Conversion of FAD to FMN and Riboflavin in Plasma: Effects of Measuring Method

Abstract

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°C were lower than those at 37°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°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 · min/l, respectively. Using excess EDTA under strict temperature and light control may be useful for assessment of vitamin B 2 in the in vivo study.

Pharmacokinetic Profile of Flavin Adenine Dinucleotide, Flavin Mononucleotide and Riboflavin Following Intravenous Administration of Riboflavin or Its Coenzymes in Rats

“…The activity of enzymes that hydrolyze FAD and FMN has been shown to inhibit under these conditions as reported previously. 6) No less than 95% of FAD and FMN in EDTA-treated plasma remained at 30 min at 4 • C (data not shown).…”

“…With regard to FAD, each flavin level was calculated from the data from our recent study. 6) The values established by the LF method were slightly lower. This underestimation might be partially due to the low recovery rate of LF from the plasma.…”

“…Sample Analyses --These plasma samples were analyzed for FAD, FMN and RF using GF as an internal standard (IS) by HPLC with a fluorescence detector, as described previously. 6) In a 1 ml test tube, 60 µl ice-cold methanol was added to 20 µl plasma to precipitate proteins. IS solution was added to the tube, it was vortex-mixed for 1 min and then centrifuged for 1 min at 10000 × g to separate proteins.…”

“…As for FAD (a), each flavin level was calculated from the data from our recent study. 6) administration of RF appeared to decrease with time in a biexponential pattern, ie, a rapid phase of elimination was followed by a slower terminal phase. Changes in the concentrations of RF were almost the same compared with those after the intravenous administration of FMN.…”

“…[3][4][5] We also previously reported that hydrolysis rates of FAD in rat and human plasma were very rapid. 6) In order to overcome this difficulty, we investigated an effective extraction method of flavins in plasma without hydrolysis of FAD and FMN. Hydrolysis of FAD was markedly inhibited when ethylenediamine-N, N, N , N -tetraacetic acid (EDTA) was added to the plasma.…”