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Significance of uric acid as a nitrogenous waste in vertebrate evolution.

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Significance of Uric Acid as a Nitrogenous
Waste in Vertebrate Evolution*
By ALEXANDER
B. GUTMAN,M.D.
Dept. of Medicine, Mount Sinai Hospital
DR. GUTMAN:It has generally been taken for granted that the inborn error
of metabolism underlying primary gout is to be sought in one of the enzyme::
concerned specifically with de novo purine biosynthesis, or else in some abnormality of renal tubular transfer of uric acid, but recent developments suggest that the search should be broadened to include an anomaly in the metabolism of amino acids. The first hint of this came when the initial experiments
with glycine-Nl5 administration to gouty subjects disclosed greater than normal
isotope incorporation into uric acid in some cases, indicating that “in the gouty
subject an unusually large uricotelic component may o ~ c u r , ”and
~ ~that
~ “a
disproportionate quantity of simple nitrogen and carbon precursors of uric
acid is diverted from the main metabolic channels culminating in urea and
carbon dioxide formation to pathways leading to urate formation.”3 More recently it has been suggested that in primary gout there may be a defect in the
utilization specifically of the amino acid, glutamine, for the formation of ammonia by the kidney, the excess glutamine being recycled and utilized instead
for synthesis of extra urea and uric acid, and excretion as s ~ c h .In~ this
, ~ view,
overproduction of uric acid in primary gout would be the result of an intrinsic
error, not in d e novo purine biosynthesis per se but in amino acid (glutamine)
metabolism, with increased formation of uric acid as one of the secondary
consequences.
Whatever the inborn metabolic defect( s ) of primary gout may prove ultimately to be, it seems appropriate at this juncture to define more clearly the
relationships between uric acid metabolism and amino acid metabolism, not
only in man, a rather special case, but, for better perspective, over the wide
range of evolutionary development. The first point to be made in this connection is that consistently throughout the phylogenetic scale, including man, uric
acid is elaborated ultimately from amino acids almost exclusively. At all levels
of biological organization (plants, bacteria, yeasts, birds, mammals, etc. ) de
novo biosynthesis of inosinic acid, the initial purine formed de novo, involves
uniformly (with few minor deviations) derivation of N-9 and N-3 from the
amide nitrogen of glutamine; N-7, C-5 and C-4 from glycine; N-1 from aspartic
acid; C-2 and C-8 from the P-carbon cf serine, by way of tetrahydrofolic acid
derivatives; and C-6 from bicarbonate, of which some proportion originates
ultimately from amino acid carbon.6 Thus of the nine atoms that make up the
framework of the uric acid molecule, six are contributed by amino acids di*Based on studies supported in part by a grant-in-aid, A-162, from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, 17. S. Public
Health Service.
614
ARTHRITIS
AND RHEUMATISM,
VOL. 8, No. &PART 1 (OCTOBER),
1965
URIC ACID AS
A NITROGENOUS WASTE
615
rectly, two indirectly, and one in part indirectly. The metabolism of uric acid,
and of purines generally, may therefore be regarded as really an extension of
amino acid metabolism, and will be discussed here in that light.
The adult, full-grown animal does not ordinarily store surplus amino acid
metabolites in the sense that carbohydrate ingested in excess is stored as
glycogen, or ingested fat as triglyceride, but on an adequate diet is in nitrogen
equilibrium: all nitrogen absorbed in excess of what is required to replace
wear-and-tear losses in proteins, purines and all other necessary nitrogenous
compounds must be eliminated as one or another nitrogenous waste; and if
not in one form, then in another. To maintain nitrogen balance, a variety of
intricate metabolic mechanisms are brought into play. The simplest scheme,
f o l l o ~ e dby a number of primitive organisms, and to some extent all the way
up the phylogenetic scale, is to eliminate the excess amino acids intact. For
the most part, however, the amino acids provided in the diet are rapidly removed from the circulation by the liver chiefly, and those that are not utilized
intact for syntheses, or after biotransformation to other amino acids, are degraded. The first step in degradation generally is removal of the a-amino group
by transamination and/ or oxidstive deamination. The liver contains a number
of transaminases, each specific for the reaction between a particular amino
acid and a-ketoglutaric acid, leading to the formation of glutamic acid, which
is then oxidized, probably by glutamic acid dehydrogenase, to yield ammonia.
In the case of glycine, oxidative deamination occurs directly through the action
of glycine oxidase (D-amino acid oxidase); in the case of glutamine, deamidation occurs by the action of glutaminase; and certain other amino acids are
deaminated by other enzymatic reactions.
However liberated from amino acids, the ammonia formed must be gotten
rid of promptly, since accumulation of this toxic compound would be lethal.
