An improved LC-MS/MS method for the quantification of prostaglandins E(2) and D(2) production in biological fluids

Abstract

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 8h 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.

Figure 1

Scheme for metabolism of arachadonic acid to form prostaglandins E₂ and D₂.

Figure 2

Negative ion electrospray product ion tandem mass spectra of PGE₂ and PGD₂ (10 ng/mL).

Figure 3

Negative ion electrospray LC-MS-MS chromatograms obtained using reversed phase HPLC and collision-induced dissociation with MRM of PGE₂, PGD₂ and d₄-PGE₂. A) PGE₂ and PGD₂ standards at 10 ng/mL (28.4 nM); B) internal standards d₄-PGE₂ and d₄-PGD₂ at 10 ng/mL (28.1 nM) after extraction; (C) PGE₂ but not PGD₂ was detected in human alveolar lung epithelial A549 cells; and (D) PGD₂ and only a trace amount of PGE₂ were detected in murine RAW 264.7 cells.

Figure 4

PGE₂ production by A549 cells stimulated by IL-1β. Only PGE₂ concentrations in the cell culture medium are shown, since no PGD₂ was detected. The data are the mean ± SD, n = 3. (A) PGE₂ formation over time after stimulation by IL-1β (10 ng/mL); (B) effect of IL-1β concentration on PGE₂ formation after 24 h; (C) Western blotting for COX-1, COX-2, L-PGDS, H-PGDS, cPGES, mPGES-1, and mPGES-2 in whole cell lysates from A549 cells. β-actin is shown as a control to indicate equal protein loading in all lanes. Changes in protein expression over time were detected resulting from stimulation by IL-1β (10 ng/mL).

Figure 5

PGE₂ and PGD₂ production by RAW 264.7 cells stimulated by LPS (mean ± SD, n = 3). (A) Formation of PGE₂ and PGD₂ over time following treatment with LPS (1 μg/mL); (B) change in prostaglandin levels 6 h after treatment with different concentrations of LPS; and (C) Western blotting for COX-1, COX-2, L-PGDS, H-PGDS, cPGES, mPGES-1, and mPGES-2 in whole cell lysates from RAW 264.7 cells. β-actin is shown to indicate equal protein loading in all lanes. Changes in protein expression over time were detected after stimulation by LPS (1 μg/mL).