What is the Methylation Pathway?

by Yusuf M. Saleeby, MD

Methylation a very hot topic these days. And to answer the question “Does our world revolve around methylation?” Well a resounding YES would be the answer. If it were not for methylation then we would cease to exist as a species. A few terms to define before we go deep into methylation and the methylation pathways and its impact on our health and well being.

First of all some basic chemistry nomenclature. The methyl group is a carbon atom with three attached hydrogen atoms. This group plays a very significant role in our body’s ability to utilize nutrients and turn genes on and off. The importance of this simplest of chemical structures has an overwhelming impact on organism function. Deficiencies in this methyl group will impact gene expression and transportation of very important chemicals and compounds through cells. Defects in the methylation pathway result in damage to end organ systems on a cellular level. Those system injuries can often be manifest as cardiovascular disease; diabetes; coagulation pathway disruption; thyroid dysfunction; neurotransmitter imbalances; inflammation; immune dysfunction leading to chronic infection and cancer expression; neurodegenerative disorders; psychiatric disorders and premature aging. If however, defects in methylation are identified early with specific testing, interventions can be implemented to reduce the effects of deficiencies and defects. This can usually be accomplished with select nutritional interventions to restore a functional pathway.

The methylation pathway is integrally interfaced with the Kreb’s Cycle and allows for the utilization of folates, by converting to a methylated folate that can cross the blood brain barrier. The methylation pathway helps in the clearance of elevated (and atherogenic) levels of homocysteine. It aids in the production of neurotransmitters, s-adenosyl methionine, and glutathione. Glutathione is one of the most important free radical scavengers around. Methylcobalamine (a methylated version of vitamin B12) and methylenetetrahydrofolate (MTHF, a metabolite of folate/folic acid), magnesium, zinc and methionine (an amino acid) combine to make S-Adenosyl-Methionine (SAMe). SAMe is a critical player in the production and regulation of neurotransmitters. An Italian immigrant to America is credited with the discovery of SAMe and the methylation pathway. Dr. Giulio Cantoni in 1952 isolated SAMe in his lab at the NIH and became the first to realize that the source of the methyl group in this process stemmed from methionine. His work was critically important in our understanding of the process whereby the neurotransmitter norepinephrine is methylated to become epinephrine.

The methylation cycle is so critically important to healthy bodily function that those who express a genetic mutation under environmental stressors (epigenetics) will suffer a breakdown in the normal cycle and develop some serious medical conditions. The focus of late has been on the genes that code for the production of an enzyme central to the proper function of the methylation cycle or pathway. The gene is the MTHFR (methylenetetrahydrofolate reductase enzyme) site located at the end of the short arm of chromosome 1 (1p36.3). Mutations of this gene carry the designation of C677T and A1289C. We all carry two copies of this gene and we inherit a single copy from each of our parents. One would hope that both copies inherited would be normal, but occasionally there are mutations that occur and are passed down from parent to child. A single mutation would be a heterozygous single nucleotide polymorphism (SNP) where one nucleic acid base is substituted for another. In the case of C677T a Thymine base (T) is substituted for the normal Cytosine (C). When two SNPs occur than two aberrant bases are found on the gene, this is referred to as a homozygous mutation. Gene expression will then be limited and the coding for the protein, in this case a very important metabolic enzyme is disrupted. Limited production of this enzyme causes disruption of the pathway. So if a person has one or more SNP on their MTHFR gene the resultant genotype has ramifications along the methylation pathway causing a hindrance in health on a number of fronts.

The methylation pathway is essential for the production of purines and pyrimidines, used to production of new DNA and RNA in our cells. As cells die and need to be replaced (literally millions of cells every minute) DNA and RNA need to be produced. If the process is slowed down new cell production cannot keep up with cell death (apoptosis). We must note that DNA and RNA synthesis are directly dependent on this particular biological pathway. Mutations or defects in this pathway will result in aberrant creation, replacement or repair of DNA and/or RNA and the result is usually expressed as disease or a myriad of illnesses.

