journ. Chem. Soc, 1862, vol, xv., p. 8. • Journal de Pliarm. et de Cliim., Tome 18. Cited from Maly's Jahresber., Bd. 18. S 24. * Compt. rend. soc. biol., Tome 41. Cited from Maly's Jahresber., Bd. 20, S. 180 ' Zeitscbr. f. physiol. Chem . Bd. 20. ESTIMATION OF URIC ACID. 479 If a drop of uric acid dissolved in sodium carbonate is placed on a piece of filter-paper which has been previously treated with silver- nitrate solution, a reduction of silver oxide occurs producing a brownish-black or, in the presence of only 0.002 milligramme uric acid, a yellow spot (Schiff's ' test). Preparation of Uric Acid from Urine. Filtered normal urine is treated with 30-30 c. c. of 25^ hydrochloric acid for each litre of urine. After forty-eight hours collect the crystals and purify them by redissolving in dilute alkali, decolorizing with animal charcoal and reprecipitating with hydrochloric acid. Large quantities of uric acid are easily obtained from the excrements of serpents by boiling them with dilute caustic potash until no more ammonia is developed. A current of carbon dioxide is passed through the filtrate until it barely has an alkaline reaction; dissolve the separated and washed acid potassium urate in caustic potash, and precipitate the uric acid by addition of an excess of hydrochloric acid to the filtrate. Quantitative Estimation of Uric Acid in the urine. As the older method as suggested by Heintz, even after recent modifica- tions, gives inaccurate results, we will not give it in detail. Salkowski " and Ludwig's' method consists in precipitating by silver nitrate the uric acid from the urine previously treated with magnesia-mixture, and weighing the uric acid obtained from the silver precipitate. Uric-acid determinations by this method are often performed according to the suggestion of E. Ludwig, which requires the following solutions: 1. An AMM0NIACA.L 8II.VKR-NITRATK solution, which Contains in one litre 26 grms. silver nitrate and a quantity of ammonia sufBcient to complfctply re- dissolve the precipitate produced by the first addition of ammonia. 2. Mag- nesia MIXTOKB. Dissolve 100 gmis. crystallized magnesium chloride in water and add enough ammonia so that the liquid smells strongly of it, and enough ammonium chloride to dissolve the precipitate and dilute to 1 litre. 'A. SODiUM-stTLPHiDB SOLUTION. Dissolve 10 grms. caustic soda whicli is free from nitric acid and nitrous acid in 1 litre of water. One half of this solution is completely saturated with sulphuretted hydrogen and then mixed with the other half. The concentration of the three solutions is so arranged that 10 c. c. of each is sufficient for 100 c. c. of the urine. 100-200 c. c, according to concentration, of the filtered urine freed from proteid (by boiling after the addition of a few drops of acetic acid) are poured into a beaker. In another vessel mix 10-20 c. c. of the silver solution with 10-20 c. c. of the niagnesia mixture and add ammonia, and when necessary also some ammonium chloride, until the mixture is clear. This solution is added to the ' Annal. d Chem. u. Pharm., Bd. 109. ' Virohow's Arch., Bd. 52, and PflOger's Arch., Bd. 5. » Wien. med. Jahrb., 1884, and Zeitschr. f. analyt. Chem., Bd. 24. 480 TEE URINE. nrine while stirring, and the mixture allowed to stand quietly for half an hoar. The precipitate is collected on a filter, washed with ammoniacal water, and then returned to the same beaker by the aid of a glass rod and a spirt-bottle, without destroying the filter. Kow heat to boiling 10-20 c. c. of the alkali-sulphide solution, which has previously been diluted with an equal volume of water, and allow this solution to flow through the above filter into the beaker containing the silver precipitate, wash with boiling water, and warm the contents of the beaker on a water-bath for a time, stirring con- stantly. After cooling filter into a porcelain dish, wash with boil- ing water, acidify the filtrate with hydrochloric acid, evaporate to about 15 c. c, add a few drops more of hydrochloric acid, and allow it to stand for 34 hours. The uric acid which has crystallized is collected on a small weighed filter, washed with water, alcohol, ether, and carbon disnlphide, dried at 100-110° C. and weighed. For each 10 c. c. of watery filtrate we must add 0.00048 grm. uric acid to the quantity found directly. Instead of the weighed filter- paper a glass tube filled with glass-wool as described in other hand- books may be substituted (Ludwig). Too strong or continuous heating with the alkali sulphide must be prevented, otherwise a part of the uric acid may be decomposed. Gkovb ' recommends a solution of potassium iodide instead of the alkali sulphide, thus making the washing with carbon disulphide unnecessary. Cameeee '' has modified this method in certain points, and he determines the nitrogen in the silver precipitate (a-uric acid = uric acid contami- nated with xanthin bodies) and also the uric acid isolated by Sal- kowski-Ludwig's method {— b-nric acid). Hatceaft's Method.' 25 c. c. of the urine are first treated with 1 grm. bicarbonate, then made strongly alkaline by ammonia, and lastly precipitated by an ammoniacal silver solution. The carefully washed precipitate is dissolved in 30-30^ nitric acid and n this solution titrated with a — -- sulphocyanide solution according to Volhaed's method. Each c. c. of this solution corresponds to 0.00168 grpi. uric acid. This method has been modified in certain points by Heemanjs"* and Czapek,' which last titrates with alkali sulphide the silver salts remaining in solution in the urine after the precipitation of the uric acid by a known volume of ammoniacal silver solution of known strength. The advantage of Hatceaft's method is the ease and rapidity with which it can be performed, and it is therefore recommended for clinical purposes. For exact ' Journ. of Physiol., Bd. 12, ' Zeitschr. f. Biologie, Bdd. 27 u. 28. ' Zeitschr. f analyt, Chem., Bd. 25. * Z itschr. f. pliysiol. Chem., Ed. 12. » lUd. , Bd. 12, S. 502. OXALIC ACID. 481 determinations it is not quite reliable, because the amount of silver in the precipitate of silver urate is not constant (Salkowski'). Haycraft's method gives the same results as Salkowski-Lud- wig's method in pure uric-acid solutions. With the urine Hayceaft's method gives on the contrary too high results, which is in part due to the fact that the silver solution precipitates from the urine other bodies, such as xanthin bases, besides the uric acid. Since the value of this method has been the subject of much adverse criticism, we will not give further particulars.' In regard to Fokkek's ' method we refer the reader to more exhaustive text-books. Hopkins's ' method is based on the fact that the uric acid is completely precipitated from the urine as ammonium urate on saturating with ammoniam chloride. The urine is saturated with ammonium chloride (for each 100 c. c. urine add 30 grms. ammonium chloride) and filter after two hours. Wash with a saturated solution of ammonium chloride, and transfer the precipi- tate from filter to a small beaker by means of boiling water, and decompose it with hydrochloric acid and heat. The uric acid which separates is determined by weighing it as such, or by titration with potassium permanganate. This simple method gives as good results as Salkowski-Ludwig's method. Keugee and Wulfe's method will be treated of in connection with xanthin bases in the urine. OxAiiTEic Acid, C,H4Nj04 = (CONjH.) CO.COOH. This acid, whose rela- tion to uric acid and urea has been spoken of above, occurs only as traces in the urine as ammonium salts. This salt is not directly precipitated by CaClj and NHj , bat after boiling, when it is decomposed into urea and oxalate. In preparing oxaluric acid from urine the latter is filtered through animal char- coal. The oxalurate retained by the charcoal may be obtained by boiling with alcohol. Oxalic Acid, C,H,0„ or Aqqtt' occurs under physiological conditions in very small amounts in the urine, about 0.03 grm. in 24 hours (Fuebeingee '). According to the generally accepted view it exists in the urine as calcium oxalate, which is kept in solu- ' Pflilger's Arch., Bd. 5; also Salskowski and Jolin, Zeitschr. f. physiol. Chem., Bd. 14. ' In regard to the literature on this subject see Huppert-Neubauer's Ham- analyse. See also Lisowski, Maly's Jahresber., Bd. 20; Deroide, ibid., Bd. 21, B. 172; Groves, 1. c; and Haycraft, Zeitschr. f. physiol. Chem., Bd. 15. » Pflilger's Arch., Bd. 10. ♦ Journal of Pathology and Bacteriology, 1893, and Proceedings of Royal Society, Vol. 52. ' Deutsch. Arch. f. klin. Med., Bd. 18. 482 THE URINE. iion by the acid phosphates present. Oalciam oxalate is a frequent constituent of urinary sediments, and occurs also in certain urinary •calculi. The origin of the oxalic acid in the urine is not well known. Oxalic acid when administered is eliminated, at least in part, by the urine unchanged, and as many vegetables and fruits, such as cabbage, spinach, asparagus, sorrel, apples, grapes, etc., contain oxalic acid, it is possible that a part of the oxalic acid of the urine originates directly from the food. According to Abelbs ' this is not the case. According to him an alimentary oxaluria, that is, an elimination of oxalic acid caused by partaking of the ordinary foods containing oxalic acid, does not exist, and the soluble oxalates of the food are in all probability converted into insoluble lime-salts in the digestive tract. That oxalic acid may be formed in the animal body from proteid or fat follows from the observations of Mills '^ that oxalic acid is found in the urine of dogs after feeding with meat and fat alone. Oxalic acid is also supposed to be derived by the incomplete combustion of carbohydrates, and is also considered, but not with sufficient basis, as an oxidation product of uric acid. An increased elimination of oxalic acid may occur in diabetes. The question whether it occurs as an independent disease {oxaluria, oxalic-acid diathesis) has not been positively decided. The properties and reactions of oxalic acid and calcium oxalate are well known. Calcium oxalate as a constituent of urinary sedi ments will be described later. Detection and Quantitative Estimation of Oxalic Acid in Urine. The presence of oxalic acid in solution in urine is debermined according to the method suggested by Netjbauee,' who treats 500-600 c. c. of the urine with CaClj solution, makes alkaline with ammonia and then faintly acid with acetic acid. After 24 hours the precipitate is collected on a small filter, washed with water, treated with hydrochloric acid (which leaves the uric acid undis- solved on the filter), and washed again with water. The filtrate, including the wash-water, is treated with an excess of ammonia and allowed to stand 24 hours. Calcium oxalate separates as quadratic octahedra. The quantitative estimation is performed after the same principle. The oxalate is converted into quicklime by heat, and weighed as such. ' Wien. klin. Woohenschr. , 1892. * Virchow's Arch., Bd. 91. » Zeitschr. f. analyt. Chem., Bd. 8, S. 521. ALLANTOIN. 483 AUantoin or GLTOXTLDiuREin, C,H,N,0, or ^^/NH.CH.NH.CO.NH, . ^^ ^ ,.,, CO lUd., Bd. 10, S. 131. * Arch. f. exp. Path. u. Pharm., Bd. 6. Also Ar. Hoffmann, ibid., Bd. 7, and Kochs, Pflilger's Arch., Bd. 20. 488 TEE UEINE. decomposes, producing a red mass and a sublimate of benzoic acid, with the generation, first, of a peculiar pleasant odor of hay, and then an odor of hydrocyanic acid. Hippnric acid is easily differen- tiated from benzoic acid by this behavior, also by its crystalline form and its insolubility in petroleum ether. Ilippuric acid and benzoic acid both give Lucke's reaction, namely, they generate an intense odor of nitrobenzol when evaporated with nitric acid to dryness and when the residue is heated. Hippuric acid forms crystallizable salts, in most cases, with bases. The combinations with alkalies and alkaline earths are soluble in water and alcohol. The silver, copper, and lead salts are soluble with difficulty in water; the iron-oxide salt is insoluble. Hippuric acid is best prepared from the fresh urine of a horse or cow. The urine is boiled a few minutes with an excess of milk of lime. The liquid is filtered while hot, concentrated and then cooled, and the hippnric acid precipitated by the addition of an excess of hydrochloric acid. The crystals are pressed, dissolved in milk of lime by boiling, and treated as above; the hippuric acid is precipitated again from the concentrated filtrate by hydrochloric acid. The crystals are purified by recrystallization and decolorized, when necessary, by animal charcoal. The quantitative estimation of hippuric acid in the urine may be performed by the following, method (Bungb and Schmibde- BEKG ') : The urine is first made faintly alkaline with soda, evapo- rated nearly to dryness, and the residue thoroughly extracted with strong alcohol. After the evaporation of the alcohol dissolve in water, acidify with sulphuric acid, and completely extract by agitating (at least five times) with fresh portions of acetic ether. The acetic ether is then repeatedly washed with water, which is removed by means of a separatory funnel, then evaporated at a medium temperature, and the dry residue treated repeatedly with petroleum ether, which dissolves the benzoic acid, oxyacids, fat, and phenol, while the hippuric acid remains undissolved. This residue is now dissolved in a little warm water and evaporated at 50-60° 0. to crystallization. The crystals are collected on a small weighed filter. The mother-liquor is repeatedly shaken with acetic ether. This last is removed and evaporated; the residue is added to the above crystals on the filter, dried and weighed. Phenaceturic Acid, C,„H,,N03 = CeHt.CHj.CO.NH.CHj.COOH. This acid, whicli is produced in the animal body by a grouping of the phenylacetic acid, CoHs.CHtj.COOH, formed by the putrefaction of theproteids with glycoeoll, has been prepared from horse's urine by Salkowski,'' but it probably also occurs in human urine, ' L. c. ' Zeitschr. f. physiol. Chem., Bd. 9. See also E. and H. Salkowski, ibid., Bd. 7. ETHBRBAL aULPHUBIC A0ID8. 489 Benzoic Acid, 071160, or CbHj.COOH, is found in rabbit's urine and some- times, though in small amounts, iu dog's urine (WEYLand v. Anbbp)). Ac- cording to J aaksvkld and Stokvis' and to Kbonbckkr" it is also found in human urine iu diseases of the kidneys. The occurrence of benzoic acid in the urine seems to be due to a fermentative decomposition of hippuric acid. Such a decomposition may very easily occur in an alkaline urine or one containing proteid (Van de Veldb and Stokvis*). In certain animals— pigs and dogs — the kidneys, according to Schmibdkbbrg ^ and Minkowski, • contain a special enzyme, Schmtedbbbrg's histozym, which splits the hippuric acid with the separation of benzoic acid. Ethereal Sulphuric Acids. In the putrefaction of proteids in the intestine, phenol, whose mother-substance is considered to be tyrosin, and indol and skatol are produced. The two last-named bodies, after they have been oxidized into indoxyl and skatoxyl, pass into the urine as ethereal sulphuric acids after uniting with sulphuric acid. The most important of these ethereal acids are phenol- and cresol-sulphuriv acid — which were formerly also called phenol-forming substance — indoxyl- and shatoxyl-sulphuric acid. To this group belong also the pyrocatecliin-sulphuric acid, which only occurs in very small amounts in human urine, and hydro- chinon-sulphuric acid, which appears in the urine after poisoning with phenol, and perhaps under physiological conditions other ethereal acids occur which have not been isolated. The ethereal sulphuric acids of the urine were discovered and specially studied by Badmakn.' The quantity of these acids jn human urine is small, while horse's urine contains larger quantities. According to the determinations of v. d. Veldbn ' the quantity of ethereal sul- phuric acid in human urine in the 24 hours varies between 0.094 and 0.630 grms. The relationship of the sulphate-sulphuric acid A to the conjugated sulphuric acid B in health is on an average as 10 : 1. It undergoes such great variation, as found by BAUMAKif and Hertee' and after them by many other investigators, that it is hardly possible to consider the average figures as normal. After taking phenol and certain other aromatic substances, as also with abundant putrefaction within the organism, the elimination of ' Zeitschr. f. physiol. Chem., Bd. 4. = Arch. f. exp. Path. u. Pharm., Bd. 10. « lUd., Bd. 16. ^Ibid., Bd. 17. ' Ibid., Bd. 14, S. 379. > lUd., Bd. 17. ' PflQger's Arch., Bdd. 13 and 13. ' Virchow's Arch., Bd. 70. ' Zeitschr. f. physiol. Chem., Bd. 1. 490 THE URINE. etliereal sulphuric acid is greatly increased. On the contrary it is diminished when the putrefaction in the intestine is reduced or prevented. Por this reason it may be greatly diminished by carbo- hydrates and one-sided milk diet.' The elimination of ethereal sulphuric acid has also been diminished in certain cases by certain therapeutic agents which have an antiseptic acid; still the state- ments are not unanimous." Great weight has been put upon the relationship between the total sulphuric acid and the conjugated sulphuric acid, or between the conjugated sulphuric acid and the sulphate-sulphuric acid, in the study of the intensity of the putrefaction in the intestine under different conditions. Several investigators, P. Mullbr," Sal- KOwsKi,' and v. Nooeden,' consider correctly that this relation- ship is only of secondary value and that it is more correct to consider the absolute value. It must be remarked that the absolute values for the conjugated sulphuric acid also undergo great varia- tion, so that it is at present impossible to give the upper or lower limit for the normal value. Phenol- and p-Cresol-sulphuric Acid, C,H,.O.SO,.OH and C,H,.O.SOj.OH. These acids are found as alkali salts in human urine, in which also orthocresol has been detected. The quantity of cresol-sulphuric acid is considerably greater than phenol-sul- phuric acid. In the quantitative estimation the phenols set free from the two ethereal acids are determined together as tribrom- phenol. The quantity of phenols which are separated from the ethereal sulphuric acids of the urine amounts to 17-51 milligrammes in the 34 hours (MtJNK°). The methods for the quantitative estimation used heretofore give, according to Eumpj' and also KosSLEK and Penny, " such inaccurate results that new determin- ations are very desirable. After a vegetable diet the quantity of ' See Hirscliler, Zeitsclir. f. physiol. Chem., Bd. 10; Bieruacki, Deutsch. Arch. f. klin. Med., Bd. 49; Rovigbi, Zeitschr. f. physiol. Chem., Bd. 16; Winternitz, ibid., and Schmltz, ibid., Bdd. 17 and 19. * See Baumann and Morax, Zeitschr. f . physiol. Chem., Bd. 10; SteifE, Zeit- schr. f. klin. Med., Bd. 16; Eovighi, 1. c; Stern, Zeitschr. f. Hygiene, Bd. 13; and Bartoschewitsch, Zeitschr. f. physiol. Chem., Bd. 17. ' Zeitschr. f. klin. Med., Bd. 13. * Zeitschr. f. physiol. Chem., Bd. 12. ' Zeitschr. f. klin. Med., Bd. 17. * Pfiilger's Arch., Bd. 12. ' Zeitschr. f . physiol. Chem. , Bd. 16. » Ibid., Bd. 17. PHENOL- AND P-CRES0L-8ULPHUBia AOID. 491 these ethereal-sulphuric acids is greater than after a mixed diet. After taking carbolic acid, which is in great part converted by synthesis within the organism into phenol-ethereal-sulphuric acid, besides also pyrocatechin- and hydrochinon-sulphuric acid," and alsa when the amount of sulphuric acid is not sufficient to combine with the phenol, forming phenyl-glycuronic acid," the quantity of phenols and ethereal-sulphuric acids in the urine is considerably increased at the expense of the sulphate-sulphuric acid. An increased elimination of phenol-sulphuric acids occurs in active putrefaction in the intestine with stoppage of the contents of the intestine, as in ileus, diffused peritonitis with atony of the intestine, or tuberculous enteritis, but not in simple obstruction. The elimination is also increased by the absorption of the products, of putrefaction from purulent wounds or abscesses. An increased elimination of phenol has been observed in a few other cases of diseased conditions of the body.' The alkali salts of phenol- and cresol-sulphuric acids crystallize in white plates, similar to mother-of-pearl, which are rather freely soluble in water. They are soluble in boiling alcohol, but only slightly soluble in cold. On boiling with dilute mineral acids they are decomposed into sulphuric acid and the corresponding phenol. Phenol-sulphuric acids have been synthetically prepared by Baumann^ from potassium pyrosulphate and phenol- or p-cresol- potassium. For the method of their preparation from urine, which is rather complicated, the reader is referred to other text-books. The quantitative estimation of these ethereal sulphuric acids is doue by determining the amount of phenol which may be separated from the urine as tribromphenol. In this determination, when the urine is not specially rich in phenol, about one fourth of the total quantity in the 24 hours is used; it is acidified with concentrated hydro- chloric acid — 5 c. c. for every 100 c. c. of urine — and distilled until a portion of the distillate does not give the slightest reaction for phenol with Millon's reagent or with bromine-water. The distil- late is now carefully neutralized with soda solution (which combines with the benzoic acid, etc.) and again distilled until a portion of the distillate is free from phenol, as shown by the above-mentioned reagents. This distillate is treated with bromine-water until a per- manent yellow color is produced, and then allowed to stand for ' See Baamann, Pflilger's Arch. , Bdd. 12 and 13, audBaumann and Preusse, Zeitschr. f. physiol. Chem., Bd. 3, S. 156. ' Schmiedeberg, Arch. f. exp. Path. u. Pharm., Bd. 14. ' See G. Hoppe-Seyler, Zeitschr. f. physiol. Chem., Bd. 12. This contains also all references to the literature on this subject. 492 THE TIRINE. about 24 honrs in the cold; the crystalline precipitate is then coilected on a small weighed filter, washed with dilute bromine- water, dried over sulphuric acid without the use of a yacuum, and weighed (100 parts tribromphenol correspond to 28.4 parts phenol). It is assumed that the paracresol is first converted by the bromine- water into tribromcresol bromine, and that this is then gradually changed into tribromphenol with the discharge of carbon dioxide. As shown by Eumpf ' this is not the case, but dibromcresol is chiefly formed instead. This method is therefore not available for this and other reasons. Among the other methods which have been sug- gested, the following seems to be the most available. KossLEB and Pekitt's ' method. This method is a modification of MESSi]!. Cliem., Bd. 13 (contains older liter- ature); also Berl. klin. Woclienschr., 1893. ' Zeitschr. f. physiol. Cliem., Bd. 3, S. 254. TESTS FOR INDIOAN. 495 of air passes into a red compound, indoxyl-red, bat in the presence of oxidizing reagents is converted into indigo-blue: 3C,H,N0 + 20 = C„H„N,0, + aH,0. The detection of indican is based on this last fact. For the rather complicated preparation of indoxyl-sulphuric acid as potassium-salt from urine the reader is referred to other text- books. For the detection of indican in urine in ordinary cases the following method of Jaffe,' which also serves as an approximate test for the quantity of iudican, is sufiBcient. Jaffe's Indican Test. 20 c. c. of urine are treated in a test- tube with 2-3 c. c. chloroform and mixed with an equal volume of concentrated hydrochloric acid. Immediately after a concentrated chloride-of-lime solution or a -J^ potassium permanganate solution is added drop by drop, and after each drop the mixture is thoroughly shaken. The chloroform is gradually colored faintly or strongly blue. An excess of oxidizing reagent, especially chloride of lime, interferes with the reaction and must therefore be avoided. The test is repeated with somewhat varying amounts of oxidizing material nntil a point is found at which the maximum coloration of the chloroform takes place. From the intensity of the color the quantity of iudigo is determined. Obermater" uses fumiug hydrochloric acid containing 2-4 parts ferric chloride per litre to decompose the indican and to oxidize the indoxyl. The urine is first precipitated with not too much lead acetate and the filtrate shaken for 1-2 minutes with an equal volume of the above hydrochloric acid. The indigo blue is taken up by chloroform in this case also. According to Eosix ' some indigo-red is always formed besides the indigo-blue in Jaffe's indican test. Greater quantities of indigo-red are formed when the decomposition of the indican takes place in the warmth (see Kosenbach's urine test). An exact determination of the amount of indigo in urine is very rarely made. The methods suggested for this purpose are very complicated, and even then they are not quite accurate; therefore tJie reader is referred to other text-books for their descriptiou. Indol seems also to pass into the urine as a glycnronic acid, indoxyl-glycuronic acid (Schmiedebbrg '). Such an acid has been found in the urine of animals after the administration of the sodium-salt of o-nitrophenylpropiolic acid (G. Hoppb-Sbtler'). Skatoxyl-sulphuric Acid, C.H.NSO. or C,H,.N.O.SO,.OH. ' PflUger's Arch., Bd. 3. ' Wien. klin. Wochenscbr., 1890. 'Virebow's Arch., Bd. 123. * Arcb. f. exp. Path. u. Pharm., Bd. 14. » ZeitBchr. f. pliysiol. Chem., Bdd. 7 and 8. 496 THE URINE, The potassinm-salt of this acid seems to occur generally in human urine as a chromogen, which yields a red or violet coloring matter on decomposing with strong acids, and an oxidizing reagent. This salt has been prepared by Otto ' from diabetic human urine. Little is known of the quantity of this skatol-chromogen, to which prob- ably also the skatoxyl-glycuronic acid mast be counted, under physiological and pathological conditions. Skatoxyl-sulphuric acid originates from skatol formed by putre- faction in the intestine, which is coupled with sulphuric acid after oxidation into skatoxyl. That skatol introduced into the body passes partly as an ethereal-sulphuric acid into the urine has been shown by Briegek.'' Indol and skatol act differently, at least in dogs; indol producing a considerable amount of ethereal-sulphuric acid, while skatol only gives a small quantity (Mestee^). Skatol seems partly to pass into the urine as a shatoxyl-glycuronic acid. The potassium-salt of skatoxyl-sulphuric acid is crystalline; it dissolves in water, but with difficnlty in alcohol. A watery solution becomes deep violet with ferric chloride, and red with concentrated nitric acid. The salt is decomposed by concentrated hydrochloric acid with the separation of a red precipitate. The nature of this red coloring matter produced by the decomposition of skatoxyl- sulphuric acid is not well known; neither is the relationship existing between this and other red coloring matters in the urine known. On distillation with zinc-dust the skatol chromogen yields skatol. Urines containing skatoxyl are colored dark red to violet by Jaffe's indican test even on the addition of hydrochloric acid ; with nitric acid they are colored cherry-red, and red on warming with ferric chloride and hydrochloric acid. The coloring matter which yields skatol with zinc-dust may be removed from the urine by ether. Urines rich in skatoxyl darken when allowed to stand in the air from the surface downward, and may become reddish, violet, or nearly black. EosiN ' is of the opinion that no skatol-chromogen exists in human urine, and that the observations made heretofore were due to a confusion with indigo-red or urorosein. Salkowski' has shown that the oceurrence of skatol-carbonic acid,CaWt. N.COOH, in normal urine is probable. This is also a putrefaction product. ' Pflilger's Arch., Bd. 33. ' Ber. d. deutsch. chem. Gesellsch., Bd. \%, and Zeitschr, f. physiol. Chem., Bd. 4, S. 414. ' Zeitschr. f. physiol. Chem., Bd. 13. * L. c. ' Zeitschr. f . physiol. Chem., Bd. 9. ABOMATIO OXYAOIBS. 497 Aromatic Oxya'cids. la the putrefaction of proteids in the intestine, paraoxyphenyl-acetic acid, C,H^(OH).CH,COOH, and paraoxyphenyl-propionic acid, C,H,(OH).CjH,.COOH, are formed from tyrosin as intermediate steps, and these in great part pass unchanged into the urine. They were first detected by BAUMANiir.' The quantity of these acids is usually very small. They are increased by the same circumstances as phenol, especially in acute phosphorus-poisoning, in which the increase is considerable. A small portion of these oxyacids is combined with sulphuric acid. Besides these two oxyacids which regularly occur in human urine we sometimes have other oxyacids in urines. To these belong homogentisic acid and uroleucicacid, which form the specific constit- uents of the urine in most cases of alcaptonuria, oxymandelic acid,. found by Schultzen" and Riess " in urine in acute atrophy of the liver, oxyhydroparacumaric acid, found by BLENDEKMANif ' in the urine on feeding rabbits with tyrosin, gallic acid, which, according to Baumann,' sometimes appears in horse's urine, and hynurenic acid (oxychinolincarbonic acid), which has only been found up to- the present time in dog's urine. The first two above-mentioned oxyacids, and also homogentisic and uroleucic acids, will be treated of here. Paraoxyphenylacetic acid and p-oxyphenylpropionic acid are crystalline and are both soluble in water and in ether. The first melts at 148° C. and the other at 125° C. Both give a beautiful red coloration on being warmed with Millon's reagent. To detect the presence of these oxyacids proceed in the following way (Baumann) : Warm the urine for a while on the water-bath with hydrochloric; acid, in order to drive off the volatile phenols. After cooling shake three times- with ether, and then shake the ethereal extracts with dilute soda solution, which dissolves the oxyacids, while the residue of the phenols soluble in ether remains. The alkaline solution of the oxyacids is now faintly acidified with sulphuric acid, shaken again with ether, the ether removed and allowe.l to evap- orate, the residue dissolved in a little water, and the solution tested with Millon'S reagent. The two oxyacids are best differentiated by their different melting-points. The reader is referred to other works for the method of Isolating and separating these two oxyacids. Homogentisic acid, C,H,0, or C.H3(0H),.CH,.C00H. This acid was detected by Wolkow and Baumann. ' They isolated it ' Ber. d. deutsch. chem. Gesellsch., Bdd. 12 and 13, and Zeitschr. f. physiol. Chem., Bd. 4. ' Chem. Centralbl., 1869. 3 Zeitschr. f. Physiol. Chem., Bd. 6, S. 257. ■•iJid., Bd. 6, S. 193. ' IMd., Bd. 15. 498 THE URINE. from the urine iu a case of alcaptonuria (see below) and showed that the characteristics of so-called alcaptonuric arine in this case were due to this acid. This acid has later been found in other cases of alcaptonuria by Embden,' GARiiriER and Voikik,' and Ogden.' Qlycosuric acid, isolated Jrom alcaptonuric urine by Marshall * and recently by Geygek,' seems to be identical with homogentisic acid. Tyrosin is considered as the mother-substance of this acid. On the introduction of tyrosin in persons with alcaptonuria, Wolkow and Baumann and Bmbden' observed a greater or less increase in the quantity of homogentisic acid in the urine. According to Wolkow and Baumann this acid is formed from the tyrosin by abnormal putrefactive processes in the upper part of the intestine. Homogentisic acid is that dioxyphenyl-acetic acid derived from hydrochinon. On fusion with potash it yields gentisic acid (hydro- chinon-carbonic acid) and hydrochinon. When introduced into the intestinal tract of dogs it is in part converted into tola-hydrochinon, which is eliminated in the form of ethereal-sulphuric acid. Homo- gentisic acid has recently been prepared synthetically by BAtTMAJfir and Peakkel,' starting with gentisic aldehyde. Homogentisic acid crystallizes with 1 mol. water in large, trans- parent prismatic crystals, which become non-transparent at the temperature of the room with the loss of water of crystallization. They melt at 146.5-147° C. They are soluble in water, alcohol, and ether, but nearly insoluble in chloroform and benzol. Homo- gentisic acid is optically inactive and non-fermentable. Its watery solution has the properties of so-called alcaptonuric urine. It becomes greenish brown from the surface downward on the addition of very little caustic soda or ammonia with excess of oxygen, and on stirring it becomes quickly dark brown or black. It reduces alka- line copper solutions with even slight heat, and ammoniacal silver solutions immediately in the cold. It does not reduce alkaline bismuth solutions. Among the salts of this acid we must mention the lead salt containing water of crystallization and 34.79^ Pb. This salt melts at 214-215° C. ' Zeitschr. f. pbyslol. Chem. , Bdd. 17 and 18. ' Arch, de Physiol., (5) Tome 4. ' Zeitschr. f. physiol. Chem., Bd. 30. * See Maly's Jahresber., Bd. 17. ' Pharm. Ztg., 6 Aug. 1893, S. 488. Cited from Embden, Zeitschr. f. phys- iol. CUem. , Bd. 18. « Zeitschr. f. Physiol. Chem., Bd. 30. URINARY PIGMENTS. 499 In preparing this acid the strongly acidified urine is shaken with ether. The residue obtained on the distillation of the ether is dissolved in water, the solution heated to boiling and treated with a lead acetate solution (1 : 5), and the brown resinous precipitate quickly separated by filtration. The lead salt gradually crystallizes from the filtrate. This is decomposed by sulphuretted hydrogen and the acid obtained as crystals from the filtrate after carefully concentrating the filtrate finally in vacuo. In regard to the quantitative estimation we proceed according 71 to the suggestion of Baumann by titrating the acid with a — silver solution. As regards details of this method we must refer the reader to the original publication.' Urolencic acid, CgHioOs, is, according to Huppebt,' probably at rioxyphenol- propionic acid, (HO)3.C6H!iCHj.CH.j.COOH. This acid was first prepared by KiKK' from the urine of children with alcaptonuria. According to Wolkow and Baumann it is not identical with homogentisic acid, and has a melting- point of 133° C. Otherwise, in regard to its beharior with alkalies, with ac- cess of air, and also with alkaline copper solutions and ammoniacal silver solu- tions, it is similar to homogentisic acid. In chemical properties it is very sim- ilar to gallic acid. TTrinary Pigments and Chromogens. The yellow color of normal urine depends apparently upon several coloring matters which have not been isolated and studied. Besides these bodies, tTEOBiLix sometimes occurs in fresh normal urine, but by no means always. Instead of urobilin, normal urine often contains a mother- substance of the same, a chromogen or ueobilinogen', from which the urobilin is gradually formed by oxidation on allowing the urine to stand exposed to the air (Jaffe,* Disque,' and others). Besides this chromogen, urine contains various other bodies from which coloring matters may be produced by the action of chemical agents. Humin substances (perhaps in part from the carbohydrates of the urine) may be formed by the action of acids (v. Udranszkt') without regard to the fact that such substances . may sometimes originate from the reagents used, as from impure amyl-alcohol (v. Udeanszky and Hoppe-Setlee'). To these humin bodies ' Zeltschr. f. physiol. Chem., Bd. 16. ' Hiippert-Neubauer, Analyse des Harns, 10. Aufl., S. 246. » Brit. Med. Journal, 18S6 and 1888; Journal of Anat. and Physiol., Vol. 23. * Centralbl. f. d. med. Wissensch,, 1868 and 1869; Virchow's Arch., Bd. 47. ' Zeitschr. f. physiol. Chem., Bd. 2. *MiL., Bdd. 11 and 12. ' Hoppe-Seyler, Ber. d. deutsch. Chem. Qellsch., Bd. 18, and v. Udrfinszky, ZeitBchr. f. physiol Chem., Bd. 13. 500 THE UBINE. developed, by the actioQ of acid in normal urine when exposed t» the air must be added the trROPHAiN of Hellbe,' the various UKOMELANiKS, and other bodies described by different investigators (Plos'z/ Thudichum," Schunck*). Indigo-blue (ueoglaucin" of Hellek, ueoctaxin, ctanukin', and other coloring matters of older investigators °) is split off from the indoxyl-sulphuric acid or indoxyl-glycuronic acid. Eed coloring matters may be formed from the conjugated indoxyl and skatoxyl acids, and ueohodii^^ (Heller), UEOEUBHq- (Plos'z), ueoh^matik (Haeley'), and perhaps also ueoeosein (Nencki and Sibbee') probably have such an origin. We cannot enter into too many details of the different coloring matters obtained as decomposition products from normal urine; and as the preformed physiological coloring matters of urine have not been closely studied, we can only discuss the most carefully investigated urinary pigment, urobilin. Urobilin was first prepared from urine by Jaeee.' This color- ing matter occurs in urine especially in fevers, and it is therefore designated eebeile ueobilin by MAcMuifN.' The urobilin occurring in normal urine is somewhat different from an optical standpoint from the above, and is called koemal ueobilin by MacMunn. As above stated, a mother-substance of urobilin, a UEOBiLiNOGEN, occurs in the urine, from which urobilin is pro- duced by the action of the air. Many investigators claim that urobilin is identical with hydro- bilirubin (Malt) and corresponds to the composition Cj^H^^If^O,. Also, that urobilin is formed by a reduction of bilirubin in the intestine. The correctness of this view is disputed by others (MacMukn, Le Nobel '"). According to MacMunn, hydrobili- ' Heller's Arch. (2), Bd. 1. Cited from Happert-Neubauer, S. 326. ' Zeitsclir. f. physiol. Chem., Bd. 8. » Brit. Med. Journal, Vol. 201 (1864), and Journ. f. prakt. Chem., Bd. 104. ' Cited from Huppert-Neabauer, S. 509. ' Ibid., S. 161. ' In regard to this and other red pigments see Huppert-Neubauer, S. 557- 598. ' Journ. f. prakt. Chem. (2), Bd. 26. 8 L. c. ' Proo. Roy. Soc, Vols. 31 and 35; Ber. d. deutsch. chem. Gesellsch., Bd. 14; Journal of Physiol., Vols. 6 and 10. In regard to difEerent urobilins, see Bogomolofi, Maly's Jahresber., Bd. 23, and Eichholz, Journal of Physiol., Vol. 14. "• See Chapter VIII, on Bile-pigments. UROBILIN. 501 rnbin and urinary nrobilin are not identical bodies, because he obtained normal urobilin by the action of peroxide of hydrogen upon a solution of hsematin in alcohol containing sulphuric acid. Pigments similar to urobilin, though not identical, have been obtained from the biliary and blood coloring matters. Besides hydrobilirnbin, prepared by Maly from bilirubin, Stokvis' ■obtained a choletelin from a biliary pigment, cholecyanin, by the action of zinc chloride and tincture of iodine, or by boiling with a little lead peroxide. This choletelin acts like urobilin, but that ■obtained from bilirubin by the action of nitric acid does not. Bodies similar to nrobilin have also been obtained by Hoppe- Setlee ' by the reduction of heematin and haemoglobin with zinc and hydrochloric acid; by Le Nobel ' by treating an acid-alcoholic -or alkaline solution of hsematoporphyrin with tin or zinc; and lastly by N"encki and Siebek ^ by treating heematoporphyrin with zinc and hydrochloric acid. From the observations of Le Nobel and Nencki and Sieber it follows that these pigments artificially pre- pared from the blood-coloring matters are not identical with urinary urobilin, even though they are closely related from an optical stand- point. It must be left undecided whether these bodies are identical with each other or with the urinary urobilin, or if the observed •difEereuce is only due to a contamination with other bodies. We have numerous observations on the elimination of urobilin in disease, especially by Jaffe, Disque, Deetfuss-Beissac, CrEEHAEDT, G. Hoppe-Setlee,' and others. Because of our im- "perfect knowledge of the urobilin of the urine and the ueobilin- OIDIN (this name has been given by Le Nobel to the substance similar to urobilin artificially prepared by him) it is difficult to say anything positive in regard to the occurrence of urobilin in the urine in disease. During the absorption of large blood extrava- sations, as also in diseases connected with destruction of the blood- corpuscles or of the appearance of methsemoglobin in the blood- plasma, the urine becomes dark in color, which generally depends npon an increased elimination of urobilin. The question whether ' See Chapter VIII. ' Ber. d. deutsch. cbem. Gesellseh., Bd. 7. ' PflUger's Arch., Bd. 40. ■* Monatsbefte f. Chem., Bd. 9, and Arch. f. exp. Path. u. Pharm., Bd. 24. » In regard to the literature on this subject we refer the reader to D. Qer- hardt, " Ueber Hydrobilirnbin und seine Beziehungen zumlkterus" (Berlin 1889), and also G. Hoppe-Seyler, Virchow's Arch., Bd. 124. 502 THE URINE. it depends on an increased elimination of nrinary urobilin or, as is more probable, upon the nrobilinoidin produced from the blood-coloring matters is still doubtful. In icterus the elimination of urobilin is often increased, and indeed cases occur in which urobilin is almost the only coloring matter which can be detected in the urine (ueobilinictbeus). In these cases we are probably dealing with a urobilinoid substance produced from bilirubin in the intestinal tract by reduction. The urobilin obtained from fever urine is, according to Jaffe, amorphous, red, dingy red, or reddish yellow, according to the method of preparation. It dissolves easily in alcohol, amyl-alcohol, and chloroform, but less readily in ether. It is less soluble in water, but the solubility is augmented in the presence of a neutral salt. It may be precipitated from a solution saturated with ammonium sulphate by the addition of sulphuric acid (Mbht'). It is soluble in alkalies, and is incompletely precipitated from the alkaline solution by the addition of acid. It is partly dissolved by chloroform from an acid (watery-alcoholic) solution; alkali solutions remove the urobilin from the chloroform. Alkaline solutions of urobilin give insoluble combinations with salts of the heavy metals, such as zinc and lead. Urobilin does not give Gmelin's test for bile-pigments. Neutral alcoholic urobilin solutious are in strong concentration brownish yellow, in great dilation yellow or rose-colored. They have a strong green fluorescence. The acid-alcoholic solutions are brown, reddish yellow, or rose-red, according to concentration. They are not fluorescent, but show a faint absorption-band, y, between I and F, which borders on F, or in greater concentration extends over F. The alkaline solutions are brownish yellow, yellow, or (the ammoniacal) yellowish green, according to concentration. If some zinc-chloride solution is added to an ammoniacal solu- tion, it becomes red and shows a beautiful green fluorescence. This solution, as also that made alkaline with fixed alkalies, shows a darker and more sharply defined band, S, almost midway between h and F. The urobilins obtained from the urine by MacMunk by another method, and that obtained by Jaffe, differ from each other mainly in the following : A solution of normal urobilin becomes deeper red ' Journal de pbarm. et de chim., 1878. Cited from Maly's JaUresber., Bd.. 8, S. 269. PREPABATION AND ESTIMATION OF UROBILIN. 503 with soda, while febrile urobilin becomes yellow. The band y of normal urobilin disappears on the addition of alkali, while the corresponding band of febrile urobilin inoves towards the left. The ethereal solution of febrile urobilin shows two faint absorption-bands on each side of D which are not to be seen in the watery solution nor in the urine. Febrile urobilin is a brownish-red and the normal a yellowish-brown powder. Febrile urobilin is, according to MacMunn, converted into normal urobilin by potassium perman- ganate. In preparing urobilin from normal urine, precipitate the urine with basic lead acetate (Jaffe), wash the precipitate with water, dry at the ordinary temperature, then boil it with alcohol, and decompose it when cold with alcohol containing sulphuric acid. The filtered alcoholic solution is diluted with water, saturated with ammonia, and then treated with zinc-chloride solution. This new precipitate is washed free from chlorine with water, boiled with alcohol, dried, dissolved in ammonia, and this solution precipitated with sugar of lead. This precipitate, which is washed with water aid boiled with alcohol, is decomposed by alcohol containing sul- phuric acid, the filtered alcoholic solution is mixed with J vol. chloroform, diluted with water, and shaken repeatedly, but not too energetically. The urobilin is taken up by the chloroform. This last is washed once or twice with a little water and then distilled, leaving the urobilin, which is purified from a contaminating red coloring matter by means of ether. According to Jaffe, the coloring matter can be directly precipitated from a fev«r urine rich in urobilin by ammonia and zinc chloride, and this precipi- tate treated as above. Mbht faintly acidifies ttie urine with sulphuric acid (1-2 grms. per litre), then saturates with ammonium sulphate, washes tbe pre- cipitate on a filter with an acidified ammonium-sulphate solution, presses the filter, and extracts the coloring matter with absolute alcohol at a gentle heat after the addition of a few drops of ammonia. MacMdnn precipitates the urine with sugar of lead and basic lead acetate, decomposes the precipitate with acidified alcohol, dilutes the solution with water, shakes with chloroform, evaporates this last, and dissolves the residue repeatedly with chloroform. The method of preparation, according to MacMtjnn, is the same for both urobilins, the normal and the febrile. The color of the acid or alkaline solution, the beautiful fluores- cence of the ammoniacal solution treated with zinc chloride, and the absorption-bands of the spectrum, all serve as means of detect- ing urobilin. In fever urines the urobilin may be detected directly or after the addition of ammonia and zinc chloride by its spectrum. It may plso be detected sometimes In normal urine directly or after the urine has stood exposed to the air until the chromogen has been converted into urobilin. If it cannot be detected by means of the spectroscope, then the urine may be treated with a mineral acid and shaken with ether. The ethereal solution, directly or after 604 THE URINE. «oncentration, may be tested with the spectroscope. It is often better to dissolve the residue, after the eyaporation of the ether, in abso- lute alcohol, and use this for the spectroscopic investigation. According to Salkowski, the urobilin may be directly extracted by gently shaking with ether free from alcohol. If the urobilin cannot be detected by the above-described methods, then precipi- tate the urine with basic lead acetate, decompose the precipitate with acidified alcohol, test this solution or extract the coloring matter by dilating with water and shaking with chloroform. In the quantitative estimation of urobilin we proceed as follows, according to G. Hofpe-Sbtlek : ' 100 c. c. of the urine are acidified with sulphuric acid and saturated with ammonium sul- phate. The precipitate is collected on a filter after some time, Washed with a saturated solution of ammonium sulphate, and repeatedly extracted with equal parts alcohol and chloroform after pressing. The filtered solution is treated with water in a separa- "tory funnel until the chloroform separates well and becomes clear. The chloroform solution is evaporated on the water-bath in a weighed beaker, the residue dried at 100° C, and then extracted -with ether. The ethereal extract is filtered, the residue on the filter •dissolved in alcohol, and transferred to the beaker and evaporated, then dried and weighed. According to this method G. Hoppe- Setlbe found 0.08-0.14 grm. urobilin in one day's urine of a Jiealthy person, or an average of 0.133 grm. The real yellow pigment of urine has been only slightly investi- gated. This pigment has been called ubocheom by Gaekod,' which name has been used by Thudichum earlier to designate a mixture of pigments and other substances. The pigment isolated toy Gaeeod by a rather complicated method was amorphous brown, very easily soluble in water and ordinary alcohol, less soluble in absolute alcohol, and insoluble in ether, chloroform, and benzol. It shows no absorption -bands, and does not fluoresce on the addition of ammonia and zinc chloride. JjToerytlirin is that coloring matter which often colors the urinary sedi- ment {sedimentum lateriiium) beautifully red. It occurs especially in fevers and other diseases, but it is also found in the urine of perfectly healthy per- sons. Its solution is colored green by alkalies and, according to ZoJA,^ shows a strong absorption beginning between D and E and extending to F. This absorption consists of two bands, of which the one at P is the stronger. Uroerythrin dissolves readily in amyl alcohol. Gabrod'' has suggested a method for obtaining uroerythrin, and has given further contributions for the detection of the same. Attention is called especially to the well-known prop- erty of uroerythrin of being bleached on exposure to light. > Virchow's Arch., Bd. 124. ' Proc. of Hoy. Hoc, Vol. 55, 1894 See also Thudichum, Brit. Med. Jour- nal, 1864, Vol. 3; Journal f. prakt. Chem., Bd. 104. ' Arch. ital. di clinica med., 1893; also Centralbl. f. d. med.Wissensch., 1893. * Journal of Physiol., Vol. 17. OARBOBTDRATES IN THE URINE. 505 Volatile fatty acids, sach as formic acid, acetic acid, and perhaps also Ijutyrio acid, occur under normal conditions in human urine (v. Jaksch '), also in that of dogs and herbivora (Schotten"). The acids poorest in carbon, such as formic acid and acetic acid, are more constant in the body than those richer in carbon, and therefore the relatively greater part of these pass unchanged into the urine (Schottbn). Normal human urine contains besides these bodies others which yield acetic acid when oxidized by potassium dichromate and sul- phuric acid (v. Jaksch). The quantity of volatile fatty acids in normal urine is, according to v. Jaksch, 0.008-0.009 gnu. per 24 hours, and according to V. ROKITANSKT,' 0.054 grm. The quantity is increased by exclusive farina- ceous food (RoKiTANSKT), also in fever and in certain diseases of the liver (v. Jaksch). It is also increased in leucaemia and in many cases of diabetes (v. Jaksch). Large amounts of volatile fatty acids are produced in alkaline fer- mentation of the urine, and the quantity is 6-15 times as large as in normal urine (Salkowski''). Paraliictic Acid. It is claimed that this acid occurs in the urine of healthy persons after very fatiguing marches (Colasanti and Moscatblli''). It is found in larger amounts in the urine in acute phosphorus-poisoning or acute yellow atrophy 'of the liver (ScHtJLTZBN and RiESS'). According to the inves- tigations of Hoppe-Sbtlbr and Araki,' lactic acid, besides sugar, passes into the urine as soon as the supply of oxygen is decreased in any way. Minkowski ' has shown that lactic acid occurs in the urine in large quantities on the extir- pation of the liver of birds. Olycero-plwapliorie acid occurs as traces in the urine, and it is probably a decomposition product of lecithin. The occurrence of aucciniaacid in normal urine is the subject of discussion. Carbohydrates and Reducing Substances in the Urine. The occurrence of grape-sugar as traces in normal urine is highly prob- able, as the investigations of Brucke, Abbles, and T. Udranszkt show. The last investigator has also shown the habitaal occurrence of carbohydrates in the urine, and their presence has been positively proved by the investigations of BAUMiNX and WEDBifSKi, and especially by Baisch. Besides glucose normal urine contains, according to Baisch, another not well-studied variety of sugar, probably isomaltose, and besides this a dextrin-like carbohydrate (animal gum), as shown by Lan-dwbhs, Wbdenski, and Baisch.' Besides traces of sugar and the previously mentioned reducing substances, uric acid and creatinin, the urine contains still other reducing substances. These last are probably (Fluckigee") con- ' Zeitschr. f . physiol. Chem. , Bd. 10. « Ibid., Bd. 7. ' Wien. med. Jahrb., 1887; cited from Maly's Jahresber., Bd. 17. * Zeitschr. f. physiol. Chem., Bd. 13. ' Moleschott's Untersuch. zur Naturlehre, Bd. 14. • Chem. Centralbl. , 1869. ' Zeitschr, f. physiol. Chem., Bdd. 15, 16, 17, and 19; Irisawa, ibid., Bd. 17-. » Arch. f. exp. Path. u. Pharm., Bdd. 21 and 31. ' Zeitschr. f. physiol. Chem., Bdd. 18, 19, and 20; Treupel, ibid., Bd. 16. These articles contain references to the work of other investigators. '» Zeitschr. f. physiol. Chem., Bd. 9. 506 TEE UBINE. jugated combinations of glycuronic acid, C,Hj„0,, which closely resembles sugar. The reducing power of normal urine corresponds, according to various investigators, to 1.5-5.96 p. m. grape-sugar." Glycuronic Acid, C,H,„0, or CHO.(CH.OH),.COOH. This acid may be converted into saccharic acid, C^Hj^O,, by the action of bromine (Thierpeloer"), and it seems to occupy an inter- mediate position between this acid and gluconic acid, C,H„0,. It is a derivative of glucose, and Fischer and Piloty ' have prepared it synthetically by the reduction of saccharo-lactonic acid. Further reduction yields gnlonic acid lacton (Thieepelder). Glycuronic acid is an intermediate metabolic product, and it only occurs in the nrine when it is protected from combustion in the animal body by combining with other bodies. Such conjugated combinations with indoxyl, skatoxyl, and phenols occur probably normally in very small quantities in human urine. This acid as conjugated glycu- ronic acids passes in large quantities into the urine after the admin- istration of various therapeutic agents or certain other substances. Thus ScHMiEDEBERG and Meter ' found campho-glycuronic acid in the urine after partaking of camphor, and v. Mering ' showed the presence of urochloralic acid (see Accidental Constituents of the Urine) after the administration of chloral hydrate. According to SOHMIEDEBERG,' glycuronic acid seems to occur in cartilage because it is contained in chondrosin, a cleavage product of chondroitic- sulphuric acid. It is also found in the artist's color " jaune indien," which contains the magnesium-salt of euxanthonic acid (euxanthon-glycnronic acid). On heating this acid with water to 120-125° 0. it splits into euxanthin and glycuronic acid, and it is the most available material for the preparation of glycuronic acid (Thierfelder). Another acid, isomeric with the ordinary glycu- ronic acid, has been found in the urine in certain cases (see Acci- dental Constituents of the Urine). Glycuronic acid is not crystalline, but is obtained only as a syrup. It dissolves in alcohol and is easily soluble in water. If the watery solution is boiled for an hour, the acid is in part (20^) con- ' See Huppert-Neubauer, S. 72. ' The works of Thierfelder on glycuronic acid are found in Zeitschr. f. physiol. Chem., Bdd. 11, 13. and 15. ' Ber. d. deutsch. chem. Gesellsch., Bd. 34, S. 531. * Zeitschr. f . physiol. Chem. , Bd. 3. ' Ibid , Bd. 6. « Arch. f. exp. Path. u. Pharm., Bd. 28. oi^rcuBoiric acid. 507 verted into the anhydride gltoueon, 0,H,0., which is crystalline, soluble in water, but insoluble in alcohol. The alkali salts of this acid are crystalline. The neutral barium salt is amorphous, soluble in water, but is precipitated by alcohol. If a concentrated solution of the acid is saturated with barium hydrate, the basic barium salt separates. The neutral lead salt is soluble in water, but the basic salt is, on the contrary, insoluble. The acid is dextrogyrate and reduces copper, silver, and bismuth salts. It does not ferment with yeast. Glycuronic acid gives the fnrfurol reaction and acts like a pentose when tested with the phloroglucin-hydrochloric-acid test. With phenylhydrazin potassium glycaronate gives a flaky yellow precipitate of microscopic needles which melt at 114-115° 0. (Thierfeldee). The statements in regard to the behavior of glycuronic acid with this test are very contradictory." All conjugated glycuronic acids are laevorotatory, while glycu- ronic acid itself is dextrorotatory. They are split into glycuronic acid and the several other groups by the addition of water. A few of the conjugated glycuronic acids, such as the urochloralic acid, reduce copper oxide and certain other metallic oxides in alkaline solution, and therefore they may interfere with the detection of sugar in the urine. Glycuronic acid may be prepared from urochloralic acid or campho-glycaronic acid by boiling with a mineral acid. It may be prepared more easily by heating euxanthonic acid with water in Papin's digester to 120-125° C. for an hour and evaporating the watery solution at -f- 40° 0. The anhydride which crystallizes gradually is removed, the mother-liquor diluted with water and boiled for a time to convert a second portion of acid into anhydride, and then evaporated at about -j- 40° C. This is continued until nearly all the acid is converted into anhydride. The anhydride may then be further purified. Organic combinations containing sulphur of unknown kind, which may in small part consist of sulplwcyanides, 0.04 ((j(schbidlkn°)-0.11 p. m. (I. MuNK),* cyatin, or bodies related to it, and protein bodies, are found in Uuman as well as in animal urines. Lang* has shown that nitriles of the fatty seyies when united with hydrocyanic acid in the animal body are converted into sulpliocyanides, and pass as such into the urine. This sulpchocyanide orig- inates, it seems, from the readily cleavable, non-oxidizable sulphur of the proteid bodies, which, as Pascheles (ibid.) has shown, readily converts potas- ' In regard to literature see Hammarsten, Zeitschr. f, physiol. Chem., Bd. 19, S. 30, and Roos, ibid. , Bd. 15, S. 535. ' Pflllger's Arch., Bd. 14. » Virchow's Arch., Bd. 69. •• Arch. f. exp. Path. u. Pharm., Bd. 34. 608 TBE URINE. svaxD. cyanide into alkali sulphocyanide in an allialine reaction and at tbe tem- perature of the body. Tlie amido-acids of the fatty series in the body are probably oxidized to nitriles, which are then transformed into sulpLiocyauides by the sulphur of the proteids. The sulphur of these mostly unknown com- binations has been called " neutral," to differentiate it from the " acid " sulphur of the sulphate and ethereal-sulphuric acids iSalkowski '). The neutral sulphur in normal urine as determined by Salkowbki is \5%, by Si'ADThagbn ' 13.3- 14.5^, and by Lbpinb ^ 20^ of the total sulphur. In starvation, according to Fr. MiJLLBK,'' the absolute and relative quantities increase. According to Hbfftbr' the quantity is greater with a bread diet than with a meat diet. Excessive muscular exercise increases the eliminntion of the acid as well as the "neutral sulphur; still, according to Bbck and Bbnedikt,' the increase in neutral sulphur takes place earlier. According to Prbsch ' sulphur when introduced in the body increases the elimination of neutral sulphur; indeed, about one fourth of the sulphur absorbed in the elementary state passes intoorganic combinations, not oxidizable by nitfic acid alone. According to the investigations of W. Smith' it is probable that the most unoxidizable part of the neutral sulphur occurs as sulpho-acids. An increased elimination of neutral sulphur has been •observed in various diseases, such as pneumonia, icterus, and cystiuuria. The total quantity of sulphur in the urine is determined by fusing tbe solid urinary residue with saltpetre and caustic alkali. The quantity of neutral sulphur is determined as tbe difEerence between tbe total sulphur and the sulpliur of the sulphate and ethereal-sulphuric acids. Sulphuretted hydrogen occurs in urine only under abnormal conditions or as a decomposition product. Sulphuretted hydrogen may be produced from the neutral sulphur of the organic substances of the urine by the action of •certain bacteria (Fb. Mijllbk,' Salkowski'"). Other investigators have given hyposulphites as the source of the sulphuretted hydrogen. The occurrence of liyposulphites in normal human urine, which is asserted by Hefptbr," is ■disputed hy Salkowski" and Phesch.'* Hyposulphites occur constantly in cat's urine and, as a rule; also in dog's urine. Organic combinations containing phosplwrus (g\jcevo--ph.os^'ixoTic acid, etc.), ■which yield phosphoric acid on fusing with saltpetre and caustic alkali, are also found in mine (Lbpikb, Etmonnet, and Aubbrt'*). Enzymes of various kinds have been isolated from the urine. Among these we may mention pepsin (Bbdckb '' and others), diastatic enzyme (Cohnhbim " and others). The occurrence of rennin and trypsin in the urine is doubtful.'-'' ' Virchow's Arch., Bd. 58, and Zeitschr. f. physiol. Chem., Bd. 9. ' VircUow's Arch., Bd. 100. ' Compt. rend., Tomes 91 and 97. * Berlin, klin. Wochenschr. , 1887. ' Pflttger's Arch., Bd. 38. « Maly's Jahresber., Bd. 33, S. 338. •"Virchow's Arch., Bd. 119. * Zeitschr. f. physiol. Chem., Bd. 17. ' Berlin, klin. Wochenschr., 1887. '«2Wd., 1888. " Pfliiger's Arch., Bd. 38. " Ibid., Bd. 39. " Vircho^w's Arch., Bd. 119. "Compt. rend.; Tome 98, and Compt. rend." de la soc. de Biol., 1882 and 1884.' " Wien. Sitzungsber., Bd. 43. >» Virchow's Arch., Bd. 38. " In regard to the literature on enzymes in the urine see Huppert-Neu- bauer, p. 599. INORaANIC 00N8TITUENTS OF UHINE. M^ Substances similar to mucin (nucleoalbumin ?) from the urinary passages and the bladder are generally present in the urine, though in very small quantities. According to several investigators normal human urine also contains traces of proteid. Ptomaines and lexicomaines or poisonous substances of an unknown kind, which are often described as alkaloidal substances, occur in normal urine (PODCHET, BonCHARD, Addcco, and others). Under pathological conditions the quantity of these substances may be increased (Bouchabd, Lbpine and GUEKIN, ViLLlEES, and olhers). Within the last few years the poisonous properties of urine have been the subject of more thorough investigation, especially by BonCHARD. He found that the night urine is less poisonous than the day urine, and that the poisonous constituents of the day and night urines have not the same action. In order to be able to compare the toxidity of the urine under differt-nt conditions, Bouchard determines the urotoxic COEFFICIENT, which is the weight of rabbit in kilos which is killed by the qunntiiy of urine excreted by one kilo of the person experimented upon in 24 hours.' Baumann and v. Udranszkt ' have shown that ptomaines may occur in the urine under pathological conditions. They demonstrated the presence of the two p omaines discovered and first isolated by Bribgeb — putrescine, C^HjjNj (tetramethylendiamin), and cadaverin, CjHuNj (pentametliylendi- amin) — in the urine of a patient suffering from cystinuria and catarrh of the bladder. Cadaverin has later been found by Stadthagen and Briegkr ' in the urine in two cases of cystinuria. Bhieger, v. UdrAnszkt and Baumann, and Stadthagen have shown that not only these but other diamins occur under physiological conditions. The occurrence in normal urine of any ' ' urine poison " is denied by certain investi- gators, such as Stadthagen.^ The poisonous action of the urine, according to them, i-i due in part to the potassium salts and in part to the sum of the toxidity of the other normal urinary constituents (urea, creatluin, etc.), which have very little poisonous action individually. Many substances have been observed in animal urine which are not found in human urine. To these belong: kynurenic acid, C,oH,NOs , which is an oxychinolincarbonic acid, occurring in dog's urine ; urocanic acid, found in dog's urine ; damaluric acid and damolic acid (according to Schotten ^ probably a mixture of benzoic acid with volatile fatty acids), obtained by the distillation of cow's urine ; and lastly lithuric acid, found in the urinary con- crements of certain animals. HI. Inorganic Constituents of Urine. Chlorides. The chlorine occurring in urine is undoubtedly combined with the bases contained in this excretion ; the chief part is combined with sodium. In accordance with this, the quantity of chlorine in the urine is generally expressed as NaCl. The quantity of chlorine combinations in the urine is subject to considerable variation. In general the quantity for a healthy adult ' A complete bibliography on ptomaines and leucomaines in the urine is found in Huppert-Neubauer, p. 403. See also GriflSths, Compt. rend.. Tomes 113, 114, and 115, on ptomaines in the urine in different infectious diseases. « Zeitschr. f. physiol. Chem., Bd. 13. » Virchow's Arch., Bd. 115. * Zeitschr. f. klin. Med., Bd. 15. ' Zeitschr. f. physiol. Chem., Bd. 7. 