TWI805982B - Method for diagnosing and treating asthma by detecting or regulating a panel of internal lipid species - Google Patents

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

A method for diagnosing and treating asthma by detecting or modulating a panel of lipid species in the body

The present invention relates to a method for diagnosing asthma by using a panel of lipid species in the body, and more particularly, the present invention relates to a method for comparing the cut-off ratio value of a panel of lipid species in the body with the ratio value of lipid species in a subject To diagnose asthma. In addition, the present invention also relates to a method for treating asthma, which comprises administering an effective dose of LEP 22:6 inhibitory modulator, bovine serum albumin or human serum albumin to an individual suffering from asthma.

The symptoms of asthma include intermittent reversible airway obstruction, persistent lung inflammation and airway hyperresponse. However, due to the phenotypic heterogeneity of asthma, the etiology and molecular basis of asthma are still incomplete. Moreover, the majority of adult asthmatic lines were characterized by integrating clinical strategies and selecting various arbitrary clinical variables to characterize their asthma phenotypes. In addition, various inflammatory and molecular markers were assessed and their relationship to different phenotypic clusters. However, the underlying mechanisms for the observed phenotypic and molecular heterogeneity are still unclear, and accurate diagnosis of asthma remains a challenge, which also leads to the complexity of distinguishing its mechanism, because asthma and chronic obstructive Chronic obstructive pulmonary disease (COPD) or a combination thereof share common airway obstruction problems. Therefore, there is an urgent need to develop disease-specific markers that can improve the diagnosis and treatment of asthma.

Recently, numerous cellular and animal studies have proposed several plausible mechanisms of environmental impact, which consistently point to the direct impact of pollutants on the production of reactive oxygen species (ROS), which contribute to inflammation and tissue remodeling. Convergence of related mechanisms, in addition, increased oxidative stress response also enhances polyunsaturated fatty acids (PUFAs) and their metabolites (including early lipid peroxidation product Nε-(hexanoyl)-lysine (HEL)) of peroxidation. The result of increased oxidative stress response is related to exposure to environmental factors, so a series of enzymatic reactions releases phospholipids (PL) and enhances the metabolism of shpingolipids (SL), producing Biologically active lipid metabolites and key messaging mediators. Phospholipids are re-synthesized (de novo) and subjected to a Lands cycle cycle involving phospholipase A2 (PLA2) involved in the production of lysophospholipids and free fatty acids, and lysophospholipid acyltransferases (LPLATs). regulation of the plastic process. However, the function and regulation of phospholipids for membrane homeostasis under physiological and pathological conditions remain poorly defined. Recently, the evaluation of the role of specific numbers of lipid species (including LPA and LPC) in various biological samples obtained from patients with asthma or chronic obstructive pulmonary disease (COPD) showed that these lipids have specific roles in the expression of the disease. potential modulatory activity.

Thus, a comprehensive study of upstream and downstream lipid metabolism pathways may reveal markers that characterize asthma and differentiate it from other obstructive airway diseases. In the present invention, markers of lipid species in vivo are explored and their relationship to asthma is assessed. Thus, the present invention provides a method for analyzing the liposomes of phospholipid species in vivo through a case-control design to detect whether any lipid metabolic pathways can be revealed to characterize asthma and to differentiate asthma from other obstructive markers of respiratory diseases. The present invention utilizes LC-MS/MS and a staged method to obtain samples from patients with asthma, chronic obstructive pulmonary disease, asthma-COPD overlap syndrome (asthma-COPD overlap syndrome, ACOS), and normal healthy controls. 48 kinds of phospholipids were detected in the subjects' plasma samples. Results: In adults with asthma, a panel of eight lipid species distinguished asthma from controls and the other two respiratory diseases, among which LPE22.6 was also biologically active to elicit mast cell responses.

In one aspect, the present invention provides a method for diagnosing asthma by detecting a panel of lipid species in the body, comprising: collecting a biological sample from a subject to determine the body lipid composition of the subject; Select a cut-off ratio value from the receiver operating characteristic (ROC) curve; and compare the cut-off ratio value with the body lipid ratio value of the subject for diagnosis. In one embodiment, the biological sample includes but not limited to plasma, serum, sputum, bronchoalveolar lavage fluid and exhaled breath condensate; in a preferred embodiment, if the subject's body lipid ratio is high If the cut-off ratio is lower, the subject can be diagnosed as an asthmatic patient.

In one embodiment of the present invention, the types of lipids in the group include hydrolyzed phosphatidylethanolamine (lysophosphatidylethanolamine, LPE) 22:6, LPE20:4, phosphatidylethanolamine (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 , used to distinguish patients with asthma from those with chronic obstructive pulmonary disease (COPD) and asthma-COPD overlap syndrome (ACOS). In a preferred embodiment, the lipid species in the group includes LPE22:6, LPE20:4, SM16:0 and PE16:0/22:6 for distinguishing asthma, ACOS and COPD.

