THE UTILIZATION OF AMMONIA IN THE PROTEIN METABOLISM.

BY ALONZO EXGLEBERT TAYLOR AND A. I. RINGER.

(From the Department of Physiological Chemistry of the University oj Pennsylvania, Philadelphia, Pa.)

(Received for publication, -April 1, 1913.)

It has long been the opinion of the physiological chemist that the higher animal organism was dependent upon the amino-acids for the maintenance of protein anabolism. That plants of all orders, from the lowest to the highest, are able to synthesize pro- tein from the salts of ammonium is, of course, fully established. For the animal organism, however, the prevailing opinion seemed conclusively est.ablished to the effect that the building-stones of protein could not be synthesized from ammonia and fatty acids. Formulated in chemical terms, the animal organism was not held to possess the power of effecting the replacement of one hydrogen attached to the a-carbon of a fatty acid by an NH, radical to form an a-amino-acid.

Ammonium introduced into the body from without, formed within the tissues through reactions of metabolism, or formed within the intestinal t.ract by the action of bacteria, was held to be converted into urea or eliminated as such. It was not held to be converted into amino-acids.

Recently published experiments by Knoop,’ 13mbden2 and their confr2res have brought evidence to the effect that the animal body does possess the power of converting aliphatic and aromatic o(- ketonic acids into amino-acids. This has led Grafe and Schlapfer”

1 Ktioop: Zeitschr. .f. physiol. Chem., lxvii, p. 489, 1910. 2 Embden: Biochem. Zeitschr., xxix, p. 423, 1910. 3 Grafe and Schlilpfer: Uber Stickstoffretention und Stickstoffgleich-

gewicht bei Fiitterung von Ammoniaksalzen., Zeitschr. f. physiol. Chem., lxxvii, p. 1, 1912.

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408 Utilization of Ammonia in Metabolism

and Abderhalden4 to study the effect of ammonia on the protein metabolism of dogs. Grafe and Schlapfer came to the conclusion that dogs on a high carbohydrate diet, with low protein intake, were able to retain very considerable quantities of the N of ammo- nia (NH&l). With larger quantities of ammonium citrate, they were able to maintain nitrogenous equilibrium for a period of fifteen days. They did not notice any elimination of retained nitrogen during the after periods. Grafe and Schlapfer, therefore, reached the conclusion that the animal has the power of synthe- sizing protein from carbohydrate and ammonium. Abdcrhalden’s method was similar to Grafe’s. His first dog received, during the fore-period of eight days, 40 grams of fat, 40 grams of sugar, 40 grams of starch and 5 grams of bone ash, per day. The nitrogen balance for that period, as was to be expected, was strongly nega- tive (- 1.83 to -2.95 per day). During the following three days, ammonium carbonate was added to the diet, and the nitrogen balance was -0.23, -0.08, -0.09 gram per day. During the after period, the nitrogen elimination was not any higher than during the fore-period. Experiments with ammonium acetate did not show such marked retention, but showed a very considerable rctcntion. Abderhalden hesitatingly reached the conclusion that “the addition of ammonium salts as the only source of nitrogen to a diet consisting of carbohydrates and fats, exercises a distinct influence on the protein metabolism, by causing nitrogen reten- tion.” The relationship of this retention to the protein anabo- lism, he left an open question, suggesting, however, the probability that a reversible reaction had set in.

In later work on the subject,5 Abderhalden tends to incline more to the retention hypothesis, denying the possibility of the synthesis of protein from ammonia and carbohydrates. He writes: “It is no doubt possible in some cases to reduce the nitro- gen elimination by ammonium salts. We have not observed any

4 16. hbderhalden: Fiitterungsversuche mit vollst%ndig bis zu Amino- saurcn abgcbnutem Eiweiss und mit Ammonsalzen., Zeitschr. f. ghysiol. Cher/l., lxxviii, p. 1, 1912.

5 E. Abderhalden and Paul Hirsch: Fortgcsezte Untersuchungen iiber die synthetischen FLhigkeiten der tierischen Zellc. Versuche iiber die Ver- wertung verschiedener Stickstoffquellen im Organismus des Hundes, Zeifachr. f. physiol. Chem., lxxxii, p. 1, 1912.

