Molecular Genetics and Metabolism 71, 121–138 (2000) doi:10.1006/mgme.2000.3027, available online at http://www.idealibrary.com on
MINIREVIEW Folic Acid: Nutritional Biochemistry, Molecular Biology, and Role in Disease Processes Mark Lucock Academic Unit of Paediatrics and Obstetrics and Gynaecology, University of Leeds, D Floor, Clarendon Wing, Leeds General Infirmary, Leeds, West Yorkshire, LS2 9NS, United Kingdom Received June 27, 2000
The various coenzymes of folic acid facilitate the transfer of one-carbon units from donor molecules into important biosynthetic pathways leading to methionine, purine, and pyrimidine biosynthesis. They also mediate the interconversion of serine and glycine, and play a role in histidine catabolism. Folic acid or pteroylmonoglutamate (PteGlu), is actually a stable, synthetic analog, which is merely the parent structure of this large family of vitamin coenzymes. The molecular biology and nutritional biochemistry of this water-soluble B vitamin is currently the focus of considerable attention. In its native form, or as a dietary or pharmacological supplement, folate has been credited with a beneficial role in preventing a range of disorders. Furthermore, since folate-dependent one-carbon metabolism is the subject of genetic variability, several common polymorphisms of genes encoding folate-dependent enzymes have also now been identified as risk factors for a variety of clinical conditions. This review looks at the chemistry, disposition, metabolism, and molecular biology of folate. In particular, it focuses on the gene–nutrient interrelationship that underpins the importance of dietary folate in a variety of disease states.
This paper reviews the chemistry, metabolism, and molecular biology of folic acid, with a particular emphasis on how it is, or may be, involved in many disease processes. Folic acid prevents neural tube defects like spina bifida, while its ability to lower homocysteine suggests it might have a positive influence on cardiovascular disease. A role for this B vitamin in maintaining good health may, in fact, extend beyond these clinical conditions to encompass other birth defects, several types of cancer, dementia, affective disorders, Down’s syndrome, and serious conditions affecting pregnancy outcome. The effect of folate in these conditions can be explained largely within the context of folate-dependent pathways leading to methionine and nucleotide biosynthesis, and genetic variability resulting from a number of common polymorphisms of folatedependent enzymes involved in the homocysteine remethylation cycle. Allelic variants of folate genes that have a high frequency in the population, and that may play a role in disease formation include 677C 3 T-MTHFR, 1298A 3 C-MTHFR, 2756A 3 GMetSyn, and 66A 3 G-MSR. Future work will probably uncover further polymorphisms of folate metabolism, and lead to a wider understanding of the interaction between this essential nutrient and the many genes which underpin its enzymatic utilization in a plethora of critical biosynthetic reactions, and which, under adverse nutritional conditions, may promote disease. © 2000 Academic Press
DISCOVERY AND STRUCTURE In 1931, Lucy Wills demonstrated that yeast extract was effective against the tropical macrocytic anemia often observed during late pregnancy in India (1). Although as yet undiscovered, the critical
Folate is the generic term for a large family of chemically similar, highly labile trace compounds. 121
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The chemical structure of tetrahydrofolate and its various derivatives.
factor involved was folic acid. Several workers contributed to the isolation of this vitamin and the elucidation of its structure (2– 4). The name folic acid is derived from the Latin—folium (leaf). Native folates differ in the oxidation state of the pteridine ring, the character of the one-carbon substituent at the N5 and N10 positions, and the number of glutamic acid moieties conjugated one to another via a series of ␥-glutamyl links to form an oligo-␥-glutamyl tail. Figure 1 illustrates the component structure of reduced, native forms of folic acid. The many structural forms in which folyl-coenzymes exist has led to difficulties in elucidating the biological occurrence and complex detail inherent in folate biochemistry. DIETARY SOURCES AND ABSORPTION Man cannot synthesize folate, and thus depends on a variety of dietary sources for the vitamin. Rich sources include yeast extracts such as Marmite, liver, kidney, leafy green vegetables, and citrus fruit. However, moderate sources of the vitamin such as bread, potatoes, and dairy products are consumed in large amounts and actually provide a significant contribution to the total folate intake. Food folates exist mainly as 5-methyltetrahydro-
folate (5-methyl-H 4PteGlu n) and formyltetrahydrofolate (formyl-H 4PteGlu n) (5). 5-Methyl-H 4PteGlu n is the predominant food folate (6,7) and is readily oxidized to 5-methyl-5,6-dihydrofolate (5-methyl5,6-H 2PteGlu n) (8). In this oxidized form it may represent a substantial amount of the total food folate (5). 5-Methyl-5,6-H 2PteGlu is rapidly degraded under the mildly acid conditions which prevail in the postprandial gastric environment. Under the same conditions, 5-methyl-H 4PteGlu is relatively stable. Ascorbic acid is actively secreted into the gastric lumen, and may be a critical factor in salvaging acid labile 5-methyl-5,6-H 2PteGlu, by reducing it back to acid-stable 5-methyl-H 4PteGlu. This mechanism might be crucial in optimizing the bioavailability of food folate (9). Dietary folates are transported across the enterocyte brush border membrane by a saturable process involving an anion exchange mechanism driven by the transmembrane pH gradient. Folate is anionic at the intraluminal pH, and is exchanged for a hydroxyl anion. Absorption occurs throughout the jejunum, although it is markedly more efficient proximally. Polyglutamyl folates are hydrolyzed to folylmonoglutamates by pteroyl-␥-glutamylhydrolase, and metabolized within the enterocyte into 5-methyl-H 4PteGlu 1. This monoglutamyl folate co-
