Comprehensive Guide to Folate One Carbon Metabolism & Disorders

Folate-dependent one-carbon metabolism represents a central biochemical network facilitating the transfer and utilization of single-carbon groups (e.g., methyl, formyl, methylene). Serving as transporters for these units, folate derivatives are indispensable for nucleotide biosynthesis, amino acid homeostasis, and methylation reactions. This review examines the structural complexity, regulatory mechanisms, and disease relevance of this metabolic pathway.

Folate-mediated one-carbon metabolism (Sobral AF et al., 2024).

Molecular Architecture of Folic Acid

Chemically termed pteroylglutamic acid, folic acid represents a water-soluble B vitamin (B₉) with three functionally integrated domains:

Pteridine Ring System

• Chemical Attributes: Comprising two fused pyrazine rings, this UV-absorbing heterocycle (λmax=280 nm) enables HPLC-based quantification.
• Redox Activity: The N5 and N8 positions undergo NADPH-dependent reduction by dihydrofolate reductase (DHFR), generating bioactive tetrahydrofolate (THF).

Para-Aminobenzoic Acid (PABA)

• Structural Role: Links the pteridine ring to glutamate via an amide bond, stabilizing molecular conformation.
• Antimicrobial Target: Sulfonamide drugs inhibit bacterial folate synthesis by mimicking PABA, disrupting pathogen metabolism.

Polyglutamyl Tail

• Functional Enhancement: Native folate exists as γ-polyglutamates (2–7 residues), augmenting enzyme binding affinity (e.g., thymidylate synthase) through increased negative charge.
• Cellular Retention: Folylpolyglutamate synthase (FPGS) catalyzes polyglutamylation, prolonging intracellular retention (half-life: hours→days) and preventing efflux.

Chemical structures of folic acid, folateand various folate derivatives (Sobral AF et al., 2024).

Core Biochemical Roles

Single-Carbon Transporter

• Folate undergoes a two-step enzymatic reduction via DHFR, forming 5,6,7,8-THF. This active coenzyme binds methyl or formyl groups at its N5/N10 positions, driving nucleotide biosynthesis (e.g., dTMP, purines) and homocysteine remethylation to methionine.

Epigenetic Modulation

• By sustaining SAM pools, folate indirectly regulates DNA methylation (5-methylcytosine) and histone modifications (e.g., H3K4 trimethylation), thereby influencing chromatin architecture and gene silencing.

Therapeutic Implications

• Preventive Use: Periconceptional folate supplementation (400 μg/day) decreases neural tube defect incidence by 70%.
• Oncological Targeting: Antifolates like methotrexate suppress malignant cell proliferation through competitive DHFR inhibition.

Pharmacokinetic Profile

Absorption Mechanism

• Dietary folate is absorbed via proton-coupled folate transporters (PCFT) at the intestinal brush border, entering systemic circulation as monoglutamate derivatives.

Tissue Distribution and Metabolism

• Hepatic Storage: The liver retains 50% of total body folate, predominantly as 5-methyl-THF, which traverses the blood-brain barrier to aid neurotransmitter production.
• Cellular Retention: Polyglutamylation by folylpolyglutamate synthase (FPGS) prolongs intracellular residence, preventing efflux.

Elimination Pathways

• Non-polyglutamylated folate undergoes renal excretion via glomerular filtration, exhibiting a plasma half-life of 3–4 hours.

Pathway

Folate absorption and activation

Intestinal absorption

• Transporter mechanism: dietary folate (monoglutamate form) enters the intestinal epithelium via the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC) in the brush border of the small intestine.
• Rate-limiting factors: intestinal pH (optimal pH 5.5-6.0) and zinc ion-dependent hydrolase activity.

Intracellular activation

Reduction process

• Step 1: Folic acid is catalyzed by dihydrofolate reductase (DHFR) and NADPH is hydrogenated to produce dihydrofolate (DHF).
• Step 2: DHFR continues to reduce DHF to tetrahydrofolate (THF), which is a one-carbon unit active carrier.

Regulatory Target

• Methotrexate (MTX): competitively inhibits DHFR (Ki=0.1 nM) for leukemia and rheumatoid arthritis treatment.
• Polyglutamylation: THF ligates multiple glutamates via folate polyglutamate synthetase (FPGS) to prolong intracellular retention (half-life extended from 3 to 24 hours).

