Folate for Thought: 12 Ideas about Methylation, mostly based on Mathematical Models

1. Methylation is different in different organs.

Some of the methylation enzymes and some parts of the cycle are active in some organs but not others. In 1990, Finkelstein published animal data showing how the activity rates of methylation enzymes vary widely in 11 different organs, with some enzymes much more active in certain organs than others. [Finkelstein, J.D. (1990). Methionine metabolism in mammals. The Journal of nutritional biochemistry, 1(5), 228-237, doi:10.1016/0955-2863(90)90070-2, Table 1.]

My guess is that most everyone now could use more methylation in the liver. However, certain helpful supplements might temporarily overactivate methylation elsewhere, such as in the brain.

2. Trimethylglycine (TMG) supports liver methylation.

Trimethylglycine (TMG) might be a particularly helpful supplement because the reaction it supports occurs almost exclusively in the liver. [Id.] Thus, TMG might specifically increase methylation in the liver more than in other organs, which many people might need.

3. Allosteric interactions are important.

These are interactions between one chemical in the cycle and another chemical or enzyme which is involved elsewhere in the cycle. For a good diagram of the allosteric interactions in the methylation and folate cycles, see: Nijhout, H.F., Best, J.A., & Reed, M.C. (2018). Systems biology of robustness and homeostatic mechanisms. Wiley Interdisciplinary Reviews: Systems Biology and Medicine, e1440, doi:10.1002/wsbm.1440, Figure 3.

That article has some neat chair graphs showing how the flat part of the chair (stable homeostasis region) becomes tilted (destabilized) without the allosteric interactions [Id. Figure 8]. Similar destabilization occurs with vitamin B12 deficiency [Id. Figure 10].

The allosteric interactions are probably quite important in the feedback loops after adding supplements.

4. Folate's 3 major roles.

Folate (vitamin B9) has at least 3 major roles related to the methylation cycle:

1. Folate as methyl folate recycles homocysteine for methylation.
2. Folate in non-methylated forms is a rate-limiting reagent for DNA synthesis.
3. In an allosteric interaction, folate as methyl folate binds and deactivates GNMT (glycine N-methyltransferase) enzyme. GNMT activity is an overflow pathway that stabilizes the methylation flow.

[Id. Figure 3.]

Number 1 above seems to get the most attention, and sometimes number 2 gets attention too. Number 3 might deserve more recognition, as discussed in the next section.

5. Disabling the GNMT buffer pathway might be the primary effect of taking high-dose methyl folate supplement.

Chris Masterjohn has pointed out that even a high-dose methyl folate supplement provides only a tiny fraction of the methyl folate used by the body. The vast majority still comes from recycling via MTHFR and the folate cycle. [Masterjohn C. (2019). Your “MTHFR” Is Just a Riboflavin Deficiency. Retrieved from https://chrismasterjohnphd.com/2019/02/26/mthfr-just-riboflavin-deficiency/ .]

"GNMT is a major folate binding protein. 5mTHF [methyl folate] binds to GNMT and tends to inactivate it." [Reed, M.C., Gamble, M.V., Hall, M.N., & Nijhout, H.F. (2015). Mathematical analysis of the regulation of competing methyltransferases. BMC systems biology, 9(1), 69, doi:10.1186/s12918-015-0215-6]. Each GNMT enzyme will bind 2 methyl folate molecules. GNMT's activity seems to be partially blocked when bound to 1 methyl folate and completely shut down when bound to 2 methyl folates. [Id.]

I propose then that the reason high dosages of methyl folate have such dramatic effects for some people is that they bind GNMT enzyme and disable the GNMT methylation overflow pathway.

High-dose methyl folate could increase methylation and increase DNA synthesis and cell division. However, additionally, high-dose methyl folate might also override the stabilizing allosteric interaction with GNMT. Therefore, the entire methylation and DNA formation cycles would become prone to more fluctuation and instability. Indirectly, this could cause refeeding syndrome involving induced shortages of other nutrients.