enzyme is the plasma form of the vitamin (10,11), and is transported to peripheral tissues where it is converted by vitamin B 12-dependent methionine synthase (MetSyn) to monoglutamyl tetrahydrofolate (H 4PteGlu 1). H 4PteGlu 1 is the preferred substrate of folylpolyglutamate synthase (FPGS) which conjugates glutamate moieties to generate oligo-␥glutamyl H 4PteGlu. The product of this reaction is predominantly hexaglutamyl-H 4PteGlu (12), which along with folylpentaglutamates are the favored oligo-␥-glutamyl chain lengths for cellular reactions based on kinetic data (13). The conversion of 5methyl-H 4PteGlu 1 into H 4PteGlu 1 by vitamin B 12dependent MetSyn is thus an essential element in converting extracellular 5-methyl-H 4PteGlu 1 from dietary sources into a biologically more useful intracellular form of the vitamin that can be used in nucleotide biosynthesis (14). TRANSPORT Plasma Following absorption, 5-methyl-H 4PteGlu 1 is released into the portal circulation. Much of this folate is taken up by the liver, although some is released into the bile where it is recirculated by the enterohepatic cycle. The plasma 5-methyl-H 4PteGlu 1 level is in the region of 3–30 ng/ml. Following short periods of folate deprivation, the supply of the vitamin is maintained by folylmonoglutamate pools within the cell and enterohepatic cycle. Decreased tissue uptake leads to reduced cellular folylpolyglutamate synthesis and increased folylpolyglutamate hydrolysis to monoglutamate species. This process increases the available extracellular plasma 5-methylH 4PteGlu 1 level. 5-Methyl-H 4PteGlu 1 is also reabsorbed in the renal proximal tubule by a mechanism involving receptor-mediated endocytosis, and contributes to the availability of circulating 5-methylH 4PteGlu 1. Plasma contains pteroyl-␥-glutamylhydrolase, and any folylpolyglutamates released into plasma would be hydrolyzed to their monoglutamate forms. The role of liver is central to folate homeostasis and is fully reviewed by Steinberg (15). Thirty to forty percent of endogenous plasma folate is associated with low affinity binding proteins, primarily albumin (K d ⬃ 1 mM). Other binders include ␣2 macroglobulin and transferrin. Binding increases in folate deficiency, with a shift in binding from ␣2 macroglobulin to transferrin during pregnancy (16 –20). Plasma also contains a less abun-
dant high-affinity folate binding protein (K d ⬃ 1 nM) which increases during folate deficiency (21,22), pregnancy (23), leukemia (24), uremia (25), liver disease (26), and in the serum from umbilical cord blood (27). This high-affinity binding protein would seem to be homologous with the cellular folate binding protein which is normally attached to the cell membrane by a glycosylphosphatidylinositol anchor (see below) (28 –30). Erythrocyte Folate is incorporated into the developing erythroblast during erythropoiesis in the marrow. Early work by Iwai et al. (31) showed that human erythrocyte folate showed almost equal activity for both Pediococcus cerevisiae and Streptococcus faecalis, indicating to them that one of the folates may be an N 5,N 10anhydroformyl-H 4PteGlu or 10-formylH 4PteGlu coenzyme. In fact, even earlier studies showed the presence of oxidized 10-formylfolate (10formyl-PteGlu) among the degradation products of erythrocyte folates (32). However, in their report, Iwai et al. (31) also indicate a high Lactobacillus casei activity, suggesting the presence of 5-methylH 4PteGlu. This pattern of microbiological activity to erythrocyte folate which shows a high L.casei response coupled with low activity for S.faecalis or P.cerevisiae can be drastically changed in the direction of enhanced activity for P.cerevisiae by treatment with high levels of ascorbate. This provides the strongest possible evidence that native erythrocyte folates exist at the formyl oxidation level, since P.cerevisiae is specific for 10- and 5-formyl-H 4PteGlu and H 4PteGlu but not the oxidative degradation product 10-formyl-PteGlu or indeed 5-methylH 4PteGlu. However, upon degradation of 10-formylH 4PteGlu, the product 10-formyl-PteGlu would exhibit L.casei activity and thus explain these early findings (33). Formyl folates are unfortunately the most difficult coenzymes to quantify since all steps are controlled by complex pH-dependent and enzyme interconversions. It has been suggested that the pathway shown in Fig. 2 exists. This shows the nonenzymatic pathway for conversion of 5,10-methenyltetrahydrofolate (5,10methenyl-H4PteGlu) into the transient intermediate coenzyme, (11S)-hydroxymethylene-H4PteGlu, and subsequently into either 10-formyl-H4PteGlu or 5-formyl-H 4PteGlu, depending on protonation of the N-5 (pKa ⫽ 4.5) or N-10 (pKa ⫽ ⫺1.2) sites. It has also been proposed that both facile and enzyme (serine
Putative scheme showing the complex interrelationship between the many formyl derivatives of folic acid.
