Folate – a scoping review for Nordic Nutrition Recommendations 2023

Folate is an essential micronutrient for normal development and metabolic function, and folate deficiency is associated with an increased risk of cancer, cardiovascular disease, mental dysfuntion and negative pregnancy outcomes. When estimating folate requirements, one must consider different bioavailability and functionality between synthetic folic acid and dietary folate, together with increased needs of folate in women of fertile age, pregnant and lactating women, preterm and small for gestational age weight infants and individuals who are homozygote for the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene polymorphism. In order to achieve an adequate metabolic status based on the metabolic marker total homocysteine, and not merely the absence of clinical signs of folate deficiency, the recommended intake of folate differs according to age, pregnancy and lactation. According to the World Health Organization, a decision limit for folate deficiency in adults is serum folate level below 10 nmol/L, and in women of fertile age a red blood cell folate level below 906 nmol/L in order to prevent neural tube defects. Qualified systematic reviews along with identified relevant literature have been used for this scoping review prepared for the Nordic Nutrition Recommendations 2023.

T he aim of this scoping review is to describe the totality of evidence for the role of folate for health-related outcomes as a basis for setting and updating dietary reference values (DRVs) (Box 1).Folate is an essential micronutrient in the B vitamin complex.It is involved in different physiological processes and particularly essential for growth and foetal development because of its role in the synthesis, repair and methylation of DNA, contributing to the formation of new cells and tissues.
Folate is present in most foods, and high concentrations are found in liver, green vegetables and legumes.Folate species in food are labile to light and oxidation, and partly destroyed by cooking, compared to the chemically most stable folate form, synthetic folic acid, which is found in supplements (1).
Higher folate requirements are found in infants, children, pregnant and lactating women, patients with intestinal disease, severe skin diseases, haemolytic anaemia, patients taking antiepileptic medications and people with certain gene polymorphisms (2,3).
The significance of folate status remains conflicting for many conditions, including cancer (4), cardiovascular disease (5), asthma (6,7), mental function (8,9) and pregnancy outcomes (10,11).More than 30 years ago, the British Medical Research Council showed that maternal Popular scientific summary • Folate is a micronutrient in the B vitamin complex involved in the synthesis, repair and methylation of DNA.• Folate is particularly essential for growth and foetal development.
• Foods rich in folate include liver, green vegetables and legumes.
• Folic acid, found in supplements, is the chemically most stable folate form.
• Associations between folate status and chronic diseases, as well as mental function, and pregnancy outcomes, are conflicting.Monsen and Ueland intake of folic acid starting before pregnancy prevents most cases of infant spina bifida and anencephaly, two major neural tube defects (NTDs) (12).Mandatory food fortification with folic acid is considered a safe and cost-effective intervention to prevent NTDs; however, fortification has not been implemented in many countries, including the Nordic countries (13).
Serum folate is the primary marker of folate status in children, adults and pregnant women.The metabolic marker, plasma total homocysteine (tHcy), increases with decreasing serum folate levels.Plasma tHcy are also affected by vitamin B 12 and B 6 status, age and renal function (3).In young children, serum folate concentrations are commonly high (>20 nmol/L) (14).In adults, the World Health Organization (WHO) recommends a serum folate concentration >10 nmol/L and a red blood cell (RBC) folate >906 nmol/L in women of fertile age (15).
In a recent study, median folate intake was 246 µg per day in a Swedish adult population (18-80 years, n = 1,797), 25% had a serum folate concentrtion <10 nmol/L, and none of the women of reproductive age had erythrocyte folate concentrations associated with the lowest risk of NTDs (16).Women may reach a preventive RBC folate concentration of more than 906 nmol/L within 4 weeks of supplementation with daily intake of 800 µg folic acid (17), while a dietary folate intake of at least 350 µg per day has been considered necessary to prevent an increase in plasma homocysteine levels of the adult population (18).

