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Hieronim Jakubowski - One of the best experts on this subject based on the ideXlab platform.
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Implications for Atherosclerosis
2016Co-Authors: In Human Endothelial Cells, Arlene Bardeguez, Hieronim Jakubowski, Li Zhang, Abram AvivAbstract:Abstract—Editing of the nonprotein amino acid Homocysteine by certain aminoacyl-tRNA synthetases results in the formation of the thioester Homocysteine thiolactone. Here we show that in the presence of physiological concentrations of Homocysteine, methionine, and folic acid, human umbilical vein endothelial cells efficiently convert Homocysteine to thiolactone. The extent of this conversion is directly proportional to Homocysteine concentration and inversely proportional to methionine concentration, suggesting involvement of methionyl-tRNA synthetase. Folic acid inhibits the synthesis of thiolactone by lowering Homocysteine and increasing methionine concentrations in endothelial cells. We also show that the extent of post-translational protein homocysteinylation increases with increasing Homocysteine levels but decreases with increasing folic acid and HDL levels in endothelial cell cultures. These data support a hypothesis that metabolic conversion of Homocysteine to thiolactone and protein homocysteinylation by thiolactone may play a role in Homocysteine-induced vascular damage. (Circ Res. 2000;87:45-51.) Key Words: Homocysteine n proteins n HDL lipoproteins n endothelial cells n atherosclerosis Elevated levels of the nonprotein amino acid homocys-teine (Hcy)1 are associated with vascular disease in humans.1 However, it is not known why Hcy can be harmful. The conversion of Hcy to thiolactone as a result of an error-editing function of some aminoacyl-tRNA synthetases (AARS in Equation 1)2,3 is one feature of Hcy metabolism that may account for detrimental effects of elevated Hcy levels
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the determination of Homocysteine thiolactone in human plasma
Analytical Biochemistry, 2005Co-Authors: Grazyna Chwatko, Hieronim JakubowskiAbstract:Abstract The thioester Homocysteine–thiolactone, a reactive metabolite of Homocysteine, has been implicated in human cardiovascular disease. However, data on the levels of Homocysteine–thiolactone in humans are limited, mostly due to a lack of facile and reliable assays. Here we describe a sensitive assay for the determination of plasma Homocysteine–thiolactone and demonstrate its utility with a cohort of 60 healthy human subjects. Plasma Homocysteine–thiolactone is first separated from macromolecules by ultrafiltration and then selectively extracted with chloroform/methanol. Further purification of plasma Homocysteine–thiolactone is achieved by high-performance liquid chromatography on a cation exchange microbore column. The detection and quantification is by monitoring fluorescence after postcolumn derivatization with o -phthaldialdehyde. The limit of detection is 0.36 nM. Using this assay, Homocysteine–thiolactone concentrations in plasma from normal healthy human subjects ( n = 60) were found to vary from zero to 34.8 nM, with an average of 2.82 ± 6.13 nM. In 29 of the 60 human plasma samples analyzed, Homocysteine–thiolactone levels were below the detection limit. Homocysteine–thiolactone represented from 0 to 0.28%, on average 0.023 ± 0.05%, of plasma total Homocysteine.
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the determination of Homocysteine thiolactone in biological samples
Analytical Biochemistry, 2002Co-Authors: Hieronim JakubowskiAbstract:Abstract Homocysteine-thiolactone, a cyclic thioester of Homocysteine, is synthesized by methionyl-tRNA synthetase in all cell types. A new assay for the determination of Homocysteine-thiolactone in biological samples is described. The assay involves separation of Homocysteine-thiolactone from macromolecules by ultrafiltration. Homocysteine-thiolactone is further purified and quantified by high-pressure liquid chromatography either on a reverse phase or a cation exchange micro-bore column. The detection and quantitation are obtained by monitoring the absorbance at 240 nm, a maximum in a UV spectrum of Homocysteine-thiolactone. The sensitivity of detection is 5 pmol. This assay has been applied to bacteria (Escherichia coli and Mycobacterium smegmatis), the yeast Saccharomyces cerevisiae, cultured human vascular endothelial cells, and human plasma. The data support the conclusion that Homocysteine-thiolactone is a ubiquitous metabolite whose levels are directly related to Homocysteine levels.
