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 (greater than or equal to 30 mu M) 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 mu g/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. (C) 1997 Academic Press.