The confounding effects of light, sonication, and Mn(III)TBAP on quantitation of superoxide using hydroethidine.
Previously, we showed that hydroethidine (HE) reacts with intracellular superoxide radical anion (O2-*) to form a unique fluorescent marker product, 2-hydroxyethidium cation (2-OH-E+), that was not formed from HE reaction with other biologically relevant oxidants (H. Zhao et al. Proc. Natl. Acad. Sci. USA102:5727-5732; 2005). Here we rigorously assessed the confounding effects of light, sonication, and Mn(III)TBAP on 2-OH-E+, the HE/O2-* reaction product. Results indicate that continuous exposure to visible light induced photo-oxidation of HE to ethidium cation (E+) by a 2-OH-E+ -dependent mechanism. Treatment of HE with ultrasound, a frequently used technique to lyse cell membranes, induced 2-OH-E+ from in situ generation of O2-*. Mn(III)TBAP, a cell-permeable metal-porphyrin complex used as a catalytic antioxidant, reacts with HE to form E+. This finding provides an alternative interpretation for Mn(III)TBAP effects during the HE/O2-* reaction. In order to correctly interpret the HE reaction with O2-* in cells, it is therefore imperative that HE and HE-derived products be measured by HPLC. A new and improved HPLC-electrochemical (HPLC-EC) detection has been developed for analysis of intracellular O2-*. The HPLC-EC method is at least 10 times more sensitive than the HPLC-fluorescence technique for detecting O2-* in cells.
Zielonka
Jacek
J
Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Vasquez-Vivar
Jeannette
J
Kalyanaraman
B
B
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D008027:Light; D008345:Manganese / Q000737:chemistry*; D008665:Metalloporphyrins / Q000737:chemistry*; D015394:Molecular Structure; D009573:Nitrites / Q000737:chemistry*; D010084:Oxidation-Reduction; D010617:Phenanthridines / Q000737:chemistry*; D010787:Photosensitivity Disorders; D013010:Sonication; D013481:Superoxides / Q000032:analysis*
C472668:AEOL 10150; D008665:Metalloporphyrins; D009573:Nitrites; D010617:Phenanthridines; C517163:manganese(III) tetrakis(5,10,15,20-benzoic acid)porphyrin; D013481:Superoxides; C058428:hydroethidine; D008345:Manganese