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Homocysteine thiolactone and protein homocysteinylation in human endothelial cells implications for atherosclerosis
Circulation Research, 2000Co-Authors: Hieronim Jakubowski, Arlene Bardeguez, Li Zhang, Abram AvivAbstract:Abstract —Editing of the nonprotein amino acid Homocysteine by certain aminoacyl-tRNA synthetases results in the formation of the thioester Homocysteine thiolactone. Here we show that in the presence of physiological concentrations of Homocysteine, methionine, and folic acid, human umbilical vein endothelial cells efficiently convert Homocysteine to thiolactone. The extent of this conversion is directly proportional to Homocysteine concentration and inversely proportional to methionine concentration, suggesting involvement of methionyl-tRNA synthetase. Folic acid inhibits the synthesis of thiolactone by lowering Homocysteine and increasing methionine concentrations in endothelial cells. We also show that the extent of post-translational protein homocysteinylation increases with increasing Homocysteine levels but decreases with increasing folic acid and HDL levels in endothelial cell cultures. These data support a hypothesis that metabolic conversion of Homocysteine to thiolactone and protein homocysteinylation by thiolactone may play a role in Homocysteine-induced vascular damage. ( Circ Res. 2000;87:45-51.)
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Homocysteine thiolactone metabolic origin and protein homocysteinylation in humans
Journal of Nutrition, 2000Co-Authors: Hieronim JakubowskiAbstract:Homocysteine thiolactone, an intramolecular thioester of Homocysteine, is synthesized by methionyl-tRNA synthetase in an error-editing reaction that prevents translational incorporation of Homocysteine into proteins. The synthesis of thiolactone occurs in all human cell types investigated. An increase in Homocysteine levels leads to elevation of thiolactone levels in human cells. In cultured human cells and in human serum, Homocysteine thiolactone reacts with proteins by a mechanism involving homocysteinylation of protein lysine residues. The homocysteinylation leads to protein damage. A calcium-dependent Homocysteine thiolactonase, tightly associated with HDL in human serum, may prevent protein damage by detoxifying thiolactone.
Joy Joseph - One of the best experts on this subject based on the ideXlab platform.
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generation and initial characterization of a novel polyclonal antibody directed against Homocysteine thiolactone modified low density lipoprotein
Journal of Lipid Research, 1998Co-Authors: Eric Ferguson, Sampath Parthasarathy, Joy JosephAbstract:Elevated plasma Homocysteine (homocystein- emia) are presumed to be responsible for the development of coronary artery disease, however, the precise etiology is un- clear. We examined the possibility that the adduct formed from the reaction between Homocysteine thiolactone, a meta- bolic product of Homocysteine, and apolipoprotein B-100 ly- syl residues of low density lipoprotein (LDL) was immuno- genic. New Zealand White rabbits were immunized with this adduct at 6-week intervals. Antisera collected following the 3rd immunization was assayed for antibody titers using solid phase ELISA techniques. Titers (defined as the inverse of the greatest serum dilution in which there was a significant differ- ence ( P , 0.05) between the percentage antibody bound from the antiserum and the pre-immune serum) were ap- proximately 10 5 . In competition-based ELISAs, Homocysteine thiolactone-treated LDL competed for binding with the anti- serum, as the 50% inhibitory concentration was approxi- mately 10 m g/ml. Neither Homocysteine, homocystine (ho- mocysteine disulfide), nor Cu 2 1 -oxidized LDL competed for binding. LDL in which lysyl residues were derivatized by acety- lation or methylation were not recognized by the antiserum. Homocysteine thiolactone-treated plasma competed for bind- ing to the antiserum, whereas native plasma did not. All lipo- protein fractions from the Homocysteine thiolactone-treated plasma competed for binding to the antiserum. We con- clude that Homocysteine thiolactone-modified LDL is highly immunogenic and specific for Homocysteine thiolactone- modified lysines. The potential for using this antibody as a di- agnostic tool for measuring plasma Homocysteine concentra- tions and the implications for understanding diseases in- duced by Homocysteinemia are discussed. —Ferguson, E., S. Parthasarathy, J. Joseph, and B. Kalyanaraman. Generation and initial characterization of a novel polyclonal antibody di- rected against Homocysteine thiolactone-modified low density lipoprotein. J. Lipid Res. 1998. 39: 925-933.