hydroxymethyltransferase (SHMT)) catalyzed formation of 5-formyl-H 4PteGlu from 5,10-methenylH 4PteGlu occurs. In this case, (11R)-hydroxymethylene-H 4PteGlu (anhydroleucovorin B), formed from the 11S isomer by isomerization (or by an as yet undiscovered enzyme), breaks down into 5-formylH 4PteGlu (34). The much higher affinity of anhydroleucovorin B for SHMT, compared to 5,10-methenylH 4PteGlu, suggests that in vivo, this 11R isomer may be the preferred substrate for SHMT hydrolysis to 5-formyl-H 4PteGlu (34). This level of detail is not provided in Fig. 3. Today it is recognized that erythrocyte folate is largely 5-methyl-H 4PteGlu and formyl-H 4PteGlu (35). However, methods of folate extraction vary, and given the interconvertability, lability, and multiplicity of form that folates exhibit, it is hardly surprising that various workers using specific chromatographic methods have obtained different information on cellular folate reserves (see effect of ascorbate above). Most studies agree that the majority of erythrocyte folate is in the form of folylpolyglutamates with penta- and hexaglutamates predominating (35,36). The range for erythrocyte folate concentration obtained using radiometric binding assay varies in the region of 140 – 450 ng/ml packed cells. Erythrocyte folate has no known metabolic role and is thought to be a storage reservoir and long term buffer for maintaining folate homeostasis. It is used as a measure of folate status, which, unlike plasma levels, is not affected by recent dietary intake. Folate within senescent erythrocytes is salvaged by the reticuloendothelial system. It is then transported to
the liver and appears in the bile for redistribution to peripheral tissues via the enterohepatic cycle. Cellular Transport Cellular transport systems can be divided into two classes: the first involves membrane carriers. Several such systems have been characterized in mammalian tissue. Tumor cells and fetal tissue have one of the most rigorously studied transporter systems. The mechanism differs from that found in normal adult tissues, which exhibit a wide range of transporters with a differential affinity for various folylcoenzymes (37). Folate transport in hepatocytes is energy dependent with saturable and low-affinity nonsaturable elements (38). The basolateral membrane of rat and human liver has an electroneutral folate H ⫹ cotransporter (39,40), while the basolateral membrane of the small intestine has an anion exchange folate transporter (41). Mitochondria also possess specific folate transporters. The second class of cellular folate transporter is a specific folate binding protein which is attached to the plasma membrane by a glycosylphosphatidylinositol anchor and is largely confined to the apical membrane of certain epithelial cells (42,43). It can bind a variety of folylcoenzymes with high affinity. The protein–folate complex internalizes folate by a nonclathrin-mediated endocytotic pathway which does not involve lysosomes (44). The phrase “potocytosis” has been given to the recycling of a binding protein via vesicular structures known as caveolae (45). Lowering of the pH releases anionic folate from the carrier before its subsequent liberation from the
FIG. 3. Folate-dependent one-carbon metabolism, highlighting the homocysteine remethylation cycle and common single-nucleotide polymorphisms.
vesicle into the cytosolic milieu. The binding protein then cycles back to the plasma membrane. Some of the tissues rich in this transport system are the choroid plexus where plasma 5-methyl-H 4PteGlu is transported into the CSF, the vas deferens, renal proximal tubules, erythropoietic cells, ovary, and placental trophoblasts (43). FOLATE COENZYMES IN ONE-CARBON TRANSFER REACTIONS The metabolic role of folate is to carry one-carbon units that exist at various levels of oxidation (see Figs. 1 and 3). Five major one-carbon transfer reactions occur within the cell: conversion of serine to glycine, catabolism of histidine, and synthesis of thymidylate, methionine, and purine. These reactions take place through various electron transfer steps facilitated by specific enzyme systems and coenzymes such as FADH 2 and NADPH. Folate polyglutamylation occurs in many cells, although the liver is the body’s main store of folates (15). Folate enters the cell largely as 5-methylH 4PteGlu 1, making vitamin B 12-dependent MetSyn rate limiting for the cellular accumulation of folates, since it is the only enzyme capable of demethylating 5-methyl-H 4PteGlu 1 to produce H 4PteGlu 1, the optimum substrate for polyglutamylation via FPGS. Formation of folyltriglutamates or longer oligo-␥glutamyl forms ensures cellular retention (46). Folylpolyglutamates are generally better substrates for folate-dependent enzymes than their monoglutamyl counterparts. K m values decrease with increasing oligo-␥-glutamyl chain length (47). Many folate enzymes are multifunctional and channel one-carbon units between reactions without achieving equilibrium with the cell medium. Therefore, the oligo-␥-glutamyl chain conjugated to folate aids cellular retention, regulates reaction rate, and allows channeling of the substrate between enzymes in a way which controls biosynthetic pathways (47). The principle origin of one-carbon units in folatedependent one-carbon metabolism is the ␤-carbon of serine in a reaction catalyzed by SHMT. In this reaction, glycine is generated and H 4PteGlu is converted to 5,10-methylenetetrahydrofolate (5,10methylene-H 4PteGlu). Formiminoglutamic acid (FIGLU), the mitochondrial glycine cleavage pathway, and choline catabolism provide alternative sources of one-carbon units. A minor source in mammalian
tissue is also 10-formyl-H 4PteGlu, which can be formed from formate, H 4PteGlu, and ATP. Catabolism of Histidine FIGLU forms from the breakdown of histidine. The excretion of urinary FIGLU following a histidine load was exploited as an early test for folate deficiency. Transfer of the formimino group of FIGLU to H 4PteGlu and removal of the ⫽NH group by the bifunctional enzyme 5-formiminotetrahydrofolate cyclodeaminase/transferase yields 5,10-methenyl-H 4PteGlu. Purine and Pyrimidine Nucleotide Biosynthesis These are perhaps the most important roles of folic acid derivatives. Aminoimidazole-4-carboxamide ribonucleotide (AICAR) and glycinamide ribonucleotide (GAR) each receive a one-carbon unit from 10-formyl-H 4PteGlu which becomes carbon atoms 2 and 8, respectively, of the developing purine ring. The enzymes responsible are AICAR transformylase and GAR transformylase. In the synthesis of pyrimidine nucleotides, 5,10methylene-H 4PteGlu methylates deoxyuridylate monophosphate (dUMP) to form thymidylate monophosphate (TMP). The enzyme responsible is thymidylate synthase (TS) which is rate limiting in the elaboration of DNA. Antifolate chemotherapeutic drugs like methotrexate resemble dihydrofolate (H 2PteGlu) and therefore inhibit the enzyme dihydrofolate reductase (DHFR) which converts H 2PteGlu to H 4PteGlu. TMP formation is sensitive to depressed levels of H 4PteGlu, leading to inhibition of DNA synthesis in rapidly dividing malignant cells. Expression of TS peaks during the S phase of the cell cycle and is related to replication rate. A multi-enzyme complex termed replicase forms during S phase. This protein aggregation contains TS, DHFR, DNA polymerase, thymidine kinase, nucleoside diphosphate kinase, deoxycytidine monophosphate kinase, and ribonucleotide reductase. Increased thymidylate synthesis in actively proliferating cells elevates H 2PteGlu levels (see Fig. 3). H 2PteGlu inhibits the allosteric enzyme 5,10 methylenetetrahydrofolate reductase (510MTHFR), and provides a regulatory mechanism for ensuring that priority is given to nucleic acid synthesis over methionine formation (48). The Interconversion of Serine and Glycine SHMT catalyzes the reversible interconversion of serine and glycine. The reaction requires vitamin B 6
