Nutritional and gonadal effects on the intra-acinar profiles of low-Km and high-Km aldehyde dehydrogenase activity in rat liver

Total and low-Km aldehyde dehydrogenase (ALDH) activity was measured in 50-150 ng microdissected liver tissue samples of the entire sinusoidal length. High-Km ALDH activity was calculated by subtracting the low-Km ALDH values from the total ALDH activity. Enzyme activity was measured by a microchemical assay, using the oil-well technique with luminometric determination of NADH. The intra-acinar profiles of high-Km and low-Km ALDH activity could be demonstrated graphically for both male and female rats after 84 h of starvation, and after starvation and refeeding for 6 nights. In addition, the ALDH distribution patterns of juvenile, castrated, and castrated and testosterone-treated rats were determined. It could be demonstrated that starvation, and starvation followed by refeeding, lead to changes in enzyme activity which parallel the loss and regain of liver- and body-weight. The nutritional factors do not essentially alter the normal intra-acinar profiles. In juvenile rats, ALDH is lower by 30% in comparison with the controls, but sex-differences in the distribution profiles are not yet present. Castration has no effect on the amount of enzyme activity but the sex specific distribution profiles are less marked. The main effect of testosterone treatment is an elevation of low-Km ALDH in the perivenous zone. The characteristics of the intra-acinar profiles of high-Km and low-Km ALDH activity are discussed with respect to hepatic acetaldehyde oxidation and alcoholic liver damage.     

Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the Ki for crotonaldehyde was about 5 to 20 microM. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than 10-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low-Km mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme.     

Inhibition of mitochondrial aldehyde dehydrogenase and acetaldehyde oxidation by the glutathione-depleting agents diethylmaleate and phorone

Experiments were carried out to study the effect of two commonly used glutathione-depleting agents, diethylmaleate and phorone, on the oxidation of acetaldehyde and the activity of aldehyde dehydrogenase. The oxidation of acetaldehyde by intact hepatocytes was inhibited when the cells were incubated with diethylmaleate. Washing and resuspending the cells in diethylmaleate-free medium afforded protection against the inhibition of acetaldehyde oxidation. The oxidation of acetaldehyde by isolated rat liver mitochondria as well as by disrupted mitochondria in the presence of excess NAD+ was inhibited by diethylmaleate or phorone, indicating inhibition of the low-Km aldehyde dehydrogenase. In addition, diethylmaleate inhibited oxidation of acetaldehyde by the high-Km cytosolic aldehyde dehydrogenase. Significant accumulation of acetaldehyde occurred when ethanol was oxidized by hepatocytes in the presence, but not in the absence, of diethylmaleate. Thus, diethylmaleate blocks the oxidation of added or metabolically generated acetaldehyde, analogous to results with other inhibitors of the low-Km aldehyde dehydrogenase such as cyanamide. These results suggest that caution should be used in interpreting the effects of diethylmaleate or phorone on metabolic reactions, especially those involving metabolism of aldehydes such as formaldehyde, because, in addition to depleting glutathione, these agents inhibit the low-Km aldehyde dehydrogenase.     

Microquantitative determination of intra-acinar distribution profiles of low-Km and high-Km aldehyde dehydrogenase activity in rat liver

Microquantitative measurements of total and of low-Km aldehyde dehydrogenase (ALDH) activity with millimolar and micromolar concentrations of acetaldehyde and propionaldehyde were carried out on the livers of male and female rats. Lyophilized cryostat sections of liver parenchyma were microdissected along the entire sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ALDH activity was measured in a microbiochemical assay using the oil-well technique with luminometric determination of NADH. On the basis of single measurements, mean values of total, low-Km and high-Km ALDH activity could be calculated and the specific distribution patterns graphically demonstrated. The two substrates acetaldehyde and propionaldehyde yielded similar values of ALDH activity, the intraacinar distribution profiles of which showed characteristic sex differences. In the liver of the male rat high-Km ALDH activity has two flat peaks in the periportal and the perivenous area, while low-Km ALDH activity is almost evenly distributed throughout the acinus. In the livers of female rats, both high-Km and low-Km ALDH activity shows a continuous gradient which decreases from the periportal to the perivenous zone (pp/pv = 1.4:1). It was therefore possible to demonstrate that the maxima of alcohol dehydrogenase activity and of low-Km ALDH activity are localized in opposite parts of the liver acinus of the female rat. This heterotopy should have consequences with respect to hepatotoxicity after alcohol ingestion.     

