Orotic aciduria in two unrelated patients with inherited deficiencies of purine nucleoside phosphorylase. A Cohen, … , A J Ammann, D W Martin Jr J Clin Invest. 1977;60(2):491-494. https://doi.org/10.1172/JCI108800. Research Article The urines of two unrelated children with inherited deficiencies of purine nucleoside phosphorylase have been found to contain significant quantities of orotic acid in addition to the previously reported purine nucleosides. The data are consistent with some cell types of these immunodeficient patients being deplete of pyrophosphoribosylphosphate, a precursor of both purine, and pyrimidine nucleosides. It is suggested that the pyrophosphoribosyl-phosphate-depleted cells may be some component of the thymus-dependent immune system. Find the latest version: https://jci.me/108800/pdf CONCISE PUBLICATIONS Orotic Aciduria in Two Unrelated Patients with Inherited Deficiencies of Purine Nucleoside Phosphorylase AMOS COHEN, GERARD E. J. STAAL, ARTHUR J. AMMANN, and DAVID W. MARTIN, JR. From the Departments of Medicine, Biochemistry and Biophysics, and Pediatrics, University of California at San Francisco, San Francisco, California 94143; and the Department of Medical Enzymology, Academic Hospital, Utrecht, The Netherlands A B S T RA C T The urines of two unrelated children tion. There have been several hypotheses proposed with inherited deficiencies of purine nucleoside phos- to explain the immune dysfunction in these enzyme- phorylase have been found to contain significant deficient disorders. The two prominent hypotheses are quantities of orotic acid in addition to the previously a cyclic AMP-mediated lymphotoxicity (1), and a reported purine nucleosides. The data are consistent pyrimidine starvation as originally proposed by Green with some cell types of these immunodeficient patients and Chan (2). In a cell culture model system using being deplete of pyrophosphoribosylphosphate, a pharmacological simulation of adenosine deaminase precursor of both purine, and pyrimidine nucleo- deficiency we have eliminated the hypothesis that sides. It is suggested that the pyrophosphoribosyl- cyclic AMP mediates the toxic effects of adenosine phosphate-depleted cells may be some component of deaminase deficiency. We have also demonstrated the the thymus-dependent immune system. ability of the pyrimidine nucleoside, uridine, to re- verse the cytotoxic effects of the simulated adenosine INTRODUCTION deaminase deficiency, and of simulated purine nucleo- side phosphorylase deficiency (3). The inherited deficiency of purine nucleoside phos- In two independent laboratories we have char- phorylase in humans is associated with a severe de- acterized the disordered purine metabolism in two ficiency of thymus-derived lymphocytes or T cells unrelated patients deficient in purine nucleoside phos- but with minimally impaired or normal function of phorylase (4, 5). Both patients excrete in their urine the bone marrow-derived or B lymphocytes. The de- large quantities of inosine and guanosine and their ficiency of purine nucleoside phosphorylase seems respective 2'-deoxyribonucleosides. During our initial to be causally related to the immunodeficiency since characterizations we attempted to determine whether the inherited deficiency of adenosine deaminase, an the urines of these patients contained orotic acid. enzyme which functionally precedes purine nucleo- This intermediate in pyrimidine biosynthesis might side phosphorylase, is also associated with a T-cell be expected to accumulate if the inhibition of pyrimi- dysfunction and a variably impaired B-cell func- dine nucleotide synthesis occurred at the level of phosphoribosylation. Ishii and Green (6) and Snyder Dr. Martin is an Investigator, Howard Hughes Medical and Seegmiller (7) have independently reported the Institute. Dr. Cohen is supported by a National Institute of in Health Program Project Grant, Clinical Pharmacology- accumulation of orotic acid cultured cells treated Pharmacokinetics. with adenosine. Because of the large quantities of Received for publication 31 March 1977. purine nucleosides in the urines we were not able to The Journal of Clinical Investigation Volume 60 August 1977 -491 -494 491 assay for orotic acid, with the desired sensitivity. OROTIC ACID (pmol) Subsequently we employed a radiochemical method 0 50 100 using isotope dilution techniques to determine the .I ..I concentration of orotic acid in the urine of one purine nucleoside phosphorylase-deficient patient. Having demonstrated orotic aciduria in that patient, we then developed a high pressure liquid chromatographic system for the detection of orotic acid in the presence of high concentrations of purine nucleosides. By the latter technique we have demonstrated orotic aciduria 14C02 in the two CPM ADDED patients with purine nucleoside phos- tOTIC ACID phorylase deficiency. The administration of oral uridine to one patient diminished the excretion of orotic acid. These observations provide considerable insight as to the site of the disordered nucleotide ... .I 5I 1.0.0 metabolism. 