MS method for the quantification of prostaglandins E2 and D2 production in biological fluids

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ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 372 (2008) 41–51 www.elsevier.com/locate/yabio

An improved LC–MS/MS method for the quantification of prostaglandins E2 and D2 production in biological fluids Hongmei Cao a, Lei Xiao b, GyeYoung Park b, Xuerong Wang b, Anser C. Azim b, John W. Christman b, Richard B. van Breemen a,* a

Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, Chicago, IL 60612, USA b Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, University of Illinois, and Jesse Brown Veterans Affairs Medical Center, Chicago, IL 60612, USA Received 16 May 2007 Available online 29 September 2007

Abstract We report an improved liquid chromatography–tandem mass spectrometry (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 interday and intraday coefficients of variation were less than 5%. Both d4-PGE2 and d4PGD2 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, whereas 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. Because PGE2 is antiasthmatic and PGD2 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.  2007 Elsevier Inc. All rights reserved. Keywords: Prostaglandins; PGD2; PGE2; Cell culture; A549 cells; RAW264.7 cells; LC–MS/MS

Prostaglandins are potent biologically active lipid molecules that are produced from arachidonic acid by nearly every cell type [1,2]. Cyclooxygenases (COXs)1 are the rate-limiting enzymes for prostaglandin production.

*

Depending on subsequent isomerases and oxidoreductases distal to COX catalysis, various bioactive prostaglandins can be produced (Fig. 1), and the pattern of prostaglandin production is determined in a stimulus and cell-specific

Corresponding author. Fax: +1 312 996 7107. E-mail address: [email protected] (R.B. van Breemen). 1 Abbreviations used: COX, cyclooxygenase; COPD, chronic obstructive pulmonary disease; PGD2, prostaglandin D2; PGE2, prostaglandin E2; GC–MS, gas chromatography–mass spectrometry; ELISA, enzyme-linked immunosorbent assay; RIA, radioimmunoassay; LC–MS, liquid chromatography–mass spectrometry; LC–MS/MS, liquid chromatography–tandem mass spectrometry; L-PGDS, lipocalin prostaglandin D synthase; H-PGDS, hematopoietic prostaglandin synthase; cPGES, cytosolic prostaglandin E synthase; mPGES, microsomal prostaglandin E synthase; BHT, butylated hydroxytoluene; ATCC, American Type Culture Collection; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal calf serum; LPS, lipopolysaccharide; IL-1b, interleukin-1b; SDS, sodium dodecyl sulfate; PVDF, polyvinylidene fluoride; TBST, Tris-buffered saline containing 0.1% Tween 20; BSA, bovine serum albumin; ECL2, enhanced chemiluminescence solution; HPLC, high-performance liquid chromatography; MRM, multiple reaction monitoring; LOD, limit of detection; LOQ, limit of quantitation; CV, coefficient of variation; TLR4, Toll-like receptor 4; PGJ2, prostaglandin J2. 0003-2697/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.08.041

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LC–MS/MS method for quantification of prostaglandins / H. Cao et al. / Anal. Biochem. 372 (2008) 41–51

Cell membrane phospholipids Phospholipase A2 COOH

Arachidonic acid Cyclooxygenases 1 & 2

O O

CO COOH

Prostaglandin G2

OOH

Cyclooxygenases 1 & 2 O O

COOH

Prostaglandin H2

OH

Thromboxane A2

TXA2 synthase

PGI synthase

Prostacyclin (PGI2)

reductase isomerase HO

Prostaglandin D2 Prostaglandin F2α COOH

O

Prostaglandin E2 COOH

O

OH HO

OH

Fig. 1. Scheme for metabolism of arachadonic acid to form PGE2 and PGD2.

fashion. Alteration of the species of prostaglandins formed in tissue plays an important role in pathophysiological events of many diseases, such as chronic obstructive pulmonary disease (COPD), bronchiectasis, bronchial asthma, chronic arthritis, and atherosclerosis, as well as many types of cancer, including colon cancer and lung cancer [3–6]. The role of prostaglandins in the pathogenesis of a particular disease is complicated by the production of multiple prostaglandins by COX that have diverse biological functions. For example, prostaglandin D2 (PGD2) has strong proinflammatory and bronchoconstrictive action in human and animal models of asthma [7]. However, prostaglandin E2 (PGE2), which is also detected in bronchoalveolar lavage fluid of asthmatic patients, appears to be bronchoprotective and acts as an endogenous antiinflammatory factor [8,9]. Therefore, assessing the exact balance of PGD2 and PGE2 in the microenvironment of the airway is critical for understanding the antagonistic role of prostaglandins in the pathogenesis of airway diseases. The investigation of the role of prostaglandins in multiple disease states depends on sensitive and specific assays to measure the levels of prostaglandins in biological fluids. Gas chromatography–mass spectrometry (GC–MS) pro-