Stomach and duodenal alcohol and aldehyde dehydrogenase isozymes in Chinese

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozyme phenotypes were determined in surgical and endoscopic biopsies of the stomach and duodenum by agarose isoelectric focusing. gamma-ADH was found to be the predominant form in the mucosal layer whereas beta-ADH was predominant in the muscular layer. Low-Km ALDH1 and ALDH2 were found in the stomach and duodenum. High-Km ALDH3 isozymes occurred only in the stomach but not in the duodenum. The isozyme patterns of gastric mucosal ALDH2 and ALDH3 remained unchanged in the fundus, corpus, and antrum. The stomach ALDH3 isozymes exhibited a Km value for acetaldehyde of 75 mM, and an optimum for acetaldehyde oxidation at pH 8.5. Since the Km value was high, ALDH3 contributed very little, if any, to gastric ethanol metabolism. The activities of ALDH in the gastric mucosa deficient in ALDH2 were 60-70% of that of the ALDH2-active phenotypes. These results indicate that Chinese lacking ALDH2 activity may have a lower acetaldehyde oxidation rate in the stomach during alcohol consumption.     

Effect of acetaldehyde and cyanamide on the metabolism of formaldehyde by hepatocytes, mitochondria, and soluble supernatant from rat liver

Formaldehyde can be metabolized primarily by two different pathways, one involving oxidation by the low-Km mitochondrial aldehyde dehydrogenase, the other involving a specific, glutathione-dependent, formaldehyde dehydrogenase. To estimate the roles played by each enzyme in formaldehyde metabolism by rat hepatocytes, experiments with acetaldehyde and cyanamide, a potent inhibitor of the low-Km aldehyde dehydrogenase were carried out. The glutathione-dependent oxidation of formaldehyde by 100,000g rat liver supernatant fractions was not affected by either acetaldehyde or by cyanamide. By contrast, the uptake of formaldehyde by intact mitochondria was inhibited 75 to 90% by cyanamide. Acetaldehyde inhibited the uptake of formaldehyde by mitochondria in a competitive fashion. Formaldehyde was a weak inhibitor of the oxidation of acetaldehyde by mitochondria, suggesting that, relative to formaldehyde, acetaldehyde was a preferred substrate. In isolated hepatocytes, cyanamide, which inhibited the oxidation of acetaldehyde by 75 to 90%, produced only 30 to 50% inhibition of formaldehyde uptake by cells as well as of the production of 14CO2 and of formate from [14C]formaldehyde. The extent of inhibition by cyanamide was the same as that produced by acetaldehyde (30-40%). In the presence of cyanamide, acetaldehyde was no longer inhibitory, suggesting that acetaldehyde and cyanamide may act at the same site(s) and inhibit the same formaldehyde-oxidizing enzyme system. These results suggest that, in rat hepatocytes, formaldehyde is oxidized by cyanamide- and acetaldehyde-sensitive (low-Km aldehyde dehydrogenase) and insensitive (formaldehyde dehydrogenase) reactions, and that both enzymes appear to contribute about equally toward the overall metabolism of formaldehyde.     

Mouse alcohol dehydrogenase 4: kinetic mechanism, substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37 degrees C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD(+) is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K(m) for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

Role of cytosolic rat liver aldehyde dehydrogenase in the oxidation of acetaldehyde during ethanol metabolism in vivo.

The activity of a high-Km aldehyde dehydrogenase in the liver cytosol was increased by phenobarbital induction. No corresponding increase in the oxidation rate of acetaldehyde in vivo was found, and it is concluded that cytosolic aldehyde dehydrogenase plays only a minor role in the oxidation of acetaldehyde during ethanol metabolism.     

