title: Analysis of kinetics of dihydroethidium fluorescence with superoxide using xanthine oxidase and hypoxanthine assay.
abstract: Superoxide (O(2)                           (-)) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O(2)                           (-) is needed to better understand numerous vascular pathologies. In this study, we performed a mechanistic study by using the xanthine oxidase (XOD)/hypoxanthine (HX) assay for O(2)                           (-) generation and a O(2)                           (-) sensitive fluorescent dye dihydroethidium (DHE) for O(2)                           (-) measurement. To quantify O(2)                           (-) and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE-O(2)                           (-) interaction to understand the effect of O(2)                           (-) self-dismutation and to quantify DHE-O(2)                           (-) reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O(2)                           (-) interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169 ± 0.059 × 10(3) M(-1) s(-1) for DHE-O(2)                           (-) reaction that is ~100× slower than the reported value of 2.6 ± 0.6 × 10(5) M(-1) s(-1). In addition, the O(2)                           (-) self-dismutation has significant effect on DHE-O(2)                           (-) interaction. A slower reaction rate of DHE with O(2)                           (-) is more reasonable for O(2)                           (-) measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O(2)                           (-). Results suggest that an accurate measurement of O(2)                           (-) production rate may be difficult due to competitive interference for many factors; however O(2)                           (-) concentration may be quantified.
authors.affiliation: Department of Biomedical Engineering, Wayne State University, 5050 Anthony Wayne Dr., Detroit, MI 48202, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Chen
authors.forename: Steven C
authors.initials: SC
authors.lastname: Rogers
authors.forename: Mahendra
authors.initials: M
authors.lastname: Kavdia
mesh_terms: D004996:Ethidium / Q000031:analogs & derivatives* / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D019271:Hypoxanthine / Q000737:chemistry*; D007700:Kinetics; D013481:Superoxides / Q000737:chemistry*; D014969:Xanthine Oxidase / Q000737:chemistry*
chemical_list: C506392:2-hydroxyethidium; D005456:Fluorescent Dyes; C067883:dihydroethidium; D013481:Superoxides; D019271:Hypoxanthine; D014969:Xanthine Oxidase; D004996:Ethidium