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Folate and vitamin B12 metabolism: Overview and interaction with riboflavin, vitamin B6, and polymorphisms

Barry Shane
Abstract
This paper provides a general review on folate and vitamin B12 nutrition and metabolism and the metabolic interrelationship between these vitamins. The effects of some common polymorphisms in folate and vitamin B12 genes and the influence of vitamin B6 and riboflavin status on folate and vitamin B12 metabolism are also discussed.
H2N H N
2 3 4 5N

H2N

O H N C O C CH

N1 N
8 7

10 CH2 9

H N

O CH2 C O Glutamate

CH2

Pteridine

p-Aminobenzoic acid

Pteroic acid Folic acid, pteroylglutamate (PteGlu) H N


2 3 4 5 NH 8 7 6

O H N C O C CH

Key words: Folate, homocysteine, megaloblastic


anemia, methionine synthase, MTHFR, neural tube defects, one-carbon metabolism, polymorphisms, riboflavin, vitamin B12

O H N CH2 C O C CH

N1 HN

10 CH2 9

H N

CH2

O CH2 C O

CH2 n-1

Tetrahydrofolylpoly- -glutamate (H4PteGlun)

Folate

FIG. 1. Structures of folic acid and tetrahydrofolylpoly--glutamate

Folates comprise a family of chemically related compounds based on the folic acid structure (fig. 1) [1, 2]. Folates in tissues act as donors and acceptors of one-carbon units in metabolic reactions known as one-carbon metabolism. These one-carbon units can be at the oxidation level of methanol (5-methyl-tetrahydrofolate), formaldehyde (5,10-methylene-tetrahydrofolate), or formate (5- or 10-formyl-tetrahydrofolate or 5,10-methenyl-tetrahydrofolate). Practically all tissue folates are polyglutamate forms in which the glutamate tail is extended via the gamma-carboxyl of glutamate. Glutamate chain lengths can vary from about 4 to 10 in human tissues. Metabolism of folates to polyglutamate forms is required for their biological activity, as the polyglutamate forms are much more effective substrates for folate-dependent enzymes than are
The author is affiliated with the Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California, USA. Please direct queries to the author: Barry Shane, 329 Morgan Hall, University of California, Berkeley, CA 94720-3104, USA; e-mail: bandie@berkeley.edu.

monoglutamates, which are the transport forms of the vitamin. Conversion of folates to polyglutamates of chain length greater than three or more is also required for effective retention of folate by tissues.
Absorption, transport, and bioavailability

Folic acid does not occur in nature and is rarely found in unfortified foods, and is not an active form of the coenzyme. However, it is the most common form of folate used in supplements and in fortified food products because it is highly bioavailable, chemically stable, and is readily reduced to tetrahydrofolate, the active coenzyme form of folate. Food folates typically occur in a reduced, polyglutamyl form. Before absorption, they are cleaved to their monoglutamyl forms by a brush border glutamylhydrolase, sometimes called intestinal folate conjugase [3]. Folates are absorbed in the proximal small intestine by a saturable, pH-sensitive transporter that transports oxidized and reduced folates [4]. Most dietary folate, and folic acid in the diet, is metabolized to 5-methyl-tetrahydrofolate during its passage across the intestinal mucosa. When high doses
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Food and Nutrition Bulletin, vol. 29, no. 2 (supplement) 2008, The United Nations University.
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of folic acid or other folate forms are consumed, some appear in the peripheral circulation unchanged. The bioavailability of folic acid is close to 100% when consumed on an empty stomach. Although information about bioavailability of food folate and folic acid consumed with food is limited, the current best estimates are 50% (aggregate food folate) and 85% (folic acid) [5]. Very large doses of folic acid and other folates are well absorbed and can result in very high plasma vitamin levels. However, these fall quite rapidly as the renal threshold is exceeded and much of the dose is excreted within 24 hours. Although plasma folate levels in excess of 100 times normal can be achieved, tissue folate levels only increase marginally, often less than 2-fold due to the limited ability of tissues to metabolize these large doses to the polyglutamate forms required for folate retention. Folate monoglutamate in plasma is transported into tissues via the reduced folate carrier, encoded by the RFC gene [6]. This ubiquitously expressed carrier has poor affinity for folic acid, and is specific for reduced folates. A distinct high-affinity folate transporter known as folate-binding protein, or the folate receptor, is expressed in a more limited range of tissues. High levels of folate-binding protein are expressed in the choroid plexus, kidney proximal tubes, and placenta, and in a number of human tumors, while lower levels have been found in a variety of other tissues. This transporter is responsible for reabsorption of folate in the kidney by a receptor-mediated endocytotic process and is believed to play a similar role in folate transport in other tissues [7]. An additional cellular transporter responsible for the transport of reduced folate monoglutamates into the mitochondrion has recently been identified [8].
Tissue retention and turnover

