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Folate and Vitamins B6 and B12
C H A P T E R
39 Folate and Vitamins B6 and B12 Natalia I. Krupenko Department of Nutrition, Nutrition Research Institute, University of North Carolina Chapel Hill, Chapel Hill, NC, United States
Glossary Terms
as well as the S-adenosylmethionine (SAM)eSadenosylhomocysteine ratio. Although plasma metabolites are well-established biomarkers of the vitamin status, accurate disease diagnosis and appropriate prevention and treatment should consider that the intracellular vitamin status is modified depending on individual genetic polymorphisms. Because the OCM pathways display numerous genetic polymorphisms, optimal intakes of folate and associated vitamins within populations will vary owing to variations within relevant genes. Individuals with single nucleotide polymorphisms (SNPs), insertions (ins), or deletions (del) in genes related to folate metabolism may have increased susceptibility to certain diseases including cancer, cardiovascular disease, Alzheimer disease, and dementia.
CBS Cystathionine b-synthase Del Deletions DHFR Dihydrofolate reductase Ins Insertions MMA Methylmalonic aciduria MS Methionine synthase MTHFD1 Trifunctional C1-synthase MTHFR Methylenetetrahydrofolate reductase MTR Methionine synthase gene MTRR Methionine synthase reductase gene NTDs Neural tube defects OCM One-carbon metabolism PLP Pyridoxal phosphate RBC Red blood cells SAM S-adenosylmethionine SHMT Serine hydroxymethyltransferase SNPs Single nucleotide polymorphisms TC II Transcobalamin II TCblR Transcobalamin receptor tHcy Total homocysteine THF Tetrahydrofolate
One-Carbon Metabolism and B Vitamins
OVERVIEW One-carbon metabolism (OCM) (Fig. 39.1) encompasses a group of biochemical reactions involving the folate coenzymes, vitamin B6 (B6), and vitamin B12 (B12). As with other vitamins, folate is an essential nutrient because humans cannot synthesize this molecule and strictly depend on diet for its proper supply. The folate coenzymes are critical for fundamental cellular processes such as nucleic acid biosynthesis, DNA repair, amino acid biogenesis, and cellular methylation. Therefore, insufficient folate intake, as well as deficiency of folate pathways, are associated with increased risk for certain diseases, most notably neural tube defects (NTDs). The status of one-carbon metabolic pathways is commonly assessed by measurements of plasma levels of folate and homocysteine, Principles of Nutrigenetics and Nutrigenomics https://doi.org/10.1016/B978-0-12-804572-5.00039-2
Vitamins B9 (folate), B6 (pyridoxine), and B12 (cobalamin) (Figs. 39.2e39.4) are important constituents of metabolic pathways in humans. Derivatives of these vitamins often function as coenzymes and therefore are critical elements of numerous biochemical pathways. In some cases, complex reactions require participation of more than one coenzyme. In this regard, the biochemistry of folate-dependent pathways is inseparable from metabolism of B12 and B6. Likewise, the status of each of the three vitamins should be considered to understand the basis for certain clinical outcomes. This is exemplified by studies that implicated the inadequacy of B12 and choline in the etiology of NTDs in folatefortified populations. Folate is a common name for a group of coenzymes involved in the biosynthesis of purine nucleotides and thymidylate, and amino acid metabolism (Fig. 39.2). Folate-dependent amino acid biogenesis includes reactions of serine and glycine interconversion, degradation
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FIGURE 39.1 Pathways of one-carbon metabolism (OCM). Enzymes catalyzing conversions of folate coenzymes are dihydrofolate reductase (DHFR), serine hydroxymethyltransferase (SHMT), 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS), trifunctional C1-synthase (MTHFD1), 5,10-methenyltetrahydrofolate synthetase (MTHFS); and thymidylate synthase (TS). Enzymes related to OCM are cystathionine b-synthase (CBS), cystathionine g-lyase (CTH). Tetrahydrofolic acid (THF), 5,10-methylenetetrahydrofolic acid (THF-CH2), 5methyltetrahydrofolic acid (THF-CH3), 10-formyltetrahydrofolic acid (THF-CHO), 5,10-methenyltetrahydrofolic acid (THF-CH), dihydrofolic acid (DHF), folic acid (FA), glycine (Gly), serine (Ser), cysteine (Cys), glutathione (GSH), homocysteine (HCys), methionine (Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), betaine (Bet), dimethylglycine (DMG). Octagons designate corresponding B vitamins required for the enzyme activity; red arrows indicate the dietary input of the specific folate form, whereas red and white arrows indicate the biosynthetic pathways using folate bound one-carbon groups. Dashed blue arrows indicate alternative pathways of Hcy use. COOH
O N H O HN H2 N
H N
COOH
10
5 N
THF
N H
COOH
O
HN
COOH
N
HN N
−CH3 −CH2− −CH= −CHO −CH=NH
N H
O
H2 N
HN
Transferred groups
N
Folic acid FIGURE 39.2 Folate, vitamin B9. Structures of the synthetic provitamin, folic acid, and an active form of the coenzyme, tetrahydrofolic acid (THF). Positions, where one-carbon groups can be attached to THF, are indicated in bold. Inset indicates one-carbon groups that can be attached to the tetrahydrofolate form only: eCH3, methyl; eCH2, methylene; eCH¼, methenyl; eCHO, formyl; and eCHNH, formimino.
of glycine and histidine, and the biosynthesis of methionine from homocysteine. In these reactions, folate coenzymes function as carriers of chemical moieties called one-carbon groups (OCG) (Fig. 39.1). Accordingly, the set of folate-dependent reactions is referred to as OCM, a term commonly synonymous to folate metabolism. OCG associated with folate metabolism differ in the oxidation state of the carbon atom and include methyl (CH3-), methylene (eCH2e), methenyl
(eCH¼), formyl (HCOe), and formimino (NHCHe) groups (Fig. 39.2). In folate-dependent biochemical reactions, the folate coenzyme accepts or donates OCG and enables oxidation or reduction of the carbon that is bound to the folate molecule. Coenzymes derived from vitamin B12 (Fig. 39.3) participate in only two biochemical reactions in humans: (1) the transfer of a methyl group in the folatedependent reaction of remethylation of homocysteine
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OVERVIEW
FIGURE 39.3 Vitamin B12. Structure of cobalamin. R is the upper axial ligand, which can be hydroxy- (eOH), cyano- (eCN), methyl(-CH3), or adenosyl- (Ado). Methylcobalamin and adenosylcobalamin are active coenzyme forms of the vitamin.
Pyridoxine
Pyridoxal phosphate (PLP)
FIGURE 39.4 Vitamin B6. Structures of the vitamin pyridoxine and the active coenzyme form, pyridoxal phosphate.
to methionine (Fig. 39.1), and (2) the conversion of methylmalonylecoenzyme A (CoA) to succinyleCoA. The active forms of this vitamin acting as coenzymes are methylcobalamin and adenosylcobalamin. Deficiency of B12 will prevent both reactions, causing increased levels of circulating total homocysteine (tHcy) and methylmalonic acid. These conditions are associated with specific types of metabolic disorders, hyperhomocysteinemia, and methylmalonic acidemia (MMA, also called methylmalonic aciduria), respectively. Pyridoxal phosphate (PLP), the derivative of vitamin B6 (Fig. 39.4), is one of the most common coenzymes in the mammalian cell and is involved in numerous biochemical reactions. Among these reactions is folatedependent reversible conversion of serine to glycine, catalyzed by the enzyme serine hydroxymethyltransferase (SHMT) (Fig. 39.1). The SHMT reaction is central to
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folate metabolism because it loads OCG on tetrahydrofolate, activating it for subsequent reactions (Fig. 39.1). Thus, the three vitamins, B9, B12, and B6, are closely related through OCM. Of note, another B6-dependent pathway, the biosynthesis of cysteine (Fig. 39.1), competes with OCM for homocysteine and serine, providing an additional link between these vitamins. Additional coenzymes (flavine adenine dinucleotide, derivative of B2; nicotinamide adenine dinucleotide, derivative of B3; and pantothenic acid, or B5) also participate in OCM, but they will not be discussed here. Because of requirements of B9, B12, and B6 for fundamental cellular processes, deficiencies in these vitamins will have dramatic effects in humans, causing severe disorders or diseases. Specifically, folate deficiency is associated with megaloblastic anemia and neural tube defects and is also implicated in the increased risk for cardiovascular disease, age-related neurodegeneration, and certain types of cancer. Mandatory food fortification with the synthetic form of the vitamin, folic acid, implemented in the United States and several other countries has led to a significant drop in NTD occurrence. B12 deficiency, which is more common in vegetarians and elderly people, can be caused by insufficient dietary intake owing to malnutrition, as well as by malabsorption. The latter mechanism is associated with insufficient function of intrinsic factor, the protein responsible for B12 absorption, and is the cause of a classic autoimmune disorder, pernicious anemia. However, this type of deficiency can be overcome by intramuscular injection of the vitamin, a common clinical approach to treating patients with insufficient B12 status, as well as by high oral doses of cobalamin, Clinically evident deficiency of B6 per se is uncommon. However, studies suggest that low plasma PLP levels inversely correlate with major markers of inflammation and are associated with increased risk of coronary artery disease and chronic kidney disease. Suboptimal B6 status is also associated with impaired cognitive function, Alzheimer disease, cardiovascular disease, and different types of cancer in elderly people. Moreover, inadequacy in B6 combined with deficiencies of other B vitamins could be a much stronger contributor to etiology of certain diseases.
