N Incorporation Into Urinary Uric Acid by Adenine-8-13C in Normal and Gouty Subjects

Suppression of glycine-15N incorporation into urinary uric acid by adenine-8-13C in normal and gouty subjects

J. Edwin Seegmiller, … , John Miller, R. W. E. Watts

J Clin Invest. 1968;47(5):1193-1203. https://doi.org/10.1172/JCI105808.

Research Article

Adenine inhibited the de novo synthesis of purines in both normal and gouty man as shown by inhibition of the incorporation of glycine-15N into urinary uric acid without altering the incorporation of glycine-15N into urinary creatinine. The diminished purine synthesis did not result in a diminution in the 24 hr excretion of uric acid. This observation was explainable in part by the prompt conversion of adenine to uric acid. In addition to this direct conversion, adenine-8-13C provided a slow and prolonged contribution to urinary uric acid.

A feedback inhibition of purine synthesis by nucleotides derived from adenine provides the best interpretation of these results.

Find the latest version: https://jci.me/105808/pdf Suppression of Glycine- 5N Incorporation into Urinary Uric Acid by Adenine-8-`3C in Normal and Gouty Subjects

J. EDWIN SEEGMILLER, JAMES R. KLINENBERG, JOHN MILLER, and R. W. E. WATTS From the Section on Human Biochemical Genetics, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014

A B S T R A C T Adenine inhibited the de novo syn- by Henderson in ascites tumor cells (4). In addi- thesis of purines in both normal and gouty man tion, the administration of the purine precursor as shown by inhibition of the incorporation of 4-amino-5-imidazole carboxamide (AIC) to the glycine-15N into urinary uric acid without altering human subject has been noted to suppress incorpo- the incorporation of glycine-'5N into urinary cre- ration of glycine-15N into urinary uric acid (5). atinine. The diminished purine synthesis did not The precise mechanism of the suppression of result in a diminution in the 24 hr excretion of uric purine synthesis is not well understood. Caskey, acid. This observation was explainable in part by Ashton, and Wyngaarden demonstrated that the prompt conversion of adenine to uric acid. In adenine-containing nucleotides as well as those addition to this direct conversion, adenine-8-13C containing guanine or hypoxanthine exert an in- provided a slow and prolonged contribution to hibitory action on the enzyme responsible for the urinary uric acid. rate-controlling step of purine synthesis (Fig. 1). A feedback inhibition of purine synthesis by This enzyme, phosphoribosylpyrophosphate gluta- nucleotides derived from adenine provides the best mine amidotransferase, is responsible for the for- interpretation of these results. mation of phosphoribosylamine from glutamine and phosphoribosylpyrophosphate (6). The find- INTRODUCTION ing that adenine-14C administered in trace quantities was converted to uric acid at a very slow A suppression of purine synthesis de novo can be rate (7) suggested the possibility that larger doses induced by the preferential utilization of exoge- of adenine might be used to inhibit, in a physio- nously supplied preformed purines in a variety of logical manner, the excessive purine synthesis biological systems. This was observed by Abrams found in some patients with gout without itself in rabbit bone marrow slices (1), by Gots in bac- being degraded immediately to uric acid. terial mutants (2), by Nierlich and Magasanik in In the present study we have observed the effect mouse fibroblasts grown in tissue culture (3), and of the administration of adenine on purine synthe- of Dr. Klinenberg's present address is the University of sis de novo as measured by the incorporation California Center for the Health Sciences, Los Angeles, glycine-'5N into urinary uric acid in normal and Calif. Dr. Watts' present address is St. Bartholomew's gouty subjects. Simultaneous determination of the Hospital, London, England. Address requests for reprints dynamics of the urate pool by means of uric to Dr. Seegmiller, National Institute of Arthritis and acid-2-'4C intravenously "administered has per- Metabolic Diseases, National Institutes of Health, Bethesda, Md. 20014. mitted the appropriate correction for the extra- Received for publication 6 November 1967 and in re- renal disposition of uric acid in the various sub- vised form 3 January 1968. jects (8). Adenine-8-'3C was used in order to The Journal of Clinical Investigation Volume 47 1968 1193 5-Phosphoribosyl-l-Pyrophosphate (PRPP) + Glutamine