vides suitable sensitivity and selectivity for prostaglandin measurement but requires laborious sample preparation, including derivatization [10,11]. To overcome these limitations, antibody-based assays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) have been developed to provide higher throughput measurement of prostaglandins in biological samples [12,13]. However, antibody-based assays suffer from cross-reactivity with related compounds resulting in reduced selectivity, leading to ambiguous and possible misleading results. In a recent clinical trial, isoprostane was measured in human samples using an ELISA-based assay and was compared with the ‘‘gold standard’’ GC–MS-based assay performed in a reference laboratory as a measure of in vivo oxidant stress. The unacceptably low correlation of the results from the two analytical methods suggests that immunologically based measurements might produce misleading information regarding clinical assessment of oxidative stress in biological samples [14]. In the current article, we report another important consideration that influences comparison of prostanoids because there is sufficient differential degradation of PGD2 and PGE2 that misleading information might be obtained regarding the balance of these antagonistic prostaglandins.

LC–MS/MS method for quantification of prostaglandins / H. Cao et al. / Anal. Biochem. 372 (2008) 41–51

To overcome the limitations of GC–MS and immunoassays for prostaglandin measurements, liquid chromatography–mass spectrometry (LC–MS)-based [15–17] and liquid chromatography–tandem mass spectrometry (LC–MS/ MS)-based [18–27] methods have evolved as powerful tools for measuring prostaglandins in biological samples because of their high sensitivity, high selectivity and simplicity of sample preparation. Although five of these LC–MS or LC–MS/MS methods have measured PGD2 [15–19], none has controlled for the inherent chemical instability of PGD2 that is critical for determining accurate levels and for comparison with the values of other more stable eicosanoids. Our method addresses this important issue by incorporation of both d4-PGD2 and d4-PGE2 internal standards in the reaction mixture to accurately quantify the relative amounts of PGE2 and PGD2 in the same sample. The article by Schmidt and coworkers [18] is the only previous publication that described the detailed LC–MS/MS quantification of both PGD2 and PGE2, but their method used d4-PGD2 as the only internal standard for measuring both analytes. According to our results, this approach cannot result in accurate measurement of both PGD2 and PGE2 because the degradation rates of PGD2 and PGE2 during sample preparation and analysis are significantly different. In addition, because PGE2 and PGD2 are geometric isomers with similar fragmentation patterns during MS/MS, complete chromatographic separation is necessary for their quantitative analysis during LC–MS/MS. However, Schmidt and coworkers separated PGE2 and PGD2 by only 0.25 min, so that at higher concentrations the peaks might overlap and cause inaccurate measurements. Because of these critical issues, we developed a novel LC–MS/MS-based method to measure both PGE2 and PGD2 in biological fluids, including culture supernatant from A549 epithelial cells and RAW macrophages and other biological samples. Our method expands the repertoire of LC–MS/MS to include the sensitive, accurate, and high-throughput measurement of prostaglandins that can determine the exact balance of both PGE2 and PGD2 in biological samples. This is an important consideration in measuring prostanoids in biological fluids in clinical studies because of accumulating information that the relative balance of antagonistic prostanoids is a critical determinant of biological activity. Materials and methods Reagents PGE2, PGD2, d4-PGD2, d4-PGE2, and antibodies against COX-1, COX-2, lipocalin prostaglandin D synthase (L-PGDS), hematopoietic prostaglandin synthase (H-PGDS), cytosolic prostaglandin E synthase (cPGES), microsomal prostaglandin E synthase (mPGES)-1 and -2 for Western blots were purchased from Cayman Chemical (Ann Arbor, MI, USA). Anti-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA,

