Folic acid, vitamin B12, and DNA methylation: An update

Abstract

Epigenetics is one of the exciting and fastest expanding fields of biology; this is above genetics. Methylation is the process involved in the transfer of methyl group to amino acids, proteins, enzymes and DNA of all the cells, and tissues of the body. During cell-division low folate availability may result in decreased production of thymidine wherein uracil may be substituted in the place of thymidine in the DNA sequence. It was reported that folate and Vitamin B12 restricted diet resulted in aberrant methylation patterns. The current review was undertaken to explore the role of folic acid and Vitamin B12 in DNA methylation.

Full text

Vol 11, Issue 1, 2018
Online - 2455-3891
Print - 0974-2441
FOLIC ACID, VITAMIN B12, AND DNA METHYLATION: AN UPDATE
BHONGIR APARNA VARMA1, SRILATHA BASHETTI2*, RAJAGOPALAN VIJAYARAGHAVAN3, KUMAR SAI SAILESH4
1Department of Biochemistry, Mediciti Institute of Medical Sciences, Medchal, Telangana, India. 2Department of Biochemistry, Apollo
Institute of Medical sciences & Research, Hyderabad, Telangana, India. 3Department of Research, Saveetha University, Chennai, Tamil
Nadu, India. 4Department of Physiology, DM Wayanad Institute of Medical Sciences, Wayanad, Kerala, India.
Email: sri_happyfamily@yahoo.com
Received: 09 August 2017, Revised and Accepted: 07 October 2017
ABSTRACT
Epigenetics is one of the exciting and fastest expanding fields of biology; this is above genetics. Methylation is the process involved in the transfer of
methyl group to amino acids, proteins, enzymes and DNA of all the cells, and tissues of the body. During cell-division low folate availability may result
in decreased production of thymidine wherein uracil may be substituted in the place of thymidine in the DNA sequence. It was reported that folate
and Vitamin B12 restricted diet resulted in aberrant methylation patterns. The current review was undertaken to explore the role of folic acid and
Vitamin B12 in DNA methylation.
Keywords: DNA methylation, Folic acid, Vitamin B12.
INTRODUCTION
Epigenetics is one of the exciting and fastest expanding fields of
biology; this is above genetics. The modifications of epigenetics play
a very important role in the regulation of many cellular processes
including DNA replication, gene expression, and recombination. This
is achieved through regulatory mechanisms such as DNA methylation,
hydroxymethylation, histone modifications, chromatin remodeling,
and RNA modifications like methylation. Misregulation of epigenetic
mechanism may have adverse effects on health and may lead to
neurological disorders, developmental abnormalities and also cancer.
Therefore, epigenetic modifications are evolving as very potent
diagnostic and prognostic biomarkers in the world of medicine.
Methylation is the process involved in the transfer of methyl group to
amino acids, proteins, enzymes and DNA of all the cells, and tissues of the
body. Donation of the methyl group is very important in the regulation
of cell energy, gene expression, neurological function, detoxification (in
liver), immunity, etc. Methylation is one of the important biochemical
processes that occur in the body and is catalyzed by different enzymes.
This is a process influenced by environmental conditions, decreases
with age. Methylation depicts the quality of life in terms of diseased and
health conditions.
