bowl Cp abE ts eee Stree Mle hoyle it } ny ite Me iy! Head sacbanei ai ht i : ANIME ti } Peartoahh iS MAS Nias i Mi : itil: i + | i i tii Asa ti i i a ei | titty ih a K Ms ti if ike oats beodait ae a a A! ny \ i Af 4 ahha ‘4 + in ie iW: Bs ate t i a yi ye any qi vada tale! a ihe i a ee ni ih 1 if ee i nis tit | a ih ff i H Y, ry i i f 7 A i AID n Tar More bal RN Nh ly ff net SRN Fina | Mane Ime Cie bal gy MEA Malet temper fi Hiatt nh i Pitenrr sie abn ae nea petata Woe sai the d hfe! a eit What i wp bt sits it i K p uM rh } hee) Lab a hte } Citys Ha ab } ate ! o " iN i i i if 4 iy wi iy } ae AA ry Ara Nea Phi Vy 4 ( ; Nal aie alte 4: A vA Meu pe i He nt 4 ERASE atstugtith AR an geal nn tn ihe} ni Harte vd ; K ne dite AIRE i Lt if are h brit tated ake | te . wf UE Montel ets 94 Mi aby a eae ue Ma veet ait: We Lee FOR THE PEOPLE FOR EDVCATION FOR SCIENCE LIBRARY OF THE AMERICAN MUSEUM OF NATURAL HISTORY PROCEEDINGS 59. OG(ta)Ka aR. OF THE SOCIETY FOR EXPERIMENTAL BIOLOGY AND MEDICINE VOLUME VI 1908-1909 EDITED BY THE SECRETARY NEW YORK 1909 YRARG IG ADAG Mita AOE }YACIEMK TAREE OE ped OAS ipa ’ swe f to- V1) b- ee. | PRESS OF THE NEW ERA PRINTING COMPANY LANCASTER, PA CONTENTS: SCIENTIFIC PROCEEDINGS (30th—34th meetings) : Communications of the thirtieth meeting, October 21, 1908 Communications of the thirty first meeting, December 16, 1908. Communications of the thirty second meeting, February 17, 1909 . 5 ¢ é ‘ : : é 3 Communications of the thirty third meeting, April 21, 1009 Communications of the thirty fourth meeting, May 26, 1909 Recapitulation of the names of the authors and of the titles of the communications : 3 : EXECUTIVE PROCEEDINGS (30th—34th meetings) REGISTER OF NAMES AND ADDRESSES OF THE MEMBERS LIST OF OFFICERS : CLASSIFIED LIST OF MEMBERS ‘ INDEX OF THE SCIENTIFIC PROCEEDINGS (iii) PaGE. MOEUN TARTANA | YAPVEIN SANIT AE 4g ay As [ bh - Set la ae a oF t ; i SCIEN MEIC PROCEEDINGS. ABSTRACTS OF THE COMMUNICA'TIONS, Thirtieth meeting. College of Physicians and Surgeons, Columbia University. October 21, 1908. President Lee in the char, I (339) Studies on the chemistry of anaphylaxis. By H. GIDEON WELLS. [From the Pathological Laboratory of the University of Chicago. | Egg albumin, freed from the other proteins of egg white by repeated crystallization, produces typically the anaphylaxis re- action. It sensitizes in doses as small as one twenty-millionth of a gram, fatally in doses of one millionth of a gram, The mini- mum lethal dose for sensitized pigs is about one half a milligram by intraperitoneal injection, and about one tenth to one twentieth of a milligram when injected into the circulation, The unpurified proteins of egg white are much less active, the minimum sensitiz- ing dose being about one hundred times greater and the minimum lethal dose being five times greater than with purified egg albumin. This suggests that inhibiting substances are possibly present in crude egg white. The minuteness of the minimum sensitizing and intoxicating dose of pure protein seems to indicate conclusively that both the sensitizing and the intoxicating agent are one and the same kind of protein molecule, or else two different constituents of the same molecule. Gelatin seems to be devoid of the power of participating in the anaphylaxis reaction, either with itself or with other proteins, This may be due to its poverty in aromatic radicals ; it probably is not due to the heating that is necessary for the conversion of collagen into gelatin. Addition of tyrosin to gelatin (without (1) iS) SCIENTIFIC PROCEEDINGS (30), chemical combination) does not modify gelatin in respect to the anaphylaxis reaction. Milk does not lose its sensitizing or intoxicating power when heated to 100° for 30 minutes. If large enough doses of serum heated to the same degree are used they will sensitize guinea pigs to unheated serum. Coagulation with alcohol destroys or reduces greatly the toxicity of proteins which it renders insoluble in water (egg albumin) but not proteins that it does not render insoluble (serum albumin). Pure zein is actively and specifically toxic to guinea pigs sensi- tized with zein, although it is devoid of tryptophane and lysine. Gliadin, which contains a less quantity of aromatic radicals than almost any other protein except gelatin, has but slight power to intoxicate pigs previously injected with gliadin. lodization of different specimens of serum by a constant method did not yield constant results. The partially saturated serum proteins suffered no modification in specificity analogous to that found by Obermayer and Pick in the case of the precipitin re- action, When most nearly saturated they may lose the power of sensitizing’for the unaltered serum, but this is uncertain. Pure crystallized egg albumin may be saturated with iodine quite readily, the iodine saturating the unsaturated carbon atoms of the benzene ring. Such iodized albumin retains its specificity un- altered, but seems to lose much of its toxicity for sensitized guinea pigs, nor does it sensitize well to egg albumin. Tryptic digestion of serum furnishes further evidence of the protein nature of the substances concerned in the anaphylaxis re- action. Both sensitizing and intoxicating principles are attacked, and slowly decrease in strength as the coagulable protein disap- pears. After 59 days’ digestion of a sample of serum so that but 4.7 per cent, of the nitrogen was in a coagulable form, the sensi- tizing dose had been changed from one one-thousandth of a cubic centimeter to one fiftieth of a cubic centimeter, while 5 cubic centi- meters intraperitoneally did not intoxicate pigs previously sensi- tized to bovine serum. Digestion of serum does not affect its specificity for species, but the digested serum sensitizes much better to itself than to bovine serum, and conversely. OBSERVATIONS ON THE CLINICAL ASPECTS OF HEMOLYSIS. 3 2 (340) Further observations on the clinical aspects of hemolysis. By GEORGE W. CRILE. [From the Laboratory of the Private Ward Service of Lakeside Flospital, Cleveland, O. | All cases of tuberculosis showed reverse hemolysis, 7. ¢., normal serum hemolyzed the patient's corpuscles. In all cases serum heated to 55° C. for ten minutes prevented hemolysis. Sudden chilling of the blood according to the method of Hoover and Stone caused a marked increase in the hemolytic action. Plasma obtained by immediately centrifugalizing the serum caused little or no hemolysis in hemolytic cases. The hemolytic property reaches its. maximum about 24 hours after the blood is drawn. In the cancer group the cases in which the disease was entirely removed lost their hemolytic property in from 12 to 21 days after operation. In cases in which an incomplete operation was performed the hemolysins continued indefinitely. Yotal number of cases studied 591. Per cent. NO NMA Sis opageencaewe, ssectonarsecers 211 iINoshentolyisissessreeteenss see fo) yo cemicilinitine sa cecseseeneternses ese 70 7mbemoliysis wen eece asst Io BenitonestinmonSerecties-sceisccessen see 55 INo®hemolysisynsscecceresece: fo) Gane en Merc: see was assitan.ceasseloe satin 153 Tig OMbiemolySismemsaene cess 85 Post operative cancer cases with climicalixeemmrenGe@sesesse-see sees as II Piehentolysiseaieessa= 4) sees 100 Post operative cases without clinical recurrence 3 weeks to I5 years BNF Gyo AWOI9n25, coco ocatooos6 soo0uE 37 INohemolysisierscscsn esctee fe) MiubenculOsissemincesceceemesseecscs ses 52 48 hemolysis ., .......- fice 192 Our conclusion is that hemolysis occurs in a number of diseases. It occurs in great frequency in cancer and tuberculosis. The re- action in tuberculosis is the reverse of that of cancer. From the clinical standpoint hemolysis offers additional evidence which may be used in the diagnosis of cancer and tuberculosis. This evidence is not as yet specific. 4 SCIENTIFIC PROCEEDINGS (30). 3 (341) The behavior of alanin in metabolism. By A. I. RINGER and GRAHAM LUSK. [from the Physiological Laboratory of the University and Bellevue Hospital Medical Collcge.| Injection of 20 grams of z-alanin ina completely phlorhizinized dog resulted in the elimination of ‘‘ extra sugar” in the urine to an amount equalling 18.8 grams, or 93 per cent. of that theoretically possible, Although z-alanin is almost completely convertible into dextrose, preliminary respiration experiments indicate that it does not spare fat metabolism as effectually as does dextrose itself. This may be due to heat loss in the breaking down of alanin into simpler molecules (formic aldehyde?) and heat absorption in its construction into dextrose. A similar reasoning would serve to explain Rubner’s “ specific dynamic action ”’ of protein. 4 (342) An important source of error in Heller’s test for urinary protein. By WILLIAM WEINBERGER. (By invitation.) [from the Laboratory of Biological Chemistry of Columbia Univer- sity, at the College of Physicians and Surgeons. | Heller’s test for urinary protein is a fairly reliable one if care is taken in its application, but several urinary protein constituents give uncertain results with it. Thus, mucin fails to yield true pre- cipitation — the ‘‘ ring” is more or less opalescent and disappears on mixing. With nucleoalbumin the ring is not quite typical and is indistinct in undiluted urine. On the other hand various mis- leading factors, such as resinous acids, must be taken into account. Resinous acids may be ignored, however, if such products as Balsamum Copaive, or Santal Oil, have not been administered, or if the specific HCl test for resinous acids shows their absence. The turbidity formed with resinous acids dissolves on warming. The acids themselves may be removed by extraction with ether. In concentrated urine, as is well known, a uric acid ring may ap- pear just above the line of junction of the urine and acid, and urea IMPORTANT SOURCE OF ERROR IN HELLER’S TEST. 5 may be precipitated in the form of glittering nitrate crystals; but dilution of the urine prevents these effects. In the course of our investigation of another problem, an ad- ditional source of error has been found in urines preserved with thymol in any of the usual ways. If Heller’s test is applied to such urine after filtration, a ring will invariably appear even in the absence of protein. The ring is most marked, all other things being equal, in urines that have been treated with a solution of thymol, although it is very conspicuous in urines that have been preserved with powdered thymol. The characteristics of this ring may be briefly stated as fol- lows: A few seconds after the urine has been carefully poured upon the acid, there forms, precisely on the line of junction, a grayish white ring about 0.5 mm. high, resembling the albumin ring given by a faint trace of albumin, and gradually becoming more and more distinct, until, in some urines under conditions to be mentioned later, it presents the appearance of a heavy thick precipitate, the height of which increases continuously and renders the lower portion of the urine completely opaque. At this stage the color is somewhat different from that of a protein ring, in that it is more yellowish. Below the ring there is a greenish zone ex- tending somewhat into the acid; above it, a reddish zone smaller than the former and more contracted. The white ring is seen best in daylight reflected from a dark background, the color rings are seen best if the test-tube is held against a white surface. On slightly disturbing the layers of urine and acid the ring, if a delicate one, disappears but reappears immediately. These ef- fects can be obtained a few times in the same mixture. = —- 3 169.1 8% 179.2 ——- — 8% 181.4 9 247.8 10% 153.0 23 158.3 18 LOZs3i ze! LOSs Tee 20 220nl Zo 146.3 54 151.3 52 154.6 47 DSS Sti eS TAS AT The viscosity determinations were made at the Laboratory of the U. S. Fish Commission, Woods Holl, Mass. 9 (347) The action of bile and some of its constituents upon intestinal peristalsis and the circulation. By ISAAC OTT and JOHN C. SCOTT. [From the Laboratory of the Medico-Chirurgical College of Philadelphia. | In their experiments with a Vella fistula, Fubini and Luzzati found that a pea, fastened to a thread, passed along the bowel more quickly when ten to fifteen minutes previously they had in- jected two grams of bile. C. Eckhard,’ of Giessen, also studied the influence of the bile upon the peristaltic movement of the small intestine. Eckhard experimented upon rabbits. He used a sodium chloride bath and studied the movements of the intestine 27 seta after opening the abdomen. After the injection into the duodenum of one cubic centimeter of bile of the rabbit the duodenum remained for ten minutes in absolute rest. He injected three c.c. of bile of rabbit, calf and sheep in different parts of the small intestine with the 1 Eckhard: Centralblatt fiir Physiologie, 1889, p. 49. 14 SCIENTIFIC PROCEEDINGS (30). same result, the intestine remaining quiet fifteen to twenty minutes. If, however, the intestine remained for some time in the salt solution, it became more:sensitive to the irritant. With the injec- tion of large quantities of bile there was more frequently than before little wave-like movements, although Eckhard does not feel sure that this was the result of the bile injected. Drs. Hallion and Netter’ have studied the influence of bile on the peristalsis of the intestine. They operated on dogs curarized or narcotized by chloralose or by morphine and chloral. By a small button hole in the small (mainly in the duodenum) intestine they introduced a balloon which was connected with a water manometer by means of rubber tubing. The balloon was flexible rubber and mounted upon a metal tube perforated by a large number of lateral openings, which prevented bends of the balloon upon itself. The water manometer was connected with a Marey tambour which inscribed the movements. After the balloon was inserted the abdomen was closed by a suture and the movements registered for more than an hour, so that subsequent curves pro- duced by the bile could be closely compared. The bile was injected either into the blood or into the intestine. Ox bile was used, concentrated by desiccation at a low temperature, but sub- sequently when used diluted to its original volume by water. Then 10 c.c. of bile was injected into the rectum. At the end of four minutes repeated movements of defecation ensued, followed by an irregular rhythm for eight minutes. The intravenous injection of bile (3-7 c.c.) was by the saphenous vein. There was produced a marked diminution of peristalsis and a relaxation of tonus in the smellintestine. Immediately afterwards the contrac- tions and the tonus considerably increased. Bile put in contact with the intestinal mucous membrane exercises a local excito-motor effect upon the small intestine. Intravenously it produces the same effect, a result that is due, in part at least, to an augmented secretion of bile by the liver, induced by the cholagogue influ- ences of the injected bile. Dr. Albert Schiipbach,? working in the Hallerianum under Professor Asher, has studied the effect of bile on the movements of 1 Hallion and Netter; Comptes Rendus de Biologie, 1907, pp. 182 and 254. 2 Schiipbach : Zeztschrift fiir Biologie, xxx, pp. I-41. AcTION OF BILE AND SOME OF ITS CONSTITUENTS. 15 the small intestine. Dr. Schiipbach made experiments witha Vella fistula in two dogs; also upon the rabbit’s large intestine in situ, and by the Magnus method with the isolated intestine of the cat and also upon the rectum in dogs. In the dogs with Vella fistula he made experiments with a ball of sealing wax, with a thread attached to a little weight. The bile was injected into the intestine. He also found that psychic irrita- tions by holding ham near the nose of the animal excited increased peristalsis. When the bile was injected he noted how fast the ball moved in the intestine. The bile was mixed with physiological salt solution, or with milk, or with water. He also used the Mag- nus method of excised intestine. He concludes that bile in the dog either has no special influence upon the small intestine in normal conditions or in many of the cases has an inhibitory effect. In the case of the implantation of the gall bladder into a Vella fistula, the gall of the dog had no special effect upon the peristalsis of the small intestine. In a state of hunger and at different hours after taking nourishment, the ac- tion of the bile was indifferent or a weak inhibition of peristalsis ensued. In rabbits under ether and morphine, with the small intestine 27 situ, the bile acted in an inhibitory manner. The excised cat’s in- testine was inhibited by bile. The large intestine of the rabbit zz situ had its peristalsis increased by bile. When through an injec- tion of gall the large intestine had increased peristalsis, the small intestine remained quiet. Bile injected into the rectum of the dog called out defecation. Our experiments were made upon etherized rabbits and cats. They were thirty-eight in number. We used two methods in the study of intestinal peristalsis. The first one was that of Magnus on the excised intestine in a modified Ringer solution with oxygen bubbling through it. The other was the insertion of a rubber balloon into about the middle of the jejunum in the small intestine and in the ascending colon of the large intestine. This was con- nected with the delicate piston recorder of Dr. Schlayer, of Tibin- gen. The bile used was that of cats and of rabbits. Effect of bile on small intestine. — With the method of Magnus small and large doses locally applied decreased peristalsis. In one 16 SCIENTIFIC PROCEEDINGS (30). case, a cat, there was with one eighth of a drop an increase of tonus and a slight augmentation of peristalsis. In the balloon method, bile given per jugular or injected into the lumen of the intestinal part experimented upon decreased peristalsis. In the rabbit, with one eighth of a drop by the Magnus method a temporary, well marked inhibition of peristalsis was seen with a decrease of extent of peristalsis. In a cat by the balloon method, one half a drop per jugular increased peristalsis. In a rabbit, one quarter of a drop by the jugular produced an increased peristalsis with the balloon method. Effect of bile on the large intestine. — The effect of bile on the large intestine by the Magnus method was to decrease tonus and peristalsis of the intestine. In rabbits there was occasionally, after a =; to a 34, of a drop of bile by the Magnus method, greatly increased peristalsis. In the balloon method, bile in the cat per jugular in doses of a drop, decreased tonus and peristalsis at first, and afterwards greatly increased them. Effect of glycocholic acid upon the small intestine. —In the cat, by the balloon method, three quarters of a grain of glycocholic acid per jugular increased the tonus and the extent of the per- istaltic movements. In other cats, one half grain of glycocholic acid decreased peristalsis, using the balloon method and injecting the acid per jugular. In the rabbit, 3 of a grain of glycocholic acid by the Magnus method greatly decreased the tonus and peristalsis. Effect of glycochole acid upon the large intestine. —In the rabbit, glycocholic acid by the balloon method in one half grain doses per jugular decreased peristalsis. In a rabbit and a cat it increased peristalsis producing quite large waves. Effect of taurocholic acid on the small intestine. —In the cat, one half grain of taurocholic acid greatly increased peristalsis by the Magnus method. In the rabbit there was a momentary in- crease of tonus by the Magnus method. In the rabbit, with =, to } grain of taurocholic acid there was with the Magnus method a great decrease of tonus and peris- talsis. In the balloon method, } of a grain of taurocholic acid per jugular in the cat increased peristalsis after a temporary decrease. 17 ACTION OF BILE AND SOME OF ITS CONSTITUENTS. og ov tg gv tor ve $g 1€ 96 cv gol br tol LY 96 of oll ra 06 br vit fv orl €v gol zy gor zp gol a4 OIl ov gor Sv Ol Lb OIl gv S11 oF S11 zh QII Sv ‘3 ‘wut “20s BB LIEIC) = § Sr a1ey ules % ulvis % ues ues ulvis 3% EpIOe a1joys094]45) of 'gf°E gee of LE°€ LE-€ SE°€ Tess S1'o£'€ of € gat gze bz't of €z°¢ f2-€ 5a) Le of-g1'f gif bre 60°€ ofgo'f o1'go't gore go't zo'e Io'f oboo'f oo°f ‘Wd aunty, "2Anssa4g po0jg uo prav 2y0YI02N/H nqgqvuy °€ INAWIYadxy vir €11 Fit gil oOzI vz1 QI ool bz zl bz1 ozI boi 9zI of! of! gII ZII ZlI of1 of! ram bz1 ver ber zeI zf1 Of! ram | ‘3H uM S431 H Sv “29S SI 238 ules % uivis % uleis % ures Y uwis “prow oyoyooine 7, Lo't ZOre Lz zS°z oS’z Siz rats of 1bh'z vz ob'z Gf:z zo'z Orit :z 1f°z of 'z Gz'z E'S Oz 17'z 1Zz'z Oz'Z c1'z ZZ O@'11'Z I1'z O1'z So’z ZO'Z G1°00'% 00°00°Z ‘Wd our, *admssadd 20019 UO p12v 2170YI0AND I, 1) ‘eS LNAWI Aad XY tor czI bz1 oI! gz gz obi oS! zv1 gSI ogI og og! bS1 oSt gs “3 7 89 ‘3 z oS "3 z vs ‘3 1 6+ 3 % gr ns he Sv ns ‘das ite | Si a3ey "aANsSa4g poojg uO ag "109 “I LNAWINad XY Lovt or'Sorf So't £o'€ LS‘z o1'SS"z SS‘z Sz Lv-z or Sh'z Srz vz LE°z of SE'z ce-z Laz Zee O1‘0z'z oz'z giz lac o£ 'o1'z Ol'z go'z ZO'T 10°Z 00°00'z ‘Wd auty, 18 SCIENTIFIC PROCEEDINGS (30). Liffect of taurocholic acid on the large intestine.—By the Magnus method, 3 grain of taurocholic acid decreased peristalsis and the tonus of the large intestine. In the rabbit, } to } grain of tauro- cholic acid by the Magnus method greatly decreased the tonus and peristalsis. There is no doubt that either with the excised intestine or with the balloon method, where the nerves of the intestine are attached, that bile from 3', of a drop up to 4 c.c. primarily inhibits peri- talsis and afterwards may or may not increase peristalsis. The taurocholic and glycocholic acids have the same action as the bile itself. The experiments upon the pulse-rate and arterial tension show that bile after a few doses reduces the tension and the heart beat. Taurocholic acid reduces the heart beat and the blood pres- sure. Glycocholic acid reduces the blood pressure, but did not materially alter the pulse-rate. From an examination of our results it is apparent that bile has contradictory effects upon the small and large intestine. It is evident that with either the Magnus method or the balloon method results are antagonistic. Dr. Schtipbach believes that this may in part be explained by a psychic reaction on the intes- tine in his experiments. It will not, however, explain the con- tradictory effect in the case of the excised intestine. In 1884, in a paper on intestinal peristalsis, Ott called the ganglia in the intestine intestino-motor and intestino-inhibitory. It is probable that these ganglia have a varying antagonistic effect, and as one or the other is in the ascendant we have an increase or a decrease of peristalsis by the bile. It is evident that the circulatory changes did not have any part in the changes in intestinal persitalsis. 10 (348) The uric acid excretion of normal men. By PAUL J. HANZLIK and P. B. HAWK. [from the Laboratory of Physiological Chemistry of the Department of Animal Husbandry of the University of Illinois. | The purpose of the investigation was to observe the course of the excretion of uric acid in normal men living on an ordinary mixed diet. Each subject was allowed to select his own diet and THE Uric Acip Excrerion oF Norma MEn, 19 then was required to ingest the diet selected during the course of six periods of four days each. Ten university students served as subjects. Quarters were provided where the men could easily be observed as to certain regulations of sleep and diet. The body weights of the subjects ranged from 53.1 kg. to 76.7 kg. and their ages varied from 19 to 29 years. There were no athletes among the subjects so that no individual took excessive or violent exercise, but all lived the life of the average, normal university student. The Folin-Shaffer method for the determination of uric acid was employed. CONCLUSIONS. 1. The average daily excretion of uric acid for ten men rang- ing in age from 1g to 29 years, and fed a normal mixed diet, was 0.597 gram, a value somewhat lower than the generally accepted average of 0.7 gram for such a period. 2. The average daily protein ingestion for these same subjects, when permitted to select their diet, was 91.2 grams or 1.33 gram per kilogram of body weight. TI (349) Hemolysins in the sera of carcinoma and syphilis. By 8. PESKIND. (By invitation.) [From Dr. Peskind’s Private Laboratory, Cleveland, Oho. | A few years ago the writer commenced a research, the object of which was to determine in what diseases hemolysins commonly occurred and whether or not they were specific for these diseases. The ultimate purpose was to obtain data that one could use in diagnosis. A preliminary report was published.'. The work had to be abandoned shortly thereafter. Since then, other investigators — notably Kelling — have taken up the study of hemolysins in con- nection with their use in diagnosis. It occurred to the writer that it would be very desirable to determine the question as to the specific nature of the hemolysins found in various diseases. With this object in view, a study of 1 Peskind: American Medicine, 1903, v, p. 918. 20 SCIENTIFIC PROCEEDINGS (30). the serum in several diseases has been undertaken. In this brief report are given the results obtained in carcinoma and syphilis. The sera and corpuscles of 12 cases of carcinoma and 7 cases of tertiary syphilis were examined. In Io other cases of tertiary syphilis, the corpuscles alone were examined. The experiments were planned in groups. In each group, the corpuscles of at least one normal person, and several cases of syphilis and carcinoma were exposed to the action of sera derived from syphilitic and carcinomatous patients. The customary technic was employed, special care being taken to use only fresh specimens of blood. Equal parts of serum and of a one per cent. saline suspension of corpuscles (washed four times) were incubated at 37° C. for one or two hours, sedimented on ice over night, and compared with control tubes of the serum. In all 290 serum-corpuscle combinations were made. In this way were studied the actions of syphilitic and carcino- matous sera on their own corpuscles, on normal corpuscles, and on the corpuscles of other cases of syphilis and carcinoma. The following results were obtained : SUMMARY OF RESULTS. Out of the 12 cases of carcinoma, 4 showed the presence of hemolysins in their sera, which caused laking of the erythrocytes derived from normal human individuals. Out of the 7 cases of tertiary syphilis whose sera were ex- amined, 6 showed the presence of hemolysins which dissolved the corpuscles of normal persons. The corpuscles showed the following behavior : The corpuscles belonging to a hemolytic carcinomatous blood were found to be immune to the action of the hemolysins in its own serum or any other carcinomatous serum. The corpuscles belonging to a non-hemolytic carcinomatous blood were readily laked by a hemolytic carcinomatous serum. Similarly the corpuscles of a hemolytic luetic blood were found to be immune to the action of its own serum or any other syphilitic serum. The corpuscles of a syphilitic blood, whose serum did not contain hemolysins were laked by any hemolytic syphilitic serum. HEMOLYSINS IN THE SERA OF CARCINOMA AND SYPHILIS. 21 In every instance, it was found that the corpuscles belonging to a hemolytic carcinomatous blood were immune to the action of the hemolysins found in syphilitic serum. Conversely, the corpuscles present in a hemolytic syphilitic blood were immune to the action of the hemolysin present in carcinomatous serum. However, the corpuscles of a non-hemolytic carcinomatous blood were readily laked by hemolytic syphilitic serum and simi- larily the corpuscles of a non-hemolytic syphilitic blood were laked by a hemolytic carcinomatous serum. Judging from the behavior of the sera towards the corpuscles derived from various normal and diseased persons, one could not distinguish a hemolytic syphilitic serum from a hemolytic carcino- matous serum. This would suggest that the hemolysin found in syphilitic serum is identical with one found in carcinomatous serum. It would seem, from the above results, that there is some con- nection between the presence of a hemolysin in the blood and the immunity of the corpuscles contained in that blood. The corpuscles found in hemolytic bloods —whether from cases of carcinoma or syphilis — are immune to the action of the hemolysins in those sera. The corpuscles of non-hemolytic bloods are vulnerable and are readily laked by the hemolysins contained in either carcinomatous or syphilitic sera. The reverse proposition could be argued from the results of the above experiments. That is, if the corpuscles of a given blood are immune to the action of a hemolytic serum, then the blood in question contains a hemolysin. If the corpuscles are laked by a hemolytic serum, then the blood in question does not contain hem- olysin. The corpuscles of 10 other cases of tertiary lues (whose serum was not obtained) were tested against the hemolysins in carcino- matous and syphilitic sera. The corpuscles of 3 of these cases were easily laked by hem- olytic sera derived from carcinomatous or syphilitic persons. The corpuscles of the other 7 cases were found to be immune to the action of the hemolysins. It is reasonable to suppose that the 3 bloods with the vulner- able corpuscles did not contain hemolysins, while the 7 bloods containing immune or resisting corpuscles did contain hemolysins. 22 SCIENTIFIC PROCEEDINGS (30). It was found that the addition of 30 per cent. of normal serum was sufficient to inhibit the action of the hemolysins in both the syphilitic and carcinomatous sera. Normal serum seems to possess protective substances which inhibit the action of the hemolysins. Hemolytic sera also may possibly contain the same protective substances as normal sera. These substances, if present, would neutralize a certain amount of the hemolysin existing in the serum. If only a small amount of hemolysin be present, it would, if the above conditions actually exist, be neutralized and rendered incapable of detection by the technique used at present. The corpuscles belonging to such a serum, however, would still be im- mune to hemolytic carcinomatous and syphilitic serum. The serum of one case of tertiary lues was found to contain so little hemolysin as to be barely demonstrable. The corpuscles of this blood, however, were perfectly immune to either carcinoma- tous or syphilitic sera. The results of this research have some bearing on the subject of transfusion. Inasmuch as some cases of carcinoma possess hemolytic sera, we could not transfuse such cases with normal blood: If transfusion of such a carcinoma case be found necessary — as a preliminary to operation or for other reasons — it will be neces- sary to secure as donor a person whose blood corpuscles are im- mune to the serum of the carcinoma case and vice versa. According to our observations, the desired blood could be found in cases of tertiary lues that have just recovered from their lesions. The hemolysins found in syphilis and carcinoma appear to be true isolysins. In experimental isolysin-formation the isolysin is a reaction product of the organism in which it is formed. The corpuscles of the animal in whom the isolysin is produced are im- mune to that isolysin. No anti-hemolysins are present in the sera of such animals. The evident immunity of the corpuscles in the hemolytic syphi- litic and carcinomatous bloods towards the hemolysins in their sera indicates that the hemolysin is a true isolysin, and, like the experi- mental isolysins, is probably a reaction product of the organism in which it occurs. Some toxic substances absorbed from the dis- HEMOLYSINS IN THE SERA OF CARCINOMA AND SYPHILIS. 23 eased focus could be assumed as the exciting cause of such an iso- lysin formation. In conclusion, the writer wishes to acknowledge his indebted- ness to Dr. J. E. Tuckerman, who collaborated in the research. 