The Hidden Dangers of Excess Folate, Uncovering the Molecular Mechanisms

1. Introduction

It is estimated that up to a third of the US population is chronically exposed to high levels of dietary folate from a combination of voluntary supplementation and food fortification with folic acid. Eukaryotic cells lack the capability to synthesize folate de novo, establishing folate as an essential nutrient for higher organisms and humans. While the benefits of adequate folate intake are well documented, there is mounting evidence linking its excess with deleterious effects on human health by impacting cancer, immunity, birth outcomes, cardiovascular disease, and overall mortality. The association between excess folate and these negative outcomes is known, but the causative mechanisms are not. Therefore, understanding and unraveling the mechanisms by which excess folate from natural sources, supplementation, enrichment, and fortification of the US food supply could negatively impact health and disease is of critical importance. Recent reports focused on the areas of nutrition, folate metabolism, cancer, and aging each conclude that there are substantial knowledge gaps relating to the effects of excess folate on human health [1,2,3]. Therefore, there is a need to understand how excess folate from either the fully oxidized folic acid used in supplementation or naturally occurring reduced folate forms impact health, and to determine the underlying biological mechanisms.

This review aims to provide a narrative summary of the existing literature describing the potential effects of excess folate and discussing the possible molecular mechanisms underlying these effects. A comprehensive perspective on the potential impacts of excessive folic acid intake on health and disease is currently lacking. Additionally, there is no consensus regarding the potentially harmful role of excess folic acid supplementation, and there is a need to reconcile conflicting reports to identify gaps and areas requiring further research. By addressing some of these gaps, our aim is to contribute to a better understanding of the complex relationship between folate intake and human health (Figure 1).

2. Defining excess folate

Determining what constitutes excessive folate intake is an elusive task since there is a lack of dose response data, and comprehensive documentation of adverse effects at high folate intake are not established [1]. The recommended daily allowance (RDA) of folic acid varies depending on age, sex, and life stage. Adult men and women are recommended 400mcg of dietary folate equivalent (DFE), increasing to 500 mcg and 600 mcg DFE for breastfeeding and pregnant women respectively. The Tolerable Upper Intake Level (UL) for folic acid was set at 1,000 mcg per day for adults. However, the determination of folic acid UL relied on limited and low-quality observational case-study data [4]. Generally, folate exposure at dosages exceeding the adult UL (1000 ug/day) are considered excessive, However, this criterion does not consider chronic exposure to elevated folate intake that approach the UL and the inherent variability and mutations in folate cycle enzymes that are prevalent in humans [5,6].

Folic acid (FA), the reduced synthetic form of folate, is used almost universally in fortification and supplementation as well as when conducting most laboratory research. Women of childbearing age taking prenatal vitamins as well as children and adults taking vitamins high in folic acid are likely to exceed the age adjusted UL of FA intake due to a combination of food fortification and supplementation [7,8]. FA supplementation is linked to elevated circulating unmetabolized folic acid (UMFA) in a dose dependent manner [9]. UMFA occurs due to saturation of mechanisms needed to convert folic acid into the active reduced forms in the intestine and liver [10]. Specifically, UMFA accumulation is associated with the saturation of the rate limiting one carbon enzyme dihydrofolate reductase (DHFR). Some evidence supports the use of UMFA as marker for excess folate in humans as UMFA levels generally increases in response to supplemental folic acid intake [11,12], however, newer sensitive methods are able to detect UMFA in majority of samples irrespective of folic acid intake [13,14]. A growing body of literature linking excess FA and UMFA to potential adverse health outcomes exists, However, evidence of causality between excess FA, UMFA and various adverse effects remains inconclusive[2].

Mandatory fortification in combination with supplementation with FA led to more than doubling of USA population serum folate concentration in the last thirty years [14,15,16]. In the USA, up to 35% of the population are taking folic acid containing supplements and roughly 5 percent of the population exceed the UL of folate intake. The average folate intake is estimated to be 813mcg/day for men and 724mcg/day for women [17]. The Canadian health measures survey identified that up to 40% of participants had High folate levels (defined as RBC folate >1360 nmol/ L) [18,19,20]. Unmetabolized folic acid was detected in over 95% of Americans participating in the National Health and Nutrition Examination Survey (NHANES), with significantly higher UMFA levels recorded among supplement users and older individuals[14,21]. Similarly, high levels of FA and UMFA was found in pregnant Canadian women and umbilical cord blood, with over 97% of pregnant Canadian women having detectable plasma UMFA [22,23], and up to 26% of pregnant women exceeded the UL of FA [24].

The distinction between natural folate and synthetic folic acid is an important consideration when evaluating the potential risks of excess intake. Natural folate, mainly in the form of 5-methyl-tetrahydrofolate (5-m-THF) and to a lesser extent, 5-formyl-tetrahydrofolate (5-f-THF), are biologically active forms that do not necessitate activation by dihydrofolate reductase (DHFR) as folic acid does. Natural folate intake is constrained by the limited amount present in food sources, as well as its lower bioavailability and stability. In contrast, large doses of synthetic folic acid can be easily consumed through food fortification and supplementation, making folic acid the primary contributor to elevated folate pools in the human population. It has been suggested that the deleterious effects of excess folate could be related to factors such as low vitamin B12 status, high levels of folic acid or UMFA, or elevated total folate pools. However, evidence linking these factors to causation of deleterious effects or specific molecular mechanisms remains limited [25]. Owing to incomplete data and a lack of scientific consensus regarding the effects of excess folate, as well as the absence of accepted physiological markers linked to such effects, determining what constitutes excess folate is challenging. For this review, we will discuss folate intake exceeding the WHO recommended level of 400 µg/day and approaching or exceeding the Upper Limit (UL) of 1 mg/day. We will also draw comparisons to studies reporting conservative levels of FA supplementation. Additionally, we will examine results from human, animal, and cell culture studies that reported elevated serum, RBC folate, and/or the presence of high levels of unmetabolized folic acid.

