In vivo and in vitro evaluation of post-translational LDL apoprotein B modification induced by homocysteine

Covalent alterations of LDL by acetylation, oxidation or glycation, increase lipoprotein uptake and cholesterol deposition within cultured cells. Besides, oxidized or glycated LDL induce lipid peroxidation in cultured cells. N-homocysteinylation of LDL has been described as proatherogeinc event since Hcy-N-LDL show a cytotoxic effect that is likely related to an increase in lipid peroxidation and oxidative damage of endothelial cells and multimeric aggregation. Despite the plentiful data about the other covalent modification of LDL the S-homocysteynilation of lipoprotein has been at now slightly studied. By this work we propose the first method able to measure simultaneously all thiols linked to LDL apoprotein. This allows us to investigate on the levels of thiols bound to LDL in a group of healthy subjects to define the normal range of this thiol in vivo. Besides we describe the ability of Hcy to bind LDL in vitro also displacing the other thiols linked to apoprotein. Moreover for the first time we show the citotoxic effect of LDL on endothelial cells demonstrated by the decrease proliferation and viability of cells incubated with homocysteinylated LDL. Even though the mechanism by which Hcy-S-LDL exert their toxic effect still remains to be elucidated, our data suggest that intracellular ROS production stimulated after incubation with Hcy-S-LDL may have some relevance.