10 50 100 METHODS URINE pi Patient FIGURE 1 Isotope dilution assays of orotic acid in urines A (8, 5), and patient B (4) have been described of purine nucleoside phosphorylase-deficient patient A and previously and were not receiving drugs other than pro- normal controls. The method and materials are described phylactic antibiotics at the times of study. The urine from in the text. Urine from a normal child, *; urine from patient the Dutch child (patient B) was lyophilized and sent at known ambient temperature A, 0; quantities (picomoles) of orotic acid standard, from the Netherlands to San 0. PNP, purine nucleoside phosphorylase; CPM, count per Francisco where the analyses were done. The [carboxyl- minute. '4C]orotic acid was purchased from New England Nuclear, (Boston, Mass.), and the orotate phosphoribosyltransferase contaminated with orotidylate decarboxylase was pur- orotic acid in addition to the purine nucleoside chased from P-L Biochemicals, Inc., (Milwaukee, Wis.). components previously reported (5). All other purine and pyrimidine compounds and other rea- The high pressure liquid chromatographic gents utilized were of the highest grades commercially system available. developed for these studies is also capable of detect- Isotope dilution assay. Orotic acid in the urine was ing orotic acid at the 5 ,uM level. The tracings from a assayed by an isotope dilution technique. Aliquots of urine normal urine and the urines of the purine nucleo- were incubated at 370C for 30 min with 0.12 nmol of side phosphorylase-deficient patients are shown in [carboxyl-'4C]orotic acid (50 mCi/mmol), 0.6 nmol of pyro- Fig. 2 with the elution position of orotic phosphorylribosylphosphate (PPriboseP)l in 0.6 ml in the acid indi- presence of an excess (0.2 mglml) of orotate phosphori- cated by the arrow. From standards the concentrations bosyltransferase-orotidylate decarboxylase. The released of orotic acid were calculated and are depicted in [I4C]carbon dioxide was trapped and counted as previously Table I along with the values for four age-matched described (9). normal controls. The treatment of the patients' urines Chromatography. High pressure liquid chromatographic with assays of orotic acid in the urine were performed on an PPriboseP, and orotate phosphoribosyltransferase ALTEX high pressure system with a 152 dual wavelength contaminated with orotidylate decarboxylase elimi- ultraviolet detector. The samples (25 pl) were applied to a nated the presumed orotic acid peak with the appear- Partisil 10-SAX column (H. Reeve Angel & Co., Inc., ance of a new uridine monophosphate peak, thus Clifton, N. J.) and eluted at a flow rate of 2 ml/min with 7 mM potassium phosphate, pH 2.8. The eluates were mon- confirming the identity of the material as orotic acid. itored at 254 and 280 nm. Standard solutions of orotic During the treatment of patient A with oral uridine acid and uridine monophosphate were employed as markers. (100 mg/kg per day) the amount of orotic acid in her urine was reduced (Table I). A similar orotic acid RESULTS reduction during uridine treatment has been reported in primary orotic aciduria (10). The isotope dilution technique for determining orotic Orotidine was absent (<5 ,uM) in all urines examined. acid in the urine samples provided a technique suf- Extracts of the erythrocytes of patient A were not ficiently sensitive to detect 5 ,uM orotic acid even in detectably deficient in the conversion of [carboxyl- the presence of 10 mM inosine, as depicted in Fig. 1. "4C]orotic acid to [14C]02 in the presence of an excess The urine sample from purine nucleoside phosphoryl- (1 mM) PPriboseP. ase-deficient patient A contained approximately 60 ,uM DISCUSSION IAbbreviation used in this paper: PPriboseP, pyrophos- Orotic aciduria is recognized in several inherited phorylribosylphosphate. diseases and reflects the limited conversion of orotic 492 A. Cohen, G. E. J. Staal, A. J. Ammann, and D. W. Martin, Jr. A PNP./- B PNP-/- NOtMAL have a disorder of urea metabolism as evidenced by their tolerance of high protein diets, and their degree CONTROL of orotic aciduria is much less than that present in patients deficient in ornithine transcarbamoylase and in patients with type I orotic aciduria. Low intracellular PPribose P, the other substrate for orotate phosphori- bosyltransferase, also results in the accumulation of orotic acid (12). We have previously demonstrated that the erythrocytes of both purine nucleoside phos- phorylase-deficient