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ON THE MECHANISM OF OVERPRODUCTION OF URIC IN PATIENTS WITH PRIMARY

James B. Wyngaarden, … , Alberta E. Blair, Linnelle Hilley

J Clin Invest. 1958;37(4):579-590. https://doi.org/10.1172/JCI103641.

Research Article

Find the latest version: https://jci.me/103641/pdf ON THE MECHANISM OF OVERPRODUCTION OF URIC ACID IN PATIENTS WITH PRIMARY GOUT 1 BY JAMES B. WYNGAARDEN, ALBERTA E. BLAIR, AND LINNELLE HILLEY (From the Department of Medicine, Duke University School of Medicine, Durham, N. C., and the National Institute of and Metabolic Diseases, National Institutes of Health, Bethesda, Md.)

(Submitted for publication October 2, 1957; accepted December 26, 1957) In order to study the rate of generation of uric production of uric acid may occur by one mecha- acid in man, Benedict, Roche, Yu, Bien, Gutman, nism in primary gout and by another in prolifera- and Stetten (5-7) administered relatively large tive hematopoietic disorders of the type associated doses of -N15 orally to control and gouty with secondary gout. subjects and measured the appearance of isotope in urinary uric acid. With this technique, they METHODS demonstrated overincorporation of isotope into Materials and instruments. Glycine-1-C4, 1 to 2 mc. uric acid only in gouty subjects excreting large per mM, was purchased from Nuclear Instrument and quantities of uric acid in . Subsequently, Chemical Corporation, Chicago. Purified uricase, xan- however, excessive incorporation of tracer quan- thine oxidase, and catalase were purchased from Worth- tities of glycine-1-C14 into uric acid was demon- ington Biochemicals Corp. Guanase was prepared from rat according to Kalckar (13). Adsorption read- strated in all of seven subjects studied, regard- ings and enzymatic analyses were made in a Beck- less of the stage of severity of the disease or of man DU ultraviolet spectrophotometer. Spectra were the magnitude of urinary uric acid (8). determined in a Beckman DK ultraviolet recording These results indicated that overproduction of spectrophotometer. Chromatographic elution fractions uric acid from glycine and other small molecules were analyzed for compounds absorbing at 253 m/A in a was Gilson ultraviolet scanner and traced by an Esterline- the fundamental defect responsible for hyper- Angus recorder; fractions were collected in a Gilson uricemia in primary gout. The characteristics of volumetric fraction collector. C1' measurements were the curves of uric acid enrichment indicated that made in a Robinson gas-flow counter. excessive biosynthesis of uric acid occurred in Subjects. Pertinent data on subjects of this study are these gouty subjects without the intermediary in- given in Table I. All subjects ingested a poor diet tervention of nucleic for a preparatory period of about five days and for the (5, 7, 8). duration of the study. This diet contained about 30 mg. Since the mechanism of overproduction of uric of purine-N and 55 to 60 Gm. of protein. Glycine-1-C1" acid in primary gout has not been defined in de- was administered in solution orally with breakfast to tail, it was decided to investigate the incorpora- four control subjects, three subjects with primary gout, tion of glycine-1-C'4 into various urinary purine one subject with polycythemia vera and secondary gout, bases and to compare enrichment patterns with and one subject with myeloid metaplasia. Subjects re- ceived from 2.5 to 25 sic. of C14. those of uric acid determined simultaneously. Collections and analyses. Twenty-four hour urine From such comparisons, information was sought collections were obtained under toluene at room tem- regarding purine intermediates involved in normal perature from the second day of dietary preparation and and abnormal uric acid production. These stud- continuously thereafter for periods up to 24 days. When ies have suggested two types of mechanism result- the urinary uric acid excretion had reached a stable low in in value, glycine-1-C1' was administered. Thereafter, sam- ing uric acid synthesis man and have strength- ples were collected and stored at - 100 C. until analyzed. ened the present concepts (5, 7, 8-12) that over- Uric acid analyses on urine and serum were performed 1 This work has been supported, in part, by grants by differential spectrophotometry (14, 15) employing from the National Institutes of Health, United States purified uricase. Public Health Service, Grant No. A-1391, and from the Urinary were precipitated with and United Medical Research Foundation of North Caro- dissolved in HCl according to the Hunter and Givens lina. Preliminary reports of this work have appeared modification (16) of the procedure of Kruger and in abstract form (1-3), and certain aspects have been Schmid (17). On reduction of volume and chilling of included in a recent review article (4). the acid solution, uric acid precipitated and was col- 579 580 JAMES B. WYNGAARDEN, ALBERTA E. BLAIR, AND LINNELLE HILLEY

