Samples
A total of 19 patients were recruited from Fujian Medical University Second Affiliated Hospital between April 28, 2018 and November 20, 2018, including 10 GPB patients and 9 COPD patients. GPB was diagnosed by a respiratory physician and by an imaging physician, and COPD was diagnosed by two respiratory physicians after undertaking a pulmonary function test. Each participant provided a one-time 10 mL of venous blood sample for further experiments. After a written informed consent was obtained, all subjects were interviewed to collect their demographic information.
The study was conducted according to the principles of Helsinki declaration. The bioethical committees at Fujian Medical University Second Affiliated Hospital, China, gave written approval for the study.
Materials and lipid standards
The Internal standards kit consisting of labelled internal standards for 13 lipid classes were purchased from Sciex (Framingham, MA); MTBE and ammonium acetate were from Sigma-Aldrich Chemie GmbH (Munich, Germany). LC–MS grade acetonitrile, 2-propanol, methanol and water were purchased from Merck (Darmstadt, Germany).
Sample preparation
All patients underwent collection of 10 ml of peripheral blood in tubes with ethylenediaminetetraacetic acid (EDTA) and the blood was centrifuged at 2000 × g (4 °C) to obtain plasma. After centrifugation, the plasma was stored at -80℃ for further lipids analysis.
Lipid extraction recipes
Lipid extraction was conducted with methyl tert-butyl ether (MTBE). Liquid configuration:A phase (95% acetonitrile water): water/ acetonitrile (5:95, v/v) with 10 mM ammonium acetate; B phase (50% acetonitrile water): water/ acetonitrile (50:50, v/v) with 10 mM ammonium acetate. Pretreatment of lipid sample: Methanol (225ul) was added to a 20μl plasma sample aliquot, which was placed into a glass tube, and the tube was vortexed at maximum speed for 10s. Then, 750 μl of MTBE was added and the mixture was vortexed at maximum speed for 10s,then incubated for 30min at room temperature. Next, another 188μl of water was added and vortexed for 20s. Upon 10 min of incubation at room temperature, the sample was centrifuged at 15000rpm for 15 min at 4℃. The samples were divided into three layers from the top to the bottom , including lipid phase,water phase and solid residue. The upper (lipid) phase (700μl) was collected. Combined lipid phases were blow-dried with termovap sample concentrator. Extracted lipids were dissolved with 100ul mixture which consists of 2-propanol: acetonitrile: water (30:65:5, v/v/v).
Mass spectrometric analysis of lipids from human plasma extracts
In the present study, HPLC-QqQ-MS was used for lipids class separation (AB SCIEX QTRAP 4500 LC-MS/MS system). The Waters Acquity UPLC BEH HILIC Pore column(100 mm×2.1 mm,1.7μm) was used as stationary phase. Mobile phase A is 95% acetonitrile solution containing 10mmol/L ammonium acetate, and mobile phase B is 50% acetonitrile solution containing 10mmol/L ammonium acetate. The solvent gradient was programmed as follows: the gradient started with 0.1% of B and increased to 20% of B during 10 min, then linearly increased to 98% of B during 10min to 11 min, and 98% of B was held for 2 mins, and returning to the initial conditions 0.1% of B in 13.1 min, finally stop the analyze in 16 min, the flow rate through the column was 0.5mL/min. The column temperature was 35 ℃, the positive electrospray ionization (ESI+) mode injection volume was 1 μL, and the negative electrospray ionization (ESI–) mode injection volume was 10 μL. The curtain gas (CUR)was set to 35 psi, the ion source gas1(GS1) was 50 psi, the ion source gas2(GS2) was 60psi, and the temperature (TEM) set at 500°C. The ion spray voltage in ESI+ and ESI– mode was set at -5500V and 5500V respectively, the declustering potential (DP) was 80V, the entrance potential (EP) was 10V, and collision cell exit potential (CXP) was 15V. Samples were added to a lipid standard mixture.
Statistical Analysis
Relative ion abundances from the two examined groups of plasma extracts (COPD and GPB) were obtained by HPLC-QqQ-MS. Peak areas were then corrected by the area of the selected internal standard by exporting integrated peak area values. After correction, the processed data set was subjected to multivariate analysis by SIMCA 14.1.
Principal components analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) were applied to the data set. The PCA, an unsupervised method, was used to detect intrinsic clusters based on the lipid profile, and to maximize the identification of differences in the metabolic profiles between the groups, the OPLS-DA model was applied and performed using SIMCA 14.1 software. The variable importance in projection (VIP) value of each variable in the model was calculated to indicate its contribution to the classification. Based on obtained OPLS-DA models the VIP values of those variables greater than 1.0 are considered to be significantly different,and combine with the P value of t-test (P<0.05) allows insight into which lipid species are different between COPD and GPB.