Genetic Variations in Folate Enzymes It has become clear that the physiological effects of nutrients cannot be attributed exclusively to their intake since they are absorbed and also modified in different ways depending on an individual genotypic pattern. Numerous studies in the past two decades investigating nutrientegene interactions and their phenotypic consequences provided clues of how individual variations in genes modulate dietary input. The field of nutrigenomics underwent a rapid expansion after the
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development of novel technologies of genetic analysis and metabolomics. Although complete comprehensive analyses of these complex “omics" and epidemiologic data as well as ontology and annotation of the interactions are still missing, studies of B vitamins provided several important links to physiological and metabolic consequences of genes variation and highlighted possible need for alterations in nutritional requirements depending on genetic variation.
POLYMORPHISMS IN VITAMIN B12 PATHWAYS Transcobalamin II Insufficient dietary intake of B12 (Fig. 39.3) and the impairment of intrinsic factor secretion are not the only mechanisms causing B12 deficiency. The B12 transporting protein transcobalamin II (TC II), which delivers the vitamin from the ileum to the tissues, has a common polymorphism 776C > G in Caucasian populations leading to the replacement of a proline with an arginine (P259R). The 776C > G genotype significantly influences tissue B12 delivery and functional B12 status. Although the effect of this polymorphism on circulating levels of tHcy is not clear, healthy older adults with two mutant alleles (RR) have significantly higher levels of methylmalonic acid than do those with PP or RP genotypes. Methylmalonic acid is commonly elevated in patients who have a deficiency of the enzyme methylmalonyle CoA mutase. MethylmalonyleCoA mutase requires the B12 derivative adenosylcobalamin to catalyze the conversion of methylmalonyleCoA to succinyleCoA. This is why insufficient B12 causes the impairment of this reaction. Thus, the elevation of MMA in individuals who carry only the R259 type of TC II is indicative of the deficiency of B12. Another TC II polymorphism causing the S348F substitution was shown to correlate with plasma Hcy levels reflecting effects of B12 deficiency on homocysteine remethylation. It has been hypothesized that these conditions could be a predisease state with regard to B12 status. Direct correlation of TC II polymorphisms with cobalamin levels has also been observed. Thus, the I23V variant of the protein is associated with lower B12 levels and with pernicious anemia. To further complicate the picture, the variant of TC II receptor (TCblR) with a G220R substitution (owing to C > T polymorphism) was associated with higher serum cobalamin levels. Interestingly, this SNP is strongly linked to a neighboring SNP on the same chromosome that has been shown to have an even stronger influence. TCblR, which is found in the plasma membrane of most cell types, binds the plasma TC IIecobalamin complex and internalizes it via receptor-mediated endocytosis. Alterations in the
receptor sequence apparently impair the cellular internalization of cobalamin and increase its serum concentration. Thus, the cells will experience B12 deficiency despite the normal or elevated serum cobalamin levels.
Methionine Synthase and Methionine Synthase Reductase The second enzyme, which requires vitamin B12 for its function in humans, is methionine synthase (MS). This enzyme, which has a complex organization, catalyzes remethylation of homocysteine to methionine using the methyl group from 5-methyl-tetrahydrofolate (THFCH3). Under conditions of adequate dietary methionine, approximately 40% of homocysteine is remethylated to methionine through this pathway. In the enzyme mechanism, the cobalamin cofactor first accepts the methyl group from methyl-THF and then transfers it to homocysteine. The enzyme also binds SAM, which is the mechanism of its activation. Catalytically inactive oxidized cobalamin cofactor, which is formed every 200e1000 catalytic cycles, is reduced by the action of a specialized enzyme, MS reductase (MTRR). Overall, MS catalysis requires the B12 cofactor, the folate coenzyme, and SAM and MTRR as activators. In mice, disruption of Mtr, the gene encoding MS, results in embryonic lethality, which cannot be rescued by nutritional supplements. These findings indicate that methionine biosynthesis is an absolutely critical process that cannot be bypassed by dietary methionine supplementation. On the flip side, methionine biosynthesis by MS clears homocysteine produced by the action of adenosylhomocysteine hydrolase on Sadenosylhomocysteine, the reaction product of numerous cellular methyltransferases. Lower MS activity results in the accumulation of homocysteine. Significant associations have been found between elevated plasma homocysteine and cardiovascular disease, cerebrovascular disease, cognitive decline, bone dysfunction, pregnancy complications, and other conditions, which underscores the central role of MS in homocysteine detoxification. The number of natural variants identified for the MTR gene in humans that are associated with MS deficiency and cause methylcobalamin deficiency disorder, is relatively small. Patients with these conditions exhibit homocysteinemia, homocystinuria, and hypermethioninemia, and in many cases have megaloblastic anemia associated with neural dysfunction and mental retardation. One mutations in the MTR gene produces the enzyme with the substitution of leucine for proline (P1173L). This mutation, which affects the interaction of the enzyme with SAM, is detrimental to MS activity and causes severe cases of cobalamin deficiency. In contrast, a common SNP in MTR, 2756A>G, which replaces aspartate 919 with glycine, has a mild phenotype.
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ENZYMES OF FOLATE METABOLISM AND COMMON GENETIC VARIANTS
This SNP involves the MTRR binding domain of MS, thus affecting methylation and activation of the B12 cofactor. Although this SNP has been implicated as a risk factor of certain diseases (such as systemic lupus erythematosus, hypertension, and gastric cancers), its effect on circulating levels of homocysteine and clinical outcomes still is unclear. MS function can be also affected by genetic variants of the MTRR gene. A genetic polymorphism in this gene (66 A>G), causing the substitution of isoleucine with methionine (I22M) in the flavin mononucleotideebinding domain of the enzyme, influences circulating levels of tHcy. Caseecontrol and prospective studies indicate that this polymorphism is associated with increased risk for NTDs. Such risk could be further increased in individuals who have genetic variants in other folate pathway genes or low vitamin B12 status. For example, the double heterozygosity MTR 2756 A>G/MTRR 66 A>G was a significant risk factor for having Down syndrome. In addition, it should be recognized that there is no pool of free B12 in the body, because B12 is sequentially bound to the proteins that assist in its transport, delivery, and formation of active coenzyme forms and enzymes. Currently, more than half a dozen proteins (cblAecblG) that make active MS and malonyleCoA mutase have been identified owing to studies of patients with severe inborn disturbances in B12 metabolism. There is little information about the function of these proteins and even less about their polymorphisms, which could modulate the B12 function.
Folate Trap The reaction catalyzed by MS uses the methyl group donated by 5-methyl-THF, which is converted to THF in this reaction. This is the only pathway using 5methyl-THF because the reaction of the 5,10methylene-THF to 5-methyl-THF reduction (Fig. 39.1) is irreversible. Because B12 is required for MS activity, the vitamin deficiency impairs 5-methyl-THF use and results in its accumulation at the expense of other folate forms. This metabolic derangement is called methyl trap or folate trap and can be associated with B12 deficiency as well as low activities of MS or MTRR enzymes. Whether mutations or common genetic variants in corresponding genes can trap methyl groups has not yet been experimentally tested. However, the methyl trap, can be at least partially compensated for by increased folate intake.
