5 Hddééé 418 u I 9015 f|\,' w\ \Jm/~ H:- \ 3 nu ¢ .. I!!! ."l ‘\ “14 1" ll. .1. 5‘, l“ 1"] . 1Q {I‘D-Illi- ‘ .luod.1zlw¢- . L!- l. I .. Ivvllf .__ o * F f. . M 3 ¢ 1 I . I . . . \fiiihlJfiz. 1!. ‘ i. P. w.» _._:::::::::::::=::_ :=.:_::__-:=.:_:=:=_2::EE:-=_E:5:::1.=3::E-zz._===-=:_::-:_::=uum E_=-:::.:.:::.._..:::M_M *0 v ‘-Q-IIIIVQ'I\III-:II 'I.".I."Ill-l.~ "\ II'. I ,I--‘\I'-II.-lI-|Il‘||'I|"I-l-“-1 m MI'I...III\IIIIIO-QIP m . mm:- "m . H .l. . n H II . n I o 11. m .‘m M W m , nu . n . v .I. 7| . w m .m . S: H F "m u .l. m: . mm U Q in F m . J u \ )4 "m 0 u . w . “m n . ~ .n . "m T a“ F .m G v . n “m E H n m T “m. . u . n ..._._.=_._3Eiiwmaaiwflmm=§===5§§ 3.5m“; ._ _.__ =.=_,_==.=._=.=.=".EEEEEE-Ewq STUDIES [N CANCER AND ALLIED SdBfECTS FROM THE DEPARTMENTS OF ZOOLOGY, SURGERY CLINICAL PATHOLOGY, AND BIOLOGICAL CHEMISTRY l l I _ VOL. II. STUDIES IN CANCER AND ALLIED SUB]ECTS Conducted under the George Crocker Special Research Fund at Columbia University VOL. I. THE STUDY OF EXPERIMENTAL C A N C E R. A Review. By WILLIAM H. WOGLOM, M.D. Illustrated with many plates. In two bindings, Quarto, boards, or 8710, cloth. pp. vii + 288. Price, $5.00 net. P A T H O L O G Y. Illustrated with many plates and charts. Quarto, boards, pp. oi + 267. Price, $5.00 riet. VOL. III. FROM THE DEPARTMENTS OF ZOOLOGY, SURGERY, CLINICAL PATHOLOGY, AND BIO- LOGICAL CHEMISTRY. Illustrated With many plates. Quarto, boards, pp. ix + 308. Price, $5.00 net. VOL. IV. CONTRIBUTIONS TO THE ANATOMY AND DEVELOPMENT OF THE SALIVARY GLANDS IN THE MAMMALIA. Illustrated with many plates. Quarto, boards, pp. 2) + 364. Price, $5.00 net. COLUMBIA UNIVERSITY PRESS Sales Agents LONDON: NEW YORK: TORONTO: HENRY FROWDE LEMCKE & BUECHNER HENRY FROWDE AMEN CORNER, E.C. 30-32 Wss'r 27TH ST. 25 RICHMOND ST; W. STUDIES IN CANCER AND ALLIED SUBJECTS FROM THE DEPARTMENTS OF ZOOLOGY, SURGERY, CLINICAL PATHOLOGY, AND BIO- LOGICAL CHEMISTRY CONDUCTED UNDER THE GEORGE CROCKER SPECIAL RESEARCH FUND AT COLUMBIA UNIVERSITY VOLUME III New fink COLUMBIA UNIVERSITY PRESS All rights reserved 1913 COPYRIGHT, 1913, BY COLUMBIA UNIVERSITY PRESS. Set up and electrotyped. Published, July, 1913. Nurhuoutl 15mm J. S. Gushing Co. - Berwick & Smith 00. Norwood, Mass., U.S.A. PREFACE WHEN Mr. George Crocker made his first donation to Columbia University for the investigation of cancer, it was decided by those who had the matter in hand that the money could best be expended by making grants to special workers in the laboratories of the Col— lege of Physicians and Surgeons, and of the Department of Zoology of Columbia, in order that no time might be lost and the money put to immediate use. The present volume represents work done in the Departments of Zoology, Surgery, Clinical Pathology, and Biological Chemistry. Volume II, containing studies from the Department of Pathology, and Volume IV, containing contributions from the Depart- ment Of Anatomy, have already been published. Volume I, now in press, is a very complete review of the subject of cancer and the ad- ' vantages which have resulted from the application of the experimen— tal method to the study of this most important problem. 257800 ARTICLE I. II. III. IV. YL WL VII-I. IX. XI. XII. XIII. XIV. CONTENTS PART I DEPARTMENT OF ZOOLOGY REPORT ON THE BIOLOGICAL WORR DONE UNDER THE GEORGE CROCKER SPECIAL RESEARCH FUND, 1909—I9II. By Gary N. Calkins . . . . . . . . . . NOTES ON TUMOR TRANSPLANTATION IN GENERAL. By Gary N. Calkins, Frederick D. Bullock, George L. Rohdenburg. and Peter J. Johnson . . . . . REGENERATION AND CELL DIVISION IN URONYCHIA. Calkins EFFECTS OF MUTILATIONS BY CUTTING, ON PARAMECIUM. N. Calkins NOTES ON THE GROWTH OF TISSUES UNDER EXPERIMENTAL CON- DITIONS. By Frederick D. Bullock TUMOR TISSUE IN COLLODION SAcs. By Frederick D. Bullock. George L. Rohdenburg, and Peter J. Johnson . DATA ON TUMOR GROWTH IN RELATION To AGE AND SEX OF RODENTS By Peter J. Johnson . . . SPONTANEOUS TUMORS. By Frederick D. Bullock . . . RETROGRADING TUMORS. By Frederick D. Bullock and George L. Rohdenburg By Gary N. B y Gary INOCULATION WITH Two TYPES OF TUMORS. By Frederick D. Bul- loCk, George L. Rohdenburg, and Peter J. Johnson THE EFFECTS OF CHRONIC IRRITATION ON TISSUES. L. Rohdenburg THE EFFECTS OF CERTAIN CHEMICALS ON THE TYPE OF TISSUE RESULTING FROM CI-IRONIC IRRITATION. By George L. Rohden- burg and Frederick D. Bullock OTHER EFFORTS To BRING ABOUT CHRONIC IRRITATION—EARTH- WORK EXPERIMENTS. By Frederick D. Bullock . By George THE RELATION OF CERTAIN INTERNAL SECRETIONS TO MALIGNANT TUMORS. By George L. Rohdenburg, Frederick D. Bullock, and Peter J. Johnson . . . . . . . . . PAGE IO 30 59 62 M m 70 75 77 % N vii viii CONTENTS PART II DEPARTMENT OF SURGERY ARTICLE PAGE I. THE DEVELOPMENT OF THE BLASTODERM OF THE CHICK IN VITRO. By John E. McWhorter and Allen 0. Whipple . . . . II I 11. A RARE TUMOR OCCURRING IN A ST. BERNARD DOG. By John E. McWhorter and Allen 0. Whipple . . . . . . 117 PART III DEPARTMENT OF CLINICAL PATHOLOGY I. RESISTANCE PRODUCED IN MICE AGAINST TRANSPLANTED CANCER BY AUTO-INOCULATION OF THE SPLEEN. By William H. Woglom . 127 II. MICE IMNUNIZED SUBCUTANEOUSLY ARE RESISTANT To THE IMPLAN- TATION OF CANCER IN INTERNAL ORGANS. By William H. Woglom I32 Ill. CONTRIBUTIONS TO THE THEORY OF THE INDIVIDUALITY OF CAR- CINOMA. By William H. Woglom . . . . . . I37 PART IV DEPARTMENT OF BIOLOGICAL CHEMISTRY I. INTRODUCTION. GENERAL PROGRAMME OF THE BIOCHEMICAL INVESTI- GATIONS UNDER THE AUSPICES OF THE GEORGE CROCKER SPECIAL RESEARCH FUND, 1909-10 AND IgIo-II. By William J. Gies . I 53 II. METHODS FOR THE DIAGNOSIS OF CANCER . . . . . I60 I. The New Test for Cancer of the Stomach, with Suggested Im- provements. ByJ. W. Weinstein . . . . 161 2. The Tryptophan Test for Cancer of the Stomach, with Special Reference to the Peptidolytic Enzyme in the Saliva. By J. W. Weinstein . . . I78 3. The Glycyltryptophan and Tryptophan Tests for Cancer of the Stomach. By Charles H. Sanford and Jacob Rosenbloom . 191 4. The Importance of the Colloidal Nitrogen of the Urine in the Diagnosis of Cancer. By Max Einhorn, Max Kahn, and Jacob Rosenbloom . . . . . . . . . . 200 5. The Colloidal Nitrogen in the Urine from a Dog with a Tumor of the Breast. By Max Kahn and Jacob Rosenbloom . . 206 CONTENTS ix ARTICLE 111. ON THE COMPOSITION OF PROTOPLASM AND THE NATURE OF THE STRUCTURAL AND DYNAMIC RELATIONSHIPS OF CELL CONSTITU- ENTS AND PRODUCTS A. Studies on Isoagglutination: I. Transfusion and the Question of Intravascular Agglutina- tion.‘ By Reuben Ottenberg . . . 2. The Occurrence of Grouped Isoagglutination in the Lower Animals. By Reuben Ottenberg and S. S. Friedman . 3. T onicity in Isohemagglutination. By Morris H. Kahn and Reuben Ottenberg . ' . . . . . . 4. Isoagglutination in Dog Blood. By Reuben Ottenberg, S. S. Friedman, and I). J. Kaliski (with a discussion by Cyrus W. Field, Isaac Levin, and Reuben Ottenberg) 5. Experimental Agglutinative and Hemolytic Trausfusions. By Reuben Ottenberg, D. J. Kaliski, and S. S. F ried- man . . . . . . . . . . B. Studies of Lipins : I. On the Forms in which Lipins are combined in Cells. By Jacob Rosenbloom . . . . . . . 2. Preliminary Reports on Studies of the Difiusion of Lipins and Lipin-soluble Substances through Rubber Mem- branes. By William J. Gies I. Introduction . . . . . . II. On the Diffusibility of Biological Substances through Rubber . . . . . . A Demonstration of Osmotic Pressure exerted by Fat . . . . . . . . A Demonstration of the Diffusion of Pigments from Fat through Rubber into Fat . . . . V. Comparative Dialysis Experiments, with Demon- strations . . . . . . . Experiments on the Difi'usibility of Alkaloids through Rubber . . . . . . . 3. A Study of the Diifusibility of Lipins from Ether through Rubber Membranes into Ether. By Jacob Rosenbloom C. Influence of Cancer Extracts : I. A Study of the Influence of Cancer Extracts on the Growth of Lupin Seedlings. By Jacob Rosenbloom III. IV. VI. .INDEx . . . . . . . . . . . . PAGE 209 211 225 230 PART I DEPARTMENT OF ZOOLOGY PART I DEPARTMENT OF ZOOLOGY I REPORT ON THE BIOLOGICAL WORK DONE UNDER THE GEORGE CROCKER SPECIAL RESEARCH FUND, 1909—1911 BY GARY N. CALKINS BIOLOGICAL work under the Crocker Fund was begun on an inde- pendent basis in April, 1909. Frederick D. Bullock, M.D., was ap- pointed chief assistant and has had charge of all of the experimental work on laboratory animals. George L. Rohdenburg, M.D., joined the staff in October, 1909, and Peter J. Johnson, M.D., in Novem- ber, 1909. An animal room was fitted up in the attic of Schermerhorn Hall and the general laboratories of the Department of Zoology were used by the members of the staff. The quarters are so cramped, however, especially in the animal room, that experiments on a large scale have been impossible, and most of the work, in consequence, has been in the nature of preliminary investigations in search of openings for further and more detailed research. Exclusive of animals that have died of diseases other than cancer, or from shock, hemorrhage, etc., following operation, we have experi- mented with 920 mice, 883 rats, 2 guinea pigs, and 7 rabbits. We have used 18 diiIerent tumors. of which I7 were carcinoma and I was sarcoma. Ten were tumors from outside sources and 8 were spon- taneous or primary tumors appearing in our own animals. At least one carcinoma and one sarcoma have been running continuously in series. In the following pages special reports are submitted, bearing I 2 DEPARTMENT OF ZOOLOGY on the routine work of maintaining the series. Special deductions from the laboratory records are given in the reports by Dr. Johnson on tumor growth in relation to age and sex; by Dr. Bullock on the spontaneous tumors that have appeared in the laboratory (page 66) ; by Dr. Bullock and Dr. Rohdenburg on retrograding tumors and ' experiments in which dried and powdered material from malignant tumors was sprinkled on the retrograding tumor tissue to see if an enzyme or something of a chemical nature would stimulate the retro- grading tissue to renewed activity. The results, while partly suc- cessful, need to be confirmed by further experiments before conclu- sions can be drawn (page 70). These tumors in the regular series have been used as a reservoir of material for experimental work of one kind or another. The nature of such experimental work follows, of course, thei particular point of view of the director in charge. In the present work, the term “bio- logical” subtends so wide an angle in the field of cancer research. that an almost unlimited choice of subjects for investigation is possible. During these two years, however, we have thrown our main strength in the direction of the biological phenomena of growth, attacking the subject jointly and separately from general and special points of view. Some of the investigations have a more fitting place in the field of pure science than in that of the pathologist or clinician; others, under Dr. Rohdenburg, have brought us to the cancer bedside with the inevitable result of success and failure in the treatment of in- operable human cancer. All lines of work, however, have centered around the biological phenomena of growth, and nothing could be more clearly demonstrative of the need of team-work or the joint activity of the pathologist, chemist, surgeon, clinician, and biologist, than our results in these two years of work. The underlying biological principle which has activated our re— searches on growth and has bound them in a consistent whole, is that of the physiological balance with self-regulation, which is perfect in normal organisms, but thrown out of adjustment in diseased or cancerous organisms. We have endeavored to get light, through experimentation, on the nature of the factors involved in such regu- lation by attempts both to induce an upset in the physiological balance of normal animals and to reéstablish it in those affected by DEPARTMENT OF ZOOLOGY 3 cancer. In connection with the former, special investigations have been undertaken to study the powers of division and regeneration of single cells, and of tissue cells, both under normal and artificial con- ditions. The report by Professor Calkins (page 10) on the division and regeneration of Uroriyckia transfuga is an experimental study on cell regeneration in relation to the periods or division. It was found that the power of a single cell to regenerate is independent of the power to divide, and increases up to a certain maximum just prior to division, thus indicating the accumulation of something, possibly enzymatic in nature, in the cell until a stage is reached analogous to saturation, when regeneration begins in advance of division of the cell. The report by Dr. Bullock (page 59) on attempts to grow tissues on artificial media records negative results in the effort to study regeneration and division in tissue cells. A long series of ex- periments which he has made with different kinds of amoeba, some of 'which are new species, on artificial culture media are not_included in the present report, but will be worked up later after the experi- mental side is completed. Analogous experiments are included in the report under the heading of tumor tissue in collodion sacs (page 62). In these the members of the staff endeavored to find out if malignant tumor cells, separated from the living tissues of the host but nourished by the body fluids of the latter, would live and multiply. The experiments have not yet been done on a sufficiently large scale to warrant conclusions and must be repeated with variations, but the results point to the need of a living stroma for development of the cancer cells. In all cases the tissue, after removal from the collodion sacs, failed to grow on reinoculation. Attempts to upset the physiological balance of organisms have been systematically carried out in experiments of different kinds. Pro- fessor Calkins presents a report on the results obtained by making small mutilations in the single-celled organism Paramecium caudatum, through which the physiological balance of the organisms was com— pletely broken down and abnormal cells, monsters, etc, were formed (page 30). The remarkable response of the organism to these minute mutilations is suggestive and lends support to the theory of the relation of abnormal growths to injury in higher animals. 4 DEPARTMENT OF ZOOLOGY Analogous experiments on mammals to break down the physio- logical balance and make the organisms more susceptible to cancer are reported by Dr. Rohdenburg (page 87). In this work, the thyroid, thymus, and other internal glands were removed from ani- mals either before inoculation with cancer or after the cancer had developed. Removal of such glands did not have the effect antici- pated, for there was no apparent increase in the percentage of “ takes” due to the physiological disturbance, but, on the contrary, there was a marked increase in the percentage of spontaneous recoveries, thyroid— free animals giving 33 per cent of recoveries, thymus-free 26 per cent, testes-free 20 per cent, as against 8 per cent in the control animals. Removal of the glands after the cancer was established gave no definite results. The same experiments were repeated on animals that had been found to be immune-to cancer, and various glands were removed in the effort to break down such immunity. This was apparently successful, for 62.5 per cent of the animals developed cancer, although all but 14 per cent recovered spontaneously in from IO to 30 days. The tumors that failed to disappear were in “immune” animals in which both thymus and testes had been removed. The Obvious conclusion is that immunity had been destroyed, but here again the small numbers employed in the experiments prevent definite conclusions. Other efforts to break down the power of regulation are described in the special reports of Dr. Bullock (page 85), of Dr. Rohdenburg (page 75), and of Drs. Bullock and Rohdenburg (page 77). The first, on the effects of irritation by living organisms, and the second, on the effects of chronic mechanical irritation, record only negative results, no response on the part of the animals treated being shown. The third, on the effects of certain chemicals on normal tissues, records some interesting positive results of growths strikingly similar in some respects to malignant tumors. Experiments, finally, have been made to restore the power of regu- lation and the physiological balance of cancer-bearing animals, by treatment with thymus gland in powder form, and with extracts of this and other organs. In this work Dr. Rohdenburg had the co- operation of Dr. Gwyer Of New York, to whom we gratefully acknowl- edge our indebtedness for many favors. The experiments were first DEPARTMENT OF ZOOLO GY 5 carried out on mice and rats, and it was found that animals fed with thymus gland powder before and after inoculation with cancer Show a much smaller percentage of takes than do control animals. Extracts from various glands injected into animals previous to inoculation with cancer were found to inhibit the growth of cancer to a remark- ab1e,degree._ Conversely, tumor-bearing animals injected with thymus extrapt gave a much higher percentage of recoveries than did the contrOl animals or the animals in the regular series (see page 87). The success was so marked that treatment with thymus extract was extended by Dr. Rohdenburg to human cancer, 49 cases previously declared inoperable being treated. Of these, 16 showed no beneficial efiect of the treatment; 9 showed a slight improvement; 21 showed a marked improvement for a period only; and 3 showed complete loss of the tumor. The various lines of research here outlined have given sufficiently clear evidence to indicate valuable results on further work. The statement cannot be reiterated too often, however, that these reports embody only the results of preliminary experimentation from which no established conclusions can be drawn. II NOTES ON TUMOR TRANSPLANTATION IN GENERAL By GARY N. CALKINS, FREDERICK D. BULLOCK, GEORGE L. ROHDENBURG, AND PETER J. JOHNSON THE first regular series of mouse carcinoma was carried through 18 generations of transplantations and showed the usual history of such tumors. It was obtained from Dr. H. Zinsser and was one Of a series from an original mouse tumor previously given to Dr. Zinsser by Dr. L. Loeb of the University of Pennsylvania. The method employed in transplanting is that usually known as the trochar method. The tumor to be used for inoculation is sterilized in situ by submerging the tumor-bearing animal in bichloride of mercury I: IOOO_ for about one minute and then into 95 per cent alcohol for about the same time (one-half to one minute). The tumor is removed by sterile instruments and placed in a sterile Petri dish after weighing both tumor-bearing animal and tumor. The tumor is then cut into small pieces, all of the same size as nearly as the eye can measure them, care being taken to select only sound tissue so that each animal inoculated receives about the same amount of active tumor tissue. The skin of the animal to be inoculated is par- tially sterilized by rubbing with cotton soaked in 95 per cent alcohol. A sterile trochar 2 millimeters in diameter is thrust under the skin of the abdomen or side, the needle withdrawn, and a piece of tumor tissue to be inoculated is pushed under the skin with a sterile plunger, after which the wound is sealed with collodion. The “infectivity” of the tumor as measured by the number of “takes" with this method was about 60 per cent (see Table I). The “virulence” as measured by the rate of growth was about the same as that of the Brooklyn tumor in the Buffalo laboratory. The successive transplantations resulted in variations in the percentage of takes, which followed the same general course as the variations indi- 6 NOTES ON TUMOR TRANSPLANTATION IN GENERAL 7 cating rhythms in the growth energy as described by Bashford, Murray, and Boyen (1906), by Hertwig and Poll (1906), and by Calkins (1908). T wo such rhythms occurred during the 18 transplantations, one of approximately 160 days and the other of about 200 days. After the second period of depression, however, the vitality of the tumor cells was not regained, and after a few weak and non-virulent growths after transplantation, the tumor died out in January, 1911 (see curve). TABLE I LOEB’s TUMOR — MICE H YOUNG OLD TOTAL I ea Spontane- I T, Spontane- 1-, Spontane- “ 3% Takes ous Re- Takes ous Re- Takes ous Re- a ’75 coveries E '3" , coveries I E '3‘ coveries :1 g s 8. E 8 g Total % Total % r25 lTami % Total % 5 Total % Total % Males . . . . 34 17 50 o — 42l 19 45.2 5 26.3 76 36 47.4 5 13.6 Females . . . 38 26 68.4 4 15.4 50I 34 68 4 11.8 88 60 68.2 8 13.3 Total . . . 72 43 58.6 4 9.5 gzi 53 57.6 9 17 164 96 58.5 13 13.5 Contrary to the observed facts by Bashford (1905), F lexner and Jobling, Jensen, Borrel, Michaelis, Lewin, and others, whose observa- tions were based upon larger numbers than are here recorded, we find that the number of takes for this tumor varies but slightly in young and old animals (59.6 per cent for young, and 57.6 per cent for old). We attach no importance to this discrepancy and believe that greater numbers would probably bear out the observations of others that young animals are markedly more susceptible than old ones. One feature, however, is unmistakably shown even with the small numbers that we have on record, viz. that female mice are more sus- ceptible than male mice to this particular tumor as shown by the percentage of 68.2 per cent of takes in females, as against 47.4 per cent in males. Old males give a slightly smaller percentage than young males (45.2 and 50 per cent), while the percentage of spon- taneous recoveries is much greater in old than in young (26. 3 and 0 per cent). In females, on the other hand, our records give an equal percentage of takes in old and young (68.2 and 68.4 per cent), while the percentage of spontaneous recoveries is slightly less for the 8 NOTES ON TUMOR TRANSPLANTATION IN GENERAL old than for the young females (11.8 and 15.4 per cent). In all cases young and old mice were inoculated with equal doses of the same tumor, usually in sets of 3 or 4 each, depending upon the number of young animals at hand. The different sets, finally, were inocu— lated at different times with different transplants of the same tumor. In interesting contrast to these results are the results obtained with a rat sarcoma of F lexner-Jobling origin, which has been success- fully transplanted through 16 generations. Tumors were trans- planted in 115 rats, of which 57 were old and 58 young and 71 were males and 44 females. The general results of the transplants are shown in Table 2. Although the number of animals used is again too small to warrant safe generalizations, the results point out some suggestive lines for further observation. For example, while the percentage of takes for male and female, including old and young, was about the same (80.3 and 81.8), the percentage of takes of old females was much smaller. than for old males (50 per cent and 78 per cent), while young females appeared to be much more susceptible than young males (100 per cent and 83.3 per cent) and much more so than old females (100 per cent and 50 per cent). Again, young animals give a higher percentage of takes than old (91.4 per cent and 70 per cent). Spontaneous recoveries are more frequent in males than in females (21.1 per cent and 16.6 per cent). The difference in the two tables may indicate some biological con- nection between carcinoma and female organization which is not shared by tumors of the sarcoma type, but the small numbers in- volved permit only the suggestion of such a connection. TABLE 2 FLEXNER—JOBLING TUMOR -— RATS YOUNG 01.1) TOTALS Number Sponta- Number Total Inocu- Takes neous Re— Inocu~ Takes Recoveries Inocu- Takes Recoveries lated coveries lated lated Total % Total % Total % Total % Total % Tolal % Males 3o 25 83.3 6 24 41 32 78 6 18.7 71 57 80.3 12 21.1 Females 28 28 100 5 17.8 16 8 _52_ 1 12.5 44 36 81.8 6 16.6 Total 58 53 91.4 11 2o.7| 57 40 7o 7 17.5 115 93 80.8 18 19.3 H HM 5 I I l 17' '- 6 c Q Q ,8 1 .. Q, 7 ' u 0 19 1 -' ' a l I I 20' ~ Q, 9 t t '1 2/ ' ' ' [0 c - - : 9' 9 11 ‘ - — 23 " " lflcm 12 - - — FIG. 5. “Experiment 63/31D, showing that mice can be rendered resistant to inoc— ulation of cancer by a preliminary inoculation with their own spleens. All mice were inoc- ulated July 12, 1909, with 0.025 cubic centi- meter of Tumor 63 in the left axilla. Mice 1 to 12 are normal and untreated mice (con- trols). Mice 13 to 23 are splenectomized mice, inoculated with their own spleens 12 days before the inoculation of the tumor. When I left England, a second experi- ment was under way, the final outcome of which Dr. Bashford has been kind enough to send me. Although the immunity gained by the mice inoculated with homologous spleen is not so striking as it was in the first experiment, it is nevertheless present. time and grew very well, while others, like Tumor 239, had been under cultivation for only a short time previous to their use, and therefore might give a more del- icate indication of slight differ- ences of resistance. Special attention was paid to the following factors, of which the importance has been re- peatedly urged in papers appear- ing from this Institute :— (1) The time interval between the introduction of normal tissue and the testing of resistance. (2) The site of inoculation. (3) The varying relations be- tween the doses of normal tissue introduced and the doses of tumor employed in testing resist- ance. Because of the fact that the amount of autologous normal tissue which may be employed to induce resistance is of necessity -limited, it is impossible to in- crease the dose beyond a certain point. This difficulty may be met in two ways. A condition frequently occurs in mice which is characterized by enlargement of the spleen even to eight times its normal size. Advantage was taken of this circumstance to obtain large amounts of the animal’s own tissue, in order to compare the THE INDIVIDUALITY OF CARCINOMA I 39 action of large and small doses. Microscopic examination of these enlarged spleens showed only an increase in the splenic pulp, sepa- rating the follicles more widely than usual, and not dependent merely upon increased congestion. Although a thorough bacteriological in- vestigation of this lesion was not made, six specimens were examined, three by agar cultures and three by smears, but without the demon- stration of any microorganisms. If such an enlarged spleen is ground up without the addition of salt solution, it furnishes about 0.5 cubic centimeter of emulsion; a normal spleen, on the contrary, affords only about 0.075 cubic centimeter, while a kidney, or two testes, gives about 0.1 cubic centimeter. The amount of normal tissue introduced can be varied by combining different organs. A moderately large spleen and a kidney together give about 0.45 cubic centimeter of emulsion. The relations between the amount of normal tissue introduced and the dose of tumor used in testing resistance, when such resistance is induced by the introduc- tion of tissue from another individual of the same species, have been determined in a special series of experiments which will be published later in another contribution from this laboratory. It was found that with a suitable dose of tumor, 0.05 cubic centimeter of normal tissue sufliced to render another animal definitely resistant. The available amount of autologous tissue should, therefore, according to the above estimations, be sufliciently large to induce resistance, if it be. possible to bring about this condition by treatment with an animal’s own tissues.* It may at once he stated that the chief result of these observations has been the demonstration that introduction of an animal’s own normal tissue does not evolve resistance, even when the quantities employed are many times greater than the amount necessary to pro- duce resistance in another animal. Consideration of the results obtained (Fig. 6) shows that where mice were subjected to preliminary treatment with their own normal sized spleens, there was not the slightest trace of resistance, while * Careful observation of the site of injection showed that the animal’s own spleen, in contradistinction to that of other mice, was capable of growing and even of attaining considerable dimensions after it had apparently _been completely absorbed. The nodules formed in this way showed a normal splenic structure with pulp, lymph nodules, and smooth muscle fibers. Nevertheless, they were always ultimately absorbed. ' 33.5, MOM/“ed "n R “ma 52-70 inoculated 1'11 Riki/la #32 Control . l9. /8. /7 or /5 days prewous/y each 19 /8.,/7 or l5dqys .priermusfirea‘cb Normal mme WM [15 mm normal spleen with 1!: 01m enlaly'ed spleen. ' 10 17 24 3102),; 10 /7 24 31w; /0 17 24.31.01.911 / woo 33113, . 52 :29! Z 5! 343:3“ 53"."? , '0’ . c (i - ' 35 ’93 5" ‘ 4 I!” 3.110! 55"“ 5'!!! 3,1,2! 56!!!! c ' '1 , , '3 s7- ' I 11 7!,” " 5g§ifl 39 I,‘ 0 l! 8 . I, , .’ , J 40 f ' 0 o 1’. . ellIl "' w"" /0.',, 4/‘... 61000 " 42 I... l/ I ' . ‘ 52 I.‘ ' ( 43 '11 QQ 63 ' D I l ' ’2 ' " 44 1 a 0 a _ [3 I I V I 64 I" ' . . o a Q 45 I ' ' ' 65 15!!!! “' 65"" 47 [6 1 I I I . " _- 48 I I f 7 ’0 f ' 68 ' ' _- / '-.' ' I. I - - /fl . o 50 ' 69 _-— 19 HH 5, ____ ’0 "*— 20 I I I 1 , a 2/ CI. 22 9 'I‘o - 1001 - 2.3 l ’ ' ' .74 I I 1 v 2.5 .‘ -' Z6 '.' .' FIG. 6.— Experiment 6 3/ 45F. All mice injected in the left axilla. 27 ‘ ‘ ' ' with 0.01 cubic centimeter of tumor emulsion. The result shows com- 75 ( .1 , .. plete absence of immunity in all the animals injected with their own normal spleens and atrace of immunity in those in which enlarged“ 39' ' ' '- _ spleens were used. nr'-’ n'-" 32 ' ' 'T '- 140 THE, INDIVIDUALITY OF CARCINOMA I4I those animals which were treated with their enlarged spleens appear to have developed a slight degree of immunity. One might be tempted to conclude from this that sufficiently large amounts of an animal’s own tissue were capable of inducing resistance. But that this conclusion would be unjustified may be readily recognized from /6-28 lhoculalea’ in R.axi//a /'/.5 C/bntrja/ I /,5 days prewaus/ each Wit/7 ZZMZe/y’ZZC/éé ms "'9 a”? $7715 3%” I // IE 25 2 3003/5 ,6 {'8 2.5 20 029/3 - o O O ' z . 0 ' . l7 4” a I o g 3 ‘ . : o 0 0 1 . 4 a Q ' ‘ . . . ' . 5 0 O ‘ I ' . . . . 6 o o p I ' ' ' Q 7 I O Q 22 ' . . . 8 . . . Z; . 0 Q 9 __ 1 0 Q Q m'—t fi----' // -=----- Z6 "'""' /Z '=" """""' 27 ---_- /3 ---- 28""— /4 --—-- ,5 ______ IOCM. FIG. 7. —— Experiment I/35B. All mice injected in the left axilla with 0.02 gram of tumor (needle method). No immunity in the animals into which their own enlarged spleens were introduced. Figs. 7, 8, and 9, reproducing experiments in which mice that had been subjected to preliminary treatment with their own enlarged spleens, were later tested with three difierent tumors, after varying 142 THE INDIVIDUALITY or CARCINOMA intervals of time, with different doses, and by two methods of inocu— lation. Not even the slightest trace‘of resistance could be demon- strated; on the contrary, in Fig. 8 the tumors in the treated mice /Z'Z4 /'/70cu/4ted in .a/r/Y/a' /-/l Contrp/ l6 0’ p. . - Normq/ m/ce its coy/:2 ’gnrgggfffl/sZaeiZf/M Amway/2! /69m5 AK l/e/g/n‘ l9yms /0 l7 14‘ days /0 /7 Z4 02% I l 1‘ _ /Z .l ' i '- 3' ' H *n _ 1 8 j , l4 ‘ ‘. 4 ' g ' _ 15 1 I 5 ' “ 16 =- i. . O O 6 0 I i 7 I ' ' [8 I ' . ‘9 ' ' ' l9 1 g; 9 ' p 9 , . . 20 ' ' . l” ' ' O 2 t l i l/ i l I . ZZ -' 2 I ’ 23 ' ~ 9 /0 CM. 24. - — — FIG. 8. — Experiment 63/47H. All mice injected in the left axilla with 0.025 cubic centimeter of tumor emulsion. Result the same as in Fig. 7, except that the mice sub- jected to preliminary treatment show a slight degree of hypersensitiwness. appear to show a more active growth than those of the control animals. ' ' ' _ In the next experiment (Fig. 10), in which a fourth tumor strain was used for inoculation, only a slight degree of resistance was in- duced following the injection of the animal’s own normal spleen. In Fig. I_I there is complete absence of immunity in mice treated with their own kidney. A relatively enormous amount of material may be _obtained through THE INDIVIDUALITY OF CARCINOMA I43 9 removing the enlarged spleen and one kidney by operation. Even so large a quantity of autologo us tissue, however, does not induce any resistance, as is shown in Fig. 12. Finally, Figs. 13, and 14 represent the results of preliminary treat- I _- / 4 Control. Normal 07/09. Air yelp/rt l8y/n5‘ ./0/7Z4a’¢y/s I, ' LI ' Z ; .j 3 r 0 4 a I, .5 $ ) 6 = i _9 7 ' )0 g , I I 9 ' ' ' 'lfl ' - // 9 /Z /3 - - '- 14 —--' I5 123 [now/aim [h R.am’_//a [4 days prewous/y 866/) MM Its 01m err/gr ed 5,0/een AK Ire/g t Z/yms /0 CM. /.5 l6 l7 /6' 7.9 20 Z/ 22 Z3 [0 /7 24 023/; FIG. 9.— Experiment 91/ 23T.' All mice injected in the left axilla with 0.02 cubic Practically no immunity. ment of mice with their own testes. centimeter of tumor emulsion. Although Fig. 13 seems to present indications of a slight degree of resistance, the results of the experiment represented by Fig. 14 are in contradiction, for no resistance whatever has been developed. Summing up the results, the conclusion is inevitable that the reac- I44 THE INDIVIDUALITY OF CARCINOMA- tion which is produced by preliminary treatment with tissue from ' other animals of the same species, and which leads to the develop- ment of resistance, is not produced by preliminary treatment with autologous tissues. Occasionally, injection of the animal’s own tissues is followed by a suggestion of the refractory condition. This, h0w- ever, is either an accidental result which falls within the limits of error attaching to such experiments, or the product of causes with which we are not yet familiar. The fact that in these experiments /Z-Z3 /hocu/a[ea’ in R. aX/Y/a /-// [antral ‘ /_2 days pren'ous/ eac/I M'I/z Nor/na/ m/ce - 1!: own mar/ma sp/ee/z AK wig/7! My”: fix ire/96! /_9y/;;5 l” /7 Z4 3/ 38 days /0 /7 24 3/ 38 days I ' 0 O I . /2 , g . . . Z ’ ' ' '- ' l3 - .0 f I‘ 3 ' ' . . l4 2 O Q . 0 4 I I J . 1- /5 .I i _0 I o _6 _- a n a I /5 - . . . 6 -. , ’ '. .0 9 - ' _ 7 .0 r 0 .~ /8 . . ' . .... .- 8 § , , . . . . . _ .- 9 ' _ - c \ _ — — /0 - . . - - _V ' a _ _ - // ' l — - — . — _ — - /0 m. ' Z3 ' " "' " " FIG. 10. —— Experiment 2 38/ 2 5B. All mice injected in the left axilla with 0.01 gram of tumor emulsion. A trace of immunity after preliminary treatment with the animal’s own normal spleen. the precautions above referred to were rigidly observed, certainly excludes the following factors from having played any role: (a) amount of tissue injected; ([2) dose of tumor used for testing resist- ance ; (a) site of inoculation; (d) interval of time between introduc- THE INDIVIDUALITY OF CARCINOMA I45 tion of normal tissue and testing dose of tumor; (e) type of growth of the tumor strains used in testingv resistance. 1.9-37inoculqted in LAM/a 2£8,,Z;2r%”,5;"" I IQ I? {4.1/{324$ ,9 7‘0 3.80213 CZ -- ..._ 20 - ~00. 3’ "5 Zl - --0., ,4 .... 22-..'.'.'.' 5 ~- - -¢0 Z3 . ...‘ 6 - -0 24 . . ,.. 7 or. 25 - -..l 5. .s. 26 _ ___»‘. 9 -- - - 0 27 _. .I . .. l0 - ' '- ' l m . .00. I] . . . . a a . . . . .0 ll --- ' ' 30 __ ,, /3 ~ '- - .. 3/ -—- - ' /4 . . . . . 32 ____ . 15-... . - 33 ' -‘ /6 . . . . . V 34 - ._...- l7 . I 35 ' --__ /8-_-—_ Lu_|_r.L1_|_|_|-| 36-----, IOCM. 37 ———-— FIG. 11. — Experiment r/34E. All mice injected in the right axilla with 0.02 cubic centimeter of tumor tissuep(needle method). No immunity after previous treatment with the animal’s own kidney. The preliminary experiment, which led to the conclusion that treat- ment with autologous tissues induces resistance, demonstrated this 146 THE INDIVIDUALITY or CARCINOMA irregular appearance of immunity in an especially pronounced man- ner; but the more extensive observations now reported leave no /4 r23 m/ce inoculated 20 days prey/owl] m the /-/3 ( antral R, aX/Y/a each with its aim; Na/ma/ mice h’lO'f/C/ 4 eh/a/yea’ 5,0/6’6’!) AV #c/yht /.9yms Ar Iva/ah! / 7 pm /0 /7 [445/5 /0 /7 [4021/5 / 5 Q , ' . /4 ' ‘Q 2 ! Q Q . /5 c i J 3 s 3 Q /6 ' 4 , , , /7' ' f 5‘ 5 , ; ; I8 ' ¢ O 5 .' v Q i /9 ' l O 7 20 \ 3 . '8 ' =- Z/ 3 3 .9 ! ' ' . ' ZZ " - "' 10 - 1 25 - 1‘ ll - -' '- /Z #- - - 10 CM. a - - 1 FIG. 12. —— Experiment 6 3/471. All mice injected in the left axilla with 0.01 cubic centimeter of tumor emulsion. No immunity after preliminary treatment with the animal’s own kidney and enlarged spleen. doubt that the conclusions there drawn must be refuted as not generally applicable. After these experiments were completed, Apolant and Marks (2) discussed this same problem, but upon a much less extensive ex- perimental basis. They repeated the preliminary experiment, and found that treatment with autologous spleen did not induce resist— ance. While this conclusion coincides in general with the reSults here reported, the explanation offered by them is not justified. They be- THE INDIVIDUALITY OF CARCINOMA I47 lieve that the absence of resistance may be ascribed to the insufficient amount of tissue contained the spleen. This explanation is shown /6-Z3 inoculated in R.axi//a /-/5 Control /4 ‘ . ' Narma/ mice #30132 5222/???” 886/) Mm AK h/e/ght l5g/215 AK height /6_ gms l] /3 26 33 40 day: /2 l? 26 33 4002916 I .1 2U! - ’6 "'" _ /7 c O 3 ‘ it /6’ V. Q ’Q I! I - I I-- 4 I ' $ - - - - 5 9 me 6 "an 2’ "'"'" 7 rHH 22"“— 9 1 ' ff. /0 ' i... // " ' i ' ' /2 ' Q' ' l3 ’ ' . mm. /4 ,' 2 ' -1.' /5 ----- FIG. 13. —Experiment 63/46G. All-mice injected in the left axilla with 0.01 cubic - centimeter of tumor emulsion. Immunity of a slight degree after preliminary treatment with the animal’s own testis. to be incorrect by the experiments here described, for it has been shown in this laboratory that the amount of tissue corresponding to a normal spleen is sufficient to make other mice resistant to appro- priate doses of tumor, although the reaction does not follow pre- liminary treatment of mouse with the same, or even much larger, amounts of its own tissue. - The essential point of these experiments, which was not recog- nized by Apolant and Marks, lies rather in the fact that an animal 148 THE INDIVIDUALITY or CARCINOMA behaves quite differently when treated with its own tissue-s, than when treated with foreign tissue. The relation of an animal to its own» cells Zl-4O inoculated. in Rex/Ila /_Z dig/,9 prer/oush/ each Ifl'th lts elm testes [-20 (ant/'0! An Ire/gilt 14 m; Ahghmefhhlfyggms ' ,0 ./7 Z4 lag/3 m /7 24 slaw ' 2/ tQQT 1"3' 2'40. Z"'. ' 3!0!l 3 '99K zioioi 4 'IIK 25.‘.". 5 '!* 16:12: 6100. 27 ,3” 7"" mitt ‘8 3"1 29,é’p' 9 o 0OK 30 ". . ' 10 9000 . .’. ” ‘." 31.... 3201' u '.. H;,.. e'::- M,,__ /4:' - 35“,“ air-- %'___ %'--- 1’ '--- 3211:: e'-—- ’ -20____ 1001. 40'"- FIG. 14. — Experiment 63/48B. All mice injected in the left axilla with 0.01 cubic centimeter of tumor emulsion. No immunity after preliminary treatment with the animal’s own testis. has now been worked out in two different ways. Haaland (3, 4) has shown that an animal suffering from a spontaneous tumor cannot be made resistant to implantation of. that tumor by preliminary treat- THE INDIVIDUALITY OF CARCINOMA I49 ment with heterologous tissue of the same species. The writer has shown that a normal animal cannot be made resistant to the trans- plantation of heterologous tumors by preliminary treatment with its own tissues. Summing up these results, it follows that the cells of an animal, whether they are normal cells or those of a tumor, possess a pro- nounced individuality when compared with the cells of another animal of the same species. This fact opposes to the cure of cancer an obstacle as yet unsurmountable. The cost of these investigations was partly met by the Crocker Cancer Research Fund, of the College of Physicians and Surgeons, Columbia University, New York. BIBLIOGRAPHY I. WOGLOM. Journal of Experimental Medicine, 1:910, xii, 29. (Reprinted in this volume, p. 127.) 2. APOLANT and MARKS. Zeitsclzr. f. Immunitiitsforsclz., 1911, x, I 59. 3. HAALAND. Jour. Path. and Bact., 1910, xiv, 407. 4. HAALAND. Proc. Roy. Soc., Ser. B, 1911, lxxxiii, 532. PART IV DEPARTMENT OF BIOLOGICAL CHEMISTRY PART IV DEPARTMENT OF BIOLOGICAL CHEMISTRY I INTRODUCTION ' GENERAL PROGRAMME OF THE BIOCHEMICAL INVESTIGA— TIONS UNDER. THE AUSPICES OF THE GEORGE CROCKER SPECIAL RESEARCH FUND, 1909—1910 AND 1910—1911. By WILLIAM 1. Guns DURING the spring and summer of 1909, the writer reviewed the extensive chemical literature on the subject of cancer. The conclu- sions from that review suggested various researches along the follow- ing main lines :— 1. Studies of the chemical and nutritional factors in the resistance of suitable animals to the development of malignant tumors after appropriate inoculations. 2. Inquiries into the biochemical differences between healthy and cancerous tissues. 3. Experiments on the influence of cancer extracts on the growth of both plant and animal cells. 4. Determinations of the efiects of cancer extracts and products on local as well as systemic nutrition. It was expected that such investigations, if projected along new lines, would yield results of significant bearing on the cause of cancer. It was clearly desirable to obtain, if possible, complete answers to such questions as the following ones :— A. What chemical alterations occur locally as cancer progresses /? B. What chemical changes result locally in cancerous tissues during spontaneous recovery from the disease? 153 I 5 4 INTRODUCTION C. What chemical modifications take place systemically as cancer progresses P D. What are the chemical readjustments, systemically, as natural recovery from cancer ensues? E. What are the specific chemical interactions between the body as a whole and any cancerous tissue in it? It was proposed to begin research along these lines in the fall of 1909. Drs. Stanley R. Benedict and Jacob Rosenbloom, Associate and Assistant in Biological Chemistry, respectively, had been ap- pointed, meanwhile, to conduct the work. Unfortunately, two important conditions rendered it impossible to inaugurate the proposed studies. In the first place, the Crocker committee’s provisional plan to breed suitable animals for such work on a large scale could not be satisfactorily undertaken. Much more discouraging, however, was our inability both to gain access to satis- factory cancer supplies from human beings and to study cancer pa- tients. For over two months in the fall of 1909 we endeavored, through personal interviews and by correspondence, to obtain the cancerous tissue that was needed for some of the work we desired to do, but with- out success. Our efforts in this direction extended to practically all the hospitals in New York City, but, although individual officers were very friendly in their personal attitude toward us, all plans for the delivery of satisfactory material to us were abortive. At Roosevelt Hospital, for example, it was impossible to obtain any help of any kind under any circumstances. Without tumorous animals, without cancer patients, and without carcinomatous supplies, all our plans for direct chemical attack on the cancer problem had to be suspended. Drs. Benedict and Rosenbloom were therefore given entire freedom to inaugurate such work as they might be able to conduct with the regular laboratory facilities. They undertook jointly a study of the Gunzberg test for free hydrochloric (mineral) acid in gastric liquids containing lactic acid and other organic acids. It was their hope that this study would improve certain phases of the technic in the diag- nosis of gastric cancer. At that time, also, Dr. Benedict began a study of several chemical methods, in the expectation of devising improved processes and thus insuring greater accuracy in such cancer metab- olism studies as we or others might be able to conduct. INTRODUCTION I 5 5 f Late in 1909, it was evident that the original plans could not be executed because of our inability to secure satisfactory facilities and supplies, as has already been stated. In due recognition of the fact that cancer is a problem of cell (tissue) growth, and realizing that very little is known regarding the relationships and functions of the indi- vidual cellular constituents, either in reproduction, growth, repair, regeneration, degeneration, or other dynamic states, it seemed best to concentrate our further efforts and available resources on studies of the 'ways in which the constituents of lining cells are related to each other — studies intended to prepare us for the work of making comparative investigations of intermolecular relationships in living cancer cells. My proposal to this effect had the hearty approval of my Crocker as- sociates, Drs. Benedict and Rosenbloom. We accordingly proceeded in January (1910) with a comprehensive plan of research on the com— position of protoplasm, and the nature of the structural and dynamic rela— tionships of cell constituents and products.- Dr. Benedict was encouraged to complete the studies of the “metabolism methods which he had undertaken in the fall, and was invited, also, to give attention to the subject of intracellular relation- ships of proteins. Dr. Rosenbloom was requested to specialize on the lipins, and he cooperated in a study of new methods of isolating and differentiating cell lipins. Dr. Rosenbloom also began the preparation of a general review of present-day knowledge of cellular fatty matters.1 In addition to the work already mentioned, Dr. I. W. Weinstein con- ducted a study of the glycyltryptophan test for gastric cancer. This research resulted in the proposal of a simple substitute for the N eubauer and Fischer test. We began the second year of our work under the auspices of the Crocker fund, with a resumption of our studies of the composition of protoplasm, and the nature of the structural and dynamic relationships of cell constituents and products.2 Dr. Wm. H. Welker succeeded Dr. Benedict in research on proteins, and Dr. Ottenberg undertook in- vestigations of isoagglutination. Dr. Weinstein continued his study 1 During the latter half of the year Dr. Rosenbloom cooperated at the German Hos- pital with Dr. Einhorn in the studies which are described on page 200. 2 Very greatly to our regret Dr. Benedict resigned his Associateship at the end of the academic year of rgog—rgro. Dr. Rosenbloom was promoted to the vacancy thus created, and continued the work on the lipins. r 56 INTRODUCTION of the tryptophan test for gastric carcinoma. During the year we also enjoyed, in various phases of the work, the hearty coOperation of Ernst Boas, George D. Beal, Max Einhorn, S. S. Friedman, George A. Geiger, Samuel Gitlow, F. G. Goodridge, Max Kahn, Morris H. Kahn, David J. Kaliski, ]. L. Kantor, Charles H. Sanford, and I. B. Sidbury. The chief extension of our plan in the second year was involved in the invitation to Dr. Ottenberg to proceed with studies of isoagglu- tination. We believed that investigations of the nature and causes of isoagglutination phenomena in man and lower animals might mate- rially increase our knowledge of both intracellular chemistry and the extracellular dynamics of various intracellular products. It seemed possible that isoanti-bodies (that is, all anti-bodies directed against cells or proteins of other individuals of the same species) are of prime importance in cancer. We thought that possibly a disturbance in the production Of isoanti-bodies might be an etiological factor in cancer. Cancer cells are certainly cells of the same species as the cells of the host. In spontaneous cancer, however, the cells show, by their mode of growth and other characteristics, that they are not absolutely uni— form with the cells of the host. They are so aberrant that they may be fairly regarded as foreign cells of the same species -— as “ isocells.” In all experimental cancer they are actually cells transplanted from one individual of a given species to another of the same (or closely related) species. Since normal isoanti-bodies of at least one kind exist in man and in some of the lower animals, and as immune isoanti-bodies of another kind can be developed under certain conditions, it seems probable that various isoanti-bodies regularly play a part in the normal sequence of events in the body. It is possible that this rOle may be the preserva- tion of the individual specificity (the autospecificity as distinguished from the isospecificity). This idea is especially supported by the hereditary transmission of isoagglutinins.3 It is very probable, there- fore, that new facts on isoanti-bodies will throw much light on the autospecific aberration of cancer cells. Aside from these considerations it is possible, also, that many of the facts of so-called “ immunity ” to experimental cancer may be due to the occurrence of natural or of artificially stimulated isoanti-bodies. 