Both a previous dose-finding trial  and a dose-response trial  have discussed the relationship between homocysteine-lowering efficacy and optimal dose of FA supplementation, but without considering the possible modifying effect of MTHFR C677T polymorphism. A more recent study  evaluated the effect of MTHFR C677T polymorphism on the response to different doses of FA supplementation (0.1 mg/d, 0.4 mg/d, 4 mg/d and 4 mg/week) in northern Chinese women of childbearing age and reported that the MTHFR 677 TT genotype was an independent predictor of plasma homocysteine concentrations, irrespective of FA dose, which was consistent with our results. However, the study population consisted of young women and the FA dosage category was not optimal, being either too low (0.1 mg/d) or too high (4 mg/d). Furthermore, plasma homocysteine concentrations (7.2 μmol/L) in the study population (without FA fortification) were lower than those observed in a U.S. population  and a Chinese population , which means that this Chinese population was not a representative population in which to evaluate the homocysteine lowering effect of FA. In our study, we investigated if the change in plasma homocysteine concentration in response to two commonly used physiological doses (0.4 mg/d and 0.8 mg/d) of FA supplementation could be modified by individual MTHFR C677T and MTG A2756G polymorphisms in a multicenter, randomized, double-blind, controlled trial in hypertensive Chinese adults, whose homocysteine concentrations were comparable to the general Chinese population .
Consistent with a previous study , baseline homocysteine concentration was the major determinant of the homocysteine-lowering effect in all genotypes and FA dosage groups. Subsequently, in the present study, after 4 or 8 weeks of treatment, subjects with MTHFR 677 TT genotype had a significantly greater homocysteine-lowering response than did subjects with CC genotype in the high FA group. However, such a heightened response was not shown in the low FA group. Furthermore, when we further adjusted baseline homocysteine, subjects with TT genotype seemed to have a more attenuated homocysteine-lowering response than did subjects with CC genotype in the low FA group but not in the high FA group. The molecular mechanisms that underlie clinical remediation of enzyme variants are the ability of elevated levels of the cofactor or substrate to overcome the binding defects of Km mutants or to serve as a chemical chaperone to improve the stability of mutant enzyme variants [16, 17]. Our results further suggest that daily 0.4 mg FA supplementation may be insufficient, and that a daily dose of 0.8 mg FA may be required to lower homocysteine concentration in subjects with TT genotype.
Furthermore, after the treatment, subjects with CT genotype had significantly higher homocysteine concentrations than those with CC genotype in the low FA group, and had similar homocysteine concentrations as CC subjects in the high FA group. These results indicate that daily 0.8 mg FA also may be necessary for lowering homocysteine concentration in subjects with CT genotypes. However, although they showed a greater homocysteine lowering effect than subjects with CC genotype, subjects with TT genotype still had the highest homocysteine concentrations even after 8 weeks of high FA treatment. Therefore, other therapeutic strategies need to be developed to reduce the risk associated with hyperhomocysteinemia in TT subjects.
In fact, Malinow et al.  first reported that TT subjects experienced much greater decreases in plasma total homocysteine concentrations after receiving FA at a dose of 1 or 2 mg/d for 3 weeks than did CC subjects. Among subjects who were not previously taking multivitamins, the mean reductions in plasma total homocysteine concentrations were -20.9%, -13.1%, and -7.1% in persons with the TT, CT, and CC genotypes, respectively (P = 0.019 for TT versus CC). A similar result also has been reported in a study in Taiwan, in which 5 mg of FA daily were supplemented for 8 wks . Our results appear to be consistent with these studies. However, the study of Woodside et al.  showed that TT subjects are less responsive to the effects of FA and B-vitamin supplementation (daily 1 mg FA, 7.2 mg Vitamin B6, and 0.02 mg Vitamin B12 for 8 wks.) than CC subjects. And Ho GY et al.  also reported that MTHFR C677T did not impact the total homocysteine-lowering effect of vitamins (daily 2.5 mg FA, 25 mg Vitamin B6, and 0.5 mg Vitamin B12 for 1 year) in a study performed in Singapore. The reasons for the disparities among the results of these studies are unknown. Additional large dose-finding studies are required to further elucidate the possible contributory factors, such as folate status, ethnicity of the participants, and in particular the possible effects of concomitant vitamin B6 and/or B12.
Our study showed that neither homocysteine concentration at baseline or post-FA treatment nor the homocysteine lowering response of FA supplementation was affected by MTR A2756G polymorphism. However, there was a significant homocysteine reduction in MTR 2756 AG/GG genotype after 4 weeks of FA supplementation in the high FA (0.8 mg/d) group, but not in the low FA (0.4 mg/d) group, which means that we cannot exclude the possible role of MTR A2756G polymorphism in the homocysteine metabolic pathway.
Overall, our results suggest that daily 0.8 mg FA may be necessary to lower homocysteine concentration for Chinese hypertensive subjects with CT or TT genotype, which have important clinical and public health implications. Increased homocysteine concentration has been implicated as a risk factor for neural tube defects (NTDs) and Downs syndrome [22, 23]. A study by Wilcken et al. reported that the frequency of CT and TT genotypes in Hungary was about 45% and 11.1%, respectively . Two Hungarian intervention trials [25, 26], which used a multivitamin containing 0.8 mg folic acid, found a significant reduction in the first occurrence of urinary tract and cardiovascular abnormalities beyond the 90% reduction of NTDs. There is no evidence that 0.4 mg folic acid has a similar preventive effect on defects beyond NTDs. Our results further suggest that there may be an additional benefit if a daily dose of 0.8 mg, but not 0.4 mg, of folic acid could be used in women, particularly in populations with a high frequency of T allele (such as the Chinese) and without folic acid fortification. Furthermore, identifying TT or CT subjects and providing them with an optimal dose of folic acid also may be very important in the prevention of CVD [4, 27] in populations without folic acid fortification.
Our study has the following strengths. This was a randomized, multicenter and double blind trial. This study simultaneously assessed the effect of FA dosages and gene polymorphisms on homocysteine-lowering efficacy. Our trial studied two commonly used doses of FA, which are of important clinical and public health relevance. However, caution is needed in generalizing our findings from this hypertensive Chinese population to other populations. Furthermore, though similar results were obtained when we restricted our analyses to subjects who fully complied with the protocol during the treatment periods, we still could not fully exclude the effect of non-compliance problem on our results. Also, it should be noted that we did not measure other polymorphisms in the folate pathway, such as the MTHFR A1298C variant. Furthermore, the treatment period with FA was only 8 weeks and the sample size was rather small when sub-grouped into various groups (control, low FA and high FA) for genetic association studies. Future studies with longer treatment duration and a larger sample size are needed to confirm our results. The association between angiotensin-converting enzyme inhibitor treatment and the change of homocysteine was controversial [28, 29]. We did not observe a significant relationship between enalapril treatment and homocysteine change in our study.. Additional large sample studies also are needed to further examine the relationship of enalapril treatment with homocysteine change and the possible interactive effects between enalapril and folic acid on the change in homocysteine.