Detection of oxygen radicals during reperfursion of intestinal cell in vitro

Abstract The nitroxide OXANO· (2-Ethyl-2,5,5-trimethyl-3-oxazolidinoxyl) which in its reduced form, OXANOH (2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine), is capable of reacting with short-lived radicals, forming a secondary stable radical, was used for ESR-detection of radical production in isolated cells. The properties of OXANO· and OXANOH in terms of stability in cellular and subcellular systems, membrane permeability and effects on cellular viability were evaluated. Ischemia and reperfusion was simulated in vitro in a preparation of cells from rat intestinal mucosa by incubation at high density (4 × 10 8 cells/ml) under an atmosphere of nitrogen for 25 min and resuspended with fresh oxygenated buffer containing 5 mM Oxanoh. A significant increase in radical formation during the 15 min reperfusion period studied was obtained in cells exposed to ischemia compared to control cells incubated at normal density under an atmosphere of oxygen . The addition of 5 μM of the scavenging enzyme superoxide dismutase reduced the radical formation by 50%. The sequence of the superoxide formation was calculated as the difference in radical production in the presence and absence of superoxide dismutase.