Abstract
Superoxide plays a critical role in number of pathological conditions including cardiovascular diseases. Development of specific and reliable methods for superoxide detection is critical for investigation of these conditions. Cellular reactions of dihydroethydium (DHE) produce both the superoxide specific product 2‐OH‐ethidium (2‐OH‐E) and non‐specific oxidation product ethidium which have overlapping fluorescence spectra. Specific superoxide detection therefore is difficult. HPLC can separate ethidium and 2‐OH‐E but this method is limited by inability to detect site‐specific 2‐OH‐E and duration of the HPLC procedure. In this study, we show optimized fluorescence spectroscopy protocol allowing reliable superoxidedetection in cell free systems, subcellular fractions, intact cells and tissue. Analysis of fluorescence emission spectra showed four times higher contribution of 2‐OH‐E fluorescence comparing to ethidium when excitation was changed from 480 nm to 405 nm. Selection of specific emission enables selective detection of 2‐OH‐E with negligible contribution of ethidium. The best selectivity of fluorescence signal for detection of 2‐OH‐E at excitation at 405 and 480 nm was achieved at emission 570 nm (1:21 ethidium:2‐OH‐E ratio) and 530 nm (1:52 Ethidium:2‐OH‐E ratio), respectively. We applied these optimized conditions for on‐line superoxidedetection generated by xanthine oxidase. Fluorescent signal was inhibited by SOD supplementation confirming specific detection of 2‐OH‐E with xanthine oxidase concentration as low as 10 microunits per ml. Specificity of plate reader‐based superoxide measurements was confirmed by HPLC analysis of 2‐OH‐E and ethidium. Our study shows that limitations of HPLC in superoxide detection in biological samples can be substantially overcome by using optimized fluorescence spectroscopy.This work has been funded by National Institute of health PO‐1 HL058000.
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