-NRLF. o LIBRARY OF THE UNIVERSITY OF CALIFORNIA. BIOLOGY LIBRARY G RECEIVED BY EXCHANGE Class STUDIES OF THE INFLUENCE OF VARIOUS DIETARY CONDITIONS ON PHYSIO- LOGICAL RESISTANCE. II. THE INFLUENCE OF DIFFERENT PRO- PORTIONS OF PROTEIN IN THE FOOD ON THE PARTITION OF URINARY NITROGEN AFTER DOSAGE WITH POTASSIUM CYANID. BY WllXIAM H. WELKER, A. C., In collaboration with NORMAN E. DITMAN, M.D. DISSERTATION Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Columbia University. NEW YORK CITY 1909 STUDIES OF THE INFLUENCE OF VARIOUS DIETARY CONDITIONS ON PHYSIO- LOGICAL RESISTANCE. II. THE INFLUENCE OF DIFFERENT PRO- PORTIONS OF PROTEIN IN THE FOOD ON THE PARTITION OF URINARY NITROGEN AFTER DOSAGE WITH POTASSIUM CYANID. BY WILLIAM H. WELKER, A. C., In collaboration with NORMAN E. DITMAN, M.D. DISSERTATION Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Columbia University. NEW YORK CITY 1909 BIOLOGY Ltr : PREFACE This dissertation describes experiments which, in point of inauguration, were the second group in a series of studies begun in this laboratory in 1905 by Dr. Gies with Dr. N. B. Foster's co-operation. The results of the first study of the series have lately been published in a preliminary communication by Foster: Proceedings of the Society for Experimental Biology and Medicine, 1909, vi, p. 61. This dissertation contains data that will be included, for the most part, in a paper on certain effects of deficient oxidation, to be published from this laboratory by Ditman and Welker, and which will appear in the New York Medical Journal on or about May 15, 1909- . . ; .it is Djy(pje':ii:ctention to continue the study of the influence physiological resistance. of *vario*us" dietaj-y^ conditions on WILLIAM H. WELKER. Laboratory of Biological Chemistry of Columbia University, College of Physicians and Surgeons, January 15, 1909. Reprinted from the Neiv York Medical Journal for July n, 1908. ON SOME BIOCHEMICAL AND ANATOMICAL CHANGES INDUCED IN DOGS BY POTASSIUM CYANIDE. BY WILLIAM H. WELKER, A. C, AND NORMAN E. DITMAN, M. D., New York, {From the Laboratories of Biological Chemistry and Pathology of Columbia University, at the College of Physicians and Surgeons, New York,) I. Introduction. In connection with an investigation that was lately conducted by us in these laboratories, on diminished oxidation under certain pathological conditions, it was desirable to ascertain the effects of such dimin- ished oxidation on elimination of various nitrogen containing products in the urine. Potassium cyanide, which induces diminished oxidation, was selected for the purpose, and the six experiments described in this paper were accordingly carried out. These experiments were also intended to furnish funda- mental data for investigations of the influence of guanidin, methyl guanidin, creatin, and creatinin under conditions of diminished oxidation. We have carried out preliminary experiments along these lines, and hope to conduct them before long to com- pletion. It had been our intention to carry out several ad- ditional experiments before making our data public, but the recent appearance of a paper on this partic- ular subject, which presented results (of only a few experiments) that are not in harmony with those obtained in this study, induced us to publish at Copyright, 1908, by A. R. Elliott Publishing Company. 218494 Welker and Ditman: Biochemical and Anatomical Changes. once the observations we have made, especially as it will be some time before we shall be able to re- sume the work in this connection. 2. Conduct of the Experiments. Animals and environment. These experiments were conducted on six dogs by the general meta- bolism methods used in this laboratory. 1 Each ani- mal was apparently healthy and normal in all respects at the beginning of the experiment in which it was used. The form of the cage 2 in which the dogs were confined was very well adapted to the collection of the excreta and allowed rapid substitu- tion of urine receivers, when vomiting very closely followed the elimination of urine or vice versa. 8 The cage as well as the room was well ventilated. The animals were thoroughly accustomed to their surroundings and apparently contented before they were subjected to experimental treatment. Food. The daily diet consisted of hashed lean meat, cracker meal, lard, bone ash and water. The raw meat was preserved by refrigeration, according to the usual method in this laboratory. 4 The bone ash was mixed with the food to give the faeces a desirable consistency. 5 Periods, weights. In the records of the experi- ments, each day ended at n a. m. Just before that time the animal was weighed and the weight recorded for the day. At that time also the excreta were removed from the cage and urine receiver. The food was then given. Injection of potassium cyanide. The potassium 1 Mead and Gies: American Journal of Physiology, v, p. 106, 1901; also Gies and Collaborators: Biochemical Researches, i, p. 419 (Reprint No. 21), 1903. 2 Gies: American Journal of Physiology, xiv, p. 403, 1905. 8 Berg and Welker: Journal / Biological Chemistry, i, p. 410, 1906; Welker: American Journal of Physiology, xx, p. 358, 1907. *Gies: American Journal of Physiology , v, p. 235, 1901; Gies and Collaborators: Biochemical Researches, i, p. 69 (Reprint No. i), 1903. 