Vitamin B9 (methylfolate/5-MTHF)

Vitamin B9: Methylfolate (5-MTHF / L-Methylfolate)

1. Identity, Chemical Names, and Common Forms

Folate is a water-soluble B vitamin that is naturally present in some foods, added to others, and available as a dietary supplement. Folate, formerly known as folacin and sometimes vitamin B9, is the generic term for naturally occurring food folates and folates in dietary supplements and fortified foods, including folic acid.

Folate is the generic term for vitamin B9, a water-soluble vitamin that includes chemically similar compounds essential in periods of rapid cell growth and division, in the maintenance of new cells, and in the making of DNA and RNA.

The term "folic" is from the Latin word folium (which means leaf) because it was found in dark-green leafy vegetables. Folate (vitamin B9) refers to the many forms of folic acid and its related compounds, including tetrahydrofolic acid (the active form), methyltetrahydrofolate (the primary form found in blood), methenyltetrahydrofolate, folinic acid, folacin, and pteroylglutamic acid.

The specific subject of this article — methylfolate — is the biologically active, circulating form of vitamin B9. Some dietary supplements contain folate in the monoglutamyl form, 5-MTHF (also known as L-5-MTHF, 5-methyl-folate, L-methylfolate, and methylfolate). Its systematic chemical name is (6S)-5-methyltetrahydrofolic acid, and it is also known commercially as levomefolic acid.

It is synthesized in the absorptive cells of the small intestine from polyglutamylated dietary folate. It is a methylated derivative of tetrahydrofolate. Levomefolic acid is generated by methylenetetrahydrofolate reductase (MTHFR) from 5,10-methylenetetrahydrofolate (MTHF) and used to recycle homocysteine back to methionine by methionine synthase (MS).

In the plasma of healthy humans, 5-MTHF typically constitutes 80–90% of total folate.

Stereochemistry and Biological Activity

The letters or numbers before the name refer to the 3D chemical structure of that compound. L- and 6(S)- indicate biologically active L-methylfolate (helpful). D- and 6(R)- indicate non-biologically active L-methylfolate (not helpful). Some products may only list "Methylfolate" or "5-MTHF," without specifying which form they contain. If the label does not state L-Methylfolate, Metafolin, or Quatrefolic, it may not be the biologically active form.

Relationship to Folic Acid

Folic acid, a synthetic compound not found in nature, is used in fortification and dietary supplementation programs. To be used by the body, folic acid must be metabolized via a series of conversions into 5-methyltetrahydrofolate (5-MTHF), the bioavailable form of folate found in blood.

L-5-MTHF is the predominant naturally occurring folate in foods. Folic acid (pteroylglutamic acid, sometimes known as Vitamin B9), which is widely used for food fortification and as an ingredient in dietary supplements, is the precursor of L-5-MTHF.

Commercial Salt Forms

In the supplement and food industry, 5-MTHF is available in several stabilized salt forms. L-5-MTHF-Ca is intended for use in dry crystalline or microencapsulated form as an alternative to folic acid in foods; it is a white to light yellowish, almost odourless, water-soluble crystalline powder. Additional commercially used salt forms include the glucosamine salt (Quatrefolic®) and the sodium salt (Arcofolin®). The different salt versions (calcium, sodium, and glucosamine) of 5-MTHF are expected to be absorbed across the small intestine in a similar manner as 5-MTHF originating from the diet.

Natural Food Sources

Folate is naturally present in a wide variety of foods, including vegetables (especially dark green leafy vegetables), fruits and fruit juices, nuts, beans, peas, seafood, eggs, dairy products, meat, poultry, and grains. Folate refers to a group of water-soluble B9 vitamins found naturally in foods like leafy greens, beans, and eggs. Top folate-rich foods include kale, broccoli, citrus fruits, asparagus, bananas, beans, and liver, with beans and liver offering the highest amounts. However, natural folates are susceptible to oxidation, can rapidly lose activity in foods, and are largely destroyed by cooking — up to 90%. Moreover, they have low and incomplete bioavailability.