Glycine °e

Formate. *, r~~~~~~~~~~~~~~~~~~~~ Nucleic Acids . *. Nuccleic Acids

Guanylic Acid- Inosinic Acid - - Adenylic Ac :id

Guanosine Inosine - Adenosine It Adenine Guanine Hypoxanthine I

Uric Acid FIGURE 1 Pathway of purine biosynthesis showing feedback inhibition by purine nucleotides. assess the extent to which exogenous adenine was exchange resin was removed by filtration, adenine hydro- converted to urinary uric acid. chloride hemihydrate was crystallized from the filtrate, and recrystallized three times from hot water. The over- METHODS all yield from barium carbonate was 52%. The molar absorbency index of the synthesized adenine Adenine-8-3C - HCI - 1/2 H20, containing 9.89 atom % hydrochloride hemihydrate -8-" C (C5H5N5 -HCl /2 H20) excess 13C, was synthesized by a modification of the pro- at pH 2e= = 13,345 and the elemental analysis was as cedure of Clark and Kalckar (9-11). '3C02 was liberated follows: from barium carbonate-s'3C (71.3 mmole; 55.7 atom % excess "C, Eastman Kodak, Rochester, N. Y.) by the calculated: C, 33.47; H, 3.90; N, 38.6; Cl, 19.57 action of orthophosphoric acid, entrained in carbon diox- found: C, 33.25; H, 3.86; N, 38.9; Cl, 19.41 ide-free nitrogen, and trapped in sodium hydroxide. The (Correcting for "3C) solution was cooled to 0'C, titrated to pH 8.5 with sul- furic acid, and the 'C-labeled sodium bicarbonate was re- Glycine containing 97.96 atom %o excess "N was synthe- duced to sodium formate by hydrogen (24 hr at 80'C sized from potassium phthalimide-"N (Iso-met Corpora- and 3700 psi in the presence of 10 g of pallidized char- tion) (13, 14). Uric acid-2-"'C was synthesized as previ- coal [1 g of Pd]) (12). Formic acid-`C was isolated by ously described (15), diluted to a specific activity of steam distillation, the distillate titrated to pH 8.4 with 12.1 ,uc/mmole, and converted to the lithium salt by the sodium hydroxide, and finally evaporated to dryness to addition of an equivalent quantity of lithium carbonate. yield sodium formate-"8C. Sterile distilled water and glucose solution were added N(4,6-diamino-5-pyrimidinyl)-formamide-"C was pre- to give a final concentration of 1 mg of uric acid per ml pared from 4,5,6-triaminopyrimidine (Sigma Chemical of 5% glucose (8). This was sterilized by filtration Co., St. Louis, Mo.) (11) and converted to adenine-8-"C through a Millipore filter, packaged in 10-ml vials, and by heating at 210°C in diethanolamine under nitrogen for tested extensively for sterility and pyrogens before use. 90 min (10). After cooling the reacting mixture on ice All vials were stored at - 5°C. we neutralized the diethanolamine by the addition of an All subjects were hospitalized during the entire study equivalent quantity of concentrated hydrochloric acid and at the Clinical Center of the National Institutes of Health. isolated adenine as the picrate. Adenine picrate was con- The three gouty subjects selected were all known to have verted to adenine hydrochloride by treatment with a satu- excessive production of uric acid from previous studies rated solution of dry hydrogen chloride in anhydrous of their purine synthesis (8). A diet essentially free of acetone. The last traces of picric acid were removed by purines, containing 70 g of protein, 350 g of carbohydrate, stirring an aqueous solution of the adenine hydrochloride and 100 g of fat was given for a period of at least 5 days with Dowex - 1 X 8 (200400 mesh, Cl- form). The ion before the administration of isotopically labeled com-

