The Renal Excretion of Oxypurines

Stephen Goldfinger, … , James R. Klinenberg, J. Edwin Seegmiller

J Clin Invest. 1965;44(4):623-628. https://doi.org/10.1172/JCI105175.

Research Article

Find the latest version: https://jci.me/105175/pdf Journal of Clinical Investigation Vol. 44, No. 4, 1965

The Renal Excretion of Oxypurines *

STEPHEN GOLDFINGER,t JAMES R. KLINENBERG,4 AND J. EDWIN SEEGMILLER § (From the National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.)

The mechanism whereby oxypurines (xanthine concomitant infusions of oxypurines as well as by and hypoxanthine) are excreted by the human allopurinol administration. In addition, the ef- kidney is relevant to several areas of current in- fect of probenecid, sulfinpyrazone, and salicylate vestigation. The use of the xanthine oxidase in- on oxypurine excretion was determined by meas- hibitor allopurinol 1 (4-hydroxypyrazolo (3, 4-d) - uring the influence of these agents on the daily pyrimidine) as an adjunct to cancer chemotherapy urinary excretion of oxypurines in a patient with (1) and in the treatment of hyperuricemia asso- xanthinuria and in a patient receiving allopurinol. ciated with gout (2, 3) results in the accumula- tion of oxypurines that would normally be de- Methods graded to uric acid. The fate of these metabo- All studies were performed while subjects were main- lites is pertinent to the clinical use of such an tained on diets essentially free of purines at the Clinical agent. Study of oxypurine clearance by the nor- Center of the National Institutes of Health. Methylated mal kidney is also of considerable importance in purines in particular were avoided since they interfere helping to define the precise derangement of oxy- with the method used for the determination of oxypurines in the urine (6). Renal clearance studies were carried purine metabolism in patients with xanthinuria out on fasting individuals by standard procedures (7), (4). Dickinson and Smellie have proposed (5) with concomitant inulin infusions providing an index for that along with deficient activity of the enzyme the measurement of glomerular filtration rate. Supple- xanthine oxidase, these patients may have a de- mentary water was ingested to insure urine volumes of fect in the reabsorption of xanthine by the renal 150 to 400 ml per period. Spontaneous voiding at 30 minutes by the properly trained subject ended each col- tubule to account for the high clearance ratio of lection period. Mid-period blood specimens were col- oxypurine to creatinine found in their patient. lected in heparinized syringes, placed on ice immediately, Accordingly, a "combined renal and general meta- and centrifuged within 30 minutes of withdrawal. bolic defect" has been postulated to occur. By Two methods were employed to elevate the normally examining the renal clearance of oxypurines in low serum concentrations of oxypurines when this was normal subjects at serum concentrations approxi- desired. One consisted of the prolonged administration of allopurinol in divided doses of 400 to 800 mg daily. mating those of a xanthinuric patient, this hy- The effectiveness of allopurinol in preventing oxypurine pothesis can be adequately tested. degradation to uric acid is presented elsewhere (1-3). The In the present study, the renal clearance of oxy- second manner by which oxypurine concentrations were purines was examined in gouty subjects without increased was by infusions of xanthine or hypoxanthine. renal disease, as well as in one patient with Sodium xanthine2 and hypoxanthine,3 both over 98% pure, were dissolved in isotonic saline at a concentration xanthinuria and one normal volunteer. Serum of 2 mg per ml with warming, and sterilized by filtration. concentrations of oxypurines were augmented by The product was tested for sterility and pyrogens. that formed at room * Submitted for publication September 28, 1964; ac- Crystals during storage temperature cepted December 21, 1964. were dissolved by warming the solution before use. A t Address: Massachusetts General Hospital, Depart- priming dose of 100 mg of xanthine or hypoxanthine in ment of Medicine, Boston, Mass. a total volume of 100 ml was followed by infusion of t Address: The Johns Hopkins Hospital, Department the same oxypurine at a constant rate of 2 mg per of Medicine, Baltimore, Md. minute. § Address requests for reprints to: Dr. J. Edwin Seeg- Plasma and urinary inulin concentrations were deter- miller, University College Hospital Medical School, mined by the method of Walser, Davidson, and Orloff Medical Unit, University Street, London, W. C. 1, Eng- (8). An enzymatic spectrophotometric assay for uric land. acid was employed (9). Urinary oxypurine concentra- 1 Zyloprim, Burroughs, Wellcome & Co., Tuckahoe, 2 Mann Research Laboratories, Inc., New York, N. Y. N. Y. 3 Sigma Chemical Co., St. Louis, Mo. 623 624 S. GOLDFINGER, J. R. KLINENBERG, AND J. E. SEEGMILLER