12 (350) The effect of instilling adrenalin chloride into the mammalian eye. By W. H. SCHULTZ. (By invitation.) [From the Division of Pharmacology, Hygienic Laboratory, Washington, D. C.] Certain writers have concluded that mydriasis cannot be pro- duced by instilling adrenalin into the eye of higher animals except under pathological conditions such as lesions of the pancreas or the removal of the superior cervical ganglion. Perhaps this con- clusion results from an oversight of the antagonism existing be- tween the influence of instilled adrenalin and light stimuli when simultaneously acting upon the intact eye. At any rate the con- clusion is not supported by more recent experiments and is mis- leading when used as a basis for diagnosing certain pathological conditions. I have found that mydriasis can be produced in these animals with relative ease and certainty. In making a comparative study, however, of different degrees of susceptibility to adrenalin, due care must be taken to keep the intensity of light stimuli constant. This is essential, since in the eyes of higher mammals where the light reflex is well developed, strong light may cause the pupil to constrict to such an extent that any antagonism of this process by adrenalin may be lost sight of. For instance, by instilling adren- alin into the normal cat eye for some minutes and then examin- ing the eye in light bright enough to constrict the untreated eye to a small slit-like aperture, no difference in the drugged and un- drugged eyes can be detected ; but the same eyes examined in a dark corner may show a distinct difference in the pupils, the drugged pupil dilating more than the normal one. Thus the early dilating effect of adrenalin can be detected more easily by reduc- ing the intensity of the light stimuli. In this preliminary com- 24 SCIENTIFIC PROCEEDINGS (30). munication, however, it is not so much the time of initial dilation as it is the time required for complete antagonism of the light stimuli by adrenalin that is considered. The degree of resistance to this antagonism is perhaps best illustrated by the appended protocols, each taken from a series of experiments. Here it is shown that maximal dilation and loss of light reflex in the guinea pig results within 23 minutes after instilling 6 drops of I: 1000 solution of adrenalin chloride. After instilling 2 drops every 2 minutes into the eyes of the following, maximal dilation and loss of light reflex results: In the rabbit, within 56 minutes; the dog, 96; the cat 101 ; whereas in the monkey it requires 148 minutes ; and in normal man even longer. From the protocols it will be seen that adrenalin not only di- lates the pupil of the mammalian eye, but that in each case the stimulating effect of light of moderate intensity can be completely overcome by it. Furthermore the ease with which this antagonism is accomplished seems to depend upon the degree of development attained by the light reflex mechanism, since in animals that have a sensitive light accommodating mechanism most highly developed the period of instillation and amount of solution must be increased to produce mydriasis, whereas in animals with a reflex mechanism less sensitive to light stimuli, mydriasis is relatively easy. In con- clusion it seems that these facts must be considered in formulating a theory dealing with the dilator mechanism of the eye or with the value of the adrenalin test for certain pathological conditions. 1. Normal guinea pig. Sept. 5. Park, Davis & Co.’s 1: 10000 adrenalin chloride. 10.30 A, M. Before instillation, diameter of right and left pupil about 5 mm. 10.32 ‘* two drops instilled in right eye. 10.36 ‘* right pupil 5.8 mm., left pupil 5 mm. TO:30))) SS cc «6 mm., left pupil 5 mm. 10.43 ‘* two drops instilled into right eye. 10.45 ‘* right pupil 7 mm., left pupil 5 mm. 10.50 * cc «ee 7.8 mm., left pupil 5 mm. 10.51 ‘‘ two drops instilled into right eye. 10.55 ‘* right pupil 9 mm., left pupil 5 mm. iM igiig ty) GC <¢ « g mm., practically dilated to a maximum. 12.32 P.M. ‘ ‘* about same as at II.II. Hispiis, 09 «oe 7% mm.,, left about 5 mm. 12a «c «¢ about same as left. Maximum dilation brought on by six drops. Time required was about 23 min., at the end of which time the pupil no longer reacted to light. EFFECT OF INSTILLING ADRENALIN CHLORIDE. 25 2. Normal gray rabbit. Sept. 3. Two drops of P., D. & Co.’s adrenalin chloride in- stilled every 2 minutes. 3.40 P. M. Right pupil 6.6x 9 mm,, left 6.6 x 9.0 mm. 4.025) 6 SS Se eb. Ox 2 5mm. AsI2 *6 eee k 2. O-x 02.5), ARG Syn ace ee 13.5 x 14mm. Lastinstillation: left 7x 10.4 mm., pupil scarcely reacted to light. 3. Large adult female cat. Aug. 20, P., D. & Co, 1:1000. Instilled every 2 minutes. 10.30 A. M. Right and left pupils alike ; transverse diameter about 114 mm. Fo: 345) <6 first instillation, ringyksy slight dilation of right eye, 214 mm., left 114 mm. 12.45 P. M. maximum dilation 11.2 mm., left 114 mm., practically no reaction to light after IoI minutes, 4. Young adult female dog. Aug. 21. P., D. & Co.’s 1: 1000 sol. of adrenalin chloride; two drops every two minutes. 10.00 A, M. Right and left pupils of same diameter — about 4.2 mm, 10.04 ‘ _ first instillation. 10.40 ‘* right pupil about 5 mm., left 4.2 mm. II.oo <6 OG COMNIG"SsmmepnsS 4c SE 11.16 <* “ 66 “c 9 mm.,, “cs 4.2 “é Mfige ke) Ch GO «Tr mm., “ 4.2 * Last instillation. 4.30 12) 91 Ge SGC oe sé 2 mm., “é 5 “6 Maximum dilation was reached after about 96 minutes at which time the pupil ceased to react to fairly bright light from a skylight. By 4.30 the right pupil had not only ceased to dilate, but even in such a light as one might secure in a well-lighted room at 4.30 P. M., the drugged eye constricted much more than did the normal one. 5. Monkey. P., D. & Co.’s adrenalin chloride, 1: 1000. Two drops every two minutes. 10.30 A. M. Both pupils of the same diameter, about 2.4 mm. 10.32 ‘ first instillation. 10.46 ‘* right pupil 2.6 mm., left 2.4 mm, 10.49 “cc “ 6 3.0 mm., 6c 2.4 ‘é Onn: GE $B Hingis, CEP pe MG 1 tay id CC OG LS) abrigg, “CO Bayt UC Tinea | GG CF G0) ASA iets, UO en ie G6 LIE4O) 16° «6.0 mm., ‘ 3.0 ‘ pupils turned toward bright light. 12.00 P.M. ‘ ** 6.5 mm., ‘* 2.8 ‘* constricted to about I mm. 125200 pce Gt OS Gioia, Cpe} © GC T2240m iss cee 8.4mm., “ 3.2 ** When turned toward bright light, the left pupil constricted to less than I mm. in diameter while the right constricted but little (8.2 mm.). 1.00 P. M. Last instillation, practically maximum dilation. 5.00 ‘* In dim light, right and left pupils about the same. Maximum dilation was reached after 148 minutes at the end of which time the pupil no longer responded to bright light. Greater constriction of the right eye than the left was noticed the following morning. In normal men, the resistance is still greater than in the monkey. 26 SCIENTIFIC PROCEEDINGS (30). 13 (351) Successful canine infection with cultures of Leishmania infantum (Ch. Nicolle). By F. G. Novy. [from the Hygienic Laboratory, University of Michigan, Ann Arbor, Mich. | By the collective term Lezshmaniasis we may designate three apparently distinct diseases (1) Kala-azar or tropical splenomegaly of India and the East; (2) Oriental sore, otherwise known as Delhi, Biskra, Aleppo, etc., boil ; and (3) infantile splenic anemia. These are characterized by the presence of peculiar intracellular parasites commonly knownasthe Leishman-Donovan bodies. The work of Rogers and others has shown that the parasite of Kala- azar develops in a citrate solution, into flagellate or trypanosome- like organisms, but attempts at cultivation on blood agar have given negative results. The recent investigations of Ch. Nicolle on the parasites of Oriental sore and of infantile splenic anemia establish the important fact that the Leishman bodies found in these two diseases can be cultivated on blood agar with the same ease as in the case of many trypanosomes. Nicolle has further shown that the infantile splenic anemia can be transmitted to dogs and monkeys by injection of suspensions of the diseased tissues, but attempts to produce an infection by inoculation of the cultures of the flagellate failed. Having received through the courtesy of M. Mesnil, of the Pasteur Institute, transplants of the eighth generation of Nicolle’s flagellate, it was decided, first of all, to test in a severe way the question as to the possibility of inducing an experimental infection in animals by means of such cultures. Accordingly a dog was given, in the interval from April 13 to Sept. 21, fifteen intraperi- toneal injections of fresh vigorous cultures. The organism was grown on blood-agar at 20°, and for each inoculation the growth from a large number of tubes (8—40) was taken up in citrate solu- tion and injected. A total of 270 cultures were thus utilized in the course of five months. The dog apparently showed no effect, other than occasional leucocytosis, and microscopic examination of the peripheral blood gave negative results. CANINE INFECTION WITH LEISHMANIA INFANTUM. 27 The dog was bled and when autopsied on October 9 presented evidence of a prolonged chronic infection. The spleen was small and tough and weighed but 33 grams, the dog weighing 11.7 kilos. The liver and kidney likewise were found to be unusually hard. Microscopical examination of the spleen, liver, kidneys, lungs, and bone-marrow showed enormous numbers of typical Leishman-Donovan bodies, free and intracellular, with no sign of the flagellate form. Cultures made from the spleen and liver gave at the end of five days exceedingly rich growths of the flagellated organism. Tubes inoculated with the peripheral blood likewise gave good cultures though somewhat later, that is, on the tenth day. The latter fact indicates the value of the cultural method as a diagnostic means. It will be seen therefore that, starting out with the flagellate form, it has been possible to produce a typical infection in the dog and to recover from the infected animal, by cultural means, the parasite in the flagellated stage. Undoubtedly this result can also be obtained by employing less massive doses than was deemed necessary in this preliminary experiment. 14 (352) New apparatus designed especially to facilitate the preserva- tion of food for use in metabolism experiments.' A demonstration. By WILLIAM J. GIES. [From the Laboratory of Biological Chemistry of Columbia Unt- versity, at the College of Physicians and Surgeons. | The writer exhibited a new form of apparatus that has been very serviceable in the preservation of fresh food by refrigeration. The apparatus consists in the main of a galvanized “angle iron”’ frame constructed to support glass trays specially designed as food containers. Fresh food, ¢. g., hashed meat, may be very satis- factorily preserved, without change of general composition, by 1This method further improves the process described by the author some years ago in the American Journal of Physiology (1901, v, p. 235). See also Gies and col- laborators : Biochemical Researches, 1903, i, p. 69 (Reprint No. 1). 28 SCIENTIFIC PROCEEDINGS (30). placing it in covered trays of the kind referred to and transferring them immediately to such a frame in a freezing room. The gen- eral characters and relationships of the main parts of the apparatus are clearly shown in Figure 1. The structure and dimensions of the glass trays are indicated in detail in Figures 2 and 3. See pages 30 and 31. In the form of this apparatus now in use in this laboratory, each “angle iron” skeleton is 20 inches high, 21 inches wide and 10 inches deep. Twenty five glass trays fit snugly into as many stalls," which are just a trifle wider and longer than the trays, and are arranged in five tiers. The removable horizontal rods at the front are so arranged as to prevent the trays from falling from the frame if the latter happens to be tilted forward. The “angle iron” fixtures at the rear prevent movement of the trays in that direction when the frame is tilted backward. The glass trays are the essential parts of the apparatus and are excellent food containers. Plates of ordinary glass furnish very satisfactory covers for the contents of the trays. Such a glass lid, trimmed to fit intimately, can easily be put in place and can readily be elevated with a finger at the depression in the edge of the tray, at one end, Air tight closure may be secured by plac- ing over the tray a full-width strip of paraffined muslin before the closely fitting lid is pressed down tight upon the ledge in the tray which supports it. Paraffined muslin will not appreciably absorb moisture from the contents, nor freeze fast to the latter, and can be washed free from the slight amounts of food that may adhere to it. If sucha paraffined muslin strip is allowed to extend a little beyond one end of the tray, the protruding portion serves as a means of drawing the tray from the frame and also of lifting the lid of the tray. Such paraffined muslin strips may be used again and again. The trays are composed of thick flint-glass and therefore are able to withstand unusally rough treatment. Neither the lids nor the trays have been cracked by alternate cooling and warming between the extremes of temperature to which they have com- 1 Five additional trays may conveniently be placed directly underneath the lower tier of trays in the frame. 2Such closed trays full of meat may be kept weeks at a time in a refrigeration room without losing weight. APPARATUS TO FACILITATE PRESERVATION OF Foop. 29 monly been subjected, z. ¢., 5° C. and room temperature, nor have the lower temperatures made the glass brittle. The covered trays hold about 550 grams of hashed, expressed, lean meat, or 325 grams of cracker meal.’ The freezing of a closed trayful of lean meat is devoid of any appreciable expansive effects. This method of food preservation, where the necessary refrigera- tion facilities are at hand, offers the following special advantages : The trays are in effect bottles that rest on one side and open on the opposite side. The paraffined muslin cover and the glass lid may be removed together as easily as a stopper may be taken from a bottle. The trays can be filled or emptied easily and quickly. The thick side of the tray furnishes a very stout fulcrum for strong leverage with a heavy knife through frozen food, such as hashed meat. Consequen‘ly, frozen food in such a tray may easily be sectioned with a knife into blocks without any risk of breaking the tray. The trays are comparatively shallow. Therefore, percolation of liquid in fresh food (such as the juice in hashed meat) before freezing sets in must be very slight, if it occurs at all.*_ The influ- ence of such possible percolation on the uniformity of composition of portions removed daily is negligible, especially if the food is used in sections cut from top to bottom. The uniformity of the dimensions of the tray makes it easy to mark off very accurately given quantities of any relatively homo- geneous product. Upright partitions, of paraffined card-board for example, may be used between weighed quantities of food, placed side by side, without any danger of admixture or difficulty of removal, Since the trays can easily be marked for identification, many dif- ferent dietary mixtures can be systematically preserved at the same time in the apparatus described, and may be used separately with- out confusion, After fresh food has been frozen, trays containing it may safely be kept in an ordinary refrigerator for a day or more, thus increasing the convenience of handling material preserved in this way. 1 The covered trays hold about 575 c.c. of water. ? Thus far no visible percolation has occurred, in such trays, in meat previously ex- ressed in an ordinary ‘¢ tincture press,”’ y P 30 SCIENTIFIC PROCEEDINGS (30). 5) Fic. 1. A view of the ‘‘angle iron’ frame holding seven glass trays. Two additional trays in different positions, with their glass lids leaning against them, are shown, The white objects in the foreground are paraffined muslin strips, which are used with the glass lids to effect air tight closure of the trays. Tray I in the second stall of the first tier has a glass lid in place, which shows distinctly in the picture. The trays in the stalls diagonally downward to the right of Tray I, and also the tray in the second stall of the lowest tier, are empty and without lids, The two covered trays in the fourth and fifth stalls of the second tier contain frozen hashed meat and show the usual appearance of full trays in reserve in the frame. Trays II and III outside the frame show clearly the inside appearance of the trays. The removable horizontal rods are in position to prevent forward movement of the trays. The special turns on the ends of the rods should be noted. APPARATUS TO FACILITATE PRESERVATION OF FoopD. 31 It is needless to dwell upon the obvious fact that the apparatus described may be used very satisfactorily for many other purposes. The trays are excellent crystallization dishes, for example, and serial recrystallizations may be effected in them very conveniently. Fic. 2. A diagram of a tray with the lid out of place, to show especially the ledge upon which the lid rests when the tray is covered. The depression in the edge of the tray at one end, where the lid may readily be lifted with a finger, is also indi- cated sharply. This depression does not extend to the surface of the ledge. There- fore, when the lid is in place, the tray is completely closed. The general structure of the apparatus permits its placement in out of the way positions. The whole of it can readily be pro- tected by an easily removable impervious covering. Fic. 3. A diagram showing the relationships of the parts of the tray, including the lid, and giving the main dimensions. I am indebted to my assistants, Mr. Walter H. Eddy and Dr. Archibald FE. Olpp, for the photograph and drawings of the apparatus. Mr. Christian Seifert, of this laboratory, executed admirably the plans for the frame. The glass trays were made to order by the Whitall Tatum Co. Thirty first meeting. Rockefeller Institute for Medical Research. December 16, 1908. President Lee in the chair. 15 (353) Reply and explanation to recent criticism of my experimental study on effects of extirpation of the salivary glands on the gastric secretion. By JOHN C, HEMMETER. (By invitation.) [From the Physiologic Laboratory of the University of Maryland, Baltimore. | It is not always a congenial task to have to reply to a criticism of one’s experimental work. To many a conservative thinker, the policy contained in a remark attributed to Ludwig under a similar circumstance, “‘ Schweigen ist gold,’ may appeal as more expedient. But yet, the dignified silence may be interpreted, by the one who has advanced the criticism and even by the research worker and general student of physiology, as a tacit approval to the fault find- ing —in other words, as signifying that the criticism was deserved and the work criticised defective. I find myself in this embar- rassing position with regard to an article published in the “ Pro- ceedings of the Society for Experimental Biology and Medicine, 1908, v, pp. 114-117,’ New York, by Dr. A. S. Loevenhart and Dr. D. R. Hooker, entitled : ‘Note on the supposed presence of a gastric hormon in the salivary glands.” Although the physiology and pathology of digestion has been my life work, yet, as one of the results of many years of labora- tory teaching and training, I am loathe to insist dogmatically on any of my opinions and am ready at any moment to be corrected and to advance another step in the attainment oftruth. (‘‘ Experientia fallax, Experimenta mendax.’’)! 1 But rather than dwell upon the moral side of scientific controversy I prefer to refer to Sir Thomas Browne’s ‘‘ Religio medici,’’ 1904 edition, p. 98. (33) 34 SCIENTIFIC PROCEEDINGS (31). Especially welcome are such corrections when they emenate from such an esteemed friend and talented worker as Dr. Loeven- hart. The original worker whose results are criticised has the right, however, to demand that his special point of inquiry (‘‘ Frage- stellung’’) and all the methods of experimentation, operative, phys- iologic and chemic, shall be conscientiously repeated on, at least, an equal number of the same kind of animals, successfully nursed through the identical operative procedures. He has a right to demand a scrupulous regard for detail, and for all the finer distinc- tions made in his application of methods, some of which may have required years for their perfection in his hands and those of his associates. Let us investigate whether my friend, Dr. Loevenhart, has ful- filled these indispensable, fundamental conditions that should pre- cede destructive criticism. I sought to ascertain the effect of salivary gland extract in dogs deprived of all four pairs of salivary glands, whose gastric juice had been carefully studied before any operation of removing the glands was undertaken. Sometimes the removal had no very marked effect ; but in those dogs in which it did, I tried to ascer- tain whether the depressed gastric secretion could be restored or not by salivary gland extract. I tried to study the effect on a secretion already abnormally depressed in three series of dogs — thoroughly recovered from the operation, allowing ten days to two weeks, at least, for recovery. Dr. Loevenhart starts with normal dogs, as he supposes, and expects to raise the gastric secretion qualitatively and quantitatively above the normal. He seeks the effect of salivary gland extract in raising a supposedly normal gastric secretion to a higher acidity and proteolysis — an entirely different problem from mine. I have never published anything on the effect of salivary gland extract on the normal gastric secretion of dogs. It is not asserted that this extract can raise the gastric secretion above normal, but only that it may, under certain conditions, partially restore a gas- tric secretion that is depressed de/ow normal. Dr. Loevenhart is attempting to change a normal secretion to an abnormal (higher) one. I studied the effect in restoring an abnormal secretion to a normal one. EFFECTS OF EXTIRPATION OF THE SALIVARY GLANDS. 35 When there are four different procedures for obtaining gastric juice on the same dog within thirty minutes, and the jugular vein exposed, a cannula inserted and submaxillary extract injected in- travenously, it must not be overlooked that, with every additional interference, the animal becomes more and more disturbed and that this seriously influences his gastric secretion. The chemico-phys- ical and the neuro-physical processes of secretion are thoroughly upset unless a long time for recovery is given. This is shown in Dr. Loevenhart’s results, page 4 of his reprint, in which the total acid- ity and free HCl and the proteolytic power became less and less in specimens A, B and C; only when the psychic secretion was aroused, granting that this was not a delayed effect of injection sal. gl. extr. specimen D, was there any notable proteolysis with- out addition of acid. The notes of the beginning of experimen- tation on this dog bear the date of April 6, and the qualitative studies bear the date of April 8 — not near time enough to permit dog No. 2 to entirely recover. To expect salivary gland extract to raise the gastric secretion qualitatively and quantitatively above what is the regular standard for the average dog is to expect something abnormal —for an un- usually abundant and unusually active gastric juice is logically as abnormal as one that is unusually diminished or inactive. 1. What Dr. Loevenhart presumes is that the salivary gland extract should change a normal gastric juice to an abnormal one (from the regular amount to an unusually high amount and activity). 2. What I attempted to ascertain was whether or not an abnor- mal gastric juice could be restored to the normal (from diminished and weakened secretion to the normal). The “ Fragestellung”’ is not the same, in fact, it is highly digressing. Dr. Loevenhart observed only two animals. Nowhere does he give the date of operations, nor state the time that elapsed between the operation and first day of experimentation, nor the amount of proteolysis in millimeters of Mett tubes. Both animals were abnormal. The first dog, No. 1, he admits had distemper and was feverish, was thin and would not eat. The first observa- tions are dated November 11, 1907, and this animal died within 48 hours, 36 SCIENTIFIC PROCEEDINGS (31). All of our results were gained from dogs that lived for three to six months and then had to be killed in most cases because we had no room or facilities for keeping them during the summer vaca- tion — excepting the series of the summer of 1907 when I kept four dogs at our country home. What Dr. Loevenhart aimed at | 6 Secretion expected to rise above normal ne Line of normal gastri BE ine of n ee Sout e of normal gastric secretion ‘5 >, no extirpation 3g 4 of salivary glands —= 8 42) 5) =} n What Dr. Hemmeter attempted to ascertain. = ag Line representing nor- ‘6 8 mal gastr. secret. 8 "on pe || $a sevcceeenesnnnceneene nnnnnneensoncereneneeeneseit gE Fs 5 i gradually re- ‘BE Sie 5 stored to as ac = nearly normal oss gee Zz oa gastric \juice > -= 5 decidedly reduced qualitatively and quantitatively Dog No. 2 of Dr. Loevenhart was also abnormal. This is evi- dent from the feeble proteolysis as indicated by Mett tubes (in Dr. Loevenhart’s article they are called “‘ Metz” tubes) and the low acidity, and Dr. Loevenhart gravely states that the fluid gained by catheterization, 8.6 c.c., specimen A, contained much dark mucus (blood? and mucus). Mucus in a fasting dog’s stomach is one of the most reliable indications of gastritis. Dog No. 2 had a dis- eased stomach also. We made our salivary extract from maceration of all four pairs of canine salivary glands, even the orbital — and it is all important that this extract should be made only from salivary glands that have been functionally active immediately before their excision EFFECTS OF EXTIRPATION OF THE SALIVARY GLANDS. 37 (the dog must be made to chew bread and then rapidly etherized). I have worked with extracts of inactive glands, but so far have refrained from publishing anything concerning their effects or non- effect. In nota single instance, has the operative plan and tech- nique used by us, nor the physiologic routine of preparing the glands by functional work, nor the chemic discipline of ascertaining the proteolytic activity been punctiliously carried out by Doctors Loevenhart and Hooker. Both of the animals had diseased stomachs. In neither were the salivary glands extirpated. The entire plan of experimentation and aspect of physiologic inquiry is so fundamentally different from mine, that comparison of their work with ours is not logical, and any deductions from their work as used to interpret our results are unfortunately misapplied. It is only fair that the work of an experimentor should be judged from his most recent publication, in this case that which appeared in the Brochemische Zeitschrift (Hamburger Festschrift, Band xi, p. 238), the only complete report published by me. The short notice by which Drs. Loevenhart and Hooker judged our work was nothing but a preliminary report, and contained, as such reports occasionally do, some inaccuracies which I have taken the privilege to correct in the article published in the Bvo- chem. Zeitschr., l. c. (“ Die Wirkung der Total Extirpation Samt- licher Speicheldriisen auf die Sekretorische Funktion des Magens beim Hunde”). Even in this article the printer has allowed some wrong figures to slip into the headings of tables C, D and F, pp. 257, 258 and 259, for which I am in no way responsible, but which do not injure the main argument, especially as the editors of the Biochem. Zeitschr. politely corrected them in a subsequent Be- richtigung. In Dr. Loevenhart’s experiment on April 8, submaxillary extract was injected into dog No. 2 at about 3.10 to 3.15 P. M., the gastric juice of twenty minutes later showed a free HCl of 0.20 (titration with z/20 NaOH) but the proteolytic power with addition of acid is declared to be “‘ good.” Butat 3.30 the stomach of the same dog was catheterized and specimen A obtained after the dog was allowed to smell meat for ten minutes. This speci- men A was the most active that Loevenhart obtained. It came 35 38 SCIENTIFIC PROCEEDINGS (31). minutes after the submaxillary extract was injected. Question is: Would not this active juice have been secreted even without the efforts to cause a psychic secretion, for the salivary extract in my experience has a latent period in which it produces no very marked secretion? After that period it may come; that is, a pro- nounced secretion may come, even thirty minutes after injection of salivary extract and even if there has been no chance for psychic secretion. Pawlow, /. c., p. 70, states that in all cases the latent period after the vagus stimulation of gastric secretion may be from I5 minutes to one hour, and even more. Hitherto we have known the term “ /atent period of secretion” only in connection with the stimulation of a nerve going to gland or muscle. We are not so familiar with the use of the term “latent period” in connection with the chemical stimulation of a gland. A moment’s reflection will bring the thought nearer to us that even after nerve stimulation, pure and simple, chemical events must transpire in the gland cells which require a certain time for their elaboration. Now, if the stimulation is purely chemical, and not through a nerve, the same or similar chemical events must precede the actual outpouring of secretion. We are still ignorant of the processes that occur during the “‘ latent period,” but recent work indicates that they are partially electrical and partially of a chemical nature. We must also consider that the immediate effect of a chemical stimulation, like the immediate effect of a nerve stimulation, may be inhibited. There are so many side influences of a physical, nervous and chemical nature which control the phenomenon of the “latent period”’ that its exact nature and what transpires during it, is still a matter of speculation. It may, at first sight, seem paradoxical that the latent period of secretion after sham feeding in dogs is stated by Pawlow to be only 5 to IO-I5 minutes, and the latent period after vagus stimulation I5 minutes to one hour —for in both instances the stimulation is transmitted by one and the same nerve to the identical synapses in the gland cells. Pawlow explains this, p. 71, /. c., by his belief that in artificial stimulation of the vagus, the stomach receives the excitatory as well as inhibitive impulses, and the latter check secretion. EFFECTS OF EXTIRPATION OF THE SALIVARY GLANDS. 