In one embodiment of the present invention, the cut-off ratio of LPE22:6 relative to PE16:0/22:6 is 0.028; in another embodiment of the present invention, LPE22:6 is relative to PE18:0/22:6 The cutoff ratio value for is 0.03.

On the other hand, LPE22:6 (LPE22:6-sn2) can induce degranulation and release of LTC4 in bone marrow-derived mast cells (BMMC), and LPE22:6 The concentration of IL-13 and TGF-β concentration in the body is positively correlated with the concentration of oxidation marker-HEL in the urine.

Accordingly, the present invention further provides a method for treating asthma comprising administering to a patient in need thereof an effective dose of a therapeutic agent, wherein the therapeutic agent is an LPE 22:6 inhibitory modulator or a targetable lipid production, metabolism and active drug.

In one embodiment of the present invention, the LPE 22:6 inhibitory modulator is an antagonist for inhibiting the functions of LPE 22:6 in inducing mast cell responses and inflammatory cell responses; in another implementation of the present invention In one example, the agents that target the production, metabolism and activity of lipids include natural, synthetic, chemical and/or biological substances.

In another embodiment of the present invention, the therapeutic agent is bovine serum albumin (bovine serum albumin, BSA); in yet another embodiment of the present invention, the therapeutic agent is human serum albumin (human serum albumin) , HSA).

Other features and advantages of the present invention will be further illustrated in the following examples, and these examples are only used as examples and are not intended to limit the scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs.

Example

Other features and advantages of the present invention will be further illustrated and described in the following examples, and the examples described herein are only for illustration, not for limiting the present invention.

The practice of the present invention requires the use of conventional techniques, including cell biology and cell culture, which are within the ordinary skill of the art. Therefore, such techniques have been fully described in the prior art.

Subject group

According to the established plan, the subject group will include adult asthmatic patients in the outpatient departments of 8 medical centers, and all eligible subjects who signed the informed consent form in each hospital will be included in this trial. From 2011 to 2015, All patients who are older than/equal to 18 years old can be selected, and the patients who want to be tested must meet the following conditions: (1) At least 18 years old; (2) Diagnosed with asthma by a physician. Physicians judged asthma and its severity according to the guidelines published by the Global Initiative for Asthma (GINA) in 2008. All subjects received a pulmonary function test (pulmonary function test, PFT), which included a non-invasive tracheal spirometry, and all pulmonary function tests were performed in accordance with the guidelines of the American Thoracic Society. Physicians also refer to GINA guidelines for the diagnosis of asthma, as taught in the guidelines of the Global Initiative for Chronic Obstructive Lung Disease (GOLD). The diagnosis of COPD is that after the use of bronchodilators, if the expiratory volume in 1 second (forced expiratory volume in 1 second, FEV1)/forced vital capacity (forced vital capacity, FVC) of the spirometer is less than 0.7, it means persistent Sexual tracheal obstruction. In addition, according to the document jointly published by GINA and GOLD (available at www.ginasthma.org/local/uploads/files/ACOS_2015.pdf), patients with persistent airway obstruction and other symptoms similar to asthma and COPD are diagnosed as ACOS. At the same time, normal subjects also participated in the trial, mainly selected from volunteers and those who had undergone annual health checkups and had no respiratory diseases. Normal subjects also underwent PFT testing to verify whether they had respiratory diseases. disease. In this test, all patients and normal subjects with comorbidities such as diabetes mellitus, hyperlipidemia, or abnormal liver function that may interfere with lipid metabolism were excluded.

Asthma severity is graded according to the intensity of treatment required to achieve good asthma control. As stated in the GINA guidelines, severe asthma patients need intensive treatment to maintain good control, such as GINA step 4 (severe) or step 5 (very severe), or cannot achieve despite high-intensity treatment good control. In the present invention, asthma is further divided into four categories: mild asthma for asthma patients who need GINA Step 1-2 treatment to maintain good control, GINA Step 3 treatment for moderate, GINA Step 4 for severe asthma, and GINA Step 5 for severe asthma for very serious. Patients with severe asthma (step 4) usually receive two or more combination controller therapies (ICS, LABA, leukotriene inhibitors, long-acting sustained-release theophylline), and the treatment phase Asthma patients in Step 5 need to receive oral corticosteroids or anti-IgE antibodies in addition to the treatment of Step 4 in the above treatment phase. In addition, patient control was assessed using the asthma control test (ACT), a validated patient-completed questionnaire consisting of five parameters designed to assess asthma symptoms (daytime and nighttime), emergency Medication use and the impact of asthma on daily life, scores range from 5 (poorly controlled asthma) to 25 (completely controlled asthma), with a score of 19 or less considered "poorly controlled." Pulmonary function was measured with a pulmonary system spirometer (Hoechberg, Germany). At baseline, both FEV1 and FVC were presented as percentages of predicted values, while FEV1/FVC was presented as a ratio only. The bronchodilator response (bronchodilator response, BDR) is presented as a percentage increase relative to the FEV1 at baseline to show an absolute change in FEV1.

