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Lack of association between ADH3 polymorphism, alcohol intake, risk factors and carotid intima-media thickness
Atherosclerosis 184 (2006) 397–403
Lack of association between ADH3 polymorphism, alcohol intake, risk factors and carotid intima-media thickness P. Marques-Vidal a , C. Bal Dit Sollier b , L. Drouet b , H. Boccalon c , J.B. Ruidavets a , J. Ferri`eres a,∗ a
INSERM U558, Facult´e de M´edecine, 37 All´ees Jules Guesde, 31073 Toulouse cedex, France b Department of Hematology, Hˆ opital Lariboisi`ere, Paris, France c Department of Vascular Medicine, CHU Rangueil, Toulouse, France Received 7 January 2005; received in revised form 28 April 2005; accepted 4 May 2005 Available online 6 June 2005
Abstract Objective: We assess the relationships between alcohol dehydrogenase 3 (ADH3) polymorphism, alcohol consumption and cardiovascular risk factor levels. Methods: In a representative population sample from Southwestern France (614 men, 567 women, age 49.7 ± 8.5 years), alcohol intake was assessed by questionnaire. Results: Alcohol consumption was significantly related with higher levels of total and HDL cholesterol, triglycerides, apolipoprotein A-I in men and with higher levels of HDL cholesterol in women. Also, an inverse relationship between alcohol consumption and intima-media thickness was found in men. Conversely, in both genders, no differences were found between ADH3 genotypes regarding all cardiovascular risk factors studied and carotid intima-media thickness. Also, in both genders, no significant ADH3 × alcohol interaction was found for all variables, and further adjustment on age, body mass index, educational level, smoking status or after excluding subjects on hypolipidemic or antihypertensive drug treatment did not change the results. Conclusion: We found no interaction between the ADH3 polymorphism and alcohol intake on cardiovascular risk factor levels and atherosclerotic markers in Southwestern France. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Alcohol dehydrogenase; Polymorphism; Population sample; HDL; Alcohol consumption
1. Introduction Moderate alcohol consumption has been shown to be protective against cardiovascular disease [1–4]. The major enzymes of alcohol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). ADH is a dimeric protein with two subunits and six ADH genes have been described, of which two (ADH2 and ADH3) are polymorphic. The ADH2 polymorphism has been shown to be related to cardiovascular risk factors, namely systolic blood pressure [5,6] and triglycerides [5]. The ADH3 polymor∗
Corresponding author. Tel.: +33 5 61 52 18 70; fax: +33 5 61 32 33 32. E-mail address: [email protected] (J. Ferri`eres).
0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.05.003
phism has two alleles, gamma 1 and gamma 2 (␥1 and ␥2), and pharmacokinetic studies have shown that the homodimeric ␥1 isoenzyme has a 2.5-fold higher maximal velocity of ethanol oxidation than the homodimeric ␥2 isoenzyme [7]. Further, it has been shown that moderate drinkers who are homozygous for the slow-oxidizing ADH3 allele have higher HDL levels and a substantially decreased risk of myocardial infarction [8]. Still, those relationships between ADH polymorphisms and cardiovascular disease have been questioned [9–11], and it is currently unclear whether ADH polymorphisms do interact with alcohol consumption regarding the levels of cardiovascular risk factors or cardiovascular disease. France is characterized by a low incidence of myocardial infarction and a moderate-high intake of alcohol but, to our knowledge,
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no study on the relationship between ADH3 polymorphism and alcohol consumption on the levels of cardiovascular risk factors has ever been conducted. Thus, we used the data from the last MONICA (multinational monitoring of trends and determinants in cardiovascular diseases) population survey conducted in Southwestern France to assess the relationships between ADH3 polymorphisms, alcohol intake and cardiovascular risk factor levels. The relationships between ADH3 polymorphisms, alcohol intake and intima-media thickness, an indicator of preclinical atherosclerosis, were also assessed. 2. Population and methods 2.1. Population sample The WHO–MONICA Project is a study that monitors deaths due to coronary heart disease, myocardial infarction (MI), coronary care and risk factors in men and women aged 35 to 64 years [12–14]. It consists of 39 MONICA Collaborative Centers (MCC) in 26 countries. Each MCC is in charge of carrying out two or three population surveys on cardiovascular risk factors in the beginning and at the end of the 10-year, and possibly in the middle of the study period. The sampling strategy was to have representative probability samples within each sex and 10-year age group, at least for the age range 35–64 years. The number of eligible subjects asked to participate was 50% higher than the required number in order to obtain the necessary quota of 200 persons for each sex and 10-year age group (allowing for subjects refusing to participate or not participating for any other reason). The informed consent to participate in the study was obtained from the subjects before the survey. The last Toulouse MONICA survey started in December 1994 and ended in July 1997 and concerned both genders; participation rates were 67% for men and 59% for women. 2.2. Data collection Subjects were advised to restrain from physical exercising, smoking, eating or drinking anything other than water for at least one hour prior to the screening visit. Screening included standardized questionnaires on personal data and measurements of body height and weight and blood pressure. Body mass index (BMI) was calculated as weight (kg) height (m)−2 . Subjects were considered as smokers if they currently smoked, and the average amount of cigarettes smoked per day was assessed for each smoker. Subjects were considered as hypertensive if their systolic blood pressure was >160 mmHg and/or their diastolic blood pressure was >95 mmHg and/or they reported being on antihypertensive drug treatment. 2.3. Biological measurements Lipids were measured on plasma EDTA plasma samples using automated enzymatic assays (Boehringer, Mannheim,
