Hepatic Alcohol Dehydrogenase Activity Research Articles

Effect of thyroid hormones on the activities of hepatic alcohol metabolizing enzymes

Treatment with thyroxine or triiodothyronine for 7 days in order to simulate a hyperthyroid state results in an enhanced activity of the microsomal ethanol oxidizing system. Conversely, a decrease of hepatic alcohol dehydrogenase activity was observed under these experimental conditions, whereas hepatic catalase activity remained unchanged. These findings suggest that if chronic ethanol consumption simulates a “hyperthyroid hepatic state”, increased rates of ethanol metabolism observed following prolonged alcohol intake might therefore be attributed at least in part to an induction of microsomal ethanol oxidizing system activity in the liver.

Effect of stress by repeated immobilization on hepatic alcohol dehydrogenase activity and ethanol metabolism

The hepatic activity of alcohol dehydrogenase was increased after 7, 14 and 42 days of stress induced by immobilization of rats for 2.5 hr day . A single immobilization had no effect on the activity of alcohol dehydrogenase. Immobilization for 14 days resulted in increases in the rates of ethanol metabolism. This was not associated with changes in the activity of the microsomal ethanol-oxidizing system, microsomal catalase, cytochrome P-450, or NADPH cytochrome c reductase. A decrease in the hepatic phosphorylation potential ([ATP]/[ADP][P i]) was found to be due to a decrease in [ATP] and an increase in [P i]; however, there were no changes in O 2 consumption by liver slices or in hepatic (Na + + K +)-stimulated adenosine triphosphatase activity. The increased rate of ethanol metabolism after stress remains unexplained since alcohol dehydrogenase activity is not rate-limiting in ethanol oxidation and there were no increases in ethanol oxidation by microsomes or in mitochondrial oxidative rate.

Effect of Temperature on the Assay of Hepatic Alcohol Dehydrogenase

SummaryAn increase in the assay temperature of hepatic alcohol dehydrogenase (ADH) activity, from 25 to 50-60°, with ethanol as the substrate, led to a stimulation of enzyme activity. The stimulation of the hepatic ADH (not observed with yeast ADH) was reversible if the heated samples were allowed to cool to room temperature and then assayed at 25°. Only at the elevated temperature (50-60°) could ADH activity be detected in normal rat or human plasma. The increased sensitivity resulting from thermal activation of ADH may prove useful for studying the enzyme in crude preparations, measuring ADH activity with substrates normally providing low activity (e.g., 17-β-hydroxy-5-β-androston-3-one), or measuring serum ADH activity in patients with various types of liver injury.

FURTHER OBSERVATION ON THE LACK OF ACTIVE UPTAKE SYSTEM FOR SUBSTANCE P IN THE CENTRAL NERVOUS SYSTEM

In an attempt to assess the effects of methylmercury on ethanol metabolism, Sprague-Dawley rats were treated with a daily dose (10 mg/kg i.p.) of methylmercuric chloride for 2 consecutive days and given a test dose (0.4 g/kgi.v.) of ethanol 24 hr after the last treatment. Blood ethanol levels were measured using gaschromatography by the direct introduction of blood samples into the sample vaporizing apparatus attached to the chromatograph. While treatment with methylmercury elicited a slight retardation in the ethanol elimination from the blood during 30 to 90 min, methylmercury did not essentially alter ethanol metabolism. There was no significant change in hepatic alcohol dehydrogenase activity of methylmercury-treated rats. By contrast, the activity of alcohol dehydrogenase purified from liver or yeast was remarkably inhibited by methyl-mercury and the type of inhibition proved to be non-competitive. Moreover, the inhibited activity was reactivated easily by sulfhydryl agents. From these results, it is conceivable that methylmercury has little influence on ethanol metabolism in vivo because of its non-specific binding with sulfhydryl groups in the organism.

EFFECT OF METHYLMERCURY ON THE ETHANOL ELIMINATION FROM THE BLOOD AND THE ACTIVITY OF ALCOHOL DEHYDROGENASE

In an attempt to assess the effects of methylmercury on ethanol metabolism, Sprague-Dawley rats were treated with a daily dose (10 mg/kg i.p.) of methylmercuric chloride for 2 consecutive days and given a test dose (0.4 g/kg i.v.) of ethanol 24 hr after the last treatment. Blood ethanol levels were measured using gaschromatography by the direct introduction of blood samples into the sample vaporizing apparatus attached to the chromatograph. While treatment with methylmercury elicited a slight retardation in the ethanol elimination from the blood during 30 to 90 min, methylmercury did not essentially alter ethanol metabolism. There was no significant change in hepatic alcohol dehydrogenase activity of methylmcrcury-treated rats. By contrast, the activity of alcohol dehydrogenase purified from liver or yeast was remarkably inhibited by methylmercury and the type of inhibition proved to be non-competitive. Moreover, the inhibited activity was reactivated easily by sulfhydryl agents. From these results, it is conceivable that methylmercury has little influence on ethanol metabolism in vivo because of its non-specific binding with sulfhydryl groups in the organism.

