Effect of ALDH 2 Genetic Polymorphism on the Adaptive Change in Alcohol Metabolism Due to Continuous Moderate Alcohol Consumption in Humans

Few studies have assessed the difference in adaptive changes of alcohol metabolism in the case of chronic alcohol consumption pertaining to the genetic polymorphism of aldehyde dehydrogenase 2 (ALDH2) in humans. To evaluate the influences of ALDH2 genotypes on changes in alcohol metabolism due to continuous alcohol intake, we conducted an intervention study by setting a continuous drinking period between two abstinence periods. The subjects in this study comprised 20 25-year-old males, including 15 males carrying ALDH2*1/*1 and 16 carrying ALDH2*1/*2 genotypes. Following the abstinence period of 4 weeks (from day 1 to day 28), all subjects drank commercially available beer (500 ml) every evening for 6 weeks (from day 30 to day 71) and subsequently abstained from drinking again for 4 weeks (from day 73 to day 100). The next morning, after the end of each period, drinking tests (DTs) were performed on each subject (DT1 on day 29, DT2 on day 72, and DT3 on day 101) to examine alcohol metabolism. The subjects drank shochu (a distilled alcoholic beverage), with an ethanol dose of 0.32 g/kg, within 20 minutes after overnight fasting. The alcohol elimination rate in subjects with ALDH2*1/*1 genotype was significantly higher during DT2 after the drinking period as compared with those at both DT1 and DT3 after the abstinence periods, whereas the elimination rate in subjects with ALDH2*1/*2 genotype did not change significantly during 3 DTs. However, blood acetaldehyde levels significantly decreased in subjects with both ALDH2 genotypes during DT2 as compared with those during DT1. The physiological subjective responses to alcohol also significantly decreased during DT2 in subjects with ALDH2*1/*2 genotype. Moreover, serum lipids, gamma-glutamyltransferase (GGT), and uric acid Corresponding author. Present address: Sankyo Labo Service Corporation, Inc., Tokyo, Janpan.


Introduction
Previous studies have reported a U-shaped relationship between alcohol consumption and mortality [1]- [5] that indicates health benefits of moderate alcohol consumption as compared with its complete abstinence or heavy alcohol use.The basic knowledge about the effects of chronic alcohol consumption in different amounts on alcohol metabolism is also essential to understand physiological effects of alcohol consumption.Moreover, numerous animal studies have demonstrated an increase in alcohol metabolism following chronic alcohol intake [6]- [12].Some human studies also advocate the results of these animal studies.Misra et al. [13] reported that chronic consumption of ethanol for one month under specific dietary conditions results in acceleration in the clearance of ethanol from blood in both alcoholics and nonalcoholics.Ethanol is rapidly oxidized to acetaldehyde and acetate in a two-step process involving alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH).Furthermore, in alcoholics, an elevation of blood acetaldehyde levels was often observed [14], because of the acceleration of alcohol metabolism or the decrease of low-Km aldehyde dehydrogenase (ALDH2) activity in the liver following excessive alcohol intake [15] [16].Also in healthy subjects, the activity of ALDH2 is important to regulate the effects of alcohol consumption.Asians, who genetically lack ALDH2 activity, show facial flushing or a sense of discomfort due to remarkably high concentrations of blood acetaldehyde after drinking.Phenotype of ALDH2 deficiency (known as Asian flush) has been related to ALDH2*1/*2 and ALDH2*2/*2 genotypes, and leads to reduced alcohol consumption in these subjects.Therefore, ALDH2*2 allele is one of the important factors that prevents humans from excessive drinking and hence from developing alcoholism.However, there has been only limited information about the influences of continuous moderate consumption of alcohol on adaptive changes in ethanol and acetaldehyde metabolisms in subjects with different ALDH2 genotypes.
In this study, we aimed to clarify how ALDH2 polymorphism affects alcohol metabolism during continuous moderate alcohol consumption.For this purpose, we conducted an intervention study that sets continuous beer drinking and abstinence periods as well as examining blood samples of the subjects.

Subjects and Protocols
This study was proposed and approved by the Shiba Palace Clinic Ethics Committee.Informed consent was obtained from 82 adult Japanese men aged 20 -25 years, who participated in baseline screening.ALDH2 genotypes included ALDH2*1/*1 in 46 subjects, ALDH2*1/*2 in 26 subjects, and ALDH2*2/*2 in 10 subjects.Inclusion criteria for the intervention study were as follows: 1) 20 -25 years of age, 2) healthy males, free of chronic illness such as alcoholism, and 3) the ability to drink beer and conduct abstinence according to the protocol.Subsequently, 72 men were recruited, and 16 subjects were randomly selected from ALDH2*1/*1 and ALDH2* 1/*2 groups, respectively.ALDH2*2/*2 subjects were excluded because they have a genetic intolerance to alcohol.Analyses were finally performed using data from 15 ALDH2*1/*1 and 16 ALDH2*1/*2 carriers because one ALDH2*1/*1 subject dropped out before the beginning of the study.

