Ethanol Oxidation In Hepatocytes Research Articles

Acetylcarnitine-mediated inhibition of ethanol oxidation in hepatocytes

Carnitine-mediated prevention of ethanol-induced hepatic steatosis is related to the attenuation of ethanol metabolism by carnitine in the intact rat. Although carnitine retards ethanol oxidation in the intact animal, the in vitro activities of ethainol-metabolizing enzymes remain unaltered. Therefore, hepatocytes were targeted to understand the mechanism of carnitine effect on ethanol metabolism. Rat hepatocytes were isolated by a collagenase-perfusion technique and incubated in albumin-containing medium with ethanol in the presence or absence of added carnitine or related compounds. Ethanol oxidation was determined by the loss of ethanol as well as by the products formed. The rate of ethanol oxidation in the presence of carnitine was one-half the rate in the absence of carnitine (14 vs. 25 nmol · min −1 · million −1 cells). It took 100 times the concentration of carnitine to equal the maximal inhibition produced by acetylcarnitine and the effect of acetylcanitine was without a lag time. It is concluded that acetylcarnitine is the mediator of carnitine inhibition of ethanol oxidation.

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.

Respective roles of the microsomal ethanol oxidizing system and catalase in ethanol metabolism by deermice lacking alcohol dehydrogenase

To evaluate the roles of MEOS (microsomal ethanol oxidizing system) and catalase in non-alcohol dehydrogenase (ADH) ethanol metabolism, MEOS and catalase activities in vitro and ethanol oxidation rates in hepatocytes from ADH-negative deermice were measured after treatment with catalase inhibitors and/or a stimulator of H 2O 2 generation. Inhibition of ethanol peroxidation by 3-amino-1,2,4-triazole (aminotriazole) was found to be greater than 85% up to 3 h and 80% at 6 h in liver homogenates. Urate (1 m m) which stimulates H 2O 2 production in living systems, increased ethanol oxidation fourfold in control but not in cells from aminotriazole-treated animals, documenting effective inhibition of catalase-mediated ethanol peroxidation by aminotriazole. While aminotriazole slightly depressed (15%) basal ethanol oxidation in hepatocytes, in vitro experiments showed a similar decrease in MEOS activity after aminotriazole pretreatment. Azide (0.1 m m), a potent inhibitor of catalase in vitro, also did not affect ethanol oxidation in control cells. By contrast, 1-butanol, a competitive inhibitor of MEOS, but neither a substrate nor an inhibitor of catalase, decreased ethanol oxidation rates in a dose-dependent manner. These results show that, in deermice lacking ADH, catalase plays little if any role in hepatic ethanol oxidation, and that MEOS mediates non-ADH metabolism.

Contribution of non-ADH pathways to ethanol oxidation in hepatocytes from fed and hyperthyroid rats: Effect of fructose and xylitol

The metabolism of (1R)[1- 3H]ethanol, [2- 3H]lactate or [2- 3H]xylitol was studied in hepatocytes from fed or T 3-treated rats in the presence or absence of fructose or xylitol. The yields of tritium in ethanol. lactate, water, glycerol and glucose were determined. A simple model, describing the metabolic fate of tritium from these substrates is presented. The model allows estimation of the ethanol oxidation rate by the non-alcohol dehydrogenase pathways from the relative yield of tritium in water and glucose. The calculations are based on a comparison of the fate of the 1- proR-hydrogen of ethanol and the hydrogen bound to carbon 2 of lactate (or xylitol) under identical condition. In our calculations we have taken into account that the reactions catalyzed by lactate dehydrogenase and alcohol dehydrogenase are reversible and that lactate or ethanol labelled during the metabolism of the other tritiated substrates will contribute to the tritium found in water. The contribution of non-ADH pathways to ethanol oxidation varied from 10 to 50% and was correlated to changes in the lactate/pyruvate ratio from 80 to 500. In T 3- treated rats the activity of non-ADH pathways were greater than in fed rats for the same lactate/pyruvate ratio.