With high-dose methyl folate, one is taking over the steering wheel. Less stable biochemistry and induced imbalances of other nutrients might be likely. Some people, especially those who are quite behind in methylation and/or DNA synthesis, might need the high-dose methyl folate as a GNMT override to allow catch-up.

For chair graphs showing the destabilization of DNA synthesis and methylation upon removing the methyl folate / GNMT allosteric interaction, please see: Nijhout, H.F., Best, J.A., & Reed, M.C. (2015). Using mathematical models to understand metabolism, genes, and disease. BMC biology, 13(1), 79, doi:10.1186/s12915-015-0189-2, Figure 4.

Researchers have suggested high-dose folate as a way to override GNMT's upper limit on methylation, in the context of helping people in Bangladesh with a high methylation burden to detoxify arsenic in the drinking water. [Reed, M.C., Gamble, M.V., Hall, M.N., & Nijhout, H.F. (2015). Mathematical analysis of the regulation of competing methyltransferases. BMC systems biology, 9(1), 69, doi:10.1186/s12918-015-0215-6].

6. MTHFR and MS as gatekeepers between folate's roles, needed in balance.

Two important enzymes in the folate cycle control the allocation of the forms of folate:

• In one direction, methylenetetrahydrofolate reductase (MTHFR) enzyme converts folate from the non-methylated form (used for DNA synthesis) to the methylated form (used for methylation). Vitamin B2 (riboflavin) is the cofactor for MTHFR enzyme.
• In the other direction, methionine synthase (MS) enzyme converts folate from the methylated form (methylation) to a non-methylated form (DNA synthesis). Vitamin B12 as methylcobalamin is the cofactor for MS enzyme, and sufficient homocysteine is required too, which requires sufficient protein.

Looking at the cycle from folate's perspective, MTHFR is gatekeeper in one direction, and MS in the other direction. MTHFR and MS need to be in balance with each other. This balance allows sufficient amounts of each form of folate, methylated folate (for methylation) and non-methylated folate (for DNA synthesis).

Too much MTHFR activity relative to MS activity is quite dangerous. This can occur with B12 deficiency or protein deficiency. When too much folate remains in the methylated form, and insufficient MS activity is present to recycle it, the folate is trapped as methyl folate. It can leak from the cell because the cell membrane is permeable to methyl folate. This is the phenomenon called complete or partial "methyl trap".

For more on methyl trap, see:

• van Konyenburg, R. (2011). Glutathione & Precursors - Detox or Induced Methylb12 and Methylfolate Deficiencies?. Phoenix Rising Forums. Retrieved from https://forums.phoenixrising.me/threads/glutathione-precursors-detox-or-induced-methylb12-and-methylfolate-deficiencies.374/page-4#post-9751
• Reed, M.C., Nijhout, H.F., Neuhouser, M.L., Gregory III, J.F., Shane, B., James, S.J., ... & Ulrich, C.M. (2006). A mathematical model gives insights into nutritional and genetic aspects of folate-mediated one-carbon metabolism. The Journal of nutrition, 136(10), 2653-2661, doi:10.1093/jn/136.10.2653
• Shane, B., & Stokstad, E.R. (1985). Vitamin B12-folate interrelationships. Annual review of nutrition, 5(1), 115-141, doi:10.1146/annurev.nu.05.070185.000555
• Wagner, C., Briggs, W.T., & Cook, R.J. (1985). Inhibition of glycine N-methyltransferase activity by folate derivatives: implications for regulation of methyl group metabolism. Biochemical and biophysical research communications, 127(3), 746-752, doi:10.1016/S0006-291X(85)80006-1
• Scott, J., & Weir, D. (1981). The methyl folate trap: a physiological response in man to prevent methyl group deficiency in kwashiorkor (methionine deficiency) and an explanation for folic-acid-induced exacerbation of subacute combined degeneration in pernicious anaemia. The Lancet, 318(8242), 337-340, doi:10.1016/S0140-6736(81)90650-4.