Methods
This scoping review follows the protocol developed within the Nordic Nutrition Recommendations (NNR) 2023 project (19).The sources of evidence used in the scoping review follow the eligibility criteria described previously (20).
The main literature search for this review was performed in MEDLINE  The number of hits was 578.Only one qualified systematic review was identified for folate (21).We also identified relevant literature for this scoping review via 'snowballing'/citation chasing that was relevant for the background information.

Physiology
Dietary folates mostly occur as polyglutamyl derivatives and undergo hydrolysis in the gut to monoglutamates before intestinal absorption (22).Folates are transported across the jejunum by a carrier-mediated process, as folic acid, 5-methyltetrahydrofolate (5-MTHF) and 5-formyltetrahydrofolate (23,24).Folates can also be absorbed by diffusion, a process that is linearly related to luminal folate concentrations and can account for 20-30% of folate absorption at high folate intakes (22).
Folates taken up by the intestinal mucosal cell are reduced to THF, which can either be transferred to the portal circulation without further metabolism or methylated before being transferred.THF is taken up by the liver, methylated to 5-MTHF and 10-formyl-THF, and transported to the peripheral tissues.Folate in the plasma is transported to the tissues as monoglutamate derivatives (2).Within the cell, THF is methylated to 5-methyl-THF, which is converted to folate polyglutamates containing up to seven glutamyl residues.Polyglutamation traps folate inside the cell at concentrations higher than extracellular fluids (25).
The chemically most stable folate form is synthetic folic acid.The bioavailability of food folate is commonly estimated as 50% of folic acid bioavailability when establishing food recommendations, but this should be considered a rough estimate, as data on the bioavailability of food folate vary between 30 and 98% (1,26,27).When adults receive daily folate doses <200 µg, little or none is lost in the urine, but at higher doses in the pharmacological range, as used by pregnant women on antiepileptic medication (28), the urinary loss is considerable: 6% of a 1 mg dose, 10% of 2 mg, 50% of 5 mg and 80% of 15 mg (29).
• This paper is one of many scoping reviews commissioned as part of the Nordic Nutrition Recommendations 2023 (NNR2023) project (19).• The papers are included in the extended NNR2023 report but, for transparency, these scoping reviews are also published in Food & Nutrition Research.• The scoping reviews have been peer reviewed by independent experts in the research field according to the standard procedures of the journal.• The scoping reviews have also been subjected to public consultations (see report to be published by the NNR2023 project).• The NNR2023 committee has served as the editorial board.
• While these papers are a main fundament, the NNR2023 committee has the sole responsibility for setting dietary reference values in the NNR2023 project.
Folate requirements and recommendations for folate intake are expressed as dietary folate equivalents (DFEs) that adjust for the greater degree of absorption of folic acid compared with folate naturally found in foods (1 μg of folate equals 0.6 μg folic acid added to food or taken with food or 0.5 μg folic acid [as a supplement] taken on an empty stomach) (30).
About half of the total body folate pool (5-10 mg) is stored in the liver.Plasma folate consists almost entirely of 5-MTHF (90%) (1).A small fraction of plasma folate is bound to a folate-binding protein.
Folates act as coenzymes for enzymes that mediate single-carbon metabolism.The fully reduced form (tetrahydro-) serves as an acceptor or donor of a single-carbon unit in reactions involved in the synthesis of pyrimidines, purines, serine and methionine (2).Thymidine monophosphate is produced by the methylation of uridine monophosphate.The coenzyme delivering the necessary methyl group in this reaction is 5,10-MTHF, which may be reduced to 5-MTHF for synthesis of methionine (Met) from homocysteine (Hcy) or oxidized to 10-formyltetrahydrofolate for use in purine synthesis (2).
In the Nordic population, 5-8% have a polymorphism in the gene coding for the 5,10-methylenetetrahydrofolate reductase (MTHFR) (C677T, Ala --> Val) (31).This mutation is associated with a decreased activity of the enzyme and results in hyperhomocysteinemia, primarily when folate levels are low.It is recommended that people with MTHFR polymorphism should have a serum folate >15 nmol/L (32).

