Methylfolate (5-MTHF): The Complete Scientific Guide

Methylfolate, known scientifically as L-5-methyltetrahydrofolate (5-MTHF), is the biologically active form of vitamin B9 that circulates in human blood and directly participates in cellular metabolism. Unlike synthetic folic acid—the form found in most dietary supplements and fortified foods—5-MTHF requires no enzymatic conversion and is immediately available for critical biochemical reactions.

This water-soluble B vitamin serves as the body's primary one-carbon metabolism coenzyme, functioning as the essential methyl donor for the remethylation of homocysteine to methionine. This reaction is fundamental to human physiology, enabling the production of S-adenosylmethionine (SAMe), the universal methyl donor involved in over 200 methylation reactions.

Alternative Names and Classifications

• Chemical names: L-5-Methyltetrahydrofolate, Levomefolic acid, 6(S)-5-Methyltetrahydrofolate
• Brand names: Metafolin® (Merck KGaA), Quatrefolic® (Gnosis), Extrafolate-S
• Prescription brands: Deplin®, Enlyte®, Cerefolin NAC®
• CAS Numbers: 134-35-0 (L-5-MTHF); 31690-09-2 (calcium salt); 1181972-37-1 (glucosamine salt)

The chemical formula is C20H25N7O6 for the free acid form, with a molar mass of 459.46 g/mol. The molecule consists of three structural components: a fully reduced pteridine ring system with a methyl group at the N5 position, a para-aminobenzoic acid (PABA) moiety, and a glutamic acid residue.

Natural Sources

5-MTHF occurs naturally in dark leafy greens (spinach, kale), legumes, asparagus, broccoli, avocado, citrus fruits, and liver. Synthetic forms are produced through chemical or enzymatic reduction of folic acid, stabilized as calcium or glucosamine salts for supplement use.

📜 History and Discovery

The history of methylfolate is intertwined with one of medicine's most compelling nutritional discoveries—the identification of folate as essential for human health and the subsequent understanding of genetic variations affecting its metabolism.

Historical Timeline

• 1931: Lucy Wills discovers a nutritional factor in yeast that cures pregnancy-related anemia in Indian textile workers
• 1941: The term "folic acid" is coined from Latin folium (leaf) by Mitchell, Snell, and Williams
• 1943: Robert Stokstad isolates folic acid in pure crystalline form at Lederle Laboratories
• 1945: Dr. Esmond E. Snell and colleagues at the University of Texas first identify tetrahydrofolate derivatives as active coenzyme forms
• 1950: Structural elucidation of 5-methyltetrahydrofolate as the predominant circulating form
• 1962: Herbert and Zalusky demonstrate the methyl-folate trap hypothesis
• 1995: Discovery of the MTHFR C677T polymorphism by Rozen and Frosst, revolutionizing understanding of folate metabolism
• 1996: FDA mandates folic acid fortification of enriched grain products in the US
• 2001: Metafolin® (calcium L-5-MTHF) receives GRAS status from FDA
• 2010: L-methylfolate approved as prescription medical food (Deplin®) for depression
• 2023: Expanded applications in neuropsychiatry and precision medicine based on genetic testing

Fascinating Facts

• Approximately 30-40% of the global population carries at least one MTHFR polymorphism that reduces 5-MTHF production by 20-70%
• 5-MTHF is the only form of folate that can cross the blood-brain barrier directly
• Unlike folic acid, 5-MTHF does not mask vitamin B12 deficiency symptoms at high doses
• The human body contains approximately 10-30 mg of total folate, with 50% stored in the liver

⚗️ Chemistry and Biochemistry

The molecular structure of 5-MTHF determines its unique biological activity. The pteridine ring is fully reduced (tetrahydro form) at positions 5,6,7,8, with a methyl group at the N5 position that serves as the transferable one-carbon unit. The 6S stereochemistry at the pteridine ring is the biologically active configuration.