TABLE I Renal clearance* of oxypurine and urate relative to inulin in gouty subjects on allopurinol, 400 to 800 mg per day, as compared to a patient with xanthinuria

Plasma Plasma Subject oxypurine urate Cox Cu Ci. COx/CIn CU/C. CoX/Cu mg/100 ml mg/100 ml ml/min ml/min ml/min ml/min Gout E.T. 0.30 2.12 82.9- 13.6 112.5 0.74 0.12 6.1 R.L. 0.40 1.88 43.7 4.3 55.0 0.79 0.08 10.2 T.J.t 0.68 2.03 106.5 7.9 87.5 1.22 0.09 13.5 F.J.t 0.71 3.89 93.7 5.6 81.0 1.16 0.07 16.7 Ccr Cox/Ccr CU/ccr COx/CU Xanthinuria (12) V.P. 0.5 0.3 45.3 1.8 52.8 0.87 0.03 25.2 * The figures for each clearance study represent an average of at least three 30-minute periods. Co, = oxypurine clearance; Cu = urate clearance; Ci = inulin clearance; Ccr = creatinine clearance. t Known "overproducers" of uric acid. tions were determined spectrophotometrically by use of a plasma oxypurine concentration was too low to be meas- Beckman model DU spectrophotometer. The changes in ured accurately by this direct procedure, a modification optical density were measured at 292 m/A after addition was devised whereby the dialyzate of plasma was lyophi- of first xanthine oxidase and then uricase to an appropri- lized and dissolved in a small volume of the pyrophos- ate sample brought to a volume of 3.0 ml in 0.1 M pyro- phate buffer. After being treated with uricase to destroy phosphate buffer pH 8.0 (6). Plasma oxypurines, when preformed uric acid, 3.0 ml was added directly to cuvettes sufficiently elevated by the infusion of xanthine, were for enzymatic assay (6). Final values were expressed determined by measuring the change in optical density in terms of uric acid derived from oxypurines and not at 292 mgm after addition of xanthine oxidase. When the fractionated into relative amounts of xanthine and hy- poxanthine. Although such fractionation is theoretically TABLE II possible, as based on the different molar extinction coeffi- Oxypurine clearance relative to inulin cients of xanthine, hypoxanthine, and uric acid, this could during infusion studies not be achieved with sufficient accuracy to warrant use for calculations of their independent renal clearance. During Plasma hypoxanthine infusion studies, some conversion to xan- oxypurine concen- thine occurred, for xanthine accounted for 15 to 50% of Subject tration COX/Cin excreted oxypurines. The calculation of renal clearance mg/100 ml* in terms of total oxypurine content of plasma and urine Endogenous oxypurine (as expressed in terms of uric acid derived from oxy- concentrations purines) is valid in this situation. W.R. 0.21 0.15 Pharmacological agents known to alter urate excretion J.H.t .13 .03 were administered to a gouty subject receiving allopurinol F.J. .08 .18 to in order to their ef- .28 and a xanthinuric patient study R.J. .12 fect on daily oxypurine excretion. Each 24-hour urine During xanthine infusion collection was stored in the cold with 3.0 ml of added W.R. .48 .43 toluene. After an initial control period, divided oral W.R. on allopurinol .70 .61 doses of salicylate (1.2 to 4.8 g per day), probenecid F.J. .24 1.16 (1 to 3 g per day), and sulfinpyrazone (800 mg per day), E.T. .16 1.01 either alone or in combination, were administered. During hypoxanthine infusion W.R. .32 .69 Results W.R. on .40 .84 allopurinol Table I shows the simultaneous renal clearance J.H.t .19 1.33 rates of urate, oxypurines, and inulin in four J.H. on allopurinol .39 1.26 gouty patients receiving allopurinol. The highest R.J. .22 1.29 R.J. on allopurinol .77 1.49 plasma oxypurine concentrations were found in two brothers (T.J. and F.J.), who were shown * Milligrams uric acid derived from oxypurines. to be overproducers of uric acid by excretion t Normal volunteer; remainder of subjects have gout without overt renal disease. studies (10) and by incorporation of glycine THE RENAL EXCRETION OF OXYPURINES 625

