Biomarker, analysis method and application

Abstract

The biomarker, the analysis method and the application can be effectively used for early diagnosis, disease course monitoring and prognosis evaluation of asthma, are high in sensitivity, rapid, convenient and accurate and reliable in result, can be used for multiple times of monitoring in a clinical control period, a chronic duration period and an acute attack period of an asthma patient, and provide a basis for clinical decision making. Meanwhile, a certain basis is provided for subsequent fundamental research and clinical research, and the method has great application and research values.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and particularly to a biomarker, a method for analyzing this biomarker, and the application of this biomarker in the dynamic monitoring of the asthma course. Background Art

[0002] Bronchial asthma (asthma) is a common chronic respiratory disease, and its symptoms include recurrent wheezing, shortness of breath, chest tightness, and coughing, which significantly affect the daily life and work of patients. An acute asthma attack may lead to severe dyspnea and even endanger life. Through systematic asthma management, the frequency of symptom occurrence can be reduced, the severity of the condition can be alleviated, enabling patients to maintain a normal life and work state, thereby improving the quality of life.

[0003] Since asthma is a highly heterogeneous disease, there are significant differences in the etiology, pathological mechanisms, and treatment responses among different patients. Currently, the treatment and management of asthma mainly adopt a standardized "one-size-fits-all" method. Although it provides a basic treatment framework for most patients, it ignores individual differences, resulting in unsatisfactory treatment and control effects for some patients. Therefore, aiming at the large individual differences among asthma patients, developing personalized and precise asthma management strategies using advanced analysis methods is beneficial to achieving better disease control and improving the quality of life of patients.

[0004] Sphingolipids are a class of functional lipids and play an important role in the occurrence and development of inflammatory diseases (such as asthma). Measuring sphingolipids based on liquid chromatography-mass spectrometry (LC-MS) analysis technology can comprehensively understand the changes in sphingolipid levels and sphingolipid metabolism under different severities of the disease, and can be used to evaluate different states of the disease. Using sphingolipid biomarkers to assist in asthma management is beneficial to providing more objective and accurate disease monitoring means, assisting medical staff in distinguishing the severity of asthma, helping to formulate more individualized treatment and management plans, and improving the long-term prognosis of patients. Summary of the Invention

[0005] To overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide a biomarker that can be used for formulating an individualized treatment plan for asthma, for predicting, diagnosing, risk stratifying, monitoring, and prognosis of asthma, achieving the goal of chronic asthma management, with high sensitivity, fast and convenient, and accurate and reliable results.

[0006] The technical solution of the present invention is: This biomarker includes at least one of the following substances: Hexosylceramide d18:1 / 12:0, Hexosylceramide d18:1 / 14:0, Hexosylceramide d18:1 / 16:0, Hexosylceramide d18:1 / 20:0, Hexosylceramide d18:1 / 22:0, Hexosylceramide d18:1 / 24:0, Hexosylceramide d18:1 / 24:1, Hexosylceramide d18:1 / 26:1, Hexosylceramide d18:2 / 16:0, Hexosylceramide d18:2 / 20:0, Hexosylceramide d18:2 / 22:0, Hexosylceramide d18:2 / 24:0, Lactosylceramide d18:1 / 12:0, Lactosylceramide d18:1 / 16:0, Lactosylceramide d18:1 / 18:0, Lactosylceramide d18:1 / 20:0, Lactosylceramide d18:1 / 24:1, Lactosylceramide d18:1 / 26:0, 2-Hexosylceramide d18:2 / 16:0, Phosphorylated Ceramide d18:1 / 12:0, Phosphorylated Ceramide d18:1 / 14:0, Phosphorylated Ceramide d18:1 / 16:0, Phosphorylated Ceramide d18:1 / 18:0, Phosphorylated Ceramide d18:1 / 20:0, Phosphorylated Ceramide d18:1 / 22:0, Phosphorylated Ceramide d18:1 / 26:0, Ceramide-Type Sphingolipid d18:0 / 18:0, Ceramide-Type Sphingolipid d18:0 / 20:0, Ceramide-Type Sphingolipid d18:0 / 22:1, Ceramide-Type Sphingolipid d18:1 / 14:0, Ceramide-Type Sphingolipid d18:1 / 22:0, Ceramide-Type Sphingolipid d18:1 / 22:1, Ceramide-Type Sphingolipid d18:1 / 24:2, Ceramide-Type Sphingolipid d18:1 / 26:0, Ceramide-Type Sphingolipid d18:1 / 26:1, Ceramide-Type Sphingolipid d18:2 / 16:0, Ceramide-Type Sphingolipid d18:2 / 20:0, Ceramide-Type Sphingolipid d18:2 / 22:0, Ceramide-Type Sphingolipid d18:2 / 24:1.

