WO2021141859A1

WO2021141859A1 - Method for diagnosing and treating asthma by detecting or regulating a panel of internal lipid species

Info

Publication number: WO2021141859A1
Application number: PCT/US2021/012089
Authority: WO
Inventors: Shau-Ku Huang; Ming-Shyan Huang; Chin-Chou Wang; Chao-Chien Wu; Zhi-fu ZHENG
Original assignee: National Health Research Institutes; Kaohsiung Medical University; Kaohsiung Chang Gung Memorial Hospital
Priority date: 2020-01-06
Filing date: 2021-01-04
Publication date: 2021-07-15
Also published as: TW202141038A; TWI805982B

Abstract

The present invention provides a method for diagnosing asthma by using a panel of internal lipid species, including LPE 22:6, LPE 20:4, SM 16:0, PE 16:0/22:6, PE 18:0/22:6, PE 18:0/20:4 PE 18:0/18:2, and phosphatidylcholine (PC) 18:0/18:2, comprising comparing a cutoff ratio value of the panel of internal lipid species with internal lipid species ratio value for diagnosing. The present invention also provides a method for treating asthma, comprising administrating a therapeutically effective amount of a LEP 22:6 inhibitory regulator, bovine serum albumin or human serum albumin to a subject in need thereof.

Description

Method for diagnosing and treating asthma by detecting or regulating a panel of internal lipid species

BACKGROUND OF THE INVENTION Technical Field of the Invention [0001 ] The present invention relates to a method for diagnosing asthma by using a panel of internal lipid species. In particular, the method comprises comparing a cutoff ratio value of the panel of internal lipid species with internal lipid species ratio value for diagnosing. Furthermore, the present invention relates to a method for treating asthma comprising administrating a therapeutically effective amount of a LEP 22:6 inhibitory regulator, bovine serum albumin or human serum albumin to a subject suffering therefrom.

Background

[0002] Asthma is characterized by intermittent reversible airway obstruction, persistent pulmonary inflammation and enhanced airway hyperresponsiveness, but the current etiological and molecular basis of asthma is still incomplete, due, in part, to its phenotypic heterogeneity. Also, a significant portion of the adult asthma has been explored to characterize asthma phenotypes by integrating clinical based strategies and a variety of arbitrarily selected clinical variables. Further, various inflammatory and molecular markers and the relationships thereof with different phenotypic clusters have been evaluated. However, the underlying mechanisms of the observed phenotypic and molecular heterogeneity remain unclear. And the fact that the precise diagnosis of asthma remains a challenge makes it even more complicated, as asthma and COPD or their combination share common airflow obstruction. It highlights the need of developing disease-specific markers for an improved diagnosis and treatment.

[0003] Recently, there are studies of cellular and animal models that have suggested several plausible mechanisms underlying the environmental influence. The most consistent observation is the direct effects of pollutants on generation of reactive oxygen species (ROS) as a converging point of mechanistic impact leading to inflammatory response and tissue remodeling. Moreover, increased oxidative stress response may lead to enhanced peroxidation of polyunsaturated fatty acids (PUFAs) and their metabolites, including Ns-(hexanoyl)-lysine (HEL), an early lipid peroxidation product. As the consequence of increased oxidative stress response is related to the exposure of environmental factors, a series of enzymatic processes liberates membrane phospholipid (PL) and increases sphingolipid (SL) metabolism, generating bioactive lipid metabolites and key signaling mediators. PLs are synthesized de novo and regulated by the Lands’ cycle remodeling process involving phospholipase A2 (PLA2) in generating lysophospholipids and free fatty acids, and lysophospholipid acyltransferases (LPLATs), but their functions and regulations in membrane homeostasis under physiological and pathological conditions remain ill defined. Recent efforts in evaluating the role of a limited number of lipid species, including LPA and LPC, in various bio-specimens from patients with asthma or chronic obstructive airway disease (COPD) have suggested that they possess certain potential regulatory activities in disease expression.

[0004] Therefore, it is likely that a comprehensive study of the up- and downstream lipid metabolic pathways may reveal markers that characterize asthma and differentiate asthma from other obstructive airway diseases. In the present invention, markers of internal lipid species are explored, and the relationship thereof with asthma is assessed. The present invention provides a method for profiling the lipidomics of internal phospholipid species with a case-control design to see if any lipid metabolic pathways may reveal markers that characterize asthma and differentiate asthma from other obstructive airway diseases. Utilizing LC-MS/MS and a phased approach, a battery of 48 phospholipid species is investigated in plasma samples from subjects with current asthma, COPD, asthma-COPD overlap syndrome (ACOS) and normal healthy controls. In an adult population with current asthma, a panel of 8 lipid species is noted to discriminate asthma from control and two other respiratory diseases, in which LPE22.6 is bioactive in triggering mast cell response. SUMMARY OF INVENTION

