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 CCl(4)- 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 C(1-5) 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 CCl(4) and PBN. The HPLC-EC chromatograms of liver extracts from CCl(4)-treated rats demonstrated the formation of both the nitroxide and hydroxylamine forms of PBN/*CCl(3), 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/C(2-5) derivatives was not observed. In conclusion, we could not correlate the production of PBN-detectable alkyl radicals with the reported CCl(4)-dependent production of C(1-5) 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*.
Novakov
C P
CP
Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Feierman
D
D
Cederbaum
A I
AI
Stoyanovsky
D A
DA
D000473:Alkanes / Q000032:analysis / Q000737:chemistry*; D000818:Animals; D001194:Asbestos / Q000009:adverse effects; D001799:Blood Sedimentation; D002251:Carbon Tetrachloride / Q000009:adverse effects; D002273:Carcinogens / Q000009:adverse effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018736:DNA Adducts; D004563:Electrochemistry; D005609:Free Radicals / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry; D015227:Lipid Peroxidation; D008099:Liver / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley; D019097:Spin Trapping
D000473:Alkanes; D002273:Carcinogens; D018736:DNA Adducts; D005609:Free Radicals; D001194:Asbestos; D002251:Carbon Tetrachloride