The preventive effect of high folic acid intake against neural tube defects (NTD) is considered one of the most important nutritional discoveries in the period 1976-2006 [1]. Folate requirements are increased in life stages with amplified cell division, e.g., during pregnancy, as folate is essential during cellular replication. It is assumed that on a population level, nutritional requirements for folate, as with those for vitamin D, cannot be completely covered by a “varied diet”, as recommended by national health authorities [2]. Dietary intake is below recommendations [3] in various Western societies owing to low consumption of folate-rich foods, e.g., pulses, citrus fruits, and leafy vegetables. It is estimated that an additional intake of 50-180 µg folate would allow most to reach the recommendations [4]. Populations of low socio-economic status have limited access to folate-rich foods, and furthermore natural food folates are rather unstable molecules, so that average losses in vitamin activity of around 30% can be expected during food processing [5]. However, the only risk factor for NTD which can be directly affected is inadequate dietary folate intake [6].

The chemically most stable folate form is synthetic folic acid [7], which is cheap to produce and therefore used for dietary supplements and food fortification. The introduction of folic acid-fortified staple foods has effectively decreased the prevalence of NTD, e.g., in the USA and Canada [8]. Significantly improved stroke mortality has also been reported [9]. In most countries, the Recommended Dietary Allowance (RDA) for folate is 300 µg/day for adults and 400 µg/day for women of childbearing age. With respect to vascular function, a folate intake according to the above recommendations has been shown to be beneficial, whereas higher doses do not provide any further health benefits [10]. The recommendations are more easily met by consumption of folic acid-fortified foods, owing to the higher stability and bioavailability of synthetic folic acid compared with native food folates [11]. The bioavailability of food folate is commonly estimated at 50% of folic acid bioavailability when establishing food recommendations [2,12], but this should be considered a rough estimate, as data on the bioavailability of food folate vary between 30% [13] and 98% [14]. In the US, dietary recommendations are expressed in terms of Dietary Folate Equivalents (DFE) to account for differences in the bioavailability of native food folate and folic acid fortificant [12]. The discrepancies in folate bioavailability estimates are caused by the nature of the test food or meal (as thoroughly reviewed [15,16]), entrapping the vitamin and affecting stability and absorption, inter-subject variation, different study designs and, as recently suggested, metabolic inequities between different folate forms with respect to post-absorption plasma kinetics in the human metabolism [17,18,19]. Another important aspect is that the interpretation of bioavailability data is hampered by large methodological differences during folate quantification in clinical samples and test foods, which is complicated by lack of standardized reference methods for sample preparation and folate quantification, internal standards, and use of certified reference material [20,21,22]. Recent publications question the validity of the conventional approach used in bioavailability studies comparing folic acid with natural food folate [19], and demand approaches using whole diets rather than single foods [11] or data from meta-analyses of controlled feeding trials [23] in order to closer elucidate post-absorptive metabolism of the different folate forms. This is considered necessary for evaluating the adequacy of US RDA for folate [23], and also for estimating potential risks from mandatory folic acid fortification, as a high intake of synthetic folic acid is suggested to accelerate the development of, e.g., colorectal cancer [24,25,26].

It is commonly assumed that food folates, which mainly exist in their polyglutamyl form, are absorbed in the jejunum as monoglutamyl folates after removal of the polyglutamyl chain by intestinal γ-glutamyl hydrolase [27], and thereafter reduced and methylated in the enterocyte. Passive diffusion across the cell membrane is limited [28], and occurs only at high doses. To a minor extent folate is also absorbed in the colon; and it is suggested that colonic absorption may contribute significantly to total folate absorption [29], but it is unknown how relevant this is for maintaining folate status. However, it has been shown for humans [29,30] and pigs [31] that folates synthesized by colon bacteria are bioavailable. Absorbed folate is transported to the liver, which contains about half the body pool of folate [32,33] and retains 10-20% of absorbed folate due to the first-pass effect [15], while the rest is transported via the systemic circulation to body tissues. Some liver folate participates in the enterohepatic circulation and is secreted into bile [33,34]. However, most biliary folate is reabsorbed, supposedly to moderate between-meal fluctuations in folate supply to cells [34].