[0005] In one aspect, the present invention provides a method for diagnosing asthma by using a panel of internal lipid species, which comprises collecting a biospecimen from a subject for determining the subject’s internal lipids profile, applying a cutoff ratio value selected from receiver operating characteristic (ROC) curves of the panel of internal lipid species and comparing the cutoff ratio value with the internal lipid ratio value for diagnosing. In one embodiment, the biospecimen includes, but not limited to, plasma, serum, sputum, bronchoalveolar lavage fluids and exhaled breadth condensate. In one preferred embodiment, if the internal lipid ratio value of the subject is higher than the cutoff ratio value, the subject is diagnosed as an asthma patient. [0006] In one embodiment of present invention, the panel of plasma lipid species comprises lysophosphatidylethanolamine (LPE) 22:6, LPE20:4, phosphatidylethanolamine (PE) 18:0/22:6, PE18:0/20:4, PE16:0/22:6, PE18:0/18:2, phosphatidylcholine (PC) 18:0/18:2 and sphingomyelin (SM) 16:0 for differentiating asthma patients from asthma-COPD-overlap syndrome (ACOS) and COPD. In one preferred embodiment, the panel of internal lipid species comprises LPE22:6, LPE20:4, SM16:0 and PE16:0/22:6 for distinguishing asthma, ACOS and COPD.

[0007] In one embodiment of present invention, the cutoff ratio value is 0.028 for LPE22:6 versus PE16:0/22:6. In another embodiment of present invention, the cutoff ratio value is 0.03 for LPE22:6 versus PEI 8:0/22:6. [0008] In another aspect, LPE22:6 (LPE22:6-sn2) induces degranulation and LTCri release in bone marrow-derived mast cell (BMMC), and the level of LPE22:6 is positively correlated with the levels of internal IL-13 and TGF-b, and with the levels of a urinary oxidative marker, HEL.

[0009] Accordingly, the present invention further provides a method of treating asthma, comprising administrating a therapeutically effective amount of a therapeutic agent to a subject suffering therefrom, wherein the therapeutic agent is a LEP 22:6 inhibitory regulator or an agent targeting lipid species for their generation, metabolism and activity.

[0010] In one embodiment of present invention, the LPE 22:6 inhibitory regulator is an antagonist to suppress LPE 22:6 functions in triggering mast cell response and inflammatory cell response. In another embodiment of present invention, the agent targeting lipid species for their generation, metabolism and activity comprises natural, synthetic, chemical and/or biological materials.

[0011] In other embodiment of present invention, the therapeutic agent is bovine serum albumin (BSA). Yet in another embodiment of present invention, the therapeutic agent is human serum albumin (HSA).

BREIF DESCRIPTION OF THE DRAWINGS

[0012] Fig. 1A and IB are radar graphs illustrating the variety of a panel of 8 internal lipid species in asthma, control, ACOS and COPD. (A) A radar graph with z-score shows variance in the levels of lipid species in asthma versus control. (B) A radar graph with z-score shows variance in the levels of lipid species in asthma, COPD, ACOS and control.

[0013] Fig. 2A-2D are curve graphs showing receiver operative characteristic (ROC) curves of the eight focus-analyzed internal lipid species among control, asthma, ACOS and COPD patients. (A) 94 asthma patients and 100 controls with area under curve (AUC) value adjusted for the differences in age and habit of religious incense burning. (B) 94 asthma patients versus 49 COPD patients with AUC value adjusted for the differences in gender, age, BMI, smoking habit, passive smoking exposure at work and habit of religious incense burning. (C) 94 asthma patients versus 52 ACOS patients with AUC value adjusted for the differences in gender, age, and smoking habit. (D) 52 ACOS versus

49 COPD with AUC value adjusted for the differences in gender, age, BMI, smoking habit, and habit of religious incense burning. P < 0.0001 for each of these eight focus-analyzed lipids evaluation in all panels.

[0014] Fig. 3 is a bar graph demonstrating a blinded validation of the ratio of LPE22:6/(PE18:0/22:6) and LPE22:6/(PE16:0/22:6) in a total 89 subjects (number 1 through 89). The internal lipid levels of subjects with physician-diagnosed asthma (N=35), non-asthmatic individual (N=30) and subjects with newly diagnosed asthma (New case, N=24) are measured. The results are un-blinded and analyzed. A ratio cutoff value of 0.028 for LPE22:6 versus PE16:0/22:6 and a ratio cutoff value of 0.023 for LPE22:6 versus PE18:0/22:6 are enumerated. *: Healthy subject (#14) with ratios of 0.03 and 0.028 for LPE22:6/(PE18:0/22:6) and LPE22:6/(PE16:0/22:6), respectively. ^%: New asthma case without history of asthma, whose ratios of LPE22:6/(PE18:0/22:6) and LPE22:6/(PE16:0/22:6) are 0.01 and 0.024, respectively. ^&: Subjects #56 and #58 with current asthma and ratios of 0.029 and 0.028, respectively, for LPE22:6/(PE18:0/22:6) and 0.023 and 0.018 for LPE22:6/(PE16:0/22:6), respectively.

