This study investigated the interactive effect of the MTHFR C677T gene variant and serum folate levels on the tHcy-lowering response to short-term, varying doses of FA supplementation, in a population without mandatory FA fortification. To the best of our knowledge, this study is the first randomized, double-blinded trial to test the feasibility and effectiveness of different doses of FA supplementation for lowering tHcy and metabolites in the FA–tHcy metabolic pathway and the genetic interaction of the MTHFR C677T genotype in Chinese patients. This study found that after 8 weeks of folic acid supplementation, tHcy decreased by 9.2% in general. Combined with the results of CSPPT, which contains a similar population and found that 0.8 mg daily FA treatment increased folate levels and led to an average of tHcy levels reduction of 11%, these studies confirmed the stability of the current population.

This study explored a stable dose–effect relationship between 0 and 1.2 mg/day FA therapy on tHcy lowering. However, further increases in folate dose brought about a plateau. Wald et al. reported that serum tHcy levels decreased with increasing folic acid dosage and reached maximum efficacy at a dosage of 0.8 mg/day FA supplementation with a 23% reduction in tHcy among a population in Great Britain with ischemic heart disease [25]. A meta-analysis showed that a 1 mg/day folic acid dose generated the most tHcy-lowering efficacy, with no further reduction in tHcy levels with increasing dosages [21]. FA treatment ranging from 0.5 to 5.0 mg lowered tHcy levels by 25%. Studies that demonstrated promising results with reductions in tHcy levels have shown that the efficacy is more prominent among patients with high baseline tHcy levels or low folate levels before treatment [21]. A daily dose of approximately 400 μg is the minimum dose required for adequate tHcy reduction [26]. A 2010 study found that folic acid supplementation (0, 100, 400, 1000, or 2000 μg/day) had no dose–response relationship between FA and tHcy concentrations, but the data indicated that healthy, older adults (aged 60–90 years) can improve their folate status through supplementation [27].

This study found that the interaction between folic acid supplementation and tHcy exhibited distinct patterns for different MTHFR C677T genotypes among this population of Chinese hypertensive adults, along with different folic acid and tHcy levels at baseline across groups. In the TT group, lower doses of folic acid therapy (0.4–0.8 mg/day) were associated with maximum tHcy-lowering efficacy. The TT group had a steeper slope compared with the CC/CT group, and the fitted curve did not reach a plateau until 1.6 mg/day of folic acid supplement. The more effective tHcy-lowering capacity of folic acid therapy in the TT group can be explained by the fact that the highest baseline tHcy levels and the lowest folic acid levels were found in the TT group compared with the CC/CT group. The fitted curves for the correlation between folic acid treatment dose and changes in tHcy levels in different MTHFR C677T genotypes intersected at 1.0 mg/day of folic acid dose. In the CT group, the fitted curve reached a plateau at 1.0–1.2 mg/day of folic acid supplementation, and increased dose was found to be associated with a poorer tHcy reduction response. In the CC group, the fitted curve reached a plateau between 1.2 and 1.6 mg/day of folic acid supplement. The variability of tHcy reduction with folic acid treatment is in accordance with the results of the CSPPT, which determined an effect modification among the MTHFR genotypes in the efficacy of FA treatment. A post hoc analysis of the CSPPT found a more pronounced L-shaped curve between tHcy and serum folate levels in participants with the TT genotype compared with those with the CC and CT genotypes, requiring a higher folate level (at least 15 ng/mL) to eliminate genotypic tHcy differences. Our study results contribute to determining optimal folic acid intervention strategies in stroke risk prevention for hypertensive patients, especially in China where the effect of tHcy on first stroke is significantly modified by the methylenetetrahydrofolate reductase C677T genotype.

