Folinic acid

Folinic acid also known as Leucovorin, Levofolinic acid or 5-formyltetrahydrofolate (5-formylTHF) is a stable form of folate, which means it is similar to the vitamin Folate (vitamin B9). Folinic acid (Leucovorin) is used in combination with certain chemotherapy drugs (anti-cancer drugs) like 5-fluorouracil (5-FU) to enhance their ability to kill cancer cells or as antidote to lessen the toxic effects of certain drugs such as methotrexate that interfere with folate metabolism by inhibiting dihydrofolate reductase (DHFR) ¹, ². Dihydrofolate reductase (DHFR) is an enzyme that converts dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor ³. This process is essential for synthesizing purines and thymidylate, which are vital for DNA synthesis and other cellular processes. The human DHFR enzyme is encoded by the DHFR gene and is a key target for antifolate drugs like methotrexate, used to treat cancers and other diseases by inhibiting cell proliferation. Folinic acid (Leucovorin) is also used to treat folate-deficiency anemia ⁴.

Often, doctors use the terms “folic acid” and “folinic acid” interchangeably, but they are not the same ⁵, ⁶. Folic acid is a synthetic (man-made) oxidized version of the water soluble vitamin Folate or vitamin B9 that requires multiple enzymatic steps for activation (see Figure 4 below). Folate is a generic term referring to both natural “folates” found in many foods and particularly in leafy green vegetables and “folic acid“, the man-made oxidized folate that is used in dietary supplements and added to certain foods (fortified foods) ⁷, ⁸.

Other synthetic versions of folate include Folinic acid (Leucovorin) also known as 5-formyltetrahydrofolate (5-formylTHF) is a stable form of folate and Levomefolic acid also known as 5-methyltetrahydrofolate (5-MTHF) is a biologically active form of folate. Folic acid has no biological activity unless it is converted into folates ⁹. Folic acid is the most common form of Vitamin B9 and is a synthetic oxidized version of folate that must be metabolized by your body before it can be used (see Figure 4 below). Folic acid is converted by your body first to dihydrofolate (DHF), then tetrahydrofolate (THF) and finally to the biologically active form, levomefolic acid also known as 5-methyltetrahydrofolate (5-MTHF). Folinic acid, also known as leucovorin or 5-formyltetrahydrofolate (5-formylTHF), is a formyl derivative of tetrahydrofolate (THF). Folinic acid exists in two isomeric forms, and only the 6S-isomer [(6S)5-formylTHF also known as Isovorin (calcium levofolinate) or Fusilev (levoleucovorin calcium)] is converted to tetrahydrofolate (THF) and then levomefolic acid (5-MTHF). As a result, unless folinic acid is provided as purified 6S-isomer it does not provide a 1:1 molar equivalent source of folate relative to folic acid. Levomefolic acid also known as 5-methyltetrahydrofolate (5-MTHF) is the biologically active form of folate and the form found in your blood. Levomefolic acid (5-methyltetrahydrofolate [5-MTHF]) does not require enzymatic conversion and can be used directly by your body. The relationship between these forms of folate is summarized in Figure 4 below. Specifically, it demonstrates the point at which these ingredients enter the metabolic pathway.

Folate functions as a coenzyme (a molecule that binds to an enzyme and is essential for its activity but is not permanently altered by the reaction) or cosubstrate in single-carbon transfers in the synthesis of nucleic acids (DNA and RNA) and metabolism of amino acids ¹⁰, ¹¹, ¹².

• Nucleic acid metabolism. Folate coenzymes play a vital role in DNA metabolism through two different pathways: (1) The synthesis of DNA from its precursors (thymidine and purines) is dependent on folate coenzymes. (2) A folate coenzyme is required for the synthesis of methionine from homocysteine and the methionine is required for the synthesis of S-adenosylmethionine (SAM) ¹³. Another folate-dependent reaction, the methylation of deoxyuridylate to thymidylate in the formation of DNA, is required for proper cell division ⁷. S-adenosyl-methionine (SAMe) is a methyl group (-CH₃ or one-carbon unit) donor used in most biological methylation reactions, including the methylation of a number of sites within DNA, RNA, proteins, and phospholipids. The methylation of DNA plays a role in controlling gene expression and is critical during cell differentiation. Defects in DNA methylation have been linked to the development of cancer.
• Amino acid metabolism. Folate coenzymes are required for the metabolism of several important amino acids (the fundamental building blocks of proteins), namely methionine, cysteine, serine, glycine, and histidine. The synthesis of methionine from homocysteine is catalyzed by methionine synthase, an enzyme that requires not only folate as 5-methyltetrahydrofolate (5-MTHF) but also vitamin B12 (cobalamin). Therefore, folate and/or vitamin B12 deficiency can result in decreased synthesis of methionine and an accumulation of homocysteine. Elevated blood concentrations of homocysteine have been considered for many years to be a risk factor for some chronic diseases, including cardiovascular disease and dementia.

Isolated folate deficiency is uncommon ⁷. Folate deficiency usually coexists with other nutrient deficiencies because of its strong association with poor diet, alcoholism, and malabsorptive disorders ¹⁴. The primary clinical sign of folate deficiency or vitamin B12 deficiency is megaloblastic anemia or macrocytic anemia, which is characterized by large abnormally nucleated immature red blood cells ¹⁰, ¹⁴. Rapidly dividing cells like those derived from bone marrow are most vulnerable to the effects of folate deficiency since DNA synthesis and cell division are dependent on folate coenzymes. When folate supply to the rapidly dividing cells of the bone marrow is inadequate, blood cell division is reduced, resulting in fewer but larger red blood cells. Neutrophils, a type of white blood cell, become hypersegmented, a change that can be found by examining a blood sample microscopically. Because normal red blood cells have a lifetime in the circulation of approximately four months (120 days), it can take months for folate-deficient individuals to develop the characteristic megaloblastic anemia. Progression of megaloblastic anemia leads to a decreased oxygen carrying capacity of the blood and may ultimately result in symptoms of fatigue, weakness, and shortness of breath ⁹. Folate deficiency can also produce soreness in and shallow ulcerations on the tongue and oral mucosa; changes in skin, hair, or fingernail pigmentation; gastrointestinal symptoms; and elevated blood concentrations of homocysteine ¹⁰, ¹⁴, ¹⁵. Individuals in the early stages of folate deficiency may not show obvious symptoms, but blood concentrations of homocysteine may increase. Yet, the concentration of circulating homocysteine is not a specific indicator of folate status, as elevated homocysteine can be the result of vitamin B12 and other B-vitamin deficiencies, lifestyle factors, and kidney disease. Subclinical folate deficiency is typically detected by measurement of folate concentrations in serum/plasma or in red blood cells.

It is important to point out that megaloblastic anemia resulting from folate deficiency is identical to the megaloblastic anemia resulting from vitamin B12 deficiency, and further clinical testing is required to diagnose the true cause of megaloblastic anemia. Megaloblastic anemia symptoms include weakness, fatigue, difficulty concentrating, irritability, headache, heart palpitations, and shortness of breath ⁹, ¹⁴.

Women with insufficient folate intakes are at increased risk of giving birth to infants with neural tube defects that result in malformations of the spine (spina bifida), skull, and brain (anencephaly) ⁹. The prevalence rate of spina bifida and anencephaly (the two most common types of neural tube defects) in the United States is 5.5 to 6.5 per 10,000 births ¹⁶. Inadequate maternal folate status has also been associated with low infant birth weight, preterm delivery, and fetal growth retardation ¹⁰, ¹⁷.

Because of folate’s role in the synthesis of DNA and other critical cell components, folate is especially important during phases of rapid cell growth ¹⁸. Although the mechanism has not been fully established, clinical trial evidence shows that adequate periconceptional (the critical period of time from before conception to early pregnancy, generally encompassing a few weeks before and a few weeks after conception) folic acid consumption by women prevents a substantial proportion of neural tube defects ¹⁹, ²⁰, ²¹, ²², ²³.

Since 1998, when mandatory folic acid fortification began in the United States where vitamins, minerals, and other essential nutrients were added to food products during processing to improve its nutritional quality and provide a public health benefit, such as preventing or correcting nutrient deficiencies, neural tube defect rates have declined by 28% ¹⁶. However, significant racial and ethnic disparities persist. Neural tube defect prevalence rates are highest among Hispanic women and lowest among non-Hispanic black women. Factors that might contribute to these disparities include differences in dietary and supplement-taking practices ²⁴ as well as factors other than folate status such as maternal diabetes, obesity, and intake of other nutrients (e.g., vitamin B12), which are also believed to affect the risk of neural tube defects ²³, ²⁵, ²⁶, ²⁷. In addition, women with genetic mutation in 677C>T MTHFR gene, which is more common in Hispanics than Caucasians, Asians, and African Americans, might have an increased risk of neural tube defects ¹⁰, ¹², ²⁸, ²⁹. Another consideration is the fact that the data on neural tube defect prevalence rates were collected before 2016, when FDA approved the voluntary addition of folic acid to corn masa flour, an ingredient commonly consumed by Hispanic populations ³⁰. Whether this policy change has affected the disparities in neural tube defect rates between Hispanic women and other populations is not yet known.

During pregnancy, demands for folate increase because of its role in nucleic acid synthesis ¹⁷. To meet this need, the the Food and Nutrition Board at the National Academies of Sciences, Engineering, and Medicine increased the folate Recommended Dietary Allowance (RDA) from 400 micrograms (400 mcg) dietary folate equivalent (DFE)/day for nonpregnant women to 600 micrograms (600 mcg) dietary folate equivalent (DFE)/day during pregnancy ⁹. This level of folate intake might be difficult for some women to achieve through diet alone. The American College of Obstetricians and Gynecologists recommends a prenatal vitamin supplement for most pregnant women to ensure that they obtain adequate amounts of folic acid and other nutrients ³¹, ³².

All women of reproductive age (15–45 years) should take folic acid supplementation. For average-risk women, supplementation with 600 micrograms of folic acid (1000 mcg DFE) per day is adequate ³¹. Because it’s hard to get this much folic acid from food alone, you should take a daily prenatal vitamin with at least 600 micrograms (1000 mcg DFE) starting at least 1 month before pregnancy and during the first 12 weeks of pregnancy. Women at increased risk of neural tube defects, including women with a prior pregnancy with an neural tube defect or women with seizure disorders, should be counseled to take 4 milligrams (4 mg) of folic acid daily for at least 3 months before pregnancy and for the first 3 months of pregnancy ³³. Because of the risk of vitamin A toxicity, women who need additional folic acid should not take additional prenatal vitamins; instead, women at higher risk of neural tube defects should be prescribed additional folic acid supplements. Most prenatal multivitamins contain adequate amounts of folic acid for average-risk-women ³⁴. Prenatal vitamins use also is associated with a lower risk of miscarriage ³⁵. Moderate caffeine consumption (less than 200 mg per day) does not appear to be a major contributing factor in miscarriage or preterm birth 60.