[0015] Fig. 4A-4C show that LPE22:6 (LPE22:6-sn2) can induce mouse (C57BL/6) bone marrow-derived mast cell (BMMC) response. In Fig. 4A and 4B, LPE22:6-sn2 induces degranulation and LTC4 release in BMMCs. Cells are stimulated with 10, 20 and

50 mM of LPE22:6-sn2 for 30 min. Degranulation is monitored by measuring the release of b-hexosaminidase (Hex release) and the level of LTC4 is measured by ELISA. *p<0.05, ** p<0.01 vs. vehicle control. Data are representative of three independent experiments. As shown in Fig. 4C, LPE22:6-sn2 induces calcium influx in BMMCs. BMMCs are loaded with the Ca²⁺ indicators, Fluo-3-AM and Fura red-AM, and stimulated with 10, 20 and 50 mM of LPE22:6-sn2 (dissolved in 0.2% BSA-PBS) in Tyrode’s buffer without Ca2+ for the initial 3 min upon stimulation, and then washed and cultured in Tyrode’s buffer with Ca²⁺ (2mM). Data are representative of three independent experiments.

[0016] Fig. 5A-5D show that the functional impact of LPE22:6 on BMMC’s response is inhibited by the addition of bovine or human serum albumin (BSA and HSA, respectively). The addition of BSA inhibits LPE22:6-induced membrane perturbation in a concentration dependent manner (Fig. 5A). In addition, HSA also inhibits LPE22:6-induced membrane perturbation, but the oxidized form of HSA is unable to inhibit LPE22:6’s effect (Fig. 5B). Furthermore, addition of BSA results in significant decrease of LPE22:6-induced calcium mobilization (Fig. 5C) and degranulation (Fig. 5D) in a concentration-dependent manner. ***, 0.001 vs. vehicle; ###, 0.001 vs. LPE22:6. Data are representative of three independent experiments.

[0017] Fig. 6A-6C indicate the correlation of linear regression analysis between the levels of internal LPE22:6 with IL-13, TGF-b and HEL levels in subjects with asthma.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Other features and advantages of the present invention will be further exemplified and described in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. [0020] Examples

[0021 ] The other characteristics and advantages of the present invention are further illustrated and described in the following examples. The examples described herein are using for illustrations, not for limitations of the invention. [0022] The practice of the present invention will employ technologies comprising conventional techniques of cell biology and cell culture, which are within the ordinary skills of the art. Such techniques are explained fully in the literature

[0023] Subject population

[0024] The subject population includes, prospectively, adult asthma patients at the outpatient departments of 8 medical centers following established protocols. All eligible subjects are enrolled after signing the informed consent approved by the respective recruitment hospitals. Patients with age >18 years are enrolled from 2011 until 2015. Patients who meet the following inclusion criteria are eligible for enrollment: (1) at least 18 years of age, (2) physician-diagnosed asthma. Physician’s diagnosis of asthma and severity level are made according to the 2008 Global Initiative for Asthma (GINA) guidelines. All the participants are subjected to pulmonary function test (PFT) consisting of spirometry measurement of unprovoked airflow. The PFT is performed and interpreted according to the statement of American Thoracic Society. The diagnosis of asthma is made by a pulmonologist based on the GINA guideline. The diagnosis of COPD is confirmed by persistent airway obstruction as evidenced by post-bronchodilator forced expiratory volume in 1 second (FEVl)/forced expiratory capacity (FVC) of less than 0-7 on spirometry, as indicated in the guideline of Global Initiative for Chronic Obstructive Lung Disease (GOLD). According to the joint document issued by GINA and GOLD (available at www.ginasthma.org/local/uploads/files/ACOS_2015.pdl), patients with persistent airflow limitation and similar features of asthma and COPD are diagnosed as having ACOS. Normal control subjects are also enrolled, they are selected from volunteers and people who request annual physical examinations without respiratory diseases. The PFT is also performed on the normal subjects to verify if they are free of respiratory diseases. All patients and normal subjects with co-morbidities such as diabetes mellitus, hyperlipidemia, or with abnormalities in liver functions that could potentially interfere lipid metabolism are excluded.

