a choot ean ev et he Brea Ty ends tes * he apd ie) a deh. ie The Th Boneh : = eb wag ites Meer a OMrers MeeteL PES tes Oe Pet ae ew Ty ange one Hie ae, et e ¢ , > 7 AMS Tt) ern vibra’ ELE ay aT gigs eet ity. ay Stet rive bo pian Oe Crip taly na See fiat Teal fies set ee bat ea Ss 4) ; pate ict we * ; P ‘ Lay ps Mt et EEE Wainy tse tes wt she Daw . ¥ H aay fs 13% antl 40 Phevade i a ' . ; hes 3 ‘ ’ Mase aa dee Ca ie wareies . Bayt: ofa ay) ,) E ’ Brats | 44 at) 2 tide’ Writs ates + Pesce ict) ay Te Ean See adebriattaiy tas iat Lear 4 a en r: i ry ed oan THE AMERICAN JOURNAL OF PHY SIOZROGY EDITED FOR Che American Physiological Society BY H. P. BOWDITCH, M.D., BOSTON ’ FREDERIC S. LEE, PH.D., NEW YORK R. H. CHITTENDEN, PH.D., NEW HAVEN JACQUES LOEB, M.D., CHICAGO W. H. HOWELL, M.D., BALTIMORE W. P. LOMBARD, M.D., ANN ARBOR W. T. PORTER, M.D., BOSTON THE AMERICAN JOURNAL OF PHY SI@L OC Y VoLuME I. BOSTON, U.S.A. GINN AND COMPANY 1898 j Copyright, 1898 - By GINN AND COMPANY a , if : | — Gnitevsity Bress ; Joun Witson anp Son, Camsrince, U.S. A. CONTE NES. THE INFLUENCE OF BoRAX AND Boric ACID UPON NUTRITION, WITH SPECIAL REFERENCE TO PROTEID METABOLISM. Sy 2. H. Chittenden OLE NEG ES EE Eb. Ee CREO OME Ce Ce VARIATIONS IN DAILY ACTIVITY PRODUCED BY ALCOHOL AND BY CHANGES IN BAROMETRIC PRESSURE AND DIET, WITH A DESCRIPTION OF RECORDING MetuHops. Sy Colin C. Stewart, A.B, Toronto . . . THE INFLUENCE OF HIGH ARTERIAL PRESSURES UPON THE BLOOD-FLOW TAROUGH. THE BRAIN. By WOE ome oe te eh Ve Ae THE RECOVERY OF THE HEART FROM FIBRILLARY CONTRACTIONS. Sy VM RIAORLC I oe nh oe ys A. ER REE TO o, VeP Ob eseie ta kad mek te NOTES ON THE ELIMINATION OF STRONTIUM. Sy Horatio C. Wood, Jr., THE NUTRITION OF THE HEART THROUGH THE VESSELS OF THEBESIUS BNOMTHESCORONARY VEINS. Lys Ee yaie; <3. gas «2 ~ ata Fart ON THE RELATION BETWEEN THE EXTERNAL STIMULUS APPLIED TO A NERVE AND THE RESULTING NERVE IMPULSE AS MEASURED BY THE PEON MCU RRM DS (by CW GECHC meas sire wn. ait Saleh ON THE NATURE OF THE CARDIOPNEUMATIC MOVEMENTS. By S./. Meltzer THE FUNCTIONS OF THE EAR AND THE LATERAL LINE IN FISHES. By Ee EReCR LULA S Po Hi. sb «51, ot, hs PAR MPEED dey a) 2s aya rg Ue : THE INFLUENCE OF THE HEART-BEAT ON THE FLOW OF BLOOD THROUGH She WALE SOM THE HEART. Bye 2ePorier oie ee hs A FURTHER STUDY OF THE INFLUENCE OF ALCOHOL AND ALCOHOLIC DRINKS UPON DIGESTION, WITH SPECIAL REFERENCE TO SECRE- TION. By Rk. H. Chittenden, Lafayette B. Mendel, and Holmes C. HOTISU Bek Bn IAG EE RGN CCN A a TTR ee ee ON THE SIMILARITY OF STRUCTURAL CHANGES PRODUCED BY LACK OF OXYGEN AND CERTAIN PoIsons. By Sidney P. Budgett. . . . . THE EFFECT OF DISTENTION OF THE VENTRICLE ON THE FLOW OF BLOOD THROUGH THE WALLS OF THE HEART. By /da H. Hyde, THE CHEMICAL COMPOSITION AND NUTRITIVE VALUE OF SOME EDIBLE AMERICAN PUNGI. By Lafayette Bo Mendel «sive ss THE RESTORATION OF COORDINATED VOLITIONAL MOVEMENT AFTER NERVE CROSSING. By it... Cunningham, M.D.» ss PAGE 104 117 128 145 164 210 215 v1 Contents. PAPAIN-PROTEOLYSIS, WITH SOME OBSERVATIONS ON THE PHYSIOLOG- pace ICAL ACTION OF THE PRODUCTS FORMED. By R. H. Chittenden, Lafayette B. Mendel,and H. E. McDermott . . . »« + + « «© « 255 THE GASTRIC INVERSION OF CANE-SUGAR BY HYDROCHLORIC ACID. By S. J. Ferris and Graham Lush... 00 es) 3 5 ON THE MEASUREMENT OF MENTAL ACTIVITY THROUGH MUSCULAR ACTIVITY AND THE DETERMINATION OF A CONSTANT OF ATTENTION. By Jeannette C. Welch «0. 6 6 6 ew es na THE INFLUENCE OF BILE AND BILE SALTS ON PANCREATIC PROTEOLYSIS. By R. H. Chittenden and Altce H. Altro, B.A. « «| «. .) ee THE REINFORCEMENT OF VOLUNTARY MUSCULAR CONTRACTIONS. By Allen Cleghorn, M.D. 2 0 eee gn oe ON CERTAIN CHARACTERISTICS OF THE PRESSURE SENSATIONS OF THE HuMAN SKIN. ‘ By Gaylord P. Clark, M.D... ss 3 THE MOVEMENTS OF THE STOMACH STUDIED BY MEANS OF THE RONTGEN RAYS: By WEB Cannon. Ut iG 2 ieee sie :” Sahat oe ei CONTRIBUTIONS TO THE PHYSIOLOGY OF THE es NekyEs IN THE GUINEA-PIG. -By D. W. Harrington, A.M.,MD. . . « . «\. « 383 PHLORHIZIN DiaBeETES IN Docs. By &. A. Reilly, F. W. Nolan, and Grihiamt' Tush 6 in a ee Se VR ee On INTESTINAL ABSORPTION AND THE SALINE CATHARTICS. By George B. Wallace and Arthur R. Cushny. «> > ss THE MOVEMENTS OF THE FooD IN THE (EsopHaGus. By W.B. Cannon QUA A MOSER) 5 ie Gee a RRS I Peete A CONTRIBUTION TO THE CHEMISTRY OF CYTOLOGICAL STAINING. by Albert NLATREWS, ou) Noho ee ge Waeicth¢ : 445 NOTES ON CETRARIA ISLANDICA (ICELAND Moss). By) Ernest W. Brown, PR AD 50 iS acy 8+ | 8) ah ONS Se pg te VARIATIONS IN THE AMYLOLYTIC POWER AND CHEMICAL COMPOSITION OF HUMAN MIXED SALiva. By R&R. 1. Chittenden and A.V. Richards, THE VENOMOTOR NERVES OF THE HIND Limp. By F. W. Bancroft. . 477 An ANALYSIS OF THE ACTION OF THE VAGUS NERVE ON THE HEART. By L. Je J. Mushkens 0 ee me ee A NEw METHOD FOR THE STUDY OF THE ISOLATED MAMMALIAN HEART. By W. 7. Porter. 0.06 6s oe al De PROCEEDINGS OF THE AMERICAN PHYSIOLOGICAL SOCIETY . . . . iii-xv INDEX ©. 6 we ee ee a THE American Journal of Physiology. VOL. 1. JANUARY 3, 1898. NO. I. THE INFLUENCE OF BORAX AND BORIC ACID UPON NUTRITION. WITH SPECIAL REFERENCE TO PROTEID METABOLISM. By R. H. CHITTENDEN anv WILLIAM J. GIES. [From the Sheffield Laboratory of Physiological Chemistry, Yale University.) N view of the wide-spread use of borax and boric acid as food pre- servatives it is somewhat singular that our knowledge of the influence of these substances upon the nutritional processes of the body is so slight and uncertain. E. de Cyon,’ M. Gruber,? and J. Forster® have indeed studied the action of these agents upon pro- teid metabolism, but with results which are utterly lacking in har- mony. Thus Cyon’s work with borax seemingly indicates that pro- teid metabolism is diminished under its influence, z.¢., that borax tends to protect the consumption of proteid matter in the tissues. Gruber’s experiments, on the other hand, indicate with equal posi- tiveness that borax has no proteid sparing power, but really leads to an increase in the rate of proteid metabolism. To add to the uncer- tainty, the experiments with boric acid carried out under Forster's supervision tend to show that this agent is wholly without influence upon proteid metabolism. Obviously, conclusions which are so much at variance cannot be accepted without careful consideration. 1 Cyon: Sur l’action physiologique du borax. Comptes rendus, 1878, tome 87, p. 845. 2 GRUBER: Ueber den Einfluss des Borax auf die Eiweisszersetzung im Organ- ismus. Zeitschr. f. Biol., 1880, Band 16, p. 198. 8 FORSTER: Ueber die Verwendbarkeit der Borsaure zur Conservirung von Nahrungsmitteln. Nach Versuchen von Dr. G. H. Schlencker aus Surakarta. Archiv. f. Hygiene, 1884, Band 2, p. 75. R. H. Chittenden and Wilham /. Gres. N Cyon’s experiments were conducted simultaneously on three full- grown dogs which were fed upon a diet almost exclusively proteid. His observations were practically limited to determining changes in body-weight during short periods, with an estimation of the nitrogen of the urine. He found that during the period when borax was in- cluded in the food, the animals gained noticeably in body-weight and that less nitrogen was contained in the excreta than in the ingesta. From these very crude observations the conclusion was drawn that borax, even to the extent of 12 grams per day, may be ingested with the food, especially when the latter is essentially proteid in nature, without provoking the slightest disturbance in general nutri- tion. Further, Cyon appeared to see in his results evidence that borax, if substituted for common salt in food, will facilitate the assim- ilation of the latter and bring about a great increase in the weight of the animal. Such deductions, however, were wholly unwarranted from the data at hand, for not only were the periods of observation exceedingly short, but, as pointed out by both Gruber! and C. Voit, the animals at the beginning were much emaciated and received throughout the experiment such excessive quantities of meat that in- crease of body-weight would have inevitably followed without the presence of borax. Consequently, all that can be inferred legiti- mately from Cyon’s experiments is that assimilation and general met- abolism were not seriously affected by borax in the quantities given. In Gruber’s work more scientific methods were pursued, but it may well be questioned whether the conditions under which the ex- periments were conducted were adapted for bringing out clearly the full action of borax upon proteid metabolism. The two dogs em- ployed were fed simply upon meat and water, and were presumably in a condition of nitrogenous equilibrium. In the first experiment, where the animal received daily 1500 grams of meat and 200 c.c. of water, the daily excretion of urea in the urine varied from 75.82 grams to 110.30 grams during the six days prior to the administra- tion of borax. Then 20 grams of borax were introduced with the food, an amount so large that vomiting was at once produced, lead- ing to a loss of about 5 grams of the borax and about 100 grams of the meat, with most of the water. On this day, however, 108.20 grams of urea were excreted in the urine, although the food con- sumed was 100 grams less than the usual quantity. On the two fol- 1 GRUBER: loc. cit. * Voir: Hermann’s Handbuch der Physiologie, Band 6, Theil I, p. 165. Influence of Borax and Boric Acid upon Nutrition. 3 lowing days, without borax and with the full complement of food, the excretion of urea amounted to 109.00 and 107.60 grams respec- tively. From these results Gruber concludes that the borax increased the excretion of urea 4-6 per cent. In the second experiment, with a dog of 34 kilos body-weight, fed on a daily ration of 1100 grams of meat and 200 cc. of water, the daily excretion of urea varied from 70.86 grams to 80.60 grams for the four days of the normal period, while the administration of 10 grams of borax was accom- panied by an excretion of 82.14 grams of urea, and, on the second day following, the introduction of 20 grams of borax was accompa- nied by an excretion of 85.25 grams of urea. Further, on this latter day the volume of urine rose to 1310 c.c., while the largest daily excretion prior to this day was 1040 c.c. Gruber, therefore, con- cludes that borax does not spare proteid as Cyon asserts, but, just as in the case of common salt, sodium sulphate, and other neutral salts, it Causes an increase in the elimination of water from the body and induces therewith an increased proteid catabolism. It is not to be inferred from this statement that there is simply an increased washing out of urea from the tissues, for, as Voit! has pointed out, the amounts of urea excreted on the days following the ingestion of borax simply fall back to the neighborhood of the average for the normal period, and do not drop below that average. Gruber also con- cludes that borax has no unfavorable influence upon the assimilation of food, since the quantity of faeces, their content of solid matter and of nitrogen are within the limits of the normal elimination dur- ing periods when meat alone is fed. Further, no harmful influence, even after the ingestion of the largest dose — 20 grams —was to be observed, and the appetite of the animal was found to be undimin- ished on the days following that upon which borax was given. The objection we would make to accepting Gruber’s conclusions in their entirety is that they are based solely upon the results following the administration of two large doses of borax, 10 and 20 grams, whereas, to our mind, longer periods with a dosage of borax con- tinued for several days in succession would seemingly render the conditions much more favorable for an accurate judgment as to the character of the influence exerted by the substance on tissue changes. Further, since urea alone was determined in the urine, possible minor changes connected with the presence of the salt would naturally be overlooked. Lastly, we are inclined to the view that it is extremely i Vor: loc cit: p. 165, 4 R. H. Chittenden and William J. Gres. hazardous to draw such sweeping conclusions from one or two single experiments of this nature, especially where, as in the animal body, individual characteristics not infrequently give rise to exceptional re- sults quite foreign to those ordinarily obtainable. In Forster’s work with boric acid, Dr. Schlencker experimented on himself, using a mixed diet and taking boric acid in daily doses of I-3 grams. Each experiment consisted of three periods, of three days each, the boric acid being taken in the middle period. The conclusions arrived at were that proteid metabolism is not influenced, the excretion of-urea in the boric-acid period being midway between that of the fore and after periods. It was noticed, however, that the quantity of ethereal sulphuric acid in the urine was considerably lessened in the boric-acid period and in the period following, thus implying an inhibitory influence upon the putrefactive processes of the intestine. Further, it was observed that the volume of the faces, together with the contained nitrogen, was greatly increased under the influence of boric acid, from which it was inferred that this agent interferes with the assimilation of the food and perhaps, at the same time, gives rise to an increased secretion of mucus with a possible increase in the discharge of epithelial cells from the intestinal mucosa. This latter, however, is purely conjectural. Increased secretion of bile is also said to result from the action of boric acid. On the pulse and temperature no action was observed. It is thus quite evident that the influence of borax and boric acid on nutrition, and especially their influence on proteid metabolism, is by no means wholly settled. The preceding statements clearly em- phasize the uncertainty of our present information on the more essential features of the question before us, and we have therefore deemed it desirable to carry out, as thoroughly as possible, a series of experiments upon the action of both borax and boric acid on pro- teid metabolism and related phases of nutrition. Conduct of the Experiments. The experiments were conducted wholly upon full-grown dogs ranging in weight from 8 to 12 kilos. The animals were confined in suitable cages partially lined with gal- vanized iron and with the floor so arranged that both fluid and solid excreta could be collected in their entirety, while the upper portions of the cage were so constructed as to permit unrestricted circulation of air. In view of the length of the experiments— ranging from twenty-seven to fifty-six days each, with periods of eight to ten days duration — it seemed inadvisable as well as unnecessary to empty the Influence of Borax and Boric Acid upon Nutrition. 5 bladder each day with a catheter. Such diurnal variations as might possibly occur from incomplete emptying of the bladder at the end of the twenty-four hours would obviously be neutralized in periods of the above length, and consequently the urine was collected as naturally excreted, thus avoiding any possible disturbance of the normal condition of the bladder, etc. At the end of each twenty- four hours, the urine collected was combined, and its volume, specific gravity, etc., determined, after which the bottom of the cage was rinsed with a little distilled water and these washings added to the main fluid. The latter was then made up to some convenient volume in preparation for the daily analysis. The faeces whenever passed were collected in a weighed dish, the mass thoroughly desiccated over a water-bath, and the dry weight ascertained. The dried material was then pulverized and the nitro- gen-content as well as the ether-soluble matter determined in sample portions. The nitrogen determinations were always made in du- plicate by the Kjeldahl method and rarely varied more than 0.05 per cent. Whenever, as sometimes occurred, hair accumulated in the cage it was likewise collected and the nitrogen determined. The ether-soluble matter was determined by extraction of the dried faces in a Soxhlet-apparatus. The animals were fed during the experiments on a mixed diet composed of fresh lean beef, cracker dust, lard, and water. The meat was prepared as follows: fresh lean beef, freed as far as possible from all adherent fat and connective tissue, was run through a hashing machine, after which it was enclosed in a bag of thin cloth, placed under a heavy press, and kept there under increasing pressure for several hours, the bloody fluid which drained off being thrown away. By this method there results a mass of tissue free from surplus mois- ture, and which, when enclosed in a bottle, will keep perfectly fresh on ice for seven to ten days without separation of fluid. Several advantages accrue from this method. Thus, we have a perfectly homogeneous mixture which can be drawn from for at least a week with surety that its nitrogen-content is constant. There is therefore no necessity for a daily determination of nitrogen in this portion of the diet, for each sample can be analyzed when prepared and the data accepted as long as the meat keeps fresh. Further, meat pre- pared in this manner at different times, if subjected to essentially the same pressure, varies only slightly in its content of nitrogen. We have invariably analyzed each lot when prepared to avoid any pos- 6 R. H. Chittenden and William J. Gres. sibility of error, but, as the following results show, the differences in composition are very slight and necessitate very little alteration in the proportion of meat when changing from one lot to another. The following results are a few of the many obtained: Absolute Content of Percentage of Weight of Meat. Nitrogen. Nitrogen. 0.8703 gram 0.03041 gram O70 ye 02682 “ O7651) 02628: “ 0.7673 0.02716 gram 0:9228° § 03238 TOSOI 03723 0.8478 gram 0.03015 TOOLS 03591 0.8876 “ 03152 1.0082 gram 0.03642 gram Wray % 03783 95 1.0803 (0396) ae 1.1977 | 0.04265 gram 0.8142 02902“ 0.9793 03463“ The carbohydrate element in the diet, as already stated, was supplied by commercial cracker dust. This was purchased in large quantity and preserved in well stoppered bottles. It contained on an average 1.46 per cent of nitrogen. The lard employed was entirely free from any recognizable amount of nitrogen. The daily diet was divided into two equal portions, one-half being fed at 8 A.M. and the other half at 6P.M. When borax .or boric acid was given, the daily dose was likewise divided and given either with the food or directly after. The body-weight of the animal was taken each morning just before feeding. Each day’s urine included the fluid passed from 8 A.M. to 8 A.M. of the next day. Methods of Analysis. — Nitrogen was determined wholly by the Kjeldahl method, viz., in the daily analyses of the urine, faeces, and food material. All analyses were made in duplicate, and the figures given are based upon the average of closely agreeing results. In | analysis of the urine 5 c.c. were used for each determination, oxida- Influence of Borax and Boric Acid upon Nutrition. 7 tion being carried out in a long-necked Kjeldahl flask with Io c.c. of sulphuric acid and a crystal of cupric sulphate, thus doing away with the necessity of adding sodium sulphide in the distillation. The ammonia formed was distilled into quarter-normal hydrochloric acid, the latter being titrated with quarter-normal ammonia, using congo red as an indicator. Sulphur and phosphorus were determined in the customary manner by evaporating a given volume of the urine — 25 c.c. for each determination —in a roomy silver crucible with Io grams of pure sodium hydroxide (made from the metal) and 2 grams of potassium nitrate, igniting the residue until oxidation was complete and treating the fused mass with water. For sulphur, the mixture was acidified with hydrochloric acid, evaporated to dryness, the residue moistened with a few drops of hydrochloric acid and dissolved in hot water. The filtered solution was then precipitated in the usual manner with barium chloride, the resultant barium sulphate filtered, ignited, and weighed, thus giving data for calcula- tion of the total sulphur. For phosphorus, the aqueous extract of the oxidized urine was acidified with nitric acid, evaporated to dryness, the residue moistened with nitric acid and dissolved in warm water. From this solution the phosphoric acid was precipi- tated in the usual manner with molybdic solution and eventually transformed into ammonio-magnesium phosphate. From the weight of magnesium pyrophosphate obtained, the total phosphorus of the urine was calculated. Uric acid was determined by the well-known Salkowski-Ludwig silver method, using 100-200 c.c. of urine. Phosphoric acid was determined by Mercier’s! modification of Neu- bauer’s method, z.e¢. by titration of 50 c.c. of urine with a standard solution of uranium nitrate and tincture of cochineal as an indicator. Total sulphuric acid was estimated by diluting 25 c.c. of urine with 3-4 volumes of water, adding 5 c.c. of dilute hydrochloric acid, heating to boiling, and precipitating hot with barium chloride. The barium sulphate so obtained, after standing twenty-four hours in a warm place, was washed with hot water until free from chlorides and lastly with hot alcohol, ignited, and weighed. Combined sulphuric acid was determined by Baumann’s method, using 100 c.c. of urine” 1 See Neubauer und Vogel’s Analyse des Harns, neunte Auflage, p. 450. 27 1bid.,.p. 447; 8 R. H. Chittenden and Wilham /. Gres. Chlorine was determined in 10 c.c. of urine by Neubauer and Salkowski’s modification of Mohr’s method.’ Other methods occa- sionally made use of are referred to in their appropriate place. First Experiment. With Borax.— The animal made use of in this experiment was a short-haired mongrel bitch weighing about 12 kilos. She was brought into a condition approximating to nitre- genous equilibrium only after a preliminary period of nearly three weeks, during which time superfluous fat was lost and she became wholly accustomed to her surroundings. The daily food, at the time the experiment actually commenced, consisted of 250 grams of the prepared meat, 70 grams of cracker dust, 40 grams of lard, and 500c.c. of water. It contained 9.814 grams of nitrogen. This diet, with the above content of nitrogen, was adhered to throughout the entire experiment of twenty-seven days, the only variation being the slight changes in the amount of nitrogen, to be seen in the tables, inci- dental to the use of different lots of meat and in the employment of gelatin capsules during the borax period. These gelatin capsules, in which the borax was administered, contained 14.95 per cent of nitrogen, the four capsules used each day during the borax period containing 0.12 gram of nitrogen. This amount was naturally included in the nitrogen of the food. The experiment extended through twenty-seven days and was divided into three periods of nine days each: a fore or normal period during which no borax was given, a borax period during which 45 grams of borax (5 grams a day) were administered, and an after period when normal conditions again prevailed. During the borax period of nine days the quantity of borax given per day amounted to nearly 0.6 per cent of the total food and drink ingested, while of the solid food it formed 1.3 per cent. This dosage of borax, consid- ering the size of the animal, was fairly large, and with this particular dog considerable difficulty was experienced in inducing the animal to take it. At first the borax was simply mixed with the food, but its presence was quickly detected and the food refused, although it was eventually coaxed down, but with some difficulty. After this first day the borax was given in capsules, as already stated, and na further difficulty of this sort was experienced. Three times during the borax period, however, the animal was nauseated and vomited a portion of the food, thus showing that this quantity of borax was sufficient to disturb the physiological equilibrium of the animal. 1 See Neubauer und Vogel’s Analyse des Harns, neunte Auflage, p. 437. Influence of Borax and Boric Acid upon Nutrition. 9 The vomited matter was eventually eaten, however, later in the day, so that this occurrence did not disturb the validity of the experiment. It will be remembered that in Gruber’s experiment with a much larger dog (39 kilos) 20 grams of borax likewise caused vomiting. In his experiment, however, the entire dose of borax was taken at one time, while in our case, 2.5 grams were given in the morning and a like quantity at night. Hence, taking into account the weight of the dog, it might perhaps be argued that 0.25 gram of borax to I kilo of body-weight will produce vomiting. This, however, is very questionable, for in the above experiment the dog did not vomit until the afternoon of December 5, when she had already taken 12.5 grams of borax. In other words, the animal was without doubt suf- fering in part from the cumulative action of the salt. Thus, there was a slight attack of vomiting again on the fifth day (December 7) and a final attack on the eighth day (December 10). During the after period of nine days the animal was perfectly normal, and at the close of the period, to again test the action of the borax, 5 grams were given at one time shortly after the morning meal. Forty-five minutes afterwards the animal vomited, and this occurred three times during the forenoon. We are inclined to lay particular emphasis upon this action of the borax because it tends to show that in this first experiment the dosage of borax through the nine days’ period was as large as it well could be for this particular animal without vitiating the experiment, and that the conditions were therefore well adapted for bringing out distinctly any possible influence the borax might have upon the metabolic phenomena of the body. Further, we would call attention to the obvious advantage — in spite of the greater labor involved — of continuing experiments of this character over comparatively long periods of time. To be sure, in some cases where the substance being tested has a marked physiological action, a single dose may show at once the character of the influence ex- erted, but too often erroneous conclusions are arrived at through negligence of this precaution. Where, however, the substance under examination is given for five to ten days consecutively, with careful examination of the excreta, the chances of detecting minor influences are greatly increased, and at the same time the danger of being led astray by a single exceptional result — or by other possible errors — is greatly diminished. The following tables contain the analytical results obtained through- out the experiment. R. H. Chittenden and Willham J. Gres. ie) 8959 $668 coe el cOl9o 690 OT ey aaralt LOE TT OcL i PO" (herve 16¢'0 Sh6'L pryy Z2LC = sa0xKyq Jo uas0nIN COT L9 = 9uliQ jo ussoIjIN SIol LTOT 6101 LTO L101 STO LIOT SIOT SLOT +19'6 sasevioay Apreq 97e88 |° °° sleIoL +186 OTT F186 601 F186 Sol +18 6 OTT +186 OTL +186 OTT +186 60 F186 601 F186 601 SUIeIS snuiyy| "WYSI9 NW UadOIVIN aa ‘SHOW HOS peurqwio 5) LOIS [e}OL, ‘rnydins bs ‘ploy dQ | ‘ussoI}IN “UOTPIVIOY u3'dg sweis SO[D] ‘TOA USSOIJIN | ‘TUSIO AA ‘ANIY 1) ‘aqooyy ‘AaOg | ‘aLvVd ‘dOlddd AXYOA— LNAWIAAdXA LSA ick Influence of Borax and Borit Acid upon Nutrition. sero @90'0 6sTT 6L5°0 L9S°0 9+0'0 Léo Ol eke | iS E1IO'OL | sesvssay Apreg 6l6¢ 6$'09 sss'0 62r'01L| 6007S 660'S II+0 9+0'C6 86h) Sh] SsIT06 S[eIOL 616 € = SaxXJ Jo uasONIN ast LEV88 = Bul Jo uadoNIN Loot 89+ £90° O+r0'T SS" OS+ £90° Sé8'8 ” OZOL 709 | S OOT OL elt Il : 090° FLT 009" 1cs" 1¢0" 9+8'6 ” 9C01 86h | S OOTOL rae a Ol oS 690° 821 96S" S19" 6+0" érL OL ” ecOL nO |S OOTOT el 6 : c+0 $780 Gr tbh’ TSO" Ata’ ” +c01 coh | S OOTOL él 8 260° T6's¢ 890° 96ST tS 81s" Os0 ecS cl ” +c0L ho) || 91001 Tl L s 090° LOTT c6S" 89s" c+" £+0'01 » LEO Ocs | S ££6'6 TIL 9 LS0° £Oll LES’ ses” 6£0° £816 ” ScOl sts” |) ££6'6 cIl $ ak i 6£0° SOLO ILe 1ée c£0" 606'S ‘oulyexTy | ccol eke |S ££6'6 TIL + 7 L60°0 Leal 6820 1Z8'0 +500 aaa “PIV 1201 96L |. S £066 601 € sues Snur} I] 4aF8) Suvi SOL ‘29q ‘ued | yystam| “OS OS |-mudme| SOUT | “PPV | ug Site 58 oag -O1IN kiq | peurquiog| [eioy YI"S| -soug m9) UssOIN, | UoHovey IOA U9SOT}IN | FOTO M ‘saow 7 “ANIY 1) ‘doo ‘Ad Og ‘dolddd XVYOd— ' LNAWINddxXa LSA R. H. Chittenden and William /. Gres. 2 ‘GOlaad AALAV —“LNAWIAaAdXxa LSald £Or'0 £90°0 ZOl'T | ss90 | o8s0 +00 000°0T £09 | L866 | sedvsoay Apreq +09 ¢ OL'8S 1090 tSrOL} dcé68’s t00'S 68£°0 100°06 LovS| v8868 |° * ° sleI0L +29 E = Sa0ayq Jo uaso1IN LLE-OS = DUTT) FO uaso1IN 629'T St'SZ 690° OSTT | 919" +L" ++0° 8196 ” 6101 | 6S 9£0°0L SIL 0z i! ‘ies 890° €80T | O19" +LS 980° L856 ” LIOT | 0€9 1866 STI 61 j 690" CLL 2609: 18° 9¢0° o£7'6 7 SIO | 22s 1866 om SI 290° €IZT | 299° 299° 90° IZUOL ” 6101 | S6s 1866 SI LT $66 I Soe’ €L0° CHET gen: +69" TsO" 6tS OL 9 SIOT | 189 1866 FIT 91 ete 6t0° 8460 | 109 est Z£0" +08 L ” 9101 | Tss 1866 STI ST €80° S971 | abl 129" 6£0° LtO01 » LIOT | 169 1866 ¥IT +1 ; peas €10° Ley POUL: 68S" €s0° Ze90T 9 SIO. | 0L9 1866 E11 eI 2 $s0'0 +Z0T | 9650 | Ttr0 z+0'0 LOLS poy 6101 | SSt 1866 S11 ZI ‘ Sok snwiy] 29 sweis SO]LY 29q] : "13 ‘ds WeBONINT es ie a ee) ae "ploy 119 | “uaso.z1N | ‘uorovay TOA | ‘uaSorty | 43194 | “96S! ‘Saow ‘aNINQ) ‘aoo¥y ‘“AaOg 13 zt7on. Acid upon Nutr 2¢ Influence of Borax and Lor +29'¢ 616¢ CCS $S+ OL 7Z68'S 6c+ OL 6020'S 66901 6les 00'S 660'S OSsT'S 100'06 9+0°C6 LOe'68 +88'68 SLT'06 92£ 88 IOV xvlog d10 4 sweis sweis WasOUINt mage OS paurquro 3) mee “myd[ng ‘snioyd -soud ‘poy ona ‘UaSOIPLN ‘gourlyeg | ‘payetoxd ‘paysosu] ‘SHOW A “ANIN ‘NADOULIN TVLOL ‘AUVAWOAS TVAANAD — ‘LNAWIYAdXaA LSAlt ‘saoldad 14 R. H. Chittenden and Wilham /. Gres. Referring now to the tables containing the results of the firs experiment, it is to be noted that in the fore period of nine days th total nitrogen ingested amounted to 88.326 grams, while in the urin excreted during this period there were contained 87.185 grams c nitrogen, and in the faeces 2.122 grams, making a total of 89.30 grams of nitrogen; hence the nitrogen balance for the period nine days is—o.g8t gram. The body-weight remained practicall constant. The slight excess of nitrogen excreted over the amour ingested in this period is due possibly to lack of complete involu tion of the mammary glands!; the deficiency, however, is too sligh considering the length of the period, to need much consideratior For comparison, the results of the three periods, showing the relz tive excretion of nitrogen, may be arranged in tabular form: Fore Period. Borax Period. After Perio Nitrogen of Hood = = 3) 2-7. 88.326 90.118 89.85 Nitrogen of Urine. . . . . 87.185 .t 89.307 88.127 92.046 86.377 | 90.06 Nitrogen of Feces ... . 2.122 j 3.919 3 3.624 J Nitrogen Balance . . . — 0.981 ess) — 0.11 Ratio of Urine Nitrogen to Food Nitrogen . . . . 98.6 per cent. 97.7 per cent. 96.0 per cen It is thus evident that in this experiment, in spite of the larg doses of borax and the length of the period, proteid metabolism not modified in any noticeable degree. The amount of nitroge eliminated through the urine in proportion to the nitrogen of th food, during the borax period, differs from that of the fore peric only to a slight extent, and this difference is due apparently to diminished assimilation of the proteid food. The change in th nitrogen balance of the borax period is plainly caused by a sligl increase in the amount of feecal nitrogen, and not to increased met: bolism, thus indicating that the borax has a tendency to diminis somewhat the absorption of proteid food, or possibly leads to a increased secretion of mucus. When, however, the nitrogen of th faeces of the borax period is compared with both that of the fo: and after periods the increase is seen to be so slight that it is pe haps unwise to attach much importance to it. Certainly the bora: though given in doses sufficiently large to keep the animal on tt verge of nausea, did not in this experiment interfere greatly with tl * Marcuse: Ueber den Nahrwerth des Caseins, Pfliiger’s Archiv. f. d. ge Physiol., 1896, Band 64, p. 223. Influence of Borax and Boric Acid upon Nutrition. 15 digestion of any of the food-stuffs, since the faces of the borax period are not much greater in amount than those of the after period, though somewhat larger than those of the fore period. The weight of the animal during the twenty-seven days’ period showed a tendency to rise somewhat, z.¢., from 10.9 kilos to I1.5 kilos. This, however, is not to be attributed to a laying on of fat nor to a retention of nitrogenous matter by the body, but is the result simply of a diminished excretion of water due to the presence of the borax. The results in this connection are in direct opposition to those obtained by Gruber with single doses of borax. There is here no suggestion whatever of an increased excretion of water, but on the contrary, a very marked decrease. Thus, by refer- ence to the accompanying tables, it will be observed that during the fore period the total volume of urine excreted amounted to 5629 c.c. and the body-weight remained practically constant, 7. ¢., 10.9-I1.0 kilos. During the borax period, however, the volume of urine ex- creted fell to 4981 c.c. and the body-weight gradually rose to 11.3 kilos, while in the after period the volume of urine rose to 5427 c.c. with a constant body-weight of 11.5 kilos. It is thus quite clear that borax may decidedly check the output of water through the kidneys, and lead, as in this case, to its retention within the body. Very noticeable also, in this experiment, is the sudden change in the specific gravity of the urine, as also in the reaction of the fluid, when borax is given. Thus, in the fore period the specific gravity of the urine stood at 1017-1018, but at the opening of the borax period it rose at once to 1022-1027, dropping back, however, as the borax was discontinued. Similarly, the reaction of the normal urine was acid to litmus, but on exhibition of borax, the reaction quickly changed to alkaline. The marked rise in the specific gravity of the urine during the borax period is not due solely to diminished elimination of water nor to increase in the proportion of metabolic products, but mainly to the borax itself, which is rapidly eliminated through the urine. We have not made any special trial to ascertain how soon the borax appears in the urine after its administration, but we have observed that the urine collected on the first day of the borax period gives, after acidulation with hydrochloric acid, a strong reaction with turmeric paper for boric acid. Further, that the elimi- nation of borax through the urine is very rapid is manifest from the fact that, at the end of the borax period, the animal having received 45 grams of the salt, no trace.of a reaction could be obtained with 16 R. H. Chittenden and William J. Gres. turmeric paper on the second day of the after period. In othe words, elimination of the borax was practically complete twenty-fou to thirty-six hours after the last dose had been taken. These obser vations accord with Johnson’s statements! that borax and boric acic begin to be eliminated through the urine a short time after thei administration. While it is clear from a study of the nitrogen excretion tha proteid metabolism, under the conditions of this experiment, is no materially affected by borax, the other analytical results must no be overlooked. Thus, in the borax period the excretion of phos phorus, sulphur, total sulphuric acid, and combined sulphuric aci is slightly below that of the fore and after periods. The differ ences, however, are so small that it is perhaps unwise to draw am positive conclusions from them, other than to admit their negativ character. It can certainly be asserted with perfect safety that th borax has failed to exert any marked influence upon the excretio; of either sulphur or phosphorus. In this connection it will b remembered that Forster? found, on feeding boric acid to man, marked increase in the output of phosphoric acid. Borax, how ever, certainly fails to produce any such result, its presence in th body (of the dog) tending on the other hand to reduce the outpu of phosphorus. Further, it is evident that the slight diminution 1 the excretion of combined sulphuric acid is not sufficient to indicat any inhibitory influence upon intestinal putrefaction. Lastly, th figures obtained in connection with uric acid are such as to indicat a purely negative action. Second Experiment. With Boric Acid. — The animal experimente on was a short-haired mongrel bitch weighing 8 kilos. Nitrogenou equilibrium was quickly established on a daily diet composed of 16 grams of the prepared meat, 40 grams of cracker dust, 30 grams c lard, and 400 c.c. of water. This diet contained 6.144 grams of nitro gen and was practically adhered to throughout the experiment. Th latter was of thirty days’ duration, z.¢., three periods of ten days eack During the middle, or boric acid period, 1-2 grams of boric aci were given daily mixed with the food, the animal taking it withou 1 JOHNSON: Ueber die Ausscheidung von Borsaure und Borax aus dem menscl lichen Organismus. Jahresbericht f. Thierchemie, 1885, p. 235. See also, VIGIER Note préliminaire sur l’action physiologique du borate de soude. Comptes rendu soc. de Biol. Paris, 1883, p. 44. * ForSTER: Archiv. f. Hygiene, 1884, Band 2, p. 75. Influence of Borax and Borie Acid upon Nutrition. 17 the slightest reluctance and without any apparent effect upon the appetite. No sign of nausea or vomiting was seen. With 2 grams of boric acid per day the mixture of food and drink contained 0.31 per cent, while the dry food contained 0.86 per cent of boric acid. The total amount of boric acid given during the ten days was 14.5 grams. During the fore period of ten days the animal received a total of 61.440 grams of nitrogen. The nitrogen excreted through the urine for this period amounted to 58.119 grams, while the feces contained 3.203 grams, thus making a total of 61.322 grams of nitrogen excreted, with a nitrogen balance of +0.118 gram. Plainly, the animal was in a condition of nitrogenous equilibrium. The relative excretion of nitrogen for the three periods may be seen in the following table: fore Period. Boric Acid Period. After Period. Nitrogen of Food . . .. . 61.440 62.032 61.943 Nitrogen of Urine. . . . . 58.119) 59.600 | > 58.979 2 61.322 63.538 62.92 Nitrogen of Feces. . . . . 3.203) 3.938 S 3.944 Nitrogen Balance .. . + 0.118 — 1.506 — 0.980 Ratio of Urine Nitrogen to Food Nitrogen... . 94.5 percent. 96.7 per cent. 95.2 per cent. From these figures it would appear that there is a slight tendency toward stimulation of proteid metabolism. When it is remembered, however, that the nitrogen balance for the boric acid period, —1.506, is the result of ten days’ consecutive feeding with boric acid, it is manifest that the stimulating action is very slight, and our results may perhaps be considered as practically in accord with those reported by Forster, who found that in man ona mixed diet, boric acid in moderate doses (1-3 grams) was without influence on proteid decomposition as measured by the excretion of urea. Upon the assimilation of the proteid food there is no evidence of any action, z.é., the nitrogen content of the feces during the boric acid period is essentially the same as that of the fore and after periods. Further, the total weight of faeces for each of the three periods is so nearly the same, it is quite evident that assimilation has not been materially interfered with. In this respect our results fail to agree with those reported by Forster, who found that small doses of boric acid (1 gram in two days) given to a man on a mixed diet, and on a milk and egg diet, increased the excretion of faces; this increase being due, 2 18 R. H. Chittenden and William /. Gres. according to Forster, not to any decrease in the assimilation of fat nor to increase in the volume of the secretions, but to a decreased assimilation of the proteid food under the influence of the boric acid. This difference in our results may of course depend upon the differ. ence in the character of the animal species. In our experiment, the weight of the animal remained perfectly constant throughout the entire period of thirty days. The accompanying tables contain the various data obtained. Unlike borax, boric acid fails to produce any change in the volume of the urine. Thus, in the fore period of ten days the total volume excreted amounted to 4647 c.c., while in the boric acid period of the same length the total volume was 4665 c.c., and in the after perioc 4644 c.c. Further, there is no marked difference, to be measurec by litmus paper, in the reaction of the fluid, although, as the tables show, alkaline reaction is more common in the normal periods thar in the boric acid period. In the latter period, however, the specific gravity of the urine, as might be expected, shows a higher average than in the two normal periods. This is due, as in the case of borax to the rapid elimination of the boric acid through the urine. The latter shows the presence of the acid by the turmeric test on the first day of the boric acid period, while on the second day o the after period all trace of a reaction disappears, thus showing thai the acid is rapidly eliminated from the body and is practically com- pletely removed twenty-four to thirty-six hours after the last dose Upon the elimination of uric acid, boric acid appears to have < slight inhibitory effect, at least under the conditions of this experi- ment, but upon the excretion of total and combined sulphuric acid chlorine and phosphoric acid, no tangible effect is produced. Cer. tainly, the results in connection with combined sulphuric acid do not indicate any retarding effect upon the putrefactive processes of the intestine. In this connection it will be remembered that in Forster’s experiments on man doses of boric acid, corresponding to those used by us, apparently gave rise to a marked retardation in the amount of ethereal sulphate excreted. Asa result, Forster arrived at the con- clusion that boric acid materially reduces intestinal putrefaction. Out results, however, show no action of this kind in the dog, and we are inclined to the view that both borax and boric acid are too rapidly eliminated from the system to be very effective in the intestine. As already stated, the elimination of borax and boric acid through the Kg Influence of Borax and Boric Acid upon Nutrition. +H19 saseiaay Apieg OFF 19 s[eq1oL O+0" 020" 9£0' +20" 6¢0° 1c0° £200 sade J Jo uaso1}IN AULI-) JO uaSOIZIN TONY ” ‘aUTeHLV ” ‘PPV STOL FLOL FIOL 9I0L FOL FOL STOL FIOL clot STOT t+T9 t+H19 vad ie) va ie) H+1T9 vad ee) vad Ge) 1a Se) HrH19 vh19 61 +7 sweis snudyT] ‘uss = |} SIO A -O1}IN Aq ‘sHOw %OS OS ‘QULIOTY.D peurqmoy | [80 L ‘PIV oA) ‘ANINQ) “UdSOL}IN “UOTIIVIY 13 ‘ds ‘dolddd AXOA —*LNAWIAAdXaA AUNOOAS suieis SO[Dy "UdSOI}IN, "VYSTOM Be Te *LO8I ‘adoo.J ‘AGOg ‘aLV( R. Hl. Chittenden and Witliam /. Gres. 20 £069 saseisa y Apreq C£0'C9 S[Te}OL SIN JO UASO.NIN AULT) Jo UdSOIVIN LIOL ZIOL LTO L101 “PPV LTO OTOL SUTE TV. LTOL FIOL 9TOL 9TOT sc0'0 ‘ploy swivis snuyty sues SO[D] “OLIN ‘uas Aq IYI AA -d x ‘OUIL 80S LOS : -O[Y) =| peaurquioy)}| [0], ‘PRW 1) ‘UdSOIJIN | “UOTIOVIY ‘SAOW “ANIXQ “doy oln0g ‘adoOldadd GIOV DIYORA— * LNAWIANdxXad ANOOUS "UasOIIN ‘dooy "JYS19 A\\ “AGOg 22 5 20N. t Zz a upon Nutr Aci 2C L[nfluence of Borax and Bor [sO 9890 6160 6£L'0 1g8'0 soc | PL9'0 0680 90TT 9¢5°0 £180 9600 £66 C9 +619 £+6'19 saseiaay Apreqd S[eIOT, HOE = 61685 = SAIN JO UBSOIJIN aUTI~) JO UISOIJIN 66S "+ LOS9 OF6'+ ” ‘OUTTeNTY ‘poy ‘AUILEALV ‘POV ‘OUTLEALY ‘PPV Stol 9101 SLOL STOL L101 e1Ol STOL OTOL FIOL STO 8sl9 S89 8sl9 1409 £609 £¢o'9 £26'9 €¢o9 €¢09 €c6'9 sues snuiy!] "WYSTO AX Aq 0S ros “QUTLOT YD pourqumiog)| [eo], ‘ply ou “Ud SOI1JIN “UOTJOVIYT ‘SHOW WY ‘ANIN us ‘ds suis SO[Ty “ua SOIJIN "YB19 A ‘Golddd YALAV —'LNAWINadXad GNOOAS ‘aoo4 “AGdOg R. H. Chittenden and William J. Gites. ‘AUVWWNS IVYANAD —’*LNAWIYAdXa GQNOOdHS Fh6'f QO9"Ss 6Ft'S CLO'+ Og I1s‘9 Sos’ 6L6°8S +t9Or 0860 — £76'C9 £+6'19 "8 8 13IFV S£6'e oe"es 00S'S LIC Y Sle 6¢69°9 98¢° 009°6$ S99F COS l= ses'e9 C£0°C9 ply 20d £07 99'9b £96'L t8'¢ LS@0 Iée'9 +050 6IT'Ss Lt+9r SIT0 + cE 19 Ort 19 8 BLOW sueis a) 2) swueis uaa ‘I4q3I9M | “OF “OS SOS ee eRilve | cy | ‘pais uasOIUIN iq [e101], aulo[yD pauiquioy| ye, 311 uaso1qIN | ‘TOA sourreg | ‘poyeioxy | ‘pojsesuy ‘saOInad ‘SHOW “ANIN ‘NHOOWLIN TVLO J, Influence of Borax and Boric Acid upon Nutrition. 23 urine commences almost immediately after their ingestion, and it is very questionable, therefore, whether, with moderate doses of these substances, enough would remain unabsorbed at the lower end of the small intestine to exert much influence upon the growth and develop- ment of micro-organisms. Certainly, the faces do not ordinarily contain any appreciable amount of borax or boric acid after these substances have been administered in moderate quantities, although obviously the length of time the feeces are forming will have some influence upon their content of soluble matter. In only one instance, to be detailed later, where a very large dose of borax was given, could any decided reaction for boric acid be obtained in the faeces. Johnson? states that in the case of the human organism borax and boric acid show great irregularity in their appearance in the faeces, and that he was able to detect them in the latter only in six cases out of fourteen, although daily doses of 0.9-3.0 grams of boric acid were given. Lastly, it is to be noted that in our experiment with boric acid there is no such increase in the excretion of phosphoric acid through the urine as was observed by Forster; our results, indeed, fail to show any distinct influence exerted by boric acid upon the metabolism of phosphorized matter. _ Third Experiment. With Borax and Boric Acid.— This experiment was divided into seven periods of eight days each, thus making a total of fifty-six consecutive days during which the variations in the composition of the urine and faces were followed as before, under the influence of both borax and boric acid. The object in extending the experiment through this lengthy period was to ascertain whether prolonged treatment with borax and boric acid might not eventually result in such a disturbance of physiological equilibrium that more positive data would be obtained. With this end in view, a mongrel bitch of ten kilos body-weight was brought into nitrogenous equilib- rium, after which the urine and feeces were analyzed for eight consec- utive days, z.¢., the fore period. Borax was then given with the food for eight days, making the first borax period. This was followed by another period of eight days during which neither borax nor boric acid were administered, after which came a third period of eight days when boric acid was fed. This, in turn, was succeeded by a normal period of equal length, followed by eight days of borax treatment — 1 JoHNSON: Ueber die Ausscheidung von Borsaéure und Borax aus dem menschlichen Organismus. Jahresbericht f. Thierchemie, 1885, p. 235. 24 R. H. Chittenden and William J. Gees. the second borax period —concluding with a final after period of eight days, 7. ¢., a total of fifty-six days. By thus keeping the same animal under continuous observation for this length of time it might reasonably be expected that any cumulative action — assuming it tc exist— would be clearly manifest. Further, considerably large: daily doses of borax and boric acid were administered than in the preceding experiments. The daily diet made use of throughout the entire experimen! consisted of 160 grams of the prepared meat, 40 grams of cracket dust, 30 grams of lard, and 430 c.c. of water. Its exact content o nitrogen is shown in the table of the fore period. The total amoun' of nitrogen ingested during the fore period was 52.163 grams. The amount excreted during the same period was 51.734 grams, thu: showing a nitrogen balance for the eight normal days of +0.429 grams The dog used in this experiment, although short-haired, lost consid: erable hair daily. This was therefore collected and at the end o each period its content of nitrogen was determined and the amoun added to the nitrogen of the urine and feces, as seen in the accom. panying tables. It is interesting to note in this connection that the loss of hair in periods of eight days’ duration may be considerable: so large, indeed, that an appreciable loss of nitrogen may result Thus, in the seven periods of this experiment the total amount o hair shed was 61.98 grams, z. ¢., 8-10 grams for each period, the total nitrogen thrown off in this manner amounting to 7.856 grams These figures show that the hair shed contained 12.6 per cent o nitrogen. Obviously, in careful experiments, this source of los: cannot be overlooked. In the first borax period of eight days the daily dose of borax ranged from 2 to 5 grams, the total amount administered being 32.: grams. In the following boric acid period the daily dose rangec from I to 3 grams, a total of 17 grams of boric acid being given On commencing the second borax period the daily dose of borax wa: placed at 10 grams. This was continued for two days, but on th third day after taking the morning dose of 5 grams the animal’s ap: petite began to fail so that it became necessary to coax her consid: erably in order to have the day’s ration consumed. On this day therefore, only grams were given, but on the following day the appetite was nearly normal and 6 grams of borax were given. The dose was then raised to 10 and 8 grams daily, as shown in the tables a total of 64 grams of borax being given in this period of eight days. 25 +950 199 OZS'9 (| Sadvioay Apeq SIS’ £9L'cs ep] jo uaso1iN FeCl LIF 1 $90@ J JO UASOIUIN aul A) JO UDSO.IN Influence of Borax and Boric Acid upon Nutrition. 9f'OL Secu 798'0 8£9'0 6c8' 1 6LL°0 AV L690 186'0 é+0'0 £60'6+ FPS‘ L 19'S Ch9' $89'L Tees 6£T'L OsO'S O9T9 909 909 90+'9 90b'9 90b'9 OLL'9 OLL9 £689 66 O'OL OOr OOL TOl oxo} O'OL O'ol suivis ua sO1IN “JYSIO NY Ac] Hl OME TROL OS poutquio 7) "SHOW ‘dOINAd AUNOA—‘*LNAWINAdXaA CGAIHL snuyty ‘pry ou ‘UdSO.IN “UOTJOV9Y] sued SO[} ‘Ud SOIJIN, “"TYSTO AA “ANIUVQ ‘doo “ACOg ‘ALVC R. H. Chittenden and Witham /. Gres. 26 €26'0 £80'0 £190 L£0'0 1949 OLY |90'4 19¢'9 | sadvroay Ajreq SL’eb FLE'L c99'0 S06'F £62'0 | 9891S tore | se] sesos |° °° sreioy OSL = we} Jo ueBontN sd QITZ = SaxXY Jo uds0I}IN $EE'Sb = PULA) Jo uaSo1IN 69°02 oLeT 2 c6L c£0° £0S°Z ” ccOl =| OFS s S829 OO 9 2 aes L160 €80° L6S° OF0" TES ” ccOL | O9b ss) $829 TO S + ue 60+'0 O+0° CLE +20" L0'+ ” LTOT | O8€ S $829 TOL + 01:02 I8Z'0 cL0° sos" T¢0" 916s ” LIOL | 02S | Sb 90+'9 66 £ ide: 680'T 9¢1° L6L C40" 820°L ” 1ZOt | OLb + 90+'9 OOo é LOTT 680° FOL" c+0" Crs'9 ” SIOL | 16s k 90+'9 OOr | 1kew : Cri T 160° LL £+0" 8EL'9 » ccOL | OOF € 90+'9 TOL O€ 96°C L650 6+0°0 LO+’0 6£0°0 SZO'h “ouTexly | STOL | OOF c 90+'9 OOo! 67 suIeis snuiyq] 9'9 sues so[D] qudy ‘yy 310 Ky & 8 € ‘pro reyes ee) 16 SFOUIN Ace rapt semen er Ser MGZODIN | “VOUPERA we 4 Sa A RSSINN, ee 2 - ‘SHOW ‘ANIN ‘aoo0.J ‘Aadog | ‘aALVG ‘GOIdadd XVAOd LSAIA —"“LNAWIAHdxXad GaYIHL 27 L[nfluence of Borax and Boric Acid upon Nutrition. T£z'0 O+6'0 040°0 L09°0 1¢0°0 c609 S6b FLE9 sasvioay Ape SF8'T 99'6£ 61S *L 2980 9S8'+ 8tZ'0 PEE'OS 796E S660 ae RAE fh 6SO'L = wey Jo uaSonN CHO T == Ssooay Jo uesoIN OF LE = ULI, Jo uds0I1yINN clr’ 128 6860 $s0° 89s" 920° PSL'S ” £1ol O6b Szt'9 TOI a! Le9 06 IL LIO'T $90° 029° 620° F68'S ” FIOL Scs Str9 TOI eT aa aot 990°T 090° 98s" 620° cS6'S ” ron KO) oes Str'9 OOL él S+L'0 $s6l eer 901° eL8 9£0° Str L ” STol L6S Szt'9 OOr Il reer eee 66$'0 $90" Cock +10" LILY » FOL 06£ Str'9 ZOOL Ol eee, Ske L8T'T Li; £29" +0" TL9°Z ” OTOL 06S $829 TOL 6 rae ge 8s+0 O+0° 6br FEO" Ofer ” Zlol Of S8c'9 ZOOL 8 Ses aed O[S'0 +S0'0 SLS°0 9£0'0 L89°S ‘PPV Stor OIF $879 Vol L suieis snwi}q] oy) swv.id SO[LY Key ‘yYys10 (0 yKs es BOS ie uefonIN | “iq | ero) pauiquiog| eyo, | PPV HM | vadomn | uonseoy TOA | ‘woSonty |-7yS1ey | “4687 “SHOW J “ANTM {) ‘adooy ‘ACO “ALVC ‘dOINad AALAV LSU —"LNAWIYadXa GUYIHL “ R. H. Chittenden and William J. Gres. 28 8220 ++8'0 140°0 s6s'0 6£6 Sb 620° 88Sb UEIOe, £69 606'S LYOv Fors LLIS Mey Jo uasoijin sa0e2y Jo uasONIN auLIf) Jo uaso1IN UdSOIIN "TYS19 AA Arq OS peurquoyg ‘POV 1 UdSO1JIN “AaNIN ” PPV snuiyy] “UOT]IVAY SIO FLOL OTOL 9IOL STOT FLOL SLOT STOT O0r'9 Sasvisay Apieq OO TS 96¢'9 96¢°9 96¢°9 96¢°9 96¢°9 96¢'°9 96£°9 8cr9 ce OIL col ZOOL TOl col cOL TOL TOL s[eqoy, 43 -dg sues SOT] “UdSOIJIN “AVIA yysioyy | “468! QOtadd=GIoOv. DIWOd—-) INAWIdddxXe Galas “IOV O1NOg ‘aqdooy *“AdOg “ELV(I 29 St80 [tl9 98L°9 Of 6b 90+'9 ose ls sosvioay Aled etl 2690 col C680 1160 SNOT 6820 1L9°0 T£0° $100 Lec l Sor Str oF = Ae]p] Jo uaso.qiN Ss90xey JO uasO.NIN LL69 66+ SIs‘9 PO's OEL'S $01 LS6'E $ ‘pry ‘OUeNLV PHY ‘aUTEALVY ‘poy LO fLOl SI0L FIO £tol FLOL OLOL Slot aULIA, JO UISO.QIN Otro OL'9 Oro OL"9 Ou9 OW’9 96¢°9 969 S[P}0], sueis snug || "WYSIO MN “UdSOIIN k { Iq %OS peurquios) HOS [P10], Influence of Borax and Bort Acid upon Nutrition. “SHOW W ‘GOIWMad UALAV GANOOAS—'LNAWIYAdXaA AGYIHL ‘POV oy *UdSOITIN “UOTIIVAY a3 “ds swiss UdSO.IN ‘JUSTO AN “ANIM ‘doo ‘ACO LV R. H. Chittenden and William J. Gies. 30 6080 soo'T 089'0 clr tl 9¢"°0 8660 coll bly 1 9L0' 040° LLO’ eI 8£0" 660° cS0° 0L0°0 VOU 4 ley JO ussoIjIN Sade J JO UdSOIJINN OUIIY) JO UdSO.IN rardo} £20 scot cc0l OZOT 9COT 6c0L ‘OUNeALTY | L201 L£5v | 58 v6e9 LS9E| 49 | STIS FLE'9 FLE9 FLE'9 c6e'9 Oro OIr9 Org Olr9 SISEIOAY ATTE(T . . . £01 ZOl cOl fOr FOL £01 TOL TOI sues snwiyt| “UdSO.1IN oe ’OS poautquio, “PPV on) 3 ds ‘uasOIIN | ‘UOTOvaYy swvis SOT "uasdOI]IN ‘SHOW YJ “ANIYG ‘WYS19.A\ ‘doo ‘adOlaadd XVAOF GNOOUS —’LNAWIAadxa CGalHL ‘AGOg il! Influence of Borax and Boric Acid upon Nutrition. 1920 es8'0 +S0'0 +290 T0'0 6L4'9 90S 96£°9 sasviaay Ayre 680°% 98'S Les'9 £et'O 966'+ 8Z£'0 Oes'TS OSOF 69T'TS ne) LZR 9 . £96'0 ey] jo wasouIN 680'2Z soouy Jo uaso.jINy SLL Sb aulIA, JO UasOI}IN F6T'T 90°02 £6L°0 $90' 1¢9 +10" e991 » ctor els Stt'9 col ST ; ‘ Sth'T 280° 9S8° O+0° LI6L “PIV SOT cs9 Shy'9 col al : ates ++9'0 0¢0" STs" £+0° SsT's ‘ourexry | elLor £0S 60+'9 £01 el $680 O8 22 bhe'T $L0' T8Z° TsO" 09S"L ” 9TOT Ses bLE'9 FOL él ZL8'0 80° 8eo LEO" 6F19 ” FIOT 00S bLEe'9 FOL Il pe eg aS 66£'°0 L+0' Ltt’ c+0" +8o'+ ” TIO Cth bLe'9 FOr Ol Sv a 989'0 £40" 8s 620° FE8'S » £10L Ses FLE'9 OL 6 ake ze ++9'0 ££0'0 L9S°0 c+0'0 €1e9 ‘PPV 9TOT Ilr PLEO £OT 8 suivis snutyy] ah) sues SON | -oune 4uS19 GALS f 2 13 ‘ds “UadOIPIN Ae rio, pourquios [eo ‘PIV IU] | ‘waso1IN | ‘uoNovay TOA | waSoNIN |7481OM "£631 ‘SHOm ‘ANTI ‘aoog | ‘xaog | ‘anvq ‘doladd YaALAV GYIHL—'LNAWIYadxXyd GUYIHL £96'0 c£6'0 Lect $9c'1 6S0'T 98T'T bee'l LES'9 9T9'L 9819 vSL'9 61S" PLe'L SELL 966'+ Sg’ 096'+ COL'Y 9S8't S06 SLL'Sb €98'3S SE Ob 6£6 SF Oth’ Lr FEES £60'6+ SULRIG 7 OSOb LG98 er6E LLLE C96E T9LE 961+ IE ENO) 813 >— A i Ai PLT + 199'0 + 108'0— 6z+'0 + OfS'Ls 6€0'9¢ Of L'6b 9CO' 6b FEE'OS 989'TS sues UdSOIIN _ | Ista ; Aid ‘UdSO.IN ea [PIO.L LOWS paurquios) [BIOL “UdSOIJIN ‘aouRleq ‘pajyaIOX | 69T TS PST IS esa 1S $660 S88'0S ‘pajsosuy MIVH R. H.. Chittenden and Witham /. Gees. cq ‘SHOW W “AUVNWOAS ‘ANIN() IVAUHNAD— LINAWNIYAdxXaA "NADOULIN IVLOT, CQaulHt * 1YV * xXB10g Panel ply ouog 1dV *xXv1og yew410N ‘SaOINad Influence of Borax and Boric Acid upon Nutrition. 33 o Throughout the entire experiment of fifty-six days the animal re- mained perfectly well, kept a fairly constant body-weight, and showed no symptoms of nausea or vomiting during the administration of either borax or boric acid. The only noticeable effect was a seem- ing loss of appetite on one day, as mentioned above. At the termi- nation of the final after period, a single dose of 5 grams of boric acid was given. This resulted in vomiting 4-5 hours afterward. The relative excretion of nitrogen for the seven periods is shown in the following table: 1. 2. 3: fore Period. First Borax Period. First After Period. Nitrogen of Food . .. . 52.163 50.885 50.995 Nitrogen of Urine . . . . 49.093 ) 48.324 ) 47.430 ) Nitrogen of Feces. . . . 1.4177 51.734 2.176 ~ 51.686 1.845 > 50.334 Nitrogen of Hair . . . . 1.224) 1.186 ) 1.059 ) Nitrogen Balance. . . + 0.429 — 0.801 + 0.661 Ratio of Urine and Hair Ni- trogen to Food Nitrogen. 96.4 per cent. 97.2 per cent. 95.0 per cent. 4h 5. 6. Boric Acid Period. Second After Period. Second Borax Period. Nitrogen of Food . .. . 51.200 SIEZ52 51.154 Nitrogen of Urine. . . . 45.939) 46.438 ) 52.363 ) Nitrogen of Feces. . . . 1.822(¢ 49.026 1.465 ¢ 49.130 2.1371 ¢ 56.032 Nitrogen of Hair . . . . 1.265 J 1.227) 0.932 ) Nitrogen Balance. . . + 2.174 + 2.122 — 4.878 Ratio of Urine and Hair Ni- trogen to Food Nitrogen. 92.2 per cent. 93.0 per cent. 104.1 per cent. ihe Third After Period. Nitrogen of Food .... . 51.169 Nitropenof Urine © . =. = 48.778 Nitrogen of Feces. . . . . 2.089¢ 51.830 INTErOREn Of lati ser) = tee 0-905 Nitrogen Balance. . . . — 0.661 Ratio of Urine and Hair Nitro- gen to Food Nitrogen . . 97.2 per cent. In the first borax period of eight days with a total consumption of 32.5 grams of borax, z. ¢.,an average of 4 grams per day, there is practically no change in the rate of proteid metabolism. There is, however, a slight rise in the amount of fecal nitrogen similar to that noticed in the first experiment with borax, by which the nitrogen 3 34 R. H. Chittenden and Wilham /. Gres. balance is somewhat changed, but there is plainly no effect produced on proteid metabolism. In the second borax period, on the other hand, there is evidence for the first time of a distinct and unquestion- able influence upon proteid metabolism. In this period of eight days 64 grams of borax were administered, and under its influence the excretion of nitrogen through the urine was greatly increased. As in the other experiments, the proportion of nitrogen in the feeces was likewise increased, implying decreased assimilation of proteid food, but the nitrogen balance of — 4.878 is mainly due to direct stimulation of proteid metabolism. When, however, it is considered that to ac- complish this result a daily dose of 8 grams of borax was required, and for eight consecutive days, with a dog weighing only 10 kilos, it is very plain that proteid metabolism is not readily affected by borax. In the boric acid period of eight days, with a total dosage of 17 grams of the acid, there is some evidence of diminished proteid meta- bolism. The excretion of nitrogen through the urine is certainly diminished; there appears to be a sparing of proteid, but it is to be noticed that, in the period following, the nitrogen balance remains unaltered, which fact casts some doubt upon the assumption that the result is due solely to the acid. It is of course possible that the action of the boric acid may be continued into the after period, but this we should hardly expect in view of the rapid elimination of boric acid from the system. Further, after the second borax period, where the nitrogen balance is so noticeably disturbed, there is a quick return to the normal, the nitrogen balance for the final period dropping back to — 0.661. Consequently, while the analytical data show a retention of nitrogen during the boric acid period, thus indicating diminished proteid metabolism, we feel some hesitation in attributing the result wholly to the boric acid, particularly as the earlier experi- ment with boric acid gave essentially negative results. Especially noticeable in this experiment, as in the earlier experi- ment with borax, is the action of the latter agent in reducing the volume of the urine. [See table showing general summary.] In both borax periods the total volume of urine excreted is distinctly reduced, and the same holds true in this experiment with the boric acid. It is quite probable that the somewhat larger daily dose of boric acid made use of in the present experiment is responsible for this result, although it is possible of course that the personality of the animal may have had some influence. In the previous experiment Influence of Borax and Boric Acid upon Nutrition. 35 with boric acid, where the maximum daily dose was 2 grams, the vol- ume of the urine was unaltered. In view of these facts it is perhaps proper to consider the larger dosage of boric acid used in the present experiment as responsible for the apparent action upon proteid metabolism likewise. Also noticeable in this experiment is the influence of the larger doses of borax upon the excretion of total and combined sulphuric acid. Both of these are distinctly increased in amount during the last borax period, in harmony with the increase in proteid metabol- ism, and there is a suggestion of the same influence in the first borax period. Moreover, in the last borax period the excretion of phos- phoric acid is noticeably increased, while the elimination of uric acid is slightly diminished. It is thus plainly evident, as already stated, that while moderate doses of borax, even long-continued, are without influence upon the nutritional processes of the body, large doses may distinctly increase the rate of proteid metabolism, giving rise not only to an increased excretion of nitrogen, but also of Se piunic acid and phosphoric acid. In all of these experiments with borax there is constant evidence of an increase in the weight of the faeces during the borax periods. This increase in weight is due in part to an increased output of nitro- genous matter through this channel, but whether the latter is caused by diminished digestion and absorption of the proteid food or to a stimulation of the mucous or other secretions from the gastro-intesti- nal tract is not so clear. It has been plainly shown, however, in another connection! that while borax in moderate quantities has no inhibitory action whatever on either gastric or pancreatic digestion of proteids, larger proportions do retard the proteolytic action of both digestive fluids. Further, retardation of proteolysis with borax is much more pronounced than with boric acid; hence it seems quite probable that the mcreased bulk of faeces and the higher content of nitrogen therein during the borax periods is due mainly to slight retardation in the assimilation of proteid food. Large amounts of borax likewise interfere with the assimilation of fatty foods; a statement which does not appear to be true of boric acid. In the accompanying table [page 37] are given the results of our analyses of the dry faces, from a study of which it is plain that under the influence of large doses of borax — first and second borax 1 Chittenden: Influence of Borax and Boric Acid on Digestion. Dietetic and Hygienic Gazette, 1893, vol. 9, p. 25. 36 Yas fe etter and William J. Gres. od periods of experiment third —both the total and percentage amounts of ether-soluble matter in the faeces are greatly increased. Boric acid, on the other hand, produces no such effect. In the first exper- iment, with borax, the evidence of decreased fat absorption is less pronounced, although both the dosage of borax and the amount of fat fed were greater than in the first borax period of experiment third. Quite possibly this apparent difference in action may be due to the personality of the animal. However this may be, it is plain that large doses of borax are prone to increase somewhat the bulk of the faeces, in part by diminishing slightly the assimilation of both proteid and fatty food, and in part, we think, through a tendency to increase the secretion of mucus. Thus, we observed in the last ex- periment, during the period when the largest doses of borax were given, that the faeces were more slimy than in the normal periods, and appeared to contain more mucus than ordinarily. Further, it is to be noted that under the influence of large doses of borax there isa tendency toward diarrhoea; not very marked to be sure, but sufficient to render the discharge of feeces somewhat watery. In spite of these evidences of minor action in the intestinal tract with large doses of borax, there is no evidence whatever of any in- fluence exerted upon intestinal putrefaction, either by borax or boric acid. Even with the largest doses of borax the combined sulphuric acid of the urine is raised rather than lowered, and careful examina- tion of the urine daily with Jaffe’s indoxyl test failed to reveal any indications pointing to an inhibitory influence exerted by either borax or boric acid upon the production of indican. If, however, one studies carefully the output of combined sulphuric acid as shown in the various tables it will be noticed that the highest figures are generally obtained on the day (or the day preceding that) on which the dog defecates; while after defecation the combined sulphuric acid of the urine falls atonce. In other words, the natural obstruction of the intestine favors, as is well known, the absorption of putrefactive products, and thus leads to an increase of combined sulphuric acid in the urine. When, on the other hand, defecation occurs, the com- bined sulphuric acid of the urine is at once diminished in amount. Upon these natural fluctuations of combined sulphuric acid even the largest doses of borax and boric acid are without effect, not because these agents are without influence upon micro-organisms, but because they are too rapidly and completely absorbed from the intestine to exert much influence upon intestinal putrefaction. In only one in- Lifluence of Borax and Boric Acid upon Nutrition. 37 TABLE SHOWING CONTENT OF FAT AND OTHER ETHER- SOLUBLE MATTER IN THE FECES. EXPERIMENT I. EXPERIMENT ITI. Date. Faces. Ether-soluble Faces, Ether-Soluble Period. matter. matter, -_—_—_—_as*4"t—7 ¥ —————s? 1896. Lo ek Percent. Grams. ey Sees Percent. Grams. Wilees 2 38.15. 35.03 | 13.362 14.33 28.91 4.134 10.36 29.09 3.029 Sol os:00). 120671 24. 7163 24.68 25.23 6.227 69 29.01 7.163 60.59 30.02 18.294 2.96 : 0.840 20.10 ie 7.306 12.140 20.69 : 7.671 6.198 43.75 18.338 After 19.55 11.90 EXPERIMENT II. §.21 Date. Feces. Ether-soluble 39.66 matter. ——————— Dry weight. Grams. 6.96 23.70 1.649 11.90 17.88 2.128 10.50 16.95 1.770 USO 20182 3.602 Percent. Grams. 46.66 19.61 9.149 10.20 = 18.87 1.924 STs: 17.67 1.723 16.30 20.31 3.311 11.60 20.60 2.390 Se 9 ZA0LS Ss ae) 53.20 1967 10.467 45, 8.596 7.579 B45. 20.54 “TIO : 5.940 BD 6 26.63 2.053 S82, >. 20:28 1.789 22.115 12/25, 20:72- 2538 10.47. 20.01 2.095 5 8.954 - 10.90 19.31 2.105 d 6.028 55.60 21.04 11.699 ; 14.982 After 38 R. H. Chittenden and William J. Gies. stance were we able to detect any boric acid in the feces, viz., on June 5th, at a time when the largest doses of borax were being given; and at the close of this period the boric acid reaction could be obtained with the urine only on the first day of the after period, so rapidly was the borax passed out of the body. Lastly, attention may be called to the constant presence, in appre- ciable amounts, of uric acid in the urine of all the animals experimented with, in opposition to the older statements of Liebig+ and others that kynurenic acid may entirely replace uric acid in the urine of the dog. Our results, so far as they extend, are thus wholly in accord with the recent observations of Solomin.* We have, however, made no attempt to determine the amounts of kynurenic acid present. General Conclusions.— Moderate doses of borax up to 5 grams per day, even when continued for some time, are without influence upon proteid metabolism. Neither do they exert any specific influence upon the general nutritional changes of the body. Under no circumstances, so far as we have been able to ascertain, does borax tend to increase body-weight or to protect the proteid matter of the tissues. Large doses of borax, 5-10 grams daily, have a direct, stimulating effect upon proteid metabolism, as claimed by Gruber; such doses, especially if continued, lead to an increased excretion of nitrogen through the urine, also of sulphuric acid and phosphoric acid. Boric acid, on the other hand, in doses up to 3 grams per day, is practically without influence upon proteid metabolism and upon the general nutrition of the body. Borax, when taken in large doses, tends to retard somewhat the assimilation of proteid and fatty foods, increasing noticeably the weight of the faeces and their content of nitrogen and fat. With very large doses there is a tendency toward diarrhcea and an increased excretion of mucus. Boric acid, on the contrary, in doses up to 3 grams per day, is wholly without influence in these directions. Borax causes a decrease in the volume of the urine, changes the reaction of the fluid to alkaline, and raises the specific gravity, owing to the rapid elimination of the borax through this channel. Under no circumstances have we observed any diuretic action with either borax or boric acid. The latter agent has little effect on the volume of the urine. ' LiesiG: Annalen d. Chem. u. Pharm., Band 86, p 125. 2 SOLOMIN: Zur Kenntniss der Kynurensaure. Zeitschr. f. physiol. Chem., 1897, Band 23, p. 497. Influence of Borax and Boric Acid upon Nutrition. 39 Both borax and boric acid are quickly eliminated from the body through the urine, twenty-four to thirty-six hours being generally sufficient for their complete removal. Rarely are they found in the faeces. Neither borax nor boric acid have any influence upon the putrefac- tive processes of the intestine as measured by the amount of combined sulphuric acid in the urine, or by Jaffe’s indoxyl test. Exceedingly large doses of borax are inactive in this direction, not because the salt is without action upon micro-organisms, but because of its rapid absorption from the intestinal tract. Borax and boric acid, when given in quantities equal to 1.5-2.0 per cent of the daily food are liable to produce nausea and vomiting. Owing to the rapid elimination of both borax and boric acid, no marked cumulative action can result from their daily ingestion in moderate quantities. At no time in these experiments was there any indication of abnormality in the urine; albumin and sugar were never present. VARIATIONS IN DAILY ACTIVITY PRODUCED BY ALCO- HOL AND BY CHANGES IN BAROMETRIC PRESSURE AND DIET, WITH A DESCRIPTION OF RECORDING METHODS.