disappeared, whereas if the carbohydrate were replaced by fat instead

THE INFLUENCE OF CARBOHYDRATE AND FAT ON PROTEIN METABOLISM. IIL-THE EFFECT OF PHLORIDZIN GLYCOSURIA1. BY E. P. CATHCART AND M. ROSS TAYLOR. University of Glasgow. ONE of us (E. P. C.) has brought forward a certain amount of evidence (1) to show that the appearance of creatine in the urine is more or less intimately connected with disturbances in the metabolism of carbohydrates in the body or at any rate with the presence or absence of these substances in the food. It was demonstrated that on a carbohydrate diet the creatine in the urine, present as the result of starvation, disappeared, whereas if the carbohydrate were replaced by fat instead of the disappearance there was a rise in the output of creatine. These results naturally raised the following questions. When the body is deprived of its stored carbohydrate (1) is there an immediate appearance of creatine in the urine, and (2) if so, is this output of creatine associated with an increase in the output of total nitrogen? The condition to be investigated was thus one in which the carbohydrate metabolism for some reason, artificial or other, was defective, for example some form of glycosuria. Diabetes mellitus was of no value from the experimental standpoint as (1) the glycosuria cannot be controlled at all or only with difficulty, (2) it is very difficult to control the diet of the diabetic patient both in quality and quantity of food taken, and (3) there is always some uncertainty as the true nature of the disease is so little known-whether there is only a fault of the carbohydrate metabolism or, what is much more likely in advanced cases at least, that all three metabolisms, protein, fat and carbohydrate, are affected. One of us (M. R. T.) has investigated the output of creatine in diabetes mellittis and found that this substance is constantly present in the urine2. Phloridzin glycosuria on the other hand seemed to offer us an ideal condition for our investigation-a simple deprivation of sugar 1 A preliminary communication was made to the Physiological Society, March, 1910. 2 Paper read before the Brit. Med. Assoc., London, July 28th, 1910.

PROTEIN METABOLISM. 277 withouit any faulty utilisation of the carbohydrate and without serious disturbance of the general metabolism(2). (This latter statement is however only true if the supply of carbohydrate, either stored in the body or present in the food, be in sufficient amount.) The majority of our experiments were carried out on one dog-a collie bitch weighing about 15 kilos. Between the experiments the animal had a rest of several weeks during which it was fed on an ordinary diet and had sufficient exercise. Some of the results were confirmed on another animal-a retriever bitch of about the same weight. The two sets of results were always in fair agreement. The products estimated were total nitrogen (by the Kjeldahl method), preformed and total creatinine (by the Folin method) and sugar (by the Pavy-Fehling method). The food given to the dogs during the experimental periods was creatine and creatinine free. It will be noted that as the result of the fast, just as is the case with man, creatine appears in the urine accompanied by apparently some rise in the output of total initrogen. On the diet given the dog lost weight although there was no appearance of creatine in the urine until glycosuria was induced. The output of sugar was also accompanied by a slight rise in the output of total nitrogen, indicating perhaps that there Exp. I. The diet consisted of oatmeal 75 grms., milk 250 e.c., and water 500 c.c. The initial weight of the animal was 15 05 kilos, and the end weight 13 75 kilos. Previous to the experiment the animal was fasted for one day. Day in c.c. in grms. in grms. in gruis. in grms. Remarks 1 1000 567 *409 *015 After fast 2 750 3'2 '319 '000-3 700 3'0 '305 '000 4 925 3-3 *339 '044 15'8 *75 grm. 5 1050 4'2 336 *024 1'08 6 870 4'8 *325 '000-7 1010 2'9. '337 '000 8 850 2'2 '260 '000 - was not a sufficiency of free carbohydrate and that some of the combined carbohydrate had beeln drawn upon. The observation of Falta, Grote and Staehelin (3) supports such a view. These observers found, using a dog from which the pancreas had been removed, that the glycosuria was accompanied by an increase in the output of total nitrogen. They came to the conclusion that this increase was due to the demand for 18-3