Sources and transfer of one-carbon units

Serine metabolism: major carbon source

• Reaction mechanism: SHMT catalyzes the formation of glycine and 5,10-methylene-THF (CH₂-THF) from serine and THF.
• Subcellular division of labor: cytoplasmic SHMT1 supports nucleotide synthesis, mitochondrial SHMT2 generates NADH in hypoxia.
• Regulation: feedback inhibition of SHMT activity by high concentrations of glycine (Ki = 2 mM).

Glycine Cleavage System (GCS)

• Mitochondrial contribution: glycine is cleaved to CO₂, NH₃, and CH₂-THF catalyzed by the GCS (containing P-protein, T-protein, and L-protein), which also generates NADH.
• Disease association: defects in the GCS lead to nonketotic hyperglycinemia (NKH), which manifests as epilepsy and mental retardation.

Histidine Metabolism

Degradation pathway

• Histidine → uremic acid: Histidase catalyzes the production of uremic acid.
• Urocanic acid → iminomethylglutamate: Urocanic acid hydratase and iminomethyltransferase act sequentially to produce CH₂-THF and glutamate.
• Nutritional significance: dietary histidine contributes about 10% of the one-carbon units.

Methionine cycle: methylation core

Methyl transfer

• Methionine synthase (MS): dependent on vitamin B12 (methylcobalamin), transfers methyl from 5-methyl-THF to homocysteine to produce methionine.
• Methyl Trap: In B12 deficiency, 5-methyl-THF builds up, THF is depleted, and nucleotide synthesis is inhibited.

SAM generation and function

• Methionine adenosyltransferase (MAT): catalyzes the generation of S-adenosylmethionine (SAM) from methionine and ATP, consuming 3 high-energy phosphate bonds.
• Methylation reactions: SAM acts as a universal methyl donor and is involved in DNA (DNMT), histone (EZH2) and small molecule (phospholipid) methylation.
• SAH hydrolysis: S-adenosine homocysteine hydrolase (SAHH) breaks down SAH into homocysteine and adenosine, maintaining a SAM/SAH ratio >5.

Utilization of one-carbon units

dTMP synthesis

• Thymidylate synthase (TS): catalyzes the formation of dTMP and DHF from dUMP and CH₂-THF, a rate-limiting step in DNA replication.
• Anticancer target: metabolism of 5-fluorouracil (5-FU) to FdUMP irreversibly inhibits TS (Ki=0.1 nM).
• DHFR recirculation: reduction of DHF to THF by DHFR maintains one-carbon pool homeostasis.

Purine synthesis

C2 & C8 position modification

• C2 (formyl): 10-formyl-THF is catalyzed by AICAR formyltransferase (ATIC), modifying AICAR to produce FAICAR.
• C8 (methylene): 5,10-methylene-THF is catalyzed by GAR formyltransferase (GART), modifying GAR to generate FGAR.
• Disease association: GART mutations lead to congenital purine synthesis disorders, triggering developmental delays and immunodeficiencies.

Methylation regulatory network

• DNA methylation: DNMT catalyzes CpG island methylation (5mC), silencing transposons and oncogenes (e.g. p16).
• Histone modification: histone methyltransferases (e.g., EZH2) utilize SAM to catalyze H3K27me3 and maintain chromatin repression.
• Clinical significance: decreased SAM/SAH ratio (<2.0) is associated with hepatocellular carcinoma and Alzheimer's disease.

Regulation

Nutritional status

• Folate intake: dietary folate intake directly affects intracellular folate levels.
• Vitamins B12 and B6: Vitamin B12 acts as a coenzyme for methionine synthase and vitamin B6 acts as a coenzyme for serine hydroxymethyltransferase, which together regulate one-carbon metabolism.

Gene expression

• Expression of key enzymes: gene expression levels of key enzymes such as SHMT, TS, and MS influence the flow of one-carbon metabolic pathways.
• Epigenetic regulation: DNA methylation and histone modification affect the expression of genes related to one-carbon metabolism.

Metabolite feedback

• SAM feedback inhibition: the level of SAM, as the main donor of methylation reaction, can feedback inhibit the activity of methionine synthase.
• THF level regulation: THF level affects the supply and utilization of one-carbon units.

Physiological significance

Folate one-carbon metabolism has an important role in physiological processes, mainly involving nucleotide synthesis, amino acid metabolism, methylation reactions, and redox homeostasis. In nucleotide synthesis, the one-carbon unit is a key raw material for purine and thymidylate synthesis, which directly affects DNA and RNA synthesis. It is also closely related to the metabolism of amino acids such as serine, glycine and methionine, maintaining amino acid homeostasis. In gene expression regulation, SAM acts as a methyl donor and participates in the methylation of DNA, RNA and proteins, which in turn affects cellular functions. In addition, one-carbon metabolism is also associated with cellular redox reactions and regulates the redox state of cells.

However, abnormalities in folate one-carbon metabolism are strongly associated with a variety of diseases. In cancer, dysregulated metabolism leads to disturbances in nucleotide synthesis and methylation reactions, promotes tumorigenesis, and affects gene expression through epigenetic alterations. In cardiovascular disease, abnormalities in one-carbon metabolism elevate homocysteine levels, which are a risk factor for cardiovascular disease. In addition, abnormal methylation reactions are closely associated with the development of neurodegenerative diseases. Folate deficiency also leads to abnormalities in one-carbon metabolism, increasing the risk of birth defects such as neural tube defects.

Learn more through the following case studies:

• Although folate is essential for health, its excessive intake may lead to dysregulation of one-carbon metabolism. The ability to metabolize folic acid is limited in the body, leading to the accumulation of unmetabolized folic acid (uFA) in the body. This excessive FA intake has been linked to health problems such as gestational diabetes mellitus (GDM). Gestational diabetes mellitus (GDM) is an abnormal glucose tolerance diagnosed during pregnancy and the accompanying symptoms of hyperglycemia that usually subsides on its own after delivery. Maternal high FA intake may affect metabolic programming in the offspring by altering gene expression, DNA methylation, and downregulation of GLUT4 in adipose and muscle tissue. In addition, the mechanism of action of FA involves FA receptor α (FOLRα) signaling, which affects pancreatic β-cell secretion and proliferation. High-dose FA intake may lead to impaired MTHFR function, resulting in reduced MTHFR activity and possibly pseudo-MTHFR deficiency. In mouse studies, excessive FA intake may lead to reduced plasma betaine levels, suggesting that FA may disrupt the maternal methionine pathway, which in turn affects the ratio of SAM to SAH. There was a significant correlation between low B12 and the diagnosis of GDM, especially in the presence of high FA intake (Williamson JM ET AL., 2022).
• Daily folic acid supplementation before and during pregnancy can reduce the risk of congenital anomalies, especially neural tube defects. Higher doses of folic acid (e.g., 5 mg/day) are recommended for some high-risk groups, such as women treated with antiepileptic drugs. However, there is still no clear consensus on the optimal dose of high-dose folic acid to use and the duration of treatment. The study population was women who were at least 22 weeks pregnant and had not given birth during the study period, and women with early death or cancer diagnoses were excluded. The study covered all relevant data between the start of the pregnancy and December 31, 2017 The study population consisted of 1,465,785 women, of whom 64,485 (4.4%) received a high-dose folic acid prescription during the follow-up period. The study showed that women exposed to high-dose folic acid had a slight increase in cancer incidence during the follow-up period compared to unexposed women (208 vs. 164 cases per 100,000 person-years). Of these, breast cancer, melanoma, and uterine cervical cancer were the most common types of cancer. The study found that high-dose folic acid use was associated with an increased overall cancer risk, with a corrected hazard ratio (HR) of 1.2 (95% CI = 1.1-1.2). In particular, high-dose folic acid use was associated with an increased risk in the cancer types of non-Hodgkin's lymphoma, gastric cancer, appendix cancer, colon cancer, rectal cancer, and leukemia, but this association was attenuated after a lagged analysis of 6 months, with a persistently higher risk only for non-Hodgkin's lymphoma (aHR = 1.9) (Vegrim HM et al., 2025).
• MTR is the only enzyme known to utilize 5-methylTHF and regenerate the THF scaffold via methyl transfer reactions. Under physiological conditions, MTR activity is critical for tumor growth when 5-methyl THF serves as an exogenous source of FA. knockdown of MTR results in impaired purine synthesis, particularly in the 5-methyl THF environment, as evidenced by decreased levels of related purine precursors (e.g., GAR, AICAR, IMP).Pyrimidine synthesis is also affected, particularly with a significant decrease in dTMP levels. dTMP synthesis requires 5,10-methylene THF, but this metabolite is reduced by MTR knockdown, and MTR knockdown results in decreased levels of SAM and SAH. In some experiments, MTR-deficient cells are unable to efficiently use circulating 5-methyl THF and therefore affect tumor growth. It was found that the role of MTR in the conversion of 5-methyl THF to THF could be bypassed by supplementing with excess FA, thereby maintaining tumor growth. Cells exhibited different metabolic profiles in media with different FA sources. For example, in 5-methyl THF medium, MTX treatment resulted in only a slight decrease in intracellular folate levels, whereas DHF levels accumulated as cell proliferation declined. This suggests that the accumulation of DHF may be responsible for MTX toxicity when 5-methyl THF is the main source of folate (Sullivan MR et al., 2021).
• FA status early in life (from conception to 2 years) may have long-term effects on the neurodevelopment of offspring. The study divided rats into three groups, FA-deficient, FA-normal and FA-supplemented. Rats in each group were fed a different diet from 5 weeks of age until the end of lactation, and continued to be fed until 100 days after weaning. At various time points, the researchers measured neurobehavior, FA and Hcy levels, relative telomere length in brain tissue, and uracil adulteration in the telomeres of the offspring. In the experiment, FA-N and FA-S had higher FA levels and lower Hcy concentrations in offspring brain tissue compared to FA-D deficiency.FA-D showed longer sensorimotor reaction times, while FA-N and FA-S performed better, with the FA-S group performing even better than the FA-N group. Early in life the FA-D group resulted in shorter telomere lengths in offspring brains, whereas the FA-N and FA-S groups attenuated telomere wear and tear.FA deficiency increased dUMP levels in brain tissue, leading to uracil misaddressing in the telomeres.FA supplementation lowered the dUMP/dTMP ratio and reduced uracil misaddressing, thus attenuating telomere damage.FA deficiency resulted in lower folate levels FA deficiency resulted in decreased folate levels, increased Hcy levels in brain tissue, misincorporation of uracil into telomeres, and impeded telomere synthesis. In contrast, FA supplementation restored FA levels, lowered Hcy levels, reduced uracil misincorporation into telomeres, and assisted in telomere de novo synthesis.FA supplementation also assisted in the development of sensory-motor function, spatial learning, and memory in offspring during adolescence and early adulthood (Zhou D et al., 2022).

References

1. Williamson JM, Arthurs AL, Smith MD, Roberts CT, Jankovic-Karasoulos T. "High Folate, Perturbed One-Carbon Metabolism and Gestational Diabetes Mellitus." Nutrients. 2022 ;14(19):3930. doi: 10.3390/nu14193930
2. Vegrim HM, Dreier JW, Igland J, Alvestad S, Gilhus NE, Gissler M, Leinonen MK, Tomson T, Zoega H, Christensen J, Bjørk MH. "High-dose folic acid use and cancer risk in women who have given birth: A register-based cohort study." Epilepsia. 2025 ;66(1):75-88. doi: 10.1111/epi.18146
3. Sullivan MR, Darnell AM, Reilly MF, Kunchok T, Joesch-Cohen L, Rosenberg D, Ali A, Rees MG, Roth JA, Lewis CA, Vander Heiden MG. "Methionine synthase is essential for cancer cell proliferation in physiological folate environments." Nat Metab. 2021 ;3(11):1500-1511. doi: 10.1038/s42255-021-00486-5
4. Zhou D, Li Z, Sun Y, Yan J, Huang G, Li W. "Early Life Stage Folic Acid Deficiency Delays the Neurobehavioral Development and Cognitive Function of Rat Offspring by Hindering De Novo Telomere Synthesis." Int J Mol Sci. 2022;23(13):6948. doi: 10.3390/ijms23136948