Differential effects of the cytochrome P-450/ reductase ratio on the oxidation of ethanol and the hydroxyl radical scavenging agent 2-keto-4-thiomethylbutyric acid (KMBA)

NADPH-cytochrome P-450 reductase catalyzes a low rate of oxidation of hydroxyl radical scavenging agents such as ethanol and 2-keto-4-thiomethylbutyric acid (KMBA), in a reaction markedly stimulated by the addition of ferric-EDTA. The effect of various ratios of cytochrome P-450 (pheno-barbital-inducible isozyme)/reductase on the oxidation of ethanol and KMBA was determined. There was essentially no increase in KMBA oxidation over the range of ratios from 0.5 to 5 as compared to the reductase-catalyzed rate. High ratios actually caused some decrease in KMBA oxidation, which was especially notable under conditions of increased rates of hydroxyl radical generation (presence of increasing amounts of ferric-EDTA). This decrease at high P-450/reductase ratios could reflect tight coupling of reductase to P-450-PB, therefore decreasing electron transfer from reductase to ferric-EDTA, or could involve non-specific scavenging of OH by P-450-PB. Indeed, native, but not boiled, P-450 inhibited KMBA oxidation by the xanthine oxidase system. By contrast, the oxidation of ethanol was stimulated at all concentrations of P-450-PB, and this increase was not sensitive to desferrioxamine. However, under conditions of high rates of OH production, the ethanol oxidation profile tended to resemble the KMBA oxidation profile with respect to the effect of P-450-PB, whereas the two profiles were different under conditions of low rates of OH production. These results suggest that P-450-PB does not catalyze the oxidation of OH scavengers or promote the production of OH, even at ratios of P-450/reductase approaching those found with intact microsomes and even in the presence of excess iron-EDTA, whereas ethanol is directly oxidized by P-450-PB, as are typical drug substrates. However, the P-450-PB-dependent oxidation of ethanol can be masked under conditions in which OH production is increased.