An on-column derivatization method for the determination of homocysteine-thiolactone and protein N-linked homocysteine |
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Authors: | Rafał Głowacki Edward Bald Hieronim Jakubowski |
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Affiliation: | 1.Department of Environmental Chemistry,University of ?ód?,Lodz,Poland;2.Department of Microbiology and Molecular Genetics,UMDNJ-New Jersey Medical School, International Center for Public Health,Newark,USA;3.Institute of Bioorganic Chemistry, Polish Academy of Sciences,Poznan,Poland;4.Department of Biochemistry and Biotechnology,University of Life Sciences,Poznan,Poland |
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Abstract: | Homocysteine (Hcy) is incorporated into protein via a reaction of the thioester Hcy-thiolactone with ε-amino group of a protein lysine residue generating N-Hcy-protein. This reaction impairs and alters protein’s function and has been implicated in atherothrombotic disease. Here, we describe new high-performance liquid chromatography assays for the determination of Hcy-thiolactone, protein N-linked Hcy, and Hcy based on an on-column derivatization with o-phthaldialdehyde and fluorescence detection. The on-column derivatization generates narrow peaks, which allows fast run times (3–5 min) and facilitates determination of N-linked Hcy directly from acid hydrolysates of plasma protein. Utility of these assays was demonstrated with human urine and plasma samples. |
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