A^"^ ^' :m ■' ,-^V^ ^f^;^*- KtA%- '^'^--p fe^^f Cornell University Library RB 45.B66 The determination and occurence of amino 3 1924 003 504 788 Cornell University Library The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003504788 A CKNO WLEDGEMENT ' I 'HE author wishes to express his sincere appreciation and thanks to Professor Stanley R. Benedict, under whose supervision this work has been done, for his kindness and advice. He also wishes to thank his fellow workers in the laboratory for their interest and help. To Mrs. Bock the author is especially indebted for many hours of help rendered in preparing the manuscript. The Determination and Occurrence of Amino- Acids in the Blood A THESIS Presented to the Faculty of the Graduate School of Cornell University for the Degree of Doctor of Philosophy BY JOSEPH CARL BOCK MAY, 1917 RIVERSIDE PRESS, N. Y. CONTENTS Page Acknowledgement Introductory ^ Part I — Qualitative Methods for Amino- Acids 6-7 Method of Fischer 6 The jg-naphtalinsulfochloride Method 6 Erben's Modification 8 Embden and Reese's Modification 6 The o-naphtylisocyanate Method 6 Method of Neuberg and Kerb 7 Method of Siegfried 7 Part II^Quantitative Methods for Amino- Acids 7-14 Methods of Pf aundler and Kruger-Schmid 7 Method of van Leersaum 7 Method of Kutscher and Lohmann 8 Method of Glaessner 8 The Forraoltitration Method of Sorensen 8 Benedict-Murlin Modification 9 Method of Kober 9 The Ninhydrin Reaction 9 Folin and Denis Procedure 9 Method of Sachsse and Kormann 10 Emmerling's Modification 10 Kern's Modification 10 Method of Konig and Splittberger 10 Method of Staneck 11 Van Slyke's Method 11-14 Part III — Experimental 14-25 Comparison Between Methyl Alcohol and Trichloroacetic Acid Precipitation 14-21 (a) For Total Non-Protein Nitrogen 15 (b) For Amino- Acid Nitrogen 17-20 Removal of Trichloroacetic Acid by: (a) Vacuum Evaporation 17 (b) Direct Evaporation 17 Heat Coagulation followed by: (a) Phosphotungstic Acid 21-23 (b) Trichloroacetic Acid and Kaolin 23-25 Part IV — Occurrence of Amino-Acids 26-30 The Work of Bingel and Abderhalden 26 The Vividiffusion Method of Abel 26 The Occurrence of Amino-Acids in: (a) Mammalian Blood 27 (b) Bird Blood ..........[.[[[ 28 (c) In Plasma and Corpuscles 28 (d) In Normal Human Blood !!!!!!!!!! 29 (e) In Pathological Human Blood !!.!!.!!! 29 Part V — The Amino-Acid Nitrogen and Non-Protein Nitrogen in Nephritis and Uremia 30 Tables I-VI Insert and 81 Bibliography 32.47 INTRODUCTORY Although the amino-acids have been known since 1802, when Delaville discovered asparagine in the juice of asparagus and cabbage, it was only within the last two decades that the chemical and biological importance of these compounds has been clearly recognized. Within recent years so much attention has been given to this field that the chemistry and physiology of amino-acids constitute very distinct chapters in the science of biological chemistry. The present study was planned to extend our knowledge of the quan- tity of amino-acids in the Wood of widely varying species, including human blood, under both normal and pathological conditions. Iln order to do this it was necessary at the outset to test existing methods for the determina- tion of amincnacids in biological fluids and to modify one of the procedures heretofore available so as to have a method capable of a satisfactory degree of accuracy. It is outside the scope of the present paper to enter into a discussion of the determination and identification of individual amino-acids. The methods with which we shall concern ourselves are for the estimation of total animo-acid nitrogen. [3] PART I The older methods for the deternxination of amino-acids have either been proved deficient or they have been found of Httle value in the analysis of body fluids. However, the fundamental, principles of the older methods are briefly discussed. The, formol titration methods, the ninhydrin re- action, and especially Van Slyke's gasometric method are generally used and, therefore, fully described. It is necessary to follow certain pro- cedures in order to prepare the body fluids for analysis by these methods, and these procedures will be discussed in some detail. Fischer's procedure, which depends upon esterification of the amino- acids and the fractional distillation of the esters, has been of immense service in the qualitative separation and quantitative determination of amino-acids in protein hydrolysis mixtures. Fischer first evaporates the substrate containing the amino-acids to a syrup under reduced pressure and then adds absolute alcohol. Gaseous hydrochloric acid is passed into the liquid until solution is complete. After boiling for one hour under a reflux condenser the liquid is evaporated under strongly reduced pressure and low temperature (35 degrees C). To the residue a little water is added and then the whole mixture is well covered with ether. It is then cooled and the free hydrochloric acid is approximately neutralized with strong sodium -hydoxide. With continued cooling and shaking solid potassium carbonate is added to set free, the esters. After vigorous shaking the ether portion is poured off and the residue extracted two or three times by shaking with ether. The ether extracts are filtered, dry potassium carbonate and a little sodium sulphate are added and allowed to stand with occasional shaking for several hours. The esters are then obtained by evaporation of the ether. They can be separated by fractional distillation under reduced pressure if necessary. , Fischer's procedure has been modified several times. The more im- portant modifications, proposed by T. B. Osborne and collaborators, at- tempt to make the method quantitative. The ester method,, in any of its forms, is rather difficult and, is not quantitative where small amounts of amino-acids are concerned. The method, therefore, has not so far been applicable to the analysis of blood and urine. The following two methods have been used for biological fluids with better success by several investigators. Fischer and Bergell, and Abderhalden and Barker used j8-naphtalinsulfochlorid in alkaline solution [5] for the determination of the amino-acids. The substrates containing the amino-acids are concentrated to a small volume and are then treated with a solution of two molecules ^-naphtalinsulfochlorid in ether, for approxi- mately one molecule of amino-acid. The mixture is made slightly alkaline with potassium or sodiumhydroxide and shaken at room temperature for nine hours by a shaking machine. At intervals of one and one-half hours one gram of the reagent in a small amount of ether is added, at the same time taking care that the reaction remains alkalifie. The mixture is then allowed to stand until the two layers have separated. The ether is poured off, the lower aqueous layer is cleared by filtration and then fully satu- rated with hydrochloric acid. If amino-acids are present more or less cloudiness should be observed at this stage. The B-naphtalinsulfo amino- acids very rarely crystalize out from the cloudy solution. The solution is therefore shaken with the same volume of ether, which dissolves the pre- cipitate. This shaking is repeated twice with new portions of ether. The collected ether etxracts are united and washed with a small portion of ^ water until free from chlorides. The extract is then filtered, if necessary, and the ether evaporated. The residue is recrystallized from hot dilute alcohol. Erben proposed to saturate the acid mixture with ammonium sulfate before the ether extraction in order to obtain better yields. Although his modification improves the procedure, the results are far from quantita- tive. Embde and Reese, after removal of hippuric acid with ether, advocate the use of much higher alkalinity and longer shaking (two days) at a somewhat higher temperature (30 degrees C). The rest of the procedure is practically the same. They claim decidedly better yields with their modi- fication. The method has been used to considerable extent in urine analysis by Ignotowsky; and by Embden