39 How can we conceive of inhibitive processes to explain a long latent period of secretion, when chemical substances (for example, salivary gland extracts) are injected intravenously? By an anal- ogous experimental reasoning, we have learned (Pawlow, /. c.) that it is impossible to imitate the influence and action which the vagus exerts during normal life while digestion is going on, for our laboratory methods are far too coarse and the complexity of fibers in this magnificent highway of nerve tracks too intricate for us to single out individually functioning secretory fibers. We are not much better off when we attempt to imitate the chemistry of the internal secretion of glands, for only in a single instance has a hormon been isolated in a state that reveals its exact chemic structure. The chemic messengers are bodies of definite chemic structure which are released with unerring exactness from their producing organs ; but when we manufacture an organ extract, it is, of course, possible that we may seize the hormon (if I may still use the term) ; but unavoidably we must extract the entire tissue of the organ and as a result obtain extracts, which contain materials that stimulate, but also materials that may inhibit secretion. This occasional inhibitive effect of salivary gland extract on gastric secretion has brought to mind two ideas: either that Iam not dealing with a hormon or stimulator at all, or that there may be two kinds of chemic correlation, one that stimulates and the other that inhibits. The conception which sees an antagonistic, as well as a syner- gistic, correlation brought about by chemic messengers is at least as rational, when applied to the physiologic correlation of organs by means of chemic substances communicated to them by means of the circulation, as when applied to the correlation of organs by means of nerve elements. This relation of organs by means of reciprocal (antagonistic or synergistic) action of nerves is not new to physiologists, and has been brought home to us in a most im- pressive manner by Meltzer, not to mention Ch. S. Sherrington, New York, 1907. All of this is still hypothesis ; but this hy- pothesis has been given color (1) by the seemingly paradoxical effects of (2) such a pure substance as adrenalin, which does not always cause constriction of vessels (only when they are severed from the nerve centers) but sometimes may cause dilatation, when in normal animals a certain vascular area is intact in connection 40 SCIENTIFIC PROCEEDINGS (31). with its nerve centers,’ and of (4) gland extracts, which sometimes raise blood pressure and often lower it (sometimes after a slight previous rise), and (2) by the contradictory effects of some salivary gland extracts on gastric secretion. All of this doubt will con- tinue so long as we are compelled to deal with a complex mixture of various substances in gland extracts and not with one pure sub- stance of known composition. To this consideration belongs, also, the antagonistic phenomena reported by Lilienfeld, Morowitz and Delezenne as occurring in blood coagulation (positive and negative phase of coagulation). This is explained by Lilienfeld and also by Delezenne by the isolation from blood plates and leucocytes of two substances, one of which they term “ /euconuclein”’ which favors coagulation, and the other, ‘ /zston,” which retards coagulation. Before the isola- tion of these two substances the phenomenon of the positive and negative phase during blood coagulation appeared paradoxical, and the idea of a latent period of coagulation might have come to many an experimentor. Just so with the latent period after chem- ical stimulation of the glands; it may be due to inhibitive sub- stances in the gland extracts used, and it is possible that this delay in bringing about the effect after chemical stimulation of the gas- tric glands, may disappear with a clearer knowledge of the chem- istry of the gland extracts, and a more accurate method of pre- paring them. Besides the latent period of secretion, we must consider the neutralization of the first acid secreted by the mucus present in the stomach. Pawlow(‘“ Arbeit. d. Verdauungsdriisen,” /. c., p. 39) calls attention to what he emphasizes as “Factum,”’ namely, “ Even with a normal stomach and with a pure gastric juice 25 per cent. of its acidity can be lost through neutralization by mucus.” How much more must this neutralization take place in a stomach that, as Loevenhart states, gave “much dark mucus.’ The very efforts of catheterization increases the mucus formation, and after the submaxillary gland extract was injected, if it had any stimu- lating effect at all (I am not prepared to state whether it had or not) this much is sure, the mucus had to be neutralized before 1 This latter effect of adrenalin is not a purely chemical effect but a mixed effect of nerve and chemical phenomena, One and the same chemically pure substance cannot be claimed to contain both stimulating and inhibitive substances. EFFECTS OF ExTIRPATION OF THE SALIVARY GLANDs. 41 there could be free HCl. The extract was injected at 3.20 on April 10; at 3.25 P. M. the gastric juice was drawn by cathe- terization (8.8 c.c., specimen C). No free HCl was in it, but six minutes after the injection of salivary extract the dog was shown meat, and ten minutes after that there was a fourth catheterization (the fourth in 30 minutes). This 5.3 c.c. was active juice and Loevenhart and Hooker attribute it to psychic secretion. Considering the latent period of secretion and the time for neu- tralization by mucus, it is reasonable to inquire whether or not the injection of extract had a feeble but delayed influence, although Loevenhart and Hooker used only submaxillary extract and not that of all four pairs of glands, and did not prepare it in the man- ner I did. Concerning the inflammation (gastritis) in the stomachs of their dogs, I can very readily appreciate the difficulty, for I had been thwarted and misled by diseased canine stomachs for almost a year before we gradually learned to recognize, avoid and treat them. Evidences like these, naturally suggest that such experiments cannot be successfully carried out in a few months. I was not aware of Dr. Loevenhart’s criticism, until November 14, 1908. That there are salivary extracts that have no peptogenic effects whatever, and others that are variable, I have already stated in my article in the Brochemische Zettschr., Vol. xi, p. 251 (“ Verschieden- heiten in d. peptogenen Kraft d. Speicheldriisen Extrakten’’). Then again, the complexity of the mechanism of gastric secre- tion in dogs is such (Liochem. Zettschr., l. c., p. 253) that the ini- tial depression caused by extirpation of the salivary glands prob- ably may be gradually replaced by special efforts of the remaining sources of stimulation to the gastric glandular apparatus. This problem is far too deep and complicated to have years of laborious experimentation set aside by a casual testing of two sick dogs, as to whether a saline extract of the inactive submaxillary gland alone can cause a secretion of gastric juice in animals not deprived of their salivary glands. That there may be defects in my work I am willing to accept as a possibility, because a general knowledge of the history of physiology reveals the status that the first results of similar ex- 42 SCIENTIFIC PROCEEDINGS (31). perimental work are only in most exceptional instances without defects or errors. Such a defect in the connexus of cause and effect has recently been brought to my knowledge and, today, makes it debatable whether the name “hormon”’ is correctly applied by myself to the stimulating quality of one gland extract upon the secretion of another set of glands. The definition and conception of the hormon allows a rather wide application, it is true, but it seems to me it ought to be restricted to substances whose chemical structure is at least approximately known and that have one pre- dominant characteristic or specific effect on other glands, in which effect they cannot be replaced by extracts from other organs or tissues. This is not the case with the salivary extracts, for, as we can learn (Biochem. Zeitschr., Vol. xi, p. 253), extracts of the pyloric mucosa and of the spleen (Luciani) act in a similar manner in stimulating gastric secretion. Concerning the pepsinogenous effect of the spleen on the gas- tric secretion, I refer to the work of Tarulliand Pascucci, executed in Luciani’s laboratory and described in the latter’s splendid work, ‘‘ Physiologie des Menschen,” translated into German by Bag- lioni and Winterstein, Vol. ii, pp. 151 and 152. Onpage 153 it will be seen that the extract must be made from an active spleen, as Luciani says ‘‘a spleen that is hyperemic and swollen,’ which means, taken from a dog during the height of the digestive period. Extracts of spleen taken during the period of functional rest had no pepsinogenous effect ; but the meaning of Luciani and his pupils above mentioned is unmistakable. A chemical substance is formed in the spleen during its activity which, when brought into the circu- lation, is absorbed by the gastric glands and is capable of aug- menting the quantity of the secreted pepsin. Additional emphasis is given in these experiments to the fact that the extract should only be made from a functionally active gland. Whatever may be the final outcome of investigations concern- ing the chemical nature of the hormones, Bayliss and Starling consider that they were originally accidental by-products of the activity peculiar and proper to the organ which has produced them. Thereafter the next step in the development of a correlation is the acquisition of a sensitiveness or a responsiveness to the hormones EFFECTS OF EXTIRPATION OF THE SALIVARY GLANDS. 43 in any remote organ (‘Die Chemische Koordination der Funk- tionen des Korpers,” Ergebnisse der Physiologie, Jahrgang v, p. 670). The only word to which I could take exception in this explanation of Bayliss and Starling is the word “ accidental ”’ (“ Zufallige’”’ Nebenprodukte). I should like to enlarge this con- ception when applying it to the digestive tract, and state that the various segments of the digestive tube are correlated and co- ordinated by a sensitiveness not only to accidental products, but to the regular by-products which are known to accompany the formation of the specific products of the organs of digestion. An infirmity in the experimental logic, suggestive of a meta- bolic by-product produced in the salivary glands during activity which might be regarded as a chemical messenger to the secretory apparatus of the stomach, might be found in the occasional failure to produce total loss of gastric secretion after the salivary glands are removed. In other words, we should expect to find invariable “ Ausfalls-Erscheinungen,’ phenomena of lapse or total deficiency of gastric secretion. That these do not occur after the salivary glands are extirpated with that regularity that is necessary to justify the use of the term “ hormon,”’ is at least partially explained by the existence of several other sources wherefrom the secretory apparatus of the stomach may receive its stimulations ; these other sources have been sufficiently considered in the preceding and in the Biochenusche Zeitschrift, Vol. xi, p. 253. I do not wish to be understood as asserting that an extract of the inactive submaxillary gland alone can have an effect in raising the amount and proteolytic activity of gastric juice, but only, that, if it possibly could exert such an effect, not sufficient time was allowed after the injection in Dr. Loevenhart’s experiments to adequately test this point of inquiry. If there is anything of importance that has revealed itself to us since the publication in the Biochemische Zeitschrift, Vol. xi, p. 238, it has come through experimental study of the occasional long latent period after injection of some salivary extracts and not after others. This has suggested the existence of chemic sub- stances which inhibit or check gastric secretion. These substances, if they exist as definite chemical bodies, must be more abundant in resting, than in functionally active, salivary glands. 44 SCIENTIFIC PROCEEDINGS (31). There is nothing contradictory in the idea that one and the same gland cell in one segment of the digestive tract may contain two kinds of chemical messengers for the succeeding segment of the digestive apparatus. One kind stimulates secretion in the fol- lowing segment and a second kind inhibits or arrests it. Starling (‘‘ Recent Advances in the Physiology of Digestion,” p. 90) speaks only of hormones (from oppda, to excite, arouse or stimulate). But on reflection it must be evident that for the nor- mal regulation of life processes, it may, under certain conditions, be equally important that any process of secretion or vascular tonus should be capable of inhibition by chemical messengers. Two such diagonally opposed chemical substances which are con- cerned in coagulation have been isolated from lymphocytes by Lil- ienfeld and Delezenne, one of which /euconuclein favors coagual- tion and a second /zston which inhibits it. The leuconuclein corresponds to the hormones but the Aisfon is an inhibitor. For such chemic bodies — physiologic arresters like Azston —I would suggest the name oliones from the Greek zwivwy, to inhibit, to prevent, arrest or check. 16 (354) A critical study of the conditions under which zymase and its associated co-enzyme bring about alcoholic fermentation. By GEORGE H. A. CLOWES. [From the Agricultural Chemical Laboratory of Professor Buchner in Berlin, and the New York State Laboratory, Buffalo. | Zymase, the enzyme of yeast discovered in 1896 has since been proved by Harden and Young to consist of two parts, (1) zymase proper, an enzyme-like body possessed of high molecular com- plexity, non-diffusible and thermo-labile, and (2) a readily dif- fusible, thermo-stabile, relatively simple, chemical complex, which, for lack of a better term, has been designated as the co-enzyme of zymase. Harden and Young separated the bodies in question by dif- fusion, but owing to the paucity of their materials and the destruc- tive effect exerted by secondary causes during the lengthy process ALCOHOLIC FERMENTATION. 45 involved, it was found impossible to obtain any clear insight into the conditions of physico-chemical equilibrium obtained in this reaction. Buchner’s dauerhefe, that is to say, a preparation of pressed yeast precipitated by an excess of acetone or alcohol ether, can be prepared in large quantities and exhibits a high degree of resistance to the action of destructive enzymes. We therefore directed our attention to the preparation of dauerhefe containing as large a zymase content and as small a co-enzyme content as possible, our object being to study the effect exerted by a preparation of this nature upon fermentable sugars, when used in conjunction with varying proportions of a boiled yeast extract containing co-enzyme. It was found possible to produce a preparation of acetone dawerhefe which in itself alone possessed no fermentative activity whatsoever, but which when used in con- junction with a suitable quantity of boiled yeast extract, exhibited an unusually active fermentation, 2 grams mixed with 6 grams of sugar and 20 c.c. of extract producing from I to 2 grams of CO, in the course of 8 to 10 days. Having thus demonstrated that it is possible to obtain a stable preparation containing relatively large quantities of zymase, and also to prepare a relatively stable boiled extract of yeast contain- ing co-enzyme, a series of experiments was commenced, the object of which was to determine the effect of varying proportions of co-enzyme used in conjunction with a constant amount of zymase, and vice versa. In several series of experiments in which a con- stant amount of zymase (2 grams dauerhefe), was used in con- junction with 6 grams of sugar and from 1 to 50 units of co- enzyme, it was found that the velocities of reaction and the fermentation end results were directly proportional to the number of units of co-enzyme employed up to an optimum concentration, after which a fall in the value of both these quantities was to be observed. The same phenomenon exhibited itself when varying proportions of dauerhefe were employed with a constant amount of co-enzyme, other conditions being constant. The velocity of reaction %, is calculated from the formula A I ED aaa is 46 SCIENTIFIC PROCEEDINGS (31). where A represents the relative molecular concentration of the sugar and A — X the concentration at any given time 7. The progress of the reaction is readily followed by estimating the loss in weight of fermentation tubes due to the evolution of CO,. In all cases in which no disturbing influence has been allowed to exert an effect, this velocity of reaction is found to be constant for a period of three or four days immediately following the establish- ment of active fermentation. Provided all experiments are carried out at a constant temperature and that other conditions are main- tained on a uniform basis, the value of K is found to be directly proportional to the product of the concentrations of the zymase and its co-enzyme, according to the formula VEN VEE, kK 40) where K and XK; represent velocities of reaction and 7 and Z, con- centrations of zymase and C and C, concentrations of co-enzyme in comparative series. The accuracy of this formula over a com- paratively wide range was demonstrated by means of tables, in which the observed and calculated values of K were compared for a series of tubes in which zymase (dauerhefe) and co-enzyme (boiled yeast extract) were employed in varying proportions. Herzog, from experiments carried out previous to the discovery of the heat-resistance component, came to the conclusion that the fermentation process was to be represented by the formula K Gne m7 (a): 1 where C and C, represent comparative concentrations of zymase and x has any value from 1 to 2. Such a formula would obvi- ously only hold in those cases in which the ratios between zymase and co-enzyme are maintained on a constant basis. The formula which we have developed above gives results closely agreeing with theoretical conclusions in all cases in which sources of experi- mental error, such as the action of outside enzymes, are eliminated. LIFE-SAVING ACTION OF ESERIN. 47 17 (355) Presentation of a dog ten months after double nephrectomy and replantation of one kidney. By ALEXIS CARREL. [From the Rockefeller Institute for Medical Research. | The animal presented to the Society underwent the extirpation and replantation of the left kidney and the extirpation of the right kidney ten months ago. He is to-day in excellent health. The result shows that the perfusion of the kidney with Locke’s solution, the interruption of the renal circulation for fifty minutes and the disconnection of the renal nerves with the central nervous system do not produce any lesion of the kidney incompatible with its functions. 18 (356) A demonstration of the life-saving action of eserin in poi- soning by magnesium. By DON R. JOSEPH and S. J. MELTZER. [From the Department of Phystology and Pharmacology of the Rockefeller Institute for Medical Research. | At the May meeting of this Society, one of us (J.) reported that by the use of magnesium, certain toxic effects of physostig- min can be completely overcome. In our present communication we wish to bring out the fact that the antagonism between physo- stigmin and magnesium is mutual, at least to a certain extent. We wish to show an experiment which demonstrates that physostigmin can overcome certain toxic effects of magnesium and thus save the life of a poisoned animal. Both these rabbits (A and B) received at about the same time I.2 gram of magnesium sulphate per kilo of body weight. The jnjections were given intramuscularly in the lumbar region. Rabbit B received in addition one milligram of eserin, also intramuscularly. Rabbit A is already dead. Rabbit B is still alive ; although anes- thetic and limp, it breathes regularly and apparently is in no danger of death.’ 1 By the end of the meeting, rabbit B had recovered completely. 48 SCIENTIFIC PROCEEDINGS (31). 19 (357) The mechanical destruction of pepsin. By A. 0. SHAKLEE and S. J. MELTZER. [From the Rockefeller Institute for Medical Research.] At various times since 1884, one of us (M.) has studied the effects of shaking upon living cells, such as red blood corpuscles, bacteria, and arbacia eggs. Those experiments led to the general conclusion that shaking, by virtue of the mechanical factor, exerts a profound influence upon living organisms. In the present series of experiments we intend to investigate the changes which shaking may produce in the action of ferments. We began with the study of pepsin, the determination of which is greatly facilitated by several recently described reactions. Solutions of pepsin, partly filling long bottles, were shaken at room temperature and at a temperature of 33° C. for periods of different lengths, by means of shaking machines. Our results which we state here very briefly are unmistakable. Shaking under these conditions destroys pepsin. Even short periods greatly diminish its strength. If shaken long enough it is completely destroyed. The temperature has a marked influence upon the rate of destruction. Higher temperatures hasten the destruction. That the effect is not due to oxidation was proved by substi- tuting for the air in the bottles, hydrogen, carbon dioxide or oxygen. There was no appreciable difference in the results. It was also shown experimentally that the destruction was not brought about by any rise of temperature caused by the shaking. Maximum thermometers were fixed in the bottles perpendicular to their long axes, that is, perpendicular to the direction of shak- ing: in no case did the thermometer inside register as much as a degree higher than the thermometer outside. We have also found that the degree of shaking which occurs in the animal body is sufficient to reduce the activity of pepsin. This was determined by introducing a small bottle containing a solution of pepsin into a dog’s stomach through an esophageal fistula and permitting it to remain there for 24 hours or longer. The pepsin strength was diminished as much as 40 per cent. com- ACQUIRED RESISTANCE OF RED BLoop CELLs. 49 pared with that of the pepsin in a similar bottle kept in the ther- mostat at a temperature of 39° C. The reported results were demonstrated by the reactions for pepsin of Jacoby and Solms, of Fuld, and of Gross. 20 (358) A demonstration of the effects of CO, upon the frog’s pupil. By JOHN AUER. [From the Department of Physiology and Pharmacology of the Rockefeller Institute for Medical Research. | Frogs placed in an atmosphere of CO, gas show within thirty seconds, before any symptoms of excitement, a good constriction of the pupil. This constriction becomes almost maximal within five minutes ; there is no dilatation. The same effect is exerted by CO, gas upon excised frog’s bulbi. When the frog’s iris, in the excised bulbus or in the living animal, is under the influence of CO, gas, the powerful mydriatic effect of adrenalin is strongly reduced. Since CO, produces this myotic action upon excised bulbi, its effect must be exerted, largely at least, upon the sphincter pupille, that is, its action is peripheral. These experiments, however, do not exclude a possible central action. This myotic effect of asphyxia in frogs is interesting, as asphyxia in mammals produces chiefly dilatation. 21 (359) On the specific acquired resistance of red blood cells. By RICHARD WEIL. [From the Loomis Laboratory, Department of Experimental Pathol- ogy, Cornell University Medical School, New York City. | It is a well known fact that the serum plays a preponderating role in the immune reactions of animals. On the other hand, it has been amply shown that the immune characters of the serum 50 SCIENTIFIC PROCEEDINGS (31). may be in no proportion to the immunity of the organism as a whole. There must therefore be an additional factor, hypotheti- cally designated by Behring as cytogenetic immunity. Of this type of immunity there is the following experimental evidence. In 1898, Kossel stated that if an animal were injected with eel serum, its washed red cells manifested resistance to the hemolytic action of the latter in vitro; a fact confirmed by Camus and Gley, and by Tshistovitch. In 1908, it was shown by Morawitz and Pratt that in animals injected with phenylhydrazin, the red cells became resistant to all hemolytic agents. This they showed to be not an immune reaction, but the direct result of the chemical action of the drug on the red cells. It was the object of the present series of experiments to deter- mine whether it was possible to induce a specific resistance of the red cells to poisons injected into the animal. Dogs and rabbits were used. Among the hemolysins injected were : eel serum, and dog serum ; saponin, and digitalin ; staphylolysin, tetanolysin, and prodigiosus toxin; and phenylhydrazin. Eel serum, saponin, and phenylhydrazin were selected for routine experimentation. All of these were found to induce a severe grade of anemia. The animals were bled at intervals, and the resistance of the corpuscles, after repeated washings in salt solution, tested against a variety of destructive agents. Eel serum was found to induce occasionally a marked change inthe red cells ; saponin did so almost invariably, in case the animal survived the treatment ; phenylhydrazin did so without exception. Inthe early stage of treatment, the resistance of the red cells to all hemolytic agents, including the injected hemolysin, was diminished. In the later stages, it was increased in a characteristic manner. The red cells of animals injected with phenylhydrazin showeda marked increase of resistance to all types of hemolysins. Animals injected with eel serum and with sapo- nin came to possess a type of erythrocytes which were very resist- ant to the specific injected hemolysin, but were almost invariably more easily destroyed than normal control cells by all other hemo- lytic agencies, including anisotonic salt solutions (demonstration). In those animals injected with eel serum, which failed to develop a specific resistance of the red cells, the serum showed marked anti- hemolytic powers. It is evident, therefore, that the erythrocytes have developed a specific immunity. Butyric REACTION FOR SYPHILIS. 51 The cause of this resistance has been made the subject of further study. After testing the saponin and eel serum on a variety of red cells, the supernatant fluid was pipetted off, and tested on normal red cells. It was found that the fluid which had been in contact with resistant cells was least hemolytic, and vice versa. It is conceivable that the resistant erythrocytes may either absorb a disproportionate quantity of the hemolysin, or may con- tain a neutralizing substance. It has been customary in human pathology to judge of the resistance of red cells according to their vulnerability in anisotonic solutions of salt. The above described experiments indicate that specific resistance to a circulating toxin may be associated with marked loss of resistance to anisotonic solutions of salts. The red cells in advanced cases of cancer have been shown (Lang and others) to possess a greatly increased degree of resistance to aniso- tonic solutions. The demonstration of a hemolysin in the circu- lating blood of cancerous cases, and of an increased resistance thereto on the part of the red cells, has made it possible to prove that the resistance is specific to this hemolysin, and only accidental and occasional for the anisotonic solutions. 22 (360) The butyric reaction for syphilis in man and in the monkey. By HIDEYO NOGUCHI. [From the Rockefeller Institute for Medical Research. | In a preliminary communication’ I stated elsewhere that an increase in certain protein constituents of the blood serum and of the cerebro-spinal fluid of patients suffering from active or latent syphilis or parasyphilitic affections is a constant occurrence. I wish to describe here briefly the technique of employing butyric acid for the detection of this increase of protein. Cerebro-spinal fluid. — One or two parts? of spinal fluid* are mixed with five parts‘ of 10 per cent. butyric acid solution ° and are lNoguchi: Jour. of Exp. Med., 1909, xi, p. 84. 20.1 or 0.2 c.c. are sufficient and convenient. 3 Must not contain blood. 40.5 c.c. for the quantities above specified. 5 Best in 0.9 per cent. salt solution. 52 SCIENTIFIC PROCEEDINGS (31). heated over a flame to a brief boiling. One part’ of normal solu- tion of NaOH is then added quickly to the heated mixture and the whole is boiled once more for a few seconds. The presence of an increased content of protein in a spinal fluid is indicated by the appearance of a granular or flocculent precipitate which grad- ually settles under a clear supernatant liquid. The intensity of the reaction varies greatly according to the amount of the protein which a given specimen contains, but the granular appearance of the precipitate means a positive reaction for syphilis or parasyphi- litic affections. With normal or non-specific specimens there will be a slight opalescence or sometimes a marked turbidity which, however, does not settle out in several hours or even in 24 hours. Blood serum. One part® of clear serum is mixed with nine parts® of half saturated solution of ammonium sulphate. Upon complete precipitation, the mixture is centrifugalized and the compact deposit (globulin fractions) is separated from the super- natant fluid by decantation. The deposit is then redissolved in ten parts‘ of 0.9 per cent. salt solution, in which it easily dissolves. The globulin solution thus obtained is ready for the acidification with butyric acid. This is done by mixing one part of the solu- tion with an equal part of Io per cent. butyric acid solution. It is my custom to take 0.5 c.c. of each solution for mixing. On standing, prompt and dense turbidity begins to appear in the tubes containing the fractions of the serum of syphilitic or certain non- syphilitic patients, while those from normal serum remain quite clear after several hours, or show only slight opalescence with- out precipitation. A few words may be added here as to the results of investiga- tions made with the above methods. About 250 specimens of cerebro-spinal fluid, mostly of parasyphilitics, and about 300 speci- mens of the blood of syphilitic and parasyphilitic patients, together with many control specimens derived from patients with non- syphilitic diseases and normal persons, have been studied. 1 Namely, 0.1 c.c. in this case. 2 Usually 0.5 c.c. is sufficient and convenient. 3 Namely, 4.5 c.c. in this instance. 45 c.c. in this instance. Butyric REACTION FOR SYPHILIS. 53 Spinal fluid derived from parasyphilitic cases gives a typical reaction, becoming granular in a few minutes and sedimenting in from 10to 15 minutes. Cerebro-spinal fluid from cases of congeni- tal, tertiary or secondary syphilis gives quite constantly a positive reaction, but the intensity is usually less and two hours may be required before the characteristic granular appearance becomes manifest. Cerebro-spinal fluid from cases of cerebral or spinal syphilis gives invariably a positive reaction. Negative reaction was obtained with the spinal fluid from cases of acute anterior poliomyelitis, epilepsy, alcoholic psychosis, dementia precox, senile dementia, spastic paraplegia, lobar pneumonia and typhoid fever. On the other hand, an abundant flocculent precipitate was usually formed with the spinal fluid from cases of tubercular menin- gitis, influenza meningitis, or epidemic cerebro-spinal meningitis.* Cerebro-spinal fluid collected from two cases of hydrocephalus also gave abundant precipitation. In all of these acute inflamma- tory cases, except one of hydrocephalus, the Wassermann reaction was, however, negative. A number of post-mortem spinal fluids were examined with such results that it seems desirable to use the method as a routine diagnosis for syphilis or parasyphilitic affec- tions at autopsy. In the spinal fluid of two monkeys with active experimental syphilitic lesion at the site of inoculation, which per- sisted about 6 months, the reaction was positive. Referring to the results of examinations of the blood serum, it appears that the reaction is non-specific for syphilis, because a similar reaction can be obtained in certain cases of tuberculosis, carcinoma and Hodgkin’s disease. In view of the constancy with which an abnormally high globu- lin content attends the florid stage of syphilis and appears to be present in an early primary stage, and is present in the late second- ary and tertiary stages of imperfectly treated cases, one is thus en- abled to follow the course of an anti-syphilitic treatment. Moreover the butyric acid test is a more delicate indicator than the Wasser- mann reaction, for the latter is very frequently negative in this latter class of cases. Under conditions of adequate treatment, the globu- lin fraction of the blood serum is not increased. A negative re- 4 These acute inflammatory conditions are quickly and perfectly excluded by clini- cal and usual microscopical methods of diagnosis. 54 SCIENTIFIC PROCEEDINGS (31). action with the butyric acid test indicates either the absence of syphilitic infection or a successful cure of the disease. There is no necessary relation between the Wassermann test and the quan- tity of globulins in the luetic serum. 