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C~~~~~~~~~~~~~~~~~~~0b)~~ 0.~Cl ON THE MECHANISM OF URIC ACID PRODUCTION 581 lected for subsequent recrystallization (8, 18) and C' oxidase (, 1-methylguanine), and mi- analysis. The filtrate, containing the urinary purine gration on two-dimensional paper chromatograms (23).4 bases as soluble hydrochlorides, was then treated with The fractions representing a single peak were pooled, ammoniacal silver nitrate and stored in the cold (19). and analyzed quantitatively. Xanthine and The washed silver salts were decomposed with a minimal were analyzed with according to quantity of HCl, and the purine hydrochlorides separated Kalckar (14). was analyzed with xanthine from the AgCl precipitate by centrifugation. This silver oxidase in the presence of catalase according to Klenow precipitation step effected a further purification of the (24). Compound S, separated from adenine on paper in purine bases, which were eventually recovered in a solu- butanol- (20, 23) and eluted by downward ir- tion of HCl of known strength. This solution was dilu- rigation with 0.01 N HCl, was shown not to be a sub- ted to 0.15 N with respect to HCl and was then added strate for xanthine oxidase, so that adenine analyses to a Dowex-50-H+ column, 12X, 200 to 400 mesh. In could be conducted enzymatically on the buffered mixed the last two studies (control H. B. and gouty subject sample obtained from the column. Guanine was assayed IT. H.) urinary purines were isolated by preliminary with guanase in the presence of xanthine oxidase ac- segregation on Dowex-50-H+ according to Weissmann, cording to Kalckar (13). The methylguanine fraction Bromberg, and Gutman (20) and eventually also placed was assayed by ultraviolet absorption alone, since on the analytical column in dilute HCl. The analytical 7-methylguanine, the major constituent, is not responsive column, 2.5 cm. in diameter, contained 150 ml. of resin to guanase (25) or xanthine oxidase (26). 1-Methyl- in acid form,2 washed with till free of Cl-. The guanine, the other component, is deaminated by guanase sample was washed into the column with 50 ml. of water, (25), and the product, 1-methylxanthine, is a substrate and then elution of purines was started with 0.15 N HC1, for xanthine oxidase (27). These reactions permitted under 2 to 4 p.s.i. added air pressure. A continuous the estimate that about 10 per cent of the total methyl- frontal analysis of the elution of ultraviolet absorbing guanine fraction was the 1-methyl isomer. However, compounds was obtained with the recording UV scanner. since these compounds rarely separated. satisfactorily on After passage through the quartz cell of the absorption Dowex-50 or on paper (20, 23), the mixture was treated meter, the eluate was collected in 24 ml. fractions. as a single fraction, and expressed as 7-methylguanine. Uric acid was not retained by the resin, and the small The major goal of the present methods was isolation quantities remaining in the sample after the copper iso- of the purine bases in quantities sufficient for isotope lation procedure were quickly washed through the col- analysis. Therefore, the appropriate fractions were umn. At about tube No. 65 xanthine appeared. As soon routinely pooled and reduced to dryness in vacuo at 30° C. as xanthine was removed, the elution system was changed in a rotary evaporator. Thereafter, the residue was re- so as to provide an acid gradient for the remainder of dissolved (or suspended) in a few ml. of water and trans- the process. This technique is a modification of the step- ferred to a 12 ml. conical test tube. Xanthine was puri- wise elution procedure described by Abrams and Bentley fied by solution in 0.5 N NaOH, decolorized with Norit, (21) and Wall (22).3 A double reservoir was put in and reprecipitated with acetic acid. 7-Methyl-8-hydroxy- place in which the upper flask, a three-liter separatory guanine was recrystallized from HCl by addition of wa- funnel, containing 2.2 L. of 2.67 N HCl, was arranged ter according to Weissmann, Bromberg, and Gutman (28). to feed into the lower flask, a two-liter bottle with an out- Hypoxanthine was isolated as the silver picrate and re- let near its base, containing 2.0 L. of 0.15 N HCl, as the crystallized from concentrated HNO3 (29). 7-Methyl- contents of the latter flowed on the column. Mixing was guanine was precipitated from dilute HCl with effected within the lower chamber by means of a mag- hydroxide at pH 9 and chilling. It was purified by solu- netic stirrer, and the rate of elution was maintained at tion in 2 N HCl, decolorized with Norit, and reprecipi- about 3 ml. per minute by means of 1 to 2 p.s.i. added tated by adjustment of the filtrate to pH 9 with am- air pressure. The identity of the compounds comprising monium hydroxide.5 Adenine was isolated as the picrate the various peaks was established by comparison with known compounds as to elution sequence, spectral char- 4Xanthine, hypoxanthine, guanine, and adenine stand- acteristics at pH 2, 6 and 9, 250/260 and 280/260 m, ab- ards were available. Their spectra, and 250/260 and sorption ratios, response to xanthine oxidase (xanthine, 280/260 m, absorption ratios were obtained in HCl hypoxanthine, adenine) or to guanase in the presence of eluates from the column, which thereafter served as references for characterizations of peaks from urine 2 Dowex-50-H+ resin was prepared by preliminary samples. 7-Methylguanine was identified by comparison washing twice each with 2 N NaOH and 4 N HCl, the with spectral data published by Weissmann, Bromberg, last HCl wash being performed in the column and con- and Gutman (20). 7-Methylxanthine, 7-methyl-8-hy- tinued until ultraviolet absorbing impurities were re- droxyguanine, 1-methylhypoxanthine, N2-methylguanine, moved. 1-methylguanine and "S" were identified by their posi- 3Weissmann, Bromberg, and Gutman (20) have also tions on two-dimensional paper chromatograms, and recently developed a modification of the Wall proce- spectral data following elution from these papers, in dure for use in separation of urinary purines. Their comparison with published data (20, 23). method is similar to that described herein, except that 5The purity of the foregoing compounds was checked stronger concentrations of HCO were employed. frequently during the course of these investigations. 582 JAMES B. WYNGAARDEN, ALBERTA E. BLAIR, AND LINNELLE HILLEY

TABLE II ployed to permit direct comparison of counting values Purity of adenine picrate of purine derivatives with free bases.

