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

Molec AspectsMed. Vol. 10, pp, 147-158, 1988

0098-2997/88 $0.00 + .50 Copyright © 1988 Pergamon Press plc.

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GENETIC POLYMORPHISM OF ENZYMES OF ALCOHOL METABOLISM AND SUSCEPTIBILITY TO ALCOHOLIC LIVER DISEASE William F. Bosron, Lawrence Lumeng and Ting-Kai Li Indiana University School of Medicine and VA Medical Center, Indianapolis, IN, U.S.A.

Introduction Studies in adopted-out children of alcoholics have demonstrated convincingly that the susceptibility of individuals to alcoholism is, in part, genetically controlled (Goodwln, 1987; Cloninger, 1987). Moreover, studies in twins indicate that concordance of alcoholism and certain complications of alcohol consumption such as alcoholic cirrhosis and psychosis is greater in monozygotic than it is in dizygotic twin pairs (Hrubec et a2., 1981). The biochemical basis for these genetic effects on alcohol-related disorders has not been determined. It is likely that many genetic factors contribute to the etiologies of these disorders. In this regard, the potential role of genetic polymorphism of the two principal enzymes involved in alcohol metabolism, alcohol dehydrogenase and aldehyde dehydrogenase, has attracted considerable attention (Bosron et a2., 1986). This is mainly due to the fact that the role of these enzymes in the regulation of alcohol metabolism and the biochemical properties of the polymorphic isoenzymes have been well characterized (Vallee et al., 1983; Pietruszko, 1983; Bosron et al., 1987). In this review, we will summarize data on the mechanisms of regulation of alcohol elimination and the biochemical properties of alcohol and aldehyde dehydrogenase isoenzymes. We will also discuss the role that genetic variability in the distribution of polymorphic alcohol and aldehyde dehydrogenase isoenzymes may play in determining individual differences in ethanol oxidizing capacity and in physiological responses to the products of ethanol metabolism. Additionally, recent studies demonstrating the role that acetaldehyde may play in alcoholic liver injury will be summarized. Mechanisms Of Ethanol Elimination AbsorptlonAnd Distribution Of Ethanol The pharmacoklnetics of absorption, distribution and elimination of ethanol have been reviewed (Li, 1983). Ethanol is passively absorbed from the gastrointestinal tract and distributed in total body water. The rates of absorption and distribution have been shown to be dependent on factors such as diet, chronic alcohol consumption, smoking, age and body habitus (Reed, 1978; Li, 1983). The absorption and distribution of a moderate amount of

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ethanol is usually complete after 30-60 min. It is neither accumulated to any extent by specific organs nor bound to cellular components. Comparison of absorption and distribution rates in mono- and dizygotic twins demonstrates that about half of the variability in the peak blood alcohol concentration and the time for attainment of this peak is genetically determined (Kopun et al., 1977; Martin, 1987). After absorption and distribution of alcohol, the blood ethanol concentration decreases with time in a nearly linear, pseudozero-order manner (Wilkinson, 1980). However, the kinetics at low blood ethanol concentration, when nearly all of the ethanol has been eliminated, are not linear. The curve indicates that the elimination process actually obeys MichaelisMenten kinetics and that the pseudolinear initial rate is due to the fact that alcohol oxidizing enzymes are saturated with substrate during the initial phase of elimination (Wilkinson, 1980; Li, 1983). Many groups have reported that there is a large, 2-3 fold, variation in the pseudolinear elimination rate among individuals (Kopun et al., 1977; Reed, 1978; Li, 1983). Comparison of elimination rates in mono- and dizygotic twins indicates that about half of this variability is genetically determined (Kopun et al., 1977; Martin, 1987). The remainder of the variability is due to environmental factors such as smoking, nutritional status and the history of alcohol consumption.