Variations in alcohol metabolism: Influence of sex and age

Male and female C57BL/Ibg mice were divided into two age groups: 50–60 and 95–110 days of age. Alcohol-induced sleep time was measured subsequent to the intraperitoneal injection of a 3.5 g × kg −1 dose. The old male group had a sleep time approximately 4 times that of the young male group and approximately twice that of the old female group. Blood alcohol concentrations at time of awakening were nearly identical in all groups, indicating the difference in sleep time is not due to an altered CNS sensitivity. Measurement of in vivo alcohol disappearance rate indicates the old male group is different from the other groups because of a slower rate of alcohol metabolism. Although changes in hepatic alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities were seen, the changes do not explain the observed decrease in alcohol metabolism observed in the old male group. These data provide further evidence that hepatic ADH and ALDH activities are not rate limiting in alcohol metabolism.

Liver aldehyde and alcohol dehydrogenase activities in rat strains genetically selected for their ethanol preference

Rat strains raised by genetical selection for either high (AA strain) or low (ANA strain) voluntary ethanol consumption were compared with respect to their hepatic alcohol and aldehyde dehydrogenase activities. Liver alcohol dehydrogenase activity was lower in both sexes in the AA strain compared with the ANA strain. The NAD-dependent aldehyde dehydrogenase activity was higher in the mitochondrial and microsomal fractions and lower in the soluble fraction in the AA strain than in the ANA strain. These differences were more pronounced in females than in males. The NAD-dependent utilization of acetaldehyde in liver homogenates was higher in the AA strain in both sexes, when the initial acetaldehydehyde level was 0·40 mM, but there was no difference at 0¢13 mM acetaldehyde. It is concluded that the higher activities in the AA strain are due mainly to those aldehyde dehydrogenases of mitochondrial and microsomal fractions, which have K m -values for aldehydes in the millimolar range. The higher alcohol dehydrogenase and lower aldehyde dehydrogenase activity in livers of rats of the ethanol-avoiding ANA strain may contribute to the previously found higher acetaldehyde levels in blood and liver of rats of this strain after ethanol administration.

Effect of dietary choline and ethanol on hepatic triglycerides and activity of hepatic ethanol oxidizing enzymes in rats.

SummaryFemale rats were maintained for 21 days on a liquid diet with and without choline supplementation. Ethanol was given to half of the rats as an isocaloric substitution for dietary sucrose. Choline supplementation inhibited the accumulation of hepatic triglycerides and was required for maximum stimulation of the hepatic microsomal ethanol oxidizing system (MEOS) activity. Increase in hepatic alcohol dehydrogenase (ADH) activity did not occur.

Prevention of ethanol-mediated redox-changes in mouse liver by sodium-diethylbarbiturate

EFFECTS of barbital-pretreatment have been investigated on the ethanol-mediated redox-changes in liver, citric acid cycle activity, ethanol oxidation and on the enzymes of alcohol metabolism. Barbital-pretreatment prevents the changes in hepatic redox-state produced upon alcohol administration. Alcohol results in about threefold increase in hepatic (lactate)/(puruvate), twofold increase in (α-glycerophosphate)/(dihydroxyacetone phosphate) and (β-hydroxybutyrate)/(acetoacetate) concentration ratios in normal mice. However, in barbital-pretreated mice smaller changes in these ratios are observed. Oxidation of (1- 14C) palmitic acid to 14CO 2 in liver is inhibited by 60–70% upon ethanol administration. However, in barbital-pretreated group this effect is prevented. Barbital pretreatment also prevents the ethanol induced inhibition of glycerol utilization, and α-glycerophosphate accumulation in liver. Hepatic alcohol oxidation rate and alcohol dehydrogenase activity is decreased by 40–50% upon barbital-pretreatment. Microsomal ethanol-oxidation remains unaffected by this treatment. It is concluded that the prevention of ethanol-induced changes by barbital in hepatic redox-state and citric acid cycle function are due to the inhibition of alcohol dehydrogenase mediated oxidation of ethanol.