Pregnancy
In women of reproductive age, the WHO recommends a RBC folate threshold of <400 ng/mL (906 nmol/L) to be used as an indicator of folate insufficiency, as RBC folate concentrations above this limit will achieve the largest reduction of NTDs (15).Pregnancy is associated with higher demands for folate due to foetal growth and drainage, as well as increased folate catabolism and excretion (33,34).Serum folate levels decrease continuously during pregnancy and folate stores are depleted after 3 months or sooner if dietary supplements are not provided (35).

Lactation
During lactation, folate is preferentially taken up by actively secreting mammary glands.5-Methyltetrahydrofolate is the predominant form of folate in human milk (36).While colostrum is relatively low in folate, milk folate increases during the lactation period (37).Despite reduced maternal folate status, average milk folate levels are reported to be maintained at recommended dietary allowances for infants (38).In a 16 weeks intervention study, there were no differences in total milk folate or in unmetabolized folic acid concentration in the breast milk of women provided with either a low dose of folic acid, a [6S]-5-methyl-THF supplement, or a placebo during lactation (39).

Infants
Maternal folate deficiency is associated with low folate levels in the infants (14,40).Significantly lower folate levels at birth have also been observed in low-birth-weight (<2,500 g) ( 41) and premature infants (42).These infants also experience a fall in folate concentrations in early life (43).
In exclusively breastfed infants, plasma folate levels are reported to be elevated after the age of 2 months and are then two-to threefold higher than maternal levels (14,44,45).In formula-fed infants, more than 70% have plasma folate concentrations below the lowest concentration for breastfed infants (46).The opposite was observed in Korean infants, where the overall folate intakes in formula-fed infants were significantly higher than those in human milk-fed infants, and this was associated with significantly higher folate and lower tHcy in formula-fed infants than human milk-fed infants at 5 months (47).As the quantity of folic acid in formula milk may differ among countries, this will impact infant folate status.

Older children
Serum folate remains high up to age 12 months and then decreases to values observed in older children and adults during the first 1-3 years of life (14) (Fig. 1).

Malabsorption
Malabsorption occurs with intestinal diseases, such as celiac disease, Crohn's disease, ulcerative colitis and tropical sprue (3,48).Folate can bind to food matrices, and many foods (e.g.oranges, lentils, cabbage) contain inhibitors of the intestinal folate conjugase, which reduces folate absorption.Iron and vitamin C deficiencies are associated with impaired food folate utilization (49).