[0025] The severity of asthma is classified based on the intensity of treatment required to achieve good asthma control. As indicated by the GINA guidelines, severe asthma requires high intensity treatment, e.g. GINA Step 4 (severe) or 5 (very severe), to maintain in good control, or where good control is not achieved despite high intensity treatment. In the present invention, asthma is further stratified into four groups: asthma that requires GINA Step 1 or 2 to maintain good control is considered as mild asthma, GINA Step 3 as moderate, GINA Step 4 as severe, and GINA step 5 as very severe. Patients with severe asthma (step 4) receive more than two combination controller therapy (ICS, LABA, leukotriene modifier and sustained-release theophylline), while step 5 asthmatics are treated with oral corticosteroid or anti-IgE Abs, in addition to step 4 therapies. The control status of patients is also evaluated by using asthma control test (ACT), a validated patient-completed questionnaire with five parameters aimed to assess asthma symptoms (daytime and nocturnal), use of rescue medications, and the effect of asthma on daily functioning. The scores range from 5 (poor control of asthma) to 25 (complete control of asthma). The scores equal or less than 19 are considered as “not well controlled’. Pulmonary function is measured with a Jaeger Master screen Pulmonary System spirometer (Hoechberg, Germany). At baseline, FEV1 and FVC are expressed as percentages of the predicted values, whereas FEV1/FVC is reported only as a ratio. Bronchodilator response (BDR) is expressed as the percent increasing from baseline FEV1 and as an absolute change in FEV1.

[0026] Questionnaires are conducted for assessing demographic information, smoking habits, occupation, asthma symptoms, history of disease, medical history, living environment, clinical examination and pulmonary function test. The age at onset of asthma is self-reported. To ensure the accuracy, patients are asked to describe episodes of dyspnea, wheezing, or coughing they have experienced during childhood and puberty. In cases of uncertainty, the time of the earliest respiratory symptoms is designated as the age at onset of asthma symptoms. The smoking status of all patients is also queried and divided into three groups: current smokers, ex-smokers who have stopped smoking for at least 1 month before the initial visit, and lifetime non-smokers. Patients have one of self-reported allergic triggers (pollen, mold, dust, animals, beans, seafood, milk and eggs), allergic rhinitis and/or atopic dermatitis are defined as atopies. The non-asthmatic volunteers, who undergo routine annual physical examination and have normal lung function with no history of asthma, are also included for comparison.

[ 0027 ] Data analysis

[0028] The comparison of demographic variables (age at sample collection, gender, body mass index, atopic status, smoking habits, passive smoking exposure at home and work) and bio-monitoring data between asthma patients and controls is conducted by two sample t-test, Chi-square test and Cohen’s D effect size. For all measurements with levels below the limit of detection, the values are substituted by half of the limit of detection. To create a comprehensible overview, a radar graph is chosen to present these means for each group. In differentiating asthma patients and normal controls, two-sample t-test with false discovery rate (FDR) adjustment is used. Values of internal lipids are first transformed into Z-scores. The Receiver operating characteristic (ROC) curves and area under curve (AUC) values are generated to differentiating asthma from COPD and ACOS. The adjusted AUC values are further calculated by Logistic regressions with additional covariates of gender distribution, age, body mass index, smoking habits, the status of passive smoke exposure at work, and the habit of religious incense burning. Correlations between the lipid metabolite, LPE22:6, and cytokines, IL-13 and TGF-b, and an oxidative stress marker, HEL, are evaluated by Spearman rank correlation coefficients (rS). All analyses are performed with SAS statistical software (version 9.4, SAS Institutes Inc., Cary, NC, USA). The p-values less than 0.05 are considered as significant.

[0029] Example 1. Profiling internal lipid species of subjects [0030] Specimen collection and extraction of lipids in plasma [0031] The blood samples of subjects are collected in the physician’s outpatient office. Peripheral whole blood (20 mL) is aspired through venipuncture and collected in heparinized blood collecting tube. The plasma is isolated by density gradient centrifugation (Lymphoprep, Oslo, Norway) at 3,000 rpm for 10 min, and immediately aliquot and frozen in -80 °C freezer. Prior to lipid extraction, a 15pL PLIS aliquot is added to every 30 pL of plasma and mixed thoroughly. Thereafter, the plasma lipids are extracted by using the method of Folch et al. For the additional analysis of ceramides and LPAs, 100 pL of plasma is mixed with 10 pL each of CerlS and LPAIS and extracted using the same method. At the end of extraction, the lipid-containing organic layer is collected and transferred to a new sample vial and the solvent is completely evaporated by a gentle stream of nitrogen. The vials containing lipid pellets are filled with nitrogen, sealed, and stored at -80 °C until analysis. Immediately before LC-MS/MS analysis, the lipid pellets are dissolved in 1.5 mL of mobile phase A and thoroughly mixed for LC-MS/MS analysis. [0032] Liquid chromatography- tandem mass spectrometry (LC-MS/MS)

[0033] Plasma phospholipids are analyzed using LC-MS/MS approach where concurrent measurement of the quality control samples is also implemented. To prepare lipid internal standard solutions, a methanolic solution containing