In rodent studies, excess folate could be defined as exceeding the accepted standard baseline supplementation of 2 mg/kg diet. However, the optimal folate requirements for animals may be lower, and the activity of rate limiting one-carbon metabolism enzymes, such as dihydrofolate reductase (DHFR), are typically much higher in rodents compared to humans [26,27,28,29]. Chow diets fed to laboratory rodents can contain a variable amount of FA ranging from 2-15mg/kg diet and averaging about 8 mg/kg [28]. The reason for supplementing rodent diets with excessive amount of FA is not clear, However, we can hypothesize that suppliers of research animals and their diets defaulted to higher levels of folate supplementation to ensure rapid growth and large litter sizes.

In cell culture studies, cells are subjected to elevated levels of folates that greatly exceed the physiological levels found in tissues in order to support growth and proliferation. In human plasma, the typical physiological levels of natural folate range from 150-450 nM, while standard cell culture media contains 2,200 nM (RPMI) or 9000 nM (DMEM) of the non-physiological synthetic folic acid [30,31]. This implies that most studies utilizing standard cell culture conditions could be confounded by the supraphysiological concentrations of folic acid [32,33].

Limited research has been conducted using physiologically relevant levels of natural folate in place of folic acid. Natural folates are inherently less stable and bioavailable than folic acid, which creates technical and logistical challenges for their integration into basic research. Additionally, the widespread use of the terms “folate” and “folic acid” interchangeably has led to the misconception that these forms are equivalent. In this review, “folate” refers to all folate species, synthetic and natural, found physiologically and in foods, while “folic acid” denotes the fully oxidized synthetic folate used in supplementation and food fortification.

3. Health impact of excess folate

3.1. Pregnancy and birth related outcomes

The primary goal of the folic acid food fortification mandates that were instituted in various countries around the world is to reduce the incidence of births with neural tube defects. Folate adequacy for preventing NTD’s is critical in the first 4 weeks of gestation. Folate supplementation in the following weeks, postnatally, and during breastfeeding could potentially provide benefits to maternal and fetal health with varying and inconsistent outcomes [34]. Supplementation at the WHO recommended levels of 0.4 mg/day in the periconceptual period protects against NTD’s with largely positive health outcomes reported. However, FA intake at higher doses, especially when approaching or surpassing the upper intake limit and administered for extended durations are at times associated with deleterious pregnancy and offspring outcomes. A comprehensive review on the effects of FA supplementation on gestation and long-term health is reviewed elsewhere by Silva et al. [34]. Common prenatal vitamins in the USA typically include an FA dose of 1mg/ day with some prescribed regiments using doses up to 5mg FA/day. On average, women taking prenatal vitamins in the are likely to surpass the UL of 1000ug folate per day due to a combination of supplementation and dietary intake [35], leading to elevated levels of total folate and UMFA in maternal and fetal samples [36,37].

The impact of FA supplementation and specifically high dose FA on non NTD’s pregnancy outcomes is not clear [34]. The link between FA supplementation and oral cleft incidence is inconsistent with studies reporting a protective effect [38,39,40,41], no clear association [42,43], or an increase in oral cleft births [44]. In one study, high FA (4mg/day) did not compromise fetal growth or provide additional protection against orofacial clefts compared to 0.4mg/day [42]. Another study that used the same dose of 4 mg FA /day in early pregnancy saw increased maternal UMFA, and serum folate, with no significant effect on RBC folate or evidence of altered one carbon metabolism [11].A Cochrane review found no conclusive evidence that FA supplementation improved focused pregnancy outcomes such as preterm birth, still birth, anemia, serum and RBC folate levels [45]. In a Greek cohort, 5mg FA/day was found to be protective against preterm birth and low birth weight [46]. FA supplementation provided benefits in reducing the incidence of congenital heart defects irrespective of the timing and dosage during pregnancy, with beneficial association attributed to high dose of FA (5 and 6 mg/day) [47,48].

High folate concentration was linked to an increased risk of gestational diabetes mellitus (GDM) in Chinese cohorts [49,50]. Specifically, Higher FA dose as well as the timing and duration of the supplementation was associated with an increased risk for the development of gestational diabetes mellitus [51,52,53,54], with a significant GDM association when high FA was combined with an imbalance in vitamin B12 levels [55]. In a Spanish cohort where pregnant women were supplemented with FA in doses exceeding 1 mg/day, an association was found with decreased birth length and an increase of the risk of low birth weight [56]. Excess FA intake in late pregnancy was linked to allergy and respiratory problems in children [34]. Increased incidence of position plagiocephaly was identified in a nested case-control study performed in the Netherlands in correlation to periconceptional high doses of FA [57]. Although both favorable and unfavorable pregnancy outcomes have been observed in relation to excess folate, more research is required to identify the appropriate levels of folate supplementation needed to achieve optimal reproductive health.

3.2. Disease risk in offspring

Folate intake plays a critical role in the availability of methyl donors necessary for epigenetic programming during conception, gestation, and early development. The paradigm that maternal exposure to various stressors, dietary factors, and nutrient excess or deficiency during the prenatal period and early development can modulate the risk for developing diseases later in life is well accepted [58]. As the scientific knowledge advances, much of the etiology behind these changes has been attributed to epigenetic modifications and imprinting and is influenced by one carbon metabolism [58,59,60]. Folate and its reduced intermediates play a crucial role in the one carbon cycle and in regulating the processes involved in methylation and epigenetic modifications. High maternal FA supplementation can negatively impact health outcomes of the progeny by altering embryonic development [61], and modulating the expression of key genes involved in growth [62]. Excess FA supplementation may potentially alter gene expression and lead to aberrant epigenetic programming in the progeny[63,64]. Understanding the link between folate status in the parents, offspring, and epigenetic changes can further our understanding of the origins of adult diseases [65,66,67].

Numerous human and animal studies identified associations between excess folate intake and disease risk in offspring, though the outcomes have been inconsistent. In the Indian subcontinent, where expecting mothers are often prescribed folic acid doses as high as 5mg/day, high folate concentrations during pregnancy were linked to insulin resistance in children [68]. Results from a UK cohort of pregnant women revealed that FA supplementation after the 12th week of pregnancy impacts DNA methylation in the offspring with significant changes in the methylation of regions that regulate the insulin like growth factor 2 in infants potentially altering susceptibility to chronic diseases [65]. A systematic review by Xie et al., aimed at exploring the association between maternal folate status and the risk of obesity and insulin resistance in offspring, was unable to reach a definitive conclusion due to inconsistencies in data collected form animal and human studies [69].

