Cardiovascular Research 1990; 24: 500-503
500
Recombinant human extracellular superoxide dismutase reduces concentration of oxygen free radicals in the reperfused rat heart Mats H Johansson, Johanna Deinum, Stefan L Marklund, Per-Ove Sjoquist
dismutase (Cu-Zn SOD), catalase, allopurinoli oxypurinol and desferrioxamine have shown protective effects, reducing the degree of myocardial damage in reperfused hearts.%” In a few studies free radicals have been measured during reperfusion of postischaemic hearts, either directly in the tissue,” l 2 or by using spin trapping technique^.'^'^ In two recent studies14 l 6 using 5,5-dimethyl-l-pyrroline-Noxide (DMPO) as a spin trap, it was possible to measure a lower concentration of the oxyradical adducts when the ischaemic hearts were reperfused with buffer solutions containing Cu-Zn superoxide dismutase. In all previous studies the superoxide dismutase isoenzyme used has been the normally intracellular Cu-Zn variety. In the extracellular space the secretory extracellular superoxide dismutase (EC-SOD) is the dominant isoenzyme. l 7 EC-SOD is a tetrameric copper and zinc containing glycoprotein.’8 l 9 It is heterogeneous with regard to heparin affinity and can be divided into three subfractions: “A” without affinity, “B” with weak affinity, and “C” with relatively strong affinity to heparin. EC-SOD C forms an equilibrium between heparin sulphate in the glycocalyx of cell surfaces and the extracellular fluid After intravenous injection, EC-SOD is rapidly sequestered by the endothelial cell surfaces.21 However, the physiological importance of this enzyme Free radical generation may be one of the major factors and its surface association has not yet been clarified. involved in the process of postischaemic reperfusion In the present study, using electron paramagnetic injury. I Antiradical agents such as Cu-Zn superoxide resonance spectroscopy (EPR) on effluents from isolated rat hearts perfused with the spin trap PBN, the aim was to study if recombinant human EC-SOD C Department of Cardiovascular Pharmacology, AB Hassle, (rh-EC-SOD C)I9 exhibited antioxidant effects similar S-43183 Molndal, Sweden to those of the previously studied Cu-Zn superoxide M Johansson dismutase. Catalase was added to the perfusate J Deinurn together with the two superoxide dismutase r-0 S]oqulst Department of Clinical Chemistry, UmeH University Hos- isoenzymes since erythrocyte catalase in vivo accounts pital, S-901 85 UmeA, Sweden for a high hydro en peroxide “scavenging activity” in S L Marklund the vasculature. F3
’
Correspondence to Dr Sloquist
K m words oxygen free radicals, myocardial ischaemia, superoxide dismutase Submitted 19 September 1989 Atrepted 83’anuary 1990
Methods Rats of the Sprague-Dawley strain (280-400 g) were anaesthetised with pentobarbitone 60 mg.kg-’ intraperitoneally. Heparin (1000 IU.kg-I) was given
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Abstract Study objective - The aim of the study was to determine whether recombinant human extracellular superoxide dismutase type C (rh-EC-SOD C) has antioxidant effects similar to those of the previously studied Cu-Zn superoxide dismutase. Design - Electron paramagnetic resonance spectroscopy was used for quantification of free radical generation during reperfusion of postischaemic isolated rat hearts. The hearts were perfused with Krebs-Henseleit buffer and then subjected to 15 min global ischaemia. N-tert-butyl-a-phenylnitrone (PBN) was added as a spin trapping agent to the perfusate. Experimental material - 18 hearts from SpragueDawley rats (280-400 g) were used. Measurements and main results - Reperfusion resulted in a burst of radical formation. In presence of rh-EC-SOD C plus catalase, the PBN spin adduct concentration was only 18% (p
500
Recombinant human extracellular superoxide dismutase reduces concentration of oxygen free radicals in the reperfused rat heart Mats H Johansson, Johanna Deinum, Stefan L Marklund, Per-Ove Sjoquist
dismutase (Cu-Zn SOD), catalase, allopurinoli oxypurinol and desferrioxamine have shown protective effects, reducing the degree of myocardial damage in reperfused hearts.%” In a few studies free radicals have been measured during reperfusion of postischaemic hearts, either directly in the tissue,” l 2 or by using spin trapping technique^.'^'^ In two recent studies14 l 6 using 5,5-dimethyl-l-pyrroline-Noxide (DMPO) as a spin trap, it was possible to measure a lower concentration of the oxyradical adducts when the ischaemic hearts were reperfused with buffer solutions containing Cu-Zn superoxide dismutase. In all previous studies the superoxide dismutase isoenzyme used has been the normally intracellular Cu-Zn variety. In the extracellular space the secretory extracellular superoxide dismutase (EC-SOD) is the dominant isoenzyme. l 7 EC-SOD is a tetrameric copper and zinc containing glycoprotein.’8 l 9 It is heterogeneous with regard to heparin affinity and can be divided into three subfractions: “A” without affinity, “B” with weak affinity, and “C” with relatively strong affinity to heparin. EC-SOD C forms an equilibrium between heparin sulphate in the glycocalyx of cell surfaces and the extracellular fluid After intravenous injection, EC-SOD is rapidly sequestered by the endothelial cell surfaces.21 However, the physiological importance of this enzyme Free radical generation may be one of the major factors and its surface association has not yet been clarified. involved in the process of postischaemic reperfusion In the present study, using electron paramagnetic injury. I Antiradical agents such as Cu-Zn superoxide resonance spectroscopy (EPR) on effluents from isolated rat hearts perfused with the spin trap PBN, the aim was to study if recombinant human EC-SOD C Department of Cardiovascular Pharmacology, AB Hassle, (rh-EC-SOD C)I9 exhibited antioxidant effects similar S-43183 Molndal, Sweden to those of the previously studied Cu-Zn superoxide M Johansson dismutase. Catalase was added to the perfusate J Deinurn together with the two superoxide dismutase r-0 S]oqulst Department of Clinical Chemistry, UmeH University Hos- isoenzymes since erythrocyte catalase in vivo accounts pital, S-901 85 UmeA, Sweden for a high hydro en peroxide “scavenging activity” in S L Marklund the vasculature. F3
’
Correspondence to Dr Sloquist
K m words oxygen free radicals, myocardial ischaemia, superoxide dismutase Submitted 19 September 1989 Atrepted 83’anuary 1990
Methods Rats of the Sprague-Dawley strain (280-400 g) were anaesthetised with pentobarbitone 60 mg.kg-’ intraperitoneally. Heparin (1000 IU.kg-I) was given
Downloaded from by guest on June 7, 2016
Abstract Study objective - The aim of the study was to determine whether recombinant human extracellular superoxide dismutase type C (rh-EC-SOD C) has antioxidant effects similar to those of the previously studied Cu-Zn superoxide dismutase. Design - Electron paramagnetic resonance spectroscopy was used for quantification of free radical generation during reperfusion of postischaemic isolated rat hearts. The hearts were perfused with Krebs-Henseleit buffer and then subjected to 15 min global ischaemia. N-tert-butyl-a-phenylnitrone (PBN) was added as a spin trapping agent to the perfusate. Experimental material - 18 hearts from SpragueDawley rats (280-400 g) were used. Measurements and main results - Reperfusion resulted in a burst of radical formation. In presence of rh-EC-SOD C plus catalase, the PBN spin adduct concentration was only 18% (p