278 E. P. CATHCART AND M. ROSS TAYLOR. carbohydrate being so great that all the direct supplies having been utilised it must now be obtained indirectly, i.e. from the protein. In our experiment with the disappearance of the sugar from the urine the creatine also disappeared, the nitrogen output however still showed a slight increase. ExP. II. The diet was increased so that it now consisted of oatmeal 100 grms., milk 500 c.c., and water 700 c.c. The initial weight of the animal was 15'1 kilos, and the end weight 13'5 kilos. Day in c.c. in grms. in grms. in grms. in grms. Remarks 1 470 5'4 '482 '003 - After fast 2 950 3'9 '391 000-3 770 3-8 *398 000-4 800 3'7 *380 000-5 800 3'2 339 '000-6 1150 4'9 *458 '030 16'1 '75 grm. 7 860 2'9 *327 '000-8 550 3-2 304 '000 Here again as the restult of the injection of there was an outpuit of creatine which only lasted so long as the glycosuria persisted. There was perhaps also a very slight rise in the output of total nitrogen which may have been due to the demand for carbohydrate. The increased output may however in part depend on the diuresis. In order if possible to prevent this increase in output of total nitrogen, supposing the nitrogen is excreted solely as the result of the call for carbohydrate and not as the result of a possible direct toxic action of the, a third experiment, with a big increase of the carbohydrate intake, was carried out. Exp. III. The diet consisted of oatmeal 100 grms., tapioca 100 grms., milk 500 c.c., and water 700 c.c. The initial weight of the animal was 13'43 kilos, and the end weight 14 kilos. Day in c.c. in grms. in grms. in grms. in grms. Remarks 1 355 4'9 '374 nil 2 1700 5'9 '467 - - 3 1400 3'8 '371 4 1350 3'1 '371 5 1700 3-7 '482 27'6 '75 grm. 6 1400 3'5 '356 16'4 7 1550 5'0 '438 8 1200 3-3 '302-9 1400 3'7 '352 - -

PROTEIN METABOLISM. 279 Here it will be observed that as the result of the large intake of carbohydrate there was no rise, or at most a very slight one, in the excretion of total nitrogen. Again there was a very marked output of sugar but without any output of creatine. Therefore it may be concluded that the increase in the output of total nitrogen in the previous experinments was not due to any toxic action of the itself but was the result of the demand for sugar from some protein source. Another experiment identical with this one on another dog gave exactly the same result. These experiments conclusively show that (1) if the supply of carbohydrate be deficient in the organism creatine appears in the urine, and (2) if the supply of carbohydrate be abundant, in spite of glycosuria, creatine is not excreted. That the excretion of creatine is directly related in some fashion to the utilisation of carbohydrate is more than suggested by the observation that creatine is only excreted, in those experiments where the supply of carbohydrate in the food is not great, so long as sugar is being excreted. Our observations on the output of creatine as the result of injection of have been confirmed by Krause and Cramer (4) (working independently). The question as to whether the carbohydrate could be replaced by fat was now dealt with. We thought that by similar experiments to the above we might obtain evidence which would support the hypothesis of Chauveau that fat before utilisation is converted into sugar. The results which we have obtained up to the present do not help much towards the elucidation of this problem. Probably the explanation lies in the fact that the glycosuric episode is of much too short duration, i.e. the animal has not time to adapt its metabolism before the demand is over. Some later experiments along another line of investigation have yielded more hopeful results. Exp. IV. The diet was made up as follows: oatmeal 100 grms., lard 100 grms., milk 500 c.c., water 250 c.c. The initial weight of the animal was 15-87 kilos, and the end weight 15-80 kilos. Day in c.c. in grms. in grms. in gruis. in grms. Remarks 1 700 2-9 *265 nil nil 2 1000 4-6 -393 - - 3 700 3-8 '376 - - 4 500 2-6 *318 - - 5 900 3-2 *448 *028 12-6 *75 grm. 6 500 2-1 '309 *028 2-5 7 800 3 0 *468 8 400 1-5 *274-9 300 1'2 *204 -

