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Clinical Chemistry 48: 1043-1048, 2002;
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(Clinical Chemistry. 2002;48:1043-1048.)
© 2002 American Association for Clinical Chemistry, Inc.

Relationship between Genetic Polymorphisms of Alcohol-metabolizing Enzymes and Changes in Risk Factors for Coronary Heart Disease Associated with Alcohol Consumption

Yoshiaki Hashimoto1a, Toshifumi Nakayama2, Azusa Futamura1, Miho Omura1, Hideo Nakarai1 and Kazuhiko Nakahara1

Departments of
1 Clinical Laboratory Medicine and
2 Internal Medicine, University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.

aAuthor for correspondence. Fax 81-3-5689-0495; e-mail d01009{at}h.u-tokyo.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Participants and Methods
 Results
 Discussion
 References
 
Background: There are large individual variations in the responses of risk factors for coronary heart disease to alcohol consumption. To clarify the factors responsible for these individual variations, we studied the relationship between blood pressure, serum lipids, and uric acid and the genetic polymorphisms of alcohol dehydrogenase (ADH) 2 and aldehyde dehydrogenase (ALDH) 2 in alcohol drinkers.

Methods: We examined 133 male workers who drank >300 g of alcohol per week. Information regarding lifestyle habits was obtained by questionnaire. The ADH2 genotype was determined by PCR and subsequent digestion with MaeIII. The ALDH2 genotype was determined based on amplified product length polymorphisms.

Results: When the workers were divided into three groups: the ADH21/21, ADH21/22, and ADH22/22 groups, the mean triglycerides and {gamma}-glutamyl transpeptidase concentrations were significantly higher in the ADH22/22 group than in the ADH21/21 group. In addition, multiple logistic regression analysis showed that the frequencies of individuals whose systolic blood pressure, triglycerides, and uric acid values were in the highest one third were significantly higher in the ADH22/22 group than in the ADH21/21 group. In contrast, no difference was observed between the ALDH21/21 and (ALDH21/22 + ALDH22/22) groups with regard to the mean value of any variable and to the frequency of individuals with any variable value in the highest one third.

Conclusion: Individuals with the ADH21/21 genotype might suffer fewer negative effects of drinking.


   Introduction
Top
 Abstract
 Introduction
Participants and Methods
 Results
 Discussion
 References
 
Epidemiologic studies have consistently shown that light or light to moderate drinkers are at a lower risk of coronary heart disease (CHD)1 (1)(2)(3)(4)(5)(6). The mechanisms of this association include beneficial effects on HDL- and LDL-cholesterol, insulin sensitivity, platelet aggregation, blood coagulation, and fibrinolysis (1)(2)(3)(4)(5)(6)(7). However, drinking also has negative effects on blood pressure (8), triglycerides (9), and uric acid (10). These effects could attenuate the cardioprotective effect of alcohol. A recent study suggested that the extent of the negative effects of drinking varied on an individual basis (11). Although the mechanism of this variability is not clear, it may be mediated partly by the speed of alcohol metabolism, the types of alcoholic beverages, the regularity of drinking, and nutritional status.

The major enzymes involved in alcohol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) (12). ADH, which metabolizes ethanol to acetaldehyde, is a dimeric protein consisting of two active subunits, and six ADH genes have been characterized (13). Among them, the ADH2 and ADH3 loci are polymorphic. The ADH2 locus has three alleles: ADH21, which encodes for the ß1 subunit with low activity; ADH22, which encodes for the ß2 subunit with high activity; and ADH21 , which encodes for the ß1 subunit, which is rarely expressed in Japanese (12). The ADH3 locus has two alleles: ADH31, which encodes for the {gamma}1 subunit; and ADH32, which encodes for the{gamma}2 subunit. Because the difference in kinetic properties is much smaller between the {gamma}1 and {gamma}2 subunits than between the ß1 and ß2 subunits, the ADH2 gene polymorphism could play an important role in individual variations regarding ethanol elimination (12). On the other hand, ALDH, which converts acetaldehyde to acetate, also has multiple forms. Among them, ALDH2, with a low Km, is thought to be responsible for most acetaldehyde oxidation (12). The ALDH21 and ALDH22 genes encode the active and the inactive subunit, respectively (14)(15); therefore, the ALDH22 gene contributes to the manifestations of increased blood acetaldehyde after alcohol drinking, e.g., facial flushing, palpitations, and nausea (16)(17).

In this study, we examined whether the changes in blood pressure, serum lipids, and uric acid associated with alcohol consumption varied in relation to the polymorphisms in ADH2 and ALDH2.


