the site of ammonia formation and the role of

THE SITE OF AMMONIA FORMATION AND THE ROLE OF VOMITING IN AMMONIA ELIMINATION.

BY STANLEY R. BENEDICT AND THOMAS P. NASH, JR.

(From the Department of Chemistry, Cornell University Medical College, New York City, and the Department of Chemistry, University of

Tennessee College of Medicine, Memphis.)

(Received for publication, May 26, 1926.)

INTRODUCTION.

A few years ago the present writers (1) reported experiments which led them to conclude that the ammonia excreted in the urine is formed by the kidney. The findings leading to this conclusion were essentially the following. (1) The blood of the renal vein contains two to three times as much ammonia as does the blood of the renal artery. (2) Systemic blood shows no increase in ammonia under conditions (acid injection, phlorhizin diabetes) which lead to several hundred per cent increase in the urinary ammonia. (3) In nephritis there is no accumulation of ammonia in the blood, though all other known non-protein constituents may show a marked increase. (4) The acidosis of nephritis cannot be explained upon the assumption that ammonia formation for acid neutralization is a generalized tissue function. If ammonia production for acid neutralization is limited to the kidney, the acidosis of nephritis finds a more ready explanation. (5) The rate of blood flow through the kidney is not sufficient to account for the high ammonia content of diabetic urine.

In a recent paper from Folin’s laboratory Sidney Bliss (2) has reported experiments upon ammonia formation and elimination which led him to disagree with the view of the present writers that the kidney is of preeminent importance in the formation of urinary ammonia. Bliss concludes that ammonia formation for acid neutralization is a “generalized tissue phenomenon.” The chief evidence offered by Bliss in support of this contention is as follows: (1) That while the blood leaving the kidney does

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382 Ammonia Formation

contain about twice as much ammonia as does the blood entering the kidney, the same general finding holds for certain other organs. (2) That ammonia does accumulate in the blood during experimental or human nephritis, providing vomiting does not occur. (3) That the ammonia content of the blood of herbivorous animals (excreting an alkaline urine) is less than that of carnivora. (4) That alkali administration in the dog may result in a lowered ammonia content of the blood.

The question of the site of origin of ammonia is obviously an important one, and much interest therefore attaches to the very valuable experimental contribution to this problem which Bliss has offered. We do not agree with the theoretical interpretations which Bliss has made of many of his experiments, and feel it desirable to offer the following discussion of the question of ammonia formation, in which we shall consider our own work as well as the recent contributions of Bliss and certain other investigators.

The term ammonia formation may be used in either one of two ways. It may refer to ammonia formation even as a transition process, and without special reference to the ammonia formed for acid neutralization, or it may be employed in the more restricted sense in which we used it in our previous articles, as referring to the ammonia formed or utilized to neutralize acid in the body, and which normally appears in the urine. That ammonia formation in the first of these two senses is a general tissue process there can be little question. Certainly it is as general as is the process of deamination of the amino acids, for ammonia, at least as a transition product, must be formed wherever nitrogen is split from the amino acids. In the following discussion, as in our previous papers on this question, we shall use the phrase ammonia formation as referring to ammonia formed to neutralize acid in the organism, unless stated otherwise.

It may be of interest at the outset of our discussion to consider the quantity of ammonia in normal blood from a very general standpoint. In normal systemic human or dog blood the quantity of ammonia present does not materially exceed about 0.1 mg. per 100 cc., and is frequently well below this figure. This quantity of ammonia is considerably less than is to be found in practically any of the purest common laboratory reagents. To obtain reagents as free from ammonia as is the blood, one must


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S. R. Benedict and T. P. Nash, Jr.

almost invariably resort to repeated processes of purification. Though the ammonia content of human urine usually exceeds that of uric acid very considerably, we find that in human blood there is normally only one-twentieth to one-thirtieth as much ammonia as there is uric acid. There is no rise of the ammonia content of blood even in conditions such as diabetes which may lead to a 600 to 800 per cent increase in the urinary ammonia.