[0007] The analysis method of the biomarker includes the following steps: (1) Sample preparation and pretreatment: Collect plasma samples from asthma patients in the control period and plasma samples from severe asthma patients, prepare the internal standard working solution, precisely pipette 50 μL of the plasma sample, add 10 μL of the internal standard working solution, add 140 μL of methanol solution to precipitate proteins, vortex for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and take the supernatant; (2) Perform chromatographic detection: Use a reverse-phase high-performance liquid chromatography column as the analytical chromatographic column; select a column length of 5 - 250 mm, mobile phase A is an aqueous solution, mobile phase B is a methanol solution, the mobile phase ratio is A + B = 100%, the analysis time is 5 - 50 min, and the elution program is gradient elution from 0 - 80% of mobile phase B to 60 - 100% of mobile phase B; the mobile phase additive is 0.1% - 1% formic acid and 0 - 10 mmol / L ammonium acetate; the flow rate is 0.1 - 1.0 mL / min; the column temperature is 4 - 50 °C; the injection volume is 1 - 20 μL; (3) Perform mass spectrometric detection: Use an electrospray ionization source, positive ion detection mode, the nebulizing gas flow rate is 1 - 5 L / min, the heating gas flow rate is 5 - 20 L / min, the interface temperature is 250 - 400 °C, the desolvation temperature is 500 - 600 °C, the DL temperature is 200 - 300 °C, the drying gas flow rate is 5 - 20 L / min, and use the multiple reaction monitoring (MRM) mode for quantitative analysis of the target sphingolipid metabolites; (4) Sphingolipid metabolite analysis: Comprehensively consider the t-test, fold change comparison, and ROC analysis results of sphingolipid metabolites between groups to screen the process management biomarkers for severe asthma.

[0008] The biomarkers and analysis method of the present invention can be effectively used for the early diagnosis, disease course monitoring, and prognosis evaluation of asthma, with high sensitivity, fast and convenient operation, and accurate and reliable results. It can be monitored multiple times during the clinical control period, chronic persistent period, and acute exacerbation period of asthma patients, providing a basis for clinical decision-making, and at the same time providing a certain foundation for subsequent basic research and clinical research, having great application and research value.

[0009] The application of the biomarker is also provided for the prediction, diagnosis, risk stratification, monitoring, and prognosis of bronchial asthma.

[0010] The application of the biomarker is also provided for the prediction, diagnosis, risk stratification, monitoring, and prognosis of cough variant asthma, chest tightness variant asthma, occult asthma, and drug-induced asthma.

[0011] The application of the biomarker is also provided for a chip, test strip, or kit for asthma management, which can obtain the level of sphingolipid biomarkers in the biological sample of the test subject and be used to judge the disease status of asthma in the test subject, thereby achieving the goal of chronic asthma management. Description of the Drawings

[0012] Figure 1 It is shown as a schematic diagram of the fragmentation rule of the biomarker.

[0013] Figure 2 It is shown as the MRM map of glycosphingolipid metabolites.

[0014] Figure 3 Shown as the MRM map of phosphorylated sphingolipid metabolites.

[0015] Figure 4 Shown as the MRM map of ceramide sphingolipid metabolites.

[0016] Figure 5 Shown as the flow chart of biomarker-assisted asthma process management.

[0017] Figure 6 Shown as the bar chart comparing the between-group differences in the levels of glycosphingolipid metabolites in the plasma of asthma patients in the control period and severe asthma patients.

[0018] Figure 7 Shown as the ROC curve comparing the between-group differences in the levels of glycosphingolipid metabolites in the plasma of asthma patients in the control period and severe asthma patients.

[0019] Figure 8 Shown as the bar chart comparing the between-group differences in the levels of phosphorylated sphingolipid metabolites in the plasma of asthma patients in the control period and severe asthma patients.

[0020] Figure 9 Shown as the ROC curve comparing the between-group differences in the levels of phosphorylated sphingolipid metabolites in the plasma of asthma patients in the control period and severe asthma patients.