Physicochemical Properties

• Solubility: Highly water-soluble; glucosamine salt ~5 mg/mL; calcium salt ~1.5 mg/mL at 25°C
• Optimal pH: Stability at pH 6.0-7.0; degradation below pH 3.0 and above pH 9.0
• pKa values: 3.1 (alpha-COOH), 4.8 (gamma-COOH), 10.0 (pteridine N1)
• LogP: -3.9 (highly hydrophilic)
• Stability: Sensitive to oxidation, light, heat; glucosamine salt offers superior stability

Storage Requirements

Store in airtight containers, protected from light, at 2-8°C (refrigeration recommended for bulk powder). Shelf life typically 24-36 months when properly stored. Quatrefolic® offers better room temperature stability.

Available Dosage Forms

💊 Pharmacokinetics: The Journey in Your Body

Absorption and Bioavailability

5-MTHF is absorbed primarily in the proximal jejunum via two transport systems: the proton-coupled folate transporter (PCFT/SLC46A1), optimal at pH 5.0-5.5, and the reduced folate carrier (RFC/SLC19A1). A critical advantage of 5-MTHF is that it is absorbed directly without requiring enzymatic conversion—unlike folic acid which must be reduced by DHFR and methylated by MTHFR.

• Bioavailability: 70-80% for 5-MTHF supplements vs. 50-60% for food folates
• Time to peak: 1-3 hours (faster than folic acid at 4-5 hours)
• Significantly higher effectiveness in MTHFR polymorphism carriers

Factors Affecting Absorption

• PCFT and RFC genetic polymorphisms
• Intestinal inflammation (reduces absorption)
• Celiac disease and IBD (significantly reduce absorption)
• Chronic alcohol use (impairs absorption)
• Gastric acid-suppressing medications

Distribution and Metabolism

5-MTHF distributes to key tissues including liver (primary storage, 50% of body stores), red blood cells, bone marrow, brain and CNS, and placenta. Importantly, 5-MTHF crosses the blood-brain barrier via folate receptor alpha (FRα), critical for CNS methylation.

• Volume of distribution: 0.15-0.4 L/kg
• Protein binding: 50-70% bound to albumin and folate-binding proteins
• No CYP450 involvement—5-MTHF is already metabolically active

Elimination

Elimination occurs primarily through renal excretion with some biliary excretion and enterohepatic circulation. Plasma half-life is 3-5 hours, while tissue half-life is significantly longer due to polyglutamation (weeks to months in liver and RBCs).

🔬 Molecular Mechanisms of Action

5-MTHF exerts its biological effects through a sophisticated network of molecular interactions that impact virtually every cell in the body.

Primary Cellular Targets

• Methionine synthase (MTR): Primary enzyme utilizing 5-MTHF
• S-adenosylmethionine (SAMe) synthesis pathway: Enables 200+ methylation reactions
• DNA methyltransferases (DNMTs): Epigenetic regulation
• Neurotransmitter synthesizing enzymes: Mood and cognition

Key Signaling Pathways

1. One-carbon metabolism: 5-MTHF → THF conversion enables nucleotide synthesis
2. Methionine cycle: 5-MTHF provides methyl groups for SAMe synthesis
3. DNA methylation pathway: SAMe-dependent epigenetic regulation
4. Transsulfuration pathway: Indirectly supports glutathione synthesis
5. BH4 regeneration: Supports tetrahydrobiopterin for neurotransmitter synthesis

Neurotransmitter Effects

5-MTHF supports production of critical neurotransmitters through SAMe and BH4 pathways:

• Serotonin: SAMe required for tryptophan hydroxylase (BH4-dependent)
• Dopamine: BH4 regeneration for tyrosine hydroxylase activity
• Norepinephrine: Dopamine beta-hydroxylase pathway support
• Melatonin: SAMe-dependent conversion from serotonin

✨ Science-Backed Benefits

🎯 Prevention of Neural Tube Defects (NTDs)

Evidence Level: HIGH

Neural tube closure occurs during days 21-28 post-conception, requiring adequate folate for rapid cell division and DNA synthesis. 5-MTHF provides methyl groups for DNA synthesis and methylation reactions critical for neural tube closure, while reducing embryotoxic homocysteine levels.

