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Folate

Folate, also known as vitamin B9 and folacin, is one of the B vitamins. Manufactured folic acid, which is converted into folate by the body, is used as a dietary supplement and in food fortification as it is more stable during processing and storage. Folate is required for the body to make DNA and RNA and metabolise amino acids necessary for cell division and maturation of blood cells. As the human body cannot make folate, it is required in the diet, making it an essential nutrient. It occurs naturally in many foods. The recommended adult daily intake of folate in the U.S. is 400 micrograms from foods or dietary supplements.

Folate in the form of folic acid is used to treat anemia caused by folate deficiency. Folic acid is also used as a supplement by women during pregnancy to reduce the risk of neural tube defects (NTDs) in the baby. Low levels in early pregnancy are believed to be the cause of more than half of babies born with NTDs. More than 80 countries use either mandatory or voluntary fortification of certain foods with folic acid as a measure to decrease the rate of NTDs. Long-term supplementation with relatively large amounts of folic acid is associated with a small reduction in the risk of stroke and an increased risk of prostate cancer. There are concerns that large amounts of supplemental folic acid can hide vitamin B12 deficiency.

Not consuming enough folate can lead to folate deficiency. This may result in a type of anemia in which red blood cells become abnormally large. Symptoms may include feeling tired, heart palpitations, shortness of breath, open sores on the tongue, and changes in the color of the skin or hair. Folate deficiency in children may develop within a month of poor dietary intake. In adults, normal total body folate is between 10 and 30 mg with about half of this amount stored in the liver and the remainder in blood and body tissues. In plasma, the natural folate range is 150 to 450 nM.

Folate was discovered between 1931 and 1943. It is on the World Health Organization's List of Essential Medicines. In 2021, it was the 77th most commonly prescribed medication in the United States, with more than 8million prescriptions. The term "folic" is from the Latin word folium (which means leaf) because it was found in dark-green leafy vegetables.

Definition
Folate (vitamin B9) refers to the many forms of folic acid and its related compounds, including tetrahydrofolic acid (the active form), methyltetrahydrofolate (the primary form found in blood), methenyltetrahydrofolate, folinic acid, folacin, and pteroylglutamic acid. Historic names included L. casei factor, vitamin Bc and vitamin M.

The terms folate and folic acid have somewhat different meanings in different contexts, although sometimes used interchangeably. Within the field of organic chemistry, folate refers to the conjugate base of folic acid. Within the field of biochemistry, folates refer to a class of biologically active compounds related to and including folic acid. Within the field of nutrition, the folates are a family of essential nutrients related to folic acid obtained from natural sources whereas the term folic acid is reserved for the manufactured form that is used as a dietary supplement.

Chemically, folates consist of three distinct chemical moieties linked together. A pterin (2-amino-4-hydroxy-pteridine) heterocyclic ring is linked by a methylene bridge to a p-aminobenzoyl group that in turn is bonded through an amide linkage to either glutamic acid or poly-glutamate. One-carbon units in a variety of oxidation states may be attached to the N5 nitrogen atom of the pteridine ring and/or the N10 nitrogen atom of the p-aminobenzoyl group.

Health effects
Folate is especially important during periods of frequent cell division and growth, such as infancy and pregnancy. Folate deficiency hinders DNA synthesis and cell division, affecting hematopoietic cells and neoplasms the most because of their greater frequency of cell division. RNA transcription and subsequent protein synthesis are less affected by folate deficiency, as the mRNA can be recycled and used again (as opposed to DNA synthesis, where a new genomic copy must be created).

Birth defects
Deficiency of folate in pregnant women has been implicated in neural tube defects (NTDs), with an estimate of 300,000 cases worldwide prior to the implementation in many countries of mandatory food fortification. NTDs occur early in pregnancy (first month), therefore women must have abundant folate upon conception and for this reason there is a recommendation that any woman planning to become pregnant consume a folate-containing dietary supplement before and during pregnancy. The Center for Disease Control and Prevention (CDC) recommends a daily amount of 400 micrograms of folic acid for the prevention of NTDs. Compliance with this recommendation is not complete, and many women become pregnant without this being a planned pregnancy, or may not realize that they are pregnant until well into the first trimester, which is the critical period for reducing risk of NTDs. Countries have implemented either mandatory or voluntary food fortification of wheat flour and other grains, or else have no such program and depend on public health and healthcare practitioner advice to women of childbearing age. A meta-analysis of global birth prevalence of spina bifida showed that when mandatory fortification was compared to countries with voluntary fortification or no fortification program, there was a 30% reduction in live births with spina bifida. Some countries reported a greater than 50% reduction. The United States Preventive Services Task Force recommends folic acid as the supplement or fortification ingredient, as forms of folate other than folic acid have not been studied.