Pathways Of Ethanol Oxidation About 90% of ethanol metabolism occurs in the liver. The main pathway of ethanol metabolism is its oxidation to acetaldehyde which is catalyzed principally by NAD+-dependent alcohol dehydrogenase (ADH; EC i.I.I.I) (Hawkins et al., 1972; Li, 1983). There are two other enzyme systems that have been implicated in ethanol oxidation: the NADPH- and cytochrome P450-dependent microsomal ethanol oxidizing system (MEOS) and the H202dependent catalase. The KM of MEOS for ethanol is high, I0 mM. This ethanol-actlve form of cytochrome P450 is induced after chronic ethanol consumption, and the contribution of MEOS to ethanol metabolism is probably substantial under these circumstances (Lieher, 1984; Lieber, 1987). The contribution of catalase to ethanol metabolism is limited by the availability of H202, which is limited in liver (Hawkins et al., 1972; Li, 1983). Acetaldehyde is oxidized to acetate by the NAD+-dependent aldehyde dehydrogenase (Li, 1983; Boston et al., 1986; Agarwal et al., 1987). As will be discussed below, acetaldehyde is a highly reactive compound which can form stable adducts with amino groups in proteins and with brain biogenic amines (Peters, et al., 1987; Lin, et al., 1988). Therefore, an efficient and low KM aldehyde dehydrogenase is required to maintain plasma acetaldehyde concentrations at the low level, less than I0 ~M, observed during ethanol metabolism. Acetate is oxidized to CO 2 and H20 mostly in nonhepatic tissue.

Regulation Of Ethanol Metabolism The fact that the blood acetaldehyde concentration is maintained about lO00fold lower than the blood ethanol concentration in most individuals after a moderate dose of ethanol indicates that the alcohol dehydrogenase-catalyzed step is rate-llmitlng (Li, 1983). Acetaldehyde levels in the range of 30-100 ~M are seen only in individuals who have a genetic variant of ALDH which is low in activity (Mizoi et al., 1983; Agarwal et al., 1987) or in alcoholics with low liver ALDH activity due to liver disease (Peters et al., 1987).

Polymorphism of Alcohol and Aldehyde Dehydrogenase A major factor that controls the rate of ethanol oxidation is the rate of reoxidation of NADH to NAD + in mitochondrla (Hawkins et al., 1972; Crow et al., 1977). The amount of NADH generated during ethanol oxidation appears to exceed the capacity of liver to reoxidize it, as evidenced by the increase in both lactate/pyruvate and ~-hydroxybutyrate/acetoacetate ratios during alcohol oxidation (Crow et al., 1977; Lumeng et al., 1980). In liver slices and perfused liver, uncouplers of mitochondrial respiration, such as dlnitrophenol, and substrates which can oxidize NADH, such as pyruvate, will stimulate ethanol oxidation (Crow et al., 1977). Direct calculations of the degree of inhibition of alcohol dehydrogenase by the steady-state concentrations of NADH and acetaldehyde measured in situ in rats indicate that alcohol dehydrogenase is working at about 68% of its Vma x under these conditions (Crabb et al., 1983). Another major factor that regulates the rate of ethanol oxidation is the content and specific activity of alcohol dehydrogenase in liver (Crabbet al., 1983; C r a b b e t al., 1987) studies of the effects of diet and hormones on ADH content and alcohol elimination rates in rats indicate that the mass of ADH protein in liver is a rate-limiting factor in alcohol metabolism (Crabbet al., 1987). For example, fasting and testosterone treatment of female rats decrease ADH activity and ethanol elimination rate proportionately. Conversely, castration of male rats, estrogen treatment, opiate administration and immobilization stress increase enzyme activity and ethanol elimination rate in parallel. In mice, cis-actlng loci have been identified which control the temporal and tissue specific expression of mouse liver ADH isoenzymes (Felder et al., 1983). We have demonstrated that the twofold difference in liver ADH activity observed among inbred strains of mice correlates with a twofold difference in ADH mRNA (Patterson et al., 1987). The high activity strain of mice exhibits a long purine-pyrimidine sequence in the first intron of the Adhl gene that is missing in low activity mice (Zhang et al., 1987). It is postulated that this locus influences the rate of transcription of the Adhl gene. Perhaps this or other regulatory loci are responsive to hormonal or nutritional signals and are responsible for the regulation of enzyme expression under the above mentioned conditions. In humans, genetically determined isoenzymes of ADH have been identified which exhibit substantially different kinetic properties (Bosron et al., 1986). As will be discussed below, the genetic polymorphism of ADH isoenzymes is most likely a contributing factor to individual differences in the rate of ethanol metabolism. Alcohol Dehydrogenase Polymorphlsm Genetics