This is accomplished in different ways according to the phylogenetic and ontogenetic scale. As these have been described in detail in recent reviews,7-11they
need only be touched on briefly here.
In ammonotelic creatures, all of which live in an aqueous medium, ammonia
may be eliminated as such directly into the ambient water, as in marine and
fresh-water invertebrates, fresh-water teleosts, the lungfish when it is swimming, and (as ammonia or ammonium bicarbonate) in aquatic forms of amphibia and reptiles.12 With emergence onto land, however, diffusion of ammonia directly into the external environment is no longer possible, except by
the kidney-a subject I shall return to later. In terrestial life, the ammonia derived from amino acids is, instead, rendered innocuous by rapid fixation to
form nontoxic compounds. The chief such reservoirs of ammonia in nontoxic
form are glutamine, the r-amide of glutamic acid, formed from glutamic acid
and ammonia in a reaction catalyzed by glutamine synthetase; and carbamyl
phosphate, formed from ammonia, carbon dioxide and ATP in a reaction
catalyzed by carbamyl phosphate synthetase, with N-acetyl glutamate as cofactor. There are a number of other ways in which ammonia can be fixed
innocuously, as in the formation of asparagine (notably in plants), trimethylamine oxide (notably in teleosts), etc., and these compounds play important
roles in the elimination of nitrogen at various stages in evolution.
616
GOUT AND PURINE METABOLISM
The glutamine and (in animals possessing carbamyl phosphate synthetase )
the carbamyl phosphate formed from ammonia are utilized, as required, for a
variety of biosynthetic reactions. Glutamine participates in the de novo biosynthesis of purines, proteins, amino sugars (glucosamine) and other amino acids
(histidine); carbamyl phosphate is utilized in the de nooo biosynthesis of pyrimidines ( in forming the intermediate, ureidosuccinic acid) and amino acids
(in forming citrulline, intermediate in the elaboration of arginine) . When
there is a surplus of glutamine nitrogen, which is the usual circumstance, it is
disposed of chiefly as uric acid, urea and ammonium, in proportions which
vary greatly at different levels of the phylogenetic scale. Surplus arginine generated from an excess of carbamyl phosphate is eliminated as urea.
Of special significance in the present context is the utilization of glutamine
in the committed and rate-regulating step of the sequential reactions of de
novo purine biosynthesis which, as already mentioned, seem to have endured
virtually unchanged from the most primitive forms of life in early geologic
times. In sharp contrast are the striking differences in the extent to which the
purines so formed are degraded at various levels of the phylogenetic scaIe
(Fig. 4). Contrary to what might be expected, the lower forms of animal life
possess a full complement of enzymes necessary for degrading purines completely, and as we go up the evolutionary scale, generally speaking, the
capacity for metabolizing purines becomes more and more deficient.
Thus bacteria and some marine invertebrates and crustacea degrade purines
via uric acid, allantoin, allantoic acid and urea, all the way to ammonia and
carbon dioxide. In this sequence uric acid is merely another purine metabolite,
of no particular significance, and not an end-product of purine metabolism.
There is successive loss of urease, allantoicase, allantoinase and uricase up
the evolutionary scale. The marine teleosts and elasmobranchs, which require
relatively high plasma concentrations of urea in order to maintain themselves
hypertonic to the surrounding sea water, eliminate a substantial proportion of
their waste nitrogen as urea, Some fish, lacking allantoicase, excrete considerable quantities of allantoic acid. Uric acid is the major nitrogenous waste in
the uricotelic birds and squamatine reptiles, also in most insects, which lack
uricase, allantoinase, etc. In the ureotelic mammals, the bulk of the waste
nitrogen is eliminated as urea, but allantoin is the end-product of purine
metabolism except in the uricase-deficient primates including man, in whom
uric acid is the chief end-product of purine metabolism. In the inborn error of
metabolism of man designated xanthinuria there is a further deficiency of
xanthine oxidase, and in lieu of uric acid there is excretion of hypoxanthine
and xanthine.13 In leeches and the fresh-water mussel Anodonta, hypoxanthine
is excreted regularly because of an innate deficiency in xanthine oxidase. In
some creatures guanase is deficient, so degradation of purines does not proceed
beyond the stage of guanine, as in the spiders, and to some extent in pigs
( guanine gout ) and fish-eating birds ( guano ) .
