Also known as vitamin B₉, vitamin B_c, vitamin M, Lactobacillus casei factor, folacin, and pteroyl-L-glutamic acid, folic acid was first discovered by Wills and Mehta in 1931 as a factor in yeast (“Wills’ factor”) that corrected the macrocytic anemia of pregnant Hindu women in India (1). The factor was later isolated from spinach leaves and was given the name folic acid (Latin folium, “leaf”) by Mitchell et al in 1941, who demonstrated that it was required for growth of Streptococcus lactis R (Streptococcus faecalis) (2). In 1945, the chemical synthesis of pure crystalline folic acid was reported in the journal Science (3). Synthetic folic acid was effective in reversing megaloblastic anemia that was refractory to treatment with liver extracts, but this agent was not able to prevent or improve neurologic damage that progressed from anemia that is now known to be caused by vitamin B₁₂ deficiency.

Shortly after the discovery of folic acid as a growth-promoting factor, the development of folate antagonists as chemotherapeutics was undertaken by the Nobel Laureates Hitchings and Elion. In 1948, the folate antagonists aminopterin and, shortly thereafter, methotrexate were developed and administered to patients with childhood acute lymphoblastic leukemia and found to be effective treatments (4). This success led to the development of numerous anticancer and antimicrobial agents over the following 50 years that targeted folate-requiring enzymes. Beginning in the 1950s to the present time, folate-dependent enzymes were purified to homogeneity, biochemical pathways were elucidated, and later, their genes were cloned and structures determined. Beginning in the 1980s, the importance of folic acid in prevention of chronic diseases, certain cancers, and birth defects gained appreciation. This knowledge led to fortification of the food supply with folic acid in the United States, Canada, and other countries to prevent a class of common birth defects known as neural tube defects (NTDs). An excellent review of the history of folic acid was published in 2001 by Hoffbrand and Weir (1).

Folate is a generic term that refers to a family of water-soluble B vitamins that are found in natural food and in biologic organisms (Fig. 26.1). Folates function as a family of enzyme cofactors that carry and chemically activate single carbons (referred to as one-carbons) for biosynthetic reactions. Folate is required for the biosynthesis of ribonucleotides and deoxyribonucleotide precursors for DNA synthesis. It is also required for amino acid metabolism, including the remethylation of homocysteine to methionine, and therefore functions in the regulation of gene expression by methylation. Hence, folate cofactors are
found in virtually all forms of life. Tetrahydrofolate (THF), which is the fully reduced form of the vitamin, carries one-carbons at one of three different oxidation levels ranging from methanol to formate (5, 6). The one-carbons are covalently bound to the N5 or N10 position of THF. In the cell, five different one-carbon substituted forms of THF are present: 10-formyl-THF; 5-formyl-THF; 5,10-methenyl-THF; 5,10-methylene-THF; and 5-methyl-THF, and each of these forms is interconverted in the cell through enzyme-mediated catalysis. Folates are also modified through the addition of a glutamate polypeptide that is polymerized through unusual γ-linked peptide bonds (7). The polyglutamate polypeptide increases the affinity of folate cofactors for folate-dependent enzymes and is required to retain folates within the cell and subcellular organelles. Folic acid (see Fig. 26.1) is not a biologically active form of folate, but it can serve as a provitamin because it is converted to the reduced, natural form of folate once transported into cells. It is an oxidized form of folate generated during the oxidative degradation of folate and normally does not accumulate in cells, although most degradation of THF is irreversible with degradation products that include oxidized pterin and para-aminobenzoyl-glutamate (8). Folic acid is also a synthetic form of folate present in fortified foods and in dietary supplements.

