Phosphate-induced phosphoribosylpyrophosphate elevations to assess deranged folate and purine nucleotide metabolism.
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 X 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.
D002460:Cell Line; D003857:Deoxyuridine / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects; D005492:Folic Acid / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D002955:Leucovorin / Q000494:pharmacology; D015470:Leukemia, Myeloid, Acute / Q000473:pathology; D008297:Male; D008727:Methotrexate / Q000494:pharmacology; D010428:Pentosephosphates / Q000096:biosynthesis*; D010710:Phosphates / Q000494:pharmacology*; D010754:Phosphoribosyl Pyrophosphate / Q000096:biosynthesis*; D011687:Purines / Q000096:biosynthesis*; D013268:Stimulation, Chemical
D010428:Pentosephosphates; D010710:Phosphates; D011687:Purines; D010754:Phosphoribosyl Pyrophosphate; D005492:Folic Acid; D002955:Leucovorin; D003857:Deoxyuridine; D008727:Methotrexate