Introduction

Tetrahydrofolate (THF) or tetrahydrofolic acid is a derivative of vitamin B9 (folic acid or pteroyl-L-glutamic acid). This water-soluble vitamin is a coenzyme for metabolic reactions involving amino acid and nucleic acid formations. It participates in important single-carbon transfer reactions often referred to as one-carbon metabolism and in synthesizing several amino acids such as serine and methionine, purines and thymine, a pyrimidine. The single carbon transfer reactions are essential in the metabolism and regulation of gene expression.[1] 

Chemically folic acid consists of three structural components- para-aminobenzoic acid (PABA), a bicyclic pteridine ring, and glutamic acid. The active form of folic acid is known as tetrahydrofolic acid or tetrahydrofolate (THF or FH4), which is a result of the addition of four hydrogen atoms to 5,6,7 and 8 positions in the pteridine ring.[2]

Fundamentals

Tetrahydrofolate is involved in single carbon transfer bound to positions N5, N10, or both. The carbon sources for the one-carbon pool are serine (the major source), glycine, histidine, and tryptophan. They donate the single carbon to THF in various oxidation states and can be oxidized or reduced. Hence, folate can exist in multiple forms,, namely N10-formyl THF (most oxidized form),  N5, N10-methenyl THF,  N5, N10-methylene THF, and N5-methyl THF (most reduced form). All these forms carrying these single carbon groups are collectively known as the "one-carbon pool.” [3]

Histidine and Tryptophan are other less common one-carbon sources that result in the formation of N5, N10-methenyl THF, and N10-formyl THF, respectively. These forms are interconvertible; however, once a methyl group is formed, it is not readily oxidized back to N5, N10-methylene THF and hence N5-methyl THF tends to accumulate in the cells.[1]

Folate is a vitamin B present in green leafy vegetables, fruits, and legumes in the diet as polyglutamate.  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. It is absorbed in the jejunum and ileum after being reduced by folate reductase to N5-methyl THF which is the major form present in the blood and stored in the liver.[4]

Cellular Level

In the cells, folic acid is reduced to THF, a biologically active form, in a two-step process that requires two molecules of NADPH and the enzyme dihydrofolate reductase (DHFR). A single carbon group is transferred from serine to THF by the enzyme serine hydroxymethyltransferase and reduced to N5, N10-methylene THF. Vitamin B6 is also required in this process. Glycine is produced as a result of this reaction, which can be readily reconverted to serine using the carbon from the one-carbon pool.[1]

The carbon carried by N5, N10-methylene THF can then be transferred to deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP) by the enzyme thymidylate synthase that can be used to synthesize thymidine, at the same time recycling THF to DHF.  The resulted DHF can be reduced once again to tetrahydrofolate and N5, N10-methylene THF again to continue this cycle. 5-fluorouracil is an anti-cancer drug that is used to inhibit the proliferation of cancer cells. It inhibits the synthesis of thymine and consequently DNA synthesis by inhibiting the enzyme thymidylate synthase.[5]

Alternately, N5, N10-methylene THF can be reduced to N5-methyl-THF, a storage form of folate, with the help of riboflavin (vitamin B2) and the enzyme methylenetetrahydrofolate reductase (MTHFR). 

The methyl group from N5-methyl-THF is transferred to cobalamine (B12) to help regenerate methylcobalamin (B12) and THF. Methylcobalamine can then donate the methyl group to homocysteine and form methionine with the help of methionine synthase. Hence B12 deficiency results in increased levels of homocysteine in the blood. Additionally, methylmalonic acid levels also increase in B12 deficiency as the conversion of methylmalonyl-CoA to Succinyl CoA is catalyzed by a vitamin B12 dependent enzyme, methylmalonyl-CoA mutase. Folate deficiency does not have any effect on methylmalonic acid levels, but homocysteine levels increase as homocysteine cannot be converted to methionine due to deficiency of N5-methyl THF. B12 plays an important role in the recycling of N5-methyl THF (the storage form of folate) to THF. Deficiency of Vitamin B12 can impair this process and prevent the use of storage form of folate when needed, resulting in functional folate deficiency.[6]

Molecular Level

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. Serine is the primary carbon source in the conversion of tetrahydrofolate to N5, N10-methylene tetrahydrofolate. Glycine production also occurs in this reaction. The formation of serine requires a hydroxymethyl group from N5, N10-methylene tetrahydrofolate, and a glycine residue in the reverse reaction. [1]

Methionine Cycle

Methionine is an essential amino acid converted to S-adenosyl methionine (SAM) that participates in various methylation reactions and results in the formation of S-adenosyl homocysteine (SAH). 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. Next, the adenosyl group is removed from SAH to form homocysteine. A methyl group is donated by methylcobalamin (B12) to homocysteine, which forms methionine. This reaction also requires the enzyme methionine synthase.  Resultant cobalamin (without the methyl group) can be regenerated back to methylcobalamin by accepting the methyl group from N5-methyl THF. This process allows the recycling of N5-methyl THF to THF for reuse.[6]

Clinical Significance

Methotrexate and Folic Acid Deficiency

Methotrexate, a folic acid antagonist, is commonly used in cancer chemotherapy, inflammatory and autoimmune conditions such as rheumatoid arthritis. As an irreversible competitive inhibitor of dihydrofolate reductase, it blocks the conversion of dihydrofolate (DHF) to tetrahydrofolate (THF). It therefore prevents cellular proliferation by preventing the synthesis of thymidine, a building block for nucleic acid synthesis and thus ultimately inhibiting DNA and RNA synthesis. Therefore, all patients receiving methotrexate are given supplemental folic acid to prevent hematologic, gastrointestinal, and hepatic side effects. [7][8][9]

Megaloblastic Anemia

Deficiency of either folic acid or cobalamin (vitamin B12) can result in megaloblastic anemia due to inhibition of DNA synthesis caused by decreased availability of purines and pyrimidines (Thymidine monophosphate). This results in enlarged red blood cells and accumulation of significant, immature precursors (megaloblasts) of RBCs in the blood and bone marrow.  The folate deficiency manifests in fatigue, muscle weakness, tingling of extremities, and loss of joint position/coordination. [10][11]

Neural Tube Defects

Folic acid deficiency is a known cause of neural tube defects such as spina bifida and anencephaly. During the first few weeks of fetal development, folic acid is a critical requirement for several folate-dependent processes. Therefore it is important that pregnant women and those planning to conceive ensure adequate folic acid consumption.[12][13]

Methylenetetrahydrofolate Reductase (MTHFR) Deficiency

Mutation in the MTHFR gene results in elevated plasma levels of homocysteine (hyperhomocysteinemia) as the defect in this enzyme prevents the conversion of homocysteine to methionine. MTHFR is the rate-limiting enzyme that catalyzes the formation of 5-methyltetrahydrofolate from N5, N10-methylene tetrahydrofolate, a cosubstrate for methylation of homocysteine to methionine. Genetic testing is available to identify this condition. It is important to diagnose hyperhomocysteinemia early since it is a risk factor for atherosclerosis, venous thrombosis, myocardial infarction, and other cardiovascular conditions.[14] In addition, there are known polymorphisms associated with the MTHFR gene associated with conditions such as colon cancer. [15]

Antibiotics

Trimethoprim and sulfamethoxazole are antibiotic drugs used in combination for the treatment of certain bacterial infections. Trimethoprim inhibits DHFR and prevents the synthesis of THF. Sulfamethoxazole is a structural analog of para-aminobenzoic acid that inhibits the bacterial synthesis of DHF.[16]