and Reese. Von Bergmann and co-workers and also Howell used the same procedure for blood analysis without obtaining very conclusive results. Neuberg and Manasse used a-naphtylisocynate for the estimation of amino-acids. This compound has the advantage of being a liquid and does, therefore, not require a special solvent as does ^-naphtalinsulfo- chloride. In this method the solution containing the amino-acids is made alkaline and the reagent added. After shaking several times for three minutes (by hand) the flask is allowed to stand for thirty to forty-five minutes. The dinaphthyl carbamid which forms is filtered off, the filtrate acidified and the a-naphtylhydantoic acids recrystallized ffom dilute alcohol. Levene and Van Slyke, Loewy, Neubei^, Osborne,, and Wohlgemuth [6] report findings of amino-acids in urine with this method, Hirschstein criticized the method, claiming that the a-naphylisocyanate does not react in a very dilute solution. Neuberg and Manasse disproved Hirschstein's statement. Loewy and Neuberg suggested first evaporating the substrate to a small volume before treatment with a-naphtylisocyanate. Another method was proposed by Neuberg and Kerb but has not found much application. They precipitate the amino-acids with mercuric acetate in the presence of sodium carbonate, then remove the mercury by hydro- gen sulfide and obtain the amino-acids on evaporation. Siegfried proposed the use of 4.nitrotoluol.2.sulphonic acid for de- termination of protein cleavage products and tried the same with several amino-acids In all these methods no real quantative results were obtained. They were mainly used to show the presence of amino-acids. The amino-acids were identified after subsequent separation of the crystallized residue by special reactions, elementary analysis, by Kjeldahl or by the polariscope. PART II We shall now discuss methods which have been used for the quantitative estimation of amino-acids. All these methods were first applied to urine and some of them later to blood, especially the ones described more fully in this paper. The method of Pfaundler, with its several modifications, makes use of the fact that amino-acids are not precipitated from dilute solution by phosphotungstic acid. Kriiger and Schmid made use of the same principle, varying from the original Pfaundler method very little. Both methods are here described at the same time. The amount of phosphotungstic acid necessary for complete precipitation is first de- termined. Portions of the filtrate obtained after precipitation are heated with phosphoric acid to 150 degrees for twenty hours (in Kruger and Schmid's method with 50 per cent, sulfuric acid to 160 degrees from three to four hours in a closed tube). The amino-acids do not split off nitro- gen in the form of ammonia by this treatment. By determining the total nitrogen and the ammonia in the filtrates so treated the amino-acid nitro- gen is obtained by difference. Van Leersman modified the Pfaundler method. He found that the greatest difficulty with the method was due to the fact that the phospho- tungstic acid attacks the glass very vigorously. Leersaum treats the urine as Pfaundler does but removes the phosphotungstic acid before hydrolysis by addition of a potassium chloride solution. [7] Kutscher and Lohmann also made use of the phosphotungstic acid precipitation. They then remove the reagent with barium hydroxide and, after evaporating the filtrate, identify the amino-acid as platinum salts or picrates. Glaessner treats the liquids containing amino-acids with phosphotungs- tic acid. The filtrate obtained after precipitation is evaporated and died in vacuo at 40 to 45 degrees C. and this residue is extracted with a mixture of equal parts of ethyl and amyl alcohol. This alcohol mixture leaves the amino-acids undissolved, with the possible exception of a little tyrosin. After filtering, the nitrogen in the undissolved part is determined by Kjeldahl. This method has been tried only for several, not for all aminos acids. All the methods discussed so far have little but historical value. Schiff contributed an important chapter in amino-acid determination when he found that by treating amino-acids with a neutral formaldehyde solution it is possible to separate the amine from the acid function, the amino group forming a methylen derivative with the formaldehyde. R-CH-NH, R-CH-N=CH2 I +CH,0= i +H,0 COOH COOH Sorensen made use of this fact 'by titrating the carboxyl group, using phenolphtalein as an indicator. It must be borne in mind, however, that ammonium salts react in a sim.ilar manner as amino-acids with formalde- hyde. Therefore the formoltitration will include both ammonia and amino-acid nitrogen present in the solution. The ammonia may be de- termined separately and a correction applied, or the ammonia may be re- moved by distillation. Phosphates which interfere by obscuring the end- point are removed by barium salts. The procedure in general is as fol- lows : Into a graduated flask introduce an accurately measured amount of the liquid to be analyzed. Add phenolphtalein and barium chloride. Shake well. Barium hydoxide solution is then added, the flask filled to mark and again well shaken and allowed to stand for some time. After filtering, a definite volume of the clear fiUrate is transferred to measuring flask, neutralized to litmus with n/s hydrochloric acid and diluted to mark with freshly boiled water. In an aliquot part taken from this solution the amino-acid nitrogen (and also ammonia) is determined. For this pur- pose neutral formaldehyde and then a standard barium hydroxide solution in excess are added. The excess barium hydroxide is titrated back with standard hydrochloric acid. The endpoint is obtained by accurately matching a control sample which has been previously titrated to a certain color. Critical studies on the Sorensen-Henriques method have formed the [8] subject of many papers, the most prominent of the publications being by Malfatti, by de Jager, by Frey and Gigon and by Labbe. A distinct improvement of the Sorensen method is the modification by Benedict and Murhn. These authors precipitate the ammonia and basic substances with phosphotungstic acid and remove the excess acid by the addition of solid barium hydroxide until the phenolphtalein used as in- dicator turns to permanent pink. After filtering, an aliquot is neutralized to litmus, neutral formaldehyde is added, and the solution is titrated to a deep red color with n/5 sodium hydroxide. A micro method for amino-acid determination has been proposed by Kober. The method makes use of the property of amino-acids to dissolve cupric hydroxide in neutral or faintly alkaline medium to form a metallic complex. The copper is determined by titration with iodine. The next procedure to be discussed is based on the so-called "Ninhy- drin" reaction. Triketo-hydrindene-hydrate (ninhydrene) is a very sensi- tive reagent for amino-acids (Ruhemann). Abderhalden and collabora- tors, Halle and Lowenstein, and especially Harding, have investigated this reaction extensively. The main difficulty lies in the fact that the nin- hydrin reacts not only with amino-acid but also with proteins, proteoses, peptones, polypeptides and ammonium salts, giving different shades of color with these bodies. According to Harding, the solution to be ex- amined is made neutral to phenolphtalein. An aqueous solution of pyridine and the reagent are added and the mixture boiled. After cooling and diluting to the required volume, the color is compared with a standard color obtained by treating a solution of pure alanin in the same way. An indirect method to study the increase in the amino-acid nitrogen in the blood under certain experimental conditions has been used by Folin and Denis in their studies on protein metabolism. These authors de- termine by difference the residual non-protein nitrogen ; i. e., nitrogen other than urea in the non-protein nitrogen fraction of the blood. The nitrogen found in this way is, of course, by no means amino-acid nitro- gen alone, but the method