Oxidation of low density lipoprotein (LDL) has been shown to occur in the artery wall of atherosclerotic lesions in both animal models and human arteries. The oxidant(s) responsible for initiating this process are under intensive investigation and 15-lipoxygenase has been suggested in this context. Another possibility is that nitric oxide and superoxide, generated by cells present in the artery wall, react together to form peroxynitrite which decomposes to form the highly reactive hydroxyl radical. In the present study we have modelled the simultaneous generation of superoxide and nitric oxide by using the sydnonimine, SIN-1 and have investigated its effects on LDL. SIN-1 liberates both superoxide and nitric oxide during autooxidation resulting in the formation of hydroxyl radicals. We have demonstrated that superoxide generated by SIN-1 is not available to take part in a dismutation reaction since it reacts preferentially with nitric oxide. It follows, therefore, that during the autooxidation of SIN-1 little or no superoxide, or perhydroxyl radical will be available to initiate lipid peroxidation. We have shown that SIN-1 is capable of initiating the peroxidation of LDL and also converts the lipoprotein to a more negatively charged form. The SIN-1-dependent peroxidation of LDL is completely inhibited by superoxide dismutase which scavenges superoxide. Neither sodium nitroprusside or S-nitroso-N-acetyl penicillamine, which only produce nitric oxide, are able to modify LDL. These results are consistent with the hypothesis that a product of superoxide and nitric oxide could oxidize lipoproteins in the artery wall and so contribute to the pathogenesis of atherosclerosis in vivo.

Peroxynitrite is an oxidant which could be formed in the vasculature by the reaction of superoxide with nitric oxide. It is capable of modifying amino acid residues and of initiating lipid peroxidation. In the present study we have shown that peroxynitrite converts low density lipoprotein to a form recognized by the macrophage scavenger receptor and that this process is associated with modification of the protein and lipid, and with the oxidation of a-tocopherol to a-tocopherol quinone.

It has been proposed that lipoxygenases, specifically 15-lipoxygenase, may play an important role in promoting the oxidation of low-density lipoprotein (LDL) in the artery wall. It is well known that peroxides are unstable in the presence of transition metals, decomposing to form the alkoxy and peroxy radicals, and so initiating lipid peroxidation. To test whether lipoxygenase-derived peroxides may promote the oxidation of LDL in the presence of copper, the lipoprotein was enriched with lipid peroxides derived from the enzymic action of 5- and 15-lipoxygenases on either linoleic or arachidonic acid. All of these products were found to promote oxidation, whereas the related hydroxy fatty acids had no effect. This suggests that lipoxygenase-derived peroxides associated with the LDL particle may promote peroxidation in the presence of a suitable transition metal catalyst. This result has implications both for the mechanism of the potential pro-oxidant action of lipoxygenases in vivo and for the ex vivo assessment of the oxidizability of LDL samples isolated from different donors.

Macrophages derived from the human monocyte cell line THP-1 or isolated from the peritoneum of C3H/HEJ mice were incubated with oxidized low-density lipoprotein (LDL) and the total glutathione content (oxidized plus reduced) was measured. An initial depletion of glutathione was followed by an increase, such that after a period of 24 h the glutathione content has approximately doubled. This response required the oxidation of the lipid phase of the LDL molecule, since both native LDL and acetylated LDL had little effect on glutathione levels. The response of the cells to oxidized LDL was dependent on the extent of oxidative modification of the protein. It was also found that 4-hydroxynonenal had a similar effect on THP-1 cells, and we suggest that this or other aldehydes present in oxidized LDL causes the induction of glutathione synthesis in response to an initial oxidative stress and consequent glutathione depletion. In addition, we found that both cell types possess transferases and peroxidases capable of detoxifying aldehydes and peroxides. However, treatment of cells with oxidized LDL or 4-hydroxynonenal for a period of 24 h had no effect on the activities of these enzymes.

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