The fact that the consistently low values for blood ammonia are maintained even during conditions leading to a very high urinary ammonia suggests that the urinary ammonia has a site of formation close to where it enters the urine. Such site of formation would probably be the kidney. Acting on the basis of this obvious inference we were led to compare the ammonia content of the arterial and venous renal blood. It seemed logical that if the kidney produced the large amounts of ammonia found in the urine, traces of this compound should escape into the blood leaving the kidney, and might be demonstrable by a comparison of the ammonia content of the blood from the renal artery and vein. The results of such experiments, now confirmed from two other laboratories (2, 3), are so striking as to admit of no doubt that the kidney habitually forms ammonia in large amount. The ammonia leaving the kidney through the renal vein does not enter the blood for the purpose of neutralizing circulating fixed acid. Unless this is true the organism would be overwhelmed with ammonia in the course of a few hours, since the blood leaving the kidney contains such an excess of ammonia over that entering it. Bliss has not adopted this view, but it is obviously the only possible one unless we assume that the kidney is not, even in the normal organism, the chief site of ammonia excretion. As a result of the facts brought out in our previous papers we concluded that the kidney is of preeminent importance in formation of urinary ammonia, and that ammonia never enter.9 the blood stream for the purpose of acid neutralization. According to this view acids are transported in the blood in combination with fixed base or with protein, and are then combined with ammonia by the kidney and excreted as the ammonium salt into the urine.

On the basis of facts already fully corroborated we may take it as definitely proven that to account for the urinary ammonia we


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need look no further than the kidney. The question remains whether other tissues in the body also neutralize acid by means of ammonia. If so we must expect to find that increased acid in the circulation invariably results in increased ammonia in the circulation.

Just as we must assume that the ammonia leaving the kidney is not ‘(fixed” ammonia, so we must assume that the presence of increased ammonia in the blood leaving any organ does not, per se, show that such ammonia ever neutralizes acid. It may be converted into urea within a very short time, just as is the excess ammonia leaving the kidney and as is the ammonia entering the liver through the portal circulation. To show that ammonia formation for acid neutralization is a general tissue function it would seem necessary to show that the same conditions which lead to a great increase in the urinary ammonia also result in an increase in the circulating ammonia in the blood. We shall discuss this point later, and shall meanwhile turn to consider another aspect of the ammonia problem.

Evidence That Ammonia Formation for Acid Neutralization Is Not a Generalized Tissue Function.

The question of whether increased ammonia is demonstrable in the blood from several tissues is obviously well worthy of study, and Bliss has rendered a valuable service in his contribution to this question. It is plain that the finding of increased ammonia in the blood from several organs other than the kidney (and where extraneous complicating factors are not present) would make more plausible the view that neutralization of acid by ammonia is a general tissue process, though it would by no means prove the correctness of such a conclusion. Bliss believes that he has offered data to prove that ammonia formation for acid neutralization is a “generalized tissue phenomenon.” We believe that Bliss has been led into errors of interpretation in this connection because he has emphasized certain limited positive findings (which are complicated by other factors) and has disregarded his negative findings, though these latter have very great significance.

The experiments of Bliss on ammonia formation in various organs include a comparison of the ammonia content of systemic arterial blood with that of blood from (a) the pancreaticoduodenal


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vein, (b) the splenic vein, (c) the femoral vein, (d) the jugular vein, (e) the vein from the adrenal, (f) the renal vein, and (g) the hepatic vein. Thus the experiments of Bliss in this connection were designed to study ammonia formation in at least seven different tissues. Before discussing the results which Bliss reports we shall consider what results might have been expected, providing we keep in mind certain well known relevant facts. It would seem reasonable that if ammonia formation for acid neutra.liza- tion occurs’elsewhere than in the kidney, the organ or organs preeminent in deamination and in urea formation (liver, muscles) should show most, or at least some ammonia formation. It should also be expected, we believe, that blood drawn from close proximity to the gastrointestinal tract should very probably show increased ammonia, because it is well known that the gut constantly contains large amounts of ammonia resulting from bac- terial action and from the hydrolysis of proteins. We should therefare tend to be suspicious of the origin of ammonia contained in blood returning from close proximity to the gastrointestinal wall.