3 Such transmission was first suggested by Dr. Ottenberg in 1908. See page 236. INTRODUCTION 1 57 Failure to observe such agents may .have been due to search in the wrong directions. Dr. Ottenberg’s papers on isoagglutination, which were pub- lished during 1910—1911, are brought together on pages 210—243, inclusive. On page I 55 I have stated some of the views which led to the proposal that intracellular chemistry be made a subject of research under Crocker auspices. In this connection it may be appropriate to suggest, in further support of those views, that all diseases are expressions of intracellular disturbances. Every disease is caused, externally or internally, by modifications of the normal sequence (coOrdination) of chemical changes in the cells Of parts or all of one or more tissues (injury). I believe there are no known exceptions to this rule. It is inconceivable that cancer is an exception. The problem of cancer remains unsolved. Chemistry has been of little service in its study. I believe this state of affairs is due pri- marily to the fact that biological chemists have not given adequate attention to the composition of protoplasm and the nature of the functional (coordinated) relationships of cellular constituents. “ Mor- phologic phenomena of growth are invariably connected with chemical processes and are more or less the expression Of underlying chemical and physical change.” Our general knowledge of disease warrants the conviction that different types of injury cause different kinds of disease because diflerent discob'rdinations of intracellular constituents result therefrom. Tumors may result from intracellular derangements ——from discob'rdinations of functionally related cellular constituents. New resultants of biological forces would thus develop with effects like those that arise from injury due to external physical or chemical agents or conditions. The writer believes that the essential factor in the etiology of cancer is a stimulus to cell division, of intracellular origin, and that complete understanding of the disease awaits more definite determination of the constitution of protoplasm, and the reaction-tendencies and functional alignments of the substances pe- culiar to cells. The main results of our work are presented in the papers compris- ing the two succeeding sections. The contents of each section are indicated on pages 160 and 210 respectively. Publications of bio- 1 58 INTRODUCTION chemical research under Crocker auspices, which have not been reprinted in this volume, are listed below. I. Improved Methods for the Study of Nutrition STANLEY R. BENEDICT. A note on the estimation of total sulphur in urine. Jour- nal of Biological Chemistry, 1910, vii, p. 101. STANLEY R. BENEDICT. A method for the estimation of reducing sugars. Ibid., 1911, ix, p. 57. STANLEY R. BENEDICT. The detection and estimation of glucose in urine. Jour- nal of the American Medical Association, 1911, lvii, p. 1193. STANLEY R. BENEDICT. The occurrence and estimation of creatinin in urine. Biochemical Bulletin, 1912, ii, p. 165. WILLIAM H. WELKER. Electrical baths for use with Benedict’s method for the determination of urea. Ibid., 1912, i, p. 439. II. On the Composition of Protoplasm and the Nature of the Struc- tural and Dynamic Relationships of Cell Constituents and Products A review of the chemistry of the cell. A symposium (abstracts). Biochemical Bulletin, 1911, i, p. 65. . Introduction, including remarks on intracellular water. WM. J. GIES. . Intracellular salins. WILLIAM H. WELKER. . Intracellular carbohydrates. ERNEST D. CLARK. . Intracellular lipins. JACOB ROSENBLOOM. . Intracellular proteins. WALTER H. EDDY. . Intracellular extractives. ISIDOR GREENWALD. . Intracellular enzymes. ALFRED P. LOTHROP. . Factors in immunity. REUBEN OTTENBERG. JACOB ROSENBLOOM and WILLIAM J. GrEs. A proposed chemical classification of lipins. Biochemical Bulletin, 1911, i, p. 51. JACOB ROSENBLOOM. Preliminary analytic studies of lipins. 1. (With FREDERIC M. HANEs.) The lipins of normal and cryptorchid testes. Journal of Experimental Medicine, 1911, xiii, p. 355. 2. The lipins of the corpus luteum. Surgery, Gynecology and Obstetrics (Frank), 1911, xiii, p. 48. . 3. The lipins of the heart muscle of the ox. Proceedings of the Biological Section of the American Chemical Society: Science, 1911, xxxiv, p. 221; Biochemical Bulletin, 1911, i, p. 114. ' 4. The effects of pregnancy on the lipins of the ovary and corpus luteum of the cow. I bid. 5. A quantitative study of the lipins “of bile obtained from a patient with a biliary fistula. Ibid., 1912, ii, p. 182. OO\I @m-POOMH INTRODUCTION 1 59 6. JACOB ROSENBLOOM. The lipins of the ovary and corpus luteum of the pregnant and non-pregnant cow. Journal of Biological Chemistry, 1913, xiii, p. 511. 7. A quantitative study of certain enzymes of the ovary, uterus, and bladder of pregnant and non-pregnant sheep. Biochemical Bulletin, 1913, ii, p- 233- 8. On the absence of certain enzymes from the human chorion. I bid, 1913, ii, p. 236. GEORGE D. BEAL and GEORGE A. GEIGER. A preliminary study of the comparative difiusibility of pigments through rubber membranes. Biochemical Bulletin, 1912, ii, p. 78. ABRAHAM RAVICH and JACOB ROSENBLOOM. Chromolipins: A review of the literature of lipochromes, with studies of carrotin and ovolutein. (In press.) J. L. KANTOR and WILLIAM J. GIES. Additional experiments with the biuret reagent. Proceedings of the American Society of Biological Chemists, 191 1, ii, p. 11; Journal of Biological Chemistry, 1911, ix, p. xxvi. Also Biochemical Bulletin, 1911, i, p. 264; ii, p. 179. WILLIAM H. WELKER. On the diffusibility of protein through rubber membranes, with a note on the deterioration of collodion membranes in common ether. Biochemical Bulletin, 1912, ii, p. 70. WILLIAM J. GIEs. Preliminary reports on studies of water absorption by proteins. 1. (With JOHN L. KANTOR.) A new microscopic test for free acid (de- pending on the afiinity between collagen and water). Proceedings of the American Society of Biological Chemists, 1911, ii, p. 20; Journal of Biological Chemistry, 1911, ix, p. xvii. 2. A criticism of Fischer’s theory of edema. Biochemical Bulletin, 191 r, i, P- 2’79- ' 3. (With SAMUEL GITLOW.) On the affinity of collagen for water in the presence of acid. I bid, 1913. (In press.) WILLIAM H. WELKER. A preliminary study of the qualities of a typical collagen- acid compound. I bid, 1913. (In press.) WILLIAM J. GIEs. Modified collodion membranes for studies of diffusion. Pro- ceedings of the American Society of Biological Chemists and the Biological Section of the American Chemical Society (joint session, Washington, 1911): Science, 1912, xxxv, p. 396; Proceedings of the Society of Biological Chemists, 1912, ii, p. 75, and Journal of Biological Chemistry, 1912, xi, p. xli. II METHODS FOR THE DIAGNOSIS OF CANCER CONTENTS . The new test for cancer of the stomach, with suggested improvements. J. W. WEINSTEIN. . . . . The tryptophan test for cancer of the stomach, with special reference to peptidolytic enzyme in the saliva. J. W. WEINSTEIN . . The glycyltryptophan and tryptophan tests for cancer of the sto CHARLES H. SANFORD and JACOB ROSENBLOOM . The importance of the colloidal nitrogen of the urine in t cancer. he diagnosis of MAx EINHORN, MAX KAHN, and JACOB ROSENBLOOM mach. . The colloidal nitrogen in the urine from a dog with a tumor of the breast. MAx KAHN and JACOB ROSENBLOOM 0 PAGE 161 I78 191 200 206 I. THE NEW TEST FOR CANCER OF THE STOMACH, WITH SUGGESTED IMPROVEMENTS * By J. W. WEINSTEIN I. INTRODUCTION General Comment. — Medical means have been of no avail in the treatment of cancer. The only resort is surgery. If we succeed in radically extirpating the cancer in toto, the chances of a permanent cure are good. The importance of an early diagnosis in cancer in general, and in carcinoma ventriculi in particular, is obvious. Un- fortunately in cancer of the stomach an early diagnosis is rather the exception then the rule, and in most cases, when we learn the diag- nosis, we also learn the prognosis. The diagnosis of cancer of the stomach rests on the history of the patient; on the absence of free hydrochloric acid; on the presence of lactic acid, Boas-Oppler bacilli, a palpable tumor, and occult blood; and on the occurrence of a variable degree of motor insufliciency. No single factor is diagnostic in itself, but a combination Of these features is required for the establishment of the diagnosis. Unfortunately such a combination is not always present, and when it is present, it is not discernible at a very early stage of the disease. The Solomon test is of very little value. Any new sign that will aid in the diagnosis of carcinoma ventriculi is more than welcome. A few months ago Neubauer and Fischer 1 published their results of a new test for the diagnosis of cancer of the stomach: the so-called glycyltryptophan test. In order to comprehend the rationale of this test we must understand certain principles in physiologic chemistry. Our food, as such, cannot be assimilated by the system. In order to be absorbed most food substances must be converted into simpler * Reprinted from the J onrnal of the American Medical Association, Sept. 24, 1910, IV, pp. 1085—1091. 1 Neubauer and Fischer: Deutsch. Arch. f. klin. M ed., 1909, xcvii, 499. I61 162 NEw TESTS FOR GAsTRIC CANCER cleavage products. Thus proteins, to be absorbed by the system, must go through a series of changes, until they reach the simplest grade. This series of changes for coagulable proteins, such as al- bumin, is: (1) acid metaprotein, (2) primary proteoses, (3) secondary proteoses, (4) peptones, (5) “ polypeptids,” and finally (6) amino acids. Amino acids are the simplest cleavage products into which proteins must be broken up for assimilation by the body. The hydrolytic cleavage of the proteins into amino acids is effected by the digestive enzymes. There are numerous amino acids, such as glycin, alanin, leucin, tyrosin, tryptophan, etc., each exhibiting characteristic properties. In a normal stomach the digestion of proteins is never carried to the amino-acid stage. It is only in the intestines that ex- tended hydrolysis into amino acids occurs. Various investigators have shown that cancerous tumors elaborate a certain enzyme which exhibits strong proteolytic powers, and which is capable of converting proteins as well as simple peptids into amino acids. Although benign tumors, as well as other somatic tissues, possess similar properties, their peptid-cleaving powers are rather weak — they never equal the powerful peptidolytic action of malig- nant growths. N eubauer and Fischer have contrived to utilize this fact in a new test for cancer of the stomach. They used glycyltrypto- phan for this purpose. Glycyltryptophan is a dipeptid,2 one of the simplest types of peptids. As a synthetic product, it owes its existence to the ingenuity of Emil Fischer, who has made some of the most important chemical dis- coveries of modern times. Emil Fischer succeeded in combining various amino acids into forms that closely resemble natural proteins of the simpler types. These synthetic peptids are like proteins in responding to the hydrolytic action of proteases,3 and in yielding their corresponding component amino acids. Glycyltryptophan, as its name signifies, consists of a union of glycin and tryptophan radicals. In making their test, Neubauer and Fischer add filtered stomach contents to a solution of glycyltryptophan. The mixture is then placed in a thermostat for about twenty-four hours. At the end of 2 Dipeptid — a peptid containing only two amino-acid radicals. 3 Proteases are enzymes possessing the power of splitting up proteins or peptids, e.g. pepsin, trypsin, erepsin, etc. NEW TESTS FOR GASTRIC CANCER 163 that time a test is made for the presence of tryptophan. If trypto- phan is found, it means, according to Neubauer and Fischer, that an enzyme present in the stomach contents has converted glycyltrypto- phan into glycin and tryptophan. Such a result could not be due to normal constituents of stomach contents, e.g. pepsin, rennin, lipase, or hydrochloric acid, since these agents do not, under gastric condi- tions, split either dipeptids or proteins into amino acids. An enzyme capable of converting glycyltryptophan into glycin and tryptophan oc- curs in gastric contents only in cases of malignant growths of the stomach. Glycyltryptophan was used for this test because tryptophan, which it easily yields, may be more readily detected than any other amino acid. No protein yields a larger proportion of tryptophan. Tyrosin is perhaps the only other amino-acid which could be employed to similar advantage in such work. T yrosin may be easily detected by reason of its comparative insolubility and its consequent tendency to crystallize. A dipeptid yielding a large proportion of tyrosin, such as glycyltyrosin, might be expected, therefore, to serve as satisfactorily in this test as that particular dipeptid did in similar work recently by Abderhalden 4 in another connection. The simplicity, promptness, and delicacy of the bromin test for tryptophan, however, give the advantage to the compound favored by Neubauer and Fischer. That the Cleavage of dipeptid in the stomach in the case Of cancer is done by an enzyme secreted by the tumor, and that it is not accomplished by pepsin, was shown by Kohlenberger.5 N eubauer and Fischer’s Precise Method of Procedure. —— In apply- ing the glycyltryptophan test, an Ewald test breakfast is extracted in thirty to forty-five minutes after a meal and filtered. Glycyltryp- tophan 6 is treated with some of the filtrate. The bottle containing the mixture is placed in a thermostat for twenty-four hours. At the expiration of that period the contents are tested for the presence of 4 Abderhalden: Ztsch. f. physiol. Chem, 1909, lxii, r36. 5 Kohlenberger: Deutsch. Arch. f. klin. M ed, 1910, xcix, I48. 6 Glycyltryptophan, as prepared for the test, is a clear, watery solution. It is dispensed by Kalle & Co., Biebrich a. Rhein, all ready for use in little bottles. Each bottle contains a few minims of glycyltryptophan solution with a layer of toluol over it, as a preservative. This amount of glycyltryptophan is sufficient for one test. The bottle has a mark on it up to which the filtered stomach contents is to be added. The total capacity is about I 5 cubic centimeters. 164 NEw TESTS FOR GASTRIC CANCER tryptophan. This is done as follows: About 3 or 4 cubic centimeters of the contents of the bottle are removed with a pipette from under- neath the layer of toluol and placed in a test-tube. A few drops of 3 per cent acetic acid solution are added. Saturated aqueous solution of bromin is then added from a pipette, drop by drop, to the stomach contents in the test-tube, until a reddish violet color appears. The appearance of this color, at times a rose-red, shows the presence of tryptophan, and the test is positive. Considerable practice is required for the accurate performance of the test in the presence of very minute proportions of tryptophan, because slight excess of bromin may make the characteristic color disappear in a moment. Large excesses may impart a lemon-yellow color to the mixture. Whenever the reddish violet color merges into yellow, we know positively that there is an excess of bromin in the mixture. The opening at the tip of the pipette from which the bromin solu- tion is dropped should be a fine one, and the “ bromin water ” added very cautiously, drop by drop, until, in case reaction is positive, a reddish violet color appears. If reaction is very faint, the test-tube should be left standing for a while. In about ten minutes, in such cases, the reaction may become considerably intensified. Usually 5 to 7 drops of bromin water are required to bring out the reaction in 3 to 4 cubic centimeters of contents, but sometimes 1 to 2 drops are sufficient. When that is the case, it is well to let a single drop of bromin water fall on the wall of the test-tube instead Of into the liquid itself, in which case the volume of delivered bromin solution is reduced to a fraction of a drop, and excess is avoided. Instead of bromin solution, pure bromin vapor may be used. If a bottle containing bromin is tilted slightly over the mouth of the test-tube, heavy bromin vapor will fall into the test-tube and dissolve in the liquid; and the reaction will promptly appear to its maximum degree. Great caution must be exercised, in this treatment also, not to add an excess of the vapor, for an excess will make the reaction disappear at once, and a lemon-yellow color may be imparted to the contents. Bromin itself should be handled with great care, for it is extremely irritating to the respiratory mucous membrane. Under no circum- NEW TESTS FOR GASTRIC CANCER 165' stances should plain liquid bromin be mixed with the gastric contents to be tested. An aqueous bromin solution may be kept saturated by retaining in it a slight excess of the heavy, liquid bromin. Acetic acid is added to the mixture to be tested because the reaction appears only in an acid medium, perhaps very faintly so in a neutral one, but never in an alkaline medium. Almost all stomach contents are acid, and in testing them for tryptophan the addition of acetic acid may not be necessary. In mixtures of very low acidity the addition of acetic acid seems to serve a useful purpose. Special Sources of Error in the Glycyltryptophan Test. —Neubauer and Fischer made a series of tests in normal and pathologic cases, and were extremely gratified by the results obtained. The following sources of error in the application of the glycyltryptophan test are mentioned by them: —— 1. Presence of Tryptophan in the Stomach Contents. — Neubauer and Fischer advise tests for tryptophan in the stomach contents before glycyltryptophan is added. If tryptophan is found, the specimen of stomach contents should be rejected, they say, and another one subjected to the test. I do not agree with Neubauer and Fischer in this regard, as I shall show further on in this paper. 2. Presence of Peptid-splitting Bacteria in the Stomach Contents. ——Neubauer and Fischer have tested the effects Of pure cultures of various species of bacteria on glyclytryptophan and have found that some species are able to split off tryptophan. The addition of toluol to the material in the test is proposed by them as a safeguard against such bacterial action. The passage of the stomach contents through a filter, according to them, also removes the offending micro-organisms. This danger is greatly overestimated by Neubauer and Fischer. 3. Presence of T rypsin in the Stomach Contents. — Duodenal con— tents may regurgitate into the stomach and the trypsin may split up the glycyltryptophan. N eubauer and Fischer exclude this latter possibility by examining the stomach contents in gross for bile, which is easily recognized by its greenish color. In the presence of manifest bile another specimen must be used for the test. 4. Presence of Blood in the Stomach Contents. —— According to Neu- bauer and Fischer blood is capable of splitting up glycyltryptophan and thus serving as a source of error in the test. If the presence of 166 NEW TESTS FOR GASTRIC CANCER blood is suspected, a test such as the guaiac or benzidin test should be made for it. If occult blood is present, Neubauer and Fischer recommend that that specimen be discarded and a new one tested. The danger from the presence of occult blood is also exaggerated by N eubauer and Fischer. ‘ Lyle and Kober,7 who recently reported the results of their study of this test as applied to twenty-one cases, concluded as follows: “ Our results with this teSt have been satisfactory. A repeated nega-' tive reaction is very valuable. When the test is positive, the com- plication of a regurgitation of trypsin must be thoroughly investi- gated.” II. DESCRIPTION OF EXPERIMENTS Glycyltryptophan not Necessary for the Detection of T ryptophan- producing Protease. —— I have been engaged, during the past few mOnths, in a study of the value Of this new test for cancer Of the stomach in its various aspects, and my experience agrees with the results obtained by the former investigators. I wish, however, to propose some important modifications based on a study of sixty-three normal and pathologic cases in all forms of gastric disease (see Section V, Record of Cases). It is my conviction that the employment of glycyltryptophan is superfluous. The tryptophan reaction may be obtained without it. Every test of my series of cases was a double one (one with and one without glycyltryptophan), and glycyltryptophan was found to be unnecessary. The reason is obvious. The enzyme secreted by the cancer is potent enough to hydrolyze protein, as well as dipeptid, into amino acids; nor does it have to do it unaided, for in the vast majority of cases the associated pepsin converts accompanying protein to peptones. There were only about two or three instances in my series of cases in which the test was positive in the presence of glycyltrypto- phan, but negative in the absence of that compound? On the other hand, I had some cases in which the opposite was true —in which without glycyltryptophan a better reaction was obtained than with it. More than that, I received the impression that the addition of - glycyltryptophan may at times serve as a source of error. It is pos- 7 Lyle and Kober: New York Med. Jour., June 4, 1910, p. 1151. NEW TESTS FOR GASTRIC CANCER I67 sible that some of the bottled specimens of glycyltryptophan undergo spontaneous decomposition into the component amino acids. I no- ticed in two tests the production of a very deep red color in the pres— ence of glycyltryptophan, whereas in its absence no reaction occurred. (In one instance a clear-cut case of chronic gastritis. See Record of Cases, special Case 39.) These observations aroused my suspicion. On one of the two occasions referred to, there was a surplus of the specimen of stomach contents under examination. I then tested that specimen with a sample of glycyltryptophan from another bottle and obtained only the faintest trace of a reaction. There appears to be no other expla- nation for this exceptional result than the assumption that the first sample of glycyltryptophan contained free tryptophan. I have subjected seventeen bottled samples of our supply of glycyl— tryptophan to tests for free tryptophan. Distilled water instead of stomach contents was added to each of these bottles of glycyltrypto- _ phan.8 The tests gave entirely negative results in each case. It should not be forgotten, however, that suspicion was aroused in only two out of nearly 150 instances, and, therefore, the seventeen negative results would not exclude the correctness of my assumption that two of the samples employed contained free tryptophan. There are several advantages in excluding glycyltryptophan from the test. Glycyltryptophan is comparatively expensive.9 The toluolized samples form opaque, milky mixtures with stomach con- tents. Detection of slight proportions of tryptophan may be very difficult, often is impossible, in such opaque media. It is the clear solution only that develops, ‘as a rule, a distinct and unmistakable reaction. Filtering the opaque mixture does not do any good in most cases. The opacity persists in spite of repeated filtrations. N eubauer and Fischer advise the use of a regular Ewald test break- fast extracted in thirty to forty-five minutes. I found, however, that employment of a regular dinner and extraction of some of it at a later stage of digestion (within three or four hours) served the purpose better. I convinced myself of this in connection with speci- mens of contents from two cases that showed marked tryptophan 8 Each bottle contained suflicient glycyltryptophan for one gastric test. 9 Material for I 50 tests, imported duty-free for use in this laboratory, cost $50. 168 NEW TESTS FOR GASTRIC CANCER reactions after a regular dinner, but failed to give any reaction after an Ewald test breakfast, the stomach having been washed previously (Cases 1 and 3). Both were obstruction cases. The Ewald test breakfast is ideal for testing the secretions of the stomach, but as an aid in the test for cancer enzymes it is much inferior to a regular dinner. There is only one Class of cases for which the Ewald test breakfast may be preferred. It happens occasionally, though very rarely, in cases of stagnation, that a stomach under the influence of a heavy meal will secrete-hydrochloric acid, While the light Ewald test break- fast will not cause any secretion. It Will be pointed out later that hydrochloric acid reduces the activity of, or may destroy, the cancer enzyme. When a negative tryptophan reaction is obtained in a case of pyloric stenosis showing free hydrochloric acid, it may be desirable to wash the stomach and administer an Ewald test breakfast, so that the contents may possibly be free from hydrochloric acid, and the elicitation of the tryptophan reaction favored on that account. (Case II illustrated this point.) Neubauer and Fischer state that “ hydrochloric acid, in a strength equal to 0.36 per cent, destroys this (cancer) enzyme.” While the results of my experiments corroborate this general statement, there appear to be exceptions to it. Cases II and 16 gave marked trypto- phan reactions in the presence of considerable free hydrochloric acid. Neubauer and Fischer further advise the application of the tryp- tophan test to stomach contents before adding glycyltryptophan for the detection of the cancer enzyme. If a positive tryptophan reaction is obtained with bromin water, that specimen, they say, should be discarded and a new one secured. But why discard it? This response tells the facts in the case! I detected preformed tryptophan in untreated stomach contents repeatedly. I obtained the reaction directly and easily. I have obtained it frequently in contents from cancer cases, but in no others! Of course, if one intends to use glycyltryptophan, the course prescribed by Neubauer and Fischer is the best one to pursue, but the results of my own research warrant the conclusion that the use of glycyltryptophan for the de- tection of gastric cancer is superfluous. I tried the effect of adding to stomach contents protein such as I NEW TESTS FOR GASTRIC CANCER 169 fibrin, or food from the meal, with the idea of enhancing the reaction, but the results were negative. N eubauer and Fischer state that the tryptophan reaction must be obtained in twenty-four hours, or it is of no value, since bacteria are likely to hydrolyze the dipeptid and thus produce tryptophan. They experimented with pure cultures of micro-organisms and found that certain species possess proteolytic properties. As a safeguard against this contingency, as has already been stated, they advised the addition of toluol. My experiments have dealt, not with pure cul- tures, but with stomach contents as such. I have kept specimens of stomach contents over a month (some specimens being devoid of free hydrochloric acid), both with and without preservatives, and, except in carcinoma cases, tryptophan did not develop from the admixed glycyltryptophan. Only in two achylic cases, on very pro— longed standing, did the reaction appear in contents from non-cancerous patients. Therefore, when an untreated specimen is kept for forty- eight hours in a thermostat, there is no special danger that bacterial development of tryptophan will occur. Sometimes a faint reaction becomes more marked in forty-eight hours. The addition of preserv- atives is superfluous and inconvenient. Thymol fragments form a heavy, milky emulsion with stomach contents, which is intensified by the addition of bromin water. Toluol is somewhat better, but it likewise forms a turbid mixture with stomach contents. As I stated before, such turbidity interferes with sharp elicitation of the trypto- phan color reaction with bromin water. Chloroform and ether are best in this respect, but they seem to interfere somewhat with the reaction in certain cases. I disagree with Neubauer and Fischer in their view that blood serves as a source of error in the glycyltryptophan test. The testing of contents for occult blood is superfluous. I do not wish to be placed in the position of taking issue with N eubauer and Fischer on the proposition that blood contains proteolytic enzymes. My conten- tion is merely that blood in stomach contents, in a quantity insufficient to be betrayed by its color, is not potent enough to hydrolyze protein or glycyltryptophan into detectable quantities of tryptophan. This conclusion is sustained by observations in several cases, in which occult blood was present in the contents, as proved by blood tests, and yet 170 NEW TESTS FOR GASTRIC CANCER no tryptophan could be detected after application of the glycyltrypto- phan test. More than that, I even added blood to stomach contents by getting blood from the patient’s finger, but no tryptophan could be obtained in the mixture even after standing in a thermostat for days. This was done by me on two patients, whose cases were both anacid. I also added blood from myself and from a fellow-worker in the labora- tory to glycyltryptophan, in quantities just sufficient to impart a slight bloody tinge to the solutions, but after six days’ incubation in a ther- mostat none of the mixtures contained a trace of tryptophan. Of course, it is probable that if a large quantity of blood is added to gastric contents, tryptophan will be formed, but such contents are, in general, worthless in this connection for another reason, namely, that it is impossible to discern the Characteristic tryptophan-bromin color in a red or dark-colored medium. Occult blood, therefore, can- not serve as a source of error in the tryptophan test. Duodenal contents which have regurgitated into the stomach are likely, if sufficient in quantity, to vitiate the test, since the contained trypsin may convert proteins into amino acids and thus produce trypto- phan that might be ascribed to cancer enzyme. Unless bile is mani- festly present in the gastric contents, however, there is little or no danger in this connection, assuming, of course, that bile and pancreatic fluid always appear together. N eubauer and Fischer aptly remarked that no chemical test need be made for bile in doubtful cases. It is sufficient, they believe, to examine the contents in gross, and if bile is obviously present, as evidenced by greenish color of the contents, such a specimen should be discarded and a fresh one obtained. Occult bile could hardly be accompanied by sufficient trypsin to produce a detectable amount of tryptophan in the test. Lyle and Kober seem to be impressed by the possible influence of the' trypsin that may occur in stomach contents in this relation. From extended experience in stomach work I can say that duodenal material rarely appears in stomach contents. Duodenal fluid may occasionally be present in very trivial amounts, but it cannot then vitiate the tryptophan test. Moreover, pepsin in the presence of acid destroys trypsin. Acid itself does so. To serve as a source of error in the tryptophan test, duodenal contents must be present in large quantities, and the stomach contents must be alkaline. These NEW TESTS FOR GASTRIC CANCER I71 two conditions prevail only when there is an obstruction of the duo- denum below the papilla and in certain pathologic cases following gastro-enterostomy, in which the drainage goes from the afferent loop into the stomach, instead of into the efferent loop. I have had several such cases; two of them are included in these experiments (Cases I4. and 15). But in cases like these the presence of duodenal contents may be determined without any difficulty. After all is said, one fact stands out prominently, viz., that the glycyltryptophan test yields positive results in practically all cancer cases and hardly ever in any others, as may be seen from the reported results of the experiments by Neubauer and Fischer, Lyle and Kober, and myself. The Author’s Tryptophcm Test. —— The test, as I have modified it and now recommend it, is made as follows: Four or five hours after a regular dinner, some stomach contents are secured, filtered, and tested with bromin water for tryptophan. If present, the reaction is positive. If absent, some of the filtrate transferred to a stoppered bottle and treated with a little toluol, or better still, without a preservative, is put in the thermostat and tested again for tryptophan twenty—four to forty-eight hours later. Although the reaction very often develops at room temperature, the mixture should be kept in a thermostat for the period stated. Deficiencies of the T rypliophrm Test. —The most serious defect of the tryptophan test is its inconstancy. The reaction may be present in one specimen of stomach contents and absent in another from the same patient. The only safeguard against this uncertainty is, in case of negative results, to test three or four specimens obtained at different times. If three or four tests yield negative results, the case is most likely non-cancerous. Work now in progress may ultimately indicate the reasons for the inconstancy referred to. (See addendum.) Satisfactory results are unattainable unless the contents are practi— cally colorless. Admixture of coloring matter may make it impossible to conduct the test. The presence, therefore, of blood or coffee grounds in the contents makes it necessary to select another specimen for the test. In giving the patient a meal for the test, he should be instructed not to ingest any food-stuffs that are likely to impart color to the contents. Coffee, tomatoes, strawberries, etc., for obvious I7 2 NEW TESTS FOR GASTRIC CANCER reasons, should be excluded. Tea must not be strong, else the con- tents will be sufficiently reddish to render the test useless. (See addendum.) ' The fact that free hydrochloric acid interferes with the activity of the cancer enzyme, or may destroy it altogether, is a serious obstacle in the way of reliable employment of the tryptophan test. III. GENERAL CONCLUSIONS A critical review of the results obtained in this study of the tryptophan test convinces me that it is a valuable sign in the diagnosis of cancer of the stomach. It is a sign in itself. The other signs of cancer, such as absence of free hydrochloric acid and presence of lactic acid, occult blood, coffee grounds, etc., have no value whatever, when taken individually, since they appear in conditions other than carcinoma. They are of value only in conjunction with other data. Moreover, none of these has any negative value. In these respects the trypto- phan test is superior to any of them. True, it is inconstant; but are positive results from the Widal test, the finding of the tubercle bacilli, the finding of plasmodia, constant features? However, in matters of such importance many observations must be made before conclu- sive deductions can be drawn. I cheerfully recommend this simple tryptophan test to the profession for the further study and more com- plete determination of its real value. Is it an early sign_of cancer? I do not know! I have not been fortunate enough, as yet, to get a very early case and determine the matter, one way or the other. Much more work must be done in this connection before that important question can be answered. I wish, in this connection, to express my indebtedness to Professor William ]. Gies of the College of Physicians and Surgeons, who has placed his laboratories at my disposal and has secured the support necessary for the conduct of this rather expensive research. With his ever ready counsel and valuable suggestions he has contributed greatly to the success of my work. I also express my indebtedness to Pro- fessor Francis Carter Wood of St. Luke’s Hospital, Miss Selma Granat of the Presbyterian Hospital, Dr. Julius Rudisch of the Mount Sinai Hospital, and Drs. A. Zemansky and W. Weinberger of the Lebanon Hospital for supplying stomach contents of some very interesting cases. NEW TESTS FOR GASTRIC CANCER I7 3 IV. ADDENDUM In an endeavor to perfect my methods further, while the manu- script of this paper was in the hands of the editor, I made observations warranting the following preliminary conclusions: Pepper interferes with the tryptophan-bromin reaction; so does lemon juice. Sugar is an excellent stimulant of the secretion of cancer enzyme; beef is much better than chicken. A good meal for the tryptophan test is one con- sisting of bread and butter, with meat prepared very plainly without extra seasoning or dressing, and some very sweet, weak tea. I made about a dozen tryptophan tests recently on each of two patients (Cases 16 and I7), and, using such meals, obtained uniformly positive results in almost every test. I have secured excellent results by giving a meal consisting of white bread and very sweet weak tea without milk, the meal having been extracted in one hour. This statement is based, however, on tests on only two patients. V. RECORD OF CASES A. Cases with Contents Yielding ti Positive Tryptophan Reaction 1° CASE I. — M. G., male, aged 49. Free hydrochloric acid, 0 ; total acidity, 58. Lactic acid, present. Two tests were made for tryptophan; both were positive. Tryptophan was present in the gastric contents immediately after removal from the stomach. A test breakfast, after lavage of the stomach, failed to yield con- tents giving the tryptophan reaction even after standing several days in the ther— mostat. The patient was operated on and proved to have an extensive carcinoma originating in the pylorus. CASE 2. —— I. 8., male, aged 45. Clear-cut carcinoma. Coffee grounds. Free hydrochloric acid, 0; total acidity, r 36 to I66. Lactic acid, considerable. Im— mediately after removal of contents from the stomach a faint tryptophan reaction was directly obtained. After forty-eight hours in the thermostat, this reaction was more marked. ' CASE 3. -— S. D., male, aged 61. Clear-cut carcinoma involving the pylorus. Marked peristaltic movements. Free hydrochloric acid, 0; total acidity, 6. Lactic acid, considerable. Two tests were made for tryptophan; both were positive. CASE 4. — G. E., male, aged 54. Cancer involving the pylorus. Three tryp- 10 Every test referred to in this record as a tryptophan test, unless otherwise indicated, was a double one — with glycyltrytophan and without it. The results in each case were identical in quality unless otherwise indicated. 174 NEW TESTS FOR GASTRIC CANCER tophan tests were made, with two positive results and one negative. Free hydrochloric acid, 0 to 34; total acidity, 42 to 66. No lactic acid. CASE 5. —- G. E., male. Autopsy: cancer of the pylorus. Five tryptophan tests were made, two with stomach washings. Three Ewald test breakfasts were given. One Ewald tryptophan test was negative; the others were positive. In two tests the direct tryptophan reaction appeared after forty—eight hours’ standing in a thermostat, but not before. CASE 6. —— N. T., female, aged 46. Palpable tumor. Patient died. Clear-cut carcinoma. No free hydrochloric acid in stomach contents. Two tryptophan tests were made with positive results. In one test the reaction was Slight. CASE 7. — S. F., male, aged 73. Cancer of the pylorus. Marked stasis of food. Free hydrochloric acid varied in quantity from a trace to 24; total acidity, 88. Lactic acid, present. Four tryptophan tests were made. Two were positive and two negative. The contents that gave the negative result contained free hydro- chloric acid, 24. CASE 8. ——D. P., female. Cancer of the pylorus and posterior wall. Opera- tion. A test for tryptophan was positive. CASE 9. —— S. N ., male, aged 28. Carcinoma of the lesser curvature. Opera- tion. Free hydrochloric acid, 0; total acidity, 6 to I4. Lactic acid, present. Two tryptophan tests were made; one was positive, one was negative. CASE IO. —— G. H., male, aged 51. Carcinoma ventriculi. Free hydrochloric acid, 0; total acidity, ro. Lactic acid, present. Palpable tumor. A tryptophan test was positive. CASE rr. — S. D., male, aged 52. Carcinoma ventriculi. Marked peristaltic movements. Dilated stomach. Marked stasis. Refuses operation. Free hydro— chloric acid, 26; total acidity, 92. Lactic acid, present. A specimen of stomach contents with the above acidities showed a marked direct tryptophan reaction. On two other occasions, with acidities of free hydrochloric acid, 50 to 72 ; conbined, 24, and total, 108 to I 30, direct tryptophan reactions were positive. One month prior to this, two tryptophan tests were negative. CASE 12. — C. C., female. Suspected carcinoma. Two tryptophan tests; one positive, one negative. CASE 13. -— A. 8., male, aged 36. Free hydrochloric acid, 32; total acidity, 76. This did not appear to be a cancer case. Unfortunately the patient could not be kept under observation. One tryptophan test was made with a positive result. CASE I4. ——A. T., female, aged 33. Material from the stomach contained much bile on repeated examinations. Free hydrochloric acid, 0; total acidity, 20. The patient underwent a gastro-enterostomy for pyloric ulcer two years ago. Non-cancerous case. Two tryptophan tests were made; both positive. CASE I 5. —S. G., male, aged 52. Jejunal ulcer. Gastro-enterostomy four years ago. Non-cancerous. Material from the stomach contained much bile on repeated examinations. Free hydrochloric acid, 0 to 28; total acidity, 24 to 68. One positive tryptophan test was made. CASE 16. — R. K., male, aged 54. Case of suspected carcinoma. Free hydro- NEW TESTS FOR GASTRIC CANCER I75 chloric acid as high as 50; always present. About a dozen tryptophan tests were made and almost all were positive. CASE r7. — I. C., male, aged 46. Clear-cut case of cancer. Palpable tumor. Cachexia. Coffee grounds. Very poor motility. Free hydrochloric acid, 0 ; total acidity, I4. Lactic acid, considerable. A dozen tests for tryptophan were made; all were positive. CASE 18. — L. B., male, aged 65. Patient of Dr. Jerome M. Lynch. Clinical diagnosis by Dr. Lynch, carcinoma ventriculi. Operation by Dr. Lynch about 2% years ago for cancer of the rectum. Free hydrochloric acid, 0; total acidity, r4. Lactic acid, trace. Starch digestion, good. Occult blood, present. One test for tryptophan was made with a positive result. CASE 19. —Mrs. M., aged 54. Patient of Dr. William Weinberger. Un- doubted case of carcinoma. Operation revealed a tumor in the pyloric region. Free hydrochloric acid, absent. Lactic acid, present. Two tests were made for tryptophan by Dr. Weinberger. The first was negative; the second, positive. B. Cases without Free Hydrochloric Acid in the Stomach Contents and not Yielding Positive Tryptophan Reaction CASE 20. —S. L., male. Suspected carcinoma. Free hydrochloric acid, 0; total acidity, 48. Lactic acid, negative. One test was made for tryptophan, but with a negative result. CASE 21. —J. O’H., male, aged 41. Chronic gastritis. Free hydrochloric acid, 0; total acidity, 44—76. Lactic acid, 0. Two tryptophan tests were made; both were negative. CASE 22. — A. C., female, aged 38. Suspected carcinoma. Free hydrochloric acid, 10; total acidity, 34. Lactic acid, 0. Blood in contents. Two tests were made for tryptophan; both were negative. CASE 2 3. — C. R., female. Chronic gastritis. Free hydrochloric acid, 0; total acidity, r4. Lactic acid, 0. Several tryptophan tests were made; all were negative. CASE 24. —— A. N., female, aged 5 5. Achylia gastrica. The patient has been under my observation for the past nine years. Several examinations were made for tryptophan; all were negative. Free hydrochloric acid, 0; total acidity, 6. Lactic acid, 0. CASE 25. ——A. N ., male. Achylia gastrica. Free hydrochloric acid, 0; total acidity, 8. Lactic acid, 0. One tryptophan test was made with a negative result. CASE 26. —R. H., aged 52. Spasmodic stricture of the upper part of the esophagus. Free hydrochloric acid, trace; total acidity, 36. Several tryptophan tests were made, but all were negative. CASE 27. — I. D., male. Chronic gastritis (alcoholic). Free hydrochloric acid, 0; total acidity, r4. Lactic acid, 0. One tryptophan test was made with a negative result. 176 NEW TESTS FOR GASTRIC CANCER CASE 28. —N. F ., male. Suspected carcinoma. Free hydrochloric acid, 0; total acidity, I6. Lactic acid, 0. One tryptophan test was made with a negative outcome. CASE 29. —— R. McM., female, aged 42. Chronic gastritis. Free hydrochloric acid, 0; total acidity, 38. Lactic acid, 0. Three tests were made for tryptophan; all were negative. CASE 30. —— A. M. K., aged 36. Gastroptosis, hypoacidity. Free hydrochloric acid, trace; total acidity, 24. Lactic acid, 0. Two tryptophan tests were made; both were negative. CASE 31.11 —— O. D., female. Diagnosis; achylia gastrica. Free hydrochloric acid, 0; total acidity, 4. _ CASE 32. — E. T., female. Chronic gastritis. Free hydrochloric acid, 0; total acidity, 40. CASE 33. — B. 1., male. Chronic gastritis. Free hydrochloric acid, 0; total acidity, 25. CASE 34. — D. F., female. Diagnosis, achylia gastrica. Free hydrochloric acid, 0; total acidity, 6. CASE 3 5. —B. 5., male. Chronic gastritis. Free hydrochloric acid, 0; total acidity, 20. CASE 36. — D. P., female. Chronic gastritis. Free hydrochloric acid, 0; total acidity, 32. ' CASE 37.—L. L., male. Chronic gastritis. Free hydrochloric acid, 0; total acidity, I6. Lactic acid, 0. CASE 38. —— M. B., female. Achylia gastrica. Free hydrochloric acid, 0; total acidity, 8. C. S Pecial Case, Giving the T ryptophan Test with Glycyltryptophan, but not without it CASE 39. — B. C., male. Chronic gastritis and hyperaeidity, very marked tryptophan reaction with glycyltryptophan ; none without it. D. Cases not Yielding the T ryPtophan Reaction None of the cases in this group gave the tryptophan reaction. The tryptophan test was repeated several times in many cases. The gastric contents from all these cases had free hydrochloric acid. Six cases of hyperacidity. Three cases of Chronic gastric ulcer. Five cases of neurasthenia. 11 At least one test with glycyltrytophan and one without it was made in each of Cases 31—38, with uniformly negative results. NEW TESTS FOR GASTRIC CANCER I77 One case of contraction of gastro-enterostomic orifice four years after operation. Marked stasis of food. Dilated stomach. One case of arteriosclerosis. One case of hypernephroma. Patient died. Three cases of gastric atony, with marked stasis of food. Two cases of gastroptosis. One case of enlarged gall-bladder, probably due to calculi. One case of chronic constipation. 2. THE TRYPTOPHAN TEST FOR CANCER OF THE STOMACH, WITH SPECIAL REFERENCE TO PEP— TIDOLYTIC ENZYME IN THE SALIVA * By J. W. WEINSTEIN I. INTRODUCTION THE attention of the medical profession has recently been drawn to new ways of diagnosing cancer Of the stomach by means of tests for the simplest products of protein digestion. Emerson,1 who was among the first investigators to approach this field, found that “can- cer tissue contains a ferment which, in the thermostat as well as in the stomach, is able to digest proteins beyond the peptone (‘albumose’) stage.” He also Observed that “ this ferment is active in the presence of free hydrochloric acid.” Emerson’s discovery, coupled with the well-known fact that pepsin does not, in the stomach, conduct the cleavage of proteins beyond the stage of peptones, Offered a method of determining whether or not the stomach has been invaded by a cancerous growth. A test for cancerous enzyme, if based on Emerson’s findings, could not be applied, however, with the ease and directness which is desirable in clinical work. II. THE GLYCYLTRYPTOPHAN TEST2 About two years ago Neubauer and Fischer 3 published a new test for cancer of the stomach, which was based on this same principle, * Reprinted from the Journal of the American Medical Association, Oct. 28, 1911, lvii, 1420—1424. 1 Emerson: Deutsch. Arch. f. klin. M ed., Igor—1902, Lxxii, 4T 5. 2 In this paper the phrase “glycyltryptophan test” is invariably employed to indicate the Neubauer and Fischer process, which includes the use of glycyltryptophan. The term “tryptophan test” will be used to designate the author’s method, which does not involve the use of glycyltryptophan. See Weinstein, ]. W.: The Journal A. M. A., Sept. 24, 1910, p. 1085. 