probably will serve to indicate any marked increase in amino-acid nitrogen. The term amino-acid nitrogen for residual nitrogen should, however, be avoided. Of all the methods considered so far only the modified Sorensen method would be of use in the quantitative estimation of amino-acid nitrogen in blood. More recently a method for the determination of amino-acid nitrogen based upon the well-known reaction of the amino group with nitrous acid has come into wide-spread use. The method involves the evolution of nitrogen through the reaction between nitrous acid and the amino group [9] and the exact measurement of the nitrogen liberated. This reaction may be written as follows : R-NH2+HN02=R-OH+N,+H,0 Numerous attempts have been made to determine the amino-acid nitrogen gasometrically but it was not until Van Slyke perfected a form of apparatus that the method came into general use. The Van Slyke method has been chosen for the investigation reported in this paper, but as previous investigators have used procedures more or less similar, it seems of interest to give brief descriptions of these methods. Sachsse and Kormann decompose the substance with a mixture of potassium nitrite and sulfuric acid. The vessel in which the decomposi- tion takes place consists of a short, wide cylinder fitted with a three-holed stopper in which two small funnels with stopcocks and a delivery tube are inserted. Potassium nitrite and sulfuric acid are introduced into the cylinder and allowed to act for some time to free the apparatus and tube of air. The material for analysis is then washed into the cylinder through one of the funnels and allowed to react for some time with the nitrous acid mixture. The gas developed is collected in a burette over ferrous sulfate solution. When the reaction is completed all the gas left in the cylinder is transferred to the burette by displacement with water. The gas in the burette is washed with ferrous sulfate solution, then with potassium hydroxide and is then finally measured by inserting the burette in a cylinder filled with water. Emmerling, using the same principle as Sachsse-Kormann, allows the substances to react in vacuo, washes the gas first with potassium hydrox- ide, and finaly with ferrous sulfate. The apparatus employed is very complicated. Kern uses an apparatus similar to the one employed by, Sachsse and Kormann. Kern replaces the air in the apparatus by carbon dioxid. The carbon dioxid is then removed by evacuation and the gases obtained are finally driven out by carbon dioxid and then treated with ferrous sulfate and potassium hydrate, as in the Sachsse-Kormann method. Bohmer also uses nitrous acid but washes the gas with alkaline per- manganate solution. Konig and Splittberger likewise use nitrous acid for the deamination A flask is fitted with a three-holed stopper, carrying two tubes and a small funnel with a stopcock. The material is first introduced into the flask in a slightly alkaline solution and a solution of sodium nitrite is added. Carbon dioxid is now passed through the flask to drive out the air. The flask is then connected with a U-shaped eudiometer, one leg of which is graduated. The eudiometer is filled with alkaline permanganate solution. [ 10 ] Dilute sulfuric acid is now added to the flask containing the sample and the nitrite, and the gases which are generated are collected in the grad- uated tube of the eudiometer. The flask is finally, shaken to complete the reaction and, after standing for some time, the nitrogen is measured, bringing the two menisci in the eudiometer to the same level. Staneck uses for the decomposition of the substance a mixture, the active part of which he claims to consist mainly of nitrosylchloride. It is made by treating fuming hydrochloric acid with a sodium nitrite solu- tion. The, reagent so obtained is an orange red liquid which will keep for several days without appreciable decomposition. The apparatus con- sists of a decomposition (deaminizing) bulb, a cylinder filled with potas- sium hydrate, a burette with water jacket and leveling bulb and a Hempel pipette filled with alkaline permanganate solution. The solution to be analyzed is first introduced into the deaminizing bulb and freed of air by passing a stream of pure carbon dioxid gas through it for some time. Then the "nitrosylchloride" mixture is run into the bulb and the reaction allowed to proceed for a half-hour with occasional shaking. The gases developed pass through the potassium hydrate and collect in the upper part of the cylinder. After the reaction is completed, all the gas is driven over into the cylinder by filling the deaminizing bulb with a little of the reagent mixed with sodium chloride solution. From the cylinder the gas is transferred to the burette and from there immediately to the potassium permanganate solution. It is left in contact with the latter until no more absorption takes place. The gas is then run back into the burette and measured. The volume divided by two gives the amount of amino-acid nitrogen in cubic centimeters. Staneck analyzed a number of amino- acids and obtained very satisfactory results. He also studied the reaction of ammonium salts, urea, gelatine and other nitrogenous substances, most of which were found to give off a larger or smaller amount of nitrogen with his method. He also mixed amino-acids with other non-nitrogenous substances in order to find out if their presence had any influence on the reaction. No material change was noticed. The gasometric method of Van Slyke has been used throughout the present work and will, therefore, be discussed in detail. The amino sub- stance is decomposed by the action of nitrous acid. The nitrous acid at the same time undergoes spontaneous decomposition to nitric oxide aftd nitric acid. The nitric oxide is absorbed with alkaline permanganate solution and the residue of pure nitrogen measured in a specal gas burette. The apparatus has undergone several modifications in the course of the last few years. At present two sizes of apparatus are used. Both are the same in principle and construction, the only difference being in [11] the size. The smaller, so-called micro apparatus, was used in all the work reported in this publication. The apparatus consists of a deamin- izing bulb (Fig. i) which is connected by means of a capillary tube to a r~^ Fig. 1. gas burette, F. This burette of 3 cc. capacity is graduated in o.oi cc. divisions about i mm. apart, so that by; estimating tenths of a division the volumes can be read to o.ooi cc. The zero point of the burette is placed on a capillary near the stopcock, thus marking off the upper boundary of the gas volume measured with an error of less than o.(X)i cc. The ab- sorption of the nitrous oxide takes place in a two bulb Hempel pipette filled with a solution of potassium permanganate in a potassium hydrate , solution (KMn04 50 g., KOH 25 g. per liter). The deaminizing vessel and the Hempel pipette, respectively, are shaken by means of a motor con- nected to a driving wheel which, in turn, has a driving rod bent in such a way as to permit connection with the pipette or with the bulb. The determination is carried out in three stages. The deaminizing [12] 1 . bulb is first freed of air, then the amino substance is decomposed by nitrous acid, the nitric oxide is absorbed, and finally the nitrogen is meas- ured. An actual determination is here described; Water acidified with sulfuric acid fills the gas burette and leveling bulb, the capillary tube leading to the Hempel pipette and also the tube from f as far as c ( Fig. I ) . The stopcock c is turned so as to let air escape from D. Close a and fill A to lower mark with glacial acetic acid. Let the acid run into D, the amount of acid just about filling one-fifth of D. Close a again and fill A to upper mark with a sodium nitrite solution (30 grams NaNOg to 100 cc. water). Run the solution into D until it reaches the stopcock c, which is then turned oflf. Enough solution should be in the tube leading to A to rise above the stop cock a. Now shake the