21.1: Folate (22a.1)

Folate is a generic term referring to both natural folates and folic acid (pteroyl­monoglutamic acid, PGA), the synthetic form used in supplements and fortified food. All folate forms comprise three moieties: a pteridine; a p‑amino­benzoic acid (PABA) and a glutamate residue (Figure 22a.1).

Figure 22a.1 The structure of tetra­hydro­folate (THF)

The parent compound, PGA, is completely oxidized and not found in nature. The natural folate forms are reduced molecules, with the addition of 2 or 4 hydrogen atoms to the pteridine, giving rise to dihydro­folate or the various tetra­hydro­folate (THF) forms. THF can carry one-carbon groups attached at the N‑5 (methyl, formyl or formimino), the N‑10 (formyl) or bridging N‑5 and N‑10 (methylene or methenyl) positions of the pteridine ring, giving rise to a number of different cofactor forms of folate. Also, whereas folic acid is a mono­glutamate, containing only one glutamic acid residue, most natural food folates exist as poly­glutamate derivatives containing additional glutamate residues bound in peptide linkage to the gamma-carboxyl group.

22a.1.1 Functions of folate

Folate coenzymes are required for one-carbon meta­bolism involving the transfer and ulilization of single-carbon atom units, including methylene (CH₂), forminino (CH=NH), methyl (CH₃), methenyl (CH), and formyl (CHO) groups (Figure 22a.2).

Figure 22a.2 Overview of Folate and related B vitamins in One-carbon Metabolism. Abbreviations. DHF, dihydrofolate; DHFR, dihydrofolate reductase; DMNT, DNA methyltransferase; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; MTHFR, methylenetetrahydrofolate reductase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; THF, tetrahydrofolate. Adapted from "Folate: Micronutrient Information Center, Oregon State University" (2021)

Folate is thus essential for key biolog­ical functions, including, serine and glycine meta­bolism, histidine cata­bolism, methionine synthesis and in thymidylate and purine bio­synthesis, precursors of DNA. Folate, in the form of 5‑methylTHF, along with vitamin B₁₂, is required for the synthesis of methionine from homo­cys­teine (catalyzed by the enzyme methionine synthase), and in turn, the synthesis of S‑adenosyl­methionine (SAM). This methyl group donor is used in numerous biolog­ical methy­lation reactions, including the methy­lation of a number of sites within DNA, RNA, proteins, and phospho­lipids. The reader is referred to Bailey et al. (2015) for a detailed review of the functions of folate.

Folate inter­action with other B vitamins.For folate to function within one-carbon meta­bolism, it interacts closely with vitamin B₁₂, vitamin B₆and ribo­flavin (McNulty et al., 2019). Reduced folates enter the one-carbon cycle as THF which acquires a carbon unit from serine in a vitamin B₆‑dependent reaction to form 5, 10 methyleneTHF. This co-factor form, once formed either converted to 5‑methylTHF or serves as the one-carbon donor in the synthesis of nucleic acids, where it is required by thymidylate synthetase in the conversion of deoxy­uridine (dUMP) to deoxy­thymidine mono­phosphate (dTMP) for pyrimidine biosynthesis, or is converted to other folate forms required for purine biosynthesis. Methylene­tetra­hydro­folate reductase (MTHFR) is a ribo­flavin-dependent enzyme that catalyzes the reduction of 5, 10 methyleneTHF to 5 methylTHF, the folate form used by methionine synthase for the vitamin B₁₂-dependent conversion of homo­cys­teine to methionine and the formation of THF. Methionine is activated by ATP to form S-adenosyl­methionine (SAM), which then donates its methyl group to more than 100 methyl­trans­ferases for a wide range of substrates such as DNA, hormones, proteins, neuro­trans­mitters and membrane phospho­lipids, all of which are regulators of important physio­logical processes (Bailey et al., 2015). In summary, effective folate functioning requires essential meta­bolic inter­action with vitamins B₁₂, B₆ and ribo­flavin. Thus sub-optimal status of one or more of these B vitamins, or poly­morphisms in folate genes, can impair one-carbon meta­bolism, even if folate status is sufficient.