* By COLIN GG STEWART, A. Bz Loronne: Fellow in Physiology, Clark University. [From the Laboratory of Physiology, Clark University, Worcester, Mass., O.S.A.] N a series of papers dealing with the laws of growth Minot? has pointed out the significance of experiments on organisms as individual wholes, as leading toward the proper object and final purpose of biological investigation, —the discovery of the laws of life. Growth, anabolic and accumulating, has its reverse in all forms of katabolism, of which by far the most important are the forms which supply from day to day and from hour to hour energy for those bodily functions which may be summed up under the broad name of “activity.” If we grant that the activity of any individual organism may be an index of the sum total of its bodily conditions, then the study of the variations of that activity, and of the conditions which lead to such variations, becomes of the utmost importance. With the commencement of such a study its difficulties begin. How is it possible to arrive at an adequate estimate of such activity? The present methods of Science can measure metabolism but exact chemical analyses would be impossible in a long series of experi- ments on normal animals. It becomes then clearly necessary to make many assumptions in devising a practicable method. In the research about to be described it has been assumed that the amount of muscular energy developed, in other words the amount of work * Acknowledgments: I wish to express my obligation to Dr. Warren P. Lombard for permission to reproduce Figure 2, to Dr. C. F. Hodge, of Clark University, and to Messrs. D. Appleton and Co., for permission to adapt Figure 7, and to Dr. Hodge, for direction and assistance throughout my work. I am deeply indebted, also, to Martin Green, Esq., of Green Hill, Worcester, Mass., whose generosity and interest in the experi- ments placed barometer records at my disposal at all times —and to Mr. Jonas G. Clark, the founder of Clark University, whose permanent provision for scientific investigation alone made the work possible. Coin C. STEWART. Vartations tn Daily Activity. Al done, by any animal day by day will be an approximate expression of its susceptibility to those variable conditions which may be chosen for experimentation. But here again new obstacles arise. No ani- mal, even under conditions which may seem to be unvaried, will spontaneously show a uniform degree of activity for any series of days. Small causes, apparently inappreciable and unmeasurable, will produce changes tending to create or to destroy that feeling of bodily well-being which accompanies the proper functioning of all component parts. In studying the effect of drugs, for example, this difficulty becomes so overwhelming that it is plain that these small causes must be investigated for themselves. Weather changes, long recognized as potent, must be studied. The effects of atmospheric pressure, atmospheric moisture, temperature, light, winds and elec- trical variations must be obtained by long series of observations before we can hope to arrive at a solution of our single equation with its almost indefinite number of unknown quantities. Of the meteorological factors just mentioned, only atmospheric pressure has been taken into consideration here. The methods of the research have nevertheless been applied for the purpose of studying the effects upon activity of variations in diet, and of the administration of alcohol, with the hope of obtaining results so broad and general as to be of value in showing at least the possibilities in this comparatively new field of operation. So much for the purpose and aims of the undertaking. A word must be said as to the selection of suitable animals for the experi- ments. It seemed best to choose rats and mice, because they fill as many as possible of the requirements. They are small, cheap, easily fed and cared for; and, best of all, when placed in revolving cages they spend most of their time, when not eating or sleeping, in running. With regard to the distribution of their working periods it may be noted that the records obtained from rats show that their activity is confined entirely to the hours of the night. Beginning at from six to eight o’clock in the evening— later in summer than in winter — they are uniformly, though not continuously, active during the next eight or ten hours. Contrasted with this is the activity of the squirrel, the records of which show greater intensity, but only for an hour or two night and morning. Still another type was that of two fox-squirrels, active throughout the whole of the day and sleeping only at night. Such differences indicate that an investigation of the 42 Colin C. Stewart. comparative distribution of activity and of the relation of its intensity to its duration promises interesting and valuable results. Com- mencing, as did Hodge and Aikins,? with the protozoa, it might be possible to develop much that would bear closely upon theories of animal rhythm, rest and sleep. The following section will give a detailed account of the apparatus used, and of the general methods of procedure. Description of Apparatus and Methods. — The apparatus consists primarily of two parts : the cages in which the animals to be experi- mented upon are placed, and the various mechanisms for recording their movements in these cages. Each of the cages (see Fig. 1) used throughout the experiments on rats is cylindrical, eighteen inches long by twenty in diameter, made of fine wire netting soldered to a FIGURE I. frame of stout steel wire. The cage revolves freely on a steel rod supported by a fixed wooden frame. At one end is a wide-hinged door; from the axle is hung~a light wooden nest-box, completely closed in but for a small round opening on one side; and to the end of the nest-box next the door is fixed a detachable tin feed-box of two compartments. At the opposite outer end of the cage is an eccentric which, with each revolution of the cage in either direction, pushes aside an upright lever attached to the wooden axle support, and in so doing pulls the wire connecting the cage lever with the recording apparatus. Cages used for mice are similar in construction, except that their small size (eight inches long by five in diameter) renders them so Variations in Daily Activity. 43 light that no wooden support is needed for the axle. The axle is held in position by a clamp upon an iron standard, while a second clamp holds a bearing for the lever which, with its wire, serves as a means of communicating the motions made by the cage. To record the revolutions of the cages a simple six-inch con- tinuous-roll kymograph, with uniform motive power, was first used. A standard carrying six light wooden levers, each five inches long, is placed before the kymograph. Each lever is tipped with thin whalebone, and to each is fixed a small glass ink-well and a pen of fine capillary glass tubing. Each is connected by a wire to the lever of the corresponding animal cage. As the cage revolves the eccentric pushes back the cage lever, the wire attached to it is pulled, the pen lever is drawn down, and the pen makes a vertical mark upon the slowly travelling scroll of paper. Upon the release of the cage lever a spring fastened to the pen draws the pen, the wires and the cage lever back to their original position, and the apparatus is ready to record another revolution of the cage. Single revolutions would be indicated by single vertical lines, but when the cage is made to revolve with sufficient rapidity a con- tinuous broad band marks the duration of a series of revolutions. _An electro-magnetic time-marker, connected with a battery and a clock making short connections every minute and longer ones every hour, gives an accompanying record by means of which the duration and distribution of such periods of activity may be computed. The kymograph method just described, though invaluable in the study of the distribution of activity throughout the day in different species of animals, and even in different animals of the same species, is nevertheless a rather unreliable one for close or accurate experi- mental measurement. After a rate of cage revolution has been reached which is sufficiently great to be recorded by a continuous broad band, no greater speed of revolution can be distinguished by any feature of the tracing. In other words, of two animals one might do twice as much work as the other in the same period of time without any indication of that fact being shown upon the record of their activity. A much more exact recording method is the following, which has been used in almost all the experiments to be described. The hair spring and balance wheel of a common spring clock are removed, and to the escapement are attached, on one side a soft spring of fine brass wire, and upon the other a wire which, passing through a slit- 44 Colin C. Stewart. like opening in the clock case, is in turn attached, as was the wire in the preceding method, to the cage lever. Each revolution of the cage, as before, pulls the wire, the escapement is drawn down, and one cog of the ratchet wheel is let go. In the clocks used two of these cogs correspond to one second on the diai—seven thousand two hundred, therefore, to an hour. The clock is set at twelve, and the exact number of revolutions performed in any given time by the cage to which it is attached may, at the end of that time, be read off on the dial. This method fails to give any representation of the distri- bution of activity, but its relative accuracy in recording the total amount of work done makes it perhaps the most useful in experi- ments where daily variations due to food and drug effects are being sought for. By estimating the circumference of the cage used, and multiplying by the observed number of revolutions in any given time, one may obtain a rough estimate of the distance travelled. For example, it was noted in the course of the various experiments to be de- scribed, that a common gray rat will run normally from five to fifteen miles in a single night, one particularly active rat travelling an aggregate distance of one hundred and forty-three miles in ten days. In a third piece of recording appa- ratus, not so well adapted for prolonged experiments, but possessing in a measure the advantages of the preceding two, in- stead of turning the hands on the dial, as in the second method, a similar clock is made to wind up a cord which runs on a bobbin fastened to the axle of the hour hand. The cord is attached to a vertically moving stylographic pen which records its position on a slowly travelling roll of paper. When the cage is not revolving the pen is at rest and traces a horizontal line; but when it is revolving, the pen slowly rises as the string is wound up. By using a roll of paper carried on a kymograph at the rate of six or eight inches a day, and recording also thermometer, barometer, hygrometer and AVERAGE KNEE-JERK 29.00 bax FIGURE 2, Knee-jerk, barometer and thermometer. (Lombard.) Variations in Daily Activity. AS time, a more or less complete picture of the data for the period studied may be obtained. The Effect upon Activity of Changes in Barometric Pressure, — Lombard? has described a correspondence between the variations of average knee-jerk for a series of days, and the condition of the weather. His results (see Fig. 2) show a direct relation between knee-jerk and barometric pressure, and a more indefinite inverse relation to temperature. No effect of changes in humidity, or in electric tension, was shown. Lombard‘ has also shown, in experi- ments upon himself, that a decrease in atmospheric pressure lessens the ability to do voluntary muscular work, while an increase in pres- sure increases muscular power. High temperature, especially when associated with much humidity, decreases this ability. CAGE —— Av.0F 6 RATS Rees we BAROMETER 00 —30 in BAR. 9 | MARCH “95 APRIL | MAY FIGURE 3. Curve of the average daily activity of six gray rats, during seventy days. The dotted line shows barometric pressure. An undoubted influence of barometric variations upon the activity of the nerve muscle complex has, therefore, already been demon- strated, so that we might naturally expect to find variations in the amount of spontaneous daily work correlated with changes in atmos- pheric pressure. Turning to the experiments in which the methods 46 Colin C. Stewart. already outlined were used, we have in Figure 3 a curve, plotted in terms of cage revolutions, for seventy days, of the average daily activity of six common gray rats, full grown when caught, and there- fore uninfluenced by domestication. The dotted line shows the variations in barometric pressure during the continuance of the ex- periment. Up to April twelfth, that is during the first half of the time, the rats were perfectly normal. During the latter half, however, four of them were getting alcohol in addition to their regular food. The consequent interference with normal activity is shown in the otherwise unaccountable rise during the latter part of April and the first few days of May. This will be referred to again more fully when the alcohol experiments are being described. _30in BAR 24 \ 20 DEC."94 JAN.*95 Ficure 4. Curve of the daily activity in minutes of a single gray rat during twenty-six days. Barometer, as before, in dotted line. Figure 4 is a curve of the variations in duration of activity shown by a single gray rat during twenty-six days. The curve of baro- metric pressure is superimposed in dotted line. In each of these curves there is shown an inverse relation between the amount of daily activity and the barometric pressure. Similar results were obtained from the records of a common red squirrel, though the squirrel’s activity showed at times extreme variations from day to day with no apparent cause. Figure 5 shows curves of the variations in daily activity of three groups of white rats for one hundred and twelve days. For the first twenty-six days the record is from five pairs of rats, one pair, male and female, to a cage; for the next twenty-six days six pairs are recorded, five of them being the five pairs of the preceding days; and for the rest of the time a new series of six pairs furnish the records. Where a point in the record has been enclosed in a Variations in Daily Activity. A7 small circle, imperfect data have been used to fill out the space. The barometric pressure is recorded, as before, in dotted line. —_—. AVERAGE OF 5 PRS —— AV. OF © PRS seseeee BAROMETER 19 i) MARCH "96. APRIL AV.OF © PRS ,NEW SERIES JUNE Juty : FiGuRE 5. Curve of average daily activity of white rats: March rgth to April 7th, average of five pairs; April 8th to May 3d, of six pairs; May 4th to the end, of a new series of six pairs. Barometer in dotted line. Whatever correspondence there is between the two curves would lead to the conclusion that the effect of changes in atmospheric pressure is a direct one, as against the inverse relation shown for gray rats. A point worthy of note, however, is that just as the pressure effect is shown more clearly in the curve of the first ex- periment where the animals are perfectly normal (see Fig. 3), so in this curve, from the beginning to the point marked April eighth, and from May fourth to June first, where the records are from nor- mal rats, a correspondence is more clearly shown. During the rest of the time the same animals were being used for alcohol experi- ments, with a consequent interference as yet unexplained. Many of the daily variations in the curve are unaccounted for, but the great rise in average activity shown during the early part of July is without doubt due to the disturbing noises incident to the cele- bration of Independence Day. 48 Colin C. Stewart. Figure 6 gives another curve of the average activity of six white rats, with barometer plotted in dotted line as before. Here again the correspondence between the curves is only of doubtful value. 150 30 in. BAR. Barz APR. FicureE 6. Average activity of six white rats for fifty-three days. Barometer in dotted line. Figure 7 shows, in solid and broken line, the curves of activity of two dogs, from data obtained by Hodge® by means of a pair of ingeniously contrived pedometer watches carried by the dogs in their collars. The curves are plotted from the readings of these watches taken once a day. To the chart I have added in dotted line the curve of barometric pressure for the forty-six days of the experiment, with the result of establishing a somewhat striking cor- respondence. The curve would be improved in legibility if the records for the two dogs were averaged instead of being plotted 150_29.5 in BAROM. 30 16 MAY/'96 : JUNE FIGURE 7. Curves of the activity of two dogs, plotted in solid and broken line. Ba- rometric variations shown in dotted line for the forty-six days of the experiment. separately, as they are in the figure; but it would not show, as it does now, the remarkable similarity between the two. Other meas- urable factors are undoubtedly at work in producing variations, — factors which can be determined only by manifold repetition of such experiments. — ee. Variations in Daily Activity. 49 The foregoing charted results show an inverse relation between the amount of voluntary daily activity of gray rats and atmospheric pressure, and a direct relation between variations in barometric pressure and the activity of the two dogs. The white rats experi- mented upon showed only a doubtful direct relation between the amount of their daily activity and pressure. The experiments seem to point to the possibility of a fundamental difference in this respect between domesticated animals, independent of weather changes, and wild animals with their greater need of individual effort for self-pres- ervation and their greater interest in food supply. The Effect of Changes in Diet.— The experiments under this head were all upon white rats normally fed on a uniform diet of dog-biscuit and water. In the first experiment (see Fig. 8) six pairs of white rats, one pair to a cage, were used. The solid line shows the average daily activity of three pairs, the broken line that of the other three. 28 | Gs 19 20 3 NOV.96 DEC. Ficure 8. Feeding experiment. Each line shows a curve of the daily average of three pairs of white rats. From Dec. 8th to 19th those of the dotted line get beef and dog-biscuit, the others corn. For the rest of the time all were fed dog-biscuit. Both groups were normal up to December seventh ; then those represented by the broken line were fed fat and lean raw beef and dog-biscuit for twelve days, during which time the others got Indian corn. From the twentieth of December to the end of the experiment, twelve days again, all were fed dog-biscuit as before. The decrease in voluntary activity with the heavier diet is shown in the curve. In the second experiment (see Fig. 9) all six pairs, the same rats as were used in the preceding experiment, were fed alike. For the first twelve days they were fed on dog-biscuit, with good average records as the result. During the next fifteen days (January first to fifteenth) white bread was given, with an accompanying increase in the amount of daily work. For the next ten days (January sixteenth to twenty-fifth) bread and fat and lean beef were given, causing a manifest decrease. Then for six days (January twenty-sixth to 4 50 Colin C. Stewart. thirty-first) bread alone, as before, was fed, with again a rise. This was followed by bread and beef for seven days (February first to seventh) with a fall in activity. From the eighth of February to the end of the experiment on the first of March bread alone was fed, 00 : /00 B 15 lo 24 3 \ DEE % JANI ae Fh ees \ 26 | Cys lo WW 18 19 24 25 23 1 aaa FEB. FiGurE 9. Second feeding experiment. Curve shows average activity of six pairs of white rats. December 2oth to 31st —dog-biscuit. January Ist to 15th—bread. Janu- ary 16th to 25th —beef and bread. January 26th to 31st —bread. February tst to 7th —beef and bread. February 8th to March 1st — bread. At points marked February 18th and roth the effect of lack of food is shown; at February 24th and 25th, the ’ effect of an escape of gas, and the same at March Ist. with a resulting rise in activity. The record for this last period re- mains uncomplicated, however, during only the first eight days. On the seventeenth of February the record for the preceding night was not noted, nor were the rats fed. The records for the sixteenth and seventeenth were therefore lost, while the records for the eighteenth and nineteenth show the effect of insufficient food. Recovery was complete by the twenty-third, when unfortunately gas escaped in the room in which the experiments were carried on, and several of the rats were noticeably poisoned by it — the effect upon their activity being shown by the records of the twenty-fourth and twenty-fifth. Re- Variations in Daily Activity. 51 covery again followed, when, on the twenty-eighth, gas escaped once more, with a similar result. The next figure shows in an- other way the result of this ex- metiment (see Fig: +10). The average for each of the periods mentioned has been taken from the perfect records of that period, while the average activity for the whole time is also shown by a horizontal broken line. For the third feeding experi- ment (see Fig. 11) six male rats were selected, and all were fed on dog-biscuit for the first ten days. FicurE to. Another rendering of Figure 9, Then from the thirteenth to the showing graphically the average activity Mrdeth of March the. three for each ce the periods of the second feed- Ing experiment. whose average activity is plotted by the broken line were fed beef, cheese, sugar, chocolate, and bread, while the other three, the solid line, got bread alone. From the thirty-first of March to the nineteenth of April the conditions were reversed, those previously well fed getting the meagre diet of bread. During the last five days the conditions were again reversed, with a 1213 03! 19 20 4 3 MAR '9T APR. FicurE 11. Third feeding experiment. Each line the average of the records of three male rats. All normal to March 12th. March 13th to 3oth, full diet for those of the broken line and bread for the others. March 31st to April roth, full diet for those of the solid line, and bread for the others. April 20 to 24th, the conditions again reversed. second crossing of the lines of activity as the result. The simpler diet in all cases gives a relatively greater degree of activity. The weights of the rats were taken at the commencement of the experiment, at each change, and at the end. The following table shows the increase in weight that accompanied the decrease in activity. 52 Colin C. Stewart. March 3d. March 3Ist. April 2oth. April 25th. Commencement. After rich diet. | After diet of bread.| After rich diet. 305 g. 329 g. 310 g. 312 335 322 235 249 238 Commencement. | After diet of bread. After rich diet. | After diet of bread. Si fee 307 g. 332. g. 300 281 286 231 256 Effect of Alcohol. — Hodge,’ in the published account of his ex- periments on the physiological effect of alcohol on dogs, records the observation that the two alcohol dogs, although never intoxicated, show much less activity than the two normal controls. The use ofa pair of pedometer watches developed the fact that, of the males, the alcoholic showed during forty-six days only seventy-one per cent of the activity of the other, while with the females the alcoholic showed a percentage of only fifty-seven. In the first series of my own experiments gray rats were used. Six rats of as nearly equal weights as could be found were placed in six similar cages and kept on a normal and entirely uniform diet for longer or shorter periods before the actual commencement of the ex- periment. Averages of their activity for such periods were taken on April twelfth, 1895, and the rats were arranged in pairs in such a way that the total average activity of any one pair was as nearly equal to that of either of the other two pairs as possible. It was decided to give weak alcohol to two, strong alcohol to two, and to keep the other two as normal controls. Alcohol was administered with their food in increasing strength, according to the following schedule: Normals. . Wieakon = in Strone. .2. Variations in Daily Activity. 53 So that one pair from April twenty-seventh to the end of the ex- periment in October of the same year drank nothing but twenty per cent alcohol, while another pair had a sixty per cent solution after May fourteenth. Figure 12 shows the voluntary activity of these six rats during the experiment. The solid line is the curve of the average of the two normal rats, the dotted line that of the weak alcohol rats, and the 300 ~...- STRONG ALCOHOL : 5% 10% 15% 20% 30% 40% 507. 60% TOTHE END a WEAK ALCOHOL: 5% 10% 15% 20% TO END OF EXP. —— NORMAL ——™, od . ‘A. oo ee _) oN, Zz may 3 a 6 22 27 12 6 10 4 9 8 Ol 415161718 A 26 " ! JUL AUG. SEPT. 23 2 1 SEPT. ocT FicurE 12. Alcohol experiment upon gray rats. Solid line plots the average of two normal rats; dotted line, of two getting weak alcohol; and broken line, of two getting strong alcohol. Water given June 8, Io, 11, 14, 15, 16, 17 and 18, and July 8, 9 and Io. broken line, that of the strong. One of the rats getting the strong solution died on the seventeenth of May, the second on the twenty- sixth of June. On the eighth, tenth, eleventh, fourteenth, fifteenth, sixteenth, seventeenth and eighteenth of June, and on the eighth, ninth and tenth of July, water was substituted for alcohol. The result was a decided rise in the activity of the strong alcohol rats and no definite effect upon the weak alcohol animals. The 54 Colin C. Stewart. general falling off in activity toward the end of the experiment is no doubt partly due to the lack of variety and general insufficiency in dict. Figure 13 gives another rendering of the same curve, with the normals plotted as a level line and the others shown as varying above and below that normal. The broken line is the curve of the strong _.-.- STRONG ALCOHOL: 5% ~ /0% 15% 20% 30% 40% 50% 60% TO THE END. 7100 “ue WEAK ALCOHOL: 5% /0% 15% 20%, TO END OF EXP. —— NORMAL 9 G Hae a SEPT. 7 . OCT. FiGuRE 13. Another chart of the same experiment. Activity of normal rats taken as base line; strong alcchol rats in broken line, and weak alcohol rats in dotted line. Water given June 8, 10, 11, 14, 15, 16, 17 and 18, and July 8, 9 and ro, alcohol rats, and the dotted line that of the weak. It shows perhaps better than Figure 12 the primary rise in the activity of the rats get- ting strong alcohol, with a subsequent fall as soon as forty per cent is given, and the rise again during the month of June when water was substituted. For the animals getting weak alcohol it shows a slower rise but no subsequent fall to the level of the normals for any length of time. Figure 14 is a chart showing the result of an experiment upon six pairs of white rats, one pair, male and female, to a cage. Three Variations in Daily Activity. om pairs are normal throughout, their average daily activity being plotted in solid line. The other three are normal for twenty-nine days, until June second, when five, ten, fifteen and twenty per cent alcohol are given on four successive days, the twenty per cent alcohol — ALCOHOL —— NORMAL aay - ‘. 5,/0,/5,20% ALCOHOL TO END oe 1 fons MAY “96 JUNE JULY FIGURE 14. Alcohol experiment on white rats. Each line shows the average of three pairs — all normal until June first. Those shown in dotted line are then fed alcohol; the others, the solid line, are control animals. being continued until the end of the experiment on July third. The curve shows no decrease as the result of administering alcohol in twenty per cent solution, but rather a relative increase. This curve, too, gives an example of increase through practice —a curve of getting used to the apparatus. Figure 15 shows, for another experiment, a decrease in activity ‘following immediately upon the administration of thirty per cent err eee RSS) ere eae ALCOHOL — NORMAL 30% TOEND OF EXP. 8 | APRIL “96 MAY FicureE 15. Alcohol experiment on white rats. Each curve shows the average of three pairs. The solid line shows the activity of the normal animals, the broken line that of the alcoholic animals. Alcohol given as indicated. Points enclosed in small circles are filled in from imperfect data. alcohol. As in the preceding experiment, six pairs of white rats, three for alcohol and three for controls, were used. Points enclosed in circles are filled in from imperfect data. 56 Colin C. Stewart. CONCLUSIONS. 1. While the experiments show, for gray rats and for a red squirrel, an influence of barometric pressure on voluntary, spontaneous activity that indicates an inverse relation between the two, the curves for dogs, and possibly also for white rats, show a direct effect of atmospheric pressure in increasing voluntary activity. The difference may be due to a constant difference between domesticated and wild animals. 2. Influences other than barometric pressure are undoubtedly at work to produce variations which must be considered as normal. 3. The effect of a rich diet upon white rats is to decrease voluntary activity, while that of a plain though apparently sufficient diet is to increase it. This increase, in one experiment, has been correlated with a slight loss in weight, while the decrease was accompanied by a corresponding gain. 4. The activity of rats is markedly decreased by the administration of alcohol in thirty to sixty per cent solutions, but no decrease has been experimentally demonstrated as a result of giving a twenty per cent solution of pure alcohol. BIBLIOGRAPHY. 1. C.S. Minor: Senescence and rejuvenation. Journ. Phys., 1891, xii, pp. 97- 153; also: On certain phenomena of growing old. Proc. Am. Ass. Adv. Sc., 1890, xxxix, pp. 271-289. . C. F. HopGe and H. A. Arkins: The daily life of a protozoon: a study in comparative psycho-physiology. Am. J. Psy., 1893, vi, 4, pp- 524-533. 3- WARREN P. LomBARD: The variations of normal knee-jerk, and their rela- tion to the activity of the central nervous system. Am. J. Psy., 1887, i, I, pp. 5-71. - 4. WARREN P. LoMBARD: Some of the influences which affect the power of voluntary muscular contractions. Journ. Phys., 1890, xiii, pp. 1-58. 5. C. F. HopGe: Experiments on the physiology of alcohol, made under the auspices of the committee of fifty. Pop. Sci. Mo., 1897, March and April. 6. J. S. LEmon: Psychic effects of the weather. /Am. J. Psy., 1893, vi, 2, pp. 277-279. oz 7. PAuL Berr: La pression barométrique. Paris, i878. N THE INFLUENCE OF HIGH ARTERIAL PRESSURES UPON THE BLOOD-FLOW THROUGH THE BRAIN. By We Ey HOW EIEN: [Professor of Physiology, Fohns Hopkins University.) HE conditions controlling the circulation of blood in the brain are peculiar, and offer an intricate physical problem the solu- tion of which has been attempted from an experimental as well as from a purely theoretical standpoint. The fact that the brain is contained within a rigid box that does not permit free expansion of the organ, has led some authors to assume that dilatation of its arteries, however produced, is not followed by an increased flow of blood, as is usual in other organs, but on the contrary, under certain conditions at least, by a lessened ow. Thus, Geigel! has held upon theoretical grounds that dilatation of the arteries of the brain is ac- companied by a compression of the capillaries, owing to the fact that the expansion of the arteries causes an increase in intracranial pres- _ sure that is transmitted to the capillaries. Upon this view, therefore, dilatation of the arteries, whether produced through vaso-dilator nerve fibres or by a rise in general arterial pressure, should be followed by a lessened flow of blood through the brain, and vice versa. Grashey? in his well-known treatise upon the hydro-statics and hydro-dynamics of the cerebral circulation has also admitted a simi- lar possibility. Grashey assumes that intracranial pressure depends upon two conditions, namely, the volume of the cerebro-spinal liquid and the amount of arterial pressure. Dilatation of the arteries must lead to an increased intracranial pressure, and this being transmitted through the brain substance acts upon the part of the circulation where the intravascular pressure is least, that is, the cerebral veins. Inasmuch as the pressure within the capillaries is greater than in the veins, an increase of intracranial pressure should affect the veins and not the capillaries, as Geigel assumed. Moreover, Grashey emphasizes a fact that other authors seem sometimes to overlook, namely, that the sinuses of the brain are probably entirely protected from any direct influence of intracranial pressure by their tense and inextensible covering of dura mater. From his theoretical standpoint Grashey concludes that an increase in arterial pressure causes a greater flow 58 W. H. Howell. of blood through the brain up to a certain limit only. So soon as intracranial pressure is raised by the expansion of the arteries to such an extent as to cause occlusion of the cerebral veins, the volume of blood circulating through the brain is diminished. There follows under these circumstances what Grashey calls a vibration of the veins. The occlusion of the veins by the intracranial pressure is overcome by the consequent rise of static pressure transmitted through the capillaries, and this in turn is followed by occlusion as the pressure in the patent veins falls, and so on. A similar view has been held by other authors who have treated the subject from a theoretical standpoint, and by some of those who have investigated the matter experimentally. The differences of opinion seem to be mainly as to the level of arterial pressure at which an obstruction to the blood-flow occurs, whether it falls within the range of normal variations of pressure or is reached only under extra- ordinary conditions. This admission appears to be of the nature of a theoretical concession to the recognized peculiarities of the cerebral circulation, and it is of interest to inquire how far it is supported by actual experiments. The simple and direct method of solving the problem is to measure the outflow of venous blood from the brain under different conditions of arterial pressure. This method has been employed by a number of authors with results which seem to be uniform and quite opposed to the conclusion obtained from a theoretical consideration alone. The experiments carried out by Gaertner and Wagner,? and since practically repeated by Bayliss and Hill,t Hill and Nabarro,? Reiner and Schnitzler® and others, have shown conclusively that the flow of blood through the brain is in- creased as the general blood pressure is raised. Even maximal blood pressures produced by the action of strychnine or absinthe give only an increased outflow from the cerebral veins with no indication of even a temporary slowing. While these experiments have shown quite conclusively that a rise of blood pressure, so far as it can be produced by the normal regulat- ing mechanisms of the circulation, fails to produce the condition of a diminished blood-flow as demanded by theory, it is held by some of the authors quoted that if the arterial pressure be still further in- creased, a point must be reached at which, owing to the compression of the cerebral veins, a permanent or temporary diminution of blood- flow will result. Thus Hill” says, “ It is, however, possible that a very sudden and abnormally high rise of arterial pressure should so expand fligh Arterial Pressures upon the Blood-flow. 59 the arteries at the base of the brain as to temporarily express capillary areas and produce anemia.” It has been the object of my experiments to determine whether or not this is true. For this purpose I have used the simplest possible method. Upon dogs I have connected the arteries of the brain with reservoirs of blood, or Ringer's solution isotonic with the blood, that could be placed at any desired height, while the outflow from the brain was caught in a large test tube suspended by a spiral spring so that its movement, as it filled with blood, could be registered upon a kymographion. The details of the experiments were as fol- lows: The animal was bled to death under ether. The internal carotids were dissected out and ligated. The vertebral arteries were exposed, and cannulas filled with defibrinated blood or isotonic salt solu- tions were introduced. ‘These cannulas were connected with the reser- voirs of carefully filtered calf’s blood or Ringer’s solution. In some cases the inflow cannulas were placed in the aorta or subclavians in- stead of in the vertebrals, and the flow directed into these latter arteries by ligating the other branches. To catch the outflow of blood from the brain, cannulas were placed directly in the superior cerebral veins at their emergence from the skull, or in some cases in the external jugular vein after ligating all communicating branches except the two superior cerebral veins. The internal jugular veins on the two sides were ligated close to the skull. An attempt was also made in some cases to shut off the outflow through the occipital sinuses into the spinal plexuses, but as this involved some danger of altering the conditions of pressure within the skull the attempt was abandoned. In the experiments as made, therefore, two paths of exit were opened to the blood flowing from the brain, — one, which was not measured, into the spinal plexuses, and one through the transverse sinuses and superior cerebral veins. The outflow from the latter was measured. This sufficed for the purposes of the experiment, inasmuch as in the dog this latter path is the one through which most of the blood escapes, and the object of the experiment was simply to determine the effect of very high arterial pressures upon the rate of outflow from the sinuses. The outflow cannulas from the two sides were united to a single short tube by means of a Y piece, and this tube opened into the test tube men- tioned above. This test tube was provided below with an opening through which it could be emptied rapidly when desired. The tube was swung by a spiral of German silver wire after the manner sug- 60 W. H. Howell. gested by Bowditch for plethysmographic records, the spiral being so adjusted that the level of the liquid in the test tube remained constant. The movements of the tube as it filled with blood were recorded upon a kymographion upon which also a time record in seconds was taken simultaneously. As the movement downward of the test tube was constant and could be calibrated beforehand, data were obtained for calculating exactly the volume and the velocity of the outflow from the brain. In performing an experiment two reservoirs were connected with the inflow cannulas; one of these was placed at a sub-normal level of from 30 to 60 mm. of mercury, while the other was at a height sufficient to cause a pressure of from 300 to 500 mm. Hg. The pinchcocks connected with the lower reservoir were first opened and the rate of flow was determined at this level. This pressure was then changed suddenly to the higher level by opening the pinchcock connected with the upper reservoir and closing the one on the tubing from the lower reservoir. The pressure was then brought back to the original amount by again making connections with the lower reservoir. The experiment was usually repeated a number of times. So long as the pressure was kept low, the rate of out-flow remained constant or nearly constant for some time, but exposure to the very high pressures brought about quickly an cedematous condition of the brain which diminished markedly the total outflow or might even suppress it entirely when the arterial pressure was subsequently lowered. The direct results of the sudden change from sub-normal to supra- normal blood pressures were, however, the same in all cases; the venous outflow was increased at once to a proportional amount, and there was never any indication on the record of even a temporary blocking of the flow through the brain. The circulation through the brain under these conditions behaves in fact precisely as it does in the other organs of the body that are not enclosed in rigid cases. The nature of the results obtained are indicated by the accom- panying figures (Figs. 1 and 2) which are reproduced from the curves of two of the experiments. Examples of the actual amounts of outflow as calculated from the records are as follows : — Exp. 1. Small dog — bled to death — blood defibrinated (140 c.c.) and mixed with equal volumes of normal saline and Ringer’s solution to make 4 litres. Inflow cannulas placed in the vertebrals, outflow cannulas in the superior cerebral veins just at their emergence from the skull. fligh Arterial Pressures upon the Blood-flow. 61 Arterial pressure of 30 mm. mercury=outflow of 7.02 c.c. per min. (43 7 13 60 “ “6 — 6 (7 18.03 6c “6 (73 “cc a6 oe 380 6c “ec — “c “e 102.66 “c “cc 6c “cc 6 6. 60 “cc 6 — 66 66 10.24 66 66 66 2d series of observations on the same animal. Arterial pressure of 60 mm. mercury= outflow of 9.65 c.c. per min. 73 73 6c 400 6 “ec — be sc 85.14 (79 (73 “ — (73 1 5.85 6“ (73 6c Exp. 2. Small dog —bled to death from carotid — blood defibrinated (130 c.c.) and mixed with equal volumes of Ringer’s solution and normal saline to make 5 litres. Inflow cannulas placed in the vertebrals, outflow cannulas in the external jugulars after ligating all branches except the superior cerebrals. Arterial pressure of 30 mm. mercury= outflow of 5.26 c.c. per min. “ s Te YES ah if —— dec Am oe 30 be oe — o6 ‘ee 222 “ “ce ve 2d series of observations on the same animal. Arterial pressure of 50 mm. mercury=outflow of 2.57 c.c. per min. oe e 6 375 oe ve oo be be 70. 2 oe 6“ oe 66 6c be 50 6“ “ec — 6 od ili’ e 6c 6c Exp. 3. Large dog — bled to death from the carotids. For irrigating used freshly defibrinated blood of young calves filtered first through a single layer of muslin and then through four layers of the same. Inflow cannulas in the vertebrals, outflow cannulas in the superior cerebral veins at their emergence from the skull. Mercury manometers were also connected with the torcular and with the sub-dural space at the parietal eminence through trephine holes in the skull. Arterial pressure of 60 mm. mercury outflow of 23.4 c.c. per min. A second determination 5 min. later= “ Og are ontaee aoe of Arterial pressute of 335:nim. mercury ,“ CEN 22 rOOs need tee ice Rise of pressure in torcular cannula= 36 mm. of mercury. a ee ue “cannula in sub-dural space = 30 mm. mercury. Arterial pressure of 60 mm. mercury =outflow of 14.66 c.c. per min. 2d series of observations on the same animal — the flow meanwhile had dimin- ished greatly, owing to leakage. Arterial pressure 60 mm. mercury outflow of 1.74 c.c. per min. i ie v.(6fo) nus Et eee SE nDIG 2agiay) “shy eeascee, He Pressure in torcular increased 52 mm. mercury. 5 ‘“‘ sub-dural space increased 20 mm. mercury. The want of corre- spondence between the intracerebral and torcular pressures was evidently due to the brain being forced into the trephine hole in the case of the former, thus block- ing off the manometer. Exp. 4. Small dog — bled to death from carotid. Irrigation liquid was blood of young calves carefully filtered. This blood had been kept over night and had frozen. This fact probably accounts for the unusually rapid diminution in flow as the experiment proceeded, the red corpuscles not passing readily through the capillaries and clogging them. Inflow cannulas in the subclavians, ligatures being so placed as to leave an open path only to the vertebrals ; outflow cannulas in the superior cerebral veins at their emergence from the skull. W. H. Howell. Ov iS) Arterial pressure 60 mm. mercury = outflow not measurable with accuracy. igo”, “* Pie 0) 28.08 c.c. per min. ec 6s 320 “ce ce == be 6c 47.38 ot 6s 74 2d series on the same animal. Arterial pressure of 60 mm. mercury = outflow not measurable with accuracy. ck “ aA CO mms ee Pore A2.120¢.ce per min. wn (@) e) II I 50.02 oe oo oe Fic. 1. Record of venous outflow from brain under arterial pressures of 60 mm., 380 mm., and 60 mm. Under 60 mm. Hg. the outflow= 18.13 c.c. per min. “c 380 ““ “ “ “ — 102.66 “ “c “ Returmeton Gms span orem of =< 10.24 ee The time record at the top of the illustration is in seconds. It is evident from the data given that in all the experiments made, the blood-flow through the brain diminished as the experiment pro- ceeded, and this effect was most marked after the blood vessels had been submitted to very high pressures. The probable explanation of his fact is that the dead capillary walls permitted a rapid filtration f liquid, which rendered the brain cedematous. This condition, in- Fligh Arterial Pressures upon the Llood-filow. 63 deed, was apparent to the eye when the brain was exposed afte: submission to the high intravascular pressures. This variation from normal conditions does not, however, affect the value of the eXperi- ments so far as the main point under investigation is concerned. The Fic. 2. Record of venous outflow from brain under arterial pressures of 60 mm. and 460mm. (2d experiment.) Under 60 mm. Hg. the outflow= 1.74 C.C. per min. “ 460 ‘“ ; “ “ “ The irregularity in the beginning of the curve showing the outflow under 460 mm. arterial pressure, is owing to a stoppage of the drum, as will be seen by consulting the ‘6 ‘ ‘“ — Ss time-record above it. 64 W. H. Howell. brain was still in the unopened cranium, and the physical conditions, which have been supposed to cause a compression of the veins and a temporary or permanent slowing of the blood-flow when the arterial pressure is suddenly raised to supra-normal levels, still prevailed. Indeed, the cedematous condition of the brain should have exag- gerated this effect instead of counteracting it. Nevertheless, the records show that in all cases where the arterial pressure was suddenly raised to as much as 400 or 500 mm. of mercury, the outflow of blood from the cerebral veins increased promptly, and there was no indication at all of even a temporary blocking of the flow. When we consider this fact, together with the results obtained by several authors upon the outflow in living animals in which the arterial pressure was raised by action upon the vaso-motor mechanisms, it seems justifiable to conclude that the blood-flow through the brain is always increased by a rise of arterial pressure, no matter how great or bow sudden this rise may be, and that a compression of the veins sufficient to block or temporarily impede the blood-flow as a direct result of a sudden rise in pressure in the cerebral arteries is physically impossible. The author can indorse the conclusion drawn by Reiner and Schnitzler from their experiments that a rise of pressure in the cerebro-spinal liquid due to increase of arterial pressure cannot exceed the simultaneous intravenous pressure. Obviously the authors who have arrived at an opposite con- clusion have erred somewhere in the theoretical premises upon which their argument was based. A satisfactory treatment of all the physical factors involved in the statics and dynamics of the blood- flow through the brain is most difficult, and, perhaps, impossible in the present condition of our knowledge; but it seems to the author that in the theoretical considerations of the subject met with in physiological literature, some factors, which explain in large meas- ure the contradiction between the experimental and_ theoretical conclusions, have been more or less overlooked. In the first place the view distinctly announced by some authors and tacitly assumed by others that arterial expansion causes a compression of the large venous sinuses of the brain seems to the author to be entirely inadmissible. Grashey calls attention to the anatomical facts that make this view improbable. The venous sinuses are covered by layers of the inextensible dura mater tightly stretched in some cases across bony channels. Any one who examines the Fligh ‘Arterial Pressures upon the Bloodflow. 65 condition of these membranous walls in a fresh skull will be impressed with the opinion stated by Grashey, that the resistance to compression at these places must be so great, as compared with the cerebral veins that open into the sinuses, that any local or general increase of intra- cranial pressure must affect only the smaller veins. It seems to me, in fact, that the walls of the sinuses are practically incompressible, and that their existing structure is a beneficent adaptation to prevent any interference with the venous outflow arising from arterial expansion, since the venous system is thereby protected from compression at the point where its total cross area would be least, and intravascular pressure at its lowest, and where compression might most seriously affect the venous flow. I have attempted to demonstrate experimentally the practical incompressibility of the large sinuses, but the method that I have used and which was the only one that seemed to be conclusive, developed so many technical difficulties that I have attained so far only partial success. The method was simple in idea, but somewhat difficult of execution. It consisted in first removing the brain entirely by washing out the tissue through the foramen magnum and a trephine hole in the parietal bone. WINTERSTEIN : Berichte der deutsch. botan. Gesellsch., xi, p. 441; also Zeitschr. fir physiol. Chemie, 1894, xix, p. 521; 1895, xxi, p. 134; GILSON: La cellule, xi, ter fascicule. 8 Cf. MORNER, C. Th.: Zeitschr. fiir physiol. Chemie, 1886, x, 506. 228 The average weight of a fresh speci- men was thus : Pileus 27 grams Stem . VE Motalsweishtes yo 415 es A specimen which had _ attained the average growth weighed : Pileus 43 grams Steny hectare Meron ars Dotalsweight 2) 68) An analysis yielded the following results : Water Total solids 92.19 per cent. 7.51 =e The dry substance contained : Total nitrogen 5.79 per cent. Extractive nitrogen . 3.87 S Protein nitrogen . 1.92 a Ether extract . 3.3 os Crude fibre. Up: < Ash by Boe i eae! PRCA i; Material soluble in 85 per cent alcohol 56.3 S Coprinus atramentarius (Inky coprinus). Two separate, freshly gathered lots of this species were examined. The one (@) contained six young small specimens weighing 5.5 grams, or 0.9 gram each; the other (2) contained eight mushrooms weighing 12 grams, or 1.5 grams each. An analysis gave: a. Bee Percent. Per cent. Water 92.31 OF AZ Total solids 7.69 5.58 The dry substance contained : Total nitrogen 4.68 ef if DKeeqewies m a 2 4 Soil bo// @rude brew ame ae nO Borate Ash 16.8 20.1 Morchella esculenta (Common morel). Two lots of this species were obtained from Stockbridge, 1 PIZzs: L. B. Mendel. Mass. (@) The specimens were of full size. Thirteen morels weighed 195 grams, or an average of 15 grams each. (4) Small, young morels. An analysis gave : a. b. Percent. Percent. Water 89.54 = G24 Total solids 10.46 8.76 The dry substance contained: Total nitrogen 4 66 5.36 Extractive nitrogen 1.17 Protein nitrogen . eee Sree, Re. Ether extract.) -o 3. "-t eee ie Crude fibre’... eS eieed 9.5 Ash Perera fe LO 13.6 Material soluble in 85 per cent alcohol 29:3 In the same species Pizzi* has found 0.575 per cent nitrogen, a figure in close agreement with the above re- sults when calculated upon the fresh material, viz. (2) 0.48 per cent N; (2) 0.47 per cent N. Polyporus polyporus). obtained from Pennsylvania. analysis gave: sulphureus (Sulphury The specimens were An Water Total solids 70.80 per cent. 29.20. The dry substance contained : Total nitrogen 3.29 per cent. Extractive nitrogen . 1.06 > Protein nitrogen . 2.23 ss Ether extract <.. i: =. sence & Crude fibre . 3.0 Ash = 35) 0, a ee - Material soluble in 85 per cent alcohol oh ZS Pleurotus ostreatus (Oyster mush- room). This mushroom is obtain- able in large quantities, and though somewhat tough in texture, is univer- sally classed with the edible species. 3otanischer Jahresbericht, 1889, p. 316. Composition and Nutritive Value of Edible Fungi. Specimens gathered from a tree in New Haven contained : Water 73.70 per cent. Total solids “ 26.30 The dry substance contained : Total nitrogen 2.40 y Extractive nitrogen . 2a Protein nitrogen. . . . 1.13 < BEPeImextTACL en) «15 1.0 We (CIAWG evil) CG a mee oan: oe Ash PUR he clare cea OSE Material soluble in 85 per cent alcohol Bileo Y Clitocybe multiceps. Peck. ‘The material was collected near Boston, in June, 1897. A portion of small, young specimens was analyzed sepa- rately. The results follow: Young Fullegrown Specimens. Specimens. Per cent. Per cent. Widter 89.61 93.49 Total solids 10.39 6.51 The dry substance of the full-grown speci- mens contained: Total nitrogen 5.36 per cent. Extractive nitrogen . 3.38 Y Protein nitrogen . 1.98 es ihemextrach, 1. «,a. ~ 6:0 s Grudesibte: <%=.% 6 «916 S Ash Fie Ree Sane ena bl kes ch Material soluble in 85 per cent alcohol 51-2 75 A portion of the mushrooms was separated into stems and caps and each analyzed separately, with the following results : Stem. FPileus. Percent. Percent. Water 94.07 92.68 Total solids 5.93 eae, Total nitrogen in dry sub- stance Sank 3.92 5.84 Ash in dry substance . 12.98 10.82 229 The relatively higher content of nitrogen in the pileus corresponds with the distribution of proteid as shown by histochemical examination. In Agaricus campestris, Boletus edulis, and Boletus scaber, C. Th. Morner has found similar differences between the nitrogen content of caps and stems. flypholoma candolleanum. The specimens were obtained from East Milton, Mass., in June, 1897. A few small, young specimens were also ob- tained from Brookline, Mass. Analy- ses follow : Full-grown Younger Specimens. Specimens, Per cent. Per cent. Water 88.97 91.97 Total solids 11.03 8.03 The dry substance contained: Total nitrogen 4.28 4.44 Extractive nitrogen. 179 Protein nitrogen . ZAD tiene xtracte.w yayen 1 0 2e5 Crude fibre . 12.1 Phares Ash oe ae APO 19.9 Material soluble in 85 per cent alcohol . 44.4 Agaricus campestris (Common mushroom). Two varieties of the common mushroom were collected in New Haven. Fifteen specimens of one variety weighed 42 grams, an average weight of 2.8 grams each. The analysis gave: a. b. Percent. Percent. Water . 87.88 92.20 Total Solids IW 2 7.80 Total nitrogen in dry sub- stance aie 4.42 4.92, Ash in dry substance 11.66 17.18 1 MOrNER, C. Th.: Zeitschr. fiir physiol. Chemie, 1886, x, p. 510. * The specimens corresponded with those described under this name by Stevenson in his work on British Hymenomycetes. Mr. Hollis Webster has informed the writer that Professor Farlow is inclined to regard them as H. apendiculatum. 230 L. B. Mendel. Regarding the differences in nitro- Total nitrogen of dry sub- gen conten ap and stem, com- Se ae BERS co uient (ot a ‘ aay Ash of dry substance pare the remarks under Clitocybe multiceps. mn iy § 23 ~ Cortinarius collinitus (Smeared Marasmius oreades (Fairy-ring cortinarius). Young ee gath- mushroom). Twenty freshly gath- ered in New Haven early in Novem- ered specimens (from New Haven) ber, 1897. ‘The analysis gave : weighed 9 grams, an average weight of Water ... . . . . 91.13 percent. 0.45 gramseach. The analysis gave: Totalsoids ..... 887 % Water .. : . . .-. 44.96 percent. Total nitrogen of dry sub- TMotalySolids ya. spas neewes eo: Onn wv stance | 275 iit. 0k ea ae OROS e Digestion Experiments.—In order to procure further data regarding the nutrient value of the mushrooms, artificial digestion experiments were carried out with seven species of the fungi. The procedure was modified after the Stutzer method. About 2.5 grams dry sub- stance were treated in a flask with 100 c.c. of an artificial gastric juice, containing 0.1 gram very active scale pepsin and having an acidity of 0.35 per cent HCl. The flasks were frequently shaken, and after remaining in a thermostat at 38° C. for twelve hours, the undissolved residue was filtered off, washed free from acid, and again treated in the flask for several hours at 38° C. with 100 c.c. amylolytically active fresh chloroform-water extract of dog’s pancreas, a little chloroform being added to prevent putrefaction or fermentation. Sodium car- bonate (0.25 gram) was then added, followed by 25 c.c. of a pro- teolytically active thymolized extract of dry pancreas powder (Kiihne’s method.') At the end of seven hours the residue was again filtered on a weighed filter, washed thoroughly with hot water, and dried at 105° C. to constant weight. Undigested residue was thus determined, and the nitrogen content ascertained by the Kjeldahl method and expressed as nitrogen in residue. The results expressed in per- centages of dry substance are tabulated below. Discussion of the Analytical Data. N7trogcen and Protein. From the results obtained it is evident that the nitrogen (and proteid) content of the mushrooms (or at least those species examined) is considerably smaller than is ordinarily stated. Thus Pavy, quoting from Payen’s analyses, announces that in the dried state ‘‘ mushrooms contain 52 per cent, morels 44 per cent, white truffles 36 per cent, black truffles * See CHITTENDEN and CumMINS: Studies from the laboratory of physiologi- cal chemistry, Yale University, i, p. 109. Composition and Nutritive Value of Edible Fungi. 231 | Nitrogen Total Total : in nitrogen | nitrogen residue. | residue. solubie. insoluble. Dissolved | Undigested SPECIES DIGESTED. | substance. Per cent. Per cent. Percent. | a Per cent. Per cent. Coprinuscomatus . . .| 73.79 26.21 4.21 5) 4.69 i: atramentarius .| 71.84 28.16 2.79 3.90 Clitocybe multiceps . .; 62.43 37.57 1.96 Hypholoma candolleanum 31.98 3.63 Morchella esculenta . . bs 49.42 4.16 Pleurotus ostreatus . . RE 59.43 1.39 Polyporus sulphureus. .| 45. 55.00 31 per cent, nitrogenous matter.” ! determined not only the total nitrogen, but also the extractive (non- proteid) nitrogen as well as the nitrogen in the residue insoluble In a number of species we have after artificial gastric and pancreatic digestion. The “ protein” nitrogen multiplied — after deduction of the nitrogen in the undi- gested residue — by the factor 6.25 will give an approximation to the amount of proteid material available through the digestive processes going on in the alimentary canal, and thus throw some light on the true nutritive value of the mushrooms. It is here assumed that the nitrogenous bodies soluble in alcohol are likewise soluble in the di- gestive fluids; as to the possible presence of alcohol soluble proteids like zein, gliadin, etc., definite information is wanting at present. The first table following gives a summary of the nitrogen content of various species; in the second table the amount of available proteid has been calculated in the manner referred to. In considering the relatively high nitrogen content of the residue resisting digestion, it is to be noted that this is not necessarily derived from unattacked proteids. Winterstein? and others have shown that the ‘‘ cellulose” preparations obtained by the usual methods from various fungi contain a considerable percentage of nitrogen; thus a preparation from Boletus edulis contained 5.5 per cent N, and this sub- stance, like similar preparations from Agaricus campestris, Morchella esculenta and other forms, yields glycosamin, C,H,,0;.NH.,, on de- 1 Pavy: Food and dietetics, 1881, p. 187. * WINTERSTEIN : Zoc. cit. ; also, Berichte der deutschen chemischen Gesellschaft, 1OOA, XXVil, Pp. 31133 XXvili, p. 167. 280 composition with HCl. L. B. Mendel. It is thus allied to the chitin found in the animal kingdom; further investigation in this direction is highly desirable. The percentages are calculated on the dry substance. Total nitrogen. Per cent. | Extractive “Crude pro- tein” nitrogen. Per cent. nitrogen. Per cent. Coprinus comatus Pleurotus ostreatus . Morchella esculenta Hypholoma candolleanum Clitocybe multiceps Polyporus sulphureus . Agaricus campestris—a . He & }. Coprinus atramentarius — a a b Morchella esculenta (young) Marasmius oreades . Cortinarius collinitus . Hypholoma candolleanum (young) Nitrogen in-| Nitrogen in The percentages are calculated on the dry substance. Per cent. 3.49 Morchella esculenta Hypholoma candolleanum 2.49 Coprinus comatus Clitocybe multiceps Polvporus sulphureus . Pleurotus ostreatus . soluble in 85% alcohol. residue from digestion. Per cent. 2.05 1.16 3.87 V2a Mel) 1.92 Tals 3.49 PhS) 1.98 2.23 Nitrogen of roteid dissolved in digestion. Digestible proteid (N X 6.25). Per cent. 9.00 8.31 5.12 7.81 10.31 1.94 Wictat Composition and Nutritive Value of Edible Fungi. 233 It is of interest in this connection to compare the results obtained by C. Th. Morner! in an investigation of thirteen species of fungi common in Sweden. Nitrogenous constituents alone were considered, total N and extractive N, as well as digestible and indigestible N being determined by methods analogous to those used in the present research. Morner’s results, summarized in the following table, show a close agreement, in general, with those already given for different American species. The results are expressed as per- centage of dry substance. N soluble in pancreatic N soluble in gastric juice. Digestible Protein-N. Indigestible Protein-N. Protein-N. Extractive-N. Agaricus procerus (cap) . Ww NS an © SNS > > Gomi) Oo - Agaricus campestris (cap) g af “ (stem) . Lactarius deliciosus rs torminosus . Cantharellus cibarius . Boletus edulis (cap) << (Stem) . scaber (cap) fae (Sten). luteus Polyporus ovinus Hydnum imbricatum . os repandum Sparassis crispa . Morchella esculenta Lycoperdon Bovista Ether Extract. The amount of ether extract varied from 1.6 to 7-5 per cent in different species, as shown in the following summary of results. 1 MOrRNER, C. Th.: Zeitschr. fiir physiol. Chemie, 1886, x, p. 503. to Go aS L. B. Mendel. ETHER: EXTRACT. SPECIES. Morchella esculenta (young) Clitocybe multiceps . Morchella esculenta . Coprinus comatus Polyporus sulphureus Coprinus atramentarius Hypholoma candolleanum Pleurotus ostreatus Percentage calcu- lated on dry substance. Cholesterin. Present. Gérard ' examined the extract from Lactarius vellereus and L. pipe- ratus, and found oleic and stearic acids present both as glycerides and as free acids. Volatile fatty acids were also obtained, together with chole- sterin or a closely related body (ergosterin), and evidences of lecithin. In the present research both fats and free fatty acids were found, and cholesterin reactions were obtained in every instance, the quantitative relations apparently varying considerably in the different species. Alcohol Extract.— The following summary shows the amount of material soluble in warm 85 per cent alcohol in a number of species. ALCOHOL EX TRACK The percentages are calculated on the dry substance. Percentage of sol- uble material. Percentage of ni- trogen dissolved, Clitocybe multiceps . Coprinus comatus Hypholoma candolleanum Pleurotus ostreatus . Morchella esculenta Polyporus sulphureus 1 GERARD: Journal de pharmacie et de chimie, 1890, 5 Série, xxi, p. 408; zbzd. ISgl, xxill, p. 7. References to the earlier literature will be found in the first of these papers. Composition and Nutritive Value of Edible Fungi. 235 Inorganic constituents. — The amount of ash varied somewhat, as shown in the table below. Among the bases present, K, Na, and sometimes Ca are to be found, the K being quite abundant. Iron was always present. Of acids, phosphoric and sulphuric predomi- nated, chlorine being occasionally found. ASH. The percentages are calculated on the dry substance. Per cent. Coprinus atramentarius — @ “ce “ -—6 (young) . “ee comatus Hypholoma candolleanum—a . “ce “ce —6 (young) . Morchella esculenta — a “ “ce — 6 (young) Agaricus campestris — a ‘ 7 ‘ “ec Oo Clitocybe multiceps . “ee (stems) . 4 (pileus) . Polyporus sulphureus Marasmius oreades . Pleurotus ostreatus . Crude Fibre.— Under this name is included the residue resistant to boiling acids and alkalis, and scarcely to be considered as homogeneous in nature. The results of the analyses are tabulated below. It has already been pointed out that the cellulose of the fungi con- tains nitrogen in many instances, and Winterstein! has shown that the latter is not due to proteids or nucleins mechanically included ; 1 WINTERSTEIN: Berichte d. deutsch. chem. Gesellsch., xxviii, p. 167: Zeitschr. fiir physiol. Chemie, 1894, xxix, p. 521. 236 L. B. Mendel. the nitrogen probably belongs to the “ cellulose ” itself. All attempts to separate the nitrogenous constituent from the portion which yields sugar on hydrolysis have failed. CRUDE FIBRE. The percentages are calculated on the Per cent. dry substance. ae Hypholoma candolleanum Clitocybe multiceps . Coprinus atramentarius Morchella esculenta (young) “ “ Pleurotus ostreatus . Coprinus comatus Polyporus sulphureus Soluble Carbohydrates.—A considerable portion of the solids of the mushrooms is made up of soluble carbohydrates, while starch is ordinarily not found. Trehalose, a carbohydrate of the formula C,.H,.O1,, and resembling maltose in some respects, has been isolated from a number of species; and in an extensive series of investiga- tions Bourquelot? has described a number of carbohydrates including mannite. In order to get some idea of the amount of soluble carbohydrates present a number of experiments were carried out in the manner described under the methods of analysis. Since trehalose, for ex- ample, cannot be quantitatively converted into dextrose by hydrolysis with acids,* the results of analysis must be somewhat low. Never- theless the data may be of comparative interest as indicating a high content of soluble carbohydrate. ‘ WINTERSTEIN : Zeitschr. fiir physiol. Chemie, 1894, xix, p. 70. The refer- ences to earlier literature are given. * These investigations were published in a series of papers in the Comptes rendus and other scientific journals. * WINTERSTEIN : 1894, Joc. cit., xix, p. 77. Composition and Nutritive Value of Edible Fungt. 237 DEXTROSE FROM HYDROLYSIS OF WATER-SOLUBLE CARBOHYDRATES. The percentages are calculated on the dry substance. Per cent. Pleurotus ostreatus . Coprinus comatus Morchella esculenta . Polyporus sulphureus To what extent these soluble carbohydrates are available for absorp- tion in their natural form or after digestion it is impossible at present to say. Such qualitative tests as were made showed them to be transformed to reducing sugars rather slowly by the action of saliva. The large undigested residues (26-59 per cent) found in artificial digestions likewise suggest that they are not completely transformed in the alimentary canal. Reference may here be made to the observa- tions of Stone! in feeding experiments on animals. He found that the pentosans, which are so widely distributed in vegetable foods, are to a marked degree less digestible than the carbohydrates, with which they have usually been indiscriminately classed in analyses. After the presentation of the preceding analytical data it will scarcely be necessary to draw any elaborate comparison between the fungi and other well-known vegetable substances considered as food- stuffs. It may be well to emphasize the deficiencies of the methods commonly followed in estimating the proteid content of vegetable foods, and to call attention to the erroneous inferences which are con- sequently drawn regarding the nutrient value of these products. Thus it is not unusual in the construction of dietetic tables to multiply the weight of nitrogen obtained by 6.25 and to express the result as “crude proteids.”* But even where the precaution has been taken to remove non-proteid nitrogenous bodies by extraction with alcohol, the application of the “ proteid factor” (6.25) to the N. of the residue may be quite misleading; for our results have demonstrated that the amount of unavailable nitrogenous material — largely, if not entirely, 1 STONE: American chemical journal, 1894, xiv, p. 13. 2 Cf. WILEY: Agricultural analysis, 1897, iii, p. 543. 238 L. B. Mendel. non-proteid in nature —is frequently equivalent to over half of the non-extractive nitrogen present (cf. Table II, p. 232). When it is remembered that the various species of mushrooms examined contain from 75 to 90 per cent of water, the amount of proteid in them appears strikingly small even when calculated on the total nitrogen in the fungi. For example, Morchella esculenta, a species of average com- position as regards total solids (10.5 per cent) and nitrogenous con- stituents (0.48 per cent N) could contain as a possible maximum only three per cent of proteid, corresponding in this respect with pota- toes, peas, green corn, etc.;” the vegetarian would thus be obliged to consume several kilos of the fresh morel to obtain the daily requisite of 100 grams of proteid. The expression ‘“‘ vegetable beef- steak” accordingly seems scarcely appropriate when applied to mushrooms in a strictly chemical sense. Moreover, the comparative poverty of many species in proteids is corroborated by the results of other investigations now in progress in this laboratory, the yield of isolated substance being quite small. The fungi thus form no excep- tion to the ordinary classes of fresh vegetable foods; indeed, they take a decidedly inferior rank in comparison with many. The carbohydrate content of the fungi is relatively high; but until more is known regarding the nature and digestibility of the carbohy- drate constituents of various vegetable foods, it will be useless to draw comparisons. As dietetic accessories the edible fungi may play an important part; but investigation has demonstrated that they can- not be ranked with the essential foods. Cf. MOrNER, C. Th. : Zeitschr. fiir physiol. Chemie, 1886, x, p. 515. Cf. ATWATER, W. O.: Foods: nutritive value and cost, doc. cét., p. 27. t 2 THE RESTORATION OF COORDINATED, VOLITIONAL MOVEMENT “APTER NERVE “CROSSING.” By R. H. CUNNINGHAM, M.D. [ Demonstrator of Physiology, College of Physicians and Surgeons, Columbia University, New VYork.]