Deuterium Isotope Effects as a Tool in the Study of Ethanol Oxidation in Rat Liver Microsomes

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Abstract
The apparent kinetic deuterium isotope effect (I) on the oxidation of ethanol to acetaldehyde by washed rat liver microsomes was measured with (1-R)-[1-2H2, 1-14C]-ethanol (I1) and [1-2H2, 2-14C]-ethanol (I2) as substrates by a competitive technique involving only measurements of radioactivity. The average values were for non-induced rats, I1 = 1.57 and I2 = 2.23. When these two substrates were used with stereospecific enzymes (alcohol dehydrogenase and catalase) a small secondary effect was observed, causing I2 to be about 10% higher than I1. With non-stereospecific systems I2 was much larger than I1, and the values were connected by a simple formula. This relation in combination with use of the inhibitors, sodium azide and thiourea, made it possible to calculate tentatively the contribution to microsomal ethanol oxidation of catalase, a non-identified stereospecific enzyme, and non-stereospecific catalytic systems, as well as the isotope effects of the latter two systems. Measurements were made in microsomes from normal, phenobarbital treated, and acetone treated rats. For the stereospecific component an isotope effect of 1.4-1.5 was calculated for all three groups. For the non-stereospecific enzyme in acetone treated rats a value of 4.0 was found. Both the other groups showed a value about 2.7. The activity of the non-stereospecific system was about twice the normal in barbiturate treated, and 3 times the normal in the acetone treated group, where it contributed 70% of the total activity. The isotope effects on the changes in ethanol oxidation (the 'differential isotope effect') caused by inhibitors and activators were utilized to decide whether inhibitors were specific for a single reaction. Thus azide while inhibiting catalase completely, also inhibited other reactions. The large increase (5-6 times) in rate caused by Fe-ESDTA has an I2 of 1.6, equal to that for oxidation of ethanol by hydroxyl radicals.

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