Association of the ADH2*2 allele with reduced ethanol consumption in Jewish men in Israel: a pilot study.

OBJECTIVE: This study provides preliminary evidence on the associations between alcohol consumption patterns and polymorphisms of the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes in a Jewish population. METHOD: Two groups of Jewish men were studied--one group (n = 92) representative of the free-living population of Jerusalem and generally light consumers of ethanol and the other group (n = 53) composed of treatment-enrolled heroin dependent individuals in the same city, most with a history of heavy daily drinking. All participants were interviewed regarding sociodemographic background, present and past alcohol consumption patterns, and familial characteristics including alcohol problems among first-degree relatives. Polymorphisms of the ADH2, ADH3 and ALDH loci were determined for all participants. RESULTS: The less common allele of the ADH2 locus (ADH2*2 allele frequency approximately 20% in Ashkenazic and non-Ashkenazic members of both groups) was related to a reduced mean level of peak weekly alcohol intake in the two groups. In multiple regression models adjusting for family history of alcohol problems and other factors, the ADH2*2 allele accounted for 20% and 30% of the explained alcohol intake variance in these two groups, respectively. Results from a logistic regression indicated that the ADH2*2 allele was also related to infrequent drinking in both groups. Evidence for an independent association between the ADH3 polymorphism and alcohol consumption patterns was not found. The ALDH gene was not polymorphic in this population. CONCLUSIONS: This report describes for the first time an association between alcohol consumption patterns and a polymorphism at the ADH2 locus in a Jewish population. The relatively high frequency of the ADH2*2 allele may contribute to the seemingly lower levels of alcohol consumption and heightened sensitivity to alcohol observed among Jews.

Alcohol metabolism in Asian-American men with genetic polymorphisms of aldehyde dehydrogenase.

BACKGROUND: About half of certain Asians have a deficiency of the low-Km aldehyde dehydrogenase (ALDH2) isoenzyme. This deficiency results from inheritance of a mutant ALDH2*2 allele. OBJECTIVE: To determine whether Asian Americans with ALDH2*2 alleles differ from Asian Americans without this mutation in terms of blood levels of alcohol and acetaldehyde after ingestion of a moderate amount of alcohol. DESIGN: Double-blind, crossover study. SETTING: Private research institute. PARTICIPANTS: 35 healthy Asian-American men. Three men who became ill after alcohol ingestion and one who had outlying data were excluded. INTERVENTION: Alcoholic beverage, containing 0.56 g of alcohol per kg of body weight, or placebo beverage, containing 3 mL of alcohol, given orally on separate occasions. MEASUREMENTS: Blood levels of alcohol and acetaldehyde measured before and several times after ingestion of the alcoholic or placebo beverage. RESULTS: Participants with ALDH2*2 alleles had significantly higher blood acetaldehyde levels after ingesting alcoholic and placebo beverages than did participants with ALDH2*1 alleles, despite similar blood alcohol concentrations. CONCLUSIONS: Blood acetaldehyde levels rather than blood alcohol concentration may mediate enhanced alcohol sensitivity among Asians with ALDH2*2 alleles.

Allele frequency of ADH2 and ALDH2 among Brazilians of different ethnic groups.

We determined the genotypes of the alcohol dehydrogenase (ALDH) and aldehyde dehydrogenase (ALDH2) loci of different ethnic groups living in Brazil, using saliva DNA amplified by PCR and allele-specific oligonucleotides. Self-reports of flushing reaction after drinking were also studied. The allelic frequencies of ADH2 and ALDH2 were found to be lower than those reported other authors, which might be a result of the admixture origin of the Brazilian population. Variability in facial flushing reaction suggests that other factors play a role in the expression of alcohol-induced flushing.

Alcohol dehydrogenase polymorphisms in Native Americans: identification of the ADH2*3 allele.

Alcohol dehydrogenase (ADH) polymorphisms were evaluated among 95 Native American Mission Indians. Approximately equal frequencies of ADH3*1 and ADH3*2 alleles were found. Twelve individuals were heterozygous for ADH2*3, an allele previously identified only in persons of African origin. None of the individuals with ADH2*3 alleles was of purely Native American descent, although none had known African ancestry. These results suggest that these candidate genes deserve broader study among Native Americans and may provide increased understanding of the likely polygenic contributions to alcohol-related disorders in this population.

Alcohol dehydrogenase-2*3 allele protects against alcohol-related birth defects among African Americans.