While the clinical significance of altered methylation in response to excess folate in humans is not clear, animal studies provided a more robust link between epigenetic changes and maternal folate status. In rodents, maternal and post weaning supplementation with FA significantly impacted global and gene specific methylation in the offspring [70]. Alteration of DNA methylation and imprinting patterns was partially responsible for deleterious transgenerational effects observed when either folate deficient or excess folate diets were fed to female mice [71]. Excessive FA during pregnancy induced obesity and altered the epigenetic profile of the offspring in rats [72,73], and led to metabolic dysfunction in late adulthood [74]. Similarly, other studies found that excess perinatal folic acid supplementation caused insulin resistance, dyslipidemia, disrupted glucose metabolism and hepatic fat metabolism in both mice[75] and rat offspring [76,77,78]. In mice, excess maternal folic acid supplementation during pregnancy led to altered offspring cardiac gene expression [79].

FA intake during the periconceptual period, as well as throughout the second and third trimesters, can induce epigenetic alterations, leading to changes in the expression of genes related to brain development and function [80,81,82]. Rodent studies have shown that exposure to maternal FA supplementation can modify offspring DNA methylation profiles and gene expression in the brain, resulting in behavioral and morphological changes [83,84,85,86]. Further research is necessary to determine the effects of maternal excess folate on health and disease risk in children.

The developmental origins of health and disease hypothesis suggest that fetal programming is influenced by maternal nutrition, among other factors. Moreover, growing evidence indicates that paternal environment and diet significantly impact progeny’s health and risk of developing disease through epigenetic changes in sperm [87]. High doses of paternal folic acid supplementation was associated with increased gestational duration in births conceived through assisted reproduction [88]. Male offspring of mice fed a high-folate diet had a decreased sperm count, altered methylation in key genes, increased postnatal mortality in offspring, and reduced litter size [89]. Similarly, high-dose folic acid resulted in sperm DNA hypomethylation in both men and mice [90]. While moderate folic acid supplementation was beneficial in reducing some assisted reproduction-induced DNA methylation variance, high-dose supplementation produced deleterious effects with sex-specific outcomes [91]. These findings highlight the critical need to examine the epigenetic effects of excess dietary folate more carefully, as the influence of both maternal and paternal folate supplementation can potentially increase children’s disease susceptibility later in life.

3.3. Neurodevelopment

Excess folic acid supplementation during pregnancy is linked to both beneficial and detrimental effects on neurodevelopmental outcomes in children. Although FA supplementation is beneficial during the pre-conceptual period and early weeks of gestation by preventing neural tube defects, continued supplementation may significantly impact children’s neurodevelopment. FA supplementation at the WHO-recommended level of 400µg/d beyond the first trimester was associated with benefits on neurocognitive development in children, with positive effects on cognitive development observed at ages 7 and 11 [92,93]. In another study where high dose FA supplementation was implemented, cord blood analysis revealed significant changes in DNA methylation, exhibiting sex-specific and loci-specific alterations in genes related to neurodevelopment [94]. Doses exceeding the upper limit (≥1000 µg/d) during the periconceptual phase were linked to impaired neurocognitive development in children [95,96]. Both low (<400 µg/d) and excess FA (>1000µg/d) maternal supplementation were associated with attentional dysfunction in children aged 4-5 years[95]. High-dose FA (>5mg/day) supplementation in pregnant mothers led to poor psychomotor development in their children compared to those receiving 0.4-1mg/day [97]. In a small Greek cohort (n=58), high-dose FA supplementation (5mg/day) in early pregnancy improved children’s vocabulary development, verbal comprehension, and communication skills, while doses exceeding 5mg/day showed no benefit [46].

The relationship between FA supplementation and autism spectrum disorders (ASD) is complex and marked by contradictory findings. Recent systematic reviews found inconclusive evidence associating FA supplementation and ASD [96]. However, some studies did find a significantly increased risk of ASD with elevated maternal plasma FA [96] and linked synthetic folic acid supplementation during pregnancy to an increased autism risk [98]. Some suggested that excess FA leading to unmetabolized folic acid (UMFA) can potentially heighten ASD risk [80]. Moderate prenatal FA supplementation of at least 400µg from diet and supplements was associated with a decreased ASD risk in children [84,85,92,93], while excess FA corresponded with lower cognitive development and increased ASD risk [94,96,99]. This suggests a U-shaped relationship where both inadequate and high FA exposure could increase ASD risk [100].

Animal models offer valuable insights into the effects of excess FA on neurodevelopmental outcomes. In these models, excess FA during the prenatal period is associated with adverse impacts on behavior, memory, embryonic growth, methyl metabolism, and neurodevelopment [100]. In rodent studies, excess maternal FA supplementation resulted in deleterious behavioral abnormalities in offspring, including anxiety-like behavior, hyperactivity, impaired reversal learning, and seizure susceptibility [96,99,101]. The collected evidence indicates that the influence of excess FA on the type of behavioral abnormalities in offspring is sex-specific [83,84,86,102]. Both deficient and excess FA during pregnancy impaired cortical neurodevelopment in mice offspring[103]. In another study, moderate FA supplementation led to behavioral changes and sex-dependent alterations in one-carbon metabolism [104]. High-dose FA supplementation in mice caused neural tube defects and disrupted embryonic development [84,105,106]. Neurodevelopmental toxicity in male mouse offspring’s brain was observed when a 2.5-fold FA requirement was provided one week before mating and continued through pregnancy and lactation [107]. In rats, high FA doses administered periconceptually decreased the offspring seizure threshold [106]. High FA intake (20mg/kg) impaired rat dam’s memory and Na+, K+ - ATPase activity in the pup’s hippocampus [108]. High exposure to maternal FA in utero resulted in DNA methylation-dependent changes in the expression of hypothalamic genes involved in food intake and weight regulation [73]. These findings underscore the intricate relationship between excess FA and neurodevelopmental outcomes, emphasizing the need for more comprehensive research to elucidate the underlying mechanisms and their implications for optimizing human health.