3 N eubauer and Fischer: Deutsch. Arch. f. klin. M ed., 1909, xcvii, 499. I78 TRYPTOPHAN TEST FOR GASTRIC CANCER I79 but was applied specifically to the detection of an enzyme having the power of digesting a simple peptid. The substrate selected for this test was glycyltryptophan, a synthetic dipeptid containing the molec- ular radicals of glycin and tryptophan. Glycyltryptophan is readily hydrolyzed into these two amino acids. N eubauer and Fischer selected glycyltryptophan as the peptid substrate for their test because trypto- phan, which can readily be produced from it by enzymolysis, is the amino acid that may be detected in the easiest and most direct manner. In the test, as recommended by N eubauer and Fischer, glycyltrypto- phan is added to a sample of the stomach contents in question, and the mixture is incubated for twenty—four hours. The presence or absence of peptidolytic enzyme is then ascertained by determining, with the aid of bromin water, whether or not tryptophan has been produced. A positive result which, according to Neubauer and Fischer, could not occur through the action of anything in normal gastric juice or con- tents, indicates the presence Of a peptidolytic enzyme and implies the occurrence of carcinoma Of the stomach, whereas, according to the same authors, a negative result indicates the non-occurrence of car- cinoma. Neubauer and Fischer supported their conclusions as to the significance of this test with a series of convincing clinical observations. Naturally, this new test for cancer of the stomach promptly received a great deal of attention and interest. III. THE TRYPTOPHAN TEST I recently published 2 the results of some experiments which showed that the use of glycyltryptophan is superfluous for the detection of tryptophan-producing enzyme in gastric contents. A gastric cancer secretes enzyme which is capable of hydrating proteins into trypto- phan and other amino acids. Gastric contents always contain pro- teoses and peptones that are readily convertible by cancer enzyme into their corresponding simpler peptids and amino acids, including trypto- phan.4 Consequently, it is unnecessary to add either a specific pro- 4 It is not very likely that stomach contents ever contain mixtures of proteins which would wholly fail to yield tryptophan among the products of complete proteolysis. Gastric mucus yields tryptophan imder such conditions. Casein, egg albumin, gliadin, and prac- tically all other ordinary food proteins yield tryptophan in comparatively large propor~ tions. It is probable that the proportion of tryptophan which is produced from the pro— 180 TRYPTOPHAN TEST FOR GASTRIC CANCER tein, or a simple peptid, or glycyltryptophan, as a tryptophan-yielding substrate for the easy detection of cancer protease in stomach contents.4 It was Shown by me that plain stomach contents served more satis— factorily for this purpose than stomach contents mixed with glycyl- tryptophan. Previous observers, among them Erdman and Winternitz,5 Glaesner,6 and Volhard,7 employed stomach contents directly for a similar pur-' pose, but as incubation was not a part of the process in any of their methods, their observations, as well as their findings, have little practical bearing on the method which was recommended in my original paper. IV. WARFIELD’S STUDY OF THE ACTION OF SALIVA ON GLYCYL- TRYPTOPHAN There has been wide difference of opinion regarding the value Of the glycyltryptophan test for gastric carcinoma. A few have com- mended it highly, but a large majority have pronounced it valueless. The main Objection which has been raised against the test is that glycyltryptophan yields tryptophan in a large number Of cases in which no cancer exists, as shown at autopsy and by operation. It has been asserted that most achylia gastrica cases, whether due to carcinoma or not, give a positive reaction with glycyltryptophan. Warfield 8 has lately made observations which -help us to understand the current differences of opinion regarding the value of the glycyl- tryptophan test.9 Warfield has found that the saliva of some people, when not acid (to litmus) to begin with, is capable of decomposing teins in stomach contents, under favorable conditions for the test, is sufficient for detection in all cases except those characterized by practical absence of peptidolytic enzyme. But in such instances glycyltryptophan would also fail to yield detectable proportions of tryp- tophan. Whether the tryptophan that arises from proteins in stomach contents (in the tryptophan. test) is ever decomposed, and sufficiently reduced in quantity (through the continued action of the enzyme), to yield a negative result with bromin water cannot be decided at present. Thus far, however, all observations indicate that no uncertainty is introduced into the test in this way. 5 Erdman and Winternitz: M u'richen. med. Wchnschr., 1903, l, 982. 6 Glaesner: Berl. klin. Wchnschr., 1903, xl, 599. 7 Volhardz Mu'nchen. med. W chnschr., 1903, l, 2129. 8 Warfield: Bull. Johns Hopkins H osp., IgII, xxii, I 50. 9 In referring to “the stages in the complete digestion of coagulable proteins” Warfield states that metaproteins are “commonly called albumoses.” It is evident that in this case albumoses is a misprint for “albuminates.” TRYPTOPHAN TEST FOR GASTRIC CANCER 181 _ glycyltryptophan into glycin and tryptophan. He concluded that swallowed alkaline saliva, when mixed with neutral or faintly acid gastric liquid, imparts to the latter the power Of producing trypto- phan from glycyltryptophan, thus rendering “ the glycyltryptophan test Of no value in the diagnosis of cancer of the stomach.” Warfield has shown that the peptidolytic enzyme in saliva is destroyed by heat.10 V. THE AUTHOR’S CONFIRMATION OF WARFIELD’S RESULTS I have verified Warfield’s findings. Every specimen of saliva, except one, in a total of _eight was found to exert marked peptidolytic power on glycyltryptophan within twenty-four hours. The experi- ments in this connection were performed with glycyltryptophan and saliva directly, as a simple mixture, and also after their mixture with stomach contents, which had either been neutralized or which contained a slight proPortion of acid. Neutralization in these cases was effected with one of the following alkalies: calcined magnesia, sodium bicar- bonate, and sodium hydroxid (I per cent solution). N o attempt was made to ascertain the limit of acidity within which saliva is able to induce the production of tryptophan from glycyl- tryptophan. Warfield states that acidity greater than that of a 0.0 5 per cent hydrochloric acid solution prevents such action. VI. THE SPECIAL SIGNIFICANCE OF WARFIELD’S FINDINGS The important facts which have been disclosed by Warfield explain in part the past difliculties with the glycyltryptophan test. That they render its positive responses particularly unreliable in cancer diagnosis is Obvious, for such results may be due to swallowed saliva. On the other hand, that Warfield’s findings do not disqualify my tryptophan test for cancer Of the stomach is equally clear, since the salivary protease does not produce detectable proportions of tryptophan from the proteins in non-cancerous gastric contents. All investigators of the subject agree on the proposition that cancer secretes enzyme which is capable Of converting protein into amino 1° Dr. Gies, although suggesting the probability of ereptic and tryptic excretion by the salivary glands, thinks it possible also that the tryptophan~producing enzyme in mixed saliva is derived in part from bacteria in the mouth, especially from cavities in carious teeth. He has planned to investigate these possibilities. 182 TRYPTOPHAN TEST FOR GASTRIC CANCER acids. The tryptophan test has all the advantages and none of the disadvantages of the glycyltryptophan test. Warfield’s peptidolytic salivary enzyme is devoid of effect on the tryptophan test. I have repeatedly added saliva to normal stomach contents of different degrees of acidity, but have never been able to detect tryptophan, even after prolonged incubation of the mixtures. In one special instance a specimen of stomach contents that contained no free hydrochloric acid, and which had been carefully neutralized with sodium hydroxid (I per cent solution), was mixed with saliva and keflt in a thermostat for fourteen days. Not a trace of tryptophan could be detected in the mixture at any time during the period. A few protocols, in this and other connections, are appended : — Mrs. S. N., sufiering from chronic gastritis, has been coming to my service at the Vanderbilt Clinic for the past nine years to get hydrochloric acid, which does her good. Examination of stomach contents shows: Free hydrochloric acid, none; combined acid, none; total acidity, 4; reaction to litmus, faintly acid. GLYCY'LTRYPTOPHAN TESTS (1—3) I. Stomach contents, 20 cubic centimeters. Glycyltryptophan, I cubic centimeter.11 Saliva, IO cubic centimeters. = Marked tryptophan reaction in twenty-four hours. 2. Stomach contents, 20 cubic centimeters. Saliva (previously boiled), IO cubic centimeters. Glycyltryptophan, I cubic centimeter. = Marked tryptophan reaction within twenty-f our hours.12 3. Stomach contents, 20 cubic centimeters. Water, IO cubic centimeters. Glycyltryptophan, I cubic centimeter. =Tryptophan reaction; not so marked, however, as before, in twenty-four hours.12 TRYPTOPHAN TEST (4) 4. Stomach contents (neutralized with NaOH), 28 cubic centimeters. Saliva, 20 cubic centimeters. 11 This quantity of glycyltryptophan solution is the volume provided by Kalle & CO. (Biebrich am Rhein) for a single test in accord with the directions of N eubauer and Fischer. The volume is usually a little greater, though it is seldom more than 1.5 cubic centimeters. The exact concentration of the solution is unknown to me. 12 This positive result was due undoubtedly to the enzyme which had been introduced into the gastric contents in the saliva swallowed by the patient. TRYPTOPHAN TEST FOR GASTRIC CANCER 183 Glycyltryptophan, None. Toluol, a few drops. =No tryptophan reaction.“ The effect of saliva on Witte peptone has been tried in experiments of a similar nature — such as are outlined below: — TRYPTOPHAN TESTS (A—B) A. Witte peptone, 2 grams. Saliva (Jacob’s), 10 cubic centimeters. Water, 20 cubic centimeters. Incubation, 7 days (with toluol). = NO tryptophan reaction. B. Witte peptone, 2 grams. Saliva (Kraus’s), 10 cubic centimeters. Water, 20 cubic centimeters. Incubation, 7 days (with toluol). = No tryptophan reaction. VII. THIE AUTHOR’S FURTHER EXPERIENCE WITH THE TRYPTOPHAN TEST I have been employing the tryptophan test for over a year and have found it a valuable Sign in the diagnosis of cancer Of the stomach. The main objection to the Neubauer and Fischer test is the frequent occurrence of positive responses in non-carcinomatous contents. This is not the case with the tryptophan test. On the contrary, in the case of the tryptophan test, negative responses have occurred in some specimens of stomach contents from true cancerous cases. By means of the tryptophan test I was able recently to distinguish all the malignant from the benign states in a series of pyloric Obstruc- tion cases prior to operation. Everybody knows the unusual diffi- culties that beset the endeavor to make a differential diagnosis Of malignant from benign Obstruction of the pylorus, Since all the clinical features are the same in both conditions. I am confident that the tryptophan test outranks every other available laboratory means for the diagnosis of cancer of the stomach. Further investigation is necessary, however, before it can be said 13 This mixture was kept in a thermostat for seven days, but no trace of tryptophan could be detected at any time during that period. 184 TRYP TOPHAN TEST FOR GASTRIC CANCER that one or two positive tryptophan findings in stomach contents absolutely prove the existence of carcinoma. This degree of uncer- tainty is much less, however, than that connected with any other “ Sign of cancer.” Among the “ other signs” are absence of free hydrochloric acid from stomach contents, presence of lactic acid in stomach contents, occurrence Of occult blood in stomach contents or feces, stasis of food, and the results of such newer methods as the Solomon, meiostagmin, and hemolytic tests. Free hydrochloric acid is absent from stomach contents not only in gastric cancer, but also in chronic gastritis, pernicious anemia, exophthalmic goiter, and achylia gastrica. On the other hand, cancer of the stomach, chiefly of the py- loric region, may be accompanied by normal and even hyperacid states of the contents. Lactic acid, an important Sign, frequently fails to occur in the contents of the stomach in cancerous cases, and it is OC- casionally, present in undoubted cases of chronic gastritis. In gen- eral, lactic acid is prone to occur when free hydrochloric acid is absent, and especially, also, when the motor powers of the stomach are im— paired. BoaS-Oppler bacilli are an excellent diagnostic feature of carcinoma ventriculi, but only when present in large numbers. Un- fortunately, they appear only when the invasion of the stomach by the carcinoma is extensive. They are never present unless free hydro- chloric acid is absent and, in general, are not of very common occurrence. Occult blood in stomach contents and in feces occurs in ulcer of the gastrO-intestinal tract, cirrhosis of the liver, and also in cardiac and renal cases that are attended by congestion of the viscera. The Solo- mon, meiostagmin, and hemolytic tests are positive in conditions other than carcinoma. I have made hundreds of applications of the tryptophan test to stomach contents. I have never obtained a positive response in any undoubted non-cancerous case, except such as resulted from gastro- enterostomy, in which the ensuing artificial conditions favored the flow of intestinal contents into the stomach and thus caused the hy- drolysis of the proteins into amino acids by intestinal protease. Whether the. occurrence of tryptophan in the gastric contents af- fords an early Sign of cancer, I cannot say. Thus far I have not diagnosed a case in its early stages by means of the tryptophan test. I have had several cases, however, in which the first Sign of cancer TRYPTOPHAN TEST FOR GASTRIC CANCER 185 of the stomach was a positive response in the tryptophan test —— all other signs failing to give a clear indication. I have lately had an elderly patient (Mrs. O. R.), who suffered from digestive disturbances accompanied by low gastric acidity. Her nutrition and general con- dition were such as to preclude the diagnosis of carcinoma, and I thought I was dealing with a case of chronic gastritis. The tryptophan re- action, however, was repeatedly positive. Four to five weeks later a tumor appeared in the region of the stomach, which grew very rap- idly. The case was then diagnosed by every one who saw the patient as an undoubted case Of cancer of the stomach. - Operation was pro- posed, but the patient disappeared from observation. ' Another experience of mine related to an elderly man (N. F.) who presented himself to my service at the Vanderbilt Clinic with stomach trouble. I Obtained several positive tryptophan tests, and on the strength of these only, as the other features did not clearly indicate malignancy, I advised an operation. This was refused by the patient, who. disappeared from Observation. Three months later I traced him to Mount Sinai Hospital. There an exploratory laparotomy was performed and a lymphosarcoma of the middle of the stomach was found and resected. VIII. ANOMALOUS EXPERIENCES WITH THE TRYPTOPHAN TEST In numerous applications of the tryptophan test I have had anoma- lous experiences with several cases. The stomach contents of one patient (Case 16 of my original paper), a man of 54 with a suspicious history of carcinoma, gave repeated tryptophan tests in the presence of free hydrochloric acid. The patient felt well afterwards for a couple of months, when I lost track of him for a time. Lately, however, I have had the opportunity of again examining him. He is now in good health and apparently free from cancer of the stomach. Another anomalous case was that of a middle-aged woman, who had a typical pyloric obstruction, with marked peristaltic movements and gastrectasia. The tryptophan test was repeatedly positive. The patient refused an operation. Four months later She seemed to be some- what improved, but still had all the stigmata of pyloric obstruction. At that time two tryptophan tests applied to different specimens of I86 TRYPTOPHAN TEST FOR GASTRIC CANCER gastric contents were negative. This patient is now in the hands of a colleague who thinks that her pyloric obstruction is a benign one, because it shows no signs of further increase. We Should not forget, however, that a scirrhous carcinoma is of Slow growth and that, with a very small open passage through the pylorus, and lavage, the patient may hold her own for a long time. Another case of interest in this connection was that of Mrs. L. S., aged 64, who presented a typical history of cancer of the stomach, such as sudden onset Of illness of nine months’ duration, loss Of appetite, considerable loss of flesh and strength, vomiting, and vomiting of coffee grounds. Occult blood was found repeatedly in the stomach contents and feces. An exploratory laparotomy was performed by Dr'. A. A. Berg, who failed to find any abdominal lesion. In this case repeated tryptophan tests were negative. Once, however, it was strongly positive, but in that instance the contents had been ren- dered very faintly alkaline with magnesium oxid, and the resultant proteolysis was undoubtedly due to intestinal protease. It was in- tended, in this procedure, to enhance, if possible, the action of the cancer enzyme through alkalization, an expedient which, as was found, may possibly lead to error. Dr. Berg performed a pylorectomy on another patient, Mrs. 5., whose history and findings pointed strongly to the diagnosis of cancer of the stomach. Several tryptophan tests were positive. The patho- logic report was “ inflammatory,” and not malignant. IX. DISADVANTAGES OF THE TRYPTOPHAN TEST The tryptophan test is inconstant. Tryptophan may appear in the contents from one meal and fail to appear in the contents from the next. This implies inconstancy in the secretion of the carcinoma enzyme, but it does not indicate variability in the reactive tendencies of the reagent in the test. I saw in consultation about three months ago an old lady who had all the stigmata of cancer of the stomach (no palpable tumor, however), and I failed to get a positive tryptophan test in a specimen of stomach contents. Only one test was tried in this case. As a rule, however, several tests of different specimens of gastric contents yield a positive reaction in each case of cancer. TRYPTOPHAN TEST FOR GASTRIC CANCER 187 X. SOURCES OF ERROR IN APPLYING TIIE TRYPTOPHAN TEST There appears to be one important source of error in the tryptophan test, and that is regurgitated intestinal fluid. Boas 14 was the first to obtain duodenal contents from the stomach. He massaged the duodenum toward the stomach and Obtained a variable quantity of bile-stained fluid through a stomach tube. Later Boldyreff,15 at the instigation of Pawlow, introduced olive oil into the stomach through a tube and subsequently extracted the contents. Olive oil has the double effect of Checking gastric secretion and stimulating pancreatic secretion. It also acts as a lubricant, and back flow of intestinal contents is facilitated. By such means, duodenal contents may be secured. This line of work was taken up by a good many investigators, such as F aubel,16 Volhard,17 Molnar,18 Ohrl, and Schittenhelrn,19 and their experiences coincide in the fact that some duodenal secretion may be secured in this manner. The observations of Ohrl and Schittenhelrn indicate that under ordinary conditions spontaneous regurgitation of intestinal contents into the stomach takes place. In considering, however, the degree to which the tryptophan test may be vitiated by erepsin or trypsin, or both, we must not for- get that these enzymes are destroyed by the acid Of gastric contents. Moreover, I have repeatedly kept non-cancerous stomach contents, devoid of free hydrochloric acid, in a thermostat at 40° C. for days and even weeks, without obtaining a positive response in the trypto- phan test. It is obviously necessary, however, in every serious applica- tion of the tryptophan test, to prevent all possible regurgitation Of duodenal contents. To prevent this occurrence the stomach contents should be withdrawn gently, and also without any straining on the part of the patient. For this purpose the aspiration method instead of the expression process should be employed. A tube with thin walls and a bore not more than 10 millimeters in diameter (30 14 Boas: Zentralbl. f. klin. flied, 1889, x, 97. 15 Boldyreff: Zentralbl. f. Physiol., 1904, xviii, 457; also, Arch. f. d. ges. Physiol., 1908, cxxi, 13. 16 Faubel: H ofmez'ster’s Beitr. 2:. Chem. Phys. u. Path, 1907, x, 3 5. 1" VOlhard: Mitnchen. med. Wchnschr., I907, liv, 403. 18 Molnar: Ztschr. f. klin. M ed., 1909, lxvii, 188. 19 Ohrl and Schittenhelm: Zentralbl. f. d. ges. Phys. :1. Path. d. Stoflwechs., 1910, v, 881. I88 TRYPTOPHAN TEST FOR GASTRIC CANCER French) should be selected for the purpose. The patient should be instructed to breathe continuously during the extraction. The manip- ulation should be conducted by one who is skilled in the work and not by an untrained assistant. By disregarding these rules, by letting the patient press forcibly on the tube until he gets blue in the face, some intestinal regurgitation will probably result and may vitiate the test. Vomitus is not reliable for obvious reasons. If, in spite of all these precautions, one obtains a specimen Of stomach contents with a greenish color, and which gives the chemical tests for bile, the speci- men should be discarded and another one secured. Various authors have stated from time to time that occult blood vitiates the glycyltryptophan test. They might also presume that it vitiates the tryptophan test. In my original paper I discussed these improbabilities. I have not ignored the fact that blood contains proteases. I am certain, however, that blood, even when present (in the tryptophan test) in a quantity suflicient to impart a distinct red tinge to the contents, is not able to produce tryptophan in detect- able proportion. I have repeatedly studied this matter, from various points Of view, and have always Obtained negative tests for tryptophan in contents to which moderate amounts of blood had been added —— in one case even after incubation of the mixture for seven days. I have also Obtained negative tryptophan results in such mixtures of glycyltryptophan and blood, even after incubation for the same length of time. Of course, it is likely that the addition of large quantities of blood would effect hydrolysis of associated protein, but in such cases the tryptophan test could not be carried out, for it would be impos- sible to discern the characteristic red color resulting from the addition of the bromin. ' The presence of free hydrochloric acid does not necessarily interfere with the test. I have found tryptophan in several instances Of car- cinoma with high gastric acidities. XI. TECHNIC OF THE TRYPTOPHAN TEST The Test Meal. —— The ordinary Ewald test breakfast is not suited to the test. This has been shown repeatedly. A simple and effective test meal is a glass of water, hot or cold and very sweet, with some TRYPTOPHAN TEST FOR GASTRIC CANCER 189 white bread, or toast or biscuits. Milk may also be added. The contents are extracted in about one hOur. A regular dinner, with extraction after two to four hours, also serves the purpose. Any sub- stance that :is likely to impart color to the contents, such as strong tea, coffee, strawberries, tomatoes, etc., Should be excluded from the test meal. After their extraction a portion of the contents should be filtered and tested directly for tryptophan. If the test is positive, no further treatment is necessary. If the direct test is negative, however, the contents, filtered or unfiltered and without the addition of toluol or other preservative, should be kept in a thermostat for from twenty- four to forty-eight hours, and then tested again for tryptophan. The addition Of a preservative appears to be superfluous, for I have shown repeatedly that the bacteria which develop do not produce tryptophan in a detectable proportion.2o The tryptophan test may often be Ob- tained in positive cases without incubation, by merely keeping the contents at room temperature for from twenty-four to forty-eight hours. Incubation, however, is preferable. The Test for T ryptophan with Bromin Water. —— In completing the tryptophan test, filtered stomach contents Should be employed. A volume equal to 6 or 7 centimeters is satisfactory for the purpose. This volume in a test-tube is treated with a few drops of a 3 per cent acetic acid solution, and then saturated aqueous solution of bromin is added, drop by drop, from a pipette. The appearance Of a reddish violet or rose-red color Shows the presence of tryptophan. If, after the addition Of about 4 drops of bromin water, the expected color does not appear, the mixture Should be allowed to stand for about fifteen or twenty minutes, when the characteristic color may develop. If, by the end of that time, the rose color fails to Show, then more bromin water should be slowly added, drop by drop, until the mixture becomes yellow or until a rose-red color is imparted. If a reddish color is pro- duced, the mixture should be allowed to stand again, when the tinge may grow deeper. Considerable practice is required for the accurate performance of the test in the presence of very minute proportions of tryptophan, because a slight excess of bromin may make the character- 20 I cannot say that this would always be the case, for some bacteria and fungi that I have not thus far encountered may be able to produce detectable proportions of tryptophan under the conditions of the test. 190 TRYPTOPHAN TEST FOR GASTRIC CANCER istic color indistinguishable. Excess of bromin imparts a lemon—yellow color to the mixture. Whenever the reddish color merges into a yellow, we know positively that there is an excess of bromin in the mixture. The opening at the tip Of the pipette from which the bromin solution is dropped should be a very small one. The bromin water should invariably be added drop by drop, and the mixture should be well shaken after each addition. Bromin itself should be handled with great care, for it is extremely irritating to the respiratory mucous membrane. Under no circumstance should plain liquid bromin be mixed with the gastric contents to be tested. An aqueous bromin solution may be kept saturated by re- taining in it a slight excess of the heavy liquid bromin. Acetic acid is added to the mixture to be tested, because the reaction appears at its best in an acid medium. Alkali, by combining with the bromin, prevents the reaction with tryptophan. Almost all stomach contents are acid, and in testing them for tryptophan the addition of acetic acid may not be necessary. In mixtures of very low acidity, however, the addition of acetic acid is especially desirable. I wish, in conclusion, to express my indebtedness to Professor William J. Gies for the use of his laboratories and suggestions in my work.21 21 The following, together with references numbered 2, 3, 8, and 19, constitute a complete bibliography of the glycyltryptophan and tryptophan tests to the time of writing this paper. Abderhalden: Ztschr. f. physiol. Chem., 1909, lxii, I 36. Kohlenberger: Deutsch. Arch. f. klin. Med, 1910, xcix, 148. Lyle and Kober: New York Med. J 0ur., 1910, xci, 1151. Kuttner and Pulvermacher: Berl. klin. Wchnschr., 1910, xlvii, 2057. Ley: Berl. klin. Wchnschr., 1911, xlviii, 119. Oppenheimer: Deutsch. Arch. f. klin. Med, 1910—1911, ci, 293. Pechstein: Berl. klin. Wchnschr., 1911, xlviii, 37 5. Hall and Williamson: Lancet, London, 1911, clxxx, 731. Neubauer and Fischer: Mu'nchen. med. Wchnschr., 191 I , lviii, 674. Ehrenberg: Berl. klin. Wchnschr., 1911, xlviii, 704. 3. THE GLYCYLTRYPTOPHAN AND T RYPTOPHAN TESTS FOR CANCER OF THE STOMACH* By CHARLES H. SANFORD and JACOB ROSENBLOOM MiiLLER’s work 1 suggested that malignant tumors contain one or more proteolytic enzymes. Emerson,2 Fischer,3 and many others have Shown that such is the case. Recently Neubauer and Fischer4 and Abderhalden and his associates 5 have found that malignant growths contain an enzyme capable of splitting certain polypeptids into amino acids. N eubauer and Fischer 4 have introduced, on this basis, the so-called “ glycyltryptophan test ” for carcinoma of the stomach. * Reprinted from the Archives of Internal Medicine, 1912, ix, pp. 445—451 : April 15. (From the Laboratory of Biological Chemistry of Columbia University, at the College of Physicians and Surgeons, and the Wards and Chemical Laboratory of the German Hospital.) Shortly after the appearance of Dr. Weinstein’s first paper in this connection (see Note 7), Dr. Sanford, working at the German Hospital, made several tests with glycyl- tryptophan and with stomach contents direct, with results that did not agree with Dr. Weinstein’s main conclusions. At that time I was an associate in this laboratory on the Crocker Foundation and also, by courtesy of the committee in charge of the Crocker Special Research Fund, pathological chemist at the German Hospital. When Professor Gies was informed that Dr. Sanford’s tests did not agree with Dr. Weinstein’s findings, Professor Gies encouraged us to proceed with, and to extend, the work on the ground that the whole truth in any matter is a prime object of scientific endeavor. We continued, with this encouragement, at the German Hospital and in this laboratory, and have ob- tained the results here recorded. Professor Gies provided glycyltryptophan from the supply with which he furthered Dr. Weinstein’s work and which was obtained under the auspices of the George Crocker Special Research Fund. JACOB ROSENBLOOM, Biochemical Laboratory, College of Physicians and Surgeons. 1 Miiller: Ztschr. f. klin. Med, 1889, xvi, 496. 2 Emerson : Deutsch. Arch. f. klin. Med, 1902, lxxii, 415. 3 Fischer: Deutsch. Arch. f. klin. Med, 1908, xciii, 98. 4 Neubauer and Fischer: Deutsch. Arch. f. klin. Med, 1909, xciii, 499. 5 Abderhalden and Rona: Ztschr. f. physiol. Chem., 1909, 1x, 415; Abderhalden, Koelker, and Medigreceanu: Ztschr. f. physiol. Chem., 1909, lxii, 145; Abderhalden: Ztschr. f. physiol. Chem., 1909, hn'i, 136. 191 192 CRITICISM OF TESTS FOR GASTRIC CANCER Lyle and Kober 6 regarded the glycyltryptophan test as a very good one, especially if more than two tests on every case are carried out. Weinstein 7 has shown that the use of glycyltryptophan is unneces- sary for the detection Of a tryptophan-producing enzyme in gastric contents, since gastric contents always contain proteoses and peptones that are easily convertible by cancer enzyme into their hydrolytic products, among which tryptophan would be included. Therefore it is unnecessary to add a dipeptid as a means for the detection of a cancer enzyme. Weinstein tests for tryptophan in the stomach contents directly and after incubation. Hall and Williamson 8 have lately asserted that tryptophan does not appear in stomach contents with Ewald test-meals either before or after incubation. It does occur before and after incubation when a meat test-meal is given, but does not Show any relation to carcinoma. They conclude that the tryptophan test is valueless if the polypeptid is not added to the gastric contents. Kuttner and Pulvermacher 9 have advocated the use Of SeidenPep- tone and have shown that comparative tests with glycyltryptophan paralleled each other accurately. The tyrosin formed by the cancer enzyme from SeidenpePtone is recognized microscopically. Other studies with the glycyltryptophan test have been carried out by Ley,10 Pechstein,11 Ehrenberg,l2 Oppenheimer,13 Kohlenberger.14 Further studies have been conducted by N eubauer and Fischer,15 who found that 84 per cent of gastric cancer verified by autopsy gave positive reactions with the glycyltryptophan test; also that 75 per cent of the clinical cases Of gastric cancer, as well as 15 per cent of stomach conditions that were not cancerous, gave the test. One of the most striking things about the glycyltryptophan test has 6 Lyle and Kober: New York Med. Jour., 1910, xci, 1151. 7 W einstein: J our. Am. Med. Assn., 1910, IV, 1085. (Reprinted in this volume, p. 161.) 8 Hall and Williamson: Lancet, London, 1911, clxxxi, 7 31; J our. Path. and Bacteriol., 1910, xv, 352. 9 Kuttner and Pulvermacher: Bert. klin. W chnschr, 1910, xlvii, 2057. 1° Ley: Berl. klin. W chnschr., 1911, xlviii, 119. 11 Pechstein: Berl. klin. Wchnschr., 1911, xlviii, 375. 12 Ehrenberg: Bert. klin. Wchnschr., 1911, xlviii, 704. 13 Oppenheimer: Deutsch. Arch. f. lelin. Med, 1910—1911, ci, 293. 14 Kohlenberger: Deutsch. Arch. f. klin. Med, 1910, xcix, I48. 15 Neubauer and Fischer: Mu'nchen. med. Wchnschr., 1911 , lviii, 674. CRITICISM OF TESTS FOR GASTRIC CANCER '193 been the wide difference Of opinion regarding its value. Some pro- nounce it very good; others maintain that it is valueless and that glycyltryptophan in stomach contents yields tryptophan in a large number of cases in which no' cancer exists. The wide divergence of opinion may now be explained on the basis of Warfield’s 16 findings, who has Shown that the saliva of several persons, when not materially acid (to litmus) to begin with, has the power of decomposing glycyltryp- tophan into its constituent amino acids. Warfield’s results make it evident that swallowed neutral or alkaline saliva (to litmus), when mixed with neutral or faintly acid gastric contents, imparts to the latter the power of producing tryptophan from glycyltryptophan, thus rendering the glycyltryptophan test of doubtful value in any instance, if not wholly vitiating it. Weinstein 17 has verified Warfield’s findings, and concludes that although Warfield’s results discredit the glycyltryptophan test, they do not depreciate fhe value of the “ tryptophan ” test, because the salivary peptidase does not produce tryptophan from the proteins in non—can- cerous gastric contents. Our results with the glycyltryptophan and tryptophan tests are presented on the following pages. The tests were carried out as follows: One to two hours after the Ewald test-meal or a full meal, the stomach contents were withdrawn by Siphonage. Undue manipulation of the tube was avoided, for in- tragastric movement may lead to the reflex regurgitation of duodenal liquid. The stomach contents were filtered and tested for blood, bile, and tryptophan. One part of the filtrate was added to a solution of glycyltryptophan and incubated for twelve, twenty-four, or forty- eight hours at from 38 to 400 C. Another portion of the filtrate was incubated directly. Some Of the glycyltryptophan solution used for the test also was incubated, and thus served as a control for free trypto- phan in the glycyltryptophan solution used for the test. Each portion Of the incubated solutions was finally acidified with acetic acid (3 per cent) and then bromin water was added drop by drop. In the pres- ence of free tryptophana lilac color develops when a sufficient amount Of bromin water is added. The color disappears in a pronounced 16 W arfield: Bull. Johns Hopkins H osp., 1911, xxii, 15o. 1" Weinstein: J our. Am. M ed. Assn., 1911, lvii, I420. 1761 HEIONVO OIHISVS HOII SISEII. .IO WSIOIIRIO TABLE I EIGHTY TESTS CARRIED OUT ON THIRTY-EIGHT PATIENTS N 0. DATE EWALD FULL GLYCYL— CONTENTS CLINICAL STOMACH CON £EN'1‘S REMARKS CASE 1910 TEST- MEAL TRYPTO- AFTER IN- DIAGNOSIS TOTAL FREE . . . . . . .. . . . . . . . . .. MEAL PHAN CUBATION, ACIDITY HCl . . . . . . . . . . . . . . . . . . .. TEST TRYPIO- . . . . . . . . . . - - - - . . . . . . . . . .. PHAN .......... ..... .....-.. . . . . . . . . . . . . . . . . . . . . . . . .. TEST 1 Io/12 + . - —- Pernicious anemia 30 o . . . . . . . . . . . . 10/14 - - + —- Pernicious anemia 3 5 0 . . . . . . . . . . . . 2 10/ 12 + . - — Rheumatic myositis 3 5 0 . . . . . . . . . . . . 10/14 - - + — Rheumatic myositis 40 0 . . . . . . . . . . . . 3 10/13 + . . — Mass in right hypochon. region Mass is suggestive of carcinoma IO/ I 5 - - + —- Mass in right hypochon. region. . . . . . . . . . . . . 4 10/ I4 + . . — Pulmonary tuberculosis . . . . . . . . . . . . 10/ 15 - - + — Pulmonary tuberculosis . . . . . . . . . . . . 5 10/14 + . . Cholelithiasis . . . . . . . . . . . . 10/15 - - + . . —- Cholelithiasis . . . . . . . . . . . . . . . . 6 * 10/14 + + + Gastric crisis of tabes 90 40 Laparotomy showed a dilated stomach IO/IS - ~ + -l- + Gastric crisis of tabes 85 50 . . . . . . . . . . . . 10/ 29 + - —- Gastric crisis of tabes 80 35 At this time patient was free of the gastric pains ' pain. * Stomach contents from Case 6 gave positive glycyltryptophan and tryptophan tests during the time the patient had severe gastric On account of the clinical diagnosis an exploratory operation was carried out, which showed only a dilated stomach. Two weeks later, when the patient was free from gastric pain, the tests were not Obtained. The patient was then found to have locomotor ataxia, and we think the positive tests were due to the regurgitation of duodenal liquid (although the stomach contents gave no reaction for bile) on account of the violent gastric pain. HEIONVO ORIJ'SVO HOE SILS'EII .iIO NSIOIIIHO 561 TABLE I (Continued) No. DATE EWALD FULL GLYCYL- CONTENTS CLINICAL STOMACH CONTENTS REMARKS CASE 1910 TEST- MEAL TRYPTO- AFTER IN- DIAGNOSIS TOTAL FREE ...... . . . . . . . . . . .. MEAL PHAN CUBATION; Acmrrx HCl . . . . . . . . . . . . . . . . . . .. TEST TRYPTO- . . . . . . . . . . . . - . . . . . . - . . . .. PHAN .......-.. ..... H... ........ . . . . . . . . . . . . . . . . . . . . . . . .. TEST 10/30 + . — Gastric crisis of tabes 85 48 At this time patient was free of the gastric pains 7 10/16 —|- . . — Gastric neurosis . . . . . . . . . . . . 10/17 . . + — Gastric neurosis . . . . . . . . . . . . 8 10/17 —|— . . —- Gastric neurosis . . . . . . . . . . . . 10/ 18 . . + - Gastric neurosis . . . . . . . . . . . . . . 9 10/17 + . . - Cirrhosis of the liver 60 20 . . . . . . . . . . . . 10/18 . . + - Cirrhosis of the liver 50 10 . . . . . . . . . . . . 10 10/ 20 + . . - Gastric neurosis 90 30 . . . . . . . . . . . . 10/ 21 . . + — Gastric neurosis 80 30 . . . . . . . . . . . . II 10/ 20 + — - Carcinoma of stomach 4o 0 Operation showed carcinoma at the pylorus 10/ 22 + — — Carcinoma of stomach 3o 0 . . . . . . . . . . . . 12 10/ 22 + - — Carcinoma of stomach 3o 0 Autopsy showed carcinoma of the stomach 10/ 2 3 . . + — -— Carcinoma of stomach 4o 0 . . . . . . . . . . . . I3 10/ 2 3 + . . - Carcinoma of uterus 50 10 . . . . . . . . . . . . 10/ 24 . . + — Carcinoma of uterus 6o 25 . . . . . . . . . . . . 14 10/23 + . . - Carcinoma of uterus . . . . . . . . . . . . 10/ 24 . . + — Carcinoma of uterus . . . . . . . . . . . . 15 10/25 + . . — Pneumonia . . . . . . . . . . . . 10/ 26 . . + — Pneumonia . . . . . . . . . . . . 16 IO/ 2 5 + . . — Typhoid fever . . . . . . . . . . . . 10/ 26 + - Typhoid fever . . . . . . . . . . . . 961 HEIONVD OIHILSVS HOE SISEII. cIO WSIOIIIHO . . . . . . . . . . . . . . . . . . . . . . . .. TEST I7 10/ 27 + — — Carcinoma of stomach 40 10 Operation showed an old ulcer with constriction of the pylorus 10/ 28 . . — — Carcinoma of stomach 45 20 . . . . . . . . . . . . 10/30 — Carcinoma of breast . . . . . . . . . . . . . . . . 20 1 1/1 2 — — Carcinoma of stomach 65 0 Operation showed extensive car- cinoma Of the stomach 11/13 . . + — — Carcinoma of stomach 6o 0 . . . . . . . . . . . . I 2/30 + - Gastroptosis 40 0 . . . . . . . . . . . . 24 1 2 / 29 + . . — Atonic dilation of stomach . . . . . . . . . . . . 12/30 + — Atonic dilation of stomach . . . . . 1911 25 1/ 5 + + + Hypernephroma 70 20 Diagnosis verified at operation 1/ 6 + + Hypernephroma 80 2 5 . . . . . . . . . . . . " 26 1/ 2 . . — Gastroptosis 60 20 . . . . . . . . . . . . 1/ 3 + — Gastroptosis 70 30 . . . . . . . . . . . . TABLE I (Continued) No. DATE EWALD FULL GLYCYL- CONTENTS CLINICAL Srom ACH CONTENTS REMARKS CASF 1910 TEST- MEAL TRYPTO— ASTER IN- DIAGNOSIS TOTAL FREE . . . . . . .. . . . . . . . . .. MEAL PHAN CUEATION; ACIDITY HCl . . . . . . . . . . . . . . . . . . .. TEST TRYPTO- . . . . . . . . . . . . . . . . . . - - . . . .. PHAN ..... 18 10/ 28 + — — Carcinoma of the uterus . . . . . . . . . . . . 10/ 29 — Carcinoma of the uterus . . . . . . . . . . . . 19 10/ 29 —- — Carcinoma of breast . . . . . . . . . . . . 21 11/1 2 + . . — — Carcinoma of stomach 80 0 . . . . . . . . . . . . 11/13 + — — Carcinoma of stomach 70 0 . . . . . . . . . . . . 22 1 2 / 29 . . - Gastroptosis 40 0 . . . . . . . . . . . . 23 12/ 28 . . — — Carcinoma of stomach 3 5 0 . . . . . . . . . . . . 12/29 + — Carcinoma of stomach 40 0 . . . . . . . . . . . . 1 2 / 30 . . + — Carcinoma of stomach 4o 0 . . . . . . . . . . . . XHONVO OIHISVO HOE SIS'SII JO WSIOIIIHO L61 TABLE I (Continued) No. DATE EWALD FULL GLYCYL- CONTENTS CLINICAL STOMACH CONTENTS REMARKS CASE 1911 TEST- MEAL TRYPTO- AFTER IN- DIAGNOSIS TOTAL FREE .... . . . .. . . . . . . . . .. MEAL PHAN CUBATION; ACIDITY HCl . . . . . . . . . . . . . . . . . . .. TEST TRYPTO- . . . . . . . . . . . . . . . . . . . . . . . .. PHAN .......-.. ..... ..... ........- . . . . . . . . . . . . . . . . . . . . . . . .. TEST 27 1/ 5 + - - -- — Carcinoma of stomach 40 0 . . . . . . . . . . . . 1/ 6 - - + — — Carcinoma of stomach 3 5 0 . . . . . . . . . . . . 28 I/ 7 . + - - —— Pulmonary tuberculosis 40 0 . . . . . . . . . . . . I / 8 - . + —- Pulmonary tuberculosis 50 0 . . . . . . . . . . . . 29 1/ 7 + . . -- Lead poisoning 60 0 . . . . . . . . . . . . I/ 8 ~ + —— Lead poisoning 50 c . . . . . . . . . . .. 3o 1/ 8 + .. —- Lymphatic leukemia 50 20 . . . . . . . . . . .. 1/ 9 . . + —- Lymphatic leukemia 45 20 . . . . . . . . . . . . 31 1 / 20 + - - — —- Carcinoma of stomach 4o 0 . . . . . . . . . . . . I / 21 - - + - - Carcinoma of stomach 50 0 . . . . . . . . . . . . _ 32 1 / 29 + - - — Carcinoma of stomach I 5 0 At operation extensive carcinoma of stomach. Inoperable. 1 / 30 - - + —- —- Carcinoma of stomach 20 0 . . . . . . . . . . . . 33 2/10 + - - -- — Carcinoma of stomach 30 0 At operation extensive carci- noma of stomach 2/12 - - + — Carcinoma of stomach 40 o . . . . . . . . . . . . 2/ 16 + . . . . —- Carcinoma of stomach 70 0 . . . . . . . . . . . . 34 2/17 + + + Carcinoma of stomach 40 0 Operation showed carcinoma of stomach 2/18 - - + . . + Carcinoma of stomach 50 0 . . . . . . . . . . . . 35 2 / 20 + - - + + Carcinoma of stomach 45 0 Operation showed carcinoma of stomach 2 / 21 . . + + Carcinoma of stomach 30 0 . . . . . . . . . . . . 36 2 / 24 + - - — Carcinoma of stomach 46 0 Operation showed carcinoma of stomach 2 / 2 5 - - + — —- Carcinoma of stomach 50 0 . . . . . . . . . . . . 37 3/ 6 + . . ' — Carcinoma of stomach 46 0 , Operation showed carcinoma of stomach 3/ 7 . . + — - Carcinoma of stomach 40 0 . . . . . . . . . . . . 33 3/ 9 + . . Carcinoma of stomach 50 0 Operation showed carcinoma of stomach 3/ 9 + — — Carcinoma of stomach 46 o . . . . . . . . . . . . .- 198 CRITICISM OF TESTS FOR GASTRIC CANCER yellow when the bromin is present in excess, whereas in the absence of free tryptophan, the fluid acquires a pale yellow tint.18 Table I contains the results of eighty tests carried out on thirty- eight patients.19 We carried out at least two tests in each case, after an Ewald test-meal and a full meal, respectively. Table II summarizes the results that we have Obtained with the tryptophan and glycyltryptophan tests: —— TABLE II SUMMARY OF RESULTS OBTAINED WITH TRYPTOPHAN AND GLYCYLTRYPTOPHAN TESTS POSITIVE NEGATIVE Glycyl- Twp- Glycyl- p- tryptophan tophan tryptophan top an Cancer of the stomach, verified by opera- tion or post mortem . . . . . 2 2 9 9 Cancer of the stomach, clinical diagnosis * — — 5 5 Other diseases than cancer of the stomach 2 2 — 20 * Case 17, which clinically suggested carcinoma of the stomach, gave negative tests and operation showed an Old ulcer with constriction of the pylorus. Dr. Weinstein has reported a few results which led him to conclude that the peptidolytic enzyme in saliva is unable to produce tryptophan from Witte peptone, even after seven days’ incubation in the presence of toluol. We have found, on the contrary, that the salivary peptidase may produce tryptophan from Witte “ peptone ” and also from casein. We have also observed that this enzyme was absent from some speci- mens of saliva which were not acid to litmus to begin with. From these Observations it is hard to believe that the presence of peptidase in swallowed saliva vitiates the glycyltryptophan test without affect-- ing the validity of the tryptophan test. One would think that both 18 The incubated material should be filtered before testing for tryptophan, especially when a small amount of trytophan has been produced. We have found that the unfiltered material may react negatively, when, if filtered, the filtrate gives a faint reaction fof tryp- tophan. Dr. Weinstein has also emphasized this point. 19 We have never obtained a reaction for tryptophan in the stomach contents of any of this series of cases immediately after the contents were withdrawn, i.e. before incubation. CRITICISM OF TESTS FOR GASTRIC CANCER 199 tests are thus rendered unreliable, for proteoses and peptone are natu- rally present in the stomach contents after all test-meals. We have also found that stomach contents from three individuals (two of them children), in whose cases there was no doubt that the stomachs were entirely free from carcinoma, contained tryptophan after incuba- tion with saliva. The positive reactions could not be Obtained in these cases, unless the gastric contents were made neutral to litmus before the addition of the saliva, a result which may be explained by the fact that acidity prevents the salivary enzyme from acting. In two other specimens of normal stomach contents, tryptophan was not produced when the tests were carried out in the same way as for those cases in which we obtained a positive reaction. Dr. Weinstein states, in a footnote in his last paper, that “ Dr. Gies, although suggesting the probability of ereptic and tryptic excre- tion by the salivary glands, thinks it possible also that the tryptophan- producing enzyme in mixed saliva is derived in part from bacteria in the mouth, especially from cavities in carious teeth. He has planned to investigate these possibilities.” The variability Of the results to which we refer in the preceding paragraph accords with this view of the situation. We agree with Dr. Weinstein that the use of glycyltryptophan is unnecessary for the detection Of tryptophan—producing peptidase in gastric contents, but our findings do not permit us to believe that the tryptophan test is a reliable one for gastric carcinoma. 4. THE IMPORTANCE OF THE COLLOIDAL NITROGEN OF THE URINE IN THE DIAGNOSIS OF CANCER * By MAX EINHORN, MAX KAHN, and JACOB ROSENBLOOM SALKOWSKI 1 and Hess and Saxl 2 have recently noted the fact that certain alcohol-precipitable nitrogenous substances are increased in the urine of patients suffering from malignant disease. The proposed methods for the estimation of these substances are too laborious for clinical utility. Lately Salkowski and his pupil, Kojo,3 have elaborated a simpler method of obtaining Such nitrogenous substances,l so that now this increase in the colloidal nitrogen of the urine can be used for diagnostic purposes. It is hardly necessary to discuss the great importance Of improved means of diagnosis in cancerous conditions, especially at an early period of the growth. In fact, this is the burning question of the day: “ the early diagnosis of cancer.” A great many lives depend upon the early discovery of this condition, at a time when operation is still feasible. It therefore appeared to us appropriate to take up this subject and study the results Of Salkowski’s new test on many patients. Our patients were selected from those suffering from a distinct cancer- ous condition, from such as were troubled with other affections which, in some of the symptoms, resembled malignant disease. We also included several normal individuals and a few febrile conditions, as well as other chronic ailments, in our investigation, in order to determine the results of this test under the most varied conditions of health and disease. - * Reprinted from the American Journal of Gasiro-enterology, June, 1911. (From the Laboratory of Biological Chemistry of Columbia University, at the College of Physicians and Surgeons, and from the German Hospital, New York.) This paper was also published in the Archiv fur Verdauungs-Krankheiten, 1911, xvii, pp. 3 57—361. 