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Page 1: Relation among alcohol dehydrogenase 2 polymorphism, alcohol consumption, and levels of gamma-glutamyltransferase

Alcohol 29 (2003) 131–135

Relation among alcohol dehydrogenase 2 polymorphism, alcoholconsumption, and levels of gamma-glutamyltransferase

Michael Loewa,*, Heiner Boeingb, Til Sturmera, Hermann Brennera

aDepartment of Epidemiology, The German Centre for Research on Ageing, Bergheimer Str. 20, D-69115 Heidelberg, GermanybGerman Institute of Human Nutrition, Potsdam-Rehbrucke, Arthur-Scheunert-Allee 114-116, 14558 Bergholz- Rehbrucke, Germany

Received 24 June 2002; received in revised form 13 January 2003; accepted 13 January 2003

Abstract

In human beings, alcohol is metabolized primarily by alcohol dehydrogenase 2 (ADH2) and acetaldehyde dehydrogenase 2 (ALDH2).Whereas polymorphisms of the ALDH2 are common in Asian persons, polymorphisms of the ADH2 seem to be more important inCaucasian individuals. The aim of this study was to assess the relation among ADH2 polymorphism, alcohol consumption, and levels ofgamma-glutamyltransferase (GGT). The question was examined among 1,663 subjects (736 men and 927 women) participating in a nationalrepresentative health and nutrition survey (VERA substudy of the German National Nutrition Survey). Alcohol consumption was assessedthrough responses to a semiquantitative food frequency questionnaire (FFQ), and the ADH2 restriction fragment length polymorphism(RFLP) Mae III and GGT levels were analyzed from frozen serum samples. The relations between the polymorphism and alcoholconsumption and between alcohol consumption and GGT levels according to the polymorphism were assessed with the use of descriptivestatistics and contingency table analysis. Of the subjects studied, 2.8% were homozygous or heterozygous for the ADH2*2 allele, andhigh levels of alcohol consumption (�20 g/day) were less common among these subjects (8.5%) than among subjects with the ADH2*1allele (19.9%). Median levels of GGT increased with increasing levels of alcohol consumption. This increase tended to be stronger amongsubjects with the ADH2*2 allele than among other subjects, although differences were not statistically significant (P value for interaction � .1)given the small number of subjects with the polymorphism. These results are consistent with the hypothesis that subjects with the ADH2*2allele, on the one hand, might tend to drink less alcohol but, on the other hand, might be at increased risk of alcohol-related effects onthe liver with consumption of larger amounts of alcohol. However, this hypothesis needs to be evaluated among larger population samples.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Alcohol dehydrogenase 2; Alcohol consumption; Gamma-glutamyltransferase

1. Introduction

In human beings, more than 90% of ingested ethanolis degraded in the liver by oxidative and nonoxidativepathways. The major enzymes involved in the metabolismof ethanol are alcohol dehydrogenase 2 (ADH2; known asADH1B in a new nomenclature), aldehyde dehydrogenase2 (ALDH2), and cytochrome P450 (CYP2E2) (Bosron &Li, 1986; Lieber, 1997). Both ADH2 and ALDH2 are poly-morphic, and genetic polymorphisms have been shown tofunctionally affect alcohol detoxification.

The most important known polymorphism is theALDH2*2 allele, which is common in Asian persons. Thisallele is associated with elevated acetaldehyde levels and

* Corresponding author. Tel.: �49-6221-54-8150; fax: �49-6221-54-8142.

E-mail address: [email protected] (M. Loew).Editor: S. Borg

0741-8329/03/$ – see front matter � 2003 Elsevier Inc. All rights reserved.doi: 10.1016/S0741-8329(03)00015-6

flushing after alcohol consumption in this population (Sunet al., 1999).

In Caucasian individuals, polymorphisms of the alcoholdehydrogenase (ADH) system are probably more importantfor the effects of alcohol (Eriksson et al., 2001). The ADHin human beings is a dimeric protein consisting of twosubunits with a molecular weight of 40 kD each. Seven ADHgenes have been mapped to chromosome 4 in human beings,but functional polymorphisms have been found only forADH2 and ADH3 (Smith, 1986). The kinetic differencesamong ADH2 polymorphisms are more striking than amongADH3 polymorphisms. The ADH2*2 allele leads to a higherrate of oxidation of ethanol (Takeshita et al., 1996). TheADH2*2 allele differs from the ADH2*1 allele by a CGC/CAC substitution, resulting in an arginine/histidine exchangein the protein. The ADH3*1 allele also codes for a moreactive enzyme. Part of the reported association with an in-creased rate of alcohol oxidation in the presence of theADH3*1 allele is probably due to linkage disequilibriumbetween ADH2 and ADH3 (Borras et al., 2000).

