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, caffe ic acid, and ascorbate in terms of a sequence of redox cycles aimed at acco mplishing 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 oxidat ions 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 conti nuous-flow experiments, spectroscopy and by separation and identification o f products by HPLC. The alpha-tocopheroxyl radical EPR signal generated by UV irradiation of alpha-tocopherol-containing micelles was suppressed by ca ffeic acid and ascorbate; in the former case, no other EPR signal was obser ved at pH 7.4, whereas in the latter case, the alpha-tocopheroxyl radical E PR signal was replaced by a doublet EPR spectrum corresponding to the ascor byl radical (A(.-)). The potential interactions between caffeic acid and as corbate 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 oxid ation of ascorbate by ferrylmyoglobin). The data presented indicate that th e reductive decay of ascorbyl radical (A(.-)) and caffeic acid o-semiquinon e (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 mic ellar phase to the phase. In independent experiments, the effects of caffei c acid and ascorbate on the oxidation of two low-density lipoprotein (LDL) populations, control and alpha-tocopherol-enriched, were studied and result s indicated that alpha-tocopherol, caffeic acid, and ascorbate acted synerg istically to afford optimal protection of LDL against oxidation. These results are analyzed for each individual antioxidant in terms of thre e domains: its localization and that of the antioxidant-derived radical, it s reduction potential, and the predominant decay pathways for the antioxida nt-derived radical, that exert kinetic control on the process.