deaminizing bulb (a open, c closed) until sufficient nitric oxide gas has been formed to drive the liquid in D down to the mark. A is now closed and c opened and the deaminizing vessel shaken rapidly for two minutes. The nitric oxide evolved frees the vessel of air. The stopcocks c and f are now turned so as to connect D and F. The burette B is filled with the solution to be analyzed, taking care that the L of the stopcock is filled with the liquid in order not to introduce any air into the apparatus. The necessary amount of solution is run into D and the vessel is then shaken from three to five minutes (depending on the room temperature). For substances requiring a longer time than five minutes to completely react one merely mixes the reacting solutions and lets them stand the required length of time and then shakes in order to drive the nitrogen completely out of the solution. In case the solution should foam very badly, B is rinsed out, dried with a roll of filter paper (or alcohol and ether) and a little caprylic alcohol is added through B and the shaking continued for the required length of time. The reaction being completed, all the gas in D is dis- placed into F by liquid from A. The mixture of nitric oxide and nitrogen is driven from F into the Hempel pipette and shaken by motor. While the Hempel • pipette is being shaken the liquid in the deaminizing bulb is run out through d and the bulb cleaned by rinsing with water. The alkaline permanganate in the Hempel pipette takes up the nitric oxide and leaves only nitrogen. After shaking for one minute the nitrogen is trans- ferred to F, where it is measured. The room temperature and barometric pressure must be noted. The calculation of the weight of nitrogen gas corresponding to the volume obtained is most readily made with the aid of tables. Such tables are found in Van Slyke's article (J. Biol. Chem., 1912, 12, 284), and also in the text-books of Hawk and of Mathews. We have found it very convenient to have these tables photographed so that prints can be made from the plates whenever tables are needed in the [13] laboratory. Blank determinations must be made with every new lot of sodium nitrite used. The determination is carried out exactly as described above, water replacing the sample. PART III When amino-acid nitrogen in blood is to be determined it is necessary to remove the proteins first. The filtrate obtained by this procedure has to undergo several manipulations before it is ready for actual analysis. The methods proposed for the removal of blood proteins are very numerous, but only a- few methods have really proven successful and not all of these can be used in connection with the determination of amino- acids in blood. The main difficulty with most precipitation methods is, that the filtrates contain too large an amount of solids in solution to permit evaporation to the small volume necessary for the final determina- tion by the Van Slyke method. The methods for the removal of blood proteins which were considered of some possible use in the present work will be shortly summarized. A method which has found much use is that of Reid as recommended by Vosburgh and Richards. Phosphotungstic acid in dilute HCl is the protein precipitant in this method. The precipitated mass is hard and granular and hence may be easily filtered and washed. Rona and Michaelis introduced an entirely new method of blood precipitation, namely the use of colloids. They used kaolin and colloidal iron, giving preference to the latter. Van Slyke and Meyer adopted ethyl alcohol as a blood precipitant prior to the amino-acid detemiination. They pre- cipitate by diluting the blood with nine volumes of 95 per cent, ethyl alcohol in an accurately graduated vessel. After standing for 24 hours the solution is filtered through a dry folded filter. An aliquot of the filtrate is concentrated to a volume of 3 to 5 cc. (Van Slyke and Meyer recommend evaporation in vacuo) and used for the determination by the Van Slyke nitrous acid method. In spite of the rapidly growing literature upon the occurrence of amino-acids in blood and tissue under various conditions, little attention appears to have been given to a study of the various procedures for the preliminary removal of protein and subsequent manipulation of the solution prior to the analysis. For the most part the alcohol precipita- tion method of Van Slyke and Meyer has been followed. Recent investigations by Folin and Denis and particularly by Green- wald have indicated that amino-acids do not completely escape precipita- [U] tion by alcohol. It was therefore considered desirable to compare the results obtained by the Van Slyke method where different procedures were employed for the removal of the proteins. The method used by Folin and Denis for the precipitation of the blood proteins is open to serious objection. Methyl (or ethyl) alcohol is not a good solvent for the extraction of substances such as some amino-acids that are compara- tively insoluble therein. Folin and Denis themselves state that creatine, asparagine and tyrosine added to the blood could not be quantitatively recovered. Greenwald as a result of his study of this question concluded that alcohol precipitates some nitrogenous constituents of the blood of which 25-50 per cent, represents amino-acid nitrogen as determined by the Van Slyke method. As it was originally planned in the present work to substitute Greenwald's procedure of blood precipitation with tri- chloroacetic acid for the alcohol precipitation, a somewhat detailed study of the two methods was made. Determinations of total non-protein nitrogen were made first. Samples of blood were precipitated according to Folin and Denis by diluting the blood with nine times its volxune of methyl alcohol in a graduated flask. After standing for twO' hours, the mixture was filtered and to the filtrate a few drops of alcoholic ZnCl^ solution were added. After standing for a few minutes the precipitate which had formed was filtered off. The clear filtrate was now ready for analysis. Samples of the same blood were precipitated according to Greenwald. The blood was diluted with nine times its volume with a 2.5 per cent, solution of tricholoracetic acid and filtered after standing for 30 minutes. The filtrate was shaken with kaolin and filtered again. Aliquot parts of the filtrates were digested and distilled as described in a previous paper by Bock and Benedict. The method employed is shortly summarized here. An accurately measured amount of the re- spective filtrates representing about i cc. of blood was introduced into large Jena test tubes and evaporated to a small volume after one drop of concentrated H2SO4 had been added. One cc. of concentrated H2SO4, 0.5 g K2SO4 and three drops of a 10 per cent, solution of CuSO^ were now added and the mixture heated over a microbumer until it had be- come perfectly clear. The heating was then continued for six minutes longer. After the mixture had partially cooled it was diluted with 7 cc. of water. After adding 3 cc. of concentrated sodium hydroxide solution, the ammonia was distilled into i to 2 cc. of N/io HCl contained in a small graduated flask. The nitrogen in the distillate was determined by Nesslerization. In other cases the equivalents of 3-5 cc. of blood were used, digested as described above and then distilled into n/ioo HCl and the excess acid titrated with N/ioo sodium hydroxide using methyl red [15] as indicator. A third set of results was obtained 'by precipitating samples of blood as described above and determining the nitrogen by Kjedahl on amounts of filtrates representing 30-40 cc. of blood. The micro determinations reported (Table A) represent averages of three to seven determinations. The Kjeldahl determinations represent averages of two to three determinations. TABLE A Comparison between Alcohol Precipitation and Trichloroacetic Acid Precipi' tation of Blood. Non-Protein Nitrogen per 100 Cc. of Blood. Material. Methyl alcohol precipitap tion. Trichloro- acetic acid precipita- tion. Remarks- mg. 28.70 25.40 23.25 22.12 22.0? 