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M. Loew et al. / Alcohol 29 (2003) 131–135132

Gamma-glutamyltransferase (GGT) is a liver enzyme anda sensitive parameter for liver damage. Alcohol consumptionis known to be one of the most common causes of elevatedliver enzyme levels in the general population, and a positiveassociation between alcohol consumption and GGT levels hasbeen described after adjustment for other co-factors (Arndtet al., 1998; Steffensen et al., 1997). Especially at higherlevels of alcohol consumption (i.e., �50 g/day), the riskfor increased GGT levels is strongly greater (Steffensen etal., 1997).

In this study, we examined the questions as to whetherthe ADH2 genotype influences alcohol consumption andto what extent the well-documented association betweenalcohol consumption and GGT levels is influenced by theADH2 genotype.

2. Materials and methods

2.1. Study design and study population

This cross-sectional study is based on a random subsam-ple of participants in the German National Nutrition Survey(NVS), which was conducted between 1985 and 1989 inWest Germany among the noninstitutionalized German adultpopulation (Speitling et al., 1992). Written informed con-sent had been obtained from each human subject, and theprocedures followed were in accordance with the ethicalstandards of the responsible committee on human experi-mentation and with the Helsinki Declaration of 1975, asrevised in 1983.

Among the 11,141 participating households in the NVS,2,006 participants (862 men and 1,144 women) were ran-domly selected for further laboratory analyses (VERA sub-study). Because of implausible anthropometric data 18participants had been excluded. Because alcohol consump-tion and GGT levels might be affected by pregnancy, wefurther excluded 16 pregnant women from the currentanalysis.

2.2. Alcohol intake

Alcohol intake was assessed through responses to asemiquantitative food frequency questionnaire (FFQ). Partic-ipants were asked about their average weekly consumptionof different alcoholic beverages during the previous year.Estimates in units were given separately for beer (unit � 1l), wine (unit � 0.25 l), and spirits (unit � 20 ml). The unitswere converted into grams of ethanol per day assuming arelative weight of alcohol of 0.8 g and average alcoholcontents (volume) of 4.5% for beer, 11% for wine, and 40%for spirits. An intake of �1 unit was taken as half of therespective unit. Abstainers were defined as participantsreporting no intake of beer, wine, or spirits.

For the bivariate analysis, we categorized alcohol con-sumption into five categories: 0, �0 to �5, �5 to �10, �10

to �20, and �20 g per day. These categories approximatelyreflect quintiles of alcohol consumption.

2.3. Gamma-glutamyltransferase levels

Levels of GGT were determined by standard laboratorymethods with Eppendorf Enzymautomat 5020 and reagentssystem packs No. 14056 and No. 14057 from Merck (Spei-tling et al., 1992). We defined an abnormal GGT level as �20U/l, corresponding approximately to the 80th percentile ofthe distribution.

2.4. Genotyping of alcohol dehydrogenase 2

Genotyping of the ADH2 was performed with the use offrozen serum samples, which were available for 1,817 partic-ipants in 1998. To amplify the polymorphic part of exon 3of the ADH2 gene (Xu et al., 1988), polymerase chainreaction was conducted with primers of Groppi et al. (1990)according to a previous publication (Sturmer et al., 2002).Despite the small amounts of DNA included in the serumsamples, the genotype of the ADH2 could be successfullydetermined in 1,746 participants (1,696 persons with geno-type ADH2*1/*1, 48 persons with genotype ADH2*1/*2,and 2 persons with genotype ADH2*2/*2). Because of thesmall number of homozygotes we combined ADH2*2allele homozygotes and heterozygotes for further analyses.

2.5. Statistical methods

For the statistical analysis, we defined a final study popu-lation with complete information on genotype, GGT level,and alcohol consumption (n � 1,663). We used descriptivestatistics to characterize the study population with respectto sociodemographics and alcohol consumption habits. In abivariate analysis, we examined the distribution of alcoholconsumption according to the ADH2 polymorphism, andwe compared the alcohol consumption [grams per day (g/day)] among people with the ADH2*2 allele (homozygoteor heterozygote) and people with the ADH2*1 allele (homo-zygote) by one-sided Wilcoxon rank sum test. Furthermore,we assessed the rise of median level of GGT with increasingalcohol consumption according to the ADH polymorphismboth graphically and by contingency table analysis. A possi-ble interaction between alcohol consumption and ADH2polymorphism with respect to GGT level was assessed bythe Breslow-Day test for homogeneity.

Statistical analysis of data was performed with StatisticalAnalysis System (SAS) Version 8.0 (SAS Institute Inc., Cary,NC, USA).

