Determination of three different pools of reduced one-carbon-substituted folates. III. Reversed-phase high-performance liquid chromatography of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates and its application to the study of unlabeled endogenous pteroylpolyglutamates of rat liver

A new reversed-phase high-performance liquid chromatographic (hplc) method is described for the separation and quantitation of picomole amounts of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates. In conjunction with our previously described procedures for the differential cleavage of one-carbon-substituted, reduced folates, this hplc method provides a rapid, sensitive, and reproducible approach to the quantitation and chain-length determination of three pools of unlabeled endogenous pteroylpolyglutamates. Analysis of rat liver (n = 9) yielded the following results ( ): total folates 14.5 ± 1.0 nmol/g; folates of pool 1 (5,10-methylenetetrahydro- and unsubstituted tetra- and dihydrofolates) 2.65 ± 0.74 nmol/g; folates of pool 2 (5-methyltetrahydrofolates) 5.30 ± 0.36 nmol/g; and folates of pool 3 (5,10-methenyltetrahydro-, 10-formyltetrahydro-, 5-formyltetrahydro-, and 5-formiminotetrahydrofolates) 6.40 ± 1.60 nmol/g. Most of the folates of rat liver occur as penta- (7.60 ± 0.69 nmol/g) and hexaglutamates (6.00 ± 0.29 nmol/g). In pool 3 the hexaglutamates predominate. We also report experiments showing that folate patterns based on the amount of radioactive label incorporated after a pulse dose of ³H]folic acid differ at all times from the true steady-state pattern of unlabeled endogenous folates.