Methylation pathways

Methylation, also known as one-carbon metabolism, is a biochemical process that takes place continuously in every cell in our body and involves the addition of a methyl group to a molecule. By persistently providing methyl groups with numerous methyltransferase reactions via the ubiquitous methyl donor, S-adenosyl methionine (SAMe), the process becomes vulnerable to destabilization leading to either hypo or hypermethylation.

Unbalanced methylation is a cornerstone of premature ageing and multiple age-related degenerative diseases. Additionally, epigenetic phenomena and epigenetic nutrition, as a novelty intervention in modifying gene expression and subsequently altering ageing and age-related degenerative disorders, are essentially DNA methylation driven. Numerous studies undertaken in this field demonstrate conclusive evidence that has enabled physicians to use evolving intervention approaches for balancing methylation, a method not widely accepted by conventional medicine.

Methylation is involved in multiple biological processes and several critical functions in the body in an intricate manner that cannot easily be recognizable when unbalanced. These processes are listed as follows:

Synthesis of DNA and RNA and biosynthesis of purine nucleotides

The process is impacted by chronic methyl donor deficiency, such as Folate (Vitamin B9), leading to DNA strand brakes, apoptosis, and carcinogenesis. By using a sensitive in vitro model system (comprised of mouse prostate transgenic adenoma simulating a prostate cancer) the results have emphasized the significance of Folate restriction for genetic, epigenetic and phenotypic changes1,2.

Gene expression and silencing

DNA methylation is one of the well known epigenetic mechanisms that directly regulates gene expression and phenotypes. In healthy cells, it ensures the proper regulation of gene expression (without changing DNA sequence) and stable gene silencing.

Methylation of DNA occurs at so-called CpG sites (where cytosine lies next to guanine). The CpG sites in the regions near the promoters of genes are known as CpG islands. Recent studies have shown that DNA methylation of CpG islands, deacetylation of histone, and methylation of histone leads to gene silencing. Under normal circumstances, the majority of CpG dinucleotides are methylated, but aberrations in gene expression caused by epigenetic promoter hypermethylation of CpG islands can lead to carcinogenesis (inappropriate silencing)3,4.

Metabolism of neurotransmitters

Metabolism of biogenic amine neurotransmitters (serotonin, epinephrine, norepinephrine, dopamine, and melatonin) is highly dependent on balanced methylation. COMT (Catechol-O-Methyl Transferase) is a methylation enzyme that continuously inactivates catecholamines. COMT transfers a methyl group from SAMe to the catechol group of the neurotransmitter that is being processed, altering its chemical structure and properties, and clearing it from the system. Unbalanced methylation leads to the accumulation of the neurotransmitters mainly epinephrine and norepinephrine that cannot be sufficiently utilized during prolonged stress. Balanced methylation also provides BH4 (tetrahydrobiopterin) needed to generate neurotransmitters. BH4 delivers the co-factors necessary to turn tryptophan and tyrosine into their respective neurotransmitters serotonin and dopamine5.

Metabolism of estrogen

COMT methylates catechol estrogens to the anti-cancer molecules of methoxy-estrogens. As the level of catechol estrogens increases, the level of methoxy estrogen metabolites also increases with COMT activity. Conversely, with increasing levels of SAH (S-adenosylhomocysteine) that takes place in ‘clogged’ methylation, there is a decreased production of protective methoxy metabolites and an increased quantity of damaging catechol–estrogens that can oxidize into DNA damaging quinones and semiquinones. A well-documented study outlines the links between low COMT activity, elevated homocysteine with low SAMe:SAH ratio, and an increased risk of reproductive organ malignancy and breast cancer6.

Immune system regulation

The differentiation of immune cells, particularly T-cells; boosting the killing activity of cytotoxic T and NK cells; and Th2 cytokine regulation are all highly dependent on balanced DNA methylation. T-cells are required to help control B-cells and balance TH1 and TH2 responses that are often affected when in a state of hypomethylation7,8.