510 TOE URINE. on a mixed diet is 10-15 grms. NaCl per 34 hours. * The qnantity of common salt in the urine depends chiefly npon the quantity of salt in the food, with which the elimination of chlorine increases and decreases. The free drinking of water also increases the elimination of chlorine, which is greater during activity than during rest (at night). Certain organic chlorine combinations, such as chloroform, may increase the elimination of inorganic chlorides by the urine (Zellee," Mtlius, Kast'). lu diarrhoea, in quick formation of large transudations and exudations, also in specially-marked cases of acute febrile diseases at the time of the crisis, the elimination of common salt is materi- ally decreased. The elimination is abnormally increased in the first days after the crisis and during the absorption of extensive exu- dations. A diminished elimination of chlorine is found in disturbed absorption in the stomach and intestine, and in acate and chronic diseases of the kidneys accompanied with albuminuria. In chronic diseases the elimination of chlorine in general keeps pace with the nutritive condition of the body and the activity of the secretion of the urine. As under physiological conditions the quantity of common salt taken with the food has the greatest influence on the elimination of NaCl in disease The quantitative estimation of chlorine in urine is most simply performed by titration with silver-nitrate solution. The urine must not contain either proteid (which if present must be removed by coagulation) or iodine or bromine compounds. In tbe presence of bromides or iodides evaporate a measured quantity of the urine to drym-ss, fuse tlie residue witli saltpetre and sod i, dis olve the fused mrss in water, and remove the iodine or bromine by the addition of dilute sul- phuric acid and some nitrite, and tlioroughly shalie with carbon disulphide. Tbe liquid thus ( btained may now be titrated with silver nitrate according to VoiiHARD's metliod. Tlie quantity of bromide or iodide is calculated as the difference between the quantity of silver-nitrate solution used for the titration of tb solution of the fused mass and the quantity used for the corresponding volume of the original urine. The otherwise excellent titration method of Mohe, according to which we titrate with silver nitrate in neutral liquids, using neutral potassium chromate as an indicator, cannot be used directly on the urine in careful work. Organic urinary constituents are also precipitated by the silver-salt, and the results are therefore some- what high for the chlorine. If we wish to use this method, the organic urinary constituents must first be destroyed. For this pur- pose evaporate to dryness 5-10 c. c. of the urine, after the addition of 1 grm. of chlorine-free soda and 1-3 grms. chlorine-free salt- ' Zeitschr. f. physlol. Chem., Bd. 8. 'Ibid.. Bd. 11. CHLORIDES. 511 petre, and carefully fuse. The mass is dissolved in water, acidified faintly with nitric acid, and then neutralized exactly with pure lime carbonate. This neutral solution is used for the titration. N The silver-nitrate solution may be a — solution. It is often made of such a strength that each c. c. corresponds to 0.006 grm. CI or 0.01 grm. NaCl. This last-mentioned solution contains 29.075 grms. AgNO, in 1 litre. Freund and Toepffee ' have modified this method in that they titrate with silver nitrate in acetic-acid solution, which prevents the precipitation of the silver combinations of uric acid, xanthin bases, etc. Dilute 5 or 10 c. c. of the urine with 25 c. c. water, add 2.5 c. c. of a solution of acetic acid and sodium acetate (3^ acid and 10^ sodium acetate), and titrate after the addition of potassium chromate. Another modification has recently been sug- gested by BODTKEK." Volhaed's Method. Instead of the preceding determination, Volhaed's method, which can be performed directly on the urine, may be employed. The principle is as follows: All the chlorine from the urine acidified with nitric acid is precipitated by an excess of silver nitrate, filtered, and in a measured part the quantity of silver added in excess is determined by means of a sulphocyanide solution. This excess of silver is completely precipitated by the sulphocyanide and a solution of some ferric salt, which, as is well known, gives a blood-red reaction with the smallest quantity of sulphocyanide, is used as an indicator. We require the following eolations for this titration: 1. A silver- nitrate solution which contains 29.075 grms. AgNO, per litre and of which each c. c. corresponds to 0.01 grm. NaCl or 0.00607 grm. CI; 2. A saturated solution at the ordinary temperature of chlorine- free iron alum or ferric sulphate ; 3. Chlorine-free nitric acid of a specific gravity of 1.2; 4. A potassium-sulphocyanide solution which contains 8.3 grms. KCNS per litre, aad of which 2 c. c. corresponds to 1 c. c. of the silver-nitrate solution. About 9 grms. of potassium sulphocyanide are dissolved in water and diluted to one litre. Tlie quantity of KCNS contained in this solution is determined by the silver-nitrate solution in the following way: Measure exactly 10 c c of the silver solution and treat with 5 o. c. of nitric acid and 1-3 c. c. ot the ferric- salt solution, and dilute with water to about 100 c. o. Now the sulphocyanide solution is added from a burette, constantly stirring, until a permanent faint red coloration of the liquid takes place. The quantity of sulphocyanide found in the solution by this means indicates how much it must be diluted to be of the proper strength. Titrate once more with 10 c c. AgNOs solution and cor- rect the sulphocyanide solution by the careful addition of water until 30 c. c. exactly correspond to 10 c. c. ot the silver solution. The determination of the chlorine in the urine is performed by • Centralbl. f. klin. Med., Bd. 13, No. 38. Cited from Maly's Jahiesber., Bd. 23, S. 335. ' Zeitschr. f. physiol. Chem., Bd. 30. 512 THE URINE. this method in the following way: Exactly 10 c. c. of the urine are placed in a flask which has a mark corresponding to 100 c. c. ; 5 e. 0. nitric acid are added; dilate with about 60 c. c. water, and then allow exactly 20 c. c. of the silver-nitrate solution to flow in. Close the flask with the thumb and shake well, slide off the thumb and wash it with distilled water into the flask, and flll the flask to the 100-c. c. mark with distilled water. Close again with the thnmb, carefully mix by shaking, and filter through a dry filter. Measure off 50 c. c. of the filtrate by means of a dry pipette, add 3 c.^ c. ferric-salt solution, and allow the sulphocyanide solution to flow in until the liquid above the precipitate has a permanent red color. The calculation is very simple. For example, if 4.6 c. o. of the sulphocyanide solution were necessary to produce the final reaction, then for 100 c. c. of the filtrate (=10 c. c. urine) 9.2 c. c. of this solution are necessary. 9.3 c.c. of the sulphocyanide solution corresponds to 4.6 e. c. of the silver solution, and since 30 — 4.6 = 15.4 c. c. of the silver solution were necessary to com- pletely precipitate the chlorides in 10 c. c. of the urine, then 10 c. c. contain 0.154 grm. NaCl. The quantity of sodium chloride in the urine is therefore 1.54^ or 15.4 "/jo. If we always use 10 c. c. for the determination, and always 30 c. c. AgNO,, and dilute with water to 100 c. c, we find the quantity of NaCl in 1000 parts of the urine by subtracting the number of c. c. of sulphocyanide (E) required with 50 c. c. of the filtrate from 30. The quantity of NaCl p. m. is therefore under these circumstances = 30 — R, and the percentage of NaCl = — ~ — . The approximate estimation of chlorine in the urine (which must be free from proteid) is made by strongly acidifying with nitric acid and then adding to it, drop by drop, a concentrated silver-nitrate solution (1 : 8). In a normal quantity of chlorides the drop sinks to the bottom as a rather compact cheesy lump. In diminished quantities of chlorides the precipitate is less compact and coherent, and in the presence of very little chlorine a fine white precipitate or only a cloudiness or opalescence is obtained. Phosphates. Phosphoric acid occurs in acid urines partly as double-, MHjPO<, and partly as simple-acid, M,HPO,, phosphates, both of which are found in acid urines at the same time. Ott ' found that on an average 60^ of the total phosphoric acid was double- and 40j^ was simple-acid phosphate. The total quantity of phosphoric acid is very variable and depends on the kind and the quantity of food. The average quantity of P.O^ is in round num- bers 3.5 grms., with a variation of 1-5 grms., per 34 hours. A small part of the phosphoric acid of the urine originates from the ' Zeitschr. f. pUysiol. Cliem., Bd. 10. PH08PHA TBS. 513 baming of organic compounds, nnolein, protagon, and lecithin, within the organism. The greater part originates from the phos- phates of the food, and the quantity of eliminated phosphoric acid is greater when the food is rich in alkali phosphates in proportion to the quantity of lime and magnesia phosphates. If the food contains much lime and magnesia, large quantities of earthy phos- phates are eliminated by the excrements; and even though the food contains considerable amounts of phosphoric acid in these cases, the quantity of phosphoric acid in the urine is small. Such a condition is found in herbivora, whose urine is habitually poor in phosphates. The extent of the elimination of phosphoric acid by the urine depends not only upon the total quantity of phosphoric acid in the food, but also upon the relative amounts of alkaline earths and the alkali salts in the food. According to Peetsz,' Olsavsky and Klug " the elimination of phosphoric acid is considerably increased by intense muscular work. Prom the transformation of tissues rich in proteid or of phos- phorized nerve-substance in the body we might perhaps expect an equal relation between the nitrogen and the phosphoric acid in the urine. Many investigations have been made upon this subject, but as all the conditions which afEect the elimination of phosphoric acid are not yet sufficiently known, it is difficult to draw any definite conclusions from the observations thus far made. As the extent of the elimination of phosphoric acid is mostly dependent upon the character of the food and the absorption of the phosphates in the intestine, it is apparent that the relationship between the nitrogen and phosphoric acid in the urine can only be approximately constant with a certain uniform food. Thus, oa feeding with exclusive meat diet, as observed by Voit' on dogs, when the nitrogen and phosphoric acid (P^O,) of the food exactly reappeared in the urine and fgeces the relationship was 8.1 : 1. In starvation this relationship is changed, namely, relatively more phos- phoric acid is eliminated, which seems to indicate that besides flesh and related tissues also another tissue rich in phosphorus is largely- destroyed. The starvation experiments show that this tissue is the bone tissue. ' SeeMaly's Jahresber., Bd. 21. ' Pflilger's Arcli. , Bd. 54. » Pliysiologie des allgemeinen Stoffwechsels und der Ernahrung in L. Hermann's Handbueh, Bd. 6, Thl. 1, S. 79. 514 THE URINE. Little is known in regard to the elimination of phosphoric acid- in disease. In febrile diseases, as shown by several observations, the quantity of phosphoric acid as compared with the nitrogen is con- siderably decreased. In diseases of the kidneys the activity ^of these organs in eliminating the phosphates may be considerably dimin- ished (Pleischbk '). In meningitis, on the contrary, a marked increase in the phosphates is observed in the nrine. Teissier has described a special form of polyuria, in which large quantities of earthy phosphates, 10-30-30 grms. per 24 hours, were eliminated. This polyuria was called phosphate diabetes " by Teissier. The statements in regard to the quantity of phosphate in the urine in rachitis and in osteomalacia are somewhat contradictory. ° Quantitative estimation of phosphoric acid in the urine. This estimation is most simply performed by titrating with a solution of uranium acetate. The principle of the titration is as follows: A warm solution of phosphates containing free acetic acid gives a vrhitish-yellow precipitate of uranium phosphate with a solution of a. uranium salt. This precipitate is insoluble in acetic acid, but dissolves in mineral acids, and on this account we always add in titrating a certain quantity of sodium-acetate solution. Potassium ferrocyanide is used as the indicator, which does not act on the uranium-phosphate precipitate, but gives a reddish-brown precipi- tate or coloration in the presence of the smallest amount of soluble uranium salt. The solutions necessary for the titration are: 1. A solution of a uranium salt of which each c. c. corresponds to 0.005 grm. P5O5 and which contains 30.3 grms. uranium oxide per litre. 30 c. c. of this solution corresponds to 0.100 grm. P^O,. 3. A solution of sodium acetate; 3. A freshly prepared solution of potasr sium ferrocyanide. The uranium solution is prepared from uranium nitrate or acetate. Dissolve . about 35 grms. uranium acetate in water, add some acetic acid to facilitate solution, and dilute to one litre. The strength of this solution is determined by titrating with a solution of sodium phosphate of known strrngth (10.085 grms crystallized salt in 1 litre, which corresponds to 0.100 grm. PjOs in 50 c. c). Proceed in the same way as in the titration of the urine (see below), and correct the solution by diluting with water, and titrate again until 20 c. c. of the uranium solution corresponds exactly to 50 c. c. of the above phosphate solution. The sodium-acetate solution should contain 10 grms. sodium acetate and 10 grms. cone, acetic acid in 100 c. o. For each titration 5 c. c. of this solution is used with 50 c c. of the urine. In performing the titration, mix 50 c. c. of filtered urine in a beaker with 5 c. c. of the sodium acetate, cover the beaker with a ' Deutsch. Arch. f. klin. Med., Bd. 29. » Centralbl. f. d. med. Wissensch., 1877. ^ In regard to the elimination of phosphates in disease see Neubaner-Hup- •pert-Thomas, Harnanalyse, 9. Aufl., Semiotischer Theil, S. 255-267. SULPHATES. 515 watch-glass, and warm over the water-bath. Then allow the uraniam eolation to flow in from a burette, and, when the precipi- tate does not seem to increase, place a drop of the mixture on a porcelain plate with a drop of the potassium-ferrocyanide solution. If the amount of uranium solution employed is not sufficient, the color remains pale yellow and more uranium solution must be added; but as soon as the slightest excess of uranium solution has been used, the color becomes faint reddish brown. When this point has been obtained, warm the solution again and add another drop. If the color remains of the same intensity, the titration is ended; but if the color varies, add more uranium solution, drop by drop, until a permanent coloration is obtained after warming, and now repeat the test with another 50 c. c. of the urine. The cal- culation is so simple that it is unnecessaiy to give an example. In the above manner we determine the total quantity of phos- phoric acid in the urine. If we wish to know the phosphoric acid combined with alkaline earths or with alkalies, we first determine the total phosphoric acid in a portion of the urine and then remove the earthy phosphates in another portion by ammonia. The pre- cipitate is collected on a filter, washed, transferred in a beaker with water, treated with acetic acid, and dissolved by warming. This solution is now diluted to 60 c. c. with water, and 6 c. c. sodium- acetate solution added, and titrated with uranium solution. The diiference between the two determinations gives the quantity of phosphoric acid combined with the alkalies. The results obtained are not quite accurate, as a partial transformation of the monophos- phates of the alkaline earths and also calcium diphosphate into triphosphates of the alkaline earths and ammonium phosphate takes place on precipitating with ammonia, which gives too high results for the phosphoric acid combined with alkalies remaining in solu- tion. Sulphates. The sulphuric acid of the urine originates only to a very small extent from the sulphates of the food. A dispropor- tionally greater part is formed by the burning of the proteids con- taining sulphur within the body, and it is chiefly this formation of sulphuric acid from the proteids which gives rise to the previously mentioned excess of acids over the bases in the urine. The quantity of sulphuric acid eliminated by the urine amounts to about 2.5 grms. n,SO, per twenty-four hours. As the sulphuric acid chiefly originates from the proteids, it follows that the elimination of sulphuric acid and the elimination of nitrogen are nearly parallel, and the relationship N : 11,80, is about 5:1. A complete parallel- ism can hardly be expected, as iu the first place a part of the sulphur is always eliminated as neutral sulphur, and secondly because the low quantity of sulphur in diilerent protein bodies undergoes 516 TEE URINE. greater variation as compared with the high quantity of nitrogen con- tained therein. Generally the relationship between the elimination of nitrogen and salpharic acid, under normal and diseased condi- tions, runs rather parallel. Sulphuric acid occurs in the urine partly preformed (sulphate-sulphuric acid) and partly as ethereal- sulphuric acid. The first is designated as A- and the other as 5-sulphuric acid. The quantity of total sulphuric acid is determined in the follow- ing way, but at the same time the precautions described in other works must be observed: 100 c. c. of filtered urine are treated with 5 c. c. concentrated hydrochloric acid and boiled for fifteen minutes. While boiling precipitate with 3 c. c. of a saturated BaCl^ solution and warm for a little while until the barium sulphate has completely settled. The precipitate must then be washed with water and also with alcohol and efclier (to remove resinous substances) and then treated according to the usual method. The separate determination of the sulphate-sulphuric acid and the ethereal-sulphuric acid may be accomplished, according to Baumann's ' method, by first precipitating the sulphate-sulphuric acid from the urine acidified with acetic acid by BaCl^, and then decomposing the ethereal-sulphuric acid by boiling after the addi- tion of hydrochloric acid, and then determining the sulphuric acid set free as barium sulphate. A still better method is the following suggested by Salkowski^: 300 c. c. of urine are precipitated by an equal volume of a barium solution which consists of 3 vols, barium hydrate and 1 vol. barium-chloride solution, both saturated at the ordinary tempera- ture. Filter through a dry filter, measure off 100 c. c. of the filtrate which contains only the ethereal-sulphuric acid, treat with 10 c. c. hydrochloric acid of a specific gravity 1.13, boil for fifteen minutes, and then warm on the water-bath until the precipitate has completely settled and the supernatant liquid is entirely clear. Filter and wash with warm water and with alcohol and ether and proceed according to the generally prescribed method. The differ- ence between the ethereal-sulphuric acid found and the total quantity of sulphuric acid as determined in a special portion of nrine is considered as the quantity of sulphate-sulphuric acid. Nitrates occur in small quantities in human urine (Schonbein '), and tliey probably originate from the drinking-water and the food. According to Weyl and Citron,* the quantity of nitrates is smallest with a meat diet and greatest with vegetable food. The average amount is about 43.5 milligrammes per litre. ' Zeitschr. f. physiol. Chem., Bd. 1, S. 70. » Virchow's Arch., Bd. 79. » Journ. f. prakt. Chem., Bd. 93, S. 153. * Virchow's Arch., Bdd. 96 u. 101. AMMONIA. 617 Potassium and Sodium. The quantity of these bodies eliminated by the urine by a healthy full-grown person on a mixed diet is, according to Salkowski,' 3-4 grms. K.^0 and 5-8 grms. Na,0, with an average of about 3-3 grms. K^O and 4-6 grms. Na,0. The proportion of K to Na is ordinarily as 3:5. The quantity ■depends above all upon the food. In starvation the urine may become richer in potassium than in sodium, which results from the lack of common salt and the destruction of tissue rich in potassium. The quantity of potassium may be relatively increased during fever, while after the crisis the reverse is the case. The quantitative estimation of these bodies is performed by the gravimetric methods as described in works on quantitative analysis. Ammonia. Some ammonia is habitually found in human urine and in that of carnivora. This ammonia may represent, as above stated (page 455), on the formation of urea from ammonia, the small amount of ammonia which, because of the excess of acids formed by the combustion, as compared with the fixed alkalies, is united with such acids, and in this way is excluded from the synthesis •to urea. This view is confirmed by the observations of Coranda,' who found that the elimination of ammonia was smaller on a vege- table diet and larger on a rich meat diet than when on a mixed diet. On a mixed diet the average amount of ammonia eliminated by the urine is about 0.7 grm. NH, per twenty-four hours (Neubauek'). AU the ammonia of the urine, as above stated, is not represented by "the residue which has eluded synthesis into urea by neutralization by acids because, as shown by Stadblmann and BecKmann,* ammonia is eliminated by the urine even during the continuous administration of fixed alkalies. The experiments of many investigators* have shown that in man and carnivora no formation of urea takes place from ammonia salts with mineral acids such as ammonium chloride, but they are elimi- nated as such in the urine, while, on the contrary, in herbivora a formation of urea may take place from ammonium chloride. In herbivora the HCl of the ammonium chloride combines with fixed .alkalies, and the ammonia set free is available for the formation of ' VircUow's Arch., Bd. 53. ' Arch. f. exp. Path. u. Pharm., Bd. 13. ' Huppert Neabauer, Harnanalyse, 10. Aufl., S. 43. * Stadelmann, Einfluss der Alkalien auf den StofEwechsel dea Meuachen. Stuttgart, 1890, S. 52. ' See footnotes pas;e 455. 518 TBE a BINE. urea. This difEerence in the behavior of ammoninm chloride in carniyora and herbivora is dependent upon the difEerent behavior of the acids in the organism of these two groups of animals. The quantity of ammonia in human and carnivoral nrine is in- creased by the introduction of mineral acids, and, as shown by JoLiN,' organic acids, like benzoic acid, which is not burned in the body, act in a similar way. This depends upon the fact that the organism of these animals has the property of producing sufficient ammonia by destruction of proteids to neutralize the acids intro- duced and in this way prevent a destructive abstraction of fixed alkalies. Herbivora, on the contrary, lack this property. In them the acids introduced are neutralized by iixed alkalies ; hence th& introduction of mineral acids soon causes a destructive action oa account of the abstraction of alkalies. Acids formed iu the destruction of proteids in the body act like those introduced from without on the elimination of ammbnia. For this reason the quantity of ammonia in human and carnivoral urine is increased under such conditions and in such diseases- where an increased formation of acid takes place due to an increased metabolism of proteids. This is the case in fevers and diabetes. In the last-mentioned disease an organic acid, /3-oxy- butyric acid, is produced, which passes into the urine combined with ammonia. As the elimination of ammonia and the formation of urea stand in close relation to each other, it was expected that an iaerease in the elimination of ammonia and a decrease in the forma- tion of urea would take place in certain diseases of the liver. We have given above, on the formation of urea in the liver, the extent of agreement of this statement, and the reader is referred to the works there cited. The detection and quantitative estimation of ammonia is per- formed generally accordiug to the method ^suggested by ScHLOSiifG. The principle of this metliod is that the ammonia from a measured amount of urine is set free by lime-water in a closed vessel and N absorbed by a measured amount of — sulphuric acid. After the absorption of the ammonia the quantity is determined by titrating the remaining free sulphuric acid with a — caustic alkali. This- method gives low results, and in exact work we must proceed as. > Skand. Arch. f. Physiol., Bd. 1. qUANTirr and QUANriTAriVK GOMPOSITJOX. 519 I snggested by Bohland.' Other methods have been suggested by ScHMiEDEBERG " and by Latschenbergee.' Calcium and magnesium occur in the urine for the most part as phosphates. The quantity of earthy phosphates eliminated daily is somewhat more than 1 gr., and of this amount f is magnesium and ^ calcium phosphate. In acid urines the simple- as well as the double-acid earthy phosphates are found, and the solubility of the first, among which the calcium-salt, CaHPO,, is especially insohi- ble, is particularly augmented by the presence of double-acid alkali phosphate and sodium chloride in the urine (Ott*). The quantity of alkaline earths in the urine depends on the composition of the food. Nothing is known with positiveness in regard to the coQstant and regular change in the elimination of these substances in disease. The quantity of calcium and magnesium is determined accord- ing to the ordinary well-known methods. Iron occurs in tlie urine only in small quantities, and, as it seems from the Investigations of Kdnkbl,' Uiacosa,' Kobbrt,' and his pupils, it does not exist as a salt, but as an organic combination — in part as pigment orchromogen. The statements in regard to the quantity of iron seem to show that the quantity is very variable, from 1 to 11 milligrammes per litre of urine (Magnibb,* Gottlieb,' Kobert, and his pupils). The quantity of silicic acid, according to the ordinary statements, amounts to about 0.03 p. m. Traces of hydrogen peroxide also occur in the urine. The gases of the urine are carbon dioxide, nitrogen, and traces of oxygen. The quantity of nitrogen is not quite 1 vol. per cent. The carbon dioxide varies considerably. In acid urines it is hardly one half as great as in neutral or alkaline urines. IV. The Quantity and Quantitative Composition of Urine. A direct participation of the kidney substance in the formation of the urinary constituents is proved at least for one constituent of the urine, namely, hippuric acid. It is hardly to be' doubted that • Pflilger's Arch., Bd. 43, ' Arch. f. exp. Path. u. Pharm., Bd. 7. ' Monatshefte f. Chem., Bd. 5. *Zeitschr. f. physiol. Chem., Bd. 10. ' Sitzungsber. d. phys.-med. Gesellsch. zu Wttrzburg, 1881. Cited from Maly's Jahresber., Bd. 11, S. 246. « See Maly's Jahresber., Bd. 16, S. 213. ' Arbeiten des pharm. Instit. zu Dorpat, Bd. 7. Stuttgart, 1891. ' Ber. d. deutsch. chem. Gesellsch., Bd. 7. ' Arch. f. exp. Path. u. Pharm. , Bd. 36. 520 THE URINE. the kidneys as well as the tissues generally have a certain part to play in the formation of other urinary constituents, but their chief task consists in separating and removing urinary constituents dis- solved in the blood which have been taken up by it from other organs .and tissues. It has been shown by the experiments of numerous investigators, HeIDENHAIN, v. WiTTIOH, NuSSBAUM, NeISSBE, USTIMOWITSCH, I. MuNK, and others, that the elimination of water and the remain- ing urinary constituents is not alone produced by simple diffusion .and filtration.' It is generally conceded that the processes of nrinary secretion depend essentially upon a specific activity of the cells of the epithelium of the urinary passages, besides which also processes of filtration and diffusion take part. The process of the secretion of urine in man and the higher animals is generally con- sidered to proceed chiefly as follows: The water together with a .small amount of the salts passes through the glomeruli, while the chief part of the solids is secreted by the epithelium of the urinary passages. A secretion of solids without a simultaneous secretion of water is not possible, and therefore a part of the water must be secreted by the epithelium-cells of the urinary passages. The passage of the chief part of the water through the glomeruli is rather generally considered as a filtration due to blood-pressure. According to Heideithain, the thin cell-layers of the glomeruli iave a secretory action. The quantity and the composition of urine are liable to great variation. Those circumstances which under physiological condi- tions exercise a great influence are the following: the blood-pressure, and the rapidity of the blood-current in the glomeruli ; the quantity of urinary constituents, especially water in the blood; and lastly, (the condition of the secretory glandular elements. Above all, the quantity and concentration of the urine depend on the elimination of water. That this last may vary with the quantity of water in the blood, with changed blood-pressure, and with circulatory conditions 'is evident; but under ordinary circumstances the quantity of water eliminated by the kidneys depends essentially upon the quantity of water which is brought to them by the blood or which leaves the body by other exits. The elimination of urine is increased by drinking freely, or by reducing the quantity of water removed ' See Heidenhain, Die Harnabsonderung in Hermann's Handbuch, Bd. 5, Thl. 1, S. 379. QUANTITY. 5-21 in other ways; bnt it is decreased by a diminiBhed introduction of water, or by a greater loss of water in other ways. Ordinarily in man just as much water is eliminated by the kidneys as by the skin, langs, and intestine together. At lower temperatures and in moist air, since under these conditions the elimination of water by the skin is diminished, the elimination of urine may be considerably increased. Diminished introduction of water or increased elimina- tion of water by other means — as in violent diarrhoea, violent vomit- ing, or abundant perspiration — greatly diminishes the elimination of urine. For example, the urine may sink as low as 500-400 c. c. per day in intense summer-heat, while after copious draughts of water the elimination of 3000 c. c. of urine has been observed during the same time. The average quantity of urine voided in the course of 34 hours must undergo considerable variation; ordi- narily it is calculated as 1500 c. c. for healthy adult men and 1200 c. c. for women. The minimum elimination occurs during the night, between 2 and 4 o'clock; the maximum, in the first hours after awaking and from 1-2 hours after a meal. The qnantity of solids excreted in the coarse of 24 hours is rather constant even though the quantity of urine may vary, and it is more constant when the manner of living is regular. Therefore the percentage of solids in the urine is naturally in an inverse pro- portion to the quantity of urine. The average quantity of solids per 24 hours is calculated as 60 grms. The quantity may be cal- culated with approximate accuracy by means of the specific gravity if the second and third decimals of the specific gravity be multiplied by Hasbe's coefiicient, 2.33. The product gives the amount of solids in 1000 c. c. of urine, and if the qnantity of urine eliminated in the 24 hours be measured, the quantity of solids in the 24 hours may be easily calculated. For example, 1050 c. c. of urine of a specific gravity 1.021 was eliminated in the 24 hours; therefore the 48 9 X 1050 quantity of solids eliminated is 21 X 2.33 = 48.9, and — ' = 51.35 grms. The urine in this case contained 48.9 p. m. solids and 51.35 grms. in the daily excretion. Those, bodies which, under physiological conditions, afEect the density of the urine are common salt and urea. The specific gravity of the first i„ 2.16 and the last only 1.32, so it is easy to understand, when the relative proportion of these two bodies essen- tially deviates from the normal, why the above calculation from the 522 THE URINE. specific gravity is not exact. The same is the case when a urine poor in a normal constituent contains large amounts of foreign bodies, such as albumin or sugar. As above stated, the percentage of solids in the urine generally decreases with a greater elimination, and a very considerable excre- tion of urine {polyuria) has therefore, as a rule, a lower specific gravity. An important exception to this rule is observed in urine containing sugar {diabetes mellitus), in which there is a copious excretion of a very high specific gravity due to the sugar. In cases where very little urine is secreted {oliguria), as when the perspira- tion is profuse, in diarrhoea, and in fevers, the specific gravity of the urine is as a rule high, the percentage of solids high, and has a dark color. Sometimes, as, for example, in certain cases of albuminuria, the urine may have a low specific gravity, notwith- standing the oliguria, and be poor in solids with a light color. It is difficult to give a tabular view of the composition of urine, on account of its variation. For certain purposes the following table may be of some value, but it must not be overlooked that the results are not given for 1000 parts of urine, but only approximate figures for the quantities of the most important constituents which are eliminated in the course of 24 hours in a quantity of 1500 c. c. Daily quantity of solids = 60 grms. Inorganic constituents ^ 25 grms. Sodium chloride (NaCl) 15 grms. Sulphuric acid (H2SO4). 2,5 " Phosphoric acid (PjOs). 2.5 " Potash (KjO) 3.3 " Ammonia (NHa) 0.7 " Magnesia (MgO) 0.5 " Lime (CaO) 0.3 " Eemaining inorg. bodies 0.3 " Urine contains on a,n average 40 p. m. solids. The quantity of urea is about 20 p. m. and common salt about 10 p. m. V. Casual Urinary Constituents. The casual appearance in the urine of medicines or of urinary constituents resulting from the introduction of foreign substances into the organism is of practical importance, because such- constitu- ents may interfere in certain urinary investigations, and also because they afford a good means of determining whether certain substances have been introduced into the organism or not. From this point of view a few of these bodies will be spoken of in a following section Organic constituents = 35 grms. Urea SO.Ogrms. Uric acid 0.7 " Creatinin 1.0 " Hippuric acid 0.7 " Remaining organic bodies 2. 6 " CASUAL CONSTITUENTS. 523 (on the pathological urinary constitnents). The presence of these foreign bodies in the nrine is of special interest in those cases in which they serve to elucidate the chemical transformations certain substances undergo within the body. As inorganic substances generally leave the body unchanged, they are of very litble interest from this standpoint, but the changes which certain organic sub- stances undergo when introduced into the animal body may be studied by this means so far as these transformations are shown by the nrine. The bodies belonging to the fatty series, though not without exceptions, fall mostly into a combustion leading towards the final products of metabolism; still, often a smaller or greater part of the body in question eludes oxidation and appears unchanged in the urine. A part of the organic acids, which are otherwise burnt into water and carbonates and render the urine neutral or alkaline, may act in this manner. The volatile fatty acids poor in carbon are less easily burnt than those rich in carbon, and they therefore pass un- changed into the urine in large amounts. This is especially true of formic and acetic acids (Schotten,' Geehant and Quinqttaud '). According to Gaglio oxalic acid is not oxidized in the animal body, while Marfori ° claims that it is nearly entirely consumed. The acid amides appear not to be changed in the body (ScHULTZEN and Nencki *). A small part of the amido-acids seems indeed to be eliminated unchanged, but otherwise they are, as stated above (page 455) for leucin, glycocoll, and aspartic acid, decomposed within the body, and they may therefore cause an increased elimi- nation of urea. Sarcasm (methylglycocoU), ]S[H(CH3).CH,.C00H, also perhaps passes in small part into the corresponding urami- do-acid, methylhydantoinic acid, ]S[H,.CO.]Sr(CH,).CH,.COOH (ScHULTZEN "). Also tauHn, amido-ethylsulphonic acid, which acts somewhat differently in different animals (Salkowski °), passes in human beings, at least in part, into the corresponding uramido- acid, taurocarlamic acid, ]SrH,.CO.]SrH.C,H,.SO,.OH. A part of ' Zeitschr. f. physiol. Chem., Bd.7, S. 375. ' Compt. rend.. Tome 104. » See Maly's Jahresber. , Bd. 16, S. 402, and Bd. 20, S. 70. •• Zeitschr. f. Biologie, Bd. 8. ' Ber. d. deutsch. chem. Gesellsch., Bd. 5. See also Baumann and v. Mering, ibid., Bd. 8, S. 584, and E. Salkowski, Zeitschr, f. physiol. Chem., Bd. 4, S. 107. ' Ber. d. deutsch. chem. Gesellsch., Bd. 6, and Virchow's Arch., Bd. 58. 52,4 TBE URINE. the tauria appears as sach in the urine. In rabbits, when tanrin is introduced into the stomach, nearly all its sulphur appears in the urine as sulphuric and hyposulphurous acids. After subcutaneous injection the taurin appears again in great part unchanged in the urine. A conjugation of bodies of the fatty series with glycocoll may also occur. As shown by Jaffe and Cohk,' furfural, which is the aldehyde of pyromucic acid, when introduced into rabbits and dogs is first oxidized into pyromucic acid and then this eliminated as pyromucuric acid, C,H,N,0, after conjugation with glycocoll. In birds this behavior is difEerent, namely, in them the acid is con- jugated to another substance, ornithin, C,H.,jN,0„ which is probably diamido valerianic acid, forming pyromucinorthuric acid.' Like furfurol so is tkiophen, C,H,S, corresponding to furfuran, oxidized to fhiophenic acid, which, according to Jaffe and Levy,^ is con- jugated with ' glycocoll in the body (rabbits) and eliminated as thiophenuric acid, C,H,NSO,. Furfurol also undergoes conjugation with glycocoll [in other forms in mammals. Thus Jaffe and Cohn found that it in part combined with acetic acid, forming furfuracrylic acid, C,H30.CH:0H.C00H, which passes into the urine coupled with glycocoll as furfur acryluric acid. Conjugation with glycuronic acid occurs in certain substituted alcohols, aldehydes, and ketones {?), which probably first pass over into alcohols (Sundvik'). Chloral hydrate, Gfi\fi~S. -\- H,0, passes, after it has been converted into trichlorethyl-alcohol by a reduction, into a Isevogyrate reducing acid, urochloralic acid or trichlorethyl-glycuronic acid, C^Cl^Hj.O.HjO, (MuscuLus and V. Meeing'). Trichloriutyl-alcohol and butyl-chloral hydrate also pass into trichlorlutyl-glycuronic acid. In animals which have starved until the glycogen has disappeared from the muscles and liver and which are given chloral hydrate or dimethyl carbinol, conjugated glycuronic acids appear in the urine (Thierfeldek '). On account of these facts the albuminous bodies are considered the ' Ber. d. deutsch. chem. Gesellscli., Bd. 30. « Jaffe and R. Cohn, Md., Bd. 21, S. 3461. » Ibid., Bd. 21, S. 3458. * See Maly's Jahresber., Bd. 16, S. 76. 'Ber. d. deutsch. chem. Gesellsch., Bd. 8; also v. JVIering, Zeitschr. f. physiol. Chem., Bd. 6, and E. Ktilz, Pflilger's Arch., Bd. 28. « Zeitschr. f. physiol. Chem., Bd. 10. CASUAL CONSTITUBNTS. 525 origin of the glycuronic acid. It may perhaps originate from snch proteids, which are found widely diffused in the hody, and from which carbohydrates or near-related acids may be split. The above starvation experiments are perhaps not quite free from exceptions.' The aromatic combinations pass, as far as we know, into the urine as snch generally after a previous partial oxidation or after a synthesis with other bodies. That the benzol ring is destroyed in the body in certain cases is very probable. The fact that benzol may be oxidized outside of the body into carbon dioxide, oxalic acid, and volatile fatty acids has been known for a long time, and we may refer the reader to the investigations of Dkechsel, mentioned in the first chapter, in which this experi- menter obtained, by the electrolysis of phenol, normal caproic acid and afterward substances in which the quantity of carbon decreased constantly until he obtained the final products of metabolism. As in these experiments a splitting of the benzol ring must take place before the formation of the bodies of the fatty series, also when aromatic bodies are consumed in the animal body, we must admit that first a rupture of the benzol ring takes place with the forma- tion of fatty bodies. If this does not take place, then the benzol nucleus is eliminated with the urine as an aromatic combination of one kind or another. As the difficultly destroyed benzol nucleus can protect from destruction a substance belonging to the fatty series when conjugated with it, which is the case with the glycocoll of hippnric acid, it seems also that the aromatic nucleus itself may be protected from destruction in the organism by syntheses with other bodies. The aromatic ethereal-sulphuric acids are examples of this kind. The difficulty in deciding whether the benzol ring itself is destroyed in the body lies in the fact that we do not know all the different aromatic transformation products which may be produced by the introduction of any aromatic substance in the organism and which we must seek for in the urine. On this account it is also impossible to learn by exact quantitative estimations whether or not an aromatic substance introduced or absorbed appears again in its entirety in the urine. Certain observations render it probable that the benzol ring, as above mentioned, is at least in certain cases destroyed in the body. Schotten" and Baumann ' have found ' See Nebelthau, Zeitsclir. f. Biologie, Bd. 28, S. 130. » Zeitschr. f. pliysiol. Chem., Bdd. 7 and 8. » Ibid., Bd. 10, S. 130. In regard to tyrosin see especially Blendermann, 526 TEE URINE. that certain ami do-acids, sach as tyrosin, pJienylamido-propionic acid, and amido-cinnamic acid when introduced into the bodj cause no increase in the quantity of known aromatic substances in the urine; this makes a destruction of these amido-acids in the animal body seem probable. Juvalta ' also made an experiment on dogs with phthalic acid, and found that 57.5-68.76^ of the acid intro- duced into the body disappeared, or more correctly was not found again. According to Juvalta, this acid does not undergo any synthesis, nor does it yield any aromatic transformation products; and if this supposition be correct, we have here a proof of the destruction of tlie benzol nucleus of a part of the phthalic acid introduced into the organism of the dog. An oxidation in the side chain of aromatic compounds is often found, and may also occur in the nucleus itself. As an example, benzol is first oxidized to oxybenzol (Schtjltzen' and Nattktk'"), and this is then in part converted into dioxybenzols (Baumakk and Peetjsse'). Nuphthalin appears to be converted into oxynaph- thalin, and probably a part also into dioxynapMhalin (LESifiK and M. Nenoki'). Anilin, C,Hj.NH„ passes into paramidophenol,' which passes into the urine as ethereal-sulphuric acid, H,N.O„H,. O.SO^.OH (F. MiJLLEE'). If the aromatic substance has a side chain belonging to the fatty series, this last is geuerally oxidized. For example, toluol, C,H,.CH3 (Sohultzen and Naunyn '), ethyl-henzol, C^H^.C^Hj, and. propylbe7izol, C,Hj.O,H, (Nbncki and Giacosa'), also many other bodies are oxidized into benzoic acid. If the side chain has several members, the behavior is somewhat different. Phenyl-acetic acid, C,H,.CH,.COOH, in which only one carbon atom exists between the benzol nucleus and the carboxyl, is not oxidized, but Zeitscbr. f. pbysiol. GUem., Bd. 6; Scliotten, ibid., Bd. 7; Baas, ibid., Bd. 11; and R. Cobn, ibid., Bd. 14. ' Zeitschr. f. pbysiol. Cbem., Bd. 13. ' Reicberfs und Dii Bois-Reymond's Arcb., 1867. ' Zeitscbr. f . pbysiol. Cbem., Bd. 3, S. 156. See also Nencki and Giacosa, ibid., Bd. 4, S. 336. >■ Arcb. f. exp. Patb. u. Pbarm., Bd. 24. See also Edlefsen, Maly's Jabres- ber., Bd. 18, S. 116. ' Scbmiedeberg, Arcb. f. exp. Patb. u. Pbarm., Bd. 8. » Deutscb. med. Wocbenscbr., 1887. Cited from Maly's Jahresber., Bd. 17, S. 87. ' Reicberfs and Da Bois-Reymond's Arcb., 1867. 8 Zeitscbr. f. pbysiol. Cbem., Bd. 4. CASUAL CONSTITUENTS. 527 is eliminated after conjagation with glycocoU as joAewace^Mrie acid (Salkowski '). Phenyl-propionic acid, C,H,.CH,.CH,.COOH, with two carbon atoms between the benzol nucleus and the carboxyl is, on the contrary, oxidized into benzoic acid.' Aromatic amido- acids with three carbon atoms in the side chain, and where the NH, groap is bound to the middle one, as in tyrosin, a-oxjrphenylamido- propionic acid, C.H,(OH).CH,.CH(NHJ.COOH, and a-phenyl- amido-propionic acid, C,H,.CH3.CH(NI1,).C00H, seem to be in great part burnt within the body. Phenylamido-acetic acid, which has only two carbon atoms in the side chain, C,H5.CH(NHJ.G00H, acts otherwise, passing into mandelic acid, phenyl-glycolic acid, C.H,.CH(OH).COOH (Schotten=). If several side chains are present in the benzol nucleus, then only one is always oxidized into carboxyl. Thus xylol, C,H,(OH,)„ is oxidized into toluic acid, C,H,(CH3)C00H (Schultzeit and Nattntn'), mesitylen, C,H3(CH,)„ into mesitylenic acid, C,H,(CH,),.COOH (L. Nencki'), and cymol into cumic acid (M. Nencki and Ziegler'). Syntheses of aromatic substances with other atomic groups occur frequently. To these syntheses belongs, in the first rank, the conjugation of benzoic acid with glycocoll to form hippuric acid, first discovered by Wohlee. All the numerous aromatic substances which are converted into benzoic acid in the body are voided partly as hippuric acid. This statement is not true for all classes of animals. According to the observations of Jaffe,' benzoic acid does not pass into hippuric acid in birds, but into another nitro- genous acid, ornithuric acid, C„H,„]S[jO^. This acid yields as splitting products, besides benzoic acid, a body, ornithin, which has been spoken of on page 524. Not only are the oxyhenzoic acids and the substituted benzoic' acids (Bertagnini °) conjugated with glycocoll, forming corresponding hippuric acids, but also the above- mentioned acids, toluic, mesitylenic, cumic, and plienylacetic acids. ' ZeitscUr. f. physiol. Chem., Bdd. 7 and 9. 'See E. and H. Salkowski, Ber. d. deutsch. chem. Qesellsch., Bd. 13. ' Zeitschr. f. pbysiol. Chem., Bd. 8. ^Reichert's und Du Bois-Keymond's Arch., 1867. s Arch. f. exp. Path. u. Pharm., Bd. 1. « Ber. d. deutsch. chem. Qesellsch., Bd. 5; see also 0. Jacobsen, ibid., Bd. 13. 'iWd., Bdd. 10 and 11. * Cited from Ktibne's Lehrbuch, S. 91. 528 THE URINE. These acids are voided as toluric, mesitylenuric, cuminuric, and phenaceturic acids. It must be remarked in regard to the oxybenzoic acids that a conjugation with glycocoU has only been positively proven with sali- cylic acid and p-oxybenzoic acid (Bertagnini, Baumann and Heetee,' and others), while Baumann and Heeter find it only very probable for m-oxybenzoic acid. The oxybenzoic acids are also in part eliminated as conjugated sulphuric acids, which is especially true for m-oxybenzoic acid.' We have the investigations on ra-amidobenzoic acid in regard to the transformation of amido- benzoic acids. Salkowski' foand, as was later confirmed by R. CoHN,* that m-amidobenzoic acid passes in part into uramido- lenzoic acid, H^N.CO.HlSr.CjHj.OOOH. It is also in part elimi- nated as amidohippuric acid. The substituted aldehydes are of special interest as substances which undergo conjugation with glycocoU. According to the inves- tigations of R. CoHisr ' on this subject o-nitrobenzaldehyde when introduced into a rabbit is only in a very small part converted into nitrobenzoie acid, and the chief mass, about 90^, is destroyed in the body. According to Siebee and Smirnovt ' m-nitrobenzaldehyde passes in dogs into m-nitrohippuric acid, and according to Cohn into urea m-nitrohippurate. In rabbits the behavior is quite different according to Cohk. In this case not only does an oxida- tion of the aldehyde into benzoic acid take place, but the nitro group is also reduced to an amido group, and finally acetic acid attaches itself to the amido group with the expulsion of water, so that the final product, m-acetylamidobenzoie acid, CH3.CO.NH. C,H^. COOH, is the result. This process is analogous to the behavior of furfarol, and the reduction does not take place in the intestine, but in the tissue.' The p-nitrobenzaldehyde acts in rabbits in part like the m-aldehyde and passes in part into p-acetylamidolenzoic acid. Another part is converted into p-nitrobenzoic acid, and the urine contains a chemical combination of equal parts of these two ' ZeitscTir. f. physiol. Chem., Bd, 1, whicli also cites Bertagnini's work. ' See Baumann and Herter, 1. c, and also Dautzenberg in Maly's Jahres- ber., Bd. 11, S. 331. 3 Zeitschr. f. pbysiol. Cliem., Bd. 7. ^ lUd.', Bd. 17, S. 393. 'iW(i.,Bd. 17. 6 Monatshefte. f. Chem., Bd. 8. ■■ Zeitscbr. f. physiol. Chem., Bd. 18. CASUAL CONSTITUENTS. 529 acids. According to Siebee and Smiknow p-nitrobenzaldehyde only yields urea p-nitrohippurate in dogs." Another very important synthesis of aromatic substances is that of the ethereal-sulphuric acids. Phenols and chiefly the hydroxyl- ated aromatic hydrocarbons and their derivatives are voided as ethereal-sulphuric acids, according to Baumann, Heetek, and others." A conjugation of aromatic substances with glycuronic acid, which last is protected from burning, occurs rather of ben. Camphor, C„H„0, when given to a dog is first converted by oxidation into camphoral, C,„H„(OH)0, and by conjugation with glycuronic acid into campho-glycuronic acid (Schmiedebeeg'). The phenols, as above stated (page 491), pass in part as conjugated glycuronic acids into the urine. The same is true for the homologues of phenols, for certain substituted phenols, for naphthols, horneol, menthol, turpentine, and many other aromatic substances.* Orthonitrotoluol in dogs passes first into o-nitrobenzyl alcohol and then into a con- jugated glycuronic acid, uronitrotoluolie acid (Jaffe '). The glycu- ronic acid split off from the conjugated acid is laevogyrate and hence not identical with the ordinary glycuronic acid, but isomeric. Indol and skatol seem, as above stated (page 495 and 496), to be eliminated in the urine partly as conjugated glycuronic acids. A synthesis in which compounds containing sulphur, mercap- turic acid, is formed and eliminated conjugated with glycuronic acid, occurs when chlorine and bromine derivates of benzol are in- troduced into the organism of dogs (Baumann and Peeusse,' Jaffe '). Thus chlorbenzol combines with ctstein, an intermediary decomposition product of proteids which is closely allied to cystia (see below), forming chlorphenylmercapturic acid 0„H„C1SNO,. On boiling with mineral acid-this compound decomposes into acetic acid and chlorphenylcystein, C.H.Cl.C.H.NSO,. ' In regard to the extensive literature on glycocoll conjugations we refer the reader to O. Killiling, Ueber StofEwecbselprodukte aromatischer KOrper. In- aug.-Dias. Berlin, 1887. 2 See O. Ktlhling, 1. c. » Schmiedeberg und Meyer, Zeitschr. f. physiol. Chem., Bd. 3. * See O. Kahling, 1. c, which gives the literature up to 1887; also B. Kttlz, Zeitschr. f. Biologie, Bd. 27. » Zeitschr. f. physiol. Chem., Bd. 2. • Ibid., Bd. 5, S. 309. ^ Ber. d. deutsch. chem. Gesellsch., Bd. 12. 530 THE URINE. Pyridin, G,H,N, which does not combine either with glycuronic acid or with sulphuric acid after previous oxidation, shows a special behavior. It takes up a methyl group as found by His ' and later confirmed by Cohn,' and forms an ammonium combination, methylpyridyl-ammoniwn hydroxyl, HO.CHj.NCjHj. Methylpy- ridin (a-picolin) on the contrary passes in rabbits part in into a-pyridin carbonic acid, and is eliminated as a-pyridinuric acid after conjugation with glycuronic acid (E. Cohn °). Several alka- loidsj such as quinin, Tnorphin, and strychnin, may pass into the urine. After taking turpentine, balsam of copaiva, and resins these may appear in the urine as resin acids. DifEerent kinds of coloring matters, such as alizarin, crysophanic acid, after the use of rhubarb or senna, and the coloring matter of the blueberry, etc. , may also pass into the urine. After taking rhubarb, senna, or santonin the urine takes a yellow or greenish-yellow color, which is transformed into a beautiful red color by the addition of alkali. Phenol pro- duces, as above mentioned, a dark-brown or dark-green color which depends mainly on the decomposition products of hydrochinon and humin substances. After the use of naphthalin the urine has a dark color, and several other medicines produce a special coloration. Thus Jcairin gives often a yellowish-green hue, and the urine darkens when exposed to the air; thallin gives a greenish-brown color which is marked green in thin layers, and antipyrin gives a yellow to blood-red. After the administration of balsam of copaiva the urine becomes, when strongly acidified with hydrochloric acid, gradually rose and purple-red (Quincke*). After the use of naphthalin or naphthol the urine gives with concentrated sulphuric acid (1 c. c. concentrated acid and a few drops of urine) a beautiful emerald-green color (Penzoldt'), which is probably due to naphthol-glycuronic acid. Odorifero'tis bodies also pass into the urine. After eating asparagus the urine acquires a sickly disagree- able odor which is piobably due to methylmercaptan, according to M. Nencki." After taking turpentine the urine may have a peculiar odor similar to that of violets. ' Arch. f. exp. Path. u. Pharm., Bd. 23. ' Zeitsobr. f. physiol. Chem., Bd. 18, S. 116. 2 L. c. , « Arch. f. exp. Path. u. Pharm., Bd. 17. ' Hid., Bd. 21. 6 Ibid., Bd. 28. PBOTEID. 531 VI. Pathological Constituents of Urine. Proteid. The appearance of slight traces of proteid in the urine of apparently healthy persons has been observed in many cases by several investigators, but still we must not conceal the fact that other investigators consider these traces of proteid as the first symptoms, though very mild, of a diseased condition of the urinary apparatus, or as a symptom of a transitory disturbance in the circa- lation. Frequently traces are found in the urine of a substance similar to iiucleoalbnmin which can easily be mistaken for mucin and which is probably identical with nucleoalbumin. This sub- stance has been isolated from the papillary part of the kidneys and from the mucous membrane of the bladder by Lonkbeeg.' In diseased conditions proteid occurs in the urine in a variety of cases. The albuminous bodies which most often occur are serglobulin and seralbumin. Albumoses and peptones also sometimes occur. The quantity of proteid in the urine is in most cases less than 5 p. m. , rarely 10 p. m., and only very rarely does it amount to 50 p. m. or over. Among the many reactions proposed for the detection of pro- teid in urine, the following are to be recommended : Tlie Heat Test. Filter the urine and test its reaction. An acid urine may, as a rule, be boiled without further treatment, and only in especially acid urines is it necessary to first treat with a little alkali. An alkaline urine is made neutral or faintly acid before heating. If the urine is poor in salts, add -^ vol. of a saturated common-salt solution before boiling; then heat to boiling-point, and if no precipitation, cloudiness, or opalescence appears, the urine in question contains no coagulable proteid, bat it may contain albumoses or peptones. If a precipitate is produced on boiling, tliis may consist of proteid, or of earthy phosphates, or of both. The simple-acid calcium phosphate decomposes on boiling, and normal phosphate may separate. The proper amount of acid is now added to the urine, so as to prevent any mistake caused by the presence of earthy phosphates, and to give a better and more flocculent precipi- tate of the proteid. If acetic acid is used for this, then add 1-2-3 drops of a 25^ acid to each 10 c. c. of the urine, and boil after the addition of each drop. On using nitric acid, add 1-3 drops of the 25^ acid to each c. c. of the boiling-hot urine. On using acetic acid, when the quantity of proteid is very small, and especially when the urine was originally alkaline, the proteid. ' See page 446. 53'2 THE URINE. may sometimes remain ia solution on the addition of the above quantity of acetic acid. If, on the contrary, less acid is added, the precipitate of calcium phosphate, which forms in amphoteric or faintly acid urines, is liable not to dissolve completely, and this may cause it to be mistaken for a proteid precipitate. If nitric acid is used for the heat test, the fact must not be overlooked that after the addition of only a little acid a combination between it and the proteid is formed which is soluble on boiling and which is only precipitated by an excess of the acid. On this account the large quantity of nitric acid, as suggested above, must be added, but in this case a small part of the proteid is liable to be dissolved by the excess of the nitric acid. When the acid is added after boiling, which is absolutely necessary, the liability of a mistake is not so great. It is on these grounds that the heat test, although it gives very good results in the hands of experts, is not recommended to physicians as a positive test for proteid. A confounding with mucin, when this body occurs in the urine, is easily prevented in the heat test with acetic acid, by acidifying- another portion with acetic acid at the ordinary temperature. Mucin and nucleoalbumin substances similar to mucin are hereby precipitated. If ia the performance of the heat and nitric acid test a precipitate first appears on cooling or is strikingly increased, then this shows the presence of albumoses in the urine, either alone or mixed with coagulable proteid. In this case a further investi- gation is necessary (see below). In a urine rich in urates a precipi- tate consisting of uric acid separates on cooling. This precipitate is colored, sandy, and hardly to be mistaken for an albnmose or proteid precipitate. Heller's test is performed as follows (see page 26) : The urine is very carefully floated on the surface of nitric acid in a test-tube. The presence of proteid is shown by a white ring between the two liquids. With this test a red or reddish-violet transparent ring is always obtained with normal urine; it depends on the indigo color- ing matters and can hardly be mistaken for the white or whitish proteid ring, and this last must not be mistaken for the ring pro- duced by bile-pigments. In a urine rich in urates another compli- cation may occur, due to the formation of a ring produced by the precipitated uric acid. The uric-acid ring does not lie, like the proteid ring, between the two liquids, but somewhat higher. For this reason we may often have two simultaneous rings with urines rich in urates and yet not containing very much proteid. The disturbance caused by uric acid is easily prevented by diluting the urine with 1-3 vol. water before performing the test. The uric acid now remains in solution, and the delicacy of Heller's test is so great that after dilution only in the presence of insignificant traces of proteid does this test give negative results. In a urine very rich in urea a ring-like separation of urea nitrate may also appear. This ring consists of shining crystals, and it does not TEST FOR PBOTEIDS IN UBINE. 533 appear in the previously diluted urine. A confusion with resinous acids, which also give a whitish ring with this test, is easily pre- vented, since these acids are soluble on the addition of ether. Stir, add ether and carefully shake the contents of the test-tube. If the cloudiness was due to resinous acids, the urine becomes gradually clear and on evaporating the ether a sticky residue of resinous acids is obtained. A liquid which contains pure mucin does not give a precipitate with this test, but it gives a more or less strongly opalescent ring, which disappears on stirring. The liquid does not contain any precipitate after stirring, but is somewhat opalescent. If a faint, not wholly typical reaction is obtained with Heller's test after some time with undiluted urine, while the diluted urine gives a pronounced reaction immediately, then, as claimed by 'K. MoKNEE,' a nucleoalbumin substance is present, which is pre- vented from precipitation by the salts of the undiluted urine. In this case proceed as described below in regard to the detection of nucleoalbumin. If we bear in mind the above-mentioned possible errors and the means by which they may be prevented, there is hardly another test for proteid in the urine which is at the same time so easily performed, so delicate, and so positive as Hellee's. With this test even 0.02 p. m. albumin may be detected without diflBculty. Still the student should not be satisfied with this test -alone, but apply at least a second test, such as the heat test. In performing this test the (primary) albumoses are also precipitated. The reaction with metaphospjioric acid (see page 26) is very 'Convenient and easily performed. It is not quite so delicate and positive as Hellbb's test. The albumoses are also precipitated by this reagent. Reaction with Acetic Acid and Potassium Ferrocyanide. Treat the urine first with acetic acid until about 2^, and then add drop by drop a potassium ferrocyanide solution (1:20), carefully avoid- ing an excess. This test is very good, and in the hands of experts it is even more delicate than Hellee's. In the presence of very small quantities of proteid it requires more practice and dexterity than Hellee's, as the relative quantities of reagent, proteid, and acetic acid infiuence the result of the test. The quantity of salts ,in the urine also seems to have an influence. This reagent also precipitates albumoses. Spibglee's '' #es<. Spiegler recommends a solution of 8 parts mercuric chloride, 4 parts tartaric acid, 20 parts glycerin, and 200 parts water as a very delicate reagent for proteid in the urine. A test-tube is half filled with this reagent, and the urine allowed to flow upon its surface drop by drop from a pipette along the wall of the test-tube. In the presence of proteid a white ring is obtained ' Hygiea, Bd. 53. See Maly's Jahresber., Ld. 32, S. 341. » Wien. klin. Wochenschr., 1893, No. 3, i.nd Centralbl. f. klin. Med., 1893, Jio. 3. 534 THE URINE. at the point of contact between the two liqaids. The delicacy of this test is 1 : 350000. ^ The use of precipitating reagents presumes that the nrine to bfr investigated is perfectly clear, especially in the presence of only very little proteid. The urine must first be filtered. This is not easily done with urine containing bacteria, but a clear urine may be^ obtained, as suggested by A. Jolles,' by shaking the urine with infusorial earth. The different color reactions cannot be directly used, especially in deep-colored urines which only contain little proteid. The common salt of the urine has a disturbing action on Millok's reagent. To pjove more positively the presence of proteid, the precipitate obtained in the boiling test may be filtered, washed, and then tested with Millon's reagent. The precipitate may also be dissolved in dilute alkali and the biuret test applied to the solution. The presence of albumoses or peptones in the urine is directly tested for by this last-mentioned test. In testing the urine for proteid one must never be satisfied with one test alone, but one must at least apply the heat test and Hbllbe's test or the potassium- ferrocyanide test. In using the heat test alone the albumoses may be easily overlooked, but these are detected, on the contrary, by Hellee's test. If we are satisfied with this last test or the potas- sium-ferrocyanide test alone, we have no sufiicient intimation of the kind of proteid present, whether it consists of albamoses or coagu- lable proteid. For practical purposes several dry reagents for proteid liave been recom- mended. Besides the metaphosphoric acid may be meutione 1 : Stutz s or FiJK- bbingbr's gelatin capsules,' which contain mercuric choride, sodium chloride, and citric acid ; and Gbissler's albumin-test papers, which consist of strips of filter-paper which have been dipped in a solution of citric acid and also mer- curic-chloride and potassium-iodide solution and then dried. If the presence of proteid has been positively proved in the urine by the above tests, it then remains necessary to determine the variety. The detection of globulin and albumin. In detecting ser- globnlin the urine is exactly neutralized, filtered, and treated with magnesium sulphate in substance until it is completely saturated at the ordinary temperature, or with an equal volume of a saturated neutral solution of ammonium sulphate. In both cases a white, floccnlent precipitate is formed in the presence of globulin. In using ammonium sulphate with a urine rich in urates a precipitate consisting of ammonium urate may separate. This precipitate does not appear immediately, but only after a certain time, and it must not be mistaken for the globulin precipitate. In detecting ser- albumin heat the filtrate from the globulin precipitate to boiling- point or add about 1^ acetic acid to it at the ordinary temperature. " Zeitschr. f. anal. Chem., Bd. 29. ' In regard to this and other reagents see Huppert-Neubauer's Harnanalyse, 10. Aufl., S. 439. ALBUM08E8 AND PEPTONE IN VRTNE. ' 535 Albumoses and peptones have been repeatedly found in the urine in different diseases. Unquestionable observations are at hand on the occurrence of albumoses in the urine. The statements in regard to the occurrence of peptones ' date in part from a time when the conception of albumoses and peptones was different from that of the present day and in parb they are based upon investigations using insufficient methods. It is difficult to give anything positive m regard to the occurrence of so-called true peptone in the urine, and the study of peptonuria seems to require thorough investigation. In detecting albumoses first remove all coagulable proteids by boiling with the addition of acetic kcid. The filtrate is then tested by the biuret test, and when this gives positive results apply the three previously mentioned albumose reagents (page 34), nitric acid, acetic acid and potassium ferrocyanide, and saturation with common salt with the addition of acid. The albumoses may also be precipitated by saturating with ammonium sulphate in substance, and the detection of albumoses as well as true peptones is best per- formed by the aid of this salt. According to Devoto ' we proceed as follows: Devoto's method. The coagulable proteid is precipitated by ammonium sulphate as directed on page 29. The precipitate also contains tbe albumoses. If true peptone is present, it is found in the filtrate and may be tested for therein by means of the biuret test. The precipitate is washed with a saturated solution of ammonium sulphate and then treated with water. The coagulable proteid remains undissolved, while the albumoses dissolve and may be tested for by the biuret test. The dentero-albumose are never- theless not completely precipitated by the ammonium sulphate, and a mistaking of this for true peptone may occur. In testing for peptone in the old sense we make use of Salkowski's ' modi- fication of Hofmbister's ■* metliod. 