The Biochemistry
Folic acid is probably my favourite vitamin, right next to choline (you caught me - i love methyl donors). Folic acid is one of the water soluble B-vitamins, B9. Folates are actually a family of related compounds that are some modified form of pteroylglutamic acid. We generally hear about folate in terms of folic acid, which is the form found in folate supplements. Folates are modified by being reduced, methylated or through varying numbers of glutamate residues on their tails. Dietary folates are mainly folylpolyglutamates, containing four to seven glutamic acid residues - some folylmonoglutamates (the most bioavailable) may be present in food because most animals/plants contain gamma-glutamyl hydrolase; however, cooking destroys the activity of this enzyme. Upon absorption by enterocrytes, the enzyme gamma-glutamyl hydrolase hydrolyzes these folylpolyglutamates to folylmonoglutamates. One reason that folic acid supplements are much more bioavailable than dietary folate is because it is already a monoglutamate and no action by the enzyme is required. Once absorbed, serum folate is mainly in the 5-methyl form. Once transported to its destined tissue, it is reconverted to the polyglutamyl form - this is catalyzed by folylpolyglutamate synthase. This re-polyglutamation is used to prevent folates from passing through membranes, enhancing binding to enzymes, increasing catalytic activity, and allowing for folate to shuttle from one active site to another without leaving the enzyme's surface.
Folate is extremely important to the body because of its role in methyl metabolism/ 1-C pathways. Here, folate works alongside methionine, b12, and zinc to maintain homocysteine homeostasis and the activated state of methionine in the form of S-adenosyl-methionine (SAM). SAM is responsible for methylating the DNA Methyltransferases, DNA nucleotide synthesis, synthesis of creatine and carnitine, and virtually every other methylation event in the body. It should be noted that the 1 carbon units that methylate folates in the cell derive from serine and are transferred to THF via serene hydroxymethyltransferase - this has been pointed out as another factor that needs to be controlled for when studying the effects of folate availability, methyl group availability and genomic methylation. This pathway is also important from an oxidative stress standpoint because homocysteine can be converted to cysteine via the transsulpluration pathway, which is a constituent of Glutathione, the main aspect of the bodies natural antioxidant system. Dietary folate, as well as dietary methionine and cysteine, all interact and can affect methylation and oxidative states.
Clinical Relevance
Folic acid has historically been a major concern because of it's role in Neutral Tube Defects (NTDs). NTDs - generally spinda bifida, anencephaly, or encephalocele- occur very early in embryogenesis because the neural tube has closed off/sealed within 30 days of conception. The formation begins on day 18. There is a 12 day window within the first month after conception that is quite sensitive to folate levels (not that folate isn't important at all times throughout pregnancy). Because of this sensitivity occurring at a time when most women do not know that they are pregnant, folate levels of women 'at risk' of becoming pregnant are most important. This is why refined grains have been fortified with folic acid. Two side notes: 1. folic acid intake can mask the symptoms of pernicious anemia, and has caused some concern over folic acid fortification/supplementation. 2. obesity, and more mechanistically high blood glucose levels, also appears to be responsible for NTDs.
The current recommendations for males and females range from 150-200 ug of folate per day, with pregnant women being recommended to consume 400ug (more on the controversial aspects of this in part 2 post). Less folate is required for children and infants. The best sources of folate include yeast and yeast extracts, liver meats, legumes, citrus fruits and dark leafy greens. Folic acid levels should be tested using red blood cell folate levels to adequately reflect true, long term folate status of an individual - levels should be 160ng/ml or greater - less than 140ng/ml indicates deficiency. Deficiency can lead to higher-than-homeostatic levels of plasma homocysteine, which is a major factor in CVD risk and potentially involved in dementia and Alzheimer's. As with every nutrient, the difference between the RDA and the UL (upper limit) leaves some wiggle room, and some people may need more/less of a nutrient depending on genetic variability..
