Metabolic role of folate

Folate plays an essential role in C₁ metabolism where it acts as a cofactor in DNA synthesis and repair, methylation and amino acid reactions (Fig. 1). Within this network, folate coenzymes function in mediating the transfer and utilisation of C₁ units in metabolic pathways involving interaction with vitamin B₁₂, vitamin B₆ and riboflavin⁽Reference Bailey, Stover and McNulty²⁾. Reduced folates enter the C₁ cycle as tetrahydrofolate (THF) which acquires a carbon unit from serine in a vitamin B₆-dependent reaction and subsequently forms 5,10-methylene THF, which is required for the synthesis of nucleic acids, or converted to 5-methyl THF. Methylenetetrahydrofolate reductase is the riboflavin (FAD)-dependent enzyme that catalyses the reduction of 5,10-methylene THF to 5-methyl THF. Within the methionine cycle, 5-methyl THF is required for the remethylation of homocysteine to methionine via the vitamin B₁₂-dependent enzyme methionine synthase. Methionine, in turn, is required for the generation of S-adenosylmethionine (SAM), the essential methyl donor for innumerable genomic and nongenomic methylation reactions required for the nervous system⁽Reference Bailey, Stover and McNulty²⁾. This pathway is essential for the methylation of DNA, by DNA methyltransferases using SAM as a cofactor, which can play a key role in controlling gene expression in a process referred to under the umbrella term epigenetics⁽Reference Armstrong⁶⁾. Methylation is also essential in the synthetic pathways of neurotransmitters (dopamine, noradrenaline and serotonin), myelination and phospholipids and is thus important for normal brain function. Over the past 40 years, the association between neurology and B-vitamin status has been extensively investigated, with evidence that folate deficiency can lead to aberrant methylation and could, in turn, affect neurocognition⁽Reference Reynolds⁷⁾.

Fig. 1. (Colour online) Overview of C₁ metabolism. BHMT, betaine homocysteine methyltransferase; CBS, cystathionine-β-synthase; CTH, cystathionine gamma-lyase; DHFR, dihydrofolate reductase; dTMP, deoxythymidine monophosphate; dTTP, deoxythymidine triphosphate; FAD, flavin adenine dinucleotide; DNMT, RNA methyltransferase; MAT, methionine adenosyltransferase; MS, methionine synthase; MT, methyl transferases; MTHFR, methylenetetrahydrofolate reductase; MTHFD, methylenetetrahydrofolate dehydrogenase; SAHH, S-adenosyl homocysteine hydrolase; SHMT, serine hydroxymethyltransferase; TS, thymidylate synthase. (Adapted from James et al.⁽Reference James, Sajjadi and Tomar¹³⁴⁾ Epigenetics, nutrition and infant health. In The Biology of the First 1000 Days, (KD Karakochuk, KC Whitfield, TJ Green, K Kraemer, editors). Florida: CRC Press).

Role of folate in pregnancy

The effect of folate status on pregnancy outcomes has long been recognised since the original discovery of folate by Lucy Wills in 1931 when marmite or other yeast extracts were found to be effective for the treatment of macrocytic anaemia in pregnant women⁽Reference Wills⁸⁾; later it was discovered that the active factor was folate.

Pregnancy is recognised as a time when folate requirements are increased to sustain the demand for rapid cell division and growth of fetal, placental and maternal tissue. This reflects the critical role folate plays in DNA, RNA and protein synthesis⁽Reference Bailey, Stover and McNulty²⁾. Along with the physiological changes related to the growth of maternal and fetal tissues, there is an expansion of plasma volume by 50 % compared with an increase in the erythrocyte mass by 25 %⁽Reference Milman, Byg and Agger⁹⁾, increasing the demand for folate. Globally, the most common causes of anaemia of pregnancy (defined as a Hb concentration of less than 11 g/dl, at any point during pregnancy⁽¹⁰⁾) are iron and/or folate deficiency, arising from increased fetal requirements and frequently aggravated by decreased maternal nutrient reserves⁽Reference Lee and Okam¹¹⁾. Numerous studies have illustrated that the prevalence of folate deficiency increases with advancing gestational age⁽Reference Ackurt, Wetherilt and Loker¹²⁾. A more recent trial in later pregnancy, however, showed that the decline in serum and erythrocyte folate concentrations and increase in plasma homocysteine concentrations, that otherwise occur as pregnancy progresses, can be prevented by continued FA supplementation (0·4 mg/d) in the second and third trimesters of pregnancy⁽Reference McNulty, McNulty and Marshall¹³⁾; which in turn may prevent anaemia in later pregnancy⁽Reference Lassi, Salam and Haider¹⁴⁾.

