Spin trapping of superoxide and hydroxyl radical: Practical aspects

Abstract

Reports are regularly appearing in the literature describing spin trapping of superoxide and hydroxyl radicals from various sources. Careful scrutiny of these reports will often reveal that insufficient controls have been run to properly validate the results. Nitrones are highly reactive compounds which can form nitroxides by mechanisms other than radical trapping. Thus, investigators using spin trapping should be cognizant of the many artifacts which accompany this technique, and take care to validate their results with satisfactory controls. We have described straightforward procedures to determine whether superoxide or hydroxyl radical trapping have occurred, and which can help verify the assignment of the radical adduct. Nitrones are the only spin traps currently suitable for detection of hydroxyl and superoxide radicals. The various nitrone spin traps in current use each have advantages and disadvantages. In general, the cyclic nitrone traps such as DMPO have greater reactivity with superoxide and hydroxyl radicals, are less readily hydrolyzed, but are more susceptible to oxygen and light, and thus have lesser shelf lives. Aryl nitrones such as 4-POBN or PBN have lesser reactivity with superoxide and hydroxyl radicals, are more readily hydrolyzed, but have greater shelf lives. The stability of DMPO-OH is also greater than that of 4-POBN-OH. Thus in general, DMPO appears to be the most versatile spin trap currently available. Spin trapping is an inefficient means of detecting superoxide, due to the low rate constants for spin trapping. Spin traps possessing a β-hydrogen will also form unstable superoxide adducts. Spin trapping will, however, undoubtedly prove useful in detecting superoxide under conditions where more conventional methods, such as cytochrome c reduction, cannot be used.