A meta-analysis of folate supplementation during pregnancy reported a 28% lower relative risk of newborn congenital heart defects. Prenatal supplementation with folic acid did not appear to reduce the risk of preterm births. One systematic review indicated no effect of folic acid on mortality, growth, body composition, respiratory, or cognitive outcomes of children from birth to 9 years old. There was no relation between maternal folic acid supplementation and an increased risk for childhood asthma.

Fertility
Folate contributes to spermatogenesis. In women, folate is important for oocyte quality and maturation, implantation, placentation, fetal growth and organ development.

Heart disease
One meta-analysis reported that multi-year folic acid supplementation, in amounts in most of the included clinical trials at higher than the upper limit of 1,000 μg/day, reduced the relative risk of cardiovascular disease by a modest 4%. Two older meta-analyses, which would not have incorporated results from newer clinical trials, reported no changes to the risk of cardiovascular disease.

Stroke
The absolute risk of stroke with supplementation decreases from 4.4% to 3.8% (a 10% decrease in relative risk). Two other meta-analyses reported a similar decrease in relative risk. Two of these three were limited to people with pre-existing cardiovascular disease or coronary heart disease. The beneficial result may be associated with lowering circulating homocysteine concentration, as stratified analysis showed that risk was reduced more when there was a larger decrease in homocysteine. The effect was also larger for the studies that were conducted in countries that did not have mandatory grain folic acid fortification. The beneficial effect was larger in the subset of trials that used a lower folic acid supplement compared to higher.

Cancer
Chronically insufficient intake of folate may increase the risk of colorectal, breast, ovarian, pancreatic, brain, lung, cervical, and prostate cancers.

Early after fortification programs were implemented, high intakes were theorized to accelerate the growth of preneoplastic lesions that could lead to cancer, specifically colon cancer. Subsequent meta-analyses of the effects of low versus high dietary folate, elevated serum folate, and supplemental folate in the form of folic acid have reported at times conflicting results. Comparing low to high dietary folate showed a modest but statistically significant reduced risk of colon cancer. For prostate cancer risk, comparing low to high dietary folate showed no effect. A review of trials that involved folic acid dietary supplements reported a statistically significant 24% increase in prostate cancer risk. It was shown that supplementation with folic acid at 1,000 to 2,500 μg/day – the amounts used in many of the cited supplement trials – would result in higher concentrations of serum folate than what is achieved from diets high in food-derived folate. The second supplementation review reported no significant increase or decrease in total cancer incidence, colorectal cancer, other gastrointestinal cancer, genitourinary cancer, lung cancer or hematological malignancies in people who were consuming folic acid supplements. A third supplementation meta-analysis limited to reporting only on colorectal cancer incidence showed that folic acid treatment was not associated with colorectal cancer risk.

Anti-folate chemotherapy
Folate is important for cells and tissues that divide rapidly. Cancer cells divide rapidly, and drugs that interfere with folate metabolism are used to treat cancer. The antifolate drug methotrexate is often used to treat cancer because it inhibits the production of the active tetrahydrofolate (THF) from the inactive dihydrofolate (DHF). However, methotrexate can be toxic,  producing side effects such as inflammation in the digestive tract that make eating normally more difficult. Bone marrow depression (inducing leukopenia and thrombocytopenia) and acute kidney and liver failure have been reported.

Folinic acid, under the drug name leucovorin, a form of folate (formyl-THF), can help "rescue" or reverse the toxic effects of methotrexate. Folic acid supplements have little established role in cancer chemotherapy. The supplement of folinic acid in people undergoing methotrexate treatment is to give less rapidly dividing cells enough folate to maintain normal cell functions. The amount of folate given is quickly depleted by rapidly dividing (cancer) cells, so this does not negate the effects of methotrexate.

Neurological disorders
Conversion of homocysteine to methionine requires folate and vitamin B12. Elevated plasma homocysteine and low folate are associated with cognitive impairment, dementia and Alzheimer's disease. Supplementing the diet with folic acid and vitamin B12 lowers plasma homocysteine. However, several reviews reported that supplementation with folic acid alone or in combination with other B vitamins did not prevent development of cognitive impairment nor slow cognitive decline.