Assessment of nutrient status
Serum or plasma folate is the primary marker of folate status in children, adults and pregnant women.Intake of food before blood sampling may affect serum folate concentrations; however, most laboratories do not demand a fasting condition.The microbiological assay is considered the gold standard for both serum and RBC folate (50), but more often modern immunoassays are used in clinical laboratories.Although RBC folate is considered to be a better indicator of body stores and nutritional status, there is considerable uncertainty about the reliability of the analytical methods for RBC folate, and many laboratories do not any longer offer this analysis (51,52,53).
A higher intake of folate or folic acid is associated with a higher serum and RBC folate.After initiation of mandatory folic acid fortification in 1998, serum folate centrations more than doubled and RBC folate increased by approximately 50% in the US population (3).Initially, in folate deficiency, serum folate decreases, then plasma tHcy increases and a reduction in RBC folate becomes evident.Folate deficiency gives rise to megaloblastic changes in the bone marrow and other rapidly dividing tissues (54), hypersegmentation in neutrophils and generation of micronuclei in lymphocytes, biomarkers of chromosome breakage or loss (55).Plasma tHcy may increase to 40-50 µmol/L in severe folate deficiency.In patients who are homozygous for the C677T polymorphism in the MTHFR gene, plasma tHcy may increase to 100 µmol/L.Plasma tHcy also increases with reduced renal function and age, so it is necessary to use age-specific decision limits (56).
Many laboratories still use the 2.5 th percentile reference limit to define folate deficiency, ranging from 5 to 7 nmol/L in the Nordic countries.The reference interval is typically defined as the 95% interval between the two reference limits (2.5th and 97.5th percentiles) derived from the distribution of values from an apparently healthy reference population (57).However, a reference interval is merely a description of the folate status in a specific population and will differ according to the diet in the tested population.The mean folate concentration was 29.5 (95% confidence interval: 27.3-31.7)nmol/L in a population-based study including 750 individuals aged ≥12 years in 2017 from Brazil, where folic acid fortification of wheat and maize flours has been mandatory since 2004 (58).In Norway, where folic acid fortification has not been implemented, the mean serum folate was 18.0 (SD 13.8) nmol/L, 39% lower, in 158 Norwegian women of fertile age in 2015 (own unpublished data).
For clinical interpretation of serum folate, one must have clinical decision limits, which defines a value above or below a threshold associated with a significantly higher risk of adverse clinical outcomes or diagnostic for the presence of a specific disease (57).The decision limit may vary according to outcome.When WHO used megaloblastic anaemia as a outcome for folate deficiency, the decision limit was <6.8 nmol/L (59).When WHO used tHcy as a functional marker for folate deficiency, the decision limit was <10 nmol/L (59).The WHO considered that folate status needs to be optimal in women of fertile age to prevent NTDs and suggested that blood cell folate concentrations below 906 nmol/L (serum folate 25-27 nmol/L) should be a decision limit for deficiency in this age group (15).As Figure 2 shows, plasma tHcy starts to increase already when serum folate falls below ~25-27 nmol/L, indicating suboptimal intracellular folate stores, and increases more sharply below ~10 nmol/L, indicating biochemical deficiency (60).A similar relation between serum folate and plasma tHcy is observed in pregnant women (61).

Dietary intake in Nordic and Baltic countries
Folate is present in most foods, and high amounts are found in liver, green vegetables and legumes.The folates in foods are almost exclusively in reduced form as polyglutamyl derivatives of tetrahydrofolate (FH 4 ) (49), and reduced forms are labile to light and oxidation and partly destroyed by cooking.The bioavailability of food folate is commonly estimated to be 50% of folic acid bioavailability when establishing food recommendations, but this should be considered a rough estimate, as data on the bioavailability of food folate vary between 30 (27) and 98% (26), depending on the methodological approach used.Synthetic folic acid, which is a stable oxidized form of pteroylmonoglutamate, is considered to be more bioavailable than natural folate, with a bioavailability when taken with food of 85% and under fasting conditions close to 100% (25,62).
The average dietary intake in adults in the Nordic countries is 257 µg per day for women and 293 µg per day for men (63).It is notable that average intake in Denmark is approxiately 38% higher in both men and women compared to the other Nordic countries.Median folate intake was 246 μg per day for Swedish adults aged 18-80 years (16).
People with low folate intake, malabsorption or increased folate requirements have a risk of developing folate deficiency.Chronic alcoholism is associated with severe folate deficiency linked to poor dietary intake, intestinal malabsorption, impaired hepatic uptake with reduced storage of folates and increased renal excretion (64).Children and pregnant and lactating women have an increased demand for folate, so have patients with haemolytic anaemia, malignancy, patients undergoing renal dialysis and patients using anticonvulsant drugs (phenytoin, primidone), sulfasalazine (used in the treatment of inflammatory bowel disease), triamterene (a diuretic) or metformin (used in type 2 diabetes) (3).