[0021] Figure 10 Shown as the bar chart comparing the between-group differences in the levels of ceramide metabolites in the plasma of asthma patients in the control period and severe asthma patients.

[0022] Figure 11 Shown as the ROC curve comparing the between-group differences in the levels of ceramide metabolites in the plasma of asthma patients in the control period and severe asthma patients.

[0023] Figure 12 Shown as the structural formula of the metabolites involved in this specification. Detailed implementation manners

[0024] This biomarker comprises at least one of the following substances: hexosylated sphingolipid d18:1 / 12:0, hexosylated sphingolipid d18:1 / 14:0, hexosylated sphingolipid d18:1 / 16:0, hexosylated sphingolipid d18:1 / 20:0, hexosylated sphingolipid d18:1 / 22:0, hexosylated sphingolipid d18:1 / 24:0, hexosylated sphingolipid d18:1 / 24:1, hexosylated sphingolipid d18:1 / 26:1, hexosylated sphingolipid d18:2 / 16:0, hexosylated sphingolipid d18:2 / 20:0, hexosylated sphingolipid d18:2 / 22:0, hexosylated sphingolipid d18:2 / 24:0, lactosylated sphingolipid d18:1 / 12:0, lactosylated sphingolipid d18:1 / 16:0, lactosylated sphingolipid d18:1 / 18:0, lactosylated sphingolipid d18:1 / 20:0, lactosylated sphingolipid d18:1 / 24:1, lactosylated sphingolipid d18:1 / 26:0, 2-hexosylated sphingolipid d18:2 / 16:0, phosphorylated sphingolipid d18:1 / 12:0, phosphorylated sphingolipid d18:1 / 14:0, phosphorylated sphingolipid d18:1 / 16:0, phosphorylated sphingolipid d18:1 / 18:0, phosphorylated sphingolipid d18:1 / 20:0, phosphorylated sphingolipid d18:1 / 22:0, phosphorylated sphingolipid d18:1 / 26:0, ceramide sphingolipid d18:0 / 18:0, ceramide sphingolipid d18:0 / 20:0, ceramide sphingolipid d18:0 / 22:1, ceramide sphingolipid d18:1 / 14:0, ceramide sphingolipid d18:1 / 22:0, ceramide sphingolipid d18:1 / 22:1, ceramide sphingolipid d18:1 / 24:2, ceramide sphingolipid d18:1 / 26:0, ceramide sphingolipid d18:1 / 26:1, ceramide sphingolipid d18:2 / 16:0, ceramide sphingolipid d18:2 / 20:0, ceramide sphingolipid d18:2 / 22:0, ceramide sphingolipid d18:2 / 24:1.

[0025] The analysis method of the biomarker comprises the following steps: (1) Sample preparation and pretreatment: Collect plasma samples of asthma patients in the control period and plasma samples of severe asthma patients, prepare the internal standard working solution, precisely pipette 50 μL of the plasma sample, add 10 μL of the internal standard working solution, add 140 μL of the methanol solution to precipitate proteins, vortex for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and take the supernatant; (2) Perform chromatographic detection: Use a reverse-phase high-performance liquid chromatography column as the analytical chromatographic column; select a column length of 5 - 250 mm, mobile phase A is an aqueous solution, mobile phase B is a methanol solution, mobile phase ratio: A + B = 100%, the analysis time is 5 - 50 min, and the elution program is gradient elution from 0 - 80% of mobile phase B to 60 - 100% of mobile phase B; the mobile phase additive is 0.1% - 1% formic acid and 0 - 10 mmol / L ammonium acetate; the flow rate is 0.1 - 1.0 mL / min; the column temperature is 4 - 50 °C; the injection volume is 1 - 20 μL; (3) Perform mass spectrometric detection: Use an electrospray ionization source, positive ion detection mode, nebulizing gas flow rate of 1 - 5 L / min, heating gas flow rate of 5 - 20 L / min, interface temperature of 250 - 400 °C, desolvation temperature of 500 - 600 °C, DL temperature of 200 - 300 °C, drying gas flow rate of 5 - 20 L / min, and use the multiple reaction monitoring (MRM) mode for quantitative analysis of the target sphingolipid metabolites; (4) Sphingolipid metabolite analysis: Comprehensively consider the t-test, fold change comparison, and ROC analysis results of sphingolipid metabolites between groups to screen the process management biomarkers for severe asthma.

