The combination of high dietary methionine plus cholesterol induces myocardial fibrosis in rabbits

Limited evidence suggests that myocardial fibrosis might be associated with dietary cardiovascular risk factors. OBJECTIVE: To investigate the effects of high dietary cholesterol, methionine (the precursor to homocysteine), and the combination of the two diets on myocardial fibrosis. METHODS: Rabbits were randomly allocated into four dietary groups for 12 weeks: control (Con), 1% methionine (Meth), 0.5% cholesterol (Chol) or 1% methionine plus 0.5% cholesterol (MethChol). RESULTS: Myocardial fibrosis was not significantly increased in Chol or Meth. However, interstitial fibrosis increased by 85% (p = 0.03) and perivascular fibrosis 28-fold (p < 0.01) in the MethChol group compared to Con. CONCLUSIONS: These results suggest that high levels of dietary cholesterol or methionine alone do not significantly increase myocardial collagen content. However, the combination of the two diets does cause myocardial fibrosis. Therefore, excessive cholesterol and methionine intake may be an important pathogenic factor in the development of myocardial fibrosis.     

Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig

Alcoholic liver disease is associated with abnormal hepatic methionine metabolism and folate deficiency. Because folate is integral to the methionine cycle, its deficiency could promote alcoholic liver disease by enhancing ethanol-induced perturbations of hepatic methionine metabolism and DNA damage. We grouped 24 juvenile micropigs to receive folate-sufficient (FS) or folate-depleted (FD) diets or the same diets containing 40% of energy as ethanol (FSE and FDE) for 14 wk, and the significance of differences among the groups was determined by ANOVA. Plasma homocysteine levels were increased in all experimental groups from 6 wk onward and were greatest in FDE. Ethanol feeding reduced liver methionine synthase activity, S-adenosylmethionine (SAM), and glutathione, and elevated plasma malondialdehyde (MDA) and alanine transaminase. Folate deficiency decreased liver folate levels and increased global DNA hypomethylation. Ethanol feeding and folate deficiency acted together to decrease the liver SAM/S-adenosylhomocysteine (SAH) ratio and to increase liver SAH, DNA strand breaks, urinary 8-oxo-2'-deoxyguanosine [oxo(8)dG]/mg of creatinine, plasma homocysteine, and aspartate transaminase by more than 8-fold. Liver SAM correlated positively with glutathione, which correlated negatively with plasma MDA and urinary oxo(8)dG. Liver SAM/SAH correlated negatively with DNA strand breaks, which correlated with urinary oxo(8)dG. Livers from ethanol-fed animals showed increased centrilobular CYP2E1 and protein adducts with acetaldehyde and MDA. Steatohepatitis occurred in five of six pigs in FDE but not in the other groups. In summary, folate deficiency enhances perturbations in hepatic methionine metabolism and DNA damage while promoting alcoholic liver injury.     

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.     

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.     

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.     

Methionine-induced elevation of plasma homocysteine concentration is associated with an increase of plasma cholesterol in adult rats

BACKGROUND AND AIMS: Dietary methionine affects cholesterol metabolism in growing rats. Methionine effects on adult rats and mechanisms by which methionine alters the lipid metabolism are not fully elucidated. We investigated possible mechanisms by which dietary methionine acts on lipid metabolism of adult rats. METHODS: Male adult rats were divided into three groups (n=10) and were fed casein-based diets differing in methionine concentration (low-methionine diet: 0.96 g/kg; adequate-methionine diet: 2.22 g/kg, high-methionine diet: 6.82 g/kg) for 4 weeks. Concentrations of triacylglycerols and cholesterol in plasma and lipoproteins, concentration of homocysteine in plasma, concentration of cholesterol in liver, fecal lipid excretion, expression of hepatic HMG-CoA reductase, phosphatidylethanolamine N-methyltransferase 2 (PEMT-2) and of LDL receptor were measured. RESULTS: Rats fed the high-methionine diet had higher plasma homocysteine concentrations than rats fed the low-methionine diet (p<0.05). Although concentrations of cholesterol in plasma and lipoproteins were not different between the groups, there was a distinct positive correlation between circulating plasma homocysteine and plasma cholesterol (R(2)=0.55, p<0.001). The fecal excretion of cholesterol and bile acids was not altered by dietary methionine. The relative mRNA concentration of HMG-CoA reductase and of LDL receptor remained unaffected by dietary methionine. Gene expression of PEMT-2 was higher in rats fed the high-methionine diet than in rats fed the other diets (p<0.05). CONCLUSION: The results demonstrate that dietary methionine contributes to a rise in circulating homocysteine concentration which positively correlates with the concentration of plasma cholesterol. However, the effects of methionine on cholesterol metabolism of adult rats were relatively weak.     