Of Alcohol Dehydrogenase Isoenzymes

Mammalian alcohol dehydrogenases are dimeric zinc metalloenzymes with subunits of 40,000 daltons (Li, 1983; Bosron et al., 1986). As many as 20 different molecular forms of alcohol dehydrogenase have been identified in human liver by gel electrophoresis or isoelectric focusing (Bosron et al., 1986). Five different isoenzyme subunits (~,~,7,x,X) are encoded at five different genes (ADH1, ADH2, ADH3, ADH4, ADH5) respectively (Vallee et al., 1983; Bosron, et al., 1986; Smith, 1986; Agarwal et al., 1987). The class I isoenzymes have cathodic electrophoretlc mobility in gels at pH 7.5 to 8.6, usually have low K M for ethanol and are inhibited by ~M concentrations of 4-methylpyrazole. These isoenzymes are homodimers and heterodimers composed of a,~ and subunits, e&., as, a~, ~ , aT, ~ (Bosron et al., 1986). The class II alcohol dehydrogenase, ~-ADH, migrates anodically to the Class I isoenzymes. It

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W.F. Bosron et al.

150

has higher KM for ethanol and higher K i for 4-methylpyrazole than the Class I forms (Bosron er al., 1979; Vallee et al., 1983). Class III alcohol dehydrogenase, x-ADH, migrates an0dlcally to Class II enzymes and is not inhibited by 4-methylpyrazole. It has very high KM'for ethanol; in fact, it is not saturable with ethanol even at molar concentrations (Wagner et al., 1984). Hence, only Class I and Class II alcohol dehydrogenases appear to be involved in ethanol metabolism in humans. Studies of the electrophoretic and isoelectric focusing patterns of ADH isoenzymes present in liver and other tissues obtained from individuals belonging to different racial groups indicate that there is genetic polymorphism at two loci (Bosron et al., 1986; Smith, 1986;iAgarwa ~ et al., 2987). Three different subunlts (~I,~2,~3) are encoded by ADH2 , ADH2 and ADH2 ~, re~pectively~ and two different subunits (71, 72 ) corresponding to the ADH3 ~ and ADH3 ~ alleles, have been identified. No polymorphlc variants have been identified at ADHI. Multiple forms of ~-ADH and x-ADH have been observed (Ditlow et al., 1984; Belsswenger et al., 1985) and differences between 5' coding sequence of the ~-ADH cDNA and the amino terminal sequence of the purified ~ subunit have been reported, (J6rnvall et al., 1987b), but it is not clear that these differences are due to genetic polymorphlsm at ADH4 or ADH5. As shown in Table I, the three ADH2 and two ADH3 alleles appear with different frequencies in different populations and the five allelic Isoenzymes account for the observed variation in electrophoretic and Isoelectric focusing patterns amon~ the Class I isoenzymes (Harada et al., 1978; Bosron et al., 1986). ADH2 ~ is the predominant all~le in White Americans and Europeans, while Orientals usually have the ADH2 ~ allele. ADH2 ~ has only been identified in American and South African Blacks. The two ADH3 alleles are distributed with approximately equal frequencies in White Americans and E~ropeans, but the predominant allele in Orientals and BlackAmericans is ADH3 ~. Table I.