[0026] The biomarkers and analysis methods of the present invention can be effectively used for the early diagnosis, disease course monitoring, and prognosis evaluation of asthma, with high sensitivity, rapidity, convenience, and accurate and reliable results. They can be monitored multiple times during the clinical control period, chronic persistent period, and acute exacerbation period of asthma patients, providing a basis for clinical decision-making, and at the same time providing a certain basis for subsequent basic research and clinical research, having great application and research value.

[0027] Preferably, in the step (2), for the reverse-phase high-performance liquid chromatography column, the column length is preferably 30 - 200 mm, mobile phase A is an aqueous solution, mobile phase B is a methanol solution, mobile phase ratio: A + B = 100%, the analysis time is 5 - 20 min, and the elution program is gradient elution from 0 - 80% of mobile phase B to 80 - 100% of mobile phase B; the mobile phase additive is 0.1% - 0.5% formic acid and 0 - 1 mmol / L ammonium acetate, the flow rate is 0.1 - 0.8 mL / min, the column temperature is 10 - 50 °C, and the injection volume is 1 - 10 μL.

[0028] Preferably, in the step (2), for the reversed-phase high-performance liquid chromatography column, the column length is preferably 30 - 150 mm, the filler is octadecylsilyl-bonded silica gel, mobile phase A is an aqueous solution, mobile phase B is a methanol solution, the proportion of the mobile phases: A + B = 100%, the analysis time is 5 - 15 min, the elution program is gradient elution from 50 - 80% of mobile phase B to 80 - 100% of mobile phase B, the mobile phase additives are 0.1% formic acid and 1 mmol / L ammonium acetate, the flow rate is 0.2 - 0.4 mL / min, the column temperature: 25 - 50 °C, and the injection volume is 5 μL.

[0029] Preferably, in the step (2), the reversed-phase high-performance liquid chromatography column is Agilent Poroshell 120 EC-C18, mobile phase A is an aqueous solution, mobile phase B is a methanol solution, the proportion of the mobile phases: A + B = 100%, the analysis time is 15 min, the elution program is gradient elution from 70% of mobile phase B to 100% of mobile phase B, the mobile phase additives are 0.1% formic acid and 1 mmol / L ammonium acetate, the flow rate is 0.3 mL / min, the column temperature: 40 °C, and the injection volume is 5 μL.

[0030] The application of the biomarker is also provided for the prediction, diagnosis, risk stratification, monitoring, and prognosis of bronchial asthma.

[0031] The application of the biomarker is also provided for the prediction, diagnosis, risk stratification, monitoring, and prognosis of cough variant asthma, chest tightness variant asthma, occult asthma, and drug-induced asthma.

[0032] The application of the biomarker is also provided for a chip, test strip, or kit for asthma management, to obtain the level of the sphingolipid biomarker in a biological sample of a test subject and use it to judge the disease condition of asthma in the test subject, so as to achieve the goal of chronic asthma management.

[0033] Preferably, the biological sample is plasma, serum, whole blood, whole blood dried blood spot, dried plasma spot, urine, sputum, or exhaled breath condensate.

[0034] Preferably, the pretreatment method of the biological sample before detection is as follows: aspirate 15 - 50 μL of human whole blood or plasma and drop it onto a dried blood spot collection card, dry it at room temperature for 1 - 4 hours to obtain a dried blood spot or dried plasma spot sample; use a punch to cut one or more discs with a diameter of 3 - 8 mm from the dried blood spot; add the dried blood spot or dried plasma spot disc to a solid-phase extraction cartridge with the function of removing phospholipids / proteins, and elute it with an organic reagent containing 5% - 50% water.

[0035] The following will make a detailed description of the embodiments of the present invention.

[0036] Example 1: Glycosphingolipidomics-assisted asthma disease management.

[0037] 1 Sample source and grouping information After approval by the Ethics Committee of China-Japan Friendship Hospital, 91 plasma samples of asthma patients in the control period were collected in the outpatient department, and 78 plasma samples of severe asthma patients were collected in the inpatient department. All participants were from China-Japan Friendship Hospital. All asthma patients were diagnosed with bronchial asthma after clinical evaluation according to the "Guidelines for the Prevention and Treatment of Bronchial Asthma (2020 Edition)", and those with a history of respiratory diseases such as pulmonary tuberculosis, interstitial pulmonary fibrosis, and chronic obstructive pulmonary disease were excluded. The plasma was collected in the early morning on an empty stomach. All samples were stored in a -80 °C refrigerator for later use.