Production of phosphatidylcholine

The cell membrane has a phospholipid bilayer, and 50% of those phospholipids are phosphatidylcholine (PC), which is used as a building block for membrane structure, bile and the transportation of fats. Studies point out that the CDP-choline pathway synthesized only two or three major types of PC; whereas, the PEMT methylation pathway generates at least eight major types of PC with significant efficiency. PEMT methylation pathway supplies PC in case of nutritional deficiency of choline. This process is highly dependent on balanced methylation as it consumes three methyl groups to synthesize PC from phosphatidylethanolamine9,10.

Energy production

The methylation pathway is necessary for the synthesis of CoQ10, which, in turn, is important for its function in ATP production in the mitochondrial respiratory chain. Carnitine is another nutrient involved in mitochondrial energy assembly; its synthesis begins with the methylation of the amino acid L-lysine by SAMe. Mitochondrial fatty acid oxidation is the primary energy source for heart and skeletal muscle. Carnitine is involved in the transport of these fatty acids into the mitochondrial matrix11.

Biotransformation and antioxidant activity

Methylation is required for the removal of environmental toxins from the body. This process involves conjugating methyl groups to the toxins before removal and supporting the production of glutathione, taurine, sulfate, and cysteine. Within direct conjugation, it detoxifies xenobiotics and carcinogens including heavy metals, such as mercury, lead and arsenic. Glutathione is a sulfated protein involved in inflammatory control and reduction reactions, catalyzed by glutathione S-transferase. It is a primary endogenous antioxidant produced by the cells, contributing reliably to the neutralization of free radicals and reactive oxygen species, as well as maintaining exogenous antioxidants (vitamin C and E) in their reduced forms.

Myelination of nerve cells

Nerve myelination is a process dependent on methylation, with research showing a decreased amount of S-adenosyl methionine in the cerebrovascular fluid in an individual where demyelination is present12.

Methylation is crucial for early CNS development (neural tube defect due to maternal low folate intake) histamine metabolism, ammonia clearing, pathology of autism and more, which are related to factors other than ageing and age-related diseases.

Methylation physiology

The primary goal of the methylation cycle is to continually provide the universal methyl donor, S-adenosylmethionine (for all cellular methyltransferase reactions), for the balanced functioning of the methionine cycle.

The methionine cycle is an intricate process that comprises the regeneration of methionine from homocysteine in the methionine synthase reaction via the vitamin B-12–dependent transfer of a methyl group from 5-MTHF (5-methyltetrahydrofolate). Methionine forms S-adenosylmethionine (SAMe) via methionine adenosyltransferase activity. The removal of the methyl group from SAMe to the different ‘methyl dependent molecules’ ends in the formation of S-adenosylhomocysteine (SAH). The SAH gets reversibly hydrolyzed to homocysteine and adenosine via SAH hydrolase (SAHH) activity.

Homocysteine is then repeatedly used to regenerate methionine. This process runs smoothly provided all methylation components are available (e.g. proteins and enzyme cofactors), there are no genetic SNPs creating misbalance, environmental toxin interference, or higher demand for glutathione production.

The process of homocysteine being either re-methylated to methionine or irreversibly removed from the methionine cycle to form cysteine, glutathione, and taurine is a continuous balancing act. Two fundamental concerns of methionine cycle turnover blockage are the reduced synthesis of SAMe and diminished synthesis of glutathione as an antioxidant master (Figure 1).

Methylation and ageing

Numerous studies have concluded that a methylation deficit and unbalanced methylation could be a cause of accelerated ageing (including gray hair), cardiovascular disease, and premature arteriosclerosis, diabetes, dementia, Alzheimer’s, Parkinson’s, neurotransmitter imbalances and psychiatric disorders, chronic inflammation, abnormal immunity and autoimmunity, various cancers, insomnia, first presentation of asthma in elderly and chemical sensitivities, thyroid disease, and more13–20.