| CURSORY glance at a tabulated list of the voluminous literature! treating of the union of nerves after division will indicate that many eminent experimental investigators have zealously re-studied and discussed this subject ever since the historical experi- mental results of Cruickshank? were announced. An analysis of this voluminous physiological and surgical literature fully substantiates the present consensus of opinion that if the cut ends of the central and peripheral portions of a recently divided mixed nerve be brought into apposition, complete restoration of function may ultimately occur in the peripheral portion. Further, the results of a number of physiologists (Flourens,® Bidder,* Schiff,® Philippeau and Vulpian,® Reichert,’ Howell and Huber,’ and others) indicate that if the central end of one divided mixed nerve be sutured to the peripheral end of another mixed nerve, union of the two ends may ultimately occur, and the function of the nerve fibres of the peripheral portion be re-established. That is to say, the nerve fibres composing the peripheral portion regain their function of conductivity as well as the property of irritability. degenerated muscle fibres 1 For the full bibliography of this subject to 1892 the reader is referred to the paper of Howell and Huber, Journal of physiology, 1892, xiii, p. 335. 2 CRUICKSHANK: Philosophical transactions, 1795, xvii. See also FONTANA’S description (Sur le venin de la vipére, etc., Florence, 1781, p. 177) of Cruickshank’s experiments. 8 FLOURENS: Recherches expérimentales sur les propriétés et les fonctions du systéme nerveux, 1824, p. 272. * BrpDER: Archiv fiir Anat., Physiol., und wissensch. Medicin, 1842, p. 102; Archiv fiir Anat. u. Physiol., 1865, p. 246. ® SCHIFF: Journal de la physiologie, 1860, iii, p. 217 ° PHILIPPEAU and VULPIAN: Journal de Ia physiologie, 1864, vi, p. 421 and 474. 7 REICHERT, E. T.: American journal of the medical sciences, 1885, January. ® Howe Lt and Huser: Journal of physiology, 1892, xiii, p. 335; 1893, xiv, p. 1. 240 R. H. Cunningham. innervated by the peripheral portion of the nerve also regenerate and again contract to the stimulus of a nerve impulse. If successful union of crossed nerves can be obtained, it is evident that when the motor nerve (N) of a group of muscles (M) has united to the peripheral portion of a motor nerve (n) of a group of muscles (m), and vice versa, the group of muscles (m) must receive their nerve impulses from that group of spinal nerve cells from which the nerve impulses to the group of muscles (M) formerly emanated. Similarly, the cells of origin of the nerve (n) will supply the nerve impulses to the muscles (M). Although a very decided change in the destination of the impulses from the spinal cells of origin of the two nerves has been produced by the crossing, the central relations of those spinal cells with each other, with other groups of cells, and with the neuraxons of the cells of the cerebral cortex have not been anatomically altered by simple division and suture of the peripheral nerve trunks. Modern histological methods reveal, to some degree at least, how intricate and how wide-reaching are the connections which exist between the various central nervous mechanisms. Naturally, there- fore, the old question investigated long ago by Flourens again arises as to whether, or no, the intricately connected central nervous mechan- isms are in reality capable of adjusting themselves to the new state of affairs, so that the individual regains complete coordinate control of the muscles supplied by the crossed nerves. Further, if the nerve to a group of muscles which are rhythmically contracting and relaxing in response to rhythmical nerve impulses discharged by a certain group of nerve cells inthe medulla or in the spinal cord, be divided and crossed with the central portion of another motor nerve, will the nerve cells from which the axis cylinders of the latter arise ultimately become a rhythmically discharging group and entirely assume the function of the rhythmically discharging cells after the regeneration of the united crossed nerves and muscles? Vith a view of obtaining a definite answer to the two preceding questions, the various experiments described in this paper were performed. Previous Work on the Subject. — Although various authors, following in the footsteps of Flourens, have divided and crossed different nerve trunks, motor nerves to supposedly sensory nerves and wice versa, and mixed nerves to other mixed nerves, none of these authors has investigated the muscular movements that occur in an animal’s leg Restoration of Movement after Nerve “ Crossing.” 241 with crossed nerves when the motor cortex is electrically or other- wise stimulated. Nor do they appear to have studied the effects upon the rhythmical respiratory and other movements of the vocal cords that may follow the crossing of one recurrens with another motor nerve. Most of the earlier investigators busied themselves with the solution of the problem as to whether, or no, sensory nerves would unite to motor nerves and vice versa, with the ultimate re- establishment of the function of the united portions. It is almost needless to point out that the results of such attempts do not come within the scope of this paper. Consequently I confine myself to a brief review of those previous observations that more directly bear upon a part of my own experiments. According to Flourens’! description of his celebrated experiments, the two principal trunks of the brachial plexus ina cock were cut, and the peripheral end of the trunk supplying the upper surface of the wing was sutured to the central end of the other trunk, supply- ing the lower surface of the wing. At the end of several months the bird had regained perfect use of the extremity of the wing, which no longer dragged, and served for flying (?) as well as before the experi- ment. When the nerves were exposed, they had completely united in the order in which they had been placed, the inferior end of one nerve being continuous with the superior end of the other, and vice versa. In describing the physiological investigation of these united nerves, Flourens writes:? “I pinched the nerves above the point of their reunion, — the wing moved at once, and the animal cried; I pinched them below, and the animal felt it as before, and his wing moved again; the same thing took place, when I pinched the enlarged point of reunion. And further, when I pinched the superior nerve above the point of reunion, the muscles of the lower surface of the wing contracted; and, on the contrary, the muscles of the upper surface of the wing contracted when I pinched the inferior nerve, — always above the point of reunion.” 1 FLOURENS: Joc. cit. 2 “Te pincai ces nerfs az-dessus du point de leur réunion, l’aile se mut aussit6t, et animal cria; je les pincai au-dessows, Vanimal le sentit de méme, et son aile se mut encore; pareille chose eut lieu, quand je pincai le fozut grossi de la réunion. Et de plus, quand je pincais le nerf supérieur au-dessus du point de la réunion, c’étaient les muscles de la face inférieure de l’aile qui se contractaient; et c’était, au contraire, les muscles de la face supérieure de l’aile qui se contractaient quand je pingais le nerf inferieur, toujours aw-dessus du point de la réunion.” 16 242 R. H. Cunningham. While this result of Flourens appears to be all that one could desire, the observer neglects to state whether the action of the tensors of the patagium, the nerves of which were probably not divided, was taken into consideration and properly excluded. No mention is made of a microscopical, or even of a very careful ana- tomical examination of the tissue between the necessarily adjacent crossed trunks. If the central and peripheral ends of the nerves in reality united with each other without the formation of a single nerve fibre between the two adjacent points of suture, the fact is all the more remarkable, for no special precautions seem to have been taken at the primary operation to prevent the latter occur- rence, although such a new formation will invariably occur, accord- ing to the experience of the writer, unless prevented by some such method as is described below. In other cocks and in a duck, Flourens sutured the central end of the fifth cervical nerve to the peripheral end of the divided vagus, and, after the expiration of a number of months, divided the other vagus. All the birds died in from one to four days after the latter operation. Information regarding the return of irritability to the united nerves is not given, but evidently the vagal functions had not been re-established véa the nucleus ‘of the fifth cervical nerve. The experiments on dogs by Philippeau and Vulpian,! in which the central end of the vagus was crossed with the peripheral end of the hypoglossal and wce versa, only tend to show that mixed nerves of different origin are capable of union, and throw no light upon the positive restitution of voluntary coordinate control of the groups of muscles supplied by the above mentioned nerves. These observers concluded that although the central end of the vagus would unite to the peripheral end of the hypoglossus, the nerve fibres of the peripheral part of the hypoglossus would not recover their connec- tions with their exciting nerve-centre, and the hypoglossus would be but an instrument at the command of the functional! centre of the motor fibres contained in the cervical part of the vagus; a conclu- sion that seems to be substantiated by the results of the later experi- ments of Reichert? After suturing in five dogs the central end of one vagus to the pe- ripheral end of the hypoglossus, Reichert found, after the nerves had 1 PHILIPPEAU and VULPIAN: Journal de la physiologie, 1864, p. 421. * REICHERT, E. T.: American journal of medical sciences, 1885, January. Restoration of Movement after Nerve“ Crossing.’ 243 united, that certain areas were present in the partially atrophied half of the tongue, which contracted synchronously with inspiration or with expiration, and concluded that the motor fibres of the vagus had actually become united to similar fibres in the trunk of the hypoglossal, and that the hypoglossal fibres conveyed impulses which were peculiar to the vagus apparatus. Rawa! has obtained such remarkably incredible results after cross- ing nerves of different destination, and also nerves of special function, that one would naturally suspect that his observations and methods must be faulty. For instance, we are told in regard‘ to the cats in which the hypoglossus was sutured to the vagus and wice versa, “that of the entire number of cats only six survived. Four of these cats (Nos. 4, 7, 9, 10) had the left central stump of the hypoglossus sutured to the peripheral vagus; two, a similar crossing of the nerves on the right side. In two other cats (12 and 14) the central vagus was sutured to the peripheral hypoglossus on the left side, and in cat No. 16 on the right side.” At the expiration of 16-20 months, the right vagus was cut in cats 4 and 7, and both animals promptly died within five days. In cat 12, section of the opposite hypoglossal nerve was followed by loss of power to move the tongue. In cat 16, after the opposite hypoglossal was cut, no movements of the tongue were present, but in a few days the tongue was slowly moved, being contracted to the left, but the animal was killed at the end of six weeks. On page 310, cats 9, 10,and II are said to have died very shortly after the primary operation, although cats 9 and 10 were previously included among the six (?) cats that survived the section of one vagus. Likewise, one finds that cat No. 8, previously unin- cluded in the number of cats surviving the section of the right vagus, was operated on 16-20 months after the primary operation, and two centimetres of the left vagus were excised. Five days later, fearing to lose the animal, it was used for an experiment, for the details of which the reader is referred to the original paper. Rawa’s experi- ence leads him to conclude that (1) “after the peripheral portion of a nerve supplying a certain muscle has united to the central end of a nerve that supplies another muscle, the function of the former muscle becomes restored. (2) The direction of the voluntary motor impulses may be altered as one pleases, and the impulses will always accommodate themselves to the peripheral nerve endings.” As a result of his experiments in crossing the hypoglossal and vagus, he 1 Rawa: Archiv fiir Physiologie, 1835, p. 296. 244 R. H. Cunningham. likewise concludes, “that the central nervous mechanisms can inner- vate organs that formerly did not connect with them, as soon as those organs become connected to them by nervous conductors.” ‘ Nerve centres will, by practice, supply exactly what the peripheral organs with which they became connected require of them.” Howell and Huber! crossed the ulnar and the median nerves in dogs and succeeded in getting the crossed nerves to unite without the formation of a cicatrix, common to all the ends. They found, to quote these observers verbatim, ‘‘ that at the second day after the operation, with both median and ulnar cut on the left side high in the arm, and with the ulnar cut on the right side at the level of the elbow, there was very little evidence of any paralysis or even awkwardness.” ‘Before the end of the first week the animal was running around in perfect freedom, and the closest scrutiny could detect no awkwardness of movement except possibly in running rapidly up stairs he would frequently stumble with his front feet; but whether this was due to the unusual innervation of the muscles, or was caused by the over-zealous activity characteristic of young dogs generally, could not be determined.” The close relation between the origin and distribution of the median and ulnar nerves led these observers to remark that “a more interesting suture would probably be one between the musculo-spiral and ulnar in which centres of ori- gin of extensor fibres would be obliged to innervate flexor muscles.” They considered there was no histological or physiological obstacle to such a union, but considerable awkwardness of movement in the beginning might attend the functional use of the nerve by the animal. Judging, therefore, from the results of Howell and Huber it would appear that such nerves as the ulnar and the median, which innervate in the dog synergic groups of muscles, are not the ones to choose for crossing when it is desired to investigate the return of voluntary coordinated movements in muscles innervated by crossed nerves. The suture of two nerves supplying antagonistic groups of muscles will yield results that can be more accurately interpreted. From the preceding brief historical review it is evident that there is room for considerable doubt as to whether the central nervous mechanisms concerned in volition and codrdination will in reality adjust their nervous discharges so that a grown animal will regain full control of antagonistically acting groups of muscles after their nerve trunks have been crossed. * HOWELL and Huser: Journal of physiology, 1892, xiii, p. 335. Restoration of Movement after Nerve “ Crossing.” 245 Methods.— All the successful experiments were performed upon dogs. Intwo monkeys which I had hoped would prove more suit- able than dogs for this variety of experiment, the ulnar and the median nerves were crossed with the musculo-spiral nerve, but as the experiments were not a success, no further mention need be made of them. Ether anesthesia was employed for every operation, and all the operations except the last were performed with the strictest aseptic and antiseptic precautions. After the cerebral cortex, etc., had been investigated at the final operation, the animals were killed with an overdose of the anesthetic. The nerves were divided with a sharp razor and sutured with fine catgut prepared by the writer’s formalin method.! Usually two to four fine sutures were employed. After the crossed nerves had been sutured, broad pieces of fascia covering the neighboring pectoral and other muscles were dissected off, and both of the apposed crossed nerves were gently wrapped, for about three-quarters of an inch above and below the point of suture, in separate pieces of this thin tissue, which was then sutured with fine catgut sutures to the fascia of the adjacent muscles. In all the experiments upon the ulnar, median, and musculo-spiral nerves, the common branch from the musculo-cutaneous nerve to the median was entirely excised, its point of origin from the musculo-cutaneous being ligated with a silk liga- ture. The wound was sutured with No. 2 catgut, dressed with bi- chloride of mercury gauze, the whole limb wrapped in cotton, bandaged, and, finally, put in plaster of Paris. The plaster not only encased the toes, but also covered the shoulder, and passed around the upper part of the thorax and the lower part of the neck. The fore limb was thus kept perfectly at rest for at least three weeks. The plaster bandage was then removed, to be immediately replaced by a clean one that was allowed to remain on the dog for four to six weeks. If any tendency to ulceration became evident after the removal of the plaster, it was again applied for two to four weeks, or longer, until the vitality of the tissues had sufficiently recovered to resist external sources of irritation. Consequently, by carefully protecting the peripheral parts, the majority of the dogs did not exhibit the ulcera- tive disturbances that are very liable to occur in the unprotected skin of the wrist, toes, etc., after division of the chief nerve supply of that region. Previous to the operation, it was found that many of the dogs ' CUNNINGHAM: New York medical journal, 1895, April 20. 246 Rk. 1. Cunningham. would give the paw, and some of the remainder were easily taught to do it also; a circumstance that was later of great assistance in judging whether, or no, the recovery of codrdinated voluntary control of the muscles concerned in that movement had occurred. Other methods of testing, such as running up a flight of steps, holding a bone after the bandaging of the uninjured foot, etc., were also used. For the electrical investigation, adu Bois induction coil by Reininger, Gebbert, and Schall was employed. The primary circuit of the coil was attached to the mains of the 115-volt illuminating current with a sixteen candle-power lamp in series with the primary of the coil; .5 ampere of current was registered by the ammeter when the hammer was in action. During the electrical examination, insulating rubber was placed under the nerves to prevent the escape of current to neighboring nerves. After the animals had been killed, very careful dissections of the united nerves were made. In the animals referred to in this paper it was found, unless it is specially mentioned to the contrary, that the crossed nerves had united in the position in which they had been sutured, and that they were not united in a common cicatrix. If the adjacent united nerves were at all firmly adherent, the result was con- sidered questionable, and was thus rejected. Experiments. —I. Central portion of right ulnar sutured to the pe- ripheral end of the right median , and the central median to the distal ulnar. Dog 1.— Operation January 8, 1895. Plaster bandage removed January 12, and wound found to be healing by first intention. On allowing the dog to run about, it did not appear to limp or seem much inconvenienced by the loss of the functional use of the flexors of the right foot and wrist. Careful comparison with the left foot plainly showed that the right wrist was considerably more extended than the left one, and when the dog was standing with this foot rest- ing on the floor, a considerable part of the palmar surface of the metacarpus touched the floor. If both fore-legs were held up, move- ments of flexion and of extension of the left paw would occur, but the right paw was held in a state of moderate over-extension, the toes being slightly spread apart. When running upa flight of steps, the dog would often stumble, appearing to strike the edge of a step with the over-extended foot. Owing to the development of a few small ulcers on the plantar balls, the plaster bandage was again applied at Restoration of Movement after Nerve “ Crossing.” 247 the end of a week, over the whole limb and shoulder, with a few turns around the body. In two weeks this plaster was removed, and on February 26 another careful examination of the animal was made. At this date, the over-extension continued. The right forearm was much smaller than the left from atrophy of the flexor muscles. On putting the left paw into a small boot and giving the dog a bone, the bone frequently slipped from under the right paw by which the dog tried to steady it when he attempted to gnaw it. No movements of the flexor muscles could be detected. After anzsthetizing the animal and exposing the crossed nerves, it was found that the central median had apparently united to the distal ulnar as well as could be desired. The bulbous ends of the crossed central ulnar and distal median had separated about three millimetres, but were connected by a delicate grayish thread-like band that was found to consist of new nerve fibres. Faradic stimulation of the central median above the point of union produced movements in many of the partially exposed muscles in- nervated by the ulnar, causing ulnar flexion of the wrist and foot. Stimulation of the distal united ulnar three-quarters of an inch below the point of union also produced ulnar flexion, but not until the strength of the current had been considerably increased. Stimulation of the central ulnar with a rather strong current (10 cm.) produced a faint median flexion of the paw. The distal median had not re- covered its faradic electrical irritability, and the electrical irritability of the right distal ulnar was much less than that of the left uninjured ulnar. After exposing the sigmoid gyrus of both cerebral hemispheres, the areas for extension and for flexion of the paw were stimulated after the paw had been flexed and the arm and forearm made immovy- able by firm fixation; — extension of the paw readily followed the cerebral stimulus. Only a very slight degree of flexion of the paw could be produced by stimulating the fore limb area in the left hemi- sphere of this dog, although a stimulus sufficiently strong to produce a severe general fit was finally applied. The central ulnar and the central median nerves were then divided above the points of union and stimulated, the results being the same as before their division. The distal median was then divided and the central ulnar stimulated with a strong current; no flexion of the paw was produced. Stim- ulation of the central median readily produced ulnar flexion. Dog 2.—Similar to No. 1, but the dog was kept for seventy-five 248 R. H. Cunningham. days. Over-extension of the paw was still present, and the animal was awkward and stumbled when running up the steps. Cutaneous faradic stimulation of the flexors of the paw showed that, although the faradic irritability of those muscles had been nearly recovered, their irritability was less than that of the flexor muscles of the nor- mal left forearm. Faradic stimulation of the exposed united nerves showed that the nerves were irritable both above and below the points of union, and stimulation of the central ulnar produced well- marked contraction of the muscles innervated by the median. Ex- citation of the central median produced contraction of muscles supplied by the peripheral ulnar which had been crossed with it. Stimulation of the cortical area for flexion of the paw readily pro- duced that movement. Dog 3.—The right fore limb of this dog was kept for seven weeks in plaster. At the end of fourteen months, a moderate predominance of the extensors over the flexors of the paw was still evident when the animal was carefully examined. The dog also frequently stumbled when attempting to run rapidly up the steps, and though flexor movements of the right paw were plainly to be seen, the movements did not appear to be quite so actively made in the right leg as in the normal left one. Even after this interval of time, the toes of the right foot were still considerably separated when the animal was resting upon that foot. Electrical excitation of the flexor area of the cortex and of the exposed crossed nerves gave results similar to those met with in dog 2, and needs no further comment. The previously atrophied right flexor muscles had evidently nearly completely re- generated, for the forearms of the dog did not perceptibly differ in size nor did the quantitative faradic electrical irritability of the flexor muscles of the forearms differ much. The preceding results thus corroborate those of previous workers, in that they clearly prove that one mixed nerve may be crossed with and unite with another mixed nerve. They also clearly demonstrate that the peripheral portion of the crossed united nerve recovers its function of conductivity before it recovers the property of electrical irritability. After the nerves have united and the various groups of muscles have regenerated, nervous impulses emanating from the motor cortex of the brain are still capable of causing the cells of the spinal cord from which the central portions of the crossed nerves arise, to discharge impulses that give rise to contractions of the muscles which the crossed nerves supply. But in the dog, as is well Restoration of Movement after Nerve “Crossing.” 249 known, the main functional use of the groups of muscles that are supplied by the ulnar and the median nerves is to produce flexion of the foot, the action of the groups of muscles being synergic and also usually synchronous. Consequently, very little, if any, disturbance of voluntary coérdinated flexion of the paw would be likely to follow in the dog when the ulnar and the median nerves have been success- fully crossed, a conclusion that is fully exemplified by the result obtained in dog No. 3. II. Central end of the right musculo-spiral nerve crossed with the distal portions of the ulnar and the median, and vice versa. This operation was performed on nine dogs, but in only four dogs were the experiments successful. In one of these four dogs, No. 2, a large, powerful, restless animal, so much swelling and induration of the tissues developed on the dorsal surface of the wrist from constant attempts to walk upon this surface, that it was impossible to definitely judge whether or not the dog was able to voluntarily contract the extensor muscles of the paw. Evidently the animal was not able to extend the paw intentionally, else it would not have continually flexed the foot at each step and come down upon the dorsal surface of the wrist and foot. Subsequent electrical investigation of the nerves and of the cortical centres showed, however, that the crossed nerves had become at least partially united and regenerated, and that they had recovered their conductivity and electrical irritability. As the experiments on dogs 1, 3, and 4 yielded essentially uniform results, a description of the results obtained in dog 3 will thus apply to dogs I and 4. Dog 3.— Nerves crossed January 20, 1895, and plaster bandage kept on for two weeks. Wound healed by first intention. Plaster reapplied and kept on for four weeks. Muscles of right forearm markedly atrophied and did not respond to cutaneous faradism. In the course of a week, some contraction of the flexors of the paw, which did not fully relax when elbow was extended. The dog continually held the forearm flexed and the foot was not allowed to touch the ground. When given a bone the animal would attempt to steady it in order to gnaw it by resting the outer side of forearm and flexed foot upon the bone, but was not very successful in keeping it firm. On October 11th, the dog attempted to use the right leg for walking, but whenever he did so, walked on the back of the foot, on the outer 250 Rk. H. Cunningham. surface of which was a small ulcer. Ether was administered, the crossed nerves exposed, stimulated, and found to have united. Their electrical irritability had been recovered. Many of the flexor and ex- tensor muscles also responded to direct faradization. After closing the wound and applying an antiseptic dressing to it, and also to the ulcer on the foot, the whole limb was put in plaster with the foot extended. At the end of three weeks the plaster was removed, and the wound and the ulcer were found to be healed. From that time until June 29, 1896, the dog was frequently examined, and the muscles stimu- lated with mild faradic currents, after previously muzzling the dog, which submitted to this treatment without any especial resistance. On June 29, 1896, the forearms scarcely differed in size. The muscles of the right forearm seemed to be almost completely re- generated. The right paw was held partially flexed, but when it was carefully observed after steadying the forearm at the elbow, alternating movements of flexion or extension of that paw could be readily seen to occur. When the dog was ordered to give this paw, the animal lifted up the forearm, but instead of extending the foot, the latter was very visibly flexed. Every time the dog walked, the right leg was advanced but the paw was quickly flexed. When a bone was given to the dog, after inserting the left foot in a boot and immobilizing the left wrist by means of a small splint, the movement of the muscles of the right forearm appeared to be so extremely incodrdinated that the animal finally held the bone by resting the middle of the forearm uponit. Irregular movements of the adductors and abductors of the toes were also noticed. It should be remarked that this dog had exhibited the above movements early in February, 1896, but certainly no improvement in the codrdination of the move- ments had occurred when the above final examination was made. The dog also seemed to have recovered sensation on all surfaces of the foot, but the various tests with clips, etc., for determining whether, or not, the animal could correctly localize the position of the peripheral stimulus gave such conflicting results that I am not able to give an opinion in regard to this subject. After anzesthe- tizing the animal, exposing the motor cortex of both hemispheres, and firmly fixing both elbows so as to prevent any movement at the elbow joint, the cortical area of the right hemisphere for flexion of the wrist was stimulated with a minimal current, and then the same strength of stimulus applied to the area for flexion in the left hemi- sphere. Result: Extension of the right wrist. Stimulation of the Restoration of Movement after Nerve “ Crossing. 251 extensor area, a little further forward in the sigmoid gyrus, pro- duced flexion of the left paw. After repeating this several times the musculo-cutaneous nerve was divided, together with the various flexors and extensors of the forearm; the crossed nerves and blood- vessels being carefully dissected away and protected by cotton wet with warm normal saline solution. After firmly fixing the elbow, the cortex was again stimulated; the flexor area giving rise to contrac- tion of the extensor muscles, the extensor area to flexion of the paw, accompanied apparently by extension of the first phalanges when the current was slightly strengthened. Two minims of the French oil of absinthe were then injected into the jugular vein. Ina few minutes the usual absinthe fit occurred. During the tonic fits the left foot was extended and the right flexed. On immediately excising the small area (extensor) of the left hemi- sphere, which had been electrically determined to produce flexion of the right foot, the right foot became extended. During another fit the flexor area was excised and the exposed extensor muscles of the right foot no longer participated in the fit. The preceding results thus conclusively show that the spinal nerve cells from which the musculo-spiral and the ulnar and median motor fibres arise still preserve their connections with the cortical motor mechanisms situated in the sigmoid gyrus. As far as the cortical areas of this region are concerned, there does not seem to be the least ground for stating that these centres readjust themselves to suit the altered innervation of the groups of muscles which the two united crossed nerves supply.. Nor did five months’ practice seem to enable the adult dog to regain the functional use of the muscles of the forearm and foot, for, as I have previously re- marked, very evident and ample volitional, but incodrdinated, move- ments were visible about five months before the dogs were killed, and none of the dogs showed the least improvement in acquiring any better control of the muscles supplied by the crossed nerves. Ill. Wall the rhythmic contractions of certain groups of muscles re- appear after union of their motor nerve with the central end of a motor nerve to non-rhythmic muscles ? To investigate this question, the right recurrens was divided in three dogs as low down in the neck as possible. After carefully freeing the long peripheral portion of the recurrens, it was turned upward around the border of the inferior constrictor of the pharynx and sutured to the 252 R. H. Cunningham. central end of the hypoglossal, which had been cut close to the tongue. The central end of the recurrens was ligated with fine silk, turned toward the root of the neck, and sutured with catgut to the adjacent tissue. Before this operation, these dogs barked very frequently, but after the operation the animals were only able to utter an imperfect, hoarse, stridulous growl. The right half of the tongue was paralyzed, and soon became atrophied and fissured. At the expiration of eight months, it was noticed that the atrophic condition of the right half of the tongue was beginning to lessen, except in dog 3, and also that two of the dogs could move the muscles of that half considerably. At this date, when examined under ether, the regenerated muscles of the right half of the tongue readily responded to faradism. At the end of fourteen to fifteen months, the movements of the tongue seemed to be almost completely restored, except in dog 3, in which the right half of the tongue was permanently paralyzed. Fourteen to fifteen months after the primary operation, the dogs were etherized and the trachea divided just below the larynx. After the insertion of a tube with a short rubber pipe attached to facilitate the administration of the anesthetic, the anterior composite convolution of both hemispheres was exposed and stimulated with an electrode, the points of which were set one millimetre apart. By carefully adjusting the narcosis, using a stimulus just strong enough to cause the vocal cord to nearly ap- proach the middle line, and carefully removing fluid on the convolu- tions before applying the electrodes, it was perfectly possible to obtain from both hemispheres adduction of the left vocal cord without any accompanying movements of the tongue when the junction of the precrucial gyrus and the upper extremity of the anterior composite was stimulated. When carefully observed! from below, or from above, through the widely opened mouth, the right vocal cord was seen to be perfectly im- movable. In all of the dogs its position seemed to be about midway between adduction and abduction. When the central hypoglossal or the recurrens which had become grafted to it were stimulated, very evident movements of adduction or of abduction would occur. Some- times the cord would begin to abduct and then suddenly adduct. When the above-mentioned focus in either hemisphere was stimulated, no movement of the right vocal cord followed unless a very strong 1 In this connection, the writer wishes to thank Professor Frederic S. Lee for his kindness in carefully observing the movements of the vocal cords on various. occasions. Restoration of Movement after Nerve “ Crossing.’ 253 current (secondary at 4 cm.) was applied. With this strong stimulus, movements of the tongue and of swallowing also occurred. Minimal stimulation of the left anterior composite gyrus farther back, where it is joined by the supra-sylvian, was followed by bilateral movements of the split tongue, with adduction, or frequently with abduction, of the right vocal cord. The rhythmical movements of the left were not inter- rupted. Stimulation of the corresponding area of the right hemisphere produced bilateral movements of the tongue with moderate abduction of the right cord. The left cord did not respond. With the coil at 3 cm., adduction of the right cord occurred. The left recurrens was then divided, in order to stop the respiratory movements of the left cord, and the above-mentioned regions again stimulated. The movements of the right cord accompanying the movements of the tongue were then more striking, but stimulation of the hemispheres at Krause’s laryngeal centre did not produce a move- ment of the right cord, unless, as previously stated, a current suffi- ciently strong to produce violent efforts of swallowing was employed. After killing the dog with the anesthetic, a dissection of the united nerves disclosed the fact that not only had the sutured recurrens united to the central hypoglossal but that from the latter numerous outgrowths had grown to the base of the tongue and had evidently united with the old peripheral hypoglossal stump. The regeneration of the tongue muscles and the return of voluntary control of the tongue was thus readily explained. A search for the central end of the right recurrens disclosed the small knobbed end of this nerve about in the position in which it had been sutured; it seemed to be attached to the sterno-thyroid muscle. It had clearly not re-established any connection with the laryngeal muscles. In dog 3, in which the sutured nerves had been rolled up in a piece of fascia, the outgrowths of nerve fibres from the large hypoglossus had not succeeded in reaching the tongue and producing regeneration of its muscles. When the cortex of this dog was stimulated, no move- ments appeared in the right half of the split tongue. The right vocal cord responded as in dogs 1 and 2, and no respiratory vocal cord movements could be detected after the section of the left recurrens. Evidently, therefore, the cells of origin of the hypoglossal nerve do not assume the rhythmical functions of the cells of origin of the recur- rens when the latter nerve is made to unite to the central portion of 254 R. H. Cunningham. the former. Clearly, the nerve impulses proceeding from certain nerve centres that innervate the muscles supplied by the recurrens do not shunt off by new or by old paths to the hypoglossal nucleus, when this nucleus, or a part of it at least, is caused to become the nucleus of the recurrens. How much the less likely, therefore, that the hypoglossal nucleus should assume all the functions of the nucleus of the vagus, were that nerve united to the hypoglossus. To conclude, it is evident that in the dog the central portion of one motor nerve may unite with the peripheral portion of another motor nerve; that the cortical representation of the groups of regenerated muscles supplied by the crossed and united distal nerve is the same as the cortical representation of the groups of muscles that were pre- viously innervated by the central portion before its section; that this cortical representation of the groups, after crossing the nerves, differs from that existing before the nerves are crossed, in that the cortical impulses produce incodrdinate movements of the muscles supplied by the united crossed nerve. If two motor nerves supplying two groups of synergic muscles, whose action is to produce almost similar sim- ple movements of an articulation, be crossed, the resultant disturbance of the coérdinated mobility of those synergic groups is exceedingly slight, as regards the performance of that particular movement. When groups of muscles innervated by the crossed nerves are of widely dif- ferent functional use, antagonists, etc., the adult animal (dog) does not regain the power of performing intentional codrdinated move- ments with those muscles, although the fibres of the muscles com- pletely regenerate and recover their former irritability. Crossing the peripheral portion of the motor nerve of rhythmically contracting muscles to the central portion of the motor nerve of non- rhythmic muscles results in the permanent abolition of the rhythmic action of the former muscles. In view, therefore, of the foregoing results, it is evident that the central nervous mechanisms do not, as Rawa has claimed, adjust their impulses to suit the altered peripheral innervation, and, by practice, supply exactly what is required of them by the peripheral organs with which they become connected. PAPAIN-PROTEOLYSIS, WITH SOME OBSERVATIONS ON. THE PAVSIOEOGICAL. ACTION OF THE PRODUCTS FORMED. By R. H. CHITTENDEN, LAFAYETTE B. MENDEL, AND H. E. MCDERMOTT. [From the Sheffield Laboratory of Physiological Chemistry, Yale University.] HEN papain, the proteolytic enzyme of the papaw plant, was first subjected to careful study by Wurtz and Bouchut,? it was compared in its mode of action to trypsin, not alone because it was active in a neutral medium, but especially because of the character of the resultant products. Thus, it was stated that by the vigorous action of papain upon blood-fibrin complete peptonization resulted, with the formation of some leucin in addition. Naturally, at this time (1879) there was no differentiation of proteoses and peptones; hence all that the above statement implied was a conver- sion of the proteid into a soluble form, precipitable by alcohol and not coagulated by heat nor by acids, although the presence of leucin would certainly suggest the formation of true peptone. Later, Martin ? pointed out that the enzyme acts vigorously in the presence of sodium carbonate (0.25 per cent) and that as products of diges- tion there are formed in both neutral and alkaline solutions an inter- mediate globulin-like body, peptone, leucin, and tyrosin, the last two being formed in small quantity. Here, likewise, the word peptone must be interpreted as meaning simply soluble proteid, and not carry- ing the distinction which is now known to exist between the pro- teoses and true peptones. Still later, however, Martin‘ studied the ? An abstract of this paper was presented at a meeting of the American Physi- ological Society held at Washington, May 4, 1897. See Science, N.S.,v, June 11, 1897, p. 902. 2 Wurtz and Boucuut: Sur le ferment digestif du Carica papaya. Comptes rendus, 1879, Ixxxix, p. 425. Wurtz: Sur la papaine. Contribution a Vhistoire des ferments solubles. Ibid., 1880, xc, p. 1379; and xci, p. 787. 3 MARTIN, S. H.C.: Papain-digestion. Journal of physiology, 1884, v, p. 213- * MaArvTIN: The nature of papain and its action on vegetable proteid. Jour nal of physiology, 1885, vi, p. 337. 256 Chittenden, Mendel, and McDermote. action of papain on the several proteids occurring with the enzyme in papaw juice, and found that the globulin present there was con- verted by the enzyme into an albumose (8-phytalbumose), and that this substance was transformed into a peptone-like body, which in turn was converted into leucin and tyrosin. In this case the peptone- like body referred to was presumably a true peptone in the modern acceptance of the term. Working with a somewhat different prepa- ration of papain, the writer’ observed incidentally that in the di- gestion of blood-fibrin and coagulated egg-albumin, deuteroalbumose and true peptone predominated among the soluble products formed ; 2. €. peptone, non-precipitable by saturation with ammonium sul- phate. More recently, Osswald” has also reported that papain as studied by him, gave rise to the formation of peptone in neutral, alkaline, and acid fluids, but that digestion was most complete and rapid in a hydrochloric acid solution. With regard to the latter part of the statement, we are inclined to believe that with most proteids the solvent action of papain is greatest in the presence of sodium carbonate and bicarbonate, although a mixture containing a very little hydrochloric acid may be more active than a neutral solution of the enzyme. Much depends, however, upon the presence or absence of extraneous matters in the ferment-preparation and on the amount of proteid present by which the presence or absence of free acid is determined. This question, however, is foreign to our present subject. If the solvent action of papain on proteids is really due to con- version of the proteids into soluble albumoses and peptone, then its action must be compared with that of a true digestive enzyme and the process itself accepted as a genuine digestive process. In this connection it will be remembered that the corresponding vegetable enzyme bromelin, the proteolytic ferment of pineapple juice, is a true peptone-forming enzyme.® In fact, it resembles trypsin very closely in its ability, under suitable conditions, to transform the pro- teid undergoing digestion into true peptone. It is, of course, hardly to be expected that these vegetable enzymes will prove to be identi- ' CHITTENDEN: Papoid-digestion. Trans. Conn. Acad. Arts and Sciences, 1892, ix, p. 321. * OsswALD: Untersuchungen iiber das Papain (Reuss). Miinchener med. Wochenschr., 1894, No. 34. * CHITTENDEN: On the proteolytic action of bromelin, the ferment of pine- apple juice. Journal of physiology, 1894, xv, p. 249. Papain-proteolysts. 25-7 cal in every respect with the corresponding enzymes of animal origin. Indeed, we already know that in the action of bromelin there are cer- tain minor differences at least in the primary or side-products formed as compared with those resulting from gastric and pancreatic diges- tion. There has, however, been no reason for doubting the ability of papain to form true peptone, although it must be admitted that since exact methods of separating albumoses or proteoses from true pep- tones have come into use, no one, so far as we are aware, has isolated the pure peptone or determined the extent or rate of its formation in papain-digestion. On the contrary, within the last few years, the statement has come from several sources that papain has no power whatever to form peptone; that its solvent or digestive action on proteids is limited to the production of proteoses and that peptone is never formed. Thus, Gordon Sharp? states, that on warming co- agulated egg-albumin with one-tenth its weight of papain and a hun- dred volumes of water for eighteen hours, no peptone could be detected either by saturating the digestive mixture with ammonium sulphate and testing the filtrate with the biuret test, or by dialyzing the digestive mixture and testing the diffusate (after one hour!) with phosphotungstic acid and by the biuret test. Albumoses, however, were formed. In a second communication” the same writer states that by the action of papain upon egg-albumin and serum-albumin in neutral, acid, and alkaline solutions peptone is never formed. Further, the opinion is expressed that the formation of peptone by papain is, on biological grounds, not to be expected, since the function of the ferment in the plant consists merely in transforming proteids into soluble compounds adapted for circulation through the open vessels, whereas in pepsin-digestion, on the other hand, the products of prote- olysis must be adapted for absorption by osmosis prior to their dis- tribution and utilization in the body. Lastly, it may be mentioned that Dott? in a comparative study of papain and pepsin has likewise found that the former enzyme, unlike pepsin, is not able to form pep- 1 SHARP: Papain-digestion: Complete absence of peptone. Pharm. J. Trans- act., liii, p. 633, Edinburgh ; Abstract in Chemisches Centralblatt, 1894, i, p. 512. 2 SHARP: The action of papain upon egg- and serum-albumin in acid and alka- line solution. Pharm. J. Transact., lili, p. 757, Edinburgh; Abstract in Chem- isches Centralblatt, 1894, i, p. 830. : 3 Dorr: Comparison of the digestive action of papain and pepsin. Pharm. J. Transact., lili, p. 758, Edinburgh; Abstract in Chemisches Centralblatt, 1894, ee O31. 17 258 Chittenden, Mendel, and McDermote. tone from egg-albumin. If these statements are correct, then, obvi- ously, papain is quite different in its mode of action from other proteolytic enzymes, and the fact, if such it is, should be clearly es- tablished. There would seem to be no great difficulty in arriving at a definite conclusion regarding the matter, and the following experi- ments have been undertaken with a view to throwing some light upon the question, In a preliminary experiment, coagulated egg-albumin (from a dozen eggs) was mixed with 800 c.c. of 0.2 per cent sodium carbonate solution, I gram of commercial papain added, and the mixture, con- tained in a closed flask with a little thymol, warmed at 40° C. for three days. Further ferment action was then stopped by boiling, the undissolved matter removed by filtration, the filtrate neutralized with acetic acid, filtered from the precipitate which resulted, and further concentrated. From this concentrated fluid the proteoses were precipitated collectively and completely by saturating the fluid while boiling hot with ammonium sulphate, — carrying out the satura- tion in a neutral, acid, and ammoniacal fluid successively, as recom- mended by Kihne’ for the complete separation of proteoses from peptone. On testing this proteose-free filtrate with the biuret test, giving due heed to the necessity of adding sufficient potassium hydrox- ide to decompose all of the ammonium salt present, an intense re- action for peptone was obtained. Indeed, it was quite evident from the character of the reaction, that a fairly large percentage of true peptone had been formed. A similar experiment was tried with coagulated blood-fibrin, this form of proteid being warmed at 40° C. for two days with I gram of papain in 800 c.c. of 0.4 per cent sodium carbonate, a little thymol being present. On removal of the proteoses with ammonium sul- phate, as described above, a strong biuret reaction was obtained in the filtrate, thus showing the formation of true peptone. Obviously, one possible danger in experiments of this order, where an alkaline fluid containing so much admixed proteid is warmed at 40° C. for two or three days, is bacterial contamination by which putrefaction may be incited. In the two preceding experiments, thymol was made use of to obviate this danger, but in the next ex- periment chloroform and sodium fluoride were likewise employed, as follows : — 1 KUNE: Erfahrungen iber Albumosen und Peptone, Zeitschrift fiir Biologie, FOQ2, XIX, (ps I Papain-proteolysis. 259 160 grams fibrin ! 60 grams fibrin 60 grams fibrin 60 grams fibrin 500 c.c.0.25% NayCOsz | 500 c.c.0.25% NagC Os | 500 c.c.0.25% NagCO3 | 500 c.c. 0.257% NagCO3 5 c.c. chloroform 2.5 grams thymol 5.0 grams NaF 5.0 grams NaF 1 gram papain boiled 5 min. 1 gram papain 1 gram papain 1 gram papain These mixtures were placed in suitably stoppered flasks, shaken thoroughly to insure complete solution of the sodium fluoride, etc., and warmed at 40° C. for twenty hours, with frequent agitation. At the end of the period the mixtures were boiled and filtered, the filtrates neutralized, concentrated, and the proteoses separated as already described by saturation with ammonium sulphate. On test- ing the filtrates with the biuret test, Nos. 1, 2, and 3 gave a strong reaction for peptone, the reaction in No. 3 being apparently a little the strongest. No. 4, in which the papain was boiled prior to mixing it with the fibrin, gave a purely negative result, thus showing that the peptone reaction in the preceding mixtures could not have come from any admixture contained in the papain itself, nor in the proteid made use of, and that consequently the peptone found must have been formed in some manner during the experiment. Further, this same negative result affords evidence that the peptone detected was not formed by putrefaction; hence it must come from the proteolytic action of the enzyme, which is plainly not hindered by the presence of either chloroform, sodium fluoride, or thymol. Lastly, it should be mentioned that the striking brilliancy of the peptone reactions obtained in Nos. 1-3 precludes the possibility of any other conclusion than that a fairly large proportion of true peptone was formed. A similar series of experiments was carried out with coagulated egg-albumin, 75 grams of the moist coagulum being used in each mixture, with results wholly in accord with those just described. Further, another series in which fresh, thoroughly washed rabbit’s muscle (60 grams in each mixture) was digested gave similar results, the only difference being that in Nos. 1-3 the peptone reaction was even stronger than with the coagulated proteids, as might perhaps be expected owing to the easier digestibility of the former. It is thus quite apparent that papain is a true peptone-forming enzyme, and 1 Coagulated blood-fibrin. 260 Chittenden, Mendel, and McDermott. furthermore is able to exert this action upon various kinds of proteid matter. What now is the extent to which this formation of peptone may be carried by papain? In the digestion of proteids with pepsin-hydro- chloric acid or gastric juice it has been clearly shown that the formation of peptone rarely exceeds 50 per cent; proteoses usually predominate.’ With alkaline trypsin solution or pancreatic juice, on the other hand, the formation of peptone is much greater, although the hemipeptone formed is eventually broken down by the continued action of the enzyme into amido-acids, etc., leaving only the anti- peptone. If papain is a true peptone-forming enzyme, related more closely to trypsin than to pepsin, it follows that under favorable cir- cumstances it might be expected to produce even more than 50 per cent of peptone. It is not to be understood by this statement that papain can be compared with trypsin in rapidity of action; but merely that of the proteid dissolved by papain, under suitable con- ditions, full 50 per cent might not unreasonably be looked upon as convertible into true peptone by the continued action of the enzyme. The correctness of this view has been tested by several series of quan- titative experiments in which the proportion of proteoses and pep- tones formed has been determined as accurately as existing methods will allow. The first experiment of this nature may be described as follows: Coagulated egg-albumin, formed by pouring the whites of eggs into boiling water acidified with acetic acid, was washed thoroughly with water, pressed, and finely divided. The content of dry albumin was then determined in a sampled portion by drying at 110° C., and igniting the residue to obtain the amount of ash. By this method 10 grams of the moist coagulum were found to contain 1.9257 grams of dry proteid. Three digestive mixtures were then prepared, each containing 150 c.c. of 0.25 per cent sodium carbonate saturated with chloroform, 50 grams of the moist coagulated albumin and 0.75 gram of active papain. To obviate any error that might be introduced through the presence of albumose, etc., in the papain, a fourth mixture was prepared similar to the above, except that it contained no albumin. All four mixtures were placed in closely stoppered flasks and trans- ferred to a warm chamber, where they were kept at 38—40° C. for vary- * CHITTENDEN and AMERMAN: A comparison of artificial and natural gastric digestion, together with a study of the diffusibility of proteoses and peptone. Journal of physiology, 1893, xiv, p. 483. Papain-proteolysts. 261 ing lengths of time with occasional agitation. One was allowed to digest for 25 hours, the second was interrupted at the end of 51 hours, while the third mixture and likewise the control were continued for 75 hours. Digestion was stopped by heating the mixture to boiling. It will be noticed in these experiments that the proportion of papain employed was quite small, considering the low digestive power of the enzyme. The mixtures were analyzed as follows: The undissolved residue, made up largely of an insoluble antialbumid-like substance, together with some unaltered proteid, was collected on a weighed filter, washed thoroughly with water and lastly with alcohol, then dried at 110° C. until of constant weight. The filtrate and washings were then neu- tralized with dilute acid, and the neutralization precipitate so obtained was collected on a weighed filter, washed with water until free from salts, dried, and weighed. To determine the albumoses, the neutral filtrate and washings were concentrated to a small volume and then precipitated while still hot by saturation with pure neutral ammonium sulphate, giving heed to Kiihne’s latest modifications of the method.! The precipitate was filtered by the aid of a hot-water funnel and washed free from peptone with a hot saturated solution of ammonium sulphate.” The precipitate, together with the adherent ammonium sulphate, was then washed into a weighed capsule with hot water, the mixture evaporated to dryness, and finally dried in an air-bath at 110° C. until of constant weight. Obviously, the weight so obtained was the combined weight of the albumoses and ammonium sulphate. To ascertain the value of the latter, the mixture was treated with water containing a little hydrochloric acid, the fluid made up to a definite volume, and in an aliquot portion of the latter the sulphuric acid was determined in the usual manner by precipitation with barium chloride. From the weight of barium sulphate thus obtained the amount of ammonium sulphate was calculated and deducted from the combined weight of the albumoses and ammonium salt. The amount of true peptone formed was obtained in this experiment by deducting the combined weight of the antialbumid and undigested residue, neutralization precipitate, and albumoses from the weight of coagulated proteid used, making the necessary corrections for pro- teoses, etc., in the papain. The results from this experiment were as follows, expressed in grams : — Blac. Cite 2 Continued until the filtrate failed to show any biuret reaction. 262 Chittenden, Mendel, and McDermott. Period of digestion at go°C. . . . S. 51 hours. 75 hours. Undissolved residue . . . .. . Adoe 3.1626 Neutralization precipitate . . . . ; 0.0920 (AUbumosestn ct. eerie te lees ets ; 2.3700 5.6246 Phy PLOLeld Sed, ke eth we on ak 6275 9.6275 Reptone Lorie seein 2 an eec : 4.0029 Expressed in percentages calculated on the dry proteid used, these figures yield the following results : — Period of digestion at g4o°C.. . . 25 hours. 51 hours. 75 hours. Undissolved residue . - ... . 5.8 32.8 Neutralization precipitate . . . . ad 0.9 Albumoses Peptone In considering these figures, emphasis is to be laid upon the fact that the large percentage of undissolved residue noted above is by no means composed mainly of unaltered proteid, but is made up to a considerable extent of a peculiar alteration product which seemingly resembles antialbumid, the formation of which must involve a certain amount of energy on the part of the enzyme. Further, it is to be noted that at the end of twenty-five hours digestion, 62.5 per cent of the proteid is converted into albumoses and peptone, while of these soluble products 56.6 per cent is composed of true peptone, the remaining 43.4 per cent being made up mainly of deuteroalbumose. Moreover, it is seen that as the digestion is continued the proportion of albumoses decreases, peptones being correspondingly increased. To be sure, the figures representing the proportions of peptone formed are obtained by difference, but we see no reason why the methods pursued are not capable of yielding results substantially 1 Lost by an accident. Papain-proteolysis. 263 correct. Moreover, on testing the three ammonium sulphate-saturated filtrates containing the peptone with the biuret test, the intensity of the reactions obtained corresponded exactly with the above data. In this connection it should be mentioned that even under most favorable conditions the formation of amido-acids or other crystalline decompo- sition products by papain is very slight. A second series of experiments similar to the above next demand attention because they help make clear possibly why some observ- ers have failed to find evidence of the formation of peptone by papain. Four distinct mixtures were prepared, each containing 150 c.c. of 0.25 per cent sodium carbonate saturated with chloroform, 50 grams of moist coagulated egg-albumin, and 0.5 gram of papain. The fourth mixture, however, differed from the other three in that the papain was boiled with a portion of the fluid prior to mixing it with the albumin. Itthusserved as a control to check any possible errors that might arise from the action of the alkali alone on the proteid, or from soluble matter contained in the papain. Of greater importance, however, is the fact that the proportion of papain employed in this series of experiments was considerably less than in the previous series ; 2. é.,0.5 gram instead of 0.75 gram for every 50 grams of proteid. Furthermore, the papain was a different sample, obtained from a dif- ferent source, and had been tested solely as to its ability to dzssolve proteid matter. The several mixtures were kept at 38-40°C. for varying periods of time, one being removed at the end of 25 hours, the second at the end of 48 hours, while the third and fourth were continued for 72 hours. The mixtures were then analyzed as in the preceding case, with the following results expressed in grams: — Period of digestion at 40°C. . . . 25 hours. 48 hours. 72 hours. Undissolved residue . . ... . 3.8354 3.9759 Neutralization precipitate . . . . 0.0405 0.0387 PMIDUMOSES ss eax MaMa cm eks a) ok 2.8180 2.3218 6.6939 6.3424 Dinvgproteid usedst. i - os 1. 6.8564 6.8564 Reprone formed 5° 3 as « t 0.1625 0.5140 264 Chittenden, Mendel, and McDermott. In the control mixture, in which the papain had been boiled be- fore mixing it with the albumin, 72 hours at 40°C. resulted simply in the formation of 0.03 gram of neutralization precipitate and a trace only of albumose. The undissolved residue when dried weighed 6.9301 grams, the plus weight being due to the insoluble matter of the papain. The slight corrections made necessary by these data have been embodied in the above figures. The percentage results calculated on the dry proteid used are as follows : — Period of digestion at 40°C. . . . 25 hours. 48 hours. 72 hours. Wicdissolvyediesiduemn 2) ei) eee 55.9 SWE 54.8 Neutralization precipitation . . . 0.6 Hs 0.1 FAUIbUIMOSCS Meni =) tse tne 5 4 36.7 Peptone se. Pai mien as iene : ‘ 8.4 Here, for some reason, the formation of peptone was comparatively slight. Although the amount of papain employed in each mixture was less than the quantity used in the first series of experiments, the ratio of papain to dry proteid was much the same in the two cases. Evidently, the papain made use of in this last experiment was far less active than the preceding preparation, as shown also by the large per- centage of undissolved residue. ‘To be sure, considerable albumose was formed, but the enzyme was so lacking in vigor that extensive proteolysis was impossible, and as a result the formation of peptone progressed very slowly. Still, even under these adverse conditions, some peptone was formed — easily recognizable by the biuret reac- tion — and the proportion increased slowly with continued digestion. There is therefore even in this experiment no confirmation of the statement that papain is unable to form peptone, but merely a sugges- tion of the necessity of obtaining an active preparation of the enzyme in order to arrive at a true understanding of its proteolytic power. In a third series of experiments still another preparation of papain was employed: one which preliminary experimentation showed to be quite active. Each mixture contained 150 c.c. of 0.25 per cent sodium carbonate saturated with chloroform, 50 grams of moist Papain-proteolysis. 265 coagulated egg-albumin, and 0.75 gram of papain. A control mix- ture of albumin, etc., in which the papain was boiled to destroy its activity, was also included in the series. In this series, however, digestion at 40° C. was continued for longer periods, and it is like- wise to be noted that the ratio of dry proteid to the papain employed varied from that in the previous experiments. Further, the method of determining the albumoses and peptone was somewhat different from that previously used. Thus, after separating the undissolved residue and neutralization precipitate, the neutral fluid was con- centrated and the albumoses precipitated by saturation of the fluid in the cold with pure zinc sulphate after the method of Bomer.’ This precipitate was collected on a filter, washed thoroughly with a satu- rated solution of zinc sulphate, after which it was dissolved in water, the solution made up to a given volume, and the nitrogen determined in a fraction of the fluid by the Kjeldahl method. On multiplying the values so obtained by the factor 6.25 the amounts of albumoses pres- ent were calculated. The figures for peptone were obtained by differ- ence, but were verified by determination of the nitrogen in the zinc sulphate-saturated filtrates. This, however, was not easily accom- plished owing to the presence of so much zinc sulphate; but on dilut- ing the fluid with water we were able to determine the nitrogen in a small volume of the mixture, using the Kjeldahl] method, and on mul- tiplying the nitrogen found by the factor 6.25 we obtained values not greatly at variance with those given by difference. It is needless to say that the control mixture was treated in a similar manner and corrections made for nitrogen introduced with the papain, etc. Fol- lowing are the results obtained expressed in grams : — Period of digestion at 40° C. ; 98 hours. 144 hours. Undissolved residue .. . 1.0569 1.1136 Neutralization precipitate. 0.0127 0.0152 PANIDUIMOSES! 2 sic 7. 0.6665 0.7506 erisfall = 1.8794 Drysproteidused = 2 3. . 4.4042 4.4042 Peptone formed . . . . . 7 2.6681 2.5248 1 BOMER: Zinksulfat, ein Fallungsmittel fir Albumosen, Zeitschr. f. analyt. Chem., 1895, p. 562. 266 Chittenden, Mendel, and Mc Dermotz. Expressed in percentages, calculated on the dry proteid used, these figures lead to the following results: — Period of digestion at 40° C. 48 hours. 98 hours. Undissolved residue .. . 21.5 24.0 Neutralization precipitate. . EZ; 0.3 Albumoses . Peptone . Here we have plain evidence again of the ability of papain under suitable conditions to form relatively large quantities of peptone, the latter, in this experiment, being greatly in excess over all the other products combined. It is furthermore evident that in order to bring out the full proteolytic power of the enzyme (assuming an active preparation) it is necessary that the latter be present in fairly large proportion, z. ¢., as compared with the proteid matter. When this is the case, as in the present experiment, the element of time is of less moment. In other words, when the ratio of enzyme to proteid is suitable, the maximum digestive action under those conditions is reached in 24-48 hours, and longer exposure at 40° C. fails to in- crease the proportion of peptone formed. In illustration of this point compare the results of the first and third experiments. In conclusion we think it clearly established that papain is not only a peptone-forming enzyme, but that under proper conditions it is able to transform a large proportion of the proteid matter into true peptone. In confirmation of this statement we have been able to prepare and isolate the pure peptone in quantity sufficient to study some of its physiological properties. SOME OBSERVATIONS ON THE PHYSIOLOGICAL ACTION OF THE DEUTEROALBUMOSE AND PEPTONE FORMED BY PAPAIN. It has been generally believed for some time past that the pri- mary products which result from the proteolytic action of vegetable enzymes, as well as those formed by the action of superheated water, are somewhat different in nature from the corresponding products Papain-proteolysis. 267 formed by pepsin-acid and by trypsin. Thus, Neumeister! has shown that if atmid albumin or atmid albumose, z. ¢., the albumose formed by the action of superheated water on blood-fibrin, is injected directly into the blood of a dog it appears in the urine wholly unaltered. An ordinary albumose, however, 7. ¢., such as is formed by pepsin or trypsin, when introduced into the circulation (of a dog) appears in the urine more or less hydrated.? Thus, proto- albumose appears in the urine in part as deuteroalbumose, while if deuteroalbumose is injected into the blood it appears in the urine as peptone. Peptone, on the other hand, is eliminated wholly un- changed. Neumeister? also makes the statement that the products which result from the action of papayotin upon albuminous sub- stances are identical with those formed by the action of super- heated water. This implies that the so-called atmid products and the papayotin products are alike in their resistance to the action of pepsin, for it is assumed at least that it is the presence of this enzyme in the kidney which leads to the hydration of the ordinary albumoses during their elimination from the body. In the case of rabbits, where pepsin is wanting in the kidney, the injection of albumoses into the blood is followed by their elimination unchanged (Neumeister). Moreover, there are certain peculiarities in the chemical composi- tion of the atmid bodies,’® shared to some degree by the proteoses formed by the action of bromelin® —the proteolytic enzyme of pine- apple juice, — which lends favor to the view that these bodies are not quite identical with the proteoses, etc., formed by animal en- 1 NEUMEISTER: Ueber die nachste Einwirkung gespannter Wasserdampfe auf Proteine und iiber eine Gruppe eigenthiimlicher Eiweisskérper und Albumosen. Zeitschr. f. Biol., 1890, xxvi, p. 77. 