[THE DIAGNOSTICS OF HEREDITARY DISORDERS OF METABOLISM OF PURINES AND PYRIMIDINES IN CHILDREN USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF ELECTRO-SPRAY TANDEM MASS-SPECTROMETRY].

The article presents data concerning new technique of diagnostic of diseases of metabolism of purines and pyrimidines using high performance liquid chromatography combined with electro-spray mass-spectrometry. The procedure of analysis is described in detail: from pre-analytical stage to interpretation of data of liquid chromatography mass-spectrometry, control of quality of data analysis, mass-spectrometry parameters and chromatographic conditions of analysis of purines, pyrimidines and their metabolites. The reference values are presented for purine and pyrimidine nucleosides and bases in urine of healthy individuals. The chemical structure of purines, pyrimidines and their metabolites and examples of chromato-mass-spectrograms under various hereditary disorders of metabolism of purines and pyrimidines are presented as well. The article is targeted to pediatricians of all profiles, medical geneticists and physicians of laboratory diagnostic.

Purines and pyrimidines determination in urine using high-performance liquid chromatography.

Single purine and pyrimidine bases are involved in two fundamental metabolic pathways that lead to formation of the building stones of DNA and RNA. Purine and pyrimidine nucleotides are also critically important metabolites in many cellular functions. The main breakdown of purines and pyrimidines produces uric acid and B-minoacids, respectively. Therefore, the study of purine and pyrimidine compounds in body fluid has high clinical relevance. We report, in this work, our experience in purines and pyrimidines determination in urine from children presenting with a clinical picture suggesting an inborn these pathways.

Two siblings with classical xanthinuria type 1: significance of allopurinol loading test.

Two brothers with classical xanthinuria who lacked xanthine dehydrogenase activity were encountered. Their hypouricemia was caused by underproduction of uric acid. In their duodenal mucosa, no xanthine dehydrogenase (oxidase) activity was detected. The patients had no symptoms except for duodenal ulcer in one case. The conversion of allopurinol to oxipurinol during an allopurinol loading test for determining the type of classical xanthinuria revealed that the patients had classical type 1 xanthinuria, because aldehyde oxidase activity was present. Furthermore, the allopurinol loading test was conducted to determine the optimal examination times and specimens required for this test.

[Renal hypouricemia].

Renal hypouricemia (serum urate < 2.0 mg/dl) is the inborn disorder due to the impaired tubular urate transport resulted in an increased urate excretion. The inheritance shows an autosomal recessive trait. The prevalence of renal hypouricemia is 0.15% in the outpatients. Based on the 4-component model, by using the pharmacological inhibitor, renal hypouricemia is classified into the 4 types as follows, 1) defective presecretory reabsorption, 2) defective postsecretory reabsorption, 3) enhanced urate secretion, and 4) both defective presecretory and postsecretory reabsorption including the three subtypes. Although renal hypouricemia has asymptomatic as usual, acute renal failure and urolithiasis (hematuria) have been reported as the complication. We further introduced the novel pathophysiology of the renal hypouricemia such as the aciduria correlating to the accelerated urate excretion and the hyperoxipurinemia in this review.

Metabolism of pyrazinamide and allopurinol in hereditary xanthine oxidase deficiency.

The metabolism of pyrazinamide and allopurinol was studied in three xanthinuric patients from two families with hereditary xanthinuria to determine whether both substrates were oxidized only by xanthine oxidase or by other oxidases as well. One xanthinuric patient could neither metabolize pyrazinamide into 5-hydroxypyrazinamide nor allopurinol into oxypurinol. Two xanthinuric patients could metabolize both pyrazinamide into 5-hydroxypyrazinamide and allopurinol into oxypurinol but could not oxidize pyrazinoic acid to 5-hydroxypyrazinoic acid. These findings suggest that xanthinuria comprises at least two subgroups.

Patterns of phosphoribosylpyrophosphate and ribose-5-phosphate concentration and generation in fibroblasts from patients with gout and purine overproduction.