Relative risk of autism spectrum disorders (ASDs) was reported reduced by 23% when the maternal diet was supplemented with folic acid during pregnancy. Subset analysis confirmed this among Asian, European and American populations. Cerebral folate deficiency (CFD) has been associated with ASDs. The cerebral folate receptor alpha (FRα) transports 5-methyltetrahydrofolate into the brain. One cause of CFD is autoantibodies that interfere with FRa, and FRa autoantibodies have been reported in ASDs. For individuals with ASD and CFD, meta-analysis reported improvements with treatment with folinic acid, a 5-formyl derivative of tetrahydrofolic acid, for core and associated ASD symptoms.

Some evidence links a shortage of folate with clinical depression. Limited evidence from randomized controlled trials showed using folic acid in addition to selective serotonin reuptake inhibitors (SSRIs) may have benefits. Research found a link between depression and low levels of folate. The exact mechanisms involved in the development of schizophrenia and depression are not entirely clear, but the bioactive folate, methyltetrahydrofolate (5-MTHF), a direct target of methyl donors such as S-adenosyl methionine (SAMe), recycles the inactive dihydrobiopterin (BH2) into tetrahydrobiopterin (BH4), the necessary cofactor in various steps of monoamine synthesis, including that of dopamine and serotonin. BH4 serves a regulatory role in monoamine neurotransmission and is required to mediate the actions of most antidepressants.

Folic acid, B12 and iron
A complex interaction occurs between folic acid, vitamin B12, and iron. A deficiency of folic acid or vitamin B12 may mask the deficiency of iron; so when taken as dietary supplements, the three need to be in balance.

Malaria
Some studies show iron–folic acid supplementation in children under five may result in increased mortality due to malaria; this has prompted the World Health Organization to alter their iron–folic acid supplementation policies for children in malaria-prone areas, such as India.

Absorption, metabolism and excretion
Folate in food is roughly one-third in the form of monoglutamate and two-thirds polyglutamate; the latter is hydrolyzed to monoglutamate via a reaction mediated by folate conjugase at the brush border of enterocytes in the proximal small intestine. Subsequentially, intestinal absorption is primarily accomplished by the action of the proton-coupled folate transporter (PCFT) protein coded for by the SLC46A1 gene. This functions best at pH 5.5, which corresponds to the acidic status of the proximal small intestine. PCFT binds to both reduced folates and folic acid. A secondary folate transporter is the reduced folate carrier (RFC), coded for by the SLC19A1 gene. It operates optimally at pH 7.4 in the ileum portion of the small intestine. It has a low affinity for folic acid. Production of the receptor proteins is increased in times of folate deficiency. In addition to a role in intestinal absorption, RFC is expressed in virtually all tissues and is the major route of delivery of folate to cells within the systemic circulation under physiological conditions. When pharmacological amounts of folate are taken as a dietary supplement, absorption also takes place by a passive diffusion-like process. In addition, bacteria in the distal portion of the small intestine and in the large intestine synthesize modest amounts of folate, and there are RFC receptors in the large intestine, so this in situ source may contribute to toward the cellular nutrition and health of the local colonocytes.

The biological activity of folate in the body depends upon dihydrofolate reductase action in the liver which converts folate into tetrahydrofolate (THF). This action is rate-limiting in humans leading to elevated blood concentrations of unmetabolized folic acid when consumption from dietary supplements and fortified foods nears or exceeds the U.S. Tolerable Upper Intake Level of 1,000 μg per day.

The total human body content of folate is estimated to be approximately 15–30 milligrams, with approximately half in the liver. Excretion is via urine and feces. Under normal dietary intake, urinary excretion is mainly as folate cleavage products, but if a dietary supplement is being consumed then there will be intact folate in the urine. The liver produces folate-containing bile, which if not all absorbed in the small intestine, contributes to fecal folate, intact and as cleavage products, which under normal dietary intake has been estimated to be similar in amount to urinary excretion. Fecal content includes what is synthezized by intestinal microflora.

Biosynthesis
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
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 adds 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.

Folate deficiency is common in alcoholics, attributed to both inadequate diet and an inhibition in intestinal processing of the vitamin. Chronic alcohol use inhibits both the digestion process of dietary folate polyglutamates and the uptake phase of liberated folate monoglutamates. The latter is associated with a significant reduction in the level of expression of RFC.

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
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
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.

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
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.

Food fortification
Folic acid fortification is a process where synthetic folic acid is added to wheat flour or other foods with the intention of promoting public health through increasing blood folate levels in the populace. It is used as it is more stable during processing and storage. After the discovery of the link between insufficient folic acid and neural tube defects, governments and health organizations worldwide made recommendations concerning folic acid supplementation for women intending to become pregnant. Because the neural tube closes in the first four weeks of gestation, often before many women even know they are pregnant, many countries in time decided to implement mandatory food fortification programs. A meta-analysis of global birth prevalence of spina bifida showed that when mandatory fortification was compared to countries with voluntary fortification or no fortification program, there was a 30% reduction in live births with spina bifida, with some countries reporting a greater than 50% reduction.