50 c c. of the urine to be tested is acidified with 5 c. c. hydrochloric acid, precipitated with phospho-tungstic acid and warmed on a wire gauze. As soon as the precipitate is converted to a resinous mass the liquid is poured ofE as well as possible and the mass washed twice with distilled water. It is then dissolved in about 8 c. c. water by the aid of 0.5 c. c. caustic soda of sp. gr. 1.16 and warmed until the blue solution is de- colorized (grayish yellow or yellow). This solution is used after cooling for the biuret test by the addition of a copper solution (1-2^) drop by drop. At the present time we have no trustworthy method for the quantitative estimation of albumoses and peptones in the urine. ' In regard to the literature on albumoses and peptones in urine see Hup- pert-Neubauer-Harnanalyse, 10. Aufl., S. 466 to 493; also A. StofEregen, Ueber das Vorkommeu von PeptonimHarn, Sputum undEiter. Inaug.-Diss. Dorpat, 1891 ; H. Hirschfeldt, Ein Beitrag zur Frage der Peptonurie. Inaug.-Diss. Dorpat, 1892; and especially Stadelmann, Untersuchungen Uber die Peptonurie. Wiesbaden, 1894. ' Zeitschr. f. physiol. Chem., Bd. 15. " Centialbl. f. d. med. Wiasensch., 1894. ' 'Zeitschr. f. physiol. Chem., Bd. 4. 636 THE URINE. Quantitative Estimation of Proteid in Urine. Of all the methods proposed thus far, the coagulation method (boiling with the addition of acetic acid) when performed with suflScient care gives the best results. The average errors need never amount to more than O.Olj^, and it is generally smaller. In using this method it is best to first find how much acetic acid must be added to a small portion of urine, which has been previously heated on the water- bath, to completely separate the proteid, so that the filtrate does not respond to Heller's test. Then coagulate 20-50-100 c. c. of the urine. Pour the urine into a beaker and heat on the water- bath, add the required quantity of acetic acid slowly, stirring con- stantly, and heat at the same time. Filter while warm, wash first with water, then with alcohol and ether, dry and weigh, ash and weigh again. In exact determinations the filtrate must not give Hellee's test. The above-mentioned method of Devoto may also be used in the quantitative estimation of coagulable proteids. The error originating from the precipitation of uric acid and other urinary constituents by the ammonium snlphate is so very small in ordinary cases where the precipitate is carefully washed that it is unimpor- tant (Redelitjs '). In the presence of only Little proteid in a urine rich in uric acid it may on the contrary be quite considerable. The separate estimation of globulins and albumins is done by carefully neutralizing the urine and prepcipitating with MgSO, added to saturation (authok), or simply by adding an equal volume of a saturated neutral solution of ammonium snlphate (Hofmeistbe and Pohl"). The precipitate consisting of globulin is thoroughly washed with a saturated magnesium sulphate or half-saturated ammonium-sulphate solution, dried continuously at 110° C, boiled with water, extracted with alcohol and ether, then dried, weighed, ashed, and weighed again. The, quantity of albumin is calculated as the difference between the quantity of globulins and the total proteids. Approximate Estimation of Proteid in Urine. Of the methods suggested for this purpose none has been more extensively employed than Esbach's. Esbach's ' method. The acidified urine (acidified with acetic acid) is poured into a specially graduated tube to a certain mark and then the reagent (a 3^ citric-acid and 1^ picric-acid solution in water) is added to a second mark, the tube is closed with a rubber stopper and carefully shaken, avoiding the production of froth. The tube is allowed to stand twenty-four hours, and then the height of the precipitate in the graduated tube is read off. The reading gives directly the quantity of proteid in 1000 parts of the ' Upsala Lakarefs Ferli , Bd. 37, and Maly's Jahresber. , Bd. 23. » Arch. f. exp. Path. u. Pharm., Bd. 80. ' In regard to the literature on this method and the numerous experiments to determine its value see Huppert-Neubauer, 10. Aufl., S. 853. NUCLEOALBUMIN AND MUCIN. 537 urine. Urines rich in proteid mast first be diluted witli water. The results obtained by this method are, however, dependent upon the temperature; and a difference in temperature of 5° to 6.5° 0. may in urines containing a medium quantity of proteid cause an error of 0.2-0.3^ deficiency or excess (Chkistbnsen and Mtgge). This method js only to be used in a room in which the temperature may be kept nearly constant. The directions for the use of the apparatus accompany it. Cheistbnsek's and Mtgge's ' method. 5 c. c. of urine, after being acidified with 2 drops of acetic acid, are poured into a some- what modified burette and precipitated with a certain quantity of a 1^ tannic-acid solution and then treated with 1 c. c. of mucilage. After the addition of water to a certain mark and after inverting the tube several times a uniform emulsion is produced. A cylin- drical glass filled one half or one third with water is now placed on a white surface having a number of close black lines traced upon it, and the contents of the burette are gradually added to the water with constant stirring, until by close observation the black lines cannot even be distinguished from the white spaces. The reading of the quantity of urine emulsion employed gives directly the quan- tity of proteid in the urine. This method is claimed to give very good results. A special description accompanies each apparatus." The method proposed by Roberts and Stolnikow and further developed by Bbandbbrq, though' somewhat more difficult to perform, also gives satis- factory results. The density methods of Lawg, Hdppert, and Zahor^ are also very good. The last consists in determining the specific gravity before and after the coagulation of the proteids. Nucleoalbumin and Mucin. Nucleoalbumin seems to be a regular constituent of urine, although ordinarily it only occurs in very small quantities. Mucin is alleged to occur in small quantities under normal conditions, but appears in greater quantities in catarrhal affections of the urinary passages. There is no doubt* that case^ exist in vi'hich true mucin occurs in the urine; in most cases, nevertheless, we are doubtless dealing with a nucleoalbumin similar to mucin, which originates in the kidneys or urinary passages.' To detect mucin in urine, it must first be diluted with water to prevent a precipitation of the uric acid on subsequent addition of acid, and also to reduce the solvent action of the common salt of the urine on the mucin. Now add an excess of acetic acid. The precipitate formed is purified by dissolving in water with the addition of a little alkali and reprecipitated with acetic acid. The precipitate is tested with the ordinary mucin reagents. To avoid mistaking mucin for nucleoalbumin, which is similar to mucin, the precipitate must be tested in regard to its behavior on boiling with dilute mineral acids. If no reducing substance is formed by this treatment, it contains no mucin. To de- tect nucleoalbumin we proceed in the same manner, but it is better to re- ' See Maly's Jahresber. , Bd. 18, S. 314. ' The apparatus may be obtained from C. Knudsen in Copenhagen. • In regard to these methods see Huppert-Neubauer'sHarnanalyse, 10. Aufl., S. 845-853. < See B. Malfatti, Maly's Jahresber., Bd. 21, S. 22. ' In regard to the literature see Huppert-Neubauer, S. 540 ; Lonnberg, Upsala Lakarefs FOrh., Bd. 25 ; K. MOrner, Hygiea, Bd 53 ; Obermayer, Centralbl. f. klin. Med., Bd. 18. 538 'IHE URllSE. move the salts from tlie urine by means of dialysis (K. Mouner '). Then pre- cipitate with not too much acetic acid. To determine if the precipitate con- sists of nucleoalbumin oranucleoproteid, we test for xanthin bases after boiling with an acid. Large quantities of the precipitate are necessary for this pur- pose. Blood and Blood-coloring Matters. The urine may contain blood from hemorrhage in the kidneys or other parts of the urinary passages (hematuria). In these cases, when the quantity of blood is not very small, the urine is more or less cloudy and colored reddish, yellowish red, dirty red, brownish red, or dark brown. In recent hemorrhages, in which the blood has not decomposed, the color is nearer blood-red. Blood-corpuscles may be found in the sediment, sometimes also blood-casts and smaller or larger blood- clots. In certain cases the urine contains no blood-corpuscles, but only dissolved blood-coloring matters, hemoglobin or, and indeed quite often, methsemoglobin (hemoglobinuria). The blood-pigments appear in the urine under different conditions, as in dissolution of blood in poisoning with arseniuretted hydrogen, chlorates, etc., after serious burns, after transfusion of blood, and also in the periodic appearance of hsemoglobinuria with fever. The urine may in hemoglobinuria also have an abundant grayish-brown sediment rich in proteid which contains the remains of the stromata of the red blood-corpuscles. In animals haemoglobinuria may be produced by many causes which force free heemoglobin into the plasma. To detect blood in the urine we make use of the microscope, spectroscope, the guaiacum test, and Heller's or Hellbr-Tbich- mann's test. Microscopic Investigation. The blood-corpuscles may remain undissolved for a long time in acid urine; in alkaline urine, on the contrary, they are easily changed and dissolved. They often appear entirely unchanged in the sediment; in some cases they are dis- tended, and in others unequally pointed or jagged like a thorn- apple. In hemorrhage of the kidneys a cylindrical clot is sometimes found in the sediment, which is covered with numerous red blood- corpuscles, forming casts of the urinary passages. These formations are called blood-casts. The spectroscopic investigation is naturally of very great value; and if it be necessary to determine not only the presence but also the kind of coloring matter, this method is indispensable. In regard to the optical behavior of the various blood-pigments we must refer to Chapter VI. ' K. MBrner, Hygiea, Bd. 53. TESTS FOB BLOOD PIGMENTS. 539 Guaiacum Test. Mix in a test-tube equal volumes of tincture of guaiacum and old turpentine which has become strongly ozon- ized by the action of air under the influence of light. To this mix- ture, which must not have the slightest blue color, add the urine to be tested. In the presence of blood or blood-pigments, first a bluish-green and then a beautiful blue ring appears where the two liquids meet. On shaking the mixture it becomes more or less blue. Normal urine or one containing proteid does not give this reaction. For the explanation of this we must refer the reader to Chapter VI, page 134. Urine containing pus, although no blood is present, gives a blue color with these reagents; but in this case the tincture of guaiacum alone, without turpentine, is colored blue by the urine (Vitali'). This is at least true for a tincture that has been exposed for some time to the action of air and sunlight. The blue color produced by pus differs from that produced by blood- coloring matters by disappearing on heating the urine to boiling. A urine alkaline \)j decomposition most first be made faintly acid before performing the reaction. The turpentine should be kept exposed to sunlight, while the tincture of guaiacum must be kept in a dark glass bottle. These reagents to be of use must be con- trolled by a liquid containing, blood. This test, it is true, in posi- tive results is not absolutely decisive, because other bodies may give a blue reaction; but when properly performed it is so extremely delicate that when it gives negative results any other test for blood is superfluous. Hellee-Teichmank's Test. If a neutral or faintly acid urine containing blood is heated to boiling, we always obtain a mottled precipitate consisting of albumin and hsematin. If caustic soda is added to the boiling-hot test, the liquid becomes clear and turns green when examined in thin layers (due to haematin alkali), and a red precipitate, appearing green by reflected light, re-forms which consists of earthy phosphates and hssmatin. This reaction is called Heller's blood-test. If this precipitate is collected after a time on a small filter, it may be used for the hfemiu test (see page 143). If the precipitate contains only a little blood- coloring matter with a larger quantity of earthy phosphates, then wash it with dilute acetic acid, which dissolves the earthy phosphates, and use the residue for the preparation of Teichmann's hsemin crystals. If, on the contrary, the amount of phosphates is very small, then first add a little OaCl, solution to the urine, heat to boiling, and add simultaneously with the caustic potash some sodium-phosphate ' SceMaly's Jabresber., Bd. 18, S. 326. 540 THE URINE. solution. In the presence of only very small quantities of blood, first make the urine very faintly alkaline with ammonia, add tannic acid, acidify with acetic acid, and use the precipitate in the prep- aration of the hsemin crystals (Stbuve '). Hsematoporphyrin. Since the occurrence of hsematoporphyrin in the urine in various diseases has been made very probable by several investigators, such as Neussee, Stokvis, MacMunn, Le Nobel, Eussel, Copemaii, and others," Salkowski ° has positively shown the presence of this pigment in the urine after sulphonal intoxications. It was first isolated in a pure crystalline state by the authoe' from the urine of insane women after sulphonal intoxica- tion. According to Gaeeod ' traces of h»matoporphyrin occur regularly in normal uriaes. It is also found in the urine during different diseases, although it only occurs in small quantities. It has been found in considerable quantities in the urine after intoxi- cation with sulphonal. Urine containing hsematoporphyrin is sometimes only slightly colored, while in other cases, as for example after the use of sul- phonal, it is more or less deep red in color. The color depends in these last-mentioned cases, in greatest part, not upon hssmatorpo- phyrin, but upon other red or reddish-brown pigments, which have not been sufficiently studied. The pathogenic moment of hsemato- porphyrinuria is according to Stokvis ' an absorption and elimina- tion of the blood emptied into the intestinal tract or present there and changed into haematoporphyrin. In detecting haematoporphyrin the urine is precipitated with alkaline barium-chloride solution (a mixture of equal volumes of a barium-hydrate solution, saturated in the cold, and a 10^ barium- chloride solution according to Salkowski), or the urine is made strongly alkaline with a soda solution, according to Gaeeod, which precipitates the earthy phosphates. In both cases the hsemato- porphyrin is carried down with the precipitate, while urobilin and certain other pigments remain in solution. The washed precipitate is allowed to stand some time at the temperature of the room with ' Zeitschr. f. anal. Chem., Bd. 11. ' A very complete index of the literature on hsematoporphyrin in the urine may be found by R. Zoja, Su qualche pigmento di alcune urine, etc., in Arch. Ital. dicliu. Med., 1893. 3 Zeitschr. f. Physiol. Chem., Bd. 15. * Skand. Arch. f. Physiol., Bd. 3. ' Journal of Physiol., vols. 13 and 17. « Zeitschr. f. klin. Med. , Bd. 38. PUS IN URINE. 541 alcohol containing hydrochloric or sulphuric acid and then filtered. The filtrate shows the characteristic spectrum of haematoporphyrin in acid solution, and gives the spectrum of alkaline hsematoporphyrin after saturation with ammonia. If the alcoholic solution is mixed with chloroform and a large quantity of water added and carefully shaken, sometimes a lower layer of chloroform is obtained which contains very pure haematoporphyrin, while the upper layer of alcohol and water contains the other pigments besides some haemato- porphyrin. Baumstark ' found in a case of leprosy two cbaracteristic coloring matters in the urine, " urorubrobsematin " and " urofuscobaematin," wbicb, as tbeir names indicate, seem to stand In close relationsliip to tbe blood-coloring matters. Urorubrolmnmtiii, CjeH^NgFejOae, contains iron and shows an absorption- band in front of D and a broader one back of D. In alkaline solution It shows four band.s, behind D, at E, beyond F, and behind O. It is not soluble either in water, alcohol, ether, or chloroform. It gives a beautiful brownish-red non- dichroitic liquid with alkalies. Urofuscohmmatin, CasHioeNsOaa, which is free from iron, shows no characteristic spectrum; it dissolves in alkalies, producing a browu color. It remains to be proved whether these two pigments are related to (impure) haematoporphyrin. Melanin. In the presence of melanotic cancers dark coloring matters are sometimes eliminated with the urine. K. Morner * has isolated two pig- ments from such a urine, of which one was soluble in warm 50-75JS acetic acid and the other, on the contrary, was insoluble. The one seemed to be phymat- orhuain (see Chapter XVI). Usually the urine does not contain any melanin, but a chromogen of melanin, a melanogen. In such cases the urine gives Eibblt's reaction, becoming dark-colored with oxidizing agents such as cone, nitric acid, potassium bichromate, and sulphuric acid, as well as with free sul- phuric acid. Urint- containing melanin or melanogen is colored black by fer- ric-chloride solution (v. Jaksch'). Urorosein, so named by Nencki,'' is a urinary coloring matter, occurring in various diseases, which appears on the acidification of the urine with a mineral acid, and which is taken up by shaking with amjl-alcohol. The solution shows an absorption-band between D and E. This pigment, which is not soluble in chloroform or ether, is not identical with indigo-red. Alkalies decolorize the solution of this pigment immediately, and it is also rather quickly bleached by light. According to Zawadski ' urorosein is derived from' urobilin by oxida- tion. Uroerythrin, which gives a rose-red color to the urinary sediments especially in fevers, seems to occur also in urine under physiological condi- tions. Pus occurs in the urine in different inflammatory affections, especially in catarrh of the bladder and in inflammation of the membrane of the kidneys or the urethra. Pus is best defected by means of the microscope. The pus-cells are rather easily destroyed in alkaline urines. ■ In detecting pus we make use of Doij^ne's pus-test, which is performed in the following way : Pour off the urine from the sediment as carefully as possible, place a small piece of caustic aLkali on the sediment, and stir. If 1 Pfluger's Arch., Bd. 9. ' Zeitsolir. f. physiol. Cham., Bd, 11. > Ibid.. Bd. 13. * Nencki und Sieber, Journal f. prakt. Chem. (N. P.), Bd. 26. = Arch. f. exp. Path. u. Pharm., Bd. 38. i)4:2 THE UBINE. the pus-cells have not been previously changed, the sediment is converted by this means into a slimy tough mass. The pus-corpuscles swell up in alkaline urines, dissolve, or at least are so changed that they canaot be recognized under the microscope. The urine ia these cases is more or less slimy or fibrous, aud it is precipitated in large flakes by acetic acid, so that it may possibly be mistaken for mucin. The closer investigation of the precipitate produced by acetic acid, and especially the appearance or non-apppearaace of a reducing substance after boiling it with a mineral acid, demonstrates the nature of the precipitated substance. Urine containing pus always contains proteid. Bile-acids. The statements in regard to the occurrence of bile- acids in the urine under physiological conditions do not agree. According to Dragendorff and Hone traces of bile-acids occur in the urine; according to Mackat and v. Udeanszkt,' they do not. Pathologically they are present in the urine in hepatogenic icterus, although not always. Detection of Bile-acids in the urine. Petteitkofee's test gives the most decisive reaction; but as it gives similar color reactions with other bodies, it must be supplemented by the spectro- scopic investigation. The direct test for bile-acids is easy after the addition of traces of bile to a normal urine. But the direct detec- tion in a colored icteric urine is more diflScult and gives very misleading results; the bile-acid must therefore always be isolated from the urine. This may be done by the following method of Hoppe-Setlek, which is slightly modified in non-essential points. Hoppb-Seylee's Method. Strongly concentrarte the urine, and extract the residue with strong alcohol. The filtrate is freed from alcohol by evaporation and then precipitated by basic lead acetate and ammonia. The washed precipitate is treated with boiling alcohol, filtered hot, the filtrate treated with a few drops of soda solution, and evaporated to dryness. The dry residue is extracted with absolnte alcohol, filtered, and an excess of ether added. The amorphous or, afcer a longer time, crystalline precipi- tate consisting of alkali-salts of the biliaty acids is used in perform- ing Pettejtkofee's test. Bile-coloring matters occur in the urine in different forms of icterus. A urine containing bile-coloring matters is always abnor- mally colored — yellow, yellowish brown, deep brown, greenish yellow, greenish brown, or nearly pure green. On shaking it froths, and the bubbles are yellow or yellowish green in color. As a rule icteric urine is somewhat cloudy, and the sediment is fre- quently, especially when it contains epithelium-cells, rather strongly ' Cited from Happert-Neubauer, Harnanal^se, 10. Aufl., S. 239. TESTS FOM BILE PIGMENTS. 543 colored by the bile-pigments. In regard to the occurrence of urobilin in icteric urine see page 501. Detection of bile-coloring matters in urine. Many tests have been proposed for the detection of bile-coloring matters. Ordinarily we obtain the best results either with Gmelin's or with Huppbrt's test. Gmelin's test may be applied directly to the urine; but it is better to use Eosexbach's modification. Filter the urine through a very small filter, which is deep-colored from the retained epithe- lium-cells and bodies of that kind. After the liquid has entirely passed through apply to the inside of the filter a drop of nitric acid which contains only very little nitrous acid. A pale-yellow spot will be formed which is surrounded by colored rings which appear yellowish red, violet, blue, and green from within outward. This modification is very delicate, and it is hardly possible to mistake indican and other coloring matters for the bile-pigments. Several other modifications of Gmelin's test on the urine directly, as with concentrated sulphuric acid and nitrate, etc., have been proposed, but they are neither simpler nor more delicate than Kosbnbach's modification. Huppeet's Reaction. In a dark-colored urine or one rich in indican we do not always obtain good results with Gmelin's test. In such cases, as also in urines containing blood-coloring matters at the same time, the urine is treated with lime-water, or first with some CaGl, solution, and then with a solution of soda or ammonium carbonate. The precipitate which contains the bile-coloring matters is filtered and used for Huppekt's test (see page 236). The precipitate consisting of lime-pigments may also be shaken out with chloroform after washing in water and after being acidified with acetic acid. The bilirubin is taken up by the chloroform, which is colored yellow thereby, while the acetic-acid solution is colored green by the biliverdin. Both solutions may then be used for Gmelin's '.est (Hoppe-Setlbr), and small quantities of bile- coloring matters may be detected in this way. The lime-pigments may, according to Hilger, also be used directly for Gmelin's test in the following way: Spread them on a porcelain dish in a thin layer, and add carefully a drop of nitric acid. The reaction generally appears very beautiful. JoLLEs' Method.' Place 50 c. c. of the urine in a cylinder with a glass stopper, add a few drops of 10^ hydrochloric acid and an excess of a barium-chloride solution with 5 c. c. chloroform, and shake thoroughly for a few minutes. After about 10 minutes remove the chloroform and the precipitate by means of a pipette and place in a test-tube and heat on the water-bath at about 80° 0. ' Zeitschr. f. pliysiol. Cliem., Bd 18, S. 545. This contains the literature on all the known tests for bile-pigrments with the exception of Stokvis'a test, which may be found in Maly'a Jahresber., Bd. 18, S. 236. 544 THE URINE. After the evaporation of the chloroform carefully decant the liquid from the precipitate and allow 3 drops concentrated nitric acid con- taining -I fuming nitric acid to flow down the sides of the test-tube. In the presence of bile-pigments the characteristic colored rings are obtained, and this modification, according to Jolles, is the most delicate of all tests for bile-pigments. Stokvis's reaction is especially valuable in those cases in which the urine contains only very little bile-coloring matter together with larger quantities of other coloring matters. The test is performed as follows: 20-30 c. c. nrine are treated with 5-10 c. c. of a solu- tion of zinc acetate (1 : 5). The precipitate is washed on a small filter with water and then dissolved in a little ammonia. The new filtrate gives, directly or after it has stood a short time in the air until it has a peculiar brownish-green color, the absorption-bands of bilicyanin (see page 335). Many other reactions for bile-coloring matters in the urine have been proposed; but as the above-mentioned are sufficient, it is perhaps only necessary to give here a few of the other reactions, without entering into details. Ultzmann's reaction consists in treating about 10 c. c. of the urine with 3-4 c. c. concentrated caustic-potash solution and then acidifying with hydro- chloric acid. The urine will become a beautiful green. Smith's Reaction. Pour carefully over the urine tinpture of iodine, where- by a green ring appears between the two liquids. You may also shake the urine with tincture of iodine until it has a green color. Bhklich's Test. First mix the urine with an equal volume of dilute acetic acid and then add drop by drop a solution of sulpho-diazobenzol. The a:;id mixture becomes dark red in the presence of bilirubin, and this color becomes blaish violet on the addition of glacial acetic acid. The sulpho-diazobeiizol is prepared with 1 grm. sulphanilic acid, 15 c. c. hydrochloric acid, and 0.1 grm. sodium nitrite; this solution is diluted to 1 litre with water. Mbdicinal colobing matters produced from santonin, rhubarb, senna, etc., may give an abnormal color to the urine which may be mistaken for bile- coloring matters or, in alkaline urines, perhaps for blood-coloring matters. If hydrochloric acid is added to such a urine, it becomes yellow or pale yellow, while on the addition of an excess of alkali it becomes more or less beautifully red. Sugar in TJrine. The occurrence of traces of grape-sugar in the urine of perfectly healthy persons has been, as above stated (page 506), quite posi- tively proved. If sugar appears in the urine in constant and especially in large quantities, it must be considered as an abnormal constituent. We have given in a previous chapter several of the most important conditions which cause glycosuria in man and animals, and we must refer the reader to Chapters VIII and IX for the essential facts in regard to the appearance of sugar in the urine. In man the appearance of glucose in the urine has been ob- DETECTION OF SUGAR. 545 •«eryed in numerous and various pathological conditions, such as lesions of the brain and especially of the medulla oblongata, abnor- mal circulation in the abdomen, diseases of the heart and lunge, diseases of the liver, cholera, and many other diseases. The continned presence of sugar in human urine, sometimes in very considerable quantities, occurs in diabetes mellitus. In this disease there may be an elimination of 1 kilogramme or even more of grape-sugar during the 24 hours. In the beginning of the disease, when the quantity of sugar is still very small, the urine often does not appear abnormal. la more developed, typical cases the quantity of urine voided increases considerably, to 3-6-10 litres per 24 hours. The percentage of the physiological constituents is as a rale very low, while their absolute daily quantity is increased. The urine is pale, but of a high specific gravity, 1.030-1,040 or even higher. The high specific gravity depends upon the quantity of sugar present, which varies in different cases, but may be as high as 10^. The urine is therefore characterized in typical cases of diabetes by the very large quantity voided, by the pale color and high specific gravity, and by its containing sugar. That the urine after the introduction of certain medicines or poisonous bodies into the system contains reducing bodies, con- jugated glycuronic acids, which may be mistaken for sugar, has already been mentioned. The properties and reactions of glucose have been treated of in a previons chapter, and it remains but to mention the methods of detecting and quantitatively estimating glucose in the urine. The detection of sugar in the urine is ordinarily, in the presence of not too small quantities of sugar, a very simple task. The pres- ence of only very small quantities may make its detection sometimes very difficult and laborious. A urine containing proteid must first have the proteid removed by coagulation with acetic acid and heat before it can be tested for sugar. The tests which are most frequently employed and are especially recommended are as follows : Tkommee's Test. In a typical diabetic urine or one rich in sugar tbis test succeeds well, and it may be performed in the manner suggested on page 69. This test may lead to very great mistakes in urines poor in sugar, especially when they have at the same time normal or increased amounts of physiological con- stituents, and therefore it cannot be recommended to physicians or to persons inexperienced in such work. Normal urine contains reducing substances, such as uric acid, creatinin, and others, and ^46 THE UBINE. therefore a f^duction takes place with all urine on nsiiig fhig test;. We do not gpijerally have a separation of copper saboxide, hnt still if we vary the proportion of the alkali to the copper sulphate and boil we often have an actual separation of suboxide in normal urines, or we obtain a peculiar yellowish-red liquid due to finely (iivided hydrated suboxide. This occurs especially on the addition pf much alkali or too much copper sulphate, and by careless manipulation the inexperienced worker may therefore sometimes obtain apparently positive results in a normal urine. On the other hand, as urine contains substances, such as creatiuin and ammonia (from the urea), which in the presence of only little sugar may keep the copper suboxide in solution, he may easily overlook small quantities of sugar that may be present. Teommer's test may of course be made positive and useful, even in the presence of very small quantities of sugar, by using the modification suggested by Woem Mullek. As this modification is rather complicated, and requires much practice and exactness, it is probably rarely employed by the busy physician. The follow- ing test is to be preferred: Almen's bismuth test, which recently has been incorrectly called !Ntlakder's, test, is performed with the alkaline bismuth solution prepared as above described (page 69). For each test 10 c. c. of urine are taken and treated with 1 c. c. of the bismuth solntion and boiled for a few minutes. In the presence of sugar the urine becomes darker yellow or yellowish brown. Then it grows darker, cloudy, dark brown, or nearly black, and non- transparent. After a shorter or longer time a black deposit appears, the supernatant liquid gradually clears, but still remains colored. In the presence of only very little sugar the test is not black or dark brown, but simply deeper-colored, and not until after some time do we see on the upper layer of the phosphate precipitate a dark or black edge (of bismuth?). In the presence of much sugar a larger amount of reagent may be used without disadvantage. In a urine poor in sugar we must use only 1 c. c. of the reagent for every 10 c. c. of the urine. This test shows the presence of 1-0.5 p. m. sugar in the urine. The sources of error which interfere in Teommer's test, such as the presence of uric acid and creatiuin, entirely disappear in this test. The bismuth test is, besides, more easily performed, and it is therefore to be recommended to the physician. Small quantities of pioteid do not interfere with this test; large quantities may give rise to an error by forming bismuth sulphide, and therefore must l)e removed by coagulation. In using this method it must not be overlooked that it is, like Trommer's test, a reduction test, and it consequently may show, besides sugar, certain other reducing substances. Such bodies are certain conjugated glycuronic acids which may appear in the urine. Positive results have been obtained with the bismuth test on urine DETEGTION OP BUG AS. 647 after the use of several medicines such as rhubarb, senna, antipyrin, kairin, aalol, turpentine, and others. From this it follows that we fihould never be satisfied with this test alone, especially when the reduction is not very great. When this test gives negative results we can consider the urine as free from sugar from a clinical stand- point, and when it gives positive results other tests must be applied. Among these the fermentation test is of special value. Fermentation Test. On using this test we must proceed in various ways, according as the bismuth test shows small or large quantities. If a rather strong reduction is obtained, the urine may be treated with yeast and the presence of sugar determined by the generation of carbon dioxide. In this case the acid urine, or that faintly acidified with tartaric acid, is treated with yeast which has previously been washed by decantation with water. Pour this urine to which the yeast has been added into a Schrottek's gas- burette, or glass tabe with the open end ground, close with the thumb, and open under the surface of mercury contained in a dish. As the fermentation proceeds, the carbon dioxide collects in the upper part of the tube, while a corresponding quantity of liquid is expelled below. As a control in this case two other similar tests must be made, one with normal urine and yeast to learn the quan- tity of gas usually developed, and the other with a sugar solution and yeast to determine the activity of the yeast. If, on the contrary, we find only a faint reduction with the bismuth test, no positive conclusion can be drawn from the absence of any carbon dioxide or the appearance of a very insignificant quantity. In this case proceed in the following way: Treat the acid nrine, or the urine which has been faintly acidified with tartaric acid, with yeast whose activity has been tested by a special test on a sugar solution, and allow it to stand 24r-48 hours at the tempera- ture of the room, or, better, at a little higher temperature. After this time test again with the bismuth test, and if the reaction now gives negative results, then sugar was previously present. But if the reaction continues to give positive results, then it shows — if the yeast is active — the presence of other reducing, unfermentable bodies. There remains of course the possibility that the urine also contains some sugar besides these bodies. This possibility may be determined by the following test: Phenylhydrazin Test. According to v. Jaksch,' this test is performed in the following way: Add in a test-tube containing 8-10 c. c. of the urine two knife-points of phenylhydrazin hydrochloride and three knife-points sodium acetate, and when the added salts do not dissolve on warming add more water. The mixture is heated in boiling water and kept there for one hour to avoid a confusion with phenylhydrazin-glycuronic acid (v. Jaksch and Hihsohl). It is then poured into a beaker of cold water. If the quantity of sugar present is not too small, a yellow crystalline precipitate la now • V. Jaksch, Klin. Diagnostik, 4. Aufl., S. 375. 548 - THE URINE. obfcaiaed. If the precipitate appears amorphous, there are found, OQ looking at it under the microscope, yellow needles singly and in groups. If very little sugar is present, pour the test into a conical glass and examine the sediment. In this case at least a few phenylglucosazone crystals are found, while the occurrence of smaller and larger yellow plates or highly refractive brown globules, do not show the presence of sugar. According to y. Jaksch, this, reaction is very reliable, and by it the presence of 0.3 p. m. sugar can be detected (Rosenbeeg,' Geyer"). The valae of this test has been considerably debated, and the objection has been made that glycuronic acid also gives a similar precipitate. A confounding with glycuronic acid is, according to HiKSCHL,' not to be apprehended when it is not heated in the water-bath for too short a time (one hour). Kisteemanx * found this precaution insafficient, and Eoos' states that the phenyl- hydrazin test always gives a positive result with human urine. In doubtful cases where we wish to be quite positive, prepare the crystals from a large quantity of urine, dissolve them on the filter by pouring over them hot alcohol, treat the filtrate with water, and boil off the alcohol. If the characteristic yellow crystalline needles,, whose meltiog-point ('-i04-205° C.) is also determined, are now obtained, then this test is decisive for the presence of sugar. It must not be forgotten that laevulose gives the same osazone aa grape-sugar, and that a further investigation is necessary in certain cases. Polarization. This test differentiates between dextrose, which polarizes to the right, and laevulose, which polarizes to the left. The polariscopic investigation is of great value, especially as in maoy cases it quickly differentiates between sugar and other reduc- ing, laevogyrate substances, such as conjugated glycuronic acid. In the presence of only very little sugar the value of this test depends on the delicacy of the instrument and the dexterity of the observer; therefore this method is perhaps inferior in most cases to the bismuth test or to the phenylhydrazin test. If small quantities of sugar are to be isolated from the urine, precipitate the urine first with sugar of lead, filter, precipitate the filtrate with ammoniacal basic lead acetate, wash this precipitate with water, decompose it with H^S when suspended in water, con- centrate the filtrate, treat it with strong alcohol until it is 80 vol. per cent, filter when necessary, and add an alcoholic caustic-alkali Bolution. Dissolve the precipitate consisting of saccharates in a ' Deutscli. med. Wochenschr., 1888. » Wien. med. Presse, 1889, S. 1688. Cited from Roos, Zeitschr. f. physlo'l. Chem., Bd. 15, S. 524. » Zeitschr. f. pliysiol. Cliem., Bd. 14. * Deutsch. Arcli. t. klin. Med., Bd. 50. Cited from Maly's Jahresber., Bd. 22, S. 339. ' Zeitschr. f. physiol. Chem., Bd. \5. QUANTITATIVE ESTIMATION OF 8UGAS. 549 little water, precipitate the potash by an excess of tartaric acid, neutralize the filtrate with calcium carbonate in the cold, and filter. The filtrate may be used for testing with the polariscope as well as in the fermentation, bismuth, and phenylhydrazin tests. The presence of grape-sugar may be' detected by this same process in animal fluids or tissues from which the proteids have been removed by coagulation or by the addition of alcohol. For the physician, who naturally wants specially simple and quick methods, the bismuth test mast be especially recommended. If this test gives negative results, the urine is to be considered as free from sugar in a clinical sense. If it gives positive resalts, the presence of sugar must be controlled by other tests, especially by the fermentation test. Other tests for sugar, as, for example, the reaction with orthonltrophenyl- propjolic acid, i icric acid, diazobenzol-sulphonic acid, are superfluous. The reaction with nr-naphthol, which is a reaction for carbohydrates in general, for ^lycuronic acid and mucin, may, because of its extreme delicacy, give rise to mistakes, and is therefore not to be recommended to pliysicians. Normal urines give this test, and if the strongly diluted urine gives this reaction we may consider the presence of large quantities of carbohydrates. In these cases we get more positive results by using other tests. This test requires great •cleanliness, and it has this inconvenience, that it is very difficult to get suffi- ciently pure sulphuric acid, and sometimes indeed perfectly pure a-naphthol. Several investigators, such as v. UdeAnsky, Luther, Rods, and Theupkl,' have investigated this test in regard to its applicability as an approximate test for carbohydrates in the urine. Quantitative Estimation of Sugar in the urine. The urine for such an estimation must first be tested for proteid, and if any be present it must be removed by coagulation and the addition of acetic acid, care being taken not to increase or diminish the original volume of urine. The quantity of sugar may be determined by TITRATION with Fehling's or Knapp's solution, by fermenta- tion, or by POLAEIZATION. The titration liquids not only react with sugar, but also with certain other reducing substances, and on this account the titration methods give rather high results. When large quantities of sugar are present, as in typical diabetic urine, which generally contains a lower percentage of normal reducing constituents, this is indeed of little account; but when small quantities of sugar are present in an otherwise normal urine, the mistake may, on the contrary, be im- portant, as the reducing power of normal urine may correspond to 6 p. m. grape-sugar (see page 506). In such cases the titration method must be employed in connection with the fermentation method, which will be described later. It is to be remarked that in typical diabetic urines with considerable quantities of sugar the titration with Fehling's solution is just as reliable as with Knapp's solution. When the urine, on the contrary, contains only little sugar with normal amounts of physiological constituents, then ' See Roos and Treupel, Zeitschr. f. physiol. C'hem., Bdd. 15 u. 18. S60 ma URINE. the titration with FEHiJisrG's solution is more difficult, indeed ir* certain cases almost impossible, the results being very uncertain. In siich cases Kxapp's method gives good results, according ta WoKM MtJLLER and his pupils. ' The TiTEATiON with Fehling's solution depends on the power of sugar to reduce copper oxide in alkaline solutions. For this we formerly employed a solution which contained a mixture of copper sulphate, Eochelle salt, and sodium or potassium hydrate (Fehling's solution) ; but as such a solution readily changes, we now prepare a copper-sulphate solution and an alkaline Rochelle- salt solution separately, and mix equal volumes of the two solutions, before using. The concentration of the copper-sulphate solution is such that 10 c. c. of this solution is reduced by 0,05 grm. grape-sugar. The: copper-sulphate solution contains 34.65 grms. pure, crystallized, non-efflorescent copper sulphate in 1 litre. The sulphate is crystal- lized from a hot saturated solution by cooling and stirring ; and the= crystals are separated from the mother-liquor and pressed between blotting-paper until dry. The Eochelle-salt solution is prepared by dissolving 173 grms. of the salt in 350 c. c. water, adding 600 c. c. of a caustic-soda solution of a specific gravity of 1.13, and dilnting^^ with water to 1 litre. According to Worm Muller, these three liquids — Eochelle-salt solution, caustic soda, and water — should be^ separately boiled before mixing together. For each titration mix in a small flask or porcelain dish exactly 10 c. c. of the copper- sulphate solution and 10 c. c. of the alkaline Eochelle-salt solution and add 30 c. c. water. The urine free from proteid is diluted before the titration with water so that 10 c. c. of the copper solution requires between 5 and 10 c. c. of the diluted urine, which corresponds to between 1 and •J^ sugar. A urine of a specific gravity of 1.030 may be diluted five times; one more concentrated, ten times. The urine so diluted is poured into a burette and allowed to flow into the boiling copper- sulphate and Eochelle-salt solution until the copper oxide is com- pletely reduced. This has taken place when, immediately after- boiling, the blue color of the solution disappears. It is very- difficult and requires some practice to exactly determine this point, especially when the copper suboxide settles with difficulty. To determine whether the color has disappeared, allow the copper sub- oxide to settle a little below the meniscus formed by the surface of the liquid. If this layer is not blue, the operation is repeated, adding 0.1 c. c. less of urine; and if, after the copper suboxide has settled, the liquid has a blue color, the titration may be considered as completed. Because of the difficulty in obtaining this point exactly another end-reaction has been suggested. This consists in filtering immediately after boiling a small portion of the treated urine through a small filter into a test-tube which contains a little; ' Pfltlger's Arch., Bdd. 16 u. 33; Journal f. prakt. Chem. (N. F.), Bd. 26. QUANTITATIVE ESTIMATION OF SUGAR. 551 acetic acid and a few drops of potassium-ferrocyamde solution and water. The smallest quaatity of copper is shown by a red colora- tion. If the operation is quickly conducted so that no oxidation of the suboxide into oxide takes place, this end-reaction is of value for urines which are rich in sugar and poor in urea and which have been strongly diluted with water. In urines poor in sugar which contain the normal amount of urea and which have not been strongly diluted, a rather abundant formation of ammonia from the urea may take place on boiling the alkaline liquid. This ammonia dissolves the suboxide in part, which easily passes into oxide thereby, and besides this the dissolved suboxide gives a red color with potassium ferrocyanide. In just those cases in which the titra- tion is most difficult this end-reaction is the least reliable. Practice also renders it unnecessary, and it is therefore best to depend simply upon the appearance of the liquid. To facilitate the settling of the copper suboxide and thereby clearing the liquid, Munk ' has lately suggested the addition of a little calcium-chloride solution and boiling again. A precipitate of calcium tartrate is produced which carries down the suspended copper suboxide with it, and the color of the liquid can then be better seen. This artifice succeeds in many cases, but unfortunately there are urines in which the titration with Peeling's solution in no way gives exact results. In those cases in which only small quantities of sugar exist in a urine rich in physiological constituents it is best to dissolve a very exactly weighed quantity of pure dextrose or dextrose-sodium chloride in the urine. The urine can now be strongly diluted with water and the titration is successful. The difference between the added sugar and that found by titra- tion gives the reducing power of the original urine calculated as dextrose. The necessary conditions for the success of the titration under all circumstances are, according to Soxhlet,' the following: The cbpper-sulphate and Eochelle-salt solution must, as above, be diluted to 50 c. c. with water; the urine must only contain between 0.5$^ and 1^ sugar, and the total quantity of urine required for the reduction must be added to the titration liquid at once and boiled with it. From this last condition it follows that the titration is dependent upon minute details, and several titrations are required for each determination. It is best to give here an example of the titration. The proper amount of copper-sulphate and Eochelle-salt solution and water (total volume = 50 c.c.) is heated to boiling in a flask; the color must remain blue. The urine diluted five times is now added to the boiling-hot liquid, 1 c. c. at a time; after each addition of urine boil for a few seconds, and look for the appearance of the end- reaction. If you find, for example, that 3 c. c. is too little, but ' Vircbbw'B Arch., Bd. 105. » Journal f. prakt. Chem. (N. F.), Bd. 21. 552 , THE URINE. that 4 c. c. is too much (the liquid becoming yellowish), then the urine has not been suflBciently diluted, for it should require between 5 and 10 c. c. of the urine to produce the complete reduction. The ■urine is now diluted ten times, and it should require bebween 6 and 8 c. c. for a total reduction. Now prepare foar new tests, which are boiled simultaneously to save time, and add at one time respectively 6, 6^, 7, and 7^ c. c. of urine. If it is found that between 6^ and 7 c. c. are necessary to produce the end-reaction, then make four other tests, to which add respecbively 6.6, 6.7, 6.8, and 6.9 c. c. of urine. If in this case the liquid is still somewhat bluish with 6.7 c. c. and completely decolorized with 6.8 c. c, we then consider the average figure 6.75 c. c. as correct. The calculation is simple. The 6.75 c. c. used contain 0.05 grm. sugar, and the percentage of sugar in the dilute urine is 5 therefore (6.75 : 0.05 = 100 : x) = —^ = 0.74. But as the urine 0.75 was diluted with ten times its volume of water, the undiluted urine 5 X 10 contained -;r-wv— = 7.4^. The general formula on using 10 c. c. 6.76 5 X w copper-sulphate solution is therefore — =; — , in which n represents the number of times the urine has been diluted and k the number of c. c. used for the titration of the diluted urine. The TITRATION ACCORDING TO Kkapp depends on the fact that mercuric cyanide is reduced into metallic mercury by grape-sugar. The titration liquid should contain 10 grms. chemically pure dry mercuric cyanide and 100 c. c. caustic-soda solution of a specific gravity of 1.145 per litre. When the titration is performed as described below (according to Worm Mullbr and Otto), 20 c. c. of this solution should correspond to exactly 0.05 grm. grape-sugar. If we proceed in other ways, the value of the solution is different. Also in this titration the quantity of sugar in the urine should be between i^ and 1^, and here also the extent of dilution neces- sary must be determined by a preliminary test. To determine the end-reaction as described below, the test for excess of mercury is made with sulphuretted hydrogen. In performing the titration allow SJO c. c. of Kutapp's solution to flow into a flask and dilate with 80 c. c. water, or, when you have reason to think that the urine contains less than 0.5^ of sugar, only with 40-60 c. c. After this heat to boiling and allow the dilute urine to flow gradually into the hot solution, at first 2 c. c, then 1 c. c, then 0.5 c. c, then 0.2 c. c, and lastly 0.1 c. c. After each addition let it boil -J minute. When the end-reaction is approaching, the liquid begins to clarify and the mercury separates with the phosphates. The end-reaction is determined by taking a drop of the upper layer of the liquid into a capillary tube and then blowing it out on pure white filter-paper. The moist spot is first held over a bottle containing fuming hydrochloric acid and then QUANTITATIVE ESTIMATION OF BUGAB. 553 over strong sulphuretted hydrogen. The presence of a minimum quantity of mercury-salt in the liquid is shown by the spot becom- ing yellowish, which is seen best when it is compared with a second spot which has not been exposed to sulphuretted hydrogen. The_ end-reaction is still clearer when a small part of the liquid is filtered, acidified with acetic acid, and tested with sulphuretted hydrogeu (Otto '). The calculations are just as simple as for the previous method. This titration, unlike the previous one, may be performed not only in daylight, but also in artificial light. Knapp's method has the following advantages over Fehling's method : It is applicable even when the quantity of sugar in the urine is very small and the quantity of the other urinary constituents is normal. It is more easily performed, and the titration liquids may be kept without decomposing for a long time (Worm Mullee and his pupils "). The views of different investigators on the value of this titration method are still somewhat contradictory. Estimation of the Quantity of Sitgar by Fermentation. This may be done in various ways; the simplest, and one at the same time suflSciently exact for ordinary cases, is Egberts' ' method. This method consists in determining the specific gravity of the urine before and after fermentation. In the fermentation of sugar, carbon dioxide and alcohol are formed as chief products and the specific gravity is lowered, partly on account of the disappearance of the sugar and partly on account of the production of alcohol. Roberts found that a decrease of 0.001 in the specific gravity corresponded to 0.33^ sugar, and this has been substantiated since by several other investigators (Worm Mullbr ' and others). If the urine, for example, has a specific gravity of 1.030 before fermentation and 1.008 after, then the quantity of sugar contained therein was 22 X 0.23 = 5.06^. In performing this test the specific gravity must be taken at the same temperature before and after the fermentation. The urino must be faintly acid, and when necessary Jt should be acidified with a little tartaric-acid solution. The activity of the yeast must, when necessary, be controlled by a special test. Place 200 c. c. of the urine in a 400-c. c. flask and add a piece of compressed yeast the size of a pea, and subdivide the yeast through the liquid by shaking, close the flask with a stopper provided with a finely drawn-out glass tube, and allow the test to stand at the temperature of the room or, still better, at -f 20-25° C. After 24-48 hours the fermentation is ordinarily ended, but this must be verified by the bismuth test. After complete fermentation filter through a dry > Journal f. prakt. Chem., Bd. 26. 'PflUger's Arcli., Bdd. 16 u. 23. » Edinburgh Med. Journal, Oct. 1861; The Lancet, Vol. 1, 1863. ''■ Pttuger's Arch., Bdd. 33 u. 37. 654 THE URINE. filtesr, bring the filtrate tti tHe proper temperature, and determine the specific gravity. If the spedific gravity be determined with a good pyknometer supplied wibh a thermometer and an expansion-tube, this method, when the quantity of sugar is not less than 4-5 p. m., gives, according to Worm Mullbe, very exact results, but this has been disputed by Buddb.' For the physician the method in this form is not quite serviceable. Even when the specific gravity is deter- mined by a delicate urinometer which can give the density to the fourth decimal, we do not obtain quite exact results, because of the principal errors of the method (Budde) ; but the errors are usually smaller than those which occur in titrations made by unpractised hands. Among the methods proposed and closely tested for the quantitative estimation of sugar, we have none which are at the same time easily performed and which give positive results in other than experienced hands. When the quantity of sugar is less than 5 p. m. these methods cannot be used. Such a small quantity of sugar cannot, as above mentioned, be determined by titration directly, because the reduc- tion power of normal urine corresponds to 4-5 p. m. In such cases, according to Woem Mullbb, first determine the reduction power of the urine by titration with Knapp's solution, then ferment the urine with the addition of yeast, and titrate again with Knapp's solution. The difference found between the two titra- tions calculated as sugar gives the true quantity of sugar. Estimation" of Sugae by Polaeization. In this method the urine must be clear, not too deeply colored, and, above all, must not contain any other optically active substances besides glucose. By using a delicate instrument and with sufiicient practice very exact results can be obtained by this method. For the physician, EoBEKTS' fermentation test, which requires no expensive apparatus and no special practice, is to be preferred. Under such circum- stances, and as the estimation by means of polarization can be performed with exactitude only by specially instructed chemists^ it is hardly necessary to give this method in detail, and the reader is referred to special works for instructions in the use of the apparatus. Levulose. Laevogyrafe urines containing sugar liave been observed by Vbntzkb, Zimmek and Czapek, Sebgbn, and others.' The nature of the sub- stance causing this action is difficult to discribe exactly, but there is hardly any doubt that the urine, at least in certain cases, as in those observed by Sbb- 6BN, contains levulose. The occurrence of laevulose in the urine from Bee- eBN's patient has been made very possible by Kt'i,z.' The presence of levulose in a urine containing sugar is only probable when the urine is laevogyrate or Optically inactive, or When it shows a reduction power not corresponding (less) to the dextrorotary power, or when it contains 'TJgeskrift for Laeger. (4), Bd. 9; Pfltlger's Arcil., Bd. 40; Zeitschr. f. physicl. Cbem., Bd. 13. 2 See Huppert-Neubauer, Harnanalyse, 10. Aufl., S. 125. 3 Zeitschr. f. Biologle, Bd. 27. MILK-SUGAR AND PENTOSES. 555 no other IsBvcegyrate Substance f/tf-oxybutyrio acid, conjugated glycuronio acids, protein bodies, or cystin). Levulose ferments with yeast and yields tlie same osazuoe as glucose. Laiose is a substance found by Leo ' in diabetic urines in certain cases, and which Leo considers as a sugar. It is laevoegyrate, amorphous, and has no sweet taste, but rather a sharp and salty taste. Laiose has a reducing action on metallic oxides, does not ferment, and gives a non-crystalline, yellow- ish-brown oil with phenylhydrazin. We have no positive proof as yet that this substance is a sugar. MiLK-suGAK. The appearance of milk-sngar in the urine with engorgement of milk has been made known especially by the inves- tigations of De Sinety and F. Hofmeister." After taking large quantities of milk-sngar some lactose may be found in the urine (see Chapter IX on absorption). The positive detection of milk-sugar in the urine is difficult, because this sugar is, like glucose, dextrogyrate and also gives the usual reduction tests. If urine contains a dextrogyrate, non- fermentable sugar which reduces bismuth solutions, then it is very probable that it contains milk-sugar. It must be remarked that the fermentation test for milk-sugar is, according to the experience of LtrsK and VoiT,' best performed by using pure cultivated yeast (saccharomyces apiculutus). This yeast only ferments the glucose, while it does not decompose the milk-sugar. The most positive means for the detection of lactose is to isolate the sugar from the urine. This may be done by the following method, suggested by F. Hofmbistek:' Precipitate the urine with sugar of lead, filter , wash with water, unite the filtrate and wash-water, and precipitate with ammonia. The liquid filtered from the precipitate is again precipitated by sugar of lead and ammonia until the last filtrate is optically inactive. The several precipitates with the excep- tion of tlie first, which contains no sugar, are united and washed with water. The washed precipitate is decomposed in the cold with sulphuretted hydrogen and filtered. The excess of sulphuretted hydrogen is driven off by a current of air; the acids set free are removed by shaking with silver oxide. Now filter, remove the dissolved silver by sulphuretted hydrogen, treat with barium car- bonate to unite with any free acetic iicid present, and concentrate. Before the evaporated residue is syrupy it is treated with 90^ alcohol until a flocculent precipitate is formed which settles quickly. The filtrate from this when placed in a desiccator deposits crystals of milk-sugar, which are purified Jjy recrystallization, decolorizing with animal charcoal and boiling with CO-70^ alcohol. Pentoses. Salkowski and Jastbowitz'' found in the unne of persons addicted to the morphin habit a variety of sugar which was a pentose, and ' Virchow's Arch., Bd. 107. « Zeitschr. f. physiol. Chem. Bd. 1, S. 101, which also contains the perti- nent literature. » Carl Voit, Uber die Qlycogenbildung nach Auf nahme verschiedeuer Zuck- erarten, Zeitschr. f. Biologie, Bd. 28. *h. c. > Centralbl. f. d. med. Wissensch., 1892, Nos. 19 and 32. 656 THE URINB. yielded an osazone which melted at 159° C. Salkowski ' has observed two new cases of pentosuria. The pentose in the urine seemed to be identical with tl.e pentose obtained by Hammarsten on the cleavage of a pancreas proteid. E. KtLZ and J. Vogel* have detected pentoses in the virine of diabetics, as well as in tbat of dogs with pancreas or phlorhiziu diabetes. Concentrated hydro- chloric acid saturated with phloroglucin may be used in detecting pentoses. Add J volume of the urine to be tested to the acid and warm. In the presence of pentoses the red coloration mentioned on page 65 appears. This test is not conclusive, as glycuronic acid gives the same reaction; further investigation is therefore necessary. IifosiT occurs only rarely, aad in but small quantities, in the nrine in albuminuria and in diabetes mellitus. Alter excessive drinking of water inosit is found in the urine. According to Hoppe-Sbtlek' traces of inosib occur in all normal urines. In detecting inosit the proteid is first removed from the urine. Then con- centrate the urine on the water-bath to \ and precipitate with sugar of lead. The filtrate is warmed and treated with basic lead acetate as long as a precipi- tate is formed. The precipitate formed after 24 hours is washed with water, suspended in water, and decomposed with sulphuretted hydrogen. A little uric acid may separate from the filtrate after a short time. The liquid is filtered, concentrated to a syrupy consistency, and treated while boiling with 3-4 vols, alcohol. The precipitate is quickly separated. After the addition of ether to the cooled filtrate, crystals separate after a time, and these are purified by de- colorization and recrystallization. With these crystals perform the tests men- tioned on page 370. Acetone and Diacetic Acid. These bodies, the occurrence in the urine and formation in the organism of which have been the subject of numerous investigations, especially by v. Jaksch,* were first observed in urine in diabetes mellitus (Petebs, Kaulich, V. Jaksch, Geehakdt). Acetone may give the diabetic urine as well as the expired air the odor of apples or other fruit. According to v. Jaksch and others acetone is a normal urinary constituent, though it may only occur in very small amounts (0.01 grm. in the 24 hours). Acetone may, as found by v. Jaksch, be a by-product in lactic- acid fermentation, and this origin for the traces of acetone eliminated by the normal urine requires further proof. There is no doubt that the appearance of acetone as well as diacetlc acid is essentially caused by an increased destruction of proteid. This follows from the very marked increase in the elimination of acetone and diacetic ' Berliner klin. Wochenschr., 1895. ' Zeitschr. f. Biologie, Bd. 33. ' Handbuch d. physiol. u. pathol. chem. Analyse, 6. Auil., S. 196. * In regard to the extensive literature on acetone and diacetic acid we refer the reader to Huppert-Neubauer, Harnanalyse, 10. Aufl., and v. Noorden's Lehrb. d. Pathol, des StofEwechsels. Berlin, 1893. ACETONE AND DIAUBTIC ACID. 557 aoid during inanition (v. Jaksck," Pk. Mullek'). This is also in accord with the observations of Weight' that in diabetes no rela- tionship exists between the elimination of acetone and sugar, while there is a relationship between the elimination of acetone and nitrogen; thas on the days when most nitrogen is eliminated we find the highest resalts for the acetone, and vice versa. Abundant proteid food also increases the elimination of acetone, according to HosiGJiANN- * and v. Nookden,' apparently in the case where with a one-sided proteid food an insuflBcient supply of calories takes place, which leads to a reduction of the body-proteid. According to this view, which requires further proof, the extent of the elimi- nation of acetone and diacetic acid is not dependent npon the extent of the metabolism of proteid, but upon the quantity of destroyed body-proteid. According to this view it is also clear that an abundant elimina- tion of acetone and diacetic acid is observed, especially in such diseases in which an abundant destruction of body-proteid takes place, such as fevers, diabetes, disturbed digestion, mental debility with abstinence, cachexia, etc. It has not been proven how far the acetonuria experimentally produced by Lustig ° by lesion of the sinus fovea rhomboidalis or by excision of the solar plexus is caused by the generally disturbed condition of the animal or by other cir- cumstances. Diacetic acid has not been observed as a physiological constituent of the urine. It occurs in the urine chiefly under the same condi- tions as acetone; still we have cases in which only acetone and no diacetic acid appears. Like acetone the diacetic acid occurs often in children, especially in high fevers, acute exanthema, etc. Dia- cetic acid decomposes readily into acetone. According to Araki ' it is probably produced as an intermediate product in the oxidation of /?