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2. Historical and Traditional Use

Scientific Discovery (20th Century)

Because folate is a micronutrient rather than an herbal or botanical ingredient, its history is primarily a narrative of 20th-century nutritional science rather than ethnobotanical tradition. In the 1930s, Lucy Wills identified a "new hemopoietic factor" in yeast and liver which cured tropical macrocytic anemia in humans and experimental anemia in monkeys. Janet Watson and William B. Castle named the unknown substance, which would ultimately become a form of folate, "Wills' factor."

Further studies with this unknown substance showed that it was active against nutritional pancytopenia in monkeys and experimental anemia in chicks, leading to various designations such as vitamin M (monkey) and vitamin Bc (chick). Other factors with growth-promoting activity for microorganisms such as Lactobacillus casei were given the interim names including folic acid — in recognition of extracts from leafy greens.

Bob Stokstad isolated the pure crystalline form in 1943, and was able to determine its chemical structure while working at the Lederle Laboratories of the American Cyanamid Company. This historical research project, of obtaining folic acid in a pure crystalline form in 1945, was done by the team called the "folic acid boys," under the supervision and guidance of Director of Research Dr. Yellapragada Subbarow, at the Lederle Lab, Pearl River, New York.

Stokstad's discovery proved that the crystalline form of folic acid was composed of a pteridine ring, para-aminobenzoic acid, and glutamic acid, and was called pteroylglutamic acid. This research subsequently led to the synthesis of the antifolate aminopterin, which was used to treat childhood leukemia by Sidney Farber in 1948.

In the 1950s and 1960s, scientists began to discover the biochemical mechanisms of action for folate. Folic acid has a long history of use, in conjunction with vitamin B12, for treatment of macrocytic (or megaloblastic) anemia.

Public Health Applications and Fortification

In the US, folic acid has been added to certain grain products like bread and pasta since 1998 to help prevent deficiencies. As of 2017, 86 countries required wheat flour to be fortified with folic acid by law. 5-MTHF, short for 5-methyltetrahydrofolate, is the bioactive form of vitamin B9.

Folate is on the World Health Organization's List of Essential Medicines. In 2023, it was the 94th most commonly prescribed medication in the United States, with more than 7 million prescriptions.

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3. Key Constituents and Mechanisms of Action

One-Carbon Metabolism

Regardless of how the active form of folate 5-MTHF has been obtained, methylfolate, in concert with vitamin B12, enters one-carbon metabolism. This metabolism is a network of interrelated biochemical reactions that occurs in all of the body's cells, and it is vital for various functions, including detoxification, energy production, immune function, maintenance and regulation of genes, mood balancing, and control of inflammation. It is essential for sustaining life and for inhibiting or slowing the development of age-associated diseases.

Folate-mediated one-carbon metabolism (FOCM) is a tightly interconnected metabolic network in which tetrahydrofolates (THF) carry and chemically activate one-carbon moieties for biosynthetic reactions including de novo purine synthesis, de novo thymidylate (dTMP) synthesis, and remethylation of homocysteine to methionine.

Homocysteine Remethylation and SAM Production

MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (5-methylTHF), the main form of circulating folate and the methyl donor for homocysteine remethylation to methionine in the reaction catalyzed by methionine synthase (MTR), which transfers the methyl group from 5-methylTHF to homocysteine, forming tetrahydrofolate (THF) and methionine. Methionine is then used for the production of SAM, required for DNA and protein methylation reactions, and THF re-enters the folate pathway as an acceptor of novel one-carbon moieties.

DNA Synthesis and Cell Division

Folate functions as a coenzyme or cosubstrate in single-carbon transfers in the synthesis of nucleic acids (DNA and RNA) and metabolism of amino acids. One of the most important folate-dependent reactions is the conversion of homocysteine to methionine. Another folate-dependent reaction, the methylation of deoxyuridylate to thymidylate in the formation of DNA, is required for proper cell division. An impairment of this reaction initiates a process that can lead to megaloblastic anemia, one of the hallmarks of folate deficiency.

