De-Mystifying Methylation

Even without a science degree, you likely have come across the term methylation. It’s receiving attention in the medical community these days because of its widespread impact on health. But what exactly is methylation? In humans, this reaction occurs in every single one of our cells, except red blood cells, which gives some insight into how vital methylation is, not only for survival but for us to thrive.

Methylation is an essential biochemical process that forms the basic building blocks for hundreds of different biochemical reactions in the body. Through this pathway, folate and vitamin B12 help to form ‘methyl’ groups (CH3) that attach to proteins, modifying their function.

Why is this important? It is a way for the body to form and degrade active proteins, making sure that certain biological processes are under tight control. This avoids excess hormones, neurotransmitters, growth and cellular replication. In other words, methylation avoids complete chaos.

Methylation is important for:

  1. Regulating mood – modifying theconversion of neurotransmitters adrenalin, dopamine, serotonin, melatonin and histamine.
  2. Creating and repairing DNA – forming the base pair thymidine.
  3. Regulating gene expression – through epigenetic modification.
  4. Enhancing immune function – necessary for production of red and white blood cells, and platelets.
  5. Supplying energy to the body – forming Coenzyme Q10, carnitine and creatine.
  6. Muscle repair and protein production – for heart, skeletal and smooth muscles.
  7. Cell membrane and nerve protection – manufacturing ofphospholipids and myelin.
  8. Producing and balancing hormones – including thyroid, cortisol and estrogens.
  9. Assisting in detoxification – affecting methylation, glutathione and sulfur pathways.

To understand the methylation pathway and how it affects overall health and how you can impact it, let’s break it down. Methylation is made up of two pathways, the folate cycle and methionine cycle (Figure 1). Together these pathways work like two gears of a clock. As one moves it propels the other.

The Folate Cycle

Folate comes from the Latin word meaning foliage, found abundantly in dark leafy greens. Folate also comes in a synthetic form known as folic acid. In order to be usable by the body, folate must be converted into its active form, known as 5-MTHF.

(5-methylene-tetrahydrafolate). The body accomplishes this using the enzyme MTHFR (among others).

Methionine Cycle

Now let’s look at the other important methylation pathway, which involves homocysteine. Homocysteine is an amino acid and breakdown product of our metabolism that is toxic in high concentrations. It is an independent risk factor for cardiovascular disease, including stroke and hypertension, and strongly associated with Alzheimer’s disease and dementia. One way the body reduces levels of homocysteine is through the methionine cycle.

It uses the active form of folate (remember, 5-MTHF) and vitamin B12 (methylcobalamine) to convert homocysteine into methionine.2,4,5 Here’s a summary of what happens next.

  • Methionine then goes on to produce SAMe (S-adenosylmethionine). SAMe is what the body uses for the processes mentioned above.
  • The enzyme methionine synthase (MTR) requires both the active forms of folate and vitamin B12 to transform homocysteine into methionine. The enzyme methionine synthasereductase (MTRR) recycles vitamin B12 to ensure the active form is available.6

The figure below shows how these two cycles orchestrate the production of SAMe, using a combination of enzymes, vitamins and minerals. In addition to folate and vitamin B12, vitamins B2, B3, B6, magnesium, and zinc arenecessary to lower homocysteine and ensure proper methylation.

Figure 2-A. A closer look at the folate and methionine pathways.

Signs of Poor Methylation

If the body is low in the vitamins and minerals necessary for methylation or is under physiological stress (as in toxic exposure, inflammation, or infection), widespread symptoms may emerge. These may include depressive symptoms, poor cellular repair (poor wound healing, digestive complaints), chemical sensitivities, low energy, poor immunity and hormone imbalance. These symptoms may indicate ‘poor methylation’ status.

Signs of Excess of Methylation

Those suffering from the problems that arise from ‘poor methylation’ described above, may choose to supplement with methyl donors, such as methyl-B12, methyl-folate (5-MTHF) and SAMe. However, caution is recommended with amounts. Some may be sensitive to these forms and require additional support to prevent what could be known as ‘excess methylation’. Signs include; anxiety, poor concentration, panic disorder, insomnia, excess inflammation, aggravation of food/chemical sensitivities, and histamine intolerance.3

Inability to Methylate.

There are a number of reasons that may influence the body’s ability to methylate.

  • Poor dietary intake of folate, vitamin B12 and protein. Those who are vegetarian, vegan or have chronic digestive complains (IBD) are more likely to have deficiencies in protein and vitamin B12.8
  • Toxin accumulation, low antioxidant status and inflammation. The MTR enzyme is susceptible to heavy metals, oxidative stress, mineral deficiency and chronic infections. This signals the body to reduce SAMe production, diverting the pathway to produce glutathione.
  • Genetic variations. Discoveries in human genetics reveal how changes in the genetic code can lead to reductions in the efficiency of the methylation cycle. This leads to an increase in homocysteine and risk for disease, which we will describe in our genetic testing section.

