Electron microscopy of gene regulation: the L-arabinose operon.

Unlike normal cells, malignant rat and two simian virus 40-transformed human cell lines can neither grow nor survive in B12- and folate-supplemented media in which methionine is replaced by homocysteine. Yet three lines of evidence indicate that the malignant and transformed cells synthesize large amounts of methionine endogenously through the reaction catalyzed by 5-methyltetrahydropterolyl-L-glutamate: L-homocysteine S-methyltransferase (EC 2.1.1.13). (1) The activities of this methyltransferase were comparable in extracts of malignant and normal cells. (2) The uptake of radioactive label from [5-14C]methyltetrahydropteroyl-L-glutamic acid (5-Me-H4PteGlu) was at least as great in the malignant cells as in the normals and was nearly totally dependent on the addition of homocysteine, the methyl acceptor; furthermore, 59-84% of the label incorporated by cells was recovered as methionine.     

Reversion to methionine independence in simian virus 40-transformed human and malignant rat fibroblasts is associated with altered ploidy and altered properties of transformation

Many transformed and malignant cells, unlike normal cells, do not grow when methionine in the growth medium is replaced by its immediate precursor homocysteine [Chello, P. & Bertino, J. (1973) Cancer Res. 33, 1898-1904 and Hoffman, R. & Erbe, R. (1976) Proc. Natl. Acad. Sci. USA 73, 1523-1527]. Rare cells from those populations revert to methionine independence [Hoffman, R., Jacobsen, S. & Erbe, R. (1978) Biochem. Biophys. Res. Commun. 82, 228-234]. We report here that methionine-independent revertants of both human fibroblasts transformed by simian virus 40 and malignant rat fibroblasts concomitantly revert for some of the properties associated with the transformed state. Of the 13 methionine-independent revertants described here, 5 showed increased anchorage dependence as reflected by reduced cloning efficiences in methylcellulose; 8 showed an increased serum requirement for optimal growth; 8 showed decreased cell density in medium containing high serum; and 3 altered their cell morphology significantly. Eight of the 13 have increased chromosome numbers. All lines tested contained immunologically identifiable tumor antigen of simian virus 40. Thus by selecting for methionine independence it is possible to select for heterogeneous transformation revertants, indicating further a relationship between altered methionine metabolism and oncogenic transformation. Therefore a positive metabolic method to select for transformation revertants has been developed, and its use has resulted in selection of human transformation revertants.     

The atherogenic effect of excess methionine intake

Methionine is the precursor of homocysteine, a sulfur amino acid intermediate in the methylation and transsulfuration pathways. Elevated plasma homocysteine (hyperhomocysteinemia) is associated with occlusive vascular disease. Whether homocysteine per se or a coincident metabolic abnormality causes vascular disease is still an open question. Animals with genetic hyperhomocysteinemia have so far not displayed atheromatous lesions. However, when methionine-rich diets are used to induce hyperhomocysteinemia, vascular pathology is often observed. Such studies have not distinguished the effects of excess dietary methionine from those of hyperhomocysteinemia. We fed apolipoprotein E-deficient mice with experimental diets designed to achieve three conditions: (i) high methionine intake with normal blood homocysteine; (ii) high methionine intake with B vitamin deficiency and hyperhomocysteinemia; and (iii) normal methionine intake with B vitamin deficiency and hyperhomocysteinemia. Mice fed methionine-rich diets had significant atheromatous pathology in the aortic arch even with normal plasma homocysteine levels, whereas mice fed B vitamin-deficient diets developed severe hyperhomocysteinemia without any increase in vascular pathology. Our findings suggest that moderate increases in methionine intake are atherogenic in susceptible mice. Although homocysteine may contribute to the effect of methionine, high plasma homocysteine was not independently atherogenic in this model. Some product of excess methionine metabolism rather than high plasma homocysteine per se may underlie the association of homocysteine with vascular disease.     

Plasma homocysteine after insulin infusion in type II diabetic patients with and without methionine intolerance.