ENZYMES OF FOLATE METABOLISM AND COMMON GENETIC VARIANTS In contrast to B12, folate malabsorption rarely causes disease. Instead, folate metabolism is affected by
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polymorphisms in genes coding for folatemetabolizing enzymes. Genetic variants in these genes are common and numerous and are found in all key genes involved in folate metabolism. Epidemiologic studies attempted to link these variants to NTDs, different types of cancer, and cardiovascular and neurologic disease. So far, the most characterized genetic variants in folate metabolism are those found in methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase (DHFR). Genetic variants in MTHFR mostly affect methylation-dependent reactions whereas variants in DHFR affect the whole OCM owing to the strategic position of the enzyme in this pathway (Fig. 39.1).
Methylenetetrahydrofolate Reductase MTHFR (5,10-methylene-THF reductase) catalyzes the conversion of 5,10-methylene-THF to 5-methyl-THF (Fig. 39.1). To better understand the disposition of this reaction in folate metabolism, it should be pointed out that it is strictly irreversible. Therefore, the only way to use 5methyl-THF produced by MTHFR is by the B12-dependent remethylation of homocysteine into methionine. As discussed earlier, this reaction catalyzed by MS has a dual significance. First, it is an important source of methionine in the cell. Although methionine has several functions, a large portion of this amino acid is used to biosynthesize SAM, a universal methyl donor involved in more than 100 methylation reactions in the cell. Second, folate-dependent methionine biosynthesis contributes significantly to the removal of homocysteine. This could be an important function because homocysteine accumulation generally has a negative effect in humans and is associated with increased risk for cardiovascular diseases. In agreement with this phenomenon, a common MTHFR polymorphism, 677C > T, is associated with mild hyperhomocysteinemia and increased risk for coronary heart disease and stroke. These conditions are exacerbated in individuals with low folate status. The 677C > T polymorphism, which is the most common inborn error of folate metabolism, results in an amino acid alteration in the catalytic domain of the enzyme. The mutant MTHFR is a less stable protein that results in an overall decrease in MTHFR activity in affected individuals. Importantly, 5-methyl-THF was shown to have a protective effect on enzyme stability. The 677C>T MTHFR variant is common in European and American populations; the homozygous form is found in 5%e20% of individuals. Its clinical manifestation is not limited to hyperhomocysteinemia but includes lower levels of folate in plasma and red blood cells (RBC), a likely contributing factor to the associated pathologies, which could be diverse. Thus, in addition to cardiovascular disease, this MTHFR SNP was implicated in the
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increased risk for neural tube defects, cleft lip and palate, Down syndrome, thrombosis, schizophrenia, depression, adverse pregnancy outcomes, and cancer. Interestingly, this mutation may have a protective effect against some cancers, although mechanisms underlying this phenomenon remain elusive. Numerous studies have also shown that mild MTHFR deficiency resulting from the 677C>T polymorphism may be corrected by dietary folic acid. At the same time, severe deficiency of the enzyme can be rescued by 5-methyl-THF, but not by folic acid. In line with this finding, 5-methyl-THF was shown to increase plasma folate more efficiently than folic acid in women of either 677CC or 677TT genotype. Overall, over 50 naturally occurring mutations have been identified in the MTHFR gene, as well as nine common polymorphisms, but 677C>T remains most studied and recognized. Another relatively common MTHFR polymorphism, 1298A>C, is found in a homozygous state in 4%e12% of individuals in different populations. This variant has reduced activity, but not as dramatically as the 677C>T mutant (only to 68% of wild-type [WT] activity), and it is not thermolabile. Individuals homozygous for this SNP have normal tHcy, not different from subjects with the WT variant. The compound heterozygotes for the 1298C and 677T variants have biochemical profiles similar to those of 677T homozygotes as well as increased homocysteine. This is the why numerous studies are testing both 677C>T and 1298A>C polymorphisms for association with diseases.
Dihydrofolate Reductase DHFR is a key enzyme in folate metabolism that incorporates ingested folic acid into the reduced folate pool. Folic acid is a synthetic provitamin that requires enzymatic activation to become an active cofactor. DHFR catalyzes the two-step reduction of folic acid, first to dihydrofolate and then to the active coenzyme THF (Fig. 39.1). The enzyme is also responsible for the conversion of dihydrofolate, which is produced in the reaction of thymidine monophosphate biosynthesis, to THF (Fig. 39.1). Homozygous knockout of Dhfr in mice was shown to be embryonic-lethal. In humans, DHFR activity can vary at least sixfold between individuals, and generally is lower than in rodents. Low activity of the enzyme results in a low rate of dietary folic acid incorporation into folate pool and higher levels of unmetabolized folic acid in serum. Although negative effects of unmetabolized folic acid can be diverse and indirect, the low rate of incorporation of the vitamin to the active folate pool creates symptoms of folate deficiency even under conditions of high folic acid intake. A well-established DHFR polymorphism is a 19ebase pair (bp) deletion in the first intron, 60 nucleotides downstream from the splice donor site. This
polymorphism is highly prevalent with the reported frequency of the del/del genotype reaching 48% in some populations (20% del/del homozygosity in the US population). Because this is an intronic mutation, its likely effect is associated with transcriptional or posttranscriptional regulation of DHFR expression, which could change the protein level. Although this mechanism has not been explored, it has been demonstrated that the del/del genotype is associated with increased unmetabolized folic acid in plasma for intakes exceeding 500 mg/ d (high folate consumption), and with decreased RBC folate at intakes below 250 mg/d (low folate consumption). The effect of this polymorphism on NTDs, carcinogenesis, and cognitive function has also been investigated. Data on its role in NTDs and cognitive function are conflicting or incomplete, but there is compelling evidence that this mutation is associated with an increased cancer risk, which is further modified by the folic acid intake: (1) the DHFR 19del allele is associated with greater breast cancer risk among multivitamin users; and (2) del/del homozygous mothers taking folic acid supplements are at a higher risk for having a child with early childhood retinoblastoma. Although the molecular mechanisms underlying the biological effects of this genetic variant are not yet known, studies to date suggest that this is a functional polymorphism that alters the incorporation of folic acid into the intracellular pool of reduced folate. Most likely, the deletion diminishes the capacity of affected individuals to make active folate coenzymes from provitamin folic acid. This brings a paradoxical conclusion that a significant part of the population with the del/del genotype who have low folate status will not benefit from the increased intake of folic acid. These findings emphasize another important issue with dietary folate: the form of the vitamin used for supplementation.
Forms of Dietary Folate Natural folate obtained from a nonfortified diet is presented mostly as 5-methyl-THF. In contrast, the component of multivitamin supplements and food fortification, folic acid, is a synthetic compound not found in the nature. It is a provitamin, which requires two rounds of enzymatic reduction to THF to become an active cofactor. Because folic acid and 5-methyl-THF are functionally different, the efficiency of the natural sources of folate versus folic acid should be considered. Crosssectional analysis of numerous studies underscores the importance of the natural food folate. The advantage of 5-methyl-THF is its higher absorption rate and higher efficiency in increasing the RBC and plasma folate than folic acid. Specific concerns of supplementation with folic acid include masking of B12 deficiency, promotion of cancer and metastasis, epigenetic changes, and
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CONCLUSIONS
presence of unmetabolized folic acid in the circulation. So, should the supplementation with 5-methyl-THF be considered instead of folic acid? Two aspects have to be considered. Folic acid is proven to reduce the incidence of NTDs, however there are no such data for 5methyl-THF. Furthermore, as discussed in this chapter, the incorporation of 5-methyl-THF into the folate pool requires the B12-dependent remethylation of homocysteine. If this process is impaired owing to B12 deficiency, or by polymorphisms in the MTR/MTRR or some other genes, supplementation will be ineffective. Another viable alternative to folic acid could be supplementation with folinic acid (5-formyl-THF), which is used to treat cancer patients in combination with 5-fluorouracil. Folinic acid rapidly incorporates into the reduced folate pool upstream of MTHFR, thus avoiding both the folate trap and the necessity to be activated by DHFR. Folinic acid has been shown to bypass the DHFR deficiency effectively by correcting hematological abnormalities, normalizing the cerebrospinal fluid folate levels, and improving neurological symptoms in patients homozygous for L80F or D153V DHFR mutations, both of which result in reduced enzyme activity.
