NAD+-dependent ethanol oxidation: redox effects and rate limitation.

Effects of ethanol on interconversion of cyclohexanol and cyclohexanone was studied in isolated hepatocytes. Oxidation and reduction catalyzed by alcohol dehydrogenase were markedly inhibited and stimulated, respectively. The changed ratio between the rates indicated that the ratio of NAD+ to NADH bound to alcohol dehydrogenase decreased several hundred times. This is much more than for the NAD+ system used by, e.g., lactate dehydrogenase, and deuterium from [1,1-2H2] ethanol was incorporated in cyclohexanol much more than in, e.g., lactate. These results indicate that the coenzyme bound to alcohol dehydrogenase is not equilibrated with free coenzyme. Thus, the dissociation of NADH might be rate-limiting for ethanol oxidation. Deuterium transfer from chiral [1-2H] ethanols and [2-2H] glycerol in hepatocytes indicated that cytosolic malate dehydrogenase and lactate dehydrogenase were not completely equilibrated, whereas there was no difference in the utilization of NADH formed at alcohol dehydrogenase and at glycerol-3-phosphate dehydrogenase. Fluxes in redox reactions during ethanol oxidation may be too high for equilibration of cytosolic dehydrogenases.