Application of the capillary electrophoresis method for the study of plasma proteins homocysteinylation

Abstract

Purpose. This article describes the use of capillary electrophoresis with UV detection to determine the ratio of protein-bound homocysteine and cysteine concentrations in human plasma. Methods. Plasma samples were reduced with dithiothreitol and derivatized by thiocarbonyldiimidazole before being filtered again for purification of proteins. The pre-concentration of analytes was carried out directly in the capillary (48.5 cm in length and an inner diameter of 50 mkm) by NaOH post-injection. The eletrophoretic separation of analytes was carried out using 0.2 M ammonium acetate with 25 mM hexadecyltrimethylammonium bromide. Results. Limit of quantitation for homocysteine was 0.8 mkM, reproducible ratio of cysteine / homocysteine <5%, full analysis time 15 min. Conclusion. The ratio of bound cysteine to homocysteine is characterized by the same regularity as the ratio of their total content. It has a fairly high degree of correlation with the level of bound homocysteine and it is characterized by less variability than the level of total homocysteine. This has the advantage of use the bound cysteine/homocysteine ratio for assessing the risk of cardiovascular disease complications.