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A. E. Taylor and A. I. Ringer 409

prolonged positive nitrogen balance. Only in a few cases was it temporarily positive. It was, however, more negative in the after period. We are inclined to assume that the established nitrogen does not stand in any direct relation to the synthesis of protein. We have no reason for the assumption that the animal organism has the power of producing the different complexes for the vari- ously built amino-acids from carbohydrates, and that the admin- istered ammonia is used for their aminization. Such an assump- tion stands in contradiction to all the present conceptions of protein metabolism.”

On examining Abderhalden’s figures, one fails to notice any marked “Ausschwemmung” of the retained nitrogen in the after periods.

The experiments reported below were performed with the object of throwing light on several questions which presented themselves to us on reviewing the subject.

1. Does the ammonia combine with some rest of the carbohydrate molecule in its retention; is the carbohydrate really necessary for the demonstration of the utilization of ammonia in the protein metabolism; and does the carbohydrate give rise to the non-nitrogenous *fraction of the various amino-acids?

‘Grafe maintains throughout his studies that large quantities of carbohydrates are necessary to get a satisfactory demonstration of the utilization of ammonia, and Abderhalden has accordingly kept his animals on a high caloric intake. The results of Grafe’s and Abderhalden’s experiments are very similar. The differences are only in the degree of retention. Grafe maintains that the differences in their results are to be sought in the difference of the carbohydrate supply, and that the quantity of the carbo- hydrate fed is the determining factor.

It seems to us that the understanding of the state of retention of the nitrogen involves t’his question directly. Is the carbohy- drate a very important factor? Does the circulation of an excess of carbohydrate molecules in the blood bring about the synthesis with the ammonia to amino-acids, which either build up or spare body protein; or does the carbohydrate play a secondary role, and the ammonia the primary role?

We have attempted to solve this problem by feeding ammonium carbonate to starving dogs. If the carbohydrate is of importance,

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4x0 Utilization of Ammonia in Metabolism

and if the ammonia in its retention is tied up with the carbohydrate molecule, the results should be negative. That is, none of the ammonia nitrogen should be retained. On the other hand, if on giving ammonium carbonate, a retention of the nitrogen should occur, Grafe’s “carbohydrate” theory can safely be eliminated.

Dog 1 was permitted to fast for three days prior to the com- mencement of the experiment. Water was given ad libitum. The animal was kept in a metabolism cage, and the urine collected quantitatively. The periods of twelve hours were separated by catheterizing and washing the bladder with distilled water. The nitrogen was determined according to Kjeldahl, the ammoniaaccord- ing to Folin. The ammonium carbonate was prepared by Baker, and has the following composition: (NHJZ CO,.NII,CO,-NH,.

EXPERIMENT 1.

Twelve-hour periods

I I 8.32 1.01 0.058 II 0.88 0.065

III 0.89 0.052

IV I 8.06 /

v I

TOTAL NlTRcmEN

NHa N

1.28 0.076 -0.28

0.87 / 0.054

NITROGEN

B A L A N C E

-1.01 -0.88 -0.89

-0.87 -

REMARK8

1 gram of N in the form of ammonium carbonate given per 0s.

This experiment shows very clearly that a good part of the nitrogen (more than half a gram) was retained, and was not elimi- nated in the after period.

To avoid the criticism of too short periods, the periods of the next experiments were extended to twenty-four hours.

EXPERIMENT II

P’wenty-four-hour periods.

TOThL NrTROGIEN NHa N “,‘,‘;;zf”,“,“,” / REMARK8

I 1 17.68

~__________~

3.26 0.24 -3.26 Sixth fasting day. 2 grams of N in the form

II 4.14 0.236 of ammonium carbonate given per OS.

III 17.32 3.22 0.408

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A. E. Taylor and A. I. Ringer 411

The results of this experiment corroborate the findings of the first. Both agree in showing that a considerable part of the nitro- gen of the ammonia is retained, and that it is not eliminated in the after period.

EXPERIMENT III.

Twenty-four-hour periods. -

I .