Questionnaires were administered to assess demographics, smoking habits, occupation, asthma symptoms, medical history, drug history, living environment, clinical examination, and pulmonary function tests. Since the age of asthma onset is self-reported, in order to ensure its accuracy, patients will be asked to describe the dyspnea, wheezing, and cough experienced during childhood or adolescence. In case of uncertainty, the time point of the earliest respiratory symptoms will be It is taken as the age at the onset of asthma symptoms; at the same time, the smoking status of all patients is also concerned, and they are divided into three groups: smokers, ex-smokers who have quit smoking for at least 1 month before the first visit, and life-long non-smokers. Smoker. Patients were judged to be atopic if they had one of self-reported allergens (pollen, mold, dust, animals, legumes, seafood, milk, and eggs), allergic rhinitis, and/or atopic dermatitis. Non-asthmatic volunteers who participated in annual health checks with normal lung function and no history of asthma were also enumerated for comparison.

data analysis

Mainly use two sample t-test (two sample t-test), Chi-square test (Chi-square test), Cohen's D effect size (Cohen's D effect size) and other methods to compare the demographics between asthmatic patients and control groups The biological variables (age, sex, body mass index, heterotopia, smoking habits, passive smoking at home and workplace) and biomonitoring data, if the detection value is lower than the detection limit, the detection limit Half of the value is used as the detection data. And for an easier overall understanding, the radar chart is used to present the mean value of each group. When distinguishing asthmatic patients from normal controls, a two-sample t-test was used and adjusted for false discovery rate (FDR). First, convert the lipid value in the body into Z-scores; generate receiver operating characteristic (ROC) curve and its area under curve (AUC) to distinguish asthma from COPD and ACOS; Further logistic regression analysis was performed with covariates including gender distribution, age, body mass index, smoking habits, passive smoking status in the workplace, and religious incense burning habits to calculate the adjusted AUC value; in addition, the Spearman rank correlation coefficient ( Spearman rank correlation coefficients, rS), assessed the correlation between lipid metabolites (LPE22:6) and cytokines IL-13, TGF-β, and oxidative stress markers (HEL). All the above analyzes were performed with SAS statistical software (version 9.4, SAS Institutes Inc., Cary, North Carolina, USA), and a p-value less than 0.05 was considered significant.

Embodiment 1, analyzing the lipid species in the subject's body

Sample Collection and Extraction of Lipids from Plasma

The subjects' blood samples were collected at the doctor's clinic, and peripheral whole blood (20 mL) was drawn by venipuncture and stored in blood collection tubes containing heparin. Plasma was separated by means of density gradient centrifugation (Lymphoprep, Oslo, Norway) at 3000 rpm for 10 minutes, and blood samples were immediately aliquoted and frozen in a -80°C freezer. Before lipid extraction, 15 µL of PLIS was aliquoted to every 30 µL of plasma and mixed well, then lipids in plasma were extracted using the method of Folch et al. For further analysis of ceramide and lysophosphatidic acid (LPA), 100 µL of plasma was mixed with 10 µL of CerlS and LPAIS, and extracted using the same method; after extraction, the organic layer containing lipids was transferred to to a new sample bottle, and the solvent was completely evaporated with a gentle nitrogen flow, and then filled with nitrogen gas in the sample bottle containing the lipid, sealed and stored at -80°C for analysis. The lipid precipitate must be dissolved in 1.5 mL of mobile phase A and mixed thoroughly prior to LC-MS/MS analysis.

Liquid Chromatography Tandem Mass Spectrometer (Liquid chromatography-tandem mass spectrometry , LC-MS/MS)

Phospholipids in plasma were analyzed using LC-MS/MS methods, and measurements for quality control of samples were performed simultaneously. When intending to prepare the internal standard solution of lipids, first prepare a solution containing dimyristoylphosphatidylcholine (DMPC), Dimethyl-2-(dimethylphosphino) ethylphosphine (DMPE), dipalmitoylphosphatidylinositol, , DPPI), SM 12:0, lysophosphatidylcholine (lysophosphatidylcholine, LPC) 14:0 and LPE 14:0 methanol solution, as the internal standard (internal standard, IS) solution (PLIS). Each 15 µL PLIS solution contains 3.3 µg of DMPC, 3.1 µg of DMPE, 1.2 µg of LPE 14:0, 1.8 µg of LPC 14:0, 1.6 µg of SM 12:0 and 2 µg of DPPI. And prepare the second Cer 17:0 (10 ng/µL) methanol solution and the third LPA 14:0 (0.5 ng/µL) methanol solution for the analysis of ceramide and LPA in plasma Ceramide and LPA internal standard (CerIS, LPAIS),. In addition, 1% (v/v) of 10% methanolic butylated hydroxytoluene was added to all IS solutions to prevent lipid oxidation.

