Bian Qiaoxia, Wang Weihui, Wang Nannan, Peng Yan, Ma Wen, Dai Ronghua
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China.
PLoS One. 2016 Nov 28;11(11):e0166777. doi: 10.1371/journal.pone.0166777. eCollection 2016.
To evaluate the potential relationship between benign prostatic hyperplasia (BPH) and the arachidonic acid (AA) metabolome, a UHPLC-MS/MS method has been developed and validated for simultaneous determination of AA and its cyclooxygenase(COX) and lipoxygenase(LOX) pathway metabolites (15-HETE, 12-HETE, TXA2, 5-HETE, AA, PGI2, PGF2α, 8-HETE, PGD2, PGE2 and LTB4) in rat tissues. The analytes were extracted from tissue samples with a protein precipitation procedure and then separated on a Shim-pack XR-ODSC18 column with 0.05% formic acid in water (pH adjusted with dilute ammonia) and methanol:acetonitrile (20:80, v/v). Detection was performed on a UHPLC-MS/MS system with electrospray negative ionization (ESI) and a multiple reaction-monitoring mode. The lower limits of quantification (LLOQ) were 0.25-50 ng/mL for all of the analytes in the prostate, seminal, bladder, liver and kidney tissues. The absolute recoveries of the analytes from all of the tissues were more than 50%. By means of the method developed, the AA metabolites in tissue samples from Sham and BPH group rats were determined. The eleven biomarkers in the BPH group prostate, seminal, bladder, liver and kidney tissues were significantly higher than those of the sham group, indicating that BPH fortified the inducible expression of COX and LOX, as well as increased the production of AA and eicosanoids. The method described here offers a useful tool for the evaluation of complex regulatory eicosanoids responses in vivo.
为评估良性前列腺增生（BPH）与花生四烯酸（AA）代谢组之间的潜在关系，已开发并验证了一种超高效液相色谱-串联质谱（UHPLC-MS/MS）方法，用于同时测定大鼠组织中AA及其环氧化酶（COX）和脂氧化酶（LOX）途径的代谢产物（15-羟基二十碳四烯酸、12-羟基二十碳四烯酸、血栓素A2、5-羟基二十碳四烯酸、AA、前列环素I2、前列腺素F2α、8-羟基二十碳四烯酸、前列腺素D2、前列腺素E2和白三烯B4）。通过蛋白质沉淀程序从组织样本中提取分析物，然后在Shim-pack XR-ODSC18柱上进行分离，流动相为含0.05%甲酸的水（用稀氨水调节pH）和甲醇：乙腈（20:80，v/v）。在配备电喷雾负离子化（ESI）和多反应监测模式的UHPLC-MS/MS系统上进行检测。前列腺、精囊、膀胱、肝脏和肾脏组织中所有分析物的定量下限（LLOQ）为0.25 - 50 ng/mL。所有组织中分析物的绝对回收率均超过50%。利用所开发的方法，测定了假手术组和BPH组大鼠组织样本中的AA代谢产物。BPH组前列腺、精囊、膀胱、肝脏和肾脏组织中的11种生物标志物显著高于假手术组，表明BPH增强了COX和LOX的诱导表达，以及增加了AA和类花生酸的产生。本文所述方法为评估体内复杂的类花生酸调节反应提供了一个有用的工具。