Sample cpm/mM RESULTS Picrate of "adenine-S mixture" 782 Separation of urinary purines Picrate of pure adenine 706 A typical elution pattern is illustrated in Figure 1. Uric acid was washed off the resin with water. and recrystallized from 25 per cent acetic acid (30). Immediately following the uric acid peak, there ap- The purity of the adenine picrate was demonstrated as follows: An adenine picrate sample was redissolved in peared a small second peak (a) of unknown iden- 0.01 N HCl, and the picrate removed by repeated extrac- tity. At about tube No. 65, xanthine appeared, tions with ether (31). The was then and thereafter, following the changeover to the placed on filter paper as a band, and a descending chro- gradient elution procedure, the other purine bases matogram developed with butanol-ammonia. Following appeared sequentially as indicated in Figure 1. drying of the paper, a single band was seen in ultraviolet light opposite an adenine marker, whereas no absorbing The tube number at which a specific purine base material was seen opposite a compound S marker. The appeared seldom varied by more than two or three adenine was eluted, and its picrate again prepared. from that indicated in the figure, in about 120 The specific activities of the original and final prepara- separate sample isolations. At about tube No. tions of adenine picrate were in good agreement (Table 110, a second unknown appeared (b). Substances II). The preparation of the picrate, therefore, was an effective means of separating adenine from compound a and b exhibit only sweeping end-absorption in S for isotope analysis. the ultraviolet, and do not migrate in the two- Guanine was not present in urine in sufficient quan- dimensional paper chromatogram. Compound S tity to permit isolation as a crystalline product. There- appears to be identical with the compound so la- fore, after the quantity of guanine in the column eluate beled by Weissmann, Bromberg, and Gutman (20). was established by enzymatic analysis (average ca., 0.3 mg.), 2 to 3 mg. of carrier guanine were added, and Substance c was inconstant. It did not migrate the solution was reduced to dryness, redissolved in a few on the two-dimensional chromatogram. When ml. of water and guanine isolated by neutralization of purines are isolated by initial segregation on the solution with NaOH and chilling (30). In other Dowex-50, the elution pattern from the analytical cases also, where the quantities of purine separated from column does not contain uric acid, peak a or peak the column were very small, as in 6 to 12 hour urine samples, small quantities of the appropriate carrier were c, and small peaks or shoulders representing com- added. pounds V and W (20) are found. None of the The crystalline products were sparingly washed with iced water, dried in vacuo at 1000 C., and transferred to stainless steel planchets as suspensions in acetone or ab- solute alcohol, dried under an infra-red lamp, and counted in a Robinson gas-flow counter (32) having a background of 4 cpm, and an efficiency of 53 per cent at infinite thin- ness. Counting was conducted to less than 10 and usu- ally to less than 5 per cent error (33), and all counting values were corrected to a standard mass of 3.3 mg. per 1.54 cm.' surface area by means of self-absorption cor- rection curves. At this mass, the counting efficiency is 26 per cent. Appropriate conversion factors were em- TUBE NUMBER The values for hypoxanthine and xanthine obtained from the optical density readings of the column eluates agreed FIG. 1. CHROMATOGRAPHIC SEPARATION OF URINARY with values obtained from analyses with xanthine oxidase PURINES (14). Also, the concentrated eluates were shown to Urinary purines, isolated by sequential copper and yield but one ultraviolet absorbing spot each when silver precipitations, were separated on Dowex-50-H+ checked by two-dimensional chromatography (23). resin by elution with HCL. Initial elution was with 0.15 Furthermore, when the crystalline free bases xanthine, N HCL. After appearance of xanthine, gradient elution 7-methylguanine, and 7-methyl-8-hydroxyguanine were was begun with 2,200 ml. of 2.67 N HC1 in the upper recovered from planchets, redissolved in HCI, and re- reservoir and 2,000 ml. of 0.15 N HC1 in the lower chromatographed on paper, they appeared pure. reservoir. Fractions equal 24 ml. each. ON THE MECHANISM OF URIC ACID PRODUCTION 583 50( In order to elucidate more clearly the events occurring immediately following administration of 40(0 URIC ACID glycine, a second control subject, H. B., was given 25 ,uc. of glycine-1-C14 and fractional urine samples 2 301 E were obtained initially. Uric acid became maxi- AHYPOXANTHIINNE E. 200 _ MADENINE - mally labeled in the 6 to 17 hour sample (Figure UL 3), the earliest time a maximum has been ob- 1O served in a normal subject. However, cumulative 0. , . A( a incorporation of C14 into urinary urate in 2.75 days 1 2 3 4 5 6 7 8 9 was 0.064 per cent of the administered dose, an DAYS entirely normal value (8). Hypoxanthine en- FIG. 2. SPECIFIC ACTIVITY VALUES OF URINARY PU- richment was