Identification of 5,10-methylenetetrahydrofolate in rat bile

5,10-Methylenetetrahydrofolate (5,10-CH2-H4PteGlu) was identified as a major active reduced folate in rat bile using high-performance liquid chromatography with electrochemical detection (HPLC-ED). The identification of the folate derivative was based on the similarities in the retention-time profiles, electrochemical properties, UV absorption characteristics and demethylenation profiles of the bile folate and the synthetic standard. An HPLC-ED method was developed for the simultaneous determination of reduced folates including 5,10-CH2-H4PteGlu, tetrahydrofolate (H4PteGlu), 10-formyltetrahydrofolate (10-HCO-H4PteGlu) and 5-methyltetrahydrofolate (5-CH3-H4PteGlu) in rat bile. All peaks of the reduced folates in bile were separated using this method with a total retention time of less than 15 min. The detection limit was 0.01 ng/injection for H4PteGlu, 10-HCO-H4PteGlu and 5-CH3-H4PteGlu, and 0.02 ng/injection for 5,10-CH2-H4PteGlu at a signal-to-noise ratio of 3 and an injection volume of 100 microliters. Recoveries of synthetic folates from rat bile were higher than 90%. The distribution percentages of 5,10-CH2-H4PteGlu, H4PteGlu, 10-HCO-H4PteGlu and 5-CH3-H4PteGlu in rat bile were 29.6 +/- 7.2, 17.7 +/- 3.5, 24.4 +/- 6.5 and 28.2 +/- 7.1%, respectively, and total secretion rate of the bile reduced folates was 1514 +/- 663 ng/h (mean +/- S.D., n = 9).