High-pressure liquid chromatography separation and determination of rat liver folates

Abstract

The endogenous levels of the various folate compounds in rat liver were determined using high-pressure liquid chromatography for the rapid separation of folate monoglutamate forms with specific quantitation of the folates by microbiological analysis of eluted fractions. The eight folate derivatives that were assayable were tetrahydrofolic acid (H 4PteGlu), 5-methyl-H 4PteGlu, 10-formyl-H 4PteGlu, 5-formyl-H 4PteGlu, 5,10-methenyl-H 4PteGlu, 5,10-methylene-H 4PteGlu, H 2PteGlu, and PteGlu. New techniques for the preparation of tissues were developed in order to reduce the degradation of the folates. Tissue folates were converted to the monoglutamate form by a partially purified hog kidney polyglutamate hydrolase preparation and incubations were carried out at pH 6.0. This minimized folate degradation but still allowed for maximal polyglutamate hydrolase activity. Rapid removal of tissues was compared with freeze-clamping techniques. The major folates in rat liver were H 4PteGlu and 5-methyl-H 4PteGlu, comprising 42 and 39%, respectively, of the total liver folate pool of 27.30 nmol/g liver (about 13 μg/g liver). In addition, 10-formyl-H 4PteGlu and 5-formyl-H 4PteGlu each comprised 10% of the total folate pool. No endogenous PteGlu, H 2PteGlu, or 5,10-methylene-H 4PteGlu was detected in rat liver samples under our conditions. Distribution of 14C derived from a previous [ 14C]folic acid injection paralleled the distribution of folate as determined microbiologically after high-pressure liquid chromatography separation. The importance of these methods for the direct determination and estimation of flux of H 4PteGlu, 5-methyl-H 4PteGlu, and 10-formyl-H 4PteGlu in studies dealing with the folate system was emphasized.