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 due 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-iron ion/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H₂O₂.