BACKGROUND: A high prevalence of hyperhomocysteinemia has been reported in type II diabetic patients with documented vascular disease; hence the hypothesis that hyperhomocysteinemia may contribute to overall mortality in diabetic patients. The link between insulin and homocysteine metabolism has not been completely clarified yet; in particular, only few data are available on the effects of insulin in vivo on homocysteine metabolism in the presence of abnormalities of sulphur amino acid metabolism (methionine intolerance). MATERIALS AND METHODS: To establish whether methionine intolerance and which of its determinants could influence total plasma homocysteine in response to insulin infusion in vivo in type II diabetic patients, we submitted 18 patients (Group A) with normal and 18 patients with abnormal (hyperhomocysteinemia) (Group B) response to oral methionine load to a glucose/clamp study. At time 0, and 30, 60 and 120 minutes after hyperinsulinemia, homocysteine and methionine plasma levels were assessed. In order to evaluate the cause of methionine intolerance, all patients were assayed for fasting homocysteine-cysteine ratio (as a marker of suspected heterozygosis for cystathionine-beta-synthase deficit), MTHFR C (677)T status and homocysteine-related vitamin status (serum vitamin B (6) [PLP], vitamin B (12) and folate). RESULTS: After hyperinsulinemia, plasma methionine was reduced (by about - 30 % at 120 minutes vs. basal values) within both groups, whereas tHcy tend to decrease in group A following insulin administration (up to - 6.6 +/- 3.6 % vs. basal values at 120 minutes) with a significantly higher variability, while in patients with "methionine intolerance" (group B) tHcy tended to increase (up to + 29.05 +/- 8.3 % vs. basal values at 120 min from the clamp). Serum folic acid (7.45 +/- 2.8 vs. 4.82 +/- 2.5 nmol/L, p < 0.05), Vit. B (12) (348 +/- 78 vs. 242 +/- 65 pmol/L, p < 0.05) and PLP (84.1 +/- 23.6 vs. 50.6 +/- 32.4 nmol/L; p < 0.01) were significantly higher in group A than in group B; PLP levels significantly correlated with homocysteine after 4 h methionine load (n = 36; r = - 0.327, p < 0.05); group A showed also a significantly lower prevalence of suspected heterozygosis for cystathionine-beta-synthase deficit (1/18 [11.1 %] vs. 5/18 [33.3 %], p < 0.05) and MTHFR T allele presence (4/18 [22.2 %] vs. 11/18 [61.1 %], p < 0.01). A stepwise regression analysis with tHcy plasma level variations (event A = reduction; event B = increase) as the dependent variable showed that low serum folate and PLP levels and presence of MTHFR T allele were the variables associated with insulin-induced tHcy increase. CONCLUSIONS: Methionine intolerance may influence the effect of insulin administration on plasma homocysteine in patients affected by type 2 diabetes. To prevent a possible acute (and repeated) hyperhomocysteinemia due to insulin administration in cases of methionine intolerance, it may be useful to assess the presence of methionine intolerance (tHcy after oral methionine loading) and Hcy-related vitamin status in all patients due to be subjected to insulin therapy.     

Distinct effects of folate and choline deficiency on plasma kinetics of methionine and homocysteine in rats

Both folate and betaine, a choline metabolite, play essential roles in the remethylation of homocysteine to methionine. We have studied the effects of folate and choline deficiency on the plasma kinetics of methionine, especially remethylation of homocysteine to methionine, by means of stable isotope methodology. After a bolus intravenous administration of [(2)H(7)]methionine (5 mg/kg body weight) into the rats fed with folate-, choline-, folate + choline-deficient or control diets, the plasma concentrations of [(2)H(7)]methionine, demethylated [(2)H(4)]homocysteine, and remethylated [(2)H(4)]methionine were determined simultaneously with endogenous methionine and homocysteine by gas chromatography-mass spectrometry-selected ion monitoring. The total plasma clearance of [(2)H(7)]methionine was not significantly different among groups, suggesting that the formation of [(2)H(4)]homocysteine from [(2)H(7)]methionine was not influenced by deficiencies of folate and choline. The area under concentration-time curve of [(2)H(4)]homocysteine significantly increased in the folate- and folate + choline-deficient group as compared with the control, but not in the choline-deficient group. The time profile of plasma concentrations of [(2)H(4)]methionine in the folate-deficient group was the same as the control group, whereas the appearance of [(2)H(4)]methionine in plasma was delayed in the choline- and folate + choline-deficient group. These results suggested plasma levels of remethylated methionine were influenced by choline deficiency rather than folate deficiency.     

