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1 From the Departments of Animal Sciences and Nutritional Sciences, University of Wisconsin–Madison, Madison, WI
2 Reprints not available. Address correspondence to NJ Benevenga, Department of Animal Sciences, University of Wisconsin–Madison, 1675 Observatory Drive, Madison, WI 53706-1284. E-mail: njbeneve{at}ansci.wisc.edu.
ABSTRACT
A major focus in attempts to ameliorate homocystinuria and neural tube defects is supplementation of the diet with B vitamins. The metabolic defect in these cases may be due in part to a deficiency of methyl groups. B vitamin supplementation supports the need for enzyme cofactors but cannot provide substrate in the form of methyl groups. L-Methionine is an essential amino acid and is required for protein synthesis, but it also plays a unique role in metabolism as S-adenosylmethionine, which is the primary methyl donor in metabolism. The observation that L-homocysteine, which is produced in the metabolism of L-methionine, is remethylated 2–4 times before it is destroyed is key to understanding the possibility of a methyl group deficiency. This suggests that the requirement for methyl groups (ie, S-adenosylmethionine) may be 2–4 times that for methionine in support of protein synthesis. L-Homocysteine can be remethylated to form L-methionine by betaine or N5-methyltetrahydrofolate. Betaine and one-carbon sources that lead to the production of N5-methyltetrahydrofolate and the remethylation of L-homocysteine to form L-methionine should be considered along with B vitamin supplementation in the treatment of homocystinuria and neural tube defects.
Key Words: Betaine • methyltetrahydrofolate • homocystinuria • neural tube defects
INTRODUCTION
The negative effects of homocystinuria and neural tube defects (NTDs) may be lessened by supplements of preformed methyl groups from betaine or enhanced methyl group production in the form of N5-methyltetrahydrofolate (N5-MTHF) obtained from the folate system. Some reports concerned with homocystinuria still focus on B vitamin supplementation alone (1, 2). Vitamins can provide the enzyme cofactors that are essential for maintenance of substrate flux but not the methyl group substrate that can aid in the ultimate removal of homocysteine. Elevated plasma L-homocysteine is thought to be a causal agent for arteriosclerosis and related problems. Folic acid is the dietary supplement suggested for lessening the incidence of NTDs.
Some unique aspects of the metabolism of L-methionine must be considered before specific dietary supplements are suggested. The 2 known pathways for catabolism of methionine (ie, transsulfuration and transamination) are shown in Figure 1. Also shown are the 2 routes by which L-homocysteine can be remethylated to form L-methionine. One source of preformed methyl groups is betaine; the other is N5-MTHF. The unique metabolites produced in the metabolism of methionine by the transamination pathway (ie, -keto--methiolbutyrate, 3-methylthiopropionate, and methanethiol) are shown.
FIGURE 1.. Potential pathways of methionine catabolism showing the familiar transsulfuration pathway on the right and the transamination pathway on the left. Structures of some of the intermediates in the transamination pathway are shown. The 2 pathways for remethylation of L-homocysteine to form L-methionine by using betaine or N5-methyltetrahydrofolate are also shown. TCA, tricarboxylic acid cycle.
Studies of L-methionine metabolism in humans reported by Mudd and Poole (3) and Mudd et al (4) provided tentative estimates of rates of utilization of S-adenosylmethionine (AdoMet) by humans. These estimates were thought to be minimal because it was not certain that all reactions consuming AdoMet had been taken into account, and because, despite studies using a patient with a partial defect in sarcosine catabolism, it was not possible to make a definite estimate of the rate of choline biosynthesis. The advantage in using this patient was the patient's near-total urinary excretion of a sarcosine load, which suggested a similar total excretion of metabolically formed sarcosine.
