Peroxynitrite-mediated alpha-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), ascorbate and CO2 were used as key biologically relevant modulators of ONOO- reactivity. Although approximately 80% alpha-TOH and gamma-TOH depletion occurred within 5 min of incubation of 0.8 microM LDL with a 60 microM 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 approximately 12% of ONOO- maximally available. Physiological concentrations of.NO and ascorbate spared both alpha-TOH and gamma-TOH through independent and additive mechanisms. High concentrations of.NO 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 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 and ascorbate may determine low alpha-tocopheryl quinone accumulation in tissues despite increased ONOO- generation.
Botti
Horacio
H
Departamento de Bioqui;mica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
Batthyány
Carlos
C
Trostchansky
Andrés
A
Radi
Rafael
R
Freeman
Bruce A
BA
Rubbo
Homero
H
D001205:Ascorbic Acid / Q000494:pharmacology; D002245:Carbon Dioxide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D015227:Lipid Peroxidation / Q000187:drug effects*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D009569:Nitric Oxide / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D030421:Peroxynitrous Acid / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D024502:alpha-Tocopherol / Q000378:metabolism*; D024504:gamma-Tocopherol / Q000378:metabolism*
D008077:Lipoproteins, LDL; D002245:Carbon Dioxide; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D024504:gamma-Tocopherol; D024502:alpha-Tocopherol; D001205:Ascorbic Acid