Human Brain Glyceraldehyde-3-Phosphate Dehydrogenase, Succinic Semialdehyde Dehydrogenase and Aldehyde Dehydrogenase Isozymes: Substrate Specificity and Sensitivity to Disulfiram

Several enzymes active in the presence of NAD with acetaldehyde and propionaldehyde have been purified from human brain and characterized. The enzymes have been identified as aldehyde dehydrogenase (EC 1.2.1.3), NAD-linked succinic semialdehyde dehydrogenase (EC 1.2.1.24), and glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). Glyceraldehyde-3-phosphate dehydrogenase is extremely heterogeneous with some isozymes active with acetaldehyde, others inactive. The cytoplasmic enzyme, which is the classical glyceraldehyde-3-phosphate dehydrogenase, is inactive with acetaldehyde as substrate; the isozymes that are active with short chain aliphatic aldehydes are localized in the mitochondrial fraction. Properties of glyceraldehyde-3-phosphate dehydrogenase isozymes with respect to short chain aliphatic aldehydes and inhibition by disulfiram are described. Their Km values for acetaldehyde range from 300 to 2000 microM. All glyceraldehyde-3-phosphate dehydrogenases that are active with acetaldehyde are easily inactivated by low concentrations of disulfiram. In all cases activity regain can be obtained with 2-mercaptoethanol; in the case of two glyceraldehyde-3-phosphate isozymes (E8.5 and 9.0), activity can also be regained with cysteine and with glutathione; activity of E6.6 and E6.8 glyceraldehyde-3-phosphate dehydrogenases could not be regained with 33 microM cysteine or glutathione. Succinic semialdehyde dehydrogenase and aldehyde dehydrogenase (EC 1.2.1.3) were also inhibited by disulfiram; their activity could be regained with 2-mercaptoethanol but not with 33 microM cysteine or glutathione. Comparison of human brain succinic semialdehyde dehydrogenase and aldehyde dehydrogenase with glyceraldehyde-3-phosphate dehydrogenase shows that the activity with short chain aldehydes is not unique to aldehyde dehydrogenase; neither is sensitivity to disulfiram; activity with 3,4-dihydroxyphenylacetaldehyde appears to be a unique property of aldehyde dehydrogenase (EC 1.2.1.3).     

Human Aldehyde Dehydrogenase: Metabolism of Putrescine and Histamine

Imidazoleacetaldehyde and gamma-aminobutyraldehyde, metabolites of histamine and putrescine, respectively, have been shown to be substrates of human liver aldehyde dehydrogenase (EC 1.2.1.3) cytoplasmic (E1) and mitochondrial (E2) isozymes. The Km values at pH 7.4 and 500 microM NAD for imidazoleacetaldehyde and gamma-aminobutyraldehyde for the E1 isozyme are 40 and 800 microM, respectively, and for the E2 isozyme are 50 and 500 microM, respectively. The Km values with gamma-aminobutyraldehyde with both isozymes are high relative to Km values with acetaldehyde (50 microM for E1 and 1 microM for E2). Since activity with both imidazoleacetaldehyde and gamma-aminobutyraldehyde in crude liver homogenates paralleled that of aldehyde dehydrogenase (EC 1.2.1.3) during purification it appears likely that in the human liver this enzyme is responsible for metabolism of both compounds. If this is the case, interaction between metabolism of histamine and putrescine and that of alcohol is likely. Both imidazoleacetaldehyde and gamma-aminobutyraldehyde were synthesized in this laboratory and their stability has been investigated. Procedures for assaying aldehyde dehydrogenase employing synthetic metabolites of histamine and putrescine are provided.     

Inhibition of Human Aldehyde Dehydrogenase Isozymes by Propiolaldehyde

Propiolaldehyde--a metabolite of pargyline (Shirota et al., 1979) can function as an inhibitor or as a substrate of human aldehyde dehydrogenase (EC 1.2.1.3), dependent on conditions. In the presence of high concentration of NAD, propiolaldehyde is a substrate for both the cytoplasmic E1 isozyme and the mitochondrial E2 isozyme. The Km values are comparable to those with other short chain aldehydes; the maximal velocity is also similar for E2 but lower by about three-fold for E1. Preincubation with propiolaldehyde in the absence of NAD produces inactivation with K1 values of 1.6 microM for E1 and 1.8 microM for E2. NAD, but not propanal, protects both isozymes against inactivation with propiolaldehyde.     