maximal in the 0 to 6 hour sample RINES OF CONTROL SUBJECT C. L., FOLLOWING ORAL and its specific activity was three times that of ADMINISTRATION OF 5 /AC. OF GLYCINE-1-C" uric acid of the same specimen, and almost two times the maximal uric acid value, attained during ultraviolet absorbing compounds gives an orcinol the next 11 hour period. Hypoxanthine enrich- test (34) for pentose, so there is no suspicion that ment promptly fell to rather low specific activity any of the compounds are ribosides. Indeed, ribo- values. Adenine was also maximally labeled in sides, if present, would in all likelihood have been the 0 to 6 hour specimen and declined in isotope hydrolyzed during the initial isolation procedure. concentration thereafter. In contrast, xanthine and showed rather low Labeling of urinary purines in control subjects 7-methylguanine specific activity values in the 0 to 6 hour specimen, and Figure 2 shows the labeling pattern of hypox- passed through isotope concentration maxima in anthine, adenine, xanthine and uric acid, and of a the 6 to 17 hour period, declining strikingly im- few samples of guanine and 7-methylguanine, in mediately thereafter, The parallelisms of the hy-. control subject C. L. following administration of poxanthine and adenine curves and of the xanthine 5 uc. of glycine-l-C14. Hypoxanthine and adenine and 7-methylguanine curves are to be noted. were maximally enriched on Day 1, and thereafter These studies strongly suggest that the rapid rapidly declined to rather low levels of isotope concentration. Xanthine enrichment was also (4740) somewhat greater on Days 1 and 2 than on subse- HYPOXANTHINE quent days, but there was no clear-cut peak on 2000| URIC ACID Day 1 as with hypoxanthine and adenine. The scattered samples of guanine and 7-methylgua- nine similarly do not suggest that either was as N.2000-E XANTHINE highly labeled at any time as were hypoxanthine or adenine. Uric acid attained maximal isotope ADENINE concentration on Day 3, whereafter a slow decline ensued. Hypoxanthine was the only urinary pu-. 1000- rine base showing C14 concentration greater than 7- METHYLGUANINE that of uric acid on Day 1.

6 It appears likely that 7-methylguanine is derived from 0 guanine rather than from more complex guanine com- 2 3 pounds, since only in the free base form is there a re- DAYS placeable on N-7. Thus, the labeling patterns of 7-methylguanine are believed to reflect those of gua- FIG. 3. SPECIFIC ACTIVITY VALUES OF URINARY PU- nine, and therefore also those of guanylic acid. Data RINES OF CONTROL SUBJECT H. B., FOLLOWING ORAL shown in Figure 10, and to some extent Figure 2, sup- ADMINISTRATION OF 25 /LC. OF GLYCINE-1-C" port the interpretataions of the significance of 7-methyl- One 6, one 12, and two 24 hour urine specimens were guanine labeling values, although the interpretations are collected. Note particularly the very high initial C1' only inferential. content of hypoxanthine. 584 JAMES B. WYNGAARDEN, ALBERTA E. BLAIR, AND LINNELLE HILLEY

600 I I II 1 man may well involve primarily the same pathway employed by the in the formation of uric acid 50 >MET HYLGUA NI NE as its major excretory product of waste (29, 42). 400 Labeling of urinary purines in patients with pri- E 300 mary gout E 0. ~~HYPOXANTHINE Three patients with primary gout were studied, 200 one excreting normal quantities of uric acid in urine, one excreting amounts somewhat in ex- cess of normal, and one excreting decidedly ab- normal amounts. In Figure 4 are shown results 2 3 4 5 6 7 8 obtained in gouty subject D. W., who excreted DAYS 317 mg. of uric acid daily. The most striking FIG. 4. SPECIFIC ACTIVITY VALUES OF URINARY Pu- finding of this study was the level of 7-methyl- RINES OF GOUTY SUBJECT D. W., FOLLOWING ORAL AD- guanine enrichment on the first day, which was MINISTRATION OF 2.5 Arc. OF GLYCINE-1-C1' twice that of uric acid of that day, and one-third Note the high initial enrichment value of 7-methyl- higher than the peak uric acid value found on the guanine. second day. The enrichment of adenine in the Permission was granted by Grune & Stratton, Inc. to reproduce this figure which originally appeared in first day sample was also greater than that of uric METABOLISM, volume 6, pp. 244-268, 1957. acid. In contrast to the control studies, the first day hypoxanthine enrichment was of small mag- synthesis of labeled uric acid from glycine-1-C'4 nitude in comparison with that of uric acid in this in control subjects proceeds via hypoxanthine subject, whose fractional incorporation of ad- compounds and probably free hypoxanthine as a ministered glycine-1-C14 into urinary uric acid was major intermediate. The most direct pathway about three times normal (8). employing known reactions involves stepwise In Figure 5 are shown data on E. H., a gouty build up of glycine to (29, 35-39), subject excreting* 1,050 mg. of uric acid daily. dephosphorylation to yield (39), phos- Uric acid was maximally labeled on Day 1, and phorolytic cleavage to yield hypoxanthine (40, the cumulative incorporation of label into urinary 41), and oxidation of hypoxanthine