Are the Associations between Life-style Related Factors and Plasma Total Homocysteine Concentration Different According to Polymorphism of 5,10-Methylenetetrahydrofolate Reductase Gene (C677T MTHFR)?

Mild hyperhomocysteinemia is one of the known strong risk factors for atherosclerotic diseases, and therefore it is important to clarify factors that could determine plasma total homocysteine (tHcy) level. A cross-sectional study with a random sample of 455 Japanese rural residents aged 40-69 years was conducted in 2000 to investigate the associations of plasma tHcy concentration with 5,10-methylenetetrahydrofolate reductase (MTHFR) gene and selected life-style related factors. The frequency of the mutant allele, Valine (V) allele, was 0.40 and the prevalence of VV genotype was 14.3 %. Plasma tHcy concentration in VV was significantly higher than those in two other genotypes. There were significant inverse associations of plasma tHcy with serum folate and serum vitamin B12 (P<0.001 for trend, respectively); both being stronger in VV than in other genotypes. The number of cigarettes smoked per day was positively associated with plasma tHcy concentration. A multivariate regression analysis revealed that serum folate, serum vitamin B12, and MTHFR genotype were independently associated with plasma tHcy. The inter-individual variance of plasma tHcy was more explained by serum folate and vitamin B12 than by MTHFR genotype. Higher intakes of folate, vitamin B12, and non-smoking may be important to prevent mild hyperhomocysteinemia and the eventual atherosclerotic diseases in this Japanese rural population.


Epidemiological
evidence indicates that higher plasma concentration of total homocysteine (tHcy), a thio containing amino acid metabolized by either remethylation to metionine or transsulfuration to cysteine, is a risk factor for atherosclerotic diseases such as coronary, cerebral, and peripheral arterial diseases 1-3).To improve programs for the primary prevention of these atherosclerotic diseases, it is important to clarify factors, whether genetic or environmental, that could determine the levels of plasma tHcy.A common genetic factor, 5,10-Methylenetetrahydrofolate reductase (MTHFR) gene, and dietary factors such as folate, vitamin B12, and vitamin B6 have been suggested previously 1" 5).Although severe hyperhomocysteinemia is rare, mild hyperhomocysteinemia occurs in approximately 5 % of general population due to these factors 1, 2, 5 and may be prevented by life-style modification.
In the present cross-sectional study conducted in a Japanese rural population, we analyzed the associations of plasma tHcy concentration with polymorphism of C677T MTHFR, serum concentrations of folate and vitamins B12 and B6, daily physical activity, smoking, and alcohol drinking to examine the effects of these genetic and environmental factors on the plasma tHcy concentration.Furthermore, we analyzed associations between plasma tHcy concentrations and the environmental factors in each of MTHFR genotypes separately because the metabolic pathways of homocysteine involve MTHFR and many cofactors (e.g.folate, vitamin B12, and vitamin B6) and, therefore, the strength of association between plasma tHcy concentration and environmental factors might be affected by the MTHFR genotype.Such knowledge about so-called the gene-environment interactions would be helpful to develop an optimal program of life-style modification to control mild hyperhomocysteinemia for individuals with different MTHFR genotypes.

Study subjects
The participants were randomly selected from all residents aged 40-69 years in Shiso, a rural county located in the northwestern part of Hyogo Prefecture, Japan, in 2000.The present cross-sectional study was carried out as a part of a longitudinal study to continuously monitor the changes in life-styles and risk factors of cardiovascular diseases in this area during the last decade 6).The sample size was determined to detect a statistically significant difference of plasma tHcy levels according to MTHFR genotypes on the basis of some previous studies 1. [7][8][9][10][11], All participants were assembled in local community halls, where 213 men and 242 women completed the following examinations.
The present study was approved by the ethics review committee of Medical Research Institute, Tokyo Medical and Dental University.All the subjects were taken for written informed consent.

Assessment of life-style factors
Under a supervision of nurses or dietitians specifically trained for this study, each of the participants completed standardized questionnaires that included items about physical activity, smoking and alcohol drinking habits, and others.Smoking habit was classified into three groups as non-smokers (including ex-smokers), 1-19, and 20+ cigarettes/day.Daily alcohol drinking was also classified into three groups as 0-2.0, 2.1-4.0, and 4.1+ drinks/day, where one drink is approximately 12 grams of ethanol.Smoking and alcohol drinking were analyzed only in men because the prevalence of these was markedly higher in men than that in women.To evaluate the usual degree of physical activity, we used the intensity of physical activity as determined by a metabolic equivalents (METs) score, which was calculated as follows.Aside from sleeping time, the frequency and average duration of various types of labor and other activities on the job, including household activities, for every 2 months within the last 12 months were queried 6).One MET is as the resting metabolic rate, or approximately equivalent to 1 kcal/kg body weight per hour 12).In this report, we used the `active intensity index' that was defined as the average METs during the time except sleeping.Body fat percentage was measured by the fitness analyzer, BFT-2000

Characteristics of the study subjects
Table 1 shows the basic characteristics of the subjects participating in this study.Mean age of men and women was 49.0 ± 8.2 years and 49.7 ± 8.2 years, respectively.The mean level of plasma tHcy was significantly higher in men than in women.The frequency of V V genotype in men (16 %) was slightly higher than that in women (12.9 %) although not statistically significant.Serum folate was significantly higher in women than in men.The prevalence of male smokers (20+ cigarettes/day) was 45.4 % and that of male drinkers (4.1+ drinks/day) was 28.3 %.