   Participants and Methods
Top
 Abstract
 Introduction
 Participants and Methods
Results
 Discussion
 References
 
participants
The participants were 241 male employees from the University of Tokyo Hospital and its affiliated hospitals and institutions, who were not taking any medication for hypertension, diabetes, hyperlipidemia, and hyperuricemia. Genetic polymorphisms were examined in 133 men who drank >300 g of alcohol per week. The other 108 men were nondrinkers. Informed consent was obtained from all participants.

methods
Information regarding current medication, smoking habits, physical activity, and alcohol consumption was obtained by questionnaire as described previously (11). Alcohol consumption was recorded as the average weekly frequency and the average daily amount. The participants were asked to convert all types of alcohol consumption to number of bottles of the Japanese rice wine, sake, based on their ethanol content (11). Blood samples were collected in the morning after a fast of >=12 h. Serum triglycerides, cholesterol, HDL-cholesterol, and uric acid were measured by commercially available enzymatic assays. {gamma}-Glutamyl transpeptidase (rGTP) was measured by an optimized method based on the recommendations of the Japan Society of Clinical Chemistry. Hemoglobin A1C (HbA1C) was measured by HPLC. The reference interval for HbA1C was 4.3–5.8%. LDL-cholesterol was estimated by the Friedewald equation (18) in individuals with triglycerides <4.51 mmol/L.

Genetic polymorphisms were examined as follows. DNA was extracted from peripheral blood leukocytes by a commercially available DNA extraction reagent set (Dr.GenTLE; Takara Biomedicals). The ADH2 genotype was determined by PCR and subsequent digestion with MaeIII, according to the method of Xu et al. (19). The ALDH2 genotype was determined based on amplified product length polymorphism analysis using three oligonucleotide primers, according to the method described by Aoshima et al. (20).

statistical analysis
Data were analyzed by the Statistical Analysis System (SAS Institute). The Tukey multiple comparison test or the Student t-test was used to assess significant differences between group means. Systolic blood pressure, HDL-cholesterol, triglycerides, rGTP, and HbA1C were compared after being logarithmically transformed to allow the use of parametric tests. The two-tailed Fisher exact test was used to compare group proportions. Multiple logistic regression analysis was used to calculate odds ratios. Differences with a P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Participants and Methods
 Results
Discussion
 References
 
We first compared lifestyle habits, blood pressure, and laboratory data between the drinkers and the nondrinkers (Table 1 ). The drinkers smoked more than the nondrinkers; they also had significantly higher blood pressure, HDL-cholesterol, triglycerides, uric acid, and rGTP and significantly lower LDL-cholesterol and HbA1C.


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  		Table 1. Clinical characteristics of drinkers and nondrinkers.1

Among the 133 drinkers, the frequencies of the ADH21/21, ADH21/22, and ADH22/22 genotypes were 21.8%, 31.6%, and 46.6%, respectively, whereas those of the ALDH21/21, ALDH21/22, and ALDH22/22 genotypes were 78.9%, 20.3%, and 0.8%, respectively (Table 2 ). When the participants were divided into three groups, the ADH21/21, ADH21/22, and ADH22/22 groups, according to the ADH2 genotypes, there were no significant differences in age, body mass index, lifestyle habits, or frequency of the ALDH genotypes. The mean triglycerides and rGTP values were significantly higher in the ADH22/22 group than in the ADH21/21 group. No differences were observed among the three groups in blood pressure, total cholesterol, LDL- and HDL-cholesterol, uric acid, or HbA1C. When the participants were divided into two groups, the ALDH21/21 and the (ALDH21/22 + ALDH22/22) groups, no significant differences were observed between the two groups for any of the variables analyzed. Similar results were obtained when the values were adjusted for age, body mass index, smoking, alcohol consumption, and physical activity.


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  		Table 2. Clinical characteristics of the participants distributed according to their ADH2 and ALDH2 genotypes.1

The associations between the ADH2 or ALDH2 genotype and the risk factors for CHD were further assessed by multiple logistic regression analysis (Table 3 ). The odds ratios for the participants whose values for the difference variables were in the highest one third were calculated after corrections for age, body mass index, smoking, alcohol consumption, physical activity, and ALDH2 or ADH2 genotype. The ADH22/22 group had significantly higher odds ratios for systolic blood pressure (odds ratio, 3.1), triglycerides (odds ratio, 3.2), and uric acid (odds ratio, 4.1) compared with the ADH21/21 group. In contrast, no significant differences were observed between the two ALDH2 groups for any of the valuables analyzed.