In terms of adaptation to environment, there are good reasons why one
animal in the phylogenetic scale eliminates its excess nitrogen chiefly as uric
acid (uricotelism) and another chiefly as urea (ureotelism). For the embryonic
development of the uricotelic bird and reptile in a closed environment, the
URIC ACID AS A NITROGENOUS WASTE
617
OH
I
y 2
Guanine
Aden i ne
4 guanase 1
1( adenase 1
(
OH
OH
I
I
C
I
I1
CH
/LN
/
HO-C+
N
'
N"
I
'c-"
HC\
NC'N
( x ant hine
oxidase)
"
H
Xanthine
( xanthine oxidase 1
0
ii
HN
I
C"\
I1
( uricase
c-0
N
/
'
p,
H2rll
O'C,
+
I
COOH
Glyoxylic acid
,c,
CrO
I
/
N H Z
H
Allantoin
Uric acid
HC=O
H
O H
0 4 , ,c,
N
H
/
Hypoxanthine
1
A
CH
II
1(aliantoinase 1
C=O
I
,
al lantoicase
H2
y
O=C
I
HN-C-
NH2
y 2
COOH C = O
1
I
NH
H
-%
Allantoic Acid
2Urea
2 NH3 t C02
Fig. 4.-The
Major Steps in the Complete Degradation of Purines.
cleidoic egg, it is essential that the excreta of the embryo be eliminated with a
minimum of water, as Needham has emphasized,? and the same applies to
conservation of water in the adult birds and reptiles living in arid climes.
For this purpose uric acid is ideal because it is so insoluble in water that it can
be evacuated virtually in a solid state, and birds and reptiles possess a capacious cloaca which can deal with large quantities of semi-solid masses of uric
acid. But what is essential for survival in the uricotelic bird and squamatine
reptile would be promptly lethal for ureotelic man. If a human subject who
puts out 15 Gm. of urinary nitrogen a day in the urine as urea (32 Gm. urea)
were obliged to eliminate the equivalent amount of nitrogen as uric acid, it
618
GOUT AND PURINE METABOLISM
Table l.-ZnfltJence of Protein lntake on the Composition
of the Urinary Total Nitrogen"
Nitrogenous Waste
(Gm./day)
TotaI N
Urea N
Ammonium N
Creatinine N
Uric acid N
Undetermined N
Low-Protein Diet
High-Protein Diet
4.20
2.90
0.17
23.28
20.45
0.82
0.64
0.11
0.30
0.52
1.07
*From Bodansky, M.: Introduction to Phyeiological Chemistry, Fourth Edition, New
York, John Wiley & Sons, 1938, page 454.
0.60
would be necessary to excrete 50 Gm. of uric acid daily, an utter impossibility
because of clogging of the urinary tract. Even the relatively minor trend to
uricotelism in gout causes difficulties enough in this regard.
Ammonotelism, ureotelism and uricotelism are nevertheless quantitative and
not altogether qualitative terms. As P. P. Cohen has shown,1° the free-swimming tadpole is ammonetelic but in the course of metamorphosis develops the
enzymes necessary for urea biosynthesis, and the adult terrestial frog is ureotelic. At sea, the sea-faring turtle is largely ammonotelic, on land uric~telic.~
Even in ureotelic normal man there is a vestige of ammonotelism and uricotelism. By way of illustration we turn to Table 1, showing the distribution of urinary nitrogen in normal man taking a low-protein diet and a
high-protein diet. Of course, in ureotelic man the great bulk of surplus nitrogen on a high-protein diet is eliminated in the urine as urea, with a sevenfold
rise in urea nitrogen, but there are increases also in urinary ammonium, uric
acid and undetermined nitrogen (in large part amino acids). To be sure,
the absolute increases in these latter nitrogenous wastes are trifling in terms
of per cent of the urinary total nitrogen, but they are appreciable in relation
to the quantities excreted on a low-protein diet. Thus the renal elimination
of ammonium nitrogen in enhanced about fivefold, to keep pace with the
marked increase in titratable acidity in accommodating to the rise in urinary
excretion of H + associated with a high-protein diet. There is an approximately threefold increase in uric acid nitrogen excretion, to a substantial
figure of 1.0 Gm. urinary uric acid a day, of which only part can be ascribed
to ingestion of preformed nucleic acids incorporated in a natural high-protein
diet since the urinary uric acid excretion increases also on a diet rich in
proteins or amino acids but devoid of purines.
The data in Table 1 do not, in fact, do full justice to the quantitative significance of the persistence of vestigial ammonotelism and uricotelism in normal
man because they give figures for the composite 24-hour urinary excretion.
After oral administration of NI5-labeled aspartic acid or glycine to normal
man5J4J5 there is a sharply peaked outpouring of N15-labeled ammonium and
uric acid in the urine within the first few hours, whereas the urinary elimination of Nl5-Iabeled urea occurs more slowly and is more ~ustained.~
Shortly
after a high-protein meal, therefore, the urinary ammonium and uric acid may
URIC ACID AS A NITROGENOUS WASTE
619
be assumed to represent, transitorily, a somewhat larger proportion of the
urinary total nitrogen than would appear from the figures in Table 1.