The chromatographic method used was modified from the known hydrophilic interaction liquid chromatography (HILC) so as to be suitable for LC-MS/MS analysis of lipids. The mobile phase was delivered at a rate of 0.2 ml/min and chromatographically separated on an Ascentis® Express HILIC column (2.7 µm; 2.1 x 150 mm, catalog number: 53946-U, Supelco), and the effluent from the chromatography was reconstituted. The electrospray ion source was directed to a mass spectrometer equipped with dual ion-funnels (AmaZon X ion trap mass spectrometer; Bruker Daltonik). Due to operational limitations of the mass spectrometer, the same plasma lipid sample was analyzed with two different multiple reaction monitoring (MRM) chromatographic methods to comprehensively examine the lipid species detected in all lipid classes. lipid species. Further details on the LC-MS/MS system and the lipid classes and species monitored, as well as specific mass spectrometer standards used to identify and quantify lipid classes and species, are described below.

The LC-MS/MS system is composed of a Waters 2695 separation module and a Bruker AmaZon X ion trap mass spectrometer. The chromatographic analysis method used is improved from a known method, and the hydrophilic interaction liquid phase is used Lipids were separated using a chromatography (HILC) column (Supelco Ascentis® Express HILIC Column, 2.7 µm; 2.1 x 150 mm, catalog number: 53946-U). Mobile phase A is composed of 85% (v/v) acetonitrile (ACN), 10% methanol (MeOH) and 5% water, while mobile phase B is composed of 65% acetonitrile, 10% Methanol was composed of 25% water, and both mobile phases contained 0.04% (v/v) formic acid and 1 mM ammonium formate. To elute the major lipids, the mobile phase was delivered at a rate of 0.2 mL/min. During the eluting process, elute with 90% mobile phase A within 0-6 minutes, then decrease linearly to 70% within 6-10 minutes, and then decrease linearly within 10-16 minutes To 50%, keep at 50% within 16-20 minutes, return to 90% within 20-20.1 minutes, then maintain at 90% until the end, the whole chromatographic analysis process is about 30 minutes.

In addition, the same HILIC was also used to extract ceramide (Cer) and LPA, the composition of the mobile phase A used was the same as that of the above mobile phase A, and the mobile phase B was composed of 6.5% acetonitrile, 1% methanol and 95 % water, the concentrations of formic acid and ammonium formate in the two mobile phases are also the same as above, and the flow rate during the eluting process is also maintained at 0.2 mL/min. At the beginning of elution, elute with 90% mobile phase A, then decrease linearly to 70% within 0-2 minutes, and decrease linearly to 50% in 2-3 minutes, and then decrease to 50% in a linear manner within 3-3 minutes. Keep at 50% in 4 minutes, then drop to 20% in 4-4.1 minutes, and keep at 20% in 4.1-10 minutes, and finally return to 90% in 10-10.1 minutes, and keep at 90% % Until the end, the above-mentioned chromatographic analysis process for eluting ceramide and LPA took a total of 23 minutes.

The effluent from the chromatography column is directed to the electrospray needle of the mass spectrometer and dissociated in negative ion mode to avoid choline-containing lipid precursors due to systemic alkali metal cations The signal deviates. However, for the detection of lipids in phosphatidylethanolamine (PE), hydrolyzed phosphatidylethanolamine (LPE) class, the effluent will be dissociated in their respective chromatographic fragments in positive ion mode. The mass spectrometers are all operated in multiple reaction monitoring (MRM) mode. During the operation of LC-MS/MS, when the ion trap captures 100,000 ions or the capture time of 200 milliseconds, the mass spectrometer will perform an analysis scan. Through this setting , with at least 10 data points collected on average as the chromatographic peak elutes to ensure that a sufficient number of data points are collected to maintain chromatographic fidelity. The MS/MS mass isolation window (mass isolation window) for each monitored lipid species was set at 12C m/z ± 1 Da; 1 µg/mL delivered at 2 µL/min) to determine the dissociation and collision voltages of each lipid species, which can be individually fine-tuned if necessary, and to obtain reliable quantitative results and overcome the inherent operational limitations of ion trap mass spectrometers The limitation is that the same PL sample was analyzed twice using two different MS/MS methods (MS/MS method 1 and 2) to comprehensively include Cer and hydrolyzed phospholipids (LPA) except for the ones to be monitored. Other major PL species; in addition, Cer and LPA will be monitored using two other different MS/MS methods (MS/MS methods 3 and 4).

