Folate is a vitamin that has only recently been appreciated for its importance beyond its essential role in normal metabolism, especially for its relevance to chronic diseases and birth defects. Widely distributed among foods, particularly those of plant leaf origin, this abundant vitamin is under-consumed by those people whose food habits are scant in plant foods. Intimately related in function with vitamins B12 and B6, its status at the level of subclinical deficiency can be difficult to assess, and the full extent of its interrelationships with these vitamins and with amino acids remains incompletely elucidated. Folate deficiency is an important problem in many parts of the world, particularly where there is poverty and malnutrition. It is an important cause of anaemia, second only to nutritional iron deficiency.
Circulating folates are taken up by a process which involves the folate transporters and receptors:-RFC, which is ubiquitously expressed in tissues in which its functional characteristics vary; PCFT, which is ubiquitously expressed in the liver and other tissues; and FRs, which facilitate the uni-directional movement of folates across cell membranes. In humans, the total body content of folate is 5–10 mg, about half of which resides in the liver. The concentration of folates in erythrocytes is widely used in the assessment of folate status, as it responds to changes in intake of the vitamin.
Folate is required for the production of new erythrocytes through its functions in the synthesis of purines and thymidylate required for DNA synthesis, as well as through its function in regenerating methionine and SAM, the methyl donor for DNA methylation. Folate deficiency results in the arrest of erythropoiesis prior to the latter stages of differentiation, resulting in megaloblastic anaemia. Anaemia can have folate-responsive components in subjects with apparently normal plasma folate levels; therefore, addition of folate to iron supplements can improve the treatment of anaemia in pregnancy as well as in undernourished individuals.
Several drugs can impair the absorption and metabolism of folates. These have proven to have clinical applications ranging from the treatment of autoimmune diseases, to cancer and malaria – all conditions in which cell proliferation can be suppressed through the inhibition of a folate-dependent metabolic step.
Methotrexate This is an analogue of folate, differing from the vitamin by the presence of an amino group replacing the 4-hydroxyl group and a methyl group. These differences give methotrexate a greater affinity than the natural substrate for dihydrofolate reductase, resulting in its inhibition. Accordingly, the drug produces an effective folate deficiency, with reductions in thymidine synthesis and purine levels. This anti-proliferative effect is the basis of the role of the drug in the treatment of cancer, rheumatoid arthritis, psoriasis, asthma, and inflammatory bowel disease. Because its side effects include those of folate deficiency, methotrexate is usually used with accompanying and carefully monitored folate supplementation to reduce the incidence of mucosal and gastrointestinal side effects. Individuals with the MTHFR 677TT genotype have a higher risk of discontinuing methotrexate treatment for rheumatoid arthritis due to adverse effects. In contrast, individuals with the MTHFR 1298CC genotype typically show improved efficacy of methotrexate therapy without increased side effects.
Other Drugs Impairments in folate status/metabolism have been reported for some anticonvulsants, anti-inflammatory drugs, glycaemic control drugs (metformin), and alcohol.
The most important factor contributing to folate deficiency is insufficient dietary intake. Other factors can contribute, particularly when combined with low dietary intakes of the vitamin. Folate utilization is impaired by insufficient supplies of vitamin B12 and/or the indispensable amino acid methionine; therefore, dietary deficiencies of either of those nutrients can produce signs of folate deficiency. Thus, patients with pernicious anaemia generally have impaired folate utilization, and show signs of folate deficiency. The metabolic basis of this effect involves the methionine synthetase reaction, which is common to the functions of both folate and vitamin B12. Methionine supplements cannot correct the low circulating folate levels caused by vitamin B12 deficiency.
Folate utilization can be impaired by depletion of zinc. This is thought to indicate a need for Zn by the enzymes of folate metabolism. Studies have yielded inconsistent but mostly negative results concerning effects of folate deficiency on Zn metabolism.
Deficiencies of folate result in impaired biosynthesis of DNA and RNA, and thus in reduced cell division, which is manifested clinically as anaemia, dermatologic lesions, and poor growth in most species. Signs and/or symptoms of folate deficiency are observed among individuals consuming inadequate dietary levels of the vitamin. These effects are exacerbated by physiological conditions that increase folate needs (e.g., pregnancy, lactation, rapid growth), by drug treatments that reduce folate utilization, by aging, and by diseases of the intestinal mucosa. Folate deficiency in humans is characterized by a sequence of signs, starting with changes in circulating polymorphonuclear leukocytes within about 2 months of deprivation of the vitamin. This is followed by megaloblastic anaemia, then by general weakness, depression, and polyneuropathy. In pregnant women, the deficiency can lead to birth defects or spontaneous abortion. Elderly humans tend to have lower circulating levelsof folate, indicating that they may be at increased risk of folate deficiency. Although the basis of this finding is not fully elucidated, it appears to involve age-related factors such as food habits that affect intake of the vitamin, rather than its utilization.