We now turn to consider Bliss’ results in these experiments. Of the blood from seven different sources which he examined, only that from two sources, the kidney and the wall of the gut (pancreaticoduodenal vein), showed marked increased ammonia. The blood from the splenic vein showed a slight, though definite tendeqcy to increased ammonia. ‘But not in one of the normal dogs used in these experiments (except in one, the blood of which was taken during digestion) was there a greater increase in the ammonia of the blood of the splenic vein than might be found as a result of individual analytical variations. Anatomical findings show that the splenic vein frequently has anastomosis with veins from the pancreatic-duodenal supply. We therefore suggest as the most logical conclusion that the increased ammonia in the blood of the pancreaticoduodenal vein, and the trace of increased ammonia in the blood of the splenic vein were absorbed from the upper portion of the intestinal tract. This conclusion is especially indicated in view of the negative results with other tissues. Bliss recognizes possible contamination of the pancreaticoduodenal blood with ammonia from intestinal sources, and attempted to guard against it by pointing the needle through which the blood


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was drawn in a special direction, and by examining the duodenum and finding that it was “clean.” So crucial a point as the one here involved should have had more adequate control. Only the demonstration that analysis of the upper intestine plus contents failed to reveal appreciable quantities of ammonia would seem to meet the demands.

Thus because of the inadequate control by Bliss of the possible source of the ammonia, we must tentatively conclude that the increased ammonia in the blood taken from close to the gut was absorbed from the gut. Certainly it is notable that just as soon as Bliss experimented at a moderate distance from the wall of the gastrointestinal tract no trace of ammonia formation could longer be demonstrated, other than the two- to threefold increase found in the blood from the kidney.

We regard the failure to find any ammonia formation in the muscles, and especially in the liver, as of the utmost importance as pointing to the kidney as the sole source of ammonia for acid neutralization. Were tissues other than the kidney concerned in ammonia formation the liver might be expected to be preeminent, and the muscles should contribute. The results here were entirely negative. Yet on the evidence cited Bliss concludes that ammonia formation is “a generalized tissue phenomenon.” Must we not rather conclude that Bliss’ experiments in this connection afford definite proof that ammonia formation is not a generalized tissue phenomenon?

The Source of Ammonia Contained in Vomifus.

Bliss believes that he has demonstrated that the organism can, when the kidney function is shut down upon, excrete ammonia through the gastrointestinal tract so that ammonia can be lost from the blood and tissues by means of vomiting. This question is not especially pertinent to the point at issue in ammonia formation, but we shall discuss it because we feel that it is of definite interest, and because we can offer a few experimental data bearing upon it. Three experiments are recorded in which Bliss thinks that he has shown the loss of ammonia from the blood of partially nephrectomized dogs by vomiting. Adequate controls are lacking. No experiments are reported to show that the gastrointestinal tract of normal fasting dogs is reasonably free from


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S. R. Benedict and T. P. Nash, Jr. 387

ammonia.* We have carried out a few experiments along this line. In one dog, weight 15.6 kilos, which was fasted 3 days and given a cathartic (castor oil) we found a total of 800 mg. of ammonia nitrogen in the fluid feces collected during a period of 4 hours. This ammonia represents several times the total urinary output of ammonia for this normal animal during 24 hours of fasting. A second dog of about the same weight after 3 days of fasting yielded 93 mg. of ammonia nitrogen in one sample of fluid feces. We have not pursued the question further. The burden of proof appears to lie with anyone who, like Bliss, assumes that the ammonia coming from any portion of the gastrointestinal tract has not been formed within some portion of that tract, but is excreted from the blood stream into the gut. Two of the protocols offered by Bliss may be cited. Thus he reports ((2), Table VIII) that a dog weighing 10.1 kilos vomited 62.6 mg. of ammonia nitrogen within a period of 6.5 houra after double nephrectomy. This quantity of ammonia (nearly 10 mg. per hour) is approximately double the quantity of ammonia which would have been eliminated in the urine of a normal fasting dog weighing 10 kilos. This dog had apparently eaten some meat prior to operation, but we still conclude that the enormous ammonia output in the vomit did not represent ammonia excreted from the blood into t.he stomach. It would seem doubtful if even after nephrectomy the stomach could become so efficient an excretory organ as to equal or excel the normal kidney in this function.

In Table IX of his paper Bliss reports that a dog weighing 5.75 kilos vomited 240 mg. of ammonia nitrogen in 3 days “and not all the vomitus could be collected.” The vomited ammonia (240 mg.) represents probably quite as much of this compound as a normal animal of less than 6 kilos weight would have eliminated

1 The only controls reported by Bliss in connection with the source of the ammonia contained in the vomit of his uremic dogs are experiments in which vomiting is induced in normal dogs after introduction of urea solution into the stomach. Such experiments are hardly adequate. In sick nephrectomized animals lessened absorption may contribute to excessive accumulation of ammonia in the gut, or antiperistalsis may lead to ammonia entering the stomach from the intestine in increased amounts in such animals.