Folic acid is added to grain products in more than 80 countries, either as required or voluntary fortification, and these fortified products make up a significant source of the population's folate intake. Fortification is controversial, with issues having been raised concerning individual liberty, as well as the theorized health concerns described in the Safety section. In the U.S., there is concern that the federal government mandates fortification but does not provide monitoring of potential undesirable effects of fortification. 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. The most commonly mandatory fortified vitamin – in 62 countries – is folate; the most commonly fortified food is wheat flour.

Australia and New Zealand
Australia and New Zealand jointly agreed to wheat flour fortification through the Food Standards Australia New Zealand in 2007. The requirement was set at 135 μg of folate per 100 g of bread. Australia implemented the program in 2009. New Zealand was also planning to fortify bread (excluding organic and unleavened varieties) starting in 2009, but then opted to wait until more research was done. The Association of Bakers and the Green Party had opposed mandatory fortification, describing it as "mass medication". Food Safety Minister Kate Wilkinson reviewed the decision to fortify in July 2009, citing as reasons to oppose claims for links between over consumption of folate with increased risk of cancer. In 2012 the delayed mandatory fortification program was revoked and replaced by a voluntary program, with the hope of achieving a 50% bread fortification target.

Canada
Canadian public health efforts focused on promoting awareness of the importance of folic acid supplementation for all women of childbearing age and decreasing socio-economic inequalities by providing practical folic acid support to vulnerable groups of women. Folic acid food fortification became mandatory in 1998, with the fortification of 150 μg of folic acid per 100 grams of enriched flour and uncooked cereal grains. The results of folic acid fortification on the rate of neural tube defects in Canada have been positive, showing a 46% reduction in prevalence of NTDs; the magnitude of reduction was proportional to the prefortification rate of NTDs, essentially removing geographical variations in rates of NTDs seen in Canada before fortification.

United Kingdom
While the Food Standards Agency recommended folic acid fortification,  and wheat flour is fortified with iron, folic acid fortification of wheat flour is allowed voluntarily rather than required. A 2018 review by authors based in the United Kingdom strongly recommended that mandatory fortification be reconsidered as a means of reducing the risk of neural tube defects.

United States

In 1996, the United States Food and Drug Administration (FDA) published regulations requiring the addition of folic acid to enriched breads, cereals, flours, corn meals, pastas, rice, and other grain products. This ruling took effect on 1 January 1998, and was specifically targeted to reduce the risk of neural tube birth defects in newborns. There were concerns expressed that the amount of folate added was insufficient.

The fortification program was expected to raise a person's folic acid intake level by 70–130 μg/day; however, an increase of almost double that amount was actually observed. This could be from the fact that many foods are fortified by 160–175% over the required amount. Much of the elder population take supplements that add 400 μg to their daily folic acid intake. This is a concern because 70–80% of the population have detectable levels of unmetabolized folic acid in their blood, a consequence of folic acid supplementation and fortification. However, at blood concentrations achieved via food fortification, folic acid has no known cofactor function that would increase the likelihood of a causal role for free folic acid in disease development.

The U.S. National Center for Health Statistics conducts the biannual National Health and Nutrition Examination Survey (NHANES) to assess the health and nutritional status of adults and children in the United States. Some results are reported as What We Eat In America. The 2013–2014 survey reported that for adults ages 20 years and older, men consumed an average of 249 μg/day folate from food plus 207 μg/day of folic acid from consumption of fortified foods, for a combined total of 601 μg/day of dietary folate equivalents (DFEs because each microgram of folic acid counts as 1.7 μg of food folate). For women, the values are 199, 153 and 459 μg/day, respectively. This means that fortification led to a bigger increase in folic acid intake than first projected, and that more than half the adults are consuming more than the RDA of 400 μg (as DFEs). Even so, fewer than half of pregnant women are exceeding the pregnancy RDA of 600 μg/day.

Before folic acid fortification, about 4,100 pregnancies were affected by a neural tube defect each year in the United States. The Centers for Disease Control and Prevention reported in 2015 that since the addition of folic acid in grain-based foods as mandated by the FDA, the rate of neural tube defects dropped by 35%. This translates to an annual saving in total direct costs of approximately $508 million for the NTD-affected births that were prevented.