Hydrogen transfer between ethanol molecules during oxidoreduction in vivo.

Rates of exchange catalysed by alcohol dehydrogenase were determined in vivo in order to find rate-limiting steps in ethanol metabolism. Mixtures of [1,1-2H2]- and [2,2,2-2H3]ethanol were injected in rats with bile fistulas. The concentrations in bile of ethanols having different numbers of 2H atoms were determined by g.l.c.-m.s. after the addition of [2H6]ethanol as internal standard and formation of the 3,5-dinitrobenzoates. Extensive formation of [2H4]ethanol indicated that acetaldehyde formed from [2,2,2-2H3]ethanol was reduced to ethanol and that NADH used in this reduction was partly derived from oxidation of [1,1-2H2]ethanol. The rate of acetaldehyde reduction, the degree of labelling of bound NADH and the isotope effect on ethanol oxidation were calculated by fitting models to the found concentrations of ethanols labelled with 1-42H atoms. Control experiments with only [2,2,2-2H3]ethanol showed that there was no loss of the C-2 hydrogens by exchange. The isotope effect on ethanol oxidation appeared to be about 3. Experiments with (1S)-[1-2H]- and [2,2,2-2H3]ethanol indicated that the isotope effect on acetaldehyde oxidation was much smaller. The results indicated that both the rate of reduction of acetaldehyde and the rate of association of NADH with alcohol dehydrogenase were nearly as high as or higher than the net ethanol oxidation. Thus, the rate of ethanol oxidation in vivo is determined by the rates of acetaldehyde oxidation, the rate of dissociation of NADH from alcohol dehydrogenase, and by the rate of reoxidation of cytosolic NADH. In cyanamide-treated rats, the elimination of ethanol was slow but the rates in the oxidoreduction were high, indicating more complete rate-limitation by the oxidation of acetaldehyde.     

Genetic polymorphism of enzymes of alcohol metabolism and susceptibility to alcoholic liver disease

Differences in the pharmacokinetics of alcohol absorption and elimination are, in part, genetically determined. There are polymorphic variants of the two main enzymes responsible for ethanol oxidation in liver, alcohol dehydrogenase and aldehyde dehydrogenase. The frequency of occurrence of these variants, which have been shown to display strikingly different catalytic properties, differs among different racial populations. Since the activity of alcohol dehydrogenase in liver is a rate-limiting factor for ethanol metabolism in experimental animals, it is likely that the type and content of the polymorphic isoenzyme subunit encoded at ADH2, beta-subunit, and at ADH3, the gamma-subunit, are contributing factors to the genetic variability in ethanol elimination rate. The recent development of methods for genotyping individuals at these loci using white cell DNA will allow us to test this hypothesis as well as any relationship between ADH genotype and the susceptibility to alcoholism or alcohol-related pathology. A polymorphic variant of human liver mitochondrial aldehyde dehydrogenase, ADLH2, which has little or no acetaldehyde oxidizing activity has been identified. Individuals with the deficient ALDH2 phenotype do not have altered ethanol elimination rates but they do exhibit high blood acetaldehyde levels and dysphoric symptoms such as facial flushing, nausea and tachycardia, after drinking alcohol. Because acetaldehyde is so reactive, it binds to free amino groups of proteins including a 37 kilodalton hepatic protein-acetaldehyde adduct and may elicit an antibody response. We would predict that individuals who have low ALDH2 activity because of liver disease or because they have the inactive ALDH2 variant isoenzyme might form more protein-acetaldehyde adducts and elicit a greater immune response. These adducts may represent good biological markers of alcohol abuse and may also play a role in liver injury due to chronic alcohol consumption.     