Germany). External quality control by the MONICA reference laboratory indicated no relevant deviation. Subjects were considered as dyslipidaemic if their total cholesterol was >6.5 mmol/L and/or they reported being on hypolipidaemic drug treatment. 2.4. Intima-media thickness measurement High-resolution B-mode ultrasonography was used to detect atherosclerotic plaques in carotid and femoral arteries and to measure common carotid IMT. An ATL UM9 system (Advanced Technology Laboratories Ultramark 9 High Definition Imaging) was used with a 7.4 MHz transducer. IMT was defined as the distance between the media ± adventitia interface and the lumen ± intima interface, avoiding the sites of plaque [15,16]. IMT was measured on the right and left common carotid arteries, on the far wall exclusively: three points at two locations on each artery, proximal and middle. Thus, 12 points were determined. The mean values at the 12 sites were combined to generate an overall mean value for common carotid IMT. In 26 subjects submitted to a second IMT assessment, the differences between the mean values of the measurements performed during the two examinations were small (−0.007 mm for the right common carotid artery and −0.022 mm for the left common carotid artery) and the intra-class correlation coefficients between the two measurements were 0.64 (p < 0.001) for the right common carotid artery and 0.53 (p < 0.001) for the left common carotid artery. 2.5. Alcohol consumption Alcohol consumption was assessed by a validated questionnaire that recorded the subjects mean consumption (in units) of wine, beer, cider and spirits for each day of the week [17,18]. Intake of alcohol (expressed in ml of pure ethanol/week) was estimated from the average number of millilitres of ethanol in one unit of each type of alcoholic beverage: wine (10% or 12% alcohol, v/v) = 12 cl serving; beer (5% alcohol) = 12 cl serving; beer (6 or 8% alcohol) = 25 or 33 cl serving; cider (5% alcohol) = 12 cl serving; spirits (20% or 40% alcohol) = 2 or 6 cl serving. For each gender, alcohol consumption was further classified into three groups: teetotalers, below and above the median. 2.6. Alcohol dehydrogenase genotyping DNA was obtained from leukocytes by salting out procedure, and ADH3 genotyping was performed in duplicate for each subject using polymerase chain reaction. Subjects were classified into three categories according to the presence of the ␥2 (slow) allele: 11 homozygotes, 12 heterozygotes and 22 heterozygotes. The alcohol dehydrogenase 3 exon eight polymorphism was determined as followed: A fragment of 130 bp in the ADH3 gene was amplified by polymerase chain reaction (PCR) using the following primers (forward 5 -GCTTTAAGAGTAAATCTGTCCCC-
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3 , reverse 5 -AATCTACCTCTTTCCAGAGC-3 ) at the annealing temperature of 55 ◦ C. The PCR products were analysis after digestion by the restriction endonuclease SSpI (New England Biolabs), and the fragments were separated by electrophoresis onto a 3% agarose gel. Wild type (␥1 alle) was cut into 67 and 63 bp fragments, whereas the ␥2 allele, associated a slower metabolism of alcohol, was uncut. 2.7. Statistical analysis Statistical analysis was conducted using SAS Enterprise Guide v.2.05 (SAS Institute, Cary, NC, USA). Results were expressed as means or as numbers of subjects and (percentage) or as mean ± standard deviation (S.D.). Bivariate statistical comparisons were assessed by Analysis of variance (ANOVA) or by χ2 . Multivariate statistical analyses were conducted using the general linear model (Proc. GLM) of SAS. Statistical significance was considered for p < 0.05.
3. Results 3.1. Toulouse MONICA survey subjects’s characteristics Overall, 1,181 subjects (614 men, 567 women, age 49.7 ± 8.5 years) were assessed. Their clinical and biological
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characteristics are summarized in Table 1. Men had higher body mass index, blood pressure levels, total cholesterol, triglyceride and LDL cholesterol levels than women, whereas women had higher HDL and apolipoprotein A-1 levels than men. Men were also more frequently alcohol drinkers, and alcohol consumption was higher among male drinkers than their female counterparts. Conversely, no differences were found for age, smoking status, educational level or the distribution of the ADH3 polymorphisms. Prevalence of hypertension and dyslipidaemia were 20.4 and 35.0% in men and 18.5 and 29.7% in women, respectively. Also, 93 men and 70 women reported being on hypolipidaemic drug treatment, and 73 men and 81 women reported taking antihypertensive medication. 3.2. ADH3 polymorphism and cardiovascular risk factor levels The frequencies of the ADH3 alleles were 65% for the ␥1 and 35% for the ␥2 allele in men and 64% for the ␥1 and 36% for the ␥2 in women. The distribution of the main cardiovascular risk factor levels according to the ADH3 polymorphisms is summarized separately for each gender in Table 2. In both genders, no significant differences between ADH3 polymorphisms were found for all parameters studied, and excluding subjects on hypolipidaemic or antihypertensive drug treatment did not change the results (not shown).
Table 1 Clinical and biological characteristics of the Toulouse MONICA survey, by gender FDF
Men (n = 614)
Women (n = 567)
p
Age (years) Body mass index (kg/m2 )
49.7 ± 8.6 26.2 ± 3.7
49.7 ± 8.6 24.7 ± 4.5
0.90 <0.001
Smoking status (%) current former and never
151 (24.6) 463 (73.4)
120 (21.2) 447 (78.8)
0.16
Educational level (years) ≤6 6 <. ≤ 12 >12 Alcohol drinkers (%) Alcohol consumption among drinkers (g/week) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mmol/L) Triglycerides (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Apolipoprotein A-1 (g/L) Apolipoprotein B (g/L)
20 (3.3) 329 (53.6) 265 (43.1) 480 (78.2) 306 ± 237 135 ± 16 82 ± 10 5.89 ± 1.01 1.36 ± 0.88 1.29 ± 0.33 4.00 ± 0.93 1.56 ± 0.22 1.26 ± 0.27
14 (2.5) 313 (55.2) 240 (42.3) 298 (52.6) 125 ± 115 127 ± 19 77 ± 10 5.77 ± 1.02 1.00 ± 0.49 1.65 ± 0.46 3.67 ± 0.95 1.73 ± 0.27 1.13 ± 0.25
ADH3 polymorphism (%) −11 −12 −22
263 (42.8) 273 (44.5) 78 (12.7)
236 (41.9) 252 (44.8) 75 (13.3)
0.66 <0.001 <0.001 <0.001 <0.001 0.05 <0.001 <0.001 <0.001 <0.001 <0.001
0.93
Results are expressed as number of subjects and percentage or as mean ± S.D. Comparisons were performed by χ2 or Student’s t-test. Triglycerides were log-transformed before comparison. Alcohol consumption was assessed among drinkers only, and comparisons were performed by non-parametric test.
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Table 2 Cardiovascular risk factor levels and carotid intima-media thickness according to ADH3 genotype, by gender p
Men 11 (n = 263) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mmol/L) Triglycerides (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Apolipoprotein A-I (g/L) Apolipoprotein B (g/L) Carotid intima-media thickness (mm)
135 83 5.85 1.37 1.29 3.96 1.57 1.25 0.60
± ± ± ± ± ± ± ± ±
16 10 0.94 0.91 0.34 0.88 0.23 0.25 0.09
12 (n = 273) 134 82 5.90 1.33 1.29 4.02 1.56 1.26 0.61
± ± ± ± ± ± ± ± ±
16 10 1.03 0.90 0.32 0.96 0.21 0.27 0.12
22 (n = 78) 135 82 5.99 1.40 1.29 4.06 1.56 1.29 0.62
± ± ± ± ± ± ± ± ±
16 11 1.16 0.70 0.38 1.03 0.24 0.31 0.11
0.69 0.61 0.53 0.48 0.98 0.63 0.94 0.61 0.45
p
Women 11 (n = 236)
12 (n = 252)
22 (n = 75)
± ± ± ± ± ± ± ± ±
127 ± 19 77 ± 11 5.81 ± 1.10 0.99 ± 0.50 1.65 ± 0.45 3.72 ± 1.05 1.73 ± 0.26 1.15 ± 0.27 0.58 ± 0.08
127 ± 21 76 ± 10 5.60 ± 0.86 0.98 ± 0.53 1.56 ± 0.37 3.59 ± 0.82 1.69 ± 0.22 1.08 ± 0.23 0.57 ± 0.07
128 77 5.76 1.00 1.68 3.63 1.76 1.12 0.57
18 10 0.95 0.46 0.48 0.88 0.28 0.23 0.08
0.66 0.84 0.29 0.69 0.17 0.45 0.10 0.08 0.39
Results are expressed as mean ± S.D. Statistical analysis by ANOVA.
