Phosphate-Induced Phosphoribosylpyrophosphate Elevations to Assess Deranged Folate and Purine Nucleotide Metabolism 1

Abstract

Phosphoribosylpyrophosphate (PRPP) levels increase several-fold in HL-60 cells adapted to folate deficiency either by continuous passage in folate-deficient medium or by short-term incubation with 10⁻⁸ M methotrexate (MTX). The addition of folic acid (PteGlu) or 5-formyltetrahydrofolic acid (5-CHO-H₄PteGlu) in the form of Leucovorin normalizes this effect. The reactions for measuring PRPP levels are time and temperature dependent and are influenced by PRPP-reacting substances in undialyzed serum. Inorganic phosphate (PO₄), when added to the assay, markedly stimulates PRPP levels in HL-60 cells and can be used to stress folate-dependent PRPP utilization for purine synthesis. The integrity of the folate-dependent pathways of purine-synthesizing cells can be sensitively assessed by measurement of PRPP levels during a 2-hr assay in the presence of PO₄ in medium free of folate but containing dialyzed serum. In HL-60 cells that are folate deficient or in the presence of MTX (as low as 2 × 10⁻⁹ M), PO₄-stimulated PRPP levels remain elevated due to ineffective utilization unless folate is added to the incubation mixture. The sensitivity of this PRPP assay to metabolically assess the integrity of folate-dependent reactions in purine synthesis is comparable to that of the deoxyuridine suppression assay. Inorganic phosphate can also be used to stimulate the incorporation of purine analogs, such as 6-mercaptopurine, into intact red blood cells which may have therapeutic implications for targeting drug delivery.