9] Spin-trapping methods for detecting superoxide and hydroxyl free radicals in vitro and in vivo

Abstract

Publisher Summary Spin trapping is tool used in the study of transient free radicals. This chapter discusses spin-trapping methods for detecting superoxide and hydroxyl free radicals in vitro and in vivo. The most useful radical trap for the study of oxygen-centered free radicals is 5,5-dimethyl-l-pyrroline N-oxide (DMPO), which has been used extensively to study superoxide and hydroxyl radicals as well as peroxyl radical formation in biochemical and biological systems. The spin trapping of superoxide has been of much interest because of the involvement of superoxide in many physiological processes. DMPO-OOH (the superoxide spin adduct of DMPO) has a distinctive spectrum that is easily recognizable. However, other peroxyl adducts of DMPO will have a similar appearance. Thus, the real proof that the spectrum observed is indeed because of DMPO. OOH is gained by using superoxide dismutase (SOD) to inhibit the signal. Although the DMPO-OOH spectrum is distinctive, the spin trapping of superoxide is not without its problems. The actual reaction of superoxide with DMPO is very slow. Thus, in most superoxide-generating systems, the spin trap concentration must be quite high to outcompete the self-decay—that is, the dismutation of superoxide. The DMPO–OH adduct is the most often reported radical adduct of DMPO. Much of the interest in the spin trapping of OH is because of its formation in the superoxide-dependent Fenton reaction.