as a cofactor, and introduces the ␤ carbon of serine into the one-carbon pool at the formaldehyde level of oxidation: H 4PteGlu ⫹ serine ⫹ NAD ⫹ 7 5,10-methylene-H 4PteGlu ⫹ glycine ⫹ H 2O. 5,10-methyleneH 4PteGlu represents a crucial intermediate coenzyme state. Although 5,10-methylene-H 4PteGlu is used by 5,10MTHFR to produce 5-methyl-H 4PteGlu in a committed step which then requires B 12-dependent MetSyn to cycle homocysteine (Hcy) into methionine (46), it is also required for both TS and methylenetetrahydrofolate dehydrogenase in the synthesis of DNA thymine and purine, respectively (see Fig. 3). Thus, 5,10-methylene-H 4PteGlu is at the branch point for three important pathways (49), and its production by SHMT is therefore an important step in mammalian one-carbon metabolism. The mitochondrial glycine cleavage reaction represents another point of entry at the formaldehyde level of oxidation. This is a complex reaction in which carbon 2 of glycine is transferred to H 4PteGlu forming 5,10-methylene-H 4PteGlu and releasing NH 3. Carbon 1 is oxidized to CO 2: H 4PteGlu ⫹ glycine ⫹ NAD ⫹ 7 5,10-methylene-H 4PteGlu ⫹ CO 2 ⫹ NH 4⫹ ⫹ NADH. Two further mitochondrial enzymes that incorporate one-carbon units at the level of formaldehyde are sarcosine dehydrogenase and dimethylglycine dehydrogenase. Dimethylglycine originates from hepatic choline catabolism. One of its methyl groups is transferred to H 4PteGlu by dimethylglycine dehydrogenase and is oxidized to 5,10-methylene-H 4PteGlu. Sarcosine is the other product, and is oxidized, with the remaining methyl group being transferred to H 4PteGlu by sarcosine dehydrogenase. The products are glycine and 5,10methylene-H 4PteGlu (50). Role of Leucovorin in One-Carbon Metabolism 5-Formyl-H 4PteGlu or leucovorin is used as a rescue therapy following methotrexate treatment for cancer. 5-Formyl-H 4PteGlu is present in cells, but does not serve as a one-carbon donor in the biosynthetic reactions leading to methionine, or nucleotides. Methenyltetrahydrofolate synthetase is a unidirectional enzyme which salvages the one-carbon unit of 5-formyl-H 4PteGlu by converting it into the 5,10-methenyl-H 4PteGlu coenzyme. The same coenzyme can be recycled to 5-formyl-H 4PteGlu via a minor enzyme activity of SHMT. However it is thought that the actual substrate for SHMT may be hydrated 5,10-methenyl-H 4PteGlu (11R-hydroxymethylene-H 4PteGlu). This cyclical pathway has
been referred to as the futile cycle, and may, through the level of 5-formyl-H 4PteGlu, and its inhibitory properties, regulate other folate pathways (see Ref. 51 for full review). The Homocysteine Remethylation Cycle: FolateDependent de Novo Methionine Biosynthesis Hcy sits on the intersection of two important pathways and is regulated by several enzymes. The partitioning of Hcy between de novo methionine biosynthesis and transulphuration to cystathionine is allosterically regulated by S-adenosylmethionine (SAM) at the level of cystathionine-␤-synthase (stimulates) and 5,10MTHFR (inhibits) (52). In the Hcy remethylation cycle, 5,10-methyleneH 4PteGlu is reduced to 5-methyl-H 4PteGlu by the flavoprotein 5,10MTHFR. This is the only reaction capable of producing 5-methyl-H 4PteGlu, and in vivo, is irreversible (53). Vitamin B 12-dependent MetSyn then converts Hcy into methionine, a process that uses 5-methyl-H 4PteGlu and yields H 4PteGlu (46). This latter step also requires a recently discovered enzyme—methionine synthase reductase (MSR) which reductively activates MetSyn (54). Utilization of betaine for conversion of Hcy into methionine involves the vitamin B 12-independent enzyme betaine: homocysteine methyltransferase. De novo synthesized methionine can then be activated by ATP and the enzyme methionine adenosyl transferase to yield the methyl donor SAM, which methylates a variety of important biomolecules such as adrenalin, phosphatidylcholine, and carnitine. During this process, SAM is converted to S-adenosylhomocysteine (SAH), which is then hydrolyzed back to Hcy to recommence a new remethylation cycle (55). This is the only route for Hcy production in vertebrates. Hcy transsulphuration involves the condensation of this thiol with serine to form cystathionine, a vitamin B 6-dependent step catalyzed by cystathionine ␤-synthase. Beyond this point in the transsulphuration pathway, Hcy can no longer serve as a precursor for methionine biosynthesis. Cystathionine is then hydrolyzed to cysteine and ␣-ketobutyrate by another B 6-dependent enzyme, ␥-cystathionase. The metabolic regulation of the remethylation and transsulphuration pathways is under the influence of SAM, which coordinates the utilization of Hcy (52). SAM allosterically inhibits 5,10MTHFR while activating C␤S. Thus, when SAM levels drop, 5-methyl-H 4PteGlu formation is unre-
stricted while cystathionine formation is reduced. In this situation Hcy is conserved for methionine production. By contrast, elevated SAM enhances the transsulphuration of Hcy due to the activation of cystathioine ␤-synthase. The SAM/SAH ratio, concentration of the de novo methyl group acceptor Hcy, and specific dietary factors, particularly folate and methionine, but also vitamins B 12 and B 6, are therefore all important determinants of one-carbon metabolism and the metabolic balance between remethylation and transsulphuration pathways (52,55–59). Humans utilize more methyl groups than they consume from dietary methionine. The shortfall is made up from 5-methyl-H 4PteGlu and betaine. The continual demand for active methyl groups in the form of SAM is largely a result of creatine formation which consumes more SAM than all other transmethylations combined. In addition to coordinated allosteric regulation by SAM (59), 5-methyl-H 4PteGlu also exerts a regulatory effect on methionine metabolism. When a greater proportion of the methyl groups for production of SAM are derived de novo from 5-methylH 4PteGlu than from dietary methionine, excess 5-methyl-H 4PteGlu inhibits glycine-N-methyltransferase (GNMT) and therefore utilization of SAM (60). This conserves limited active methionine for essential methylation reactions. This mechanism (inhibition of 5-methyl-H 4PteGlu production by SAM, and inhibition by 5-methyl-H 4PteGlu of SAM utilization via GNMT) links de novo methyl group synthesis with control of the SAM/SAH regulatory “switch” and availability of dietary methionine. 5-Methyl-H 4PteGlu is also an inhibitor of porcine SHMT (61), where it may provide a feedback mechanism to further reduce 5-methyl-H 4PteGlu production for de novo methionine biosynthesis. Cellular Folate Compartmentalization The folate-dependent one-carbon transfer reactions above are compartmentalized (50). Folate pools in these two compartments are not in equilibrium. Figure 4 shows how folates are compartmentalized. Purified cytosolic and mitochondrial SHMT have similar activities but are enzymes with differing primary structures. Although folate coenzymes themselves move slowly between compartments, serine, glycine, and formate equilibrate rapidly, and an interdependence in one-carbon metabolism exists between compartments.
