Methylation and Vitamin U

 

Photo by Alonso Reyes on Unsplash

Previously we looked at the methionine cycle, one of the functions of which is to generate methylation capacity Vitamin U (S-methylmethionine) information: The methionine cycle and Vitamin U Here we'll take a closer look at methylation.

Methylation describes over 100 reactions in our body ranging from the biosynthesis of creatine and phosphatidylcholine to the methylation of cytosine residues of DNA. The universal methylation substrate is S-adenosylmethionine (SAM or SAMe), which is a form of methionine in which the methyl group has been activated and thus made suitable for transfer to acceptor molecules. Methylation is the transfer of a methyl group from a donor molecule to an acceptor molecule. Methylation capacity is maintained by the methionine cycle, which was covered previously.

The most renowned function of methylation is gene regulation, a component of epigenetics. Genes are made up of four nucleotides referred to using their initial - A,T,C,G. Methylation of particular C (cytosine) residues reduces expression of a particular gene by interfering with the binding of transcription factors involved in making the mRNA that encodes a protein. For example, the cell broadly methylates toxic genes like retroviruses that have been incorporated into our genome to silence them. When your methylation state is low, retroviruses can reactivate. Conversely, the promoters of genes that must be on at all times are usually embedded in CpG regions that are resistant to methylation.

Quantitatively, the most important methylation reaction in our body is the biosynthesis of creatine. Creatine is an amino acid used in our muscles to regenerate ATP, the major energy molecule in our body. Creatine is made in the liver from guanidinoacetate in a reaction catalyzed by guanidinoacetate methyltransferase (GAMT). This one reaction alone consumes 40-75% of our methylation capacity. Creatine travels from the liver to the muscles where it is activated by phosphorylation to produce creatine phosphate. The high-energy phosphate bond in creatine phosphate is used to regenerate ATP from ADP. The reason so much methylation capacity goes into making creatine is not just because creatine is vital, but rather that 1-3% of creatine cyclizes to form creatinine, which cannot be phosphorylated and is excreted as waste.  

Another major methylation reaction is involved in the biosynthesis of phosphatidylcholines. Although most phosphatidylcholines are made elsewhere, a significant portion is made by methylation of phosphatidylethanolamine. The enzyme phosphatidylethanolamine methyl transferase (PEMT) catalyses the transfer of three methyl groups from three SAM molecules in three consecutive steps to a single phosphatidylethanolamine molecule to produce a single molecule of phosphatidylcholine. Or said another way, phosphatidylcholine is trimethylated phosphatidylethanolamine.

Our methylation status can affect the ability to perform these functions. When the concentration of SAM is low, methylation slows down. The most common reason for a lack of methylation is insufficient methyl capacity from the diet. SAM is a reactive molecule that isn't stored in food or in our body. SAM supplements do exist, though they are relatively expensive, unstable and short-acting. The best way to meet our SAM requirements is by eating food containing nutrients that are used by our body to synthesize SAM. Our body makes SAM from methionine and ATP, with methionine supplying the methyl group. After SAM has donated its methyl group, we are left with S-adenosylhomocysteine, which in turn, is hydrolyzed to homocysteine. Homocysteine sits at an important metabolic fork. When our body is in a low methylation state, there is a tendency for homocysteine to be converted back into methionine by remethylation, even when the body may also be short of transsulfuration products such as glutathione. In contrast, when the methylation state in our body is normal, excess homocysteine is diverted into the transsulfuration pathway to form cysteine and its derivatives like glutathione and taurine.

Methionine is an essential amino acid found in protein. When methionine is remade from homocysteine, there are three nutrients that can supply the methyl group -

1) Folate/Serine
2) Choline/Betaine
3) Vitamin U

Remethylation is catalyzed by a specific enzyme for each nutrient -

1) Methionine synthase (MS) uses the cofactor B12 to transfer a methyl group from 5-methyltetrahydrofolate (folate) to homocysteine to form methionine and tetrahydrofolate.

2) Betaine homocysteine methyltransferase 1 (BHMT1) uses the cofactor zinc to transfer a methyl group from betaine to homocysteine to form methionine and dimethylglycine.

3) Betaine homocysteine methyltransferase 2 (BHMT2) uses the cofactor zinc to transfer a methyl group from Vitamin U to homocysteine to form two molecules of methionine.

We get most of our folate from vegetables and whole grains. Folate comes in various forms which our body converts into the active form MTHF. In fact, the major source of folate for many Westerners is folic acid added to processed wheat flour to make up for the loss of folate during milling. While a certain amount of folate we eat is already methylated, the main role of folate is that of a carrier. The same folate molecule accepts and donates a methyl group many times during its existence. The methyl group is supplied several enzymatic steps upstream by serine.

