On the use of fluorescence lifetime imaging and dihydroethidium to detect superoxide in intact animals and ex vivo tissues: A reassessment

Recently, DJ. Hall eta!, reported that ethidium (E+) is formed as a major product of hydroethidine (HE) or dihydroethidium reaction with superoxide (O-2(center dot-)) in intact animals with low tissue oxygen levels U. Cereb. Blood Flow Metab. 32:23-32, 2012). The authors concluded that measurement of E+ is an indicator of O-2(center dot-) 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 O-2(center dot-) 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 O-2(center dot-). 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 O-2(center dot-) reaction with HE. (C) 2013 Published by Elsevier Inc.