Rethinking the Efficacy of Natural and Synthetic Folic Acid on Human Health and Looking into a Better Alternative

Women in their childbearing age are encouraged to eat up folic acid before conception worldwide by governments to prevent defects in the neural tube.[1-10] In realm like Australia, the United States of America and Canada, there has been implementation of the programme that includes taking food fortified with folic acid to make sure that all women who may become pregnant unintentionally and those not taking supplements of folic acid do not have folic acid deficiency throughout the period of pregnancy and thus prevent neural tube defects.[11,12] In Australia, along with the consumption of folic acid-fortified food, a woman wanting to conceive has been recommended to take 400 μg of folic acid.[6] Although neural tube defects have been prevented, research shows that unmetabolised folic acid has been noted in the serum of individuals taking folic acid supplementation and/or enrolled in folic acid-fortified food programmes.[13-15] Unmetabolised folic acid in the system may lead to several adverse effects in these individuals.[16-21] NATURAL FOLATE PROVENANCE Natural folate originates in foods such as green vegetables and several fruits where it is in its reduced form.[13] Okra, papaya, jackfruits and beans are the good sources of natural folic acid. Five different varieties of Mango (Anacardiaceae, Mangifera indica) were analysed in a study for folic acid content and the values ranged from 55.8 ± 0.73 to 74.5 ± 2.09 μg/100 g.[22] A similar study done by Akilanathan et al. found that folic acid content of mango was between 60 and 138 μg/100 g.[23] Both Striegel et al. and Akilanathan et al. noted that the most miniature and unripe fruits suggested a relation between folate content and size of the fruit.[22,23] The content of folic acid in guavas (Myrtaceae, Psidium) was also analysed in studies done by Striegel et al. and Akilanathan et al. Striegel et al. studied different varieties of guavas and reported folate content extend between 43.1 ± 5.16 μg and 47.9 ± 0.57 μg/100 g in the case of unpeeled guava.[22] Akilanathan et al. found folate content in these fruits to be between 49 and 211 μg/100 g.[23] The folate found in guava was in the form of 5-CH3-H4 folate and mango; it is in the form of 5-CH3-H4 folate which is predominant and 5-CHO-H4 folate (in low amount).[22] Striegel et al. also measured the folate content of two papaya fruits (Caricaceae and Carica papaya) and found water-soluble natural Vitamin B9 from 61.6 ± 3.01 to 90.7 ± 1.24 μg/100 g. Furthermore, when they recorded the folate content of papaya pulp and seeds separately, they noted that the seed had folate content of 25.6 ± 5.91 μg/100 g and 41.2 ± 1.91 μg/100 g and the content of folate in pulp was 56.3 ± 1.48 μg/100 g and 90.8 ± 1.91 μg/100 g. In papaya, the folate was found in the form of 5-CH3-H4 folate.[22] They also measured the folate content in two jackfruits and found its content to be similar in quantity to that of mango. The folate content in jackfruit was recorded to be 83.6 ± 5.50 (1) μg/100 g and 52.9 ± 2.61 (2) μg/100 g and in the form of 5-CH3-H4 folate.[22] Okra (Malvaceae, Abelmoschus esculentus) is a vegetable that is a good source of folate, as observed in several studies.[22,24,25] Ismail et al. reported that the amount of folate in okra was 100 μg/100 g.[24] Devi et al. revealed a higher amount (81 μg/100 g) of folate in okra.[25] However, other fruits such as dragon fruit and tamarind have low folate content.[22] CONSUMPTION OF FOLIC ACID FROM FORTIFIED SOURCES AND SUPPLEMENTATION It is generally agreed on that at least 400 μg of folic acid per day must be taken more than the natural folate ingested from a normal daily diet by women in their childbearing age to lower the future neural tube defect development risk in their children.[10] It was observed that women are getting half of the daily recommended dose of folic acid from natural sources, and this led to the fortification of food (140 μg/100 g in cereals) by the United States of America in 1998 and 200 μg/100 g of wheat flour in Australia in 2009.[11,26] However, several foods that may be fortified were not controlled. Furthermore, adults and children exceeding upper tolerable limits, as well as its harmful effects, were not taken into consideration.