1194 J. E. Seegmiller, J. R. Klinenberg, J. Miller, and R. W. E. Watts pounds and throughout the period of study. All drugs Normal Male with the exception of maintenance doses of colchicine were J.ES Glycine-15N discontinued before starting the equilibration period. A A- J. ES Glycine-15N and Adenine fluid intake of at least 3 liters/day was maintained during O-O P.R. Glycine-15N 0-0 P.R. Glycine-15N and Adenine the 2-day period immediately after adenine administra- 0.-14 tion in order to minimize the hazard of renal deposition of the 2,8-dioxyadenine formed from adenine by xanthine 4 oxidase action. Such a deposition has been shown to con- 2 0.12 stitute the principal toxic effect of adenine administration in the rat (16). The largest dose of orally adminis- < 0,10 tered adenine that could be given to the rat without causing renal deposition of 2,8-dioxyadenine or other toxic manifestations was found to be 25-50 mg/kg body -0.08 weight. An additional margin of safety in translating z these studies to the human subject is the fact that the cn w 0.06 u specific activity of xanthine oxidase in human liver is only x one-fiftieth the activity of rat liver (17, 18). Adenine-8- C HCl l/2 H20, 11.25 mg/kg body weight, along with . 0.04 2 carrier adenine, 3.6 mg/kg body weight, (Sigma Chemi- 0 cal Co.), was dissolved in a minimum volume of water 4 and administered in orange juice with breakfast. Glycine- 0.02 N was diluted with carrier glycine (Fisher Scientific Co., Pittsburgh, Pa.) to 60.0 atom %o excess in order with con- 1 2 3 4 5 6 7 that data could be directly compared previous DAY trol studies performed on two subjects, G. S. and J. S., (8) and 100 mg/kg body weight was given in breakfast FIGURE 2 Effect of adenine on the incorporation of gly- milk immediately after the intravenous administration of cine-'5N into urinary uric acid in normal subjects. 8 mg of lithium urate-2-"C. A period of at least 6 months elapsed betwen control studies with glycine-m'N and urate- 2-"C and studies with glycine-'lN, urate-2-w"C, and ade- reached in control subjects (Fig. 2) by the 3rd- nine-'3C. Serum urate was determined daily (19). All 4th day at values of 0.1 and 0.13 atom% excess. urine was collected at room temperature with 3 ml of in a toluene as preservative, analyzed for uric acid (19) and Adenine administration resulted substantially creatinine content (20), and then stored at - 50 C until lower enrichment of 15N in each sample of uric isolation procedures could be carried out. Uric acid was acid. In the control studies of three gouty patients isolated from urine as previously described (21). Cre- the maximum enrichment was found on the 1st or atinine was isolated from urine as the picrate and re- 2nd day, with maximum values of 0.3-0.8 atom% crystallized as creatinine zinc chloride (22). The content of '1C and '5N in uric acid and the abundance of I5N excess, about three to six times the values found in creatinine were determined in a consolidated-Nier mass in normal subjects. Adenine administration not spectrometer after appropriate digestion of samples (23). only diminished glycine incorporation into urinary Residual '5N found in uric acid during the 2 days before uric acid but also changed the time course of 15N adenine administration was taken as the natural abundance The of 15N incorporation value for the second study. The "C content of uric acid enrichment. early peak was determined by plating uric acid on planchets and by characteristic of gouty subjects with excessive uric counting at 30% efficiency in a shielded end-window gas- acid synthesis was abolished and the time of ap- flow counter (Nuclear-Chicago Corp., Des Plaines, Ill.) pearance of maximum incorporation was delayed. with coincidence circuitry to give a background of less Although adenine administration to gouty patients than 2 cpm. Counts of all samples were corrected to a diminished the atom per cent excess 15N found in standard thickness represented by 50 mg of uric acid. Selected specimens of uric acid were degraded to al- uric acid to values approaching those found in the loxantin to separate the nitrogen atoms of positions 7 and control studies of normal subjects, they nonethe- 9 from those of 1 and 3 (24, 25). less remained substantially greater than the values found when the same control subjects were given RESULTS adenine. This effect of adenine was also reflected Adenine administration diminished the incorpora- in the total recovery of 15N in urinary uric acid tion of glycine-15N into urinary uric acid in both shown in Table I. normal and gouty subjects (Figs. 2 and 3). The The magnitude of the inhibition of glycine-15N maximum enrichment of uric acid with 15N was incorporation into urinary uric acid in response Suppression of Purine Synthesis by Adenine 1195 .0.8

0.7

u, 0.6

< 0.5 z 2 z 0.4 z to 0.) (I) 0.3 w 0.. 2 0.2 0

0.I FIGURE 3 Effect of adenine on the incorporation on '5N into urinary uric acid in gouty patients. 4 DAY to adenine varied greatly among the various sub- disposition of uric acid. The urate pool size and jects. In the normal subjects the total recovery turnover rate (Table II) were not altered sig- of 15N in urinary uric acid was reduced to 26 nificantly by the administration of adenine. The and 52%o of control values, respectively, while in close agreement of the per cent of urate turnover the gout patients it was reduced to 31-55% of excreted each day (Table II) and the per cent of control values. The 7 day total recovery in the urate-2-"4C recovered in the urine (Table I) pro- urine of the intravenously administered uric vides independent confirmation of the validity of acid-2-14C was used to correct for the extrarenal these determinations.