Inhibition of rat hepatic mitochondrial aldehyde dehydrogenase-mediated acetaldehyde oxidation by trans-4-hydroxy-2-nonenal

The hepatic oxidation of ethanol has been demonstrated to cause peroxidation of cellular membranes, resulting in the production of aldehydes that are substrates for hepatic aldehyde dehydrogenases. It was the purpose of this study to evaluate the cooxidation of the lipid peroxidation product, trans-4-hydroxy-2-nonenal, and acetaldehyde by high-affinity mitochondrial aldehyde dehydrogenase, which is of prominent importance in the oxidation of ethanol-derived acetaldehyde. Experiments were performed for determination of kinetic parameters for uninhibited acetaldehyde and 4-hydroxynonenal oxidation by semi-purified mitochondrial aldehyde dehydrogenase prepared from male Sprague-Dawley rat liver. The affinity of the enzyme for the substrate at low substrate concentrations and the Michaelis-Menten constant of mitochondrial aldehyde dehydrogenase for acetaldehyde were 25 and 10 times greater, respectively, than those determined for 4-hydroxynonenal. Coincubation of acetaldehyde with physiologically relevant concentrations of 4-hydroxynonenal (0.25 to 5.0 mumol/L) with mitochondrial aldehyde dehydrogenase demonstrated that 4-hydroxynonenal is a potent competitive or mixed-type inhibitor of acetaldehyde oxidation, with concentration of 4-hydroxynonenal required for a twofold increase in the slope of the Lineweaver-Burk plot for acetaldehyde oxidation by ALDH of 0.48 mumol/L. The results of this study suggest that the aldehydic lipid peroxidation product, trans-4-hydroxy-2-nonenal, is a potent inhibitor of hepatic acetaldehyde oxidation and may potentiate the hepatocellular toxicity of acetaldehyde proposed to be an etiological factor of alcoholic liver disease.     

Regional Distribution of Low-Km Mitochondrial Aldehyde Dehydrogenase in the Rat Central Nervous System

To clarify the regional capacity of the brain to oxidize biogenic aldehydes and ethanol-derived acetaldehyde, a quantitative immunohistochemical study of the microregional and cellular expression of low Km mitochondrial aldehyde dehydrogenase (mALDH; EC 1.2.1.3) in the rat central nervous system was undertaken, using antiserum raised in rabbit against low-Km aldehyde dehydrogenase purified from rat liver mitochondria. mALDH-specific immunoreactivity (IR) was observed to various extent in the majority of structures in all brain and spinal cord areas. Staining was strong in the extranuclear cytoplasm of neuronal and glial cell bodies but less pronounced in their processes and terminals, the conducting tracts, white matter and neuropile and in blood vessels. Immunostaining density was 2 to 3 times higher in neuronal perikarya as compared with neuropile. mALDH-positive neurons were found in all brain regions, being strongest in the inferior olive and hippocampus stratum pyramidale and weakest in substantia nigra. The percentage of morphologically identifiable ALDH-positive neurons ranged from 40% in the arcuate hypothalamic nucleus to 88% in the cerebellar Purkinje cells. A comparison of the heterogeneous expression of mALDH in various rat CNS regions and cells, as observed in the present study, with the corresponding previously published distributions of the potential acetaldehyde-producing enzymes ADH and cytochrome P450 2E1 indicates major differences, which may help in understanding potential acetaldehyde-mediated CNS effects of ethanol. Knowledge of the regional distribution of high-affinity aldehyde dehydrogenase should also throw light on the neurophysiological role of local regulation of the metabolism of biogenic aldehydes in the brain.     

