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

Abstract

A B S T R A C T 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 daily urinary uric acid excretion (gouty controls); group 3, 7 patients with well-defined enzyme abnormalities and excessive urinary uric 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 PPribose-P generation, while those with excessive PP-ri-

Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions.

“…cells, were added to 1. (20,29). In previous studies in fibroblasts, results of this method have correlated well with in vivo determinations of rates of purine synthesis de novo (20,27,30).…”

“…Fibroblasts were removed from the surface of plastic vessels (Falcon Labware, Oxnard, Calif.) by treatment with trypsin and were washed twice as described (20). The supernatant layer from the final wash was then removed by aspiration, and the cells were resuspended at a density of 5-10 x 106 cells/ml in Krebs-Ringer phosphate buffer (pH 7.4) with 5.5 mM glucose (KRPG).…”

“…Study of inherited abnormalities of human PPribose-P synthetase, in which structurally variant forms of the enzyme with excessive activity lead to increased PP-ribose-P concentration and generation in conjunction with purine nucleotide overproduction and clinical gout (10,(14)(15)(16)(17)(18)(19)(20), have provided one line of evidence supporting a role for PP-ribose-P in the regulation of the rate of purine synthesis de novo (21)(22). Among these abnormal forms of the enzyme, superactive PP-ribose-P synthetases with either diminished sensitivity to feedback inhibition (16,17) or excessive reaction velocity per molecule of enzyme (10) have been described and extensively characterized.…”

“…Among these abnormal forms of the enzyme, superactive PP-ribose-P synthetases with either diminished sensitivity to feedback inhibition (16,17) or excessive reaction velocity per molecule of enzyme (10) have been described and extensively characterized. To date, the clinical manifestations of all the males bearing superactive forms of this enzyme, the structural gene for which has been mapped on the X-chromosome (23)(24)(25), have been restricted to gouty arthritis, uric acid urolithiasis, and/or renal insufficiency first manifested in early adulthood (15,18,20).…”

“…; and H.B., an adult male with gout whose purine nucleotide and uric acid overproduction is associated with excessive fibroblast and erythrocyte PP-ribose-P synthetase activity (18). Fibroblasts were grown in monolayer to confluence (corresponding to greater than -1.5 x 10O cells/75 cm2 culture flask) under conditions described (20). For each fibroblast strain, biochemical determinations were made in duplicate on at least three separate occasions between the 6th and 14th passages in culture.…”