The methylation processes require two cycles - Cycle A:
S- adenosylmethionine (SAM) and Cycle B- folate cycle. The most
stable synthetic form of Vitamin B9 is folic acid (FA), and the natural
form is termed as folate. The natural dietary folates are polyglutamated
consisting of six glutamate molecules linked together by peptide
bonds. Mainly there are two dietary folates, 5-methyltetrahydrofolate
(5-MeTHF) and 10- formyltetrahydrofolate (10-formyl THF). In
the gut, before to the absorption polyglutamates are hydrolyzed
to monoglutamates by the enzymes γ-glutamyl hydrolases and is
absorbed [1,2]. FA with monoglutamyl residues gets converted to
the biologically active form known as THF by the reducing reaction
(first it is converted to dihydrofolate (DHF) and then to THF). These
reduction reactions are catalyzed by the single NADPH-dependent
enzyme DHF reductase (DHFR). THF receives the one-carbon (C1)
units from various donors such as serine, glycine, and histidine during
catabolic reactions and can transfer them to specific acceptors for the
synthesis of various compounds such as purines, methionine, choline,
formyl-methylated tRNA, thymidylate, and serine. The addition of one
carbon unit with a simultaneous reduction will produce 5-MeTHF, the
main circulating form in the blood. Protein carriers and the tissue-
specific folate receptors carry 5-MeTHF into the cells, where they get
accumulated and are transformed to polyglutamates. Polyglutamates
cannot traverse biological membranes by passive diffusion. Thus,
polyglutamylation serves to sequester folate in the cells in which it is
required. The enzymes involved in folate metabolism have a higher
affinity for polyglutamates than to monoglutamates. FA is involved in
remethylation process wherein, 5-MeTHF donates a methyl group for
homocysteine in the presence of the enzyme methionine synthase,
converting homocysteine to methionine liberating THF. This THF
directly gets converted into 5,10-methylene THF by the action of the
enzyme serine hydroxymethyltransferase which is present both in
mitochondria and cytosol.
5-MeTHF is used in remethylation of homocysteine to methionine in
the presence of methionine synthase in all the tissues except red blood
cells. This process requires a cofactor the Vitamin B12 (cobalamin).
Methylcobalamin is the donor of a methyl group to homocysteine and
the transfer mediated through THF getting converted to 5-MeTHF.
After donating the methyl group, methylcobalamin is converted back
to cobalamin. Methylcobalamin is reproduced from cobalamin by
receiving the methyl group from 5-MeTHF. Methionine is an essential
amino acid converted to active methionine, i.e., SAM also known
as the universal donor. SAM is the active donor of a methyl group
for various methylation reactions that occur in the body such as
methylation of nucleic acids, proteins, lipids, neurotransmitters, and
creatine synthesis [3]. SAM gets converted to S-adenosyl homocysteine
(SAH) after donating the methyl group to the acceptors further, gets
hydrolyzed to adenosine and homocysteine by the action of the enzyme
SAH hydrolase (SAHH). The decrease in 5-MeTHF or cobalamin made
lead to the accumulation of homocysteine the potent inhibitor of various
methyltransferases. Thus, cobalamin and folate are together involved in
methylation process. An absence of cobalamin leads to the cessation of
the reaction and build-up of methyltetrahydrofolate (MeTHF) known
as “folate trap.” Therefore, MeTHF tends to accumulate in Vitamin B12
(cobalamin) deficiency, leads to depletion of other coenzyme forms that
are needed for nucleotide synthesis. This is how folate trap hypothesis
© 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2018.v11i1.21892
Review Article

18
Asian J Pharm Clin Res, Vol 11, Issue 1, 2018, 17-20
Varma et al.
explains the anemia of cobalamin deficiency, but it cannot account for
the neurological manifestations of pernicious anemia.
METHODS
A detailed review of published literature from Google, PubMed, and
MEDLINE was performed and analyzed.
Folate DNA methylation
FA, when consumed in fortified foods or supplements, is primarily
metabolized to 5-methyl THF that behaves similar to natural dietary
folate. Initially, FA is reduced to DHF in the presence of the enzyme
DHF reductase, further converted to THF and enters the folate pool. In
some cases, the oxidized form of FA may appear in circulation with an
increase in DHF reductase enzyme [4]. The coenzyme THF is converted
to 5,10-methyleneTHF by the enzyme serine hydroxymethyltransferase
requires Vitamin B6. Further MTHFR irreversibly reduces it to 5-methyl
THF. This is a key reaction for the maintenance of the methyl flux
essential for the remethylation of homocysteine to methionine in the
presence of Vitamin B12-dependent methionine synthase. Methionine
is converted to SAM, an active methyl donor wherein numerous
SAM-dependent reactions play regulatory roles by affecting gene
transcription, genome stability [5] and localization of protein [6], etc.
Along with folate, many other dietary nutrients such as Vitamin B6,
Vitamin B12, riboflavin (Vitamin B2), and choline are required for
the maintenance of one carbon flux and normal formation of SAM,
homocysteine remethylation, and DNA methylation. DNA methylation
and one carbon metabolism work under tight regulatory control.