5 Steel and Gies: American Journal of Physiology, xx, p. 343, 1907. Welker and Ditman: Biochemical and Anatomical Changes. cyanide used in these experiments was a sample of Kahlbaum's purest ("K"). Whenever used, the cyanide was rapidly weighed and a I per cent, solu- tion prepared. This solution was protected from direct light when not in use. Only fresh solutions were employed. The injections were made sub- cutaneously in the lumbar region. The animals were in no instance put deeply under the influence of the cyanide by the amounts injected. Analytic methods. No analyses were made of the food materials used in these experiments, but great care was taken to secure a sufficient supply of each of the various ingredients to last throughout an entire experiment. Uniformity of diet from begin- ning to end of each experiment was thus assured. The amount of ammonia in the urine was deter- mined daily in all experiments except the fifth, in which experiment the dog was on a relatively high plans of protein nutrition. In the case of that ani- mal, it was found by experiment that the urine which was preserved with thymol, showed little ten- dency to undergo change of ammonia content even after several days, and ammonia was accordingly determined in combined daily urines. The remain- ing determinations were made on "period urines." These period urines were prepared by combining aliquot fractions of the daily volumes. In the case of the first dog the volumes of the "period urines" were so low that they were diluted with equal quan- tities of water, in order that the usual amounts could be used for the various determinations. The meth- od used for the determination of ammonia in the urine was Folin's. 8 Total nitrogen was determined by the use of the Kjeldahl method. The urine was decomposed by means of concentrated sulphuric acid and small quantities of copper sulphate, a method long in satis- 6 Folin: Zeitschrift fur physiologische Chemie, xxxii, p. 161, 1902. Welker and Ditman: Biochemical and Anatomical Changes. factory use in this laboratory. The digestion was continued for thirty to forty-five minutes after the mixture had assumed a true greenish tint. On cool- ing, in each case the mixture was colorless. The alloxuric nitrogen was determined by pre- liminary precipitation according to the Salkowski 7 method, followed by treatment of the washed pre- cipitate, together with the nitrogen-free filter paper, by the A rn stein 8 method for the removal of ammonia nitrogen, i. e., by adding water and magnesium ox- ide to the paper and precipitate, and boiling the mix- ture. To facilitate matters this was done in a large Kjeldahl flask. After all the ammonia had been driven off, the residual nitrogen was estimated by the usual Kjeldahl method. Urea was determined by the Folin 9 method ; allan- toin by the Loewy 10 method ; uric acid by the Folin- Shaffer 11 method ; creatinin by the Folin 12 method. Summaries of observations and results in the experi- ments with animals on medium planes of pro- tein nutrition (Tables I IV.). Table L First Dog. Preparatory period. The daily diet of this ani- mal consisted of meat, 97 grammes ; 13 cracker meal, 26 grammes ; lard, 20 grammes ; bone ash, 10 grammes ; water, 225 c.c. The initial weight of the animal was 6.5 kilos. The preparatory period con- tinued for seventeen days. 7 Salkowski: PHuger's Archiv, Ixix, p. 268, 1897. 8 Arnstein: Zeitschrift fur physiologische Cheinie, xxiii, p. 417, 1897; Centralblatt fur die medizinischen Wissenschaften, xxxvi, p. 257, 1898. 9 Folin: Zeitschrift fur physiologische Chemie, xxxii, p. 504, 1901; xxxvi, p. 333, 1902; xxxvii, p. 548, 1903. 10 Loewy: Archiv fur experimentelle Pathologic und Pharmakologie. 44, p. 19, 1900. "Folin and Shaffer: Zeitschrift fur physiologische Chemie, xxxii, p. 552, 1901. 12 Folin: Ibidem, xli, p. 223, 1904. 1:5 About 15 grammes per kilo; approximately 0.53 gramme of ni- trogen per kilo. Welker and Ditman: Biochemical and Anatomical Changes. Table I. First Dog. FIRST PERIOD. NORMAL CONDITIONS, JULY 5 TO 13, 1907. 8 5-94 5-94 5-94 5-92 5-92 5-96 5-95 5-93 5-97 170 130 i85 95 170 21 20 22 21 24 28 26 19 26 ..2 53 Acid. Faintly acid. Acid. Amphoteric. Acid. _c c g Slf ft 0.1196 0.0912 0.1582 0.1034 0.0926 0.1165 0.0885 0.1276 ^ H 3*. SECOND PERIOD. EFFECTS OF POTASSIUM CYANIDE (SMALL DOSES), JULY 14 TO 25, 1907. II.... Acid 12 . . . . 5 91 I 6O ll o 0986 20 e og u 14 ... 5-95 ... 5-93 . 5.96 no 90 1 20 28 29 28 Faintly alkaline. Amphoteric 0.1127 0.0924 0.1233 15 26 12 17 !g ... 5-97 e qi 130 20 Acid o.i353 21 2O 19 . . . . . . . 591 s 22 Amphoteric. O.I2I I 10, 20 26 Neutral I e 21. . 5.94 14 ; 20 O.IOI3 II THIRD PERIOD. U FURTHER EFFECTS OF POTASSIUM CYANIDE (RELA- TIVELY LARGE DOSES), JULY 26 TO 29, 1907. 23 40 1 60 Acid 0.0351 18 3 3 24. . 25 . . 90 170 29 Faintlv alkaline. Acid 0.0629 o 1388 8 25 3 "3 "The urine of this period contained a small quantity of the liquid portion of the vomit. 15 Vomited sixteen minutes after injection. A few c. c. of the liquid part of the vomit were unavoidably mixed with some of the urine in the receiver. The solid vomitus was eaten again, shortly afterward when offered to the animal. 16 Vomited twenty minutes after injection. Ate all vomitus later. "Vomited seven minutes after injection. Ate all vomitus later. "Vomited twenty minutes after injection. Ate all vomitus later. Welker and Ditman: Biochemical and Anatomical Changes. Table I. First Dog Continued. FOURTH PERIOD. FURTHER EFFECTS OF POTASSIUM CYANIDE (IN- CREASED DOSES), JULY 30 TO AUGUST 6, 1907. i di 5 ^ C re " I C p .2 oa !s S> 26 27 . . . 5-86 S 81 1 60 I 2O 22 26 Faintly acid. Acid. o.i 1 66 0.0723 13 3.5 19- - 28 S.8e 60 48 0.0940 IO 20 35 2Q S8S I IO tt o. 1 024 20 o- 3c 30 n 5.85 5.85 120 27 c e Alkaline. Faintly alkanie. 0.0906 o. 1 043 18 1 o 32 145 OO 24 Alkaline. o.i 390 25 3 33. . s'.so I SO 20 .Neutral. O.I 023 30 22o"e FIFTH PERIOD. AFTER PERIOD, AUGUST 7 TO 16, 1907. je 3-"0 e 80 126 I ii o 081 5 23 si::::: . 5 80 1 *5 3C ii o 1 534 20 . . 37 ' 5-77 190 jg (i o. 1343 23 . . 38 e 80 26 ii on. . . c 80 I SO 26 . . 40 5.80 no 23 Alkaline. o 0633 22 5 80 42 43 5-73 e 74 115 I2O 29 Amphoteric. Alkaline 0.0862 12 I c "Vomited repeatedly, beginning eighteen minutes after injection, ending fifteen minutes afterward. Ate all vomitus later. 20 Vomited eighteen, and again thirty-four minutes after injection. Ate all vomitus later. 21 Vomited several times thirty minutes after injection. Ate all vomitus shortly afterward. 22 Diarrhcea occurred near the end of the day. Welker and Ditman: Biochemical and Anatomical Changes. *J'C QJ W +- *1 *^ " i O O\ 5 coo N o^ t tx '0-= S fj N M C ft O PL, ^vovo'od o\o\ C . Is EH?S O i- O POOO (-< 2 60006 00000 PM C cj c5 H! ^ U M u >>J 1 Jg i .s" 55 Q 1 vJlTROGl 52 66666 too oo fxin W tc^iococo 2 O M 66666 lj F-* ** 00 M w ^ INARV SOOOOO.K ^ H H O o 5 P 2 to d ^ M \6 55SSII H t.^joj^oioo ?J Cl N' N N 2 00000000000* "? 5 ^ H [j^ CO CO K ( g t> vo rt N to 0\ ^ tOOO txOO ? 0*0 ] j ^ ts -4- n h ~-i S 9. *? ^" StS M M d 6 M o ' " "" " jJJ g ?> f s u M w O M < bo " S NOOVO to N <0 txO ' C"** ""*" B o* ^^ " cT N- CO t N N w ^ "o 8 1". ! N ON CO M p^ s be o ci co co co c >, Ih lll'rl 18,-S !i' : I T3 _C Jlo 1 II 1 rsg> M ^g'>> ^^SEi^ Welker and Ditman: Biochemical and Anatomical Changes. Table III. Second Dog. Preparatory period. The initial weight of this animal was 8.87 kilos. The preparatory period con- tinued for 13 days. The daily diet consisted of the following: Meat, 135 grammes; 23 cracker meal, 36 grammes ; lard, 27 grammes ; bone ash, 10 grammes ; water, 315 c.c. FIRST PERIOD. NORMAL CONDITIONS, OCTOBER 21 TO 29, 1907. At .2 -> $ 000 odd * C g g*g ooo - d d d odd 6^ SI 3 ^>fe OO a aj a O^ 00 ^ ... ... O n. g . 00 OOO OOO O^yOOO .5 *o aJ c E cj S MOOCO w q o > d d d .2S in*-- c P to o N - *i c H -. b - f) i ^; l ~ 1 !^ Welker and Ditman: Biochemical and Anatomical Changes. Discussion of the chemical results of the experi- ments with animals on medium planes of pro- tein nutrition (first and second dogs, Tables I-IV}. The injection of small doses of cyanide in the case of the first dog (Period II, Tables'l and II) caused a decrease in the average daily excretion of total nitrogen in the urine. There was a corresponding decrease in the amounts of all the various forms of nitrogen named in the Tables (I and II), except that of the purin bases, which was increased. The analytic results for the third period (Tables I and II), in which the daily doses of the cyanide were 3 nigs, per kilo, are not uniform enough to tell a definite or concordant story. The period was per- haps comparatively too short for the full registra- tion of the effects that may have been induced by the cyanide. Then, too, contamination of the urine with vomit may have introduced some of the ap- parent uncertainties of significance in the results. In the fourth period, during which the doses of cyanide were 3^ mgs. per kilo, the average daily ex- cretion of total nitrogen was less than that of the first or normal period. This was also true of the various other forms of nitrogen, except the "unde- termined nitrogen," which increased in amount in this period. The daily amount of nitrogen in purin bases, which had risen considerably in the first dos- age period and fallen in the next, was so small as to be practically nil in the third dosage period. If cyanide has a tendency, as claimed by previous observers, to increase nitrogenous elimination, we should expect to find an exhibition of this effect in the fourth period, during which comparatively large doses of cyanide were given, unless the cyanide treatment in the preceding periods had altered the basis for further comparisons. As indicated above, however, decrease rather than increase was the rule, except in the third period. In the after period slight 10 Welker and Ditman: Biochemical and Anatomical Changes. increases in most of the figures in the tables are to be noted. During the period of cyanide injection in the ex- periment on the second dog, the average daily quan- tities of total nitrogen, of the nitrogen of urea, of that of purin bases, and of creatinin, were increased, while the quantities of nitrogen of ammonia, allan- toin, uric acid, and the undetermined fraction were decreased. The average dose of cyanide for this dog was only about 2 mg. per kilo. No vomiting ever oc- curred, and from all outward appearances the effects of the treatment were much less marked than in the case of the first. In spite of this fact, however, the total nitrogen elimination from this dog increased, but from the first dog decreased, with cyanide treat- ment. Histological data First and second dogs. Microscopical examination of the viscera of these dogs showed the following: First dog. Kidney The uriniferous tubules contained a large amount of degenerated cellular matter, which was granular in appearance. The epithelium of the convoluted tubules was slightly swollen and irregular in outline, with a few areas of degeneration. There was slight proliferation of connective tissue along the course of the small bloodvessels. Liver This was the seat of a degenerative pro- cess of irregular intensity in its distribution, with no definite relation to the periphery of the lobule or the central vein. The degeneration was albu- minous in type and moderate in intensity many liver cells being simply the seat of extreme vacuoli- zation. The nuclei were unchanged. Heart Normal. Spleen Normal. Pancreas Normal. n Welker and Ditman: Biochemical and Anatomical Changes. Second dog. Kidney There was moderate al- buminous degeneration of the tubular epithelium, which was most marked in the convoluted tubules. Some contained casts. There were numerous areas of small round cell proliferation along the blood- vessels and about the glomeruli. A few of these areas were infiltrated with pus cells. Liver There was well marked albuminous de- generation, uniform in distribution, with marked vacuolization of the cells. There was no conges- tion. The nuclei were unchanged. Heart Normal. Spleen Normal. Pancreas- Normal. Summaries of observations and results in the ex- periments with animals on low planes of protein nutrition (Tables V-X). Table V. Third Dog. Preparatory Period. Table showing the decreasing quantities of meat fed the animal daily in bringing it gradually down to a low plane of protein nutrition. (Initial daily diet: Meat, 96 grammes; 24 cracker meal, 32 grammes ; lard, 24 grammes ; bone ash, 8 grammes ; water, 280 c.c.) Date. Body weight, kilos. 8.00 Weight of meat, grammes. 96 8.00 96 7.93 96 7.Q2 96 8.OO qi 8.00 86 1 f ' 7-92 81 7.88 76 uly 18 . .. 7 90 71 .... 7-9O 66 7.92 61 7.86 56 uly 22 7.82 25 S6 24 About 12 grammes per kilo; approximately 0.42 gramme of ni- trogen per kilo. ~ >5 About 7 grammes per kilo; approximately 0.25 gramme 01 ni- trogen per kilo. 12 Welker and Ditman: Biochemical and Anatomical Changes. The only ingredient in the diet that was subject to quantitative change during the preparatory period was the meat. Two days previous to the beginning of the fore period and from then on to the end of the experiment, the quantity of meat fed daily was 56 grammes. The other ingredients were as fol- lows : Cracker meal, 32 grammes ; lard, 24 grammes ; bone ash, 8 grammes ; water, 280 c.c. Table VI. Third Dog. FIRST PERIOD. NORMAL CONDITIONS, JULY 23 TO 28, 1907. 4 p 7.81 290 ii Acid. 0.1135 10 2 7.78 260 ii 3 7-74 290 10 4 7-82 195 13 5 7.81 245 TO 6 7-73 340 10 O.I2II 27 O.II9I II O.I 1 22 8 O.0973 22 0.0978 18 SECOND PERIOD. EFFECTS OF POTASSIUM CYANIDE, JULY 29 TO AUGUST 5, 1907- 7 7.67 300 12 Acid. 0.1413 32 8 7-65 303 10 0.2946 .. i 9 7-65 255 10 0.1432 31 1.5 10 7.65 223 ii 0.5027 17 1.5 ii 7.64 295 ii Alkaline. 0.4606 20 2 12 7.65 255 ii 0.2871 23 2.5 13 7-55 290 12 Acid. 0.1986 39 - 6 3 14 7.55 270 ii Alkaline. 0.1960 9 2.5 THIRD PERIOD. AFTER PERIOD, AUGUST 6 TO 13, 1907- 16. . . /-O" 0.1404 20 1 7 ... 7.65 205 o.i 123 IO 18... . . . . 7.63 255 1 1 n O.I222 Alkaline 1 1 02 "?O 20 ... 21 C s O.O5OI 6 21 ... 7.70 290 ii Acid. 0.1803 JO 20 This quanitity of potassium cyanide caused vomiting about one- half hour after injection, also mild diarrhoea, followed by marked prostration. Later the animal appeared to be normal and ate all of the vomitus. 13 Welker and Ditman: Biochemical and Anatomical Changes. ^ ^ 00 S ^ ". T3.C M M o" o^- M co4 M CO vo ^t to 000 SSSSR w. oo odd odd p^ 1> . U sX S5 4 H * s S oTS ovo^ ||*8. I-' U M \O to 00 M M P 5^ ON O ON M fj M O H hr ^ .: 000 ( S v^ o^4 H TO Sf a H? .2 0) 10 M tX C S S|ag =3 gg| 1 JSS| Iff aoS" Ssl 27 The differences between the results of the determinations of the alloxuric nitrogen and the uric acid were so slight as to ^warrant the conclusion that the uric acid nitrogen was the only purin nitro- gen eliminated in quantities sufficiently large for consideration. 14 Welker and Ditman: Biochemical and Anatomical Changes. Table VIII. Fourth Dog. Preparatory Period, October n to November 2, 1907. Table showing the diminishing quantities of meat fed to the animal in bringing it gradually down to the low nitrogen plane. (Initial daily diet: Meat, 1 60 grammes; cracker meal, 40 grammes; lard, 30 grammes; bone ash, 10 grammes; water, 350 c.c.) I 4 I | i j I ii II I li II Q K3 3& tt CSS S S> Oct. ii 9.48 160 Oct. 22 8.76 105 Oct. 12 9.26 28 iss Oct. 23 8.71 29 ioo Oct. 13 9.05 150 Oct. 24 8.64 95 Oct. 14 9.10 145 Oct. 25 8.75 90 Oct. 15 9.06 140 Oct. 26 8.61 85 Oct. 16 8.90 135 Oct. 27 8.58 80 Oct. 17 8.89 130 Oct. 28 75 Oct. 18 8.87 125 Oct. 29 8.53 70 Oct. 19 8.85 120 Oct. 30 8.57 70 Oct. 20 8.73 115 Oct. 31 8.59 70 Oct. 21 8.79 no Nov. i 8.39 70 Nov. 2 8.52 80 7O The meat was the only ingredient that was sub- ject to quantitative change during the preparatory period. At the beginning of the fore period, and throughout the whole experiment thereafter, 70 grammes of meat were given daily with the fol- lowing amounts of the remaining ingredients of the diet : Cracker meal, 40 grammes ; lard, 30 grammes ; bone ash, 10 grammes ; water, 350 c.c. 28 About 17 grammes per kilo; approximately 0.6 gramme of nitrogen per kilo. 29 About 11.5 grammes per kilo; approximately 0.4 gramme of nitrogen per kilo. 30 About 8.25 grammes per kilo; approximately 0.3 gramme of nitrogen per kilo. IS Wclker and Ditman: Biochemical and Anatomical Changes. Table IX. Fourth Dog. FIRST PERIOD. NORMAL CONDITIONS, NOVEMBER 3 TO 7, 1907- I & c i I 1 f ., f. ig| tgljl * > g rt^"? U.2 'C'o '^{.0 *CgS 'Cg-Sj O P5^2 P>cj Pco Prtvs P<5B6 i 8.41 423 ii Acid. 0.2085 19 2 8.52 200 09 Faintly alkaline. 