The Action of Hydrogen Peroxide on the Formation of Thiobarbituric Acid-Reactive Material From Microsomes, Liposomes Or From Dna Damaged By Bleomycin Or Phenanthroline. Artefacts in the Thiobarbituric Acid Test

Abstract

Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H2O2 caused a loss of cytochrome P-450 but not of cytochrome b5. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H2O2 induces lipid peroxidation, but this was found to be due largely or completely to an effect of H2O2 on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H2O2 inhibited ascorbate/Fe(III)-induced microsomal lipid peroxidation, but part of this effect was dues to an action of H2O2 in the TBA test itself. H2O2 also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu2+/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-ion iron/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H2O2.