The methionine connection: homocysteine and hydrogen sulfide exert opposite effects on hepatic microcirculation in rats

Increased intrahepatic resistance in cirrhotic livers is caused by endothelial dysfunction and impaired formation of two gaseous vasodilators, nitric oxide (NO) and hydrogen sulfide (H(2)S). Homocysteine, a sulfur-containing amino acid and H(2)S precursor, is formed from hepatic methionine metabolism. In the systemic circulation, hyperhomocystenemia impairs vasodilation and NO production from endothelial cells. Increased blood levels of homocysteine are common in patients with liver cirrhosis. In this study, we demonstrate that acute liver perfusion with homocysteine impairs NO formation and intrahepatic vascular relaxation induced by acetylcholine in methoxamine-precontracted normal livers (7.3% +/- 3.0% versus 26% +/- 2.7%; P < 0.0001). In rats with mild, diet-induced hyperhomocystenemia, the vasodilating activity of acetylcholine was markedly attenuated, and incremental increases in flow induced a greater percentage of increases in perfusion pressure than in control livers. Compared with normal rats, animals rendered cirrhotic by 12 weeks' administration of carbon tetrachloride exhibited a greater percentage of increments in perfusion pressure in response to shear stress (P < 0.05), and intrahepatic resistance to incremental increases in flow was further enhanced by homocysteine (P < 0.05). In normal hyperhomocysteinemic and cirrhotic rat livers, endothelial dysfunction caused by homocysteine was reversed by perfusion of the livers with sodium sulfide. Homocysteine reduced NO release from sinusoidal endothelial cells and also caused hepatic stellate cell contraction; this suggests a dual mechanism of action, with the latter effect being counteracted by H(2)S. CONCLUSION: Impaired vasodilation and hepatic stellate cell contraction caused by homocysteine contribute to the dynamic component of portal hypertension.     

Abnormal hepatic methionine and glutathione metabolism in patients with alcoholic hepatitis

BACKGROUND: Abnormal methionine metabolism occurs in animals fed ethanol and in end-stage cirrhotic patients. Expected consequences of these abnormalities include reduced hepatic S-adenosylmethionine and glutathione (GSH) levels, impaired transmethylation, and reduced homocysteine catabolism, resulting in the often-observed hyperhomocystinemia in cirrhotic patients. These parameters have not been examined simultaneously in patients with less advanced alcoholic liver disease. METHODS: Six patients hospitalized for alcoholic hepatitis were studied. Plasma was analyzed for homocysteine, methionine, and GSH levels. Liver biopsies diagnosed acute alcoholic hepatitis and underlying fibrosis. Liver specimens were processed for messenger RNA (mRNA) levels and various metabolites and were compared with those of six normal controls. RESULTS: Three patients had cirrhosis, and three had only portal fibrosis. Plasma levels of homocysteine and methionine were increased in two of the three patients with cirrhosis but not in the patients with fibrosis. All patients had markedly lower plasma GSH levels (mean +/- SD: 0.27 +/- 0.19 microM, which is at least 10-fold lower than the normal range). Hepatic S-adenosylmethionine levels were reduced by 50%, whereas methionine, GSH, and cysteine levels were reduced by 70-80%. The mRNA levels of most enzymes involved in methionine metabolism and GSH synthesis were decreased, whereas albumin expression was unchanged. Despite the well known induction of cytochrome P450 2E1 in chronic alcoholics, its mRNA levels were nearly 70% lower in these patients. CONCLUSIONS: In alcoholic hepatitis, abnormal hepatic gene expression in methionine and GSH metabolism occurs and often contributes to decreased hepatic methionine, S-adenosylmethionine, cysteine, and GSH levels. It may be important to replenish these thiols in patients hospitalized with alcoholic hepatitis.     