status, whole body folate turns over quite slowly with a half-life in excess of 100 days [10]. Urinary excretion of intact folate accounts for only a very small proportion of this turnover. Over 99% of tissue folate is in the polyglutamate form. The actual mechanism of catabolism is poorly understood but primarily involves cleavage at the C9-N10 bond to generate p-aminobenzoylpolyglutamates and a pterin moiety [11]. The p-aminobenzoylpolyglutamates are hydrolyzed to the monoglutamate by a lysosomal glutamylhydrolase and acetylated, and then excreted in urine as N-methylaminobenzoyl-monoglutamate. The pterin moiety is excreted in bile and appears in the feces. Feces contain very high levels of folate, but most of this arises from bacterial synthesis in the lower gut. Studies in rodents have shown that some of this bacterially synthesized folate is bioavailable [12]. Whether folate synthesized by gut bacteria in humans is also bioavailable has not been determined.
Biochemistry and functions

Folate coenzymes are involved in three major interrelated metabolic cycles in the cytosol of cells [1, 2]. These cycles are required for the synthesis of thymidylate and purines, precursors for DNA and RNA synthesis, and for the synthesis of methionine from homocysteine and the interconversion of serine and glycine. 5,10-Methylene-tetrahydrofolate plays a central role in these cycles, as it can be used directly for thymidylate synthesis, or it can be reduced to 5-methyltetrahydrofolate in the methionine synthesis cycle, or can be oxidized to 10-formyl-tetrahydrofolate to be used in purine synthesis. Although these synthetic cycles are located in the cytosol, mammalian cells also contain a large mitochondrial folate pool, which is also involved in the provision of one-carbon precursors for cytosolic one-carbon metabolism.
Serine-glycine interconversion

Folate monoglutamate transported into cells is metabolized to polyglutamate forms by the enzyme folylpolyglutamate synthetase. Mammalian cells contain mitochondrial and cytosolic isozymes encoded by a single gene [9]. In the cytosol, much of the entering folate is metabolized to the 5-methyl-tetrahydrofolate derivative which is a poor substrate for folylpolyglutamate synthetase. This has to be metabolized to tetrahydrofolate via the methionine synthase reaction before effective polyglutamylation and tissue retention is achieved. As the flux of 5-methyl-tetrahydrofolate through the methionine synthetase reaction is quite limiting, particularly when the cell contains high levels of 5-methyltetrahydrofolate polyglutamate, much of the newly absorbed folate would not be retained by the tissue and would appear in the circulation predominantly as 5-methyl-tetrahydrofolate. Under normal conditions of dietary intake and

Serine is the major provider of one-carbon units for folate-dependent one-carbon metabolism [1, 2]. It donates its -carbon to tetrahydrofolate to generate glycine and 5,10-methylene-tetrahydrofolate. The reaction, which is catalyzed by the PLP-dependent enzyme serine hydroxymethyltransferase (SHMT), is at near equilibrium and can be easily reversed. Mammalian tissues contain two distinct isozymes of SHMT, one cytosolic and one mitochondrial, encoded by different genes [13]. Recent studies suggest that the mitochondrial isozyme, which is expressed in all tissues, may be responsible for the generation of the majority of one-carbon units required for cytosolic one-carbon metabolism [1416]. 5,10-Methylene-tetrahydrofolate generated via the mitochondrial SHMT reaction is oxidized to 10-formyl-tetrahydrofolate and then hydrolyzed to formate. The formate produced exits the

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Folate and vitamin B12 metabolism

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mitochondria and the one carbon is reincorporated into the cytosolic one-carbon pool as 10-formyltetrahydrofolate. 10-Formyl-tetrahydrofolate can be reduced to 5,10-methylene-tetrahydrofolate in the cytosol via the action of a reversible C1 tetrahydrofolate synthase enzyme. The tissue distribution of the cytosolic isozyme of SHMT is more limited than the mitochondrial enzyme; the cytosolic isozyme is highly expressed in liver and kidney and in rapidly replicating cells. A major role of the hepatic cytosolic SHMT enzyme may be to synthesize serine from glycine for use in gluconeogenesis.
Nucleotide synthesis

Folate is required for the synthesis of thymidylate, a nucleotide required specifically for the synthesis of DNA. Thymidylate synthase catalyzes the transfer of the onecarbon group from 5,10-methylene-tetrahydrofolate to the 5-position of dUMP and its reduction to a methyl group to generate dTMP (fig. 2). The folate molecule also provides the reducing component in this reaction and the tetrahydrofolate is oxidized to dihydrofolate.
Homocysteine Methyl-B12 MS 5-Methyl-H4PteGlun NADP+ NADPH + H+ dUMP dTMP TS Glycine H2PteGlun