Genetic mutations of the component parts of the methylation pathway such as MTHFR genotypes, emotional stress with high cortisol releases, or physical stress by environmental chemicals or toxins can affect the cycle and produce overt signs of disruption. The systems most affected are the bone marrow’s erythrocytes and lymphocytes, as well as neural tissues and hair follicles (all aforementioned are the rapidly growing and dividing cells of our bodies). Therefore, mutations can cause abnormal CBC counts and neurological disorders. Even loss of hair growth and thickness can be impacted.

The effects of a disrupted Methylation Pathway can affect heart health. We know that mutations of MTHFR gene can impact serum homocysteine levels which can lead to coronary artery disease. It also impacts the production of the cardiac supporting coenzyme Q10 via a pathway requiring SAMe for production of coQ10. CoQ10 is also a required component in ATP production in the mitochondria of our cells. These mitochondria are the little power plants of our cells and any dysfunction can wreck havoc on our system. Another required element is a compound called carnitine. Carnitine is necessary for mitochondrial function and energy production especially in cardiac and skeletal muscles. Carnitine interestingly enough is dependent on the methylation of the amino acid L-lysine by SAMe. Carnitine production is yet another connection to the Methylation Pathway. Both coQ10 & carnitine, dependent on the Methylation Pathway, are responsible for proper energy production within the mitochondria. In each of our cells we can witness the impact of mutations in our DNA and/or environmental stressors with resultant failures of optimal energy production.

The impact on the brain and mood are also to be considered based on MTHFR genotype. Neurotransmitters are reliant on the blood-brain barrier crossing methylenetetrahydrofolate (MTHF), which helps synthesize dopamine, epinephrine and norepinephrine, serotonin and melatonin. If a person has a mutation of the MTHFR gene (C677C being normal and C677T being abnormal) it can result in a depress production of the MTHF-reductase enzyme that converts folates or folic acid to the useful MTHF. Therefore, treatment with the vitamin metabolite L-MethylFolate in high doses may be of more benefit than some modern day antidepressants (Prozac®, Celexa®, Wellbutrin®, etc.). Medical Foods such as Deplin® (high dose L-methylfolate) is an FDA approved and prescriptive therapy.

More detail about the process follows. Folic Acid or folates once absorbed and within the intestinal wall are converted to monoglutamate entities by an enzyme called alpha-L-glutamyl transferase. Once there this monoglutamate folate derived compound is converted to MTHF (essentially the active form of folate) it is utilized by the brain and trimonoamine neurons to created neurotransmitters. So, additional folic acid is not the cure here if one has disruption of MTHFR. The treatment would be MTHF to bypass the defects in the Methylation Pathway due to genome or a toxin disrupting the pathway. This therapy may be helpful in depressed and anxious patients who have not responded to traditional antidepressant therapy. Folate levels in the blood may be normal but mutation in MTHFR production and levels may be found on genomic profiling, MTHFR activity assays are not clinically available, although monitoring homocysteine levels can indicate a problem and a means of tracking progress.

Dopamine the neurotransmitter responsible for focus, concentration, short term memory, and sleep patterns, is the product of tyrosine to dopamine metabolism with the key factor of methylfolate as a driving force. Dopamine further affects the balance of epinephrine and norepinephrine. SAMe again part of the Methylation Pathway is a principle methyl donor for the production of these neurotransmitters. We can trace this back again to the link to MTHF. Not only are neurotransmitters the direct result of our Methylation Pathway, but also the protective sheath surrounding nerves called myelin is dependent on it working correctly. Methylation of amino acids stabilizes the myelin sheath against degradation.

The immune system is not immune to the workings of the Methylation Pathway. No pun intended here. In actuality, T-cell synthesis requires the methylation cycle to generate new T-cells and bolster the activity and immune function of B-cells. There is a regulatory balance between T & B-cells that keeps our immune function in balance. When methylation is interrupted or defective, B-cells create antibodies on a more vicious level to compensate for lack of T-cell levels and this often times runs amuck creating the immune-cells-attacking-self scenario or autoantibiodies or autoimmune dysfunction. In cases of autoimmune disorder, methylation dysfunction results in improper T-cell function and levels, and when the system is corrected with proper methylation function, appropriate immune function returns.

