Dihydrofolate and tetrahydrofolate-derivates – formerly called vitamin B9 – are involved in multiple processes often in combination with other vitamins. They serve as C1-donors or -receivers and through this function as enzyme activators (cofactors). Deficiency of folate causes certain anaemia types and when occurring before or during pregnancy neural tube defects and often death of the foetus. Folate concentrations below 6.8 nmol/l and 317 nmol/l in serum and erythrocytes, respectively, indicate folate deficiency. Homocysteine concentrations destabilize with folate concentrations below 10 nmol/l and 340 nmol/l in serum and erythrocytes, respectively (EFSA NDA, 2014). For prevention, some countries started fortification programs successfully (McNulty et al., 2019). Recommendations vary between countries yet pregnant women are advised to secure 600 μg dietary folate equivalents (DFE)/day and lactating women 500 μg DFE/day to maintain adequate folate concentrations (EFSA NDA, 2014, ods.od.nih.gov, 2019-0-19).
Folate is an umbrella term for several natural water-soluble pteoryl-polyglutamic acids derived from plants and foods. They belong to the B-vitamin complex and a former name for folate was "vitamin B9". Biological activity lies in the natural folate derivates, yet these are chemically instable, have poor bioavailability, and are damaged through exposure to UV light, oxygen or high temperatures. In the nutrition context, folic acid describes a synthetic monoglutamate that is chemically stable. Subsequently, folic acid can be used to fortify foods or as food supplement (EFSA NDA, 2014, McNulty et al., 2019).
Folic acid can be reversibly converted via the partially oxidised intermediate dihydrofolate (DHF) to the fully reduced folate cofactor form with vitamin function, that is tetrahydrofolic acid (THF). However, our cells contain several derivates of tetrahydrofolic acid and these differ in the one-carbon groups that are bound to their core structure (EFSA NDA, 2014). These derivates can be interconverted by energy-dependent enzymatic steps from one form into the next. Through these conversions, one-carbon groups are transferred from one molecule to the next. This pathway is called the "one carbon-" or "C1 metabolism" (McNulty et al., 2019).
The intermediate molecule in the conversion from folic acid to tetrahydrofolate is dihydrofolate (DHF) (EFSA NDA, 2014). The tetrahydrofolate derivates present in most cells carry a methyl- or formyl-(one carbon) group. These are predominantly (EFSA NDA, 2014):
DHF and THF-derivates are cofactors and essential for DNA, RNA and protein synthesis, cell division, red blood cell generation, and affect hundreds of enzymatic processes. In periods of rapid growth, elevated concentrations are needed to prevent DNA damage, impaired DNA replication or -cell division (EFSA NDA, 2014).
Folate deficiency at time of fertilisation and early pregnancy is associated with neural tube defects such as spina bifida or anencephaly, two congenital anomalies that affect the spine and the brain. The defects occur during the 3rd and 4th week of pregnancy and can lead to impairment of the spinal cord at best or death of the foetus or neonate (medlineplus.gov, McNulty et al., 2019). The folate metabolism is closely linked to that of other vitamins particularly cobalamin (VitB12), pyridoxine (VitB6) and thiamine (VitB2) and other nutrients like choline and methionine. An imbalance can lead to increased homocysteine concentrations and its detrimental effects (Škovierová et al., 2016). Pregnancy-related complications associated with hyperhomocysteinaemia or -urea include next to neural tube defects also preeclampsia, placental disruption, loss of embryo or foetus early in the pregnancy, intrauterine growth retardation of the child and subsequent low birth weight (McNulty et al., 2019). Low folate concentrations during pregnancy have also been associated with impaired neurocognitive development of the offspring and gestational supplementation with improved cognitive performance of the children aged three and six years (McNulty et al., 2019).
Since cobalamin and folate overlap in the same pathways, for example that of homocysteine, a cobalamin deficiency can cause folate deficiency. A folic acid supplementation can in turn mask the presence of the cobalamin deficiency. High homocysteine concentrations in combination with high serum yet low erythrocyte folate concentrations are indicative of cobalamin deficiency. Cobalamin deficiency is also linked to neurological dysfunction, which can become irreversible if left untreated (EFSA NDA, 2014, McNulty et al., 2019).
