Characterization of aggregated low density lipoproteins induced by copper-catalyzed oxidation

Oxidation of low density lipoproteins (LDL) has been shown to lead to enhanced uptake by macrophages mediated by the scavenger receptor. In the present study, changes in LDL induced by copper-catalyzed oxidation were investigated using gel permeation chromatography (GPC), and the results were compared with several parameters of oxidized LDL (ox-LDL). When LDL at 200 micrograms/ml was oxidized with 10 microM Cu2+ at 37 degrees C for up to 24 hours, increases in thiobarbituric acid-reactive substances and electrophoretic mobility were first observed within 3 hours. An increase in fluorescence and a decrease in intact apolipoprotein B (apoB) were than observed in parallel with an increase in 125I-LDL degradation by macrophages after 6 hours. Finally, LDL aggregation separated by liquid chromatography was observed after 24 hours. The aggregated and monomeric fractions of ox-LDL were analyzed and the results compared with the monomeric fraction of native LDL. Both fractions of ox-LDL contained hardly any intact apoB and showed an intense fluorescence. The electrophoretic mobility increment of aggregated ox-LDL was almost half that of monomeric ox-LDL, yet the lysine residues of aggregated ox-LDL were more extensively decreased than those of monomeric ox-LDL. Degradation of aggregated ox-LDL by macrophages showed a slightly greater increase than that of monomeric ox-LDL. GPC analysis is a useful method to estimate the LDL aggregation, and these results provide a basis to investigate the formation of aggregated LDL.