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This cross-sectional study included 51 patients with asthma who were treated at our hospital between January 2015 and December 2015. All of these patients met the diagnostic criteria for asthma, as specified in the Guidelines for Prevention and Management of Bronchial Asthma[1]. Patients with complicated chronic obstructive pulmonary disease, bronchiectasis, pneumonia, obstructive sleep apnea-hypopnea syndrome, malignant disease, acute or chronic respiratory failure, or severe cardiovascular disease were excluded. The study was approved by the Ethics Committee of the Peking University Third Hospital (approval number 2014071). All participating patients provided written informed consent.
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Demographic data, including sex and age, body mass index, inhaled steroid doses, and Asthma Control Test (ACT) scores were recorded. All subjects underwent standard pulmonary function tests using spirometry (Elite series, MGC Diagnostics, St Paul, MN, USA). The percent predicted forced expiratory volume in 1 second (FEV1%pred) and FEV1/forced vital capacity (FVC) were recorded. The eosinophil count was measured as part of routine peripheral blood testing, and 2 mL of peripheral blood was collected to detect the serum phospholipid profile. Induced sputum was also obtained for cell and differential counts.
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Preparation of Serum Two milliliters of peripheral venous blood was collected without anticoagulant. The serum was separated (3, 000 r/min, centrifugal radius 13 cm, 5 min) and stored in a refrigerator at −80 ℃. The patients were fasted for more than 12 h before collection of samples and completed a food frequency questionnaire for analysis of the influence of diet on the test results.
Lipid Extraction First, 100 μL of plasma was taken (10 μL of the storage solution of the lipidomic internal standard mixture were added), mixed with 400 μL of frozen methanol (75%), and sonicated for 2 min. Next, 1 mL of methyl t-butyl ether (MTBE) was added. The sample was then vortexed for 1 h at room temperature, after which 250 μL of H2O was added, and the sample was allowed to stand for 10 min to separate. The sample was then centrifuged at 12, 000 ×g for 10 min at 4 ℃. Next, the supernatant was removed and blown dry in a new tube. Finally, the supernatant was dissolved in 100% methanol, filtered, and placed into sample bottles. The storage solution of the lipidomic internal standard mixture consisted of 20 μg/mL each of phosphatidylcholine (PC, 17:0/17:0), phosphatidylethanolamine (PE, 17:0/17:0), phosphatidylglycerol (PG, 17:0/17:0), phosphatidylinositol (PI, 17:0/17:0), phosphatidic acid (17:0/17:0), and phosphatidylserine (17:0/17:0).
Determination of the Serum Phospholipid Profile by Liquid Chromatography-mass Spectrometry An Acquity UPLC liquid chromatograph and an UPLC BEH C18 column (1.7 μm, 100 × 2.1 mm id; Waters, Milford, MA, USA) were used, with a column temperature of 25 ℃ and a flow rate of 0.25 mL/min. Solution A contained 60% acetonitrile (5 mmol/L ammonium acetate) and solution B contained isopropanol:acetonitrile (9:1); gradient: 0-3 min 15% B; 3-15 min 15%-99% B; 15-17 min 99% B; 17-19 min 99%-15% B; and 19-20 min 15% B. A 5500 QTRAP mass spectrometer (AB Sciex, Farmingham, MA, USA) was used, and the ion source was Turbo V Ion Spray electrospray ionization. The scan mode was multiple reaction monitoring and the ion source parameters were as follows: CUR, 40 psi; GS1, 30 psi; GS2, 30 psi; IS, −4500 V; CAD, medium; and temperature, 350 ℃. The scanning strategy used for multiple reactions monitoring of the glycerophospholipids is shown in Table 1.