[21,27,28] Natural folate versus synthetic folic acid metabolism Naturally occurring folic acid is in the polyglutamate form, which is converted to mono-glutamate by the enzyme glutamate carboxypeptidase II (present in the brush border of the intestine). The folate crosses the apical brush border through a proton-coupled transporter and reduced folate carrier.[29-31] The food-sourced folate is converted chiefly to 5-methyltetrahydrofolate in the gastrointestinal and hepatic system. The reduced folate carrier has a higher affinity for folate in its reduced form, like 5-methyltetrahydrofolate. This carrier is noted all through the intestine and found in high concentrations in the liver and placenta.[30] The proton-coupled folate (found in the duodenum and jejunum brush border) functions optimally in an acidic pH and has a higher affinity for folic acid than for reduced folates.[16,30,32] Reduced folates are converted very well to 5-methyltetrahydrofolate. However, since it is the first-pass biotransformation and absorption for folic acid, the variation between folic acid and reduced folate metabolism depends on its dose.[14,33] After absorption, the folate passes through the hepatic portal vein to the liver.[30] Natural folates are not necessarily converted into 5-methyltetrahydrofolate in the intestine but must be metabolised in the liver before being released into bile or blood.[31] Although natural folates are directly converted to 5-methyltetrahydrofolate, the synthetic form of folic acid depends on an enzyme known as dihydrofolate reductase for reduction.[21] This enzyme’s activity is slow and saturates quickly. Thus, when the dose of folic acid is more than the enzyme’s ability or if there is polymorphism, there may be a rise in plasma-free folic acid or unmetabolised folic acid [Figure 1].[31] The 5-methyltetrahydrofolate is significant for synthesising DNA and increasing cell division during the development of the foetus.[30]Figure 1: The difference in the metabolism of natural folic acid and synthetic folic acid. DHFR: Dihydrofolate reductase; UMFA: Unmetabolised folic acid; MTHFR: Methylenetetrahydrofolate reductase; MTHF methyltetrahydrofolate; 5-MTHF: 5-methyltetrahydrofolate. This figure has been drawn with the premium version of BioRender (https://biorender.com/accessed on 15th December 2023) with license number AK267V4SEG. Image credit: Rahnuma Ahmad.The uptake of 5-methyltetrahydrofolate in the developing brain and placenta employs folate receptor-α. Activities that depend on folate, such as uterus enlargement, tissue growth and placenta development, lead to upregulation of the folate receptor-α during pregnancy.[30,34] The methyl group of 5-methyltetrahydrofolate promotes methylation of DNA and is, therefore, directly connected to the embryo’s viability.[30,35] Deficiency of 5-methyltetrahydrofolate would thus lead to hampering of methylation of DNA, breaking of chromosomes, impairment of ovarian follicular development and raised pregnancy loss risk.[34] The metabolisation of folate can be deficient in individuals carrying the methylenetetrahydrofolate gene polymorphism, leading to a decrease in 5-methyltetrahydrofolate.[36-38] Bioavailability of folic acid It had been assumed that synthetic folic acid bioavailability was better than that of natural folic acid based on a small-scale study, in which it was noted that natural folate (a reduced form) had bioavailability about 50% of that of synthetic folic acid.[10] However, other studies have since found that the bioavailability of natural folate ranges from 78% to 98% of folic acid in food.[39,40] Another study found that natural folate had the same bioavailability as synthetic folate.[31] It is essential to understand the metabolism of folate, considering the influencing factors for natural folate such as folate loss during cooking, enzyme action in brush border, intestinal luminal pH and nutrients that affect absorption.[31,40-42] Bioavailability of natural folate needs to be appropriately estimated to make an accurate estimation of the amount of synthetic folic acid that needs to be added to food. This may help policy-makers to come to a proper decision that will prevent harmful adverse effects amongst consumers due to an overdose of folic acid (synthetic).