Study Design
All subjects abstained from alcohol for 4 weeks (from day 1 to day 28) in period 1 (Figure 1).During the subsequent period (period 2), subjects drank 500 ml of commercially available beer every day at dinnertime for 6 weeks (from day 30 to day 71).Finally, subjects abstained again from alcohol consumption for 4 weeks (from day 73 to day 100) (period 3).Drinking tests (DTs) were conducted on days 29 (DT1), 72 (DT2), and 101 (DT3) and changes in alcohol metabolism and physiological situations due to continuous drinking or abstinence were assessed.Subjects refrained from eating for at least 10 h prior to each DT, and after measurements of weight, water content of the body, blood pressure and heart rate, they were given 0.32 g of ethanol/kg body weight in the form of commercially available shochu (distilled alcoholic beverage), which was diluted to 16% (v/v) alcohol in water.Blood samples were then collected from the cubital vein before and after alcohol ingestion on each experimental day.

Analytical Techniques
Blood samples were mixed with 0.5 N perchloric acid and the supernatants were prepared by centrifugation.Subsequently, blood ethanol and acetaldehyde concentrations were measured using a head space gas chromatography according to the procedures described by Okada and Mizoi [17], and blood acetate concentration was measured using a high-performance liquid chromatography (Shimadzu organic acid determination system; Shi-madzu, Japan) fitted with an ion-exclusion column and a conductivity detector [18].Blood ethanol elimination rate (β value; mg/ml/h) was calculated from the slope of pseudolinear decline in the blood ethanol concentration curve.
Serum gamma-glutamyltransferase (GGT, U/l), uric acid (mg/dl), and lipid concentrations (mg/dl) (triglyceride, total-cholesterol, LDL-cholesterol, HDL-cholesterol) were determined by the routine measurements in a clinical laboratory after fasting and before the start of each DT.Atherogenic index was calculated using the following formula: (total-cholesterol − HDL-cholesterol)/HDL-cholesterol [19].To assess subjective sensations to alcohol, subjects were required to select a score from 0 -10 grading the sensations of face flush or changed heart beats before and after alcohol consumption in each DTs.

Statistical Analysis
Statistical analyses were conducted using Dr. SPSS II software (SPSS Inc.).Data were presented as mean ± SD.Maximum blood acetaldehyde and acetate levels at DTs 2 and 3 were expressed relative to those of DT1.Differences between DTs were identified using repeated measures ANOVA and the Scheffe's post-hoc test, and were considered significant when p < 0.05.Different letters in the figures indicate statistically significant differences between means (Scheffe's test, p < 0.05).Differences between ALDH*1/*1 and ALDH2*1/*2 groups were identified using Student's t-test and were considered significant when p < 0.05.

Subject Characteristics
Recruited subjects who completed the whole process (periods 1 -3 and DTs 1 -3) included 15 males with ALDH2*1/*1 genotype and 16 with ALDH2*1/*2 genotype.As shown in Table 1, BMI and water content of the body did not differ between 3 DTs in ALDH2*1/*1 subjects, whereas BMI was greater at DTs 2 and 3 than at DT1 in ALDH2*1/*2 subjects.BMI and body water contents did not differ between the genotype groups (Table 1).Blood pressure (systolic, diastolic) did not differ between DTs in subjects of either ALDH2 genotype (data not shown).

Blood Ethanol Concentration and Ethanol Elimination Rate
Maximal blood ethanol concentration (BAC max) and the blood elimination rate (β value) were calculated using blood ethanol concentration curves at each DT (Table 2 and Figure 2).BAC max showed no differences between ALDH2*1/*1 and *1/*2 groups, as well as between DTs.However, whereas β value was significantly higher during DT2 than at DTs 1 and 3 in subjects with ALDH2*1/*1, it did not vary between DTs in subjects carrying ALDH2*1/*2 genotype.Particularly, β value was higher in ALDH2*1/*1 than in ALDH2*1/*2 subjects during DT2.

Blood Acetaldehyde and Acetate Concentrations
In accordance with the results of previous reports [20], blood acetaldehyde levels were significantly higher in subjects with ALDH2*1/*2 than in subjects with ALDH2*1/*1 during all DTs (Data not shown).However, the maximal blood acetaldehyde concentrations (BACH max) were significantly less during DTs 2 and 3 than during DT1 in both ALDH2 genotype groups (Figure 3).Consequently, the maximal blood acetate concentrations (BACE max) were significantly increased during DTs 2 and 3 in subjects with ALDH2*1/*2, and were similarly but insignificantly increased in subjects with ALDH2*1/*1 (repeated ANOVA; p = 0.069).Moreover, BACE during DT3 was significantly lower in subjects with ALDH2*1/*2 than in subjects with ALDH2*1/*1 (Data not shown).