LC-MS/ MS Run Lipid Class Internal Standards Parent Ion Daughter Ion CE 1 Phosphatidylcholine (PC) PC 17:0/17:0 [M+H]+ 184.1 45 1 Alkyl-phosphatidylcholine [PC(O)] PC 17:0/17:0 [M+H]+ 184.1 45 1 Phosphatidylcholineplasmalogen (PCP) PC 17:0/17:0 [M+H]+ 184.1 45 1 Phosphatidylethanolamine (PE) PE 17:0/17:0 [M+H]+ NL141 31 1 Alkylphosphatidylethanolamine [PE(O)] PE 17:0/17:0 [M+H]+ NL141 31 1 Phosphatidylethanolamineplasmalogen (PEP) PE 17:0/17:0 [M+H]+ NL141 31 1 Phosphatidylserine (PS) PS 17:0/17:0 [M+H]+ NL185 29 1 Phosphatidylglycerol (PG) PG 17:0/17:0 [M+NH4] NL189 25 1 Phosphatidylinositol (PI) PI 17:0/17:0 [M+NH4] NL277 43 1 Lysophosphatidylcholine (LPC) PC 17:0/17:0 [M+H]+ 184.1 31 2 Lysophosphatidylethanolamine (LPE) PE 17:0/17:0 [M-H]- 196.0 -29 2 Lysophosphatidylserine (LPS) PS 17:0/17:0 [M-H]- NL87 -22 2 Lysophosphatidylglycerol (LPG) PG 17:0/17:0 [M-H]- NL228 -26 2 Lysophosphatidylinositol (LPI) PI 17:0/17:0 [M-H]- NL316 -40 Note.LC-MS/MS, liquid chromatography-tandem mass spectrometry. Table 1. Glycerophospholipid Scanning Strategy Using Liquid Chromatography-mass Spectrometry
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The steps used for sputum induction cytology[10] were as follows. Before nebulization, the patients were instructed to gargle with water, blow their nose, inhale nebulized sodium chloride 3%, and expectorate into a sterile sputum container after 20-30 min. An equivalent volume of dithiothreitol 0.4% was added, and the sample was incubated at 37 ℃ for 30 min. The sample was centrifuged for 5 min at 2, 000 r/min (centrifugal radius 13 cm). The cells were sorted after Wright-Giemsa staining.
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Asthma was divided into the following groups[3]: eosinophilic type (≥ 3%) and non-eosinophilic type (< 3%) according to the ratio of eosinophils to leukocytes in induced sputum; eosinophilic type (≥ 3%) and non-eosinophilic type (< 3%) according to the ratio of eosinophils to leukocytes in blood; eosinophilic type (≥ 300 cells/μL) and non- eosinophilic type (< 300 cells/μL) according to the absolute value of eosinophils in blood; and neutrophilic type (≥ 71%) and non-neutrophilic type (< 71%) according to the ratio of neutrophils to leukocytes in induced sputum.
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The metabolomics analysis tools[11] on the http://www.metaboanalyst.ca/ website were used to compare the metabolic profiles of glycerophospholipids between the different inflammatory subtypes of asthma. Partial least squares discriminant analysis (PLS-DA) was performed on the patients' samples. The different metabolites were screened by methods including variable importance in projection and loading weights. All statistical analyses were performed using the SPSS 19.0 software package (IBM Corp. Armonk, NY, USA). The asthma type diagnostic accuracy was evaluated using the subject working curve method. A P-value ≤ 0.05 was considered statistically significant.
Patients
Collection of Demographic and Clinical Data
Serum Phospholipid Tests
Sputum Induction Cytology
Classification of Inflammatory Subtypes of Asthma
Statistical Analysis
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Fifty-one patients with asthma and an average age of 43.3 ± 14.0 years were included. The FEV1%pred was 92.3% ± 16.0%, and the FEV1/FVC was 73.3% ± 10.8% (Table 2). One hundred and twenty-nine phospholipids in 14 categories were detected as follows: 21 types of phosphatidylcholine (PC), 15 types of alkylphosphatidylcholine [PC(O)], 8 types of phosphatidylcholine plasmalogen (PCP), 12 types of phosphatidylethanolamine (PE), 4 types of alkylphosphatidylethanolamine [PE(O)], 2 types of phosphatidylethanolamine plasmalogen (PEP), two types of phosphatidylglycerol (PG), 11 types of phosphatidylinositol (PI), 11 types of lysophosphatidylcholine (LPC), 12 types of lysophosphatidylethanolamine (LPE), 5 types of hemolytic phosphatidylserine (LPS), 11 types of lysophosphatidylglycerol (LPG), 9 types of lysophosphatidylinositol (LPI), and 6 types of lysoalkylphosphatidylcholine [LPC(O)].
