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Fig. 35. Decomposition of dimeric GSH-DNIC by superoxide generated by xanthine oxidase in 0.1 mM phosphate buffer, pH 7.4. (curve 1) Kinetics of the EPR signal decrease in the solution of 0.25 mM GSH-DNIC + 0.2 U/ml xanthine oxidase + 1 mM xanthine; (curve 2) 600 U/ml catalase was added to the solution 1; and (curve 3) 600 U/ml catalase + 150 U/ml superoxide dismutase were added to the solution. Dimeric GSH-DNIC is diamagnetic and cannot be detected with EPR directly. For detection, 1.6 mM cysteine was added to transform EPR-silent dimeric GSH-DNIC into monomeric EPR-detectable Cys-DNIC monomers. (From Ref. [98].)

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Fig. 35. Decomposition of dimeric GSH-DNIC by superoxide generated by xanthine oxidase in 0.1 mM phosphate buffer, pH 7.4. (curve 1) Kinetics of the EPR signal decrease in the solution of 0.25 mM GSH-DNIC + 0.2 U/ml xanthine oxidase + 1 mM xanthine; (curve 2) 600 U/ml catalase was added to the solution 1; and (curve 3) 600 U/ml catalase + 150 U/ml superoxide dismutase were added to the solution. Dimeric GSH-DNIC is diamagnetic and cannot be detected with EPR directly. For detection, 1.6 mM cysteine was added to transform EPR-silent dimeric GSH-DNIC into monomeric EPR-detectable Cys-DNIC monomers. (From Ref. [98].)

produced in the tissues. The destruction of DNIC by peroxynitrite was shown by our group [131]. The destruction by superoxide was demonstrated only recently [98]. Fig. 35 presents the kinetics of the decomposition of GSH-DNIC by superoxide as generated enzymatically by xanthine oxidase. With 0.2 U/ml xanthine oxidase and 1 mM xanthine, ca 80% of DNIC was lost after 4 min. The decomposition could be prevented completely by the addition of catalase (600 U/ml) + superoxide dismutase (SOD) (150 U/ml). Catalase alone attenuated the DNIC degradation for 50%. These experiments prove that superoxide and hydrogen peroxide can destroy DNIC complexes. Together with peroxynitrite, these compounds keep the basal levels of endogenous DNIC below the detection limit for EPR.

These results were corroborated by in vitro experiments on protein-bound DNIC with KO2 as superoxide donor (Vanin, unpublished data). Solutions of DNIC-BSA were generated from BSAby adding low-molecular DNIC (1:20) with cysteine, phosphate or thiosulfate (forming DNIC-BSA-1, DNIC-BSA-2 or DNIC-BSA-3, respectively). The addition of equimolar KO2 in the presence of catalase resulted in the instantaneous loss of EPR signal intensity from the solutions of DNIC-BSA-1, DNIC-BSA-2 or DNIC-BSA-3. The signal intensities were reduced by a factor 3, 2 or 1.3, respectively. In the presence of SOD, no loss of DNIC was observed.

It is important to note that the stability of DNIC-BSA depends on the type of thiol ligand. We noted that incorporation of thiosulfate anions into the DNIC greatly enhanced the stability of DNIC-BSA against destruction by superoxide or hydrogen peroxide in vitro. This observation was reminiscent of the high stability of low-molecular DNIC with thiosulfate ligands previously observed in vivo: thiosulfate-DNICs have much longer lifetime in mice than low-molecular DNICs with ligands like cysteine or glutathione [99]. The mice had been injected with bacterial lipopolysaccharide (LPS) to stimulate the synthesis of iNOS, and subsequently received a dose of preformed Cys-DNIC or GS-DNIC. The injection resulted in the formation of some protein-bound DNICs in various tissues of the animals. These animals have strong generation of NO by iNOS due to the LPS. Surprisingly, the yields of protein-bound DNICs in liver proved lower (!) than yields in controls where low-molecular DNICs were injected without LPS. This situation was reversed when thiosulfate ligands were used. With the injection of thiosulfate-DNIC, the yields of protein-bound DNIC in the LPS-treated mice were threefold higher than in controls. Clearly, the thiosulfate ligands afford far better protection against endogenous superoxide or hydrogen peroxide in inflammated animals than the usual thiols like glutathione.

Subsequent experiments [99] exploited the high stability of thiosulfate-DNIC to give the first direct proof that endogenous DNICs may form from NO released by NOS. As before, endogenous NO production was stimulated by injecting the mice with LPS. The boost of endogenous NO was confirmed by NO trapping with iron-diethyldithiocarbamate (Fe-DETC) complexes (Fig. 36, trace 1).

The endogenous NO production could be eliminated by the administration of the NOS inhibitor Nœ-nitro-L-arginine (NNLA). This inhibition led to a decrease in the concentration of these complexes to nearly zero (Fig. 36, curve 2). Formation of DNIC was not observed in these animals (Fig. 36, curve 3). Significant quantities of DNIC could be detected in liver

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