Dietary Folate Equivalent (DFE) is defined as ⁷:

• 1 mcg dietary folate equivalent (DFE) = 1 mcg food folate
• 1 mcg dietary folate equivalent (DFE) = 0.6 mcg folic acid from fortified foods or dietary supplements consumed with foods (1,000 mcg folic acid from fortified foods or dietary supplements is equivalent to 1,667 mcg DFE because 0.6 mcg folic acid = 1 mcg DFE)
• 1 mcg dietary folate equivalent (DFE) = 0.5 mcg folic acid from dietary supplements taken on an empty stomach

Factors for converting mcg dietary folate equivalent (DFE) to mcg for supplemental folate in the form of 5-MTHF have not been formally established ⁷.

Food folates are in the potent tetrahydrofolate (THF) form and usually have additional glutamate residues, making them polyglutamates ¹⁰. Folic acid, the synthetic folate, is the fully oxidized monoglutamate form of folate (vitamin B9) that is used in fortified foods and most dietary supplements ⁷. Some dietary supplements also contain folate in the monoglutamyl form, 5-MTHF also known as L-5-MTHF, 5-methyl-folate, L-methylfolate, and methylfolate ⁷.

Heating during cooking destroys folic acid ³⁶. Folate is absorbed in the jejunum (the 2nd part of your small intestine) by active and passive transport mechanisms across the intestinal wall ³⁶. Folate or folic acid is a water-soluble type of vitamin B. This means it is not stored in the fat tissues of the body. Leftover amounts of the vitamin leave the body through the urine. The body has about 1,000-20,000 mcg of folate stores, and adults need about 400 mcg/day to replenish the daily losses. Folate deficiency may take 8-16 weeks to become evident ³⁶.

Folate is poorly stored, and folate deficiency can develop in weeks to months in persons with folate-deficient diets. Most of the serum folate is present in the inactive 5-methyltetrahydrofolate (5-methyl THF) form ³⁶. Upon entering cells, 5-methyltetrahydrofolate (5-methyl THF) demethylates to tetrahydrofolate (THF), the biologically active form of folate that is involved in folate-dependent enzymatic reactions. Cobalamin (vitamin B12) serves as a co-factor for this demethylation to occur, and in the absence of vitamin B12, folate is “trapped” inside cells as 5-methyltetrahydrofolate (5-methyl THF). Tetrahydrofolate (THF) is involved in the formation of many coenzymes in metabolic systems, particularly for purine and pyrimidine synthesis, nucleoprotein synthesis, and maintenance in red blood cells formation ³⁷. The deficiency of folate, as a result, leads to impairment of cell division, accumulation of toxic metabolites, and impartment of methylation reactions required for regulation of gene expression, resulting in megaloblastic anemia, which is characterized by large, abnormally nucleated red blood cells ¹⁴, ¹⁰.

The total body content of folate is estimated to be 10 to 30 mg; about half of this amount is stored in the liver and the remainder in blood and body tissues. A serum folate concentration is commonly used to assess folate status, with a value above 3 nanograms (ng)/mL indicating adequacy ³⁸. This indicator, however, is sensitive to recent dietary intake, so it might not reflect long-term status. Erythrocyte folate concentration provides a longer-term measure of folate intakes, so when day-to-day folate intakes are variable—such as in people who are ill and whose folate intake has recently declined—it might be a better indicator of tissue folate stores than serum folate concentration ³⁹. An erythrocyte folate concentration above 140 ng/mL indicates adequate folate status ³⁹, although some researchers have suggested that higher values are optimal for preventing neural tube defects ⁴⁰.

A combination of serum or red blood cell concentration and indicators of metabolic function can also be used to assess folate status. Plasma homocysteine concentration is a commonly used functional indicator of folate status because homocysteine levels rise when the body cannot convert homocysteine to methionine due to a 5-methyl-THF deficiency. Homocysteine levels, however, are not a highly specific indicator of folate status because they can be influenced by other factors, including kidney dysfunction and deficiencies of vitamin B12 and other micronutrients ⁴¹. The most commonly used cutoff value for elevated homocysteine is 16 micromoles/L, although slightly lower values of 12 to 14 micromoles/L have also been used ³⁹.

Folate-deficiency anemia is most common during pregnancy. Other causes of folate-deficiency anemia include alcoholism and certain medicines to treat seizures, anxiety, or arthritis.

The symptoms of folate-deficiency anemia include:

• Fatigue
• Headache
• Pale skin
• Sore mouth and tongue

The latest research reveals the following about folic acid deficiency ³⁶:

• There may be a link between elevated homocysteine (a marker for an increased risk for arteriosclerosis) and folate deficiency.
• A lowering of the risk of stroke but not adverse cardiac event when hyperhomocysteinemia is corrected with folic acid
• Reduction in the incidence of neural tube defects with folic acid supplementation during pregnancy.
• Lack of folic acid during pregnancy may increase the risk of diabetes-associated congenital disabilities and autism.
• Maternal folic acid during pregnancy may lower the risk of childhood leukemia.
• Folic acid supplementation may increase the risk of cancer.

If you have folate-deficiency anemia, your doctor may recommend taking folic acid vitamins and eating more foods with folate.

Folic acid is available in multivitamins and prenatal vitamins, supplements containing other B-complex vitamins, and supplements containing only folic acid. Common doses range from 680 to 1,360 mcg dietary folate equivalent (DFE) (400 to 800 mcg folic acid) in supplements for adults and 340 to 680 mcg DFE (200 to 400 mcg folic acid) in children’s multivitamins ⁴². About 85% of supplemental folic acid, when taken with food, is bioavailable ¹¹, ¹⁴. When consumed without food, nearly 100% of supplemental folic acid is bioavailable ⁷.

Figure 1. Folinic acid (Leucovorin)

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Figure 2. Folic acid

Footnotes: Folic acid (pteroylglutamic acid) is a complex chemical compound whose molecule contains three components: a pteridine derivative (2-amino-4-hydroxy-6-methylpteridine), p-aminobenzoic acid (PABA) and glutamic acid ⁴⁴.

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Figure 3. Folic acid biologically active derivatives

Footnotes: (A) dihydrofolate (DHF); (B) tetrahydrofolate (THF); (C) 5-methyltetrahydrofolate (5-methylTHF)

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Figure 4. Folic acid metabolism

Footnotes: This diagram shows the process by which folate and folic acid is used for DNA methylation (a biochemical reaction resulting in the addition of a methyl group [-CH₃] to another molecule). The methylenetetrahydrofolate reductase (MTHFR) 677C/T genetic mutation reduces enzyme activity ⁴⁶ and may help to divert the available methyl (-CH₃) groups from the DNA methylation pathway toward the DNA synthesis pathway ⁴⁷, ⁴⁸, ⁴⁹. The pathway is complex and highly regulated with feedback loops and interactions not shown in the schematic. Gene names for enzymes are in italics and cofactors (compounds that are essential for the activity of an enzyme) are in parentheses.

Abbreviations: DHF = dihydrofolate; DHFR = dihydrofolate reductase; DNMT = DNA methyltransferase; dTMP = thymidylate; dUMP = deoxyuridine monophosphate; MS = methionine synthase; MTHFR = methylenetetrahydrofolate reductase; SAH = S-adenosylhomocysteine; SAM = S-adenosylmethionine; SHMT = serine hydroxymethyltransferase; THF = tetrahydrofolate; TS = thymidylate synthase.

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Figure 5. Folate functions

Footnotes: The only function of folate coenzymes (a molecule that binds to an enzyme and is essential for its activity but is not permanently altered by the reaction) in the body appears to be in mediating the transfer of one-carbon (-CH₃ methyl group) units ⁵¹. Folate coenzymes act as acceptors and donors of one-carbon units in a variety of reactions critical to the metabolism of nucleic acids and amino acids ⁵².

[Source ⁵³ ]

Figure 6. Folate and methionine cycle

Footnote: Folate and methionine cycle. The Folate cycle begins with the conversion of dietary folate (vitamin B9) into dihydrofolate (DHF) then tetrahydrofolate (THF) by dihydrofolate reductase (DHFR). Next, tetrahydrofolate (THF) is converted to 5, 10-methylene-THF by serine hydroxymethyltransferase (SHMT) before being reduced into 5-methyl-THF (5-mTHF) by methylenetetrahydrofolate reductase (MTHFR). As part of the methionine cycle, 5-methyl-THF (5-mTHF) donates its carbon group to convert homocysteine (Hcy) to methionine (Met), which is catalyzed by methionine synthase (MS) and requires vitamin B12 as a cofactor, hence initiating Methionine cycle. In turn, methionine (Met) is used by methionine adenosyltransferase (MAT) to generate S-Adenosyl-Methionine (SAM) – the principal donor of methyl groups for DNA and proteins methylation. Thus, SAM is used by different methyltransferases, resulting in S-adenosylhomocysteine after its demethylation. Finally, S-adenosylhomocysteine hydrolase (SAHH) mediates deadenylation of S-adenosylhomocysteine to hcysteine, enclosing the methionine cycle. Homocysteine can be used by cystathionine synthase (CBS), which converts it to cystathionine. In turn, cystathionine is a substrate for cystathionine gamma-lyase (CTH), which uses it for synthesis of cysteine. Cysteine is required for the synthesis of proteins as well as for generation of taurine and glutathione, the latter is one of the critical molecules for redox homeostasis.

Abbreviations: Ado = adenosine; ATP = adeno-sine triphosphate; vitamin B6, vitamin B9, vitamin B12; Cbs = cystathionine beta synthase; DHF = dihydrofolate; DHFR = dihydrofolate reductase; Cys = cysteine; GSH = glutathione; Hcy = homocysteine; MAT = methionine adenosyltransferase; Met = methionine; 5–methylTHF = 5, 10–methyleneTHF; MTHFR = methylenetetrahydrofolate reductase or 5,10-methylene-trahydrofolate reductase; MS = methionine synthase, SAM = S-adenosyl-methionine; SAH = S-adenosylhomocysteine; SHMT = hydroxymethyltransferase; THF = tetrahydrofolate.

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Figure 7. Homocysteine metabolism

Footnote: Schematic representation of pathways of homocysteine metabolism. The metabolism of homocysteine, an intermediate in the metabolism of sulfur-containing amino acids, provides an example of the interrelationships among nutrients necessary for optimal physiological function and health. Healthy individuals utilize two different pathways to metabolize homocysteine (Figure 3). One pathway (methionine synthase) synthesizes methionine from homocysteine and is dependent on both folate and vitamin B12 as cofactors. The other pathway converts homocysteine to another amino acid, cysteine, and requires two vitamin B6-dependent enzymes. Thus, the concentration of homocysteine in the blood is regulated by three B-vitamins: folate (vitamin B9), vitamin B12 (cobalamin), and vitamin B6 (pyridoxine) ⁵⁵. In some individuals, riboflavin (vitamin B2) is also involved in the regulation of homocysteine concentrations.

Abbreviations: DHFR = dihydrofolate reductase; THF = tetrahydrofolate; SHMT = serinehydroxymethyltransferase; MTHF = methylenetetrahydrofolate; MTHFR = 5,10-methylene-THF reductase; ATP = adenosine triphosphate; MAT = methionine adenosyltransferase; ADP = adenosine diphosphate; SAM = S-adenosylmethionine; SAH = S-adenosylhomocysteine; BHMT = betaine-Hcy S-methyltransferase; CBS = cystathionine beta-synthase; CSE = cystathionase; GSH = glutathione; H2S = hydrogen sulphide

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What is the difference between Folinic acid and Folic acid?

