Determination of three different pools of reduced one-carbon-substituted folates: I. A study of the fundamental chemical reactions

Abstract

The reduced one-carbon-substituted derivatives of folic acid can be grouped in three pools according to their response to acid treatment. Pool 1 is made up of N 5, N 10-methylene-tetrahydrofolic acid and unsubstituted dihydro- and tetrahydrofolic acid which at pH 1.0 and subsequent exposure to air cleave to p-aminobenzoylglutamic acid. Pool 2 is made up by the acid-stable N 5-methyl-tetrahydrofolic acid, and pool 3 includes N 5, N 10-methenyl-tetrahydrofolic acid, N 10-formyltetrahydrofolic acid, N 5-formyltetrahydrofolic acid, and N 5-formiminotetrahydrofolic acid, all of which convert to the stable N 5, N 10-methenyl-tetrahydro form when acid treated. Conditions are described to selectively cleave the C 9-N 10 bond of the folates of pool 1, pools 1 + 2, and pools 1 + 2 + 3. The cleaved pools are quantitated as the Bratton-Marshall azo dyes of p-aminobenzoylglutamate. The uncleaved pools are converted to Bratton-Marshall-negative products. Pool 1 is determined by converting pool 2 to 4a-hydroxy-5-methyltetrahydrofolic acid and pool 3 to N 10-formylfolic acid, both Bratton-Marshall negative, by 10% hydrogen peroxide oxidation at pH 6.0. Pools 1 + 2 are cleaved with 0.015% hydrogen peroxide and 0.1% potassium permanganate at pH 9.0 which convert the N 5-methyltetrahydrofolic acid to the acid-cleavable N 5-methyl-dihydrofolic acid. Pool 3 oxidizes to the Bratton-Marshall-negative N 10-formylfolic acid. Pools 1 + 2 + 3 are cleaved by first reducing pool 3 to N 5-methyltetrahydrofolic acid with sodium borohydride followed by oxidation at pH 9.0 to its acid-labile dihydro form. Determination of the poly-γ-glutamyl chain length of each pool is possible by chromatographing the azo- p-aminobenzoylpolyglutamates with authentic synthetic markers.