Homocysteine remethylation is folate-dependent and requires
SAM as an important regulator for this process. Increase in SAM
inhibits MTHFR this reduces 5-methylTHF synthesis further hinders
homocysteine remethylation. In contrast, remethylation is favored with
low concentrations of SAM and SAM-dependent methyltransferase is
inhibited by SAH [7,43,44]. Therefore, for the maintenance of normal
DNA methylation, there should be a continual conversion of SAH to
homocysteine [8] and increased plasma concentration of homocysteine
is associated with increased concentration of SAH which in-turn
associated with hypomethylation of global DNA [9]. The common
genetic variant 677C-T modifies the activity of MTHFR and reduces the
formation of 5-methylTHF [10].
DNA methylation and low folate status
The studies on the association of low folate with increased risk of
NTDs, cardiovascular disease and multiple cancers are well established
but, the mechanism leading to these disorders is yet unclear [11-13].
During cell-division low folate availability may result in decreased
production of thymidine wherein uracil may be substituted in the place
of thymidine in the DNA sequence. This may increase the frequency of
chromosomal breaks to repair the defect made by the mutagenic event.
This was studied by a tissue culture where the MTHFR-TT genotype
shows the formation of increased micronuclei as a result of multiple
chromosomal breakages occurred under low folate conditions [14].
The effects of supplementation of FA may promote or prevent cancers,
stated in many studies. In human cancers, the DNA methylation is
dysregulated. It shows either hypermethylation or hypomethylation
stating that the association of DNA methylation with tumor is cell or
tissue or organ-specific. A study showed genome-wide hypomethylation
but found 5% hypermethylated patterns defining the characteristics of
the specific type of human tumor. These altered DNA methylation cause
chromosomal instability and silencing of tumor suppressor genes [16].
However, it is very important to note that decrease in folate status may
result either in hyper or hypomethylation leading to the misregulation
in the complex system. (Shelnutt et al., 2004) On controlled feeding
studies observed the association of low folate concentration with
reduced DNA methylation in older women but not in younger female
adults (Rampersaud et al., 2000; Jacob et al., 1998). Jacob and Shelnutt
both had observed that the folate intake during repletion resulted in
increased DNA methylation and the increase was limited to MTHFR
TT genotype. Friso et al. (2002) and Pufulete et al. (2005) also found
the same trend of lowered DNA methylation with low serum folate
concentration. These studies taken together suggest that the genotype,
age, duration, and magnitude of exposure should be considered as the
response of global DNA methylation to the folate status is different at
different conditions. In the above studies, variations in DNA methylation
were found between the sexes and ages (El-Maarri et al., 2007); (Fraga
et al., 2005) justifying the observations of different methylation
patterns with low and high folate status.
MTHFR is an essential enzyme involved in the irreversible conversion
of 5,10 MethyleneTHF to 5-methylTHF thus, playing important role
in DNA synthesis, DNA methylation and maintenance of balanced
nucleotide pool (Friso et al., 2002; Kim et al., 1999). Analyses of MTHFR
polymorphisms are included to investigate the folate status and DNA
methylation in humans. As many observational studies had established
the association of low folate with hypomethylated DNA in subjects with
homozygous MTHFR C677TT genotype. The biochemical mechanism
behind this is the MTHFR C677TT polymorphism causes thermolability
thereby reducing the MTHFR activity by lowering 5-methylTHF levels
and leading to the accumulation of 5,10methyleneTHF, increase
in plasma homocysteine levels and finally changing the cellular
composition of one-carbon folate derivatives stating that there is a
greater risk of global DNA hypomethylation in the women carrying
MTHFR T allele as it is involved in the impairment of enzyme activity
modulating both gene and genome-specific DNA methylation
(La Merrill et al. 2012).