Mouse alcohol dehydrogenase 4: kinetic mechanism,substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37°C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD⁺ is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K_m for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

Evaluation of a role of acetaldehyde in the mechanism of inhibition of p-nitroanisole O-demethylation in isolated hepatocytes by ethanol

The rate of p-nitroanisole O-demethylation is markedly inhibited by ethanol. To evaluate a role of acetaldehyde in the inhibition by ethanol, a comparison was made of the effects of ethanol and acetaldehyde on the metabolism of p-nitroanisole by isolated liver cells. No effect on the metabolism of p-nitroanisole was found at low concentrations of acetaldehyde (<0.5 mm), whereas inhibition occurred at high concentrations (1 mm). In fact, acetaldehyde was not any more inhibitory than crotonaldehyde, which is a poor substrate for the low-K_m mitochondrial aldehyde dehydrogenase. Cyanamide, an inhibitor of acetaldehyde oxidation, did not prevent the inhibition by ethanol. Crotonol, an alcohol that does not change the mitochondrial redox state, in contrast to ethanol, proved to be a more effective inhibitor of the metabolism of p-nitroanisole than ethanol. Greater sensitivity to crotonol was also found in isolated microsomes and may reflect hydrophobic effects by crotonol, relative to ethanol. These results suggest that although high levels of acetaldehyde can be inhibitory, physiological levels of acetaldehyde did not affect the metabolism of p-nitroanisole. It is unlikely that acetaldehyde itself plays a major role in the mechanism by which ethanol inhibits the metabolism of p-nitroanisole. The inhibition of p-nitroanisole O-demethylation by ethanol was prevented by pyruvate or fructose, but not by xylitol, sorbitol, or lactate. All these substrates by themselves stimulated metabolism of p-nitroanisole. Pyruvate and glyceraldehyde (which arises from the metabolism of fructose) can oxidize cytosolic NADH. These results suggest that the generation of cytosolic NADH from the oxidation of ethanol, the subsequent requirement for substrate shuttles to transfer NADH into the mitochondria, and redox inhibition of the citric acid cycle, interfere with the transport of NADPH out of the mitochondria, and consequently with drug metabolism.     

Amidino-substituted aromatic heterocycles as probes of the specificity pocket of trypsin-like proteases.

The effect of pargyline on the uptake of acetaldehyde (in the presence of pyrazole) by isolated rat liver cells was studied after incubating the liver cells for 0, 10, 30, 45, and 60 min with 0.40, 1.30, and 2.6 mm pargyline. Without any incubation period, pargyline had no effect on acetaldehyde uptake. With increasing time of incubation, there was a progressive increase in the extent of inhibition of acetaldehyde uptake by pargyline. This suggests the possibility that pargyline is metabolized to the effective inhibitor or the incubation period allows pargyline to reach its site(s) of action. Pargyline was also a more effective inhibitor of the uptake of lower concentrations of acetaldehyde, e.g., 0.167 mm, than of higher concentrations (1.0 mm) of acetaldehyde, especially after short incubation periods or when pyrazole was omitted from the reaction medium. After a 20- to 30-min incubation period, pargyline inhibited the control rate of ethanol oxidation by the liver cells, as well as the accelerated rate of ethanol oxidation found in the presence of pyruvate or an uncoupling agent. Pargyline had no effect on hepatic oxygen consumption. During ethanol oxidation, a time-dependent release of acetaldehyde into the medium was observed. Pyruvate, by increasing the rate of ethanol oxidation, increased the output of acetaldehyde five- to tenfold. Pargyline increased the output of acetaldehyde two- to threefold, despite decreasing the rate of ethanol metabolism by the liver cells. These data indicate that pargyline inhibits the low K_m aldehyde dehydrogenase in intact rat liver cells and that this enzyme plays the major role in oxidizing the acetaldehyde which arises during the metabolism of ethanol. Although most of the acetaldehyde generated during the oxidation of ethanol is removed by the liver cells in an effective manner, changes in the activity of aldehyde dehydrogenase or the rate of acetaldehyde generation significantly alter the hepatic output of acetaldehyde.     

The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose.

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.     