Absorption and transport of folate. Folate absorption takes place by an active process, primarily from the proximal part of the jejunum. Before absorption, the poly­glutamate forms of folate are decon­jugated to the mono­glutamate form in the gut by the intestinal brush-border enzyme glutamate carboxy­peptidase II (GCPII), more commonly termed “folate conjugase”. The mono­glutamates are then taken up by the mucosal cells. Folates require transporters to cross cell membranes; these include the reduced folate carrier (RFC), the proton-coupled folate trans­porter (PCFT), and the folate receptor proteins, FRα and FRβ. Folic acid is a mono­glutamate and thus does not require decon­jugation before uptake by intestinal cells.

Most of the folate mono­glutamates absorbed from the gut are transported to the liver (the major storage organ for folate) and are reconverted intra­cellularly within hepato­cytes to poly­glutamate derivatives by the enzyme folyl­poly­glutamate synthetase. These folate poly­glutamates are stored in the liver or converted to 5‑methyl­tetra­hydro­folate for secretion into the bile, and then reabsorbed by way of the entero­hepatic circulation. This recircu­lation process may account for as much as 50% of the total folate that reaches the periph­eral tissues. Circulating folate in blood is found in the mono­glutamate form, predominantly as 5‑methyl­tetra­hydro­folate.

22a.1.2 Deficiency of folate in humans

Severe deficiency of folate leads to megalo­blastic anemia, presenting as fatigue, weakness, and shortness of breath owing to a low red blood cell count. This condition is hemato­logically characterized by the presence of immature, enlarged nucleated cells, reflecting impaired DNA synthesis as a result of folate depletion. Rapidly prolif­erating cells are especially sensitive to abnormalities in DNA synthesis. Hence, the manifes­tations of folate defi­ciency appear first in the hemato­poietic system and then in the epithelial cell surfaces and the gonads. Abnormal cell replication in the hemato­poietic system, manifested by hyper­segmented neutro­phils, is one of the earliest morpho­logical changes. Later, megalo­blasts appear in the bone marrow and macro­cytes in the peripheral blood. Some additional signs and symptoms that have been reported with clinical folate defi­ciency include fatigue, angular cheilosis, anorexia, insomnia, glossitis, recurrent aphthous ulcers, and pallor of the skin and mucous membranes. Of note, the megalo­blastic anemia resulting from folate defi­ciency is identical to that resulting from vitamin B₁₂ defi­ciency, and therefore specific bio­marker testing is essential to provide a differential diagnosis (See Section 22a.2).

Causes of low and deficient folate status. Folate defi­ciency arises from various causes, relating either to increased requirements, reduced avail­ability, or both. Pregnancy is a time when folate requirement is greatly increased in order to sustain the demand for rapid cell replication and growth of fetal, placental and maternal tissue (McNulty et al., 2019). Gastro­intestinal conditions, such as celiac disease, can also lead to deficient folate status through chronic malab­sorption. Certain drugs, including phenytoin and primidone (anti­convulsants) and sulfa­salazine (used in inflammatory bowel disease), are also associated with folate defi­ciency through adversely affecting folate meta­bolism. Like­wise, heavy alcohol consumption and smoking are also linked with lower folate status (Bailey et al., 2015).

Prevalence of defi­ciency. Folate defi­ciency occurs commonly in pregnant and lactating women in low- and middle-income countries, where it is reported to occur in > 20% of women of repro­ductive age (Rogers et al., 2018). Here, dietary intakes of folate are often inadequate to meet the high requirements of pregnancy.

In high-income countries, clinical folate defi­ciency is less common (due largely to the beneficial effects on folate status of supple­men­tation and/or food fortifi­cation), but subclinical defi­ciency (indicated by low serum and RBC folate concen­trations) is widespread, particularly in women of reproductive age, especially if pregnant and lactating, in European countries. Low folate status has also been reported in low birth weight and premature infants (Scholl & Johnson, 2000) and adolescents of low socio­economic status (Bailey et al., 2015). Serum and RBC folate concen­trations typically decrease throughout pregnancy; however, supple­men­tation with folic acid prevents this decline and can thus prevent the occurrence of megalo­blastic anemia of pregnancy (Blot et al., 1981; McNulty et al., 2013). Estimates of folate defi­ciency can however vary consid­erably across different populations and population sub-groups, depending on the method used and the cutoff points applied to bio­marker measures of folate status (Bailey et al., 2015).