Folic acid is a synthetic (man-made) oxidized version of the water soluble vitamin Folate (vitamin B9) or Folacin that requires multiple enzymatic steps for activation (see Figure 4 above). Folate is a generic term referring to both natural “folates” found in many foods and particularly in leafy green vegetables and “folic acid“, the man-made oxidized folate that is used in dietary supplements and added to certain foods (fortified foods) ⁷, ⁸. Other synthetic versions of folate include Folinic acid (Leucovorin) also known as 5-formyltetrahydrofolate (5-formylTHF) is a stable form of folate and Levomefolic acid also known as 5-methyltetrahydrofolate (5-MTHF) a biologically active form of folate. Folic acid has no biological activity unless it is converted into tetrahydrofolate (THF) ⁹.

Folic acid is the most common form of Vitamin B9 and is a synthetic oxidized version of folate that must be metabolized by your body before it can be used. Folic acid is converted by your body first to dihydrofolate (DHF), then tetrahydrofolate (THF) and finally to the biologically active form, 5-methyltetrahydrofolate (5-MTHF) also known as levomefolic acid.

Folinic acid, also known as leucovorin or 5-formyltetrahydrofolate (5-formylTHF), is a man-made molecule, a formyl derivative of tetrahydrofolate (THF) that is readily converted to 5-10 MTHF (5,10-methylenetetrahydrofolate) and 5-methyltetrahydrofolate (5-MTHF) a biologically active form of folate without requiring the action of the enzyme methylenetetrahydrofolate reductase (MTHFR) (see Figure 4 above).

Folinic acid exists in two isomeric forms, and only the 6S-isomer [(6S)5-formylTHF also known as Isovorin (calcium levofolinate) or Fusilev (levoleucovorin calcium)] is converted to tetrahydrofolate (THF) and then levomefolic acid (5-MTHF) the biologically active form of folate. As a result, unless folinic acid is provided as purified 6S-isomer [(6S)5-formylTHF also known as Isovorin (calcium levofolinate) or Fusilev (levoleucovorin calcium)] it does not provide a 1:1 molar equivalent source of folate relative to folic acid.

Levomefolic acid also known as 5-methyltetrahydrofolate (5-MTHF) is the biologically active form of folate and the form found in your blood. Levomefolic acid (5-methyltetrahydrofolate [5-MTHF]) does not require enzymatic conversion and can be used directly by your body. The relationship between these forms of folate is summarized in Figure 4 above. Specifically, it demonstrates the point at which these ingredients enter the metabolic pathway.

Your body needs folate to make DNA, other genetic material, several amino acids, as well as in methylation reactions (a biochemical reaction resulting in the addition of a methyl group [-CH₃] to another molecule). Folic acid and folate also help your body make healthy new red blood cells. Red blood cells carry oxygen to all the parts of your body. If your body does not make enough red blood cells, you can develop anemia. Anemia happens when your blood cannot carry enough oxygen to your body, which makes you pale, tired, or weak. Also, if you do not get enough folic acid, you could develop a type of anemia called folate-deficiency anemia ⁴.

Folinic acid mechanism of action

Folates (naturally occurring vitamin B9) and folic acid (synthetic folate) are not biologically active and must be converted into tetrahydrofolate (THF) through dihydrofolate reductase (DHFR). Folinic acid (Leucovorin or 5-formyltetrahydrofolate [5-formylTHF]) does not require dihydrofolate reductase (DHFR) for conversion into tetrahydrofolate (THF) ⁵. Tetrahydrofolate (THF) and its derivatives then participate in thymidylate and purine synthesis as they are essential in carrying out one-carbon transfer reactions in the body ⁵⁷. These reactions are essential in the generation of nucleic acids, the regulation of gene expression, and the overall stability of the genome ⁵⁷. Serine, methionine, histidine, and glycine metabolism also depend on such substrates and reactions. Therefore, folinic acid ultimately plays a crucial role in normal metabolism and gene regulation ⁵. Furthermore, folinic acid is essential for synthesizing methionine from homocysteine, as this is a methylation reaction ⁵.

When consumed, food folates are hydrolyzed to the monoglutamate form in the gut prior to absorption by active transport across the intestinal mucosa ³⁹, ¹⁰. Passive diffusion also occurs when pharmacological doses of folic acid (the man made folate) are consumed ³⁹. Before entering the bloodstream, the monoglutamate form is reduced to tetrahydrofolate (THF) and converted to either methyl or formyl forms. The main form of folate in plasma is 5-methyl-THF (5-MTHF). Folic acid can also be found in the blood unaltered known as unmetabolized folic acid, but whether this form has any biological activity or can be used as a biomarker of status is not known ⁵⁸.

Some dietary supplements also contain folate in the monoglutamyl form, 5-methylenetetrahydrofolate or 5-methyl-folate (5-MTHF), also known as L-5-MTHF, L-methylfolate, and methylfolate ⁷. For some people with an methylenetetrahydrofolate reductase polymorphism (MTHFR polymorphism), supplementation with 5-methylenetetrahydrofolate (5-MTHF) might be more beneficial than with folic acid ⁵, ⁵⁹. The bioavailability of 5-methylenetetrahydrofolate (5-MTHF) in supplements is the same as or greater than that of folic acid ⁶⁰, ⁶¹, ⁶², ²⁸. However, conversion factors between mcg and mcg dietary folate equivalent (DFE) for 5-MTHF (5-methyl-folate) have not been formally established.

Folate and its coenzymes require transporters to cross cell membranes. Folate transporters include the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT), and the folate receptor proteins, folate receptor alpha (FRα) and folate receptor beta (FRβ). Folate homeostasis is supported by the ubiquitous distribution of folate transporters, although abundance and importance vary among tissues ⁶³. Proton-coupled folate transporter (PCFT) plays a major role in folate intestinal transport since mutations affecting the SLC46A1 gene encoding PCFT cause hereditary folate malabsorption. Defective PCFT (proton-coupled folate transporter) also leads to impaired folate transport into the brain. Folate receptor alpha (FRα) and reduced folate carrier (RFC) are also critical for folate transport across the blood-brain barrier when extracellular folate is either low or high, respectively. Folate is essential for the proper development of the embryo and the fetus. The placenta is known to concentrate folate to the fetal circulation, leading to higher folate concentrations in the fetus compared to those found in the pregnant woman. All three types of receptors have been associated with folate transport across the placenta during pregnancy ⁶⁴.

Pharmacokinetics

• Absorption: Folinic acid is rapidly and almost completely absorbed following oral administration. Bioavailability is dose-dependent ⁶⁵.
• Distribution: IV folinic acid administration results in a greater volume of distribution than oral, with oral dosing showing a lower maximum blood concentration primarily due to first-pass metabolism ⁶⁵.
• Metabolism: Folinic acid is metabolized in the liver and gastrointestinal tract and has an active metabolite. The average half-life is approximately 6 hours.
• Elimination: Excretion is 80% to 90% in urine and 5% to 8% in the feces.

Folinic acid uses

Indications for folate supplementation include preventing several diseases, such as ulcerative colitis, neural tube defects, and cognitive dysfunction in older patients ⁵. Doctors consider folinic acid or leucovorin (5-formyltetrahydrofolate [5-formylTHF]) supplementation superior to folic acid supplementation because folinic acid can reach higher concentrations in plasma and function in the face of defective folate metabolism ⁵, ⁶⁶.

The 2 main roles of folinic acid in clinical practice are to counteract the effects of folic acid antagonists (antifolates) and enhance the impact of fluoropyrimidines ⁶⁵. The former function is possible because folinic acid can enter cells through the reduced folate carrier and subsequently be converted to tetrahydrofolate despite the presence of methotrexate, thereby “rescuing” these cells from methotrexate toxicity ⁶⁷.

Most folic acid antagonists (antifolates) share a similar mechanism of action that includes the inhibition of dihydrofolate reductase (DHFR), the enzyme responsible for generating the functional tetrahydrofolate molecule. Folinic acid does not require dihydrofolate reductase (DHFR) to convert into its active derivatives. In this setting, folinic acid is an antidote that rescues these cells from the chemotherapeutic toxicities of folate antagonists such as methotrexate ⁵⁷.

While folinic acid acts as antidote to the adverse effects of methotrexate, folinic acid also functions to enhance the anti-cancer effects of the drug fluorouracil (5FU). In the cell, fluorouracil (5FU) converts to fluoro-deoxy uridylic acid, a molecule that inhibits thymidylate synthase. Thymidylate synthase is an enzyme that is important in DNA repair and replication. The functional derivative of folinic acid, 5-10 MTHF (5,10-methylenetetrahydrofolate), stabilizes the bound fluoro-deoxy uridylic acid to thymidylate synthase ². This interaction yields a ternary complex known as the thymidylate synthase 5-fluorodeoxyuridine monophosphate-methylenetetrahydrofolate complex, which inhibits thymidylate synthase ². Increased cellular amounts of folinic acid derivatives lead to increased stability of the aforementioned inhibitory complex, which leads to a depletion of thymidylate synthesis and disrupts DNA synthesis and repair ⁶⁵, ⁶⁶.

Folinic Acid FDA-Approved Indications

• Folinic acid enhances the cytotoxic effects and effectiveness of the cancer drug fluorouracil (5FU) in the cell by stabilizing its binding to its target enzyme ⁶⁸. This finding has revolutionized the treatment of several cancers, most notably colorectal cancer, which, according to the American Cancer Society, represents the third leading cause of cancer-related death in both sexes ⁶⁹. The combination of folinic acid with fluorouracil (5FU) is an FDA-approved indication for the palliative treatment of colorectal cancer. Specifically, patients with advanced, nonresectable adenocarcinoma of the colon who are receiving treatment with folinic acid and 5-FU display more prolonged progression-free survival and response rates ⁶⁸. High-dose folinic acid + fluorouracil (5FU) regimens can also be used as adjuvant therapy for resectable colorectal cancer. Folinic acid aids in producing a significant increase in disease-free survival compared to patients who receive treatment with surgical resection alone ⁷⁰
• Folinic acid (leucovorin) is helpful as an antidote to folic acid antagonists or antifolates (ie, methotrexate, trimethoprim [an antibiotic] and pyrimethamine [an antimalarial drug]), which are class of drugs that inhibit the use of folic acid (folate) by cells to make DNA, particularly by blocking the enzyme dihydrofolate reductase (DHFR) and may kill cancer cells ⁷¹, ⁶⁷. Frequently referred to as “leucovorin rescue” or “folinic acid rescue therapy” relates to folinic acid administration with the chemotherapy drug methotrexate. Methotrexate works by inhibiting dihydrofolate reductase (DHFR), shutting down folate metabolism and killing fast-dividing cancer cells. Giving folinic acid bypasses this blockade, “rescuing” healthy cells from the toxic effects of methotrexate therapy and reducing side effects. Methotrexate has a variety of indications in the treatment of various cancers and autoimmune disorders. Therefore, all conditions requiring methotrexate therapy, especially in high dosages, benefit from folinic acid as rescue therapy ⁶⁷. As an antidote, folinic acid limits bone marrow suppression, gastrointestinal toxicity, kidney toxicity, and neurotoxicity that can result secondary to high dosages of methotrexate and other folic acid antagonists ⁶⁷. Study results show that administering folinic acid after weekly doses of methotrexate reduces the incidence of transaminitis (an elevated level of liver enzymes called transaminases (specifically AST and ALT in the blood, indicating liver injury or inflammation), gastrointestinal complications, and stomatitis (oral ulcers) ⁷². Folinic acid supplementation during methotrexate therapy is so essential that chemotherapy protocols using methotrexate also include detailed recommendations regarding folinic acid rescue administration ⁶⁷. For reducing toxicity and counteracting the effects of high-dose methotrexate, folinic acid is FDA-approved in both adults and children.
• The intravenous formulation of folinic acid (leucovorin calcium) is also FDA-approved for use in adults and children to treat megaloblastic anemia in patients who have normal vitamin B12 levels and in whom oral therapy is not possible ⁷³.
• Folinic acid is also approved to treat folate-deficiency-associated megaloblastic anemia.