Contribution of “substrate shuttles” in the transport of extramitochondrial reducing equivalents by hepatic mitochondria from chronic alcohol-fed mice

Effects of chronic alcohol treatment have been investigated on the rates of extramitochondrial NADH utilization by hepatic mitochondria in the presence or absence of “malate-aspartate shuttle,” oxidation of ethanol, α-glycerophosphate, and the activity of succinic dehydrogenase, along with the changes in the intrahepatic distribution of aspartate aminotransferase. The rates of blood alcohol clearance, hepatic alcohol dehydrogenase activity, and NADPH-dependent microsomal ethanol oxidation were also studied after different time intervals of alcohol withdrawal from chronically alcohol-fed animals. Hepatic mitochondria from chronically ethanol-fed mice (ethanol withheld 20 hr before sacrifice) utilized extramitochondrial NADH at rates 25–40% higher than the corresponding pair-fed controls. Addition of malateaspartate shuttle components to mitochondria from control and ethanol-fed groups resulted in 70 and 90% stimulation of NADH utilization, respectively. Mitochondria from both groups showed respiratory control upon ADP addition (state 3). Preincubation with amino-oxyacetate or hydrazine, which inhibit aspartate aminotransferase activity, prevented the stimulatory effect of malate-aspartate shuttle on NADH utilization. Mitochondria from livers of chronic ethanol-fed mice in the presence of reconstituted malate-aspartate shuttle showed 30–40% higher utilization of ethanol than the corresponding pair-fed control animals. The rate of mitochondrial α-glycerophosphate utilization by alcohol-fed animals was significantly higher than the control group. Succinic dehydrogenase activity measured as an index of mitochondrial permeability in the absence of Ca 2+ showed 85% higher activity in alcoholtreated group than the control animals. Chronic ethanol feeding for 4 weeks resulted in an increase in the activity of hepatic aspartate aminotransferase in the cytoplasmic fraction and a corresponding decrease in the mitochondrial fraction. Alcohol withdrawal from chronic alcohol-fed animals resulted in a decrease in the blood alcohol clearance rate after 10 days. Furthermore, a lack of correlation was observed between the rates of blood alcohol clearance and the activity of hepatic alcohol dehydrogenase on one hand, and between the rates of blood alcohol clearance and the microsomal ethanol-oxidizing activity on the other.

Ethanol sleep times and hepatic alcohol and aldehyde dehydrogenase activities in mice

The roles of hepatic alcohol and aldehyde dehydrogenase activities in the determination of individual differences in ethanol-induced sleep time were investigated in a genetically heterogeneous population of mice. Both dehydrogenase activities correlated signigicantly with sleep time when a 90-min interval was allowed between ethanol challenge and biochemical assay, but not when a 42-day interval was allowed. Alcohol dehydrogenase activity appears to be important in the determination of sleep time presumably because this enzyme catalyzes the rate limiting step in ethanol metabolism. Aldehyde dehydrogenase appears to be somewhat less important in the determination of sleep time than is alcohol dehydrogenase. Increases in alcohol dehydrogenase activity due to a single challenging dose of ethanol were also investigated in three inbred mouse strains. Significant increases were observed in the C57BL/6J strain, but not in the BALB/cJ and DBA/2J strains. The peak effect was observed at 90 min post-injection.

The redox-state in relation to ethanol metabolism by rat and guinea pig liver in vitro

Effect of hepatic redox-state on the rate of ethanol metabolism and citric acid cycle activity has been studied in rat and guinea pig liver. Cytoplasmic (NADH) (NAD) ratio in guinea pig liver is about 8-fold higher than in rat liver. Although, hepatic alcohol dehydrogenase (EC 1.1.1.1) activity is similar in the two species, ethanol metabolism by rat liver in vitro, is about 3 times greater than by guinea pig liver. Addition of ethanol results in a 3-fold increase in the hepatic redox-state in the rat but causes a small increase in the guinea pig. Ethanol-induced increase in hepatic (NADH) (NAD) ratio, results in a 40–60% inhibition of 14CO 2 formation from various radioactive substrates in the rat. However, in guinea pig liver where this ratio is high, 14CO 2 formation is already lower by 30–50% than in rat liver and ethanol addition does not result in a further decrease. The inhibitory effect of ethanol on 14CO 2 formation is also observed in rat kidney cortex slices. However, this effect can be abolished by the addition of methylene blue or pyrazole. This study shows that in normal liver cytoplasmic redox-state regulates the rate of ethanol metabolism and that the activity of hepatic alcohol dehydrogenase is not a rate-limiting factor. Furthermore, the inhibitory effect of ethanol on CO 2 production and therefore on citric acid cycle activity is a consequence of increased (NADH) (NAD) ratio in liver.

Elevation of alcohol dehydrogenase activity in the subclinically scorbutic guinea-pig.