Effects of long-term administration of methionine on vascular endothelium in rabbits

BACKGROUND AND AIM: We studied the effects of long-term methionine administration on the vascular endothelium of Japanese white rabbits. METHODS AND RESULTS: Eleven rabbits were divided into a control group (n = 6) and a methionine-fed group (n = 5), and reared for 22 weeks. Blood samples were collected at baseline and after 22 weeks for the measurement of serum homocysteine and cysteine, serum lipids and serum superoxide dismutase activity. At the end of experiments, the animals were sacrificed, and the thoracic aorta was removed for the measurement of isometric tension and histopathological examination. The blood samples taken from the methionine group in the 22nd week showed slight but significant increases in serum homocysteine and cysteine levels (Hcy: 13.7 +/- 1.4 vs 21.0 +/- 4.9, p < 0.01; Cys: 241.6 +/- 37.8 vs 342.6 +/- 35.0, p < 0.01). In the isometric tension experiments, the methionine group had a significantly decreased (p < 0.01) vasodilatation reaction induced by acetylcholine, an endothelium-dependent vasodilator. The histopathological examination (immunostaining in response to eNOS and tissue factor) showed significant increases in endothelium expression in the methionine group before atherosclerotic changes appeared. CONCLUSIONS: The above results suggest that vascular endothelial dysfunction played an important role in the atherosclerosis occurring after excess methionine feeding.     

Effects of polymorphisms of methionine synthase and methionine synthase reductase on total plasma homocysteine in the NHLBI Family Heart Study

The metabolism of homocysteine requires contributions of several enzymes and vitamin cofactors. Earlier studies identified a common polymorphism of methylenetetrahydrofolate reductase that was associated with mild hyperhomocysteinemia. Common variants of two other enzymes involved in homocysteine metabolism, methionine synthase and methionine synthase reductase, have also been identified. Methionine synthase catalyzes the remethylation of homocysteine to form methionine and methionine synthase reductase is required for the reductive activation of the cobalamin-dependent methionine synthase. The methionine synthase gene (MTR) mutation is an A to G substitution, 2756A-->G, which converts an aspartate to a glycine codon. The methionine synthase reductase gene (MTRR) mutation is an A to G substitution, 66A-->G, that converts an isoleucine to a methionine residue. To determine if these polymorphisms were associated with mild hyperhomocysteinemia, we investigated subjects from two of the NHLBI Family Heart Study field centers, Framingham and Utah. Total plasma homocysteine concentrations were determined after an overnight fast and after a 4-h methionine load test. MTR and MTRR genotype data were available for 677 and 562 subjects, respectively. The geometric mean fasting homocysteine was unrelated to the MTR or MTRR genotype categories (AA, AG, GG). After a methionine load, a weak positive association was observed between change in homocysteine after a methionine load and the number of mutant MTR alleles (P-trend=0.04), but this association was not statistically significant according to the overall F-statistic (P=0.12). There was no significant interaction between MTR and MTRR genotype or between these genotypes and any of the vitamins with respect to homocysteine concentrations. This study provides no evidence that these common MTR and MTRR mutations are associated with alterations in plasma homocysteine.     

Oral challenge with a methionine load in patients with inflammatory bowel disease: a better test to identify hyperhomocysteinemia

BACKGROUND: Patients with inflammatory bowel disease have an increased risk of thrombosis. Hyperhomocysteinemia is one of the factors that have been related to thromboembolic complications. Patients with hyperhomocysteinemia and normal fasting homocysteine levels can be identified with an oral methionine load. We studied homocysteine levels in patients with IBD during fasting and after methionine load to determine the true prevalence of hyperhomocysteinemia and its relation with thrombotic events. METHODS: Prospective analysis of homocysteine levels in consecutive patients with IBD during fasting and 6-8 hours after an oral methionine load. Levels of folate and vitamin B12 were also determined. History of thrombotic events were recorded. RESULTS: Eighty-two patients with IBD, 56 with UC and 26 with CD were included. Eighteen patients (22%) had hyperhomocysteinemia during fasting. Mean levels of homocysteine after methionine load were 20.4 +/- 18.1 micromol/l (range, 1-79.7 micromol/l), and 43 patients (52%) had hyperhomocysteinemia (> or =20 micromol/l) after methionine load. Six patients (7.3%) had history of thrombosis. The homocysteine levels during fasting and after methionine load were significantly higher in patients with thrombotic events than in patients without thrombosis (15.5 +/- 3.7 micromol/l vs. 6.6 +/- 6.5 micromol/l; P = 0.002; 44.5 +/- 20.9 micromol/l vs. 18.4 +/- 16.5 micromol/l; P < 0.001, respectively). CONCLUSIONS: There is a higher prevalence of hyperhomocysteinemia in IBD patients than previously thought, this can be identified with an oral challenge of a methionine load. Hyperhomocysteinemia increases the risk of thromboembolic complications in patients with IBD.     