No adverse effects of high oral doses of folate have been reported in animals. Inconsistent results have been reported concerning the effects of high folate levels (1 to 10 mg doses) on human epileptics; some have indicated increases in the frequency or severity of seizures and reduced anticonvulsant effectiveness, whereas others have shown no such effects.
SOURCES OF FOLATE
Folates occur in a wide variety of foods of both plant and animal origin. Liver, mushrooms, and green, leafy vegetables are rich sources of folate in human diets. The folates in foods and feedstuffs are almost exclusively in reduced form as tetrahydrofolic acid (FH4). Very little free folate (folyl monoglutamate) is found in foods or feedstuffs. Analyses of foods have revealed a wide distribution of general types of polyglutamyl folate derivatives, including 5-methyl-FH4 and 10-formyl-FH4 as well as FH4.
Most folates in foods and feedstuffs are easily oxidized, and therefore are unstable to oxidation under aerobic conditions of storage and processing. Under such conditions (especially in the added presence of heat, light, and/ or metal ions), FH4 derivatives can readily be oxidized to the corresponding derivatives of dihydrofolic acid (FH2) (partially oxidized) or folic acid (fully oxidized), some of which can react further to yield physiologically inactive compounds. Substantial losses in the folate contents of food can occur as the result of leaching in cooking water when boiling (losses of total folates of 22% for asparagus and 84% for cauliflower have been observed), as well as oxidation, as described above. Due to such losses, green leafy vegetables can lose their value as sources of folates despite their relatively high natural contents of the vitamin.
The biological availability of folates in foods has been difficult to assess quantitatively. Estimates are variable among foods, but generally indicate bioavailabilities of about half that of folic acid; a recent study found a relatively high (80%) aggregate bioavailability of a mixed diet. In general, folates appear to be less well utilized from plant-derived foods than from animal products (1). Several factors affect the biologic availability of food folates: Folates can bind to the food matrices; many foods contain inhibitors of the intestinal brush border folate conjugase and/or folate transport; folate vitamers vary in biopotency; deficiencies of iron and vitamin C status are associated with impaired utilization of dietary folate. Vitamin C has also been shown to enhance the utilization of 5-methyl-FH4 by preventing its oxidative degradation to 5-methy-FH2, which does not enter the folate metabolic pool. Interactions of these factors complicate the task of predicting the bioavailability of dietary folates.
The gut microflora can synthesize folate in amounts that approximate daily dietary needs and studies have shown that folate can be absorbed across the human colon (2). Because the majority of food folates occur as reduced polyglutamates, they must be cleaved to the mono- or diglutamate forms for absorption. This is accomplished by the action of folyl conjugases. Folyl conjugase inhibitors have been identified in certain foods: cabbage, oranges, yeast, beans (red kidney, pinto, soy), lentils, and black-eyed peas. This also appears to be the basis for the low bioavailability of folate in orange juice. Folate absorption can also be reduced by certain drugs, including cholestyramine (which binds folates), some anticonvulsants, aspirin, and other salicylates, as well as several non-steroidal anti-inflammatory drugs.
Dietary folates are absorbed in deconjugated form, i.e., as folic acid, 5-methyl-FH4, and 5-formyl-FH4.13 These vitamers are actively transported by three protein transporters: The reduced folate carrier (RFC), proton-coupled folate transporter (PCFT) and folate receptors (FRs), which form folate-binding proteins, FBPs. Folic acid can also be absorbed passively, apparently by diffusion. Folates of dietary origin are absorbed and transported to the liver as FH4, which is converted to the methylated form and transported to the peripheral tissues. Erythrocytes contain greater concentrations of folate than plasma; typically 50–100 nmol/l. These stores are accumulated during erythropoiesis; the mature erythrocyte does not take up folate. The folate levels of both plasma and erythrocytes are reduced by cigarette smoking; smokers show plasma folate levels that are more than 40% less than those of non-smokers and a similar situation exists in alcoholics.
FOLATE IN HEALTH AND DISEASE
High doses of folate (e.g., 400 μg/day intramuscular; 5 mg/day oral) have been shown to correct the megaloblastic anaemia of pernicious anaemia patients, who are deficient in vitamin B12. This phenomenon renders megaloblastic anaemia not useful for diagnosing either vitamin deficiency without accompanying metabolic measurements: FIGLU (elevations indicate folate deficiency) and methylmalonic acid (MMA) (elevations indicate vitamin B12 deficiency). Supplemental folate does not mask the irreversible progression of neurological dysfunction and cognitive decline of vitamin B12 deficiency (3); however, those signs develop over a longer period of time than the anaemia produced by the same deficiency. In fact, folate supplementation has been shown to exacerbate the cognitive symptoms of vitamin B12 deficiency.
