Gene markers for alcohol-metabolizing enzymes among recombinant inbred strains of mice with differential behavioural responses towards alcohol

The genetic variability of alcohol dehydrogenase (C₂ isozyme), aldehyde dehydrogenase (A₂ isozyme) and aldehyde oxidase (A₂ isozyme) has been examined among recombinant inbred strains of mice which have been previously studied concerning their differential behavioural responses towards alcohol. The results showed no correlation between biochemical phenotype for these loci and behavioural response.     

Aldehyde dehydrogenase isozyme deficiency and alcohol sensitivity in four different Chinese populations

Four different populations of China were studied regarding aldehyde dehydrogenase isozyme variation and incidence of alcohol sensitivity. While Korean and Mongolian minorities in the north showed an isozyme I deficiency with a frequency of about 25 and 30%, 45-50% of Zhuang and Han were deficient, respectively. Adverse reactions after alcohol drinking were mainly reported by those subjects who showed the lack of aldehyde dehydrogenase isozyme I.     

Aldehyde dehydrogenase polymorphism in North American, South American, and Mexican Indian populations.

While about 40% of the South American Indian populations (Atacameños, Mapuche, Shuara) were found to be deficient in aldehyde dehydrogenase isozyme I (ALDH2 or E2), preliminary investigations showed very low incidence of isozyme deficiency among North American natives (Sioux, Navajo) and Mexican Indians (mestizo). Possible implications of such trait differences on cross-cultural behavioral response to alcohol drinking are discussed.     

Aldehyde oxidase and alcohol dehydrogenase genetics in the mouse. New alleles for the Aox-2 and Adh-3 loci

The genetic variability of one of the liver isozymes of aldehyde oxidase (AOX-B₂ or AOX-2) and the stomach isozyme of alcohol dehydrogenase (ADH-C₂) has been examined among strains of mice. Evidence is presented for a fourth allele of Aox-2 and a third allele of Adh-3 . The hybrid allozyme pattern for mouse liver AOX was consistent with a dimeric subunit structure for this enzyme.     

Purification and molecular properties of mouse alcohol dehydrogenase isozymes

Alcohol dehydrogenase isozymes from mouse liver (A2 and B2) and stomach (C2) tissues have been purified to homogeneity using triazine-dye affinity chromatography. The enzymes are dimers with similar but distinct subunit sizes, as determined by SDS/polyacrylamide gel electrophoresis: A, 43000; B, 39000, and C, 47000. Zinc analyses and 1,10-phenanthroline inhibition studies indicated that the A and C subunits each contained two atoms of zinc, with at least one being involved catalytically, whereas the B subunit probably contained a single non-catalytic zinc atom. The isozymes exhibited widely divergent kinetic characteristics. A2 exhibited a Km value for ethanol of 0.15 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length; C2 also exhibited this broad specificity for alcohols but showed a Km value of 232 mM for ethanol. These isozymes also showed broad substrate specificities as aldehyde reductases. In contrast, B2 showed no detectable activity as an aldehyde reductase for the aldehydes examined, and used ethanol as substrate only at very high concentrations (greater than 0.5 M). The isozyme exhibited low Km and high Vmax values, however, with medium-chain alcohols. Immunological studies showed that A2 was immunologically distinct from the B2 and C2 isozymes. In vitro molecular hybridization studies gave no evidence for association between the alcohol dehydrogenase subunits. The results confirm genetic analyses [Holmes, Albanese, Whitehead and Duley (1981) J. Exp. Zool. 215, 151-157] which are consistent with at least three structural genes encoding alcohol dehydrogenase in the mouse and confirm the role of the major liver isozyme (A2) in ethanol metabolism.     