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generation and initial characterization of a novel polyclonal antibody directed against Homocysteine thiolactone modified low density lipoprotein
Journal of Lipid Research, 1998Co-Authors: Eric Ferguson, Sampath Parthasarathy, Joy Joseph, Balaraman KalyanaramanAbstract:Elevated plasma Homocysteine (Homocysteinemia) are presumed to be responsible for the development of coronary artery disease, however, the precise etiology is unclear. We examined the possibility that the adduct formed from the reaction between Homocysteine thiolactone, a metabolic product of Homocysteine, and apolipoprotein B-100 lysyl residues of low density lipoprotein (LDL) was immunogenic. New Zealand White rabbits were immunized with this adduct at 6-week intervals. Antisera collected following the 3rd immunization was assayed for antibody titers using solid phase ELISA techniques. Titers (defined as the inverse of the greatest serum dilution in which there was a significant difference (P < 0.05) between the percentage antibody bound from the antiserum and the pre-immune serum) were approximately 10(5). In competition-based ELISAs, Homocysteine thiolactone-treated LDL competed for binding with the antiserum, as the 50% inhibitory concentration was approximately 10 microg/ml. Neither Homocysteine, homocystine (Homocysteine disulfide), nor Cu2-oxidized LDL competed for binding. LDL in which lysyl residues were derivatized by acetylation or methylation were not recognized by the antiserum. Homocysteine thiolactone-treated plasma competed for binding to the antiserum, whereas native plasma did not. All lipoprotein fractions from the Homocysteine thiolactone-treated plasma competed for binding to the antiserum. We conclude that Homocysteine thiolactone-modified LDL is highly immunogenic and specific for Homocysteine thiolactone-modified lysines. The potential for using this antibody as a diagnostic tool for measuring plasma Homocysteine concentrations and the implications for understanding diseases induced by Homocysteinemia are discussed.
Eric Ferguson - One of the best experts on this subject based on the ideXlab platform.
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generation and initial characterization of a novel polyclonal antibody directed against Homocysteine thiolactone modified low density lipoprotein
Journal of Lipid Research, 1998Co-Authors: Eric Ferguson, Sampath Parthasarathy, Joy JosephAbstract:Elevated plasma Homocysteine (homocystein- emia) are presumed to be responsible for the development of coronary artery disease, however, the precise etiology is un- clear. We examined the possibility that the adduct formed from the reaction between Homocysteine thiolactone, a meta- bolic product of Homocysteine, and apolipoprotein B-100 ly- syl residues of low density lipoprotein (LDL) was immuno- genic. New Zealand White rabbits were immunized with this adduct at 6-week intervals. Antisera collected following the 3rd immunization was assayed for antibody titers using solid phase ELISA techniques. Titers (defined as the inverse of the greatest serum dilution in which there was a significant differ- ence ( P , 0.05) between the percentage antibody bound from the antiserum and the pre-immune serum) were ap- proximately 10 5 . In competition-based ELISAs, Homocysteine thiolactone-treated LDL competed for binding with the anti- serum, as the 50% inhibitory concentration was approxi- mately 10 m g/ml. Neither Homocysteine, homocystine (ho- mocysteine disulfide), nor Cu 2 1 -oxidized LDL competed for binding. LDL in which lysyl residues were derivatized by acety- lation or methylation were not recognized by the antiserum. Homocysteine thiolactone-treated plasma competed for bind- ing to the antiserum, whereas native plasma did not. All lipo- protein fractions from the Homocysteine thiolactone-treated plasma competed for binding to the antiserum. We con- clude that Homocysteine thiolactone-modified LDL is highly immunogenic and specific for Homocysteine thiolactone- modified lysines. The potential for using this antibody as a di- agnostic tool for measuring plasma Homocysteine concentra- tions and the implications for understanding diseases in- duced by Homocysteinemia are discussed. —Ferguson, E., S. Parthasarathy, J. Joseph, and B. Kalyanaraman. Generation and initial characterization of a novel polyclonal antibody di- rected against Homocysteine thiolactone-modified low density lipoprotein. J. Lipid Res. 1998. 39: 925-933.
