QUANTITATIVE MEASUREMENT OF SUPEROXIDE GENERATION USING THE SPIN TRAP-(DIETHOXYPHOSPHORYL)-5-METHYL-1-PYRROLINE-N-OXIDE

Measurement and quantitation of superoxide by electron paramagnetic re sonance (EPR) using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DM PO) have been limited by the short half-life of the superoxide adduct DMPO-OOH (similar to 50 s at pH 7). Recently a beta-phosphorylated nit rone, -(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), was developed and reported to form a more stable superoxide adduct with a half-life of similar to 15 min. We evaluated the use of DEPMPO for qu antitative measurement of superoxide in chemical and biochemical syste ms. To estimate the efficiency of trapping, EPR oximetry was used to m easure oxygen consumption and the intensity of the DEPMPO-OOH signal t o measure superoxide generation and adduct decay. With the superoxide generating systems, riboflavin/light and xanthine/xanthine oxidase. DE PMPO trapped similar to 65% of the superoxide produced. The efficiency of superoxide trapping by DEPMPO was compared to the commonly used cy tochrome c reduction method. When superoxide production was >20 mu M, cytochrome c detected similar to 100% of the superoxide produced, whil e DEPMPO trapped 60 to 70%. However, EPR detection with DEPMPO was 40- fold more sensitive than cytochrome c. Thus, DEPMPO is an efficient sp in trap which enables specific and sensitive detection and quantitatio n of superoxide generation. (C) 1997 Academic Press.