[43] The bioavailability of 5-methyltetrahydrofolate should also be compared with that of synthetic folate, as studies have found the bioavailability of the two to be equivalent to each other.[27,44] Even though synthetic folic acid may be more bioavailable, it is not sufficiently metabolised to form 5-methyltetrahydrofolate. This is because its metabolism depends on the dihydrofolate reductase enzyme that becomes saturated easily beyond 200–300 μg dose of folic acid instead of converting to dihydrofolate or tetrahydrofolate. Hence, intact folic acid accumulates in the serum.[14,45-47] This increase in amount may inhibit the formation and reuptake of 5-methyltetrahydrofolate.[33] DOSES OF FOLIC ACID AND UPPER TOLERABLE LIMIT Fortification of food with folic acid is mandatory in some countries, and since it is freely available, inexpensive and stable, it is used widely for fortification of food and supplementation.[7,43,48-50] However, there is no restriction on the several foods that often added nutrients, especially pre-packed food by manufacturer to attract consumers and maximise profit. This may result in the intake of folic acid levels to exceed the daily requirement of 100 μg/day.[11,12,51-56] Governments promote the intake of folic acid supplements and the consumption of natural folates and other fortified food during pregnancy and preconception. The Australian Health Department recommends a dosage of 400–500 μg in a day for such women, which they include in prenatal multi-vitamins, whereas in the United States, recommendation of 800 μg/day of folic acid has been recommended by the Centres for Disease Control and Prevention.[5-7] In Canada, prenatal multivitamins contain a minimum of 1000 μg of folic acid.[54,57,58] There is also a routine prescription of 4–5 mg of folic acid for women who are at a high pregnancy loss risk or have a prior history of neural tube defect-affected pregnancy in the United States, Canada and Australia.[6-9,59] As per total food folate daily folate equivalent (DFE) (a method for comparing folate provided to the human body by natural and synthetic folate), the folate from folic acid from natural sources is 1.7 times less than that from fortified food. For example, a food serving of green leafy vegetables provides 100 μg of folate, equivalent to 100 μg of DFE. In contrast, fortified food with 100 μg of folic acid provides about 170 μg DFE.[43] There is an upper tolerable limit for synthetic folic acid, which does not exist for natural folate. No upper limit is recognised for the forms that exist in food from nature. The Institute of Medicine of the United States of America has set the upper tolerable limit for folic acid at 1000 μg.[10,33] Synthetic folic acid in high doses has a masking effect in the case of Vitamin B12, and a study noted that folic acid in excess results in a rise in intact folic acid.[13,16,19,21,60,61] Un-biotransformed folic acid accumulates when folic acid does not get metabolised to 5-methyltetrahydrofolate due to the limited activity of dihydrofolate reductase enzyme, and it may appear within the circulation when even 200 μg folic acid is consumed in a day.[45,62] Several adverse health conditions are connected to not metabolically transformed folic acid, which includes cleft lip, autism in children, decreased functioning of the brain as well as lowering somatosensory and motor processing in children, cancer, cytotoxicity related to natural-killer cells, anaemia, asthma, impairment of cognition and adverse effects of the cardiovascular system [Figure 2].[21,61,63-67]Figure 2: Illustrates the adverse effect on human health resulting from the accumulation of unmetabolised folic acid in the blood when synthetic folic acid fails to convert to dihydrofolate due to saturation of the rate-limiting enzyme dihydrofolate reductase. DHFR: Dihydrofolate reductase this figure has been drawn with the premium version of BioRender (https://biorender.com/accessed on 15th December 2023) with license number HQ267V2GAA. Image credit: Rahnuma Ahmad.Women exposed to high doses of folic acid may suffer from adverse outcomes of health, which makes it necessary to understand clearly all the sources from which women in the stages of preconception and pregnancy consume folate.[43] The primary reason for exceeding the 1000 μg a day (upper tolerable limit) in 2.4%–7% of women of motherhood age was the contribution of 47.5%–57% of folate ingesting through the supplementation.