ENZYMATIC DECONJUGATION OF ERYTHROCYTE POLYGLUTAMYL FOLATES DURING PREPARATION FOR FOLATE ASSAY - INVESTIGATION WITH REVERSED-PHASE LIQUID-CHROMATOGRAPHY

Erythrocyte (RBC) folates occur mainly as 5-methyltetrahydrofolate pol yglutamates. Determination of RBC folate concentration requires an ini tial deconjugation of these polyglutamates. In this study, existing HP LC methods were adapted to investigate the rate and extent of this dec onjugation process. The action of endogenous plasma pteroylpolyglutama te hydrolase activity was strongly affected by the conditions of sampl e preparation, with pH of the incubation mixture more critical to effe ctive deconjugation than incubation time. Dilution of whole blood with 10 g/L ascorbic acid yielded fast hydrolysis of long-chain polyglutam ates, and total conversion to 5-methyltetrahydrofolate monoglutamate o ccurred after 90 min of incubation at 37 degrees C. In contrast, dilut ion of whole blood with 10 g/L sodium ascorbate, with up to 90 min of incubation at 37 degrees C, yielded a mixture of polyglutamates of 5-m ethyltetrahydrofolate (glu(n) = 1-8). As documented by direct HPLC ana lysis and in concurrent assays with Lactobacillus casei, acidification provided by ascorbic acid can have dramatic effects on the measuremen t of RBC folates.