Serine, the major source of one-carbon units, dimethylglycine, and sarcosine, which are products of choline metabolism, enter mitochondria and produce 5,10-methylene-H 4 PteGlu which can generate 10-formyl-H 4 PteGlu for mitochondrial protein synthesis. Surplus formate can efflux back to the cytosol side of the mitochondrial membrane via the 10-formyltetrahydrofolate synthase reaction (50). THE ROLE OF FOLIC ACID IN HEALTH AND DISEASE Over the past decade, interest in the health benefits of folic acid has increased considerably. This was initially because of its role in preventing neural tube defects (NTD) like spina bifida (62). More recent interest in this B vitamin has arisen through its relationship with the potentially atherogenic thiol, Hcy, and consequently the beneficial role it might play in treating occlusive vascular disease (OVD) (63– 66). In fact, the health benefits of folate nutrition extend well beyond these important conditions. The various disorders now thought to be under the influence of either folate status and/or allelic variation in genes coding for folate-dependent enzymes include not only NTD and OVD, but other midline defects such as cleft palate (67), several cancers (cervical, bronchial, colon, and breast) (68 –71), Alzheimers disease (72), affective disorders (73), Down’s syndrome (74), unexplained recurrent early pregnancy loss, and preeclampsia (75,76). Most of these disorders can be explained within the context of folate-dependent one-carbon transfer reactions involving methionine, purine, and pyrimidine biosynthesis. However, the precise underlying cause is most probably linked to (a) one or more common gene polymorphisms of the Hcy remethylation cycle that alters cellular folate disposition, (b) low intakes of dietary folate, or (c) impaired DNA elaboration and/or gene expression linked to folate metabolism. In fact, it is highly likely that a combination of these factors (and as yet undiscovered gene mutations) may come into play and precipitate disease. The following account attempts to integrate recent findings in this field, and update our understanding of folate status by addressing the varied mechanisms through which folate ameliorates disease.
Cellular compartmentalization of folate metabolism.
Occlusive Vascular Disease Associated with Elevated Homocysteine Elevated plasma and urinary Hcy levels result from several inherited and nutritional diseases that affect Hcy remethylation and transsulphuration. The plasma Hcy range in normal subjects is quoted as 7–24 mmol/L, with urinary levels in the same range. Plasma Hcy exists in sulphydryl and mixed disulphide form. Homocystinuria, as an inborn error of metabolism, was first described by Carson and Neill (77). Mudd and co-workers later showed a deficiency of cystathionine ␤-synthase in liver biopsies taken from homocystinuric individuals (78). Other rare enzyme deficiencies leading to elevated Hcy were subsequently discovered. Homozygotes for this cystathionine ␤-synthase defect experience mental
retardation, thromboembolism, and premature OVD which presents at any age (79). Human and animal studies clearly link plasma Hcy level with vascular disease; sustained Hcy treatment in primates results in changes that mimic those observed in early human arteriosclerosis (80). Clinical studies support the experimental data, and are consistent in their findings, which indicate patients with OVD have higher blood Hcy than individuals with no disease. Despite this, most patients with vascular disease had values within what had been considered to be the normal range (81,82). A profound reciprocal relationship exists between blood Hcy and blood B vitamins (particularly folate). Because of this, folate supplements, especially when in combination with vitamins B 6 and B 12, may offer a preventative measure against OVD (83,84). It has
been calculated that 9% of male and 54% of female coronary artery deaths in the United States (around 50,000 deaths/year) could be prevented by mandatory fortification of grain products with 350 g PteGlu/100 g food (85). In 1998, the US-FDA mandated that folate fortification at 140 g PteGlu/100 g grain product be instituted. This is estimated to increase folate intake by 70 –120 g/day. The potential efficacy of further increasing this level of fortification is currently under debate. A few studies failed to find an association between plasma Hcy and OVD (86,87), however, sufficient evidence now exists to support such an association (63– 66). Even modest elevations in plasma Hcy have a pathological effect on vascular endothelium. Hcy is therefore an independent risk factor for arteriosclerosis and venous thrombosis, although the precise mechanisms involved are unclear. The prooxidant activity of this thiol may inhibit production of endothelin-derived relaxation factor and activate quiescent vascular smooth muscle cells. However, at physiological concentrations, Hcy may inhibit the vascular endothelial cell cycle at or before the G1-S junction. This inhibition seems to be mediated by a drop in carboxyl methylation, membrane association, and activity of p21 ras, a G1 regulator (88). Further interest exists in Hcy because of its association with a gene that encodes an important folatedependent allosteric enzyme (5,10MTHFR) that may link folate to OVD via regulation of plasma Hcy levels (89). A common mutation of the gene coding for this enzyme (677C 3 T-MTHFR) affects approximately 10% of people and is associated with elevated plasma Hcy. It is also associated with a tiered reduction in MTHFR activity between genotypes (89), indicating incomplete dominance at the biochemical level. The 677C 3 T-MTHFR variant in which a C-to-T substitution at nucleotide 677 converts an alanine to a valine (A222V) residue is easily identified using PCR followed by restriction enzyme digestion of the amplified product with Hinf 1. Guenther et al. showed that folate coenzymes stabilize 677 C 3 T MTHFR by preventing the polymorphic enzyme from relinquishing its flavin cofactor (90). Their model supports the treatment of hyperhomocysteinemia with folate, and provides an elegant example of a nutrient– gene interrelationship which may have profound health implications. Although small increases in Hcy are considered an independent risk factor for OVD (91,92), and despite Kang et al. showing that the variant 677C 3 T