Considerable variation in offspring outcome is observed after intrauterine alcohol exposure. The underlying mechanism may include genetic diversity in the enzymes responsible for alcohol metabolism. Of the known genetic polymorphisms, differences at the alcohol dehydrogenase-2 locus (ADH2) are likely most critical because the resulting enzymes are >30-fold different in their kinetic constants. To test whether differences in maternal or offspring ADH2 genotype are determinants of risk for alcohol-related birth defects, maternal-infant pairs (n = 243) were enrolled on the basis of maternal alcohol intake during pregnancy and maternal ADH2 genotype. Infant outcome was measured using the Bayley Scales of Infant Development Mental Index (MDI) at 12 months of age. Drinking during pregnancy was associated with lower MDI scores but only in the offspring of mothers without an ADH2*3 allele (P < .01, analysis of variance, post hoc). The offspring of drinking women with at least one ADH2*3 allele had MDI scores similar to those of nondrinking women of either ADH2 genotype. Lower MDI scores were associated with the three-way interaction among increasing alcohol intake and maternal and offspring absence of the ADH2*3 allele (P < .01, multiple linear regression). We suggest that the protection afforded by this allele is secondary to its encoding of the high-Km/high-Vmax ADH beta3 isoenzyme, which would provide more efficient alcohol metabolism at high blood alcohol concentrations. These observations are supportive of alcohol, rather than acetaldehyde, being the more important proximate teratogen and are the first observations of a specific genetic explanation for susceptibility differences to alcohol-related birth defects.

Alcohol elimination in Native American Mission Indians: an investigation of interindividual variation.

The high prevalence of alcohol abuse and alcohol dependence among Native Americans may be mediated by unique environmental and genetic factors in this population. One factor that may influence the development of alcoholism is variability in alcohol metabolism. To determine factors that contribute to differences in alcohol elimination rates within a Native American population, this study evaluated healthy southern California Mission Indian men between the ages of 18 and 25 years. Each man drank a dose of alcohol, 0.56 g/kg of body weight as a 20% by volume solution, at approximately 9:00 AM after eating a low-fat breakfast and having fasted overnight. The drink was consumed within 7 min, and the concentrations of alcohol in blood were determined before and at 15, 30, 60, 90, 120, and 150 min after beverage ingestion. Rates of alcohol elimination were calculated from the pseudolinear slope of the blood alcohol versus time curve. The influences of estimated body water, recent drinking history, recent smoking history, polymorphism at the ADH2 and ADH3 loci, family history of alcoholism, and percentage Native American heritage on alcohol elimination rate were determined using multiple regression analyses. Estimated body water accounted for a significant proportion of the variance in alcohol elimination rate. There was also a nonsignificant trend for subjects with an ADH2*3 allele (n = 6) to have faster rates of alcohol elimination than those with ADH2*1 alleles only (n = 33). Given the high prevalence of alcoholism and alcohol-related health problems among Native Americans, the results from this study suggest that evaluation of alcohol metabolism and genotypes of the alcohol-metabolizing enzymes in Native American populations merits further study.

Investigator-observed alcohol-induced flushing but not self-report of flushing is a valid predictor of ALDH2 genotype.

OBJECTIVE: This article presents data on the validity of using self-report of alcohol-induced flushing and actual investigator-observed flushing following alcohol challenge to predict ALDH2 genotype in Asian-American men. METHOD: Men between the ages of 21 and 25 years who were of Chinese, Japanese or Korean descent completed questionnaires about their drinking history and their alcohol-induced flushing history and associated symptoms. Fifty men selected for participation in the study were genotyped for alleles of ALDH2 and individually tested on two separate occasions following oral administration of placebo and 0.75 ml/kg (0.56 g/kg) alcohol. Facial flushing was assessed at baseline and at intervals over a 150-minute period after drinking using observational ratings. RESULTS: By comparing the results of ALDH2 genotype with investigator-observed flushing and with previous self-report of facial flushing, it was found that investigator-observed flushing is both a sensitive (100%) and specific (96%) predictor of ALDH2 genotype, whereas self-report of facial flushing is a sensitive (100%) but not a specific (68%) predictor of ALDH2 genotype. CONCLUSIONS: The results suggest that investigator-observed flushing provides a valid estimate of ALDH2 genotype, but that self-report of facial flushing is biased, giving a substantial overestimate. Due to an increase in error variance, studies that rely solely on self-report of flushing will more often lead to the conclusion that no association exists. This study supports the importance of using ALDH2 genotype, rather than self-report of flushing or ALDH2 phenotype, when examining factors associated with differences in drinking behavior, response to alcohol and risk for alcoholism or alcohol-related disease among Asians.

Simultaneous measurement of ethanol and ethyl-d5 alcohol by stable isotope gas chromatography-mass spectrometry.

