Morrow J D, Zackert W E, Yang J P, Kurhts E H, Callewaert D, Dworski R, Kanai K, Taber D, Moore K, Oates J A, Roberts L J
Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.
Anal Biochem. 1999 May 1;269(2):326-31. doi: 10.1006/abio.1999.4008.
The isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical-catalyzed peroxidation of arachidonic acid. The first series of IsoPs characterized contained F-type prostane rings analogous to PGF2alpha. One F-ring IsoP, 15-F2t-IsoP (8-iso-PGF2alpha) has been shown to be formed in abundance in vivo and to exert potent biological activity. As a means to assess the endogenous production of this compound, we developed a method to quantify the major urinary metabolite of 15-F2t-IsoP, 2,3-dinor-5,6-dihydro-15-F2t-IsoP (2,3-dinor-5, 6-dihydro-8-iso-PGF2alpha), by gas chromotography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an 18O2-labeled derivative for use as an internal standard. After purification, the compound was analyzed as a pentafluorobenzyl ester trimethylsilyl ether. Precision of the assay is +/-4% and accuracy is 97%. The lower limit of sensitivity is approximately 20 pg. Levels of the urinary excretion of this metabolite in 10 normal adults were found to be 0. 39 +/- 0.18 ng/mg creatinine (mean +/- 2 SD). Substantial elevations in the urinary excretion of the metabolite were found in situations in which IsoP generation is increased and antioxidants effectively suppressed metabolite excretion. Levels of 2,3-dinor-5, 6-dihydro-15-F2t-IsoP were not affected by cyclooxygenase inhibitors. Thus, this assay provides a sensitive and accurate method to assess endogenous production of 15-F2t-IsoP as a means to explore the pathophysiological role of this compound in human disease.
异前列腺素（IsoPs）是由自由基催化花生四烯酸过氧化反应生成的一系列新型类前列腺素（PG）化合物。首批被鉴定的异前列腺素含有与前列腺素F2α类似的F型前列腺环。一种F环异前列腺素，15-F2t-异前列腺素（8-异前列腺素F2α）已被证明在体内大量生成并具有强大的生物活性。作为评估该化合物内源性生成的一种手段，我们开发了一种方法，通过气相色谱/负离子化学电离质谱法定量15-F2t-异前列腺素的主要尿液代谢产物2,3-二降-5,6-二氢-15-F2t-异前列腺素（2,3-二降-5,6-二氢-8-异前列腺素F2α）。这种代谢产物经化学合成并转化为18O2标记的衍生物用作内标。纯化后，该化合物作为五氟苄基酯三甲基硅醚进行分析。该检测方法的精密度为±4%，准确度为97%。灵敏度下限约为20 pg。在10名正常成年人中发现该代谢产物的尿液排泄水平为0.39±0.18 ng/mg肌酐（平均值±2标准差）。在异前列腺素生成增加的情况下，发现该代谢产物的尿液排泄量大幅升高，而抗氧化剂可有效抑制代谢产物的排泄。2,3-二降-5,6-二氢-15-F2t-异前列腺素的水平不受环氧化酶抑制剂的影响。因此，该检测方法为评估15-F2t-异前列腺素的内源性生成提供了一种灵敏且准确的方法，以此来探究该化合物在人类疾病中的病理生理作用。