Epigenetics and DNA Methylation

Dietary folate is the most extensively studied micronutrient in animal and epidemiological DNA methylation research. Folate is reduced to dihydrofolate (DHF) and subsequently to tetrahydrofolate (THF), serving as a single-carbon donor in the form of 5-methyl THF. Consequently, 5-methyl THF feeds into the one-carbon metabolism cycle by donating its methyl group to homocysteine, converting it to methionine.

5-MTHF is the main form of dietary folate and represents the predominant physiologic form of folate found in blood and in umbilical cord blood; the availability of 5-MTHF contributes to the conversion of methionine to SAM (S-adenosylmethionine), the universal effector for methylation. After the release of a methionyl group, S-adenosylhomocysteine (SAH) and homocysteine accumulation exert feedback inhibition on the MS enzyme, inhibiting methylation.

Neurotransmitter Synthesis

Adequate folate status is necessary for methylation reactions that sustain tetrahydrobiopterin (BH4) and SAM — both required for synthesis of serotonin, dopamine, and norepinephrine. In other words, low L-methylfolate availability can indirectly impair neurotransmitter synthesis, which may contribute to depressive symptoms or treatment resistance in some patients.

The MTHFR Enzyme and Genetic Polymorphisms

Among common MTHFR genetic variants, C677T and A1298C are the most studied. The C677T homozygous (TT) genotype can reduce enzyme activity by about 70%, while heterozygotes (CT) show about a 30–40% reduction. The A1298C variant generally has milder effects and rarely elevates homocysteine on its own.

A number of consecutive steps in the two cycles are subject to mutations due to single nucleotide polymorphisms (SNPs) in the methyltetrahydrofolate reductase (MTHFR) enzyme that affect the efficiency of the cycles by decreasing MTHFR activity, compromising methylation reactions via the reduced availability of methionine, and the accumulation of homocysteine.

MTHFR is an enzyme required for the formation of 5-methyltetrahydrofolate (5-MTHF), a form of folate able to cross the blood-brain barrier and which is necessary as a substrate for the remethylation of homocysteine to methionine by methionine synthase.

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4. Scientific Evidence by Area of Use

4.1 Neural Tube Defects (NTD) Prevention

A crucial role demonstrated for folic acid is to help prevent neural tube defects (NTDs). This is the best-established clinical application for folate supplementation broadly.

Research efforts have been productive in the identification of environmental factors, such as periconceptional folic acid supplementation, that modulate risk for the development of NTDs. Studies of the folic acid biosynthetic pathway led to the discovery of an association between elevated levels of homocysteine and NTD risk.

Regarding 5-MTHF specifically for NTD prevention: More studies are definitely still needed to establish 5-MTHF as a safe and effective therapeutic approach comparable with folic acid. Moreover, there is a lack of clinical studies that evaluate the efficacy of 5-MTHF supplementation in the prevention of NTDs. Despite the potential value of 5-MTHF as an alternative to folic acid, clinical studies would be urgently needed to support the efficacy, dosage, timing, and/or safety of its use as a supplement.

Current evidence does not demonstrate a clear advantage of 5-methyltetrahydrofolate (5-MTHF) over folic acid for most pregnant women in preventing neural tube defects, though 5-MTHF may offer theoretical benefits for specific populations with reduced folic acid metabolism.

Regarding homocysteine levels, total plasma homocysteine levels decreased significantly in both subjects receiving 1 mg folic acid or 5-MTHF over time; however, no differences in total homocysteine levels nor abortion rates were observed between the groups.

Evidence strength: Strong for folate (as a class) in NTD prevention; insufficient direct evidence specific to 5-MTHF supplementation versus folic acid for NTD prevention in clinical trials.

4.2 Homocysteine Lowering and Cardiovascular Risk

5-MTHF, the predominant form of folate in plasma and red cells, is a substrate for the methionine synthase and vitamin B12 (methylcobalamin)-mediated conversion of homocysteine to methionine. Suboptimal 5-MTHF availability leads to an increase in circulating homocysteine (hyperhomocysteinaemia), which has been associated with many diseases and health complications including cardiovascular disease.