Metabolism of retinaldehyde and other aldehydes in soluble extracts of human liver and kidney

Purification and characterization of enzymes metabolizing retinaldehyde, propionaldehyde, and octanaldehyde from four human livers and three kidneys were done to identify enzymes metabolizing retinaldehyde and their relationship to enzymes metabolizing other aldehydes. The tissue fractionation patterns from human liver and kidney were the same, indicating presence of the same enzymes in human liver and kidney. Moreover, in both organs the major NAD(+)-dependent retinaldehyde activity copurified with the propionaldehyde and octanaldehyde activities; in both organs the major NAD(+)-dependent retinaldehyde activity was associated with the E1 isozyme (coded for by aldh1 gene) of human aldehyde dehydrogenase. A small amount of NAD(+)-dependent retinaldehyde activity was associated with the E2 isozyme (product of aldh2 gene) of aldehyde dehydrogenase. Some NAD(+)-independent retinaldehyde activity in both organs was associated with aldehyde oxidase, which could be easily separated from dehydrogenases. Employing cellular retinoid-binding protein (CRBP), purified from human liver, demonstrated that E1 isozyme (but not E2 isozyme) could utilize CRBP-bound retinaldehyde as substrate, a feature thought to be specific to retinaldehyde dehydrogenases. This is the first report of CRBP-bound retinaldehyde functioning as substrate for aldehyde dehydrogenase of broad substrate specificity. Thus, it is concluded that in the human organism, retinaldehyde dehydrogenase (coded for by raldH1 gene) and broad substrate specificity E1 (a member of EC 1. 2.1.3 aldehyde dehydrogenase family) are the same enzyme. These results suggest that the E1 isozyme may be more important to alcoholism than the acetaldehyde-metabolizing enzyme, E2, because competition between acetaldehyde and retinaldehyde could result in abnormalities associated with vitamin A metabolism and alcoholism.     

Liver alcohol dehydrogenase and aldehyde dehydrogenase in the Japanese: isozyme variation and its possible role in alcohol intoxication.

Forty autopsy livers from Japanese individuals were studied concerning alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozymes using electrophoretic and enzyme assay methods. A remarkably high frequency (85%) was found for the atypical ADH phenotype. The gene frequencies of ADH22 and ADH32 were .625 and .05, respectively. The usual ALDH phenotype showed two major isozyme bands, a faster migrating (low Km for acetaldehyde) and a slower migrating isozyme (high Km for acetaldehyde). Fifty-two percent of the specimens had an unusual phenotype of ALDH, which showed only the slower migrating isozyme. The usual phenotype was inhibited about 20%--30% by disulfiram and the unusual type up to 90%. Such a high incidence in the Japanese of the unusual phenotype, which lacks in the low Km isozyme, suggests that the initial intoxicating symptoms after alcohol drinking in these subjects might be due to delayed oxidation of acetaldehyde rather than its higher-than-normal production by typical or atypical ADH.     

Aldehyde dehydrogenase of the Mongolian gerbil, Meriones unguiculatus.

The aldehyde dehydrogenase (Aldehyde:NAD(P) oxidoreductase E.C. 1.2.1.3. and 1.2.1.5) phenotype in several tissues of the Mongolian gerbil, Meriones unguiculatus, has been established. The tissue distribution of gerbil aldehyde dehydrogenase is similar to that of the rat, with liver possessing the majority of the aldehyde dehydrognease activity. Male kidney and testis possess significantly more activity than female kidney and ovary. The substrate and co-enzyme specificity of gerbil liver aldehyde dehydrogenase is also similar to that of rat and mouse liver. Gel isoelectric focusing resolves one major gerbil liver aldehyde dehydrogenase isozyme at pI 5.3. Mouse liver is resolved into two major isozymes at pIs 5.3 and 5.6 and rat liver aldehyde dehydrogenase into one major isozyme at pI 5.4. Gerbil liver aldehyde dehydrogenase is functional over a broad pH range with an optima at pH 9.0. Rat and mouse liver aldehyde dehydrogenase possess sharp pH optima at pH 8.5.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde reductase, aldehyde oxidase and xanthine oxidase from horse tissues

Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (AHD), aldehyde reductase (AHR), aldehyde oxidase (AOX) and xanthine oxidase (XOX) extracted from horse tissues were examined. Five ADH isozymes were resolved: three corresponded to the previously reported class I ADHs (EE, ES and SS) (Theorell, 1969); a single form of class II ADH (designated ADH-C2) and of class III ADH (designated ADH-B2) were also observed. The latter isozyme was widely distributed in horse tissues whereas the other enzymes were found predominantly in liver. Four AHD isozymes were differentially distributed in subcellular preparations of horse liver: AHD-1 (large granules); AHD-3 (small granules); and AHD-2, AHD-4 (cytoplasm). AHD-1 was more widely distributed among the horse tissues examined. Liver represented the major source of activity for most AHDs. A single additional form of NADPH-dependent AHR activity (identified as hexonate dehydrogenase), other than the ADHs previously described, was observed in horse liver. Single forms of AOX and XOX were observed in horse tissue extracts, with highest activities in liver.     

An enzymatic model for rat liver alcohol dehydrogenase in the presence of low-Km aldehyde dehydrogenase

Four isoenzymes of aldehyde dehydrogenase were partially purified from rat liver mitochondria by hydroxylapatite chromatography and gel filtration. While three forms display low affinity for acetaldehyde, the fourth is active at extremely low aldehyde concentrations (Km less than or equal to 2 microM) and allows the oxidation of the acetaldehyde formed by catalysis of alcohol dehydrogenase at pH 7.4. Different models of alcohol dehydrogenase have been examined by analysis of progress curves of ethanol oxidation obtained in the presence of low-km aldehyde dehydrogenase. According to the only acceptable model, when the acetaldehyde concentration is kept low by the action of aldehyde dehydrogenase, NADH no longer binds to alcohol dehydrogenase, but acetaldehyde still competes with ethanol for the active site of the enzyme. The seven kinetic parameters of the two enzymes (four for alcohol dehydrogenase and three for aldehyde dehydrogenase) and the equilibrium constant of the reaction catalyzed by alcohol dehydrogenase have been determined by applying a new fitting procedure here described.     

Effect of glucose on beta-adrenergic induced downregulation of insulin receptor binding in human fat cells

Population genetics followed by purification suggested that "null" mutation in the mitochondrial E2 isozyme of human aldehyde dehydrogenase (EC 1.2.1.3) occurred in the Oriental individuals who are sensitive to alcohol. This report demonstrates that the Oriental E2, thought to be a "null" mutant, is catalytically active and except for maximal velocity and isoelectric point, identical with Caucasian E2 isozyme. The data presented are not inconsistent with mutation but preclude active site of the enzyme as the point at which alteration has occurred; they are, however, inconsistent with "null" mutation.     

Structural mutation in a major human aldehyde dehydrogenase gene results in loss of enzyme activity.

Most Caucasians have two major liver aldehyde dehydrogenase isozymes, ALDH1 and ALDH2, while approximately 50% of Orientals have only ALDH1 isozyme, missing the ALDH2 isozyme. A remarkably higher frequency of acute alcohol intoxication among Orientals than among Caucasians could be related to the absence of the ALDH2 isozyme, which has a low apparent Km for acetaldehyde. Examination of liver extracts by two-dimensional crossed immunoelectrophoresis revealed that an atypical Japanese liver, which had no ALDH2 isozyme, contained an enzymatically inactive but immunologically cross-reactive material corresponding to ALDH2, beside the active ALDH1 isozyme. Therefore, the absence of ALDH2 isozyme in atypical Orientals is not due to regulatory mutation, gene deletion, or nonsense mutation, but must be due to a structural mutation in a gene for the ALDH2 locus, resulting in synthesis of enzymatically inactive abnormal protein.     