1-C'4 into urinary uric acid (11). The rate of TABLE III clearance of oxypurines was far greater than that Effect of oxypurine infusion on the clearance of uric acid. In fact, the oxypurine clearance ex- of urate relative to inulin ceeded the inulin clearance in the two patients Cu/Ciu Effect of during infusion (T.J. and F.J.) who had the highest plasma oxy- CU/CIn Infused oxy- oxypurine (per cent purine concentrations. Subject Control purine infusion change) The renal clearance of endogenously derived R.J. 0.099 Hypoxanthine 0.095 -4 oxypurines (Ox), urate (U), and creatinine J.H. 0.108 Hypoxanthine 0.107 -1 W.R. 0.028 Hypoxanthine 0.036 +29 (Cr) in a xanthinuric patient has been previously W.R. 0.028 Xanthine 0.029 +4 and is presented in the same J.F. 0.101 Xanthine 0.117 +16 reported (12) 0.116 +21 Table. If we assume that the endogenous cre- E.T. 0.096 Xanthine atinine clearance approximates inulin (In) clear- ance in the absence of frank renal failure (13), the Infusion of either xanthine or hypoxanthine re- clearance ratio for oxypurines (Co,/Ccr) in the sulted in much higher clearances than were ob- xanthinuric patient is in the same range as those tained at endogenous levels. In some cases (E.T., found in gouty patients in whom the plasma oxy- F.J., J.H., and R.J.) Co./Cin values exceeding purine concentrations had been increased to com- 1.0 occurred at lower plasma concentrations with parable levels by administration of allopurinol. infusion of oxypurine than were observed with Table II compares the endogenous clearance administration of allopurinol. However, in three of oxypurines with those obtained when infusions instances when allopurinol was administered dur- of xanthine and hypoxanthine were administered ing an oxypurine infusion study (W.R. X2, and in order to elevate their plasma concentration. R.J.), it resulted in further augmentation of oxy-

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I 0 20 30 40 50 60 70 80 DAY FIG. 1. EFFECT OF URICOSURIC AGENTS ON OXYPURINE EXCRETION IN A PATIENT WITH TOPHACEOUS GOUT RECEIVING ALLOPURINOL, 800 MG PER DAY. 626 S. GOLDFINGER, J. R. KLINENBERG, AND J. E. SEEGMILLER

180 151 |140203 177 9] Average excretion of L I ..LI L LJJ loxypurines/period 300i E Sa/icy/ate 4.8gm/day EEZ=i5i] &,Ifinpyrozone 800mg/day Probenecid (Gm/day)