Several studies have specifically validated that changes in DNA methylation patterns are associated with biological ageing and affect human lifespan. Studies found that DNA methylation changes due to an epigenetic drift are fundamental to the ageing process. Both hypermethylation and hypomethylation have been associated with the progressive accumulation of epigenetic damage due to environmental factors. This phenomenon demonstrates that the epigenetic similarities between young individuals are lost over time leading to different phenotypic expression and ageing-associated DNA methylation changes in the elderly (studies on identical twins). Additionally, with ageing, methylation capacity decreases by 20–30% even without any apparent disease21,22.

Unbalanced methylation

Both hypermethylation or hypomethylation can affect a person’s health. Global DNA hypomethylation is a potential factor in reducing genome stability and increasing chromosomal aberrations. Specific DNA hypomethylation can be seen in many diseases, such as autoimmunity. Site-specific hypermethylation of promoter regions is found in both ageing and tumour genesis.

Factors contributing to
unbalanced methylation
Single nucleotide polymorphism (SNPs)

The most important genes governing methylation are MTHFR A1298C and C677T, COMT V158M and H62H, AHCY, MTR, MTRR, GST, MAO-A R297R, NOS D298E, SHMT, PEMT, VDR/Taq and VDR/ Fokl, and CBS. The importance of MTHFR  in domineering methylation has been supported by numerous studies23,24.

The main role of MTHFR is to generate 5-methyltetrahydrofolate, which is then utilized by MTR (methionine synthase) to re-methylate homocysteine back into methionine for the renewal of SAMe. When MTHFR SNPs are present, homocysteine cannot be re-methylated, SAMe decreases and the methyl cycle subsequently failS causing premature ageing and age-related neurodegenerative disorders (Parkinson’s, arteriosclerosis, ischemic stroke, cancers, diabetes) usually with a high homocysteine level25,26.

MTHFR C677T positive individuals have trouble using dietary folic acid to re-methylate homocysteine as they cannot efficiently convert dietary folic acid into the 5-MTHF form.

The presence of various methylation SNPs concomitantly has the cumulative effect of causing health problems. For example, the presence of MTRR /66/AG/GG and /H595Y/ increases the risk of breast, prostate, and pancreatic cancer, as well as gastric cancer in Asian populations alongside obesity. However, if present with MTHFR C677T, CBS, and MTR, MTRR can increase the risk of other unbalanced methylation health risks due to a collective effect27.

Describing all SNPs is beyond the purpose of this article, but understanding that their presence will impact methylation, especially in conjunction with environmental factors, is essential in determining methylation capability.

Nutritional factors

Nutritional factors required for methylation can affect the methylation rate and outcome if deficient. These are methionine, choline, glycine, glutamine, cysteine, betaine, phosphatidylcholine, and vitamins as well as cofactors, such as riboflavin, pyridoxalphosphate, B12, folate, niacin, zinc, magnesium, molybdenum, vitamin C, and vitamin D. It is well known how folic acid fortification in the USA and routine prescription of folic acid in pregnant women worldwide decreased neural tube defect by manipulating the methylation nutritional input. Over the years, folic acid fortification has become excessive, and it now adversely affects methylation due to the high levels of folic acid found in food. Dietary folate from leafy greens gets hydrolyzed in the intestines in order to enter the folate cycle as THF (tetrahydrofolate). Folic acid, in synthetic form, on the other hand, needs to be reduced in the liver twice by the much slower DHFR enzyme before it can enter the folate cycle as THF (Figure 2). More importantly, non-metabolised synthetic folic acid blocks DHFR, which is needed for its reduction but also prevents MTHFR activity (one of the most important enzymes for the SAMe production.) Adding 5-MTHF instead of synthetic folic acid can overcome all the above-related blockages, with studies demonstrating that the folate status undeniably improved after such intervention28,29.

However, adding methyl donors is not always an easy and simple task as methylation dynamic depends on other participants and interplays. We sometimes witness methyl donors over-sensitivity after supplying 5-MTHF or SAMe specifically in individuals suffering from co‑existent inflammation, high free radical formation and stress (‘clogged’ methylation).

