[Determination of prostaglandin F2alpha release in the rat spinal cord upon hydroxyl free radical damage by high performance liquid chromatography].
As prostaglandin F2alpha 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 PGF2alpha in HPLC with a detectable limit of 10-15 fmol for PGF2alpha. In this work we optimized its reaction conditions. Thus the PGF2alpha 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, KHCO3, Br-DMEQ and 18-crown-6-ether at 50 degrees C for 30min in the dark. The corresponding fluorescent derivatives produced were separated on a C8 column (Phase-Sep Ltd.), 5microm, 4.6mm x 150mm. Stepwise elution with different ratios of A and B was carried out. 30:10:60 of CHsCN:CH3OH:H2O 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 lambdaEX 370nm and lambdaEM 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 OH causes release of arachidonic acid products in vivo. We did this by (1) generating OH radical in vivo in rat spinal cord by administering H2O2 and FeCl2/EDTA through two parallel microdialysis fibers so they mixed in the cord, and (2) analyzing PGF2alpha in microdialysates in response to OH generation by HPLC. We utilized dialysis fibers of < or = 220microm 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 PGF2alpha during OH generation by Fe/H2O2 is given. Typical chromatogram of the dialysate collected from one animal is illustrated. Prostaglandin F2alpha dramatically increased in response to hydroxyl radical generation from undetectable (basal level) to about 333 +/- 166nmol/L (SD, n = 5) in 90min, Prostaglandin F2alpha was undetectable when either H2O2 or FeCl2/EDTA was administered alone in control experiments, demonstrating that its formation was caused by generated hydroxyl radical.
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Beijing Institute of Chemical Fiber Technology, Beijing, 100029.
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015237:Dinoprost / Q000032:analysis* / Q000378:metabolism*; D004195:Disease Models, Animal; D006801:Humans; D017665:Hydroxyl Radical / Q000009:adverse effects*; D008297:Male; D051381:Rats; D013116:Spinal Cord / Q000737:chemistry* / Q000378:metabolism; D013119:Spinal Cord Injuries / Q000139:chemically induced / Q000378:metabolism*
D017665:Hydroxyl Radical; D015237:Dinoprost