Off-Label Non-FDA-approved uses for folinic acid

• Treating Breast Cancer: Similar to its use in colorectal cancer, folinic acid has also been shown to potentiate the effects of fluorouracil (5FU) in breast cancer. This regimen is currently non-FDA approved, partly because there is insufficient evidence that folinic acid can increase the therapeutic index of fluorouracil (5FU) in breast cancer and because many such clinical trials are ongoing ².
• Regimens including folinic acid and fluorouracil (5FU) also have non-FDA-approved indications for treating unresectable/advanced gallbladder and biliary tree carcinoma, gastric cancer, squamous cell carcinoma of the head and neck, and resectable pancreatic cancer ⁷⁴, ⁷⁵, ⁷⁶, ⁷⁷.
• Combination chemotherapy regimens that include folinic acid have been effective in various non-Hodgkin lymphomas. Methotrexate and folinic acid rescue, combined with other chemotherapy agents (ie, doxorubicin, cyclophosphamide), have shown a complete response rate of 84% in treating diffuse large cell lymphoma ⁷⁸.
• For patients who require prophylactic therapy for toxoplasmosis and who are unable to tolerate sulfamethoxazole/trimethoprim, folinic acid is recommended in combination with clindamycin and pyrimethamine as an alternative. Folinic acid, in conjunction with pyrimethamine and sulfadoxine, is another option ⁷⁹.
• For patients requiring prophylactic treatment for Pneumocystis jirovecii pneumonia and who cannot tolerate sulfamethoxazole/trimethoprim, an alternative therapy can be used that includes folinic acid in combination with pyrimethamine and dapsone, among other combinations ⁸⁰.
• Management of Mitochondrial Disorders: Folinic acid is used in some cases to support cellular energy production pathways.
• Specialized Supplementation: Folinic acid is increasingly being used in supplements targeting individuals with the MTHFR gene mutation and in Cerebral Folate Deficiency and Autism Spectrum Disorder, especially in those patients that are positive for folate receptor autoantibodies.
• Serum plasma levels of homocysteine and folate derivatives have an inverse relationship. In particular, the methionine cycle, which uses homocysteine as a substrate, is sensitive to folate deficiency. Therefore, plasma homocysteine levels are markedly increased when cells have functional depletion of folinic acid ⁵. In patients with hyperhomocysteinemia, folinic acid has been found to reduce the plasma level of homocysteine, particularly in patients on hemodialysis ⁸¹. This finding suggests that folinic acid intake may reduce the risk of cardiovascular disease. Furthermore, folinic acid levels can be an indirect indicator of homocysteine levels ⁵.
• Patients with repeated nitrous oxide exposure or requiring treatment with long-term nitrous oxide may benefit from folinic acid prophylaxis to prevent bone marrow suppression ⁸².

Folinic acid in Pregnancy

Everyone needs folic acid to be healthy. But folic acid is especially important for women of reproductive age (15–45 years):

Before and during pregnancy, folic acid protects your unborn child against serious birth defects called neural tube defects that result in malformations of the spine (spina bifida), skull, and brain (anencephaly) ⁹. The prevalence rate of spina bifida and anencephaly (the two most common types of neural tube defects) in the United States is 5.5 to 6.5 per 10,000 births ¹⁶. Inadequate maternal folate status has also been associated with low infant birth weight, preterm delivery, and fetal growth retardation ¹⁰, ¹⁷. These birth defects happen in the first few weeks of pregnancy, often before a woman knows she is pregnant. Folic acid might also help prevent other types of birth defects and early pregnancy loss (miscarriage). Since about half of all pregnancies in the United States are unplanned ⁸³, experts recommend all women get enough folic acid via supplements even if you are not trying to get pregnant. For average-risk women, supplementation with 600 micrograms (600 mcg) of folic acid per day is adequate ³¹. Because it’s hard to get this much folic acid from food alone, you should take a daily prenatal vitamin with at least 600 micrograms (600 mcg) folic acid starting at least 1 month before pregnancy and during the first 12 weeks of pregnancy. Women at increased risk of neural tube defects, including women with a prior pregnancy with an neural tube defect or women with seizure disorders, should be counseled to take 4 milligrams (4 mg) of folic acid daily for at least 3 months before pregnancy and for the first 3 months of pregnancy ³³. Because of the risk of vitamin A toxicity, women who need additional folic acid should not take additional prenatal vitamins; instead, women at higher risk of neural tube defects should be prescribed additional folic acid supplements. Most prenatal multivitamins contain adequate amounts of folic acid for average-risk-women ³⁴. Prenatal vitamins use also is associated with a lower risk of miscarriage ³⁵. Moderate caffeine consumption (less than 200 mg per day) does not appear to be a major contributing factor in miscarriage or preterm birth 60.

Not getting enough folic acid can lead to a type of anemia called folate-deficiency anemia. Folate-deficiency anemia is more common in women of childbearing age than in men.

Preterm birth, congenital heart defects, and other congenital anomalies

According to observational studies, folic acid supplementation might increase mean gestational age and lower the risk of preterm birth ¹⁰, ⁸⁴. In addition, folic acid in combination with a multivitamin supplement helps minimize the risk of congenital heart defects, possibly because heart tissue development depends on cells that require large amounts of folate ¹⁰, ⁸⁵.

The authors of a large population-based cohort study of about 98% of all births in Canada from 1990 to 2011 concluded that folic acid fortification of foods was associated with an 11% reduction in the rate of nonchromosomal congenital heart defects ⁸⁶. In a population-based case-control study in Atlanta involving 3,987 infants, congenital heart defects were 24% less common in the infants of women who took multivitamins containing folic acid during the periconceptional period than in the infants of women who did not ⁸⁷. A case-control study in California in 866 infants had similar results ⁸⁸. However, it is not possible to determine whether the findings from these studies could be attributed to components of multivitamins other than folic acid.

Studies have also found associations between the use of folic acid in combination with multivitamin supplements and reduced occurrence at birth of urinary tract anomalies, oral facial clefts, limb defects, and hydrocephalus, but the results of these studies have been inconsistent ⁸⁵.

Additional research is needed to fully understand the extent to which maternal consumption of folic acid might affect the risk of these adverse birth outcomes. However, folic acid’s established role in preventing neural tube defects and possibly other birth defects underscores its importance during the periconceptional period.

Metabolic diseases

Folinic acid (leucovorin), a tetrahydrofolic acid derivative, is used in the clinical management of rare inborn errors that affect folate transport or metabolism ⁸⁹. Such conditions are of autosomal recessive inheritance, meaning only individuals receiving two copies of the mutated gene (one from each parent) develop the disease. Folinic acid can be administered orally, intravenously, or intramuscularly.

Hereditary folate malabsorption

Hereditary folate malabsorption is caused by mutations in the SLC46A1 gene that provides instructions for making a protein called the proton-coupled folate transporter (PCFT) ⁹⁰, ⁹¹. The proton-coupled folate transporter (PCFT) protein is found within the membrane of cells, where it helps transport folates into the cell. PCFT (proton-coupled folate transporter) is primarily found in cells that line the walls of the small intestine. These cells have fingerlike projections called microvilli that absorb nutrients from food as it passes through the intestine. Based on their appearance, groups of these microvilli are known collectively as the brush border. PCFT (proton-coupled folate transporter) is involved in transporting folates from food across the brush border membrane so they can be used by the body ⁹². PCFT (proton-coupled folate transporter) is also found in the brain, where it is involved in the transport of folates between the brain and the surrounding fluid (cerebrospinal fluid) ⁹². Patients with hereditary folate malabsorption due to SLC46A1 gene mutations present with low to undetectable concentrations of folate in their serum and cerebrospinal fluid (CSF); anemia (often macrocytic), low platelets count (thrombocytopenia), and/or low number of all blood cells (pancytopenia); impaired immune responses that increase susceptibility to infections; and a general failure to thrive ⁹³, ⁹⁰. Neurologic symptoms, including developmental delays, cognitive disorders, and seizures, have also been observed ⁹⁴, ⁹⁰. Early treatment with intramuscular or high-dose oral folinic acid (leucovorin or 5-formyltetrahydrofolate [5-formylTHF]) or, preferably, the active isomer of 5-formyltetrahydrofolate (calcium levofolinate [Isovorin] or levoleucovorin calcium [Fusilev]) readily corrects the systemic folate deficiency and, if the dose is sufficient, can achieve cerebrospinal fluid (CSF) folate levels that prevent or mitigate the neurologic consequences of hereditary folate malabsorption ⁹⁰, ⁹⁵, ⁹⁶. The active, physiologic, isomer of 5-formyltetrahydrofolate [5-formylTHF] is (6S)5-formylTHF also known as Isovorin (calcium levofolinate) or Fusilev (levoleucovorin calcium); it is available for intramuscular administration. The biologic impact of the active isomer is twice that of folinic acid (leucovorin or 5-formyltetrahydrofolate [5-formylTHF]) when the dose is the same ⁹⁰. Isovorin (calcium levofolinate) or Fusilev (levoleucovorin calcium) is the preferred form of folate for treating hereditary folate malabsorption, if it is available and cost is not a limiting factor. The active isomer should be utilized especially when there is refractory neurologic disease. Complete reversal of the systemic consequences of folate deficiency is easily achieved. While correction of the neurologic consequences is more difficult, favorable neurologic outcomes are possible when adequate treatment is initiated promptly after birth ⁹⁷. Severe neurologic and cognitive complications, including seizures, are invariably due to delay in diagnosis and implementation of treatment to achieve adequate cerebrospinal fluid (CSF) folate levels.

Dosing is aimed at achieving CSF folate trough concentrations as close as possible to the normal range for the age of the affected individual (infants and children have higher CSF folate levels than adults) ⁹⁰. Because hereditary folate malabsorption is rare, controlled studies to establish optimal treatment have not been possible. The oral dose of folinic acid (leucovorin or 5-formyltetrahydrofolate [5-formylTHF]) required to overcome the loss of the proton-coupled folate transporter (PCFT)-mediated intestinal folate absorption appears to vary among individuals ⁹⁰. The dose required to obviate the neurologic consequences is much higher than that needed to correct the systemic folate deficiency. The dose should be guided by its effect on trough CSF folate concentrations. The endpoint is CSF folate concentrations as close as possible to the normal range for the affected individual’s age.

