A Rapid, Widely Applicable Screen for Drugs that Suppress Free Radical Formation in Ischemia/Reperfusion

Substantial injury can occur during reoxygenation of previously ischemic tissue in many experimental models, as the result of the generation of oxygen-derived free radicals. To test the antiradical activity of potentially protective compounds in this setting, we developed a simple screening system, applicable to fresh biopsy specimens, in which warm ischemia and reoxygenation of excised tissue are performed in vitro. Tissue production of malondialdehyde (MDA) equivalents is used as a nonspecific-but-sensitive marker of oxygen radical damage. Test compounds with putative antiradical activity are added prior to the reoxygenation phase, and their ability to suppress MDA production is an index of activity in preventing reoxygenation injury. Comparison with ischemic but not reoxygenated controls confirms the oxygen-dependent nature of the effect. Standard positive controls of known effective agents, such as butylated hydroxytoluene or deferoxamine, provide a reference for the activity of the test compound. The method is applicable to surgical biopsy specimens in veterinary and human medicine

Effect of Oxygen Concentration on the Formation of Malondialdehyde-Like Material in a Model of Tissue Ischemia and Reoxygenation

This study was conducted to explore the functional relationship between oxygen concentration during tissue reoxygenation after ischemia and the extent of postischemic lipid peroxidation, an indicator of reoxygenation injury. Excised rat liver or kidney tissue was rendered ischemic for 1 h at 37°C, minced into 1 mm3 fragments, and then reoxygenated for 1 h in flasks of buffered salt solution containing various amounts of oxygen. Production of malondialdehyde-like material (MDA) was measured to indicate lipid peroxidation. MDA production was minimal at oxygen tensions less than 10 mmHg, increased sharply from 10 to 50 mmHg, and plateaued at approximately 100 mmHg. A similar functional relationship was produced by a simple mathematical model of free radical mediated lipid peroxidation in biological membranes, suggesting that MDA production is indeed caused by free radical oxidation of membrane phospholipids and that the oxygen effect is governed by simple competition between chain propagation and chain termination reactions within the membrane. These experimental and analytical results confirm that relatively low concentrations of oxygen arc sufficient to produce oxidative damage in post-ischemic tissues

Methylene Blue as an Inhibitor of Superoxide Generation by Xanthene Oxidase: A Potential New Drug for the Attenuation of Ischemia/Reperfusion Injury

Tissue oxidases, especially xanthine oxidase, have been proposed as primary sources of toxic oxygen radicals in many experimental models of disease states. Among these, ischemia-reperfusion injury may be of the greatest clinical interest. In this paper we propose the use of methylene blue as a means of suppressing the production of superoxide radicals, O2, by acting as an alternative electron acceptor for xanthine oxidase. Previous work has indicated that methylene blue accepts electrons from xanthine oxidase at the iron-sulfur center. Initial experiments in our laboratory demonstrated that (1) pairs of electrons from each enzymatic oxidation are transferred to methylene blue, (2) the reduction of methylene blue can be achieved by model iron-sulfur centers, similar to the iron-sulfur center of xanthine oxidase, (3) reduced methylene blue auto-oxidizes to produce H2O2 directly, rather than O2, and (4) methylene blue is effective at non-toxic levels (2-5 mg/kg) in preventing free radical damage to liver and kidney tissues in an in vitro model of ischemia and reoxygenation. Accordingly, we propose that methylene blue may represent a new class of antioxidant drugs that competitively inhibit reduction of molecular oxygen to superoxide by acting as alternative electron acceptors for tissue oxidases. We have termed these agents “parasitic” electron acceptors

Evidence That Free Fatty Acid-Iron Complexes Directly Initiate Lipid Peroxidation in Vitro and in Vivo: A New Mechanism of Oxidative Stress

Through a series of biochemical and histochemical experiments we explored the novel hypothesis that iron and free fatty acids, liberated after tissue injury, combine to form liposoluble complexes that directly initiate lipid peroxidation. The addition of 100 M ferric iron to 30 mM linoleate suspensions at pH 7.4 produced time dependent lipid peroxidation, measured as conjugated diene formation. Complexes of 100 M ferric iron and 600 M pentanoate also initiated formation of conjugated dienes in linoleate suspensions and formation of malondialdehyde-like materials in rat liver slices. A histochemical stain for free fatty acids revealed positive reactions within cell membranes in traumatized regions of rat liver tissue that underwent compression injury followed by thirty minutes of blood perfusion, but not in nontraumatized control regions. The diaminobenzidine-H2O2 histochemical reaction for iron, revealed increased levels of redox cyclable iron in the membranes and the cytoplasm of traumatized hepatocytes. We propose that traumatic injury initiates cascades leading to liberation of iron from storage proteins and free fatty acids from membranes, which combine, distribute to the lipid domains of cell membranes, and directly initiate lipid peroxidation

Binary systems and their nuclear explosions

Peer ReviewedPreprin

Drotrecogin alfa (Activated) in adults with septic shock

There have been conflicting reports on the efficacy of recombinant human activated protein C, or drotrecogin alfa (activated) (DrotAA), for the treatment of patients with septic shock.In this randomized, double-blind, placebo-controlled, multicenter trial, we assigned 1697 patients with infection, systemic inflammation, and shock who were receiving fluids and vasopressors above a threshold dose for 4 hours to receive either DrotAA (at a dose of 24 μg per kilogram of body weight per hour) or placebo for 96 hours. The primary outcome was death from any cause 28 days after randomization.At 28 days, 223 of 846 patients (26.4%) in the DrotAA group and 202 of 834 (24.2%) in the placebo group had died (relative risk in the DrotAA group, 1.09; 95% confidence interval [CI], 0.92 to 1.28; P=0.31). At 90 days, 287 of 842 patients (34.1%) in the DrotAA group and 269 of 822 (32.7%) in the placebo group had died (relative risk, 1.04; 95% CI, 0.90 to 1.19; P=0.56). Among patients with severe protein C deficiency at baseline, 98 of 342 (28.7%) in the DrotAA group had died at 28 days, as compared with 102 of 331 (30.8%) in the placebo group (risk ratio, 0.93; 95% CI, 0.74 to 1.17; P=0.54). Similarly, rates of death at 28 and 90 days were not significantly different in other predefined subgroups, including patients at increased risk for death. Serious bleeding during the treatment period occurred in 10 patients in the DrotAA group and 8 in the placebo group (P=0.81).DrotAA did not significantly reduce mortality at 28 or 90 days, as compared with placebo, in patients with septic shock. (Funded by Eli Lilly; PROWESS-SHOCK ClinicalTrials.gov number, NCT00604214.)