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Effect of ethanol on the redox state of the coenzyme bound to alcohol dehydrogenase studied in isolated hepatocytes.

ABSTRACT: 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.

SUBMITTER: Cronholm T

PROVIDER: S-EPMC1148579 | biostudies-other | 1987 Dec

REPOSITORIES: biostudies-other

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Project description:1. Hepatocytes isolated from starved rats and incubated without other substrates oxidized ethanol at a rate of 0.8-0.9mumol/min per g wet wt. of cells. Addition of 10mm-lactate increased this rate 2-fold. 2. Quinolinate (5mm) or tryptophan (1mm) decreased the rate of gluconeogenesis with 10mm-lactate and 8mm-ethanol from 0.39 to 0.04-0.08mumol/min per g wet wt. of cells, but rates of ethanol oxidation were not decreased. From these results it appears that acceleration of ethanol oxidation by lactate is not dependent upon the stimulation of gluconeogenesis and the consequent increased demand for ATP. 3. As another test of the relationship between ethanol oxidation and gluconeogenesis, the initial lactate concentration was varied from 0.5mm to 10mm and pyruvate was added to give an initial [lactate]/[pyruvate] ratio of 10. This substrate combination gave a large stimulation of ethanol oxidation (from 0.8 to 2.6mumol/min per g wet wt. of cells) at low lactate concentrations (0.5-2.0mm), but rates remained nearly constant (2.6-3.0mumol/min per g wet wt. of cells) at higher lactate concentrations (2.0-10mm). 4. In contrast, owing to the presence of ethanol, the rate of glucose synthesis was only slightly increased (from 0.08 to 0.12mumol/min per g wet wt. of cells) between 0.5mm- and 2.0mm-lactate and continued to increase (from 0.12 to 0.65mumol/min per g wet wt. of cells) with lactate concentrations between 2 and 10mm. 5. In the presence of ethanol, O(2) uptake increased with increasing substrate concentration over the entire range. 6. Changes in concentrations of glutamate and 2-oxoglutarate closely paralleled changes in the rate of ethanol oxidation. 7. In isolated hepatocytes, rates of ethanol oxidation are lower than those in vivo apparently because of depletion of malate-aspartate shuttle intermediates during cell preparation. Rates are returned to those observed in vivo by substrates that increase the intracellular concentration of shuttle metabolites.

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Effect of ethanol on the redox state of the coenzyme bound to alcohol dehydrogenase studied in isolated hepatocytes.

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