Cancer
The potential impact of folate on cancer risk has been evaluated with conflicting findings, as various studies have demonstrated increased, risk, no effect and decreased risk.So the relation between folic acid and dietary folate intake, folate status and cancer still remains an unresolved issue (65).Evidence from animal and human studies suggests that folic acid supplementation may prevent neoplastic initia tion, but may promote the progression of established pre cancerous lesions (4).
Elevated tHcy levels and folate deficiency, as determined by serum folate level, were associated with increased overall risk of cancer in a meta-analysis of 83 case-control studies (66).Folate level was inversely associated with most cancer types except prostate, bladder, pancreatic and breast (66).Folic acid supplementation and higher serum levels are associated with increased risk of prostate cancer.Gene polymorphisms may impact cancer risk in certain ethnic groups (67).
The differences in bioavailability and metabolism of synthetic folic acid and natural dietary folate as well as variation in the baseline characteristics of subjects and different methods of folate status assessment in various studies have been suggested as reasons for the controversies regarding colorectal cancer prevention versus promotion (65).Both randomized controlled trials (RCT) and cohort studies have, however, shown beneficial effects of both supplementary folic acid and dietary folate on the primary prevention of colorectal adenomas (68,69,70,71).A recent systematic review including a total of 24 cohort studies involving 37,280 patients and 6,165,894 individuals showed that high folate intake was associated with a reduced risk of colorectal cancer, particularly in people with middle or high alcohol consumption.However, the authors concluded that this still needs to be further confirmed (72).

Cardiovascular disease
An adequate dietary folate intake (i.e. according to the recommendations) has been inversely associated with both severe and subclinical cardiovascular disease outcomes (73).A systematic review of RCTs from 2016 indicates a 10% lower risk of stroke and a 4% lower risk of overall cardiovascular disease with folic acid supplementation.Folic acid supplementation had no significant effect on risk of coronary heart disease (5).A meta-analysis which included search for both folate and vitamin B 12 found that homocysteine lowering with B-vitamins among high vascular risk patients who are not taking antiplatelet therapy was related to a significant reduction (29%) in overall stroke risk (74).In the China Stroke Primary Prevention Trial, daily supplementation with 0.8 mg folic acid reduced the incidence of a first stroke by 21%, with greater benefit in those with lower folate levels or higher homocysteine levels (75).

Mental health
In 2008, a Cochrane review concluded that there is no consistent evidence that folic acid, with or without vitamin B 12 , has a beneficial effect on cognitive function of unselected healthy or cognitively impaired older Serum folat, nmol/L Change in plasma tHcy, μmol/L people (9).However, in Swedish adolescents, higher folate intake and lower homocysteine level have been associated with improved achievement in school, and this effect was consistent after correcting for parental education and other confounders (76).A Norwegian study on 2,189 elderly community subjects followed for 6 years showed higher memory scores in those with higher serum folate (77).A recent meta-analysis found that low folate status was associated with an increased risk of cognitive decline or dementia, whereas folate supplementation protected against the development of dementia (78).After controlling for vitamin B 12 , creatinine, demographic variables and depressive symptom score, a US study from 2005 concluded that RBC folate was directly associated with cognitive function scores and inversely associated with dementia in 1,789 people aged ≥60 years exposed to folic acid fortification (79).A Norwegian study including 2,203 people aged 72-74 years, unexposed to mandatory folic acid fortification, showed that plasma folate was associated with cognitive performance.Among the elderly participants with vitamin B 12 concentrations in the lower range, the association between plasma folate and cognitive performance was strongest (8).

Obesity
There is an increasing prevalence of obesity in most parts of the world (80), including the Nordic countries (81).Body mass index (BMI) has been inversely correlated with concentrations of folate in healthy children aged 2 months to 18 years (82), in women of fertile age (83), pregnant women (84) and non-pregnant adults (85).