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  		Table 3. Prevalences and odds ratios for the participants for whom values for the variables listed were in the highest one third.


   Discussion
Top
 Abstract
 Introduction
 Participants and Methods
 Results
 Discussion
References
 
The present study indicates that the ADH2 genotype, but not the ALDH2 genotype, might be involved in individual variations in blood pressure, triglycerides, and uric acid in relation to alcohol consumption. This is the first report showing a relationship between ADH2 genotype and risk factors for CHD. The findings suggest that the most appropriate amount of drinking for cardioprotection differs among individuals with different ADH2 genotypes. The distribution of the ADH2 genotypes is reported to be quite different in Japanese, Chinese, African Americans, and Brazilians (12). This may be one of the reasons for the considerable inconsistency regarding the amount of alcohol consumption that provides cardioprotective effects (5). The ratio of ADH to ALDH is believed to play a more important role in alcohol metabolism in the case of light to moderate alcohol consumption (21). At higher consumption, other pathways may play a more important role. Because the drinkers in the present study were moderate to heavy drinkers, it will be important to examine light to moderate drinkers. Furthermore, it will be interesting to examine the relationship between the ADH2 genotype and other risk factors for CHD, such as insulin resistance, platelet aggregation, fibrinogen, and plasminogen activator inhibitor 1.

It is not clear at present what mediates the association between the ADH2 genotype and such responses to alcohol drinking. Our results support those of reports showing no association between blood pressure and the ALDH2 genotype (22)(23). Because the ALDH2 genotype largely influences blood acetaldehyde concentrations after alcohol consumption (24), the presence of acetaldehyde in the blood is probably not the chief cause of increases in those risk factors associated with drinking. In contrast, the involvement of the ADH2 genotype in individual variations in such responses to drinking suggests an important role of the ADH2 isoenzymes in differences in alcohol metabolism. It is reported that there is a two- to threefold variation in alcohol elimination rate (25)(26)(27). A study in mono- and dizygotic twins showed that approximately one-half of this variability is attributable to genetic factors (28). The only ADH genes exhibiting polymorphisms are ADH2 and ADH3, and the kinetic properties show only a small difference among ADH3 isoenzymes (12). Therefore, it is reasonable to assume that the ADH2 genotype accounts for the differences in alcohol elimination rates. Although drinkers with the ADH22/22 genotype who had a high alcohol elimination rate had higher triglycerides and rGTP concentrations, this can not be attributed to acetaldehyde accumulated in the blood, as described above; it may be caused by metabolic changes accompanying the oxidation of alcohol to acetaldehyde by ADH, such as an increase in the NADH:NAD ratio (29), increases in the concentrations of reactive oxygen species (30)(31), and other factors, although there is no evidence against this hypothesis.

Our study participants who were moderate to heavy drinkers showed significantly higher frequencies of the ADH21/21 and ALDH21/21 genotypes and lower frequencies of the ALDH21/22 and ALDH22/22 genotypes compared with Japanese, including drinkers and nondrinkers, who participated in other studies (17)(32)(33). This is probably because our study groups included only drinkers. This idea is supported by studies involving individuals with alcoholism or alcoholic liver diseases, who showed frequencies of the ADH2 and ALDH2 genotypes similar to those observed in the present study (34)(35)(36). A high prevalence of the ADH21/21 genotype in drinkers also suggests that the ADH genotype plays an important role in alcohol metabolism after heavy drinking, although one published study showed that the ADH genotype does not influence the alcohol elimination rate after moderate drinking (24).

In conclusion, we showed that the ADH2 genotype influences the responses of blood pressure, triglycerides, and uric acid to alcohol consumption. We do not believe that this influence was mediated by the acetaldehyde concentration in the blood because the ALDH2 genotype did not influence those variables. The present findings suggest that the amount of alcohol intake that provides cardioprotective effects varies on an individual basis. Further studies in a larger population, including drinkers and nondrinkers, will be needed to confirm that the findings obtained from the present study are correct and that the ADH genotype does not influence the risk factors for CHD in nondrinkers.


   Acknowledgments
 
This study was supported in part by grants from the Health Science Center Foundation, the Smoking Research Foundation, and the Clinical Pathology Research Foundation of Japan.


   Footnotes
 
1 Nonstandard abbreviations: CHD, coronary heart disease; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; rGTP, {gamma}-glutamyl transpeptidase; and Hb, hemoglobin.


   References
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 Abstract
 Introduction
 Participants and Methods
 Results
 Discussion
 References
 

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