-

I II

III IV

V VI

VII VIII

IX X

XI XII

XIII XIV xv

XVI

XVII XVIII

XIX

xx XXI

XXII

XXIII XXIV xxv

XXVII

8.95 8.85 8.79 8.69 8.74 8.66 8.58 8.48 8.44 8.36 8.20

7.80

7.48

7.32

6.98 6.95

I

2.43 -2.43 2.32 -2.32 2.39 -2.39 2.21 -2.21 2.13 -2.13 1.96 -1.96 2.00 -2.00 2.45 -2.45 2.16 -2.16 3.12 -0.82 1.90 -1.90 1.75 -1.75 2.13 +0.17 1.81 -1.81 1.62 -1.62

2.74

1.83 1.72

-0.44

-1.83 -1.72

3.40 -1.10

2.91 -2.91 2.37 -2.37

3.97 -1.67

2.00 1.76 4.02 2.16

~- !

i

-2.00 -1.76 A.72 -2.16

REMARKS

Eighth fasting day.

73 grams of meat = 2.3 grams of N.

2.3 grams of N in the form of meat.

2.3 grams of N in the form of am- monium carbonate per OS.

2.3 grams of N in form of ammo- nium carbonate given subcutane-

I ously.

2.3 grams of N in the form of urea given per OS.

2.3 grams of N in the form of urea.

This experiment, in addition to the fact that it corroborates the first two, also shows that the nitrogen of ammonia may affect the nitrogen balance almost to the same extent as does the feeding of meat. Ammonia administered subcutaneously is eliminated quan- titatively. Urea administered per OS is eliminated quantitatively.

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412 Utilization of Ammonia in Metabolism

The outcome of these experiments proves very conclusively that the nitrogen of ammonia can be retained in the system even in the absence of carbohydrates, and Grafe’s theory can therefore be discarded. The ammonia is undoubtedly utilized in some other way. That it is not retained as an end product, urea, we believe to be demonstrated by the fact that a corresponding dose of urea is completely eliminated within twenty-four hours.

The second question that presented itself was: Will ammonia nitrogen be retained in the case of glucosuria, where the utilization of carbohydrates is almost completely lost?

Two experiments were performed on completely phlorhizinized dogs. The results are as follows:

EXPERIMENT IV.

Twelve-hour periods.

PERIOD TOTAL NITROGEN CLUCOLIE D:N NHoN 1 REILIARHC)

I 8.20 11 8.06 --.I --- ---~ i 2 grams of NH3 N.

III 8.30 I

EXPERIMENT V.

I ~ 6.65 25.93 3.89 0.35 II 6.66 25.88 3.87 0.46

III 7.40 26.36 3.42 0.32 3 grams of N as ammonia. IV 6.21

These two experiments show that a diabetic animal has the power of retaining ammonia nitrogen to a much larger extent than does the starving animal.

How can we expbin these peculiar retentions of nitrogen? Can we bring them into harmony with known facts, or must we seek some other interpretation?

hbderhalden’s latest assumption, that the nitrogen is retained temporarily, is not borne out by his own figures. The fact that there happened to be an increase in the nitrogen elimination on a starvation day immediately following the ammonia feeding, is not sufficient to prove the point. It werely illustrates the necessity of carefully separating the urine at the end of each twenty-four hours, which both Abderhalden and Grafe seemed to conside!

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A. E. Taylor and A. I. Ringer 413

unnecessary, and which makes the interpretation of their results considerably more difficult. Grafe’s theory that the carbohy- drates pass into union with the nitrogen of the ammonia is here proven to be erroneous. The most probable answer to the above questions, and the one most strongly in harmony with well-known physiological and chemical facts, is the following: the ammonia, because of its high concentration in the tissues, most probably in the intestinal wall and liver, reverses the process of deaminization. By catabolism of protein in the cells, we understand a cleavage of protein into the constituent amino-acids that make up the pro- tein molecule. These amino-acids are believed to be deaminized, the amino radical going into urea, and the non-nitrogenous frac- tion either broken down and burnt directly or utilized in the synt’hesis of glucose, glycogen and fat. It is at the point of this deaminization that we believe the ammonia to exert its influence. It is now well established that the non-nitrogenous part of the deaminized amino-acids are either or-hydroxy or ar-ketonic acids, both of which when administered into the body or perfused through the liver have the power of combining with the amino radical.