Dimyristoylphosphatidylcholine (DMPC),

Dimethyl-2-(dimethylphosphino)ethylphosphine (DMPE), dipalmitoylphosphatidylinositol (DPPI), SM 12:0, LPC 14:0, and LPE 14:0 is prepared as the internal standard (IS) solution for the analysis of major plasma phospholipids (PLs). Every 15pL ofPLIS solution contained 3-3 pg of DMPC, 3 1 pg of DMPE, 1-2 pg of LPE 14:0, 1-8 pg of LPC 14:0, 1-6 pg of SM 12:0, and 2 pg of DPPI. A second methanolic solution of Cer 17:0 (10 ng/pL), and a third methanolic solution of LPA 14:0 (0-5 ng/pL) are also prepared as the IS for ceramides (CerlS) and LPAs (LPAIS) for the analyses of plasma ceramides and LPAs. To prevent oxidation of lipids, 1% (v/v) of 10% methanolic butylated hydroxytoluene (BHT) solution is added in the vehicle for all the IS solutions.

[0034] The chromatographic method is modified from the previously reported method of hydrophilic interaction liquid chromatography (HILIC) for the application of LC-MS/MS analysis of lipids. The mobile phase is delivered at 0.2 ml/min, and the Ascentis® Express HILIC column (2.7 pm; 2.1 x 150 mm, Cat. No.: 53946-U, Supelco) is used for chromatographic separation. The chromatographic outflow is introduced to the electrospray ionization source of a mass spectrometer equipped with dual ion-funnels (AmaZon X ion trap mass spectrometer; Bruker Daltonik). Due to the operational limitation of the mass spectrometer, one plasma lipid sample is analyzed twice by using the same chromatographic method coupled with two different multiple reaction monitoring (MRM) methods for acquiring the comprehensive coverage of all the lipid species detected in every lipid class. Details on the LC-MS/MS system and the monitored lipid classes and species, the salient mass spectrometric criteria for the identification and quantitation of lipid classes and species are described below. [0035] The LC-MS/MS system consists of a Waters model 2695 separation module coupled with a Bruker AmaZon X ion trap mass spectrometer. The chromatographic method is modified from a previously reported method in which a hydrophilic interaction liquid chromatography (HILIC) column (Supelco Ascentis® Express HILIC column, 2.7 pm; 2.1 x 150 mm, Cat. No.: 53946-U) is used for the separation of lipids. The mobile phase A consists of the mixture of 85% (v/v) acetonitrile (ACN), 10% methanol (MeOH), and 5% of H2O, whereas mobile phase B is composed of the mixture of 65% ACN, 10% MeOH, and 25% H2O. Both mobile phases contain 0.04% (v/v) formic acid and 1 mM of ammonium formate. For the elution of major lipids, the mobile phase is delivered at 0.2 mL/min. During elution, mobile phase A is delivered at 90% from 0-6 min and then linearly decreases to 70% from 6-10 min, followed by another linear decrease to 50% from 10-16 min; after holding at 50% for 16-20 min, it is stepped back to 90% from 20-20.1 min and held at 90% until the end of the run. The entire chromatographic run is 30 min.

[0036] For the elution of ceramides (Cer) and LPAs, the same HILIC column is used. The mobile phase A composition is identical to the above mobile phase A, while the mobile phase B consists of 6.5% ACN, 1% MeOH and 95% H2O. The formic acid and ammonium formate concentrations in these two mobile phases are also identical to the above formula. The flow rate is also maintained at 0.2 mL/min during elution. Mobile phase A is delivered at 90% at the beginning of the elution run, followed by a linear decrease to 70% from 0-2 min, then a steep linear reduction to 50% from 2-3 min, then held at 50% from 3-4 min, then stepped down to 20% from 4-4.1 min and held at 20% from 4.1-10 min, and stepped back to 90% from 10-10.1 min and held at 90% until the end of the run. The entire chromatographic run for the elution of ceramides and LPAs is 23 min.

[0037] The outflow of the chromatographic column is directed to the electrospray needle of the mass spectrometer and ionized under negative ion mode to avoid the bias of choline-containing lipid precursor signal by the systemic presence of alkali metal cations. However, for the detection of lipids in phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (LPE) classes, the outflow is ionized under positive ion mode at their respective chromatographic segments. The mass spectrometer is operated under multiple reaction monitoring (MRM) mode. During the LC-MS/MS run, the mass spectrometer performs one analytical scan when the ion trap has either collected 100,000 ions, or 200 ms of collection time has elapsed. Using this setting, at least 10 data points can be evenly collected along the elution time of the chromatographic peak so to ensure a sufficient number of data points are collected to retain the chromatographic fidelity. The mass isolation window for MS/MS is set at the 12C m/z ± 1 Da of each monitored lipid species. The ionization voltage and collision voltage of each lipid class is determined first by the direct infusion of its respective internal standard (IS) solution (1 pg/mL delivered at 5 pL/min with a syringe pump), then fine-tuned individually when necessary. In order to obtain reliable quantitative results and to overcome the inherent operational limitation of the ion trap mass spectrometer, one PL sample is analyzed twice by using two different MS/MS methods (MS/MS Method 1 and 2) for acquiring a complete coverage of all the major PL species except Cer and lysophosphatidic acids (LPAs) intended to monitor. For the monitoring of Cer and LPAs, another two different MS/MS methods (MS/MS Method 3 and 4, respectively) are also utilized.