The reported oral dose of folinic acid (leucovorin or 5-formyltetrahydrofolate [5-formylTHF]) associated with a “good” outcome is approximately 150-200 mg daily ⁹⁸. Much higher doses have been used as well ⁹⁰. A reasonable starting oral dose of folinic acid (leucovorin or 5-formyltetrahydrofolate [5-formylTHF]) in an infant could be 50 mg or 10-15 mg/kg given daily as a single dose, with subsequent dosing dependent on correction of the systemic signs of the disorder and achievement of an adequate cerebrospinal fluid (CSF) folate level ⁹⁰.

Note: Normal CSF folate is ~100 nmol/L for infants to age two years, decreasing to ~75 nmol/L by age five years and to ~65 nmol/L by age 19 years ⁹⁹.

The intramuscular dose required to achieve adequate serum and cerebrospinal fluid (CSF) folate levels is much lower than the oral dose. With intramuscular injections of approximately 1 mg/day of folinic acid (leucovorin or 5-formyltetrahydrofolate [5-formylTHF]), the anemia, immunologic, and gastrointestinal manifestations will fully resolve. However, the endpoint for treatment is based on achieving an adequate cerebrospinal fluid (CSF) folate level to mitigate the neurologic consequences of hereditary folate malabsorption, which will require much higher folate doses. It would appear that the maximum achievable cerebrospinal fluid (CSF) folate levels are in the range of 40-50 nmol/L ¹⁰⁰, ⁹⁷, ¹⁰¹, ¹⁰². For instance, with a folinic acid (leucovorin or 5-formyltetrahydrofolate [5-formylTHF]) oral dose of 28 mg/kg/day versus a parenteral dose of 2 mg/kg/day (both racemic), the CSF folate levels were 16 nmol/L and 43 nmol/L, while the blood levels were ~800 nmol/L and ~2,000 nmol/L, respectively ¹⁰¹, ⁹⁵.

Cerebral Folate Deficiency (CFD)

Cerebral Folate Deficiency (CFD) also called cerebral folate transport deficiency or neurodegeneration due to cerebral folate transport deficiency is a neurological syndrome in which development is usually normal in the first year of life, but at approximately 2 years of age, affected children start to lose mental and motor skills (psychomotor regression) ¹⁰³, ¹⁰⁴, ¹⁰⁵, ¹⁰⁶, ¹⁰⁷, ¹⁰⁸. Mutations in the FOLR1 gene cause cerebral folate transport deficiency. The FOLR1 gene provides instructions for making a protein called folate receptor alpha (FRA). The folate receptor alpha (FRA) protein is found within the cell membrane where it attaches (binds) to folate, allowing folate (vitamin B9) to be brought into the cell. Folate receptor alpha (FRA) is produced in largest amounts in the brain, specifically in an area of the brain called the choroid plexus. This region releases cerebrospinal fluid (CSF), which surrounds and protects the brain and spinal cord. Folate receptor alpha (FRA) is thought to play a major role in bringing folate from the bloodstream into brain cells. It transports folate across the choroid plexus and into the CSF (cerebrospinal fluid), ultimately reaching the brain. In the brain, folate is needed for making myelin and chemical messengers called neurotransmitters. Both of these substances play essential roles in transmitting signals in the nervous system. Additionally, folate is involved in the production and repair of DNA, regulation of gene activity (expression), and protein production.

FOLR1 gene mutations result in a lack of folate receptor alpha (FRA) protein or malfunctioning of folate receptor alpha (FRA) protein. As a result, folate from the bloodstream cannot be transported into the cerebrospinal fluid (CSF). Without folate, many processes in the brain are impaired, leading to the neurological problems typical of cerebral folate transport deficiency.

Even though there may be normal folate levels in the serum and red blood cells, evaluation of the cerebrospinal fluid (CSF) shows a decreased level of 5-methyltetrahydrofolate (5MTHF) ¹⁰⁷. The brain may appear normal on an MRI, but in some affected children, a loss of white matter in the brain (leukodystrophy) may be seen ¹⁰⁷. Frontotemporal atrophy and impairment of the protective layer that surrounds nerve fibers in the brain and spinal cord (subcortical demyelination) can be seen as early as 18 months ¹⁰⁷.

The symptoms of cerebral folate deficiency may begin as early as four to six months of age with irritability and sleep problems (insomnia) ¹⁰⁷. Delays in development may be noted including slow head growth, low muscle tone (hypotonia), ataxia, loss of voluntary movement (dyskinesia), constant contracted muscles (spasticity), speech complications, and epilepsy ¹⁰⁷. Additional signs may involve visual disturbances, hearing loss and autistic features ¹⁰⁷.

Some early symptoms of cerebral folate deficiency are intellectual disability, speech difficulties, and development of recurrent seizures in a third of affected children. Motor issues such as tremors and lack of muscle control or coordination of voluntary movements (ataxia) can become severe.

Neurological abnormalities, along with visual and hearing impairments, have been described in children with cerebral folate deficiency. Autism spectrum disorder (ASD) is present in some cases. Cerebral folate deficiency has also been described in adults presenting with neurological symptoms ¹⁰⁹.

Cerebral folate deficiency condition can be treated with folinic acid (leucovorin calcium). Oral treatment with folinic acid (leucovorin) has been shown to improve symptoms and stabilize the level of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid (CSF). Folinic acid can enter the brain and normalize the level of folate coenzymes and has been shown to normalize folate concentrations and improve various social interactions in cerebral folate deficiency condition, including mood, behavior, and verbal communication in children with autism spectrum disorder (ASD) ¹⁰³, ¹¹⁰, ¹¹¹.

The overall outcome of cerebral folate deficiency seems to depend on the age at which treatment is initiated, the earlier the treatment, the better outcome. Supplementation with folic acid is not recommended because it is associated with adverse effects such as producing epileptic seizures. No serious adverse effects have been recorded during leucovorin calcium treatment. A milk-free diet in combination with leucovorin has been reported to improve symptoms, especially when used in the early stages of the disease.

Dihydrofolate reductase (DHFR) deficiency

Dihydrofolate reductase (DHFR) is the NADPH-dependent enzyme that catalyzes the conversion of dihydrofolate (DHF) to tetrahydrofolate (THF), an essential step in the synthesis of precursors of DNA, including glycine and purines, and the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) (see Figure 4 above) ¹¹², ¹¹³. Dihydrofolate reductase (DHFR) is also required to convert folic acid (a synthetic folate [vitamin B9] not found in nature) to dihydrofolate (DHF). Dihydrofolate reductase (DHFR) deficiency is characterized by megaloblastic anemia and cerebral folate deficiency causing intractable seizures and mental deficits ¹¹⁴, ¹¹⁵, ¹¹⁶, ¹¹⁶, ¹¹⁷, ¹¹⁸. Although folinic acid treatment can alleviate the symptoms of DHFR deficiency, early diagnosis is essential to prevent irreversible brain damage and improve clinical outcomes ¹¹⁴, ¹¹⁵.

Autism spectrum disorder

Autism spectrum disorder (ASD) also known as autism, is a lifelong neurodevelopmental disorder that affects how you behave and interact with the world ¹¹⁹, ¹²⁰, ¹²¹, ¹²². People with autism have difficulty with communication and interacting with other people as well as repetitive behaviors and limited interests. The classification and diagnosis of autism spectrum disorder (ASD) was changed in 2013 to include conditions previously known as autistic disorder, Asperger’s syndrome, and pervasive developmental disorder not otherwise specified ¹²³. However, The term ‘Asperger’s syndrome’ is no longer used. Health professionals used to think of autism as a condition that ranged from high functioning to severe. People who were diagnosed with Asperger’s syndrome would now be diagnosed with autism spectrum disorder. “Spectrum” means that autism affects people differently. There is a wide range of symptoms and levels of support needed by people with autism. People with autism will all have different experiences and need different levels of support. Along with some challenges, a person with autism will also have a range of strengths. People with autism are “neurodivergent”. This means their brains work in ways that are different from what is ‘typical’ or common, but it is not necessarily a problem. The term neurodivergent also describes people with many other conditions, such as dyslexia and attention deficit hyperactivity disorder (ADHD).

The main symptoms related to autism fall into 2 broad areas:

• difficulty with social interactions and communication
• limited and repetitive behaviours and interests

The features of autism usually start in infancy, but they may not be noticeable until the age of 18 months or older. Sometimes autism is only noticed much later in life.

The causes of autism spectrum disorder are not clear, but genetic and environmental factors (including infections) and prenatal exposure to certain drugs, pollutants, and pesticides are believed to play a role ¹²⁴, ¹²⁵, ¹²⁶, ¹²⁷, ¹²⁸, ¹²⁹, ¹³⁰, ¹³¹, ¹³², ¹³³.

Emerging evidence suggests that folic acid supplementation before or around the time of conception might reduce the risk of autism spectrum disorder or reduce the potentially increased risk of autism spectrum disorder from prenatal exposure to certain drugs and toxic chemicals. The mechanism of these potential benefits is unknown, but it might be related to folic acid’s role in DNA methylation, which, in turn, can affect neurodevelopment ¹³⁴, ¹³⁵, ¹³⁶.

Some, but not all, observational studies have shown associations between maternal use of folic acid and/or multivitamin supplements before and/or during pregnancy and lower risk of autism spectrum disorder in the women’s offspring. For example, the prospective Norwegian Mother and Child Cohort Study that included 85,176 children age 3.3 to 10.2 years found that children of mothers who took up to 400 mcg per day folic acid during all or part of the time from 4 weeks before to 8 weeks after the start of pregnancy were 39% less likely to have autistic disorder than those whose mothers did not take the supplements ¹³⁷. The results showed no significant associations, however, between folic acid supplementation and Asperger’s syndrome or pervasive developmental disorder not otherwise specified. In a U.S. population-based, case-control study of 837 children, those born to mothers who consumed a mean of 600 mcg folic acid per day or more from supplements and fortified breakfast cereals during the first month of pregnancy had a 38% lower risk of autism spectrum disorder than those of mothers who consumed less than 600 mcg per day ¹³⁸. This association was strongest for mothers and children with the genetic mutation involving 677C>T MTHFR gene (a common genetic variant where a change occurs in the MTHFR (methylenetetrahydrofolate reductase) gene, which produces an enzyme critical for processing folate and homocysteine). Similarly, a 2018 case-control cohort study of 45,300 Israeli children demonstrated a significantly decreased risk of autism spectrum disorder in children of mothers who took folic acid and/or multivitamin supplements before and/or during pregnancy ¹³⁹. In contrast, a longitudinal, population-based cohort of 35,059 pregnant Danish women and their children found no association between periconceptional folic acid or multivitamin use and autism spectrum disorder ¹⁴⁰.