3.3. ADH3polymorphism, alcohol consumption and cardiovascular risk factor levels Multivariate adjustment for age, body mass index, educational level and smoking status (and menopausal status for women) did not lead to any significant interaction in both genders, and also after excluding subjects on hypolipidaemic drug treatment (not shown). The effect of alcohol consumption (in tertiles) on the distribution of the main cardiovascular risk factor levels
according to ADH3 polymorphisms is summarized separately for each gender in Tables 3a and 3b. Although alcohol consumption was significantly related with higher levels of total and HDL cholesterol, triglycerides, apolipoprotein AI and lower levels of intima-media thickness in men and with higher levels of HDL cholesterol in women, no significant ADH3 × alcohol interactions were found for all variables studied, and similar results were obtained when alcohol consumption was treated as a continuous variable, when the analysis was restricted to drinkers or when subjects
Table 3a Cardiovascular risk factor levels according to ADH3 genotype and alcohol consumption, men Alcohol Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Total cholesterol (mmol/L)
Triglycerides (mmol/L)
HDL cholesterol (mmol/L)
LDL cholesterol (mmol/L)
Apolipoprotein A-I (g/L)
Apolipoprotein B (g/L)
Carotid intima-media thickness (mm)
11 (n = 263)
12 (n = 273)
22 (n = 78)
p
Non-drinkers <246 g/week ≥246 g/week
133 ± 2 135 ± 2 137 ± 2
133 ± 2 132 ± 2 136 ± 2
133 ± 4 133 ± 3 136 ± 3
0.98
Non-drinkers <246 g/week ≥246 g/week
81 ± 1 83 ± 1 83 ± 1
81 ± 1 81 ± 1 83 ± 1
78 ± 3 82 ± 2 83 ± 2
0.82
Non-drinkers <246 g/week ≥246 g/week
5.70 ± 0.13 5.86 ± 0.10 5.92 ± 0.10
5.75 ± 0.13 5.78 ± 0.10 6.10 ± 0.10
5.42 ± 0.26 6.04 ± 0.21 6.18 ± 0.16
0.35
Non-drinkers <246 g/week ≥246 g/week
1.29 ± 0.11 1.27 ± 0.08 1.54 ± 0.09
1.24 ± 0.11 1.23 ± 0.08 1.48 ± 0.08
1.13 ± 0.23 1.24 ± 0.18 1.60 ± 0.14
0.43
Non-drinkers <246 g/week ≥246 g/week
1.22 ± 0.04 1.29 ± 0.03 1.33 ± 0.03
1.21 ± 0.04 1.30 ± 0.03 1.34 ± 0.03
1.21 ± 0.09 1.46 ± 0.07 1.22 ± 0.05
0.07
Non-drinkers <246 g/week ≥246 g/week
3.95 ± 0.12 4.00 ± 0.09 3.92 ± 0.10
3.98 ± 0.12 3.94 ± 0.09 4.13 ± 0.09
3.69 ± 0.24 4.02 ± 0.19 4.23 ± 0.15
0.31
Non-drinkers <246 g/week ≥246 g/week
1.49 ± 0.03 1.58 ± 0.02 1.61 ± 0.02
1.49 ± 0.03 1.56 ± 0.02 1.61 ± 0.02
1.46 ± 0.06 1.61 ± 0.04 1.56 ± 0.03
0.58
Non-drinkers <246 g/week ≥246 g/week
1.23 ± 0.03 1.24 ± 0.06 1.28 ± 0.03
1.24 ± 0.03 1.23 ± 0.03 1.31 ± 0.03
1.20 ± 0.07 1.23 ± 0.05 1.36 ± 0.04
0.68
Non-drinkers <246 g/week ≥246 g/week
0.58 ± 0.01 0.61 ± 0.01 0.61 ± 0.01
0.60 ± 0.01 0.58 ± 0.01 0.63 ± 0.01
0.60 ± 0.03 0.61 ± 0.03 0.64 ± 0.02
0.26
Results are expressed as mean ± S.D. The p column refers to the test of the ADH3 × alcohol interaction factor.
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Table 3b Cardiovascular risk factor levels according to ADH3 genotype and alcohol consumption, women Alcohol Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Total cholesterol (mmol/L)
Triglycerides (mmol/L)
HDL cholesterol (mmol/L)
LDL cholesterol (mmol/L)
Apolipoprotein A-I (g/L)
Apolipoprotein B (g/L)
Carotid intima-media thickness (mm)
11 (n = 236)
12 (n = 252)
22 (n = 75)
p
Non-drinkers <96 g/week ≥96 g/week
128 ± 2 129 ± 3 127 ± 2
126 ± 2 128 ± 2 126 ± 2
131 ± 3 125 ± 4 120 ± 5
0.48
Non-drinkers <96 g/week ≥96 g/week
77 ± 1 77 ± 2 77 ± 1
77 ± 1 75 ± 1 77 ± 1
78 ± 2 74 ± 2 75 ± 3
0.91
Non-drinkers <96 g/week ≥96 g/week
5.69 ± 0.09 5.80 ± 0.15 5.84 ± 0.12
5.83 ± 0.10 5.82 ± 0.11 5.76 ± 0.13
5.54 ± 0.16 5.72 ± 0.21 5.54 ± 0.28
0.85
Non-drinkers <96 g/week ≥96 g/week
0.97 ± 0.04 0.92 ± 0.07 1.10 ± 0.06
1.05 ± 0.05 0.94 ± 0.05 0.96 ± 0.06
1.08 ± 0.08 0.88 ± 0.10 0.88 ± 0.13
0.24
Non-drinkers <96 g/week ≥96 g/week
1.64 ± 0.04 1.61 ± 0.07 1.79 ± 0.05
1.55 ± 0.04 1.65 ± 0.05 1.81 ± 0.06
1.54 ± 0.07 1.65 ± 0.09 1.50 ± 0.13
0.24
Non-drinkers <96 g/week ≥96 g/week
3.61 ± 0.09 3.77 ± 0.14 3.58 ± 0.11
3.81 ± 0.09 3.74 ± 0.10 3.53 ± 0.12
3.52 ± 0.15 3.68 ± 0.19 3.64 ± 0.26
0.65
Non-drinkers <96 g/week ≥96 g/week
1.73 ± 0.03 1.73 ± 0.04 1.83 ± 0.03
1.68 ± 0.03 1.74 ± 0.03 1.81 ± 0.03
1.66 ± 0.04 1.74 ± 0.05 1.67 ± 0.07
0.46
Non-drinkers <96 g/week ≥96 g/week
1.11 ± 0.02 1.13 ± 0.04 1.13 ± 0.03
1.19 ± 0.02 1.15 ± 0.03 1.09 ± 0.03
1.09 ± 0.04 1.08 ± 0.05 1.07 ± 0.07
0.35
Non-drinkers <96 g/week ≥96 g/week
0.56 ± 0.01 0.57 ± 0.01 0.59 ± 0.01
0.58 ± 0.01 0.60 ± 0.01 0.57 ± 0.01
0.57 ± 0.01 0.58 ± 0.02 0.56 ± 0.03
0.42
Results are expressed as mean ± S.D. The p column refers to the test of the ADH3 × alcohol interaction factor.