Frequency of ADH Alleles in Different Populations

Population

ADH21

ADH22

ADH23

ADH31

ADH32

(~i)

(~2)

(~3)

(71)

(72)

White American

>95%

<5%

<5%

50%

50%

White European

90

I0

<5

60

40

Chinese, Japanese

35

65

<5

95

5

Black American

85

<5

15

85

15

The investigation of the potential relationships between ADH polymorphism and alcohol elimination rate, genetic predisposition to alcoholism or susceptibility to alcohol-related diseases has been hampered by the need to use liver biopsy tissue to determine the enzyme phenotype. This problem has now been overcome. Direct genotyping is now possible by amplifying DNA in vitro with the polymerase chain reaction and distinguishing the different ADM alleles by use of specific ollgonucleotlde probes directed at single base pair differences among them (Xu et al., 1988). This method can identify all three of the ADH2 and the two ADH3 alleles using leukocyte DNA.

C a t a l y t i c And S t r u c t u r a l P r o p e r t i e s Of A l c o h o l Dehydrogenases

Polymorphism of Alcohol and Aldehyde Dehydrogenase

151

Over the past i0 years, methods have been developed for the purification of all variant forms of ADH. The catalytic and structural properties of these isoenzymes have been characterized (Vallee et al., 1983; Bosron et al., 1987; J6rnvall et al., 1987a). The steady-state kinetic constants for ethanol oxidation at pH 7.5 of the homodimeric forms of ADH containing all of the different ~ and 7 subunits are summarized in Table II. Of particular interest are the kinetic differences among isoenzymes with E1 , ~2 and 83 subunits (Bosron e t a ] . , 1983; Yin et al., 1984; Burnell e t a ] . , 1987). The 70-fold difference in KM for NAD +, 700-fold difference in K M for ethanol, and 40-fold difference in Vma x for ethanol oxidation among homodimeric isoenzymes with these subunits could be part of the basis of the inherited differences in the pharmacoklnetics of ethanol elimination. We would also expect Black Americans and Africans with the-high-KM-for-ethanol ~3 isoenzymes to exhibit nonlinear elimination kinetics at high ethanol concentration. Table II.

Kinetic Constants of the ~ ~I~i

and 77 Isoenzymes at pH 7.5

~2~2

Km, NAD +, pM

7.4

Km, ethanol, mM

0.049

Vmax, min "I

9.2

P3~3

180 0.94 400

7171

7272

530

7.9

8.7

34

1.0

0.63

270

87

35

As shown in Table III, assays performed under near-physiological conditions and with substrate concentrations comparable to blood alcohol levels of i00 and 200mg% (22 and 44 mM, respectively) demonstrate that ~2~2 is about 20-times more active in ethanol oxidation than ~i~i. This may account for the greater ethanol elimination ~ate observed in Orientals, as compared with Caucasians, because the ADE2 is observed more frequently in Orientals than ADH2 and the converse is true for Caucasians (Table I) (Agarwal e t a ] . , 1987). Similarly, the 4-7 fold higher ethanol oxidation rate for ~3~3 compared with ~i~i (Table III) suggests that Blacks may also have higher elimination rates than Caucasians. The 2.5-fold variation in Vma x of ethanol oxidation between 7171 and 7272 (Table II) might also contribute to differences in ethanol elimination rates ~oth within and between the different racial groups, because the ADH3 ~ and ADH3 alleles appear with different frequencies (Table I). Table III.

Ethanol Oxidation Rates by Human ~ Ethanol

ADH Isoenzymes

Ethanol Oxidation, Units/mg protein

Pl/~I

/~2,82

P3,83

22

0.2

3.9

0.9

44

0.2

4.0

1.5

The Class, I (a, ~, 7) subunits show more than 90% sequence identity as determined both by amino acid and DNA sequencing (J6rnvall et al., 1987a: J6rnvall et al., 1987b). The ~ subunit differs from E1 and 71 at 24 and 28 out of 374 residues, respectively. The three ~ subunits differ from each other by a single amino acid. Both of the variable residues, at positions 47 and 369, are located in the NAD(H) binding site (J6rnvall et al., 1984; Burnell et al., 1987). These substitutions may account for the observed differences in K M for NAD + and the high Vma x of ethanol oxidation by ~2~2 and ~3~3 relative

