Ascorbic Acid And The Flavin-containing Monooxygenase.

Abstract

The effect of ascorbic acid deficiency on the flavin-containing monooxygenase (FMO) and the biochemical mechanism by which ascorbate participates with this enzyme were investigated. The N-oxidation of dimethylaniline (DMA) was reduced by 50% in ascorbate deficient guinea pig hepatic 12,000 g supernatant. Although the cytochrome P-450 electron transport system (P-450-MO) is known to be reduced in ascorbate deficiency, this reduction in DMA metabolism was associated with the FMO by the following criteria. In both ascorbate supplemented and deficient guinea pig 12,000 g supernatant, SKF525A and n-octylamine did not inhibit DMA metabolism. Phenobarbital induction did not increase the rate of DMA metabolism. In addition, the hepatic supernatant fraction heated to 50(DEGREES)C was unable to metabolize DMA, but greater than 80% of the P-450-MO activity was retained. Also, at pH 9.4, 84% of DMA N-oxidation was maintained while P-450-MFO activity was reduced by 50%. Kinetic studies with purified FMO indicated no significant change in the apparent K(,m) of DMA, NADPH, or FAD in ascorbate supplemented or deficient animals. Following purification of ascorbate deficient guinea pig FMO by DEAE-cellulose and blue agarose chromatography 15% of the total microsomal enzyme was dependent on the addition of exogenous FAD for optimal activity. In contrast, only 5% of the total microsomal enzyme purified from ascorbate supplemented animals required exogenous FAD. Furthermore, there was an enhanced sensitivity to substrate inhibition with the purified ascorbate deficient guinea pig FMO. Also, purified ascorbate supplemented guinea pig FMO was stable for 4 weeks at -20(DEGREES)C, while the ascorbate deficient enzyme was inactivated. A decrease in the quantity of ascorbate deficient guinea pig FMO compared to ascorbate supplemented was suggested by reduced protein staining intensity with SDS-PAGE at 56,000 daltons and a marked reduction in total FMO activity recovered from blue agarose chromatography. Weight loss in combination with ascorbate deficiency was also investigated. Ascorbate deficient guinea pigs on a food restricted regimen had only 20% of the FMO activity compared to ascorbate supplemented animals on an identical food restricted regimen.