23 (361) The quantitative separation of leucin from valin. By D. D. VAN SLYKE and P. A. LEVENE. [From the Rockefeller Institute for Medical Research. ] Of the known amino-acids determined in semi-quantitative esti- mations of final proteolytic products, leucin and its relatives, iso- leucin and valin, have proven unusually difficult to prepare pure in even approximately quantitative amounts. The separation of these substances, because of their close physical and chemical similarity, has offered almost insurmountable difficulties to previous investi- gators. The acids form isomorphous mixtures which are absolutely inseparable by crystallization ; and their esters have so nearly the same boiling points that they cannot be fractionated by distillation. Because of these difficulties, most investigators have not attempted to separate the mixture, but have reported the entire mass as leucin. Fischer’ states that all the figures reported from his laboratory for leucin in protein hydrolyses refer to this mixture. Ehrlich? has recently reported a method for separating the three substances, but it involves a long process, large losses, and the racemization of the isoleucin and valin. We have been able to separate the leucin isomers readily from valin in quantitative amounts. The method, which is very simple, rests on the fact that if a molecular lead acetate solution is added to an ammoniacal solution of the leucin-valin mixture, the leucins are precipitated as analytically pure Pb(C,H,,O,N),. If too great an excess of lead acetate is added, a portion of the valin may also be precipitated. Consequently, the mixture is first analyzed, an estimate of the proportion of leucin calculated from the carbon content, and 20 per cent. excess of the theoretical amount of lead 1 Fischer: Unters. iiber Aminos., Polypeptide, und Proteine, p. 67. 2 Ehrlich: Bioch. Zeitschr., 8,'399, 1908. SEPARATION OF LEUCIN FROM VALIN. 55 acetate used for precipitation. The valin is obtained analytically pure by freeing the filtrate from lead with H,S, evaporating to dry- ness, and washing with absolute alcohol. A slight amount of valin dissolves, but is regained by evaporating the washings. The following is a typical separation, the material being a por- tion obtained by tryptic digestion of casein and fractional distilla- tion of the amino-acid esters. 12.546 g. of the mixture was used. Analysis showed 52.79 per cent. C, 9.55 per cent. H. The mixture was suspended in 80 c.c. of boiling water. The flask was removed from the flame and 20 c.c. of concentrated aqueous ammonia added. The flask was loosely stoppered, and shaken gently until the acids were dissolved. The leucin was then pre- cipitated with 25 c.c. of M/1 lead acetate. The cooled solution was filtered, and the precipitate washed with 50 c.c. of dilute ammonia. 8.955 g. of lead salt, equivalent to 5.025 g. of leucin, and 7.322 g. of valin were obtained analytically pure, making 12.347 g. from the original 12.546 g. Analytic data: Lead salt: (1) 44.25 per cent. Pb; (2) 44.36 per cent. Pb. Calculated for Pb(C,H,,O,N),, 44.29 per cent. Pb. Valin: 51.44 per cent. C; 9.42 per cent. H. Calculated for CATO, NG 5 t-24uper cent. € ; 9.47. per cent. Fi. The specific rotation of the valin was [a]”pD-+ 26.51°. The pure active substance has the rotation + 28.8°. The product was partially racemized, the usual result of long tryptic digestion. The lead-leucin salt contains a mixture of leucin and isoleucin. Levene and Jacobs’ have shown that these isomers can be readily separated in the absence of valin. The complete separation of the two leucins from valin, therefore, renders the systematic separation of all three comparatively easy of accomplishment. This is of im- portance, not only for protein analysis, but also for the preparation of pure active valin and isoleucin, a task which has hitherto been extremely difficult. The work will be reported in full in the Biochemische Zeitschrift. 1 Levene and Jacobs: Bioch. Zettschr., 9, 231, 1908. 56 SCIENTIFIC PROCEEDINGS (31). 24 (362) Further studies on the constitution of inosinic acid. By W. A. JACOBS and P. A. LEVENE. [from the Rockefeller Institute for Medical Research. | In a former article’ on the constitution of the inosinic acid ob- tained from beef extract, we have demonstrated that by acid hydro- lysis there is formed an intermediate product, a pentose phosphoric acid, which we isolated as a well crystallized barium salt. From the fact that this body showed strong reducing properties, it is evident that the aldehyde group is free, and the phosphoric acid is bound, ester-like, on one of the hydroxyl groups of the pentose. As the inosinic acid itself does not reduce Fehling’s solution, it is at once obvious that the hypoxanthin contained in its molecule must be bound as in a glucoside on the aldehyde group. We also mentioned that upon alkaline hydrolysis we were able to isolate a small quantity of a silver compound of a purin-pentose complex which gave all the qualitative tests for such a body. Meanwhile it came to our notice that Haiser and Wenzel? had obtained a compound of hypoxanthin and a pentose from karnin to. which they gave the name zzostn. We have succeeded, by heating the barium salt of inosinic acid in water solution in a sealed tube at 125°—130°, in obtaining a mixture from which we have isolated a substance which in all respects corresponds with Haiser and Wenzel’s inosin. From this substance we obtained a levorotatory pentosazone. Furfurol distillation yielded the phloroglucid required by a pentose. 1 Levene and Jacobs: Berichte d. deut. chem. Gesell. , 41, 2703 (1908). 2 Haiser and Wenzel: Monatshefte fiir Chemie, 29, 157 (1908). PERMEABILITY OF THE CELL PLAsMA MEMBRANE. 57 25 (363) The significance of changes in the permeability of the plasma membrane of the living cell in the processes of stimulation and contraction. By RALPH §. LILLIE. [From the Physiological Laboratory, Zoélogical Department, Unt- versity of Pennsylvania. | The general facts indicating that stimulation is dependent on a temporary increase in the permeability of the surface layer or plasma membrane of the irritable element are as follows : A. The nature of the motile process in such plants as Mimosa, Dionea and the Cynarez, where the movement depends on a sud- den loss of turgor. Such a change indicates either (1) a sudden decrease in the concentration of the osmotically active substances within the cell due to chemical action, or (2) a sudden loss of impermeability relatively to the osmotically active substances. The latter explanation is almost certainly the correct one. B. The identity of the electrical change accompanying stimu- lation in motile plant cells with that observed in irritable animal tissues (Burdon-Sanderson), indicating a fundamental similarity in the conditions of stimulation in the two classes of organisms. C. The fact that the post-mortem increase in permeability is accompanied by contraction in muscle cells; the same is, of course, true of motile plant organs where the movement depends on loss of turgor. D. The nature of the electrical change accompanying stimu- lation. If the irritable element represents a concentration-cell in which a semi-permeable membrane (the plasma membrane) greatly diminishes the velocity of the anion, while leaving that of the cation practically unaltered (Ostwald-Bernstein membrane theory), any marked increase in permeability relatively to the anion must result in a fall of the potential difference between exterior and interior of the irritable element. Such an electrical change actu- ally occurs on death or injury of the element ; also momentarily during stimulation. A demonstrable increase in permeability occurs at death; inferentially, therefore, the same change occurs during stimulation. 58 SCIENTIFIC PROCEEDINGS (31). E. The fact that an irritable tissue loses irritability for a variable usually brief) period after stimulation (refractory period), — indi- cating that at that time the property of semi-permeability, on which electrical stimulation depends (Nernst), —is temporarily lost. The special observations presented in this paper are as follows: 1. Arenicola larve are stimulated intensely by pure isotonic solutions of various salts (NaCl, KCl, NH,Cl, LiCl, SrCl,, BaCl,), contracting to half their length for several seconds when first in- troduced into the solution. At the same time a yellow pigment contained in the cells of the organism diffuses freely to the exterior and colors the solution. 2. Solutions which do not produce this strong initial contrac- tion do not cause such loss of pigment. Isotonic CaCl, and MgCl, (especially the latter) are instances. In these solutions the muscles lose the power of contraction and the organism becomes stiff and motionless (though still propelled by the cilia which remain active). Addition of small quantities of CaCl, to a NaCl solution prevents the strong initial contraction and loss of pigment (antitoxic action). 3. MgCl, and similarly acting solutions appear to decrease the permeability of the tissues, and so prevent the ionic transfer on which stimulation depends. The general action of anzsthetics consists in decreasing the normal permeability ; stimulating agencies, on the other hand, have the reverse effect. 4. Strong solutions of fat-solvents (chloroform, ether, benzol, (etc.) produce a contraction of the muscles accompanied by loss of pigment, even in 7/2 MgCl,. This effect is to be referred to an alteration of the lipoid substances in the plasma membrane. Such alteration, if slight, decreases permeability (anzsthetic action in low concentrations) ; if extreme, it produces the reverse effect, with re- sulting stimulation. 5. The hypothesis is presented that the chemical effect of the above changes in permeability depends essentially on their influence in varying the rate at which carbon dioxid leaves the cell. The velocity of the oxidative energy-yielding processes whose end- product is CO, is thus varied with the rate of removal of this latter substance from the system ; this velocity is accordingly increased during the increased permeability of stimulation, and is decreased LysINns, PRECIPITINS, AGGLUTININS., OPSONINS. 59 during anesthesia or inhibition, This view is supported by a con- sideration of the electrical changes accompanying inhibition and stimulation, respectively (‘ positive’’ and “ negative variations’’). 26 (364) On the relative concentration of lysins, precipitins, agglutinins, opsonins and related substances in the different body fluids of normal and immune animals. By F. ©. BECHT and J.R. GREER. (By invitation.) [from the Hull Physiological Laboratory of the University of Chicago. | The present work was suggested by some of our previous work with Prof. Carlson on the physiology of lymph. It was suggested that it would be of profit to establish the differences between serum and the other body fluids in their content of anti- bodies of various kinds in normal and immune animals, with the hope that it would have some bearing upon the problem of lymph formation, and also upon the origin of these antibodies. Thus far the hemolysins, hemagglutinins, bacterial agglutinins, bacterial opsonins, hemopsonins, and precipitins have been studied in the serum, neck lymph, thoracic lymph, pericardial fluid, cerebrospinal fluid, and aqueous humor. The bacterio-lysins are also under consideration. The results with normal dogs have been the following : Hemo- lysins are found in the serum, thoracic lymph, and neck lymph in the normal animal. Serum and thoracic lymph contain them in almost equal quantities, with a slight balance in favor of the serum. The hemolytic power of the neck lymph is much lower than that of the serum, and is almost entirely wanting in the lymph which is secured without massage. In two of seven cases there was a small amount of hemolysis in the pericardial fluid, in the remain- ing cases there was no laking when the fluid was free from ery- throcytes. There is no hemolysis in the cerebrospinal fluid, ex- cept in one case where there was a trace of free hemoglobin. There was no hemolysis in the aqueous humor. The hemagglutinins run parallel with the hemolysins except that they act in higher dilutions than the latter. 60 SCIENTIFIC PROCEEDINGS (31). Bacterial agglutinins are found in the serum, thoracic lymph, and neck lymph. The two lymphs were of equal strength, and the serum was approximately ten times as strong. Pericardial fluid, cerebro-spinal and aqueous humor were entirely lacking in these anti-bodies. No precipitins for rabbit serum are found in any of the body fluids of the normal dog. The hemolytic power of the serum and thoracic lymph of dogs immune to typhoid is slightly higher than that of the same fluids of the normal animal. No increase has been noted in the other fluids examined. The hemagglutinating power is increased in the serum, neck lymph, thoracic lymph, and pericardial fluid, particu- larly in the latter. The bacterial agglutinating power of the serum, neck lymph, and thoracic lymph is much increased. The pericardial fluid and aqueous humor are inactive or at most show only a trace. The cerebro-spinal fluid has not yet been tested. In dogs rendered immune to rabbit blood we have not yet noted any very marked increase in hemolysins over those of the normal animal. There was a marked increase of the hemaggluti- nins in the serum, neck lymph and thoracic lymph, and in one case in the pericardial fluid and aqueous humor. Immunization to rabbit blood has no effect on the bacterial agglutinins. The injec- tion of defibrinated rabbit blood did not produce precipitins for rabbit serum in any of the body fluids of the animal. The work on bacterial opsonins and hemopsonins in both normal and immune animals has been entirely unsatisfactory. The work is being continued and extended to cats and rabbits. Later, lymph will be collected from various organs and extracts from various tissues made with the hope of finding the source of these antibodies. Parallel experiments are being made on the number and kind of leucocytes in the various body fluids. Drerary CONDITIONS ON PHYSIOLOGICAL RESISTANCE. OI 27 (305) Studies of the influence of various dietary conditions on phys- iological resistance. I. The influence of different pro- portions of protein in the food on resistance to the toxicity of ricin and on recupera- tion from hemorrhage.' By NELLIS B. FOSTER. [fromthe Laboratory of Biological Chemistry of Columbia Univer- sity, at the College of Physicians and Surgeons. | It was planned to compare, in these experiments, the behavior of two sets of dogs under variously induced pathological conditions. The experiments were conducted on two pairs of dogs, each pair being kept under identical conditions, so far as they could be con- trolled, except with respect to the food. One animal of each pair was given /zbeval amounts of protein in the daily diet, the other received barely sufficient protein to provide for the necessary nitrog- enous metabolism, the remaining ingredients of the food for each animal being uniformly equal to the daily amounts ordinarily given per kilo, in this laboratory, to perfectly healthy dogs. No attempt was made to maintain equal caloric values in the diets. In each experiment the animal was fed on a diet of hashed lean meat, cracker meal and lard, the meat being gradually increased or dimin- ished to a high or low plane, according to the plan in each case and before the particular pathological condition was induced. It was a part of the plan of the work to keep the animal in each instance on a high or low plane of protein nutrition for a con- siderable period before the pathological phase was brought into the experiment. Such a course is not only desirable but essential, for if, as has been claimed, a diet rich in protein exercises a dele- 1 This study was begun during the summer of 1905, at Dr. Gies’ suggestion and thas been carried forward from time to time under his direction and with the aid of a grant from the Rockefeller Institute. I am also indebted to Dr. Flexner for impor- tant suggestions. The work has been frequently interrupted by researches in other directions and has been beset by unusual experimental difficulties. Although planned to be the first of a series of investigations, in point of publication it is the second from this laboratory on the general subject stated above. See Dissertation by Welker, Columbia University, 1908. It is Dr. Gies’ intention to continue investigation along these lines, 62 SCIENTIFIC PROCEEDINGS (31). terious influence on the animal organism, that effect is not ordi- narily an immediate one, but rather the slow perversion of organic function induced by chronic misuse. Again, if benefit results from a special diet under any condition, time may be required to make that benefit clearly distinguishable. The dogs used in these ex- periments were under daily observation for periods of at least two months and in several cases for four months before inauguration of the pathological phase. In testing the physiological resistance of the dogs in these experiments, use was made of two methods: (1) hypodermic in- jection of the toxin, ricin; and (2) the withdrawal of definite amounts of blood from one of the large arteries. Ricin was selected because it produces some symptoms which are analogous to those of acute infections, namely, fever, cardio-vascular embar- rassment and marked prostration. As in the case of infections, ricin also causes noticeable stimulation of metabolism, as is shown by the increased elimination of nitrogen and sulfur after its injec- tion. The disadvantages in its use are that immunity quickly arises and that the material is so very toxic that no latitude is per- mitted for minor individual peculiarities in animals. By experi- ment I milligram per kilo was found to be the maximum non- lethal dose for the commercial sample used. EXPERIMENTS WITH RICIN. Six dogs were subjected to inoculation with ricin. Of these, three were on a high plane of protein nutrition, the daily amount of food containing uniformly in each case 1.4 to 2.0 grams of N per kilo of body weight. After inoculation with ricin (1 mg. per kilo), all of these three specially well-fed animals died. A fourth dog was on a medium plane of protein nutrition, 1.1 gram. of N per kilo, and this dog survived the inoculation. Of the two remain- ing animals of this group, both on a low plane of protein nutri- tion, one died and one survived. The dog which survived received the protein equivalent of 0.35 gram of N per kilo of body weight during the experimental period and the one that died, 0.37 gram of N per kilo. In order to test the effect of exercise, the former of these two dogs, after the completion of the above mentioned experiment, was DiETARY CONDITIONS ON PHYSIOLOGICAL RESISTANCE. 63 allowed to run at large in the laboratory for six months (July 17, 1906, to Jan. 10, 1907), no effort being made to collect excretions for analyses. During this time the protein content of the diet was gradually dropped to an equivalent of 0.27 gram of N per kilo. The animal was painfully thin but appeared to be in excellent health. She was lively during the warm months of summer and autumn, but with the advent of colder weather she became less and less active and lost weight, although the laboratory was adequately heated. It finally became evident that the diet must be changed. The portion of meat in the diet was gradually in- creased, but a diarrhea supervened, which proved fatal. HEMORRHAGE EXPERIMENTS. Although hemorrhage is one of the crudest factors for the determination of physiological resistance, it has, nevertheless, the advantage of being a real test, inasmuch as the production of new blood is an exemplary reparative process. Hemorrhage, unlike the injection of ricin, can also be gauged, to some degree at least, to meet the individual resistance of the animal, as I have found in these experiments. The procedure was uniform throughout this part of the work. At the first hemorrhage from each animal, the endeavor was made to take blood equivalent to 4 per cent. of the body weight. After an interval of four days this procedure was repeated; and again eight days after the initial hemorrhage, making three hemorrhages at successive intervals of four days. All operations were conducted under ether narcosis. Six dogs were experimented on in this manner, three upon high, three upon low, planes of protein nutrition, and four survived. Of the two which died, one had been on a low plane of protein nurition (0.4 gram of N per kilo) and one ona high nitrogen plane (1.4 gram per kilo). Control animals on more extreme dietary conditions, withstood the effects of greater hemorrhages. Therefore, it is evident, I think, that these two deaths must be assigned to reasons outside the realm of this research. In the case of the dog on the high plane of protein nutrition, the cause is not far to seek; the dog was a collie, apparently pretty well bred, and it is a matter of common knowledge that these dogs have very low resistance. The dog that died on a low plane of protein nutrition was a fox terrier mongrel. 64 SCIENTIFIC PROCEEDINGS (31). I. Outlines of the Experiments. Nature of the experiment. Dog No. N of food per kilo, Ricin. Hemorrhage.* o Raetle Brame; 1 mg. per | Per’cent. of body weight. kilo of body weight. I 2 3 I 0.35 ae one aes ... | Very sick: Survived. II 1.10 a Survived, III 0.37 — Died. IV 1.5 + Died. Vv 3.37 —for I mo. + Over fed : 0.51+—for 20 ds. Died. VI 0.40 4- a0 ae a07 Died. VII 0.44 pe Ba8 2.5 eos Died. VIIl B57) a 4.0 | 4.3 3.1 | Survived. IX 1.4 ee 3.8 2.3 0.6 | Died. - 0.35 ee 4.2 4.0 4.0 | Survived. 0.81 6 4 3.7. | Survived. XII ay Pops 0.23 ie a5 2.9 | Survived. * The numerals above the figures for percentage indicate the number of the hemor- rhage in the series applied to the dog in question. + The food was dropped to this equivalent at once after two days of fasting follow- ing pronounced gastro-intestinal disturbances. Il. General Summary. Survived, 2 High plane of protein nutrition — 6 dogs Died, Totals (I — XII) 5 ist urvive Low “cc “e “ 5 dogs Died, High plane of protein nutrition — 3 dogs Died, Ricin experiments Survived, 1 Low uC Ke Ke 2 dogs Died, I Survived, 2 High plane of protein nutrition — 3 dogs | Loe | Died, I Hemorrhage experiments Survived, 2 Low e 66 as 3 dogs Died, I In order to reduce individual idosyncrasy to its lowest terms the last experiment of the series was conducted on two dogs from DieTARY CONDITIONS ON PHYSIOLOGICAL RESISTANCE. 65 the same litter. These animals were half-breed Scotch terriers, about two years old, that had lived together under the same con- ditions until they were brought to the laboratory. One was grad- ually accustomed to a diet containing only 0.2 gram of N per kilo, the other to food containing 0.8 gram of N per kilo. As a matter of further interest the opsonic index of these dogs was several times estimated, using Staphylococcus aureus that had been ren- dered pathogenic to dogs. This index was, and remained, the same for both animals. The final hemorrhages were survived by both animals and, so far as one might estimate, about equally well. The accompanying tables present an outline of the experi- ments and show details not mentioned above. yin oUt ih a AGC Lt ia nay thee : wi lbs i i ‘ ip i be , fi Tine MARU LSMON Pye i PY, te ra Thirty second meeting. New York University and Bellevue Hospital Medical College. February 17, 1909. President Lee in the chatr. 28 (366) A method for the direct observation of normal peristalsis in the stomach and intestines. By YANDELL HENDERSON. [Zrom the Physiological Laboratory of the Yale Medical School. | When the abdomen is opened peristalsis ceases. Because of this fact all previous investigators have found difficulty in making observations directly upon the motility of the stomach and intes- tines. The peristalsis which has been seen differs considerably from that which the radiographs of Cannon have shown to be the normal movements. Failure of peristalsis, as Meltzer especially has pointed out, is one of the characteristic and important phenomena of surgical shock. I have advanced the hypothesis’ that the cause of shock is acapnia. By applying this theory to the problem of maintain- ing normal peristalsis after laparotomy, the following simple and effective method was devised. After the administration of a moderate dose of morphin, the animals (dogs of about 10 kilos) were anesthetized with chloroform. So far as possible the hyper- pnoea of the initial stage of anesthesia was avoided. The ab- domen was laid open the entire length of the mid line. The omen- tum was cut out, and the viscera moved sufficiently to bring into view the upper colon, lower ileum, and the greater curvature of the stomach from the pre-antral groove to the pylorus. A sheet of transparent celluloid was inserted under the body wall and over the viscera; and the air in the space back of this window was washed out with a stream of carbon dioxide gas. To the trachea was attached a tube 15 mm. in diameter and 2 meters in length. Blood gas analyses showed that the blood-gases were thus main- 1Y, Henderson: American Journal of Physiology, 1908, xxi, 126. (67) 68 SCIENTIFIC PROCEEDING (32). tained normal, — not asphyxial in respect either to the oxygen or carbon dioxide contents. When the stomach was distended with air, and the large intestine and lower ileum with bread mush, movements in these three parts of the alimentary canal were seen identical with those shown by the radiographs of Cannon. In the stomach a deep constriction developed at the pre-antral groove every 15 seconds and moved toward the pylorus where it disap- peared as its successor was developing. In the colon there was active anti-peristalsis. In the ileum vigorous rhythmic segmenta- tion was seen. In other experiments I have found that animals under ordinary operative conditions develop, and remain in, a state of acapnia. This lowered carbon dioxide content of the blood and tissues, by inducing loss of tonus, is the cause of the failure of peristalsis after laparotomy. The essential point in the above described method is the prevention of acapnia. 29 (367) Studies on the effects of carbon mon-oxide poisoning. By A. I. RINGER. (By invitation.) [From the Physiological Laboratory of the New York University and Bellevue Hospital Medical College. | If an animal be allowed to breathe an atmosphere containing carbon mon-oxide, it will soon present a series of circulatory, respiratory, cerebral and metabolic disturbances, which, if carried too far, will result in death. These disturbances are believed to be brought about by the reduction of the oxygen-carrying capacity of the blood, due to the formation of the relatively stable carbon mon-oxide-hemoglobin, thus producing a state of progressive as- phyxiation of the tissues. The severity of these disturbances de- pends entirely upon the degree of asphyxiation; and, with the exception of some individual peculiarities in a few of the twenty- one dogs that I have experimented upon under anesthesia, all pre- sented the same symptoms at the same stage of asphyxiation. In nine experiments the following subjects were studied in their relationship to the degree of saturation of the hemoglobin with carbon mon-oxide: (1) the pulse, (2) the blood pressure, EFFECTS OF CARBON MON-OXIDE POISONING. 69 (3) the number of respirations per minute, (4) the volume of air respired per minute; in twelve experiments: (1) the physical symptoms, (2) the point of onset of coma, (3) the point of death. As soon as the animal begins to breathe the carbon mon-oxide there is an immediate acceleration of the pulse, which steadily gains in frequency until 45-50 per cent. of the hemoglobin is saturated with carbon mon-oxide. Then the pulse rate is at its maximum. After that it declines gradually until a point is reached between 71-74 per cent. of carbon mon-oxide saturation, when the pulse rate falls abruptly and the heart ceases to beat. The blood pressure in about half the cases was found to go gradually downward in spite of the markedly increased rate in the heart beat. In the rest of the cases there was a preliminary rise in pressure of about 10-15 mm. of Hg, which reached its maxi- mum when 45-50 per cent. was saturated with carbon mon-oxide. This was followed by a gradual decline, until when about 67 per cent. of the hemoglobin was saturated, there was an abrupt fall in blood pressure. The number of respirations per minute, taking two character- istic examples, was found to increase from a normal of 19 and 22 to 32 and 45, respectively, when about 50 per cent. of the hemo- globin was saturated with carbon mon-oxide. In other cases there was no increase in the rate of respiration at all, but there was a marked increase in the depth of each inspiration. The volume of air respired per minute, however, increased in all cases as soon as the animal began to breathe the carbon mon-oxide. It presented a curve with an ascending and descending limb. The maximum ventilation of the lungs took place when about 45 per cent. of the hemoglobin was combined with carbon mon-oxide. After that it declined gradually. At about 65 per cent. saturation, however, breathing became irregular, sometimes of the Cheyne-Stokes variety. Respiration always stopped about one to three minutes before the heart ceased beating. When about 40 per cent. of the dog’s hemoglobin is deprived of its oxygen carrying capacity, the animal begins to get weak. It cannot stand on its legs. It isin a state of general indifference. It does not partake of any food or drink, though it may be hungry. It is seized with vomiting and occasionally has convulsive spells. 70 SCIENTIFIC PROCEEDINGS (32). It responds to a call very slowly, and sometimes falls into a sleep, from which it is aroused with difficulty. Actual coma sets in at a point immediately following the decline in the pulse rate, or when about 50 per cent. of the hemoglobin is saturated with carbon mon-oxide. This has been found to be constant in all cases. Death also takes place in all dogs at about the same point of saturation. Nota single one reached the 75 per cent. mark. All died between 71 and 74 per cent. of saturation of the hemoglobin with carbon mon-oxide. Neither size, weight nor strength seemed to influence the point of death. From the foregoing it is seen that the symptoms of carbon mon-oxide poisoning may be conveniently divided into three stages: First or Compensatory Stage, which lasts up to 50 per cent. saturation; second or Stage of Depression which lasts up to about 70 per cent. saturation; third or Stage of Collapse. SYMPTOMS OF THE FIRST OR COMPENSATORY STAGE. Gradual increase in pulse rate. High blood pressure. Spasmodic attacks of vomiting. Slight dyspnoea at first — more marked at the end. Muscular weakness. Drowsiness, indifference and deep sleep from which dog can be aroused. Om pw YS & SYMPTOMS OF THE SECOND OR STAGE OF DEPRESSION. 1. Ushered in by clonic convulsion and muscular rigidity which lasts for a few minutes. 2. Deep narcosis from which animal cannot be aroused. 3. Pulse declines gradually in rate, tension and volume. 4. Respiration either rapid and shallow, or slow and deep; it is more or less regular. 5. Absolute loss of sensation. SYMPTOMS OF THE THIRD OR STAGE OF COLLAPSE. Pulse slow, irregular and of low tension. Respiration irregular. Loss of tone of sphincters. Conjunctival reflex lost. Death due to respiratory paralysis. wm Bw NN EFFECTS OF CARBON MON-OXIDE POISONING, 71 | Typical experiment of first series.— February 19,1908. Dog’s weight 9.7 Kg. Ether anesthesia. Time. Respiration, 2.15 P. M 22 Zeree 18 I per cent. CO respired 2535m «fi 24 2.50 ‘<6 36 3-058 ff 40 Cpe, 45 S235) ae 41 3:45) 5 40 B50) 6 36 Spey fe 32 BS Sin is 8 irregular 4.05 6c Io “c 4.08 Death & fe) o a 2D Ep A i am Volume of air Sa Palen Al oiedpe |. deg 7 & minute. $ 2 BA 5S (23) 0 Fs 140 90 1,750 148 go 1,925 164 92 2,000 20 180 96 2,125 204 | 104 2,250 47 194 94 2,250 166 | 88 2,100 54 120 72 1,925 82 62 1,650 66 56 40 1,175 68 | eae 75° 73 Typical experiment of second series.