Efforts to define gender- and ethnic-dependent differences in ethanol first-pass metabolism by gastric mucosa and liver have been limited by a lack of analytical tools that distinguish ethanol concurrently administered by oral and intravenous routes. A stable isotope gas chromatography-mass spectrometry method for simultaneous measurement of ethanol and ethyl-d5 alcohol in serum was developed to meet this need. The assay was linear from 1 to 30 mmol/L. The limit of quantification was 1 mmol/L. Analytical imprecision (CV) was <10%. Analytical recovery was >90%. Specificity was based on retention time and reproducibility of ion ratios. The assay was free from interference by other volatile alcohols. Simultaneous oral administration of ethanol and ethyl-d5 alcohol produced nearly identical pharmacokinetic profiles. Simultaneous oral ingestion of ethanol and intravenous infusion of ethyl-d5 alcohol, adjusted for gastric emptying time, revealed decreased bioavailability of ethanol by the oral route. The method described is sufficient to study the first-pass metabolism of ethanol.

ADH2 gene polymorphisms are determinants of alcohol pharmacokinetics.

The class I hepatic alcohol dehydrogenases (ADHs) are primarily responsible for ethanol metabolism in humans. Genetic polymorphism at the ADH2 locus results in the inheritance of isozymes of strikingly different catalytic properties. The most common ADH2 allele, ADH2*1, encodes the low K(m) isozyme subunit beta 1. The ADH2*3 allele encodes a high-activity isozyme subunit of alcohol dehydrogenase, beta 3, identified in approximately 25% of African-Americans. The Vmax of beta 3 beta 3-ADH is 30 times greater than that of the beta 1 beta 1-ADH. Therefore, we hypothesized that the rate of ethanol metabolism, an important factor in the toxicity of ethanol, in persons with beta 3-containing ADH, either beta 3 beta 3- or beta 1 beta 3-ADH, would be faster than that of persons with only beta 1 beta 1-ADH. We tested this hypothesis with ethanol administered orally to healthy, young African-Americans. Three hundred and twenty-six African-American men and women were genotyped using polymerase chain reaction amplification of their leukocyte DNA followed by hybridization with allele-specific probes. One hundred twelve volunteers, selected by genotype, received an oral dose of ethanol designed to produce a blood ethanol concentration of 80 mg/dl (0.080 g/dl), when the blood alcohol concentration-time curve was extrapolated back to time 0. Ethanol metabolic rates (beta 60s) were determined in the 112 subjects from the slope of the pseudolinear portion of the blood ethanol concentration-time curves. The mean beta 60 of African-Americans having beta 3-containing ADH isozymes had significantly faster ethanol elimination rates than those with only beta 1 beta 1-ADH isozymes. There were no significant differences in body weight, ethanol intake in the week before testing, peak breath ethanol concentration, time to peak, or volume of distribution between the genotype groups. Within each of these groups, men had lower ethanol disappearance rates than women. These results demonstrate in vivo the kinetic differences of ADH2 isozymes that may influence individual risk for the effects of ethanol.

Cimetidine inhibition of human gastric and liver alcohol dehydrogenase isoenzymes: identification of inhibitor complexes by kinetics and molecular modeling.

Cimetidine, an H2-receptor antagonist, is one of the most commonly prescribed drugs in the world. It has been reported to increase blood alcohol concentrations in drinking individuals. To determine if this increase could be due to inhibition of alcohol dehydrogenase activity, the effect of the drug on ethanol oxidation by gastric sigma sigma alcohol dehydrogenase and liver beta 2 beta 2, pi pi, and chi chi alcohol dehydrogenase isoenzymes was observed. Cimetidine inhibited all isoenzymes studied except chi chi; the chi chi isoenzyme showed no inhibition up to 5 mM cimetidine. Inhibition of the alcohol dehydrogenase isoenzymes by the H2-receptor antagonists nizatidine, ranitidine, and famotidine was negligible. Docking simulations with the beta 2.NAD+.4-iodopyrazole X-ray structure indicated that cimetidine fit well into the substrate binding site. The substitution on the thiazole ring of nizatidine, however, prevented docking into the binding site. Cimetidine inhibition of ethanol oxidation by sigma sigma and beta 2 beta 2 was competitive with varied ethanol, exhibiting Ki values of 2.8 +/- 0.4 mM and 0.77 +/- 0.07 mM, respectively. Cimetidine inhibition of ethanol oxidation by pi pi was noncompetitive with varied ethanol (Ki = 0.50 +/- 0.03 mM). Inhibition of ethanol oxidation by sigma sigma and beta 2 beta 2 with varied NAD+ was competitive. These results, together with the cimetidine inhibition kinetics of acetaldehyde reduction by sigma sigma and beta 2 beta 2, with either varied NADH or varied acetaldehyde, are consistent with cimetidine binding to two enzyme species. These species are free enzyme and the productive enzyme.NAD+ complex.