Plasma tHcy and C677T MTHFR genotypes
The frequency of mutant allele (V allele) of MTHFR was 0.40; the frequencies of three genotypes were AA 34.3%, AV 51.4%, and VV 14.3%.The distribution of the genotypes did not differ from the values expected for the Hardy-Weinberg equilibrium ( X2 = 2.09, df = 1, P = 0.48).Age-and sex-adjusted LSM of plasma tHcy level in MTHFR mutant genotype (VV) was significantly higher than those in two other genotypes (Table 2).

Plasma tHcy and life-style factors
Table 3 shows LSM of plasma tHcy (log and non log-trans-    formed values) by tertile of selected life -style factors .Both serum folate and serum vitamin Bit were inversely correlated with plasma tHcy; smoking was positively correlated to plasma tHcy.There were no statistically significant associations of plasma tHcy with serum vitamin B6, alcohol drinking , BMI, and physical activity .
Associations between plasma tHcv and life-style factors according to MTHFR genotypes As shown in Table 4, an inverse association between plasma tHcy and serum folate was stronger in the order of V V (std B = -0 .42),AV (std B = -0.33), and AA (std B = -0.17)genotype, where the difference in the strength of association , expressed as std B, was statistically significant between VV and AV or AA genotype.Partial R2 implied that the inter-individual variance of plasma tHcy was more explained by the level of serum folate (partial R2=0.075) than by MTHFR genotypes (partial R2=0.011).Similarly, an inverse association between plasma tHcy and serum vitamin Bra was a little stronger in VV (std B=-0.29) than in AV (std 13=4 15) or in AA (std B =-0.11) , but the difference of std B was not statistically significant among three genotypes.No association was observed between plasma tHcy and serum vitamin B6 in any genotype .
Plasma tHcy was higher in smokers , particularly in those with 20+ cigarettes/day than in non-smokers , but this was statistically significant only in VV genotype .Alcohol drinking, BMI, and physical activity were not related to plasma tHcy in any MTHFR genotype.

Multivariate analysis of association of plasma tHcy with MTHFR genotype and selected life-style factors
As shown in Table 5 , MTHFR VV genotype, serum folate , and serum vitamin Biz were independently associated with plasma tHcy.The interaction of serum folate x V V genotype also indicated that serum folate was more strongly associated with plasma tHcy in VV genotype than in AA genotype even in the multivariate analysis .Partial R2 of serum folate (0.0652) and that of serum vitamin Biz (0 .0165) were larger than that of MTHFR genotype (0.0092) .We added smoking habits as an independent variable to this model at the first step , but the association between smoking and tHcy was not significant (P=0.18)and, therefore, we did not include smoking in the final analysis shown in Table 5 .