Pregnancy increases the need for folate to meet the increasing demands of the placenta and foetus, which can put women at increased risk of folate deficiency. Accordingly, several studies in apparently well-nourished populations have demonstrated the value of periconceptional folate supplementation in increasing placental weight and foetal growth rate, and reducing the risk of low birth weight. Folate, an essential factor in the support of normal cell division, is a key factor affecting embryogenesis and normal development.
For nearly four decades, adequate folate status has been linked to reduced risks of abnormalities in early embryonic development and, specifically, to risk of malformations of the embryonic brain and/or spinal cord, collectively referred to as NTDs. These linkages have involved observations of high incidences of low folate status among women with NTD birth compared to women with normal birth outcomes. Additional support came from the production of NTDs in animal models by the folate antagonist aminopterin. Several clinical intervention trials have tested the hypothesis that periconceptional supplemental folate can reduce NTD risk (4-6). One of these, a large, well-designed, multi-centre trial conducted by the British Medical Research Council, found that a daily oral dose of 4 mg of folic acid reduced significantly the incidence of confirmed NTDs among the pregnancies of women at high risk for such disorders. Several subsequent studies have shown that periconceptional supplementation of folate can reduce the risk of NTDs. These have included trials conducted in the US, which found folate supplements (400–4,000 μg) effective in preventing NTDs in women with prior NTD pregnancies (7). While folate supplements do not appear to affect NTD case-fatality rates, reductions in NTD incidence are associated with reductions in neonatal deaths (8). A meta-analysis of eight observational studies indicated that folate supplementation was associated with a 46% reduction in NTD risk, which was associated with a 13% reduction in neonatal deaths. A systematic review of 14 folate intervention trials pointed out that not all NTD cases can be prevented by folate; that analysis suggested that a rate of 8–10/10,000 live births or abortions would appear to involve factors not affected by increasing folate intakes (9). The protective effect of folate supplementation may be limited to a subset of subjects with defective folate metabolism due to MTHFR mutations. The MTHFR 677TT genotype is associated with NTD risk, but the effect appears to be dependent on folate status. In a recent analysis, TT homozygosity was associated with a five-fold increase in NTD risk for mothers not using multivitamin supplements, but was without effect for mothers using supplements (10).
Other Birth Defects
Evidence is inconsistent for associations of low folate status and risks of other congenital defects, including orofacial clefts, and defective development of limbs and the heart. Homocysteinemia has been associated with increased risks of hypertension, pre-eclampsia, and placental abruption. Evidence suggests that MTHFR 677TT genotype elevates risk of Down syndrome in individuals also carrying a mutation in methionine synthase reductase (11).
In 1969, McCully pointed out a relationship between elevated plasma homocysteine levels and risk of occlusive vascular disease. Epidemiologic studies subsequently showed associations of moderately elevated plasma Hcy and risks of coronary, peripheral, and carotid arterial thrombosis and atherosclerosis; venous thrombosis; retinal vascular occlusion; carotid thickening; and hypertension (12). A meta-analysis of the results of 27 cross-sectional and case– control studies (13) attributed 10% of total coronary artery disease to homocysteinemia. Homocysteinemia and the resulting homocysteinuria have been associated with increased risks of cardiovascular disease, recurrent early pregnancy loss, and hip fracture. Accumulation of Hcy can occur through its elevated production from methionine and, probably to a lesser extent, its impaired disposal through transsulfuration to cystathionine. Both result in homocysteinemia, can have congenital causes, and can be related to nutritional status with respect to vitamin B6, vitamin B12, and folate.
Experimental folate deprivation has been shown to cause elevated plasma Hcy concentrations, and the use of folate-containing multivitamin supplements has been associated with low mean plasma Hcy levels. One prospective, community-based study found plasma Hcy to be strongly inversely associated with plasma folate level (which was positively associated with the consumption of folate-containing breakfast cereals, fruits, and vegetables) but not plasma levels of vitamin B12 and pyridoxal phosphate (14).
Individuals with the MTHFR 677TT genotype have slightly lower levels of folate and slightly higher levels of Hcy compared to other genotypes (15). The TT genotype has been identified as a risk factor for carotid intima-media thickening, itself a risk factor to vascular disease. Low riboflavin status has been shown to be a significant determinant of another risk factor, high blood pressure, in the TT genotype. Dietary intakes of polyunsaturated fatty acids (PUFAs) have been found to be a significant covariate with the MTHFR C677T and A1298T genotype in affecting plasma Hcy level (16).