[0038] Table 1 Sample grouping information

[0039] 2 Experimental instruments and materials 2.1 Experimental instruments: Shimadzu LC-MS 8040CL liquid chromatography-mass spectrometry system.

[0040] 2.2 Experimental materials: Methanol (mass spectrometry grade, batch number: 222609) was purchased from Fisher Company, chloroform (chromatography grade, batch number: 015167) was purchased from Beijing Maida Technology Co., Ltd., isopropanol (chromatography grade, batch number: L190U32) was purchased from Beijing J&K Scientific Co., Ltd., ammonium acetate (mass spectrometry grade, batch number: MKCJ6670) was purchased from Sigma Company, formic acid (mass spectrometry grade, batch number: 214911) was purchased from Sigma Company, pure water (batch number / Lot#: 522022TW, 320420TJ) was purchased from Hangzhou Wahaha Company, and C16 ceramide-d7 reference standard Cer(d18:1-d7 / 16:0) (product number: 1ST005012D7, batch number: FS1640915) was purchased from Tianjin Aladdin Industrial Corporation.

[0041] 3 Experimental methods 3.1 Analytical method 3.1.1 Chromatographic conditions Chromatographic column: Agilent Poroshell 120 EC-C18 (2.1×50 mm 1.9 μm), mobile phase A: water (containing 0.1% formic acid, 1 mmol / L ammonium acetate), mobile phase B: methanol (containing 0.1% formic acid, 1 mmol / L ammonium acetate), mobile phase gradient is shown in Table 2, injection volume: 5 μL, column temperature: 40 °C.

[0042] Table 2 Mobile phase gradient elution program

[0043] 3.1.2 Mass spectrometry conditions Ion source: DUIS-8040; Detection mode: positive ion mode; Ion pairs and related parameter settings are shown in Table 3; Interface: ESI, nebulizing gas flow rate (N): 3 L / min, heating gas flow rate: 10 L / min, interface temperature: 300 °C, desolvation temperature: 526 °C, DL temperature: 250 °C, heating block temperature: 400 °C, drying gas flow rate: 10 L / min.

[0044] Using the multiple reaction monitoring (MRM) mode, according to the mass spectrometry fragmentation rules of glycosphingolipid metabolites ( Figure 1 ), 27 glycosphingolipid metabolites were predicted. The retention times and related mass spectrometry parameters of each sphingolipid metabolite are shown in Table 3. The MRM chromatograms of glycosphingolipid metabolites in the plasma of asthma patients are shown in Figure 2 .

[0045] Correspondence between compound names and abbreviations: Hexosylceramide d18:1 / 12:0: HexCer(d18:1 / 12:0), Hexosylceramide d18:1 / 14:0: HexCer(d18:1 / 14:0), Hexosylceramide d18:1 / 16:0: HexCer(d18:1 / 16:0), Hexosylceramide d18:1 / 18:0: HexCer(d18:1 / 18:0), Hexosylceramide d18:1 / 18:1: HexCer(d18:1 / 18:1), Hexosylceramide d18:1 / 20:0: HexCer(d18:1 / 20:0), Hexosylceramide d18:1 / 20:1: HexCer(d18:1 / 20:1), Hexosylceramide d18:1 / 22:0: HexCer(d18:1 / 22:0), Hexosylceramide d18:1 / 22:1: HexCer(d18:1 / 22:1), Hexosylceramide d18:1 / 24:0: HexCer(d18:1 / 24:0), Hexosylceramide d18:1 / 24:1: HexCer(d18:1 / 24:1), Hexosylceramide d18:1 / 26:0: HexCer(d18:1 / 26:0), Hexosylceramide d18:1 / 26:1: HexCer(d18:1 / 26:1), Hexosylceramide d18:2 / 16:0: HexCer(d18:2 / 16:0), Hexosylceramide d18:2 / 20:0: HexCer(d18:2 / 20:0), Hexosylceramide d18:2 / 22:0: HexCer(d18:2 / 22:0), Hexosylceramide d18:2 / 24:0: HexCer(d18:2 / 24:0), Dihexosylceramide d18:2 / 16:0: Hex2Cer(d18:2 / 16:0), Sulfated Hexosylceramide d18:1 / 16:0(OH): SHexCer(d18:1 / 16:0(OH)), Lactosylceramide d18:1 / 12:0: LacCer(d18:1 / 12:0), Lactosylceramide d18:1 / 16:0: LacCer(d18:1 / 16:0), Lactosylceramide d18:1 / 18:0: LacCer(d18:1 / 18:0), Lactosylceramide d18:1 / 20:0: LacCer(d18:1 / 20:0), Lactosylceramide d18:1 / 24:0: LacCer(d18:1 / 24:0), Lactosylceramide d18:1 / 24:1: LacCer(d18:1 / 24:1), Lactosylceramide d18:1 / 26:0: LacCer (d18:1 / 26:0).