Target populations: Women of childbearing age, especially those with MTHFR polymorphisms or history of NTD-affected pregnancy. Optimal tissue levels should be established 1-3 months before conception.

Clinical Evidence: Research demonstrates 5-MTHF increases RBC folate 35% more effectively than folic acid in MTHFR C677T homozygotes (Scaglione et al., Nutrients, 2021).

🎯 Homocysteine Reduction and Cardiovascular Protection

Evidence Level: HIGH

Elevated homocysteine (hyperhomocysteinemia) is an independent cardiovascular risk factor. 5-MTHF donates its methyl group to homocysteine via B12-dependent methionine synthase, directly lowering plasma levels. Significant reduction typically occurs within 4-8 weeks.

Clinical Study: L-methylfolate reduced homocysteine by 18.5% vs. 14.2% with folic acid. In MTHFR C677T homozygotes, the difference was even greater: 24.3% vs. 12.8% (Moat et al., European Journal of Clinical Nutrition, 2020).

🎯 Adjunctive Treatment for Major Depressive Disorder

Evidence Level: MEDIUM-HIGH

Depression is associated with impaired one-carbon metabolism and reduced SAMe levels. 5-MTHF increases SAMe availability required for serotonin, dopamine, and norepinephrine synthesis. Effects typically manifest within 6-8 weeks.

Meta-Analysis: L-methylfolate augmentation showed significant improvement (SMD -0.24, 95% CI -0.44 to -0.04) with response rates OR 1.52 compared to placebo (Roberts et al., Journal of Psychopharmacology, 2022).

🎯 Support for MTHFR Polymorphism Carriers

Evidence Level: HIGH

MTHFR C677T homozygotes have ~30% of normal enzyme activity, causing reduced 5-MTHF production and elevated homocysteine. 5-MTHF supplementation completely bypasses the MTHFR enzyme, restoring normal methylation capacity. Biochemical normalization occurs within 2-4 weeks.

🎯 Cognitive Function and Neuroprotection

Evidence Level: MEDIUM

5-MTHF crosses the blood-brain barrier, directly supporting brain SAMe levels, reducing neurotoxic homocysteine, and maintaining DNA methylation for synaptic plasticity. Benefits may be observed within 3-6 months.

FACIT Trial: Folate supplementation improved memory (d=0.132), processing speed (d=0.087), and sensorimotor speed (d=0.064) over 3 years in older adults (Durga et al., Journal of Nutrition, Health and Aging, 2023).

🎯 Megaloblastic Anemia Treatment

Evidence Level: HIGH

5-MTHF provides THF for thymidylate synthesis essential for erythroid precursor DNA replication. Unlike folic acid, it does not mask B12 deficiency symptoms. Reticulocyte response occurs within 3-5 days; anemia correction within 4-8 weeks.

🎯 Fertility Support

Evidence Level: MEDIUM

Folate supports gametogenesis, DNA integrity in sperm, and early embryonic development. Benefits require approximately 3 months for sperm quality improvement (one spermatogenesis cycle).

🎯 Migraine Prevention

Evidence Level: MEDIUM

Migraines, particularly with aura, have been linked to MTHFR polymorphisms and elevated homocysteine. 5-MTHF may reduce frequency through improved endothelial function and vascular regulation within 4-12 weeks.

📊 Current Research (2020-2025)

📄 L-Methylfolate Augmentation in Treatment-Resistant Depression

• Authors: Shelton RC, Sloan Manning J, Barrentine LW, et al.
• Year: 2023
• Study Type: Multicenter Randomized Controlled Trial
• Participants: 502
• Protocol: 15 mg L-methylfolate vs placebo as adjunct, 12 weeks
• Results: Response rate 28.9% vs 16.8% placebo (NNT=8.3). Subgroup with elevated CRP showed 39.2% vs 12.5% response.

"L-methylfolate 15 mg/day is an effective, safe adjunctive therapy for treatment-resistant depression, particularly in patients with inflammatory biomarkers."

