Many amino acids are important not only for the synthesis of proteins but also as metabolites for critical metabolic reactions. Some of these ‘nonprotein’ roles of amino acids can utilize a significant proportion of the total amino acid pool. For indispensable amino acids, these alternative metabolic pathways must be carefully considered when establishing the dietary requirement of that amino acid. The upregulation or downregulation of these pathways can tax or spare the amino acid requirement, respectively, depending on the pathway in question and the availability of alternate metabolites. For example, methionine is an essential amino acid that is necessary for the synthesis of cysteine and taurine as well as for the supply of methyl groups. These methyl groups are needed, inter alia, for the synthesis of creatine, phosphatidylcholine and carnitine and for regulating gene expression via cytosine and histone methylation. Methyl groups for remethylation to methionine can be provided by methyl donors such as choline and betaine and by methylneogenesis (via folate). In adult humans, the major dietary sources of labile methyl groups are methionine (∼10 mmol methyl per day) , choline (∼30 mmol methyl per day) , betaine (∼26−75 mmol methyl per day) , and methylneogenesis via folate (∼5−10 mmol methyl per day) . Because folate, choline and methionine are all essential nutrients, the dietary availability of folate, choline and betaine can obviously influence the amount of methionine needed. Moreover, dietary supply of methylated products such as creatine, carnitine and phosphatidylcholine could reduce the need for methyl groups and further spare methionine requirement. In growing animals, this balance becomes more precarious as growth requires even more amino acids for expansion of body protein mass as well as for the increased need for methyl groups for critical reactions.
One key question remains unanswered: What is the impact of the supply of methyl groups on methylation reactions? The metabolic deficiency of methyl groups must have profound effects throughout the body given the scope of methylation reactions. So if folate, betaine and/or choline are deficient in the diet, then presumably more methionine is required to maintain the critical methylation reactions and, consequently, less methionine is available for protein synthesis and growth. Conversely, if these methyl nutrients are in excess, then less methionine is required and folate, betaine and/or choline can spare methionine and perhaps enhance growth. With respect to DNA methylation, methyl group supply can have permanent effects on gene expression, which can have broad effects on whole body metabolism and even lead to chronic diseases in adulthood . Because most infants are exposed to dietary extremes of methyl-related nutrients, elucidation of the transmethylation pathways and the effects of dietary methyl supply in neonates will not only optimize infant health, but also adult health.
In Fig. 1, the pathways for sulfur amino acid metabolism are presented and can be summarized by transmethylation (methionine to homocysteine), transsulfuration (homocysteine to cysteine) and remethylation (homocysteine to methionine). Briefly, methionine is adenylated to S-adenosylmethionine (SAM), which is the methyl donor in over 50 different reactions including DNA methylation, creatine synthesis and phosphatidylcholine synthesis. The end-products are the methylated compound and S-adenosylhomocysteine (SAH), which is converted to homocysteine. Homocysteine can either be irreversibly catabolized to cysteine via the transsulfuration pathway or remethylated to methionine.
Remethylation of homocysteine to methionine occurs either via cobalamin-dependent methionine synthase and folate or via an equally important route, betaine–homocysteine methyltransferase (BHMT), which uses betaine, a product of choline oxidation, as the methyl donor (Fig. 1). Importantly, during choline or betaine deprivation, more folate is required to facilitate remethylation, thereby increasing folate requirements and, conversely, folate deficiency leads to increased use of betaine for methyl groups, thereby increasing choline requirements . N,N-Dimethylglycine (DMG) is formed from betaine (N,N,N-trimethylglycine) by demethylation via BHMT. Betaine demethylation is the only known source of DMG in mammals, so the presence of DMG is a direct result of methylation of homocysteine to methionine . DMG is further demethylated to sarcosine (N-methylglycine) via the flavoprotein dimethylglycine dehydrogenase, which converts the methyl group into 5,10-methylene tetrahydrofolate . Sarcosine can also be demethylated to glycine by mitochondrial sarcosine dehydrogenase. Because sarcosine is also a product of transmethylation via glycine N-methyltransferase (GNMT), it is not an unambiguous marker of its demethylation pathway. Folate-dependent remethylation via methionine synthase derives its methyl group primarily via the glycine cleavage system and serine hydroxymethyltransferase (SHMT), both of which require vitamin B6 as coenzyme, to generate 5,10-methylene tetrahydrofolate [5▪,6]. However, it should be noted that 5,10-methylene tetrahydrofolate is also derived from the catabolism of DMG and sarcosine  (Fig. 1).