3. Results

The main characteristics of the study population are pre-sented in Table 1. The study population comprised 1,663persons: 736 men and 927 women. The mean age was 43.2

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M. Loew et al. / Alcohol 29 (2003) 131–135 133

Table 1Characteristics of study population

Age (yr), mean (S.D.) 43.2 (15.7)Weight (kg), mean (S.D.) 71.8 (13.5)Height (cm), mean (S.D.) 169.6 (9.1)Body mass index (kg/m2), mean (S.D.) 25.0 (4.2)Sex, N (%)

Male 736 (44.3)Female 927 (55.7)

Average alcohol consumption (g/day), N (%)0 383 (23.0)�0–�5 282 (17.0)�5–�10 345 (20.7)�10–�20 327 (19.7)�20 326 (19.6)

Genotype of ADH2, N (%)ADH2*1/*1 (homozygote) 1,616 (97.2)ADH2*1/*2 (heterozygote) 45 (2.7)ADH2*2/*2 (homozygote) 2 (0.1)

GGT (U/l)Median (interquartile range) 11.2 (8.2; 17.8)�20, N (%) 1,334 (80.2)�20, N (%) 329 (19.8)

ADH2 � Alcohol dehydrogenase 2; GGT � gamma-glutamyltransferase

years. The mean body weight and body mass index were71.8 kg and 25.0 kg/m2, respectively. According to the FFQ,1,337 persons had an alcohol intake of �20 g/day, and 326persons consumed �20 g/day. The majority (97.2%) ofstudy participants were homozygous for the ADH2*1 allele.Only 45 persons were heterozygous (ADH2*1/*2), and 2persons were homozygous, for the ADH2*2 allele. A GGTlevel of �20 U/l was observed in 19.8% of participants.

Of the 326 subjects who consumed �20 g/day, 250 weremen (34.0% of 736 men) and 76 were women (8.2% of927 women).

Table 2 shows the distribution of alcohol consumptionin five categories according to ADH2 genotype. Among thegroup of individuals who consumed �20 g of alcohol per

Table 2Alcohol consumption per day according to alcohol dehydrogenase 2(ADH2) genotype

ADH2*2 allele(heterozygote or ADH2*1 allelehomozygote) (homozygote)

Averagealcoholconsumption(g/day) N % N %

0 13 27.7 370 22.9�0–�5 9 19.1 273 16.9�5–�10 8 17.0 337 20.9�10–�20 13 27.7 314 19.4�20 4 8.5 322 19.9

Mean (median) 8.5 (5.1) 11.6 (6.3) P � .1*alcoholconsumptionper day(g/day)a

aConsumption for all study subjects with indicated ADH2 allele.*One-sided Wilcoxon rank sum test.

day, the proportion of people who were heterozygous or ho-mozygous for the ADH2*2 allele (8.5%) was lower thanthe proportion of people who were homozygous for theADH2*1 allele (19.9%). Accordingly, the mean alcohol con-sumption was lower in individuals who were heterozygousor homozygous for the ADH2*2allele than in individuals whowere homozygous for the ADH2*1 allele, although this dif-ference was not statistically significant (P � .1 in one-sidedWilcoxon rank sum test).

In Fig. 1, we present the association between alcoholconsumption (in five categories according to grams of alco-hol consumed per day) and GGT levels according to ADH2genotype. For the four categories of consumption up to 20 gof alcohol per day, median GGT levels essentially did notrise with increasing amount of alcohol consumed, neitherin the group with the ADH2*1/*1 genotype nor in the groupwith the ADH2*2 allele. For participants who consumedmore than 20 g of alcohol per day, however, there was arise in GGT level. This increase was more pronounced inthe group with the ADH2*2 allele than in the group with theADH2*1/*1 genotype (median GGT levels of 28.7 and14.3 U/l, respectively), although median levels of alcoholconsumption within that category were similar in bothgroups.

The number (and percentage) of individuals with an ele-vated GGT level (�20 U/l) was assessed according to twocategories of alcohol consumption (�20 or �20 g/day) asa study population and by ADH2 genotype. The results areshown in Table 3. Of the total population (1,663), 326consumed �20 g of alcohol per day, and of these subjects,30.4% (99) had an abnormal GGT level (i.e., �20 U/l). Ofsubjects with ADH2*1/*1 genotype, 29.8% (96 of 322) hadan abnormal GGT level. Of subjects with the ADH2*2 allele,75% (3 of 4) had an elevated GGT level. Despite this differ-ence, the interaction between genotype and alcohol con-sumption was not statistically significant (P � .1), given thesmall number of subjects with the polymorphism.