In order to distinguish and quantify specific lipid species, Xia and Jemal's MS/MS fragmentation protocol was used to analyze choline-containing lipid species, such as phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin ( SM), and the major lipid species were eluted with the mobile phase, formate addition ions (ie [M+45]-) were the only detectable precursor ions for these choline-containing lipids. In order to confirm and quantify the plasma phospholipids, [M-15]- (i.e. [M+ formate-60]-) fragment ion was selected as the fragment ion for quantification, and the fatty acid fragment ion was used to confirm the fat of the precursor In order to confirm phosphoinositide (PI), the m/z 241 fragment ion derived from the phosphoinositide head group was used as the fragment ion for confirmation, and the phosphoinositide precursor missing the sn-2 part It is used for quantification; the confirmation and quantification of phosphatidylethanolamine (PE) and hydrolyzed phosphatidylethanolamine (LPE) is based on the 141 DA neutral loss of the precursor ion in the positive ion mode, and can pass through the loss Precursor ion fragmentation of their respective aliphatic acid moieties or additional LC-MS/MS analysis in negative ion mode further validated the aliphatic acid composition of these ethanolamine-containing phospholipids (PL). The confirmation and quantification of the Cer species is accomplished by the MS3 tandem mass spectrometer of the precursor. The [MH ₂ O]+ product ion from the initial MS/MS reaction was chosen as a precursor for MS3 confirmation to monitor the m/z 264.5 fragment ion as the ceramide-labeled fragment; and the m/z 153.2 product ion as the hydrolysis Phospholipid (LPA) labeling for confirmation and quantification of LPA species. The amount of each lipid species is determined by the ratio of its quantitative fragments to the area under the extracted ion chromatogram (EIC) of its respective internal standard (IS), and multiple LC-MS/MS experiments are performed To ensure that the amount of IS in the plasma sample is sufficient so that the peak area ratio between the monitored lipid species and their respective IS falls within the range of 1:10 to 10:1, and through simultaneous quality control of each batch of plasma samples analysis to monitor the quality of the analysis.

Example 2. Evaluation of a set of eight lipids that can characterize asthma

In order to confirm the molecular characteristics of asthma, a liposomal analysis was performed in which a subset of asthmatic subjects ( N=365) and the control group (N=235), the sampling rates were 31% and 16%, respectively. In this data set, demographic comparisons were carried out by means of two sample t-test, Chi-square test, and Cohen's D effect size. The results showed that There were significant differences in both heterotopic subjects and lung function, with values around 0.0045 (=0.05/11, Bonferroni multiple comparison adjustment). Table 1. Demographic comparison between 365 asthmatic patients and 235 controls Asthma (n=365) Normal control (n=235) P-value* Cohen's D Standard ^& Gender, N (%) male 149 (40.82) 111 (47.23) 0.12 0.13 female 216 (59.18) 124 (52.77) Age (mean ± standard deviation) 55.96 (±14.79) 53.6 (±13.6) 0.05 0.16 Body mass index (mean ± standard deviation) 25.11 (±4.13) 24.27 (±3.42) 0.01 0.20 Smoking Habit, N (%) never smoked 282 (77.53) 181 (77.02) 0.15 0.01 ever smoked 56 (15.34) 28 (11.91) 0.10 smoking 26 (7.12) 26 (11.06) 0.15 Secondhand smoke from households, N (%) 227 (62.19) 158 (67.24) 0.21 0.10 Secondhand smoke from work, N (%) 150 (41.1) 78 (33.19) 0.05 0.16 Allergic rhinitis, N (%) 241 (66.03) 46 (19.57) <0.001 0.98 Atopic dermatitis, N (%) 12 (3.29) 7 (2.98) 0.83 0.02 FVC% (mean ± standard deviation) 86.28 (±18.46) 85.46 (±14.83) 0.55 0.04 FVE1% (mean ± standard deviation) 78.15 (±21.5) 87.62 (±14.99) <0.001 0.44 FVE1/ FVC % (mean ± standard deviation) 73.83 (±11.9) 85.09 (±9.74) 0.0009 0.95 *Two-sample t-test and chi-square test. ^& Cohen's D effect size; a value greater than 0.2 is considered to be different.

A phased approach was used to assess the composition of phospholipids (PL) and sphingolipids (SL) in vivo through a case-control study. In the discovery phase, a group of 48 types of phospholipids and sphingolipids were initially screened, 18 of which could significantly distinguish asthma (N=35) from controls (N=30), including Lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), hydrolyzed phosphatidylethanolamine (LPE), and sphingomyelin (SM) had a false discovery rate (FDR)-adjusted p value of less than 0.0001 (Data not shown). ; Among them, there are 8 kinds of lipid types, 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, SM16:0, showed significant differences in the validation phase analyzing an additional 94 asthmatic patients versus 100 controls. It is worth noting that, through Z-score analysis, it was shown that compared with the control group, the patterns of the 8 lipid composition differences were different in asthmatic patients. In asthmatic patients, LPE22:6, LPE20:4, SM16:0, PC18: The performance of 0/18:2 is more prominent than that of normal subjects; on the other hand, PE18:0/22:6, PE18:0/20:4, PE16:0/22:6, PE18 of normal subjects The expression of :0/18:2 was more obvious than that of asthmatic patients (as shown in Figure 1A).

