Development of Analytical Methods Coupled to Microdialysis Sampling for Studying Biomarkers of Oxidative Stress

Abstract

This research describes the development of analytical methods based upon separation techniques coupled to microdialysis sampling for the detection of potential biomarkers of oxidative stress. The research focused on two biomarkers, glutathione and the hydroxyl radical (.OH). Microdialysis sampling was utilized for the continuous monitoring of these biomarkers in rat liver and heart. Oxidative stress was induced, causing the generation of reactive oxygen species (ROS). It was hypothesized that ROS generation would increase in biological systems due to induced oxidative stress. In one investigation, a capillary electrophoresis (CE) method with ultra-violet (UV) detection was developed employing pH-mediated stacking, an on-column preconcentration technique, to detect two forms of glutathione, reduced glutathione (GSH) and oxidized glutathione (GSSG), simultaneously in microdialysates. The detection limits necessary for measuring GSH and GSSG in microdialysates with UV detection could not be achieved without the use of pH-mediated stacking. The method was utilized to monitor glutathione in microdialysates from rat liver that was subjected to chemically induced oxidative stress using adriamycin. GSSG concentration was found to increase from the basal concentration as a result of oxidative stress, suggesting increased antioxidant activities during oxidative stress. For the indirect detection of .OH in microdialysates, a trapping agent, 4-hydroxybenzoic acid (4-HBA), was employed. Indirect determination involved the trapping of .OH by 4-HBA and then detection of the radical adduct, 3,4-dihydroxybenzoic acid (3,4-DHBA), by the developed CE-UV method. In vitro studies of .OH generation systems (e.g., UV photolysis of H2O2, the Fenton reaction, and hypoxanthine/xanthine oxidase systems with and without superoxide dismutase) demonstrated the capability of 4-HBA to trap .OH and the ability of the CE-UV method to detect 3,4-DHBA in the reaction products. Finally, an in vivo investigation was performed using CE-UV and high performance liquid chromatography (HPLC) method with electrochemical (EC) detection to detect 3,4-DHBA in microdialysates from rat heart where 4-HBA was delivered through the microdialysis probe to trap .OH. Increased generation of 3,4-DHBA was observed in microdialysates of rat heart subjected to physically induced oxidative stress (ischemia-reperfusion), suggesting increased generation of ROS during oxidative stress.