thermolabile enzyme is also a risk factor for OVD (93), not all recent studies have shown the recessive tt genotype to be a significantly greater risk for OVD (91,94,95). Despite these studies, several reports do, however, link the tt genotype to OVD in specific population groups (95). Birth Defects Randomized studies (62,96,97) now support earlier work which showed that periconceptional supplementation with folic acid as PteGlu prevents NTD (98,99). However, the precise mechanism(s) responsible for NTD still remain unclear. The etiology of these birth defects is recognized as multifactorial, with both genetic and environmental elements. Although folic acid is the best known environmental factor, low vitamin B 12 levels have also been implicated in NTD pregnancy (100,101). Several studies show that an elevation in Hcy and increased frequency of 677C 3 T-MTHFR are both associated with NTD (102,103,104), however, research indicates that some Hcy probably originates from a locus other than 677C 3 T-MTHFR; Lucock et al. showed an increase in Hcy in NTD mothers (104), while the same patients did not have an increased frequency of the tt genotype; OR 0.98 (0.19 – 6.49) (35), yet they exhibited a significantly altered folate metabolism that was clearly not due to the 677C 3 T-MTHFR polymorphism (35). Several research groups studying the disposition of B vitamins and Hcy in blood of affected individuals have identified the remethylation cycle, including its MetSyn locus as an area where the underlying metabolic problem(s) in NTD may exist (100,105). However, no mutations of the MetSyn gene yet studied represent an increased risk for NTD. One of the most common mutations of MetSyn is 2756A ⬎ G MetSyn (106). Although 2756A ⬎ G MetSyn appears not to be a risk factor for NTD (107), the A 3 G transition at bp 2756, converting an aspartic acid into glycine (D919G), occurs at the penultimate site of an extended helix. This helix runs from the cobalamin domain to the SAM binding domain within prokaryotic MetSyn, and is reasonably homologous to the human form. Since glycine is a helix breaker, and aspartic acid a helix maker, the glycine substitution could affect the enzymes secondary structure with significant functional consequences. Despite this, the 2756A ⬎ G polymorphism frequency is similar in NTD and controls in a number of recent studies (35,106,107). One report does, however, suggest that
this mutation may make a moderate, but significant, contribution to clinical conditions that are associated with elevated Hcy (108). Many genes coding for folate-dependent enzymes have now been studied for mutations that might account for NTD. Of these, only 677C 3 T-MTHFR (102,103), a second MTHFR mutation (A to C substitution at bp 1298) (109) and a polymorphism of MSR (A to G substitution at bp 66, converting an isoleucine to a methionine residue, I22M (110) currently represent increased risk for an affected pregnancy. However, 677C 3 T-MTHFR only accounts for a small proportion of all NTDs (111), while 66A 3 G-MSR increases the risk of NTD only when cobalamin is low or in the presence of the former 677C 3 T-MTHFR mutation (110). Combined heterozygosity for 1298A 3 C-MTHFR and 677C 3 T-MTHFR was found in 28% of NTDs versus 20% of controls giving an odds ratio of 2.04. This combined heterozygosity risk factor therefore accounts for a proportion of folate-related NTDs not explained by 677C 3 T-MTHFR alone (109). Despite these findings, not all studies have been able to show an increased risk for NTD in the presence of the 677C 3 T-MTHFR mutation (35,112,113). Other potential gene mutations that could account for NTD have been investigated and include 5,10methylenetetrahydrofolate dehydrogenase (MTHFD). However, single-strand conformation polymorphism analysis of the MTHFD gene followed by sequencing found only two amino acid substitutions which in neither case were deemed causal for the majority of NTD pregnancies (114). A further enzyme, SHMT, may be of interest in the future, since it represents the major entry point of single carbon units into folate-dependent one-carbon metabolism. Currently, the author is unaware of any work relating mutations of this gene to NTD pregnancy. Changes in the intracellular folate profile associated with an altered equilibrium in the Hcy remethylation cycle due to common polymorphisms of folate/vitamin B 12 metabolism may have important effects on folate-dependent biosynthetic reactions and could therefore have primary or secondary involvement in the etiology of NTD. In fact, since the remethylation cycle enzymes—MTHFR, MSR, and MetSyn—are responsible for generating 5-MethylH 4PteGlu and converting it to a biologically more useful form of the vitamin that can be used in nucleotide biosynthesis, we are dealing with a critical nexus of one-carbon metabolism. In particular, it can be predicted that a problem anywhere within
this locus would lead to a redistribution of cellular folate derivatives, and reduced cellular folate levels (46). Indeed, differences in the composition of cellular folates between NTD and control mothers have been described (35). Although it has yet to be determined whether Hcy is simply a marker, or has any causal role in NTD, studies have shown that NTD Hcy levels are only mildly elevated above normal compared to many non-NTD homozygous 677C 3 T-MTHFR individuals studied, whose levels are often 2– 4x higher than wildtypes. In one study, typical median Hcy values were 6.5 and 8.2 mol/L for control and NTD, respectively (104), compared to what can often be in excess of 20 mol/L for homozygous 677C 3 T-MTHFR individuals. It is recommended that all women planning a pregnancy should take an extra 400 g folate per day. Many pregnant women are not aware that they should have taken folate supplements before conception, although awareness campaigns are helping this situation, with GPs and other health professionals playing an important role in disseminating information to women of child bearing age. Unfortunately, those groups most likely to have low folate intakes (smokers and young women) are probably the least likely to make contact with health professionals before pregnancy or indeed to plan their pregnancies at all. Current views on the prevention of NTD advocate a population