Deuterium D(V/K) isotope effects on ethanol oxidation in hepatocytes: importance of the reverse ADH-reaction

The kinetic deuterium isotope effect, D(V/K), on ethanol oxidation was measured on hepatocytes from rat and pig by the radiometric competitive method using 14C-labelled ethanol containing deuterium in the (1-R)-position. The corrected D(V/K) values of 2.68 and 2.80 for rat and pig hepatocytes respectively were significantly different, suggesting differences in the amount of non-ADH ethanol oxidizing activity. The apparent isotope effects declined rapidly with time when acetaldehyde was present in the medium as a result of the reduction to ethanol of the [14C]-acetaldehyde formed from the double labelled ethanol by alcohol dehydrogenase (ADH). Fructose and cyanamide caused the acetaldehyde concentration during ethanol oxidation to increase by entirely different mechanisms, and the isotope effect to decrease with time, as did also the addition of acetaldehyde. The apparent first order rate constant for the reverse ADH reaction, assuming the reactants to be acetaldehyde and the ADH-NADH complex, was determined by two methods giving comparable results. In the presence of semicarbazide, which removes acetaldehyde, the isotope effect was nearly constant. This was the case also when the acetaldehyde concentration was very low (less than 1 microM) for other reasons, as in hepatocytes from starved animals. A mathematical formula describing the expected decrease of the apparent isotope effect with time was derived. The different response of pig and rat hepatocytes to addition of fructose (the 'fructose effect') is suggested to be caused by differences in activity of aldehyde dehydrogenases in the two species.