Microdialysis sampling combined with electron spin resonance for superoxide radical detection in microliter samples

Quantitation of superoxide radical (O-2(.-)) production at the site of radical generation remains challenging. Microdialysis sampling is an advantageous tool for sampling from localized environments. It is difficult to combine electron spin resonance (ESR) spin traps with microdialysis because O-2(.-) adducts with common nitrone spin traps have shorter half-lives than typical microdialysis collection times. Furthermore, typical dialysate samples (5-15muL) suffer significant sensitivity loss when diluted for detection in a conventional ESR flat cell (200,A). To overcome these difficulties, a cyclic hydroxylamine, 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H), which produces a stable nitroxide radical (PP.) product upon reaction with O-2(.-) was employed. Capillary cells (1.4 muL effective volume) coupled with a loop-gap resonator were utilized to measure PP. in microliter microdialysis samples (LOD 0.36 pmol). A xanthine/xanthine oxidase (X/XO) model system provided sustained O-2(.-) production. When PP-H was included in the X/XO medium external to the microdialysis probe, a relative recovery of 22.1 +/- 1.1 and 57.2 +/- 5.7% for PP. was achieved at perfusion fluid flow rates of 0.5 and 1.0 muL/min, respectively. The respiratory burst in interferon-gamma and zymosan-stimulated RAW 264.7 macrophages was also investigated.