Redox cycles of caffeic acid, alpha-tocopherol, and ascorbate: implications for protection of low-density lipoproteins against oxidation

This study addresses the dynamic interactions among alpha-tocopherol, caffeic acid, and ascorbate in terms of a sequence of redox cycles aimed at accomplishing optimal synergistic antioxidant protection. Several experimental models were designed to examine these interactions: UV irradiation of alpha-tocopherol-containing sodium dodecyl sulfate micelles, one-electron oxidations catalyzed by the hypervalent state of myoglobin, ferrylmyoglobin, and autoxidation at appropriate pHs. These models were assessed by ultraviolet (UV) and electron paramagnetic resonance (EPR), entailing direct- and continuous-flow experiments, spectroscopy and by separation and identification of products by HPLC. The alpha-tocopheroxyl radical EPR signal generated by UV irradiation of alpha-tocopherol-containing micelles was suppressed by caffeic acid and ascorbate; in the former case, no other EPR signal was observed at pH 7.4, whereas in the latter case, the alpha-tocopheroxyl radical EPR signal was replaced by a doublet EPR spectrum corresponding to the ascorbyl radical (A*-). The potential interactions between caffeic acid and ascorbate were further analyzed by assessing, on the one hand, the ability of ascorbate to reduce the caffeic acid o-semiquinone (generated by oxidation of caffeic acid by ferrylmyoglobin) and, on the other hand, the ability of caffeic acid to reduce ascorbyl radical (generated by autoxidation or oxidation of ascorbate by ferrylmyoglobin). The data presented indicate that the reductive decay of ascorbyl radical (A*-) and caffeic acid o-semiquinone (Caf-O*) can be accomplished by caffeic acid (Caf-OH) and ascorbate (AH-), respectively, thus pointing to the reversibility of the reaction Caf-O* + AH- <--> Caf-OH + A*-. Continuous-flow EPR measurements of mixtures containing ferrylmyoglobin, alpha-tocopherol-containing micelles, caffeic acid, and ascorbate revealed that ascorbate is the ultimate electron donor in the sequence encompassing transfer of the radical character from the micellar phase to the phase. In independent experiments, the effects of caffeic acid and ascorbate on the oxidation of two low-density lipoprotein (LDL) populations, control and alpha-tocopherol-enriched, were studied and results indicated that alpha-tocopherol, caffeic acid, and ascorbate acted synergistically to afford optimal protection of LDL against oxidation. These results are analyzed for each individual antioxidant in terms of three domains: its localization and that of the antioxidant-derived radical, its reduction potential, and the predominant decay pathways for the antioxidant-derived radical, that exert kinetic control on the process.