Quantitative measurement of superoxide generation using the spin trap 5- (diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide

Abstract

Measurement and quantitation of superoxide by electron paramagnetic resonance (EPR) using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) have been limited by the short half-life of the superoxide adduct DMPO-OOH (~50 s at pH 7). Recently a β-phosphorylated nitrone, 5- (diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), was developed and reported to form a more stable superoxide adduct with a half-life of ~15 min. We evaluated the use of DEPMPO for quantitative measurement of superoxide in chemical and biochemical systems. To estimate the efficiency of trapping, EPR oximetry was used to measure oxygen consumption and the intensity of the DEPMPO-OOH signal to measure superoxide generation and adduct decay. With the superoxide-generating systems, riboflavin/light and xanthine/xanthine oxidase, DEPMPO trapped ~65% of the superoxide produced. The efficiency of superoxide trapping by DEPMPO was compared to the commonly used cytochrome c reduction method. When superoxide production was >20 μM, cytochrome c detected ~100% of the superoxide produced, while DEPMPO trapped 60 to 70%. However, EPR detection with DEPMPO was 40-fold more sensitive than cytochrome c. Thus, DEPMPO is an efficient spin trap which enables specific and sensitive detection and quantitation of superoxide generation.