Characterization of novel radicals from COX-catalyzed arachidonic acid peroxidation

The peroxidation of arachidonic acid (AA) catalyzed by cyclooxygenase (COX) is a well-known free radical-mediated process that forms many bioactive products. Because of a lack of appropriate methodologies, however, no comprehensive structural evidence has been found previously for the formation of COX-mediated and AA-derived free radicals. Here we have used a combination of LC/ESR and LC/MS with a spin trap, alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone (POBN), to characterize the carbon-centered radicals formed from COX-catalyzed AA peroxidation in vitro, including cellular peroxidation in human prostate cancer cells (PC-3). Three types of radicals with numerous isomers were trapped by POBN as ESR-active peaks and MS-active ions of m/z 296, 448, and 548, all stemming from PGF(2)-type alkoxyl radicals. One of these was a novel radical centered on the carbon-carbon double bond nearest the PGF ring, caused by an unusual beta-scission of PGF(2)-type alkoxyl radicals. The complementary nonradical product was 1-hexanol, another novel beta-scission product, instead of the more common aldehyde. The characterization of these novel products formed from in vitro peroxidation provides a new mechanistic insight into COX-catalyzed AA peroxidation in cancer biology.