A 13-week randomized, double-blind clinical trial enrolled 149 free-living persons with mild hyperhomocysteinemia. Subjects received daily 200 µg from a natural folate-rich diet, 200 µg [6S]-5-methyltetrahydrofolate (5-MTHF), 200 µg folic acid, or placebo. Participants were stratified according to their MTHFR genotype.

In the SU.FOL.OM3 trial, 2,381 patients with a personal history of cardiovascular disease were randomly assigned to one of four groups: B-vitamins alone (560 µg of 5-methyl-THF, 3 mg of vitamin B6, and 20 µg of vitamin B12), n-3 fatty acids alone, B-vitamins and n-3 fatty acids, or placebo. Participants were followed up for 4.7 years. Total homocysteine decreased by 26.3% during the first year (p < 0.0001), then steadily increased throughout the 5 years. However, at the end of follow-up, that increase was smaller among TT than among CT or CC subjects.

A randomized, double-blind, placebo-controlled trial by Venn et al. and Lamers et al. demonstrated that supplementation with either L-5-MTHF or folic acid equally reduced plasma total homocysteine concentrations in healthy women (as cited in peer-reviewed literature), with comparable efficacy between the two forms.

Prinz-Langenohl et al. demonstrated that 5-MTHF supplementation is not affected by MTHFR gene polymorphism. In another chronic bioavailability study, Litynski et al. showed a significant prolonged effect in reducing homocysteine levels at six months after ceasing treatment with 5-MTHF in homozygous individuals (TT), at 12.1 ± 2.5 as compared to 16.9 ± 6.8 for folic acid (P < .01).

A 2024 randomized, double-blind, placebo-controlled trial specifically investigated the effect of methylfolate-containing B-vitamin formulations: Exploring the link between genetic polymorphisms in folate metabolism genes (MTHFR, MTR, and MTRR) and cardiovascular disease, this study evaluated the effect of B vitamin supplements (methylfolate, pyridoxal-5′-phosphate, and methylcobalamin) on homocysteine and lipid levels. In a randomized, double-blind, placebo-controlled trial, 54 patients aged 40–75 with elevated homocysteine and moderate LDL-C levels were divided based on MTHFR, MTR, and MTRR genetic polymorphisms. Over six months, they received either a combination of methylfolate, P5P, and methylcobalamin, or a placebo. Methylfolate, P5P, and methylcobalamin supplementation tailored to genetic profiles effectively reduced homocysteine and LDL-C levels in patients with specific MTHFR, MTR, and MTRR polymorphisms, particularly with homozygous minor allele polymorphisms.

Critically, impaired remethylation of homocysteine to methionine can lead to mild hyperhomocysteinemia, which is a biochemical marker of cardiovascular risk. However, clinical trials have not shown that lowering homocysteine reduces cardiovascular events.

Evidence strength: Moderate to strong for 5-MTHF lowering homocysteine (a surrogate marker), particularly in MTHFR TT genotype carriers. Evidence is insufficient to establish that homocysteine lowering by 5-MTHF translates into reduced cardiovascular events.

4.3 Depression and Mood Disorders

Systematic reviews suggest that adjunctive L-methylfolate modestly improves symptoms in major depressive disorder.

A landmark pair of multicenter, randomized, double-blind, parallel-sequential trials (Papakostas et al., 2012) investigated L-methylfolate augmentation in SSRI-resistant major depressive disorder: The authors conducted two multicenter sequential parallel comparison design trials to investigate the effect of L-methylfolate augmentation in the treatment of major depressive disorder in patients who had a partial response or no response to SSRIs. In the first trial, 148 outpatients with SSRI-resistant major depressive disorder were enrolled in a 60-day study divided into two 30-day periods. Patients were randomly assigned, in a 2:3:3 ratio, to receive L-methylfolate for 60 days (7.5 mg/day for 30 days followed by 15 mg/day for 30 days), placebo for 30 days followed by L-methylfolate (7.5 mg/day) for 30 days, or placebo for 60 days. The study concluded that adjunctive L-methylfolate at 15 mg/day may constitute an effective, safe, and relatively well tolerated treatment strategy for patients with major depressive disorder who have a partial response or no response to SSRIs.