Beyond MTHFR

The body is smart and usually has a number of different ways to accomplish an end goal. This avoids the possibility of severe biochemical road blocks that may be detrimental to survival. Methylation is no exception. The body has several additional pathways to reduce homocysteine levels.

Alternate Methylation Pathways

The alternate pathways to reduce homocysteine include the betaine pathway and the transsulfuration pathway.

Betaine Pathway

Betaine, also known as trimethylglycine (TMG) is an alternate route for the body to reduce homocysteine levels. The enzyme betaine-homocysteine methyltransferase (BHMT) requires zinc and betaine to function. Choline is also important to the process.

Transsulfuration Pathway

If the body needs to produce more antioxidants or increase detoxification it can degrade homocysteine into glutathione. This is through the transsulfuration pathway, whereby homocysteine is broken down into cysteine using the CBS enzyme.9 The enzyme cystathione-beta-synthase (CBS) requires vitamin B6.

Methylation connects hundreds of biochemical processes, and as such, may have a huge impact on overall health. It is essential to identify possible errors in this process and the key vitamins and minerals necessary to correct these alterations, ultimately reducing risk of disease.

Figure 2-B. A closer look at the transsulfuration and betaine pathways.

Is Methylation an Issue for You – Find out with Testing

To assess overall methylation status, there are some simple tests that are helpful, including blood, urine and genetic analyses.9,10 Standard labs can test for the following: homocysteine, complete blood count (CBC), red blood cells (RBC), folate, RBC magnesium, vitamin B12, Methylmalonic acid (for adenosyl B12 deficiency), zinc, organic acids urine testing for B vitamin status.

Genetic Testing

One way to determine your susceptibility to poor methylation is through genetic testing. Genetic variations are responsible, not only for how different we look from one another, but also for our biochemical individuality. For example, studies reveal that two people eating the same amount of folate show different levels of folate in the blood. Why? Some people are less efficient at using dietary folate and are subsequently at a greater risk of folate deficiency. The reason for these differences are due to changes in the gene known as MTHFR.

There are approximately 23,000 genes within the human genome, made up of DNA, and represented by a set of ‘letters’ (A, C, T and G) that define the genetic code. Similar to a blueprint, which are instructions for an architect on what to build, the genetic blueprint contains instructions to the cell on what biological building blocks to construct.

Genetic testing examines an individual’s genetic blueprint and more specifically the changes within this blueprint that lead to one’s biochemical individuality. One way genetic variation may occur is through mutations in the genetic code, and the most common form of mutation in humans is a SNP.

What are SNPs? Single nucleotide polymorphisms, or SNPs, are “mutations” or changes in the genetic code. Changes to the genetic code can alter the gene product and therefore affect the cell’s biological building blocks. One example is the SNP C677T in the MTHFR gene. This SNP changes the genetic code from a C to a T, reducing the MTHFR enzyme efficiency by up to 70%. This subsequently leads to a reduction in 5-MTHF (active folate) availability and an increase in homocysteine. Ultimately this leads to an increase in certain diseases.11

How Genetic Mutation Testing Can help

SNP testing allows individuals and their doctors to gain a better understanding of what’s happening on a genetic and cellular-level, including methylation.

It can identify one’s susceptibility to potential problems with methylation, risk for disease and direct specific testing and treatment to compensate for inborn genetic errors.

With regards to methylation, genetic testing can help to determine a number of solutions:

•      Identify an ideal form of vitamin B12: Those with certain variationsof the COMT gene may experience anxiety with the ‘methyl’ forms of B12. A blend of hydroxy and adenosyl B12 are preferably forms to avoid this reaction.

•      Ways of taking of vitamin B12: Those with a certain version of the FUT2 gene are susceptible to vitamin B12 deficiency and may benefit from sublingual from B12.12,13

•      Daily dosage of folate: Those withthe slow version of the MTHFR gene are susceptible to folate deficiency and require a minimum intake of 400mcg of folate per day.14

•      Specific treatments: Those withmultiple at-risk variants within the methylation are at risk for high homocysteine. If homocysteine levels are high, these individuals can benefit from taking specific minerals (zinc, magnesium, copper) and vitamins (vitamins B2, B3, B6, 5-MTHF and B12).15

  • Disease prevention: Identifying anincrease risk in disease can provide a huge opportunity to make the necessary lifestyle and dietary changes to significantly reduce the risk of getting that disease. With such a plethora of good advice, genetic testing helps decide what advice will have the biggest effect on your health.


1. Fava, M., and Mischoulon, D. (2009). Folate in depression: efficacy, safety, differences in formulations, and clinical issues. J. Clin. Psychiatry 70 Suppl 5, 12–17.

