【摘要】 研究了荧光衍生化试剂Ｂｒ－ＤＭＥＱ与前列腺素（ＰＧＦ２α）的反应条件及其荧光产物的色谱分离条件。方法灵敏度高，最小检测量为１０ｆｍｏｌ。并且利用微透析技术和Ｆｅｎｔｏｎ反应直接在脊髓内产生羟基游离基，测定了中枢神经系统受到羟基游离基损伤时ＰＧＦ２α的释放。更多还原

【Abstract】 As prostaglandin F 2α is present in biological materials, and plays an important physiological role at trace level in the living body, then, highly sensitive determination of PGs is required. Various fluorescence derivatization reagents have been proposed for the determination of PGs. The 3 bromomethyl 6,7 methylenedioxyl 1 methyl 2(1H) quinoxalinone was found to be a highly sensitive fluorescence derivatization reagent for PGF 2α in HPLC with a detectable limit of 10 15 fmol for PGF 2α In this work we optimized its reaction conditions. Thus the PGF 2α was extracted from the microdialysates with ethyl acetate at pH 3.0 3.5 following which the extracts were evaporated to dryness. The residue was derivatized by adding acetonitrile, KHCO 3, Br DMEQ and 18 crown 6 ether at 50℃ for 30min in the dark. The corresponding fluorescent derivatives produced were separated on a C 8 column (Phase Sep Ltd.), 5μm, 4.6mm×150mm. Stepwise elution with different ratios of A and B was carried out. 30∶10∶60 of CH 3CN∶CH 3OH∶H 2O constituted A solution and 35∶30∶35 made B solution. The A/B(97/3) was first run for 25 min and A/B(50/50)for the next 15min. Then the column was equilibrated with A/B(97/3) for 20min before the next sample injected. Fluorescence detector was used at λ EX  370nm and λ EM  455nm,and flow rate of 2.0mL/min. Because the most evidence for a role of free radicals in tissue damage is indirect, we attempt to determine whether O·H causes release of arachidonic acid products in vivo. We did this by (1) generating O·H radical in vivo in rat spinal cord by administering H 2O 2 and FeCl 2/EDTA through two parallel microdialysis fibers so they mixed in the cord, and (2)analyzing PGF 2α in microdialysates in response to O·H generation by HPLC. We utilized dialysis fibers of ≤220μm external diameter including their coating except for a 2mm dialysis zone which was coated with a thin layer of silicon rubber. When the animal was clamped, two microdialysis fibers glued together were inserted through the cord until the dialysis zone just placed in the gray matters of the cord. The time course of changes in levels of PGF 2α during O·H generation by Fe/H 2O 2 is given. Typical chromatogram of the dialysate collected from one animal is illustrated. Prostaglandin F 2α dramatically increased in response to hydroxyl radical generation from undetectable (basal level) to about 333±166nmol/L (SD, n=5) in 90min, Prostaglandin F 2α was undetectable when either H 2O 2 or FeCl 2/EDTA was administered alone in control experiments , demonstrating that its formation was caused by generated hydroxyl radical.更多还原