We have attempted to test this theory in the following way: Grafe, Abderhalden and the writers, in the experiments reported above, administered all the ammonium carbonate in a rather con- centrated form. This causes the ammonia to be absorbed rapidly, and to exert a high mass action. It was thought that, by giving the ammonia in very high dilution and in small quantities at a time, the mass action might be eliminated.

The subject of this experiment was a man of about 70 kilos body weight, in medium flesh. The diet consisted of 200 grams of starch, 200 grams of cane sugar, 5 grams of table salt and 3000 cc. of water. The starch was the best of several preparations; the one used contained only 0.065 gram of nitrogen in the day’s ration. The diet could fairly be termed nitrogen-free. It contained 1600 calories, not enough to place the individual in caloric equilibrium, but enough to spare body protein effectively. The water was con- sumed with the food, three times daily. The subject was placed on this diet for a fore-period of five days. The urine and feces were collected only during the last three days of the fore-period. The urine was analyzed for total nitrogen by the method of Kjeldahl, ammonia and creatinine by the methods of Polin, urea

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414 Utilization of Ammonia in Metabolism

by the latest method of Benedict, and the purine bodies by succes- sive precipitation with silver nitrate and copper bisulphate, the final precipitates being analyzed for their nitrogen content by the method of Kjeldahl. The feces were dried in the usual way.

On the sixth day ammonium carbonate was administered in three doses with the food in dilute solution, the salt being dissolved in the water taken with the meal (750 cc.). The ingestion for the day was 11.37 grams, ‘containing (by analysis) 2.8 grams of nitro- gen. On the following day, 11.6 grams of ammonium carbonate were ingested in a similar manner, containing 2.86 grams of nitro- gen. The two following days constituted the after period, the diet being maintained.

Such doses of ammonium carbonate are very irritating to the intestinal tract. Diarrhea set in, so that a marking of the stools became impossible. The f&es of the entire period were collected, therefore, and analyzed as one. This introduced an error, or at least an opportunity for error, since the degree of resorption of ammonium carbonate could not be definitely fixed. As will be later detailed, no error of consequence was committed, as the nitrogen content of the feces was not abnormally high. We had hoped to extend the after period, since we wished this period to be so long as to absolutely exclude any later elimination of retained nitrogen. However, when the low figure for the nitrogen output for that day became known, the experiment was discontinued, largely on account of the intestinal distress, which made it advisa- ble to seek another diet. Other symptoms of the action of ammo- nium than those described were not noted; in particular, such high doses had no effect, upon the circulation.

The following table contains the analytical data of the experi- ment. All values are expressed in gram;.

EXPERIMENT VI.

DhY on EXPERIMENT ~---.?LdLL

3 4

Urinary N.. ................. 8.86 6.64 5.54 6.84 6.39 4.66 Fecal N ..................... 0.76 0.76 0.76 0.76 0.76 0.76 TotalNoutput.. ........... 9.62 7.40 6.30 6.60 7.16 5.42 Urea N ...................... 6.58 4.07 3.47 3.62 4.61 2.27 Ammonia N ................. 0.54 0.73 0.41 0.42 0.41 0.37 CreatinineN.. .............. 0.64 0.66 0.68 0.66 0.64 0.67 Purine N .................... 0.050 0.045 0.045 0.046 0.070 0.05! Rest N ...................... 1.05 1.13 0.94 1.10 0.70 1.5

--. 9

3.75 0.76 4.w I.81 0.41 0.66 0.040 0.83

--,

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A. E. Taylor and A. I. Ringer 415

The average fecal nitrogen, 0.76 per day, cannot be considered in any way above the normal. Obviously, the ammonium car- bonate was fully resorbed. And the diarrhea made apparently no difference in this resorption. If there was any effect, there was possibly an increase in the secretory nitrogen eliminated, though, as stated, 0.76 gram of nitrogen per day cannot be con- sidered high; it is within the usual limits for normal figures.

Strikingly constant are the figures for creatinine and purine and, with the exception of one day, of ammonia also. There is a short rise in the purine on one day, the second day of the test, leading possibly to the inference of increased nuclear catabolism in the liver, as the result of, or associated with, the presence of the ex- cess of ammonium carbonate in the liver.