1 Salkowski: Berl. klin. Woch., 1910, xlvii, p. 1746. 2 Hess and Saxl: Beit/ra'ge zur Carcinomforschung, 1910, Heft II. 3 Salkowski and Kojo: Berl. klin. Woch., 1910, xlvii, p. 2297. 200 URINARY COLLOIDAL NITROGEN 201 The method for obtaining the colloidal nitrogen is conducted as follows : — T o 100 cubic centimeters of the well-mixed, filtered, 24-hour speci- men of urine, zinc sulphate is added in sufficient quantity to effect saturation.4 The saturated liquid is allowed to stand for twenty- four hours. It is then filtered through an ashless paper, and the pre- cipitate is washed five times on the paper with saturated zinc sulphate solution.5 The paper and precipitate are then placed in a Kjeldahl flask and the nitrogen content determined by the Kjeldahl method. The nitrogen in 5 cubic centimeters of the urine is also determined by the Kjeldahl method, and the ratio of the nitrogen in the zinc sulphate precipitate to the total urinary nitrogen is computed.6 TABLE I * NORMAL CASES TOTAL COLLOIDAL COLLOIDAL NAME OF NITROGEN IN NITROGEN 1N NITROGEN As NO- PATIENT DATE DIAGNOSIS 100 CC. or 100 CC. or PER CENT OF UR INF. URINE TOTAL NITROGEN Gram Gram 1 Dr. S. I, 23 Normal .7951 .0102 1.2 2 Dr. S. I, 26 Normal .9352 .0174 1.8 3 Dr. R. I, 22 Normal .8260 .0164 2.0 4 ' Dr. R. 1, 24 Normal .7986 .0138 1.73 5 Dr. B. 1, 28 Normal .9420 .0201 2.13 6 Dr. B. I, 29 Normal .8976 .01968 2.19 7 Dr. C. 1, 30 Normal .9120 .0178 1.95 8 Dr. C. I, 31 Normal .8870 .0172 1.94 9 Dr. K. 2, 26 Normal .6048 .013 56 2.2 *The table contains the data of 9 urinary examinations of 5 normal individuals. The accompanying tables contain the results we have obtained with the above method. 4 The urine is first tested for protein, which, if found, is removed by means of heat coagulation after the addition of a few drops of dilute acetic acid solution. 5 We have found that this washing is sufficient to remove other nitrogenous substances that would be present in the precipitate. After the washing process no nitrogen is evolved when the precipitate is put in a nitrometer and decomposed by means of hypobromite solution. 6 We have also compared “this precipitation with the lead subacetate precipitation as described by Salkowski, and found that the method gave almost the same results. 202 URINARY COLLODAL NITROGEN The nitrogen values for the zinc sulphate precipitate as compared with the total nitrogen (Table I) varied from 1.2 per cent as a minimum to 2.2 per cent as a maximum, with an average of 1.9 per cent of the total nitrogen. Salkowski and Kojo found in the normal cases which they investi- gated in this connection that 1.75 per cent was the average for normal individuals by the zinc sulphate method. In the results (Table II) for thirty-five urinary examinations of cases of cancer (21 cases of carcinoma, 2 sarcoma, and 1 case of hyper- nephroma) the per cent of the total nitrogen of the urine present as colloidal nitrogen was much larger than the normal average in all cases except those numbered 18, 32, 43, and 44. Even in those cases the colloidal nitrogen was slightly increased. The values for the per cent of colloidal nitrogen in the total nitrogen varied, in this series of cases, from 2. 3 per cent as a minimum to 8.5 per cent as a maximum, with an average of 4.5 per cent. Salkowski and Kojo, in the series of cancer cases studied by them, found 2. 53 per cent as a minimum and 4.26 per cent as a maximum. The results in Table III Show that the colloidal nitrogen is also increased in the urine of other diseases than cancer, as shown by cases 45—49, 50, 53, 56, 58—60, 62—65, and 67—68. _ Case No. 69 is particularly interesting on account of the fact that the clinical diagnosis fluctuated between cancer of the liver and a benign affection of the liver; the low per cent of colloidal nitrogen (1.5 per cent) seemed to point to a benign affection, a diagnosis which was later Shown to be correct. Our data for normal individuals Show that the percentage Of colloidal nitrogen in the total urinary nitrogen varied from 1.2 per cent as a minimum to 2.19 per cent as a maximum, with an average of 1.9 per cent. For the different cases of disease the per cent of colloidal nitrogen is small: a minimum of 1.1 per cent and a maximum of 2.1 per cent, with the exception Of one case-of each of the following dis- eases: chronic myocarditis, with lack of compensation; aneurysm of the aorta, tuberculosis of the kidney, cerebrospinal syphilis, progres- sive muscular atrophy, renal stone, typhoid fever (second week Of the disease), diabetes mellitus (two cases), cellulitis of the hand, acute lymphatic leukemia, and pernicious anemia. In one case of chronic URINARY COLLOIDAL NITROGEN 203 TABLE II* CASES OF MALIGNANT DISEASE TOTAL COLLOIDAL Cgummn NO_ NAME OF DATE DIAGNOSIS NITROGEN NITROGEN AS égioéigT PA IIENT ‘ IN 100 cc. IR 100 cc. or URINE 0F URINE OF TOTAL NITROGEN Grams Gram 10 Dr. W. 1, 26 Cancer of oesophagus 1.2600 .07140 5.6 11 Dr. W. 1, 26 Cancer of bladder .8232 .04424 5.3 12 Mr. B. I, 24 Cancer of pelvic glands .6048 .02828 4.6 13 Mr. C. I, 24 Cancer of stomach .7214 .04872 6.7 I4 Mr. B- I, 23 Cancer of pelvic glands .7516 .04060 5.4 15 Mr. B. I, 24 Cancer Of stomach 1.3066 .03660 3.2 16 Mr. L. I, 25 Cancer of stomach 1.1797 .04780 4.05 17 Mr. B. 1, 27 Cancer of stomach .9072 .04560 5-°3 18 Mr. L. 1, 26 Cancer of stomach 1.2544 @2912 2-3 19 Mr. B. 1, 28 Cancer of stomach 3904 @3556 3-9 20 Mr. R. 1, 23 Cancer of stomach .9576 .08230 8. 5 21 Mr. C. 1, 29 Cancer of rectum 1.2992 ‘07340 5~6 22 Mr. K. 1, 18 Cancer of bladder 1.0584 .06272 5.9 23 Mrs. P. 1, 16 Cancer of uterus .9352 03416 3-5 24 Mr. Z. 1, 14 Cancer of stomach $064 @4820 5-9 25 Mr. G. 1, I2 Carcinomatosis #896 @3136 3-9 26 Mr. K. I, 18 Cancer of stomach 1.1928 .05236 4-3 27 Mr. S. 1, 26 Cancer of rectum .4872 .02856 6.06 28 Mr. R. I , 30 Cancer of stomach .9632 .06880 7.10 29 Mr. S. I, 28 Cancer of rectum .4424 .02070 4.6 30 Mrs. P. - 1, 30 Cancer of uterus 1.0024 .04060 4.05 31 Mr. S. 1, 29 Sarcoma of thigh 1.0584 .05210 4.9 32 Mr. D. 2, 2 Sarcoma of thigh 1.3160 .03024 2.3 33 Mr. K. 2, 4 Cancer of rectum 1.1088 .05376 4.8 34 Mr. S. 2, 3 Cancer of prostate .8129 .03864 4.7 35 Mr. K. 2, 3 Cancer of stomach .9240 .03472 3.7 36 Mr. S. 2, 2 Cancer of stomach .5264 .01876 3.5 37 Mr. S. 2, 4 Hypernephroma .9576 .03444 3.6 38 Mrs T 1, 27 Cancer of breast .8288 .02604 3.1 39 Mr. K 1, 6 Cancer Of stomach 1.1928 .05236 4. 3 40 Mr. J. 2, 12 Cancer of liver .8008 .03136 3.9 41 Mr. B. 2, 12 Cancer of stomach .9128 .04116 4. 5 42 Mr. S. 2, 14 Cancer of stomach .6664 .02828 4.2 43 Mr. K. 2, 14 Cancer of stomach 1.0236 .02688 2.6 44 Mr. M. 2, 14 Cancer of stomach .9744 .02268 2.3 * REMARK: Table II embodies the results of 35 urinary examinations of 24 patients with cancer of different organs. 204 URINARY COLLOIDAL NITROGEN. gastritis, at the height of an acute attack, the proportion of colloidal nitrogen was 2.6 per cent of the total urinary nitrogen, while two weeks later it was only 1.2 per cent. Case No. 55 (benign rectal stric- ture), with 1.8 per cent of colloidal nitrogen, is interesting when com- pared with cases Nos. 21, 27, 29, and 33 — all Cases of cancer of the rectum in which the colloidal nitrogen is greatly increased. TABLE III * VARIOUS DISEASES (NOT CANCEROUS) TOTAL COLLOIDAL NAME OF NITROGEN (1212:2215; NITROGEN As PATIEM DATE DIAGNOSIS 18:18:15;- m 100 CC. ZiRTgifLr OF URINE NITROGEN Grams Gram Miss L. 1, 20 Chronic myocarditis 1.1899 .0428 3.6 Miss L. 1, 23 Chronic myocarditis .81732 .03977 4.8 Miss L. 1, 24 Chronic myocarditis .9763 .0367 3.7 Miss L. 1, 26 Chronic myocarditis 1.1984 .0344 2.9 Miss L. 2, 28 Chronic myocarditis .5376 .0344 6.4 Mr. H. 2, 4 Chronic gastritis .6832 .0179 2.6 Mr. H. 2, 28 Chronic gastritis 9 1.0472 .01356 1.2 Mr. S. 2, 4 Alcoholic gastritis 1.1088 .01288 1.1 Mr. W. 2, 8 Aneurysm of the aorta 1.0864 .03388 3.1 Mr. M. 2, 20 Syphilis aneurysm .6832 .01484 2.1 Mr. G. 2, 22 Benign rectal stricture .7296 .01708 1.8 Mr. G. 2, 14 Tuberculosis of the kidney .5096 .01316 2.5 Mr. A. 1, 20 Pulmonary tuberculosis .7784 .0154 1.9 Mr. M. 2, 8 Cerebrospinal syphilis 1.136 .03416 3.0 Mr. P. 2, 8 Progressive muscu. atrophy .07168 .02744 3.8 Mr. G. I, 28 Renal stone 1.204 .0426 3.5 Mr. K. 1, 26 Typhoid fever(convalesc’t) 1.2208 .0272 1.7 Mr. S. 2, 18 Typhoid fever (2d week) .8904 .04844 5.4 Mr. K. 2, 12 Diabetes mellitus 1.4672 .0655 4.4 Miss K. 2, 10 Diabetes mellitus 1.4602 .06272 4.2 Mr. L. 1, 20 Cellulitis of the hand .7056 .02072 2.9 Mr. F. I, 22 Influenza 1.6464 .03446 2.0 Mr. M. 2, 15 Acute lymphatic leukemia 1.5400 .06 5 52 4.2 Mr. F. 2, 24 Pernicious anaemia .9184 .05096 5.5 Mr. S. 2, 24 Hypertrophic cirrhosis of the liver .8344 .1232 1. 5 Mr. B 2, 28 Varicose ulcers of the leg 1.1008 .01176 1.08 * REMARK: The data in Table III relate to 26 urinary specimens from 21 cases. URINARY COLLOIDAL NITROGEN 20 5 In Table II (cancer cases) the proportion of colloidal nitrogen in the total urinary nitrogen is always higher than the normal, varying from 2.3 per cent to as much as 8. 5 per cent in one case, while the great ma- jority show from 3. 5 per cent to 5 per cent, with an average of 4.5 per cent for the series of 24 cases. The high figures for the per cent of colloidal nitrogen in the total urinary nitrogen which have been found for all of the 24 malignant cases are certainly of great‘significance. The other diseases for which we have found a high colloidal nitrogen value can, as a rule, be easily differentiated from cancer. It is probable, therefore, that this new test may prove to be a real aid in the diagnosis of cancerous affections 5. THE COLLOIDAL NITROGEN IN THE URINE FROM A DOG WITH A TUMOR OF THE BREAST* By MAX KAI-IN and JACOB ROSENBLOOM IN 1892 TOpfer1 found that the urine of patients suffering from cancer contained a very large amount of* “extractive substance.” This “ extractive substance” he calculated by first determining the total nitrogen, and then subtracting from this amount the sum Of the nitrogen values for the urea, uric acid, and ammonia contained in the same urine. Bondzynski and Gottlieb,2 five years later, reported that the oxyproteic acid nitrogen was 2 to 3 per cent of the total nitrogen. Salkowski,3 and Hess and Saxl,4 using different procedures in their researches, came to the conclusion that the oxyproteic acid or. the alco- hol-precipitable substances are increased in the urine of human beings suffering from carcinoma. Salkowski and Kojo,5 in a preliminary communication, recently suggested several methods for the determination of colloidal nitrogen in the urine. A year later Kojo published the results of a comparative study of the various procedures suggested in this connection.6 Ein- horn, Kahn, and Rosenbloom7 studied the zinc sulphate-precipitable, colloidal, nitrogenous material from the urine of normal subjects as well as from the urine Of carcinomatous patients, and came to the con- clusion that the amount of colloidal nitrogen was invariably increased in subjects with carcinomatous growths. The writers lately embraced * Reprinted from the Biochemical Bulletin, 1912, ii, p. 87. 1 TOpfer: Wiener klin. W 0chenschrift, 1892, v, p. 49. 2 Bondzynski and Gottlieb: Zcntralbl. f. d. med. Wissen., I897, xxxv, p. 577. 3 Salkowski: Berliner klin. W och., 1910, xlvii, p. 1746. 4 Hess and Saxl: Beitrage zur Carcinomforschung, 1910, Part II. 5 Salkowski and Kojo: Berl. klin. W och., 1910, xlvii, p. 2297. 6 Kojo: Zeitschr. f. physiol. Chem., 1911, lxxiii, p. 416. 7 Einhorn, Kahn, and Rosenbloom: Amer. J ourn. of Gastro-enterology, 1911,i, p. 2; and Archiv f. Verdarntngs—Krankheiten, 1911, xvii, p. 557. 206 URINARY COLLOIDAL NITROGEN 207 an opportunity to study the colloidal nitrogen output in the urine of a dog with a large tumor. The dog upon which this study was made had a hard calcified growth about the Size of an orange in one of the breasts. The tumor involved the nipple and the breast tissue for some distance around the nipple. Several metastatic deposits were present along the “ breast lines.” MicrosCopic examination of sections of the original growth and of the metastatic infiltrations, according to several pathologists who examined them, indicated that the tumor was a chondroma which had undergone carcinomatous degeneration. Other pathologists, on the contrary, believed the growth to be of a benign nature, with the histological structure of a chondroma. For the determination of colloidal nitrogen the alcoholic precipita- tion method of Salkowski was used, with modifications, as follows: 8— The total nitrogen was determined in 5 cubic centimeters of the urine by the Kjeldahl process. Two portions of 100 cubic centimeters each of the urine were evaporated in a porcelain dish over a gently steaming water bath till they were of the consistency of thin syrup. The residues were then taken up in 100 cubic centimeters of alcohol (98.5 per cent) and throughly stirred. The alcoholic extracts were then filtered through ashless filter papers, and the precipitates washed with alcohol. We determined the effect of dialysis upon this alcohol-precipita- ble, so-called “ colloidal” nitrogenous material. Most colloidal sub- stances fail to dial yze through the very best grade of parchment paper. Only that fraction of the alcoholic precipitate which would remain in— diffusible under certain conditions of dialysis could be called colloidal at the present stage of our knowledge of the subject. Accordingly, the two precipitates on the ashless filter paper were treated as follows:— The precipitate on one filter paper, together with the filter, was placed in a Kjeldahl flask, digested with sulphuric acid, and the nitro- gen determined in the usual way. The second precipitate and filter paper were placed with water in a bag of the finest grade of parch- 8 Before subjection to analysis the urine was first tested for protein, which, if found, was removed by means of heat coagulation aided by the addition of a few drops of dilute acetic acid solution. 208 URINARY COLLOIDAL NITROGEN ment paper and dialyzed for forty-eight hours. The liquid in the bag was then analyzed quantitatively for nitrogen. The appended summaries present the results obtained for urine from the dog with the breast tumor and also for urine from several ' normal dogs. In the Salkowski method for the determination of “ colloidal” nitrogen (as the results in the summary Show) indiffusible nitrogenous substance is precipitated as “ colloidal” nitrogen. It has not yet been shown that such ditfusible nitrogenous matter in the colloidal precipi- tate is true colloidal material. A. DATA PERTAINING To THE URINE OF NORMAL DOGS PERCENTAGE OF TOTAL NITROGEN COLLOIDAL NITRO- ,1? (fillimgéiisii INDIFFUSIBLE TOTAL NITROGEN No. IN 100 CC. or GEN IN 100 cc. Ag COLLOIDAL‘ COLLOIDAL As INDIFFUSIBLE URINE \ 0F URINE NITROGF“ NITROGEN COLLOIDAL " NITROGEN Grams Gram Gram 1 2.3045 .0437 1.85 .02775 1.2 2 3.2051 .03142 .98 .01634 .5 3 .8590 .0172 2.002 .01202 1.4 4 1.6436 .0214 1.28 .01841 1.1 B. DATA PERTAINING To THE URINE OF THE DOG WITH A TUMOR OF THE BREAST 5 a. 4.0088 .3392 8.40 .0939 2.3 5 b. 6.3034 ' .3897 6.10 .2293 3.6 5 c. 4.4591 .3210 7.10 .0767 1.7 5d. 3.6862 .3294 8.10 .0817 2.2 5e. 3.1414 .0958 3.04 .0867 2.7 5f. 3.9642 .3566 8.90 .1175 2.8 5g. 2.5139 .4617 13.10 .1342 3.6 The results demonstrate that the “ colloidal ” nitrogen, both before and after dialysis, was greater in amount in the urine of the dog with the tumor than that in the urines from normal dogs. It is desirable to study the effect of dialysis upon the “ colloida ” nitrogenous substances in the urine of cancer patients. III ON THE COMPOSITION OF PROTOPLASM AND THE NATURE OF THE STRUCTURAL AND DYNAMIC RELATIONSHIPS OF CELL CONSTITUENTS AND PRODUCTS * CONTENTS A. Studies on I soagglutination . Transfusion and the question of intravascular agglutination. REUBEN OTTENBERG . The occurrence of grouped isoagglutination in the lower animals. REUBEN OTTENBERG and S. S. FRIEDMAN . . . . . . . . . . . . . . Tonicity in isohemagglutination. MORRIS H. KAHN and REUBEN OT- TENBERG . Isoagglutinationindog blood. REUBEN OrrENBERo, S. S. FRIEDMAN, and D. J. KALISKI. (With a discussion by CYRUS W. FIELD, ISAAC LEVIN, and REUBEN OTTENBERG) . . . . . . . . . . . . . . . . Experimental agglutination and hemolytic tranfusions. REUBEN OTTEN- BERG, D. J. KALISKI, and S. S. FRIEDMAN B. Studies of Lipins . On the forms in which lipins are combined in cells. JACOB ROSENBLOOM . Preliminary reports on studies of the diffusion of lipins and lipin—soluble substances through rubber membranes. WILLIAM 1. (km . . . . . A study of the diflusibility of lipins from ether through rubber membranes into ether. JACOB ROSENBLOOM . . . . . . . . . C. Influence of C ancer Extmcts . A study of the influence of cancer extracts on the growth of lupin seed- lings. JACOB ROSENBLOOM PAGE ZII 225 230 236 243 260 278 ' 287 292 A. STUDIES ON ISOAGGLUTINATION 1. TRANSFUSION AND THE QUESTION OF INTRAVAS— CULAR AGGLUTINATION * By REUBEN OTTENBERG INTRODUCTION THIS paper offers a solution of a special problem in isoagglutination; namely, the question whether transfusion is permissible between per- sons Whose sera agglutinate the red blood cells of each other. It also shows why such transfusions are not regularly followed by serious symptoms. The isoagglutination of human red blood cells was discovered independently by Landsteiner and by Shattock in 1900. Many workers at first regarded the phenomenon as one of pathological significance. Halban, Ascoli, and others, however, showed that isoagglutination occurred with a large proportion of normal bloods, and Landsteiner discovered the remarkable fact that all human bloods can be divided into three sharply defined groups, according to the way in which they interagglutinate. To these groups was subsequently added a fourth, in- dependently discovered by several observers. The groups are as follows (see Table I) : — The serum of the first group, designated in this paper as group I, possesses the power of agglutinating the red cells of members of all the other groups, but the red cells of members of group I are not agglutinated by any human serum. This group includes about 50 per cent of all persons examined. The serum of members of the second group (group II) can agglutinate the cells of persons belonging to the third and fourth groups, but not of other mem- bers of the second group itself nor, of course, of group I. The cells of members of the second group can be agglutinated by sera of individuals of group I and group III only. The third group is the reciprocal of the second group; its serum agglutinates cells of persons belonging to members of the second and fourth groups. Its cells are agglutinated by sera of the second and first groups. Members of this group sometimes show slight individual irregularities, the cells now and then failing to * Reprinted from the Journal of Experimental Medicine, 1911, xiii, 425. (From the Pathological Laboratory of Mount Sinai Hospital and the Laboratory of Biological Chemistry of Columbia University, College of Physicians and Surgeons, New York.) 211 212 TRANSFUSION AND AGGLUTINATION be agglutinated by the sera of some members of the second group, although being agglutinated by others, and the sera occasionally agglutinating the cells of some, but not all, other members of the third group itself. ' The fourth group, whose members are relatively rare, is characterized by possessing no agglutinin for human red cells and by its cells being agglutinable by the sera of all other groups. TABLE I INTERAGGLUTINATION REACTIONS or TEN INDIVIDUALS Sera GROUPS I II III IV I z 3 4 8 6 7 9 10 5 I _ __ _ __ _ _ __ _ _ __ Cells I . 2 _ — _ — _ _ _ _ '— 3 __ _ __ _ _ __ _ _ __ 4 _ __ _ _ __ __ _ _ __ Cells II 8 + + + + — + + +\ + g - 6 + + + + - — — — - — 7 + + + + + — — - — — Cells III . 9 + + + + + _ __ _ _ _- 10 + + + + + — ~ - — Cells IV - 5 + + + + + + + - P - Landsteiner himself suggested that the groupings could be explained by as- suming the existence of two agglutinins, of which the second group possessed one, the third group the other, the first group both, and the fourth group neither. In each case the cells are susceptible only to that agglutinin which does not exist in the individual’s own serum. (Moss has, in a recent paper, made the quite unnecessary assumption that there are three agglutinins.) The group characteristics are permanent for each individual throughout his life. When concentrated, the agglutinins act almost instantaneously; when diluted, they act more slowly. They act in the cold as well as at high tempera- tures, and they are relatively thermostable. Hektoen, who first worked out many of their characteristics, has shown that the agglutinin is absorbed by the cells which it agglutinates. These agglutinins, like other serum agglutinins, are probably associated with the euglobulin of the blood. I have recently found that when agglutinative serum in collodion sacs is dialyzed against running water, the precipitate which forms in the serum can be redissolved in saline solution, and contains the agglutinin in only slightly weakened concentration. TRANSFUSION AND AGGLUTINATION 213 The peculiar groupings are not only permanent with the individual, but they are hereditary. In 1908, in a paper 1 by Dr. A. A. Epstein and the author, we stated that these agglutinins were inherited and followed the Mendeiian law. At that time I had examined only five families and was unable to prove com- pletely the assertion. Recently Von Dungern and Hirschfeld have examined seventy-two families and have conclusively proved that the agglutinins are hered- itary and follow Mendel’s law. Landsteiner, Hektoen, and, in fact, almost all who have written on the subject, as well as Crile in his work on transfusion, have expressed the opinion that isoagglutination might be a danger in transfusion. Never- theless, the surgeons have performed a large number of transfusions without making any serum tests, and in most of the cases there have been no accidents. There have been accidents and untoward symp- toms, however, both in published and unpublished cases.2 The only direct observations on the present question are those in a recent communication by W. Schultz.3 Schultz made intravenous injections in ten cases of small amounts ( 5 to 2 50 cubic centimeters) of defibrinated blood. In his first case the serum of the patient was agglutinative as well as hemolytic toward the donor’s red cells. After the injection of 50 cubic centimeters of blood there was a collapse, followed by vomiting, diarrhea, fever, and edema of the face and hands. In case 6, however, in which there was neither agglutination nor hemoly- sis between the two bloods there was also a collapse after the injection of only 5 cubic centimeters of blood. In case 4 there was mutual agglutination but no hemolysis. Schultz, made cautious by the first experience, injected only 5 cubic centimeters of defibrinated blood. No ill effects followed. Schultz concluded that agglutinative blood is an absolute contra-indication to transfusion. The numerous experiments by Flexner, Pearce, Coca, and others, demonstrating that various lesions and symptoms, including death, could be produced by the agglutinative sera or agglutinable cells of foreign species, do not bear directly on the present subject. 1 Epstein and Ottenberg: T r. of the New Y ark Path. Soc., 1908, viii, 117. 2 The results of a study of transfusions with regard to agglutination and hemolysis tests made at Mount Sinai Hospital during the past two years, will be published sepa- rately by Dr. Kaliski and the author. 3 Schultz: Berl. klin. Wchnschr., 1910, xlvii, I407. 214 TRANSFUSION AND AGGLUTINATION EXPERIMENTAL PART I have found that the explanation of the inconsistency between the very sharp test-tube agglutination, and the frequent absence of symptoms on transfusion of agglutinative blood, depends on the quan— titative relations between agglutinin and agglutinable cells, and non-agglutinable cells. While performing some experiments for the purpose of determining the bulk of agglutinable cells that is necessary for complete absorption of all the agglutinin from a given serum, I noticed that when the amount of red cells exceeded a certain ratio to the amount of serum, all the agglutinin was absorbed without, however, any agglutination tak— ing place, so far as the naked eye could determine. This was confirmed by several experiments, of which one only is referred to in Table II. In this experiment blood cells belonging to group II were obtained in normal salt solution containing I per cent of sodium citrate. The cells were washed once in salt solution, then again sedimented by centrifuging for about ten minutes at high speed. All the supernatant fluid was decanted, and the concentrated cells were used in the experiment. An actual count (with Thoma- Zeiss hemocytometer) showed 10,000,000 red blood cells per cubic millimeter. It has been calculated (P. Weber) that there is only space for 13,000,000 human red cells in one cubic millimeter, so the suspension used might be designated as a 7 5 per cent suspension. The serum used was of class I. Its exact agglutinative titer was not determined, but the serum was known to be active in a dilution of at least I to 12. The mixtures were made in the proportions stated, kept at room temperature for thirty minutes, again thor- oughly mixed, and kept in an ice box over night. On the following day ‘the serum was pipetted off and its agglutinative power tested against a 5 per cent suspension of the same supply of red cells. From Table II it may be seen that one volume of the cell suspension absorbed all the agglutinin from at least sixteen volumes of serum, and that as one volume of cells was still completely agglutinated by thirteen volumes of serum, the cells were able to absorb more agglutinin than was actually necessary to agglutinate them. Controls with non- agglutinable cells showed no absorption of agglutinin. On the other TRANSFUSION AND AGGLUTINATION 2 I 5 hand, above this concentration of one part of cells to thirteen parts of serum, the agglutination diminished in intensity, all the agglutinin being still absorbed; and-when the concentration of four volumes of cells to six volumes of serum was reached, agglutination could no longer be observed, either macroscopically or on microscopic examinations of a drop of the mixture. It was at first thought that in these concen- trated cell mixtures the agglutinin was distributed among the cells in such a way that too little of the active substance went to each cell to produce the visible physical change known as agglutination. Later, however, when the work was repeated and the microscopic examina- tions were made in a different way, namely, by diluting the mixture with an excess of salt solution or of Hayern’s fluid, it was seen that some agglutination also occurred in the more concentrated mixtures, but that the clumps were extremely small (four to five cells) and had been masked previously by the excess of densely concentrated non-agglu- tinated cells. TABLE II THE ABSORPTION or AOOLUTININ BY AGGLUTINABLE CELLS VOLUMES AGGLUTINATION ABSORPTION or AGGLUTININ Serum Cells 4 6 — All absorbed 5 5 —- All absorbed 6 4 — All absorbed 7 3 + (microscopic) All absorbed 8 2 + (naked eye) All absorbed 10 I almost complete All absorbed I 3 I complete All absorbed I4 I complete All absorbed I 5 I complete All absorbed 16 I complete All absorbed I7 I complete Partly absorbed 18 I complete Partly absorbed 19 I complete Not absorbed 20 I complete Not absorbed 21 I complete Not absorbed In transfusion, however, two other substances may accompany agglutinable cells and agglutinative serum (plasma). These are non- agglutinative serum (or plasma) and non-agglutinable cells. The 216 TRANSFUSION AND AGGLUTINATION question therefore arises whether non-agglutinative serum exerts any hindering effect on agglutination. Hektoen states that the addition of increasing quantities of serum from one group to sera of the other groups does not hinder agglutination of the proper corpuscles. The author has reéxamined the question by directly comparing the results of dilution with saline solution and dilution with neutral (non- agglutinative) serum (Table III). TABLE III AGGLUTINATION IN DILU'I‘ED AND IN MIXED SERA j— PROPORTIONS or ACTUAL AGGLUTINATION _ DILUTION or Diluent Susgfgion ACTIVE SERUM With Saline as Diluent With Serum as Diluent 2 7 3 1 : 6 Complete: 2 or 3 Complete: numerous cltunps small clumps 1 5 2 I : 8 Complete: 2 or 3 Complete: numerous clumps small clumps 2 13 5 I : 10 Complete: 2 or 3 Complete: numerous clumps small clumps I 8 3 I : 12 Complete: 2 or 3 Complete: numerous clumps small clumps 2 19 7 I : 14 Complete: 2 or 3 Complete: numerous clumps small clumps I 11 4 I : 16 Complete: 2 or 3 Complete: numerous clumps small clumps 2 25 9 1 : 18 Complete: 2 or 3 Complete: numerous clumps small clumps I 14 5 _ I : 20 Numerous small Very fine clumps clumps I I7 6 I : 24 Fine clumps Clumps seen only through microscope I 20 7 I : 28 Very fine clumps Clumps seen only through microscope 1 23 8 I : 32 Clumps visible only No agglutination through microscope 1 26 9 I : 36 No agglutination No agglutination Two specimens of blood (labeled E and K) obtained by defibrination were used, the one of class II and the other of class III.4 The cells of one (K) were washed and made up to a 5 per cent suspension in 4 It was not determined which specimen was of class III; and the determination would be of no significance in the present instance. TRANSFUSION AND AGGLUTINATION 21'] 0.9 per cent saline solution. Two parallel series of dilutions of the agglutinative serum E were then made; in one series saline solution was the diluent; in the other, neutral (non-agglutinative) serum K. T 0 each test tube was added exactly the same proportion of cell suspension; namely, one fourth of the total volume. The actual dilution of the agglutinative serum is shown in the fourth column of Table III. It is seen that when saline solution was the diluent, agglutination was still observable at a dilution of I to 32 ; when serum was the diluent, it occurred at a dilution of I to 28. There is then only an extremely slight interference, if any, on the part of neutral serum. The qualitative differences between the two series, however, are more significant than the quantitative. Where saline solution was the diluent, there were, in all the dilutions below I to 20, only two or three large compact clumps. Where serum was the diluent, on the other hand, there were very many small clumps. The second question, whether the presence of non-agglutinable cells in the mixture interferes with agglutination, was investigated by making mixtures of whole defibrinated bloods in varying proportions, instead of serum and washed cells. These mixtures imitated then, as closely as can be done in a test tube, the actual conditions in trans- fusion (Table IV). TABLE IV MIXTURES OF AN AGGLUTINATIVE AND AN AGGLUTINABLE WHOLE BLOOD. DEFIBRI- NATED BLOOD OF CLASS I (AGGLUTINATIVE SERUM, NON—AGGLUTINABLE CELLS) AND OF CLASS II (AOOLU'IINABLE CELLS, NON-AGGLUTINATIVE SERUM) PROPORTIONS AGGLUTINATION * Blood (class II) Blood (class I) I 9 + 2 8 + 3 7 + 4 6 + 5 5 + (greatest number of clumps) 6 4 + (smaller number of clumps) 7 3 i + (only a few clumps) 8 2 — 9 I — * The clumps were all microscopic in size. 2 I 8 TRAN SFU SION AND AGGLUTINATION The mixed bloods were thoroughly shaken at once and again in about thirty minutes, at room temperature. They were then put on ice and Observations were made at the end of twenty-four hours. None of the mixtures exhibited macroscopic agglutination; all of them formed, when Shaken, thick emulsions. Through the micro- scope it was seen, however, that microscopic agglutination had oc— curred in seven of the nine mixtures. The presence of non-agglutinable cells in the mixtures, then, did not prevent agglutination, but did change entirely the nature of the agglutination;—- instead of a few large clumps, innumerable microscopic clumps were produced. This was particularly true of the first five mixtures in the series; in the other mixtures the absorption of agglutinin by the excess of agglutinable cells present also played a part; and in the last two, they apparently prevented agglutination, as in the experiments referred to in Table II. This, of course, is extremely significant for transfusion; a few large clumps might cause serious or fatal embolism. A considerable number of microscopic clumps, becoming plugged in widely scattered capillaries, might cause few or no symptoms. The microscopic appear- ances were controlled by making a Similar series of mixtures of each of the specimens of blood used with another blood of its own class. In none of these were any clumps observed. From this experiment it is seen, then, that when the proportion of agglutinative to agglutinable blood is small (I to 4 or less), no agglutination occurs, all the agglutinin being absorbed by the excess of substratum. When the proportion is reversed, agglutination occurs but is microscopic. When the bloods are mutually aggluti- native (one of class II, the other of class III), one finds, likewise, that the blood Shows no agglutination to the naked eye, but shows microscopic clumps in all proportions (Table V). In this instance both of the inhibitory factors are in play; either the one or the other variety of red cells being always present in too great a pro- portion to be agglutinated completely. The excess of cells func- tionates as non-agglutinable, and interferes (probably mechani- cally) with the agglutination of the other species of red cells, which, in many of the mixtures, are present in small amounts compared to their agglutinative serum, and would otherwise be agglutinated com- pletely. TRANSFUSION AND AGGLUTINATION 219 There is no doubt, then, that concentrated non-agglutinable cells interfere with the agglutination of actually agglutinable cells pres- ent in the same mixture. This interference is probably mechanical; it does not occur when both varieties of cells are present in small amounts. Additional experiments were performed to determine whether similar conditions prevail when one or the other blood is very anemic, TABLE V MIXTURES OF MUTUALLY AGGLUTINATIVE BLOODS PROPORTIONS NONE OF THE MIXTURFS SHOW'ED ANY AGGLUTINATION '10 THE NAKED EYE, BUT ALL DID Blood 3 (Class III) Blood 3 (Class II) MICROSC°PICALLY * 9 I Numerous small compact clumps 8 2 Small clumps of 10 to 30 cells 7 3 A few small clumps 6 4 A few small clumps 5 5 A few small clumps 4 6 A few small clumps 3 7 A few small clumps 2 8 A few small clumps I 9 More numerous compact clumps, 20 to 24 cells * The microscopic examinations were made by diluting with Hayem’s fluid, in which further agglutination cannot occur. as is practically always the case in patients requiring transfusion. From the defibrinated whole bloods of normal people, artificial anemic bloods were made by pipetting off one volume of the sedimented red cells and suspending it in nine volumes of the corresponding serum. These anemic bloods then contained less than one million red cells per cubic millimeter. Mixtures were made (Table VI) in such a way that in one series (A) the anemic blood was agglutinative, in the other (B) the anemic blood was agglutinable. From the results in series B it appears that, if the anemic patient’s cells are agglutinable by the donor’s serum, a considerable amount (at least one fourth as much as the total blood volume of the patient) can be transfused be- fore the danger of intravascular agglutination occurs. If, as in A, the contrary is true, and the anemic patient’s blood is agglutinative 220 TRANSFUSION AND AGGLUTINATION toward the full-blooded donor’s cells, the likelihood of intravascular agglutination is much greater and disappears only when a large amount (more than the volume of the patient’s blood) is transfused. The same should be true for mutually agglutinative bloods. This would depend, of course, to a considerable extent on the ra- pidity of mixing of transfused blood. The assumption need not be made that mixing occurs very rapidly, because when the binder- ing factors are present — dilution of agglutinative serum, excess of agglutinable cells, interference by non-agglutinable cells — any agglu- tination that does take place occurs very slowly. TABLE VI SERIES A MIXTURES or ANEMIC BLOOD No. IO (ACCLUTINATIVE, CLASS I) AND FULL BLOOD No. I (AOCLUTINABLE, CLASS 11) PROPORTIONS AGGLUTINATION Anemic No. 10 Full No. I 9 1 Fine clumps seen with naked eye 8 2 Fine clumps seen with naked eye 5 5 Microscopic chimps only 2 8 Doubtful, a few small clumps I 9 N0 clumps SERIES B MIXTURES or FULL BLOOD NO. IO (ACCLUTINATIVE, CLASS I) AND ANEMIC BLOOD No. 1 (AGGLUTINABLE, CLASS 11) PROPORTIONS (MICROSCOPIC CLUMPING ONLY) Full No. 10 Anemic No. I I 9 No clumps 2 8 No clumps 5 5 Many Sharply defined clumps 8 2 Smaller number of clumps 9 1 Very few clumps, but all sharply defined TRANSFUSION AND AGGLUTINATION 221 Above and beyond all the considerations presented here, there remains a possibility that the isoagglutinins are substances which, like fibrin ferment, are formed only after the blood leaves the ves- sels. This, though possible, seems highly improbable. The agglu- tinins are present no matter how the cells and serum are obtained, —— whether by defibrination, ordinary clotting, citration, and after the use of oxalate or hirudin. Furthermore, I-have recently found them to _ occur in most transudates and exudates. CLINICAL PART I have recently seen several transfusions which confirm the view that in the presence of an excess of agglutinable cells all the agglu- tinin can be absorbed without doing harm. In the first case the patient was a young woman with a bleeding gastric ulcer. Her blood belonged to the second or third aggluti- native class, while that of the donor, who was chosen in the emer- gency, belonged to the first; that is, the donor’s serum agglutinated the patient’s cells, but his cells were not agglutinated by the patient’s serum (the same instance as that in Table VI, series B). Sufficient blood was transfused to carry the patient’s hemoglobin from 18 at the beginning to 40 per cent at the end of the transfusion. By cal- culation, this must have been about forty per cent as much as the patient’s own blood volume.5 No untoward symptoms occurred, and the patient subsequently recovered. On examining the patient’s serum, obtained thirty-six hours after the transfusion, it was found to contain no agglutinin of class I; it did not agglutinate her cells obtained either before or after the transfusion. All the agglutinin of class I which had been transfused into her had disappeared. A confirmation of the view expressed here is also furnished by a a case recently described by I. G. Hopkins.6 The transfusion was instituted for the relief of an extreme anemia resembling the pri- mary pernicious type. The patient’s serum agglutinated the donor’s 5 Patient’s hemoglobin, at beginning = 18 per cent; at end = 40 per cent. Donor’s hemoglobin, at beginning = 90 per cent. Let the relation of volume transfused to the patient’s original blood volume = x : I; then 18 + 90 x = 40 (x + I) and x = %% = 0.44. ‘ 8 Hopkins : Arch. Int. M ed., 1910, vi, 270. 222 TRANSFUSION AND AGGLUTINATION cells, but the donor’s serum did not agglutinate the patient’s cells (the more dangerous instance, as Shown in Table VI, series A). Im- mediately following transfusion, smears of the peripheral blood Showed large numbers of polymorphonuclear leucocytes containing red blood cells. After the transfusion, the patient was incontinent of feces, and irrational. Six hours later he developed hemiplegia and coma. Nine hours after “the transfusion he died. “The post-mortem serum did not agglutinate the donor’s cor- puscles.” “The agglutinins present when the serum was first ex- amined were evidently bound.” Hopkins is “ unable to determine the meaning of the blood-picture in this case ” and believes that “what was observed was probably phagocytosis of the transfused red cells by the phagocytes of the recipient.” We have here a case, then, in which apparently all the agglutinin in the body was absorbed by the transfused red cells. It seems at least probable that the death was due to intravascular agglutination, and that the phagocyted red cells were such as had already been affected by the agglutinative serum and were in effect foreign bodies, although on account of the hindering factors mentioned in the present article, they had not yet become actually agglutinated. Since the publication of Hopkins’ paper I have had the .oppor- tunity of studying the blood of three transfusions with regard to phagocytosis. In the first two cases a number of candidates were examined, and a donor was chosen who belonged to the same agglu- tinative group as the patient. In these cases no phagocytosis of red cells was observed.7 In the third case the surgeon elected to disregard the blood tests. The patient’s serum was agglutinative toward the donor’s red cells, but the donor’s serum was not agglu- tinative to the patient’s cells. The patient’s serum was also very slightly hemolytic. Blood smears made at the end of transfusion showed that many of the polynuclear leucocytes were phagocyting red cells in the periph- eral circulation of the patient. The leucocytes contained from one to three red cells each. No agglutinated red cells could be seen 7 For one of these cases I am indebted to Dr. Kaliski, with whom I shall at a later date publish more detailed accounts of all the cases referred to. TRAN SFUSION AND AGGLUTINATION 22 3 in the smears, nor were any seen in a fresh drop of blood diluted with normal salt solution containing sodium citrate. The examination of cells and serum after the transfusion showed a remarkable condition of affairs (Table VII). TABLE VII P1 = Patient’s blood taken before transfusion. P2 = Patient’s blood taken at end of transfusion. D = Donor’s blood. Sera P1 P2 D Cells P1 _ _ f _- P2 + — — D + - — The serum after transfusion no longer agglutinated the donor’s cells; as in the two cases above, the agglutinin had all been absorbed. On the other hand, the patient’s cells after transfusion were agglu- tinated markedly by his own serum obtained before transfusion, but not, of course, by his own serum after transfusion. This proves definitely that the donor’s cells in the patient’s circulation had not lost their agglutinability, but that most of them were not aggluti- nated Simply because there was not enough agglutinin. The absence of phagocytosis of red cells in two transfusions of non-agglutinable blood, and its occurrence in two transfusions of agglutinable blood, indicate clearly that there is a close connection between agglutination and phagocytosis. It further makes it prob- able that the transfusion of agglutinable blood, even if no accident occurs, is useless, as the agglutinable cells are foreign and do not remain in the patient’s circulation.8 8 Since writing the above, I have had an opportunity to confirm further these views in two cases. One was a transfusion between persons of the same agglutination class. No phagocytosis was seen. The other was a transfusion in which the bloods were mutually agglutinative. Extensive intravascular phagocytosis of erythrocytes occurred. Other observations on these cases will be published later. 224 TRANSFUSION AND AGGLUTINATION CONCLUSIONS 1. Intravascular agglutination can occur, and is the probable cause of occasional untoward symptoms, or even death, following transfusion of agglutinative blood. In the majority of cases, how- ever, it does not occur, or if it does, it causes no symptoms. This is dependent on the influence of three factors: (1) concentration of the agglutinin ; (2) absorption of the agglutinin by an excess. of agglutinable’cells ; (3) interference with agglutination by an excess of non-agglutinable cells, so that when clumps occur, they are micro- scopic in size. ' 2. If, for a given transfusion, a non—agglutinative donor, i.e. a donor whose blood is of the same agglutinative class as the patient’s, cannot be obtained, then it is safer to use a person whose serum'is agglutinative toward the patient’s cells than one whose cells are agglutinated by the patient’s serum. 3. Tests for agglutination, 'as well as for hemolysis, ought to be made before transfusion. When time does not permit this, one has to weigh the possible dangers of agglutination or hemolysis against the dangers of letting the patient go without transfusion. 4. Agglutinable cells when transfused are taken up by the phago- cytes in the patient’s blood; and, for this reason, the transfusion of - agglutinable blood, even when no accident happens, can be expected to do little permanent good. 2. THE OCCURRENCE OF GROUPED ISOAGGLUTINA— " TION IN THE LOWER ANIMALS * BY REUBEN OTTENBERO AND S. S. FRIEDMAN IN the previous paper of this series,1 the isoagglutinins which occur in nearly all human bloods, and according to which all human beings can be divided into four sharply separated groups, were discussed. For some time it has seemed to us that a constant and hereditary characteristic like this grouping must have some very fundamental significance, and is not likely to be limited to one species of animal. Throughout the literature it is stated that normal isoagglutinins occur only in human beings. Von Dungern has recently Shown that immune isoagglutinins can be developed in dogs by treating them with each other’s blood, and that two isoagglutinable substances occur in dog bloods. He has also shown, with the method of absorp- tion of agglutinins from animal sera by human red blood cells, that the serum of many animals contains agglutinins which have the same selective activity on human cells as that shown by human isoagglutinins. Likewise he has demonstrated by the absorption method with human sera and the red cells of other animals, that the cells of many other animals contain agglutinable substances identical in their suscepti- bilities with the isoagglutinable substances of human cells. Grouped isoagglutination in animals seems to have escaped ob- servation. EXPERIMENTAL OBSERVATIONS We have investigated the occurrence of isoagglutination in thirty- two rabbits and in eleven steers. Blood of Rabbits. —The rabbits were taken in groups of about *Reprinted from the Journal of Experimental Medicine, 1911, xiii, 531. (From the Laboratory of Biological Chemistry Of Columbia University, at the College of Physi- cians and Surgeons, and the Pathological Laboratory of Mount Sinai Hospital, New York.) ~ 1 Ottenberg : .7 our. Exper. M ed., 1911, xiii, 425. (This volume, page 211.) 225 226 GROUPED ISOAGGLUTINATION ten, and the serum of each rabbit was tested against the red blood cells of all the other rabbits in the group. Suspensions (3 or 5 per cent) of washed red blood cells in 0.9 per cent salt solution were used; and in general one volume of cell suspension was mixed with three volumes of serum. The mixtures were made in capillary pipettes of three to five millimeters in diameter, ‘using the modified Wright technique as described by Epstein and Ottenberg.2 In the first set of examinations of rabbits, the tubes were sealed with paraffin and kept at room temperature for twenty-four hours. In the remaining series, the tubes were put in a thermostat for one hour at 37° C., and then in an ice box for twenty-four hours. The results were about the same. TABLE I Sera GROUPS I II III IV KIND OF RABBIT 3 6 I2 I3 I4 I 9 Io II 7 3 — —- —- — — — —- — —- — White CellsI . . 6 — —— — —— — — — — — — White 12 — — — — — —— — — — — Hare 13 + ++ + — — — — - — — Black Cells II . 14 + ++ + — —— +? — — — — White I — + — — — — — — -— — White 9 ++ + ++ + ++ ++ — — — — Black Cells III . IO ++ + + +? + + — —- — — White 11 ++ ++ ++ — +++ + — — - — Hare Cells 1v . 