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).
Patsoukis
Nikolaos
N
Department of Biology, University of Patras, Patras, 26500, Greece.
Papapostolou
Ioannis
I
Georgiou
Christos D
CD
D000818:Animals; D016477:Artifacts; D002417:Cattle; D005456:Fluorescent Dyes / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000737:chemistry; D007962:Leukocytes / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism*; D010617:Phenanthridines / Q000737:chemistry*; D010783:Photometry / Q000379:methods*; D012680:Sensitivity and Specificity; D013481:Superoxides / Q000032:analysis* / Q000737:chemistry
D005456:Fluorescent Dyes; D010617:Phenanthridines; D013481:Superoxides; C058428:hydroethidine; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D009195:Peroxidase