3.4. Immune function and allergies.

Folate plays a crucial role in maintaining a healthy immune system, influencing both innate and adaptive immune responses. Adequate folate status is essential for optimal immune function, while deficiency or excess of folate can lead to immune dysregulation. Folate is required for the production and maintenance of fast proliferating white blood cells and is necessary for the maturation of lymphocytes. Folate plays a role in regulating inflammation and the production of pro-inflammatory cytokines [109]. Folate deficiency has been associated with impaired immune cell function and increased levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), which can contribute to impaired immunity, chronic inflammation, and the development of inflammatory diseases [110]. There is evidence that folate status is linked to the development and progression of inflammation and autoimmune diseases [111,112,113]. However, the impact of FA supplementation on inflammatory markers and associated diseases in humans remains inconclusive [114].

Excessive folate intake can negatively impact innate immunity. In postmenopausal women, high levels of folic acid intake through supplements and a folate-rich diet were found to reduce NK cell cytotoxicity [115]. Unmetabolized folic acid (UMFA) was inversely associated with NK cell cytotoxicity in the same study [115]. Prenatal and periconceptual FA supplementation and particularly excess FA was associated with an increased risk of childhood asthma in several studies [116,117,118,119,120,121], However, others could not find a clear association [122,123,124,125]. Interestingly, late pregnancy FA supplementation as well as the extended duration of the supplementation (over 6 months) was associated with the highest risk of childhood asthma [63,121]. Similarly, FA supplementation in the first trimester of pregnancy correlated with a higher probability and severity of bronchiolitis compared to non-supplemented mothers in the USA [126]. FA supplementation of pregnant women was associated with an increased risk of wheeze and lower respiratory tract infection in the offspring up to 18 months of age in a Norwegian cohort [127]. Conversely, others reported that high plasma folate in mid pregnancy was linked to decreased odds of wheezing at age 3 [128]. In a recent systematic review and meta-analysis, Chen et al. concluded that FA intake can be a risk factor for respiratory tract allergies in infants and children, however, a significant association was found only for doses not exceeding 400 ug/day, while the risk effect decreased with increasing FA dose. Moreover, the association between FA supplements and children’s risk of allergic diseases was significantly increased only in countries without FA fortification. This increase in risk was more pronounced in cases where mothers used folic acid supplements [129]. Others reported that high folate and B12 levels during pregnancy were associated with a higher prevalence of atopic dermatitis in children [130], while third trimester FA supplementation was associated with eczema in children but no other allergic outcomes [131].

In preclinical models, high dose FA (50mg/kg-diet) administered intraperitoneally to a murine model of colitis improved associated inflammation [132], however, others found that a high methyl donor diets including 5mg/kg-diet FA increased susceptibility to colitis in offspring [133,134]. Similarly, excess FA supplementation to rat dams (20mg/kg diet) exacerbated weight gain and inflammation [135]. Our own research demonstrated that high folic acid exposure negatively affected human lymphocyte stress response, genomic stability, and DNA methylation in both in-vitro and in-vivo conditions [136,137].The relationship between FA supplementation and immune-related outcomes is complex, with varying results depending on factors such as supplementation timing, dosage, food fortification, and more. Further research is needed to elucidate the potential mechanisms underlying these associations and to determine the optimal FA supplementation strategies for minimizing immune-related risks.

3.5. Carcinogenesis

Due to the importance of folate cycle in DNA replication and repair, inadequate consumption of folate is considered an independent risk factor for cancer. A low folate diet leads to uracil misincorporation into the DNA leading to single and double stranded breaks [138]. On the other hand, excess folate may increase cancer risk in the presence of preneoplastic lesions [4]. In rapidly proliferating pre-neoplastic or transformed cells, excess folate could help sustain high rates of growth by providing thymidylate and purines for DNA synthesis.

Colorectal cancer (CRC) is the second most prevalent cancer in western societies, and it is closely associated with folate intake. Following the fortification mandate, a small increase in the incidence of colorectal cancer in the USA and Canada was observed [139]. This association has proven controversial as data from human and animal studies yielded contradictory results. Higher folate intake in natural and synthetic forms was associated with decreased risk of CRC in women participating in the nurses’ health study [140]. Others found that vitamin B12 and folic acid supplementation significantly increased CRC incidence [139] and increased overall cancer incidence and mortality [141]. Similarly, a meta-analysis found a borderline significant increase of overall cancer risk with FA supplementation[142]. Another independent meta-analysis of RCT’s did not find any significant effect of FA supplementation on overall cancer or colorectal cancer risk [143]. Supplementation of FA at 1mg/d was associated with increased multiplicity of colorectal adenomas, advanced lesions, and increased risk of prostate cancer in some studies [144,145,146]. However, In a recent systematic review, High folate intake was associated with reduced risk of colorectal cancer is the USA and Europe, particularly among people with middle or high alcohol consumption [147].

The association of folate intake with the incidence of non-CRC cancers is also inconsistent and is reviewed elsewhere [148]. Higher intake of folate was associated with a reduced risk of squamous cell carcinoma of the head and neck [141], esophageal [149,150,151], oral [152], pancreatic [153,154], and bladder cancers [155,156]. We should note that the higher intake in these studies were unlikely to represent excess folate intake and the analysis was not stratified to isolate the effects of intake that approach or exceed the UL. No clear association between high folate intake and lung cancer was observed in a meta-analysis [157], However, a study from Poland found that high systemic folate levels (RBC folate concentrations above 506.5 nmol/l) increased lung cancer risk in heavy smokers[158]. High intake of folate was not protective against ovarian cancer [159]. Despite an overall marginally negative association between endometrial cancer and folate intake, at higher intake levels ranging between 205-987mcg/day, cancer risk increased in a dose dependent manner suggesting a threshold effect [160].

Conflicting associations were found between folate intake and breast cancer risk [161,162,163]. In a meta-analysis of by Chen et al., a U-Shaped relationship was observed in prospective studies where moderate folate intake (153-400mcd/day) decreased breast cancer risk compared to <153mcg/day, while intake >400mcg had no effect [162]. In another analysis by Zhang et al., moderate folate intake between 200-320 mcg/day lowered the risk of breast cancer, while a significantly increased risk was observed with a daily folate intake exceeding 400mcg [161]. recent studies concluded that high intake of one carbon related vitamins including folic acid may be protective against estrogen-receptor-negative/progesterone receptor negative tumors and in individuals with moderate to high alcohol consumption [163,164].