Genetic Variation and Folate
A number of factors affect folate uptake and its downstream byproducts. Folate metabolism has a large environmental component, via its interactions with other dietary constituents. However, a number of genetic factors affecting folate requirements, utilization and disease have been identified. When considering the genetics of folate, we must look consider both folate receptors/transporters and the enzymes involved folate metabolism - there are a number that have been identified and I'd imagine that several have not been - because it is so difficult to parse out the environmental confounders to find true statistical association, here i'll discuss the most understood/widespread variants:
1. MTHFR - Methylene Tetrahydrofolate Reductase is required for the reduction of 5,10 Methylene THF to 5 Methyl THF, the metabolite that is necessary for re-methylating homocysteine. Normal functioning variants include 677 C/T (has been implicated in a number of conditions but conclusive evidence lacks) or 677C/C; a less functional variant occurs in individuals with the MTHFR 677 T/T genotype. This genotype leads to a phenotype where the enzyme is less stable and remethylation of homocysteine is much slower, though it appears that higher folate intake may overcompensate for reduced enzyme activity. This T/T variant results in hyperhomocysteinemia, previously mentioned to be a risk factor for CVD. It is important to note that this genotype does not destine one for hyperhomocysteinemia - the overall haplotype of an individual matters .The variant has epistatic interactions with other folate-related SNPs such as A1298C MTHFR, G80A reduced folate carrier, A2756G Methionine Synthase and A66G methionine synthase. Dietary intake of riboflavin can also affect levels of homocystein by increasing FAD concentrations and stabilizing the T/T variant (1) - small, individual studies associated riboflavin intake with better CVD-related outcomes from individuals with this variant. Intake of choline and betaine, two other dietary methyl donors, can also confound the deleterious effects of this variant by providing methyl groups to donate to S-adenosyl homocysteine --> SAM. These two MTHFR variants aren't all bad - a number of studies have found them to be conditionally protective against breast cancer in postmenopausal women (2), rectal and colon cancer (3, 4). A potential explanation for this protection lies in the increased availability of folate for nucleotide synthesis due to reduced MTHFR synthesis, as well as reduced methylation in CpG sites of the p53 tumor suppressor gene (5)- a major hallmark of cancer is genome wide hypomethylation but site specific hypermethylation. However, for individuals who have early onset breast cancer and are undergoing treatment with cyclophosphamide, methotrexate and fluorouracil, TT homozygosity appears to lead to severe toxicity ( Toffoli, 2000)
- you might be thinking to yourself, hey if these variants are protective against later in life diseases but reduce MTHFR activity, why are they so prevalent in the human population?? Interestingly, there is evidence for a selection gradient in these polymorphisms (6,7). It appears that there is some sort of advantage conferred by TT homozygosity when dietary folate intake is adequate. This reproductive advantage is not known, partly because of the widespread heterogeneity amongst the distribution of this variant in modern human populations- it has been suggested to be involved with resistance to malaria and also implicated in the evolution of skin pigmentation - more conclusive research is needed.
2. four genes encode human folate receptors - Folate Receptor 1 has received the most attention, though variation in this gene, for humans, are not strongly associated with Neural Tube defects or other morbidities. This receptor has been shown to exhibit loss of function mutations in individuals with cerebral folate transport deficiency (8).
Folate is an extremely important example of how nutrition, genetics, evolution and disease are all deeply intertwined. I found it quite difficult to keep my thoughts organized with the overwhelming information out there on this topic and its genetic variants. While this post addressed the biochemistry and nutrigenetic perspective, part 2 will elucidate more of folate's role in Nutrigenomics, particularly Genomic Methylation (epigenetics) and interactions with the microbiota.