[0038] For the differentiation and quantitation of class-specific lipids, the MS/MS fragmentation scheme of Xia and Jemal for choline-containing lipid species such as phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs), and sphingomyelins (SMs) is adapted. Using the mobile phase for the elution of major lipid classes, the formate-adducted ions (i.e., [M+45]-) are the only detectable precursor ions for these choline-containing lipids. For the confirmation and quantitation of these PLs, the [M-15]- (i.e., [M+formate-60]-) fragment ion is selected as the quantitative fragment ion and the fatty acyl fragment ions is chosen to confirm the fatty acyl composition of the precursors. For the confirmation of Pis, the m/z 241 fragment ion derived from the phosphoinositol head group is practiced as the confirmative fragment ion, whereas the fragment of PI precursor losing the sn-2 moiety is used for quantitation. The confirmation and quantitation of PEs and LPEs are based on the neutral loss of 141 Da from the precursor ions under positive ion mode. The fatty acyl compositions of these ethanolamine-containing PLs are verified by the fragment of each precursor ion losing its respective fatty acyl moiety, or by additional LC-MS/MS analyses under negative ion mode. The confirmation and quantitation of Cer species are based on the MS3 tandem mass spectrometry of the precursors. The [M-H20]+ product ion from the initial MS/MS reaction is selected as the precursor for the MS3 confirmation which monitors the m/z 264.5 fragment ion as the signature fragment of ceramides. For the confirmation and quantitation of LPA species, the m/z 153.2 product ion is chosen as the signature of LPA. The amount of each lipid species is determined by the ratio of the area under the extracted ion chromatograph (EIC) of its quantitative fragment to that of its respective IS. Several pilot LC-MS/MS runs are conducted to ensure that the amounts of IS included in the plasma sample are adequate to confer the peak area ratio between monitored lipid species and its respective IS falling within the range of 1:10 to 10:1. The quality of analysis is monitored via the analysis of quality control samples concurrently performed with each batch of plasma samples.

[0039] Example 2. Evaluating a panel of eight lipid characterizing asthma

[0040] To identify molecular signatures of asthma, lipidomics analyses are conducted, wherein a subset of the cases (N=365) and the controls (N=235) are systematically selected from a total 1,163 subjects with current asthma and of 1,493 controls with sampling rate of 31% and 16%, respectively. In this dataset, demographical comparison by two-sample t-test, Chi-square test and Cohen’s D effect size reveal significant differences in the distribution of atopic subjects and lung function at level of 0.0045 (=0.05/11, Bonferroni multiple comparison adjustment). Table 1. Demographic comparisons between 365 asthma patients and 235 controls

*Two-sample t-test and Chi-square test.

^&Cohen’s D effect size; values greater than 0.2 are considered to be different.

[0041] Using a case-control design, the profiles of internal PLs and SLs with a phased approach are investigated. In the discovery phase, a panel of 48 internal phospholipid and sphingolipid species is initially screened and 18 of those are found significant, including LPCs, PEs, LPEs and SMs, in differentiating asthma (N=35) and controls (N=30) with False discovery rate (FDR)-adjusted p values <0.0001 (data not shown). Among them, a cluster of 8 lipid species, including LPE22:6, LPE20:4, PE18:0/22:6, PE18:0/20:4, PE16:0/22:6, PE18:0/18:2, PC18:0/18:2, and SM16:0, shows significance in the validation phase with analyses of additional 94 asthma patients and 100 controls. Notably, analysis by Z-score revealed distinct patterns of 8-lipid profile in asthma versus control. In asthma patients, the cluster of LPE22:6, LPE20:4, SM16:0 and PC 18:0/18:2 is more prominent than normal subjects; on the other hand, the cluster of PE18:0/22:6, PE18:0/20:4, PE16:0/22:6 and PE18:0/18:2 of normal subjects is more distinguished than asthma patients (as shown in Fig. 1 A).

[0042] As asthma, COPD and ACOS sharing common clinical features, plasma samples from 49 and 52 subjects with COPD and ACOS are included for comparison. Z-score analysis showing that the 8 focus-analyzed lipid species all show significance in differentiating asthma from COPD and ACOS (Fig. IB), wherein a panel of seven lipid species, including LPE 22:6, LPE 20:4, SM 16:0, PE 18:0/22:6, PE 18:0/20:4, PC 18:0/18:2 and PE 16:0/22:6, distinguishes asthma from COPD, while three lipid species, SM 16:0, PC 18:0/18:2 and PE 18:0/18:2, are distinguishable between asthma and ACOS. Further, comparison of COPD versus ACOS shows significant difference for a panel of five lipid species, including LPE 22:6, LPE 20:4, PC 18:0/18:2, PE 18:0/22:6 and PE 18:0/20:4.

