Aspects of Folate and Tetrahydrofolate Chemistry and Metabolism in the Rat

Abstract

Most of the work in the literature dealing with folate metabolism has been done using unlabelled folates, analysis being by microbiological assay. This method is not highly specific or quantitatively accurate: this thesis reports results on the synthesis of 14 c- labelled folates, and the use of these compounds in investigation of folate metabolism in the rat. The synthesis of (±)-5-formyltetrahydrofolic acid -(2-14C), (±)-5,10-methenyltetrahydrofolic acid -(2-14C), (±)-10-formyltetrahydrofolic acid -(2-14C), and 10-formylfolic acid -(2-14C) from folic acid -(2-14C) is described. These compounds, and commercial 5-( CH, )- methyltetrahydrofolic acid, were given orally to rats at doses of 3 and 30 μg/ke. Quantitative and qualitative assay of urine, liver, and faeces showed that 5-formyltetrahydrofolate was largely unmetabolised: this compound was also given i.v., and showed even less metabolism. 5,10-methenyltetrahydrofolate was more extensively metabolised, but depended on dose, being less for small doses. 10-formyltetrahydrofolate was almost completely metabolised, both diastereoisomers apparently being utilised: this was true also for 5-(14C-methyl) tetrahydrofolic acid, the methyl group being rapidly lost and a small amount exhaled as 14CO2. 10-formylfolate was not metabolised; it gave a large amount of excreted pteridines, not observed with the other compounds, It inhibited folic acid metabolism in humans, but was ineffective as a growth inhibitor of R1 lymphoma in mice. Folic acid -(2-14C) given to tumour-bearing animals was metabolised more than in normals: absorption of the vitamin at low doses was impaired iin some cases, An unknown folate found in rat urine by previous work (Dransfield 1972) was not identified. Rat livers were extracted after folate administration to investigate any conversion into polyglutamates,. Complex and variable chromatograms were obtained with established extraction procedures, the results being interpreted as artifact formation during the extraction process, Elevated L. casei assays after treatment of monoglutamate with conjugase were found in one case, Aspects of the in vitro chemistry of folates which are important in metabolic studies, and which are ill-defined in the literature, were also studied. Folic acid, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate and 5,10-methenyltetrahydrofolate were autoclaved under conditions commonly used for extraction of folate from natural material. In presence of antioxidant, little decomposition was found apart from the expected formyltetrahydrofolate interconversions. Folic acid and 5-methyltetrahydrofolate were photolysed in the presence and absence of riboflavin. An unusual decomposition of 5-methyltetrahydrofolate to folic acid in presence of riboflavin was found. Formyltetrahydrofolates were subjected to differing pH values, and their rates of isomerisation studied. 5- and 10-formyltetrahydrofolates were stable above pH 4; below this value, 10-formyltetrahydrofolate was cyclised more rapidly than 5-formyltetrahydrofolate. 5,10-methenyltetrahydrofolate was hydrolysed above pH 5, the rate depending on buffer species and the concentration. The results are discussed in terms of the pH changes in the alimentary canal.