The first and most important observation made by Mudd et al that, in the metabolism of L-methionine, on average, L-homocysteine is remethylated between 2 and 4 times before it is irreversibly removed by formation of cystathionine (see Figure 1). The degree to which L-homocysteine is remethylated appears to be dependent on the ingestion of dietary L-methionine. Consumption of "normal" amounts of L-methionine (ie, 7.1 mmol/d) resulted in the remethylation of L-homocysteine on average 2 times. Limiting consumption of L-methionine to approximately one-half of the "normal" amounts (ie, 3.3 mmol/d) resulted in a doubling of L-homocysteine remethylation to 4 times. Raising dietary L-methionine intake to 17.2 mmol/d resulted in halving L-homocysteine remethylation to 1 time. Others have used tracer methods in human volunteers to assess the remethylation of L-homocysteine in response to supplemental betaine—a provider of methyl groups through the action of betaine-homocysteine methyltransferase (EC 2.1.1.5) and one-carbon units via the folate system (5)—or with adequate L-methionine or limited L-methionine with added cysteine (6). Results of these tracer method studies are difficult to interpret because the betaine effect was not consistent and because limiting L-methionine consumption to 20% with added cysteine resulted in a calculated reduction of remethylation to one-half, which would be unexpected on the basis of reports by Mudd and various coauthors. The reports of Mudd and Poole (3) and Mudd et al (4) and findings by others (7-9) in patients with genetically determined defects in the tetrahydrofolate-dependent methylation of L-homocysteine make it clear that remethylation of L-homocysteine is a normal and variable feature of L-methionine metabolism. That methyl flux through AdoMet may exceed 4 times the dietary L-methionine intake suggests that the formation of AdoMet plays a critical role in the overall metabolism of L-methionine. Betaine and the folate one-carbon system are the only sources of the methyl groups used in the reformation of L-methionine and thus AdoMet, which is the primary methyl donor in metabolism.
One can create a competition for one-carbon sources and thus develop a methyl deficiency in chickens by feeding a high-protein or high-amino-acid diet (10-12). Because nitrogen excretion in the chicken is via uric acid and because uric acid biosynthesis requires 2 folate one-carbon intermediates (10-formyl-tetrahydrofolate and N5-N10methenyltetrahydrofolate) for its biosynthesis, a competition for folate one-carbon intermediates can result in a deficiency of methyl carbons. Feeding twice the required amount of amino acid increased the choline (a methyl donor via betaine) requirement by 34% (10). Increasing dietary protein from 13% to 64% tripled the choline requirement (11). The growth depression caused by increasing soy from 25% to 50% of the diet was reversed by adding choline, and the growth depression caused by adding 15% L-glutamic acid to a 25% soy diet was reversed by adding betaine (12). In general, a metabolic deficiency of methyl groups would be expected to result in serious complications. A decrease in the remethylation of L-homocysteine to form L-methionine would reduce the metabolic availability of L-methionine and other essential metabolites, such as AdoMet, spermidine and spermine, choline, carnitine, creatine, and methylhistidine and would be expected to result in undermethylated or abnormally methylated DNA. A metabolic methyl deficiency can lead to liver cancer, possibly due to undermethylation, abnormal DNA methylation, or both (13, 14).
It seems that a dietary supplement of methyl groups should be considered, because supplemental folate, vitamin B-6, and cyanocobalamin provide cofactors for metabolism and not the one-carbon substrates that are needed. Published reviews focused more on B vitamin supplements and did not consider the possibility of a metabolic deficiency of methyl groups. A review of homocysteine metabolism (2) focused on the role of the B vitamins in the metabolism of homocsyteine in homocystinemia and homocystinuria. In another review, the potential beneficial effect of betaine supplementation in the treatment of homocystinuria was not covered (15). Another review of sulfur amino acid metabolism covered many aspects of L-methionine and cysteine metabolism but did not address the potential role of AdoMet-independent pathways (ie, transamination; see Figure 1) of L-methionine catabolism (16). A pathway for L-methionine catabolism that does not reform L-homocysteine is a crucial element in the role of betaine therapy for homocystinemia and homocystinuria that are due to a cystathionine ß-synthase (EC 4.2.1.22) deficiency (17). For betaine to have a long-term effect of lowering blood L-homocysteine concentrations in patients with a cystathionine ß-synthase deficiency, a route or routes of L-methionine catabolism other than the transsulfuration pathway must be available. In these patients, metabolism of L-methionine by the transsulfuration pathway results in the reformation of L-homocysteine and not in its removal. An alternative pathway for L-methionine catabolism involving transamination was proposed by Steele and Benevenga (17). In the metabolism of L-methionine by the transaminative pathway (see Figure 1 in 17), -keto--methiolbutyrate is produced, and this is converted into a unique intermediate 3-methylthiopropionate (probably as the coenzyme A form). The unique intermediate, 3-methylthiopropionate, was isolated and identified. Production of carbon dioxide from the methyl carbon of methionine via the transaminative pathway was not dependent on the production of AdoMet.