Aldehyde dehydrogenase in alcoholic subjects

Hepatic aldehyde dehydrogenase activity is depressed in alcoholic liver disease and may account for the observation that alcoholics develop high blood acetaldehyde concentrations following ethanol. To determine whether this is a specific defect in alcoholics, aldehyde dehydrogenase was studied in liver tissue obtained from three groups of subjects. Group I comprised 30 patients with alcoholic liver disease, Group II consisted of eight subjects with liver disease unrelated to alcohol abuse and Group III was a control group of 10 individuals with no significant liver disease. Mean hepatic aldehyde dehydrogenase activity was significantly lower in Group I than in Groups II or III [4.9 +/- 0.6 (mean +/- S.E.), compared to 10.2 +/- 1.8 and 12.4 +/- 1.1 nmoles of acetaldehyde oxidized per min X mg of protein, respectively]. Aldehyde dehydrogenase activity in Group II was relatively well maintained. Aldehyde dehydrogenase activity was found in cytosolic and mitochondrial fractions of liver homogenates. In alcoholic subjects, cytosolic aldehyde dehydrogenase activity was not more depressed than was mitochondrial aldehyde dehydrogenase. Isoelectric focusing demonstrated a single mitochondrial isoenzyme and a single cytosolic isoenzyme in most cases in Group III. In contrast, multiple cytosolic isoenzymes were consistently found in liver tissue from Group I subjects. These findings suggest that depressed aldehyde dehydrogenase activity in alcoholic subjects is not a consequence of liver disease.     

Subcellular localization of aldehyde dehydrogenase isozymes in human liver

The subcellular distribution of aldehyde dehydrogenase (ALDH) isozymes in human liver was studied by isoelectric focusing and biochemical procedures in biopsied liver specimens obtained during surgical procedures. Four types of ALDH isozymes (ALDH I, II, III and IV) were identified in human liver by isoelectric focusing. In 6 of the 13 livers examined, ALDH I was not detected, indicating that about half of the Japanese people may be classified as the unusual type. ALDH I, which exhibits a low Km with respect to acetaldehyde (Ac-CHO), was located mainly in the mitochondrial and cytosolic fractions. ALDH II (high Km for Ac-CHO) was found to be localized mainly in the microsomal and cytosolic fractions. ALDH III and IV (very high Km for Ac-CHO) were localized in all fractions, except for ALDH III in the microsomal fraction. Biochemical analysis indicates that low Km ALDH activity was localized in the mitochondrial and cytosolic fractions, while high Km and whole ALDH activities were detected in all 3 fractions. More than 80% of the low Km, high Km and whole ALDH activity was found in the cytosolic fraction. These distribution patterns were quite different from those in rats. These results indicate that the results obtained in animal experiments cannot be directly applied to humans and that the main site of Ac-CHO oxidation in the human liver is in the cytosol.     

Genetic Variants of Enzymes of Alcohol and Aldehyde Metabolism

The distribution of genetic variants (or gene markers) for alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, and aldehyde reductase isozymes has been examined among 12 inbred strains of mice. Electrophoretic variants are described for the major liver and stomach alcohol dehydrogenase isozymes (ADH-A2 and C2); liver, kidney, and stomach aldehyde dehydrogenase isozymes (AHD-1; AHD-2; AHD-4); a liver-specific aldehyde reductase (AHR-A2); and a liver aldehyde oxidase isozyme (AOX-2). Genetically determined activity variants were observed for a testis-specific aldehyde dehydrogenase (AHD-6); liver and kidney aldehyde reductase isozymes (AHR-3 and AHR-4); and the major liver AOX isozyme (AOX-1). These variants may serve as useful gene markers in alcohol research involving animal model studies with inbred strains in mice.     

Daidzin suppresses ethanol consumption by Syrian golden hamsters without blocking acetaldehyde metabolism.

Daidzin is a potent, selective, and reversible inhibitor of human mitochondrial aldehyde dehydrogenase (ALDH) that suppresses free-choice ethanol intake by Syrian golden hamsters. Other ALDH inhibitors, such as disulfiram (Antabuse) and calcium citrate carbimide (Temposil), have also been shown to suppress ethanol intake of laboratory animals and are thought to act by inhibiting the metabolism of acetaldehyde produced from ingested ethanol. To determine whether or not daidzin inhibits acetaldehyde metabolism in vivo, plasma acetaldehyde in daidzin-treated hamsters was measured after the administration of a test dose of ethanol. Daidzin treatment (150 mg/kg per day i.p. for 6 days) significantly suppresses (> 70%) hamster ethanol intake but does not affect overall acetaldehyde metabolism. In contrast, after administration of the same ethanol dose, plasma acetaldehyde concentration in disulfiram-treated hamsters reaches 0.9 mM, 70 times higher than that of the control. In vitro, daidzin suppresses hamster liver mitochondria-catalyzed acetaldehyde oxidation very potently with an IC50 value of 0.4 microM, which is substantially lower than the daidzin concentration (70 microM) found in the liver mitochondria of daidzin-treated hamsters. These results indicate that (i) the action of daidzin differs from that proposed for the classic, broad-acting ALDH inhibitors (e.g., disulfiram), and (ii) the daidzin-sensitive mitochondrial ALDH is not the one and only enzyme that is essential for acetaldehyde metabolism in golden hamsters.     