Betaine is abundant in whole grains. It functions as an osmoprotectant to the plant, so is found in most grains with the notable exception of rice, most of which is grown underwater. The major source of betaine in the typical American diet is bread, especially whole grain. In addition, a substantial amount of betaine is derived from choline. Choline is found in most whole foods, especially eggs, meat and whole vegetables. It is mainly used in phosphatidylcholine synthesis. Extra choline is broken down to make betaine. 

Betaine has three methyl units available that can feed into methylation pathways at various points. Betaine donates a methyl unit to homocysteine to produce methionine and dimethylglycine in a reaction catalyzed by betaine homocysteine methyltransferase. Dimethylglycine donates a methyl unit to tetrahydrofolate to produce sarcosine (methylglycine) and methylenetetrahydrofolate in a reaction catalyzed by dimethylglycine dehydrogenase. Sarcosine also donates a methyl unit to tetrahydrofolate to produce methylenetetrahydrofolate, this time in a reaction catalyzed by sarcosine dehydrogenase. The latter two reactions activate folate, which enables it to methylate homocysteine. 

Vitamin U (S-methylmethionine) from plants has two methyl units to donate as its reaction with homocysteine not only converts homocysteine into methionine, but itself is transformed into a second molecule of methionine. The reaction is catalyzed by the enzyme BHMT2 and mostly happens in the liver and kidneys. The major dietary source of Vitamin U are fresh vegetables and fruit.

Does Vitamin U contribute to methylation status in people? Assuming a maximum of 250 mg Vitamin U is taken each day from the diet (from a generous one liter of cabbage juice), that's 1.25 mmoles of Vitamin U or 2.5 mmoles of methyl groups. In a typical diet, methionine contributes 10 mmoles, choline contributes 30 mmoles, betaine contributes 26-75 mmoles, and folate (via methylneogenesis) contributes 5-10 mmoles of methyl groups per day. Assuming these estimates are correct, Vitamin U plays quite a modest role in the methylation status of people. However, it should be taken into consideration that not all of these nutrients give up their methyl groups. Methionine incorporated into proteins and choline used to make phosphotidylcholine are examples. 

For a vegan with a low protein diet and whose staple grain is rice, the contribution from Vitamin U is more significant as a percentage, though this diet may be low in total methylation capacity. It's not that Vitamin U is not a quality source of methyl groups - it's just that it's low in abundance in a natural diet compared to the other methyl donors. The fact that mammals have an enzyme devoted to its use demonstrates the importance Vitamin U has in the diet. To increase the contribution of Vitamin U towards methylation in people whose diet contain low concentrations of methyl groups, it is possible that larger amounts of Vitamin U via supplementation could be effective.

References

https://www.ncbi.nlm.nih.gov/pubmed/17209172

https://www.ncbi.nlm.nih.gov/pubmed/23196816

https://www.ncbi.nlm.nih.gov/pubmed/23661599

https://www.ncbi.nlm.nih.gov/pubmed/17413090

https://www.ncbi.nlm.nih.gov/pubmed/1128236

https://pubmed.ncbi.nlm.nih.gov/30871166/

Allergies and Vitamin U

At this time of the year, allergies are a seasonal problem for many people. With the coming of pollen also comes itchy eyes, a runny nose, an annoying cough and maybe more serious conditions such as asthma or hives. Cells in our immune system called mast cells produce histamine, which triggers an inflammatory response by binding H1 receptors. Our blood vessels dilate and fill with fluid to help get rid of allergens and to counteract the narrowing causes by the build up of mucus. Unfortunately, our bodies tend to overreact and produce way too much histamine. Antihistamines like loratadine (Claritin), cetirizine (Zyrtec), diphenhydramine (Benadryl) work by blocking the binding of histamine to these receptors.

Our body has a few mechanisms that can remove excess histamine from our body. One of the most important is the enzymatic action of histamine N-methyltransferase. HNMT catalyzes the methylation of histamine using the universal methyl donor S-adenosylmethyltransferase (SAM) as its source of methyl groups. Methylated histamine can no longer bind to the H1 receptor and cannot trigger more inflammation. Methylhistamine is removed from our body in our urine. People with polymorphisms in the gene encoding HNMT often present with a runny nose, hives and peptic ulcer disease.

Vitamin U is a natural support for decreases in methylation capacity caused by allergies. Vitamin U carries two methyl groups that contribute to the formation of SAM. Taking Vitamin U in the form of fresh cruciferous or stalky vegetables, or as a supplement, helps replenish methylation capacity when you are struck by allergies. Allergens can have a draining effect on the whole body, with low methylation capacity reducing our ability to maintain good health and can lead to low methylation conditions such as peptic ulcers and histamine intolerance.