Studies in rodents revealed a complex and sometimes contradictory relationship between folic acid intake and tumorigenesis. The difference in models, timing, duration, dose, and experimental design led to inconsistent findings in rodents. The impact of folic acid supplementation on colorectal cancer in rodents was reviewed in details elsewhere [165]. Notably, in some rodent studies that used high doses of folic acid (8mg FA kg⁻¹), supplementation led to increased occurrence of preneoplastic lesions and CRC [149,150,151]. Increased proliferation of colorectal epithelia and intake of folate after the development of neoplastic lesions was hypothesized to be responsible for the observed increased risk. In an inducible breast cancer mouse model, high FA (10 mg kg⁻¹) significantly increased tumor volume compared to baseline diets (2mg/kg)[166].

Moderately high FA supplementation (5mg FA kg⁻¹) prior to and during pregnancy and lactation in rats increases the risk and multiplicity of mammary adenocarcinomas in the offspring and reduced global DNA methylation [167]. In another rat model, FA supplementation (ranging from 5-10mg FA kg⁻¹) promoted the progression of established mammary tumors [168]. Restricting FA in the diet lowered the incidence of neuroblastoma in the offspring of a predisposed pregnant mice model receiving a low FA diet [169]. In a chemically inducible prostatitis model, maternal folic acid supplementation at a dose of 2mg/kg in rats led to a heightened incidence and severity of inflammation in their offspring [140]. This is especially relevant to the observation that folic acid supplementation was associated with increased risk of prostate cancer in humans [170]. However, further research is needed to validate the effects and explore the underlying mechanisms.

In cell culture studies, FA depletion and supplementation enhanced the transformation of keratinocytes infected by the human papilloma virus [171,172]. Furthermore, depletion and over-supplementation of FA led to increased progression, migration, and invasion of the hepatocarcinoma HepG2 cells [173]. In human lymphoblastoid cell lines, we found that excess FA can mimic folate restriction by similarly impacting oxidative stress response, DNA damage repair, and DNA methylation [136]. The relationship between excess folic acid intake and carcinogenesis presents complex and often contradictory findings. Further research is necessary, particularly to investigate the effects of high levels of folate intake, and better understand the underlying mechanisms and potential risks. It is crucial to consider human genetic polymorphisms, genetic backgrounds, diets, environment, smoking, and other factors that may confound study outcomes. By addressing these factors, future studies can help elucidate the optimal levels of folate intake for various populations and contribute to more accurate public health recommendations.

3.6. Morbidity and mortality.

Numerous studies have sought to determine the relationship between high folate intake, chronic disease risk, and overall mortality, but the results remain inconclusive. A systematic review by Colapinto et al. observed a negative association between high folate levels and adverse health outcomes. However, the variability in study methods, designs, and high folate cutoffs prevented definitive conclusions [18]. A large UK population cohort (n=115664) found that higher dietary folate intake correlated with lower CVD risk and overall mortality[174], and a similar trend was noted in a recent NHANES survey data analysis [175]. Other studies, however, did not find a significant association between high folic acid intake and overall mortality [176,177]. A prospective analysis of 2011-2014 NHANES data revealed a higher mortality risk associated with increased serum folate levels, such as 5-mTHF, UMFA, non-methyl folate, and MeFox (a biologically inactive degradation product of 5-mTHF), as well as with 5-mTHF insufficiency [178]. Another cohort study based on 1999-2010 NHANES data reported a modestly elevated all-cause and cardiovascular mortality in participants with high folate levels (>40 nmol/L) [179].

The impact of high folate levels on overall mortality remains unclear, but some studies have offered insights into the relationship between folate intake and non-cancer health outcomes, particularly metabolic and cardiovascular diseases. A recent systematic review and meta-analysis found that folate supplementation improved fasting glucose, insulin resistance, and insulin levels but had no effect on diabetes or HbA1c [180]. A large meta-analysis of randomized controlled trials did not establish a connection between folic acid supplementation and reduced cardiovascular risk, although a modest benefit was observed in stroke prevention[181]. However, high folate intake was associated with an increased risk of thyroid and endocrine disorders [5,181]. Studies in rats comparing the effects of 5-m-THF and folic acid supplementation on metabolism showed that high doses of either form led to distinct adverse and harmful effects [182]. Research comparing the effects of high intakes of natural and synthetic folate on health and disease is of particular significance, given that very few comparative studies have been conducted in human and animal models. Future investigations into the consequences of excess folate should consider the impacts of supplementation with natural folates, such as 5-m-THF and 5-formyl-THF, in comparison to folic acid.

Folic acid supplementation is commonly used to treat hyperhomocysteinemia, a condition characterized by elevated homocysteine levels [178,183]. Homocysteine is a risk factor for stroke, cardiovascular disease, cognitive function, and adverse pregnancy outcomes [184,185,186,187]. In two separate studies, folic acid supplementation at 0.5 and 5 mg per day were similarly effective at reducing homocysteine levels[188,189]. A dose-finding interventional trial concluded that long-term doses as low as 0.2 mg of folic acid or lower effectively lowered homocysteine levels, with no further significant reduction observed at higher doses[190]. However, a high dose of folic acid (5 mg/day) for three months failed to normalize homocysteine levels in over 40% of participants in a recent study [191].

The relationship between high folate intake, chronic disease risk, and overall mortality remains unclear due to inconsistent results from various studies. Caution must be exercised in interpreting these findings, as the studies employed different methodologies and high folate cutoffs. Given the equivocal results, additional research is needed to better understand the complex relationship between high folate intake and its potential impact on chronic disease risk and overall mortality.

