Different mechanisms are progressively recruited to promote Cu(II) reduction by isolated human low-density lipoprotein undergoing oxidation

The kinetics of Cu(II) reduction and its relationship to the process of low density lipoprotein (LDL) oxidation were investigated in isolated human LDL incubated with CuSO 4 by using the Cu(I) chelator and indicator dye bathocuproine disulfonate (BC). The inclusion of BC in the incubation medium containing isolated LDL and different concentrations of CuSO 4 revealed a biphasic kinetics of Cu(II) reduction consisting of an early phase followed by a plateau phase and a subsequent extensive reduction phase. The amount of Cu(I) formed during the early phase, as well as the rate of its generation, were strictly dependent on both the level of Cu(II) available (saturation was observed at 20 and 50 μM CuSO 4) and the concentration of α-tocopherol within native LDL particles. Artificial enrichment of LDL with different concentrations of α-tocopherol led to a parallel increase of both the amount of Cu(II) reduced and the rate of reduction. The late phase of Cu(II) reduction was strictly related to the availability of copper but was largely independent from α-tocopherol. Neither the amount of Cu(I) generated nor the rate of generation were saturated at concentrations of copper up to 100 μM. Comparable results were obtained by adding BC at different time-points to the LDL-copper mixture, in order to measure at the same time-points both the true rate of Cu(II) reduction and the generation of TBARS during the dynamic process of LDL oxidation. The rate of Cu(II) reduction was already high during the lag-phase of the LDL oxidation profile and progressively decreased as α-tocopherol concentration decreased. The subsequent increase in the rate of Cu(II) reduction paralleled the formation of TBARS during the extensive LDL oxidation phase. These results suggest that different mechanisms of Cu(II) reduction, namely α-tocopherol-dependent and independent (likely lipid peroxide-dependent), are progressively recruited during copper-promoted LDL oxidation.