The effects of oral methionine and homocysteine on endothelial function

BACKGROUND—Raised homocysteine is a risk factor for vascular disease. Homocysteine is formed from methionine, and dietary manipulation of homocysteine in primates and humans with oral methionine is associated with endothelial dysfunction. A cause-effect relation has not been clearly established.
AIM—To study the effect of oral methionine and then oral homocysteine on endothelial function.
METHODS—22 healthy adults were recruited for two randomised crossover studies, each containing 11 subjects. Endothelial function was determined by measuring forearm blood flow in response to intra-arterial infusion of acetylcholine (endothelium dependent) and sodium nitroprusside (endothelium independent). Subjects received methionine or placebo (study 1), or homocysteine or placebo (study 2). Methionine and homocysteine were determined at baseline and t = 4 hours. Endothelial function was determined at four hours. The responses to the vasoactive substances are expressed as the area under the curve of change in forearm blood flow from baseline.
RESULTS—Study 1: plasma methionine and homocysteine concentrations increased significantly versus placebo. The increases were associated with a reduction of endothelium dependent responses (mean (95% confidence interval), arbitrary units), from 48.8 (95% CI 36.4 to 61.2) to 29.9 (95% CI 18.0 to 41.1), p < 0.04; endothelium independent responses were unchanged. Study 2: homocysteine concentration increased significantly while methionine remained unchanged. Endothelium dependent responses were reduced from 34.6 (95% CI 20.6 to 48.6) to 22.8 (95% CI 12.0 to 33.6), p < 0.03.
CONCLUSIONS—Homocysteine and not methionine is responsible for the changes in endothelial function. This supports the hypothesis that homocysteine promotes atherosclerosis by inducing endothelial dysfunction.


Keywords: homocysteine; methionine; endothelial function; plethysmography     

Effect of folic acid and betaine on fasting and postmethionine-loading plasma homocysteine and methionine levels in chronic haemodialysis patients

OBJECTIVES: To study fasting and postmethionine-loading (increment and decrement) plasma homocysteine levels in end-stage renal disease (ESRD) patients in relation to B-vitamin status and after folic acid treatment without or with betaine. DESIGN: Plasma total homocysteine (tHcy) and methionine levels were measured in chronic haemodialysis patients after an overnight fast, and 6 and 24 h after an oral methionine load (0.1 g kg-1). The patients were subsequently randomized to treatment with folic acid 5 mg daily with or without betaine 4 g daily, and the loading test was repeated after 12 weeks. The patients were then re-randomized to treatment with 1 or 5 mg folic acid daily for 40 weeks, after which a third loading test was performed. SETTING: Haemodialysis unit of university hospital and centre for haemodialysis. SUBJECTS: Twenty-nine consecutive maintenance (> 3 months) haemodialysis patients, not on folic acid supplementation, 26 of whom completed the study. RESULTS: At baseline, the mean fasting, the 6 h postload and the 6 h postload increment plasma tHcy levels were increased as compared with those in healthy controls (46.8 +/- 6.9 (SEM), 92.8 +/- 9.1 and 46.0 +/- 4.2 mumol L-1, respectively) and correlated with serum folate (r = -0.42, P = 0.02; r = -0.61, P = 0.001 and r = -0.54, P = 0.003, respectively), but not with vitamin B6 or vitamin B12. At week 12, these variables had all decreased significantly. Betaine did not have additional homocysteine-lowering effects. At week 52, fasting and postload tHcy levels did not differ significantly between patients on 1 or 5 mg folic acid daily. Plasma tHcy half-life and plasma methionine levels after methionine loading were not altered by folic acid treatment. CONCLUSIONS: In chronic haemodialysis patients, fasting as well as postmethionine-loading plasma tHcy levels depend on folate status and decrease after folic acid therapy. Increased postload homocysteine levels in these patients therefore do not necessarily indicate an impaired transsulphuration capacity only; alternatively, folate may indirectly influence transsulphuration. The elucidation of the complex pathogenesis of hyperhomocysteinaemia in chronic renal failure requires further investigation.     

