Congestive heart failure (CHF), a type of cardiovascular disease (CVD), is a complex clinical syndrome with symptoms and signs that result from any structural or functional impairment of ventricular filling or the ejection of blood [1]. Presently, approximately 6 million individuals aged 20 and above in the United States have CHF [2]. Folate is an essential nutrient required for complex biochemical reactions such as nucleotide synthesis and methyl group transfer [3, 4]. Hyperhomocysteine, which is mainly caused by folate deficiency [5], has been widely demonstrated to increase the risk of CHF [5, 6, 7, 8, 9, 10]. The existing body of evidence substantiates that folate intake may reduce plasma homocysteine (Hcy) concentrations [11, 12]. Furthermore, research has revealed a marked contrast in folate consumption between CHF patients and healthy subjects [13].

All dietary folate functions biologically through absorption and conversion to active forms of folate in the body. Red blood cell (RBC) folate is a reliable biomarker of long-lasting folate status and is the endorsed gold standard by the World Health Organization [14]. Notably, a study found that compared with the lowest quintile of RBC folate, the highest quintile was associated with higher CVD mortality [15]. However, prior studies have not yet quantified the relationship between this biomarker and CHF risk. Therefore, it remains unclear whether and to what extent RBC folate concentration is associated with CHF risk. To address this question, our study employed data from a cross-sectional analysis of the National Health and Nutrition Examination Survey (NHANES) to elucidate the intricate association between RBC folate levels and risk of CHF.

Based on data from a nationally representative United States survey across 10 years (2011–2020), we investigated the relationship between RBC folate and the prevalence of CHF. To the best of our knowledge, this is the first study to extensively explore this relationship using a world-class dataset. Notably, our findings suggest the dual nature of RBC folate’s impact on CHF risk: either excessive levels (the highest tertile) or deficiency in RBC folate might increase the risk of CHF.

Multivariate-adjusted logistic regression analysis revealed several factors associated with an increased risk of CHF, including high RBC folate concentrations (the highest tertile or $>$2757 nmol/L), folate deficiency (317 nmol/L), insufficient dietary folate (mcg 40 µg), older age, male sex, smoking status, DM, hyperlipidemia, hypertension, history of stroke, and annual household income under $\$$35,000. While further discussion is warranted for each identified factor, this research focused specifically on delineating the relationship between RBC folate concentrations and CHF risk.

A novel finding of this study is that the prevalence of CHF was higher in the highest tertile group compared to the lowest tertile group of RBC folate concentration. Our findings remained consistent and robust across both univariate and multivariate logistic regression models, as well as subgroup analyses, demonstrating an increased risk of CHF associated with the highest tertile of RBC folate concentrations. However, it is important to note that this trend was not consistently observed within certain subgroups in the Mexican-American or male populations. Interestingly, there was no interaction between all subgroups. Therefore, we determined whether the threshold at which elevated RBC folate concentrations would increase the risk of CHF in male and Mexican-American populations would not be consistent with other subgroups. To test this hypothesis, we divided the male and Mexican-American populations into four groups by the quartiles of RBC folate and then reran the model. We found that the highest quartile of RBC folate increased the risk of CHF compared with the lowest quartile (OR = 2.26; 95% CI: 1.12–4.56) in male populations, and a similar trend was also found in Mexican-Americans (OR = 3.49; 95% CI: 1.00–12.14). Thus, the conclusion that high levels of RBC folate may increase the risk of CHF is consistent and reliable. The results of the quantitative analyses of RCS were also consistent. The risk of CHF was low and relatively stable up to a predicted RBC folate level of 2757 nmol/L, but then began to increase rapidly, suggesting that excessive RBC folate may increase the risk of CHF. Although no studies have found that high levels of RBC folate may increase the risk of CHF, one study showed that high levels of RBC folate may increase CVD mortality [15]. A study from NHANES spanning six cycles from 2003 to 2014 with a total of 14,234 participants with high-risk factors for CVD found that compared with the lowest quintile of RBC folate, the highest quintile was associated with higher CVD mortality (hazard ratio: 1.40, 95% CI: 1.02–1.93; p = 0.030) [15].

Research suggests that high RBC folate concentrations may increase the risk of CHF possibly due to high folate levels impairing normal folate physiological function [25]. Excessive folate will accumulate in the circulation if it is not utilized, thus reducing the formation of thymidylate and leading to the inhibition of aberrant DNA methylation [26] and DNA synthesis [25]. Another possible reason is that high concentrations of RBC folate may inhibit folate-dependent enzymes for which RBC folate is a substrate, thereby affecting normal biochemical reactions [27]. Furthermore, excessive RBC folate can induce the cytotoxicity of natural killer (NK) cells [28]. The activation of NK cells can lead to the secretion of proinflammatory cytokines [29], possibly resulting in increased risk of CVD and all-cause mortality [30, 31, 32]. These factors may represent mechanisms underlying the harmful effects of a folate overdose, although this still needs to be verified.