[0046] Table 3 Retention Times and Related Mass Spectrometry Parameters of Glycosylated Sphingolipid Metabolites

[0047] 3.2 Preparation Method of Internal Standard Working Solution Weigh 1 mg of ceramide internal standard Cer(d18:1-d7 / 16:0) and place it in a glass vial. Add 0.5 mL of methanol-chloroform (1:1, v / v), and vortex and ultrasonicate until the sample is dissolved to obtain a single-standard stock solution of Cer(d18:1-d7 / 16:0) at 2 mg / mL. Seal the vial mouth with a sealing film to prevent solvent evaporation. Pipette 1 μL of Cer(d18:1-d7 / 16:0) with a concentration of 2 mg / mL into a 10 mL volumetric flask, and add isopropanol to the scale to obtain a working solution of Cer(d18:1-d7 / 16:0) at a concentration of 0.1 μg / mL.

[0048] 3.3 Sample Pretreatment Method Precisely pipette 50 μL of plasma, add 10 μL of the internal standard working solution, add 140 μL of methanol solution to precipitate proteins, vortex for 10 min, centrifuge at 12000 rpm for 10 min, and take the supernatant for injection analysis.

[0049] 3.4 Data Analysis Apply the Shimadzu Labsolution series workstation to perform peak extraction and integration on the target sphingolipid metabolites and internal standards, and use the peak area ratio of the target analyte to the internal standard for subsequent data processing. In this study, the sensitivity and specificity of sphingolipid metabolites in diagnosing acute asthma were comprehensively investigated through the receiver operating characteristic curve (ROC curve) analysis. In large-scale data analysis, if the area under the ROC curve is greater than 0.7, it can be considered that the index has good diagnostic ability; in the case of progressive significance p < 0.05, if the area under the ROC curve is greater than 0.65, it can also be considered that the index has good diagnostic ability, and it is defined as a candidate biomarker. The ROC curve and independent sample t-test were calculated and plotted using the Graphpad Prism 10.2.3 statistical analysis software.

[0050] 4. Analysis of Experimental Results Analyze the determination results of glycosylated sphingolipid metabolites in plasma samples of asthma patients in the control period and severe asthma patients. Considering the t-test of glycosylated sphingolipid metabolites between groups, the comparison of change multiples, and the ROC analysis results, screen the process management biomarkers for severe asthma. The inter-group difference comparison of glycosylated sphingolipid metabolite levels in plasma of asthma patients in the control period and severe asthma patients is shown in Table 4 below and Figure 4 、 Figure 5, HexCer(d16:1 / 20:0) (AUC = 0.9977, p < 0.0001), HexCer(d16:1 / 24:0) (AUC = 0.7343, p < 0.0001), HexCer(d18:1 / 12:0) (AUC = 0.7857, p < 0.0001), HexCer(d18:1 / 14:0) (AUC = 0.8636, p < 0.0001), HexCer(d18:1 / 16:0)(AUC = 0.8436, p < 0.0001), HexCer(d18:1 / 20:0) (AUC = 0.8316, p < 0.0001), HexCer(d18:1 / 22:0) (AUC = 0.8677, p < 0.0001), HexCer(d18:1 / 24:0) (AUC = 0.7209, p < 0.0001), HexCer(d18:1 / 24:1) (AUC = 0.8805, p < 0.0001), HexCer(d18:1 / 26:1) (AUC = 0.9200, p < 0.0001), HexCer(d18:2 / 16:0) (AUC = 0.8284, p < 0.0001), HexCer(d18:2 / 20:0) (AUC = 0.8225, p < 0.0001), HexCer(d18:2 / 22:0) (AUC = 0.7743, p < 0.0001), HexCer(d18:2 / 24:0) (AUC = 0.7574, p < 0.0001), LacCer(d18:1 / 12:0) (AUC = 0.7867, p < 0.0001), LacCer(d18:1 / 16:0)(AUC = 0.9039, p < 0.0001), LacCer(d18:1 / 18:0) (AUC = 0.7884, p < 0.0001), LacCer(d18:1 / 20:0) (AUC = 0.7072, p < 0.0001), LacCer(d18:1 / 24:1) (AUC = 0.6744, p < 0.0001), LacCer(d18:1 / 26:0) (AUC = 0.7003, p < 0.0001), Hex2Cer(d18:2 / 16:0) (AUC = 0.8163, p < 0.0001) have good diagnostic discrimination ability and can be used for the process management of asthma.