Pregnancy outcomes
A Cochrane review reported consistent results showing that folic acid, alone or in combination with vitamins and minerals, prevents NTD, but does not have a clear effect on other birth defects (86) or pregnancy outcomes (10).The US Preventive Services Task Force (87) reviewed systematically results from RCTs on supplementation in pregnancy and NTDs confirming previous conclusions on protective effects.As demands for folate increase during pregnancy, the mother is at risk of developing folate deficiency throughout pregnancy.Folate deficiency has been associated with anaemia and peripheral neuropathy in mothers (88).Recent reviews have found limited evidence for an association between folate status or folic acid supplementation in pregnancy and offspring neurodevelopment (89,90).However, continued folic acid supplementation beyond early pregnancy, which is currently recommended to prevent NTD, however, is reported to benefit neurocognitive development of the child (91).Use of folic acid supplements during pregnancy was associated with improved neurodevelopment in 4-year-old Spanish children when adjusting for sociodemographic and behavioural factors (92).The absence of folic acid supplementation in early pregnancy was associated with a higher risk of behavioural problems in the offspring at 18 months of age (93).A detrimental effect of high dosages of folic acid supplements (>5,000 vs. 400-1,000 μg/ day) during pregnancy on psychomotor development after the first year of life has also been reported (94).A more recent Norwegian study found a 23% increased risk of asthma in children aged 7 years whose mothers had a folate intake of >578 μg per day in pregnancy (7); however, the evidence for an association between folate intake or status in pregnancy and offspring risk of asthma and allergy appears inconclusive (95).There also appears to be inconclusive evidence for an association with protection against hypertensive disorders in pregnancy (96).There is evidence from India of an increased risk of insulin resistance and of obesity in children of women with high serum folate and low serum B 12 (97).The high serum folate in this population might, however, be secondary to vitamin B 12 deficiency, which causes an increased amount of functionally inactive methylated folate in the blood.

Toxicity
The European Food Safety Authority's (EFSA) upper tolerable intake level of folic acid is set to 200 μg per day for children aged 1-3 years, 300 μg per day for children 4-6 years, 400 μg per day for children 7-10 years, 600 μg per day for children 11-14 years, 800 μg per day for children 15-17 years, 1,000 μg per day for adults >17 years and pregnant and lactating women (98).
Observations indicating adverse effects from excess folic acid intake, elevated serum folate and unmetabolized folic acid concentrations remain inconclusive (99).Although harmful effects in elderly with low vitamin B 12 status have been reported in several countries, as reviewed recently (100), the data do not yet provide the evidence needed to affect public health recommendations (99).
Adverse effects are exclusively reported from use of the synthetic compound folic acid and no adverse effects have been associated with the consumption of excess folate from foods (101).Only intake of folic acid in excess of 5,000 µg per day may mask haematological manifestations of cobalamin deficiency, as well as antagonize anticonvulsant therapy and affect zinc physiology (4).
Unmetabolized folic acid is detected in nearly all serum samples from US children, adolescents and adults (102).Concerns have been raised about the potentially untoward effects of unmetabolized synthetic folic acid with regard to cancer, depression and cognitive impairment (103).In postmenopausal women unmetabolized folic acid, but not total folate, in plasma has been found to be related to a decrease in natural killer cell cytotoxicity (104).The causative role for unmetabolized folic acid in this study has been questioned (3), and there are still gaps in understanding the factors that contribute to unmetabolized synthetic folic acid accumulation in plasma and the metabolic effects (99).

Requirement and recommended intakes
The EFSA concluded in 2014 for adults on an average requirement (AR) of 250 μg DFE per day and a population reference intake (PRI) of 330 μg DFE per day.This was based on the folate intake required to maintain folate adequacy characterised by serum folate of ≥ 10 nmol/L and RBC folate concentrations of 340 nmol/L.They assumed a coefficient of variation (CV) of 15% to account for the additional variability associated with the higher requirement for folate in individuals with the MTHFR 677TT genotype (105).
A dietary folate intake of at least 350 µg per day has been considered necessary to prevent an increase in plasma homocysteine levels of the adult population (18).In a study on elderly men and women (66-94 years), a gradual decrease in plasma tHcy from ~11.0 to ~8.5 µmol/L was evident with increasing folate intake ranging from 160 to ~850 µg per day (106).An intake of ~300 µg folate per day was associated with a plasma tHcy of ~10 µmol/L, indicating that a higher folate intake than recommended by NNR2012 (AR for adults: 200 µg/day and RI to 300 µg/day) may improve folate status.