TABLE I Effect of Adenine on the Recovery over a 7 Day Period of 14C, 1'C, and 15N in Urinary Uric Acid of Normal and Gouty Subjects

Administered adenine-8-13C Administered glycine-15N Administered uric acid-2-14C Uncorrected Corrected % of U Uncor- Corrected G (G/U) X 100 Control rected (JI/U) (Ad/C) Subject C Ad H X100 C Ad C Ad X100

Control P.R. 73 80 14.5 18.1 0.099 0.048 0.116 0.060 52 J.S. 65 75 14.0 18.7 0.16 0.044 0.25 0.059 24 Gout G.S. 48 38 12.3 32.4 0.40 0.098 0.83 0.26 31 T.B. 78 81 32.0 39.5 1.09 0.46 1.40 0.57 41 R.J. 67 70 24.4 34.9 0.69 0.40 1.03 0.57 55 Lithium urate-2-14C was administered intravenously and glycine-15N orally with and without administration of adenine-8-18C. C, control study; Ad, study with adenine administration; U, recovery of uric acid-2-14C; H, recovery of BC from adenine-8-1uC; G, recovery of 'IN from glycine-15N. 1196 J. E. Seegmiller, J. R. Klinenberg, J. Miller, and R. W. E. Watts TABLE I I Effect of Adenine Administration on the Urate Pool Size and Turnover Rate of Normal and Gouty Subjects

Turnover Excretion uric Excreted Serum urate Urate pool size, A Turnover rate, K Turnover, KA acid, E E/KA X 100

Subject C Ad C Ad C Ad C Ad C Ad C Ad

mg/100 ml mg pools/day mg/day mg/day % Control P.R. 4.9 6.3 1088 1670 0.53 0.47 580 786 435 597 75 76 J.S. 4.2 4.8 1008 1230 0.67 0.53 729 650 536 480 74 74 Gout G.S. 11.9 11.5 2660 2560 0.49 0.41 1274 1050 529 444 41 42 T.B. 10.3 10.8 2700 3020 0.57 0.60 1540 1820 1242 1453 81 80 R.J. 10.8 10.5 2050 2520 0.80 0.77 1645 1950 1159 1295 70 66

C, control; Ad, study with adenine administration.

Glycine initially contributes primarily to the tions. Adenine administration diminished the spe- 7 nitrogen position of the purine ring and only cific contribution of 15N to position 7 and 9 to secondarily to the remaining nitrogens through a a greater extent than the contribution to positions labeling of the body ammonia and glutamine pools. 1 and 3 of the purine ring in three of four patients Suppression of purine synthesis during the period Table III). The incorporation of glycine-15N into of maximal labeling of the body's glycine pool urinary creatinine was not affected significantly should, therefore, suppress principally the primary by adenine administration (Table IV). contribution of glycine to position 7 of uric acid. The suppression of glycine-'5N incorporation The secondary contribution to the remaining 1, 3, into urinary uric acid was not reflected in a net and 9 positions from the glutamine and ammonia decrease in the serum urate concentration or in pools would be expected to be diminished to a the 24 hr excretion of uric acid (Fig. 4). In fact, lesser extent since the glutamine and ammonia urinary uric acid excretion was substantially pools show a more prolonged pattern of labeling greater during the day of adenine administration with 15N (26, 27). Degradation of uric acid than during the control study in four of the five allowed specific determination of 15N enrichment subjects. at the 1, 3 and, by difference, at the 7, 9 posi- A possible explanation for the transient increase

TABLE III Distribution of 15N in Positions 1, 3 as Compared to 7, 9 of Uric Acid

Atom per cent excess 15N Ratio 15N position 7, 9/ Total Position 1. 3 Position 7, 9 position 1. 3 Subject C Ad C Ad C Ad C Ad Control P.R. 0.0837 0.0355 0.0284 0.0169 0.1390 0.0541 4.89 3.20 J.S. 0.117 0.0325 0.0370 0.0177 0.197 0.0473 5.30 2.67 Gout G.S. 0.0873 - 0.0585 - 0.1161 - 1.98 T.B. 0.6008 0.2070 0.3651 0.1440 0.8365 0.2700 2.29 1.88 R.J. 0.2899 0.1354 0.1535 0.0626 0.4263 0.2082 2.78 3.33