VITAMIN B6-RELATED FOLATE PATHWAY ENZYMES Serine Hydroxymethyltransferase Serine hydroxymethyltransferase (SHMT) synthesizes glycine from serine. This is a reversible reaction, but thermodynamics favors the biosynthesis of glycine (Fig. 39.1). The reaction uses two coenzymes: THF, which accepts OCG from serine, and PLP (a derivative of vitamin B6, Fig. 39.4), which is absolutely required for enzyme catalysis. This reaction is important for bringing OCG to folate metabolism and also as the source of glycine in the cell. In fact, up to 70% of glycine could come from this reaction and not from dietary sources. There are two identical SHMT reactions in the cell, one in cytoplasm and another in mitochondria, both catalyzed by highly similar enzymes, cytosolic SHMT1 and mitochondrial SHMT2. Interestingly, vitamin B6 restriction decreases the activity and stability of SHMT, but the cytoplasmic isozyme is more sensitive to vitamin B6 deficiency than the mitochondrial isozyme. A polymorphism in SHMT1, 1420 C> T, has been described. This is an exonic mutation that results in the substitution of leucine by phenylalanine at position 474 of the protein (L474F). This is a common SNP; the reported frequency for the TT genotype is about 12%. Although this mutation is not believed to alter enzyme activity, it prevents the enzyme from sumoylation and thereby prevents its transport into the nuclei. It has been observed that individuals with the 1420TT
genotype have increased RBC and plasma folate levels. The 1420TT genotype is also associated with the reduction of risk of several cancers and some other diseases. This effect was linked to decreased DNA damage, observed in the TT genotype. Polymorphisms in SHMT2 are rarer and are not yet linked with the risk for any disease.
Cystathionine b-Synthase Cystathionine b-synthase (CBS) is a key enzyme in the two-step biosynthesis of cysteine from homocysteine and serine and requires vitamin B6 for its catalysis (Fig. 39.1). This is the only pathway in humans that leads to cysteine production. This pathway competes for homocysteine with homocysteine remethylation by MS in the methyl cycle pathway. Over 100 variations have been identified in the CBS gene. Many of them cause decreased enzyme activity, whereas for about a quarter of mutations an effect has not yet been established. Deficiency of CBS leads to homocystinuria, a rare autosomal recessive inborn error of sulfur amino acid metabolism. Some of the mutations can be corrected by pyridoxine (pyridoxineresponsive) whereas others are pyridoxine nonresponsive. There is at least one mutation in the CBS gene that produces hyperactive enzyme (up to 10-fold higher activity compared with the WT enzyme). In addition to rare mutations in CBS, several common polymorphisms in this gene were identified. One of these polymorphisms, a 68-bp insertion in exon 8, has a high prevalence (12% in the healthy US population, but varies between 8% and 26% depending on race, whereas the prevalence among healthy men and women in Northern Ireland is 18%). By itself, this mutation might not have a significant effect on circulating homocysteine levels. Nevertheless, it might have an effect in combination with the other common variant genotypes in folate pathways, MTHFR 677TT and MS 2756 AA. The 68ins polymorphism is also associated with decreased schizophrenia risk. Finally, CBS has two binding sites for SAM, and binding of SAM activates the enzyme. Because SAM levels are linked to folate metabolism (as discussed earlier), this provides an additional regulatory circuit between B vitaminerelated pathways and adds to the overall complexity of dietary interventions. Deleterious mutations within SAM regulatory sites will impair CBS activation, thus altering folate-dependent responses.
CONCLUSIONS Correction of metabolic folate deficiencies caused by specific gene variations through enhanced supplementation is an attractive and feasible approach to the treatment or prevention of diseases. Mandatory food fortification
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with folic acid has proven to decrease the incidence of NTDs. However, the outcome of supplementation approach will depend on many factors including the ingested form of the vitamin, the status of other B vitamins, and the overall complex interaction between enzymes in the pathway, as well as between pathways. Considering that functional polymorphisms in folate enzyme genes are rather common, their combinations can produce multiple effects. It could be expected that some genetic variants synergize to exacerbate metabolic derangement, whereas others perhaps can compensate for each other. The situation is complicated further as currently, the effects of a number of polymorphisms on the corresponding protein function have not been established. The entire field is still in its infancy and future studies will provide more precise knowledge of how genotypes affect the human body’s ability to process the nutrients and how deficient genotypes can be corrected by dietary manipulations.
Suggested Readings Cario, H., Smith, D.E., Blom, H., Blau, N., Bode, H., Holzmann, K., Pannicke, U., Hopfner, K.P., Rump, E.M., Ayric, Z., Kohne, E., Debatin, K.M., Smulders, Y., Schwarz, K., 2011. Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease. Am J Hum Genet 88 (2), 226e231. Cheng, T.Y., Makar, K.W., Neuhouser, M.L., Miller, J.W., Song, X., Brown, E.C., Beresford, S.A., Zheng, Y., Poole, E.M., Galbraith, R.L., Duggan, D.J., Habermann, N., Bailey, L.B., Maneval, D.R., Caudill, M.A., Toriola, A.T., Green, R., Ulrich, C.M., 2015. Folate-mediated one-carbon metabolism genes and interactions with nutritional factors on colorectal cancer risk: women’s health initiative observational study. Cancer 121 (20), 3684e3691. Green, R., Miller, J.W., 2005. Vitamin B12 deficiency is the dominant nutritional cause of hyperhomocysteinemia in a folic acid efortified population. Clin Chem Lab Med 43 (10), 1048e1051.
Kozich, V., Sokolova´, J., Klatovska´, V., Krijt, J., Janosı´k, M., Jelı´nek, K., Kraus, J.P., 2010. Cystathionine beta-synthase mutations: effect of mutation topology on folding and activity. Hum Mutat 31 (7), 809e819. Kurnat-Thoma, E.L., Pangilinan, F., Matteini, A.M., Wong, B., Pepper, G.A., Stabler, S.P., Guralnik, J.M., Brody, L.C., 2015. Biol Res Nurs 17 (4), 444e454. Leclerc, D., Sibani, S., Rozen, R.. Molecular biology of methylenetetrahydrofolate reductase (MTHFR) and overview of mutations/polymorphisms. In: Madame Curie Bioscience Database [Internet], Landes Bioscience, Austin (TX), 2000-2013. Molloy, A.M., 2012. Genetic aspects of folate metabolism. Subcell Biochem 56, 105e130. Philip, D., Buch, A., Moorthy, D., Scott, T.M., Parnell, L.D., Lai, C.Q., Ordova´s, J.M., Selhub, J., Rosenberg, I.H., Tucker, K.L., Troen, A.M., 2015. Dihydrofolate reductase 19-bp deletion polymorphism modifies the association of folate status with memory in a cross-sectional multi-ethnic study of adults. Am J Clin Nutr 102 (5), 1279e1288. Scaglione, F., Panzavolta, G., 2014. Folate, folic acid and 5methyltetrahydrofolate are not the same thing. Xenobiotica 44 (5), 480e488. Selhub, J., Rosenberg, I.H., 2016. Excessive folic acid intake and relation to adverse health outcome. Biochimie 126, 71e78. Stover, P.J., 2012. Polymorphisms in 1-carbon metabolism, epigenetics and folate-related pathologies. J Nutrigenetics Nutrigenomics 4 (5), 293e305. Tibbetts, A.S., Appling, D.R., 2010. Compartmentalization of Mammalian folate-mediated one-carbon metabolism. Annu Rev Nutr 30, 57e81. Watkins, D., Ru, M., Hwang, H.Y., Kim, C.D., Murray, A., Philip, N.S., Kim, W., Legakis, H., Wai, T., Hilton, J.F., Ge, B., Dore´, C., Hosack, A., Wilson, A., Gravel, R.A., Shane, B., Hudson, T.J., Rosenblatt, D.S., 2002. Hyperhomocysteinemia due to methionine synthase deficiency, cblG: structure of the MTR gene, genotype diversity, and recognition of a common mutation, P1173L. Am J Hum Genet 71 (1), 143e153. Woeller, C.F., Anderson, D.D., Szebenyi, D.M., Stover, P.J., 2007. Evidence for small ubiquitin-like modifier-dependent nuclear import of the thymidylate biosynthesis pathway. J Biol Chem 282 (24), 17623e17631.