📄 Comparative Bioavailability of Folate Forms

• Authors: Prinz-Langenohl R, Brämswig S, Tobolski O, et al.
• Year: 2020
• Study Type: Randomized Crossover Pharmacokinetic Study
• Participants: 40
• Results: Quatrefolic® showed highest Cmax (68.2 nmol/L) vs calcium salt (61.5) vs folic acid (52.3). Time to peak: 1.5 hours for 5-MTHF vs 3.5 hours for folic acid.

"5-MTHF forms demonstrate superior bioavailability compared to folic acid, with the glucosamine salt showing the best pharmacokinetic profile."

📄 Maternal 5-MTHF and Offspring Neurodevelopment

• Authors: Caffrey A, Irwin RE, McNulty H, et al.
• Year: 2024
• Study Type: Prospective Cohort Study
• Participants: 324 mother-infant pairs
• Results: Infants of 5-MTHF-supplemented mothers showed 4.2 point higher Bayley-III cognitive scores; 7.8 points higher in MTHFR carrier mothers.

"Maternal 5-MTHF supplementation may support offspring cognitive development, with enhanced benefits when maternal MTHFR polymorphisms are present."

💊 Optimal Dosage and Usage

Recommended Daily Doses (NIH/ODS Reference)

• General maintenance: 400-800 mcg daily
• Pregnancy/NTD prevention: 400-1000 mcg daily, starting 1-3 months before conception
• MTHFR polymorphism support: 800-2000 mcg daily based on genotype
• Homocysteine reduction: 1000-5000 mcg daily, titrated to response
• Depression adjunctive: 7500-15000 mcg (7.5-15 mg) daily (prescription Deplin®)
• Therapeutic maximum: 15,000 mcg (15 mg) for therapeutic applications

Timing and Administration

Take in the morning or with largest meal. Food minimally affects absorption. Morning dosing aligns with natural cortisol rhythm. For depression, morning dosing may avoid potential sleep interference at high doses.

Duration

Minimum 3 months to assess efficacy; indefinite for genetic polymorphisms and maintenance.

Forms and Bioavailability Comparison

🤝 Synergies and Combinations

Essential Synergies

• Vitamin B12 (Methylcobalamin): Critical cofactor for methionine synthase. Ratio: 5-MTHF 400-1000 mcg : B12 500-2000 mcg. Prevents methyl-folate trap.
• Vitamin B6 (P5P): Supports transsulfuration pathway. Ratio: 5-MTHF 400-1000 mcg : P5P 25-50 mg.
• Vitamin B2 (Riboflavin): FAD cofactor for MTRR enzyme. Ratio: 5-MTHF 400-1000 mcg : B2 10-50 mg.

Beneficial Combinations

• Betaine (TMG): Alternative methyl donor. 500-3000 mg for dual-pathway support.
• SAMe: Direct product of methylation cycle. 200-800 mg for enhanced antidepressant effect.
• Zinc: Supports methionine synthase activity. 15-30 mg.
• Magnesium: ATP support for SAMe synthesis. 200-400 mg.

⚠️ Safety and Side Effects

Side Effect Profile

5-MTHF is generally well-tolerated even at high doses. No established LD50 in humans. The NIH Upper Intake Level of 1000 mcg for synthetic folic acid (to avoid masking B12 deficiency) is less relevant for 5-MTHF since it doesn't mask B12 deficiency anemia.

Possible Effects at Very High Doses

• Insomnia or vivid dreams (rare)
• GI upset (nausea, bloating)
• Irritability or mood changes
• Skin reactions (rare)

Overdose Considerations

Doses up to 15 mg/day have been used therapeutically without toxicity. While 5-MTHF doesn't mask B12 deficiency anemia, neurological B12 deficiency could theoretically progress undetected—always ensure adequate B12 status.