Vitamin U is not a drug: it will not stop a runny nose dead in its tracks like antihistamines can. Nor will Vitamin U be effective in treating anaphylactic shock. If you have a severe allergic reaction, please immediately rush to the hospital for treatment. Vitamin U simply aids our body's natural mechanism for removing excess histamine. Ensuring your dietary intake of Vitamin U is adequate will complement drugs in your battle with seasonal and persistent allergies.

Further reading

• https://pubmed.ncbi.nlm.nih.gov/30744146/
• https://pubmed.ncbi.nlm.nih.gov/17490952/

Neural tube defects and Vitamin U

By Centers for Disease Control and Prevention - Centers for Disease Control and Prevention, Public Domain, https://commons.wikimedia.org/w/index.php?curid=30509337

 

Summary - Vitamin U may play a supportive role in correct neural tube formation, a process that depends on the methylation of dUMP to form dTMP via the folate cycle. The folate and methionine cycles work together to meet the methylation needs of the body. Low cellular methylation status diverts methyl groups from the folate cycle to the methionine cycle, thereby decreasing dTMP synthesis and increasing the risk of neural tube defects. Dietary methyl donors that enter the methionine cycle directly (e.g. methionine, betaine, Vitamin U) support neural tube formation by reducing this methyl group drain. While folate is the most important factor affecting neural tube formation, folate intake may not be sufficient in and of itself due to other factors such as polymorphisms within these two pathways and intake of other components that contribute to methylation homeostasis. Talk to your dietitian before conception about designing a diet that reduces the risk of neural tube defects in your baby.

Neural tube defects (NTDs) are a collection of medical conditions in which the neural tube of the baby does not form completely during early development, exposing the spinal cord and/or brain with permanent disability resulting. Two common neural tube defects are spina bifida (spine) and anencephaly (brain). 

What causes NTDs? Like many conditions, NTDs result from a combination of environmental and genetic factors. The most common environment factor is the insufficient intake of folate by the mother just before and during the first few weeks of pregnancy. Folate is a vitamin (B9), and is therefore an essential component of one's diet. In the 1950s it was noted that pregnant women taking anti-folate drugs to treat cancer gave birth to babies afflicted with congenital abnormalities like NTDs (Safi 2012). It was established that folate is essential for embryonic development, that women whose folate levels were low were at greater risk of having babies with NTDs, and that supplementation by the mother-to-be with folate substantially reduced the risk of NTD occurrence (Smithells 1980; Wald 1991). Folate supplementation is the most effective way to prevent NTDs, reducing risk by 50-70%. Consequently, medical authorities recommend young women ensure their diet contains 400 ug of folate per day through food and supplements in case they become pregnant.

How does folate prevent neural tube defects? The function of folate is to transfer methyl groups generated from serine to a range of molecules throughout the cell. In embryos, these methyl groups are essential to make nucleotides required for DNA synthesis. There are many compounds that are methylated as a result of the action of folate. However, there is one for which there is evidence that a shortage of results in neural tube defects - dTMP (deoxythymidylate). There are four nucleotides used to make DNA - abbreviated to A, T, C, G. dTMP is a precursor in the formation of dTTP, usually shortened to T. In humans, the nucleotide dTMP is made from dUMP by the transfer of a methyl group from folate. Embryos supplemented with dTTP do not develop neural tube defects despite very low folate levels, indicating that dTMP shortage is the causal factor (Leung 2013). At conception, nucleotides must be synthesized in utero, and therefore a shortage of folate results in low production of dTMP, which in turn results in NTDs.

Figure 1 - A simplified depiction of the folate cycle. The enzymes responsible for catalyzing each step are -

1. Serine hydroxymethyltransferase

2. Thymidylate synthase

3. Dihydrofolate reductase

4a. 5,10-methylenetetrahydrofolate dehydrogenase NADP+

4b. 5,10-methenyltetrahydrofolate cyclohydrolase

4c. Formate-tetrahydrofolate ligase

5. Phosphoribosylaminoimidazolecarboxamide formyltransferase

6. Methylenetetrahydrofolate reductase

7. Methionine synthase

N.B. 4a-c are three components of MTHFD1

Which folate should you take and where should you get it from? 

First a note on nomenclature. The term 'folate' is commonly used to refer to any of the components of this pathway plus other forms like folic acid and folinic acid. Naturally-occurring folate is a mixture of these forms, with the exception of folic acid, which is a non-natural, oxidized version that is relatively stable and therefore used to fortify food in which natural folate has been removed or degraded. Vitamin supplements usually contain either folic acid or 'methyl folate' or 'activated folate', which usually refers to 5-methyltetrahydrofolate.