High folate and DNA methylation
Many studies on folate insufficiency and the effect of low folate in
humans are well established. Low folate has detrimental effect on
the embryo and increases the risk of NTD’s, increases the possibility
of long-term risk of diabetes and also may lead to many other health
outcomes [26-29]. High folate concentrations and increased folate
supplementation had shown contradicting results in different in
controlled feeding trial studies. Certain studies have shown the
association of high folate with high global DNA methylation and reduced
risk of cancer [30,31]. A recent study had shown reversal effect that
is, the increased folate supplementation resulted in stimulation and
progression of existing tumors and altered normal DNA methylation
patterns [32-34]. As DNA methylation is a regulatory process depends
on tissues, sequence of DNA, genome region, stage of transformation,
degree, duration and exposure of folate intervention, timing, and other
regulatory proteins and enzymes involved in the process. Hence, we
should include all these factors to study the effect of high folate, whether
high folate leads to increased risk or benefit.
Folate, Vitamin B12, and placental DNA methylation
Placenta is an important organ. Proper development and function of
placenta are crucial for the growth, health, and survival of developing
fetus. Many studies had established links between epigenetic changes
in the placenta and the risk of disease in gestation and early life (Kim
et al., 2009). On examination of the epigenetic changes occurred in the
placenta had evolved interest in the research of biomarkers of exposure,
pathogenesis of the disease and the biology of the development of the
disease [36]. Presently, many studies on nutrition during pregnancy
and placental outcomes are taken up to understand the basis of disease
seen in early or later in life. Nutrition and epigenetic changes are the
emerging topic of interest in the present scenario to understand the
effects of increased supplementation of micronutrients like FA and
learning the importance of balancing the different micronutrients in
the diet to avoid unbalanced nutritional disorders and other health
complications later in life. One such study is carried on rats to know the
effect of FA supplementation in utero on the epigenetic changes in the
offspring. It was observed that maternal dietary folate during pregnancy
led to placental DNA hypomethylation and showed that there is a
significant correlation between folate levels of placenta and placenta
genomic DNA methylation. On the other hand, the study also stated
the importance of maintaining the ratio of folate and Vitamin B12. As

19
Asian J Pharm Clin Res, Vol 11, Issue 1, 2018, 17-20
Varma et al.
it has a significant role in determining genomic methylation patterns.
Wherein, high folate in the absence of Vitamin B12 resulted in placental
DNA hypomethylation [37]. It has also shown the association of
maternal folate deficiency with DNA hypomethylation [38]. Yet, another
animal study in sheep with maternal folate and Vitamin B12 restricted
diet resulted in aberrant methylation patterns, i.e., only 4% of cytosine-
guanine dinucleotide islands (CpG islands) out of 1400 CpG islands
were methylated. Along with hypomethylation the adult male offspring
also showed increased adiposity, altered immune function, high blood
pressure, and insulin resistance [39].
A study conducted by Kulkarni et al. [37] on the adverse effects
caused by excess FA supplementation in the presence or absence of
Vitamin B12 deficiency and correlated it with global DNA methylation
patterns. The team observed a reduction in the levels of global DNA
methylation on excess maternal FA supplementation with low plasma
Vitamin B12 concentration. They also observed the effect of Vitamin
B12 deficiency with excess or normal FA levels on docosahexaenoic
acid (DHA) levels. In this study, the team for the first time had identified
the DHA plays an important role in one-carbon metabolism, influencing
the placental global DNA methylation. Further learned that on the
supplementation of omega three fatty acids, the DHA levels in placenta
got increased and the DNA methylation levels were reverted back to
that of the control group. This study suggested that the altered ration of
FA and Vitamin B12 during pregnancy have effect on DNA methylation
thereby influence imprinting in the embryo and could be associated
with adverse pregnancy outcomes. These epigenetic changes caused,
may alter the gene expression and could be carried throughout the
lifespan of an individual [40].
Till date, there are only a few studies performed to learn the association
of FA and Vitamin B12 and together their effect on thyroid hormones.
The well- established knowledge regarding the two micronutrients
(folate and Vitamin B12) is (i) deficiency of Vitamin B12 is very common
in hypothyroidism (ii) both the micronutrients are very important
for fetal development and the imbalance ratio of Vitamin B12 and FA
(high folate with low Vitamin B12) may lead to small for gestational
age infants [42-44] and other detrimental effects on the growing fetus,
and (iii) both folate and Vitamin B12 are important along with other
micronutrients such as Vitamin B6, choline to prevent NTD’s, and
proper methylation process to occur.