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generation and initial characterization of a novel polyclonal antibody directed against Homocysteine thiolactone modified low density lipoprotein
Journal of Lipid Research, 1998Co-Authors: Eric Ferguson, Sampath Parthasarathy, Joy Joseph, Balaraman KalyanaramanAbstract:Elevated plasma Homocysteine (Homocysteinemia) are presumed to be responsible for the development of coronary artery disease, however, the precise etiology is unclear. We examined the possibility that the adduct formed from the reaction between Homocysteine thiolactone, a metabolic product of Homocysteine, and apolipoprotein B-100 lysyl residues of low density lipoprotein (LDL) was immunogenic. New Zealand White rabbits were immunized with this adduct at 6-week intervals. Antisera collected following the 3rd immunization was assayed for antibody titers using solid phase ELISA techniques. Titers (defined as the inverse of the greatest serum dilution in which there was a significant difference (P < 0.05) between the percentage antibody bound from the antiserum and the pre-immune serum) were approximately 10(5). In competition-based ELISAs, Homocysteine thiolactone-treated LDL competed for binding with the antiserum, as the 50% inhibitory concentration was approximately 10 microg/ml. Neither Homocysteine, homocystine (Homocysteine disulfide), nor Cu2-oxidized LDL competed for binding. LDL in which lysyl residues were derivatized by acetylation or methylation were not recognized by the antiserum. Homocysteine thiolactone-treated plasma competed for binding to the antiserum, whereas native plasma did not. All lipoprotein fractions from the Homocysteine thiolactone-treated plasma competed for binding to the antiserum. We conclude that Homocysteine thiolactone-modified LDL is highly immunogenic and specific for Homocysteine thiolactone-modified lysines. The potential for using this antibody as a diagnostic tool for measuring plasma Homocysteine concentrations and the implications for understanding diseases induced by Homocysteinemia are discussed.
Changyi Chen - One of the best experts on this subject based on the ideXlab platform.
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ginkgolide a attenuates Homocysteine induced endothelial dysfunction in porcine coronary arteries
Journal of Vascular Surgery, 2006Co-Authors: Wei Zhou, Andy Courson, Hong Chai, Alan B. Lumsden, Changyi ChenAbstract:Background Homocysteine is an independent risk factor for atherosclerosis. The objective of this study was to investigate whether ginkgolide A (GA), a major constituent of Ginkgo biloba , could block Homocysteine-induced endothelial dysfunction in porcine coronary arteries. Methods Porcine coronary artery rings were assigned to six treatment groups: control; Homocysteine (50 μmol/L); low-dose (50 μmol/L) or high-dose (100 μmol/L) GA; and Homocysteine plus low-dose or high-dose GA. After 24 hours' incubation, the rings were analyzed for vasomotor function in response to a thromboxane A2 analogue (U46619), bradykinin, and sodium nitroprusside. Endothelial nitric oxide synthase (eNOS) was studied by using real-time polymerase chain reaction and immunohistochemistry analysis. Superoxide anion production was assessed by chemoluminescence analysis. Results Endothelium-dependent relaxation (bradykinin) was significantly reduced in ring segments treated with Homocysteine as compared with the control ( P P Conclusions Homocysteine significantly impairs endothelium-dependent vasorelaxation through oxidative stress and downregulation of eNOS in porcine coronary arteries. GA effectively prevents Homocysteine-induced endothelial dysfunction and molecular changes in porcine coronary arteries. This study underscores the potential clinical benefits and applications of GA in controlling Homocysteine-associated vascular injury and cardiovascular disease.
Per Magne Ueland - One of the best experts on this subject based on the ideXlab platform.
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Homocysteine and cardiovascular risk the perils of reductionism in a complex system
Clinical Chemistry, 2012Co-Authors: Per Magne Ueland, Joseph LoscalzoAbstract:Patients with genetic defects causing severe hyperHomocysteinemia have a high rate of mortality from cardiovascular diseases at an early age, irrespective of the specific genetic lesion. Long-term therapy with B vitamins or betaine in patients with homocystinuria due to cystathionine β-synthase deficiency decreases Homocysteine and reduces the rate of vascular events by 90%. Kilmer McCully studied the vascular pathology in young patients who died with homocystinuria and in 1969 proposed that a moderate increase in circulating Homocysteine could be involved in the pathogenesis of vascular occlusive disease in the general population, i.e., the so-called Homocysteine theory of atherosclerosis. This theory was investigated in the early 1990s in many clinical and epidemiologic studies. A metaanalysis of studies of Homocysteine concentration and vascular risk concluded that an increase of 0.68 mg/L (5 μmol/L) in plasma Homocysteine had an impact on cardiovascular risk that was equivalent to a 19-mg/dL (0.5-mmol/L) increase in total cholesterol. In general, the risk estimates from the prospective studies are weaker than those obtained from case–control and cross-sectional studies (1, 2), yet they clearly support plasma Homocysteine as an independent