Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturated fatty acid.
Electron paramagnetic resonance (EPR) spin trapping was used to detect lipid-derived free radicals generated by iron-induced oxidative stress in intact cells. Using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), carbon-centered radical adducts were detected. These lipid-derived free radicals were formed during incubation of ferrous iron with U937 cells that were enriched with docosahexaenoic acid (22:6n-3). The EPR spectra exhibited apparent hyperfine splittings characteristic of a POBN/alkyl radical, aN = 15.63 +/- 0.06 G and aH = 2.66 +/- 0.03 G, generated as a result of beta-scission of alkoxyl radicals. Spin adduct formation depended on the FeSO4 content of the incubation medium and the number of 22:6-enriched cells present; when the cells were enriched with oleic acid (18:1n-9), spin adducts were not detected. This is the first direct demonstration, using EPR, of a lipid-derived radical formed in intact cells in response to oxidant stress.
North
J A
JA
Department of Biochemistry, College of Medicine, University of Iowa, Iowa City 52242.
Spector
A A
AA
Buettner
G R
GR
D002460:Cell Line; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005231:Fatty Acids, Unsaturated / Q000032:analysis / Q000378:metabolism*; D005609:Free Radicals; D006801:Humans; D007951:Leukemia, Myeloid; D009589:Nitrogen Oxides; D011725:Pyridines; D013113:Spin Labels
D003497:Cyclic N-Oxides; D005231:Fatty Acids, Unsaturated; D005609:Free Radicals; D009589:Nitrogen Oxides; D011725:Pyridines; D013113:Spin Labels; C043438:alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone; C017245:5,5-dimethyl-1-pyrroline-1-oxide