Periconceptional use of folic acid might reduce the potentially increased risk of autism spectrum disorder in children exposed to certain drugs and neurotoxins in utero ¹³¹, ¹³², ¹³³. An analysis of data from the Norwegian Mother and Child Cohort Study, which included 104,946 children, found that children exposed to antiepileptic drugs (known to reduce folate in the body) in utero were 5.9 to 7.9 times more likely to have autistic traits at age 18 and 36 months if their mothers did not take folic acid periconceptionally than if they did ¹³¹. In addition, the severity of autistic traits was inversely associated with both maternal plasma folate concentrations and folic acid doses. Similarly, in a U.S. study of 712 children, mothers exposed to any indoor pesticide during pregnancy who had folic acid intakes of 800 mcg or more per day during the first month of pregnancy were 1.7 times more likely to have a child with autism spectrum disorder than women with the same folic acid intakes who were not exposed to indoor pesticides ¹³². The risk of autism spectrum disorder was even higher (2.5 times) if the women were exposed to indoor pesticides and had daily folic acid intakes of less than 800 mcg, suggesting that folic acid might attenuate the potentially increased risk of autism spectrum disorder from pesticide exposure.

Overall, the evidence to date suggests a possible inverse association between mothers’ periconceptional folic acid intakes and risk of autism spectrum disorder in their offspring. However, most, if not all, of the currently available data are observational, and confounding weakens the ability to demonstrate causal inference. Additional research and validation in other studies are needed before firm conclusions can be drawn.

Cancer

Several epidemiological studies have suggested an inverse association between folate intakes and status and the risk of colorectal, lung, pancreatic, esophageal, stomach, cervical, ovarian, breast, bladder, and other cancers ¹⁰, ¹⁴¹, ¹⁴², ¹⁴³. Research has not established the precise nature of folate’s effect on carcinogenesis, but scientists hypothesize that folate might influence cancer development through its role in one-carbon metabolism and subsequent effects on DNA replication and cell division ¹⁴³, ¹⁴⁴. Evidence also indicates that folate might play a dual role in cancer initiation and progression ¹⁴⁵. That is, folate might suppress some types of cancer during the early stages of development, whereas high doses of folic acid taken after preneoplastic lesions have been established might promote cancer development and progression.

Results from clinical trials involving folic acid supplementation have been mixed. In addition, most trials have included other B-vitamins (frequently at doses well above RDA levels) and sometimes other nutrients, making it difficult to disentangle the effects, if any, of folic acid alone. For example, in a trial in France, 2,501 people with a history of cardiovascular disease received daily supplements of 560 mcg folic acid, 3 mg vitamin B6, and 20 mcg vitamin B12 for 5 years ¹⁴⁶. The researchers found no association between B-vitamin supplementation and cancer outcomes. In a combined analysis of two trials in Norway (where foods are not fortified with folic acid), supplementation with 800 mcg/day folic acid plus 400 mcg/day vitamin B12 for a median of 39 months in 3,411 people with ischemic heart disease increased cancer incidence rates by 21% and cancer mortality rates by 38% compared with no supplementation ¹⁴⁷. Findings from these Norwegian trials have raised concerns about folic acid supplementation’s potential to raise cancer risk.

The most thorough research has focused on folate’s effect on the development of colorectal cancer and its precursor, adenoma ¹⁴³, ¹⁴⁸. Several epidemiological studies have found inverse associations between high dietary folate intakes and the risk of colorectal adenoma and cancer ¹⁴⁹, ¹⁵⁰, ¹⁵¹, ¹⁵². For example, in the NIH-AARP Diet and Health Study, a cohort study of more than 525,000 people age 50 to 71 years in the United States, individuals with total folate intakes of 900 mcg/day or higher had a 30% lower risk of colorectal cancer than those with intakes lower than 200 mcg/day ¹⁵⁰. Other studies, however, have found no significant associations between dietary folate intakes ¹⁵³, ¹⁵⁴ or circulating folate concentrations ¹⁵⁵, ¹⁵⁶ and colorectal cancer risk.

Several clinical trials have examined whether supplemental folic acid (sometimes in combination with other B-vitamins) reduces the risk of colorectal adenoma in individuals with or without a history of adenoma. In the Women’s Antioxidant and Folic Acid Cardiovascular Study, which included 1,470 older women at high risk of cardiovascular disease, daily supplementation with 2,500 mcg folic acid, 50 mg vitamin B6, and 1,000 mcg vitamin B12 did not affect rates of colorectal adenoma during 7.3 years of intervention and about 2 years of postintervention follow-up ¹⁵⁷. A pooled analysis of three large clinical trials (one in Canada, one in both the United States and Canada, and one in both the United Kingdom and Denmark) found that folic acid supplementation for up to 3.5 years neither increased nor decreased adenoma recurrence rates in people with a history of adenoma ¹⁵⁸. However, in one of the studies included in the analysis, folic acid supplementation (1,000 mcg/day) significantly increased the risks of having three or more adenomas and of noncolorectal cancers, although it had no effect on colorectal cancer risk ¹⁵⁹.

Folic acid supplementation also had no effect on the risk of all cancer types combined in the pooled analysis of three clinical trials cited above ¹⁵⁸. Similarly, a meta-analysis of 13 randomized trials showed no statistically significant effects of folic acid supplementation (median daily dose of 2,000 mcg) over an average treatment period of 5.2 years on overall cancer incidence or the incidence of colorectal, lung, breast, prostate, or other cancers ¹⁶⁰.

Some research has found associations between folic acid supplementation and increased cancer risk. In a randomized clinical trial investigating osteoporotic fracture incidence in 2,919 participants age 65 years or older with elevated homocysteine levels, those who received 400 mcg folic acid plus 500 mcg vitamin B12 and 600 International Units (IU) vitamin D3 for 2 years reported a significantly higher cancer incidence, especially of colorectal and other gastrointestinal cancers, than those who received only 600 IU vitamin D3 ¹⁶¹. In addition, a 2018 prospective study found that folic acid intake from fortified foods and supplements was positively associated with a risk of cancer recurrence among 619 patients with non–muscle-invasive bladder cancer, whereas natural folate intakes showed no significant association ¹⁶². Higher plasma folate concentrations have also been associated with an increased risk of breast cancer in women with a BRCA1 or BRCA2 mutation ¹⁶³. A secondary analysis of the study by Cole and colleagues ¹⁵⁹ found that folic acid supplementation significantly increased the risk of prostate cancer ¹⁶⁴. Subsequent research has shown an association between increased cancer cell proliferation and higher serum folate concentrations in men with prostate cancer ¹⁶⁵. A meta-analysis of six randomized controlled trials that included a total of 25,738 men found that the risk of prostate cancer was 24% higher in men receiving folic acid supplements than those taking a placebo ¹⁶⁶.

The mixed findings from clinical trials, combined with evidence from laboratory and animal studies indicating that high folate status promotes tumor progression, suggest that folate might play dual roles in cancer risk, depending on the dosage and timing of the exposure. Modest doses of folic acid taken before preneoplastic lesions are established might suppress cancer development in healthy tissues, whereas high doses taken after the establishment of preneoplastic lesions might promote cancer development and progression ¹⁴⁸, ¹⁶⁷, ¹⁶⁸, ²⁰, ¹⁶⁹. This hypothesis is supported by a 2011 prospective study that found an inverse association between folate intake and risk of colorectal cancer only during early preadenoma stages ¹⁷⁰.

A 2015 expert panel convened by the National Toxicology Program and the National Institutes of Health Office of Dietary Supplements concluded that folic acid supplements do not reduce cancer risk in people with adequate baseline folate status ¹⁷¹. The panel also determined that the consistent findings from human studies that supplemental folic acid has an adverse effect on cancer growth justify additional research on the effects of folic acid supplementation on cancer risk ¹⁷¹. Several important questions about these effects remain, including the dose and timing of folic acid supplementation that might exert tumor-promoting effects and whether this effect is specific to synthetic folic acid or other forms of folate ¹⁴⁵.

Overall, the evidence to date indicates that adequate dietary folate intake might reduce the risk of some forms of cancer. However, the effects of supplemental folic acid on cancer risk are unclear, especially among individuals with a history of colorectal adenomas or other forms of cancer. More research is needed to fully understand how dietary folate and supplemental folic acid affect cancer risk and whether their effects differ by timing of exposure.

Cardiovascular disease and stroke

An elevated homocysteine level has been associated with an increased risk of cardiovascular disease ¹⁰. Folate and other B vitamins are involved in homocysteine metabolism, and researchers have hypothesized that these micronutrients reduce cardiovascular disease risk by lowering homocysteine levels ¹⁷², ¹⁰.

Folic acid and vitamin B12 (cobalamin) supplements lower homocysteine levels. However, these supplements do not actually decrease the risk of cardiovascular disease, although they appear to provide protection from stroke ¹⁷³, ¹⁷⁴, ¹⁷², ¹⁷⁵, ¹⁷⁶, ¹⁷⁷, ¹⁷⁸, ¹⁷⁹, ¹⁸⁰, ¹⁸¹. For example, in 5,442 U.S. women age 42 or older who were at high risk of cardiovascular disease, daily supplements containing 2,500 mcg folic acid, 1 mg vitamin B12, and 50 mg vitamin B6 for 7.3 years did not reduce the risk of major cardiovascular events ¹⁷⁶. In a substudy of 300 participants, the supplementation also had no significant effects on biomarkers of vascular inflammation ¹⁸², but it did lower homocysteine levels by a mean of 18.5% ¹⁷⁶. Another clinical trial included 5,522 patients age 55 years or older with vascular disease or diabetes from various countries (including the United States and Canada) that had a folic acid fortification program and some that did not ¹⁷⁵. Patients received 2,500 mcg folic acid plus 50 mg vitamin B6 and 1 mg vitamin B12 or placebo for an average of 5 years. Compared with placebo, treatment with B vitamins significantly decreased homocysteine levels but did not reduce the risk of death from cardiovascular causes or myocardial infarction. Supplementation did, however, significantly reduce the risk of stroke by 25%.

In a large trial in regions of China without folic acid fortification among 20,702 adults with hypertension but no history of stroke or myocardial infarction, supplementation with 800 mcg folic acid plus 10 mg enalapril (used to treat high blood pressure) for a median of 4.5 years significantly reduced the risk of stroke by 21% compared with enalapril alone ¹⁷⁹. The effect was more pronounced in participants with the lowest baseline levels of plasma folate. An analysis of 10,789 participants from this trial found that folic acid supplementation significantly reduced the risk of stroke by 73% among those who had a low platelet count and an elevated homocysteine level (increasing their risk of stroke) but had no significant effect on participants with a high platelet count and low homocysteine level ¹⁸³. These findings suggest that folic acid supplementation might primarily benefit those with insufficient folate levels, which are less common in countries, such as the United States, with folic acid fortification ¹⁸⁴.

The authors of a 2012 meta-analysis of 19 randomized controlled trials that included 47,921 participants concluded that B-vitamin supplementation has no effect on the risk of cardiovascular disease, myocardial infarction, coronary heart disease, or cardiovascular death, although it does reduce the risk of stroke by 12% ¹⁷². Likewise, the authors of the third update of a Cochrane Review of the effects of homocysteine-lowering interventions on cardiovascular events concluded that folic acid supplementation alone or in combination with vitamin B6 and vitamin B12 does not affect the risk of myocardial infarction or death from any cause, but it does reduce the risk of stroke ¹⁸⁵. Three other meta-analyses have also found that folic acid is effective for preventing stroke, especially in populations exposed to no or partial folic acid fortification ¹⁸¹, ¹⁸⁶, ¹⁸⁷.