4. Mechanisms Underlying the Adverse Effects of Excess Folate Intake

Several potential mechanisms have been proposed to explain the effects of excess folate. In this section, we will discuss known potential molecular and genetic mechanisms that are hypothesized to mediate the deleterious effects observed in human and animal studies. (Figure 2)

4.1. Excess folate and B12 dependent mechanisms

The aging population is particularly at risk for vitamin B12 deficiency, and there is concern that folic acid (FA) supplementation might mask and exacerbate the severe, potentially irreversible neurological damage caused by B12 deficiency [183,184,192,193]. Excess FA can conceal hematological and neurological symptoms of B12 deficiency, worsening the condition and delaying diagnosis, leading to irreversible morbidities [3,194]. Reynolds et al.’s comprehensive review highlighted evidence that long-term exposure to excess FA, even at doses below the upper limit (0.5-1 mg), combined with B12 deficiency, can lead to neurological harm and cognitive decline [192]. FA supplementation may increase the demand for vitamin B12, exacerbating its depletion, pernicious anemia, and associated neurological and hematological decline [195,196,197]. Following fortification mandates in countries like the United States and Australia, epidemiological studies found a link between excess folic acid and cognitive decline in older adults at risk of B12 deficiency [198,199,200,201]. However, there is still no consensus regarding the role of excess FA in potentiating neurological and cognitive impairment in individuals with low or deficient B12 status [202,203].

A B-vitamin imbalance resulting from high FA combined with inadequate B12 may lead to a functional folate deficiency, contributing to insulin resistance and gestational diabetes mellitus [204]. In pregnant rats, high FA supplementation combined with a B12-deficient diet led to oxidative stress in both the mothers and pups, compared to the same supplementation with adequate B12 status [205]. Excess FA supplementation alongside B12 deficiency during pregnancy and lactation resulted in diet-dependent metabolic impairment in female rat offspring [206]. Similar studies in rats demonstrated that adequate B12 levels are required to mitigate the potential harmful effects of high FA on DNA methylation and neurodevelopment in offspring [207,208].

Mechanistically, the paradoxical combination of excess folate with B12 deficiency leads to unique metabolic situation known as the 5-Methyltetrahydrofolate Trap. The primary enzymatic reaction implicated in the methyl trap is catalyzed by methionine synthase (MTR), a vitamin B12 (cobalamin)-dependent enzyme. MTR catalyzes the conversion of homocysteine to methionine, with 5-m-THF acting as the methyl donor. During this process, 5-m-THF transfers a methyl group to cobalamin, generating methylcobalamin, which subsequently transfers the methyl group to homocysteine, yielding methionine and tetrahydrofolate (THF). THF is essential for purine and pyrimidine synthesis, which are required for DNA and RNA production. In situations of vitamin B12 deficiency, MTR activity is compromised, leading to an accumulation of 5-m-THF, as the enzyme is unable to utilize it as a methyl donor, leading to the phenomenon known as the methyl trap (Figure 3). This sequestration of folate in the form of 5-m-THF results in a functional folate deficiency, as the intracellular pool of THF is depleted. The reduced availability of THF impairs the synthesis of purines and pyrimidines, ultimately affecting DNA and RNA synthesis. Consequently, this disruption in nucleotide synthesis manifests as megaloblastic anemia, characterized by large, immature erythrocytes. Additionally, the vitamin B12 deficiency and the subsequent impairment of MTR activity leads to an accumulation of homocysteine, as it cannot be converted to methionine. Elevated homocysteine levels have been associated with an increased risk of cardiovascular disease, neurodegenerative disorders, and other adverse health outcomes.

The complex interplay between excess folate and B12 deficiency presents a significant challenge in understanding the potential consequences on health, particularly in vulnerable populations such as the elderly and pregnant women. The 5-MTHF trap phenomenon illustrates the critical need for a balanced intake of these essential B-vitamins and highlights the importance of monitoring their levels to prevent detrimental health outcomes. Nevertheless, further research is required to study the long-term effects of this phenomenon and determine the optimal balance of these nutrients. Such studies will be instrumental in establishing evidence-based guidelines and devising effective strategies to prevent, diagnose, and manage the health consequences arising from the interplay between excess folate and B12.

4.2. Mechanisms Related to the One-Carbon Cycle

The one-carbon metabolism plays a crucial role in numerous cellular processes, including DNA synthesis, repair, and methylation. Excess folic acid can disrupt the balance of this metabolic cycle, leading to various adverse health effects. The mechanisms related to the one-carbon cycle can be further divided into the following categories:

4.2.1. Pseudo-MTHFR Deficiency

MTHFR is an enzyme that catalyzes the conversion of 5,10-methylenetetrahydrofolate (5,10-MTHF) to 5-methyltetrahydrofolate (5-MTHF), the biologically active form of folate. This conversion is a critical step in the folate metabolic pathway, as 5-MTHF participates in the re-methylation of homocysteine to methionine. high folic acid intake can lead to an accumulation of unmetabolized folic acid in the bloodstream. The presence of excess folic acid could competitively inhibit the enzymes involved in the conversion of other forms of folate, such as dihydrofolate reductase (DHFR), which converts dietary folic acid to dihydrofolate (DHF) and subsequently to tetrahydrofolate (THF). This competitive inhibition can exacerbate an existing MTHFR deficiency by further disrupting the folate metabolic pathway, leading to a functional folate deficiency despite adequate folate intake. Polymorphisms in the MTHFR gene are associated with reduced enzyme activity and increased health risks. In humans C677T and A1298C polymorphism are common with an estimated 20-40% of the US population harboring one or both mutations [6]. Nevertheless, MTHFR inhibition by excess folate is not exclusively linked to cases where genetic polymorphisms are present. Even in the absence of known MTHFR polymorphisms, elevated levels of folic acid can still result in a pseudo-MTHFR deficiency, which hinders the conversion of 5,10-methylenetetrahydrofolate to 5-MTHF [61,107,209,210,211]. This pseudo-deficiency can disrupt the one-carbon cycle and negatively affect methylation reactions, potentially leading to various health issues, including abnormal embryonic growth and neurodevelopment [210].