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39 Folate and Vitamins B6 and B12 Natalia I. Krupenko Department of Nutrition, Nutrition Research Institute, University of North Carolina Chapel Hill, Chapel Hill, NC, United States
Glossary Terms
as well as the S-adenosylmethionine (SAM)eSadenosylhomocysteine ratio. Although plasma metabolites are well-established biomarkers of the vitamin status, accurate disease diagnosis and appropriate prevention and treatment should consider that the intracellular vitamin status is modified depending on individual genetic polymorphisms. Because the OCM pathways display numerous genetic polymorphisms, optimal intakes of folate and associated vitamins within populations will vary owing to variations within relevant genes. Individuals with single nucleotide polymorphisms (SNPs), insertions (ins), or deletions (del) in genes related to folate metabolism may have increased susceptibility to certain diseases including cancer, cardiovascular disease, Alzheimer disease, and dementia.
CBS Cystathionine b-synthase Del Deletions DHFR Dihydrofolate reductase Ins Insertions MMA Methylmalonic aciduria MS Methionine synthase MTHFD1 Trifunctional C1-synthase MTHFR Methylenetetrahydrofolate reductase MTR Methionine synthase gene MTRR Methionine synthase reductase gene NTDs Neural tube defects OCM One-carbon metabolism PLP Pyridoxal phosphate RBC Red blood cells SAM S-adenosylmethionine SHMT Serine hydroxymethyltransferase SNPs Single nucleotide polymorphisms TC II Transcobalamin II TCblR Transcobalamin receptor tHcy Total homocysteine THF Tetrahydrofolate
One-Carbon Metabolism and B Vitamins
OVERVIEW One-carbon metabolism (OCM) (Fig. 39.1) encompasses a group of biochemical reactions involving the folate coenzymes, vitamin B6 (B6), and vitamin B12 (B12). As with other vitamins, folate is an essential nutrient because humans cannot synthesize this molecule and strictly depend on diet for its proper supply. The folate coenzymes are critical for fundamental cellular processes such as nucleic acid biosynthesis, DNA repair, amino acid biogenesis, and cellular methylation. Therefore, insufficient folate intake, as well as deficiency of folate pathways, are associated with increased risk for certain diseases, most notably neural tube defects (NTDs). The status of one-carbon metabolic pathways is commonly assessed by measurements of plasma levels of folate and homocysteine, Principles of Nutrigenetics and Nutrigenomics https://doi.org/10.1016/B978-0-12-804572-5.00039-2
Vitamins B9 (folate), B6 (pyridoxine), and B12 (cobalamin) (Figs. 39.2e39.4) are important constituents of metabolic pathways in humans. Derivatives of these vitamins often function as coenzymes and therefore are critical elements of numerous biochemical pathways. In some cases, complex reactions require participation of more than one coenzyme. In this regard, the biochemistry of folate-dependent pathways is inseparable from metabolism of B12 and B6. Likewise, the status of each of the three vitamins should be considered to understand the basis for certain clinical outcomes. This is exemplified by studies that implicated the inadequacy of B12 and choline in the etiology of NTDs in folatefortified populations. Folate is a common name for a group of coenzymes involved in the biosynthesis of purine nucleotides and thymidylate, and amino acid metabolism (Fig. 39.2). Folate-dependent amino acid biogenesis includes reactions of serine and glycine interconversion, degradation
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FIGURE 39.1 Pathways of one-carbon metabolism (OCM). Enzymes catalyzing conversions of folate coenzymes are dihydrofolate reductase (DHFR), serine hydroxymethyltransferase (SHMT), 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS), trifunctional C1-synthase (MTHFD1), 5,10-methenyltetrahydrofolate synthetase (MTHFS); and thymidylate synthase (TS). Enzymes related to OCM are cystathionine b-synthase (CBS), cystathionine g-lyase (CTH). Tetrahydrofolic acid (THF), 5,10-methylenetetrahydrofolic acid (THF-CH2), 5methyltetrahydrofolic acid (THF-CH3), 10-formyltetrahydrofolic acid (THF-CHO), 5,10-methenyltetrahydrofolic acid (THF-CH), dihydrofolic acid (DHF), folic acid (FA), glycine (Gly), serine (Ser), cysteine (Cys), glutathione (GSH), homocysteine (HCys), methionine (Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), betaine (Bet), dimethylglycine (DMG). Octagons designate corresponding B vitamins required for the enzyme activity; red arrows indicate the dietary input of the specific folate form, whereas red and white arrows indicate the biosynthetic pathways using folate bound one-carbon groups. Dashed blue arrows indicate alternative pathways of Hcy use. COOH
O N H O HN H2 N
H N
COOH
10
5 N
THF
N H
COOH
O
HN
COOH
N
HN N
−CH3 −CH2− −CH= −CHO −CH=NH
N H
O
H2 N
HN
Transferred groups
N
Folic acid FIGURE 39.2 Folate, vitamin B9. Structures of the synthetic provitamin, folic acid, and an active form of the coenzyme, tetrahydrofolic acid (THF). Positions, where one-carbon groups can be attached to THF, are indicated in bold. Inset indicates one-carbon groups that can be attached to the tetrahydrofolate form only: eCH3, methyl; eCH2, methylene; eCH¼, methenyl; eCHO, formyl; and eCHNH, formimino.
of glycine and histidine, and the biosynthesis of methionine from homocysteine. In these reactions, folate coenzymes function as carriers of chemical moieties called one-carbon groups (OCG) (Fig. 39.1). Accordingly, the set of folate-dependent reactions is referred to as OCM, a term commonly synonymous to folate metabolism. OCG associated with folate metabolism differ in the oxidation state of the carbon atom and include methyl (CH3-), methylene (eCH2e), methenyl
(eCH¼), formyl (HCOe), and formimino (NHCHe) groups (Fig. 39.2). In folate-dependent biochemical reactions, the folate coenzyme accepts or donates OCG and enables oxidation or reduction of the carbon that is bound to the folate molecule. Coenzymes derived from vitamin B12 (Fig. 39.3) participate in only two biochemical reactions in humans: (1) the transfer of a methyl group in the folatedependent reaction of remethylation of homocysteine
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OVERVIEW
FIGURE 39.3 Vitamin B12. Structure of cobalamin. R is the upper axial ligand, which can be hydroxy- (eOH), cyano- (eCN), methyl(-CH3), or adenosyl- (Ado). Methylcobalamin and adenosylcobalamin are active coenzyme forms of the vitamin.