18.31 26.30 21.10 mff. 37,60 34; 70 26.25 25.90 23.30 24.30 29.55 25 60 By micro Kjeldahl. Distillates Nessler* It it tt Ox " (defibrinated) tt it tt ized. e|ica. Calcu- lated. Ox blood mg. 5.13 5 07 mg. 10.44 9.53 mg. 5.31 4.46 mg. 6.28 6.28 mg, 6.89 6.43 mg. 12.59 12.71 mg. 5.70 6.24 mg. 6 28 *l K 6.28 frothing will occur. Fatty substances which are taken up by the alcohol give trouble in the Van Slyke apparatus, causing the mixture to froth and also make the subsequent cleaning of the deaminizing bulb very troublesome. The precipitation with trichloroacetic acid was carried out as de- [20 1 scribed before. Table D compares the results obtained by the two methods on different bloods. The recovery of amino-acids added to the blood by the two procedures was also investigated. The amino-acid solu- tions used were obtained by hydrolyzing pure casein with strong HQ as described by Fischer, E. (Untersuchungen, etc.). After removing the HQ as far as possible by vacuum distillation, the solution was diluted to a convenient concentration of amino-acid nitrogen and treated by the Van Slyke method. As ammonia is formed in the hydrolysis of the casein, it is necessary to remove this before the determination of the amino-acid nitrogen is made. The results obtained by the methyl alcohol precipitation method are invariably lower, and the recovery of added amino-acids is not so com- plete as in the case of the trichloroacetic acid procedure. The latter procedure gives satisfactory results, but is somewhat troublesome in certain stages of the manipulation. Filtration is very slow even if a fluted filter is used, the use of suction is not advisable due to the nature of the precipitate and the alternative process of centrif uging such large volumes (300-500 cc.) is not always convenient. Another procedure was therefore sought. It has been pointed out previously that most precipitation methods leave too large an amount of solids in the filtrate. Therefore a procedure was adopted providing for preliminary coagu- lation of the proteins by heat in a faintly acid solution and evaporation of the filtrates to a small volume. The trace of proteins escaping the first precipitation is then removed by a precipitant which does not ap- preciably increase the amount of salts in the final solution. The heat coagulation was carried out as suggested in Benedict's uric acid method. The following procedure is recommended : Into a flask introduce approximately 0.3 gm. of ground soy bean (20 mesh), add 3 to 5 cc. of water and i cc. of a 3 per cent, solution of NaHjPO^, and let stand for a few minutes with occasional shaking. Run in a measured amount of blood (from 30 to 50 cc.) and let stand at room temperature for 30 minutes. Heat 0.01 N acetic acid to boiling in a casserole, using five volumes of acid for one volume of blood. Run the blood from the flask slowly into the boiling acid and with constant stirring boil for one-half minute, Add the same amount of boiling water, using this also' to rinse the flask. Boil with stirring for i minute. Filter through a folded filter and wash the casserole three times with small portions of water (30 cc), heating the water in the casserole in which the original coagulation took place and using a rubber-tipped stirring rod. The filtrate is boiled down [21] rapidly over a free flame to about lo cc. in a casserole. The contents of the casserole are now quantitatively transferred to a small graduated flask or cylinder, choosing the size so as to obtain nearly the volume of the original blood. Wash the casserole with the smallest possible amount of hot water three times. The volume in the flask or cylinder, after the final wash water has been added, should not be more than about three- fourths of the final volume. At this stage of procedure different protein precipitants were tried. The first was a solution of colloidal iron (5 per cent. Merck). This method, while removing protein completely, requires a little experience to obtain filtrates, which can be evaporated to a small volume without getting cloudy. It was therefore abandoned for the present. The next precipitant tried was phosphotungstic acid. The filtrate obtained by the heat coagulation procedure was evaporated to a small volume as de- scribed above and transferred to a 50 cc. graduated flask, the total volume at this stage being about 40 cc. The filtrate in the flask was heated on a water bath and a hot solution of phosphotungstic acid ( 10 g. phospho- tungstic acid in 100 cc. of 2 per cent. HQ) was added. The mixture was heated for 15 minutes on a water bath, cooled, made up to volume and filtered. The filtrate was made distinctly alkaline to phenolphtalein by gradual addition of small amounts of solid bariumhydroxide. After filtering, the liquid was made slightly acid to litmus with HCl. An ali- quot part was evaporated to a suitably small volume and the amino-acid nitrogen determined by the Van Slyke method. In some cases the filtrate obtained after the precipitation with phosphotungstic acid was allowed to stand for 24 hours before treating with bariumhydroxide. Although a TABLE F- Amino-Acid Nitrogen per 100 Cc. of Blood (Sheep). Heat coagulation followed by phospho- tuDgstic acid precipitation. Heat coagulation followed by trichloro- acetic acid precipitation. 4.64 4.13 4.37 4,48 3.25 3.12 Blood 3.18 " + amino-acid 8 . Difference 5.42 Calculated 9,32 7.60 6.34 [22; slight precipitate was formed on longer standing, there was no material change in the araino-acid contents. The precipitates obtained by the phosphotungstic acid procedure are very coarse and easy to filter, but the results are too low. As the phosphotungstic acid precipitant may possibly be used in some later work, some of the results obtained are given in Table F. The re- sults are compared with another procedure which will be discussed shortly. The third precipitant tried was trichloroacetic acid followed by kaolin, as suggested by Greenwald. The filtrate from the heat coagula- tion after being evaporated to a small volume and transferred to a grad- uated flask or cylinder is treated with trichloroacetic acid. Introduce into the graduated flask enough solid trichloroacetic acid to make an ap- proximately 3 per cent, solution. For this purpose the acid is either weighed out on a small scale in a little glass scoop or watch-glass and washed into the cylinder with a little water, or if several determinations are made, a 50 per cent, solution of trichloroacetic acid is kept on hand and the corresponding amount of this solution is added with Mohr pipette. After making the solution up to volume, let it stand for 20 to 30 minutes. Shake with 2 gm'. of kaolin, centrifuge, and run the super- natant liquid through a drj" filter paper, because a little kaolin always sticks to the side of the centrifuge tube above the liquid level and is car- ried along when the liquid is poured out. An aliquot part of the filtrate is transferred to a small flask, and a few beads and a drop of alizarin indicator are added. The liquid is brought to boiling over a micro burner and kept boiling very slowly (simmering) until the indicator turns. The flask is removed from the flame and enough (i or 2 cc.) of i.o N potassium hydroxide is added to make the liquid distinctly alka- line. Boil for I to 2 minutes, taking care that it does not boil over, be- cause at this stage slight frothing and bumping occur. Make distinctly acid with acetic acid and boil down to the smallest possible volume. The liquid is now ready for the amino-acid apparatus. It is either trans- ferred directly to the burette of the Van Slyke apparatus, washing the flask with very little water, or first transferred to a small accurately graduated text-tube and made to a definite volume. From this tube dupli- cates can be measured out by means of the burette of the amino-acid apparatus. The latter procedure is especially recommended where large amounts of blood are available. The heat-trichloroacetic acid method gives filtrates which rarely ex- hibit any tendency to froth, when