The best source of natural folates are green leafy vegetables, although all vegetable sources (including fruit and grains) are reasonable sources. Folic acid is a form of folate that is often added to processed grains that have been polished, e.g. wheat flour, white rice. For most people, it makes little difference whether their folate is derived from natural or synthetic sources. The important factor is getting the right amount. However, some people do not metabolize synthetic folic acid effectively and as such can actually suffer from a deficiency in functional folate even when their serum levels of folic acid seem sufficient (Bailey and Ayling 2009). Sometimes too much folic acid can even cause fertility problems (Cornet 2019). 

There are other components in the folate cycle that also have an effect on neural tube formation, albeit to a lesser extent. For example, vitamin B12 is an essential cofactor for the enzymatic conversion of 5-MTHF to THF, and low B12 levels have been linked to NTDs (Li 2009). Another example is Vitamin B6, which is a coenzyme for serine hydroxymethylfolate transferase. Cobalt is a component of B12 and therefore is vital for methionine synthase function.

Folinic acid is given to people who have overdosed on methotrexate. Folinic acid (5-formyltetrahydrofolate; drug common name - leucovorin) is converted into 5,10-methenyltetrahydrofolate by MTHFS. It is given to patients who have overdosed with methotrexate because it enters the folate downstream of DHFR, thereby enabling the generation of tetrahydrofolate required to make thymine for DNA synthesis and new cell growth (thymineless death)

Where does genetics fit in? While folate supplementation reduces risk, unfortunately genetic polymorphisms in the mother are responsible for the majority of NTDs cases (Copp 2013). Mutations of genes in the folate cycle and other branches of one carbon metabolism (methylation) are particularly relevant. There are many enzymatic steps in the folate cycle, and each enzyme is encoded by a gene. Polymorphisms are nucleotide changes (mutations) in these genes that differ from that found in the majority of people. Most polymorphisms have no significant physiological effect by themselves, but may have an effect in combination. However, there are a few polymorphisms that do correlate with the occurrence of NTDs, such as MTHFR C677T and MTHFD1 R653Q (Copp 2013). 

Genetic testing is becoming an increasing-popular tool to identify polymorphisms. Though it is tempting to do so, it is important to not assume that polymorphisms necessarily result in reduced physiological function. Human physiology is not fully understood and often contains redundancies that can mask over minor metabolic blocks. The correct way to establish whether there are deficiencies within the folate cycle is through biochemical analysis of the various folate metabolites. Biochemical analysis in combination with genetic analysis is used by specialists to establish whether there is a functional deficit in the mother that results in heightened risk of NTDs in her baby. Consult with your doctor or dietitian to determine if you have polymorphisms, and if you do, whether these actually affect your metabolism (often they don't) and what measures can be taken to relieve any metabolic blocks.

Aside from the folate cycle, there is another cycle that is even more important in meeting our methylation needs. The methionine cycle is the way in which the methyl donor S-adenosylmethionine (SAM) is generated (methionine cycle). SAM is the methyl donor for just about all methylation reactions, with the notable exception of those in the folate cycle. It has been shown that a functioning methionine cycle is essential for correct neural tube formation (Leung 2017). When there is a shortage of methyl groups in the methionine cycle (low S-adenosylmethione:S-adenosylhomocysteine), methyl groups are directed from the folate cycle into the methionine cycle. Instead of being used to make nucleotides, 5,10-methylenetetrahydrofolate is reduced by MTHFR to make 5-methyltetrahydrofolate, which donates its methyl group to the methionine cycle in a reaction catalyzed by methionine synthase. The folate molecule is conserved within the folate cycle, but must be remethylated. When maternal folate levels are low and flux through the folate cycle is already slow, this shunt may reduce methylation capacity in the folate cycle to critical levels. 

The methionine cycle obtains a large amount of its methyl groups from the folate cycle. This shunt operates at moderate levels most of the time - this is actually a normal process. In addition, the methionine cycle obtains methyl groups from methionine, betaine and Vitamin U. Similar to the methionine synthase reaction, betaine and Vitamin U donate methyl groups to homocysteine via enzyme-catalyzed reactions. Importantly, methyl groups in the methionine cycle cannot enter the folate cycle. For example, SAM from the methionine cycle cannot substitute for 5,10-MeTHF required for dTMP synthesis. Low maternal methionine levels pre- and post-conception are associated with a heightened risk of neural tube defects (Shaw 2004). There is evidence that betaine entering the methionine cycle reduces the flow of methyl groups from the folate cycle, which in principle should support 5,10-MeTHF levels (Benevenga 2007).