280 E. P. CATHCART AND M. ROSS TAYLOR. This experiment was an exact duplicate of Exp. III. except that the 100 grms. of tapioca were replaced by 100 grms. of lard, and therefore as a result the caloric value of the diet was increased by about one-half The result however was not the same. The glycosuiria was much less intense and it was accompanied by quite a marked excretion of creatine. As regards the output of total nitrogen it is difficult to decide whether it was affected or not as there were marked fluctuations in the output of. the urine which of course of itself might quite easily affect the nitrogen output. As it was possible, although hardly probable, that a still larger intake of fat might lead to results similar to tllose obtained with the carbohydrate another experiment was carried out. It will be noted that it was soon evident that the amouint of carbohydrate present in the diet was insufficient for the mnetabolic needs even before the glycosuria was induced. Tlais is of course quite in keeping with the results obtained by one of us (E. P. C.) on mian (I.c.). There was curiously enough as the result of the injection no rise in the output of nitrogen (further evidence that the previous increases were not due to the toxic action of the ) and no very definite increase in the output of creatine. The glycosuria was quite well marked. There was apparently little or no acidosis as the result of the fat feeding as evidenced by the absence of acetone fiom the uirine. Krause and Cramer (I.c.) also observed this freedom from acidosis. Exp. V. The diet consisted of oatmeal 50 grms., lard 150 grms., milk 500 c.c., water 250 c.c. The initial weight was 16-85 kilos, and the end weight 16-25 kilos. pased nitrogen creatinine Creatine Sugar Day in c.c. in grms. in grms. in grms. in grms. Remarks 1 600 2'9 *415 *000 - No acetonne 2 500 2'5 '366 '021-3?.54 0/0 *081 0/ '007 /0 - Part of urine lost 4 600 3.3 '528 *036-5 800 3'0 *568 *036 21'6 *75 grm. 6 300 2'4 '247 '011 7'8-7 800 3'4 '487.035 - - 8 650 2'2 '436 *020 In the next experiment it was decided to increase the amount of carbohydrate present id the diet to discover, if possible, how much carbohydrate was necessary to prevent the output of creatine before the glycosuria was induced. Exp. IV. had shown that apparently 100 grms. of oatmeal were sufficient, and here 75 grms. were tried.

PROTEIN METABOLISM. 281 Exp. VI. The diet was made up as follows: oatmeal 75 grms., lard 150 grms., milk 500 c.c., water 250 c.c. The initial weight of the animal was 16 kilos, and the end weight 17 kilos. Day in c.c. in grms. in grms. in grmis. in grms. Remarks 1 500 4-5 '284 *031 nil No acetone 2 500 3'0 307 '025 - - 3 400 2'8 *396 '067 - - 4 300 1'7 *297 *024 - - 5 900 3'6. '611 '104 16'2 *75 grm. 6 400 2'5 '404 '038 6o0-7 500 2-8 *436 '031 - - 8 600 3'8 '406 '032 -- Here, as in Exp. V., creatine was steadily excreted before the glycosuria was induced, in other words 75 gryis. of carbohydrate proved to be insufficient. With the induction of the glycosuria however not only was there a rise in the output of nitrogen but there was also a very decided transitory rise in the output of,reatine. Again, if acetone is to be regarded as an index of acidosis none was present as the acetone tests were consistently negative. Further it will be noted that here there was quite a definite rise in body-weight. Evidently although the fat could be stored it could not minister to the metabolic needs in the same way as carbohydrate. It would appear then that fat in the food, even in considerable amount, does not prevent protein catabolism, in other words under the conditions and within the period of the present experiments fat cannot replace carbohydrates. It is not a question of the caloric needs of the animal being unsatisfied as the intake of calories on the fat diets was farhigher than on the carbohydrate and much in excess of the animal's own requirements. It may be that fat can replace a certain amount of the carbohydrate provided of course that there still be a sufficiency of carbohydrate in the diet to carry on certain essential metabolic processes, one of which, according to RoseTnfeld, is the proper combustion of fats. It is evident that the carbohydrates possess like the proteins quite a wellmarked specific action. In the remarks on the various experiments the output of creatinine was not touched on. If, however, the various tables be studied it will be observed that in every case along with the output or rise in the output of creatine there is a corresponding rise in the output of creatinine sometimes only slight but for the most part marked. This observation lends furt'her support to the view that these two substances are, as has been

282 E. P. CATHCART AND M. ROSS TAYLOR. contended by many but denied by others, intimately connected. The rise in the output of creatinine takes place not only in actual amount but also, in certain experiments, as the following table demonstrates, in percentage amount. An examination of the percentage output of creatinine nitrogen does not yield a great deal except that there appears to be on the whole some slight connection between the rise in the output of total nitrogen and the rise in the output of creatinine. Perecntage of total nitrogen. Exp. Creatinine nitrogen Creatine nitrogen Exp. Creatinine nitrogen Creatine nitrogen I 3-76 IV 4-5 3-7 *5 5-2 *3 30 *16 5-4 *6 2a5 5'757 II 39 - V 5 9 *4 3-4.3 7-0 *46 4-1 - 3-8.2 4-2.4 III 4*4 - VI 6'4 *6 4 8 6-3 1.0 3.7-60 5 3-2 5.7.4 In conclusion another experiment was carried out to test whether the output of creatine might not be due simply to the increased catabolism of muscle or other creatine-containing tissues as the result of acidosis. It is of course well known that acidosis can lead to an increase in the ouitput of nitrogen. Attention has been drawn to the fact that there was no evidence of acidosis, judging at any rate by the absence of acetone, in the fat experiments already carried out. It will be noted here that as usual there was no output of creatine during the carbohydrate period and that the output of total nitrogen was very steady. This outpuit of total nitrogen remained steady from the commencement of the fat diet until the alkali was given. Whether the changes observed be due to the action of the alkali or merely to the alteration in the amount of urine secreted it is at present inmpossible to decide. On the first day of the alkali there was a sharp fall, although not by any means a- very great one, followed later by a marked rise. On the day following the commencement of the fat diet the first trace of creatine appeared, the amount present rapidly increasing with, at the same time, a steady rise in the output of preformed creatinine. With