C, control study; Ad, study with adenine. Atom per cent excess 15N in position 1, 3 was determined in alloxantin derived from uric acid, while that in position 7, 9 was calculated by difference. Suppression of Purine Synthesis by Adenine 1197 TABLE IV o 3 Control *m Adenine Effect of Adenine Administration on the Recovery of NORMAL ADEN/NE Glycine-15N in Urinary Creatinine J.S.

Per cent ON recovered in urinary creatinine over a 7 day period 50ojjjg Subject Control Adenine Control P.R. 0.27 0.28 J.S. - 0.24 P.R. - 0 5 Gout 500 G.S. - 0.22 T.B. 0.30 0.31 R.J. 0.27 0.23 14 ' GOUT, G.S. ti2 m in urinary uric acid excretion was found in the prompt conversion of adenine-8-13C to urinary uric acid shown in Fig. 5. The magnitude of the 18C z 0 enrichment of urinary uric acid was similar for wa 0kmin . both normal and gouty subjects. As a result, the ---- R.J. Z ..o total recovery of 13C in urinary uric acid was 1500 largely determined by the daily uric acid production (Table I). The atom per cent excess 13C in 4 urinary uric acid reached a maximum on the 1st or 2nd day in each patient and the subsequent decline in isotope abundance between the 3rd and 7th day followed first-order kinetics, as shown by the linear relationship when the values for isotope abundance were plotted on a logarithmic scale with time (Fig. 6). Comparison of the decline in isotope abundance for both 13C and 14C determined on the same samples of uric acid shows that the 500- 13C values declined much more slowly than did the 14C values in each patient. The numerical val- 01 1000o 01234567~~~~~~1 ues calculated for the first-order rate constant for decline in 13C abundance is shown in Fig. 6. This constant was the same in the normal subjects and in two of the three gouty patients. However, one of the gout patients, T. B., showed a value almost 0 1 2 3 4 5 6 7 twice that found in control subjects, which indi- DAYS cated a more rapid turnover of the pool labeled FIGURE 4 Serum urate concentration and urinary uric by adenine. acid excretion during study periods with and without DISCUSSION adenine administration. Incorporation of glycine-15N into uric acid could reflected in a change in the incorporation of be diminished by several different mechanisms: glycine into other glycine metabolites such as (a) Alteration in the dynamics of the glycine pool. creatinine. The fact that adenine administration Such an alteration in the glycine pool should be did not alter the incorporation of glycine-15N into 1198 J. E. Seegmiller, J. R. Klinenberg, J. Miller, and R. W. E. Watts 0.50

0.45

0.40 0 4 Y 0.35

< z 0.30

Z 0.25 u en w3 0.20 0 wx l 0.15 0

0.10

incorporation of adenine-'5C into 0.05 FIGURE 5 The urinary uric acid in normal subjects and gouty patients. The enrichment of urinary uric acid with 'SC 0 is shown for the 7 days after its administration. DAY urinary creatinine provides evidence against this though repression of enzymes of purine synthesis mechanism. has been shown in bacterial systems, such a mecha- (b) A simple dilution of isotopically labeled uric nism for control of purine synthesis in mammalian acid precursors by the added adenine. Such a cells has not yet been demonstrated. Evidence for dilution should not alter the relative contribution the existence of a feedback control mechanism has of the 15N to the 1 and 3 positions as compared to been presented (4-6) and activation of such a the 7 and 9 positions. The 15N enrichment at the mechanism offers the most likely explanation for 7,9 positions was decreased to a greater extent our results. than at the 1,3 positions in three of the four sub- The suppression of glycine-15N incorporation jects. Furthermore adenine administration altered which resulted from adenine administration was the time course as well as the magnitude of 15N comparable to that produced by administration of incorporation into urinary uric acid. 4-amino-5-imidazole carboxamide (AIC) (5). (c) Depletion of' intracellular 5-phosphoribosyl- The early peak of 15N incorporation which char- 1-pyrophosphate (PRPP) by adenine administra- acterizes gout patients who have excessive uric tion. Recent studies have shown that adminis- acid production was abolished by both substances. tration of much larger quantities of orotic acid, The response of the gout patients to adenine and another substrate known to utilize PRPP in its to AIC shows that the excessive purine synthesis metabolism, did not alter the incorporation of 15N found in these patients is capable of being modu- glycine into uric acid.' lated. The derangements leading to the excessive (d) Inhibition of purine synthesis de novo. Al- purine synthesis in these patients must, therefore,

