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

Previous reports proposed that peroxynitrite (ONOO-) oxidizes alpha-tocopherol (alpha-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 alpha-TOH and gamma-tocopherol (gamma-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)-N-.), ascorbate, and CO2 were used as key biologically relevant modulators of ONOO- reactivity. Although similar to80% alpha-TOH and gamma-TOH depletion occurred within 5 min of incubation of 0.8 muM LDL with a 60 muM bolus of ONOO-, an equimolar infusion of ONOO- over 60 min caused total consumption of both antioxidants. gamma-Tocopherol was preserved relative to alpha-TOH, probably due to gamma-tocopheroxyl radical recycling by alpha-TOH. alpha-TOH oxidation in LDL was first order in ONOO- with similar to12% of ONOO- maximally available. Physiological concentrations of (NO)-N-. and ascorbate spared both alpha-TOH and gamma-TOH through independent and additive mechanisms. High concentrations of (NO)-N-. and ascorbate abolished alpha-TOH and gamma-TOH oxidation. Nitric oxide protection was more efficient for alpha-TOH in LDL than for ascorbate in solution, evidencing the kinetically highly favored reaction of lipid peroxyl radicals with (NO)-N-. than with alpha-TOH as assessed by computer-assisted simulations. In addition, CO2 (1.2 mM) inhibited both alpha-TOH and lipid oxidation. These results demonstrate that ONOO- induces alpha-TOH oxidation in LDL through a one-electron free radical mechanism; thus the inhibitory actions of (NO)-N-. and ascorbate may determine low alpha-tocopheryl quitione accumulation in tissues despite increased ONOO- generation. (C) 2003 Elsevier Inc. All rights reserved.