on hypolipidaemic or antihypertensive drug treatment were excluded (not shown). Finally, no relationship was found between ADH3 polymorphisms and the prevalence of hypertension or dyslipidaemia, or between ADH3 polymorphisms and the presence of antihypertensive or hypolipidaemic drug treatment (not shown).
4. Discussion Few studies have assessed the relationships between ADH3 polymorphisms, alcohol consumption and cardiovascular risk factors. This study enabled the assessment of those relationships in a representative population sample of Southwestern France, a region characterized by a low level of cardiovascular disease [15,19,20] and a regular intake of alcohol [17,18,21]. The frequencies of the ADH3 alleles were comparable to that observed in other caucasian populations [8] and were in Hardy–Weinberg equilibrium (p = 0.93 for men and p = 0.90 for women). In this study, no individual effect of the ADH3 polymorphism on blood pressure levels was found in both genders, and
those findings did not change after excluding subjects on antihypertensive drug treatment. Also, no relationship between hypertensive status and ADH3 polymorphism was found. Further, no significant interaction between the ADH3 polymorphism and alcohol consumption on blood pressure levels was found, either individually or after adjusting on age, body mass index, educational level and smoking status. Thus, our findings indicate that there is no interaction between ADH3 polymorphisms and alcohol consumption on blood pressure levels. A recent report indicated that subjects homozygous for the ␥2 allele and consuming at least one drink per day had the highest plasma HDL levels [8]. Conversely, and in agreement with other findings [10], no such relationship was found. Rather, subjects homozygous for the ␥2 allele tended to present with the lowest HDL cholesterol levels in both genders, and those findings did not change after multivariate adjustment or after excluding subjects on antihypertensive or hypocholesterolemic drugs. Thus, our data indicate that, at least in this population sample, ADH3 polymorphism does not influence the effect of alcohol consumption on HDL cholesterol levels. Alcohol consumption has been shown to increase HDL levels by various mechanisms: increase in lipoprotein lipase activity [22] or in synthesis and secretion
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of apolipoprotein A–I [23]; accelerated turnover of VLDL [24]; thus, it would be expected that the slow alcohol metabolizers (homozygotes for the ␥2 allele) would present with higher HDL cholesterol levels, which was not case. A possible explanation is the fact that the alcohol – HDL relationship might not be linear but concave, with a threshold effect [25]. Those findings could explain the increase in HDL levels in one study with a relatively low total alcohol consumption [8] compared to others [10], thus suggesting a threshold effect on the relationship between alcohol consumption and the ADH3 polymorphism on HDL levels. Still, further studies are needed to better assess this point. To our knowledge, no study has ever assessed the effect of alcohol and ADH3 polymorphism on carotid intima-media thickness, a marker of atherosclerosis. Our data indicate that ADH3 is not related to this variable, and also to other atherosclerotic risk factors such as LDL, apolipoprotein B or triglycerides. Thus, it is unlikely that this polymorphism might be a determinant for the development of atherosclerosis, as it has been suggested previously [8]. Also, conflicting results regarding the relationship between alcohol consumption and IMT have been published: negative (protective) association [26]; no association [27] or an association depending on the amount [28] or the pattern [29] of alcohol consumption. Thus, the exact relationships between alcohol consumption and atherosclerosis await further investigation. This study has several limitations: alcohol consumption was assessed by questionnaire, which might have led to reporting bias by some subjects; still, excluding subjects on sick leave did not change the results (not shown). Also, total alcohol consumption was divided into tertiles, which might have blurred the relationships between alcohol consumption, ADH3 polymorphism and cardiovascular risk factors; still, no significant interactions were found using alcohol consumption as a continuous variable, divided into quintiles or when the analysis was restricted to subjects who consumed less than the median (not shown). Further, mean alcohol consumption was significantly higher than reported in other studies [8]; still, comparing subjects with more than the equivalent of one drink per day to the other groups or restricting the analysis to postmenopausal women did not change the results (not shown). Finally, our sample size was comparable to or even higher than that of other studies on the relationships between ADH polymorphisms and cardiovascular risk factors [5,6,8–10]. In summary, we found no interaction between the ADH3 polymorphism and alcohol intake on cardiovascular risk factor levels and atherosclerotic markers in this representative population sample from Southwestern France.
Acknowledgements We would like to thank all the investigators of the Toulouse MONICA center for their invaluable contribution in the careful collection and validation of the data. We would
like to thank the “Institut National de la Sant´e et de la Recherche M´edicale” (INSERM), the “Direction G´en´erale de la Sant´e (DGS)”, “ONIVINS”, the “Fondation de France”, the “F´ed´eration Franc¸aise de Cardiologie”, the Parke-Davis and Bayer pharmaceuticals, and CERIN for their financial supports enabling this work.