The dihydrofolate generated has to be reduced back to tetrahydrofolate before it can be reutilized in one-carbon metabolism in a reaction catalyzed by dihydrofolate reductase (DHFR). Thymidylate synthetase activity is only expressed in replicating cells and is highest in fastgrowing cells. Consequently, drugs targeted to DHFR, such as methotrexate, have proven to be effective chemotherapeutic agents as they are selective inhibitors of rapidly growing cells [17, 18]. Folate coenzymes are also used in two steps of de novo cytosolic purine biosynthesis. The C-8 and C-2 positions of the purine ring are derived from 10-formyl-tetrahydrofolate in reactions catalyzed by glycinamide ribonucleotide transformylase and 5-amino-4-imidazolecarboxamide ribonucleotide transformylase (fig. 2).
Methionine synthesis

The methylation of homocysteine to produce methionine uses 5-methyl-tetrahydrofolate as the methyl donor in a reaction catalyzed by methionine synthase, one of only two vitamin B12-dependent enzymes in mammals [2, 19] (fig. 3). 5-Methyl-tetrahydrofolate
Methionine

H4PteGlun Serine Glycine NADP+

Methionine cycle
MTHFR SHMT

5,10-Methylene-H4PteGlun

MTHFD

NADPH + H+ Hystidine

5,10-Methylene-H4PteGlun

Thymidylate cycle

SHMT Serine

Purine cycle

CYH

H2O

Formate

NADPH + H+

DHFR

H4PteGlun

H4PteGlun

GART AICART

10-Formyl-H4PteGlun

NADP+

FGAR FAICAR

GAR AICAR

FIG. 2. Metabolic cycles of cytosolic one-carbon metabolism. SHMT, serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; MS, methionine synthase; TS, thymidylate synthase; DHFR, dihydrofolate reductase; MTHFD, methylenetetrahydrofolate dehydrogenase; CYH, methenyltetrahydrofolate cyclohydrolase; GART, GAR formyltransferase; AICART, AICAR formyltransferase; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; FAICAR, formyl-aminoimidazole carboxyamide ribonucleotide; AICAR, aminomidazole carboxamide ribonucleotide; GAR, glycinamide ribonucleotide; FGAR, formyl-glycinamide ribonucleotide
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is generated from 5,10-methylene-tetrahydrofolate in a reaction catalyzed by the flavoprotein methylenetetrahydrofolate reductase (MTHFR). Methionine can be metabolized to S-adenosylmethionine, which acts as the methyl donor in many reactions, including the methylation of DNA, histones and other proteins, neurotransmitters, and phospholipids, and the synthesis of creatine. These methylation reactions play important roles in development, gene expression, and genomic stability. S-Adenosyl-homocysteine, the product of methylation reactions, is a potent inhibitor of many methyltransferases and is catabolized by hydrolysis to adenosine and homocysteine. Homocysteine in liver and kidney can also be converted back to methionine via the folate-independent betaine-homocysteine methyltransferase (BHMT), which catalyzes the transfer of one of the methyl groups of betaine to homocysteine to generate methionine and dimethylglycine [2]. Betaine arises from choline oxidation in liver mitochondria. BHMT is present in high concentrations in liver, and limited studies in humans indicate that up to 30% of homocysteine remethylation may occur through this reaction [20]. In the liver and kidneys, homocysteine can also be metabolized to cysteine via the transsulfuration pathway, which involves two PLP-dependent enzymes, cystathionine -synthase and cystathionase. In most other tissues, homocysteine is exported to the circulation or is reconverted back to methionine via the folateRemethylation
Serine SHMT Glycine PLP THF

dependent methionine synthase reaction. Methionine is a dietary-essential amino acid, as mammals lack the ability to synthesize homocysteine, its carbon skeleton, and because homocysteine is not normally present in the diet. It is often the most limiting amino acid in human diets. Methylation reactions account for a large proportion of the methyl group intake in humans, and the methionine synthase reaction allows salvage of its backbone after its use for methylation. The folate-dependent methionine cycle is very sensitive to inadequate folate status. When folate status is poor, the decreased ability to remethylate cellular homocysteine results in an increased plasma homocysteine level, and the plasma total homocysteine level is an indirect indicator of folate insufficiency [21, 22].

Vitamin B12
Vitamin B 12 (cobalamin) consists of a central cobalt atom surrounded by a heme-like planar corrin ring structure, with the four pyrrole nitrogens coordinated to the cobalt (fig. 4). It contains a phosphoribo-5,6-dimethylbenzimidazolyl side group, with one of the nitrogens linked to the cobalt by coordination at the bottom position. When bound to enzymes, this lower axial ligand is usually replaced by an active site histidyl residue. The upper axial position can be occupied by a number of different ligands,
Transmethylation

Methionine MATI/II Glycine

AdoMet
B12 BHMT

X
GNMT

MS CH2-THF MTHFR

AdoHcy
Homocysteine SAHH C S PLP

CH3-X
Sarcosine

FAD

CH3-THF

Adenosine

Cystathionine Cystathioninase PLP

Transsulfuration
Cysteine

FIG. 3. Methionine synthesis and transmethylation cycles and transsulfuration pathway in liver. THF, tetrahydrofolate; FAD, flavin adenine dinucleotide; PLP, pyridoxal phosphate; B12, vitamin B12; AdoMet, adenosylmethionine; AdoHcy, adenosylhomocysteine; SHMT, serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; MS, methionine synthase; MATI/II, methionine adenosyltransferase I and II; SAHH, AdoHcy hydrolase; CS, cystathionine -synthase; BHMT, betainehomocysteine methyltransmethylase; glycine NMT, N-methyl transferase
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Folate and vitamin B12 metabolism


