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

Jacobo Ghitis, Carol Schreiber, Samuel Waxman

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3 Scopus citations


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.

Original languageEnglish
Pages (from-to)90-95
Number of pages6
JournalExperimental Biology and Medicine
Issue number1
StatePublished - Oct 1987
Externally publishedYes


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