Too little MTHFR activity could be problematic too. Chris Masterjohn and Alex Leaf recently proposed that genetic variations in MTHFR are unimportant if one has sufficient vitamin B2 (riboflavin), which many people might not have.
[Leaf A. (2019). Supplement riboflavin to fix the MTHFR polymorphism. Retrieved from https://alexleaf.com/2019/02/11/supplement-riboflavin-to-fix-the-mthfr-polymorphism/ ]
[Masterjohn C. (2019). Your “MTHFR” Is Just a Riboflavin Deficiency. Retrieved from https://chrismasterjohnphd.com/2019/02/26/mthfr-just-riboflavin-deficiency/ ]

MTHFR enzyme, and the very common genetic variations in it, have been widely publicized and gotten a bad rap. However, the popular concern about MTHFR gene mutations seems to overlook folate's multiple roles, and MTHFR enzyme's job as a partitioner or gatekeeper. For data about how the partitioning of folate forms varies with an MTHFR mutation, see: Misselbeck, K., Marchetti, L., Field, M.S., Scotti, M., Priami, C., & Stover, P.J. (2017). A hybrid stochastic model of folate-mediated one-carbon metabolism: Effect of the common C677T MTHFR variant on de novo thymidylate biosynthesis. Scientific reports, 7(1), 797, doi:10.1038/s41598-017-00854-w, Table 6.

In protein or B12 deficiency, lower MTHFR may be helpful to conserve some folate in non-methylated form for DNA synthesis and prevent methyl trap. The genetic variations in MTHFR may be nature's way of experimenting with different partitions between the methylated and non-methylated forms of folate.

7. Allosteric interactions contributing to glutathione trap.

Freddd and others taking active B12 and folate supplements have described catastrophic worsening of symptoms when adding glutathione or glutathione precursors such as N-acetylcysteine (NAC). Rich van Konyenburg gave a brilliant explanation of why glutathione binding to unescorted vitamin B12 might halt MS enzyme function and trigger methyl trap. [van Konyenburg, R., Freddd (2011). Glutathione & Precursors - Detox or Induced Methylb12 and Methylfolate Deficiencies?. Phoenix Rising Forums. Retrieved from https://forums.phoenixrising.me/threads/glutathione-precursors-detox-or-induced-methylb12-and-methylfolate-deficiencies.374/page-4#post-9749 and subsequent posts on the same page.]

The glutathione-B12 interaction would be an example of an allosteric interaction. Though Rich's explanation likely covers the most important part, I want to suggest there might be other allosteric interactions involved too.

Oxidative stress might trap the added glutathione as GSSG (glutathione disulfide, oxidized form of glutathione). The GSSG might allosterically inhibit MAT enzyme (reducing SAM-e formation). The oxidative stress itself might promote CBS enzyme (more homocysteine leaving the methylation cycle) and also inhibit MS enzyme (less recycling of homocysteine for methylation). [Reed, M.C., Thomas, R.L., Pavisic, J., James, S.J., Ulrich, C.M., & Nijhout, H.F. (2008). A mathematical model of glutathione metabolism. Theoretical biology and medical modelling, 5(1), 8, doi:10.1186/1742-4682-5-8.]

All these changes could block methylation, especially if B12 were in short supply. In turn, folate would be trapped as methyl folate and leak from the cell, and transsulfuration to make more reduced glutathione (GSH) would be blocked too. The trapped GSSG might continue blocking all the cycles until cleared from the body.

Freddd said that all those reporting catastrophic effects of glutathione supplements were already supplementing active B12s and most also were taking methyl folate. [Freddd (2011). Glutathione & Precursors - Detox or Induced Methylb12 and Methylfolate Deficiencies?. Phoenix Rising Forums. Retrieved from https://forums.phoenixrising.me/threads/glutathione-precursors-detox-or-induced-methylb12-and-methylfolate-deficiencies.374/page-4#post-9752.]

