Radical production from the interaction of ozone and PUFA as demonstrated by electron spin resonance spin-trapping techniques

Abstract

There is considerable evidence that indicates that a fraction of the damage caused by ozone to cellular systems involves radical-mediated reactions. The most direct method for probing the mechanism by which ozone reacts with target molecules such as polyunsaturated fatty acids (PUFA) involves the use of electron spin resonance (ESR). In 1968, Goldstein et al. reported that ESR signals were observed when 40 ppm ozone in air is bubbled through linoleic acid ( Arch. Environ. Health 17, 46–49). We have repeated this experiment and have performed several experiments modified from this design; in none of these do we observe ESR signals. We have studied the reaction of ozone with PUFA at −78°C using spin traps. Spin traps themselves react with ozone, but the following protocol avoids that reaction. (1) Ozone (20–200 ppm) in air or oxygen-free ozone is allowed to bubble through the sample in Freon-11 in an ESR tube at −78°C; no ESR absorption is observed. (2) Unreacted ozone is flushed out with argon or nitrogen. (3) The spin trap in Freon-11 is added to give a 0.1 m solution, still at −78°C; no ESR signal is observed. (4) The tube is allowed to warm slowly. At about −45°C, the ESR spectra of spin adducts appear. Using this method with methyl linoleate we observe spin adducts of alkoxy radicals and also a signal that is consistent with a carbon radical with one α-H (as would be expected if the trapped radical were the pentadienyl radical produced by hydrogen abstraction from linoleate). We hypothesize that an intermediate is formed from the reaction of ozone with PUFA that is stable at −78°C but decomposes to form radicals at about −45°C. We tentatively identify the intermediate as a trioxide (ROOOH, ROOOR, or R-CO-OOOH) on the basis of analogies and its temperature profile for decomposition to radicals. It appears reasonable to suggest that the reaction(s) responsible for the production of radicals under these low-temperature conditions also occurs at room temperature. Although the low-temperature intermediate cannot be observed at ambient temperatures, radicals from it could be responsible for the effects on autoxidation that are induced by ozone.