Interference of non-specific peroxidases in the fluorescence detection of superoxide radical by hydroethidine oxidation: a new assay for H2O2.

The present study shows that hydroethidine (HE), used for in-vivo qualitative fluorescent detection of superoxide anion, can be also oxidized by H2O2 via non-specific peroxidase (horseradish peroxidase and myeloperoxidase) catalysis, forming fluorescent oxidation products. These products give broad excitation/emission peaks (490-495/580-600 nm) near the excitation/emission peaks (475/580 nm) of the HE-superoxide oxidation product, and this may pose serious interference problems to the fluorescent detection of the superoxide radical. The study suggests cautionary use of the HE-superoxide anion assay mainly for detection of reactive oxygen species. A byproduct of this study was the development of a simple and sensitive HE-horseradish peroxidase assay for the in-vitro quantification of H2O2 in biological tissues with a sensitivity of 1 micromol L(-1).