2 NEUMEISTER: Ueber die Einfiihrung der Albumosen und Peptone in den Organismus. /é7d., 1888, xxiv, p. 272. 3 NEUMEISTER: J/67d., xxvi, p. 82. * Since this paper was written, there has appeared an article by E. Salkowski, “ Ueber die Einwirkung des iiberhitzten Wassers auf Eiweiss,” Zeitschr. f. Biol., 1897, Xxxiv, p. I90 (Jubelband zu Ehren von W. Kiihne), in which it is stated that the atmidalbumose formed by him from blood-fibrin was not resistant to the action of either pepsin, trypsin, or bacteria, thus differing widely from Neumeister’s product. ®° CHITTENDEN AND MEARA: A study of the primary products resulting from the action of superheated water on coagulated egg-albumin. Journal of physi- ology, 1894, xv, p. 50. 5 CHITTENDEN: The proteolytic action of bromelin, the ferment of pineapple Juice. /ézd@., 1894, xv, p. 249. 268 Chittenden, Mendel, and McDermott. zymes. Consequently, it seemed desirable to study with some care the physiological behavior of the albumoses and peptone resulting from papain-digestion with a view to ascertaining what differences of a physiological nature, if any, exist between the latter products and those resulting from animal enzymes. As has already been pointed out, the soluble products which are formed in the digestion of coagulated egg-albumin with papain are mainly deuteroalbumose and peptone. These were prepared in con- siderable quantity by digesting the coagulated albumin from four dozen hen’s eggs with 9 grams of papain in 2 litres of 0.25 per cent sodium carbonate for 48 hours at 40° C. in the presence of chloroform. The resultant fluid freed from insoluble matter and neutralization pre- cipitate was concentrated to a small volume and the albumoses precip- itated by saturation with ammonium sulphate, boiling hot, from a neutral, acid, and alkaline reacting fluid. The precipitate so obtained was dissolved in water, the fluid carefully neutralized, and then dia- lyzed in running water until wholly free from ammonium sulphate and other salts. The solution was then filtered from a little insoluble mat- ter (heteroalbumose, dysalbumose) concentrated to a small volume, and a portion tested for protoalbumose by saturation of the neutral fluid with rock salt. No precipitate whatever was obtained, conse- quently the entire volume of fluid was brought to a syrup and the deuteroalbumose precipitated with strong alcohol. After thorough washing with alcohol and ether, the substance was dried at 100° C. making about 20 grams of pure deuteroalbumose. To obtain the peptone, the ammonium sulphate-saturated filtrate from the albumoses was treated with 50 per cent alcohol, thereby pre- cipitating a large portion of the ammonium salt, while the residual sulphate was removed from the filtrate, after freeing from alcohol, by treatment with barium hydroxide followed by barium carbonate. On evaporating the final filtrate to a syrup and treating with alcohol, the peptone was precipitated more or less gummy, after which it was dehydrated by successive treatments with absolute alcohol and ether, and finally dried at 100° C. About 10 grams of pure peptone were obtained. Mode of Experimentation. — Our study of the physiological action: of the deuteroalbumose and peptone formed above was limited to ascertaining their effects on blood-coagulation, their influence on blood-pressure, and their elimination by the kidneys. In all of the ex- periments dogs were employed, the animals always being anesthetized. Papain-proteolysts. 269 Most generally this was accomplished by means of a mixture of equal parts of chloroform and ether, although in some of the experiments morphine sulphate was injected hypodermically followed by the ad- ministration of chloroform and ether. In the few cases where mor- phine was used, it was employed in the proportion of 1 centigram of morphine sulphate for each kilo of body-weight. The albumose or peptone was introduced either into the left fem- oral vein or into the facial vein through a cannula connected with a burette. The substance, in the proportion of 0.5 gram per kilo of body-weight, was dissolved in 0.7 per cent sodium chloride solution, the volume of the fluid injected ranging from 30 c.c. to 50 c.c. and never exceeding the latter. The fluid was warmed to 40° C. To observe the rate at which the blood coagulated, portions about 5 c.c. each were withdrawn at stated intervals from the right femoral artery through a cannula inserted in that vessel, the blood being col- lected in slender test-tubes to observe the time of coagulation. Each time the blood was withdrawn from the artery the first portion passing out was discarded. Moreover, the cannula was removed and cleaned after each withdrawal of blood. Blood-pressure was registered at the carotid artery, or in some instances at the left femoral artery, using a Hiirthle spring manometer and a Baltzar kymographion driven at a slow rate. Influence on Coagulation of the Blood. — The effects of deutero- albumose and peptone on the coagulation of the blood were observed in eight experiments on dogs ranging in weight from 5 to 11.5 kilos. In the first experiment the dosage of albumose was 0.33 gram per kilo of body-weight, but in four other experiments the dosage was increased to 0.5 gram per kilo, which proportion was likewise used in the three experiments with peptone. Following are the results obtained : — FIRST EXPERIMENT. Dog, 9 kilos. 3 grams deuteroalbumose in 37 c.c. 0.7 per cent NaCl. Injection lasted 2 min. 45 sec. The normal blood coagulated in 3 minutes. Blood withdrawn 3 minutes after injection of albumose coagulated in 30 min. - “ee “ 9 “ee “ “ “ 25 “ee Ser; ‘“ 92 “ ‘ TG ‘ oF « zs aS 28 us ce ‘s < 1-2 hours. ' The figure given for the coagulation-time of the normal blood is the average of 2-3 determinations. 270 Chittenden, Mendel, and McDermott. SECOND EXPERIMENT. Dog, 6.5 kilos. 3.25 grams deuteroalbumose in 50 c.c. 0.7 per cent NaCl. Injection lasted 3 minutes. The normal blood coagulated in 10 minutes. Blood withdrawn 1] minute after injection of albumose coagulated in ] hr. 27 min. ‘ “ + . ‘ : & Vey St es “ 8 - ra a s Ly Oe Sh wie i e “ g 1 te “ “ 18 “ “ “c “ il “ 10 “c : eos 220 ; ‘ ¢ ¢ 0 oee “ a : é ‘ 0 aD Eae i Fey 1! BAG a ‘ ‘ " 0 rae THIRD EXPERIMENT. Bitch, 7.2 kilos. 3.5 grams deuteroalbumose in 50 c.c. 0.7 per cent NaCl. Injection lasted 1 minute. The normal blood coagulated in 9 minutes. Blood withdrawn 2 min. after injection of albumose was uncoagulated at the end of 18 hrs. “ “ 8 “ “c “ “ “ “ “6 “ “ 25 ‘6 “ “ “ “c “ “c “ “ 46 “cc “c “c “ “ “ “ec FOURTH . EXPERIMENT. Dog, 7 kilos. 3.5 grams dewteroalbumose in 50 c.c. 07 per cent NaCl. Injection lasted ] min. 15 sec. The normal blood coagulated in 3.5 minutes. Biood withdrawn 6 min. after injection of albumose was uncoagulated at the end of 36 hrs. “ “ IY “cc “ “c ““ “ “ ““ “c “ce 18 “ “ce “ “ “ “ec “ “ “ 24 “ Ts “ «ec “ “ “ % ss 34 - of u 3s coagulated within 5% hrs. “ “ 42 “c “ “ “ “ “c 3 “ FIFTH EXPERIMENT. Dog, 11.6 kilos. 5.6 grams deuteroalbumose in 40 c.c. 0.7 per cent NaCl. Injection lasted 45 seconds. The normal blood coagulated in 9 minutes. Blood withdrawn 2 minutes after injection of albumose coagulated in 7% hours. “ “ 4 6“ “c “ “c “ “ce “ « 7 “ “ “ “ “ “ .s s 12 ef 5s ‘ : “2 hrs:s6umime “c “ 7 “ec “ “ “ “ O. Mt ei eae 73 “ 26 “ “ “ “« «& QO « 42 « ‘“ “ 36 6s ‘ “ “ “ QO « 20. * “ce “ 45 “e “ce “ec “e “ @) “ 23 “c “ “ 55 a4 ““c “c “c “ec (0) “ 13 e “ “ 65 é “ “ “ “ (0) “c Gas “ “é 75 “ ‘ «“ “ “ (0a Byes “ “ 85 “c ‘ “ “ 0) « 2 “ce Papain-proteolysis. zy ak SIXTH EXPERIMENT. Bitch, 5 kilos. 3.7 grams fefptone in 30 c.c. 0.7 per cent NaCl. Injection lasted 40 seconds. The normal blood coagulated in 3 minutes. Blood withdrawn 5 minutes after injection of peptone coagulated in 6 hours. a7 “ 9 “ “ “e ““ “ “ee “ sc 15 ‘ “ec “ “cc “cc “c a fs 24 ss sf eS eS Y 1 hour. “ «“ 55 ‘ & ‘ « “ 45 minutes. “ “ 65 “e “ce “ce oe “ 40 “ce “ “ce 7d “6 “ “e “é “ 9 “cc SEVENTH EXPERIMENT. Bitch, 5.5 kilos. 2.75 grams feptone in 30 c.c.0.7 per cent NaCl. Injection iasted 30 seconds. The normal blood coagulated in 1.5 minutes. Blood withdrawn 3 minutes after injection of peptone coagulated in 3-10 hours. “e “ee Dog, 5 kilos. 7 “ce “ce “ec “ce “ “ 13 ts 4 & . § ee 18 s us « « ss a 39 es * “ rt * - 50 s us ¢ se «76 minutes. 60 se ss es Hem 0 lpn 80 oe es “ ‘s A S10 gr 90 & Ly “ Dae 98 Me s +30% “ hase 0.5238 47.62 105.7 rs ‘ +5.0% “ Sie 0.6750 32.50 12.2 A critical examination of the foregoing results shows us first that the bile salts from ox bile have no very great influence in either direction upon pancreatic proteolysis. In two experiments (31 and Influence of Bile on Pancreatic Proteolysis. 227 33) there is some evidence of acceleration, while in one experiment retardation is more noticeable. With pure sodium glycocholate (3 per cent) relative proteolytic action is reduced from 100 to 87. In Experiment 47, to be quoted later, stimulation of proteolysis is quite marked. In considering these results, however, in their bearing on the influence of fresh bile, it is to be remembered that 3-5 per cent of these bile salts are equivalent to the addition of 40-50 per cent of the original bile. With regard to the apparent difference in action of the several samples of ox bile salts we are inclined to attribute this to variations in the proportion of glycocholate and taurocholate present. Pure glycocholate, other influences excluded, seems to have a greater inhibitory action than the mixed salts, and possibly the more pro- nounced retardation seemingly characteristic of the salts from pig’s bile is due to the fact that the salts are mainly glycocholates. Still it is to be observed that the samples of salts from pig’s bile vary con- siderably in the intensity of their action, and this independently of their acidity, for when the latter is neutralized the same retarding effect is still produced. Somewhat noticeable also is the difference in intensity of action of the neutralized bile salts (from pig’s bile) when added to the pancreatic juice of the same species as contrasted with the result obtained when the salts are added to the pancreatic extract from another species (contrast Experiments 38 and 39). The results collectively certainly warrant the conclusion that the isolated bile salts taken by themselves do not exert any very marked stimulation of pancreatic proteolysis. They may, on the other hand, give rise to some retardation, — an effect which is seemingly more characteristic of the salts from pig’s bile than of those common to ox bile. It is to be noted, however, that the salts from pig’s bile were not so pure chemically as the crystallized salts separated from ox bile, but fre- quently showed an acid reaction. The above somewhat unsatisfactory results have served to strengthen our conviction that such limited action as normal bile exerts on pan- creatic proteolysis is the result mainly of influence on the reaction of the digestive mixture, and that many agencies other than the specific bile salts are concerned. No doubt, some of these are more or less antagonistic to each other. Thus, pig’s bile, as has been frequently stated by many observers, is liable to be extremely viscid, but the vis- cidity is not always conspicuous; at times the bile is quite limpid. This viscidity is due, in great part at least, to a mucin or nucleoal- 328 Chittenden and A loro. bumin, precipitable by alcohol, and we have found that when this substance is removed from the bile there is a noticeable difference in the influence of the fluid on proteolysis. We may cite the following experiment: Fresh pig’s bile, very viscid, having a specific gravity of 1036, an acidity of 0.58, and an alkalinity of 1.15, was treated with five volumes of strong alcohol, the precipitate filtered off, and washed with alcohol. The united filtrate and washings were then evaporated for the removal of the alcohol, and the fluid made up with distilled water to the original volume. The acidity was now 0.48, and the alkalinity 0.90. The action of a portion of the original fresh bile and of the bile freed from nucleoalbumin, etc., on proteolysis was then tested. Experiment 40. With fresh pig’s bile. Neutral extract of ox pancreas. Per cent of Bile. Undaigested residue. Fibrin digested. Relative proteolytic action. 0 0.5777 gram 42.23 per cent 100.0 0.25 0.6088 39.12 92.6 0.50 0.6241 37.59 89.0 1.00 0.7000 30.00 71.0 2.50 0.7118 28.82 68.2 5.00 0.6496 35.04 82.9 10.00 0.7235 27.65 65.4 Experiment 44. With bile freed from nucleoalbumin. Neutral extract of ox pancreas. Per cent of Bile, Undigested residue. Fibrin digested. Relative proteolytic action. 0) 0.6446 gram 35.54 per cent 100.0 0.25 0.6556 34.44 96.9 0.50 0.6651 33.49 ore 1.00 0.7007 29.93 84.2 2.50 0.7351 26.49 74.5 5.00 0.7217 27.83 78.3 10.00 0.6598 34.02 SSy It is noticeable from these two experiments that the removal of the nucleoalbumin, with possibly some of the inorganic salts from pig’s _ bile, diminishes in a general way the retarding effect of the latter on proteolysis. Somewhat noticeable also is the peculiar relationship in the rise and fall of proteolysis under the influence of different per- centages of the two samples. With ox bile an attempt was made to separate the fluid into three distinct fractions, using methods which would presumably cause little or no change in the nature or composition of the various constituents. For this purpose 440 c.c. of fresh ox bile, containing 12.4 per cent of solid matter, were evaporated to a very thick syrup on the water-bath and precipitated with absolute alcohol. The small precipitate which Influence of Bile on Pancreatic Proteolysis. 220 resulted was filtered off, washed thoroughly with alcohol, and then dried over sulphuric acid. It weighed 2.27 grams. The alcoholic filtrate was treated with a large volume of ether, the precipitated bile salts filtered off, washed thoroughly with ether, and dried. The alcohol- ether filtrate was allowed to evaporate, and finally brought to com- plete dryness on the water-bath. The effect of these three fractions on pancreatic proteolysis was then determined in the usual manner. The bile salts and the residue from the alcohol-ether filtrate were readily soluble in water, but the alcoholic precipitate was not com- pletely soluble. Following are the results obtained : — Experiment 42. With a neutral extract of ox pancreas. Per cent of Bile Constituents. Undigested residue. Fibrin digested. pro ae ean (0) as ge ace hae er a a 0.4217 gram 57.83 per cent 100.0 OSmBiletsaltsie i. 5 va.) ome 0.4533 54.67 94.5 1.0 aoe te 0.4452 55.48 O59. 15 € Si ieeromee tc 0.4415 55.85 96.5 OZ5eAlcoholicsp:py i's -- 2% Bile salts. 0.6359 36.41 Chiles ‘i : y tte i . 0.6230 37.70 80.0 L[nfluence of Bile on Pancreatic Proteolysis. 308 Experiment 48. With extract of pig’s pancreas. Bile salts from pig’s bile. Relative Character of the fluid. Undigested residue. Fibrin digested. proteolytic action. Neutralemra yews) oil Ggcueey Minor e es 0.1755 gram 82.45 percent 100.0 Proteids combined with HC] — Half saturated (O03 )9, ELC) 0.2337 76.63 92.9 Be # “« + 0.5% Bile salts, 0.4433 55.67 67.5 Y F “ + 1.0% Es 0.4039 59.61 72.2 K ee “ + 2.0% a 0.4958 50.42 61.1 & me “ + 3.0% ss 0.4992 50.08 60.7 Experiment 49. With extract of pig’s pancreas. Bile salts from pig’s bile. The salts somewhat acid in reaction. Salicylic acid used to combine with the proteids. Relative Character of the fiuid. Undigested residue, Fibrin digested. idee ytic action. IGHLEAN Gol tiie We) rca ye ge wis ie ow - S08 gram 69.59 per cent 100.0 se cee S EpDILEsSAaltSe Masia G20 fs sell Ys 0.5338 46.62 66.9 Proteids combined with acid — Wholly saturated “ (00647)! 9) S795 42.05 60.4 Ee a Sioa silersaltse 0.7608 Zoe 34.3 Half és ef KOL03 277) eee 0.4296 57.04 81.9 = “ + 3% Bile salts. 0.6773 SEB 46.3 Quarter “ ff (OOM) oe 0.3608 63.92 91.8 ‘ = “« + 3% Bile salts. 0.5591 44.09 63.3 Experiment 50. With extract of pig’s pancreas. Bile salts from pig’s bile made perfectly neutral. Salicylic acid used to combine with the proteids. Relative Character of the fiuid. Ondigested residue. Fibrin digested. proteolytic action. INIGUULEGEI ae Uae tala JPR aa mer 0.2132 gram 78.68 per cent 100.0 Proteids combined with acid — Half saturated “ (0.053% acid) . 0.6038 39.62 50.3 Me om “+ 0.5% Bile salts . 0.7659 Zon 29:7 - a “+ 1.0% is : 0.8120 18.80 22.6 € oH “+ 2.0% : 0.8319 16.81 21.3 a ss a sOYs . : 0.8900 11.00 139 Experiment 51. With extract of ox pancreas. Fresh pig’s bile. Salicylic acid used to combine with the proteids. Relative Character of the fluid. Undigested residue. Fibrin digested. proteolytic 2 action. IN Grater Meee seis mis: Ags cl hy Tentetyas es 0.4125 gram 58.75 per cent 100.0 Proteids combined with acid — Half saturated sf (0.038% acid) . 0.7111 28.89 * 49.1 = ~ Se OYA Janus 4G 0.7242 27.58 469 % a rae ele Oye a ee 0.7283 Zhe lel 46.2 as = er aU ie 20 cee 0.7662 23.38 Boi B a ae NOM Fg og 0.7854 21.46 36.5 s Md © LIOR 2 S- 7819 21.81 37.1 334 Chittenden and Albro. Experiment 52. Neutral extract of ox pancreas. Fresh ox bile. In this experiment the proteids of the pancreatic extract were not treated with acid, but sufficient acid was added to the fibrin to saturate it, or half saturate it (as tested by tropzolin oo), prior to addition of the pancreatic extract. Relative Conditions. Ondigested residue. Fibrin digested. proteolylic action. Neutralsbibrinv.e icp meeie) oer eegeceia aueere 0.3643 gram 63.57 per cent 100.0 Fibrin saturated with acid (5 c.c. 0.2% HCl), 0.5755 42.45 68.3 3 ‘s ci} OO, D116 eeuen ars 0.5490 45.10 70.9 32 dayeilyg (2s CO OVAGA SKC). 0.4690 53.10 83.5 ae nae we HE ey lett ad-nig Pe 0.4980 50.20 78.9 In only one of these experiments (Experiment 47) do we see any distinct suggestion of aid to pancreatic proteolysis when bile or bile salts are added to a pancreatic extract containing combined acid. Combined acid alone tends to retard proteolysis, and the addition of bile to such mixtures as a rule increases still further the extent of retardation. Our results afford no confirmation whatever of the view that bile greatly aids pancreatic juice in its proteolytic action on acid fibrin. Neither are we inclined to believe “ that pancreatic juice, plus bile, plus hydrochloric acid, can accomplish more work in pro- teolysis than can any other known pancreatic mixture.’! If such were the case we fail to see why some evidence of such favorable action should not appear in our results. The inhibitory action of acids alone, and of acids and bile combined, on pancreatic pro- teolysis is not in our judgment to be looked upon as unfavorable to the normal digestive processes of the small intestine. What right have we to assume that the conditions existent in the normal duo- denum are such as to require pancreatic proteolysis to take place in the presence of acid, either free or combined? The combined or free acid which passes from the stomach through the pylorus is with- out doubt quickly removed by absorption or destroyed by neu- tralization. The evidence is certainly in favor of the view that the contents of the duodenum are generally alkaline. This question has been admirably discussed in a recent paper by Moore and Rockwood,? in which also a large number of experimental data are offered, showing that in many animals at least, under different forms of diet, the con- tents of the intestine from pylorus to cecum react alkaline. In some cases, to be sure, the contents closely adjacent to the pylorus were 1 RACHFORD and SOUTHGATE: Joc. cit. * Moore and Rockwoop: Journal of physiology, 1897, xxi, p. 373. L[nfluence of Bile on Pancreatic Proteolyses. a35 found to be acid, but when this was the case the acidity was usually limited to a few inches. Hence, we are inclined to believe that pan- creatic proteolysis as it occurs in the normal intestine takes place, to a great extent, in the presence of a neutral or alkaline reaction, and that under such conditions the proportion of bile ordinarily present is not inimical to the process. THE REINFORCEMENT OF VOLUNTARY MUSCULAR CONTRACTIONS. By ALLEN CLEGHORN, M.D. [From the Laboratory of Physiology in the Harvard Medical School.) N 1890 Bowditch and Warren! found that the knee-jerk was I accelerated or reinforced when the interval between the various sensory stimuli used in their experiments and the blow on the patellar tendon was less than three-tenths second; when this interval exceeded three-tenths second an inhibitory or negative result was obtained. Their results were so uniform that it seemed desirable to ascertain whether sensory stimuli would exert a similar influence upon volun- tary muscular contractions, and to that end the following experiments were undertaken. The subject of the experiment contracted his muscles rhythmically every three seconds. The signal for contraction was the sound of a metronome beating every half second. The contractions were ' recorded by means of Mosso’s ergograph and were registered on a revolving drum. Each contraction was communicated by the recording lever of the ergograph to a Crermaksclecttic) doublewlevet.49 Ehe reader may be reminded that this most useful piece of mechanism (Fig. 1) was designed to record the limits of oscil- lation of a moving body without being otherwise affected by the movements of the body. By it an electric current can be either made or broken at the moment when the movement has reached its full extent. It consists of two levers (C) and (I) (Fig. 2), each connected with one pole of a battery (F). Both levers move on the same axis(H). The first lever (C) is U-shaped and freely movable. It embraces the second lever. The latter (I) is stationary, except FIGURE I. * BowpbitcH and WARREN : Journal of physiology, 1890, xi, p. 25. Reinforcement of Voluntary Muscular Contractions. 337 when either elevated or depressed by the action of the first. Each arm of the first lever can be made either a conductor or a noncon- ductor by means of small screws (B and C), tipped with platinum or ivory respectively. This lever is connected with the ergograph (K) at L. A movement of the recording arm (A) of the ergograph (K), which is connect- ed with the long or “Lt lever (C)vab Is, brings one of the arms of the ‘“‘U”’ into con- tact with the indiffer- ent, 4. ¢é., stationary lever I. Should this be the arm carry- ing the ivory-pointed screw no connection is made, but if it be the arm with the plat- inum-pointed screw the current is at once completed. By means of this instrument and an automatic short- circuiting key (E), which it was subsequently found necessary to introduce, the stimuli were controlled in a definite manner. Thus, when A was depressed or in a position of rest, 7.¢., the position it occupied when the muscles were completely relaxed, C was raised, through its connection at L, and made to turn on the axis at H, and so to pull on the cord (J) attached to the key E and hence to short- circuit itself automatically. Without this arrangement unavoidable vibrations would have broken the circuit through the sensitive con- tact of a and b and thus have caused the signal marker (B) and the electro-magnet (G) to act. With this arrangement, however, when A began to ascend, C was lowered and contact made between a and b, while the spring at E pulled open the short-circuiting key, the cord J between C and E being relaxed, and so allowed the current to pass a—b; consequently the ivory-tipped screw c — by elevating C and so drawing b away from a— broke the circuit 22 FIGURE 2. Diagram of the double-lever and the recording apparatus. 338 Allen Cleghorn. as soon as the recording lever (A) of the ergograph (K) began to descend.! The stimuli used in these experiments were light flashed into the eye, a sudden sound, and induction shocks applied to the skin. For the retinal stimulus a flash from a thirty-two candle-power electric lamp was suddenly brought to bear on the eyes, which were in dark- ness, by means of Czermak’s lever, the subject at the beginning of his muscular contraction breaking the current? and so by means of the electro-magnet (G) releasing a catch which let fall the shutter of the box containing the electric lamp. The sound stimulus was effected in the same way, except that the breaking of the circuit re- leased a hammer which struck upon a tin disk connected with the ears by a stethoscope. Similarly, in the case of the stimulation of the skin, the breaking of the circuit opened the short-circuiting key in the secondary circuit of a du Bois-Reymond inductorium, hammer in action, and so let a series of induction shocks pass to the subject through common sponge electrodes fastened to the left arm (the right made the contractions). The point at which the reinforcing stimuli were made was indicated on the drum by an electric signal marker. The subject usually contracted against the weight of two kilogrammes. The electric current was under the immediate control of the operator, by means of the short-circuiting key (D), which cut out the action of Czermak’s lever and the automatic key (E); conse- quently no stimulus could take place except at the will of the opera- tor, for the circuit could not be broken by Czermak’s lever unless D was open. The operator closed the circuit at irregularly varying intervals so that the subject might not know when the stimulus was to be applied. Fig. 2 illustrates the electrical connections for the three stimuli of sound, light, and the induction shock. It was found that a sensory stimulus applied just as the muscle was. beginning to contract, caused an increase in the height of the con- traction (Fig. 3). At this point in the research Hofbauer*® published experiments similar to my own. He used sound as the reinforcing stimulus and ' For a full account of the double lever see CZERMAK, J. H.: Der electrische Doppelhebel, Leipzig, 1871. * The contact screws of Czermak’s lever were here reversed, the lower one (c) being the conductor; the previous description applies to the arrangement in the experiments to be described presently. * HOFBAUER: Archiv f. d. ges. Physiol., 1897, xviii, p. 546. Reinforcement of Voluntary Muscular Contractions. 339 obtained practically the same results. In both researches the aug- mentation was particularly noticeable as fatigue set in and the con- tractions grew smaller. The most important feature noticed in my experiments was the fact that the relaxation following a stimulated contraction was de- FIGURE 3. One-fourth original size. Reinforcement of voluntary contraction by sensory stimuli. The sensory stimulus was applied at the breaks in the horizontal line beneath the contractions. The load was one kilo. cidedly quicker and more complete than that following a normal or unstimulated one, even when the stimulated contraction showed no signs of augmentation, and as Hofbauer’s paper fully covered the ground of the augmented contractions, attention was now turned to the relaxation phenomena. Czermak’s levers were now so arranged that the reinforcing stimulus should be made at the moment the subject began to relax from a contraction.!. With this arrangement it was found that the duration of relaxation is very considerably shortened when the subject is stimulated at the moment of relaxation, as the accompanying tracings show. So marked was this phenomenon that whereas the average time of a whole unstimulated or normal muscle- curve was about one second, the time of a contraction reinforced at the moment of relaxation averaged only 0.65 second, the difference (0.35 second) being taken from the time of relaxation. The stimu- lated relaxation is not only quicker than the normal but also more complete; the end of a normal relaxation is slow; usually it exhibits one or two very long curves before reaching the base line, which it approaches in a very gradual manner. Relaxation under the in- fluence of the stimulus, on the contrary, shows nothing of this, but is 1 The contact screw was changed from c to b, c being now the non-conductor ; thus electric contact was preserved during the contraction period but broken as soon as relaxation began. See the description of the apparatus and its connections on page 337. 340 Allen Cleghorn. J a sudden sharp drop directly to the base line and sometimes beyond it. Fig. 4 shows this clearly, the broken-line curve being the stimu- lated one. In the course of the research it was noticed that the relaxation of some of the subjects was quicker than that of others, while the height of their normal contraction varied also, some persons giving a strong, high contraction while others gave a small, feeble one. Again, some re- acted in a more decisive way than others, and in a few instances the sub- ject gradually became accustomed to the stimuli and reacted more slowly than at first. In some cases, finally, it was observed that the next con- FIGURE 4. One-fifth original size. The : < : curves record voluntary muscular traction following the reinforced one contractions (load, one kilo). In exhibited the same phenomenon (see the broken curve, during the period er face Fig. 5), but this occurred in a manner marked by the rise in the line just eee : above the time record. a faradic t°© itregular to enable ‘conclusignsias current was applied to the skin. be drawn from it. (he breaks in this curve were The table on page 341 shows ata painted in after the curve was Ze . : , glance the effect of the various stimuli drawn. The lowest tracing gives the time in fifths of seconds. on muscular relaxation. Each of the three parts of the table is compiled from one tracing, in each case taken from a different individual, and is typical of the results obtained from all the other tracings. The same number of normal and stimulated contractions are given in order to facilitate comparison. In the first section of the table light was the reinforcing stimulus; it was applied at the beginning of muscular vedaxation. From this portion we can see that in a normal muscular movement the period of muscular re- laxation is slightly longer (equal in two cases) than the time of contraction, while in the movements with simultaneous retinal stim- ulation the reverse is the case, the time of relaxation being con- siderably shorter than the time of contraction (see Fig. 3). With sound as the stimulus, the same results are noticed, the short- ening of the relaxation time being again marked. The same effect is gained by a simple cutaneous sensory stimulus (electric). No attempt was made to find the results of using powerful shocks. Reinforcement of Voluntary Muscular Contractions. 341 The duration (in seconds) of voluntary muscular contraction and relaxation (1) without simultaneous stimulation, and (2) with simultaneous visual, auditory, or cutaneous stimuli at the beginning of relaxation. INDUCTION SHOCKS TO THE SKIN. Stimulus Stimulus Stimulus No stimulus. | atbeginning of] No stimulus. | atbeginning of] No stimulus. | atbeginning of relaxation. relaxation. relaxation. -| Relaxa-|Contrac-| Relaxa- fContrac-| Relaxa-|Contrac- 6 -| Relaxa-|Contrac-| Relaxa- tion. tion. | tion. tion. tion. tion. : on. tion. tion, Ae AVERAGES- It appears, therefore, that the phase of relaxation after voluntary muscular contraction is shortened by a sensory stimulus applied at the beginning of relaxation. I pass now to a discussion of the cause of this interesting phenom- enon. It is necessary to inquire first whether the shortening of the relaxation phase (or acceleration of relaxation) is due to a reflex contraction of the extensors of the forearm, now relaxing from their contraction as antagonists during the contraction of the flexor muscles. Such a reflex contraction of the extensors would forcibly elongate the relaxing flexors and thus shorten their phase of relaxation. The solution of this new problem required that the contractions of both extensors and flexors should be registered simultaneously, —a difficult task, as it proved, necessitating an addition to the apparatus already in use. The wrist being fixed as before in Mosso’s ergograph, the subject flexed his arm so as to bring the upper arm at right angles to the forearm. Two upright supports firmly screwed to the base board of the ergograph embraced the elbow and upper arm, which were then tightly bandaged in this position, the bandage in- cluding the uprights and the upper arm so that no portion of the 342 Allen Cleghorn. limb except the finger in connection with the ergograph could move. The button of a Marey receiving tambour was placed in contact with the integument over the extensors, the tambour itself being supported from below. Connection was now made with a recording tambour, the lever of which carried a Pfliiger’s marker which wrote vertically on the drum; this did away with the difference that would result were one curve drawn by a lever moving perpendicularly and the other (extensor) curve drawn by a lever which moved in the arc of a circle. Over twenty different subjects were examined, and rather more than five hundred contractions were taken from each. The tracings obtained by this method all showed the same characteristics and demonstrated the fact that the antagonistic extensors and the flexors relax in the same way. In other words, the stimulus seemed to affect it | el | i } i" WN Ni UN J \ : \ \, Joh | V/ \ | / r eee awe FIGURE 5. One-fourth original size. The uppermost curve was drawn by the flexors of the forearm. The second curve was drawn by the extensors. The breaks in the third curve indicate the stimuli. The lowermost curve is the time in fifths seconds. the extensors in a similar manner to the flexors. Figure 5 shows the action of both sets of muscles under the influence of the reinforcing stimulus of sound. The tracing is especially valuable as the subject was contracting against the weight of only one kilo, thus minimizing any influence which the weight might have on the relaxation of the muscles. In this tracing the points of the recording levers were accurately placed in the same vertical line. It is seen from these results that the acceleration of the relaxation phase cannot be ex- plained by the reflex contraction of the extensors of the forearm. A second explanation suggests itself. The relaxation phase in voluntary contraction may perhaps be regarded as the gradual sub- sidence of the contraction-discharge from the motor neurons. If such a process were inhibited by the stimulation of the retina, the auditory heinforcement of Voluntary Muscular Contractions. 343 apparatus, or the skin, the flexor muscles would at once cease to oppose the extending force exerted by the load which they had raised. The passive muscles would then be rapidly extended by the load, and the relaxation curve would fall more steeply, as actually observed. Instances of the reflex inhibition of motor discharges in consequence of sensory excitation are sufficiently numerous. The interesting experiment of Bubnoff and Heidenhain! may be cited as an example. These observers found that in some stages of morphine poisoning the subminimal electrical excitation of a cortical motor field produces strong tonic contractions lasting for some time. Ifa sensory stimulus, such as gently stroking the skin, or blowing on the face, is now applied, the muscles at once relax. The contraction process in the central neurons is in this case cut short by the sensory stimulus. Attractive as this explanation of the acceleration of relaxa- tion witnessed in my experiments may be, there are reasons which seem to preclude its acceptance. An examination of Fig. 5 will show that a muscle weighted with one kilo relaxed as quickly as one weighted with three kilos. Had the contraction process been sud- denly inhibited, the extension of the still partially shortened flexors —now no longer in receipt of the motor discharge from the central neurons — would have been more rapid with the greater load. In my experiments, an increase in the load was not followed by an increase in the rapidity of relaxation. If the quickening of relaxation is not to be explained by the con- traction of antagonists or by the passive extension of the muscles in consequence of the reflex inhibition of the contraction process in the phase of sinking energy, —and these explanations cannot be recon- ciled with my results, —there seems to be no way of avoiding the con- clusion that the quickening is due to the augmentation of an active relaxation process. That the phase of sinking energy in the contrac- tion process is as much an active conversion of stored power as is the phase of rising energy has been long considered probable. Indeed, instances are not wanting of a modification of the contraction process by the stimulation of peripheral nerves. Richet? and Biedermann® have thus relaxed the contracted muscles of a crayfish claw and the 1 BuBNOFF and HEIDENHAIN: Archiv f. d. ges. Physiologie, 1881, xxvi, p. 137. 2 RICHET: Cong. périod internat. d. sc. méd. Compt. rend., 1879, Amst. ; 1880, vi, p- 554-560. 3 BIEDERMANN: Sitzungsber. der kénigl. Acad. der Wissensch. zu Wien, 1887, REV Digs 344 Allen Cleghorn. sartorius muscle of a dog to which veratrine had been given. Kaiser! tetanized the nerve of a muscle that had been brought into a state of tonic contraction by exciting its nerves with glycerine, and observed relaxation. Wedensky ? finds that an ordinary nerve muscle prepara- tion may be made either to relax or to contract according to the strength of the stimulus employed. Many other instances could be cited. Further, we possess ample evidence that cerebral influences may directly cause relaxation in muscular tissue. A familiar example is the action of the inhibitory fibres of the vagus on the heart. Mus- cular relaxation has even been obtained by direct cortical stimulation. Thus Bubnoff and Heidenhain? found that stimulation of the motor fields of contracted muscles, and indeed of other fields as well, would produce relaxation. Sherrington and Hering* have also obtained muscular relaxation by stimulating cortical motor areas. It is with these experiments that my own results should probably be placed, for they show that in the human subject sensory stimuli modify re- flexly the relaxation from voluntary muscular contraction as well as the contraction itself. The result of my experiments, then, favors the view that the accele- ration of relaxation is due to the augmentation of an “ active”’ relaxa- tion process, rather than to the inhibition of the contraction process, but it would perhaps, in the present state of knowledge, be unsafe to make too positive a statement regarding the nature of the phase of sinking energy in muscular contraction. In conclusion I desire to express my appreciation of the many valu- able suggestions of Professor Bowditch, under whose direction this work was done. SUMMARY. 1. A sensory stimulus applied at the beginning of a voluntary con- traction increases the height of the contraction. 2. The relaxation following a contraction with intercalated sensory stimulus is quicker and more complete than when no stimulus is given. 3. This acceleration of relaxation is not due to augmentation of the contraction of the antagonistic muscles, for the relaxation of the ex- KAISER: Zeitschrift fiir Biologie, 1891, xxviii, p. 423. WEDENSKY: Archives de physiologie, 1891, iii, p. 687. BUBNOFF and HEIDENHAIN: Joc. cit. 1 2 3 * SHERRINGTON and HERING: Archiv f. d. ges. Physiol., 1897, Ixviii, p. 222. Reinforcement of Voluntary Muscular Contractions. 345 tensors does not visibly differ in rapidity and extent from the relaxa- tion of the flexor muscles. 4. The acceleration of relaxation cannot be ascribed to the sensory stimulus inhibiting the discharge from the motor neurons and thus permitting the rapid passive extension of the muscles by the load of the ergograph, for the acceleration does not increase with the increase of the load. 5- In the present state of knowledge, the acceleration is best explained as an augmentation of an active relaxation process by the sensory stimulus. ON CERTAIN CHARACTERISTICS OF. THE PRESSURE SENSATIONS OF THE HUMAN SKIN. By GAYLORD P. CLARK,’ M.D. Professor of Physiology, Syracuse University. [From the Physiological Laboratory of the University of Leipzig.] BY pressure sensations are to be understood those sensations which are provoked by non-painful mechanical stimuli applied to the skin. It appears that such stimuli must produce a local deformation of the skin in order to be effective. In Meissner’s experiment of immersing the hand in mercury of the temperature of the skin, a considerable amount of pressure is exerted upon the skin of the sub- merged part, but no sensations are there provoked. In this case there is sufficient pressure to stimulate, but, a large surface being subjected to the same pressure, there is no deformation and consequently no sensation. Von Frey ! has shown that mechanical stimuli of threshold strength are felt only at the moment of their application, the continuation and end of the stimulus not being perceived; that is, the sensation van- ishes very soon after the application of the weight, and the removal of the weight is not noticed. He has shown, further, that stimuli above the threshold strength may be felt as continuing sensations, although the intensity of the sensation diminishes, the unloading being always more difficult to perceive than the loading. The sensa- tion may outlast the stimulus, probably as a result of the deformation of the skin, which remains for some time. Finally, von Frey has demonstrated that the effectiveness of the stimulus depends upon certain factors in addition to the strength of the stimulus; namely, upon the size and position of the surface stimulated and the rapidity with which the stimulus is applied. In order to determine more clearly the value of the physiological factors involved, von Frey devised test hairs (Reizhaare), which fur- nish a very circumscribed mechanical stimulus, capable of gradation, and thus measure quite accurately the number, position, and relative * von Frey: Abhandl. d. math. - physikal. Cl. d. kénigl. Sachsischen Gesellsch. d. Wissensch., 1896, xxiii, p. 175. . The Pressure Sensations of the Human Skin. 347 ‘ sensitiveness of the points designated by Blix! as “ pressure points.” These “test hairs” consist of short pieces of hair, preferably from the human head, glued by one end at right angles to the end of a small stick. For each hair two measurements are made; a microm- eter measurement of the diameter of the cross-section, from which the area of the cross-section is calculated; and the weight which the hair can lift when its free end is brought to bear on a scalebeam. The later measurement determines the “‘ power” of the hair. It has been found that approximately the maximal power can be obtained without undue bending of the hair, and further, that the area of the cross-section and the power remain quite constant for long periods of time. The “ pressure” of the hair is its power per unit of surface, that is, the quotient of its power divided by the area of its cross-sec- tion. Variety in the sectional surface of the different hairs used and variety in the power of different hairs of the same sectional surface can be obtained by taking pieces of hair of different length. By means of the test hairs it is found that the sensations of pres- sure are provoked only at certain so-called ‘“ pressure points” which are constant in their location but which vary in the number distributed to equal areas of skin. The pressure points are therefore separated by intervals which are not sensitive to this form of stimulus, and these intervals increase in size as the sensitive points are more scattered. The pressure points are fatigued by rapidly repeated stimuli, but soon regain their normal sensitiveness when left to them- selves. Upon the haired portion of the skin, estimated to amount to about 95 per cent of the whole, it has been found that the pressure points correspond to the number of the hairs, the point being located over the hair sac not far from the spot where the hair pierces the epidermis; —the hairs as a rule grow obliquely out of the skin. Such pressure points are subject to stimulation by movements of the hairs — which act as levers — as well as by the deformation pro- duced by the direct contact of objects with the surface of the skin. With such small surfaces as those of the test hairs it has been observed that physiological effectiveness is not proportional to the amount of pressure used. Test hairs of greater surface and power are more effective than those of smaller surface and less power not- withstanding their hydrostatic pressure may be the same. Test hairs, the power of which is proportional not to the surface of the applied end, but to the radius of that surface, are found to be of equal physi- 1 Buix: Zeitschrift fiir Biologie, 1884, xx, p. I4I. 348 Gaylord P. Clark. ological value. From this it is assumed that the organs thus stimu- lated are not superficial in their location but lie somewhat deeper, the effect of the deformation upon the deeper lying structures being more marked when the surface over which the pressure is applied is in- creased. With larger surfaces than those of the test hairs the deform- ation always attains its maximum effect at the deeper levels of the skin, and then the physiological effectiveness of the deformation-pro- ducing stimulus becomes proportional to its pressure. During the summer of 1897 I had the opportunity of co-operating with Professor von Frey in the Physiological Institute of the University of Leipzig in some extension of the investigations along the line on which he has been engaged, and I gratefully acknowledge my indebt- edness to him.! The first object of the research thus jointly under- taken was to determine whether deformation caused by traction (Zug), —which is opposite in direction to that produced by pressure (Druck), —excites the same organs that have been shown to be called into action by pressure, or whether the skin contains also organs which react to traction. It is evident that external objects in contact with the skin produce chiefly the deformations of pressure rather than those of traction, but a brief consideration will show that the tissues of the skin may be subject to pressure, or to changes of pressure, by movements of the underlying structures of the motor apparatus, and that the terminal organs of the so-called pressure sense may be thereby excited... The skin presents a very uneven surface, here convex, there concave, as it conforms to the varying contour of the bones and muscles over which it is stretched. A change of position of these structures must necessarily change the natural tissue-pressure in the overlying skin, increasing that of convex areas when their convexity is increased, and diminishing it when their convexity is diminished: increasing that of concave areas when their concavity is decreased and diminishing it when their concavity is increased. Variations in pressure corresponding to those caused by the deformation of local traction as well as that of local pressure from external objects, but due to body movements, may therefore play a réle in exciting the nerve organs of the skin, and the impulses thus provoked may be assumed to contribute to our knowledge of the position and the condition of a part, a function ascribed by some to “ common sensibility.” 1 The chief results of our observations have been briefly reported by von Frey to the kénigliche Sachsische Gesellschaft der Wissenschaften in Leipzig (Berichte, Aug. 2, 1897). The Pressure Sensations of the Human Skin. 349 Our observations were directed to very small and to large surfaces on the left wrist and thumb, and to the effects of momentary and con- tinued stimuli of different degrees of intensity. The results are given first for very small surfaces, then for large surfaces. Very Small Surfaces. — The method of investigation was as fol- lows: — The pressure points on the triangular non-haired area at the distal end of the anterior surface of the left forearm (which we may term for the sake of brevity the left wrist) were very carefully sought out by the aid of suitable test hairs. Each point was then marked with a minute drop of silver nitrate solution. The physio- logical character of the surface to be stimulated was thus determined so far as the pressure sense is concerned, and the stimulus could be applied as desired directly to the chosen sensitive point or points, or, as the pressure points in this particular locality are widely scat- tered, to a non-sensitive area. In order to ensure fixation of the surface to be tested and at the same time the comfort of the person upon whom the tests were to be made (the “ Reagent,” as we shall term him), the left forearm was held in a plaster of Paris form moulded to fit the forearm from the elbow to the tips of the fingers, leaving its anterior surface exposed sufficiently to allow the forearm to be drawn out and inserted at will. The stimuli were applied by means of a double-arm thin wooden lever 20 or 30 centimetres in length, the axis of which was supported by a heavy and practically immovable stand. The arms of the lever being of unequal length, the lever was brought into equilibrium by a rider placed upon the shorter and remote arm. A light straw attached at right angles to the end of the longer arm served to transmit its movements to the surface of the skin to which the stimulus was to be applied. The surface of the free end of the light straw, the area of which was 0.3 mm.” to 0.5 mm.?, was glued to the skin, various kinds of adhesive substances being used (Le Page’s ‘“‘ Liquid Glue,” Collodion, etc.). Weighting the longer arm of the lever—that towards the skin — served to produce pressure; weighting the shorter arm served, on account of the adhesion of the straw, to produce traction. Disturb- ing oscillations of the lever, and the consequent rapidly changing deformations of the skin, when the weights were brought to bear upon one or the other of the arms of the lever, were avoided by suspending the weights by rubber bands tied to the lever arms. Sometimes two equal weights were suspended at the same time from each arm of the lever at equal distances from its axis, the lever being 350 Gaylord P. Clark. thus left in equilibrium, and one weight was allowed to produce its effect by quickly raising the other with the hand. During the tests ‘care was taken to avoid all external disturbance, and the Reagent sat with closed eyes in the most comfortable position possible, atten- tive to the locality of the skin to be stimulated. The word “now” warned him when a stimulus was about to be applied, and the attention was then especially concentrated. Two protocols of such tests follow, one having been made upon an isolated sensitive pressure point, the other between previously located points upon a surface not sensitive to pressure: — June 4, 1897. Reagent C. End of straw glued upon a sensitive pressure point on the volar side of the left wrist. Surface loaded: 0.3 mm?. Actual Reply of Reagent to Stimulus of weight in grams. Pressure. Traction. 10 Pressure (after 20 sec.) weaker ; (after 30 sec.) vanished. Pressure, stronger than before; remains. Nothing. Traction ? Pressure, continuing, not strong; after 30 sec., vanished. Touch, vanishes very quickly. Touch, weak, only momentary. Pressure, rather strong, soon diminishing, after 20 sec. ap- parently gone, then again per- ceived, finally definitely van- ished. Touch, very weak; after a few seconds, gone. Touch (better sensation of de- formation), stronger than be- fore, lasting longer (about 20 sec.). Touch, continues some seconds. Traction, probably; after 20 sec., gone. Pressure? Not strong, The Pressure Sensations of the Hluman Skin. 3518 June 5,1897. Reagent C. End of straw glued upon aspace between three pressure points on the volar side of the left wrist. Surface loaded: 0.3 mm?. Actual Reply of Reagent to a Stimulus of weight in grams. Pressure. Traction. Touch, momentary but distinct. Touch, the same strength but lasting longer. Pressure, distinct and lasting ; after 20 sec., weaker ; after 35 sec., uncertain ; after 50 sec., nothing. Touch, momentary pressure. Touch, momentary. Touch, momentary, weaker than before. Touch, momentary, somewhat more distinct. Touch, pressure distinct but momentary. Touch, distinct and lasting ; after Io sec., weaker ; after 20 sec., vanished. Touch, very momentary and somewhat weak. Touch, momentary; after 30 sec., gone. Touch, momentary, but as strong as before. Pressure, continuing, but not long; after a few seconds, gone. From the foregoing and other similar tests it was found that the so-called pressure points shown to be sensitive to the deformation caused by pressure are equally sensitive to the deformation caused by traction, and, what is most striking, it was seen that with very small surfaces there is inability to determine the direction of the deformation, that is, to distinguish between pressure and traction, the sensation being that simply of a deformation even with strengths of stimulus which are very marked and the action of which is long continued. Pressure and traction each produced compara- tively quick fatigue, the duration of the sensation falling markedly 352 Gaylord P. Clark. short of that of the stimulus, and the removal of the stimulus being unperceived. Attention was next turned to the determination of the effect of fatigue produced by a long continued pressure stimulus upon momentary traction stimuli following immediately after the removal of the fatiguing load. The stimulus used to produce fatigue con- sisted of a 400 gram weight hung upon the pulley of the axis of the lever. The distance of the weight from the axis was 75 that between the axis and the straw by which the load was transmitted to the skin. Thus the actual weight upon the tested surface was 10 grams. The sectional area of the end of the straw being 0.5 mm.?, the actual pressure was 20 grams to the square millimetre. The momentary stimuli were obtained by means of small double-arm thin wooden levers arranged at right angles to the main lever already described and so placed that they could be made to strike upon it on each side of and at equal distances (e. g., about ;5 the lever length) from its axis. These levers were moved by weights hung upon the pulley of the axis on the side towards the main lever, and the height of their stroke was determined by an adjustable screw in a post placed under the remote arm of each lever to serve as a stop. This arrangement permitted the selection of a stroke that could provoke a weak or a distinct sensation as desired; and, by depress- ing the remote arm till it touched the stop and then suddenly releasing it, any number of uniform momentary stimuli could be applied. An isolated, sensitive pressure point was selected and the end of the straw glued upon it. The following protocols set forth the results obtained from two series of tests in which momentary stimuli of pressure and then of traction were applied, the fatigue in both cases being caused by pressure. The results of such tests as those here given in detail show that with very small surfaces repeated momentary pressure or traction stimuli of a uniform strength just above the threshold value always excite a similar sensation, if the pause between successive stimuli is sufficient to avoid fatigue. The results show also that if a strong pressure-producing stimulus be allowed to act sufficiently long to produce fatigue and then removed and momentary rhythmical stimuli immediately applied, the latter are not at first perceived, but soon begin to be felt, although imperfectly, and with an increasing distinct- ness inconstant in degree. Several minutes may elapse before the The Pressure Sensations of the Human Skin. 353 sensations regain their original uniform strength. It was shown further that the fatigue produced by pressure is as effective in im- July 3, 1897. Reagent C. End of straw glued upon a very-sensitive pressure point on the radial side of the left wrist. Surface loaded: 0.5 mm?. Fatiguing stimulus 20 grams mm*. Momentary or “stroke” stimuli as near threshold strength as possible. A.— Momentary or stroke stimulus applied as a pressure stimulus. ae Stimulus. Statements of Reagent. Stroke stimulus applied several times. | Felt each time, but weak. Fatiguing stimulus applied. Weaker. Uncertain. Nothing. Fatiguing stimulus removed. Nothing. Stroke stimulus applied. No. Uncertain. No. No. Yes, very weak, Uncertain. Yes, weak. Uncertain. Yes, weak. Yes, weak. No. No. Yes, weak. No. Yes. Yes, very weak. Yes. Yes, Uncertain. Yes, weak. Yes. , it becomes more distinct. Yes, quite constant and distinct. f 354 Gaylord P. Clark. July 3, 1897. Reagent C.— Continued. B.— Momentary or stroke stimulus applied as a traction stimulus. Time. Stimulus. Statements of Reagent. Stroke stimulus applied several times. | Felt each time, weak but dis- tinct, about as strong as pressure stimulus in A. Fatiguing stimulus applied. Not completely vanished. Uncertain, variable. Fatiguing stimulus removed. Nothing. Stroke stimulus applied. Uncertain. No. No. Uncertain. No. No. No. Uncertain. Yes, but very weak. Yes, a little more distinct. Very weak. Yes. Yes, still weak. Uncertain. Uncertain. No. No. Yes. The Pressure Sensations of the Human Skin. 355 pairing the sensations provoked by subsequent momentary traction stimuli as those called out by pressure stimuli of the same strength. Therefore the pressure which causes fatigue for pressure also causes an equal fatigue for traction. Large Surfaces. — It has been shown thus far that with very small surfaces points most sensitive to pressure are also most sensitive to traction; that their sensitiveness to each kind of stimulus is approxi- mately the same; that fatigue produced by pressure is fatigue for subsequent traction stimuli as well as for pressure stimuli; and that even with strong and continued stimuli producing deformation in one or the other direction there is inability to determine the direction of the stimulus, that is, to distinguish between pressure and traction. Tests were now made upon larger surfaces. A cork disc with a sec- tional area of 50 mm.” was slipped on to the free end of the straw used in the foregoing tests and glued upon the surface to be tested. Observations were first made to determine the effect of momentary stimuli applied to the skin upon the most convex part of the ball of the left thumb. Such stimuli were obtained by the “stroke levers” arranged as in the foregoing tests and could be changed at will from pressure to traction. These observations were followed by others with continued stimuli of different strengths. Following is a protocol: — July 7, 1897. Reagent F. End of cork glued on the convex surface of the ball of the left thumb. Surface loaded: 50 mm?. Very strong momentary stimuli by “stroke levers.” Kind Statements of Kind Statements of of Stimulus. Reagent. of Stimulus. Reagents. Pressure. Yes. Pressure. Yes. Traction. Yes, the same. Traction. Strong. Traction. Yes. Pressure. Strong. Pressure. Wiese Traction. Strong. Pressure. Weak. Pressure. Perhaps weaker. Intervals 5 to 10 sec. Traction. Strong. Traction. Strong. (3) ) n =) ec ae 2 ve) n a a > ~ o ~ S Le Pressure and traction not distinguished. 356 Gaylord P. Clark. ’ Continued stimuli. Instead of “stroke levers,” weights were hung on main lever four- tenths of its length distant from its axis. Duration of stimulus from 5 to 10 seconds, with intervals of several seconds. With 100 grams. With 100 grams acting as pressure or traction according to the pleasure of the observer and without previous information to the person upon whom the observations were made, 10 such stimuli were each correctly judged as to the direction of the stimulus. With 20 grams. Kind of Stimulus. | Statements of Reagent. | Kind of Stimulus. | Statements of Reagent. Pressure. Uncertain. Traction. Traction. Traction. Perhaps traction. Pressure. Traction, uncertain, Traction. Perhaps traction. Pressure. Also traction. Pressure. Pressure. Traction. Also traction. Pressure. Uncertain. Repeated tests of the above character made on both of us upon the wrist as well as upon the thumb and with stimuli of different strengths, showed that with large surfaces momentary stimuli of pressure and traction even of marked strength, provoking distinct sensations, could not be distinguished as to their direction, a deformation undeter- mined in character being in each case alone perceived. When stimuli were continued instead of momentary it was found that with smaller weights, that is, with a diminution in the strength of the stimuli, the ability to judge of the character of the deformation was also impaired. In order to obtain equality in the rapidity of application of the con- tinued stimuli and thus eliminate the influence of that factor, which had previously been shown to be an important one in the effective- ness of pressure stimuli, the following arrangement of the apparatus already described was made. The weights hanging on rubber bands attached to each side of the axis of the main lever were supported by the two levers previously used as “stroke levers” but now placed under the main lever. The ends of the arms that supported the weights rested upon a shelf attached to a horizontal clock-work kymograph drum. Rotation of the drum carried the ends of the levers down and The Pressure Sensations of the Human Skin. 357 allowed the weights which they supported to act upon the main lever. By placing a sufficiently heavy rider upon the remote arm of either lever that lever could be held in place so that only one weight would be brought to act upon the main lever when the drum was set in motion. The excursion of the drum was limited to about one-fourth revolution. The stimulus was removed by turning the drum back by the hand to its former position, the shelf lifting the end of the lever and thereby the weight. A counterpoise clamped on the opposite side of the drum served to offset the effect of the weights upon the shelf when the drum was at rest and a stimulus was not being applied. In the above manner a continued pressure or traction stimulus of any desired strength could always be applied with the same rapidity. A protocol of a test made under such conditions is presented below. The tests showed clearly that a certain degree of strength is essen- tial to a correct judgment of the kind of deformation produced by continued stimuli upon large surfaces. By gradually increasing the strength of stimulus, the size of the surface stimulated and the rapid- ity of application of the stimulus remaining constant, it was found that the ability to distinguish the direction of the deformation ap- peared with a certain increase of weight, not suddenly but gradually, some still undetermined difference in the character of the stimulus being first noticed. With stimuli of sufficient strength a correct judgment as to their direction could always be formed: — July 16, 1897. Reagent F. End of cork glued upon the convex surface of the ball of the left thumb. Surface loaded: 10 mm”. Continued stimuli 30 grams, two-tenths lever length from axis of main lever, corresponding to an actual load of 6 grams. Duration of stimulus, 10 seconds. Kind of Stimulus. Statements of Reagent. Pressure. Distinct excitation, continuing; not determinable whether pressure or traction, perhaps traction. Traction. The same, but weaker, lasting a very short time. Traction. More distinct, does not last very long; believe it is pressure. More distinct than second stimulus, about as long as first. Pressure. Distinct, lasting ; different from the preceding exci- tation, perhaps traction. Pressure. Distinct and lasting; cannot say what it is. Traction. Weaker, very weak, almost momentary. 358 Gaylord P. Clark. 30 grams, pressure side 2 spaces from axis; actual load 6 grams. 30 grams, traction side 2'4 spaces from axis; actual load 7.5 grams. Kind of Stimulus. Statements of Reagent. Traction. Quite distinct, not very strong, impossible to dis- tinguish. Pressure. Distinct, lasting; not determinabie whether pressure or traction, perhaps traction. Pressure. Distinct, lasting, gradually vanishing; perhaps pres- sure, quite uncertain. Traction. Distinct, lasting ; not determinable. Traction. Yes, distinct and lasting; perhaps pressure. Pressure. Exactly the same as to intensity and character. All stimuli notably of equal strength. (It is to be noted that the removal of the stimulus was not perceived, and that the sensation outlasted the stimulus.) CONCLUSIONS. Collectively the tests show that the recognition of the direction of a deformation, that is, the ability to distinguish between pressure and traction, depends upon the size of the surface stimulated, the duration of the stimulus, and the strength of the stimulus. The perception of a deformation is therefore a simpler psychological process than the recognition of its character. The impulses provoked by variations in pressure appear to contain in themselves no determination of the direction of the exciting deformation. That determination is gained by a combination of impulses of some intensity, of more than momen- tary duration, and arising from a not too limited area of the skin. Our demonstration that the points most sensitive to pressure are equally sensitive to traction; that the impulses produced by stimuli in either direction provoke simply sensations of deformation without indications as to its direction; that a stimulus which causes fatigue for pressure stimuli causes also fatigue for traction stimuli; and that the strength of the stimulus, the rapidity of its application, and the size and the location of the surface to which it is applied, influence equally the effectiveness of traction and of pressure stimuli — makes it probable that the organs in the skin which are stimulated by pressure are also stimulated by traction. THE MOVEMENTS OF THE STOMACH STUDIED BY MEANS OF THE RONTGEN RAYS.! By W. B. CANNON. [from the Laboratory of Physiology in the Harvard Medical School.) Page We yp bairoduetory iteratare:! “9S Bee ee a) US aah ieee heme thod yess 91) ade a ARG OZ III. The anatomy of the eee Eade its iacers to athe alien Ais 1 304. IV. The normal movements of the stomach. . . . ..... . . 365 t.. Movements of the pyloric pare 55 29 ee oes 367 2. Movements-of the pyloric sphincter’ . 2 2). 2 \4').'. ! 368 a. ‘Activity of the Cardia€ portiom.:) 0) 2 ne) wats sa os OS A 3z7O V. The movements of the stomach in vomiting . . Sate eee, 2 y/s: VI. The effect of the movements of the stomach on the had Peeice Ou eyes: Vit. Salivary cipestiom in’ the stomach... 2.09). of. a ss Se) BO VIII. The inhibition of stomach movements during emotion . . . . . 380 oS the stomach gives no obvious external sign of its workings, investigators of gastric movements have hitherto been obliged to confine their studies to pathological subjects or to animals subjected to serious operative interference. Observations made under these necessarily abnormal conditions have yielded a literature? which is full of conflicting statements and uncertain results. The only sure conclusion to be drawn from this material is that when the stomach receives food, obscure peristaltic contractions are set going, which in some way churn the food to a liquid chyme and force it into the in- testines. How imperfectly this describes the real workings of the stomach will appear from the following account of the actions of the organ studied by a new method. The mixing of a small quantity of subnitrate of bismuth with the food allows not only the contractions of the gastric wall, but also the movements of the gastric contents ’ The first account of this work was given at the meeting of the American Phys- iological Society, in May, 1897 (see Science, June 11, 1897); and the later results were presented at the meeting of the Society in December, 1897. A summary of the results was published in the Proceedings of the American Physiological So- ciety, this Journal, 1898, i, p. xiii. A report of the research was also made to the Boston Society of Medical Sciences, February 15, 1898. * POENSGEN (Die motorische Verrichtungen des menschlichen Magens und ihre Storungen, Strassburg, 1882,) gives a comprehensive review of the literature to that date. 360 W. B. Cannon. to be seen with the R6ntgen rays in the uninjurea animal during nor- mal digestion. An unsuspected nicety of mechanical action and a surprising sensitiveness to nervous conditions have thereby been disclosed. I. INTRODUCTORY LITERATURE. The early writings on the subject of gastric movements are charac- terized by general inferences from physical laws and from the anatom- ical structure of the stomach. According to Galen,! the stomach had four functions: to draw the food from the mouth ( facuwltas attractrix), to retain the food (/facultas retentrix) during the process of chemical digestion ( facultas alteratrix), and, finally, to pass the changed mate- rial onward (facultas expultrix). In later writings the facultas at- tractrix failed to appear as one of the functions of the stomach. Fallopius,” in the sixteenth century, changed the notion of the facad- tas retentrix by suggesting that the pylorus alone performed this office, and that the muscles of the gastric wall could help only by remaining quiet. Thus the facu/tas alteratrix and the facultas expul- trix are left as true gastric functions. It is with the latter activity and its effects that this paper is concerned. The ideas of the early writers concerning the pylorus and cardia are of interest. The cardia, they were agreed, is closed during nor- mal digestion in order to keep the food from re-entering the cesoph- agus. The pylorus they looked upon as the ruler of the actions of the stomach. Such names as pylorus (keeper of the gate), janitor justus, and rector, which the first investigators gave to the sphincter, indicate their theories of its functions.. The passage of chyme into the duodenum, the keeping of undigested food in the stomach, the act of vomiting, were all dependent, they believed, an the “will” of the pylorus.’ : No substantial advance was made beyond these hypotheses until the beginning of the eighteenth century, when Wepfer and Schwartz applied the experimental method to the study of the gastric move- ments and laid the foundation of a more accurate knowledge. Wep- fer* vivisected wolves, dogs, and cats, and observed constrictions following stimulation of the stomach. He remarked a general con- 1 GALEN: Opera omnia. Leipzig, 1822, iii, pp. 275, 281. 2 FALLOPIUS: Opera omnia; observationes anatomice. Frankfort, 1600, Pp: 412: 8 VAN HELMONT: Opera omnia. Frankfort, 1707, p. 215. * WEPFER: Historia cicute aquatice. Basel, 1679, p. 152 ef seg. Lhe Movements of the Stomach. 361 traction of the pyloric part in vomiting (pp. 152, 168, and 250), and noted peristaltic and antiperistaltic movements passing over the organ. About the middle of the stomach he frequently saw a deep constriction. The investigations of Schwartz! are more valuable in that his search was for the normal action of the muscular coats. The movements, as he observed them, were generally only slight. They began either at the pylorus and passed to the left, half-way to the cardia, or started at the fundus and went to the pylorus. The con- tractions and relaxations, following one another, formed larger or smaller depressions and elevations, z. é., more or less definite waves. Near the middle of the last century, Haller,? after confirming the results obtained by Schwartz and Wepfer, summarized his knowledge of the motor functions of the stomach as follows: In general, con- traction alternates with relaxation, so that the stomach is, now here, now there, made narrower by longitudinal or transverse depressions ; then in these same places relaxation and bulging occur (pp. 260-262, and p. 276). So long as both apertures are closed the food is driven hither and thither by the shifting movements. It first takes a definite direction when the cardia or the pylorus opens. Ifthe cardia opens, there is an antiperistalsis followed by regurgitation and vomiting (p. 281). If, on the contrary, the pylorus relaxes, a contraction, starting at the cesophagus, pushes the contents of the stomach into the duodenum. The pylorus allows the passage of fluids, but if it be stimulated by over distention or by hard pieces of food, it closes tightly (p. 277). . Such was the knowledge of gastric movements in Haller’s time. A comparison of his descriptions with those in any standard work on physiology published ten or fifteen years ago will show that, despite very many researches, little advance had been made. Examinations of animals and men with gastric fistulas, studies of the stomach through the atrophied abdominal wall, and vivisection, have yielded numerous results, but these have not been harmonious, and have led to much controversy. Prominent in this mass of material as a val- uable contribution are Beaumont’s careful observations through the gastric fistula of Alexis St. Martin. Beaumont’s work has recently been confirmed by Hofmeister and Schiitz, who, with Rossbach, Hirsch, Openchowski, and others, have presented during the last twelve years 1 B. SCHWARTZ in Haller’s Dissertationes anatomice. Gdttingen, 1746, i, pp. g Pp 337-338. ? HALLER: Elementa physiologiz. Berne, 1764, vi, p. 260 e¢ seg. 262 W. B. Cannon. oO much new and interesting information. Since, however, it will con- duce to clearness to set forth the results of these investigations in connection with my own work, their consideration will be deferred until later. It will then appear that these later investigations, like the earlier researches, disagree as to the details of the stomach movements. Such differences in results are the proper outcome of the abnormal conditions under which the studies have been conducted. Obviously, in order to see the natural movements of the stomach, the organ should be observed in its natural state, and not after it has been dis- turbed by removal from the abdomen or by the adhesions and losses of substance incident to gastric fistulas. As a means of watching the gastric motor activities under normal circumstances, Dr. H. P. Bowditch, in the autumn of 1896, suggested the use of the Rontgen rays. The present paper is the result of the work thus far completed. The kind assistance and stimulating counsel of Dr. Bowditch throughout the investigation are gratefully acknowledged. II. THE METHOD. The method consists in mixing subnitrate of bismuth—a harm- less, non-irritating powder — with the food, and observing the move- ments of the swallowed mass by means of the Rontgen rays.. As is now generally known, the picture thrown on the fluorescent screen by the Rontgen rays is one of shadows of varying intensity; the denser the substance, the darker the shadow. There is nothing in the structure of the stomach to cause it to cast a different shade from that of its neighboring organs. But the dense bismuth powder, uniformly mixed with the food that fills the stomach, throws the dark shadow of the stomach contents on the screen, and the changes in the shape of the outlines indicate the intrinsic movements of the organ. The animal used throughout the research was the cat. The meal given before making an observation consisted of from fifteen to eighteen grams of dry bread, softened to a mushy mass by milk, hot water, or thin gravy, and mixed with from one to five grams of subnitrate of bismuth, according to the purpose in hand. One or two grams of the bismuth compound produce a dim shadow of the stomach within which may be clearly seen the darker forms of food containing a larger amount of the substance; three grams are enough for ordinary observations; four or five grams are needed to The Movements of the Stomach. 363 see the passage of food from the pylorus. The cat was usually kept from eating for at least twelve hours before an observation, in order that the stomach might be wholly free from contents transparent to the X-rays. The construction of the holder on which the cat was tied is shown in the diagram (Fig. 1). It consisted of a framework supporting a sheet of black cotton cloth. The frame was made of two side pieces each 80 cm. long and 2.5 cm. square, connected at either end by blocks 2.5 cm. thick, 12.5 cm. wide, and 16 cm. long. The black cloth, which sagged for the com- fort of the cat, was held by strips és 7 Sarr Peeve fa aw of wood nailed to the inner face 7 —F sa Oma of the frame. Through the side pieces were holes 0.6 cm. in di- ameter, and 5 cm. apart. Each of the leather nooses securing the legs went down through one of these holes, and up through another, in which it was made fast by forcing a pointed peg into the hole with it. The cat's head was held by two pegs, one on either side FIGURE I. of the neck, joined above by a leather thong. One of the pegs was movable and could be put in any of the three holes, 3, 4.5, or 6 centimetres from the other peg, according to the thickness of the cat’s neck. For seeing the regular movements of the stomach the cat was tied back downward, with the fore paws in nooses at either side, and with the hind legs stretched out and fastened to the holder at the cat's right. For watching the passage of food from the pylorus, the hind legs were both fastened to the left side of the frame, so that the cat lay on her left flank. Most of the female cats would lie on the holder by the hour without making any attempt to break away or manifesting any signs of discomfort. In marked contrast was the behavior of the males. Almost without exception they seemed worried when fastened down. The interesting effects of these different ways of reacting to novel surroundings will be described later. The cat-holder was supported at either end. Belowit at a distance of 19 cm. was placed the tube generating the Rontgen rays. This tube had a self-regulating device for maintaining a uniform vacuum, —very useful in that it allowed long observations with rays of uni- form intensity. A Topler-Holtz machine, run by a small motor, produced the electrical discharge through the tube. This apparatus 364 W. B. Cannon. was placed behind the holder. The light from the tube and the machine was shut off from the observer by drapings of black cloth, so that in the dark room where the work was carried on it was possible to use an open fluorescent screen with sides only two centimetres high. This plan was found especially valuable in that it permitted tracing the outlines of the stomach on tissue paper laid over the fluorescent surface. Ill. THE ANATOMY OF THE STOMACH AND ITS. RELATIONS TO THE SHADOW. It must be constantly borrre framintthat-the shadows described in this research are cast by the gastric contents, — not by the stomach itself. Therefore the movements of the organ are not seen directly, but are indicated by their effect on the contained food. Variations in the length and breadth of the stomach can be inferred from changes in the outline of the shadow, but variations in the front-to-back diameter of the organ must be judged from changes in the intensity of the shadow. The form of the active stomach soon after food has been taken is shown in outline in Figure 2. Since the several parts of the stomach are to be mentioned frequently, it will be well to recall them here in their relations to the outline. The larger, cardiac part of the organ lies to the left of a line through wx. Into it the cesophagus opens through the cardiac sphincter, or cardia, at c. The pyloric part, ie. Which includes all of the stomach situated at the right of a line w 2, is closed by the pylorus at 7. This part has two divisions; the antrum at the right of the line y 2, and the preantral part of the pyl- oric portion, or middle region of the stomach, between the lines wa and yz. The lesser curvature corresponds approximately to the anterior border of the shadow c wp, the greater curvature to the more extensive sweep, ¢ f, along the posterior border. The wall of the cat’s stomach consists of three coats, but as this paper deals only with the functions of the muscular coat, that alone will be described. The gastric muscular fibres are disposed in three sets: an outer longitudinal layer, a middle circular layer, and a set Right. * Post. FIGURE 2. The Movements of the Stomach. 365 of inner oblique fibres. The longitudinal fibres continue those of the cesophagus, and, radiating over the cardiac end, become more marked along the curvatures than on the front and back surfaces. Over the antrum they lie in a thick, uniform layer. The circular fibres form a complete investment, and are arranged in rings at right angles to the curved axis of the stomach. Towards the pyloric end they become denser and stronger, and at the pylorus form a thick bundle, the py- loric sphincter. Separating the antrum from the rest of the stomach, at y %, is a special thickening of the circular fibres, called by the early writers! the “transverse band,’ and described by Hofmeister and Schiitz? as the “sphincter antri pylorici.” The oblique fibres start from the left of the cardiac orifice, and pass as two strong bands along the anterior part of the dorsal and ventral surfaces, giving off fine fasciculi to the circular musculature; towards the antrum they gradually disappear. The musculature of the stomach consists of smooth muscle fibres, the chief physiological characteristics of which are slowness of con- traction, rhythmic alternation of contraction and relaxation, and a very great tonicity, or power of prolonged contraction. The action of these muscles in the process of gastric digestion is now to be considered. IV. THE NORMAL MOVEMENTS OF THE STOMACH. Since the time of Haller the chief contributors to the knowledge of the mechanics of the stomach have been Beaumont, Hofmeister and Schiitz, and Rossbach. Beaumont’s famous investigations on Alexis St. Martin are recorded in almost all general works on physiology. Through a gastric fistula he introduced a thermometer-tube and observed how it was affected by the motions of the stomach. His conclusions are as follows: “The circular or transverse muscles contract progressively from left to right. When the impulse arrives at the transverse band, this is excited to a more forcible contraction, and, closing upon the alimen- tary matter and fluids contained in the pyloric end, prevents their regurgitation. The muscles of the pyloric end, now contracting upon the contents detained there, separate and expel some portion of the chyme. . . . After the contractile impulse is carried to the pyloric 1 BEAUMONT: Physiology of digestion. Burlington, 1847, p. 104. * HOFMEISTER and Scuttz: Archiv fiir exper. Pathol. und Pharmakol., 1886, SRD 7s 366 W. B. Cannon. extremity, the circular band and all the transverse muscles become relaxed, and a contraction commences in a reversed direction, from right to left, and carries the contents again to the splenic extremity to undergo similar revolutions.” ? In close accord with Beaumont’s description of the activities of the human stomach are the records of the investigations on the stomach of dogs by Hofmeister and Schiitz.2 They removed the stomach from the body and placed it ina moist chamber, kept at body-heat and covered with glass. Under such conditions the organ remained active for from sixty to ninety minutes. A typical movement is de- scribed by these observers as composed of two phases. In the first phase a constriction of the circular fibres, deeper on the greater cur- vature, starts a few centimetres from the cardia and passes towards the pylorus. As the constriction proceeds it increases in strength until a maximum is reached about two centimetres in front of the an- trum. This annular contraction, called by Hofmeister and Schiitz the *‘preantral constriction,” closes the first phase. Immediately there- after the strong sphincter antri pylorici, or transverse band, contracts. Now, while the preantral constriction is relaxing, the sphincter antri pylorici tightens still more, and the antrum is shut off from the rest of the stomach. As soon as this has occurred a general contraction of the muscles of the antrum follows. Relaxation begins at the sphincter antri pylorici and progresses slowly toward the pylorus; it is sometimes accompanied by an antiperistaltic movement. Although Rossbach ® also used dogs his results vary considerably from those of Hofmeister and Schiitz. This discrepancy is possibly accounted for by a difference in method, for Rossbach left the stomach in the body. The dogs were treated with morphia and curare, and the abdomen was then widely opened, so that the move- ments could be clearly seen. When the stomach was full Rossbach saw deep constrictions begin near the middle and pass in waves to the pylorus. At first these movements were weak: later, however, they became more vigorous. The fundus remained in tonic contraction about its contents and took no part in the peristalsis. Before attempting to explain the difference in the records of these observers I shall give an account of what may be seen in a cat by use of bismuth subnitrate and the Rontgen rays. | BEAUMONT: Joc. cit., p. 106. HOFMEISTER and SCHUTZ: Joc. cit., p. I. 1 8 ROssBACH: Deutsches Archiv fiir klinische Medicin, 1890, xlvi, p. 296. The Movements of the Stomach. 367 I. Movements of the Pyloric Part. — Within five minutes after a cat has finished a meal of bread, there is visible near the duodenal end of the antrum a slight annular contraction which moves peristaltically to the pylorus: this is followed by several waves recurring at regular intervals. Two or three minutes after the first movement is seen, very slight constrictions appear near the middle of the stomach, and, pressing deeper into the greater curvature, course slowly towards the pyloric end. As new regions enter into constriction, the fibres just previously contracted become relaxed, so that there is a true moving wave, with a trough between two crests. When a wave swings round the bend in the pyloric part the indentation made by it deepens; and as digestion goes on the antrum elongates and the constrictions run- ning over it grow stronger, but, until the stomach is nearly empty, they do not entirely divide the cavity. After the antrum has length- ened, a wave takes about thirty-six seconds to move from the middle of the stomach to the pylorus. At all periods of digestion the waves recur at intervals of almost exactly ten seconds. So regular is this rhythm that many times I have been able to determine within two or three seconds when:a minute had elapsed simply by counting six simi- lar phases of the undulations as they passed a given point. It results from this rhythm that when one wave is just beginning, several others are already running in order before it. Between the rings of constric- tion the stomach is bulged out, as shown in the various outlines in Figures 3, 4, and 5. The number of waves during a single period of digestion is larger than might possibly at first be supposed. In a cat that finished eating fifteen grams of bread at 10.52 A.M., the waves were running regularly at 11.00 o’clock. The stomach was not free from food until 6.12 P.M. During that time the cat was fastened to the holder at intervals of half an hour and the waves were always observed, following one another in slow and monotonous succession. At the rate of three hundred and sixty an hour, approximately two thousand six hundred waves passed over the antrum during that single digestive period. From the above review, it will be manifest that my observations of the movements of the pyloric part agree closely with those of Ross- bach, but differ considerably from the harmonious results of the work of Beaumont, and Hofmeister and Schiitz. Beaumont’s methods, however, may be justly criticised on the ground that the thermometer- tube which he held in the stomach was wholly unlike food and very liable to bring about unwonted contractions in so sensitive an organ 368 W. B. Cannon. as the stomach. Further, the movements observed by Hofmeister and Schiitz, as Ewald has pointed out,! may easily have resulted from the abnormal stimulus due to lack of blood —a potent cause of per- istalsis. And it will be shown later that the accounts given by these investigators describe very well the actions of the stomach when stimu- lated by an unusual irritant. In this connection it may be added that since the publication of the preliminary notice of my work,? Roux and Bathazard,? using the Rontgen rays, have published the results of observations on the stomachs of the dog and man, similar to those thus far described in this paper. The fact that my observations and those of Roux and Bathazard were conducted under normal conditions, and that the conditions of Rossbach’s experiments were more nearly normal than those of the other observers mentioned, warrants the conclusion that the pyloric part has a more important function than that of merely expelling the contents of the stomach into the intestines. After summarizing the description given by Hofmeister and Schiitz, Ewald, for a przorz rea- sons, declares: ‘‘I cannot accept this view. The plain fact that the pyloric portion secretes a strongly digesting fluid containing pepsin and hydrochloric acid, proves it to be an important part for the pep- tonizing function of the stomach.” * The account of the remarkable manner in which the pyloric portion performs this function must be deferred until the movements of other parts of the stomach have been considered. 2. Movements of the Pyloric Sphincter. — Rossbach® mars _ his otherwise careful work by declaring that the pylorus is tightly closed during the whole digestive period of from four to eight hours; and that then the sphincter relaxes and the peristaltic waves empty the stomach. That this is not the normal action of the sphincter has been shown by several observers. Hirsch® watched dogs with duodenal fistulas and saw food come from the stomach at intervals. of one-fourth of a minute to several minutes. Roux and Bathazard ’ 1 EwALp: Lectures on digestion. London, 1891, p. 66. 2 CANNON: Science, June 11, 1897, p. 902. 3 Roux et BATHAZARD: Comptes rendus de la soc. de biologie, 1897, Io S, iv, pp- 704, 785, and Archives de physiologie, 1898, 5 S, x, p. 85. * EWALD :, lo: Cif. 107, 5 RossBACH: Deutsches Archiv fiir klinische Medicin, 1890, xlvi, p. 317- ®° HirscuH: Centralblatt fiir klin. Medicin, 1892, xiii, p. 994. 7 Roux et BATHAZARD: Comptes rendus de la soc. de biologie, 1897, 10 S,. AVA DAO Se The Movements of the Stomach. 369 maintain that in dogs food enters the duodenum at the completion of each wave of constriction. Observations on the cat, however, do not support their view, but agree rather with the statement of Hirsch. In cats fed with bread mixed with subnitrate of bismuth, ten or fifteen minutes elapse after the first constriction in the antrum before any food can be seen in the duodenum. When food does appear it is spurted through the pylorus and shoots along the intestine for two or three centimetres. Not every constriction-wave forces food from the antrum. On one occasion, about an hour after the movements began, three consecutive waves were seen, each of which squirted food into the duodenum. The pylorus remained closed against the next eight waves, opened for the ninth, but closed once more against the tenth and eleventh. For each of the four succeeding waves the sphincter relaxed, but blocked the food brought by three constrictions that followed; and in this irregular way the food continued passing from the stomach. Near the end of gastric digestion, when the con- strictions are very deep, it may be that the pylorus opens for every wave. When a hard bit of food reaches the pylorus, the sphincter closes tightly and remains closed longer than when the food is soft. This action of the sphincter was shown by giving with the regular food of the cat a dry, hard pellet of equal parts of starch paste and bismuth subnitrate, about the size of a pea. The food itself contained merely enough bismuth to throw a dim shadow, near the centre of which the pellet could be clearly seen as a dark object. The continual passing of the contraction-waves finally brought the little ball to the pylorus. When it arrived there, five grams of bismuth subnitrate were intro- duced into the stomach through a tube in the cesophagus. This was done in order that the food passing into the intestines after the ball came to the pylorus, might be distinguished from that which had gone on before. By kneading the stomach the bismuth was distributed, as shown by the uniformly black shadow. The pellet could still be seen nearthe end of the antrum when the constrictions passed over it. Now, although the waves continued to run regularly, the very black food did not gather in the intestines in sufficient amount to be recog- nized until forty-two minutes after it had been introduced. And when, finally, the food did show itself in the intestines, its shadow contrasted strongly with that of the food which had already passed on. The slowness of the expulsion is not to be regarded as wholly due to the 24 370 W. B. Cannon. FIGURE 3. hard mass. No doubt the knead- ing of the stomach mixed the con- tents of different parts of the organ and brought to the pylorus food not yet sufficiently digested to be passed by that selective sphincter. But this does not explain the whole delay. Food similar to that given here except that it contained no hard particles has usually been seen as small masses in the intestines within fifteen minutes after being swallowed. N. D Ba S oe oN = fe) -~ Ss & DOG IT: Dextrose. Glycogen in the liver = 0.392 gr. | Weight in keg. of urine Amount ! 6? hours’ urine. relation between the sum of the dextrose and nitrogen, we obtain 550.6 grams D: 147.8 grams N:: 3.72: 1. By adding to this nitrogen that derived from the feeces (84 X 0.23) we obtain the following ratio: 550.6 grams D: 149.7 grams N:: 3.67: 1. According to this latter ratio the amount of sugar obtainable from proteid may be 58.7 per cent of the molecule. This great sugar excretion is accompanied by a highly remarkable rise in the proteid metabolism above that in fasting. Thus on the third day of fasting 4.17 grams of nitrogen were eliminated, whereas 398 Reilly, Nolan, and Lusk. on the fifth day of fasting and the second of diabetes the nitrogen in the urine amounted to 18.76 grams, an increase of 450 per cent. Another experiment shows an increase of 540 per cent and two more an increase of 340 per cent each. A parallel to this rise in proteid metabolism is only to be found in the literature of phosphorus poisoning, and indeed the cause of the high metabolism, namely, the non-burning of the carbohydrates, would seem to be identical in the two cases. In the case of diabetes the sugar is removed and its well-known sparing influence over proteid metabolism is eliminated.1. The explanation of the similar metabolism in phos- phorus poisoning which seems most plausible to us is that the proteid sugar instead of being burned is converted quantitatively into fat, with the resulting high proteid metabolism noted in fatty degeneration. Experimental evidence upon this part of the subject is being now obtained. It will be noted above that there is no difference in the sugar ex- cretion whether 2.5 grams or 5 grams of phlorhizin be administered every eight hours. Fat.— Feeding 25 grams of lard does not increase the sugar excretion, a result which was to be expected from the work of Moritz and Prausnitz.? Behavior of Ingested Sugars. — The discovery of this intense form of diabetes in dogs led to the question of the behavior of different sugars after they had been fed to such dogs. In the following experiment 100 grams of lean meat was fed every eight hours from the fourth day of diabetes, and the dog being very fat bore the experiment extremely well. The idea was to obtain D: N::3.75:1, and then to feed the various monosaccharides which enter into physiologi- cal consideration, namely dextrose, levulose, and galactose. The “extra sugar” in the urine of these dogs can be estimated by multiplying the nitrogen in the urine by 3.75 (which represents sugar from proteid) and subtracting this from the total sugar found for the day. Any such “extra sugar” would be derived from the sugar fed. The sugar dissolved in water was readily taken by the dogs and was fed in portions of eight grams every six hours, commencing immediately after the urine had been drawn off in the morning. 1 Lusk: loc. cét., p.973; also Lusk: Zeitschrift fiir Biologie, 1890, xxvii, p. 459. * Moritz and PRAuSNITZ: Zeitschrift fiir Biologie, 1890, xxvii, p. 81. ye —, ek ee el te ee Cee 399 Phlorhizin Diabetes in Dogs. ‘UD AIS SeM 9UOU TOY M “W'v b pue ‘uaais o10M sueis zc uay “wv Z pur Fr ye ydaoxa ‘samoy aaayy A199 UIzIYIO[Yd wes UO paAtaoeI Sop oy} ‘1z pur ‘oz ‘61 [Udy "ALO = aposnut Arezunjoa ut uasood [ry I¢'0 = swieis 966'0 = 19AT] ul uasooATDH ‘br judy saiye smmoy 9 Araaa uiztytoyyq suis Z—'T]] DOC ‘Wea L9 86+'0 a ake ese Lee ‘yeoyM| 0's 262'1 as 00'F 6£°01 » ” 8 OZI'T | S6I'T 00's SS°6 ‘asojouyes 18 +7 + Io OL Oso'T 68'L Ch'b Soll ‘yea Zs 002'T . $S'e 166 ‘aSO[NAI| “13 ZO'bZ + IVI S'S sel ord, Let SIT yea 16 SLZ'1 zs'¢ Soll ‘ASO[NAGT “13 COE + IVAW es [esl Te'sT S6'r 6L ZI ‘RaW ae OT seis toe STet ‘ASO.1XAp “15 $7 + IO vl LSST | 64% 99°S SLI » ” 6S Ssl'% moe 68'¢ 06°21 ‘smoy g AI9A9 JLOU “13 COT L9 (00:04 | atyats 9¢'b OF eT ‘aSO1}Xap SWIVIS 1 /°EZ £8 StO'T | zS'0z +9 698 oi 96 0660 | +901 LS'b 616 i ae OIL | +se0 | Os'€z oF'6 2+ ey iatie Is cgH'0 é Pike ee eae es sté0 aera ana bow ‘poo “ofan | %0%a Sate ‘NEC. | ‘ueSontN SOL 6€ Sth 90S orze 6b O'r+ ‘rejod Aq d Stb LST ove 19‘ Th E Siskel 6L Lb a OSET 6S°IS 6'+% OST Ose ‘ SScl SS'Ts = SSSI OS tr “a OF9T LO€9 O'SZ eL8I PLS ace S9LT 99°99 eet OE? 620 athe 0002 89°8S 3'SZ OS6L wes ay OLIT 0 OF aa SScl S6S8E ee $98 50 eae 997 tks CLE SSP 9 UL *9S01}x9q] "TYSIO MA aur jo JUNOULY T:6L,¢:: N ‘+ q = Shep yeoutr xis sv] Jo osvisAy fautin ,sinoy g IL6S8T ‘8% [Udy We ‘9 ‘SC ‘+1 ‘CT L681 ‘21 tady 400 Reilly, Nolan, and Lusk. Dextrose. On the first day of dextrose feeding, 20.82 grams of extra sugar was present in the urine, following an ingestion of 23.77 grams of dextrose. The experiment was however inconclusive, since the fasting ratio between dextrose and nitrogen had not been obtained. The day before the second feeding of dextrose, the ratio between dextrose and nitrogen was 3.89:1; on the sugar day, 5.66:1; and on the following day 3.94:1. The extra sugar here was 22.49 grams following an ingestion of 24 grams of dextrose. A third experiment is taken from the unpublished work of Messrs. T. S. McDermott and W. E. Ray done in this laboratory and is given below. DOG IV.— Weight 10 kg. 1 gram Phlorhizin every 8 hours. Extra su- Date. Dextrose. | Nitrogen. ae gar. Nov. 18, 1897 36.72 9.63 19, 29.66 7.04 20 48.40 10.29 { 150 grams meat + 11.76 ( grams dextrose. 21, 41.52 10.89 aaciske 150 grams meat. Here we have on the sugar day a ratio of 4.70:1 and an excre- tion of 9.82 grams of extra sugar following upon an ingestion of 11.76 grams. We can conclude from our experiments that the dextrose fed is very nearly quantitatively eliminated. These experiments prove also that, within these limits at least, very little loss takes place through fermentation. Levulose. After feeding 24.05 grams of levulose, 15.31 grams of extra sugar appeared in the urine. The extra sugar could not be destroyed by heating the urine for several hours at 100° with 8 per cent hydrochloric acid. It therefore was not levulose. The reading in the saccharimeter was lower than it should be. Two causes might. have contributed to this: first, the levo-rotary substances of phlo- rhizin urines discovered by Cremer; ! second, personal error, it being the first time this particular saccharimeter was used, and the solution being so weak that a difference of 0.2 per cent would account for the- discrepancy between 63.27 grams and 57.5 grams. * CREMER: Zeitschrift fiir Biologie, 1898, xxxvi, p. 115. Phlorhizin Diabetes in Dogs. AOI On the second feeding of 24.02 grams of levulose, 7.42 grams of extra sugar appeared in the urine, and here the totals of dextrose by Allihn’s method and by polarization were respectively 51.85 and 49.4 grams. After ingestion of levulose is seen therefore its well- known partial conversion into dextrose, which in phlorhizin diabetes is quantitatively eliminated. It is especially remarkable that the burning levulose does not affect the proteid metabolism. Possibly the levulose may be burned by the sugar-hungry cells of the villus as soon as it is absorbed, and therefore its influence on metabolism is not widespread, but limited to certain localities. Minkowski! has shown that levulose if fed to diabetic dogs may increase the glycogen in the liver. In virtue of the small amounts of levulose used in our experiments, it seems improbable that it could reach the liver before combustion, and we would suggest that possibly the epithelium of the villus likewise may have the power for this conversion. Galactose. Results similar to those obtained after feeding levulose were found on feeding galactose. In both cases of galactose feeding the extra sugar consisted of dextrose. This was determined by the fact that the specific rotation of the solution indicated the same amount of dextrose as was shown by the method of Allihn. For dex- trose (a), = + 52.6°, for galactose, + 83.8°. Galactose may therefore in part be converted into dextrose. Phosphates. — It has been stated by von Ackeren? that phosphates in diabetic urine are present above the amount normally pertaining to the proteid decomposition. The phosphates were therefore deter- mined in the phlorhizin urine by titration with uranium nitrate. The results show that after fifteen days of diabetes there is no marked change from the usual ratio between phosphates excreted and proteid metabolism. This result is in confirmation of the experiments of Tenbaum®? in diabetes mellitus, who also finds the calcium excretion to be unchanged. | Glycogen. — Phlorhizin diabetes fails to remove glycogen entirely | from the liver and muscles. Glycogen in the liver of Dog II amounted £0. 10.392) Gtams — .O.0%per cent; in the liver of Dog III,.0.936 grams = 0.13 per cent, and in the muscle of the hind leg the large amount of 0.37 per cent was found. The dogs were killed while 1 MINKOwSKI: Archiv fiir exper. Pathol. und Pharmakol., 1893, xxxi, p. 165. 2 Von ACKEREN: See von NoorDEN: Pathologie des Stoffwechsels, 1893, p. 416. 8 TENBAUM: Zeitschrift fiir Biologie, 1896, xxxiii, p. 379. 26 402 Reilly, Nolan, and Lusk. under the influence of morphine, which prevents loss of glycogen from struggling. More frequent administration of phlorhizin, z. e., once every three instead of once every six hours, does not change the ratio D: N in the urine. Sugar production from Meat and Gelatine.— We have seen that feeding meat in small quantities during the day does not change the ratio D: N. Experiments were now tried with larger amounts of meat and with gelatine to seek for the ratio under these circum- stances. On the days of meat feeding the urine was analyzed in fractions of equal periods, as will be described later. The general results for twenty-four hours urine are given below, two dogs having been experimented upon. DOG V.—1 gram Phlorhizin every § hours after Feb. 22. | Amount of urine ee Dextrose. | Nitrogen. in cc. sata Feb. 22, 1898 7 12.8 serene Pag 23, ae 6.88 13.49 14.01 21.11 : 500 grams meat. 11.53 14.52 4 60 grams gelatine. , 1898 skates 4.20 OF 63.95 18.39 : 500 grams meat. Mar. 3, 1898) 6 hours’ urine§ 10.6 6.93 1.68 Average for last eight days: D:N::3.63:1. Rise in N excretion during starvation = 560%. Before entering into the detailed discussion of the experiments, a few brief remarks on the theory of the method of proteid destruction within the organism would seem to be necessary. The well known theory of Voit! maintains that there is a preliminary cleavage of the 1 Voir: Hermann’s Handbuch der Physiologie, 1881, vi, I, p- 295. Phlorhizin Diabetes in Dogs. 403 DOG VI.—2 grams Phlorhizin every 8 hours after Feb. 6. | | Amount | Weight | of urine : : in kg. in cc. Dextrose. | Nitrogen.| D:N. Feb. 3, 1898 eit 36.71 105 grams gelatine. 870 grams meat. 105 grams gelatine. } B 15282 1O0IC 20 grams levulose. A, 238% . 5 ei 5704 1 Albuminuria. 3 Some lost through mixture with feces. 216 hours’ urine. 46 hours’ urine. proteid molecule in metabolism into a nitrogenous radicle, and into a non-nitrogenous radicle, each of which may subsequently be burned within the cells at different times. The experiments of Feder?! have shown that after feeding meat to a starving dog the quantity of nitro- gen in the urine rises, attaining a maximum at the sixth to the eighth hour, and then continuously sinks, so that at the fourteenth hour most of the nitrogen corresponding to the meat eaten has been elim- inated. The excretion of CO. under these circumstances is, however, much more evenly distributed over the course of the twenty-four hours. Voit? concludes therefore that there is an early cleavage of the proteid molecule through which little energy is liberated; that there is a rapid combustion of the nitrogenous radicle, as shown by the elimination of the nitrogenous end products in the urine; 1 FEDER: Zeitschrift fiir Biologie, 1881, xvii, p. 541. 2 Voir: Zeitschrift fiir Biologie, 1891, xxviii, p. 291. 404 Reilly, Nolan, and Lusk. and that the non-nitrogenous radicle which contains the major part of the potential energy of the molecule may in part be temporarily stored either as glycogen or as fat, and be fed to the tissues more evenly during the course of the twenty-four hours as need requires. Now the indications are that phlorhizin acts quickly, removing sugar from the organism as soon as it is produced. If Voit’s non-nitrog- enous portion of the proteid consist of dextrose and the idea of its quick cleavage from proteid be true, then on feeding meat to a phlo- rhizin dog more sugar should appear in the urine of the first hours after feeding than corresponds to the nitrogen eliminated. The results of one experiment after feeding 500 grams of meat to a fasting diabetic dog, and collecting the urine first in two periods of three hours each, and then in one of six hours, are given below. The urine was, of course, obtained through a catheter and the bladder was carefully washed with water. The meat was eaten within one minute. DOG V.—Urine of Feb. 26. Amount in cc. | Dextrose. | Nitrogen. Preceding 3 hours (estimated) . . eee 5.96 1.75 ISG SeMOUrS' os, ae. ra eee if 12.43 jeSw 202 SIMOUNS!. cave ts lo oes 14.70 od! 3 hours ts... A eee ae eee E23 At hes OUTS acy yo nen ee on 11.23 Following 3 hours (estimated) . . Beier: 6.34 We start here with the estimated decomposition during three hours of fasting. During three hours after feeding meat the ratio in the urine equals D:N:: 4.92: 1, and in a subsequent period of three hours the ratio reads 3.91: I, whereas during the next six hours it sinks to 2.92: 1. This however is followed by a period in which the original fasting quantity of dextrose and nitrogen with the original ratio are approximately attained. If we take the whole period of twelve hours when meat was fed and compare it with the foregoing and following periods of like duration we discover the following relations : — Phlorhizin Diabetes in Dogs. 405 Dextrose. Nitrogen. Fasting, 12 hours (estimated) . . 23.87 7.00 After meat feeding, 12 hours . . 49.59 14.00 Subsequent U2 hours; =~ . = = .- bE Ziel lal Since the meat period of twelve hours was followed by a period entirely similar to the fasting period from which the start was made, it follows that the meat was absorbed and burned almost entirely within the first twelve hours after feeding. And this table shows that in the aggregate the ratios D:N in all three periods are the same. The sugar of eaten proteid is therefore entirely eliminated in phlo- rhizin diabetes, but it may be eliminated before the nitrogen belonging to it. Hence it is probably one of the more immediate cleavage products of the proteid molecule in metabolism. It is impossible at the present writing to state whether the sugar in the urine of the above periods of three hours represents the amount of sugar formed within the three hours, and immediately eliminated, or whether the sugar excretion represents the maximum limit of renal activity, and hence its comparatively even distribution over the hours named. The ability to gradually eliminate the sugar formed was illustrated in two experiments similar to the above but somewhat less satisfac- tory in their results. One was a subsequent experiment four days later on the same dog, when the general tone of the animal was less favorable. That the absorption and destruction of the meat within twelve hours was not due to special accident in the first experiment is shown by the fact still to be discussed that the dog was able to repeat the performance with 60 grams of gelatine two days later. In the meat experiment on dog VI the animal was not very strong, and subsequent to the experiment the ratio fell to approximately 2.8: 1, which as will be explained later probably indicates a decreased power of the kidneys. The conditions in the following two experiments were therefore distinctly less favorable for the quick removal of sugar through the kidney. That certain amounts of sugar may be pro- tected from combustion in the organism is illustrated by the experi- ment on a fasting diabetic rabbit, whose urinary sugar was increased 406 Reilly, Nolan, and Lusk. after convulsions (which brought about a conversion of glycogen into sugar) this sugar being eliminated not immediately within the first hour, but gradually during six hours.! The following results have been obtained from the urines of meat- fed dogs after dividing the urine into separate portions corresponding to two hours each. Dog V received 500 grams of meat, Dog VI 870 grams at one meal. DOG Vr Urine of March 2. Urine of Feb. 12. ING ec) SID HING > N. 1D Ne Preceding 2 hours (estimated) : eae 4:22 3.42 [llse elie a Ma Ig Nig 5. 4 7.49 1.92 4.82 Saya ium aimee acvNtie sal Sek 69 | 4.05 2.07 = aye Sd Dihowts icon. 1. Ree ee ao) | 3118 182 | 2.58 Hth 2 hours es ay elute ee eels : . | 2.60 S57 247 Sth 2 hours On en aes f 28. | 33.08 3.50 2.19, OtoeZsHOULSH ey eee : 2.15 | Bali, : ial 2.39 Following 2 hours (estimated) : ! Ko 33:95 1.60 ous 1 The urine of the first ten minutes was voided outside the cage and lost. 2 A part of the urine was lost through admixture with faces. The curve of nitrogen excretion is not essentially different from that obtained by Feder in normal fasting dogs after feeding meat. In the first two hours in both of the above experiments we find a large quantity of sugar in the urine showing the high ratios 7.49: I and 4.82: 1. The complete excretion of the sugar, however, extends over some little time, which is probably due to deficient power of the kidney to remove the sugar, as has been explained above. ‘ Lusk: Zeitschrift fiir Biologie, 1898, xxxvi, p. 82. Dextrose fed to rabbits in large excess is burned in phlorhizin diabetes. The protecting power is there- fore limited. Whether a similar power to burn carbohydrates when fed in excess is present in total phlorhizin diabetes in dogs is at present unknown to us. Phlorhizin Diabetes in Dogs. 407 For further clearness the following summary is added : — Fasting 12 hours Meat 12 hours Following 12 hours Following 6 hours . Following 24 hours Total subsequent to meat. These experiments, especially the first one made on Dog V, give a full confirmation of Voit’s views. There is a quick preliminary cleav- age of proteid into sugar and an unknown nitrogenous radicle. The sugar amounts to about 60 per cent of the proteid molecule, and con- tains not far from 60 per cent of its physiologically available energy. Through the action of phlorhizin the dextrose may be rapidly re- moved, whereas normally it is a substance which in excess may be stored as glycogen, or may even contribute to the formation of fat, both of which substances may be subsequently burned in the cells as need requires. Absence of Putrefaction.— The fact that, generally speaking, in the cases of phlorhizin diabetes in dogs, there is no appreciable change in the ratio D: N, whether meat be fed or whether the dog be fasting, would indicate complete absence of putrefaction, for it is hardly pos- sible that sugar could be produced within the organism from leucin, tyrosin, and other amido bodies formed in putrefaction. Exceptional Cases. — We have remarked in the case of Dog VI the decrease in the ratio D: N to the basis found in rabbits of 2.8: 1. This was accompanied by albuminuria. Only one other similar case has come to our notice, and that is taken from the work of Messrs. McDermott and Ray, done in this laboratory upon a fasting dog. The dog weighed 8.5 kg., and like the former dog had albumin in the urine. The albuminuria suggests renal disease, but why, in the apparent absence of any chemical difference, one fraction of the sugar in pro- 408 Reilly, Nolan, and Lusk. teid should behave differently from the other fraction, remains a mys- tery. This peculiarity seems to bring added proof of the at least - partially renal character of phlorhizin diabetes noted by Zuntz.! Phlerhizin diabetes cannot be wholly renal in character, for some unknown influence must protect sugar from combustion. DOG VII.—1 gram Phlorhizin every eight hours after Dec. 19. Dextrose. Nitrogen. 3.23 7.34 [eshours?-e eee ee 10.36 SelakeneS co on Rae 5. 2.84 lGshours yc). wee 5.39 Gelatine. — It has already been shown in work on the rabbit? that both nitrogen and sugar rise in the urine after feeding gelatine, and that nearly the proteid ratio is maintained between the two. More exact determinations have been possible with dogs, and among sev- eral attempted by us may be mentioned the two following. In the first case 105 grams of French gelatine (containing 14.91 grams N), and in the second case 60 grams (8.52 grams N) were fed. The gelatine was stirred into hot water and on cooling was broken in fragments and moistened with water containing 0.5 — 0.3 grams meat extract (=0.05 — 0.03 grams N). The mass was eaten greedily. In the first experiment the day commenced at 8.45 A. M., and at 9.15 A. M. about one quarter of the gelatine was eaten, at 3 P. M. about one half, and the remainder at 4.55 P.M. In the second experiment the whole mass was eaten at one time immediately at the commencement of the day. Both of these experiments show an increased elimination of sugar and nitrogen after feeding gelatine. In the first experiment the ratio D:N fell from 3.86 in fasting to 3.30 on the gelatine day and rose to 3.42 on the following fasting day. The results in the second ex- periment are more convincing because here the ratio hardly changes in the two fasting periods, and it will be noticed that it also remains 1 ZuNTz: Archiv fiir Physiologie, 1895, p. 570. 4 Lusk oe: cz. Phlorhizin Diabetes in Dogs. 409 Dextrose. | Nitrogen. ESS: boc! dass 5c be Ccceed ee fel OO 022 Me kSIOF: ui ee Maeda LON ie 50.23 14.66 10, pee Ae) Pre) Sete kt 79.66 23.78 : 105 grams gelatine. Doc V. Dextrose. | Nitrogen. Feb. 27, 1898, 12 hours (estimated) : ( WAINOUES Bb. dc Buss ‘ 60 grams gelatine. 28, ) 12 hours Mar. 1, 1898, 12 hours (estimated) unaltered during the intervening period when gelatine was fed. We may therefore conclude that the same large percentage of dextrose is obtainable from the metabolism of gelatine as from proteid. In this second experiment the whole of the gelatine was apparently absorbed and destroyed within twelve hours after feeding, because the amount of nitrogen is similar in each of the fasting periods. A very considerable amount of proteid was evidently spared from destruction by the burning gelatine. The twelve hours urine after gelatine feeding shows the presence of 9.7 grams of N. The gelatine con- tained 8.4 grams N, and if 8 grams of this appeared in the urine, then an amount of proteid corresponding to only 1.7 grams N would have been burned during the twelve hours against 5.76 grams N found for the twelve hours preceding. This would indicate a sparing of proteid to the extent of 69 per cent by the gelatine. The considerable sparing power of burning gelatine over proteid is well known. Theoretical Considerations as to the Constitution of Proteid. — The discovery that the proteid molecule yields on cleavage in meta- bolism an amount of sugar equal to 58.7 per cent (as near as we have been able to determine) leaves on the other hand a nitrogen-containing radicle in which the carbon and nitrogen would appear in the atomic ratio of 2.2 of C to 1 of N. It is evident from this that the nitrog- enous radicle cannot in metabolism yield much leucin or tyrosin, which require much carbon, whereas glycocoll and the sulphur-con- 410 Reilly, Nolan, and Lusk. taining taurin are theoretically possible. It will further be remem- bered that modern theory regarding the synthesis of proteid in plants, supposes the formation of proteid from sugar through union with asparagin and glutamin. Asparagin contains 2 of C, glutamin 2) of C to 1of N. It is also impossible to ignore the fact that the carbo- hydrate portion may be even larger than 58.7 per cent, and may possi- bly contain the radicles of levulose, of galactose, or even of pentoses, substances which may in part escape elimination in phlorhizin dia- betes. Again, there is evidence that the glucoside phlorhizin which yields dextrose in the laboratory is destroyed in the body. Hence the sugar of all compounds of dextrose, even in phlorhizin diabetes, may not appear in the urine. SUMMARY. 1. Frequent subcutaneous injections of phlorhizin in fasting dogs establish ultimately the ratio in the urine of Dextrose: Nitrogen: : 3.75: 1, which indicates a production of 60 grams of dextrose from 100 grams of proteid. Taking the faecal nitrogen into consideration, the amount of dextrose obtained from proteid may be more accu- rately estimated at 58.7 per cent. 2. The proteid metabolism may increase above that in simple fasting to an extent as high even as 560 per cent. 3. Dextrose fed in phlorhizin diabetes is quantitatively eliminated. Levulose and galactose are not eliminated as such, but only in so far as they are converted into dextrose. 4. Feeding fat does not affect the ratio. 5. Feeding meat does not affect the ratio for the day, but the sugar from eaten proteid may be eliminated before the nitrogen belonging to it, on account of an early preliminary cleavage of the molecule. 6. Gelatine yields the same amount of sugar as proteid does. Gelatine spares much proteid from metabolism. 7. Intestinal putrefaction and fermentation can only slightly have affected the proteid or dextrose which were fed in our experiments. THE American Journal of Physiology. VOL, I. JULY 1, -1808. NO. IV. ON INTESTINAL ABSORPTION AND THE SALINE CATEARTICS# By “GEORGE B. WALLACE ann ARTHUR R. CUSHNY. [from the Laboratory of Pharmacology in the University of Michigan.] N his well-known paper on the absorption of salt solutions from the intestine Heidenhain? came to the conclusion that two distinct factors were involved in the process, —the osmotic pressure of the solution, and the “physiological activity” of the epithelium. The latter induces a constant current from the lumen of the bowel towards the blood vessels. Hamburger ? accepts Heidenhain’s account of the osmotic action, but attempts to show that his “ physiological activity” is really a combination of the effects of certain physical forces. These are molecular imbibition; the intra-intestinal pressure, which induces filtration; and the suction induced by the blood current through the intestinal vessels, similar to that observed with the ordinary suction pump of the laboratory. Hamburger’s explanation of the intestinal absorption therefore involves an obscure process —the molecular imbibition — but differs from Heidenhain’s in not involving the living cell. Heidenhain performed a few experiments with solutions of the saline cathartic magnesium sulphate, from which he found that the water was much more slowly absorbed than from corresponding solutions of sodium chloride. He explains this retarded absorption by supposing that the sulphate lessens the ‘ physiological activity ” and that movement of its solutions is therefore controlled more by Received by the editors May 5. 1898. HEIDENHAIN: Arch. f. d. ges. Physiol., 1894, lvi, p. 579. 1 3 HAMBURGER: Archiv fiir Physiologie, 1896, p. 428. AI2 G. B. Wallace and A. R. Cushny. their osmotic pressure than is the case when solutions of non-purga- tive substances such as common salt are employed. In some expe- riments in which sodium sulphate or sodium fluoride was added to solutions of sodium chloride the absorption was also retarded for the same reason, each of these salts weakening the “ physiological activity,” although the fluoride is much more powerful than the sulphate. Of the factors involved in Hamburger’s scheme, only one — the molecular imbibition — can be affected by a change in the salt con- tained in the solution, for the filtration and the suction of the blood current remain unchanged. Hamburger made a few experiments to satisfy himself that molecular imbibition could occur in dead tissues, but a much more extensive investigation of the subject had been made earlier by Hofmeister.! From Hofmeister’s results it would appear that colloid substances (gelatine), and pieces of dried tissue, such as the wall of the bladder, are by no means indifferent to the solutions in which they are soaked. Thus much more of a solution of sodium chloride, and of the salt itself, was imbibed than of solutions of some other salts including some of the saline cathartics. The more recent work of Hedin? to which we shall return later, indicates that the red blood cells also imbibe more freely the solutions of certain salts than those of others for which they seem to have less affinity. We felt that some light might be thrown on the process of intestinal absorption by a more accurate definition of the group of saline cathartics, and we have for this purpose compared the rate of absorp- tion from the intestine of a large number of salt solutions with that of a one per cent sodium chloride solution. On looking over the list of salts which are generally regarded as saline cathartics it is apparent that the anion, or acid constituent of the salt, is generally the deter- mining factor. Thus sodium sulphate, potassium sulphate, sodium phosphate, potassium tartrate, potassium-sodium tartrate, potassium citrate, sodium citrate, potassium ferrocyanide, and sodium ferrocy- anide, are all looked upon as cathartics, while sodium chloride, potas- sium chloride, sodium acetate, potassium acetate, etc., are believed to be indifferent so far as action on the bowel is concerned. It is evident therefore that the anion or acid constituent is the deter- ' HOFMEISTER: Archiv fiir exper. Pathol. und Pharmakol., 1890, xxvii, p. 3953 1891, XXviil, p. 210. * HEDIN: Arch. f. d. ges. Physiol., 1898, Ixx, p. 525. Intestinal Absorption and the Saline Cathartics. 413 mining factor here, for the cations K and Na occur in both groups. As regards the magnesium salts the basic constituent or cation would also seem to be involved, for while in magnesium sulphate and mag- nesium citrate the purgative anion might explain the effects, these salts are generally believed to be more active than the corresponding salts of the alkalies, and in addition magnesium chloride, magnesium oxide, and magnesium carbonate have also some cathartic action; the presumption is therefore strong that the Mg ion is not indifferent as the K and Na ions are. We took up first the question of the purgative anions by compar- ing the rate of absorption of a large number of salts of soda. A preliminary note of our results was published in the Journal of the Boston society of medical sciences, January 18, 1898. Almost sim- ultaneously, Hober! published an account of his investigations on the absorption of a number of salts, dwelling particularly upon the effect of the basic constituent. We have therefore confined our examina- tion of the cations to a few experiments which were carried out mainly to confirm his results, which we have much pleasure in doing. In order to compare the influence of the salts on the unknown factor in absorption, whether this be physiological activity or molecular imbibition, it is of course necessary to eliminate the influence of the known factor, osmosis, by using solutions of the same osmotic pressure. This may be most easily accomplished by forming solutions which cause an equal depression of the freezing point. Our point of de- parture was a solution of about one per cent sodium chloride, which gave a depression of the freezing point (A) of 0.59 — 0.64° C., esti- mated by Beckmann’s apparatus. The other salts employed were dissolved in water, the freezing point of each solution determined, and more water or salt added until A approached that of the sodium chloride solution. We are therefore unable to state the percentage composition of most of our solutions, but append the depression of the freezing point to each one (see protocols, page 429). The varia- tion in the A of the solutions may at first sight seem to be consider- able, but the osmotic pressure never varied more than five per cent above or below the average; and when the solution of a salt showed any marked departure from the rate of absorption of the standard sodium chloride solution, care was taken to reduce the error arising from this variation in the osmotic pressure to a minimum. As a ‘matter of fact a considerable variation in the osmotic pressure may 1 Hoper: Arch. f. d. ges. Physiol., 1898, Ixx, p. 624, issued March 3. 414 G. B. Wallace and A. R. Cushny. be allowed without causing any appreciable difference in the rate of absorption, owing to the unavoidable errors of the method.!’ Thus a solution of NaCl A 0.67 was found to disappear as rapidly as one of A 0.61. | In our first experiments we used rabbits, but we could obtain no satisfactory results, as the absorption from the intestine seems to be extremely irregular in these animals. This may be partly due to the fact that it is impossible to empty the stomach and bowel by fasting of reasonable duration, and partly perhaps to the rabbit's intestine being more sensitive to handling than that of the other animals used. The cat’s intestine gave somewhat better results, but here also the absorption was often irregular. The great majority of our experiments were performed on dogs, and we have left those on the rabbit entirely out of account, while the results obtained from the cat’s intestine have always been controlled by others on the dog. The dogs and cats were anesthetized by the subcutaneous injec- tion of morphine, followed by the administration of chloroform acetone by the mouth. In some cases ether cr chloroform was given by inhalation, instead of chloroform acetone. In every case the animal fasted for 36-48 hours before the operation, and in our later experiments we obtained the best results from animals which had fasted for three or four days. This preliminary fasting appears to be of great importance in any experiment in which a regular absorption is necessary. In all our experiments two or more intestinal loops were used. They were ligatured off in the usual way. We are quite aware of the objections to this method, for Hay? showed that ligation is liable to cause irritation of the bowel. Owing to the limited supply of dogs, however, we could choose only between using several short loops, or using one long loop a number of times, and we soon found that the necessary manipulations rendered a loop very unreliable after three or four injections. We have attempted to avoid the disadvan- tages of our method by using a series of controls. Thus, the ex- periment was commenced by ascertaining the rate of absorption of the standard solution in all the loops, and this control was repeated whenever any solution was found to deviate considerably from the ' In Experiments 21 and 23, solutions of salts which differed considerably in their osmotic pressure were used for a purpose apart from the general scope of the work. * Hay: Saline cathartics, Edinburgh, 1884. Intestinal Absorption and the Saline Cathartics. 415 ‘standard solution in rate of absorption. In this way any abnormality developed in an individual loop could be recognized. In addition one loop was injected each time with the standard solution, and we could thus eliminate any errors due to the general condition of the animal, and also those arising from the manipulation, for the control loop was treated in exactly the same manner as the others. In esti- mating the rate of absorption of any solution we have taken into consideration not only the variation in the particular loop in which it was contained, but also any change in the control loop. Our loops were much shorter than those of most other investigators, varying from 25 to 45 cm., but it seems to us that the unabsorbed fluid can be much more completely removed from these short loops than from the longer ones, and that one source of error may thus be lessened. On the other hand, the short loops have the disadvantage that greater irritation is produced by the close proximity of the ligatures; but we believe that our system of controls has reduced very greatly the error arising from this source as well as the error due to the different rate of absorption in different parts of the bowel (Heidenhain). A glass cannula was passed into each loop, fixed by a ligature, and closed by a short indiarubber tube and clamp. Insome of the early experi- ments the loops were washed out before and after each injection of salt solution, but we found that the additional manipulation caused a larger error than that arising from the use of unwashed loops, and this procedure was abandoned in our later work. The loops were emptied by gently stripping them, and were not exposed to the air longer than was absolutely necessary in order to empty them completely. The salts examined fall naturally into groups as follows: 1. The halogen salts: NaCl, NaBr, Nal, NaFl. Exp. 17, 18, 19. Of these salts the bromide seemed to be absorbed as rapidly as the chloride, while the iodide was sometimes absorbed more slowly. In Experiment 17, however, all three salts disappeared equally from the loops, and in this experiment it is noted that a fresh iodide solution prepared that morning was used. It seems possible that the diver- gence in Experiments 18 and 19 was due to the use of an older solu- tion, in which some decomposition had occurred. H6ber found the chloride the most easily absorbed of the three, then the bromide, and last the iodide. In our experiments we did not observe such marked differences in the rate of absorption as appear in his protocols, and in fact are inclined to hold that very little difference exists between these salts in regard to the rate of absorption of their solutions. The 416 G. B. Wallace and A. R. Cushny. fluoride, on the other hand, is absorbed with great difficulty, as is: shown in Experiment 19 and in others not included in the published protocols. It always caused more or less congestion and inflammation of the loop, as is evidenced by the absence of absorption of the standard solution from the loop afterwards. Heidenhain also found that the presence of even a small percentage of fluoride in a salt solution retarded its absorption. The chloride, bromide, and iodide may thus be classed among the indifferent salts, while the fluoride has a very distinctly retarding effect on absorption. 2. Other inorganic salts : NazSO,, Na(C,Hs)SOs, NaNOs;, Na,H PO,, NaH.PO,, MgSO.:, KNO;, K(C,:H,)SO,,1 CNH.).5O,. Experiments T, 2,9, 10, 12, 17, 18, 20, 21, 22, 23, 24. The simple silpiavesmmee absorbed much more slowly than the chlorides, as has been observed by a number of workers on the subject. No evidence of inflammatory reaction was observed, and the loop rapidly returned to its normal condition when the solution was removed. Sodium ethyl-sulphate has been used occasionally as a mild saline purge, and from our experi- ments it would seem to stand midway between sodium chloride and sodium sulphate. No simple sulphate was contained in the fluid injected or in the residue. The nitrate solutions are more slowly absorbed than the chlorides, but much more rapidly than the sul- phates, as was observed also by Hober. The use of the nitrates was followed in one of our experiments (Exp. 18) by very distinct signs of irritation and inflammatory reaction, the residue being blood- stained and containing large quantities of mucus. The nitrates are generally looked upon as being more irritant to the alimentary canal than such salts as the chlorides and sulphates, and although in the second experiment (Exp. 24) no signs of irritation of the bowel were present, we think it questionable whether the fluid found at the end of the experiment was really due to the lack of absorption or to an effusion into the bowel. The two phosphates proved almost iden- tical in the rate of absorption, and may best be classed with sodium sulphate. 3. Ferrocyanides and ferricyanides. Experiments 21, 22, 23. These two salts seem to be absorbed as slowly as the sulphates. The - ferricyanide solution contained no ferrocyanide when injected, but the residue gave a copious precipitate of Prussian blue, indicating that much of the ferricyanide had been reduced to the ferrocyanide, The effect is therefore probably due to the latter salt. 1 This salt was found to contain a considerable quantity of ordinary sulphate. Intestinal Absorption and the Saline Cathartics. 417 4. Salts of the fatty acids; sodium formate, acetate, propionate, butyrate, valerate, caproate, cenanthylate, and caprylate. Experi- medio, 11, 13, r4, 15, 10, 1. Phe fiest sim of these salts are absorbed as rapidly as the chloride. The cenanthylate disappears somewhat more slowly, although in Experiment 15 this is not the case. The caprylate is somewhat more rapidly absorbed than the sulphate. The lactate (Experiments II and 13) seems to lie midway between the chloride and sulphate of soda. 5. Oxalic acid series: Oxalate, malonate, and succinate of sodium. Experiments 3, 7, 8, 13, 25. The oxalate is but little absorbed, and always induces congestion and inflammation, from which the loop does not soon recover. It therefore resembles the fluoride. The malonate and succinate solutions are scarcely absorbed, but do not cause inflammation, and the loop recovers after the solution is removed. 6. Tartrate, citrate, and malate of sodium: Experiments 3, 5, 6, 7, 8, 13, 14. The solutions of these three salts are absorbed at about the same rate as those of the sulphates. The first two are well-known saline cathartics. 7. Salts of the aromatic acids: Salicylate, ortho-phthalate, and para- phthalate of soda. Experiments 12, 17. The phthalates seem to disappear more slowly than the chlorides, but do not retard absorp- tion to the same extent as do the sulphates. The salicylate was also slowly absorbed, but was used in only one experiment, which is insufficient to determine its exact position. 8. The metallic tons: Sodium, potassium, ammonium, magnesium, barium, calcium. In all the experiments except 23, 24, and 25 these are combined with —SO,, —NO;, —FeCy;, and —FeCy, ions. In Experiments 23, 24, and 25, their chlorides and calcium acetate are compared. No difference could be detected in the behavior of the potassium and sodium salts. The ammonium chloride solution dis- appeared in Experiment 25 more rapidly than the standard sodium chloride solution, while in Experiment 24 it was absorbed at least as quickly; but here the sodium chloride solution was also éntirely absorbed, so that it is impossible to state which was taken up the more rapidly from the bowel in this experiment. The salts of the alkaline earths were absorbed much more slowly than the correspond- ing salts of the alkalies. As regards the cations, therefore, our results are practically identical with those of Hober. 27 418 G. B. Wallace and A. R. Cushny. The soda salts can thus be arranged into four fairly distinct groups, according to the rate of absorption of their solutions. TABLE: I. IV. Chloride. Fluoride. Bromide. Iodide. Ethyl-Sulphate. Sulphate. Nitrate. Phosphates. Ferrocyanide. Ferricyanide. Formate. (Enanthylate. Caprylate. Acetate. Lactate. Propionate. Butyrate. Valerate. Caproate. Malonate. Oxalate. Succinate. Tartrate. Citrate. Malate. Salicylate. Phthalates. The solutions of the salts of column I are all absorbed equally rapidty. Those of column II vary more or less in their behavior, but are generally absorbed more slowly than those of I. Those of IIf disappear very slowly, but, as a general rule, do not impair the absorption of the loop permanently, while the solutions of IV scarcely lessen at all in amount and evidently injure the loop seriously, for the solutions subsequently injected are only slowly absorbed or may even Lntestinal Absorption and the Saline Cathartics. 419 increase in amount. We are not inclined to look upon this as differ- entiating the salts of IV from those of III qualitatively but only quantitatively, for the subsequent absorption was sometimes im- paired by the salts of III, and Hay also found that strong solu- tions of sodium sulphate reduced the absorption of sodium chloride afterwards. This table resembles in many features that given by Hofmeister to indicate the relative power of different salts to precipitate egg albu- min.! His tables may be abbreviated by arranging the salts in two columns. It must be premised that the cations seem to have more influence here than in the intestine. fons with little or no power of lons with greater power of precipitation. precipitation. Chlorides. Sulphates. Bromides. Phosphates. Iodides. Acetates. Nitrates. Citrates. Some acetates and chromates. Tartrates. Chromates. In experiments on the precipitation of gelatine with neutral salts he obtained similar results, and also in those on the precipitation of colloid iron oxide, while the sodium oleate solutions behave differ- ently in regard to some salts.” Hofmeister ® found that gelatine plates absorbed less fluid when soaked in sulphate, tartrate, citrate, or acetate solutions than in chlorides, chlorates, nitrates, or bromides. Here, as in his experi- ments on the precipitation of egg albumin, the results are calculated for normal solutions so that they may be considered as isotonic except in so far as the dissociation of the salt is concerned. His experiments on the imbibition by pieces of the bladder wall were unfortunately carried out with percentage solutions and cannot be utilized for comparison. The same is true of Limbeck’s * experiments on the diuretic action of salts. It will be seen that Hofmeister’s results do not quite correspond with ours, although they bear a very close resemblance. The most 1 HOFMEISTER: Archiv fiir exper. Pathol. und Pharmakol., 1888, xxiv, p. 247. 2 HOFMEISTER: /did., 1888, xxv, p. I. 3 HOFMEISTER: /67d., 1891, xxvili, p. 210. * LimBeck: Archiv fiir exper. Pathol. und Pharmakol., 1888, xxv, p. 69. 