History
In the 1920s, scientists believed folate deficiency and anemia were the same condition. In 1931, researcher Lucy Wills made a key observation that led to the identification of folate as the nutrient required to prevent anemia during pregnancy. Wills demonstrated that anemia could be reversed with brewer's yeast. In the late 1930s, folate was identified as the corrective substance in brewer's yeast. It was first isolated via extraction from spinach leaves by Herschel K. Mitchell, Esmond E. Snell, and Roger J. Williams in 1941. The term "folic" is from the Latin word folium (which means leaf) because it was found in dark-green leafy vegetables. Historic names included L. casei factor, vitamin Bc after research done in chicks and vitamin M after research done in monkeys.

Bob Stokstad isolated the pure crystalline form in 1943, and was able to determine its chemical structure while working at the Lederle Laboratories of the American Cyanamid Company. This historical research project, of obtaining folic acid in a pure crystalline form in 1945, was done by the team called the "folic acid boys", under the supervision and guidance of Director of Research Dr. Yellapragada Subbarow, at the Lederle Lab, Pearl River, New York. This research subsequently led to the synthesis of the antifolate aminopterin, which was used to treat childhood leukemia by Sidney Farber in 1948.

In the 1950s and 1960s, scientists began to discover the biochemical mechanisms of action for folate. In 1960, researchers linked folate deficiency to risk of neural tube defects. In the late 1990s, the U.S. and Canadian governments decided that despite public education programs and the availability of folic acid supplements, there was still a challenge for women of child-bearing age to meet the daily folate recommendations, which is when those two countries implemented folate fortification programs. As of December 2018, 62 countries mandated food fortification with folic acid.

Animals
Veterinarians may test cats and dogs if a risk of folate deficiency is indicated. Cats with exocrine pancreatic insufficiency, more so than dogs, may have low serum folate. In dog breeds at risk for cleft lip and cleft palate dietary folic acid supplementation significantly decreased incidence.

title: Folate


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Biotin

Biotin (also known as vitamin B7 or vitamin H) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name biotin, borrowed from the German Biotin, derives from the Ancient Greek word ('life') and the suffix "-in" (a suffix used in chemistry usually to indicate 'forming'). Biotin appears as a white, needle-like crystalline solid.

Chemical description
Biotin is classified as a heterocyclic compound, with a sulfur-containing tetrahydrothiophene ring fused to a ureido group. A C5-carboxylic acid side chain is appended to the former ring. The ureido ring, containing the −N−CO−N− group, serves as the carbon dioxide carrier in carboxylation reactions. Biotin is a coenzyme for five carboxylase enzymes, which are involved in the catabolism of amino acids and fatty acids, synthesis of fatty acids, and gluconeogenesis. Biotinylation of histone proteins in nuclear chromatin plays a role in chromatin stability and gene expression.

Dietary recommendations
The US National Academy of Medicine updated Dietary Reference Intakes for many vitamins in 1998. At that time there was insufficient information to establish estimated average requirement or recommended dietary allowance, terms that exist for most vitamins. In instances such as this, the academy sets adequate intakes (AIs) with the understanding that at some later date, when the physiological effects of biotin are better understood, AIs will be replaced by more exact information. The biotin AIs for both males and females are:

Australia and New Zealand set AIs similar to the US.

The European Food Safety Authority (EFSA) also identifies AIs, setting values at 40 μg/day for adults, pregnancy at 40 μg/day, and breastfeeding at 45 μg/day. For children ages 1–17 years, the AIs increase with age from 20 to 35 μg/day.

Safety
The US National Academy of Medicine estimates upper limits for vitamins and minerals when evidence for a true limit is sufficient. For biotin, however, there is no upper limit because adverse effects of high biotin intake have not been determined. The EFSA also reviewed safety and reached the same conclusion as in the United States.

Labeling regulations
For US food and dietary supplement labeling purposes the amount in a serving is expressed as a percent of daily value. For biotin labeling purposes 100% of the daily value was 300 μg/day, but as of May 27, 2016, it was revised to 30 μg/day to bring it into an agreement with the adequate intake. Compliance with the updated labeling regulations was required by January 1, 2020, for manufacturers with US$10 million or more in annual food sales, and by January 1, 2021, for manufacturers with lower volume food sales. A table of the old and new adult daily values is provided at Reference Daily Intake.

Physiology
Biotin is a water-soluble B vitamin. Consumption of large amounts as a dietary supplement results in absorption, followed by excretion into urine as biotin. Consumption of biotin as part of a normal diet results in urinary excretion of biotin and biotin metabolites.