4.2.2. Altered folate cycle and Methylation Patterns

Folate is vital for the generation of S-adenosylmethionine (SAM), the primary methyl donor for DNA methylation. However, excess folic acid can disrupt the balance of SAM and its demethylated product, S-adenosylhomocysteine (SAH). This disruption may lead to global DNA hypomethylation, resulting in genomic instability, altered gene expression, and an increased risk of diseases. Excess folic acid intake can cause an imbalance in the distribution of folate coenzymes and derivatives, leading to disruptions in the one-carbon cycle thereby impeding methylation reactions, embryonic growth, and neurodevelopment [210]. High FA levels were linked to dysregulation of intracellular one carbon metabolism [212]. Both deficient and excess maternal folic acid in mice have been shown to cause alterations in offspring’s folate metabolism, favoring DNA synthesis over methylation reactions and leading to cytoarchitectural defects and behavioral abnormalities [103]. Similarly, both folate deficiency and excess supplementation similarly compromised folate dependent pathways, resulting DNA damage, cell cycle arrest, and impaired hematopoiesis in mice [213]. Excessive FA led to abnormal cardiac development and defects in folate metabolism similarly to folate depletion using the antifolate drug methotrexate in a zebrafish model. Interestingly, both FA excess and insufficiency resulted in similar phenotypic and genotypic changes impacting methylation levels and expression of cardiac development related genes [214]. Both excess paternal FA supplementation and FA depletion in mice resulted in a similarly deleterious transgenerational outcomes potentially stemming from altered methylation of imprinted sperm genes [89]. Similarly, paternal FA supplementation in chicken induced transgenerational changes in glucose and lipid metabolism [210]. Excess FA administered during pregnancy, lactation, and post weaning impacted DNA methylation, gene expression, behavior, neural development, and disrupted folate metabolism in mice offspring [83,84,85,215,216,217].

4.2.3. Accumulation of Unmetabolized Folic Acid (UMFA)

High intake of folic acid can result in the accumulation of UMFA in the bloodstream, as the body’s natural capacity to convert folic acid to its active form, 5-methyltetrahydrofolate (5-MTHF), becomes saturated [1,218]. This excess UMFA can compete with and inhibit key enzymes in the one-carbon cycle, such as dihydrofolate reductase (DHFR) and methylenetetrahydrofolate reductase (MTHFR) [219]. As a result, essential reactions in the cycle can be disrupted, affecting DNA synthesis and repair, as well as methylation processes. However, UMFA is not known to accumulate in biological tissues and inside cellular compartments. Unlike reduced folate, UMFA has low affinity for the reduced folate carrier transporter (RFC) and is not a substrate for the enzyme folate polyglutamate synthase (FPGS) [220,221]. Since UMFA cannot participate in one carbon cycle metabolism and binds weekly to folate enzymes, it is also plausible that the potential effects of UMFA could be mediated by non-canonical pathways independent of one carbon metabolism.

4.3. Non-Canonical pathways

4.3.1. Folic acid specific pathways

Folic acid is the fully oxidized form of folate. It is a pre-vitamin that requires a 2-step reduction by DHFR to THF, the metabolically active form participating in the one carbon cycle. However, FA can bind to and be transported into tissues by the folate receptor (FOLR) and the proton coupled folate transporters (PCFT). Excess intake of FA is linked to saturation of the capacity of DHFR leading to accumulation of UMFA. DHFR activity in humans is variable and limited compared to lab rodents [218]. Unlike natural folates that are polyglutamated and retained within the cells, Folic acid in its unmetabolized form is not known to accumulate in tissues. Studies have shown that UMFA circulates at a low levels in individuals not taking supplementation, with more sustained elevation of UMFA recorded in adults ingesting 200ug to 400ug of FA daily [10]. Higher levels of UMFA generally correlate with increased folic acid intake [222,223]. Intakes of FA that surpass the UL are associated with an increased likelihood of having elevated UMFA levels [224,225,226,227,228].

The presence of UMFA is of interest as a potential causative factor in mediating the deleterious effects of excess FA. However, there are significant gaps in understanding the mechanisms involved [229]. one possible mechanism of interest involves the role of folate receptor (FOLR) in binding FA and mediating its transport and triggering potential downstream effectors. Folate receptors have much higher affinity to FA compared to natural folates. The FOLRα facilitates the intake of FA through receptor meditated endocytosis. FOLRα is ubiquitously expressed in the epithelia of normal tissues (kidney, placenta, blood-brain barrier), as well as being overexpressed in many cancers such as ovarian, breast, and colon cancers [230]. The role of FOLRα in mediating tumorigenesis as well as the associated physiological function in cancer are not clear [166,231]. In the presence of FA, FOLRα could translocate into the nucleus and function as a transcription factor impacting genes responsible for cellular pluripotency [222,223]. In fact, FA bound FOLR may function as signaling molecule activating downstream targets such as the pro-oncogene STAT3, extracellular signal-regulated kinases (ERK) kinases, and cellular Sarcoma (SRC) pathways [224,225,226,227,228].

Folic acid, but not reduced folate were linked to regulation of neurogenesis in the ventral hippocampus in rats [229]. Folic acid could also be involved in modulating endothelial NO synthase activity via post translational modifications [230]. A study in piglets found that a high FA diet altered the expression of liver proteins involved in oxidative responses, metabolic regulation, and cancer pathways compared to a control diet (30mg FA/kg vs 1.3 mg FA/kg diet) [232]. Periconceptual excess FA led to upregulated Fos expression in mice [83], and activation of β-catenin (Wnt pathway) in the brain of male mouse offspring [108]. In studies that compared the effects of supplementing excess synthetic and natural folate in rats, the authors observed contrasting metabolic shifts leading to weight gain, leptin dysregulation, and increased food intake int the female offspring when dams where fed excess 5-m-THF compared to excess FA, with FA significantly impacting neurotransmitter signaling genes [182,233]. Taken together, these studies suggest the existence of non-canonical pathways that could mediate the effects of excess folate, both synthetic and natural independently of one carbon cycle. Additional exploratory work and research is needed to fully elucidate the mechanisms involved.

4.3.2. Mechanisms related to elevated folate

Determining the specific effects of excess folic acid intake from elevated folate status is a difficult task. In individuals with elevated folate status the widespread use of folic acid supplementation contributes significantly to folate intake, compared to natural dietary sources. Naturally occurring folate in unfortified foods generally is characterized by its limited content, stability, and bioavailability compared to synthetic folic acid. Consequently, individuals who consume excessive amounts of folate are more likely to be supplemented with synthetic folic acid, potentially surpassing their relative intake of natural dietary folate. Determining whether exogenous folic acid intake or the resulting elevated folate status is responsible for the observed effects is a significant challenge. Nonetheless, studies investigating the relationship between high folate levels and adverse health outcomes managed to identify possible underlying mechanisms.