The cancer connection with the Methylation Pathway is simply a situation of massive reduction in the methylation process with over and under methylation of particular genes that turn on and off tumor growth (tumor suppressor genes). Methyl donation via SAMe is able to suppress or inactivate certain chemicals or hormones that can cause a rise in cancer expression. Some estrogens can cause cancers and methylation of estrogens reduces this effect. Colorectal cancers (CRC) are linked to the Methylation Pathway. Mutations in the MTHFR gene and methionine synthase (MTR A2756G) and thymidylate synthase (TS enhancer region) and others have been implicated in CRC. Methylation plays a role in our immune system to identify cancer cells and in particular NKT-cells to recognize and destroy cancer and potential pre-cancer cells. Therefore, those with poor methylation function are more susceptible to cancers.

Environmental toxins and hormone disrupters are also implicated in cancer and again we see the utility of the Methylation Pathway and methylation playing a part in detoxifying. Methylation is critical in the process of removal of toxins and the production of glutathione. Glutathione is a highly effective free radical scavenger and helps control inflammation at the cellular level. It acts as one of the liver’s chief detoxifiers. Derangement of the Mehtylation Pathway impacts the speed in which we age. Methylation and methyl donation tend to sequester with aging. Maintenance of our body’s ability to methylate, will stave off degenerative diseases of end organs associated with the aging process.

We have all hear of the implementation of folic acid or folate in the prevention of neural tube defects in the expectant mother. The recommendation of a prenatal vitamin high in folic acid has been around for quite a while. Further research has shown that mothers to be who possess a MTHFR gene mutation and disruption of the methylation pathway are more apt to have a miscarriage and children with neuropsychiatric disorders. Supplementation with MTHF will help lessen the risks of losing a baby, as well as reduce spina bifida and other neuronal disorders. Mutation in the methionine synthase reductase gene, as well as elevations in homocysteine raise the risk for children born with Down’s syndrome and is implicated in ADD. Folate supplementation in a prenatal vitamin is only worthwhile when you have functional MTHF Reductase to process the substrate folate to a usable form. However, mothers who have mutations are not protected by just folate, they then require the end-product.

As you can see from the diagram (below) of the Methylation Pathway and Kreb’s cycle it is dependent on several compounds and elements such as vitamin B12, P-5-P (Pyridoxal-5-Phosphate) aka vitamin B6, folate or folic acid, magnesium (Mg), zinc (Zn), copper (Cu), and molybdenum (Mb), tetrahydrobiopterin (BH4), and dihydrobiopterin (BH2). BH2 is recycled into BH4 and is instrumental in the production of neurotransmitters. All this is dependent on methylation and the Methylation Pathway and that is what keeps our world turning. Clinically available genomic testing for MTHFR genotype mutations is available by simple blood or buccal mucosa DNA sampling.

– – –

Yusuf M. Saleeby, MD is an integrative medical physician who specializes in early detection of potential health altering processes by the use of advanced biomarkers. His focus is on nutritional medicine, proper thyroid function and identifying genotype polymorphisms that cause disease to assist his patients in better health and outcomes. He is medical director for PHC.


Arakawa Y, Watanabe M, et.al., Association of polymorphisms in DNMT1, DNMT3A, DNMT3B, MTHFR and MTRR genes with global DNA methylation levels and prognosis of autoimmune thyroid disease. Clin Exp Immunol. 2012 Nov;170(2):194-201. PubMed PMID: 23039890.

Cho SE, Hong KS, Shin GJ, Chung WS. The methylenetetrahydrofolate reductase C677T gene mutation is associated with hyperhomocysteinemia, cardiovascular disease and plasma B-type natriuretic peptide levels in Korea. Clin Chem Lab Med. 2006;44(9):1070-5. PubMed PMID: 16958597.