Pyridoxine
Pyridoxal phosphate (PLP)
FIGURE 39.4 Vitamin B6. Structures of the vitamin pyridoxine and the active coenzyme form, pyridoxal phosphate.
to methionine (Fig. 39.1), and (2) the conversion of methylmalonylecoenzyme A (CoA) to succinyleCoA. The active forms of this vitamin acting as coenzymes are methylcobalamin and adenosylcobalamin. Deficiency of B12 will prevent both reactions, causing increased levels of circulating total homocysteine (tHcy) and methylmalonic acid. These conditions are associated with specific types of metabolic disorders, hyperhomocysteinemia, and methylmalonic acidemia (MMA, also called methylmalonic aciduria), respectively. Pyridoxal phosphate (PLP), the derivative of vitamin B6 (Fig. 39.4), is one of the most common coenzymes in the mammalian cell and is involved in numerous biochemical reactions. Among these reactions is folatedependent reversible conversion of serine to glycine, catalyzed by the enzyme serine hydroxymethyltransferase (SHMT) (Fig. 39.1). The SHMT reaction is central to
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folate metabolism because it loads OCG on tetrahydrofolate, activating it for subsequent reactions (Fig. 39.1). Thus, the three vitamins, B9, B12, and B6, are closely related through OCM. Of note, another B6-dependent pathway, the biosynthesis of cysteine (Fig. 39.1), competes with OCM for homocysteine and serine, providing an additional link between these vitamins. Additional coenzymes (flavine adenine dinucleotide, derivative of B2; nicotinamide adenine dinucleotide, derivative of B3; and pantothenic acid, or B5) also participate in OCM, but they will not be discussed here. Because of requirements of B9, B12, and B6 for fundamental cellular processes, deficiencies in these vitamins will have dramatic effects in humans, causing severe disorders or diseases. Specifically, folate deficiency is associated with megaloblastic anemia and neural tube defects and is also implicated in the increased risk for cardiovascular disease, age-related neurodegeneration, and certain types of cancer. Mandatory food fortification with the synthetic form of the vitamin, folic acid, implemented in the United States and several other countries has led to a significant drop in NTD occurrence. B12 deficiency, which is more common in vegetarians and elderly people, can be caused by insufficient dietary intake owing to malnutrition, as well as by malabsorption. The latter mechanism is associated with insufficient function of intrinsic factor, the protein responsible for B12 absorption, and is the cause of a classic autoimmune disorder, pernicious anemia. However, this type of deficiency can be overcome by intramuscular injection of the vitamin, a common clinical approach to treating patients with insufficient B12 status, as well as by high oral doses of cobalamin, Clinically evident deficiency of B6 per se is uncommon. However, studies suggest that low plasma PLP levels inversely correlate with major markers of inflammation and are associated with increased risk of coronary artery disease and chronic kidney disease. Suboptimal B6 status is also associated with impaired cognitive function, Alzheimer disease, cardiovascular disease, and different types of cancer in elderly people. Moreover, inadequacy in B6 combined with deficiencies of other B vitamins could be a much stronger contributor to etiology of certain diseases.
Genetic Variations in Folate Enzymes It has become clear that the physiological effects of nutrients cannot be attributed exclusively to their intake since they are absorbed and also modified in different ways depending on an individual genotypic pattern. Numerous studies in the past two decades investigating nutrientegene interactions and their phenotypic consequences provided clues of how individual variations in genes modulate dietary input. The field of nutrigenomics underwent a rapid expansion after the
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development of novel technologies of genetic analysis and metabolomics. Although complete comprehensive analyses of these complex “omics" and epidemiologic data as well as ontology and annotation of the interactions are still missing, studies of B vitamins provided several important links to physiological and metabolic consequences of genes variation and highlighted possible need for alterations in nutritional requirements depending on genetic variation.
POLYMORPHISMS IN VITAMIN B12 PATHWAYS Transcobalamin II Insufficient dietary intake of B12 (Fig. 39.3) and the impairment of intrinsic factor secretion are not the only mechanisms causing B12 deficiency. The B12 transporting protein transcobalamin II (TC II), which delivers the vitamin from the ileum to the tissues, has a common polymorphism 776C > G in Caucasian populations leading to the replacement of a proline with an arginine (P259R). The 776C > G genotype significantly influences tissue B12 delivery and functional B12 status. Although the effect of this polymorphism on circulating levels of tHcy is not clear, healthy older adults with two mutant alleles (RR) have significantly higher levels of methylmalonic acid than do those with PP or RP genotypes. Methylmalonic acid is commonly elevated in patients who have a deficiency of the enzyme methylmalonyle CoA mutase. MethylmalonyleCoA mutase requires the B12 derivative adenosylcobalamin to catalyze the conversion of methylmalonyleCoA to succinyleCoA. This is why insufficient B12 causes the impairment of this reaction. Thus, the elevation of MMA in individuals who carry only the R259 type of TC II is indicative of the deficiency of B12. Another TC II polymorphism causing the S348F substitution was shown to correlate with plasma Hcy levels reflecting effects of B12 deficiency on homocysteine remethylation. It has been hypothesized that these conditions could be a predisease state with regard to B12 status. Direct correlation of TC II polymorphisms with cobalamin levels has also been observed. Thus, the I23V variant of the protein is associated with lower B12 levels and with pernicious anemia. To further complicate the picture, the variant of TC II receptor (TCblR) with a G220R substitution (owing to C > T polymorphism) was associated with higher serum cobalamin levels. Interestingly, this SNP is strongly linked to a neighboring SNP on the same chromosome that has been shown to have an even stronger influence. TCblR, which is found in the plasma membrane of most cell types, binds the plasma TC IIecobalamin complex and internalizes it via receptor-mediated endocytosis. Alterations in the
receptor sequence apparently impair the cellular internalization of cobalamin and increase its serum concentration. Thus, the cells will experience B12 deficiency despite the normal or elevated serum cobalamin levels.
Methionine Synthase and Methionine Synthase Reductase The second enzyme, which requires vitamin B12 for its function in humans, is methionine synthase (MS). This enzyme, which has a complex organization, catalyzes remethylation of homocysteine to methionine using the methyl group from 5-methyl-tetrahydrofolate (THFCH3). Under conditions of adequate dietary methionine, approximately 40% of homocysteine is remethylated to methionine through this pathway. In the enzyme mechanism, the cobalamin cofactor first accepts the methyl group from methyl-THF and then transfers it to homocysteine. The enzyme also binds SAM, which is the mechanism of its activation. Catalytically inactive oxidized cobalamin cofactor, which is formed every 200e1000 catalytic cycles, is reduced by the action of a specialized enzyme, MS reductase (MTRR). Overall, MS catalysis requires the B12 cofactor, the folate coenzyme, and SAM and MTRR as activators. In mice, disruption of Mtr, the gene encoding MS, results in embryonic lethality, which cannot be rescued by nutritional supplements. These findings indicate that methionine biosynthesis is an absolutely critical process that cannot be bypassed by dietary methionine supplementation. On the flip side, methionine biosynthesis by MS clears homocysteine produced by the action of adenosylhomocysteine hydrolase on Sadenosylhomocysteine, the reaction product of numerous cellular methyltransferases. Lower MS activity results in the accumulation of homocysteine. Significant associations have been found between elevated plasma homocysteine and cardiovascular disease, cerebrovascular disease, cognitive decline, bone dysfunction, pregnancy complications, and other conditions, which underscores the central role of MS in homocysteine detoxification. The number of natural variants identified for the MTR gene in humans that are associated with MS deficiency and cause methylcobalamin deficiency disorder, is relatively small. Patients with these conditions exhibit homocysteinemia, homocystinuria, and hypermethioninemia, and in many cases have megaloblastic anemia associated with neural dysfunction and mental retardation. One mutations in the MTR gene produces the enzyme with the substitution of leucine for proline (P1173L). This mutation, which affects the interaction of the enzyme with SAM, is detrimental to MS activity and causes severe cases of cobalamin deficiency. In contrast, a common SNP in MTR, 2756A>G, which replaces aspartate 919 with glycine, has a mild phenotype.
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ENZYMES OF FOLATE METABOLISM AND COMMON GENETIC VARIANTS
This SNP involves the MTRR binding domain of MS, thus affecting methylation and activation of the B12 cofactor. Although this SNP has been implicated as a risk factor of certain diseases (such as systemic lupus erythematosus, hypertension, and gastric cancers), its effect on circulating levels of homocysteine and clinical outcomes still is unclear. MS function can be also affected by genetic variants of the MTRR gene. A genetic polymorphism in this gene (66 A>G), causing the substitution of isoleucine with methionine (I22M) in the flavin mononucleotideebinding domain of the enzyme, influences circulating levels of tHcy. Caseecontrol and prospective studies indicate that this polymorphism is associated with increased risk for NTDs. Such risk could be further increased in individuals who have genetic variants in other folate pathway genes or low vitamin B12 status. For example, the double heterozygosity MTR 2756 A>G/MTRR 66 A>G was a significant risk factor for having Down syndrome. In addition, it should be recognized that there is no pool of free B12 in the body, because B12 is sequentially bound to the proteins that assist in its transport, delivery, and formation of active coenzyme forms and enzymes. Currently, more than half a dozen proteins (cblAecblG) that make active MS and malonyleCoA mutase have been identified owing to studies of patients with severe inborn disturbances in B12 metabolism. There is little information about the function of these proteins and even less about their polymorphisms, which could modulate the B12 function.