One area that has received significant research attention pertains to the effects of excess folate on carcinogenesis. The precise causal mechanism underlying folic acid’s role in promoting carcinogenesis remains unclear. Excess folate could elevate cancer risk when preneoplastic lesions are present [4]. In rapidly proliferating pre-neoplastic or transformed cells, an abundance of folate could support accelerated growth by supplying thymidylate and purines for DNA synthesis [234]. Furthermore, folate availability has a direct and substantial influence on DNA methylation. Disturbances in folate levels, arising from either low or high folate status, can lead to hypo- or hypermethylation, resulting in epigenetic instability [235,236,237]. Disruption of normal DNA methylation patterns can lead to the dysregulation of gene expression and genomic instability, ultimately contributing to the initiation and progression of cancer. DNA hypomethylation is considered an early event in colon carcinogenesis [238]. Excess FA is capable of inducing hypomethylation in cultured human cells [239], and alteration in methyl metabolism in mice [104,209]. Work in multiple models suggest that both folate deficiency and excess FA can disrupt the folate cycle and the connected methionine cycle leading to dysregulated methylation [103,210,240,241]. In our investigations involving human lymphocytes, we observed that high folic acid intake relative to natural folate, can influence DNA methylation, disrupt genomic stability, and diminish the capacity to withstand with oxidative stress [27,136]. Excess folic acid supplemented to cancer cell organoid, can counteract methionine dependency, thus promoting cancer stem cell formation [242]. In mice, a high FA diet promoted hepatocellular carcinoma development through stabilization of the methionine cycle enzyme MATIIα. In prostate cancer, folate was found to be directly involved in oncogenic signaling pathways, specifically the PI3K-AKT-mTOR cascade [243]. In our study exploring the impact of folate restriction on colon carcinogenesis, we observed a significant increase in mTOR signaling in mice supplemented with folic acid compared to those without supplementation [26].

The role of excess folate in genomic methylation and epigenetic reprogramming is not limited to cancer etiology. As previously mentioned, excessive folate intake during pregnancy has been linked to both adverse and favorable birth outcomes, as well as an elevated risk of disease in offspring, as demonstrated in both human and animal studies. Folic acid supplementation impacts one-carbon metabolism during embryogenesis, which in turn leads to changes in genome wide and gene specific methylation patterns [82]. In rodent models, excess maternal folic acid supplementation has been demonstrated to alter methylation and expression of imprinted genes, histone modifications, and heterochromatin assembly, However the underlying epigenetic mechanism is still unknown [71,82]. In humans, maternal FA supplementation was linked to changes in DNA methylation of genes related to brain development with beneficial effects on cognitive development [80,92,93]. The in-vivo and in-vitro mechanisms through which excess maternal FA supplementation can influence neurological development were reviewed elsewhere [244], detailing behavioral, morphological, and molecular changes in the offspring’s brain exposed to FA over-supplementation. A recent investigation into some of the underlying mechanisms discovered significant changes in DNA methylation of neurodevelopmental genes, which play a crucial role in transcriptional regulation [245]. Notably, these modified epigenetic patterns persisted throughout an individual’s life, as demonstrated in the Aberdeen Folic Acid Supplementation Trial [246]. Although both animal and human studies have identified a correlation between excessive maternal FA intake and metabolic abnormalities in offspring, a clear causative link with the modified epigenetic landscape has not yet been established [68,76,77,78,247,248]. Further research is required to establish if epigenetic reprogramming is partially or wholly responsible for both the beneficial and adverse effects of excess folate.

Both folate deficiency and excess are known to impact the immune system and its function which could result in increased susceptibility to inflammation, chronic diseases, and cancer. Lymphocytes are especially sensitive to changes in the folate pool due to high proliferation rate, making them a robust biomarker for folate status [138,251]. Excess folic acid intake is also linked to impaired natural killer cell activity among postmenopausal women, healthy adults, and aged mice [115,249,250]. Both folate deficiency and excess impaired metabolism and hematopoiesis in mice [213]. Our findings in human lymphocytes aligned with these observations, as we identified impaired DNA repair and oxidative stress response under conditions of excess folic acid and high folic acid to natural folate intake ratio, both in-vitro and in-vivo settings, respectively [136,137]. Others found that folic acid supplementation could promote a proinflammatory transcriptomic milieu in the colon [251,252], and, that excess FA increased inflammation in response to high fat diet in rats [253]. It remains an open question whether the observed perturbations in the immune system and inflammatory response are responsible for some of the deleterious effects of excess folate.

Recently, the one-carbon cycle has been identified as a central regulator of organismal aging [254,255]. Metabolomic analysis of long-lived C. elegans and mouse models revealed common signatures and regulation of the one-carbon folate cycle. In a robust analysis, Annibal et al. demonstrated that supplementing long-lived C. elegans mutants with physiological levels of folate partially abrogated the lifespan extension, while RNAi knockdown of the key enzyme DHFR extended it in the wild-type worms [254]. This work highlighted the folate cycle as a shared causal mechanism for longevity. In our preliminary work with mice, we observed increased longevity in wild-type C57/Bl6 mice on a folic acid-restricted diet compared to those receiving folic acid supplemented diet (86% vs. 70% survival rate at 690 days, respectively). Similarly, our pilot work revealed that exposing C. elegans nematodes to the DHFR inhibitor methotrexate significantly increased the worm’s lifespan, while supplementing the media with either folic acid, 5-formyl-THF (leucovorin), or 5-methyl-THF significantly shortened lifespan in the wildtype N2 worms in a dose-dependent manner.

5. Conclusions

As researchers, including ourselves, strive to unravel the conserved and intricate dynamics governing the relationship between folate, health, and longevity, a crucial question arises: could exposing the human population to excessive levels of folate potentially accelerate aging and increase the risk of disease?

The key to answering this question lies in identifying causal underlying mechanisms that can robustly link excess folate intake to the observed detrimental effects. Achieving this goal requires substantial research efforts and collaboration across multiple scientific disciplines to identify and address the existing knowledge gaps. The importance of pursuing such research cannot be overstated, as these gaps in knowledge may be unknowingly and insidiously compromising the health of hundreds of millions of people in the USA and worldwide who are consuming excess folate.