7 - ' - - _ - - _ - - - White In those tubes in which the more marked agglutinations occurred, the phenomenon could be observed after about twenty to thirty minirtes. In the majority of cases, agglutination was seen only when the tubes were reéxamined after twenty-four hours. Most of the tests were examined microscopically also, but only aggluti- nation which could be seen with the naked eye was recorded. In general, the agglutination results were not as striking as those with human blood. Tables I and II give the data arranged in groups. 2 Ottenberg and Epstein : Arch. I nt. M ed. , 1909, iii, 467. GROUPED IS OAGGLUTIN ATION 2 2 7 TABLE II Sera GROUPS I II III IV KIND or RABBIT 25 31 37 22 35 21 29 24 26 28 4o 25 — —- —- — - —- — —— — -— — White Cells I ‘ . 3r — -— — — -— — — — — — — White 37 — "' - - — — — — — — — Gray Cells II . 22 + ++ ++ - — - - — — — —— White 35 + ++ + ? + 7’ ~— — —— + P — — — Gray Cells III . 21 + ++ +++ ++ + p - ?+ + P — — — White 29 +P ++ ++ + - — - - _ +2 _ White 24 — — — — — -— — — — — —— White Cells IV . 26 _ I? _ — — — — — — - —- White 28 — — — — — — — — - — — Whlte 4o -— — — — — — — — —- — — White Each series of observations was repeated and substantially the same results were obtained. It is seen that the bloods divide themselves naturally into four groups. In group I the serum is agglutinative toward all agglu— tinable cells, but the cells are non-agglutinable. In group II the serum agglutinates cells of group III, but the cells are agglutinable only by the serum of group I. In group III the serum is generally non— agglutinative; the cells are agglutinable by the sera of groups I and II. In group IV the serum is not agglutinative, and the cells are not agglutinable (like the embryonic state of human blood). There was no relation of the grouping to the race or color of the animals. It was probably an accident that all the animals of group IV were white. It is clear that the facts can be explained by the assumption that there are two agglutinins (which we may designate x and y) and two agglutinable substances (X and Y). Group I possessed agglutinins x and y, but no agglutinable substance. Group II possesses agglu- tinin y and agglutinable substance X. Group III possesses agglu— tinable substances X and Y, but no agglutinin (with possibly occasional exceptions). Group IV possesses neither agglutinable substance nor agglutinin. 228 GROUPED ISOAGGLUTINATION Studies of the effects of dilutions were made with a number of the sera. In general they produced decided agglutination up to a dilution of 1 in 4 (when a quarter of the total volume of the mix- ture was 5 per cent cell suspension), but perceptible agglutination was observed with several of them up to a dilution of 1 in 10. It is prob— able that because of their feeble action these agglutinins have been overlooked. ' Blood of Steers. —— The blood of fifteen steers was defibrinated. Four which Showed some laking were rejected. The other eleven were tested for isoagglutination with the same technic that was used in the rabbit experiments. The resultant agglutinations were much heavier than those obtained with rabbit blood; in fact they were quite as striking as human isoagglutinations. TABLE III Sera GROUPS I II III 6 II 2 3 5 8 10 12 7 9 13 6 _ _ _ _ __ _ _ _ _ _ _ CellsI II _ _ _ _ _ _ _ _ _ _ _ 2 ++ ++ — — - - — — — — — 3 ++ ++ — - — — — — — — — C11 II 5 + + — — - - - - __ _ _ e s 8 + ++ - - - - - _ - _ _ Io +++ ++ — — — — —- — —- - - 12 ++ +++ — — — — — — - - - 7 _ _- _ __ _ _ _- _ -- _ _ CellsIII 9 — — — -— — - _ _ __ _ __ I3 — — — — —— —— —- — — — — When the results are arranged in tabular form, the bloods of the steers are seen to fall into three groups (Table III). Group I is agglu- tinative, but not agglutinable. Group II is agglutinable, but not agglutinative. Group III is neither agglutinable nor agglutinative. The grouping of these eleven bloods can be explained by assum- ing that there iS one isoagglutinin and one isoagglutinable substance. GROUPED ISOAGGLUTINATION 2 29 Bloods of group I possess agglutinin, but no agglutinable substance. Bloods of group II contain agglutinable substance, but no agglu- tinin. Bloods of group III contain neither agglutinable substance nor agglutinin. It is possible that examination of a larger number of animals may reveal the existence of a second agglutinin and a second agglutinable substance. CONCLUSION Grouped isoagglutination is not limited to man, but is much more widespread than has been hitherto suspected. It occurs in the bloods of steers and rabbits. It seems probable that it will be found to occur in the bloods of other animals. Just how many of the isoagglu- tinins and the isoagglutinable substances in different species are re- spectively identical is still to be determined. The work is being con- tinued with other animals. 3. TONICITY IN ISOHEMAGGLUTINATION* BY MORRIS H. KAHN AND REUBEN OTTENBERG IN the introduction to the first paper in the present series,1 an out- line was given of the main facts concerning the isoagglutination of human bloods, and the division of human bloods into four sharply defined varieties or groups, according to the way in which they ag- glutinate each other. For the present purpose these groups may be briefly summarized as follows: In group I the serum aggluti— nates the cells of all other groups, but the cells are not agglutinable. In group II the serum agglutinates the cells of groups III and IV, and the cells are agglutinable by the sera of groups I and III. In group III the serum agglutinates the cells of groups II and IV, and the cells are agglutinable by sera of groups I and II. In group IV the serum is non-agglutinative, and the cells are agglutinable by sera of all other groups. Three years ago Gay attempted to Show that a relation existed between the resistance of human red blood cells to laking by hypo- tonic solutions and their isoagglutinative grouping. In an exami- nation of twelve bloods he found that corpuscles not agglutinated by any sera (i.e. those belonging to group I) are more susceptible to laking by saline solutions of low percentage content than are other corpuscles, and that definite tonicity groups exist corresponding to agglutination groups. He suggested that, as sera are assumed to be of the same tonicity as the contents of the red cells,2 the differ- ences in tonicity might be the cause of the agglutinations. * Reprinted from the Journal of Exirerimental Medicine, 1911, xiii, p. 536. (From the Pathological Laboratory of Mount Sinai Hospital and the Laboratory of Biological Chemistry of Columbia University, at the College of Physicians and Surgeons, New York. 1 O)ttenberg : Jonr. Exper. M ed., 1911, xiii, 425. (This volume, page 211.) 2 This assumption is not entirely warranted, as the work of Theobald Smith and others has Shown. 230 TONICITY IN ISOHEMAGGLUTINATION 2 3 I The hypertonicity of sera belonging to the first group would ex- plain their power Of agglutinating cells of the other groups. The hypertonicity of the serum of one of the agglutinable groups would explain its agglutination of the cells of the other group. But, as Gay himself points out, it would not explain the reverse interaction between the groups, which also occurs. Agglutination groupings are permanent for the individual. They are hereditary. Isoagglutinative serum is active at a considerable dilution —— generally up to about I in 32 — with ordinary physiolog— ical saline solution. The resistance of human red cells is known to vary widely in disease and under special conditions, such as follow the ingestion of salts (Sutherland and McCay). These important facts led us to doubt whether so simple an explanation as Gay offers can account for the phenomena. We therefore determined to reex- amine the subject and repeat Gay’s experiments. The technic which we followed was practically identical with that of Gay. With a clean, dry, hollow needle, fifteen to twenty :cubic centimeters of blood were obtained from an arm vein and de- fibrinated with a few glass beads in a perfectly clean bottle. (Gay used a rod instead of beads, and obtained a somewhat larger amount of blood.) After the cells had sedimented during six hours in an ice box, all the supernatant clear serum was pipetted off and the sedimented cells were used for the tonicity tests. Bloods of which the serum showed any trace of laking were discarded. It is not necessary to describe here again the well—known method of making the agglutination tests and of arranging the bloods in their correct groups. The resistance of the red blood cells was tested by Hamburger’s method, as used by Gay. A series of salt solutions ranging in con- centration from 0.38 per cent to 0.57 per cent of sodium chlorid was prepared. In the first experiments, the difference between the suc- cessive concentrations was 0.03 per cent, as in Gay’s work. Later it was made 0.02 per cent. All precautions were taken to make the solutions accurate and constant. Chemically pure sodium chlorid dried to constant weight was used, and fresh solutions were pre- pared for each new group of cases tested. Evaporation was care- fully prevented. 232 TONICITY IN ISOHEMAGGLUTINATION - In testing each blood for tonicity,_a series of small test tubes was arranged, each tube containing five cubic centimeters of one of the graded salt solutions. The sedimented unwashed red cells were shaken, to insure uniform distribution, and 0.1 cubic centimeter of the suspension was added to each tube. The tubes were shaken and allowed to stand at room temperature for a short time. Read- ings were made then, and again after the tubes had been in an ice box for twelve hours. The amount of hemolysis was estimated for each tube of the series by comparing its color with the colors of a standard series made up separately with each blood. This scale was prepared by laking 0.4 cubic centimeter of the sedimented red cells in twenty cubic centi- meters of distilled water (the same ratio of blood as that in the tests). This was designated 100 per cent hemolysis, and dilutions of this were then made to represent 80, 70, 60, 40, 30, 20, 10, 5, and 2. 5 per cent hemolysis. Each of these solutions was put in a tube of the same caliber as that of the tubes used in the tests. In order to facilitate our discussion, we reproduce Gay’s 3 tables. GAY’S RESULTS NON—AGGLUTINABLE CELLS. CLASS I NaCl SOLUTION (PER CENT) .39 .42 .45 .48 .51 AMOUNT or HEMOLYSIS (PERCENTAGE) Blood No. 18 . . . . 100 75 40 5 5 Aug. 26 100 100 60 35 10 Sept. 24 Blood NO. 22 . . . . 100 90 75 50 2.5 Aug. 18 100 40 I 5 5 2. 5 Aug. 26 Blood No. 34 . . . . 80 i 80 3 5 I5 10 Blood No. 35 . . . . 95 50 15 10 5 Blood No. 38 . . . . 95 35 IO 10 2.5 3 Gay : Jonr. Med. Research, 1907—8, xvii, 330. TON ICI'I‘Y 1N ISOHEMAGGLUTINATION 2 3 3 GAY’S RESULTS (Continued) ACCLUTINABLE CELLS. CLASS II NaCl SOLUTION (PER CENT) .39 .42 .45 .48 .51 AMOUNT or HEMOLYSIS (PERCENTAGE) Blood No. 17 . . . . 90 65 25 15 5 Aug. 18 90 50 I5 5 2.5 Aug. 26 60 65 I 5 5_ 5 Sept. 24 Blood No. 26 . . . . 85 35 10 5 5 Aug. 18 40 10 2.5 2.5 2.5 Aug. 26 _ 8s as s s 5 Sept- 24 Blood No. 30 . . . . 7o 70 10 5 2. 5 Blood N0. 33 . . . . 60 50 10 5 2. 5 Blood No. 37. . . . 7o 45 25 5 0 AGGLUTINABLE CELLS. CLASS HI NaCl SOLUTION (PER CENT) .39 .42 .45 .48 .51 AMOUNT or HEMOLYSIS (PERCENTAGE) Blood No. 23 . . . . 65 25 5 5 5 Aug. 18 80 I 5 5 5 2.5 Aug. 26 50 40 IO 5 0 Sept. 24 Blood No. 25 . . . . 60' I5 20 5 5 Gay’s conclusions from these results are as follows: “(1) That human cor- puscles which are not agglutinated by foreign human sera are uniformly more susceptible to laking by salt solution of low percentage than are corpuscles which are agglutinated by foreign isosera. (2) That definite groups as regards resist- ance to salt solutions are present, which groups coincide with the groups classi- fied according to agglutination. (3) That the resistance of a given blood does not change apparently within a month or more, and probably corresponds to the relatively unchanging relations manifested in isoagglutination.” Gay’s tables do not entirely justify his conclusions. as an examina- tion of the data will Show. Thus the first two of five bloods belonging to class II exhibited, on several occasions, only the most minute differ- ences from the last three of the five in class I. The differences between the same blood on several dates are, in some cases (e.g. N o. 26), as great as the differences between bloods in the different classes.4 4 Relations must be ascertained from general comparisons of the results for one blood with the results for another blood. The irregularities are so numerous that a comparison of different bloods as tested by any particular salt solution involves contradictions. As a 234 TONICITY IN ISOHEMAGGLUTINATION Our own work consisted of the examination of the blood of twenty- two persons. In our first group of Six cases (Table I), the three TABLE I SODIUM CELORID SOLUTION (PER CENT) ‘42 '45 '48 -SI -54 ~57 AMOUNT OF HEMOLYSIS (PER CENT) 25 5 0 o 0 0 Group I . . . . . 20 2.5 0 0 o 0 40 5 0 0 0 0 Group H _ _ ‘ 60 10 2.5 o 0 0 60 7 2.5 o 0 0 Group III . . . . . I 50 15 2.5 0 o 0 classes showed a definite grouping as regards corpuscular tonicity. The blood in class I, however, proved to be the most resistant; that is, it was the least hemolyzable by hypotonic saline solution. This, strangely enough, is exactly the reverse of Gay’s finding. The two groups of agglutinable cells showed little difference in saline con- centration from each other, those of class II being least resistant. Our second series (Table II) consisted of the bloods of six other individuals. Here, the blood of the first group (only one individ— ual in this group) is most easily hemolyzed, and the agglutinable cells evidence most resistance. TABLE II SODIUM CHLORID SOLUTION (PER CENT) [ .40 I .42 .44 I .46 l .48 AMOUNT OF HEMOLYSIS (PER CENT) GroupI. . . . . . . . . 80 50 20 10 2.5 50 4O 20 15 5 50 20 IO 5 2. 5 G . roup H 80 6o 20 5 0 60 10 10 2. 5 0 GroupIII . . . . . . . . 6o 25 15 2.5 2.5 rule, readings of more than 70 per cent hemolysis cannot be accurate, because the intensity of the colors makes such readings extremely uncertain. These remarks apply to our own as well as to Gay’s results. TONICITY IN ISOHEMAGGLUTINATION 2 3 5 In our third group of ten cases (Table III), the first or non-ag- glutinable group Showed instances of both low and high tonicity. Here, the second and third groups showed high tonicity, the reverse of Gay’s findings. TABLE III SODIUM CHLORID SOLUTION (PER CENT) .38 - .40 .42 l .44 \ .46 AMOUNT OF HEMOLYSIS (PER CENT) 60 40 20 10 o 50 40 I 5 5 0 40 30 10 0 Group I ' 80 60 20 5 o 80 60 40 10 b 5 80 70 40 10 5 70 60 30 I 5 5 GroupII. . . . . . . . . 80 40 40 10 5 90 80 60 20 10 Group III . . . . . . . . 90 80 60 40 20 Correlating our results, we see that instances of high and of low tonicity occur with about equal frequency in the groups of agglu- tinable and non-agglutinable cells. We therefore conclude that there is no support for the opinion that isoagglutination of human blood is due Simply to variations of molecular concentration. BIBLIOGRAPHY DECASTELLO AND STURLI. M iinchen. med. W chnschr., 1902, xlix, 1900. DONATH. Wien. klin. Wchnschr., 1900, xiii, 497. HALBAN. Wien. klin. Wchnschr., 1900, xiii, 545. HALBAN and LANDSTEINER. M zinc/zen. med. W chnschr., 1902, xlix, 473. HAMBURGER. Cenlralbl. f. Physiol., 1893, vii, 161, 656. HEKTOEN. J our. Infect. Dis., 1907, iv, 297. LANDSTEINER. Cenlralbl. f. Baht, Ite A M., 1900, xxvii, 361 ; W ien. klin. Wchnschr. 1901, xiv, 1132; hliinchen. med. W chnschr., 1902, xlix, 1905. LANDSTEINER and LEINER. Centralbl. _f. Bakt., Orig., 1905, XXXViii, 548. LANDSTEINER and RICHTER. Ztschr. _f. M ed.-Beamte, 1903, xvi, 85. MOSS. Bull. Johns Hopkins Hosp, 1910, xxi, 63. PESKIND. Am. Jour. Med. Sc., 1904, cxxvii, 1011. THEOBALD SMITH. Jonr. Med. Research, 1904, xii, 385. SUTHERLAND and MCCAY. Biochem. J our., 1910, v, I. 7 4. ISOAGGLUTINATION IN DOG BLOOD* BY REUBEN OTTENBERC, S. S. FRIEDMAN, AND D. ]. KALISKI (WITH A DISCUSSION BY CYRUS W. FIELD, ISAAC LEVIN, AND REUBEN OTTENBERC) RECENTLY two of us reported that normal isoagglutination could be detected in the blood of rabbits and in the blood of steers.1 We desire now to report one additional fact, viz., the occurrence of iso- agglutination between the bloods of normal dogs. Isoagglutination of human blood has been known for about ten years. Ever since Landsteiner’s work it has been known that all human beings can be divided sharply into four groups according to the agglutinative power of their blood serum for each other’s blood cells. There are two isoagglutinins and two corresponding iso- agglutinable substances. One group of human beings has in the blood serum both agglutinins, and in the blood cells no agglutinable sub- stances (that is, the cells are not agglutinable). Another group of persons has the first agglutinin and the second agglutinable substance. The third group has the second agglutinin and the first agglutinable substance. The fourth group has both agglutinable substances and no agglutinin. These peculiarities are a permanent characteristic of the blood of each individual. Before this society, in 1908, in a paper with Dr. Epstein, I announced my belief that these character- istics were hereditary and were inherited according to Mendel’s law. At that time I had examined only five families and could not definitely prove the point. Recently von Dungern and Hirschfeld have proved that human isoagglutinable substance is hereditary according to Mendel’s law. Von Dungern has also shown by the method of ab- sorption that many of the heteroagglutinins of animals’ serum can * Reprinted from the Proceedings of the New York Pathological Society, 1911, xi, PP- 49—56- 1 Ottenberg and Friedman : Journal of Exterimental Medicine, 1911, xiii, p. 531. (This volume, page 22 5.) ‘ 236 ISOAGGLUTINATION IN DOG BLOOD 237 be identified with one or the other of the human isoagglutinins, and that many of the heteroagglutinable substances of animals’ red cells are closely Similar to the isoagglutinable substances of human blood. Actual iSoagglutination in lower animals has been overlooked, how- ever, probably because most of the agglutinations are very weak and require a special technic for their demonstration. In a previous study on the quantitative relationship of aggluti- native serum and agglutinable red cells it was found that an excess of cells absorbed a small amount of agglutinin without agglutination being visible at all. Therefore in the present studies the method used was that of mixing very large proportions of serum with very small proportions of red blood cells. While natural isoagglutinins had not been described before our work, immune isoagglutinins had been developed in dogs by von Dungern and I-Iirschfeld. We have repeated their work and have developed immune isoagglutinins in two out of Six dogs injected. The mutual agglutinations of these six dogs are shown in Table I. Whether these immune agglutinins are exaggerations of the natural agglutinins which (with an improved technic) we subsequently discovered, we cannot state at present. TABLE I “IMMUNIZED ” DOGS Sera A B C D E F B —— — + + — - C —— — — + — - Cells D -— — — -— — -— E —— — — + — — F __ _ __ ._ _ _ In looking for normal isoagglutination between the bloods of different dogs we were at first unsuccessful, and only succeeded when we adopted for our technic the mixing of fifteen parts of serum with one part of defibrinated blood. (The ease with which the dogs’ cells 238 ISOAGGLUTINATION IN DOG BLOOD laked in normal saline solution was one of the causes of failure in the early experiments.) The mixtures were made in capillary pipettes of two to five millimeters diameter, using a modified Wright technic (as described by Epstein and Ottenberg, Archives of Internal Medicine, May, 1905). The reason for the use of this technic was that the work could be done with small quantities of blood, and particularly that in the thin layer of dilute cell suspension in a small pipette it was possible to see fine agglutination which escaped observation in ordinary test tubes. Many precautions are necessary in order to insure accuracy in the results. The glass tubes must be thoroughly cleaned and washed in distilled water, and dried. After the mixtures are made the open ends of the pipettes are sealed with paraffine to prevent drying. The mixtures are generally Observed for one hour at room temperature. At the end of this time with dogs’ blood, agglutination when it occurs can usually be seen. A column of fluid about one inch long is found most convenient, and it is important to have the air bubbles (which by rotation of the tube are used as mixers) of a uniform, rather small, Size. All the tests reported here were made in duplicate; that is, two of us making independent readings found practically the same results. AS stated above, the agglutination between the bloods of untreated dogs is feeble as compared with the isoagglutination of human bloods or of immunized dogs. By thorough mixing the clumps can be made to disappear, but generally they form again if the mixture is allowed to stand. Ten or twelve dogs were bled at a time, and their bloods tested against each other at once. AS can be seen in the chart, the aggluti- nations which occur admit of a certain grouping, but, as in the case of rabbits’ bloods, there are irregular agglutinations which do not fit well in the classification, and the groupings can therefore be regarded as only tentative. The one definite division of the bloods is into those whose serum is non-agglutinative and those whose serum agglutinates some other dog’s cells. Agglutinability of the cells seems to be ir- regular. In general those animals whose sera agglutinate the cells of most of the other dogs have cells which are agglutinated by the sera of only a small number of the other dogs —— and vice versa. Auto- agglutination was never observed, though it was tested for regularly. ISOAGGLUTINATION IN DOG BLOOD 2 39 TABLE 11* Does, MARCH 29 Sera I 6 8 11 4 9 To 2 3 5 12 I _ __ __ _ _ _ _ _ _ _ __ 6 + —- —- — - — - — — - -- 8 __ ._ __ __ __ _ ._ __ __ _. __ n - - — + — — — - - - 4 + + + + — + + — - — — Cells . . . . . 9 + + + -|- - _ + __ _ _ __ 10 + -— — — + — — —— — — — 2 + — - — + - — - - — — 3 + + + + — - — — — — — s + + + + - — — - - — — 12 1+ + + + + — — — -— — I — TABLE III DOGS, APRIL 22 Sera II 18 8 13 I7 4 2 lacs 16 5 4 14 II — — — + —- + — — — — — — 18 + - —— — —— + — — —- — — — 8 + — - — —— —- —- —- —— — — — 13 - - - - — + - — - - - - 17 — — -- — — -— -— — — — — — Cells 4 + + _ _ _ _ _ _ _— _ — _— 2 -— — — — + — — — — — —- 15 — — '— - + + — — - — — — 16 — — —- — — + — — — — — — s + + + — - — -— — - - - - 9 + + + — — — — — — — - — 14 + — — — — + — — —— — -— — * The heavy lines represent a tentative grouping, although the authors do not believe that sharply defined groups, such as are found in human beings, exist in either dogs or rabbits. 240 ISOAGGLUTINATION IN DOG BLOOD AS to the permanence of these peculiarities of dogs we can say little at present. Most of the tests were repeated with blood obtained after two or three days and were found to be unchanged. In a series of dogs (Table III), examined on April 22, were six dogs which had been examined on March 29 (Table II). In the meantime several of the dogs had been used in various transfusion experiments. It will be seen by direct comparison of the record of the Six surviving dogs (Table IV), that only two new interagglutinations appeared, and only one of the former agglutinations was lost out of thirty combinations. Whether these changes were due to the treatment which the dogs re- ceived, we are not at present prepared to say. TABLE IV SIX SURVIVING DOGS ON APRIL 22 AS COMPARED WITH SAME DOGS ON MARCH 29 * Sera (March 29) 8 II 4 9 2 5 8 __ __ ._ _ _. __ II — —- + — - - 4 + + — + — — Cells . 9 + + _ _ _ _ 2 — — + — — ~— 5 + + — — — - (April 22) 8 II 4 9 2 5 8 — + — -— — — II — - + __ _ __ 4 — + — - — — Cells . 9 + . + _ _ _ _ 2 — — + — '- "- s + + — — - - * Several of the dogs had been used in transfusion experiments in the interim. ISOAGGLUTINATION IN DOG BLOOD 241 Discussion Dr. C yrns W. Field.——I must say very frankly that I would not trust the results obtained with such a method. In working with agglu- tinins, as with all immune bodies, one cannot be too careful. In the first place, in agglutination, the quality of the glass makes a great deal of difference. Unless one used glass tubes of the very best quality, which had been washed under known conditions most carefully and tested, one could not accept the results at all, because one may get curious results if the technic is not of the best. Dr. Isaac Levin. —Dr. Ottenberg’s paper presents the results of a comparative study, and the faults of the technic, if there are any, are identical for all sets of experiments. The errors would con- sequently be neutralized on the analysis of the results. If I understand Dr. Ottenberg correctly, the presence of aggluti- nation is not constant in the dogs. The phenomenon may be present at a certain time and then disappear. I should like to know whether there is a method by the aid of which we can change the phenomenon at will. For instance, if two dogs were tested, one immediately after and the other before a meal, whether the property of isoagglutination of the serum would be different. The second question is whether the blood serum of a dog possesses the property of homoagglutination. The study of this property as indicating the interrelationship between cells and blood serum of the same animal may be of some aid in the investigatiOn of the patho- genesis of malignant tumors. Dr. Reuben Ottenberg—In regard to Dr. Field’s criticism, there is a certain amount of truth in what he says. Notice, however, in regard to these experiments that they have all been repeated with Substantially the same results. Some of the irregularities may have been due to such causes as he suggests. The method is not ideal. I have tried out the method pretty thoroughly in human iso- agglutination and hemolysis tests, of which we have done many hun- dreds in the past three years. With this method the results have always agreed with the well-known peculiarities of human isoaggluti- nation, and when hemolysis occurred, it could always be confirmed by repetition of the test. In these agglutinations in the blood of various 2 4 2 ISOAGGLUTINATION IN DO G BLOOD animals, generally far more delicate than in the blood of human beings, we probably do make an occasional mistake. But on the whole the tests come out quite constant, and they control one another, so that we feel confident that the results are all right. With regard to Dr. Levin’s second question, autoagglutination has been stated to occur in pernicious anemia. I have often looked for it myself, but have never found it. Probably what has been called ag- glutination in these cases is simply the clumpy appearance of any very anemic blood as it runs from the needle prick. Landsteiner has indeed described autoagglutination of normal human cells, but the highly artificial conditions of his experiment which included bringing the mixtures down to the freezing point, render it highly improbable that the phenomenon which he observed have any relation to ordinary agglutination. As Landsteiner him— self points out, autoagglutinins, even if formed, would be extremely difficult, if not impossible .to demonstrate, because being always in the presence of a large number of susceptible cells, the agglutinins would be absorbed as rapidly as formed. With regard to Dr. Levin’s first question, the only methods that I know of changing these properties in the living animal are by im— munization or by absorption. Immunization I have already referred to. I recently saw a human blood transfusion which illustrated this absorption of agglutinin in vivo. Before the transfusion the patient’s serum was agglutinative to the donor’s cells, but not the donor’s serum to the patient’s cells. Serum obtained from the patient after the transfusion no longer agglutinated the donor’s cells; all the agglu- tinin had been absorbed by the donor’s transfused cells. On the other hand the patient’s blood cells collected after the blood transfusion were agglutinated by his own serum saved from before the trans- fusion (because, of course, the patient’s cells after the transfusion consisted largely of donor’s cells, susceptible to patient’s original agglutinin). These conditions persisted for several days in this case. Human isoagglutination seems to vary in intensity from time to time, but there are no accurate studies on this question, and the causes of the variation are unknown. (I can add from recent work with Dr. Friedman that rabbit isoagglutinins Seem to be far less constant than those of dogs or human beings). 5. EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC T RANSFUSIONS * By REUBEN OTTENBERG, D. J. KALISKI, and S. S. FRIEDMAN TWO years ago the authors determined to find out by experiment what would happen when hemolytic or agglutinative blood was trans- fused directly between two animals of identical species. Although the work had to be interrupted for extraneous reasons over a year ago, and on this account the Series of experiments is incomplete, the results so far, while not leading to final conclusions, offer so much of interest that it iS thought worth while to present them now. OBSERVATIONS ON ISOAGGLUTINATION IN DOGS The first difliculty was the choice of animals. The smaller animals, such as rats and rabbits, in which the authors have been able to find isoagglutinins, were not available on account of the difficulty of doing direct transfusions between them. In dogs, the ideal experimental animals, previous workers (like the authors at first) had repeatedly failed to find isoagglutinins or isohemolysins. After several failures, however, the technical difliculties which had previously interfered with the demonstration of such anti-bodies were, at least partly, overcome; and it was then readily demonstrated that isoagglutination and iso- hemolysis occasionally occur between the bloods of apparently normal dogs. The technical difliculty to be overcome was the fragility of dog- blood-cells which were found to lake more or less when suspended in salt solution of any strength. Various concentrations (between 0.6 per cent and 2.0 per cent) of sodium chloride were tried with the same result. In the experiments, therefore, salt solution was avoided en- tirely, the cells being simply suspended in their own sera. The technic * Reprinted from the Journal of Medical Research, 1913, xxviii, p. 141. (From the Biochemical Laboratory of Columbia University, at the College of Physicians and Sur- geons, and the Pathological Laboratory of Mount Sinai Hospital.) The authors wish to thank Dr. W. Thalhimer for his assistance with the histological part of the work. 243 244 EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRANSFUSIONS finally adopted was to mix one volume of defibrinated blood with nineteen volumes of the serum whose effect on the blood-cells was to be tested. In this way ten or twelve animals were taken at a time and their bloods reciprocally tested. Dog-blood cells, even when sus- pended in their own serum, frequently showed slight hemolysis, prob— ably due to the insult of defibrination. On this account note was made of hemolysis only when it was so prompt and marked as to leave no doubt that it was caused by the foreign serum. When about ten dogs were tested at a time some interagglutinations were always found. These varied in intensity, but in general were very weak as compared with the intense and complete isoagglutination of human blood-cells under similar conditions. About the weaker ag— glutinations there was naturally some doubt. Thus, with the first series of eleven dogs, two of the_authors made Simultaneous but inde— pendent observations. Their reading agreed in 113 out of the 121 mixtures. In the table below, and in most of the work, only those tests were recorded as positive in which two independent readings gave a positive result. Macroscopic agglutination only was taken account of (Table I). TABLEI 411151221911 March 29 1911 Sera Sera 1118813174215165914 16811102493512 11---+-+-—---— 1---—--———-—- 18+----—-+—--———-‘> e+——————————— s+-------—--- s-----—---—— 13---_-.+------ 11---—--+———— 17-- --—------ CeH310+-——--+—-—— Calls4++—-—?—-—--——— 2+—————+—-—— 2—--——+——-——— 4+++++——+——— 15—-——++———-—-— 9+++++-—————— 16————-—+———-—— 3++++—— —-—— 5+++————-.>—--—— 5++++—-——-——— 9+++———?'1---— 12++++——+—--———- 14+————+—————- Inter-agglutinations of dog bloods. It was found that only the first readings, within Six hours of the time when the mixtures were made, were reliable. Later readings were confusing and inconstant. Agglutination generally appeared in one- EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRANSFUSIONS 24.5 half to one hour at room temperature or in the ice-box. At 37° C. the fragility of the cells was so marked that incubation at this tem- perature was given up. Hemolysis was, however, frequently noted even at room temperature. When the clumps were broken up by shaking, they reformed only in the case of the stronger agglutinations. AS to the permanency of the peculiarities in these animals, final conclusions cannot be drawn, because all the dogs that were kept for any length of time were used for transfusions. In general, however, most of the interagglutinations persisted, but some of them under- went changes. Thus, of six dogs surviving from March 29th to April 22d, the agglutinations were as follows (Table II) :— TABLEII March 29 1911 April 22 1911 Sera sera 8114925 8114925 s—--—-——— s—+—-——-— 11---+-—— . 11—-+—-— C°||$4++—+—— 'CeIls4-+____ 9++—-—— 9++—-—?— 2——+——- 2--+--— 5++—'-—— 5+?-———— Inter-agglutinations of blood from six dogs after an interval of 26 days. (The other dogs of the first lot died in the interim of an epidemic of distemper.) In the interim dog 8 had twice been transfused from dog 2, dog 11 once from dog 3 and once from dog 5, and dog 4 once from dog 1. The changes observed, as, for instance, the failure of the cells of dog 4 to be agglutinated by the sera of dog 8 and dog 9 on the second occasion, and the appearance of the new agglutination of the cells of dog 8 by the serum of dog II on the second occasion, do not seem to be expli- cable either on the supposition that agglutinins were absorbed by the cells transfused or that new immune isoagglutinins were developed. They seem to bear no relation to the transfusions. A similar phe- nomenon was noted also in the later experiments. Thus four dogs were repeatedly examined and transfused between June 25 and Dec. 20. (Table III). Here dog D was transfused into dog E four times be- tween July 18 and Sept. 12, and dog F into dog C four times between July 26 and Sept. 6. The transfusions in the reverse directions, 246 EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRANSFUSIONS namely, of dog E into dog D and dog C into dog F, were done respec- tively on Sept. 26 and Sept. 16. The Changes observed in this series were generally the addition of new agglutinations rather than the loss of those already present; but again the changes do not seem to be directly related to the transfusions. That some of the changes may possibly have been due to greater accuracy of observation in the later experiments is possible, as there is only one change between Aug. 26 and Dec. 20, a period of four months. The readings of these two dates were both made in duplicate; those of the earlier date were not. The one additional agglutination on Dec. 20, not present on Aug. 26, is easily explained as due to an induced anti-body, because dog D, whose serum showed on this date the new agglutinin for the cells of dog E, had been transfused with the blood of dog E on Sept. 26. TABLEIII June 25 July 26 Aug. 22 Sera Sera Sera CDEF ODEF CDEF o—--— o— - o- + CellsD — — — — CellsD — — CellsD — -— E——'-— E --— E _— F++—- F? -— F-l- — Aug. 26 Sept. 26 Dec_ 20 Sera Sera sera ODEF CDEF ODEF C—+—+ O— -|- C-_-|-__|_ Ce|IsD——- — — CellsD —- — ceHsD ___._ E———+ E —— E—+—+ F++—— F+ — F++—— An effort was made to group the bloods according to agglutinations, as is seen in the charts above. Many bloods have what might be termed family resemblances to one another, but no definite groups seem to occur. On repeating their former work with rabbits the authors have also failed to find the grouping which they at first thought was present. In some as yet unpublished studies on rat blood 1 the same thing was observed. 1 Made by one of the authors in association with Dr. R. A. Lambert. EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRANSFUSIONS 247 Imgebrigtsen, in a recent publication, reports that cat bloods also occasionally contain isoagglutinins, which do not seem to occur in groups. In dogs, rabbits, and rats the isoagglutinations are weak as compared with those of human blood, and the failure to group may pos- sibly be due to the difficulties of correct observation. In steers (of which the authors have, however, observed only one series) the agglu- tinations are as intense as in human bloods, and here there is a sharp division into three groups. OBSERVATIONS ON ISOHEMOLYSIS IN DOGS For the reasons given above the observations on hemolysis were not very Satisfactory. Nevertheless, it was repeatedly demonstrated that true isohemolysis does occasionally occur between the bloods of dif- ferent dogs. Undoubtedly some instances of this were overlooked, as, on account of the fragility of the cells, the mixtures were only in- cubated at room temperature. It is of importance to remember this in interpreting the results of the transfusions. Instances of isohe- molysis are referred to in the descriptions of transfusions that follow. TECHNIC AND PLAN OF THE TRANSFUSIONS Twenty-two transfusions were made, all by the direct artery to vein, intima to intima method, with the aid of the Elsberg canula. A hypodermic injection of morphine was given first and cocaine was used as local anaesthetic. AS repeated transfusions were done between the same dogs different arteries had to be used. These were the femoral and brachial or axillary. The large superficial veins of the extremi- ' ties were by far the best and could be used repeatedly. Except in certain instances which will be referred to in detail presently, the dogs withstood the operation (and, in the case of the donors, the blood loss) very well. It was generally not feasible to prevent some infec- tion, but only one dog was lost by wound infection. Both the donor of blood and the recipient were weighed carefully before and after each transfusion, and the amount of blood transfused was thus ap- proximately ascertained. In the first series of twelve transfusions the dogs were all kept in a large room and allowed to run about. In the second series of ten transfusions on four dogs, the animals were 248 EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRANSFUSIONS much more closely observed; they were kept separate, in metabolism cages, and blood counts and urine examinations were made before and after each transfusion, and the presence or absence of agglutina- tion was determined before each transfusion. - In the first series 18 dogs were used and it was thus possible to select several between which the agglutination was strong. The first series was affected by an epidemic of distemper which killed a majority of the animals before the experiments could be concluded. In the second series of experiments on only four dogs, the number of possible combi- nations was, of course, much smaller and there were no strong aggluti— nations. This probably explains the difference between the results of the two series. The plan was to do an equal number of agglutinative transfusions and of control experiments (i.e. between animals which had no agglutinins or hemolysins for each other’s blood). The transfusions were done at intervals of one to three weeks so as to give the dogs time to recover between operations and to develop new anti-bodies if such could be developed. In the agglutinative transfusions the dogs were chosen in such a way that the agglutinative serum was in the recipient, the ag- glutinable cells in the donor. In a previous paper one of the authors has shown that under these circumstances intravascular agglutination, if it occurs at all, is most likely to occur. In several transfusions of the second series, however, the bloods were mutually agglutinative (although very weakly so). RESULTS OF THE TRANSFUSIONS 1. Control Exteriments AS will be Seen from the accompanying tabulations, the control experiments Showed uniformly negative results (transfusions in which dogs 8, I5, and E were recipients). There were no symptoms of any kind. In the second series of experiments, the urine, which was col— lected and examined, chemically and microscopically, was free from blood and casts, and also albumin, excepting traces occasionally, fol- lowing transfusion. No new hemolytic anti-bodies appeared between any of the dogs used in these control experiments. One new agglu- tination developed as noted above (between dogs D and E). TABLE IV.—DATA PERTAINING To THE FIRST SET or TRANSFUSIONS DONOR RECIPIENT TRAFNSIPSJSION TRENSEUSION TRANgIJISION TRIANSIIEU‘SION REMARKS l 3 brown dog Yellow mon- March 29 April 15 May 12 May 25 Date 10,360 gm. grel bitch 75 cc. 70 cc. 60 cc. 280 cc. Amount trans- (first transfu- 9380 gm. fused sion) 9380 10,380 9490 8710 Weight before 5 brindle bull ' - transfusion bitch No symp- N 0 symp- Hematuria Hematuria, Result 12,080 gm. toms toms Bloody etc. (other 3 trans- stools Died fusions) (Dog preg- (Had been (Pups had been Note Transfusions in nant) nursing taken away) which the serum of pups about the recipient agglu— , 1 week ) tinated and in some 4 fox terrier dog 1 bull dog April 12 instances laked the 13,300 gm. 14,080 gm. 300 cc. cells of the donor 14,080 No Symp- toms 14 Shaggy brown 4 fox terrier April 27 June 7 mongrel bitch dog 300 cc. 200 cc. 15,040 gm. 12,550 gm. 12,550 8620 Hematuria, Hematuria, etc. etc. very Sick Killed when moribund 2 young black 8 black dog April 7 April 13 April 29 . dog 14,520 gm. 10,810 250 cc. 320 cc. 50 cc. 10,810 10,130 10,140 No symp- No symp- No symp- Control transfusions. toms toms toms N0 aggluhmauon or ‘ 16 gray bitch 15 tan and April 25 May 5 hem01Y$1$ 14,340 gm. white dog 30 cc. 420 cc. (first transfu- 11,760 gm. 11,760 12,070 sion) No symp- No symp- 13 Shaggy yel- toms toms low dog - ’1 13,070 gm. EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRANSFUSIONS 249 2. Agglutinatioe and H emolytic Transfusions In three dogs repeated transfusions of blood whose cells were ag- glutinable by the recipient’s serum were made. Of these the first two dogs (dog 11 and dog 4) Showed the most interesting results. Dog. 11 (Table IV). Dog 3 was chosen as donor for dog 11 because, among all the agglu- tinative mixtures, the agglutination of the cells of dog 3 by the serum of dog 11 was one of the strongest. The serum of dog 3 had no effect on the cells of dog 11. Dog 3 died of distemper after the first transfusion and the experiment was continued with dog 5 as donor for the subsequent transfusions. The blood-cells of this dog were strongly agglutinated by the serum of dog 11. After the first two transfusions dog II showed no untoward symp- toms whatever. The urine unfortunately was not examined. After the third transfusion (60 cubic centimeters of blood having been trans- fused) dog 11 was observed to be very sick. She trembled and showed great muscular weakness. There was an intense hematuria which lasted about twenty-four hours. The dog recovered from these symp- toms, however, and was again transfused nine days later. This last transfusion 'on dog 11 was intensely interesting. Before the transfusion the blood of the two dogs was tested again. The serum of dog 1 I was found completely to agglutinate and partly to lake the cells of dog 5 within an hour at room temperature, and Slightly to agglutinate them in five minutes. The serum of dog 5 neither ag- glutinated nor laked the cells of dog 11. Both the agglutinin and the hemolysin present were extremely feeble in the test-tube (unless, as is likely, there was some inhibiting substance in the serum of dog 5). Four dilutions (5, i. a, and 3%) of serum 11 were made. using serum 5 as diluent. None of these dilutions agglutinated or laked the cells of dog 5. (The mixtures of 20 parts of serum with one of deflbrinated blood mentioned in the first part of this paper were of course used.) The result was all the more surprising. Two hundred and eighty cubic centimeters of blood were trans- fused. During the transfusion dog 11 became Sick at once. Her breathing became rapid and labored. Her head and extremities trembled and twitched. An hour after the transfusion these symp— 2 50 EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRAN SFUSIONS toms were still present and there was oozing of blood from the wound. The wound was opened; there was, however, no bleeding point, the oozing being capillary. It was noted that the blood clotted very Slowly and imperfectly. The dog was catheterized and 20 cubic centimeters of thick, black, very bloody urine were obtained. Microscopically the abundant sediment showed masses of granular débris. No red blood- cells were recognizable. A smear of the dog’s blood made at this time showed an unexpected blood picture. The spread Showed a very large number of nucleated red blood-cells. To 100 leucocytes there were counted 164 nucleated red cells, of which Seven were megaloblasts. The differential leucocyte-count is given in Table V. The nucleated red blood-cells were generally pear-Shaped and often polychroma- tophilic; the remaining red blood-cells were normal in shape, but varied considerably in size; macrocytes were common. Many cells showed polychromatophilia. Blood serum of the dog obtained at this time (one hour after the transfusion) was only slightly blood-tinged. The clotting phenomena of a tube of blood collected from the ear vein at this time were also of interest. The blood clotted Slowly, and when the first clot formed, it was removed and a secondary plasmatic clot formed. This phenom- enon may have been connected with the hemorrhagic tendency, as Shown by the wound oozing. Six hours after the transfusion there was still oozing from the wound. The following day the dog died, and an au— topsy was done at once. Autopsy : Dog 11.