Relationship between homocysteine and superoxide dismutase in homocystinuria: possible relevance to cardiovascular risk.
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A modest homocysteine elevation is associated with an increased cardiovascular risk. Marked circulating homocysteine elevations occur in homocystinuria due to cystathionine beta-synthase (CbetaS) deficiency, a disorder associated with a greatly enhanced cardiovascular risk. Lowering homocysteine levels reduces this risk significantly. Because homocysteine-induced oxidative damage may contribute to vascular changes and extracellular superoxide dismutase (EC-SOD) is an important antioxidant in vascular tissue, we assessed EC-SOD and homocysteine in patients with homocystinuria. We measured circulating EC-SOD, total homocysteine (free plus bound), and methionine levels during the treatment of 21 patients with homocystinuria, 18 due to CbetaS deficiency, aged 8 to 59 years, and 3 with remethylating defects. We measured total homocysteine by immunoassay, EC-SOD by ELISA, and methionine by amino acid analysis and assessed interindividual and intraindividual relationships. There was a significant, positive relationship between EC-SOD and total homocysteine. For the interindividual assessment, levels were highly correlated, r=0.746, N=21, P<0.0001. This relationship was maintained after taking into account intraindividual patient variation (r=0.607, N=62, P<0.0001). In 2 newly diagnosed CbetaS-deficient patients, treatment that lowered the markedly elevated pretreatment homocysteine level (from 337 to 72 and from 298 to 50 micromol/L) reduced the associated elevated EC-SOD in each by 50%. EC-SOD and methionine levels were unrelated (r=0.148, n=39, P=0.368). The positive relationship between circulating EC-SOD and homocysteine could represent a protective antioxidant response to homocysteine-induced oxidative damage and contribute to reducing cardiovascular risk in homocystinuric patients. EC-SOD levels may be relevant to the pathogenesis of vascular disease in other patient groups. (+info)
Familial thrombophilia associated with homozygosity for the cystathionine beta-synthase 833T-->C mutation.
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Severe hyperhomocysteinemia due to cystathionine beta-synthase (CBS) deficiency is a strong risk factor for premature cardiovascular disease. Among untreated patients, approximately 50% have suffered a thromboembolic event by 30 years of age. We report on 3 sisters with severe hyperhomocysteinemia due to homozygosity for the CBS 833T-->C mutation. These patients, who displayed no other known thrombophilic predisposition, had suffered single or multiple venous thrombosis before CBS deficiency was diagnosed relatively late in life. In this family, homozygosity for the 833T-->C mutation was associated with a mild phenotype with respect to other sequelae of CBS deficiency. Consequently, our results indicate that most cases with this genotype may remain undiagnosed. Investigated family members heterozygous for the 833T-->C mutation displayed normal total homocysteine in plasma (tHcy) levels, even when they were homozygous for the methylenetetrahydrofolate reductase 677C-->T polymorphism. The prevalence of homozygosity for the 833T-->C mutation has previously been estimated at no less than 1:20 500 in our population. Because a reduction of the severely elevated levels of tHcy in CBS deficiency reduces cardiovascular risk and because homozygosity for the 833T-->C mutation is more prevalent than previously thought, our results emphasize the importance of measuring tHcy routinely in thrombophilia screening. (+info)
Accurate and rapid "multiplex heteroduplexing" method for genotyping key enzymes involved in folate/homocysteine metabolism.
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BACKGROUND: Hyperhomocysteinemia, which is often associated with low folate status, is an independent risk factor for cardiovascular diseases and several other pathologies. The four most common functional polymorphisms in genes involved in folate/homocysteine metabolism are methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, methionine synthase (MS) A2756G, and cystathionine beta-synthase (CBS) 844ins68. The pathogenic impact of these variants is under active investigation in many laboratories. However, conventional genotyping methods, mostly using PCR followed by restriction enzyme digestion, often are compromised by partial fragment digestion. There is, therefore, a need to develop more reliable approaches to genotyping the above polymorphisms that may be applied in large-scale studies. METHODS: Sequence-specific heteroduplex generators for each of the MTHFR and MS single nucleotide polymorphisms were generated by site-directed mutagenesis. These were subcloned into a single construct, pHcyHG-1, which could be multiplexed with a simple PCR amplification across the CBS 844ins68 polymorphic site to generate composite genotype-specific banding patterns from individual genomic DNA samples that could be electrophoretically resolved. RESULTS: The "multiplex heteroduplexing" method yielded unambiguous MTHFR, MS, and CBS genotypes in a single-tube reaction that could be analyzed in a single gel run. CONCLUSIONS: This method permits unambiguous genotyping of the four most common functional variants of enzymes involved in folate/homocysteine metabolism. It is rapid, reproducible, and inexpensive, and requires no special preparative or analytic facilities; consequently, it will facilitate large-scale studies of the genetic basis of hyperhomocysteinemia and the many pathologies that have been associated with this phenotype. (+info)
Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia.