Variables Results Sex (male/female), n 17/34 Age, years 42.0 ± 13.5 Body mass index (kg/m2) 23.8 ± 3.0 ACT score 18.3 ± 5.7 FEV1%pred (%) 92.3 ± 16.0 FEV1/FVC (%) 73.3 ± 10.8 Note.ACT, Asthma Control Test; FEVl, forced expiratory volume in 1 second; FEV1%pred, FEVl expressed as a% of the predicted value; FVC, forced vital capacity. Continuous variables are shown as the mean ± standard deviation. Table 2. Patient Demographics and Disease Severity
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Thirty-nine of the 51 patients enrolled underwent a sputum induction test. According to the sputum cell count, eosinophils accounted for ≥ 3% of leukocytes in the sputum of 23 subjects and for < 3% in 16 subjects. The ACT scores were lower in the eosinophilic group than in the non-eosinophilic group; the difference was not statistically significant (P = 0.062; Table 3).
Variables Inflammatory Subtypes P-value EOS in sputum ≥ 3% (n = 23) EOS in sputum < 3% (n = 16) Sex (male/female), n 6/17 7/9 0.250 Age, years 42.2 ± 15.0 41.6 ± 12.7 0.888 ACT score 16.7 ± 6.3 20.3 ± 5.0 0.062 ICS (regular/irregular), n 14/7 10/6 0.793 Blood EOS (%) > 3 15 6 < 3 6 8 Blood EOS (/μL) > 300 9 2 > 300 12 11 EOS in blood ≥ 3% (n = 29) EOS in blood < 3% (n = 17) Sex (male/female), n 11/18 5/12 0.672 Age, years 42.7 ± 14.3 42.8 ± 12.0 0.986 ACT score 17.3 ± 6.1 18.8 ± 5.3 0.424 Inhaled ICS (regular/irregular), n 19/8 10/5 0.895 EOS in blood ≥ 300 cells/μL (n = 17) EOS in blood < 300 cells/μL (n = 29) Sex (male/female), n 4/13 12/17 0.372 Age, years 44.9 ± 13.2 41.5 ± 13.5 0.405 ACT score 15.5 ± 5.3 19.2 ± 5.6 0.033 Inhaled ICS (regular/irregular), n 10/6 19/7 0.712 Sputum neutrophils ≥ 71% (n = 19) Sputum neutrophils < 71% (n = 20) Sex (male/female), n 7/12 6/14 0.651 Age, years 43.5 ± 11.7 40.5 ± 16.0 0.514 ACT score 18.1 ± 6.0 18.4 ± 6.2 0.901 Inhaled ICS (regular/irregular), n 12/7 12/6 0.823 Note.Twelve patients had no sputum cell differential count results. Five patients did not have blood routine tests. The data are shown as the mean ± standard deviation except for patient sex. EOS, eosinophils; ACT, Asthma Control Test; ICS, inhaled corticosteroids. Table 3. Patient Demographics and Disease Severity between Different Inflammatory Subtypes
Multivariate statistical analysis of the serum glycerophospholipid results in the sputum eosinophilic group (≥ 3%) and non-eosinophilic group (< 3%) was performed. A heat map (Figure 1A) showed the metabolic profiles of the glycerophospholipid in each sample; hierarchical cluster analysis showed that there was no obvious similarity in the metabolic profiles in each group, and there was no significant difference between the two subgroups. The PLS-DA score plot (Figure 1B) showed that the two groups could not be separated perfectly by the PLS model, indicating that the difference between the two groups was not reflected in the whole glycerophospholipid metabolism proflie. Therefore, we performed a univariate analysis. A volcano plot (Figure 1C) showed that there were three glycerophospholipid species with significant changes: PC(O) 32:0, LPG 22:6, and LPG 20:5, indicating a significant difference between the two subgroups. A receiver-operating curve (ROC) curve analysis (Figure 1D) was performed with the three major significantly different glycerophospholipids as the test variables and sputum eosinophils ≥ 3% as the state variable. The areas under the curve (AUCs) were 34.8% [95% CI (17.4%, 52.1%)], 72.0% [95% CI (56.0%, 88.1%)], and 74.7% [95% CI (57.4%, 92.0%)], respectively. The AUCs for LPG 22:6 and LPG 20:5 were ≥ 70%; therefore, they can be considered as biomarkers for a diagnosis of eosinophilic asthma. The ROC curve was shown in Figure 1D (Figure 1E for the histogram). The results of the comparison are shown in Table 4.