Women of reproductive age
To promote optimal NTD risk reduction at the population level, the WHO recommends that the RBC folate concentrations should be above a threshold of 906 nmol/L (400 ng/mL) in women of reproductive age (15).It is considered that an RBC folate concentration below this level indicates folate insufficiency and suboptimal NTD prevention.An RBC level of 906 nmol/L corresponds to a plasma/serum folate concentration threshold of 25.5 nmol/L (107).This level coincides with increased genomic stability and stable plasma tHcy concentrations (Figure 2) (108).
As far from all pregnancies are planned, an RI of 400 µg per day for all women of reproductive ages is considered necessary to provide adequate folate status of women experiencing unplanned pregnancies.It has been shown that women may reach a preventive RBC folate concentration of more than 906 nmol/L within 4 weeks of supplementation with daily intake of 800 µg folic acid (17).The prevalence of having a RBC folate <906 nmol/L was 35% after 40 weeks with a daily folic acid supplement of 140 µg and 18% with 400 µg (109).Median folate intake was 227 μg per day for Swedish women of reproductive age (16).Finnish studies report mean folate intakes in women of reproductive ages ranging from 215 to 230 μg per day (65,66) Pregnant women In NNR2004, the recommended intake during pregnancy was set to 500 µg per day.This was based on previous studies indicating that 400-500 µg per day was considered sufficient to meet the increased requirement from fast-growing foetal tissues during pregnancy (67) and the recommendation was kept unchanged in NNR2012.
The EFSA Panel considers that it is not possible to set an AR for pregnancy and proposed an adequate intake (AI) for folate for pregnancy at 600 μg DFE per day based on a study on maintenance of serum and RBC folate concentrations in pregnancy (105).This study reported that a total of 450 µg per day of dietary folate in addition to synthetic folic acid was sufficient to maintain folate status in pregnant women.This level of intake was considered equivalent to ~600 µg per day dietary equivalents, assuming 50 and 75% availability of dietary folate and synthetic folic acid consumed with meals, respectively (110).
In the Norwegian MoBa study, women who were regular folic acid supplement users had a total mean intake of folate of 615 (SD 270) µg per day of which mean 275 (SD 95) µg per day came from diet alone (111).In pregnancy week 18, women from the MoBa study, with a regular intake of folic acid supplement from 4 weeks before pregancy to week 17, had median serum folate 15.7 (IQR 9.4-23.1)nmol/L compared to median 10.2 (IQR 7.3-16.6)nmol/L in irregular users and median 5.7 (IQR 4.3-7.7)nmol/L in non-users (112), showing that a regular intake of folic acid supplements is necessary to achieve an adequate folate status.However, even among regular supplement users, less than 25% had a serum folate in the range of 25.5 nmol/L, considered optimal for NTD protection.Based on this, recommendations on folate intake should be adjusted.Additionally, folate supplementation should if necessary be combined with vitamin B 12 supplementation, in view of the commonly found low B 12 status in pregnancy and the risk of harm to the child of high folate and low B 12 (113).This recommendation applies especially to women who are vegans and not currently taking a B 12 supplement (114).

Lactation
The NNR2012 recommended 500 µg per day to lactating women, and this amount was also considered to allow replenishment of stores before a possible new pregnancy.This was based on the following reasoning: the concentration of folate in human milk varies throughout the lactation period and is highest between 3 and 6 months (115).Smith and co-workers reported the average concentration of folate in human milk to be 85 µg/L (116).Based on a daily milk production of 0.75 L and a bioavailability of 50%, the diet should contain approximately 100 µg of extra folate.
The EFSA added an additional intake of 130 μg DFE per day to the AR for non-lactating women considered to cover folate losses via breast milk, and a PRI of 500 μg DFE per day was derived for lactating women (105).