to uric acid uric acid was some four times normal (8). Hy- (14, 27). One would anticipate that enrichment poxanthine labeling was also maximal on Day 1, values of xanthine would reflect its role as product and its specific activity curve seemed to parallel of hypoxanthine and precursor of uric acid in this 600 I sequence. A possible explanation of the pattern . II I I. observed is that hypoxanthine once bound by xan- 500 URIC ACID thine oxidase is not appreciably displaced from the until oxidized to uric and 400 / fully acid, S HYPOXANTHINE that urinary xanthine is derived from organs not E actively participating in rapid urate synthesis, or * 300 perhaps primarily from guanine rather than from ff 200 hypoxanthine. The early maxima of adenine, 7-methylguanine and xanthine suggest that all of the newly formed may be subject in part to cleavage reactions, and that several path- 2 3 4 5 6 7 8 9 10 contribute to of uric acid. DAYS ways may early labeling FIG. 5. SPECIFIC ACTIVITY VALUES OF URINARY PU- However, the initial specific activity data suggest RINES OF GOUTY SUBJECT E. N., FOLLOWING ORAL AD- that inosinic acid cleavage may normally be quan- MINISTRATION OF 5 1AC. OF GLYCINE-1-C' titatively the most important of these pathways. This subject excreted an average of 1,054 mg. of uric Thus, the direct synthesis of uric acid in normal acid daily during this study. ON THE MECHANISM OF URIC ACID PRODUCTION 585 times normal (20). Three samples of this purine were successfully isolated, and specific activity val- ues were similar to those of other purines on Days 2 to 4. These studies suggest that the prompt exces- E 7- Methylguanine sive synthesis of uric acid found in gouty sub- nucleo- ypoxonthine jects may involve early cleavage of purine tides, as in normal subjects. The high early la- 21000 Adnne 7 ety- Hyrxganin beling of 7-methylguanine in two gouty subjects, not found in the control subjects, is of interest. The observation that urinary excretion of 7-methyl- 8-hydroxyguanine may be increased during acute demonstrated again in 0 gouty attacks (11, 43), 2 3 4 patient H. H., further directs attention toward DAYS guanine compounds in this disease. FIG. 6. SPECIFIC ACTIVITY VALUES OF URINARY PU- In order to determine to what extent the RINES IN GOUTY SUBJECT H. H., FOLLOWING ORAL AD- 7-methylguanine enrichment pattern might be MINISTRATION OF 25 /AC. OF GLYCINE-1-C" specific for patients with primary gout, it was that of uric acid. Nevertheless, the hypoxanthine decided to conduct similar studies on patients specific activity values were at all times consider- with known overproduction of uric acid involving ably less than those of uric acid. It was, however, excessive turnover of nucleoproteins. appreciated that an extremely rapid turnover of the hypoxanthine pool in both gouty subjects Labeling of urinary uric acid in patients with pro- D. W. and E. H. might have yielded highly la- liferative hematopoietic disorders beled uric acid, as observed, and that the maximal Since the quantities of uric acid excreted by pa- labeling of urinary hypoxanthine might not be tient D. D., a female with myeloid metaplasia, and discernible within a 24 hour sample. For this by patient J. H., a male with polycythemia and reason, a third gouty subject was studied in whom secondary gout, were considerably greater than fractional urine samples were obtained during the those of control subjects, the C" enrichment data first day. Figure 6 shows data obtained on H. H., a gouty 1400- subject excreting an average of 685 mg. of uric 200 acid per day. Uric acid enrichment was maximal between 6 and 12 hours following oral administra- 9 1000. tion of 25 uc. of glycine-1-C"4, and the cumulative & incorporation of C14 into urinary uric acid was 800_ 0.19 per cent in four days, a value about 2.5 times normal (8). The labeling of other purines was greatest in 7-methylguanine, hypoxanthine, and adenine. However, the shapes of the curves sug- gest that specific activity values were probably higher during the initial hours than is reflected in 2 4 6 8 10 12 14 16 18 20 the pooled 0 to 6 hour sample. It is to be noted DAYS that in this patient, as in patient D. W., the early FIG. 7. SPECIFIC ACTIVITY VALUES OF URINARY URIC specific activity values of 7-methylguanine are high ACID IN MALE CONTROL W. W., AND A MALE SUBJECT compared with results in the two control sub- WITH POLYCYTHEMIA VERA AND SECONDARY GOUT, J. H., AND IN FEMALE CONTROL SUBJECT E. S., AND A FEMALE jects. In this subject, studied during the waning SUBJECT WITH MYELOID METAPLASIA AND HYPERURI- days of a prolonged attack of acute gouty arthritis, CEMIA, D. D. the urinary excretion of 7-methyl-8-hydroxygua- Values have been adjusted to a standard dose of 20 /Ac. nine was about 5 mg. per day, a value two to three of glycine-l-C1' 586 JAMES B. WYNGAARDEN, ALBERTA E. BLAIR, AND LINNELLE HILLEY phases of uric acid production were studied in patient D. D. Labeling of urinary purine bases in a patient with

