title: The carboxyproxyl-derived spin trap (CP-H) is an appropriate detector-compound for oxidative stress.
abstract: 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 degrees C. It could be demonstrated that at least 3.0 +/- 0.5 microM *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 microM 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.
authors.affiliation: Institut für Pathophysiologie der Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Adam
authors.forename: H
authors.initials: H
authors.lastname: Loertzer
authors.forename: P
authors.initials: P
authors.lastname: Fornara
authors.forename: H J
authors.initials: HJ
authors.lastname: Brömme
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000737:chemistry*; D003497:Cyclic N-Oxides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D007511:Ischemia; D007668:Kidney / Q000378:metabolism*; D008297:Male; D018384:Oxidative Stress; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000737:chemistry*
chemical_list: C523522:1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxylate; D015415:Biomarkers; D003497:Cyclic N-Oxides; D017382:Reactive Oxygen Species