Because asthma, COPD, and ACOS share common clinical features, plasma samples from 49 COPD patients and 52 ACOS patients were included for comparison. Z-score analysis showed that the 8 key lipid species can significantly distinguish asthma from COPD and ACOS (Figure 1B), including 7 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, PE 16:0/22:6, can distinguish asthma from COPD; and the three lipids Types, including SM 16:0, PC 18:0/18:2, and PE 18:0/18:2, can distinguish asthma from ACOS; in addition, when comparing COPD and ACOS, there are also significant differences in the five lipid types , including LPE 22:6, LPE 20:4, PC 18:0/18:2, PE 18:0/22:6, PE 18:0/20:4.

By a staged approach, this panel of 8 in vivo lipid species was shown to be a possible candidate molecule for characterizing asthma and distinguishing asthma from controls. The influence of lipid metabolism on asthma was also confirmed by integrating the characteristics of lipid in vivo (as a new endophenotype form) to identify a phenotypic subset of patients with asthma. In addition, the compositional profiles of phospholipids and sphingolipids found in the subject population further suggest selective liposomal reorganization in adult asthmatic patients.

Embodiment three, calculation 8 A receiver operating characteristic for the main analysis of lipid species (ROC) The curve and its area under the curve (AUC) , to be used from the control group, ACOS , COPD asthma patients

ROC curves were generated with in vivo lipid datasets, and AUC values for each lipid class were calculated. The AUC of each lipid class was adjusted according to its gender distribution, mean age, body mass index, smoking habits, passive smoking in the workplace, and religious incense burning habits. Adjusted AUC results showed that LPE 22:6, LPE 20:4, SM 16:0, PE 16:0/22:6 could distinguish asthma patients from controls, ACOS, COPD (Fig. 2A, 2B, respectively , 2C), at the same time, ACOS and COPD can also be distinguished (Figure 2D).

In the unadjusted ROC analysis, after selecting the cut-off value, three different group-wise comparisons were used to calculate the discriminant value. Table 2 summarizes the cutoff values used and their sensitivities and specificities for distinguishing asthma from controls, COPD, and ACOS. The results showed that the cut-off values of LPE 22:6 and LPE 20:4 showed high sensitivity (100% and 97.87%, respectively) and specificity (99% and 100%, respectively) in distinguishing asthma from controls; When distinguishing asthma from COPD, 7 kinds of phospholipids (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, PE 16:0/22:6), also showed high sensitivity (ranging from 80.85%-100%) and specificity (ranging from 86.17%-100%); When differentiating asthma from ACOS, a cutoff of 3 lipid classes (comprising SM 16:0, PC 18:0/18:2, PE 18:0/18:2) had the highest discriminatory power, showing high sensitivity (range fall in 86.15%-97.87%) and specificity (range falls in 89.36%-100%); in addition, 5 kinds of lipids used to distinguish COPD and ACOS (including LPE 22:6, LPE 20:4, PC 18: 0/18:2, PE 18:0/22:6, PE 18:0/20:4) with the highest sensitivity (range 98.08%-100%) and specificity (range 94.23%-100%). These results suggest that the 8 key lipids analyzed have great potential as differential markers for distinguishing asthma from COPD or ACOS, and distinguishing ACOS from COPD. Table 2. Select cut-off values from the ROC curves of 8 key phospholipids, and evaluate their sensitivity and specificity by ROC analysis Types of Phospholipids Asthma vs. control group Asthma vs. COPD Asthma vs. ACOS COPD vs. ACOS Cutoff value (µM) sensitivity specificity Cutoff value (µM) sensitivity specificity Cutoff value (µM) sensitivity specificity Cutoff value (µM) sensitivity specificity LPE 22:6 4.05 100 99 4.05 100 97.96 5.68 69.23 60.64 3.12 98.08 95.92 LPE 20:4 4.60 97.87 100 7.37 92.55 91.84 15 88.46 53.19 10.1 100 65.92 SM 16:0 321.43 82 71 182.13 100 100 256.23 97.87 100 100.88 83.67 69.23 PC 18:0/18:2 299.90 40.42 81 206.04 80.85 87.76 443.75 96.15 89.36 431.76 100 100 PE 18:0/22:6 146.79 57 77.66 178.65 97.96 86.17 72.8 68.09 73.08 132 100 94.23 PE 18:0/20:4 50.83 82 47.87 127.71 89.8 87.23 32.64 79.79 86.54 74 100 100 PE 16:0/22:6 71.35 89 64.89 163.81 83.67 91.49 77.99 32.98 82.69 106.63 89.8 94.23 PE 18:0/18:2 10.16 81 59.57 15.29 91.84 79.79 20.08 96.15 90.43 30.21 65.38 83.67

