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 o f low density lipoprotein (LDL) oxidation were investigated in isolate d human LDL incubated with CuSO4, by using the Cu(I) chelator and indi cator dye bathocuproine disulfonate (EC). The inclusion of BC in the i ncubation medium containing isolated LDL and different concentrations of CuSO4, revealed a biphasic kinetics of Cu(II) reduction consisting of an early phase followed by a plateau phase and a subsequent extensi ve reduction phase. The amount of Cu(I) formed during the early phase, as well as the rate of its generation, were strictly dependent on bot h the level of Cu(II) available (saturation was observed at 20 and 50 mu M CuSO4) and the concentration of alpha-tocopherol within native LD L particles. Artificial enrichment of LDL with different concentration s of alpha-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) re duction was strictly related to the availability of copper but was lar gely independent from alpha-tocopherol. Neither the amount of Cu(I) ge nerated nor the rate of generation were saturated at concentrations of copper up to 100 mu M. Comparable results were obtained by adding EC at different time-points to the LDL-copper mixture, in order to measur e at the same time-points both the true rate of Cu(II) reduction and t he generation of TEARS during the dynamic process of LDL oxidation. Th e rate of Cu(II) reduction was already high during the lag-phase of th e LDL oxidation profile and progressively decreased as alpha-tocophero l concentration decreased. The subsequent increase in the rate of Cu(I I) reduction paralleled the formation of TEARS during the extensive LD L oxidation phase. These results suggest that different mechanisms of Cu(II) reduction, namely alpha-tocopherol-dependent and independent (l ikely lipid peroxide-dependent), are progressively recruited during co pper-promoted LDL oxidation.