Ethanol feeding of micropigs alters methionine metabolism and increases hepatocellular apoptosis and proliferation

Chronic alcoholism is associated with increased cancer risk that may be related to ethanol-induced alterations in methionine and deoxynucleotide metabolism. These metabolic relationships were studied in micropigs fed diets for 12 months that contained 40% ethanol or cornstarch control with adequate folate. Ethanol feeding altered methionine metabolism without changing mean terminal liver folate levels. After initial equilibration to diet, ethanol feeding significantly increased monthly serum homocysteine levels while reducing serum methionine levels over the time course of the experiment. After 12 months, hepatic methionine synthase activity and the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) were significantly reduced in ethanol-fed animals, whereas the ratio of liver deoxyuridine triphosphate (dUTP) to deoxythymidine triphosphate (dTTP) was increased and correlated inversely with methionine synthase activity. These findings were associated with increased frequency of hepatocytes with apoptotic bodies and positivity for proliferating cell nuclear antigen (PCNA) in livers from ethanol-fed minipigs. These studies suggest that chronic ethanol feeding perturbs methionine metabolism by impairment of methionine synthase activity, resulting in deoxynucleoside triphosphate (dNTP) imbalance, increased apoptosis, and regenerative proliferation. These biochemical alterations may provide a promoting environment for carcinogenesis during long-term ethanol exposure. (Hepatology 1996 Mar;23(3):497-505)     

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.     

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.     

Homocysteine and lipid metabolism in atherogenesis: effect of the homocysteine thiolactonyl derivatives, thioretinaco and thioretinamide

In order to study the relation of homocysteine and lipid metabolism to atherogenesis, rabbits were fed a synthetic atherogenic diet and treated with parenteral thioretinaco (N-homocysteine thiolactonyl retinamido cobalamin), thioretinamide (N-homocysteine thiolactonyl retinamide) or homocysteine thiolactone hydrochloride. All three substances were found to increase dietary atherogenesis. Thioretinaco and thioretinamide increase total homocysteine of serum, but there is no effect of parenteral homocysteine thiolactone hydrochloride on serum homocysteine. The synthetic diet with corn oil significantly lowers serum homocysteine, compared either to baseline chow diet or to the synthetic diet with butter. Atherogenesis is correlated with total homocysteine, total cholesterol and LDL + VLDL cholesterol, and serum homocysteine is correlated with total cholesterol, LDL + VLDL, and HDL cholesterol in the total sample. Both synthetic diets elevate serum cholesterol, triglycerides and LDL + VLDL, but not HDL, compared to baseline values. Thioretinamide causes significant elevation of cholesterol and LDL + VLDL, compared to controls. The results show that increased dietary saturated fat and cholesterol cause deposition of lipids within the arteriosclerotic plaques produced by homocysteine, converting fibrous to fibrolipid plaques. Facilitation of atherogenesis is attributed to the effect of homocysteine on artery wall, either from parenteral homocysteine or from the increased synthesis of homocysteine from methionine, produced by thioretinaco and thioretinamide.     

