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 therefore limited due to its very low concentration in the lipid phase. For the detection of lipid-derived radicals, three derivatives of DEPMPO with increasing degree of lipid solubility have been investigated: 5-(di-n-propoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DPPMPO), 5-(di-n-butoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DBPMPO), and 5-(bis-(2-ethylhexyloxy)phosphoryl)-5-methyl-1-pyrroline N-oxide (DEHPMPO). As compared with the spin trap DMPO, the half-lives of the respective superoxide adducts were clearly higher in aqueous solutions of the spin traps, which facilitates qualitative ESR measurements. The stability of the superoxide spin adducts formed with the various lipophilic spin traps in aqueous buffer were similar to those observed with DEPMPO (half-life: 7-11 min.). In model experiments using Fe(3+)-catalyzed nucleophilic addition of methanol or tert-butanol to the respective spin trap the respective alkoxyl radical adducts were formed in aqueous solution as transient species in the presence of high concentrations of the alcohol. Upon dilution with water the alkoxyl group was substituted by water, giving the respective hydroxyl adduct of the spin trap. Care must therefore be taken when Fenton-type reactions are used for the generation of radicals such as the use of Fe(2+) complexes with phosphate or DTPA or inactivation of iron by addition of "Desferal" (Novarti's Pharma GmbH, Vienna, Austria) after a short incubation time. Addition of Fe(2+) under anaerobic conditions to an aqueous suspension of linoleic acid hydroperoxide and the spin trap resulted in the detection of three different species: a carbon-centered radical adduct, an acyl radical adduct, and the hydroxyl adduct. In the presence of oxygen a different species was observed with DEPMPO, DPPMPO, and DBPMPO, which was only slightly suppressed upon the addition of SOD, possibly the respective spin adduct of either the alkylperoxyl radical or, in analogy to DMPO, a secondary alkoxyl radical.
Stolze
K
K
Institute of Pharmacology and Toxicology, Veterinary University of Vienna, Vienna, Austria.
Udilova
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N
Nohl
H
H
D003497:Cyclic N-Oxides / Q000138:chemical synthesis / Q000737:chemistry*; D004355:Drug Stability; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry; D066298:In Vitro Techniques; D015227:Lipid Peroxidation; D008055:Lipids / Q000737:chemistry*; D013113:Spin Labels / Q000138:chemical synthesis*; D013481:Superoxides / Q000737:chemistry
D003497:Cyclic N-Oxides; D005609:Free Radicals; D008055:Lipids; D013113:Spin Labels; D013481:Superoxides; C098505:5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide