Biochemistry, Tetrahydrofolate

Article Author:
Elysia Tjong
Article Author:
Manjari Dimri
Article Editor:
Shamim Mohiuddin
7/23/2020 11:53:50 AM
PubMed Link:
Biochemistry, Tetrahydrofolate


Tetrahydrofolate or tetrahydrofolic acid is a folic acid derivative that serves as a coenzyme for metabolic reactions involving amino acids and nucleic acids. The term "folate" denoted a water-soluble B-complex vitamin that functions in the transfer process of single-carbon groups at different stages of oxidation and essential for biosynthesis of certain amino acid, purine bases  and thymine which is a type of pyrimidine base. Chemically folic acid contains three components: PABA(para amino benzoic acid, pteridine ring and glutamic acid. Folic acid usually contains one glutamic residue which is known as PGA( pteroyl-glutamic acid). The active form of folic acid is called as tetrahydrofolic acid or more popularly known as tetrahydrofolate(THF or FH4). In presence of 2 mole of NADPH and with the help of an enzyme known as dihydrofolate reductase folic acid is converted to tetrahydrofolate. Hydrogen atoms are added in positions 5,6,7 and 8 of  folic acid to converted it into tetrehydrofolic acid. This reducing equivalent used to provide by two moles of NADPH.


Tetrahydrofolate is involved in one-carbon metabolism, which includes the synthesis of thymidylate, purines, and pyrimidines for DNA synthesis. Tetrahydrofolate acquired this one carbon units mainly from amino acids serine, histidine, and glycine as well as they also used to obtain it from formic acid and formaldehyde. These one carbon groups those attached to their carrier tetrahydrofolate is collectively nomenclate as "one-carbon pool." Due to the attachment of these carbon units with tetrahydrofolate, they can be either oxidized or reduced. For these attachments, folate can be present in various chemical forms.  It is also used to remethylate homocysteine to form methionine and S-adenosylmethionine (SAM).


Folate is a B vitamin present in plants and obtained in our diets as polyglutamates. [1] The synthetic analog of folate is folic acid. It is also a synthetic oxidized dietary supplement that plays no direct biological role nor is it considered biologically active [2]. It is absorbed in the jejunum and ileum after being reduced by folate reductase.[2] Folate/folic acid can also undergo reduction to dihydrofolate, which can undergo another reduction reaction to produce the biologically active coenzyme, tetrahydrofolate. The enzyme dihydrofolate reductase is responsible for both reduction reactions.[2]

A single carbon group from serine is added to tetrahydrofolate to be reduced to 5,10-methylene tetrahydrofolate and glycine by methylenetetrahydrofolate reductase (MTHFR), which is the first step in the cycle.[1] 5,10-methylene tetrahydrofolate can then donate that carbon atom to form thymidine or can be modified into 5-methyl-tetrahydrofolate or 10-formyl-tetrahydrofolate, both of which are taken up by the liver.[1]

The one-carbon transfer is essential in the production of dTMP. 5,10-methylene tetrahydrofolate is involved in the reductive methylation of dUMP to dTMP with the enzyme thymidylate synthase[3] Also, tetrahydrofolate acts as a coenzyme for this reaction by donating a methyl group to the alpha carbon.[4] The resulting products are dTMP, which participates in DNA synthesis and dihydrofolate, a one-carbon carrier. Dihydrofolate can be reduced to tetrahydrofolate and 5,10-methylene tetrahydrofolate again to continue this cycle in synthesizing more dTMP.[1]

When the formaldehyde group of 5-methyl-tetrahydrofolate is oxidized, it forms 10-formyl-tetrahydrofolate - 10-formyl-tetrahydrofolate can hydrolyze back to tetrahydrofolate and formate.[3] It can also participate in the synthesis of purine bases in the cytosol and the formylation of mitochondrial initiator methionyl-tRNA (MET-rRNA).[5]


Tetrahydrofolate serves as a single-carbon donor use in enzymatic reactions, such as the synthesis of amino acids and nucleic acids. It also participates in homocysteine metabolism.


Tetrahydrofolate can convert to carbon-donating forms of folate, 5,10-methylene tetrahydrofolate, and 10-formyl-tetrahydrofolate. Serine and glycine can transfer 1-carbon groups to tetrahydrofolate, producing 5,10-methylenetetrahydrofolate.[1] 5,10-Methylene tetrahydrofolate can convert to 5-methyltetrahydrofolate which can partake in the synthesis of S-adenosylmethionine (SAM).[1]

Serine and glycine cycle: Serine is a non-essential amino acid that can be obtained from supplements and in our diet. Glycine can form in our tissues from serine.[6] Serine is the primary source of carbon in the conversion of tetrahydrofolate to 5,10-methylenetetrahydrofolate.[7] Glycine production also occurs in this reaction. The formation of serine requires a hydroxymethyl group from 5,10-methylene tetrahydrofolate and a glycine residue in the reverse reaction.[8]

Methionine cycle: 5,10-methylene tetrahydrofolate can be converted to 5-methyl-tetrahydrofolate by methylene-tetrahydrofolate reductase through the donation of an oxidized carbon group. 5-methyl tetrahydrofolate donates a methyl group to homocysteine to regenerate methionine. The enzyme methionine synthase catalyzes this step and utilizes vitamin B12 (cobalamin) as a cofactor. The enzyme methionine adenyltransferase (MAT) assists with the conversion of methionine, along with ATP, to S-adenosyl methionine (SAM).[1] SAM acts as a methyl donor, where it can donate single carbons to assist with the production of nucleic acids, proteins and neurotransmitters, and other methyltransferase reactions. More specifically, SAM supports the creation of the lipid head group of phosphatidylcholine which is a major constituent of cell membranes.[9]

Clinical Significance

Methotrexate, a folic acid antagonist, is commonly used for initial and ongoing use in cancer chemotherapy, rheumatoid arthritis and as a non-surgical treatment for ectopic pregnancies.[10] The mechanism of action of methotrexate involves the irreversible competitive inhibition of dihydrofolate reductase, which decreases the formation of intracellular tetrahydrofolate. Methotrexate also undergoes polyglutamine which inhibits methotrexate from leading the cell so that it can build up intracellularly for future use.[11] Polyglutamation also affects folate and recycled DHF, therefore, causing increased intracellular levels. Folate cofactors are important to purine synthesis, where the administration of methotrexate can ultimately inhibit DNA and RNA synthesis.[11] Therefore, all patients receiving methotrexate should be treated with folic acid or folinic acid daily to prevent hematologic, gastrointestinal and hepatic side effects.[12]

A deficiency in either folic acid or cobalamin (vitamin B12) can cause megaloblastic anemia, where megaloblasts are present due to inhibition of DNA synthesis.[7] Megaloblastic anemia leads to impairment of red blood cells, which can result in different manifestations such as fatigue, tingling of extremities and loss of joint position/coordination.[7] A deficiency in folic acid or cobalamin can lead to a buildup of folates in the form of 5-methyltetrahydrofolate, which cannot be demethylated by methionine synthesis; this leads to a depletion of other folates and S-adenosyl-methionine.[13][14]

Another consequence of folic acid and vitamin B12 is the increased risk of neural tube defects, such as spina bifida, in newborns. The demand for folate increases during pregnancy due to the need for increased cell division, and also, some mothers may not have adequate folate intake.[2] Research ash identified another cause related to genetic mutation. The mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene results in elevated plasma homocysteine levels that can be detectable.[15] This mutation has also been found to cause decreased plasma levels of folate and decreased vitamin B12 levels.[15] As a result, folic acid supplementation (400 mcg/day) has been the recommendation for pregnant mothers. A recent study also suggested that this supplementation may help prevent memory impairment and brain-derived neurotrophic factor (BDNF) imbalance.[16]

Patients can go a long time with masked cobalamin deficiency. Excess dietary folates can replenish levels of 5-methyltetrahydrofolate, 5,10-methylene-tetrahydrofolate, and tetrahydrofolate, which then allows for normal DNA production despite low cobalamin concentration. Because of this, patients may not have symptoms associated with megaloblastic anemia.[14][17] However, there will still be inadequate regeneration of S-adenosyl methionine.

Methylenetetrahydrofolate reductase (MTHFR) deficiency is a genetic condition that results in elevated plasma levels of homocysteine (hyperhomocysteinemia). This enzyme is involved in the conversion of homocysteine to methionine.[18] Genetic testing of the MTHFR gene may be done to confirm the diagnosis of this condition. However, blood tests may be an alternative test to measure total homocysteine levels in aiding diagnosis. It is important to diagnose hyperhomocysteinemia early as it is a risk factor for atherosclerosis, venous thrombosis, myocardial infarction, and other cardiovascular conditions.[19] Patients with this deficiency may take a low dose of folic acid to help reduce and normalize their homocysteine levels.[20]


