Direct evidence for in vivo hydroxyl-radical generation in experimental iron overload: an ESR spin-trapping investigation.

Although the hydroxyl radical is often implicated as the species responsible for the initiation of oxidative damage in iron-overload conditions, no ESR evidence for the formation of the radical in vivo has been reported. We have employed a secondary radical-trapping technique in which the hydroxyl radical reacts with dimethyl sulfoxide to form the methyl radical, which is then detected as its adduct of the spin trap N-t-butyl-alpha-phenylnitrone in the bile of animals given an intragastric dose of ferrous sulfate. The identity of this adduct was verified by isotope-substitution techniques. We show that unless measures are taken to inactivate the iron excreted in the bile of treated animals, reactions between iron, oxygen, dimethyl sulfoxide, N-t-butyl-alpha-phenylnitrone, and bile components lead to the formation of artifacts during sample collection.