0.0412 28 3 8.48 360 ii Acid. 0.1637 14 4 8.39 394 10 0.1545 19 S 8.33 386 12 0.1945 : 5 SECOND PERIOD. EFFECTS OF POTASSIUM CYANIDE, NOVEMBER 8 TO 13, 1907- 6 8.29 300 ii Acid. 0.1446 29 i 7 8.28 335 10 0.1233 ii i-5 8 8.25 335 ii 0.1360 30 2. 9 8.20 254 07 0.0654 18 31 2.5 10 8.29 311 13 0.1545 IS 32 2 II 8.23 366 II O.I905 20 83 2 THIRD PERIOD. AFTER PERIOD, NOVEMBER 14 TO 20, 1907. 11 .. - 8 21 * 316 00 * o 0987 6 14. . . . 8 13 I 4 15 .... .. 8 16 1 1 o 1228 p 16 .. 8.14 360 IO o 1400 1 1 17. ., .. 8.16 285 06 Faintly acid. 0.1376 36 T8 , 8.IQ ^86 OS Acid. 0.1267 IS Discussion of the chemical results of the experi- ments with animals on low planes of protein nutrition (third and fourth dogs, Tables V-X). The average daily total urinary elimination of nitrogen in these two experiments failed to show a concordant tendency. In the case of the third dog (Tables VI-VII) there was a slight increase dur- ing the cyanide period, while, on the other hand, there was a decrease in the case of the fourth dog (Table IX-X) during the cyanide period. The 81 Vomited repeatedly during the first half hour after injection. Refused all offers of vomitus, but ate the same with the regular meal on the following day. 32 Appetite diminished. Vomited within twenty minutes after in- jection. Ate all of vomitus later. 33 Vomited about fifteen minutes after injection. Ate the vomitus with the meal on the following day. 16 Welker and Ditman: Biochemical and Anatomical Changes. ; rrt 1 8 i-i N o o vooo . " co N r-s 5 Tg S m invq "NO ? t ^ J.cSdd 900 (I, w coo\co , " o q q odd o o o q q o odd rtoM-iN mmri- -.gNCOCo OOO K ^-4-5oOOO .Sjjqqq q q q Sw 150 } }" ^000 odd oPHoodd S S* .sfs 56 ^^S bob -^ N - < M gno\mtx "S^n moo H . ^j M o\ w H ^rtNcoin qqq r2Sc WM 52 !?oflddd >ddd ^ fU g ej < S o 3 S"e lg^ Q ^1? H Ci J- 3 i-sS 1 ^^ 1 ? *?". w . *-> a N com w M N 6 |3 odd d 6 6 rt3K ON \VO\ III Isl ss Welker and Ditman: Biochemical and Anatomical Changes. nitrogen of ammonia was markedly increased in the case of the third dog during the cyanide period (Tables VI-VII), but was diminished slightly in the experiment on the fourth dog (Tables IX-X). The average daily elimination of urea was very slightly increased during the cyanide period in the experiment on the third dog, but in terms of per cent, of the total nitrogen it decreased markedly. During the same period in the experiment on the fourth dog, however, the average daily amount of urea nitrogen, as well as its per cent, of the total nitrogen, decreased decidedly. The other notable result of these two experiments was the sharp in- crease in the average daily amount of the undeter- mined nitrogen in each experiment. The remaining results of these two experiments are diametrically opposed to each other. Histological data Third and fourth dogs. The microscopical examination of the viscera of these dogs showed the following: Third dog. Kidney There was congestion of the cortical area and slight swelling and irregu- larity of outline of the cells of the convoluted tubules. Liver This was the seat of a diffuse degenera- tive process, uniform in intensity in all parts of the liver lobules. The nuclei were intact, but the cyto- plasm was the seat of an extreme albuminous de- generation, which was so excessive that only a few areas of granular cytoplasm remained and the out- lines only of the cells persisted. There were ir- regular areas of congestion. Heart Normal. Spleen Normal. Pancreas Normal. Fourth dog. Kidney There was slight swelling of the epithelial cells of the cortical tubules, with irregularity of outline. There was a little exudate, Scattered irregularly through the cortex were areas of small round cell infiltration. Most of these 18 Welker and Ditman: Biochemical and Anatomical Changes. areas contained a number of pus cells. They oc- curred along the course of the bloodvessels, but the glomeruli were not involved. Liver There was well marked albuminous de- generation, uniform in distribution, with marked vacuolization of the cells. There was no congestion. The nuclei were normal. Heart Normal. Spleen Normal. Pancreas Normal. Summaries of observations and results in the ex- periments with animals on high planes of pro- tein nutrition (Tables XI-XVI}. Table XL Fifth Dog. Preparatory period. Table showing the increas- ing quantities of meat fed the animal in raising it to the high nitrogen plane. (The initial diet of this animal consisted of meat, 1 20 grammes; cracker meal, 32 grammes; lard, 24 grammes; bone ash, 10 grammes; water, 280 c.c.) Body weight, Weight of meat, Date. kilos. grammes. July July July I:::::::::::::::: 8.25 8.21 8.08 3*120 140 160 uly 8 7-92 1 80 uly 9 7.85 200 uly 10 7-92 220 uly it 7-90 240 uly 12 7-94 240 ulv 13 8.00 2 4 uly 14 8.00 240 ulv 15 8.02 260 July Tuly 16 17 8.03 8:09 280 300 July 18 8.13 320 luly 19 8.17 340 luly 20 8.22 360 July July 21 22 8.31 8.37 380 3 380 The meat in the daily diet was increased twenty grammes each day, excepting on July I2th, I3th, 34 About 15 grammes per kilo; approximately 0.53 gramme of ni- trogen per kilo. SH About 30 grammes per kilo; approximately 1.05 gramme of ni- trogen per kilo. 38 About 45 grammes per kilo; approximately 1.58 gramme of ni- trogen per kilo. 19 Welker and Ditman: Biochemical and Anatomical Changes. and I4th, ending with July 2ist, when the daily amount was 380 grammes. The other ingredients remained constant in quantity throughout the entire experiment. Table XII. Fifth Dog. FIRST PERIOD. NORMAL CONDITIONS, JULY 23 TO 29, 1907. M!S 8.37 8.40 8.50 8.48 8-53 8.58 8.63 (. lid 418 450 410 400 440 Faintly acid. SECOND PERIOD. EFFECTS OF POTASSIUM CYANIDE, JULY 30 TO AUGUST 2, 1907. 