CH3 CH3 CH CO 2 C C CH CH2 N C C N Co3+ N C C C CH3 CH CH2 C CH CH3 C CH3 CH2 CO NH2 Corrin ring

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NH2 NH2 CO CO CH2 CH2 NH2 CH2 CO NH2

CH2 CH C C N H3C C H3C CH N CH2 C C CH3

NH2

CO CO NH

CH2 CH2

CH2

Aminoisopropanol CH3

CH2 CH O O P O HO C H O O N CH N CH CH3 C C CH3 Dimethylbenzimidazole

C C

Ribose 3-phosphate CH HO CH2

C H CH

CH

FIG. 4. Structure of vitamin B12

including methyl, hydroxyl, and 5-deoxyadenosyl groups. In what is commonly known as vitamin B12, the upper ligand is a cyano group (cyanocobalamin) [2].
Absorption, transport, and bioavailability

The bioavailability of food vitamin B12 varies depending on the amount of vitamin B12 in the diet but normally averages around 50% [23]. Vitamin B12 in food is bound to protein and is released in the stomach by the acid environment and by proteolysis of binders by pepsin. The released vitamin B12 initially binds to R-binders, which are dietary proteins that have affinity for vitamin B12. The stomach contains specialized parietal cells that contain the H+, K+-ATPase that produces gastric acid. In humans, these cells also secrete a 50-kDa glycoprotein called intrinsic factor (IF) that can bind vitamin B12. As the vitamin B12-R-binder complexes pass through the small intestine, the R-binders are hydrolyzed by pancreatic proteases, and the freed vitamin B12 binds to IF. Sufficient IF is released following a meal to bind 2 to 4 g of vitamin B12. Vitamin B12 is absorbed via receptors located at the distal ileum at the end of the small intestine. The IF-receptor (cubulin) recognizes the IF-vitamin B12 complex, not vitamin B12 or unligated IF, and the complex is internalized by a receptor-mediated endocytotic process [24]. The endosomes fuse with lysosomes, the IF is degraded, and the vitamin B12 is released into the cytosol. Vitamin B12 is released from the gut epithelial cells as a complex bound to a 38-kDa protein called transcobalamin II (TC-II). This process of absorption across the gut epithelium takes about 3 to 4 hours [25]. The 460-kDa IF receptor (cubulin) is also expressed in the kidney and in the yolk sac of animals [26]. The reason why it should be expressed in the kidney is not clear, but may

be related to its wide specificity for ligands. Cubulin interacts with a second protein, amnionless (AMN), which shows a similar tissue distribution and appears to be required for the apical localization of cubulin in polarized cells [27]. Vitamin B12 can also be absorbed by a diffusion-like process, but this is very inefficient: less than 1% of a vitamin B12 dose can be absorbed by this process, but this can be quantitatively important if high doses are provided to individuals who lack IF or malabsorb vitamin B12 in food. The TC-II-vitamin B 12 complex carries newly absorbed vitamin B12 around the body and provides tissues with vitamin B12. TC-II-B12 is transported into tissues by receptor-mediated endocytosis, in this case via a receptor that recognizes TC-II. The nature of the TC-II receptor is controversial, as a number of different-size proteins have been reported in the literature as the putative receptor. In the kidney, a very large protein (megalin) is responsible for the reabsorption of TC-II-bound B12. Megalin is also found in the yolk sac and, like cubulin, shows a wide specificity for ligands [28]. However, the TC-II-B12 receptor in liver and most other tissues is not thought to be megalin. Once endocytosed, the TC-II-B12 complex is degraded in the lysosome, and the free vitamin B12 is transported out of the lysosome to the cytosol [29]. The lysosomal vitamin B12 transporter has not been characterized. The half-life of the TC-II-vitamin B12 complex in plasma is about 6 minutes. Plasma contains two additional vitamin B12binding glycoproteins or R-binders called haptocorrin (transcobalamin I, TC-I) and transcobalamin III (TC-III). They are less specific than TC-II and also bind B12 analogs. Plasma turnover of these transcobalamins is much slower than that of TC-II. Although newly absorbed vitamin B12 is associated with TC-II, about 75% of the plasma vitamin