Regulation of 5-Methyltetrahydrofolate:Homocysteine Methyltransferase Activity by Methionine, Vitamin B12, and Folate in Cultured

Rapid growth of BHK cells in methioninedeficient medium required supplementation with homocysteine, B(12), and over 40-fold greater levels of folic acid than growth in methionine-supplemented medium. The activity of the B(12)-dependent 5-methyltetrahydrofolate: homocysteine methyltransferase was studied in extracts of BHK cells grown in media containing various concentrations of the compounds of the enzyme reaction. The methyltransferase activity increased over 4-fold when B(12)-deficient deficient medium was supplemented with optimal levels of B(12); this increase was not prevented by puromycin. Addition of homocysteine to growth medium containing methionine, B(12), and folic acid was without effect. However, methyltransferase activity increased 2.5- to 4.0-fold further beyond the highest levels obtained in the presence of methionine, B(12), and folic acid when homocysteine was substituted for methionine in the growth medium. This increase was blocked by puromycin and was not due to removal of feedback inhibition of activity by the product methionine. These results suggest that methyltransferase activity may be regulated in part by derepression of the enzyme's synthesis on substitution of the substrate homocysteine for the product methionine.     

Homocysteine catabolism: levels of 3 enzymes in cultured human vascular endothelium and their relevance to vascular disease.

Elevated plasma homocysteine enhances the risk of thrombosis and premature arteriosclerosis. We have assessed the activity of the 3 prime enzymes of homocysteine metabolism in cultured human venous endothelial cells, in a study of their possible protective roles. In cells from 4 individuals, cultured in Dulbecco's modified Eagle medium, the mean activity +/- S.D. of cystathionine beta-synthase (nmol of product/h per mg of cell protein, at 37 degrees C) was 3.58 +/- 3.11 at pH 8.6. The assay used was our newly developed amino acid analyser-based procedure. The activity of 5-methyltetrahydrofolate:homocysteine methyltransferase at pH 7.4 was 4.12 +/- 1.25 and betaine:homocysteine methyltransferase (BHMT) was undetectable (< 1.4 nmol/h per mg protein). Cells were also cultured in a medium aimed at stimulating methionine biosynthesis, containing methionine-deficient Dulbecco's modified Eagle medium to which L-homocystine (100 mumol/l) and methylcobalamin (1 mumol/l) had been added. In these cells 5-methyltetrahydrofolate:homocysteine methyltransferase activity increased to 7.95 +/- 1.45, P < 0.001, there was a non-significant decrease in cystathionine beta-synthase activity to 2.16 +/- 1.52 and BHMT activity was still undetectable. These cells were more resistant to in vitro homocysteine-induced detachment than were cells from the same line cultured in Dulbecco's modified Eagle medium alone. Our findings establish that human endothelial cells express 2 of the 3 primary enzymes of homocysteine catabolism. They suggest that persons who are deficient in cystathionine beta-synthase or 5-methyltetrahydrofolate:homocysteine methyltransferase activity may not only develop homocysteinemia, but also have vascular endothelium which is more susceptible to damage by homocysteine than persons with normal enzyme levels.     

A common polymorphism in methionine synthase reductase increases risk of premature coronary artery disease

BACKGROUND: Methionine synthase reductase (MTRR) catalyzes the regeneration of methylcobalamin, a cofactor of methionine synthase, an enzyme essential for maintaining adequate intracellular pools of methionine and tetrahydrofolate, as well as for maintaining homocysteine concentrations at nontoxic levels. We recently identified a common A-->G polymorphism at position 66 of the cDNA sequence of MTRR; this variant was associated with a greater than normal risk for spina bifida in the presence of low levels of cobalamin. OBJECTIVE: To investigate whether the polymorphism was associated with alterations in levels of homocysteine, folate, and vitamin B12, and with risk of developing premature coronary artery disease (CAD), in a population of individuals presenting for cardiac catheterization procedures. METHODS: We screened 180 individuals aged < 58 years with angiographically documented coronary-artery occlusions or occlusion-free major arteries for the presence of the 66A-->G MTRR polymorphism using a polymerase-chain-reaction-based assay. RESULTS: We identified a trend in risk of premature CAD across the genotype groups (P = 0.03) with a sex-adjusted relative risk of premature CAD equal to 1.49 (95% confidence interval 1.10-2.03) for the GG versus AA genotype groups. There was no difference in fasting levels of plasma total homocysteine, serum folate, and vitamin B12 among the three MTRR genotypes. CONCLUSIONS: Our findings suggest that the GG genotype of MTRR is a significant risk factor for the development of premature CAD, by a mechanism independent of the detrimental vascular effects of hyperhomocysteinemia. This association needs to be confirmed in other studies.     