Figure 1. (A) Heat map. (B) Partial least squares discriminant analysis score chart. (C) Volcano map. (D) Receiver-operating characteristic curve for LPG 22:6 and LPG 20:5. (E) Histogram. Group 1 and 2 represent asthmatic patients with sputum eosinophils (EOS) ≥ 3% and < 3% respectively. The heat map in (A) compares the glycerophospholipid profiles of patients in the asthma subgroups with sputum EOS ≥ 3% and < 3%. Each square represents a glycerophospholipid, and the color indicates the content of glycerophospholipid in the serum. The higher the serum content, the darker the color (red is upregulated, and blue is downregulated). The PLS-DA score plot in (B) shows an overall difference in the metabolic glycerophospholipid profiles of patients in the asthma subgroups with sputum EOS ≥ 3% and < 3%. The red dots represent the group with sputum sputum EOS ≥ 3%, and the green dots represent the group with sputum EOS < 3%. The volcano plot in (C) shows the glycerophospholipids with significant differences between the subgroups with sputum EOS ≥ 3% and < 3%. The glycerophospholipids with significant changes in this figure are PC-O 32:0, LPG 22:6, PC 38:5, and LPG 20:5. The ROC curve for LPG 22:6 and LPG 20:5 in (D) and has an AUC ≥ 70%. (E) Is a histogram showing LPG 22:6 and LPG 20:5 in the asthma groups with sputum EOS ≥ 3% and < 3% (*P < 0.05).
Glycerophospholipid FC Log2 (FC) P-value EOS in sputum ≥ 3% (n = 23) EOS in sputum < 3% (n = 16) PC (O 32:0) 0.593 -0.754 0.009 LPG 22:6 3.572 1.836 0.022 PC 38:5 0.845 -0.243 0.029 LPG 20:5 1.387 0.471 0.039 EOS in blood ≥ 3% (n = 29) EOS in blood < 3% (n = 17) PC (O 34:2) 0.785 -0.348 0.007 PC 34:3 0.826 -0.276 0.014 PCP 34:1 0.796 -0.324 0.033 PE (O 40:7) 0.446 -1.155 0.052 EOS in blood ≥ 300 cells/μL (n = 17) EOS in blood < 300 cells/μL (n = 29) LPS 20:4 0.434 -1.204 0.014 PEP 40:6 2.148 1.103 0.023 LPI 18:0 2.616 1.387 0.024 PG 34:2 1.333 0.414 0.036 PI 40:5 2.110 1.078 0.037 LPG 22:6 2.591 1.374 0.040 PC 34:3 0.865 -0.209 0.060 Note.Twelve patients had no sputum cell differential count results. Five patients did not have blood routine tests. LPG, lysophosphatidylglycerol; PC, phosphatidylcholine; PC(O), alkylphosphatidylethanolamine; PE(O), alkylphosphatidylethanolamine; PCP, phosphatidylcholine plasmalogen; LPE, lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; PEP, phosphatidylethanolamine plasmalogen; PG, phosphatidylglycerol; PI, phosphatidylinositol. Table 4. Comparison of Serum Glycerophospholipids between Patients with Different Inflammatory Subtypes
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Forty-six of the 51 patients underwent routine blood tests. Blood eosinophils accounted for ≥ 3% of total leukocytes in 29 patients and < 3% in 17 patients (Table 3).
A multivariate statistical analysis of the serum glycerophospholipid results in the blood eosinophilic group (≥ 3%) and non-eosinophilic group (< 3%) was performed. A heat map (Figure 2A) shows the metabolic profiles of glycerophospholipid in each sample; hierarchical cluster analysis showed that there was no obvious similarity in the metabolic profiles of each group, and there was no significant difference between the two subgroups. The PLS-DA score plot (Figure 2B) showed that the two groups could not be separated by the PLS model, indicating that the difference between the two groups was not reflected in the whole glycerophospholipid metabolism spectrum. Therefore, we performed the following univariate analysis. A volcano plot (Figure 2C) showed that PC(O 34:2), PC 34:3, PCP 34:1, and PE(O 40:7) were significantly different between the two subgroups. ROC curve nalysis was performed using these four major significantly different glycerophospholipids as the test variables and blood eosinophils ≥ 3% as the state variable. The AUCs were 34.2% [95% CI (21.7%, 52.3%)], 34.3% [95% CI (25.8%, 57.6%)], 34.1% [95% CI (23.2%, 54.3%)], and 40.7% [95% CI (22.4%, 54.4%)], respectively, and did not reach the 70% threshold. A comparison of these results is shown in Table 4.