Where does Vitamin U fit into all this? It should be emphasized that there has not been any scientific research testing whether Vitamin U supplementation can prevent NTDs. The studies have simply not been done. However, there is some genetic evidence that suggests that Vitamin U may play a role in correct neural tube formation. 

• Vitamin U supplies methyl groups to mammals via its reaction with homocysteine to form methionine catalyzed by the enzyme BHMT2. This is very similar to that of betaine, though whether Vitamin U plays this role in embryonic tissue has not been investigated. 

• Vitamin U is abundant in vegetables, the benefits of which have been long known in preventing neural tube defects. While the presence of folate is most likely the primary factor, it is possible that some of the benefits conferred by eating vegetables are due to the provision of methyl groups from Vitamin U.

• A preconception diet rich in methionine reduces the prevalence of neural tube defects. Vitamin U is essentially methionine with an extra methyl group. One molecule of Vitamin U actually supplies two molecules of methionine, one being the newly-methylated homocysteine, the other being the demethylated Vitamin U.

• Studies have shown that methylation in the embryo is supplied by methyl groups from both the folate cycle and betaine. If we assume that methionine synthase and BHMT1 contribute to embryonic methylation, then Vitamin U is also likely to make a contribution. It is logical that the benefits of green vegetables in preventing neurological abnormalities is due to the combined effects of folate, betaine and Vitamin U, with the emphasis on folate.

Summary

• Folate is absolutely necessary at some level to provide the embryo nucleotides during the first weeks following conception. It cannot be replaced by other molecules.
• Folate requirements may be lowered as long as adequate levels of methyl groups are provided by methionine and betaine from the methionine cycle. 
• Though its role in fetal development has not been investigated, it is likely that Vitamin U has a similar role to that of methionine and betaine, and would be of greater importance for people whose diet is low in protein and fat such as vegans.

Further Reading

• Shaw et al 2004 https://pubmed.ncbi.nlm.nih.gov/15234930/
• Zhang et al 2015 https://pubmed.ncbi.nlm.nih.gov/25466894/
• Froese et al 2019 https://pubmed.ncbi.nlm.nih.gov/30693532/
• Halsted et al 2002 https://pubmed.ncbi.nlm.nih.gov/12122204/
• Shin et al 2010 https://pubmed.ncbi.nlm.nih.gov/20220206/
• Bailey and Ayling 2009 https://pubmed.ncbi.nlm.nih.gov/19706381/
• Cornet et al 2019 https://pubmed.ncbi.nlm.nih.gov/31205715/
• Huennekens 1969 https://pubmed.ncbi.nlm.nih.gov/4891866/
• Purohit et al 2007 https://pubmed.ncbi.nlm.nih.gov/17616758/
• Duncan et al 2013 (1) https://pubmed.ncbi.nlm.nih.gov/23857220/ 
• Duncan et al 2013 (2) https://pubmed.ncbi.nlm.nih.gov/23143835/
• Teng et al 2012 https://pubmed.ncbi.nlm.nih.gov/23014492/
• Rinehart and Greenberg 1948 https://pubmed.ncbi.nlm.nih.gov/18859393/
• Leung 2013 https://pubmed.ncbi.nlm.nih.gov/23935126/
• Leung 2017 https://pubmed.ncbi.nlm.nih.gov/29141214/
• Blom 2006 https://pubmed.ncbi.nlm.nih.gov/16924261/
• Benevenga 2007 https://pubmed.ncbi.nlm.nih.gov/17413090/
• Bertolo and McBreairity 2013 https://pubmed.ncbi.nlm.nih.gov/23196816

Vitamin U is metabolized by the enzyme BHMT2

Summary - BHMT2 is the enzyme that catalyzes the assimilation of Vitamin U into our body. Made at high levels in the liver and kidneys, BHMT2 catalyzes the transfer of a methyl group from Vitamin U to homocysteine. This reaction plays an important role in the maintenance of healthy glutathione levels by contributing to an optimal methylation state, which drives existing and newly-formed homocysteine into the transsulfuration pathway and towards glutathione synthesis.

Vitamin U is a nutrient that is ubiquitous and abundant in vegetables and fruit. It is a noted dietary mucin secretagogue that has been shown to play an important role in healing and preventing peptic ulcers. It appears that Vitamin U interacts directly with the cells lining the stomach and induces secretion through a non-receptor mediated mechanism, different to that used by other secretagogues (more). 

