Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

Role of metals in oxygen radical reactions

Trace levels of transition metals can participate in the metal-catalyzed Haber-Weiss reaction (superoxide-driven Fenton reaction) as well as catalyze the oxidation of ascorbate. Generally ascorbate is thought of as an excellent reducing agent; it is able to serve as a donor antioxidant in free radical-mediated oxidation processes. However, as a reducing agent it is also able to reduce redox-active metals such as copper and iron, thereby increasing the pro-oxidant chemistry of these metals. Thus ascorbate can serve as both a pro-oxidant and an antioxidant. In general, at low ascorbate concentrations, ascorbate is prone to be a pro-oxidant, and at high concentrations, it will tend to be an antioxidant. Hence there is a crossover effect. We propose that the "position" of this crossover effect is a function of the catalytic metal concentration. In this presentation, we discuss: (1) the role of catalytic metals in free radical-mediated oxidations; (2) ascorbate as both a pro-oxidant and an antioxidant; (3) catalytic metal catalysis of ascorbate oxidation; (4) use of ascorbate to determine adventitious catalytic metal concentrations; (5) use of ascorbate radical as a marker of oxidative stress; and (6) use of ascorbate and iron as free radical pro-oxidants in photodynamic therapy of cancer.

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Catalytic metals, ascorbate and free radicals: combinations to avoid.

The peroxidation of membrane phospholipids induced in vitro by ascorbic acid or by dialuric acid (hydroxybarbituric acid) does not occur in the absence of traces of metal ions. Peroxidation induced by adding iron salts to phospholipids can either be promoted or inhibited by the chelators EDTA, diethylenetriaminepenta-acetic acid and bathophenanthrolinesulphonate, depending on the ratio [chelator]/[iron salt]. The iron chelator desferrioxamine inhibits peroxidation at all concentrations tested, and it also inhibits the iron-catalysed formation of hydroxyl radicals (OH.) from superoxide (O2-.). Since desferrioxamine is approved for clinical use, it might prove a valuable tool in the treatment of inflammation, poisoning by autoxidizable molecules and radiation damage.

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Inhibition of the iron-catalysed formation of hydroxyl radicals from superoxide and of lipid peroxidation by desferrioxamine

Ferritin and superoxide-dependent lipid peroxidation.

1. Homogenates of rat liver, spleen, heart and kidney form lipid peroxides when incubated in vitro and actively catalyse peroxide formation in emulsions of linoleic acid or linolenic acid. 2. In liver, catalytic activity is distributed throughout the nuclear, mitochondrial and microsomal fractions and is present in the 100000g supernatant. Activity is weak in the nuclear fraction. 3. Dilute (0.5%, w/v) homogenates catalyse peroxidation over the range pH5.0-8.0 but concentrated (5%, w/v) homogenates inhibit peroxidation and destroy peroxide if the solution is more alkaline than pH7.0. 4. Ascorbic acid increases the rate of peroxidation of unsaturated fatty acids catalysed by whole homogenates of liver, heart, kidney and spleen at pH6.0 but not at pH7.4. 5. Catalysis of peroxidation of unsaturated fatty acids by the mitochondrial and microsomal fractions of liver is inhibited by ascorbic acid at pH7.4 but the activity of the supernatant fraction is enhanced. 6. Inorganic iron or ferritin are active catalysts in the presence of ascorbic acid. 7. Lipid peroxide formation in linoleic acid or linolenic acid emulsions catalysed by tissue homogenates is partially inhibited by EDTA but stimulated by o-phenanthroline. 8. Cysteine or glutathione (1mm) inhibits peroxide formation catalysed by whole homogenates, mitochondria or haemoprotein. Inhibition increases with increase of pH.

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Wills Ed

Papers

Mechanisms of lipid peroxide formation in animal tissues.