The carboxyproxyl-derived spin trap (CP-H) is an appropriate detector-compound for oxidative stress

Reperfusion of ischemic tissue disturbs the balance between reactive oxygen species (ROS) and the cellular antioxidative defense. This imbalance is known as oxidative stress. In this study the spin trap 3-carboxy-2,2,5,5-tetramethylpyrrolin-1-hydroxide (CP-H) with its ESR-detectable paramagnetic analogue 3-carboxy-2,2,5,5-tetramethylpyrrolin-1-oxyl (•CP) was analyzed in vitro and in vivo. In preliminary in vitro experiments we studied the interaction of CP-H with reactive compounds like hydroxyl radicals (•OH) and alkylperoxyl radicals (ROO•) which are formed during organ reperfusion or tissue reoxygenation. The increase in the peak intensity of the ESR signal of the •CP-radical was used as a measure for CP-H oxidation by the above-mentioned oxidizing radicals. It could be clearly shown that •OH as well as ROO• induce CP-H oxidation. The intensity of the ESR signal (•CP) depends on the concentration of the applied oxidant. In a further set of in vitro experiments we analyzed some factors influencing the stability of the generated •CP. Cellular reductants are able to interact with many radicals whereby their paramagnetic signal intensity decreases. We could show that glutathione (GSH) up to 5 mM does not influence •CP concentration. On the other hand, ascorbate at a concentration of 0.6 mM significantly reduces 55% of •CP within 60 min to the ESR-silent CP-H. At 1 mM ascorbate the •CP derived ESR signal is reduced within 60 min by 90%. Lower concentrations of ascorbate (0.1–0.3 mM) do not significantly decrease signal intensity within 1 h. Homogenization of ischemic rat kidney in the presence of an air-equilibrated buffer obviously induces the formation of oxidizing radicals which in turn are able to convert diamagnetic CP-H into paramagnetic •CP. The intensity of the formed •CP was analyzed in a 600 g supernatant with ESR spectroscopy at 25°C. It could be demonstrated that at least 3.0 ± 0.5 μM •CP is formed 15 min after starting tissue homogenization and reoxygenation. Subsequent measurements of the •CP concentration indicated that its signal intensity continuously decreases. After 75 min a residual •CP concentration of 0.7 ± 0.3 μM was monitored. Removal of mitochondria from the homogenate by centrifugation at 6,000g decelerates the disappearance of •CP but does not block it completely. In summary it could be shown that the marker (CP-H) is able to indicate the formation of oxidizing radicals during reoxygenation of ischemic tissue. This method underestimates the amount of produced oxidizing radicals. One reason for this is the reduction of •CP by some cellular reductants. Other reasons will be discussed. We assume that the used method allows a nearly real-time determination of radical production during organ reoxygenation.