Yan Edwin B, Unthank Jessica K, Castillo-Melendez Margie, Miller Suzanne L, Langford Steven J, Walker David W
Dept. of Physiology, Fetal & Neonatal Research Group, Monash University, Clayton, Victoria 3800, Australia.
J Appl Physiol (1985). 2005 Jun;98(6):2304-10. doi: 10.1152/japplphysiol.00617.2004. Epub 2005 Feb 17.
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 mul/min with artificial cerebrospinal fluid containing 5 mM TA, and samples were collected every 30 min. Fluorescence measured in 10 mul 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.
羟基自由基（·OH）是在严重缺氧、窒息或局部缺血过程中产生的一种活性氧，可导致细胞死亡，进而造成脑损伤。胎儿在子宫内窒息期间，其大脑中可能会产生·OH。·OH的半衰期极短，需要使用水杨酸或苯丙氨酸等捕获剂来进行检测，但其羟基化衍生物要么不稳定，要么是内源性产生的，要么在微量透析样本的小体积中难以测量。在本研究中，我们使用了对苯二甲酸（TA），其羟基化会产生一种稳定且具有高荧光性的异构体（激发波长为326nm；发射波长为432nm）。利用芬顿反应产生的·OH进行的体外研究表明，羟基化的TA迅速形成（<10秒），抗漂白（荧光变化<5%），并且能够检测到<0.5皮摩尔的·OH。在体内研究中，我们将微量透析探针植入胎羊的矢状旁皮质进行研究。探针以2微升/分钟的速度用含有5毫摩尔TA的人工脑脊液灌注，并每30分钟收集一次样本。在10微升透析液中测得的荧光显著高于未灌注TA的探针流出液中的荧光。高效液相色谱分析表明，透析样本中的荧光完全是由于TA的羟基化所致。因此，本研究表明，有可能将TA用作捕获剂，在体外和体内检测低浓度的·OH，并且胎儿脑组织中存在低浓度的·OH，且会随时间波动。
