HYDROGEN TRANSFER BETWEEN ETHANOL MOLECULES DURING OXIDOREDUCTION INVIVO

Rates of exchange catalyzed by alcohol dehydrogenase were determined in vivo in order to find rate-limiting steps in ethanol metabolism. Mixtures of [1,1-2H2]- and [2,2,2-2H3]ethanol were injected with bile fistulas. The concentrations in bile of ethanols having different numbers of 2H atoms were determined by GLC-m.s. after the addition of [2H6]ethanol as internal standard and formation of the 3,5-dinitrobenzoates. Extensive formation of [2H4]ethanol indicated that acetaldehyde formed from [2,2,2-2H3]ethanol was reduced to ethanol and that NADH used in this reduction was partly derived from oxidation of [1,1-2H2]ethanol. The rate of acetaldehyde reduction, the degree of labeling of bound NADH and the isotope effect on ethanol oxidation were calculated by fitting models to the found concentrations of ethanols labeled with 1-42H atoms. Control experiments with only [2,2,2-2H3]ethanol showed that there was no loss of the C-2 hydrogens by exchange. The isotope effect on ethanol oxidation appeared to be about 3. Experiments with (1S)-[1-2H]- and [2,2,2-2H3]ethanol indicated that the isotope effect on acetaldehyde oxidation was much smaller. Both the rate of reduction of acetaldehyde and the rate of association of NADH with alcohol dehydrogenase were nearly as high or as higher than the net ethanol oxidation. The rate of ethanol oxidation in vivo is determined by the rates of acetaldehyde oxidation, the rate of dissociaton of NADH from alcohol dehydrogenase, and by the rate of reoxidation of cytosolic NADH. In cyanamide-treated rats, the elimination of ethanol was slow but the rates in the oxidoreduction were high, indicating more complete rate-limitation by the oxidation of acetaldehyde.