— March 2, 1908. Dog’s weight 6.6 Kg. Dog placed in air-tight cage through which the gas mixture was driven by means of .bellows, operated by a water pressure engine. The ventilation of the cage was 5,000 c.c. of air per minute. Time, Respiration. Pulse. General condition. 10.30 A. M. | 16 104 | Dog in good condition. TOss5e ° 0.4 per cent. CO respired TROON ES 16 104 | Dog comfortable and quite active. 11.45 6c 16 116 “c “6 73 66 “ec 12.00 “ec 18 124 “cc “c “ec 6c ce TiZesOn Lee Mo eX: 140 | Lies quietly; slightly drowsy. T2rAGe aes 21 170 | Drowsiness marked. rifers) UG 18 182 | Slight convulsion and vomiting. 20m iS 16 208 | Responds to sensory stimulus after long latent period. Hegiey 1 16 192 | Convulsions of clonic type. Pain 0 17 196 | Slightly comatose; when called he only opens his eyes. 2:05, <6 16 180 | Coma quite deep. Xo) WG — 166 | All muscles and sphincters relaxed. ZAG te — — | Taken out of cage. Blood sample shows 52 per cent. of the Hb saturated with CO. Dog placed in warm place near radiator. Braineemss Respiration slow and deep, 10-12 per minute. 4.00 ‘§ Dog began to move about. A250 at Able to stand up and walk but falls frequently, due to lack of coérdi- nation. alOnn se Walked about fairly well, but very slowly. Next morning Dog perfectly well. 72 SCIENTIFIC PROCEEDINGS (32). Experiments have also shown that a dog with as much as 69 per cent. of hemoglobin tied up with carbon mon-oxide which corresponds to the end of the second stage, can be resuscitated if proper treatment be instituted promptly. 30 (308) Intestinal excretion during diarrhea. By GEORGE B. WALLACE and HUGO SALOMON. [From the Laboratory of the Von Noorden Clinic, Vienna.) Analyses were made of the faeces of a number of patients with diarrheas of different origin. During one period of observation the patients were on the Schmidt-Strassburger diet, during a sec- ond period the diet consisted of 250 grm. sugar daily. In those cases where there was present an ulcerative process in the intes- tine — tuberculosis, carcinoma—the amount of nitrogen in the feces was markedly increased — being from 1.7 to 4. grm. daily on the sugar diet. In cases of severe catarrhal inflammation it was not over 1.5 grm.; in light catarrh it was within normal lim- its. The fat and carbohydrate elimination showed no such strik- ing differences although it was highest where an ulcerative condi- tion was present. Of the inorganic constituents the alkali excre- tion was fairly parallel to that of nitrogen. The other inorganic constituents were increased by the ulcerative processes but in some instances were increased equally where ulcerations were absent. The most striking result of the analyses is the high nitrogen excretion which occurs in ulcerative processes in the intestine. 31 (369) The vascularity of the kidney as influenced by sensory ‘ impulses. By R. BURTON-OPITZ and DANIEL R. LUCAS. [From the Physiological Laboratory, Columbia University. ] Quantitative determinations of the blood-flow through the left kidney were made with the aid of the stromuhr of Burton-Opitz. On stimulation of the central end of the sciatic nerve, a slight de- INFLUENCE OF TEMPERATURE ON HEMOLYSIS. 73 crease in the vascularity of this organ was observed. This decrease apparently followed a tonic contraction of the blood vessels and not a true constriction as is produced, for example, by stimulation of the corresponding splanchnicus major. It seemed to be merely a tonic reaction of the kidney against the high systemic blood pressure which follows stimulation of the sciatic. Similarly, the application of cold compresses across the back in the region of the kidneys, reduced the blood-flow through this organ, while hot compresses increased the flow. As the tempera- ture of the organ itself, or of the tissues in its immediate vicinity, was not changed by the compress, these variations in the vascu- larity of the kidney must have been produced reflexly. Stimulation of the distal ends of the vagi, below the point where the cardiac branches are given off, did not change the blood flow. The vagus, therefore, appears to carry no efferent vaso- motor impulses to the kidney. 32 (370) The influence of temperature on hemolysis in hypotonic solutions. By PAUL A. LEWIS. [From the Antitorzin Laboratory of the Massachusetts State Board of Flealth. | Hemolysis in hypotonic solutions is progressively increased as the temperature is decreased from thirty seven degrees centigrade to five degrees centigrade. In order to bring out this fact, that modification of Hamburger’s method for testing the resistance of erythrocytes, which was introduced by Theobald Smith, was used. The solutions were brought to the required temperature and then the corpuscles were added. The differences are present both at the points of beginning and complete hemolysis, but are only well marked at the intermediate points. This accounts for results obtained by Hamburger (1887 and 1903) who held that tempera- ture within these limits was without influence. The effect of temperature is the same whether sodium chloride or cane-sugar is used to give tonicity to the fluid. The corpuscles 74 SCIENTIFIC PROCEEDINGS (32). of the horse, rabbit, guinea-pig, calf, and sheep are equally affected. The differences become well marked after a few minutes’ exposure to the different temperatures, and thereafter bringing them to one temperature fails to equalize the hemolysis even after many hours. The effect, then, is on the corpuscle rather than on the surround- ing fluid and is exerted chiefly in the first moments of exposure. Temperatures above 37° C. act variously according to the particular species whose blood is used. Horse corpuscles give distinctly more hemolysis at 42° C. than at 37° C. Thecor- puscles of the guinea-pig and the calf give still less hemolysis at 43° C. than at 37 °oG: 33 (371) A carcinoma of the rat (Flexner-Jobling) considered from the standpoint of immunity. By F. P. GAY. [From the Laboratory of the Cancer Commission of Harvard University. | Experiments have been in progress for the last year and a half with the Flexner-Jobling rat tumor for the purpose of gaining some insight as to the normal and artifically produced conditions of resistance to this tumor. The tumor as originally described by Flexner and Jobling was a sarcoma and later became carcinomatous in structure. It has shown no marked variations in histological structure during the eight generations which we have cultivated it. White rats from different dealers varied considerably in their susceptibility to in- oculation with this tumor. Animals from the most susceptible source gave 100 per cent. of ‘“‘takes’’ whereas the next most sus- ceptible strain gave only 50 per cent. Following inoculation into the region of the axilla metastases occur regularly in the lungs but rarely in the adjacent lymph-nodes. The time of occurrence of metastases would seem to be relatively constant in the most susceptible rats. Metastases occur later and at more irregular intervals in less susceptible animals. The tumor may be transplanted from the metastases and such “‘metastatic’’ tumors would seem after several generations to have INFLUENCE OF TEMPERATURE UPON PEPSIN. 75 become somewhat more “virulent” in that such tumors grow more rapidly and produce more extensive metastases of a more epithelial type. Animals that have failed to take the first inoculation of tumor are very seldom susceptible to a second or third implantation. The blood serum of such refractory animals gives no reaction of fixation with cancer extract, and when injected simultaneously with cancer in susceptible rats leads to no prevention of the growth of the tumor. The refractory blood, however, when injected pre- viously to or at the same time with the tumor in naturally insus- ceptible rats gives a larger percentage of ‘takes’”’ than in control animals. If a tumor is removed during the “ premetastatic’’ period a secondimplanted tumor seldom grows. Subsequent to this period a second implanted tumor does grow. When a primary growing tumor is left and a second implanted during the premetastatic period, not only does the second tumor fail to grow but the first tumor entirely disappears in many in- stances. In cases in which the resorption of original tumor was. incomplete, from lack of sufficient time the primary tumor showed regressive changes in the nature of cell degeneration or a marked increase of connective tissue stroma. A reaction of fixation was found with the blood of a few ani-. mals with tumor during the premetastatic period but never during: metastatic period. The premetastatic period then would seem to be characterized by an active defence on the part of the animal body and during this period reimplantation of tumor increases this resistance to such an extent that the original tumor is destroyed. When this period is passed metastases occur and a second im- planted tumor grows. 34 (372) Influence of temperature upon pepsin. By A. 0. SHAKLEE. [From the Laboratory of the Department of Physiology and Phar- macology of the Rockefeller Institute for Medical Research. ] While studying the destruction of pepsin by shaking pepsin solutions it was thought essential to make some study of the spon- 76 SCIENTIFIC PROCEEDINGS (32). taneous deterioration that was seen to take place in solutions stand- ing at corresponding temperatures, and also at body temperature. One per cent. solutions of a commercial pepsin in hydrochloric acid (0.1 per cent., 0.25 per cent. and 0.5 per cent.) were kept in glass bottles at approximately the following temperatures: 5° C., 20° C., 33° C., 37° C. From time to time samples were tested by Fuld’s method, modified somewhat to decrease the error. The following results were obtained for solutions in 0.25 per cent. hy- drochloric acid at 37° C.: Duration of heating DEE be 7 . Te in days. t(1—4) 0.5 days. 19.5 per cent. -48 0784 31 Ls 53 Os 35 . 54 ae SOS far 54 2.0 ‘ 53 oe -56 GO 73 - 54 hey, 80 oe 50 230 es 86 Ot 51 These results seem to indicate that the destruction of pepsin under the conditions described takes place in accordance with the law of the bimolecular reaction, and the formula which seems to apply is: X "Cie ola where X represents the destruction, in hundredths of the original in time 4, and A represents a constant. 35 (373) Synthesis of uric acid. By NELLIS B. FOSTER and JAMES C. GREENWAY. [from the Laboratory of Biological Chemistry of Columbia Univer- sity, at the College of Physicians and Surgeons, and the Wards of the New York Hospital. | The possibility of a synthesis of uric acid from lactic acid and urea has been considered by a number of investigators, but when these substances are taken by way of the stomach, all researches alike have failed to disclose any evidence of uric acid synthesis in SERUM DIAGNOSIS OF SYPHILIS. Tix mammals. The object of this study was first, to repeat the earlier experiments of Minkowski; second, to find out if slight changes of method would perhaps serve to show a synthesis of uric acid ; and third, to consider the problem experimentally in relation to a man suffering from chronic gout. In gout it is possible that an abnormal synthesis of uric acid occurs, and also, since the uri- colytic powers of the gouty organism are less active than normal, a synthesis masked in a normal person might be evident in a per- son suffering from gout. The results of the experiments may be briefly summarized as follows : when lactic acid is administered to a normal man who has been fed on apurin free diet, there is no resulting increase of uric acid in the urine, even when the amounts of lactic acid are very large —. é., 20 grams ina dose. Ina dog ona purin free diet (milk, eggs, and rice), following the hypodermic injection of lactic acid, and of lactic acid and urea, there was in both instances a slight increase in the percentage of total nitrogen excreted as uric acid. The absolute amounts were also slightly increased as were those of allantoin. These figures are difficult to interpret and we are not prepared to assert without further investigation that there is a synthesis of uric acid in the manner described. In a case of chronic gout the effects of the lactic acid and urea were entirely obscured by the irregularity of nitrogen excretion ; periods of nitrogen retention and excretion making it impossible to estimate the effects of the treatment. 36 (374) Some critical considerations on the serum diagnosis of syphilis. By HIDEYO NOGUCHI. [From the Rockefeller Institute for Medical Research. | In its application to the detection of syphilis antibody the Bordet-Gengou phenomenon of complement fixation has received but little consideration in its quantitative aspect. As will pres- ently be pointed out it is only by respecting the quantitative rela- tions of all reagents concerned that the test becomes reliable and delicate. Even with an adequate quantity of antigen, blood cell suspension and the patient’s serum the detection of the antibody 78 SCIENTIFIC PROCEEDINGS (32). by means of complement fixation may or may not be successful according to whether or not appropriate amounts of hemolytic amboceptor and complement are employed. A large excess of either one of these two reagents can prevent the test from reveal- ing the presence of the antibody. While it is easy for a serologist to see why complement should be used in definite and uniform quantity, not every worker seems to be conscious of the disturbing effects which are exerted by an excess of the amboceptor. In view of the overlooking of certain principles of hemolysis by most of the investigators of the present time a brief consideration of this particular subject seems to be advisable. For the sake of con- venience I take the example of the antisheep hemolytic amboceptor for illustrating the influence of an excessive amount of the ambo- ceptor upon the phenomenon of complement fixation. The effects exerted by the excessive sensitization is two-fold. The first effect is to augment gradually the activity of guinea-pig’s complement by increasing doses of the amboceptor, until a maximum is reached. Thus in the presence of one unit of amboceptor 0.1 c.c. of the complement is usually required to produce complete hemolysis. By using four, eight and twenty units of the ambo- ceptor the same effect is obtainable with 3, } and }, of the 0.1 c.c. of complement respectively. For this reason it is impossible to demonstrate a partial fixation of complement by using more than several units of the amboceptor, and when several units are em- ployed the test suffers in delicacy. The second effect is still more disturbing than the first. It depends upon partial dissociation of the complement from its combination with the antigen and anti- body compound. A quantity of syphilis antibody just sufficient to fix 0.1 c.c. of the complement against two units of the ambo- ceptor is no longer efficacious to hold back the complement from partial liberation against the influence brought on by more than four units of the amboceptor. The fixation of the complement by two and three units of syphilis antibody respectively is also quite ineffective to prevent hemolysis when ten and twenty units of the amboceptor are added. Under these conditions the test fails to indicate the presence of any syphilis antibody although it is really present. When eight units of syphilis antibody are employed the fixation of complement becomes so firm that twenty units of the amboceptor can no longer bring about its liberation. Serum DIAGNosIS OF SYPHILIS. 79 From the foregoing it becomes at once evident that any system of the complement fixation test in which definite and appropriate amounts of these two vitally important reagents are not employed is not delicate and accurate enough to be a reliable diagnostic measure. Referring to the method of Wassermann I may state that it has all the disadvantages arising from the presence of unknown, but often considerably large amounts of natural antisheep ambo- ceptor contained in human serum. Wassermann was quite una- ware that the natural antisheep amboceptor is capable of being reactivated by guinea-pig’s complement, and hence he recommended the use of two units of immune antisheep amboceptor of the rabbit with the view of obtaining complete hemolysis. The reactivability of the natural amboceptor by this complement has been dis- covered since by Bauer who, in turn, proposed to utilize the natural amboceptor and dismiss the use of the immune ambo- ceptor. By systematic examination of more than 100 speci- mens of human sera in regard to the content of natural antisheep amboceptor I found that it varies from almost none to as many as twenty units in 0.1 c.c., the quantity usually employed for each tube in the fixation test. Thus the method of Wassermann is destined to give unreliable and inaccurate reactions. Bauer’s modi- fication is just as inaccurate as the original as it relies upon unknown amounts of the natural amboceptor alone. The method which I have recently perfected is also a comple- ment fixation test and differs from the Wassermann method in employing an antihuman hemolytic system instead of an antisheep hemolytic system. Thus the human blood corpuscles are to be hemolysed by means of an antihuman amboceptor prepared in the rabbit and the complement of the guinea-pig. I use two units of the amboceptor for each tube. In this new system the danger of introducing any uncalculated amount of the hemolytic amboceptor is absolutely excluded. As the fixation test is carried out with definite and uniform amounts both of the complement and the amboceptor, the results obtained with different specimens and at different occasions are all comparable with one another. The sharpness of the reaction enables one also to follow the fluctuation of the antibody content even to a fraction of one unit. 80 SCIENTIFIC PROCEEDINGS (32). Just as I had finished my experiments Tschernogubow published an article in which he stated that he successfully employed an antihuman amboceptor in combination with human complement. As he made no statement as to the source and strength of his antihuman amboceptor no judgment can be made on his method. It is rather striking to observe that the amount of the amboceptor he employed was 0.25 c.c. for each tube, in contrast to 0.002 in my method. It may not be entirely out of place to mention here some essential reasons why I use guinea- pig’s serum as complement. According to my observations the amount of complement in human serum varies considerably in different individuals. In the majority of specimens 0.1 to 0.03 c.c. of the fresh serum contain about enough complement to produce complete hemolysis with ten units of the amboceptor, while the same quantities do not cause any marked hemolysis when two units of the amboceptor are employed. Thus ten units of the amboceptor are to be used as a necessary amount for utilizing human serum as complement. Now in regard to the quantity of each specimen of human serum to be used for complement it is essential to determine the exact strength by a preliminary titration, because if we use some excess of complement the test turns out completely negative. It appears probable that human complement, like that of the rabbit, is not very sensitive to the fixing action of the antigen-antibody of syphilis. In this respect guinea-pig’s complement is excellent The method which Tschernogubow recommended is to collect a few drops of a patient’s blood in saline solution and use the sus- pension both for the complement and corpuscles at the same time. But, this does not permit one to make any estimation of the complement content of the blood. Moreover, there is no direct way of ascertaining whether any inhibition which may be observed with a given specimen is due to the anticomplementary property of antigen alone or to the combined action of the antigen and syphilis antibody, because the complement and antibody exist in the same serum side by side, if this latter is present at all. Again the relying upon human complement makes impossible the testing of any specimen of blood which has been allowed to stand for several days, as the activity of complement rapidly diminishes and NiTrRoGENouS METABOLISM IN CHRONIC NEPHRITIS. 8I finally disappears under these conditions. His method is not applicable to cerebrospinal fluid. In contrast to the use of human complement the use of guinea- pig’s serum does not possess one of the disadvantages enumerated. The quantity of complement is always uniform and definite. The human complement does not affect the reaction, because the amount present is too trifling to be of any influence. It is possible moreover to employ for the tests an old specimen of blood, dried or moist, by my method, since guinea-pig’s complement is used. Unlike Tschernogubow’s the present method enables one to repeat the test in case of need. Before leaving the subject I would like to point out certain advantages from the technical standpoint of the method which I recommend. The quantity of patient’s serum’ required for the test is only two capillary drops (one for each of the two tubes) and no preliminary inactivation at 56° C. is necessary. The hemo- lytic indicator is readily prepared from the patient or a normal person by mixing the blood with physiological salt solution in the ratio of one drop of the blood to 4 c.c. of the saline solution, I c.c. of such suspension being used for each tube. The antigen, com- plement and amboceptor can be used either in liquid or in dried form. This latter, as prepared on filter paper slips, can be pre- served permanently under ordinary conditions at room temperature and be employed in place of the corresponding liquid reagents. In this simple form the test should have a wide application to the sero-diagnosis of syphilis and as a measure and control of the efficient treatment of the disease. 37 (375) On nitrogenous metabolism in chronic nephritis. By D. MANSON, L. KRISTELLER and P. A. LEVENE. [from the Chenucal Laboratory of the Montefiore Hospital, New York. | The present work represents the results of observations on the character of the nitrogenous metabolism in a patient who was 'Tn case of cerebrospinal fluid use 0.2 c.c, for each tube. 82 SCIENTIFIC PROCEEDINGS (32). placed on a diet containing a sufficient supply of calories and a limited proportion of protein, so that his kidneys could readily re- move the end products of catabolism. That the capacity of the kidneys were not overtaxed may be concluded from the fact that the patient remained practically in nitrogenous equilibrium for four months. On this patient it was attempted to establish whether or not the abnormal, as v. Noorden termed it “‘bizarre”’ nitrogen elimination observed in course of nephritis is conditioned exclu- sively by faults of elimination. CONCLUSIONS. 1. The elimination-capacity of the kidneys of the patient was established by placing the patient on a diet containing a low pro- portion of protein and a sufficient supply of calories. To this standard diet varying quantities of urea were added, and the rate of the nitrogen output was measured. The output of nitrogen on the standard diet was generally about 5.5 grm. and the addition of 1.5 to 5.0 grm. of nitrogen in form of urea caused a rise in the output never exceeding 6.25 grm. Thus it was concluded that an intake of nitrogen below 7.0 grm. was the most suitable for the condition of our patient. 2. Comparing the rate of elimination of nitrogen after the administration of glycin, alanin, asparagin, with that after adminis- tration of urea, one notes a slower rate after the administration of the first two acids, and an equal rate after administration of aspar- agin (probably owing to the presence of an acid amid group in the molecule). 3. After administration of excessive protein in addition to the standard diet, one notes a much lower rate of nitrogen elimination than one should expect to find in a normal man, on the basis of the work of Falta. 4. Of the total nitrogen removed by our patient in excess over that on the standard diet, 80 per cent. was in form of urea, while in normal man, as calculated from the tables of Folin, the proportion of urea varies between 90 and 100 per cent.; in a normal dog the proportion is always 100 per cent. 5. On the basis of these observations it was concluded that in our patient the rate of conversion of protein into simple nitroge- nous substances and into urea is below the normal. FERTILIZING AND CYTOLYTIC EFFECT OF Soap. 83 6. The patient remained for four months in a condition of nitrogenous equilibrium, and otherwise in good health, on a diet containing about 6.5 grm. of nitrogen and 3,000 calories, which were ultimately reduced to 2,500 calories to prevent a constant gain in weight. 38 (376) The formation of gluconic acid by the olive-tubercle organism and the function of oxidation in some microorganisms. By CARL L. ALSBERG. [From the Office of Poisonous Plant Investigation, Bureau of Plant Industry, U. S. Department of Agriculture. | The olive-tubercle organism, Bacterium savastanot, recently de- scribed by Erwin F. Smith,' when grown in the presence of glucose and an excess of calcium carbonate, converts the greater part of the glucose into calcium gluconate. The amount of energy liberated thereby is exceedingly great in comparison to the weight of the organisms. This is to be explained by the fact that the energy requirements of microorganisms are very much greater than those of higher forms, partly because of the dispro- portion between the body surface and the body volume of micro- organisms, and partly because microorganisms exist in a medium which is an excellent conductor of heat. 39 (377) On the fertilizing and cytolytic effect of soap. By JACQUES LOEB. [rom the Phystological Laboratory of the University of California. | It has been shown by experiments on the eggs of sea-urchins, starfish, and annelids that the artificial membrane formation is the act which causes the unfertilized egg to develop. The agencies which cause the artificial membrane formation, as a rule, injure the egg. For the eggs of the starfish and certain other annelids 1Erwin F. Smith: Recent Studies of the Olive-Tubercle Organism. U.S. De- partment of Agriculture, Bureau of Plant Industry, Bulletin No. 131, Part IV, Wash- ington, 1908. 84 SCIENTIFIC PROCEEDINGS (32). this injury is rather slight, and these eggs are able to develop into larve without any further treatment. In the egg of the Califor- nian sea-urchin this secondary, injurious effect connected with the artificial membrane formation is more severe and demands a further treatment of the egg. This consists in preventing the eggs from developing for about from two to three hours after the membrane formation, by depriving them of oxygen or by preventing oxida- tions in the egg through the addition of a trace of potassium cyan- ide. During this time the egg is able to recuperate from the in- jurious effects of the membrane formation and is able to develop perfectly normally into a pluteus if transferred into normal sea- water. In my first experiments with this method, four years ago. not more than ten per cent. of the eggs could be caused to develop in this way. I have recently found that by a slight improvement of the method all the eggs can be caused to develop into larve, The segmentation is as a rule as normal as if the eggs were fer- tilized by sperm. A second method of overcoming the injurious effect caused by the artificial membrane formation consists in put- ting the eggs for from 10 to 40 minutes into hypertonic sea-water. This method also causes all the eggs to develop. These experiments showed that the process of membrane for- mation is the real cause which starts the development of the unferti- lized egg ; and the question therefore arose what the nature of this process is. My recent experiments have shown that the agencies which cause hemolysis also cause the membrane for- mation and the development of the unfertilized egg. I have thus been able to show that saponin, solanin, digitalin, bile salts, fatty acids, alkalis, hydrocarbons, ether and alcohols and the blood serum of not too closely related forms cause the membrane forma- tion of the unfertilized egg and its subsequent development. There remained only one cytolytic substance which seemed to form an exception, namely, soap, but experiments which I have recently carried out have shown that it is possible to cause the membrane formation and subsequent development of the egg with sodium oleate. If the unfertilized eggs of the sea-urchin are put into a mixture of 50 c.c. N/2 sodium chloride + 0.2 c.c. N/1to sodium oleate, the eggs form no membrane, nor do they undergo cytolysis ; but DEPRESSION OF THE FREEZING PoINT OF WATER. 85 if they are transferred into sea-water they form membranes and a smaller percentage of them undergoes cytolysis. If the eggs re- main a short time only in the soap solution, they all form mem- branes, but few cytolyze after being transferred into sea-water ; if they remain for a longer time, they all form membranes but cyto- lysis follows very soon after the membrane formation. The question arises, why do the eggs form their membrane only after they are transferred into sea-water? This is due to the alkaline reaction of the sea-water. If we make the sea-water faintly acid by the addition of hydrochloric acid no egg forms a membrane or undergoes cytolysis after being transferred into sea- water, and if we make the solution of sodium oleate in sodium chloride slightly alkaline by the addition of sodium hydroxide the eggs form membranes while they are in the soap solution. If we allow the soap solution to act only long enough to cause the membrane formation, but not long enough to cause cytolysis, the eggs can be caused to develop larve. We may from all these experiments draw the inference that the development of the resting egg is caused by a superficial or mild cytolysis, and that the spermatozoon must carry a cytolytic substance into the egg, possibly a trace of higher fatty acid. 40 (378) On the depression of the freezing point of water due to dissolved caseinates. By T. BRAILSFORD ROBERTSON and THEODORE C. BURNETT. [From the Rudolph Spreckels Physiological Laboratory of the University of California. | The question whether or not proteins possess, in solution, a definite osmotic pressure has been the subject of much controversy. The original investigations of Graham! appeared to indicate that colloids in general exert a high osmotic pressure. Subsequent investigators, however, attribute these results to an admixture of crystalloids and the investigations of Sebanejew,? Tamman,?® 1Graham: Phil. Trans. Roy. Soc., 1861, cli, 183. 2Sebanejew: Berichte d. deut. chem, Gesell., 1890, xxiii, 87; 1891, xxiv, 558; xxvi, 385. Sebanejew and Alexandrow: Journ. of the Russian Phys.-chem. Soc., 1891, p. 7; quoted after Maly’s Jahresber. f. Tierchem., 1891, xxi, 11. ’Tamman: Ze?t. f. physikal. Chem., 1896, xxi, 180. 86 SCIENTIFIC PROCEEDINGS (32). Dreser,’ Koeppe’ and others indicate that when they are carefully freed from associated inorganic substances the cryoscopic depres- sion due to dissolved proteins is negligible, while Reid? finds that proteins purified by repeated recrystallization, resolution and re- crystallization frequently possess, in solution,‘ no measurable osmotic pressure ; and he concludes that provided every precaution be taken to exclude impurities (among which he includes inorganic constituents) from the protein solution it will be invariably found to possess no osmotic pressure whatever and that the osmotic pressures observed in solutions incompletely purified are due, not to protein, but to the associated impurities. It appears to us that many of the above-quoted observations and conclusions are vitiated by the fundamentally erroneous con- ception that the inorganic constituents which are found associated with proteins are invariably present as impurities and not in a state of chemical combination. The manner in which this assumption vitiates conclusions regarding the molecular weight (estimated from the depression of the freezing-point or directly from the osmotic pressure) of proteins will be clear from the following considera- tions: Bases and acids have been demonstrated to form definite salts of a constant composition with casein, serum globulin and pro- tamin, and there can be no doubt whatever that similar compounds are formed with other proteins. In solutions of casein and of serum globulin it can be shown that as the neutral point is ap- proached the alkali-binding power becomes less and from a variety of data it can be shown that this phenomenon is due to a polymeri- zation of the protein molecule according to equations of the type: HXOH + HXOH = HXXOH + H,0* so that at or in the neigh- borhood of the neutral point molecular aggregates are formed of such dimensions that, in the cases of the proteins mentioned, the solu- tion assumes the character of a suspension and the protein is pre- cipitated ; addition of acid or alkali shifts the equilibrium in the direction of the lower complexes and the protein goes into solution again in the form of a salt. Similar phenomena may be safely as- 1Dreser: Arch. f. exper. Path. und Pharm., 1892, xxix, 314. 