Conversion of FAD to FMN and riboflavin in plasma: effects of measuring method.

The stability of flavin adenin dinucleotide (FAD) in plasma was studied under a low-intensity light and FAD was found to be converted to flavin mononucleotide (FMN) and riboflavin (RF) in both human and rat plasma. The hydrolysis rates of FAD in plasma at 4 degrees C were lower than those at 37 degrees C. In addition, the hydrolysis rates were markedly inhibited when EDTA, known as an anticoagulant, was added to plasma. These results indicated that plasma samples in pharmacokinetic studies should be pretreated with EDTA, extracted at the earliest convenience and lower temperature like 4 degrees C to keep a high stability. The pharmacokinetic study after intravenous administration of FAD at a dose of 500 nmol/kg as FAD in rats was performed with plasma samples after addition of EDTA under strict light and temperature control. A measurable amount of FAD in plasma together with rapid conversions of FAD to FMN and RF were observed in rat plasma. The AUC values (mean+/-S.D. of 4 rats) for FAD, FMN and RF were 707+/-378, 3643+/-958 and 30095+/-3544 nmol x min/l, respectively. Using excess EDTA under strict temperature and light control may be useful for assessment of vitamin B2 in the in vivo study.