Inhibition of copper-induced LDL oxidation by vitamin C is associated withdecreased copper-binding to LDL and 2-oxo-histidine formation

Oxidatively modified low-density lipoprotein (LDL) has numerous atherogenic properties, and antioxidants that can prevent LDL oxidation may act as ant iatherogens. We have previously shown that vitamin C (L-ascorbic acid, AA) and its two-electron oxidation product dehydro-l-ascorbic acid (DHA) strong ly inhibit copper (Cu)-induced LDL oxidation. These findings are unusual, a s AA is known to act not only as an antioxidant, but also a pro-oxidant in the presence of transition metal ions in vitro, and DHA has no known reduci ng capacity. Here we report that human LDL (0.4 mg protein/ml) incubated wi th 40 mu M Cu2+ binds 28.0 @ 3.3 Cu ions per LDL particle (mean @ SD, n = 1 0). Go-incubation of LDL with AA or DHA led to the time- and concentration- dependent release of up to 70% of bound Cu, which was associated with the i nhibition of LDL oxidation. Incubation of LDL with Cu and AA or DHA also le d to the time-dependent formation of 2-oxo-histidine, an oxidized derivativ e of histidine with a low affinity for Cu. Addition of free histidine preve nted the formation of the LDL-Cu complexes and inhibited LDL oxidation, des pite the fact that Cu remained redox-active. Interestingly, histidine was m ore effective than AA or DHA at limiting Cu binding to LDL, but at low conc entrations AA and DHA were more effective than histidine at inhibiting LDL oxidation. These data suggest that there are at least two types of Cu bindi ng sites on LDL: those that bind Cu in a redox-active form critical for ini tiation of LDL oxidation, and those that bind Cu ina redox-inactive form no t contributing to LDL oxidation. The former sites may be primarily histidin e residues of apolipoprotein B-100 that are oxidized to 2-oxo-histidine in the presence of Cu and AA or DHA, thus explaining, at least in part, the un usual inhibitory effect of vitamin C on Cu-induced LDL oxidation. (C) 1998 Elsevier Science Inc.