A microbiologic disc assay for 5-methyltetrahydrofolate in the presence of methotrexate

High-dose methotrexate-citrovorum factor rescue regimens are used extensively for the treatment of human malignancies. Since the rescue agent citrovorum factor is rapidly converted to 5-methyltetrahydrofolate, it is desirable to follow the distribution kinetics of both of these coenzymes to assess the selective therapeutic effectiveness of the regimen. The assay methods using methotrexate-resistant strains of Lactobacillus casei ATCC 7469 (LC/A), Streptococcus faecium var. durans ATCC 8043 (SF/A), and Pediococcus cerevisiae ATCC 8081 (PC/A) have been developed to determine the concentrations of 5-methyltetrahydrofolate and other folates in the presence of methotrexate. Since LC/A determines the total folates, SF/A deterimines the total folates except 5-methyltetrahydrofolate, and PC/A by our procedure determines only the most stable form of reduced folates, citrovorum factor, the quantitation of both 5-methyltetrahydrofolate and citrovorum factor can very easily be achieved by differential assays. This report describes the determination of as low as 10 ng/ml of 5-methyltetrahydrofolate in the presence of as high as 500 microgram/ml of methotrexate. The assay method described here is currently used in studying the pharmacokinetics of 5-methyltetrahydrofolate in patients with meningeal neoplasms.