From these considerations the relevant fate of amino acid nitrogen ingested
in surplus by normal man may be surmised as follows. The great bulk of the
excess ammonia derived therefrom is converted to carbamyl phosphate for the
formation of urea at a sustained rate and excretion as such in the urine. Some
of the excess ammonia evidently is rapidly converted to glutamine for prompt
regeneration of ammonia by the kidneys and conversion to uric acid in the
liver, with elimination of these minor nitrogenous wastes in the urine.
According to Pitts,16 glutamine is not only the main source of ammonia production by the kidneys but virtually the sole source, and not only the amide
nitrogen but also the amino nitrogen of glutamine is so utilized. Liberated by
the glutaminases of the kidney, the ammonia diffuses freely across the renal
tubular epithelium along a gradient of hydrogen ion concentration. If the
tubular fluid of the proximal and/or distal nephron is acid, which is the usual
circumstance, ammonia is “trapped there as ammonium ion and eliminated
in the urine as ammonium salts; the analogy with ammonotelism in lower forms
is all the more pertinent here because the luminal fluid which bathes the renal
tubular cells provides, in effect, an external aqueous environment. That proportion of the ammonia which returns to the peritubular circulation is recycled, to be converted in the liver largely to urea for elimination as such
in the urine.
It has already been mentioned that glutamine is a prime substrate in the
first committed and rate-regulating step in de nooo purine biosynthesis, in
which the amide group of glutamine irreversibly displaces the pyrophosphate
of 5-phosphoribosylpyrophosphate to give 5-phosphoribosylamine, a reaction
catalyzed by a specific amidotransferase.GA surplus of available glutamine ac? presumably in vivo, to yield a surplus
celerates this reaction in v i t r ~ , ~and
of inosinic acid and subsequently formed purines. In vivo, by a regulatory
mechanism not yet defined in man, but presumably closely resembling that
described by Magasanik in bacteria,ls the surplus inosinic acid and/or the
guanylic and adenylic acids derived therefrom, is diverted to the “shunt” reactions through which uric acid is directly formed by way of hypoxanthine and
xanthine. This is uricotelism in the same sense that excess amino acid nitrogen
is disposed of as uric acid by the bird and squamatine reptile, and emphasizes
again that, even in man, uric acid should in the final analysis be considered a
nitrogenous waste of amino acid metabolism-by way of purine metabolism,
to be sure.
Uric acid is the chief but by no means the only end-product of amino acid
metabolism by way of purine metabolism in man. Human urine contains small
quantities of a variety of other free purine bases of endogenous origin, in an
aggregate amount of about 30 mg./day.1g,20These include hypoxanthine,
xanthine, guanine, adenine and 6-succinoaminopurine; and the methylated
purine bases 7-methylguanine, 8-hydroxy-7-methylguanine, l-methylguanine,
N2-methylguanine and l-methylhypoxanthine, that presumably are metabolites
of the methylated purine components of s-RNA. Also, small quantities of
620
GOUT AND PURINE METABOLISM
adenosine have been recovered from human urine and inosine and guanosine
have been reported to occur. Moreover, an appreciable proportion of the total
daily production of uric acid in man is excreted via the gut, where it is degraded by the bacterial flora and eliminated not as uric acid but as ammonia
and carbon dioxide.21
I made reference earlier to a s ~ g g e s t i o nthat
~ ~ ~in primary gout there may
be an innate defect in renal production of ammonia. Dr. Y u and I have recently made additional studies on this point, comparing the urinary elimination
of ammonium and titratable acid, at the same (acid) urine pH, in 97 gouty
subjects and 46 nongouty control subjects. Only gouty subjects who were free
of renal disease, so far as could be detected by the conventional criteria, were
selected for study, and as paired controls only those nongouty subjects were
used who excreted distinctly acid urine. It was difficult to find a sufficient
number of suitable nongouty control subjects because of the distinct and persistent acidity of the urine in most patients with primary gout. This undue
urinary acidity, which thus far has not been accounted for, is an important
factor in the predisposition of the gouty to uric acid stone formation, in an
incidence more than a thousandfold greater than in the adult population at
large.
When compared in the fasting, postabsorptive state, under conditions of
ordinary dietary metabolic acid loads, there was much dispersion in the figures
for urinary ammonium excretion, at the same (acid) urine pH, within both
the normal group ( a s has been the experience of others) and the gouty group,
with considerable overlap between the two groups. However, in the 83 gouty
subjects who excreted urine of pH less than 5.7 the mean urinary ammonium
excretion was significantly less than in the nongouty controls, whereas the
mean excretion of titratable acid was not different. Consequently there was a
mean net deficit in elimination of metabolic acid under these conditions. This
was small, averaging 8 pEq./min.