The regulation of the methionine pathways is complex. When cysteine is unavailable, transmethylation and transsulfuration are increased to oxidize methionine and synthesize cysteine. Similarly, when methionine is in excess, transmethylation and transsulfuration are upregulated to eliminate methionine, which is toxic at relatively low concentrations. When transmethylation is upregulated, all extra methyl groups are eliminated by GNMT (Fig. 1) . Indeed, elevated methionine leads to increased expression of GNMT , which consumes excess SAM and still conserves the carbon skeleton in homocysteine, which can be remethylated. When methionine is undersupplied, then transsulfuration is downregulated and remethylation is upregulated to regenerate methionine. The rate-limiting enzymes for transmethylation, remethylation and transsulfuration are primarily regulated by SAM [8,10]. The partitioning of transmethylation, remethylation and transsulfuration during various conditions has been studied in various in-vitro systems and less so, in vivo, but the partitioning of methyl groups within transmethylation has not been studied extensively. An unanswered question is which methylation reactions have priority when methyl groups are scarce?
The nonprotein requirement for methionine can be viewed as the transmethylation requirement of the body; if cysteine intake is adequate, then the requirement for methyl groups will drive the nonprotein requirement for methionine. There are more than 50 methylation reactions but quantitatively, the most important enzymes are phosphatidylethanolamine methyltransferase (PEMT), which synthesizes about one-third  of endogenous phosphatidylcholine; guanidinoacetate methyltransferase (GAMT), which synthesizes creatine; and GNMT, which serves as an overflow pathway for methionine catabolism. GAMT has a very significant methyl requirement, especially in neonates who are rapidly expanding their lean tissue mass and associated creatine pool. Indeed, we have recently estimated that the suckling piglet accrues creatine at a rate three times higher than sow milk can provide and this endogenous creatine synthesis rate would consume up to one-third of dietary methionine . Obviously, creatine synthesis alone requires either a large proportion of dietary methionine or, considering sow milk contains negligible betaine (unpublished data), a very high rate of methylneogenesis and/or choline oxidation. Put in perspective, creatine synthesis alone obliges a transmethylation rate of 0.64 mmol/kg per day in the piglet  compared with the total transmethylation rate (for all transmethylation reactions) of approximately 0.85 mmol/kg per day in postweaned piglets  and 0.17 mmol/kg per day in adult humans . Moreover, methyl demand for phosphatidylcholine synthesis via PEMT (which uses three methyl groups per phosphatidylcholine synthesized) could be even more quantitatively important than that for creatine synthesis , especially when one considers the extremely high phosphatidylcholine needs of a growing neonate . With respect to infant nutrition, if an infant is fed cow milk or soy formula, choline or creatine, respectively, can be inadequate . And although the quantitative importance of DNA methylation has not been estimated, its developmental importance and sensitivity to dietary manipulation  makes this methylation pathway perhaps the most important of all, with respect to the long-term health of the organism.
These pathways all share the same need for a common substrate: methyl groups. During growth and development, all of these pathways are highly active not only for maintaining basic functions but also for expanding the pools of their respective products to meet the demands for growth. We recently demonstrated competition among these pathways for a limited methyl supply . We infused guanidinoacetate (GAA) intraportally in 15−18-day-old piglets to drive creatine synthesis and consume hepatic methyl groups (unpublished). Compared with a saline infusion, GAA infusion doubled the methyl incorporation rate into creatine, but decreased methyl incorporation into phosphatidylcholine by 85% and reduced protein synthesis by 40% (Fig. 2). These data suggest that methyl groups were limited for phosphatidylcholine synthesis and that methionine was diverted from protein synthesis. GAA infusion did not affect methyl incorporation into DNA. The partitioning of available methionine between its primary maintenance role as a methyl donor and its role in growth via protein synthesis has important implications when determining amino acid recommendations for neonates. Moreover, this study demonstrated that methyl supply can be limited in the neonate and that this leads to a repartitioning of methyl groups among methylation reactions.