43

USA), and citric acid and butylated hydroxytoluene (BHT) were obtained from Sigma–Aldrich (St., Louis, MO, USA). Ammonium acetate, acetonitrile, and methanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Deionized water was produced using a MilliQ water purification system (Millipore, Bedford, MA, USA). Cell culture The human lung epithelial cell line A549 (ATCC no. CCL-185) and the mouse macrophage cell line RAW 264.7 (ATCC no. TIB-71) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Han’s F-12 medium was used to culture A549 cells, and Dulbecco’s modified Eagle’s medium (DMEM) was used to culture RAW cells. Each cell culture medium was supplemented with 10% fetal calf serum (FBS), 100 U/ml penicillin, and 100 lg/ml streptomycin. All cells were incubated in 5% CO2 humidified air at 37 C. A549 cells and RAW cells were grown to approximately 80 to 90% confluency in six-well plates. Cells were incubated overnight in serum-free media before changing to fresh serum-free media containing the stimuli. A549 and RAW cells were stimulated to activate COX-2 production and prostaglandin synthesis. Lipopolysaccharide (LPS, Sigma) was used to stimulate RAW cells, whereas human interleukin-1b (IL-1b, Calbiochem, La Jolla, CA, USA) was used to stimulate A549 cells. In time-dependent experiments with A549 cells, 10 ng/ml of IL-1b was used to stimulate the cells, and the cell supernatants were collected at 0, 2, 6, 24, and 48 h. In the dosedependent experiments, A549 cells were stimulated for 24 h with IL-1b at concentrations of 0, 0.1, 1, 10, 30, and 100 ng/ml before collecting the supernatants. In similar experiments, RAW cells were stimulated with 1 lg/ml of LPS and the cell supernatants were collected at 0, 4, 6, and 24 h, or RAW cells were stimulated for 6 h with LPS at 0, 1, 10, and 100 lg/ml before the supernatants were collected for analysis. Western blot assay Cells were cultured as described above and were treated with either LPS (1 lg/ml) or IL-1b (10 ng/ml) for various time points prior to being harvested in cell lysis buffer (Cell Signaling, Danvers, MA, USA). Cell lysates were sonicated and centrifuged at 10,000g for 10 min. Equal amounts (30 lg) of supernatant were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% (w/v) nonfat dried milk and then were incubated with the respective primary and horseradish peroxidase conjugated secondary antibodies in TBST with 4% bovine serum albumin (BSA). The blots were washed three times

44

LC–MS/MS method for quantification of prostaglandins / H. Cao et al. / Anal. Biochem. 372 (2008) 41–51

with TBST after the primary and secondary antibody incubations. Proteins were revealed using enhanced chemiluminescence solution (ECL2, Amersham International, Amersham, UK). Sample preparation Supernatants from cell culture wells were collected and stored at 80 C until analysis. For each analysis, a 500ll aliquot was spiked with 20 ll of d4-PGE2 and d4PGD2 (100 ng/ml each) as internal standards. Next, 40 ll of 1 M citric acid and 5 ll of 10% BHT were added to prevent free radical-catalyzed peroxidation. PGE2 and PGD2 were extracted by adding 2 ml of hexane/ethyl acetate (1:1, v/v) followed by vortex mixing for 1 min. After centrifugation at 4 C, the upper organic phase was removed and saved. The extraction procedure was repeated twice more, and the organic phases were combined, evaporated to dryness under a stream of nitrogen, and reconstituted in 200 ll methanol/10 mM ammonium acetate buffer (1:3, v/v) (pH 8.5) prior to LC–MS/MS analysis. Stability was evaluated by using low (2 ng/ml) and high (20 ng/ml) concentrations of PGE2 and PGD2 spiked into cell culture medium. At room temperature, one set of spiked media samples was stored for 0, 4, 8, or 26 h either in the dark or under normal room illumination. A second set of samples was stored at 20 C for 0, 2, or 4 weeks, and a third set was subjected to three freeze–thaw cycles consisting of freezing for 24 h at 80 C, followed by thawing for 30 min at room temperature, and then refreezing at 80 C. To determine whether the absolute amounts of PGE2 and PGD2 will be affected by using only d4-PGE2 or d4PGD2 as an internal standard, we spiked PGE2, PGD2, d4-PGE2, and d4-PGD2 standards (10 ng/ml) into different solutions. All samples were prepared in duplicate. After sample preparation and LC–MS/MS analysis, the ratios of the analytes to both internal standards were calculated and compared. LC–MS/MS The high-performance liquid chromatography (HPLC) system consisted of Shimadzu (Columbia, MD, USA) LC-10A pumps with a Leap (Carrboro, NC, USA) HTS PAL autosampler. Separation of PGE2 and PGD2 was carried out using a Luna (Phenomenex, Torrance, CA, USA) phenyl-hexyl analytical column (2 · 150 mm, 3 lm) with a 10-min linear gradient from 21 to 50% acetonitrile in 10 mM ammonium acetate buffer (pH 8.5) at a flow rate of 200 ll/min. The injection volume was 10 ll. Negative ion electrospray tandem mass spectrometric analysis was carried out using an Applied Biosystems (Foster City, CA, USA) API 4000 triple quadrupole mass spectrometer at unit resolution with collision-induced dissociation and multiple reaction monitoring (MRM). The source temperature was 350 C, the electrospray voltage was 4200 V,