shaken in the deaminizing bulb. Should frothing occur, for some reason, caprylic alcohol, as recommended [23] 'by Van Slyke is very efficient. The best caprylic alcohol which we have been able to obtain at present gives such high corrections for the blanks that it should not be used without purification. For that purpose the alcohol is shaken twice (best in a separatory funnel) with a mixture of glacial acetic acid and NaNOa solution (30 gm. in 100 cc. of HjO), the acid and the nitrate being in the proportion of i -.5. The alcohol is then washed with a little water two or three times, transferred to a distilling flask, a very small fraction of sodiumhydroxide added, and distilled un- der reduced pressure. The caprylic alcohol so purified shows a negligible increase in the blank figures. Table G-a shows a comparison between the direct trichloroacetic acid precipitation and the heat coagulation followed by the trichloroacetic acid precipitation. The corresponding results were obtained from the same blood each time. Table G-b shows the recovery of amino-acids added to the blood. TABLE s . el. Amino-AHd Nitrogen per 100 Cc. of Blood. Material. Trichloroacetic acid precipitation. Heat coagulatioD followed by trichloroacetic acid precipi- tation. Sheep blood (oxa- lated) mg. 7 82 779 7 50 7 60 8 33 7.43 Sheep blood (oxa- lated) . 7.60 Sheep blood (oxa- lated) Calf • blood (oxa- lated) 6.34 7.24 Pig blood (oxalated) . 8.37 fe.' Recovery of Added Amino-Adds. Sheep blood (oxa- lated) , Calf • blood (oxa- lated) pa 7.50 7.60 16.33 16.85 IB a 8.83 9.25 o 9 32 9 40 6 34 7.24 ■§1 15 84 16.44 9 50 9 24 ■a o 9 "32 9 40 The use of heat coagulation prior to amino-acid determination might seem objectionable on account of possible hydrolysis of protein during [24] the process. Greenwald has shown in his publication that no splitting off of nitrogen takes place with his procedure, but here the first precipi- tation takes place in the cold. A glance at Table G-a shows that the heat coagulation-trichloroacetic acid procedure gives even slightly lower results than the direct Greenwald procedure. Recently Folin and Denis have stated that : "All reagents involving heating are useless, because by heat (half an hour in a water bath), the nitrogen of normal blood fil- trates may be increased to twice the real value." No figures are offered in substantiation of this statement. According to the statement of Folin and Denis, we should expect that the heat coagulation procedure would show much higher results in amino-acid nitrogen because the supposed increase in nitrogen would to a large extent be derived from protein hydrolysis. The above-mentioned comparison seemed convincing but an ad- ditional experiment was made to furnish further proof. Blood was pre- cipitated with methyl alcohol according to Folin and Denis. Another sample of the same blood was precipitated according to Greenwald, and a third part of the blood was coagulated by heat and after evaporation treated with trichloroacetic acid and kaolin exactly as described above. On the filtrates, obtained by these three procedures, Kjeldahl determina- tions were made, using such volumes of filtrates as toi represent about 40 cc. of blood, and the determination was repeated with three different samples of blood. Table H shows the results obtained. TABLE H Non-Protein Nitrogen per 100 Cc. of Blond (Pig). Meth.vl alcohof precipitation. Trichloroacetic acid precipitation. Huat coagulation followed by tricliloroacetic acid precipi- tation. 22.93 29.92 28.15 mg. 23 88 32.03 30.81 mg. 23.60 30.80 29 22 The a:bove presented facts indicate clearly that the use of alcohol as a blood precipitant in the determination of amino-acid nitrogen in blood is undesirable and the use of trichloroacetic acid as a precipitant preceded by heat coagulation of the blood proteins has been adopted for a study of the quantitative occurrence of amino-acid nitrogen in the blood of various species. [25] PART IV The data thus far available upon the occurrence of amino-acids in blood are comparatively few. In the literature of only a few years ago one will frequently find the statement that amino-acids occur only in "traces" in the blood. Bergmann, studying the non-protein nitrogen of the blood finds substances which react with /3-naphtalinsulfochloride. Howell made the same observation on blood dialysates, but neither of these two authors were able to actually identify any amino-acids. Bingel found glycocoll in blood. He proceeded as follows : 5 liters of ox blood were diluted with 5 liters of water. 10 liters of 2 per cent. HQ and 10 liters of 5 per cent. Hjg'Clj solution were added and after standing for some time the whole was filtered. The excess of mercury was removed by HjS. An aliquot part of the filtrate was evaporated to a small volume and then treated with /8-naphtalinsulfochloride. Bingel obtained 0.35 g. yS-naphtalinsulfoglycocoU in 10 liters of blood. Neuberg and Strauss using the naphtylisocyanate method found small amounts of amino-acids in pathological bloods and sera. Abderhalden separated amino-acids from blood. He used whole blood first, but in the latter part of his ex- periment used plasma and serum only. Abderhalden removed the pro- teins by boiling the blood or the plasma with 15 times the volume of water. After 15 minutes boiling one per cent, acetic acid was added drop by drop to the boiling mixture until coagulation was complete. The coagulum was extracted by boiling several times with water and finally rubbed in a mortar with hot water in order to remove as much of the amino-acids as possible. For each liter of serum there were finally 50 liters of filtrate. The filtrate was evaporated under reduced pressure. By use of the estermethod it was possible to prove the presence of amino- acids, but the attempts to identify individual amino-acids were not suc- cessful. By using different precipitation methods, Abderhalden finally succeeded in identifying several amino-acids. No quantitative estima- tion was made. Instead of removing the blood proteins by coagulation or precipita- tion, the non-protein bodies have been separated from the blood by dialysis. Abderhalden has used this method in part of the work just mentioned. But he and other investigators dialyzed the blood after it has been removed from the body. Abel and co-workers introduced a dializing arrangement, which makes it possible to remove diffusible sub- stances from the blood of a living animal without actual withdrawal of the blood from the body. The method is known as vividiflFusion. The animal is first treated with hirudin or leech extract. The blood from an artery is sent through a series of collodion tubes surrounded by physio- [26] logical salt solution. Substances which are diifusible through collodion tubes will pass from the blood into the outer liquid and accumulate there. After passing through the dialyzer the blood returns through' a vein. Abel and co-workers analyzed the dialysates so obtained and found amino-acids to be present. They positively identified valine, alanine and histidine. The total non-protein nitrogen in a 112 hours dialysate was found to be 20 g. and of this 1.5 g. were amino-acid nitrogen as deter- mined by Van Slyke's method. The above-mentioned methods prove the presence of amino-acids in blood but do not give any quantitative results. The data thus far available upon the quantitative occurrence of amino- acid nitrogen in the blood oi different species are far from complete. Furthermore, many of the figures heretofore reported have been obtained after preliminary removal of the blood proteins with alcohol, a procedure which has been shown to be undesirable from the standpoint of accuracy. It was, therefore, believed that it would be of interest tO' make a fairly complete survey of the question of the quantity of amino-acid nitrogen in various bloods, making use of one method throughout, which had been found to be the most accurate available. Blood from various animals as well as normal and pathological human blood was obtained and the