Body composition: an important determinant of homocysteine and methionine concentrations in healthy individuals

ObjectiveHomocysteine is a sex-related risk factor for cardiovascular disease but the reason for dimorphism is unclear. It has been hypothesized that fat-free mass is an independent determinant of the circulating homocysteine and methionine concentrations.MethodsThe relationship of homocysteine to body composition was investigated in 52 healthy middle-aged 40–60 year olds. Plasma total homocysteine, methionine, folates, vitamins B6 and B12 concentrations were measured with fat mass, bone mineral content, lean body mass, fat-free mass, body water compartments and resting energy expenditure by Anthropometry, Dual X-ray Absorptiometry, Bioimpedentiometry and Indirect Calorimetry.ResultsMen had higher homocysteine (+28%) and methionine (+18%) concentrations than women, but a similar ratio between the two concentrations. Men also had higher lean body mass and fat-free mass. Homocysteine and methionine concentrations were significantly related to fat-free mass, lean body mass, total, extracellular and intracellular water both in simple correlations and in multivariate models including age, smoking habits and vitamin concentrations. Fat-free mass related measures explained the sex effect on homocysteine and methionine concentrations but not the ratio of homocysteine to methionine concentrations.ConclusionIn healthy middle-aged adults homocysteine concentration independently relates to fat-free mass and to water components. Homocysteine and methionine concentrations increase together in relation to the proportion of fat-free mass but their ratio is unrelated to fat-free mass.     

Influence of methionine synthase (A2756G) and methionine synthase reductase (A66G) polymorphisms on plasma homocysteine levels and relation to risk of coronary artery disease

BACKGROUND: Elevated plasma total homocysteine (tHcy) is increasingly being recognized as a risk factor for coronary artery disease (CAD) and other defects. Recent genetic studies have characterized molecular determinants contributing to altered homocysteine metabolism. Our objectives were therefore to confirm the relationship of tHcy with CAD and to examine the importance of genetic influence on tHcy in the coronary angiograms and conventional cardiovascular risk factors recorded in 230 subjects. We also determined the genotype frequencies distribution of the A2756G transition of the B12-dependent methionine synthase (MTR) gene and the A66G mutation of the methionine synthase reductase (MTRR) gene. RESULTS: Patients with CAD (n=151) had significantly higher tHcy concentrations than control subjects (15.49 +/- 2.75 micromol/l vs. 11.21 +/- 3.54 micromol/l, P < 0.001). Hyperhomocysteinaemia (tHcy > or =15 micromol/l) was a risk factor for CAD [RR = 4.07, 95% CI: 2.21 - 7.47, P < 0.001]. The homocysteine concentrations were significantly different between smokers and non-smokers, at 15.63 +/- 3.10 vs. 12.45 +/- 3.84 micromol/l, P < 0.05. In addition, smokers with hyperhomocysteinaemia demonstrated a markedly increased risk of CAD (OR = 2.50, 95% CI: 1.67 - 3.32, P < 0.05) compared with non-smokers with normal homocysteine.The 2756G and the 66G allele contribute to a moderate increase in homocysteine levels (P = 0.008 and P = 0.007, respectively), but not to CAD (P > 0.05). Combined MTR and MTRR polymorphisms, the 2756AG + 66AG and the 2756AG + 66GG were the combined genotypes that were a significant risk factor for having hyperhomocysteinaemia (14.4 +/- 2.8 micromol/l, OR = 2.75, IC 95% = 1.21 - 6.24, P=0.016 and 17.9 +/- 4.1 micromol/l, OR = 6.28, IC 95% = 1.46 - 12.1, P = 0.021, respectively). Statistic analysis using the UniANOVA test shows that these two polymorphisms have an interactive effect circulating homocysteine levels (P < 0.05). CONCLUSION: Our data suggest that moderately elevated tHcy levels are prevalent in our population and are associated with an increased risk for CAD. This study provides evidence that the MTR A2756G and MTRR A66G polymorphisms significantly influence the circulating homocysteine concentration. In addition, the MTR and MTRR genes may interact to increase the risk for having hyperhomocysteinaemia.     