Symptoms of folate deficiency include fatigue, irritability, forgetfulness, diarrhoea, grey hair, glossitis, oral or peptic ulcers, and poor growth in children (medlineplus.gov). Advanced deficiency shows in haemolytic and megaloblastic anaemia, which is the most common diagnostic symptom, later accompanied by low counts of granulocytes and platelets, and the associated symptoms of anaemia (tiredness, heart palpitations, headaches, concentration difficulties, light-headedness, pale skin, and enlarged spleen)(EFSA NDA, 2014, medlineplus.gov).
Recent intake of folate is reflected by a single measurement in serum or plasma. As single measure, they do not provide insights on folate status. For folate status, repeated measures over several weeks should confirm folate concentrations in circulation or this parameter should be combined with other markers for folate status. As repeated measure, serum/plasma folate concentrations do not reflect dietary intake but the cellular equilibrium of this water-soluble compound. It takes about three months of supplementation to reach equilibrium (EFSA NDA, 2014).
Erythrocyte (red blood cell) folate concentrations are a reliable biomarker for folate status. They reflect 50% of total folate stores. However, because cell turnover is about 120 days, these counts reflect a long-term deficiency. It takes several months for this marker to be affected (EFSA NDA, 2014, McNulty et al., 2019).
Homocysteine in combination with the other readouts is a marker for folate deficiency whereas urinary folate is not . Because of the multi-vitamin interplay in homocysteine homeostasis, homocysteine is not a specific marker. For folate, the interplay is inverse and non-linear: homocysteine concentrations increase when circulating folate concentrations are low, yet when circulating folate concentrations are adequate homocysteine concentrations plateau. This plateau can be reached with circulating folate concentrations of more than 4.4 ng/ml (10 nmol/l) or erythrocyte concentrations above 150 ng/ml (340 nmol/l). (EFSA NDA, 2014).
The conversion for dietary food equivalents (DFE) has been introduced because folic acid has a higher bioavailability than naturally occurring folate-derivates. Accounting for this difference 1 μg DFE corresponds to 1 μg folate from foods yet only 0.6 μg folic acid from supplements or fortified foods. Subsequently, 1 μg folic acid corresponds to 1.7 μg DFE. For a supplement that is to be taken on an empty stomach, the conversion of 1 μg DFE equals 0.5 μg folic acid applies (EFSA NDA, 2014).
Adults should eat about 330-400 μg DFE per day. For pregnant women or those that are likely to conceive, the European Food Safety Agency and the National Institutes of Health recommend an intake of 600 μg DFE/day preferably prior to fertilisation and 500 μg DFE/ day during lactation (ods.od.nih.gov, 2019-0-19, EFSA NDA, 2014). The World Health Organisation recommends at least 400 μg DFE during pregnancy (WHO, 2016); under certain circumstances a once weekly oral dose of 2800 μg DFE has been suggested (WHO, 2016). For infants, an upper limit could not be defined and the Average Intake derives from folate concentrations in breast milk (ods.od.nih.gov, 2019-0-19, EFSA NDA, 2014).
Vegetables with dark green leaves such as spinach or mustard greens, asparagus, and Brussel sprouts, legumes ((dried) beans and peas), liver, mushroom, poultry, pork, shellfish, nuts, peanuts and citrus fruits are rich in folate (ods.od.nih.gov, 2019-0-19, medlineplus.gov). Some foods may be enriched in folic acid as part of a national fortification program. These are most commonly flour (wheat, cornmeal, pasta or rice) or cereals or in form of food supplements and are established in more than 80 countries (ods.od.nih.gov, 2019-0-19, McNulty et al., 2019). However, even when following a balanced dietary pattern with foods rich in folate it is difficult to reach recommended intake values. In countries where a nation-wide folate fortification of foods is not practiced, women who are sexually active or wish to become pregnant are advised to supplement folic acid (McNulty et al., 2019, DGE-ÖGE-SGE, 2019).