In 24-hour collections of urine, a small mean deficit was again noted in
gouty as compared to nongouty subjects, but the excretion of titratable acid
was increased sufficiently to wipe out any difference in the elimination of
metabolic acid. After administration of ammonium chloride, the urinary excretion of both ammonium and titratable acid increased in the gouty, but significantly less than in the nongouty, leaving a peak deficit in elimination of
metabolic acid averaging 16.2 mEq./day.
It is presumed that this modest deficiency in urinary ammonium excretion
in many patients with primary gout, apparently due to selective impairment
of renal production of ammonia from glutamine, contributes to the undue
acidity of the urine characteristic of the disorder, hence also to the striking
tendency to uric acid urolithiasis. It is further assumed that, in the interest
of nitrogen balance, the surplus glutamine is recycled for conversion in the
liver to extra urea and uric acid, in accord with the results of isotope studies
previously r e ~ o r d e d .I~ should
,~
point out that while the quantity of nitrogen
involved in the deficit in urinary ammonium excretion is trivial in relation to
the total urinary nitrogen excretion, it is large in relation to the urinary uric
acid nitrogen excretion, and not much surplus glutamine utilization for de
621
URIC ACID AS A NITROGENOUS WASTE
novo purine biosynthesis would be required to bring about discernible overproduction of uric acid.
In concluding, I have endeavored in the preceding remarks to portray primary gout as but an incident in the evolutionary development of mechanisms
for the maintenance of nitrogen equilibrium. In this instance the machinery
has gone somewhat awry, as a result of a genetic mutation, and it is suggested
that in the ensuing adjustments in amino acid (glutamine) metabolism, hyperuricemia has developed, with the clinical consequences we designate as primary gout.
REFERENCES
1. Bcnedict, J. D., Roche, M., Yii, T. F.,
Bien, E. J., Gutman, A. B., and
Stettcn, Dew., Jr.: The Incorpmation of Glycine Nitrogen into Uric
Acid in Normal and Gouty Man.
Metabolism 1:13, 1952.
2. Bmedict, J. D., Yu, T. F., Bien, E. I.,
Gutman, A. B., and Stetten, Dew.,
Jr.: A Further Study of the Utilization of Dietary Glycine Nitrogen for
Uric Acid Synthesis in Gout. J. Clin.
Invest. 32:775, 1953.
3. Gutmm, A. B., and Yd, T. F.: Gout, a
Derangement of Purine Metabolism.
Ada. in Int. bled. 5:227, 1952.
4. Gutman, A. B., and Yii, T. F.: On the
Nature of the Inborn Metabolic Err o r ( ~ )of Primary Gout. Trans. Asm.
A m . Physic. 86:141, 1963.
5 . Gutman, A. B., and Yd, T. F.: An Abnormality of Glutamine Metabolism
in Gout. Am. J. Med. 35820, 1963.
6. Buchanan, J. M.: The Enzymatic Synthesis of the Purine Nucleotides,
Haraey Lectures, 54:104, 1960.
7. Needham, J.: Bioclzmdstry and Morphogenesis. Cambridge, Cambridge
University Press, 1942.
8. Florkin, M.: Biochemical Evolution.
New York, Academic Press, 1949.
9. Prosser, C. L.: Comparative Animal
Physiology, Philadelphia, Saunders
1950.
10. Cohen, P. P., and Brown, G. W., Jr.:
Ammonia Metabolism and Urea Biosynthesis, in Comparative Bwchemistry, Florkin, M. and Mason, H. S.
(eds.), Vol. 2. New York, Academic
Press, 1960.
11. Baldwin, E.: A n Introduction to Comparativc Biochemistry, Fourth Ed.
Cambridge, Cambridge University
Press, 1964.
12. Coulson, R. A., and Hernandez, T.:
Biochemistry of the Alligator. ,4
Study of Metabolism in Slow Motion.
Baton Rouge, Louisiana State University Press, 1964.
13. Engelman, K., Watts, R. W. E., Klinenberg, J. R., Sjoerdsma, A., and Seegmiller, J. E.: Clinical, Physiological
and Biochemical Studies of a Patient with Xanthinmia and Pheochromocytoma. Am. J. Med. 373339,
1964.
14. Wu, H.: Relative Concentrations of
N15 in Urinary Ammonia N and Urea
N After Feeding N15-labeled Cunipounds. J. Gen. Physiol. 34:403,
1951.