The methyl folate could have destabilized the methylation cycle by binding GNMT, as discussed above. The high dose B12s also might have an unknown destabilizing effect.

Thus, the other supplements might have caused a predisposition to fall off the homeostasis chair when given glutathione. "All homeostatic mechanisms, including the long-range interactions discussed here, can be broken if inputs become too extreme" [Reed, M.C., Gamble, M.V., Hall, M.N., & Nijhout, H.F. (2015). Mathematical analysis of the regulation of competing methyltransferases. BMC systems biology, 9(1), 69, doi:10.1186/s12918-015-0215-6].

It's a sort of a double jeopardy -- the high-dose supplements can fix things but risk ending up worse -- a risk some will want to take.

8. At the edge of the homeostasis chair.

Increasing levels of toxins and environmental stresses today might predispose us to more biochemical imbalances and diseases now:

[M]utations or environmental changes can place an individual closer to the edge of the homeostatic region, thus predisposing that individual to deleterious effects caused by additional mutations or environmental changes. Mutations and environmental variables can also reduce the size of the homeostatic region, thus releasing potentially deleterious cryptic genetic variation. These considerations of mutations, environment, homeostasis, and escape from homeostasis help to explain why the etiology of so many diseases is complex.

[Nijhout, H.F., Best, J., & Reed, M.C. (2014). Escape from homeostasis. Mathematical biosciences, 257, 104-110, doi:10.1016/j.mbs.2014.08.015].

"Escape from homeostasis," the title of the above journal article, might be a good characterization of the pattern of weakened bodies today. On the first day of high school biology class, my teacher told us that if we didn't know the answer to a biology question, try "Increase surface area" or "Maintain homeostasis." Today, escape from homeostasis has become quite common as humans face new challenges.

In some cases, the escape from homeostasis may have a single primary cause, such as a major genetic defect like the cobalamin-lettered diseases. I'm guessing that much more common is that a plethora of stresses of the modern world, including toxins, EMF radiation, nutritionally depleted food and water, more emotional stress, and many others, together, can combine to knock a person off the homeostasis chair.

The mechanism for falling off the homeostasis chair is probably more complex than we can explain in most cases. Therefore, Dr. Larry Wilson's large-scale approach makes sense to me: strengthen the body and rebuild its self-healing ability in many ways, including foods, supplements, lifestyle, and detox procedures suited to the modern world. [E.g. Wilson L.D. (2018). Chronic Fatigue Syndrome or Myalgic Encephalopathy (ME). Retrieved from http://drlwilson.com/Articles/CHRONIC%20FATIGUE.HTM . Note that many of Dr. Wilson's ideas are fascinating but controversial, and are beyond the scope of this reference.]

In other words, regardless of causation for escaping from homeostasis, one could make many diet, supplement, lifestyle, and detox procedure steps to return back closer to the stable seat of the multi-dimensional homeostasis chair graph.

9. Nobel Prize for B12.

Google Books has a copy of Dorothy Crowfoot Hodgkin's remarkable work on the structure of vitamin B12. [Hodgkin, D.C. (1958). X-ray Analysis and the Structure of Vitamin B 12. In Fortschritte der Chemie organischer Naturstoffe/Progress in the Chemistry of Organic Natural Products/Progrès dans la Chimie des Substances Organiques Naturelles (vol. XV pp. 167-220). Springer, Vienna. doi:10.1007/978-3-7091-7162-2_4. Retrieved from https://books.google.com/books?id=1F6yBgAAQBAJ.]

Hodgkin won the 1964 Nobel Prize in Chemistry in part for revealing B12's intricate structure. Unfortunately, she and the researchers she cited probably made a mistake in accepting cyanide as an intrinsic part of B12. This might have led to cyanocobalamin becoming accepted as the standard form of B12, rather than the natural coenzyme forms methylcobalamin and adenosylcobalamin. Cyanocobalamin continues to be used today in multivitamins, fortified foods, and "vitamin B12" supplements.