[0043] Through a phased approach, a panel of 8 internal lipid species shows promise as candidate molecular characterizing asthma and discriminating asthma from control. Integrating internal lipid signatures, a new form of endotype, in identifying phenotypic subsets of patients with asthma is lending the support for the contribution of lipid metabolites to asthma. Further, the discovery of phospholipid and sphingolipid signatures in subject population suggests the existence of selective lipidomic remodeling in adult asthma.

[0044] Example 3. Calculating receiver operative characteristics (ROC) curves from the eight focus-analyzed lipid species and the area under curve (AUC) values thereof for differentiating asthma patients from controls, ACOS, and COPD.

[0045] The dataset of internal lipids is used to generate the ROC curves and calculate the AUC values of each lipid species. The AUC values for each individual lipid species is either unadjusted or adjusted, based on gender distribution, average age, body mass index, smoking habits, the status of passive smoke exposure at work, and the habit of religious incense burning. The adjusted AUC results indicate that LPE 22:6, LPE 20:4, SM 16:0, and PE 16:0/22:6 are the lipid species capable of differentiating asthma patients from controls (Fig. 2A) and from ACOS and COPD (Figs. 2B and 2C, respectively) and also distinguishing ACOS and COPD (Fig. 2D). [0046] From the unadjusted ROC analyses, the cut-off values are selected, and their discriminative values are calculated in three different group-wise comparisons. Table 2 summarizes the cut-offs and their sensitivity and specificity in differentiating asthma from control, COPD and ACOS. The results show that in differentiating asthma from control, the cut-off values of LPE 22:6 and LPE 20:4 show high sensitivity (100% and 97.87%, respectively) and specificity (99% and 100%, respectively). For discriminating asthma from COPD, the cut-off values of a panel of seven phospholipid species, including LPE 22:6, LPE 20:4, SM 16:0, PE 18:0/22:6, PE 18:0/20:4, PC 18:0/18:2 and PE 16:0/22:6, show high sensitivity (range, 80.85%-100%) and specificity (range, 86.17%-100%); while for distinguishing asthma from ACOS, the cut-offs of three lipid species, SM 16:0, PC 18:0/18:2 and PE 18:0/18:2, are the most discriminative with high sensitivity (range, 96.15%-97.87%) and specificity (range, 89.36%-100%). Furthermore, to distinguish COPD and ACOS, the cut-offs of five lipid species, including LPE 22:6, LPE 20:4, PC 18:0/18:2, PE 18:0/22:6 and PE 18:0/20:4, show the highest levels of sensitivity (range, 98.08%-100%) and specificity (range, 94.23%-100%). These results suggest that these eight focus-analyzed lipids are promising markers for the differentiation between asthma and COPD or ACOS and for the distinction between ACOS and COPD. Table 2. Cut-off values selected form ORC curves for 8 focused-analyzed phospholipids and the respective sensitivity and specificity from the ROC analyses.

Atorney Docket No. 5382/0325PWO2

[0047] It is particularly noted that the ratio of LPE22:6/(PE18:0/22:6) or LPE22:6/(PE16:0/22:6) is significantly increased in asthmatics compared to the controls, suggesting imbalanced Lands’ cycle in asthmatic patients. In the subsequent blinded validation study of plasma samples from a total 89 subjects consisting of patients suspected to have asthma (N=35), non-asthmatic individual (N=30) and patients with newly diagnosed asthma (N=24). The lipid levels of specimens are measured and labeled in a blinded fashion, and the results are though unblinded analysis. Based on a ratio cutoff value of 0.028 for LPE22:6 versus PE16:0/22:6, among the 32 samples with ratio below the cut-off, 29 are from controls, one with newly diagnosed asthma (#83) and two with established asthma (#56 and #58), with overall sensitivity and specificity being 97% and 96%, respectively (Fig. 3). Similarly, a ratio cutoff value of 0.023 for LPE22:6 versus PE18:0/22:6 also shows high sensitivity (96%) and specificity (95%; Fig. 3), in which the ratio for a subject with newly diagnosed asthma (#83), the ratio is 0.01, whereas for a healthy subject #14, the ratio is 0.03. It is interestingly noted that for the healthy subject (#14) with FEV1 at 81% of predicted and at 88% after bronchodilator administration, the ratios are 0.028 and 0.03, respectively, for LPE22:6/(PE16:0/22:6) and LPE22 : 6/(PE 18 : 0/22 : 6) .