'~5000- myeloid metaplasia In Figure 10 are plotted the specific activity Q- 4000- values of adenine, guanine, 7-methylguanine, hy- z poxanthine, xanthine and uric acid over a 15 day period following administration of 20 Pc. of gly- a-, cine-1-C4 to subject D. D. The specific activities of all purine bases exceeded that of uric acid on Day 1, xanthine by about 15 per cent, hypoxan- thine by twofold, guanine and 7-methylguanine by more than threefold, and adenine by more than '2' ' 4 ' 6 '8 '10 12 '14 '16' '18' '20 sixfold. There is an immediate sharp decline in DAYS specific activity of these bases such that xanthine FIG. 8. TOTAL C"4 IN DAILY URIC ACID SAMPLES and 7-methylguanine reach low values by the sec- Subjects as described in legend of Figure 7. In ad- ond day. Minimal values of adenine and hypoxan- dition, data on a second male control subject, C. L., have been included. Note the secondary phases of uric acid thine are not reached for five to seven days, and enrichment in subjects D. D. and J. H. these results are presumably a reflection of the slow rate of turnover of the adenine pool (44). are plotted in terms of concentration (Figure 7) Uric acid reached maximal enrichment on Day 3 and total quantityC"cof (Figure 8) in daily sam- and thereafter declined in C14 concentration. ples of urinary uric acid,7 and in terms of cumu- The secondary phase of uric acid enrichment dem- lative excretion of C"s in uric acid (Figure 9). onstrated above is now seen to be a reflection of Although in both patients there is marked early secondary maxima occurring also in the various enrichment of uric acid, in neither is the initial purine bases. The parallelism of the enrichment (first day) synthesis of uric acid-C14 clearly curve of 7-methylguanine (and guanine) with greater than in control subjects, as is regularly that of xanthine, and of adenine with that of hy- found in patients with primary gout (8). In poxanthine, are worthy of note, as is the lack of a both patients D. D. and J. H. there is, however, close correlation between the shapes of the hypox- evidence for a secondary phase of incorporation of anthine and xanthine curves themselves. These C14 into uric acid, maximal on about the eleventh relationships have certain implications regarding to thirteenth days (Figure 8). This second phase precursor-product relationships. Suffice it to of enrichment is in good agreement with the time say here that Figure 10 is believed to present of occurrence of isotope maxima in uric acid fol- lowing glycine-N15 administration (9, 10, 12). a 1.0 The purine intermediates involved in these two 0.8

7Wide fluctuations in uric acid excretion occurred in 0.6 patients D. D. and J. H. Low outputs on Days 9 and 10 in D. D. and Day 9 in J. H., as well as high outputs o z 0.4 on Days 7 and 17 in J. H., have perhaps introduced for- tuitous effects in Figure 8. The difference in C' uric acid output between control and experimental subjects is most a. 0 remarkable on Days 10 and 11. Although the data on 2 4 6 8 10 12 14 16 18 20 control subjects have not been extended beyond 11 days DAYS in the present study, other studies have shown that there FIG. 9. CUMULATIVE INCORPORATION OF CU INTO URI- is no secondary late accentuation of uric acid labeling in NARY URIC ACID OF SUBJECTS DESCRIBED IN LEGENDS OF control or primary gouty subjects (12) such as has FIGURES 7 AND 8 been demonstrated here and elsewhere (12) in patients Data are plotted in per cent of administered dose ver- with myeloproliferative disorders. sus time. ON THE MECHANISM OF URIC ACID PRODUCTION 587 evidence for two classes of pathways of uric acid synthesis, differing remarkably in rate, but in- 2600 volving the same intermediates. The rapid proc- 1400 ADE NINE ess, yielding significant labeling of purine bases on 1200 URI ACID Day 1, is reflected in a rapid enrichment of uric a Cooo acid. The slower process, yielding secondary E a maxima in purine bases 7 to 11 days later, is in 70 YPTKAGUANINE TI turn reflected in a second week enrichment peak 600 This late process is quite consistent 400 in uric acid. XATHINEHPCNHN with known time relationships of ribonucleic acid 200 turnover (45, 46), although other mechanisms, 1 2 3 4 5 6 7 8 9 0 1I 12 13 4 15 such as reutilization of glycine moieties released DAYS from protein, might also be entertained. FIG. 10. SPECIFIC ACTIVITY VALUES OF URINARY PU- RINES IN D. D., A PATIENT WITH MYELOID METAPLASIA WHO RECEIVED 20 ,uC. OF GLYCINE-1-C14 ORALLY DISCUSSION Note particularly the high initial specific activity values From the knowledge that purine bases are not of all purine bases and the secondary maxima occurring formed de novo, but are rather generated as ribo- on Days 7 to 12, illustrating two distinct mechanisms for tides (35, 36, 38, 39, 47-49), it follows that the synthesis of labeled uric acid. Permission was granted by Grune & Stratton, Inc. to prompt appearance of highly labeled purine bases reproduce this figure which originally appeared in ME- in urine results from cleavage of newly formed TABOLISM, volume 6, pp. 244-268, 1957. nucleotides. There is evidence for the existence of this mechanism in all subjects of the present labeling of guanine in subject