152

W.F. Bosron et aL

to ~i~i (Table II), since the rate-limiting step in ethanol oxidation by alcohol dehydrogenase is the dissociation of NADH. The two 7 isoenzymes differ at residues 271 and 349. The substitution of Gin for Arg-349 might affect Vma x by changing the NAD(H) dissociation rate (H66g et al., 1986). Aldehyde Dehydrogenase Polymorphlsm

Genetics Of Aldehyde Dehydrogenase Isoenzymes Four different groups of aldehyde dehydrogenase isoenzymes have been identified by isoelectric focusing of human liver tissue and staining for aldehyde oxidizing activity (Harada et al., 1980). Two forms, ALDHI and ALDH2, have been purified and characterized (Pietruszko, 1983; Johnson et al., 1987). ALDHI is the major cytosolic enzyme form and it has high KM for acetaldehyde, about i00 pM. ALDH2, the major mitochondrial enzyme form, has low KM for acetaldehyde, about I pM, and it is undoubtedly responsible for the majority of acetaldehyde oxidation. These two forms appear to be homotetramers with no common subunits. The ALDHI and ALDH2 subunits have been sequenced (J6rnvall et al., 1987a) and cDNAs have been isolated and sequenced (Hsu et al., 1985). The two forms are closely related; there is 68% sequence identity between the subunits. ALDH3 and ALDH4 have very high KM for acetaldehyde, about i mM; hence, they probably do not participate in acetaldehyde oxidation (Harada et al., 1980; Pietruszko, 1983). Differences in electrophoretlc or isoelectric focusing patterns of aldehyde dehydrogenases have been observed between Oriental and Caucasian populations (Agarwal et al., 1981). About 50% of Japanese and Chinese lack the active form of ALDH2 when gels are stained for acetaldehyde oxidizing activity. In these subjects, an immunoreactive band has been identified which migrates cathodically to the active ALDH2 Isoenzyme (Impralm et al., 1982; Johnson et al., 1987). Amino acid sequencing has shown that Glu-487 in the normal active isoenzyme is replaced by Lys in the inactive variant form (Hsu et al., 1985; J6rnvall et al., 1987a). The function of this residue in the catalytic mechanism of ALDH2 has not been determined. The ALDH2 phenotype can be determined by isoelectrlc focusing of hair root extracts (Agarwal et al., 1981). A summary of the distribution of the ALDH2 deficient phenotype in different populations is shown in Table IV (Goedde et al., 1986; Boston et al., 1988). Besides Orientals, it only appears with high frequency in South American Indians. North American Indians do not exhibit the ALDH2 deficient phenotype. Recently, Hsu and Yoshida have reported that individuals with the variant phenotype also can be identified by probing white cell D N A w i t h an ollgonucleotide probe directed toward the single base change giving rise to the Glu to Lys-487 substitution (Hsu, et al., 1987). It is assumed that individuals with the deficient phenotype are homozygous for the ALDH2 ~ variant allele and those with the normal phenotype are homozygous for the normal AI_~H2 allele or heterozygous for both. The relationship between physiological responses to alcohol consumption and ALDH genotype now can be studied using either white cell DNA or hair root extracts. Another variant ALDH phenotype has been observed in a only few Orientals. They lacked the active ADLHI band on electrophoresls (Yoshlda et al., 1983). The molecular nature and distribution of this abnormality has not been determined.

Polymorphism of Alcohol and Aldehyde Dehydrogenase

Table IV. Distribution of ALDH2 Phenotypes in Populations.

Population

North American Indian: Sioux Navajo Pueblo Sioux Navajo Oklahoma South American Indian: Atacame~os Mapuche Shuara Asian: Japanese Caucasian: Germans Negro: Black Americans

