Apolipoprotein B carbonyl formation is enhanced by lipid peroxidation during copper-mediated oxidation of human low-density lipoproteins

To determine whether lipid peroxidation is required for apolipoprotein B (apoB) carbonyl formation of human low-density lipoproteins (LDL) during copper-mediated oxidation, we investigated oxidation of native and probucol- preloaded LDL by measuring thiobarbituric acid-reactive substances (TBARS) and apoB carbonyls. Probucol was used because it is known to inhibit lipid peroxidation, but not protein modification. During copper-mediated oxidation, apoB carbonyls formed in a time-dependent manner; high copper concentrations (≤30 μM) resulted in saturation of apoB carbonyl content. ApoB carbonyl formation and lipid peroxidation were linearly related during incubation of LDL with copper for 3 h. During Cu²⁺-mediated LDL oxidation of probucol- LDL, TBARS production was very low, nonetheless apoB carbonyls increased significantly, and vitamin E was depleted. Bovine serum albumin (fatty acid free; BSA) oxidation in the presence of trace amounts of LDL, linoleic acid, or tert-butyl hydroperoxide was used to further understand the role of lipid peroxidation in apoB carbonyl formation. Protein carbonyl formation during BSA incubation with copper (either Cu⁺ or Cu²⁺) was trivial; however, further addition of linoleic acid (1:1, m/m), trace amounts of LDL (10 μg/ml), or tert-butyl hydroperoxide (1:1, m/m) markedly increased protein carbonyl formation. These results demonstrate that lipid peroxidation enhances copper-mediated carbonyl formation and suggest that copper ions react with LDL lipid hydroperoxides producing the necessary reactive species.