approach using food fortification of grain products with PteGlu at source. Intervention studies have recently shown that periconceptional folate–multivitamin supplements can reduce other major congenital abnormalities such as conotruncal cardiovascular malformations, defects of the urinary tract, congenital hypertrophic pyloric stenosis, and congenital limb deficiencies, although the rate of cleft lip and palate were not lowered by multivitamins (115). A more recent study, however, showed that recessive homozygosity for 677C 3 T-MTHFR is more frequent in individuals with isolated cleft palate, and the authors suggest this could be etiologically important (67). Role of Folate in the Development of Cancer Several explanations can be put forward for the beneficial role that folate seems to have in the etiology of malignant conditions. As alluded to earlier, 5-methyl-H 4PteGlu is a critical cofactor in the de novo biosynthesis of SAM, which is the body’s primary methyl donor. The SAM-dependent methyl-
ation of specific CpG sites on DNA regulates gene expression. Since the methylation of CpG clusters associated with promoter regions tend to silence gene expression, impaired folate-dependent de novo methionine biosynthesis may lead to methyl group deficiency, altering the normal control of protooncogene expression (116). A second route by which folate may influence malignant transformations occurs where a generalized folate depletion precipitates the misincorporation of uracil into DNA, causing chromosome breakages (117). This event is particularly sensitive to the 5,10-methylene-H 4PteGlu level, since this coenzyme is required by thymidylate synthase for converting dUMP 3 dTMP. An increasing number of specific cancers are being linked to folate status: An association between folate and premalignant cervical dysplasia was demonstrated by Butterworth (68). Kamei et al. (69) showed that folate could reduce squamous metaplasia of the bronchial epithelium, while interest is presently focused on the role of dietary folate and mutations in genes coding for folate-dependent enzymes in relation to colon cancer (70). In particular, the A222V amino acid substitution arising from the common 677C 3 T-MTHFR polymorphism may offer protection against colon cancer under certain conditions (118). This may be due to the mutation increasing the 5,10-methylene-H 4PteGlu level and reducing the likelihood of uracil misincorporation into DNA. Similarly, Skibola et al. (119) have shown that individuals with 677TT-MTHFR and both homozygous recessive and heterozygous 1298A 3 CMTHFR have a decreased risk of acute lymphoblastic leukemia (but not acute myeloid leukemia), indicating that folate depletion may play a role in the development of this form of cancer. Some of the most current findings on folate and cancer development indicate that low levels of the vitamin are a risk for breast cancer when associated with high alcohol intake (71). It has also been shown that a threshold exists for vitamin B 12 and increased breast cancer risk among postmenopausal women (120). The observation of a parallel decline in NTD and gastric cancer which exhibit a familial association indicate a common etiology which may possibly be folate related (121). Folate and Affective Disorders Parenteral treatment with pharmacological 5-methyl-H 4PteGlu has been used by some research-
ers as a treatment for mood disorders (73). It is unclear whether the folate effect is mediated via a direct impact on neuronal membranes, or whether 5-methyl-H 4PteGlu acts indirectly through an alteration in neurotransmitter metabolism—methyltransferases such as catechol-O-methyltransferase, hydroxyindole-O-methyltransferase, and phenylethanolamine-N-methyltransferase are crucial for the synthesis of neuronal products. SAM, produced de novo from 5-methyl-H 4PteGlu, is the methyl donor for all these important enzymes. Further evidence for a neuronal role for folate is indicated by plasma 5-methyl-H 4PteGlu being taken up at the choroid plexus and concentrated in the cerebro-spinal fluid. Down’s Syndrome Since evidence exists that abnormal folate and methyl group metabolism can lead to genomic hypomethylation and abnormal chromosome segregation, James et al. (74) hypothesized that the 677C 3 T-MTHFR polymorphism may be a risk factor for maternal meiotic nondisjunction and Down’s syndrome (trisomy 21) in young mothers. Results from their study showed that folate metabolism is abnormal in mothers of these children, and that this may be explained in part by the 677 nucleotide substitution. Alzheimer’s Disease A recent study indicates that OVD may contribute to the cause of Alzheimers disease. Since elevated plasma Hcy is a risk factor for vascular disease, it may also be relevant to Alzheimers disease. Clarke et al. (72) showed that low blood levels of folate and vitamin B 12, and elevated Hcy were indeed associated with Alzheimers disease. They demonstrated that the stability of Hcy levels over time and lack of relationship with duration of symptoms argue against these findings being a consequence of the disease. Folate Metabolism in Preeclampsia, Recurrent Miscarriage, and Low Birth Weight Folate is a critical nutrient in maintaining normal cell growth and division and is thus important in pregnancy. Studies have shown an inverse correlation between maternal Hcy and birth weight (122,123). There is a also a direct association between both maternal serum folate and red cell folate and birth weight (124,125). It is unclear whether the link between elevated Hcy levels in mother or fetus