Sample Size

Percent with Deficient Phenotype

33 34 7 90 56 63

0 0 0 5 2 16

133 64 99

43 41 42

184

44

300

0

20

0

Relationship Between ALDH2 Polymorphlsm And Acetaldehyde Toxic Reactions The most striking relationship between a polymorphism in an enzyme of alcohol metabolism and a physiological response to alcohol consumption is the alcohol-flush reaction exhibited by those Orientals with the ALDH2 deficient phenotype. These individuals have very high blood acetaldehyde levels (about 30 ~M) after the administration of low doses of ethanol, e.g., 0.4 g/kg ethanol (Mizoi et al., 1983; Agarwal, et al., 1987). Immediately after drinking, they exhibit facial flushing, tachycardia and the same type of dysphoric reactions observed for individuals drinking while they are taking Antabuse, an ALDH inhibitor. Those Japanese who have the deficient ALDH2 phenotype do not have an alcohol elimination rate which is different from Japanese who have the normal phenotype (Mizoi et al., 1983; Agarwal et al., 1987). Hence, the content of active ALDH2 does not appear to be a rate-limiting factor for ethanol elimination rate. Interestingly, the frequency of alcoholism in those Japanese with the ALDH2 deficient phenotype is significantly lower than that for Japanese with the normal phenotype (Harada et al., 1983). Thus, the ALDH-dependent toxic reaction to acetaldehyde appears to be a negative risk factor for alcoholism. This is the first demonstration of a relationship between alcohol drinking behavior and the genetic variability of an enzyme involved in alcohol metabolism. F o r m a t i o n Of P r o t e i n A c e t a l d e h y d e Adducts

Acetaldehyde is considerably more reactive than ethanol and it has been implicated in the initiation as well as the perpetuation of hepatotoxiclty due to alcohol abuse. Plasma acetaldehyde concentrations are usually
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W . F . Bosron et aL

studies in vitro have shown that acetaldehyde can react with plasma proteins, albumin, erythrocyte membrane proteins, tubulin, hepatic proteins, a number of enzymes with critical lysine residues and hemoglobin (Sorrell, et al., 1988). The exact chemical structure of these proteinacetaldehyde adducts is not fully defined but the initial step most likely involves the formation of a Schiff's base. Whether proteins can form adducts with acetaldehyde in vivo has been a key question. In the rat, hepatic concentrations of acetaldehyde may reach 16 to i00 ~M after alcohol administration. Recently, we have detected the formation in vivo of a protein-acetaldehyde adduct in liver when rats were fed alcohol chronically (Lin, et al. 1988). Israel and his coworkers (Israel, et al. 1986) and Hoerner et al. (Hoerner, et at., 1986) have reported high serum antibody tlters directed against protein-acetaldehyde adducts in mice given alcohol chronically and in alcoholic patients with and without liver disease; however, our report on the detection of a 37,000 dalton liver proteinacetaldehyde adduct in alcohol-fed rats represents the first on an antigen that perhaps is responsible for eliclting the antibody response. This 37 kilodalton protein-acetaldehyde adduct was detected in the soluble protein fraction of livers from rats fed a liquid diet (AIN76) containing 5% ethanol for seven weeks. The proteins were separated by SDS-polyacrylamide gel electrophoresis and the protein-acetaldehyde adduct identified by antibodies that recognize acetaldehyde adducts as an epitope. This protein-acetaldehyde adduct appeared in the liver of rats fed ethanol for as short a period as one week. Intraperitoneal injections of ethanol (2g/kg body weight) at 8-hour intervals to rats over a 24-hour period did not produce any detectable adduct in liver. Incubation of liver homogenates from control rats with acetaldehyde without sodium cyanoborohydride for 4 hour also failed to generate any adduct. Therefore, the formation of the 37 kilodalton protein-acetaldehyde adduct in vivo is specifically dependent on chronic alcohol consumption. The specificity in formation of this protein-acetaldehyde adduct is puzzling but has clear implications in the mechanism of alcohol-related liver injury by autoimmume mechanisms. Summary 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, ~-subunit, and at ADH3, the 7-subunit, are contributing factors to the genetic variability in ethanol elimination rate. The recent development of methods for genotyplng 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 tachycardla, after drinking alcohol. Because acetaldehyde is so reactive, it binds to free amino

Polymorphism of Alcohol and Aldehyde Dehydrogenase groups of proteins including a 37 kilodalton hepatic proteln-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 and may also play a role in liver injury due to chronic alcohol consumption.

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