Vitamin U doesn't just interact with the stomach. In fact, much of Vitamin U is absorbed by the small intestine and taken to the liver where it is metabolized. It is assimilated into our body via the methionine cycle (more). The enzyme that is responsible for Vitamin U assimilation is BHMT2 (Betaine Homocysteine Methyl Transferase 2). BHMT2 catalyzes the transfer of a methyl group from Vitamin U to homocysteine to produce two molecules of methionine. These products enter the methionine cycle where they donate methyl groups to form the universal methyl donor SAM and eventually are converted back into homocysteine. The fate of homocysteine is determined by the methylation status in the cell. A low SAM:SAH ratio results in homocysteine awaiting the appearance of new methyl donors in the form of Vitamin U, betaine and methyl folate for reentry into the methionine cycle. A high SAM:SAH ratio results in homocysteine entering the transsulfuration pathway, eventually forming cysteine and glutathione.

BHMT2 is well-expressed in the liver and kidneys (top 5% of proteins in these organs by abundance) (more) and is expressed at low levels in many other tissues throughout our body (more). 

High expression kidney, liver

Moderate expression thyroid, adrenal, pancreas, gallbladder, ovaries, rectum

Low expression nasopharynx, bronchus, stomach, duodenum, small intestine, colon, testis, epididymis, prostate, endometrium, fallopian tubes, heart muscle, skeletal muscle

Examples

BHMT2 is expressed at low, but measurable levels in the gastric glands. Not expressed in the gastric pits or muscle layer (more). 

BHMT2 is expressed at moderate levels in the thyroid gland (more).

BHMT2 is expressed at moderate levels in the mucosa lining the gallbladder (more).

BHMT2 is expressed at high levels in the renal tubules, but not in the renal glomeruli (more).

BHMT2 is expressed at high levels in hepatocytes, but not in bile duct cells (more).

To what extent these antibody stains indicate function of BHMT2 remains an open question. If the low expression of BHMT2 in the stomach is actually responsible for the protection afforded by Vitamin U, then the similar levels of expression in other tissues may indicate that Vitamin U has a physiological function in those tissues too. This remains to be scientifically investigated. 

How was BHMT2 discovered?

Around 1940, scientists at the Lankenau Hospital Research Institute in Philadelphia were investigating the effects of oxidation on the uptake and metabolism of proteins. Gerrit Toennies was a chemist focusing on making forms of methionine and cysteine that had undergone oxidation to varying degrees. Mary Bennett fed these oxidized amino acids to rats to further our understanding of how animals use sulfur amino acids. They found that when the milk protein casein was chemically oxidized, it was no longer a viable source of protein for rats. Most proteins are made up of 20 types of amino acids. The scientists discovered that methionine and tryptophan were the two types of amino acid that were irreversibly oxidized (more).

During these studies, Toennies made a methionine derivative that was a little different. By reacting methionine with methyl iodide, methionine was methylated at the sulfur again, converting the sulfur into a sulfonium. Bennett fed this methionine sulfonium to rats in place of methionine. For 5 days, the rats did not grow. On the 6th day, the rats suddenly started growing at the same rate as the control rats being fed methionine (more).

What happened to the rats? Bennett suggested that the rats "may have developed a special mechanism for taking care of the extra methyl group" that allowed the sulfonium to be converted into methionine. This special mechanism was probably an enzyme that at that time had yet to be discovered. In 1959, Shapiro and Yphantis revealed that the liver expresses an enzyme that converts Vitamin U into methionine by methyl transfer to homocysteine (more). The gene encoding this activity was characterized in 2000 by Chadwick and named BHMT2 due to its resemblance to another liver enzyme BHMT1 (more). It was shown by the Garrow lab in 2008 that purified BHMT2 enzyme catalyzed the reaction between Vitamin U and homocysteine (more). 

It was hypothesized by Toennies that sulfoniums could have a biological role and it was speculated that methylmethionine sulfonium could exist naturally. In 1954, this hypothesis was confirmed by McRorie and others, extracting this compound from cabbage. Interestingly, the latter linked the chemical properties of their newly-discovered compound with those of a recently-discovered Vitamin U, and proposed that this compound was a source of methionine as well as a methyl donor (more). 

Does expression of BHMT2 depend on the presence of Vitamin U? From the studies of Bennett, it would seem that a lack of methionine might induce BHMT2 expression in rats rather than the presence of Vitamin U. For comparison, expression of the related enzyme BHMT1 in rat liver is induced 4-fold when methionine levels are low/choline normal and an additional 2-fold in the presence of betaine (more). However, not much research has gone into addressing BHMT2 expression, especially in humans. 

The methionine cycle and Vitamin U

Summary - The methionine cycle is a multistep enzymatic process than enables Vitamin U to be used as a source of methyl groups vital for gene regulation and the regeneration of creatine/ATP in muscles, as well as its use as a precursor of glutathione required to fight oxidative stress and inflammation.

