A Novel Method for in Vivo Hydroxyl Radical Measurement by Microdialysis in Fetal Sheep Brain, in Utero

Hydroxyl radical (OH •) is a reactive oxygen species produced during severe hypoxia, asphyxia or ischemia that can cause cell death resulting in brain damage. Generation of OH • may occur in the fetal brain during asphyxia in utero. The very short half-life of OH • requires use of trapping agents such as salicylic acid or phenylalanine for detection, but their hydroxylated derivatives are either unstable, produced endogenously, or difficult to measure in the small volume of microdialysis samples. In the present study we used terephthalic acid (TA), hydroxylation of which yields a stable and highly fluorometric isomer (excitation, 326nm; emission, 432nm). In vitro studies using OH • generated by the Fenton reaction showed that hydroxylated terephthalic acid formed quickly (< 10 sec), was resistant to bleaching (< 5% change in fluorescence), and permitted detection of < 0.5 pmol OH •. In vivo studies were performed in fetal sheep using microdialysis probes implanted into the parasagittal cortex. The probe was perfused at 2 µl/min with artificial CSF containing 5 mM TA and samples collected every 30 min. Fluorescence measured in 10 µl of dialysate was significantly greater than in the efflux from probes perfused without TA. HPLC analysis showed that the fluorescence in dialysis samples was entirely due to hydroxylation of TA. Thus, this study shows it is possible to use terephthalic acid as a trapping agent for detecting low concentration of OH • both in vitro and in vivo, and that low concentrations of OH • are present in fetal brain tissue and fluctuate with time.