Botti Horacio, Batthyány Carlos, Trostchansky Andrés, Radi Rafael, Freeman Bruce A, Rubbo Homero
Departamento de Bioqui;mica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
Free Radic Biol Med. 2004 Jan 15;36(2):152-62. doi: 10.1016/j.freeradbiomed.2003.10.006.
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.
先前的报告提出，过氧亚硝酸根（ONOO⁻）通过双电子协同机制氧化α-生育酚（α-TOH）。相比之下，ONOO⁻通过过氧键均裂产生的自由基氧化酚类。为了解低密度脂蛋白（LDL）中α-TOH和γ-生育酚（γ-TOH）氧化的动力学和机制（直接氧化与自由基氧化），我们将LDL暴露于一次性添加或持续输注的ONOO⁻中。一氧化氮（·NO）、抗坏血酸盐和二氧化碳被用作ONOO⁻反应性的关键生物学相关调节剂。尽管在0.8微摩尔/升的LDL与60微摩尔一次性添加的ONOO⁻孵育5分钟内，约80%的α-TOH和γ-TOH被消耗，但在60分钟内等摩尔输注ONOO⁻会导致两种抗氧化剂完全消耗。相对于α-TOH，γ-生育酚得以保留，这可能是由于α-TOH对γ-生育酚自由基的循环利用。LDL中α-TOH的氧化对ONOO⁻呈一级反应，约12%的ONOO⁻可被最大程度利用。生理浓度的·NO和抗坏血酸盐通过独立且相加的机制保护α-TOH和γ-TOH。高浓度的·NO和抗坏血酸盐可消除α-TOH和γ-TOH的氧化。计算机辅助模拟评估显示，LDL中·NO对α-TOH的保护比对溶液中的抗坏血酸盐更有效，这表明脂质过氧自由基与·NO的反应在动力学上比与α-TOH的反应更有利。此外，二氧化碳（1.2毫摩尔/升）可抑制α-TOH和脂质氧化。这些结果表明，ONOO⁻通过单电子自由基机制诱导LDL中α-TOH的氧化；因此，尽管ONOO⁻生成增加，但·NO和抗坏血酸盐的抑制作用可能决定了组织中α-生育醌的低积累。