and low birth weight is mediated through defective placental function, although this is clearly a possibility. Studies have also described increased Hcy in women with placental abruption or infarction as compared to normal controls (123,126,127). Further to this, preeclampsia, a condition characterized by defective placentation, is also associated with elevated Hcy levels (75,76). Collectively, these are all pathological conditions associated with notable maternal and fetal morbidity and mortality. Indeed, even low birthweight in itself is associated with OVD in later life (128). The common 677C 3 T-MTHFR polymorphism may be clinically relevant in these conditions. In women with preeclampsia there is higher carriage of the mutant T allele and representation of the recessive genotype (129). This association is also seen in women with unexplained recurrent early pregnancy loss (130). Clearly, folate status combined with specific common polymorphisms of genes coding for folate-dependent enzymes maybe important determinants of pregnancy outcome. Folate Deficiency Overt folate deficiency and its associated anemia are now rare, and largely confined to the developing world where it affects a small proportion of the population. Folate deficiency can also arise through malabsorption caused by coeliac disease, where sensitivity to the flour protein, gliadin, leads to villus atrophy. A similar phenomenon occurs in tropical sprue (131). Further causes of folate deficiency are alcohol consumption (131) and anticonvulsant therapy (131), with several studies looking at the effects of phenytoin on folate status. Folate deficiency leads to inadequate nucleic acid synthesis and impairs cellular division. The effect of folate and vitamin B 12 deficiency are the same and are most profound in the hemopoietic tissue of bone marrow followed by epithelial tissues of the skin, gut mucosa, and genito-urinary system. Erythroblasts enlarge and fail to divide properly— circulating erythrocyte numbers drop and become macrocytic. Eventually, leukocytes and platelets become affected. This condition is known as megaloblastic anemia, and is sometimes associated with pregnancy due to an increased demand for folate. Vitamin B 12 is essential for MetSyn activity. H 4PteGlu, the precursor of cellular folates, cannot be regenerated from 5-methyl-H 4PteGlu if the avail-
ability of B 12 is compromised. If this happens, folate is trapped as 5-methyl-H 4PteGlu and since reversal of 5,10MTHFR is energetically unfavorable (53), nucleotide biosynthesis is reduced. A shortage of vitamin B 12 therefore impairs DNA synthesis and a megaloblastic anemia clinically indistinguishable from that due to folate deficiency arises. This locking up of folate in its 5-methyl-H 4PteGlu form is often referred to as the “methylfolate trap hypothesis” (132), and is exacerbated by a reduced intake of methionine, since levels of SAM drop, preventing allosteric inhibition of MTHFR, which causes further accumulation of 5-methyl-H 4PteGlu (46). At the time of writing worries have been voiced regarding the potential masking of pernicious anemia due to mandatory fortification of food with PteGlu. FORTIFICATION OF FOOD WITH FOLIC ACID At the cellular level, individual folate coenzymes respond to changes in folate availability. In one study (104), folate supplementation (400 g/day) for 1 month caused a 29% increase in total formylfolate and a 35% drop in total methylfolate (n ⫽ 13). The response of red cell folates to supplementation also varied according to the number of glutamate moieties conjugated to the pteroic acid. Most of the decline within the methylfolate pool was due to a loss in hexaglutamates, with a minor loss of pentaglutamates. By contrast, mono- to tetraglutamates of methylfolate actually became more abundant. This may represent the first human study to show that oligo-␥-glutamyl chain length is sensitive to a changing folate status. In vitro studies support the idea that glutamate chain length diminishes as a response to increased folate supply and vice-versa (47). The most likely purpose of extending the folyloligo-␥-glutamyl chain in folate depletion is an increased avidity for folate-dependent enzymes. Such a mechanism would enable folate-dependent pathways, like nucleotide biosynthesis, to be maintained in the face of perturbations in folate availability. It has been suggested by policy makers that mandatory fortification of food aimed at increasing the average daily folic acid intake of every woman by 400 g/day should be implemented, the average intake currently being 200 g/day. The folate analogue used in tablet form and in fortified foods is PteGlu. Unlike most natural forms of the vitamin, this synthetic coenzyme is cheap to produce and
highly stable. The body is reasonably efficient at metabolizing PteGlu into monoglutamyl 5-methylH 4PteGlu, the plasma form of the vitamin. However, since PteGlu does not occur naturally, and as research shows that the absorption and biotransformation of PteGlu is saturated at doses in the region of 266 – 400 g (133,134), the possibility is raised of a lifetime exposure to unmetabolized PteGlu, with mandatory fortification at 400 g/day. Such exposure may present no health risk at all, however, we can’t know this with 100% certainty. Other issues have also been raised regarding concerns over mandatory fortification masking the diagnosis of B 12 deficiency in pernicious anemia, along with the associated and serious progression of neurological sequelae. Therefore, taking account of the possible risks, we have available a simple population measure that could lead to an enormous improvement in our health and well being, and perhaps it is not so much whether we should fortify, but by how much. The benefits of supplementation are clearly alluded to above, and simply cannot be ignored.
3. 4. 5.
SUMMARY Initial interest in folic acid arose through its role in preventing neural tube defects such as spina bifida. More recently its relationship with homocysteine and OVD has taken center stage, although its potential benefits may extend beyond these important clinical conditions to embrace other birth defects, a number of different cancers, dementia, affective disorders, Down’s syndrome, and serious conditions affecting pregnancy outcome. The beneficial effect of maintaining a healthy folate status in these conditions can be explained largely within the context of folate-dependent pathways leading to methionine and nucleotide biosynthesis, and genetic variability arising from a number of common polymorphisms of folate-dependent enzymes involved in the Hcy remethylation cycle. At the time of writing, allelic variants of folate genes discovered that have a high frequency in the population include 677C 3 T-MTHFR, 1298A 3 C-MTHFR, 2756A 3 G-MetSyn, and 66A 3 G-MSR. Future endeavour will undoubtedly uncover variability in other genes encoding folate-dependent enzymes, and lay bare further gene–nutrient interrelationships that underpin a variety of disease states.
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