8" 8.65 510 22 Neutral 53 s "i 9 8.77 390 24 Amphoteric 43 89 o io 37 8.70 475 22 " 44 4 i ii 8.63 165 35 Acid o 41 o THIRD PERIOD. RECOVERY FROM EFFECTS OF POTASSIUM CYANIDE, AUGUST 3 TO 7, 1907. 44 8 65 9 2 43 JO 36 Acid Alkaline 68 14 . . 8 72 ov 26 \cid 8 80 3OO 45 ' 16 8 82 C70 40 87 The small doses of cyanide injected on the eighth and tenth days (July 3oth and August ist) set up violent disturbances (ap- parently because of this high protein diet), so that it was impossible to continue the cyanide treatment without decreasing the protein in the food. The daily quantity of meat was accordingly lowered to 300 grammes (about 34 grammes per kilo; approximately 1.19 gramme of nitrogen per kilo), on the seventeenth day (August 8th), and this amount was fed daily thereafter to the end of the experi- ment. ^No apparent effects. 89 Vomiting occurred some time after feeding. All of the vomitus was eaten later. *No apparent effects. ^Vomiting occurred: all offers of same were refused. ^Vomited during the night. No vomit in the urine. 43 Urine contained fluid vomit. ^Diminished appetite, mild diarrhoea. 45 Vomited after feeding. Urine contained fluid vomit. Ate solid vomitus later. ^Vomited. Urine contained trace of fluid vomit. 20 Welker and Ditman: Biochemical and Anatomical Changes. FOURTH PERIOD. PREPARATORY PERIOD WITH REDUCED AMOUNT OF MEAT IN THE DAILY DIET, AUGUST 8 TO 16, 1907. I 17 18 19 20 21 22 23 24 25 ' 8.75 8.79 8.85 8.88 8.85 8.88 8.90 8.89 8.88 420 320 410 432 475 390 380 445 465 23 30 19 23 18 25 22 22 23 Amphoteric Faintly acid 40 51 48 40 51 37 57 37 37 FIFTH PERIOD. EFFECTS OF POTASSIUM CYANIDE, AUGUST 17 TO 18, 1907. 8.73 345 21 Alkaline 22 47 o.s 8.88 440 21 60 o 26 27 SIXTH PERIOD. NORMAL CONDITIONS, AUGUST 19 TO 23, 1907. 28 ........ 8.89 460 21 Faintly acid 57 29 30 31 32 8.90 8.90 8.93 8.96 450 445 455 430 Amphoteric Faintly acid Amphoteric SEVENTH PERIOD. EFFECTS OF POTASSIUM CYANIDE IN RELATIVELY SMALL DOSES, AUGUST 24 TO 30, 1907. 33 34 35 36 37 38 39 8.92 9-00 8.92 8.94 8.94 9.00 8.94 485 490 525 455 320 555 495 Alkaline Amphoteric Acid Faintly acid Acid Faintly acid Faintly acid 0.5 0.5 -5 i 1.5 EIGHTH PERIOD. EFFECTS OF POTASSIUM CYANIDE IN INCREASED DOSES, AUGUST 31 TO SEPTEMBER 4, igo?. 48 40 8.97 415 22 Acid 24 49 2 41 8.98 510 21 35 2 42 8.97 500 23 46 43 8.97 475 20 40 44 9-03 S 2 21 50 2 47 yomited within the hour following injection. Some of the fluid vomitus ran into the urine. The treatment was discontinued and a new normal period inaugurated on the twenty-eighth day. 46 No analytical work was done on the urine of this period because of the contamination of the urine with fluid vomit. 49 Vomited during the night. Urine contained fluid vomit. 21 Welker and Ditman: Biochemical and Anatomical Changes. NINTH PERIOD. NORMAL CONDITIONS, SEPTEMBER 5 TO 10, 1907. 4? > 9 t s 8 I B -f. - .1- '-i 8 & is !!> o 111 i| Itl >c/3 f >0i;s fe bo P- &S 45 9.10 345 26 Acid 40 46 9.04 502 21 Faintly alkaline 55 47 9.06 505 21 Faintly acid o 48 9.10 500 22 Acid 23 49 9.11 435 20 Acid 56 50 9.10 485 20 Amphoteric 53 TENTH PERIOD. EFFECTS OF POTASSIUM CYANIDE IN MODERATE DOSES, SEPTEMBER n TO 15, 1907. 51 9.20 430 20 Faintly acid 33 2 52 9.20 410 24 Amphoteric 64 2 53 9.21 393 25 Faintly alkaline 40 2 54 9.19 513 22 Amphoteric 48 2 55 9-21 475 23 33 2 ELEVENTH PERIOD. AFTER PERIOD, SEPTEMBER 16 TO 20, 1907. 56 9.18 485 22 Acid 52 57 9-20 455 24 35 58 9-25 420 24 29 59 9-20 530 21 30 60 9.20 470 21 42 22 Welker and Ditman: Biochemical and Anatomical Changes. w w 3 <* ^"g * o -too -< j-x gj| Mn6 ooooo p^ocococooco ^ S S- ^ 1 vo^^^vg w J g ^ 1 N . 1 1 Jj jj, O O O O O u 5 ^ 1 s IS c Tf ci vo o o\ 'O Os^O VO M* O C VO OO 10 f5\O S*^ o rt o o o q o '"- 60 ooooo w c o'oo'o'o' odd do oP- g 1 TlTROGEI NI^O\0 H .'VOOiH OSOO 1 <^ ^^ ddddd > ddddd HfLiudddi-id C/} & ^ a a 2 4) tx txOO >-" O ^ 10 wOOvO N ,jr g M O\ * <*5 1-1 C^ ** S P N ^ * t N C| ti ^ * ""** aw osoo os g^S ooso s ^ M bj ^r- # f^* T w iv^ ^ S^SSo -TJ-^M^VO ^ < M dddo'd P^o co io to to co >< ^ O N VO C^ C^ rfowvovo ^J ^ _T c r) IH cc f^) dad o\d E-H M T *' c >> h >> >. X >. o *rt o o3 i> S ^(UCjlUu "rtiyWOW o ^ wo S Grto^O Cj^OcSO OOw OO WO i-J rs'x'x'd s*x* Welker and Ditman: Biochemical and Anatomical Changes. Table XIV. Sixth Dog. Preparatory period. Table showing the increasing quantities of meat fed the animal daily in raising it gradually to a high plane of protein nutrition. (The initial daily diet was made up as follows: Meat, 1 08 grammes 60 ; cracker meal, 36 grammes; lard, 27 grammes; bone ash, 9 grammes; water, 315 c.c.) S * > 4- S i iJ fl s & |i Q M^ > W Q MS M Oct. 12 8.10 B1 i28 Oct. 19 7.95 268 Oct. 13,., 8.09 148 Oct. 20 8.04 B3 288 Oct. 14. 