Folate Trap The reaction catalyzed by MS uses the methyl group donated by 5-methyl-THF, which is converted to THF in this reaction. This is the only pathway using 5methyl-THF because the reaction of the 5,10methylene-THF to 5-methyl-THF reduction (Fig. 39.1) is irreversible. Because B12 is required for MS activity, the vitamin deficiency impairs 5-methyl-THF use and results in its accumulation at the expense of other folate forms. This metabolic derangement is called methyl trap or folate trap and can be associated with B12 deficiency as well as low activities of MS or MTRR enzymes. Whether mutations or common genetic variants in corresponding genes can trap methyl groups has not yet been experimentally tested. However, the methyl trap, can be at least partially compensated for by increased folate intake.
ENZYMES OF FOLATE METABOLISM AND COMMON GENETIC VARIANTS In contrast to B12, folate malabsorption rarely causes disease. Instead, folate metabolism is affected by
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polymorphisms in genes coding for folatemetabolizing enzymes. Genetic variants in these genes are common and numerous and are found in all key genes involved in folate metabolism. Epidemiologic studies attempted to link these variants to NTDs, different types of cancer, and cardiovascular and neurologic disease. So far, the most characterized genetic variants in folate metabolism are those found in methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase (DHFR). Genetic variants in MTHFR mostly affect methylation-dependent reactions whereas variants in DHFR affect the whole OCM owing to the strategic position of the enzyme in this pathway (Fig. 39.1).
Methylenetetrahydrofolate Reductase MTHFR (5,10-methylene-THF reductase) catalyzes the conversion of 5,10-methylene-THF to 5-methyl-THF (Fig. 39.1). To better understand the disposition of this reaction in folate metabolism, it should be pointed out that it is strictly irreversible. Therefore, the only way to use 5methyl-THF produced by MTHFR is by the B12-dependent remethylation of homocysteine into methionine. As discussed earlier, this reaction catalyzed by MS has a dual significance. First, it is an important source of methionine in the cell. Although methionine has several functions, a large portion of this amino acid is used to biosynthesize SAM, a universal methyl donor involved in more than 100 methylation reactions in the cell. Second, folate-dependent methionine biosynthesis contributes significantly to the removal of homocysteine. This could be an important function because homocysteine accumulation generally has a negative effect in humans and is associated with increased risk for cardiovascular diseases. In agreement with this phenomenon, a common MTHFR polymorphism, 677C > T, is associated with mild hyperhomocysteinemia and increased risk for coronary heart disease and stroke. These conditions are exacerbated in individuals with low folate status. The 677C > T polymorphism, which is the most common inborn error of folate metabolism, results in an amino acid alteration in the catalytic domain of the enzyme. The mutant MTHFR is a less stable protein that results in an overall decrease in MTHFR activity in affected individuals. Importantly, 5-methyl-THF was shown to have a protective effect on enzyme stability. The 677C>T MTHFR variant is common in European and American populations; the homozygous form is found in 5%e20% of individuals. Its clinical manifestation is not limited to hyperhomocysteinemia but includes lower levels of folate in plasma and red blood cells (RBC), a likely contributing factor to the associated pathologies, which could be diverse. Thus, in addition to cardiovascular disease, this MTHFR SNP was implicated in the
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increased risk for neural tube defects, cleft lip and palate, Down syndrome, thrombosis, schizophrenia, depression, adverse pregnancy outcomes, and cancer. Interestingly, this mutation may have a protective effect against some cancers, although mechanisms underlying this phenomenon remain elusive. Numerous studies have also shown that mild MTHFR deficiency resulting from the 677C>T polymorphism may be corrected by dietary folic acid. At the same time, severe deficiency of the enzyme can be rescued by 5-methyl-THF, but not by folic acid. In line with this finding, 5-methyl-THF was shown to increase plasma folate more efficiently than folic acid in women of either 677CC or 677TT genotype. Overall, over 50 naturally occurring mutations have been identified in the MTHFR gene, as well as nine common polymorphisms, but 677C>T remains most studied and recognized. Another relatively common MTHFR polymorphism, 1298A>C, is found in a homozygous state in 4%e12% of individuals in different populations. This variant has reduced activity, but not as dramatically as the 677C>T mutant (only to 68% of wild-type [WT] activity), and it is not thermolabile. Individuals homozygous for this SNP have normal tHcy, not different from subjects with the WT variant. The compound heterozygotes for the 1298C and 677T variants have biochemical profiles similar to those of 677T homozygotes as well as increased homocysteine. This is the why numerous studies are testing both 677C>T and 1298A>C polymorphisms for association with diseases.
Dihydrofolate Reductase DHFR is a key enzyme in folate metabolism that incorporates ingested folic acid into the reduced folate pool. Folic acid is a synthetic provitamin that requires enzymatic activation to become an active cofactor. DHFR catalyzes the two-step reduction of folic acid, first to dihydrofolate and then to the active coenzyme THF (Fig. 39.1). The enzyme is also responsible for the conversion of dihydrofolate, which is produced in the reaction of thymidine monophosphate biosynthesis, to THF (Fig. 39.1). Homozygous knockout of Dhfr in mice was shown to be embryonic-lethal. In humans, DHFR activity can vary at least sixfold between individuals, and generally is lower than in rodents. Low activity of the enzyme results in a low rate of dietary folic acid incorporation into folate pool and higher levels of unmetabolized folic acid in serum. Although negative effects of unmetabolized folic acid can be diverse and indirect, the low rate of incorporation of the vitamin to the active folate pool creates symptoms of folate deficiency even under conditions of high folic acid intake. A well-established DHFR polymorphism is a 19ebase pair (bp) deletion in the first intron, 60 nucleotides downstream from the splice donor site. This
polymorphism is highly prevalent with the reported frequency of the del/del genotype reaching 48% in some populations (20% del/del homozygosity in the US population). Because this is an intronic mutation, its likely effect is associated with transcriptional or posttranscriptional regulation of DHFR expression, which could change the protein level. Although this mechanism has not been explored, it has been demonstrated that the del/del genotype is associated with increased unmetabolized folic acid in plasma for intakes exceeding 500 mg/ d (high folate consumption), and with decreased RBC folate at intakes below 250 mg/d (low folate consumption). The effect of this polymorphism on NTDs, carcinogenesis, and cognitive function has also been investigated. Data on its role in NTDs and cognitive function are conflicting or incomplete, but there is compelling evidence that this mutation is associated with an increased cancer risk, which is further modified by the folic acid intake: (1) the DHFR 19del allele is associated with greater breast cancer risk among multivitamin users; and (2) del/del homozygous mothers taking folic acid supplements are at a higher risk for having a child with early childhood retinoblastoma. Although the molecular mechanisms underlying the biological effects of this genetic variant are not yet known, studies to date suggest that this is a functional polymorphism that alters the incorporation of folic acid into the intracellular pool of reduced folate. Most likely, the deletion diminishes the capacity of affected individuals to make active folate coenzymes from provitamin folic acid. This brings a paradoxical conclusion that a significant part of the population with the del/del genotype who have low folate status will not benefit from the increased intake of folic acid. These findings emphasize another important issue with dietary folate: the form of the vitamin used for supplementation.