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P. Marques-Vidal et al. / Atherosclerosis 184 (2006) 397–403 [17] Ruidavets J, Ducimeti`ere P, Arveiler D, et al. Types of alcoholic beverages and blood lipids in a French population. J Epidemiol Community Health 2002;56:24–8. [18] Ruidavets J, Teissedre P, Ferri`eres J, et al. Catechin in the Mediterranean diet: vegetable, fruit or wine? Atherosclerosis 2000;153: 107–17. [19] Ferri`eres J, Amouyel P, Arveiler D, et al. The MONICA Project: trends in coronary disease frequency and severity in France. In: Davignon J, Fruchart JC, Ordovas JM, editors. Omega-3, lipoproteins and atherosclerosis. Paris: John Libbey Eurotext; 1996. p. 1–5. [20] Tunstall-Pedoe H, Kuulasmaa K, M¨ah¨onen M, et al. Contribution of trends in survival and coronary event rates to changes in coronary heart disease mortality: ten-year results from 37 WHO MONICA Project populations. The Lancet 1999;353:1547–57. [21] Marques-Vidal P, Arveiler D, Evans A, et al. Patterns of alcohol consumption in middle-aged men from France and Northern Ireland. The PRIME Study. Eur J Clin Nutr 2000;54:321–8. [22] Belfrage P, Berg B, Hagerstrand I, et al. Alterations of lipid metabolism in healthy volunteers during long-term ethanol intake. Eur J Clin Invest 1977;7:127–31. [23] Dashti N, Franklin A, Abrahamson DR. Effect of ethanol on the synthesis and secretion of apo A-I- and apoB-containing lipoproteins in HepG2 cells. J Lipid Res 1996;37:810–24.
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Lack of association between ADH3 polymorphism, alcohol intake, risk factors and carotid intima-media thickness P. Marques-Vidal a , C. Bal Dit Sollier b , L. Drouet b , H. Boccalon c , J.B. Ruidavets a , J. Ferri`eres a,∗ a
INSERM U558, Facult´e de M´edecine, 37 All´ees Jules Guesde, 31073 Toulouse cedex, France b Department of Hematology, Hˆ opital Lariboisi`ere, Paris, France c Department of Vascular Medicine, CHU Rangueil, Toulouse, France Received 7 January 2005; received in revised form 28 April 2005; accepted 4 May 2005 Available online 6 June 2005
Abstract Objective: We assess the relationships between alcohol dehydrogenase 3 (ADH3) polymorphism, alcohol consumption and cardiovascular risk factor levels. Methods: In a representative population sample from Southwestern France (614 men, 567 women, age 49.7 ± 8.5 years), alcohol intake was assessed by questionnaire. Results: Alcohol consumption was significantly related with higher levels of total and HDL cholesterol, triglycerides, apolipoprotein A-I in men and with higher levels of HDL cholesterol in women. Also, an inverse relationship between alcohol consumption and intima-media thickness was found in men. Conversely, in both genders, no differences were found between ADH3 genotypes regarding all cardiovascular risk factors studied and carotid intima-media thickness. Also, in both genders, no significant ADH3 × alcohol interaction was found for all variables, and further adjustment on age, body mass index, educational level, smoking status or after excluding subjects on hypolipidemic or antihypertensive drug treatment did not change the results. Conclusion: We found no interaction between the ADH3 polymorphism and alcohol intake on cardiovascular risk factor levels and atherosclerotic markers in Southwestern France. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Alcohol dehydrogenase; Polymorphism; Population sample; HDL; Alcohol consumption
1. Introduction Moderate alcohol consumption has been shown to be protective against cardiovascular disease [1–4]. The major enzymes of alcohol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). ADH is a dimeric protein with two subunits and six ADH genes have been described, of which two (ADH2 and ADH3) are polymorphic. The ADH2 polymorphism has been shown to be related to cardiovascular risk factors, namely systolic blood pressure [5,6] and triglycerides [5]. The ADH3 polymor∗
Corresponding author. Tel.: +33 5 61 52 18 70; fax: +33 5 61 32 33 32. E-mail address: [email protected] (J. Ferri`eres).
0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.05.003
phism has two alleles, gamma 1 and gamma 2 (␥1 and ␥2), and pharmacokinetic studies have shown that the homodimeric ␥1 isoenzyme has a 2.5-fold higher maximal velocity of ethanol oxidation than the homodimeric ␥2 isoenzyme [7]. Further, it has been shown that moderate drinkers who are homozygous for the slow-oxidizing ADH3 allele have higher HDL levels and a substantially decreased risk of myocardial infarction [8]. Still, those relationships between ADH polymorphisms and cardiovascular disease have been questioned [9–11], and it is currently unclear whether ADH polymorphisms do interact with alcohol consumption regarding the levels of cardiovascular risk factors or cardiovascular disease. France is characterized by a low incidence of myocardial infarction and a moderate-high intake of alcohol but, to our knowledge,
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no study on the relationship between ADH3 polymorphism and alcohol consumption on the levels of cardiovascular risk factors has ever been conducted. Thus, we used the data from the last MONICA (multinational monitoring of trends and determinants in cardiovascular diseases) population survey conducted in Southwestern France to assess the relationships between ADH3 polymorphisms, alcohol intake and cardiovascular risk factor levels. The relationships between ADH3 polymorphisms, alcohol intake and intima-media thickness, an indicator of preclinical atherosclerosis, were also assessed. 2. Population and methods 2.1. Population sample The WHO–MONICA Project is a study that monitors deaths due to coronary heart disease, myocardial infarction (MI), coronary care and risk factors in men and women aged 35 to 64 years [12–14]. It consists of 39 MONICA Collaborative Centers (MCC) in 26 countries. Each MCC is in charge of carrying out two or three population surveys on cardiovascular risk factors in the beginning and at the end of the 10-year, and possibly in the middle of the study period. The sampling strategy was to have representative probability samples within each sex and 10-year age group, at least for the age range 35–64 years. The number of eligible subjects asked to participate was 50% higher than the required number in order to obtain the necessary quota of 200 persons for each sex and 10-year age group (allowing for subjects refusing to participate or not participating for any other reason). The informed consent to participate in the study was obtained from the subjects before the survey. The last Toulouse MONICA survey started in December 1994 and ended in July 1997 and concerned both genders; participation rates were 67% for men and 59% for women. 2.2. Data collection Subjects were advised to restrain from physical exercising, smoking, eating or drinking anything other than water for at least one hour prior to the screening visit. Screening included standardized questionnaires on personal data and measurements of body height and weight and blood pressure. Body mass index (BMI) was calculated as weight (kg) height (m)−2 . Subjects were considered as smokers if they currently smoked, and the average amount of cigarettes smoked per day was assessed for each smoker. Subjects were considered as hypertensive if their systolic blood pressure was >160 mmHg and/or their diastolic blood pressure was >95 mmHg and/or they reported being on antihypertensive drug treatment. 2.3. Biological measurements Lipids were measured on plasma EDTA plasma samples using automated enzymatic assays (Boehringer, Mannheim,