Subcellular localization of the F1 and F2 isozymes of horse liver aldehyde dehydrogenase

The subcellular distribution and relative amounts of the two isozymes, F1 and F2, of aldehyde dehydrogenase (EC 1.2.1.3) which were recently purified to homogeneity from horse liver (Eckfeldt, J., et al. (1976) J. Biol. Chem.251, 236–240) have been investigated. A fresh horse liver homogenate was fractionated on DEAE-cellulose. The results indicate that approximately 60% of the total pH 7.0 acetaldehyde dehydrogenase activity is due to the F1 isozyme and 40% is due to the F2 isozyme. Several horse livers were then fractionated into subcellular components using a differential centrifugation method. Based on the disulfiram (Antabuse) inhibition and the aldehyde concentration dependence of the enzymatic activity, it appears that the disulfiram-sensitive F1 isozyme (K_m acetaldehyde ? 70 μm) is primarily cytosolic and the disulfiram-insensitive F2 isozyme (K_{m acetaldehyde} ? 0.2 μm) is primarily mitochondrial. Fluorescence studies showed that the acetaldehyde dehydrogenase of the intact mitochondria could utilize only the endogenous pyridine nucleotide pool and not externally added NAD. Also, the ethanol dehydrogenase activity was found to be nearly 10 times the total acetaldehyde dehydrogenase activity when assaying a horse liver homogenate at pH 7.0 and with saturating substrates. The significant differences between this work and the results reported in rat liver are discussed with respect to the physiological importance of the cytosolic and mitochondrial aldehyde dehydrogenase during the ethanol oxidation in vivo.     

Biochemical genetics of aldehyde dehydrogenase isozymes in the mouse: Evidence for stomach- and testis-specific isozymes

Electrophoretic and activity variants have been observed for stomach and testis aldehyde dehydrogenases, respectively, among inbred strains of the house mouse (Mus musculus). Genetic evidence was obtained for two new loci encoding these isozymes (designated Ahd-4 and Ahd-6, respectively, for the stomach and testis isozymes) which segregated independently of a number of mouse gene markers, including Ahd-1 (encoding mitochondrial aldehyde dehydrogenase) on chromosome 4, ep (pale ears), a marker for chromosome 19, on which Ahd-2 (encoding liver cytosolic aldehyde dehydrogenase) has been previously localized, and Adh-3 (encoding the stomach-specific isozyme of alcohol dehydrogenase) on chromosome 3. Recombination studies have indicated, however, that Ahd-4 and Ahd-6 are distinct but closely linked loci on the mouse genome. An extensive survey of the distribution of Ahd-1, Ahd-2, Ahd-4, and Ahd-6 alleles among 56 strains of mice is reported. No variants have been observed, so far, for the microsomal (AHD-3) and mitochondrial/cytosolic (AHD-5) isozymes previously described. This study, in combination with previous investigations on mouse aldehyde dehydrogenases, provides evidence for six genetic loci for this enzyme.     

Inducibility of liver cytosolic aldehyde dehydrogenase activity in various animal species

1. The inducibility of hepatic cytosolic aldehyde dehydrogenase activity was studied in rat, mouse, guinea pig, chicken, frog, salamander and rainbow trout, by using two different types of inducers of drug metabolism. 2. Phenobarbital (a type I inducer of drug metabolizing enzymes) increased total liver cytosolic aldehyde dehydrogenase activity (up to 20-fold) in a genetically defined substrain of responsive rats (RR) and only slightly, if at all, in a non-responsive substrain (rr). On the contrary, both types of rats showed a highly induced aldehyde dehydrogenase activity after treatment with methylcholanthrene (a type II inducer). Phenobarbital is affecting mainly an isozyme of aldehyde dehydrogenase which is best measured with propionaldehyde as the substrate and NAD as the coenzyme (P/NAD). 3. Administration of phenobarbital to mice produced only a slight increase (2-fold) in the P/NAD aldehyde dehydrogenase activity. 4. Methylcholanthrene treatment caused a 2-fold increase of the hepatic P/NAD aldehyde dehydrogenase activity in the chicken. 5. In the guinea pig, phenobarbital produced an approximate 3-fold increase of the P/NAD activity. Methylcholanthrene had a similar effect, although to a lesser extent. 6. In the salamander, a 4-fold increase was detected in the enzyme activity measured with benzaldehyde as the substrate and NADP as the coenzyme (B/NADP), after treatment with either phenobarbital or methylcholanthrene. 7. The hepatic aldehyde dehydrogenase activities were found unchanged in the rainbow trout, after treatment with phenobarbital or 2,3,7,8-tetrachlorodibenzo-p-dioxin. 8. The rat model remains the only one examined that shares with human hepatocytes strong inducibility of the B/NADP aldehyde dehydrogenase isozyme upon treatment with polycyclic aromatic hydrocarbons.     

