Abstract
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.
Highlights
Electron Paramagnetic Resonance Spin Trapping Using Intact Cells propagation radical reactions
Many aspects of the competing mechanisms involved in lipid peroxidation are known from investigations with chemical systems, liposomes, and subcellular fractions, few studies have examined the generation of lipid radicals in Electron paramagnetic resonance (EPR) spin trap- the intact cell
Spin Trapping of Lipid-deriued Radicals-When intact U937 cells enriched with DHA were incubated with 80 p~ FeS0, in the presence of the spin trap POBN,a carboncentered spin adduct was observed (Fig. lD, aN= 15.63 +- 0.06 G and aH= 2.66 +- 0.03 G)
Summary
Formed during incubation of ferrous iron with U937 Materials-5,5-Dimethyl-l-pyrrolineN-oxide (DMPO), a-(4-pyricells that were enriched with docosahexaenoic acid dyl 1-oxide)-N-tert-butylnitrone(POBN),and chelating resin (C(22:6n-3). Spin adduct formation depended on the FeSO, content of the incubation medium and the number of 226-enriched cells present; when the cells were enriched with oleic acid (18:ln-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. The cell lipids were modified by the addition of 10 FM DHA or oleic acid to the growth medium. 1.05 G ; time constant, 1.3 s; scan rate, 60 G/335 s; receiver gain, 1X lo
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