420 G. B. Wallace and A. R. Cushny. striking difference is in the behavior of some of the acetates, which precipitate proteids and other colloids and prevent the imbibition of gelatine plates much more than the chlorides do, while in our experi- ments solutions of the acetates and chlorides are equally rapidly absorbed from the intestine. In spite of this and of some other minor differences which may be found by comparing Hofmeister’s original tables with ours, there is a very striking similarity in our results, — most of those salts which precipitate egg albumin and prevent the permeation of gelatine plates also retard the absorption of fluid from the intestine. This would seem to support the view that these salts act as saline cathartics not through their lessening the “ physiological activity” of the intestinal wall, as Heidenhain supposed, but through their being devoid of some general relation to colloid substances, organized or unorganized. This would not entail the belief that absorption from the intestine is a purely physical process, for the suggested explana- tion only covers the absorption of the fluid into the epithelium, and does not attempt to account for its transmission to the bloodvessels. On the other hand some facts point to the opposite conclusion, namely, that the reaction of the intestinal epithelium to the salts is not due to the general physical properties of colloids. Thus Hedin! investigated the behavior of the red cells of the blood in solutions of various ammonium salts, and found that they were permeated with- out resistance by the chloride, bromide, sulpho-cyanate, oxalate, ferro- and ferricyanide, lactate, and ethyl-sulphate, while the sul- phate, phosphate, tartrate, and succinate penetrated them with diff- culty. These results present much greater contrasts to ours than Hofmeister’s do, for while the ions that penetrate the red blood cells with difficulty also prevent the absorption of fluids by the intes- tinal wall, several ions that permeate the blood corpuscles with ease act as cathartics (oxalates, ferrocyanides), and others stand mid- way between the cathartics and the indifferent ions (ethyl-sulphate, lactate). It is evident therefore that the colloids of the red blood cells and those of the intestinal epithelium differ very considerably in their relations to different anions, although there are some common features. This conclusion is confirmed by the fact that the red cells are permeated only with the greatest difficulty by the fixed alkali ions, whereas comparatively little resistance is offered by the intestine. 1 HEDIN: Joc. cit. Intestinal Absorption and the Saline Cathartics. 421 Again, Leathes and Starling* found that the pleural endothelium absorbed solutions of magnesium sulphate and sodium sulphate as rapidly as those of sodium chloride, so that here the cell contents present yet another variation in their affinities. Lastly Pohl,?, Young,’? and others have investigated the precipita- tion of colloid carbohydrates by neutral salts and find a considerable variation in their relations. Pohl states that the sulphate of ammo- nia precipitates a larger number of these than the phosphate of ammonia or the acetate of potash, while these again act on a larger number than the sulphate of magnesium. The conclusion seems inevitable that while a general resemblance may exist in the relation of the neutral salts to the different groups of colloid bodies, the details vary with each individual colloid. This differentiation of salts into two series, —the one permeating the intestinal epithelium, the other apparently repelled by it, naturally demands explanation, and we have therefore attempted to find some further characters common to the cathartic salts and not possessed by the indifferent salts. Loeb? has recently advanced the view that the action of some sub- stances may be determined by the number of dissociated ions, and by their velocity. The amount of dissociation can scarcely be ex- pected to have much importance, however, where identical effects are obtained with two salts which vary so greatly in their dissocia- tion as the chloride and acetate of sodium. Dr. K. Guthe of the physical laboratory had the kindness to ascertain the relative electrolytic conductivity of our solutions, and we found that it bore no relation to their behavior in the intestine. For example, the sodium chloride solution gave a deviation of the electrometer of 158, the acetate of 97.5, the fluoride of 110, the oxalate of 155, the tar- trate of 137, and the sulphate of 235. The purgative fluoride and oxalate therefore stand between the indifferent acetate and chloride. The variation in the velocity of the anions is also apparently without significance, for as it decreases with an increase in the atomic weight the purgative caprylate ion must have a smaller velocity than the indifferent caproate or acetate, while the oxalate ion on the other hand must have a greater velocity than the succinate, which how- ever is less purgative. LEATHES and STARLING: Journal of physiology, 1895, xviii, p. 106. POHL: Zeitschr. f. physiol. Chemie, 1890, xiv, p. 151. YounG: Journal of physiology, 1897, xxi, p. xvi. 1 3 4 Loes: Arch. f. d. ges. Physiol., 1897, lxix, p. 1. 422 G. B. Wallace and A. R. Cushny. The physical differences of the solutions do not present any rela- tion to the differences in their action, then, and we have sought for some pharmacological property common to the cathartics, and not possessed by the indifferent salts. As regards the oxalate and fluoride (4th column, table I), this might be found in their action as general protoplasmic poisons; the connection between this and their action on the bowel is rendered more plausible by the fact that the addition of quinine hydrochlorate, a well known protoplasmic poison, to the standard solution prevents absorption and causes con- gestion and irritation, although the quantity added is too small to alter the osmotic pressure, It is more difficult to find any relation between the substances of the third column, for while the tartrate and citrate are undoubtedly poisonous when injected into the blood, the sulphate has little or no such effect. Most of these salts are dibasic or tribasic, while those of the first column are monobasic; but the significance of this fact is lessened by the presence of the phthalates in the second column, and of the caprylates in the third. The lower members of the acetic acid series permeate freely, but a sudden change occurs when the cenanthyiate and caprylate are reached. This would suggest that the increasing size of the mole- cule influenced the rate of absorption, but this does not hold good in other cases, for the malonate and succinate — the higher members of the oxalic acid series — are less cathartic than the lowest homo- logue, the oxalate. The second column is even less homogenous than the third. The cenanthylate may be looked upon as bridging the gap between the permeating simpler members and the purgative higher members of the acetic acid series, while. the lactate and salicylate bear some relation to each other in both being oxy-acids. The phthalate and ethyl-sulphate, on the other hand, might have been expected in the third column, for the former is a dibasic salt, like most of the other salts of the third column, while the ethyl-sulphate might be expected to resemble the simple sulphate. One curious relation, which struck us early in our experiments, and which determined to some extent the direction of our work, was that existing between the behavior of the ions in the intestine and the solubility of the corresponding calcium salts. The solubility of some of these salts has not been determined, and we have therefore ascertained them by shaking the calcium salt in Intestinal Absorption and the Saline Cathartics. 423 water for three or four hours and estimating the amount of the salt dissolved in a given quantity of the filtered solution by evaporating and weighing. The results of the estimations made by others and by ourselves are given in the following table, in which the figures in the first column give the number of grams of salt dissolved in 100 c.c. water, while the figures in the second column give the tempera- ture at which the estimation was made. We have selected tempera- tures at 40° C. where possible, so as to approach the conditions in the body more nearly. TABLE, Of. Grams of Calcium Salt dissolved tn 100 c.c. of water at the temperature given. Calcium iodide®. . . J Calcium cenanthylate 7 bromides =. i | malate . chloride? : d ferrocyanide . Tpihatel eyes 550% phthalate (ortho) propionate? . able i malonate 2 ACStAte a ane sulphate! . fOTMALE EIS ira} Z | 39. caprylate . butyrate® . . : ; citrate . lactateare tenes areytes subeete tartrate valerate = 2: 5. | 40.0 hydric phosphate §| Caproate i. |). ns | 40.0 fluoride’ . succinate”. . SiS |) Gy oxalate RAUPENSTRAUCH: Monatshefte fiir Chemie, 1885, vi, p. 579. 2 MICZYNSKI: 767d., 1886, vii, p. 2.55 3 KRASNICKI: 767d., 1887, viii, p. 595. FURTH: 267d., 1888, ix, p. 308. 5 KEPPICH: 7bzd., 1888, ix, p. 589. § DEAZATHY : zbid., 1893, xiv, p. 250. LANDAU: 707d., 1893, Xiv, p. 707. CoMEyY: Dictionary of chemical solubilities, London, 1896. On comparing Table I and Table II, it will be observed at once that the most soluble calcium salts are those formed by combinations with the indifferent ions (first column, table I), while the cathartic salts of the third column form very much less soluble salts with 424 G. B. Wallace and A. R. Cushny. calcium and the fluoride and oxalate (fourth column) are entirely insoluble. This is remarkably exemplified by the behavior of the acetic series, for while the first six members of this series are in- different in the intestine and form fairly soluble salts with lime, the seventh (cenanthylic) is slowly absorbed and rather insoluble, and the eighth (caprylic), which is very insoluble, acts in the same way as the sulphates. In the same way the least permeating member of the oxalic acid series (oxalic) forms an absolutely insoluble lime salt, while the less cathartic higher members form more soluble compounds with calcium. Some exceptions to the general rule undoubtedly exist, apart from the nitrates, which we do not regard as of the same class as the others. Thus the succinate of calcium is more soluble than the cenanthylate, and yet sodium succinate is more cathartic, while the phthalates are less soluble’ and yet appear in the second column. The lactate and salicylate also form rather soluble lime salts and yet appear to be somewhat slowly absorbed. Another exception is the ethyl-sulphate, which forms a very soluble lime salt, but it seems not impossible that this body may in part be decomposed in the course of absorption, in which case the sulphate formed would retard absorption. Similarly, the ferricyanide of calcium is soluble, but the sodium salt is reduced to the ferrocyanide in the intestine and therefore retards absorption. It is to be remarked, however, that no very soluble lime salt is formed by the really cathartic group of ions (third and fourth col- umns, table I), while no acid forming insoluble lime salts is found in the first column. The exceptions cited above all fall into the second column, which is a makeshift group of substances neither entirely indifferent nor sufficiently slowly absorbed to entitle them to a place among the distinctly cathartic salts. Besides, it is very evident that the property which prevents the absorption of certain ions, and at the same time renders their combinations with lime insoluble, is not the only determining factor in absorption, for quinine hydrochlorate in traces prevents absorption. These exceptions therefore do not seem to us to invalidate the general result, namely, that acids which form insoluble salts with calcium act as * The phthalates of calcium are said to differ considerably in solubility, but we found that the two phthalates precipitate lime water in the same degree of dilution. The quantity at our disposal did not admit of more accurate chemical examination. Intestinal Absorption and the Saline Cathartics. 425 cathartics when combined with ordinarily indifferent bases such as the alkalies. The question at once arises whether the connection between these two properties is a causal one, 2. e., whether the cathartic salts are slowly absorbed because they precipitate calcium in the intestinal wall. It is needless to say that this is a possible explanation, for the precipitation of calcium has been shown to have a very con- siderable effect in such processes as the coagulation of the blood and of milk. The importance of calcium in the nutrition of the heart and of developing ova (Ringer), in the contraction of muscle (Locke), in the irritability of nerve fibres (Howell), and in the growth of plants (Loew) is generally recognized,1 and we are tempted to suppose that in the absorption from the bowel the calcium plays a similar rdle. We feel however that our experiments are not sufficient to allow of a positive statement, and must leave the question open for the present. Howell’s work on the action of oxalates on the heart left him in the same position of uncertainty as to whether the effects were due to a precipitation of calcium or to some specific action of the oxalates.! In this relation we may mention that in a number of experiments which we have performed on the tortoise heart the sulphate of sodium seemed to have the same effect as the acetate, while the citrate was extremely poisonous. In the account of our results hitherto we have tacitly assumed that the salt failed to permeate the intestinal wall. This assumption is based upon results obtained by Hay, and more lately by Kovesi? and confirmed by our own observations that a considerable amount of the cathartic salt remains in the fluid in the intestine. Some salt un- doubtedly disappears, but not nearly so much as when solutions of chloride of sodium or of any other indifferent salt are used. The depression of the freezing point (A) of the residue remains un- changed if the solution was originally isotonic, as were most of our solutions. If on the other hand a hyperisotonic solution is injected, the A slowly declines to about .61 (that of the blood), while if a hypistonic solution is used, a concentration of the fluid sets in until the A again approaches that of the blood. This is in accord with Kovesi’s results on the rabbit’s intestine, but does not conflict as he supposes with Heidenhain’s results obtained with sodium chloride 1 HOWELL: Journal of physiology, 1894, xvi, p. 476. 2 Kovesi: Centralblatt fiir Physiologie, 1897, xi, p. 553. 3G. G. B. Wallace and A. R. Cushny. solutions, for the alteration in the A of the intestinal contents is evi- dently due in both cases to the osmotic interchange of fluid and salt with the blood, which Heidenhain fully recognized. In many of our experiments a considerable amount of mucus was present in the residual fluid, but this was not constant, and there did not seem more mucus in the residue of the cathartic solutions than in that of the standard solution. In many of the intestinal loops tape- worms were present, and in these there seemed more mucus than elsewhere. Hay! is inclined to look upon the increased secretion of mucus by the intestine under sodium sulphate as of some importance in retarding absorption, and this explanation has been again brought forward by Fusari and Marfori.2, We are not disposed to look upon the secretion of mucus as of much importance in determining the absorption or non-absorption of the cathartic solutions. The objection may always be brought against the method we have adopted that the conditions are so abnormal that no inferences as to the behavior of the uninjured intestine can be drawn. On the other hand no accurate results can be obtained by measuring the fluid in the feeces after the use of one of the purges, because the amount of fluid in the bowel previously is unknown. We have therefore at- tempted to determine the action of these purgatives by comparing the amount of fluid which escaped from a cecal fistula after the administration by the stomach of isotonic solutions of various salts. A medium sized dog was chloroformed, the abdomen laid open, and a loop of intestine immediately above the termination of the ileum sewed into the wound. Four days later, when complete adhesion had occurred, and the wound was rapidly healing, the loop was opened. A week later, the exami- nation of the action of different salts was commenced. The animal received no food in the morning and in the afternoon a measured quantity of sodium chloride (A .615) was administered by the stomach tube and the amount of fluid passed by the fistula during the next hour measured. When no more fluid was passed an equal amount of an isotonic solution of another salt was given in the same way, and the fluid escaping by the fistula again measured. The results confirmed those obtained by the other method, but the investiga- tion could not be carried far as the animal died, apparently from having been exposed to great cold during the night of Dec. 25. 1 Hay: Saline cathartics, 1884, p. 69. 2 Fusari and MARFoRI: Atti della acad. delle scienze med. e nat. in Ferrara, 1894; cited from Centralbl. f. innere Medicin, 1894, p. 1245. Intestinal Absorption and the Saline Cathartics. 427 Our results were as follows: Dec. 21, 1897. Experiment 1. Injected into stomach 100 c.c. NaCl A .615. 15° Some solid matter evacuated with a few c.c. fluid. 60’ Total amount discharged 5 c.c. fluid and faecal matter. Experiment 2. Injected too c.c. sodium citrate A .62. 15’ Some faecal matter discharged and fluid began to appear. 60’ Total amount of fluid discharged = 70 c.c. Dec. 22. Experiment 1. Injected 100 c.c. NaCl A .615. 60’ ‘Total amount discharged = 4 c.c. Experiment 2. Injected roo c.c. sodium acetate A .615. 60’ Total amount discharged = o. Experiment 3. Injected 100 c.c. sodium phthalate (ortho) A .62. 60° Total amount discharged = o. Dec. 23. Experiment 1. Injected too c.c. NaCl A .615. 60’ Total amount discharged = o. Experiment 2. Injected So c.c. § sodium phthalate (ortho) A .62. ! sodium phthalate (para) A .56. 60’ Total amount discharged = o. Dec. 24. Experiment 1. Injected 100 c.c. NaCl A .615. 60° Total amount discharged = o. Experiment 2. Injected 100 c.c. Na,SO, A .62. 60’ Total amount discharged = 75 c.c. The whole of the solutions of sodium chloride, sodium acetate, and sodium phthalate was absorbed in the course of its passage through the stomach and small intestine, while three fourths of the citrate and sulphate solutions reached the large intestine, and in the normal animal would have gone to increase the fluidity of its contents. We think that this demonstrates conclusively the method of action of the dilute solutions of the cathartics such as are found in some of the natural mineral waters. They do not necessarily increase the fluid of the bowel, but merely fail to be absorbed, and thus render the faeces more fluid and more easily moved through the large intestine. 428 G. B. Wallace and A. R. Cushny. CONCLUSIONS. 1. The absorption of the salts of the fixed alkalies varies with the anion, those acids which form insoluble calcium salts tending to retard absorption more than others. 2. The behavior of these salts in the intestine has much in common with their action on unorganized colloid matter, as they tend to pre- cipitate colloids in solution and are less imbibed than other salts by undissolved colloids. 3. But no complete analogy in their behavior towards the tissues in general exists, for several of the cathartic salts permeate the red corpuscles freely and others are absorbed rapidly from the serous membranes. 4. As regards the cations, ammonium is absorbed more rapidly than the fixed alkali ions, while those of the alkaline earths are very slowly taken up by the intestinal epithelium. 5. Dilute solutions (isotonic) of the saline cathartics retard the absorption of fluid from the stomach and small intestine, and thus act by rendering the contents more watery and more easily moved through the lower parts of the alimentary canal. Protocols of the experiments are given on pages 429-434. 429 L[ntestinal Absorption and the Saline Cathartics. VST papelfur uonnjos jo yunowy Ud Sp = doo] yore jo yysus'T IOPN a}eIVIV LEN aqVIPIe LUN IOPN ‘6 ‘AON —G pOROR SI payealur uoynjos jo junowy Md Sf = door yva jo yuIT OEE peyoalur uonnjos jo Junowy ud Sp = dooy yore jo yBuUs'T OS papelfur uonnjos jo junowy Md Sp = dooy yovs Jo yysue'T ayeyooVeEN IDeN quowriedxy IO®N a]v]IDVEN 0€ Of |? ‘9 “AON — f Juoutredxg TOPN IDBN a}e[eXOVN AVIV LEN a}vTeXxXOQUN KOLAN I ‘V'AON —@& OS V2 OPK Oka | quow1iodxg FOSTeN POSEN FOSS TORN IOPN TDRN ¢ 0° 6S" OF 6° ‘yeq — LOg1 ‘Z “AON —B 09° | 2 Og | 9 OcmalE2 quowriodxy ‘9°90 ST = doo] yore O}UT poyolur uornpos Jo juNOW Vy "Wo Sp = door yore Jo WySua'T] SYIVUIYY TORN TDO®N ID®N ‘yep — 46gI ‘I ‘AON —T 09 O¢ Oe, | 2 quowriedxy ONPTSa yy V UPS enpisey| V UPS ‘III 4007 ‘II dooT anpisay| V WeS ‘T 100] soqn -uTW ul ou G. B. Wallace and A. R. Cushny. “UOT}NIOS yes yeul1ou YIM poysem sdoo'T 0°9 6% = pajoelur junowy ud OF = door yove Jo yASuUaT “uoTyN[OS yes yeutuou YIM payseam sdooy 0°0 ¢Z = payoofur junowy ‘wld OE = doo] yove jo yySua'T ‘uoTjN[OS yes [euou YIM poyseam sdooT ‘99 6g = poyelur junowy 0G) Cele —— SIiy yf (aloxelayy i TONG — coon Ve yyouey ud $'Zp = ‘J dooT ‘uorqoafur 0} snorasid uoljnyos qes [eULIOU YIM paysem sdoo'y 0'0 ST = payolur junowy "Ud Cf = dooy yova jo YysuaT SSAVULI YY eee = OvOes i Aes S 0 6's sia) eb ges O61 95° FO dH°eN o¢ P O'S? OS Od°H®N OT Of FOdHN OL SLD} DONG ee |e OP Sg! IOPBN 0'6 S19’ IOeN Kd sy HOEANE | Ole" SON slo! IDPN Ore ish OLAN Ol S19 OIG || (OE Nh ‘Bog — L6g1 ‘gi ‘AON — & Juewtsedxg O'Se | SZS |eveUCTeEINeEN OGL | Seo: o7L[PINVN 06 S19 TOENS |) COGS |r O€¢ | SLS | eyeUOTeIVeN Saal |) reey 978 [PEN GL SLS): ONAN I (Oke OL 719° TDN OF S19" IOPN 0% S19 TORING Ocala ‘Soq — Z6g1 ‘z1 ‘AON —gQ Juewmtredxg SLT S29" | 9381} LEN S'1é 6S IDeN| O'%2 | S29" | B38TPEXOVPN | OF | 2 O€ oO | =9yeJIVEN OOF | S729 SYETEXOVPN | .67€ 6S" IO®N | OF Y OZ C9 | 97e}29VEN O¢¢2 | SZ 9JETEXOVEN 07 6S IDeN | OF q TOOL || ol IOeN 0% 6S ID®eN| O¢ 6S" TORN | OF || 7 ‘BSoq — 46g1 ‘11 AON — J, Juowrsedxg Dera e Tt eee rate O'9T 6S IOPN OST 19 | 981tDeN | OF | ? ae oh| eae he O'9T FS" OFLA DCN oy) 6S [DENT 0S az sl NP aha ee ONS enon Lo) SJEIWOVN [VISE | o 6S IDENT Of ie? ‘ye9q — L6gI ‘Ol “AON — g quemtiedxg anpIsoy| V Wes anpisoy| V [eS anpisey | VV Wes saqn -UTUL ur ‘III doo, ‘II doo] *] doo'y owty, L 26S Cathart line ‘09 Cg = poaqoalur junowy ‘wo OF = doo] yova Jo yysuaT dqLIPIV TEN aye1a[e ARN IO®N “‘UOTN[OS yes Jeurtou yA paysem sdooT ‘0°90 Sg = payoalur yunowy ‘m9 OF = doo] yove Jo YSuaT 09 6g = paqoalur yunowy ‘wo OF = doo] yova Jo y\SuaT uoljnyos yes Teuiou YIM paysem sdoo'T (ered) STeTeUeC che N| TOBN (vied) SELENITE N TO®N 9}e2DVUN a1VJIIVEN dVIIIVAEN | aie -uotdorgen | ayerAqngen aye -uotdo1gen IDPN (OY}10) aqeeyy den IDPN (OY}10) ayepeqyy den IDPN ID®N TDO®N LOBN AI S19) SiL9: S19" ‘Boq — Z6g1 ‘1 09q — Zt TORN IO®N IDO®N IDO®N O€ 0€ of Oe quowrrod xm a}vOV TUN aqyvoidegrN ayeadqngen a}VWIO FEN avr TUN ayepArdegen 9}e]20VeUN a}V}OIVEN Se Of O€ O€ ‘6c ‘AON — TT Juowmrs0dxy ’Od H*eN JOURN ¥0a°H®N O£ O€ enal Absorption and the Sa L[ntest O'S OL lo S19” [IDOPN [99 0° 10 STO IOPN | O€ ‘4yeq — ZOgI ‘Or ‘AON — OT JuomtTIedxa ee . . ¢ 99 ST = poqoalur junowy DO € 0 - aes 0° S i 8 95 | *OdH*eN "Wd Cf = dooy yee Jo ysUIG anpisey| anpisoy| V Wes anpisay| V Wes soqn -UTUL ul SYIVUIDY G. B. Wallace and A. R. Cushny. 432 peyoelur gunowy = dooy yore jo ysue'T — ‘SHUVNAY Ff) cz = tw OF Sede a | cae ie ; hee O€ C9 I®N OL ey LOENG Ocul? OST 19 IO®N O'r Iter LANE OF We [OPN os 19° IOVANT | Oke Ih 23° Sst | 09° | aretdideSen sg | szo | -AyjueusDeN| O'€ 19 | owordeDeN] O's 19" TOP NOE a | 2 c+ 19° ID®N OL 19° TD®N O€ Wer TOPN sy I ey TOUNG en Ocmeae ‘Bog — 9691 ‘Or ‘qa — LT Juewtedxg SS aa eee ee ‘99 6g = paqoalurjunoury “wo Og = doo] yova Fo yysuey — ‘SHUVNAY eel sst| 09 | orethadegen! 06 | S29 | -AqueUDeEN| 0's 19 | awoideDoeN| 08 19" IOeN} OF | 2 OL 19° [DPN os (er ID®N OL ey IDeN s'9 wey TORING 0 con es eae | ; Ost | 09 | aaephadeSen SOL | szo | -AyueugDeN| 0's 19 | ayeordeDeN| 0°8 19’ IDeN| OF | 9 00 19° IDeN 00 19° IO®N 0'0 ey IDOPN 00 Wey IOPN) | OF ee ‘Bog — 9681 ‘6 ‘qaq — gt quomrredxg ‘99 6Z = payoefur junowy “wo Sh = dooy yova jo yysuaT — ‘SHUVWAY Onc wale . . . . OL 09° ayepAide je N . ee ee eitaltens o¢ Pp oy ; OT V9 IO®N 0'8 49° (LORAIN || Oke |} 9°] ak. s“t | szo | -AyjueugpeN | SZ'T 19 | eywoideaeN| OF | 2 ‘ SLT y9 IOPN| ST +9" DENG Oe he ‘Bog — g6g1 ‘2 ‘qaq — ¢T Juommioedzg ‘99 6g = payoafuryunomy ‘wo O¢ = doo] Yyova jo yysuay — ‘SuUVNAY OOD Sy: ID®N OCI 99° | ayeuotdor1geN Sint 19 | aepfadepen Or (Ae) ayeyoe TUN | O€ | ? OOT | S19 IDeN OL S19 ID®N Ses S19 IOeN OT S19 TOUNG Ore O6T | 6 o7eTTOeN SST | SLg° | a7euo[eWeN (eral sc) aje[eWeN ive | ate IOXeN| OF | 2 PIO OT |oS19 IO®N |'9°9 G0 Jo STF IOPN 9 O'S 1oSTF IO®N 1°99 S'0 | o STI [LOVAING|| Oke Me ‘Boq — 4091 ‘V ‘99d — FI quowIIed xy anpisoy| VY yes enpisayt| V yes anpisoy| VY Wes anpisey}| V Wes soyn ——————— “UL ul ‘AI d00'T ‘III 100'T ‘TI d00'T ‘I dooT oui, | 433 20S. me Cathart 2ue ad the Sal. 2OnN QU l Absorpt I[ntestina 70S"H°0RN 1OeN ¥OS%eN IOeN oo $o9 Ses’ S29" IOH our “wind + TO®N _ 1IOeN Fost(FHN) IO®N ID@N OMANI IeN IDO®N O¢ = payoolur junowy ey Sco" 69 $9 ‘uvAOLL19 o. WORN TOSCH*O)EN IO®N O'L2 O'+ C+ O€ €9° S69 Soo" S29" ‘WO Of} = door yova Jo yASuNT — ‘sHUVWAY apruvAsoiay yy] o¢ | 2 OPN Whe || TOEN | Olan? JROVZINN| (Oke ff 2 ‘Boq — ger ‘Si yore — Tg JuowrIedxg” FOS aa1j sutequoo Fos* F494 $9° 09 $9 IDH 9ul “und + [DeN IO®N FOS*eN IDPN 09 CZ = paqoolur junowy +9" , IOeN COM OS CEs) on $9" IOeN A 8 8 S 0 0 0) ‘wd OF = doo] yova Jo YASUI] — 'SHUVWAY OIEING | Oe) TORING Ocoee. LOIN | OK 8 ‘S0q — QOer ‘r yOIey — 0g quomrredxg peqelur JUNOWY Zo | aqeyAorpegen +9" IOeN : IqeN IOeN +9" (Ay +9" $9" ‘B0q — g6gI oooo olsohcehte) "ud YE = doo] yore jo ySuaT — ‘suNNVINAY IO®N | OF IZN| OF IO®N | OF TORN! OF | ? ‘SI ‘qoy — 6T JuemIIedxy ‘0°90 ¢Z = payalur junowy $9" SONtN ob9 IO®N anpisey| ON pIsay Vv wes 0%6 9 SZ" +9 ot 9 ‘80q — S6gr ‘wd OE = doo] yova Fo yJSUNT — ‘SHYVWNAY ey og | 9 IDEN | OG=e a2 ‘Ol ‘qay — QT Juoursredxg anpIsoy}| Vv “AI doo'T ‘TI] doo'Ty, anpiseyy| V es son ‘T] adooT ‘] dooT -UTU ur aul, G. B. Wallace and A. R. Cushny. 434 29 OF = pezoafur juno ‘wo Sp = door yove jo yySUe'] — ‘SHAVNAY IOPHN TOeN|{ O'9T | 09" POINT eee Se Sly | 2 ayeuTIIuSeN j | 9} euU0[e NEN O'S Sco" IO®N ! LOE NGO cee [D@N : IO®N ‘kL $9" TORN TORING EO See IOeN - | ajeuronsen LT AGZO: d}CUOTLIAEN ; a}E[PXOVN | OF | 9 IDPN | i } IOPN ‘ll $9" IO®N $79" TOENG RO Sane ‘Bog — g6g1 ‘gi [ludy — ¢g quourrsdxg Y) (XS == peraei junowy ‘woos = [I] dooy f'wo cp = J] dooT a be dooy jo yysuayT — ‘SHAUVWAY $79" IOeN 2ONM SONPN| OF | 2 $zo" 1OUN OPN ; IOBeN| OF | 2 S+9° id FO Sad Nt ayeJIVeO : lora| O€ | 2 Sz" IOeN ID*HN WAS Fo hl ek 0) Sn $79" IOeN [OPN TOPE OL es Ve” ae _— Pie ‘6c OIL — HZ JuomIedxg = poyofur yunowy ‘wo cp = door yova jo yysuaT — ‘SHUVNAY Fossn 8 9" Hosm| ste | Le | wedoomeay| Of | 2 IO®N I " IOeN $79" LEN Caan oa Z SLE os ‘uvhd0.La UN ; . f “1O51N Oe Sco" TEN OLS 7 00 Sco" OVINE Che ‘Bog — 9691 ‘cz yolr — gg juewtiedxg [OPN ; 1OeN = payoafurjunowy “wd Sf = doo] yove jo ySuaT — ‘SNAVNAY FoS(*H20)eN ; i ‘uvfdI18q 3 679" IOeN - | 5 “uvh00.19 JM | o¢ IOPN : $c9" IOeN SyAc} 1IO®N ae IDEN | O€ "OS*°N | [FOSCH®OIEN | 0" S29" FOS*EN De | 500) [IOeN| OF (OPN 129" 0 $29" IOeN |'2°9 SOT | 0 $29 IOeN| OF | ? “Bog — g6g1 ‘Iz your — gg quewtredxg Vv ‘ anpisey| anpisey| Vv Wes anpisay| Vv eS | soqn -ULUL ul ‘AI 4007 ‘IIL d00'T ‘TI 4007] ‘I dooT oul THE. MOVEMENTS OF THE FOOD IN THE CHSOPHAGUS: By W. B. CANNON anp A. MOSER. [from the Laboratory of Physiology in the Harvard Medical School.] 5 ema movements of deglutition, in common with many other physiological processes, were explained by the older physiolo- gists on anatomical grounds. Thus Magendie! divided the act into three parts, corresponding to the anatomical regions of the mouth, pharynx, and cesophagus. The muscles of each of these divisions were considered the active agents in propelling the food onward. The function of moving the mass to the pharynx was variously ascribed to the tongue itself, to the mylohyoid muscles, and to gravity. For the second part, the movement through the pharynx, there was more unanimity of opinion, since the constrictors, espe- cially the middle and lower, were evidently concerned. Direct observations on the movement of swallowed masses in the cesophagus were first made by Mosso? The cesophagus of a dog was laid bare and a transverse incision made through it, or a piece of it excised. A small wooden ball was placed in the canal below the excised part, and the animal was then stimulated to swallow. One or two seconds after the contraction of the pharyngeal muscles a peristaltic wave began to traverse the cesophagus. This wave did not stop at the point of excision, but in due time reappeared below and carried the ball to the stomach. Thus the act was shown to be controlled by the central nervous system. Peristalsis was so plainly the motive power that the action was never doubted. Yet this belief was soon to be questioned. In 1880, Falk and Kronecker? studied the movements in the mouth and pharynx, and advanced the theory that deglutition was accomplished by the rapid contraction of the muscles of the mouth. During the act of swallowing the air-tight buccal cavity shows a manometric pressure of 20 centimetres of water. The same pressure was demonstrated to be present also in the cesophagus, but not in MAGENDIE: Précis élémentaire de physiologie. Paris, 1836, i, p. 63. Mosso: Moleschott’s Untersuchungen, 1876, xi, p. 331. i 2 3 FALK AND KRONECKER: Archiv fiir Physiologie, 1880, p. 296. 436 W. B. Cannon and A. Moser. the stomach. This pressure was considered sufficient to force food through the cesophagus before the peristaltic wave traversed it. Another argument for rapid descent was found in the fact that cold water can be felt in the epigastric region almost immediately after being swallowed. Further, when strong acids pass through the cullet, they corrode but small parts of it, and not the entire mucous membrane, as would be the case were the acid carried to the stomach by peristalsis. In the same year, in confirmation of the above results the well known experiments of Kronecker and Meltzer! were published. A rubber balloon, connected by a tube to a Marey tambour, was placed in the pharynx, and another balloon, similarly connected, was intro- duced a varying distance into the cesophagus. When water was swallowed the increased pressure in the pharynx was transmitted to the first tambour, which traced a curve on a rotating drum. Almost instantly thereafter the cesophageal balloon was compressed, causing the second tambour to write its curve below the first. This second curve was supposed to mark the passage of the food through the cesophagus. After a varying number of seconds the cesophageal balloon recorded another curve, caused by a peristaltic wave which carried to the stomach any fragments left in the canal. To demonstrate that the first curve of the cesophageal balloon was caused by the passage of the swallowed liquid, Meltzer devised another experiment. A strip of blue litmus paper was placed in a stomach tube, opposite the side openings at the lower end. A long thread attached to the paper ran through the tube to the other end. The tube was now passed into the lower end of the cesophagus and an acid drink swallowed. Ifthe litmus paper was pulled away from the side openings a second after the beginning of swallowing, it was found distinctly reddened, showing a rapid descent of the swallowed liquid. Reference to this experiment will be made later. From these observations Kronecker and Meltzer concluded that liquids and semi-solids are not carried to the stomach by peristalsis, but are squirted down the cesophagus by the rapid contraction of the muscles of the mouth. For this purpose the mylohyoids alone are sufficient, since the middle and inferior constrictors can be cut without interfering with the act. The succeeding peristalsis is of use merely in gathering up adhering fragments and carrying them to the stomach. 1 KRONECKER AND MELTZER: Archiv fiir Physiologie, 1880, p. 446. Movements of the food tn the Esophagus. 437 To determine whether the cardia offered any resistance to this rapid passage into the stomach, Meltzer! tried another method. Ifa stethoscope is placed over the epigastrium during the swallowing of liquids, a sound can be heard from six to seven seconds after the rise of the larynx. The sound is caused by the passage of the swallowed mass, liquid and air, through the tonically contracted cardia. Ina few cases the sound is heard immediately after swallowing, showing a probable insufficiency of the cardia. These phenomena led Kronecker and Meltzer? to modify their previous views. They now maintained that the mass is not squirted by the mylohyoids directly into the stomach, but halts a short distance above the cardia. Here it remains until carried into the stomach by the succeed- ing peristalsis, about six or seven seconds after the beginning of swallowing. The care with which these experiments were conducted has won general assent to their results. But the methods employed are not beyond criticism. Primarily it may be said that swallowing with one or more balloons and a stomach tube in the canal is not normal de- glutition. Moreover, semi-solids were found to yield less readily to pressure than liquids, and even to be delayed in their descent.? Again nearly all the work was done with liquids and semi-solids; solids are not even mentioned. The investigators themselves de- clared that their results were true for liquids and semi-solids only, and admitted that a dry bolus could not be so swallowed. Yet the indiscriminate use of such terms as “liquid,” “‘ swallowed mass,” and “bolus,” easily leads to an inference that the results of these investi- gations are true for the swallowing of food of all consistencies. A difference in rate, however, certainly exists in respect to consistency, and it was to discover the actual movement of solids, semi-solids, and liquids in the normal cesophagus that the present work was undertaken. Over a year and a half ago it was suggested by Prof. H. P. Bow- ditch that if some substance opaque to the Rontgen rays were swal- lowed, it could be seen in its passage to the stomach and the nature of its movement thus determined. Anesthesia could be dispensed with, — a desirable condition, since observers had found that it inter- 1 MELTZER: Centralbl. fiir die med. Wissenschaften, 1883, p. 1. 2 KRONECKER AND MELTZER: Archiv fiir Physiologie, 1883, Suppl. Bd., p. 337, 351. 3 KRONECKER AND MELTZER: 70id., p. 337. 438 . W. B. Cannon and A. Moser. ered greatly with the deglutition reflex.t It would be unnecessary to open either the abdominal or the pleural cavity. The reflex stimu- lus of food moreover would be better than electrical stimulation of the superior laryngeai nerve. In short, the animal would swallow normal food under practically normal conditions. At Dr. Bowditch’s suggestion and with his valuable assistance — which we gratefully acknowledge — we made the following series of experiments. To render the swallowed mass opaque subnitrate of bismuth was used. The salt is tasteless, practically inert, and can be fed in large quantities without harm. In order that observations could be made by more than one person, all experiments were conducted in a dark room. On the side of the animal opposite the Crookes tube was placed an open fluorescent screen on which the different tissues of the animal were outlined with varying degrees of light and shade. Among these shadows the swallowed mass appeared as a darker object, and thus its motion could be studied. For the first experiments the goose was selected. The head and neck were held stationary by a tall pasteboard collar which allowed free movement of the head without constriction of the neck. The fluorescent screen was placed against this collar at a uniform dis- tance of thirty centimetres from the tube. When a bolus of corn meal ° mush mixed with bismuth was placed in the pharynx it descended slowly and regularly, and occupied about twelve seconds in passing over a distance of fifteen centimetres. The screen was marked at intervals of two centimetres with cross lines, by means of which the relative rate in different parts of the cesophagus could be studied. A vibrator marking tenths of a second was interrupted whenever the bolus crossed a line. An average of over one hundred such observations showed that the rate became slightly slower as the bolus proceeded. In order to test liquids, molasses was mixed with bismuth to such a consistency as to drop easily from a glass rod. When this was fed with a pipette it passed slowly and regularly down the cesopha- gus, clearly by peristalsis. The rate was about the same as for solid food. In both these experiments, the addition of water would some- times cause irregularities in the descent. Microscopic sections from four different parts of the cesophagus of the goose showed no histo- logical difference. In the experiments on the cat, the animal was placed on its back and 1 MELTZER: Journal of experimental medicine, 1897, ii, p. 457. Movements of the ood in the Qsophagus. 439 ao left side on a holder. The extremities were secured by straps. The head was held between two upright rods connected above by a thong; this allowed free movement of the head without resistance to the passage of food. Shreds of meat dipped in bismuth were ordinarily masticated and swallowed without difficulty. For soft solids bread and milk were used, so fluid as to be easily drawn up into a pipette. The insolubility of the bismuth salt rendered the study of liquids more difficult. Strong solutions of potassic iodide and other salts and suspension of bismuth in acacia and molasses were tried; but a simple mixture of milk and bismuth, shaken in a test tube and imme- diately drawn up into a pipette, was found most practicable. Inasmuch as the movement of these different foods varied in differ- ent parts of the cesophagus, it will be convenient to divide the latter into three sections. The first or cervical portion extends from the pharynx to the thorax, the second or thoracic from here to the lower half of the heart, and the third comprises the rest of the canal. The relative length of these three parts is about in the ratio of 9:8:6. The beginning of deglutition was noted by one observer by a fin- ger on the larynx; the same observer called out when the bolus arrived at the thorax, heart, and stomach respectively, while the other observer noted the time. The movement of solids will first be considered. The descent the entire way was by peristalsis, but the rapidity varied. The duration of the movement in the cervical portion was two and a half seconds, and in the thoracic region a little less than two seconds. At the lower end of the heart there was some- times a slight pause. In the lower section, from the heart to the stomach, the movement was decidedly different. The rate was always very slow. The distance was less than one-third of the entire canal, yet the time consumed in this part ranged from six to seven seconds, or three-fifths of the entire time of descent. The character of the movement here was also peculiar. Whereas in the upper sections the passage was uniform and regular, with a slight accelera- tion in the thoracic region, here it was apparently irregular, for the bolus descended about one centimetre with each inspiratory move- ment of the diaphragm, and remained stationary or descended very slightly during expiration. Thus a series of hitches seemed to carry the bolus to the cardia. A probable explanation of this peculiar movement is that the stomach and lower cesophagus were pulled down with each descent of the diaphragm. This would make the movement appear irregular although it was really a slow peristalsis. 440 W. B. Cannon and A. Moser. It may be well to remark here that this movement was invariably observed in the cat with every kind of food. Semi-solids, namely, a mush of bread and milk, descended in the same way as solids; but the rate was slightly faster in the upper cesophagus, for the bolus took about a’second less to reach the car- diac level. From here the rate was the same as with solids. For liquids one and a half to two seconds sufficed for the descent to the midheart region. Here there often occurred a long pause — from a few seconds to a minute or more. Then the cesophagus apparently contracted above the liquid, which slowly passed on to the stomach as already described. Sometimes it seemed as if a swallowing move- ment, evidenced by a rise of the larynx, started the peristaltic wave. Again, several swallows would succeed one another before the liquid passed on. A few times the bismuth and milk seemed strung out along the cesophagus; some more liquid descending would gather this up, and the whole mass assuming an ovoid form would move into the stomach. Thus in the cat the total time for deglutition varies from nine to twelve seconds. The lowest section presents no change ascribable to a difference in consistency, while in the upper sections the rate does slightly increase with the more liquid character of the food. In experiments on the dog, bismuth enclosed in capsules or wrapped in shreds of meat was fed as the solid. The general phe- nomena were as follows. With the rise of the larynx there was a quick propulsive movement of the bolus, which descended rapidly for a few centimetres, sometimes as far as the clavicle. From this point the rapidity was diminished; yet no pause was observed; the bolus simply moved more slowly. This rate was then continued to the stomach without a slackening of speed in the diaphragmatic region,’as was observed in the cat. Semi-solids moved in the same way as solids. The total time of descent from larynx to stomach was from four to five seconds. Liquids gave even a more decided squirt in the beginning of the movement. To render the cesophagus as lax and free as possible, the head of the dog was released from the upright rods and held by the hands after the food was placed in the mouth. Sometimes the liquid descended rather rapidly as far as the heart, at other times no further than the clavicle; then without a pause it passed on slowly and regularly, reaching the stomach in about the same time as solids and semi-solids. Movements of the Food in the @sophagus. 441 Thus in the dog and cat but little variation was seen in the swal- lowing of liquids and solids. The liquids pass somewhat faster in the upper cesophagus. But in some animals the difference of rate with foods of varying consistency is much more marked. In the horse, for instance, mere observation shows a decided variation in the rate of movement in the cesophagus. Liquids shoot along the gullet, while solids move clearly by peristalsis. To determine the rate of solids one hand was placed on the larynx of a horse to note the beginning of swallowing and the other hand near the shoulders, where the bolus could be easily felt in its passage. The time con- sumed by the bolus in passing over a certain distance was measured by a stop watch. The rate obtained for solids, such as hay or grain, was from thirty-five to forty centimetres a second. For semi-solids, a mixture of bran and water was made, thin enough to run easily between the fingers. Each bolus was watched by a separate observer with a separate watch. The average rate obtained was the same as for solids. Liquids in the horse pass with a rapidity too great to be affected by peristalsis. Another force must be sought. Among the various muscles supposed to be effectual in moving food into the pharynx, the mylohyoids were shown by Meltzer! to be essential. The sty- loglossi were cut by him without much interference with deglutition, but section of the mylohyoid nerves rendered the act impossible. The activity of these muscles in the horse during swallowing is easily perceived by the hand. Their energetic contraction is a sufficient explanation of the rapid passage of water through the cesophagus. The motion here is more than five times as rapid as that of solids and semi-solids. Meltzer’s experiment to measure the rate of liquids in man by passing a stomach tube containing litmus paper was repeated by us with some modifications. Congo red paper was used, since it is more sensitive than litmus; it also furnishes a means of differentiating between mineral and organic acids, as the discoloration produced on Congo red by mineral acids is removed by ether. It was thus possible to distinguish between the discoloration produced by gas- tric regurgitation and that produced by the swallowed liquid. For the swallowed liquid one-half per cent lactic acid was found most satisfactory, as the color produced by it on Congo red test paper is almost instantly discharged in ether. By this method the paper 1 KRONECKER and MELTZER: Archiv fur Physiologie, 1880, p. 299. 442 W. B. Cannon and A. Moser. was found discolored within half a second after the rise of the larynx, certainly too short a period for a peristaltic wave to carry the liquid to the neighborhood of the cardia. The X-ray method lends itself less successfully to the study of deglutition in man than in the other ‘animals we have studied. The thickness of the thorax, the distance of the cesophagus from the surface, and the relation to dense tissues, render the observation of a swallowed mass difficult, especially when the mass is in rather rapid motion. The few observations which we have to report were made on a seven year old girl placed in the sitting posture. Gelatine capsules containing bismuth were used for solids, and were traced to a point below the heart. The motion was very regular, and ap- parently due to peristalsis, for the bolus descended without a hitch or irregularity of any kind. Sometimes the capsule became fixed in the upper cesophagus at about the level of the second rib. Re- peated swallows of water would fail to dislodge it. An interesting point was noted here. With each attempt at swallowing, the capsule would rise slightly as if the cesophagus was pulled up with the rise of the larynx; then the capsule would descend to its former position. Semi-solids —a mush of bread and milk—could be seen about as far as solids, z. ¢. to just below the heart. The motion of the mushy bolus was the same as with solids, except that the rapidity was perhaps slightly greater. It should be noted here that with the human subject, as well as with the horse, our results for semi-solids differ from those derived by Meltzer’s method; for according to his statements semi-solids,. like liquids, are squirted down the cesophagus and are not pro- pelled by peristalsis, as has been the case in our observations. Liquids — bismuth and water —were seen only in the neck and upper thorax. Here there was a decided squirt. With the rise of the larynx the liquid was seen to pass rapidly through the pharynx and well down into the thoracic cesophagus before it was lost to observation. The rate, however, by estimation was less than that of liquids in the horse. There remains to be considered Meltzer’s latest investigation, in which he endeavored to ascertain whether liquids remain above the cardia till the arrival of the peristalsis, or ooze down before. An experimental answer was secured by Meltzer by the following 1 MELTZER: Journal of experimental medicine, 1897, ii, p. 453. Movements of the Food in the GQ sophagus. 443 a method. The abdominal and gastric walls of an anesthetized dog were incised and a tube (vaginal speculum) introduced. Through this the entrance of food into the stomach could be observed directly. In repeated experiments no liquid was seen to pass through the cardia before the arrival of the peristaltic wave. An incision through the diaphragm near its anterior origin showed that the swallowed liquid was not squirted as far as a point an inch above the diaphragm. To observe the cesophagus nearer its beginning, the upper three ribs were resected on the left side. Thus the swallowed liquid was seen to shoot along the cesophagus before any peristalsis reached this point. The resection of the fifth rib exposed the cesophagus half way between the bifurcation of the trachea and the diaphragm. Here a bulging was sometimes observed immediately after the begin- ning of the act, and the swallowed mass remained there until a peris- taltic wave carried it down. If the mass swallowed was small, or was projected with moderate force, it might not even reach as far as the bifurcation. From these experiments Meltzer concluded that in animals as in man, liquid food is not carried down the cesophagus by peristalsis, but is thrown rapidly into a deep part of the canal. The depth reached depends on the quantity swallowed, the force used, and the tonicity of the lower part of the cesophagus. The difference between these methods of Meltzer and those em- ployed in our experiments has already been mentioned; and merely his results, which were obtained with liquids alone, need be con- sidered here. According to our observations on the dog, there was no distinct pause at any part of the canal. The movement simply became slower, and continued at this rate until the stomach was reached. Neither was the rate through the diaphragmatic part of the cesophagus slower than through the thoracic. The quick propulsive movement noticed in the dog was observed with solids and semi- solids as well as with liquids, but the liquids descended further down the canal before the movement changed to the slower peristalsis. While this difference was evident to the eye, the total time consumed by liquids in passing from pharynx to stomach was not. enough shorter than the time for solids and semi-solids to be determined by our measurements. A44 W. B. Cannon and A. Moser. SUMMARY. The phenomena of cesophageal deglutition as determined by our experiments may then be described as follows: — There is a difference in swallowing according to the animal and the food which is used. In fowls the rate is slow and the movement always peristaltic, without regard to consistency. A squirt-movement with liquids is manifestly impossible, as the parts forming the mouth are too hard and rigid. With this diminution of propulsive power in the mouth there is observed a greater reliance on the force of gravity. The head is raised each time after the mouth is filled, and the fluid by its own weight trickles into the cesophagus, through which it is carried by peristalsis. In the cat the movement is always peristaltic and slightly faster than in fowls. A bolus takes from nine to twelve seconds in reaching the stomach. Liquids move somewhat more rapidly than semi-solids in the upper cesophagus. In the lower or diaphragmatic part the rate is very much slower than above, and is the same for liquids as for solids. In the dog the total time for the descent of a bolus is from four to five seconds. The food is always propelled rapidly in the upper cesophagus and moves more slowly below. This rapid movement is frequently continued further with liquid food. No distinct pause was observed when the movement of the bolus changed from the rapid to the slower rate. In man and the horse liquids are propelled deep into the cesoph- agus at a rate of several feet a second by the rapid contraction of the mylohyoid muscles. Solids and semi-solids are slowly carried through the entire cesophagus by peristalsis alone. m& CONTRIBUTION TO) THE CHEMISTRY. OF CYTOLOGICAL STAINING: By ALBERT MATHEWS. [From the Zovlogical Laboratory of Columbia University.] T has long been known to histologists that different elements of the cells and tissues show affinity for different stains. Many nuclei, some mucins, and hyaline cartilage stain powerfully in methyl green, Bismarck brown, thionin, and other basic stains, while other nuclei and most cytoplasmic elements show a decided preference for eosin, acid fuchsin, acid green, and other acid stains. The nature of the chemical reactions upon which this elective staining power rests has never received adequate attention. It is several years since Ehrlich? classified all stains as ‘‘ acid,” ‘‘ basic,” and ‘“‘ neutral,” yet it is still uncertain upon just what properties the affinity of chromatin for basic dyes and cytoplasm for acid dyes really depends. It is still not “uncommon to find in cytological works methyl green and other basic stains regarded as microscopical reagents for the detection of chro- matin, and some cytoplasmic bodies because of their affinity for basic dyes have been looked upon as chromatin or derivatives of chromatin. The first observations on the possible chemical basis of the staining reactions of chromatin were made by Miescher,? who found that nucleinic acid, a component of chromatin, formed green insoluble salts with methyl green. Lilienfeld? called attention to the same fact and referred the affinity of the chromatin for basic stains to the for- mation of these salts. Lilienfeld* also advanced our knowledge by showing that albumin stained pre-eminently in the acid stains and nucleinic acid only in the basic. In studying the artificial nucleins he found that they possessed a varying affinity for acid or basic stains according as the nucleinic acid was more or less completely saturated with albumin. He also observed that egg albumin precipitated by 1 EHRLICH: Archiv fiir Physiologie, 1879, p. 571. 2 MIESCHER: Verhandl. d. naturf. Gesellsch. in Basel, 1274, vi, p. 138. 8 LILIENFELD: Archiv fiir Physiologie, 1893, p. 391. 4 LILIENFELD: Archiv fiir Physiologie, 1893, p. 554. 446 Albert Mathews. alcohol stained neither in acid nor in basic stains. Lilienfeld believed that the affinity of the cytoplasm for acid stains was due to its con- taining much albumin, but he made no suggestion as to the nature of the combination of the stain with the albumin molecules. He fell into error in supposing that albumin stained only in the acid stains. It will be shown farther on that under suitable conditions the albumin molecule may be made to combine also with the basic stains. In practical experience histologists have observed that sections stained in acidified solutions of the Biondi-Ehrlich mixture take chiefly the acid stain, while in alkaline solutions they take the basic. No expla- nation of the cause of this phenomenon has been offered, so far as | am aware. The present paper presents the results of experiments which give some indication I believe of the probable nature of these affinities, and which also show how far cytological stains may be used as accurate micro-chemical reagents. It must be understood at the outset that the results here recorded of experiments on egg albumin, albumoses, and peptones can be directly applied only to such sections of tissues as have been killed and fixed in alcohol or acid media free from metallic salts such as mercuric and platinic chlorides; and further that the conclusions do not apply to those staining processes which probably involve the precipitation of the coloring matter in the tissue, such as the iron-hematoxylin method. I. EXPERIMENTS ON ALBUMOSES. A. The acid stains. — Physiological chemists are aware that albu- min and the albumoses react like weak bases, and that they will combine with free acids. If acetic, hydrochloric, or sulphuric acid is added to a solution of albumoses it may be shown by appropriate methods that the acids have chemically combined with the albumoses, although no precipitate is thrown down. Many other free acids enter into similar combinations with the albumins and albumoses, but form insoluble compounds, thus precipitating the albumoses from solution. If a solution of picric acid is brought into a solution of albumose a precipitate consisting of the picric acid combination of the albumose is thrown down. The same kind of reaction ensues with meta-phosphoric, molybdic, wolframic, phosphor-wolframic, tannic, stearic, or chromic acid. Only the free acids will combine with the albumoses. If a neutral solution of the salts of the above- mentioned acids is added to a neutral solution of the albumoses no Pate items races The Chemistry of Cytological Staining. 447 reaction occurs. A few drops of acetic or hydrochloric acid are necessary to call forth the reaction. On the addition of acetic acid to a mixture of albumose and sodium picrate, a precipitate consisting of the picric acid combination of albumose appears at once. Probably the reason is that the acetic acid sets free the picric acid, which at once combines with the albumose molecule. It occurred to me that the so-called ‘‘acid” stains, which are generally the sodium salts of sulfonic acids, probably combine with the albumin molecule in the same manner as the above mentioned acids. Experiment fully bore out this hypothesis, for I found that the acid stains possess the same albumin-precipitating powers as sodium picrate or wolframate. The addition of acid fuchsin, acid green, nigrosin, anilin blue black, erythrosin, congo red, carminate of soda, methyl] blue, indigo carmine, or other acid stain to a solution of albumoses or albumin gives no reaction. If, however, a few drops of dilute acetic acid be added to the mixture of albumose and acid stain, the colored combination of the stain with the albumose is at once precipitated. One can indeed use this test for detecting the presence of albumin or albumoses in solution or for distinguish- ' ing between acid and basic stains, as the basic stains do not give this reaction. This reaction of the acid stains indicates beyond doubt that these stains when in acidulated solutions will enter into chemical combination with the albumose or albumin molecule like any other acid. Inasmuch as it is probable that the free acids enter one or more of the basic NH, groups of the albumin molecule, the acid stains also probably enter this group. B. The basic stains. — The basic stains react with the albumoses very differently from the acid. The former stains are generally the chlorides or hydrochlorates of colored organic bases. They react as might be expected like other organic bases. It is well known that in alkaline solution many metals may be made to form combinations with the albumin molecule. If for instance lead ace- tate is brought into a neutral solution of albumoses or albumins no reaction occurs. If now the solution be made slightly alkaline with sodium carbonate a precipitate is formed consisting of a lead compound of albumin. It is probable that the lead enters the albumin molecule in a different place from the acids already mentioned, and that it enters the hydroxyl of the phenol group, since gelatine and protamin, which lack this group, are not precip- itated by basic lead acetate. Many organic bases react like lead. a 448 Albert Mathews. Protamin, histon, and quinine — strong organic bases — precipitate albumin and the albumoses in alkaline solution. The basic aniline colors react similarly. They may in this manner be made to form colored combinations with albumin and the albumoses. If basic fuchsin, methyl green, thionin, safranin, or other basic stains (with the possible exception of vesuvin), are brought into a neutral or slightly acid solution of the albumoses, no reaction takes place. If on the other hand they be brought into solutions of the albumoses made slightly alkaline with sodium carbonate a flocculent, colored precipitate consisting of the albumose in com- bination with the dye is thrown down. This reaction may be used to distinguish the basic from the acid dyes. Vesuvin is precipitated . by sodium carbonate alone, but if albumoses are present it is possible, though I have not specially examined the matter, that the precipitate is a combination of vesuvin with the albumose. These experiments prove that many of the basic dyes enter into chemical combination with the albumose molecule when in alkaline solutions, forming insoluble colored compounds. They show also that in acid or neutral solution this reaction does not occur. To sum up: (1) The acid stains will combine with albumoses only in acid solutions. (2) Under such circumstances they form combinations similar to picric or other acid combinations with albumoses, and probably enter one or more NH: groups in the albumose molecule. (3) The basic stains will combine with the albumoses only in alkaline solution, when they form insoluble colored compounds. The basic dyes react in this respect like basic lead acetate, protamin, histon, or other organic bases. (4) The basic stains probably enter the hydroxyl of the phenol group of the albumose molecule, since they will not precipitate gelatine. II. COAGULATED EGG ALBUMIN. A. The acid stains.—Coagulated egg albumin reacts toward the acid stains like the albumoses. If egg albumin coagulated by heat or alcohol be brought into neutral or alkaline solutions of the acid dyes the albumin will not stain. It is true that it will imbibe a certain amount of color and will appear stained, but this color is easily and quickly removed by washing in water. If on the other hand pieces of coagulated albumen be brought into solutions of the acid stains which have been slightly acidulated with acetic acid the albumin a en The Chemistry of Cytological Staining. 449 stains instantly and intensely. The color cannot be removed even by prolonged washing. A most striking contrast is shown by two pieces of coagulated albumin, one of which has been immersed in a neutral, the other in an acid solution of acid fuchsin. After washing, the former will be found to be colorless, the latter a brilliant red. B. The basic stains.— Towards the basic stains coagulated albumin reacts on the whole like the albumoses. Egg albumin coagulated by heat is normally alkaline. If its alkalinity be neutralized or if it be brought into a slightly acid solution of the basic dyes it will stain but slightly. Its power of staining under such circumstances I believe to be due to some other constituent than the albumin, possibly to the mucoid matter present. If however the coagulated egg albumin without neutralization be brought into neutral or slightly alkaline solutions of the basic dyes methyl green, thionin, safranin, methylen blue, or toluidin blue, it stains with great intensity and instantane- ously. This may be most strikingly seen in the case of thionin or safranin. If two pieces of coagulated egg albumin be brought the one into slightly acid and the other into alkaline solutions of thionin, the stain poured off after a few seconds, and the albumin washed in water, the piece that has been in the alkaline solution will be an intense purple, the other barely tinged with color. These reactions clearly indicate that the staining of coagulated albumin depends on chemical combinations similar in all respects to those which the albumoses enter into with the same stains. In neu- tral solution, neutral coagulated albumin combines neither with acid nor basic stains; in alkaline solutions, it combines only with the basic; in acid solutions, only with the acid stains. IIJ. CARMINIC ACID, HAZMATINE, AND THE ACTION OF ALUMINIUM. Paul Mayer! has shown that carminate of soda and hematine are plasma stains, as are the acid aniline colors, whereas the aluminium salts of these acids are chromatin stains, as are the basic aniline colors. Carminate of soda and hematine react towards the albumoses like the acid statns. In neutral or alkaline solutions they do not combine with the albumoses or albumins; in acid solution they precipitate the albumoses at once. Freshly prepared hematoxylin will not stain tissues, and corresponding with this I find that fresh solutions 1 Mayer: Mittheil. a. d. zoolog. Stat. Neapel, 1892, x, p. 170. By 450 | Albert Mathews. of hematoxylin will precipitate the albumoses neither in acid, neu- tral, nor alkaline solutions. As aluminium gives carminic acid and hamatine the staining properties of the basic aniline colors, it is of interest to see how these salts react towards the albumoses. I found that the aluminium salts of these acids (Mayer’s carmalaun and hemalaun) would not precipitate the albumoses in neutral or acid solutions. Thus they differ completely from the sodium salts. In alkaline solutions of the albumoses the addition of solutions of car- malaun or hamalaun caused heavy flocculent colored precipitates. It is possible that the precipitate was simply the stain which is insolu- ble in alkaline solutions, but it is also possible that it was the stain in combination with the albumose. In any case the aluminium salts of carminic acid and hematine no longer react toward the albumoses or tissues like acid stains, but like basic stains. This is possibly due to the strong basicity of the aluminium, and its tendency to form double acid salts. It will probably be found, I believe, that the aluminium salts of the acid aniline colors stain like the basic dyes. IV. THE STAINING OF SECTIONS. The foregoing experiments suggest that the affinity of sections of tissues for stains depends upon reactions similar to the above. So far as I have experimented, the results have fully confirmed this sug- gestion, but the formation of salts by acids of the tissues with the basic dyes also comes into play. We will consider this first. A. The basic dyes in neutral solution.— The basic dyes in neutral or acid solution will not combine either with albumin or the albumoses. It is clear from this that the affinity shown by chromatin, cartilage, and mucin for such dyes when in neutral solution must depend on something else than the albumin molecule. The suggestion of Miescher and Lilienfeld that the affinity of chromatin for the basic dyes depends on the nucleinic acid indicates the essential cause of the staining reactions of the elements just mentioned. Besides the albumin molecules they contain, mucin, chromatin, and hyaline cartilage have little else in common than the presence in each of organic acids in salt combinations with strong bases. There can be little doubt that the basic dyes in neutral solution will stain any element of the tissue which contains an organic acid in a salt com- bination with a strong base. That methyl green in neutral or acid solutions stains those chro- matins in which the nucleinic acid exists in a salt form is shown by a The Chemistry of Cytological Staining. 451 its striking affinity for the chromatin of some spermatozoa, thymus gland cells, leucocytes, cells of the spleen, and the red blood corpus- cles of birds, and by its slight affinity for the cells of the vertebrate pancreas. Inthe thymus gland, leucocytes, and red blood corpuscles, Kossel! has shown the chromatin to be composed largely of the histon salt of nucleinic acid. In the spermatozoa of the fish and sea- urchin, Miescher,? Kossel,? and the author‘ have shown the chromatin to be either a histon or protamin salt. In the pancreas on the other hand nucleinic acid exists in a much firmer combination. Lilien- feld’s* observations on the artificial nucleins confirm this also. He found that the artificial nucleins stained in methyl green so long as they were not saturated with albumin. So soon as the acid became saturated with albumin, the nuclein showed a preponderating attrac- tion for the acid stains. This is strong evidence that the acid stain enters the albumin molecule, while the basic enters the nucleinic acid molecule in these nucleins. Cytoplasmic bodies with an affinity for basic dyes also indicate that these dyes will stain elements containing the salts of other organic acids. Hyaline cartilage possessing an affinity for such dyes consists, according to Schmiedeberg,® largely of the potassium or other salt of the chondroitin-sulphuric acid. Many mucins have the same power of staining in basic stains. The chemistry of mucins is not well known, but many of them, at any rate, react distinctly acid.7 Many other acids which are possibly present in the cell form insoluble colored salts with the basic dyes. If a basic dye is added to neutral soap solutions a flocculent, colored precipitate consisting probably of the colored salt of palmitic or stearic acid is thrown down. Neutral solutions of thyminic acid, a derivative of nucleinic acid, or of the pseudo-nucleinic acid derived from the yolk of hen’s eggs show similar reactions. These considerations permit us to formulate the following con- clusions as to the staining powers of the basic stains. In slightly 1 See LILIENFELD: Zeitschr. f. physiol Chemie, 1894, xviii, p. 473. * MiescueEr: Archiv fir exper. Pathol. und Pharmakol., 1896, xxxvii, p. Too. 8 KossEL: Zeitschr. f. physiol. Chemie, 1896, xxii, p. 176. * MATHEWS: Zeitschr. f. physiol..Chemie, 1897, xxiii, p. 399. 5 LILIENFELD: Archiv. fiir Physiologie, 1893, p. 301. ° SCHMIEDEBERG, O.: Archiv fiir exper. Pathol. und Pharmakol., 1891, xxviii, P. 355: * HAMMARSTEN: Zeitschr. f. physiol. Chemie, 1888, xii, p. 189; also Lehrbuch der physiologischen Chemie, ii Aufl., 1896, p. 139. 452 Albert Mathews. acid or neutral solutions the basic dyes will stain any element of the tissue which contains an organic acid in a salt combination with a strong base. In no sense are these dyes a test for nucleinic acid or chromatin. All conclusions in regard to the origin of cytological elements from chromatin or their similarity to chromatin based on the staining reaction are hence worth very little. In neutral or acid solutions the basic stains may be used, I believe, as micro-chemical tests of some accuracy for the detection of the salts of organic acids. B. The basic stains in acid and alkaline solutions.