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

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.