[0048] Example 4. Functions of LPE22:6 in triggering mast cell response.

[0049] To explore the potential impact of LPE22:6 in regulating cellular function, mouse bone marrow-derived mast cells are used as a model. The results show that significant induction of b-hexosaminidase release, a preformed granule mediator, and leukotriene C4 (LTC4), a lipid mediator, is found in mast cells (Figs. 4A and 4B, respectively), concomitant with increased calcium influx, not calcium mobilization, from the intracellular store (Fig. 4C). [0050] On the other hand, the addition of bovine serum albumin (BSA) or human serum albumin (HSA) is found to be able to alleviate LPE22:6-induced membrane perturbation. Cells are treated with or without BSA (1% or 4%) or HSA (1%) for 10 min, then stimulated with 50 mM LPE22:6 for 1 min in the presence of FM1-43, a fluorescence dye that preferentially inserts into loosely packed membranes. The results suggest that the addition of BSA inhibits LPE22:6-induced membrane perturbation (Fig. 5A). In addition, HSA also inhibits LPE22:6-induced membrane perturbation, but the oxidized form of HSA is unable to inhibit LPE22:6’s effect (Fig. 5B).

[0051] Furthermore, the addition of BSA results in significant decrease of LPE22:6-induced calcium mobilization (Fig. 5C) and degranulation (Fig. 5D) in a concentration-dependent manner. These results, collectively, demonstrate that intervention of LPE22:6’s effect on cellular response by albumin may provide a viable approach in alleviating its lipotoxicity

[0052] Also, the level of LPE22:6 is positively correlated with the levels of plasma IL-13 and TGF-b (r=0.287, p=0.029 and r=0.302, p=0.021, respectively; Figs. 6A and 6B, respectively), and with the levels of a urinary oxidative marker, HEL (Spearman correlation, r=0.25, p=0.001; Fig. 6C).

[0053] In summary, a panel of internal lipid species, including LPE 22:6, LPE 20:4, SM 16:0, PE 16:0/22:6, PE 18:0/22:6, PE 18:0/20:4 PE 18:0/18:2, and phosphatidylcholine (PC) 18:0/18:2, is demonstrated capable of separating asthma from COPD and ACOS. Furthermore, receiver operating characteristic analyses of the lipid panel reveal that the respective cutoff values can distinguish asthma from COPD and ACOS. In additional, a lysophosphorylethanolamine (LPE) specie, LPE22:6, is particularly prominent, correlating with the levels of an oxidative stress marker, Ne-(hexanoly)-lysine (HEL), IL-13 and TGF-bI in subjects with asthma, and is found to trigger degranulation and LTC4 release in mast cells, which is reversible by the addition of serum albumin. Therefore, the panel of plasma phospholipid species revealed by the present invention may be used as markers to characterize asthma and uniquely differentiate asthma from other obstructive airway diseases..

Claims

1. A method for diagnosing asthma by using a panel of internal lipid species, which comprises collecting a biospecimen from a subject for determining the subject’s internal lipids profile, applying a cutoff ratio value selected from receiver operative characteristics (ROC) curves of the panel of internal lipid species to the subject’s internal lipid ratio value, and comparing the cutoff ratio value with the internal lipid ratio value for diagnosing.

2. The method of claim 1, wherein the biospecimen comprises plasma, serum, sputum, bronchoalveolar lavage fluids and exhaled breadth condensate.

3. The method of claim 1, wherein the panel of internal lipid species comprises lysophosphatidylethanolamine (LPE) 22:6, LPE20:4, phosphatidylethanolamine (PE) 18:0/22:6, PE18:0/20:4, PE16:0/22:6, PE18:0/18:2, phosphatidylcholine (PC)

18:0/18:2 and sphingomyelin (SM) 16:0 for differentiating asthma patients from ACOS and COPD.

4. The method of claim 2, wherein the panel of internal lipid species comprises LPE22:6,

LPE20:4, SM16:0 and PE16:0/22:6 for distinguishing asthma, ACOS and COPD.

5. The method of claim 1, the cutoff ratio value is 0.028 for LPE22:6 versus PE16:0/22:6

6. The method of claim 1, wherein the cutoff ratio value is 0.03 for LPE22:6 versus PEI 8:0/22:6.

7. A method of treating chronic airways diseases, including asthma, which comprises administrating a therapeutically effective amount of a therapeutic agent to a subject suffering therefrom, wherein the therapeutic agent is a LEP 22:6 inhibitory regulator, bovine serum albumin, human serum albumin, or an agent targeting lipid species for their generation, metabolism and activity.

8. The method of claim 6, wherein the LEP 22:6 inhibitory regulator is an antagonist to suppress LEP 22:6 functions in triggering mast cell and inflammatory cell response.

9. The method of claim 7, wherein the agent targeting lipid species for their generation, metabolism and activity comprises natural, synthetic, chemical and biological materials.