——-The body weighed 9 kilos. It was still warm. There was black clotted blood in the wound. The subcutaneous fat was of a peculiar and intense yellow color (jaundice P). Pleura and lungs were negative. Of the heart chambers, only the right ventricle was distended; it contained black clotted blood. The heart muscle showed no blood imbibition. The stomach contained undigested food. The small and large in- testines were empty and showed nothing abnormal. In the mesen- tery there were several, bright red, flame-Shaped hemorrhages. There was also one such hemorrhage in the distribution of the pancreatico- duodenal vessels, and one in the neighborhood of the portal vein, as well as several on the surface of the gall bladder. The mesenteric glands were large (pea-Sized). EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRAN SFUSIONS 2 SI The pancreas was pale. The spleen was of a dark purple color and very hard. It weighed 41 grams. The Malpighian bodies were very prominent. The liver weighed 252 grams and showed on the surface and on section dark purple and irregular mottlings. The gall blad- der was filled with thick black bile. The adrenals showed nothing abnormal. The urinary bladder contained black altered blood; the mucous membranes and the urethra Showed nothing abnormal. The kidneys were very hard and almost ebony black in color. The two together weighed 42 grams. On section they showed concen- tric rings of black and gray. The outer rim of the cortex was black, shading down to gray at the inner part. The bases of the pyramids were of the deepest black color and shaded gradually to yellow at the papillae. The renal arteries and veins were not thrombosed. Microscopic Examination: Dog 11. Spleen. Section shows large Malpighian corpuscles with marked hyperplasia. There is moderate congestion. Prominent throughout the section in both reticular spaces and blood sinuses are large numbers of large mononuclear phagocytes filled with red blood-cells, fragments of red blood—cells, and crystalline blood-pigment, some of them with the remains of nuclei. In addition there are large numbers of a very unusual type of giant cell. These cells are from thirty to forty micra in diameter. They lie free in the large blood sinuses, are found in the reticular network, or completely fill the small capillary-like sinuses. They vary greatly in outline from round or oval to very irregular, with pseudopod-like processes. The protoplasm is unusually large in amount and takes a very light, almost homogeneous pink stain with eosin. The nuclei stain very densely with hematoxylin. Some of the nuclei are round or oval, but most of them are extremely polymorphous, resembling an exaggerated nucleus of a polymorphonuclear leucocyte. Sections stained with Giemsa stain Show large numbers of nucleated red cells of the normoblastic type. Most of these are found in the blood sinuses and capillaries ; many, however, are found in the reticular spaces, but Show no definite arrangement around a germinal center. Quite a number of these cells Show nuclei in the process of amitotic division. Liver. There is an extreme grade of quite uniform cloudy swell- ing. The blood-vessels Show an uneven congestion; here and there 2 5 2 EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRANSFUSIONS are sinuses tremendously engorged, and in some places the sinuses have the appearance of being surrounded by hemorrhagic infiltration. In the portal septa there is marked infiltration, chiefly with round cells, but also with some polymorphonuclears. The larger branches of the portal vein are filled with blood, containing great numbers of leucocytes, and many mononuclear macrophages filled with red blood- cells. A few of the large branches of the portal vein are filled with hyaline, thrombus-like material. Scattered throughout the section are a moderate number of the large giant cells with dense—staining nuclei already described as occurring in the spleen. A few of these are found lying free in the large branches of the portal vein. Most of them are found flattened out in the sinusoids of the liver between the columns of liver cells. In this Situation they are relatively deficient in protoplasm, but no bare nuclei are found. Adrenal. The adrenal shows an hemorrhagic infiltration in its capsule. The cortical cells have a slight hydremic appearance. Kidneys. There is an intense congestion and an extreme grade of cloudy swelling. Many of the convoluted and collecting tubules seem to be filled with hemoglobin casts. In many of the smaller arteries and capillaries the blood-cells which are present appear as a homo- geneous hyaline-like mass the color of hemoglobin. This is probably to be interpreted as a fused mass of red blood-cells. Lungs. The air spaces are collapsed. The blood-vessels are Slightly distended; there are no definite thrombi. A large number of mononuclear phagocytes containing red blood-cells are found through- out the section, many of them scattered throughout the lumen of the blood-vessels, but some giving the impression of endothelial cells which have become phagocytes. A moderate number of the giant cells described at length under spleen and liver are found wedged in the small capillaries or lying free in the small arteries. The bronchi and other structures of the lung are normal. Dog 4 (Table In this set of only two transfusions dog 4 was the recipient, dog 14 the donor. As will be seen by reference to the chart, dog 4 had been used previously as a donor in one transfusion. Three days after the transfusion in which he was donor, dog 4 had a secondary wound hemor- rhage from which he promptly recovered. He was, therefore, in all EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRANSFUSIONS 2 5 3 probability at the time of the transfusion ten days later (in which he was recipient for the first time) somewhat anemic. His behavior and appearance at this time were normal. Tests made five days before the transfusion showed that the blood serum of dog 4 was markedly agglutinative to the cells of dog 14 (see Table I). For the reasons given previously no notes were made on hemolysis at that time, but from the outcome it is quite possible that hemolysis may have been present. Three hundred cubic centimeters of blood were transfused. While the transfusion was going on, the appearance of dog 4 changed suddenly. He became excited, whereas usually the dogs lay perfectly quiet under the influence of cocaine and morphine. He whined, breathed rapidly and heavily, and his hind legs trembled violently. For twenty-four hours after the transfusion he had an intense hematuria. On the third day after the operation his urine Showed no hemoglobin. He con— tinued to look ill for several weeks, and on this account no further transfusion was done for five weeks. By the end of this time he had apparently recovered in health, and his behavior was normal; smears of his blood, however, showed great numbers of nucleated red blood- cells and other abnormal forms. Before the Second and final transfusion the action of the two dog bloods on each other was re'éxamined. It was found that the serum of dog 4 was very strongly agglutinative, but not at all hemolytic to the cells of dog 14 in 30 minutes at room temperature (June 17; a fairly warm day). The serum of dog 14 had no effect on the cells of dog 4. Two hundred cubic centimeters of blood were transfused. During the transfusion dog 4 again became obviously sick and distressed. A specimen of his blood collected just at the end of transfusion Showed clear, very yellow serum; no laking. This serum agglutinated the cells of dog 14 just as strongly as before transfusion. The cells ob- tained from dog 4 at the end of transfusion were agglutinated slightly by his own serum obtained before transfusion. Six hours after the operation dog 4 was very sick and scarcely able to stand. Muscular tremor was very pronounced. He had passed no urine, but about four ounces of dark, manifestly bloody stool. Eighteen hours after the transfusion he had passed 200 cubic centi- meters of black, bloody urine. Microscopically the urine contained 2 54 EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRANSFUSION S great masses of disintegrated red blood—cells, blood casts, some leuco- cytes, and epithelial cells. He also passed another black stool. Twenty-one hours after the operation the dog was plainly moribund. He was therefore killed rapidly with an overdose of ether; and an autopsy was performed at once. Autobsy : Dog 4. — A well-nourished male fox terrier weighing 8.7 kilos. The subcutaneous fat was well developed and yellow in color. The muscles contracted when cut. The blood was dark, flowed freely, and clotted naturally. Blood serum obtained at autopsy was not in the least laked, but was of a deep yellow color (jaundice P). The lungs were negative. . The heart was moderately distended. All the chambers were filled with dark, recent clots. The liver weighed 390 grams. Its surface was markedly mottled with blotches of yellow and dark blue. There was some capillary hemorrhage in places under the capsule, and there was evidence of some local peritonitis (fibrin layer) between the lobes. On section the organ was greatly congested and less mottled than on the surface. Minute light yellow points (focal necroses P) could be made out. The gall bladder was distended with dark bile. The spleen was large, weighing 54 grams. It was tense and firm in consistency and bluish black in color. On section the Malpighian corpuscles were prominent. The kidneys were large and firm and weighed together 81 grams. On section the cortex was seen to be dark in color, Shading gradually to a paler color at the apices of the pyramids. The outlines of the pyramids were not well defined, but their radial striations showed plainly. The bladder contained about 100 cubic centimeters of brown-black urine. The stomach contained bile and mucus. The duodenum showed pale submucous hemorrhages, but was empty. The lower part of the small intestine and the entire large intestine were full of soft, bloody stool. Microscopic Examination: Dog 4. Spleen. This section presents very much the same appearance as that of the spleen of dog 11. The multinuclear cells are only about half as numerous, but occur in EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRANSFUSIONS 2 S 5 the same locations. A larger number of nucleated reds are present than in the spleen of dog 11; and in several places these seem to have a distinct tendency to occur at the periphery of what looks like a germinal center for this type of cell. The central portion of this structure is occupied by large mononuclear cells. The gradations or developmental stages between these large mononuclear cells and the nucleated red cells cannot be made out, so that it cannot be defi- nitely decided whether the nucleated red cells are positively formed in the spleen or are simply deposited there from the blood stream. Liver. Practically all of the lobules Show a fairly marked chronic passive congestion and central necrosis. All of the liver cells seem slightly smaller than normal, are rather granular, and contain large numbers of small and medium sized vacuoles which give the cells an hydropic appearance. Diffusely scattered throughout the entire section, and forming small collections at the necrotic centers of the lobules are many polymorphonuclear leucocytes. No multinuclear giant cells are seen in this section. N ucleated red blood—cells are seen throughout the blood-vessels. Adrenals. The epithelial elements are swollen and edematous. There are some deposits of blood—pigment and some small hemorrhagic areas in the capsule, but none in the gland itself. Kidneys. There is an extreme cloudy Swelling and an albuminous secretion in the convoluted and collecting tubules. The blood-vessels are moderately engorged. Many of the epithelial cells lining the con- voluted tubules are infiltrated with finely granular or slightly crystalline hemoglobin material. Within an occasional convoluted tubule one finds small hemoglobin casts or blood casts and some of the tubules contain ghosts of red blood-cells. The capillaries of the glomerular tufts are engorged with blood. In the cortex there are small areas of focal necrosis. In these areas the epithelial cells are disintegrating and their nuclei Show karyorrhexis. Lungs. The blood-vessels are moderately engorged and stand out very clearly. Scattered here and there in the capillaries are found multinuclear giant cells, few in number, and of the same type as oc- curring in the spleen. Blood smears of dog 4 both before and after the last transfusions Showed enormous numbers of nucleated red blood-cells, chiefly nor— 2 5O EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRAN SFUSIONS moblasts, but some megaloblasts. No blood counts were made, but differential counts of the smears stained with Giemsa stain showed that before the last transfusion the erythroblasts greatly exceeded the leuco- cytes (see Table V). Their relative decrease in number after the transfusion was apparent, not real, as the leucocytes (with which their numbers were compared) were, to judge from the smears, greatly in- creased in number. Variations in the size, of red blood-cells with nu- merous macrocytes and some polychromatophilia, were noted. Phag— ocytosis of red blood-cells by leucocytes was carefully searched for in all of the sniears, but was found in none.2 A smear of the blood of dog 14, donor to dog 4, made before the last transfusion, showed that he also had nucleated red blood-cells, but in very small numbers as compared with dog 4. Ten blood smears of dogs used in the control transfusions were examined (see Table VI). Five of these dogs were recipients. Only one showed, after trans- fusion, any nucleated red cells at all, and these in very small numbers, as compared with the great numbers seen in the blood of dog 11 and dog 4. This one dog, which showed a few nucleated red blood-cells (dog A), was, at the time, fatally ill with septic infection, following gangrene of the leg from ligature of the femoral vein. Of the five smears from donor dogs two likewise showed a very small number of nucleated red blood-cells after the transfusion. Dog C (Table VI). This set of five transfusions between two dogs F and C (parallel with five control transfusions between two other dogs D and E) was in- tended to extend the observations made in the first series of transfusions. Unexpectedly the results, as far as symptoms of blood destruction were concerned, were completely negative. This was possibly due to the fact that the agglutination of the donor’s cells, by the recipient’s serum present at the beginning of the transfusions, was exceedingly weak; in fact, it remained feeble during the four months throughout which the two dogs were under observation, and on one occasion it could not be detected at all. An interesting fact is that in the course of the trans- fusions the serum of the donor dog acquired the power of agglutinating 2 It iS unfortunate that more complete blood examinations were not made. Smears were Simply made as a matter of routine, stained and set aside to be studied some months later. TABLE V. —DATA PERTAINING To BLOOD SMEARS NUCLEATED RED BLOOD DIERERENTIAL LEUCOCYTE COUNT CELLS a if: g '13 if g 1- m 2 a. H 1:! H .4 1- Doc DATE SEE ggé g? SS Ea 8 3 i 8 a a: 5 E s a $2 2 ‘i S if 53 °‘ S 8 °‘ it! 4* June 7 Recipient, before last transfusion 127 3 56 44 o 0 4* June 7 Recipient, after last transfusion 99 5 83 17 0 0 4* June 7 Recipient, 6 hours after last transfusion 52 1 93 5 2 0 4* June 8 Recipient, post mortem 54 2 85 I3 2 0 11 May 25 Recipient, after last transfusion 150 1 50 46 4 0 3 April I Donor to 11 after 1st transfusion I 0 70 30 0 0 14 June 7 Donor to 4 before last transfusion 14 0 78 18 4 0 C July 27 Recipient, 24 hours after 1st transfusion 1 0 80 15 5 0 C Aug. 9 Recipient, before 2d transfusion 0 0 75 14 1 1 0 C Aug. 10 Recipient, 24 hours after 2d transfusion 0 0 81 15 4 0 C Aug. 22 Recipient, before 3d transfusion 0 0 72 25 3 0 C Sept. 6 Recipient, before 4th transfusion o 0 73 23 4 0 C Sept. 16 (As recipient), before 5th transfusion 0 0 83 26 1 0 F Aug. 9 Donor to C, before 2d transfusion 0 0 72 22 6 0 F Aug. 22 Donor to C, before 3d transfusion o 0 80 I 5 5 0 F Sept. 6 Donor to C, before 4th transfusion 0 0 82 15 3 0 E July 18 Recipient, after 1st transfusion 0 0 65 31 1 3 E July 19 Recipient, 1 day after 1st transfusion 0 0 66 14 20 0 E Aug. I Recipient, before 2d transfusion 1 0 58 34 7 O E Aug. 2 Recipient, I day after 2d transfusion 0 0 76 20 4 0 E Aug. 17 Recipient, 1 day after 3d transfusion 0 0 70 18 2 0 E Sept. 12 Recipient, before 4th transfusion 0 0 76 21 3 0 E Sept. 13 Recipient, 1 day after 4th transfusion 1 0 80 17 3 0 D July 18 Donor, before 1st transfusion 0 0 62 28 9 1 D July 19 Donor, I day after Ist transfusion 0 0 54 33 I3 0 D Aug. 2 Donor, 1 day after 2d transfusion 0 0 62 27 10 I D Aug. 14 Donor, before 3d transfusion I 0 80 19 I O D Sept. 12 Donor, before 4th transfusion 0 0 82 I4 4 0 A June 11 Recipient, I day after transfusion 0 0 85 10 4 0 A June 12 Recipient, dying of septic infection 2 0 80 15 4 1 B June 10 Donor to A, after transfusion 0 0 64 31 ° 0 June 12 Donor, 2 days after transfusion 3 0 56 43 1 ° 8 April 13 Recipient, after 2d transfusion 0 0 84 I5 1 0 * Dogs that had hematuria after transfusion. EXPERIMENTAL AGGLUTINATIV E AND HEMOLYTIC TRANSFUSIONS 2 the cells of the recipient. We can offer no satisfactory explanation of this. It was on this account that after the four symptomless trans- fusions from dog F into dog C had been done according to the original plan, the reverse transfusion was done, viz. from dog C into dog F. Although the agglutination of donor’s cells by recipient’s serum was in this transfusion fairlysharp, no symptoms occurred. There was dis- appearance of the agglutinin from the serum of the recipient at once after transfusion, due probably to its absorption by the susceptible cells. A similar phenomenon has been observed by one of us in several human transfusions. It is interesting to note that this phenomenon did not occur in dog 4, which showed severe symptoms. CONCLUSIONS 1. By a suitable technic, iso-agglutination and isohemolysis can be demonstrated to occur between the bloods of different dogs. Iso- agglutinins, and possibly isohemolysins, occur naturally, and it is pos- sible that the immune isoagglutinins produced by von Dungern and Hirschfeld are merely intensifications of these. N o sharp grouping (such as would indicate a limited number of agglutinable substances and of agglutinins) could, however, be made out in the naturally oc— curring agglutinins. Natural (as distinguished from immune) isoag- glutination is, however, a relatively weak phenomenon. 2. Isohemolysis and isoagglutination are closely connected with each other in dogs, as Moss and others have shown them to be in human blood. In our observations hemolysis neveDoccurred without agglutination. Apparently isohemolysins may be developed de nooo by the repeated transfusion of agglutinable cells, but they are never developed by the transfusion of non-agglutinable cells. 3. Hemolysis in the body is far more intense than in the test-tube. We have made the same observation in the case of a human trans- fusion (unpublished), and similar experimental findings have been published by Muir and M’Nee. 4. The direct transfusion of blood Whose red cells can be aggluti— nated and laked by the recipient’s serum is followed by destruction of the transfused blood with an intense intoxication. It is not yet clear whether agglutination plays any part in this result, or whether it is due entirely to hemolysis. TABLE VI—DATA PERTAINING To THE SECOND SET or TRANSFUSIONS FIFTH , , SECOND THIRD FOURTH TRANSFUSION DONOR RECIPE“ rms'r TRANSFUS‘ON TRANSFUSION TRANSFUSION TRANSFUSION (Reverse : Donor REMARKS became recipient) D mongrel Ebrindle July 18 Aug. I Aug. 16 Sept. 12 Sept. 26 Date bun dog 400 cc. 110 cc. 40 cc. 80 cc. Amount 480 cc. Amount E = 12,400 E = 12,830 E = 14,500 E = 10 500 E = 10,270 Weight 15,300 gm- 12,4008111- ’ D = 12,790 _ —— -——-— — Agglutination Control — —— —-— —— —— Hemolysis trans ——-— —— —— g-—-— —— Symptoms fusions RBC 5,500,000 RBC 3,300,000 D, Donor WBC 10,000 (before) RBC 8,300,000 RBC 8,000,000 Recipient WBC 11,200 16,000 (before) neg. neg. neg. neg. neg. Urine Recipient after) ' F mongrel C _fOX- July 26 Aug. 0 Aug. 22 Sept. 6 Sept. 16 Date bull dog terner dog 380 400 210 170 300 Amount C = 10,550 C = 8 420 C = 8,490 C = 8 700 Weight 12,700 10,550 ’ F = 1’2,450 Agglut. of F cells by C Very feeble ag- No agglut. of F Weak agglut. of Agglutination serum slight or doubt- glut. of F cells cells by C F cells by C ful by C serum and serum serum No agglut. of C cells by of C cells by F Distinct agglut. Distinct agglut. _ F serum serum of C cells by of C cells by Agglgtl" F serum F serum na ve . .._ ' trans_ Hemolysrs fusions RBC July 27 wigopéo 7,300,000 7,300,000 9,400,000 Dog C 16,400 15,400 6,600 9,200 10,500,000 RBC 6,300,000 3,700,000 6,300,000 RBC 7,000,000 Dog F WBC 22,000 before 7,900,000 after neg. (dog C) faint trace albu- neg neg. neg. (dog F) Urine (recipient) men edema of face —— —— —- —-- Symptoms SNOISIIEISNVEICL OIIA'IOWGIH (INV GIAIIVNIIII'IOOV TVLNHWIEIEIcIXEI 886 EXPERIMENTAL AGGLUTINATIVE AND HEMOLYTIC TRANSFUSIONS 259 5. A very remarkable blood picture, presenting many of the mor- phological forms peculiar to pernicious anemia, is produced when the blood of another animal of the same species is destroyed in the cir- culation. (Similar blood pictures have been observed by Bunting and others to follow anemia produced by hemolytic poisons.) In our experiments this was not due to anemia, as the animal’s own blood was not destroyed, and there was no reason to believe they were anemic. The changes must have been due to some peculiar toxic effect, on the bone-marrow, of hemolytic blood destruction. B. STUDIES OF LIPINS 1. ON THE FORMS IN WHICH LIPINS ARE COMBINED IN CELLS* BY JACOB ROSENBLOOM CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . 260 11. Classifications of the lipins . . . . . . . . . . . . . . _. . 260 III. Compounds of lipins . . . . . . . . . . . . . . . . . . - 267 1. With inorganic substances . . . . . . . . . . . . . . 267 2. With tissue metabolites . . . . . . . . . . . . . . . 267 3. With fatty acids . . . . . . . . . . . . . . . . . 268 4. With amino acids . . . . . . . . . . . . . . . . . 269 5. With carbohydrates . . . . . . . . . . . . . . . . 269 6. With proteins . . . . . . . . . . . . . . . . . 272 7. With alkaloids . . . . . . . . . . . . . . . . . . 275 8. With toxins . . . . . . . . . . . . . . . . . . . 276 IV. General Bibliography . . . . . . . . . . . . . . . . . 277 I. INTRODUCTION FIFTY years ago Kletzinski coined the term “ lipoid ” as a convenient designation of fat-like substances. For years the term was ignored. In 1901, however, Overton reintroduced it in connection with a dis- cussion of the theory of narcosis which he and Meyer proposed. At present, the term “lipoid” is generally used to indicate any of the “fat-like” biological substances which dissolve in ether or Similar solvents, and which may be extracted from animal and plant parts with these reagents. Unfortunately the basis for this usage is in- definite and uncertain. ‘ II. CLASSIFICATIONS OF THE LIPINS The classification of lipins that is given on page 261 is a-classification which depends primarily on definite chemical distinctions. Its accep- tance would prevent much of the uncertainty and indefiniteness involved in latter-day discussion of fats and the substances physically, chemically, and biologically resembling them. * An abstract of this paper was published in the Biochemical Bulletin, 1911, i, p. 75. 260 INTRACELLULAR LIPINS 261 'A PROPOSED CLASSIFICATION OF LIPINS1 LIPINS ARE ORGANIC SUBSTANCES OF WIDE BIOLOGICAL DISTRIBUTION. IN PRAC- TICALLY ALL TIONS. CASES THEY ARE INSOLUBLE IN CONCENTRATED NEUTRAL SALINE SOLU- THEY ARE SOLUBLE IN HOT ALCOHOL OR IN WARM ETHER, 0R, As IS USUALLY THE CASE, IN BOTH. I. Natural Aliphatic Lipins A. SIMPLE LIPIN S. Natural aliphatic fats, waxes, soaps, and also the acids and alcohols (except glycerol) represented in them. Excepting the alcohols, these compounds yield soaps with hot concentrated solutions of caustic alkali, or alcoholate, or both. All of these substances con- tain carbon, hydrogen, and oxygen, but are free from phosphorus sulphur, and nitrogen. (Ammonium soaps are exceptions, of course, in the matter of nitrogen content.) a. FATTY _ACIDS, such as butyric acid and palmitic acid (formic and acetic b. SALT 1. Soaps. Waxes. 2. acids excepted); various unsaturated acids, such as oleic acid and linoleic acid; and hydroxy acids of all these types. S AND ESTERS of the aliphatic acids comprising Group I, A, a. Inorganic salts, such as potassium caproate and sodium stearate. Esters of aliphatic monohydroxy and dihydroxy alcohols, such as cetyl palmitate (in spermaceti wax) and myricyl palmitate (in beeswax). (See Group II, B —— carbocyclic esters.) ' 3. Fats and fatty (“fixed”) oils — “ Glycerides”: Esters of the trihydroxy alcohol, glycerol, such as tributyrin, tripalmitin, and triolein. c. ALCOHOLS (monohydroxy and dihydroxy) of the kinds obtainable from B. CONJU waxes, such as cetyl alcohol, carnaubyl alcohol, and myricyl alcohol. a. PROTEOLIPINS. b. GLYCOLIPINS. GATE LIPINS. Natural compounds of simple lipins with non-lipin substances or radicals. All of these compounds contain carbon, hydrogen, and oxygen; and practically all of them contain nitrogen. These substances, with those in Group II, A, constitute the group of so-called “lipoids” — a term Without a definite chemiCal basis. (Protagon is a mechanical mixture containing several conjugate lipins.) Compound lipins containing protein radicals (“lecitho- proteins”), such as lecithalbumins and ovovitellins. (These products may be mechanical mixtures of proteins and lipins.) Compound lipins that are free from protein radicals, but which contain carbohydrate radicals, such as the “cerebrogalacto- Sids” represented by phrenosin (“cerebron”). The substances in this group are free from phosphorus. (Thudichum’s “cerebrosids.”) (See Group I, B, d.) 1 Rosenbloom and Gies : Biochemical Bulletin, 1911, i, p. 52. 262 INTRACELLULAR LIPINS c. PHOSPHOLIPINS. Compound lipins that are free from protein and carbo- hydrate radicals, but which contain phosphoric acid radicals, such as lecithin, cuorin, and sphingomyelin. (Thudichum’s “phosphatids.”) d. GLYco-PHOSPI-IOLIPINS. 'Phospholipins which contain carbohydrate radicals, such as carnaubon and various phytolecithins. (Jecorin appears to be a phospholipin-carbohydrate mixture.) II. Natural Carbocyclic Lipins A. STEROLS. Natural terpeno-alcoholic derivatives. All of these substances contain carbon, hydrogen, and oxygen, but they are free from phos- phorus, sulphur, and nitrogen. They resist saponification (“non- saponifiable fat”), but form esters (Group II, B). Leading members of the group are cholesterol, isocholesterol, koprosterol, and sitosterol. These substances are Abderhalden’s “sterins ” and, with the conjugate lipins (Group I, B), constitute the group of so-called “lipoids.” B. ESTEROLS. Natural terpeno-aliphatic waxes; soap-yielding sterol esters (Group II, A), such as cholesteryl palmitate in lanolin and cholesteryl oleate in blood. C. CHOLATES. Terpeno-acid derivatives of hepatic origin, probably from sterols or sterol radicals (page 56). - a. CHOLIC ACIDS (and their simple biological salts), such as cholic acid and choleic acid. b. BILE ACIDS (and their biological salts), such as glycocholic acid and tau- rocholic acid. III. Natural Lipins of Undetermined Constitution A. CHROMOLIPINS. Pigments which cannot be saponified and which are comparatively unaffected tinctorially by caustic alkali, such as lipo- chromes. B. MISCELLANEOUS LIPINS. Substances of uncertain qualities or doubtful existence, such as krinosin and bregenin. IV. Artificial Lipins Many laboratory products such as triacetin, lead oleate, cholesterol benzoate, sodium cholesterylate, strychnin lecithate, stearyl alanate, creatin lecithate, and potassium kephalate. In line with the recent general acceptance of the term “protein” to indicate all albuminous and albuminoid substances we have proposed the use of the word “ lipin ” to signify all fat and fat-like (“lipoid”) compounds. The Simplification and clarification which our proposed classification INTRACELLULAR LIPINS 263 introduce may readily be seen when the several previous attempts in this direction are reviewed in some detail. Earlier Classifications of the Lipins Thudichum’s classification of the so-called lipoids was one -of the first to be presented. It is given below in its essential details : —— r. PHOSPHATIDSZ Containing nitrogen and phosphorus, besides carbon, hydrogen a. and oxygen. Monophosphatids, N: P: : I : r. I. Lecithin. 2. Kephalin. 3. Paramyelin. 4. Myelin. (Sphinogomyelin acid). Diamino-monophospkatids, N : P : : 2 : I. r. Amidomyelin. 2. Amidokephalin. 3. Sphingomyelin. 4. Apomyelin. Diamino—diphosphatid, N: P: : 2: 2. r. Assurin. Phosphosulpkatids. r. Cerebrosulphatid. 2. Cerebrin acids. N iti'o gen- free monoplzosphatids. r. Lipophosphoric acid. 2. Butophosphoric acid. 3. Kephalophosphoric acid from kephalin. 2. NITROGEN—CONTAINING BUT PHOSPHORUS-FREE PHOSPHATIDS. (Z. b. Cerebrosids or cerebrogalactosids. r. Phrenosin. 2. Kerasin. Cerebrin acids. c. d. 3. SUBSTANCES CONTAINING ONLY CARBON, HYDROGEN, AND OXYGEN. I. Cerebrinic acids. 2. Spharocerebrin. 3. Other ill-defined substances. Cerebrosulphatids (contain sulphur). Amidolipotids 0r nitrogen-containing fats. r. Breginin. 2. Krinosin. a. Cholesterins. b. Phrenosterins. 264 INTRACELLULAR LIPINS 1 Bang, following Thudichum’s lead in many respects, has proposed the following classification of the whole group of lipins : -- r. FATS: Contain, only carbon, hydrogen, and oxygen; free from nitrogen and phosphorus (aliphatic series). 2. CHOLESTERINS: Contain only carbon, hydrogen, and oxygen; free from ni- trogen and phosphorus (aromatic series). 3. PHOSPHATIDS: Besides carbon, hydrogen, and oxygen, contain nitrogen and phosphorus. _ A. UNSATURATED PHOSPHATID. ' i. M 0noamino-monophosphatids, N: P : : 1 : r. a. Lecithin. b. Kephalin. c. Paramyelin. d. Vesalthin. M 0noamino-diphosphatids, N: P: : r : 2. a. Cuorin. b. Liver phosphatid. c. Egg-yolk phosphatid. Triamino-diphosphatids, N: P z: 3 : I. a. Sahidin. b. Kidney phosphatid. B. SATURATED PHOSPHATIDS. i. Diamiiio-monophosphatids, N: P: : 2 : I. . Sphingomyelin. Aminomyelin. Apomyelin. Muscle diamino-monophosphatid. Egg-yolk diamino-monophosphatid. Dog pancreas diamino-monophosphatid. T riamin0-m01i0ph0sp/zatids, N: P z: 3 : I. a. Neottin. b. Carnaubon. Protagon. C. PHOSPHATIDS OF INDEFINITE QUALITIES. 4. CEREBROSIDS: Contain, besides carbon, hydrogen, and oxygen, nitrogen but no phosphorus. Phrenosin. Kerasin. Cerebron. Cerebrin and homocerebrin. . Pyosin and phygenin. 5. LIPOIDS OF UNKNOWN CONSTITUTION. *9 5115‘ vs @9996?“ INTRACELLULAR LIPINS 265 Rosenheim has effectively contributed to simplification in this connection by proposing the appended classification of the “ lipoids. ” I. CHOLESTEROL GROUP! Contain carbon, hydrogen, oxygen; are free from phos- phorus and nitrogen. A. Lipochromes. B. Phytosterol. C. Cholesterol. 2. CEREBRO—GALACTOSIDSZ Free from phosphorus, but contain nitrogen, besides carbon, hydrogen, and oxygen. 3. PHOSPHATIDSZ Contain nitrogen and phosphorus, besides carbon, hydrogen, _ and oxygen. A. M 0n0ami1z0—monoplzospkatids, N : P : : I : I. a. Lecithin. b. Kephalin. c. Vesalthin. Diamino-monopliosphatids, N: P z: 2: I. a. Sphingomyelin. Triamino-monophosplzatids, N: P z: 3: I. a. Neottin. Triamino-diplzospkatids, N: P :: 3: 2. a. Substance from ox-kidney (Frankel and N ogueira). M 0noamino-diphosphatids, N: P z: I: 2. a. Cuorin. ' P199?“ Rosenheim has also proposed the rejection of the following names :— Cerebrote } Cerebric acids Mixtures of various lipins.2 Protagon Cerebrin Pseudo-cerebrin } Identical with phrenosin.2 Cerebron Homocerebrin (identical with kerasin). Myelin I heartily “agree with Rosenheim in the desirability of discarding these terms. Leathes suggests the following comparatively simple terminology for some of the lipins. r. Phospkolipins: Compounds of fatty acids that contain nitrogen and phosphorus. A. Lecithin . B_ Kephalim }Lecrthans. 2 These propositions accord with those previously made by Gies in other connections. 266 INTRACELLULAR LIPINS C. Cuorin. D. Sphingomyelin. E. Substances related to these (A—D), but less definitely characterized. 2. Galactolipins: Compounds of fatty acids that contain nitrogen and galactose, but no phosphorus. They correspond to cerebrins, cerebrosids, or cerebrogalactosids of previous classifications. 3. Lipins: Compounds of fatty acids containing nitrogen but no phosphorus or carbohydrate groups. They correspond to amidolipotids and cerebrin acids (Thudichum). Cramer has lately proposed the following classification, which, like all previous groupings, has much to commend it, but which fails to answer practical demands. 1. Phosphorus-containing fats: Glycero-phosphatids: nitrogen-containing gly- cerin-esters of phosphoric and fatty acids. “Lecithin,” “ kephalin,” etc. 2. Phosphorus—containing waxes: N itrogen-containing esters of phosphoric and fatty acids with unknown alcohols. “Sphingomyelin” (“provisional”). 3. Galactophosphatids: Nitrogen-containing esters of phosphoric and fatty acids with galactose and alcohol groups. “Carnaubon.” 4. Cerebrosids: Nitrogen-containing %ters of fatty acid and galactose. They contain no phosphoric acid: (a) Cerebron; (b) Cerebrin; (c) Homocere- brin. 5. Phosphocerebrosids: Cerebrosids with phosphorus-containing groups. a. Protagon. One can readily understand the serious difficulties attending work in this field with so many uncertain classifications and so many doubts about the nature of most of the substances encountered. In this paper I shall discuss the lipins and their various combinations in terms of the classification printed on page 261. The lipins are receiving more and more attention as we gain more definite knowledge of cell dynamics. As Bang has well said, it may be that the term “carrier of life” which has been applied to intracellular proteins may just as well be given to the intracellular lipins. The fact that so many have thought that protoplasm is protein has favored neglect of the intracellular lipins. Our knowledge of proteins, and especially of the intracellular states and conditions of proteins, is very meager. Our knowledge of the intracellular relationship of lipins is even less definite. INTRACELLULAR LIPINS 267 I shall discuss lipin combinations under the following general heads: I. With inorganic substances. 5. With carbohydrates. 2. With tissue metabolites. 6. With proteins. 3. With fatty acids. 7. With alkaloids. 4. With amino acids. 8. With toxins. III. COMPOUNDS OF LIPINS I. With inorganic substances. —— Glikin and also Rosenheim and Tebb have found varying amounts of iron in the so-called protagon. T hudi- chum and Roscoe also found the same. Glikin detected iron in various vegetable and animal fats, which could not be removed by water containing hydrochloric acid. Winterstein and Stegman found 6.7 per cent of calcium in a vegetable phospholipin. Thudichum also found calcium in kephalin. Thierfelder and Stern found in one of their egg kephalin preparations, 1.03 per cent of calcium. Lecithin combines with acids and bases; silver oxid forms well- defined compounds. Lecithin also combines with many salts of organic acids, such as sodium lactate, oxybutyrate, potassium acetate, and sodium benzoate. Lecithin also supposedly forms addition products with salts of the heavy metals such as HgCl-z and PtCl4, but these- combinations are not true addition products, since decomposition of the lecithin takes place in their production, and Gilson claims that cholin is split off. Koch and his collaborators have recently determined the amount of sodium and potassium in various lecithan preparations, and think that the presence of potassium in cells is due toits affinity for certain lecithans. It cannot be said with certainty that the inorganic matter which has been found in various lipins, exists in true chemical union. Porges and Neubauer have shown that lecithin in ether solution takes up water. This may account for the solution of some inorganic matter in ether solutions of lecithin, even though the inorganic material is insoluble in pure ether. There is also the possibility that the inor- ganic matter is present merely by adsorption. However, Ostwald claims that we cannot draw any hard and fast distinction between chemical affinity and adsorption. 2. With tissue metabolites. — Koch has recently described the preparation of compounds of lecithan with various tissue metabolites, 268 INTRACELLULAR LIPINS such as urea, creatin, creatinin, glucose, lactic acid, etc., but it is difficult to say whether these preparations represent true chemical compounds, adsorption prdducts, or mechanical mixtures. 3. With fatty acids.——Conradi in 177 5 and Gren in 1788 noted the existence in gall stones of a fatty substance and called it “ gall-stone fat.” Fourcroy subsequently classified it along with spermaceti and adipocere. Chevreul, in 1815, demonstrated certain differential quali- ties of this substance and called it cholesterin. This substance, now preferably called cholesterol on account of its alcohol nature, combines very readily with many fatty acids. The esters of cholesterol with oleic and palmitic acids exist preformed in animal tissues, while that with stearic acid has not been identified positively in organisms. Bondet, in 1883, found cholesterol-oleate in the blood serum and called it “ seroline,” but Hiirthle was the first to identify and study the substance. Wolff and Bang have found this ester in chemical combinations with euglobulin in abdominal ascitic fluid due to a carcinomatous growth. This ester is also found normally and pathologically in many portions of the body, in the kidneys, liver, arterio-sclerotic arteries, etc., but is absent from the brain. Aschoff has described a preparation of the ester as small globules or crystals examined under the microscope (myelin). This ester is also found very largely in the wool fat of sheep (lanolin). Some of the esters of cholesterol show a crystalline fluid phase (fluid crystals) and the identification of such substances in the kidneys by Panzer .was the first time such crystals were found in the animal or- ganism. Drechsel and Winogradoff have found a silicic acid ester of cholesterol in the feathers of birds. Abderhalden and Gressel have lately prepared three compounds of cholesterol with iodized fatty acids : a-iodopropionyl-cholesterol, B-iodopropionyl-cholesterol, and di-iodo— elaidyl-cholesterol. It is interesting to note that the hemolysis and anemia that occur with the presence in the intestine of the worm, Bothriocephalns latns, may be caused by cholesterol-oleate from this .organism. This ester is powerfully hemolytic, which property is due to its unsaturated fatty acid radicals. The hemolytic action of saponin, and also of agaricin and tetanolysin, is prevented by cholesterol because these substances form combina- INTRACELLULAR LIPINS 269 tions with the cholesterol, the product being unable to act on the cor- puscle. It is the alcoholic hydroxyl group in cholesterol, not the unsaturated portion that induces hemolysis, according to Hausmann. 4. With amino acids. ——No compounds of amino acids with lipins have been described as occurring in cellular material. Abderhalden and Guggenheim have prepared compounds of glycerol with tyrosin, and a di-palmityl bromo-isovaleryl glycerol which gives the acrolein test. Abderhalden and Funk have prepared compounds of choles— terol with various amino acids and compounds of cholesterol with the palmityl, stearyl, and lauryl, radicals; also compounds of palmityl and stearyl with various amino acids. 5. With carbohydrates. ——A compound of lecithin containing car- bohydrate was first described by Drechsel as occurring in the livers of horses and dolphins. Baldi has found the same kind of substance in the livers and spleens of various animals, in the muscles and blood of horses, and in human brain. Drechsel called this substance jecorin. He found it soluble in ether, insoluble in alcohol, and very hygroscopic. Bang has prepared combinations of lecithin with glucose, fructose, maltose, lactose, and sucrose. He thought his preparation of lecithin- glucose was identical with Drechsel’s jecorin. He prepared this com- pound by evaporating a solution of lecithin and glucose in alcohol. He found the product soluble in ether and benzol, slightly soluble in alcohol, and that with water it formed an opalescent solution. The combination was not constant in composition. One molecule of lecithin was found to be associated in several preparations, 'with r, 2, 9, and 5. 5. molecules of glucose. The glucose gradually separated from the product in ether solution. On dialysis, the sugar passed quantitatively into the dialysate. Mayer has analyzed such products (lecithin with glucose), with the results given in the appended summary. Lncrrnm-Gwcoss Lncmmr Grucosr. Per Cent Per Cent Per Cent C 38.7 65 40 H 9.29 10.8 6.7 N 1.09 r .8 P .66 3.9 2 7O INTRACELLULAR LIPINS It is not easy to decide that lecithin forms chemical combinations with carbohydrates, especially with glucose. The product with glucose has received most attention, for it is well known that lecithin facilitates the solution of glucose in ether and that glucose favors the solution of lecithin in fluids like blood. Thudichum has shown that sphingomyelin can hold kerasin in solution and vice versa. If the sphingomyelin is precipitated by CdCl2, the kerasin also separates. Here there is no question of a chemical combination; one substance exerts an influence on another, thereby modifying its solubilities. Baskofi’ has found that if alcoholic solutions of lecithin and of glu- cose be evaporated to dryness together, the residue that dissolves in ether contains glucose as well as lecithin. The percentages of glu- cose and lecithin vary according to conditions. If the ether solution of the residue be precipitated by alcohol, three times as much sugar is contained in the precipitate as in the material left in solution. The amount of fatty acid obtained by saponifying the precipitate is considerably less than it should be if the precipitate were a simple combination of lecithin and glucose. Baskofl thinks that the evapora- tion of lecithin and glucose together in alcohol solution results, not in the formation of a chemical compound of the two unaltered substances, but in changes in the unstable lecithin which are at present not fully understood. Such facts have made the individuality of jecorin a doubtful matter. Thus, jecorin is said to have been isolated from blood. It is hard to say, however, that this substance preéxisted there, because blood contains lecithin and sugar, and since both are extracted by alcohol, it is clear that, in the mere drying of the extract, lecithin-glucose prod- ucts may be formed. Bang has also shown that by the addition of glucose to blood serum one can increase the yield of lecithin-glucose. However, it is possible that dissociable compounds of lecithin with glucose can exist in blood and other tissues of the body. It is very probable that the jecorins which have been prepared from various organs are not identical. Manasse has isolated from the adrenals a jecorin which showed reducing power only after extended hydrolysis, whereas liver jecorin reduced directly, without hydrolysis. The properties ascribed to liver jecorin by different investigators are very contradictory. Thus, the product prepared by Siegfried and INTRACELLULAR LIPINS 2 7 I Marx was water-soluble, whereas the majority of other investigators declared jecorin to be water-insoluble. Mayer’s jecorin yielded 18.2 per cent of glucose; that of Meinertz, 14 per cent. On the other hand Offer prepared, from liver, a jecorin which was free from carbohy- drate. Meinertz found jecorin fermentable, while Jacobsen did not. The data for quantitative elementary composition of liver jecorin are very discordant, as the following table shows :— Aurnon Bxsxorr Dnncnsnr. MAYER Smcmmn BALDI MANASSE BERNADSKY AND MARX C 50-39 51-32 55.79 39-7 46-88 41-45 48-63 H 7.29 8.11 4.14 6.4 7.81 7.1 7.68 N 3.12 2.86 2.59 5.2 4.36 —-— 2.67 P 2.89 3.2 1.37 1.9 2.29 4.4 2.88 S 1.82 1.42 1.17 2.2 2.14 1.8 1.57 Na 2.87 2.72 3.54 5.9 4.36 Cl 0.49 trace Ash 12.79 12.09 P:N 1:2.38 1:1.8 1:4.19 126.08 123.87 1:2.04 Hammarsten found 1.41 per cent of sulphur and 1.04 per cent of phosphorus in a preparation of jecorin from the bile of a polar bear. The disparities in the data for the elementary quantitative com- position of liver jecorin products raises the question whether the liver contains different jecorins or whether jecorin is a mechanical mixture. Baskoff has prepared several different jecorins from the liver. He claims that, by a careful method of preparation, it is possible to obtain a product of constant composition containing 14 per cent of sugar and a considerable quantity of incorporated inorganic matter. He also claims that the phospholipin in combination with the sugar is not lecithin, but a member of the diamino-monophosphatid group. Sieg- fried and Marx have isolated several jecorin preparations, the chemical content of which varied very greatly. Their values for nitrogen, phos- phorus, sulphur, and sodium were given as follows :— FRACTION 1 FRACTION 2 FRACTION 3 l FRACTION 4 F mcrron 5 N 455 4-55 2-29 4-59 1-45 P 3.16 2.40 .64 .72 .38 S 1.84 1.74 .49 .66 .39 Na 5.10 2.05 1.22 2.04 1.13 2 7 2 INTRACELLULAR LIPINS Mayer and T erroine think that jecorin is a product of the simul- taneous precipitation of glucose and lecithalbumin, and that the prop- erties which differentiate jecorin from lecithalbumin, especially the precipitability, depend upon the contained glucose. Siegfried and Marx do not believe that jecorin is a definite chemical substance, but Mayer believes that jecorin is a compound of lecithin with glycuronic acid. Manasse has proved that the cleavage products of lecithans may be produced from jecorin by decomposition; the resultant sugar he identified as glucose. Baskoff has found both ether-soluble and ether-insoluble jecorins in the liver. Jecorin is precipitated by concentrated salt solutions and yields precipitates with copper acetate and with silver salts. It is not yet. settled whether jecorin is a chemical individual. It is not improbable that jecorin is a mixture of different substances, of which sugar and lecithin are the most conspicuous. Winterstein and Hiestand have found that in lipin preparations from cereals, the carbohydrate present is more firmly united than in arti- ficial lecithin-sugar compounds. Galactose, glucose, and small quanti- ties of pentose and methylpentose were found to be present in their lipin preparations. The amount of contained carbohydrate varies and may be as high as 16 per cent, reckoned as glucose from the amount of copper reduced. Bloor has described the synthesis of carbohydrate esters of the higher fatty acids and prepared the compound mannid distearate, C6H802(C18H3502)2, formed from stearic acid and mannite. This synthesis suggests the probability that such combinations occur in the animal organism. ' 6. With proteins. —— Valenciennes and Fremy demonstrated that organic phosphorus was present in ichthulin. Hoppe—Seyler then prepared vitellin from egg. This substance was the first of the so- called lecithin-protein compounds. Later, vitellin was regarded as a globulin, and now Hammarsten considers it a phosphoprotein. The foundation of this view involves the question whether all the phos— phorus in vitellin is present as phospholipin or not. The earlier analyses showed that phosphorus remains in the substance if it is not extracted sufficiently with alcohol. However, all the later investiga— tors came to the conclusion that the phosphorus cannot be completely INTRACELLULAR LIPINS 2 7 3 removed by alcohol and ether. Hammarsten found a phosphorus con- tent of 074 per cent for a vitellin preparation after complete treat- ment with alcohol and ether. This content on daily renewed extrac- tion sank to 0.4 5 per cent, and after two months’ continuous extraction was 0.39 per cent. This shows how difficult it is to remove the phos- pholipin completely. Liebermann has found that it is very difl'icult to remove lecithin, by extraction with ether and alcohol, from an artificial mixture of lecithin and protein. On account of these facts it is difficult to say whether vitellin is a compound of lecithin with protein or a phos- phoprotein. Its reactions, solubility in dilute salt solutions and in- solubility in water, do not support the phosphoprotein idea. Its coagulation by heat is also against the phosphoprotein view. How- ever, the formation of pseudonuclein, by peptic digestion, indicates phosphoprotein qualities, although, according to Liebermann, leci- thalbumins also have this property. Osborne and Campbell think that vitellin represents a mixture of compounds of protein matter with lecithin, containing from 15 to 30 per cent of lecithin. They call these compounds lecithin- nucleovitellins. The lecithin thus combined is not removed by ether, but readily by alcohol. Osborne also claims that lecitho- proteins have not been isolated from plants, and satisfactory evidence of their existence has not yet been brought forth. Schulze and Likiernik, and Schulze and Winterstein, assume the presence of lecithoprotein in seeds from the fact that a part of the lecithin always remains undissolved when the powdered seeds are extracted with ether. Hoppe-Seyler thought that vitellin was a chemical compound of lecithin with protein and believed it was impossible to remove the lecithin without changing the vitellin. He claimed that it contained 25 per cent of lecithin, while Gross found 30 to 32 per cent of phos— phorus, which, on the arbitrary assumption that all the_phosphorus is contained as lecithin, amounts to 8 per cent of lecithin. Erlandsen and others have shown that egg yolk contains a diamino-phospholipin which is combined with protein, whereas the contained lecithin is free. It is well known that certain diamino-phospholipins which are soluble in ether cannot be directly extracted from tissues by ether before treat- 274 INTRACELLULAR LIPINS ment with alcohol, a fact indicating that they probably exist in com* bination with proteins. Bang thinks that vitellin is a phosphoprotein. He believes that the substance occurs preformed as a compound of protein with a diamino-phospholipin and not with lecithin. Kossel thinks that leci- thin can form true chemical combinations with proteins. Liebermann has given the name lecithalbumins to certain compounds of lecithin and protein which he obtained from the kidney, gastric mucous mem- brane, lungs, spleen, and liver. He obtained them by digesting the organs for 2 to 3 days with pepsin-HCl and considered the residue to be lecithalbumin. His later preparations were purified by dissolving this residue in soda and precipitating with acid. He found that the lecithin was not removed from these compounds by simple extraction with alcohol and ether. These products are probably not immediate constituents of the cells, for they were obtained after subjecting the cells to a very severe process of hydrolysis. They yielded no purin bases on hydrolysis. It is difficult to understand how Liebermann separated lecithalbumin from nucleins. In his method of preparation, any lecithin present would have been decomposed because the materials were treated for several days with 0.2 per cent HCl at 40° C. and later with boiling acid-alcohol for 2 to 3 hours. The preparations did not show a constant chemical composition. ' According to Liebermann all the phosphorus in his lecithalbumin occurs as lecithin ; in one case it was 3 per cent. This compound had to contain at least 80 per cent of lecithin, but nothing of the kind could be extracted from it by means of boiling alcohol. However, although Liebermann’s evidence in favor of the chemical individuality of these compounds is not conclusive, the possibility that this type of compound occurs in cells cannot be disregarded. It is well known that a tissue may contain as much as 18 to 20 per cent of fat, while microscopically it is very evident that the fat is “ masked,” or bound, in some way in the tissue, apparently with protein. In order to obtain the full amount of fat from a tissue, one is obliged, after the ordinary extrac- tions with fat solvents, to subject the tissue to proteolysis and to ob- tain the released lipin from among the products of the digestion. Many investigators ascribe to lecithin compounds of globulin the milk-like appearance of the chyle-forming fluids. Compounds of INTRACELLULAR LIPIN S 2 7 5 cholesterol with globulin have also been described. Sedlmayer claims that the lecithin present in yeast is a lecithalbumin. Boggs and Morris, in some work on experimental lipemia in rabbits, obtained a milky serum which, under 1000 diameters magnification, could be seen to contain droplets that did not stain with osmic acid or Sudan III. On treating the serum with ether it was but slightly altered. The same was true after treatment with various other fat solvents; but, after the addition of ammonium oxalate to precipitate the calcium, and washing with ether, a large residue of fat could be obtained. This suggested that the fat was present in the serum as a protein-calcium- lecithin combination, which was broken up by the precipitation of the calcium. It is also interesting to note that Hoppe-Seyler considered a crys- tallized chlorophyll derivative, “ chlorophyllan,” obtained by ex- tracting grass with boiling alcohol, to be a lecithin derivative in which the fatty acid radicals are replaced by acid chromophoric groups —— radicals of what he called chlorophyllanic acid. Magnesium also seemed to be an integral constituent. Stoklasa, and Schunck and Marchlewski, however, doubt the validity of Hoppe-Seyler’s conclu- sions in this matter. Ulpiani and Lelli claimed to have isolated from brain tissue a sub- stance which they called paranucleoprotagon, which was supposed to be a compound of paranuclein with protagon. Gies and his co-workers have shown that protagon is not a chemical individual, but represents a mixture of lipins. Gies and Steel have shown that Ulpiani and Lelli’s paranucleoprotagon is not what it was believed to be. Gies and Steel obtained incidental results which indicate the presence of a compound of protein with lipin in brain tissue. It is Dr. Gies’ intention to make a further study of this matter at an early opportunity. 