Minimizing tocopherol-mediated radical phase transfer in low-density lipoprotein oxidation with an amphiphilic unsymmetrical azo initiator.

The antioxidant alpha-tocopherol (alpha-TOH) has been found to act as a pro-oxidant under many in vitro conditions. The observed tocopoherol-mediated peroxidation (TMP) is dependent on two primary factors. (1) Chain transfer: alpha-TO. radical reacts with lipid to form lipid peroxyl radicals. (2) Phase transfer: alpha-TOH can transport radical character into the lipoprotein. Given the limitations of existing initiators, there is a need for new compounds that avoid the requirement for alpha-TOH to act as a phase-transfer agent. We report here a study showing that the new unsymmetrical azo compound, C-8, initiates LDL lipid peroxidation without requirement for alpha-TOH. This initiator provides a steady source of free amphiphilic peroxyl radicals that efficiently initiates oxidation of alpha-TOH-depleted LDL at a rate comparable to that reported for the very reactive hydroxyl radical (.OH). With other initiators tested, unsymmetrical C-12 and C-16 and symmetrical C-0 and MeOAMVN, alpha-TOH-depleted LDL displayed significant resistance to oxidation. Results indicate that the amphiphilic nature of the unsymmetrical initiators increases their partitioning into lipoprotein depending on the hydrocarbon chain length, and the symmetrical azo initiators C-0 and MeOAMVN primarily remain in the aqueous phase. Evidence suggests that even when the phase-transfer activity of alpha-TOH is limited, with the use of an initiator such as C-8, the mechanism of peroxidation remains controlled by TMP chain-transfer activity.

Design of unsymmetrical azo initiators to increase radical generation efficiency in low-density lipoproteins.

Lipid peroxidation studies often employ the use of azo initiators to produce a slow, steady source of free radicals, but the lack of initiators capable of efficiently generating radicals in lipid regions has created persistent problems in these investigations. For example, experiments with symmetrical lipophilic or symmetical hydrophilic azo initiators increasingly suggest that their initiation mechanisms in low-density lipoproteins (LDL) rely upon the presence of alpha-tocopherol to mediate peroxidation. We report here the synthesis and study of the new unsymmetrical azo compounds SA-1, SA-2, C-16, C-12, and C-8 that decompose over a range of convenient temperatures and improve radical generation efficiency and access to lipid compartments. The half-life for decomposition (tau(1/2)) of the unsymmetrical initiators at 37 degrees C in methanol covered a range of 121 hours for SA-1, 77 hours for SA-2, and approximately 25 hours for the series C-16, C-12, and C-8. Agarose gel electrophoresis of LDL incubated with these unsymmetrical initiators supports the conclusion that the initiators associate with lipoprotein without disrupting integrity of the particle. The unsymmetical initiator C-8 when compared to symmetical hydrophilic initiator C-0 is capable of providing increased peroxidation of LDL, as monitored by formation of cholesteryl linoleate oxidation products and consumption of alpha-tocopherol. Efficiency of radical generation in lipophilic and hydrophilic compartments was found to be represented with the use of the radical scavenger combination alpha-tocopherol and uric acid, but not with the use of N,N'-Diphenyl-p-phenylenediamine (DPPD) and uric acid. These unsymmetrical initiators, when compared to the widely used symmetrical azo initiators, provide an advantage of free radical production, lipophilic access, and constant radical generation in the investigation of lipid peroxidation in low-density lipoproteins.