Observational studies have suggested that low maternal folate status is also associated with an increased risk of other adverse pregnancy outcomes including pre-eclampsia, gestational hypertension and preterm delivery⁽Reference Lindblad, Zaman and Malik^15–Reference Czeizel, Puhó and Langmar¹⁷⁾, while improved folate may help to prevent these conditions. In a study of 3000 Canadian women, supplementation of multivitamins containing FA was associated with a reduced risk of pre-eclampsia⁽Reference Wen, Chen and Rodger¹⁸⁾, while in a study of 215 Korean pregnant women, FA supplementation was associated with significantly lower risk of pre-eclampsia⁽Reference Kim, Ahn and Ryu¹⁹⁾. In contrast, one very recent international randomised trial found that high dose of FA in pregnancy did not reduce pre-eclampsia in high-risk pregnancy⁽Reference Wen, White and Rybak²⁰⁾. Notably, this study did not account for the common MTHFR C677 T polymorphism which is associated with a significantly increased risk of hypertension and hypertension in pregnancy⁽Reference McNulty, Strain and Hughes²¹⁾.

The demand for folate is also increased during lactation to support neonatal growth and development⁽Reference Tamura and Picciano¹⁶⁾. Folates are actively transported across the mammary epithelium, thereby allowing breast-milk folate concentration to be maintained and preventing folate insufficiency in breast-fed infants, but this is at the expense of maternal folate status in the absence of supplementation⁽Reference Mackey and Picciano²²⁾. Only in the case of frank maternal folate deficiency is milk folate reported to decline to critically low concentrations⁽Reference Metz, Zalusky and Herbert²³⁾.

Neural tube defects and other congenital abnormalities

Since the early 1990s, conclusive evidence has existed that periconceptional FA supplementation prevents the first occurrence⁽Reference Czeizel and Dudas⁴⁾ and recurrence of NTD⁽³⁾. This has led to worldwide recommendations that have been in place since 1992⁽²⁴⁾, for women of childbearing age to take 0·4 mg/d FA from before pregnancy until the end of the first trimester. NTD, including spina bifida, anencephaly, encephalocele and hydrocephalus, are major birth defects that can lead to miscarriage, stillbirth, or to lifelong and usually severe disabilities. NTD are the largest group of anomalies of the central nervous system characterised by incomplete closure of the embryonic neural tube and are among the most significant congenital causes of morbidity and mortality in infants worldwide⁽Reference Zaganjor, Sekkarie and Tsang^25, Reference Botto, Moore and Khoury²⁶⁾. The conclusive evidence of the protective effect of FA comes from two randomised controlled trial (RCT), the first of which demonstrated that periconceptional supplementation at 4 mg/d FA in women with a history of NTD (n 1195), decreased the recurrence by 70 %⁽³⁾. The second trial by Czeizel and Dudas⁽Reference Czeizel and Dudas⁴⁾ in over 4000 women, showed that periconceptional multiple micronutrient supplementation containing 0·8 mg/d FA prevented the first occurrence of NTD.

These intervention studies, although not designed to test birth defects other than NTD, yielded additional information on other congenital abnormalities. The aforementioned Czeizel and Dudas trial⁽Reference Czeizel and Dudas⁴⁾ was the subject of a subsequent analysis which found that the total rate of all major congenital abnormalities (including heart defects, oral facial clefts and urinary tract anomalies) were significantly reduced in women using FA-containing multivitamins during the periconceptional period⁽Reference Czeizel²⁷⁾. Consistent with these findings, FA fortification in Canada has been associated with an 11 % reduction in the prevalence of overall congenital heart defects⁽Reference Liu, Joseph and Luo²⁸⁾. A recent meta-analysis of fifteen studies from countries worldwide reported a decreased risk of cleft lip, with or without cleft palate, when orofacial cleft prevalence was examined in pre- v. post-FA fortification periods⁽Reference Millacura, Pardo and Cifuentes²⁹⁾.

Offspring brain development

Recent advances in behavioural neuroscience have shown the important roles that nutrition plays in brain development⁽Reference Fernstrom^30–Reference Nyaradi, Li and Hickling³³⁾. Brain development begins at the very early stages of fetal life and continues after birth through early life. Initially, brain cells are formed followed by cell migration and differentiation, and the development of synapses to enable cells to communicate with one another⁽Reference Irwin, Pentieva and Cassidy³⁴⁾. Myelin is the supportive tissue that surrounds and protects the nerve axons and facilitates communication. Nutrient deficiencies, such as inadequate folate intake, can interfere with early brain development and function, resulting in neuroanatomical, neurochemical, or neurometabolic changes that are expressed by restricting the myelination and synaptic connectivity⁽Reference Black³⁵⁾ as well as changes in tissue levels of neurotransmitters (e.g. serotonin, dopamine, norepinephrine and acetylcholine). The functional consequences of these alterations vary, depending on the specific nutritional deficiency and the timing of the deficiency relative to the development of the neurological structures⁽Reference Roffman³⁶⁾. The last trimester of pregnancy until 2 years after birth, is a critical period of rapid growth and development of certain regions of the brain such as cortical and subcortical grey matter⁽Reference Isaacs^32, Reference Hasegawa, Houdou and Mito^37, Reference Gilmore, Shi and Woolson³⁸⁾.

