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INNOVATIVE METHODOLOGY

Novel method for in vivo hydroxyl radical measurement by microdialysis in fetal sheep brain in utero

¹Department of Physiology, Fetal & Neonatal Research Group, and ²School of Chemistry, Monash University, Clayton, Australia

Submitted 17 June 2004 ; accepted in final form 14 February 2005

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, 326 nm; emission, 432 nm). In vitro studies using ·OH generated by the Fenton reaction showed that hydroxylated TA formed quickly (<10 s), 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 cerebrospinal fluid containing 5 mM TA, and samples were collected every 30 min. Fluorescence measured in 10 µl of dialysate was significantly greater than in the efflux from probes perfused without TA. High-performance liquid chromotography analysis showed that the fluorescence in dialysis samples was entirely due to hydroxylation of TA. Thus this study shows that it is possible to use TA as a trapping agent for detecting low concentrations of ·OH both in vitro and in vivo and that low concentrations of ·OH are present in fetal brain tissue and fluctuate with time.

terephthalic acid; Fenton reaction; fetus; microdialysis

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