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B12 (mainly methyl-Cbl) is associated with TC-I. In healthy individuals, total TC-II levels are 2- to 3-fold higher than TC-I levels. However, TC-I is about 90% saturated with vitamin B12, while only about 10% of TC-II is holo-TC-II. Because of its much faster turnover, TC-II delivers about 4 nmol/day of vitamin B12 to tissues, with a maximum capacity of about 6 nmol/day, while TC-I turnover accounts for only 0.1 nmol/day [30]. Although the role of TC-I is not entirely clear, this 70-kDa glycoprotein transfers vitamin B12 to the liver. Liver contains a nonspecific asialoglycoprotein receptor that can mediate uptake of TC-I and TC-III. Most of the body store of vitamin B12, estimated at about 2 to 3 mg, is in the liver. 5-Deoxyadenosylcobalamin, attached to methylmalonylCoA mutase, is the major form of the vitamin in liver, whereas methylcobalamin is the major form in plasma. The vitamin is excreted via the urine and via the bile. Normally, the enterohepatic circulation results in effective reuptake, via the IF receptor, of biliary vitamin B12. Turnover rates of whole body vitamin B12 have been estimated at about 0.1%/day.
Biochemistry and functions

in many of these steps in vitamin B12 metabolism have been described [34].
Methionine synthase

Mammals need vitamin B12 as a cofactor for two enzymes, cytosolic methionine synthase, which utilizes methylcob(III)alamin as the cofactor, and mitochondrial methylmalonyl CoA mutase, which uses 5-deoxyadenosylcob(III)alamin as its cofactor [3133]. Vitamin B12 is released into the cytosol of cells as hydroxycob(III)alamin and is reduced to cob(I)alamin. Cob(I)alamin is then methylated to methylcob(III) alamin after binding to methionine synthase. Alternatively, vitamin B 12 can be transported into the mitochondria and reduced, and the 5-deoxyadenosyl ligand added from ATP in a reaction catalyzed by a deoxyadenosyltransferase. Rare human genetic defects
Co1+ 5-Methyl-H4PteGlun Cob(I)alamin
..

Methylcobalamin is a cofactor for the previously described folate-dependent methionine synthase involved in homocysteine remethylation (fig. 5). Methionine synthases are Zn metalloproteins and contain three domains: a catalytic domain containing the binding sites for 5-methyl-H4PteGlun and homocysteine, a vitamin B12 domain in which the vitamin B12 cofactor binds, and an accessory protein domain [35]. Most of the methionine synthase in mammalian tissues is normally present as the holoenzyme form, containing a tightly bound vitamin B12 cofactor. The cob(I) alamin cofactor is methylated by 5-methyl-H4PteGlun, generating enzyme-bound methylcob(III)alamin and releasing H4PteGlun, and then methylcob(III)alamin transfers its methyl group to homocysteine to generate methionine. Heterolytic cleavage of the cobalt-carbon bond regenerates the enzyme-bound cob(I)alamin cofactor. The cofactor is occasionally oxidized to the nonfunctional cob(II)alamin form during catalysis. The enzyme is reactivated by methionine synthase reductase, an accessory protein that catalyzes the AdoMet and NADPH-dependent reductive methylation of enzyme-bound cob(II)alamin to methylcob(III) alamin. Methionine synthase reductase contains binding sites for NADPH, FAD, and FMN and belongs to the P450 reductase protein family [36].
Methylmalonyl CoA mutase

Mitochondrial -oxidation of dietary odd-chain fatty acids produces propionyl CoA in addition to acetyl CoA. Propionyl CoA is converted to D-methylmalonyl CoA in a reaction catalyzed by the biotin-dependent propionyl CoA carboxylase. Propionyl CoA and methylmalonyl CoA can also arise during catabolism

Methionine CH3 S COO CH2 NH3


+

Cob(II)alamin

Co2+ AdoMet + NADPH | H AdoHcy NADP+

(CH2)2

Accessory proteins

SH (CH2)2

COO CH NH3
+

H4PteGlun

Methylcob(III)alamin CH3 Co3+

Homocysteine

FIG. 5. Methionine synthase reaction


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Folate and vitamin B12 metabolism

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of isoleucine, valine, methionine, and threonine. A racemase converts D-methylmalonyl CoA to L-methylmalonyl CoA, and the 5-deoxyadenosylcobalamin dependent methylmalonyl CoA mutase catalyzes the conversion of L-methylmalonyl CoA to succinyl CoA. The mutase reaction involves the breakage and migration of a carbon-carbon bond. All these reactions occur in the mitochondria, and the succinyl CoA formed has several potential fates, including entry into the citric acid cycle and heme biosynthesis. In liver, conversion of propionyl CoA to succinyl CoA allows the carbon skeletons of some amino acids, as well as propionyl CoA from odd-chain fatty acid metabolism, to be used for gluconeogenesis [2].
Metabolic basis for folate and vitamin B12 deficiency symptoms
Megaloblastic anemia