Mitochondria as the Primary Site of Acetaldehyde Metabolism in Beef and Pig Liver Slices

Aldehyde dehydrogenase (ALDH) is the major enzyme involved in the oxidation of acetaldehyde. It has been shown that the liver enzyme is located in both cytosol and mitochondria. It has not been established where the subcellular oxidation of acetaldehyde occurs in species other than rat. Using slices isolated from beef and pig livers and selectively inhibiting the mitochondria enzyme with cyanamide or the cytosolic enzyme with disulfiram, it was possible to address this question. It was found that with both beef and pig liver slices 60% of the oxidation was catalyzed by the mitochondrial ALDH and 20% by the higher Km cytosolic enzyme. The remainder of the metabolism was the result of non-ALDH involvement. Furthermore, any decrease in the level of the low Km mitochondrial aldehyde dehydrogenase activity resulted in a decreased rate of acetaldehyde oxidation showing that its activity governed the rate of acetaldehyde oxidation. These were the same conclusions previously reached using rat liver tissue slices. Thus, it appears that for all mammalian tissue, mitochondria is the primary location of acetaldehyde oxidation.     

Canine Liver Aldehyde Dehydrogenases: Distribution, Isolation, and Partial Characterization

Canine liver aldehyde dehydrogenases (ALDH) (aldehyde:NAD oxidoreductase; EC 1.2.1.3) are analogous to enzymes identified in human and other mammalian liver tissue in regard to subcellular localization, affinity for substrates, inhibition by disulfiram, and effects of magnesium ions on enzyme activity. Aldehyde dehydrogenase activity is distributed in the mitochondrial, microsomal, and cytosolic fractions of the cell. Four isoenzymes designated ALDH IA, IB, IIA, and IIB have been isolated from canine liver via ammonium sulfate fractionation, ion-exchange chromatography, and affinity chromatography. Based on cell fractionation followed by enzyme isolation, ALDH IA and IB appear to be extramitochondrial whereas ALDH IIA and IIB appear to be mitochondrial in origin. ALDH IA has a high Km for acetaldehyde (3 mM) and propionaldehyde (4 mM). ALDH IB and IIA have Km values for acetaldehyde and propionaldehyde in the range of 4-60 microM. ALDH IIB has the lowest Km of the four isoenzymes for acetaldehyde and propionaldehyde (1-3 microM). All four isoenzymes have Km values for NAD in the range of 4-70 microM. ALDH IB and IIA are sensitive to inhibition by disulfiram whereas ALDH IA and IIB are resistant. Magnesium ions inhibit ALDH IA, IB, and IIA whereas ALDH IIB activity is stimulated approximately 2-fold. Magnesium ions do not affect molecular weight estimates of the isoenzymes as determined by gel filtration chromatography.     

Human Blood Acetaldehyde Levels: With Improved Methods, a Clearer Picture Emerges

New reliable methods for the determination of acetaldehyde in human blood, either from separated plasma or from acid-precipitated whole blood, demonstrate that the blood of healthy Caucasians contains at most only extremely small amounts of acetaldehyde (less than 1 microM) after moderate alcohol intoxication. On the other hand, among about 50% of the Japanese population ethanol ingestion results in elevated blood acetaldehyde levels (10-50 microM) with consequent unpleasant cardiovascular responses such as facial flushing and tachycardia, apparently because of a lack of one of the acetaldehyde-oxidizing aldehyde dehydrogenase isozymes. Elevated acetaldehyde levels may eventually occur also among intoxicated Caucasian alcoholics, primarily as a consequence of abuse-induced loss of hepatic aldehyde dehydrogenase activity, but accentuated by an accelerated ethanol oxidation rate. The elevation is probably reversible, since no acetaldehyde is seen in alcoholics after abstinence and hospital treatment. There is thus little evidence that an elevation of acetaldehyde could serve as a marker for predisposition for alcoholism.     