Subjective Physiological Responses to Alcohol and Blood Biochemistry Markers
Sensations of face flush and change in heart beat were recorded after 1 h post alcohol consumption at each DT (Figure 4), because blood ethanol, acetaldehyde and acetate levels were close to maximal at this time.Sensation scores were very low in the ALDH2*1/*1 group and did not differ significantly between DTs.In comparison, both sensation scores were very high in subjects with ALDH2*1/*2 and were significantly lower at DTs 2 and 3 than at DT1. "Face numbness", "dizzy" and "warm cheeks" also indicated similar tendencies in subjects with ALDH2*1/*2 (data not shown).
To examine the influence of continuous drinking and abstinence on physiological conditions, the blood biochemistry markers (serum GGT, uric acid, triglyceride, total-cholesterol, LDL-cholesterol, HDL-cholesterol) and the atherogenic index were analyzed under fasting conditions before the start of each drinking test (Table 3).Although no abnormal values in biochemical markers were observed in any of the subjects, significant differences were identified between DTs in subjects with ALDH2*1/*1, whose GTT and uric acid levels increased during DT2, compared with those at DTs 1 and 3, whereas total-cholesterol and LDL-cholesterol decreased during DTs 2 and 3.In ALDH2*1/*2 subjects, only HDL-cholesterol levels increased during DT2.These changes of blood lipids resulted in falls of atherogenic index during DT2 compared with other DTs in both ALDH genotypes, though the baselines were different between two genotypes (repeated ANOVA).No blood biochemistry markers differed significantly between ALDH2 genotypes during all DTs.