Absorption
Biotin in food is bound to proteins. Digestive enzymes reduce the proteins to biotin-bound peptides. The intestinal enzyme biotinidase, found in pancreatic secretions and in the brush border membranes of all three parts of the small intestine, frees biotin, which is then absorbed from the small intestine. When consumed as a biotin dietary supplement, absorption is nonsaturable, meaning that even very high amounts are absorbed effectively. Transport across the jejunum is faster than across the ileum.

The large intestine microbiota synthesize amounts of biotin estimated to be similar to the amount taken in the diet, and a significant portion of this biotin exists in the free (protein-unbound) form and, thus, is available for absorption. How much is absorbed in humans is unknown, although a review did report that human epithelial cells of the colon in vitro demonstrated an ability to uptake biotin.

Once absorbed, sodium-dependent multivitamin transporter (SMVT) mediates biotin uptake into the liver. SMVT also binds pantothenic acid, so high intakes of either of these vitamins can interfere with transport of the other.

Metabolism and excretion
Biotin catabolism occurs via two pathways. In one, the valeric acid sidechain is cleaved, resulting in bisnorbiotin. In the other pathway, the sulfur is oxidized, resulting in biotin sulfoxide. Urine content is proportionally about half biotin, plus bisnorbiotin, biotin sulfoxide, and small amounts of other metabolites.

Factors that affect biotin requirements
Chronic alcohol use is associated with a significant reduction in plasma biotin. Intestinal biotin uptake also appears to be sensitive to the effect of the anti-epilepsy drugs carbamazepine and primidone. Relatively low levels of biotin have also been reported in the urine or plasma of patients who have had a partial gastrectomy or have other causes of achlorhydria, as well as burn patients, elderly individuals, and athletes. Pregnancy and lactation may be associated with an increased demand for biotin. In pregnancy, this may be due to a possible acceleration of biotin catabolism, whereas, in lactation, the higher demand has yet to be elucidated. Recent studies have shown marginal biotin deficiency can be present in human gestation, as evidenced by increased urinary excretion of 3-hydroxyisovaleric acid, decreased urinary excretion of biotin and bisnorbiotin, and decreased plasma concentration of biotin.

Biosynthesis
Biotin, synthesized in plants, is essential to plant growth and development. Bacteria also synthesize biotin, and it is thought that bacteria resident in the large intestine may synthesize biotin that is absorbed and utilized by the host organism.

Biosynthesis starts from two precursors, alanine and pimeloyl-CoA. These form 7-keto-8-aminopelargonic acid (KAPA). KAPA is transported from plant peroxisomes to mitochondria where it is converted to 7,8-diaminopelargonic acid (DAPA) with the help of the enzyme, BioA. The enzyme dethiobiotin synthetase catalyzes the formation of the ureido ring via a DAPA carbamate activated with ATP, creating dethiobiotin with the help of the enzyme, BioD, which is then converted into biotin which is catalyzed by BioB. The last step is catalyzed by biotin synthase, a radical SAM enzyme. The sulfur is donated by an unusual [2Fe-2S] ferredoxin. Depending on the species of bacteria, Biotin can be synthesized via multiple pathways.

Cofactor biochemistry
The enzyme holocarboxylase synthetase covalently attaches biotin to five human carboxylase enzymes:
 * Acetyl-CoA carboxylase alpha (ACC1)
 * Acetyl-CoA carboxylase beta (ACC2)
 * Pyruvate carboxylase (PC)
 * Methylcrotonyl-CoA carboxylase (MCC)
 * Propionyl-CoA carboxylase (PCC)

For the first two, biotin serves as a cofactor responsible for transfer of bicarbonate to acetyl-CoA, converting it to malonyl-CoA for fatty acid synthesis. PC participates in gluconeogenesis. MCC catalyzes a step in leucine metabolism. PCC catalyzes a step in the metabolism of propionyl-CoA. Metabolic degradation of the biotinylated carboxylases leads to the formation of biocytin. This compound is further degraded by biotinidase to release biotin, which is then reutilized by holocarboxylase synthetase.

Biotinylation of histone proteins in nuclear chromatin is a posttranslational modification that plays a role in chromatin stability and gene expression.

Deficiency
Primary biotin deficiency, meaning deficiency as a consequence of too little biotin in the diet, is rare, because biotin is contained in so many foods. Subclinical deficiency can cause mild symptoms, such as hair thinning, brittle fingernails, or skin rash, typically on the face.

Aside from inadequate dietary intake (rare), deficiency of biotin can be caused by a genetic disorder that affects biotin metabolism. The most common among these is biotinidase deficiency. Low activity of this enzyme causes a failure to recycle biotin from biocytin. Rarer are carboxylase and biotin transporter deficiences. Neonatal screening for biotinidase deficiency started in the United States in 1984, with many countries now also testing for this genetic disorder at birth. Treatment is lifelong dietary supplement with biotin. If biotinidase deficiency goes untreated, it can be fatal.