PROTEIN METABOLISM. 283 ExP. VII. The dog was fed for the first five days on a carbohydrate diet made up as follows: oatmeal 75 grms., tapioca 100 grms., milk 500 c.c., water 500 c.c., and then during the subsequent days of the experiment on a fat diet: oatmeal 75 grms., lard 100 grms., milk 500 c.c., water 500 c.c. After the output of the creatine on the fat diet was pronounced the animal was givena, by means of the stomach-tube, two large doses of sodium carbonate on successive days. Throughout the fat period there was neither diacetic acid nor acetone present in the urine at any time..o~~~~~~~~~~~~~~~~~~~~b i be ca 15-40 1 700 Acid 182'0 2'7 *457-2 1800,, 187-2 3'0 '491 - - 3 1300,, 88-4 2'0-434 4 1800,, 98'5-2S8-412 - 1400,, 95'2-2'1-365 - 15'45 6 650 Alk. very faint 82'2 2-0 *339 '009 7 700 Slt. acid 72.8 2-3.373 *058 8 600 Alk. faint 76-8 2'0 *441 047 9 700 Slt. acid 1008-157 2-2 *465 043 10 460 64-6 *076 1P8 401 *023 Na2CO320grms. 4p.m. 11 1000 Alk. marked IL64-0 *122 2'8 *608 '045 Na2CO 15 grms. 4 p.m. 12 950 13 700 Alk. 15'70 14 650 Amphoter Remarks Carbohydrate period Fat period began previous day Free carbonate in urine 95.0? 1'7 '400 *014 Free carbonate in urine 70'0? 1'8-464 '015 Free carbonate (trace) in urine 106-6 '199 2X9 *555 the administration of the alkali there was a sharp fall particularly in the output of creatine not so marked in the creatinine, followed, as in the case of the total nitrogen, by a sharp rise both of creatine and creatinine, and agairn followed by a fall in the output of both these substances. The fall in the case of creatine went so far that two days later it was questionable whether or no any creatine were present, on the other hand in the case of creatinine there was a steady increase in output. At the same time the total acidity of the urine (bv Folin's method) and the output of ammonia (as estimated by the formol titration method) were estimated. The total acidity fell when the diet was changed from carbohydrate to fat, and throughout, even after the large doses of alkali when free carbonate was found in the urine, there was no very great variation. The ammonia was estimated from the day preceding the administration of the alkali. As the result of the first dose of carbonate there was a marke(d fall of some 50 per cent. in the output followed by a rise on the day following. This was succeeded by two days when no

284 E. P. CATHCART AND M. ROSS TAYLOR. ammonia could be detected or if present then in the merest traces, to be followed in turn by a sudden rise in the output. No full explanation of this curious rate of output can be offered. We think however that it may be concluded fromn this and the previous experiments that the acidosis has little or nothing to do with bringing about the output of creatine. CONCLUSIONS. (1) In glycosuria, if carbohydrate be not present in the food in sufficient amount, there is an excretion of creatine. (2) This carbohydrate cannot be replaced by fat. (3) The output of creatine persists so long and only so long as the glycosuria lasts. (4) There is no apparent causal relationship between acidosis and the output of creatine. REFERENCES. (1) Cathcart. Journ. of Physiol. xxxix. 311. 1909. (2) a. Moritz und Prausnitz. Zeit. f. Biol. xxvii. 93. 1890. b. Stiles and Lusk. Amer. Journ. of Physiol. x. 67. 1903. (3) Falta, Grote und Staehelin. Hofmeister's Beitr. x. 197. 1907. (4) Krause and Cramer. Journ. of Physiol. (Proc. Physiol. Soc.) XL. lxi. 1910. E. P. C. is responsible for the injections of and other experimental work. The expenses of this investigation were defrayed by a grant from the Carnegie Trustees to whom our best thanks are due.