Our findings suggest a dose-dependent relationship between folate levels and folic acid supplementation. With increasing doses of folic acid supplementation, folate levels were elevated with no observed plateau, while the decreases in tHcy levels showed a significant plateau. The underlying mechanism can be explained by the differences in physical and chemical properties of synthetic folic acid. Folates are water-soluble vitamins that provide one-carbon units for the regulation of gene expression, nucleotide synthesis, and production of amino acids and neurotransmitters [28]. Available forms of synthetic folic acid include FA, folinic acid (formyl tetrahydrofolate), and 5-methyltetrahydrofolate (5-MTHFR). Folic acid from fortified foods and supplements differs from folate from natural sources, in that it requires dihydrofolate reductase (DHFR) for conversion to tetrahydrofolate to be active in one-carbon metabolism [29]. In areas that have FA fortification or synthetic FA recommendation [30, 31], circulating unmetabolized FA (UMFA) and 5-methyl-THF accounts for about 4% and 85% of total folate, respectively [32].

In a large national study with randomly selected US adults, both low and high serum total folates (total folate, UMFA, non-methyl folate, 5-mTHF, and MeFox [pyrazino-s-triazine derivative of 4ahydroxy-5-methyltetrahydrofolate]) were associated with a higher risk of all-cause, cardiovascular disease (CVD), and cause-specific mortality, including 5-mTHF insufficiency [33]. A study of major depressive disorder found that L-5-MeTHF improved symptoms in treatment-resistant major depressive disorder [34]. The MIREC (Maternal–Infant Research on Environmental Chemicals) study observed that the consumption of FA supplements by women resulted in a significantly increased total folate in breast milk. However, the increase in total milk folate was attributed to higher UMFA concentration, but not to reduced folates [35]. The higher proportion of UMFA in breast milk compared to 5-methylTHF in women consuming 400 μg FA daily suggests that higher doses exceed the physiological capacity to metabolize FA, resulting in preferential absorption of FA in breast milk. Our study population was characterized by low baseline levels of folate and with no FA fortification. The presence of a plateau in tHcy lowering within the range of 0.8–1.2 mg/day dose of folic acid therapy and the constant increase in folate levels for all doses indicate that caution should be taken for the use of higher folic acid doses for general population supplementation.

Folic acid therapy is frequently simultaneously considered with other B vitamins in metabolic cycles. The interpretation of clinical trial results for vitamin therapy to reduce tHcy levels is heavily reliant on folate levels, B12 status, and renal function [36, 37]. Most previous studies have adopted multiple vitamin B supplements with folate as the main component, for it has been shown that folate contributes the most to tHcy lowering, and additional supplements of B12 and B6 can lower tHcy levels by 7% [38].

No sex differences on the association between folic acid therapy and tHcy changes were observed in this study. A sensitivity analysis in the population with > 80% adherence showed the same patterns as the main study result.

Even in countries where fortified folic acid supplementation policies exist, most people still rely on food supplements, and a smaller proportion is aware of or adheres to the recommended daily intake levels [39]. Variation have been noted between countries in terms of the utilization, awareness, and beliefs toward FA supplement policies. Our study results highlighted the importance of evaluating and monitoring the utilization of supplements during antenatal care to facilitate appropriate usage. However, a systematic review demonstrated that in countries with mandatory food fortification policies, women who take FA supplements may surpass the upper tolerable limit of FA [40]. A recent study also found that excessive folic acid intake in parental mice increased DNA mutations and epigenetic changes in offspring embryos [41]. The problems of folic acid dosage, form, suitable population, and duration of supplementation have been plagued and are still ongoing.

Mammals, with the absence of folate biosynthesis, primarily meet their folate requirements through the diet. Historically, folate deficiency caused by poor nutrition has been one of the most widespread vitamin deficiencies and has persisted in countries that do not have mandatory FA fortification [42]. Future studies are critically needed to determine the optimal approaches in various pathological conditions. This study showed a precise dosage recommendation for a rural Chinese population with H-type hypertension, depending on the MTHFR C677T genotype.

This RCT has some limitations. The sample size was modest, although adequate power was expected based on the power estimation, for addressing the primary and secondary outcomes. This trial had only 8 weeks of treatment and follow-up. Although this duration is adequate for our primary and secondary outcomes, we were unable to evaluate long-term outcomes such as stroke incidence. The current analysis does not specifically confirm the proportion of people who might have actually met the target tHcy level window. Efforts are needed to try to increase the proportion of the whole population meeting the standards. To achieve a higher proportion of people with reduced levels, future analysis and research are required.