In the majority of patients with gout and excessive uric acid production, underlying enzyme abnormalities have not been identified. In the present study, measurement of both the rate of generation and concentration of phosphoribosylpyrophosphate (PP-ribose-P) and the concentration of ribose-5-phosphate in cultured cells were undertaken to establish a classification of purine overproducers to direct study of additional enzyme defects. Fibroblasts were cultured from 24 individuals assigned to 4 groups: group 1, 5 normal controls; group 2, 5 patients with gout and normal dialy urinary uric acid excretion (gouty controls); group 3, 7 patients with well-defined enzyme abnormalities and excessive urinary acid excretion (4 with hypoxanthine-guanine phosphoribosyltransferase deficiency and 3 with excessive PP-ribose-P synthetase activity); and group 4, 7 patients with gout and excessive uric acid excretion but without grossly abnormal activities of the above enzymes in erythrocyte lysates. In all 14 fibroblast strains from patients showing excessive production of uric acid (groups 3 and 4), rates of purine synthesis de novo and PP-ribose-P concentrations exceeded values for cells from control groups. Cells from group 3 patients with hypoxanthine-guanine phosphoribosyltransferase deficiency showed normal PP-ribose-P generation, while those with excessive PP-ribose-P synthetase activity demonstrated increased generation of this regulatory substrate. All strains from group 3 patients had normal ribose-5-phosphate concentrations. Five cell strains from group 4 patients showed one of the two patterns of abnormalities in these measurements seen in strains from group 3 patients: two resembled hypoxanthine-guanine phosphoribosyltransferase-deficient cells, and three resembled cells with excessive PP-ribose-P synthetase activity. Analyses of erythrocyte enzyme preparations from two of these patients in group 4 have led to identification of a kinetic variant of each enzyme as predicted from the foregoing patterns. Two additional group 4 cell lines that showed increased ribose-5-phosphate concentrations in addition to increased PP-ribose-P concentrations and generation were classified in a separate subgroup, since in the individuals excessive purine synthesis appeared to result from increases ribose-5-phosphate concentration, leading to increased availability of PP-ribose-P. No abnormality in either hypoxanthine-guanine phosphoribosyltransferase or PP-ribose-P synthetase has been found in erythrocyte preparations from one patient so classified.

Recent advances in the identification of enzyme abnormalities underlying excessive purine synthesis in man.

Work is continuing in the attempt to increase knowledge of the regulation of the rate of purine synthesis in man by means of an analysis of biochemical alterations leading to purine overproduction among patients with gout. The authors are now assessing the frequency of kinetic mutations in enzymes whose alterations already have been associated with increased purine synthesis. Efforts in this regard have been rewarded by the identification of a new form of alteration leading to partial deficiency of HGPRT and a kinetic variant of PRPP synthetase with increased affinity for ribose-5-phosphate. In order to identify new forms of enzyme abnormalities associated with excessive purine synthesis, the value of a proposed classification scheme requiring measurement of PRPP and ribose-5-phosphate concentration and generation is being assessed in cultured fibroblasts. It is hoped that the results of these measurements will lead to the identification of additional kinetic variants of presently known enzyme abnormalities and will help to identify new classes of mutants in the regulation of human purine metabolism. The excessive purine synthesis that underlies the hyperuricemia of a substantial number of patients with gouty arthritis reflects alterations in the normal mechanism regulating the rate of purine nucleotide synthesis. The study of such purine "overproducers" has provided insight into the nature of this regulatory mechanism and has underscored the diversity of specific genetic and biochemical aberrations affecting it. Despite these advances, however, less than 10% of all patients with gout and excessive purine production can presently be accounted for by known enzyme abnormalities (1). Recognition that current knowledge of the regulation of the rate of purine nucleotide synthesis in man is incomplete has provided the authors impetus leading to the studies described here, which are preceded by a brief review of background.