2Koeppe: Arch. f. d. ges. Physiol., 1896, \xii, 571. 3Reid: Journ. of Phystol., 1904, xxxi, 438. *T. Brailsford Robertson : Journ. of Physical Chem., 1908, xii, 473. DEPRESSION OF THE FREEZING POINT OF WATER. 87 sumed to occur in other protein solutions, although the polymeri- zation of the protein, which occurs when the uncombined protein is set free, may not result in actual precipitation. The elaborate precautions which have been taken by many observers to free the protein under investigation from accompanying inorganic sub- stances, have, therefore, defeated their own ends by converting the protein into molecular aggregates so enormous as to possess a necessarily immeasurably small osmotic pressure. Since it appears probable, therefore, that the dissolved sa/¢s of proteins may exert a measurable osmotic pressure in solution, and hence, cause an appreciable lowering of the freezing-point of water in which they are dissolved, we have undertaken a series of deter- minations of the lowering of the freezing-point of water, which is brought about by dissolved (neutral) caseinates. The solutions are made up as follows: Alkali of a given con- centration is shaken up with excess of casein until no more casein will dissolve and the solution is then filtered through rapid-filter- ing paper. The resulting solution is a solution of the “neutral caseinate’”’ of the base and is neutral to litmus.’ The cryoscopic depression is estimated in the usual way. The following are the results which have so far been obtained : Experimental error of determination + 0.0025°. Base. 7c oricentea ionic! pase A Indicating a concentration of NH,OH m[50 0.045 m4 ae m | 33-3 -055 m [33-6 KOH m [50 +0325 m [57 a ¥ 0375 m | 49-3 “ m [33-3 .0425 m | 43-7 Ue fs :0475 m [38.9 (i m [20 05 m [37 at es -075 m [24.6 GK mis 5) m [18.5 LiOH m/59-5 .03 m [61.6 “ m [39.6 -045 m/ 41 “ m [23.8 .O7 m [26.4 OG m/17.8 .08 m [20.3 Since these solutions are neutral and no inorganic substance is introduced save the base employed to dissolve the casein it is evi- dent that the compounds of bases with casein cause, in solution in !T, Brailsford Robertson: Journ, of Biol, Chem., 1907, ii, 336. 88 SCIENTIFIC PROCEEDINGS (32). water, a definite cryoscopic depression. In harmony with deduc- tions from titration- and conductivity-data’ the results are such as indicate that casein behaves towards bases, essentially as a mono- basic acid possessing a molecular weight, in solutions neutral to litmus, of approximately 2,000. 41 (379) The daily excretion of bacteria in the feces of healthy men. By W. J. MACNEAL, LENORE L. LATZER and JOSEPHINE E. KERR. [From the Laboratory of Physiological Chemistry, Department of Animal Husbandry, University of Illinois, Urbana, 11.) During the past year we have examined at intervals of about two weeks the fecal bacteria of each individual ina group of twelve men who were the subjects of a prolonged metabolism experiment. These men were fed a mixed diet, in quantity according to their choice at the beginning of the experiment. Altogether we have examined bacteriologically 266 stools. The quantity of bacteria in each of these stools was estimated by two different methods of microscopic counting and in about half of them the quantity of bacterial dry substance was also estimated by the gravimetric method of Strasburger. In the individual examinations the largest number of bacteria observed was 816 x 10° bacterial cells per gram fresh feces, 2,642 x 10° bacterial cells per gram dry feces. The smallest number of bacteria counted was 124 x 10° per gram fresh feces, 983 x 10° per gram dry feces. By the gravimetric method the largest quantity of bacterial dry substance observed was 42.53 per cent. of the fecal dry substance or 13.2 per cent. of the moist feces. The smallest quantity of bacterial dry substance observed was 14.03 per cent. of the fecal dry substance or 2.6 per cent. of the moist feces. Theaverage of all examinations was 375 x 10° bac- terial cells per gram fresh feces; 1,587 x 10° bacterial cells per gram dry feces; bacterial dry substance in fecal dry substance, 1T. Brailsford Robertson: Journ. of Physical Chem., 1908, xii, 479, etc. DaiLty EXxcRETION OF BACTERIA IN THE FECES. 89 26.89 per cent.; bacterial dry substance in moist feces, 7.0 per cent. The average daily quantity of fecal dry substance and of fecal nitrogen was calculated from the analysis of eight-day period collections, and the average daily bacteria and bacterial nitrogen calculated upon these quantities, regarding the bacterial content of the dry substance of the single stool, upon which the bacteria were determined, as representative of the feces for that period. Calcu- lated in this way the greatest values found were 58 x 10” bacterial cells per day by count, and 9.15 grams bacterial dry substance, containing 1.006 grams nitrogen, per day by the gravimetric method. The smallest values were 14 x 10” bacterial cells and 1.87 grams bacterial dry substance, containing 0.194 gram nitro- gen per day. The average of all examinations was 32 x 10” bacterial cells, and 5.34 grams bacterial dry substance, containing 0.585 grams of nitrogen, per day. There was considerable individual variation among the different subjects of the group. For example, the average of all examina- tions of Subject H was 40 x 10” bacterial cells and 7.26 grams dry bacterial substance, containing 0.819 gram nitrogen, per day. The average of all examinations of Subject B was 26 x 10” bac- terial cells and 3.56 grams bacterial dry substance, containing 0.393 grams nitrogen, per day. The nitrogen contained in the bacteria varied from 66.8 per cent. to 23.3 per cent. of the total fecal nitrogen, the average of all examinations being 46.3 per cent. Individual variation in this respect was also considerable. In Subject I the average of all examinations showed 57.4 per cent. of the total fecal nitrogen contained in the bacteria; in Subject K this quantity was 32.1 per cent. These two subjects were the ones who showed the two extremes (66.8 per cent. and 23.3 per cent., above) for the single examinations. A detailed account of these experiments will be published in a short time in The Journal of Infectious Diseases. go SCIENTIFIC PROCEEDINGS (32). 42 (380) Further studies on the constitution of inosinic acid. By WALTER A. JACOBS and P. A. LEVENE. [From the Rockefeller Institute for Medical Research. | In a former paper’ we have already communicated that the inosin which we obtained from inosinic acid was identical with that obtained from karnin by Haiser and Wenzel.?, We have now suc- ceeded in isolating from inosin the pentose in a crystalline state. The properties of this sugar are as follows: Melting point 87° C. Its rotation in aqueous solution is (2), = — 19°.4. The osazone melts at 163°-164° C. and shows a rotation when 0.2 gram are dissolved in Io c.c. of a mixture of four parts pyridine to six parts of alcohol of (2),= —0°.92. The benzylphenylhydrazone melts at 128° C. and in absolute alcoholic solution rotates (d) >= —26°.46. We therefore conclude that this sugar is neither xylose nor arabinose as stated by Neuberg and Brahm®* and Bauer ‘ respec- tively. We hope, by further study, to establish its exact nature. 43 (381) The effect of heat on the anaphylactic properties of proteins. By JOHN F. ANDERSON and M. J. ROSENAU. [From the Hygienic Laboratory, P. H. and M. H. S., Washington, D. C.] We have demonstrated that horse serum, egg-white and milk when dried, then heated and redissolved, possess unaltered powers of sensitizing and poisoning guinea-pigs in the sense of hypersus- ceptibility. The above named substances, when thoroughly dried, were heated to 130° C. for two hours, 150° C. for ten minutes, or 170° C. for ten minutes. We have previously shown that both the sen- sitizing and toxic properties of liquid horse serum are gradually in- 1 Proc. Soc. Exp. MED. AND BIOL., 1909, vi, 56. Ber. d. deutschen chem. Gesell., 1909, xlii, 335. 2 Monatshefte fiir Chemie, 1909, xxix, 157. 3 Ber. d. deutschen chem. Gesell., 1908, xli, 3376. * Beitraze zur chem. Physiol. und Path., 1908, x, 345. Skin REACTION IN CARCINOMA. gI fluenced by heat and are practically destroyed at about 100° C. The difference probably depends upon coagulation of the protein and consequent failure of absorption. Dried sensitive guinea-pig blood serum, containing anaphylac- tin, withstands at least 100° C. for ten minutes. 44 (382) A skin reaction in carcinoma from the subcutaneous injection of human red blood cells. By CHARLES A. ELSBERG. [From the Mount Sinai Hospital. ] Numerous investigators have shown that if the blood serum of a patient suffering from carcinoma be mixed with normal human red blood cells hemolysis occurs. The reaction takes place in from 50 per cent. to 80 per cent. of patients with malignant disease. It occurs with considerable frequency in tuberculosis, and more rarely in other diseases. It occurred to the writer, that, by the injection of red blood cells under the skin of the carcinoma patient, it might be possible to produce a local reaction at the site of the injection. Logically, a local hemolysis should take place. Theoretically, such a reaction might prove to be a delicate one ; it might give more positive and definite results than the test-tube method. In the technique which is used for the test-tube method the presence and degree of hemo- lysis is indicated by the amount of laking of the red cells — that is, by the amount of hemoglobin which has been set free. The tube reaction gives no evidence of other substances than the hemoglo- bin which have been liberated. A small amount of hemolysin in the serum which is being tested might not be capable of detection. If normal human blood cells are injected under the skin of a patient whose serum is hemolytic, fresh quantities of hemolysin would be continually carried to the cells, and therefore even a small amount of hemolysin might cause hemolysis of the cells. Every organic substance which was set free would enter the tissues and might there have its effect. Accordingly, after some experimentation, normal blood was in- 92 SCIENTIFIC PROCEEDINGS (32). jected under the skin of patients suffering from carcinoma or other disease. In patients with malignant disease, a decided local reaction was observed. The technique employed was a very simple one. Under aseptic precautions, blood was aspirated from the median basilic vein of a normal individual (preferably a child), every possible precaution being taken that the indi- vidual was healthy and free from hereditary or acquired disease. The blood was defibrinated, and the cells washed four times in normal saline solution, care being taken that the washings and centrifuging was thoroughly done. A 20 per cent. emulsion of the red cells in normal saline solution was made and kept in the ice-box for 24-48 hours before it was used. Five minims of this 24-48 hour old suspension of washed red blood cells were sub- cutaneously injected into the anterior surface of the forearm of the patient. In the patients in whom a “‘ reaction’ was obtained, the following changes were noted in the skin at the site of the injection. Six to eighteen hours after the injection, the affected area was slightly raised and slightly tender, it had a more or less well defined margin, it measured from two to four centimeters and it was of a somewhat dusky red color. The changes in the skin reached their maximum within one or two hours, and the red area then began to fade, rapidly or slowly. Eight to twenty four hours after the injection, the skin lesion had either entirely disappeared, or more often, a brownish, bluish or lemon-yellow discoloration remained, which persisted for a number of days. In the patients who did not show this reaction, there was either nothing to be seen at the site of the injection excepting the needle puncture, or a brownish discoloration of the skin, or a bluish dis- coloration, as is often seen after a hypodermic injection. My investigations of this cutaneous lesion are very incomplete, I have given thirty four injections to twenty patients with known carcinoma, and every one of the cases had a positive reaction. In most of the patients several injections were given of different blood cells. With succeeding injections, the reaction was either less marked or failed to appear. Of four patients with known sarcoma, three gave a positive reaction. Injections were given to over one hundred normal individuals or to those suffering from diseases other than sarcoma or carcinoma. These included a SKIN REACTION IN CARCINOMA. 93 variety of diseases, such as nephritis, tuberculosis of lungs, bladder, kidneys (7 cases), leukemia, syphilis (2 cases), benign tumors, acute and chronic inflammatory affections, etc. In all but three cases, the reaction was negative. One patient with a septic endocarditis gave a suspicious, but not a positive reaction, Two other injections made in this patient were negative. This case was among the earliest of our series, and therefore must be accepted with caution. A patient who had been operated on for a large rapidly growing lymphangioma of the suprapubic region and had several large angiomata of the thigh with a large post- operative hematoma of the scrotum, gave a marked positive reaction. A patient with gastric symptoms, absence of hydro- chloric acid and presence of lactic acid, with a loss of thirty pounds in weight, gave a positive reaction. The operation failed to show any carcinoma. I obtained no reaction in several patients in whom malignant disease was suspected but in whom no malignant disease was found at operation or at autopsy, and have obtained positive reactions in several patients in whom carcinoma was not suspected, but in whom carcinomatous disease was found at operation. Jaundice seemed to have no influence upon the appearance of the reaction. In order to gain a more definite idea of the causation of the skin changes, normal defibrinated blood was laked with distilled water, and, after the tubes had been centrifuged, the supernatant fluid was injected into a number of patients. For purposes of con- trol, hypodermic injections of sterile distilled water were given. All of the patients, whether suffering from malignant disease or not, who received hypodermic injections of laked blood, presented a skin lesion similar to that which was observed in carcinoma patients after the injection of washed red blood cells. This seemed to show that in the patients with carcinoma the reaction from the blood cell injection was due to a local hemolysis. In the normal individual or one suffering from some other disease, on the other hand, there was no hemolysis and therefore no reaction. When, however, the hemolyzable substances extracted from the red cells by laking were injected, the characteristic local lesion was ob- served. I have not yet been able to obtain pure human hemo- globin for injection purposes. 94 SCIENTIFIC PROCEEDINGS (32). A fuller account of the technique employed and a more exact description of the cutaneous lesions observed, of the possible value of red blood cell mixtures, z. ¢., of suspensions of the mixed red blood cells of several individuals, of the possible value of some animal red blood cells for injection purposes, of the significance of the persistence or disappearance of the reaction after an opera- tion, and many other aspects of the subject will be discussed in a future communication. The number of injections thus far given is far too small to allow of positive conclusions as to the reliability of this method for diagnostic purposes. The results obtained thus far have been striking. The purpose of this preliminary report is to call attention to the fact that it is possible to cause a local hemolysis in the living body by the subcutaneous injection of washed normal human red blood cells, and that in patients with malignant disease, especially carcinoma, a characteristic and easily recognizable local skin lesion is caused by this injection. Thirty third meeting. Cornell University Medical College, New York City. April 21,1909. President Lee in the chair. 45 (383) The vascularity of the spleen as influenced by single nerves of the plexus lienalis. By R. BURTON OPITZ. [From the Physiological Laboratory of Columbia University. | The blood supply of the spleen was determined by means of a stromuhr inserted into the splenic vein. The average for a dog of 18.0 kilos body weight, with a spleen weighing 100 grm., amounted to 0.97 c.c. per second, the velocity of the blood stream to 42.0 mm. per second, the venous pressure to 10.0 mm. Hg. Vaso-constrictory influences were obtained on stimulation of either splanchnicus major, or of the plexus lienalis. Single nerves of the plexus, designated as «, 8, 7 and 0 also showed strong vaso-constrictory powers. Thus, it was possible to trans- fer from 30 to 50 c.c. of blood from the spleen into the systemic circuit by moderate stimulation of either one of the nerves just mentioned. The removal of so large a quantity of blood from this organ by the constriction following the stimulation, resulted in a rise in general blood pressure. On the venous side the vaso-constrictions made themselves felt by : 1. A quick sharp rise in the venous return and venous pressure. 2. A gradual decrease in the blood-flow and pressure. 3. A slow adjustment toward normal values. (95) 96 SCIENTIFIC PROCEEDINGS (33). 46 (384) An experimental study of the influence of kidney extracts and of the serum of animals with renal lesions upon the blood pressure. By RICHARD M. PEARCE, M.D. [from the Carnegie Laboratory of the University and Bellevue Hospital Medical College, New York City.] I. Extracts of the rabbit’s kidney injected into the rabbit cause a slight increase in blood pressure which is barely more than that due to the mechanical effect of the injection. 2. Extracts of the dog’s kidney injected into the dog cause a decided fall in pressure ; an equal fall may be caused by the dog’s urine. A series of control experiments indicates that the fall caused by the kidney extract may be due to the urinary salts which it contains. 3. Extracts of cat’s kidney cause a risein pressure ; as the cat’s urine causes a fall, this rise in pressure indicates the possibility of a kidney extract containing a pressor substance which cannot be influenced by the depressor substance of the urine. 4. Rabbit’s kidney which in the rabbit produces a slight rise when injected into the dog causes a drop comparable to that caused by the dog’s kidney itself. Similarly the dog’s kidney, which in- jected into the dog causes a drop, produces in the rabbit a rise analogous to that produced by rabbit’s kidney. It is evident there- fore that these pressor and depressor substances of the kidneys in question do not have a constant effect on all animals as do the extracts of the adrenal gland. 5. Extracts of kidneys which are the seat of various forms of nephritis cause the same effect as extracts of normal kidneys. 6. The serum of dogs with considerable reduction of kidney substance causes a slight fall in pressure ; the serum of dogs with spontaneous nephritis gives divergent results, as does also the serum of rabbits with various forms of acute nephritis. The serum of dogs with chromate nephritis causes a slight rise, while that of dogs with uranium nephritis produces a sharp and decided fall in pressure. Although there is no uniformity in these results, their OBSERVATIONS ON EFFECT OF ASPHYXIA AND CURARE. 97 general character, and especially the experience with uranium and chromate sera of the dog, suggests that pressure-disturbing sub- stances are present in the serum as the result of the kidney lesion. The very slight evidence of the constant presence of a pressor substance, however, offers little support to the theory that such a substance is furnished by the diseased kidney or is due to dis- turbances of metabolism caused by disease of the kidney. 47 (385) Further observations on the effect of asphyxia and curare on the reducing power of the blood after section of the hepatic nerves in dogs. By J. J. R. MACLEOD. [From the Laboratory of Physiology, Western Reserve University, Cleveland. | In a previous communication on this subject (Macleod, American Jour. of Phys., 1909, X XIII, 278) tt was concluded that section of the hepatic nerves does not prevent the establishment of hypergly- cemia as a result of asphyxia and curare poisoning (Table III, p. 293, Zoc. cit.). The conclusion was based on the results of three experiments in which asphyxia was practiced, and in two of which marked hyperglycemia was observed ; and on one in which curare was injected. Subsequent experiments of the same nature have yielded results which do not corroborate the above conclusion for asphyxiated animals, but do so for those which are curarized. The following table gives the results of these experiments: No. and nature Per cent. of reducing substance in blood. of experiments. Before. After. 102 asphyxia 0.104 ig i aa. 104 asphyxia {eae 0.262 (60 min.), 105 asphyxia Reais 0.176 (45 mm, ). 107 asphyxia 0.113 Neate ae Bae 0.304 (30 min. ). 108 curare 0.265 2383 (45 min. ). 0.354 (75 min. ). 109 curare 0.178 0.334 (40 min. ). 110 curare 0.146 eee es a 98 SCIENTIFIC PROCEEDINGS (33). 48 (386) Toxin-antitoxin mixtures as immunizing agents. By WILLIAM H. PARK and EUGENE FAMULENER. [From the Research Laboratory of the Department of Health, New York City. | Ehrlich early suggested that injections of partially neutralized diphtheria toxin produced active immunity. This was demon- strated by Wernicke, Dreyer and Madsen, Morgenroth, ourselves and others. Smith in a recent article suggested the use of such mixtures in the immunization of children. The possibility of such a practical application has suggested to us some experiments with especial reference to the safety and effectiveness of the injections. The proportion of toxin to antitoxin in the mixture required to produce immunization. —In May, 1903, one of us reported some experiments in which one set of horses were injected with mixtures containing toxin .66 of L+ dose for each unit of antitoxin and another with .66 of L+ for each 4 units of antitoxin. Three large injections produced in the first series an average of 150 units, in the second an average of 3 units in each c.c. of serum. Smith injected three guinea pigs with 1 unit plus 1 L+ dose, 2 units plus 1.3 L+ dose, and 2 units plus 1 L+ dose respec- tively. The litters born from the first and second animals showed marked immunity at theend of eight months. The litter from the third animal showed slight immunity at three months and none at six months. These experiments indicate that while even a proportion of 6 units of antitoxin to 1 L+ of toxin produces slight immunity, the toxin must be in proportion of more than 1 L+ dose to 2 units to give marked and lasting effects. This brings us to the question of the safety of such mixtures. The work of Morgenroth suggests that mixtures which are toxic for one species are toxic for all. There is a difference, however, among animals as to the amount of natural immunity and perhaps therefore as to the development of serious symptoms or death from a given quantity of toxin-antitoxin mixture. Even if all guinea pigs lived, therefore, there would be a slight uncertainty in infants. ANTIPERISTALSIS IN RELATION TO TUBERCLE BACILLI. 99 In a series of tests we found that the least proportion of diph- theria toxin which was necessary to give lasting immunity was not quite harmless in guinea pigs. Thus of four guinea pigs receiving a mixture of 1 unit plus } L+ the two larger remained perma- nently well, while the two smaller finally died of paralysis. Four guinea pigs receiving one half the quantity of the same mixture all remained alive. Two other series receiving still larger quantities of the same mixture acted as the first lot. Some of these guinea pigs received six months later two fatal doses of toxin without serious poisoning. It is interesting to note that three of these animals received repeated injections of toxin in increasing amounts, until finally 6,000 fatal doses were given in one injection. The blood of the animals at this time contained from 25 to 30 antitoxin units per cubic centimeter. It is possible that the toxin used in these experi- ments which was produced by Culture No. 8 may have more tend- ency to promote late paralysis than that from other cultures such as used by Smith. 49 (387) Antiperistalsis in its relation to tubercle bacilli and other bac- teria in the alimentary tract. By ALFRED F. HESS. [trom the Research Laboratory of the Department of Health, New York City. | Fifteen years ago Griitzner showed that charcoal, starch and similar substances when introduced in normal salt solution into the rectum, ascended in the intestinal tract and after from four to six hours could be demonstrated in the stomach. His work was confirmed by some and refuted by others. This problem has of late assumed a new aspect inasmuch as some workers have claimed that when bacteria are introduced in the same way, within a short time they ascend by means of the antiperistaltic action of the alimentary tract to the stomach, cesophagus and thence into the respiratory tract. The most recent report of this phenomenon comes from the Kaiserliche Gesundheitsant, which concludes that this antiperistaltic movement must be considered, not only in exper- 100 SCIENTIFIC PROCEEDINGS (33). imental studies on tuberculosis, but also in the prophylaxis against infectious diseases ; more especially in the disinfection in cases of cholera and typhoid fever, where the sputum may in this way be contaminated. The experimental facts claimed are: When Bacillus prodigiosus, tubercle bacilli or other bacteria are suspended in from 10 to 20 c.c. of salt solution and are injected by rectum into an adult rabbit, they may be recovered by culture or animal inoculation an hour later from the small intestine, stomach, cesophagus, trachea, lungs and other organs. These results are interpreted as proving con- clusively that these bacteria, after traversing the gastro-intestinal tract, have entered the trachea and lungs, and thence have been transported throughout the body. The technique previously employed was, in brief, to keep the animal securely fastened following the injection, so that he could not lick himself, to kill him after a variable period, and then under the strictest asepsis to make numerous cultures from the contents of the alimentary tract, from the macerated organs, and from the heart’s blood. It appeared that the foregoing facts could, for the most part, be conceded, and nevertheless be open to quite a different interpre- tation. With this question in view I undertook the following series of experiments. The technique which I made use of differs in two respects from that used by others: (a) Large amounts of blood obtained for examination were drawn from the ear vein or jugular vein, previous to pithing the animal; (4) care was taken to avoid contact with blood in the cultures made from the hollow viscera. In addition it was found necessary to introduce other experiments. The following is a summary of my results : Of four experiments in which the animals were killed within three hours of the rectal injection, in three bacteria were not pres- ent above the ileo-czcal valve ; in one they were obtained in the small intestine after seventy minutes ; in no instance could they be cultivated from the stomach or cesophagus. Furthermore of four other experiments where twenty-four hours was allowed to elapse before killing the animal, in one the tubercle bacillus was demonstrated by means of inoculation to be ANTIPERISTALSIS IN RELATION TO TUBERCLE BACILLI. IOI present in the stomach. In the three others the injected bacteria were not found in the alimentary tract. In the various experiments bacilli were recovered from the lungs, liver and kidneys, urine and mesenteric glands. In three of four experiments Bacillus prodigiosus was cultivated from the blood within three hours of the rectal injection, and once twenty-four hours later. In two instances, where attempt was made tubercle bacilli were found in the blood twenty-four hours after they had been introduced into the rectum. Forty-five and fifty milligrams of a bovine culture had been injected, and 2 or 3 c.c. of blood was inoculated into each of six guinea pigs in both instances. In none of these experiments was the organism found in every blood culture, and in many of them the heart’s blood tested proved to be sterile. The result of these blood cultures suggested the injection of the bacilli directly into the circulation with an injury as to their subsequent distribution in the body. This work is still unfinished, but it may be of interest to note in this connection that one hour after injecting one forty-eight hour agar culture of Bacillus pro- digiosus, this organism was found in the small intestine, and that three hours following the injection of 130 mg. of tubercle bacilli they were demonstrated in the stomach and small intestine. That these tubercle bacilli did not enter the alimentary tract by way of the lungs was shown by two experiments in which the pylorus was ligated previous to the intravenous inoculation and the bac- teria were found in the small intestine. In these instances the bacteria either passed into the lumen of the stomach or intestine from without, or entered by means of the bile passages. Further experiments are being carried out to determine this question. After it was shown that the bacteria entered the blood follow- ing introduction into the rectum, it seemed necessary to inquire whether they were excreted by the salivary glands and could possibly in this way enter the upper part of the alimentary tract. To this end three experiments were carried out on dogs. Rectal injections of Bacillus prodigiosus were given in the manner de- scribed and two or three hours later the secretion from the parotid gland was obtained by means of a capillary tube inserted in the opening of Stenson’s duct. In most instances this is easy to carry 102 SCIENTIFIC PROCEEDINGS (33). out and five to ten cubic centimeters of saliva can thus be obtained after giving the dog 1/100 gr. of pilocarpine subcutaneously. None of the cultures made from the saliva showed Bacillus pro- digiosus ; however, I do not believe that this route is absolutely excluded. One clinical test was made. A “typhoid-carrier’’ who is known to have had typhoid bacilli in her stools for some years, at times almost in pure culture, but at present in the ratio of about ten per cent. of the total number of faecal bacteria, was submitted to an examination. If the bacteria ascend from the intestine then typhoid bacilli should be found in her stomach. This patient was starved for eight hours; at the end of the period her mouth was washed with sterile salt solution, and the washings tested for typhoid bacillii Her stomach, which was found to be empty after this period, was accordingly washed out and the washings, which were of neutral reaction, likewise plated on Conradi-Dri- galski media. Neither of these fluids was found to contain typhoid bacilli, nor indeed colon bacilli. From these experiments I conclude that bacteria injected by way of the rectum into rabbits are not carried in a viable state above the small intestine, and that they do not enter the respira- tory tract by this route. In fact their presence in the small in- testine may at times be due not to antiperistalsis, but to excretion from the blood or the bile. Furthermore; where experiment has showed them to be present in the lungs, the trachea and the cesophagus, they have entered these organs by way of the blood stream. 