CONCLUSION
Nutrition and epigenetic changes are the emerging topic of interest in the
present scenario to understand the effects of increased supplementation
of micronutrients like FA. The available information is that the increased
folate supplementation had a suppressive effect on thyroid hormones
(T3 and T4) and may possibly lead to motivational deficits and memory
impairments in adolescent rats and also observed that the animals
were functionally hypothyroid during the administration of folate. The
increased levels of folate supplementation alone may be harmful not
only during fetal development and on growing infant but also in the
adolescent period. If the same effect implies in humans with increased
folate supplementation during pregnancy, i.e., high folate intake may
lead to suppression of maternal plasma thyroid hormonal levels, would
have alarming implication on the health of the fetus.
REFERENCES
1. Stover PJ. Physiology of folate and vitamin B12 in health and disease.
Nutr Rev 2004;62:S3-12.
2. Beaudin AE, Stover PJ. Insights into the metabolic mechanisms
underlying folate-responsive neural tube defects: A mini review. Birth
Defects Res 2009;85:274-84.
3. Finkelstein JD. Metabolic regulatory properties of S-adenosylmethionine
and S-adenosylhomocysteine. Clin Chem Lab Med 2007;45:1694-9.
4. Bailey RL, Mills JL, Yetley EA, Gahche JJ, Pfeiffer CM, Dwyer JT, et al.
Unmetabolized serum folic acid and its relation to folic acid intake from
diet and supplements in a nationally representative sample of adults
aged > or =60 y in the United States. Am J Clin Nutr 2010;92:383-9.
5. Miranda TB, Jones PA. DNA methylation: The nuts and bolts of
repression. J Cell Physiol 2007;213:384-90.
6. Winter-Vann AM, Kamen BA, Bergo MO, Young SG, Melnyk S,
James SJ, et al. Targetting ras signaling through inhibition of carboxyl
methylation: An unexpected property of methotrexate. Proc Natl Acad
Sci U S A 2003;100:6529-34.
7. Hoffman DR, Marion DW, Cornatzer WE, Duerre JA. S-adenosylmethionine
and S-adenosylhomocystein metabolism in isolated rat liver. Effects
of L-methionine, L-homocystein, and adenosine. J Biol Chem
1980;255:10822-7.
8. James SJ, Melnyk S, Pogribna M, Pogribny IP, Caudill MA. Elevation
in S-adenosylhomocysteine and DNA hypomethylation: Potential
epigenetic mechanism for homocysteine-related pathology. J Nutr
2002;132:2361S-6.
9. Yi P, Melnyk S, Pogribna M, Pogribny IP, Hine RJ, James SJ. Increase
in plasma homocysteine associated with parallel increase in plasma
S-adenosylhomocyateine and kymphocyte DNA hypomethylation.
J Biol Chem 2000;102:29318-23.
10. Bailey LB, editor. Folate in Health and Disease. Boca Raton, FL: Taylor
and Francis; 2009.
11. Erickson JD. Folic acid and prevention of spina bifida and anencephaly.
10 years after the U.S. public health service recommendation. MMWR
Recomm Rep 2002;51:1-3.
12. Lamprecht SA, Lipkin M. Chemoprevention of colon cancer by
calcium, vitamin D and folate: Molecular mechanism. Nat Rev Cancer
2003;3:601-14.
13. Holmquist C, Larsson S, Wolk A, Faire U. Multivitamin supplements
are inversely associated with risk of myocardial infarction in men
and women-stockholm heart epidemiology program (SHEEP). J Nutr
2003;133:2650-4.
14. Kimura M, Umegaki K, Higuchi M, Thomas P, Fenech M. Methylene-
tetrahydrofolate reductase C677T polymorphism, folic acid and
riboflavin are important determinants of genome stability in cultured
human lymphocytes. J Nutr 2004;134:48-56.