Many co-factors important for balanced methylation cannot simply be added on the assumption that their effect will cause increased methylation. For example, riboflavin that is fundamental for MTHFR activity can be affected by an under-active thyroid, and its impact can then be compromised. Simply adding riboflavin without checking thyroid function will result in a possible failure to balance methylation. Therefore, an integrative approach while understanding the interaction between all methylation participants is crucial. Similarly, adding certain co-factors after comprehensively investigating methylation can create significant immediate improvement in some patients. For example, adding molybdenum and ceasing dairy product ingestion (containing xanthine oxidase, which uses up molybdenum) in patients with high sulfite, immediately improved symptoms.

Environmental toxicity

Environmental toxicity can affect DNA methylation (epigenetic effect) and metabolic methylation, predominantly by using methyl donors for detoxification processes (Figure 3). Some of these toxins can block specific methylation enzymes (mycotoxins preventing glutathione production from cysteine) diminishing the global methylation process. Various studies have proven the adverse effect of heavy metals, such as mercury, cadmium, arsenic, and lead as well as the effect of persistent organic pollutants, such as phthalates, bisphenol A, benzene jet fuel, mould, and pesticides30. Several epigenetic mechanisms, such as DNA methylation, histone modifications, and microRNA expression can change genome functions when exposed to environmental toxins. For example, smoking has pro-ageing effects by inducing DNA methylation changes to genes involved in age-related diseases, such as cardiovascular disease and cancer. Recent evidence suggests that arsenic increased the toxicity of specific methylated intermediate metabolites, and that cadmium exposure has been associated with hypermethylation-dependent silencing of DNA repair31. Extensive investigation has shown that plastics, such as bisphenol A (BPA) and phthalates that are widely used in daily products negatively affect human health. The epigenetic impact of BPA and phthalates (hypomethylation) was demonstrated in yellow mice and was prevented by maternal dietary supplementation with a methyl donor like folic acid (Figure 4)32.

Methylation disruptors

An aspect not often considered in conventional medicine that significantly affects methylation is the impact of medication. Their use is accompanied by substantial ignorance, perhaps helping with some problems but blocking methylation in the process. It is well documented how methotrexate affects methylation by inhibiting DHFR (folate metabolism) and affecting B12 malabsorption (GI toxicity, hepatotoxicity and alopecia in MTHFR positive patients)33. Many other drugs interfere with folate metabolism: carbamazepine and phenytoin are folate antagonists; antimalarial drugs, TMP-sulfamethoxazole, triamterene, and sulfasalazine are DHFR inhibitors; and oral contraception increase folate requirements. Antacids, PPI, and cholestyramine alter B12 absorption that is necessary for methionine recycling, and nitrous oxide inhibits methionine synthase and can be very dangerous in people with MTHFR SNP that already have compromised methylation. Niacin and theophylline limit pyridoxal kinase that activates vitamin B6 necessary for methylation. Niacin also depletes SAMe in a manner similar to alcohol, but sometimes this can be helpful; for example, in cases of oversensitivity with methyl donors, the quickest way of reducing this issue would be via the use of high doses of niacin.

Internal methylation interplay: SAMe competition and metabolic priorities

Methylation is an exceptionally complex process with an internal interplay, where competitive needs for SAMe, environmental factors, current health state, and methylation ‘players’ continuously impact the process. Understanding methylation dynamics is therefore essential when attempting to balance methylation.

The internal interplay refers to specific interactions that exist within the methylation process and these always follow the same rule.

High SAH (S–Adenosylhomocysteine) that get created when SAMe is spend as a methyl donor has a negative impact on COMT (Catechol-O-Methyltransferase). This assumes impaired Catecholamine metabolism leads to overstimulation of adrenoreceptor mediated physiological responses in CNS and CVS. It also damages vascular endothelial cells and catechol-containing neurons by the production of excessive semiquinones. A similar impact occurs in estrogen metabolism where a high formation of semiquinones and DNA damaging quinones increase the risk of breast cancer34. SAH build up also inhibits SAMe dependant DNA methyltransferases (DNMTs) leading to accelerated ageing and malignancies while contributing to multiple degenerative diseases, such as Alzheimer’s35. It is clear that a low SAMe:SAH ratio is undesirable when assessing methylation capacity.

