L-Homocysteine

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Hieronim Jakubowski - One of the best experts on this subject based on the ideXlab platform.

  • Implications for Atherosclerosis
    2016
    Co-Authors: In Human Endothelial Cells, Arlene Bardeguez, Hieronim Jakubowski, Li Zhang, Abram Aviv
    Abstract:

    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

  • the determination of homocysteine thiolactone in human plasma
    Analytical Biochemistry, 2005
    Co-Authors: Grazyna Chwatko, Hieronim Jakubowski
    Abstract:

    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.

  • the determination of homocysteine thiolactone in biological samples
    Analytical Biochemistry, 2002
    Co-Authors: Hieronim Jakubowski
    Abstract:

    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.

  • homocysteine thiolactone and protein homocysteinylation in human endothelial cells implications for atherosclerosis
    Circulation Research, 2000
    Co-Authors: Hieronim Jakubowski, Arlene Bardeguez, Li Zhang, Abram Aviv
    Abstract:

    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.)

  • homocysteine thiolactone metabolic origin and protein homocysteinylation in humans
    Journal of Nutrition, 2000
    Co-Authors: Hieronim Jakubowski
    Abstract:

    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.

  • generation and initial characterization of a novel polyclonal antibody directed against homocysteine thiolactone modified low density lipoprotein
    Journal of Lipid Research, 1998
    Co-Authors: Eric Ferguson, Sampath Parthasarathy, Joy Joseph
    Abstract:

    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.

  • generation and initial characterization of a novel polyclonal antibody directed against homocysteine thiolactone modified low density lipoprotein
    Journal of Lipid Research, 1998
    Co-Authors: Eric Ferguson, Sampath Parthasarathy, Joy Joseph, Balaraman Kalyanaraman
    Abstract:

    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.

  • generation and initial characterization of a novel polyclonal antibody directed against homocysteine thiolactone modified low density lipoprotein
    Journal of Lipid Research, 1998
    Co-Authors: Eric Ferguson, Sampath Parthasarathy, Joy Joseph
    Abstract:

    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.

  • generation and initial characterization of a novel polyclonal antibody directed against homocysteine thiolactone modified low density lipoprotein
    Journal of Lipid Research, 1998
    Co-Authors: Eric Ferguson, Sampath Parthasarathy, Joy Joseph, Balaraman Kalyanaraman
    Abstract:

    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.

Sampath Parthasarathy - One of the best experts on this subject based on the ideXlab platform.

  • generation and initial characterization of a novel polyclonal antibody directed against homocysteine thiolactone modified low density lipoprotein
    Journal of Lipid Research, 1998
    Co-Authors: Eric Ferguson, Sampath Parthasarathy, Joy Joseph
    Abstract:

    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.

  • generation and initial characterization of a novel polyclonal antibody directed against homocysteine thiolactone modified low density lipoprotein
    Journal of Lipid Research, 1998
    Co-Authors: Eric Ferguson, Sampath Parthasarathy, Joy Joseph, Balaraman Kalyanaraman
    Abstract:

    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.

Balaraman Kalyanaraman - One of the best experts on this subject based on the ideXlab platform.

  • generation and initial characterization of a novel polyclonal antibody directed against homocysteine thiolactone modified low density lipoprotein
    Journal of Lipid Research, 1998
    Co-Authors: Eric Ferguson, Sampath Parthasarathy, Joy Joseph, Balaraman Kalyanaraman
    Abstract:

    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.