and the declustering potential was 55 V. Nitrogen was used as the collision gas at 22 eV, and the dwell time was 1000 ms/ion. During MRM, both PGE2 and PGD2 were measured by recording the signal for the transition of the deprotonated molecules of m/z 351 to the most abundant fragment ion of m/z 271. The MRM transition of m/z 355 to 275 was monitored for the internal standards d4-PGE2 and d4-PGD2. Data were acquired and analyzed using Analyst software (version 1.2, Applied Biosystems). Standards for calibration curves and quality control measurements were prepared by spiking 500-ll aliquots of cell culture medium with measured amounts of PGE2 and PGD2. These standards were then processed as described above, including the addition of d4-PGD2, d4PGE2, citric acid, and BHT. The concentrations of PGE2 and PGD2 in these standards ranged from 0.10 to 500 ng/ml. The linearity of the MRM response was determined by plotting the peak area ratio (y) of the analytes to the internal standards versus the nominal concentration (x) of the analytes in blank cell culture medium. The calibration curves were obtained by using weighted (1/x2) least squares regression analysis. Results and discussion Method validation The deprotonated molecules of PGE2 and PGD2 were detected at m/z 351 during negative ion electrospray mass spectrometry. Collision-induced dissociation of the [M-H] ions produced abundant fragment ions of m/z 333, 315, 271, and 233 for both species, corresponding to [M-H-H2O] , [M-H-2H2O] , [M-H-2H2O-CO2] , and [M-H-hexanal-H2O] , respectively [28,29]. The product ion tandem mass spectra of PGE2 and PGD2 are shown in Fig. 2. Because the most abundant product ion of both analytes was m/z 271, the mass transition of m/z 351 to 271 was selected for their quantitative analysis during LC–MS/ MS and the corresponding transition of m/z 355 to 275 was monitored for the deuterated internal standards d4-PGD2 and d4-PGE2. Because PGD2 and PGE2 are geometrical isomers that show similar fragmentation patterns, complete chromatographic separation with excellent peak shape is essential for accurate quantitation. Our chromatographic method, based on the use of a phenyl-hexyl stationary phase, achieved complete and reproducible separation of 1.4 min for these isomers (Fig. 3) compared with only 0.25 min for the method of Schmidt and coworkers [18]. Furthermore, the mobile phase was compatible with MS/MS detection using negative ion electrospray. Mobile phases containing 0.5% acetic acid or 0.1% formic acid were investigated, and although the peak shape could be improved, these additives diminished the separation between PGD2 and PGE2. Isocratic mobile phases were investigated and found to provide good separation of these isomers, but the peak shape of PGD2 was too broad for quantitative

LC–MS/MS method for quantification of prostaglandins / H. Cao et al. / Anal. Biochem. 372 (2008) 41–51

45

HO 271

100

COOH 315 O 333

Relative abundance

50

OH

PGD2

[M-H]-

233

351

0

O 271

100

COOH HO

OH

315

50

PGE2

[M-H]351

233 333 0 200

220

240

260

280

300

320

340

360

380

m/z Fig. 2. Negative ion electrospray product ion tandem mass spectra of PGE2 and PGD2 (10 ng/ml).

100

50 0

MS-MS relative response

10 100

PGE2 PGD2

A 3

5

7

MRM 351

9

271

11

13

PGE2-d4 MRM 355

B

50

27 275

PGD2-d4

0 100

50

3

C

5

7

9

11

13

PGE2 MRM 351

271 27

0 3

5

7

100

50

9 PGD2

11 MRM 351

D

13 271

PGE2 0

3

5

7

9

11

13

Retention time (min) Fig. 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. (D) PGD2 and only a trace amount of PGE2 were detected in murine RAW 264.7 cells.

analysis and the limits of detection (LODs, defined as signal/noise ratios of 3:1) of both analytes were inferior to the gradient methods. Finally, a linear gradient from 10 mM ammonium acetate (pH 8.5) to acetonitrile was found to provide optimum separation, peak shape, and sensitivity. As shown in Fig. 3, PGD2 and PGE2 were separated by 1.4 min, so that there was no overlap between the two peaks. To ensure that no interfering species coeluted with

either peak, the supernatants from six different unstimulated batches of RAW 264.7 and A549 cells were analyzed using LC–MS/MS. No interfering peaks were observed at the PGE2 and PGD2 retention times of 5.9 and 7.3 min, respectively. The calibration curves for PGE2 and PGD2 were linear (r2 > 0.999) over the entire concentration range tested from 0.10 to 500 ng/ml. The LOD was 20 pg/ml (0.2 pg on-column) for both PGE2 and PGD2, which is superior to pre-