Evaluation of the Role of Acetaldehyde in the Actions of Ethanol on Gluconeogenesis by Comparison with the Effects of Crotonol and Crotonaldehyde

Liver cells from fasted rats oxidized ethanol and crotonol at identical rates. Ethanol and crotonol increased the cytosolic lactate/pyruvate ratio to the same extent, however only ethanol increased the mitochondrial B-hydroxybutyrate/acetoacetate ratio. The rate of oxidation of crotonaldehyde by liver cells was 30% to 50% of the rate of oxidation of acetaldehyde. Cyanam-ide, which is especially inhibitory towards the low Km mitochondrial aldehyde dehydrogenase, inhibited the oxidation of acetaldehyde to a greater extent than it inhibited the oxidation of crotonaldehyde. Acetaldehyde, but not crotonaldehyde, increased the B-hydrox-ybutyrate/acetoacetate ratio; both aldehydes produced some increase in the lactate/pyruvate ratio. Consequently, differences between the effects of ethanol and crotonol on the mitochondrial redox state may relate to differences between the metabolism of their respective aldehydes. Intact mitochondria oxidized crotonaldehyde at about 10% the rate found with acetaldehyde. In deox-ychoiate-disrupted mitochondria, in the presence of external NAD+, crotonaldehyde was oxidized at rates less than one-fourth the rate found with acetaldehyde. Cyanamide inhibited the oxidation of acetaldehyde to a greater extent than the oxidation of crotonaldehyde. These results suggest that crotonaldehyde is a poor substrate for the low Km mitochondrial aldehyde dehydrogenase. In isolated hepatocytes, acetaldehyde stimulated glucose production from pyruvate, but inhibited gluconeogenesis from glycerol, xylitol, and sorbitol. Crotonaldehyde had no effect on glucose production from these substrates, indicating that the effects of acetaldehyde were due to the metabolism of acetaldehyde in the mitochondria. Ethanol stimulated glucose production from pyruvate, whereas crotonol was without effect. The stimulation by ethanol, and the lack of effect by crotonol, appears to be due to changes in the mitochondrial redox state produced as a consequence of the further oxidation of acetaldehyde, but not crotonaldehyde. Crotonol and ethanol inhibited glucose production from glycerol, xylitol, and sorbitol, suggesting that changes in the cytosolic redox state play the major role in the effect of the alcohols on glucose production from these substrates. However, the inhibition by ethanol was consistently 10% to 15% greater than the inhibition by crotonol, suggesting that acetaldehyde contributes to the effects of ethanol. These differences between ethanol and crotonol are consistent with a role for acetaldehyde metabolism in the actions of ethanol. Crotonol may be a useful aid in determining the mechanism whereby ethanol alters henatic functions.     

Genetic variability of enzymes of alcohol metabolism in human beings

Alcohol is eliminated from the body almost entirely by hepatic metabolism, first to acetaldehyde, then to acetate, and finally to carbon dioxide and water. The time course of elimination is best described by Michaelis-Menten kinetics, and rates of elimination following standard doses of ethanol vary among subjects as much as three-fold. Studies comparing rates of elimination in identical and fraternal twins have shown that about half of the variability is attributable to genetic factors. The principal enzymes responsible for ethanol metabolism are alcohol dehydrogenase and aldehyde dehydrogenase. The reaction catalyzed by alcohol dehydrogenase is the rate-limiting step of the pathway. Human livers contain multiple isoenzymes of alcohol dehydrogenase, which are dimeric molecules arising from the association of two subunits encoded by five different structural genes. Genetic polymorphism at two of these gene loci has been described, and all known homo- and heterodimeric forms of the isoenzymes have now been isolated and characterized. Notably, some of them differ quite strikingly in reactivity toward ethanol. Thus a basis for the genetic variability in alcohol metabolic rate can be found in the kinetic properties of the alcohol dehydrogenase isoenzymes. The efficient oxidation of acetaldehyde by hepatic aldehyde dehydrogenase is essential for ethanol oxidation to continue over time, because the equilibrium of the alcohol dehydrogenase reaction favors the conversion of acetaldehyde to ethanol. Acetaldehyde is a very toxic substance the removal of which makes possible the consumption of large quantities of ethanol frequently imbibed by alcoholics. There are also multiple molecular forms of aldehyde dehydrogenase in liver, and the mitochondrial form is the one principally responsible for acetaldehyde oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired acetaldehyde metabolism in patients with non-alcoholic liver disorders.

