Abstract
Lipid-derived free radicals were detected by electron paramagnetic resonance (EPR) spectrometry when cultured endothelial cells attached to Cytodex beads were exposed to iron-induced oxidant stress in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). Radical adduct formation was enhanced greatly when the cells were supplemented during growth with polyunsaturated fatty acids. The largest EPR signal intensity was observed in cells enriched with docosahexaenoic acid (DHA) or eicosapentaenoic acid, but enhanced radical adduct production also occurred after exposure to arachidonic, alpha-linolenic, gamma-linolenic, or linoleic acids. Radical adduct formation increased as the DHA content of the cells increased and approached a maximum after only 6 h of exposure to DHA. Ascorbic acid, acting as a pro-oxidant, enhanced radical adduct formation in cells enriched with DHA. The EPR signal intensity was reduced when the cells were tested 6 h after replacement of the DHA-enriched medium with a medium containing 5-20 microM oleic acid, indicating that the increased endothelial responsiveness to oxidant stress is reversible. Likewise, when U937 monocytes enriched with DHA were exposed subsequently to 20 microM oleic acid, a 35-45% decrease in radical adduct formation also occurred. These findings suggest that the endothelium may become more susceptible to oxidative injury when it is exposed to elevated amounts of polyunsaturated fatty acids. However, the effect appears to be temporary. The protective action of oleic acid against oxidant stress is not confined to the endothelium; it applies to monocytes as well.
Highlights
Lipid-derived free radicals were detected by electron paramagnetic resonance (EPR) spectrometry when cultured endothelial cells attached to Cytodex beads were exposed to ironinduced oxidant stress in the presence of the spin trap a(4-pyridyl-l-oxide)-N-tert-butylnitrone (POBN)
Detection of lipid radicals O u r EPR spin trapping studies demonstrated the formation of radical adducts when endothelial cells adherent to microcarrier beads were subjected to oxidant stress induced by exposure to 100 p M Fez+.Figure 1 shows EPR spectra obtained with Porcine pulmonary artery endothelial cells (PPAEC) and the spin trap POBN
No spin adducts were detected if PPAEC enriched with docosahexaenoic acid (DHA) were not exposed to Fez+(Fig. 1B)
Summary
Lipid-derived free radicals were detected by electron paramagnetic resonance (EPR) spectrometry when cultured endothelial cells attached to Cytodex beads were exposed to ironinduced oxidant stress in the presence of the spin trap a(4-pyridyl-l-oxide)-N-tert-butylnitrone (POBN). Likewise, when U937 monocytes enriched with DHA were exposed subsequently to 20 pM oleic acid, a 35-45% decrease in radical adduct formation occurred These findings suggest that the endothelium may become more susceptible to oxidative injury when it is exposed to elevated amounts of polyunsaturated fatty acids. - - - Supplementary key words electron paramagnetic resonance free radicals lipid peroxidation docosahexaenoicacid oleic acid iron POBN spin trapping U937 monocytes. Recent work with U937 monocytes, a cultured cell line derived from a human monocytic leukemia [12], has demonstrated the formation of carbon-centered free radicals during lipid peroxidation induced by iron [13] These radicals were detected by electron paramagnetic resonance (EPR) spectrometry when a suspension of intact cells was incubated with ferrous ions (Fez') in the presence of a spin trap, a-(4-pyridyl-l-oxide)-N-tertbutylnitrone (POBN).
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