Evaluation of the Forrester-Hepburn mechanism as an artifact source in ESR spin-trapping

Nitrone spin traps such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are commonly used for free radical detection. Though proven examples are rare, artifact formation must be considered. For example, the Forrester-Hepburn mechanism yields the same radical adduct as that formed by genuine radical trapping. A hydroxylamine is formed by nucleophilic attack of the substrate on DMPO and subsequently oxidized to the respective nitroxide radical. One potential candidate for this artifact is the sulfur trioxide radical adduct (DMPO/(•)SO(3)(-)), as detected in spin-trapping experiments with horseradish peroxidase and sulfite. It has previously been shown by NMR experiments that the hydroxylamine intermediate does indeed form, but no direct proof for the ESR artifact has been provided. Here, we used isotopically labeled DMPO with horseradish peroxidase and ferricyanide to test for the Forrester-Hepburn artifact directly in a spin-trapping experiment. Besides sulfite, we investigated other nucleophiles such as cyanide, cysteine, and glutathione. Neither sulfite nor biological thiols produced detectable spin-trapping artifacts, but with cyanide the relatively weak signal originated entirely from the nucleophilic reaction. The hydroxylamine intermediate, which is more abundant with cyanide than with sulfite, was identified as cyano-hydroxylamine by means of 2D NMR experiments. Although our study found that spin trapping provided authentic free radical signals with most of the substrates, the occurrence of the Forrester-Hepburn mechanism artifact with cyanide emphasizes the importance of isotope measurements with nucleophile substrates.