High-performance liquid chromatographic determination of the distribution of naturally occurring folic acid derivatives in rat liver

Procedures which allow extraction and quantitation of labile, reduced folic acid derivatives in rat liver have been developed. These procedures entail extraction of hepatic folates at 100°C in 2% ($\text{w}\text{v}$) sodium ascorbate, 0.2 m 2-mercaptoethanol, pH 7.85. The extract was treated with conjugase to hydrolyze folate polyglutamates and reverse-phase, ion-pair high-performance liquid chromatography was used to separate the resulting monoglutamates which were measured by microbiological assay using Lactobacillus casei. Experiments with HPLC-purified standard derivatives, so treated, showed excellent stability of tetrahydropteroylglutamic acid (H₄PteGlu), 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5-methyl-H₄PteGlu, and pteroylglutamic acid (PteGlu). Under these conditions, approximately 56% of H₂PteGlu was recovered unchanged while about 27% was converted to PteGlu; 5,10-methylene-H₄PteGlu was quantitatively recovered as H₄PteGlu. These procedures were applied to the task of measuring the distribution of naturally occurring folate cofactors in rat liver. These results indicated that rat liver folates have the following compositions: 5-methyl-H₄PteGlu, 37.2%; H₄PteGlu, 32.7%; 10-formyl-H₄PteGlu, 22.6%; and 5-formyl-H₄PteGlu, 7.7%. Experiments with ³H]PteGlu injection showed that all hepatic folates had the same specific radioactivity as determined by radioassay and L. casei assay, indicating that L. casei exhibited the same growth response to all the folates detected in rat liver.