[1] Locasale JW, Serine, glycine and one-carbon units: cancer metabolism in full circle. Nature reviews. Cancer. 2013 Aug;     [PubMed PMID: 23822983]
[2] Milman N, Intestinal absorption of folic acid - new physiologic & molecular aspects. The Indian journal of medical research. 2012 Nov     [PubMed PMID: 23287118]
[3] Stover PJ, One-carbon metabolism-genome interactions in folate-associated pathologies. The Journal of nutrition. 2009 Dec;     [PubMed PMID: 19812215]
[4] Carreras CW,Santi DV, The catalytic mechanism and structure of thymidylate synthase. Annual review of biochemistry. 1995;     [PubMed PMID: 7574499]
[5] Vickers TJ,Murta SM,Mandell MA,Beverley SM, The enzymes of the 10-formyl-tetrahydrofolate synthetic pathway are found exclusively in the cytosol of the trypanosomatid parasite Leishmania major. Molecular and biochemical parasitology. 2009 Aug;     [PubMed PMID: 19450731]
[6] Reynolds E, Vitamin B12, folic acid, and the nervous system. The Lancet. Neurology. 2006 Nov;     [PubMed PMID: 17052662]
[7] Ebara S, Nutritional role of folate. Congenital anomalies. 2017 Sep;     [PubMed PMID: 28603928]
[8] Miyo M,Konno M,Colvin H,Nishida N,Koseki J,Kawamoto K,Tsunekuni K,Nishimura J,Hata T,Takemasa I,Mizushima T,Doki Y,Mori M,Ishii H, The importance of mitochondrial folate enzymes in human colorectal cancer. Oncology reports. 2017 Jan;     [PubMed PMID: 27878282]
[9] Kinney AJ,Moore TS, Phosphatidylcholine Synthesis in Castor Bean Endosperm : I. Metabolism of l-Serine. Plant physiology. 1987 May     [PubMed PMID: 16665410]
[10] Funk RS,van Haandel L,Becker ML,Leeder JS, Low-dose methotrexate results in the selective accumulation of aminoimidazole carboxamide ribotide in an erythroblastoid cell line. The Journal of pharmacology and experimental therapeutics. 2013 Oct     [PubMed PMID: 23887097]
[11] Hagner N,Joerger M, Cancer chemotherapy: targeting folic acid synthesis. Cancer management and research. 2010 Nov 19;     [PubMed PMID: 21301589]
[12] Shea B,Swinden MV,Ghogomu ET,Ortiz Z,Katchamart W,Rader T,Bombardier C,Wells GA,Tugwell P, Folic acid and folinic acid for reducing side effects in patients receiving methotrexate for rheumatoid arthritis. The Journal of rheumatology. 2014 Jun     [PubMed PMID: 24737913]
[13] Aslinia F,Mazza JJ,Yale SH, Megaloblastic anemia and other causes of macrocytosis. Clinical medicine     [PubMed PMID: 16988104]
[14] Yadav MK,Manoli NM,Madhunapantula SV, Comparative Assessment of Vitamin-B12, Folic Acid and Homocysteine Levels in Relation to p53 Expression in Megaloblastic Anemia. PloS one. 2016;     [PubMed PMID: 27780269]
[15] van der Put NM,Thomas CM,Eskes TK,Trijbels FJ,Steegers-Theunissen RP,Mariman EC,De Graaf-Hess A,Smeitink JA,Blom HJ, Altered folate and vitamin B12 metabolism in families with spina bifida offspring. QJM : monthly journal of the Association of Physicians. 1997 Aug     [PubMed PMID: 9327028]
[16] Deniz BF,Confortim HD,Deckmann I,Miguel PM,Bronauth L,de Oliveira BC,Barbosa S,Cechinel LR,Siqueira IR,Pereira LO, Folic acid supplementation during pregnancy prevents cognitive impairments and BDNF imbalance in the hippocampus of the offspring after neonatal hypoxia-ischemia. The Journal of nutritional biochemistry. 2018 Oct     [PubMed PMID: 30064014]
[17] Mills JL,Molloy AM,Reynolds EH, Do the benefits of folic acid fortification outweigh the risk of masking vitamin B{sub}12{/sub} deficiency? BMJ (Clinical research ed.). 2018 Mar 1;     [PubMed PMID: 29496696]
[18] Holmes MV,Newcombe P,Hubacek JA,Sofat R,Ricketts SL,Cooper J,Breteler MM,Bautista LE,Sharma P,Whittaker JC,Smeeth L,Fowkes FG,Algra A,Shmeleva V,Szolnoki Z,Roest M,Linnebank M,Zacho J,Nalls MA,Singleton AB,Ferrucci L,Hardy J,Worrall BB,Rich SS,Matarin M,Norman PE,Flicker L,Almeida OP,van Bockxmeer FM,Shimokata H,Khaw KT,Wareham NJ,Bobak M,Sterne JA,Smith GD,Talmud PJ,van Duijn C,Humphries SE,Price JF,Ebrahim S,Lawlor DA,Hankey GJ,Meschia JF,Sandhu MS,Hingorani AD,Casas JP, Effect modification by population dietary folate on the association between MTHFR genotype, homocysteine, and stroke risk: a meta-analysis of genetic studies and randomised trials. Lancet (London, England). 2011 Aug 13;     [PubMed PMID: 21803414]
[19] Wald DS,Law M,Morris JK, Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ (Clinical research ed.). 2002 Nov 23     [PubMed PMID: 12446535]
[20] Guttormsen AB,Ueland PM,Nesthus I,Nygård O,Schneede J,Vollset SE,Refsum H, Determinants and vitamin responsiveness of intermediate hyperhomocysteinemia (> or = 40 micromol/liter). The Hordaland Homocysteine Study. The Journal of clinical investigation. 1996 Nov 1     [PubMed PMID: 8903338]