References:
Brody, T., Nutritional Biochemistry, 2nd addition. 1999. UC Berkley
Toffoli et al. MTHFR gene polymorphism and severe toxicity during adjuvant treatment of early breast cancer with cyclophosphamide, methotrexate and fluorouracil. 2000. Annals of Oncology - Letters to the Editor
1. http://www.ncbi.nlm.nih.gov/pubmed/12560354
2. http://www.ncbi.nlm.nih.gov/pubmed/15598763
3. http://www.ncbi.nlm.nih.gov/pubmed/15829374
4. http://clincancerres.aacrjournals.org/content/9/2/743.full
5.. http://www.ncbi.nlm.nih.gov/pubmed/16177213
6. http://www.biomedcentral.com/1471-2350/9/104#B17
7. http://www.ncbi.nlm.nih.gov/pubmed/16522920
8. http://omim.org/entry/613068
Folic acid is probably my favourite vitamin, right next to choline (you caught me - i love methyl donors). Folic acid is one of the water soluble B-vitamins, B9. Folates are actually a family of related compounds that are some modified form of pteroylglutamic acid. We generally hear about folate in terms of folic acid, which is the form found in folate supplements. Folates are modified by being reduced, methylated or through varying numbers of glutamate residues on their tails. Dietary folates are mainly folylpolyglutamates, containing four to seven glutamic acid residues - some folylmonoglutamates (the most bioavailable) may be present in food because most animals/plants contain gamma-glutamyl hydrolase; however, cooking destroys the activity of this enzyme. Upon absorption by enterocrytes, the enzyme gamma-glutamyl hydrolase hydrolyzes these folylpolyglutamates to folylmonoglutamates. One reason that folic acid supplements are much more bioavailable than dietary folate is because it is already a monoglutamate and no action by the enzyme is required. Once absorbed, serum folate is mainly in the 5-methyl form. Once transported to its destined tissue, it is reconverted to the polyglutamyl form - this is catalyzed by folylpolyglutamate synthase. This re-polyglutamation is used to prevent folates from passing through membranes, enhancing binding to enzymes, increasing catalytic activity, and allowing for folate to shuttle from one active site to another without leaving the enzyme's surface.
Folate is extremely important to the body because of its role in methyl metabolism/ 1-C pathways. Here, folate works alongside methionine, b12, and zinc to maintain homocysteine homeostasis and the activated state of methionine in the form of S-adenosyl-methionine (SAM). SAM is responsible for methylating the DNA Methyltransferases, DNA nucleotide synthesis, synthesis of creatine and carnitine, and virtually every other methylation event in the body. It should be noted that the 1 carbon units that methylate folates in the cell derive from serine and are transferred to THF via serene hydroxymethyltransferase - this has been pointed out as another factor that needs to be controlled for when studying the effects of folate availability, methyl group availability and genomic methylation. This pathway is also important from an oxidative stress standpoint because homocysteine can be converted to cysteine via the transsulpluration pathway, which is a constituent of Glutathione, the main aspect of the bodies natural antioxidant system. Dietary folate, as well as dietary methionine and cysteine, all interact and can affect methylation and oxidative states.
Clinical Relevance
Folic acid has historically been a major concern because of it's role in Neutral Tube Defects (NTDs). NTDs - generally spinda bifida, anencephaly, or encephalocele- occur very early in embryogenesis because the neural tube has closed off/sealed within 30 days of conception. The formation begins on day 18. There is a 12 day window within the first month after conception that is quite sensitive to folate levels (not that folate isn't important at all times throughout pregnancy). Because of this sensitivity occurring at a time when most women do not know that they are pregnant, folate levels of women 'at risk' of becoming pregnant are most important. This is why refined grains have been fortified with folic acid. Two side notes: 1. folic acid intake can mask the symptoms of pernicious anemia, and has caused some concern over folic acid fortification/supplementation. 2. obesity, and more mechanistically high blood glucose levels, also appears to be responsible for NTDs.
The current recommendations for males and females range from 150-200 ug of folate per day, with pregnant women being recommended to consume 400ug (more on the controversial aspects of this in part 2 post). Less folate is required for children and infants. The best sources of folate include yeast and yeast extracts, liver meats, legumes, citrus fruits and dark leafy greens. Folic acid levels should be tested using red blood cell folate levels to adequately reflect true, long term folate status of an individual - levels should be 160ng/ml or greater - less than 140ng/ml indicates deficiency. Deficiency can lead to higher-than-homeostatic levels of plasma homocysteine, which is a major factor in CVD risk and potentially involved in dementia and Alzheimer's. As with every nutrient, the difference between the RDA and the UL (upper limit) leaves some wiggle room, and some people may need more/less of a nutrient depending on genetic variability..