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.     

Aldehyde dehydrogenase (EC 1.2.1.3): comparison of subcellular localization of the third isozyme that dehydrogenates gamma-aminobutyraldehyde in rat, guinea pig and human liver.

1. Subcellular fractionation of rat, guinea pig and human livers showed that aldehyde dehydrogenase metabolizing gamma-aminobutyraldehyde was exclusively localized in the cytoplasmic fraction in all three mammalian species. 2. Total gamma-aminobutyraldehyde activity of aldehyde dehydrogenase was found to be ca 0.41, 0.3 and 0.24 mumol NADH min-1 g-1 tissue, respectively in rat, guinea pig and human liver, with more than 95% of activity in the cytoplasm. 3. Partially purified cytoplasmic isozyme from rat liver showed similar chromatographic behavior and kinetic properties to the E3 isozyme isolated from human liver. 4. The rat isozyme was insensitive to disulfiram (40 microM) and to magnesium (160 microM) and had Km values of 5 microM (pH 7.4) for gamma-aminobutyraldehyde, 7.5 microM (pH 9.0) for propionaldehyde and 4 microM (pH 7.4) for NAD.     

Betaine aldehyde oxidation by spinach chloroplasts

Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H₂O₂-mediated reaction cannot be ruled out.     

Human aldehyde dehydrogenase. Activity with aldehyde metabolites of monoamines, diamines, and polyamines

Two isozymes (E1 and E2) of human aldehyde dehydrogenase (EC 1.2.1.3) were purified to homogeneity 13 years ago and a third isozyme (E3) with a low Km for gamma-aminobutyraldehyde only recently. Comparison with a variety of substrates demonstrates that substrate specificity of all three isozymes is broad and similar. With straight chain aliphatic aldehydes (C1-C6) the Km values of the E3 isozyme are identical with those of the E1 isozyme. All isozymes dehydrogenate naturally occurring aldehydes, 5-imidazoleacetaldehyde (histamine metabolite) and acrolein (product of beta-elimination of oxidized polyamines) with similar catalytic efficiency. Differences between the isozymes are in the Km values for aminoaldehydes. Although all isozymes can dehydrogenate gamma-aminobutyraldehyde, the Km value of the E3 isozyme is much lower: the same appears to apply to aldehyde metabolites of cadaverine, agmatine, spermidine, and spermine for which Km values range between 2-18 microM and kcat values between 0.8-1.9 mumol/min/mg. Thus, the E3 isozyme has properties which make it suitable for the metabolism of aminoaldehydes. The physiological role of E1 and E2 isozymes could be in dehydrogenation of aldehyde metabolites of monoamines such as 3,4-dihydroxyphenylacetaldehyde or 5-hydroxyindoleacetaldehyde; the catalytic efficiency with these substrates is better with E1 and E2 isozymes than with E3 isozyme. Isoelectric focusing of liver homogenates followed by development with various physiological substrates together with substrate specificity data suggest that aldehyde dehydrogenase (EC 1.2.1.3) is the only enzyme in the human liver capable of catalyzing dehydrogenation of aldehydes arising via monoamine, diamine, and plasma amine oxidases. Although the enzyme is generally considered to function in detoxication, our data suggest an additional function in metabolism of biogenic amines.