8.00 168 Oct. 21 8.00 308 Oct. 15 8.06 188 Oct. 22 8.00 328 Oct. 1 6 8.00 208 Oct. 23 8.07 348 Oct. 17 8.00 228 Oct. 24 8.10 M 348 Oct. 18 8.00 B2 248 The daily portion of meat was increased 20 grammes per day ending with October 23d, on which day the weight of meat in the diet was 348 grammes. This weight of meat was fed daily, to- gether with the weights of the rest of the ingre- dients as specified above, to the end of the experi- ment. The animal was fed this diet from October 23d to November 2d (eleven days), before the actual observation of normal conditions was com- menced. 50 About 13 grammes per kilo; approximately 0.46 gramme of ni- trogen per kilo. 51 About 1 6 grammes per kilo; approximately 0.56 gramme of ni- trogen per kilo. 62 On this day the bone ash was increased to 15 grammes. All the other ingredients except the meat remained constant. 53 About 32 grammes per kilo; approximately 1.12 gramme of ni- trogen per kilo. 64 About 43 grammes per kilo; approximately 1.51 gramme of ni- trogen per kilo. 24 Welker and Ditman: Biochemical and Anatomical Changes. Table XV. Sixth Dog. FIRST PERIOD. NORMAL CONDITIONS, NOVEMBER 3 TO 7, 1907. 1 3 * * M I -4 r w J . Jgu ^.tol is II" IS 111 |||i il?t pqS S>d S~ D q q odd 1-1 N o q odd o ft o odd x! si 5 M O o ft II c H o * o o tx in w in m en S it- M tx i( N VO * cs m S w ti ^ N c o ^ ^ rt tx tx O W HI >-< W r ^ d d d <; t- 1 PH g KH' ^ M > M > EH w. < j-j < 2 s S HI < 0\ 0\m Q o oo w VO K*^ s a ^* ON CO ^ CO CO \ E 2 cj d d CM y N COCO >< < M ^ CO "s :x ! CO txO VO x3 *" ts * * " COOO O 4J 3 M3 O\ O\ tS* q qoo rt- CO . . . tT 1 U 00 XvS Jx d o c fel > .2 3 a 11 1& ; l&? c rt o M fiB J rt tS ||| ^Q^S O LJ S 52 HH 26 Welker and Ditman: Biochemical and Anatomical Changes. In the experiment on the fifth dog, the urea ni- trogen fell slightly during the period (VII) in which relatively small doses of cyanide were inject- ed, and also during the period in which compara- tively moderate doses of cyanide (X) were admin- istered. On the basis of its proportion of the total nitrogen, the urea nitrogen in the urine of the fifth dog fell slightly in the first of the two periods re- ferred to, but rose in the latter period. During the cyanide period of the experiment on the sixth dog (Tables XV-XVI) the urea nitrogen rose in quantity. The allantoin nitrogen was diminished after ad- ministration of comparatively small doses of cyan- ide in the fifth dog, but was increased after mod- erate doses in the same animal. The allantoin ni- trogen was diminished during the cyanide period of the experiment on the sixth dog. In the experiment on the fifth dog the uric acid nitrogen was fairly constant throughout, but in the case of the sixth dog the eliminated amount in the cyanide period was larger than that of the first period, while the excreted quantity in the after period was greater than the combined amounts of uric acid nitrogen in the fore period and the cyan- ide period. In both experiments, creatinin nitrogen appeared to be diminished by the cyanide treatment. The "undetermined nitrogen" was but little af- fected by small doses of cyanide in the fifth dog, but was decreased markedly in the same animal after treatment with moderate doses of cyanide. The same decrease in undetermined nitrogen ap- peared after the cyanide treatment of the sixth dog. Histological data Fifth and sixth dogs. The microscopical examination of the viscera of these dogs showed the. following: Fifth dog. Kidney There was marked conges- 27 Welker and Ditman: Biochemical and Anatomical Changes. tion, with well marked albuminous degeneration of the epithelium of the convoluted tubules. The epithelium was swollen and in places disintegrating. The lumina of the tubules contained a large amount of granular matter. The nuclei were intact. Some glomeruli were much shrunken and were composed almost entirely of connective tissue cells. In the neighborhood of some glomeruli and along the course of some blood vessels were large areas, rather sharply circumscribed, of small round cell proliferation. Liver. This was the seat of mild parenchymatous degeneration, slightly more marked about the cen- tral vein than in the periphery of the lobule. The degeneration, which was albuminous in nature, was slight and accompanied by very moderate vacuoli- zation of the cells. The congestion was not marked. Heart Normal. Spleen Normal. Pancreas Normal. Sixth dog. Kidney The cortex was much con- gested and the tubular epithelium was the seat of marked albuminous degeneration, which was most evident in the convoluted tubules. The cells were greatly swollen and granular, and irregular in out- line. The lumina contained granular matter. No distinct proliferation of connective tissue could be made out. The glomeruli were normal. Liver There was mild albuminous degeneration of the liver cells, slightly more marked about the central vein than in the periphery of the lobule. There was very slight vacuolization of the cells. There was no congestion. Heart Normal. Spleen Normal. Pancreas Normal. Historical. The first investigation of the influence of potas- sium cyanide upon nitrogenous metabolism was made by Loewy." In a preliminary communica- "Loewy: Centralblatt fur Physiologic, xix, p. 856, 1906. 