Genetic Variation and Folate
A number of factors affect folate uptake and its downstream byproducts. Folate metabolism has a large environmental component, via its interactions with other dietary constituents. However, a number of genetic factors affecting folate requirements, utilization and disease have been identified. When considering the genetics of folate, we must look consider both folate receptors/transporters and the enzymes involved folate metabolism - there are a number that have been identified and I'd imagine that several have not been - because it is so difficult to parse out the environmental confounders to find true statistical association, here i'll discuss the most understood/widespread variants:
1. MTHFR - Methylene Tetrahydrofolate Reductase is required for the reduction of 5,10 Methylene THF to 5 Methyl THF, the metabolite that is necessary for re-methylating homocysteine. Normal functioning variants include 677 C/T (has been implicated in a number of conditions but conclusive evidence lacks) or 677C/C; a less functional variant occurs in individuals with the MTHFR 677 T/T genotype. This genotype leads to a phenotype where the enzyme is less stable and remethylation of homocysteine is much slower, though it appears that higher folate intake may overcompensate for reduced enzyme activity. This T/T variant results in hyperhomocysteinemia, previously mentioned to be a risk factor for CVD. It is important to note that this genotype does not destine one for hyperhomocysteinemia - the overall haplotype of an individual matters .The variant has epistatic interactions with other folate-related SNPs such as A1298C MTHFR, G80A reduced folate carrier, A2756G Methionine Synthase and A66G methionine synthase. Dietary intake of riboflavin can also affect levels of homocystein by increasing FAD concentrations and stabilizing the T/T variant (1) - small, individual studies associated riboflavin intake with better CVD-related outcomes from individuals with this variant. Intake of choline and betaine, two other dietary methyl donors, can also confound the deleterious effects of this variant by providing methyl groups to donate to S-adenosyl homocysteine --> SAM. These two MTHFR variants aren't all bad - a number of studies have found them to be conditionally protective against breast cancer in postmenopausal women (2), rectal and colon cancer (3, 4). A potential explanation for this protection lies in the increased availability of folate for nucleotide synthesis due to reduced MTHFR synthesis, as well as reduced methylation in CpG sites of the p53 tumor suppressor gene (5)- a major hallmark of cancer is genome wide hypomethylation but site specific hypermethylation. However, for individuals who have early onset breast cancer and are undergoing treatment with cyclophosphamide, methotrexate and fluorouracil, TT homozygosity appears to lead to severe toxicity ( Toffoli, 2000)
- you might be thinking to yourself, hey if these variants are protective against later in life diseases but reduce MTHFR activity, why are they so prevalent in the human population?? Interestingly, there is evidence for a selection gradient in these polymorphisms (6,7). It appears that there is some sort of advantage conferred by TT homozygosity when dietary folate intake is adequate. This reproductive advantage is not known, partly because of the widespread heterogeneity amongst the distribution of this variant in modern human populations- it has been suggested to be involved with resistance to malaria and also implicated in the evolution of skin pigmentation - more conclusive research is needed.
2. four genes encode human folate receptors - Folate Receptor 1 has received the most attention, though variation in this gene, for humans, are not strongly associated with Neural Tube defects or other morbidities. This receptor has been shown to exhibit loss of function mutations in individuals with cerebral folate transport deficiency (8).
Folate is an extremely important example of how nutrition, genetics, evolution and disease are all deeply intertwined. I found it quite difficult to keep my thoughts organized with the overwhelming information out there on this topic and its genetic variants. While this post addressed the biochemistry and nutrigenetic perspective, part 2 will elucidate more of folate's role in Nutrigenomics, particularly Genomic Methylation (epigenetics) and interactions with the microbiota.
References:
Brody, T., Nutritional Biochemistry, 2nd addition. 1999. UC Berkley
Toffoli et al. MTHFR gene polymorphism and severe toxicity during adjuvant treatment of early breast cancer with cyclophosphamide, methotrexate and fluorouracil. 2000. Annals of Oncology - Letters to the Editor
1. http://www.ncbi.nlm.nih.gov/pubmed/12560354
2. http://www.ncbi.nlm.nih.gov/pubmed/15598763
3. http://www.ncbi.nlm.nih.gov/pubmed/15829374
4. http://clincancerres.aacrjournals.org/content/9/2/743.full
5.. http://www.ncbi.nlm.nih.gov/pubmed/16177213
6. http://www.biomedcentral.com/1471-2350/9/104#B17
7. http://www.ncbi.nlm.nih.gov/pubmed/16522920
8. http://omim.org/entry/613068
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