3 50 (388) The action of soaps on the pneumococcus. By SIMON FLEXNER and RICHARD V. LAMAR. [From the Rockefeller Institute for Medical Research, New Vork.] The object of the study to be reported briefly is the ascertain- ing of the manner in which the pneumococcus is disposed of in the body of infected animals that recover. The animal experiments were made on full grown rats. A strain of pneumococcus fatal to them in 1/10,000 of a cubic centimeter of a twenty-four hour bouillon culture wasemployed. Strong solutions (1 to 5 per cent.) INFLUENCE OF SHAKING UPON TRYPSIN AND RENNIN. 103 of soap (sodium oleate) precipitate the diplococci in an adherent mass which afterwards undergoes complete solution in water or salt solution. Solutions of soap of a strength of 1 to 10,000 do not produce visible changes in the bacterial suspensions but reduce slightly the number of viable cocci. Solutions of I to 15,000 or I to 20,000 do not affect the viability in cultures but reduce some- what the virulence. At the same time the diplococci appear some- what swollen but not otherwise altered. Untreated diplococci begin to multiply at once in the peritoneal cavity of rats. The treated diplococci at first almost entirely dis- appear from the cavity and begin to multiply after eight or more hours and cause death at a later period than the controls. There is a greater emigration of leucocytes in the case of the treated cocci than in that of the controls. There is little or no phago- cytosis. Normal goat serum does not affect the process appre- ciably ; but immune goat serum prevents multiplication of the treated cocci and brings about recovery of the rats but, under the conditions of the experiment, not of the control rats injected with untreated cocci. Phagocytosis does not play a direct part in the recovery. The experiments can be repeated zz vitro with approximately similar results. The soap-treated cocci are subject to serum lysis, while the untreated are not, and the lysis is not assisted but rather hindered by the presence of living leucocytes. The study is being continued. 51 (389) The influence of shaking upon trypsin and rennin and a com- parison of this influence with that upon pepsin. By A. 0. SHAKLEE and 8. J. MELTZER. [From the Department of Physiology and Pharmacology of the Rockefeller Institute for Medical Research, New York. | At the December meeting of this Society we mentioned our studies of the effects of shaking upon ferments and reported that pepsin can be practically destroyed by shaking. Our studies were extended to other digestive ferments and we wish now to report very briefly that shaking proves to be very injurious also to trypsin 104 SCIENTIFIC PROCEEDINGS (33). and to rennin. Trypsin was tested by the casein method of Gross and the rennin was determined by the method of Blum and Fuld with slight modifications which will not be discussed here. Both ferments were shaken at room temperature, and at 33°C.; also the influence upon the results of different rates of shaking and of changes in other conditions were investigated. We shall, however, state here only that the destructive effect of shaking upon trypsin and rennin is, as for pepsin, distinctly increased by increasing the rate of shaking and by increasing the temperature at which the shaking is carried on. There is a pronounced difference in the resistance to shaking between pepsin and trypsin under the condi- tions thus far studied, the latter being more readily affected. The destructibility of rennin runs practically parallel with that of pepsin. 52 (390) The influence of sodium and calcium upon direct and indirect muscle irritability and their mutual antagonistic actions. By DON R. JOSEPH and §S. J, MELTZER. [from the Department of Physiology and Pharmacology of the Rockefeller Institute for Medical Research, New York. | By the researches of Kithne, Biedermann, Ringer, Loeb, and many others, it is established that solutions of sodium chloride cause rhythmical movements of the muscles of the frog, and that the addition of a small quantity of calcium will stop them. By the researches of Locke, Carslaw, Cushing, Poljakoff and Overton, it is further established that solutions of sodium chloride abolish indirect irritability, and that the addition of a small dose of calcium restores it. There has been very little work done on the primary effect of calcium upon the direct and indirect irritability of the skeletal muscles of the frog and there are practically no researches on the action of sodium upon the primary effects of calcium. In our experiments, sodium and calcium chlorides were em- ployed in M/1o solutions and were introduced by infusion through the abdominal aorta according to the method described by Cushing.’ The graphic records were obtained from the gastro- 1Cushing: American Jour. of Physiol., 1902, vi, 77. EFFECTS OF MAGNESIUM UPON MEDULLA. 105 cnemius and the sciatic plexus was stimulated at about one minute intervals by two consecutive shocks (make and break) from an induction current. In agreement with the above mentioned statements, we found that sodium chloride reduces indirect (curare-like action) and reduces moderately also direct (Poljakoff) irritability. Both are promptly restored by the addition of a small dose of calcium. As a new fact we may mention that the irritability is more readily abolished in cooled frogs. Although calcium restores indirect irritability when abolished by sodium, it ado/ishes indirect irritability when injected primarily. The dose necessary is considerably smaller than that of sodium for the same effect. Again, the indirect irritability thus abolished by primary infusion of calcium can be restored by sodium of which, however, a larger dose is required than of calcium in a secondary injection for a similar purpose. Calcium also reduces or abolishes direct irritability, which again can be restored by sodium. The loss of indirect and direct irrita- bility by calcium is not exactly parallel. Cooling seems to favor the effects also of calcium. In these experiments neither sodium nor calcium exclusively increased or decreased the irritability. Both depressed in primary infusion and were mutually antagonistic in secondary infusions. 53 (301) The effects of local applications of chloride and sulphate of magnesium upon the centers in the medulla compared with those of sodium chloride. By J. AUER and 8. J. MELTZER. [ From the Department of Physiology and Pharmacology of the Rocke- Seller Institute for Medical Research. | The three salts were applied in molecular solutions to the exposed medulla oblongata of rabbits. Both salts of magnesium abolished sooner or later all the functions depending upon the centers located in the medulla, the average time until a complete effect took place being fifteen minutes. Respiration stopped and 106 SCIENTIFIC PROCEEDINGS (33). blood-pressure came down to forty or thirty millimeters of mercury and sometimes even lower. Strong stimulations of the sciatic nerve had now no effect upon blood-pressure. After curarin and strychnin were given stimulation of the sciatic caused some rise (spinal centers). Electric stimulation of the superior laryngeal nerves or mechanical stimulation of the pharynx caused no de- glutition. Injection of fluid into the cesophagus caused no con- traction of that organ (no secondary peristalsis). Intravenous in- jection or local application of calcium did not restore these func- tions. In a few cases spontaneous respiration returned after a few hours of continuous artificial respiration. Sodium chloride had no depressing effect; on the contrary, there was a moderate stimulating effect upon the respiration and blood-pressure. There was a strikingly stimulating effect upon the center of deglutition ; for eight or ten minutes the animal had to swallow every ten or fifteen seconds. 54 (392) Respiration by continuous intrapulmonary pressure without the aid of muscular action. By J. AUER and 8. J. MELTZER. [From the Department of Physiology and Pharmacology of the Rockefeller Institute for Medical Research. | Investigations of the nature of the mechanism which keeps up the respiration in the underpressure and overpressure methods of Sauerbruch and of Brauer led to the discovery that the respi- ration can be kept up for hours by a continuous stream of air equal to fifteen or twenty millimeters of mercury without the aid of any muscular action. The only requirement is that the air stream must reach at least the bifurcation. If the air is intro- duced simply through a tracheal cannula, as in the Brauer method, and curare is given, the animal dies in a few minutes. Our object was attained in three ways. In one method a slit was made in the trachea and a glass tube, filling out about two thirds of the trachea was introduced to the tracheal bifurcation or even a short distance into the right bronchus. Air entered through this tube and returned through the slit in the trachea and through the THE PRopUCTION OF KIDNEY INSUFFICIENCY. 107 mouth and nose. In the second method a short tracheal cannula was tightly ligated into the upper part of the trachea and a nar- row tube was introduced through a small slit in the lower part of the trachea into the right bronchus. The air entered through the tracheal cannula and had to reach the lower end of the glass tube before it could make its exit. Finally in a third method a long O’Dwyer tube bent at right angles was introduced into the larynx, the pharynx and mouth were packed with gauze, and a long soft rubber catheter was introduced through the O’Dwyer tube deep into the trachea so that its lower end reached the bifurcation. By means of a T-tube the air entered through the O’ Dwyer tube into the trachea and had no other escape than through the side open- ings at the lower end of the catheter (the air passed through an ether bottle; the animals also received morphin). By any of these methods the animals (dogs and rabbits) continued to live for a long time after their muscular action was completely elimi- nated by curare. The thorax was wide open in most of the ex- periments and the widely distended lungs showed only the vibra- tions due to the heart beats. In many cases the lungs lost their pink color. Opening the ether bottle for a second or two per- mitted a momentary collapse of the lungs and in an instant they again looked pink. Besides the principle which is demonstrated by this new ob- servation and the possibility of its practical application, it offers a very convenient method for the study of the heart movements without any interference from the respiratory movements. 55 (393) Note on the production of kidney insufficiency by reduction of the arterial circulation of the kidney. By ALEXIS CARREL. [From the Laboratories of the Rockefeller Institute for Medical Research, New York. | In order to obtain an insufficiency of the renal functions, I attempted to find a method simpler and more practical than the reduction of renal substance used by Tuffier, Bradford, Pearce and others. This new method consists of reducing the renal circula- 108 SCIENTIFIC PROCEEDINGS (33). tion by ligature or stenosis of the branches of the renal artery. The operation is harmless and very simple. The results obtained by Dr. Janeway show that it is efficient. 56 (394) A modification of the Riva-Rocci method of determining blood- pressure for use on the dog. By THEODORE C. JANEWAY. [From the Rockefeller Institute for Medical Research. | Previous studies of the blood-pressure changes in living animals, by repeated direct measurements from the femoral or carotid, while accurate from the standpoint of the blood-pressure at the moment of observation, have been of very limited value. When used as a means of following the changes occurring over long periods of time, as in the study of experimental kidney insuffi- ciency, it is questionable whether a single pre-operative reading, with several post-operative ones, afford in themselves any basis for the conclusions drawn. The figures given by Volkmann for the blood-pressure of different animal species show readings from the dog of 104, 123, 143, 157, 166 and 172 mm., a variation so wide that, in the light of our knowledge of the fluctuations of blood-pressure in man, it suggests strongly the fallacy of any con- clusions drawn from a comparison of two or three measurements at long intervals. To obtain some more definite idea of the changes occurring from day to day in experimental animals, I have endeavored to apply to the dog the commonly employed clinical methods. After various attempts, the most satisfactory method was found to be a modified Riva-Rocci cuff applied to the lower foreleg, the pulse being palpated in the artery at the bend of the ankle or in the plantar aspect of the paw. A rubber bag 7.5 x 15 cm., with a slightly larger outer leather cuff, will fit almost any dog, the fore- leg being not less than 8 cm. in length, and from II to £4 cm. in circumference in a large number of laboratory dogs examined. For small dogs a cuff 5 x 11 cm. is adequate. Measurements are greatly facilitated by using a pressure bottle connected with the cuff and the manometer through valves operated by foot pedals, BLOOD-PRESSURE CHANGES. 109 leaving both hands free, in place of the usual rubber bulb or Politzer bag as the source of pressure. The greatest difficulty is the satisfactory palpation of the small pulse in the foot. In certain dogs it is impossible, and animals must be selected that possess a reasonably large and superficial artery. It is impossible to appreciate the return of the pulse after obliteration, but with practice, if the animal can be kept quiet, the obliteration of the pulse can be appreciated within perhaps 10 or I5 mm. limits of error, always on the side of under-estimation. When the foot is cold, it should be wrapped in warm cloths to dilate the vessels, before taking readings. A number of experiments, in which I have followed the pres- sure changes during an operation coincidently with a direct carotid tracing, show that one can follow fairly rapid and marked fluctua- tions of blood-pressure in this way, with reasonable certainty. The results have no absolute, but, I am convinced, a real relative value. For the solution of such problems as the one studied by Passler and Heineke, and which Carrel and I are engaged in, I believe that frequent approximate blood-pressure observations are of more significance than a few isolated, though accurate, measurements. 57 (395) Note on the blood-pressure changes following reduction of the renal arterial circulation. By THEODORE C. JANEWAY. [From the Rockefeller Institute for Medical Research. | Of the various workers who have studied the effects of reduc- tion of kidney substance, only Passler and Heineke record system- atic blood-pressure observations. They were able to make direct measurements in the femoral on five dogs before and after operation, and reported a rise in pressure in all, the smallest increase being I5 mm., the greatest 29 mm., and the average 21.5 mm. These figures are based on the comparison of single readings before operation with one or more after operation, and are open to the objections I have previously urged. Because of the small number of reported observations in this field, I hope to be pardoned for presenting my still very incomplete studies at this time, in order 110 SCIENTIFIC PROCEEDINGS (33). that I may demonstrate one of the animals now living with reduced kidney substance and hypertension. I have made blood-pressure readings, by the rough method previously described, on twenty-three dogs, over a period of fifteen months. Asa guide to normal readings in the dog I have figures from twelve dogs that were in good health, several of these being finally checked by direct carotid tracings. In these twelve dogs the average pressure, calculated from a number of readings on each, lay between g1 and 119 mm., the highest individual reading being 130 mm., and the lowest 85 mm. A number of observa- tions made before operation on the ten nephrectomized dogs showed an average blood-pressure between g0 and 117 mm.; highest reading was 135 mm., lowest 80 mm. These readings average lower than those obtained in man, but the method as applied to the dogs is more comparable to the results of Gartner in man, since the artery used is more peripheral, and the pressure within it more subject to fluctuations due to variations in local vaso- motor tone. As I have already said, however, the errors are all on the side of too low readings; therefore, with a sufficient number of pre-operative readings to give a fair average, the finding of a marked rise in blood-pressure subsequently cannot be attributed to errors inherent in the method. The dogs studied were operated on by Dr. Carrel as already described, with the exception of one after the method used by Bradford, Passler,and Pearce. Four died from too extreme reduc- tion of the arterial blood supply of inanition, and one of an abscess unrelated to the operation. All showed a slight rise in pressure in the first three to seven days, with subsequent fall, except one dog that died of extensive resection in four days. The most strik- ing of the fatal cases showed the following : Blood-pressure, mm. Hg. Dog 19. Maximum. Minimum, Average. Before operation, 1'5/ dayS.....--cscscseseetsoeseees 110 100 106 Afterioperation;i2 (dayS.c.c-ssssersnentacensscese=> 135 120 127 Terminalipertods 14/days is cmeccrsssese-ee-e- 2-2) LLO 70 83 Five dogs are still living, one having been operated on 105 days ago, the others ashorter time. This dog, No. 12, shows the following clear result: ACUTE INSUFFICIENCY OF RIGHT HEART. ae Blood-pressure, mm. Hg. Maximum, Minimum. Average. Before operation, 45 days .......sscsssesovsscevceoseers 110 80 fofe) After as Hirst periods) AS dAaySis.ccccecsaccess 120 100 III Gt Ks second period, 26 days............. 140 IIo 121 “ 6 third period, 31 days ............6 150 110 125 ae cs Whole perntOdiswcc.ssscctieses+eeorrses 120 He has gained from 12,770 grm. to 16,250 grm. and his urine has been free from albumin for three weeks, but is increased in quan- tity. A still more marked hypertension has been obtained in Dog 20, as the following table shows : Blood-pressure, mm. Hg. Maximum. Minimum, Average. Betorevoperationy:-23/GAYSiccsessesoessecseses esses 135 95 117 After GG IS BO ceaphosonecobosonooosgeceet 175 130 150 This dog is excitable and single readings are liable to vary somewhat on this account, but the comparison of the averages, based on eleven observations before and twenty-one after operation, or of either the highest or lowest readings, all show a true hyper- tension asa result of the reduction of functioning kidney substancs by this method. This dog has a persistent albuminuria, with casts and red blood cells, and a daily urine quantity of about 500 c.c. The three remaining dogs have not been under observation long enough, or have not had sufficient reduction of circulation, to give definite results as yet. 58 (396) The effect of experimental acute insufficiency of the right heart upon the volume of the organs. By H. C. THACHER, M.D. [From the Laboratory of the Medical Clinic in Titbingen.| If a small balloon be introduced into the right auricle or ven- tricle, its inflation interferes with the action of the right heart and renders the heart insufficient to perform its normal work. The effect of this upon the systemic circulation should be nearly simi- lar to that caused by acute cardiac insufficiency in general. The changes in the volumes of the brain, liver, spleen, kidney, and extremity resulting from such cardiac obstructions were registered by oncometry in rabbits, cats and dogs. The carotid blood-pressure, which was always registered as an I12 SCIENTIFIC PROCEEDINGS (33). index of the degree of circulatory disturbance produced, falls abruptly to a lower level when the balloon is inflated. It then remains fairly constant until a final collapse occurs just before exitus. The liver and brain increase at once in volume as the result of an acute passive dilatation caused mechanically by the increased venous pressure. In the other organs and extremity a moderate similar passive dilatation can be demonstrated if, but only if, they have been put ina state of active contraction before the cardiac obstruction is made. Thus when previously contracted directly, by the intravenous injection of adrenalin, or reflexly, by sensory irritation, their volume-curve rises during the period of cardiac insufficiency. But unless thus previously contracted, the kidney, spleen, intestine, and extremity decrease promptly in volume when the heart is obstructed. This decrease overshadows the relatively slight effects of the increased venous pressure, so that the presence of the latter is only manifest in a short “additional fall” of the volume-curve which occurs just at the moment when the obstruc- tion is removed. This ‘additional fall” is synchronous with the drop in the venous pressure, and occurs before the organs begin to return to their normal size. The decrease in volume of these organs, on the other hand, does not correspond to the fall of arterial blood-pressure, but may continue for as much as five minutes after the latter has reached its lowest point. It is due rather to an active contraction of the arteries and capillaries tending to compensate for the blood lost from the circulation by stagnation in the veins, liver, and brain. The foregoing work was conducted in the laboratory of the Medical Clinic in Tubingen. I desire here to express my grati- tude to Professor Romberg and his first assistant, Dr. Schlayer. Thirty fourth meeting. The Rockefeller Institute for Medical Research. May 26, 1909. President Lee in the chair. 59 (397) The comparative toxicity of sodium chloride and of staining solutions upon the embryo of Fundulus. By ELIZABETH COOKE and LEO LOEB. [from the Laboratory of Experimental Pathology of the University of Pennsylvania; and from the Marine Biological Laboratory, Woods Fiole. | What substances enter cells and upon what conditions the entrance of various substances into cells and the permeability of organized animal membranes generally depends, is as yet only very imperfectly understood. The following experiments may not be without interest in this connection. In studying the toxicity of stains upon star-fish eggs, we find thionin, Bismarck brown, methylene blue and neutral red all to be very poisonous, if the solutions are exposed to light. Among these, neutral red is perhaps less poisonous than the other stains. Solutions of eosin are very much less toxic than the other sub- stances. Thionin, Bismarck brown, methylene blue and neutral red easily penetrate into ova and stain them in a characteristic way. LEosin does not stain living cells, but only enters in combi- nation with the dead protoplasm. Very different is the degree of toxicity of these stains towards the eggs of /undulus. Here, Bismarck brown, thionin, methylene blue and eosin are devoid or almost devoid of toxic action, whilst neutral red alone possesses any marked degree of toxicity, if the solution acts in the light. And the latter stain is likewise the only one able to enter the healthy ova of Funxdulus and to stain certain parts of the embryo. We are therefore justified in the conclusion that in the case of stains the toxicity of these substances towards (113) 114 SCIENTIFIC PROCEEDINGS (34). ova is dependent upon and is an indicator of their combination with the protoplasm of certain cells of the embryo. Now, it is not without interest to state that the toxicity of neutral red varies according to the stage of development at which the eggs are exposed to the influence of the staining solutions. Ova immersed in a solution of neutral red and exposed to the light, inside of sixteen hours after fertilization are most severely affected ; ova exposed approximately twenty to thirty hours after fertilization are somewhat more resistant, and ova which are exposed to the light as late as two to four days after fertilization are affected only to a very slight degree. Correspondingly, we find that the older the embryo becomes, the less is it liable to be stained with neutral red and in embryos five days old we usually find almost the whole embryo unstained with the exception of the newly developed liver which appears in an orange-yellow color. We may therefore conclude that the embryos of Fundulus and their cells become less and less permeable for neutral red as the development advances and that its toxicity decreases cor- respondingly. A curve of toxicity almost parallel to that of neutral red we find in the case of isotonic sodium chloride solutions. During the first sixteen hours isotonic solutions of sodium chloride are extremely toxic to the embryo of /undulus ; from twenty to thirty hours there is noticeable a certain decrease in toxicity ; while embryos two to four days old develop in 5/8 N. sodium chloride solutions almost as well as in sea water. We see, therefore, that the similarity of the curves is very great and inasmuch as in the case of the neutral red the variations in toxicity seem to depend upon variations in the staining ability of this substance and therefore probably upon the permeability of certain membranes or of the protoplasm of cer- tain cells to the stain, we may assume as the most plausible ex- planation that in the case of sodium chloride the variations in toxicity also depend upon the permeability of certain organized structures to the latter substance, and that therefore the conditions of permeability in the embryo of /indulus depend upon the same conditions in the case of the lipoid soluble neutral red and in the. case of lipoid insoluble inorganic salts, a conclusion which is at. INFLUENCE OF CALCIUM CHLORIDE UPON URINE. 115 variance with the views of Overton and Hoeber, but agrees with the observations made by Jacques Loeb, Robertson and by the botanist, Ruhland. We are well aware of the number of variable factors which are to be taken into account in the interpretation of these phenomena which may perhaps later necessitate a somewhat more complicated explanation; but we believe that comparative studies in the toxicity of stains and of various other substances will prove to be of value in the elucidation of the problems of cell per- meability and of the cause of toxicity. 60 (398) The influence of calcium chloride and of adrenalin upon the secretion of urine and upon absorption from the peritoneal cavity. By MOYER §S. FLEISHER and LEO LOEB. From the Laboratory of Experimental Pathology of the Universit Jf 14 Sy, LV of Pennsylvania. | I. Intraveneous injection of calcium chloride diminishes the secretion of urine. Porges and Pribram ascribed this effect to the lowering of blood pressure which follows the intravenous injection of this substance. Our experiments, we believe, show such an interpretation to be erroneous for the following reason : If we add adrenalin to sodium chloride solutions the blood pressure rises during the intravenous injection of this fluid and we also find a noticeable increase in diuresis under the influence of adrenalin. If we now add calcium chloride to the adrenalin- sodium chloride mixture the blood pressure remains likewise very high during the intravenous injection and the ultimate fall due to the influence of calcium chloride is delayed for a considerable time, but notwithstanding the high blood pressure produced by adrena- lin which is in itself a substance favorable to diuresis, the addition of calcium chloride again causes a marked decrease in diuresis. The effect of calcium chloride in diminishing the secretion of urine can therefore not be ascribed to its action on the blood pressure, but to some other condition, most probably to its direct influence upon the epithelial cells of the kidney, an interpretation originally given by John B. MacCallum. 116 SCIENTIFIC PROCEEDINGS (34). II. As Exner and Meltzer and Auer found, the intravaneous (Meltzer and Auer) and intraperitoneal (Exner) injection of adrena- lin delays the absorption of fluoresceine and other substances from the tissues, from the peritoneal cavity and from the blood vessels. On the other hand, we found that adrenalin has a distinctly accel- erating effect upon absorption of isotonic sodium chloride solutions from the peritoneal cavity, if adrenalin is injected repeatedly intra- peritoneally during a period of two and a half hours. This accel- erating effect is absent in nephrectomized animals; it is, however, noticeable in rabbits injected twenty hours previously with uranium nitrate and is still indicated in animals injected with uranium nitrate three days before testing the absorptive power. Adrenalin also causes an increase in the secretion of urine and the improved absorption might therefore perhaps be ascribed to the increased elimination of fluid through the kidneys. Such an interpretation seems to be strengthened, if we consider that in nephrectomized animals this effect of adrenalin is absent. On the other hand, in individual experiments, parallelism between the ab- sorption from the peritoneal cavity and the degree of diuresis fre- quently is absent. In experiments concerning the effect of coffeine upon the absorption from the peritoneal cavity we found that after injection of coffeine the absorption from the peritoneum may be very slight notwithstanding a very strong diuresis. We also notice that during the first period of the action of uranium nitrate the in- creased diuresis is not accompanied by a corresponding increase in absorption. Notwithstanding these possible objections, at the present we cannot yet exclude the possibility that the improvement in absorp- tion from the peritoneal cavity under the influence of adrenalin is due to the diuretic action of this substance. The difference in absorption from the peritoneal cavity which we notice in experi- ments with animals after nephrectomy on the one hand and after administration of uranium nitrate on the other hand, is of interest and may perhaps bea causative factor in the edema which develops in animals injected with uranium nitrate. Furthermore these experiments suggest that adrenalin may improve the absorption of water, but at the same time retard the absorption of sodium chloride from the peritoneal cavity. We have begun experiments, in order to decide this question. STUDIES ON THE LiFE CYCLE oF PARAMECIUM. Ly 61 (399) Observations on uricolysis, with particular reference to the “uric acid infarcts” of the newborn. By H. GIDEON WELLS and HARRY J. CORPER. [From the Pathological Laboratory of the University of Chicago. | Mendel and Mitchell demonstrated that in the embryo pig the enzymes concerned with purin metabolism appear at different stages of development, the uricolytic power not appearing until after birth and being feeble during the first months of extra-uterine life. If the same late development of uricolytic power were present in the human fetus it would explain the occurrence of deposits of urates in the kidneys of newborn infants. Schittenhelm and Schmidt alone have studied uricolysis by infantile and fetal tissues, and have claimed to get active uricolysis. This result is questionable, because later work by Kunzel and Schittenhelm indicate absence of uricolysis by adult tissues. We have found no evidence what- ever of uricolytic activity on the part of fetal tissues at any stage of development, nor of adult tissues. The latter observation is in harmony with the negative results obtained by Wiechowski in ex- periments 27 vitro and 7 vivo, and indicates that the human body has little if any power to destroy uric acid. The statements in the older literature that allantoin is found in the urine of pregnant women has been disputed by Wiechowski, and our failure to demonstrate uricolysis by human placenta as well as other fetal or adult human tissues points in the same direction. Additional observations are the demonstration of active urico- lysis by the liver of the guinea pig, absence of uricolysis by spleen, bone marrow and probably the leucocytes of the dog, and the apparent absence of inhibitory power of dog serum upon uricolysis by dog liver. 