amino-acid nitrogen determined. In most cases the blood proteins were removed by heat coagulation followed by precipitation with trichloroacetic acid and kaolin as described above. The heat coagvtlation gives clear and easily filterable precipitates with freshly drawn blood ; i. e., blood not over 24 hours old. If the blood has been drawn for more than that time, it often happens that the filtrate from this precipitation is not entirely clear and the solution filters very slowly. The same thing has been found with laked or frozen blood. In such cases the blood must be precipitated with nine volumes of 2.5 per cent, trichloroacetic acid and treated as described above. The blood of larger animals, such as the ox, calf, sheep and pig, was obtained from slaughter houses and immediately (i to 2 hours after slaughtering) used for analysis. The blood of cats and dogs was obtained from the carotid artery. The blood was drawn about 12 hours after the last feeding. Normal human blood was drawn from a vein in the forearm (usually the median basilic). The normal and most of the pathological bloods were drawn 3 to 4 hours after breakfast. The blood of birds was obtained by cutting the throat and letting the blood run into a bottle containing potassium oxalate. 40 tO' 50 cc. of animal and normal human blood were used for the analysis. The amounts of pathological bkxjds obtained from the wards varied greatly, but with few exceptions there were always more than 15 cc. used for analysis. These bloods [2T] were delivered in small 'bottles and in order to prevent waste, the blood was weighed out instead of measured, pouring it into the previously tared flask .containing the soy bean mixture. The weight in gm. was divided by 1.06, the mean specific gravity of human blood. For the sake of comparison the results are tabulated at the end of the paper. Table i shows the amino-acid nitrogen content of the blood of various mammals, including the ox, sheep, pig, cat and dog. The figures for each species are constant. Van Slyke and Meyer using the alcohol precipitation method, find from' 3 to 5 mg. of amino- acid nitrogen per 100 cc. of blood of dogs which had been fasting from 20 to 24 hours. Costantino reports findings of 10 mg. of amino-acid nitrogen in 100 gm. of blood obtained from dogs during full digestion. His analysis of pig blood shows 10 mg. of amino-acid nitrogen per 100 gm. of blood. Costantino uses the Sorensen formol titration method after drying the blood at 70° C. and extracting with 10 per cent, alcohol in the presence of barium salts. Gyorgy and Zunz using the alcohol precipita- tion method (removing urea by treatment with soy bean and subsequent aeration) find 4.8 mg. of amino-acid nitrogen in 100 cc. of blood obtained from dogs fasted for 24 hours. In the present work, dog 'blood was found to contain an average of 7.47 mg. of amino-acid nitrogen per 100 cc. of blood, and pig blood 8.43 mg. per 100 cc. of blood. These higher figures in the dog are probably due to the more complete extraction of the amino-acid nitrogen obtained in the present work. We cannot ex- plain Costantino's high results for pig blood. The amino-acid nitrogen of bird blood (Table II) is, roughly speak- ing, three times as high as that of mammals. The individual and gross variations are in proportion to those of mammalian blood. Costantino, using the formol method finds 20 mg. of amino-acid nitrogen per 100 gm. of turkey blood, agreeing closely with the findings in the present paper. The distribution of the amino-acid nitrogen between plasma and corpuscles in certain species was studied by Costantino and by Gyorgy and Zunz. Costantino finds that serum and corpuscles during fastings are constant in their amino-acid nitrogen. His findings show 4.4 mg. of amino-acid nitrogen per 100 gm. of dog serum and 100 gm. of the cor- puscles of ox blood containing 3.2 mg. of amino-acid nitrogen. Turkey blood analyzed by Costantino showed 3 mg. of amino-acid nitrogen in 100 gm. of serum and 34 mg. in 100 gm. of corpuscles. Gyorgy and Zunz find 1.7 mg. of amino-acid nitrogen in the plasma of 100 cc. of dog blood and 3.1 mg. in the corpuscles of 100 cc. of the same blood, the corpuscle content being calculated by difference. In the present work the whole blood was first analyzed. Samples of [28] equal volumes were centrifuged for one-half hour at high speed. The plasma which had separated out was withdrawn by means of a pipette, physiological salt solution added in its place, and the sample was mixed and centrifuged again. This procedure was repeated four times. After the clear supernatant fluid had been withdrawn down to the light colored layer of leukocytes, the corpuscles were laked with water, transferred to a flask, and the liquid was diluted approximately to the same volume as the plasma plus the washings. As the laked blood gives trouble with the heat coagulation procedure the direct trichloroacetic acid precipita- tion was used after treatment with soy bean. Table III shows the re- sults obtained from mammalian and bird blood. In mammalian blood the corpuscles show slightly higher figures than the plasma. The dif- ference in bird blood is pronounced, the corpuscles containing about two-thirds of the total amount of amino-acid nitrogen. These findings agree in general with those of the authors mentioned above. Costantino studied the permeability of corpuscles for amino-acids. He first determined the amino-acid contents of serum and corpuscles. He then adds to another part of serum a measured amount of amino- acid solution, then the corresponding amount of corpuscles and mixes well. After standing for one hour, the mixture is centrifuged and the amino-acid nitrogen of serum and corpuscles determined. It was found that the corpuscles had taken up a large percentage of the amino-acids added to the serum, the maximum amount being taken up amounting to about 45 mg. N per looo cc. of corpuscles. In view of the fact that the corpuscles contain a large proportion of the amino-acid nitrogen of the blood and in view of the probability that any increase in amino-acid nitrogen will be more noticeable in the corpuscles than in the serum, the corpuscles should be included in experimental or clinical work on the nitrogen content of the blood. The normal htunan bloods are reported in Table IV. It will be noted that the figures are remarkably constant for different individuals. The average figure is 7.13 mg. with a maximum of 7.9 mg. and a minimum of 6.13 mg. per 100 cc. of blood, approximately those of other mammals. Sex seems to be without influence. The placental blood tends to be dis- tinctly higher. Numerous reports have been published on the variations in the non-protein nitrogenous constituents of the blood in disease, but only isolated attempts have been made to find out whether or not the amino-acids in the blood were subject to variations under pathological conditions and these few attempts have met with rather indifferent success. Kaplan reported a decreased amino-acid nitrogen in luetic sera. [29] He uses the serum without any previous removal of proteins. The amount of serum used was from 2.5 to 4 cc. and the amino-acid nitro- gen obtained from these amounts were measured with the (50 cc.) burette of the Van Slyke apparatus. Ellis, Cullen and Van Slyke using alcohol for protein precipitation and the micro apparatus of Van Slyke failed to confirm Kaplan's results. The wide variations reported by Kaplan are apparently due to erroneous technique. Pettibone and Schlutz report findings on children's blood. These authors use the method as described by Van Slyke and Meyer. The results on normal children show a variation from 2.05 mg. to 4.59 mg. of amino-acid nitrogen per 100 cc. of blood. There are no consistent or characteristic variations reported in the pathological blood examined by these authors. The pathological bloods (Table V) were taken at random from ward cases in Bellevue Hospital and thus serve to show the possible variations found in a wide variety of conditions. In these cases variations occur from 