Trapping of fatty acid allyl radicals generated in lipoxygenase reactions in biological fluids by nitroxyl radical

Abstract

Nitroxyl radicals can trap fatty acid allyl radicals on ferric-lipoxygenases at lower oxygen content, which are an intermediate in the lipoxygenase reaction. In the present study, we examined whether nitroxyl radical-trapping of fatty acid allyl radicals on the enzyme proceeds in biological fluids with abundant antioxidants. The fatty acid allyl radical-nitroxyl radical adducts were quantified by HPLC with electrochemical detection (HPLC-ECD); the adducts in eluate degraded into nitroxyl radical by passing through heating coil at 100 degrees C, and then nitroxyl radical was detected by electrochemical detector. Soybean 15-lipoxygenase and nitroxyl radical (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl, CmDeltaP) were mixed with rat serum prepared from fresh venous blood, and the solution was stood at 37 degrees C for 1 h. One volume of the solution was mixed with 5 vols of cold acetonitrile. After centrifugation, the supernatant was subjected to HPLC-ECD. Arachidonate allyl radical-CmDeltaP adducts as well as linoleate allyl radical-CmDeltaP adducts were detected in the solution, and the content of these adducts remarkably increased in the presence of phospholipase A(2). It is proved for the first time that nitroxyl radical traps fatty acid allyl radicals generated in the lipoxygenase reaction in biological fluid without competition from endogenous antioxidants.