animals, including humans, cannot synthesize folate and therefore must obtain folate from their diet. all plants and fungi and certain protozoa, bacteria, and archaea can synthesize folate de novo through variations on the same biosynthetic pathway. the folate molecule is synthesized from pterin pyrophosphate, para-aminobenzoic acid, and glutamate through the action of dihydropteroate synthase and dihydrofolate synthase. pterin is in turn derived in a series of enzymatically catalyzed steps from guanosine triphosphate (gtp), while para-aminobenzoic acid is a product of the shikimate pathway. bioactivation biotransformation of folic acid into folinic acids where r = para-aminobenzoate-glutamate. all of the biological functions of folic acid are performed by thf and its methylated derivatives. hence folic acid must first be reduced to thf. this four electron reduction proceeds in two chemical steps both catalyzed by the same enzyme, dihydrofolate reductase. folic acid is first reduced to dihydrofolate and then to tetrahydrofolate. each step consumes one molecule of nadph (biosynthetically derived from vitamin b3) and produces one molecule of nadp. mechanistically, hydride is transferred from nadph to the c6 position of the pteridine ring. a one-carbon (1c) methyl group is added to tetrahydrofolate through the action of serine hydroxymethyltransferase (shmt) to yield 5,10-methylenetetrahydrofolate (5,10-ch2-thf). this reaction also consumes serine and pyridoxal phosphate (plp; vitamin b6) and produces glycine and pyridoxal. a second enzyme, methylenetetrahydrofolate dehydrogenase (mthfd2) oxidizes 5,10-methylenetetrahydrofolate to an iminium cation which in turn is hydrolyzed to produce 5-formyl-thf and 10-formyl-thf. this series of reactions using the β-carbon atom of serine as the carbon source provide the largest part of the one-carbon units available to the cell. alternative carbon sources include formate which by the catalytic action of formate–tetrahydrofolate ligase add a 1c unit to thf to yield 10-formyl-thf. glycine, histidine, and sarcosine can also directly contribute to the thf-bound 1c pool. drug interference a number of drugs interfere with the biosynthesis of thf from folic acid. among them are the antifolate dihydrofolate reductase inhibitors such as the antimicrobial, trimethoprim, the antiprotozoal, pyrimethamine and the chemotherapy drug methotrexate, and the sulfonamides (competitive inhibitors of 4-aminobenzoic acid in the reactions of dihydropteroate synthetase). valproic acid, one of the most commonly prescribed epilepsy treatment drugs, also used to treat certain psychological conditions such as bipolar disorder, is a known inhibitor of folic acid, and as such, has been shown to cause birth defects, including neural tube defects, plus increased risk for children having cognitive impairment and autism. there is evidence that folate consumption is protective. function tetrahydrofolate's main function in metabolism is transporting single-carbon groups (i.e., a methyl group, methylene group, or formyl group). these carbon groups can be transferred to other molecules as part of the modification or biosynthesis of a variety of biological molecules. folates are essential for the synthesis of dna, the modification of dna and rna, the synthesis of methionine from homocysteine, and various other chemical reactions involved in cellular metabolism. these reactions are collectively known as folate-mediated one-carbon metabolism. dna synthesis main articles: purine metabolism and pyrimidine metabolism folate derivatives participate in the biosynthesis of both purines and pyrimidines. formyl folate is required for two of the steps in the biosynthesis of inosine monophosphate, the precursor to gmp and amp. methylenetetrahydrofolate donates the c1 center required for the biosynthesis of dtmp (2′-deoxythymidine-5′-phosphate) from dump (2′-deoxyuridine-5′-phosphate). the conversion is catalyzed by thymidylate synthase. vitamin b12 activation simplified schematic diagram of the folate methionine cycle methyl-thf converts vitamin b12 to methyl-b12 (methylcobalamin). methyl-b12 converts homocysteine, in a reaction catalyzed by homocysteine methyltransferase, to methionine. a defect in homocysteine methyltransferase or a deficiency of b12 may lead to a so-called "methyl-trap" of thf, in which thf converts to methyl-thf, causing a deficiency in folate. thus, a deficiency in b12 can cause accumulation of methyl-thf, mimicking folate deficiency. dietary recommendations because of the difference in bioavailability between supplemented folic acid and the different forms of folate found in food, the dietary folate equivalent (dfe) system was established. one dfe is defined as 1 μg of dietary folate. 