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Ammonia Formation

through the kidneys during 3 days of fasting. One cannot but speculate as to whether any of this enormous ammonia output in the vomit came from the blood and tissues. Of this animal Bliss writes “the striking reduction from 0.25 mg. to 0.14 mg. was made within a period of 20 minutes, coincident with the elimination of 63.8 mg. of ammonia nitrogen in the vomitus, vomiting being induced by forcing fluids by stomach tube.” Here Bliss assumes that some 60 times the total ammonia contained in the circulation of this animal at any one time was excreted into the stomach within a short time. Obviously there can be no such rate of excretion of ammonia into the stomach, even though the entire body of this animal had contained such an amount of ammonia to be excreted. Like the experiment of Bliss’ cited above, this one suffers because of the discrepancy between the ammonia found and the quantity which could be reasonably expected were it de- rived from the blood. Another doubtful inference to be noted in the discussion by Bliss of the vomiting experiments is his conclusion that the vomiting act per se is the necessary factor in ridding the organism of ammonia. If the excretion of ammonia into the gut takes place when the activity of the kidneys is shut down upon one would expect that the stomach should act like the urinary bladder, and that the vomiting act should not be sud- denly effective in lowering the blood ammonia, any more than washing out the urinary bladder will result in a sudden lowering of the blood urea. We feel that Bliss has not demonstrated the excretion of ammonia into the gut under any circumstances. Antiperistalsis may be a factor in sick nephrectomized or uremic animals, whereby ammonia is carried in greater quantity from the intestine into the stomach than would occur in normal animals. Vomiting might contribute to a lowered blood ammonia because of a decreased rate of ammonia absorption following vomiting. It would seem that when one is dealing with ques- tions of ammonia within the orgarism, and within the gastrointestinal tract at the same time, the greatest caution should be exercised in reaching conclusions.

The Blood Ammonia in Nephritis and in Uremia.

Bliss gives an extended series of experiments and a considerable amount of discussion to show the accumulation of ammonia in the


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blood in human and in experimental nephritis. Apparently he thinks that the entire question of the production of urinary ammonia by the kidney hinges upon this point. We are not at all prepared to adopt this view. We merely pointed out in our original paper that the generally negative findings as to the accumulation of ammonia in the blood in nephritis can best be explained upon the basis that ammonia for acid neutralization is formed in the kidney. We are still of this opinion. We were unable to demonstrate the accumulation of ammonia in the blood of nephrectomized dogs, entirely irrespective of whether they vomited or not. In a series of carefully conducted experiments Dorothy Russell (4) found no accumulation of ammonia in the blood of human nephritics. There is every reason to doubt that these cases were vomiting more ammonia than they absorbed from the gastrointestinal tract.

Very recently Van Slyke and collaborators (5) have reported an extended investigation of the acid ammonia ratio in the urine and the ammonia content of the blood in nephritic cases as compared with normal individuals. Their studies cover a wide range of types and degrees of nephritis. In discussing their tind- ings Van Slyke and collaborators state:

“The ammonia content of the blood was as low or lower in those forms of nephritis with diminished ammonia output as in normal individuals. The fall in ammonia excretion in glomerulonephritis appears, therefore, due not to ammonia retention in the body, but to failure to form ammonia in proportions normally related to those of the acid metabolites. The concurrence of diminished ammonia formation with diminished kidney function is compatible with the conclusion of Nash and Benedict that ammonia is formed in the kidney.”

Plainly these findings cannot be reconciled with the view that ammonia formation is a general tissue process, unless we are to accept the wholly improbable and unwarranted conclusion that so soon as the kidney becomes damaged ammonia is excreted into the gut while the acids with which it is combined are returned to the circulation to be eliminated by the kidney.

