title: On the use of fluorescence lifetime imaging and dihydroethidium to detect superoxide in intact animals and ex vivo tissues: a reassessment.
abstract: Recently, D.J. Hall et al. reported that ethidium (E(+)) is formed as a major product of hydroethidine (HE) or dihydroethidium reaction with superoxide (O2(-)) in intact animals with low tissue oxygen levels (J. Cereb. Blood Flow Metab. 32:23-32, 2012). The authors concluded that measurement of E(+) is an indicator of O2(-) formation in intact brains of animals. This finding is in stark contrast to previous reports using in vitro systems showing that 2-hydroxyethidium, not ethidium, is formed from the reaction between O2(-) and HE. Published in vivo results support the in vitro findings. In this study, we performed additional experiments in which HE oxidation products were monitored under different fluxes of O2(-). Results from these experiments further reaffirm our earlier findings (H. Zhao et al., Free Radic. Biol. Med. 34:1359, 2003). We conclude that whether in vitro or in vivo, E(+) measured by HPLC or by fluorescence lifetime imaging is not a diagnostic marker product for O2(-) reaction with HE.
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA; Institute of Applied Radiation Chemistry, Lodz University of Technology, 90-924 Lodz, Poland.
authors.forename: Radoslaw
authors.initials: R
authors.lastname: Michalski
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology, 90-924 Lodz, Poland.
authors.forename: Bartosz
authors.initials: B
authors.lastname: Michalowski
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA; Institute of Applied Radiation Chemistry, Lodz University of Technology, 90-924 Lodz, Poland.
authors.forename: Adam
authors.initials: A
authors.lastname: Sikora
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Jacek
authors.initials: J
authors.lastname: Zielonka
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: balarama@mcw.edu.
authors.forename: Balaraman
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D001931:Brain Mapping; D004996:Ethidium / Q000031:analogs & derivatives*; D005456:Fluorescent Dyes / Q000008:administration & dosage*; D019255:NADPH Oxidases / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D013481:Superoxides; D019255:NADPH Oxidases; D004996:Ethidium