Folate deficiency is usually due to a dietary insufficiency, but can also arise from malabsorption syndromes. The classical symptom of folate insufficiency is megaloblastic anemia, a condition reflecting deranged DNA synthesis in the erythropoietic cells [19, 37]. Megaloblastic changes occur in all fast-growing tissues such as the marrow and the gut epithelia. Megaloblastic cells contain close to twice the normal DNA content and the DNA is partially fragmented. Many cells are arrested in the G2 phase just prior to mitosis. Cells that divide often undergo apoptosis. The defect in DNA synthesis in folate deficiency has been ascribed to defective thymidylate synthesis under these conditions with a resulting increase in uracil misincorporation into DNA. Removal of uracil by the repair enzyme uracil DNA glycosylase, and a decreased repair of the gaps produced by this enzyme, would lead to an increase in double-stranded DNA breaks under these conditions [38]. Megaloblastic anemia is also a symptom of impaired vitamin B12 status. This condition, which is more prevalent in the elderly, usually results from malabsorption of vitamin B12. However, the anemia that results is identical to that of folate deficiency. Classical pernicious anemia is caused by an inability to absorb vitamin B12 as a result of lack of production of IF. The disease is age-related, and is usually due to destruction of the parietal cells in the stomach, which is caused by an autoimmune disease in which antibodies to the H+, K+-ATPase or to IF are produced [39]. Because body vitamin B12 stores are usually ample at the onset of the disease and the turnover of body stores is slow, it can take many years before deficiency symptoms become apparent. The destruction of the parietal cells decreases acid production, which also impairs release of dietary vitamin from protein binders. The prevalence of untreated pernicious anemia in elderly people has been estimated to be about 2%. Malabsorption of dietary vitamin B12

also can arise from any condition of decreased acid production or pancreatic insufficiency. The prevalence of atrophic gastritis in subjects 50 years of age or older has been estimated at 10% to 30%, and many elderly people suffer from Helicobacter pylori infection. These conditions result in an impairment of food vitamin B12 absorption because of an impaired ability to release the vitamin from protein binders, while absorption of crystalline (synthetic) vitamin B12 in the absence of food is unimpaired [40]. In vitamin B12 deficiency, the vitamin B12-dependent methionine synthase enzyme is inactive and cytosolic folate is trapped as 5-methyl-tetrahydrofolate at the expense of other folate coenzyme forms required for one-carbon metabolism such as thymidylate synthesis, leading to a functional folate deficiency or methyl trap in the cell [19]. As 5-methyl-tetrahydrofolate is a poor substrate for folylpolyglutamate synthetase, the ability of tissues to accumulate folate is reduced and the functional folate deficiency is compounded by a drop in cellular folate levels. As the defective DNA synthesis in pernicious anemia is caused by an induced secondary folate deficiency, high levels of folate cause a hematological response in patients with megaloblastic anemia due to vitamin B12 deficiency, but folate is ineffective in preventing the severe neurological pathologies associated with vitamin B12 deficiency.
Subacute combined degeneration (SCD)

The mechanism behind the demyelination disease that occurs in some vitamin B12-deficient subjects is not understood. Most experimental animals do not develop neurological symptoms when placed on a vitamin B12-deficient diet, in part because it is difficult to eliminate gut bacterial synthesis of vitamin B12 as a source of the vitamin. Although rarely used as a model, the South African fruit bat does display neurological abnormalities when made vitamin B12-deficient, as do nonhuman primates [41]. As mammals have only two vitamin B12-dependent enzymes and methionine synthase is the locus of the defect-causing anemia, attention has been focused on the role of methylmalonyl CoA mutase in the etiology of the neurological defects. Vitamin B12 deficiency causes accumulation of methylmalonyl CoA in the mitochondria, an elevation in circulating methylmalonate, acidosis, and elevated methylmalonate excretion. The accumulation of mitochondrial methylmalonyl CoA depletes the CoA pool available for other mitochondrial enzymes and metabolites, and the increased propionyl CoA can be incorporated into long-chain fatty acids in place of acetyl CoA. A role for the accumulation of unusual fatty acids in myelin as a reason for the demyelination has been proposed, but evidence for this is not convincing [41]. Humans with severe genetic impairment of the mut locus encoding the mutase suffer from a variety of severe clinical

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conditions and metabolic abnormalities, but do not develop subacute combined degeneration of the spinal cord or megaloblastic anemia. Monkeys treated with nitrous oxide, which inactivates methionine synthase, develop neurological disease similar to that observed in humans, and symptom development is retarded by methionine supplementation [42], suggesting that the neurological disease of vitamin B12 deficiency may also result from a defect in methionine synthase (in this case, because of the impairment in methionine synthesis). In the few known cases of genetic defects in methionine synthase or in the enzymes responsible for methylcobalamin synthesis, the patients exhibit the expected megaloblastic anemia and some also exhibit demyelination. Similarly, some patients with defects in methylenetetrahydrofolate reductase exhibit the same neurological symptoms as are observed in vitamin B12 deficiency. These patients do not exhibit megaloblastic anemia, because a defect in the reductase prevents the formation of 5-methyl-H4PteGlun and increases folate coenzyme availability for nucleotide synthesis [43, 44]. Although the mechanism responsible for the neurological disease of vitamin B12 deficiency is not understood, current evidence supports the hypothesis that it may be related to defective methionine synthesis. Recent studies using the gastrectomized rat as a model for extreme vitamin B12 malabsorption have resulted in rodents that exhibit demyelination and other symptoms of the subacute combined syndrome. Multiple biochemical disturbances have been observed in these animals, including an increase in TNF- and reduced EGF [45, 46]. No mechanism is known for a direct effect of vitamin B12 on TNF expression and it remains to be established whether the effects noted are specific for vitamin B12 deficiency or whether they are secondary to the total gastrectomy.
Recommended dietary intakes and effects of high doses

