Determination of Superoxide and Ascorbyl Radicals in the Circulation of Animals under Oxidative Stress

Quantitation of the superoxide radical and its related metabolites in vivo is practically difficult predominantly because of their short biological half-lives. Though oxidized cytochrome c (cyt c) has been used for determining superoxide radicals in vitro, it cannot be used for in vivo analysis because of its low specificity as an electron acceptor and rapid disappearance from the circulation. To measure superoxide radicals and related metabolites in normal and pathologic subjects, we have synthesized a cyt c derivative (SMAC) with prolonged half-life in the circulation (T1/2 = 130 min) by conjugating acetylated cyt c with poly(styrene-co-maleic acid) butyl ester (SM). An SM-conjugated superoxide dismutase (SM-SOD) with prolonged in vivo half-life was also synthesized. When injected intravenously to the rat, SMAC was rapidly reduced in the circulation of normal rats. The rate of SMAC reduction was markedly increased by intravenous administration of menadione, a compound capable of redox cycling and generating superoxide. The rate of SMAC reduction was not inhibited by a large dose of SM-SOD (27,000 unit/kg) in both normal and menadione-treated animals. The rate of SMAC reduction also increased in animals which were administered alloxan, a diabetogenic agent. In contrast to the experiments with menadione, the alloxan-enhanced reduction of SMAC was significantly inhibited by SM-SOD. Kinetic analysis using ascorbate oxidase suggested that ascorbyl radical was principally responsible for the SM-SOD-insensitive reduction of SMAC. Streptozotocin, another diabetogenic agent, failed to increase the rate of SMAC reduction. Thus, the effect of streptozotocin on the redox state of animals and the mechanism of its diabetogenic action might differ from those of alloxan. Combined use of SMAC and SM-SOD might permit quantitative studies on the occurrence of ascorbyl and superoxide radicals in the circulation of animals challenged with oxidative stress.