It has been almost 24 years since mandatory folic acid fortification was introduced in the United States. The prevalence of folate deficiency persisted at 1% over the entire 25-year period [23]. Our study included only 48 participants with folate deficiency. After performing multivariate logistic regression analysis with CHF, the resulting CI was wide. Thus, our study did not present sufficient evidence that folate deficiency can increase the risk of CHF. Nevertheless, previous studies suggest that folate deficiency may increase the risk of CHF [5, 33]. And folate is a free radical scavenger that acts as an antioxidant, protecting the organism from damage caused by the accumulation of free radicals [34]. Its antioxidant role is very important in CVD [35]. In addition to oxidative stress, a deficiency of one or more B vitamins (including folate) may impair ATP production. ATP depletion causes the weakening of cardiac muscle, which eventually leads to HF [36]. A prospective cohort study found that elevated total Hcy concentration independently predicted the risk of occurrence of CHF in adults [9]. Hyperhomocysteinemia can damage the endothelium and blood vessel wall, having a negative impact on the mechanisms underlying CHF, such as oxidative stress and inflammation [37]. Some animal experiments have shown that hyperhomocysteinemia might cause myocardium fibrosis and diastolic dysfunction in rats [38, 39]. In addition to high Hcy, some studies have shown that uric acid (UA) is positively associated with an increased risk of incident CHF [40, 41]. However, folate and its derivatives may reduce UA by inactivating xanthine oxidoreductase, the enzyme responsible for the oxidation of hypoxanthine to xanthine and xanthine to UA [42]. Thus, folate may also reduce the risk of CHF by lowering UA. It is not difficult to deduce that folate deficiency may therefore increase the risk of CHF. It is worth mentioning that the low RBC folate concentration in our study was just relative to the total population, which is the concentration after folic acid fortification, and not equivalent to folate deficiency. Therefore, the low RBC folate concentration in our study may not increase the risk of CHF. This may be the reason why the risk of CHF is not greater at the lower levels of RBC folate in the RCS curve.

This study showed that insufficient DFE (400 mcg) intake also increased the risk of CHF, consistent with previous prospective studies that reported the inverse association of increasing dietary folate intake with lower CVD mortality [15, 43]. Some studies have shown that increased DFE intake also reduces the risk of hyperuricemia [44] and hyperhomocysteinemia [45], which may ultimately reduce CHF risk. However, the cardioprotective effect did not increase proportionally as DFE intake was above the UL. Notably, the correlation between RBC folate concentration and DFE was found to be weak (Spearman’s r = 0.14, p 0.001), as RBC folate is influenced by numerous factors such as fortified products or supplements containing folic acid [46], folate requirements, absorption [46], and polymorphisms in folate metabolizing enzymes [47, 48], sex [46], age [49], and smoking [50]. Furthermore, in a study conducted by the NHANES, even though DFE intake remained the same, there was a notable increase in RBC folate concentrations as individuals aged [51]. Older subjects are more likely to develop CHF. In our study, 61.5% of CHF patients were older than 65 years of age, and these CHF patients still had higher RBC folate concentrations even with insufficient folate intake. Therefore, we should focus on DFE in the elderly to avoid excessive intake and an increase in CHF risk.

Our analyses found that folate deficiency exacerbates the risk of CHF. This illustrates the protectiveness and effectiveness of the folic acid fortification programs. In consideration of this, we believe that folic acid fortification should continue to be vigorously implemented. Following the RDA, we found that insufficient DFE is associated with an increased risk of CHF, thus requiring clinicians, health managers, and nurses to promote people to achieve the RDA. However, considering the two sides of RBC folate for CHF, to avoid excessive folate intake and high RBC folate concentration, there is a critical need for large-scale clinical research to identify safe DFE and RBC folate concentration intervals. In light of the prevalence of CHF and the associated morbidity and mortality, nutritional strategies to promote appropriate amounts are important areas for further research [52]. Furthermore, DFE and RBC folate levels should also be considered in the overall management of patients with CHF.

This study had several limitations. Firstly, our findings can be generalized to the United States population only. Secondly, the present study had a cross-sectional design. Thus, our findings are limited to the potential association but not causation between CHF and RBC folate, and the underlying mechanism remains elusive. Thirdly, excluding about 50% of participants due to missing data is a limitation which could potentially impact the final quantitative results. Finally, there is a restriction in particular due to the sensitivity of self-reported CHF. Patients did not report having CHF until they went to the hospital and were diagnosed with CHF by a doctor, and patients with less severe CHF who were not hospitalized and who did not have a definitive CHF diagnosis may have been unaware they have CHF and may not have reported CHF.

The risk of CHF may be increased either by high RBC folate concentrations (highest tertile of RBC folate or $>$2637 nmol/L) or by folate deficiency. Considering the two sides of the association between RBC folate and CHF, there is a need for large-scale clinical research to better investigate if the association between RBC folate and CHF is a cause-effect relationship, what are the underlying pathophysiological basis, as well as to identify optimal DFE and RBC folate concentration intervals.

CHF, congestive heart failure; RBC, red blood cell; DFE, dietary folate equivalent; NHANES, National Health and Nutrition Examination Survey; RCS, restrictive cubic spline; OR, odds ratio; CI, confidence interval; ATP, adenosine triphosphate; Hcy, homocysteine; MCQ, Medical Conditions Questionnaire; RDA, recommended daily allowance; UL, tolerable upper intake level; CDC, Centers for Disease Control and Prevention; BMI, body mass index; DM, diabetes mellitus.

All data is open source and all data is available from the website at the following address: https://wwwn.cdc.gov/Nchs/Nhanes/.

LBW designed the research study. LBW, FCY, and TXY performed the research. JXY and XXW provided help and advice on study design and research performance. LBW, ZNS, JRS, and YPZ analyzed the data. LBW and FCY wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

We would like to thank Zhang Jing (Shanghai Tongren Hospital) for his work on the NHANES database. His outstanding work involving the nhanesR package and webpage significantly facilitated our exploration of the NHANES database. We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.

This research was funded by the National Natural Science Foundation of China and the Natural Science Foundation of Zhejiang Province, grant number: NSFC81400192, NSFC82070050, LY19H020002.

The authors declare no conflict of interest.