1 Kelley, W. N., F. M. Rosenbloom, and J. E. Seeg- 2 Seegmiller, J. E., and L. Laster. Effect of 4-amino-5- miller. Effect of orotic acid on incorporation of glycine imidazole carboxamide on the incorporation of glycine N'5 into urinary uric acid. In preparation. N'5 into urinary uric acid in gout. In preparation. Suppression of Purine Synthesis by Adenine 1199 ATOM % EXCESS 13C IN URINARY URIC ACID ATOM % EXCESS 13C IN URINARY URIC ACID 00 Lo 10 o aq (P V ! N - o0O O O0 O o)r -o 0 0 0 o00 0 0 0 O6 * o o 0o0 o 0 0 a o-4 U En

.4-i

Ca cis

Cd~ C)d 0 0 ca,

=W t C 0 0 0 0 0 0 0 0 0 0 0 Uo 0 0 0 0 c 0 0 to N - 0D 0 0 0 U-) CQ N - 0 to C - U- (NIW / SiNnOO) 0._ aiov olun AIVNifn NI Otl AIIAIIlV' 3HIA3dS (3JflNV4/SiNnoD) aiOv oifl AiVNl~n NI °b lO40AJIAA11 3IIJ)3dS ~40

°4- Ca

ATOM % EXCESS 13C IN URINARY URIC ACID ATOM % EXCESS 13C IN URINARY URIC ACID --U

co 0D < 00 CZ co (0 V CN -0 0 C C = 00 _ 0oo C o C u C 0 z 0 *c . a _ C. U0 r- (0 Ca, a-Y

Q a

U-)

04 -- -Cd

a'- C)4-4 0

C\_(.0 *4-icd

0 0> 0 0 o 0 LO o to Ni 0 0D 0D 0 0D 0 0 0D (3NlNIV/ S ANnoo tO C'- - amowln k8VN18n Nl JO lAIIA113 0I1313dS (3NlNIUSIANnoV) Otl aiov oidn AVUNid1Nl °bl AI1A110t DI1013dS z I-

z 0 0 a 4 0 iK

r z c: z

L. 0 I- F: 0 4

w a- CD)

4 DAY (c) FIGURE 6 Continued. constitute a difference in quantitative aspects of patient and in the other gout patients, than in the regulatory mechanism rather than a qualitative normal subjects, as shown by the lower ratio of loss of the ability to respond to these agents. isotope abundance in positions 7 and 9 of uric acid Support for this view has come from studies in as compared to 1 and 3 during control studies and which excessive purine synthesis has been asso- after adenine administration (27). This type of ciated with a complete absence of the enzyme hy- 15N distribution has been taken as indirect evi- poxanthine phosphoribosyltransferase (PRTase) dence of a postulated deficiency of glutaminase (E. C. 2.4.2.8) in children with a familial neuro- (30). However, no defect in glutaminase has yet logical disease (28) and with a partial deficiency been found (31). The presence of this lower ratio, of the same enzyme in certain patients with gout in a patient who has a deficiency of PRTase, sug- who produce excessive quantities of uric acid (29). gests that the abnormal distribution of 15N is Patient R. J. had such a partial deficiency. The related to the presence of excessive uric acid pro- activity of the enzyme PRTase in his erythrocytes duction in some less specific manner that is not was only 1 %o of normal values (29). This enzyme immediately apparent. deficit interferes with the reutilization of hypoxan- Despite the decrease in glycine-15N incorpora- thine and guanine for nucleotide formation (Fig. tion into urinary uric acid the total amount of 1) and may be correlated with the earlier appear- uric acid appearing in the 24 hr urine was not ance of maximal 13C labeling of urinary uric acid decreased by adenine administration in the present in this patient and an earlier decline in isotope study. On the contrary, a transient increase in abundance than was observed in the other patients urinary uric acid above values found in control (Fig. 5). The relative contribution of 15N from subjects was observed during the 2 days after the glutamine pool was significantly greater in this adenine administration in four of the five subjects