We should expect theoretically that whenever, as in uremia, there is a marked accumulation of urea in the blood there may be some slight accumulation of ammonia. Bliss, in speaking of the blood ammonia values in his nephritic dogs says, “among them-


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selves they grade up as the non-protein nitrogen values do.” Bar- nett and Addis (6) obtained very high values for the blood ammonia in rabbits after giving large quantities of urea. While the analytical method employed by Barnett and Addis may be open to criticism, the ammoniavalues obtained (up to 11 mg. per 100 cc. of blood) are so veryhigh compared to their controls, as to admit of little doubt that great increases in the urea content of the blood may lead to an increase in the ammonia. We believe that this is quite to be expected. The trace of ammonia circulating in normal blood represents, we believe, the equilibrium which the tissues usually maintain between urea and ammonia.2 Hence a great increase in the blood urea might be expected to result in some increase in the blood ammonia. We were unable to find such increase in our dogs, but apparently Bliss has succeeded in doing so in uremic animals and in human cases of uremia.

The Blood Ammonia in Herbivora and in Dogs after Alkali Administration.

Bliss believes that the finding that herbivorous animals have a lower blood ammonia speaks against the view that the kidney is preeminent in ammonia formation. We fail to see the force of this argument. In the dog much larger amounts of ammonia

2 In his discussion, and again in his summary, Bliss suggests “a shift of emphasis from the ammonia content of venous blood to that of arterial.” We were not aware that the ammonia content of venous blood has had undue attention. In any event since the ammonia values of systemic arterial and venous blood are identical, as shown in Bliss’ work as well as our own, we fail to see any gain in a shift of attention from one to the other. On page 117 of his paper Bliss says: “Table VI is a comparison of carotid with jugular blood. Normal dogs show no difference between the ammonia content of carotid and jugular blood.” But on page 127 of the same paper, in discussing the “Significance of Arterial Values” Bliss gives a somewhat different interpretation of Table VI. Here he says: “Table VI gives the carotid values for a representative portion of our dogs. In non-nephritics the values are generally lower and in the nephritics (due to acidosis) higher. This would necessarily be the case if the ammonia of arterial blood is there on its way to the kidney for excretion.” There is nothing very clear to us in this apparently contradictory discussion. But we might suggest that if, as Bliss assumes, ammonia is freely excreted into the gut in nephritic dogs, arterial values for ammonia should average higher than venous values in such animals. Apparently the contrary is true.


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are formed in the kidney and find passage into the blood than is the case in herbivorous animals. It is also quite probable that the lower protein content of the food of herbivora results in absorption of less ammonia from the gut. We have already sug- gested that the ammonia normally present in the circulation represents an equilibrium between urea and ammonia. Obviously when, as in herbivora, less ammonia reaches the circulation, we should expect a generally lower blood ammonia. If one injects ammonium carbonate continuously into the circulation of an animal one should expect to find more ammonia in the blood of such an animal, even though the great bulk of the ammonium carbonate is converted into urea. This is what we believe occurs in the dog as compared with the rabbit or goat. That the kidney in these herbivorous animals forms no appreciable amount of ammonia has already been demonstrated (2, 3). A quite similar condition occurs in the dog when enough alkali has been given to lower ammonia production by the kidney. We did not obtain clear cut results in such experiments, but we did not infer that our findings especially corroborated or controverted our general theory. We reported the inconclusive findings, and stated that further work was necessary to determine the effect of alkali administration. Apparently Bliss has succeeded in giving enough alkali to diminish the ammonia in the renal vein and in the general circulation. Such findings, like those with the goat and rabbit, are quite in agreement with the view that the kidney alone is responsible for ammonia production.

It is of interest to point out further in this connection that the urinary ammonia may be increased in amount under special conditions which result in a simultaneous increase in the alkali reserve of the tissues. Hendrix and Sanders (7) have reported that fasting dogs show an increased excretion by kidney of both titratable acid and ammonia after injections of disodium phos- phate and of sodium hippurate, either of which salts increases the available alkali in the blood [as indicated by an increased carbon dioxide-combining capacity) and probably also in the other tissues. Of these results Hendrix and Sanders say:

“The older conception of the formation of urinary ammonia assumes that it reacts with acids formed in the tissues and is then excreted by the kidney. . . . . If the increase of urinary ammonia observed in this study


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did not have its origin in the kidney, some new explanation for the formation in other tissues of the animal body must be found.”

Bliss reports experiments with urease and ammonium carbonate injections to show the toxicity of ammonium salts. Obviously a finding of high toxicity of ammonium salts is in harmony with the view that the tissues do not form ammonium salts. We feel, however, that Bliss was unfortunate in his selection of ammonium carbonate for this study. The carbonate of ammonium is strongly hydrolyzed in solution, so that its effects cannot safely be attrib- uted to the ammonium ion. Studies with the ammonium salts of hydrochloric, lactic, or butyric acids might have afforded more satisfactory evidence of the effect of the ammonium ion in the circulation.