-oxybutyric acid in the organism. The three bodies appearing ' Ueber Acetonurie und Diaceturie. Berlin, 1885. ' Bericbt ilber die Ergebnisse des an Cetti ausgef tthrten Hungerversuohes. Berlin, klin. Wocbenschr., 1887. »See Maly's Jabresber., Bd. 21, S. 404. * Zar Enstehung des Acetous. Diss. Breslau, 1886. Cited from v. Noorden, Lebrb., S. 177. ' L. c, S. 78. 6 Centralbl. f. Pbysiol., Bd. 6. 'Zeitacbr. f. pbysiol. Chem., Bd. 18. 558 THE URINE. in the urine, acetone, diacetic acid, and oxybatyrio acid, stand in close relationship to each other. Acetone, dimethyl ketone, C,H,0 or CO.(CH,)„ is a thin water- clear liquid boiling at 56.5° C. and with a pleasant odor of fruit. It is lighter than water, with which it mixes in all proportions, also with alcohol and ether. The most important reactions for acetone are the following: Libben's ' Iodoform Test. When a watery solution of acetone is treated with alkali and then with some iodine-potassium-iodide solution and gently warmed a yellow precipitate of iodoforqi is formed, which is known by its odor and by the. appearance of the crystals (six-sided plates or stars) under the microscope. This' reaction is very delicate, but it is not characteristic of acetone. Gunning's" modification of the iodoform test consists in using an alcoholic solution of iodine and ammonia instead of the iodine dis- solved in potassium iodide and alkali hydrate. In this case, besides iodoform, a black precipitate of iodide of nitrogen is formed, but this gradually disappears on standing, leaving the iodoform visible. This modification has the advantage that it does not give any iodoform with alcohol. On the other hand, it is not quite so delicate, but still it detects 0.01 milligramme acetone in 1 o. c. Eeynold's " mercuric-oxide test is based on the power of acetone to dissolve freshly precipitated HgO. A mercuric-chloride solution is precipitated by alcoholic caustic potash. To this add the liquid to be tested for acetone, shake well and filter. In the presence of acetone the filtrate contains mercury, which may be detected by ammonium sulphide. This test has about the same delicacy as Gunning's test. Legal's' Sodium-nitroprusside Test. If an acetone solution is treated with a few drops of a freshly prepared sodium-nitro- prusside solution and then with caustic-potash or soda solution, the liquid is colored ruby-red. Creatinin gives the same color; but if we saturate with acetic acid, the color becomes carmine or purplish red in the presence of acetone, but yellow and then gradually green and blue in the presence of creatinin. If we use ammonia instead of the caustic alkali (Lb Nobbl), the reaction takes place with ' Annal. d. Chem. u. pbarm., Suppl. Bd. 7. ' Gunning, by Bardy, Journ. de pbarm. et chim. (5), Tome 4. ' Cited from Huppert-Neubauer, Harnanalyse, 10. Aufl. , S. 60. * Breslauei- arztl. Zeitsohr. , 1883. DUOBTIC ACID. 559 acetone, but not with creatinin.' Leoal's test indicates even 0.1 milligrm. acetone. Penzoldt's' indigo test depends on the fact that orthonitro- benzaldehyde in alkaline solution with acetone yields indigo. A warm saturated and then cooled solution of the aldehyde is treated with the liquid to be tested for acetone and then with caustic soda. In the presence of acetone the liquid first becomes yellow, then green, and lastly indigo separates; and this may be dissolved with a blue color by shaking with chloroform. 1.6 milligrms. acetone can be detected by this test. MaIiERBA. ' uses a solution of dimethylparaphenylendiamin as a reagent for acetone. It gives a red liquid which has an absorption-spectrum very similar to oxyhsemoglobin. Diacetic acid, or aceto-acetic acid, C.H.O, or C,H,O.CH,.COOH. This acid is a colorless, strongly acid liquid which mixes with water, alcohol, and ether in all proportions. On heating to boiling with water, and especially with acids, this acid decomposes into carbon dioxide and acetone, and therefore gives the above-mentioned reactions for acetone. It differs from acetone in that it gives a yiolet-red or brownish-red color with a dilute ferric-chloride solu- tion. This color decreases even at the ordinary temperature within 24 hours, and more quickly on boiling. It differs in this from phenol, salicylic acid, acetic acid, or sulphocyanides. Detection of Acetone and Diacetic Acid in the urine. Before testing for acetone test for diacetic acid, and as this acid gradually decomposes on allowing the urine to stand, the urine must be as fresh as possible. In the presence of diacetic acid the urine gives the so-called Gekhakdt's reaction, showing a wine-red color on the addition of a dilute, not too acid, ferric-chloride solution. Treat 10-50 c. c. of the urine with ferric chloride as long as it gives a precipitate, filter the precipitate of ferric phosphate, aud add some more ferric chloride to the filtrate. Iq the presence of the acid a claret-red color is produced. After this heat a second, similar portion of the faintly acid urine to boiling, and repeat the test on cooling, which should now give negative results. A third portion of urine is acidified with sulphuric acid and shaken with ether (which takes up the acid). Now shake the removed ether with a very dilute watery solution of ferric chloride, and the watery layer becomes violet-red or claret-red. The color disappears on warming. ' According to the author this statement is not correct. » Arch, f . klin. Med. , Bd. 84. » Atti della E. Academ. med. chirurg. di Napoli, Anno 48, Nuova Serie, 660 TEE URINE. K. MoBNEE ' suggests that in testing for diacetic acid in the urine the urine be treated with a little KI and Pe,Cl, in excess and heated. In the presence of diacetic acid very irritating vapors of iodoacetone (?) are developed. In the absence of diacetic acid the acetone may be tested for directly. This may be done directly on the urine by Pbstzoldt's test. This test, which is only approximate, is only of value when the urine coatains a considerable amount of acetone. For a more accurate test we distil at least 250 c. c. of the urine faintly acidified with sulphuric acid, care being taken to have a good condensation. Most of the acetone is contained in the first 10-30 c. c. of the dis- tillate. This distillate is tested for acetone by the above tests. In testing for acetone in the simultaneous presence of diacetic acid, first make the urine faintly alkaline, and shake it carefully with ether free from alcohol and acetone in a separatory funnel. The removed ether is then shaken with water, which takes up the acetone, and then the watery liquid is tested. The quantitative estimation of acetone in the urine is done by converting it first into iodoform. The urine is acidified with acetic acid (according to Huppert, 1-2 c. c. 50^ acetic acid for every 100 c. c. urine) and distilled, The iodoform formed is deter-, mined in the distillate either gravimetrically according to Keamee or colorimetrically, according to v. Jaksch. It is best to proceed according to the method as suggested by Messingee and Hup- PEET.' They determine the quantity of acetone by determining the quantity of iodine necessary in the formation- of iodoform by titration. In regard to this method and its execution we refer the reader to Huppeet-Neubauee.° yS-Oxybutyric Acid, C.H.O, or CH3.CH(0H).CH,C00n. The appearance of this acid in the urine was first positively shown by Minkowski,' Kulz' and STADBLMANiir.' It occurs especially in difficult cases of diabetes, but it has also been observed in scarlet fever and in measles (KtJLz), in scurvy (Minkowski), and in dis- eases of the brain with abstinence (KtJLZ). /?-oxybutyric acid is undoubtedly derived from an abnormal destruction of body-proteid, and it therefore occurs in the urine in inanition, cachexia, etc. /3-oxybutyric acid is accompanied by diacetic acid in the urine, ' Skan. Arch. f. Physiol., Bd. 5. ' Huppert-Neubauer, Harnanalyse, 10. Aufl., S. 760, which also contains the description of other methods and summary of the literature. 3 L. c, 10. Aufl. * Arch. f. exp. Path, u. Pharm., Bd. 18 u. 19. ' Zeitschr. f. Biologie, Bdd. 20 u. 33. • Arch. f. exp. Path. u. Pharm., Bd. 17. /S-OXYBUTYSIC AOID. 561 • -while on the other hand the last-mentioned acid occurs in the urine without the first. /J-oxybatyric acid forms an odorless syrup which mixes readily with water, alcohol, and ether. This acid is optically active and indeed laevogyrate, and it therefore interferes with the estimation of sugar in the uriae by means of polarization. It is not precipi- tated either by basic lead acetate or by ammoniacal basic lead acetate. On boiling with water, especially in the presence of a mineral acid, this acid decomposes into a-CKOTONic acid, which melts at 71-72° C, and water: CH,.CH(OH).CH,.COOH = H,0 -|- CH,.CH:CH.COOH. It yields acetone on oxidation with a chromic-acid mixture. Detection of fi-Oxybutyric Acid in the urine. If a urine is still Isevogyrate after fermentation with yeast, the presence of oxy- butyric acid is probable. A farther test may be made, according^ to KtJLz, by evaporatiugthe fermented urine to a syrup, and, after the addition of an equal volume of concentrated sulphuric acid,, distilling directly without cooling. «-crotonic acid is produced which distils over, and, after collecting in a test-tube, crystals^ which melt at + 73° C, separate on strongly cooling. If no- crystals are obtained, then shake the distillate with ether, and test- the melting-point of the residue obtained after evaporating the- ether which has been washed with the water. According to Minkowski the acid may be isolated as a silver-salt.' Ehiii,ich'b ' Urine Test. Mix 350 c. c. of a solution whicli contains 50 c. c. HCl and 1 gnu. salpbanilic acid in one litre with 5 c. c. of a ^% solution of sodium nitrite (wbich produces very little of the active body, sulphodiazobeu- zol) In performing this test treat the urine with an equal volume of this mixture and then supersaturate wiih ammonia. Normal, urine will become yellow thereby, or orange after the addition of ammonia (aromatic oxyacids may sometimes give after a certain time red azo bodies which color the upper layer of phosphate sedimeni). In pathological urines we sometimes Lave (and! this is the cLiaracteristic diazo reaction) a primary yellow coloration, with a very marked secondary red coloration on the addition of ammonia, and the froth is also tinged with red. The upper layer of the sediment becomes green- ish. The body wbich gives this reaction is unknown, but it occurs especially in the urine of typhoid patients (Ehblioh). Views are divided in regard to- the significance of this reaction. RosENBACu'B urine test, which consists in adding nitric acid drop by drop- to the boiling-hot urine and obtaining a claret-red coloration and a bluish- red foam on shaking, depends upon the formation of indigo substances, especially indigo red.' Fat in the urine. The elimination of a urine which in appearance and rich- ness in fat resembles chyle is called chyluria. It contains habitually proteid, ' Arch. f. exp. Path. u. Pharm., Bd. 18, S. 35 ; Zeitschr. f. anal. Chem., Bd. 24, S. 153. ' Zeitschr. f. Win. Med., Bd. 5. » See Rosin, Viichow's Arch., Bd. 123. 562 THE UBINE. SiOcl often fibrin. Cliyluria occurs mostly in the inhabitants of the tropics. Lipuria, or the elimination of fat with the urine, may appear in apparently liealthy persons, sometimes with and sometimes without albuminuria, in preg- nancy, and also in certain diseases, as in diabetes, poisoning with phosphorus, and fatty degeneration of the kidneys. Fat is usually detected by the microscope. It may also be dissolved with ether, and it may always be detected by evaporating the urine to dryness and extracting the residue with ether. Cholbstbhin is also sometimes found in the urine in chyluria and in a few other cases. Leucik and Tteosin. These bodies are found in the urine, especially in acute yellow atrophy of the liver, in acute phosphorus- poisoning, and in difficult cases of typhoid and smallpox. Detection of Leucin and lyrosin. Tyrosin occurring as sediment may be identified by means of the microscope ; but if a positive proof is desired, a recrystallization of the same from ammonia or ammoniacal alcohol is neces- sary. I'o detect both these bodies when tbey occur in solution in the urine, pro- ceed in the following manner : The urine free from proteid is precipitated by basic lead acetate, the lead removed from the filtrate by HgS, and concentrated as much as possible. The residue is extracted with a small quantity of absolute alcohol to remove the urea. The residue is then boiled with faintly ammoniacal alcohol, filtered, the filtrate evaporated to a small volume and allowed to crys- tallize. If no tyrosin crystals are obtained, then dilute with water, precipitate again with basic lead acetate, and proceed as before. If tyrosin crystals now separate, they are filtered, and the filtrate still further concentrated to obtain the leucin crystals. Cystin (0,H,NSOJ,. This body is, according to BAUMAifK,' to be considered as disulphide, „° y Cs' „ /^\ -vrTi' ' of the previously mentioned cystein, C3H,NS0j (page 539). Cystein TT P \ /STT itself is a-amldothiolactic acid, „° /^\ pnnTT' Cystin is con- verted into cystein by nascent hydrogen and is reconverted into cystin by oxidation. BAUMANisr and Goldmaun ° claim that a substance similar to cystin occurs in very small amounts in normal urine. This sub- stance occurs in large quantities in the urine of dogs after poisoning •with phosphorus. Oystin itself is found with positiveness, and even -fchen very rarely, only in urinary calculi and in pathological arines, from which it may separate as a sediment. Cystinuria occurs •oftener in men than in women, and cystin seems to be an abnormal splitting product of the proteids, BAUMANif and t. Udkanszky ' ' Zeitschr. f. Physiol. Chem., Bd. 8. In regard to the literature on cystin see Breuzinger, ibid., Bd. 16, S. 553. - Zeitschr. f physiol. Chem., Bd. 12. ^Ibid.. Bd. 13 VT8TIN. 563 iound ill urine in cystinaria the two diamins, cadaverin (penfca- methjlendiamin) and putrescin (tetramethylendiamin), which are produced in the putrefaction of proteids. These two diamins were also found in the contents of the intestine in cystinuria, while under normal conditions they are not present. The author therefore considers that perhaps some connection exists between the formation of diamins in the intestine, by the peculiar putrefac- tion in cystinuria, and cystinuria itself. Cadaverin has also been found in the urine in cystinaria by Stadthagen and Brieger.' Cystin has also been found in ox-kidneys, in- horse's liver (Drechsel"), and as traces in the liver of a drunkard. KClz' •once observed the occurrence of cystin during the digestion of fibrin with pancreas. Cystin crystallizes in thin, colorless, six-sided plates. It is not soluble either in water, alcohol, ether, or acetic acid, but dissolves in mineral acids and oxalic acid. It also dissolves in alkalies and in ammonia, but not in ammonium carbonate. Cystin is optically active and strongly laevorotatory. If cystin is boiled with caustic alkali it decomposes, yielding among other products alkali sulpbides, which may be detected by lead acetate or sodium nitroprusside. On treating cystin with tin and hydrochloric acid, only a little sul- phuretted hydrogen is evolved and cystein is produced. On shaking a solution of cystin in an excess of caustic soda with benzoyl- chloride a voluminous precipitate of benzoyl-cystin is produced {Battmanu and Goldmann '). On heating on platinum foil, cystin does not melt, but ignites and burns with a bluish-green flame accompanied by a peculiar sharp odor. On warming with nitric acid cystin dissolves with decomposition and leaves a reddish- brov/n residue on evaporation which does not give the murexid test. Cystein hydrochloride gives a nearly insoluble precipitate having the composition 3(C3H,NSO,) + SHgCl, with mercuric chloride. Baumakn and BoRissow ' have based a method for the quantitative estimation of cystin on this behavior. They first reduce the cystin by zinc and hydrochloric acid. Cystin is easily prepared from cystin calculi by dissolving them in alkali carbonate, precipitating the solution with acetic acid, and > Berl. klin. Wochenscbr., 1889. ' Du Bois-Keymond's Arch., 1891. « Zeitsuhr. f Biolofjie, Bd. 27. * Zeitscbr f. physiol CLern., Bd. 13. Hbul., Bd. 19, S. 511. 564 THE UBINB. redissolving the precipitate in ammonia. The cystin crystallizes on the spontaneous evaporation of the ammonia. The cystin dissolved in the urine is detected, in the absence of proteid and sulphuretted hydrogen, by boiling with alkali and testing with lead salt or sodium nitroprusside. To isolate cystin from the urine, acidify the urine strongly with acetic acid. The precipitate containing cystin is collected after 34 hours and digested with hydrochloric acid, which dissolves the cystin and calcium oxalate, leaving the uric acid undissolved. Filter, supersaturate the filtrate with ammonium car- bonate, and treat the precipitate with ammonia, which dissolves the cystin and leaves the calcium oxalate. Filter again and precipitate with acetic acid. The precipitated cystin is identified by the microscope and the above-mentioned reactions. Cystin as a sedi- ment is identified by the microscope. It must be purified by dissolving in ammonia and precipitating with acetic acid and then tested. Traces of dissolved cystin may be detected by the produc- tion of benzoyl-cystin, according to Baumann and Goldmann. VII. Urinary Sediments and Calculi. Urinary sediment is the more or less abundant deposit which is found in the urine after standing. This deposit may consist partly of organized and partly of non-organized constituents. The first, consisting of cells of various kinds, yeast-fungus, bacteria, sperma- tozoa, casts, etc., must be investigated by means of the microscope, and the following only applies to the non-organized deposits. As above mentioned (page 447), the urine of healthy individuals, may sometimes, even on voiding, be cloudy on account of the phosphates present, or become so after a little while because of the geparation of urates. As a rule, urine just voided is clear, and after cooling shows only a faint cloud (nubecula), which consists of BO-called mucus, a few epithelium-cells, mucous corpuscles, and nrate particles. If an acid urine is allowed to stand, it will gradually change; it becomes darker and deposits a sediment con- sisting of uric acid or urates, and sometimes also calcinm-oxalate crystals, in which yeast-f ungas and bacteria are often to be seen. The cause of this change, which the earlier investigators called " ACID FBBMBlirTATIOK OF THE UEIKE," is, according to SCHEEBE,' the mucus, which acts like an enzyme or ferment, producing an acetic-acid or lactic-acid fermentation, precipitating free uric acid or acid urates. According to Neubauee," an actual acid fermen- 1 Annal. d Cbem. u. Pharm., Bd. 43 (1843). ' Neubauer und Vogel, Analyse des Hams (1876). URINARY SEDIMENTS AND CALCULI 665 tation may occur in diabetic urine, but this seems to occur only veiy seldom, and according to Eohmann^ ' an acid fermentation of the urine in Scheeer's sense does not occur under normal condi- tions. According to VoiT and Hofmann ' a separation of free uric acid and acid urates may be produced, without any increase in the acid reaction, by an exchange of the di-hydrogen alkali phosphate» with the alkali urate on cooling and on standing. Simple acid phosphate and, according to the conditions, acid urate or free uric acid are formed. A gradual precipitation of uric acid may occur not only without an increase in the acid reaction, but, because of the alkaline reaction of the simple acid-alkali phosphate, it may occur with a simultaneous decrease of the same. Eohmanst has presented objections to this statement. He claims that a steady decrease of the acid reaction, without formation of ammonia, caused by the above-mentioned transformation of phosphates and urates does not take place. The acid reaction does not decrease until the ammonia increases. According to Bbkce Jones ' the precipitation of the uric acid and urates has another cause. He claims that the urine contains hyperacid salts, so-called quadriurates (seepage 478), which gradually split into uric acid and biurates. Earlier or later, sometimes only after several weeks, the reaction of the original acid urine changes and becomes neutral or alkaline. The urine has now passed into the " alkaline ebementation," which consists in the decomposition of the urea into carbon dioxide and ammonia by means of lower organisms, micrococcus ureae, bacteria ureae, and other bacteria. Musculus* has isolated an enzyme from the micrococcus ureae which decomposes urea and is soluble in water. During the alkaline fermentation volatile fatty acids, especially acetic acid, may be produced, chiefly by the fermentation of the carbohydrates of the urine (Salkowski °). A fermentation by which nitric acid is reduced to nitrons acid, and another where sulphuretted hydrogen is produced, may sometimes occur. If the alkaline fermentation has only advanced so far as to render the reaction neutral, then we often find in the sediment ' Zeitschr. f . physiol. Chem. , Bd. 5. » Sitzungsber. d. k. b. Aiad. d. Wissensch., 1867, Bd. 2, S. 379. Cited from. RShmann, 1. c. 8 Joum. Chem. Soc, Vol. XV. p. 8. -•Pflilger's Arcb., Bd. 13. •• Zeitsclir. f. physiol. Chem., Bd. 13. 566 TEB URINB. fragmeats of nrie-acid crystals, sometimes covered with prismatie Crystals of alkali urate; dark-cOlored spheres of ammonium urate» often crystals of calcium oxalate, and sometimes crystallized calcium phosphabe are also found. Crystals of ammonium-magnesium phos- phate (triple phosphate) and sphei-ical ammonium urate are specially- characteristic of alkaline fermentation. The urine in alkaline fer- mentation becomes paler and is often covered with a fine membrane which contains amorphous calcium phosphate and glistening crystals, of triple phosphate and numerous micro-Organisms. Non-organized Sediments. Uric Acid. This acid occurs in acid urines as colored crystals- which are identified partly by their form and partly by their property of giving the murexid test. On warming the urine they are not dissolved. On the addition of caustic alkali to the sediment the crystals dissolve, and when a drop of this solution is placed on a microscope-slide and treated with a drop of hydrochloric acid, small crystals of uric acid are obtained which are easily seen under the microscope. Acid Urates. These only occur in the sediment of acid or neutral urines. They are amorphous, clay-yellow, brick-red, rose- colored, or brownish red. They differ from other sediments in that they dissolve on warming the urine. They give the murexid test, and small microscopic crystals of uric acid separate on the addition of hydrochloric acid. Crystalline alkali urates occur very rarely in the urine, and as a rule only in such as have become neutral bat not alkaline by the alkaline fermentation. The crystals are some- what similar to those of neutral calcium phosphate; they are not dissolved by acetic acid, however, but give a cloudiness therewith due to small crystals of uric acid. Ammonium urate may indeed occur as a sediment in a neutral urine which at first was strongly acid and has become neutralized by the alkaline fermentation, but it is only characteristic of am- moniacal urines. This sediment consists of yellow or brownish, rounded spheres which are often covered with thorny-shaped prisms and, because of this, are rather large and resemble the thorn-apple. It gives the murexid test. It is dissolved by alkalies with the development of ammonia, and crystals of uric acid separate on the addition of hydrochloric acid to this' solution. N0N-0R6AXTZED SEDIMENTS. 567 Calcium oxalate occurs in the sediment generally as small, shining, strongly refractive quadratic octahedra, which on micro- scopical examination remind one of a letter-envelope. The crystals can only he mistaken for small, not fully developed crystals of ammonium-magnesiam phosphate. They differ from these by their insolubility in acetic acid. The oxalate may also occur as flat, oval, or nearly circular disks with central cavities which from the side appear like an hour-glass. Calcium oxalate may occur as a sedi- ment in an acid as well as in a neutral or alkaline urine. The quantity of calcium oxalate separated from the urine as sediment depends not only upon the amount of this salt present, but also upon the acidity of urine. The solvent for the oxalate in the urine seems to be the double-acid alkali phosphate, and the greater the quantity of this salt in the nrine the greater the quantity of oxalate in solution. When, as above mentioned (page 565), the simple- acid phosphate is formed from the double-acid phosphate, on allowing the urine to stand, a corresponding part of the oxalate may be separated as sediment. Calcium carbonate occurs in considerable quantities as sediment in the urine of herbivora. It occurs in but small quantities as a sediment in human urine, and in fact only in alkaline urines. It either has almost the same appearance as amorphous calcium oxalate, or it occurs as somewhat larger globules with concentric bands. It dissolves in acetic acid with the generation of gas, which differentiates it from calcium oxalate. It is not yellow or brown like ammonium urate, and does not give the mnrexid test. Calcium svlpluite occurs very rarely as a sediment in strongly acid urines. It appears as long, thin, colorless needles, or generally as plates grouped together. Calcium Phosphate. The calcium tkiphosphatb, Oa^POj,, which occurs only in alkaline urines, is always amorphous and occurs partly as a colorless, very fine powder and partly as a mem- brane consisting of very fine granules. It differs from the amor- phous urates in that it is colorless, dissolves in acetic acid, but remains undissolved on warming the urine. Calcium diphos- phate, CaHPO. + 2H,0, occurs in neutral or only in very faintly acid urine. It is found sometimes as a thin film covering the urine, and sometimes as a sediment. In crystallizing the crystals may be single, or they may cross one anotlier, or they may be arranged in groups of colorless, wedge-shaped crystals whose wide end is sharply otiS THE URINE. defined. These crystals differ from crystalline alkaline urates in that they dissolve without a residue in dilute acids and do not give the mnrexid test. Ammonium-magnesium phosphate, tkiple phosphate, may separate of course from an amphoteric urine in tiie presence of a sufficient quantity of ammonium salts, but it is generally character- istic of a urine become ammoniacal through alkaline fermentation. The crystals are so large that they may be seen with the unaided eye as colorless glistening particles in the sediment, on the walls of the vessel, and in the film on the surface of the urine. This salt forms large prismatic crystals of the rhombical system (coffin-shaped) which are easily soluble in acetic acid. Amorphous magnesium triphosphate, Mg3(PO,)3, occurs with calcium triphosphate in urines rendered alkaline by a fixed alkali. Crystalline magnesium phos- phate, Mgj(PO,), + 22E[,0, has been observed in a few cases in Jiuman urine (also in horse's urine) as strongly refractive, long rhombical plates. Kyestein is the film which appears after a little while on the surface of the urine. This coating, which was formerly considered as chaiacteristic of urine in pregnancy, contains various elements, such as fungus, vibriones, «pithelium-cells, etc. It often contains earthy phosphates and triple phosphate crystals. As more rare sediments we find cystin, tyrosin, hippurio aeid, xantMn, Jicematoidin. In alkaline urine blue cry.stals of indigo may also occur, due to M, decomposition of indoxyl-glycuronic acid. Urinary Calculi. Besides certain pathological constituents of the urine, all those urinary constituents which occur as sediments take part in the formation of the urinary calculi. Ebstein ' considers the essential difference between an amorphous or crystalline sediment in the iurine on one side and urinary sand or large calculi on the other to be the occurrence of an organic frame in the last. As the sediments which appear in normal acid urine and in a urine alkaline through fermentation are different, so also are the urinary calculi which appear under corresponding conditions. If the formation of a calculus and its further development take place in an undecomposed urine, it is called a primakt formation. If, on the contrary, the urine has undergone alkaline fermentation and the ammonia formed thereby has given rise to a calculous formation by precipitating ammonium urate, triple phosphate, and ' Die Natur und Behandlung der Harnsteine. Weisbadeu, 1884. URINARY OALOnLI. 569 earthy phosphates, then it is called a SECOiirDAEY formation. Such a formation takes place, for instance, when a foreign body in the bladder produces catarrh accompanied by alkaline fermentation. We discriminate between the nucleus or nuclei — if such can be seen — and the different layers of the calculus. The nucleus may be essentially different in different cases, for quite frequently it consists of a foreign body introduced into the bladder. The calculus may have more than one nucleus. In a tabulation made by Ultzmann of 545 cases of urinary calculi, the nucleus in 80.9^ of the cases consisted of uric acid (and urates); in 5.6j^, of calciam oxalate; in 8.6^, of earthy phosphates; in 1.4^, of cystin; and in 3.3^, of some foreign body. During the growth of a calculus it often happens that, for some reason or other, the original calculus-forming substance is covered with another layer of a different substance. A new layer of the original substance may deposit on the outside of this, and this process may be repeated. In this way a calculus consisting origi- nally of a simple stone may be converted into a so-called compound stone with several layers of different substances. Such calculi are always formed when a primary formation is changed into a secon- dary. By the continued action of an alkaline urine containing pus, the primary constituents of an originally primary calculus may be partly dissolved and be replaced by phosphates. Metamorphosed urinary calculi are formed in this way. Uric-acid calculi are very abundant. They are variable in size and form. The size of the bladder-stone varies from that of a pea or bean to that of a goose-egg. Uric-acid stones are always colored ; generally they are grayish yellow, yellowish brown, or pale red- brown. The upper surface is sometimes entirely even or smooth, sometimes rough or uneven. Next to the oxalate calculus, the uric- acid calculus is the hardest. The fractured surface shows regular concentric, unequally colored layers which may often be removed as shells. These calculi are formed primarily. Layers of uric acid sometimes alternate with other layers of primary formation, most frequently with layers of calcium oxalate. The simple uric-acid calculus leaves very little residue when burnt on platinum foil. It gives the murexid test, but there is no material development of ammonia when acted on by caustic soda. Avimonium-urate calculi occur as primary calculi in new-born or nursing infants, rarely in grown persons. They often occur as 570 THE UniNE. a secondary formation. The primary stones are small, with a pale- yellow or dark-yellowish surface. When moist they are almost like dough ; in the dry state they are earthy, easily crumbling into a pale powder. They give the murexid test, and develop mucli ammonia with caustic soda. Calcium-oxalate calculi are, next to uric-acid calculi, the most abundant. They are either smooth and small (hemp-seed calculi) or larger, of the size of a hen's egg, with rough, uneven surface, or their surface is covered with prongs (mulbekrt calculi). These calculi produce bleeding easily, and therefore they often have a dark-brown surface due to decomposed blood-coloring matters. Among the calculi occurring in man these are the hardest. They dissolve in hydrochloric acid without developiug gas, but are not soluble in acetic acid. After gently heating the powder it dissolves in acetic acid with frothing. After strongly heating the powder it is alkaline, due to the production of quick- lime. Phosphate Calculi. These, which consist mainly of a mixture of the normal phosphate of the alkaline earths with triple phos- phate, may be very large. They are as a rule of secondary forma- tion, and contain besides these phosphates also some ammonium urate and calcium oxalate. These calculi ordinarily consist of a mixture of these three constituents, earthy phosphate, triple phos- phate, and ammonium urate, surrounding a foreign body as a nucleus. Their color is variable— white, dingy white, pale yellow, sometimes violet or lilac-colored (from indigo-red). The surface is always rough. Calculi consisting of triple phosphate alone are seldom found. They are ordinarily small, with granular or radiated crystalline fracture. Stones of simple-acid calcium phosphate are also seldom obtained. They are white and have a beautiful crystal- line texture. The phosphatic calculi do not burn up, and the powder dissolves in acid without effervescence, and the solution gives the reactions for phosphoric acid and alkaline earths. The triple-phosphate calculi generate ammonia on the addition of an alkali. Galeium-ca/rbonate calculi occur chiefly in herbivora. They are seldom found in man. They have mostly chalky properties, and are ordinarily white. They are completely or in great part dissolved by acids with effervescence. Cysiin calculi occur but seldom. They are of primary formation, of various sizes, sometimes attaining the size of a hen's egg. They have a smooth or rough suriace, are white or pale yellow, and have a crystalline fracture. They urinaut calculi. 571 are not very bard ; they burn up almost entirely on platinum foil, burning with a bluish flame. They give the above-mentioned reactions for cystiu. Xanthin calculi are very rarely found. They are also of primary forma- tion. They vary from the size of a pea to that of a hen's egg. They are whitish, yellowish brown or cinnamon-brown in color, medium hard, with amorphous fracture, and on rubbing appear like wax. They burn up com- pletely when heated on platinum foil. They give the xanthin reaction with nitric acid and alkali, but this must not be mistal^en for the murexidtest. JJrostealith calculi have only been observed a few times. In the moist state th^y are soft and elastic at the temperature of the body, but in the dry state they are brittle, wi;h an amorphous fracture and waxy appearance, 'i'hey burn with an illuminating flame wLen heated on platinum foil, and jrenerate an odor similar to resin or shellac. Such a calculus, investigated by Krukbn- BERG,' consisted of paraffine derived from a paratiine bougie used as a sound on the patient. Perhaps the urostealith calculi observed in other cases had a similar origin, although the substances of which they consisted have not been closely studied. Horbaczewski has recently analyzed a case of urostealith which, to all appearances, was formed in the bladder. This calculus con- tained 25 p. m. water, 8 p. m. inorganic bodies, 117 p. m. bodies insoluble in ether, and 850 p. m. organic bodies soluble in ether, among which were 515 p. m. free fatty acids, 335 p. m. fat, and traces of chloresterin. The fatty acids consisted of a mixture of stearic, palmitic, and probably myristic acids. Horbaczewski ' has also analyzed a bladder-stone which contained 958.7 p. m. cholesterin. Fibrin calculi sometimes occur. They consist of more or less changed fibrin coagulum. On burning they develop an odor of burnt horn. The chemical investigation of urinary calculi is of great practical importance. To make such an examination actually instructive it is necessary to investigate separately the difEerent layers which con- stitute the calculus. For this purpose saw the calculus, which has been wrapped in paper, with a fine saw so that the nucleus is sawed through and accessible. Then peel ofE the difEerent layers, or, if the stone is to be kept, scrape ofE enough of the powder from each layer for examination. This powder is then tested by heating on platinum foil. It must not be forgotten that a calculus is never entirely burnt up, and also that it is never so free from organic matter that on heating it does not carbonize. Do not, therefore, lay too great stress on a very insignificant unburnt residue or on a very small amount of organic matter, but consider the calculus in the former case as completely burnt and in the latter as not burnt. When the powder is in great part burnt up, but a significant quantity of unburnt residue remains, then the powder in question contains as a rule urates mixed with inorganic bodies. In such cases remove the urate with boiling water, and then test the filtrate for uric acid and the expected bases. The residue is then tested according to the following schema of Heller, which is well adapted to the investigation of urinary calculi. In regard to more detailed examination the reader is referred to special works on the subject. ' Chem. Untersuch. z. wissensch. Med., Bd. 2. Cited from Maly's Jahresber., Bd. 19, S. 432. ' Zeitschr. f . physiol. Chem. , Bd. 18. 572 TEE URINE. On heating the powder on platinum foil it Does not burn Does burn The powder when treated with HGl With flame Wi ;hout flame The powder Does not effervesce M 1^ •^ s gives the murexid test Tiie gently heated ? c-g »&i powder with HC < f° CD CO g £vd The powder The powder when moistened with a little KHO o CD a> on C p cr ^B- p 1 ' *^ when treated with KHO gives CD !2( > !z! CQ § ffiB p " |i.3 2.1 p^B g » B 3 bundant ammonia, acid or HCl. This itate with ammonia fn P o g 3 » 88 °&- p-p 3 p Us 1 B B 5. 1 1 i 2 B p p- log 1" c 3 CO ^ & 0-2 1 IK! ^ "SI S'3 2 S-. 2.e- CO « CO 2 a 't 8 a, 5' S gco § 3 i' m 2, aw f !? i: § to iJ l§ 5-^ 2 5. 2 * II 2 P- ^ CD CO HI &| H te) Q O • >^ ci W > c^ riple phosphate with unknown am earthy phosphate) II B p" II so, n 5 o so, (0 » a B 1 1 1 5 i i so a B B § c B p ? 2. • o,-. S » c B 2-i- a S. c o «. 1^ s.a CHAPTEE XVI. THE SKIN AND ITS SECRETIONS. In the strncture of the skin of man and vertebrates many differ- ent kinds of substances occur which have already been treated of, snch as the constituents of the epidermis formation, the connective and fatty tissnes, the nerves, muscles, etc. Among these the different horn-formations, the hair, nails, etc., whose chief constit- uent, keratin, has been spoken of in another chapter (Chap. II), are of special interest. The cells of the horny formation show, in proportion to their age, a different resistance to chemical reagents, especially fixed alkalies. The younger the horn-cell the less resistance it has to the action of alkalies; with advancing age the resistance becomes greater, and the cell-membranes of many horn-formations are nearly insoluble in caustic alkalies. Keratin occurs in the horn-formation mixed with other bodies, from which it is isolated with difBliulty. Among these bodies the mineral constituents in many cases occupy a prominent place because of their quantity. Hair leaves on burn- ing 5-70 p. m. ash, which may contain in 1000 parts 230 parts alkali sulphates, 140 parts calcium sulphate, 100 parts iron oxide, and even 400 parts silicic acid. Dark hair on burning seems gen- erally, but not always, to yield more iron oxide than blond. The nails are rich in calcium phosphate, and the feathers rich in silicic acid. The granules occurring in the stratum granulosum of the skin consist of a substance which has been called eleidifi, and which is considered as an intermediate step in the transformation of the protoplasm into keratin. The chemical nature of this substance is unknown. The skin of invertebrates has been the subject, in a 'few cases, of chemical investigation, and in these animals various substances 573 574 THE SKIN AND ITS 8ECBETI0N8. have been found, of which a few, though little studied, are worth discussing. Among these bodies tunicin, which is found especially in the tunic of the tunicata, and ' the widely diffused cliitin, found in the cuticle-formation of invertebrates, are of interest. Tunicin. Cellulose seems, according to the investigations of Ambronn,' to occur rather extensively in the animal kingdom in the arthropoda and the mollusks. It has been known for a long time as the tunic of the tunienta, and this animal cellulose was called tunicin by Bbethblot.' According to the recent inve tigations of Wintbestein * there does not seem to exist any marked difference between tunicin and ordinary cellulose. On boiling with dilute acid tunicin yields dextrose, as shown first by Fbanchimont * and later confirmed by WiMTKKBTEIN. CMtin is not found in vertebrates. In invertebrates chitin is alleged to occur in several classes of animals; but it can only be positively asserted that true, typical chitin is found only in articu- lated animals, in which it forms the chief organic constituent of the shell, etc. According to Kkawkow' chitin of the shell, etc., does not seem to occar free, but in combination with another substance, probably a proteid-like body. According to Sundwik ° the composition of chitin is probably C,„H,,jN,Ojg + niJLfi), where n may vary between 1 and 4, and it is probably an amine derivative of a carbohydrate, with the general formula w(0,,Hj„0,„). According to Krawkow' chitin shows different origins by its unequal behavior with iodine, and he there- fore concludes that there must exist quite a group of chitins, which seern,,to be amine derivatives of diiferent carbohydrates such as dexttose, glycogen, dextrins, etc. Chitin is decomposed on boiling with mineral acids and yields, as shown by Ledderhose,' glucosa- mine and acetic acid. Schmiedeb,erg ' therefore considers chitin as a probable acetyl acetic acid combination of glucosamine. If, as previously mentioned (page 346), the chondroitic-sulphuric acid contains a glucosamine group, as made probable by the investiga- tions of Schmibdeberg, then, according to Schmiedeberg, glu- cosamine forms the bridge which leads from the chitin of lower ' Maly's Jahresber., Bd. 20, S. 318. ^ Annal. de chiiu. et phys., Tome 56, Compt. rsAd., Tome 47. « Zeit^chr. f. hysiol. Chem., Bd. 18. * Ber. de deutsch. chem. Gesellsch., Bd. 13. ' Zeitsclir. f. Biologie, Bd. 29. ' Zeitschr. f. physiol. Ohem., Bd. 5. ' L. c. " Zeitschr. f. physiol. Chem., Bdd. 2 u. 4. • Arch. f. exp. Path. u. Pharm., Bd. 28. CHITIN. 575 animals to the cartilage of higlier organized beings. According to the recent investigations of GiLsbisr' and Wintbkstein " several fungi seem to contain chitin instead of cellulose. On heating chitin with alkali and a little water to 180° C. a cleavage takes place, according to Hoppe-Setlee and Akaki/ with the formation of a new substance, cJdtosan, C,^H„N^O,„, which retains the shape of the original chitin and the splitting off of acetic acid. Chitosan is dissolved by dilute acids, also acetic acid, and is colored violet by a dilute iodine solution. It splits into acetic acid and glucosamine by the action of hydrochloric acid. On heating with acetic anhy- dride it is converted into a chitin-like substance, which is not iden- tical with chitin and contains at least three acetyl groups. In the dry state chitin forms a white, brittle mass retaining the form of the original tissue. It is insoluble in boiling water, alcohol, , ether, acetic acid, dilute mineral acids, and dilute alkalies. It is soluble in concentrated acids. It is dissolved without decomposing in cold concentrated hydrochloric acid, but is decomposed by boil- ing hydrochloric acid. When chitin is dissolved in concentrated sulphuric acid and the solution dropped into boiling water and then boiled, we obtain a substance (glucosamine or glucose) which reduces copper suboxide in alkaline solutions. According to Keawkow the various chitins behave differently with iodine or with sulphuric acid and iodine in that some are colored reddish brown, blue, or violet, while others are not colored at all. Chitin may be easily prepared from the wings of insects or from the shells of the lobster or the crab, the last mentioned having first been extracted by an acid so as to remove the lime-salts. The wings or shells are boiled with caustic alkali until they are white, afterward washed with water, then with dilute acid and water, and lastly extracted with alcohol and ether. If chitin so prepared is dissolved in cold, concentrated sulphuric acid and diluted with cold water, then pure chitin separates out, having been set free from the combination with the other body (Keawkovt). Hyalin is the chief organic constituent of the walls of hydatid cysts. From a cliemical point of view it stands close to chitin, or between it and the pro- teid In old and more transparent sacs it is tolerably free fri m mineral bodies, but in younger sacs it contains a great quantity (16^) of lime-salta (carbonate, phosphate, and sulphate). ' Compt. rend., Tome 120. • Ber. d. deutsch. chem. Gesellsch., 1894-1895. • Zeitschr. f. pbysiol. Chem., Bd. 20. 5Y6 THE SKIN AND ITS SECRETIONS. According to LiJCKB ' its composition is; C H N From old cysts 45.3 6.5 5.2 43.0 From young cysts 44.1 6.7 4.5 44.7 It differs from keratin on tlie one hand and from proteids on the other by the absence of sulphur, also by its yielding, when boiled with dilute sulphuric acid, a variety of sugar in large quantities (50^), which is reducing, fermenta- ble, and dextrogyrate. It differs from ohitin by the property of being gradu- ally dissolved by caustic potash or soda, or by dilute acids ; also by its solu- bility on heating with water to 150° C. The coloring matters of the shin and horn-formations are of different kinds, but have not been mach studied. Those occurring in the Malpighian layer of the skin, especially of the negro, and the black or brown pigment occurring in the hair belong to the group of coloring matters which have received the name melanins. Melanins. This group includes several different varieties of amorphous black or brown pigments which are insoluble in water, alcohol, ether, chloroform, and dilute acids, and which occur in the skin, hair, epithelium-cells of the retina, in sepia, in certain patho- logical formations, and in the blood and urine in disease. Of tliese pigments- there are a few, such as the melanin of the eye and that from the melanotic sarcomata of horses, the hippomelanin (Nencki aud Beedbz"), which are soluble with difficulty in alkalies, while others, scich as the pigment of the hair and the coloring matter of certain pathological swellings in man, the phymatorusin (Nbncki and Beedez) , are easily soluble in alkalies. Among the melanins there are a few, for example, the choroid pigment, which are free from sulphur; others, on the contrary, as the pigment of the hair and of horse-hair, are rather rich in sulphur (3-4^), while the phymatorusin found in certain swellings and in the urine (Nencei and Bekdez, K. Mornbr ') is very rich in sulphur (8-10^). Whether any of these pigments, especially the phymatorusin, contains any iron or not is an important though disputed point, for it leads to the question whether these pigments are formed from the blood-coloring matters. The pigment, phymatorusin, isolated by Neji^cki and Beedez from melanotic sarcomata, is, according to them, free from iron and is not a deriva- tive of haemoglobin. K. Moenee and later also Beandl and L. Peeifeee * found, on the contrary, that this pigment did contain ' Virchow's Arch. , Bd. 19. « Arch, f . exp. Path. u. Pharm. , Bdd. 20 u. 24. ^ Zeitschr. f. physiol. Chem., Bd. 11, which contains all the older litera- ture, and Bd. 12. * Zeitschr. f . Biologie, Bd. 26. MELANINS. 577 iron, and they consider it as a derivative of the blood-pigments. The difficulties which attend the isolation and purification of the melanins have not been overcome in certain cases, while in others it is questionable whether the final product obtained has not another composition than the original coloring matter, owing to the ener- getic chemical processes resorted to in its purification. Under such circumstances it seems that a tabulation of the analyses of different melanin preparations made up to the present time are of secondary importance. Among the above-mentioned bodies belonging to the melanin group, the phymatorusin prepared by Nencki and Siebee from melanotic sarcomata, and that prepared by K. Moenee from the sarcomata and the urine of a patient, seem to be of special interest. Phymatorusin is an amorphous dark-brown pigment soluble in alka- lies or alkali carbonates, but insoluble in warm 50-75j^ acetic acid. In alkaline solution it shows no absorption-bands. According to Nencki and Siebee it is free from iron, but Moene:^, on the contrary, claims that it does contain iron. Moenee found for this coloring matter from tumors (A) and from urine (B) the following composition calculated on the substance considered as ash-free: A B c 55.3^—56.13 55.76 H 5.65— 6.33 5.95 N 12.30 12.37 S 7.97 9.01 Fe 0.063—0.081 0.30 Nencki and Siebee have also shown that other melanins, not identical with phymatorusin, occur in melanotic sarcomata of man. The investigations of Beandl and Pfeiffee seem to lead to a. similar conclusion. The coloring matter or matters of human hair contain a low quantity of nitrogen, 8.5^ (Siebee '), and a variable but high quan- tity of sulphur, 3.71—4.10^. The considerable quantity of iron oxide found in the ash does not seem to belong to the pigments. In addition to the coloring matters of the human skin we may also here treat of the pigments found in the skin or epidermis-formation of animals. The beautiful color of the feathers of many birds depends in certain cases on purely physical causes (interference-phenomena), but in other cases on col- oring matters of various kinds. Such a coloring matter is the amorphous red' dish violet turaein, which contains 1% copper, and whose spectrum is very similar to that of oxyhaemoglobin. Kkukenbkkg ' found a large number of coloring ' Arch. f. exp. Path. u. Pharm., Bd. 20. « See Physiol, Studien, Abth. 5, u. 2, Eeih. Abth. 1, s! 151, Abth. 2, S. 1, und Abth. 3, S. 128. S78 TEE SKIN AND ITS SECBBTI0N8. matters in birds' feathers, namely, zooerythrin, zoofulvin, turacoverdi/rt, zoorw- Mn, psiUacofulviri, and others wliicli cannot be enumerated here. Totronerythrin, so named by Wdbm,' is a red amorphous pigment, which is soluble in alcohol and ether, and which occurs in the red warty spots over the eyes of tlie heath-cock and the grouse, and which is very widely spread among the invertebrates (Hallibdeton,' Db Mbeejkowski,' MacMdnn *). Besides tetronerythrin MAcMtnsN found in the shells of crabs and lobsters a blue col- oring matter, cyanoerystallin, which turns red with acids and by boiling water. Haematopwphyrin, according to MacMdnn,' also occurs in the integu- ments of certain lower animals. In addition to the coloring matters thus far mentioned a few others found in certain animals (though not in the skin) will be spoken of. Carminic acid, or the red coloring matter of cochineal, has the composition CitHibOio. It gives sugar on boiling with acids, but this does not correspond with the recent statements of Libbbkmann.' The beautiful purple solution of ammonium carminatehas two absorption-bands between 2> and .& which are similar to those of oxyhaemoglobin. These bands lie nearer to B! and closer to- gether, and are less sharply defined. Purple is the evaporated residue from the purple- violet secretion, caused by the action of the sunlight, from the so- called " purple gland " of the tunic <'f certain species of murex a.nA purpura. Its chemical nature has not been investigated. Among the remaining coloring matters found in invertebrates we may men- tion blue sientorin, acUniochrom, boneilin, polyperythrin, peutamnin, ante- donin. crusiaceorubin, janthinin, and clUoropliyU. Sebum when freshly secreted is an oily semi-fluid mass which solidifies on the upper surface of the skin, forming a greasy coating. The quantity is very different in different persons. Hoppb- Setlee ' has found a body similar to casein, besides albumin and fat, in the sebum. Cholesterin is also found in this fat, and in especially large quantities in the vernix caseosa. The solids of the sebum consist chiefly of fat, epithelium-cells, and protein bodies; the vernix caseosa consists chiefly of fat. On account of the generally diffused view that wax of the plant epidermis serves as protection for the inner parts of the fruit and plant, LiEBEBiOH * has suggested that the combinations of fatty acids with monatomic alcohols are the reason for the resistance property of the waxes as compared with the glycerin fats. He also qonsiders that the cholesterin fats play the r61e of a protective fat in the animal kingdom, and he has been able to detect cholesterin fat in human skin and hair, in vernix caseosa, whale-bone, tortoise- ' Zeitschr. f. wissensch. Zool., 1871. Cited from Maly's Jahresber., Bd. 1, S. 52. ' Journal of Physiol., Vol. 6. ' Compt. rend., Tome 93. ■•Proc. Roy. Soc, 1883. » Quart. Journ. of Micros. Sc., 1877, and Journal df Physiol., Vol.^. * Ber. d. deutsch. chem. Geseilsch., Bd. 18. 'Physiol. Chem.,S. 760. • Virchow's Arch., Bd. 121. SEBUM AND CERUMEN. 5^9 shell, cow's horn, the feathers and beaks of several birds, the prickles of the hedgehog and porcupine, the hoofs of horses, etc. He draws the following conclusion from this, namely, that the cholesterin fats always appear in combination with the keratinous substance, and that the cholesterin fat, like the wax of plants, serves as protection for the skin-surface of animals. Cerumen is a mixture of the secretion of the sebaceous and sweat glands of the cartilaginous part of the outer organs of hearing. It contains chiefly soaps and fat, and besides these a red substance easily soluble in alcohol and with a bitter-sweet taste. The preputial secretion, smegma prmputii, contains chiefly fat, also cholesterin and ammonium soaps, which probably are produced from decomposed urine. The hippuric acid, benzoic acid, and calcium oxalate found in the smegma of the horse have probably the same origin. We may also consider as a preputial secretion tlie eastoreum, wUicli is se- creted by two peculiar glandular sacs in the prepuce of the beiiver. This eas- toreum is a mixture of pri)teids, fat, resins, traces of ph. nol (volatile oil), and a non-nitrogenized body, castorin, crystallizing in four-sided needles from alco- hol, insoluble in cold water, but somewhat soluble in boiling water, and whoso composition is little known. , Wool-fiU, or the so-called fat-sweat of sheep, is a mixture of the secretion of the sudoriparous and sebaceous glands. We find in the watrry extract a large quantity of potassium which is combined with organic acid, volatile aud non- volatile fatty acids, benzoic acid, pheuol-sulpliuric acid, lactic acid, malic acid, succinic acid, and others. The fat contains among other bodies abundant quantities of ethers of fatty acids with cholesterin and isocholes- terin. The secretion of the coccygeal glands of ducks and geese contains a body similar to casein, besides albumin, nuclein, lecithin, and fat, but no sugar (Db JOKGB '). Poisonous bodies have been found in the secretion of the skin of the salamander and the toad respectively, samandarin (Zalbsky ') and bufidin (JOBNABA and Cabali '). The Sweat. Of the secretions of the skin, whose quantity amounts to about ^ of the weight of the body, a disproportioDally large part consists of water. Next to the kidneys, the skin in man is the most important means for the elimination of water. As the glands of the skin and the kidneys stand near to each other in regard to their functions, they may to a certain extent act vica- riously for one another. The circumstances which influence the secretion of sweat are very numerous, and the quantity of sweat secreted must consequently ' Zeitschr. f. physiol Chem., Bd. 3. ' Hoppe-Seyler's Med chem, Untersuch., S. 85. » Riv. di Bologna, 1873. Cited from Maly's Jahresber., Bd. 3, S. 64. 580 THE 8KIN AND ITS SECRETIONS. vary very considerably. The secretion differs for different parts of the skin, and it has been stated that the perspiration of the cheek, that of the palm of the hand, and that under the arm stand to each other as 100 : 90 : 45. From the unequal secretion on different parts of the body it follows that no results as to the quantity of secretion for the entire surface of the body can be calculated from the quantity secreted by a small part of the skin in a given time. In determin- ing the total quantity a stronger secretion is as a rule produced, and as the glands can with difficulty work for a long time with the same energy, it is hardly correct to estimate the quantity of secretion per 24 hours from a strong secretion enduring only a short time. The perspiration obtained for investigation is never quite pure,, but contains cast-off epidermis-cells, also cells and fat-globules from the sebaceous glands. Filtered sweat is a clear, colorless fluid with a salty taste and of different odors from different parts of the body. The physiological reaction is acid, according to most statements. Under certain conditions also an alkaline sweat may be secreted (Teumpt and Luchsinger,' Hetjss"). An alkaline reaction may also depend on a decomposition with the formation of ammonia. According' to a few investigators the physiological reaction is alkaline, and an acid reaction depends, according to these investiga- tors, upon an admixture of fatty acids from the sebum. Moeiggia ' found that the sweat from herbivora was ordinarily alkaline, while that from carnivora was generally acid. According to Smith* horse's sweat is strongly alkaline. The specific gravity of human sweat is 1.003-1.005. Perspiration contains 977.4-995.6 p. m., average 988.3 p. m., water, and. 4.4-22.6 p. m., average 11.80 p. m., solids. The organic bodies are neutral fats, cholesterin, volatile fatty acids, traces of proteid (according to Lecleec " and Smith ° habitually in horses, according to Gaube ' regularly in man, and according to > Pfluger's Arch., Bd. 18. « Monatsbefte f. prakt. Dermat., Bd. 14. Cited from Maly's Jahresber. Bd. 33, S. 193. ' Molescliott, Untersueh. zur Naturlire, Bd. 11 ; also Maly's Jahresber. Bd. 3, S. 136. < Journal of Physiol., Bd. 11. In regard to the older literature on sweat see Hermann's Handbucb, Bd. 5, Thl. 1, S. 431 u. 543. ' Compt. rend., Tome 107. «L. c. « Maly's Jahresber., Bd. 23, S. 193. TEB SWEAT. 581, Leube ' sometimes after hot baths, in Bright's disease, and after the use of pilocarpin), also creatinin (CAPRAincA'), aromatic oxyadds, ethereal-sulphuric acids of phenol and skatoxyl (Kast'), but not of indoxyl, and lastly urea. The quantity of urea has been determined by ARGUTi2srsKT.* In two steam-bath experiments, in which in the course of J and f hour respectively he obtained 325 and 330 c. c. sweat, he found 1.61 and 1.24 p. m. urea. Of the total nitrogen of the sweat in these two experiments 68.5 and 74.9^ respectively belong to the urea. From Argutinsky's experiments, and also from those of Cramer,' it follows that of the total nitrogen a portion not to be disregarded is eliminated by the sweat. This portion was indeed 12% in an experiment of Cramer at high tem- perature and powerful muscular activity. Cramer has also found ammonia in the sweat. In nrgemia, and in ischuria in cholera, urea may be secreted in such quantities by the sweat-glands that crystals ■deposit upon the skin. The mineral bodies consist chiefly of sodium chloride with some potassium chloride, alkali sulphate, and pbosphate. The relative quantities of these in perspiration differ materially from the quantities in the urine (Fatre,° Kast). The relationship, according to Kast, is as follows : Chlorine . Phosphate Sulphate perspiration 1 • 0.0015 0.009 urine 1 0.1320 0.397 Kast found that the proportion of ethereal-sulphuric acid to the sulphate sulphuric acid in sweat was 1 : 12. After the admin- istration of aromatic substances the ethereal-sulphuric acid does not increase to the same extent in the sweat as in the urine (see Chapter XV). Sugar may pass into the sweat in diabetes, but the passage of the bile-col- oring matters has not been positively shown in this secretion. Benzoic add, succinic acid, tartaric acid, iodine, arsenic, mercuric chloride, and quinine pass into the sweat. Uric acid has also been found in the sweat in gout, and eystin in cystinura. ChTomhidrosls is the name given to the secretion of colored sweat. Some- times sweat has been observed to be colored blue by indigo (Bizio'), by 1 Leabe, Virchow's Arch., Bdd. 48 u. 50, and Arch. f. klin. Med., Bd. 7. « Maly's Jahresber., Bd. 13, S. 190. » Zfcitschr. f. physiol. Chem., Bd. 11, S, 501. * Pflilger's Arch., Bd. 46. ' Arch. f. Hygiene, Bd. 10. • Compt. rend.. Tome 35, and Arch, gengr. de m6d., 1853 (Ser. 5), Vol. 2. 'Wien. Sitzungsber., Bd. 39. 