Spin trapping of lipid radicals with DEPMPO-derived spin traps: Detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase

The spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms a superoxide adduct with a half-life of almost 15 min. DEPMPO is very hydrophilic and its use for the detection of radicals in the lipid phase ( lipid-derived radicals and superoxide generated in the lipid phase) is ther efore limited due to its very low concentration in the lipid phase. For the detection of lipid-derived radicals, three derivatives of DEPMPO with incr easing degree of lipid solubility have been investigated: 5-(di-n-propoxyph osphoryl)-5-methyl-1-pyrroline N-oxide (DPPMPO), 5-(di-n-butoxyphosphoryl)- 5-methyl-1-pyrroline N-oxide (DBPMPO), and 5-(bis-(2-ethylhexyloxy)phosphor yl)-5-methyl-1-pyrroline N-oxide (DEHPMPO). As compared with the spin trap DMPO, the half-lives of the respective superoxide adducts were clearly high er in aqueous solutions of the spin traps, which facilitates qualitative ES R measurements. The stability of the superoxide spin adducts formed with th e various lipophilic spin traps in aqueous buffer were similar to those obs erved with DEPMPO (half-life: 7-11 min.). In model experiments using Fe3+-c atalyzed nucleophilic addition of methanol or tert-butanol to the respectiv e spin trap the respective alkoxyl radical adducts were formed in aqueous s olution as transient species in the presence of high concentrations of the alcohol. Upon dilution with water the alkoxyl group was substituted by wate r, giving the respective hydroxyl adduct of the spin trap. Care must theref ore be taken when Fenton-type reactions are used for the generation of radi cals such as the use of Fe2+ complexes with phosphate or DTPA or inactivati on of iron by addition of "Desferal" (Novarti's Pharma GmbH, Vienna, Austri a) after a short incubation time. Addition of Fe2+ under anaerobic conditio ns to an aqueous suspension of linoleic acid hydroperoxide and the spin tra p resulted in the detection of three different species: a carbon-centered r adical adduct, an acyl radical adduct, and the hydroxyl adduct. In the pres ence of oxygen a different species was observed with DEPMPO, DPPMPO, and DB PMPO, which was only slightly suppressed upon the addition of SOD, possibly the respective spin adduct of either the alkylperoxyl radical or, in analo gy to DMPO, a secondary alkoxyl radical. (C) 2000 Elsevier Science Inc.