Apolipoprotein E levels in vegetarians

Vegetarians are known to have low lipoprotein lipid and apolipoprotein AI and B levels. Since dietary cholesterol has recently been shown to have important effects on apolipoprotein E (apo E) metabolism, we measured plasma apo E levels in three groups of vegetarians. Group I (n = 36) consumed < 10 mg cholesterol daily and 42% of calories as fat (P:S ratio 2.6). Group II (n = 10) and Group III (n = 18) consumed 97 and 179 mg cholesterol daily, and 35% of calories as fat (P:S ratios 0.7 and 0.9) respectively. Compared to control values, vegetarian plasma cholesterol and triglyceride levels were decreased by 10%–30% and 30%–55%. Plasma apo E levels were decreased equally in all groups by 35% (2.4 ± 0.1 mg/dl versus 3.6 ± 0.1 mg/dl, p < .001). Plasma apo E levels were increased in parallel with lipid levels in pregnant vegetarians but were not different from non-lactating vegetarians in postpartum lactating women. Decreased apo E levels did not correlate with relative body weight, P:S ratio or intake of fat, carbohydrates or protein. Since all vegetarian diets studied were low cholesterol diets, decreased cholesterol intake may contribute to the low apo E levels. The apparent modification of apo E metabolism by vegetarian diets may be important in mediating effects of lipid lowering diets on atherogenesis.     

High dietary methionine plus cholesterol exacerbates atherosclerosis formation in the left main coronary artery of rabbits

Although mild hyperhomocysteinemia is a risk factor for cardiovascular events and mortality, there is no evidence to suggest that mild hyperhomocysteinemia stimulates coronary artery atherosclerosis formation. OBJECTIVE: To compare the development of coronary artery atherosclerosis in rabbits following the induction of hyperhomocysteinemia and hypercholesterolemia through diet, and whether the combination of these risk factors exacerbated atherosclerosis formation. METHODS: New Zealand White rabbits were fed for 12 weeks either a control diet, a 1% methionine diet (Meth), a 0.5% cholesterol diet (Chol) or the combination of the two diets (MethChol). Using volumetric stereological techniques, we quantitated the volume of intima, media and lumen of the left main coronary artery (LMCA). RESULTS: Atherosclerosis was present in the Chol group, and increased in the MethChol group. There was no atherosclerosis in the control or Meth groups. CONCLUSIONS: These results underscore the difference in the atherogenicity of hypercholesterolemia alone and mild hyperhomocysteinemia alone. Thus, we suggest that isolated mild hyperhomocysteinemia is not a risk factor for the initiation of coronary artery atherosclerosis formation over a short period of time, but may act in conjunction with other risk factors to further increase plaque formation.     

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.     

蛋氨酸对兔同型半胱氨酸水平及动脉粥样硬化的影响

目的　观察高蛋氨酸摄入对实验动物同型半胱氨酸 (Homocysteine Hcy)水平及动脉组织的影响。方法　雄性新西兰兔 2 0只随机分成 2组 :对照组和蛋氨酸组 ,每组 10只 ,分别喂以普通兔饲料及普通兔饲料中添加 0 .5 %的蛋氨酸 ,喂养 6个月后 ,测定血浆 Hcy、血栓烷 B2 (TXB2 )、6 -酮 -前列腺素 F1α(6 - Keto- PGF1α)水平 ,光镜检测主动脉组织学改变。结果　实验后 ,蛋氨酸组断食 2 h和 7h的血浆 Hcy浓度 (分别为 2 36 .5± 5 9.75μmol/ L ,6 7.83± 2 2 .17μm ol/ L )均明显高于对照组 (分别为 10 .6 2± 1.4 2μm ol/ L ,11.0 6± 1.2 3μmol/ L ) ,(P<0 .0 0 1) ;蛋氨酸组主动脉出现典型的动脉粥样硬化损伤 ;血浆 TXB2 水平和 TXB2 / 6 - Keto- PGF1α升高。结论　过量蛋氨酸摄入可引发高 Hcy血症 ,并造成主动脉粥样硬化损伤     