15. Gutman, A. B., Yd, T. F., Adler, M.,
and Javitt, N. B.: Intramolecular Distribution of Uric Acid-Nl5 after Administration of Glycine-Nl5 and Ammonium-Nl5 Chloride to Gouty and
Nongouty Subjects. J. Clin. Invest.
41 :623, 1962.
16. Pitts, R. F.: Renal Production and Excretion of Ammonia. Am. J. Med. 36:
720, 1964.
17. Sonne, J. C., Lin, I., and Buchanan, J.
M.: Biosynthesis of the Purines. IX.
Precursors of the Nitrogen Atoms of
the Purine Ring. J. Biol. Chem. 220:
369, 1956.
18. Magasanik, B.: Biosynthesis of Purine
and Pyrimidine Nucleotides, in The
Bacteria. I. C. Gunsalus and R. Y.
Stanier (eds.), Vol. 111, p. 295, New
York, Academic Press, 1962.
19. Weissmann, B., Broniberg, P. A., and
Gutman, A. B.: The Purine Bases of
Human Urine. I. Separation and
Identification. J. Biol. Chem. 224:
407, 1957.
20. Weissmann, B., Bromberg, P. A., and
622
Gutman, A. B.: The Purine Bsses of
Human Urine. 11. Semi-Quantitative
Estimation and Isotope Incorporation. J. Biol. Chem. 224:423, 1957.
21. Sorensen, L. B.: The Elimination of
COUT AND PURINE METABOLISM
Uric Acid in Man Studied by Means
of C14-labeled Uric Acid. Uricolysis.
Scand. J. Clirr. Lab. Inuest. 12,
supp. 54, 1960.
Discussion
DR. BERLINER:
Teleological argument is always dangerous and distasteful,
and I have good reason to believe that this is particularly so in the area of gout.
There is not much sense in making uric acid in the first place, and if it is made,
there is no sense in holding on to any of it, as man appears to do. And if YOU
are going to regulate uric acid production, I can’t think of a poorer way to do
it than to depend on what is left over from ammonia production. This is what
disturbs me about the hypothesis that it is a defect in ammonia formation
which causes overproduction of urate in gout, since ammonia formation is an
extremely variable phenomenon, depending on circumstances. Ammonia excretion may vary 20- or 30-fold in the same individual within a few hours if
the pH of the urine is changed, and ammonia formation will change by a
further factor of 4 or 5 if the diet is shifted from one which gives an alkaline
residue to one which yields an acid residue. As Dr. Gutman has indicated, the
gouty subject receiving an acidifying load puts out far more ammonia than
the normal subject not taking an acidifying load.
All this would imply that there should be tremendous variation in the rate
at which uric acid is produced. Anyone who took a dose of alkali for an appreciable period ought to develop gout.
DR.STETTEN:I think that the etiology of disease should not be confused with
the wisdom of the body. Indeed we have been deceived, I think, by the axiom
that the body always is wise. Clearly, the body is stupid on occasion, and this
stupidity manifests itself in disease. It is equally clear that uric acid is related
to two major disorders-namely, gout and uric acid calculus-and perhaps
others of which I am not aware. So we must not seek wisdom here at all.
DR. BERLINER:I am simply pointing out that if uric acid production were
dependent upon the quantity of ammonia excreted in the urine, there should
be other manifestations of this relationship which are not apparent. The administration of alkali should be reflected in an alteration in uric acid metabolism such as occurs in gout, if indeed this is responsible for gout.
DR. MACASANIK:
In reference to Dr. Berliner’s remarks about teleology, we
are actually talking about evolution, not teleology, but I think it is entirely
respectable, in biology, to indulge in teleology. When we say that the cell
handles something in a certain way because that is good for the cell, this is
really shorthand for saying that there was strong evolutionary pressure over -a
long period of time to select certain mechanisms that were necessary for the
survival of the cell-the cells that did not possess these particular advantages
would be eliminated.
As Dr. Stetten has pointed out, disease is a state in which things just don’t
work out quite right, and obviously in many cases they do not work out quite
right. In the evolutionary process all kinds of residual mechanisms persist,
URIC ACID AS A NITROGENOUS WASTE
623
of which uric acid production may be one. As Dr. Gutman has indicated, uric
acid plays an important role in certain organisms, in which uric acid production makes good sense, and this process is not quite eliminated in higher forms,
in which the production of uric acid may make little or no sense.
I know too little about the problem of ammonia excretion to venture an
opinion as to what is possible and what is not, but I think that the critical
point here is whether glutamine accumulation will speed up purine metabolism, which is what Dr. Gutman’s theory implies. Unfortunately, for various
reasons we could not, in our studies on bacteria, determine whether the first
reaction of purine biosynthesis, in which glutamine participates, is very dependent on glutamine concentration. Perhaps Dr. Wyngaarden might know
about this in other systems. However, this is how I would expect the normal
controls to operate-that the normal feedback inhibition would be counteracted
by glutamine, and purine biosynthesis would be accelerated-if Dr. Gutman’s
idea were to prove to be correct.