Forms of Dietary Folate Natural folate obtained from a nonfortified diet is presented mostly as 5-methyl-THF. In contrast, the component of multivitamin supplements and food fortification, folic acid, is a synthetic compound not found in the nature. It is a provitamin, which requires two rounds of enzymatic reduction to THF to become an active cofactor. Because folic acid and 5-methyl-THF are functionally different, the efficiency of the natural sources of folate versus folic acid should be considered. Crosssectional analysis of numerous studies underscores the importance of the natural food folate. The advantage of 5-methyl-THF is its higher absorption rate and higher efficiency in increasing the RBC and plasma folate than folic acid. Specific concerns of supplementation with folic acid include masking of B12 deficiency, promotion of cancer and metastasis, epigenetic changes, and
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presence of unmetabolized folic acid in the circulation. So, should the supplementation with 5-methyl-THF be considered instead of folic acid? Two aspects have to be considered. Folic acid is proven to reduce the incidence of NTDs, however there are no such data for 5methyl-THF. Furthermore, as discussed in this chapter, the incorporation of 5-methyl-THF into the folate pool requires the B12-dependent remethylation of homocysteine. If this process is impaired owing to B12 deficiency, or by polymorphisms in the MTR/MTRR or some other genes, supplementation will be ineffective. Another viable alternative to folic acid could be supplementation with folinic acid (5-formyl-THF), which is used to treat cancer patients in combination with 5-fluorouracil. Folinic acid rapidly incorporates into the reduced folate pool upstream of MTHFR, thus avoiding both the folate trap and the necessity to be activated by DHFR. Folinic acid has been shown to bypass the DHFR deficiency effectively by correcting hematological abnormalities, normalizing the cerebrospinal fluid folate levels, and improving neurological symptoms in patients homozygous for L80F or D153V DHFR mutations, both of which result in reduced enzyme activity.
VITAMIN B6-RELATED FOLATE PATHWAY ENZYMES Serine Hydroxymethyltransferase Serine hydroxymethyltransferase (SHMT) synthesizes glycine from serine. This is a reversible reaction, but thermodynamics favors the biosynthesis of glycine (Fig. 39.1). The reaction uses two coenzymes: THF, which accepts OCG from serine, and PLP (a derivative of vitamin B6, Fig. 39.4), which is absolutely required for enzyme catalysis. This reaction is important for bringing OCG to folate metabolism and also as the source of glycine in the cell. In fact, up to 70% of glycine could come from this reaction and not from dietary sources. There are two identical SHMT reactions in the cell, one in cytoplasm and another in mitochondria, both catalyzed by highly similar enzymes, cytosolic SHMT1 and mitochondrial SHMT2. Interestingly, vitamin B6 restriction decreases the activity and stability of SHMT, but the cytoplasmic isozyme is more sensitive to vitamin B6 deficiency than the mitochondrial isozyme. A polymorphism in SHMT1, 1420 C> T, has been described. This is an exonic mutation that results in the substitution of leucine by phenylalanine at position 474 of the protein (L474F). This is a common SNP; the reported frequency for the TT genotype is about 12%. Although this mutation is not believed to alter enzyme activity, it prevents the enzyme from sumoylation and thereby prevents its transport into the nuclei. It has been observed that individuals with the 1420TT
genotype have increased RBC and plasma folate levels. The 1420TT genotype is also associated with the reduction of risk of several cancers and some other diseases. This effect was linked to decreased DNA damage, observed in the TT genotype. Polymorphisms in SHMT2 are rarer and are not yet linked with the risk for any disease.
Cystathionine b-Synthase Cystathionine b-synthase (CBS) is a key enzyme in the two-step biosynthesis of cysteine from homocysteine and serine and requires vitamin B6 for its catalysis (Fig. 39.1). This is the only pathway in humans that leads to cysteine production. This pathway competes for homocysteine with homocysteine remethylation by MS in the methyl cycle pathway. Over 100 variations have been identified in the CBS gene. Many of them cause decreased enzyme activity, whereas for about a quarter of mutations an effect has not yet been established. Deficiency of CBS leads to homocystinuria, a rare autosomal recessive inborn error of sulfur amino acid metabolism. Some of the mutations can be corrected by pyridoxine (pyridoxineresponsive) whereas others are pyridoxine nonresponsive. There is at least one mutation in the CBS gene that produces hyperactive enzyme (up to 10-fold higher activity compared with the WT enzyme). In addition to rare mutations in CBS, several common polymorphisms in this gene were identified. One of these polymorphisms, a 68-bp insertion in exon 8, has a high prevalence (12% in the healthy US population, but varies between 8% and 26% depending on race, whereas the prevalence among healthy men and women in Northern Ireland is 18%). By itself, this mutation might not have a significant effect on circulating homocysteine levels. Nevertheless, it might have an effect in combination with the other common variant genotypes in folate pathways, MTHFR 677TT and MS 2756 AA. The 68ins polymorphism is also associated with decreased schizophrenia risk. Finally, CBS has two binding sites for SAM, and binding of SAM activates the enzyme. Because SAM levels are linked to folate metabolism (as discussed earlier), this provides an additional regulatory circuit between B vitaminerelated pathways and adds to the overall complexity of dietary interventions. Deleterious mutations within SAM regulatory sites will impair CBS activation, thus altering folate-dependent responses.
CONCLUSIONS Correction of metabolic folate deficiencies caused by specific gene variations through enhanced supplementation is an attractive and feasible approach to the treatment or prevention of diseases. Mandatory food fortification
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with folic acid has proven to decrease the incidence of NTDs. However, the outcome of supplementation approach will depend on many factors including the ingested form of the vitamin, the status of other B vitamins, and the overall complex interaction between enzymes in the pathway, as well as between pathways. Considering that functional polymorphisms in folate enzyme genes are rather common, their combinations can produce multiple effects. It could be expected that some genetic variants synergize to exacerbate metabolic derangement, whereas others perhaps can compensate for each other. The situation is complicated further as currently, the effects of a number of polymorphisms on the corresponding protein function have not been established. The entire field is still in its infancy and future studies will provide more precise knowledge of how genotypes affect the human body’s ability to process the nutrients and how deficient genotypes can be corrected by dietary manipulations.
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Kozich, V., Sokolova´, J., Klatovska´, V., Krijt, J., Janosı´k, M., Jelı´nek, K., Kraus, J.P., 2010. Cystathionine beta-synthase mutations: effect of mutation topology on folding and activity. Hum Mutat 31 (7), 809e819. Kurnat-Thoma, E.L., Pangilinan, F., Matteini, A.M., Wong, B., Pepper, G.A., Stabler, S.P., Guralnik, J.M., Brody, L.C., 2015. Biol Res Nurs 17 (4), 444e454. Leclerc, D., Sibani, S., Rozen, R.. Molecular biology of methylenetetrahydrofolate reductase (MTHFR) and overview of mutations/polymorphisms. In: Madame Curie Bioscience Database [Internet], Landes Bioscience, Austin (TX), 2000-2013. Molloy, A.M., 2012. Genetic aspects of folate metabolism. Subcell Biochem 56, 105e130. Philip, D., Buch, A., Moorthy, D., Scott, T.M., Parnell, L.D., Lai, C.Q., Ordova´s, J.M., Selhub, J., Rosenberg, I.H., Tucker, K.L., Troen, A.M., 2015. Dihydrofolate reductase 19-bp deletion polymorphism modifies the association of folate status with memory in a cross-sectional multi-ethnic study of adults. Am J Clin Nutr 102 (5), 1279e1288. Scaglione, F., Panzavolta, G., 2014. Folate, folic acid and 5methyltetrahydrofolate are not the same thing. Xenobiotica 44 (5), 480e488. Selhub, J., Rosenberg, I.H., 2016. Excessive folic acid intake and relation to adverse health outcome. Biochimie 126, 71e78. Stover, P.J., 2012. Polymorphisms in 1-carbon metabolism, epigenetics and folate-related pathologies. J Nutrigenetics Nutrigenomics 4 (5), 293e305. Tibbetts, A.S., Appling, D.R., 2010. Compartmentalization of Mammalian folate-mediated one-carbon metabolism. Annu Rev Nutr 30, 57e81. Watkins, D., Ru, M., Hwang, H.Y., Kim, C.D., Murray, A., Philip, N.S., Kim, W., Legakis, H., Wai, T., Hilton, J.F., Ge, B., Dore´, C., Hosack, A., Wilson, A., Gravel, R.A., Shane, B., Hudson, T.J., Rosenblatt, D.S., 2002. Hyperhomocysteinemia due to methionine synthase deficiency, cblG: structure of the MTR gene, genotype diversity, and recognition of a common mutation, P1173L. Am J Hum Genet 71 (1), 143e153. Woeller, C.F., Anderson, D.D., Szebenyi, D.M., Stover, P.J., 2007. Evidence for small ubiquitin-like modifier-dependent nuclear import of the thymidylate biosynthesis pathway. J Biol Chem 282 (24), 17623e17631.
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