An in vitro hydroxyl radical generation assay for microdialysis sampling calibration

A xanthine oxidase hydroxyl radical ((OH)-O-.)-generating system was created for sustained in vitro production of (OH)-O-.. This assay was coupled with microdialysis sampling to elucidate the factors that influence microdialysis calibration during radical trapping. A (OH)-O-. trapping agent, 4-hydroxybenzoic acid, was included either in the microdialysis perfusion fluid or in the medium external to the microdialysis probe. Xanthine oxidase enzymatic activity was reproducible and had an average activity measured by UV absorbance of produced uric acid of 0.037 +/- 0.005 DeltaAU/min (n = 5). A considerable amount of variance in the rate and amount of the product, 3,4-dihydroxybenzoic acid (3,4-DHBA), was observed when one microdialysis probe was placed in the reaction mixture. When two microdialysis probes were placed in the reaction mixture, a greater rate and amount of 3,4-DHBA was observed. Different concentrations of 3,4-DHBA were obtained between quiescent and stirred systems. (C) 2002 Elsevier Science (USA).