15. Herman JG, Baylin SB. Gene silencing in cancer in association with
promoter hypermethylation. N Engl J Med 2003;349:2042-54.
16. Rooij SR, Painter RC, Phillips DI, Osmond C, Michels RP, Godsland IF,
et al. Impaired insulin secretion after prenatal exposure to the dutch
famine. Diabetes Care 2006;29:1897-901.
17. Roseboom T, Rooij S, Painter R. The dutch famine and its long-term
consequences for adult health. Early Hum Dev 2006;82:485-91.
26. Berry RJ, Li Z, Erickson JD, Li S, Moore CA, Wang H, et al.
Prevention of neural-tube defects with folic acid in China. China-U.S.
collaborative project for neural tube defect prevention. N Engl J Med
1999;341:1485-90.
27. Painter RC, Rooij SR, Bossuyt PM, Simmers TA, Osmand C,
Barker DJ, et al. Early onset of coronary artery disease after prenatal
exposure to the dutch famine. Am J Clin Nutr 2006;84:322-7.
28. Hsiung DT, Marsit CJ, Houseman EA, Eddy K, Furniss CS,
McClean MD, et al. Global DNA methylation level in whole blood
as a biomarker in head and neck squamous cell carcinoma. Cancer
Epidemoil Biomarkers Prev 2007;16:108-14.
29. Piyathilake CJ, Macaluso M, Alvarez RD, Chen M,
Badiga S, Siddiqui NR, et al. A high degree of LINE-1 methylation
in peripheral blood mononuclear cells, a one-carbon nutrient related
epigenetic alteration, is associated with a lower risk of developing
cervical intraepithelial neoplasia. Nutrition 2011;27:513-9.
30. Smith AD, Kim YI, Refsum H. Is folic acid good for everyone? Am J
Clin Nutr 2008;87:517-33.
31. Ulrich CM, Potter JD. Folate and cancer-timing is everything. J Am
Med Assoc 2007;297:2408-9.
Kim YI. Folic acid supplementation and cancer risk: Point. Cancer
Epidemiol Biomarkers Prev 2008;17:2220-5.
36. Maccani MA, Marsit CJ. Epigenetics in the placenta. Am J Reprod
Immunol 2009;62:78-89.
37. Kulkarni A, Dangat K, Kale A, Sable P, Chavan-Gautam P, Joshi S.
effects of altered maternal folic acid, vitamin B12 and docosahexaenoic
acid on placental global DNA methylation patterns in wistar rats. PLoS
One 2011;6:e17706.
38. McKay JA, Waltham KJ, Williams EA, Mathers JC. Folate depletion
during pregnancy and lactation reduces genomic DNA methylation in
murine adult offspring. Genes Nutr 2011;6:189-96.
39. Sinclair KD, Allegrucci C, Singh R, Gardner DS, Sebastin S,
Bispham J, et al. DNA methylation, insulin resistance, and blood
pressure in offspring determined by maternal periconceptional B vitamin
and methionine status. Proc Natl Acad Sci U S A 2007;104:19351-6.
40. Watreland RA, Jirtle RL. Early nutrition, epigenetic changes at

20
Asian J Pharm Clin Res, Vol 11, Issue 1, 2018, 17-20
Varma et al.
transposons and imprinted genes, and enhanced susceptibility to adult
chronic diseases. Nutrition 2004;20:63-8.
42. Dwarkanath P, Barzilay JR, Thomas T, Thomas A, Bhat S, Kurpad AV.
High folate and low vitamin B-12 intakes during pregnancy are
associated with small-for-gestational age infants in South Indian
women: A prospective observational cohort study. Am J Clin Nutr
2013;98:1450-8.
43. Sarheed O, Ramesh K, Shah F. In vitro evaluation of dissolution profile
of two commercially available folic acid preparations. Int J Pharm
Pharm Sci 2015;7:473-5.
44. Antakli S, Sarkees N, Sarraf T. Determination of water-soluble vitamins
B1, B2, B3, B6, B9, B12 and C ON C18 column with particle size
3 µm in some manufactured food products by HPLC with UV-dad/FLD
detection. Int J Pharm Pharm 20157:219-24.