H₂O₂ detection with 10-acetyl-3,7-dihydroxyphenoxazine: comparison with homovanillic acid

H₂O₂ is assumed to be produced and involved in several (patho-)physiological processes. For the determination of low amounts of H₂O₂ formed in biological systems sensitive and reliable assays are necessary. Exploring suitable detection systems for mitochondrial H₂O₂ production different enzyme-catalyzed redox reactions were tested. By horseradish peroxidase (HRP) and H₂O₂, homovanillic acid (HVA) and 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) were enzymatically oxidized to the fluorescent HVA dimer (λ_ex = 312 nm; λ_em = 420 nm) and resorufin (λ_ex = 563 nm; λ_em = 587 nm), respectively. The specificity of the assays was confirmed by catalase which dose-dependently inhibited the H₂O₂-induced fluorescence increase. Amplex Red and HVA were applied to the H₂O₂-generating glucose/glucose oxidase system and compared for their sensitivity. Albumin, which is frequently added to mitochondrial or cell incubation media, significantly decreased the fluorescence intensity of the Amplex Red oxidation product while the fluorescence intensity of the HVA dimer was significantly increased. This effect was observed in 0.3 M sucrose and was lacking in 0.15 M KCl. The mitochondrial H₂O₂ formation was studied in antimycin A-inhibited succinate-respiring rat heart mitochondria in the presence and absence of superoxide dismutase which catalyzes the dismutation of primarily produced superoxide radicals to H₂O₂. Amplex Red turned out to be a more sensitive analytic tool for H₂O₂ detection than HVA.