Oxidized LDL is characterized by adduct formation between apolipoprotein (apo)B-100 in the low density lipoprotein (LDL) particles and reactive aldehydes such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), which are decomposition products of lipid peroxidation. Since LDL isolated from human atherosclerotic lesions was also shown to have interacted with HNE and MDA and to have undergone oxidation, we initiated a study to structurally and functionally characterize LDL modification with both HNE and MDA and to compare these characteristics with those of LDL modified individually with HNE and MDA. Using concentrations of LDL and HNE that alone resulted in extensive particle aggregation, modification at pH 7.4 with HNE plus MDA at 20 mM and above, resulted in the prevention of particle aggregation and less aggregation of apoB-100. Increases in electrophoretic mobility and blockage of exposed lysine residues on apoB were approximately additive for LDL modified at pH 7.4 with 6 mM HNE and MDA up to 20 mM compared to LDL modified individually with HNE or individually with MDA. When 125I-labeled LDL modified at pH 7.4 with HNE and increasing amounts of MDA was incubated with J774 macrophages, the doubly modified LDL showed a linear increase in degradation and in ACAT stimulation. LDL modified with MDA alone showed first a reduction in degradation and ACAT stimulation, due to reduced recognition by the LDL receptor. This was followed by an increase in degradation when further modification with MDA was performed at pH 6.4, but no or little increase in degradation when performed at pH 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)