cardiovascular risk factor. The possible role of Homocysteine in the development of vascular disease inspired research on the biochemical and molecular effects on the vasculature of Homocysteine and the determinants of circulating Homocysteine concentrations. Experimental studies revealed numerous Homocysteine effects relevant to the pathogenesis of vascular disease, including induction of endothelial dysfunction, inhibition of biological methylation through accumulation of S -adenosylHomocysteine, homocysteinylation and posttranslational modification of proteins, increased oxidant stress, and decreased availability of nitric oxide (1). In addition, plasma Homocysteine positively correlates with a variety of biochemical parameters, clinical traits, and lifestyle factors associated with increased cardiovascular risk, including dyslipidemia, increased blood pressure, impaired renal function, a sedentary …
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plasma Homocysteine as a risk factor for vascular disease the european concerted action project
JAMA, 1997Co-Authors: Ian Graham, Per Magne Ueland, L Daly, Helga Refsum, Killian Robinson, L Brattstrom, Roberto Palmareis, G H J Boers, Richard G Sheahan, Bo IsraelssonAbstract:Context. —Elevated plasma Homocysteine is a known risk factor for atherosclerotic vascular disease, but the strength of the relationship and the interaction of plasma Homocysteine with other risk factors are unclear. Objective. —To establish the magnitude of the vascular disease risk associated with an increased plasma Homocysteine level and to examine interaction effects between elevated plasma Homocysteine level and conventional risk factors. Design. —Case-control study. Setting. —Nineteen centers in 9 European countries. Patients. —A total of 750 cases of atherosclerotic vascular disease (cardiac, cerebral, and peripheral) and 800 controls of both sexes younger than 60 years. Measurements. —Plasma total Homocysteine was measured while subjects were fasting and after a standardized methionine-loading test, which involves the administration of 100 mg of methionine per kilogram and stresses the metabolic pathway responsible for the irreversible degradation of Homocysteine. Plasma cobalamin, pyridoxal 5'-phosphate, red blood cell folate, serum cholesterol, smoking, and blood pressure were also measured. Results. —The relative risk for vascular disease in the top fifth compared with the bottom four fifths of the control fasting total Homocysteine distribution was 2.2 (95% confidence interval, 1.6-2.9). Methionine loading identified an additional 27% of atrisk cases. A dose-response effect was noted between total Homocysteine level and risk. The risk was similar to and independent of that of other risk factors, but interaction effects were noted between Homocysteine and these risk factors; for both sexes combined, an increased fasting Homocysteine level showed a more than multiplicative effect on risk in smokers and in hypertensive subjects. Red blood cell folate, cobalamin, and pyridoxal phosphate, all of which modulate Homocysteine metabolism, were inversely related to total Homocysteine levels. Compared with nonusers of vitamin supplements, the small number of subjects taking such vitamins appeared to have a substantially lower risk of vascular disease, a proportion of which was attributable to lower plasma Homocysteine levels. Conclusions. —An increased plasma total Homocysteine level confers an independent risk of vascular disease similar to that of smoking or hyperlipidemia. It powerfully increases the risk associated with smoking and hypertension. It is time to undertake randomized controlled trials of the effect of vitamins that reduce plasma Homocysteine levels on vascular disease risk.
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total Homocysteine in plasma or serum methods and clinical applications
Clinical Chemistry, 1993Co-Authors: Per Magne Ueland, Sally P. Stabler, Helga Refsum, M R Malinow, A Andersson, R H AllenAbstract:Total Homocysteine is defined as the sum of all Homocysteine species in plasma/serum, including free and protein-bound forms. In the present review, we compare and evaluate several techniques for the determination of total Homocysteine. Because these assays include the conversion of all forms into a single species by reduction, the redistribution between free and protein-bound Homocysteine through disulfide interchange does not affect the results, and total Homocysteine can be measured in stored samples. Total Homocysteine in whole blood increases at room temperature because of a continuous production and release of Homocysteine from blood cells, but artificial increase is low if the blood sample is centrifuged within 1 h of collection or placed on ice. Different methods correlate well, and values between 5 and 15 mumol/L in fasting subjects are considered normal. Total Homocysteine in serum/plasma is increased markedly in patients with cobalamin or folate deficiency, and decreases only when they are treated with the deficient vitamin. Total Homocysteine is therefore of value for the diagnosis and follow-up of these deficiency states and may compensate for weaknesses of the traditional laboratory tests. In addition, total Homocysteine is an independent risk factor for premature cardiovascular diseases. These disorders justify introduction of the total Homocysteine assay in the routine clinical chemistry laboratory.