Germany). External quality control by the MONICA reference laboratory indicated no relevant deviation. Subjects were considered as dyslipidaemic if their total cholesterol was >6.5 mmol/L and/or they reported being on hypolipidaemic drug treatment. 2.4. Intima-media thickness measurement High-resolution B-mode ultrasonography was used to detect atherosclerotic plaques in carotid and femoral arteries and to measure common carotid IMT. An ATL UM9 system (Advanced Technology Laboratories Ultramark 9 High Definition Imaging) was used with a 7.4 MHz transducer. IMT was defined as the distance between the media ± adventitia interface and the lumen ± intima interface, avoiding the sites of plaque [15,16]. IMT was measured on the right and left common carotid arteries, on the far wall exclusively: three points at two locations on each artery, proximal and middle. Thus, 12 points were determined. The mean values at the 12 sites were combined to generate an overall mean value for common carotid IMT. In 26 subjects submitted to a second IMT assessment, the differences between the mean values of the measurements performed during the two examinations were small (−0.007 mm for the right common carotid artery and −0.022 mm for the left common carotid artery) and the intra-class correlation coefficients between the two measurements were 0.64 (p < 0.001) for the right common carotid artery and 0.53 (p < 0.001) for the left common carotid artery. 2.5. Alcohol consumption Alcohol consumption was assessed by a validated questionnaire that recorded the subjects mean consumption (in units) of wine, beer, cider and spirits for each day of the week [17,18]. Intake of alcohol (expressed in ml of pure ethanol/week) was estimated from the average number of millilitres of ethanol in one unit of each type of alcoholic beverage: wine (10% or 12% alcohol, v/v) = 12 cl serving; beer (5% alcohol) = 12 cl serving; beer (6 or 8% alcohol) = 25 or 33 cl serving; cider (5% alcohol) = 12 cl serving; spirits (20% or 40% alcohol) = 2 or 6 cl serving. For each gender, alcohol consumption was further classified into three groups: teetotalers, below and above the median. 2.6. Alcohol dehydrogenase genotyping DNA was obtained from leukocytes by salting out procedure, and ADH3 genotyping was performed in duplicate for each subject using polymerase chain reaction. Subjects were classified into three categories according to the presence of the ␥2 (slow) allele: 11 homozygotes, 12 heterozygotes and 22 heterozygotes. The alcohol dehydrogenase 3 exon eight polymorphism was determined as followed: A fragment of 130 bp in the ADH3 gene was amplified by polymerase chain reaction (PCR) using the following primers (forward 5 -GCTTTAAGAGTAAATCTGTCCCC-
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3 , reverse 5 -AATCTACCTCTTTCCAGAGC-3 ) at the annealing temperature of 55 ◦ C. The PCR products were analysis after digestion by the restriction endonuclease SSpI (New England Biolabs), and the fragments were separated by electrophoresis onto a 3% agarose gel. Wild type (␥1 alle) was cut into 67 and 63 bp fragments, whereas the ␥2 allele, associated a slower metabolism of alcohol, was uncut. 2.7. Statistical analysis Statistical analysis was conducted using SAS Enterprise Guide v.2.05 (SAS Institute, Cary, NC, USA). Results were expressed as means or as numbers of subjects and (percentage) or as mean ± standard deviation (S.D.). Bivariate statistical comparisons were assessed by Analysis of variance (ANOVA) or by χ2 . Multivariate statistical analyses were conducted using the general linear model (Proc. GLM) of SAS. Statistical significance was considered for p < 0.05.
3. Results 3.1. Toulouse MONICA survey subjects’s characteristics Overall, 1,181 subjects (614 men, 567 women, age 49.7 ± 8.5 years) were assessed. Their clinical and biological
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characteristics are summarized in Table 1. Men had higher body mass index, blood pressure levels, total cholesterol, triglyceride and LDL cholesterol levels than women, whereas women had higher HDL and apolipoprotein A-1 levels than men. Men were also more frequently alcohol drinkers, and alcohol consumption was higher among male drinkers than their female counterparts. Conversely, no differences were found for age, smoking status, educational level or the distribution of the ADH3 polymorphisms. Prevalence of hypertension and dyslipidaemia were 20.4 and 35.0% in men and 18.5 and 29.7% in women, respectively. Also, 93 men and 70 women reported being on hypolipidaemic drug treatment, and 73 men and 81 women reported taking antihypertensive medication. 3.2. ADH3 polymorphism and cardiovascular risk factor levels The frequencies of the ADH3 alleles were 65% for the ␥1 and 35% for the ␥2 allele in men and 64% for the ␥1 and 36% for the ␥2 in women. The distribution of the main cardiovascular risk factor levels according to the ADH3 polymorphisms is summarized separately for each gender in Table 2. In both genders, no significant differences between ADH3 polymorphisms were found for all parameters studied, and excluding subjects on hypolipidaemic or antihypertensive drug treatment did not change the results (not shown).
Table 1 Clinical and biological characteristics of the Toulouse MONICA survey, by gender FDF
Men (n = 614)
Women (n = 567)
p
Age (years) Body mass index (kg/m2 )
49.7 ± 8.6 26.2 ± 3.7
49.7 ± 8.6 24.7 ± 4.5
0.90 <0.001
Smoking status (%) current former and never
151 (24.6) 463 (73.4)
120 (21.2) 447 (78.8)
0.16
Educational level (years) ≤6 6 <. ≤ 12 >12 Alcohol drinkers (%) Alcohol consumption among drinkers (g/week) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mmol/L) Triglycerides (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Apolipoprotein A-1 (g/L) Apolipoprotein B (g/L)
20 (3.3) 329 (53.6) 265 (43.1) 480 (78.2) 306 ± 237 135 ± 16 82 ± 10 5.89 ± 1.01 1.36 ± 0.88 1.29 ± 0.33 4.00 ± 0.93 1.56 ± 0.22 1.26 ± 0.27
14 (2.5) 313 (55.2) 240 (42.3) 298 (52.6) 125 ± 115 127 ± 19 77 ± 10 5.77 ± 1.02 1.00 ± 0.49 1.65 ± 0.46 3.67 ± 0.95 1.73 ± 0.27 1.13 ± 0.25
ADH3 polymorphism (%) −11 −12 −22
263 (42.8) 273 (44.5) 78 (12.7)
236 (41.9) 252 (44.8) 75 (13.3)
0.66 <0.001 <0.001 <0.001 <0.001 0.05 <0.001 <0.001 <0.001 <0.001 <0.001
0.93
Results are expressed as number of subjects and percentage or as mean ± S.D. Comparisons were performed by χ2 or Student’s t-test. Triglycerides were log-transformed before comparison. Alcohol consumption was assessed among drinkers only, and comparisons were performed by non-parametric test.