582 THE SKIN AND ITS SECRETIONS. pyocyanin, or by ferro-phospliate piOLLMANN '). True blood-sweat, in which blood-corpuscles exude from the openings of the glands, have also been ob- served. The exchange of gas through the skin in man is of very little- importance compared with the exchange of gas by the langs. The absorption of oxygen by the skin, which was first shown by EegjS^atjlt and Eeiset, is very small. The quantity of carbon dioxide eliminated by the skin increases with the rise of temperature (AuBEET," Eohkig/ FuBiiiri and Eonchi'). It is also greater in light than in darkness. It is greater during digestion than when fasting, and greater after a vegetable than after an animal diet (FuBiNi and Eonchi). The quantity calculated by various inves- tigators for the entire skin surface in 24 hours varies between 2.23 and 32.8 grms.' In certain animals, as in frogs, the exchange of gas through the skin is of great importance. As the exchange of gas through the skin in man and mammalia is very small, it follows that the injurious and dangerous effects caused by covering the skin with varnish, oil, or the like can hardly depend on a prevented exchange of gas. After varnishing the skin there is a considerable loss of heat, and the animal quickly dies. If the animal, on the contrary, be guarded from this loss of heat, it may be saved, or at least kept alive for a longer time. This effect was supposed to be due to a poisoning caused by a reten- tion of one or more substances of the perspiration {perspirabile retentum) , accompanied by fever and increased loss of heat through the skin; but this statement has not been substantiated. This phenomenon seems to be due to other causes, and at least in certain animals (rabbits) death seems to ensue from the paralysis of the vaso-motor nerves. In anastomosis the loss of heat through the skin eeems to be increased to such an extent thatthe animal dies from the lowered temperature. ' Wurzb. med. Zeitsch., Bd. 7, S. 351. Cited from Gorup-Besanez, Lehrb., 4. Aufl., S. 555. « Pfluger's Arch., Bd. 6. "Deutsch. klin., 1872, S. 309. * Moleschott's Metersuch. zur Naturhhre., Bd. 13. ' See Hoppe-Seyler, Physiol. Chem. S. 580. CHAPTEE XVII. CHEMISTRY OF RESPIRATION. Dtjking life a constant exchange of gases takes place between the animal body and the sarronnding medium. Oxygen is inspired and carbon dioxide expired. This exchange of gases, which is called respiration, is brought about in man and vertebrates by the nutri- tive fluids, blood and lymph, which circulate in the body and which are in constant communication with the outer medium on one side and the tissue-elements on the other. Such an exchange of gaseous constituents may take place wherever the anatomical conditions offer no obstacle, and in man it may go on in the intes- tinal tract, through the skin, and in the lungs. As compared with the exchange of gas in the lungs, the exchange already mentioned which occurs in the intestine and through the skin is very insig- pificant. For this reason we will discuss in this chapter only the exchange of gas between the blood and the air of the lungs on one side, and the blood and lymph and the tissues on the other. The first is often designated external respiration, and the other internal respiration. In this chapter we will accordingly first discuss the gases of the blood and lymph, and then the exchange of gas in the lungs and tissues. The quantitative circumstances of the exchange of gas stand in such close relationship to metabolism in general that they will be treated of in the last chapter, on the income and output of the body under different conditions. Only the chief points in the methods commonly employed for measuring the exchange of gas will be mentioned. I. The Gases of the Blood. Since the pioneer investigations of Magnus and Lothae Meyek the gases of the blood have formed the subject of repeated, careful investigations by prominent experimenters, among whom we must mention first C. Ludwig and his pupils and E. Pfluger and 584 CHEMISTRY OF RESPIRATION. his school. By these investigations not only has science been enriched by a mass of facts, but also the methods themselves have been made more perfect and accurate. In regard to these methods, as also in regard to the laws of the absorption of gases by liquids, dissociation, and other questions belonging here, the reader is referred to complete text-books on physiology, on physics, and on gasometric analysis. The gases occurring in blood under physiological conditions are oxygen, carton dioxide, and nitrogen. The last-mentioned gas is found only in very small quantities, on an average of 1.8 vol. per cent. The quantity is here, as in all following experiments, calcu- lated for 0° C. and 760 mm. pressure. The nitrogen seems to be simply absorbed into the blood, at least in great part. It appears to play no part in the processes of life, and its quantity varies but slightly in the blood of different blood-vessels. The oxygen and carbon dioxide behave otherwise, as their quantities have significant variations, not only in the blood from different blood-vessels, but also because many conditions, such as a difference in the rapidity of circulation, a different temperature, Test and activity, cause a change. In regard to the gases they .contain the greatest difference is observable between the blood of the arteries and that of the veins. The quantity of oxygen in the arterial blood of dogs is on an average 22 vols, per cent (Pflugek). In human blood Sbtschenow found about the same quantity, namely, 21.6 vols, per cent. Lower figures have been found for rabbit's and bird's blood, respectively 13.2^ and 10-15^ (Walter, Joltet). Venous blood has very variable quantities of oxygen. Ludwig and Sczelkow found 6.8^ oxygen in the venous blood of resting muscles, and a still smaller quantity in the venous blood of active muscles. Oxygen is entirely absent from blood after asphyxiation, or occurs only as traces. The venous blood of the glands seems, on the contrary, daring secretion to be richer in oxygen than ordinary venous blood. By summarizing a great number of analyses by different experimenters Zuntz has calculated that the venous blood of the right side of the heart contains on an average 7.15^ less oxygen than the arterial blood. The quantity of carlon dioxide in the arterial blood of dogs is 30 to 40 vols, per cent (Ludwig, Setschenow, Pfluger, P. Bert, and others), most generally about 40$^. Setschen-qw found 40.3 vols, per cent in human arterial blood. The quantity of carbon OXYGEN ABSORPTION. 585 dioxide in venous blood varies still more (Ludwig, Pfluger and their pupils, P. Bert, Mathief and Ukbain, and others). According to the calculations of Zuntz the venous blood of the right side of the heart contains about 8.2^ more carbon dioxide than the arterial. The average amount may be put down as 48 vols, per cent. Holmgeen found in blood after asphyxiation even 69.21 vols, per cent carbon dioxide." Oxygen is absorbed only to a small extent by the plasma or serum, in which Pfluger found but 0.26?^. The greater part or nearly all of the oxygen is loosely combined with the hsemoglobin. The quantity of oxygen which is contained in the blood of the dog corresponds closely to the quantity which from the activity of the haemoglobin we should expect to combine with oxygen, and also the quantity of haemoglobin in canine blood. It is difficult to ascertain how far the circulating arterial blood is saturated with oxygen, as immediately after bleeding a loss of oxygen always takes place. Still it seems to be unquestionable that it is not quite completely saturated with oxygen in life. The question whether ozone occurs in the blood is to be answered decidedly in the negative. It is not only impossible to detect ozone in the blood, but the possibility of the occurrence of ozone in the fluids and tissues is even a priori to be denied. Ozone acts as nascent oxygen; and as easily oxidizable substances occur in the organism which combine with nascent oxygen, ozone, if such a formation should take place at all, would be destroyed instantly. But such a formation of ozone in the animal body cannot be admitted. Ozone may indeed be formed by slow oxidation, since the nascent oxygen formed in consequence combines with neutral oxygen, forming ozone; but in the animal organism the nascent oxygen mast be combined with the oxidizable substances before it can form ozone. It was formerly believed that the hagmoglobin acted as an " ozone-exciter," possessing the property of converting the inactive oxygen of the air into ozone. The red blood-corpuscles can by themselves also give a blue color with tincture of guaiacum, which is markedly seen when this tincture is dried on blotting-paper and a drop of blood previously dilated with 5-10 vols, water is added. • All the figures given above may be found in Zuntz's " Die Gase des Blutes" in Hermann's HandbucU d. Physiol., Bd. 4, Tbl, 8, S. 33-43, which also contains detailed statements and the pertinent literature. 586 CHEMISTRY OF RESPIRATION. According to Pfluger,' we are here (iealing (see page 134) with a decomposition and gradual oxidation of haemoglobin, in which processes the neutral oxygen is split, setting free oxygen atoms. The carbon dioxide of the blood occurs in part, and indeed, according to the investigations of Alex. Schmidt," Zuntz,' and L. Fbedekicq,' to the extent of at least one third, in the blood- corpuscles, and also in part, and in fact the greatest part, in the plasma and seram respectively. The carbon dioxide of the red corpuscles is loosely combined, and the constitaent uniting with the CO, of the same seems to be the alkali combined with phosphoric acid, oxyhaemoglobin or haemo- globin and globulin on one side and the haemoglobin itself on the other. That in the red corpuscles alkali phosphate occurs in such quantities that it may be of importance in the combination with carbon dioxide is not to be doubted, and we must admit that from the diphosphate, by a greater partial pressure of the carbon dioxide, monophosphate and alkali carbonate are formed, while by a lower partial pressure of the carbon dioxide the mass action of the phos- phoric acid comes again into play, so that, with the carbon dioxide becoming free, a re-formation of alkali diphosphate takes place. It is generally admitted that the blood-coloring matters, especially the oxyhaemoglobin, which can expel carbon dioxide from sodium carbonates in vacuo, act like an acid; and as the globulins also act like acids (see below), this body. may also occur in the blood-cor- puscles as an alkali combination. The alkali of the blood-corpuscles must therefore, according to the law of mass action, be divided between the carbon dioxide, phosphoric acid, and the other con- stituents of the blood-corpuscles which are considered as acid acting, and among these especially the blood-pigments, as the globulin can hardly be of importance because of its small quantity. By greater mass action or greater partial pressure of the carbon dioxide, bicar- bonate must be formed at the expense of .the diphosphates and the , other alkali combinations, while at a diminished partial pressure of the same gas, with the escape of carbon dioxide, the alkali diphos- phate and the other alkali combinations must be re-formed at the cost of the bicarbonate. ' Pfliiger's Arch., Bd. 10, S. ?52. ' Ber. d. k. sSchs. Gesellsch. d.Wissensch., Matb.-phys. Klasse, Bd. 19, 1867. 2 Centralbl. f. d. med. Wissensch., 1867, S. 529. ■* Recherches sur la constitution du Plasma sanguin, 1878, p. 50-51. CARBON DIOXIDE OF TSE BL00D-C0SPU80LB8. 58T Hemoglobin must nevertheless, as the investigations of Set- SCHENOW ' and Zontz," and especially those of Bohk ' and Torup,* have shown, be able to hold the carbon dioxide loosely combined even in the absence of alkali. Bohe has also found that the dissociation curve of the carbon-dioxide haemoglobin corresponds essentially to the curve of the absorption of carbon dioxide, on which ground he and Tobup consider the haemoglobin itself as of importance in the binding of the carbon dioxide of the blood and not its alkali combinations. In regard to this question the condi- tions are not quite clear. If carbon dioxide is allowed to act on haemoglobin, it unites (Bohe, Tokup) with the colored atomic group of the haemoglobin, splitting ofE proteid, and from this haemoglobin, so decomposed, oxyhaemoglobin cannot be formed by the action of oxygen. According to Bohr, for each gramme of haemoglobin at -|- 18.4° C. and a pressure of 30 mm. 2.4 c. cm. carbon dioxide are combined.; and since in the arterial blood nearly all the haemoglobin exists as oxyhaemoglobin, it is difficult to- understand how the heemoglobin can be of any great importance in the binding of the carbon dioxide of the blood. According to the recent investigations of Bohr ' this condition is explained by the property of the haemoglobin to take up both gases, carbon dioxide and oxygen, simultaneously and independently of each other. It takes place, as admitted by Bohr, by the oxygen probably uniting with the pigment nucleas, and the carbon dioxide with the proteid component. The chief part of the carbon dioxide of the blood is found in the blood-plasma or the blood-serum, which follows from the fact that the serum is richer in carbon dioxide than the corresponding blood itself. By experiments with the air-pump on blood-serum it has been found that the chief part of the carbon dioxide contained in the serum is given off in a vacuum, while a smaller part can be pumped out only after the addition of an acid. The red corpuscles also act as an acid, and therefore in blood all the carbon dioxide is expelled in vacuo. Hence a part of the carbon dioxide is firmly chemically combined in the serum. ' Centralbl. f. d. med. Wissensoli., 1877. See also Zuntz in Hermann's. Handbuch, 8.76. ' L c, S. 76. » See Maly's Jahresber., Bd. 17, S. 115. *lUd.. S. 115. » See foot-note 4, p. 139. 588 OHEMISTRY OP RESPIRATION. Absorption experiments with blood-serum have shown ns further that the carbon dioxide which can be pumped out is in great part loosely chemically combined, and from this loose combination of the carbon dioxide it necessarily follows that the serum must also contain simply absorbed carbon dioxide. For the form of binding of the carbon dioxide contained in the serum or the plasma we find the three following possibilities: 1. A part of the carbon dioxide is simply absorbed; 2. Another part is loosely chemically combined; 3. A third part is in firm chemical combination. The quantity of simply absorbed carbon dioxide has not been exactly determined. Setschbis^ow ' considers the quantity in dog- serum to be about yi,- of the total quantity of carbon dioxide.- According to the tension of the carbon dioxide in the blo6d and its absorption coefficient, the quantity seems to be still smaller. The quantity of firmly chemically combined carbon dioxide in the blood-serum depends upon the quantity of simple alkali car- bonate in the serum. This quantity is not known, and it cannot be determined either by the alkalinity found by titration, nor can it be calculated from the excess of alkali found in the ash, because the alkali is not only combined with carbon dioxide, but also with other bodies, especially with proteid. The quantity of firmly chemically combined carbon dioxide cannot be ascertained after pumping out in vacuo without the addition of acid, because to all appearances certain active constituents of the serum, acting like acids, expel carbon dioxide from the simple carbonate. The quantity of carbon dioxide not expelled from dog-serum by vacuum alone without the addition of acid amounts to 4.9 to 9.3 vols, per cent, according to the determinations of Pfluger.' From the occurrence of simple alkali carbonates in the blood- serum it naturally follows that a part of the loosely combined carbon dioxide of the serum which can be pumped out must occur as bicarbonate. The occurrence of this combination in the blood- serum has also been directly shown. In experiments with the pump, as well as in absorption experiments, the serum behaves in other ways as a solution of bicarbonate, or carbonate of a corre- sponding concentration; and the behavior of the loosely combined carbon dioxide in the serum can be explained only by the occurrence ' Centralbl. f. d. med. Wissensch., 1877, No. 35. ' E. Pfliiger, Ueber die Koblensaure des Blutes. Bonn, 1864. S. 11. Cited from Zuntz in Hermann's Handbuch, S. 65. CARBON DIOXIDE OF THE HERUM. 589 of bicarbonate in tlie serum. By means of vacuum the serum always allows much more thau one half of the carbon dioxide to be expelled, and it follows from this that in the pumping out not only may a dissociation of the bicarbonate take place, but also a conver- sion of the double sodium carbonate into a simple salt. As w£ know of no other carbon-dioxide combination besides the bicarbon- ate in the serum from which the carbon dioxide can be set free by simple dissociation in vacuo, we are obliged to assume that the serum must contain other faint acids, in addition to the carbon dioxide, which contend with it for the alkalies, and which expel the carbon dioxide from simple carbonates in vacuo. The carbon dioxide which is expelled by means of the pump and which, without regard to the simple absorbed quantity, is generally designated as " loosely chemically combined carbon dioxide," is thus only obtained in part iu dissociable loose combination; in part it originates from the simple carbonate's, from which it is expelled in vacuohj other faint acids. These faint acids are thought to be in part phosphoric acid and in part globulins. The importance of the alkali phosphates for the carbon dioxide combination has been shown by the investigations of Fbknet; but the quantity of these salts in the serum is, at least in certain kinds of blood, for example in ox-serum, so small that it can hardly be of importance. In regard to the globulins Set- SCHENOW is of the opinion that they do not act as acids themselves, but form a combination with carbon dioxide, producing carboglobn- linic acid, which unites with the alkali. According to Sertoli,' whose views have lately found a supporter in Toeup, the globulins themselves are the acids which are combined with the alkali of the blood-serum. In both cases the globulins would form, directly or indirectly, that chief constituent of the plasma or of the blood- serum which, according to the law of the action of masses, contends with the carbon dioxide for the alkalies. By a greater partial pressure of the carbon dioxide the latter deprives the globulin alkali of a part of its alkali, and bicarbonate is formed; by low partial pressure the carbon dioxide escapes, and the bicarbonate is abstracted by the globulin alkali. In the foregoing it has been assumed that the alkali is the most essential and important constituent of the blood-serum, as well as of the blood in general, in uniting with the carbon dioxide. The ' Hoppe-Seyler, Med. cheiii. Untersuch. 590 OHEMISTBT OF RE8PIBATI0N. fact that the quantity of carbon dioxide in the blood greatly dimin- ishes with a decrease in the quantity of alkali strengthens this assumption. Such a condition is foand, for example, after poison- ing with mineral acids. Thus Walter ' fonnd only 2-3 vols, per cent carbon dioxide in the blood of rabbits into whose stomachs hydrochloric acid had been introduced. In the comatose state of diabetes mellitus the alkali of the blood seems to be in great part saturated with acid combinations, /3-oxybntyric acid (Stadel- MAKX," Minkowski), and Minkowski ' found only 3.3 vols, per cent carbon dioxide in the blood in diabetic coma. In the above we have emphasized the fact that the oxygen in the blood occurs in a dissociable combination with the haemoglobin, and that for the formation of this combination, oxyhsemoglobin, a distinct partial pressure of the oxygen is necessary for every variation in temperature. Also that the carbon dioxide of the blood, that which is contained in the blood-corpuscles as well as that in the plasma, occurs mostly in combinations which are dependent to a great extent upon the partial pressure of the carbon dioxide. Hence for the study of the exchange of gases between the blood and the alveolar air on one side, and the blood and the tissues on the other, special regard must be paid to the question as to how far this exchange of gases is the result of the law of diffusion and how far other forces take part in it; also the tension of the oxygen and the carbon dioxide is of the greatest importance. 'Sot these reasons it is best to treat of these questions in that section of this chapter dealing with the exchange of gas in the lungs and tissues. Oases of the Lymph and Secretions. The gases of the lymph are the same as in the blood-serum, and the lymph stands close to the blood-serum in regard to the quantity of the various gases, as well as to the kind of carbon dioxide combi- nation. The investigations of Daenhaedt and Hensek'ou the gases of human lymph are at hand, but it still remains a question whether the lymph investigated was quite normal. The gases of ' Arch. f. exp. Path. u. Pharm., Bd. 7. « Ibid., Bd. 17. ' Mittlieil. a. d. med. Klink in KOnigsberg, 1888, and Arch. f. exp. Path. u. Pharm , Bd. 18. * Virchow's Arch., Bd. 37. GASES OF THE LTMPH AND THE 8E0RETI0N8. 591. normal dog-lymph were first investigated by the author.' These gases contained traces of oxygen and consisted of 37.4-53. Ij^ CO, and 1.6^ N at 0° C. and 760 mm. Hg pressure. Aboat one half of the carbon dioxide was firmly chemically combined. The quantity was greater than in the serum from arterial blood, but smaller than from venous blood. The remarkable observation of BucHifEE ' that the lymph col- lected after asphyxiation is poorer in carbon dioxide than that of the breathing animal is explained by Zuntz ' by the formation of acid immediately after death in the tissues, and especially in the lymphatic glands, and this acid decomposes the alkali carbonates of the lymph in part. The secretions with the exception of the saliva, in which Pfluger' and Kulz' found respectively 0.6 and 1^ oxygen, are free from oxygen. The quantity of nitrogen is the same as in blood, and the chief mass of the gases consists of carbon dioxide. The quantity of this gas is chiefly dependent upon the reaction, i.e., upon the quantity of alkali. This follows from the analyses of Pflugek.' He found 19^ carbon dioxide removable by the air- pump and 54^ firmly combined carbon dioxide in a strongly alka- line bile, but on the contrary 6.6^ carbon dioxide removable by the air-pump and 0.%% firmly combined carbon dioxide in a neutral bile. Alkaline saliva is also very rich in carbon dioxide. As average for two analyses made by Pflugee ' of submaxillary saliva of a dog we have 27. 5j^ carbon dioxide removable by the air-pump and 47.4^ chemically combined carbon dioxide, making a total of 74. 9j^. KiJLZ " found a maximum of 65.78^ carbon dioxide for the parotid saliva, of which 3.31^ was removable by the air-pump and 63.47^ was firmly chemically combined. From these and other statements on the quantity of carbon dioxide relnovable by the air-pump and chemically combined in the alkaline secretions it follows that bodies > Ber. d. k. sSchs. Gesellsch. d. Wissenaeh., Math.-phys. Klasse, Bd. 23, 1871. • Arbeiten a. d. pUysiol. Anstalt zu Leipzig, 1876. » Hermann's Handbuch, Bd. 4, ThI. 3, S. 85. • Pflilger's Arcb., Bd. 1. » Zeitscbr. f . Biologie, Bd. 23. • Pflftger's Arch., Bdd. 1 u. 3. ' L. c. » L. c. It seems as if Cillz's reaalta were not calculated at 760 mm. H^, bat rather at 1 mm. 592 CHEMiaTRT OF RESPIRATION. occur in them, although not in appreciable quantities, which are analogous to the albuminous bodies of the blood-serum and which act like faint acids. The acid or at any rate non-alkaline secretions, urine and milk, contain, on the contrary, considerably less carbon dioxide, which is nearly all removable by the air-pump, and a part seems to be loosely combined with the sodium phosphate. The figures found by Pflugeb for the total quanbity of carbon dioxide in milk and urine are 10 and 18.1-19.7^ respectively. EwALD ' has made investigations on the quantity of gas in pathological transudations. He found only traces, or at least only very insignificant quantities, of oxygen in these fluids. The quantity of nitrogen was about the same as in blood. The quantity of carbon dioxide was greater than in the lymph (of dogs), and in certain cases even greater than the blood after asphyxiation (dog's blood). The tension of the carbon dioxide was greater than in venous blood. In exudations the quantity of carbon dioxide, especially that firmly combined, increases with the age of the fluid, while, on the contrary, the total quantity of carbon dioxide, and especially the quantity firmly combined, decreases with the quan- tity of pus-corpuscles. II. The Exchange of Gas between the Blood on the one hand and Pulmonary Air and the Tissues on the Other. In the introduction (Chapter I, p. 3) it was stated that we are to-day of the opinion, derived especially from the researches of Pflugek and his pupils, that the oxidations of the animal body do not take place in the fiuids ^d juices, but are connected with the form-elements and tissues. It has, it is true, been shown by Alex. Schmidt' and Pflugek' that oxidations take place in the blood, although only to a slight extent; but these oxidations depend, it seems, upon the form-elements of the blood, hence it does not con- tradict the above statement that the oxidations occur exclusively in the cells and chiefly in the tissues. ' C. A. Ewald, Arch. f. Anat. u. Physiol., 1873 and 1876. ' Ber. d. k. sSchs. Gesellsch. d. Wissensch., Math.-pbys. Klasse, Bd. 19, 1867, and CentralbL f. d. med. Wissensch., 1867, S. 356. » Centralbl. f. d. med. Wissensch., 1867, S. 722. THE RESPIRATORY EXOHANOE OF GAB. 593 The gaseous exchange in the tissues, which has been designated internal respiration, consists chiefly in that the oxygen passes from the blood in the capillaries to the tissues, while the chief bulk of the carbon dioxide of the tissues originates therein and passes into the blood of the capillaries. The exchange of gas in the lungs, which is called external respiration, consists, as we learn by a comparison of the inspired and expired air, in the blood taking oxygen from the air in the lungs and giving ofE carbon dioxide. What kind of processes take part in this doable exchange of gas? Is the gaseous exchange simply the result of an unequal tension of the blood on one side and the air in the lungs or tissues on the other ? Do the gases pass from a place of higher pressure to one of a lower, according to the laws of dijEEusion, or are other forces and processes active ? These questions are closely related to another question as to the tension of the oxygen and carbon dioxide in the blood and in the air of the langs and tissues. Oxygen occurs in the blood in a disproportionately large part as oxyhaemoglobin, and the law of the dissociation of oxyhsemoglobin is of fundamental importance in the study of the tension of the oxygen in the blood. If we recall that, according to BoHR, what we generally call oxyhaemo-^ globin is a mixture of haemoglobins, which for one and tlie same oxygen pressure can unite with different quantities of oxygen, and also, as shown by Siegfried, that there exists, besides the oxyhaemoglobin, another dissociable oxygen combination of haemoglobin, namely, pseudohaemoglobin, it seems that we have several important preliminary questions to solve before we come to a discussion of the dissociation conditions of oxyhaemoglobin. As the above statements are in part contradicted and in part not sufficiently proven, and as also, according to Hufnbr, no difference exists between a oxyhaemoglobin solution and a solution of blood-corpuscles in regard to its delivery of oxygen, we are justified in setting the above statements aside for the present and only taking up the generally accepted aud authoritative assertions. for the understanding of the laws by which the oxygen is taken up by the blood in the alveoli of the lungs the investigations on the dissociation. of oxyhEemoglobin are important, and especially those which relate to the dissociation at the temperature of the body are of great physiological importance. Several investigators have experimented on this subject, especially G. IIufner.' He has proven an important fact, namely, that a freshly prepared solu- tion of pure oxyhaemoglobin crystals does not act unlike freshly ' Du Bois-Reymond's Arch., 1890. Hilfner here gives also his older re- searches on this subject. &94 OSEMISmr OF HESPlMATIOJr. defibrinated blood as regards tbe dissociatioii of oxyhasmoglobin. He also showed that the dissociation is dependent upon the concen- tration, namely, that at a given pressure a dilate solution is more strongly dissociated than a more concentrated solution. He found : for solutions containing 14^ oxyhaemoglobin that the dissociation at -f 35° 0. and an oxygen partial pressure of 75 mm. Hg was only very insignificant and only little stronger than with a partial pressure of 152 mm. In the first instance 96.89^ of the total pigment Was present as oxyhsgmoglobin and 3.11^ as haemoglobin, while in the other case, at 152 mm. pressure, the respective figures Were 98.42 and 1.58^. The dissociation becomes stronger first with an oxygen partia;l pressure of about 75 mm. Hg and down- wards, and a corresponding increase in the quantity of reduced haemoglobin; but even wibh an oxygen partial pressure of 50 mm. Hg the quantity of haemoglobin was only 4.6^ of the total pigment. From these and older researches by HirPiTEK ' which were made at 35 or 39° 0. it follows that the partial pressure of the oxygen may be reduced to one half of the atmospheric air without influenc- ing essentially the quantity of oxygen in the blood or a correspond- rng solution of oxyh moglobin. "We can also conclude from the quantity of oxygen or oxyhaemoglobin in the arterial blood that the tension of the oxygen in the arterial blood must be relatively higher. Based on the investigations of several experimenters, sach as P. Bert,'' Heetee,' and Hufkbe, who experimented partly on living animals and partly with haemoglobin solutions, we generallv consider the tension of the oxygen in arterial blood at the tempera- ture of the body equal to an oxygen partial pressure of 75-80 mm. Hg. We must now compare these figures with the tension of the oxygen in the air of the lungs. Numerous investigations as to the composition of the inspired atmospheric air as well as the expired air are at hand, and we can say that these two kinds of air at 0° 0. and a pressure of 760 mm. Hg have the following average composition in volume per cent: Oxygen. Nitrogen. Carbon Dioxide. Atmospheric air 20.96 79.02 0.03 Expired air 16.03 79.59 4.38 ' L. c. ' Paul Bert, La pre=sion barometrique. Paris, 1878. • Zeitschr. f. physiol. Cliem., Bd. 8. THE MESPIRATORT EXCIIANOE OF 0A8. 595 The partial pressure of the oxygen of the atmospheric air corre- sponds at a normal barometric pressure of 760 mm. to a pressure of 159 mm. Hg. The loss of oxygen which the inspired air suffers in respiration amounts to about 4.93^, while the expired air contains about one hundred times as much carbon dioxide as the inspired air. The expired air is therefore a mixture of alveolar air with the residue of inspired air remaining in the air-passages; hence in the study of the gaseous exchange in the lungs we must first consider the alveolar air. We have no direct determination of the composi- tion of the alveolar air, but only approximate calculations. Prom the average results found by Vieeordt ' in normal respiration for the carbon dioxide in the expired air, 4.63^^, Zuntz ' has calculated ■the probable quantity of carbon dioxide in the alveolar air as equal to 5.44^. If we start from this value, with the assumption that the quantity of nitrogen in the alveolar air does not essentially differ from the expired air, and admit that the quantity of oxygen in the alveolar air is 6^ less than the inspired air, we find that the alveolar air contains 14.96^ oxygen, corresponding to a partial pressure of 114 mm. Hg. We have several direct determinations as to the composition of the alveolar air of dogs which show that the alveolar air is not much richer in carbon dioxide than the expired air. By means of the lung catheter, an apparatus constructed by Pfldgbk, his pupils Wolffbbeg' and Nussbaum* have investi- gated the composition of the alveolar air of dogs. By the introduc- tion of a catheter of a special construction into a branch of a bronchia the corresponding lobe of the lung may be hermetically sealed, while in the other lobes of the same lung and in the other lung the ventilation remains unhindered, so that no stowing of carbon dioxide takes place in the blood. When the cutting off lasts so long that a complete equalization between the gases of the blood and the cut-off air of the lungs is assumed, a sample of this air of the lungs is removed by means of the catheter and analyzed. In the air thus obtained from the lungs Wolffbekg and Ntjssbaum found an average of 3.6^ CO,. Nussbaum has also determined the ' See Zdntz in Hermann's Handbucb, Bd. 4, TKl. 2, S. 105. » Hermann's Handbucb, Bd. 4, Tbl. 2, g. 106. " Pflilger's Arch., Bdd. 5 u. 6. Wien. SitaungSilper., Bd. 33, and Ann*l. d. Chem. u. Pharm., Bd. 108. » Zeitsclir. f. pUysiol. Chem., Bd. 8. » Pfluger's Arcb , Bd. 13. * Ueber die Wirkungen der verdUnnten Luft auf den Organismus. Berlin, 1883, 600 CEEMISTBY OF RESPIRATION. pressure of 378-365 mm. ib was a little diminished, and only at a lowering of the pressure to 300 mm. was a considerable decrease of the oxygen observed. The question how it is possible for man and animals to live for any length of time in high altitudes with a diminished oxygen pressure is important in this connection. In this regard Viault ' has called attention to the fact that the number of blood-corpuscles is greater in such individuals. Thus the llama, according to him, has about 16 million blood-corpuscles per cubic millimetre. By observations on himself and other persons, as well as on animals, YiAULT found the first effect of living in high altitudes is a con- siderable increase in the number of red corpuscles, which in his own case amounted to 5-8 million. The quantity of heemoglobin is on the contrary, according to Viault, increased only in narrow limits on living for some time in the mountains, but the hemo- globin is divided among so many more blood-corpuscles, and there- fore a much greater surface comes in contact with oxygen. Con- trary to the statement of Viault, Muntz ' has found that among the above-mentioned conditions a considerable increase in the quan- tity of iron and haemoglobin in the blood takes place. Eggbe ' found among the influences of high climates a considerable increase in the number of blood-corpuscles as well as in the quantity of hemoglobin, while Koeppe,^ on the contrary, observed a diminution of the latter besides a great increase in the number of blood-cor- puscles. Eegxakd ' has observed a considerable increase in the quantity of haemoglobin in a guinea-pig which was enclosed in a receiver for a whole month with a diminution of pressure corre- sponding to a height of 3000 metres. The tension of the carbon dioxide in the blood has been determined in different ways by Pelugee and his pupils, Wolff- berg," Steassbubg,' and Nussbaum.' According to the aerotono- metric method the blood is allowed to flow directly from the artery ' Compt. rend., Tomes 111, 113 et 114. ' Compt. rend., Tome 113. ' Cited from Maly's Jaliresber., Bd. 33. * Ibid., Bd. 23. ' Compt. rend. Soc. de Biol., 1893. Cited from Centralbl. f. Physiologie, Bd. 7, 1893. « Pflilger's Arch. , Bd. 6. ' Ibid.. Bd. 6. » Ibid.. Bd. 7. CARBON DIOXIDE TENSION IN THE BLOOD. 601 or vein through a glass tube whict contains a gas mixture of a known composition If the tension of the carbon dioxide in the blood is greater than the gas mixture, then the blood gives up carbon dioxide, while in the reverse case it takes up carbon dioxide from the gas mixture. The analysis of the gas mixture after passing the blood through it will also decide if the tension of the carbon dioxide in the blood is greater or less than in the gas mixture; and by a sufl&ciently great number of determinations, especially when the quantity of carbon dioxide of the gas mixture corresponds as nearly as possible in the beginning to the probable tension of this gas in the blood, we may learn the tension of the carbon dioxide in the blood. According to this method the carbon-dioxide tension of the arterial blood is on an average 2.8^ of an atmosphere, corresponding to a pressure of 31 mm. mercury (Steassbubg '). In the blood from the pulmonary alveoli Nussbaum' found a carbon-dioxide tension of 3.81^ of an atmosphere, corresponding to a pressure of 38.95 mm. mercury. Steassburg, who experimented on tracheot- omized dogs in which the ventilation of the lungs was less active and therefore the carbon dioxide was removed from the blood with less readiness, found in the venous blood of the heart a carbon- dioxide tension of bA-jl, of an atmosphere, corresponding to a partial pressure of 41.01 mm. mercury. Another method is the catheterization of a lobe of the lungs (see page 695). In the air thus obtained from the lungs Nussbaum and WOLFFBERG found an average of 3.6^ CO,. Nussbaum, as previously mentioned, has also determined the carbon-dioxide tension in the blood of the pulmonary alveoli in a case simnltaneous with the catheterization of the lungs. He found nearly identical results, namely, a carbon-dioxide tension of 3.84^ and 3.81^. Bohr in his experiments above mentioned (page 596) has arrived at other results in regard to the carbon-dioxide tension. In eleven experiments with inhalation of atmospheric air the carbon- dioxide.tension in the arterial blood varied from to 38 mm. Eg, and in five experiments with inhalation of air containing carbon dioxide from 0.9 to 57.8 mm. Hg. A comparison of the carbon-dioxide tension in the blood with the bifurcated air gave in several cases a greater carbon-dioxide pressure in the air of the lungs than in the blood, and as maximum this difference amounted to 17.2 mm. in favor of the air of the lungs in the experiments with inhalation of ' L. c. ' L. c. 602 03P!MIBTR7 OF RESPIRATION. atmospheric air. As the alveolar air is richer in carbon dioxide than the bifurcated air, this experiment unquestionably proves, according to Bohe, that the carbon dioxide has migrated against the high pressure. In opposition to these investigations, Fredebicq ' in his above- mentioned experiments, obtained the same figures for the carbon- dioxide tension in arterial peptone blood as Pflugee and his pupils found for normal blood. The low figures obtained by Bohe for the carbon-dioxide tension appear remarkable when we recall that Geandis " found in peptone blood which Lahousse ' and Blach- STEIN * had shown was poor in carbon dioxide a high carbon-dioxide tension. A certain importance has been ascribed to oxygen in regard to the elimination of carbon dioxide in the lungs, in that it has an expelling action on the carbon dioxide from its combinations in the blood. This statement, first made by Holmgeen,' has recently found an advocate in Weeigo.' This investigator has made ingenious experiments on living animals in which he allows both lungs of the animal to breathe separately, the one with hydrogen and the other with pure oxygen or a gas mixture rich in oxygen. He invariably found a greater carbon-dioxide tension in the air sucked from the lungs in the presence of oxygen, and he draws the con- clusion from his experiments that the oxygen passing from the lung alveoli into the blood raises the carbon-dioxide tension. According to "Wbrigo, by this action the oxygen is a powerful factor in the elimination of carbon dioxide, and therefore it is not necessary to assume a specific action of the lung itself in these processes. Ztjntz ' has suggested important objections to the investigations of Weeigo, but they have not been substantiated by experiment; hence the question is still open. Also in regard to the elimination of carbon dioxide in the lungs we have no striking reason for abandoning the commdn view that the carbon dioxide simply passes from the blood into the air of the lungs according to the laws of diffusion. ' Centralbl. f. Physiol., Bd. 7. • Du Bois-Reymond's Arch., 1891, S. 499. > Ibid., 1889, S. 77. *md., 1891, S. 394. ' Wien. Sitzungsber. , Math.-nat. Klasse, Bd. ^. « Pflttger's Arch., Bdd. 51 u. 53. ' Ibid.. Bd. 53. INTERNAL RESPIRATION. 603 From what has been said above (page 593) in regard to the internal respiration we derive that it consists chiefly in that in the capillaries the oxygen passes from the blood into the tissues, while the carbon dioxide passes from the tissues into the blood. The assertion of Estor and Saint Pieree ' that the quantity of oxygen in the blood of the arteries decreases with the remoteness from the heart has been shown as incorrect by PFLiroEE,' and the oxygen tension in the blood on entering the capillaries must be higher. As compared with the capillaries the tissues are to be con- sidered as nearly or entirely free from oxygen, and in regard to the oxygen a considerable difference in pressure must exist between the blood and tissues. The possibility that this difference in pressure is sufficient to supply the tissues with the necessary quantity of oxygen is hardly to be doubted. In regard to the carbon-dioxide tension in the tissue we must assume a priori that it is higher than in the blood. This is found to be true. Steassbueg ' found in the urine of dogs and in the bile a carbon-dioxide tension of 9j^ and 7^ of an . atmosphere, respectively. The same experimenter has, further, injected atmos- pheric air into a ligatured portion of the intestine of a living dog and analyzed the air taken out after some time. He found a carbon-dioxide tension of 7.7^ of an atmosphere. The carbon- dioxide tension in the tissues is considerably greater than in the venous blood, and there is no objection to the view that the carbon dioxide simply diffuses from the tissues to the blood according to the laws of diffusion. That a true secretion of gases occurs in animals follows from the composi- tion and beliavior of the gases in the swimming-bladder of fishes. These gases consist of oxygen and nitrogen with only small quantities of carbon dioxide. In fishes which do not live at any great depth the quantity of oxygen is ordinarily as high as in the atmosphtre, while fishes which live at great depths may, according to BiOT and others,* contain considerably more oxy- gen and even above 80^. MOBEA0'has also found that after emptying the swimming-bladder by means of a trocar new air collected after a time, and this air was richer in oxygen than the atmosplieiic air and contained even 85% oxvgen B0HR,« who has proved and confirmed these statements, also found that this collection is under the influence of the nervous system, because on • Joam. de I'anat. de la physiol.. Tome 2, 1865. ' PflUger's Arch., Bd. 1. ' Pflilger's Arch., Bd. 6. • See Hermann's Handb., Bd. 4, Thl. 2, S. 151. • Compt. rend.. Tome 57, S. 37 u. 816. » Journ. of Physiol., Vol. 15. See also Hiifner, Du Bois-Reymond's Arch., 1892, 604 CHEMISTRY OF RESPIRATION. the section of certain branches of pneumogastric nerve it is discontinued. It is beyond dispute that we have here a secretion and not a diffusion of oxygen. , Several methods have been suggested for the study of the quantitative relationship of the respiratory exchange of gas. We must refer the reader to other text-books for more details of these methods, and we will only here mention the chief traits of the most important methods. Rbgnault and Rbiset's Method. According to this method the animal or person experimented upon is allowed to breathe in an enclosed space. The car- bon dioxide is removed from the air, as it forms, by strong caustic alkali, from which the quantity may be determined, while the oxygen is replaced continu- ally by exactly measured quantities. This method, which also makes possible a direct determination of the oxygen used as well as the carbon dioxide pro- duced, has since been modified by other investigators, such as Ppluqee and his pupils, Seegen and Nowak, and Hoppe-Seyjlbr.' Pettkukopbe's Method.^ According to this method the individual to be experimented upon breathes in a room through which a current of atmospheric air is passed. The quantity of air passed through is carefully measured. As it is impossible to analyze all the air made to pass through the chamber, a small fraction of this air is diverted into a branch line during the entire experiment, carefully measured, and the quantity of carbon dioxide and water determined. From the composition of this air the quantity of water and carbon dioxide contained in the large quantity of air made to pass through the cham- ber can be calculated. The consumption of oxygen cannot be directly deter- mined in this method, but may be indirectly by difference, which is a defect in this method. Speck's Method.' For briefer experiments on man Speck has used the following: He breathes into two spirometer receivers on which the gas volume can be read off very accurately, through a mouthpiece with two valves, closing the nose with a clamp. The air from one of the spirometers is inhaled through one valve, and the expired air passes through the other into the other spirometer. By means of a rubber tube connected with the expiration tube an accurately measured part of the expired air may be passed into an ab- sorption tulie and analyzed. ZuNTZ and Qeppbrt's Metliod.* This method, which has been improve 1 by Ztjntz and his pupils from time to time, consists in the following: The individual being experimented upon inspires pure atmospheric air through a very wide feed-pipe leading from the open air, the inspired and the expired air being separated by two valves (human subjects breathe with closed nose by means of a soft rubber mouthpiece, animals through an air-tight tracheal canula). The volume of the expired air is measured by a gas-meter, and an aliquot part of this air collected and the quantity of carbon dioxide and oxygen determined. As the composition of the atmospheric air can be considered as constant within a certain limit, the production of carbon dioxide as well as the consumption of oxygen may be readily calculated (see the works of Zuntz and his pupils). . Hanriot and Kichet's method ' is characterized by its simplicity. These ' In regard to this method see Zuntz in Hermann's Handb. , Bd. 4, Thl. 3 and Hoppe-Seyler in Zeitschr. f. physiol. Chem., Bd. 19. ' See Zuntz, 1. c. ' Speck, Physiologie des mensohlichen Athmens. Leipzig, 1893. ■* Pflilger's Arch., Bd. 42. See also Magnus-Levy in Pflilger's Arch. Bd. 55 S. 10, in which the work of Zuntz and his pupils is cited. ? Compt. rend., Tome 104. LUNG 8 AND rilEIR EXPEOTOItATIONS. 605 investigators allow the total air to pass tlirouffh tliree gas-meters, one after the other. The first measures the inspired air, whose composition is known. The second gas-meter measures the expired air, and the third the quantity of the expired air after the carbon dioxide has been removed by a suitable appa- ratus. The quantity of carbon dioxide produced and the oxygen consumed can be readily calculated from these data. Appendix. The Lungs and their Expectorations. Besides proteid bodies and the albuminoids of the connective substance group, lecithin, taurin (especially in ox-lnngs), iXricacid, and inosit have been found in the lungs. Poulet ' claims to have found a special acid, which he has called pul mo tartaric acid, in the lung tissue. Glycogen occurs abundantly in the embryonic lung, but is absent in the adult lung. The black or dark brown pigment in the lungs of human beings and domestic animals consists chiefly of carbon, which originates from the soot in the air. The pigment may in part also consist of melanin. Besides carbon, other bodies, such as iron oxide, silicic acid, and clay, may be deposited in the lungs, being inhaled as dust. Among the bodies found in the lungs under pathological condi- tions we must specially mention albumoses and peptones (in pneu- monia and suppuration), glycogen, a faintly dextrorotatory carbohydrate difEering from glycogen found by Pouchbt' in consumptives, and finally also cellulose, which, according to Feeund,' occurs in the lungs, blood, and pus of persons with tuberculosis. C. W. Schmidt * found in 1000 grms. mineral bodies from the normal'hnman lung the following: NaCl 130, K,0 13, Na^O 195, CaO 19, MgO 19, Fe.O, 32, P,0, 485, SO, 8, and sand 134 grms. According to Oidtmann ' the lungs of a l4-day-old child contained 796.05 p. m. water, 198.19 p. m. organic bodies, and 6.76 p. m. inorganic bodies. The expectoration is a mixture of the mucous secretion of the respiratory passages, of saliva and buccal mucus. Because of this its composition is very variable, especially under pathological con- ' Cited from Maly's Jahresber., Bd. 18, S. 248. « Compt. rend., Bd. 96. » Wien. med. Jahrb., 1886. Cited from Maly's Jahresber., Bd. 16, S. 471. * Cited from v. Oorup-Besanez, Lehrbuch, 4. Aufl., S. 727. ' Ibid. , S. 732. 606 CHEMI8TRY OF RESPIRATION. ditions when various products mix with it. The chemical constit- uents are, besides the mineral substances, chiefly mucin with a little proteid and nuolein substance. Under pathological conditions albumoses and peptones, volatile fatty acids, glycogen, Charcot's crystals, and also crystals of cholesterin, hsematoidin, tyrosin, fat, and fatty acids, triple phosphates, etc., have been found. The form constituents are, under physiological circumstances, epithelium-cells of various kinds, leucocytes, sometimes also red blood-corpuscles and various kinds of fungi. In pathological con- ditions elastic fibres, spiral formations consisting of a muciu-like substance, fibrin coagulum, pus, pathogenic microbes of various kinds, and the above-mentioned crystals occur. CHAPTER XVIII. METABOLISM' WITH VARIOUS FQODS, AND THE DEMAND FOR FOOD IN MAN. The conversion of chemical tension into living energy, which characterizes animal life, leads, as previously stated in Chapter I, to the formation of relatively simple compounds — carbon dioxide, urea, etc. — which leave the organism, and which, moreover, being very poor in potential energy, are for this reason of no or very little value for the body. It is therefore absolutely necessary for the con- tinuance of life and the normal course of the functions of the body that the organism and its different tissues should be supplied with new material to replace that which has been exhausted. This is accomplished by means of food. Those bodies are designated as food which have no injurious action upon the organism and which replace those constituents of the body that have been consumed in metabolism or that can prevent or diminish the consumption of such constituents. Among the numerous dissimilar substances which man and ani- mals take with the food all cannot be equally necessary or have the same value. Some perhaps are unnecessary, while others may be indispensable. We have learned by direct observation and a wide experience that besides the oxygen, which is necessary for oxida- tion, the essential foods for animals in general, and for man espe- cially, are water, mineral bodies, proteids, carioliydrates, and fats. It is also apparent that the various groups of food-stuffs neces- sary for the tissues and organs must be of different importance ; ; thus, for instance, water and the mineral bodies have another value than the organic foods, and theSe again must vary in importance ■ The translator will use in th& following pages for tlie German word " Btoffwechsel" Dr. Burdon-Sanderson's translation (SjUabus of Lectures, IS^), eichaitge (^ nloUriat, add at the same time the more general terdl " metabolism." 607 608 • METABOLISM. among themselves. The knowledge of the action of various nutri- tive bodies on the exchange of material from a qualitative as well as a quantitative point of view must be of fundamental importance in determining the value of difEerent nutritive substances relative to the demands of the body for food under various conditions and also in deciding many other questions — for instance, the proper nutrition for an individual in health and in disease. Such knowledge can only be attained by a series of systematic and thorough observations, in which the quantity of nutritive ma- terial, relative to the weight of the body, taken and absorbed in a given time is compared with the quantity of final metabolic products which leave the organism at the same time. Researches of this kind have been made by several investigators, but above all should be mentioned those made by Bischoff and VoiT, by Pettjenkofer and VoiT, and by VoiT and his pupils. It is absolutely necessary in researches on the exchange of material to be able to collect, analyze, and quantitatively estimate the excreta of the organism, so that tliey may be compared with the quantity and composition of the nutritive bodies taken up. In the first place, one must know what the habitual excreta of the body are and in what way these bodies leave the organism. One must also have trustworthy methods for the quantitative estimation of the same. The Organism may, under physiological conditions, be exposed to accidental or periodic losses of valuable material — such losses as only occur in certain individuals, or in the same individual only at a certain period ; for instance, the secretion of milk, the production of eggs, the ejection of semen or menstrual blood. It is therefore apparent that these losses can only be the subject of investigation and estimation in special cases. The regular and constant excreta of the organism are of the very greatest importance in the study of metabolism. To these belong, in the first place, the true final metabolic products — caebok diox- iDe, urea (uric acid, hippuric acid, creatinin, and other urinarv constitijei^ts), and a part of the water. The remainder of the water, the mineral bodies, and those secretions or tissue-constituents MUCUS,' DIGESTIVE FLUIDS, SEBUM, SWEAT, and EPIDERMIS FOR- MATIONS— which are either poured into the intestinal tract, or se- creted from the surface of the body, or broken off and thereby lost for the body, also belong to the constant excreta. EXCRETA OP THE ANIMAL BODY. ' (509 The remains of food, sometimes indigestible, sometimes digest- ible but not acted upon, contained in the faeces, which vary con- siderably in quantity and composition with the nature of the food, also belong i.o the excreta of the organism. Even though these remains, which are never absorbed and therefore are never consti- tuents of the animal fluids or tissues, cannot be considered as excreta of the body in a strict sense, still their quantitative esti- mation is absolutely necessary in certain experiments on the exchange of material. The determination of the constant loss is in some cases accom- panied with the greatest diflBculties. The loss from the detached epidermis, from the secretion of the sebaceous glands, etc., cannot be determined with exactness without difficulty, and therefore — as. they do not occasion any mentionable loss because of their small quantity — they need not be considered in quantitative experiments on metabolism. This also applies to the constituents of the mucus, bile, pancreatic and intestinal juices, etc., occurring in the contents of the intestine, and which, leaving the body with the faeces, cannot be separated from the other contents of the intestine and thereforei cannot be quantitatively determined separately. The uncertainty which, because of the intimated difficulties, attaches itself to the results of the experiments is very small as compared to the variation which is caused by different individualities, different modes of liv- ing, different foods, etc. No general but only approximate values- can therefore be given for the constant excreta of the human body. The following figures represent the quantity of excreta for 34 hours of a grown man weighing 60-70 kilos on a mixed diet. The numbers are compiled from the results of different investigators. Q-ramniPs. Water 2500-3500 Salts (with the urine) 20-30 Clarbon dioxide 750-900 Urea _. 20-40 Other nitrogenous urinary constituents 2-5 SoliJs in the excrements 30-50 These total excreta are approximately divided among the various excretions in the following way — but still it must not be forgotten that this division may vary to a great extent under various external circumstances : by kespiration about 32^, by the evapobatiok FEOM THE SKIN 17^, with the URiKE 46-47^, and with the excre- ments 5-9j^. The elimination by the skin and lungs, which is sometimes differentiated by the name " perspieatio insensibtlis " 610 METABOLISM. from the visible elimination by the kidneys and intestine, is on an average about 50^ of the total elimination. This proportion, only ■quoted relatively, is subject to considerable variation, because of the great difference in the loss of water through the skin and kid- neys under different circumstances. About 90^ of the water in carnivora is excreted through the iidneys. In herbivora 60j^ of the water is eliminated by the ex- crements, which are 30-50^ of the total excreta. In man only a smaller fraction of the water (about 5^) is eliminated with the faeces, and the great mass of the water is divided between the kid- neys, lungs, and skin. The nitrogenous constituents of the excretions consist chiefly of urea, or uric acid in certain animals, and the other nitrogenous urinary constituents. A disproportionally large part of the nitrogen leaves the body with the urine, and, as the nitrogenous constitu- ents of this excretion are final products of the metabolism of pro- teids in the organism, the quantity of proteids transformed in the body may be easily calculated by multiplying the quantity of nitrogen in the urine by the coefficient 6.25 (-ij^y- = 6.35), if we admit that the proteids contain in round numbers 16^ nitrogen. Still another question is whether the nitrogen leaves the body only with the urine or by other channels. This last is habitually J;he case. The discharges from the intestine always contain some nitrogen which has a twofold origin. A part of this nitrogen de- pends upoii undigested or non-absorbed remnants of food, and another part on the non-absorbed remains of digestive secretions — bile, pancreatic juice, intestinal mucus — and of epithelium -cells of the mucous membrane. It follows that a part of the nitrogen of faeces has this last-mentioned origin from the fact that discharges from the intestine occur also in complete inanition. If the question to be decided is, how much of the nitrogenous todies is assimilated in certain modes of nutrition or after taking a certain quantity of food, then naturally the quantity of nitrogen originating from the food and leavihg the body with the excre- ments must be subtracted from the total quantity of nitrogen taken up with the food. To obtain the quantity of nitrogen leaving the body with the excrements it is necessary to subtract from the total quantity of nitrogen of the excrements the quantity of nitrogea coming from the digestive tract itself and its secretions, and the amount of this last must be known. ELIMINATION OF NITROGEN. 611 It is obvious that exact results which answer for all times can- not be given for that part of the nitrogen which has its origin in the digestive canal and fluids. It may not only vary in different individuals, but also in the same individual after more or less active secretion and absorption. In the attempts made to determine this part of the nitrogen of the excrements it has been found that in man, on non-nitrogenous or nearly nitrogen-free food, it amounts in round numbers to somewhat less than 1 grm. per 24 hours (KiEDER,' RuBNEE.') During starvation, in which a smaller quantity of digestive secretions is eliminated, it is less. Mullee ' found in his observations on the faster Cetti that only 0.3 grm. nitrogen was derived from the intestinal canal. Nitrogen also leaves the body through the horn formation. The quantity which is lost in this manner is, though it cannot be ex- actly determined, insignificant. Man loses only about 0.03 grm. nitrogen daily by means of the hair and nails (Moleschott*) and about 0.3-0.5 grm. by the scaling off of the skin. The quantity of nitrogen which leaves the body under ordinary circumstances by the perspiration must be so small that, like the loss by the horny structure, it need not be considered in experiments on the exchange of material. The elimination of nitrogen by the perspiration need only be considered in cases of profuse sweating. The view was formerly held that in man and carnivora an elim- ination of gaseous nitrogen took place through the skin and lungs, and because of this, on comparing the nitrogen of the food with that of the urine and faeces, a nitrogen deficit occurred in the vis-' ible elimination. This question has been the subject of much discussion and of numerous investigations. The conclusion has been drawn from the respiration researches of Regnault and Reiset' that also an exhalation of nitrogen takes place. Seegen and Nowak ' especially have recently endeavored to prove the correctness of this con- clusion. Such an experiment is, however, accompanied with so ' Zeitscbr. f. Biologie, Bd. 20. * Ibid., Bd. 15. » Berl. klin. Wocbenschr., 1887. * Untersuch. zur Naturlebre, Bd. 12. ' Aunal. d. Chim. et Phys. (3), Tome 27, and Annal. d. Chem. a. Pbann.. Bd. 73. * Wien. Sitzungsber., Bd. 71, and Pflilger's Arch., Bd. 25. 612 METABOLISM. many difficulties, and there are so many sources of error, that it can scarcely be considered as conclusive. In fact, Pettenkofer and VoiT ' have demonstrated the existence of errors in the experi- ments of Seegen and Nowak. On the other hand, Pfluger and Leo ' have found no appreciable exhalation of nitrogen in rabbits." Also many investigators, especially Pettenkobek and VoiT,' have shown by experiments on man and animals that with the proper quantity and quality of food we can bring the body into nitroge- nous equilibrium, in which the quantity of nitrogen voided with the urine and fseces is equal or nearly equal to the quantity con- tained in the food. The experiments made by Gkuber in Voit's institute seem to be especially conclusive on this point. Geubee ' fed a dog seven- teen days on meat which in all contained 368.53 grms. nitrogen, and he found in the same time 368.28 grms. nitrogen in the urine and faeces. In later experiments ' he found a difference varying only between — 0.21^ and -(- 1^. From this and other experiments ■we may conclude with VoiT that a deficit of nitrogen does not exist; or it is so insignificant that in experiments upon metabolism it need not be considered. In investigations on the metabolism of proteids in the body, ordinarily, it is only necessary to consider the nitrogen of the urine and faeces, but it must be remarked that the nitrogen of the urine is a measure of the extent of the metabolism of the proteids in the body, while the nitrogen of the faeces (after deducting somewhat less than 1 grm. on mixed diet) is a measure of the non-absorbed part of the nitrogen of the food. In the oxidation of the proteids in the organism their sulphur is oxidized into sulphuric acid, and on this depends the fact that the elimination of sulphuric acid by the urine, which in man is only to a small extent derived from the sulphates of the food, makes nearly equal variations as the elimination of nitrogen by the urine. If we consider the amount of nitrogen and sulphur in the proteids as 165^ and 1^ respectively, then the proportion between the nitrogen of the proteids and the sulphuric acid, H,SO„ produced by their ' Zeitschr. f. Biologie, Bd. 16. ' Pflager's Arch. , Bd. 26. ' Zuntz und Tacke, Maly's Jahresber. , Bd. 16, S. 361. * See Voit' in Hermann's Handbucb, Bd. 6, TL. 1. » Zeitschr. f. Biologie, Bd. 16. « Ibid. ; Bd. 19. CALCULATING THE EXTENT OF METABOLISM. 6 13 burning is in the ratio 5.2 : 1, or about the same as in the Urine (see page 515). The determination of the quantity of sulphuric acid eliminated with the urine gives us an important means of con- trolling the extent of the transformation of proteids, and such a control is especially important in cases in which we wish to study the action of certain nitrogenous non-albuminous bodies on the metabolism of proteids. A determination of the nitrogen alone is not sufficient in such cases. If it is found, on comparing the nitrogen of the food with that ■of the urine and faeces, that there is an excess of the first, this means that the body has increased its stock of nitrogenous substances — proteids. If, on the contrary, the urine and faeces contain more nitrogen than the food taken at the same time, this denotes that the body is giving up part of its nitrogen — that is, a part of its own proteids has been decomposed. We can, from the quantity of ni- trogen, as above stated, calculate the corresponding quantity of pro- teids by mutiplying by 6.25. Usually, according toVoiT's propo- sition, the nitrogen of the urine is not calculated as decomposed proteids, but as decomposed muscle-substance or flesh. Lean meat contains on an average about 3.4^ nitrogen; hence each gramme of nitrogen of the urine corresponds in round numbers to about 30 grms. flesh. The assumption that lean meat contains 3.4j^ nitrogen is arbitrary, as specially shown by Ppltjgek,' and the relationship of N : C in the proteids of dried meat, which is of great importance in certain experiments on metabolism, is given different by various experimenters, namely, 1 : 3.22 — 1 : 3.68. Aegutinsky ' found in ox-flesh, after complete removal of fat and subtraction of glycogen, that the relationship was 1 : 3.34. A disproportionally large part of the carbon leaves the body as carbon dioxide, which escapes chiefly through the lungs and skin. The remainder of the carbon is eliminated in the form of organic combinations by the urine and faeces, in which the quantity of car- bon can be determined by elementary analysis. The quantity of gaseous carbon dioxide eliminated may be determined by means of Pettenkofek's respiration apparatus, or by other methods as de- scribed in the preceding chapter. By multiplying the quantity of carbon dioxide found by 0.273 we obtain the quantity of carbon eliminated as CO,. If we compare the total quantity of carbon > Pfltiger's Arch., Bd. 51, S 339. « Ibid. , Bd. 55. 