Discussion
The mitochondrial enzyme ALDH2 is subjected to the genetic polymorphism of ALDH2 in Mongoloid popula-tions.The variant ALDH2*2 gene produces inactive ALDH2 with a Glu487Lys substitution, and is responsible for flushing symptoms after alcohol intake [21].Thus, the ALDH2*2 allele is an important factor that prevents from drinking excessively and hence from developing alcoholism [22].However, the influences of ALDH2 polymorphism on adaptations of ethanol and acetaldehyde metabolism to chronic moderate alcohol consumption remain poorly understood in normal subjects.Thus, in the present interventional study, alcohol metabolism was monitored across continuous beer drinking and abstinence periods, and the effects of ALDH2 polymorphism were investigated.ALDH2 polymorphism is known to affect the rate of alcohol metabolism [20] and the daily consumption of alcohol [23], which also in turn affects the rate of alcohol metabolism [24].Thus, alcohol metabolism was compared between subjects with ALDH2*1/*1 and ALDH2*1/*2 genotypes after a 4-week abstinence period, a subsequent 6-week drinking period, and then another 4-week abstinence period.
Previous studies showed that chronic alcohol consumption causes dose-dependent increase in alcohol elimination rates, reflecting the acceleration of alcohol metabolism [6]- [13].In this study, the consumption of 500 ml beer per day for 6 weeks increased β value in subjects with ALDH2*1/*1, indicating that continuous intake of alcohol induces adaptive increases in alcohol metabolism even when the consumption is moderate.In contrast, no such adaptive increases were observed in subjects with ALDH2*1/*2.The suppression of ADH activity by acetaldehyde has been demonstrated to result in partial inhibition of alcohol metabolism in both animal and kinetic studies [25] [26].Because ADH1 accounts for about 70% of systemic alcohol metabolism [27], resistance to adaptive changes in alcohol metabolism in ALDH2*1/*2 subjects may be due to their high levels of blood acetaldehyde, even under conditions of moderate drinking.Moreover, previous studies showed faster alcohol metabolism in subjects with ALDH2*1/*1 than in subjects with ALDH2*1/*2 [20].β values in this study were also greater in the subjects with ALDH2*1/*1 than in the subjects with ALDH2*1/*2 after continuous drinking.However, no difference was observed between the two genotypes after each abstinence period.It is also known that humans with the ALDH2*1/*1 genotype drink more in daily life than those with ALDH2*1/*2 genotype [28].Thus, the present data may suggest that differences in the rate of alcohol metabolism between two genotypes reflect differences in daily consumption of alcohol.Further studies are needed to assess the influence of ALDH2 polymorphism on alcohol metabolism.
Blood acetaldehyde concentrations were found to decrease following continuous moderate intake of alcohol in both subjects with ALDH2*1/*1 and ALDH2*1/*2.Moreover, these low levels were maintained after a second abstinence, leading to maintenance of high blood acetate levels.In agreement with these metabolic changes, subjective physiological responses to alcohol, as indicated by sensation scores on face flush and change in heart beats, decreased following continuous drinking, particularly in ALDH2*1/*2 carriers.These observations further indicate that the continuous moderate intake of alcohol increases the rate of acetaldehyde metabolism to lower blood acetaldehyde levels.In agreement, Tomita et al. [29] showed that although both low-and high-Km mitochondrial ALDHs were decreased by liquid diets containing 4% ethanol for one week in mice, they were adaptively increased after 4 weeks.
Few studies have assessed the influence of ALDH2 polymorphism on changes in body composition following continuous moderate consumption of alcohol.Our present data demonstrated that BMI and body water contents are increased after continuous beer consumption in subjects with ALDH2*1/*2, but not in subjects with ALDH2*1/*1.The increase in BMI in subjects with ALDH2*1/*2 may not reflect an increase in caloric intake because body water contents were also elevated in these subjects during the study.Persistent pharmacological stresses caused by higher acetaldehyde levels might trigger such adaptive phenomena in subjects with ALDH2*1/*2, although detailed mechanisms remain unknown.
Positive influences of moderate drinking on health remain controversial, although it is accepted that chronic excessive alcohol consumption increases various disease risks.We evaluated the health influences of continuous moderate beer consumption by analyzing serum GGT, uric acid, and lipid contents and the atherogenic index in healthy subjects in this study.Interestingly, changes in these markers following continuous beer consumption differed between subjects with ALDH2*1/*1 and ALDH2*1/*2 genotypes.GGT and uric acid were more affected in subjects with ALDH2*1/*1 than ALDH2*1/*2 by the continuous drinking.Although the atherogenic index (TC − HDL-C)/(HDL-C) decreased following continuous drinking in both ALDH2 genotypes, the causality of the decrease of atherogenic index differed between the genotype groups.LDL-cholesterol decreased together with TG contents in subjects with ALDH*1/*1, whereas HDL-cholesterol increased in subjects with ALDH2*1/*2.It cannot be denied that the difference in the baseline may have reflected differences in caloric intake between the genotype groups during the study period, because no calorie surveys were performed in the present study.Nonetheless, our study demonstrated that influences of continuous moderate drinking are different between ALDH2*1/*1 and ALDH2*1/*2 carriers, not only on alcohol metabolism but also on blood biochemistry markers.The differences of these adaptive changes between two genotypes are mainly due to the differences in their blood acetaldehyde levels.It is considered that the increases in BMI and HDL-cholesterol observed only in ALDH2*1/*2 subjects are the results of adaptation to the higher levels of blood acetaldehyde.On the other hand, acetaldehyde metabolism was adaptively accelerated, regardless of the different genotype of ALDH2, and consequently, subjective physiological responses to acetaldehyde decreased especially in ALDH2*1/*2 subjects.However, alcohol metabolism is accelerated only in ALDH2*1/*1 subjects, but not in ALDH2*1/*2 subjects.Several blood biochemistry markers, such as GGT, uric acid, LDL-cholesterol and total-cholesterol, were also adaptively changed only in ALDH2*1/*1 subjects.These adaptive changes observed only in ALDH2*1/*1 subjects may be disturbed and become unclear by the high levels of blood acetaldehyde in ALDH2*1/*2 subjects.Further investigations are required to assess the influence of ALDH2 polymorphisms on physiological and pathological effects of chronic alcohol consumption.

Figure 1 .
Figure 1.Flowchart of participant selection and the intervention study.

Figure 3 .Figure 4 .
Figure 3.The maximal blood acetaldehyde (BACH max) or acetate concentrations (BACE max) after ethanol ingestion (0.32 g/kg) at each drinking test.(A) BACH max; (B) BACE max.Results are expressed the means (relative value) and standard deviation.Relative value is the ratio of the measured value at DT2 or 3 to the measured value at DT1. Means with different subscripts are significantly different at p < 0.05 by repeated ANOVA at each DT.

Table 1 .
Changes in body composition status during the abstinence and the drinking periods.
Values are means (standard deviation).Means with different subscripts are significantly different at p < 0.05 in the each index.

Table 2 .
The maximal blood ethanol concentrations (BAC max) after ethanol ingestion (0.32 g/kg) at each drinking test (DT).
Figure 2. Changes in ethanol disappearance rate from the blood (β value) after ethanol ingestion (0.32 g/kg) at each drinking test (DT).Results are expressed the means and standard deviation (Mean ± SD).Means with different subscripts are significantly different at p < 0.05 by repeated ANOVA in each DT.*p < 0.05 by Student's t-test between two ALDH2 genotypes at each DT.

Table 3 .
Changes in serum GGT, uric acid, lipid concentrations and atherogenic index during the abstinence and the drinking period.Values are means (standard deviation).Means with different subscripts are significantly different at p < 0.05 in the each index.