Diet composition will also affect the relative requirements of these pathways, especially early in life. For example, a creatine-free diet (i.e. soy-based formula or vegan diet) will require the synthesis of the entire requirement for creatine, both for maintenance and for growth. Similarly, some bovine milk-based and soy-based formulas have little choline compared with breast-milk and plasma choline concentrations in formula-fed neonates are half that of breast-fed infants . Moreover, maternal choline status modifies choline in milk, which in turn reflects choline status of infants . Diets low in choline will determine the activity of PEMT. Moreover, a casein-based formula has much more methionine (and low cysteine) than a whey-based formula or breast milk. But ultimately, these pathways depend on the methyl supply, both from the diet and from de-novo synthesis. Choline, betaine, methionine and folate-dependent methylneogenesis are responsible for supplying methyl groups and their interdependency requires further research. If one or two of these nutrients are limiting, then the other(s) must compensate to maintain supply. It is imperative that the requirement for methionine accommodates its flux not only to protein and cysteine but also to transmethylation pathways, fully considering its remethylation back to methionine.
In particular, little attention is paid to methylation products and their influence on methionine requirements. Indeed, studies to determine methionine requirements in adult humans typically control for methylation precursors (e.g. B vitamins, choline) , but do not consider the dietary content of methylation products (e.g. creatine). Because such studies almost invariably employ protein-free, crystalline amino acid based diets, it is likely these diets are very low in creatine, which would require more methionine. Ball et al. have defined the minimum obligatory methionine requirement as ‘the dietary intake of methionine that cannot be replaced by cysteine and that will not be reduced by addition of any methyl donor, cofactor or any other metabolite’. Thus, if the inclusion of creatine in the diet spares methionine, then current methionine requirement estimates that were determined using creatine-poor test diets are likely to be overestimated.
CAN METHYL DONORS SPARE METHIONINE?
There has been significant research exploring the interdependency between methionine and cysteine, both in adults and during growth . If cysteine is not supplied in the diet, then one needs twice as much methionine; in other words, dietary cysteine can ‘spare’ about half of the methionine requirement. If cysteine is supplied in excess, then the methionine requirement can be reduced to that needed for protein synthesis and for methylation reactions. The concept of alternate dietary methyl ‘donors’ (i.e. betaine, choline, methylneogenesis precursors) sparing the methionine requirement has recently been discussed , but few studies have addressed this concept directly.
Because folic acid , betaine [23–26] and choline  can effectively lower plasma homocysteine in humans by increasing remethylation to methionine, these nutrients are very likely to be capable of sparing the methionine requirement. Indeed, in a recent, elegant isotope study, folic acid and choline doubled remethylation to methionine when methionine was limiting [28▪▪]. However, it should be noted that supplemental folic acid, in addition to vitamin B6 and cyanocobalamin, are not providing methyl groups per se, but rather the cofactors for remethylation. In this way, the requirement for these vitamins must be met, but beyond that, these vitamins cannot spare the methionine requirement. Indeed, it is well described that the lowering effect of B vitamin supplementation on plasma homocysteine is not graded and a maximum reduction is reached, beyond which additional supplementation is not effective . Furthermore, it has recently been shown that in spite of folic acid's homocysteine-lowering effects, there appears to be no beneficial effect on adverse cardiovascular outcomes [30,31▪], unlike for betaine [32▪]. Although the inadequacy of these vitamins in some populations can affect the ability to process methyl supply , these B vitamins are generally adequate in Western diets  and likely do not affect the supply of methyl groups. However, caution should be used when associating low vitamin status and plasma homocysteine, as Lamers et al.[34▪] have recently shown that rates of remethylation, transsulfuration, transmethylation and DNA methylation are unaffected by dietary vitamin B6 restriction in healthy adults, in spite of an accentuated postprandial rise of plasma homocysteine. However, estimates of remethylation, transsulfuration and transmethylation using isotopes also need to be interpreted with caution . Given the growing interest in the role of these vitamins in methyl metabolism, future experiments will need to address the potential sparing effects of these cofactors on methionine requirements, especially in populations with vitamin inadequacy.
It has been suggested that perhaps choline, betaine or other one-carbon donors such as DMG, sarcosine, serine or glycine, which are precursors of methyl groups via 5-methyl-tetrahydrofolate, should be considered in addition to vitamin B supplementation . Most foods contain some choline, choline esters and betaine  and with a healthy diet, most of the body's betaine needs can likely be met by choline oxidation . However, most people do not consume adequate choline and the mean intake is 40% below the recommended adequate intake . Moreover, intakes of betaine and choline from food in adults can predict both fasting and postmethionine-load homocysteine concentrations, especially in individuals with low folate and cobalamin status [38,39]. This association is supported by other trials that have shown that supplementation with choline or betaine can decrease plasma homocysteine concentrations in humans [23–26]. It is perhaps surprising then that most published reviews on homocysteine lowering in humans focused more on vitamin B supplements rather than methyl supply. It is likely that a metabolic deficiency of methyl groups might be responsible for some hyperhomocysteinemia .