Excessive SAMe inhibits MTHFR and slows down methylation, which leads to other negative consequences. Excessive homocysteine inhibits the enzyme that hydrolyzes SAH to homocysteine protecting it from a further increase in homocysteine but also increasing SAH build up36.

SAMe competition is an ongoing process that is unique to each individual and continuously adapts depending on immediate demands. Examples of these demands are: environmental toxicity, high catecholamine requirement during stress, estrogen dominant states, certain medications, high histamine clearance demand but also increased need for energy production, and phosphatidylcholine that uses more SAMe molecule then others. Balancing methylation is the only way to keep these requirements satisfied. Occasionally adding or removing some of these burdens can spare SAMe and ensure that it is used for critical functions.

Methylation dynamics commonly adapts and modifies to the body’s ongoing requirements and metabolic priorities. Excessive oxidative damage, inflammation, physical and psychological stress, gut and microbiome issues, insulin resistance often affect methylation balance. Inflammation distresses methylation via inflammation-signalling molecules that interfere with DNA methylation and, indirectly, by changing insulin signalling and increasing oxidative stress that use homocysteine through transsulfuration, rather than recycling it back for SAMe production. It is a challenge to balance methylation under-activation and up-regulation of NFKb and high ROS production as bodily ‘insight’ focuses methylation towards overcoming it. It should also be a priority for a physician to clarify this before attempting to balance methylation37.

Methylation assessment

From the above discussion, we can assume that assessing methylation requires an integrative and holistic evaluation of the body functions that can potentially affect methylation. Furthermore, there is no typical marker for evaluating unbalanced methylation, which makes the assessment even more challenging. There are several tests that can be performed to estimate methylation capacity, such as homocysteine level; SAMe:SAH ratio; serum folate and vitamins B6, B12, and B2; organic acids, evaluating inflammation levels, oxidative stress, adrenal and insulin function, estrogen level, thyroid activity, heavy metal intoxication, gut and microbiome issues, use of medication, and alcohol and environmental toxicity, are all critical in assessing methylation.

Symptom questionnaires can help identify problems in methylation well before unbalanced methylation leads to apparent disease. Methylation SNPs that govern susceptibilities are essential to locate in order to modify methylation concerns prior to disease or ageing setting-in.

Numerous studies have demonstrated the role of homocysteine in ageing and age-related diseases. There is a significant association between high homocysteine and cardiovascular disease, including arteriosclerosis, heart attacks, and cerebrovascular insult. It is supposed that hyperhomocysteinemia causes endothelial cell damage and a reduction of blood vessel plasticity. This process is mediated by several mechanisms, such as oxidative stress, inhibition of endothelial nitric oxide (eNOS), NFkb activation, and inflammation.

Furthermore, there has been a possible suggestion that hyperhomocysteinemia has a role in the pathophysiology of CNS neurodegenerative disorders. Several studies confirmed that homocysteine could trigger neuronal damage via oxidative stress, stimulation of inflammation, DNA damage and activation of pro-apoptotic factors. These may be connected to changes in mental health such as dementia, Alzheimer’s, and Parkinson’s disease and depression. There are indications that hyperhomocysteinemia can be responsible for a global DNA hypomethylation. Low homocysteine can also be a pathological sign like in CBS up-regulation that is often seen in fibromyalgia and chemical sensitivities.  Homocysteine is one of the markers of methylation that has been accepted by conventional medicine and is widely used. However, for improved evaluation of methylation balance, SAMe:SAH ratio is recommended as it defines the methylation potential of the cell (Figure 5)38–42.

Methylation balancing interventions

The major challenge of the balancing act is to manage the methylation act without inducing hypo or hypermethylation as these are harmful to health. The first step without attempting to add, correct or manipulate any part of methylation is to clear underlying problems. The clearing process involves all interventions to remove anything that is not substantially supporting methylation (but rather blocking it), such as inflammation, oxidative stress, environmental toxicity, heavy metals, mycotoxins and stress, which is often sufficient to rebalance methylation.

