Peroxynitrite-mediated α-tocopherol oxidation in low-density lipoprotein: a mechanistic approach

Previous reports proposed that peroxynitrite (ONOO−) oxidizes α-tocopherol (α-TOH) through a two-electron concerted mechanism. In contrast, ONOO− oxidizes phenols via free radicals arising from peroxo bond homolysis. To understand the kinetics and mechanism of α-TOH and γ-tocopherol (γ-TOH) oxidation in low-density lipoprotein (LDL) (direct vs. radical), we exposed LDL to ONOO− added as a bolus or an infusion. Nitric oxide (•NO), ascorbate and CO2 were used as key biologically relevant modulators of ONOO− reactivity. Although 80% α-TOH and γ-TOH depletion occurred within 5 min of incubation of 0.8 μM LDL with a 60 μM bolus of ONOO−, an equimolar infusion of ONOO− over 60 min caused total consumption of both antioxidants. γ-Tocopherol was preserved relative to α-TOH, probably due to γ-tocopheroxyl radical recycling by α-TOH. α-TOH oxidation in LDL was first order in ONOO− with 12% of ONOO− maximally available. Physiological concentrations of •NO and ascorbate spared both α-TOH and γ-TOH through independent and additive mechanisms. High concentrations of •NO and ascorbate abolished α-TOH and γ-TOH oxidation. Nitric oxide protection was more efficient for α-TOH in LDL than for ascorbate in solution, evidencing the kinetically highly favored reaction of lipid peroxyl radicals with •NO than with α-TOH as assessed by computer-assisted simulations. In addition, CO2 (1.2 mM) inhibited both α-TOH and lipid oxidation. These results demonstrate that ONOO− induces α-TOH oxidation in LDL through a one-electron free radical mechanism; thus the inhibitory actions of •NO and ascorbate may determine low α-tocopheryl quinone accumulation in tissues despite increased ONOO− generation.