More specifically, compared with the control group, the ratio of LPE22:6/(PE18:0/22:6) or LPE22:6/(PE16:0/22:6) was significantly increased in asthmatic patients, Indicates that the Lands cycle is out of balance in asthmatic patients. In the follow-up blind validation of 89 subjects, including asthmatic patients (N=35), non-asthmatic individuals (N=30) and patients newly diagnosed with asthma (N=24), the detection and labeling The determination of the lipid level of the samples was done in a blinded manner, while the analysis of the results was not blinded. According to the cut-off ratio value of 0.028 for LPE22:6 to PE16:0/22:6, a total of 32 samples had ratio values below this cut-off ratio value, 29 of which were from the control group and 1 from a patient with newly diagnosed asthma (# 83) and 2 from patients with confirmed asthma (#56, #58), overall, the sensitivity and specificity were 97% and 96%, respectively (Fig. 3). Similarly, the cut-off ratio value of 0.023 for LPE22:6 to PE18:0/22:6 also had high sensitivity (96%) and specificity (95%; Figure 3), among patients with newly diagnosed asthma (#83) Interestingly, the healthy subject (#14) had an expected FEV1 of 81%, and after administration of bronchodilators It was 88%, and the ratios of LPE22:6/(PE16:0/22:6) and LPE22:6/(PE18:0/22:6) were 0.028 and 0.03, respectively.

Example 4 , LPE22:6 Can trigger a mast cell response

In order to explore the potential effect of LPE22:6 on regulating cell function, mast cells derived from mouse bone marrow were used as a model. The results showed that a significant release of β-hexosaminidase (β-hexosaminidase , a pre-formed granule medium) and leukotriene C4 (LTC4, a lipid medium) (see Figure 4A, 4B, respectively), and was accompanied by an increase in calcium influx from intracellular stores, rather than calcium Ions move (Figure 4C).

On the other hand, membrane perturbation caused by LPE22:6 could be alleviated by administration of bovine serum albumin (BSA) or human serum albumin (HSA) ( FIG. 5A ). Cells were treated for 10 min with or without BSA (1% or 4%) or HSA (1%), followed by FM1-43, a fluorescent stain that preferentially inserts into loose membranes , stimulated with 50 µM LPE 22:6 for 1 min. The final results showed that the addition of BSA could inhibit the membrane perturbation caused by LPE22:6, but the oxidized HAS could not inhibit LPE22:6 (Fig. 5B).

In addition, calcium ion mobilization ( FIG. 5C ) and degranulation ( FIG. 5D ) induced by LPE22:6 were significantly reduced by administration of BSA in a concentration-dependent manner. These results suggest that interfering with the effects of LPE22:6 on cellular responses through albumin can be used as a method to reduce its lipotoxicity.

In addition, the concentration of LPE22:6 is also correlated with the concentration of IL-13 and TGF-β in plasma (as shown in Figure 6A and 6B, r=0.287, p=0.029 and r=0.302, p=0.021), the oxidation rate in urine The sex marker HEL (as shown in Figure 6C, Spearman correlation, r=0.25, p=0.001) showed a positive correlation.

Based on the above results, a group includes 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 lipid species in vivo can be used to distinguish asthma patients from COPD patients from ACOS patients; The receiver operating characteristic analysis performed showed that their respective cut-off values could be used to distinguish asthma patients from COPD patients and ACOS patients; in addition, LPE22:6 in the lysophosphatidylethanolamine (LPE) category was special, and it was compared with The concentrations of oxidative stress markers Ne-(hexanoyl)-lysine (HEL), IL-13, and TGF-β1 in asthmatic patients are correlated, and can induce degranulation of mast cells and release of LTC4, but can be cured by administration of serum white The protein reverses the response of mast cells. Therefore, the panel of plasma phospholipids provided by the present invention can be used as a marker to characterize asthma and be useful for distinguishing asthma patients from other obstructive airway diseases.

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Figures 1A-1B are radar charts showing changes in lipid composition in a group of 8 species in asthmatic, control, ACOS and COPD patients. Figure 1A is a radar chart with z-scores showing the difference in lipid species composition in patients with asthma compared with controls; Next, differences in lipid species composition.

