Superoxide radical scavenging activity of idebenone in vitro studied by ESR spin trapping method and direct ESR measurement at liquid nitrogen temperature

Abstract

The radical scavenging activity of oxidized and reduced idebenone (ID-O and ID-H, respectively) against superoxide radical (O2(-*) was studied in vitro using two methods: (1) O2(-*) radicals were generated enzymatically in a hypoxanthine (HPX)-xanthine oxidase (XOD) system and detected by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trapping. Superoxide dismutase and other scavengers added to this system competed to various extents with DMPO to trap O2(-*) radicals, resulting in a decrease of the ESR signal intensity of the DMPO-OOH spin adduct. ID-O reacted about 12-fold quicker (k = 4.48 x 10(4) M(-1)s(-1)) with the O2(-*) radicals than ID-H (k = 3.62 x 10(3) M(-1)s(-1)) x (2) O2(-*) radicals were generated chemically in potassium superoxide (KO2)-crown ether system. Quinoid compounds reacted with the O2(-*)radicals to form semiquinone radicals that could be observed by ESR. At liquid nitrogen temperature (-196 degrees C), the ESR signal of O2(-*) radicals could be observed directly, thus allowing us to estimate the scavenging activity of ID-O and ID-H. These experiments also revealed that ID-O possesses an O2(-*) radical scavenging activity, whereas ID-H reacts quantitatively much slower. Analyzing various quinone compounds, it has been established that the O2(-*) radical scavenging process is a reversible, most probably oscillating, monovalent electron transfer from superoxide to the quinone, and that the O2(-*) radical scavenging activity depends on the redox potential, i.e., on the actual state of oxidation of the quinones.