In order to determine the specificity of abnormalities of alcohol metabolism in patients with alcoholic liver disease, blood acetaldehyde concentrations after oral ethanol challenge and the activities of alcohol metabolising enzymes in liver biopsy samples have been determined in patients with alcoholic liver disease and a wide variety of non-alcoholic liver disorders. Significant decreases in hepatic cytosolic aldehyde dehydrogenase activity were associated with significant increases in acetaldehyde concentrations after ethanol in both patient groups compared with control subjects. There was a significant correlation between hepatic cytosolic aldehyde dehydrogenase and mean blood acetaldehyde concentration 30-180 min after ethanol ingestion (y = 17.4-0.45x; r = -0.56; p less than 0.01) confirming the importance of this enzyme in controlling blood acetaldehyde concentrations. These findings suggest that disturbances in alcohol metabolism in patients with alcoholic liver disease are the consequence of liver damage rather than a specific abnormality predisposing to alcohol induced liver injury.     

The Effect of Cyanamide on Acetaldehyde Oxidation by Isolated Rat Liver Mitochondria and on the Inhibition of Pyruvate Oxidation by Acetaldehyde

Compared to other substrates, the oxidation of pyruvate by isolated mitochondria is especially sensitive to inhibition by acetaldehyde. It is not known whether this inhibition represents a direct effect of acetaldehyde or requires the metabolism of acetaldehyde. Experiments were therefore carried out in the presence of cyanamide, an inhibitor of aldehyde dehydrogenase. After a brief incubation period, cyanamide inhibited the state 4 and state 3 rate of acetaldehyde (0.1–1.0 mM) oxidation by isolated rat liver mitochondria. Little inhibition was found in the absence of the incubation period. Maximum inhibition was found at cyanamide concentrations of 0.01 to 0.033 mM. Cyanamide also inhibited the activity of aldehyde dehydrogenase assayed in disrupted mitochondrial fractions. The inhibition by cyanamide was specific since cyanamide did not affect mitochondrial oxidation of succinate, glutamate, or pyruvate. Acetaldehyde inhibited the state 3 rate of pyruvate oxidation by liver mitochondria. Despite preventing acetaldehyde oxidation, cyanamide did not prevent the inhibition of pyruvate oxidation by acetaldehyde. These results indicate that (a) cyanamide can be used as an effective in vitro inhibitor of acetaldehyde oxidation and (b) the unique sensitivity of pyruvate oxidation to acetaldehyde represents a direct effect of acetaldehyde on pyruvate dehydrogenase.     

Genetic polymorphism of human liver alcohol and aldehyde dehydrogenases, and their relationship to alcohol metabolism and alcoholism

It is now widely accepted that the various pharmacologic and addictive consequences of alcohol consumption are related to the tissue concentration of ethanol or its metabolic products. The oxidative metabolism of ethanol in liver is principally catalyzed by alcohol dehydrogenase and aldehyde dehydrogenase. Both of these enzymes exist in multiple molecular forms, and genetic models have been proposed to account for the multiplicity of isoenzymes. Alcohol dehydrogenase subunits are encoded at five different gene loci, and genetic polymorphism occurs at two alcohol dehydrogenase loci. Variant isoenzymes produced at the two polymorphic alcohol dehydrogenase loci account for the differences in enzyme electrophoretic patterns observed among individuals. Some of these variant isoenzymes exhibit widely different kinetic properties, and this may account for the 2- to 3-fold variation in alcohol elimination rate among individuals. Since the protein sequence of several of the alcohol dehydrogenase subunits has been determined and several of the alcohol dehydrogenase genes has been cloned, some of the structural changes which give rise to differences in catalytic and electrophoretic properties are now known. Genetic polymorphism also occurs at the aldehyde dehydrogenase gene locus which encodes the mitochondrial low Km for acetaldehyde aldehyde dehydrogenase isoenzyme. The variant isoenzyme exhibits little or no catalytic activity. Individuals with this "null" variant have higher than normal blood acetaldehyde levels and exhibit an alcohol-flush reaction which appears to be a deterrent to heavy drinking and alcoholism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and Characterization of Beef and Pig Liver Aldehyde Dehydrogenases