2. Obeid, R. (2013). The Metabolic Burden of Methyl Donor Deficiency with Focus on the Betaine Homocysteine Methyltransferase Pathway. Nutrients 5, 3481–3495.

3. Miller, A.L. (2008). The Methylation, Neurotransmitter, and Antioxidant Connections Between Folate and Depression. 13, 11.

4. Xiao, Y., Su, X., Huang, W., Zhang, J., Peng, C., Huang, H., Wu, X., Huang, H., Xia, M., and Ling, W. (2015). Role of S-adenosylhomocysteine in cardiovascular disease and its potential epigenetic mechanism. Int. J. Biochem. Cell Biol. 67, 158–166.

5. Miller, J.W. (1999). Homocysteine and Alzheimer’s disease. Nutr. Rev. 57, 126–129.

6. Seremak-Mrozikiewicz, A., Bogacz, A., Deka-Pawlik, D., Klejewski, A., Wolski, H., Drews, K., Karasiewicz, M., and Czerny, B. (2016). The polymorphisms of methionine synthase (MTR) and methionine synthase reductase (MTRR) genes in pathogenesis of preeclampsia. J. Matern. Fetal Neonatal Med. 1–17.

7. Murphy, T.M., O’Donovan, A., Mullins, N., O’Farrelly, C., McCann, A., and Malone, K. (2015). Anxiety is associated with higher levels of global DNA methylation and altered expression of epigenetic and interleukin-6 genes. Psychiatr. Genet. 25, 71–78.

8. Oussalah, A., Guéant, J.-L., and Peyrin-Biroulet, L. (2011). Meta-analysis: hyperhomocysteinaemia in inflammatory bowel diseases: Meta-analysis: hyperhomocysteinemia in inflammatory bowel diseases. Aliment. Pharmacol. Ther. 34, 1173–1184.

9. Morris, A.A.M., Kožich, V., Santra, S., Andria, G., Ben-Omran, T.I.M., Chakrapani, A.B., Crushell, E., Henderson, M.J., Hochuli, M., Huemer, M., et al. (2017). Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency. J. Inherit. Metab. Dis. 40, 49–74.

10. Sobczyńska-Malefora, A., and Harrington, D.J. (2018). Laboratory assessment of folate (vitamin B9) status. J. Clin. Pathol. 71, 949–956.

11. Liang, S., Zhou, Y., Wang, H., Qian, Y., Ma, D., Tian, W., Persaud-Sharma, V., Yu, C., Ren, Y., Zhou, S., et al. (2014). The Effect of Multiple Single Nucleotide Polymorphisms in the Folic Acid Pathway Genes on Homocysteine Metabolism. BioMed Res. Int. 2014.

12. Paul, C., and Brady, D.M. (2017). Comparative Bioavailability and Utilization of Particular Forms of B12 Supplements With Potential to Mitigate B12-related Genetic Polymorphisms. Integr. Med. Clin. J. 16, 42–49.

13. Wacklin, P., Tuimala, J., Nikkilä, J., Sebastian Tims, Mäkivuokko, H., Alakulppi, N., Laine, P., Rajilic-Stojanovic, M., Paulin, L., de Vos, W.M., et al. (2014). Faecal Microbiota Composition in Adults Is Associated with the FUT2 Gene Determining the Secretor Status. PLoS ONE 9.

14. Li, W.-X., Dai, S.-X., Zheng, J.-J., Liu, J.-Q., and Huang, J.-F. (2015). Homocysteine Metabolism Gene Polymorphisms (MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G) Jointly Elevate the Risk of Folate Deficiency. Nutrients 7, 6670–6687.

15. Chuang, C.Z., Boyles, A., LeGardeur, B., Su, J., Japa, S., and Lopez-S, A. (2006). Effects of riboflavin and folic acid supplementation on plasma homocysteine levels in healthy subjects. Am. J. Med. Sci. 331, 65–71.

Dr. Robyn Murphy

About The Author

Dr. Robyn Murphy is the Clinical Research Advisor for AOR and a practicing naturopathic doctor, public speaker and researcher. Immensely passionate about educating both healthcare professionals and the public about advances in genetics and integrative medicine, Dr. Murphy strives to empower individuals with the proper tools and information to significantly change their health. As Scientific Advisor for DNA Labs, Dr. Murphy is the co-developer of lifestyle genetic testing with several published articles in medical journals. She is the past Associate Medical Director of a lifestyle genetics company and co-founder of The IBS Academy. Dr. Murphy holds a Bachelor of Science from the University of Alberta and Doctor of Naturopathic Medicine from CCNM, with advanced training and certifications in functional gastroenterology, hormone therapy (BHRT), biological medicine and advanced medical herbalism.

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