Phytoestrogen α-zearalanol improves vascular function in ovariectomized hyperhomocysteinemic rats

Previous studies have showed that phytoestrogen α-zearalanol (α-ZAL) could antagonize homocysteine (Hcy) induced endothelin-1 (ET-1) expression, oxidative stress and apoptosis in human umbilical vein endothelial cells in vitro, however, its effect on vascular function in vivo remains to be determined. This study was designed to investigate the effects of α-ZAL on vascular function in ovariectomized (OVX) hyperhomocysteinemia (HHcy) rats and explore the mechanisms involved primarily. HHcy rat model was induced by diets containing 2.5% methionine (Met) for 12 weeks. Forty adult female Wistar rats were assigned randomly into five groups: (1) Con; (2) Met; (3) OVX+Met; (4) OVX+Met+α-ZAL; (5) OVX+Met+17β-E(2) (17β-estradiol). Blood was collected to analyze plasma estradiol, Hcy and ET-1. Thoracic aortas were isolated to detect its response to phenylephrine (PE) and acetylcholine (ACh) or sodium nitroprusside (SNP). Aortas eNOS expression was determined by Western blot. Thoracic aortas histological characterization was analyzed by optical microscope and scanning electron microscope (SEM). Rat plasma Hcy was significantly elevated after fed with 2.5% methionine diets, and ovariectomy aggravated this elevation. Phytoestrogen α-ZAL or 17β-E(2) could attenuate this elevation. Plasma ET-1 levels increased significantly in ovariectomized HHcy rats, and supplement with α-ZAL or 17β-E(2) could reverse these changes. In rats of OVX+Met group, PE elicited significantly greater contraction in a dose-dependent manner in endothelium-intact thoracic aortas rings; ACh elicited significantly less percentage relaxation. These effects were significantly attenuated by supplement with α-ZAL or 17β-E(2). There was no significant difference between groups in relaxation induced by SNP whether endothelium intact or not. Thoracic aortas morphology study also showed severe endothelium injury in ovariectomized HHcy rats, both α-ZAL and 17β-E(2) could attenuate this change. Aortas eNOS expression was decreased in ovariectomized HHcy rats, and supplement with α-ZAL or 17β-E(2) could reverse these changes. These findings demonstrated that α-ZAL could effectively alleviate the impairment of endothelial cells and improve vascular function in ovariectomized HHcy rats by decreasing plasma Hcy and antagonizing decreasing of aortas eNOS expression. This protective effect is somewhat similar with 17β-E(2).     

Reduced availability of endogenously synthesized methionine for S-adenosylmethionine formation in methionine-dependent cancer cells.

Methionine (Met) dependence--i.e., the inability of cultured cells to grow when Met is replaced by its immediate precursor homocysteine (Met-Hcy+ medium)--is a frequent component of the oncogenically transformed phenotype. Normal cells, on the other hand, grow in this medium. There have been reports [Hoffman, R. M. & Erbe, R. W. (1976) Proc. Natl. Acad. Sci. USA 73, 1523-1527; Hoffman, R. M., Jacobsen, S. J. & Erbe, R. W. (1978) Biochem. Biophys. Res. Commun. 82, 228-234] of normal or higher rats of Met biosynthesis in Met-dependent cells and a postulation that Met-dependent cells are deficient in utilization of endogenously synthesized Met as opposed to exogenously supplied Met. To answer the critical question of what biochemical reaction(s) requires preformed Met in Met-dependent cels, we labeled cells with Met-free [35S]Hcy or [35S]Met and determined the levels of Met, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy). We report here experiments that demonstrate that Met-dependent cells synthesize a normal amount of endogenously synthesized Met and are deficient in utilizing this Met for AdoMet synthesis. In contrast, exogenously supplied Met is utilized normally for AdoMet biosynthesis. The ratio of AdoMet to AdoHcy is low in Met-dependent cells growing in Met-Hcy+ medium but is normal in Met+Hcy- medium. We determined that the low AdoMet/AdoHcy ratio probably limits growth of Met-dependent cells in Met-Hcy+ medium.     

