Microdialysis monitoring of reactive oxygen species

Accurate quantitation of reactive oxygen species such as hydroxyl radical (HO•) and superoxide radical (O2•- ) at oxidative stress sites remain challenging. Microdialysis sampling can be advantageous for collecting radicals produced from living tissues because it allows continuous monitoring of the localized environment.; Microdialysis sampling of HO• using 4-hydroxybenzoic acid (4-HBA) is becoming a well-established procedure. However, there has been no published analysis that relates the concentration of the indirectly trapped product, 3,4-dihydroxybenzoic acid (3,4-DHBA), to HO• level external to the microdialysis probe. In chapter 2, a sustained in vitro production of HO• was provided by xanthine oxidase (XO) system and 4-HBA was included either in the perfusion fluid or in the external medium to elucidate the factors that influence calibration during radical trapping. XO enzymatic activity was reproducible but a considerable amount of variance in the rate and amount of 3,4-DHBA was observed when one microdialysis probe was placed in quiescent reaction mixture. Results from two-probe setup and stirred systems were compared.; It has been considered very difficult to combine electron spin resonance (ESR) spin traps with microdialysis because commonly used nitrone traps have shorter half-lives for their radical adducts (1 to 8 min) than typical dialysates collection times. Furthermore, typical dialysates (<15 muL) will suffer significant sensitivity loss when diluted for detection in a conventional ESR flat cell (200 muL). Chapter 3 assessed feasibility of combining a stable spin trap 5-methyl-5-phenylpyrroline-1-oxide (MPPO) for HO• sampling since MPPO-OH adduct has a reported half-life of 76 minutes, Four prevalent products were found being produced between MPPO and various HO • generation systems (Fenton system and UV photolysis of H 2O2). A comprehensive approach combining ESR, LC-MS, LC-MS/MS and electrochemistry was used to determine both the origin and the chemical nature of these MPPO-derived products. Chapter 4 used a cyclic hydroxylamine, 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H) for microdialysis sampling of O2•-. PP-H produces a stable nitroxide radical upon reaction with O2•- . Capillary cells (effective volume 1.4 muL) coupled with a loop-gap resonator was utilized to overcome the detection volume limitation. Reproducible results were obtained for different microdialysis sampling settings. Respiratory burst in stimulated macrophages was also investigated.