An improved LC-MS/MS method for the quantification of prostaglandins E2 and D2 production in biological fluids
An improved LC-MS/MS method for the quantification of prostaglandins E2 and D2 production in biological fluids
复制标题
DOI：
10.1016/j.ab.2007.08.041
复制
发表时间：
2008-01-01
影响因子：
2.9
通讯作者：
van Breemen, Richard B.
中科院分区：
文献类型：
--
作者：
Cao, Hongmei;Xiao, Lei;van Breemen, Richard B.
我们报道了一种改进的LC-MS/MS分析方法，可以准确地测定细胞培养上清液和其他生物液中的前列腺素D-2(PGD(2))和前列腺素E-2(PGE(2))。每种前列腺素的检出限均为20pg/ml(0.20pg，0.55fmol柱上)，日内、日间变异系数均小于5%。以d(4)-PGE(2)和d(4)-PGD(2)作为替代标准，控制分析物的差示损失和降解。稳定性研究表明，为了准确地测定PGD(2)，样品制备时间应小于8h，而制备时间不影响PGE(2)的测定，因为它在生物样品中具有较高的稳定性。作为该方法的应用，测定了A549细胞和RAW 264.7细胞培养上清液中的前列腺素D(2)和前列腺素E(2)。人肺泡细胞系A549产生前列腺素E(2)，但不产生前列腺素D(2)，而小鼠巨噬细胞系RAW 264.7产生前列腺素D(2)，仅有微量的前列腺素E(2)。这一直接比较表明，COX-2基因的表达可以导致上皮细胞和巨噬细胞产生不同的PGD(2)和PGE(2)。由于PGE(2)具有平喘作用，而PGD(2)具有促喘作用，因此我们推测肺内上皮细胞和巨噬细胞产生二十烷基类物质的平衡与慢性阻塞性肺疾病(COPD)、支气管扩张、哮喘和肺癌的发病有关。(C)2007 Elsevier Inc.保留所有权利。
We report an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that accurately measures prostaglandins D-2 (PGD(2)) and E-2 (PGE(2)) in cell culture supernatants and other biological fluids. The limit of detection for each prostaglandin was 20 pg/ml (0.20 pg, 0.55 fmol on-column), and the interday and intraday coefficients of variation were less than 5%. Both d(4)-PGE(2) and d(4)-PGD(2) were used as surrogate standards to control for differential loss and degradation of the analytes. Stability studies indicated that sample preparation time should be less than 8 h to measure PGD(2) accurately, whereas preparation time did not affect PGE(2) measurement due to its greater stability in biological samples. As an application of the method, PGD(2) and PGE(2) were measured in culture supernatants from A549 cells and RAW 264.7 cells. The human lung alveolar cell line A549 was found to produce PGE(2) but no PGD(2), whereas the murine macrophage cell line RAW 264.7 produced PGD(2) and only trace amounts of PGE(2). This direct comparison showed that COX-2 gene expression can lead to differential production of PGD(2) and PGE(2) by epithelial cells and macrophages. Because PGE(2) is antiasthmatic and PGD(2) is proasthmatic, we speculate that the balance of production of these eicosanoids by epithelial cells and macrophages in the lung contributes to the pathogenesis of chronic obstructive pulmonary disease (COPD), bronchiectasis, asthma, and lung cancer. (C) 2007 Elsevier Inc. All rights reserved.