A systematic review and meta-analysis (2022) analyzed 9 articles (N=6,707 patients): Qualitative assessment suggests that adjunctive L-methylfolate improved antidepressant response. In the meta-analysis of categorical Hamilton Rating Scale for Depression-17 response (three studies, N=483), adjunctive L-methylfolate was associated with improved outcomes. The conclusion was that adjunctive L-methylfolate may have modest efficacy in antidepressant-treated adults with MDD.

A separate systematic review and meta-analysis of folate (including L-methylfolate or folic acid) as adjunct to SSRI/SNRI: The initial search revealed 293 articles with 6 randomized control trials ultimately meeting inclusion criteria. That meta-analysis found a significant benefit for adjunctive therapy compared to monotherapy. Only when the evidence was restricted to folate at a dose of <5 mg/day or methylfolate at a dose of 15 mg once daily as an adjunct to SSRI therapy was there a significant benefit compared with placebo. All evidence was graded as low or very low quality for each outcome.

The rationale for the neuropsychiatric effects involves MTHFR and neurotransmitter pathways: Dysregulation of the MTHFR pathway affects the expression of neuropsychiatrically relevant genes (e.g., those involved in neuroplasticity and mood regulation), ultimately leading to poor antidepressant response and persistent depressive symptoms characteristic of treatment-resistant depression.

Evidence strength: Moderate evidence for adjunctive 5-MTHF (particularly at 15 mg/day) in SSRI-resistant major depressive disorder; most studies graded low or very low quality. Evidence for monotherapy is insufficient.

4.4 MTHFR Deficiency (Severe/Rare Genetic Form)

A case series investigated patients with severe MTHFR deficiency: Patients with severe methylenetetrahydrofolate reductase (MTHFR) deficiency cannot make 5-MTHF and have extremely low levels in the CSF. As a consequence, methylation reactions in the CNS are compromised, and this is likely to play an important role in the neurological abnormalities that occur in MTHFR deficiency. Although treatment with oral betaine can remethylate homocysteine to methionine in the liver, betaine crosses the blood-brain barrier poorly, and CSF levels of methionine remain low. Three patients with severe MTHFR deficiency (enzyme activity ≤1% of controls) had undetectable levels of CSF 5-MTHF at diagnosis and while on treatment with either folic acid or calcium folinate.

Rare and severe MTHFR mutations lead to MTHFR deficiency, an autosomal recessive inborn defect characterized by extremely high homocysteine levels in the urine and plasma, causing developmental delay, eye disorders, thrombosis, and osteoporosis.

Evidence strength: 5-MTHF (mefolinate) is specifically relevant for severe MTHFR deficiency; evidence derives primarily from case series and small clinical observations.

4.5 Pregnancy, Fertility, and Reproductive Health

Prescription of folic acid to women in the preconception period and during pregnancy is a consolidated practice. All women and teen girls who could become pregnant should consume 400 mcg of folic acid daily from supplements, fortified foods, or both, in addition to the folate they get from following a healthy eating pattern.

In dietary supplements, folate is usually in the form of folic acid, but methylfolate (5-methyl-THF) is also used. Dietary supplements containing methylfolate might be better than folic acid for individuals who have a certain mutation in a gene called MTHFR.

While best recognized for pregnancy and fertility support, recognition is growing that folate also benefits cardiovascular health, mood, and cognition.

Evidence strength: Strong for folate as a class in periconceptional neural tube defect prevention (Grade A recommendation by USPSTF). Evidence that 5-MTHF is superior to folic acid for general NTD prevention is currently insufficient; 5-MTHF may have advantages in individuals with impaired folic acid metabolism.

4.6 Cancer — Association with Folate Status

The most thorough research has focused on folate's effect on the development of colorectal cancer and its precursor, adenoma. Several epidemiological studies have found inverse associations between high dietary folate intakes and the risk of colorectal adenoma and cancer.