In 1998, the US Food and Nutrition Board (FNB) of the National Academy of Sciences, and a joint FAO/WHO Expert Consultation on Human Vitamin and Mineral Requirements, reviewed their recommendations for folate intake. Both groups used plasma and red blood cell folate concentrations as primary indicators of folate adequacy, and plasma total homocysteine concentrations as an additional indicator of folate status. The FNB set the folate Estimated Average Requirement (EAR) and Recommended Dietary Allowance (RDA) for adults as 320 g and 400 g of dietary folate equivalents (DFE)/day, based mainly on controlled studies in which subjects received a folate-deplete diet supplemented with various levels of folic acid [5]. An RDA of 600 g of DFE/day was recommended for pregnant women, but higher levels may be required to minimize the risk of birth defects. To reduce the risk of neural tube defects (NTD), it was recommended

that women of childbearing age who are capable of becoming pregnant consume 400 g of folic acid/day from supplements and/or fortified food in addition to their food folate intake. The recommended intake for lactation is based on an increase in the EAR to cover the amount of folate secreted in milk; the RDA is 500 g of DFE/day. Recommendations for children were extrapolated from adult EAR on the basis of metabolic body weight. Adequate intake (AI) levels for infants were based on the folate content of milk of well-nourished mothers. The AI for infants during the first 6 months after birth (65 g/day or DFE) was based on a mean daily intake of 0.78 L of milk that contains 85 g of folate/L. The AI for infants age 7 through 12 months was extrapolated from that for other age groups. Increases in dietary intake of folate do not affect maternal milk folate levels. As no information is available on the bioavailability of milk folate in infants compared with that of folic acid added to formula, the use of DFE levels to calculate how much folic acid to add to formula would be inappropriate. Folate fortification in the United States has added an average of about 220 g folic acid (380 g DFE) to the diet [47] and has greatly decreased the incidence of folate deficiency and approximately halved the incidence of fasting hyperhomocysteinemia [22]. Marginal vitamin B12 status has now become a more important factor in the population-attributable risk of fasting hyperhomocysteinemia than folate status. NTD rates have decreased by about 20% in the United States [48, 49], a population that started with a fairly low underlying rate and about one-third of which took folate supplements and would receive little extra benefit from food fortification. Whether rates would decrease further with increased levels of folate fortification has been the subject of much controversy. In Canada, where fortification levels are similar to the United States, NTD rates have decreased over 50% in regions that had higher initial prevalence than the United States [50]. Better ascertainment may also partly explain these differences. Folic acid doses of up to 15 mg in healthy adults have not been associated with any reported serious adverse effects but there have been limited studies looking at possible adverse effects of high intakes. The FNB recommended 1,000 g as an upper limit for folic acid intake by adults 19 years and older, including pregnant and lactating women [5]. This upper limit was not related to any known toxicity of folate. Instead, the concern was the possible masking of vitamin B12 deficiency anemia and limited information, primarily anecdotal, that folic acid might exacerbate the neurologic effects of vitamin B12 deficiency. The FNB set upper limits for children and adolescents by adjusting the adult limit on the basis of relative body weight. No upper limit was set for infants due to lack of adequate data. The FNB also recommended that food (or maternal milk) be the only source of folate for infants. Some

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concerns have recently been raised because of the role of folate status in epigenetic processes such as DNA and histone methylation. Coat color in the yellow agouti mouse, which harbors a transposon in the agouti gene, was influenced by increased folate and methyl group intakes due to increased methylation of the transposable element, suggesting a possible deleterious effect on genome stability [51]. The FNB estimated the adult EAR for vitamin B12 on the basis of hematological evidence and serum vitamin B12 values [23]. The current EAR is 2 g/day of vitamin B12 for adults, and the RDA was set at 2.4 g of vitamin B12 /day. An intake of 2.6 g/day is recommended for pregnant women to allow for fetal deposition of 0.1 to 0.2 g/day, and an intake of 2.8 g is recommended for lactating women to allow for secretion of vitamin B12 in milk. Because of the high incidence of malabsorption of food vitamin B12 in elderly people, it was recommended that subjects older than 50 years meet the RDA by taking foods fortified with vitamin B12 or by taking a supplement. EAR for children was extrapolated from adult values based on metabolic body weight, and the RDA was set as 1.2 times the EAR. AI for infants is based on the vitamin B12 content of milk of well-nourished mothers (0.42 g/L). No toxicity of high doses of vitamin B12 has been reported, and milligram doses are used to treat pernicious anemia with no apparent side effects.
Polymorphisms in folate and vitamin B12 genes