Beef liver cytosolic, mitochondrial, and pig liver mitochondrial aldehyde dehydrogenases (ALDH) had been purified to homogeneity. The two mitochondrial enzymes as with other mammalian mitochondrial enzymes had properties very similar to that of the corresponding human enzyme. These include immunological as well as basic kinetic properties such as low Km for aldehyde, activation by Mg2+ ions, and lack of inhibition by disulfiram. A major difference between these two enzymes and the human mitochondrial enzyme was that they contained an N-terminal-blocked amino acid. Cytosolic ALDHs from human and horse liver have been shown to possess an N-acetyl serine as the N-terminal residue; beef cytosolic ALDH was also found to be blocked. Tissue preparations and subcellular fractions from beef or pig liver could be used to study acetaldehyde oxidation. This is the subject of the accompanying paper (Cao Q-N, Tu G-C, Weiner H, Alcohol Clin Exp Res 12:xxx-xxx, 1988).     

Identification and Selective Precipitation of Human Aldehyde Dehydrogenase Isozymes Using Antibodies Raised to Horse Liver Aldehyde Dehydrogenase Isozymes

Aldehyde dehydrogenase (ALDH) enzymes from human liver homogenates were recognized in immunoblotting experiments and precipitated in Ouchterlony double diffusion gels by antibodies raised to the horse liver mitochondrial and cytosolic ALDH isozymes. The antibody raised to the cytosolic horse liver ALDH (alpha HC) has been shown to be specific for cytosolic ALDH isozymes, while the antibody raised to the horse liver mitochondrial ALDH (alpha HM) precipitated both mitochondrial and cytosolic ALDH isozymes. It was possible to selectively remove the cytosolic ALDH from a homogenate of a liver sample from a Caucasian by preincubation with alpha HC; the remaining mitochondrial enzyme was then precipitated by alpha HM in double diffusion gels. The experiments were repeated with a liver sample from an Oriental, presumed to have been alcohol sensitive since no active mitochondrial ALDH was found. The precipitation of a relatively inactive mitochondrial enzyme by alpha HM from a cytosolic ALDH-free sample confirmed previous reports of the existence of a mitochondrial ALDH in tissue from an alcohol-sensitive Oriental. The results of immunoblotting experiments confirm the co-migration, in electrophoresis, of the cytosolic and mitochondrial ALDHs from the liver of an alcohol-sensitive Oriental. The results reported here, together with previous observations, indicate that the antibodies raised to horse liver ALDH isozymes can be used to determine the subcellular location of ALDH isozymes in various human tissues, including frozen tissue samples which are not amenable to subcellular fractionation.     

Human class II (pi) alcohol dehydrogenase has a redox-specific function in norepinephrine metabolism.

Studies of the function of human alcohol dehydrogenase (ADH) have revealed substrates that are virtually unique for class II ADH (pi ADH). It catalyzes the formation of the intermediary glycols of norepinephrine metabolism, 3,4-dihydroxyphenylglycol and 4-hydroxy-3-methoxyphenylglycol, from the corresponding aldehydes 3,4-dihydroxymandelaldehyde and 4-hydroxy-3-methoxymandelaldehyde with Km values of 55 and 120 microM and kcat/Km ratios of 14,000 and 17,000 mM-1 X min-1; these are from 60- to 210-fold higher than those obtained with class I ADH isozymes. The catalytic preference of class II ADH also extends to benzaldehydes. The kcat/Km values for the reduction of benzaldehyde, 3,4-dihydroxybenzaldehyde and 4-hydroxy-3-methoxybenzaldehyde by pi ADH are from 9- to 29-fold higher than those for a class I isozyme, beta 1 gamma 2 ADH. Furthermore, the norepinephrine aldehydes are potent inhibitors of alcohol (ethanol) oxidation by pi ADH. The high catalytic activity of pi ADH-catalyzed reduction of the aldehydes in combination with a possible regulatory function of the aldehydes in the oxidative direction leads to essentially "unidirectional" catalysis by pi ADH. These features and the presence of pi ADH in human liver imply a physiological role for pi ADH in the degradation of circulating epinephrine and norepinephrine.