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The confounding effects of light, sonication, and Mn(III)TBAP on quantitation of superoxide using hydroethidine. Free Radic Biol Med 2006 Oct 01;41(7):1050-7 PMID: 16962930

Pubmed ID

16962930

Abstract

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.

Author List

Zielonka J, Vasquez-Vivar J, Kalyanaraman B

Authors

Balaraman Kalyanaraman PhD Chair, Professor in the Biophysics department at Medical College of Wisconsin
Jeannette M. Vasquez-Vivar PhD Professor in the Biophysics department at Medical College of Wisconsin
Jacek M. Zielonka PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin




Scopus

2-s2.0-33748291680   40 Citations

MESH terms used to index this publication - Major topics in bold

Animals
Cattle
Chromatography, High Pressure Liquid
Light
Manganese
Metalloporphyrins
Molecular Structure
Nitrites
Oxidation-Reduction
Phenanthridines
Photosensitivity Disorders
Sonication
Superoxides
jenkins-FCD Prod-336 69ef4a6b262d135130251597d5d39873903802b5