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Mechanism of ActionKey BenefitsDosage & FormsIn the Singular FormulaSafety & PrecautionsScientific StudiesFrequently Asked Questions
Vitamin B9

Vitamin B9

Acide folique · Folate · 5-MTHF · L-Méthylfolate · Folic acid

VitaminsGenomic stability

Vitamin B9, or folate, is a methyl donor that supplies the building blocks needed to make and repair DNA. Its most robust effect concerns the blood: folate contributes to normal blood formation. The second pillar is homocysteine: folate contributes to its normal metabolism, and a meta-analysis of 25 randomised trials measures a 20% reduction at 400 µg per day. Folate also contributes to normal amino acid synthesis, to normal psychological function, to the normal function of the immune system, to the process of cell division and to the reduction of tiredness and fatigue. Eight physiological functions in all, every one of them resting on the same role as a carrier of methyl groups. With age, DNA methylation patterns drift, and this epigenetic instability blurs gene expression. In the laboratory, the availability of methyl groups is one of the parameters that shape those patterns.

Last updated: August 11, 2026

Mechanism of Action

Active vitamin B9 (5-MTHF) feeds into the folate cycle, a metabolic crossroads where DNA synthesis and remethylation converge. 5-MTHF donates its methyl group to homocysteine, converting it into methionine, an amino acid that subsequently generates S-adenosylmethionine (SAMe). SAMe is the body's primary methyl donor: it participates in the methylation of DNA, proteins and neurotransmitters.

In parallel, folates supply the one-carbon units required for the synthesis of purine and pyrimidine bases, the building blocks of DNA. This dual role explains why rapidly renewing tissues (blood cells, digestive mucosa) are the first affected when folate status declines.

Key Benefits

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    Better-renewed blood: folate contributes to normal blood formation. A 24-week controlled trial in 144 women aged 19 to 33 measures a continuous rise in folate stored inside red blood cells on 416 µg of methylfolate per day.

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    Lower blood homocysteine: folate contributes to normal homocysteine metabolism, an amino acid whose level rises when methylation falters. A meta-analysis of 25 randomised trials in 2,596 adults measures a 20% reduction at 400 µg per day, which is 90% of the maximum attainable effect.

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    Cells that renew correctly: folate plays a role in the process of cell division. It supplies the one-carbon units (small chemical fragments) essential for faithfully copying DNA across all tissues.

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    Amino acids produced normally: folate contributes to normal amino acid synthesis. It is through folate that homocysteine becomes methionine again, and that serine and glycine interconvert.

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    Supported immune defences: folate contributes to the normal function of the immune system. The multiplication of lymphocytes (the white blood cells of defence) depends directly on folate availability to build DNA.

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    Preserved psychological function: folate contributes to normal psychological function. Nervous tissue consumes methyl groups continuously, just as rapidly renewing tissues do.

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    Less fatigue: folate contributes to the reduction of tiredness and fatigue. This role follows on from the one it plays in making red blood cells, which carry oxygen to the tissues.

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    A better-supported pregnancy: folate contributes to maternal tissue growth during pregnancy. Needs rise with the rapid renewal of the tissues involved.

Dosage & Forms

Three forms of vitamin B9 coexist on the market. Folic acid, the most common synthetic form, goes through four enzymatic steps before reaching the active form. Folinic acid (5-formylTHF) partially bypasses this cascade but remains an intermediate form. Calcium L-5-methyltetrahydrofolate is the directly bioactive form, identical to the one circulating in human plasma.

European dietary reference values are set at 200 µg per day, and the maximum allowed in a food supplement in France at 500 µg. On homocysteine, a meta-analysis of 25 randomised trials measures a 13% reduction at 200 µg per day, 20% at 400 µg and 23% at 800 µg.

The two forms have been compared in the same people, at equivalent amounts. In 144 women followed for 24 weeks, methylfolate raises folate stored inside red blood cells more than folic acid does, while the fall in homocysteine does not differ. In 60 pregnant women taking 0.6 mg for 16 weeks, that gap is not found. The constant point is the amount of unmetabolised folic acid left in plasma, roughly half as much with methylfolate.

In the Singular Formula

Inclusion rationale

Folates contribute to normal blood formation, to normal homocysteine metabolism, to normal amino acid synthesis, to normal psychological function, to the normal function of the immune system, to the reduction of tiredness and fatigue, and play a role in the process of cell division. Essential for DNA synthesis and methylation, folates are particularly active in rapidly renewing tissues: bone marrow, intestinal mucosa, and during embryonic development. Synthetic folic acid must be converted to 5-MTHF by the enzyme MTHFR before it can perform its biological functions. The 5-MTHF form selected in the formula is already at the end of that conversion: it is the form in which folate circulates in human plasma, whatever form is ingested. In the methylation cycle, B9 works in synergy with vitamin B12 (methylcobalamin) and vitamin B6 (P-5-P), also present in the formula, to ensure optimal remethylation of homocysteine.

Selected form

Patented active

Vitamin B9 as calcium L-5-methyltetrahydrofolate (5-MTHF), stabilised crystalline form. 5-MTHF is the biologically active form of folate, directly usable by the body. Unlike synthetic folic acid, it requires no enzymatic reduction by MTHFR (methylenetetrahydrofolate reductase), an enzyme whose activity is reduced in a significant portion of the population due to common genetic polymorphisms. Folate contributes to maternal tissue growth during pregnancy and to the normal function of the immune system.