This is a simple depiction of the four-step methionine cycle in our body. In the first step, the adenosyl group of ATP is transferred to methionine to form S-adenosylmethionine (SAM), thereby activating the methyl group of methionine. In the second step, SAM donates its methyl group to a range of acceptor molecules (notably DNA, guanidinoacetate, and phosphatidylethanolamines), also yielding S-adenosylhomocysteine (SAH). In the third step, the adenosyl group SAH is removed by hydrolysis leaving homocysteine. In the fourth step, homocysteine is either remethylated using one of three methyl donors to reform methionine (step 4a) or is directed into the transsulfuration pathway to form cystathionine (step 4b).

The methionine cycle has a myriad of functions including -

1. The generation of methylation capacity, 

2. The biosynthesis of cysteine as a component of proteins and glutathione, and as a precursor to taurine and hydrogen sulfide, 

3. The biosynthesis of polyamines from SAM. 

The most important function of the methionine cycle is to generate methylation capacity. A measure of our body's methylation capacity is the SAM:SAH ratio, i.e. the relative amounts of the two intermediates. If this ratio is low (below 4), the first enzyme in transsulfuration (cystathionine beta synthase) will have low activity and homocysteine will be remethylated to reenter the methionine cycle (step 4a). This tendency will continue until the ratio is above 4, at which point the relatively high concentration of SAM activates cystathionine beta synthase (CBS) resulting in excess homocysteine being funneled into the transsulfuration pathway. 

Another important function of the methionine cycle is the biosynthesis of cysteine via transsulfuration. Transsulfuration adds cysteine to that obtained from our diet as a component of protein (~50%). Cysteine is used as a building block in human proteins, is the catalytic center of the master antioxidant glutathione as well as acting as a precursor to molecules such as taurine and hydrogen sulfide. Increased oxidative stress will result in activation of CBS activity via allosteric binding by glutathione and transcriptional upregulation by hydrogen sulfide, nitric oxide and carbon monoxide. However, despite the negative health effects of high homocysteine levels (associated with cardiovascular disease) and low glutathione levels (associated with inflammation), the maintenance of methylation capacity trumps that of the provision of transsulfuration products.

The most common cause of a low SAM:SAH is a shortfall in the supply of dietary methyl donors. Other causes of low flux include shortages in vitamins that help catalyze reactions (e.g, folate, B12, B6), mutations in genes that encode enzymes involved in catalysis (e.g. MTHFR, CBS), and very low calorie diets.

There are several nutrients that can contribute methyl groups to the methionine cycle. Aside from methionine, which enters the methionine cycle directly, the other dietary methyl donors enter the methionine cycle via methylation of homocysteine. There are three enzymes known to catalyze this reaction in humans, with each enzyme acting upon a single methyl donor molecule. Other molecules that can contribute methyl groups must do so indirectly. Consequently, the three classes of dietary methyl donor are characterized by the enzyme that catalyzes the reaction with homocysteine and its substrate -

1. Betaine:homocysteine methyltransferase 1 (BHMT1) and betaine (trimethylglycine or TMG)
2. Methionine synthase (MS) and folate (5'-methyltetrahydrofolate or MTHF)

3. Betaine:homocysteine methyltransferase 2 (BHMT2) and Vitamin U (S-methylmethionine)

Betaine (trimethyl glycine) has three methyl groups, one of which is transferred to homocysteine to form methionine and dimethyl glycine. The other two methyl groups contribute to methylation, though via assimilation through the folate cycle. Dimethyl glycine dehydrogenase catalyzes the transfer of a methyl group from dimethyl glycine to tetrahydrofolate to produce 5, 10-methylenetetrahydrofolate. The other product, methyl glycine (sarcosine) yields the last methyl group to tetrahydrofolate in a similar reaction catalyzed by the homologue sarcosine dehydrogenase.

Betaine is plentiful in whole grains, with the notable exception of rice (betaine is an osmoprotectant in plants and it appears that under the wet conditions in which rice is usually grown betaine formation is suppressed). Betaine is also produced in our body from choline, which is abundant in the fatty component of food as phosphatidylcholine. Consequently, food with more naturally occurring fat such as meat, eggs, dairy and nuts are the richest sources of choline, with produce and grains contributing a lesser amount.

The active form of folate (Vitamin B9) is 5'-methyltetrahydrofolate, which supplies a methyl group to homocysteine to yield methionine and tetrahydrofolate. Once folate has donated its methyl group, it must be remethylated in the folate cycle to be reused. The primary source of these methyl groups is serine. Contrary to popular belief, folate itself is a minor dietary source of methyl groups. Even taking supplements labelled "methyl folate" or "activated folate" or eating green leafy vegetables provides minimal extra methylation substrate. With regards to its role in methylation, folate is better thought of as a carrier molecule analagous to homocysteine rather than as a methyl source.