Expression and kinetic characterization of recombinant human stomach alcohol dehydrogenase. Active-site amino acid sequence explains substrate specificity compared with liver isozymes.

A full-length 1966-base pair clone of the human class IV alcohol dehydrogenase (sigma-ADH) was isolated from a human stomach cDNA library. The 373-amino acid sigma-ADH encoded by this cDNA was expressed in Escherichia coli. The specific activity of the recombinant enzyme for ethanol oxidation at pH 7.5 and 25 degrees C, calculated from active-site titration of NADH binding, was 92 +/- 9 units/mg. Kinetic analysis of the catalytic efficiency (kcat/KM) of recombinant sigma-ADH for oxidation of primary alcohols indicated broad substrate specificity. Recombinant human sigma-ADH exhibited high catalytic efficiency for oxidation of all-trans-retinol to all-trans-retinal. This pathway is important in the synthesis of the transcriptional regulator all-trans-retinoic acid. Secondary alcohols and 3 beta-hydroxysteroids were inactive with sigma-ADH or were oxidized with very low efficiency. The KM of sigma-ADH for ethanol was 25 mM, and the KM for primary straight chain alcohols decreased substantially as chain length increased. There are important amino acid differences in the alcohol-binding site between the human class IV (sigma) and human class I (beta) alcohol dehydrogenases that appear to explain the high catalytic efficiency for all-trans-retinol, the high kcat for ethanol, and the low catalytic efficiency for secondary alcohols of sigma-ADH relative to beta 1-ADH. For example, modeling the binding of all-trans-retinol in the human beta 1-ADH structure suggested that coordination of retinol to the active-site zinc is hindered by a loop from residues 114 to 120 that is at the entrance to the alcohol-binding site. The deletion of Gly-117 in human sigma-ADH and a substitution of Leu for the bulky Tyr-110 appear to facilitate retinol access to the active-site zinc.

Gender differences in alcohol metabolism. Physiological responses to ethanol.

A gender difference in alcohol pharmacokinetics has been suggested to explain why women are more vulnerable to ethanol's toxic effects. The results of animal experiments suggest that females exhibit higher alcohol metabolic rates than males as a result of hormonal differences. Experimental results examining gender differences in human alcohol metabolism have been inconsistent; the diversity of experimental protocols and variety of pharmacokinetic parameters reported have made comparisons of these studies very difficult. Variability in alcohol metabolic rate between individuals of the same sex is often significant, preventing an assessment of gender differences in some studies. This chapter attempts to summarize the findings of studies from the last decade that examined the role of gender and sex hormone differences on ethanol metabolism in men and women. The role of body composition, genetic factors, gastric and hepatic alcohol dehydrogenase, and gastric absorption in creating gender differences in alcohol metabolism is discussed. Suggestions are offered that may result in better cross-study comparisons and more consistent experimental results.

Allele frequencies of the preproenkephalin A (PENK) gene CA repeat in Asians, African-Americans, and Caucasians: lack of evidence for different allele frequencies in alcoholics.

Evidence from animal models and from recent reports on the efficacy of the opioid antagonist naltrexone in the treatment of alcoholism suggests that the endogenous opioid systems may play a role in alcohol seeking behavior. The gene encoding preproenkephalin A (PENK) is flanked at its 3' end by a polymorphic (CA)n repeat. We determined the allele frequencies for this locus in samples of Chinese and Atayal living in Taiwan, Caucasians living in the United States and Byelorussia, and African-Americans living in the United States. We compared the allele frequencies of nonalcoholics in each population with those of alcoholics with or without alcohol-induced organ pathology. There was no difference in allele frequencies within any racial group when alcoholics with or without organ pathology were compared; there was also no difference in allele frequency between nonalcoholics and alcoholics within the two Asian populations, Caucasians, or African-Americans. There were highly significant differences in the frequency of the various length polymorphisms between the Asian, Caucasian, and African-American samples.

Low frequency of the ADH2*2 allele among Atayal natives of Taiwan with alcohol use disorders.