Acetaldehyde oxidation in rat liver mitochondria, action of Mg2+, ATP and rotenone on acetaldehyde oxidation in intact mitochondria, and on some purified aldehyde dehydrogenase isozymes

In intact rat liver mitochondria acetaldehyde is oxidized by three functionally distinct dehydrogenase systems. Two of these reduce intramitochondrial nicotinamide adenine dinucleotide (NAD): one is operative with micromolar acetaldehyde concentrations and is stimulated by Mg2+, the other is operative with millimolar acetaldehyde concentrations and is stimulated by adenosine 5'-triphosphate (ATP). The third system reduces added NAD and is stimulated by rotenone. Connected to these systems, three aldehyde dehydrogenase isozymes (ALDH) have been purified: a low-Km ALDH activated by Mg2+, a high-Km ALDH activated by ATP and Mg2+, a high-Km ALDH activated by rotenone. The properties of some isozymes are affected by detergents. Thus, deoxycholate augments the stimulation of low-Km isozyme by Mg2+ and confers sensitivity to Mg2+ and ATP on one of the high-Km isozymes. A fourth isozyme has been purified. Its affinity for acetaldehyde is so low that it is very unlikely that acetaldehyde is the physiological substrate.     

Differences in hepatic and metabolic changes after acute and chronic alcohol consumption.

Hepatic metabolism of ethanol to acetaldehyde by the alcohol dehydrogenase pathway is associated with the generation of reducing equivalents as NADH. Conversely, reducing equivalents are consumed when ethanol oxidation is catalyzed by the NADPH dependent microsomal ethanol oxidizing system. Since the major fraction of ethanol metabolism proceeds via alcohol dehydrogenase and since the oxidation of acetaldehyde also generates NADH, an excess of reducing equivalents is produced. This explains a variety of effects following acute ethanol administration, including hyperlactacidemia, hyperuricemia, enhanced lipogenesis and depressed lipid oxidation. To the extent that ethanol is oxidized by the alternate microsomal ethanol oxidizing system pathway, it slows the metabolism of other microsomal substrates. Following chronic ethanol consumption, adaptive microsomal changes prevail, which include enhanced ethanol and drug metabolism, and increased lipoprotein production. Severe hepatic lesions (alcoholic hepatitis and cirrhosis) develop after prolonged ethanol consumption in baboons. These injurious alterations are not prevented by nutritionally adequate diets and can therefore be ascribed to ethanol rather than to dietary inadequacy.     

Bioelectronic sniffers for ethanol and acetaldehyde in breath air after drinking

Two kinds of bioelectronic gas sensors (bio-sniffer) incorporating alcohol oxidase (AOD) and aldehyde dehydrogenase (ALDH) were developed for the convenient analysis of ethanol and acetaldehyde in expired gas, respectively. The sniffer devices for gaseous ethanol and acetaldehyde were constructed by immobilizing enzyme on electrodes covered with filter paper and hydrophilic PTFE membrane, respectively. The AOD and ALDH sniffers were used in the gas phase to measure ethanol vapor from 1.0 to 500 ppm, and acetaldehyde from 0.11 to 10 ppm covering the concentration range encountered in breath after alcohol consumption. Both bio-sniffers displayed good gas selectivity which was attributed to the substrate specificity of the relevant enzymes (AOD and ALDH) as gas recognition material. From the results of physiological application, the bio-sniffers could monitor the concentration changes in breath ethanol and acetaldehyde after drinking. The ethanol and acetaldehyde concentrations in expired air from ALDH2 [-] (aldehyde dehydrogenase type 2 negative) subjects were higher than that of the ALDH2 [+] (positive) subjects. The results indicated that the lower activity of ALDH2 induced an adverse effect on ethanol metabolism, leading to ethanol and acetaldehyde remaining in the human body, even human expired air.     