[0051] Table 4 Comparison of between-group differences in the levels of glycosphingolipid metabolites in plasma of asthma patients in the control period and severe asthma patients

[0052] Example 2: Phosphorylated Sphingolipidomics Assisted Asthma Disease Management.

[0053] 1 Sample Source and Grouping Information: The same as in Example 1.

[0054] 2 Experimental Instruments: The same as in Example 1.

[0055] 3 Experimental Methods 3.1 Analytical Method 3.1.1 Chromatographic Conditions: The same as in Example 1.

[0056] 3.1.2 Mass Spectrometry Conditions Ion Source: DUIS-8050; Detection Mode: Positive Ion Mode; Ion Pairs and Related Parameter Settings are shown in Table 5; Interface: ESI, Nebulizing Gas Flow Rate (N): 3 L / min, Heating Gas Flow Rate: 10 L / min, Interface Temperature: 300 °C, Desolvation Temperature: 526 °C, DL Temperature: 250 °C, Heating Block Temperature: 400 °C, Drying Gas Flow Rate: 10 L / min.

[0057] The multiple reaction monitoring (MRM) mode was used to predict 11 phosphorylated sphingolipid metabolites according to the mass spectrometry fragmentation rules of phosphorylated sphingolipid metabolites ( Figure 2 ). The retention times and related mass spectrometry parameters of each sphingolipid metabolite are shown in Table 5. The MRM chromatogram of phosphorylated sphingolipid metabolites in the plasma of asthma patients is shown in Figure 3 .

[0058] Correspondence between Compound Names and Abbreviations: Phosphorylated Sphingolipid d18:0 / 16:0: CerP (d18:0 / 16:0), Phosphorylated Sphingolipid d18:0 / 18:0: CerP (d18:0 / 18:0), Phosphorylated Sphingolipid d18:1 / 12:0: CerP (d18:1 / 12:0), Phosphorylated Sphingolipid d18:1 / 14:0: CerP(d18:1 / 14:0), Phosphorylated Sphingolipid d18:1 / 14:1: CerP(d18:1 / 14:1), Phosphorylated Sphingolipid d18:1 / 16:0: CerP(d18:1 / 16:0), Phosphorylated Sphingolipid d18:1 / 18:0: CerP(d18:1 / 18:0), Phosphorylated Sphingolipid d18:1 / 20:0: CerP(d18:1 / 20:0), Phosphorylated Sphingolipid d18:1 / 22:0: CerP(d18:1 / 22:0), Phosphorylated Sphingolipid d18:1 / 26:0: CerP(d18:1 / 26:0), Sphingosine-1-phosphate.

[0059] Table 5 Retention Times and Related Mass Spectrometry Parameters of Phosphorylated Sphingolipid Metabolites

[0060] 3.2 Preparation method of internal standard working solution: The same as in Example 1.

[0061] 3.3 Sample pretreatment method: The same as in Example 1.

[0062] 3.4 Data analysis: The same as in Example 1.

[0063] 4. Analysis of experimental results The determination results of phosphorylated sphingolipid metabolites in plasma samples of asthma patients in the control period and severe asthma patients were analyzed. Considering the t-test, fold change comparison and ROC analysis results of phosphorylated sphingolipid metabolites between groups, biomarkers for the process management of severe asthma were screened. The between-group differences in the levels of phosphorylated sphingolipid metabolites in the plasma of asthma patients in the control period and severe asthma patients are shown in Table 6 and Appendix Figure 6 、 Figure 7 , CerP(d18:1 / 12:0) (AUC = 0.8145, p < 0.0001), CerP(d18:1 / 14:0) (AUC = 0.7653, p < 0.0001), CerP(d18:1 / 14:1) (AUC = 0.8408, p < 0.0001), CerP(d18:1 / 16:0) (AUC = 0.6629, p = 0.0011), CerP(d18:1 / 18:0) (AUC = 0.8590, p < 0.0001), CerP(d18:1 / 20:0) (AUC = 0.8829, p < 0.0001), CerP(d18:1 / 22:0) (AUC = 0.9424, p < 0.0001), CerP(d18:1 / 26:0) (AUC = 0.8321, p < 0.0001), Sphingosine1-phosphate(AUC = 0.7981, p < 0.0001) have good diagnostic discrimination ability and can be used for the process management of asthma.