46

LC–MS/MS method for quantification of prostaglandins / H. Cao et al. / Anal. Biochem. 372 (2008) 41–51

vious LC–MS assays for PGE2 that reported, for example, LODs of 1 or 1.3 pg injected on-column [26,27] (it should be noted that PGD2 was not measured in these previous studies). The limit of quantitation (LOQ, defined as a signal/noise ratio of 10:1) was 100 pg/ml (1 pg on-column) for both compounds, which is similar to the LOQs of 1.2 and 2.5 pg on-column for PGE2 and PGD2, respectively, reported by Schmidt and coworkers using LC–MS/MS with a different chromatography system [18]. The fact that Schmidt and coworkers reported a higher LOQ for PGD2 might be due to the instability of PGD2, but we found that the LOQ and LOD should be identical for both PGE2 and PGD2 when using identical analytical conditions and fresh PGD2 solutions. The recovery of PGE2 and PGD2 from the cell supernatants was determined at three different concentrations (1, 10, and 100 ng/ml) by comparing the peak areas of spiked and processed cell supernatants with the corresponding standard solutions (matrix free) analyzed without extraction. The extraction efficiencies for PGE2 and PGD2 were identical over the entire range of concentrations. The average recoveries of PGE2 and PGD2 were 92.0 ± 4.9% and 77.0 ± 3.0%, respectively. The lower recovery of PGD2 probably was caused by selective degradation of this prostaglandin (described below). Quality control samples representing low, medium, and high concentrations (0.5, 3, and 10 ng/ml, respectively) were used to evaluate the accuracy and precision for PGE2 and PGD2 measurement on 3 consecutive days. Nine replicates of each quality control sample were analyzed together with a set of calibration standards. Based on these measurements, the intraday and interday precision values were determined as the coefficient of variation (CV) of these analyses, and accuracy was expressed as a percentage of the nominal concentration (Table 1). The intraday precision ranged from 0.57 to 4.52%, and the accuracy was 97.2 to 100.8%. The interday precision was 1.26 to 2.43% with an accuracy of 99.4 to 100.4%. These results indicate that this method showed excellent accuracy and precision. PGD2 is inherently less stable in physiological solutions than is PGE2 [30]. The instability of PGD2 was reported in

1983 by Fitzpatrick and Wynalda [31], who determined that albumin catalyzes the dehydration of PGD2 in vitro to three products. Furthermore, Ito and coworkers [32] described difficulties in production of a high-affinity PGD2-specific antibody that has a high fidelity. However, we are unaware of other published articles that have described inaccuracies in quantification of PGD2 due to its chemical instability. The results of our stability studies of PGE2 and PGD2 are shown in Table 2. In cell culture medium, PGE2 was stable for at least 24 h at room temperature or for 4 weeks at 20 C. In contrast, 10% of PGD2 had degraded after just 8 h at room temperature, and 40% had degraded after 26 h. After 4 weeks in cell culture medium at 20 C, the level of PGD2 had dropped by 70%. Light did not affect the stability of either PGE2 or PGD2 for at least up to 26 h. Because PGD2 is relatively unstable compared with PGE2 in cell culture media, the analysis of these prostaglandins should be carried out promptly, preferably within 8 h. Our data indicate that if quantitative analysis of PGD2 cannot be carried out soon after sample collection, samples are best stored at 80 C but not at 20 C. It should be noted that although Schmidt and coworkers [18] reported that both PGE2 and PGD2 were stable at room temperature in a solution of methanol, water, and formic acid, they did not report measurement of the stability of these prostaglandins in biological fluids or under different experimental conditions that would have indicated the instability of PGD2. Our data in Table 3 show that regardless of whether they were incubated in water/methanol (1:1), 10% FBS, or FBS-free medium, PGD2 and d4PGD2 were much less stable than PGE2 and d4-PGE2. Regardless of the composition of the physiological or solvent solution, the stability results were similar for all of the prostanoids. Although the ratios of PGD2 to d4PGD2 and of PGE2 to d4-PGE2 remained essentially

Table 2 Stability of PGE2 and PGD2 under different storage and handling conditions Stability test

Table 1 Interday and intraday reproducibility of the LC–MS/MS quantitative analysis of PGE2 and PGD2 in the culture medium of human lung A549 epithelial cells Analyte Concentration (ng/ml) Intraday (n = 3) Nominal

Measured (means, n = 9)

Interday (n = 9)

Freeze–thaw

0.50 3.00 10.00

0.50 3.01 9.94

3.19 0.86 0.57

99.5 99.9 100.8

1.68 1.26 1.35

100.2 100.4 99.4

PGD2

0.50 3.00 10.00

0.49 2.96 10.02

3.72 2.35 4.52

97.2 100.2 100.3

2.08 2.43 2.04

98.6 98.6 100.2

PGE2 (% remaining)

PGD2 (% remaining)