Overall, the available evidence suggests that supplementation with folic acid alone or in combination with other B-vitamins reduces the risk of stroke, especially in populations with low folate status, but does not affect other cardiovascular endpoints.

Dementia, cognitive function, and Alzheimer’s disease

Most observational studies conducted to date have shown positive associations between elevated homocysteine levels and the incidence of both Alzheimer’s disease and dementia ¹⁸⁸, ¹⁸⁹, ¹⁹⁰, ¹⁹¹, ¹⁶⁷, ¹⁵, ¹⁹². Scientists hypothesize that elevated homocysteine levels might have a negative effect on the brain via numerous mechanisms, including cerebrovascular ischemia leading to neuronal cell death, activation of tau kinases leading to tangle deposition, and inhibition of methylation reactions ¹⁹¹. Some, but not all, observational studies have also found correlations between low serum folate concentrations and both poor cognitive function and higher risk of dementia and Alzheimer’s disease ¹⁹¹, ¹⁶⁷, ¹⁸⁹, ¹⁸⁸, ¹⁹³.

Despite this evidence, most clinical trial research has not shown that folic acid supplementation affects cognitive function or the development of dementia or Alzheimer’s disease, even though supplementation lowers homocysteine levels. In one randomized, double-blind, placebo-controlled trial in the Netherlands, 195 people age 70 years or older with no or moderate cognitive impairment received 400 mcg folic acid plus 1 mg vitamin B12; 1 mg vitamin B12; or placebo for 24 weeks ¹⁹⁴. Treatment with folic acid plus vitamin B12 reduced homocysteine concentrations by 36% but did not improve cognitive function. In another clinical trial in older adults (mean age 74.1 years) with elevated homocysteine levels, supplementation with 400 mcg folic acid plus 500 mcg vitamin B12 and 600 IU vitamin D3 for 2 years lowered homocysteine levels but did not affect cognitive performance compared with 600 IU vitamin D3 alone ¹⁹⁵.

As part of the Women’s Antioxidant and Folic Acid Cardiovascular Study, 2,009 U.S. women age 65 years or older at high risk of cardiovascular disease were randomly assigned to receive daily supplements containing 2,500 mcg folic acid plus 1 mg vitamin B12 and 50 mg vitamin B6 or placebo ¹⁹⁶. After an average of 1.2 years, B-vitamin supplementation did not affect mean cognitive change from baseline compared with placebo. However, in a subset of women with a low baseline dietary intake of B vitamins, supplementation significantly slowed the rate of cognitive decline. In a trial that included 340 individuals in the United States with mild-to-moderate Alzheimer’s disease, daily supplements of 5,000 mcg folic acid plus 1 mg vitamin B12 and 25 mg vitamin B6 for 18 months did not slow cognitive decline compared with placebo ¹⁹⁷.

A secondary analysis of a study in Australia (which did not have mandatory folic acid fortification at the time of the study) found that daily supplementation with 400 mcg folic acid plus 100 mcg vitamin B12 for 2 years improved some measures of cognitive function, particularly memory, in 900 adults age 60 to 74 years who had depressive symptoms ¹⁹⁸. Another meta-analysis included 11 randomized controlled trials in more than 20,000 older adults (mean age 60–82 years) that administered 400 to 2,500 mcg folic acid plus 20–1,000 mcg vitamin B12 in 10 trials and 3–50 mg vitamin B6 in 8 trials for 0.3 to 7.1 years. The supplementation significantly lowered homocysteine levels but did not affect cognitive aging, global cognitive function, or specific cognitive domains (including memory, speed, and executive function) ¹⁹⁹.

Several large reviews have evaluated the effect of B vitamins on cognitive function. Most of the authors concluded that supplementation with folic acid alone or in combination with vitamins B12 or B6 does not appear to improve cognitive function in individuals with or without cognitive impairment ²⁰⁰, ²⁰¹, ²⁰², ²⁰³. Some noted, however, that when researchers took baseline homocysteine and B-vitamin status into account, B-vitamin supplementation slowed cognitive decline in individuals at high risk of cognitive decline ¹⁹¹, ¹⁹². For example, one trial in the Netherlands administered either 800 mcg folic acid or placebo daily for 3 years to 818 participants age 50–70 years with elevated homocysteine levels (13 micromol/L or higher) and normal vitamin B12 levels ²⁰⁴. Folic acid supplementation reduced homocysteine concentrations by 26% and significantly improved global cognitive function, memory, and information processing speed compared with placebo, but it did not affect sensorimotor speed, complex speed, or word fluency.

Additional clinical trials are needed to better understand the effects of folic acid supplementation on cognitive function and cognitive decline.

Depression

Low folate status has been linked to depression and poor response to antidepressants in some, but not all, studies. The possible mechanisms are unclear but might be related to folate’s role in methylation reactions in the brain, neurotransmitter synthesis, and homocysteine metabolism ²⁰⁵, ²⁰⁶. However, secondary factors linked to depression, such as unhealthy eating patterns and alcohol use disorder, might also contribute to the observed association between low folate status and depression ²⁰⁷.

In an ethnically diverse population study of 2,948 people age 15 to 39 years in the United States, serum and erythrocyte folate concentrations were significantly lower in individuals with major depression than in those who had never been depressed ²⁰⁷. An analysis of 2005–2006 NHANES data found that higher serum concentrations of folate were associated with a lower prevalence of depression in 2,791 adults age 20 or older ²⁰⁵. The association was statistically significant in females, but not in males. However, another analysis showed no associations between folate intakes from both food and dietary supplements and depression among 1,368 healthy Canadians age 67–84 years ²⁰⁶. Results from a study of 52 men and women with major depressive disorder showed that only 1 of 14 participants with low serum folate levels responded to antidepressant treatment compared with 17 of 38 with normal folate levels ²⁰⁸.

A few studies have examined whether folate status affects the risk of depression during pregnancy or after childbirth. A systematic review of these studies had mixed results ²⁰⁹. One study included in the review among 709 women in Singapore found that compared with women with higher plasma folate concentrations (mean 40.4 nmol/L [17.8 ng/mL]) at 26–28 weeks’ gestation, those with lower plasma folate concentrations (mean 27.3 nmol/L [12.0 ng/mL]) had a significantly higher risk of depression during pregnancy but not after giving birth ²¹⁰. Another study of 2,856 women in the United Kingdom found no significant associations between red blood cell folate levels or folate intakes from food and dietary supplements before or during pregnancy and postpartum depressive symptoms ²¹¹. More recently, a cohort study of 1,592 Chinese women found a lower prevalence of postpartum depression in women who took folic acid supplements for more than 6 months during pregnancy than in those who took them for less time ²¹².

Studies have had mixed results on whether folic acid supplementation might be a helpful adjuvant treatment for depression when used with traditional antidepressant medications. In a clinical trial in the United Kingdom, 127 patients with major depression were randomly assigned to receive either 500 mcg folic acid or placebo in addition to 20 mg of fluoxetine daily for 10 weeks ²¹³. Although the effects in men were not statistically significant, women who received fluoxetine plus folic acid had a significantly greater improvement in depressive symptoms than those who received fluoxetine plus placebo. Another clinical trial in the United Kingdom randomized 475 adults with moderate to severe depression who were taking antidepressant medications to either 5,000 mcg folic acid or placebo daily for 12 weeks in addition to their antidepressants ²¹⁴. Measures of depression did not improve in participants taking folic acid compared with those taking placebo. The authors of a systematic review and meta-analysis of four trials of folic acid (<5,000 mcg/day in two trials; 5,000 mcg/day in two trials) in combination with fluoxetine or other antidepressants in patients with major depressive disorder concluded that less than 5,000 mcg/day folic acid might be beneficial as an adjunct to serotonin reuptake inhibitor (SSRI) therapy ²¹⁵. The authors noted, however, that this conclusion was based on low-quality evidence. Another meta-analysis of four clinical trials found that 500–10,000 mcg folic acid per day for 6–12 weeks as an adjunctive treatment did not significantly affect measures of depression compared with placebo ²¹⁶.

Other studies have examined the effects of 5-MTHF supplementation as an adjuvant treatment to antidepressants, and results suggest that it might have more promise than folic acid ²¹⁵, ²¹⁶, ²¹⁷, ²¹⁸. In a clinical trial in 148 adults with major depressive disorder, supplementation with 7,500 mcg/day 5-MTHF for 30 days followed by 15,000 mcg/day for another 30 days, both in conjunction with SSRI treatment, did not improve measures of depression compared with SSRI treatment plus placebo ²¹⁹. However, in a subsequent trial with the same study design in 75 adults, supplementation with 15,000 mcg/day 5-MTHF plus SSRI treatment for the full 60 days did significantly improve depression compared with SSRI treatment plus placebo ²¹⁹.

The authors of a systematic review and meta-analysis of three trials of 5-MTHF (<15,000 mcg/day in one trial, and 15,000 mcg/day in two trials) in combination with fluoxetine or other antidepressants, concluded that 15,000 mcg/day 5-MTHF might be an effective adjunct to SSRI therapy in patients with major depressive disorder, although they noted that this conclusion was based on low-quality evidence ²¹⁵. In addition, evidence-based guidelines from the British Association for Psychopharmacology ²¹⁷ and the Canadian Network for Mood and Anxiety Treatments ²¹⁸ state that 5-MTHF might be effective as an adjunct to SSRI treatment for depressive disorders.

Additional research is needed to fully understand the association between folate status and depression. Although limited evidence suggests that supplementation with certain forms and doses of folate might be a helpful adjuvant treatment for depressive disorders, more research is needed to confirm these findings. In addition, many of the doses of folate used in studies of depression exceed the Tolerable Upper Intake Level (maximum daily intake unlikely to cause adverse health effects) and should be taken only under medical supervision.

Folinic acid dosage

Folinic acid (leucovorin) is available in 5, 10, 15, and 25 mg tablets. Injectable folinic acid (leucovorin) formulations are 10 and 20 mg/mL solutions. Folinic acid is usually compounded with calcium, so it should not be administered intravenously at rates >160 mg/min ². Folinic acid is also not to be administered intrathecally ²²⁰.

Due to the wide range of folinic acid indications and administration guidelines, specific protocols are available that clinicians should follow; the dosing regimens below are general and are not a replacement for checking the facility protocols. In general, folinic acid is compounded with leucovorin calcium (especially for FDA-approved indications) and can be administered intramuscularly, intravenously, or orally ². The timing, dosage, and route of folinic acid administration depend on the desired outcome for the particular indication ⁶⁵.

Adult Dose

For high-dose methotrexate leucovorin rescue, the folinic acid (leucovorin) dosage is typically 15 mg orally, IM, or IV every 6 hours for 10 doses, starting 24 hours after initiating methotrexate. The maximum oral dose is 25 mg; IM or IV doses may be higher.

• In patients with delayed methotrexate administration, the dosage is 15 mg orally, IM, or IV every 6 hours. Continue until the methotrexate level <0.05 micromolar ⁶⁷.