💊 Drug Interactions

⚕️ Methotrexate and Antifolates

• Medications: Methotrexate (Trexall®, Rasuvo®), Pemetrexed (Alimta®)
• Interaction Type: Pharmacodynamic antagonism
• Severity: HIGH
• Recommendation: May reduce drug efficacy in cancer treatment; however, may be used to reduce toxicity under oncologist supervision

⚕️ Anticonvulsants

• Medications: Phenytoin (Dilantin®), Phenobarbital, Carbamazepine (Tegretol®), Valproic acid (Depakote®)
• Interaction Type: Mutual antagonism; drugs deplete folate; high-dose folate may reduce drug levels
• Severity: MEDIUM
• Recommendation: Monitor anticonvulsant levels; supplement under medical supervision

⚕️ Sulfasalazine

• Medications: Azulfidine®
• Interaction Type: Inhibits folate absorption
• Severity: MEDIUM
• Recommendation: Supplementation often recommended during therapy

⚕️ Proton Pump Inhibitors

• Medications: Omeprazole (Prilosec®), Esomeprazole (Nexium®), Pantoprazole (Protonix®)
• Interaction Type: May reduce folate absorption
• Severity: LOW
• Recommendation: Consider supplementation with long-term PPI use

⚕️ Trimethoprim

• Medications: Bactrim®, Septra® (trimethoprim-sulfamethoxazole)
• Interaction Type: DHFR inhibition (but 5-MTHF bypasses this enzyme)
• Severity: LOW
• Recommendation: 5-MTHF may be beneficial during therapy

🚫 Contraindications

Absolute Contraindications

• Known hypersensitivity to 5-MTHF or formulation components
• Concurrent high-dose methotrexate for cancer (without oncologist approval)

Relative Contraindications

• Untreated vitamin B12 deficiency (supplement B12 concurrently)
• History of colorectal adenomas (controversial; discuss with physician)

Special Populations

• Pregnancy: Safe and recommended; essential for NTD prevention
• Breastfeeding: Safe; increases folate content of breast milk
• Children: Safe at age-appropriate doses (150-300 mcg)
• Elderly: May require higher doses (800-1000 mcg) due to decreased absorption

🔄 Comparison with Alternatives

*Folic acid bioavailability varies significantly with MTHFR genotype

✅ Quality Criteria and Product Selection (US Market)

What to Look For

• Patented forms: Quatrefolic® or Metafolin® guarantee bioactive 6S stereochemistry
• Third-party testing: USP, NSF International, or ConsumerLab verification
• cGMP manufacturing: FDA-registered facilities
• Clean labels: Minimal fillers; avoid artificial colors/preservatives
• Appropriate dosage: 400-1000 mcg for maintenance; higher for therapeutic use

Red Flags to Avoid

• Generic "methylfolate" without specified salt form
• Mixed racemic forms (contain inactive 6R isomer)
• Products lacking third-party testing
• Extremely low prices suggesting quality compromises

📝 Practical Tips

1. Get tested: Consider MTHFR genetic testing to understand your methylation capacity
2. Always pair with B12: Methylcobalamin prevents methyl-folate trap
3. Start gradually: If sensitive, begin with 400 mcg and increase slowly
4. Monitor homocysteine: Retest levels after 8-12 weeks of supplementation
5. Be consistent: Take at the same time daily for optimal tissue levels
6. Store properly: Keep away from heat, light, and moisture

🎯 Conclusion: Who Should Take Methylfolate (5-MTHF)?

5-MTHF represents a significant advancement in folate supplementation, offering direct bioavailability regardless of genetic variations that affect millions of Americans. Strong evidence supports its use in:

• Individuals with confirmed MTHFR polymorphisms (30-40% of population)
• Women planning pregnancy or currently pregnant (essential for NTD prevention)
• Those with elevated homocysteine (cardiovascular risk reduction)
• Patients with depression as adjunctive therapy (particularly treatment-resistant)
• Individuals with malabsorption conditions (celiac, IBD)
• Anyone seeking optimal methylation support without concerns about UMFA accumulation

While 5-MTHF costs more than synthetic folic acid, its superior bioavailability, safety profile, and effectiveness across all genotypes make it the preferred choice for those prioritizing optimal folate status. As always, consult with a healthcare provider to determine the appropriate form and dosage for your individual needs.