Neural tube defects. Maternal folate nutrition before and in early pregnancy is known to play a critical role in fetal development. Notably, conclusive scientific evidence published 30y ago shows that folic acid supple­men­tation with folic acid (the synthetic form of the vitamin) in early pregnancy protects against both first occurrence (Czeizel & Dudás, 1992) and recurrence (MRC, 1991) of neural tube defects (NTDs). These major birth defects occur as a result of a failure of the neural tube to close properly in the first few weeks of pregnancy, leading to death of the fetus or newborn, or to various disabilities involving the spinal cord, the most common form of which is spina bifida. The conclusive evidence that folic acid can prevent NTD has led to clear folic acid recommendations for women of reproductive age which are in place worldwide. It is important to appreciate, however, that the risk of NTD is increased when maternal folate status is low, but not necessarily within the range typically classed as folate defi­ciency. In a large prospective study of women in Ireland (where rates of NTD are among the highest in the world), a woman's risk of having a child with an NTD was found to be strongly associated with pregnancy RBC folate in a continuous dose-response relation­ship (Daly et al., 1995).

In practice, implementing folic acid recommendations so that women and their babies benefit, is somewhat challenging. Although mandatory folic acid fortif­ication of foods has proved to be highly effective in reducing NTD wherever it has been introduced (in 84 countries worldwide), elsewhere preventable NTDs are not being prevented including in European countries. Notably, one recent study estimated that from 1998 to 2017, a total of 95,213 NTD pregnancies have occurred amongst 104 million births in 28 European countries; a prevalence of 0.92 per 1,000 births (Morris et al., 2021). This study concluded that failure to implement mandatory folic acid fortif­ication in the 28 European countries continues to cause NTD to occur in almost 1,000 preg­nancies every year.

The precise mech­anism explaining the beneficial effects of peri­concep­tional folic acid against NTD remains uncertain, though proposed mech­anisms have focussed on factors that impair normal folate meta­bolism, including poly­morphisms in folate genes. Among the latter, an increased risk of NTD is most strongly associated with the 677C>T variant in the gene encoding the folate-meta­bolising enzyme methylene­tetra­hydro­folate reductase (MTHFR), as reported in most studies and meta-analyses (Botto & Yang, 2000; Vollset & Botto, 2004). Auto­anti­bodies against folate receptors are also implicated in preg­nancies affected by NTD (Rothenberg et al., 2004). Also, although low maternal folate is considered the major contributing factor in NTD, convincing evidence shows that low vitamin B₁₂ is an independent risk factor in NTD (Molloy et al., 2009). In addition, apart from preventing NTD, there is good evidence that peri­concep­tional folic acid use may prevent congenital heart defects in infants (van Beynum et al., 2010), and possibly orofacial clefts (Bailey et al., 2015).

22a.1.3 Food folate sources, bio­avail­ability and dietary intakes

Folates are widely distributed in animal and plant foods, but only certain foods are rich sources, including liver, yeast, green leafy vegetables, asparagus, beans, legumes, where they exist primarily as poly­glutamates, containing up to nine glutamate residues attached to the p‑amino­benzoic group of the molecule (Figure 22a.1). In contrast, folic acid, the synthetic vitamin, is a mono­glutamate, containing only one glutamic acid residue in its structure. Also, unlike folic acid, which is a fully oxidized molecule, natural folates are reduced at the 5, 6, 7 and 8 positions of the pteridine ring and so are prone to oxidative cleavage at the C9‑N10 bond producing two degradation products, a pteridine and p‑amino­benzoyl­glutamate, both of which are inactive and cannot be biolog­ically converted to any active folate form.