4.5 mg. to 30 mg. per 100 cc. of blood. The most pronounced variations from the normal were found in nephritis. Of three typhoid cases, two are decidedly below normal. Jaundice, cardiac cases, car- buncles, rheumatic fever, hyperthyroidism, and cirrhosis of the liver show an increase over the normal. The remaining cases investigated give the same results as normal bloods. PART V In view of the fact that almost no observations are available upon both the non-protein nitrogen and amino-acid nitrogen in nephritis and uremia, aside from four cases reported without detail by N. B. Foster, it was thought of interest to find out whether the amino-acid nitrogen tends to parallel the total non-protein nitrogen in nephritic blood. In cases in which complete history and diagnosis were available the total non-protein nitrogen of the blood was determined as described above. The findings are reported in Table VI. From the results it seems apparent that the amino-acid nitrogen does not necessarily parallel the total non-protein nitrogen. While an increase in total non-protein nitrogen is followed in most cases by a higher amino-acid nitrogen, these variations are by no means proportional. Thus, it may be noted in Case No. 50 that an increase of about fifty per cent, in the total non-protein nitrogen is accompanied by a decrease in the amino-acid nitrogen. Again, in Cases Nos. 119, 122 and 124 it is evident that the total non-protein nitrogen does not parallel the amino- acid nitrogen. It is interesting to note in Cases Nos. 78, no and 124 that in uremia there seems to be a lower amino-acid nitrogen than in the earlier stages of the nephritis, though the total non-protein nitrogen may be increased. There is apparently no marked difference in the cases of interstitial and parenchymatous nephritis reported in this paper. A more complete survey of this question will be undertaken in the near future. rsoi J. C. Bock 195 TABLE 1. Amino-Acid Nitrogen per 100 Cc. of Blood. Sample No. Source. N* Sample No. Source. N* mg. mg. 4 Ox (oxalated). 6.17 42 Pig (defibrinated). 8.37 125 tt * iC 6.22 43 It tt 8.49 10 tl It 6.43 Average . 8.43 5 (I it 6.66 125a " (defibrinated). 6.67 14d Cat tt 8.12 9 (oxalated). 6.89 14b it it 8.13 3 t( iC 7.04 14g it tt 8.64 Average . 6.68 14a Average . tt tt 9.83 8.68 100 Calf (defibrinated). 6.29 8 " (oxalated). 6.66 97 Dog it 6.68 18 " (defibrinated). 6.81 52 t( ii 6.87 18a tl it 7.60 113 " " 6.87 Average . 6.84 51 ii it 7.15 114 U it 7.47 6 Sheep (oxalated). 6.84 116 ii ti 7.88 13 " defibrinated). 7.50 98 it ti 8.37 12 It tl 7.79 99 tt '' 8.48 11 It it 7.82 Average . 7.47 7 " (oxalated). 8.19 Average . 7.63 * The data are arranged in sequence for each group. Amino-Acid Nitrogen per wu \jc. oj mooa [uxaiaieaj. Sample No. Source. N* mi- 26 Chicken. 17.81 36 tt 19.84 23 It 20.64 24 " 21.32 25 it 21.58 110 tt 21.93 107 tt 23.78 Averasre . 20.99 35 Duck. 20.22 32 tt 20.88 34 it 20.95 33 it 21.55 27 it 22.98 Average. • 21.32 101 Turkey. 20.00 102 Goose. 16.97 104 It 17.16 105 tt 18.49 103 tt 19.20 106 tt 21.18 Average 18.60 ' The data are arranged in sequence for each group. TABLE III. Sample No. Source. Amino-acid nitrogen per 100 cc. of blood. Whole blood. Plasma. Corpuscles. mff. mg. mg. 100 Calf. 5.69 (3. 06 \3.13 3.59 3.48 100a « 6.30 r3.33 \3.33 3.84 3.28 106 Goose. 21.18 ("6.60 \6.07 14.78 15.95 107 Chicken. 23.78 f6.63 \7.97 14.20 16.00 110 tt 21.93 5.31 19-. 12 125 Ox. (■6.22 \6.77 3.05 2.99 4.70 4.87 196 J. C. Bock 197 TABLE IV. Amino-Acid Nitrogen per 100 Cc. of Human Blood. Sample No. Sex. N* Normal venous blood. Average. 47 15 57 60 58 77 83 86 21 30 55 48 16 22 19 65 56 46 cT d" c? cT cf d" d" cf 9 d" d' d" d" d' 9 9 6.13 6.40 6.58 6.75 6.78 7.00 7.01 7.11 7.18 7.27 7.29 7.31 7.38 7.38 7.58 7.66 7.67 7.90 7.13 Placental blood. 20 6.78 38 7.51 28 8.90 31 9.76 138 11.80 139 12.15 Average. 9.48 * The data are arranged in sequence for each group. 198 • Amino- Acid Nitrogen TABLE V. Amino-Add Nitrogen per 100 Cc. of Pathological Human Blood. £ p N. Diagnosis. ¥ N. Diagnosis. mg. mg. 39 7.78 Syphilis. 85 8.07 Nephritis. 40 7.98 tl 87 6.97 Cardionephritis. 44 6.54 Ulcer on penis. 88 8.27 Rheumatic fever. 45 8.34 No symptoms. 89 7.01 Cardiac. 49 8.49 Nephritis. 90 9.38 Nephritis. 50 8.38 ct 91 5.72 Carcinoma of liver. 53 7.11 Cardionephritis. 92 7.50 Nephritis. 54 17.50 Nephritis, alcoholism. 93 6.52 tc 59 6.77 No record. 94 8.02 tt 61 7.27 Nephritis. 95 11.28 Parenchymic nephritis. 62 8.78 No record. 108 8.13 tt tt 63 8.31 Jaundice. 109 8.04 Nephritis. 64 7.25 'Nephritis. 110 8.49 tt 66 8.62 Cardiac. 111 7.55 Parenchymic nephritis. 67 7.80 Diabetes. 112 8.69 Cardionephritis. 68 7.86 Cardionephritis. 115 8.87 tt 69 6.47 Chronic nephritis. 117 6.66 Nephritis. 70 6.93 Typhoid. 118 8.24 Parenchjrmic nephritis. 71 8.70 Gout, rheumatism. 120 10.98 Nephritis. nephritis. 121 9.82 tt 72 5.88 Typhoid. 122 11.17 Colic, uremia. 73 5.45 it 123 8.41 Cardionephritis. 74 4.45 Nephritis. 124 29.98 Uremia. 75 15.10 tc 126 9.78 tt 76 7.52 Arteriosclerosis. 127 7.25 Nephritis. 78 9.05 Nephritis. 128 7.45 Delirium tremens. 79 5.90 Uremia. 129 9.91 Nephritis. 80 6.05 Nephritis. 130 10.45 Cirrhosis of liver and 81 6.65 It cardiovalvular case. 82 8.20 Carbuncles. 131 .9.58 Hjrperthyroidism. 17 6.89 Nephritis. 132 6.46 Parturition. 84 6.24 te 133 6.63 Diabetes. INTERSTITIAL NEPHRITIS Case No. REMARKS Mg. per 100 cc. of blood Total non-protein Amino- acid N SO Male, 36 years, chronic interstitial nephritis, cardiovalvular disease, anemia, B. P. 218/195, died Blood sample taken 28 days after the first one (9 days before death) 68.50 102.87 8.38 6.89 75 Male, 52 years, chronic interstitial nephritis, oedima, glottidis, B. P. 210/90, died 153.16 15.10 78 Male, 45 years, chronic interstitial nephritis, myocardial degeneration, cirrhosis of liver, uremia, B. P. 196/78, died (Sample taken 24 days before death) 147.14 9.05 109 Male, 55 years, chronic interstitial nephritis, uremia, B. P. 210/110, improved 60.51 8.04 110 Male, 47 years, chronic interstitial nephritis, uremia, B. P. 2S0/160, died (Sample taken 2 months before death) 66.49 8.49 119 Male, 29 years, chronic interstitial nephritis, cardiovalvular disease, pericarditis, B. P. 200/110, died (Sample taken 28 days be- fore death) 166.02 18.95 122 Male, 37 years, chronic interstitial nephritis, uremia, chronic lead poisoning, B. P. 180/144, died (Sample taken 4 days be- fore death) 238.00 11.17 124 Male, 49 years, chronic interstitial nephritis, cirrhosis of liver, chronic cardiovalvular disease, B. P. 210/120, died Sample taken 3 days after the first one (day of deathl 206.50 201 84 29.98 Q 78 PARENCHYMATOUS NEPHRITIS 54 Male, 38 years, chronic parenchymatous neph- ritis, alcohol poisoning, coma, furuncu- losis, improved 166.79 17.50 94 Male, 40 years, chronic parenchymatous neph- ritis, chronic cardiovalvular disease, B. P. 220/140, improved 70.00 8.03 137 Male, 29 years, chronic parenchymatous neph- ritis, B. P. 140/95, improved 28.08 12.06 95 Male, 27 years, parenchymatous nephritis, B. P lRft/11'i imt^rnvpd First samnle 86.04 63.05 38.12 32.26 11.28 8.13 7.55 8.24 Second sample (6 days later) BIBLIOGRAPHY The references are arranged alphabetically according to authors. After the names of the author follows the year, then the number of the volume and Anally the page. Some of the latest European publications may not be included in this bioliography on account of the present difUculties to obtain mail from abroad. Abel, J. J., Rowntree, L. G., and Turner, B. B. : J. Pharm. and Exp. Ther., 1914, 5, 275 and 611, On the removal of diffusable substances from the circulating blood of living animals by dialysis. Abderhalden, E. : Z. f. physiol. Chem., 1903, 37, 484, Hydrolyse des krystallisierten Oxyhsemoglobins aus Pferdeblut. — Z. f. physiol. Chem., 1903, 37, 495, Hydrolyse des krystallisierten Serumal- bumins aus Pferdeblut. — Z. f. physiol. Chem., 1903, 37, 499, Hydrolyse des Edestins. — Z. f. physiol. Chem., 1903, 38, 557, Familiare Cystindiathese. — Z. f. physiol. 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