The use of spin traps to investigate site-specific formation of free radicals in low-density lipoprotein oxidation.

Introduction It is currently believed that the oxidatively modified LDL plays a key role in the development of atherogenesis [ 1-31. Abundant data suggest that the oxidative modification of low-density lipoprotein (LDL) is mediated by lipid-derived free radicals, lipid hydroperoxides, or aldehydes derived from them [4-lo]. Although several oxidizing species have been shown to initiate the oxidative modification of LDL (Table l), the exact nature of oxidant species or enzymes responsible for oxidation of LDL in vivo still remains unknown. LDL oxidation has been shown to be sitespecific [25]. In order to fully understand the mechanism of oxidative modification of LDL, it is essential to investigate and characterize the sitespecific formation of oxidants and their reactions. For example, the water-soluble radical traps may simply scavenge free radicals generated in the aqueous phase, without affecting LDL modification. In contrast, the lipid-soluble scavengers should react with lipid-derived radicals that are more target-specific. The water-soluble reductants, such as ascorbic acid, can regenerate vitamin E in LDL via a recycling mechanism [26,27]. Electron spin resonance (e.s.r.) is the only physical technique that can provide unambiguous structural information on free radicals. Direct e.s.r. has been used to detect phenoxyl radicals formed during oxidation of LDL [26, 271. Direct e s r . has also provided unequivocal evidence for the reaction between LDL-associated a-tocopheroxyl, probucol phenoxyl radicals and ascorbic acid [26, 271. The technique cannot, however, be used to detect transient LDL lipid-derived radicals. The e.s.r.-spin trapping technique must be resorted to in this case [28-301. Spin traps are diamagnetic organic molecules containing either nitroso (-N=O) or nitrone (=\fi-o) functional groups. The spin-trapping