0 200

wCf) z I FE

- x 0 10QF Li

I 0 2 0 30 40 DAY FIG. 2. EFFECT OF URICOSURIC AGENTS ON OXYPURINE EXCRETION IN A PATIENT WITH XANTHINURIA. purine clearance, probably by promoting further shown in Figure 2. Probenecid and sulfinpyra- increases in the plasma concentration of the in- zone again produced a depression of oxypurine fused material. Of a total of six oxypurine in- excretion, but as before, the most striking reduc- fusion studies, urate clearance increased signi- tion resulted from the combination of sulfinpyra- ficantly during three (Table III). zone and salicylate. Urate excretion by this The effect of probenecid, sulfinpyrazone, and patient, which averaged less than 10 mg per 24 salicylate administration on the urate and oxy- hours, was too variable at these low levels to be purine excretion of a gouty subject treated with demonstrably altered by the drugs. allopurinol is shown in Figure 1. Probenecid and sulfinpyrazone produced a moderate decrease in Discussion oxypurine excretion concomitant with their ex- Despite the structural similarities of the oxy- pected uricosuric action. When salicylate, 4.8 purines xanthine and hypoxanthine to uric acid, g per day, was added to the sulfinpyrazone regi- the dynamics of their renal excretion appear to men, there was significant urate retention, in be different. Oxypurine clearance relative to keeping with previous observations (14). At that of inulin greatly exceeded the normal Cu/C1n the same time, oxypurine excretion was signi- ratio of 0.03-0.12 (10, 15, 16), and in some ficantly reduced to the lowest values encountered instances was observed to be greater than 1.0. during the study. Subsequent withdrawal of This would suggest that reabsorption of filtered sulfinpyrazone unveiled the known uricosuric ac- oxypurines is less efficient and/or that tubular tion of large doses of salicylate, and at this time, secretion plays a more vigorous role in the ex- oxypurine excretion rose to levels approximating cretion of oxypurines as compared to that of uric those seen with the other uricosuric drugs. Low acid. doses of salicylate that produced urate retention The report by Dickinson and Smellie (5) of did not significantly alter oxypurine excretion a xanthinuric patient with a renal clearance of when compared to the final control period. oxypurines that was 85%o of creatinine clearance The effect of the same drugs on the excretion is entirely consistent with our findings (12). of oxypurines by a patient with xanthinuria is Their conclusion that this rapid clearance repre- THE RENAL EXCRETION OF OXYPURINES 627 sents a defect peculiar to xanthinuria is based effective in controlling the hyperuricemia of gout on their values for normal subjects of 10 to 20%. (2, 3). Plasma concentrations of urate have de- Although the data from which this normal range creased by as much as 7 mg per 100 ml; how- was calculated are not presented in their article, ever, the concentration of plasma oxypurines has the studies were performed at low endogenous not exceeded 1 mg per 100 ml. This is doubtless concentrations of plasma oxypurines characteristic due to the more efficient renal clearance of oxy- of control subjects (17). When we elevated this purines, although a reduction in total purine syn- concentration to the levels found in xanthinuria, thesis has also been suggested (2). In either a concomitant increase in oxypurine clearance event, the rapid clearance of oxypurines can be to the same range seen in xanthinuria occurred. considered to be protective against the danger Rapid oxypurine clearance was also observed of their reaching saturation levels in the plasma. when plasma concentrations were augmented by Renal lithiasis from xanthine stone formation infusing xanthine or hypoxanthine. This counters might be anticipated in patients taking allopurinol the possible objection that clearance values ob- chronically since this is a complication of the tained in the presence of a xanthine oxidase in- constitutional lack of xanthine oxidase in xanthi- hibitor (allopurinol) are spurious because it may nuria. However, no well-documented instances block an analogue of xanthine oxidase postulated of this complication from allopurinol therapy have to function as a permease for facilitating renal been reported to date. tubular reabsorption (5). Our findings, there- fore, do not support the view that xanthinuric Summary patients have a combined renal and general meta- bolic defect. The renal clearance of oxypurines was meas- When uricosuric drugs were administered in ured in humans by standard techniques. Rela- conventional dosage to an allopurinol-treated sub- tively low values were observed at endogenous ject and to one with xanthinuria, oxypurine ex- plasma oxypurine concentrations. When these cretion