Suppression of Purine Synthesis by Adenine 1201 studied, and this was greater than could be normal range provides evidence of metabolic het- accounted for by the fraction of administered ade- erogeneity among even those gout patients who nine which appeared in urinary uric acid. Possible have an excessive production of uric acid. explanations of this observation include a mild uricosuric effect induced by adenine or its metabo- REFERENCES lites, or an enhanced degradation of tissue nucleo- 1. Abrams, R. 1951. Some factors influencing nucleic tides induced by adenine. An increase in urinary acid purine renewal in the rat. Arch. Biochem. Bio- uric acid was also found during the 2 days after phys. 33: 436. 2. Gots, J. S. 1957. Purine metabolism in bacteria. V. AIC administration and was attributed to a direct Feed-back inhibition. J. Biol. Chem. 228: 57. conversion of AIC to uric acid (5). 3. Nierlich, D. P., and B. Magasanik. 1965. Regulation The rapid conversion of adenine-8-13C to uric of purine ribonucleotide synthesis by end product acid-13C found in this study is different from the inhibition. The effect of adenine and guanine ribonucleotides on the 5'-phosphoribosylpyrophosphate slow conversion of adenine-14C to urinary uric amidotransferase of aerobacteraerogenes. J. Biol. acid previously reported (7). This discrepancy is Chem. 240: 358. undoubtedly related to the fact that a trace dose 4. Henderson, J. F. 1962. Feedback inhibition of purine of adenine-8-14C was used in the earlier study biosynthesis in ascites tumor cells J. Biol. Chem. whereas a loading dose of adenine-8-13C was used 237: 2631. 5. Seegmiller, J. E., L. Laster, and DeW. Stetten, Jr. in the current study. Evidently under the condi- 1955. Incorporation of 4-amino-5-imidazole-carboxa- tions we used, a substantial portion of the adenine mide-4C" into uric acid in the normal human. J. Biol. was degraded by a fairly direct pathway to uric Chem. 216: 653. acid to account for the prompt appearance of 13C 6. Caskey, C. T., D. M. Ashton, and J. B. Wyngaarden. in urinary uric acid as well as the increased uric 1964. The enzymology of feedback inhibition of glutamine phosphoribosylpyrophosphate amidotransferase acid excreted on the day adenine was administered. by purine ribonucleotides J. Biol. Chem. 239: 2570. As shown in Fig. 1, there is no adenine deaminase 7. Wyngaarden, J. B., J. E. Seegmiller, L. Laster, and to catalyze the direct deamination of free adenine A. E. Blair. 1959. Utilization of hypoxanthine, to hypoxanthine in mammalian tissue. The degra- adenine, and 4-amino-5-imidazolecarboxamide for uric dation to uric acid must, therefore, have occurred acid synthesis in man. Metabolism. 8: 455. after conversion of the adenine to its nucleotide by .8. Seegmiller, J. E., A. I. Grayzel, L. Laster, and L. Liddle. 1961. Uric acid production in gout. J. Clin. adenine phosphoribosyltransferase (A-PRTase) Invest. 40: 1304. or to its nucleoside by purine nucleoside phos- 9. Clark, V. M., and H. M. Kalckar. 1950. A synthesis phorylase. Although patient R. J. has only 1% of adenine labelled with "C. J. Chem. Soc. 1029. of normal PRTase activity, he has a very active 10. Abrams, R., and L. Clark. 1951. Transformylation in A-PRTase and so would be expected to incorpo- the synthesis of adenine-8-C". J. Am. Chem. Soc. rate adenine into nucleotides very effectively. 73: 4609. 11. Balis, M. E. 1957. Adenine 8-C". Biochem. Prep. 5: In addition, the incorporation of a portion of 62. adenine-8-13C into tissue nucleic acids and other 12. Murray, A., III, and D. L. Williams. 1958. Organic tissue nucleotides is a possible explanation for the Synthesis with Isotopes. Interscience Publishers, New continued slow contribution of 13C to urinary uric York. (Pt. 1) . acid. A similar slow contribution of 13C to urinary 13. Schoenheimer, R., and S. Ratner. 1939. Studies in uric acid was observed when AIC-1'C was ad- protein metabolism. III. Synthesis of amino acids ministered (5). The decline in abundance of '3C containing isotopic nitrogen. J. Biol. Chem. 127: 301. 14. Muller, A. F., and W. Bauer. 1953. Uric acid pro- in urinary uric acid after adenine administration duction in normal and gouty subjects determined by followed an exponential decline described by the N'5 labeled glycine. Proc. Soc. Exptl. Biol. Med. formula dI/dt = K'I. Where I is isotope abun- 82: 47. dance, K' is a constant, and t is time. All subjects 15. Cavalieri, L. F., V. E. Blair, and G. B. Brown. showed K' values in the same range except T. B., 1948. The synthesis of uric acid containing isotopic who showed a almost twice that of either nitrogen. J. Am. Chem. Soc. 70: 1240. value 16. Philips, F. S., J. B. Thiersch, and A. Bendich. 1952. control subject or the other gout patients. This Adenine intoxication in relation to in vivo formation discordance in K' values as well as the fact that and deposition of 2,8-dioxyadenine in renal tubules. G. S. and T. B. each had a PRTase activity in the J. Pharmacol. Exptl. Therap. 104: 20.