62 (400) Studies on the life cycle of Paramecium. By LORANDE LOSS WOODRUFF. [From the Sheffield Biological Laboratory of Yale University. | A year ago I reported to this society the results obtained up to that time on the life cycle of Paramecium when subjected to a 118 SCIENTIFIC PROCEEDINGS (34). varied environment. I wish now to bring the results up to date (May 26, 1909). A culture of Paramecium aurelia (caudatum) was started on May 1, 1907, with a “ wild”’ individual isolated from a laboratory aquarium, and during the twenty-five months which have elapsed since that time it has been under daily observation. Infusions of hay and grass together with any material that may be found in the normal habitat of Paramecium have been employed as a culture medium. The possibility of contamination by cysts or “wild” Paramecia has been eliminated by boiling the infusion. Daily iso- lation of an individual from each of the various lines of the culture has enabled an accurate record of the division rate to be kept and has precluded the possibility of endogamous conjugation. So far the culture has attained the 1,185th generation. The average rate of division for the entire period is over one and a half divisions per day. The average rate has not fallen during any ten-day periods as low as one division in two days, while during several ten-day periods it has averaged over two and a half divisions per day. Marked physiological depression has not been indicated by the rate of division and consequently special stimuli have not been employed to ‘‘ rejuvenate” the organisms. The results thus far obtained certainly show that the life cycle of Paramecium when subjected to a varied environment may be of very great duration, and, I believe, strongly suggest that the life history may be of unlimited duration. 63 (401) Immunity to various species of trypanosomes induced in mice by the cure of experimental infections. By B. T. TERRY. [From the Rockefeller Institute for Medical Research. | When properly treated a temporary immunity was secured against the organisms of surra of India, surra of Mauritius, caderas, dourine and nagana. The immunity was specific in the sense that it was active against the species cured but not against any other. The immunity varied with the virus, the medicament, the time at which the immunity tests were begun, the number of the tests, the ImmuNITY TO VARIOUS SPECIES OF TRYPANOSOMES. eae) intervals between them and the natural variations in the mice employed. Against the parasites of surra of India, surra of Mauritius, caderas, nagana, and a toluidin blue resistant strain of nagana, an immunity was produced which was strong enough to prevent one or more subsequent inoculations of virus from infecting. The im- munity to dourine was less strong. In producing immunity to surra of India a single injection of dichlorbenzidine plus amidonaphtol disulphonic acid, 1, 8, 3, 6, or “Cl,” was more efficient than one of acetylatoxyl or arseno- phenylglycin. Even when the medicaments apparently prevented infection an immunity was produced. It is interesting that two injections of a mixture of “Cl” and acetylatoxyl has completely protected a normal mouse against an inoculation with surra of India given six days after the second treatment. The mixture protected about twice as long as either of its two constituents used alone had done. The immunity reaction distinguished with sharpness organisms supposed to have had a common origin, namely, surra of India and surra of Mauritius. Occasionally, however, these two species im- munized against each other. With equal clearness the reaction distinguished between organisms known to have had a common origin, ¢. g., the toluidin blue resistant strain and the parafuchsin resistant strain. When mice were immunized to the toluidin blue trypanosomes, an inoculation with the same virus failed completely, while the tests with the parafuchsin resistant strain and with all others, infected and killed. By means of the immunity reaction it was apparently possible to separate in purity organisms that had been mixed zx vitro. By inoculating a mixture of surra of India and surra of Mauritius into mice immune to surra of India an infection with surra of Mauritius was obtained. The surra of India was separated by inoculating the mixture into mice immune to surra of Mauritius. Ina similar way caderas and surra of India were separated. When mice infected with a mixture of two species of trypano- somes were cured, an immunity to both was produced. In securing prolonged specific immunity, frequent injections of the virus at close intervals were of value. A mouse cured of 120 SCIENTIFIC PROCEEDINGS (34). caderas by a single injection of trypanred acquired an immunity that lasted 50 days. Another mouse cured of surra of Mauritius by acetylatoxyl and “Cl” hadastrongimmunity. In the 46 days that followed treatment it received eleven inoculations of virulent surra of Mauritius trypanosomes but did not become infected. When next tested for its immunity (311th day), it was apparently hypersensitive to infection. It was killed in 8 days by a greatly attenuated surra of Mauritius strain. The attenuation was appar- ently due to long continued passage through guinea pigs. One of the two controls inoculated with this virus died on the twentieth day. The other carried the trypanosomes for I11 days, recov- ered spontaneously, and is still alive (184th day). 64 (402) The leucin fraction of proteins. II. By DONALD D. VAN SLYKE and P. A. LEVENE. [From the Rockefeller Institute for Medical Research, New York. | In a previous communication’ we have described a method for quantitatively separating leucin and isoleucin from valin, the leucin isomers being precipitated as normal lead salts. It has since been found that from the specific rotation of the mixture of leucin and isoleucin, as obtained analytically pure from the lead salts, the percentage of each can be accurately calculated. The rotation of d-leucin in 20 per cent. hydrochloric acid is + 36.8°, of l-leucin + 15.6°. Consequently the proportions are calculated by the formule : R—15.6 per cent. d-isoleucin = ReGen 2) Zee 6.8—R per cent. d-leucin = sa iS 5 ) (R representing specific rotation in 20 per cent. hydrochloric acid). The specific rotations of the isomers are unaffected by the boiling acid used for hydrolysis, but are affected by boiling with alkalies. Consequently the method is not applicable to products of alkaline hydrolysis, which however is seldom used. .) These Proceedings, 1909, vi, 54. “ CALVIN,” VAHLEN’S ACTIVE PRINCIPLES OF ERGOT. 121 Analysis of the leucin fractions of casein and edestin gave the following results, those obtained by Abderhalden being given for comparison. The figures represent yield in grams from 100 grams of protein. Casein. Edestin. V-S. and L. Abderhalden, V-S.andL. Abderhalden. Leuci 8 eee 7 = 10.5 8.1 20.9 Isoleucin, 1.51 Valin, 6.69 1.0 5.6 0.45 Total ‘‘leucin fraction,” 16.04 11.5 ne Ey) 21.35 Our figures are based on analytically pure products. It is evident that Abderhalden missed from 80 to go per cent. of the valin which was undoubtedly calculated in with the leucin, as the two are not separable by the methods previously available. Still other prod- ucts in the necessarily crude mixture may account for the high yield from edestin. 65 (403) «“ Clavin,” Vahlen’s active principle of ergot. By DONALD D. VAN SLYKE. [From the Rockefeller Institute for Medical Research, New York. ] In a recent paper Vahlen’ describes the isolation of the two constituents of clavin, a crystalline substance isolated by him from ergot. By means of their copper salts two substances, leucin and “clavin base’”’ of the formula C,H,,O,N, were separated from the clavin. From analysis of clavin it appeared to consist of leucin and clavin base in molecular proportions, and was regarded as a salt-like combination, similar to that in which some alkaloid bases are found, the leucin acting as acid, the clavin base as base. Vahlen regards clavin as the active principle of ergot in stimulat- ing contraction of the uterus, and the clavin base as the active constituent of clavin. From the description of clavin, it appeared similar in both physical and chemical properties to the mixture of leucin and valin obtained from proteins. A sample of Vahlen’s clavin was obtained from Merck and submitted to the process devised for determination of leucin, isoleucin, and valin in the presence of one another.” The 1 Arch. f. exper. Path. u. Pharm., 1909, |x, 42. ? Levene and Van Slyke: These Proceedings, 1909, vi, 54. 122 SCIENTIFIC PROCEEDINGS (34). clavin, which when purified free from ash had the properties de- scribed by Vahlen, consisted entirely of these three amino acids, the latter being isolated analytically pure: 2.02 grams of clavin gave 0.79 gram of leucin, 0.45 gram isoleucin, 0.75 gram valin. We have not yet determined whether any of these amino acids has the pharmacological effect assigned to clavin. 66 (404) Some effects of sodium benzoate. By DANIEL R. LUCAS. (By invitation.) [trom the Laboratory of Biological Chemistry of Columbia Uni- versity, at the College of Physicians and Surgeons. | This research was suggested to me by different experiences with sodium benzoate when taken by mouth in the following ways : A. Pure (1) as crystalline salt, or (2) in aqueous solution. B. In neutral or alkaline solutions, or in mixtures rich in fat, carbo- hydrate or protein, ¢. g., milk. C. With vegetable or fruit acids (1) hoi, as in tomato soup, or (2) cold, as in canned plums, oranges, lemons, etc. D. In beverages containing high percentages of organic acids, ¢. g., cider, lemonade, grape juice, wine, etc. E. In mixtures containing inorganic acids, e. g., artificial gastric juice. Brunton has studied the effects of benzoic acid on enzymes and bacteria. The strong inhibiting effect of this substance on their ac- tivity is in striking contrast to the slight effect of sodium benzoate. Doepner has shown that fairly large quantities of sodium benzoate (2 per cent.) did not prevent the development of Proteus vulgaris and, in strengths equal to 0.5 per cent., only slightly retarded the development of B. enteritidis, B. fluorescens and B. coli. Fleck found that benzoic acid in concentrations equal to 0.6-0.7 per cent. caused marked inhibition of yeast fermentation and that the inhib- iting action was markedly decreased by the amount of protein present. Lehman observed that meat extract putrefies in the pres- ence of I to 2 per cent. of sodium benzoate, but less benzoic acid acts more strongly antiputrefactive when the reaction of the medium is markedly acid. The action of sodium benzoate under markedly acid conditions is the same as the actionof benzoic acid. Under such conditions the action of the benzoate diminishes with decrease of SoME EFFECTS OF SODIUM BENZOATE. 123 acidity. Chassevant and Garnier found that 1.4 gram of benzoic acid per kilo was fatal to guinea pigs in five to seven hours ; larger doses (2 grams per kilo) did not necessarily kill sooner. The results of my own work may be briefly summarized as follows : Effects on microorganisms. —Sodium benzoate, in concentrations of about I per cent., preserves fruits and vegetables which are strongly acid. Crystals of free benzoic acid often appear in such mixtures. Sodium benzoate (1 per cent.) added to weakly acid fruits and vegetables does not preserve them well. Sodium ben- zoate (I per cent.) added to fruits and vegetables, the acidity of which has been neutralized, does not preserve them. Pure apple juice, containing 0.1 per cent. of sodium benzoate, developed mould after ten days; commercial benzoated cider under the same conditions, without the further addition of benzoate, did not de- velop mould or otherwise undergo degeneration. Effects on taste. — Acid fruit juices containing I per cent. of so- dium benzoate, have a biting taste, an effect due to the liberated benzoic acid. Milk or alkaline vegetables treated with sodium benzoate (1 per cent.) do not taste of benzoic acid at any time during the first twenty-four hours after the treatment. After twenty-four hours, however, acid decomposition begins in milk in spite of the presence of 1 per cent. of sodium benzoate, when the mixture tastes distinctly of benzoic acid. Sips of 1 c.c. of orange juice, to which 1 per cent. of sodium benzoate has been added, cause burning in the posterior part of the mouth, the throat, the esoph- agus and stomach, with gastric discomfort, belching, uneasi- ness of the bowels and excessive passage of gas by rectum. Experiments on men with cider. Pure cider. —Pure apple juice tastes sweet, bland; produces no stinging sensation in the throat; and is ordinarily enjoyed and well borne in volumes equal to 1,000 to 2,500 c.c. (ingested during a period of two or three hours). It is diuretic in action and, in amounts varying from 1,000 to 2,000 c.c., causes laxation of the bowels. This effect frequently depends on the rapidity with which it is ingested ; it does not ordinarily cause laxation even when taken in large amounts, if ingested little by little. The average amount of pure apple juice consumed during an evening by adult males who 124 SCIENTIFIC PROCEEDINGS (34). had free access to it was about 1,200 c.c. (twenty subjects). When from 1,000 to 2,500 c.c. of pure apple juice are con- sumed neither headache, nausea, albuminuria nor sub-normal temperature is produced. The specific gravity of the urine is greatly decreased when a liter of pure unfermented cider is con- sumed but the volume is markedly increased within forty-five minutes after its ingestion. The forty gallons of pure apple juice consumed by the human subjects of my investigation contained considerable apple pulp and 2.716 grams of free acid (calculated as acetic acid) per 1,000 c.c. Benzoated cider. — Twenty four subjects were observed in the first experiment. Twelve received pure apple juice; twelve received samples of the same apple juice containing 0.1 per cent. of sodium benzoate. As none of the subjects knew that they were to receive at that time anything but pure apple juice, unfavorable psychological influences were eliminated from the experiments. Each subject received three question blanks to be filled out by himself daily as long as any symptoms lasted, which, I am assured, was done faithfully in every instance. In comparison with those who received pure cider, the men who drank the benzoated apple juice exhibited the following special symptoms: Burning taste, fulness in the head, headache, nervous- ness, nausea, vomiting, itching of the skin, unusual perspiration, ir- regularity of bowels (constipation usually) decreased flow of urine, increased specific gravity of the urine, and albuminuria. | Excessive amounts of hippuric acid were eliminated, especially during the first few hours, after ingestion of the benzoated apple juice. Apple juice to which a small amount of sodium benzoate is added becomes sweeter to the taste, but astringent, stinging, and irritating to mucous membranes. The presence of 0.5 per cent. of sodium benzoate renders cider quite unpalatable, but the pres- ence of 0.1 per cent. may be overlooked by subjects not acquainted with the taste of pure apple juice. Ifthe apple pulp is previously filtered from the juice the effects of added benzoate become much more evident. A liter of such filtered cider, containing 0.2 to 0.3 per cent. of sodium benzoate, caused albuminuria within three hours almost without exception in the largest and soundest picked subjects. However, I myself Some EFFects OF SopiuM BENZOATE. 125 was able to ingest 1,000 c.c. of apple juice containing 0.5 per cent. of sodium benzoate, without any albuminuria arising. The amount of hippuric acid in the urine was very large for the first few hours. The secretion of urine was very much reduced for twelve hours, while I suffered from some of the other symptoms above men- tioned, although as a subject in a former investigation I ingested as much as 6 grams per day, for three successive days, in milk on a full stomach, without the slightest discomfort. Small doses of sodium benzoate given with acid substances to patients with albuminuria aggravated this condition and caused alarming symptoms, classical of nephritis—for six days there- after in one subject. Experiments on dogs. First experiment. — One dog weigh- ing 3.5 kilos fasted for twenty four hours and was then given I gm. of sodium benzoate, decomposed with the theoretical amount of hydrochloric acid to form sodium chloride and free benzoic acid, with no excess of hydrochloric acid. In thirty minutes the animal showed evidences of muscular weakness and nausea, lay quietly and breathed in a laborious manner. This continued for six hours. On the next day, twenty four hours after the previous dose, the animal was given 4 gm. of sodium benzoate with a sufficient amount of hydrochloric acid to de- compose it into benzoic acid, plus 120 c.c. of 0.2 per cent. citric acid. The animal became very weak in one hour, respirations were reduced to nine per minute, and were very labored. Tonic and clonic convulsions began one hour and fifteen minutes after the dose was given. The animal, after several hard convulsions, died two hours and twenty minutes after administration. The autopsy showed congestion of various organs. There was very pronounced congestion of the kidneys, stomach and intestines, with ulceration in places. The liver and lungs also showed evi- dences of infarcts. Second experiment. — Two dogs had been fed on dog biscuits and water for several weeks, and then fasted for thirty six hours. Animal No. 1.— The first animal was a male, weighing 3.5 kilos. He was given a mixture of 3.5 gm. of sodium benzoate, 50 c.c. of water, 0.65 c.c. of concentrated hydrochloric acid (sp. gr. 1.19) and 100 c.c. of citric acid (0.2 per cent.). The animal 126 SCIENTIFIC PROCEEDINGS (34). became quite uneasy after receiving the dose. At the end of an hour he showed great muscular weakness and tremor. Animal No. 2.— The weight of the second animal was 4.25 kilos. It was given a mixture of 100 c.c. of 0.2 per cent. citric acid, 50 c.c. of water and 0.85 c.c. of concentrated hydrochloric acid (sp. gr. 1.19). This animal was entirely unaffected. The same experiments were repeated on the same animals the next day; the results were practically identical. The animal (1) that received the free acid-benzoic acid mixture, however, was more prostrated than on the previous day and showed general stiff- ness of the muscles. At the end of six hours it was chloroformed and autopsied, when it was found that the stomach contained “ coffee ground”’ material. There were ecchymotic areas and some places appeared to be ulcerated slightly. The intestines showed marked congestion here and there and appeared to be slightly ulcerated in places throughout. The grumous material in the stomach and intestines gave a strong guaiac test and was undoubt- edly modified blood. The liver and lungs showed considerable congestion with some evidences of infarcts. The kidneys were cyanotic, the cortex very much congested, while the medulla was pale and anemic. Further investigation is contemplated, especially on the influ- ence of nephrectomy on the toxicity of benzoic acid. I am indebted to many members of the Purdue University Alumni Association of New York City for volunteering as subjects in this investigation and thus making it possible for me to carry out experiments ona large number of individuals. The Secretary, Mr. Leslie Hustable, Mr. Ray C. Ewry, Mr. R. W. Parks, Mr. F. M. Waltz and Mr. H. Worsham of that organization have given me special assistance in various ways. I am also indebted to Drs. A. E. Olpp and Matthew Steel, and Messrs. Herzfeld and Bisch for cooperation, and to Drs. Foster, Mosenthal and Rosenbloom for criticism and suggestions. Professor Gies has given me all the facilities of his laboratory for the conduct of this research, as well as valuable criticism and suggestions. IMPROVEMENT OF THE FoLIn METHOD. rey, 67 (405) An improvement of the Folin method for the determination of urinary ammonia nitrogen. By MATTHEW STEEL. (By invitation.) [From the Laboratory of Biological Chemistry of Columbia Univer- sity, at the College of Physicians and Surgeons. | In the fall of 1907, during the progress of a metabolism re- search on “the influence of magnesium sulphate on metabolism,” * anomalous results were obtained in our quantitative determinations of urinary ammonia, whenever the magnesium salt was injected either subcutaneously or intravenously into the animal.” These anomalous results were found to be due to the facts that the magne- sium was eliminated into the urines in question in relatively large quantities, as ammonio-magnesium phosphate and that the re- sultant deposits of crystalline triple phosphate were not thoroughly decomposed by sodium carbonate, as used in the Folin method, whereby ammonia, in variable amounts, remained in its solid form as triple phosphate in the urines under investigation. We, therefore, sought another method that would liberate all the ammonia from ammonio-magnesium phosphate without producing ammonia from such compounds as urea in the urine. None, however, was found that fulfilled both conditions. Consequently, we were obliged either to devise a new method, or else modify the Folin process, so as to make it liberate the ammonia from triple phosphate. We chose the latter course. Our attempt was to find some alkali which would liberate all the ammonia from ammonio-magnesium phosphate and whichat the same time would not convert into ammonia any of the amino or imino radicals in the various organic compounds in the urine. Varying quantities of milk of lime, baryta water, and sodium hydroxide were added separately to weighed amounts of triple phosphate. These mixtures were aérated as usual. The results obtained made it evident that neither milk of lime nor baryta water, 1Steel: These Proceedings, 1908, v, 132; /ournal of Biological Chemistry, 1908, v, 85. ?Steel and Gies: These Proceedings, 1908, v, 134; Journal of Biological Chem- istry, 1908, v, 71. 128 SCIENTIFIC PROCEEDINGS (34). even in large amounts, was capable of liberating all the ammonia from triple phosphate, whereas sodium hydroxide, in comparatively small amounts, discharged the ammonia completely. The first condition was, therefore, solved. It now remained for us to ascer- tain whether sodium hydroxide would produce ammonia from such amino compounds as urea. After many preliminary trials, in some strictly comparative tests on normal urines, I found that from 0.5 gramto 1 gram of sodium hydroxide, plus about 16 grams of sodium chloride, gave results that were in perfect accord with those of the Folin method. Over fifty comparisons were made. In oder to ascertain positively whether the modified method would produce ammonia from non-ammoniacal radicals, weighed samples of urea, uric acid, glycocoll, taurin, leucin, tyrosin and hippuric acid were added separately and also collectively to 25 c.c. portions of urine and the results compared with the figures for the ammonia obtained from equal volumes of the original urine. In no case was any increase obtained. The tests were repeated, but 20 c.c. of a standard solution of ammonium chloride were substi- tuted for the 25 c.c. of the urine. In these cases, also, no increase of ammonia output was obtained. As a final test of the efficiency of the modified method the ammonia content was determined in a normal urine by both the Folin method and the modified method. Then to equal volumes of the original urine 0.5 gram samples of triple phosphate were added, and the ammonia contents again determined by both methods. The ammonia was also determined in separate portions of the triple phosphate. The results of these directly comparative tests showed that, with the modified method, the ammonia obtained from the urines to which the triple phosphate had been added exactly equaled the total amount of ammonia obtained from the corresponding urine and the triple phosphate separately whereas, wth the Folin method, only from 70 to 80 per cent. of the total amount of this ammonia was recovered. OBSERVATIONS ON METABOLISM. 129 68 (406) The depressor substance of dog’s urine; its disappearance in experimental acute nephritis. By RICHARD M. PEARCE. [From the Carnegie Laboratory of the New York University and Bellevue Hospital Medical College. | The urine of a normal dog when injected intravenously into another dog in doses of three cubic centimeters causes an immedi- ate fall in blood pressure varying from 25 to 96 mm. of Hg. This effect, constant for normal urine, is not always obtained when the urine from a chromate or uranium nephritis of the third to fifth day is used. It is still obtained, however, in arsenic and cantharidin nephritis of the same periods. This difference suggests that in the tubular lesions of chromate and uranium nephritis, which are characterized by extensive epithelial destruction, some substance normally eliminated is retained while in the glomerular nephritis caused by arsenic and cantharidin poisoning this retention does not occur. The elimination of the depressor substance would ap- pear therefore to be a function of the tubular epithelium. In animals with experimental nephritis of the tubular type the disappearance of the depressor substance from the urine is fre- quently associated with a lowering of the blood pressure which would appear to indicate that the retained depressor substance has a definite effect on the general blood pressure. This observation is not based however on blood pressure determinations on the same animal before and after the development of nephritis but by con- trasting the pressure in animals with tubular nephritis with that of normal animals and those with glomerular nephritis. The nature of the depressor substance has not been determined. 69 (407) Observations on the metabolism of a subject of diabetes. By PHILIP A. SHAFFER. [from the Laboratory of Pathological Chemistry, Department of Experimental Pathology, Cornell Medical College. | The subject of the observations was a patient in the service of Dr. Warren Coleman in Bellevue Hospital. Different known 130 SCIENTIFIC PROCEEDINGS (34). diets were used and the urine was analyzed over a period of more than three months. A portion of the results of the last part of the period is given in the accompanying table. The unusual fea- tures of these results will be briefly discussed. Between twenty and eighty grams of tofa/ B-oxybutyric acid (the acetone and diacetic acid being calculated as f-oxybutyric acid) were excreted each day for at least eighty days without any signs of impending coma. For more than two months with this severe acidosis the subject showed practically complete carbo- hydrate intolerance. As shown in the table the diet was then changed to 255 grams of oatmeal. On this diet the acidosis and the glycosuria decreased very much, the former ultimately disap- pearing. This comparatively sudden transition from a condition of severe to one of mild diabetes is very striking and appears to con- firm the good results obtained by von Noorden and others with his so-called ‘oat cure.” The dependence of /-oxybutyric acid production upon the amount of fat in the food was very clearly shown throughout the observations. The amount of total $-oxybutyric acid excreted varied in general with the amount of fat eaten, which is not usually the case. Note the effect of the addition of butter to the oatmeal diet. The amount of food-fat appears to have determined, in this instance, the amount of fat burned. An increase in the amount of fat in the food which caused an increased acidosis appeared to result also in a damage to the carbohydrate tolerance ; with the increase of acidosis there was a very marked decrease in the amount of sugar burned. This phe- nomenon is just the reverse of that so often observed, z. ¢., the decrease of acidosis with an increased burning of sugar, and again emphasizes the close inter-dependence between the metabolism of carbohydrate and fat. The observations were terminated by the death of the patient. The immediate cause of death was not definitely established but was probably due to the phthisis which complicated the diabetes. There was no sign of coma and in view of the absence of acidosis and of the great improvement in carbohydrate tolerance we can scarcely believe that the diabetes was immediately responsible. The blood obtained at autopsy contained only traces of S-oxybutyric acid, the whole blood containing about 0.3 gram. There was no OBSERVATIONS ON METABOLISM. 131 suppression of urine, over 500 c.c. being passed in the last five hours. The urine of the last two days is of interest; there was no acetone and no increase in glycosuria, but a very great increase in ammonia and in total nitrogen indicating a marked ante-mortem increase in protein katabolism. Sugar was determined by titration with Fehling’s or Pavy’s solutions; and f-oxybutyric acid and its derivatives by the writer’s method. Foep. Urine. | SS Total NH. Fat. N. NH,—N. Sugar. fB-oxybutyric acid, Average for 5 days...... 256 164 19.0 4.6 328 19.0 Average for 2 days...... 180 187 19.0 293 24.7 165 18 255 gm. oatmeal.......... ps Le = 32 Es fol 1.5 94 1.6 s¢ ut 5.8 I.I 72 0.9 sose-eene-o oe Cornell University Medical College. PAMULENER, Th2 Woo ncocsc osscnccesserce Department of Health, New York City. FIELD, (CYRUS |W ioe ee. 5-aonasnlere cscs soccer University of Louisville. FISHER: MARTIN GEeoree- ccc see cee ereee tenons Oakland College of Medicine. ELEXNER, | SIMON) .cc-scemeetecesnaed Rockefeller Institute for Medical Research. FLOURNOY, THOMAS! +. .52.c05cs- fo... o..ce ees case eee eee ee Columbia University. MAYER, ALFRED.....--:2:25-223 Marine Laboratory of the Carnegie Institution, Tortugas, Fla. MEETZER, |S. ss ecnaieee tec coseeeset Rockefeller Institute for Medical Research. MENDEL, LAFAYETTE 'B.....50. ccs-conc-aracsepanceee eee Yale University. Mervin, ANOEPH::22-.-see0e Pathological Institute, Wards Island, New York. Mrver, Gustave Mi 2 oo .ocecocsscccnanc- neeeeeoe ee eee Columbia University. MORGAN, THOMAS T= 22.--. = .dsnpase- eke aeaee a eee eee Columbia University. MORSE, MAX 5 2205 o.ccc. Secece center ee City College of New York. MURLEIN, JOHN R. 2.0 .< 2 ee eno eee St. Francis Hospital, New York City. SHAPPER,- Pair Aq. .- ccesnenae-e-sarn ae -Cornell University Medical College. Rott oF MEMBERSHIP. I51 HIERN MIANT mEDEONIR Va Gar cenisnticiiais aisiersitclaairale avielsinicieitinocieieis/sinie'sie Columbia University. STOVE O Ness: © HOARY ESS SEG or rata sats ais Siesevwiejarn ova a blaiesiareioiaie seine sia'ere Baltimore Medical College. SS VANCED pee GELIE, ORUANIG Dore eres reseratatota vislovedatcisieicraisiets sigs seiela’s. io/eiouans siss'ewieiars Harvard University. SOLLMANNE oe HORATED eects ener se cieicteeisiis Western Reserve University, Cleveland. SS OUI VANE DD org EGS cre clei recicic(esiovcinieiic rs cis eis ches wisleletioveltaie’simoveieetelelesie's Harvard University. SMEWARIH GEORGE: Nii iescreccivatjecces Western Reserve University, Cleveland. SMDIBES GEER C Yia Grol sieeeloielsieisteisisiioles actesisel Massachusetts Institute of Technology. SP OGIWARID 8: CEUAGS Ns rissa anjois siaiais duleisloleieisaterepe sia woreelereten’s Cornell Medical School. STOOKEY, LyMAn B. ......... University of Southern California, Los Angeles. STOREY web OMAGH Avsiocsistetaissarinicsicic