1. Because it has been shown that a majority of alcoholics are subclinically scorbutic, the metabolism of ethanol was studied in subclinically-scorbutic guinea-pigs. 2. Hepatic alcohol dehydrogenase activity was raised maximally by ethanol within 2 days. 3. In twenty-three subclinically-scorbutic guinea-pigs fed ethanol for 2 weeks, the alcohol dehydrogenase activity (±SD) was 11·5 ± 1·2 units/g of liver protein compared with 8·6 ± 0·6 units/g of liver protein in twenty-three healthy animals fed ethanol. 4. The NAD+/NADH ratio in subclinically-scorbutic guinea-pigs and healthy guinea-pigs fed ethanol, shows that there is more NAD+ available for oxidation of alcohol in subclinically-scorbutic guinea-pigs. These results may explain the increased tolerance of alcoholics to alcohol.

Effect of carbon tetrachloride treatment on ethanol metabolism

Administration of a single dose (2.5 ml/kg body weight, p.o.) of carbon tetrachloride to rats was found to cause a marked decrease in activity of the hepatic microsomal ethanol oxidizing system (MEOS). As early as 6 hr after CCl 4 administration 50 per cent decrease of MEOS activity was observed; this decrease amounted to 58 and 63 per cent at 10 and 20 hr respectively. With identical CCl 4 treatment, there was no change in hepatic alcohol dehydrogenase activity. At 20 hr, when the reduction of MEOS was greatest, there was no significant effect of the CCl 4 on the rate of ethanol uptake by liver slices or on the rate of ethanol metabolism in vivo as measured in the whole body or as estimated from the rate of decrease of blood ethanol concentration. It is, therefore, suggested that MEOS does not play a significant role in ethanol metabolism in vivo in the rat.

Ethanol metabolism in rats treated with ethyl-α-p-chlorophenoxyiso-butyrate (Clofibrate)

Clofibrate treatment (60 mg/day/100 g body wt im. for nine days) increased the liver to body weight ratio of the rats by 71 %. The activity of mitochondrial α-glycerophosphate dehydrogenase in the liver of clofibrate-treated rats was five times greater than in normal rat liver. The amounts of cytochrome a and mitochondrial protein per gram of liver wet weight were not changed. Clofibrate treatment increased the oxygen consumption of liver slices by 16 %. Ethanol increased the lactate/pyruvate ratio (an index of the redox state in the cytosolic compartment of the liver cell) in perfused normal livers and only slightly in the livers from clofibrate-treated rats. This effect of clofibrate is similar to that of thyroxine and seems to be due to enzyme induction, as diethylaminoclofibrate hydrochloride added in vitro did not prevent ethanol-induced increase in hepatic lactate/pyruvate ratio. Clofibrate treatment in rats significantly increased the disappearance rate of ethanol. The activity of hepatic alcohol dehydrogenase per gram of liver wet weight of per milligram of soluble protein was not changed by clofibrate treatment.

The effect of whiskey and low-protein diets on hepatic enzymes in rats

Chronic administration of whiskey to adult female rats reduced hepatic alcohol dehydrogenase activity. Isocitric dehydrogenase and glutamic pyruvic transaminase activities were also decreased.

Liver Disease, Hepatic Alcohol Dehydrogenase Activity, and Alcohol Metabolism in the Human

Liver Disease, Hepatic Alcohol Dehydrogenase Activity, and Alcohol Metabolism in the Human

Ribonucleic acids II. Improved preparation of rat-liver ribonucleic acid

The glycolethers 2-methoxyethanol (2-ME), 2-ethoxyethanol (2-EE), and 2-butoxyethanol are widely used organic solvents with teratogenic, spermatotoxic, and hematotoxic effects due to the respective alkoxyacetic acid metabolites formed via alcohol dehydrogenase (ADH). ADH displays sexually dimorphic activities in adult rats, and is probably at least in part under the control of testosterone. The aim of this study was to investigate whether induction of ADH is also sex-dependent. Ethanol, 2-ME, and 2-EE were tested as inducers of hepatic and gastric ADH in female, male, and castrated male rats. The activity of hepatic ADH was higher in female than in male rats, while the activity of gastric ADH was higher in male than in female rats. The activities of ADH increased with increasing chain length of the glycolethers and alcohols. Castration of male rats led to a female pattern of ADH activity, i.e. increased activity of hepatic ADH and decreased activity of gastric ADH. Ethanol had no inducing effect on hepatic ADH in either male or female rats. 2-ME and 2-EE caused an increase in the activity of hepatic ADH in male and castrated male rats only. The present data demonstrate a different expression of ADH isoenzymes in male and female rats, and a sex-dependent induction of ADH isoenzymes. The different possible regulatory mechanisms for the different ADH isoenzymes require further investigation.