It would seem to me that the level of ammonia formation at any one moment
would depend on two principal reactions, if glutamine is a major source of
ammonia: the rate at which glutamine is split, and the rate at which ammonia
is converted to urea. So the argument that under certain conditions of acidity
there is more or less accumulation of ammonia may not really be very pertinent to Dr. Gutman’s hypothesis. If it were shown that, under certain conditions, ammonia accumulates and is rapidly excreted, but simply because urea
is not formed rapidly enough, uric acid would not be formed in excess as long
as the glutamine level fell. The critical point is whether or not there is defective or inhibited conversion of glutamine to ammonia.
DR. BERLINER:
There is information at least on one point in this connection.
Less ammonia is formed from glutamine when the urine contains little ammonia. It is not just a matter of forming ammonia and taking it off and converting it to urea. Less ammonia is formed.
DR.GUTMAN:
As Dr. Berliner has pointed out, the excretion of ammonium
by the kidney is very variable, depending on the fluctuations in urine pH
and other factors. There is nevertheless a reciprocal relationship between the
urinary excretion of nitrogen as ammonium and as urea, since if acidosis is induced by administration of an acidifying agent, the ensuing increase in urinary
ammonium nitrogen is accompanied by a proportionate decrease in the elimination of nitrogen as urea. In this way nitrogen balance is preserved. What
we are suggesting in gout is simply an analogous reciprocal relationship between the urinary excretion of nitrogen as ammonium and as uric acid, although on a much smaller scale because comparatively little nitrogen is eliminated as uric acid.
Whether or not there is any increase in uric acid formation by the liver
when the urine is alkaline and little ammonium is excreted in the urine would
depend in part, if our ideas are correct, on how much ammonia and how
much intact glutamine is recycled by the kidneys. Recycled ammonia is converted in the liver to urea, not uric acid, as studies with Nlj-labeled ammonium
salts have shown. Most of the recycled glutamine also is converted ultimately
to urea, but some presumably would be converted to uric acid, as our isotope
624
GOUT AND PURINE METABOLISM
studies suggest. If we assume that in the kidney of gouty subjects there is a
deficiency, let us say of glutaminase I, I would expect that more intact glutamine would be recycled and that more uric acid would be produced in the
liver. This also is what our isotope studies suggest.
DR. WYNGAARDEN:
I can comment briefly on Dr. Magasanik‘s question. The
Km of the amidotransferase for glutamine is about
The level of glutamine that exists in plasma is well below that, and in the linear portion of the
velocity-substrate curve, One would anticipate that an increase in glutamine
concentration would promote phosphoribosylamine synthesis, but if there is an
increase in the steady-state level of glutamine in gout it is not reflected in
the plasma level. Dr. Stanton Segal and I looked at plasma glutamine levels
some time ago, and discovered that they were normal in gouty subjects,2 in
contrast to an earlier claim that they were low.
The data on ammonia excretion in gout are very interesting. Others have
also looked at excretion of ammonia in hyperuricemic subjects, some of whom
were gouty. There are data published by Atsmon, DeVries and coworkers in
the Israeli literature, references to which are given in their recent book on
uric acid stones.” In addition there are data from Oliver Wrongs laboratory,
which are in press, in the proceedings of a renal conference, held in Budapest a
year or so ago. Both of these groups find that ammonia excretion under acid
load conditions is normal in hyperuricemic subjects with or without gout, in
the absence of some indication of renal insufficiency. In Wrongs study, ammonia production was normal unless creatinine clearance was less than 65 ml.
per min. A low ammonia-titratable acidity ratio has been known to occur with
renal damage since the studies of Lawrence Henderson in 1915. I wonder if
Dr. Gutman could tell us more about the renal status of the patients that comprise his series?
REFERENCES
1. Caskey, C. T., Ashton, D. M., and
U’yngaarden, J. B.: The Enzymology
of Feedback Inhibition of Glutainine
Phosphoriboaylpyrophosphate Amidotransferase by Purine Ribonucleotides.
J. B i d . Chpm. 239:2570, 1964.
2. Segsl, S . , and Wyngaarden, J. B.: Plas-
ma Glutamine and Oxypurine Content
in Patients with Gout. Proc. SOC. Exper. Biol. and Mcd. 88:342, 1955.
3. Atsmon, A., DeVries, A,, and Frank, M.:
U ~ Acid
c
Lithiasis. Amsterdam, Elsevier Publishing Co., 1963.
