10-formyltetrahydrofolate exists in pig plasma

We determined plasma folate concentrations by microbiological assay and hig h-performance liquid chromatography with electrochemical detection (HPLC-EC D). We observed a marked difference between total folate (53.4 +/- 21.0 nM, n=12) obtained by microbiological assay and the sum (26.7 +/- 9.9 nM, n=12 ) of tetrahydrofolate (H(4)PteGlu) and 5-methyltetrahydrofolate (5-CH3-H(4) PteGlu) detected by HPLC-ECD. In order to elucidate this difference, microb iological assays were performed with fractions of the HPLC eluate of pig pl asma. Heat or acid treatment was used for deproteinization. Pig plasma afte r heating showed three peaks of folate activity on the chromatogram. Those retention times were identical to those of 10-formyltetrahydrofolate (10-HC O-H(4)PteGlu), H(4)PteGlu and 5-CH3-H(4)PteGlu. It was suggested that the e xistence of 10-HCO-H(4)PteGlu produced the difference (26.7 +/- 16.0 nM, n= 12) between total folate by microbiological assay and the sum of H(4)PteGlu and 5-CH3-H(4)PteGlu by HPLC-ECD. After acid treatment, the peak with rete ntion time similar to 10-HCO-H(4)PteGlu became smaller than that in heat tr eatment, and at the same time a peak in addition to 10-HCO-H(4)PteGlu, H(4) PteGlu and 5-CH3-H(4)PteGlu appeared on the chromatogram. This peak exhibit ed the same retention time as that of 5,10-methenyltetrahydrofolate (5,10-C H=H(4)PteGlu). H(4)PteGlu and 5-CH3-H(4)PteGlu in pig plasma were completel y recovered after heating under ascorbate concentration at 100 mM (pH 7.4). These results also supported the existence of 10-HCO-H(4)PteGlu in pig pla sma. Our data indicates that 10-HCO-H(4)PteGlu exists in pig plasma as a ma jor folate derivative in addition to H(4)PteGlu and 5-CH3-H(4)PteGlu. (C) 2 000 Elsevier Science Inc.