Docosatetraenoyl LPA is Elevated in Exhaled Breath Condensate in Idiopathic Pulmonary Fibrosis

Sydney B Montesi; Susan K Mathai; Laura N Brenner; Irina A Gorshkova; Evgeny V Berdyshev; Andrew M Tager; Barry S Shea


BMC Pulm Med. 2014;14(5) 

In This Article


Study Subjects

Subjects with IPF were identified from those being cared for in the Massachusetts General Hospital (MGH) outpatient pulmonary clinic or inpatient pulmonary consult service. For inclusion in this study, subjects had to meet criteria for a diagnosis of IPF based on the recent joint consensus statement of the American Thoracic Society (ATS), European Respiratory Society (ERS), Japanese Respiratory Society (JRS), and Latin American Thoracic Association (ALAT).[5] Controls were recruited through the Partners Healthcare System Research Study Volunteer Program (RSVP). Controls were non-smoking individuals at least 50 years of age without a history of chronic lung disease. Study approval was obtained through the Partners Institutional Review Board, and informed consent was obtained on all subjects. Eleven IPF subjects and eleven controls were included in this study. EBC was obtained on all subjects, and plasma was obtained on all 11 IPF patients and 10 of the controls.

Exhaled Breath Condensate (EBC) Collection

EBC was collected using the handheld RTube™ exhaled breath condensate collector (Respiratory Research, Inc.), according to the manufacturer's instructions, and following the ATS/ERS methodological recommendations for EBC collection.[29] Collection was performed during 10 minutes of tidal breathing, with a nose clip in place, using a cooling chamber pre-cooled to -20°C. EBC samples were placed in aliquots and immediately frozen and stored at -80°C until analysis.

Plasma Collection

Blood was obtained via venipuncture into tubes containing CTAD (citrate-theophylline-adenosine-dipyridamole) additive, in order to potently inhibit platelet activation, as activated platelets are known to release abundant amounts of LPA.[30] Within 30 minutes of collection, whole blood was centrifuged at 1500 × g for 15 minutes to obtain plasma, which was then placed in aliquots and immediately frozen and stored at -80°C until analysis.

Lipid Extraction

EBC samples were subjected to lipid extraction using the modified Bligh and Dyer method as described.[31,32] Briefly, lipid extraction was initiated by adding 2 ml methanol and 1 ml chloroform to 0.5 ml EBC, followed by the addition of 2 pmol C17-LPA (internal standard; Avanti Polar Lipids, Alabaster, AL, USA). Extraction was allowed for 30 minutes with the samples kept on ice. Then, phase separation was achieved by adding 1 ml chloroform and 1.3 ml 0.1 N HCl with vigorous vortexing. The chloroform phase was collected, the solvent was evaporated under a stream of nitrogen gas, and residues were dissolved in methanol and transferred into autosampler vials for LC-MS/MS analysis.

Measurement of LPA Species by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS)

LPA levels were determined using electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS) with an AB Sciex 5500 QTRAP hybrid triple quadrupole/ion trap mass spectrometer coupled with an Agilent 1200 liquid chromatography system. Lipids were separated on Ascentis Express C8 (75 × 2.1 mm, 2.7 um) column using methanol:water:HCOOH, 60:40:0.5, v/v with 5 mM NH4COOH as solvent A and acetonitrile:chloroform:water:HCOOH, 80:20:0.5:0.5, v/v with 5 mM NH4COOH as solvent B. LPA molecular species were analyzed in negative ionization mode with declustering potential and collision energy optimized for each LPA molecular species. Individual saturated and unsaturated LPA molecular species (16:0-, 17:0-, 18:0-, 18:1-, and 20:4-LPA, all obtained from Avanti Polar Lipids, Inc., Alabaster, AL) were used as reference compounds. 17:0-LPA was used as the internal standard, and LPA quantitation was performed by creating standard curves with variable amounts of each available LPA molecular species versus fixed amount of the internal standard (17:0-LPA). Total lipid extract from fetal bovine serum was used as a source of otherwise unavailable LPA molecular species to determine their chromatographic behavior and parameters of ionization and collision-induced decomposition, and the quantitation of these LPA molecular species was achieved via the use of the best possible approximation from the standard curves obtained with available individual LPA standards. The identification of LPA molecular species was achieved via monitoring for selected transitions from molecular to product (m/z 153) ions specific for each LPA molecular species, and by the analyte retention time identified by the available LPA standards and by comparing with LPA extracted from bovine serum.

Statistical Analyses

Statistical analysis was performed using Prism 6.0 (GraphPad Software, Inc.). Differences in LPA levels between IPF patients and controls were analyzed for statistical significance using a two-tailed Student's t-tests or Mann Whitney tests for parametric and nonparametric data, respectively. To adjust for multiple comparisons, we used the Bonferroni method to calculate the accepted α (Type I) error rate for each individual comparison performed, keeping the family-wise error rate at 0.05. Therefore, for EBC LPA levels, in which 9 different LPA species measured were measured, p values ≤ 0.0055 (0.05/9) were considered statistically significant. For plasma LPA levels, in which 13 different LPA species were measured, p values ≤ 0.0038 (0.05/13) were considered statistically significant.