From an inspection of the figures for the total urinary nitrogen and urea, it is clear that there was a rise, associated with the ingestion of the ammonium carbonate, but which was not at all commensurate with the input. The greatest part of the admin- istered ammonia was retained in the system, and was not eliminated in the after period. This shows that the ammonia is retained even when given in dilute form. It does not, however, vitiate the theory of the reversible action of ammonia on deaminization. For dilute as the ammonia was when administered, we have no means of telling at what rate and in what concentration it was absorbed. We hope to test this again by dividing the ammonia into very small doses, administered at very short intervalsduring the day.

The curve yields a graphic reproduction of the data. The heavy continuous line represents the actual figures for urinary nitrogen. The dotted curve for the two days indicates approximately what would have been the curve of elimination had no ammonium carbonate been administered. The dashed line indicates what would have been the curve of elimination had all the nitrogen of the ingested ammonia been eliminated. It is clear that the larger fraction of the nitrogen has been retained.

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416 Utilization of Ammonia in Metabolism

4 5 6 7 8 9 DAY OF EXPERIMENT.

SUMMARY.

Three experiments were performed on starving dogs, and the nitrogen output studied. Ammonium carbonate was given per OS, and it was found that a considerable part of the nitrogen was retained and failed to be eliminated in the after period. When given subcutaneously, it was promptly eliminated.

The administration of urea was followed by a complete elimi- nation of all the nitrogen.

Ammonia administered to diabetic dogs was also retained, but to a larger extent than in normal dogs.

Ammonia given to man on a protein-free diet (0.065 gram per day) was retained to the extent of about two-thirds.

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A. E. Taylor and A. I. Ringer 4’7

These experiments corroborate the findings of Grafe and Abder- halden, and also show that the presence of carbohydrates is not an obligatory factor in the retention of nitrogen from ammonia.

It is suggested that the ammonia nitrogen is retained because of a reversed reaction that leads to combination with the a-ketonic or a-hydroxy-acids to form amino-acids which may be used in the upbuilding or sparing of body protein. This may be Zustrated in the simple reaction for the deamihization of alanine.

CHs CH, I

CHNHz + Hz0 fg I

CHOH + NHa I I COOH COOH

Like every reaction, this reaction must be reversible under appro- priate conditions. What the station of equilibrium in the animal body may be, we have no way of knowing. But according to the interpretation of the retention that we are inclined to accept, with the administration of large amounts of ammonia this reaction is reversed, probably in the liver. Whether such a situation as this might arise outside of the experiment, normally or pathologi- cally-the formation of such amounts of ammonia as to reverse the reaction-is problematical. It is possible that such concen- tration of ammonia within the portal system could never occur naturally, in health or disease. In that event, this experiment would simply demonstrate the possibility of reversion of reaction in the animal body and add another illustration to the long list of demonstrations of the validity of physico-chemical laws within living bodies. We have used alanine as the simplest illustration of the reaction; and in view of the omnipresence of lactic acid within the organism, it is possible that the retention occurs largely or entirely in this state. The body could then make use of this alanine to the same extent and in the same way that alanine derived from the hydrolysis of protein is utilized.

If one does not incline to the view that the ammonia is retained in combination, one must assume that it is retained either in the state of ammonia or urea. The latter appears unlikely, in view of the prompt and complete elimination of urea in the direct experiment. The retention of ammonia as such, in the form of a salt, appears to us quite unlikely.

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418 Utilization of Ammonia in Metabolism

Since the completion of this paper one more experiment upon a dog was performed by Dr. L. Jonas of this department, which corroborates our previous findings.

EXPERIMENT VII.

Twenty-four-hour periods.

PERIOD TOTAL N N BALANCE REMLRKS

I 3.59 -3.59 1::

/ 3.64 4.04 -3.64 -4.04

IV 3.52 -3.52 V 3.86 -2.81 1.04 grams N as ammonium carbon-

ate. VI 3.54 -3.54

VII 3.15 -3.15

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Alonzo Englebert Taylor and A. I. RingerTHE PROTEIN METABOLISM

THE UTILIZATION OF AMMONIA IN

1913, 14:407-418.J. Biol. Chem. 

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