New methods for the study of folate coenzymes: endogenous polyglutamate patterns of subcellular hepatocyte fractions and of regenerating rat liver.

A procedure has been developed that permits the quantitation of three different pools of one-carbon-substituted folates with the simultaneous determination of their corresponding poly-gamma-glutamyl chain lengths. Pool 1 is made up of N5,N10-methylene-tetrahydrofolates and unsubstituted tetrahydro and dihydrofolates. Pool 2 is made up solely of N5-methyl-tetrahydrofolates and pool 3 includes the folates with one-carbon-substituents at the formyl oxidation level: N5,N10-methenyl-tetrahydrofolates, N10- and N5-formyl-tetrahydrofolates, and N5-formimino-tetrahydrofolates. Conditions have been found that permit the selective cleavage of the C9-N10 bond of the folates of pools 1, 1 + 2, and 1 + 2 + 3. The cleaved folates are quantitated as the Bratton-Marshall azo-dyes of the p-amino-benzoyl-poly-gamma-glutamates (AzoGlun) after converting the uncleaved pools to Bratton-Marshall negative products. Determination of the poly-gamma-glutamyl chain length of the cleaved pools is carried out by high pressure liquid chromatography of the AzoGlun comparing the elution position of each peak with that of authentic synthetic markers. Quantitation of the individual peaks is obtained by integration and by reference to an internal synthetic standard (usually AzoGlu3). To increase the sensitivity of the procedure and make it applicable to plasma or needle biopsy specimens we have developed a synthesis for [3H]-N'-naphthylethylenediamine. This compound is the one coupled to the diazotized p-aminobenzoyl-poly-gamma-glutamates to form the AzoGlun in the Bratton-Marshall test. The new methods do not require labeling the animal's folates by the prior injection of radioactive folic acid thus permitting the study of endogenous folate patterns. Applied to rat liver these methods have shown that pool 3 accounts for 44.3 +/- 7.7%, pool 2 for 36.7 +/- 4.7%, and pool 1 for 18.5 +/- 5.8% of the total folates. The pentaglutamates made up about 52% and the hexaglutamates about 42% of the folates. Hepta and tetraglutamates account for approximately 2.8 and 3.5% respectively. The cytosolic folates are predominantly hexaglutamates whereas pentaglutamates predominate in the heavy mitochondrial fraction. Profound changes in the relative proportions of the polyglutamates were observed 24 h after partial hepatectomy. There are marked increases in hepta and hexaglutamates and decreases in penta and tetraglutamates. These results support the hypothesis that changes in polyglutamate chain length are related to the regulation of one-carbon metabolism.