The Blood Ammonia and the Urine Ammonia during the Transportation and Excretion of Abnormally Large

Quantities of Acid.

Bliss fails to consider adequately what is, we believe, the main issue in the ammonia problem. Thus in some of Bliss’ protocols we note the injection or feeding of hydrochloric acid, and the use of phlorhizinized animals. Apparently these experiments were made in connection with the study of the question of ammonia formation. Yet Bliss does not mention the results of such experiments in discussing the protocols where they are recorded, and we find very little reference to them later in the paper. It is therefore desirable to emphasize that the experiments of Bliss in this connection corroborate our own similar earlier experiments in that they fail to show any ammonia increase in the blood following procedures which enormously increase the ammonia in the urine. Concerning the effect of acids upon the blood ammonia Bliss states : “We have shown that a physiological influx of acids does increase the concentration of ammonia in the blood when means are devised for showing it.” The exact significance of this statement is not clear to us, but apparently it refers to some results obtained by Bliss with uremic animals or with human cases of uremia.3 In

3 Bliss mentions one or two experiments in which very high blood values for ammonia are obtained within 0.5 hour or so after double ureter ligation. We know of no theory of ammonia formation which would account for such


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S. R. Benedict and T. P. Nash, Jr. 393

any event it evades the main question, and does little to clear up the problem involved. Fortunately we do not have to con- cern ourselves with what is meant by a “physiological influx of acids,” nor need we limit ourselves to uremia, with its extraneous complicating factors, as the only condition suited to the study of acid neutralization in the organism. Through the introduction of mineral acid into the circulation, or through the use of phlorhizin, the urinary ammonia can be increased 400 to 800 per cent above the fasting level. Yet under these conditions there is no increase whatever in the blood ammonia. In regard to no other known constituent of blood and urine can this finding be duplicated.

In commenting on his failure, as well as our own, to find increased ammonia in the blood in phlorhizin diabetes Bliss says:

“Our results with phlorhizin-poisoned dogs are in agreement with those obtained by Nash and Benedict, but we see no argument here for localizing ammonia formation in the kidney. The plasma CO, values of 38.1 to 54.1 reported by them, in spite of high urinary ammonia values, cannot be inter- preted as indicating a severe phlorhizin acidosis, and the normal values obtained by them and us are quite to be expected in the absence of a marked acidosis.”

Such a statement shows a complete failure to appreciate what is probably the most fundamental and important point in connection with the problem of ammonia formation. If one who, like Bliss, has presumably given the question of ammonia formation some special consideration can so entirely fail to grasp the significance of enormous urinary ammonia values without increased blood ammonia values, we deem it important to discuss the point here involved in detail.

The position taken by Bliss assumes that acids can be transported as ammonium salts through the general circulation to the kidney in quantity sufficient to increase urine ammonia several hundred per cent above the normal, and yet under such conditions only the normal blood values for ammonia are “to be expected,”

great accumulation within such a short time. Other blood constituents do not show accumula,tion within such a short period. It may be noted that the kidneys were still present in these animals and perhaps absorption of urine contributed to the unaccountable results obtained.


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until acidosis appears, though acidosis simply indicates that excess ammonia production has ceased or is not sufficient to neutralize the circulating acid!

Our view of ammonia production by the kidney involves the corollary that acids are transported in the blood not in combination with ammonia, but in combination with fixed base or with protein. The kidney then forms ammonia, combines it with the acid and excretes the resulting ammonium salt in the urine. The older view, still held by Bliss, is that the tissues in general neutralize the acids with ammonia, and that the resulting ammonium salt is transported in the blood and excreted by the kidney. Acidosis cannot, according to this view, be the determining factor for increase of ammonia in the blood. Increased quantity of circulating acid combined with ammonia would be the sole determining influence to raise the blood ammonia. Thus according to this view a demonstrable increase of acid in the blood, and which causes an increase of ammonia in the urine, must result in an increase in the blood ammonia. According to the view we have taken, increase of acid in the blood should not result in increase of ammonia in the blood. Thus according to our view only the urinary ammonia reflects acid transported in the blood. Ac- cording to the older view both blood and urine ammonia values must increase following excess acid in the circulation.

