An improved LC-MS-MS method for the quantification of prostaglandins E2 and D2 production in biological fluids

摘要

We report an improved LC-MS-MS assay that accurately measures prostaglandins D2 (PGD2) and E2 (PGE2) 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 inter-day and intra-day coefficients of variation were less than 5%. Both d4-PGE2 and d4-PGD2 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 PGD2 accurately, whereas preparation time did not affect PGE2 measurement due to its greater stability in biological samples. As an application of the method, PGD2 and PGE2 were measured in culture supernatants from A549 cells and RAW 264.7 cells. The human lung alveolar cell line A549 was found to produce PGE2 but no PGD2 while the murine macrophage cell line RAW 264.7 produced PGD2 and only trace amounts of PGE2. This direct comparison showed that COX-2 gene expression can lead to differential production of PGD₂ and PGE₂ by epithelial cells and macrophages. Since PGE₂ is anti-asthmatic and PGD₂ is pro-asthmatic, we speculate that the balance of production of these eicosanoids by epithelial cells and macropahges in the lung contributes to the pathogenesis of COPD, bronchiectasis, asthma, and lung cancer.