— It has been shown above that in acid or neutral solutions the basic stains will not unite with albumin, but in alkaline solution will combine with the albumin molecule. To test the staining reactions of tissues in the basic dyes in the light of this fact, pieces of liver, kidney, and voluntary muscle of the frog were placed in neutral and acidulated ninety-five per cent alcohol. The acidulated alcohol contained one per cent of acetic acid. The tissues were imbedded in paraffine and cut as usual. The fixation was excellent. In staining, all dyes were used in weak aqueous solutions, and the sections were well washed in water before and after immersion in the stain. Sections were left in the dyes from a few seconds to three minutes. If brought into strong solutions of the dyes the tissues imbibe a considerable amount of stain, I presume by a physical process, but this may be entirely removed from the cytoplasm by a comparatively short bath in water or alcohol. The liver, kidney, and muscle fixed in neutral or acid alcohol give purely chromatin stains with neutral solutions of the dyes vesuvin, methyl green, methylen blue, safranin, toluidin blue, thionin, and dahlia. In his Vade-Mecum, Lee, speaking of methyl green, insists again and again that the stain must be slightly acidulated with acetic acid. With all basic dyes, I have found the result better if a neutral solu- tion is taken, though slight acidification seems to do nothing more than to diminish somewhat the intensity of the stain. In either case a pure chromatin stain is obtained. When used in alkaline solutions, the basic stains react otherwise. It has been shown that in alkaline solutions the basic dyes combine with albumin. I find that sections of the above mentioned tissue, if immersed for an instant in one-tenth per cent sodium carbonate solu- tion before staining or if stained in solutions of the basic stains made slightly alkaline with sodium carbonate show the cytoplasm deeply be i wale ——_ The Chemistry of Cytological Staining. 453 stained, as well as the chromatin. The stain, even in the cytoplasm, is in such firm combination that it is exceedingly difficult, if not impossible, to wash it out. In this manner the cytoplasm of these cells may be stained a bright green with methyl green, brilliant red with safranin, a deep blue with methyl blue or toluidin blue, and purple with thionin. Vesuvin alone seems to be an exception. These reactions, which are identical with those of the albumoses, show that in alkaline solution many of the basic dyes will combine with the albumin molecule whether in cytoplasm or nucleus. As we have already seen, they probably enter the phenol group of this molecule. The basic dyes in alkaline solution may thus be used for the detection of albumins in the cell, and indeed of albumins possess- ing a phenol or tyrosin group. C. ‘The acid stains.— The acid stains do not combine with albu- min in neutral or alkaline solutions, but only in acid solution. The - tissues show the same reaction. Tissues hardened in neutral alcohol will not stain in neutral or alkaline solutions of the acid stains, even such intense stains as acid fuchsin. If brought into concentrated aque- ous solutions of these stains, the sections imbibe a certain amount of stain, more or less difficult to remove by washing. If such sections be run rapidly through the alcohols they will appear stained. That the stain in such sections is not in chemical combination is shown by the fact that in dilute solutions of the stains such imbibition is exceed- ingly slow or wholly lacking, and also by the fact that even after im- mersion in concentrated staining solutions the stain may be entirely removed by washing some time in water. If, on the other hand, sections of tissues hardened in neutral alcohol are washed before staining with one per cent acetic acid or are brought into acidulated solutions of the acid stains, indigo carmine, carminate of soda, nigro- sin, methyl blue, erythrosin, acid green, congo red, orange G., and acid fuchsin, the cytoplasm stains instantly and intensely. The stain cannot be washed out. The observation that sections of such tissues as liver, kidney, and muscle will not stain in neutral acid fuchsin appears at first glance to be contrary to the common experience that séctions will stain in non- acidulated solutions of this color. The contradiction is only appa- rent. Nearly all fixing reagents are acid, and the free acid undoubt- edly combines with the albumin of the protoplasm. Having acid already in combination it is not necessary to acidulate the acid stains, for the sodium of the stain probably unites with the acid derived from 454 Albert Mathews. the fixing fluid, and the acid stain replaces this in the albumin mole- cule. That this is true is shown by the fact that the same tissues hardened in acid alcohol stained readily in neutral solutions of the acid stains. This staining reaction of the tissues with the acid stains, corre- sponding as it does with the reactions of the albumoses and albumin, enables us to conclude that the acid stains enter into chemical com- bination with the albumin molecule in protoplasm and probably with an NH, group in that molecule. The acid stains may be used in acid solution on tissue hardened in alcohol or acetic-alcohol, as micro-chemical reagents for the detection of albumin in the cell ele- ments, with the proviso that there may be other unknown basic sub- stances in protoplasm forming similar compounds, and that possibly in some cases the albumin may already be in such combination with other substances that it will not unite with the acid stains. The observations here recorded by no means elucidate all the phenomena of staining, but, I believe, they indicate one method of attacking the problem. It would be interesting to know what influ- ence the introduction of mercury or other metals and of acids into the albumin molecule may exert on its staining properties. Until this is known the results and conclusions of the present paper cannot be applied to tissues fixed in corrosive sublimate, Hermann’s fluid, and many other fixing fluids. NOLES ON -CEERARTA, ISEANDICA (ICELAND: -MOSS)- BY ERNEST “WwW. BROWN, PH.5; [From the Sheffield Laboratory of Physiological Chemistry, Yale University.] ROM early times lichens have been utilized as articles of diet for man and domestic animals.!_ First among them in importance as a food-stuff is ‘‘ Iceland moss’”’ (Cetraria islandica), which seems to have recommended itself because of its large content of carbohydrate matter, the so-called lichen-starch. In its natural form this lichen contains bitter constituents, and these must be removed by treatment with water or weak alkalies before the material can be made into bread, as has been the custom in some northern countries. Rabbits almost invariably refuse to eat the lichen unless it has been rendered more palatable as described. With reference to the real dietetic value of Cetraria islandica, the following analysis of the commercial material will afford some data.? Analysis of Cetraria tslandica (dried at 105°C.) ut alamitrorenin foie) ee See ose) A ts) ae LOE DO ENCENt: IB ReraCbINe NTLTOPEM EON 2 os) sa) ste ise et ee, om OEE | Ss “ Protein” nitrogen O32 Ether extract % 1-2 ‘ Crude fibre gi ui Ash 5 te snd ate doa Hes 2.2 of Material soluble in $5 per cent alcohol . 16.1 ss Soluble carbohydrates (as dextrose) 43.3 e After successive treatment with gastric juice and amylolytically and proteolytically active pancreatic juice at 38° C. only 32 per cent of the material used was dissolved. The residue resisting digestion contained practically all the original nitrogen (0.55 per cent) of the lichen. It will be observed that the quantity of proteids present must be small at most. The buik of the material is made up of soluble car- bohydrates. The latter were early made the subject of chemical in- Cf. ALBERT SCHNEIDER: A text-book of general lichenology, 1897. The methods of analysis employed were essentially the same as described by L. B. MENDEL: This journal, 1808, i, p. 226. * This consisted of free fatty acids (0.4 per cent) and saponifiable fat (0.62 per cent). 1 2 456 EL. W. Brown. vestigation. Without attempting to recite the older and somewhat conflicting. observations, we may refer to the more recent results of Honig and St. Schubert.!’ These investigators conclude that extracts of Cetraria, obtained with hot water, contain two carbohydrates. The chief one of these, lichenin, forms a difficultly soluble jelly in cold water, an opalescent solution in hot water, is not colored blue by iodine, and does not rotate polarized light; on boiling with dilute acids lichenin yields crystallizable dextrose in addition to dextrins. The second carbohydrate, called lichenin-starch, is regarded by these authors as a soluble modification of ordinary starch. It has also been called isolichenin.2, Munk? states that lichenin is most nearly related chemically to starch, and that it probably undergoes the same fermentative changes in the alimentary canal as are produced by boiling with dilute acids. The following experiments by the writer confirm in part and extend previous observations. Lichenin. — Preparation. — The dry assorted Iceland moss was. heated in a steam sterilizing apparatus for several hours with a con- siderable quantity of water, and the extract then filtered on hot water funnels. The cool filtrates deposited a thick jelly which was thrown upon filters and allowed to drain. The gelatinous mass was redis- solved in hot water and reprecipitated repeatedly until the cold fil- trates as well as the jelly no longer gave any blue coloration with iodine. The gelatinous substance was next treated with warm alcohol until all coloring matter was removed, then extracted with ether and dried. There resulted an almost white, tasteless, odorless powder, soluble in hot water, insoluble in cold water, free from nitrogenous matter, and yielding about one-half per cent of ash. Flydration by dilute acid. —In each trial a weighed quantity of lichenin was boiled for twelve hours with two per cent hydrochloric acid, and the resultant sugar determined in the neutralized fluid by the Allihn gravimetric method. The specific rotation was likewise ascertained and osazones were prepared. ‘ HONIG UND ST. SCHUBERT: Sitzungsb. d. k. Akad. d. Wissenschaften zu Wien, 1887, xcvi, 2 Abth., p. 685. The older literature is referred to here. Cf. also BEILSTEIN: Handbuch der organ. Chemie, 3'¢ Auflage, i, p. 1098. 2 Cf. BEILSTEIN, Joc. cit., p. 1099. ’ Munk, J. und C. A. EwaLp: Die Ernahrung des gesunden und kranken Menschen, 1895, p. 102; also C. Voir: Die Ernahrung. Hermann’s Handbuch der Physiologie, 1881, vi, p. 413. a Cetraria Islandica (Iceland Moss). AGT I. 1.0936 grams lichenin (ash-free) yielded on hydration 1.097 grams dextrose. Assum- ing a hydration equivalent to that of starch, 1.0936 grams lichenin should yield 1.215 grams sugar. II. (az) Ina solution of hydration products containing 1.53 per cent sugar (determined as dextrose), in a 200 mm. tube an average of five polariscopic readings gave a rota- tion of + 1.6%. Then (a)p=+ 52.2°. (4) Ina solution containing 0.51 per cent sugar in a 220 mm. tube, an average of six readings gave a rotation of +0.6°. Then (a)p=+ 53.1°. The specific rotation of dextrose, (a)p=-+ 52.5°. III. The osazones of the sugar formed were prepared with phenylhydrazin in the usual manner, and recrystallized four times from alcohol. M. p. 199°— 201° C. The melting point of phenylglucosazone = 204° C. The experiments thus indicate an almost complete hydration of lichenin, analogous in its results to the conversion of ordinary starch. Action of enzymes and dilute HCl,.—In order to determine the possible fate of ingested lichenin in the alimentary canal, the be- havior of the carbohydrate towards the ordinary amylolytic enzymes was reinvestigated. The following typical experiments are selected from the protocols. I. A one per cent solution of lichenin in boiling water was prepared and placed in a bath at 38°C. Most of the material stays in solution; a portion separates out at this temperature. Saliva was added and the solution was tested for reducing sugars from time to time, with Fehling’s solution. No reaction was obtained after forty-five minutes. To one portion ordinary starch paste (one per cent) was now added. The solution reached the “achromic point” to iodine solution! in ove minute and sugar was abundantly formed, thus showing that there was nothing present inhibitory to the action of the enzyme. The other portion of the original fluid was unchanged even after several hours. I. A very active diastase preparation likewise failed to transform the lichenin to reducing sugar during an hour’s action at 38-40° C. II. To a one per cent lichenin paste was added an amylolytically active pancreatic extract (alcoholic). No sugar was formed, while the unimpaired activity of the enzyme was demonstrated as in Experiment I. Iv. The ash from one gram of lichenin was added to a small quantity of starch paste. There was no inhibition of the subsequent action of saliva. V. A one per cent lichenin paste was treated with saliva for an hour at 38° C. No sugar was formed. The solution was then precipitated with alcohol and the precipitate redissolved in water. The action of saliva was again tried, with the usual negative result. These operations were repeated four times with similar effects. From experiments like the above it must be concluded that the ordinary amylolytic enzymes have no noticeable action on lichenin. Berg? is reported to have obtained similar results with saliva, malt diastase, pancreatic extract, and gastric juice. Since it has been shown that cane-sugar is readily inverted in the stomach by the 1 Cf. GAMGEE: Physiological chemistry of the animal body, 1893, ii, p. 57. 2 BERG: Abstract in Jahresbericht der Chemie, 1873, p. 848. 458 £.. W. Brown. gastric juice! and experiments in this laboratory have shown that inulin — likewise resistant to enzymes—is partly transformed to reducing sugar by the action of dilute HCl (0.2-0.4 per cent), the following experiment was tried. A one per cent lichenin paste was treated with an equal volume of 0.4 per cent HCl and kept at 38° C. for twelve hours. The test for sugar was negative. The mixture was carefully neutralized and treated with amylolytic pancreatic extract. No sugar was formed. Acid of 0.3, 0.4, and 0.5 per cent strength also gave negative results. Glycogen is likewise resistant to the action of these acids at 38° C. Feeding experiments. — In view of the behavior of lichenin already recorded, it seemed desirable to ascertain whether this carbohydrate would give rise to a formation of glycogen in the liver as has been found by Miura? to occur after inulin feeding. Miura’s experiments were followed as a type and protocols are given below. Two rabbits, weighing 2.2 and 2.3 kilos respectively, were starved for six days. The control animal (2.3 kilos) was killed and the glycogen content of the liver found by the Briicke-Kiilz method to be 0.286 gram (0.7 per cent). The cther rabbit (2.2 kilos) received ten grams of lichenin, suspended in warm water, in five portions through the stomach sound at intervals of two hours. Twelve hours after the last portion was fed the animal was killed. The glycogen-content of the liver was found to be 0.086 gram (0.25 per cent). Another rabbit of 2 kilos, likewise starved, was fed about eight grams of lichenin in several doses. The animal was accidentally killed immediately after a portion had been fed. The liver did not contain a weighable amount of glycogen. The writer has not succeeded in finding rabbits that would eat any considerable quantity of the lichen itself, even after extraction with potassium carbonate to remove the bitter taste. Further experiments with larger quantities of lichenin are desirable. Isolichenin. This carbohydrate, to which is due the blue iodine- reaction in the filtrates from the lichenin preparation, has received little investigation. It is in some respects closely related to soluble starch. The amount present in the lichen is decidedly less than the amount of lichenin, and a micro-chemical study shows it to be distributed through the cell walls of both the cortical and medullary portions of the plant. Micro-chemical reactions for cellulose give negative results. Preparation. — The filtrates from the lichenin were concentrated in vacuo at a low temperature (35°-40°C.). If any remaining lichenin settled out on cooling it was filtered off and the solution was treated with several volumes of alcohol. The somewhat gummy precipitate was redissolved in hot water and again cooled. Further traces of lichenin were removed by filtration from the concentrated fluid; the 1 FERRIS and Lusk: This journal, 1898, i, p. 277. 2 MiurA, K: Zeitschr. fiir Biologie, 1895, xxxli, p. 255. 3 Cf. BERG: Joc. cit. ; HONIG und ST. SCHUBERT: Joc. cit. Cetraria [slandica (Lceland Moss). 459 isolichenin was reprecipitated with alcohol, extracted with alcohol and ether, and reduced to an almost white powder, containing 0.4 per cent ash. This preparation dissolves with difficulty in cold water, readily in hot water, from which it does not separate on cool- ing. With iodine solution it gives a blue coloration. Hydration by dilute acid. —The following data were obtained by the methods already indicated for lichenin. I. 1.021 grams isolichenin (ash-free) yielded on hydration 1.125 grams dextrose. Assum- ing a hydration equivalent to that of starch, the yield of dextrose should have been 1.134 grams. I. (2) Ina solution of hydration products containing 1.23 per cent sugar (determined as dextrose) in a 200 mm. tube, an average of six polariscopic readings gave a rota- tion of + 1.259. Then (a)p>=+ 50.8°. (2) Ina solution containing 1.13 per cent sugar in a 200 mm. tube, an average of six polariscopic readings gave a rotation of + 1.17°. Then (a)p=+ 51.7°. The specific rotation of dextrose, (a)p= + 52.5°. Ill. The osazones of the sugar formed were prepared and recrystallized four times from alcohol. M. p. 199° C. The crystals resemble those of phenylglucosazone in appear- ance and solubility. The hydration products of the isolichenin thus correspond closely in behavior with those obtained from the lichenin of the same plant. Action of enzymes and dilute HCl. — Honig and St. Schubert? sub- jected this carbohydrate to the action of malt diastase at 60°C. for several hours. They observed a rapid disappearance of the iodine reaction and formation of dextrin-like substance precipitable by alcohol. From such observations they class isolichenin — their lichen- starch —with soluble starch. The writer has further studied the action of saliva, diastase, and pancreatic extract. Typical experi- ments are given below. I. A one per cent isolichenin solution was treated at 38° C. with saliva. The “achromic point” was reached in about one minute, no erythro-dextrin stage being detected. Digestion was continued for an hour. The solution, tested from time to time, gave a slight reduction (with Fehling’s solution) which did not increase in amount. Ny- lander’s reagent gave no test for dextrose. The solution was precipitated with alcohol and the filtrate gave no reaction for sugars after removal of the alcohol. The precipitate of dextrin-like substance gave a slight reduction.2_ A flocky blue precipi- tate was always present in the test. Towards diastase and amylolytic pancreatic extract isolichenin showed similar behavior. Il. Isolichenin was treated with varying strengths of HCl (0.2-0.5 per cent) at 38° C. for twelve hours. No sugar was obtained in any instance. HO6niG und St. SCHUBERT: J/oc. cit., pp. 694-696. 2 MuscuLus and v. MERING (Zeitschr. fiir physiol. Chemie, 1876, ii, pp. 410- 419) obtained from glycogen and starch achroodextrins which likewise slightly reduce Fehling’s solution. 460 E. W. Brown. The unusual behavior of isolichenin towards amylolytic enzymes — the formation of dextrins without sugars— recalls the formation (from glycogen) of dystropo-dextrin, an achroodextrin resisting the further action of enzymes.! The peculiar carbohydrates of Gataria islandica are doubtless merely types of those occurring in numerous other varieties of this group of plants. 1 SEEGEN: Archiv f. d. ges. Physiol., 1879, xix, p. 106; TEBB, M. C.: Journal of physiology, 1898, xxii, p. 428. ; VARIATIONS IN THE AMYLOLYTIC. POWER AND CHEMICAL COMPOSITION OF HUMAN MIXED SALIVA. By R. H. CHITTENDEN anp A. N. RICHARDS, B. A. [From the Sheffield Laboratory of Physiological Chemistry, Yale University.] INCE saliva is the product of secretory glands having their periods of comparative rest and activity, it follows quite naturally that this secretion might be expected to show variations in amylolytic power at different periods of the day: z.e., that the secretion obtained after a period of glandular activity might possess less starch-digesting power than the secretion coming from glands which have been in a state of rest— due mainly to variations in the proportion of active enzyme present. Further, the well-known sensitiveness of the amy- lolytic enzyme to changes of reaction suggests also the possibility of fluctuations in amylolytic power dependent primarily upon changes in the proportion of alkaline-reacting salts contained in the secretion. In spite of the large amount of work of a chemico-physiological nature done upon saliva, these questions have received very little attention. During the past year, however, Hofbauer? in an interest- ing communication has presented a series of results, bearing on the daily fluctuations in the amylolytic power of saliva, but his observa- tions were limited solely to determination of the starch-digesting power at different periods of the day without regard to any pos- sible relationship between the amylolytic power and the chemical composition of the secretion. His results, however, show clearly that human mixed saliva does fluctuate in amylolytic power through- out the twenty-four hours, and further that the starch-digesting power of the saliva secreted before breakfast, for example, is greater than that of the secretion collected after breakfast. Our results afford distinct confirmation of the general truth of this observation. Hof- bauer states in his paper that the only previous work bearing upon 1 A summary of some of the results contained in this paper was presented at the Meeting of the American Physiological Society in December, 1897, and published in the Proceedings of the Society, this Journal, 1898, ii, p. iii- * HOFBAUER: Archiv f. d. ges. Physiol., 1897, Ixv, p. 503. 462 Chittenden and Richards. this subject is that by Chittenden and Ely.’ The latter work, however, has no bearing whatever upon the question of possible variation in the amylolytic power of the secretion at different periods of the day. Indeed, in the paper in question it is distinctly stated that ‘‘ the saliva was collected generally an hour or two after breakfast,’ with the distinct object of avoiding possible variations in composition due to the period of collection. The sole object of that investigation was to ascertain whether there is any connection between possible variations of alkalinity and the amylolytic power of saliva. ‘The results there reported afford no indication whatever of the relative amylolytic action of the secretion for different periods of the day, since the fluids studied were invariably collected at essentially the same hour. It was ascertained, however, that the alkalinity of mixed saliva as meas- ured by titration with a standard acid, using cochineal as an indicator, was fairly constant for a given individual at a given period of the day (9-10 A.M.), while saliva from different individuals may show a con- stant difference in alkalinity, although in the majority of cases the alkalinity varied only within narrow limits. In amylolytic action, however, there were no corresponding differences; fluctuations were observed, but within too narrow limits to indicate any tangible rela- tion between the two factors. It has become the custom to assume that the alkalinity of saliva, as indicated by its reaction toward litmus paper, is due more or less to the presence of sodium carbonate. Thus, in the latest text-book of physiology the statement? is made that “ the alkalinity of saliva de- pends upon the presence of sodium carbonate. In man and in the dog the percentage of this salt varies from 0.08 to 0.19 per cent.” So far as we are aware, however, there is no justification for this statement. In the earlier work from this laboratory ? it was stated that the average alkalinity for fifty-one samples of human mixed saliva was 0.08 per cent, ‘‘expressed in the form of sodium carbonate.” Further, in all the tabulated results contained in that paper, the alkalinity, as meas- ured by titration with standard acid in the presence of cochineal as an indicator, was carefully expressed as “ equivalent in Na,CQOs,” this being done to avoid any positive statement as to the exact cause of the alkalinity. Further, in the oft-quoted work of Werther the al- CHITTENDEN and Ey: American chemical journal, 1883, iv, p. 329. Text-book of physiology, edited by E. A. Schafer, 1898, vol. i, p. 504. CHITTENDEN and ELy: American chemical journal, 1883, iv, p. 333- WERTHER: Archiv f. d. ges. Physiol., 1886, xxxviii, p. 293. Variations in the Power and Composition of Saliva. 463 kalinity of the saliva of the dog was determined by titration with decinormal sulphuric acid with litmus as an indicator: a method which obviously would throw no light upon the cause of the alkalinity. Moreover, in at least some of the tables containing his results the percentage of alkalinity is expressed as ‘alkalinity calculated as NaeCOs.” Examination of a large number of samples of human mixed saliva obtained from different individuals at different periods of the day convinces us that, under normal conditions at least, human saliva never contains the least trace of sodium carbonate. Toward litmus, lacmoid, etc., human saliva constantly reacts alkaline, but with phe- nolphthalein it invariably shows an acid reaction, and a certain amount of a decinormal alkali solution is required to bring out an alkaline re- action with this indicator. Further, phenolphthalein is an extremely sensitive reagent for sodium carbonate; a solution containing 0.001 per cent of sodium carbonate will give a pink color when brought in contact with a solution of phenolphthalein. With human saliva, however, we have never obtained any color reaction with phenol- phthalein whatever; the solution invariably remains colorless, thus proving that the alkalinity indicated by litmus must be due to some acid salt or salts, like the hydrogen alkali phosphates, with possibly some alkali bicarbonate. The submaxillary saliva of the dog, how- ever, obtained by stimulation of the chorda tympani is usually, at least, faintly alkaline to phenolphthalein;! consequently this fluid may owe its alkalinity in part to sodium carbonate. These facts, which admit of easy confirmation, are worthy of some consideration, since they have an important bearing upon the normal conditions govern- ing enzyme action. I. RELATIVE ALKALINITY AND ACIDITY OF HUMAN SALIVA BEFORE AND AFTER EATING. In this series of experiments the saliva was collected from one individual, stimulation of the secretion being effected by chewing a small piece of rubber. About 15 c.c. of fluid were collected each time. The portion collected before breakfast was obtained at 7.30 A. M., half an hour before eating, while the portion collected after eating was obtained fifteen minutes after the close of the meal. The alkalinity 1 CHITTENDEN ; Science, n. s., 1897, v, p. go2. Also CHITTENDEN, MENDEL, and JACKSON: This Journal, 1898, i, p. 174. 464 Chittenden and Richards. was determined by titrating the saliva (5 c.c.) with a decinormal solu- tion of sulphuric acid, using lacmoid as an indicator, while the acidity was determined by the use of a decinormal solution of sodium hydroxide, the indicator being phenolphthalein. The alkalinity was calculated in terms of sodium carbonate, and is also expressed as milligrams of H2SO, (absolute) required to neutralize 1 gram of saliva. The degree of acidity is expressed as milligrams of NaOH (absolute) required to neutralize 1 gram of saliva. Following are the results obtained : — ALKALINITY. ACIDITY. Time. Expressed | Milligrams H.SO,| Milligrams NaOH as Na,COs. to neutralize to neutralize Per cent. I gram saliva. I gram saliva. Before Breakfast After Breakfast . 0.163 0.127 0.78 0.61 Before Breakfast After Breakfast . 0.193 0.130 0.93 0.64 3efore Breakfast After Breakfast . 0.142 0.122 0.69 0.59 Before Breakfast After Breakfast . 0.132 0.132 0.64 0.64 Before Breakfast After Breakfast . 0.173 0.127 0.83 0.61 Before Breakfast After Breakfast . 0.148 0.132 0.71 0.64 Before Breakfast At > per 0.168 0.81 After Breakfast. <<... OAT 0.61 Before Breakfast . .. . 0.122 0.59 0.11 After Breakfast . 0.122 0.59 Before Breakfast ee) 0.148 0.71 After Breakfast. : <0.0% 0.137 0.66 Betore Winner 2) ee 0.132 0.64 0.08 Afters Dinner, “. 2) ae. See 0.142 0.69 0.02 Before Dinner. ... . 0.168 0.81 0.08 After Dinner 700. eee 0.158 0.76 0.08 Béetore Dinners). | eee 0.153 0.73 0.06 AYvter Dinner ac soe eee | 0.158 0.76 0.02 ati Variations in the Power and Composition of Saliva. 465 A glance at these results shows that the alkalinity of saliva, as in- dicated by lacmoid, is noticeably greater in most cases in the fluid secreted after a night’s rest, before breakfast, than in the secretion obtained after the glandular activity induced by the morning meal. Before and after dinner, however (1 P. M.), this distinction is less conspicuous. It is also interesting to note that the average alkalin- ity, expressed in terms of sodium carbonate, is somewhat higher -with lacmoid as an indicator than with litmus or cochineal; a fact which would be expected in view of the presence of the hydrogen alkali phosphates contained in the fluid. It is likewise to be seen that the average acidity as indicated by phenolphthalein, though less con- spicuous, is also inclined to diminish after eating. II. RELATIVE ALKALINITY AND AMYLOLYTIC POWER OF HUMAN SALIVA BEFORE AND AFTER EATING. In this series of experiments the main object was to ascertain whether there are noticeable variations in the amylolytic power of saliva before and after eating and whether such variations, if existent, run parallel with variations in the alkalinity. As in the previous experiments, the saliva was collected by chewing a small piece of rubber. Amylolytic power was determined as follows; 5 c.c. of the filtered saliva were diluted with distilled water to 50 c.c.; 10c.c. of the diluted fluid were then added to 1 gram of pure arrowroot starch made into a paste with goc.c. of water, and the mixture kept at 38° C. for half an hour. Amylolysis was then stopped by boiling the fluid, after which the solution, when cool, was made up to 150 c.c. with water and the reducing sugar determined by the Allihn Method, using 25 c.c. of the sugar-containing solution. The results are expressed as milli- grams of maltose formed from I gram of starch by 1 c.c. of saliva. The data obtained are given on the next page. From these results it would appear that saliva secreted after a period of glandular inactivity, as before breakfast, is ordinarily possessed of greater amylolytic power than the secretion obtained after eating; results which accord closely with Hofbauer’s observations. Before and after dinner (1 P. M.), however, the difference in amylolytic power is less pronounced; a fact which might be expected in view of the short period for recuperation between the breakfast and dinner and because of the more or less constant stimulation of the salivary glands during the waking hours. Further, we see in these results a suggestion of some degree of relationship between the percentage of 466 Chittenden and Richards. ALKALINITY. AMYLOLYTIC POWER. Collector. Time. Expressed | Milligrams H,SO, Milligrams Maltose as Na,COs, to neutralize formed by I cc. Per cent. I gram saliva. saliva. saat 3efore Breakfast 0.173 0.83 523.4 After Breakfast 0.127 0.61 511.8 Before Breakfast 0.168 0.81 630.6 After Breakfast 0.127 0.61 583 8 Before Breakfast 0.148 0.71 562.2 After Breakfast 0.132 0.64 485.4 Before Breakfast 0.122 0.59 620.4 After Breakfast 0.122 0.59 534.6 Before Breakfast 0.148 0.71 549.0 After Breakfast 0.137 0.66 510.5 Before Breakfast Sieses fees 209.4 After Breakfast oc Slee 224.4 Before Breakfast 585.0 After Breakfast 468.6 Before Breakfast eas as 621.0 After Breakfast Spee Le eae 537.6 Before Dinner | 549.6 After Dinner l 536.4 Before Dinner 582.0 After Dinner 564.6 Before Dinner mt com 570.0 After Dinner Sty Ste 562.8 Before Dinner ; 599.8 After Dinner L 606.6 Before Dinner 4 as Cave 594.0 After Dinner sentee agente 547.2 alkaline salts contained in the saliva and its amylolytic power. Be- fore breakfast, for example, the content of alkaline salts and the starch- digesting power of the secretion are greater than in the fluid secreted after glandular activity. At first glance, then, it might seem that the variations in amylolytic action noticed above are due to changes in the proportion of alkaline salts. The objection to this view, however, is that it associates the higher degree of amylolytic power with the Variations in the Power and Composition of Saliva. 467 higher percentage of alkalinity, whereas numerous trustworthy ex- periments tend to show that saliva manifests its highest degree of digestive power in a perfectly neutral fluid! Consequently, if the above variations in amylolytic action are primarily due to changes in the proportion of alkaline-reacting salts, then the higher degree of amylolysis should be connected with the lower degree of alkalinity. As the reverse is true, the more plausible and natural explanation of the results is that the higher degree of amylolysis is connected pri- marily with the presence of larger amounts of the amylolytic enzyme, and as this is presumably connected with the outpouring of a more concentrated secretion a corresponding increase in alkaline-reacting salts might naturally be expected. Further, in harmony with the latter view it is to be noticed that the secretions obtained before and after breakfast fail to show any close parallelism between the varia- tions in amylolytic power and variations in alkalinity. Thus, the most marked differences in digestive power are frequently seen with salivas which show only a slight difference in alkalinity, and on the other hand marked differences in alkalinity may be associated with minor differences in amylolytic power. III. ALKALINITY, AMYLOLYTIC POWER, AND COMPOSITION OF HUMAN SALIVA BEFORE AND AFTER EATING. In view of the preceding results, the following set of experiments was tried in which, in addition to alkalinity and amylolytic power, the proportion of dry solids and inorganic salts of the saliva was likewise determined. The dry solids were determined by simply drying a weighed amount of the filtered saliva— usually five grams—on a water-bath and heating at 105° C. in an air-bath until of constant weight. The inorganic salts were then determined by careful ignition of the residue. In some of the following experiments relative amylolytic action was determined by Robert’s? method, the method being based on the different lengths of time which solutions of different amylolytic power require to digest a certain amount of starch paste to the achromic point. The results obtained by this method are expressed in minutes; 2. ¢., the number of minutes which elapse from the time the diluted saliva is added to the starch paste until the appearance of the achromic point. 1 LANGLEY and Eves: Journal of physiology, 1883, iv, p. 18. CHITTENDEN and SMITH: Studies in physiol. chemistry, Yale University, 1885, i, p. 8. 2 See Gamgee’s Physiological chemistry of the animal body, vol. 2, p. 56. 468 Chittenden and Richards. Following are the results obtained : — LKALINITY. A : AMYLOLYTIC POWER. Total | Organic} Inorganic solids. | matter. salts. Mg.H,SO,4 ' c.c. |As NayCOg] to neutral- | Mg. maltose Wea as eee Per cent. | ize 1 gram | formed by Breakfast. saliva. I c.c. saliva. Collector. Per cent.|Per cent.| Per cent. Before 0.158 0.76 649.2 1.02 0.77 0.24 After B 0.122 0.59 601.2 0.51 0.33 0.17 Issiois || 2 0.163 0.78 651.0 0.86 0.58 0.28 After 0.112 0.55 615.6 0.51 0.30 0.21 3efore 0.122 0.59 467.4 0.44 0.23 0.22 After : 0.102 0.49 491.4 0.40 0.19 0.21 Before 0.081 0.39 43.01 037 0.21 0.16 After 0.096 0.46 50.0 0.39 0.24 0.15 Before | ; 0.153 0.73 12.0 0.45 0 30 015 After 0.158 0.76 15.0 0.53 0.34 0.19 Before 0.137 0.66 13.0 0.32 0.15 0.17 After 0.132 0.64 8.0 0.37 0.21 0.16 1 Tn this and the two following experiments amylolytic power was determined by Robert’s method. In these results we have a suggestion of the same general tendency toward decrease of amylolytic power and lowered content of alkaline salts in the saliva secreted after the morning meal, while as accom- panying results we see corresponding fluctuations (although not in all cases) in the proportion of total solids, organic matter, and inorganic salts, thus bearing out the view that the variations in amylolytic power are connected mainly with changes in the general concentration of the secretion. At the same time it is to be observed that the above differences in composition and amylolytic power are much more marked with the individual R than with A, B, and D. In fact, with the latter three individuals there is very little difference in composi- tion in the saliva before and after the morning meal, and further in the third experiment with R the amylolytic power after the meal is greater than that of the saliva secreted before eating. These results have led to another series of experiments having in view especially the determination of the fluctuations in the character of: the saliva throughout the day. —s EE ee Vartations in the Power and Composition of Saliva. 469 IV. VARIATIONS IN THE COMPOSITION AND AMYLOLYTIC POWER OF HUMAN SALIVA THROUGHOUT THE DAY. In the first series of experiments under this head the saliva studied was collected by chewing a piece of rubber. The mid-day dinner was omitted; breakfast, however, was taken at 7.50 A. M. and supper at 6.40 P.M. Samples of saliva were analyzed every hour or two throughout the day. Following are the results obtained : — Alkalinity | Amylolytic Total! \Oxeanic Inor- calculatedas| power. Salis matter. | S27c Na,COs. Milligrams i ‘|. salts. Per cent. maltose. Per cent.| Per cent.) Per cent. 0.112 574. BS 0.29 0.30 3.15, Breakfast 25 10.00 to 10.15 11.00 to 11.18 P.M. 12.00 to 12.13 12.45 to 12.55 2.00 to 2.15 3.00 to 3.12 4.00 to 4.13 5.00 to 5.14 7.00 to 7.25, Supper 8.30 to 8.45 29 10.40 to 10.55 | 28 Here, as in the preceding experiments, there is noticeable the same diminution of amylolytic power, alkalinity, and content of solid matter, etc., in the mixed saliva secreted directly after the morning meal. Of special significance, however, is the marked variation in the values throughout the day, thereby suggesting the existence of a normal curve of secretion. Thus, after the morning meal the saliva shows the effect of the stimulation by its lower content of solids, etc. Soon after, however, there is an upward tendency; the curve rises, and amylolytic power is increased as well as the alkalinity, together with the total solids and organic matter. The inorganic salts, on the 470 Chittenden and Richards. other hand, still remain low. Towards noon time, amylolytic power sinks very greatly, and there is a corresponding drop in the propor- tion of organic solids, although the alkalinity and inorganic salts still remain fairly high. After this, amylolytic power gradually rises, reaching the maximum again at 5 P. M. with a corresponding rise in alkalinity, total solids, etc. Supper at 7 P. M. apparently causes a slight fall in amylolytic power, together with a fall in the solid matter secreted. At 10.40 P. M. amylolytic power shows a still greater fall, although alkalinity and solid matter are increased in amount. How far are the preceding variations in the secreted saliva due to the combined influence of taking food and the mechanical stimulation incidental to mastication of the rubber, and how far to a natural variation in the composition of the secretion? This question we have endeavored to answer by noting the variations in the saliva on a day when food was abstained from, and by collecting the saliva without movement of the jaws. This was accomplished by simply resting the head on the hands, with the mouth downwards, and allowing the saliva to drip into a beaker without any unnecessary movement.’ In this way 15-20 c.c. of saliva were collected in half an hour. Following are the results obtained : — Alkalinity Volume | calculated as Time. saliva. Na COs. Crce | Amylolytic | : In- power. Total | Oa organic Milligrams solids: mat) ean Per cent. maltose. Percent. Per cent.| Per cent. Midnight | 11.45 to 12.15 : 0.081 490.4 0.38 | 0.23 A.M. | 6.40 to 7.30 0.088 572.4 0.631 | 0.47 | 9.30 to 10.00 : 0.092 558.6 11.00 to 11.30 0.071 381.0 P.M. 12.25 to 12.50 0.102 441.0 Ze tom Zits 0.092 347.4 4.00 to 4.2 0.091 416.4 5.115 to. 5.5 0.102 423.0 7.00 to 7.25, Supper 8.30 to 9.00} 20 oo2 | 4033 0.43 028 0.15 1 This result is of somewhat questionable accuracy, having been obtained with a very small amount of saliva. 1 See HOFBAUER: Joc. Ccit., p. 503. CO Variations in the Power and Composition of Saliva. 471 A study of these results shows clearly that when the stimulating influences of food and mastication are withdrawn, conspicuous altera- tions in the composition and physiological action of the saliva are still found, as though there might be a normal curve independent of the fluctuations induced by stimuli. Thus at 11.30 A. M. there is seen the same fall in amylolytic power that was so conspicuous in the preceding experiment. Further, the saliva secreted at 2.15 P. M. shows a diminution in amylolytic power, as noticeable as the diminu- tion frequently observed after a hearty meal. It is thus quite evident that in the absence of food and other stimulation hourly changes in the amylolytic power of mixed saliva may occur just as marked as those noticed in the saliva secreted before and after breakfast. Vari- ations in alkalinity, total solids, etc. are not so prominent. It is to be noticed, however, from the last series of experiments, that in the absence of breakfast there is no great variation in the amylolytic power of the saliva secreted between 6.40 and 11.00 A. M.; conse- quently we may accept the conclusion, justified by the results of most of our experiments, that the taking of food, as at breakfast, tends to lower the starch-digesting power of the saliva secreted some time thereafter. This being so it seems probable that other forms of stimulation may likewise give rise to a change in the composition and physiological action of mixed saliva. V. INFLUENCE OF VARIOUS STIMULI ON THE COMPOSITION AND AMYLOLYTIC POWER OF HUMAN SALIVA. In this series of experiments the attempt was made to ascertain how far the character of the stimulus modifies the properties of mixed saliva. The special stimuli employed were ether, alcohol, whiskey, and gin. The first two were taken into the mouth in the form of vapor, and the saliva allowed to trickle from the mouth with- out motion of the jaws, the fluid so obtained being compared with saliva resulting from the mechanical stimulation produced by chew- ing a piece of rubber. With whiskey and gin, the mouth was well rinsed with the fluid and the saliva collected by allowing it ‘to flow from.the corner of the mouth. The control experiments with water were made in the same way; 27.¢., the mouth was rinsed with water and the saliva allowed to trickle forth. Finally, for the sake of comparison and to ascertain how far two samples of saliva obtained at such close intervals, under similar forms of stimulation, differ from 472 Chittenden and Richards. each other, four control experiments were tried with water and rub- ber alone. Following are the results obtained : — ere ant Inor- | Alkalinity Amylolytic Total | Organic | ganic Volume | calculated power. ‘ime. stin s. iva | = sae ids. _ : Date. Time Stimulus.| saliva las NasCO3. Milligrams solids.) matter. salts maltose. Per Per Per cent. cent. cent. Per cent. A.M. Dec. 3 11.05-11.30, Rubber : 582.6 0.63 0.31 | 0.32 11.30-11.50 Ether ; 624.6 0.76 0.54 | 0.22 9 9.50-10.10, Rubber aeaiene 562.8 0.54 030 | 0.24 10.10-10.30 Ether ie Ore 498.6 0.54 0.29, 70-31 13 11.40-12.00, Rubber | 472.2 0.41 | 0.21 0.20 p.m. 12.00-12.35 Alcohol 510.6 0.43 0.19 0.24 A.M. 10.00-10.30, Water : 0.32 0.19 0.13 10.30-11.00! Whiskey 5 : 0.42 0.29 0.13 10.15-10.40, Water | : 0.34 | 0.20. | 0.14 10.45-11.20) Gin : 0.53 0.36 0.17 10 20-10.38) Ether 6. 0.32 0.16 0.16 10.45-10.55) Rubber 577. 0.52 0.24 0.28 20 11.15-11.48| Water | | 606. 0.68 | 055 | 0.13 p.m. 12.15-12.45) Water 0 | 038 | O20 sam Jan. 11 3.03- 3.35; Water : 0.30 0.16 0.14 4 05- 4.40) Water 532. 0.35 0.21 0.14 A.M. | «43 11.25-11.40, Rubber 571. 0.49 0.26 0.23 p.m. 12.10-12.26 Rubber ; 0.47 0.24 0.23 A.M. “ 14 10.38-10.58 Rubber | Sie | 0.50 0.27 0.23 11.30-11.45 Rubber | : | 0.51 0.26 0 25 A glance through these results shows at once certain marked dif- ferences in the character of the saliva obtained under the different conditions specified. Thus, saliva which flows from the mouth after the latter has been rinsed once with water invariably shows a lower degree of alkalinity, and generally contains a smaller percentage of solid matter, than the secretion obtained by the other methods. In amylolytic power, however, there is great variation; some samples showing a relatively strong amylolytic action, while others with essentially the same degree of alkalinity are much weaker in their Variations in the Power and Composition of Saliva. 473 starch-digesting power. Simple mastication of rubber has a marked influence in raising the content of alkaline salts in the saliva, as well as the total inorganic constituents, and there is a tendency toward increase in amylolytic power although the latter is not constant. As to the influence of alcohol, ether, gin, and whiskey, there is, we think, no question that these agents taken into the mouth change the character of the secretion, increasing its alkalinity, amylolytic power, and content of solid matter. This is certainly true if the secretion so obtained is compared with the saliva flowing from the mouth without stimulation of any kind. Saliva, however, secreted under the stimulation produced by chewing rubber, is, as we have seen, comparatively concentrated, and the difference between the secretion resulting from that method and the fluid coming from ether, alcohol, and other like forms of excitation, without mechanical stimulation, is not so decisive in the above experiments as to make the matter quite clear, especially in view of the fact that two portions of saliva obtained one after the other, by the same method of stimula- tion, are liable to show marked differences in composition and reac- tion. Particularly noteworthy is the fact that of two portions of saliva collected one after the other by mechanical stimulation (chew- ing rubber) or by simply allowing the saliva to flow from the mouth after once rinsing the latter with water, the latter portion of saliva is, as a rule, more concentrated and possessed of higher amylolytic power than the portion first secreted. It is thus obvious that great care must be exercised in drawing deductions from the . composition and amylolytic action of mixed saliva when the latter is so prone to vary under what seem to be essentially the same forms of stimulation. It is furthermore equally obvious that the possible causes to which the above variations may be attributed are many, since there are involved three distinct sets of glands in addition to the buccal glands of the mouth cavity. Hence, in- crease or decrease in amylolytic power, as well as in the general concentration of the secretion, may involve simply an alteration in the relative activity of the individual glands and not be connected primarily with any specific stimulation of metabolic or secretory activity. However this may be, it is quite clear that the natural variations in the character of the mixed saliva, indicated by the results of the last four experiments of the above series, render it necessary to use great 474 . Chittenden and Richards. caution in arranging the conditions under which the experiments are tried. We have therefore repeated the above experiments, choosing for the collection of the saliva a time of day when we have found the mixed saliva most constant in composition; viz., between 9.30 and 10.30 A.M. To be sure, there are variations in the compo- sition and starch-digesting power of successive portions of saliva collected by the same method at this period, but they are relatively small; quite small, indeed, as compared with the variations liable to occur at other periods of the day. The truth of this statement is illustrated by the two following experiments, in which the saliva was collected without stimulation, simply allowing it to tow from the mouth. Alkalinity | Amylolytic | Total | Organic) In- Volume Power. Solids. | constit- | organic } saliva Nag . | Milligrams uents. salts. Gc; maltose. Per cent. Percent.) Percent.) Per cent: A.M. 9.32 to 10.06 | 21.0 0.0816 Soe 0.50 0.31 0.19 10.15 to 10.42 | 22.0 0.0918 549.0 0.46 0 29 0.17 0.0918 573.6 0.49 0.31 0.18 0.1122 613.8 0.68 0.51 0.17 Thus, the two portions collected between 9.32 and 10.42 A.M. are essentially alike, while the two fractions secreted between 5.00 and 5-50 P.M., all without stimulation, are more dissimilar. Adopting the morning hour as the better time for collection, experiments were tried with alcohol, ether, chloroform, whiskey, and gin, comparing in each case the saliva obtained under their influence with the secretion coming without stimulation of any kind. The exact method pursued in the case of the control, z. ¢., with water, was to rinse the mouth once with distilled water after which the saliva was simply allowed to drop from the mouth into a beaker. With ether and chloroform the mouth was filled once with the vapor and the saliva then allowed to flow spontaneously into a receptacle without any motion of the jaws. With the alcohol, gin, and whiskey 10 c.c. of the fluid were taken into the mouth, held a moment, and then ejected, after which the saliva was collected as in the other cases. Lastly, an experiment was tried (Feb. 15) by chewing rubber as a stimulant, and comparing the NE Stimulus. Vol. saliva ic: A.M. 7 | 10:05-10:32 10.37-10.56 9.37-10.05 10.11-10.32 9.53-10.18 10.27-10.47 9.40-10.07 10.14-10.36 9.52-10.16 10.21-10.27 9.33-10.03 10.10-10.34 Water 407, Alcohol Water |Ether Water ‘Chloroform /Water Whiskey 'Water ‘Rubber Water | (Gin Alkalinity | as NagCO;| Per cent. Amylolytic Power. Milligrams maltose. Variations in the Power and Composition of Sakva. saliva so obtained with a control secreted without stimulation. lowing are the results obtained: Total solids. Per | cent. | 475 Fol- Organic constit- uents. Per cent. 0.0714 0.1122 0.0612 0.1122 0.0816 0.0714 0.0714 0.1020 0.0816 0.1530 0.0714 0.1020 480.6 514.2 566.4 558.6 604.2 0.42 | 0.43 0.42 | 0.54 0.51 0.69 0.39 0.50 0.38 0.58 0.49 0.57 0.22 0.26 0.25 0.29 0.33 0.48 0.25 0.31 0.21 0.26 0.33 0.39 0.18 0.18 Water Water 0.30 0.31 Sido Bhs) 10.01-10.24 0.0714 0.0714 From these results it would seem quite clear that the several agents employed, with the exception of chloroform, give rise to a marked increase in the content of alkaline-reacting salts in mixed saliva. Mechanical stimulation, as by chewing rubber, however, is even more effective than the chemical stimuli employed, although it must not be overlooked that in the above experiments the action of alcohol, ether, whiskey, etc., is necessarily of short duration. Further, there is evidence in most of the results of an increase in amylolytic power, as well as in the content of solid matter under the influence of the stimuli. It is thus safe to assert that alcohol and alcoholic fluids not only stimulate the flow of saliva, but that they also tend to increase the concentration and amylolytic power of human mixed saliva, — results which are in close accord with the action of these fluids upon the secretion of the sub-maxillary saliva of the dog.t. Further, simple mechanical stimulation, as mastication, may also 1 See CHITTENDEN, MENDEL, and JACKSON: This journal, 1898, i, p. 167. 476 Chittenden and Richards. increase the amylolytic power of mixed saliva. Lastly, it should be mentioned that the saliva resulting from the above forms of stimu- lation, excepting mechanical stimulation, is much more viscid than the fluid secreted spontaneously, evidently from a higher content of mucin. SUMMARY. Human mixed saliva contains normally no sodium carbonate what- ever; the alkalinity indicated by litmus, lacmoid, etc., is due to hydrogen alkali phosphates, with possibly some alkali bicarbonate. Mixed saliva invariably reacts acid to phenolphthalein. The alkalinity of mixed saliva, as indicated by lacmoid, is greater before breakfast than after the morning meal; a conclusion which stands in direct opposition to the statement frequently made that “the alkalinity (of mixed saliva) is least when fasting, as in the morning before breakfast, and reaches its maximum with the height of secretion during or immediately after eating.” } Saliva secreted after a period of glandular inactivity, as before breakfast, manifests greater amylolytic power than the secretion ob- tained after eating, as observed by Hofbauer. Corresponding with this increase in amylolytic power occurs an increase in the proportion of alkaline-reacting salts, but the increased amylolysis is due primarily to an increase in the amount of active enzyme contained in the saliva, Mixed saliva, whether collected by mechanical stimulation or col- lected without effort, shows a natural tendency to vary both in composition and in amylolytic power throughout the twenty-four hours, and apparently independent of the taking of food. Between ‘7.00 and I1.00 A.M., however, in the absence of food the secretion is remarkably constant. Mechanical stimulation, as chewing a tasteless substance, and alcohol, ether, gin, whiskey, etc., taken into the mouth, all lead to the outpouring of a secretion richer in alkaline-reacting salts and in amylolytic power than the secretion coming without stimulation. Mixed saliva resulting from stimulation with ether, alcohol, etc., con- tains a much larger proportion of mucin than the secretion coming without stimulation, being noticeably thick and viscid. This quality is not apparent in the saliva resulting from mechanical stimulation. 1 Text-book of physiology, edited by E. A. SCHAFER, 1898, i, p. 344. ‘THE VENOMOTOR NERVES OF THE. HIND LIMB. By F. W. BANCROFT. [From the Laboratory of Physiology in the Harvard Medical School.] LTHOUGH several investigations of the venomotor nerves of other regions, particularly the portal vein, have been pub- lished, the literature of the venomotor nerves of the hind limb is lim- ited to the single paper of Thompson.!' On stimulating the sciatic nerve or the spinal cord of four dogs, Thompson observed that the superficial veins of the hind limb were constricted. The constriction ‘did not extend throughout the vein exposed, but was limited to short sections, between which the diameter remained unchanged. ‘The same result was obtained in four of the five rabbits used. In my experiments rabbits and cats were employed. The cat is much more satisfactory than the rabbit. The sciatic nervé was severed under ether and the peripheral end stimulated with a weak in- terrupted induction current while the superficial veins on the outside of the hind limbs were examined. Contractions of the skeletal muscles were prevented by curare. At first the aorta was ligated before stimulation — to exclude the possibility of a decrease in the -diameter of the observed vein in consequence of constriction of the arteries of the limb.2, The veins were kept covered by the skin when not actually under examination. Closing the aorta did not cause any marked decrease in the diameter of the vein, but merely a flatten- ing and general flabbiness throughout its extent. The stimulation on the other hand caused a marked constriction, which was quite irregu- larly localized. Usually the constricted segments were short, but occasionally a piece ten or twenty millimetres in length would con- tract uniformly. After a brief exposure to the air the contractions were more variable than at first, parts that had formerly contracted now often failing. More uniform results were gained when the vein was kept from drying and cooling by irrigation with warm normal saline solution. A flap of skin was raised to form a small reservoir for the saline 1 THompson: Archiv fiir Physiologie, 1893, p. 102. * The closing of the aorta was omitted in the later experiments on cats, as it ~was found to make no essential difference in the result. 478 fr. W. Bancrofe. solution, in which the vein lay exposed. The part contracted by stimulation was now much longer. Thus in the rabbit the vein oc- casionally contracted uniformly over a length of seventy millimetres, and contractions of thirty to forty millimetres were the rule. In the cat, the length usually contracting was even greater. But even with the warm saline solution the phenomena were not constant, some parts tiring rapidly and failing to constrict after several stimulations, while others continued with hardly diminished vigor. The position of the latter was usually the same in different individuals. A part of the vein about twenty millimetres in length —just before the vein leaves the surface and passes between the underlying muscles to enter the pelvic cavity — never contracted in any of the rabbits. This part is probably supplied with constrictor fibres through some nerve other than the sciatic, or else the constrictor fibres leave the sciatic on the central side of the point stimulated. The character of the contraction admits no doubt that it is caused by the vasomotor nervous mechanism. Usually the change in size is considerable, and there is no difficulty in determining whether the vein is constricted or not. Simple inspection of the vein, however, cannot determine with certainty the smaller changes of calibre or the exact time of their beginning. At first an interrupted current that is just distinct on the tongue will usually decrease the diameter of the vein one-third, and sometimes will obliterate the lumen so that no blood can be seen; but the venomotor apparatus is soon tired and then a stronger stimulus is necessary to produce a decided contrac- tion. The latent period is quite long, varying from about ten to twenty or even to thirty seconds. Stronger and more frequent induction shocks decrease the latent period and increase the con- striction. Having determined the presence of venomotor fibres in the sciatic nerve, the next step was to trace them from the spinal cord. For this purpose only cats were used, as they endure the operation much better than rabbits. The animals were anesthetized with ether dur- ing the preparation of the nerves and the vein. The stimulation, which was limited to the peripheral segment of the nerves, was done under curare. The characteristic changes in the vein occurred whether the animal was completely or incompletely under the in- fluence of the drug, but in order to make sure that no activity of the voluntary muscles was responsible for the constriction no results were considered as final unless the curarization was complete. The Venomotor Nerves of the Hind Limo. 479 The part of the hind extremities the veins of which have been par- ticularly examined in determining the course of the venomotor fibres is the lateral surface of the crus, and the pes. All the veins in the former and most of those in the latter region have been seen to con- tract at one time or another. Since the diagonal vein in the lower part of the crus was the most reliable, the majority of the ob- ‘servations were made in its immediate neighborhood so as to have ‘smaller and fewer cuts in the skin. The veins of the thigh and the medial surface of the crus are apparently less sensitive, for I have as yet not seen them contract; but the number of observations on these veins was small. To determine the origin of the venomotor fibres from the spinal cord the roots were cut and the peripheral segments stimulated within the vertebral canal. In order to facilitate the operation both the anterior and the posterior roots were tied together outside the dura mater. The cord was removed in the region stimulated so that the possibility of leakage of the current to the cord was excluded. Neg- ative results were never accepted as evidence of the absence of ven- omotor fibres in the nerve stimulated unless the stimulation of some other spinal nerve, or of the sciatic, gave constriction of the vein and thus proved that the vasomotor apparatus was in working order. The venomotor fibres to the hind limb, as may be seen from Table I, may be demonstrated in the I to IV lumbar nerves, but in no case were they found in more than three of these in any one animal, and in about half the cases they were found in only two of the nerves. The greatest constriction in every animal but one followed stimula- tion of the III lumbar nerve. In this exceptional case, the IV lum- bar nerve was the most efficient. As this was the only instance in which the lumbo-sacral plexus was of Langley’s posterior type! it may be that in this type the IV nerve is commonly the most effective. The nerves that produced the most vigorous contraction also influ- enced a greater length of the vein. There was no definite localiza- tion of the area supplied by one nerve, such as was observed later when the gray rami communicantes were stimulated. From the spinal cord the venomotor fibres enter the sympathetic system. It is @ priori probable that their course is through the white rami of the spinal nerves, the stimulation of which produces contraction. The highest part of the sympathetic that has given any contraction of the vein is immediately below the III lumbar ganglion. 1 LANGLEY: Journal of physiology, 1894, xvii, p. 296. 480 fF. W. Bancroft. TABLE I. VENOMOTOR FIBRES IN THE SPINAL NERVES. ——— Lumbar. Sacral. Character of | ; i j = nol i Plexus. Number of Experiment. iv Median. Ant. O denotes that no venomotor fibres run in the nerve designated, c that stimula- tion of the nerve gives a contraction of the vein. The exponents 1, 2, 3, indicate the strength of the contraction, 1 standing for the strongest. Langley’s (Yournal of Physiology, 1894, xvii, p. 296) classification of the different types of the lumbo-sacral plexus is followed. The limit below which no venomotor fibres enter the sympathetic cannot be determined with certainty because it is masked by the fibres descending the sympathetic trunk from the upper white rami. Thus the constriction obtained by stimulation near what should be the lower limit cannot be used as evidence, for it may be the result of the stimulation of these descending fibres which have entered the sympathetic higher up. From the III to the VI lumbar ganglion the venomotor fibres are found in the main trunk of the sympathetic; they have not yet begun to leave the sympathetic by the gray rami. The evidence for this. The Venomotor Nerves of the Hind Limo. 481 consists in the fact that the stimulation of any part of the main trunk of the sympathetic between the III and the VI lumbar ganglia was always followed by contraction, when the cat was in good condition, and section of the main trunk below the point of stimulation always prevented subsequent contraction. This evidence is conclusive, but it may be added that stimulation of the inferior mesenteric ganglia or any of the nerves connected with it invariably gave negative results. Let us now inquire by what rami the venomotor fibres leave the sympathetic. The results of stimulating the gray rami communi- cantes of the spinal nerves forming the lumbo-sacral plexus are brought together in Table II. The rami were not stimulated directly, but the main trunk of the sympathetic was cut above and below the ganglion the ramus of which it was desired to investigate, and stimu- lated above the ganglion. In the case of the sacral rami, however, it was found inexpedient to cut the main trunk below the ganglia, so that the contractions recorded stand not only for these rami but also for any lower ones that may contain venomotor fibres. But on ac- count of the general absence of these fibres in the II sacral and their occasional absence in the I sacral ramus there is no likelihood of their occurrence in any nerves below the II sacral. In stimulating the II sacral the general method was also deviated from in another re- spect. Instead of cutting and stimulating the sympathetic below the I sacral ganglion, which would have been difficult, the nerve was first stimulated above the I sacral ganglion and then its ramus severed, or easier still the whole spinal nerve severed, and stimulation repeated at the same place. It will be seen from Table II that the venomotor fibres reach the sciatic by the rami to the VI and VII lumbar and the I and II sacral nerves. In the same animal, two, or more frequently three, rami contain these fibres, but in no case have they been found in all four rami. In every case the VII lumbar ramus contained venomotor fibres, while the VI lumbar and I sacral contained them in about eighty-five per cent of the cases. In one instance only was the II sacral found to give a contraction, but here, although the leneth of vein influenced was but one or two millimetres, the constriction was very distinct. The most noticeable feature of the contractions obtained by stimu- lating the gray rami is their local nature. While the constriction upon stimulating the sciatic or sympathetic is several centimetres in 482 F. W. Bancroft. TABLE I. Rami to Lumbar and Sacral Number Spinal Nerves. Character of of rin , : : ey lige Ha Plexus. Experiment e ei A s XXII XXXII > OO NU XXXIV XL XLI XLITI ALIN 3 DGIVisege te are ? ! Ant. SRUIEAWAl , Z Ant. 14 thoracic vert. MEV left oe ae oe el Osteen XLVII right XLVIII left XLVIII right DXCES TENS eae BY e Post. a 1b Che 1 os c? Post. b O denotes that the ramus was stimulated and no contraction obtained, although the stimulation of other nerves produced contraction. C means that the contraction was obtained by stimulating the ramus indicated. Exponents a, p, mean that the an- terior or posterior veins only contracted; where no exponent is given, the condition of the cat was such that the localization observed was probably not significant. length, the constriction caused by the stimulation of some of the rami is but a few millimetres long, and the region affected by one ramus is frequently different from that affected by another. The VII lumbar ramus controls a greater portion of the veins examined than any of the others, though occasionally the I sacral may equal or even exceed it in importance. The region controlled by the VI lumbar ramus is almost invariably quite small, and is confined to the anterior part of the leg. The transition from the contracting to the The Venomotor Nerves of the Hind Limb. 483 inert region is often most abrupt, so that there is not the least diffi- culty in tracing the distribution of the fibres; but it may also be so gradual that it cannot be definitely located. When the contracting regions are well marked off from the inert ones it can be seen that sometimes the regions controlled by the VI and VII rami overlap, and that sometimes one stops almost exactly where the other begins.! But probably more frequent than either of these two arrangements is the one in which the VII ramus constricts the whole of the region that is affected, the anterior part of the same region being also controlled by the VI ramus. The relations between the VII Jumbar and I sacral rami are not so definite, though occasionally similar phenomena are observed. In fact all the contractions of the posterior veins are usually less definite and clear-cut. The constancy in the control of the anterior veins by upper rami is somewhat surprising in view of the variability in other respects. The only decided deviation from this control was in the II sacral ramus (Exp. XLVIII, left). Even in this case, however, the other side of the same animal possessed the normal arrangement. The most variable quantity in the whole process is the size of all the regions that contract no matter what nerves are stimulated. From a good many cats no contraction at all can be obtained, and from this wholly negative result to the condition in which stretches of eight to ten centimetres contract strongly and uniformly there is every gradation in the size of the contracting region. Even in this varia- bility, however, there is the constant feature that whenever there is any contraction at all it is almost sure to occur at about the middle of a superficial vein on the lateral side of the lower end of the crus, extending from the posterior edge of this member diagonally down- wards and forwards to the upper extremity of the foot. Whena greater part of the vein contracts it is usually this same region which contracts most strongly; and it is also to this place that fibres from both the VI and VII rami are distributed. Another variable feature, which may depend somewhat upon the one just discussed, is the number and arrangement of the rami that produce a contraction. A rather close direct correlation between these and the anterior or posterior arrangement of the plexus would be expected, but Table II shows that there is no such correlation, so far as the small number of observations will allow us to judge. 1 In several such cases I have subsequently stimulated the sciatic and found that it caused contraction of both these sharply differentiated regions. 