Effect of vitamin E on resistance vessel endothelial dysfunction induced by methionine

We tested if vitamin E, a fat-soluble antioxidant, prevents resistance vessel endothelial dysfunction caused by methionine-induced hyperhomocysteinemia in humans. Moderate elevations in plasma homocysteine concentrations are associated with atherosclerosis and hypertension. Homocysteine causes endothelial dysfunction possibly through several mechanisms. No previous study has tested if a fat-soluble antioxidant can prevent endothelial dysfunction caused by experimental hyperhomocysteinemia. Ten healthy subjects participated in a 2 x 2 factorial, double-blind crossover study, receiving L-methionine (100 mg/kg at -6 hours) or vehicle, with and without vitamin E (1,200 IU at -13 hours). Endothelial function of forearm resistance vessels was assessed using forearm blood flow responses to brachial artery administration of endothelium-dependent and endothelium-independent agents. Forearm resistance vessel dilatation to acetylcholine was significantly impaired 7 hours after methionine (placebo, 583 +/- 87% vs methionine 30 +/- 68%; p <0.05). Dilatation to bradykinin was also impaired (placebo, 509 +/- 54% vs methionine 289 +/- 48%; p <0.05). Methionine did not alter vasodilatation to the endothelium-independent vasodilators, nitroprusside, and verapamil. Methionine-induced impairment of resistance vessel dilatation to acetylcholine and bradykinin (p <0.05 vs placebo) was prevented by administration of vitamin E (acetylcholine, p = 0.004; bradykinin, p = 0.004; both vs methionine alone). Experimentally increasing plasma homocysteine concentrations by oral methionine rapidly impairs resistance vessel endothelial function in healthy humans and this effect is reversed with administration of the fat-soluble antioxidant, vitamin E.     

Mid-trimester amniotic fluid methionine concentrations: a predictor of birth weight and length

The association between body size at birth and risk of later cardiovascular disease is thought to be a consequence of metabolic changes that accompany slow growth in utero. The metabolism of methionine and homocysteine has been investigated in relation to cardiovascular risk and has also been assigned an important role in organogenesis and normal fetal growth. We determined concentrations of cobalamin, folate, methionine, cysteine, cystathionine, and the marker of B-vitamin function, homocysteine, in 625 samples of amniotic fluid obtained in the second trimester from normal pregnancies. Both vitamins and metabolites varied according to gestational age. The most noticeable observation was that methionine in amniotic fluid during gestational weeks 13 to 17 strongly predicted final birth weight and length. Metabolism of methionine may be a critical factor affecting fetal growth.     

Lack of significant effects of methionine loading on endothelial function in elderly volunteers

OBJECTIVE: To test the hypothesis that an acute increase in plasma homocysteine concentration (Hcy) produced by methionine loading is associated with an acute decrease in brachial artery blood flow measured by flow-mediated dilatation (FMD) using forearm plesthysmography. DESIGN: A double-blind, cross-over, placebo controlled design was used and FMD of the brachial artery, plasma Hcy, plasma methionine, total cholesterol, high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, plasma triglyceride, oxidised LDL, apolipoproteins (Apo) A1 and B and C reactive protein (CRP) were measured between 12 and 20 hours after methionine loading or placebo. RESULTS: Between 12 and 20 hours, after a methionine loading test, acute hyperhomocysteinaemia had no significant effect on mean FMD compared to placebo (57.08+/-6.18ml/100ml/min versus 63.46+/-5.87ml/100ml/min, p<0.5). The mean age of the eight subjects was 71.5+/-6.9 years. Twelve hours after methionine, mean triglyceride concentration was significantly increased by 23.0% compared to placebo (1.51+/-0.47mmol/l versus 1.23+/-0.44mmol/l, p<0.02). CONCLUSION: In elderly volunteers, acute hyperhomocysteinaemia induced by methionine loading resulted in no significant late impairment of endothelial function although further investigation is recommended. Acute hyperhomocysteinaemia resulted in a significant increase in plasma triglyceride concentration.