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Homocysteine is a risk factor for the development of atherosclerosis and its thrombotic complications. We have employed an animal model to explore the hypothesis that an increase in reactive oxygen species and a subsequent loss of nitric oxide bioactivity contribute to endothelial dysfunction in mild hyperhomocysteinemia. We examined endothelial function and in vivo oxidant burden in mice heterozygous for a deletion in the cystathionine beta-synthase (CBS) gene, by studying isolated, precontracted aortic rings and mesenteric arterioles in situ. CBS(-/+) mice demonstrated impaired acetylcholine-induced aortic relaxation and a paradoxical vasoconstriction of mesenteric microvessels in response to superfusion of methacholine and bradykinin. Cyclic GMP accumulation following acetylcholine treatment was also impaired in isolated aortic segments from CBS(-/+) mice, but aortic relaxation and mesenteric arteriolar dilation in response to sodium nitroprusside were similar to wild-type. Plasma levels of 8-epi-PGF(2alpha) (8-IP) were somewhat increased in CBS(-/+) mice, but liver levels of 8-IP and phospholipid hydroperoxides, another marker of oxidative stress, were normal. Aortic tissue from CBS(-/+) mice also demonstrated greater superoxide production and greater immunostaining for 3-nitrotyrosine, particularly on the endothelial surface. Importantly, endothelial dysfunction appears early in CBS(-/+) mice in the absence of structural arterial abnormalities. Hence, mild hyperhomocysteinemia due to reduced CBS expression impairs endothelium-dependent vasodilation, likely due to impaired nitric oxide bioactivity, and increased oxidative stress apparently contributes to inactivating nitric oxide in chronic, mild hyperhomocysteinemia. (+info)
Genetic characterization of the metK locus in Escherichia coli K-12.
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Three independently isolated metK mutants have been shown to have leisions lying between speB and glc near 57 min on the Escherichia coli chromosome. Two deletions result in a lack of the metC gene product but neither extends into the metK glc region. The three metK mutations are recessive to the wild-type allele carried on the KLF16 episome. (+info)
Folate dependence of hyperhomocysteinemia and vascular dysfunction in cystathionine beta-synthase-deficient mice.
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Hyperhomocysteinemia is a risk factor for stroke, myocardial infarction, and venous thrombosis. Moderate hyperhomocysteinemia is associated with impaired endothelial function, but the mechanisms responsible for endothelial dysfunction in hyperhomocysteinemia are poorly understood. We have used genetic and dietary approaches to produce hyperhomocysteinemia in mice. Heterozygous cystathionine beta-synthase-deficient mice (CBS +/-), which have a selective defect in homocysteine transsulfuration, and wild-type (CBS +/+) littermates were fed either a control diet or a diet that is relatively deficient in folic acid for 6 wk. Plasma total homocysteine was 5.3 +/- 0.7 microM in CBS +/+ mice and 6.4 +/- 0.6 microM in CBS +/- mice (P = 0.3) given the control diet. Plasma total homocysteine was 11.6 +/- 4.5 microM in CBS +/+ mice and 25.1 +/- 3.2 microM in CBS +/- mice (P = 0.004) given a low-folate diet. In mice fed the control diet, relaxation of aortic rings in response to the endothelium-dependent vasodilator acetylcholine did not differ significantly between CBS +/+ mice and CBS +/- mice. In contrast, in mice fed a low-folate diet, maximal relaxation to acetylcholine was markedly impaired in CBS +/- mice (58 +/- 9%) compared with CBS +/+ mice (84 +/- 4%) (P = 0.01). No differences in relaxation to the endothelium-independent vasodilator sodium nitroprusside were observed among the four groups of mice. These data indicate that CBS-deficient mice are predisposed to hyperhomocysteinemia during dietary folate deficiency, and moderate hyperhomocysteinemia is associated with marked impairment of endothelial function in mice. (+info)
Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli.
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Significant derepression of serine hydroxymethyltransferase is observed when metE or metF mutants of Escherichia coli K-12 are grown on D-methionine sulfoxide instead of L-methionine. The derepression is not prevented by addition of glycine, adenosine, guanosine, guanosine, and thymidine to the growth medium of methionine-limited metF cells showing that the effect is not due to a secondary deficiency of these nutrients. On the other hand, methionine-limited growth of a metA mutant leads to derepression of met regulon enzymes, but only a marginal increase in serine hydroxymethyltransferase activity. A prototrophic metJ strain grown on minimal medium has about the same serine hydroxymethyltransferase as the wild type. The enzyme activity of the metJ strain is not influenced by methionine, but it is partially repressed by glycine, adenosine, and thymidine. metK strains have about twice as much serine hydroxymethyltransferase activity as wild-type cells when grown on minimal medium; but when both types of cells are grown on medium supplemented with glycine, adenosine, guanosine, and thymidine, their enzyme activities are about the same. The results show that methionine limitation can lead to depression of serine hydroxymethyltransferase, but that the regulatory system is different from the one which controls the methionine regulon. (+info)
Functional properties of the active core of human cystathionine beta-synthase crystals.
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Human cystathionine beta-synthase is a pyridoxal 5'-phosphate enzyme containing a heme binding domain and an S-adenosyl-l-methionine regulatory site. We have investigated by single crystal microspectrophotometry the functional properties of a mutant lacking the S-adenosylmethionine binding domain. Polarized absorption spectra indicate that oxidized and reduced hemes are reversibly formed. Exposure of the reduced form of enzyme crystals to carbon monoxide led to the complete release of the heme moiety. This process, which takes place reversibly and without apparent crystal damage, facilitates the preparation of a heme-free human enzyme. The heme-free enzyme crystals exhibited polarized absorption spectra typical of a pyridoxal 5'-phosphate-dependent protein. The exposure of these crystals to increasing concentrations of the natural substrate l-serine readily led to the formation of the key catalytic intermediate alpha-aminoacrylate. The dissociation constant of l-serine was found to be 6 mm, close to that determined in solution. The amount of the alpha-aminoacrylate Schiff base formed in the presence of l-serine was pH independent between 6 and 9. However, the rate of the disappearance of the alpha-aminoacrylate, likely forming pyruvate and ammonia, was found to increase at pH values higher than 8. Finally, in the presence of homocysteine the alpha-aminoacrylate-enzyme absorption band readily disappears with the concomitant formation of the absorption band of the internal aldimine, indicating that cystathionine beta-synthase crystals catalyze both beta-elimination and beta-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by cystathionine beta-synthase. (+info)