A series of reports (18-21) showed that, in patients with hypermethioninemia due to inadequate activity of methionine adenosyltransferase or with homocystinuria due to cystathionine ß-synthase deficiency, the intermediate products of the transaminative pathway (-keto--methiolbutyrate, 3-methylthiopropionate, methanethiol, and products of methanethiol metabolism) became of quantitative importance only after the plasma concentration of methionine exceeded 300–350 µmol/L. Gahl et al (20) and Blom et al (21) studied a patient who was apparently homozygous for a mutation of methionineadenosyltransferase 1A, as later described by Hazelwood et al (22), but who possessed a functional methionineadenosyltransferase II. With a dietary intake of 11.2 mmol L-methionine/d, this patient was estimated to form 14.9 mmol AdoMet/d. The intake of L-methionine sulfur was balanced by daily urinary excretions of 2.3 mmol of intermediates of the transamination pathway (ie, 20% of the L-methionine intake), 2.7 mmol methionine and methionine sulfoxide, and 6.2 mmol inorganic sulfate (20). How much of the sulfate was formed via the transsulfuration pathway and how much via transamination and possibly other pathways remains an open question. Is it possible that the unknown pathway(s) identified in 3-methylthiopropionate dilution studies described below may be of significance in man?
Dilution studies reported in Figure 2 of the report by Steele and Benevenga (17) found that the addition of 3-methylthiopropionate at concentrations varying from 2.5 to 25 mmol/L resulted in suppressed conversion of the methyl carbon of L-methionine to carbon dioxide to 15% and 55% of the activity of the controls in rat and monkey liver homogenates, respectively. Thus, it seems that the contribution of the transaminative pathway to L-methionine methyl group catabolism, based on 3-methylthiopropionate inhibition of carbon dioxide production from L-methionine methyl, varied by animal and may vary because of other effectors. Because the inhibitory effect of 3-methylthiopropionate was constant from 2.5 to 20 mmol/L and from 7.5 to 20 mmol/L in rat and monkey in vitro liver homogenate systems, respectively, it appears, on the basis of the inability to completely inhibit carbon dioxide production from the methyl carbon of L-methionine, that yet another pathway (ie, in addition to transamination) exists for the conversion of the L-methionine methyl to carbon dioxide. The interpretation of the effects of a diluting pool of 3-methylthiopropionate here is the same as that used by Christensen (23) in studies of the identification of separate amino acid transporters with the use of specific structural amino acid inhibitors. Could this residual capacity account in man for L-methionine catabolism that is not due to the transaminative pathway?
A review of the recent literature on dietary folate supplementation to reduce plasma L-homocysteine concentrations found a diminishing-returns response to graded amounts of supplemental folate (0.2, 0.4, or 0.8 mg/d) (24). A maximum reduction of 23% was seen at a folic acid supplementation of 0.8 mg/d. The addition of vitamin B-12 to folic acid supplementation resulted in an additional reduction of 7%. The addition of vitamin B-6 to folate and vitamin B-12 did not result in a further reduction of plasma L-homocysteine. I suggest that supplemental betaine or one-carbon sources such as dimethylglycine, sarcosine, L-serine, or glycine, which are precursors of methyl groups via N5-MTHF, should be considered for patients with any of the 3 major forms of homocystinuria. Betaine is known to be a specific methyl donor in the conversion of L-homocysteine to L-methionine. As seen above, supplemental B vitamins provide enzyme cofactors and will not alone provide the methyl-carbon substrates required for remethylation of L-homocysteine to form L-methionine.