Myelination of the brain, which is most intensive from mid-gestation through the second year of life but continues through puberty, may be specifically vulnerable to B-vitamin deficiency⁽Reference Black³⁵⁾. In infants, B-vitamin deficiencies have been associated with demyelination and brain atrophy⁽Reference Lövblad, Ramelli and Remonda³⁹⁾. Thus the continuation of FA supplementation after the first trimester (i.e. after the recommended period for the prevention of NTD) may also be an important period for optimal folate status and prenatal brain development⁽Reference Irwin, Pentieva and Cassidy^34, Reference Roffman^36, Reference McGarel, Pentieva and Strain⁴⁰⁾. Thus maternal folate nutritional status can influence both structural and functional development of the brain⁽Reference Georgieff, Brunette and Tran⁴¹⁾, while folate insufficiencies in pregnancy may result in lasting changes in brain function.

Food folates, folic acid and bioavailability

There are three options to achieve optimal folate status in individuals and in populations, namely through naturally-occurring food folates, fortified foods and supplements. Food folates exist predominantly in the polyglutamyl form and are converted to monoglutamates for absorption, whereas FA, the synthetic vitamin form found in fortified foods and supplements, is a monoglutamate. In addition, natural folates are reduced molecules, whereas FA is fully oxidised⁽Reference Bailey, Stover and McNulty²⁾. As a result, naturally occurring food folates show incomplete bioavailability compared with FA at equivalent levels of intake⁽Reference McNulty and Pentieva⁹⁴⁾. Apart from their limited bioavailability once in the body, food folates are inherently unstable during cooking, and this can substantially reduce the folate content of this food before they are even ingested⁽Reference McKillop, Pentieva and Daly⁹⁵⁾. In contrast, FA provides a highly stable and bioavailable form of the vitamin. The bioavailability of FA is assumed to be 100 % when ingested as a supplement, while FA in fortified food is estimated to have about 85 % the bioavailability of FA supplements⁽Reference Pfeiffer, Rogers and Bailey⁹⁶⁾. Folate intakes and recommendations in the USA and other countries are therefore now expressed as Dietary Folate Equivalents, a calculation that considers the greater bioavailability of FA compared with naturally occurring food folates⁽⁹⁷⁾.

Owing to the instability and poor bioavailability of natural food folates, the potential to optimise folate status through food folates alone is relatively ineffective, whereas intervention with FA supplements or FA fortified food has been shown to be highly effective in optimising folate biomarker status in women of reproductive age⁽Reference Cuskelly, McNulty and Scott⁹⁸⁾.

Folic acid recommendations for neural tube defect prevention

Once conclusive evidence had become available to show that FA supplementation could prevent the first occurrence⁽Reference Czeizel and Dudas⁴⁾ and recurrence of NTD⁽³⁾, committees worldwide produced recommendations for women of childbearing age to take 0·4 mg/d FA from before pregnancy until the end of the first trimester^{(24, 99)}. These recommendations, which have been in place for almost 30 years, are challenging for a number of reasons. An estimated 50 % of pregnancies are unplanned⁽Reference Mills and Dimopoulos¹⁰⁰⁾ and for many women, the very early stage of pregnancy (when the neural tube is closing) may have passed before supplementation is even started. Therefore, in many cases, the malformations of NTD may have occurred before a woman even knows that she is pregnant.

There is evidence from nearly 500 000 pregnant women that only 31 % took FA supplements before pregnancy, with women under 20 years of age and non-caucasian women being the least likely to take FA as recommended⁽Reference Bestwick, Huttly and Morris¹⁰¹⁾. Therefore, the public health measure of recommending FA supplements before pregnancy has substantial limitations and is putting young women and those in ethnic minorities at a particular disadvantage⁽Reference Bestwick, Huttly and Morris¹⁰¹⁾. Research from Northern Ireland⁽Reference McNulty, Pentieva and Marshall¹⁰²⁾ and the Republic of Ireland⁽¹⁰³⁾ indicates low levels of compliance among Irish women surveyed during pregnancy, between 14 and 44 % reporting to have taken FA supplements as recommended in the periconceptional period. This is of concern given that the benefit of FA supplementation in preventing NTD is confined to those women (the minority) who follow the recommendations correctly.