1202 J. E. Seegmiller, J. R. Klinenberg, J. Miller, and R. W. E. Watts 17. Watts, R. W. E., J. E. M. Watts, and J. E. Seeg- 25. Cavalieri, L. F., and G. B. Brown. 1948. The mecha- miller. 1965. Xanthine oxidase activity in human nism of the oxidation of uric acid studied with tissues and its inhibition by allopurinol (4-hydroxy- isotopic nitrogen as a tracer. J. Am. Chem. Soc. 70: pyrazolo (3,4-d)pyrimidine). J. Lab. Clin. Med. 66: 1242. 688. 26. Benedict, J. D., M. Roche, T. F. Yfi, E. J. Bien, 18. Richert, D. A., and W. W. Westerfeld. 1951. Xan- A. B. Gutman, and DeW. Stetten, Jr. 1952. Incorpo- thine oxidases in different species. Proc. Soc. Exptl. ration of glycine nitrogen into uric acid in normal Biol. Med. 76: 252. and gouty man. Metabolism. 1: 3. 19. Liddle, L., J. E. Seegmiller, and L. Laster. 1959. The 27. Gutman, A. B., T. F. Yu, M. Adler, and N. B. enzymatic spectrophotometric method for determina- Javitt. 1962. The intramolecular distribution of uric tion of uric acid. J. Lab. Clin. Med. 54: 903. acid-N'5 after administration of glycine-N'5 and am- 20. Taussky, H. H. 1954. A microcolorimetric determina- monium-N' chloride to gouty and non-gouty subjects. tion of creatine in urine by the Jaffe reaction. J. Biol. J. Clin. Invest. 41: 623. Chem. 208: 853. 28. F. M. and W. N. 21. Grayzel, A. E., J. E. Seegmiller, and E. Love. 1960. Seegmiller, J. E., Rosenbloom, Kelley. 1967. Enzyme defect associated with a sex- Suppression of uric acid synthesis in the gouty human linked human neurological disorder and excessive by the use of 6-diazo-5-oxo-L-norleucine. J. Clin. Science, Invest. 39: 447. purine synthesis. 155: 1682. 22. Benedict, S. R. 1914. Studies in 29. Kelley, W. N., F. M. Rosenbloom, J. F. Henderson, creatine and creatinine and metabilism. I. The preparation of creatine and creati- J. E. Seegmiller. 1967. A specific enzyme defect nine from urine. J. Biol. Chem. 18: 183. in gout associated with overproduction of uric acid. 23. Rittenberg, D. 1947. The preparation of gas samples Proc. Natl. Acad. Sci. 57: 1735. for mass spectrographic isotope analysis. In Prepara- 30. Gutman, A. B., and T. F. Yfi 1965. Uric acid metabo- tion and Measurement of Isotopic Tracers. D. W. lism in normal man and in primary gout. New Engl. Wilson, A. 0. C. Nier and S. P. Reimann, editors. J. Med. 273: 252. Edward Brothers, Ann Arbor. 31. 31. Pollak, V. E., and H. Mattenheimer. 1965. Glutami- 24. Nightingale, D. 1943. Alloxantin dihydrate. Org. nase activity in the kidney in gout. J. Lab. Clin. Med. Syn. 23: 6. 66: 564.

Suppression of Purine Synthesis by Adenine 1203