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Critical Evaluation of DHE Fluorescence and Cytochrome c Absorbance Measurement of Superoxide in the Presence of Nitric Oxide and Superoxide Dismutase
Author(s) -
Rogers Steven Clay,
Kavdia Mahendra
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.617.1
Subject(s) - superoxide , chemistry , superoxide dismutase , nitric oxide , hydrogen peroxide , xanthine oxidase , reactive oxygen species , peroxynitrite , cytochrome c , biochemistry , oxidative stress , enzyme , mitochondrion , organic chemistry
Excessive superoxide levels can disrupt vascular homeostasis and lead to increased formation of hydrogen peroxide, peroxynitrite and decreased vasodilation. An accurate detection of superoxide in vivo is necessary to better understand a number of vascular pathologies. In this study, we measure the effect of nitric oxide and superoxide dismutase (SOD), which are two of the most effective scavengers of superoxide in vivo, presence on the fluorescence and absorbance measurement of superoxide. We used xanthine oxidase/hypoxanthine and sodium nitroprusside to produce superoxide and NO, respectively. A microplate reader was used for two common methods to measure superoxide: the fluorescence of dihydroethidium (DHE) and absorbance of ferricytochrome‐c. SOD significantly decreased superoxide concentrations in both the absorbance and fluorescence assays. NO showed only an effect on superoxide production at higher concentrations in the fluorescence measurements. Kinetic analysis of DHE‐superoxide interaction resulted in a reaction rate of 1.1 × 10 3 M −1 s −1 that is ~250× lower than the previously reported reaction rate of 2.6 × 10 5 M −1 s −1 . The results suggest that accurate in vivo measurements of superoxide production may be extremely difficult due to competitive interference from varying NO and SOD levels, however the superoxide concentration may be quantified. Supported by: AHA 0530050N & NIH R01 HL084337.