Diagnosis
Low serum and urine biotin are not sensitive indicators of inadequate biotin intake. However, serum testing can be useful for confirmation of consumption of biotin-containing dietary supplements, and whether a period of refraining from supplement use is long enough to eliminate the potential for interfering with drug tests. Indirect measures depend on the biotin requirement for carboxylases. 3-Methylcrotonyl-CoA is an intermediate step in the catabolism of the amino acid leucine. In the absence of biotin, the pathway diverts to 3-hydroxyisovaleric acid. Urinary excretion of this compound is an early and sensitive indicator of biotin deficiency.

Deficiency as a result of metabolic disorders
Biotinidase deficiency is a deficiency of the enzyme that recycles biotin, the consequence of an inherited genetic mutation. Biotinidase catalyzes the cleavage of biotin from biocytin and biotinyl-peptides (the proteolytic degradation products of each holocarboxylase) and thereby recycles biotin. It is also important in freeing biotin from dietary protein-bound biotin. Neonatal screening for biotinidase deficiency started in the United States in 1984, which as of 2017 was reported as required in more than 30 countries.

Profound biotinidase deficiency, defined as less than 10% of normal serum enzyme activity, which has been reported as 7.1 nmol/min/mL, has an incidence of 1 in 40,000 to 1 in 60,000, but with rates as high as 1 in 10,000 in countries with high incidence of consanguineous marriages (second cousin or closer). Partial biotinidase deficiency is defined as 10% to 30% of normal serum activity. Incidence data stems from government mandated newborn screening. For profound deficiency, treatment is oral dosing with 5 to 20 mg per day. Seizures are reported as resolving in hours to days, with other symptoms resolving within weeks. Treatment of partial biotinidase deficiency is also recommended even though some untreated people never manifest symptoms. Lifelong treatment with supplemental biotin is recommended for both profound and partial biotinidase deficiency.

Inherited metabolic disorders characterized by deficient activities of biotin-dependent carboxylases are termed multiple carboxylase deficiency. These include deficiencies in the enzymes holocarboxylase synthetase. Holocarboxylase synthetase deficiency prevents the body's cells from using biotin effectively and thus interferes with multiple carboxylase reactions. There can also be a genetic defect affecting the sodium-dependent multivitamin transporter protein.

Biochemical and clinical manifestations of any of these metabolic disorders can include ketolactic acidosis, organic aciduria, hyperammonemia, rash, hypotonia, seizures, developmental delay, alopecia and coma.

Use in biotechnology
Chemically modified versions of biotin are widely used throughout the biotechnology industry to isolate proteins and non-protein compounds for biochemical assays. Because egg-derived avidin binds strongly to biotin with a dissociation constant Kd ≈ 10−15 M, biotinylated compounds of interest can be isolated from a sample by exploiting this highly stable interaction. First, the chemically modified biotin reagents are bound to the targeted compounds in a solution via a process called biotinylation. The choice of which chemical modification to use is responsible for the biotin reagent binding to a specific protein. Second, the sample is incubated with avidin bound to beads, then rinsed, removing all unbound proteins, while leaving only the biotinylated protein bound to avidin. Last, the biotinylated protein can be eluted from the beads with excess free biotin. The process can also utilize bacteria-derived streptavidin bound to beads, but because it has a higher dissociation constant than avidin, very harsh conditions are needed to elute the biotinylated protein from the beads, which often will denature the protein of interest.

Interference with medical laboratory results
When people are ingesting high levels of biotin in dietary supplements, a consequence can be clinically significant interference with diagnostic blood tests that use biotin-streptavidin technology. This methodology is commonly used to measure levels of hormones such as thyroid hormones, and other analytes such as 25-hydroxyvitamin D. Biotin interference can produce both falsely normal and falsely abnormal results. In the US, biotin as a non-prescription dietary supplement is sold in amounts of 1 to 10 mg per serving, with claims for supporting hair and nail health, and as 300 mg per day as a possibly effective treatment for multiple sclerosis (see § Research). Overconsumption of 5 mg/day or higher causes elevated concentration in plasma that interferes with biotin-streptavidin immunoassays in an unpredictable manner. Healthcare professionals are advised to instruct patients to stop taking biotin supplements for 48 h or even up to weeks before the test, depending on the specific test, dose, and frequency of biotin uptake. Guidance for laboratory staff is proposed to detect and manage biotin interference.