484 F. W. Bancrofe. The whole path of the venomotor fibres from their origin in the spinal cord to their termination in the veins of the hind limb is apparently made up of two neurons. The cell body of one of these neurons lies in the spinal gray matter; its axis-cylinder process, as I have shown, passes through the anterior root of one of the I ea 9 Med FE or IV lumbar nerves and the corresponding white ramus into the sympathetic chain, down which it runs for a certain distance, as described above. The cell body of the second or peripheral or sym- pathetic neuron lies in one of the sympathetic ganglia. The position of these ganglia was determined by Langley’s nicotine method. After painting the III, IV, and V sympathetic ganglia, the stimulation of the pre-ganglionic fibres still causes constriction of the veins. The peripheral nerve cells are consequently not in these ganglia. Paint- ing the VI and VII ganglia, however, renders the stimulation of pre- ganglionic fibres ineffective — the veins do not constrict. It is in one or both of these ganglia, then, that the peripheral venomotor neurons for the veins examined have their cells of origin, and it is here that the axis-cylinder process of the spinal venomotor neuron ends. This at least is true of all the cases I have examined, but it is possible that in Langley’s more posterior types of the plexus some peripheral neuron cells may lie in the I and II sacral ganglia. This is suggested by the course of the post-ganglionic fibres. The post-ganglionic fibres usually leave the sympathetic by the sray ramus immediately below the ganglion in which their cells of origin are situated. Thus when the sympathetic trunk is cut both above and below either the VI or the VII lumbar ganglion, the stimu- lation of the pre-ganglionic fibres between the section and the gan- slion causes constriction. When, however, the ganglion is painted with nicotine the stimulation is usually, but not always, ineffective. This shows that the cells of the distal venomotor neurons are usually in the ganglion just above the ramus through which the fibres leave the sympathetic, but that occasionally they are located in a ganglion higher up. Since it has already been shown that stimulation of the I sacral gray ramus usually, and of the II exceptionally, causes con- striction, and since, as has just been pointed out, the cells of origin of post-ganglionic fibres are usually situated in the ganglion immediately above the gray ramus in which they are contained, it follows that peripheral neuron cells may be situated in the I and II sacral ganglia, although I have not been able to demonstrate them with the nicotine method. The Venomotor Nerves of the Hind Limo. 485 In general the arrangement of the venomotor nerves here described corresponds to that of the arterial vasomotor and sweat fibres of the hind limb.! The location of the ganglion cells and the course of the fibres through the gray rami is the same as that of the arterial vaso- motor fibres, except that the stimulation of the II sacral ramus does not usually produce a contraction of the veins; but on the other hand the origin of the venomotor fibres from the spinal cord is more restricted. Bayliss and Bradford,? experimenting on the dog, found vasomotor fibres in the XI thoracic to the III lumbar spinal nerves, and Langley, who used cats, found them in the XII thoracic to the IV lumbar, whereas I have found them only in the I to IV lumbar nerves and have obtained the maximum effect from the III lumbar. It is evident, therefore, that the fibres to the superficial veins of the hind limb originate from the lower end of the region supplying all the vasomotor nerves for that member. In conclusion I wish to express my thanks to Dr. W. T. Porter, at whose instance this work was undertaken and under whose direction it was carried on. 1 Compare LANGLEY: Journal of physiology, 1891, xii, p. 347; zb¢d., 1891, xii, P- 375; zbzd., 1894, xvii, p. 296. 2 BAYLIiss and BRADFORD: Journal of physiology, 1894, xvi, p. Io. AN ANALYSIS OF THE ACTION OF THE VAGUS NERVE ON THE HEART. By L. J. J. MUSKENS. [From the Laboratory of Physiology in the Harvard Medical School.| Page T. ‘Methods. of investigation 6 25.0 aie! 0) ese) ee 1, The stimulation of the vagus® «) .= 0) 0 6) 6 6) he oe ee 2. The method of recording contractions . . . .... +. + + «+ +» 490 3. The preparation of the experimental animal. . . . 492 II. The effect of vagus excitation on the interval between the airaeaene oF the several parts.af.thebeamie oy 6 S08 ic ae se ee ee 1. The,auriculo-ventricular interval... . . |. 2 ).ceee 2 493 2. The sino-auricular interval . .. . - 495 3. The interval between the contractions of ane different ‘iar ‘Of ‘he sinus . 497 III. The influence of the vagus nerve on the force of the heart-beat . . . . . . 497 1. On the:force of the, ventricle. (Fie 2 os 0 es 1. os, 6 Oe 2. On the-force.of theaunicles = 555) ee oe re 3. On the force of the sinus. . . : Meas 86 SOS IV. The vagus influence on the frequency meh mia the sinus contracts. . . 500 V. The action of the vagus explained by its influence on the conduction of the Cardiac excitation wave =. FIGURE I L. J. J. Muskens. The probability of the vagus increasing or di- minishing the resistance to the conduction of the excitation wave has been demonstrated above, and this action of the vagus suggests that the nerve may also regulate the resistance to the discharge of the excitation wave. The latter hypothesis is a simple and, to my mind, easily accepted explanation of the alterations in rhythm often observed when the vagi are stimulated; the threshold value of the ex- citation discharge is altered under vagus in- fluence. It should be stated here that an increase in the contraction interval also occurs apparently independently of the vagi in badly nourished hearts. Thus, in the exhausted frog’s heart, especially after loss of blood, very interesting changes in the duration of the contraction interval may be observed. In Fig. 12, the auricle (lower curve) beats with perfect regularity. The contractions of the ventricle are in groups of three. In each group, the auriculo-ventricular interval in- creases from nearly 0.7 to I.1 secs. | @ie sm tervals are 0.94, 1.07, dropped beat; 0.77, 1.01, I.10,° 1.14, dropped beat; 0.307 i-@ieainusm dropped beat. The sino-auricular interval is also some- times lengthened in badly nourished hearts. Fig. 13 is an example. In this case the sinus beat with absolute regularity (lower curve), but the sino-auricular interval increased until the excitation wave was blocked entirely. The intervals are 1.38, 1.74, dropped beat; 1.16, 1.38, 1.56, dropped beat; 1:16, aigepuiages dropped beat? 1.16,° 1.387 cte It seems highly probable that the periodic loss of one beat in the cases illustrated by Figs. 12 and 13 is a consequence ofthe Action of the Vagus Nerve on the Fleart. 509 continuous increase of the contraction interval. Gaskell ! and Engelmann? have shown that in the heart every contraction reduces the conducting power of the contracting part. In the record be- fore us the auricle contracts with perfect rhythm. At each auricular contraction an excitation wave passes over the ventricle. On the arrival of the first excitation, the ventricle contracts. Its con- ducting power is lowered by this contraction. Recovery is slow because of imperfect nutrition. The next excitation from the regularly working auricle is delayed by the difficult conduction. Hence the second auriculo-ventricular interval is greater than the first. The excitation from the third auricular beat is delayed like the two pre- ceding ones. The third auriculo-ventricular inter- val is longer than the second, as the second was longer than the first, for to the delay of the first interval is added the delay of the second. This process goeson. The auriculo-ventricular interval grows longer with each cardiac cycle, as the delay of each is added to the summed delays of its pre- decessors. Finally, the interval between the beat of the auricle and ventricle exceeds a certain limit, as in the case of vagus excitation (page 493), and a ventricular beat is dropped. The period is complete. The loss of this beat gives time for the conducting power to reach its former level. The next auriculo-ventricular interval is about the ‘QZIS [ULSIIO 94} Spalyj-omy, “C1 ANNI length of the first interval of the preceding series, and a new period begins. This explanation of periodic pulses is of especial interest, for the reason that in badly nourished human hearts, for example in myocarditis and arterio-sclerosis, similar irregularities are not in- frequent.? It is probable also that many of the ? GASKELL: Journal of physiology, 1883, iv, p. 97. 2 ENGELMANN : Archiv f. d. ges. Physiol., 1896, Ixii, p- 543- 3 Compare ENGELMANN: /0zd., p. 553- ‘SNUIS OY} JO asOY} “AMO OY} fapLINe 94} JO SUOT}OVI]UOD 9Y} Sp109a1 9AAND soddn ayy, 510 L./. J. Muskens. periodic groups of Luciani (recently studied by Oehrwall') will be thus explained. In conclusion I desire to express my grateful appreciation of the valuable criticism of Dr. H. P. Bowditch. I am also greatly indebted to Dr. W. T. Porter for assistance in the preparation of my paper for the press. SUMMARY. 1. The sino-auricular and auriculo-ventricular contraction intervals are usually lengthened by vagus excitation; sometimes, however, they are diminished; the one may be increased, while the other is dimin- ished. The vagus effect quickly reaches a maximum and then slowly decreases. : 2. The interval between the contractions of different parts of the sinus is sometimes increased by vagus excitation, so that the dif- ferent parts are dissociated and beat at measurably different times. 3. The force of contraction of the sinus and auricle is frequently diminished by vagus excitation. 4. The vagus does not diminish the force of ventricular contraction in the frog, except when the normal nutrition of the heart is disturbed by the loss of blood or otherwise. 5. The frequency of sinus contraction is usually diminished by the stimulation of the vagus; at times it is increased. 6. The various actions of the vagus nerve just enumerated, together with Bowditch’s staircase, interference, and various forms of irregular pulse, can be readily explained by variations in the transmission of the cardiac excitation. 1 OEHRWALL: Skandinav. Archiv fiir Physiologie, 1898, viii, p. I. ANEW SEETHOD POR THE SfUDY* OF THE ISOLATED MAMMALIAN HEART. By aWie el. PORKGIER: [From the Laboratory of Physiology in the Harvard Medical School.] HE isolation of the mammalian heart was first accomplished by H.N. Martin,! in the Biological Laboratory of the Johns Hop- kins University. Normally warmed defibrinated blood entered the right auricle and right ventricle from a reservoir at normal venous pressure. The right ventricle pumped the blood through the lungs, where it was oxygenated, artificial respiration being maintained for that purpose. The pulmonary veins brought the arterialized blood to the left heart, and the ieft ventricle drove it into a tall tube tied into the aorta, whence the blood was returned to the venous reser- voir. The height of the liquid column in this tube determined the pressure against which the ventricle worked, and this “ arterial press= ure” could be regulated at will. The advantages of Martin’s method are great. The heart is fully isolated from all other organs except the lungs, and works under conditions closely approximating the normal state. Indeed, for many purposes the original plan of Martin is superior to those since advocated. The chief objection to it has been the difficulty of secur- ing blood enough. It is necessary to use dog’s blood for the dog’s heart, cat’s blood for the heart of the cat, etc., and several animals have to be sacrificed in order to secure a sufficient quantity for one perfusion. In 1890, Martin and Applegarth? published an important modifi- cation of the procedure just described. In the new method “all the branches of the aorta, except the coronary arteries, are ligated. The vene cave are also ligated. In the aorta itself is placed a cannula, which is connected with a Mariotte’s flask, raised a sufficient height above the organ. The defibrinated blood from the flask fills the 1 MARTIN: Studies from the biological laboratory of the Johns Hopkins University, 1881, ii, p. 119. 2 MarTIN and APPLEGARTH: Studies from the biological laboratory of the Johns Hopkins University, 1890, iv, p. 275. 512 Wo 1. Porier. connecting tubes, the aorta, and the coronary arteries at a constant pressure, which, of course, is quite independent of the force and the frequency of the heart-beat. The blood taking the coronary circuit, on reaching the right auricle, proceeds to the corresponding ventri- cle, and from it through the lungs to the left auricle. This blood is, therefore, the only blood entering the cavities of the heart or passing through the lungs unless there be some inefficiency of the aortic semi-lunar valves. That the cavities of the heart are not distended with more blood is not found to influence the normal character of its beat, which continues rhythmically and forcibly for three or four hours.” Arnaud, in 1891, injected defibrinated blood into the aorta of a rabbit the heart of which had ceased to beat, and saw co-ordinated contractions return. The following year Hédon and Gilis? made similar injections in a dog and in an executed man, and secured co-ordinated beats. Langendorff,’ in 1895, modified the method of Martin and Apple- garth by omitting the lungs, receiving the coronary blood from the right heart into a dish or beaker. The omission of the lungs per- mits the heart to be removed from the body, an advantage for cer- tain purposes. Langendorff’s modification is however open to the objection that the blood cannot be so satisfactorily oxygenated as when it passes through the lungs. Moreover, the removal of the heart prevents stimulation of the extrinsic cardiac nerves. My own experiments on the isolated heart began nearly a year before the publication of Langendorff’s first paper. They were at first directed to the discovery of a method by which the warm- blooded heart could be maintained in rhythmic, forcible contraction by thoroughly oxygenated blood supplied in the normal way, namely, through the right auricle to the right ventricle, thence to the left auricle and left ventricle, and so to the right auricle again. This is not the place to speak of the many devices which were employed one after the other in the attempt to secure a really satisfactory oxy- genation of the blood. It is enough to state that none of these devices succeeded in thoroughly oxygenating in a sufficiently short * ARNAUD: Archives de physiologie, 1891, p. 396. 2 HEDON and Gits: C. r. de la soc. de biologie, Paris, 1892, p. 760. LANGENDORFF : Archiv f. d. ges. Physiol., 1895, xi, p. 292. Langendorff’s modification has recently been employed in altered form by RUMKE: Geneeskun- dige Bladen uit Kliniek en Laboratorium, Harlem, 1897, (iv) x, p. 201. 3 The Lsolated Mammalian Heart. 513 time the quantity of blood required for the successful perfusion of the warm-blooded heart. The attempt was therefore temporarily given over and the method of Martin and Applegarth employed. My first arrangement of Martin and Applegarth’s method agreed with Langendorff’s modification in omitting the lungs, but differed from Langendorff’s in many other respects. All the branches of the aorta except the coronary arteries were tied. The aorta was kept filled with blood from a reservoir at constant pressure. The semi- lunar valves being closed by the constant high aortic pressure, the blood was forced through the coronary arteries into the right heart; a very small quantity entered the left heart through the vessels of Thebesius. The force and frequency of the contractions of the left ventricle were recorded by a Hiirthle manometer connected with a tube passed into the left ventricle through the left auricular appendix and mitral valve. The blood flowing through the coronary vessels into the right heart escaped through the pulmonary artery on to a registering apparatus, so that the volume of the coronary circulation, excepting the very small quantity reaching the left heart through the vessels of Thebesius, was recorded. With this method the fact that stimulation of the vagus diminishes the flow through the coronary arteries was discovered,! an experi- ment recently repeated by Maas? in Langendorff’s laboratory. Curves showing simultaneously the intraventricular pressure and the diminu- tion in the volume of the coronary circulation in vagus excitation, obtained by this method, were shown to the American Physiological Society at its meeting in Boston in December, 1896. One of the curves is to be found in this Journal, 1898, i, p. 160. Another was published in the American Text-book of Physiology, 1896, p. 453, to illustrate the influence of the vagus on the frequency of ventricular contraction.®? This is the first instance in which a graphic record of the volume of the coronary circulation has been obtained; and the first in which the intraventricular pressure in the isolated heart has been recorded. With this method also the relation of the volume of the coronary 1 PORTER: Boston med. and surg. journal, 18¢6, cxxxiv, p. 39. 2 Maas: Archiv f. d. ges. Physiol., 1898, Ixxi, p. 399. 3 In reproducing this curve, the line recording the volume of the coronary cir- culation was cut out because unnecessary to the illustration of the vagus action on the frequency of the heart; this curve and the one published in the American Journal of Physiology were from the same experiment, March 26, 1896. 514 W. 7. Porter. circulation to the frequency and force of the ventricular contraction in the isolated heart of the cat was studied. Curves showing the main result of this investigation were published in the American Text-book of Physiology, 1896, p. 476. A detailed account is to be found in the Journal of Experimental Medicine.! In the method last described the animal and the recording appara- tus were placed in a huge warm chamber kept at a constant temper- ature. After a time, I discarded the warm chamber and designed in its stead the apparatus afterwards used by Miss Hyde in her study of - the effect of distention of the ventricle on the flow of blood through the walls of the heart. The new method is described in her commu- nication in this Journal.? Meanwhile, I had found that any part of the ventricle of the dog’s heart, even the ganglion free apex, will beat for hours if supplied with defibrinated dog’s blood through its nutrient artery. By this means the whole ventricle, as well as the apex of the ventricle, was for the first time fully isolated from the rest of the heart and kept in powerful, rhythmic, long-continued contraction.? This method has now been constantly used in this laboratory during fourteen months, and can be highly commended. Dogs are usually employed. The animal is anesthetized with ether, bled from the left carotid artery, the blood defibrinated, and filtered through glass wool. Mean- while, warm 0.8 per cent sodium chloride solution is allowed to flow into the right jugular vein. After a short interval, the dog is bled again from the carotid artery, and the blood defibrinated as be- fore. The heart is now rapidly removed and placed still beating in a beaker of warm saline solution. Often the beats are so vigorous that the heart with each ventricular systole springs more than an inch from the bottom of the beaker. Thus the organ is self-cleansed from blood. A glass cannula is now tied into the coronary artery supplying the area the contractions of which are to be studied, and the part of the heart wall supplied by the artery is cut out. The cannula bearing the attached ventricular segment is filled with defi- brinated blood and joined to a glass tube passing through the rubber stopper of a small glass chamber. A small adjustable clamp sup- 1 MAGRATH and KENNEDY: Journal of experimental medicine, 1897, li, p. 13. 2 Miss I. H. HypE: This journal, 1898, i, p. 215. 8 PORTER: Journal of experimental medicine, 1897, ii, p. 391; preliminary account in Journal of the Boston Society of Medical Sciences, 1897, i, issued March to, 1897. The Lsolated Mammalian Fleart. 515 ports the upper margin of the piece of heart to keep its weight from dragging on the nutrient artery. The chamber is provided with a thermometer. The neck of the chamber passes through a rubber stopper in the floor of a galvanized iron water tank, the sides of which rise above the top of the chamber. A wire attached by a hook to the lower end of the piece of ventricle passes through the neck of the chamber and is fastened to a lever of the second class, the writ- ing-point of which traces the contraction curve, usually magnified seven times, upon a Baltzar drum. Within the water tank also is placed a litre flask filled with defibrinated blood. ‘The contents of this flask are kept at any desired pressure by means of a pressure- bottle. The blood passes from the blood-flask to the heart-chamber and through the cannula in the coronary artery into the ventricular segment, from which it escapes through the severed veins into the lower part of the chamber and runs out into a tall beaker placed to receive it. The blood-flask, the heart-chambey, and the tubes con- necting the two are surrounded by a large volume of water at any desired temperature. The water tank is thirty-six centimetres long, twenty-one broad, and twenty-five deep. A window in the front per- mits a view of the heart as it contracts. The advantages of this method for studying the fundamental prop- erties of cardiac muscle, the action of animal extracts and drugs upon the heart, and certain other problems, are very great. A large dog’s heart affords two and sometimes three separate apex-preparations each with a nutrient artery large enough for a practicable cannula. Several preparations of the basal portion of the ventricle can also be obtained from the same heart. The experiment scarcely ever fails. The perfused piece seldom refuses to beat, and if it does another piece from the same heart can be used. The relatively very large quantity of perfusion fluid allows the circulation to continue for a long time; there is no troublesome turning of stopcocks at frequent intervals. Often the quantity of blood together with the small num- ber of vessels to be supplied makes it unnecessary to perfuse the heart twice with the same blood. The preparation can be made with all care. There is no hurry. Even pieces left for more than an hour will usually beat when perfused. The long survival makes it pos- sible to prolong experiments through most of the day, a fresh piece of ventricle being taken when the one in use wears out. This change of pieces we have found of value in testing the effect of poisons and animal extracts. 516 W. T. Porter. Such are the various methods of isolating the mammalian heart. At best, they leave much to be desired. They fail to realize the long- deferred hope that the mammalian heart shall be made to beat like the heart of the frog in a Williams apparatus, contracting for hours while fed on a simple perfusion fluid. This result is reached by the following procedure. It will be remembered that the great obstacle to the perfection of the methods of isolating the mammalian heart has been the difficulty of properly oxygenating the nutrient blood. Oehrwall’ has shown that even the batrachian heart contracts much more powerfully when surrounded with pure oxygen. Since the publication of Oehrwall’s paper many plans for the use of oxygen in the isolation of the warm- blooded heart have been tried by the present writer without avail. In every instance the mechanical difficulties of keeping considerable quantities of blood thoroughly oxygenated have been too: great. Not until the discovery that the mammalian heart would beat with a blood-supply much less than is ordinarily thought to be essential,’ ‘and the further discovery that this relatively small quantity may be effectively supplied to the heart muscle through the veins of Thebe- sius and the coronary veins ® did the problem seem once more prac- ticable. On returning to the attack, I determined to feed the heart through these veins in an atmosphere of oxygen, and, if this were not enough, to increase the oxygen pressure, in the hope of thereby facilitating oxidation in the manner taught by Haldane.* The following experiment was accordingly performed. May 2, 1898. A cat was bled and the blood defibrinated and filtered through glass wool. Cannulas were tied into the right auricular appendix, the pulmonary artery, and the aorta. The cannula in the right.auricular ap- pendix led through a Williams valve to a small reservoir of blood. ‘The pulmonary and aortic cannulas were each connected with glass tubes which rose to a short distance above the blood-reservoir and then turned to dis- charge their contents into the reservoir itself. All the heart vessels except the two arteries mentioned were ligated. The arrangement therefore was closely similar to that of the frog’s heart in a Williams apparatus. ‘The heart with its several tubes was now placed in a strong glass cylinder immersed in warm water. ‘The top of the cylinder was provided with a stout brass cap 1 OEHRWALL: Archiv fiir Physiologie, 1893, Suppl. Bd., p. 4o. * MAGRATH and KENNEDY: Journal of experimental medicine, 1897, ii, p. 13. 8 PRATT: This journal, 1808, i, p. 86. * HALDANE: Journal of physiology, 1895, xviii, p. 211. The Isolated Mammatan Freart. 51 perforated by two tubes. One was a T-tube the side branch of which led to a large metal cylinder containing oxygen under high pressure, while the other branch was provided with a stop-cock opening into the atmospheric air. The second tube led to a pressure gauge. So soon as the oxygen pressure began to rise, the heart, which had ceased to beat, began to contract with great vigor. Surrounding the heart with oxygen even at the pressure of the atmosphere was distinctly helpful, but the contractions became decidedly stronger and more frequent as the oxygen pressure rose. A pressure of about two atmospheres was that ordinarily employed, but as high as four atmospheres was occasionally tried. The blood coursed from the reservoir into the right side of the heart. Each beat of the right ventricle drove blood in a stream through the tube in the pulmonary artery back into the reservoir. The Williams valve prevented regurgitation from the right heart. The heart muscle was nourished through the veins of Thebesius and the coronary veins. A small quantity of blood found its way through foramina Thebesii into the left auricle and ventricle, whence it was pumped by the latter out through the aorta. The vigorous beating of the heart continued from early in the morning until late in the afternoon, when the experiment was broken off. The contractions were vigorous also at room temperature. Two conclusions may be drawn from this experiment : — (1) An atmosphere of oxygen is of advantage in maintaining the contractions of the isolated mammalian heart. (2) A heart fed simply through the veins of Thebesius and the coronary veins will maintain strong, rhythmic contractions for many hours if supplied with oxygen at high tension. The first thought suggested by these statements is whether the mammalian heart, like the frog’s heart, will beat when fed on serum alone, provided that a sufficient supply of oxygen is furnished. The experiment was accordingly repeated on other hearts, but the blood was replaced by serum obtained by centrifugalizing defibrinated blood. As was expected, the absence of corpuscles was readily borne by the heart. Continued rhythmic contractions were obtained with the serum alone, so soon as the oxygen tension rose to about two atmospheres. It follows that the mammalian heart fed through the vessels of Thebesius and the coronary veins with blood-serum alone will main- tain rhythmical contractions for hours when surrounded by oxygen at high tension. | The ease with which this remarkable result was obtained encour- aged the hope that even isolated pieces of the ventricle would beat if 518 W. T. Porter. fed with serum through a branch of the coronary artery. It was @ priort almost certain that this would be the case, were the piece of ventricle supplied with serum at the normal blood-pressure. But to force serum through a coronary artery at the normal pressure requires a pressure-apparatus difficult of control in an extrinsic pressure of two atmospheres. Even were this difficulty overcome, the rate of flow through a piece of ventricle fed at fairly high pressure is rapid and a large volume of serum would be required. Now, a sufficiently large volume of serum cannot be obtained from a single animal, and it is somewhat disadvantageous to use the blood of other animals, even of the same species. It seemed best, then, to attempt perfusion at a very low arterial pressure, trusting that even this slight driving force would carry serum enough through the capillaries to produce and maintain contractions. The complete success of this undertaking is shown in the following experiment. June 17, 1898. A cat, anesthetized with ether, was bled from the left carotid artery, the blood defibrinated, diluted one-half with 0.8 per cent NaCl solution, and the serum separated in a centrifugal machine. An hour after the heart had ceased to beat it was removed from the chest, a cannula tied into the ramus descendens of the left coronary artery, and the part of the ventricle supplied by this branch cut away. The cannula was joined to a vessel containing 50 c.c. of the cat’s serum, and placed in a glass cylinder connected with an oxygen reservoir. The height of the column of serum above the piece of ventricle was about 25 centimetres. The flow was approxi- mately at the rate of one drop per second. ‘The temperature was that of the room, about 25°C. The oxygen pressure was now raised to nearly two atmospheres. In a very few minutes the piece of ventricle began to beat with regularity and force, and these strong and rhythmical contractions con- tinued so long as the supply of serum was kept up. When the serum ceased to pass, the ventricle ceased to beat. This experiment permits the further conclusion that even tsolated portions of the mammalian ventricle supplied through their nutrient arteries with a small quantity of serum at very low pressure will main- tain rhythmical, long-continued, forceful contractions when surrounded by oxygen at high tension. 1 Similar results have been since attained with the isolated apex of the dog’s heart. ————— INDE L Os WOU, BSORPTION, intestinal, 411. Activity, daily, methods of record- ing, 40. , variations produced by alcohol, baro- metric changes, and diet, 4o. ALBRO, ALICE H. See CHITTENDEN, R. H., and ALICE H. ALBRO, 307. Albumose, excretion by kidneys, 274. ——,, physiological action, 266. Alcohol, effect on distemper, xv. ——,, effect on voluntary muscular power in fatigue, xv. ——,, effect on young dogs, xv. Alcoholic beverages, influence on digestion, 184. American Physiological Society, Proceed- ings, iil. Amylolytic power of saliva, variations, iii. Anesthesia, by ether in rectum, viii. Aorta, blood currents in, xiv. Attention, determination of a constant of, 283. ANCROFT, F. W. The venomotor nerves of the hind limb, 477. Barrows, F. W. The effect of inanition on the structure of nerve cells, xiv. Bile, chemical reaction, 317. , influence on pancreatic proteolysis, 307. Biological problems of to-day, xv. Blood-pressure, influenced by albumose and peptone, 275. Borax, its influence on nutrition, 1. Boric acid, its influence on nutrition, 1. Brain-circulation, influenced by high arterial pressures, 57. Brown, E. W. Notes on Cetraria islan- dica (/celand moss), 455. BubcettT, S. P. On the similarity of struc- tural changes produced by lack of oxygen and certain poisons, 210. ALCIUM SALTS, solubility, 423. Cane-sugar, inversion in stomach, 277. CANNON, W. B. See MOSER and CANNON, Xll. CANNON, W. B. The movements of the stomach, studied by means of the Rontgen rays, 350, Xili. CANNON, W. B., and A. Moser. ‘The movements of the food in the cesopha- Cathartics, 411. Cetraria islandica (Zeeland moss), 455. CHITTENDEN, R. H. Variations in the amylolytic power of saliva and their rela- tion to the chemical composition of the secretion, ili. CHITTENDEN, R. H., and ALICE H. ALBRO. The influence of bile and bile salts on pancreatic proteolysis, 307. CHITTENDEN, R. H., and W. J. Gigs. The influence of borax and boric acid upon nutrition, with special reference to proteid metabolism, I. CHITTENDEN, R. H., L. B. MENDEL, and H. C. Jackson. A further study of the intluence of alcohol and alcoholic drinks upon digestion, with special reference to secretion, 164. CHITTENDEN, R. H., L. B. MENDEL, and H.E. McDErRmortT. Papain-proteolysis, with some observations on the physio- logical action of the products formed, 2 - CHITTENDEN, R. H., and A. N. RICHARDS. Variations in the amylolytic power and chemical composition of human mixed saliva, 461. CLARK, G. P. On certain characteristics of the pressure sensations of the human skin, 346, xi. CLEGHORN, A. The reinforcement of vol- untary muscular contractions, 336. gus, 435. Cardiopneumatic movements, 117. | 520 Coagulation of blood influenced by albu- mose and peptone, 269. Color vision, xv. Contraction, static, methods of determining, 284. Coronary circulation, volume of, method of recording, 215. Coronary pulse-wave, 152. Coronary veins, nourish the heart, 86. Coronary vessels compressed in systole, 145. CUNNINGHAM, R. H. The restoration of coérdinated volitional movement after nerve “crossing,” 239. Cusuny, A. R. See WALLACE and CuSH- NY, 411. EGLUTITION, 435, xii. ——, studied with Rontgen rays, 435, xii. Distemper, affected by alcohol, xv. Depressor-nerve in guinea-pig, 393- Development, affected by alcohol, xv. Diabetes, 395. Digestion by papain, 255. Digestion, influenced by alcohol, 164. Digestion of cane-sugar, 277. Dynamograph, 284. AR, function in fishes, 128. Electrodynamometer, 106. Ether-anesthesia by the rectum, viii. ATIGUE, affected by alcohol, xv. Fatty degeneration, metabolism in, v. Ferris, S. J., and G. Lusk. inversion of cane-sugar by hydrochloric acid, 277. Fibrillary contractions of heart, 71, 99. Friction-machine, 294. Fungi, composition and nutritive value, 225. ALVANOMETER, Rowland, 106. Gastric cannula, ror. GirEs, J. See CHITTENDEN and GIES, Tf. GREENE, C. W. On the relation between the external stimulus applied to a nerve and the resulting nerve impulse as meas- ured by the action current, 104. Guinea-pig, cardiac nerves in, 383. The gastric L[ndex. ALLOCK, W. (with F. S. Muckey). The action of the larynx in the pro- duction of voice, vi. HARRINGTON, D. W. Contributions to the physiology of the cardiac nerves in the : guinea-pig, 383. Heart, conduction of excitation wave, 502. , contraction interval, 493. ——,, force, 497. ——, distention influences intramural flow, aay ——,, fibrillary contractions, 71. , frequency, 500. , influenced by vagus nerve, 486. , intramural circulation increased by systole, 157. , mammalian, method of isolating, 93, 2055 Lie ——,, nerves in guinea-pig, 383. , nutrition through vessels of Thebesius. and coronary veins, 86, 516. , oxygen at high tension replaces red corpuscles, 516. Hopce, C. F. Influence of alcohol upon voluntary muscular power in conditions. of fatigue, xv. Influence of alcohol upon the young in dogs, and upon the severity of an at- tack of distemper, xv. Howe i, W. H. The influence of high arterial pressures upon the blood-flow through the brain, 57. Hype, Ipa H. The effect of distention of the ventricle on the flow of blood through the walls of the heart, 215. ; Hydrochloric acid inverts cane-sugar, 277. | (Scenes moss, 455: Inanition, effect on nerve cells, xiv. Intestinal absorption, 411. ACKSON, H. C. See CHITTENDEN, MENDEL, and JACKSON, 164. Kee acid, excretion of, xv. | age nance action in voice-production, vi. Lateral line in fishes, function, 128. Ler, F. S. The functions.of the ear and the lateral line in fishes, 128. Lichenin, 456. Index. Logs, J. The biological problems of to- day : physiology, xv. Lung, redistention after collapse, ix. Lusk, G. See Ferris and Lusk, 277. See REILLY, NOLAN, and Lusk, 395. —. On metabolism in fatty degenera- tion, v. ATHEWS, A. A contribution to the chemistry of cytological staining, 445. McDermott, H. E. See CHITTENDEN, MENDEL, and McCDERMOTT, 255. MELTZER, S. J. A new pleural cannula 7 situ, XV. A simple method for the redistention of the collapsed lung, ix. Ether-anzsthesia by the rectum, viii. On the nature of the cardiopneumatic movements, II7. MENDEL, L. B. See CHITTENDEN, MEN- DEL, and JACKSON, 164. See CHITTENDEN, McDERMOTT, 255. Some experiments on the excretion of kynurenic acid, xv. The chemical composition and nu- tritive value of some edible American fungi, 225. Mental activity, measurement through.mus- cular activity, 283. Metabolism, in fasting animals, 395. , in fatty degeneration, v. —,, in phlorhizin diabetes, 395. Mosgr, A. See CANNON and MOSER, 435. Moser, A., and W. B. CANNON. The movement of food in deglutition, xii. Muckey, F.S. See HaLitock and Muc- KRY, Vi. Muscle, active relaxation, 343. Muscular contractions, reinforcement, 336. Muscular power, affected by alcohol, xv. Mushrooms, composition and _ nutritive value, 225. MuskeEns, L. J. J. An analysis of the ac- tion of the vagus nerve on the heart, 486. and ss MENDEL, N ERVE cells, affected by inanition, xiv. Nerve-crossing, restoration of move- ment after, 239. Nerve impulse, relation to external stimu- lus, 104. Nerves of veins, 477. NoLan, F. W. See REILLY, NOLAN, and LUSK, 395. 521 SOPHAGUS, movements of food in, 435: ? Oxygen, lack of, causes changes in protozoa, 210. pPerceEans proteolysis, influenced by bile and bile salts, 307. Papain-proteolysis, 255. PaTtTEN, W. A basis for a theory of color vision, xv. Peptone, physiological action, 266. , excretion, 274. Phlorhizin diabetes, 395. Pleural cannula, xv. PorTeER, W. T. A new method for the study of the isolated mammalian heart, Sil. New experiments on the mammalian heart, xiv. The influence of the heart-beat on the flow of blood through the walls of the heart, 145. The recovery of the heart from fi- brillary contractions, 7T. Pratt, F. H. The nutrition of the heart through the vessels of Thebesius and the coronary veins, 86. Pressure sensations of human skin, 346, xi. Proceedings of the American Physiological Society, iii. Proteid, its constitution, 409. Proteid metabolism, influenced by borax and boric acid, 1. Proteolysis, influenced by bile, 307. Protozoa, structure changed by lack of oxygen and by certain poisons, 210. Pulse, irregularities explained, 508. ECTUM, anesthesia by ether, viii. Reinforcement of voluntary muscular contractions, 336. REILLY, F. H., F. W. NoLAN, and G. Lusk. Phlorhizin diabetes in dogs, 395. RICHARDS, A. N. See CHITTENDEN and RICHARDS, 461. ALIVA, amylolytic power, 461, iii. S , chemical reaction, 463. , composition, 461, ili. secretion, influenced by alcohol, 164. , submaxillary of dog, composition, 166. , variations in amylolytic power, 461, iii- 522 L[ndex. Salivary digestion in stomach, 379. Skin, pressure sensations, 346, xi. Staining, chemistry of, 445. STEWART, C. C. Variations in daily activ- ity produced by alcohol and by changes in barometric pressure and diet, with a description of recording methods, 4o. Stomach, absorption of alcohol from, 205. ——,, anatomy in cat, 364. ——,, digestion of cane-sugar, 277. ——,, duration of digestion, 202. ——, inhibited by emotions, 380. —— -movements, 359, xiii. , reaction of contents, 378. , Salivary digestion in, 379. Strontium elimination, 83. Sugar production, 402. HEBESIUS, vessels of, anatomy, 87. Torcular pulse, 68. AGUS-NERVE in guinea-pig, 384. , influence on heart, 486. , method of stimulating, 488. Vasomotor nerves. See Venomotor. Veins, nerves of, 477. Veins of Thebesius, anatomy, 87. ——,, nourish the heart, 86, 516. Venomotor nerves of hind limb, 477. Ventricle, distention checks flow of blood through walls of heart, 215. Voice production, vi. Vomiting, 373. ALLACE, G. B., and A. R. CusHny. On intestinal absorption and the sa- line cathartics, 411. WELCH, JEANNETTE C. On the measure- ment of mental activity through muscular activity, and the determination of a con- stant of attention, 283. Woop, H. C., Jr. Notes on the elimina- tion of strontium, 83. PROCEEDINGS OF THE AMERICAN _PHYSIO-— EOGICAL. SOCIE EN: TENTH ANNUAL MEETING: CORNELL UNIVERSITY, DECEMBER 28 and 29, 1897. PROCEEDINGS OF THE AMERICAN PHYSIOLOGICAL BOCIE BY. VARIATIONS IN THE AMYLOLYTIC POWER OF SALIVA AND THEIR RELATION TO THE CHEMICAL COMPOSITION OF THE SECRETION. By R. H. CHITTENDEN. IN some experiments conducted in 1882! an attempt was made to ascertain whether there is any definite relationship between, the amylolytic power of human saliva and its degree of alkalinity. In the experiments then recorded, alkalinity was determined by titration with a standard acid, using cochineal as an indicator, while amylolytic power was estimated by determining the quantity of sugar formed from a definite amount of starch under given conditions. The results led to the conclusion that such variations in amylolytic power as saliva ordinarily shows, are not associated with corresponding variations in the degree of alkalinity. In a recent paper by Hofbauer,? the above results are referred to with the statement that they constitute the only data recorded bearing on the amylolytic power of human saliva at different periods of the day. This statement, however, is quite misleading, for in our paper it is distinctly stated that ‘‘the saliva was collected gen- erally an hour or two after breakfast,’ no attempt having been made to ascertain variations in amylolytic power for different periods of the day; indeed, in practically all of our experiments at that time, the saliva was collected at a convenient period after breakfast. The average alkalinity expressed in terms of sodium carbonate of fifty- one samples of saliva was found by the above method to be 0.08 per cent, the extremes being 0.052—0.163. We would now call atten- tion to the fact that human saliva, while ordinarily alkaline to litmus 1 CHITTENDEN and Exy: On the alkalinity and diastatic power of human saliva. American chemical journal, 1883, iv, p. 329. 2 HOFBAUER, L.: Tagliche Schwankungen der Eigenschaften des Speichels. Archiv f. die ges. Physiol., 1897, lxv, p- 503. iv Proceedings of the American Phystological Society. or lacmoid, is almost invariably acid to phenolphthaléin, hence such alkalinity as it possesses, is due not to sodium carbonate but mainly to alkaline phosphates, acid phosphates being likewise present. Experi- ments made in our laboratory by Mr. A. N. Richards show that human mixed saliva, using lacmoid as an indicator, requires on an average 0.7 milligram H.SO, to neutralize the alkalinity of I gram of the secretion. Expressed in terms of sodium carbonate, this would be equal to an alkalinity of 0.14 per cent. With phenolphthaléin as an indicator, on the other hand, 1 gram of saliva requires on an average 0.06 milligram NaOH to neutralize the acid salts present. It has also been found that the alkalinity as indicated by lacmoid and the acidity as indicated by phenolphthaléin are both noticeably greater in the saliva collected before breakfast than in the secretion collected after breakfast. Further, in conformity with Hofbauer’s results, we find,‘as a rule, that the amylolytic power of saliva coming from glands which have been in a state of rest for some time, z. ¢., collected be- fore breakfast, is greater than that secreted an hour after breakfast. We are not inclined, however, to consider that the increased amylo- lytic power of saliva secreted before breakfast, for example, is to be attributed directly to the increased alkalinity, for occasional results show that amylolysis may be more pronounced with saliva having a comparatively low degree of alkalinity. The true explanation is to be found in the greater concentration of the secretion coming from the glands which have been in a state of inactivity; 7. ¢., such secretion contains a larger amount of solid matter with a corresponding increase in the proportion of amylolytic enzyme, etc. The results of a single experiment may be cited : — Amylolytic Solids. Organic Time. Alkalinity.? matter. 7-10-7.30 A.M. 163 % nls 0.86% 0.58% 9.00-9.30 “ } 5s 0.51 0.30 Numerous results similar to the above testify to the truth of the foregoing statement. Somewhat noticeable also is the influence of 1 Before and after breakfast. Determined by ;!5 normal H,SO, with lacmoid as an indicator and expressed as sodium carbonate. 3. ote: Expressed as milligrams of maltose formed from I gram of starch. Tenth Annual Meeting. Vv different stimuli upon the amylolytic power and chemical composi- tion of human saliva. Experiments have been made with ether and chloroform vapor, alcohol, whiskey, and gin, the secretion obtained under their influence being compared with that resulting from mechanical stimulation, etc. The results thus far obtained tend to show that the above agents cause the secretion of a fluid richer in amylolytic enzyme and having a higher content of solid matter. The details of the experiments will be published in the next number of this Journal. ON METABOLISM IN FATTY DEGENERATION. By GRAHAM LUSK. MANY years ago Voit declared his belief in a preliminary cleavage of the proteid molecule within the organism into a nitrogenous por- tion and a non-nitrogenous portion, which were subsequently burned within the cells, often at different times. To the non-nitrogenous por- tion belonged the sugar of the starving diabetic, and it likewise fur- nished fat in fatty degeneration. Through the subcutaneous injection of phlorhizin in dogs a ratio of sugar to nitrogen as 3.75 is to I has been established in the laboratory of the writer. This signifies that the proteid molecule may yield 60 per cent of dextrose. Accompany- ing this intense form of diabetes may be seen in the starving dog a rise of 450 per cent in the proteid decomposition, an effect probably due to the non-combustion of the sugar produced. The only case parallel to this in the extent of its proteid decomposition lies in phosphorus poisoning, where a similar increase is present. The question arises, is not this high proteid metabolism in phosphorus poisoning likewise due to the non-burning of the sugars, consequent upon their quantitative conversion into fat? In other words, may not the 60 grams of dextrose obtainable from every 100 grams of proteid be converted into fat in cases of acute fatty degeneration? In a first experiment upon a diabetic dog the ratio in the urine was found to be Dextrose: Nitrogen = 3.75: 1. During the administra- tion of the phlorhizin, phosphorus oil was also given, with the idea of reducing possibly the sugar in the urine by means of its conversion into fat. No decrease in the sugar followed, although the dog died with every symptom of phosphorus poisoning. This experiment, vi Proceedings of the American Physiological Soctety. however, does not disprove the idea that in fatty degeneration the sugar from proteid is converted into fat, for the phlorhizin may have protected the sugar immediately upon its formation from any further change. A second experiment made the subject clearer. A starving dog was poisoned with phosphorus; all the symptoms, including a high rise in proteid metabolism, were manifest. Under these circum- stances, if proteid sugar is being converted into fat there should be no sugar present in the body. Now, the action of phlorhizin is first to sweep the body clear of sugar, as is indicated by the high ratio of sugar to nitrogen observed always on the first day of phlorhizin ad- ministration, even after long fasting. If now in the dog poisoned with phosphorus no sugar was present and we administered phlorhizin, no excess of sugar should be eliminated; only that belonging to the proteid decomposition for the time being should be eliminated. The result obtained conformed with this theoretical expectation. The ratio in the urine was Dextrose: Nitrogen = 3.65: 1. This indi- cates that in phosphorus poisoning there is no sugar present in the dog. Either one of two conditions may here be possible: either the sugar is burned as soon as formed, or it is converted into another substance. That it is immediately burned is improbable on account of the high proteid metabolism ; — its burning would reduce proteid metabolism. The sugar must, therefore, have been converted into another substance or into fat. It seems reasonable to conclude that in acute fatty metamorphosis of the cell the dextrose formed from proteid in the cytoplasm may be quantitatively converted into fat. THE ACTION OF THE LARYNX IN THE PRODUCTION OF VOICE. By W. HALLOCK (with F. S. MUCKEY). THE organ of voice-production is essentially a string, not a reed instrument. The two fundamental reasons for this conclusion are: first, the agencies for the control of pitch are the agencies that control the pitch of a string, namely, tension, length, and weight; secondly, the quality of the tone produced is the quality of the tone of a string. Voice-production and voice-modification (articulation) are managed by distinct, independent sets of muscles, the former by the intrinsic Tenth Annual Meeting. vil laryngeal muscles, the latter by the extrinsic muscles; and neither set should be permitted to usurp or interfere with the functions of the other. In the correct production of voice there should be no registers. The three agencies for the control of pitch are mediated by the intrinsic laryngeal muscles only. They should act simultaneously, independently, evenly, and gradually, and produce a smooth and continuous rise in pitch from the lowest tone to the highest, the action and operation of the larynx being the same throughout. If the extrinsic muscles are allowed to come into action and pull upon the larynx, the latter is distorted and the delicate action of the arytenoid cartilages is absolutely blocked. It then becomes necessary to rely entirely on change of tension to control pitch, and of the three factors this is the most difficult of control, because the pitch is directly pro- portional to only the square root of the tension of the cords, whereas it is inversely proportional to the length and weight. Under these conditions registers arise, owing to the imperfect codperation of and coordination between the intrinsic and extrinsic muscles, and the cords are seriously strained by the high tension to which they must be submitted in the effort to produce the high tones. This abnormal strain results in impairment of the muscle-structure, and then in faulty approximation of the vocal bands, with all the evil consequences thereof. The most pernicious of all habits in voice-production is this of permitting the large and powerful extrinsic muscles to usurp the duties of the delicate intrinsic muscles and prevent their action, while unable themselves to accomplish the same results. The classic investigations of Helmholtz, Konig, and many others, have proved that in the human voice the sound consists of a fundamental or pitch tone, accompanied by one or more of a series of overtones, the quality of the voice being dependent upon the latter. By photograph- ing the movements of sensitive flames we have been able to analyze tones and thus to verify completely the general correctness of the visual and oral observations of Helmholtz and Konig. Our photographs give an impersonal impartial record of the “ string overtones” in the voice, and their modification of quality, not only in different: voices, but in different vowel sounds in the same voice. In order to reinforce a tone, a cavity must have a fixed size, shape, and opening. The vibrations must be able to pass in as well as out at the opening. Reinforcement by chest-resonance is impossible, for two reasons especially: the chest is a cavity of varying size even vii Proceedings of the American Physiological Society. during a single breath, and it is essentially a closed cavity. The air in the chest may, and does vibrate, — so it does in the wind-box of an organ, — but these vibrations cannot reinforce the tone produced externally. The antra and sinuses are also useless for resonant reinforcement. DEMONSTRATION : ETHER-ANA!SSTHESIA BY THE RECTUM. By S. J. MELTZER. A SMALL bottle half filled with ether, and closed with a cork perfo- rated by a glass tube, was placed in a water-bath at a temperature above the boiling point of ether, The ether vapor generated was led into the rectum by means of a metal tube provided with a number of side openings, besides an aperture at the end, and connected with the ether bottle by means of rubber tubing. As ether boils at a point below the temperature of the body (about 35°C.), the introduced vapor remains in the intestinal canal in a gaseous state, and is there readily absorbed. The rate of absorption in this place is by no means comparable with that of the absorption of ether by the lungs. The absorption, however, is greatly facilitated by an increase of the intra-intestinal pressure, which can be easily accomplished by increas- ing the temperature of the water-bath, and thus introducing rapidly large quantities of ether vapor into the intestines. It must be borne in mind that the favorable as well as the dangerous state of anzs- thesia depends upon the amount of ether present at one time in the blood, and this depends not only upon the rate of absorption, but also upon the rate of the excretion from the body. Ether is always excreted through the lungs. If too large quantities of ether vapor are rapidly thrown into the intestines, not only will absorption be increased, but the enormously developing meteorism may seriously impair the respiration, and considerably diminish the excretion of the ether, and thus cause death. On the other hand, the excretion of ether by the lungs is apparently more completely accomplished when the vapor is introduced into the rectum than when inhaled by the lungs, as in the latter case the ether has to be exhaled into an atmos- phere already saturated with ether. If the temperature of the water is not too high, and if care is taken to remove frequently the ether bottle from the water-bath, the ether anzesthesia by the rectum is a safe and convenient method for certain laboratory purposes. Thus, a rab- Tenth Annual Meeting. 1X bit can be narcotized in a few minutes, and can be kept in a state of anesthesia for many hours without the aid of a special assistant. The peristalsis usually removes the surplus of gas from the intestines, if the gas is not generated too rapidly, and a moderate meteorism can easily be removed by gentle massage. The absorption seems to take place in the rectum, at least there was no ether present in the small intestines in cases of complete anzsthesia from a moderate generation of ether vapor. In dogs between twenty minutes and half an hour is required for a thorough anesthesia. But during this period there seems to be no danger whatsoever for the animal. An injection of morphine facilitates the result without increasing the danger. The rectal tube should be fastened so as to prevent its expulsion by the peristalsis, and the ether bottle should be kept higher than the rectum, in order to prevent the contamination of the ether by intestinal contents. Anesthesia by the rectum has the following advantages: If performed properly it is by far less dangerous to the animal than anesthesia by inhalation. It requires little attention, and no special assistant. It does away with all the reflexes affecting respiration, heart-beat, and blood-pressure, which are such disturbing elements in anesthesia by inhalation. Rectal anesthesia was suggested by Pirogoff for surgical operations as early as 1849. It did not come into practical use until the begin- ning of the eighties, when it was tried abroad and in this country. The slow procedure, the tenesmus, and the possibility of meteorism prevented its general use. Some experiments were made on animals, but only for testing its practicability for surgical purposes. So far as I know, no previous attempt to introduce it for laboratory purposes, has been made. DEMONSTRATION: A SIMPLE METHOD FOR THE REDIS- TENTION OF THE COLLAPSED LUNG. BY S. jj. MEETZER: THE method mostly employed for the redistention of the collapsed lung (in animals) is the sucking out of the air from the pleural cavity. But any one who has had extensive experience with this method knows how unsatisfactory it is. In many cases a piece of the lung is firmly sucked into the cannula, or if the opening in the chest is x Proceedings of the American Physiological Soczety. made in the sixth intercostal space, or lower, it often happens that even the diaphragm is sucked into it. With the aid of my pleural cannula I have demonstrated on a rabbit a simple method for the redistention of the collapsed lung, and the re-establishing of negative pressure in the pleural cavity. The protruding nozzle of the cannula is connected with a Miiller’s valve. Then the hand is placed upon the abdomen, and the stomach and the liver are pressed into the thorax while the trachea is being compressed. As the air of the compressed, non-collapsed lung cannot escape through the trachea, it enters into the collapsed lung and distends it. By this distention, and by the pressure from below, the air is driven out of the perfo- rated pleural cavity, while the valve prevents the entrance of air. oe ADA ARAL Ura eos Wo Ne, Cane FIGURE I. When now the stopcock of the cannula is closed, the tube leading to the valve removed, and the nozzle connected with a manometer, the latter immediately shows a negative pressure. In the experi- ment illustrated by Fig. 1 the nozzle of the cannula was connected with a Marey’s tambour. The straight line was drawn under normal atmospheric pressure; all above the line is at positive, and all below at negative pressure. The undulations at the left were obtained from the pleural cavity while it contained air. The expiration was always positive. Then the lung was distended, and the air driven out by the method described above, and the cannula again connected with a Marey’s tambour. Both expiration and inspiration were now below the line of the atmospheric pressure. Tenth Annual Meeting. xl ON CERTAIN CHARACTERISTICS OF THE PRESSURE SENSATIONS OF THE HUMAN SKIN. By G. BP. CLARK. VoN FREy has shown that the effectiveness of non-painful me- chanical stimuli, in exciting the so-called sense of pressure of the human skin, depends upon certain factors in addition to the strength of the stimulus, namely, the rapidity of its application, the size of the surface to which it is applied, and the locality of the skin stimu- lated. He determines the value of the physiological factor, the so-called ‘ pressure-points,” of any skin surface, by the use of test- hairs (Ketzhaare), the pressure of which is calculated from careful measurements of the applied surface and the power, 27. ¢., the weight which each can balance on the scales. Finding that test-hairs of greater surface and power are more effective physiologically than those of smaller surface and power, but of the same hydrostatic pressure, he assumes that the nerve organs concerned in the pressure sense are situated somewhat deeply in the skin. An object of the research here reported was to determine whether the same organs in the skin, which have been shown to be called into action by the de- formation caused by pressure (Druck), are also excited by that caused by traction (Zug), or whether other organs are concerned. The movements of the structures underneath the skin may evidently change the tissue pressure of the skin, either increasing or diminish- ing it, according to the kind of movement and the relation of the skin to the part moved. Changes of pressure, corresponding to those of pressure or traction from without upon the surface of the skin, may thus arise. Tests were made upon very small (0.3 to 0.5 mm”. ) and large (10 to 50 mm?.) surfaces on the left wrist and thumb, and with momentary and continued stimuli of different strengths. The stimuli were applied by means of a double-arm wooden lever in equilibrium, the end of one arm being connected by a very light straw with the surface of the skin to be stimulated, the end of the straw, or of a cork disc, which was slipped on to it when increase of surface was desired, being glued to the skin. The forearm of the person upon whom the tests were made was held in a plaster of Paris mould. Weighting or striking the arm of the lever between its axis and the skin served to produce pressure; weighting or striking the opposite arm produced traction. It was found that the so-called xii Proceedings of the American Physiological Soczety. ‘‘pressure-points”’ most sensitive to pressure are also most and equally sensitive to traction; that with very small surfaces (0.3 mm?.) there is inability to distinguish between pressure and traction, even with strong and continued stimuli; and that fatigue produced by a strong continued pressure stimulus is fatigue for effects of subsequent momentary traction as well as pressure stimuli. With large surfaces (50 mm?.) it was found that with momentary stimuli, even of marked strength, there is inability to distinguish between pressure and trac- tion, and that continued stimuli may be of insufficient strength to enable one to distinguish those of pressure from those of traction. Collectively the tests showed that ability to distinguish between pressure and traction depends upon the size of the surface stimu- lated, the duration of the stimulus, and the strength of the stimulus; that it is not an inherent quality of the impulse excited in the nerve organs of the skin by the changes of pressure. It having been found that the points most sensitive to pressure are also most sensitive to traction; that simple sensations of deformation are provoked by simple stimuli in either direction; that fatigue for pressure is also fatigue for traction; and that the factors, strength of stimulus, ra- pidity of application, size of surface to which the stimulus is applied, and locality of skin stimulated are of the same value in the effective- ness of traction stimuli as they have previously been found to be in that of pressure stimuli; —it is assumed that the same nerve organs in the skin are excited by both kinds of stimuli. THE MOVEMENT OF FOOD IN DEGLUTITION. By A. MOSER anv W. B. CANNON. [Reported for H. P. BOWDITCH by W. T. PORTER.) By mixing subnitrate of bismuth with the bolus, the passage of the food along the cesophagus can be seen with the Roentgen rays. In the cat, solid and mushy boluses are carried down by peristalsis, the descent being more rapid in the upper thoracic region than in the neck or below the level of the heart. Liquids descend faster than solids or soft solids as far as the level of the heart, but often remain there for several minutes before a peristaltic wave pushes them into the stomach. In man, solids and soft solids are likewise forced down the cesophagus by peristalsis. a ee ee Se Tenth Annual Meeting. xl THE MOVEMENTS OF THE STOMACH, STUDIED BY MEANS OF THE ROENTGEN RAYS. By W. B. CANNON. [Reported for H. P, BOWDITCH by W. T. PORTER.] THE conclusions reached in this investigation are as follows: (1) By mixing a-harmless powder, subnitrate of bismuth, with the food, the movements of the stomach can be seen by means of the Roentgen rays. (2) The stomach consists of two physiologically distinct parts: the pyloric part and the fundus: over the pyloric part, while food is present, constriction-waves are seen continually coursing towards the pylorus; the fundus is an active reservoir for the food, and squeezes out its contents gradually into the pyloric part. (3) The stomach is emptied by the formation, between the fundus and the antrum, of a tube along which constrictions pass. The con- tents of the fundus are pressed into the tube, and the tube and antrum are slowly cleared of food by the waves of constriction. (4) The food in the fundus is not moved by peristalsis, and conse- quently it is not mixed with the gastric juice; it can therefore undergo salivary digestion in this region for a considerable period without being disturbed. The food in the pyloric portion is first pushed forward by the running wave, and then by pressure of the stomach wall is returned through the ring of constriction; thus the food is thoroughly mixed with gastric juice and is forced by an oscillating progress to the pylorus. (5) The pylorus does not open at the approach of every wave, but . only at irregular intervals. The arrival of a hard morsel causes the sphincter to close tightly, thus materially interfering with the passage of the already liquified food. (6) Solid food remains in the antrum to be rubbed by the con- strictions until triturated, or to be softened by the gastric juice, or later it may be forced into the intestine in the solid state. (7) The constriction-waves have, therefore, three functions: the mixing, trituration, and expulsion of the food. . (8) At the beginning of the act of vomiting the gastric cavity is separated into two parts by a constriction at the beginning of the antrum; the cardiac portion is relaxed and the spasmodic contrac- tions of the abdominal muscles force the food through the opened cardia into the cesophagus. xiv Proceedings of the American Physiological Soctety. (9) The stomach movements are inhibited whenever the animal shows signs of anxiety, rage, or distress. The full paper will be published in the next number of this Journal. NEW EXPERIMENTS ON THE MAMMALIAN HEART. By W. T. PORTER. I. The recovery of the whole heart from fibrillary contractions ; see this Journal, vol. i, page 71. II. The effect of the beat of the heart upon the flow of blood through the walls of the heart; see this Journal, vol. i, page 145. III. A method for the study of the blood-currents at the root of the aorta. A small cylinder, covered with lead foil, and of the same specific gravity as the blood, is fastened by a very short thread to the end of a probe and passed through the carotid artery and aorta to a position just above the semilunar valves. The movements of the cylinder are those of an equal mass of blood. They may be watched with the Roentgen rays after the removal of the ribs. THE EFFECT OF INANITION ON THE STRUCTURE OF NERVE CELLS. By F. W. BARROWS. THE researches to be described were undertaken in order to find out by what structural alterations, if any, a starved nerve cell may be distinguished from one that is well nourished. In each of three experiments, three rats of the same sex, and similar in weight and general condition, were kept in mechanical cages side by side. Kymograph records gave a continuous history of the activities of each animal during the experiments, together with the temperature and atmospheric pressure for each moment of time. A study of these records shows that fatigue as well as starvation is a strong factor in producing the effects noted. Upon the death of the famished rat, the control rat was weighed and killed. The tissues of the famished and control animals selected for comparison were treated together in the manner described by Dr. Hodge in his work on Fatigue. By this method, the tissues of the normal and famished Tenth Annual Meeting. Xv animals received exactly the same treatment from the moment of dis- section until they were mounted together on the same slide. Micro- scopical comparison and measurement of normal and famished nerve cells from the occipital cortex, spinal ganglia, and cord, shows : — (1) A decided shrinkage in size of the cells and nuclei in the fam- ished animals, averaging about 20 per cent, and a still greater shrink- age in the nucleoli. (2) An evident exhaustion of the substance of famished cells, as shown by their faint staining with osmic acid and the notable ab- sence of nuclei and nucleoli. The protoplasm of these cells shows a very fine vacuolation, not so marked as that described by Rosenbach for starving animals, and by Hodge for extreme fatigue. In the brains of famished rats the pericellular lymph spaces are consider- ably enlarged. THE COMPOSITION AND NUTRITIVE VALUE OF SOME EDIBLE AMERICAN FUNGI. By Larayerre B. MENDEL. See this Journal, vol. i, p. 225. SOME EXPERIMENTS ON THE EXCRETION OF KyYNURENIC ACID. By L. B. MENDEL. DEMONSTRATION: A NEw PLEURAL CANNULA zy srru. By S. J. MELTZER. A description of the cannula will be published in this Journal. DEMONSTRATION: THE NUTRITION OF THE MAMMALIAN HEART THROUGH THE VESSELS OF THEBESIUS. By W. T. PorTer (for F. H. Pratr). See this Journal, vol. i, p. 86. A Basis FoR A THEORY OF CoLor Vision. By W. PatTeEn. INFLUENCE OF ALCOHOL UPON THE YOUNG IN DOGS AND UPON THE SEVERITY OF AN ATTACK OF DISTEMPER. By C. F. HObGE. Read by title. INFLUENCE OF ALCOHOL UPON VOLUNTARY MUSCULAR POWER IN CONDI- TIONS OF FatTicuE. By C. F. Hopce. Read by title. THE INFLUENCE OF BILE AND BILE SALTS ON PANCREATIC PROTEOLYSIS. By R. H. CHITTENDEN. Read by title. This paper will appear in the next issue of this Journal. THE BIoLoGiIcAL PROBLEMS OF To—Day: PuystoLocy. By J. LOEs. oY y ‘ iv Ae reds sy > Pa ae | ee tree + “<.* 5 ¢ 5 en ) a aha? ‘ 2 + = 1 / « ‘ Y iT @ f i iv rod fru Yar i] j v7, ee a ae ae (Ph a eae Nit | * caane® |i iN ; BINDING SECT. MAR4 1966 QP American Journal of Physiology 1 aD Vel COPpec Biologica| & Medica] Serials PLEASE DO NOT REMOVE CARDS OR SLIPS FROM THIS POCKET UNIVERSITY OF TORONTO LIBRARY STOR SB MSV SEHE LY SOV es Yri she See ee Peete en gee oR cate op sp men - oe oF ae ee) Fi : i Bie ee sad ¢ PANS ry 4 APLC Y We 4 gb 4 ee? oat ’ , bog da ie t ryt at ms ee } “eihety seaees f ‘ 4 ek t Mi ty 14:8 4 Kittens 0 Ae . ahs 4, f Sy ties ons at fe Pe 4S tee \ he NEEL Ape Tes hee te te Voy rh bey Ths are.) ay 78 KOR ised eeheat i) a pt te peat ivi