Conversely, data from Norwegian trials have raised safety signals: Supplementation with 800 mcg/day folic acid plus 400 mcg/day vitamin B12 for a median of 39 months in 3,411 people with ischemic heart disease increased cancer incidence rates by 21% and cancer mortality rates by 38% compared with no supplementation. Findings from these Norwegian trials have raised concerns about folic acid supplementation's potential to raise cancer risk. Whether this applies to 5-MTHF similarly has not been established in dedicated trials.

Evidence strength: Epidemiological associations only. The relationship between folate and cancer risk is complex and context-dependent (protective at adequate intake, potentially promotional at high supplemental doses or in the setting of pre-existing neoplastic lesions). Data specific to 5-MTHF supplementation and cancer outcomes are lacking.

4.7 Cognitive Function and Dementia

Decreased folate levels can be related to the development of neuropsychiatric diseases including Alzheimer's disease, Parkinson's disease, and depression. The NIH ODS specifically identifies dementia, cognitive function, and Alzheimer's disease as areas in which folate might play a role, along with depression, cardiovascular disease and stroke, NTDs, cancer, and autism spectrum disorder.

Evidence strength: Preliminary and mixed. Associations between low folate status and cognitive decline are observed in epidemiological literature, but whether supplementation with 5-MTHF specifically improves cognitive outcomes in clinical trials has not been well established.

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5. Body Systems Associated with Vitamin B9 / 5-MTHF

• Hematopoietic system: Impairment of the thymidylate synthesis reaction initiates a process that can lead to megaloblastic anemia, one of the hallmarks of folate deficiency.
• Nervous system: 5-MTHF is a form of folate able to cross the blood-brain barrier and which is necessary as a substrate for the remethylation of homocysteine to methionine by methionine synthase.
• Cardiovascular system: Suboptimal 5-MTHF availability leads to an increase in circulating homocysteine, which has been associated with cardiovascular disease. There is also evidence to suggest that 5-MTHF deficiency may be a cardiovascular risk factor independent of homocysteine.
• Reproductive system / fetal development: Folate and its metabolites play a critical role in DNA synthesis and methylation, and inadequate or imbalanced folate status is strongly associated with neural tube defects and other adverse health outcomes.
• Epigenome / gene expression: One-carbon metabolism is a network vital for maintenance and regulation of genes, mood balancing, and control of inflammation.

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6. Bioavailability and Pharmacokinetics

When consumed, food folates are hydrolyzed to the monoglutamate form in the gut prior to absorption by active transport across the intestinal mucosa. Passive diffusion also occurs when pharmacological doses of folic acid are consumed. Before entering the bloodstream, the enzyme dihydrofolate reductase reduces the monoglutamate form to THF and converts it to either methyl or formyl forms. The main form of folate in plasma is 5-MTHF.

The Food and Nutrition Board (FNB) developed dietary folate equivalents (DFEs) to reflect the higher bioavailability of folic acid than that of food folate. At least 85% of folic acid is estimated to be bioavailable when taken with food, whereas only about 50% of folate naturally present in food is bioavailable.

Regarding 5-MTHF vs. folic acid bioavailability specifically, the literature is nuanced. A double-blind crossover study in 13 men (Pentieva et al.) compared 500 µg folic acid and 500 µg [6S]-5-MTHF as single oral doses: The study demonstrated a rapid increase in plasma folate concentration, finding total equivalence in the pharmacokinetic parameters analyzed (Maximum Plasma Concentration (Cmax), time to Cmax (Tmax), and Area Under the Curve (AUC)).

Owing to the structural differences between the folic acid and 5-MTHF molecules, it is worth questioning whether there are any differences in their bioavailability. Nevertheless, the studies carried out so far do not show relevant differences in the bioavailability of these compounds. However, at higher doses, advantages for 5-MTHF emerge: 5-MTHF, when consumed as a dietary supplement or in fortified foods, has higher bioavailability than folic acid when ingested at concentrations greater than 0.4 mg per day, as the reduced form does not need to undergo the enzymatic reduction process. In addition, the mechanism by which folic acid is reduced to THF has been shown to saturate easily, thus further limiting bioavailability relative to 5-MTHF.