[4,56,68,69] The consumption of about 400–1000 μg of folic acid per day through supplements was observed.[43] A study reported that 24% of women take 400 μg of folic acid daily,[70] whereas in another investigation, it was about 78% of women.[69] About 20% of women were observed to be taking folic acid supplementation of 1000 μg per day.[69,71] A higher percentage of gravid womenfolk were exposed to added extra amount of folic acid in comparison to women who were not pregnant. Studies found 92% to 72% of folic acid consumption in pregnant women.[70,72] In several studies, folic acid-based supplementation was the reason for pregnant women crossing the upper limit of folic acid.[53,55,57,58,65,73-78] Studies found that the upper limit for folic acid was exceeded in 33.4%,[73] 40%,[76], 25%[57] 87%,[74] 83%–85%,[45,58] 96%[53] and 100%[75] of women. The excess consumption of folic acid was noted to occur in the first 3 months of gestation.[77] A mean dose of folic acid consumption per day was found to be 1000 μg/day,[58] 878 μg/day[76] and above 2000 μg/day.[53,65] The DFE level for women of childbearing age was observed to range from 864 μg DFE to 1778 DFE,[1,71] and in pregnant women, DFE went from 1451 μg DFE to 2181 μg DFE.[73,74] Yet again, the DFE intake mainly occurred in the first trimester of pregnancy.[53,58] Unmetabolised folic acid was found in 93% of cord blood samples and 97% of women in the first trimester.[58] RECOMMENDING 5-METHYLTETRAHYDROFOLATE INSTEAD OF SYNTHETIC FOLIC ACID The saturation point of metabolism of folic acid appears to be exceeded upon consuming fortified food, and during pregnancy, it almost always crosses the upper limit. It is also recognised from earlier research that consuming natural folates only may not be sufficient to meet the demands for healthy foetal development.[43] Therefore, 5-methyltetrahydrofolate may be considered instead of synthetic folic acid. It does not accumulate like synthetic folic acid[30,31] and can raise folic acid levels in the blood to avert neural tube defects such as synthetic folic acid.[29,59,79-82] It was noted to expand, more effectively, the plasma levels of folate compared to folic acid.[82] The 5-methyltetrahydrofolate can raise the serum folate levels rapidly when 7.5 mg is consumed every 12 h for 4 days and replenish stores of folic acid in women suffering from insufficiency in a few days.[81] Various studies have noted no adverse effects of consuming high doses of 5-methyltetrahydrofolate (17 mg per day for 12 weeks; 15 mg; 7.5 mg) in animal studies and studies done on pregnant women as well as patients with depression.[83-86] Folate status may be more effectively improved by 5-methyltetrahydrofolate and is likely a better substitute for folic acid in those suffering from repeated loss of pregnancy and infertility.[29,59] Optimum levels of folate needed to decrease the hazard of neural tube defect or help prevent miscarriages through adequate DNA methylation may not be achieved by the consumption of natural folates alone. However, intake of food that has been fortified with synthetic folic acid puts women at risk of having not biotransformed folic acid accumulation in their system. Further supplementing with synthetic folic acid above the upper tolerable limit may lead to adverse effects on health, decreased activity of methyltetrahydrofolate, masking of Vitamin B12 and lessening in DNA methylation that can cause adverse outcomes of pregnancy such as loss of pregnancy. Thus, policy-makers and researchers in the field of public health need to find an appropriate dose for folic acid or find an alternative to ensure adequate folate levels and avoid the build-up of not biotransformed folic acid levels to fulfil the goal of a good health outcome for both foetus and mother. Author contributions All authors contributed significantly to the work, whether in the conception, design, utilisation, collection, analysis and interpretation of data or all these areas. They also participated in the article’s drafting, revision or critical review, gave their in final approval for the version that would be published, decided on the journal to which the article would be submitted and made the responsible decision to be held accountable for all aspects of the work.

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