The measurement of erythrocyte folate in women of reproductive age is a useful way to assess NTD risk within populations on the basis of the known continuous dose–response inverse relationship between maternal erythrocyte folate concentrations and NTD⁽Reference Daly, Kirke and Molloy¹⁰⁴⁾. On this basis, the WHO has established guidelines for optimal erythrocyte folate concentrations of 906 nmol/l in women of reproductive age for the prevention of NTD⁽¹⁰⁵⁾. In the UK, where there is voluntary (but not mandatory) fortification of foods with FA, the percentage of women with insufficient erythrocyte folate concentrations (<906 nmol/l) to prevent folate-responsive NTD is estimated to be 83 % in Northern Ireland, 81 % in Scotland and 79 % in Wales⁽¹⁰⁶⁾. Also, evidence from the National Adult Nutrition Survey in Ireland showed that non-consumers of FA from fortified food or supplements were at particularly high risk of suboptimal folate status, again using the cut-point of 906 nmol/l erythrocyte folate to define optimal status⁽Reference Hopkins, Gibney and Nugent¹⁰⁷⁾.

Folic acid fortification and neural tube defect prevalence

The indisputable protective role of FA in the prevention of NTD, coupled with the low compliance of women to FA recommendations, has stimulated the option of mandatory FA fortification, a policy now in place in over 80 countries worldwide⁽⁵⁾. Mandatory food fortification requires food manufacturers to add FA to certain foods (e.g. starch or grain products), whereas voluntary fortification allows FA to be added to foods at the discretion of manufacturers. A systematic review and meta-analysis of global birth prevalence of spina bifida by FA fortification status found that spina bifida prevalence was significantly lower in studies from countries where FA fortification was mandatory (3·4 per 10 000 live births) compared with countries where fortification was voluntary or non-existent (4·8 per 10 000 live births)⁽Reference Atta, Fiest and Frolkis¹⁰⁸⁾. Furthermore, evidence has shown that in 2017 only 18 % of FA-preventable NTD cases globally were prevented, resulting in 230 000 children with either spina bifida or anencephaly⁽Reference Kancherla, Wagh and Johnson¹⁰⁹⁾. In the USA, after the implementation of mandatory FA fortification, reported rates of NTD prevalence decreased by 35 %, from 10·7 to 7·0 NTD per 10 000 live births, preventing over 1300 NTD annually⁽Reference Parker, Mai and Canfield^110–Reference Crider, Qi and Devine¹¹²⁾. In Canada, the prevalence of NTD decreased by 46 %, from 15·8 per 10 000 births before FA food fortification to 8·6 per 10 000 births after implementation of mandatory fortification⁽Reference De Wals, Tairou and Van Allen¹¹³⁾.

In contrast, public health strategies based on promoting increased awareness of the benefits of FA supplementation, as in place in European countries, have been shown to be largely ineffective⁽Reference Khoshnood, Loane and De Walle^114–Reference Obeid, Pietrzik and Oakley¹¹⁶⁾. Analysis of European data showed that average infant mortality with congenital anomaly was 1·1 per 1000 births, with higher rates where termination of pregnancy is illegal (Malta 3·0 and Ireland 2·1)⁽Reference Boyle, Addor and Arriola¹¹⁷⁾. These rates have shown no downward trend over time, even with the introduction of government recommendations for FA usage, and NTD rates in 2011 were found to be comparable with that in 1991 (about 0·9 per 1000 births)⁽Reference Khoshnood, Loane and De Walle^114, Reference Botto, Lisi and Robert-Gnansia¹¹⁵⁾. As a result, one report investigating the period 2000–2010 estimated 1·6-fold higher NTD prevalence in European countries compared with countries with mandatory food fortification in place⁽Reference Obeid, Pietrzik and Oakley¹¹⁶⁾.

Ireland is recognised as having one of the highest rates of NTD-affected pregnancies in the world and there are concerns that the incidence of NTD is increasing in recent years⁽Reference Botto, Lisi and Robert-Gnansia¹¹⁵⁾. In 2016, following an extensive review, the Food Safety Authority of Ireland scientific committee published an updated report recommending that mandatory fortification of bread or starch with FA should be implemented⁽¹⁰³⁾. Similarly, The UK Scientific Advisory Committee on Nutrition has recently confirmed its longstanding advice that mandatory fortification of cereal flours with FA should be introduced for the prevention of NTD⁽¹¹⁸⁾. Legislation to implement mandatory FA fortification has yet to be introduced in either country.