1 μg of folic acid supplement counts as 1.7 μg dfe. the reason for the difference is that when folic acid is added to food or taken as a dietary supplement with food it is at least 85% absorbed, whereas only about 50% of folate naturally present in food is absorbed. national institutes of health (u.s.) nutritional recommendationsµg dfe per day for rda, µg folic acid for tolerable upper intake levels(ul) age infants children and adults pregnant women lactating women (ai) (ul) (rda) (ul) (rda) (ul) (rda) (ul) 0–6 months 65 none set – – – – – – 7–12 months 80 none set – – – – – – 1–3 years – – 150 300 – – – – 4–8 years – – 200 400 – – – – 9–13 years – – 300 600 – – – – 14–18 – – 400 800 600 800 500 800 19+ – – 400 1000 600 1000 500 1000 the u.s. institute of medicine defines estimated average requirements (ears), recommended dietary allowances (rdas), adequate intakes (ais), and tolerable upper intake levels (uls) – collectively referred to as dietary reference intakes (dris). the european food safety authority (efsa) refers to the collective set of information as dietary reference values, with population reference intake (pri) instead of rda, and average requirement instead of ear. ai and ul are defined the same as in united states. for women and men over age 18 the pri is set at 330 μg/day. pri for pregnancy is 600 μg/day, for lactation 500 μg/day. for children ages 1–17 years the pris increase with age from 120 to 270 μg/day. these values differ somewhat from the u.s. rdas. the united kingdom's dietary reference value for folate, set by the committee on medical aspects of food and nutrition policy in 1991, is 200 μg/day for adults. safety the risk of toxicity from folic acid is low because folate is a water-soluble vitamin and is regularly removed from the body through urine. one potential issue associated with high doses of folic acid is that it has a masking effect on the diagnosis of pernicious anaemia due to vitamin b12 deficiency, and may even precipitate or exacerbate neuropathy in vitamin b12-deficient individuals. this evidence justified development of a ul for folate. in general, uls are set for vitamins and minerals when evidence is sufficient. the adult ul of 1,000 μg for folate (and lower for children) refers specifically to folic acid used as a supplement, as no health risks have been associated with high intake of folate from food sources. the efsa reviewed the safety question and agreed with united states that the ul be set at 1,000 μg. the japan national institute of health and nutrition set the adult ul at 1,300 or 1,400 μg depending on age. reviews of clinical trials that called for long-term consumption of folic acid in amounts exceeding the ul have raised concerns. excessive amounts derived from supplements are more of a concern than that derived from natural food sources and the relative proportion to vitamin b12 may be a significant factor in adverse effects. one theory is that consumption of large amounts of folic acid leads to detectable amounts of unmetabolized folic acid circulating in blood because the enzyme dihydrofolate reductase that converts folic acid to the biologically active forms is rate limiting. evidence of a negative health effect of folic acid in blood is not consistent, and folic acid has no known cofactor function that would increase the likelihood of a causal role for free folic acid in disease development. however, low vitamin b12 status in combination with high folic acid intake, in addition to the previously mentioned neuropathy risk, appeared to increase the risk of cognitive impairment in the elderly. long-term use of folic acid dietary supplements in excess of 1,000 μg/day has been linked to an increase in prostate cancer risk. food labeling for u.s. food and dietary supplement labeling purposes the amount in a serving is expressed as a percent of daily value (%dv). for folate labeling purposes 100% of the daily value was 400 μg. as of the 27 may 2016 update, it was kept unchanged at 400 μg. compliance with the updated labeling regulations was required by 1 january 2020 for manufacturers with us$10 million or more in annual food