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Table 2 Cardiovascular risk factor levels and carotid intima-media thickness according to ADH3 genotype, by gender p
Men 11 (n = 263) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mmol/L) Triglycerides (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Apolipoprotein A-I (g/L) Apolipoprotein B (g/L) Carotid intima-media thickness (mm)
135 83 5.85 1.37 1.29 3.96 1.57 1.25 0.60
± ± ± ± ± ± ± ± ±
16 10 0.94 0.91 0.34 0.88 0.23 0.25 0.09
12 (n = 273) 134 82 5.90 1.33 1.29 4.02 1.56 1.26 0.61
± ± ± ± ± ± ± ± ±
16 10 1.03 0.90 0.32 0.96 0.21 0.27 0.12
22 (n = 78) 135 82 5.99 1.40 1.29 4.06 1.56 1.29 0.62
± ± ± ± ± ± ± ± ±
16 11 1.16 0.70 0.38 1.03 0.24 0.31 0.11
0.69 0.61 0.53 0.48 0.98 0.63 0.94 0.61 0.45
p
Women 11 (n = 236)
12 (n = 252)
22 (n = 75)
± ± ± ± ± ± ± ± ±
127 ± 19 77 ± 11 5.81 ± 1.10 0.99 ± 0.50 1.65 ± 0.45 3.72 ± 1.05 1.73 ± 0.26 1.15 ± 0.27 0.58 ± 0.08
127 ± 21 76 ± 10 5.60 ± 0.86 0.98 ± 0.53 1.56 ± 0.37 3.59 ± 0.82 1.69 ± 0.22 1.08 ± 0.23 0.57 ± 0.07
128 77 5.76 1.00 1.68 3.63 1.76 1.12 0.57
18 10 0.95 0.46 0.48 0.88 0.28 0.23 0.08
0.66 0.84 0.29 0.69 0.17 0.45 0.10 0.08 0.39
Results are expressed as mean ± S.D. Statistical analysis by ANOVA.
3.3. ADH3polymorphism, alcohol consumption and cardiovascular risk factor levels Multivariate adjustment for age, body mass index, educational level and smoking status (and menopausal status for women) did not lead to any significant interaction in both genders, and also after excluding subjects on hypolipidaemic drug treatment (not shown). The effect of alcohol consumption (in tertiles) on the distribution of the main cardiovascular risk factor levels
according to ADH3 polymorphisms is summarized separately for each gender in Tables 3a and 3b. Although alcohol consumption was significantly related with higher levels of total and HDL cholesterol, triglycerides, apolipoprotein AI and lower levels of intima-media thickness in men and with higher levels of HDL cholesterol in women, no significant ADH3 × alcohol interactions were found for all variables studied, and similar results were obtained when alcohol consumption was treated as a continuous variable, when the analysis was restricted to drinkers or when subjects
Table 3a Cardiovascular risk factor levels according to ADH3 genotype and alcohol consumption, men Alcohol Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Total cholesterol (mmol/L)
Triglycerides (mmol/L)
HDL cholesterol (mmol/L)
LDL cholesterol (mmol/L)
Apolipoprotein A-I (g/L)
Apolipoprotein B (g/L)
Carotid intima-media thickness (mm)
11 (n = 263)
12 (n = 273)
22 (n = 78)
p
Non-drinkers <246 g/week ≥246 g/week
133 ± 2 135 ± 2 137 ± 2
133 ± 2 132 ± 2 136 ± 2
133 ± 4 133 ± 3 136 ± 3
0.98
Non-drinkers <246 g/week ≥246 g/week
81 ± 1 83 ± 1 83 ± 1
81 ± 1 81 ± 1 83 ± 1
78 ± 3 82 ± 2 83 ± 2
0.82
Non-drinkers <246 g/week ≥246 g/week
5.70 ± 0.13 5.86 ± 0.10 5.92 ± 0.10
5.75 ± 0.13 5.78 ± 0.10 6.10 ± 0.10
5.42 ± 0.26 6.04 ± 0.21 6.18 ± 0.16
0.35
Non-drinkers <246 g/week ≥246 g/week
1.29 ± 0.11 1.27 ± 0.08 1.54 ± 0.09
1.24 ± 0.11 1.23 ± 0.08 1.48 ± 0.08
1.13 ± 0.23 1.24 ± 0.18 1.60 ± 0.14
0.43
Non-drinkers <246 g/week ≥246 g/week
1.22 ± 0.04 1.29 ± 0.03 1.33 ± 0.03
1.21 ± 0.04 1.30 ± 0.03 1.34 ± 0.03
1.21 ± 0.09 1.46 ± 0.07 1.22 ± 0.05
0.07
Non-drinkers <246 g/week ≥246 g/week
3.95 ± 0.12 4.00 ± 0.09 3.92 ± 0.10
3.98 ± 0.12 3.94 ± 0.09 4.13 ± 0.09
3.69 ± 0.24 4.02 ± 0.19 4.23 ± 0.15
0.31
Non-drinkers <246 g/week ≥246 g/week
1.49 ± 0.03 1.58 ± 0.02 1.61 ± 0.02
1.49 ± 0.03 1.56 ± 0.02 1.61 ± 0.02
1.46 ± 0.06 1.61 ± 0.04 1.56 ± 0.03
0.58
Non-drinkers <246 g/week ≥246 g/week
1.23 ± 0.03 1.24 ± 0.06 1.28 ± 0.03
1.24 ± 0.03 1.23 ± 0.03 1.31 ± 0.03
1.20 ± 0.07 1.23 ± 0.05 1.36 ± 0.04
0.68
Non-drinkers <246 g/week ≥246 g/week
0.58 ± 0.01 0.61 ± 0.01 0.61 ± 0.01
0.60 ± 0.01 0.58 ± 0.01 0.63 ± 0.01
0.60 ± 0.03 0.61 ± 0.03 0.64 ± 0.02
0.26
Results are expressed as mean ± S.D. The p column refers to the test of the ADH3 × alcohol interaction factor.