2 ng/ ml

2 ng/ ml

20 ng/ ml

20 ng/ ml

106

99

105

107

Room temperature, dark

0h 4h 8h 26 h

100 98 98 90

100 96 100 97

100 100 95 60

100 91 92 59

Room temperature, light

0h 4h 8h 26 h

100 104 110 101

100 106 107 107

100 96 90 52

100 98 87 53

Storage ( 20 C)

0 weeks 2 weeks 4 weeks

100 93 99

100 104 109

100 58 33

100 64 28

% CV Accuracy % % CV Accuracy %

PGE2

Time point

LC–MS/MS method for quantification of prostaglandins / H. Cao et al. / Anal. Biochem. 372 (2008) 41–51 Table 3 Evaluation of the relative stabilities of PGE2, PGD2, d4-PGE2, and d4PGD2 at 23 C in different solvents and cell culture media Sample description

Ratio PGE2/d4PGE2

PGE2/d4PGD2

PGD2/d4PGD2

PGD2/d4PGE2

1.001

1.00

1.00

1.00

0.98

1.09

1.01

0.91

1.01

1.14

1.02

0.91

1.08

1.47

1.12

0.81

Medium containing 10% FBS, 0 h Medium containing 10% FBS, 4 h Medium containing 10% FBS, 8 h Medium containing 10% FBS, 24 h

1.00

1.00

1.00

1.00

1.05

0.97

0.88

0.95

1.01

1.71

1.12

0.67

1.15

5.07

1.30

0.31

FBS-free medium, 0 h FBS-free medium, 4 h FBS-free medium, 8 h FBS-free medium, 24 h

1.00 0.78 1.10 1.19

1.00 3.62 2.51 6.62

1.00 1.42 1.30 1.41

1.00 0.31 0.57 0.24

1:1 Methanol/Water, 0h 1:1 Methanol/Water, 4h 1:1 Methanol/Water, 8h 1:1 Methanol/Water, 24 h

Note. All samples were prepared and analyzed in duplicate using LC–MS/ MS, and the mean concentration values were used to calculate the ratios. The initial concentration of each prostanoid was 10 ng/ml, and each ratio was normalized to 1.00 for the 0-h time point.

constant (Table 3), the ratio of PGE2 to d4-PGD2 increased while the ratio of PGD2 to d4-PGE2 decreased due to the instability of PGD2. Therefore, the quantification of PGD2 would be most accurate when d4-PGD2 and d4PGE2 are used as surrogate standards for PGD2 and PGE2, respectively. Because sample preparation and analysis can require up to 2 h, substantial inaccuracy in the measurement of PGE2 or PGD2 could occur if surrogate standards are not used for both compounds and the accuracy of the determination of the relative and absolute amounts of PGE2 and PGD2 would be insufficient for clinical studies. Therefore, we strongly recommend that this issue be considered when measuring PGD2 in biological samples where accurate levels or comparison with PGE2 influences the interpretation of the data. Studies that have not considered this issue should be interpreted with caution because the results may be misleading. PGE2 and PGD2 production by epithelial cells and macrophages To illustrate the potential importance of employing our LC–MS/MS assay, we measured PGE2 and PGD2 produced by the human lung alveolar epithelial cell line A549 and the murine macrophage cell line RAW in response to COX-2 induction. The time-dependent and dose-dependent responses of the A549 cells were measured following treatment with IL-1b because these cells are not