sales, and by 1 january 2021 for manufacturers with lower volume food sales. a table of the old and new adult daily values is provided at reference daily intake. european union regulations require that labels declare energy, protein, fat, saturated fat, carbohydrates, sugars, and salt. voluntary nutrients may be shown if present in significant amounts. instead of daily values, amounts are shown as percent of reference intakes (ris). for folate, 100% ri was set at 200 μg in 2011. deficiency main article: folate deficiency folate deficiency can be caused by unhealthy diets that do not include enough vegetables and other folate-rich foods; diseases in which folates are not well absorbed in the digestive system (such as crohn's disease or celiac disease); some genetic disorders that affect levels of folate; and certain medicines (such as phenytoin, sulfasalazine, or trimethoprim-sulfamethoxazole). folate deficiency is accelerated by alcohol consumption, possibly by interference with folate transport. folate deficiency may lead to glossitis, diarrhea, depression, confusion, anemia, and fetal neural tube and brain defects. other symptoms include fatigue, gray hair, mouth sores, poor growth, and swollen tongue. folate deficiency is diagnosed by analyzing a complete blood count (cbc) and plasma vitamin b12 and folate levels. a serum folate of 3 μg/l or lower indicates deficiency. serum folate level reflects folate status, but erythrocyte folate level better reflects tissue stores after intake. an erythrocyte folate level of 140 μg/l or lower indicates inadequate folate status. serum folate reacts more rapidly to folate intake than erythrocyte folate. since folate deficiency limits cell division, erythropoiesis (production of red blood cells) is hindered. this leads to megaloblastic anemia, which is characterized by large, immature red blood cells. this pathology results from persistently thwarted attempts at normal dna replication, dna repair, and cell division, and produces abnormally large red cells called megaloblasts (and hypersegmented neutrophils) with abundant cytoplasm capable of rna and protein synthesis, but with clumping and fragmentation of nuclear chromatin. some of these large cells, although immature (reticulocytes), are released early from the marrow in an attempt to compensate for the anemia. both adults and children need folate to make normal red and white blood cells and prevent anemia, which causes fatigue, weakness, and inability to concentrate. increased homocysteine levels suggest tissue folate deficiency, but homocysteine is also affected by vitamin b12 and vitamin b6, renal function, and genetics. one way to differentiate between folate deficiency and vitamin b12 deficiency is by testing for methylmalonic acid (mma) levels. normal mma levels indicate folate deficiency and elevated mma levels indicate vitamin b12 deficiency. elevated mma levels may also be due to the rare metabolic disorder combined malonic and methylmalonic aciduria (cmamma). folate deficiency is treated with supplemental oral folic acid of 400 to 1000 μg per day. this treatment is very successful in replenishing tissues, even if deficiency was caused by malabsorption. people with megaloblastic anemia need to be tested for vitamin b12 deficiency before treatment with folic acid, because if the person has vitamin b12 deficiency, folic acid supplementation can remove the anemia, but can also worsen neurologic problems. cobalamin (vitamin b12) deficiency may lead to folate deficiency, which, in turn, increases homocysteine levels and may result in the development of cardiovascular disease or birth defects. sources the united states department of agriculture, agricultural research service maintains a food composition database from which folate content in hundreds of foods can be searched as shown in the table. the food fortification initiative lists all countries in the world that conduct fortification programs, and within each country, what nutrients are added to which foods, and whether those programs are voluntary or mandatory. in the us, mandatory fortification of enriched breads, cereals, flours, corn meal, pastas, rice, and other grain products began in january 1998. as of 21 december 2018, 81 countries required food fortification with one or more vitamins. the most commonly fortified vitamin – as used in 62 countries – is folate; the most commonly fortified food is wheat flour, followed by maize flour