A 2025 randomized, double-blind, single-dose crossover study of a dicholine salt of (6S)-5-methylTHF found: The incremental area under the curve (iAUC0-8h) was significantly higher after the administration of (6S)-5-MethylTHF-2Chol compared to folic acid (1.64-fold, P < 0.0001, for total folate and 2.56-fold higher for (6S)-5-MethylTHF, P < 0.0001).

Naturally occurring 5-MTHF has important advantages over synthetic folic acid — it is well absorbed even when gastrointestinal pH is altered and its bioavailability is not affected by metabolic defects.

Regarding plasma levels after oral dosing: In a study of 21 subjects with coronary artery disease, peak plasma levels were reached in one to three hours following oral or parenteral administration. Peak concentrations were found to be more than seven times higher than folic acid (129 ng/ml vs. 14.1 ng/ml). L-methylfolate is water-soluble and primarily excreted via the kidneys.

A key pharmacological advantage: After consuming folic acid, some unmetabolized folic acid (UMFA) remains in the circulation. 5-MTHF does not cause unmetabolized folic acid (UMFA) to rise in plasma, which has been associated with the risk of cancer and autism spectrum disorder.

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7. Dosage Forms and Doses Reported in Studies

Folate is available in multivitamins and prenatal vitamins. It is also available in B-complex dietary supplements and supplements containing only folate. In dietary supplements, folate is usually in the form of folic acid, but methylfolate (5-methyl-THF) is also used.

The recommended dietary allowances (RDAs) for folate are 400 µg/day for adults and 600 µg/day for women of childbearing age.

Doses reported in clinical studies vary considerably by indication:

• Homocysteine lowering / metabolic support: A 13-week randomized trial used 200 µg [6S]-5-methyltetrahydrofolate (5-MTHF) per day. The SU.FOL.OM3 trial used 560 µg of 5-methyl-THF per day combined with 3 mg of vitamin B6 and 20 µg of vitamin B12.
• Depression augmentation: In the Papakostas et al. trials for SSRI-resistant major depressive disorder, patients received L-methylfolate at 7.5 mg/day for 30 days followed by 15 mg/day for 30 days. Except for one study, the main dose used in meta-analyses of L-methylfolate for depression was 15 mg adjunctive L-methylfolate.
• Hyperhomocysteinemia (high-dose cycling): Two studies administered 5-MTHF at 15 mg per day for up to one consecutive month. One study (Ambrosino et al., 2015) demonstrated no adverse effects related to supplementation with 5-MTHF at 15 mg per day in patients with mild or moderate hyperhomocysteinemia (5-MTHF cycled, one month of dosing followed by two months of withdrawal for a total of two years).
• Periconceptional NTD prevention (general population): The U.S. Preventive Services Task Force (USPSTF) strongly recommends that all women planning or capable of pregnancy take a daily supplement containing 0.4 to 0.8 mg (400 to 800 µg) of folic acid. This recommendation is for folic acid; specific dosing guidance for 5-MTHF formulations in NTD prevention has not been formally established by major bodies.
• Range across clinical trials overall: Seven clinical trials identified tested 5-MTHF, L-5-MTHF, or [6S]-5-MTHF at doses ranging from 0.4–1.13 mg per day. No adverse effects were reported in any study; however, not all studies included monitoring for such in the methodology.

Factors for converting mcg DFE to mcg for supplemental folate in the form of 5-MTHF have not been formally established.

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8. Safety Considerations and Drug Interactions

Tolerable Upper Intake Level (UL)

Based on the metabolic interactions between folate and vitamin B12, the Food and Nutrition Board (FNB) established a UL for the synthetic forms of folate available in dietary supplements and fortified foods. The FNB did not establish a UL for folate from food because high intakes of folate from food sources have not been reported to cause adverse effects.

