Reduction of copper, but not iron, by human low density lipoprotein (LDL). Implications for metal ion-dependent oxidative modification of LDL.

Cell-mediated oxidative modification of human low density lipoprotein (LDL), most likely an important early step in atherosclerosis, requires redox active metal ions such as copper or iron. We have previously shown that iron-dependent, in contrast to copper-dependent, oxidative modification of LDL requires superoxide, a physiological reductant. In the present study, we sought to explain these discrepant results. LDL was incubated at 37 degrees C with Cu2+ (10 microM) and bathocuproine (BC, 360 microM), an indicator molecule which specifically complexes Cu+, but not Cu2+. In a time- and concentration-dependent manner, LDL reduced Cu2+ to Cu+. An LDL concentration as low as 10 micrograms of protein/ml (about 20 nM) reduced about 7 microM Cu2+ within 1 h of incubation. Complexation of the Cu+ formed under these conditions with BC significantly inhibited oxidative modification of LDL, as assessed by agarose gel electrophoresis. Preincubation of LDL with N-ethylmaleimide had no effect on the rate and extent of Cu2+ reduction nor LDL oxidation, indicating that free sulfhydryl groups associated with apolipoprotein B are not involved. Addition of either superoxide dismutase or catalase or increasing the alpha-tocopherol content of LDL from 11.8 +/- 3.0 to 24.4 +/- 2.8 nmol/mg of protein also had no significant effect on the kinetics of Cu2+ reduction by LDL. In contrast, incubation of LDL with Fe(3+)-citrate (10 microM) and the indicator bathophenanthroline (BP, 360 microM) resulted in no significant Fe2+ formation, even at LDL concentrations as high as 200 micrograms of protein/ml. However, incubation of LDL with Fe(3+)-citrate and an enzymatic source of superoxide led to rapid formation of Fe2+ and consequent oxidative modification of LDL. Addition of BP inhibited iron-mediated LDL oxidation under these conditions. Our results indicate that reduced metal ions are important mediators of LDL oxidation, and that LDL specifically reduces Cu2+, but not Fe3+. These data, therefore, help explain why copper, in addition to being chemically more reactive, is more potent than iron at mediating LDL oxidation.