DR. LEVITIN:I was about to make the same point, namely, that the decrease
in urinary ammonium excretion in gout might be a manifestation of early gouty
nephropathy. However, I don’t think that this possibility would be excluded by
a normal blood urea nitrogen or creatinine clearance in any particular instance, because ammonia output may diminish and titratable acid increase in
acquired renal tubular disease, and these changes can occur well within the
spectrum of a normal blood urea nitrogen and creatinine clearance.
DR. GUTMAN:
I cannot comment on Wrong‘s data, which I have not seen,
but I am familiar with the Israeli studies, which refer to Dr. Henneman’s
work on uric acid stone formation in nongouty normouricemic subjects, not to
hyperuricemic or gouty subjects. Dr. Henneman’s finding of reduced urinary
URIC ACID AS A NITROGENOUS WASTE
6
Z
ammonium excretion in nongouty uric acid stone formers has been confirmed
in some laboratories but not by deVries and his coworkers in Israel.
In regard to the possibility that the reduced urinary excretion of ammonium
described in gout was due to acquired renal tubular damage, all of the subjects
studied had no detectable renal damage, a serum non-protein nitrogen less
than 40 mg. per cent, and creatinine clearances within the normal range for
their age. All those with detectable renal damage naturally were excluded from
the study. In general, while there are, of course, many patients with gout who
have marked renal disease, our experience has been that in most instances renal function, as estimated by the usual measures, is remarkably well preserved
in relation to age-and I would include in this the renal tubular transfer of
urate. Most of the reports in the literature indicate what Dr. Wyngaarden has
said of Dr. Wrong’s findings, namely that renal excretion of ammonium is not
appreciably reduced, if the urine is acid, unless there is enough renal damage
to cause significant reduction in renal hemodynamics, and some investigators
stress the persistence of the renal capacity to produce ammonia despite extensive renal damage. However, it is conceivable that the reduction in urinary
ammonium excretion in our gouty subjects was due to very subtle, acquired
damage to the tubules; we can neither demonstrate nor exclude this possibility.
DR. HENNEMAN:
The nongouty subjects with uric acid stone that we studied
and found had a defect in renal formation of ammonia were in the older age
group. Our series was not well controlled as far as age is concerned because we
couldn’t find any uric acid stone formers who were young. How about the age
factor in your studies, Dr. Gutman?
DR. GUTMAN:
The age of our gouty subjects averaged 48 years, and that of
our control subjects 35 years, with much overlap between the two groups. We
had great difficulty getting older nongouty persons to serve as controls. I
cannot exclude the possibility that age played a role in some of our older gouty
subjects.
DR. HENNEMAN:
Just two more comments: The first is that we did not find
any change in urinary uric acid excretion in our uric acid stone patients when
they were given alkali for periods up to six days. The second is that, with Dr.
Wyngaarden, we studied the rate of uric acid production in some patients with
uric acid stone and a defect in renal excretion of ammonia. We found the rate
of uric acid production to be normal.
DR. YU: I would like to emphasize that the most important factor in determining the rate of renal excretion of ammonium is the urine pH, and our
comparisons between gouty and nongouty subjects were made not only when
the urine was distinctly acid in both, but also at the same acid pH. Another
relevant factor is the total urinary nitrogen output, which also was comparable
in the two groups. None of our subjects was taking any acid or alkali during
the control studies. As already made clear, only persons with normal renal
function, as judged by the usual criteria, were included in the study.
DR. SORENSEN:In the isotope studies by Drs. Gutman and Yii that were reported in the American Journal of Medicine in 1963 they included as a control
a nongouty subject who had persistently alkaline urine and also a serum urate
level and urinary uric acid excretion somewhat higher than the other nongouty
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G O U T AND PURINE METABOLISM
control subjects. I would like to ask whether you have measured the urinary
uric acid excretion and uric acid turnover in the same person when the urine is
acid and again when it is alkaline. If, as you imply, more glutamine is utilized
for purine synthesis when the urine is alkaline and less ammonium is excreted,
it might be rather dangerous to alkalinize the urine in gouty subjects who have
a low urinary excretion of uric acid, since this would lead to further overproduction of uric acid.
DR. GUTMAN:We have previously reported that the urinary excretion of
uric acid is slightly less when the urine is made acid and slightly more (some
10 per cent) when the urine is alkalinized. At the time this was interpreted to
mean that there may be a modest nonionic back-diffusion of uric acid in the
tubules, and that this is somewhat increased by acidification and reduced by
alkalinization of the urine. This interpretation may not be correct, but the pH
of the urine may have something to do with the fluctuations in serum urate
that occur in gout, even when the purine and protein content of the diet is
well regulated. I cannot say at the moment.
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