An ESR and HPLC-EC assay for the detection of alkyl radicals

The correlation of lipid peroxidation with release of alkanes (RH) is considered a noninvasive method for the in vivo evaluation of oxidative stress. The formation of RH is believed to reflect a lipid hydroperoxide (LOOH)-dependent generation of alkoxyl radicals (LO.) that undergo, beta -scission with release of alkyl radicals (R-.). Alternatively, R-. could be spin-trapped with a nitrone before the formation of RH and analyzed by ESR. Extracts from the liver and lung of CCl4- and asbestos-treated rats that were previously loaded with nitrones exhibited ESR spectra suggesting the formation of iso-propyl, n-butyl, ethyl, and pentyl radical-derived nitroxides. In biological systems, various nitroxides with indistinguishable ESR spectra could be formed. Hence, experiments with N-tert-butyl-alpha -phenylnitrone (PBN) for spin trapping of R-. were carried out in which the nitroxides formed were separated and analyzed by HPLC with electrochemical detection (EC). The C1-5 homologous series of PBN nitroxides and hydroxylamines were synthesized, characterized by ESR, GC-MS, and HPLC-EC, and used as HPLC standards. For in vivo generation and spin trapping of R-., rats were loaded with CCl4 and PBN. The HPLC-EC chromatograms of liver extracts from CCl4-treated rats demonstrated the formation of both the nitroxide and hydroxylamine forms of PBN/(CCl3)-C-., as well as the formation of a series of unidentified PBN nitroxides and hydroxylamines. However, formation of PBN adducts with retention times similar to these of the PBN/C2-5 derivatives was not observed. In conclusion, we could not correlate the production of PBN-detectable alkyl radicals with the reported CCl4-dependent production Of C1-5 alkanes. We speculate that the major reason for this is the low steady-state concentrations of R-. produced because only a small fraction of LO. undergo,beta -scission to release R-..