Excess dietary methionine decreases indices of copper status in the rat

Two groups (n = 5) of male weanling Wistar rats were housed individually and fed copper (Cu)-deficient (0.5 mg Cu/kg) diets either with or without methionine supplementation (18 g/kg) for 49 days. Plasma caeruloplasmin (EC 1.16.3.1) and erythrocyte superoxide dismutase (EC 1.15.1.1, CuSOD) activities were measured in blood. Tissue Cu levels and the activities of cytochrome c oxidase (EC 1.9.3.1, CCO) and CuSOD were measured in the heart and liver. Hepatic activities of the sulfhydryl-sensitive enzymes, creatine kinase (EC 2.7.3.2), fumarase (EC 4.2.1.2) glutathione S-transferase (EC 2.5.1.18) and lipoamide dehydrogenase (EC 1.6.4.3) were also measured. Apart from cardiac CCO activity all of the measured indices of Cu status were found to be significantly (p less than 0.05) decreased in the methionine supplemented rats. Although fumarase activity was significantly (p less than 0.05) decreased in the methionine-supplemented animals compared with controls, the activities of the other sulfhydryl-sensitive enzymes were not significantly decreased. These results suggest that some of the toxic effects of excess dietary methionine may be mediated through interference with copper metabolism rather than through the previously postulated inhibition of sulfhydryl-sensitive enzymes by metabolites of methionine.     

Homocysteine and methionine metabolism in renal failure

Renal insufficiency is invariably accompanied by elevated plasma concentrations of the sulfur-containing and potentially vasculotoxic amino acid homocysteine. There is a strong relationship between glomerular filtration rate and plasma homocysteine concentration. Unlike creatinine, however, homocysteine is avidly reabsorbed in the renal tubules, and its urinary excretion is minimal. There is no evidence that homocysteine is actively removed by the human kidney. In renal insufficiency, plasma concentrations of S-adenosylmethionine, S-adenosylhomocysteine, cystathionine, cysteine, and sulfate are elevated, pointing to a remethylation or distal transsulfuration/oxidation block as the cause of hyperhomocysteinemia in renal failure. Stable isotope techniques have shown that both whole-body homocysteine remethylation and methionine transmethylation are decreased in renal failure, whereas homocysteine transsulfuration seems intact. Metabolic homocysteine clearance (i.e., transsulfuration relative to plasma homocysteine) is decreased to a major extent. These metabolic disturbances in renal failure can only be partially restored with current treatments. Folic acid treatment lowers plasma homocysteine concentration and increases remethylation and transmethylation rates. Plasma homocysteine, however, is not normalized, and metabolic homocysteine clearance by transsulfuration remains impaired. According to the currently available data, effective normalization of plasma homocysteine can only be obtained when its metabolic clearance through transsulfuration is restored.     

Folic acid reduces adhesion molecules VCAM-1 expession in aortic of rats with hyperhomocysteinemia