Homocysteinemia can respond to supplementation of folic acid. Reduction of serum Hcy in response to supplemental folate is linear up to daily folate intakes of about 0.4 mg, particularly for individuals with relatively high serum Hcy levels (17). Greater efficacy may be realized when folate is given in combination with vitamin B12. A meta-analysis of 25 randomized controlled trials showed that daily intakes of 0.8 mg folic acid are required to realize maximal reductions in plasma Hcy levels. A meta-analysis of eight trials involving 37,485 subjects randomized to folate and/or other B vitamins were used as the intervention agents showed that a 25% reduction in circulating Hcy level for five years was not associated with any reductions in cardiovascular events (or death from any cause) (18). In one trial, cardiovascular disease patients who received a combined supplement of folate, vitamin B6, and vitamin B12 showed increased risk for subsequent myocardial infarction (19). Meta-analyses of trials that used folate as the single intervention agent yielded inconsistent results: one found the vitamin to improve flow-mediated dilatation, suggestive of enhanced vascular function (20); the other found folate supplementation to be of no benefit in reducing stroke risk (21). These findings have cast doubt on the role of Hcy in the etiology of cardiovascular disease. The controversy involves questions as to whether homocysteinemia may be a cause or a marker of disease, and whether serum Hcy may be an indicator of metabolic status. It has been suggested that the cardiovascular pathogenesis may actually involve another factor: the metabolic precursor of Hcy, S-adenosylhomocysteine (SAH). SAH is reversibly converted with Hcy, the equilibrium favouring SAH. Therefore, homocysteinemia would be expected to lead to elevated levels of SAH, which appears to be present at only very low amounts in the circulation in normal circumstances.
Folate is required to maintain Hcy at low levels in the central nervous system. Hyperhomocysteinemia has been associated with increased risks of psychiatric and neurodegenerative disorders. Studies have shown several ways Hcy can be neurotoxic: through inhibition of methyltransferases involved in catecholamine methylation by the metabolite S-adenosylhomocysteine and through oxidative stress resulting from the production of reactive oxygen species generated by the oxidation of Hcy. Folate may also be directly involved in the regulation of neurotransmitter metabolism. The MTHFR 677 TT genotype appears to be associated with increased risk of neurological disorders. Such patients are also likely to be exposed to anticonvulsants that antagonize folate metabolism.
Cognitive Function Homocysteinemia is associated with age-related cognitive decline and risk of developing dementia; however, analyses of the NHANES data suggest that these outcomes are associated with low vitamin B12 status and not low folate status (22). In fact, they suggest that high folate status may exacerbate the neuropsychiatric effects of low vitamin B12 status. A meta-analysis of randomized clinical trials concluded that folic acid yielded no beneficial effects on measures of cognition within three years of supplementation (23).
Depression Mood changes and other symptoms of depression have frequently been observed in folate deficiency. These symptoms are associated with homocysteinemia; it has been suggested that they reflect Hcy-induced cerebral vascular disease and neurotransmitter deficiency. Studies have shown that individuals of the MTHFR 677TT genotype have increased risks of depression (24). A systematic review of randomized clinical trials suggested that folic acid may benefit the treatment of depression (25).
A randomised double blind pilot study to assess the effect of 1 mg folic acid supplementation of cholinesterase inhibitors (ChI) in a six month double-blind placebo-controlled study of patients with Alzheimer’s Disease (AD) and to assess whether outcome measures were affected by changes in homocysteine levels suggests that response to ChI in patients with AD may be improved by the use of folic acid possibly due to the relationship between changes in homocysteine levels (26).
Homocysteinemia can produce skeletal signs. These include knock-knees and unusually high arches of the foot in children, with subsequent development of long limbs and osteoporosis. Studies of the relationship of plasma Hcy level and bone health in humans have yielded inconsistent results, although some studies have found high plasma Hcy to be associated with reduced bone mineral density and increased fracture risk (27). A randomized trial that used a combined supplement of folate, vitamin B6, and vitamin B12 found no effect on biomarkers of bone turnover in older adults, despite the efficacy of that treatment in reducing plasma Hcy levels. Some studies have indicated that the MTHFR genotype may be related to bone health. Individuals with the 677TT genotype have been found to have higher risks of fracture and lower bone mineral density, independent of any effects of Hcy. However, not all studies have detected such relationships. That other factors are involved is suggested by a study that found 677TT women to show reduced bone mineral density only if they also had relatively low intakes of folate, vitamin B12, and riboflavin.
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Keywords :-folic acid, folate, pernicious anaemia, neural tube defects, cardiovascular disease, cognitive function, depression, alzheimer’s disease, bone health