28 Welker and Ditman: Biochemical and Anatomical Changes. tion he stated that on the day of poisoning, and on the day after, the nitrogen elimination was marked- ly increased. The output of amino-acid nitrogen also seemed to be decidedly increased. In a second paper on this subject, Loewy 86 stated that the total quantity of excreted nitrogen did not increase in all cases. He concluded that the plane of nutrition has some relation to the manner in which cyanide affects nitrogenous metabolism. That conclusion is in accord with the prior results obtained by Frankel and Geppert 57 in their re- searches on diminished oxygen supply. They es- tablished a similar relation between increased ni- trogen elimination arid the plane of nutrition. The increase of excreted nitrogen was always least in fasting animals, and invariably appeared later. As the first series of experiments in this study were nearing completion, we were informed that a similar investigation was being conducted by Richards and Wallace. 58 Their published results do not show any marked difference in the effects of cyanide on two dogs, on different planes of nu- trition. Richards and Wallace observed an in- crease in the total of excreted nitrogen and urea nitrogen, with no increase of ammonia nitrogen in the urine. Creatinin was markedly diminished in the urine on the days of severe poisoning. The un- determined nitrogen increased in the urine on the days of severe poisoning. Conclusions. Potassium cyanide, injected subcutaneously in gradually increasing daily amounts from one half to three and a half milligrammes per kilo, through periods ranging from four to twelve days, failed to cause any marked increase in the total elimination of nitrogen in the urine. In six out of the nine 58 Loewy: Biochemische Zeitschrift, iii, p. 439, 1907. "Frankel and Geppert. Cited by Loewy, loc. sit. ^Richards and Wallace: Journal of Biological Chemistry, iv, p. 179, 1908. 2Q Welker and Ditman: Biochemical and Anatomical Changes. cyanide periods, however, the total amount of ni- trogen excreted in the urine was actually dimin- ished. In the case of the three other periods the increase observed was very slight. Possibly the urinary excretion of nitrogen was not materially increased in these experiments, after the cyanide injections, because the doses were too small to produce convulsions or to cause the asso- ciated conditions which may markedly augment catabolism, and which may have been largely re- sponsible for the increased elimination of nitrogen that was observed by some of our predecessors in such studies. The plane of protein nutrition seems to influ- ence the degree to which subcutaneous injections of cyanide affect the elimination of some of the forms of nitrogen in the urine. A comparison of the re- sults for urea and undetermined nitrogen in the urines of the animals on high and low planes of protein nutrition makes it evident that the percent- age of urea nitrogen increased and that of the undetermined products decreased in the urines from the dogs on high protein nutrition planes, whereas the reverse in each case was noted for the urines from the dogs on low protein nutrition planes. Further work will be necessary before the open questions that remain can be answered with assur- ance. We wish to express our most heartfelt thanks to Professor William J. Gies for his kind advice and guidance in the conduct of these experiments. BIOGRAPHICAL William Henry Welker received his early education in the public schools and prepared for college at Perkiomen Seminary and Ursinus College. He taught public school at Klinesville, Pa., during the terms of 1898-99 and 1899-1900. He graduated from Perkiomen Seminary in the spring of 1900, entered Lehigh University in the fall of 1900, and graduated from Lehigh in 1904, receiving the degree of Analytical Chemist. He was undergradu- ate instructor in general chemistr} 7 at Lehigh in 1902-03 and 1903- 04, and undergraduate instructor in physics in 1903-04. He was appointed Assistant in Chemistry at Lehigh University at the time of his graduation in 1904, but resigned during the summer of 1904 to accept the position of Assistant in Physiological Chemistry in Columbia University, at the College of Physicians and Surgeons. He served in this capacity until Dec. i, 1906, when, in addition, he became Pathological Assistant in Obstetrics at the College of Physicians and Surgeons. Recently he resigned both these positions to accept that of Demonstrator of Physiological Chemistry at the University of Pennsylvania. PUBLICATIONS Experiments to determine the influence of the bromides of barium and radium on protein metabolism. (With William N. Berg). Journal of Biological Chemistry, 1906, i, p. 371. A contribution to our knowledge of the effects of urinary pre- servatives on urinary analysis. New York Medical Journal, 1907 (September 21), p. 552. A simple electrical annunciator for use in metabolism experi- ments and in connection with filtration, distillation and similar operations. American Journal of Physiology, 1907, xx, p. 358. On some biochemical and anatomical changes induced in dogs by potassium cyanid. (With Norman E. Ditman). New York Medical Journal, 1908 (July n), p. 59. UNTVEESITY OF CALIFOENIA LIBEAEY, BEEKELEY THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW Books not returned on time are subject to a fine of 50c per volume after the third day overdue, increasing to $1.00 per volume after the sixth day. 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