Figures 2A-2D are graphs showing receiver operating characteristic (ROC) curves for the eight key analyzed in vivo lipid classes in control, asthma, ACOS, and COPD patients. Figure 2A is a graph of 94 asthmatic patients and 100 controls, in which the area under curve (AUC) value has been adjusted according to age and religious incense habit; Figure 2B is a graph of 94 asthmatic patients and 49 COPD patients The patient's curve, in which the area under the curve has been adjusted according to gender, age, BMI, smoking habits, passive smoking at work and religious incense habits; Curve, where the area under the curve has been adjusted according to gender, age and smoking habits; Figure 2D is the curve of 52 ACOS patients and 49 COPD patients, where the area under the curve has been adjusted according to gender, age , BMI, smoking habits and religious incense habits were adjusted. In all graphs, the P values for the lipid assessments for the 8 focused analyzes were less than 0.0001.

Figure 3 is a bar graph showing the ratio of LPE22:6/(PE18:0/22:6) to LPE22:6/(PE16:0/22:6) in 89 subjects (labeled 1-89) blind test. Lipid composition was measured in physician-diagnosed asthma patients (N=35), non-asthmatic individuals (N=30), and newly diagnosed asthmatic subjects (new cases, N=24), and the results were based on non-asthmatic Blind way analysis. The cutoff ratio of LPE22:6 to PE16:0/22:6 is 0.028, and the cutoff ratio of LEP22:6 to PE18:0/22:6 is 0.023. *: Healthy subject (#14), with LPE22:6/(PE18:0/22:6), LPE22:6/(PE16:0/22:6) ratio values of 0.03 and 0.028, respectively; %: no For new asthma cases with a history of asthma, the ratios of LPE22:6/(PE18:0/22:6) and LPE22:6/(PE16:0/22:6) were 0.01 and 0.024, respectively; &: Recipients with asthma Subjects #56 and #58 had LPE22:6/(PE18:0/22:6) ratios of 0.029 and 0.028, and LPE22:6/(PE16:0/22:6) ratios of 0.023 and 0.018.

Figures 4A-4C show that LPE22:6 (LPE22:6-sn2) can induce the response of bone marrow-derived mast cells (BMMC) in mice (C57BL/6). In Figures 4A and 4B, LPE22:6-sn2 induced degranulation and LTC4 release in BMMC. The cells were treated with 10, 20, and 50 µM LPE22:6-sn2 for 30 minutes, and then the degranulation effect was detected by measuring β-hexosaminidase (β-hexosaminidase), and the concentration of LTC4 was measured by ELISA, where * indicates the relative Compared with the blank control group, p<0.05, ** indicates p<0.01 compared with the blank control group. As shown in Figure 4C, LPE22:6-sn2 induces calcium influx into BMMC. First co-culture BMMC with Ca ²⁺ indicator, Fluo-3-AM and Fura red-AM, and then add 10, 20, 50 µM LPE22:6-sn2 (dissolved in 0.2% BSA-PBS) stimulated the cells for the first 3 minutes, then washed with Tyrode's buffer containing calcium ions, and incubated in it. The experimental data are the data of three independent experiments.

Figures 5A-5D show that administration of bovine or human serum albumin (BSA or HSA) inhibits the functional effects of LPE22:6 on BMMC. BSA can inhibit the membrane perturbation (membrane perturbation) caused by LPE22:6 in a concentration-dependent manner (Fig. 5A). In addition, HAS can also inhibit the membrane perturbation caused by LPE22:6, but the oxidized HAS cannot inhibit the effect of LPE22:6 (Figure 5B); moreover, after administration of BSA, it can significantly reduce the effect of LPE22:6 The calcium ion mobilization (calcium mobilization) (Figure 5C) and degranulation (Figure 5D) were concentration-dependent. *** indicates p<0.001 compared with the blank control; ### indicates p<0.001 compared with LPE22:6, and the experimental data are the data of three independent experiments.

6A-6C show the linear regression analysis correlation between the concentration of LPE22:6 and the concentration of IL-13, TGF-β and HEL in asthmatic patients.

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Claims (5)

A method of identifying asthma in vitro using a panel of lipid species comprising: collecting a biological sample from a subject to assess lipid profile in the subject; operating a recipient of the lipid species from the panel The cut-off ratio value selected by the characteristic (ROC) curve is applied to the body lipid ratio value of the subject; and comparing the cut-off ratio value with the body lipid ratio value to identify whether the subject is asthma; wherein the lipid type in the group includes 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 sphingolipid (SM) 16:0 are used to distinguish asthmatic patients from ACOS and COPD patients. The method according to claim 1, wherein the biological sample comprises plasma, serum, sputum, bronchoalveolar lavage fluid and exhaled breath condensate. The method according to claim 1, wherein the lipid species in the group includes LPE22:6, LPE20:4, SM16:0, PE16:0/22:6, and is used to distinguish asthma patients, ACOS patients and COPD patients. The method according to claim 1, wherein the cut-off ratio is 0.028 of LPE22:6 to PE16:0/22:6. The method according to claim 1, wherein the cut-off ratio is 0.03 of LPE22:6 to PE18:0/22:6.

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