Effect of ethanol on the redox state of the coenzyme bound to alcohol dehydrogenase studied in isolated hepatocytes.
Hepatocytes were isolated from fed female rats and incubated with a redox indicator system consisting of cyclohexanone and unlabelled or perdeuterated cyclohexanol. The concentrations and deuterium contents of these were measured by g.l.c. and g.l.c.-m.s. of oxime t-butyldimethylsilyl derivatives. The equilibrium composition represented the redox state of the coenzyme bound to alcohol dehydrogenase, since 4-methylpyrazole inhibited the interconversion. Reduction appeared to be catalysed to a small extent also by an NADPH-dependent aldehyde reductase. The NADH/NAD+ ratio on alcohol dehydrogenase was 3 orders of magnitude higher in the presence of ethanol than in its absence. This redox shift has the degree expected from reported kinetic constants. The shift was due both to a decreased rate of oxidation and to an increased rate of reduction in the indicator system. The results indicate that the redox effect of ethanol on the free NAD system is due to efficient removal of acetaldehyde from a near-equilibrium system consisting of ethanol, acetaldehyde and bound coenzymes, together with dissociation of NADH from the enzyme. The effect on the redox state of the bound coenzyme was less marked when the ethanol was deuterated at C-1, indicating an isotope effect. The 2H excess in the cyclohexanol formed was about 70% of that in the [1,1-2H2]ethanol. This dilution, which is caused by binding of free NADH to the enzyme, indicates that reoxidation of cytosolic NADH partly limits the rate of ethanol oxidation.
Department of Physiological Chemistry, Karolinska Institute, Stockholm, Sweden.
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Cronholm
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D003067:Coenzymes / Q000378:metabolism; D003511:Cyclohexanols / Q000378:metabolism; D003512:Cyclohexanones / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D005260:Female; D000077604:Fomepizole; D008401:Gas Chromatography-Mass Spectrometry; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D003067:Coenzymes; D003511:Cyclohexanols; D003512:Cyclohexanones; D011720:Pyrazoles; D009243:NAD; D000431:Ethanol; C036468:cyclohexanone; D000077604:Fomepizole; D000426:Alcohol Dehydrogenase