Folate bio­avail­ability. Bioavail­ability refers to the proportion of ingested nutrient that is absorbed and available for meta­bolic processes. Naturally-occurring food folates are only partially bioavail­able (McNulty & Pentieva, 2004). Natural food folates are reduced molecules, and therefore are inherently unstable outside living cells. In addition, the ease with which folates are released from different food matrices and their conversion to the mono­glutamate form before uptake by intestinal cells can vary greatly. Folate bio­avail­ability from different food sources is also dependent on the presence of certain dietary constituents, that may enhance folate stability during digestion, or inhibit bio­avail­ability owing to specific inhibitors of deconjugation. Thus, the bio­avail­ability of food folates from a mixed diet is limited and highly variable. In addition, natural food folates (particularly green vegetables) can be unstable during cooking, and this can substantially reduce the folate content of a food before it is ingested (McKillop et al., 2002). This is an additional factor that further contributes to the limited potential for natural food folates to positively influence folate status (Cuskelly et al., 1996).

Dietary folates. In contrast to the natural folate forms which have limited stability and bio­avail­ability, folic acid provides a highly stable and bio­available vitamin form. The bio­avail­ability of folic acid is assumed to be 100% when ingested as a supple­ment, while folic acid in fortified food is estimated to have about 85% the bio­avail­ability of supple­mental folic acid (Pfeiffer et al., 1997). These differences have led to the develop­ment of “dietary folate equivalents” or DFE values; more details are given in Section 8a. Briefly, expressing dietary folate intakes and recommend­ations in DFE terms, allows an adjust­ment for the differences in bio­avail­ability between natural food folates and the synthetic vitamin. The DFE is defined as the quantity of natural food folate plus 1.7 times the quantity of folic acid in the diet; this definition is based on the assumption that the bio­avail­ability of folic acid added to food is greater than that of natural food folate by a factor of 1.7 (Institute of Medicine, 1998). This estimation is largely dependent on a meta­bolic study in non-pregnant women that estimated the bio­avail­ability of food folates to be 50% relative to that of folic acid (Sauberlich et al., 1987), and other evidence, mentioned above, showing that folic acid added to food has 85% of the bio­avail­ability of free folic acid (Pfeiffer et al., 1997).

Fortified foods as a source of folate. Food fortif­ication, the process of adding essential micro­nutrients to food, plays an important role in facilitating a more optimal nutritional status in individuals and populations. In the case of folate, it plays a crucial role. Folic acid, the folate form used for food fortif­ication, is cheap to produce, very stable once added to foods and highly bioavailable when ingested. Thus, depending on national fortif­ication policy and/or access to folic acid-fortified foods, the folate status of populations can vary greatly from one country to the next This is reflected in differences in health outcomes (notably in relation to NTD risk). Food fortif­ication may be under­taken on a voluntary or mandatory basis. Voluntary fortif­ication, whereby folic acid is added to foods such as break­fast cereals at the discretion of the manufac­turer, is permitted in most European countries. This results in higher folate intake and status (Hopkins et al., 2015), but the benefit is limited only to consumers who choose to eat the fortified food products. However, when fortif­ication is undertaken on a mandatory (population-wide) basis, including in the USA, Canada, Australia and Chile, it has proved to be highly effective, not only in increasing folate status and reducing folate defi­ciency (Yang et al., 2010), but also in reducing NTD in that country (Honein et al., 2001, Lopez-Camelo et al., 2005, De Wals et al., 2007, Sayed et al., 2008). Mandatory fortif­ication is now in place in 85 countries worldwide, both high‑ and low-middle income countries

Effects of high intakes of folate. High folate intakes are generally not assoc­iated with any adverse effects. However, there are concerns of potential adverse effects of excess intakes of folate acid, the synthetic vitamin form. Excessive folic acid intake consti­tutes exposure doses that exceed the Tolerable Upper Intake Level (UL) of 1000µg/d for adults, as set by the U.S. Institute of Medicine (1998).

Historically the concern regarding excess folic acid focused on the potential to mask pernicious anemia and exacerbation of the clinical effects of vitamin B₁₂ defi­ciency. More recently other concerns have been raised, including potential adverse effects on cancer risk, birth outcomes, and other diseases. However, a recent report from a 2019 expert workshop tasked with reviewing this research area, concluded that there is an insufficient body of evidence to support adverse human health outcomes as a result of high intakes of folic acid. None­theless, these experts called for further research to determine the safety of excess folic acid intake (Maruvada et al., 2020).