7. With alkaloids. — Cholesterol reacts with the various saponins. A specially well-defined compound is that between cholesterol and digitonin, a substance formed by the interaction of one molecule of each of the substances without the splitting off of water. Bing and Koch have described the preparation of compounds of lecithin with various alkaloids and glycosids. They consider that the selective action of various drugs is due to their chemical aflinity for lecithins and similar substances. 276 INTRACELLULAR LIPINS Vernon thinks that alcohol, ether, and chloroform enter into loose combinations with the lipins and other colloidal constituents of the tissues by means of “ molecular valences.” There are many known cases of combination, such as salts and their water of crystallization, of colloidal sulphides and hydrogen sulphide, of lecithin and dextrose, in which, as Moore has pointed out, the combining molecules are completely saturated in respect of their atomic valences, but in which exact stoichiometric relationships exist between the numbers of the combining molecules. Frequently, also, the union is productive of exothermic phenomena. The molecules may therefore possess inde— pendent molecular valences by means of which they combine with other molecules. When crystalloids effect such molecular combina- tions with colloids, exact stoichiometric relationships are not observed as a rule, because of the disproportionate mass of the colloidal aggre- gate to which each crystalloidal molecule is united. Then, too, there may be molecular dissociation, or again a part of the mass of colloid ~ present may not be involved in the reaction. 8. With toxins. — The poison of the cobra has active hemolytic powers, but corpuscles washed free from serum are not hemolyzed unless something from the serum, which is soluble in alcohol or in ether, is added. Since the addition of lecithin makes the poison act on washed corpuscles, Kyes regards the hemolytic agent as a combina- tion of the venom with the lecithin present in the serum. Kyes has analyzed this compound and finds that it agrees closely with mono- stearyl lecithin. Manwaring also claims that the combination between lecithin and the hemolysin is a true chemical union, but Bang thinks that the venom merely adsorbs the hemolysin. Sachs believes that the union is chemical and that the cobralecithid is a derivative of lecithin which is entirely devoid of free venom. Bang claims that he was unable to get the result when he used a lecithin prepared from egg yolk, 'while from commercial lecithin he found it possible to pre- pare the cobralecithid of Kyes, without the toxic properties. At present it is impossible to decide that the substance called cobra- lecithid is a true chemical compound. It is well known that the poison of tetanus travels along the nerve trunks to reach the nerve centers. Wassermann and Takaki have shown that in the nerve centers, tetanus combines with some com-- INTRACELLULAR LIPINS 277 ponent of the nerve tissue, since the toxin can be rendered innocuous if mixed with an emulsion of brain pulp. It was shown by Takaki that if tetanus poison be mixed with phrenosin (Thierfelder’s cere- bron), a substance present in the myelin sheath of nerves and of the nature of a complex lipin, 50 milligrams of the phrenosin will neutralize between two and three hundred lethal doses of the toxin. He also showed that cerebronic acid, which can be produced from phrenosin by hydrolysis, is three times as active as phrenosin itself. IV. GENERAL BIBLIOGRAPHY BANG. Biochemie der Zelllipoide. Erg. d. Physiol., 1907, vi, p. 131; 1909, viii, p. 463. BANG. Die biologische Bedeutung der Lipoidestoffe. Erg. d. inn. Med. u. Kin- derheil., 1909, iii, p. 447. BANG. Chemie und Biochemie der Lipoide. Bergman. Wiesbaden, 1911. BANG. Phosphatide. Biochem. Handlexikon. 1911, iii, p. 22 5. BRAHM. Fette und Wachse. Biochem. Handlexikon, 1911, ii, p. 1. FRANKEL. Gehirnchemie. Erg. d. Physiol., 1909, viii, p. 212. GLIKIN. Fette und Lipoide. Hand. d. Biochem., 1909, i, p. 91. HIESTAND. Beitrage zur Kenntniss der pflanzlichen Phosphatide. Ziirich, 1906. KANITZ. Die Lipoide. Hand. (1. Biochem., 1910, ii, part 1, p. 235. LEATHES. The fats. Monographs on biochemistry. 1910. OVERTON. Studien iiber die Narkose. Jena, 1901. ROSENFELD. Fettbildung. Erg. d. Physiol., 1902, i, p. 651; 1903, ii, p. 50. SOHULZE and WINTERSTEIN. Phosphatide. Hand. d. Bioch. Arbeitsmethoden, 1910, ii, p. 256. THUDICHUM. Die chemische- Konstitution des Gehirns des Menschen und der Tiere. Tiibingen. 1901. WINDAUS. Sterine. Biochem. Handlexikon, 1911, iii, p. 268. 2. PRELIMINARY REPORTS ON STUDIES OF THE DIF— FUSION OF LIPINS AND LIPIN—SOLUBLE SUB—- STANCES THROUGH RUBBER MEMBRANES* By WILLIAM 1. Guns CONTENTS PAGE I.Introduction....................278 II. On the diffusibility of biological substances through rubber . . . . . 281 HI. A demonstration of osmotic pressure exerted by fat . . . . . . . 282 Iv. A demonstration of the diffusion of pigment from fat through rubber intofat.....................283 V. Comparative dialysis experiments, with demonstrations . . . . . . 284 VI. Experiments on the diffusibility of alkaloids through rubber . . . . 285 I. INTRODUCTION WHEN I proposed to my Crocker colleagues that we undertake a study of intracellular chemistry (page 1 5 5), I realized that new ana- lytic methods and unconventional experimental procedures were pre- requisites for material advance in this as in any other chemical re- lation. The greatest obstacle in the path of progress in intracellular chemistry is the evident lability of the essential intracellular constitu- ents. Our best chemical methods increase this predicament because each is essentially anti-biological in character. Biochemical dis- coOrdinations are enforced whenever any of our present chemical processes is effectively applied to protoplasmic material. In reflecting on the properties and possible coOrdinations of intra- cellular lipins, it seemed probable that such lipins might be separated from protoplasmic material with the least chemical violence, and iso- lated with the least possible alteration of their qualities, if they could be removed by dialysis.1 When this idea first came to mind, however, execution of its essential feature appeared to be impossible. I be- lieved that the diffusion of a solute depends very largely on chemical * Reprinted from the Biochemical Bulletin, 1912, ii, p. 5 5. 1 After preliminary desiccation by treatment with anhydrous sodium sulphate or other suitable process. 278 DIFFUSIBILITY OF LIPINS THROUGH RUBBER 279 affinity between the separating membrane and the solvents on both sides of the partition. In that view, it seemed highly improbable that any of the ordinary membranes, except possibly collodion, could be of service in the dialysis of lipins under any circumstances. Collo- dion appeared to possess favorable qualities because of its solubility in common lipin solvents and its possible affinity for the latter under conditions of dialysis.2 Collodion is the only one of the available membranes which, while soluble in ether-alcohol solutions, freely permits the passage of salins, ex- tractives, carbohydrates, and proteins from aqueous solutions to water, or to aqueous solutions, outside, and vice versa. At first thought this suggested special availability of collodion for the work in mind. On the other hand lipins could not be expected to dialyze through collo- dion in the presence of much water and, as preliminary dehydration seemed an inevitable necessity for the dialysis of lipins from cellular matter, the permeability of collodion membranes to water-soluble sub- stances did not appear, after all, to imply any practical advantages for the diffusion of lipins. I also recalled the fact that, in some ex- periments in another relation, we found that collodion was occasionally rendered defective by ether when the latter was used as a preservative of aqueous solutions undergoing dialysis.3 Continuing actively to consider these matters from one viewpoint and then another, I thought of rubber as a possible choice of mem- brane. Recalling the well-known fact that rubber swells very markedly in ether and even in ether vapor, I assumed that the rubber expands in ether under such conditions because ether dissolves in the rubber or combines with it. This was but the prelude to the deduction that if ether dissolves in or combines with rubber, ether would also carry dissolved lipins with it into a rubber membrane; and if ether were on the opposite side of such a membrane, to work inwardly under such conditions, ether currents would develop; and lipins would pass from the solution of higher concentration to that of the lower, and there accumulate until an equilibrium was established. 2 Collodion is a serviceable membrane for such purposes. Experiments with collodion will be described elsewhere. 3 In some experiments which Professor Welker has conducted at my request, we have found that the disintegrative effect of ether on collodion membranes may be due to con- tained alcohol and other impurities. (See page 28 5 .) 280 DIFFUSIBILITY or LIPINS THROUGH RUBBER This conception was so attractive that I proceeded at once to state it to Dr. Rosenbloom and, with his coOperation, immediately tested it. The solid residue from an evaporated ether extract of egg yolk offered the greatest advantages for a preliminary test. We accord- ingly made an ether solution of such a yellow residue, transferred the deep yellow solution to a rubber condom, immersed and supported the latter in ether in a stoppered bottle, and almost immediately ob- served diffusion currents as well as the rapid egress of lipochrome. Fat and cholesterol were easily detected in the diffusate. Assuming that this prompt positive result might be due to defects in the rubber, we made many tests to satisfy ourselves that the ob- servations were or were not what they appeared to be. Dr. Rosen- bloom gave very earnest attention to this phase of the matter for some time and established the fact that we were dealing, except in a few cases of obviously imperfect membranes, with true diffusion phenomena. The original experimental observations were made on March 1, 1910. At that time I was ignorant of similar results of previousrwork with rubber membranes, although I recalled rather vaguely the fact that Kahlenberg had made use of such membranes in another con— nection. The references to Kahlenberg’s work which are given in the C hernisches Zentralblatt [1906 (2), pp. 1391 and 1772], the only ones we could find on this subject at that time, satisfied us that if we extended these experiments, the observations of a previous observer would not be repeated.4 A month or two after the work was inau- gurated we also saw a late reference to the well-known fact, regarding the swelling of rubber in lipin solvents, on which our work was based.5 Four months later we demonstrated these findings at a meet- ing of the American Society of Biological Chemists (see page 287). The succeeding sections of this paper present reprinted preliminary 4 The references to which I allude gave the substance of a paper in the Transactions of the Wisconsin Academy of Sciences, Arts, and Letters, 1905, xv (1), pp. 209—272, entitled: “On the nature of the process of osmosis and osmotic pressure, with observations concern- ing dialysis.” The results with which our own could be directly compared were the follow- ing ones: Copper oleate was found to diffuse from benzene through a rubber membrane into benzene; and camphor diffused from pyridin, alcohol, and toluol through rubber membranes into the same solvents, respectively. A recent study of Kahlenberg’s paper in the original makes it evident that our results may be explained on the theory of difiusion which Kahlenberg has done much to render convincing. 5 Flack and Hill: Journal of Physiology, 1910, xl, p. xxxiii. DIFFUSIBILITY or LIPINS THROUGH RUBBER 281 reports on various portions of the studies which thus far have devel- oped from the observations described above. It is my intention to discuss each section of the work, and additional experiments, in de- tail at the earliest opportunity, when I hope to dwell more particularly on the significance of such results for the student of the functions of cell membranes, and for the investigator of the coOrdinations and equilibria in intracellular affairs. 11. ON THE DIFFUSIBILITY or BIOLOGICAL SUBSTANCES THROUGH RUBBER 6 The writer and his associates have found that many ether-soluble substances of biological origin, such as fat and cholesterol, pass readily from ether solutions through rubber membranes into ether when the mechanical conditions for such diflusions are favorable. Lecithans appear to be wholly indiffusible. Many substances which are soluble in fatty oils, chloroform, al- cohol, acetone, ethyl acetate, and other solvents of similar powers, or in mixtures of such solvents, promptly diffuse through rubber under suitable conditions. COllodion is one of the products which appears to be indiffusible under such circumstances. When an ordinary ethereal solution of collodion (containing 24 per cent of alcohol) is dialyzed in a rubber condom against ether in a closed vessel, the alcohol rapidly passes to the exterior and the collodion gradually gelatinizes. Liquid accumulates in the bag under these conditions. Various inorganic substances difluse through' rubber under the conditions mentioned above. Ferric sulphocyanate readily passes from ether solution through rubber into ether. The writer inaugurated these studies, with Dr. Rosenbloom’s co- operation,7 in the hope of devising improvements in the methods for the isolation of lipins. The work is progressing along several lines, especially with reference to methods of isolation and purification, and to osmosis. (See page 287.) 6 Gies: Proceedings of the Biological Section of the American Chemical Society: Science, 1911, xxxiv, p. 223; Biochemical Bulletin, 1911, i, p. 125. 7 Rosenbloom and Gies: Proceedings of the American Society of Biological Chemists, 1911, ii, p. 8; Journal of Biological Chemistry, 1911, ix, p. xiv. See page 287 of this volume. 282 DIFFUSIBILITY OF LIPINS THROUGH RUBBER III. A DEMONSTRATION OF OSMOTIC PRESSURE EXERTED BY FAT 8 In the first of two demonstrations, a cylindrical rubber bag, 1% inches in diameter and 8 inches long, was lowered into an oiled muslin bag of about the same dimensions. The rubber bag was then filled to overflowing with olive oil. The rubber bag expanded, as the oil filled it, to the full length and width of the muslin sheath. The sheath prevented further extension of the rubber bag and imparted rigidity to the osmometer that was ultimately constructed. The double bag, full of oil and with its mouth wide open, was then raised so as to inclose about an inch of the lower end of a long glass tube which was firmly supported vertically above the demonstration table. The glass tube was 5 feet long and its bore was 4 millimeters in diameter. Ligatures were tightly secured around the neck of the double bag against the immersed lower end of the vertical tube. The bag then hung directly from the end of the tube. The bag and its sheath were in a tightly distended condition and a stationary column of oil an inch high in the tube was visible above the protruding edge of the Sheath. ' The tube and bag were then lowered into a salt-mouth liter bottle on the table until the bag almost touched the bottom of the bottle. The height of the bottle and the length of the bag were nearly equal. The tube was then marked with a label on the plane of the oil meniscus just above the neck of the bag, and enough ether was poured into the bottle to provide immersion for the bag to the depth of an inch. For a mo- ment no change in the volume of oil was apparent, and the lateral pressure of the ether was obviously without mechanical effect. But in a minute or two downward diffusion currents were visible along the surface of the bag and oil rose rapidly in the tube. After the initial effects of the ether had been shown, the bottle was filled with ether containing Sudan III, and a 5—foot vertical extension of the same bore was added to the upright glass tube. In a moment the upward movement of the liquid was accelerated. The demon- stration was started at about 9 P.M. At 10 P.M. the osmotic pres- sure had carried the column of oily fluid to the top of the 10-foot tube, and liquid continued to run rapidly from the upper orifice until the 8 Rosenbloom and Gies : Proceedings of the Society for Experimental Biology and M edi- cine, 1911, viii, p. 71. DIFFUSIBILITY or LIPINS THROUGH RUBBER 283 apparatus was dismantled after the adjournment of the meeting, at about 11.30 P.M. . During the progress of the demonstration, Sudan III diffused rapidly from the exterior, through the rubber, to the very top of the rising column of fluid, before any of the liquid passed out of the upper open- ing. Oil diffused rapidly through the rubber into the ether. The second demonstration was essentially the same in principle and technic as the first. Instead of a 10-foot upright tube, however, the authors substituted an L tube with an inside diameter of 6 millimeters. The vertical extension of the tube was 17 inches, the horizontal ex- tension was only 3 inches. The latter extension was drawn out to a narrow bore in an inclined plane, to facilitate direct delivery of any liquid that might pass through that end of the tube. When partial immersion of the bag first occurred there was no visible response, but, in a minute or two, oil began to rise in the tube. The bag was then completely covered with ether. The upward move- ment proceeded rapidly, and in about an hour nearly 200 cubic centi- meters of liquid passed through the upper orifice into a graduated cylinder which was supported underneath it to catch the overflow. The authors are engaged in a study of various relationships that are suggested by the demonstrated phenomena. Iv. A DEMONSTRATION OF THE DIFFUSION OF PIGMENTS FROM FAT THROUGH RUBBER INTO FAT9 The writer has found that many fat-soluble pigments, such as Sudan III and Scarlet R, diffuse readily from solid and liquid fats through rubber into various solid and liquid media, among them both solid fat and oil. Thus, when Sudan III is dissolved in melted lard, the red liquid poured into a rubber bag, the bag supported in melted lard in a bottle, and the apparatus promptly immersed in ice water — the fatty matter will congeal before any Sign of pigmentary diffusion occurs, but, in a few hours, a reddish tinge will develop outside of the bag, and on each successive day for several weeks fur- ther extension of the pigmented matter may be witnessed, until the whole of the external lard is deeply suffused with red. This process 9 Gies: Proceedings of the Society for Experimental Biology and Medicine, 1911, viii, p. 73. 284. DIFFUSIBILITY OF LIPINS THROUGH RUBBER takes place quite rapidly when the lard and apparatus are kept in a thermostat at 40° C. ' CONTENTS OF THE RUBBER BAG NATURE OF THE “arms Rm Oil Pigment SUSPENDED 1 Olive oil Scarlet R Olive oil Visible diffusion of the pigment oc- curred promptly 2 Cocoanut oil Scarlet R Cocoanut oil Visible diffusion of the pigment oc~ curred promptly _ 3 Lard oil Sudan III Lard oil Visible difiusion of the pigment oc- curred promptly 4 Paraflfin oil Sudan III Paraffin oil Visible diffusion of the pigment oc- curred promptly 5 Olive oil Sudan III Ether Visible diffusion of the pigment oc- curred almost immediately The demonstrations were intended to exhibit a few instances of such pigmentary diffusions as occur speedily enough at room tem- perature to yield positive results within an hour. The appended summary indicates briefly the precise nature and results of the dem- onstrations (including two control tests —4 and 5), which were made with thin rubber bags in ordinary glass bottles. The bags were securely supported in the bottles, and the mixtures were shaken occasionally during the demonstration. The bags were found, after the adjournment of the meeting, to be without defects. Numerous related experiments are now in progress. V. COMPARATIVE DIALYSIS EXPERIMENTS, WITH DEMONSTRATIONS 10 When dry bags of rubber, gold-beater’s skin, parchment, and collo- dion, each containing olive oil and Sudan III, are separately immersed in olive oil, and the remaining conditions of the environment are uniform, diffusion of the pigment promptly occurs through rubber, but does not take place at all through any of the other three mem- branes. When the bags are lifted from the oil, washed externally 1° Goodridge and Gies: Proceedings of the Society for Experimental Biology and Medicine, 1911, viii, p. 74. DIFFUSIBILITY OF LIPINS THROUGH RUBBER 285 with ether, and then immersed in ether,11 thelpigment quickly passes through the rubber, but diffuses very slowly if at all through the remaining membranes. Successive immersions of moist impermeable membranes (gold- beater’s skin and parchment) in alcohol and ether, for different pe- riods of time, fail to render the treated membranes more permeable to Sudan III than before. The authors demonstrated the general facts in this connection pertaining to rubber and gold-beater’s Skin. Experiments along these lines, with additional membranes, pig- ments, and liquid media, are in progress, in an effort to obtain further knowledge of the functions of membranes in diffusion. VI. EXPERIMENTS ON THE DIFFUSIBILITY OF ALKALOIDS THROUGH RUBBER 12 Rosenbloom and Gies have found that various ether-soluble sub- stances, when dissolved in ether and placed in rubber bags immersed in ether, readily pass through the rubber membranes thus imposed.18 We have found that various alkaloids and some related substances readily diffuse through rubber under such conditions. Our experiments were conducted as follows : A moderate quantity of the pure ether-soluble substance was mixed with 15 to 25 cubic centi- meters of ether.“ This mixture was poured through a funnel into a new air-tight rubber condom in such a way as to preclude the possibility of overflow upon the external surface. The bag was then immersed in about 50 cubic centimeters of ether in a narrow salt-mouth bottle 7 inches high. With the bag suspended at full extension in this position, its mouth was about an inch above the opening in the bottle. 11 In experiments which the senior author has been conducting with Dr. Welker’s cobperation, it has been found that collodion bags are disintegrated by ether containing more than about I. 5 per cent of alcohol. Pure ether does not dissolve or in any way dis- organize collodion membranes. A collodion bag containing pure ether may be immersed for a week or more in pure ether without undergoing any appreciable deterioration. 12 Sidbury and Gies: Proceedings of the Society for Experimental Biology and Medicine, 1911, viii, p. 104. 13 Rosenbloom and Gies: Journal of Biological Chemistry, 1911, ix; Proceedings of the American Society of Biological Chemists, p. xiv (December, 1910); also, Proceedings of the Society for Experimental Biology and Medicine, 1911, viii, p. 71. 1‘ Substances which did not dissolve readily were triturated with ether in a mortar. .286 DIFFUSIBILITY OF LIPINS THROUGH RUBBER The protruding condom was supported in the neck of the bottle by a tightly fitting cork stopper, which also served to keep the bag closed. After a diffusion period of convenient length (sometimes 2 to 5 days),15 the condom was removed from the bottle, the ether diffusate was poured into a porcelain dish, and the ether completely removed by evaporation on a steam bath. At least one appropriate test was then applied to the residue.16 Meanwhile, the ether solution in the condom was removed. A large volume of water was then poured into the suspended bag, which, during its distention by the water, was carefully examined for signs of leakage. In a few instances defective membranes temporarily rendered the outcome doubtful. All results with such bags were ignored, of course. Each of the tests, even after reliable positive responses, was repeated at least once with a new rubber bag. The substances named below (the complete list of those already tested in this connection) are readily diffusible under the conditions of these experiments : —— A. Apomorphin, atropin, brucin, caffein, cocain, codein, colchicin, coniin, morphin, narcein, narcotin, nicotin, physostigmin, quinin, strychnin, veratrin. B. Acetanilid, antipyrin, phenacetin, picric acid, picrotoxin, pyram- idon, salicylic acid. Experiments with other solvents, and with additional substances of alkaloidal type, will be added to this series. 15 Some of the alkaloids pass through rubber almost immediately under the conditions of these experiments. 1° In the experiments with nicotin, the “tobacco odor” of the concentrated liquids was very pronounced. 3. A STUDY OF THE DIFFUSIBILITY OF LIPINS FROM ETHER THROUGH RUBBER MEMBRANES INTO ETHER* BY IAOOB ROSENBLOOM MANY experiments, in completion of the diffusion work I have been doing in collaboration with Dr. Gies,1 have been performed to deter- mine the diffusibility or non-diffusibility of lipins and similar sub— stances (page 280). Such data must obviously be obtained in detail, if any attempt to devise methods for the isolation and purification of lipins by dialysis through rubber can be successful. I present here briefly the essential results of the work already com- pleted in this connection. In the experiments described below, ordinary rubber condoms were used as diffusion membranes.2 Various kinds of “ sheet rubber,” known as “ pure Mexican plantation rubber,” and fumed with carbon- disulphid, were found to be good membranes for this kind of work, but besides allowing fats, fatty acids, soaps, cholesterol and lipochrome to diffuse through it, this sheet rubber also permits the passage of lecithans under the conditions to be described, although the lecithans pass through the sheet rubber very slowly compared with other lipins such as fat and cholesterol. Condoms do not permit the diffusion of lecithans under the conditions of the tests to be described, and they were preferred for this work for that reason. The cause of the Observed difference in permeability is unknown to us, but will soon be made the subject of special inquiry. The substances to be tested in the diffusion experiments were dis- solved in 100 to 200 cubic centimeters of ether (anhydrous and distilled over sodium), the concentrations of the solutions varying from 0.5 * Reprinted from the Biochemical Bulletin, 1912, ii, p. 64. 1 Rosenbloom and Gies: Proceedings of the American Society of Biological Chemists, 1911, ii, p. 8; Journal of Biological Chemistry, 1911, ix, p. xiv. 2 Befqre the condoms were used for this purpose, they were placed in fresh portions of ether daily for several days, to free them from the powder adherent to them. This is especially important when one proposes to test the dialysates for phosphorus, since the adherent powder has been found to contain phosphorus. 287 288 DIFFUSIBILITY OF LIPINS THROUGH RUBBER to 5 per cent. The solution or suspension was carefully poured through a funnel into a new air-tight rubber condom in such a way as to pre- clude the possibility of overflow upon the external surface. The bag was then immersed in from too to 200 cubic centimeters of pure ether in a wide-mouthed bottle of convenient size and suspended loosely by a thin cord held securely between the stopper and neck of the tightly stoppered bottle. The bottle was kept well stoppered through- out the whole of each test to prevent egress of ether and ingress of dust and _other extraneous matter. I. Ether Extract of Egg-yolk. — Within five minutes after ether extract of egg-yolk is subjected to the diffusion treatment described above, the lipochrome appears in the dialysate, diffusion currents being visible about the same time. The following substance can be detected in the dialysate after short periods of dialysis: fat, fatty acid, cholesterol, and lipochrome. The lecithans do not pass through the condoms, even during prolonged periods of dialysis. We tested for lecithans in the dialysate by analyzing the evaporation residue for phosphorus by the fusion and Neumann methods, and by seeking an “ acetone precipitate ” in the concentrated ether solution after the addition of electrolyte (sodium chlorid). Sometimes a positive phos- phorus test was obtained from a dialysate which did not yield an “ ace- tone precipitate.” In such cases, it was found that this result was due to the presence of glycerophosphoric acid in the dialysate. If to a solution of lecithans, which, after dialysis in a condom, does not yield a phosphorus compound to the dialysate, one adds some glycero- phosphoric acid, and then dialyzes this solution through the same rubber condom, glycerophosphoric acid appears in the difiusate. 2. Ether Extract of Brain. — The dialysate from ether extracts of brain contained fat, fatty acid, and cholesterol. Lecithans failed to dialyze. 3. Ether Extract of Heart Muscle —— Fat, fatty acid, lipo- chrome, and cholesterol were detected in the dialysate from ether extracts of the heart muscle of oxen. Lecithans did not diffuse. 4. Ether Extract of Kidney and Liver (Dog). —Fat, fatty acid, lipochrome, and cholesterol appeared in the diffusates from ether extracts of dog kidneys and livers. Lecithans did not dialyze. 5. Ether Extract of Blood (Dog). —— Fat, fatty acid, lipochrome, and DIFFUSIBILITY or LIPINS THROUGH RUBBER 289 cholesterol occurred in the dialysates from ether extracts of dog blood. N o lecithans dialyzed. . 6. Ether Extract of Carrots. —— The coloring matter dialyzes very rapidly from ether extracts of carrots. A small amount of fat was also present in the dialysate. 7. Ether Extract of X anthoma (S kin). — The yellow coloring matter dialyzes very quickly from an ether extract of xanthomatous skin, but it faded at the end of twelve hours. 8. Ether Extract of Cerumen. — Cholesterol, fat, and fatty acid were present in the dialysate from ether extracts of cerumen. Neither the coloring matter nor the lecithans diffused. 9. Ether Extract of Yeast. —— The dialysates from ether extracts of yeast exhibited a peculiar opalescence, even at the end of six weeks’ dialysis. A small amount of fat dialyzed, but lecithans did not diffuse. IO. The following Substances or special Products, when subjected to diffusion by the method described above, were found to be diffusible :— Ethyl butyrate Lactic acid Urochrome 3 Ethyl acetate Beta-hydroxy-butyric acid Sudan III Oleic acid Acetone Olive oil stained with Sudan HI Palmitic acid Glycerol 4 Caseinogen (Lipochrome and fat dialyzed) Stearic acid Cholesterol (from brain, egg- Sodium stearate5 yolk, and gall-stones) Acetic acid- Cholesterol-acetate Sodium palmitate 5 Formic acid Cholesterol-benzoate Potassium stearate 5 Propionic acid Butter (fresh and rancid) Potassium palmitate 5 Butyric acid Mutton tallow Valerianic acid Olive oil Lead oleate In some special experiments we found 6 that cholesterol benzoate, cholesterol stearate, cholesterol oleate and cholesterol palmitate, when dissolved in ether, readily diffuse through rubber into ether. 3 Ether-alcohol solution (equal amounts) dialyzed against ether-alcohol. 4 When ether-alcohol solutions of glycerol are dialyzed against ether-alcohol, and alcohol solutions of glycerol are dialyzed against ether, the dialysates contain glycerol. 5 Treated with water, then with alcohol to the point of precipitation, then with ether until a precipitate was produced. The filtrate was dialyzed against water, alcohol, and ether in the same proportions. 6 Boas and Rosenbloom: Proceedings of the Society for ExjJerimentaZ Biology and Medi- cine, 1911, viii, p. 132. 290 DIFFUSIBILITY OF LIPINS THROUGH RUBBER Cholesterol stearate with a molecular weight of 652.6r diffuses, whereas the various lecithans, with molecular weights considered to be 770 to 785, do not. If we assume that the diffusion of a sub- stance depends on the size of its molecules, the above facts strengthen Hiestand’s conclusion that the molecular weight of egg-yolk lecithin is 1446, which figure he obtained by a molecular weight determination. 11. I ndifiusible substances. -—— The following substances, when subjected to diffusion by the method described above, were found to be indiffusible.7 Sodium chlorid Lecithans from yeast Lecithans from brain Lecithans from wheat embryo Lecithans from egg-yolk Kephalin from brain Lecithans from heart muscle Cuorin from heart muscle Lecithans from pig testicle Compound of lecithin with platinic Lecithans from liver and kidney chlorid Koch8 has lately described the preparation of various compounds with lecithans, but it is uncertain whether these compounds are colloidal adsorptions, mechanical mixtures, or true chemical compounds. It seemed of interest to study the behavior of these substances in ether solution, when subjected to dialysis in rubber bags suspended in ether. The preparations used in these experiments were made according to the method described by Koch. For the dialysis tests the solu- tions of the lecithan compounds were evaporated to dryness at 38° and the residues triturated with ether. The extracts were filtered, and the filtrates placed inside of rubber bags and dialyzed against ether for thirty-seven days. The dialysates were tested every week to see if the substance combined with the lecithan diffused. Compounds of lecithin with glucose, lactic acid, strychnin, digi- tonin, salicin, urea, creatin, creatinin, and caffein were prepared. It was found that the glucose and lactic acid dialyzed completely, the strychnin, digitonin, and salicin dialyzed partially, while urea, creatin, creatinin, and calfein did not dialyze at all.9 7 We have found that lecithans prepared by the Zuelzer,Bergell, or Roaf and Edie method when dialyzed always yielded traces of cholesterol and often fat to the dialysate. 8 Koch and collaborators: Journal of Pharmacology and Experimental Therapeutics, 1910, xii, 239-269. 9 Boas and Rosenbloom: Loc. cit. DIFFUSIBILITY OF LEINS THROUGH RUBBER 291 It was thought that some of the various substances which did not diffuse might do so in the presence of a considerable amount of diffu- sible material, but on dialyzing various mixtures of the above-named indifiusible substances with varying amounts (up to 15 grams) of neu- tral fat, fatty acid, cholesterol, or olive oil, no diffusion of lecithans occurred. When solutions of lecithans are subjected to dialysis by the method described above, they take up a great deal of ether, and the volume of liquid in the bag is greatly increased. We have demonstrated that lipins exert strong osmotic pressure. (See page 282.) We have also placed ether solutions of lecithans with cholesterol and fat in closed rubber bags suspended in Soxhlet extractors. Soxhlet extraction in the usual way failed to remove lecithan from the bag under these conditions. These findings favor the development of a method for the thorough removal of impurities from lecithan solutions. It is perhaps superfluous to add that the results already mentioned may be obtained by placing the solution to be tested outside the rubber bag and allowing dialysis to take place into pure ether contained in the bag. SUMMARY OF GENERAL CONCLUSIONS I. Most lipins, chief among them fat, fatty acid, soaps, cholesterol, cholesterol-esters, lipochrome, and various other ether-soluble sub- stances, diffuse from ether solution through rubber membranes into ether. _ 2. Sodium chlorid, lecithans prepared from various sources, kephalin, cuorine, and the compound of platinum with lecithin, do not diffuse under such conditions. 3. One or more of the diffusible substances in these experiments may be dialyzed from solutions containing them, together with one or more of the indiifusible ones, without inducing any of the latter to pass through the membrane. C. INFLUENCE OF CANCER EXTRACTS I. A STUDY OF THE INFLUENCE OF CANCER EXTRACTS ON THE GROWTH OF LUPIN SEEDLINGS * BY JACOB ROSENBLOOM 1 INTRODUCTION. —— One Of the peculiar effects Of cancer is the resul— tant cachexia. There have been many efforts to find, in cancer tissue, a poison that might account for the characteristic condition. It has been claimed that the cachexia is due to pressure by the growing tumor on the blood-vessels and consequent interference with adjacent circulation, with development of areas of necrosis, autolysis, and pro- duction of hemolytic and toxic substances. Riilf 2 considers that proteases are impOrtant factors in the causa— tion of cancer cachexia. Bard 3 found that blood is rapidly hemolyzed in hemorrhagic carcinomatous exudates in serous cavities, which is not the case in exudates under other conditions. Kullmann 4 Observed that extracts of carcinoma contain hemolytic substances that are active in oioo and in vitro, soluble in alcohol and water, and toxic for all varieties of corpuscles. Micheli and Donati 5 also found hemolytic substances in eight Of sixteen tumors, of which five hemolyzed all varieties of corpuscles and three acted on some varieties only. They thought the hemolytic substances result from autolysis of the tumors, as it is well known that certain hemolytic substances occur among the products of autolysis of normal tissues. Mtiller 6 claimed, from the * Reprinted from the Biochemical Bulletin, 1913, ii, 229—232. 1 This paper presents the results of a preliminary study that was begun, at Dr. Gies’ suggestion, as a part of the plan of biochemical research described on page 1 53. ' 2 Riilf : Zeit. f. Krebsforsch., 1906, iv, 417. 3 Bard : La semaine med, 1901, xxi, 201. 4 Kullmann : Zeit. f. klin. Med, 1904, liii, 293. 5 Micheli and Donati : Riforma med, 1903, xix, 1037. 6 Miiller: Zeit. f. klin. Med, 1889, xvi, 496. 292 INFLUENCE OF CANCER EXTRACTS ON LUPIN SEEDLINGS 293 results of a study of nitrogenous metabolism in cancer patients, that in cachexia of cancer there is toxogenic destruction Of protoplasm inde~ pendent of nutrition, i.e., a specific toxic effect Of cancerous tissue. Miiller’s results have been confirmed by other workers,7 but cumulative research has shown that the cases with normal pro- tein catabolism exceed in number those with increased protein catabolism.8 According to the prevailing opinion c'ancer cachexia is not specific, but is the same as the cachexia of other conditions. It has been impos- sible to show the occurrence in cancer tissue of any substance that would account for the cachexia of this disease.9 In the experiments described below, we studied some of the effects of extracts of cancer tissue on the growth of lupin seedlings, in the hope that this procedure for the detection of toxic and stimulating substances might yield significant results. Experimental. Preparation of lupin seedlings. — Lupin seeds were soaked in water overnight. Seeds Of the same size were then selected and planted in wet moss. After three, or four days the seed- lings were taken from the moss, the coat of each removed, and the sprout rinsed with distilled watei'l The root was carefully measured on a millimeter'scale. The seedlings were then fastened on glass rods drawn out at one end to form a sharp-pointed L and suspended in per- forated cork covers over 400 cubic centimeter Jena beakers, each con- taining 200 cubic centimeters of water and 5 cubic centimeters of boiled or unboiled cancer extract prepared as described below. “Control” seedlings were suspended in distilled water. The glass rods were so adjusted that the roots were immersed in the liquid, but the cotyledons were not in contact with it. Four seedlings were suspended in each beaker. At intervals of 20 hours all the seedling roots were measured.10 7 Char. Annal., 1891, xvi, 138; Arch. prov. de Med, 1899, March; Arch. f. Verdau— ungskn., 1899, v, 540; Riv. wen. d sci. Med, 1899, xvi, 31; Zeit.f klin. Med, 1897, xxxiii, 385. 8 Zeit. f. Krebsforsch., 1904, i, 199; Salkowski Festschrift, Berlin, 1904, 75; Fifth Ann. Rept. Cancer Lab., New York State Dept. of Health, 1903,—1904. 9 Blumenthal : Salkowski F estschrift, Berlin, 1904. 1° True and Gies: Bulletin of the Torrey Botanical Club, 1903, m, p. 390; Rose: Bio- chemical Bulletin, 1911, i, 428. 294 INFLUENCE OF CANCER EXTRACTS ON LUPIN SEEDLINGS DATA SHOWING EFFECTS OF EXTRACTS OF CANCEROUS AND NORMAL TISSUES ON THE GROWTH OF LUPIN SEEDLINGS I. Extract of Bone Sarcoma Rate of growth per plant in millimeters Lupin seemings ' Unboiled extract Boiled extract 1st 20 hr. 2d 20 hr. 1st 20 hr. 2d 20 hr. A . 14 19 18 30 B . 12 28 13 38 C . 20 22 1 5 24 D . . . . 17 4O 16 30 Average . . . . 16 27 16 30 Control (average) . 5 8 6 8 II. Extract of Fibroma of Uterus A . 8 12 6 10 B . 6 12 8 10 C . 8 IO 8 12 D . . . . 6 8 8 16 “ Average . . . . 7 10.5 7.5 12 Control (average) . 8 IO 8 11 III. (a). Extract of a Carcinoma of the Breast A 18 9 12 8 B 12 7 12 6 C . . . . . . . . 15 8 9 8 D . . . . . . . . 1 5 8 1 5 8 Average . . . . . . . I5 8 12 7.5 Control (average) . . . . l 17 7 16 8 III. (b). Extract of Normal Breast Tissue near the Cancer 11 A . 20 8 26 6 B . I8 6 17 8 C . 24 8 24 8 D . . . . . 22 6 2 5 7 Average . . . . . . . 21 7 r 23 7.3 III. (c). Extract of Pectoral Muscle Removed at Operation 11 A . 14 7 26 9 B . 26 6 18 9 C . . . . . . . . 14 11 26 12 D . . . . . . . . 24 8 24 10 Average . . . . . . . 19.5 8 23.5 10 11 “ Control” figures are given in section III (a) INFLUENCE OF CANCER EXTRACTS ON LUPIN SEEDLIN GS 29 5 Preparation of cancer extracts. —-— Fresh cancerous tissue, direct from the operating room, was minced, then triturated with sand and water, and the thin mixture frequently shaken for about an hour. The liquid was strained through gauze, then filtered. Portions of this filtered extract (boiled or unboiled) were used in the manner indicated above. Data pertaining to growth. — The accompanying summary pre— sents the result of this study. General conclusion. —-— The extracts failed to inhibit growth of the seedlings. The Observed acceleration of growth was probably due to inorganic salts in the extracts. It is possible, of course, that deleterious action by cancer toxins was neutralized or overcome by the stimulating power of associated nutrient or other substances. This particular point requires special investigation.12 12 Additional studies in this series will be published by Gies and collaborators. [The titles of papers and reports on biochemical studies under Crocker auspices, which have not been reprinted in this volume, are listed on page 158.] INDEX Abderhalden, Use of tyrosin by, 163, 190; on a peptidolytic enzyme, 191 ; on compounds Of cholesterol, 268, 269 Abdominal organs, Successful inoculation of, I35 Abnormal growths, so. 36, 37, 40—45, 49, 56, 57 Achylia gastrica, Free hydrochloric acid absent in, 184 Agar, Failure of tissues to grow on, 59—60 Agglutinable blood, Transfusion of, useless, 223, 224 Agglutinable substances, Two, in rabbits’ blood, 227 ; one, in blood of steers, 228—29; in human blood, 236 Agglutination, in diluted and in mixed sera, 216; action of non-agglutinable cells on, 217—18, 224; macroscopic and microscopic, 218, 219; connection between phagocytosis and, 223, 224; should be tested for, 224 Agglutination, intravascular, Transfusion and the question of (R. OTTENBERG) 211—24 Agglutinative and hemolytic transfusions, Ex— perimental (R. OTTENBERG, D. I. KALISKI, and S. S. FRIEDMAN) 243—59 Agglutinative bloods, Mixtures of mutually, normal and anemic, 219—20 Agglutinin, Quantitative relations between, and agglutinable and non-agglutinable cells, 214— 221; absorption of, 214—15, 218, 221, 224, 237; concentration of, 215, 224; one, in blood Of steers, 228-29; absorption of, in viva, 242 Agglutinins, Landsteiner assumed existence of two, 212, 236; absorption of, from animal sera, by human red blood cells, 22 5; two, in rabbits’ blood, 227 Alkaloids, Compounds of lipins with, 275—76; diifusibility of, through rubber, 285-86 Alveolar spindle cell sarcoma, Dog tumor shows as an, 120. Amino acids, 162, 179; compounds with, 269 Amylopsin, Use of, 97—98 Anemia, pernicious, Free hydrochloric acid absent in, 184 ’ Anemia, Transfusion for relief of, fatal, 221—22 Anemic bloods, Experiments with, 219-20 Animals experimented on, 1 ; young, more sus- ceptible than Old, 7; plan for breeding, fails, I54 Animals, Higher, pnits of organization, 51 Animals, lower, Isoagglutination in the, 22 5-29; overlooked, 237 Apolant, on mouse tumors, 66 Apolant and Marks, on inoculation with autolo- gous spleen, 146—48, 149 Aschoff, on myelin, 268 Ascoli, on isoagglutination, 21 1 Autoagglutination, Landsteiner on, 242 Auto-cells, To render animals resistant to, the problem, 137 Auto-inoculation of the spleen in mice, 127—3 1, 137—49 Autologous normal tissue, Use of, limited, 138; inoculation with, does not evolve resistance, 139’ 1:45—47) 149 Autopsies of dogs, 2 50, 254 Autospecificity, 156 Axilla of normal mice, Inoculation of tumor into, 133,136,138,I4O—48 3.. Mrs., Case of, 107 Balance, Physiological, in normal organisms, 2—5; upset by mutilation Of cell, 51—52, 55, 58; restoration of, not easy, 56, 58 Balbiani, Studies of, in regeneration, 10, 31, 58; on Paramecium, 46; on cause of monsters, 48 Baldi, on Drechsel’s jecorin, 269 Bang, Classification of lipins proposed by, 264; on application of term “carrier of life,” 266 ; on cholesterololeate, 268 ; combinations of lecithin, 269; on vitellin, 274; papers by, 277 Bard, on cancer exudates, 292 Barsky, Dr. M. H., exposed rats to Roentgen rays, 81 Bashford, E. F., on immunity, 97; on tumors in lower animals, 117, 124; on disintegration of cells, 130; thanks to, 131 Bashford, E. F., Murray, and Cramer, 131 Baskoif, an alcoholic solutions of lecithin and of glucose, 27o; liver jecorins of, 271 Beal, George D., r 56 ; joint author, 159 Beard and Mackenzie, Use of trypsin and amy- lopsin by, 97—98 Beebe and Crile, Treatment of cancerous dogs by, 97 Bell on atrophy of thyroid gland in carcinoma, 87.129 Benedict, Stanley R., Studies of, 154, 155, 158; resigned, 155 n 2 Berg, A. A., Operations by, 186 297 2998 INDEX Bibliography of auto-inoculation, 131, 149 of biochemical research, 158—59 of colloidal nitrogen of the urine, 200, 206 of dog tumors, 124 of glycyltryptophan and tryptophan tests. 