Translational Research On Lowering Homocysteine And Prevention Of Cardiovascular Disease

Background: Stroke has been the leading cause of death in China, and its morbidityand mortality have been rapidly rising, particularly in rural areas. Previous studies havereported that hypertension and hyperhomocysteinemia are the two most importantmodifiable risk factors for stroke. Furthermore, though the importance of translationalresearch has been suggested for years, no simple and effective methodology for thedevelopment of translational research has been established in China.Objectives: We aimed to provide a series of clinical evidence for appropriate andeffective folic acid supplementation in prevention of cardiovascular disease, and furtherestablish a set of methodology for translational research in China. We first (study1)conducted a series of meta-analyses of relevant randomized controlled trials to evaluatthe effect of folic acid supplementation in stroke prevention, cardiovascular disease(CVD) prevention in ESRD or Advanced Chronic Kidney Disease (ACKD), and on theprogression of atherosclerosis as measured by carotid intima-media thickness (CIMT)and then investigated the optimal dose of folic acid in lowering plasma homocysteineconcentration (study2: MTHFR C677T and MTR A2756G Polymorphisms and theHomocysteine Lowering Efficacy of Different Doses of Folic Acid in HypertensivesChinese Adults; study3: Effect of Folic Acid Intervention on the Change of SerumFolate Level in Hypertensive Chinese Adults: Do MTHFR and MTR GenePolymorphisms Affect Therapeutic Responses?;) and address the question of whetherfolic acid supplementation can affect serum alanine aminotransferase(ALT) level inhypertensive Chinese adults (study4: Effect of Folic Acid Intervention on ALT Concentration in Hypertensives without Known Hepatic Disease: A randomized,double-blind, controlled trial;).Methods: Study1: Relative risk (RR) was used to measure the effect of folic acidsupplementation on risk of stroke and CVD. Summary estimates of weighted meandifferences (WMDs) were obtained to measure the effect of folic acid supplementationon the progression of CIMT. Meta-regression and subgroup analyses were performed toidentify the source of heterogeneity. Study2-4: a total of480subjects with mild ormoderate essential hypertension were randomly assigned to three treatment groups:1)enalapril only (10mg, control group);2) enalapril-FA tablet [10:0.4mg (10mg enalaprilcombined with0.4mg of FA), low FA group]; and3) enalapril-FA tablet (10:0.8mg, highFA group), once daily for8weeks. Blood samples collected at baseline and at week8ofthe trial were used for the measurement of glucose, lipids and ALT. Plasma/serumsamples collected at baseline, week4and week8were used for measurement ofhomocysteine and folate. The MTHFR C677T and MTR A2756G polymorphisms weregenotyped by PCR-RFLP method.Results:Study1: Overall, folic acid supplementation reduced the risk of stroke by8%(n=55,764;RR:0.92;95%CI:0.86-1.00, P=0.038). In the10trials with no or partial folic acidfortification (n=43,426), the risk of stroke was reduced by11%(0.89;0.82-0.97,P=0.010). Within these trials, a greater beneficial effect was observed among trials withlower percent use of statins or higher percent hypertension. In the remaining5trialsconducted in populations with folic acid fortification (n=12,338), folic acidsupplementation had no effect on stroke risk (1.03;0.88-1.21, P=0.69);When pooling the seven trials, folic acid therapy reduced the risk of CVD by15% (n=3886; RR:0.85;95%CI:0.76to0.96, P=0.009). A greater beneficial effect wasobserved among those trials with a treatment duration>24months, a decrease inhomocysteine level>20%, and no or partial folic acid fortification. In the correspondingcomparison groups, the estimated RRs were attenuated and insignificant.This analysis included2,052subjects from ten folic acid randomized trials with thechange in CIMT reported as one of the end points. Our analysis showed that folic acidsupplementation significantly reduces the progression of CIMT (WMD:-0.04;95%CI:-0.07,-0.02; P<0.001), particularly in those trials with baseline CIMT levels≥0.8mm, areduction in the homocysteine concentration≥30%and in subjects with chronic kidneydiseaseor high CVD risk but not in subjects who were generally healthy with onlyelevated homocysteine concentrations. Furthermore, meta-regression analysis of thedata showed that the baseline CIMT levels (P=0.011) and the percent reduction ofhomocysteine (P<0.001) were positively related to the effect size.Study2: The prevalence of MTHFR C677T polymorphisms677CC,677CT, and677TT were0.256,0.497, and0.247, respectively. The prevalence of the2756AA,2756AG, and2756GG genotypes were0.820,0.171, and0.090, respectively. After4or8weeks of treatment, homocysteine concentrations were reduced across all genotypesand FA dosage groups, except in subjects with MTR2756AG/GG genotype in the lowFA group at week4. However, compared to subjects with MTHFR677CC genotype,homocysteine concentrations remained higher in subjects with CT or TT genotype in thelow FA group (P<0.05for either of these genotypes) and TT genotype in the high FAgroup (P<0.05). Furthermore, subjects with TT genotype showed a greaterhomocysteine-lowering response than did subjects with CC genotype in the high FAgroup (median ratio of homocysteine at week8to that at baseline: CC0.89vs. TT:0.74,P=0.005), but not in the low FA group (median ratio at week8: CC0.89vs. TT0.87, P=0.989).Study3: After4or8weeks of treatment, increases in serum folate were seen across allgenotypes and folic acid dosage groups. However, compared to subjects with677CCgenotype, subjects with CT or TT genotype in the low FA group and TT genotype in thehigh FA group still had significantly lower folate concentrations, particularly females. Inthe low FA group, subjects with CT or TT genotype showed an attenuated responsecompared to subjects with CC genotype (median ratio of folate at week8to that atbaseline: CC,1.953vs. CT,1.755or TT,1.637, P<0.01for both). Such an attenuatedresponse was not observed in the high FA group. Yet, only in the high FA group didserum folate appear to reach a plateau after4weeks of treatment in all3MTHFR677genotypes and the MTR2756AG/GG genotype.Study4: We found a significant reduction in ALT level in the high FA group [median(25th percentile,75th percentile),-0.6(-6.9,2.0) IU/L, P=0.0008]. Compared with thecontrol group, the high FA group showed a significantly greater ALT lowering responsein men [median ALT ratio (ALT at week8to ALT at baseline)(25th percentile,75thpercentile):0.93(0.67,1.06) vs.1.00(0.91,1.21), P=0.032]; and in participants withelevated ALT(ALT>40IU/L) at baseline [0.67(0.38,1.00) vs.0.94(0.77,1.11),P=0.008]. There was no difference in ALT lowering between the control and the lowFA group.Conclusions:(1) Our analysis indicated that folic acid supplementation is effective in strokeprevention and in reducing the progression of CIMT. Furthermore, folic acid therapycan also reduce CVD risk in patients with ESRD/ACKD. (2) We demonstrated that MTHFR C677T polymorphisms can not only affect serumfolate levels and hcy concentrations at the baseline and post-folic acid treatment, butalso therapeutic responses to various dosages and durations of folic acidsupplementation. A daily dose of0.8mg folic acid was recommended for subjects withMTHFR677CT or TT genotype.(3) Compared to treatment with10mg of enalapril alone, a daily dose of10mgenalapril combined with0.8mg of folic acid showed a beneficial effect on serum ALTlevel, particularly in men and in participants with elevated(>40IU/L) ALT.(4) Based on these results, we establish a set of methodology for translational researchin China.