614 METABOLISM. eliminated in various ways with the carbon contained in the food we obtain some idea as to the transformation of the carbon compounds. If the quantity of carbon in the food is greater than in the excreta, then the excess is deposited; while if the reverse be the case it shows a corresponding loss of bodily substance. The nature of the substances here deposited or lost, whether they consist of proteids, fats, or carbohydrates, is learned from the total quantity of nitrogen of the excretions. The corresponding quan- tity of proteids may be calculated from the quantity of nitrogen, and, as the average quantity of carbon in the proteids is known, th& quantity of carbon which corresponds to the decomposed proteids may be easily ascertained. If the quantity of carbon thus found i& smaller than the quantity of the total carbon in the excreta it is then obvious that some other nitrogen-free substance has been con- sumed besides the proteids. If the quantity of carbon in the pro- teids is considered in round numbers as 53^,' then the relation between carbon (53) and nitrogen (16) is as 3.3 : 1. If we multi- ply the total quantity of nitrogen eliminated by 3.3 the excess of carbon in the eliminations over the product found represents the carbon of the decomposed non-nitrogenous compounds. For in- stance, in the case of a person experimented upon, 10 grms. nitro- gen and 200 grms. carbon were eliminated in the course of 24 hours ; then these 62.5 grms. proteid correspond to 33 grms. carbon, and the difference, 200 — (3.3 X 10) = 167, represents the quantity of carbon in the decomposed non-nitrogenous compounds. If we start from the simplest case, starvation, where the body lives at the expense of its own substance, then, since the quantity of carbohy- drates as compared to the fats of the body is extremely small, in such cases in order to avoid mistakes the assumption must be made that the person experimented upon has only used fat and proteids. As animal fat contains on an average 76.5^ carbon, the quantity of transformed fat may be calculated by multiplying the carbon by — - = 1.3. In the case of the above example the person experi- mented upon would have used 62.5 grms. proteids and 167 X 1.3- = 217 grms. fat of his own body in the course of the 24 hours. Starting from the nitrogen balance, we can calculate in the same- way whether an excess of carbon in the food as compared with the quantity of carbon in the excreta is retained by the body as pro- ' This figure is perhaps a little too high. RESPTRATORY QUOTIENT. 615 teids or fat or as both. On the other hand, with an excess of carbon in the excreta we can calculate how much of the loss of the substance of the body is due to a consumption of the proteids or of fat or of both. The quantity of water and mineral bodies voided with the urine and faeces can easily be determined. The quantity of water elimi- nated by the skin and lungs may be directly determined by means of Pettenkofek's apparatus. The quantity of oxygen taken up is calculated as the difference between the weight of the individual before the experiment plus all the directly determined substances taken in, and the final weight of the individual plus all his excreta. The oxygen may, according to the methods given in the pre- ceding chapter, be directly determined, and such a determination with the simultaneous estimation of the carbon dioxide eliminated is of great importance in the study of metabolism. On comparing the inspired and the expired air we learn, on meas- uring them when dry and at the same temperature and pressure, that the volume of the expired air is less than that of the inspired air. This depends upon the fact that not all of the oxygen appears again in the expired air as carbon dioxide, because it is not only used in the oxidation of carbon, but also in part in the formation of water, sul- phuric acid, and other bodies. The volume of expired carbon dioxide is regularly less than the volume of the inspired oxygen, and the CO" relation — =r-, which is called the respiratory quotient, is generally less than 1. The magnitude of the respiratory quotient is dependent upon the kind of substances destroyed in the body. In the combustion of pure carbon one volume of oxygen yields one volume of carbon dioxide, and the quotient is therefore equal to 1. The same is true in the burning of carbohydrates, and in the exclusive decomposition of carbohydrates in the animal body the respiratory quotient must be approximately 1. In exclusive metabolism of proteids it is 0.73, and with the decomposition of fat it is 0.7. In starvation, as the animal draws on its own flesh and fat, the respiratory quotient must be a close approach to the latter figure. The respiratory quotient therefore gives important explanations on the quality of the material decomposed in the body, naturally with the supposition that the elimination of carbon dioxide, independent of the formation of carbon dioxide, is not infiuenced by special conditions, such as alternation of the respiratory mechanism. 6 1 6 METABOLISM. It is also possible in systematized experimentation so to carry on the metabolism experiments that the decomposition material of the body — as shown by the respiratory quotient — remains qualitatively the same, at least for a short time. In such experiments it has been shown, especially by Zuntz ' and his pupils, that the extent of oxygen consumption may be taken as a measure for the action of different influences on the extent of metabolism. This possibility is based on the fact proven by Pfluger ' and his pupils, and by VoiT,' that the consumption of oxygen within wide limits is inde- pendent of the supply of oxygen, and is exclusively dependent upon the oxygen demand of the tissues. For certain reasons ' the con- sumption of oxygen gives indeed a better conclusion than the elimination of carbon dioxide as to the extent of exchange of material and energy ; but as the same quantity of oxygen (100 grms. ) con- sumes different quantities of fat, carbohydrates, and proteids in the body — namely, 35, 84.4, and 74.4 grms. respectively — the respira- tory quotient must also be determined, in order to ascertain the nature of the substance burnt in the body, by the simultaneous determination of the elimination of carbon dioxide. I, Potential Energy and the Relative Nutritive Value of Various Organic Foods. With the organic foods the organism receives a supply of poten- tial energy which is converted into living force in the body. This potential energy of the various foods may be represented by the amount of heat which is set free in their combustion. This quan- tity of heat is expressed as calories, and a small calorie is the quan- tity of heat necessary to warm 1 grm. water from 0° to 1° C. A large calorie is the quantity of heat necessary to warm 1 kilo water 1° (3. Here and in the following pages large calories are to be under- stood. We have numerous investigations by different experimenters, such as Fbankland, Dawilewski, Eubster,' Bekthblot,°Stoh- ' See Chapter XVII, foot-note 4, p. 604. « Pflttger's Arcli., Bdd. 6, 10 u. 14 ; Finkler, ihid., Bd. 10 ; Pinkler and Oertmann, ibid., Bd. 14. > Zeitscbr. f. Biologie, Bdd. 11 u. 14. ■• See Ad. Magnus-Levy, Pflilger's Arch., Bd. 55, S. 7. ^ Zeitschr. f. Biologie, Bd. 31, where the works of Frankland and Danilewski are cited. • Compt. rend., Tomes 109, 104, 110. OALOBIFIO VALUE OF FOODS. 617 MANX,' and others, on tlie calorific value of different foods. The following results, which represent the calorific value of a. few nutri- tive bodies on complete combustion outside of the body to the highest oxidation products, are taken from Stohmann's' latest work; „ . Calories. Casein 5 86 Ovalbumin 5 74 Conglutin 5 48 Proteid (average) g.71 Animal tissue-fat , 9 5q Butter- fat 923 Cane-sugar .'... s'gg Lactose 3 95 Dextrose 3.74 Siaich 4.19 Fat and carbohydrates are completely burnt in the body, and we can therefore consider their combustion equivalent as a measure of the living force developed by them within the organism. We generally designate 9.3 and 4.1 calories for each grm. of sub- stance as the average for the physiological calorific value of fats and carbohydrates respectively. Tlie proteids act differently from the fats and carbohydrates. They are only incompletely burnt, and they yield certain decom- position products, which, leaving the body with the excreta, still represent a certain quantity of potential energy which is lost for the body. The heat of combustion of the proteids is smaller within the organism than outside of it, and they must therefore be specially determined. For this purpose Efbneb ' fed a dog on washed meat, and he subtracted from the heat of combustion of the food the heat of combustion of the urine and faeces, which corresponded to the food taken plus the quantity of heat necessary for the swelling up of the proteids and the solution of the urea. Eubnee has also tried to determine the heat of combustion of the proteids (muscle-pro- teids) decomposed in the body of rabbits in starvation. According to these investigations, the physiological heat of combustion in cal- ories for each gramme of substance is as follows : 1 Rrm. of the Dry Substance. Calories. Proteids from meat 4.4 Muscle 4.0 Proteids in starvation 3.8 Fat (average for various fats) 9.3 Carbohydrates (calculated average) 4.1 ' Zeitschr. f. Biologie, Bd. 31. ' L. c. » Zeitschr. f. Biologie, Bd. 21. 618 METABOLISM. The physiological combustion value of the various foods belong- ing to the. same group is not quite the same. It is, for instance, 3.97 calories for a vegetable proteid, conglutin, and 4.43 calories for an animal proteid body, syntonin. According to Eubnee we may consider the normal heat value per 1 grm. of animal proteid as 4.23 calories, and of vegetable proteid as 3.96 cal- ories. When a person on a mixed diet takes about 60^ of the proteids from animal foods and about 40^ from vegetable foods, we may consider the value of 1 grm. of the proteid of the food as about 4.1 calories. The physiological value of each of the three chief groups of organic foods, by their decomposition in the body, is in round numbers as follows : Calories. 1 grm. proteid 4. 1 1 " fat 9.3 1 ' ' carboliydrate 4. 1 As will be shown, the fats and carbohydrates may decrease the metabolism of proteids in the body, while, on the other hand, the quantity of proteids in the body or in the food acts on the meta- bolism of fat in the body. In physiological combustion the various foods may replace one another to a certain extent, and it is there- fore important to know in what proportion they can replace one another. The investigations made by Rubstee have taught that this, if it relates to the force and heat production in the animal body, is in proportions that correspond with the figures of the heat value of the same. This is apparent from the following table. In this we find the weight of the various foods equal to 100 grms. fat, a part determined from experiments on animals and a part calcu- lated from figures of the heat values. Table 1. 100 grms. fat are equal to or isodynamic with: From Experiments From the Difference, on Animals. Heat Value. percent. Syntonin 325 213 -j^ 5.6 Muscle-flesli (dried) 243 235 -j- 4.3 Starcli 232 229 -|- 1.3 Cane-sugar 234 335 -0 Grape-sugar 356 253 —0 Prom the given isodynamic value of the various foods it follows that these substances replace one another in the body almost in exact ratio to the potential energy contained in them. Thus in round numbers 237 grms. proteid and carbohydrate are equal to or METAB0U8M IN STARVATION. 619 isodynamic with 100 grms. fat in regard to source of energy, because each yields 930 calories on combustion in the body. By means of recent very important calorimetric investigations RuBNER ' has shown that the heat produced in an animal in several series of experiments extending over 45 days corresponded to within 0.47$^ with the physiological heat of combustion calculated from the decomposed body and foods. This isodynamic law is of fundamental value in the study of meta- bolism and nutrition. By this law it is possible to consider the processes of metabolism as more uniform. The quantity of energy in the foods may be used as a measure for the total consumption of energy, and the knowledge of the quantity of energy in the foods must also be the basis for the calculation of dietaries for human beings under various conditions. II. Metabolism in Starvation. In starvation the decomposition in the body continues uninter- ruptedly, though with decreased intensity ; but, as it takes place at the expense of the substance of the body, it can only continue for a limited time. When an animal has lost a certain fraction of the mass of the body death is the result. This fraction varies with the condition of the body at the beginning of the starvation period. Fat animals succumb when the weight of the body has sunk to ^ of the original weight. Otherwise, according to Chossat," animals die as a rule when the weight of the body has sunk to | of the orig- inal weight. The period when death occurs from starvation not only varies with the varied nutritive condition at the beginning of starvation, but also with the more or less active exchange of material. This is more active in small and young animals than in large and older ones, but different classes of animals show an un- equal activity. Children succumb in starvation in 3-5 days after having lost \ of their bodily mass. Grown persons, as observed on Succi,' may starve for 20 days without lasting injury ; and we have statements of even over 40-50 days' starvation. Dogs can live witliout food from 4-8 weeks, birds 5-20 days, snakes more than half a year, and frogs more than a year. • Zeitscbr. f. Biologie, Bd. 30. ' Cited from Voit in Hermann's Handbuch, Bd. 6, Thl. 1, S. 100. « See Luciani, Das Hungern. Hamburg u. Leipzig, 1890. 620 METABOLISM. In starvation the iveight of the body decreases. The loss of weight is greatest in the first few days, and then decreases rather uniformly. In small animals the absolute loss of weight per day is naturally smaller than in larger animals. The relative loss of weight — that is, the loss of weight of the unit of the weight of the body, namely, 1 kilo — is, on the contrary, greater in small animals than in larger ones. The reason for this is that the smaller animals have a greater surface of body in proportion to their mass than larger animals, and the greater loss of heat caused thereby must be replaced by a more active consumption of material. It follows from the decrease in the weight of the body that the absolute extent of metabolism must diminish in starvation. If, on the contrary, we refer the extent of the metabolism to the unit of the weight of the body, namely, 1 kilo, we find that this quantity re- mains nearly unchanged during starvation. The investigations of ZuNTZ, LBHMAiTif, and others ' on Cetti showed on the 3d to 6th day of starvation an average consumption 4.65 c. c. oxygen per kilo in one minute and on the 9th to 11th day an average of 4.73 c. c. The calories, as a measure of the metabolism, fell on the 1st to 5th day of starvation from 1850 to 1600 calories, or from 32.4 to 30 per kilo, and he remained nearly unchanged, if we refer to the unit of bodily weight. As the metabolism in starvation takes place at the expense of the constituents of the body, it must take place in essentially the same way in both carnivora and herbivora. As the food of the herbivora is ordinarily richer in carbohydrates and non-nitrogenous nutritive bodies than that of the carnivora, so in starvation the body of the herbivora becomes relatively richer in proteids. On thjs account the elimination of nitrogen is increased in herbivora in the first part of the period of starvation. In carnivora the elimination of nitrogen decreases, as a rule, immediately at the beginning of the starvation, and in the later periods only small quantities of nitrogen are voided by herbivora as well as by carnivora. The extent of the metabolism of proteids, or the elimination of nitrogen by the urine, which is a measure for the same, does not show in carnivora any uniform decrease during the entire period of starvation. During the first few days the elimination of nitrogen is greatest, and the quantity of the same depends essentially upon the amount of proteids in the organism and the nature of the food ' Berlin, klin. Wochenschr., 1887. METABOLISM IN STARYAriON. fi21 previously taken. The richer the body is in proteids from the food previously taken the greater is the metabolism of proteids, or, in other words, the elimination of nitrogen during the first days of starvation is greater. The rapidity with which the elimination of nitrogen decreases in the first days depends also, according to Voit, upon the proteid condition of the body. It decreases more quickly — that is, the curve of the decrease is more sudden — the first days of starvation, as a rule, the richer the food was in proteids which was taken before starvation. This condition is apparent from the fol- lowing table. This table contains three different starvation experi- ments made by Voit ' on the same dog. This dog received 2500 grms. flesh daily before the first series of experiments, 1500 grms. flesh daily before the second series, and a mixed food relatively poor in nitrogen before the third series. Table II. ^ , -,. ,. Grammes of Urea Eliminated in the Twenty-four Hours. Day of Starvation, g^^ j gg,. jj gg,. m 1 60.1 36.5 13.8 2 24.9 18.6 11.5 3 191 15.7 10.3 4 17.3 14.9 12.3 5 13.3 14.8 13.1 6 13.3 13,8 13.6 7 12.5 13.9 11.3 s". ■.!.■. 10.1 13.1 10.7 Other conditions, such as varying quantities of fat in the body, have an influence on the rapidity with which the nitrogen is eliminated during the first days of starvation. After the first few days the elimination of nitrogen, as is seen in the above table, is more uniform, and as the starvation proceeds it decreases as a rule very slowly and uniformly. Cases also occur in which the elimina- tion of nitrogen becomes constant in these stages, and towards the end, indeed, the elimination of nitrogen increases. This so-called premortal increase always occurs as soon as the adipose tissue in the body has sunk to a certain point, and it also depends on the fact that as soon as the fat is consumed a corresponding increase in the decomposition of proteids is necessary for the generation of heat as well as of other forms of living force. Besides the proteids the fat occurring in the body is also decom- posed in starvation. Since fat has a diminishing infiuence on the destruction of proteids (see further on), the elimination of nitrogen > Physiol, des StoifwechBels, etc., in Hermann's Handbuch, Bd. 6, Thl. 1, S. 89. 622 METABOLISM. in starvation is less in fat than in lean individuals. For instance, only 9 grms. of urea were voided in twenty-four hours during the later stages of starvation by a well-nourished and fat person suffer- ing from disease of the brain, while I. Mttkk found that 20-29 grms. urea were voided daily by Oetti,' who had been poorly fed. Like the destruction of proteids during starvation, the decom- position of fat proceeds uninterruptedly. The decomposition of fat does not show' so great and rapid a decrease in the first days of starvation as the destruction of proteids. Pettenkofer and Voix found, for instance, in a starving dog the following losses of pro- teids and fat from the body on different days of starvation: Table III. T».,, Loss of Loss of """■ Flesh. Calories." Fat. Calories. 3 341 aOI-S 86 7998 5 167 145.6 lOB 957.9 8 138 120.1 99 920.7 The consumption of fat on the second day, when the decom- position of proteids was considerable, was indeed less than in the following days. The reason for this was that the animal had pre- viously been fed with abundant quantities of meat (2500 grms.). If the exchange is expressed as calories we find for the fifth and eighth days of starvation that 13.2^ and 11.5^ respectively of the total cal- ories was covered by the decomposition of proteids and 86.8^ and 85.5^ by the decomposition of fat. Other observations on animals as well as man ha've led to a similar result, and we can assume that in starvation ordinarily the greatest part of the expenditure is replaced by the decomposition of fat and only a small part by the decomposition of proteids. The investigations on the exchange of gas in starvation have shown, as previously mentioned, that the absolute extent of the same is diminished, but that, when the consumption of oxygen and elimination of carbon dioxide is calculated on the unit of weight of the body, 1 kilo, this quantity quickly sinks to a minimum and then remains unchanged, or on the continuation of the starvation may indeed rise. It is a generally known fact that the body tem- perature of starving animals remains rather constant without show- ing any appreciable decrease during the greater part of tlie star- 'L. 0. ' The calorie,? of tlie decomposed proteids were calculated by the author assuming that the flesh contains ZA% nitrogen as proteids. METABOLISM IN STARTATION. 623 vation period. The temperature of the animal first sinks a few days before death and starvation death occurs at about 33-30° C. From what has been said on the respiratory quotient it follows that in starvation it is about the same as with exclusive ts^i and meat as food, namely, about 0.7. This is often the case, but it may also be indeed lower, 0.65 — 0.50, as observed in the cases of Cetti and Succi. As explanation for this unexpected behavior we admit of a storage of incompletely oxidized substances in the body during starvation. Water passes uninterruptedly from the body in starvation even when none is given. If the quantity of water in the tissues rich in proteids is considered as 70-80^, and the quantity of proteids in the same 20j^, then for each gramme of destroyed proteids about 4 grammes of water are set free. A special increase in the demand for water does not seem to occur in starving animals. The mineral svbstances leave the body uninterruptedly in star- vation until death, and the influence of the destruction of tissues is plainly perceptible by their elimination. Because of the destruction of tissues rich in potassium the proportion between potassium and sodium in the urine changes in starvation, so that, contrary to the normal conditions, the potassium is eliminated in proportionally greater quantities. Mukk also observed in Cbtti's' case a relative increase in the phosphoric acid and calcium in the urine during starvation, which was due to an increased exchange of bone-sub- stance. Table IV. Pigeon (Chossat). Male Cat (Voir). Adipose tissue 93 per cent. 97 per cent. Spleen 71 Pancreas 64 Liver 52 Heart 45 Intestine 42 Muscles 43 Testicles Skin 33 Kidneys 33 Lungs 22 Bones I'i' Nervous system 2 67 17 54 3 18 31 40 21 26 18 14 3 The question as to the participation of the diflerent organs in the loss of weight of the body during starvation is of special interest. To illustrate this question we have given above the results of Chos- < L. e. 624 METABOLISM. sat's ' experiments on pigeons and those of Voit on a male cat.. The results are percentages of weight lost from the original weight of the organ. The total quantity of blood, as well as the quantity of solids con- tained therein, decreases, as Pantjm " has shown, in the same pro- portion as the weight of the body. The statements in regard to the loss of water by different organs are somewhat contradictory; according to LuKJAsrow," it seems that the various organs act somewhat differently in this respect. The above-tabulated results cannot serve as a measure of the metabolism in the various organs during starvation. For instance, the nervous system shows only a small loss of weight as compared with the other organs, but from this it must not be concluded that the exchange of material in this system of organs is least active. The condition may be quite different; for one organ may derive its nutriment during starvation from some other organ and exist at its expense. A positive conclusion cannot be drawn in regard to the activity of the metabolism in an organ from the loss of weight of that organ in starvation. The knowledge of metabolism during starvation is of the greatest importance in the study of nutrition, and it forms to a certain extent the starting-point for the study of metabolism under different physiological and pathological conditions. To answer the question whether the metabolism of a person in a special case is abnormally increased or diminished it is naturally very important to know the average extent of metabolism of a healthy person under the same circumstances for comparison. This quantity can be called the abstinent value, namely, the extent of metabolism used in absolute bodily rest and inactivity of the intestinal tract. As measure of this quantity we determine according to Geppert-Zuktz the extent of gaseous exchange, and especially the consumption of oxygen, of a person lying down, best sleeping, in the early morning and at least 12 hours after a light meal, not rich in carbohydrates. The gas volume reduced to 0° C. and 760 mm. Hg pressure is calculated on 1 kilo of body weight and for 1 minute. The results vary between 3 and 4.5 for the consumption of oxygen and between 2.5 and 3.5 ' Cited from Voit in Hermann's Handbuohi Bd. 6, TUl. 1, S. 96 u. 97. ' Virehow's Arch., Bd. 29. • ZeitscUr. f. physiol. Chem., Bd. 13. DEPOSITION OF FLESH. 641 kilo) Kkuq was close to nitrogenous equilibrium for six days. He then increased the nutritive supply to 4300 cal. = 71 cal. per kilo for 15 days by the addition of fat and carbohydrate, and in this time 309 grms. proteid, corresponding to 1455 grms. flesh, was spared. Of the excess of administered calories in this case only 5% was used for flesh deposit and 95^ for fat deposit. As the large, excessive quantity of food was only transitorily and reluctantly eaten, this experiment, as T. Noorden has correctly emphasized, has placed the diflBculty of flesh deposition in another light. We must admit ■with v. NooKDEK that it is impossible to produce a permanent flesh deposit in man by overfeeding, and that it is not possible to make a person muscle-strong by excessive feeding. Flesh deposition is, according to v. N^oobden, a function of the specific development energy of the cells and the cell-work to a much higher extent than the excess of food. Therefore we observe, according to v. Noorden, abundant flesh deposition (1) in each growing body; (2) in those no longer growing but whose body is accustomed to increased work (hypertrophy of the muscles by work) ; (3) whenever, by previous insufficient food or by disease, the flesh condition of the body has been diminished and is com- pensated by abundant food. The deposition of flesh is in these cases an expression of the regenerative energy of the cells. The experiences of cattle-raisers show that in food-animals a flesh deposit does not occur, or at least is only inconsiderable, on over-feeding. The individuality and the race of the animal is of importance for flesh deposition. As a direct formation of fat is denied, and if it does occur it is only very insignificant, the most essential requisite for a fat deposi- tion mast be an overfeeding with non-nitrogenous nutritive bodies. The extent of fat deposition is determined by the excess of admin- istered calories over those used. If a large part of the caloric de- mand is covered by proteid, then a greater part of the simultane- ously given non-nitrogenous food-stuffs is spared, i.e., XTsed for fat deposition. But as proteid and fat are expensive nutritive bodies as compared with carbohydrates, the supply of greater quantities of car- bohydrates is important for fat deposition. The body decomposes less substance at rest than during activity. Bodily rest, besides a proper combination of the three chief groups of organic foods, is therefore also an essential requisite for an abundant fat deposit. The fat formed in fat deposition originates, as stated above. M2 METABOLISM. entirely from the carbohydrates according to Pelugee's doctrine. In this fat-formation, as suggested by Haneiot ' and Pf^ugee,' a; splitting off of carbon dioxide takes place from the carbohydrates. This carbon dioxide, which in excessive feeding with carbohydrates is expired, has, according to Pflugee, a double origin. It is in part split ofE from the carbohydrates in the formation of fat, and it originates in part from the combustion of carbohydrates. This behavior explains the circamstance that after partaking large quan- tities of carbohydrates the respiratory quotient, as first shown by Hankiot and then also by M. Blbibteetj,' was raised under cir- cumstances to 1.2-1.3. Action of certain other Bodies on Metabolism. Water. If a quantity in excess of that which is necessary is introduced into the organism, the excess is quickly and principally elimiaated with the urine. This increased elimination of urine causes in fasting animals (Voit,' Foestee"), but not to any appreciable degree in animals taking food (Sbegen,' Salkowski and Munk,' Matee,' Dubblie'), an increased elimination of urea. The reason for this increased elimination is sought for in the fact that the abundant drinking of water causes a complete washing out of the urea from the tissues. Another view, which is defended by VoiT, is that because of the more active current of fluids after taking large quan- tities of water an increased metabolism of proteids takes place. VoiT considers this explanation the correct one, although he does not deny that by the abundant administration of water a more com- plete washing out of the urea from the tissues takes place. In regard to the action of water on the formation of fat and its metabolism, the view that free drinking of water is favorable for the deposition of fat seems to be generally admitted, while taking only very little water acts against its formation. Salts. The excretion of urine, even when no great quantities of water are taken, is increased by common salt, and the elimination ' Compt. rend.. Tome 114. » Pflilger's Arch., Bd. 53, S. 45. »Jbid.,Bd. 56. * Untersucli. uber den Einfluss des Kochsalzes, etc. Milnchen, 1860. ' Cited from Voit in Hermann's Handbucb, Bd. 6, S. 153. * Wien. Sitzungsber. , Bd. 63. ' Virchow's Arcb., Bd. 71. » Zeitscbr f. Idin. Med., Bd. 3. « Zeitscbr. f. Biologie, Bd. 28. ACTION OF SALTS AND ALCOHOL. 643 of urea is also increased at the same time. The same two possibili- ties may be considered for this last as in the action of water on the excretion of urea. The experiments continued for a long time by VoiT, in which the absolute increase of the elimination of urea was considerable (106 grms. in 49 days), render the conclusion probable that common salt somewhat increases the metabolism of the pro- teids. DuBELiE has obtained contrary results which he considers was due to giving the animal large quantities of common salt. It is possible that the decomposition activity of the cells maybe reduced on giving large quaatities of salt. Certain other salts, such as potas- sium chloride, sodium sulphate, sodium phosphate, sodium acetate, saltpetre, and ammonium chloride, also seem to act like common salt. Sodium borate and the sodium salts of salicylic and benzoic acids also seem to have an increased action on the metabolism of proteids. Alcohol. The question as to how far the alcohol absorbed in the intestinal canal is burnt in the body, or whether it leaves the body unchanged by various channels, has been the subject of much dis- cussion. To all appearances the greatest part of the alcohol is burnt. According to Bodlandek," 1.18^ of the alcohol taken is eliminated with the urine, 0.14^ by the evaporation from the skin, and 1.6j^ with the expired air. The remainder, or about 97^, is burnt in the body. As the alcohol is in greatest part burnt in the body and has a high calorific value (1 grm. = 7 cal.), then the question arises whether it acts sparingly on other bodies, and whether it is to be considered as a nutritive body. The investiga- tions made to decide this question have led to no decisive result. In the experiments on the elimination of nitrogen in human beings sometimes a diminished (Hammond, E. Smith, Obeenieb), some- times an unchanged (Paekes and Wollowicz"), while in other cases an increased (Poestee and Eometn ') elimination of nitrogen was observed after the administration of small amounts of alcohol. In the recent experiments of Stammbeich and v. NooEDENr* alcohol could only replace the isodynamic quantity of non-nitrog- enous food-stufEs, without an essential influence on the proteid > Pflttger's Arch., Bd. 33. « In regard to the older investigations see Voit in Hermann's Handbach, Bd. 6, S. 170. » Maly's Jahresber., Bd. 17, S. 400. < V. Noorden, Alkobol als Sparmittel. Berlin, klin. Wochenscbr., 1891. 644 METABOLISM. condition of the body, in a food richer in proteid than ordinarily. MiUKA ' coald not find any sparing action oa proteids by alcohol in. his experiments, and according to him alcohol cannot replace the sparing action of carbohydrate on proteid. Fokkbr ' and I. Munk * after the administration of small quantities of alcohol to dogs found a diminished, and after large quantities an increased, metabolism of proteids. Chittb]n'den, Nokeis, and E. Smith' make the statement, baaed on their experiments with 1.9, 3.3, and 3.7 c. c. alcohol per kilo of dog per diem, that alcohol acts like a non- nitrogenous nutritive body in regard to its sparing action on proteids. Many observations have been made on animals in regard to the extent of exchange of gas after taking alcohol. The results in these cases are somewhat different, depending upon the size of dose and the kind of animal. In an investigation on the human body Zuntz and Bekdez,' and also Geppert,' observed no essential change in the respiratory exchange of gas after small, non-intoxicating doses of alcohol. As alcohol is in greatest part burnt up in the body and the exchange of gas is nevertheless not essentially raised, it seems as if the alcohol diminishes the combustion of other bodies and thereby has a sparing value. Corresponding to this, as is well known, a deposition of fat may take place in the body under the influence of alcohol. The nutritive value of alcohol may be of essential importance only in certain cases, as large quantities of alcohol taken at once or the continued use of smaller quantities has injurious action on the organism. Alcohol may therefore be con- sidered as a nutritive body only in exceptional cases, and it other- wise must be considered as an article of luxury. Coffee and tea have no positively proved action on the exchange of material, and their importance lies chiefly in their action upon the nervous system. It is impossible to enter into the action of various therapeutic agents upon metabolism. ' Zeitschr. f. klin. Med., Bd. 30. Cited from Maly's Jahresber., Bd. 23, S. 461. '' Cited from Voit in Hermann's Handbuch, Bd. 6, S. 170. 3 Du Bois-Reymond's Arch., 1879, S. 163. * Journal of Physiology, Vol. 13. 5 See Maly's Jahresber., Bd. 7, S. 343. • Arch. f. Path. u. Pharm., Bd. 32. DEPENDENCE ON OTHER CONDITIONS. (545 V. The Dependence of Metabolism on Other Conditions. The previously mentioned so-called abstinence value, i.e., the extent of metabolism -with absolute bodily rest and inactivity of the intestinal tract, serves best as a starting-point for the study of metabolism under various external circumstances. The metabolism going on under these conditions leads in the first place to the pro- dnction of heat, and it is only to a subordinate degree dependent upon the work of the circulatory and respiratory apparatus and the activity of the glands. According to a calculation by Zuntz,' only 10-20$^ of the total calories of the abstinence valae belongs to the circulation and respiration work. The extent of the abstinence value depends in the first place upon the heat production necessary to cover the loss of heat, and this heat production is in turn dependent upon the relationship between the weight of body and the surface of the body. Weight of Body and Age. The greater the mass of the body the ^eater the absolute consumption of material; while on the con- trary, other things being equal, a small individual of the same species ■of animals metabolizes absolutely less, but relatively more as com- pared with the unit of the weight of the body. It must be remarked that we mean flesh weight when we say body weight. The extent of the metabolism is dependent upon the quantity of living cells, and a very fat individual therefore decomposes less substance per kilo than a lean person of the same weight of body. In women, who generally have less bodily weight and a greater quantity of fat than men, the metabolism in general is smaller, and the latter is ordi- narily about ^ of that of men. Otherwise sex does not seem to "have any special influence on the exchange of material. The essential reason why small animals decompose relatively more substance, i.e., as calculated on the kilos of the body, than large ones is that the smaller animals have greater bodily surface in proportion to their mass. On this account the loss of heat is greater, which causes increased heat production, i.e., a more active metabolism. This is also the reason why young individuals of the same kind ahow a relatively greater decomposition than older ones. Rubnbk, ' ' Cited from v. Noorden's Lelirbuch, etc,, S. 97. ' Zeitschr. f. Biologfie, Bdd. 31 u. 19, 646 METABOLISM. whom we have to thank especially for oar knowledge ia regard to the bearing of the relative sarfacial development on the extent of metabolism, has given us the following table on this point with respect to man : Table X. Calories in 24 Hours Calories per after Sub- Calories in Surface in Square traotine the 24Hours Square Centimetre Heat of Com- per Kilo. Centimetres. of bustion of Surface. the Faeces. Children weigliing 4.03 kilos.. 368 91.3 3013 1231 11.8 " .. 966 81.5 7191 1343 16.4 " .. 1218 78.9 7681 1579 23.7 " .. 1411 59.5 10156 1389 30.9 " .. 1784 57.7 18123 1473 40.4 •■ .. 2106 52.1 14491 1452 Man ' 67.0 " .. 3843 43.4 20305 ■ 1399 If we exclude the smallest, actively growing children, in whose- case special conditions govern, we find that the heat production for the unit of surface of body varies only a few per cent from the average of 1447 cal. We see how the relative extent of surface decreases with an increase in the mass of the body. Correspondent with this the metabolism per kilo of body weight also decreases, and it is smallest in adults. A similar result was obtained by Richet' in his investigations- on the elimination of carbou dioxide, in dogs of various sizes, as elucidated in the following table : Table XI. Average Weight of Body in Kilos. COj eliminated in Grammes per Kilo in 1 Hour. Surface of Body in Square Centimetres. CO, eliminated in Grammes per 1000 Square Centimetres. 24.0 13.5 11.5 9.0 6.5 5.0 31 2.3 1.036 1.210 1.380 1.506 1.634 1.688 1.964 3.365 9296 6273 5656 4816 8920 3382 2341 1936 2 6.1 2.60 381 2.81 2.69 3 57 2.71 2.70 ' Arch, de Physiol. (5), Bd. 2. WEIGHT OF BODY AND AGE. 647 The raising of the metabolism which is necessary to cover the loss of heat because of the relatively larger surface of body in small animals is due, according to Richet, to the influence of the nervous system, which may be reduced by chloral hydrate. In the last case the quantity of carbon dioxide produced per kilo in dogs of various sizes is nearly the same. The question whether the active metabolism in young animals depends upon a more active decomposition in the cells than in older animals is still undecided. As the total calories exchanged per kilo of body weight in young animals is greater than in older ones, this difference must be seen in measuring as well the exchange of gas as the elimination of nitrogen. This is true, and we give here Camekek's' figures on the elimination of urea in children. Tablb XII. Age. Weight of Body in Kilos. Urea in grms. Per Day. Per Kilo» U years 10.80 12.10 1.35 3 " 13.30 11.10 0.90 5 " 16.20 12.37 0.76 7 '■ 18.80 14.05 0.75 9 " 25.10 17.27 0.69 12^ ' 32.60 17.79 0.54 15 " 35.70 17.78 0.50 In adults weighing about 70 kilos about 30-35 grms. urea per day are eliminated, or 0.5 grm. per kilo. At about 15 years of age^ the destruction of proteids per kilo is about the same as in adults. The relatively greater metabolism of proteids in young individuals is explained partly by the fact that the metabolism of material in general is more active in young animals, and partly by the fact that young animals are as a rule poorer in fat than those full grown. As the metabolism may be kept at its lowest point by absolute rest of body and inactivity of the intestinal tract, it is manifest that work and the taking up of food have an important bearing on the extent of metabolism. Resi and Work. During work a greater quantity of potential energy is converted into living force, i.e., the metabolism is increased more or less on account of work. As explained in a previous chapter (XI) work, according to the generally accepted view, has no material influence on the elimi- ' Zeitschr. f. Biologie, Bdd. 16 n. 30. 648 METABOLISM. nation of nitrogen. It is nevertheless true that several investigators in certain cases have observed an increased elimination of nitrogen; but these observations have been explained in other ways. For instance, work may, when it is connected with violent move- ments of the body, easily cause dyspnoea, and this last, as Feam'- KEL ' has shown, since diminution of the oxygen supply increases the proteid metabolism, may cause au increase in the elimina- tion of nitrogen. In other series of experiments the quantity of carbohydrates and fats in the food was not sufficient; the supply of fat in the body was decreased thereby, and the destruction of proteids was correspondingly increased. Work may also increase the appetite, and an increase in the elimination of nitrogen may be caused by the gfpater quantity of proteids taken. According to the generally accepted views muscular activity has hardly any influ- ence on the metabolism of proteids. On the contrary, woik has a very considerable influence on the elimination of carbon dioxide and the consumption of oxygen. This action, which was first observed by Lavoisier, has recently been confirmed by many investigators. Pettenkofek and VoiT ' have made investigations on a full-grown man as to the metabolism of the nitrogenous as well as of the non-nitrogenous bodies during rest and work, partly while fasting and partly on a mixed diet. The experiments were made on a full-grown man weighing 70 kilos. The results are contained in the following table : Table XIII. Consumptiop of Proteids. Fat. Carbohydrates. CO, eliminated. O coDsumed. w.stin^ (Rest 79 309 ... 716 761 ^"^""^•■- J Work 75 380 ... 1187 1071 Mixeaaiet^ ^^j.^ ^3^ 173 352 1309 980 In these cases work did not seem to have any influence on the destruction of proteids, while the gas exchange was considerably increased. ZuNTZ and his pupils Lehmann ' and Katzenstein * have made very important investigations on the extent of the exchange > Vlrchow's Arch., Bdd. 67 u. 71. ' Zeitschr. f. Blologie, Bd. 2. » Maly's Jaliresber., Bd. 19, S. 418. * Pfliiger's Arcb., Bd. 49. LACK OF WATMll AND MINERAL SUBSTANOES. 625 c. c. for the carbon dioxide. As average we can accept 3.81 c. c. oxygen and 3.08 c. c. carbon dioxide.' The extent of proteid destruction cannot be determined in transient experiments, aad for these reasons only the valaes foand after several days of starvation are useful. In the starvation experi- ments on Cetti and Succi the eliminatioa of nitrogen per kilo in the fifth to the tenth starvation day was 0.150-0.203 grm. N. III. Metabolism with Inadequate Nutrition. The food may be quantitatively insufl&cient, and the final result is absolute inanition. The food may also be qualitatively insnflB- cient or, as we say, inadequate. This occurs when any of the necessary nutritive bodies are absent in the food, while the others occur in suflBcient or perhaps indeed in excessive amounts. Lack of Water in the Food. The quantity of water in the organism is greatest during foetal life, and then decreases with increasing age. Naturally, the quantity differs in various organs. The tissue in the body being poorest in water is the enamel, which is almost free, containing only 2 p. m. water, tbe teeth about 100 p. m., the fatty tissues 60-120 p. m. The bones with 140-440 p. m. and the cartilage with 540-740 p. m. are somewhat richer in water, while the muscles, blood, and glands with 750 to more than 800 p. m. are still richer. The quantity of water is even greater in the animal fiuids (see preceding chapter), and the adult body contains in all about 630 p. m. water.' If we bear in mind that two thirds of the animal organism consists of water; that water is of the very greatest importance in the normal, physical composi- tion of the tissues; moreover that all fiow of juices, all exchange of substance, all supply of nutrition, all increase or destruction, and all discharge of the products of destruction are dependent upon the presence of water; besides this, that by its evaporation it is an im- portant regulator of the temperature of the body,— we perceive that water must be necessary for life. If the loss of water be not replaced by fresh supplies sooner or later, the organism succumbs. Lack of Mineral Substances in the Food. We are chiefly indebted to LiEBiG lor showing that the mineral substances are just as neces- > These figures are taken from v. Noorden's Lehrbuoh der Path, des Stoff- ■wechsels, S. 94. » See Voit in Hermann's Handbuch, Bd. 6, Thl. 1, S. 345. 626 METABOLISM. aary for the normal composition of the tissues and organs, and for the normal coarse of the processes of life, as the organic constituents of the body. The importance of the mineral constituents is evident from the fact that there is no animal tissue or animal fluid which does not contain mineral substance, and also from the fact that certain tissues or elements of tissues contain regularly certain min- eral substances and not others, which explains the unequal division of the potassiam and sodium compounds in the tissues and fluids. With the exception of the skeleton, which contains about 220 p. m. mineral bodies (Volkmann '), the animal fluids or tissues are poor in inorganic coustituents, and the quantity of such only amounts, as a rule, to about 10 p. m. Of the total quantity of mineral sub- stances in the organism, the greatest part occurs in the skeleton, 830 p. m., and the next greatest in the muscles, about 100 p. m. (Volkmann). The mineral bodies seem to be partly dissolved in the fluids and partly combined with organic substances. In accordance with this the organism persistently retains, with food poor in salts, a part of the mineral substances, also such as are soluble, as the chlorides. On the burning of the organic substances the mineral bodies com- bined therewith are set free and may be eliminated. It is also ad- mitted that they in part combine with the new products of the burning, and also that they in part are attached to organic nutritive bodies poor in salts or nearly salt-free, which are absorbed from the intestinal canal and are thus retained (VoiT, Fokster''). If this statement be correct, it is possible that a constant supply of mineral substances with the food is not absolately necessary, and that the amount of inorganic bodies which must be administered is insignificant. The question whether this be so or not has not, especially in man, been sufficiently investigated; but generally we consider the need of mineral substances by man as very small. It may, however, be assumed that man usually takes with his food a considerable excess of mineral substances. Investigations on animals in regard to the action of an insuffi- cient supply of mineral substances with the food have been made by several investigators, especially Foestek. He observed, on experi- menting on dogs and pigeons with food as poor as possible in ' See Voit in Hermann's Handbuch, Bd. 6, Thl. 1, S. 353. ^ Zeitschr. f . Biologie, Bd. 9. See also Voit in Hermann's Handbuch, Bd. 6, Tbl. 1, S. 354. LACK OF MINERAL SUBSTANCES. 627 mineral substances, a very suggestive disturbance of the functions of the organs, especially the muscles and the nervous system, and death resulted after a time, indeed earlier than in complete starva- tion. In opposition to these observations Bungb' has suggested that the early death in these cases was not caused by the lack of mineral salts, but more likely by the lack of bases necessary to neutralize the sulphuric acid formed in the burning of the proteids in the prganism, which must be then taken from the tissues. In accordance with this view, Bunge and Lunin ' also found on experimenting on mice that animals which received nearly ash-free food with the addition of sodium carbonate were kept alive twice as long as animals which had the same food without the addition of sodium carbonate. Special experiments also show that the carbon- ate cannot be replaced by an equivalent amount of sodium chloride, and that to all appearances it acts by combining with the acids formed in the body. The addition of alkali carbonate to the other- wise nearly ash-free food may indeed delay death, but cannot prevent it, and even in the presence of the necessary amount of bases death results for lack of mineral substances in the food. In the above series of experiments made by Bungb the food of the animal consisted of casein, milk-fat, and caue-sugar. While milk alone was an adequate and sufficient food for the animal, BuNGE found that the animal could not be kept alive longer by food consisting of the above constituents of milk and cane-sugar with the addition of all the mineral substances of milk, than with the food mentioned in the above experiments with the addition of alkali carbonate. The question whether this result is to be explained by the fact that the mineral bodies of milk are chemically combined with the organic constituents of the same and can be assimilated only in such combinations, or whether it depends on other condi- tions, Bungb leaves undecided. These observations, however, show how difficult it is to draw positive conclusions from experiments made thus far with food poor in salts. Further investigations on this subject seem to be necessary. With an insufficient supply of clilorides with the food the elimi- nation of chlorine by the urine decreases constantly, and at last it may stop entirely while the tissues still persistently retain the chlo- rides. These last are, at least in part, combined in the body with. ' LeUrbucb d. pbysiol. Chem., 1. Aufl., S. 103. ' Ihid., and Zeitscbr. f. pbysiol. CUem., Bd. 5. 628 METABOLISM. the organic substances which retain them. The great importance of snch a retention of chlorides by the tissues is apparent if we bear in mind that the NaCi is not only a solvent for certain albuminous, bodies, or a material for the elaboration of the gastric juice, but that it is also of the greatest importance as a so-called indifferent Salt for the preservation of the normal consistency and the physio- logical imbibition relation of the tissues. If there be a lack of sodium as compared with potassium, also if there be an excess of potassium compounds in any other form than KCl, the potassium combinations are replaced in the organism by NaOl, so that new potassium and sodium compounds are produced which are voided with the urine. The organism becomes poorer in NaCl, which therefore must be taken in greater amounts from the outside (Bunge). This occurs habitually in herbivora, and in man with vegetable food rich in potash. For human beings, and espe- cially for the poorer classes of people who live chiefly on potatoes and foods rich in potash, common salt is, under these circumstances^ not only a condiment, but a necessary addition to the food (Bungb"). Lack of Alkali Carbonates or Bases in the Pood. The chemical processes in the organism are dependent upon the presence of alka- line-reacting tissue-fluids, whose alkaline reaction is due to alkali carbonates. The alkali carbonates are also of great importance not only as a solvent for certain proteid bodies and as constituents of certain secretions, such as the pancreatic and intestinal juices, but they are also a means of transportation of the carbon dioxide in the blood. It is therefore easy to understand that a decrease below a certain point in the quantity of alkali carbonate must endanger life. Such a decrease not only occurs with lack of bases in the food which accelerates death by a relatively too great production of acids by the burning of the proteids (see above: Buh^ge and Lunik), but it also occurs when an animal is given dilute mineral acids for a certain time. In herbivora the fixed alkalies of the tissues combine with the mineral acids, and the animal succumbs after a time. In carnivora (and in man) the bases of the tissues are obstinately retained; the mineral acids unite with the ammonia produced by the decomposition of the proteids or their cleavage products, and carnivora can therefore be kept alive for a longer time. Lack of Earthy Phosphates. With the exception of the impor- ' Zeitschr. f. Biologie, Bd. 9. LACK OF IRON. 629 tance of the alkaline earths as carbonates and principally as phos- phates in the physical composition of certain structures, such as the bones and teeth, their physiological importance is nearly unknown. The occarrence of earthy phosphates in all proteids, and the great importance of the earthy phosphates in the passage of the proteids from a soluble to a coagulabie and solid state, make it probable that the earthy phosphates play an important part in the organization of the proteids. The action which an insufficient supply of alkali- earths with the food causes is connected with the interesting ques- tion as to the effect of this lack upon the bony structure. This action, as well as the various results obtained by experiments on young and old animals, has already been spoken of in Chap. X, to which we refer the reader. Lack of Iron. As, iron is an integral constituent of haemoglobin, indispensable for the introduction of oxygen, so iron is an indispen- sable constituent of the food. In iron starvation iron is continually eliminated, even though in diminished amounts (Dietl,' v. Hos- LiN," and others). From the observations of v. Hoslin on dogs it seems that an inadequate supply of iron with the food causes an insufficient formation of haemoglobin. A special result of the lack of iron is chlorosis, which the physician has often to contend with •and whose origin is not really a lack of iron in the food, but more likely an incomplete assimilation and absorption of the foods contain- ing iron (Bdnge). The iron-salts as such seem not to be absorbed at all in the intestinal canal, or only to a very small extent, so that it is questionable whether their absorption has any importance worth noting. It seems more probable that the absorption of iron from the food takes place in the form of protein bodies (nucleo- albumin) containing iron (Bukse) ; and the importance of the iron- salts in preventing the lack of hemoglobin consists chiefly, accord- ing to BuNGE,' in that these salts counteract the decomposition in the intestine of the protein bodies containing iron, with a splitting •off of iron as iron sulphide. In the absence of proteid bodies in the food the organism must nourish itself by its own proteid substances, and on such nutrition it must earlier or later succumb. By the exclusive administration ■of fat and carbohydrates the consumption of proteids in these cases ' Wien. Sitzungsber., Bd. 71, Abth. 3, 1875. ' Zeitsclir. f. Biologie, Bd. 18. » Zeitscbr. f. pbysiol. Chem., Bd. 9. 630 METABOLISM. is redaeed, for by an exclusive fat and carbohydrate diet the meta- bolism of proteids may indeed be smaller than in complete starvation (HiESCHFELD,' KUMAGAWA," KlEMPEEER,' MuNK,' KOSEKHEIM,^ and others). In conformity with this the animal may be kept alive longer by food containing only non-nitrogenous bodies than in complete starvation. The absence ol fats and carbohydrates in the food affect carniv- ora and herbivora somewhat differenbly. It is unknowu whether carnivora can be kept alive for any length of time by food entirely free from fat and carbohydrates. But ib has been positively demon- strated that they can be kept alive a long time by feeding exclusively wibh meat freed as much as possible from visible fat (Pflugee')- Human beings and herbivora, on the contrary, cannot live for any length of time on such food. On one side they lose the property of digesting and assimilating the necessarily large amounts of meat, and on the other a distaste for large quantities of meat or proteids. soon appears. IV. Metabolism with Various Foods. For the carnivora, as above stated, meat as poor as possible in fat may be a complete and sufficient food. As the proteids more- over take a special place among the organic nutritive bodies by the quantity of nitrogen they contain, it is proper that we first describe- the exchange of material with an exclusively meat diet. Metabolism with food rich in proteids, or feeding only witb meat as poor in fat as possible. By an increased supply of proteids the metabolization of proteids and the elimination of nitrogen is increased, and this in proportion to the supply of proteids. If a certain quantity of meat has oeen given as food daily to car- nivora and the quantity is suddenly increased, an increased meta- bolism of proteids or an increase in the quantity of nitrogen elimi- nated is the result. If we feed the animal daily for a certain time with larger quantities of the same meat, we find that a part of the proteids accumulates in the body, but this part decreases from day ' Virchow's Arch., Bd. 114. » Ibid.. Bd. 116, ' Zeitschr. f. klin. Med., Bd. 16. * Du Bois-Reymond's Arch., 1891. 'Ibid , S. 341 and Pfluger's Arch., Bd. 54. • Pflager's Arcb., Bd. 50. ■WITH FOOD HIGH IN PROTEIDS. 631 to day, while there is a corresponding daily increase in the elimina- tion of nitrogen. In this way a nitrogenous equilibrium is estab- lished, that is, the total quantity of nitrogen eliminated is eqaal to the quantity of nitrogen in the absorbed proteids or meat. If, on the contrary, an animal which is in nitrogenous equilibrium, having been fed on large quantities of meat, is suddenly fed with a small quantity of meat per day, then the animal gives up its own bodily proteids, the amount decreasi ng f rom day to day. The elimination of nitrogen and the metabolism of proteids decrease constantly, and the animal may in this case also pass into nitrogenous equilibrium or nearly into this condition. These relations are illustrated by the following table (Voit ') : Table V. Orms. of Meat in the Food per Day. Before the Test. During the Test. 500 1500 1500 1000 Grms. of Flesh metabolized in Body per Day. 1 1222 1153 2 1310 1086 3 4 5 6 1390 1410 1440 1450 1088 1080 1037 7 1500 In the first case (1) the metabolism of flesh before the beginning of the actual experiment on feeding with 500 grms. meat was 447 grms., and it increased considerably on the first day of the experi- ment, after feeding on 1500 grms. meat. In the seconcicase (2), in which the animal was previously in nitrogenous equilibrium with 1500 grms. meat, the metabolism of flesh on the first day of the experiment, with only 1000 grms. meat, decreased considerably, and on the fifth day a nearly nitrogenous equilibrium was obtained. During this time the animal gave up daily some of its own proteids. Between that point below which the animal loses from its own weight and the maximum, which seems to be dependent upon the digestive and assimilative capacity of the intestinal canal, a carniv- ora may be kept in nitrogenous equilibrium with varying quantities of proteids in the food. The supply of proteias, as well as the proteid condition of the body, affects the extent of the proteid metabolism. A body which has become rich in proteids by a previous abundant meat diet must, 1 Hermann's Handbach, Bd. 6, TM. 1, S. 110. (332 METABOLISM. to prevent a loss of proteids, take np more proteid with the food than a body poor in proteids. Pettenkofbe and VoiT have made investigations on the meta- bolism of fat with an exclusively albuminous diet. These investi- gations have shown that by increasing the quantity of proteids in the food the daily metabolism of fat decreases, and they have drawn the conclusion from these experiments, as detailed in Chapter X, that even a formation of fat may take place under these circumstances. The objections presented by Pfluree against these experiments are also mentioned in this chapter, and Kuma- GAWA ' has recently published a new and important investigation on this subject. KuMAGAWA caused two dogs of the same litter to fast for over 30 days in order to remove the body fat. One of the dogs (the control dog) was then killed and the total fat determined. The other animal received meat poor in fat (with a known quantity of ether extractives, glycogen, nitrogen, water, and ash) in as large quantities as it could endure, and this feeding with meat was continued (about 50 days) until a marked increase in bodily weight had taken place. The quantity of nitrogen in the urine and faeces daring this period was also determined, and finally the animal was killed and the total quantity of fat determined. The results were that the fat formed daring the period of feeding corresponded exactly with the quantity existing in the meat fed to the animal and formed from the glycogen of the meat. In this case no fat formatiofi from proteid was found, and according to Kumagawa the animal body under normal circumstances has no ability of forming fat from proteid. According to Pfluger's doctrine, which has received support from these investigations, the proteid can inflaence the formation of fat only in an indirect way, namely, in that it is consumed instead of the non-nitrogenous bodies and hence the fat and fat-forming carbohydrates are spared. If sufiicient proteid is introduced iu the food to satisfy the total nutritive requirements, then the decomposi- tion of fat stops; and if also non-nitrogenoas food is taken at the same time, this is not consumed, bat is stored up in the animal body, the fats as such, and the carbohydrates at least in great part as fat. ' Zur Frage der Fettbildung aus Eiweiss im ThierkSrper. Mittheil. der med. Fakultat der kaiserl. Japan. Universitat zu Tokio, Bd. 3, No. 1, 1894. ■TISSUE AND GIROULATINO PBOTEIDS. 633 Pfluger calls the "nutritive requirement" as the smallest quantity of lean meat which produces nitrogenous equilibrium without causing any decomposition of fat or carbohydrates. At rest and at an average temperature it is found for dogs to be 2.073 grms. nitrogen (in meat fed) per kilo of flesh weight (not bodily weight, as the fat, which often forms a considerable fraction of the weight of the body, cannot as it were be used as dead measure). Even when the supply of proteid is in excess of the nutritive requirements, Pfluger has found that the proteid metabolism increases with an increased supply until the limit of digestive power is reached, which limit is about 3600 grms. meat with' a dog weigh- ing 30 kilos. In these experiments of Pflugek's all of the excess of proteid introduced was not completely decomposed, but a part was retained by the body. Pflugek therefore defends the proposi- tion " that an exclusive proteid supply, without fat or carbo- hydrate, does not exclude a proteid fattening." From what has been said on proteid metabolism in starvation and with one-sided proteid food it follows that the proteid meta- bolism in ,the animal body never stops, that the extent is dependent in the first place upon the extent of proteid supply, and that the animal body has the property, withia wide limits, of accommodating the proteid metabolism to the proteid supply. These and certain other peculiarities of proteid metabolism have led VoiT to the view that all proteids in the body are not decom- posed with the same ease. VoiT differentiates the proteids fixed in the tissue-elements, so-called organized proteids, tissue-proieids, from those proteids which circulate with the fluids in the body and its tissues and which are taken up by the living cells of the tissues from the interstitial fluids washing them and destroyed. These circulating proteids are, according to Voit, more easily and quickly destroyed than the tissue-proteids. When, therefore, in a fasting animal which has been previously fed with meat an abundant and quickly decreasing decomposition of proteids takes place, while in the further course of starvation this proteid metabolism becomes less and more uniform, this depends upon the fact that the supply of circulating proteids is destroyed chiefly in the first days of starva- tion and the tissue-proteids in the last days. The tissue-elements constitute an apparatus of a relatively stable nature, which has the power of taking proteids from the fluids washing the tissues and digesting them, while a few proteids, the 634 METABOLISM. tissne-proteids, are ordinarily disorganized to only a small extent, about 1^ daily (Voit). By an increased supply of proteids the activity of the cells and their ability to decompose nutritive proteids is also increased to a certain degree. When nitrogenous equilibrium is obtained after increased supply of proteids, it denotes that the decomposing power of the cells for proteids has increased so that the same quantity of proteids is metabolized as is supplied to the body. If the proteid metabolism is decreased by the simnltaneous administration of other non-nitrogenous foods (see below), a part of the circulating proteids may have time to become fixed and organ- ized by the tissues, and in this way the mass of the flesh of the body increases. Daring starvation or with lack of proteids in the food the reverse takes place, for a part of the tissue proteids is converted into circulating proteids which are metabolized, and in this case the flesh of the body decreases. Voit's doctrine has been severely attacked by Pflugbb.' Pplugek states, basing his statement on an investigation made by one of his pupils, Schondokfb," that the extent of proteid destruc- tion is not dependent upon the quantity of circulating proteids, but upon the nutritive condition of the cells for the time beiag — a view which is not very contradictory of Voit's doctrine, if the author does not misunderstand P-flugek's statement. Voit° has, as is known, stated that the conditions of the destruction of substances in the body exist in the cells, and also that the circulating proteid, likewise according to Voit, is first metabolized after having been taken up by the cells from the fluids washing them. The organized proteid, which is fixed by the cells and has become a part of the same, is destroyed less readily, according to Voit, than the proteid taken up by the cells from the nutritive fluid, which serves as material for the chemical construction of the very much more com- plicated organized proteids. This nutritive proteid, which circu- lates with the fluids before it is taken up by the cells, and which can exist in store in the cells as well as in the fluids, which corre- sponds to Voit's view, has been called circulating proteid or supply proteid by him. It is clear that these names may lead to misunder- standing, and therefore too much stress should not be put on them. The most essential part of Voit's doctrine is the supposition that > Pflugei's Arch., Bd. 54. ^ Ihid., Bd. 54. » Zeitschr. f. Biologie, Bd. 11. NUTRITIVE VALUE OF GELATIN. 