47

responsive to LPS. The lack of response to LPS by A549 cells probably is due to the absence of the Toll-like receptor 4 (TLR4) in this cell line. The results or our experiment are summarized in Fig. 4. The A549 cells produced significant quantities of PGE2, but PGD2 could not be detected in response to treatment with IL-1b. Maximum PGE2 production occurred 24 h after treatment (Fig. 4A), and 1 ng/ml of IL-1b produced the same level of stimulation as did 10, 30, and 100 ng/ml (Fig. 4B). These data indicate that pulmonary epithelial cells manufacture PGE2, which has antiinflammatory biological effects, but not PGD2, which is proasthmatic. These results indicate that formation of PGE2, an antiinflammatory bronchodilator, is the prevailing product of COX-2 in lung epithelial cells [33,34]. Similar results were observed using primary mouse tracheal epithelial cells (data not shown), indicating that the formation of PGE2 in lung epithelium might be biologically relevant to the pathogenesis of asthma. In contrast, RAW macrophages express COX-2 and synthesize PGD2 in response to multiple biological stimuli that include LPS and growth factors. Unlike epithelial cells, which produced mostly PGE2 in response to treatment with IL-1b, RAW cells produce mainly PGD2 in response to treatment with LPS via the COX-2 enzymatic pathway [35]. We examined the response of COX-2 enzymatic activity after LPS stimulation in RAW macrophages by measuring PGE2 and PGD2 in the cell culture supernatant. As shown in Fig. 5, RAW 264.7 cells produced both PGD2 and PGE2, but PGD2 is the major product in response to treatment with LPS. Maximum production of PGD2 was observed 6 h after treatment with LPS (Fig. 5A), and the most effective dosage of LPS was 0.1 lg/ml (Fig. 5B). Only trace amounts of PGD2 and PGE2 were detected in the cell culture medium of unstimulated macrophages. These results indicate that PGD2, a proinflammatory bronchoconstrictor, is the major prostaglandin product of RAW macrophages [33,36]. Similar results have been observed using primary mouse bone marrow-derived macrophages (data not shown), and these findings are similar to those of Rouzer and coworkers [37]. For comparison with our LC–MS/MS measurements, COX-1, COX-2, L-PGDS, H-PGDS, cPGES, mPGES-1, and mPGES-2 protein levels were measured by Western blotting in whole cell lysates from A549 cells or RAW cells in response to IL-1b (Fig. 4C) or LPS (Fig. 5C) treatment, respectively. COX-2 is inducible in both cell lines. In A549 cells, COX-2 protein was detectable after 2 h of IL-1b stimulation and reached a maximum level after 6 h. COX-2 gene expression declined after 6 h and returned to baseline by 48 h. Our results are consistent with those of Newton and coworkers [34], who showed that COX-2 gene expression in A549 cells increased after IL-1b treatment. In RAW cells stimulated by LPS, COX-2 protein expression reached its peak after 24 h, whereas PGD2 production reached a maximum concentration at 6 h and declined thereafter. The difference in these time courses might be the result of the chemical instability of PGD2, which can spontaneously

48

LC–MS/MS method for quantification of prostaglandins / H. Cao et al. / Anal. Biochem. 372 (2008) 41–51 40

PGE2 (ng/ml) (n

A 30 20 10 0

0

2

6

24

48

Stimulation time (h)

40

PGE2 (ng/ml) (ng/ml

B 30 20 10 0

Control

0.1

1

10

30

100

[IL-1β] (ng/ml)

C COX-1

← 70 kDa

COX-2

← 72 kDa

L-PGDS

← 25 kDa

H-PGDS

← 25 kDa

cPGES

←23 kDa

mPGES-1

←16 kDa

mPGES-2

← 33 kDa

IL-1β (h)

0

2

Actin

6

24

48 ← 43 kDa

Fig. 4. PGE2 production by A549 cells stimulated by IL-1b. Only PGE2 concentrations in the cell culture medium are shown because no PGD2 was detected. The data are the means ± SD (n = 3). (A) PGE2 formation over time after stimulation by IL-1b (10 ng/ml). (B) Effect of IL-1b 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. b-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-1b (10 ng/ml).

dehydrate to form prostaglandin J2 (PGJ2) [31,38] and then D12-PGJ2 through albumin catalysis [31,39]. PGD2 levels reflect the sum of the rates of de novo synthesis and decomposition. In contrast, PGE2 accumulated in the cell culture medium because PGE2 is chemically stable under physiological culture conditions [37]. Our Western blot data (Fig. 4C) show that A549 cells produce abundant constitutive cPGES and mPGES-2, a small amount of inducible mPGES-1, but very little immunoreactive H or L-PGDS.

Interestingly, COX-2 was induced in the A549 cells, but we were unable to detect immunoreactive COX-1. These data are consistent with our findings that A549 cells produce abundant amounts of PGE2 but essentially no PGD2. We interpret these data as indicating that COX-2 is coupled with the PGES pathway in IL-1b-stimulated A549 cells. In contrast, RAW cells constitutively express both L-PGDS and H-PGDS and all three isoforms of PGES: cPGES, mPGES-1, and mPGES-2 (Fig. 5C). In

PGE2 and PGD2 (ng/ml)

LC–MS/MS method for quantification of prostaglandins / H. Cao et al. / Anal. Biochem. 372 (2008) 41–51

400

49

A PGE2 PGD2

300 200 100 0 0

4

6

4

PGE2 and PGD2 (ng/ml)

Stimulation time (h) 500

B

400 300 200 100 0 0

0.10

1.00

10.0

[LPS] (μg/ml)

C COX-1

← 70 kDa kD

COX-2

← 72 kDa kD

L-PGDS

← 25 kDa

H-PGDS

← 25 kDa

cPGES

← 23 kDa

mPGES-1

← 16 kDa

mPGES-2

← 33 kDa

LPS (h)

0

4

Actin

6

24 ← 43 kDa

Fig. 5. PGE2 and PGD2 production by RAW 264.7 cells stimulated by LPS (means ± SD, n = 3). (A) Formation of PGE2 and PGD2 over time following treatment with LPS (1 lg/ml). (B) Change in prostaglandin levels 6 h after treatment with different concentrations of LPS. (C) Western blotting for COX1, COX-2, L-PGDS, H-PGDS, cPGES, mPGES-1, and mPGES-2 in whole cell lysates from RAW 264.7 cells. b-Actin is shown to indicate equal protein loading in all lanes. Changes in protein expression over time were detected after stimulation by LPS (1 lg/ml).

