Faculty Collaboration Database

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

Date

09/12/2006

Pubmed ID

16962930

DOI

10.1016/j.freeradbiomed.2006.04.017

Scopus ID

2-s2.0-33748291680 (requires institutional sign-in at Scopus site)   50 Citations

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 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

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