We believe that the point brought up here is altogether the most important and fundamental one in connection with the problem of ammonia formation. Hence we look upon the results obtained in a study of this question as crucial in deciding between the two views of ammonia formation.

Fortunately the conditions are ideal for studying the point whether excess acids which appear in the urine combined with ammonia also circulate in the blood combined with ammonia.

The method for determination of ammonia is perhaps the most delicate and sensitive chemical analytical method in existence. By its proper use any increase, slight or marked, of ammonia in the blood can probably be more surely demonstrated than can the increase of any other blood constituent. Condit,ions are readily produced in the organism (acid injection, phlorhizin diabetes, diabetes mellitus) where the urinary ammonia can be increased 500 to 800 per cent above the fasting level. In both phlorhizin


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diabetes and diabetes mellitus the presence of increased acid radi- cle in the blood may be readily demonstrated, thcugh the analytical methods for diacetic and p-hydroxybutyric acids are very crude as compared with the method for ammonia detection and estimation. If the acids are being transported in combination with ammonia there can be no difficultyin demonstrating that fact. Our results showed that blood carrying increased acid, in quantity sufficient to cause a tremendous increase in urine ammonia, showed no more ammonia than did the blood in simple fasting. Bliss has now confirmed these findings.

In such experiments on acid transportation there are no complicating factors. The issue is clear and simple and the findings are equally clear and simple. The acids are demonstrably increased in both blood and urine.4 The ammonia is increased only in the urine. Hence the acids are not transported as ammonium salts, and all the increased urinary ammonia must be formed in the kidney. It is only a reasonable assumption that the same mech- anism which can meet the strain of ammonia formation for the excess acids, performs the same function under normal conditions. That the kidney does constantly form ammonia we have shown by direct analysis of blood entering and leaving this organ. In two other laboratories (2, 3) this finding has been confirmed. Bliss has offered the further corroborative evidence that no ammonia is formed in the liver or muscles. Hence we regard the view that formation of ammonia is preeminently a function of the kidney as now definitely proven beyond question. No known facts are

4 Van Slyke and Fitz (S) report that in the blood of human diabetics they have found as high as 250 mg. of total acetone bodies (calculated as acetone) per 100 cc., while patients under ordinarily good control show 10 60 40 mg. of acetone bodies per 100 cc. of blood. If as much as 10 mg. of acetone bodies (calculated as acetone) were transported in 100 cc. of blood combined with ammonia, the ammonia nitrogen figure for the blood would have to reach at least 2 mg. per 100 cc. In the more severe types of cases the figures would thus have to be increased at least ten or twenty times (to 200r 40 mg. per 100 cc.) if the circulating acid is in combination with ammonia in the blood. Yet in no cases of diabetes, mild or severe, is there increase of ammonia in the blood above the normal level of considerably less than 0.2 mg. per 100 cc. Obviously then, the acids which circulate in the blood in diabetes, and which are eliminated in the urine chiefly in combination with ammonia, do not circulate in the blood in such combination.


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in serious disagreement with this view. It adequately explains findings which can be explained upon no other basis. The most cogent argument so far advanced against the view is its novelty.

BIBLIOGRAPHY.

1. Nash, T. P., Jr., and Benedict, S. R., J. Biol. C&m., 1921, xlviii, 463; 1922, Ii, 183.

2. Bliss, S., J. Biol. Chem., 1926, lxvii, 109. 3. Loeb, R. F., Atchley, D. W., and Benedict, E. M., J. Biol. Chem., 1924,

Ix, 491. 4. Russell, D. S., Biochem. f., 1923, xvii, 72. 5. Van Slyke, D. D., Linder, G. C., Hiller, A., Leiter, L., and McIntosh,

J. F.,-J. &in. Ik., 1926, ii, 255. 6. Barnett, G. D., and Addis, T., J. Biol. Chem., 1917, xxx, 41. 7. Hendrix, B. M., and Sanders, J. P., J. Biol. Chem., 1923-24, lviii, 503. 3. Van Slyke, D. D., and Fita, R., J. BioZ. Chem., 1917, xxxii, 495.


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Stanley R. Benedict and Thomas P. Nash, Jr.AMMONIA ELIMINATION

AND THE RÔLE OF VOMITING IN THE SITE OF AMMONIA FORMATION

1926, 69:381-396.J. Biol. Chem.

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