Most methionine in our diet is found as a component of protein, which requires extensive digestion by a slew of enzymes to release methionine as an amino acid before it can enter the methionine cycle. Vitamin U (S-methylmethionine) is methionine with an extra methyl group, although unlike methionine, Vitamin U is rarely a component of proteins. It supplies a methyl group to homocysteine yielding two molecules of methionine. Vitamin U is abundant in vegetables and fruits, especially cruciferous (e.g. cabbage, kale) and stalky (e.g. celery, asparagus) vegetables.

The degree to which these methyl donors contribute to the methionine cycle is dependent upon our diet. In a diet rich in protein and fats, methionine and choline will be major sources. In a diet in which more calories are gleaned from whole grains, betaine will make a greater contribution. Folate and Vitamin U will make larger contributions in diets rich in fresh produce.

References

1. Finkelstein (1990) https://pubmed.ncbi.nlm.nih.gov/15539209/
2. Mudd and Poole (1975) https://pubmed.ncbi.nlm.nih.gov/1128236/
3. Benevenga (2007) https://pubmed.ncbi.nlm.nih.gov/17413090/
4. Bertolo and McBrearity (2013) https://pubmed.ncbi.nlm.nih.gov/23196816/
5. Olszewski (1989) https://pubmed.ncbi.nlm.nih.gov/2930611/
6. Reed (2008) https://pubmed.ncbi.nlm.nih.gov/18442411/
   
7. Filipcev (2018) https://pubmed.ncbi.nlm.nih.gov/29596314/
8. Dai (2020) https://pubmed.ncbi.nlm.nih.gov/32503840/

Cabbage Juice Heals Peptic Ulcers

In the 1940s and 1950s, Dr Garnett Cheney from Stanford discovered that having his peptic ulcer patients drink one liter of fresh cabbage juice daily healed their stomach and duodenal ulcers 3-6 times faster than a bland diet alone (Cheney 1949; Cheney 1952; Cheney 1956). Chronic ulcers disappeared in 1-2 weeks depending on their severity. Dr Cheney was of the opinion that peptic ulcers resulted from a deficiency of a nutritional factor he termed Vitamin U, which was later identified as the amino acid S-methylmethionine, a plant product. While cabbages are a particularly rich source of Vitamin U, Cheney found that all raw plant products contain some Vitamin U. In addition to healing existing ulcers, Dr Cheney found that Vitamin U also prevented the formation of new ulcers.

How do ulcers form? Ulcers result from an imbalance in the digestive system between protective and destructive factors. An alkaline bilayer of mucus containing mucin protects the wall of the digestive tract from harsh elements such as stomach acid, infection by bacteria living in the digestive system, NSAIDs, and dietary factors like high salt and alcohol. In modern times, NSAIDs increasingly contribute to ulcer formation by inhibiting the mucus-stimulating function of our body's prostaglandins. When the mucus layer is depleted, these harsh elements irritate the epithelial cells lining the digestive tract causing inflammation and enabling deep infection. 

Ulcers are usually treated with combinations of proton pump inhibitors or H2 blockers that reduce acid production, antibiotics to treat bacterial infections (particularly Helicobacter pylori in the stomach), antacids to neutralize acid, and mucosal protectants such as prostaglandin mimics. Unfortunately, these only provide a temporary solution to the problem. Ulcers return soon after cessation of treatment. Excessive stomach acid is usually not the root problem, nor is H. pylori infection. Most people with ulcers produce a normal amount of stomach acid, and half the world's population has H. pylori yet remain ulcer-free. These facts indicate that while acid and infection contribute to the formation of ulcers, other factors are at play.

Given the findings of Dr Cheney, it's likely that eating a diet rich in Vitamin U promotes good gastrointestinal health. But how does Vitamin U work? In later studies, it was shown that Vitamin U has four properties that help maintain a healthy gut.

1. Stimulating the release of mucin into the mucus layer, thereby protecting the walls from acid and bacterial infection (Watanabe 1996; Watanabe 2000; Salim 1991).
2. Reducing inflammation by acting as a precursor to glutathione, the master antioxidant of the human body, via its conversion to cysteine (Szegedi 2008).
3. Coordinating with other nutrients such as methionine, folate, B12, betaine, choline, SAMe and B6 to supply vital methyl groups required for optimal health (Suzue 1967).

Increasing the Vitamin U content of one's diet in combination with reducing the intake of elements that deplete the protective mucus layer has been shown to improve ulcerative conditions in the digestive system. A diet rich in fresh vegetables, vegetable juice and fruit, and low in salt, alcohol and sugars is a good approach for restoring the mucus bilayer in many people.