History
In 1916, W. G. Bateman observed that a diet high in raw egg whites caused toxic symptoms in dogs, cats, rabbits, and humans. By 1927, scientists such as Margarete Boas and Helen Parsons had performed experiments demonstrating the symptoms associated with "egg-white injury." They had found that rats fed large amounts of egg-white as their only protein source exhibited neurological dysfunction, hair loss, dermatitis, and eventually, death.

In 1936, Fritz Kögl and Benno Tönnis documented isolating a yeast growth factor in a journal article titled "Darstellung von krystallisiertem biotin aus eigelb." (Representation of crystallized biotin from egg yolk). The name biotin derives from the Greek word ('to live') and the suffix "-in" (a general chemical suffix used in organic chemistry). Other research groups, working independently, had isolated the same compound under different names. Hungarian scientist Paul Gyorgy began investigating the factor responsible for egg-white injury in 1933 and in 1939, was successful identifying what he called "Vitamin H" (the H represents Haar und Haut, German for 'hair and skin'). Further chemical characterization of vitamin H revealed that it was water-soluble and present in high amounts in the liver. After experiments performed with yeast and Rhizobium trifolii, West and Wilson isolated a compound they called co-enzyme R. By 1940, it was recognized that all three compounds were identical and were collectively given the name: biotin. Gyorgy continued his work on biotin and in 1941 published a paper demonstrating that egg-white injury was caused by the binding of biotin by avidin. Unlike for many vitamins, there is insufficient information to establish a recommended dietary allowance, so dietary guidelines identify an "adequate intake" based on best available science with the understanding that at some later date this will be replaced by more exact information.

Using E. coli, a biosynthesis pathway was proposed by Rolfe and Eisenberg in 1968. The initial step was described as a condensation of pimelyl-CoA and alanine to form 7-oxo-8-aminopelargonic acid. From there, they described three-step process, the last being introducing a sulfur atom to form the tetrahydrothiophene ring.

Multiple sclerosis
High-dose biotin (300 mg/day = 10,000 times adequate intake) has been used in clinical trials for treatment of multiple sclerosis, a demyelinating autoimmune disease. The hypothesis is that biotin may promote remyelination of the myelin sheath of nerve cells, slowing or even reversing neurodegeneration. The proposed mechanisms are that biotin activates acetyl-coA carboxylase, which is a key rate-limiting enzyme during the synthesis of myelin, and by reducing axonal hypoxia through enhanced energy production. Clinical trial results are mixed; a 2019 review concluded that a further investigation of the association between multiple sclerosis symptoms and biotin should be undertaken, whereas two 2020 reviews of a larger number of clinical trials reported no consistent evidence for benefits, and some evidence for increased disease activity and higher risk of relapse.

Hair, nails, skin
In the United States, biotin is promoted as a dietary supplement for strengthening hair and fingernails, though scientific data supporting these outcomes in humans are very weak. A review of the fingernails literature reported brittle nail improvement as evidence from two pre-1990 clinical trials that had administered an oral dietary supplement of 2.5 mg/day for several months, without a placebo control comparison group. There is no more recent clinical trial literature. A review of biotin as treatment for hair loss identified case studies of infants and young children with genetic defect biotin deficiency having improved hair growth after supplementation, but went on to report that "there have been no randomized, controlled trials to prove efficacy of supplementation with biotin in normal, healthy individuals." Biotin is also incorporated into topical hair and skin products with similar claims.

The Dietary Supplement Health and Education Act of 1994 states that the US Food and Drug Administration must allow on the product label what are described as "Structure:Function" (S:F) health claims that ingredient(s) are essential for health. For example: Biotin helps maintain healthy skin, hair and nails. If a S:F claim is made, the label must include the disclaimer "This statement has not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure, or prevent any disease."

Animals
In cattle, biotin is necessary for hoof health. Lameness due to hoof problems is common, with herd prevalence estimated at 10 to 35%. Consequences of lameness include less food consumption, lower milk production, and increased veterinary treatment costs. Results after 4–6 months from supplementing biotin at 20 mg/day into daily diet reduces the risk of lameness. A review of controlled trials reported that supplementation at 20 mg/day increased milk yield by 4.8%. The discussion speculated that this could be an indirect consequence of improved hoof health or a direct effect on milk production.

For horses, conditions such as chronic laminitis, cracked hooves, or dry, brittle feet incapable of holding shoes are a common problem. Biotin is a popular nutritional supplement. There are recommendations that horses need 15 to 25 mg/day. Studies report biotin improves the growth of new hoof horn rather than improving the status of existing hoof, so months of supplementation are needed for the hoof wall to be completely replaced.

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title: Biotin