Sex differences, alcohol dehydrogenase, acetaldehyde burst, and aversion to ethanol in the rat: a systems perspective

Individuals who carry the most active alcohol dehydrogenase (ADH) isoforms are protected against alcoholism. This work addresses the mechanism by which a high ADH activity leads to low ethanol intake in animals. Male and female ethanol drinker rats (UChB) were allowed access to 10% ethanol for 1 h. Females showed 70% higher hepatic ADH activity and displayed 60% lower voluntary ethanol intake than males. Following ethanol administration (1 g/kg ip), females generated a transient blood acetaldehyde increase ("burst") with levels that were 2.5-fold greater than in males (P < 0.02). Castration of males led to 1) an increased ADH activity (+50%, P < 0.001), 2) the appearance of an acetaldehyde burst (3- to 4-fold vs. sham), and 3) a reduction of voluntary ethanol intake comparable with that of na?ve females. The ADH inhibitor 4-methylpyrazole blocked the appearance of arterial acetaldehyde and increased ethanol intake. Since the release of NADH from the ADH.NADH complex constitutes the rate-limiting step of ADH (but not of ALDH2) activity, endogenous NADH oxidizing substrates present at the time of ethanol intake may contribute to the acetaldehyde burst. Sodium pyruvate given at the time of ethanol administration led to an abrupt acetaldehyde burst and a greatly reduced voluntary ethanol intake. Overall, a transient surge of arterial acetaldehyde occurs upon ethanol administration due to 1) high ADH levels and 2) available metabolites that can oxidize hepatic NADH. The acetaldehyde burst is strongly associated with a marked reduction in ethanol intake.     

Effects of alcohol consumption on mitochondrial aldehyde dehydrogenase isoenzymes in rat liver

The effects of long-term and short-term exposure of rats to ethanol on aldehyde dehydrogenase (ALDH) activity in the liver mitochondria were investigated. The specific activities of mitochondrial high Km ALDH and low Km ALDH after the prolonged administration of ethanol were both increased to levels about 2.5 times that of the control group. In contrast, high Km and low Km ALDH showed maximum activity 12 h after administration of a single large dose of ethanol, increasing 21 and 4.4 times, respectively, over the level in the control group. When ethanol was administered for a long time, the two ALDH isoenzyme levels showed approximately the same increase, while the high Km ALDH level was more significantly increased than the low Km ALDH level after a single large dose. These results suggest that the high Km ALDH level of the outer membrane was increased as a result of a transient increase in the level of acetaldehyde around the liver mitochondria after a single large dose of ethanol, and that high Km ALDH plays an important role in acetaldehyde metabolism. However, when ethanol was administered for a long time, the mitochondria were exposed to low concentrations of acetaldehyde over a long time, leading to an increase in levels of low and high Km ALDH in the matrix.     

Molecular Cloning, Characterization, and Potential Roles of Cytosolic and Mitochondrial Aldehyde Dehydrogenases in Ethanol Metabolism in Saccharomyces cerevisiae

The full-length DNAs for two Saccharomyces cerevisiae aldehyde dehydrogenase (ALDH) genes were cloned and expressed in Escherichia coli. A 2,744-bp DNA fragment contained an open reading frame encoding cytosolic ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitochondrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino acids were identified as the putative leader sequence. The ALDH5 gene was located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was found to be essentially NADP dependent, while the ALDH5 enzyme could utilize either NADP or NAD as a cofactor. The activity of ALDH1 was stimulated two- to fourfold by divalent cations but was unaffected by K⁺ ions. In contrast, the activity of ALDH5 increased in the presence of K⁺ ions: 15-fold with NADP and 40-fold with NAD, respectively. Activity staining of isoelectric focusing gels showed that cytosolic ALDH1 contributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest. Neither ALDH5 nor the other ALDH-like proteins identified from the genomic sequence contributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in the genome of strain TWY397 separately or simultaneously. The growth of single-disruption Δald1 and Δald2 strains on ethanol was marginally slower than that of the parent strain. The Δald1 Δald2 double-disruption strain failed to grow on glucose alone, but growth was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermentation. The double-disruption strain grew very slowly on ethanol. The role of mitochondrial ALDH5 in acetaldehyde metabolism has not been defined but appears to be unimportant.