To investigate effects of supplementation of folic acid on the expression of adhesion molecules VCAM-1 in the aortas of rats with hyperhomocysteinemia. Thirty male SD rats (200 +/- 20 g) were invided into 3 groups (n = 10 for each group): control group(Control), high Met group(Met) and Met plus Folate group(Met + Folate), fed. for 45 days. Plasma Hcy levels were higher with the high-methionine diet (140.68 +/- 36.87 micromol/L vs 6.47 +/- 1.10 micromol/L in control rats) an effect which was reduced by folate. Respectively, the aortic expression of adhesion molecules VCAM-1 at protein and mRNA levels were higher in the Met groups than those in the control groups or the Met + Folate groups. A high methionine diet for 45 days was sufficient to induce hyperhomocysteinemia. Folate supplementation prevented elevation of Hcy levels in the blood, and reduced expression of the adhesion molecule VCAM-1. Hyperhomocysteinemia is now regarded as one of the important risk factors for cardiovascular and cerebralvascular disorders.[Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med 1998; 38(15):1042-50.] Several plausible mechanisms for Hcy-induecd atherosclerosis have been proposed. These include endothelial dysfunction, enhancement of oxidative stress, reduction in NO bioavailability, and augmentation of thrombus formation.[Holven KB, Holm T, Aukrust P, et al. Effect of folic acid treatment on endothelium-dependent vasodilation and nitric oxide-derived end products in hyperhomocysteinemic subjects . Am J Med 2001;110(7):536-42; Guba SC, Fonseca V, Fink LM. Hyperhomocysteinemia and thrombosis. Semin Thromb Hemost 1999;25(3):291-309.] However, the precise molecular mechanism is still unclear. Recent reports have suggested a role for inflammatory processes in the pathogenesis of atherosclerosis.[Gerard C, Rollins BJ. Chemokines and disease. Nat Immunol 2001;2(2):108-15.] Dysfunction of endothelial cells is the key process promoting inflammatory reactions. On injury, endothlial cells are capable of producing various cytokines that participate in inflammatory reactions in the arterial wall. Although results from in vitro studies suggest that Hcy, at pathophysiological concentrations, stimulates chemokine expression in vascular cells, it is unknown whether hyperhomocysteinemia can initiate similar changes, leading to enhanced momocyte adhesion/binding to the vascular endothelium in vivo.[Zeng X, Dai J, Remick DG, Wang X. Homocysteine mediated expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human monocytes. Circ Res 2003;93(4):311-20.] On the basis of the potential pathogenic role of chemokines in atherogenesis, the objective of the present study was to investigate that homocsteine may exert its effect in part though adhesion molecules VCAM-1 and that folic acid supplementation may downregulate these inflammatory responses. Male Sprague-Dawley rats (bred from animal centers of Tongji Medical College, Huazhong Science and Technology University) aged 8 weeks were divided into 3 groups(n=10 for each group) and maintained for 45 days on the following diets before the experiments: (1) regular diet; (2) high-metheionine diet, consisting of regular diet plus 1.7% methionine; and (3) high-methionine plus folate -rich diet, consisting of regular diet plus 1.7% methionine and 0.006% folate.[Boisvert WA, Curtiss LK, Terkeltaub RA. Interleukin-8 and its receptor CXCR2 in atherosclerosis. Immunol Res 2000;21(2-3):129-d37.] Plasma and serum samples wee colleced and stored at -80 degrees C after 45 days until analysis. The plasma homocysteine concentration of rats in three groups were determined by high-pressue liquid chromatography. To detect the endothelial expression of adhesion molecules VCAM-1, the thoracic aorta was isolated and dived into segments. These segments were immersion-fixed in 10% neutral-buffered formalin overlight and then embedded in paraffin. Sequential 5 mum paraffin-embedded cross sections were prepared. Immunohistochemical analyisis was performed to detect vascular cell adhesion molecule(VCAM)-1, The fixed cryosections were immediately blcked in 10% horse serum and phosphate baffered saline(PBS) at room temperature for 30 min. Goat polyclonal andibodies against rat VCAM-1(Santa Cruz Biotechnology) were diluted 1:100 in PBS and incubated with the cryosections for 1 h of room temperature. After three washes, the sections were incubated with biotin-conjugated rabbit anti-goat immunoglobulins(Dako) at 1:250 dilution in PBS. After three washes, the samples were mounted in 90% glycerol-PBS. Photographs were taken by use of a light microscope at a mignification of x200.     

Acute administration of methionine and/or methionine sulfoxide impairs redox status and induces apoptosis in rat cerebral cortex

Metabolic Brain Disease - High plasma levels of methionine (Met) and its metabolites such as methionine sulfoxide (MetO) may occur in several genetic abnormalities. Patients with hypermethioninemia...