Before attempting to add methyl donors, assessment of symptoms of unbalanced methylation and co-factors deficiencies should be performed. Low zinc or magnesium are common findings as well as the low activity of riboflavin in such a prevalent hypothyroid state. These corrections come without risk of inducing hyper or hypomethylation.

Use of methyl donors especially 5-MTHF has become very popular among integrative medicine physicians, especially in MTHFR positive individuals. Significant improvements were observed in transmethylation metabolites and glutathione redox status after treatment with methylcobalamin and folinic acid in some children with autism. Many studies suggested that folate supplementation may be crucial for methylation maintenance in individuals with MTHFR polymorphisms. Novel experiments researching maternal methyl donor supplementation in utero demonstrated the impact of early nutrition in influencing the epigenome. Using folate and B12 in mild cognitive decline patients with high homocysteine confirmed an improvement compared to individuals with normal homocysteine. In the past decade, conflicting evidence has emerged suggesting that excessively supplementing methyl donors especially 5-MTHF can have an adverse effect on DNA methylation (e.g., DNA hypermethylation) and its impact is more complicated than previously understood43–45. However, supplementing folate and other methylation nutrients from whole food have never been shown in studies to harm DNA methylation; in fact, it demonstrated the same effect on lowering homocysteine levels as 5-MTHFR and had a positive effect on DNA methylation. Methylation diet is a novelty approach in safely modifying and balancing methylation according to a genetic and clinical assessment of methylation47. In a simplified portrayal, a methylation diet should include food that is toxin, hormone, antibiotic, GMO and heavy metals free. Diet should be rich in plant-based components preferably ‘rainbow-colored’ that is enhanced with deferent phytonutrients and cruciferous vegetables, adequate protein intake and fat consumption (fish, olive oil, avocado, etc.)46,47.

In addition to diet, lifestyle interventions such as physical activity, caloric restriction, and high antioxidant intake may counteract DNA methylation changes contributing to ageing48–51.

Another safe approach would be via an intervention to spare and thereby make available SAMe to donate sufficient methyl groups; for example, by adding phosphatidylcholine.

As epigenetic technologies and nutri-epigenetics continue to emerge, incorporating nutrition and DNA methylation becomes vital in creating epigenetically- focused therapeutic and preventative strategies in combating ageing. Several studies demonstrated an epigenetic effect of specific nutrients and bioactive food components on the regulation of DNA methylation via SAMe and directly on DNA methyltransferases expression. These bioactive food components, such as EGCG, resveratrol, curcumin, and isoflavone genistein are now recognized as methylation balancing factors and can be used in preventing ageing and age-related degenerative diseases.  For example, curcumin-mediated DNA methylation modifications are recognized as cancer chemo-prevention (colon cancer studies), and EGCG demonstrated in studies certain hypermethylated genes in cancer and ageing underwent re-expression under its influence48–50.

Conclusion

Methylation is a complex process influenced by multifactorial internal and external factors that require an in-depth and encompassing holistic assessment. Simply by adding methylation nutrients, co-factors and donors will not necessarily positively drive methylation as expected and may undergo hypermethylation, for example. The ultimate goal is to achieve balanced methylation which is attainable by going back to basics:  via the use of methylation supporting diet and lifestyle interventions. And indeed epigenetic clock analysis on diet and lifestyle factors has proven that our lifestyle influences our epigenetic age51.

Acknowledgements

  Declaration of interest None

  Opening image © Christoph Bock, Max Planck Institute for Informatics

Figures 1 © Sly Nedic, 2  Adapted from Fan BJ, Chen T, Grosskreutz C et al. Lack of association of polymorphisms in homocysteine metabolism genes with pseudoexfoliation syndrome and glaucoma. Molecular Vision 2008; 14:2484-249; 3 Adapted from Baccarelli A, Bollati V. Epigenetics and environmental chemicals. Curr Opin Pediatr. 2009; 21(2):243-51; 4 Adapted from Singh S, Shoei-Lung Li S. Epigenetic Effects of Environmental Chemicals Bisphenol A and Phthalates. Int J Mol Sci. 2012; 13(8): 10143–10153; 5 Sly Nedic

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