C. L. and of 7-methyl- study, although there is no way of determining guanine 6 in subject H. B. suggest that the from the data of this paper whether ribotides or guanylic acid cleavage pathway accounts for a deoxyribotides are primarily involved. Insofar smaller portion of the early labeling of uric acid. as the bases generated are hypoxanthine, xanthine, In the patient with myeloid metaplasia, the very or guanine, mechanisms exist for their prompt high specific activity values of guanine com- oxidation to uric acid. Hypoxanthine is readily pounds, and of adenine, suggest that direct path- oxidized to uric acid via xanthine by xanthine oxi- ways involving nucleotides of guanine and ade- dase (14, 27). Guanine is deaminated by guanase nine were quantitatively of greater than usual to form xanthine (13), which is then converted to significance. In view of the recognized overproduc- uric acid by xanthine oxidase. Adenine, however, tion of nucleic acids in this subject, the high label- cannot be directly deaminated in man (50) so that ing of bases derived from these nucleotides is not free adenine is converted to uric acid only follow- surprising. Weissmann, Bromberg, and Gutman ing reincorporation as riboside (40) or ribotide (28) have observed initial high N15 labeling of (51) and further metabolism via inosine or ino- 7-methylguanine, 7-methyl-8-hydroxyguanine and sinic acid (39). adenine in a patient with polycythemia vera given Since it is known that nucleoside phosphorylase glycine-N 5, so that the isotope relationships ob- attacks inosine and much more readily served in subject D. D. may be common in patients than (40, 41, 52) or xanthosine (53), with proliferative hematopoietic disorders. it might be anticipated that stepwise cleavages of The major goal of this study was the acquisition inosinic acid to hypoxanthine and of guanylic acid of information regarding mechanisms by which to guanine, with subsequent conversion of the free persons with primary gout synthesize uric acid bases to uric acid, would constitute major path- excessively. Time relationships suggest that over- ways of the shunt mechanism. The isotope data production of uric acid by these subjects occurs in the control subjects accord with these anticipa- without the intermediary intervention of nucleic tions. In both subjects the initial high labeling of acids (5, 7, 8). The data presented suggest fur- hypoxanthine provides strong indication that ther that rapid synthesis of labeled urate in these cleavage of inosinic acid constitutes a major path- patients occurs via cleavage pathways, way for direct synthesis of uric acid. The early as in normal subjects and patients with myelo- 588 JAMES B. WYNGAARDEN, ALBERTA E. BLAIR, AND LINNELLE HILLEY proliferative disorders, but to a quantitatively ex- uric acid generation in primary gout have previ- cessive extent, and possibly at an accelerated rate, ously been discussed (8). It was demonstrated in that even short initial collection periods failed that the quantity of glycine administered as gly- to reveal the true maximal initial enrichment. cine-N'5 was in excess of tracer levels, and that Furthermore, in both the gouty patients and the the magnitude of early incorporation of label into subjects with myeloid metaplasia, the labeling of uric acid was percentually low as a consequence. guanine compounds appeared to be greater and to The results obtained in patients with proliferative occur earlier than in normals. Since in patients hematopoietic disorders (D. D. and J. H.) with with myeloproliferative disorders a generalized glycine-1-C4 are also different from those reported overproduction of nucleotides is of necessity pres- with glycine-N15. With the N15 technique the ent as an accompaniment of the accelerated turn- early phase of isotope incorporation is not as much over of nucleic acids, the similarity of the initial in evidence, a rather smooth increase in isotope labeling patterns of urinary purines in the patient concentration of uric acid usually being seen, with myeloid metaplasia and in subjects with reaching a maximum at about 10 to 15 days (9, primary gout suggests that the patterns observed 10, 12). The studies with tracer doses of gly- in primary gout may also be a reflection of over- cine-1-C'4 have, however, shown clearly that there production of various nucleotides from glycine and are two major phases to this incorporation proc- other small molecules. Again, the possibility that ess. Although the results of both processes have deoxyribotides may participate must be kept in been interpreted in terms of known reactions, mind. At any rate, it appears unlikely that the accessory pathways of nucleotide and purine syn- metabolic error of primary gout consists solely of thesis leading to production of uric acid have not an accentuation of a single cleavage pathway. been excluded. The time lapses between peak en- The data of the present paper implicate overpro- richments of primary precursor purines and uric duction of uric acid probably via inosinic acid, and acid in some studies, e.g., control subject C. L. abnormal