Hepatic Alcohol Dehydrogenase Research Articles

Decreased in vivo acetaldehyde oxidation and hepatic aldehyde dehydrogenase inhibition in C57BL and DBA mice treated with carbon tetrachloride

Male C57BL 6J (C57) and DBA 2J (DBA) mice were dosed with 500 μl/kg ig CCl 4, 24 hr before a 3.25 g/kg ip dose of ethanol. The relative bioavailability of CCl 4 was similar in both mouse strains. CCl 4 pretreatment produced genotypically related decreases in blood ethanol elimination rates with DBA mice showing the greater decrease. Blood acetaldehyde concentrations were significantly elevated in the CCl 4-pretreated animals of both strains. DBA CCl 4-pretreated mice exhibited significantly higher blood acetaldehyde concentrations than C57 mice 1, 2, 3, and 4 hr after ethanol administration. CCl 4 pretreatment resulted in acetaldehyde elevations of approximately threefold in C57 mice four- to fivefold in DBA mice. Four hours after the ethanol dose, animals were sacrificed for enzymatic analyses of hepatic aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activity. The specific activities and activity per gram of cytosolic and mitochondrial ALDH were decreased in both mouse strains. ADH activity was not significantly altered by CCl 4 pretreatment. Liver necrosis, as measured by serum alanine aminotransferase activity, was similar in both strains (approx fivefold increase). These data indicate that CCl 4 increased blood acetaldehyde concentrations by inhibiting hepatic cytosolic and mitochondrial ALDH and that the degree of blood acetaldehyde elevation is different in the two inbred mouse strains C57 and DBA.

Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Effect of chronic ethanol administration.

In young (4-week-old) male and female spontaneously hypertensive (SH) rats, ethanol metabolic rate in vivo and hepatic alcohol dehydrogenase activity in vitro are high and not different in the two sexes. In males, ethanol metabolic rate falls markedly between 4 and 10 weeks of age, which coincides with the time of development of sexual maturity in the rat. Alcohol dehydrogenase activity is also markedly diminished in the male SH rat and correlates well with the changes in ethanol metabolism. There is virtually no influence of age on ethanol metabolic rate and alcohol dehydrogenase activity in the female SH rat. Castration of male SH rats prevents the marked decrease in ethanol metabolic rate and alcohol dehydrogenase activity, whereas ovariectomy has no effect on these parameters in female SH rats. Chronic administration of testosterone to castrated male SH rats and to female SH rats decreases ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in mature males. Chronic administration of oestradiol-17beta to male SH rats results in marked stimulation of ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in female SH rats. Chronic administration of ethanol to male SH rats from 4 to 11 weeks of age prevents the marked age-dependent decreases in ethanol metabolic rate and alcohol dehydrogenase activity, but has virtually no effect in castrated rats. In the intoxicated chronically ethanol-fed male SH rats, serum testosterone concentrations are significantly depressed. In vitro, testosterone has no effect on hepatic alcohol dehydrogenase activity of young male and female SH rats. In conclusion, in the male SH rat, ethanol metabolic rate appears to be limited by alcohol dehydrogenase activity and is modulated by testosterone. Testosterone has an inhibitory effect and oestradiol has a testosterone-dependent stimulatory effect on alcohol dehydrogenase activity and ethanol metabolic rate in these animals.

Psychotropic drug metabolism in fetal alcohol syndrome.

Simultaneous intake of ethanol with chlorpromazine (thorazine), an antipsychotic drug, leads to about 60% decrease in the chlorpromazine removal from the rat blood. Studies with liver homogenates showed that ethanol inhibits the metabolism of this drug by about 50%. The inhibitory effect of ethanol on the metabolism of chlorpromazine can be largely abolished by pyrazole (2 mM) preincubation. Prolonged maternal ethanol consumption during pregnancy and lactation leads to a decrease in chlorpromazine metabolism in the fetal (30%), neonatal (46%) and maternal livers. Prolonged maternal ethanol intake also leads to an increase in the (UDPG)/(UDPGA) ratio in the suckling neonatal liver and the maternal liver. Simultaneous acute administration of ethanol (2g/kg) with psychotropic drugs such as chlordiazepoxide (librium), diazepam (valium), chlorpromazine (thorazine) or meprobamate (equanil) to pregnant or non-pregnant rats led to a decrease in the blood alcohol clearance rates. In another group of nonpsychotropic drugs tested, tolbutamide (orinase) produced the most pronounced (47%) decrease in blood alcohol clearance rates. This decrease was found to be accompanied by the inhibition of hepatic alcohol dehydrogenase.