Crott, JW., et. al., Methylenetetrahydrofolate reductase C677T polymorphism does not alter folic acid deficiency-induced uracil incorporation into primary human lymphocyte DNA in vitro
Carcinogenesis. 2001; 22(7): 1019-1025.

Crott, JW, et. al., Moderate folate depletion modulates the expression of selected genes involved in cell cycle, intracellular signaling and folate uptake in human colonic epithelial cell lines, J Nutritional Biochem, 2008 (19):5;328-335

Garilli, B., MTHFR Mutation: A Missing Piece in the Chronic Disease Puzzle
http://www.drbiancagarilli.com, June, 2012, Vol.13,No.2.

Gokcen C, Kocak N, Pekgor A., Methylenetetrahydrofolate Reductase Gene Polymorphisms in Children with Attention Deficit Hyperactivity Disorder. Int J Med Sci 2011; 8(7):523-528.
Gupta A, Datta M, Shukla GS. Cerebral antioxidant status and free radical generation following glutathione depletion and subsequent recovery. Mol Cell Biochem. 2000 Jun;209(1-2):55-61. PubMed PMID: 10942201.
Guthikonda S, Haynes WG. Homocysteine: role and implications in atherosclerosis. Curr Atheroscler Rep. 2006 Mar;8(2):100-6. Review. PubMed PMID: 16510043.

Henderson, LM, Hulse, JD, and Henderson, LL, (1980) Purification of the enzymes involved in the conversion of trimethyl-lysine to trimethylaminobutyrate. Carnitine Biosynthesis, Metabolism, and Functions (Frenkel and McGarry, eds.), 35–43, Academic Press, Inc., New York.
Ketterer B., Glutathione S-transferases and prevention of cellular free radical damage. Free Radic Res. 1998 Jun;28(6):647-58. Review. PubMed PMID: 9736316.

Kim S, Lim IK, et.al., Biological methylation of myelin basic protein: enzymology and biological significance. Int J Biochem Cell Biol. 1997 May;29(5):743-51. Review. PubMed PMID: 9251242.

Lewis SJ, Lawlor DA, et.al., The thermolabile variant of MTHFR is associated with depression in the British Women’s Heart and Health Study and a meta-analysis. Mol Psychiatry. 2006 Apr;11(4):352-60. Review. PubMed PMID: 16402130.

McCully KS., Chemical pathology of homocysteine. I. Atherogenesis. Ann Clin Lab Sci. 1993 Nov-Dec;23(6):477-93. Review. PubMed PMID: 8291902.

Miller AL. The methylation, neurotransmitter, and antioxidant connections between folate and depression. Altern Med Rev. 2008 Sep;13(3):216-26. Review. PubMed PMID: 18950248.

MTHFR genotype testing, eStatLabs. http://www.estatlabs.com/methylenetetrahydrofolate-reductase-mthfr-dna-mutation-genomic-testing, (Accessed 7/2014).

Oransky, I, Obituary of Giulio Cantoni, The Lancet, 2005;366,(9489):888, doi:10.1016/S0140-6736(05)67305-9.

Sadighi Z, Butler IJ, Koenig MK. Adult-onset cerebral folate deficiency. Arch Neurol. 2012 Jun;69(6):778-779. PubMed PMID: 22371854.

Skoric D, Ivana J, Tanja R, et.al., Methylenetetrahydrofolate reductase and glutathione S-tranferase gene polymorphisms in secondary mixed phenotype acute leukemia: a case report. J Pediatr Hematol Oncol. 2014 Apr;36(3):e152-4. PubMed PMID: 24276031.

Stahl, SM, Depression and Bipolar Disorder: Stahl’s Essential Psychopharmacology, 3rd Edition, 2008, Cambridge.

Steward, KL, Methylation Pathway, http://www.drkendalstewart.com (accessed 7/2014)

Zhang Y, Zhao M, et.al., Impaired DNA methylation and its mechanisms in CD4(+)T cells of systemic lupus erythematosus. J Autoimmun. 2013 Mar;41:92-9. Review. PubMed PMID: 23340289.