DISCUSSION
Higher plasma tHcy is known as one of the strong risk factors for coronary heart disease and atherothrombotic stroke .A meta-analysis of 27 studies which investigated the relation between fasting tHcy levels and coronary artery disease (CAD) yielded an odds ratio of 1.6 for men and 1.8 for women for every 5 µmol/l increase in plasma tHcy level , and also showed that a 5 p nol/l increase in plasma tHcy elevated CAD risk by as much as an increase in total cholesterol of 20 mg/dl '8).A more recent review article indicated that the relative risk associated with a 5 pmol/l increment in the tHcy level was 1.7 (95% CI, 1.5-1.9)for coronary heart disease, 1.9 (95% CI, 1.6-2.3)for cerebrovascular disease, and even higher for peripheral artery disease 19).Most of these epidemiological studies demonstrated that such risks were independent of other known risk factors.In our study, there were no significant associations between plasma tHcy and other major traditional cardiovascular risk factors such as SBP, DBP, TC, HDL-C, body fat percentage (data not shown), and BMI.This suggested that elevated plasma tHcy could be an independent risk factor for CAD in this Japanese rural population, although we did not examine the relationship between tHcy and risks of CAD .Therefore, it would be important to conduct more cross-sectional studies like ours to gain the knowledge that would allow a more advanced discussion to develop control measures against elevated plasma tHcy.
Inherited disorders (MTHFR thermolability), which are present in approximately 5% of a general population 20) , can alter enzyme activity in the transsulfurration and remethylation pathways ', 7. 19.21,22), and these may result in elevated tHcy levels.The C677T MTHFR gene was shown to be responsible for the thermolabile enzyme 17,21,22,23) and the mutation altered the enzyme activity in the transsulfurration and remethylation pathways 1. 7, 19.21,22).In our study, the C677T MTHFR genotype mutation, particularly homozygous, was significantly associated with the elevated plasma tHcy .This finding was almost consistent with previous Japanese studies conducted with a relatively small number of individuals 17,22) or CAD patients 8) .In addition, Frosst et al .17) reported that the VV genotype resulted in reducing the activity of MTHFR enzyme and raising the level of plasma tHcy in French Canadian subjects .Several Japanese studies 9. 10, 11) showed that MTHFR mutation was significantly associated with the risk of CAD although such an association was not observed in several studies conducted in the US and Australian populations  .
A multivariate linear regression analysis revealed that there was a significant inverse association between plasma tHcy and serum folate levels in individuals with any MTHFR genotype .I nterestingly, we found that this inverse association was significantly stronger in VV genotype than in AA genotype .This f inding supported the hypothesis that folate might stabilize the MTHFR enzyme activity 171; furthermore it may be consistent with the observation in which the oral supplementation of folic acid normalized hyperhomocysteinemia due to thermolabile MTHFR 27.28).Our results also supported that when folate concentration was high , this common genetic variant had little effect on tHcy levels 7, 24,29,30).The present study demonstrated significant associations of MTHFR V V genotype with not only higher tHcy levels but also lower serum folate levels .This finding was consistent with the NHLBI Family Heart Study 7) and other studies 24.31).MTHFR catalyzes the synthesis of 5 -Table 4. The effects of combinations between MTHFR genotypes and selected life-style factors onto the plasma total homocysteine concentration.
To be continued.Several studies demonstrated an inverse relationship between vitamin Bit status and plasma homocysteine levels [34][35] although the strength of the correlation was not as strong as that observed between folate status and homocysteine levels.
Our result also showed that plasma tHcy was significantly and inversely associated with serum vitamin B12 concentration.
Vitamin B12 in the form of methycobalamine is required as Alcohol drinking was not associated with the level of plasma tHcy.Some studies observed that there was a modest association between alcohol intake and fasting tHcy, and significant positive associations were seen between tHcy and consumption of hard liquor and red wine, but not of beer 5°, 51).In our study, the majority of alcohol beverages consumed was beer.Therefore, the lack of association between alcohol drinking and plasma tHcy might be due to the type of alcohol beverages consumed in this population.
BMI was not associated with plasma tHcy and MTHFR genotype.This result supported to Gudnason et al 22), although Wilcken et al 4 observed that BMI was significantly associated with MTH R mutation.BMI may be related to intake of total energy and hence other nutrients such as folate, vitamin B12, and Bb.However, adjustment for BMI virtually unchanged the associations of folate, vitamin B12, and B6 with plasma tHcy (data not shown).Unlike some other studies 5 '11,41'48), there was no association between plasma tHcy and daily physical activity in our study.We further need to study for elucidating other potential confounding factors to explain this result.
In the multivariate analysis, MTHFR mutant genotypes, serum folate, and serum vitamin B12 were independently associated with plasma tHcy levels.Although homocysteine level was independently influenced by both genetic (MTHFR) and environmental (folate and vitamin B12) factors, the partial R2 implied that the effects of folate and vitamin B12 on the tHcy level were larger than that of MTHFR gene.Furthermore, there was a significant gene-environmental interaction between MTHFR gene and serum folate.Therefore, future studies on the relationship between MTHFR gene and tHcy should be necessarily done considering such environmental factors.
Since the measurement of homocysteine is currently expensive, a mass screening for mild hyperhomocysteinemia may be impracticable.However, it would be a valuable advice for any people to take foods rich in folate (e.g., green soybeans, leafy and dark green vegetables, citrus fruits, citrus juice, and legumes) and vitamin B12 (e.g., seaweed, shell-fish, fin-fish, and poultry) 5253.54), and to quit smoking.
Values are means ± SDs or percentages.* P< 0 .05for comparison between sexes.+ Means ± SDs of log-transformed values $ 722 = 2.09, df = 1, P = 0.48 for comparison with Hardy-Weinberg equilibrium among all subjects.I One drink is approximately 12 grams of ethanol.

Table 1 .
Basic characteristics of the subjects in Shiso, a Japanese rural county, in 2000.

Table 2 .
Plasma total homocysteine level according to MTHFR genotype .

Table 3 .
Plasma total homocysteine and selected life-style factors.

Table 5 .
Association of plasma tHcy with MTHFR genotypes and selected life-style factors in