Abstract

Background

The beneficial effects of folate have been observed under different conditions, but the available evidence on inflammation and reduction of cardiovascular disease (CVD) in type 2 diabetes mellitus (T2DM) is limited. The study aimed to explore the effects of folate on inflammation and homocysteine amongst individuals with T2DM.

Methods

PubMed, Scopus, and Cochrane Library were used to search for evidence. A random-effect model meta-analysis through Review Manager (version 5.4) and metaHun was performed. Results were reported as standardized mean differences (SMD) and 95% confidence intervals graphically using forest and funnel plots.

Results

Data from 9 trials with 426 patients living with T2DM were analyzed. Folic acid supplementation significantly revealed a large effect size on homocysteine levels compared to placebo, SMD = −1.53, 95%CI (−2.14,−0.93), p < 0.05. Additionally, we observed a medium marginal effect size on C-reactive protein (SMD = −0.68, 95%CI (−1.34, −0.01), p = 0.05). However, no significant effect on tumor necrosis factor-α (SMD = −0.86, 95%CI (−2.65, 0.93), p = 0.34), and interleukin-6 (SMD = −0.04, 95%CI (−1.08, 1.01), p = 0.95) was observed.

Conclusion

Evidence analyzed in this study suggests that folic acid supplementation in T2DM reduces homocysteine and may mitigate CVDs. However, its effect on inflammation is inconclusive.

Discussion

To the best of our knowledge, this is the first comprehensive meta-analysis of RCT to evaluate the effect of folic acid supplementation on homocysteine and inflammation in adult patients with T2DM. We found that folic acid supplementation was associated with a reduction in homocysteine levels. The observed SMD (1.52) was large effect, suggesting anti-homocysteine properties. Additionally, there was a marginal effect of folic acid on CRP without a significant effect on TNF-α and IL-6 in patients with T2DM. Subgroup analysis showed that the folate effect on homocysteine was more pronounced at a higher dose (10 mg) than 5 mg supplementation. However, it is important to note that only one trial used 10 mg compared to 5 mg that used 5 mg of folic acid. It was evident that studies with a sufficient sample size (50 and above patients) had a more pronounced effect than those with a smaller sample size. Although folic acid supplementation at both short and medium periods reduced homocysteine, the reduction was more pronounced at short periods (0–4 weeks) compared to medium periods (8–12 weeks). Our research reveals lower homocysteine levels in individuals living with T2DM receiving folate supplements, indicating a potential decrease in the risk of CVD. We are confident with the evidence synthesized in this study as the evidence showed moderate certainty in homocysteine. In T2DM, insulin resistance and associated impaired kidney function results in an elevated homocysteine level [43,44,45,46,47]. This elevation promotes the development of CVD complications associated with T2DM.

Interestingly, the evidence from this study shows that folic acid supplementation can reduce homocysteine by converting it into methionine, lowering the risk of cardiovascular complications in T2DM patients [26]. Notably, previous evidence has shown that folic acid supplementation can reduce homocysteine levels in patients living with T2DM by increasing the 5-methyltetrahydrofolate intracellular pool [37]. This effect is crucial as an elevated level of homocysteine can damage blood vessels and contribute to the development of CVD [45]. Therefore, any strategies therapeutically that reduce homocysteine may assist in alleviating CVD among T2DM. In obese children, similar trends have been observed when administering a minimum of 1 g of folic acid, leading to a substantial reduction in homocysteine levels [48]. Similar findings are observed in gestational diabetes, as 1 mg and 5 mg of folic acid supplementation significantly reduced homocysteine levels [49]. Although the pathological and physiological mechanisms of these conditions differ, these findings demonstrate the efficacy of folate supplementation across diverse conditions and age groups. A non-randomized trial in menopausal T2DM women also showed that 800 µg of folate significantly reduced homocysteine levels. However, this study revealed an inverse correlation between folic acid and homocysteine (r = −0.4876, p-value = 0.0134) [50]. Although the mechanism by which folate reduces homocysteine in T2DM is poorly documented, it is assumed that this is associated with the role of folate in one-carbon metabolism. Folic acid supplementation increases the availability of one-carbon units, which then promotes the remethylation of homocysteine to methionine [51]. This subsequently results in a decrease in homocysteine levels in the body. While such benefits are acknowledged, contrasting findings from other studies suggest a possible risk of CVD in T2DM, even with folic acid supplementation [52]. These findings suggest a limitation in the beneficial effect of folate, especially in T2DM. It is assumed that folate deficiency impairs the conversion of homocysteine to methionine, resulting in homocysteine accumulation in the blood [53]. High homocysteine levels are associated with an increased risk of CVDs and other health problems.