The section of Hodgkin's original research that covers the cyanide group appears on page 179.

10. Folic acid as chemotherapy.

Concentration of the DHF (dihydrofolate) form of folate is usually very low. Chemotherapy blocks DNA synthesis by blocking the DHFR (dihydrofolate reductase) enzyme. [Nijhout, H.F., Reed, M.C., Budu, P., & Ulrich, C.M. (2004). A mathematical model of the folate cycle new insights into folate homeostasis. Journal of Biological Chemistry, 279(53), 55008-55016, doi:10.1074/jbc.M410818200.]

Folic acid uses a lot of DHFR to enter the folate cycle, requiring 2 reduction steps by DHFR. [Powers, H.J. (2007). Folic acid under scrutiny. British Journal of Nutrition, 98(4), 665-666, doi:10.1017/S0007114507795326.]

In the long term, perhaps folic acid may inhibit DNA synthesis just as chemo drugs do. Some people’s bodies can handle it, but some might be able to activate very little folic acid, and the folic acid and DHF accumulates. Perhaps the excess folic acid and DHF exhausts or blocks the DHFR enzyme. The accumulation might produce disturbances to DNA synthesis, and eventually perhaps even cancer. It might be reasonable to think of folic acid — the most common form of folate supplement and food fortification — as long-term chemotherapy.

11. Lamb might be helpful.

Since going back to eating meat, I've found lamb to be one of the most helpful foods. Though less common than beef in the United States, I'm guessing lamb may be very high in carnitine in a good form.

12. Bistability of liver methylation: Occasional large animal feasts might help catch up on methylation.

Martinov et al proposed a mathematical model with two stable steady states of liver methylation in which SAM-e supply equals SAM-e consumption:

• In the low-methionine steady state, the MAT-I enzyme makes most of the SAM-e, the methylation reactions regulate the flow of SAM-e to meet the methylation needs, and there is little to no methylation in the glycine to sarcosine overflow pathway.
• In the high methylation steady state, the MAT-III enzyme makes most of the SAM-e, SAM-e concentration increases by an order of magnitude (10x), methylation of glycine to sarcosine by GNMT increases in flow to absorb the overabundance of SAM-e, and the levels of the other methylation reactions stay constant. The high methylation steady state permits an increased flow through the transsulfuration pathway to produce increased cysteine and the antioxidant glutathione.

For more on bistability of liver methylation, please see:

• Martinov, M.V., Vitvitsky, V.M., Banerjee, R., & Ataullakhanov, F.I. (2010). The logic of the hepatic methionine metabolic cycle. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1804(1), 89-96, doi:10.1016/j.bbapap.2009.10.004.
• Korendyaseva, T.K., Kuvatov, D.N., Volkov, V.A., Martinov, M.V., Vitvitsky, V.M., Banerjee, R., & Ataullakhanov, F.I. (2008). An allosteric mechanism for switching between parallel tracks in mammalian sulfur metabolism. PLoS computational biology, 4(5), e1000076, doi:10.1371/journal.pcbi.1000076.
• Martinov, M.V., Vitvitsky, V.M., Mosharov, E.V., Banerjee, R., & Ataullakhanov, F.I. (2000). A substrate switch: a new mode of regulation in the methionine metabolic pathway. Journal of theoretical biology, 204(4), 521-532, doi:10.1006/jtbi.2000.2035.

The high-methionine mode might give our livers a chance for helpful methylation and transsulfuration catch-up. We might get into this catch-up mode with occasional animal feasts with a large amount of meat, as native peoples might have eaten.

Conclusion

Hope these ideas about methylation have been helpful to you! If you have any comments, questions, or want to talk about it more, please email me.