Folate is a vitamin and therefore must be acquired from the diet. Folate nutritional status is supported by the intake of the vitamin found in natural foods as well as dietary supplements and fortified foods (9). The best dietary sources of natural folate include fresh fruits, leafy green vegetables, yeast, liver, and legumes (10). Natural folates found in food are chemically labile and readily undergo irreversible oxidative degradation during food preparation and cooking. 5-Methyl-THF and formyl-substituted THF are the primary forms of folate present in natural foods, and they are also among the more stable forms of the vitamin. Folic acid, the synthetic, fully oxidized, and stable provitamin, is present in dietary supplements and fortified food (see Fig. 26.1). Folic acid has greater bioavailability than natural food folate because of its chemical stability and lack of a polyglutamate moiety, which impairs absorption across the intestinal epithelium (11). Once transported into the cell, folic acid is reduced to dihydrofolate (DHF) and subsequently THF by the enzyme DHF reductase (DHFR), and once fully reduced, folic acid is indistinguishable from natural food folate. Low levels of DHFR expression may result in the appearance of folic acid in the serum of individuals with high levels of folic acid intake (9). Evidence indicates that the total DHFR activity is highly variable among individuals—a finding that may indicate a variable capacity to metabolize folic acid among individuals (12).

The recommended intakes of folate established by the Food and Nutrition Board of the Institute of Medicine are shown in Table 26.1 (13). Dietary folate requirements are expressed as dietary folate equivalents (DFEs) because of the need to adjust for the increased bioavailability of folic acid compared with natural food folate (11). Folic acid is estimated to be 1.7 times more bioavailable than natural food folate. The recommended dietary allowance (RDA) for both men and women is 400 µg/day DFEs. The requirement for women of childbearing age is 400 µg of folic acid from fortified foods and supplements, in addition to food folate consumption from a varied diet (13). The tolerable upper intake level for adults was set at 1000 µg/day of folic acid exclusive of food folate, based on concerns that elevated intake of folic acid would exacerbate neurologic consequences of vitamin B₁₂ deficiency.

The United States and Canada mandated the addition of folic acid at a level of 140 µg/100 g of product to enriched flour in 1998 to achieve a predicted intake of 100 µg/day folic acid to reduce the incidence of NTDs (14). Before folic acid fortification, the median folate intake from food was estimated to be 250 µg/day. Population intake levels increased by 529 µg DFE/day between the interval 1998 to 1994 (before fortification) and 1999 to 2000 (after fortification) and then decreased by 135 mg between 1999 to 2000 and 2003 to 2004 (14). Folic acid fortification increased serum and red blood cell folate concentrations in the United States and decreased total plasma homocysteine levels by 6% to 13% (15, 16).

Folate absorption across the intestinal epithelium occurs in the acidic environment of the upper small intestine through the proton-coupled folate transporter (PCFT) (17), which was originally and probably incorrectly discovered as a heme transporter. Loss of PCFT function is associated with severe folate malabsorption, a finding indicating that it functions as the primary folate transporter in the gut. Only folate monoglutamates are bioavailable and absorbed. During digestion, the γ-glutamyl polypeptide of natural food folate is hydrolyzed to generate folate monoglutamate forms through a reaction catalyzed by the enzyme γ-glutamyl hydrolase. Folates circulate in serum as monoglutamate derivatives primarily in the form of 5-methyl-THF. In circulating red blood cells, 5-methyl-THF polyglutamates are the primary form of folates, although individuals with polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene accumulate 10-formyl-THF in red blood cells (18). Transport into cells occurs primarily through the reduced folate carrier. Once transported into cells, folate monoglutamate derivatives are either converted to their polyglutamate forms by the addition of a γ-glutamyl polypeptide, usually consisting of five to nine glutamate residues in the cytoplasm or transported into mitochondria as monoglutamate derivatives and converted to polyglutamate forms in that compartment. The glutamate polypeptide serves to retain the vitamin within mitochondria and within the cell.

THF polyglutamates function as coenzymes that donate or accept one-carbons in an integrated network of biosynthetic and catabolic reactions involved in nucleotide and amino acid metabolism. Collectively, the network is commonly referred to as folate-mediated one-carbon metabolism. Folate metabolism is compartmentalized in the cytoplasm and nucleus (Fig. 26.2A) and mitochondria (Fig. 26.2B) (6). Each of these intracellular compartments is associated with specific metabolic pathways, and the compartments are interdependent through the exchange of common intermediates including formate, serine, and glycine (see Fig. 26.2B) (5, 19). Folate-mediated one-carbon metabolism also requires the water-soluble vitamins riboflavin (vitamin B₂), niacin (vitamin B₃), choline, pantothenic acid (vitamin B₅), pyridoxal phosphate (vitamin B₆), and cobalamin (vitamin B₁₂) for its function (6) (see Fig. 26.2).