Formula dosage

0 to 500 µg.

Dose expressed as active substance, excluding excipients and carriers of the raw material.

Synergies in the formula

Vitamin B9 participates in a methylation cycle that relies on several cofactors present in the Singular formula. Vitamin B12 (methylcobalamin) catalyses the transfer of the methyl group from 5-MTHF to homocysteine: without B12, folate remains trapped in its methylated form and the cycle stalls. Vitamin B6 (pyridoxal-5'-phosphate) operates in the transsulfuration pathway, the second route for homocysteine degradation, directing this amino acid towards cysteine and glutathione synthesis. Glycine belongs to the same one-carbon metabolism. The interconversion between serine and glycine feeds the folate pool with the one-carbon units that B9 then carries. Iron benefits indirectly from this cooperation: optimal folate status supports normal red blood cell formation, the primary vehicle for iron in the bloodstream.

Linked Biomarkers

Total vitamin B12HomocysteineAlkaline phosphatase (ALP)Mean Corpuscular VolumeVitamin B9Hemoglobin

Safety & Precautions

Vitamin B9 in 5-MTHF form has a favourable tolerability profile at nutritional doses. The European upper safety limit is 1,000 µg per day in adults. Reviewed in 2023, it applies to the combined intake of folic acid and methylfolate salts.

That limit rests on one precise point: a high folate intake can normalise certain blood markers while vitamin B12 status remains insufficient. Maintaining adequate B12 status during folate supplementation is therefore advisable. Calibration draws on the measured vitamin B12 result.

Individuals taking methotrexate or antiepileptic medications consult a healthcare professional before supplementing, as folates may interfere with the mechanism of action of these molecules.

Supplementation is not recommended for children under 3 years without medical advice. No toxicity has been reported at standard nutritional doses.

Scientific Studies

AuthorsYearTypeJournal

Dose-dependent effects of folic acid on blood concentrations of homocysteine: a meta-analysis of the randomized trials

Meta-analysis of 25 randomised trials using individual data from 2,596 subjects. Homocysteine lowering follows the dose: 13% at 200 µg per day, 20% at 400 µg, 23% at 800 µg. The authors conclude that 200 and 400 µg deliver 60% and 90% of the maximal effect respectively.

Red blood cell folate concentrations increase more after supplementation with [6S]-5-methyltetrahydrofolate than with folic acid in women of childbearing age

Double-blind, randomised, placebo-controlled trial in 144 women aged 19 to 33 over 24 weeks. At equivalent amounts, 416 µg of methylfolate raises red blood cell folate more than 400 µg of folic acid. No plateau was reached for that marker within 24 weeks.

Supplementation with [6S]-5-methyltetrahydrofolate or folic acid equally reduces plasma total homocysteine concentrations in healthy women

Same cohort of 144 women, same 24-week duration, different endpoint: homocysteine lowering does not differ between 400 µg of folic acid, 416 µg of methylfolate and 208 µg of methylfolate. On this endpoint, neither form nor dose makes a difference.

Supplementation with (6S)-5-methyltetrahydrofolic acid appears as effective as folic acid in maintaining maternal folate status while reducing unmetabolised folic acid in maternal plasma: a randomised trial of pregnant women in Canada

Randomised trial in 60 pregnant women, 0.6 mg per day for 16 weeks. Red blood cell and serum folate do not differ between the two forms, unlike the 2006 trial. Plasma unmetabolised folic acid is however roughly halved with methylfolate, a difference whose biological relevance the authors describe as unclear.

[6S]-5-methyltetrahydrofolate increases plasma folate more effectively than folic acid in women with the homozygous or wild-type 677C-->T polymorphism of methylenetetrahydrofolate reductase

Single-dose crossover trial in 24 women (16 TT, 8 CC genotypes): methylfolate increases plasma folate more effectively than folic acid, and the gap is the same in both genotypes. Follow-up covers 8 hours and plasma folate only, with no clinical endpoint.

Scientific opinion on the tolerable upper intake level for folate

Revision of the upper safety limit for folate. The value of 1,000 µg per day in adults is retained, and it applies to the combined intake of folic acid and the two authorised methylfolate salts. The point used to set it is the progression of neurological signs in people whose vitamin B12 status is insufficient.

Effects of folic acid supplementation on overall and site-specific cancer incidence during the randomised trials: meta-analyses of data on 50,000 individuals

Meta-analysis of individual data from 13 randomised trials and 49,621 participants followed for 5.2 years on average, at doses of 0.5 to 5 mg per day. No significant effect on overall cancer incidence, nor on any specific site.

Efficacy of Folic Acid Therapy in Primary Prevention of Stroke Among Adults With Hypertension in China: The CSPPT Randomized Clinical Trial

Randomised trial in 20,702 hypertensive adults over 4.5 years: folic acid at 0.8 mg per day, added to enalapril in both arms, cut the risk of a first stroke by 21%. The dose is 800 µg, above the maximum allowed in a food supplement in France, and the population lived in a country without folic acid fortification of flour.

Homocysteine-lowering by B vitamins slows the rate of accelerated brain atrophy in mild cognitive impairment: a randomized controlled trial

Controlled trial (VITACOG) in 271 randomised older adults, with 168 MRI scans analysed over 2 years. A combination of folate 0.8 mg, vitamin B12 0.5 mg and vitamin B6 20 mg slowed the rate of brain atrophy by 30%. The effect cannot be attributed to any one of the three vitamins on its own.

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