635 the ■ food proteid of the cells is more easily destroyed than the organized, real protoplasmic proteid, and this statement can hardly, for the present, he considered as refuted or exactly proven. This question is intimately connected with another, namely, whether the food proteids taken up by the cells are metabolized as such or whether they are first organized. The investigations of Panum ' and Falck ' on the transitory progress of the elimination of urea after a meal rich in proteids throws light on this question. From the investigations on a dog it was found that the elimination of urea increases almost immediately after a meal rich in proteids, and that it reaches its maximum in about six hours, when about one half of the quantity of nitrogen corresponding to the administered proteids is eliminated. If we also recollect that, according to an observation of Schmidt-Mulheim ' on a dog, about 37j^ of the given proteids are absorbed in the first two hours after the meal and about 59^ in the course of the first six hours, we may then infer that the increased elimination of nitrogen after a meal is due to a metabolizatiou of the digested and assimilated proteids of the food not previously organized. If we admit that the metabolized proteid must have been organized, then the greatly increased elimination of nitrogen after a meal rich in proteids supposes a far more rapid and comprehensive destruction and reconstruction of the tissues than has been generally admitted and not proven. It has been stated above that other foods may decrease the metabolism of proteids. Gelatin is such a food. Gelatin and the gelatin-formers do not seem to be converted into proteid in the body, and this last cannot be entirely replaced by gelatin in the food. For example, if a dog is fed on gelatin and fat, its body sustains a loss of proteids even when the quantity of gelatin is so large that the animal, with an amount of fat and meat containing just the same quantity of nitrogen as the gelatin in question, may remain in nitrogenous equilibrium. On the other hand, gelatin, as VoiT,* Panum and Obeum' have shown, has a great value as a means of sparing the proteids, and it may decrease the metabolism of proteids to a still greater extent than fats and carbohydrates. ' Nord. med. Arkiv., Bd. 6. » Cited from Voit in Hermann's Handbuch. Bd. 6., Thl. 1, S. 107. ' Du Bois-Reymond's Arch., 1879. •L. c.,S. 123. . ' Nord. med. Arkiv.. Bd. 11 636 METABOLISM. This is apparent from the following sammary of Voit's experiments on a dog: Table VI. Food per Day. Flesh. Meat. GelatiD. Fat. Sugar. Metabolized. On tlie Body. 400 300 450 - 50 400 250 439 - 39 400 200 256 +44 I. MusTK ' has later arrived at similar results by means of more decisive experiments. lie found in dogs that on a mixed diet which contained 3.7 grms. proteid per kilo of body, of which hardly 3.6 grms. was metabolized, nearly f could be replaced by gelatin. The same dog metabolized on the second starvation day three times as much proteid as with the gelatin feeding. Muif k states also that gelatin has a much greater sparing action on proteids than the fat or the carbohydrates. This ability of gelatin to spare the proteids is explained by VoiT by the statement that the gelatin is decomposed instead of a part of the circulating proteids, whereby a part of this last may be organized. Gelatin may also decrease somewhat the consumption of fat, although it is of less value in this respect than the carbohydrates. The question of nutritive value oi peptones stands in close rela- tion to the nutritive value of the proteids and gelatin. The early investigations made by Maly, Plos'z and Gtekgtat, and Adam- KiEWicz " have led to the conclusion that an animal with food which contains no proteids besides peptones may not only preserve its nitrogenous equilibrium, but its proteid condition may even increase. According to recent, more exact investigations of PoLLiTZEK, ZuNTZ,' and MuNK ' the albumoses and peptones have the same nutritive value as proteids, at least in short experiments. According to Pollitzek this is true for different albumoses as well as for true peptone. Contrary to this view Voit * is of the opinion that the albumoses and peptones can replace the proteids only for a short time, not indefinitely. According to Voit the albumoses and peptones, like gelatin, may, by their ability to spare proteid, ' Pflllger's Arch., Bd. 58. » Cited from page 339. ' See Maly's Jaliresber., Bd. 19, S. 353 u. 403. *L. c, S. 894. WITH MIXED BTET. 637 entirely or nearly arrest the consumption of proteid, but cannot pass into proteid. From experiments made by Weiske ' and others on herbivora it appears that asparagin may spare proteid in such animals. In carnivora (I. Munk") and in mice (Voit and Politis') it was found that asparagin does not seem to hare any sparing action on the proteids,' or only a very slight action. It is nob known how ib acts in man. Metabolism on a Diet consisting of Proteid, with Fat and Carbohydrate. Pat cannot arrest or prevent the metabolism ofpro- teids; but ib can decrease ib, and so spare the proteids. This is apparent from the following table of Voit.' A is the average for three days, and B for six days. Table VII. Food. Flesb. A B Meat. Fat. 1500 1500 150 Metabolized. On the Body. 1513 - 13 1474 + 34 According to Voit the adipose tissue of the body acts like the food-fat, and the proteid-sparing effect of the former may be added to that of the latter, so thab a body rich in fab may nob only remain in nibrogenoas equilibrium, but may even add to the store of bodily proteids, while in a lean body with the same food conbaining the same amount of proteids and fab there would be a loss of proteids. In a body rich in fat a greater quantity of proteids is protected from metabolism by a certain quantity of fat than in a lean body. Because of the sparing action of fats an animal by the addition of fab to its food may, as is apparent from the tables, increase its proteid condition with a quantity of meat which is insuflScient to preserve nitrogenous equilibrium. Like the fats the carbohydrates have a sparing action on the proteids. By the addition of carbohydrates to the food the carni- vor not only remains in nitrogenous equilibrium, but the same quantity of meat which in itself is insufficient and which without ' Zeitschr. f. Biologie, Bdd. 15 u. 17 and Centralbl. f. d. med. Wissensch., 1890, S. 945. ' Virchow's Arch., Bdd. 94 u. 98. ' Zeitsclir. f. Biologie, Bd. 38. ' See Manthner, ibid., Bd. 38, and Gabriel, ibid., Bd. 39, and Volt, ibid., 8. 135. ' See Voit in Hermann's Handbuch, Bd. 6, S. 130. 638 METABOLISM. carbohydrates would cause a loss of weight in the body may with the addition of carbohydrates produce a deposit of proteids. This is apparent from the following table ' : Table VIII. Food. Flesh. Meat. Fat. Sugar. Starch. Metabolized. On the Body. 500 250 558 - 58 500 300 466 + 34 500 300 505 - 5 800 350 745 + 55 800 300 773 + 27 2000 200-300 1792 --208 3000 250 ■ 1883 --117 The sparing of proteid by carbohydrate is greater, as shown by the table, than by fats. According to Voit the first is on an average 2fo and the other 7^ of the administered proteid, without a previous addition of non-nitrogenous bodies. Increasing quantities of carbohydrates in the food decrease the proteid metabolism more regularly and constantly than increasing quantities of fat. The law as to the increased proteid metabolism with increased proteid supply applies also to food consisting of proteid with fat and carbohydrates. In these cases the body tries to adapt its proteid metabolism to the supply; and when the daily calorie supply is completely covered by the food, the organism can, within wide limits, be in nitrogenous equilibrium with different quantities of proteid. The upper limit to the possible proteid metabolism per kilo and per day has only been determined for herbivora. It is not known for human beings, and its determination is from a practical standpoint of secondary importance. What is more important is to ascertain the lower limit, and on this subject we hare several investigations on man as well as animals by Hieschebld, Kumagawa, Klem- PEKEE, MuNK, EoSENHEiM,' and others. It follows from these investigations that the lower limit of proteid needed for human beings for a week or less is about 30-40 grms. proteid or 0.4-0.6 grm. per kilo with a body of average weight, v. Nooeden"' con- siders 0.6 grm. proteid (assimilated proteid) per kilo and per day as the lower limit. The above-mentioned figures are only valid for short series of experiments; still we have the observations of ' Voit in Hermann's Handbnch, Bd. 6, S. 148. ' See foot-notes 1-5, page 630. » Grandriss einer Method ik der StofEwechseluntersucbungen. Berlin, 1892. WITH MIKED DIET. 639 E. VoiT and Constantinidi ' on the diet of a vegetarian in which the proteid condition was kept nearly but not completely maintained with about 0.6 grm. proteid per kilo. According to Voit's normal figares, which will be spoken of below for the nutritive need of man, an average working man of about 70 kilos weight on a mixed diet requires about 40 calories per kilo (two calories or net calories, namely, the combustion value of the assimilated foods). In the above experiments with food very poor in proteid the demand for calories was considerably greater, as for instance in certain cases it was 51 (Kcmagawa) or even 78.5 calories (Klempeeee). It therefore seems as if the above very low supply of proteid was only possible with great waste of non-nitrogen- ous food; but in opposition to this we must recall that in Voit and CoNSTANTiN^iDi's experiments on the vegetarian, who for years was used to a food very poor in proteid and rich in carbohydrate, the calories only amounted to 43.7 per kilo. It is an open question how a nitrogenous eqailibrium can exist also on a diet very poor in nitrogen, when the need of calories is only just covered by the total supply. In Munk's and Eosenheim's experiments on dogs the food poor in proteids must have raised the total supply of calories consider- ably. These experiments also teach that in dogs the continuous administration for a long time of food poor in proteid has an action on the health of the animal and may even cause death. In the experiments recently published by Eosexheim, which extended over two months, 2 grms. proteid per kilo of body was not sufficient to keep the animal healthy although the heat value of the food taken up amounted to 110 calories per kilo. The very important question as to the conditions for the deposi- tion of fat and flesh on the body stands in close connection to what has just been said in regard to foods consisting of proteid and non- nitrogenous food-stuffs. In this connection we mast recall in the first place that all fattening presupposes an overfeeding, i.e., a supply of food-stuffs which is greater than that metabolized at the same time. In carnivora, as shown by the investigations of Voit and Pflugek, a very inconsiderable metabolized proteid, in proportion to the deposition of flesh, may take place with exclusive meat food. In man and herbivora, on the contrary, the demand for calories ' C. Voit, Zeitschr. f . Biologie, Bd. 25. 64-0 METABOLISM. may not be covered by proteid alone, and the question as to the con- ditions of fattening with a mixed diet is of importance. These conditions have also been studied on carnivora, and here, as VoiT has shown, the relationship between proteid and fat (and carbohydrates) is of great importance. If considerable fat is given in proportion to the proteid of the food, as with average quantities of meat with considerable addition of fat, then nitrogenous eqnilib- riam is only slowly attained and the daily deposit of flesh, though not large, but quite constant, may be considerable in the course of time. If, on the contrary, much meat besides proportionally little fat is given, then the deposit of proteid with increased metabolism is smaller day by day, and nitrogenous equilibrium is attained ia a few days. In spite of the daily, somewhat larger deposit, the total flesh deposit is not considerable in these cases. The following experiment of VoiT may serve as example: Table IX. Number of Days of ISxpeiimenca- Food. Total Deposit of Flesh. Daily Deposit of Flesh. Nitrogenbus tiuii. Meat, p^niis. Fat, grms. Equilibrium 32 7 500 1800 250 250 1792 854 56 122 not attained attained The greatest absolute deposition of flesh in the body was obtained in these cases with only 500 grms. flesh and 350 grms. fat, and even after 32 days the nitrogenous equilibrium had not occurred. On feeding with 1800 grms. meat and 250 grms. fat the nitrogenous equilibrium occurred after 7 days; and' though the deposition of flesh per day was greater, still the absolute deposit was not one half as great as in the former case. Inasmuch as the quantity of proteids does not decrease below a certain amount, it seems that the most abundant and most lasting deposition of flesh is obtained with a food which does not contain too much proteids in proportion to the fat. The same is also true of a diet consisting of proteids and carbohydrates. The experiments of Krug ' on himself, under the direction of V. NoOEDBN, give us information as to the practicability of flesh deposition in man. With abundant food (3590 cal. = 44 cal. per ' Cited from v. Noorden's Lehrbtich der Path, des Stoffwechsels. Berlin, 1893, S. 120. Missing Page Missing Page Missing Page Missing Page Missing Page Missing Page Missing Page Missing Page REST AND WORK. 649 of gas as a measure of metabolism during work and cansed by work, using Zuntz-Geppert's method (see page 604). These iuvestiga- tioQs not only show the important influence of muscular work oa the decomposition of material, but they also show in a very instructive way the relationship between the extent of metabolism of material and useful work of various kinds. We can only refer to these im- portant investigations, which are of special physiological interest. The action of muscular work on the gas exchange does not alone appear with hard work. From the researches of Speck,' who has also made very meritorious studies on the exchange of gas in man under various conditions, we learn that even very small, apparently quite unessential movements may increase the produc- tion of carbon dioxide to such an extent that by not observing these, as in numerous older experiments, very considerable errors may creep in. The quantity of carbon dioxide eliminated during a working period is uniformly greater than the quantity of oxygen taken up at the same time, and hence a raising of the respiratory quotient was formerly usually considered as caased by work. This rise does not seem to be based upon the kind of chemical processes going on during work, as we have a series of experiments made by ZuifTZ, LEHiTANU, and Katzenstein in which the respiratory quotient remained almost wholly unchanged in spite of work. According to LoEWT ' the combustion processes in the animal body go on in the same way in work as in rest, and a raising of the respiratory quotient (irrespective of the transient change in the respiratory mechanism) takes place only with insufficient supply of oxygen to the muscles, as in continuous fatiguing work or short excessive muscular activity, also with local lack of oxygen caused by excessive work of certain groups of muscles. This varying condition of the respiratory quotient has been explained by Katzenstbik ' by the statement that during work two kinds of chemical processes act side by side. The one depends upon the work which is connected with the pro- duction of carbon dioxide also in the absence of free oxygen, while the other brings about the regeneration which takes place by the tak- ing up of oxygen. When these two chief kinds of chemical processes make the same progress the respiratory quotient remains unchanged 1 Speck, Physiologie des menscUliclien Athmens. Leipzig, 1892. » Pflager'S Arch., Bd. 49. ' Ibid., Bd. 49. 650 _ METABOLISM. during work; if by hard work the decomposition is increased as compared with the regeneration, then a raising of the respiratory quotient takes place. In sleep metabolism decreases as compared with that during waking, and the most essential reason for this is the muscular inactivity during sleep. The investigations of Rubnbr ' on a dog, and of LoEWT " on human beings, teach us that if the muscular work is eliminated the metabolism during waking is not greater than in sleep. The action of light also stands in close connection to the question of the action of muscular work. It seems positively proven that metabolism is increased under the influence of light. Most investi- gators, such as Speck,' Loeb,* and Ewald,' consider that this increase is due to the movements caused by the light or an increased muscle tonus. Fubini and BEif edicenti ' assume that the in- crease in metabolism due to light is independent of the movements. They base this assumption on experiments made on hibernating animals. Mental activity does not seem to have any influence on meta- bolism. Action of the External Temperature. In cold-blooded animals the production of carbon dioxide increases and decreases with the rise and fall of the surrounding temperature. In warm-blooded animals this condition is the reverse. By the investigations of LuDwiG and SANDERS-Bziir, Pflugbk and his pupils, and Duke Charles Theodore of Bavaria and others,' it has been demon- strated that in warm-blooded animals the change in the external temperature has different results according as the animal's own heat remains the same or changes. If the temperature of the animal sinks, the elimination of carbon dioxide decreases ; if the tem- perature rises, the elimination of CO, increases. If, on the contrary, the temperature of the body remains unchanged, then the elimina- tion of carbon dioxide increases with a lower and decreases with ' Ludwig-Pestschrift, 1887. ' Berlin, klin. Wochensclir., 1891, S. 434. » L. c. * Pflilger's Arch., Bd. 42. ' Journal of Physiol., Vol. 13. « See Maly's Jahresber., Bd. 33, S. 395. ' The pertinent literature may be found cited by Volt in Hermann's Hand- buch, Bd. 6, and also by Speck, 1. c. INFLUENCE OF EXTERNAL TEMPERATURE. 651 a higher external temperature. This fact may be explained, according to Pfluqek and Zuntz, by the statement that the low temperature, by exciting a reflex action in the sensitive nerves of the skin, causes an increased metabolism in the muscles witli an increased production of heat, affecting the temperature of the body, while with a higher external temperature the reverse takes place. The experiments made on animals are somewhat uncertain for several reasons, but the determinations of the oxygen absorption, as well as the elimination of CO,, made by Speck ' and Lobwt " on human beings, have shown that cold does not produce any essential increase in the metabolism of man. The irritation caused by cold may reflexly cause a forced respiration with an action on the gas exchange, and weak reflex muscular movements, such as shivering, trembling, etc., may cause an insignificant increase in the elimination of carbon dioxide; in complete muscular inactivity cold seems to cause no increased absorption of oxygen or increased metabolism. According to Lobwt the most essential thing in the regulation of heat under the influence of cold is, not an increased prodaction of heat, but rather a diminished loss of heat by contrac- tion of the skin and its vessels. Metabolism is increased by the partaking of food, and Zuntz' has calculated that in man the consumption of oxygen is raised on an average 15^ for about 6 hours after taking a moderately hearty meal. This increase in the metabolism is caused, according to the generally accepted view of Speck, probably only by the increased work of the digestive apparatus on the partaking of food. Fick* claims that the increased metabolism is due to the oxidation of the circulating, combustible material (proteid). This view, as shown by Magnus-Lbvt,' is not correct; but still Levy inclines to the vjew that besides the digestion work the proteids may possibly also have a specific exciting action on metabolism. ' L. c. « Pflilger's Arch., Bd. 46. ' Zuntz and Levy, Beitrag zur Kenntniss der Verdaulichkeit, etc., des Erodes. Pflilger's Arch., Bd. 49. * Sitzungsber. d. Wfirzb. phys.-med. Gesellsch., 1890. ' Pflilger's Arch., Bd. 55, contains the pertinent literature. 652 METABOLISM. VI. The Need of Food by Man under Various Conditions. Various attempts have been made to determine the daily quantity of organic food needed by man. Certain investigators have calculated, from the total consumption of food by a large number of similarly fed individuals, soldiers, sailors, laborers, etc., the average quantity of food required per head. Others have cal- culated the daily demand of food from the quantity of carbon and nitrogen in the excreta. Others again have calculated the quantity of nutritive material in a diet by which an equilibrium was main- tained in the individual for one or several days between the con- sumption and elimination of carbon and nitrogen. Lastly, others still have quantitatively determined during a period of several days the organic nutritive substances consumed daily by persons of vari- ous occupations who chose their own food, by which they were well nourished and rendered fally capable of labor. Among these methods a few are not quite free from reproach, and others have not as yet been tried on a sufficiently large scale. Nevertheless the experiments collected thus far serve, partly because of their number and partly because of the methods, to correct and control one another, and also serve as a good starting- point in determining the diet of various classes and similar questions. If the quantity of nutritive substance taken daily be converted iato calories produced daring physiological combustion, we then obtain some idea of the Sum of the chemical potential energy which under varying conditions is introduced into the body. It must not be forgotten that the food is never completely absorbed, and that undigested or unabsorbed residues are always expelled from the body with the faeces. The gross results of calories calculated from the food taken must therefore, according to Eubker,' be diminished at least 8^. The following summary contains certain examples of the qaantity of food which is consumed by individuals of various classes nader different coaditlons. In the last column we also find the quantity of living force which corresponds to the quantity of food in question, calculated as calories, with the above-stated correction. ' Zeitsehr. f. Blologie, Bd. 21, S. 379. NEED OF FOOD BT MAN. 653 The calories are therefore net results, while the figures for the nutritive bodies are gross results. Table XIV. Proteids. Fat. j,^gj^°gg Calories. Authority. Soldier during peace 119 40 529 3784 Platfair.' " light service 117 35 447 2434 Hildeshbim. " infield 146 46 504 2852 Latorer 130 40 550 2903 Molbschott. " at rest 137 72 352 2458 Pbttenkofer & Voir. Cabinet-maker (40 years). 131 68 494 2835 Fokster.* Young physician 127 89 3B3 2602 " 134 102 293 2476 Laborer 133 95 422 3902 English smith 1.76 71 666 3780 Platpair. pugilist 288 88 93 2189 Bavarian wood-chopper.. 135 208 876 5589 Liebig.- Laborer in Silesia 80 16 553 2518 Mbinert." ■ Seamstress in London. . . 54 29 392 1688 Playfaik. Swedish laborer 134 79 485 3019 Hultgren & Landbr- Japanese student 83 14 623 3779 Eijkman.' [grbn.* " shopman 55 6 394 1744 Ta-wara.!* It is evident that persons of essentially different weight of body who live under unequal external conditions must need essentially different food. It is also to be expected (and this is confirmed by the table) that not only the absolute quantity of food consumed by various persons, but also the relative proportion of the various organic nutritive substances, shows considerable variation. Eesults for the daily need of human beings in general cannot be given. For certain classes of human beings, such as soldiers, laborers, etc., results may be given which are valuable for the calculation of the daily rations. Based on extensive investigations and a very wide experience, VoiT has proposed the following average quantities for the daily diet of adults: Proteids. Fat. Carbohydrates. Calories. For men 118 grms. .56 grms. 500 grms. 2810 But it should be remarked that these statements relate to a man weighing 70 to 75 kilos and who was engaged daily for ten hours in not too fprtiguing labor. ' In regard to the older researches cited in this table we refer the reader to Voit ill Hermann's Handbuch, Bd. 6, S. 519. ' Ibid, and Zeitschr. f. Biologic, Bd. 9. ' Armee- and Volksernahrung. Berlin, 1880, * Investigations on the food of Swedish laborers with free selected diet. Stockholm, 1891. * Cited from Kelner and Mori in Zeitschr. f. Biologic, Bd. 35. 654 METABOLISM. The quantity of food required by a woman engaged in moderate work is about | that of a laboring man, and we may consider the following as a daily diet with moderate work : Proteids. Fat. Carbohydrates. Calories. For women 94 grms. 45 grms. 400 grms. 2240 The proportion of fat to carbohydrates is here as 1 : 8-9. Such a proportion occurs often in the food of the poorer classes, while the ratio in the food of wealthier persons is 1 : 3-4. The maximum quantity of carbohydrates in the food mast, according to Voit, not be above 500 grms. ; and as the carbohydrates besides constitute the chief part of the often very bulky vegetable foods, it has been sug- gested and is' desirable on this and other grounds to increase the quantity of fat at the expense of the carbohydrates in such rations. But because of the high price of fat such a modification cannot always be made. In examining the above numbers for the daily rations it must not be forgotten that the figures for the various nutritive bodies are gross results. They consequently represent the quantity of the nutritive bodies which mast be taken in, and not those which are really absorbed. The figures for the calories are, on the contrary, net results. The various foods are, as is well known, not equally digested and absorbed, and in general the vegetable foods are less completely used up than animal foods. This is especially true of the proteids. When, therefore, Voit, as above stated, calculates the daily quantity of proteids needed by a laborer as 118 grms., he starts with the supposition that the diet is a mixed animal and vegetable one, and also that of the above 118 grms. about 105 grms. are absorbed. The results obtained by Pflugee and his pupils Bleibtbeu " and Bohland" for the extent of the metabolism of proteids in man with an optional and sufficient diet correspond well with the above figures, when the unequal weight of body of the various persons experimeuted upon is sufficiently considered. As a rule, the more exclusively a vegetable food is employed, the smaller is the quantity of proteids in the same. The strictly vegetable diet of certain people, as of the Japanese and that of the so-called vegetarians, is therefore a proof that, if the quantity of food be sufficient, a person may exist on considerably ' Pfluger's Arch., Bd. 36. * lMd.,'BA. 38. NEED OF FOOD BY MAN. 655 smaller quantities of proteida than VoiT suggests. It. follows from the investigations of Hirschfeld, Kumagawa., and Klempbebk (see page 638) that a nearly complete or indeed a complete nitrog- enous equilibrium may be attained by the sufflsient administration of non-nitrogenons nutritive bodies with relatively very small quantities- of proteids. If we bear in mind that the food of people of different countries varies greatly, and that the individual also takes essentially different nourishment according to the external conditions of living and the influence of climate, it is not remarkable that a person accustomed to a mixed diet cannot exist for a long time on a strictly vegetable diet deficient in proteids, even though not especially difficult to digest. No one doubts the ability of man to adapt himself to a heterogeneonsly composed diet when this is not too difficult of digestion and is sufficient; but this ability does not seem sufficient reason for essentially altering the figures suggested by Voit. Although man may be satisfied under certain circumstances with a lower quantity of proteid than that calculated by Voit, still it does not follow that such a diet is also the most serviceable. Voit's fig- ures are only given for certain cases or certain categories of human beings. It is apparent that other figures must be taken for other cases, and it is evident that the daily ration given by Voit as neces- sary for a laborer must be altered slightly for other countries because of the existing conditions in middle Europe, where Voit made his investigations. For example, Hultgeen' and Landergrbn have shown in very careful investigations that the laborer in Sweden with moderate work and an average body weight of 70.3 kilos, with optional diet, partakes 134 grms. proteid, 79 grms. fat, and 522 grms. carbohydrates. The quantity of proteid partaken of is here greater than is necessary according to Voit. If we compare the figures of Table XIV with the average figures proposed by Voit for the daily diet of a laborer, it would seem at the first glance as if the consumed food in certain cases was con- siderably in excess of the need, while in other cases, as for instance for the seamstress in London, it was entirely insufficient. A posi- tive conclusion cannot, therefore, be drawn if we do not know the weight of the body, as well as the labor performed by the person, and also the conditions of living. It is certainly true that the amount of nutriment required by the body is not directly propor- tional to the bodily weight, for a small body consumes relatively 656 METABOLISM. more substance than a larger one, and varying quantities of fat may also cause a difference ; but a large body, which must maintain a greater quantity, consumes an absolutely greater quantity of sub- stance than a small one, and in estimating the nutritive need one must also always consider the weight of the body. According to VoiT, the diet for a laborer with 70 kilos bodily weight requires 40 calories for each kilo. As several times stated above, the demands of the body for nourishment vary with its varying conditions. Among these con- ditions two are especially important, namely, labor and rest. la a previous chapter, in which muscular labor was spoken of, it was seen that the generally accepted view is that non-nitrogenous food is the most essential, if not the exclusive, source of muscular force. As a natural sequence it is to be expected that in activity the non-nitrogenous foods before all must be increased in the daily rations. Still this does not seem to hold true in daily experience. It is a well-known fact that hard-working individuals — men and animals — require a greater quantity of proteids in the food than less active ones. This contradiction is, however, only apparent, and it depends, as VoiT has shown, upon the fact that individuals used to violent work are more muscular. For this reason a person perform- ing severe muscular labor requires food containing a larger propor- tion of proteids than an individual whose occupation demands less violent exertion. Another question is, how should the relative and absolute quantity of food be changed if increased exertion be demanded of one and the same individual ? An answer based upon experience may be found in statistics concerning the maintenance of soldiers in peace and in war. Many such statements are obtainable. In a critical examination of the s.ime it is found that in war rations the quantity of non-nitrogenous bodies as compared to the proteids is only increased in exceptional cases, while usually the reverse is the case. Even in these cases the actual proportion does not correspond with the theoretical demand, upon which, however, too great stress must not be placed, since in the case of soldiers in the field many other circumstances are to be considered, such as the volume and weight of the food, etc, etc., which cannot here be more closely discussed. The following table shows the average results of soldiers' rations in war and peace from NEMD OF FOOD BY MAN. 657 tho data given for various countries.' These average results al?o include the figures for Sweden. Tablk XV. A. Peace Eation. B. War Bation. Proteids. Fat. Carb. Proteids. Fat. Garb. Minimum 108 22 504 126 38 484 Maximum 165 97 731 197 95 688 Mean 130 40 .551 146 59 557 Sweden (proposed).... 179 102 591 202 137 565 if we do not consider the very abundant rations proposed for the soldier in Sweden, and if we only adhere to the above mean figures, we obtain the following results for the daily rations : Proteids. Fat. Carb. Calories. In peace 130 40 551 2900 Inwar 146 59 557 8250 If we calculate the fat in its equivalent quantity of starch, then the relation of the proteids to the non-nitrogenous foods is : In peace 1 : 4.97 In war , 1:4.79 The proportion is nearly the same in both cases; the slight difference which occurs shows a trifling relative increase in the proteids in the war ration. On the contrary, as is especially apparent from the total of the calories, the total quantity of nutri- tive bodies is greater in the war than in the peace ration. As more work requires an increase in the absolute quantity of food, so the quantity of food must be diminished when little work is performed. The question as to how far this can be done is of importance in regard to the diet in prisons and poorhouses. We give below the following as example of such diets : Table XVI. Proteids. Fat. Carb. Calories. Prisoner (not working) 87 22 305 1667 Schuster.' " .... 85 30 300 1709 Voit. Man in poorhouse 92 45 332 1985 Fobster.' Woman in " 80 49 266 1725 The figures given by Voit are, according to him, the lowest ' Germany, Austria, Switzerland, France, Italy, Bussia, and the United States, ' See Voit, Untersuchung der Kost. MUnchen, 1877. S. 142. » J6i(?., S. 186. 658 METABOLISM. figures for a non-working prisoner. He considers the following as the lowest diet for old non-working people : Proteids. ' Fat. Carb. Calories. Men 90 40 350 2200 Women 80 35 300 1733 In calculating the daily diet it is in most cases sufficient to ascertain how much of the various nutritive substances must be daily administered to the body to keep it in the proper condition to per- form the work required of it. In other cases it may be a question of improving the nutritive condition of the body by properly selected food ; but we also have cases in which we desire to diminish the mass or weight of the body by an insufficient nutrition. This is especially the case in obesity, and all the dietaries proposed for this purpose are chiefly starvation cures. The oldest and most generally known diet cure for corpulency is that of Harvey/ which is ordinarily called the Banting method. The principle of this cure consists in increasing, as far as possible, the consumption of the accumulated fat of the body by as limited a supply of fat and carbohydrates as possible and a simultaneous increased supply of proteids. A second cure, called EBSTBiif's ' cure, is based on the assumption (not correct) that the fat of the food is not accumulated in a body rich in fat, but is completely burnt. . In this cure large quantities of fat are therefore allowed in the food, while the quantity of carbohydrates is diminished very materially. The third cure, called Oektel's ' cure, is based on the correct view that a certain quantity of carbohydrates has no greater influence in the accumulation of fat than the isodynamic quantities of fat. In this cure, therefore, carbohydrates as well as fat are allowed, provided the total quantity of the same is not so great as to hinder the decrease in the fatty condition. A greatly diminished supply of water is also one of the features of Oertel's cure, especially in certain cases. The average quantity of the various nutritive substances supplied to the body in these three cures is as follows, and we give also for comparison in the same table Voit's diet necessary for a laborer : Proteids. Fat. Carb. Calories. Habvbt-Banting's cure 171 8 75 1066 . Bbbtbin's cure 102 85 47 1391 Okrtbl's " 156 22 72 1124 " (max.) 170 44 114 1557 Laborer, according to VoiT 118 56 500 2810 ' Banting, Letter on Corpulence. London, 1864. ' Ebstein, Die Fettliebigkeit und ihre Bebandlung. 1882. ' Oertel, Handbuch der allg. Therapie der Kreislaufst6rungen. 1884. STARVATION CUBES. 659 If the fat in all cases is recalculated in starch, then the propor- tion of the proteids to the carbohydrates is: Harvey-Banting's cure 100 : 54 Ebstbin's cure 100 ■ 246 Oertkl's " 100: 80 " "(max.) 100:139 Laborer 100 : 540 In all these cures for corpulence the quantity of non-nitrogenous bodies is diminished as compared with the proteids; but chiefly the total quantity of food, as is shown by the number of calories, is considerably diminished. Haetet-Banting's cure differs from the others in a relatively very much greater quantity of proteids, while the total number of calories in it'is the smallest. On this account this cure acts very quickly; but it is therefore also more dangerous and more difiicnlt to accomplish. In this regard Ebstein's and Oeetbl's cures (especially Oektbl's), having a greater variation in the selection of food, are better. As the adipose tissue has a proteid-sparing action, we have to consider in using these cures, especially Bantiko's, that the destruction of proteids in the body is not increased with the decrease in the adipose tissue, and one must therefore carefully watch the elimination of nitrogen by the urine. All diet cures for obesity are moreover, as above stated, starvation cures; and if the daily quantity of food required by an adult man, represented as calories, is in round numbers 2500 calories (according to the average figures found by Foestbr in the case of a physician), then one immediately sees what a considerable part of its own mass the body must daily give up in the above cures. This reminds us of the great care necessary in employing these cures; but each special case should be conducted with regard to the individuality, the weight of the body, the elimination of nitrogen in the urine, etc., etc., and always under strong control and only by physicians, never by a layman. A closer discussion of the many conditions which must be considered in these cases does not enter into the plan and scope of this work. 660 FOOD TABLES. TABLE I.— FOODS.' 1. Animal Foods. 1000 Farts contain f^m ■is Relationship of a. Flesh withotjt Boneb. Fat beef Beef (average fat ') Beef» Corned beef (average fat) Veal Horse, salted and smoked — Smoked ham Pork, salted and smoked * Flesli from hare " " chicken " partridge " " wild duck , b. Flbsh with Bones. Fat beef Beef, average fat' •Beef, slightly corned Beef, thoroughly corned Mutton, very fat " average fat Pork, fresh, fat Pork, corned, fat Smoked ham e. Fishes. River eel, fresh, entire Salmon, " " Anchovy, " " Flounder, " " .. River perch," " Torsk, " '•• 183 196 190 218 190 318 255 100 233 195 353 246 156 167 175 190 135 160 100 120 200 89 131 128 145 100 166 98 120 115 80 65 365 660 11 93 14 31 141 83 93 100 333 160 460 540 300 330 67 39 14 2 1 11 18 18 117 13 125 100 40 13 11 14 12 9 15 85 100 8 10 5 60 70 6 10 11 11 8 8 640 688 672 550 717 493 280 130 744 701 719 711 544 585 480 430 437 520 365 200 340 150 150 16 180 88 150 70 80 90 352 333 469 333 489 333 580, 250 440' 450 455 4.'50 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 90 50 63 53 42 30 143 660 5 48 6 13 90 49 53 53 246 100 460 450 150 346 56 31 9 3 1 a & a ' The results in the following tables are chiefly compiled from the summary of AlmSk and of KBnio. As " waste " we here designate that part of the foods which is lost in the preparation of the food or that which is not used by the body; for instance, the bones, sliin, egg-shell, and the cellulose in the vegetable foods. 3 Meat such as is ordinarily sold in the markets in Sweden. ■ Beef such as is delivered by large purveyors to public institutions in Sweden. < Pork, chiefly from the breast and belly, such as occurs in the rations of Swedish ■oldiers. ANIMAL FOODS. t)61 TABLE l.—FOOD^.—{ContvnmA.) Animal Foods. Pike, fresh, entire Herring, salted, entire Anchovy, " " Salmon (side), salted Eabeljau (salted haddock). . . Codfish (dried ling) " (dried torsk) J^h-meal from variety of Gaottb d. Inner Obgaks (Fresh). £rain Beef -liver Beef-he.art Heart and lungs of mutton. . . . Veal-kidney •Ox-tongue (fresh) Blood from various animals (average results) e. Other Animal Foods. Kind of pork-sausage (Mett- wurst) Same for frying Butter Lard Meat extract Oow's milk (full) " " (skimmed) Buttermilk Cream Cheese (fat) " (poor) Whey cheese (poor) Hen's egg, entire " •' without shell Tolk of egg White" " 1000 Farts contain 82 140 116 200 246 532 665 736 116 196 184 163 221 150 182 190 220 7 3 304 35 35 41 37 230 334 89 106 133 160 103 1 140 43 108 4 5 10 7 103 56 92 106 38 170 150 160 850 990 35 7 9 257 270 66 70 93 107 307 7 11 50 50 38 35 40 50 456 4 5 6 100 107 132 178 106 59 87 50 55 15 175 7 7 7 6 60 50 56 8 10 13 8 461 450 100 280 340 100 334 460 472 257 116 170 770 720 714 721 728 670 807 610 565 119 7 217 873 901 905 665 400 500 329 654 756 520 875 Relationship of 400 100 100 100 150 135 100 100 100 100 100 100 100 100 100 100 100 100 100 1 100 37 54 1 1 1 1 89 28 50 65 17 113 1 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 79 73 12100 33000 100 20 22 695 117 19 79 192 7 100 143 143 93 95 17 15 512 4 4 7 662 FOOD TABLES. TABLE I.— FOODS.— (CoraW?iM6(i.) 2. Vegetable Foods. "Wheat (grains) Wheat-flour (fine) " (very fine) Wheat-bran Wheat-bread (fresh) Macaroni. , Eye (grains) Rye-flour Rye-bread (dry) " " (fresh, coarse) " (fresh, fine) Barley (grains) Scotch barley Oat (grains) Oat (peeled) Corn Rice (peeled for boiling) French beans Peas (yellow or green) Flour from peas Potatoes Turnips Carrot (yellow) Cauliflower. Cabbage Beans Spinach Lettuce Cucumbers Radishes Edible mushrooms (average).. . Same dried in the air (average), Apples and pears Various berries (average) Almonds Cocoa lOPO Farts contain 123 110 93 150 88 90 115 115 114 77 80 111 110 117 140 101 70 233 330 370 20 14 10 35 19 37 31 14 10 12 33 319 4 5 343 140 2 3- 17 10 11 39 10 3 17 15 30 10 14 31 10 60 60 ,58 7 31 15 15 3 2 2 4 2 1 5 3 1 1 4 35 587 480 676 740 768 439 550 768 688 730 725 480 514 654 720 563 660 656 770 537 530 520 200 74 90 50 49 66 83 22 60 412 180 90 73 180 18 8 3 50 17 8 18 30 15 16 11 36 ■5 30 30 17 2 36 35 25 10 7 10 8 12 6 19 10 4 7 9 61 3 6 39 50 140 120 120 130 330 181 140 110 110 400 370 140 146 130 100 140 146 137 150 135 760 893 873 904 900 888 908 944 956 934 877 160 833 849 54 55 26 3 6 193 5 32 20 16 17 11 48 7 100 20 28 5 37 60 45 8 10 15 1 12 8 7 6 8 18 133 31 50 66 95 Belationship of 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 loo 100 100 100 100 100 100 100 100 100 100 100 100 100 14 11 13 26 11 3 15 13 18 14 18 19 9 51 43 57 10 9 7 6 10 14 20 16 11 4 16 31 10 8 13 13 333 343 549 654 835 293^ 625 853 600 626 634 623 634 589- 654 481 471 662 1100- 231 240 193 1030- 539 900 200 358 344 106 157 230- 317 188 188 3350- 1800 30 129' MALT AND ALCOHOLIC LIQUOBS. 663 TABLE II.-MALT LIQUORS. 1000 Farts b^ Weight contain S as -S'R Porter. . , Beer (Swedish) " (Swedish export). . . Draught- beer Lagerbeer Bock-beer Weiss-beer Swedish " Svagdricka". . 871 887 885 911 903 881 916 945 76 13 65 73 2 1.5 1.7 4 33 TABLE III. -WINE AND OTHER ALCOHOLIC LIQUORS. 1000 Parts by Weight contain ^ ^ £a s 5 sl ^ y jj 1 1^ 1 ill 1 ^ 883 94 23 6 5.9 2.0 863 115 23 4 5.0 2.0 776 90 134 115 6.0 1.0 1.0 801 94 105 87 6.0 1 2.0 808 130 72 51 7.0 9.0 3.0 795 170 35 15 5.0 6.0 p.o 774 164 62 40 4.0 3.0 3.0 791 156 53 33 5.0 3.0 B.O 790 164 46 35 5.0 4.0 4.0 479 363 460 550 443-590 333 260-475 I. Bordeaux wine White wine (Rheingau). Champagne Rhine wine (sparkling). Tokay Sherry Port- wine Madeira Marsala Swedish punch Brandy French cognac Liqueurs 60-70 664 INDEX TO SPECTRUM PLATE. SPECTEUM PLATE. 1. Absorption spectrum of a solution of oxyhmmogloMn. 2. Absorption spectrum of a solution of hcemoglobin, obtained by the action of an ammoniacal ferro-tartrate solution ou an oxybaemoglobin solution. 3. Absorption spectrum of a faintly-alkaline solution of meiJuemoglobin. 4. Absorption spectrum of a solution of Juematin in ether containing oxalic acid. 5. Absorption spectrum of an alkaline solution of hcematin. 6. Absorption spectrum of an alkaline solution of hmmochromogen, obtained by the action of an ammoniacal ferro-tartrate solution on an alkaline- haematin solution. 7. Absorption spectrum of an acid solution of urobilin. 8. Absorption spectrum of an alkaline solution of urobilin after the addition of a zinc-chloride solution. 9. Absorption spectrum of a solution of lutein (ethereal extract of the egg-yolk). B C D E b * I-5' k y ft 4 >o // js lilllillllllllllilllliillHiliiiiliiiiltiiitnil F G /tf /g /|r /S to so Qi S3 23 ?* |?5 a /? HIZ ", /? I n ra IV El a- I lllll|llllll|l| | ||||[|||lllll(|l[ s. ■=■ yS TrTr|PipiTp[iii |i iii|iiii|i i i ij iiii|i ii|m |iiii|i i ii | iii i |ii i i | i i ii|mqiiiipTT[iiii^ }' 5 6 f 13 & iO il IB Itfi lY, 16 16 Al 18 }9 ZO Sf 22 ' 23 2V 2S BCD E t F INDEX. Absorption, 326—341 , importance of cells in, 330, 340, 341 , action of putrefactive processes in the intes- tine on, 320 Absorption ratio, 147 Acetic acid in intestinal contents, 314 in gastric juice, 264 contents, 264, 283, 288 , passage of, into the urine, 505, 523 Acetone, 558 in blood, 176 in urine, 556, 557 Aoetonuria, 556, 557 Acetyl-amido-benzoic acid, 528 Acetylene, compound with haemoglo- bin, 140 Acholia, pigmentary, 242 Achromatin, 96 Achroo-dextrin, 78, 256 Acid albuminates, 16 , properties, 32, 33 , formation in peptic digestion, 271, 272 Acid amides, behavior in the animal body, 523 Acid rigor, 375 Acids, organic, behavior in the ani mal body, 449, 505, 517, 518, 523 Acidity of urine, 448 — 451 of the gastric contents, 284 of the muscles, 360, 376 Acrite, 67 Acrolein, 82 Acrolein test, 82, 85 Aeroses, 67 Acrylic acid, action on the elimination of uric acid, 473 Acrylic acid diureid. See Uric acid. Actinioehrom, 578 Adamkiewicz's reaction, 27 Adelomorphic cells, 261 Adenin, 102 , properties, reaction, and oc- currence, 107 , urine, 484 Adenylic acid, 99 Adhesion, importance in blood coagu- lation, 155 Adipocere, 355 iEgagropila, 325 Aerotonometer, 597, 600 Alanin, 57 Albumins, 18 , general properties, 30. See also the various albu- mins. Albumin, detection in urine, 531 , quantitative estimation, 536 See Proteids. Albuminates, 18 , properties and reactions, 32, 33 , ferruginous albuminate in the spleen, 201 Albuminoids, 18, 49 in cartilage, 347 666 INDEX. AlbumoidSj 18, 49 in the crystalline lens, 401 Albuminous bodies. See Proteid bodies. Albuminous glands, 251 Albumoses, IS , general properties, 33 — 42 , formation in putreiaction of proteid, 314 , formation in pepsin diges- tion, 271 , formation in trypsin di- gestion, 302 , nutritive value, 636 , absorption, 327 , transformation of, into proteid, 329 5 occurrence of, in the blood, 328 , occurrence in the urine, 535 Alcapton and alcaptonuria, 493, 498, 499 Alcohol. See Ethyl alcohol. Alcoholic fermentation, 9, 68 in intestine, 313 in milk, 429 Aldepalmitic acid, 424 Aldoses, 60, 63 Aleuron grains, 411 Alexines, 16, 179 Alkali albuminate, 18 , properties and re- actions, 32, 33 , occurrence in the yolk of the egg, 412 occurrence in the brain, 390 occurrence i n smooth muscles, 389 , Lieberkuhn's, 32 Alkali carbonate, physiological impor- tance, 628 , action on the secre- tion of gastric Juice, 263 Alkali carbonate, occurrence. See the various tissues and fluids. Alkali phosphates in urine, 448, 477, 513 , occurrence. See the various tissues Alkali urates, 447, 478 in calculi, 569 in sediments, 447, 566 Alkaline earths in urine, 519 in bones, 349 , insufficient supply of, 351, 629 Alakaline fermentation of urine, 565 Alkaloids, action on the muscles, 375 , passage of, into the urine, 530 , retention by the liver, 206 Alimentary glycosuria, 220, 332 Alimentary oxaluria, 482 Alizarin in the urine, 530 , feeding with, 351 Alizarin blue, behavior in the tissues, 5 Allantoic fluid, 483 Allantoin, properties and occurrence, 483 , in transudations, 191, 194, 419 , formation from uric acid, 472 Alloxan, 472, 478 AUyl alcohol, relationship to forma- tion of glycogen, 214 Almen-Bottger's sugar test, 69, 546 Almen's guaiacum blood test, 539 Amanitin, 93 Ambergris, 326 Ambrain, 326 Amido-acids, relation to formation of uric acid, 475 , relation to formation of urea, 455, 523 , formation in putrefac- tion, 23, 314 from protein substances, 20—22, 50, 54, 56, 57, 302, 314 in trypsin digestion, 302 INDMX. 667 Amido-acetio acid. See GlyoocoU. Aniido-benzoic acid, behavior in the animal body, 528 Amido-caproic acid. See Leuciu. Amido-cinnamic acid, 526 Amido-ethylen-lactic acid. See Serin. Amido-oxyethyl-sulphonie acid. See Taurin. Amido-phenyl-acetic acid, behavior in body, 527 Amido-phenyl-propionic acid, forma- tion in the putrefaction of proteids, 23, 486 Amido-phenyl-propionic acid, behavior in organism, 526, 527 Amido-succinic acid. See Aspaitij acid. Amidulin, 76, 256 Ammonia, estimation of, 518 formation in proteid putre- faction, 314 from protein substances, 20, 21, 302, 314 in trypsin digestion, 302 , occurrence in blood, 176 , occurrence in the urine, 448, 454, 458, 459, 517, 518 Ammonia elimination after adminis- tration of mineral acids, 517, 518 Ammonia elimination in diseases of the liver, 454, 459 Ammonia elimination after extirpa- tion of the liver, and atrophy ex- periments, 459, 475 Ammonium chloride, relation to forma- , tion of urea, 517 , action on metab- olism, 643 Ammonium salts, relation to formation of glycogen, 214 , relation to uric-acid formation, 475 , relation to urea formation, 455, 457 Ammonium-magnesium phosphate in urinary calculi, 569, 570 Ammonium-magnesium phosphate in urinary sediments, 568 Ammonium urate in urinary calculi, 569 Ammonium urate in urinary sedi- ments, 566 Amniotic fluid, 419 Amphicreatinin, 369 Amphopeptone, 35 Amyl nitrite, poisoning with, 177 Amylodextrin, 76 Amyloid, 57 , vegetable, 79 Amyloid degeneration, bile in, 242 Amyloid degeneration, chondroitin- sulphuric acid in the liver, 344 Amylolytic enzymes, 13, 255, 297 Amylopsin, 297 Amylum. See Starch. Anssmia, pernicious, 174 Anhydride theory of glycogen forma- tion, 215 Anilin, behavior in the animal body, 526 Anisotropous substance, 361 Antedonin, 578 Anthrax protein, 19 Anthrax spores, behavior with gastric juice, 282 Antialbumose, 35 Antimony, passage of, into milk, 443 , action on elimination of nitrogen, 453 Antipeptone, 35 in meat extract, 368 Antipyrin, relation to formation of glycogen, 214 ^ , action on urine, 530 Apatite, 349 Approximate estimation of proteid in urine, 536 Aqueous humor, 195 Arabinoses, 61, 65, 80 , relation to formation of glycogen, 213 Arabit, 61 Arachidic acid in butter, 424 INDEX. Arachnoidal fluid, 190 Arbutin, relation to glycogen forma- tion, 214 , behavior in animal body, 493 Arginin, 50 Aromatic compounds, behavior in ani- mal body, 525—531 Arsenic, passage of, into milk, 443 in perspiration, 581 , action on the elimination of nitrogen, 453 Arsenious acid, action on pepsin diges- tion, 270 Arsenuretted hydrogen, poisoning with, 244—247, 538 Arterin, 130 Ascitic fluids, 193 Asparagin, relation to proteid synthe- sis, 23 , relation to formation of glycogen, 214 , nutritive value, 637 Asparaginic acid. See Aspartic acid. Asparagus, odoriferous bodies of, in the urine, 530 Aspartic acid, relation to formation of uric acid, 475 , relation to formation of urea, 455 , formation from proteid, 21, 302, 307 , behavior in organism, 455, 475, 523 Asphyxiation, blood in, 5, 130, 154, 585 Assimilation. See Absorption. Assimilation limit, 220, 333 Ass's milk, 434 Atmidalbumin, 36 Atmidalbumose, 36 Atmid substances, 36 Atropin, action on the elimination of uric acid, 473 , action on the secretion of saliva, 259 Auto-intoxication, 15 Auto-oxidation, 8 Bacteria ureae, 565 Bactericidal action, 16, 179 Banting cure, 658, 659 Bases, nitrogenous, from proteids, 21 Beeswax, 87 Benzaldehyde, oxidation, 4 , substituted aldehyde, behavior in animal body, 528 Benzoic acid, formation from protein substances, 24, 54, 486 , passage of, into the per- spiration, 581 , behavior in the organ- ism, 3, 486, 527 , occurrence in the urine, 489 , action on metabolism, 643 , substituted benzoic acids, action in body, 527 Benzol, behavior in the animal body, 525, 526 Benzoyl-amido-acetic acid. See Hip- puric acid. Benzoyl-chloride, behavior with car- bohydrates, 70, 212 , behavior with eys- tin, 563 Benzoyl-cystin, 563 Bezoar-stone, 325 Bifurcated air, 596, 601 Bile, 222—250 , general chemical properties, 224 , analysis of, 239, 240 , antiseptic action of, 318, 319, 320 , constituents of, 225, 236 in diseases, 241 , diastatic action of, 238, 309 , influence upon proteid digestion, 312, 313 , influence upon the emulsification of fats, 311 , influence upon the secretion of bile, 224 INDEX. 669 Bile, influence upon the absorption of ■ fats, 312, 336 , influence upon the splitting of neutral fat, 310 , influence upon trypsin digestion, 301, 311 , quantity of, 222 , absorption of, 224, 339 , transit of foreign bodies into the, 241, 242 , occurrence of, in urine, 339, 542 — 544 , occurrence of, in contents of stom- ach, 283, 312 , occurrence of, in meconium, 323 , composition of, 239, 240 , chemical formation of, 242 — 247 , secretion of, 222 Bile-acids, 225—230 in blood, 176, 242 in pus, 200 in urine, 339, 542 , absorption of, 339 , Pettenkofer's test for, 226 Bile-mucus, 224 Bile-pigments, 233—239 , origin and formation of, 242—247 , reactions of, 234, 235, 543, 544 , passage into urine of, 543, 544 , occurrence in blood- serum, 125, 176 , occurrence in egg-shells, 416 Bile-salts, 225 Bilianic acid, 229 Biliary calculi, 247, 248 Biliary fistulse, 222 , influence on putrefac- tion in the intes- tine, 319 , influence on the want of food, 319 Bilieyanin, 233, 235, 237 Bilifulvin, 233 Bilifuscin, 233, 237, 248 Bilihumin, 233, 237 Biliphoein, 233 Biliprasin, 233, 237 Bilirubin, 233 , relationship to the blood- pigments, 145, 244, 245 , relationship to haematoidin, 145, 233, 244 , properties, 234 , occurrence, 233 occurrence in the corpora lutea, 406 , occurrence in urine, 542 , occurrence in the placenta, 419 Bilirubin-calcium, 233, 247 Biliverdin, properties, 236 , occurrence, 208 , occurrence in egg-shells, 416 , occurrence in excrements, 332 , occurrence in urine, 542 , occurrence in the placenta, 419 Birotation, 64 Bismuth, passage of, into the milk, 443 Bitch's milk, 434 Biuret, 459 Biuret, reaction, 27, 459 Blister fluid, 196 Blood, 111-179 , general behavior. 111, 152, 153 , coagulation of, 153 — 161 , gases of. See Chemistry of respiration. Chapter XVII. , quantitative analyses, 163 — 169 , arterial and venous, 130, 169, 584, 585, 596, 601 , defibrinated, 112 in asphyxiation, 5, 130, 151, 5^'5 , quantity in the body, 177 , detection, chemico-legal, 145 , behavior in starvation, 172, 624 , composition under abnormal conditions, 173—177 , composition under physiological conditions, 169—173 670 INDEX. Blood in urine, 538—540 in gastric contents, 283 , transfusion of, 173, 178 , loss of, 177 Blood-casts, 538 Blood-clot (placenta sanguinis), 112, 153 Blood-corpuscles, white, 149, 150, 174 , white, behavior in the coagulation of blood, 150, 156—158 , white, relationship to the formation of uric acid, 476 , red, 128—130 , red, in urine, 538 , red, composition, 127, 168, 174 Blood-pigments, 130—149 in bile, 242 in urine, 538 — 540 Blood-plasma, 113—122 , composition of, 126, 107 Blood-plates, 149, 151 , importance in coagula- tion, 156 Blood-serum, 112, 122—127 , globulicidal action, 178 , composition of, 126, 167 Blood-spots, 145 Blood-sweat, 582 Blue stentorin, 578 Bone and bony tissue, 348 — 354 in starvation, 513, 623 Bone-earths, 349, 350 Bone, softening of, 352 Bonellin, 578 Borax, action on metabolism, 643 on trypsin digestion, 301 Bcfrneol, 529 Bottcher's spermin crystals, 404 Bottger-Almen's test for sugar, 69, 546 Bowman's disks, 361 Brain, 390—402 Bread, behavior in the stomach, 277 , excrement after, 321 Bromadenin, 103 Bromanil, 24 Bromhypoxanthin, 103 Bromine compounds, passage of, into the saliva, 260 Bromoform, 24 Brunner's glands, 289 Buccal mucus, 253 Buffy coat, 153 Bufidin, 579 Bull, spermatozoa of, 405 Bursse mucosae,, 197 Butalanin, 57 Butter-fat, 425, 435 , calorific value of, 617 , absorption of, 336 Buttermilk, 434 Butyl alcohol, behavior in animal body, 524 Butyl-chloral hydrate, behavior in ani- mal body, 524 Butyric acid in contents of stomach, 283, 288 in gastric juice, 264 in milk fat, 424, 435 Butyric-acid fermentation, 5, 69 in intestine, 316 Byssus, 18, 57 Cadaverin, 14 CafiFein, action on the muscles, 375 Calcium, lack of, in food, 352, 629 , occurrence. See various tis- sues and fluids. Calcium salts, significance for the co- agulation of blood, 116, 117, 159 , significance for the co- agulation of milk, 426 , significance for the co- agulation of muscle plasma, 363 See also various cal- cium salts. Calcium carbonate in urine, 447 in urinary calculi, 570 INDEX. 671 Calcium carbonate in urinary sedi- ments, 567 in bones, 349, 350, 353 in tartar, 261 Calcium formate, enzymotio decompo- sition, 11 Calcium oxalate in urine, 481, 483 in urinary sediments, 482, 567 in urinary calculi, 570 ■Calcium phosphate, relation to co- agulation of fibrinogen, 117, 118, 159 Calcium phosphate, relation to coagu- tion of casein, 426 Calcium phosphate, occurrence in in- testinal calculi, 325 Calcium phosphate in urine, 447, 512, 513, 519 Calcium phosphate in urinary sedi- ments, 567 Calcium phosphate in urinary calculi, 570 Calcium phosphate in salivary calculi, 261 Calcium sulphate in urinary sediments, 567 Calories of the food, 617, 618 of various dietaries, 653 Campho-glycuronic acid, 506, 529 Camphor, behavior in the body, 506, 529 Camphoral, 529 Cane-sugar, 72, 73 , inversion of, 290, 309, 332 , caloric value of, 617 , absorption of, 339 , b e h a vi o r to intestinal juice, 290 , behavior to gastric juice, 272 Capillary endothelium, secretory sig- nificance of, 187, 189 Capric acid, 424, 435 Caproic acid, formation from phenol, 7, 525 Caproic acid, occurrence in fatty tis- sue, 354 , occurrence in milk-fat, 424, 435 Caprylic acid, 424 Caramel, 68, 73 Carbamic acid, 467 in the blood, 125, 456 in the urine, 456, 467 ( poisonous action of, 456 Carbamic-acid ethylester, 467. See Urethan, 467 Carbolic acid, action on pepsin diges- tion, 270 See also Phenol. Carbolic urine, 493 Carbohaemoglobin, 139 Carbohydrates, 59 — 80 , importance for the formation of fat, 358 , importance for fh e formation of glyco- gen, 214, 215 , importance for muscu- lar activity, 378, 384, 385 , action on the metab- olism of proteids, 630, 639 , action on putrefaction, 318, 490 , absorption of, 332, 334 , inadequate supply of, 630 See also the various carbohydrates. Carbon dioxide in the' blood, 583 — 590 in diabetes, 590 in poisoning vifith min- eral acids, 590 in the intestine, 314 316 in the Tymph, 182, 590 in the stomach, 278 in the muscles in ac- tivity and at rest, 378, 384 in rigor mortis, 376 672 INDEX. Carbon dioxide in secretions, 591 in transudations, 592 , binding of CO in the blood, 586—590 , action on the secretion of gastric juice, 262 , action on the secre- tion of pancreatic juice, 296 , tension in blood, 601, 602 , tension in tissues, 603 , tension in the lymph, 182 , tension in transuda- tions, 592 elimination, depend- ence on the exter- nal temperature, 650, 651 elimination in ac- tivity and at rest, 378, 379, 384, 647— 650 elimination by the skin, 582 elimination in vari- ous ages, 646, 647 haemoglobin, 139 Carbon-monoxide poisoning, 139, 176, 372 Carbon-monoxide poisoning, action en lactic acid formation, 372 Carbon-monoxide poisoning, action en the elimination of nitrogen, 453 Carbon-monoxide poisoning, action on the elimination of sugar, 220, 372 Carbon-monoxide blood test, Hopfe Seyler's, 139 Carbon-monoxide hsemoglobin, 133, 140 Carbon-monoxide methsemoglobin, 139 Carminic acid, 578 Carnic acid, 366, 368 Carniferrin, 363 Cariiin, 102, 367 in urine, 484 Carp, sperma of, 100, 106, 406 Cartilage, 343—348 Cartilage, amount of ash in, 347 , behavior to gastric juice, 271, 276 , behavior to pancreatic juice, 307 Cartilage gelatin, 53, 343 Cartilage of the knee-joint, 347 Casein, origin, 420, 441 from woman's milk, 436 from cove's milk, 425 , quantitative estimation, 431 , behavior with rennet, 273, 426, 436 , behavior with gastric juice, 270, 277, 427, 436 , caloric value of, 617 , phosphorus of, 427 Caseinogen, 427 Caseoses, 36 Castor bean, 15 Castoreum, 579 Castorin, 579 Cataract, 402 Catheterization of the lungs, 595, 601 Cat's milk, 434 Cells, animal, 88—110 Cell constituents, primary and second- ary, 89 Cell fibrinogen, 102 Cell globulin, 90, 129 Cell membrane, 92 Cell nucleus, 96 , relation to fibrin coagu- lation, 151, 156, 157 Cellulose, 79 , fermentation of, 310, 317 , occurrence in tuberculcsis, 605 , a c t i o n on absorption of foods, 331 Cement, 353 Cerebrin, 72, 394 , properties and behavior, 394, 395 in pus, 199 Cerebrosides, 392, 393 Cerebrospinal fluid, 195 Cerolein, 87 INDEX. 673 Cerotic acid, 87 Cerumen, 579 Cetin, 86 Cetyl alcohol, 87 ChalaztL, 413 Charcot's crystals, 175, 404, 606 Charge of the stomach with pepsin, 275 of the pancreas with pepsin, 202 Cheese, 277, 427 Cheno-tauroeholic acid, 228 Chief cells, 261, 274, 275 Children's urine, 447, 454, 483 Chitin, 56, 58, 574 , behavior in trypsin digestion, 308 Chitosan, 575 Chloral hydrate, absorption, 339 , behavior in animal body, 506, 524 Chlorate, poisoning with, 136, 538 Chloraaol, 24 Chlorbenzol, behavior in animal body, 529 Chlorides, elimination by the urine, 127, 509, 510 , elimination by the sweat, 580, 581 , action on proteid metab- olism, 642, 643 , insufficient supply of, 627 See also the various fluids and tissues. Chlorocruorin, 148 Chloroform, action on the elimination of chlorides, 510 , action on the muscles, 375 Chlorophan, 399 Chlorophyll, 2 Chlorosis, 174 Chlorphenyleystein, 529 Chlorphenylmereapturic acid/ 529 Chlorrhodinic acid, 200 Cholagogues, 224 Cholalic acid, 228 , relation to cholesterin, 248 Cholanic acid, 230 Cholecyanin, 234, 235 Choleglobin, 246 Choleic acid, 230 CholepyiThin, 233 Cholera, blood in, 173, 175, 176 , contents of intestine in, 324 , sweat in, 581 , ptomaines in, 14 Cholera bacilli, behavior in gastri: juice, 282 Cholesterilin, 248 Cholesterin, 248 in expectorations, 606 in bile, 238, 239, 240 in biliary calculi, 248 in the brain, 391, 397 in the urine, 562 , importance of, in the life processes of the cells, 96 Cholesterin calculi, 248 Cholesterin fat as protective fat, 578 Cholesterin-propionic ester, 249 Choletelin, 233, 236 , relation to urobilin, 501 Cholin, 15, 93, 238 Cholohaematin, 237 Choloidic acid, 230 Chondrigen, 343 Chondrin, 56, 343 , in pus, 200 Chondrin balls, 346 Ghondrosin from ehondroitin sulphuric acid, 345, 506 from gelatinous sponges, 47 Chondroitic acid, 344 ehondroitin, 345 Chondroitin-sulphuric acid, 344, 506, 574 Chondromucoid, 47, 344 Chorda saliva, 252 Choroid coat, 402 , pigment of, 576 Christensen and Mygge's method for the approximate estimation pf pro- teid in urine, 537 Chromatin, 96 Chromhidrosis, 581 Chromogens in urine, 499 674 INDEX. Chromogens in the supra-renal capsule, 205 Chrysophanic acid, action on urine, 530 Chyle, 180—183 Chyloperieardium, 192 Chyluria, 561 Chyme, 276 , investigation, 284 — 289 Chymosin, 13, 272, 426 in urine, 508 Cinnamic acid, behavior in the animal body, 486 Citric acid in milk, 425, 433, 437 Cleavage processes. See Splitting proc- esses. Coagulation of the blood. 