Effect of glucagon on ethanol oxidation in isolated rat liver cells

Addition of glucagon 5 min after ethanol was found to stimulate the rate of ethanol oxidation in hepatocytes isolated from starved rats. This stimulation is of the same order of magnitude as that mediated by asparagine. The glucagon effect is suppressed by antiproteolytic agents such as insulin or NH4Cl. The stimulating effect of glucagon on ethanol oxidation is probably linked to enhanced proteolysis and an elevated glutamate level in the hepatocytes.

The role of hydrogen translocating shuttles during ethanol oxidation in hepatocytes from euthyroid and hyperthyroid rats.

A quantitative study of the contribution of the malate-aspartate and glycerol-3-phosphate cycles to the translocation of reducing equivalents from cytosol to mitochondria during ethanol oxidation has been made in hepatocytes from euthyroid and hyperthyroid rats. 1. In hepatocytes from euthyroid rats both cycles have an almost equal capacity and their relative contribution to total hydrogen transport to the mitochondria depends on the conditions chosen. 2. In hepatocytes from hyperthyroid rats maximal rates of ethanol oxidation were significantly lower than in hepatocytes from euthyroid rats, even though the capacity of the glycerol-3-phosphate cycle was increased. This was due to a decreased activity of alcohol dehydrogenase.

Effect of natural amino acids on ethanol oxidation in isolated rat hepatocytes

The effects of the various naturally occurring amino acids on ethanol oxidation in hepatocytes from starved rats was systematically studied. In order to minimize the non ADH pathways, the ethanol concentration used was 4 mmol/litre, the amino acids being added at the same concentration. In hepatocytes from fasted rats, alanine, arginine, asparagine, aspartate, citrulline, cysteine, glutamate, glutamine, glycine, histidine, hydroxyproline, ornithine and serine increase significantly ethanol consumption. The stimulatory effect of glutamine being much less pronounced than the asparagine one and proline being devoid of action, the influence of ammonium chloride addition on ethanol consumption in the presence of these amino acids was studied. Ammonium chloride determines an enhancement of ethanol oxidation in these conditions, the results showing no apparent correlation between intracellular glutamate concentration and ethanol oxidation rate, contrarily to previous data. In hepatocytes from fed rats, only alanine, asparagine, cysteine, glycine, hydroxyproline, ornithine and serine increase ethanol oxidation, although to a lesser extent than in cells from starved rats.

Ethanol oxidation by isolated hepatocytes from ethanol-treated and control rats; factors contributing to the metabolic adaptation after chronic ethanol consumption

Chronic consumption of ethanol by rats produced a fatty liver and resulted in a pronounced increase in the rate of ethanol oxidation by isolated hepatocytes. Despite the increase in ethanol oxidation, oxygen consumption with several substrates was not enhanced after chronic ethanol treatment. Ouabain, an inhibitor of the (Na + + K + )-ATPase activity, did not abolish the increase in the rate of ethanol oxidation. About 40–50 per cent of the increase in ethanol oxidation persisted after inhibition of alcohol dehydrogenase, mitochondrial oxygen consumption or the malate-aspartate shuttle. The addition of substrates for the malate-aspartate shuttle slightly increased the rate of ethanol oxidation in hepatocytes from control and ethanol-treated animals. The increased rate of ethanol oxidation was not abolished by the uncoupling agent dinitrophenol. which by itself had little effect on ethanol oxidation. In the presence of aspartate or α-glycerophosphate, dinitrophenol augmented the rate of ethanol oxidation; in the presence of glutamate, the rate of ethanol oxidation was doubled by dinitrophenol. However, the higher rate of ethanol oxidation after ethanol consumption was still found in the presence of various combinations of substrate shuttles, with or without dinitrophenol. Pathways independent of alcohol dehydrogenase may contribute, at least in part, to the increase in ethanol oxidation found after chronic ethanol consumption. It is concluded that ethanol oxidation may be enhanced after chronic ethanol consumption without the estabishment of a hypermetabolic state of the liver.