RAW cells, we detected constitutive levels of immunoreactive COX-1 and inducible levels of COX-2. In contrast to A549 cells, in RAW cells the predominant product is PGD2 rather than PGE2, and this seems to indicate a functional coupling of COX-1 and COX-2 with PGDS. In conclusion, an improved and accurate LC–MS/MS assay has been developed to measure PGD2 and PGE2 in

cellular supernatants and other biological fluids. Compared with GC–MS, HPLC–UV, ELISA, and previous LC–MS/ MS methods, our method shows a superior balance of selectivity, speed, and accuracy. Complete chromatographic separation of PGD2 and PGE2 were obtained with excellent linearity of response, accuracy, reproducibility, LODs, and LOQs comparable or superior to previous

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LC–MS/MS method for quantification of prostaglandins / H. Cao et al. / Anal. Biochem. 372 (2008) 41–51

methods. To the best of our knowledge, previous studies have not evaluated the relative stabilities of PGE2 and PGD2 in biological samples. In contrast to the stability of PGE2, PGD2 was found to substantially degrade in cell culture media after 8 h at room temperature or after 2 weeks at 20 C. As a result, sample handling for PGD2 measurement should not exceed 8 h, and storage of biological samples should be of minimal duration and at least at 80 C. Our data indicate that d4-PGE2 and d4-PGD2 should be used together as surrogate standards for the accurate quantitative analyses of PGE2 and PGD2, respectively, because of their significant rates of degradation. Finally, A549 cells were found to produce PGE2 but not PGD2, and RAW cells produced PGD2 but very little PGE2. This direct comparison of the formation of PGE2 and PGD2 by these types of cells shows that COX-2 gene expression in epithelial cells and macrophages results in different products that can contribute to pathogenesis of COPD, bronchiectasis, asthma, and other lung-related diseases, including bronchogenic lung cancer. Acknowledgments This research was supported by the Department of Veterans Affairs and grants HL-075557 and HL-66196 from the National Heart, Lung, and Blood Institute (to J.W.C.), American Heart Association National Scientist Development grant 0230279N and University of Illinois at Chicago Campus Research Board grant S06-118 (to L.X.), and National Institutes of Health grant P50 AT00155 jointly funded by the Office of Dietary Supplements, the National Center for Complementary and Alternative Medicine, and the Office for Research on Women’s Health (to R.B.v.B.). We thank Dejan Nikolic, Ruxana Sadikot, Magdalena Ornatowska, Biji Mathew, and Jeff Dahl for helpful discussions and assistance. References [1] A.N. Hata, R.M. Breyer, Pharmacology and signaling of prostaglandin receptors: Multiple roles in inflammation and immune modulation, Pharmacol. Ther. 103 (2004) 147–166. [2] C.D. Funk, Prostaglandins and leukotrienes: Advances in eicosanoid biology, Science 294 (2001) 1871–1875. [3] R.S. Peebles Jr., K. Hashimoto, J.D. Morrow, R. Dworski, R.D. Collins, Y. Hashimoto, J.W. Christman, K.H. Kang, K. Jarzecka, J. Furlong, D.B. Mitchell, M. Talati, B.S. Graham, J.R. Sheller, Selective cyclooxygenase-1 and -2 inhibitors each increase allergic inflammation and airway hyperresponsiveness in mice, Am. J. Respir. Crit. Care Med. 165 (2002) 1154–1160. [4] H. Wolff, K. Saukkonen, S. Anttila, A. Karjalainen, H. Vainio, A. Ristime`aki, Expression of cyclooxygenase-2 in human lung carcinoma, Cancer Res. 58 (1998) 4997–5001. [5] M.A. Ia¨niguez, J.L. Pablos, P.E. Carreira, F. Cabraˆe, J.J. GomezReino, Detection of COX-1 and COX-2 isoforms in synovial fluid cells from inflammatory joint diseases, Br. J. Rheumatol. 37 (1998) 773–778. [6] M.E. Burleigh, V.R. Babaev, J.A. Oates, R.C. Harris, S. Gautam, D. Riendeau, L.J. Marnett, J.D. Morrow, S. Fazio, M.F. Linton, Cyclooxygenase-2 promotes early atherosclerotic

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