Brain contains a highly diversified complement of molecular species of a mitochondria-specific phospholipid, cardiolipin (CL), which - due to its polyunsaturation - can readily undergo oxygenation. Here, we used global lipidomics analysis in experimental traumatic brain injury (TBI) and showed that TBI was accompanied by oxidative consumption of polyunsaturated CL and accumulation of more than 150 new oxygenated molecular species in CL. RNAi-based manipulations of CL-synthase and CL levels conferred resistance of primary rat cortical neurons to mechanical stretch - an in vitro model of traumatic neuronal injury. By applying the novel brain permeable mitochondria-targeted electron-scavenger, we prevented CL oxygenation in the brain, achieved a substantial reduction in neuronal death both in vitro and in vivo, and markedly reduced behavioral deficits and cortical lesion volume. We conclude that CL oxygenation generates neuronal death signals and that its prevention by mitochondria-targeted small molecule inhibitors represents a new target for neuro-drug discovery.

. Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am J Physiol Cell Physiol 287: C895-C902, 2004. First published August 11, 2004 10.1152 10. /ajpcell.00028.2004 was demonstrated that superoxide oxidizes dihydroethidium to a specific fluorescent product (oxyethidium) that differs from ethidium by the presence of an additional oxygen atom in its molecular structure (Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vásquez-Vivar J, and Kalyanaraman B. Free Radic Biol Med 34: 1359 -1368. We have adapted this new HPLC-based assay to quantify this product as a tool to estimate intracellular superoxide in intact tissues. Ethidium and oxyethidium were separated using a C-18 column and quantified using fluorescence detection. Initial cell-free experiments with potassium superoxide and xanthine oxidase confirmed the formation of oxyethidium from dihydroethidium. The formation of oxyethidium was inhibited by superoxide dismutase but not catalase and did not occur upon the addition of H2O2, peroxynitrite, or hypochlorous acid. In bovine aortic endothelial cells (BAEC) and murine aortas, the redox cycling drug menadione increased the formation of oxyethidium from dihydroethidium ninefold (0.4 nmol/mg in control vs. 3.6 nmol/mg with 20 M menadione), and polyethylene glycolconjugated superoxide dismutase (PEG-SOD) significantly inhibited this effect. Treatment of BAEC with angiotensin II caused a twofold increase in oxyethidium formation, and this effect also was reduced by PEG-SOD (0.5 nmol/mg). In addition, in the aortas of mice with angiotensin II-induced hypertension and DOCA-salt hypertension, the formation of oxyethidium was increased in a manner corresponding to superoxide production estimated on the basis of cytochrome c reduction. Detection of oxyethidium using HPLC represents a new, convenient, quantitative method for the detection of superoxide in intact cells and tissues.oxyethidium; hypertension; menadione; angiotensin II; endothelium IT HAS BECOME EVIDENT that mammalian cells produce reactive oxygen species (ROS) and that these can serve as signaling molecules that modulate events such as enzyme phosphorylation, cell growth, hypertrophy, and programmed cell death. When produced in excessive amounts, ROS can contribute to cellular dysfunction (9). Overproduction of ROS has been implicated in diverse diseases such as cancer, hypertension, atherosclerosis, Alzheimer's disease, lung injury, and aging (20).The production of ROS is mediated by a variety of mammalian enzymes that are capable of reducing molecular oxygen. While occasional enzymes such as glucose oxidase and xanthine oxidase are capable of performing a two-electron reduction of oxygen to form hydrogen peroxide, the most common scenario is a one-electron reduction, leading to formation of superoxide (O 2 Ϫ ⅐). O 2 Ϫ ⅐ can in turn serve as a progenitor for other ROS such as hydrogen peroxide, peroxynitrite, and the hydroxyl radical. In vascular cells, increased ...

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Abstract-Oxidative stress contributes to the pathogenesis of atherosclerosis. p22phox -based NAD(P)H oxidases exist in the vessel wall, acting as important superoxide-generating systems in the vasculature. Some studies have identified reduced atherosclerosis in the presence of the C242T CYBA polymorphism, whereas others have not. Because vascular p22 phox is identical to neutrophil p22 phox , we studied the association between the C242T, A640G, and Ϫ930 A/G CYBA polymorphisms and the quantity of superoxide produced from neutrophils isolated from healthy adults to determine if these polymorphisms had any functional impact on NADPH oxidase function. Neutrophils were isolated from 90 subjects by Percoll density gradient centrifugation. Genotypes were determined by polymerase chain reaction (PCR) and restriction mapping, as well as real-time PCR. The oxidative burst was stimulated with phorbol 12-myristate 13-acetate. Superoxide was quantified using the superoxide dismutase inhibitable oxidation of the spin probe hydroxylamine 1-hydroxy-3-carboxy-pyrrolidine, detected by electron paramagnetic resonance. Superoxide production was significantly affected by the C242T polymorphism, being 8.7Ϯ0.7, 7.9Ϯ0.6, and 5.9Ϯ1.2 mol/L per minute per 10 6 neutrophils for the C242T CC, CT, and TT genotypes, respectively (PϽ0.05). In contrast, the A640G and the Ϫ930

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