Betaine supplementation results in 2 positive changes—first, a lowering of L-homocysteine and, second, the production of L-methionine, which may be in short supply. High-dose dietary betaine supplements have been shown to be successful (25) and are listed by the Physicians Desk Reference–Health (26) as an effective treatment for all 3 forms of homocystinuria. Wilcken and Wilcken (27) concluded that betaine supplementation of homocystinuric patients "effectively lowers circulating homocyst(e)ine, even to suboptimal levels, markedly reduces cardiovascular risk in patients with cystathionine ß-synthase deficiency, and ... contributes importantly to this in pryidoxine-nonresponsive patients. Betaine as additional therapy is safe and effective for at least 16 y."
With respect to NTDs, I suggest that their occurrence may result from the deficiency in a methyl group or methionine. As suggested earlier, B vitamin supplements (folate and vitamins B-6 and B-12) can be converted into enzyme cofactors and may increase the metabolic ability to provide one-carbon or methyl units (or both), but B vitamin supplementation cannot overcome a substrate deficiency. Because it is the fetus who is affected, I assume that all supplemental B vitamins and methyl group sources have to pass through the placenta to be of use to the developing neonate.
Two reviews of the clinical manifestation of a N5-10-MTHF reductase deficiency showed elevated L-homocysteine in blood and urine along with plasma L-methionine concentrations that were 50% of those in controls (7, 28). This folate system defect results in a one-carbon deficiency that affects the remethylation of L-homocysteine and hence results metabolically in a deficiency in methyl group or methionine (or both) and clinically in abnormal brain function, as shown by motor and gait abnormalities. In one of these reviews (7), it was found that autopsy of these patients showed dilated cerebral ventricles, internal hydrocephalus, low brain weight, and demyelination. Mental retardation was mentioned in relation to 75% of the patients (7). Both of these reviews indicated that supplemental betaine had the advantage of reducing circulating L-homocysteine and raising the concentration of L-methionine (7, 28). Thus, the clinical and metabolic consequences of the inherited disorder, a N5-10-MTHF reductase deficiency, result in a deficiency in methyl group or methionine (or both) that can be corrected by betaine supplements. Additional evidence of the importance of L-methionine in brain development is found in studies with cultured rat embryos (29, 30), in which neural tubes failed to close in rat embryos cultured in cow serum unless L-methionine was supplemented at a concentration of 25 µg/mL in media. These observations may in part be due to the lower concentration of methionine in cow serum (1/3 that of rat serum). Supplemental choline has also been shown to result in partial closure of the neural tube (28). In a report using the chick embryo as a model, Afman et al (31) showed that inhibition of transmethylation delayed neural tube closure. One wonders whether, during embryo development, the requirement for methionine, methyl groups, or sulfur (or all 3) is higher in brain than in other organs and whether a tissue-specific deficiency of methyl groups occurs.
In their reevaluation of the benefits of the folic acid–supplementation program in the United States, Gross et al (32) presented dose-response data indicating that 100 and 200 µg folic acid/d would be expected to decrease NTDs by 13–22% and 23–41%, respectively. In a review of folic acid supplementation and birth defects, Green (33) found that preconception folic acid supplementation resulted in a 50–70% reduction in the incidence of NTDs. Daly et al (34) projected that folic acid supplementation at 400 and 1000 µg/d would reduce the incidence by 48% and 53%, respectively. Whereas folic acid supplementation has clearly decreased the incidence of NTDs, folic acid supplementation alone does not appear to lower the incidence by more than one-half. Recently, Blom et al (35) came to essentially the same conclusion: "Biochemical, genetic and epidemiologic observations have led to the development of the methylation hypothesis, which suggests that folic acid prevents neural tube defects by stimulating cellular methylation reactions." Therefore, my take-home message is that betaine or one-carbon sources (eg, dimethylglycine, sarcosine, L-serine, or glycine) that lead to the production of N5-MTHF should be considered along with folate as part of dietary supplementation when the possible occurrence of NTDs is of concern.
ACKNOWLEDGMENTS
The author had no personal or financial conflict of interest.
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