Fortification of foods in Europe is allowed with folic acid (pteroylmonoglutamic acid), calcium‑L‑methylfolate and others. Folate concentrations for infants and toddlers are separately regulated for infant and follow-on formulas as well as for cereal-based- and baby-foods (EFSA NDA, 2014, EU/2016/127). In 2020, EFSA supported the use of calcium‑L‑methylfolate also in infant- and follow-on formulas because of high bioavailability and lack of safety concerns (EFSA NDA, 2020).
Balogun OO, da Silva Lopes K, Ota E, Takemoto Y, Rumbold A, Takegata M, Mori R. Vitamin supplementation for preventing miscarriage. The Cochrane database of systematic reviews 2016; 2016(5):CD004073. at: https://pubmed.ncbi.nlm.nih.gov/27150280/
De-Regil LM, Peña-Rosas JP, Fernández-Gaxiola AC, Rayco-Solon P. Effects and safety of periconceptional oral folate supplementation for preventing birth defects. The Cochrane database of systematic reviews 2015; (12):CD007950. at: https://pubmed.ncbi.nlm.nih.gov/26662928/
DGE-ÖGE-SGE. D-A-C-H-Referenzwerte für die Nährstoffzufuhr. 2. Auflage, revidierte Ausgabe, bearbeitete Ausgabe. Bonn: Deutsche Ges. f. Ernährung; 2019. at: https://www.dge-medienservice.de/d-a-ch-referenzwerte-fur-die-nahrstoffzufuhr.html
EFSA NDA. Dietary Reference Values for folate. EFSA Journal 2014 [status of: 2020 Nov 10]; 12(11). at: https://www.efsa.europa.eu/en/efsajournal/pub/3893
EFSA NDA. Dietary Reference Values for Nutrients. Summary. Report. EFSA Journal 2017:1–92. at: https://www.efsa.europa.eu/en/supporting/pub/e15121
EFSA NDA. Calcium l-methylfolate as a source of folate added for nutritional purposes to infant and follow-on formula, baby food and processed cereal-based food. EFSA Journal 2020; 18(1):e05947. at: https://www.efsa.europa.eu/en/efsajournal/pub/5947
EU/2016/127. Commission Delegated Regulation (EU)2016/127 of 25 September 2015 supplementing regulation (EU) No 609/2013 of the European Parliament and the Council as regards the specific compositional and information requirements for infant formula and follow-on formula and as regards requirements on information relating to infant and young child feeding. [status of: 2019 Aug 22]. at: https://eur-lex.europa.eu/eli/reg_del/2016/127/2019-06-12
McNulty H, Ward M, Hoey L, Hughes CF, Pentieva K. Addressing optimal folate and related B-vitamin status through the lifecycle: health impacts and challenges. The Proceedings of the Nutrition Society 2019; 78(3):449–62. at: https://pubmed.ncbi.nlm.nih.gov/31155015/
medlineplus.gov. Health Information from the National Library of Medicine [status of: 2020 Nov 23]. at: https://medlineplus.gov
ods.od.nih.gov. Folate: Office of Dietary Supplements: National Institutes of Health; 2019-0-19 [status of: 2020 Nov 23]. at: https://ods.od.nih.gov/factsheets/Folate-Consumer/
Škovierová H, Vidomanová E, Mahmood S, Sopková J, Drgová A, Červeňová T, Halašová E, Lehotský J. The molecular and cellular effect of homocysteine metabolism imbalance on human health. International journal of molecular sciences 2016; 17(10). at: https://pubmed.ncbi.nlm.nih.gov/27775595/
WHO. Optimal serum and red blood cell folate concentrations in women of reproductive age for prevention of neural tube defects: Guideline. Geneva, Switzerland: World Health Organization; 2015. 38 p. [status of: 2020 Nov 9]. at: https://www.who.int/nutrition/publications/guidelines/optimalserum_rbc_womenrep_tubedefects/en/
WHO. WHO recommendations on antenatal care for a positive pregnancy experience. 1st ed. Geneva, Switzerland: WHO Press; 2016. (WHO Publications) [status of: 2020 Feb 14]. at: https://www.who.int/reproductivehealth/publications/maternal_perinatal_health/anc-positive-pregnancy-experience/en/back