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Table 3b Cardiovascular risk factor levels according to ADH3 genotype and alcohol consumption, women Alcohol Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Total cholesterol (mmol/L)
Triglycerides (mmol/L)
HDL cholesterol (mmol/L)
LDL cholesterol (mmol/L)
Apolipoprotein A-I (g/L)
Apolipoprotein B (g/L)
Carotid intima-media thickness (mm)
11 (n = 236)
12 (n = 252)
22 (n = 75)
p
Non-drinkers <96 g/week ≥96 g/week
128 ± 2 129 ± 3 127 ± 2
126 ± 2 128 ± 2 126 ± 2
131 ± 3 125 ± 4 120 ± 5
0.48
Non-drinkers <96 g/week ≥96 g/week
77 ± 1 77 ± 2 77 ± 1
77 ± 1 75 ± 1 77 ± 1
78 ± 2 74 ± 2 75 ± 3
0.91
Non-drinkers <96 g/week ≥96 g/week
5.69 ± 0.09 5.80 ± 0.15 5.84 ± 0.12
5.83 ± 0.10 5.82 ± 0.11 5.76 ± 0.13
5.54 ± 0.16 5.72 ± 0.21 5.54 ± 0.28
0.85
Non-drinkers <96 g/week ≥96 g/week
0.97 ± 0.04 0.92 ± 0.07 1.10 ± 0.06
1.05 ± 0.05 0.94 ± 0.05 0.96 ± 0.06
1.08 ± 0.08 0.88 ± 0.10 0.88 ± 0.13
0.24
Non-drinkers <96 g/week ≥96 g/week
1.64 ± 0.04 1.61 ± 0.07 1.79 ± 0.05
1.55 ± 0.04 1.65 ± 0.05 1.81 ± 0.06
1.54 ± 0.07 1.65 ± 0.09 1.50 ± 0.13
0.24
Non-drinkers <96 g/week ≥96 g/week
3.61 ± 0.09 3.77 ± 0.14 3.58 ± 0.11
3.81 ± 0.09 3.74 ± 0.10 3.53 ± 0.12
3.52 ± 0.15 3.68 ± 0.19 3.64 ± 0.26
0.65
Non-drinkers <96 g/week ≥96 g/week
1.73 ± 0.03 1.73 ± 0.04 1.83 ± 0.03
1.68 ± 0.03 1.74 ± 0.03 1.81 ± 0.03
1.66 ± 0.04 1.74 ± 0.05 1.67 ± 0.07
0.46
Non-drinkers <96 g/week ≥96 g/week
1.11 ± 0.02 1.13 ± 0.04 1.13 ± 0.03
1.19 ± 0.02 1.15 ± 0.03 1.09 ± 0.03
1.09 ± 0.04 1.08 ± 0.05 1.07 ± 0.07
0.35
Non-drinkers <96 g/week ≥96 g/week
0.56 ± 0.01 0.57 ± 0.01 0.59 ± 0.01
0.58 ± 0.01 0.60 ± 0.01 0.57 ± 0.01
0.57 ± 0.01 0.58 ± 0.02 0.56 ± 0.03
0.42
Results are expressed as mean ± S.D. The p column refers to the test of the ADH3 × alcohol interaction factor.
on hypolipidaemic or antihypertensive drug treatment were excluded (not shown). Finally, no relationship was found between ADH3 polymorphisms and the prevalence of hypertension or dyslipidaemia, or between ADH3 polymorphisms and the presence of antihypertensive or hypolipidaemic drug treatment (not shown).
4. Discussion Few studies have assessed the relationships between ADH3 polymorphisms, alcohol consumption and cardiovascular risk factors. This study enabled the assessment of those relationships in a representative population sample of Southwestern France, a region characterized by a low level of cardiovascular disease [15,19,20] and a regular intake of alcohol [17,18,21]. The frequencies of the ADH3 alleles were comparable to that observed in other caucasian populations [8] and were in Hardy–Weinberg equilibrium (p = 0.93 for men and p = 0.90 for women). In this study, no individual effect of the ADH3 polymorphism on blood pressure levels was found in both genders, and
those findings did not change after excluding subjects on antihypertensive drug treatment. Also, no relationship between hypertensive status and ADH3 polymorphism was found. Further, no significant interaction between the ADH3 polymorphism and alcohol consumption on blood pressure levels was found, either individually or after adjusting on age, body mass index, educational level and smoking status. Thus, our findings indicate that there is no interaction between ADH3 polymorphisms and alcohol consumption on blood pressure levels. A recent report indicated that subjects homozygous for the ␥2 allele and consuming at least one drink per day had the highest plasma HDL levels [8]. Conversely, and in agreement with other findings [10], no such relationship was found. Rather, subjects homozygous for the ␥2 allele tended to present with the lowest HDL cholesterol levels in both genders, and those findings did not change after multivariate adjustment or after excluding subjects on antihypertensive or hypocholesterolemic drugs. Thus, our data indicate that, at least in this population sample, ADH3 polymorphism does not influence the effect of alcohol consumption on HDL cholesterol levels. Alcohol consumption has been shown to increase HDL levels by various mechanisms: increase in lipoprotein lipase activity [22] or in synthesis and secretion
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of apolipoprotein A–I [23]; accelerated turnover of VLDL [24]; thus, it would be expected that the slow alcohol metabolizers (homozygotes for the ␥2 allele) would present with higher HDL cholesterol levels, which was not case. A possible explanation is the fact that the alcohol – HDL relationship might not be linear but concave, with a threshold effect [25]. Those findings could explain the increase in HDL levels in one study with a relatively low total alcohol consumption [8] compared to others [10], thus suggesting a threshold effect on the relationship between alcohol consumption and the ADH3 polymorphism on HDL levels. Still, further studies are needed to better assess this point. To our knowledge, no study has ever assessed the effect of alcohol and ADH3 polymorphism on carotid intima-media thickness, a marker of atherosclerosis. Our data indicate that ADH3 is not related to this variable, and also to other atherosclerotic risk factors such as LDL, apolipoprotein B or triglycerides. Thus, it is unlikely that this polymorphism might be a determinant for the development of atherosclerosis, as it has been suggested previously [8]. Also, conflicting results regarding the relationship between alcohol consumption and IMT have been published: negative (protective) association [26]; no association [27] or an association depending on the amount [28] or the pattern [29] of alcohol consumption. Thus, the exact relationships between alcohol consumption and atherosclerosis await further investigation. This study has several limitations: alcohol consumption was assessed by questionnaire, which might have led to reporting bias by some subjects; still, excluding subjects on sick leave did not change the results (not shown). Also, total alcohol consumption was divided into tertiles, which might have blurred the relationships between alcohol consumption, ADH3 polymorphism and cardiovascular risk factors; still, no significant interactions were found using alcohol consumption as a continuous variable, divided into quintiles or when the analysis was restricted to subjects who consumed less than the median (not shown). Further, mean alcohol consumption was significantly higher than reported in other studies [8]; still, comparing subjects with more than the equivalent of one drink per day to the other groups or restricting the analysis to postmenopausal women did not change the results (not shown). Finally, our sample size was comparable to or even higher than that of other studies on the relationships between ADH polymorphisms and cardiovascular risk factors [5,6,8–10]. In summary, we found no interaction between the ADH3 polymorphism and alcohol intake on cardiovascular risk factor levels and atherosclerotic markers in this representative population sample from Southwestern France.
Acknowledgements We would like to thank all the investigators of the Toulouse MONICA center for their invaluable contribution in the careful collection and validation of the data. We would
like to thank the “Institut National de la Sant´e et de la Recherche M´edicale” (INSERM), the “Direction G´en´erale de la Sant´e (DGS)”, “ONIVINS”, the “Fondation de France”, the “F´ed´eration Franc¸aise de Cardiologie”, the Parke-Davis and Bayer pharmaceuticals, and CERIN for their financial supports enabling this work.
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