Alterations of hepatic alcohol metabolizing enzyme activities due to thyroid hormones.

In order to achieve a hyperthyroid state, rats were treated for 7 days with thyroxine (150 microgram/100 g BW) or triiodothyronine (10 microgram/100 g BW). This regimen resulted in an enhanced activity of the microsomal ethanol oxidizing system. In addition, 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 functional "hyperthyroid hepatic state", increased rates of ethanol metabolism observed following prolonged alcohol intake might therefore be attributed at least in part to an induced activity of the microsomal ethanol oxidizing system in the liver.

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.

Methanol poisoning I. The role of formic acid in the development of metabolic acidosis in the monkey and the reversal by 4-methylpyrazole

The administration of methanol (3 g/kg) to rhesus and pigtail monkeys produced signs and symptoms similar to those described for methanol poisoning in man. These were a mild central nervous system depression, a latent period of 8–12 hr when no signs were observed, followed by a severe metabolic acidosis leading to coma and death 12–33 hr after the initial administration. The gradual development of metabolic acidosis coincided with the accumulation of formic acid in the blood, and the decrease of bicarbonate in the plasma. There was an increase in the anion gap during the period of metabolic acidosis, and formic acid concentration accounted for about one-half of the increase observed. Therefore, formic acid was a major, but not the only, determinant of the metabolic acidosis. Administration of 4-methylpyrazole (50 mg/kg), a potent inhibitor of monkey hepatic alcohol dehydrogenase, produced a 75% inhibition of the rate of [ 14C]methanol metabolism to 14CO 2 in the monkey. During the first 36 hr following the administration of 4-methylpyrazole and methanol, no metabolic acidosis developed, no formate accumulated in the blood, and no signs of toxicity were observed. After a single dose of 4-methylpyrazole and methanol, the toxic syndrome was delayed by about 36 hr in the monkey, after which time the onset of metabolic acidosis and the accumulation of formic in blood was noted. The use of the monkey as a model for the study of methanol poisoning is presented, and the possible use of 4-methylpyrazole in the treatment of methanol poisoning is implicit.

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.

Inhibition by nicotinic acid of hepatic steatosis and alcohol dehydrogenase in ethanol-treated rats

A single intoxicating dose of ethanol (6.0 g/kg) depressed the NAD: NADH 2 ratio and increased the NADPH 2: NADP ratio in rat liver. Nicotinic acid (NA) treatment prevented the accumulation of nonesterified fatty acid (NEFA), total fats, and neutral fats in the liver of rats given ethanol (EtOH); it also kept blood EtOH elevated. We postulate that NA acted by blocking EtOH oxidation—a block apparently exerted by inhibition of alcohol dehydrogenase (ADH), mediator of the principal oxidative pathway of ethanol metabolism. It was inferred that NA inhibits the enzyme by binding at the adenosine diphosphate ribose site since the addition of Zn 2+ in vitro failed to reverse the NA inhibition of rat liver ADH. Blockage at the aforementioned site would thus inhibit coenzyme (NAD) binding to the apoenzyme, resulting in a block in the EtOH oxidation.

Metabolism of riboflavin: reduction of 7,8-dimethyl-10-formylmethylisoalloxazine by an enzyme in liver.

1. The riboflavin metabolite, 7,8-dimethyl-10-formylmethylisoalloxazine, has been shown to be reduced to the corresponding alcohol, 7,8-dimethyl-10-(2′-hydroxyethyl)isoalloxazine, by an enzyme present in the soluble fraction of liver and kidney.2. The enzyme is present in these organs in a number of species but is especially prevalent in the tissues of ruminants. It appears to be different from the normal hepatic alcohol dehydrogenase in that it uses NADPH rather than NADH as cofactor.

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.

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.

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.

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.