labeling of 7-methylguanine. Recent (Figure 2) and patient D. D. (Figure 10), raise data from Gutman's laboratory (12, 43) impli- such possibilities. Potential reactions of this type, cate altered of 6-succinoaminopurine e.g., involving an intermediary role of uric acid (the aglycone of the intermediate ribotide in riboside, have been discussed (4). It would seem the inosinic-adenylic acid transformation), and more likely, however, that these isotope relation- 7-methyl-8-hydroxyguanine (of unknown origin) ships, as well as the lack of conventional precursor during the acute gouty attack. Thus, the gouty product concentration relationships of hypoxan- derangement would appear to be complex, indeed, thine, xanthine, and uric acid, or of guanine and and probably to involve several compounds at the xanthine, are manifestations of the complexities of nucleotide level. It is not possible at present to kinetics of body pools of intermediates, and of the formulate a hypothesis based on a single missing limitations of information obtainable from urinary or deficient enzyme that would adequately ex- products having diverse origins. Thus these plain these findings. It may be, however, that studies, although suggestive of types of mecha- the metabolic defect of primary gout involves pri- nisms involved in uric acid synthesis, will have marily a defect in regulation of rates of nucleotide to be supplemented by more detailed studies of synthesis rather than a specific missing reaction. reactions conducted in isolated tissue systems. The findings of Seegmiller, Laster, and Stetten Such studies are now in progress. (54) that aminoimidazole carboxamide given con- comitantly with glycine-N15 reduces the incorpora- SUMMARY tion of N15 into urinary uric acid in both normal 1. Procedures are described for isolation from and gouty persons, but frequently less so in the urine of hypoxanthine, xanthine, adenine, guanine, gouty, indicates that purine nucleotide synthesis 7-methylguanine and 7-methyl-8-hydroxyguanine in man is subject to regulatory mechanisms which in crystalline form suitable for measurement of C14 may be defective in primary gout. content. Possible reasons for the differences obtained 2. These purine bases have been isolated fol- with glycine-NI5 and glycine-1-C14 in studies of lowing administration of glycine-1-C04 to control ON THE MECHANISM OF URIC ACID PRODUCTION 589 subjects and patients with primary gout or mye- zation of dietary glycine nitrogen for uric acid syn- loid metaplasia. Specific activity values have been thesis in gout. J. clin. Invest. 1953, 32, 775. compared with of uric acid 7. Stetten, D., Jr. Recent contributions to the under- those over variable pe- standing of the metabolic defect in gout. Geriatrics riods of time in normal subjects and in patients 1954, 9, 163. with disorders of . 8. Wyngaarden, J. B. Overproduction of uric acid as 3. Two major mechanisms exist by which the cause of in primary gout. J. uric acid is generated in man. In addition to the clin. Invest. 1957, 36, 1508. traditional reactions by which of nu- 9. Yii, T. F., Wassermann, L. R., Benedict, J. D., Bien, E. J., Gutman, A. B., and Stetten, D., Jr. A cleic acids gives rise to purine bases susceptible of simultaneous study of glycine-N1" incorporation subsequent conversion to uric acid, there appear into uric acid and heme, and of Fe5' utilization, in to be nucleotide cleavage reactions which yield a case of gout associated with polycythemia sec- highly labeled purine bases in urine within hours ondary to congential heart disease. Amer. J. Med. after administration of glycine-1-C14. 1953, 15, 845. 10. Laster, L., and Muller, A. F. Uric acid production 4. It is proposed that in normal and gouty sub- in a case of myeloid metaplasia associated with jects the synthesis of labeled uric acid from gly- gouty arthritis, studied with N`5-labeled glycine. cine-1-C14 occurs primarily via the "shunt" mecha- Amer. J. Med. 1953, 15, 857. nism; in contrast, in the patient with myeloid 11. Gutman, A. B., Yui, T. F., and Weissmann, B. The metaplasia, a second mechanism, presumably that concept of secondary gout; relation to purine me- tabolism in polycythemia and myeloid metaplasia. of augmented nucleic acid turnover, accounted for Trans. Ass. Amer. Phycns 1956, 69, 229. the late (second week) synthesis of considerable 12. Yui, T. F., Weissmann, B., Sharney, L., Kupfer, S., additional labeled uric acid. and Gutman, A. B. On the biosynthesis of uric acid 5. On the basis of the patterns of labeling of uri- from glycine-N1" in primary and secondary poly- nary purines in patients with primary gout, and a cythemia. Amer. J. Med. 1956, 21, 901. comparison of these patterns with the initial purine 13. Kalckar, H. M. Differential spectrophotometry of purine compounds by means of specific . labeling pattern of a patient with myeloid meta- III. Studies of the enzymes of purine metabolism. plasia and generalized overproduction of nucleo- J. biol. Chem. 1947, 167, 461. tides, it is proposed that patients with primary 14. 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