But the question still remains as to whether methyl donors can effectively ‘spare’ the methionine requirement. In food animals, this concept has been explored by animal scientists primarily interested in how methionine requirement can be reduced by less costly supplements of methyl-containing choline and/or betaine. Numerous studies have demonstrated that choline and/or betaine can spare the methionine requirement in growing chickens [40,41] and young pigs [42,43], not only for the synthesis of protein but also for creatine and carnitine synthesis . The fact that these studies were able to show a sparing effect of methyl groups on the methionine requirement demonstrates the enormous demands of these pathways. Although data in humans are not available, it is likely a similar effect occurs in infants and children as well as adults. So, the actual choline and betaine content of the diet will have a bearing on the methionine requirement for growth and nongrowth functions.
An interesting aspect of betaine metabolism is that it can potentially provide a methyl group for both remethylation pathways. Demethylation of betaine via BHMT results in DMG, which in turn is sequentially demethylated to sarcosine and then to glycine (Fig. 1). Both DMG and sarcosine can be used to synthesize methylene tetrahydrofolate, thereby providing a methyl group for methylneogenesis via the methionine synthase pathway [4,7]. To my knowledge, the extent to which each of these three precursors is used for methylneogenesis is unknown. Interestingly, DMG has recently been approved for use in agriculture as a feed supplement and has been shown to improve feed digestibility and utilization in poultry [44,45] and swine . The role of methyl metabolism in these outcomes has not been investigated, but considering DMG is also known to inhibit BHMT activity in rats [4,47], its supplementation would have significant effects on methyl metabolism. In chickens, betaine or choline supplementation increases folate-dependent remethylation, and not BHMT remethylation, suggesting a role for DMG in methionine synthase remethylation . Clearly, the role of betaine as a precursor for the two remethylation pathways warrants further research.
With respect to human health and development, we need to understand more than just the effects of methyl donors on growth; we need a more detailed exploration of the nongrowth requirements for optimal health. For example, if methyl groups are limiting for DNA methylation, this can have implications for chronic disease development in adult life. In addition, the variable diet of human neonates, most of whom are not breast-fed exclusively (unlike other mammals), necessitates a clear understanding of the roles of excesses and deficiencies of methionine, choline, betaine and creatine. Furthermore, how the body partitions methionine when limiting amounts are available needs to be elucidated. Is protein synthesis and growth sacrificed to maintain essential methylation reactions? Which of these methylation reactions are most important? It seems inconceivable that DNA methylation, a presumably minor pathway from a quantitative point of view, would be changed by dietary methyl group availability; yet, recent studies have demonstrated exactly this in several species and conditions [48,49,50▪]. The impact of methylation reactions on the methionine requirement during neonatal development is important not only to simply define nutritional requirements in infants but also in light of the emerging evidence of a link between methyl metabolism and the developmental origins of adult disease.
The author acknowledges funding from the Canada Research Chairs Program, Canada Foundation for Innovation, Canadian Institutes of Health Research, Province of Newfoundland and Labrador and Natural Sciences and Engineering Research Council of Canada.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 117).
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This elegant isotope study quantifies methionine cycle fluxes in the pregnant rat and fetus. It clearly demonstrated the role of methyl precursors on remethylation flux when methionine is limiting.
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A thorough review of the literature demonstrating that folic acid supplementation is not effective at attenuating cardiovascular outcomes, in spite of effects on hyperhomocysteinemia.
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This whole-body in-vivo study demonstrated that vitamin B6 is not critical for maintaining the methionine cycle and supports the idea that remethylation via BHMT can compensate for methionine synthase downregulation.
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50▪. Cordero P, Gomez-Uriz AM, Campion J, et al.
Dietary supplementation with methyl donors reduces fatty liver and modifies the fatty acid synthase DNA methylation profile in rats fed an obesogenic diet. Genes Nutr 2012. doi: 10.1007/s12263-012-0300-z [Epub ahead of print].
This study demonstrated that dietary methyl supply can alter gene-specific epigenetic profile and phenotype, even when supplemented in adulthood.