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Determination of 3 different pools of reduced 1 carbon substituted folates 3. reversed phase high performance liquid chromatography of the azo dye derivatives of p aminobenzoyl poly gamma glutamates and its application to the study of unlabeled endogenous pteroyl poly glutamates of rat liver



Determination of 3 different pools of reduced 1 carbon substituted folates 3. reversed phase high performance liquid chromatography of the azo dye derivatives of p aminobenzoyl poly gamma glutamates and its application to the study of unlabeled endogenous pteroyl poly glutamates of rat liver



Analytical Biochemistry 120(2): 323-329



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-.gamma.-glutamates. In conjunction with previously described procedures for the differential cleavage of 1-carbon-substituted, reduced folates, this HPLC method provides a rapid, sensitive, and reproducible approach to the quantitation and chain-length determination of 3 pools of unlabeled endogenous pteroylglutamates. Analysis of rat liver (n = 9) yielded the following results (.hivin.x .+-. SE): 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. Folate patterns based on the amount of radioactive label incorporated after a pulse dose of [3H]folic acid after at all times from the true steady-state pattern of unlabeled endogenous folates.

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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-gamma-glutamates and its application to the study of unlabeled endogenous pteroylpolyglutamates of rat liver. Analytical Biochemistry 120(2): 323-329, 1982

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