190, 191—93 ——-———— of immunization, 136 of irritation of tissues, 76, 84 of lipin compounds, 277 of mutilations of Paramecium, 58 of relation of internal secretions to tu- mors, 109 of tonicity in isohemagglutination, 235 Biochemical investigations, General programme 0L 1909—II,Iss—59 Biological chemistry, Department of, 153—242 Biological substances, Diffusibility of, through rubber, 281 Biological work done, 1909—11, Report on, 1—5 Biology, Department of, 1—109 Blastoderm of the chick, Development of, in vitro, 111—16; earliest phenomena of, 115—16 Blastomycetes, San Felice produced tumors by injection of, 85 Blood, cellular elements of the, Resistance con- ferred by the, 127 Blood, Occult, in cancer of stomach, 161, 172, 184; in stomach contents, 165—66; test for, superfluous, 169-70; does not vitiate tryp- tophan test, 188 Blood cells, Circulation of the, in chick embryo, observed, 116 Blood cells, red, Isoagglutination of human, 211, 225, 236; number Of, 214; Of other animals, 22 5; resistance of, to laking, 230—31 Blood picture, A remarkable, 2 59 Blood smears, Table of, 256 Bloodgood, Joseph C., on bone tumors, 120, 121, 124 Bloods, All human, divided into four groups, 211, 230, 236; groupings hereditary by the Mendelian law, 213 Bloor, on synthesis of carbohydrate esters, 272 Blumenthal, Paper by, 293 n 9 Boas, Ernest, 156; Obtained duodenal contents from stomach, 187 Boas and Rosenbloom, Paper by, 289 n 6 Boas-Oppler bacilli, in cancer of stomach, 161, 184 Boggs and Morris, on experimental lipenia in rabbits, 27 5 Boldyreif, Use of Olive Oil by, 187 Bondet, on seroline, 268 Bondzynski and Gottlieb, on urine of cancer patients, 206 Bone sarcoma, Extract of, 294 Borrel, Inoculation of liver, spleen, and brain by, 128,131 Borroughs, Successof, 59 Borst, M., on tumors of the long bones, 124 Bothriocephalus latus, cause of hemolysis and anemia, 268 Brahm, Paper by, 277 Brain, Ether extract of, 288 Breast tissue near the cancer, Extract of, 294 Bridré, Inoculation experiments of, 128, 131 Bromin, as test for tryptophan, 163—65, 179, 189—90.Ios Brooklyn tumor in Buffalo laboratory, 6 Bullock, Frederick D., 1, 2, 3, 4, 6—9 BULLOCK, FREDERICK D., Notes on the growth of tissues under experimental conditions, 3, 59—61 —— Other efforts to bring about chronic irri- tation, 85-86 Spontaneous tumors, 66—69 see also ROHDENBURG, GEORGE L., and BULLOCK, FREDERICK D., and G. L. ROHDEN— BURG, Retrograding tumors, 70—72 BULLOCK, FREDERICK D., G. L. ROHDENBURG, and P. I. JOHNSON, Inoculation with two types of tumor, 73—74; tumor tissue in col- lodion sacs, 3, 62—63 Bunting, Blood pictures observed by, 259 C., Miss, Case of, 106 Cachexia, The characteristic, of cancer, 292-93 CALKLNS, GARY N., Effects of mutilations by cutting on Paramecium, 3, 30—58, 77—78 Regeneration and cell division in Urony- chia, s, 10—29, 70, 77 Report on biological work done 1909-1 1, I—s (and others), Notes on tumor transplan— tation in general, 6—9 Campbell, on vitellin, 273 Cancer, Biological phenomena of growth of, 2; relation of cell-cutting to, 51—53 ; connection Of irritation and, 55; reputed cures Of, 97; treatment of human cases of, with gland extracts, 5, 102—9; sometimes an apparent cure of, 109; real problem of cure of, 137; an insurmountable obstacle, 149; conclusions from review of chemical literature on the subject of, 153—54; a prob- lem of cell growth, 155; methods for diag- nosis of, 160—208; importance of early diagnosis of, 200 Cancer, diagnosis of, Importance of the colloidal nitrogen of the urine in the (M. EINHORN, M. KAHN, and J. ROSENBLOOM) 200—5 Cancer, spontaneous, Cells of, aberrant, 156 Cancer, transplanted, Resistance against, in mice, produced by auto-inoculation of the spleen, 127—31; refuted by later experiments, I37—49 Cancer cachexia, 292—93 Cancer cells, of same species as cells of host, r56; autospecific aberration of, 156 INDEX 299 Cancer enzymes, Proteolytic powers of, 162, 166, 179, 181; sugar stimulates secretion of, 173; Emerson on, 178, 191 Cancer extracts, Influence of, on growth of plant and animal cells, 153; on growth of lupin seedlings, 292—95; preparation of, 295 Cancer extracts and products, Effects of, on local and systemic nutrition, 153—54 Cancer of the stomach,. The glycyltryptophan and tryptophan tests for (C. H. SANFORD and _T. ROSENBLOOM) 191—99 The new test for (I. W. WEIN- STEIN) 161—77: Factors of diagnosis of, 161; the glycyltryptophan test, 161—65 ; change of proteins into cleavage products, 162; proteolytic powers of cancer enzyme, 162—63; method of procedure, 163—65; sources of error, 165—66; experiments show- ing glycyltryptophan not necessary, I66— 171; positive results, 171; the trypto- phan test, 171-72; conclusions, 172—73; record of cases, 173—77 The tryptophan test for, with special reference to peptidolytic enzyme in the saliva (I. W. WEINSTEIN) 178—90: The glycyltrypto- phan test, 178—79; tryptophan test, 179—80; Warfleld’s study and findings, 180—83; expe- riences with tryptophan test, 183—86; dis- advantages, 186—88; technic, 188—90 Cancer patients, Inability to gain access to, 154 Cancer research, The basic problem in, 77 Carbohydrates, Compounds of lecithin with, 269—72 Carcinoma and female organization, 8 Carcinoma, Contributions to the theory of the individuality of, 137—49 human, Hodenpyl’s case Of, 97 ———-— of the breast, Extract of, 294 Scirrhous, of slow growth, 186 ventriculi, Diagnosis Of, 161 Carcinomata, Rat, received, 9 Carrel, Alexis, Success of, 59 Carrel and Burrows, Method Of Obtaining plasma, 113 “Carrier Of life,” Application of the term, 269 Carrots, Ether extract of, 289 Cazin and Heimeter on chronic irritation, 75 Cell, abnormal, Division of the, 37, 58 Cell, Division energy of the, 9; in Urony- chia, 10—29; general remarks on, 25—29; in Paramecium, 32, 54—57, 58 Cell, A review of the chemistry of the, 158 Cell, Studies in physiology of the, 10; delicate Cobrdination of parts of, 31; balanced phys- iological condition of the, 48, 53; Hertwig and Popoff on, 48—51; mutilation of, upsets balance, 51—52, 54—55 Cell constituents and produéts, Relationships of, 155, 157; papers on the, 158-59, 209 Cell division, Uncontrolled, in cancer, 77; a stimulus to, of intracellular origin, 157 Cell growth, Cancer a problem of, 155 Cell proliferation, Epithelial, produced experi- mentally, 85 Cells, Concentrated non-agglutinable, interfere with agglutination, 219 Cells, Division and regeneration of, 3, 10—29; independent phenomena, 51, 54, 58, 77 Cells in depression, a misnomer, 52—53 Cells, Influence of cancer extracts on growth of, 153; relationships of constituents of living, I55 Cells, normal or tumor, Pronounced individ- uality of, 149 Two races of, in cut Paramecium, 37 Cerumen, Ether extract of, 289 Chemicals, Effects of, on cell protoplasm, 53—54, 55; after chronic irritation, 77—86 Chevreul, on cholesterin, 268 Chick, Development of the blastoderm of the, in vitro, III—16; vascular system in action, 111; the incubator, 112; the plasma, 113— 1 14 ; factors preventing prolonged growth, 1 14 ; growing embryos, 114 ; isolated spaces, channels and plexus, 115—16 Child, on movements in regeneration, 47 Chimney sweeps, Cancer in, 55 Chlorophyllan, from grass, 27 5 Cholesterol, Compounds of, 268 Cholesterol-oleate found in many portions of the body, 268 Chromatin, Bodies of, in Uronychia, 12, 29 Chromolipins, Paper on, 159 Ciliate, Holotrichous, see Paramecium Ciliates, Hypotrichous, II; division of, 13—14, 30—31 Cirrhosis of the liver,-Occult blood in cases of, I84 Cirri of Uronychia, 11 ; movements produced by, 12; absorbed, 13; new, 13, 19; precocious, 28 Clark, Ernest D., Intracellular carbohydrates, 158 Classification of the lipins, 260—66: Proposed by Rosenbloom and Gies. 261—62; earlier, by Thudichum, 263; by Bang, 264; by Rosen- heim, 265; by Leathes. 265—66; by Cramer, 266 Clinical Pathology, Department of, 127—49 Clubroot in vegetables, 55 Cobralethicid, Opinions on, 276 Coca, Experiments of, in transfusion, 213 Cohen, on result of removing the ovaries, 87, 109 Collodion, a serviceable membrane for diffu- sion, 279; indiffusible, 281 Collodion sacs, Experiments on tumor tissue in. 62—63 300 INDEX Colloidal nitrogen, The, in urine from a dog with a tumor of the breast (MAX KAHN and J. ROSENBLOOM) 206—8 of the urine, Importance of the, in the diagnosis of cancer (MAX EINHORN, M. KAHN, and J. ROSENBLOOM) 200-5; Salkowski’s new tests for, 200; method, 201 ; normal cases, 201 ; malignant cases, 203; non-cancerous diseases, 204 —————-— Salko wski method for determination of, 207, 208 Columbia University, Experiments at, 30, 33 Condoms, Rubber, used as difiusion membranes, 287 Conradi, on “gall-stone fat,” 268 Contributions to the theory of the individuality of carcinoma (W. H. WOOLOM) 137—49 Cramer, Classification of lipins suggested by, 266 Crile, on danger in transfusion, 213 Crocker Cancer Research Fund of College of P. and 8., I31, I36, 149 Crown galls, Similarity of, with cancer, 55—56 Culture media, Attempts to grow tissues on, 3; employed by Reed, 59 Curative agents, X substance found in, 99—100; a common active substance in, 100 Cutting, retards cell division, 27, 5o; difficult with Paramecium, 31—33, 57 Cytoplasm, Condition in, changed, relation of, to nucleus, 49—51 28-29; Decastello and Sturli, on tonicity, 235 Delafield’s hematoxylin, 15 Delage, Yves, Thanks to, IO Development of the blastoderm of the chick in vitro (J. E. MCWHORTER and A. 0. W111?- PLE) 111—16 : The apparatus, 112—13; the technic, 113—14; observations, 114-16 Dialysis, of “colloidal” nitrogenous material, 207—8; should be tried on urine of cancer patients, 208; some problems of, 278—81; experiments, 284—85 Difiusible substances, 289 Diffusion, Modified collodion membranes for studies Of, 159, 279; through rubber, 279—86, 287—91 Dipeptid, defined, 162 n 2 Disease, an expression of intracellular disturb- ance, 157 Division energy, 9; independent of power of regeneration, 51, 54, 58, 77; of specific cells enhanced in tumor growth, 53; artificial increase of, 53; deviation or suppression Of, by mutilation, 54—55, 77; stimulated by irritants, 55—56, 78 Division of Uronychia, 12—14 Dog blood, Isoagglutination in, 236—41, 243—47; ether extract Of, 288—89 Dog-blood cells, Fragility of, 243, 245 Dog, Rare tumor in a St. Bernard, 117—24 Dog tumor tissue, Transplantation of, 122; culture of, in vitro, 122—24 Dog with a t1imor of the breast, Colloidal nitro- gen in urine from a, 206-8 Dogs, ideal experimental animals, 243 ; isoag- glutination in, 243—47; isohemolysis in, 247 ; technic and plan of transfusions in, 247—48; results, 248—57 ; conclusions, 2 57—59 Donath, on tonicity, 23 5 Doses of normal tissue and of tumor, Relation of, 138, 142, 144 Drechsel, on a silicic acid ester of cholesterol in bird feathers, 268; on jecorin, 269 Duodenum, Regurgitation Of contents of, I87— 188 Earthworm experiments, 85—86 Eddy, Walter H., Intracellular proteins, 158 Effects of certain chemicals on the type of tissue resulting from chronic irritation (G. L. ROHDENBURG and F. D. BULLOCK) 77—86 Effects of chronic irritation on tissues (G. L. ROHDENBURG) 4, 75—76 Effects of mutilation by cutting on Paramecium (G. N. CALKINS) 3, 30—58 Egg albumen, Experiments in injection of, 80—83 Egg, Removal of blastoderm from the, 113 Ehrenberg, on tests for gastric cancer, 190, 192 Ehrlich, on tumor mixtures, 73; on immunity, 91,97 Ehrlich Laboratory, Studies of tumors at the, 66 Einhorn, Max, 156 EINHORN, Max, Max KAHN, and JACOB ROSEN- BLOOM, The importance of the colloidal nitro- gen of the urine in the diagnosis Of cancer, 200—5, 206 Embryo skin, emulsion of, Inoculation with, 132 Embryos of chick, Attempts to grow, in vitro, 114 Emerson, on cancer enzyme, 178, 191 Encapsulation in bone tumors, 118, 121 Enderlein transported organs, 6O Enzyme activity inhibited by X substance, 101 ; preceded by stimulation Of, 102 Enzymes, The digestive, 162 Enzymes, Proteolytic, generated in malignant tumors, 162, 166, 178, 179, 181, 191 Epstein, A. A., and R. Ottenberg, on aggluti- nins, 213 Erdman and Winternitz, Observations of, 180 Erepsin destroyed by the acid Of gastric con- tents, 187 Euplotidaa, Family of, 11 Ewald test breakfast, 163, I67, 188, 192; in- ferior to regular dinner in cancer tests, 168 Extirpation of abnormal cells, 56 F., Mr., Case of sarcoma of liver, 107 F., Mrs, Carcinoma of breasts, 107 INDEX 3c>1 Fat, Osmotic pressure exerted by, 282—83 Fatty acids, Compounds of lipins with, 268-69 Faubel, on duodenal regurgitation, r87 Fibroma of uterus, Extract of, 294 Field, Cyrus W., Criticism Of experiments on dog blood, 241 Fischer, Emil, on infiltrative growth, 55; experi- ments of, with Scharlach R, 78; combined amino acids into synthetic peptids, 162 ; on proteolytic enzymes, 191 Flack and Hill, Paper by, 280 n 5 Flexner, Simon, Experiments of, on transfusion, 213 F lexner and Jobling, on heated cancer cells, 130, 131 Flexner-Jobling tumor, Inoculation with, 88 Flexner-Jobling tumor-rats, 8 Food, Stasis of, a sign of cancer, 184 Form, a stable characteristic Of Paramecium, 47—48 Frankel, Paper by, 277 F remy, on phosphorus in ichthulin, 272 F riedlander, Carl, on motility of giant cells, 123, 124 Friedman, S. S., 156; joint author, 159, 225, 236,243 Frontonia leucas, Cutting experiments of Popofl on, 50 Funk, on compounds of cholesterol, 269 Futterer, on chronic irritation, 75 Gall-stone fat, 268 Galleotti and Pentamelli, on metaplasia, 85 Gastritis, Chronic, 184 Gay, on tonicity groups, 230—31; tables Of, 239-"33 Gaylord and Clowes, on immunity, 97 Geiger, George A., 156 ; joint author, 159 Germ glands, Observations Of Sticker on ab- sence of, 87 Giant tumor cells in dog tumor, 119—21; cul- ture growth of, 122—23; features of, 123-24 GIES, WILLIAM JOHN, General programme of the biochemical investigations, 1909—11, 153— 159 ————— Papers by, 158—59, 281 n 6, 283 n 9 ; thanks to, 172, 190; on bacteria in carious teeth, 181 n 10; encouraged Sanford and Rosenbloom, 191 11; joint author, 261 n, 281 n 7, 284 n 10, 285 n, 287 n 1,293 n 10 Preliminary reports on studies of the diffusion of lipins and lipin soluble substances through rubber membranes, 278—86 Gies and Steel, on Ulpiani and Lelli’s paranu- cleoprotagon, 275 Gilbert and Rogers, on chronic irritation, 75 Gitlow, Samuel, 156 ; joint author, 159 Glaesner, Observations of, 180 Gland extracts, 5; attempts to produce immu- nity by use of, 88, 93—94, 109; treatment of tumor-bearing animals with, 95; isolation of the active substance of, and study of mode of action, 97—102; treatment of human cases with, 102—9 Gland-free animals, Results of second tumor inoculation of, 92 Glands, Experiments in removal of, 4, 60-61, 78—81 ; effects of removal, on infectivity, 88— 89, 109; removal of, after inoculation with cancer, 88, 90—91, 109; efiects of, on immu- nity, 88, 91—92 Glandular tissues, Transportation of, 60-61 Glikin, found iron in protagon, 267; paper by, 277 Globulin, Lecithin compounds of, 274—75 Glycyltryptophan, a dipeptid, 162, I79; cleav- age Of, by cancer enzyme, I63, 179 ; used for cancer test, 163, 179; method of procedure, 163—65, 179; sources of error in test, 165—66 ; use of, superfluous, 166—70, 179, 192, 199; expensive, 167; gives positive results, 171; action of saliva on, 180—81; value of test questioned, 180, 181; main Objection to, 182 ; wide difference of opinion on, 192—93 Glycyltryptophan and tryptophan tests, The, for cancer of the stomach (CHARLES H. SAN- FORD and JACOB ROSENBLOOM) 191—99: Opinions of authorities, 191—93 ; how tests were carried out, 193, 198; tests tabulated, 194-97; results, 198—99; conclusions, 199 Glycyltyrosin, 163 Goiter, exophthalmic, Free hydrochloric acid absent in, 184 Goodridge, F. G., 156 Goodridge and Gies, Paper by, 284 n 10 Granat, Selma, Thanks to, 172 Greenwald, Isidor, Intracellular extractives, 15 Gren, on “gall-stone fat,” 268 Gressel, on compounds of cholesterol, 268 Gross, on the phosphorus in vitellin, 273 Growth Of tissues under experimental conditions, 59 Gruber, Studies of, in regeneration, 10 Gt'mzberg test for free hydrochloric acid, A study of the, 154 Gwyer, Dr. Fred, Treatment with thymus gland by, 4, 87, 109; extracts prepared by. 93; X substance isolated by, 98—100 H., Mr., Case of carcinoma of the pancreas, 107 Haaland, on disintegrated tumor or normal cells, 130; on resistance to implantation of tumor, 148—49 Halban, on isoagglutination, 211, 23 5 Hall and Williamson, on tests for gastric cancer, 190; on tryptophan in stomach contents, 192 Hamburger’s method of testing resistance of red blood cells, 231 ; on tonicity, 23 5 Hammarsten, on jecorin from bile of a polar bear, 271; on vitellin, 272—73 3c>2 INDEX Hanau and Moreau, Discovery of transplantable mouse tumor by, 66 Hanes, Frederic M., joint author, 158 Harrison’s researches on cell growth, 53, 59; success with lymph and plasma, 62, 111 Harvard Cancer Laboratory, Virulent tumor from, 9 Heart-beat of chick embryo in plasma, 111, 115, 116 Heart muscle (ox), Ether extract of, 288 Hektoen, on absorption Of agglutinin by cell it agglutinates, 212 ; on danger in transfusion, 213; on agglutination, 216, 235 Hemolysis, how estimated, 232; tables of, 232— 235'; tests for, 241; more intense in the body than in the test-tube, 257 Hemolytic test, The, 184 Hertwig’s theory of cell division, 27, 29, 48—50, 51 Hess and Saxl, on urinary colloidal nitrogen, 200, 206 Heteroagglutinins of animals’ serum identified with human isoagglutinins, 236—37 Hiestand, on lipin preparations from cereals, 272; paperby,277 Hirschfeld, joint author, 213, 257 Hodenpyl, Case of human carcinoma under, 97 Hofer, Studies of, in regeneration, 10 Holmes, on regeneration, 47 Homoagglutination in dogs queried, 241 Hopkins, J. G., Case of transfusion described by, 221—22 ‘ Hoppe-Seyler, prepared vitellin from egg, 272; on vitellin, 273; on chlorophyllan, 275 Howard and Schultz, on changed ratio, 52 Hiirthle, on cholesterol-oleate, 268 Hutchinson, on relation of arsenic to carcinoma, 5 Hydrochloric acid, Absence of free, in cancer of the stomach, 161, 172, 184; free, may destroy cancer enzyme, 168, 172 ; increases its activ- ity, 178; cases without, not yielding positive tryptophan reaction, 175 ; does not interfere with tryptophan test, 188 Hypersensibility produced, 130, 142 Imgebrigsten on isoagglutinins in cat bloods, 247 Immune state, General distribution of the, questioned, 132; confirmed, I36 Immunity, destroyed, 4; attempt to produce, by use of gland extracts, 88, 93—94; effects on, of removal of internal glands, 91—92; general findings on, 97; gained by inoculation with homologous spleen, 128, 129; refuted, I37— 149; no, by inoculation with kidney, 142; facts of, may be due to isoanti-bodies, 156 Imperial Cancer Research Fund, Results of experiments by, 127, 128, 129; laboratory of, 131 Incubator, The modified, 112—13; microscope and camera attachments to, 112—13 Indilfusible substances, 29o Infectivity, Measurement of, 6; effect of removal of glands on, 89 Inoculation of homologous tissue ineffective against development after transplantation of tumor, 129—30; not effective at any time, 137—49 Inoculation of rats with tumor after removal of different glands, 87—89; before removal, 90—91 Inoculation, Time, site, and dose, important factors in, 138, 141—42, 144—45 . Inoculation with retrograding tumors, 70—72; with parasites to produce cancer, 77 Inoculation with two types of tumors (F. D. BULLOCK and others) 73-74 Inorganic substances, Compounds Of lipins with, 267 Interagglutination reactions of ten individuals tabulated, 212 Interagglutinations of dog bloods and cells, 244—47 Internal organs protected from implantation of cancer by subcutaneous immunization, 132—36 Intracellular derangements, 157 Irritation, cause of proliferation Of cells, 55 Irritation, chronic, Effects of, on tissues, 75—76; effects of chemicals after, 77—86; other efforts to bring about, 85—86 Isoagglutination in dog blood (REUBEN OTTEN= BERG, S. S. FRIEDMAN, and D. J. KALIsKI) 236—40; discussion, 241—42; immune agglu- tinins in six dogs, 237 ; technic of experiments, 238; tables of agglutinations, 239—40 The occurrence of grouped, in the lower animals (R. OTTENBURG and S. S. FRIEDMAN) 225—29: Four groups in bloods of rabbits, 225-28 ; three groups in bloods of steers, 228—29 Studies on, 157, 209 ; solution of a special problem in, 211—24, 230, 236; a danger in transfusion, 213; of human blood not due to variations of molecular concentra— tion, 235; human, varies in intensity, 242; h1d0g8,243—47,257 Isoagglutinins, Hereditary transmission of, I56, 236; possible character of, 221; in human bloods, 225; Von Dungern on immune, in dogs, 225, 257; identity of, in different species, not determined, 229; two, 236; immune, developed in dogs, 237 Isoanti-bodies, defined, 156; of prime importance in cancer, 156; development of immune, 156 Isoeells, 156 Isohemolysis in dogs, 247, 257 Isospecificity, 156 J ecorin, found by Drechsel, 269; various opin- ions on, 270—72; composition of, 271 INDEX 303 JOHNSON, PETER J., Data on tumor growth in relation to age and sex of rodents, 64—65 I, 2; see also BULLOCK, FREDERICK D., CALKINS, GARY N ., and ROHDENBURG, GEORGE L. K., Mr., Cancer case of, 106 Kahlenberg, Experiments of, with membranes, 280 Kahn, Max, 156 ; joint author, 200 KAnN, MAx, and JACOB ROSENBLOOM. The col— loidal nitrogen in the urine from a dog with a tumor of the breast, 206—8 Kahn, Morris, H., 156 KABN, MORRIS H., and REUBEN OTTENBERG, Tonicity in isohemagglutination, 230-3 5 Kaliski, David J., 156; joint author, 159, 236, 243; case of transfusion by, 222 n 7 Kangri cancer, 55 Kanitz, Paper by, 277 Kantor, John L., 156; joint author, 159 Kidney and liver (dog), Ether extract of, 288 Kidney and spleen, Emulsion of, used, 139 Kidney, NO immunity in mice treated with own, 142 Kidney trouble in a dog, 117 Kidneys of mice inoculated to test subcutaneous immunity, 132—36; tumors in normal and immunized mice, 135—36 Kjeldahl method of determining the nitrogen content, 201, 207 Kletzinski, coined the term “lipoid,” 26o Knauer, transported organs, 60 Knife, The, in extirpation of cancers, 56 Koch, on sodium and potassium in lecithan preparations, 267; on compounds of lecithin, 267—68, 290 Kohlenberger, on cancer enzyme, 163, 190, 192 Kojo, on study of colloidal nitrogen in urine, 206; joint author with Salkowski, 200, 206 Kossel, on true chemical combinations Of lecithin with proteins, 274 Kraus, Ranzi, and Ehrlich, on distribution of immunity, I32, 136 Kullmann, on extracts of carcinoma, 292 Kuttner and Pulvermacher, 190; on tests, 192 Kyes, on CObralecithid, 276 Laboratories, The general, 1 Laboratory of Imperial Cancer Research Fund, 66 Lactic acid, Presence of, in cancer of stomach, I61, 172, 184; occurrence of, 184 Laking, Resistance of red blood cells to, 230—31 Lambert, R. A., on motility of giant cells, 123, 124 Lambert and Hanes, on cells, 53, 58, 59 Landsteiner, Discovery of isoagglutination of human red blood cells by, 211, 236 ; on danger in transfusion, 213; papers by, 23 5 Langhans, Th., on motility of giant cells, 123, 124 Laparotomy, Exploratory, performed, 186 Leaf, on chronic irritation, 75 Leathes, Terminology for some lipins suggested by, 265-66; paper by, 271 Lecithans, indiffusible, 281, 287 Lecithin, Experiments in injection of, 79, 80, 82—83; combinations of, 267—76 Lemon juice interferes with tryptophan test, 173 Leucocytes in treated tumors, 96, 98, 100 Leucocytes, Polymorphonuclear, containing red cells, 222 Levin, Dr. Isaac, Queries of, on agglutination in dogs, 241 Lewin, K. B., on regeneration, 16 11 Lewis, Carl, Experiments of, in growing tissue, 62 ; on new growths in the dog, 117, 124 Ley, on tests for gastric cancer, 190, 192 Liebermann, on vitellin, 273; on lecithalbumin, 274 Lip, Cancer of, in smokers, 55 Lipin, Proposed use of the term, 262 Lipins and lipin-soluble substances, Diffusion of, through rubber membrances (W. J. GIEs) 278—86: Problems of dialysis, 278—81; dif- fusion Of biological substances through rubber, 281; osmotic pressure exerted by fat, 282— 283; diffusion Of pigments from fat through rubber into fat, 283—84; comparative dialysis experiments, 284—85; diffusibility of alkaloids through rubber, 285—86 Lioins, The diffusibility of, from ether through rubber membranes into ether (J. ROSENBLOOM) 287—91: Condoms as diffusion membranes, 287; diffusible substances, 288—90; indiflus- ible substances, 290—91; conclusions, 291 Lipins, Intracellular, might be called “carriers of life,” 266; isolation of, 281 Lipins, On the forms in which, are combined in cells (JACOB ROSENBLOOM) 260—77: Classi- fications, 260—66; compounds vn'th inorganic substances, 267; with tissue metabolites, 267—68; with fatty acids, 268—69; with amino acids, 269; with carbohydrates, 269— 272; with proteins, 272—75; with alkaloids, 27 5—76 ; with toxins, 276—77; bibliography, 271 Lipins, Papers on, 155, 158—59, 209, 260, 278, 287 Lipoid, Origin of the term, 260 Lipoids, Reaction of tissues to injections of, 80, 83 Locke’s solution, Use of, 113—14 Loeb, L., Mouse carcinoma of, 6—8; grew epithe- lium on blood serum agar, 59; on retrograding tumors, 70 Lothrop, Alfred P., Intracellular enzymes, 158 Low, Stuart, Removal of thyroid by, 87, 109 304 INDEX Lumen, A, in isolated spaces of chick embryo, 115; in the channels, 116 Lupin seedlings, Influence of cancer extracts on growth of (J. ROSENBLOOM) 292—95 Lyle and Kober, on glycyltryptophan test, 166, 171, 190, 192 Lymph as a culture medium, 59 McCone transplanted organs, 60 M’Nee, Observations of, 2 57 MCWHORTER, JOHN B., and ALLEN O. WHIPPLE, The development of the blastoderm of the chick in vitro, 111—16 —— A rare tumor occurring in a St. Bernard dog, 117—24 Manasse, The jecorin of, 270; on jecorin, 272 Manwaring, on cobralethicid, 276 Marine Biological Laboratory at Roscoff, France, o, 10, so, 33 Mathews, on the pancreas cell, 53 Maximow, Alex., on motility of giant cells, 123— 124 Mayer, Analysis of lecithin combinations by, 269; jecorin of, 271; on jecorin, 272 Meal, An effective test, 188—89 Mechanism of cell extremely delicate, 77 Medulla of long bones, Names applied to tumors of the, 120 Meinertz, The jecorin of, 271 Meiostagmin test, The, 184 Mendel’s law, Isoagglutination characteristics of blood groups hereditary according to, 236 Metabolism, Experiments to disturb, 80—83; products of protein, 84 Metaprotein, Acid, 162 Metastasis rare in tumors of long bones, 124 MetazoOn a unit of organization, 51—52 Mice immunized subcutaneously are resistant to the implantation of cancer in internal organs (W. H. WOGLOM) 132—36 Mice inoculated with carcinoma, Tumor growth in, 64, 65 Mice, Resistance to cancer produced in, by auto- inoculation of the spleen, 127-31; refuted by later experiments, 137—49 Mice, Susceptibility of, 7—8, tumors in, 9 Michaelis, on killed cancer cells, 130, 131 Micheli and D0nati,. on hemolytic substances in tumors, 292 Molnar, secured duodenal secretion, 187 Monster forms from cutting of Paramecium, 30, 40—45, 57; Balbiani on cause of, 48 Moore, on combination of molecules, 276 Moss, on three agglutinins, 212; paper by, 235, 257 Motility of giant cells, 123—24 Motor insufliciency, in cancer of stomach, I61 Mouse carcinoma, Transplantation of a Loeb’s, 6—8 127 ; primary Mouse embryos, Resistance produced by inocu- lation of, 128 Mouse, macerated, Extract of, 93 Mouse tissues, Inoculation of homologous and heterologous, elicit same resistance, 128—31 Mouse tumor resembling human carcinoma, 66 Movements of Uronychia, 12 Muir, Experimental observations of, 257 Miiller, on proteolytic enzymes, 191 ; on ca- chexia of cancer, 293 Murray, J. A., on dog tumors, 117, 124 Myeloma, 120 Necrosis, in dog tumor, 118 Neubauer and Fischer test, The, 155, 178—79, 190, 191, 192; results of, 161—63; method of procedure, 163—65; sources of error in, 165— 166 ; experiments, 166—70; yields positive results, 171; objection to, 183 Nichols, on transplanted ovaries, 60 Nuclei, numerous in giant cells, 123 Nucleinic acid, Experiments in injection Of, 80- 83 Nucleus-protoplasmic relation, 49—51 N ussbaum, Studies of, in regeneration, 10 Nutrition, Effects of cancer extracts and prod- ucts on, 153; papers on improved method for study of, 158 Offer, The liver jecorin of, 271 Ohrl and Schittenhelrn, on regurgitation of intestinal contents, 187 Olive oil, Effect of, on gastric and pancreatic secretions, 187 Oppenheimer, on ‘tests for gastric cancer, 190, 192 Osborne and Campbell, on vitellin, 273 Osmotic pressure exerted by fat, 282—83 Osteitis in bone tumors, 119, 120 Osteoclasts in dog tumor, 119, 121, 123 Ottenberg, Reuben, Investigations of, 155, I56, 157, 158, 159 ; joint author with M. H. Kahn, 23o Ottenberg, Reuben, and Epstein, on the Wright technique, 226; on hereditary agglutinins, 236 OTTENBERG, REUBEN, Transfusion and the ques- tion of intravascular agglutination, 211—24 OTTENBERG, REUBEN, D. J., KALISKI, and S. S. FRIEDMAN, Experimental agglutinative and hemolytic transfusions, 243—59 OTTENBERG, REUBEN, and S. S. FRIEDMAN, The occurrence of grouped isoagglutination in the lower animals, 225—29, 236 OTTENBURG, REUBEN, S. S. FRIEDMAN, and D. J. KALISKI, Isoagglutination in dog blood, 236-40, 241—42 Ovaries, Cohen on removal Of the, 87 Ovary, Transplantation of, 60—61 Overton, used term “lipoid,” 260; paper by, 277 INDEX 305 Oxyproteic acid substances in urine of cancer patients, 206 Pancreas cell, Mathews on the, 53 Pancreas gland, Extract of, 93 Panzer found esters of cholesterol in the kidneys, 268 Paraflin, Chronic irritation by means of, 80—84 Paramecium, Effects of cutting on, 3, 30—58; power Of regeneration poor in, 31, 57; dim- culty of cutting, 31—33; division rate of, normal, 32; giant forms of, selected. 33; mortality of, 35, 57; monsters, 40—45, 57; form a stable characteristic of, 47—48; a divi- sion zone in, 57; normal cells from monsters, 57—58; physiological balance destroyed, 51, 58 Parasites in cells, 56 Pearce, Experiments of, in transfusion, 213 Pechstein, on tests for gastric cancer, 190, 192 Pectoral muscle removed at operation, Extract of, 294 Pepper interferes with the tryptophan-bromin reaction, 173 Pepsin, Action of, on proteins, 178 Peptids, 162, 179 Peptid-splitting bacteria in stomach contents, 165 Peptones, 162, 178; in gastric contents, 179 Peskind, Paper by, 235 _~ Phagocytosis, Cases for study of, 222—23; con- nection between agglutination and, 223, 224; intravascular, of erythrocytes, 223 n 8 Phosphorus, influences activities of the cell, 79 Photomicrography, Device for, 113 Phrenosin, neutralizes the poison Of tetanus, 277 Physicians furnishing cancer cases, 105 Pigments, Diffusion of, from fat through rubber into fat, 283—84 Pituitary glands, Extract of, 93 Plasma, as a culture medium, 59, 111; method of obtaining, from fowl, 113 ; blood from dog for, 118 Plexuses, developed from spaces and channels in blastoderm, 115, 116 Polypeptids, 162 Popofi, on cell division, 48—51, 58 Porges and N eubauer, on lecithin, 267 Precautions observed in experiments, 138, 144— I45 Preservatives, Addition of, superfluous, 169 Proteases, defined, 162 n 3; detection of tryp- tophan-producing, 166, 186 Protein, Diffusibility of, through rubber—mem- branes, 159; use of the term, 262; protoplasm thought to be, 266 Proteins, Studies of, 155; water absorption by, 159; cleavage products of, 162; action of pepsin on, 178; action of cancer enzymes on, 162, 179; hydrolysis of, into amino acids by intestinal protease, I84 ; intracellular, called “carriers of life,” 266; compounds of lipins with, 272—75 Proteoses, Primary and secondary, 162; gastric contents, 179 Protoplasm, Research on composition of, 155, 157; papers on, 158—59, 209; thought to be protein, 266 Protozoa, Studies of regeneration in, 10; effects of cutting on, 46 Purdy’s method for determining sugar content, 101 Pylorectomy performed, 186 Pylorus, Case Of obstruction of the, 185—86 in Q., Mrs., Cancer case of, 105—6 Rabbits, injected with Scharlach R., 78—79; seldom hosts of malignant tumors, 79; four groups in bloods of, 22 5—28; isoagglutinins of, less constant than those of dogs or humans, 242, 247 Rat, macerated, Extract of, 93 Rat sarcoma, A Flexner—Jobling, transplanted, 8—9 Rats, inoculated with sarcoma, Tumor growth in, 64—65; injected with Scharlach R, 79; iodine-fed thyroid-free, 96; isoagglutinations weak in, 247 Ravich, Abraham, and J. Rosenbloom, Chromo- lopins, 159 Recoveries, Spontaneous, in gland-free animals, 89, 9°, 91 Reed, grew bone marrow, 59 Regeneration and cell division distinct processes, 51, 54, 58, 77 Regeneration and cell division in Uronychia (G. N. CALKINS) 3, 10—29, 30—31 ; little or no regeneration, 15—17, 23—2 5; greater power and rate of, 17—19; normal, 19—22 ; greatest at time Of division, 25, 28, 54; limitation of maximum, 27; activating substance in, 29 Regeneration in protozoa, 10; in Paramecium, 100011.31, as, 37; percentage of, 46, 48, 57 Regulation in metazoa proceeds from the organism as a unit, 53 Reicher, used suprarenal extract, 98 Relation of certain internal secretions to malig- nant tumors (G. L. ROHDENBURG and others) 4. 87—109 Researches suggested, 153 Resistance produced in mice against transplanted cancer by auto-inoculation of the spleen (W'. W. WOGLOM) 127—31; refuted by later experiments, 137—49 Resistance to transplanted tumors effected by injection of heterologous normal tissue, 137, 143—44- Rhythms in growth energy, 7 Ribbert, C., transported organs, 60; on tumors of medulla of long bones, 121, 124 3cx5 INDEX Rockefeller Institute, Carcinoma from the, 9; investigations at, 53 I Roentgen ray cancers due to X-rays, 55 Roentgen rays produced first artificial cancers in the human, 81 Rohdenburg, George L., 1, 2, 4 ROHDENBURG, GEORGE L., Effects of chronic irritation on tissues, 4, 75—76 ROHDENBURG, GEORGE L., and F. D. BULLOCK. Effects of certain chemicals on the type of tissue resulting from chromic irritation, 4, 77—86 ROHDENBURG, GEORGE L., and P. J. JOHNSON, The relation of internal secretions to malig- nant tumors, 4, 87—109 ROHDENBURG, GEORGE L., see also BULLOCK, FREDERICK D., and Roosevelt Hospital, Impossible to obtain any help from the, 154 Roscoe, found iron in protagon, 267 Roscoff, Marine Biological Laboratory at, 9, 10, so, 33 ROSENBLOOM, JACOB, On the forms in which lipins are combined in cells, 260—77 A study of the diffusibility of lipins from ether through rubber membranes into ether, 287—91 A study of the influence of cancer extracts on the growth of lupin seedlings, 292—95 Studies of, 154,155, 158, I59, 191 n, 260; on diffusion phenomena, 280; joint author, 289 n 6 ROSENBLOOM, JACOB, and WILLIAM J. Gms, A proposed classification Of lipins, 261—62 ; papers by, 281 n7, 282 n8, 285 n 13, 287 n1 ROSENBLOOM, JACOB, see EINHORN, MAx, and; SANFORD, CHARLES H., and; and KAHN, MAX, and Rosenfeld, Paper by, 277 Rosenheim, Classification of “lipoids” proposed by, 265; found iron in protagon, 267 Rubber membranes, Diffusion of lipins through, 278—86, 287—91 Rudisch, Julius, Thanks to, 172 Riilf, on cancer cachexia, 292 Sachs, on cobralethicid, 276 St. Bernard dog, A rare tumor occurring in a (J. E. MCWHORTER and A. O. WHIPPLE) 117— 124 Saliva, Peptidolytic power of, 180—81, 193; has no effect on tryptophan test, 182, 193; effect Of, on Witte peptone, 183, 198 Salkowski, on urinary colloidal nitrogen, 200, 206; and Kojo, 202, 206; alcoholic precipi- tation method of, 207 San Felice, produced tumors by injecting blas- tomycetes, 85 Sanford, Charles H., 156 SANFORD, CHARLES H., and JACOB ROSENBLOOM, The glycyltryptophan and tryptophan tests for cancer of the stomach, 191—99 Sarcoma, predominates over other types, 73; replaced by carcinoma, 74; giant cell, 120; myeloid, 120 Scharlach R, Experiments with, 78—79 Schtine, Inoculation with mouse embryos, by, 128, 131 Schultz, W., on transplanted ovaries, 60; cases of transfusion by, 213 Schulze and Likiernik, on lecithoprotein in seeds, 273 Schulze and Winterstein, on lecithoprotein in seeds, 273; paper by, 277 Secretions, internal, Relation of certain, to ma- lignant tumors, 87—109 Sedlmayer. on the lecithin in yeast, 275 Seidenpeptone, Kuttner and Pulvermacher on use of, 192 - Sera of rabbits, 226-27; Of steers, 228—29; of human bloods groups, 230—32; assumed tonicity Of, 230 Serum, Agglutinative, and non-agglutinative, 215—16; activity of isoagglutinative, 231 Serum, of the several blood groups, Action of the 211—12, 230; in Schulz’s cases, 213; in experi- ments, 214—16; agglutination in diluted and mixed sera, 216—17; Of a gastric ulcer patient, 221 ; of patient of J. G. Hopkins, 221—23; large proportions of, used, 237 Serum, Resistance not produced by the, 127 Shattock, discovered isoagglutination of human red blood cells, 211 Sidbury, J. B., 156, 285 n Siegfried and Marx, The liver jecorin of, 270— 271; jecorin preparations, 271; on jecorin, 272 Smith, Irwin, produced tumors in plants, 85 Smith, Theobald, on tonicity, 230, 23 5 Solomon test, The, of little value in cancer of the stomach, 161, 184 Spaces, Isolated, in area pellucida of blastoderm, 115; become channels, then plexuses, 115 Spleen, auto-inoculation of the, Resistance against transplanted cancer produced by preliminary, 127—31; (refuted, 137—49); inef- fective after transplantation of tumor, 129— 130; enlargement of, in mice, common, 138—39 Spleen, Extract of, 93 Spleens, Emulsification of, 128; use of enlarged, with kidney, 139 Splenic tissue, Damaged homologous, inefiective to protect, 130—31 Staining, Delafield’s hematoxylin test for, 15 Steers, Three groups in bloods of, 228—29; agglutinations in, 247 Sticker, Anton, Researches of, on retrograding tumors, 70; observations on absence of germ glands, 87, 109; on immunity, 97; on dog tumors, 117, 124; on zonal immunity, 136 INDEX 307 Stomach, cancer of the, New test for, 161-77 Stomach contents, Method Of testing, 163—65; disturbing factors in, 165—66; bacteria in, 169; blood in, 169—70; duodenal contents in, 170-71; use of plain, 166, 180; method of withdrawing, 187—88, 193; should be filtered, 198 n 18 Stroma, A living, necessary for development of cancer cells, 3 ; Of dog tumor, 119 Studies in cause of cancer proposed, 153—54 ; reasons for giving up, 154 Sugar stimulates secretion of cancer enzymes, 1 73 Suprarenal extract, Use of, by Reicher, 98 Surgery, Department of, 111—49 Surgery, the only resort in treatment of cancer, 161 Sutherland and McCay, on tonicity, 23 5 T., Mrs., Cancer case of, 106 Takadiastase, Experiments with, 101—4 Takaki, on tetanus poison, 276—77 Takes, Tables of, 7, 8 Tebb, found iron in protagon, 267 Teeth, Bacteria in cavities of, 181 . Test cases tabulated, 194—97 Testes, Extract of, 93; Walker on use of emul- sion of, 100; no resistance in mice inoculated with own, 143 Testis, NO resistance elicited by inoculation of, 128 Tetanus, The poison of, 276—77 Thierfelder and Stern found calcium in an egg Kephalin preparation, 267 Thudichum, Classification of so-called lipoids by, 263; found iron in protagon, 267; on sphin- gomyelin. 270; paper by, 277 Thymus gland, Removal of, 4, 88—92 ; treatment with, in powder form, 4, 87, 93, 94; extract of, 5, 93, 94—95, 102—3; powerfully influenced by Roentgen rays, 81 ; use of extract of, in 49 cases of human cancer, 104—5 Thyroid gland, Removal of, 4, 88, 89—92; transportation of, 60; extract of, 93 Tissue, cancerous, Impossible to Obtain, 154 Tissue, Heterologous, when ineffective, 148—49 Tissue metabolites, Compounds of lipins with, 267—68 Tissue of dog tumor microscopically examined, 118—20 Tissues, Notes on the growth of, under experi- mental conditions (F. D. BULLOCK) 59—61 Tissues, Reaction of, to various lipoids, 83—84; biochemical differences between healthy and cancerous, 153 Tonicity in isohemagglutination (M. H. KAHN and R. OTTENBERG) 230-35: Gay’s experi- ments repeated, 231; his conclusions not justified, 233—35 ; ~ bibliography, 235 TOpfer, on urine of cancer patients, 206 Toxins, Combination of lecithin with, 276—77 Transfusion and the question of intravascular agglutination (REUBEN OTTENBERG) 211—24: From groups of agglutinating sera, 211—13; experimental part, 214—21; clinical part, 221— 223; conclusions, 224 Transfusions, Experimental agglutinative and hemolytic, 159, 243—59: Isoagglutination in dogs, 243—47 ; isohemolysis in dogs, 247; technic and plan of the transfusions, 247; results, 248—57; autopsies, 250, 254; con- clusions, 257—59 Trotter, on bone tumors, 120 True and Gies, Paper by, 293 n 10 Trypsin, Use of, 97; experiments with, by Drs. Weil and Feldstein, 101 ; in stomach contents, 165, 170—71; destroyed by the acid, 187 Tryptophan, Bromin test for, 163—65, 189—90, 193; presence of, in stomach contents, 165, 179; reaction obtained without using gly- cyltryptophan, 166, 168; bacterial develop- ment of, 169; modified test for, 171—72, 179— 180; valuable, 172, 173; cases, 173—75 ; cases not yielding, 175—77 Tryptophan test, The, for cancer of the stomach, with special reference to peptidolytic enzyme in the saliva (J. W. WEINSTEIN) 178—90: Neubauer and Fischer’s test, 178-79; War- field’s study and findings on saliva, 180—83; experiences with, 183—86; disadvantages of, 186; sources of error in applying, 187—88; technic of, 188—90; bibliography, 190, 192; Sanford and Rosenbloom on, 199 Tumor, 63, 239, 138 Tumor, Emulsion of, I32, 137 Tumor, primary malignant, Efforts to produce a, 77 Tumor, A rare, occurring in a St. Bernard dog (J. E. MCWHORTER and A. O. WBIPPLE) 117— 124: Clinical history, 117—20; macroscopical findings, 118; microscopical findings, 118—20; tumors of long bones, 120; transplanted to other dogs, 121—22; results noted, 122—24; bibliography, 124 Tumor-bearing animals, Treatment Of, with thymus gland, 88, 94—95 Tumor growth and the problem of organization, 53; in relation to age and sex Of rodents, 2, 64—65 Tumor strains employed, 137—38 Tumor tissue in collodion sacs (F. D. BULLOCK and others) 62—63 Tumor transplantation, Notes on, in general (CALKINS and others) 6—9; of a Loeb’s mouse carcinoma, 6—8; of a Flexner—Jobling rat sarcoma, 8—9 Tumors, heterologous, Auto-tissues ineffective against, 149 Tumors, malignant, Experiments with powder of, 70—72; first artificial, in the human, pro- inoculated, 128, 129, 308 INDEX duced by Roentgen rays, 81; relation of internal secretions to, 87-109; chemical and nutritional factors in resistance to inoculated, 153 Tumors, Retrograding (F. D. BULLOCK and G. L. ROHDENBURG) 2, 7o-72 Tumors, Spontaneous (F. D. BULLOCK) 2, 66— 69; carcinomata, 66 Tumors used, 1—2; in mice, 9; due to constant irritation, 52; inoculation with two types of, 73—74; histological studies of treated, 88, 96; in medulla of long bones, 120; may result from intracellular derangements, 157 Tyrosins, as a test, 163 Tyzzer, Dr., Virulent carcinoma tumor received from, 9 Ulcer of the gastro-intestinal tract, Occult blood in cases of, 184 Ulpiani and Lelli, on paranucleoprotagon, 27 5 ; Gies and Steel, on, 27 5 Units of organization, 51—52; regulatory pro- cesses of, disorganized, 54 Urine from dog with a tumor, Colloidal nitrogen in the, 206—8 Urine, Importance of the colloidal nitrogen of the, in the diagnosis of cancer, 200—5; pro- tein removed from the, 201 n 4 Uronychia, Regeneration and cell division in, 3, Io—29; description, 10—12, 49; movements, 12; division, 12—14; experimental cuttings, 14—25; general remarks, 2 5-29; table, 28 Uterine fibroid tissue, Extract of, 93 Valenciennes, on phosphorus in ichthulin, 272 Vascularity in bone tumors, 121 Vegetative cells of Paramecium, 39; monster forms from fragments of, 40—45 Vegetative period, Regeneration feeble in the, 26, 29 Vein, kinked, Result of a, in chick embryo, I r5—I6 Vernon, on molecular valences, 276 Verworn, Studies of, in regeneration, IO Virulence, Measurement of, 6 Vitality, Varying degrees of, in Paramecium, 47; no explanation of, 48 Vitellin, first of the lecithin-protein compounds, 272 ; various opinions on, 272—74 Vitro, Development of chick embryo in, 111—r6; same as in 0210, Ir4 Volhard, Observations of, I80 ; regurgitation, r87 Von Dungern and Hirschfeld, on agglutinins, 213, 225, 236—37, 257 on duodenal W., Mrs., Carcinoma of the uterus, 107—8 Walker, H. D., on malignant growths due to earthworms, 85; on immunity, 97; on use of emulsion of testes, 100 UNIV. CF Wallengren, on hypotrichous ciliates. 12—13, 28 Warfield, Study of the action of saliva on gly- cyltryptophan, 180—81, 193; significance of findings of, 181—82; verified, 193 Weber, B., on number of human red cells in one cubic millimeter, 214 Weil and Feldstein, Trypsin experiments by, 101 Weinberger, W., Thanks to, 172 WEINSTEIN, JULIUS W., The new test for cancer of the stomach, 161—77: Neubauer and Fischer’s glycyltryptophan test, 161—65; method, 163—65; sources of error, 165—66; description of experiments, 166-71; the tryptophan test, 171—72; general conclusions, 172—73; record of cases, 173—77 ' The tryptophan test for cancer of the stomach, with special reference to pepti- dolytic enzyme in the saliva, 178—90 ; 198 Studies of, 155 Welker, William Henry, Research work of, 155, 158, 159, 279 n s WHIPPLE, ALLEN 0., see MCWHORTER, JOHN E. Windaus, Paper by, 277 Winogradoff, on a silicic acid ester of cholesterol in bird feathers, 268 Winternitz, Erdman and, 180 Winterstein and Hiestand, on lipin preparations from cereals, 272 Winterstein and Stegman found calcium in a phospholipin, 267 Witte peptone, Efiect of saliva on, 183, 198 Woerom, WILLIAM H., Contributions to the theory of the individuality of carcinoma, 137— I49 Mice immunized subcutaneously are resistant to the implantation of cancer in internal organs, 132—36 Resistance produced in mice against transplanted cancer by autoinoculation of the spleen, 127—3r Wolff, on cholesterol-oleate, 268 Wood, Francis Carter, Thanks to, 172 Wright technique, The, 226, 228 X substance isolated by Gwyer, 98—100; ex- periments on action of, roo—2 ; does not cause direct death of cancer cell, 102; common to many tissues, Iog Xanthin, Experiments in injection of, 80—83; a stimulant to proliferation, 84 Xanthoma (skin) Ether extract of, 289 Yeast, Ether extract of, 289 Zemansky, A., Thanks to, I72 Zinsser, H., Tumor obtained from, 6 CHARLES ALEXANDER NELSON .. !CHlGAN'--' glllllllllelllfllll 362 5 ll“ “"“llmulllln ' lll . . . .6 a. i , . i i . . ,IVQi ,.. ii . 2 .LNJH ,,w»Q-rvirhe.ln,,lruiawnu,£er’t\ .1302: 9,2: ., ail-$1.1... i i c w. A. -.- p1,:- gives- , b , . ’i _ $1 If; 21,. . , . . i ; _ " Iflifiikavflf k..m¢- . . , x..