diminished. The precise manner by which concentrations were increased, by direct infusion this effect is mediated is unknown. Gutman, or by allopurinol therapy, oxypurine/inulin clear- Yii, and Berger (18-20) have proposed that in ance ratios increased considerably and at times the case of urate excretion, uricosuric agents block exceeded 1.0. A patient with xanthinuria cleared the tubular reabsorption of uric acid which, ac- oxypurines at a normal rate when allowance was cording to their model, normally handles all that made for her high endogenous plasma oxypurine is filtered. On the other hand, such agents are concentration. Uricosuric agents were found to considered to interfere primarily with tubular reduce the renal excretion of oxypurines in a pa- excretion of urate when they "paradoxically" tient with xanthinuria and in a subject who was exert a urate-retentive effect when given in low receiving allopurinol. dosage. At present, there is insufficient informa- tion available to construct such a bidirectional References model for the handling of oxypurines by the renal tubule. Whether uricosuric agents promote renal 1. Elion, G. B., S. Callahan, H. Nathan, S. Bieber, R. W. Rundles, and G. H. Hitchings. Potentiation retention of oxypurines by inhibiting excretion by by inhibition of drug degradation: 6-substituted tubular cells, by providing more reabsorptive sites purines and xanthine oxidase. Biochem. Pharma- for oxypurines that had been previously occupied col. 1963, 12, 85. by urate, or via still other mechanisms, cannot be 2. Rundles, R. W., J. B. Wyngaarden, G. H. Hitchings, G. B. Elion, and H. R. Silberman. Effects of a answered on the basis of the evidence presented xanthine oxidase inhibitor on thiopurine metabo- in this communication. It is noteworthy that lism, hyperuricemia and gout. Trans. Ass. Amer. probenecid has been shown to exert no influence Phycns 1963, 76, 126. on oxypurine clearance at endogenous serum oxy- 3. Klinenberg, J. R., S. E. Goldfinger, and J. E. Seeg- miller. The effectiveness of the xanthine oxidase purine concentrations (21). inhibitor allopurinol in the treatment of gout. In Recent reports have shown that allopurinol is preparation. 628 S. GOLDFINGER, J. R. KLINENBERG, AND J. E. SEEGMILLER 4. Dent, C. E., and G. R. Philpot. Xanthinuria. An 13. Brod, J., and J. H. Sirota. The renal clearance of inborn error (or deviation) of metabolism. Lan- endogenous "creatinine" in man. J. clin. Invest. cet 1954, 1, 182. 1948, 27, 645. 5. Dickinson, C. J., and J. M. Smellie. Xanthinuria. 14. Yfi, T. F., P. G. Dayton, and A. B. Gutman. Mu- Brit. med. J. 1959, 2, 1217. tual suppression of the uricosuric effects of sulfin- 6. Klinenberg, J. R., S. E. Goldfinger, K. H. Bradley, pyrazone and salicylate: a study in interactions be- and J. E. Seegmiller. An enzymatic spectrophoto- tween drugs. J. clin. Invest. 1963, 42, 1330. metric method for the determination of xanthine 15. Gutman, A. B., and T. F. Yu. Renal function in and hypoxanthine. In preparation. gout. Amer. J. Med. 1957, 23, 600. 7. Smith, H. W. The Kidney, Structure and Function 16. Nugent, C. A., and F. H. Tyler. Renal excretion in Health and Disease. New York, Oxford Uni- of uric acid in patients with gout and in nongouty versity Press, 1951. subjects. J. clin. Invest. 1959, 38, 1890. 8. Walser, M., D. G. Davidson, and J. Orloff. The 17. Dickinson, C. Personal communication. renal clearance of alkali-stable inulin. J. clin. In- J. vest. 1955, 34, 1520. 18. Gutman, A. B., T. F. Yui, and L. Berger. Tubular 9. Liddle, L., J. E. Seegmiller, and L. Laster. The secretion of urate in man. J. clin. Invest. 1959, enzymatic spectrophotometric method for deter- 38, 1778. mination of uric acid. J. Lab. clin. Med. 1959, 54, 19. Yui, T. F., and A. B. Gutman. Study of the paradoxi- 903. cal effects of salicylate in low, intermediate and 10. Seegmiller, J. E., A. I. Grayzel, R. R. Howell, and high dosage on the renal mechanisms for excre- C. Plato. The renal excretion of uric acid in gout. tion of urate in man. J. clin. Invest. 1959, 38, J. clin. Invest. 1962, 41, 1094. 1298. 11. Seegmiller, J. E., A. I. Grayzel, L. Laster, and L. 20. Yui, T. F., and A. B. Gutman. Paradoxical reten- Liddle. Uric acid production in gout. J. clin. In- tion of uric acid by uricosuric drugs in low dos- vest. 1961, 40, 1304. age. Proc. Soc. exp. Biol. (N. Y.) 1955, 90, 542. 12. Engelman, K., R. W. E. Watts, J. R. Klinenberg, A. 21. Gjorup, S., and H. Poulsen. Effects of probenecid, Sjoerdsma, and J. E. Seegmiller. Clinical, physio- cinchophen, and colchicine on the plasma concen- logical, and biochemical studies of a patient with tration and renal excretion of oxypurine in pa- xanthinuria and pheochromocytoma. Amer. J. tients with gout. Acta pharmacol. (Kbh.) 1955, Med. 1964, 37, 839. 11, 343.