Figure 2. (A) Heat map. (B) Partial least squares discriminant analysis score chart. (C) Volcano map. Group 1 and 2 represent asthmatic patients with blood eosinophilis (EOS) ≥ 3% and < 3% respectively. The heat map in (A) compares the metabolic profiles of glycerophospholipids in patients with blood EOS ≥ 3% and < 3%. The PLS-DA score plot in (B) shows the overall differences in the metabolic glycerophospholipid profiles in patients with blood EOS ≥ 3% and < 3%. The volcano plot in (C) shows the glycerophospholipids with significant differences between the subgroups with blood EOS ≥ 3% and < 3%. The phospholipids that vary significantly in this figure are PC(O 34:2), PC 34:3, PCP 34:1, and PE (O 40:7).
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Seventeen patients had an absolute eosinophil value ≥ 300 cells/μL and 29 had a value < 300 cells/μL. The ACT level in the blood eosinophilic group was significantly lower than that in the blood non-eosinophilic group (P < 0.05; Table 3).
A multivariate statistical analysis of the serum glycerophospholipid results in the blood eosinophilic group (≥ 300 cells/μL) and non-eosinophilic group (< 300 cells/μL) was performed. A heat map (Figure 3A) showed the metabolic profiles of the glycerophospholipids in each sample, but the hierarchical cluster analysis showed that there was no obvious similarity in the metabolic profiles of each group, and there was no significant difference between the two subgroups. The PLS-DA score plot (Figure 3B) showed that the two groups could not be separated by the PLS model, indicating that the difference between the two groups was not reflected in the whole glycerophospholipid metabolism profile. Therefore, we performed a univariate analysis. A volcano plot (Figure 3C) showed a significant difference in LPS 20:4, PEP 40:6, LPI 18:0, PG 34:2, PI 40:5, LPG 22:6, and PC 34:3 between the two subgroups. An ROC curve analysis was performed using these seven major significantly different glycerophospholipids as the test variables and blood eosinophils ≥ 300 cells/μL as the state variable. The AUCs were 32.8% [95% CI (16.4%, 49.2%)], 66.8% [95% CI (50.6%, 83.0%)], 61.8% [95% CI (45.1%, 78.4%)], 66.2% [95% CI (50.7%, 81.6%)], 63.0% [95% CI (46.6%, 79.3%)], 61.1% [95% CI (43.8%, 78.3%)], and 43.9% [95% CI (26.6%, 61.3%)], respectively, and did not reach the 70% threshold. A comparison of these results is shown in Table 4.
Figure 3. (A) Heat map. (B) Partial least squares discriminant analysis score chart. (C) Volcano map. Group 1 and 2 represent asthmatic patients with blood eosinophils (EOS) ≥ 300 cells/μL and < 300 cells/μL respectively. The heat map in (A) compares the metabolic profiles of glycerophospholipids in patients with blood EOS ≥ 300 cells/μL and < 300 cells/μL. The PLS-DA score plot in (B) indicates an overall difference in the metabolic glycerophospholipid profiles between the two subgroups. The volcano plot in Figure (C) shows significant differences in LPS 20:4, PEP 40:6, LPI 18:0, PG34:2, PI 40:5, LPG 22:6, and PC 34:3 between the two subgroups.
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Among the 39 patients who underwent a sputum induction test, sputum neutrophils accounted for ≥ 71% of total leukocytes in 19 patients and < 71% in 20 patients (Table 3). A multivariate analysis of the serum glycerophospholipid results in the sputum neutrophilic group (≥ 71%) and sputum non-neutrophilic group (< 71%) was performed. A heat map (Figure 4A) shows the metabolic glycerophospholipid profiles in each sample; the hierarchical cluster analysis showed that there was no obvious similarity in the metabolic profiles in each group, and there was no significant difference between the two subgroups. The PLS-DA score plot (Figure 4B) showed that the two groups could not be separated by the PLS model, indicating that the difference between the two groups was not reflected in the entire metabolic glycerophospholipid spectrum. Therefore, we performed a univariate analysis. A volcano plot (Figure 4C) showed that there was no significant difference between the two subgroups.
Figure 4. (A) Heat map. (B) Partial least squares discriminant analysis score chart. (C) Volcano map. Group 1 and 2 represent asthmatic patients with sputum neutrophils (NEU) ≥ 71% and < 71% respectively. The heat map in (A) compares the metabolic glycerophospholipid profiles in patients with sputum NEU ≥ 71% and < 71%. The PLS-DA score plot in (B) shows the overall differences in the metabolic glycerophospholipid profile between the two subgroups. The volcano plot in (C) shows no significant difference in the metabolic glycerophospholipid profile between the two subgroups.