For leucovorin rescue for methotrexate overdose, the dosage is typically 10 mg/m² orally, IM, or IV every 6 hours, starting ASAP after overdose or within 24 hours if delayed methotrexate elimination.

For folate antagonist overdose:

• For pemetrexed: 50 mg/m2/dose IV every 6 hours for 8 days. Start with 100 mg/m2/dose IV for the first dose.
• For trimethoprim or pyrimethamine: Give 5 to 15 mg orally, IM, or IV daily until the restoration of hematopoiesis.

For colorectal cancer combination therapy with 5-FU, clinicians should follow specific administration schedules as individual protocols vary. In general, when used for combination therapy, folinic acid is administered as an IV bolus or short IV infusions (minutes to hours) ⁶⁶. Dosing may require adjustments based on toxicity.

• With 5-FU 370 mg/m²/day: Leucovorin 200 mg/m²/day administered IV on days 1 to 5 of a 28-day cycle. Give 2 cycles, then continue 28-day cycles or extend to 35-day cycles.
• With 5-FU 425 mg/m²/day: Leucovorin 20 mg/m2/day administered IV on days 1 to 5 of a 28-day cycle. Give 2 cycles, then continue 28-day cycles or extend to 35-day cycles.

Folate-deficiency associated megaloblastic anemia:

• Dosing is <1 mg IV daily; maximum dose 1 mg daily.

Children Dose

As with adult dosing recommendations, individual protocols require close adherence, as guidelines differ depending on the desired outcome and particular indication. However, there are similar generalities in both patient populations. For both methotrexate toxicity and combination therapy with 5-FU, the previously mentioned adult administration recommendations are similar for the pediatric patient population ⁶⁶.

For high-dose methotrexate leucovorin rescue:

• In patients with standard methotrexate administration, the dosage is 10 mg/m²/dose, orally, IM, or IV every 6 hours for 10 doses, starting 24 hours after initiating methotrexate. The maximum oral dose is 25 mg; IM or IV doses may be higher.
• In patients with delayed methotrexate administration, the dosage is mg/m²/dose orally, IM, or IV every 6 hours. Continue until the methotrexate level <0.05 μM ⁶⁷.

Leucovorin rescue for methotrexate overdose:

• Dosage is 10 mg/m²/dose orally, IM, or IV every 6 hours, starting ASAP after overdose or within 24 hours if delayed methotrexate elimination.

For folate antagonist overdose:

• For trimethoprim or pyrimethamine: Give 5 to 15 mg orally, IM, or IV daily until the restoration of hematopoiesis.

Folate-deficiency-associated megaloblastic anemia:

• Dosing is <1 mg IV daily; maximum dose 1 mg daily.

Special Patient Populations

• Liver impairment: Dosing in hepatic impairment is undefined.
• Kidney impairment: Dosing for patients with renal impairment, including those on dialysis, is undefined.
• Pregnant women: Folinic acid/leucovorin may be used during pregnancy; human data shows no known risk of fetal harm.
• Breastfeeding considerations: Folinic acid can be used by breastfeeding women. No human data is available, but based on the drug’s properties, infant harm is not expected.
• Older patients: No data specific to leucovorin prohibits or limits its use in older patients.

Folinic acid side effects

Along with its needed effects, folinic acid may cause some unwanted effects. Although not all of these side effects may occur, if they do occur they may need medical attention.

Check with your doctor immediately if any of the following side effects occur.

Rare side effects:

• Skin rash, hives, or itching
• Wheezing

Check with your doctor as soon as possible if any of the following side effects occur:

Rare side effects reported with use in treatment of cancer:

• Convulsions (seizures)

Other side effects not listed may also occur in some patients. If you notice any other effects, check with your healthcare professional.

Drug interactions

When nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin or ibuprofen, are taken in very large therapeutic dosages (i.e., to treat severe arthritis), they may interfere with folate metabolism. In contrast, routine use of nonsteroidal anti-inflammatory drugs (NSAIDs) has not been found to adversely affect folate status. The anticonvulsant (antiepileptic), phenytoin, has been shown to inhibit the intestinal absorption of folate, and several studies have associated decreased folate status with long-term use of the anticonvulsants, phenytoin, phenobarbital, and primidone ²²¹. However, few studies controlled for differences in dietary folate intake between anticonvulsant users and nonusers. Also, taking folic acid at the same time as the cholesterol-lowering agents, cholestyramine and colestipol, may decrease the absorption of folic acid ²²². Methotrexate is a folic acid antagonist used to treat a number of diseases, including cancer, rheumatoid arthritis, and psoriasis. Some of the side effects of methotrexate are similar to those of severe folate deficiency, and supplementation with folic or folinic acid is used to reduce antifolate toxicity. Other antifolate molecules currently used in cancer therapy include aminopterin, pemetrexed, pralatrexate, and raltitrexed ⁶, ²²³. Furthermore, a number of other medications have been shown to have antifolate activity, including trimethoprim (an antibiotic), pyrimethamine (an antimalarial), triamterene (a blood pressure medication), and sulfasalazine (a treatment for ulcerative colitis). Early studies of oral contraceptives (birth control pills) containing high doses of estrogen indicated adverse effects on folate status; however, this finding has not been supported in more recent studies that used low-dose oral contraceptives and controlled for dietary folate ²²⁴.

Health Risks from Excessive Folinic Acid

No adverse effects have been associated with the consumption of excess folate from food. Concerns regarding folate safety are limited to synthetic folic acid intake e.g. folinic acid. Some health experts have been concerned that high intakes of folate supplements might mask vitamin B12 deficiency until its neurological consequences become irreversible ²²⁵, ²²⁶, ²²⁷, ²²⁸, ²²⁹, ²³⁰, ¹⁷². Vitamin B12 deficiency is a condition caused by low levels of vitamin B12 (cobalamin) in the body, leading to symptoms like megaloblastic anemia, fatigue, weakness, pale skin, nerve problems (numbness, tingling), mood changes, and in severe cases, neurological damage. Vitamin B12 deficiency may affect a significant number of people, especially older adults. One symptom of vitamin B12 deficiency is megaloblastic anemia, which is indistinguishable from that associated with folate deficiency. There is concern that large doses of folic acid given to an individual with an undiagnosed vitamin B12 deficiency could correct the megaloblastic anemia without correcting the underlying vitamin B12 deficiency, leaving the individual at risk of developing irreversible neurologic damage. Such cases of neurologic progression in vitamin B12 deficiency have been mostly seen in case studies at folic acid doses of 5,000 mcg (5 mg) and above. In order to be very sure of preventing irreversible neurologic damage in vitamin B12-deficient individuals, the Food and Nutrition Board of the US National Academy of Medicine advises that all adults limit their intake of folic acid (supplements and fortification) to 1,000 mcg (1 mg) daily (Table 1). The Food and Nutrition Board also noted that vitamin B12 deficiency is very rare in women in their childbearing years, making the consumption of folic acid at or above 1,000 mcg/day unlikely to cause problems ⁹. Furthermore, there are limited saefty data on the effects of large folic acid doses ²³¹.

However, there are concerns that high folic acid intakes might accelerate the progression of preneoplastic lesions, increasing the risk of colorectal and possibly other cancers in certain individuals ¹⁰, ¹², ¹⁴⁸, ¹⁶⁸, ²⁰. In addition, intakes of 1,000 mcg (1 mg) per day or more of folic acid from supplements during the periconception period have been associated with lower scores on several tests of cognitive development in children at age 4–5 years than in children of mothers who took 400 mcg to 999 mcg ²³².

Intakes of folic acid that exceed the body’s ability to reduce it to tetrahydrofolate (THF, the biologically active form of folic acid) lead to unmetabolized folic acid in the body, which has been linked to reduced numbers and activity of natural killer cells, suggesting that it could affect the immune system ²³³, ²³⁴. A small study conducted in postmenopausal women also raised concerns about the effect of exposure to unmetabolized folic acid on immune function ²³³. Recent observational studies have found no link between circulating maternal unmetabolized folate during pregnancy and allergic disease in infancy ²³⁵ or autistic traits or language impairment in early childhood ²³⁶. In a small, randomized, open-label trial in 38 women of reproductive age receiving 30 weeks of daily multivitamin supplements, daily supplementation with either 1.1 mg or 5 mg of folic acid resulted in the transient appearance of unmetabolized folic acid in blood over the first 12 weeks of supplementation ²³⁷. However, unmetabolized folic acid concentrations returned to baseline levels at the end of the study, suggesting that adaptive mechanisms eventually converted folic acid to reduced forms of folate.

Some scientists have hypothesized that unmetabolized folic acid might be related to cognitive impairment among older adults (≥60 years) ²²⁶, ²³⁸. These potential negative health consequences are not well understood and warrant further research ¹⁰, ¹⁴¹. The use of supplemental Levomefolic acid (5-methyltetrahydrofolate [5-MTHF] the most active form of folate) may provide an alternative to prevent any potential negative effects of unconverted folic acid in older adults.

Studies have found unmetabolized folic acid in blood from children, adolescents, and adults ¹⁰, ²³⁴, ²³⁹, ²⁴⁰; breastmilk ²⁴¹; and cord blood from newborns ²⁴², ²⁴³. Limited research suggests that single doses of 300 mcg or 400 mcg folic acid (a common amount in folic acid-containing supplements or servings of fortified foods, such as breakfast cereals) result in detectable serum levels of unmetabolized folic acid, whereas doses of 100 mcg or 200 mcg do not ²⁴⁴, ²⁴⁵. In addition, a dose-frequency interaction appears to occur in which smaller amounts of folic acid consumed more frequently produce higher unmetabolized folic acid concentrations than the same total dose consumed in larger, less frequent amounts ²⁴⁶.

Based on the metabolic interactions between folate and vitamin B12, the Food and Nutrition Board established a Tolerable Upper Intake Level (maximum daily intake unlikely to cause adverse health effects) for the synthetic forms of folate available in dietary supplements and fortified foods (Table 1) ⁹. The Food and Nutrition Board did not establish a Tolerable Upper Intake Level (maximum daily intake unlikely to cause adverse health effects) for folate from food because high intakes of folate from food sources have not been reported to cause adverse effects ⁹. Unlike the Recommended Dietary Allowance (RDA), the Tolerable Upper Intake Level (maximum daily intake unlikely to cause adverse health effects) are in mcg, not mcg DFE. For folic acid, 1,000 mcg is equivalent to 1,667 mcg DFE because 0.6 mcg folic acid = 1 mcg DFE ⁷. The ULs do not apply to individuals taking high doses of supplemental folate under medical supervision ⁷.

Table 1. Tolerable Upper Intake Levels (ULs) for Folate from Supplements or Fortified Foods

Age	Male	Female	Pregnancy	Lactation
Birth to 6 months	Not possible to establish*	Not possible to establish*
7–12 months	Not possible to establish*	Not possible to establish*
1–3 years	300 mcg	300 mcg
4–8 years	400 mcg	400 mcg
9–13 years	600 mcg	600 mcg
14–18 years	800 mcg	800 mcg	800 mcg	800 mcg
19+ years	1,000 mcg	1,000 mcg	1,000 mcg	1,000 mcg

[Source ⁷ ]

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