A large number of variants in folate genes and some in genes involved in vitamin B12 metabolism have been investigated, although only a few have been definitively shown to influence metabolism and/or disease risk. A common polymorphism (677CT, Ala to Val) in the MTHFR cDNA has been the most extensively studied [52]. Prior to the cloning of the MTHFR gene, studies had shown that some patients with vascular disease expressed a variant MTHFR enzyme that was heatlabile. Subjects homozygous for the 677T allele have lower lymphocyte MTHFR levels, lower plasma folate levels, higher plasma homocysteine levels, decreased global DNA methylation, an increased proportion of nonmethylfolate in their red blood cells, and, in some studies, a higher risk of vascular disease [5355]. Depending on the population, anywhere from 5% to 25% are homozygous and up to 50% heterozygous for this polymorphism, but the major adverse effects are seen only in the homozygous val/val variant. The ala to val change in the protein lowers the affinity of the enzyme for its FAD cofactor but does not affect the affinity of the enzyme for its substrates. Apoprotein lacking FAD is unstable, and the metabolic effects of this polymorphism are due to lower amounts of enzyme rather than an abnormal enzyme activity [56]. Folate stabilizes the enzyme by decreasing the rate of

loss of FAD. Although it is expected that metabolic and adverse effects of this polymorphism would primarily affect people with poorer folate status, the level of folate intake at which differences between the val/val and ala/ ala variants become insignificant currently is not known. In addition, elevated homocysteine in val/val subjects requires both poorer folate and riboflavin status but not necessarily levels of the vitamins that have traditionally been considered indicative of deficiency [57]. Riboflavin deficiency is considered rare in the United States. However, levels that are normally considered adequate may be insufficient for val/val subjects. It is likely that genetic polymorphisms, particularly common ones such as this, are responsible for much of the variation in nutrient requirements among individuals. Homozygosity for offspring with the MTHFR 677T allele has also been shown to be a risk factor for NTD, with the risk increasing very significantly in comparisons of offspring of mothers with poor folate status [58]. Carrying this particular allele can account for about 15% of the population-based attributable derived risk. However, a number of studies have suggested that the T allele is protective against certain cancers and that the major protective effect is in subjects with good folate status [59]. The reason for this is unclear, as it would be expected that differences in MTHFR levels with the different variants would disappear in subjects with good folate status. It has been speculated that possession of the T allele directs more of the one-carbon flux into nucleotide synthesis, which may allow more efficient DNA replication and/or repair, and reduce methionine synthesis and DNA methylation, but this remains to be demonstrated. However, poor folate status is known to be associated with an increased risk of certain types of cancer, including colon cancer. A second polymorphism in MTHFR (1298AC) has also received much attention. Although no biochemical phenotype has been established for this variant, it has been reported to influence cancer risk [60]. Many studies on this polymorphism have been confounded because it is in linkage disequilibrium with 677CT, with the variant appearing on the C allele, and the variant is practically never found in cis with the 677T variant. Variants in other folate genes for which no biochemical phenotype have thus far been established have been reported to influence homocysteine levels and/or disease risk. These include methionine synthase A3150G associated with reduced homocysteine levels [61] and cytosolic SHMT 1420CT which influences vascular disease risk, the variant having a strong protective effect in subjects with a MTHFR 677C background while increased risk was observed in subjects with a MTHFR 677T background [62]. Finally, a variant in C1 THF synthase (1958GA) has been established as a maternal risk factor for NTD and for second-trimester pregnancy losses [63].

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B. Shane

Variants in vitamin B12 genes have received less attention. A 259P R variant in the TC-II protein has been reported to alter plasma vitamin B12 levels and the proportion of holo to total TC-II [64].
Interaction with riboflavin and vitamin B6

Many of the enzymes involved in folate and vitamin B12 metabolism are flavoproteins. As described above, the 677T variant of MTHFR has a reduced affinity for FAD, and MTHFR protein levels are reduced and plasma homocysteine levels elevated in subjects with relatively poorer folate and riboflavin status, i.e., at levels that would not normally be considered indicative of vitamin deficiency. PLP is a cofactor for cytosolic and mitochondrial SHMT, but there is no evidence to suggest

that activities of these enzymes would be compromised in subjects with poorer vitamin B6 status, although this has not been rigorously investigated. Impaired vitamin B6 status has little effect on fasting homocysteine levels, but an elevation is noticed in population-based studies, which often include nonfasted blood samples. The increase in plasma homocysteine following a meal or a methionine load is exacerbated by reduced vitamin B6 status, whereas folate or vitamin B12 status has little effect on this rise. Transmethylation and transulfuration pathways are stimulated under these conditions, and this reflects that PLP is a cofactor for cystathionine -synthase and cystathioninase, the two enzymes involved in the transsulfuration pathway from homocysteine to cysteine [2]. Cystathioninase appears to be particularly sensitive to vitamin B6 deficiency.

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