Primary deuterium and tritium isotope effects upon V/K in the liver alcohol dehydrogenase reaction with ethanol

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Abstract
The primary isotope effect upon V/K when ethanol stereospecifically labeled with deuterium or tritium is oxidized by [horse] liver alcohol dehydrogenase was measured between pH 6 and 9. The deuterium isotope effect was obtained with high reproducibility by the use of 2 different radioactive tracers, i.e., 14C and 3H, to follow the rate of acetaldehyde formation from deuterium-labeled ethanol and normal ethanol, respectively. Synthesis of the necessary labeled compounds is described in this and earlier work referred to. V/K isotope effects for both tritium and deuterium was measured with 3 different coenzymes, NAD+, thio-NAD+ and acetyl-NAD+. With NAD+ at pH 7, D(V/K) [deuterium V/K] was 3.0 and T(V/K) [tritium V/K] was 6.5. With increasing pH, these values decreased to 1.5 and 2.5 at pH 9. The intrinsic isotope effect evaluated by the method of Northrop (1977) varies little with pH. It amounts to .apprx. 10 with NAD+ and .apprx. 5 with the coenzyme analogs. Commitment functions and their dependence upon pH calculated in this connection appear to be in agreement with known kinetic parameters of liver alcohol dehydrogenase. This assay method was applied in vivo in the rat. Being a noninvasive method because only trace amounts of isotopes are needed, it may yield information about alternative routes of ethanol oxidation in vivo. In naive rats at low concentrations of ethanol, it confirms the discrete role of the non-alcohol dehydrogenase systems.