The EFSA (2023) Panel acknowledged the differences in the pharmacokinetics between reduced folates and folic acid, noting that there is no indication that 5-MTHF salts would impart B12-masking effects. Conversely, the Panel also concluded that the safety of 5-MTHF-glucosamine or l-5-MTHF-Ca above the UL for folic acid (1 mg per day) could not be determined, due to a lack of new data.

Masking of Vitamin B12 Deficiency

Taking large amounts of folate supplements might hide a vitamin B12 deficiency because these supplements can correct the anemia that the vitamin B12 deficiency causes but not the nerve damage that the vitamin B12 deficiency also causes. Additional research is needed to determine whether reduced folates, such as 5-MTHF, impart the same vitamin B12-related effects as supplemental folic acid (leading to increased risk of neurological symptoms) and to what extent.

Adverse Effects at High Doses (Folic Acid Data)

Although folic acid has a water-soluble nature and rarely produces toxicity as excessive amounts tend to be eliminated through urine and it does not accumulate in tissues, adverse reactions might occur at high amounts, such as diarrhoea, nausea, abdominal cramps, bloating and gas, rash, insomnia, zinc deficiency, psychotic behaviour, seizures, bitter taste in the mouth, hyperactivity, irritability, or excitability. Of note, these adverse reactions are reported as rare or at unknown frequency, even for drugs containing 5 mg of folic acid.

Unmetabolized Folic Acid (UMFA) — Advantage of 5-MTHF

After consuming folic acid, some unmetabolized folic acid (UMFA) remains in the circulation. 5-MTHF does not cause unmetabolized folic acid (UMFA) to rise in plasma, which has been associated with the risk of cancer and autism spectrum disorder. This property is considered a potential safety advantage of 5-MTHF, though its clinical significance has not been conclusively established in human trials.

Interaction: Methotrexate

Methotrexate (MTX) is a folic acid antagonist and cell cycle-specific antimetabolite. Co-administration of methotrexate with vitamin preparations containing folic acid, folinic acid (leucovorin), or their derivatives may diminish the therapeutic effects of methotrexate. By extension, 5-MTHF, being the active folate form, may similarly interact and should be used with caution alongside methotrexate without medical oversight.

Interaction: Antiepileptic Drugs

Folate supplements can interact with several medications. Individuals taking these medications on a regular basis should discuss their folate intakes with their health care providers. Several antiepileptic drugs (e.g., phenytoin, carbamazepine, valproic acid, and primidone) are known to lower folate levels through various mechanisms including interference with folate absorption, and conversely, high-dose folate can reduce plasma concentrations of these drugs, potentially affecting seizure control.

Interaction: MTHFR Polymorphisms — A Clinical Consideration

The C677T homozygous (TT) genotype can reduce enzyme activity by about 70%. For individuals carrying reduced-function MTHFR variants, standard folic acid supplementation may be less effective, as it depends on the impaired enzyme for conversion to 5-MTHF. Other studies suggest that folate insufficiency due to MTHFR deficiency is bypassed by 5-MTHF supplementation.

Populations at Risk of Folate Inadequacy

Although most people consume adequate amounts of folate, certain groups, including women of childbearing age and non-Hispanic Black women, are at risk of insufficient folate intakes. Even when intakes of folic acid from dietary supplements are included, 19% of female adolescents age 14 to 18 years and 17% of women age 19 to 30 years do not meet the estimated average requirement (EAR). Similarly, 23% of non-Hispanic Black women have inadequate total intakes, compared with 13% of non-Hispanic White women.

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References

• NIH Office of Dietary Supplements — Folate: Fact Sheet for Health Professionals
• Pizzorno L. Active Folate Versus Folic Acid: The Role of 5-MTHF (Methylfolate) in Human Health. Integrative Medicine 2022; PMC9380836
• Martínez-Morata I et al. Supplementation with Folic Acid or 5-Methyltetrahydrofolate and Prevention of Neural Tube Defects: An Evidence-Based Narrative Review. Nutrients 2024; PMC11435031
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