Genetic variation at two polymorphic alcohol dehydrogenase loci, ADH2 and ADH3, and at the polymorphic mitochondrial aldehyde dehydrogenase locus, ALDH2, may influence the risk of developing alcoholism by modulating the rate of elimination of ethanol and the rate of formation and elimination of acetaldehyde. Populations differ in allele frequencies at these loci. We determined the genotypes at all three of these loci in Atayal natives of Taiwan. The frequencies of ADH2*2, ADH3*1, and ALDH2*1 alleles (0.91, 0.99, and 0.95, respectively) were significantly higher among the Atayal than among a predominantly Han Chinese population from Taiwan. Among the Atayal, the group with alcohol use disorders (alcohol dependence and alcohol abuse) had a significantly lower frequency of the ADH2*2 allele (0.82) than those without alcohol use disorders (0.91). The ADH2*2 allele encodes the beta 2 subunit; isozymes containing beta 2 subunits oxidize alcohol faster in vitro than the beta 1 beta 1 isozyme encoded by ADH2*1. Thus, the simplest explanation for these data is that individuals with a beta 2 isozymes have a higher rate of ethanol oxidation, which is a deterrent to alcohol abuse and dependence in some individuals. The Atayal with alcohol use disorders also had a lower frequency of ALDH2*2 than the controls; this allele is known to be responsible for the alcohol-flush reaction among Asians, and thereby deters drinking.

Purification and partial amino acid sequence of a high-activity human stomach alcohol dehydrogenase.

To understand the relative importance of alcohol dehydrogenase (ADH) isoenzymes in gastric ethanol metabolism, a stomach-specific ADH (sigma-ADH) was purified to homogeneity from human transplant donor and surgical tissues, and its activity for ethanol oxidation was examined. The enzyme from these tissues had a specific activity at pH 10 of approximately 70 units/mg, about 10 times that reported by Moreno and Parés (J. Biol. Chem. 266:1128-1133, 1991). The enzyme exhibited a high Km for ethanol at pH 7.5 and 10 (29 and 5.2 mM, respectively). This high-activity sigma-ADH isoenzyme migrated on starch and isoelectric focusing gels to a position slightly anodic to the liver pi pi isoenzyme. It was subjected to digestion by endoproteinases, and approximately 40% of the protein was sequenced. The sigma-ADH exhibited 75%, 68%, and 62% sequence identity to the human class I (beta 1), II (pi), and III (chi) isoenzymes, respectively, and 61% identity to the deduced ADH6 amino acid sequence. Phylogenetic analysis indicated that precursors to this high-activity sigma-ADH and the class I isoenzymes diverged more recently than precursors to the class II and III isoenzymes, after reptilian and avian divergence. The high-activity sigma-ADH isoenzyme therefore represents a distinct class of ADH (class IV), more closely related in evolution to the class I isoenzymes than to the other known human isoenzymes.

Alcohol and aldehyde dehydrogenase polymorphisms and alcoholism.

The alcohol-flush reaction occurs in Asians who inherit the mutant ALDH2*2 allele that produces an inactive aldehyde dehydrogenase enzyme. In these individuals, high blood acetaldehyde levels are believed to be the cause of the unpleasant symptoms that follow drinking. We measured the alcohol elimination rates and intensity of flushing in Chinese subjects in whom the alcohol dehydrogenase ADH2 and ALDH2 genotypes were determined. We also correlated ADH2, ADH3, and ALDH2 genotypes with drinking behavior in 100 Chinese men. We discovered that ADH2*2 and ADH3*1, alleles that encode the high activity forms of alcohol dehydrogenase, as well as the mutant ALDH2*2 allele were less frequent in alcoholics than in controls. The presence of ALDH2*2 was associated with slower alcohol metabolism and the most intense flushing. In those homozygous for ALDH2*1, the presence of two ADH2*2 alleles correlated with slightly faster alcohol metabolism and more intense flushing, although a great deal of variability in the latter was noted.

Subjective feelings of alcohol intoxication in Asians with genetic variations of ALDH2 alleles.

Asian-American men who possess ALDH2*2 alleles and who experience a facial flush after consuming alcohol were carefully matched on drinking history and demographic variables with nonflushing Asian males with only ALDH2*1 alleles. Each man was tested following placebo and a challenge dose of 0.75 ml/kg alcohol. Following alcohol, flushers reported experiencing significantly more positive feelings of intoxication than nonflushers, despite equivalent blood alcohol concentrations. These data suggest that Asians who flush after drinking, particularly those with ALDH2*1/2*2 genotype, have a more intense, although not necessarily a more negative, response to alcohol than comparable nonflushing Asians. This alcohol sensitivity reaction that many Asian flushers experience may contribute to their lower tendency to drink excessively, even though their response to alcohol is not predominantly negative.