EPR detection of lipid-derived free radicals from PUFA, LDL, and cell oxidations

Abstract

We have used the spin trap 5,5-dimethyl-pyrroline-1-oxide (DMPO) and EPR to detect lipid-derived radicals (L d •) during peroxidation of polyunsaturated fatty acids (PUFA), low-density lipoprotein (LDL), and cells (K-562 and MCF-7). All oxygen-centered radical adducts of DMPO from our oxidizable targets have short lifetimes (<20 min). We hypothesized that the short lifetimes of these spin adducts are due in part to their reaction with radicals formed during lipid peroxidation. We proposed that stopping the lipid peroxidation processes by separating oxidation-mediator from oxidation-substrate with an appropriate extraction would stabilize the spin adducts. To test this hypothesis we used ethyl acetate to extract the lipid-derived radical adducts of DMPO (DMPO/L d •) from an oxidizing docosahexaenioc acid (DHA) solution; Folch extraction was used for LDL and cell experiments. The lifetimes of DMPO spin adducts post-extraction are much longer (>10 h) than the spin adducts detected without extraction. In iron-mediated DHA oxidation we observed three DMPO adducts in the aqueous phase and two in the organic phase. The aqueous phase contains DMPO/HO a N ≈ a H ≈ 14.8 G) and two carbon-centered radical adducts (a N 1 ≈ 15.8 G, a H 1 ≈ 22.6 G; a N 2 ≈ 15.2 G, a H 2 ≈ 18.9 G). The organic phase contains two long-chain lipid radical adducts (a N ≈ 13.5 G, a H ≈ 10.2 G; and a N ≈ 12.8 G; a H ≈ 6.85 G, 1.9 G). We conclude that extraction significantly increases the lifetimes of the spin adducts, allowing detection of a variety of lipid-derived radicals by EPR.