111, 112, 115 — 119, 154 — 163, 169, 170, 171, 175 .intravascular, 162 of milk, 422, 426, 436 of muscle-plasma, 361, 363, 376 Cobalt hydrocarbonate, behavior to gastric juice, 264 Coccygeal glands, 579 Cochineal, 578 Coefficient, HSser'a, 521 , respiratory, 384, 615, 623 , urotoxic, 509 Coffee, action on metabolism, 644 Collagen, 18, 53, 342, 343, 346, 348 Collidin, 14 Colloid, 47, 407, 408 Colloid corpuscles, 407 Colloid cysts, 407 Coloring matters. See Pigments. Colostrum of woman's milk, 438 of cow's milk, 433 Colostrum corpuscles, 433, 442 Combustion, physiological, 6 Comma bacillus, behavior in gastric juice, 282 Compound proteids, 18, 43 — 49 in protoplasm, 91, 101, 292, 420 Conchiolin, 18, 57 Concrements. See various calculi. Cones of the retina, pigment of, 398 Conglutin, calorific value, 517 Connective tissues, 342 Copaiva balsam, action on the urine, 530 Copper in the blood, 125, 168 in the bile, 238 in biliary calculi, 248 in hsemocyanin, 148 in protein substances, 17 in turacin, 577 Cornea, 348, 402 Cornein, 18, 57 Corriicrystallin, 57 Corpora lutea, 406 Corpulence, diet cures for, 658, 659 Corpuscula amylacea, 395 Cow's milk, 421—434 , general behavior, 42?, 422 , analysis of, 430 — 433 , constituents, inorganic, 432—433 , constituents, organic, 423 —430 , checking action on putre- faction, 318, 490 , coagulation with rennet, 273, 422, 426 , behavior in the stomach, 276, 281, 282 , composition of, 432 — 434 Cream, 434 Creatin, relation to the formation of urea, 367, 454 , relation to muscular activity, 381, 384 , properties and occurrence, 366, 367 Creatinin, relationship to muscular ac- tivity, 381, 384, 467 , properties and occurrence, 467 , zinc chloride, 468 Cresol, 22, 314, 489, 490 Cresol-sulphuric acid, 489, 490 Crotonic acid, 561 Crotyl alcohol, relationship to forma- tion of glycogen, 214 INDEX. 675 Cruor, 112 Crusocreatinin, 369 Crustaceorubin, 578 Crusta inflammatoria or phlogistica, 153, 175 Crystalbumin, 401 Crystalfibrin, 401 Crystallin, 18, 401 Crystalline lena, 400—402 Cumic acid, 527 Cmninuric acid, 528 Curare poisoning, action on muscular tonus, 378 , action on elimina- tion of sugar, 220 Qyanmetheemoglobin, 140 Cyanocrystallin. 417, 578 Cyanogen in proteid molecule, 4 Cyanuric acid, 459, 471 Cyanurin, 500 Cymol, 527 <^stein, 529, 562 , conjugation in animal body, 529 Cystin, properties, 562 , occurrence in urine, 507, 509, 562 , in urinary calculi, 570 , in urinary sediments, 568 , in sweat, 581 , in trypsin digestion, 563 Cystinuria, 14, 509, 562 Cysts, tapeworm, 196 , ovarial, 406 — 410 , thyroid, 204 Cytin, 102 Cytoglobin, 18, 91, 102, 157 Cytoplasm, 90 Cytosin, 100 Damaluric acid, 509 Damolic acid, 509 Dehydrocholalic acid, 229 Dehydrocholeio acid, 230 Delomorphic or parietal cells, 261, 273, 275 Denige's reaction for uric acid, 478 Dentin, 350, 353 Descemet's membrane, 47, . 348 Desoxycholalic acid, 229, 230 Deuteroalbumose, 36, 40, 535 Deuteroelastoae, 52 Deuterogelatose, 55 Devoto's method of determining the quantity of proteid, 29, 535 Dextrins, 77, 78 , formation from starch, 78, 256 , loading the stomach with, 275 , occurrence in the contents of the stomach, 277 in muscles, 271 in portal blood, 170, 332 Dextrin-like substances in the urine, 505 Dextrose, 67 — 71 in the blood, 123 170, 217, 218—220 in the urine, 123, 218, 544r— 554 in the lymph, 181 in the muscles, 371 , preparation of, 71 , caloric value of, 617 , detection of, 71, 544—549 , reactions of, 68, 69, 70 , absorption of, 339, 340 , quantitative estimation in , the urine, 549 — 554 Diabetes mellitus, 219, 220, 221, 293, 544 , elimination of NHs by the urine in, 518 , relation of the liver to, 219—221 , relation of the pan- creas to, 221, 293 , to elimination of sugar, blood in, 176, 221 , quantity of sugar in the blood in, 176, 219 676 INDEX. Diabetes mellitus, urine in, 447, 522, 544 , carbon dioxide in the blood in, 590 , oxybutyric acid in tiie blood in, 590 , oxybutyric acid in the urine in, 518, 560 Diacetic acid, 559 in urine, 556, 557 Diagonal disks of the muscles, 361 Diamid, poisoning with, 483 Diamins in the urine, 14, 509, 563 in the intestinal contents, 14, 563 Diailiido-acetic acid, 21 Diamido-caproic acid, 21 Diamido-valerianie acid, 524 Diarrhoea, 324, 334 , action on the quantity of urine, 522 Diastatic enzymes, 12, 256, 297. See also Enzymes. Diastase in the blood, 124 Dicalcium casein, 425 Diet for various classes of people, 653 Diet cures for corpulence, 658 Digestion, 251—341 Digestibility of food-stuffs, 279, 280, 330, 331, 334, 335 Digestion leucocytosis, 172, 473, 476 Dimethyl carbinol, behaviot in animal body, 524 Dimethylketone. See Acetone. Dioxyaceton, 67 Dioxybenzol, 526 Dioxynaphthalih, 526 Disaccharides, 72 in urine, 333, 655 Distearyllecithin, 93 Distribution of blood in the organs, 179 Doeglic acid, 85 Dog's milk, 434 Dolphin milk, 434 Donne's pus test, 541 Dotterplattchen, 24, 411 Dulcite, 61 , relation to glycogen forma- tion, 214 Dysalbumose, 36 Dyslysine, 230 Dyspeptone, 271 Dyspnoea, action on proteid trans- formation, 453, 648 Earthy phosphates, elimination by the urine, 513, 514, 519 , solubility in pro- teid fluids, 353 , occurrence in bone- ash, 348—350 , occurrence in cal- culi, 247, 261,. 325, 570 , occurrence in sedi- ments, 566—568 See also various, earthy phos- phates. Ebstein's diet cure, 658 Eichinochrom, 148 Echinococcus cysts, cyst wall, 575 , cyst Contents, 19& Eck's fistula, 456 Eel, serum of, 126 , flesh, 387 Eigg, 410 , hen's, 410 — 419 , absorption in the intestine, 331 , incubation, 418 E^g albumin (see Ovalbumin), 413 Efeg-shell, 416 Ehrlich's test for bile-pigments, 544 urine test, 561 Eiselt's reaction, 541 Elaidic acid, 85 Elaidin, 84 Elastin, 18, 51 , behavior to gastric juice, 271 , behavior to trypsin, 307 Mastin albumoses, 52 Elastin peptone, 52 Electrosyntheses, 7 INDEX. 677 Eleidin, 573 Elephant bones, 349 Elephant va\\\ 434 Elephant tusk, 354 EUagic acid, 326 Emulsin, 12 Emydin, 417 Enamel, 353 Encephalin, 392, 394 Endolymph, 402 Energy, potential of, food-stuffs, 616— 619 jEnzymes, in general, 10 — 13 , diastatic, in pancreatic juice, 296, 297 , diastatic, in blood, 124, 125, 217 , diastatic, in bile, 238, 309 , diastatic, in urine, 508 , diastatic, in the liver, 217, 218 , diastatic, in lymph, 181 , diastatic, in muscles, 366 , diastatic, in the secretion of, the mucous membrane of the intestine, 289, 290 , diastatic, in saliva, 255 , proteolytic, in the mucous membrane of the intestine,; 290 ! , proteolytic, in the urine, 508 , proteolytic, in the stomach, 261, 264, 265 , proteolytic, in the pancreas, 296, 299, 300 , proteolytic", in the plant king- dom, 265 , proteolytic, in the lower ani- mals, 265 , steatolytic, 13, 297, 298, 299 , coagulating. See Fibrin fer- ment and Rennin. , urea splitting, 565 Epiguanin, 484 Episarkin, 102, 485 Erucic acid absorption, 335 , synthesis from erucin, 335 Erythrit, relation to glycogen forma- tion, 214 Erythro-dextrin, 78, 256 Erythropsin. See Visual purple. Esbaeh's estimation of proteid, 536 urea, 466 Esters, action on the pancreatic juice, 298 Ethal, 87 Ether, action on blood, 128 , action on secretion of gastric juice, 262 , action on the muscles, 375 , action on the secretion of pan- creatic juice, 295 Ethereal sulphuric acids in the bile, 240 Ethereal sulphuric acids in the urine, 314, 489—496, 525, 529 Ethereal sulphuric acids in sweat, 581 Ethereal oils, action on muscles, 375 Ethyl alcohol, formation in intestine, 313 , absorption, 339 , passage of, into milk, 443 , behavior in animal or- I ganism, 643 , action on secretion of i gastric juice, 262 , action on the muscles, 375 , action on metabolism, 643 , action on digestion, 270, 280 Ethyl benzol, behavior in organism, 526 Ethylen glycol, relationship to forma- tion of glycogen, 214 Ethylenimin. See Spermin. Ethylidene-lactic acid, 371. See also other lactic acids. Euxanthic acid, 500 Euxanthin, 506 Excrements, 320—324 in dogs with biliary fis- tula, 319 678 INDEX. Excrements in starvation, 611 , elimination of water with, 610 Excreta of the body, 608—616 , division among the various excretions, 609 Excretin, 323 Excretolic acid, 323 Exostosis, 352 Expectorations, 605, 606 Extinction coefficient, 147, 148 Extracellular action of enzymes, 11 Exudations, 188—197 Eye, 397^03 Faeces. See Excrements. Fat, origin in the body, 355—358, 632, 633 , general properties, detection, and occurrence of, 81 — 87 , emulsification of, 290, 298, 299, , 310, 311, 334, 336—338 in blood-serum, 122, 172, 175 in chyle, 183 in yolk of egg, 412 in pus, 199 in excrements, 336, 338 in fatty tissue, 354 in bile, 238, 239, 240 in the brain, 391 in the urine, 561, 562 1 in the bones, 350 in milk, 422, 423, 424, 431, 433, 434, 435, 442 , caloric value of, 616, 617 ' , nutritive value of, 616—619, 621, 637—642 , rancidity, 83 , absorption of, 334 — 336, 341 , behavior to intestinal juice, 290 , behavior to gastric juice, 278 ' , behavior to pancreatic juice, 298, 337 , saponification of, 82, 85, 298, 310, 338 , action on the secretion of bile, 223 Fat-metabolism in activity and at rest, 383—385 in starvation, 621, 622 ' with various foods, 630, 632, 637—644 Fat-cells, 354 Fat-sweat, 579 Fatty acids, general properties, detec- tion and occurrence, 82—86 , absorption of, 334 — 336 , synthesis to neutral fats, 335, 355 Fatty degeneration, 208, 356 Fatty infiltration, 208 Fatty series, behavior of the respective members in the animal body, 523 Fatty tissue, 354 , behavior with gastric juice, 272, 278 Feathers, 49, 577 Fehling's solution, 69, 549—552 Fellic acid, 230 Fermentation, 5, 10, 64, 68 in the intestine, 313 in urine, 505, 564, 565 in contents of stomaehv 277, 281, 283 See also various fer- mentations, Alcohol ferme.:tation, etc. Fermentation test in the urine, 547, 553 Fermentation lactic acid, properties,, occurrence; etc., 371, 373 Fermentation lactic acid in the brain,^ 392 Fermentation lactic acid in the stom- ach contents, 277 Fermeiitation lactic acid in the gas': re juice, 264 Fermentation lactic acid, formation of, in the souring of milk, 422 Fermentation lactic acid in urine fer- mentation, 564 Fermentation lactic acid, detection of, in stomach contents, 285 INDEX. 679 Ferments, in general, 10. See also various enzymes. Fever, elimination of ammonia in, 517, 518 , elimination of uric acid, 474 , elimination of urea, 454 , elimination of potassium salts, 517 , metabolism of proteids in, 454, 474 Fibres, elastic, in sputum, 606 , reticulate, 342 Fibrin, 18, 112 , occurrence of, in transudations, 188, 191—196 , properties of, 114 , Henle's, 403 Fibrin coagulation, 114—119,153—163 Fibrin calculi, 325 Fibrin digestion, 267—271 Fibrine soluble. See Serglobulin. Fibrin ferment, 13, 115, 116, 117, 157— 163 Mbrin formation (see Fibrin coagula- tion), 114—119, 153—163 Fibrin globulin, 117, 122 Fibrinogen, 18, 91, 102, 113, 158, 160, 161, 181, 190 Fibrinolysis, 115 Fibrinoplastic substance. See Ser- globulin. libroin, 18, 57 Filtration, relation to absorption, 340 Fish-eggs, 24, 417 Fish-bones, 351 Fish-scales, 105 Fish air-bladder, 105, 603 Flesh, metabolism of, in starvation, 621 , metabolism of, with various foods, 630—642 , accumulation of, with various foods, 630, 631, 633, 634, 636, 638—641 Flesh quotient, 388 Fluorine in bones, 349 in enamel, 354 Fly-maggots, formation of fat in, 357 Food, influence of, on the secretion of intestinal juice, 289 , influence of, on the secretion of bile, 223 , influence of, on the secretion of gastric juice, 262, 263 , influence of, on the secretion of pancreatic juice, 295 , influence of, on the elimination of ammonia, 517 , influence of, on the elimination of uric acid, 473 , influence of, on the elimination of urea, 452, 453, 621 , influence of, on the elimination of CO , 615, 622 2 , influence of, on the elimination of mineral bodies, 510, 512,517 , influence on metabolism, 625 — 642 rich in proteid, 630—637 , mixed, 637—642 , insufficient, 625—630 Food-stuffs, necessary, 607 , heat of combustion of, 616—619 Formaldehyde, formation of sugar from, 67 Formic acid in butter, 424 in gastric contents, 288 , p a s s a g e of, into the urine, 505, 523 Formose, 67 Frog's eggs, membrane of, 44 Fructose, 60, 61, 63, 66, 67, 71, 77 in urine (see Lsevulose), 554 Fruit-sugar. See Fructose. Fumaric acid, 24 Fundus-glands, 261, 273 Fungi, glycogen therein, 210 Furbringer's albumin reagent, 534 Furfuraeryluric acid, 524 Furfurol from glycuronic acid, 507 from pentoses, 65 , relation to Pettenkofer's test for bile-acids, 226 , reagent for urea, 459 , behavior in the body, 524 680 INDEX. ftscin, 399, 400 Galactonio aeid, 72 Galactose, 61, 66, 72, 80, 428 from cerebrin, 394 from vegetable bodies, 443 , relation to glycogen forma- tion, 216 Gallic acid in urine, 497 Gallois'a inoait test, 370 Gas, exchange of, with various ages, 646, 647 , exchange of, by the skin, 582 , exchange of, in starvation, 615, 622, 624 , exchange of, in various condi- tions of the body, 384, 622, 624, 643, 644 , exchange of, in muscles, 376, 378, 384 , exchange of, with various foods, 643, 644 , exchange of, abstinent value of, 624, 625, 645 Gases of the blood, 583—590 of the intestinal contents, 316 of the bile, 241, 591 of the urine, 519, 592 of the hen's egg, 417 — 419 of the lymph, 182, 590 of the milk, 433, 592 of the muscles, 375, 376, 378, 384 of the transudations, 190, 592 from woman's milk, 438 Gastric catarrh, 283 Gastric contents. See Chyme. Gastric fistula, 262 Gastric juice, 262 , secretion of, 262, 263 , estimation of acidity, 284, 286—288 5 relation to intestinal pu- trefaction, 320 , artificial, 267 , action of, 34, 35, 267 — 270,276—283,427,436 Gdatin, 54 Gelatin, relation to the formation of glycogen, 214 , putrefaction of, 54, 314 , nutritive value of, 636 , behavior to gastric juice, 271 , behavior to pancreatic juice, 307 Gelatin-forming substances (see Col- lagen), 53 Gelatin peptones, 55 Gelatin sugar. See GlycocoU. Gelatinous tissue, 343 Gentisic acid, 498 Gentisic aldehyde, 498 Germ of the hen's egg, 410 Globin, 140 Globulicidal bodies in serum, 178 Globulins, 18 , general properties, 30 in urine, 534 in protoplasm, 90 See also the various globu- lins. Globulin-plates, 151 Globuloses, 36 Gluease in the blood, 124 Glucocyanhydrin, 61 Glucoheptose, 61 Gluconic acid, 61 Gluco-proteins, 30 Glucosamin from chitin, 574 in cartilage, 345 Glucosan, 68 , Glucose. See Dextrose. Glucosoxime, 61 Glutamic acid, 21 Gluten protein, 42 Glutin. See Gelatin. Glycerin, relation to the formation of glycogen, 214 , action on the elimination of uric acid, 473 , solvent for enzymes, II Glycerin aldehyde, 67 Glycero-phosphoric acid, 93, 176, 201, 238 in urine, 505, 508 INDEX. 681 •Glycm. See GlycoeoU. Glycocholic acid, 225, 226, 227, 240 , properties of, 227 , quantity in e x c r e- ments, 317 in various animal biles, 241 , absorption of, 340 , behavior in the pu- trefaction in the intestine, 317 GlyeocoU, properties of, 231 , formation from gelatin, 54, 314 , formation from other pro- tein substances, 54r— 56 , relation to the formation of uric acid, 471, 475 , relation to the formation of urea, 455, 523 , syntheses with, 3, 485, 486, 524, 527 Glycogen, 77, 89, 210—219 , origin of, 213—217 , general chemical behavior, 211, 212 , relation to muscular activ- ity, 378—385 , relation to rigor mortis, 376 , relation to the formation of sugar, 217—222 , occurrence of, in sputum, 606 , occurrence of, in muscles, 370 , occurrence of, in the lungs, 605 , occurrence of, in p r o t o - plasm, 90, 95, 150, 199 -Glycolysis, 123, 181, 294 Glycolytic enzyme, 124 Glyeo-proteids, 18, 31, 43, 92 Glycosuria, 123, 219, 220, 544 Glycosuric acid, 498 Glycuron, 507 Olycuronic acid, relation to glycogen formation, 214 , properties of, 506 Glycuronie acid, conjugated, 491, 4®3, 496, 506 , conjugation of, in the body, 524, 529 , origin of, 524 Glyozyl diureid. See Allantoin. Gmelin's test for bile-pigment, 235 test for bile-pigment in urine, 543 Goat-milk, 434 Goose-fat, absorption of, 335 Gout, elimination of uric acid in, 472, 474 Graafian follicles, 406 Grape-moles, 419 Gravimetric estimation of proteid in urine, 536 Guaiacum blood test, 539 Guanin, properties and occurrence, 105 in urine, 484 , quantity in liver, 208 , quantity in pancreas, 292 , quantity in sperma, 406 Guanin calcium, 105 Guanin gout, 105 Guano, 105, 472 Guano bile-acids, 227 GuanOvulit, 417 Gulonic acid lacton, 506 Gulose, 66, 71 Gums, various, 65 Gum, animal, 45 in urine, 505 Hsemataerometer, 596 Hsematin, 141 , relation to bilirubin, 245 , relation to urobilin, 245, 499 , properties of, 141 Hsematinometer, 146 Haematoblasts, 151 Hsematochlorin, 419 Haematocrit, 165 Haematocrystallin. See Oxyhsemo- globin. Hsematoidin, 145 , relation to bilirubin, 145, 233, 243, 244 682 INDEX. Esematoidin, properties of, 145 , occurrence in expectora- tions, 606 , occurrence in corpora lu- tea, 406 , occurrence in excre- ments, 322 J occurrence in sediments, 568 Esematogen, 411, 417 Heematoglobulin. See Oxyhsemoglobin. Hsematolin, 144 Hsematoporphyrin, relation to biliru- bin, 144, 245 , relation to urobi- lin, 501 , properties of, 144 J occurrence of, in urine, 540 in lower animals, 578 Hsematoporphyrinuria, 540 Hsematuria, 538 Haemerythrin, 148 Hsemin, 142, 143 Hsemin crystals, 142, 143, 144, 540 Heemochromogen, 131 , properties of, 140 , occurrence in mus- cle, 365 Hsemocyanin, 148 Haemoglobin, 43, 135 , properties and behavior, 135 , quantity in blood, 130, 131, 169—174 , quantitative estimation, 148 , behavior in trypsin di- gestion, 308 See also Oxyhsemoglobin and the combinations of haemoglobin with other gases. Heemoglobinuria, 538 Hsemometer, 148 Haemosiderin, 246 Haeser's coefficient, 521 Hair, 49, 573 Hair-ash, 573 Hair-balls, 325 Hair-pigments, 576, 577 Half rotation, 64 Haptogen-membrane, 423 Heat, action on metabolism, 645, 646, 650, 651 of combustion: of food-Btuffs, 617—619 , loss of, by the skin, 582, 620, 645, 646 Heat development in plants, 2 Helicpproteid, 18, 47 Heller's albumin test, 26 albumin test for urine, 532 Heller-Teichmann's blood test, 539 Hemialbumose, 35 Hemicelluloses, 80 ■ Hemicollin, 55 Hemielastin, 52 Hemipeptone, 35 Hemp-seed calculi, 570 Hen's egg, 410—418 , incubation of, 418, 419 Heteroalbumose, 35 Heteroxanthin, 102 in urine, 484 Hexobioses, 72 High elevations, action on the blood, 600 Hippomelanin, 576 Hippuric acid, 485 , properties and reac- tions, 487 5 formation in the or- ganism, 3, 436, 487, 488, 527 , cleavage of, 485, 489 , occurrence, 486 , occurrence as s e d i - ments, 568 Histon, 101, 158, 162, 203 Histozyme, 489 Hofmann's tyrosin test, 305 Holothuria, mucin of, 47 Homocerebrin, 392, 394 Homogentisic acid, 493, 497, 498 INDEX. 683 Hopkins's method for the estimation of uric acid, 481 Hoppe-Seyler's carbon-monoxide test, 139 xanthin test, 105 Horn, 49, 573, 579 Horn substance in the gizzard of birds, 51 See also Keratin. Huckleberries, coloring matter of, in urine, 530 Humin substances in urine, 499, 530 Humor, aqueous, 195 Huppert's reaction for bile-pigments, 235 reaction for bile-pigments in urine, 543 Hyalines, 47 of the walls of hydatid cysts, 575 of Eovida's substance, 91, 129, 150, 199, 403 Hyalogen, 47 Hyalomucoid, 400 Hyaloplasm, 90, 96 Hydatid cysts, 575 Hydracrylic acid, 371 Hydrsemia, 173 Hydramnion, 419 Hydrazone, 62 Hydrobilirubin, 234 , relation to urobilin, 245, 317, 501 , formation in putre- faction, 317 Hydrocele fluids, 194 Hydrocinnamic -acid, behavior in the body, 486 Hydrochinon, 493, 530 Hydrochinon-sulphuric acid, 489, 491 Hydrochloric acid, secretion of, in the stomach, 263, 274, 277, 283 , anti-fermentive ac- tion of, 282 , action on the se- cretion of pep- sin, 262 HydrocTiloric acid, action on the py- lorus, 279 , quantity in gas- tric juice, 264 , quantitative esti- mation in gaa- t r i c contents, 286 , reagents for free, 285, 286 , action on proteid, 21, 27, 32. 268, 272 Hydrogen in putrefactive and fermen- tive processes, 5, 314, 316 Hydrogen peroxide in urine, 519 , decomposition of, by enzymes, 12 Hydrolytic splittings, in general, 9, 10. See also the various split- tings. Hydronephrosis fluid, 446 Hydroparacumaric acid in putrefac- tion in the intestine, 305, 314 Hydrophenoketon, 7 Hydrocyanic acid, action on pepsin digestion, 270 , action on trypsin, digestion, 301 Hyoglycocholic acid, 227 Hypalbuminosis, 175 Hyperalbuminosis, 175 Hyperglycsemia, 220 Hyperinosis, 175 Hypinosia, 175 Hypnotics, relation to glycogen for- mation, 214 Hyposulphurous acid in the urine, 508, 524 Hypoxanthin, relation to uric-acid formation, 475 , properties, 106 , quantity in the liver, 208 , quantity in the mus- cles, 366 , quantity in the pan- creas, 292 684 INDEX. Hypoxanthin, quantity in the sperma (seeSarkin), 406 , transition into tlie urjne, 484 Ichthidin, 411, 417 Ichthin, 417 Ichthulin, 18, 47, 411, 417 Icterus, 222, 246, 247 , blood, 176 . , urine, 502, 542 Immunity against infection, 16 Indican, urine, 493 — 495 , elimination in sta^-vation, 317, 494 , elimination in diseases, 494 Indican test, Jaffa's, 495 , Obermayer's, 495 Indigo, 493 in the sweat, 581 Indigo blue, 495, 500 Indol, properties, 314, 315 , formation from proteida, 22 , formation in putrefaction, 314, 317, 489, 494 . Indophenol blue, behavior in the tis- sues, 5 Indoxyl, 489, 494 Indoxyl-glyeuronie acid, 493, 495, 529 Indoxyl red, 495 Indoxyl-sulphuric acid, 489, 493 — 495 Inosinic acid, 366 Inosit, properties and occurrence, 369 in urine, 565 , relation to the formation of glycogen, 214 Internal 'respiration, 583, 593 Intestine, putrefaction processes in, 313—320 , absorption in, 318, 320, 327—341 , digestion processes in, 309 —314 Intestinal calculi, 325 Intestinal contents, 309 — 325 Intestinall fistula, 289 Intestinal gases, 314, 316 Intestinal juice, 289—291 Intestinal mucous membrane, 289 Intracellular action of enzymes, 11, 73 Inulin, 71, 76 as a glycogen-former, 214 Inversion, 10, 73, 272, 290, 309, 332 Invertin, 74, 257, 297 Invert-sugar, 10, 73 Iodine combinations, passage of, into the milk, 443 , passage of, into the sweat, 581 , passage of, into the saliva, 260 Iodoform test. Gunning's, 558 , Liebe-i's, 558 Iron in the blood, 125, 167, 168 in the blood-pigments, 131, 141, 143, 146, 245, 246 in the bile, 238, 241 in the urine, 519 in the liver, 183, 184, 238, 245, 246 in the milk, 433, 437, 443 in the spleen, 201, 202 in the muscles, 374 in new-born, 201, 210, 440 in protein substances, 17, 19, 31, 32, 98, 99, 201, 207 in cells, 109, 110 , elimination of, 238, 245, 246, 519 , quantity of, in bitch's milk and new-born dogs, 440 , absorption of, 173, 629 , grains rich in, of the spleen, 201 Iron salts, elimination by the urine, 519 , action on the blood, 173 , action on trypsin diges- tion, 301 , action on absorption, 173, 629 Iron starvation, 629 Ischuria in cholera, 581 Isocholesterin, 250 Isodynamic law, 618, 619 INDEX. 685 Isoglucosamin, 63 Isomaltose, 72, 74, 78, 256, 297 in urine, 505 Isosaccharin, relation to glycogen formation, 214 Isotonic relationship, 164 Isotropous substance, 361 Ivory, 354 Jaffa's indican ttst, 495 ereatinin reaction, 469 Janthinin, 578 Japanese, nourishment of, 653, 654 Jaune indien, 506 Jecorin, 95, 123, 201 , properties and occurrence, 208 Jequirity bean, 15 Jolles's reaction for bile-pigments, 543 Kairin, action on the urine, 530 Kephalines, 391 Kephir, 434 , preventive action on putre- faction, 318 Kerasin, 392 Keratins, 18, 49, 573 , properties of, 49, SO , behavior with gastric juice, 271 , behavior with pancreatic juice, 308 Keratinose, 50 Ketoses, 60, 63 Kidneys, 446 , relation to formation of uric acid, 476 , relation to formation of urea, 457 , relation to formation of hip- puric acid, 487 Kjeldahl's method of determining ni- trogen, 461, 466 •Knapp's titration method, 552 . Knop-Hufner's method for determin- ing urea, 466 Kumyss, 434 Kyestein, 568 Kynurenic acid, 497, 509 Laborer, diet, 653—658 Lactalbumin, 18, 428 Lactates, 373 Lactic acids, 371 in the intestine, 313 in the urine, 380, 505 in the bones, 352 in the gastric juice, 264 , relation to the formation of uric acid, 475 See also Paralactic and Fermentation lactic acid. Lactic-acid fermentation, 69, 27/, 283, 332, 371, 422, 429 in intestine, 309, 371 in urine, 564 in stomach, 277, 283 in milk, 422, 429 Lacto-caramel, 428 Lacto-globulin, 428 Lacto-protein, 428 Lactose. See Milk-sugar. Ijffivo-lactic acid, 371 Laiose, 555 Lanolin, 250, 579 Lard, absorption of, 336 Latebra, 410 Laurie acid in butter, 424 in spermaceti, 86 Laxatives, action on the blood, 175 , action on the secretion of intestinal juice, 289 , action of, 324 Lead in blood, 168 in the liver, 210 , passage of, into milk, 443 Lecithalbumins, 31 , relation to the secre- tion of gastric juice, 274 , relation to the secre- tion of urine, '446 686 INDEX. Lecithin, properties, occurrence, etc., 93 , action on the coagulation of blood, 160 , putrefaction of, 94, 317 , behavior in muscular activ- ity, 381 Legal's reaction for acetone, 558 Legumin from peas, 42 Lens (see Crystalline lens), 400 , capsule of the, 47, 400 , fibres of, 401 Leo's method for the determination of acidity, 288 sugar, 555 Lethal, 86 Leucaemia, blood in, 103, 174 , secretion of uric acid, 202, 474, 476 , xanthin bases in, 103, 174, 202, 483 Leuceine, 20 Leuein, 20, 21, 50—57, 302 , relation to the formation of uric acid, 475 , relation to the formation of urea, 455, 523 , preparation of, 305 , properties of, 302—304 , transition into the urine, 562 , behavior in the body, 455, 523 Leucinic acid, 303 Leucinimid, 24 Leucocytes, relation to absorption, 330 , relation to formation of uric acid, 475 in the thymus gland, 91, 92 See also the white blood- corpuscles. Leucomaines, 15 in urine, 509 in muscles, 369 Leuconuclein, 159, 162, 203 Levulose, relation to the formation of glycogen, 216, 221 , absorption of, 332 , behavior in diabetics, 221 j Levulose, occurrence in urine, 554 See also Fructose. Levulinic acid, 66, 428 Lichenin, 77 Lieberkiihn's alkali-albuminate, 32 glands, 289 Lieberman's reaction for proteids, 27 Lielbermann-Burchard's eholesterin re- action, 249 Liebig's titration method for urea, 461 Ligamentum nuchse, 51 Lignin, 79 Linseed oil, feeding with, 355 Lion's urine, 471 Lipsemia, 175 Lipacidsemia, 175 Lipanin, absorption of, 335 Lipochromes, 125, 412 Lipuria, 562 Lithobilic acid, 326 Lithofellic acid, 326 Lithium in the blood, 168 Lithium lactate, 374 Lithium urate, 478 Lithuric acid, 509 Liver, 206—210 , relation to the formation of uric acid, 475 — 477 , relation to the formation of urea, 455, 457, 458 , blood of the, 169, 206, 217 , proteids in, 207 , fat in, 207, 208 , amount of sugar in, 217 , atrophy of, acute yellow, 458 , atrophy of, elimination of am- monia in, 458 " , atrophy of, elimination of urea in, 458 , atrophy of, elimination of leu- ein and tyrosin in, 562 , atrophy of, elimination of lac- tic acid in, 372, 505 , extirpation of, elimination of ammonia, 457, 474 , extirpation of, elimination of uric acid, 474 INDEX. fisr Ijver, extirpation of, elimination of lactic acids, 372, 474, 505 , extirpation of, action on the formation of bile, 242, li44 , cirrhosis of, ascitic fluid in, 193, 194 , cirrhosis of, action on the elimination of ammonia and urea, 458 Limgs, 605 , catheter for, 595 Lutein, 412 in corpora lutea, 406, 412 in yolk of the egg, 412 in serum, 125 relation to heematoidin, 145 406 Lymph, 180—188 Lymphagogues, 180, 185—188 Lymphatic glands, 200 Lymph-cells, quantitative composi- tion of, 203 See also White blood- corpuscles. Lymph-fibrinogen. See Tissue-fibrino- gen. Lysatin, 21, 454 Lysatinin, 21, 50, 52, 54, 56, 302, 313 Lysin 21, 50, 52, 54, 56, 302, 313, 454 Mackerel, flesh of, 387 Madder, feeding with, 351 Magnesium in urine, 513, 519, 522 in bones, 349 in muscles, 374, 386 See also the various tis- sues and fluids. Magnesium phosphate in intestinal calculi, 325 in urine, 513, 519 i n urinary calculi, 569, 570 in urinary sediments, 568 in bones, 349 Magnesium soaps in excrements, 322 Malaria, 176 Malerba's acetone reaction, 559 Malt diastase, 257 Maltodextrin, 78 Maltose, 74 , relation to starch, 78> 256, 297 , absorption of, 332 , relation to glycogen forma- tion, 216 , relation to intestinal juice, 257, 309, 332 Mammary glands, 420, 441, 443 Man in poorhouse diet, 657 Mandelic acid, 527 Mannite, 61, 67 , relation to glycogen forma« tion, 214 Mannose, 67, 71 Mannoso-cellulose, 80 Mare's milk, 434 Margarine and margarie acid, 84 Marsh-gas in intestine, 314, 317 in putrefaction, 22,314,317 in fermentation of cellu- lose, 317 in the decomposition of lecithin, 94, 317 Meat, consumation of, in intestinal canal, 330 , caloric value, 617, 618 , digestibility, 279, 280 , composition, 356, 357, 386— 388 See muscles in general. Meconium, 323 Melansemia, 176 Melanins, relation to blood-pigments, 246 , properties and occurrence, 576—578 in the eye, 400 in the urine, 541 Melanogen in the urine, 541 Melanotic sarcoma, pigments of, 576, 577 - Melebiose, 75 688 INDEX. Mellitseinia, 176 Melissyl alcohol, 87 Membraiiin, 47, 348, 400 Menstrual blood, 170 Menthol, behavior in animal body, 529 Mercaptiiric acids, 529 Mercury salts, passage of, into milk, 443 , passage of, into sweat, 581 , action on ptyalin, 258 , action on trypsin, 301 Mesitylein, behavior in animal body, 527 Mesitylenic acid, 527 Mesitylenuric acid, 528 Metabolism, dependence upon exter nal temperature, 422, 650 in various ages, 645, 646 in work and rest, 377 — 385, 648 in the dififerent sexes, 645 in starvation, 619 — 625 ■with different foods, 630 —642 in sleep and awaking, 650 , calculation of the extent of, 612—616, 624 Metalbumin, 407, 408 M*taphoephoric acid, constituent of nucleins, 98 , as reagent for proteids, 26, 533 Metheemoglobin, 136 in blood after poison- ing, 177 in urine, 538 Methal, 86 Methane, formation in putrefaction, 22, 314, 317 Methylenitan, 67 Methyl glycocoll. See Sarcosin. Methyl guanidin, 367, 469 Methyl-guanidin-acetic acid. See Creatin. Methyl-hydandoinic acid, 523 Methyl hydantoin, 471 Methyl indol. See Skatol. Methyl mercaptan in proteid putre- faction, 22, 314, 316 in urine, 530 Methyl pyrtdin, behavior in the or- ganism, 530 Methyl-pyridyl-ammonium hydroxide, 530 Methyluramin, 367, 469 Methyl-uric acid, 471 Microorganisms in intestinal tract, 13, 282, 313, 321 Micrococcus restituens, 329 Micrococcus urese, 565 Milk, 420—444 , secretion of, 441 — 443 , consumation of, in intestine,. 330, 337, 338 , blue or red, 444 , anti-putrefactive action, 318, 490 in disease, 443 , passage of foreign bodies into, 443 , behavior in the stomach, 276, 281, 436 See also the different varieties- of milk. , human, 435—439 , human, behavior in stomach, 276, 436 , human, composition of, 436,437 of blondes, 439 of brunettes, 439 Milk-fat, 424, 434, 435 , analysis of, 424 , formation, 442 Milk-globules of cow's milk, 422, 423 of human milk, 435 Milk-plasma, 424 Milk-sugar, 73, 428 , relation to glycogen for- mation, 216 INDEX. 689 Milk-sugar, properties, 428, 429 , fermentation, 277, 422, 429 , inversion, 290, 332, 429 , calorie value, 617 , quantitative estimation, 432 , absorption, 332 , passage of, into the urine, 429, 555 , origin, 420, 442 Millon's reagent, 27 Mineral acids, alkali-removing action, 448, 517, 590, 626 , anti-fermentive action, 282 , action on the elimina- tion of ammonia, 517, 628 Mineral bodies eliminated in starva- tion, 623 , insufficient supply of, 625 , behavior in the or- ganism, 626 See the various fluids, tissues, and juices. Mitoplasm, 96 Mixture of the nitrogenous substances in the urine, 454, 473, 474 Modified proteid bodies, 30 Mohr's titration method for estimat- ing chlorine, 510 Monosaccharides, 60 M5rner and SjSqvist's method of esti- mating urea, 466 method of esti- mating acids, 286 Moore's test for sugar, 68 Morphin, passage of, into the urine, 530 , passage of, into the milk, 443 Mucie acid, 77, 428 , relation to glycogen for- mation, 214 Mucilages, vegetable, 77, 79 Mucin, 18, 45 in sputum, 606 in connective tissue, 342 in urine, 509, 537 in salivary glands, 44, 252 , detection of, in the urine, 537 Mucin-like substances in bile, 225 in urine, 509, 537 in the kidneys, 446 in the thyroid gland, 204 in the synovial fluid, 196 Mucoids, 18, 47 in ascitic fluids, 191, 193 in the vitreous humor, 343, 400 in the cornea, 348 Mucinogen, 44, 252 Mucin peptone, 271 Mucous glands, 44, 251 Mucous membranes of the stomaeh, 261 Mucous tissue, 343 Mucus of the bile, 224, 225 of the urine, 447, 509, 532, 537 of the synovial fluid, 196 Mulberry calculus, 570 Murexide test, 478 Muscles, striated, 360—388 , non-striated, 388 , blood of the, 170, 378, 384, 584 , chemical processes in work and at rest, 377 — 385 in rigor, 375—377 , proteids of, 361—366 , extractives of, 366 — 375 , pigments of, 365 , fat of, 374, 383, 386, 387 , gases of, 375, 377, 384 , caloric value of, 617 , mineral bodies, 374, 386 , quantity of water, 387 , composition of, 386 690 INDEX. Muscle-fibres, 360 Muscle-pigments, 365 Muscle-plasma, 361, 362 , coagulation of, 362, 363, 376, 388 Muscle-serum, 362 Muscle-stroma, 365 Muscle-sugar, 371 Muscle-syntonin, 365 Muscular energy, origin of, 384 Muscular work, chemical processes in the muscles, 377 — 385 , influence on the urine, 448, 468, 471, 508 , influence on metabo- lism, 377—385 Musculin, 18, 364 Mussels, glycogen of, 210 Mustard-seed oil, action on the secre- tion of pancreatic juice, 296 Mutton-fat, feeding with, 355 , absorption of, 335, 336 Mycb-protein, 19 Myeline, 391 Myeline forms, 94, 391 Mygge and Christensen's estimation of proteid, 537 Myoalbumose, 364 Myoalbumin, 364 Myoglobulin, 364 Myohsematin, 365 Myosin, 362 in leucocytes, 150 Myosinogen, 363 Myosin ferment, 363 Myosinoses, 36 Myricin, 87 Myricyl alcohol, 87 Myristic acid in butter, 424, 435 in the bile, 238 Myxoedema, 204, 343 Nails, 49, 573 Naphthalin, action on the urine, 530 , behavior in the animal body, 526 Naphthol, reagent for sugar, 70, 549 , behavior in the animal body, 529, 530 Naphthol-glycuronie acid, 529, 530 Narcotics, relation to glycogen forma- tion, 214 Native proteids, 29 Navel cord, mucin of, 46, 343 Neossin, 47 Nerves, 390, 397 Neuridin, 392, 395, 410 Neurin, 93 in suprarenal capsule, 205 in protagon, 392 Neurochitin, 397 Neurokeratin, 49, 390, 397 Neutral fats. See Fats. Nicotin, action on quantity of CO in the stomach, 278 Nitrates in the urine, 517 Nitric-oxide hsemoglobin, 140 Nitrogen, free, in blood, 584 , free, in intestine, 316 , free, in stomach, 278 , free, in secretions, 591 , free, in transudations, 592 , combined, quantity of, in the intestinal evacua- tions, 610, 611 , combined, quantity of, in meat, 388, 613 , combined, quantity of, in the urine, 454 , estimation of, in the urine, 461, 465 elimination in work and rest, 381—384, 647, 648 elimination in starvation, 620—622 elimination with different foods, 630—642 elimination by intestinal evacuations, 610, 611 elimination by the urine, 454, 513, 515, 610, 612 elimination by the epi- dermis, 611 INDEX. 691 Nitrogen elimination by the sweat, 581, 611 elimination, relationship to the elimination of phos- phoric acid, 513 elimination, relationship to the elimination of sul- phuric acid, 513 in meat, 388 Nitrogenous equilibrium, 612 Nitrogenous equilibrium with differ- ent foods, 630 — 642 Nitrogenous deficit, 611 Nitro-benzaldehyde, behavior in the animal body, 528 Nitro-benzoio acid, 24, 528 Nitro-benzyl alcohol, 529 Nitro-cellulose, 79 Nitro-hippuric acid, 528 Nitroso-indol nitrate, 315 Nitro-phenyl-propiolic acid, reagent for sugar, 70 Nitro-phenyl-propiolic acid, behavior in the body, 494, 495 Nitro-toluol, behavior in the animal body, 529 Nitro-tyrosin nitrate, 305 Nubecula, 447, 564 Nucleic acid, 48, 91, 96, 97, 99, 100 , combination with haemo- globin, 131 , combination with pro- tamin, 405 Nuclein bases, 98, 103 in sperma, 405, 406 Nucleins, 48, 91, 96, 97, 98 , relation to formation of uric acid, 475 Nuclein plates, 151 Nucleo-albumins, 18, 31 in the bile, 225 in the urine, 509, 537 in the kidneys, 446 in protoplasm, 31, 91 in the synovial fluid, 196 Nucleo-albumins in transudations, 189, 190, 192 , behavior to pepsin, digestion, 31, 270, 427 Nucleo-histon, 18, 92, 101 , relation to the coagu- lation of blood, 158, 159 Nucleo-proteids, 18, 31, 43, 48 in the mammary glands, 420 in the pancreas, 48, 292 in protoplasm, 91 in the cell nucleus, 96, 101 , behavior to pepsin digestion, 48, 101, 270 Nutrition requirements, 633 of man, 652 —659 Nylander's reagent. See AZm6n-B8tt- ger's sugar test. Obermayer's indican test, 495 ObermuUer's cholesterin reaction, 250 Odoriferous bodies in the urine, 530 CEldema, subcutaneous, fluid from, 196 Oertel's cure for corpulency, 658, 659 Oleic acid, 81, 84 Olein, 81, 84 Oligsemia, 173 Oligocythsemia, 173 Oliguria, 522 Olive oil, absorption of, 335 , action on the secretion of bile, 223 Onuphin, 47 Ooeyanin, 416 Oorodein, 416 Opium, passage of, int(^ milk, 443 Optograms, 399 Organic acids, behavior in the animal body, 505, 518, 523 Organized proteida, 633, 634 Organs of generation, 403 — 419 692 INDEX. Organs, loss of weight in starvation, 623 Ornithin, 21, 524, 527 Orthonitro-phenyl-propiolic acid. See Mtro-phenyl-propiolic acid. Osazones, 62 Osmosis, relation to absorption, 340 Osone, 62 Ossein, 348, 352 Osteomalacia, 352, 353 , lactic acid in the urine in, 372 Osteoporosis. See Osteosclerosis. Osteosclerosis, 352 Otoliths, 402 Ovalbumin, 18, 414 , behavior in the animal body, 121, 415 Ovarial cysts, 406 — 410 Ovaries, 406 Ovglobulin, 414 Ovomucoid, 415 — 418 Ovovitellin, 18, 410 Oxalate of lime. See Calcium oxalate. Oxalate calculi, 570 Oxalates, action on blood coagulation, 112 Oxalic acid in the blood, 176 in the urine, 481, 482 , behavior in the animal body, 481, 523 Oxalic-acid diathesis, 482 Oxaluria, 482 Oxaluric acid, 472, 481 Oxamid, 20 Oxidations, 1—9, 134, 221, 234, 314, 454, 456, 474, 489, 494, 499, 523, 525, 526, 529, 585 Oxidation ferment, 8 Oxyacids, formation in putrefaction, 314 , passage of, into the urine, 314, 497 , passage of, into the sweat, 581 Oxybenzols, 526 Oxybenzoie acid, behavior in the ani- mal body, 527, 528 Qxybutyric acid in the blood, 590 , passage of, into the urine, 518, 560 Oxygen absorption in work and rest, 378, 384 in starvation, 622,, 624 by the skin, 582 Oxygen, activity of, 4, 7, 134, 585 in the blood, 584, 585, 594,. 596, 597, 599 in the intestine, 316 in the lymph, 182, 590 in the stomach, 278 in the swimming-bladder of fishes, 603 in secretions, 590 — 592 in transudations, 592 , combination of, in the blood,. 132, 133, 585, 593, 597 , tension of, in the blood, 593 —597 , tension of, in the expired air,, 594, 595 , action of CO on the tension of, 600, 60f, 602 , lack of, action on proteid destruction, 372, 453 , lack of, action on elimina- tion of lactic acid, 372, 505 , lack of, action on elimina- tion of sugar, 372, 505 , specific quantity, 597 Oxygen carriers, 8, 134 Oxygen consumption in the blood,. 136, 585 Oxyhsematin, 141 Oxyhsemocyanin, 148 Oxyhsemoglobin, 132 , dissociation of, 132,. 593 , properties and reac- tions, 133, 134 , quantity of, in the blood, 130, 131, 169—174 , quantity of, in the muscles, 365 INBBX. 693 Oxyhsemoglobin, passage of, into the urine, 538 , behavior with gas- trie juice, 272 , behavior with tryp- sin, 308 Oxyhydro-paracumaric acid, 497 Oxynaphthalin, 526 Oxynitro-albumin, 24 Oxyphenyl-acetie acid, 305, 314, 497 Oxyphenyl-amido-propionic acid. See Tyrosin. Oxyphenyl-propionic acid, 22, 314, 497 Oxyproto-sulphonic acid, 24 Ozone, 3, 585 Ozone exciter, 134, 585 Ozone transmitter, 134 Palmitic acid, 84 Palmitic-acid ether, 87 Palmitin, 83 Pancreas, 291, 292 , relation to glycolysis, 123, 294 extirpation, action on ab- sorption, 331, 332, 334 extirpation, action on elimi- nation of sugar, 221, 292 , charge of, 202 , change during secretion, 292, 308 Pancreas proteid, 48 Pancreas rennin, 307 Pancreatic juice, 294 , secretion, 295, 296, 308, 309 , enzymes of, 12, 297 —302 , action on foods, 297 —302, 307, 311 312, 331, 332, 337, 338 Paracasein, 427 Parabanic acid, 103, 472 Paracresol, formation in proteid pu- trefaction, 314 Paraglobulin. See Serglobulin. Parahsemoglobin, 134 Paralactic acid, 371, 372 in blood, 125, 168 , relation to the forma- tion of uric acid, 475 , properties and occur- rence, 371 , formation from gly- cogen, 373—377 in osteomalacia, 352 J production of, in muscles during ac- tivity, 380, 384 , production of, in rigor mortis, 375, 376 in deficiency of oxy- gen, 372, 380, 505 in animals with ex- tirpated livers, 372, 505 , passage of, into the urine, 475, 505 Paralbumin, 407, 409 Paramidophenol, 526 Paramucin, 409 Paramyosinogen, 362, 364 Paranuclein, 31, 97 Paranucleon, 369 Parapeptone, 271 Para-oxyphenyl-acetic acid, 305, 314, 497 Para-oxyphenyl-propionic acid, 22, 314, 497 Paraxanthin, 102, 484 in the urine, 484 Parietal or delomorphic cells, 261, 273, 275 Parotid, 251 Parotid saliva, 254 Parovarial cysts, 410 Peas, absorption of, in the intestine, 334 Pemphigus chronicus, 196 Penicillum glaucum, 303 Pentacrinin, 578 Pentamethylendiamin. See Caday- 694 INDEX. Pentosanes, 65 Pentoses, 65, 77 , relation to glycogen forma- tion, 213 in the urine, 65 in the pancreas, 65, 555 Pentosuria, 556 Piepsin, 264, 265—267 , properties, 266 , detection in the gastric con- tents, 283 , quantitative estimation of, 268 , occurrence in the urine, 339, 508 , occurrence in the muscles, 366 , action on proteid, 267 , action on other bodies, 271, 272 Pepsin digestion, 267, 269—272, 27ft , products of, 33, 34, 41, 270, 271, 272 Pepsin glands, 261 Pepsin-hydrochloric acid, 272 Pepsinogen, 261, 275 Pepsin test, 268 Peptochondrin, 346 Peptones, 33—42 in putrefaction, 21, 314 in pepsin digestion, 33^1, 271 in trypsin digestion, 33 — 41, 302 , assimilation of, 327 — 330 , relation to amylolysis, 258 , preparation of, 40 , nutritive value of, 330, 636 , absorption of, 327—330 , passage into the urine, 327, 535 Peptone-plasma, 111, 156, 160 , carbon-dioxide ten- sion, 602 Peptonuria, 534 Pericardial fluid, 189, 191 Perilymph, 402 Period of incubation, 418 Peritoneal fluid, 189, 193 Petspiratio insensibilis, 609 Pettenkdfer's test for bile-acids, 226 respiration apparatus, 604 Phacozymase, 401 Phaseomannit, 369 Phenaceturic acid, 488, 527 Phenols, elimination by the urine, 489 —493, 529 in starvation, 317 , estimation in the Urine, 490 —492 , action on the urine, 493, 530' , electrolysis of, 6, 525 , formation in putrefaction, 22, 314, 489, 490, 529 . , behavior in the animal body, 314, 489 Phenol-glycuronic acid, 491, 529 Phenol-sulphuric acid in the urine, 489 — 492^ 529 in the sweat, 581 Phenyl-acietic acid, formation in pu- trefaction, 22, 314 , behavior in the body, 488, 526, 527 Phenyl-amido-acetie acid, behavior in the body, 527 Phenyl-amido-propionic acid, 23 Phenyl-amido-propionic acid, behavior in the body, 526, 527 Phenyl-glucoSazone, 62, 70 Phenyl-hydrazine test, 70 in the urine, 507, 547 Phenyl-lactosazone, 429 Phenyl-propionic acid, formation in putrefaction, 22, 23, 314, 486 Phenyl-propionic acid, behavior in the body, 486, 527 Philothion, 8 Phlebin, 130 Phlorhidrin, 219 Phlorhidzin diabetes, 219 INDEX. 695 Phlorogiucin as reagent, 285 Phosphocarnic acid, 368 Phosphates in the urine, 512 — 515, 531, 566 — 568. See also the various phosphates. Phosphate calculi, 517, 518 Phosphate diabetes, 514 Phospho-glyeo-proteid, 47 Phosphoric acid, elimination by the urine, 512 — 515 , formation in mus- cular aetiyity,381 , physiological impor- tance, 109 Phosphorized combinations in the urine, 508 Phosphorus poisoning, action on elim- ination of am- monia, 458 , action on elim- ination o f urea, 458 , action on elim- ination o f lactic acid, 505 , fatty degenera- tion caused by, 356 , change in the urine, 458, 505, 562 Phrenosin, 394 Phthalic acid, behavior in the body, 526 PhyiB^,toruain, 57fi, 577 in the urine, 541 Physetoelie acid, 87 Phytovitellin, 42 a-Picolin, behavior in the animal body, 530 Picric acid, reagent for proteid, 27, 536 , reagent for creatinin, 469 , reagent for sugar, 70, 469 Pigments of the eye, 397 — 400 of the blood, 130—149 of the blood-serum, 125, 412 of the corpora lutea, 406 Pigments of egg-shells, 416 of the fat-cells, 354 of bile, 224, 233—239, 241, 243 of the urine, 499—504 of the skin, 576—578 of the lobster, 417, 578 of the muscles, 365, 366 of lower animals, 578 of bird-feathers, 577, 578 of medicinal drugs in the urine, 530, 544 Pig's milk, 434 Pike, flesh of, 388 , stomach of, 267 Pilocarpin, action on secretion of in- testinal juice, 289 , action on CO elimination 2 in the stomach, 278 , action on secretion of pan- creatic juice, 296 , action on the sweat, 581 , action on the saliva, 259 , action on the elimination of uric acid, 473 Piperazin, solvent for uric acid, 477 Piqllre, 220 Piria's tyrosin test, 305 Placenta, 419 Plants, chemical processes in the same, 1, 2 Plasma. See Blood-plasma. Plasmoschisis, 156 Plastin, 90, 96, 101 Plattner's crystallized bile, 225 Plethora polycythsemia, 173 Pleural fluid, 189, 192 Plexus ccEliacus, relation to acetonu- ria, 557 , relation to sugar formation, 293 Plums, influence on the elimination of hippuric acid, 486 Poikilocytosis, 174 Polaristrobometer, 29 Polycythsemia, 173, 178 Polysaccharides, 75 Polyperythrin, 578 696 INDEX. Polyuria, 514, 522 Pork-fat, absorption of, 335 Portal-vein blood, 169, 217, 328 Potassium combinations, elimination in fevers, 517 Potassium combinations, elimination in starvation, 517, 623 Potassium combinations, elimination by the urine, 517, 623 Potassium combinations, elimination by the saliva, 260 Potassium combinations, division in the form elements and fluids, 109 Potassium chlorate, poisoning with, 136, 177 Potassium phosphate in yolk of the egg, 413 in muscles, 375 in cells, 109 Potassium sulphocyanide in the urine, 507 Potassium sulphocyanide in the sa- liva, 253, 254 Potatoes, consumation of, in the in- testine, 334 Preglobulin, 91, 102, 157 Preputial secretion, 579 Prisoners, food-ration for, 657 Propepsin, 275 Propyl benzol, behavior in the body, 526 Propylen glycol, relation to glycogen formation, 214 Prostatic calculi, 406 Prostatic secretion, 403 Prostetic group, 48 Protagon, 95, 199, 391, 392, 397 Protamin, 405 Proteid, separation from fluids, 29 , approximate estimation in the urine, 536 , circulating and organized, 633 , action on glycogen forma- tion, 214, 215, 216 , living and dead, 4 , detection of, 26, 27 Proteid, detection of, in the urine, 531—537 , quantitative estimation of, 28 , quantitative estimation of, in the urine, 536 — 538 , quantitative estimation of, in milk, 430-432 , absorption of, 326—332 , passage of, into the urine, 372, 531 , heat of combustion of, 617, 618 , digestibility in gastric juice, 269, 280—282 , digestibility in pancreatic juice, 301, 302 Proteid bodies, in general, 17 — 29 , poisonous, 15, 42 , summary of the va- rious, 17, 29—43 , vegetable, 42 See also the various proteid bodies of the tissues and fluids. Proteid fattening, 633 Proteid of the hen's egg, 413 Proteid metabolism in work and rest, 38i— 385, 647, 648 in starvation, 620 —621 at various ages, 647 with different food, 629—642 Proteid putrefaction, 13, 14, 22, 314 — 320, 486, 489—496 Protein substances, 17 — 58. See also Individual protein bodies. Protein chromogen, 22, 302 Proteoses, 33, 36. See also Albumoses. Prothrombin, 116, 157, 159, 160 Protic acid, 366 Protocatechuic acid, behavior in the body, 493 Protogelatose, 55 INDEX. 697 Protoplasm, 89 Pseudohsemoglobin, 130, 136 Pseudomucin, 47, 408 in ascitic fluids, 193 in the gall-bladder, 240 Pseudonucleins, 31, 97 from casein, 427, 436 from vitellin, 411 Pseudoxanthin, 369 Psittacofulvin, 578 Ptomaines, 13, 14, 22 in the urine, 509, 563 Ptyalin, 255 , behavior with hydrochloric acid, 257, 277, 309 , action on starch, 255 — 259 Pulmotartaric acid, 605 Purple, 578 Purple eruorin, 135 Pus, 95, 197—200 , blue, 200 in urine, 541 Pus-cells, 198 Pus-serum, 197 Putrescin, 14 in intestine, 563 in the urine, 509, 563 Pyiri, 192, 198, 200 Pyinic acid, 200 Pyloric glands, 261, 273 pyloric secretion, 275 Pyocyanin, 200 in sweat, 581 Pyogenin, 199, 393 Pyosin, 199, 393 Pyoxanthose, 200 Pyridin, behavior in the body, 530 a-Pyridinic acid, 530 fr-Pyridin-carbonic acid, 530 Pyrocatechin, 493 , occurrence in urine, 493 , occurrence in supra- renal capsule, 205 • , occurrence in transuda- tions, 191, 195 Pyrocatechin-sulphuric acid, 489, 492 Pyromucic acid, 524 Pyromucin-ornithuric acid, 524 Pyromucuric acid, 524 Quadriurates, 478, 565 Querclt, relation to glycogen forma- tion, 214 Quinic acid, behavior in animal body, 486 Quinin, passage of, into urine, 530 , passage of, into sweat, 681 , action of, on the elimination of uric acid, 473 , action of, on the spleen, 203 Quotient, respiratory, 384, 599 Rachitis, bones in, 352, 353 KaflBnose, 75 Rape-seed oil, feeding with, 355 Reduction processes, 1, 2, 5, 7, 234, 316, 358, 486, 500, 524 Reducing substances, formation in pu- trefaction and fermentation, 5, 316 Reducing substances, occurrence in the blood, 5, 123 Reducing substances, occurrence in the intestine, 316, 500, 501 Reducing substances, occurrence in the urine, 505 Reducing substances, occurrence in transudations, 191, 195 Rennin, 13, 264, 272, 276, 426 , detection of, in stomach con- tents, 284 , detection of, in pancreas, 307 , transition into the urine, 508 Rennin-cells, 261 Rennin-glands, 261 Rennin zymogen, 261, 273, 276 Reserve cellulose, 71, 80 Resin acids, transition into the urine, 530, 533 Respiration, external, 583, 593, 594— 603 , Internal, 583, 603 with increased air-prea- sure, 598 with diminished air-pres- sure, 598 698 INDEX. Respiration. See also Exchange of Gas under various condi- tions, in the hen's egg, 418 of plants, 2 See Chapter XVII, on the Chemistry of res- piration and on Ex- change of gas. Respiratory quotient, 384, 599 Rest, metabolism during, 377, 378 — 385 Reticulin, 18, 56, 342 Retina, 398 Reversion, 73 Rhamnose, 59, 65, 66 , relation to glycogen for- mation, 213 Rhodizonic acid, 370 Rhodophan, 399 Rhodopsin, 398 Rhubarb, action on the urine, 530, 544 Rib-cartilage, 347 Ribose, 66 Rigor mortis of the muscles, 375, 388 Ring faeces, 321 Roberts' method for estimating sugar, 553 Rodents, bile-acids of, 227, 241 Rods of the retina, pigments of, 398 Rosenbach's urine test, 495 Rovida's hyaline substance, 91, 129, 150, 199, 403 Rye bread, consumation in the intes- tine, 331, 334 Saccharic acid, 61, 506 , relation to glycogen formation, 214 Saccharin, relation to glycogen forma- tion, 214 Saccharogen in the mammary glands, 443 Salicylic acid, action on pepsin diges- tion, 270 , action on metabolism, 643 Salicylic acid, action on trypsin di- gestion, 301 , behavior in the animal body, 528 Saliva, 251—261 , secretion of, 259, 260 , mixed, 254 , physiological importance of, 260 , behavior in the stomach, 260, 278, 309 , various kinds of, 252, 253, 254 , action of, 257, 258 , composition of, 259 Salivary calculi, 261 Salivary diastase. £ee Ptyalin. Salivary glands, 251 . Salmon, flesh of, 387 , sperma of, 405, 406 Saltpetre, action on metabolism, 643 Salts, absorption of, 339. See also the various salts. Salt-plasma, 112 Salts of vegetable acids, behavior in. the organism, 449 Samandarin, 579 Santonin, action on the urine, 530, 544 Saponification of neutral fats, 82, 85, 298, 310, 338 Sarcolactic acid. See Paralactic acid. Sarcolemma, 360 Sarcosin, 366 , behavior in the animal body, 523 Sarkin. See Hypoxanthin. Schreiner's base, 404 Schweitzer's reagent, 79 Sclerotica, 402 Scyllit, 201 Sebacic acid, 85 Sebum, 578 Sedimentum lateritium, 447, 478, 504 Sediments. See Urinary sediments. Semen, 403 ISemiglutin, 55 Seminose. See Mannose. Senna, action on the urine, 530, 544 Seralbumin, 18, 120 INDEX. t)»y Seralbumin, detection in the urine, 535 , quantitative estimation, 122, 536 , behavior in the animal body, 121, 415 Serglobulin, 18, 119 , importance in the co- agulation of the blood, 157 , detection in the urine, 534 , quantitative estimation, 120, 536 Sericin, 18, 57 Sericoin, 37 Serin, 57 Serous fluids, 188—197 Serum. See Blood-serum. Serum casein. See Serglobulin. Sharks, bile of, 225, 238 , urea in bile of, 452 Sheep-milk, 434 Shell-membrane of the hen's egg, 49, 416 Silicic acid in feathers, 573 in urine, 519 in the hen's egg, 413, 416, 418 Silk gelatin, 57 Sinkalin, 93 Sinistrin, animal, 48 Skatol, 22, 314, 315 , formation in putrefaction, 22, 314, 489, 496 , behavior in the animal body, 314, 489, 496, 529 Skatol-acetic acid, 23 Skatol-amido-acetic acid,. 23 Skatol-carbonic acid, 496 Skatol-pigment, 496 Skatoxyl, 315, 496 Skatoxyl-glyeuronic acid, 496, 529 Skatoxjl-sulphuric acid, 489, 496 in sweat, 581 Skeletins, 56 Skeleton at various ages, 350 Skin, 573—582 Skin, excretion through the, 578, 579 —582, 609—613 Sleep, metabolism in, 650 Smegma praeputii, 579 Snail mucin, 45 Snake poison, 15 Soaps in blood-serum, 123 in chyle, 183, 335 in pus, 199 in faeces, 321, 322, 338 in bile, 225, 238 , importance in the emulsifica- tion of the fats, 298, 311, 338 Sodium alcoholate as a saponification agent, 86 Sodium bicarbonate, action on the se- cretion of pancreatic juice, 295 Sodium chloride, elimination by the urine, 127, 509, 510 , elimination by the sweat, 581 , physiological impor- tance, 627, 628 , quantitative e s t i- mation, 510 — 512 , influence on the quantity of urine, 642 , influence on the elimination o f urea, 643 , influence on the secretion of gas- trie juice, 274 , influence on the secretion of pan- creatic juice, 295 , influence on absorp- tion, 340 , behavior with food rich in potash, 628 , insufficient supply of, 127, 274 , action on pepsin di- gestion, 270 , action on trypsin digestion, 301 YOO INDMX. Sodium combinations, elimination by the urine, 517 Sodium combinations, division -among the form elements and fluids, 109 Sodium combinations. See also the various tissues and fluids. Sodium fluoride, antiseptic action of, 11 Sodium idodide, absorption of, 339 Sodium phosphate in the urine, 448, 513 , action on metabo- lism, 643 Sodium salicylate, action on secretion of bile, 224 Sodium sulphate, absorption of, 340 , action on proteid metabolism, 643 Soldiers, diet of, 656, 657 Source of muscular energy, 384, 385 Sparing theory, 215 Specific rotation, 64 Spectrophotometry, 147, 148 Sperma, 403 Spermaceti, 86 Spermaceti oil, 86 Spermatin, 406 Spermatocele fluids, 194 Spermatozoa, 404, 405 Spermin, 404 Spermin crystals, 404 Sputum, 605, 606 Spider excrement, guanin therein, 105 Spider poison, 15 Spiegler's reagent, 533 Spirographin, 47 Spirogyra, 109 Spleen, 200— 203 , relation to blood formation, 202 , relation to uric-acid forma- tion, 202, 475, 476 , relation to digestion, 202 , blood of, 170 , pulp of, 475 Splitting processes, in general, 1, 2, 9. See also the various enzymes and ferments. Spongin, 18, 57 Spongioplasm, 90 Staphylococcus, behavior with gastric juice, 282 Starch, 75 , hydrolytic cleavage by intes- tinal juice, 290 , hydrolytic cleavage by pan- creatic juice, 297 , hydrolytic cleavage by saliva, 256, 257 , caloric value of, 617 , absorption of, 332, 334 , behavior in the stomach, 276 Starch cellulose, 76 Starch granulose, 76 Starvation, action on blood, 172, 177 , action on bile secretion, 223 , action on urine, 318, 453, 467, 473, 486, 494, 517 , action on elimination of indican, 317 , action on secretion of pancreatic juice, 295 , action on elimination of phenol, 317 , action on metabolism, 615, 616—619 , quantity of nitrogen in excrements in, 611 , death from, 619 Starvation cures, 658, 659 Steapsin, 298 Stearic acid, 83 Stearin, 83 , absorption of, 335 Stercobilin, 234, 322 Stethal, 86 Stomach, importance in digestion, 281, 282 , self-digestion of, 282, 283 , digestion in, 276 — 283 Stomachic glands, 261 Streptococcus, behavior with gastric juice, 282 Stromafibrin, 129 Stroma of the blood-corpuscles, 129 INDEX. 701 stroma of the muscle, 365 Struma cystica, 204 Strychnin, passage of, into the urine, 530 Sublingual gland, 251 Sublingual saliva, 253 Submaxillary gland, 251 Submaxillary mucin, 45 Submaxillary saliva, 252 Succinic acid in intestine, 313 in the spleen, 201 ' in transudations, 191, 195 in the thyroid gland, 204 passage of, into the urine, 505 passage of, into per- spiration, 581 Sugar, absorption of, 332—334, 340 , syntheses of varieties of sugars, 61, 62, 66 , relation to muscular activity, 379, 380, 384 See also the various varieties of sugars. Sugar formation with lack of oxygen, 372 in the liver, 217, 218, 293, 294 after extirpation of the pancreas, 221, 293, 294 Sugar tests in the urine, 544 — 549 Sulphocyanides in the urine, 507 Sulphonal intoxication, urine in, 540 Sulphur in proteid bodies, 19 in the urine, 507, 508 , elimination of, during work, 383, 508 , neutral and acid, in the urine, 508 , behavior in the organism, 508, 509 Sulphur methsemoglobin, 139 Sulphuretted hydrogen in putrefac- tion in the intestine, 314, 316 Sulphuretted hydrogen in the urine, 508 Sulphuric acid, ethereal and sulphate in the urine, 489, 490, 516 ', elimination of, during work, 383 , elimination of, by the urine, 448, 515 , elimination of, by the sweat, 580, 581 , estimation of, 516 , relation to elimina- tion of nitrogen, 515, 612 Suprarenal capsule, 205 , bile-acids therein, 243 Sweat, 579—581 , excretion of, 579 , action on the urine, 448, 451, 521 Swimming-bladder of fishes, gases of, 603 Swimming-bladder of fishes, guanin of, 105 Sympathetic saliva, 252 Synovial fluid, 196 Synovin, 196 Syntheses, 1, 2, 6 of ethereal sulphuric acids, 314, 489, 493, 494, 496, 529, of conjugated glycuronic acids, 491, 496, 506, 524, 529 of uric acid, 471, 472, 475 of urea, 452, 456 of hippurie acid, 3, 4S6 of varieties of sugars, 63, 66 in the liver,206,215,456,475 Syntonin, 32, 365 , caloric value of, 618 Talonic acid, 72 Talose, 66, 72 Tapeworm cyst, 196 Tartar, 261 Tartaric acid, relation to glycogen formation, 214 702 INHMX, Tartaric aeid, passage of, into the sweat, 581 Tatalbumin, 413 Taurin, 231 , behavior in the animal body, 523, 524 Tauro-carbamic aeid, 523 Taurocholic acid, 227 , quantity in differ- ent biles, 240, 241 , occurrence in me- conium, 323 , decomposition in the intestine, 317 Tea, action on metabolism, 644 Tears, 402 Teeth, 353 Teiehmann's crystals, 142, 539 Tendon mucin, 45, 342 Tendon synovia, 196 Tension of the CO in the blood, 600— 2 603 in the tissues, 603 in the lymph, 182 in the blood, 593 —599 Terpen-glycuronic acid, 529 Turpentine, action on the secretion of bile, 224 , action on the urine, 529, 530 , behavior in the animal body, 529 Tetanin, 14 Tetronerythrin, 148, 578 Tiestis, 403 Tewfikose, 428 Thallin, action on the urine, 530 Theobromin, 102 Theophyllin, 102 Thiolaetie aeid, 50 Thiophen, 524 Thiophenic acid, 524 Thiophenuric acid, 524 Thrombin, 116, 157, 159, 160 Thrombosin, 118, 159 Thymin, 100 Thymlnic acid, 100 Thymus, 203 Thyreoidea, 204 Thyroid gland, 204 Thyreoproteine, 204 Tissue-fibrinogen, 91, 102, 161 Toluhydrochinon, 498 Toluol, behavior in the animal body, 486, 526 Tolurie acid, 528 Toluylendiamin, poisoning with, 247 Toluylic acid, 528 Tonus, chemical, of the muscle, 377 Tooth tissue, 353, 625 Tortoise, bones of, 349 Tortoise-shell, 49 Toxalbumins, 16, 42 Toxins, 14, 206 Transfusion of blood, 173, 178 Transudations, 180, 188—197, 592 Transudation into the intestine, 324 Trehalose, 75 Tribromacetic acid, 24 Tribrom-amido-benzoic acid, 24 Tricalcium casein, 425 Trichlor-acetic acid as reagent, 27, 213 Triehlor-butyl alcohol, behavior in the animal body, 524 Triehlor-butyl-glycuronic aeid, 524 Trichlor-ethyl-glycuronie acid. See Urochloralic acid. Trinitro-albumin, 24 Triolein, 84 Tripalmitin, 83 Triple phosphate in urinary sediments, 566, 568 in urinary calculi, 569, 570 Tristearin, 83 Trommer'e test for sugar, 69, 546 test for sugar, behavior with glycuronic acid, 507 test for sugar, behavior with uric acid, 478 test for sugar, behavior with creatinin, 469 Trypsin, 296, 299 , action on proteids, 301 INDMX. 703 Trypsin, action on other bodies, 307 Trypsin digestion, 21, 301 , action of various conditions on, 301 , products of, 30Si Trypsin zymogen, 299, 308 y> Trytophan, 22, 302 Tuoercle virus, behavior with gastric juice, 282 Tuberculin, 43 Tubo-ovarial cysts, 409 Tunicin, 574 Turacin, 577 Turacoverdin, 578 Typhotoxin, 14 Tyrosin, 304 in the urine, 562 ' in sediments, 562, 568 , detection of, 305, 568 , origin of, 21, 22, 302, 314 , , behavior in putrefaction, 314, 487, 489, 498 , behavior in ' the animal body, 526, 527 Tyrosin-sulphuric acid, 305 Uraemia, blood in, 176 , bile in, 241 , gastric contents in, 283 , sweat in, 581 Uramido-acids, 523 Uramido-benzoie acids, 528 Urates, 478 in sediments, 447, 564, 565 Urea, 452 elimination in work and rest, 382, 384, 385 elimination in starvation, 452, 621 elimination in children, 454, 647 elimination in disease, 453, 458, 517, 518 elimination after different foods, 452, 630, 631, 632, 637, 638, 642, 644 , progress of elimination after a meal, 635 , properties and reactions, 459 Urea, formation and origin, 451—469 , quantitative estimation, 461 — 467 , cleavage by ferments, 459, 565 , synthesis of, 452, 454 — 456 , occurrence in blood, 124, 170, 171 , occurrence in bile, 238, 241 , occurrence in vitreous humor, 400 , occurrence in the liver, 455, 457, 458 , occurrence in muscles, 366 Urea nitrate, 460 Urea oxalate, 460 Ureometer, Esbach's, 466 Urethan. See Carbamicacid ethyl- ester, 467 Uric acid, 471 , relation to urea, 471, 475 , properties and reactions, 477—479 , formation in the organism, 474^-477 from ammonia, 474 , relation to leueoeytosis, 475 , relation to the spleen, 202, 475, 476 , quantitative estimation, 479-^81 synthesis of, 471 , behavior in the body, 474, 483 , occurrence, 472 , occurrence in the sweat, 581 , occurrence in sediments, 447, 564, 568 Uric-acid calculi, 569 Urieaeidaeraia, 176 Urinary calculi, 568 — 572 Urinary pigments, 499 — 505, 540, 541 , medicinal, 544 Urinary sand, 568 Urinary sediments, 447, 566 — 568 Urine, 445—572 , excretion of, 519— 52& 704 INDEX. Urine, constituents, anorganic, 509 — 619 , constituents, poisonous, 509 , constituents, organic, patho- logical, 531—564 J constituents, physiological, 452 —509 , constituents, casual, 522 — 530 , color of, 447, 499, 500, 522, 530, 538, 541—544 , solids, calculation of their quantity, 521 , solids, percentage of same, 522 , alkaline fermentation, 459, 505, 565 , acid fermentation, 564 , gases of, 519 , quantity of, 519—522 , physical properties of, 445 — ■ 452 , reaction of, 447—451, 459, 460 , degree of acidity, 448 : , determination of degree of acidity, 449 , specific gravity, 451, 452, 521, 522 , specific gravity, determination of same, 451 , passage of foreign bodies into, 522—530 , composition of, 522 Urine indicaii,. 493 Urine indigo, 493, 496 Urine poison, 509 Urine stones. See Urinary calculi. Urine sugar. See Dextrose. Urinometer, 451 Urobilin, 499, 500—504 , relationship to bilirubin, 234, 245, 500 , relationship to choletelin, 501 , relationship to hsematin, 245 , relationship to haematopor- phyrin, 501 , relationship to hydrobiliru- bin, 234, 323, 500 Urobilin icterus, 502 Urobilinogen, 499, 500 Urobilinoidin, 501 Urocanic acid, 509 Urochloralic acid, 506, 524 Urochrom, 504 Urocyanin, 500 Uroerythrin, 504, 541 Urofuscohaematin, 541 Uroglaucin, 500 Urohsematin, 500 Uroleucic acid, 497, 499 Uromelanins, 500 Uronitro-toluolic acid, 529 Urophsein, 500 Urorubin, 500 Urorubrohsematin, 541 Urorosein, 500, 541 Urostealith, 571 Uroxanthin, 493 Urohodin, 500 Ureids, 20, 471, 472, 483 Uterine milk, 419 Utilization of foods,. 330, 332, 335 Valerianic acid, 21, 354 Varnishing the skin, 582 Vegetable gums, 77, 79 Vegetable mucilage, 77, 79 Vegetable myosin, 42 Vegetable proteids, 42 Vegetarians, food of, 639, 654 , excrements of, 321 Venesection, 168, 176 Vernix caseosa, 578 Vesicatory blisters, contents, 196 Visual purple, 398 Visual red, 398 Vitellin, 18 in yolk of egg, 410 in protoplasm, 91 Vitellolutein, 412 Vitellorubin, 412 Vitelloses, 36 Vitreous humor, 343, 400 Water drinking, action on the elimi- nation of chlo- rides, 510 INDEX. 705 Water drinking, action on the elimi- nation of uric acid, 473 , action on the elimi- nation of urea, 642 , action on the deposi- tion of fat, 642 , action on the secre- tion of urine, 520, 521 Water elimination by the urine, 520 —522, 609, 610 elimination by the skin, 579, 610 elimination in starvation, 623 , importance for the animal body, 625 , quantity of, in the various organs, 623 , lack of, in the food, 623 , absorption of, 339 Wax, 87 in plants, 579 Weyl's reaction for creatinin, 469 Wheat bread, absorption of, 334 Whey, 422 WTiey proteid, 427 White of egg, 413 , calorific value, 574 Witch's milk, 439 Woman's milk. See Human milk. Wool-fat, 250, 579 Work, action on chlorine elimination, 510 , action on elimination of phos- phoric acid, 513 , action on elimination of sul- phuric acid, 383, 508 , action on the need for food, 656, 657 Work, action on metabolism, 377, 383 —385 Xanthin, 104 in the urine, 484 in urinary sediments, 568 , quantity in the liver, 208 , quantity in the pancre:;s, 292 , detection and quantitative estimation, 108, 109 Xanthin bases, in general, 102 , relationship to uric- acid formation, 203, 475 , behavior to muscular activity, 382 , occurrence in the blood, 103 , occurrence in the urine, 484 Xanthin calculi, 571 Xantho-creatinin, 369, 381, 470 Xanthophan, 399 Xanthoproteic acid, 24 Xanthoproteic acid, reaction, 27 Xylol, behavior in the animal body, 527 Xyloses, 65, 66 , relation to glycogen forma- tion, 213 Yeast-cells, proteids in, 48 Yolk of the hen's egg, 410 Zinc in the bile, 238 in the liver, 210 , passage of, into milk, 443 Zoofulvin, 578 Zoonerythrin, 578 Zoorubin, 578 Zymogens. 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