Table 3Gamma-glutamyltransferase (GGT) levels in relation to twocategories of alcohol consumption for study population as a whole andaccording to alcohol dehydrogenase 2 (ADH2) genotype

AlcoholGGT�20 U/lconsumption

Population (g/day) N1a N2

b (%)c

Total (1,663) �20 1,337 230 17.2�20 326 99 30.4

ADH2*1 allele �20 1,294 222 17.2(homozygote) �20 322 96 29.8

ADH2*2 allele �20 43 8 18.6(heterozygote orhomozygote)

�20 4 3 75.0

aTotal study subjects in each group.bNumber of study subjects in each alcohol consumption group with

GGT �20 U/l.cPercentage of study subjects in the respective alcohol consumption

group with GGT �20 U/l.

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M. Loew et al. / Alcohol 29 (2003) 131–135134

Fig. 1. Alcohol consumption and gamma-glutamyltransferase (GGT) levels according to alcohol dehydrogenase 2 (ADH2) genotype. Data are presentedas median GGT level in five alcohol consumption categories for individuals homozygous for the ADH2*1 allele (solid line) and individuals homozygousor heterozygous for the ADH2*2 allele (dotted line).

4. Discussion

In the current study, we examined the relation betweenADH2 genotype and alcohol consumption. We found a lowerpercentage of subjects with high alcohol consumption (�20g/day) in the group with the ADH2*2 allele (homozygotesand heterozygotes) than in the group with ADH2*1/*1 geno-type, although this difference was not statistically significant.This result is consistent with a finding from another studythat the ADH2*2 allele may be protective against drinkingbecause of the unpleasant consequences of elevated acetalde-hyde levels (Eriksson et al., 2001). These effects are probablymore pronounced with higher classes of amounts of alcoholconsumed (i.e., �50 g/day) (Borras et al., 2000; Muramatsuet al., 1995).

In the published literature, there are many reports regard-ing the ADH2 polymorphism and its role in metabolizingalcohol. In particular, association of the ADH2*2 allele withflushing has been reported (Takeshita et al., 1996). Resultsof a number of studies seem to indicate that the ADH2*2allele protects against alcohol abuse and alcoholism in Asianpersons (Muramatsu et al., 1995; Shen et al., 1997) andCaucasian populations (Borras et al., 2000; Whitfield et al.,1998). In a Russian population, a negative association hasbeen described between the ADH2*2 allele and alcoholmisuse (Ogurtsov et al., 2001). The ADH2*2 allele may evenbe protective against fetal alcohol syndrome, probably bypreventing the mother from drinking during pregnancy(Eriksson et al., 2001).

Findings obtained from the current study would be consis-tent with a possible interaction between the ADH2 genotypeand alcohol consumption with respect to GGT level: Therise in GGT level associated with higher levels of alcohol

consumption might be more pronounced in the group withthe ADH2*2 allele than in the wild-type group. This findingseems plausible from a pathophysiological point of view:The higher activity of the enzyme of the ADH2*2 allelemay lead to a faster oxidation of alcohol and therefore tohigher concentrations of acetaldehyde in the liver. Acetalde-hyde is hepatotoxic, and an increased risk of liver cirrhosisdue to alcohol misuse has been observed in subjects withthe ADH2*2 allele (Ogurtsov et al., 2001). We believe that, todate, the results of the current study are the first to indicatea possible interaction between alcohol consumption and el-evated GGT levels, a sensitive parameter for liver damage(Penn & Worthington, 1983) with respect to ADH2 polymor-phism in a population-based sample. Again, however, thisinteraction was not significant, given the small number ofpersons with the ADH2 allele.

The main limitation of the current study is the smallnumber of persons with the ADH2*2 allele, especially inhigher alcohol consumption categories, despite the largeoverall sample size. The low prevalence of persons withthe ADH2*2 allele (2.8% homozygotes or heterozygotesin the current study) was similar to the prevalence reportedpreviously for other European population samples, whichranged from 0% in France, 2.4% in Germany, and 3.0% inPoland to 7.5% in Sweden and 10.8% in Spain (Borraset al., 2000).

Although the results of the current study are in agreementwith prior expectations on the basis of pathophysiologicalconsiderations, the power of the study to verify a poten-tial interaction between alcohol consumption and the ADH2polymorphism was insufficient. To confirm such an interac-tion, larger samples are needed in future studies.

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M. Loew et al. / Alcohol 29 (2003) 131–135 135

Acknowledgments

The study was supported in part by the Federal Ministryand Technology Grant Nos. 704752, 704754, and 704766,and by the faculty of medicine of the university of Ulm(P583, P589).

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