In the cell nucleus, tetrahydrofolate-polyglutamate (H₄-folate) is the substrate for both nucleotide synthesis and the methylation cycle (Figure 1), by providing or accepting one-carbon atoms [35]. 5-methyltetrahydrofolate (5-CH₃-H₄folate) donates a methyl group to vitamin B12, which transfers it to homocysteine, avoiding accumulation of homocysteine in the cell and maintaining synthesis of amino acids. The first signs of deficiency appear in tissues with rapid cell turnover and during life stages with increased cell division, such as pregnancy or growth [36], and can result in megaloblastic anemia by impaired synthesis of red blood cells [33].

It is estimated that about 0.3-0.8% of the folate pool is excreted daily, mainly in the form of catabolites not affected by intake [32,39]. Renal excretion of intact folate is as low as ~5% after physiological doses [34,40,41,42], but it increases at higher folate intake [37]. In human feces, about 400 nmol/day folate can be found, originating mainly from bacterial production, lysed enterocytes, and gastrointestinal secretions (e.g., bile) [34,43]. Little to at most 20% of fecal folate is estimated to derive from non-absorbed food folate [34,44,45,46,47].

Genetic polymorphisms of key enzymes and carriers can affect cellular folate metabolism and result in decreased enzyme activity, increased folate requirements [48,49,50], and an increased risk of NTD [51]. Furthermore, vitamin B12 deficiency reduces the activity of methionine synthase (MS; Figure 1), resulting in cellular accumulation of homocysteine and 5-methyltetrahydrofolate according to the methyl trap theory [37].

Accurate estimation of dietary folate bioavailability is a prerequisite for the formulation of nutrition recommendations, but is still a challenge. Folate absorption is defined as the in vivo process by which folate proceeds from the site of administration to the site of measurement (usually plasma) [52]. Folate bioavailability is defined as the fraction of ingested folate that is absorbed and can be used for metabolic processes [16]. Findings from early bioavailability studies until the mid-1990s discussed by others indicate the need for further investigations on folate bioavailability and influencing factors [53].

Long-term (Table 1 and Table 2) and short-term (Table 3) human trials, conducted during the past 15 years, have produced inconclusive findings regarding dietary folate bioavailability, complicating the interpretation of data by different definitions for the term bioavailability and differing study designs. Furthermore, difficulties with respect to the exact quantification of ingested folate dose from food samples and in clinical samples limit the validity of many studies [19,54]. Although there are recommendations for the extraction of food samples using trienzyme treatment [55,56,57], this approach is used in only some studies [13,14,45,58,59,60,61,62,63,64]. Others apply dienzyme [46] or monoenzyme [18,40,42,65] extraction prior to folate quantification by microbiological assay or HPLC (LC-MS or LC-FLD). Information is sometimes missing [17,66,67], or dietary folate doses are determined by calculation [19,68,69,70] (Table 1 ,and ).

This summary of existing information shows that only limited folate bioavailability data are available for vegetables, fruits, cereal products and fortified foods. It also shows the difficulties in assessment. No safe conclusion can be drawn as to whether intervention with food folate results in improved folate status. Additionally, it is not possible to estimate the size of the required minimum intervention dose of food folate to achieve improvement of status, as the status parameters are affected to different extents.

However, the data highlight differences in the metabolic handling of folic acid and reduced folates in humans, as evidenced by differing acute post-absorptive plasma kinetics and different effects on fasting plasma folate concentrations after long-term intervention. It is still unclear whether these findings have implications for human health, and they need to be considered in recommendations regarding folic acid fortification of food. As it is not expected that the more expensive and less stable bioactive diastereoisomer (6S)-5-methyltetrahydrofolate (Metafolin^®) will be commonly used for fortification purposes, synthetic folic acid reference doses are appropriate for evaluation of the effectiveness of folic acid-fortified foods at population level.

An immediate consequence is our recommendation to revise the classical approach of using folic acid as the reference dose in estimating the plasma kinetics and relative bioavailability of food folate. The design of future studies on relative folate bioavailability faces the challenge of accounting for the bias from metabolic handling of folate compounds, and existing findings have to be interpreted with caution.