Infants and children
The calculated folate intake for infants from birth to 6 months of age was estimated by the EFSA to be 64 μg per day, based on a mean folate concentration of mature breast milk ~80 μg/L (range 45-99 μg/L) (105) and mean breast milk intake per day the first 6 months ~0.8 L per day.In a study based on healthy, well-nourished lactating mothers and infants published in 1980, the mean breast milk folate level was 141.4 μg/L and total daily folate intake for breastfed infants was assessed at 14-25 μg/kg body weight (117).Approximately the same levels were reported in a more recent study from Korea (47).Mean breast milk folate contents ranged from 88.4 to 160.6 μg/L with an overall mean of 128.0 μg/L, and the contents peaked at 2 months postpartum.Folate intake in the infants ranged between 100 and 140 μg per day during the first 12 months.Serum folate in the infants at age 5 months was mean 72 (SD 39) nmol/L and at 12 months mean 76 (SD 40) nmol/L with adequate plasma tHcy levels of mean 4.4 (SD 1.5) and 3.5 (0.8) μmol/L at 5 and 12 months, respectively (47).Mainly breastfed Norwegian infants have high serum folate levels the first 6 months of life [median 31.6 (IQR 21.3-43.3)nmol/L], indicating that folate content in breast milk is adequate (14).
For infants aged 7-11 months, the EFSA recommended an AI of 80 μg DFE per day, by extrapolating upwards from the estimated folate intake in exclusively breastfed infants, considering the metabolically active body mass [median weight or infants at 3 months (6.1 kg) and at 9 months (8.6 kg)] (105).
For older children, the EFSA extrapolated the ARs from the AR for adults using allometric scaling and growth factors and considering differences in reference weights.PRIs ranging from 120 μg DFE per day for age group 1-3 years to 330 μg DFE per day for age group 15-17 years were derived (105).
In a study from Germany, only children who ate food enriched with folic acid had a folate intake corresponding to recommended EFSA intake.For age group 6-12 months, the folate intake was ~105 μg DFE per day and increased to 323 μg DFE per day in age group 15-18 years, compared to infants who did not eat folic acid-enriched food: ranging from ~63 μg DFE per day to 164 μg DFE per day from 6-12 months to 15-18 years (118).
During the first 2 months of life, exclusively breastfed low-birth-weight (<2,500 g) and/or preterm infants (≤32 gestational weeks) could be at risk for folate deficiency, especially when mothers are smokers and/or do not receive folic acid supplementation during pregnancy (119).

Integration
Recommended intake of folate for various age groups must depend on what is considered an adequate status according to levels of serum folate and the metabolic marker tHcy and not only on the absence of clinical signs of folate deficiency.Based on the recommended clinical decision levels indicating deficency, serum folate needs to be >10 nmol/L in adults and red cell folate >906 nmol/L in women of fertile age.
When estimating folate requirements, one must consider different bioavailability and functionality between synthetic folic acid and dietary folate, along with increased needs of folate in women of fertile age, pregnant and lactating women, preterm and small for gestational age weight infants and individuals who are homozygote for the MTHFR gene polymorphism.Adequate levels of both folate and vitamin B 12 are necessary for an optimal intracellular metabolism, and folate status needs to be reviewed together with serum vitamin B 12 .

Fig. 2 .
Fig. 2. Change in plasma tHcy in relation to serum folate in adults >16 years with normal renal function and serum cobalamin >275 pmol/L (n = 12,988) by generalized additive models (GAMs).The values on the y-axis represent the difference from mean plasma tHcy.Published with permission of the Journal of the Norwegian Medical Association (60).