Although there was a marginal effect on hs-CRP (p = 0.05), no significant effect of folic acid supplementation on other markers of inflammation was observed. This was shown by no significant effect on TNF-α and IL-6 following supplementation with folic acid compared to placebo. A reduction in hs-CRP following folic acid supplementation reveals, to some extent, the beneficial effect of folic acid as an anti-inflammatory agent. However, as not all inflammatory markers were reduced, the findings are thus inconclusive. Among some factors contributing to the challenge in elucidating conflicting findings regarding the impact of folic acid on inflammation is the limited number of trials conducted. The inability of folic acid to improve some markers of inflammation indicates that it does not exhibit anti-inflammatory properties. For instance, Spoelstra-de Man et al., [23] reported no effect of folic acid on hs-CRP, IL-6, and TNF-α. The same findings were observed by Title et al. [27], however, only TNF-α and hs-CRP were investigated, and no effect was observed. Despite these null findings, another trial observed a significant effect of folic acid on hs-CRP in T2DM, as demonstrated by a significant decrease in hs-CRP within the folic acid group before and after folic acid supplementation. This same trend was also observed when folic acid supplementation was compared to placebo [24]. The latter supports our findings as we observed a reduction in hs-CRP with a medium to large effect size (Cohen d = 0.68). Other researchers reported a significant decrease in IL-6 and TNF-α following folic acid supplementation compared to placebo, suggesting the anti-inflammatory effects of folic acid in T2DM [38]. These findings differ from our overall findings in this study as we reported no significant effect of folic acid on TNF-α and IL-6. Another study showed a significant change between baseline and post-treatment on hs-CRP, however, there were no significant changes between the folic acid and placebo groups [38]. In obese children, 1 mg of folic acid has proven to offer an anti-inflammatory effect, as demonstrated by a significant decrease in IL-6, TNF-α, and IL-8 [48].

Similarly, El-khodary et al. [37] also showed a significant decrease in hs-CRP between baseline and post-treatment (p = 0.008). The same trial reported a significant decrease in hs-CRP following three months of folic acid supplementation compared to placebo (p = 0.005). This study also showed a positive correlation between homocysteine and hs-CRP (r = 0.308, p = 0.002). Due to these contradicting results on inflammation, the effect of folic acid on inflammation is not clear, other researchers have suggested that folic acid may be involved in the reduction of hs-CRP by reducing homocysteine and oxidative stress. For instance, Talari et al., [24] reported an increased adjusted glutathione (GSH) following folic acid supplementation in T2DM compared to placebo.

Additionally, folic acid exhibits anti-insulinemic activities [54, 55] may further alleviate inflammation by suppressing the synthesis of inflammatory cytokines. It is important to note that while the benefits were not observed in this study, this might be attributable to the number of trials analyzed primarily because observational differences were noted. Previous evidence suggests that homocysteine promotes the expression of inflammatory markers by increasing the activation of nuclear factor kappa β (NF-kβ) and poly-adenosine diphosphate (ADP) ribose polymerase. Therefore, we speculate that folic acid anti-homocysteine properties may alleviate inflammation by inhibiting the activation of NF-κβ and ADP and thus suppressing the expression of inflammatory markers [56, 57]. Evidence from in vitro studies has also shown that folic acid may reduce inflammation by inhibiting the phosphoinositide 3-kinases (PI3K)/hypoxia-inducible factor 1-alpha (HIF-1α) pathway [58]. Even though the reduction in homocysteine following folate supplementation is normally accompanied by a reduction in CRP and subsequent deactivation of NF-κβ and low IL-6 and TNF-α, the contradictory findings observed in our study may be due to few trials and sample size across the trials analyzed in this study.

Strength and limitation

The present analyses exclusively examined evidence from randomized trials, considered to provide high clinical evidence. Notably, there was a low risk of bias observed across various domains in the risk of bias assessment, indicating that the quality of the studies was satisfactory. The GRADE tool was also employed to evaluate the overall quality of the analyzed evidence, and it was categorized as either moderate or very low in one outcome due to the small sample size.

Furthermore, a comprehensive subgroup analysis was performed, considering various confounding factors. The I² statistics revealed moderate heterogeneity. For transparency, the study was registered with PROSPERO, and the experienced researchers adhered to PRISMA guidelines, boosting confidence in the reliability of the current findings. However, it is crucial to acknowledge certain limitations in our study, such as few relevant trials, indicating a minimal sample size of only 426 patients living with T2DM. Moreover, existing trials have employed varying quantitative methodologies, introducing potential differences in sensitivity and specificity, especially with the use of ELIZA AND HPLC for the determination of homocysteine.

Conclusion and future recommendations

The findings from nine trials involving a sample of 426 participants in this study indicate that folic acid supplementation in T2DM may reduce homocysteine levels, a potential biomarker for CVDs. However, due to the limited number of trials analyzed, null effects were observed concerning some of the inflammatory markers. It is crucial to interpret the conclusions of our study with caution, emphasizing the need for further trials with adequate sample sizes.

Considering the limitations acknowledged in this study, we propose recommendations for future investigations into folic acid in T2DM, particularly focusing on inflammation. We suggest that forthcoming RCTs use sufficient sample sizes and adhere to the reporting guidelines outlined in the consolidated standards of reporting trials (CONSORT). Additionally, these trials should adhere to standardized methodologies, implementing an accurate randomization process, blinding of personnel and participants. Furthermore, we emphasize the necessity for high-quality meta-analyses to comprehensively elucidate the benefits of folic acid supplementation in managing T2DM.