[0064] Table 6 Between-Group Difference Comparison of Phosphorylated Sphingolipid Metabolite Levels in Plasma of Asthma Patients in the Control Period and Severe Asthma Patients

[0065] Example 3: Ceramidomics-Assisted Asthma Disease Management.

[0066] 1 Sample source and grouping information: The same as in Example 1.

[0067] 2 Experimental instruments: The same as in Example 1.

[0068] 3 Experimental methods 3.1 Analytical methods 3.1.1 Chromatographic conditions: the same as in Example 1.

[0069] 3.1.2 Mass spectrometry conditions Ion source: DUIS-8050; Detection mode: positive ion mode; Ion pairs and related parameter settings are shown in Table 7; Interface: ESI, nebulizing gas flow rate (N): 3 L / min, heating gas flow rate: 10 L / min, interface temperature: 300 °C, desolvation temperature: 526 °C, DL temperature: 250 °C, heating block temperature: 400 °C, drying gas flow rate: 10 L / min.

[0070] The multiple reaction monitoring (MRM) mode was used to predict 24 ceramide sphingolipid metabolites according to the mass spectrometry fragmentation rules of ceramide sphingolipid metabolites ( Figure 1 ). The retention times and related mass spectrometry parameters of each sphingolipid metabolite are shown in Table 7. The MRM chromatogram of ceramide sphingolipid metabolites in the plasma of asthma patients is shown in Figure 4 .

[0071] Correspondence between compound names and abbreviations: Ceramide sphingolipids d18:0 / 18:0: Cer(d18:0 / 18:0), Ceramide sphingolipids d18:0 / 20:0: Cer(d18:0 / 20:0), Ceramide sphingolipids d18:0 / 22:0: Cer(d18:0 / 22:0), Ceramide sphingolipids d18:0 / 22:1: Cer(d18:0 / 22:1), Ceramide sphingolipids d18:0 / 24:0: Cer(d18:0 / 24:0), Ceramide sphingolipids d18:0 / 24:1: Cer(d18:0 / 24:1), Ceramide sphingolipids d18:1 / 14:0: Cer(d18:1 / 14:0), Ceramide sphingolipids d18:1 / 16:0: Cer(d18:1 / 16:0), Ceramide sphingolipids d18:1 / 18:0: Cer(d18:1 / 18:0), Ceramide sphingolipids d18:1 / 20:0: Cer(d18:1 / 20:0), Ceramide sphingolipids d18:1 / 22:0: Cer(d18:1 / 22:0), Ceramide sphingolipids d18:1 / 22:1: Cer(d18:1 / 22:1), Ceramide sphingolipids d18:1 / 24:0: Cer(d18:1 / 24:0), Ceramide sphingolipids d18:1 / 24:1: Cer(d18:1 / 24:1), Ceramide sphingolipids d18:1 / 24:2: Cer(d18:1 / 24:2), Ceramide sphingolipids d18:1 / 26:0: Cer(d18:1 / 26:0), Ceramide sphingolipids d18:1 / 26:1: Cer(d18:1 / 26:1), Ceramide sphingolipids d18:2 / 16:0: Cer(d18:2 / 16:0), Ceramide sphingolipids d18:2 / 20:0: Cer(d18:2 / 20:0), Ceramide sphingolipids d18:2 / 22:0: Cer(d18:2 / 22:0), Ceramide sphingolipids d18:2 / 24:0: Cer(d18:2 / 24:0), Ceramide sphingolipids d18:2 / 24:1: Cer(d18:2 / 24:1), Dihydrosphingosine: Sphinganine, Sphingosine: Sphingosine.

[0072] Table 7 Retention times and related mass spectrometry parameters of ceramide sphingolipid metabolites

[0073] 3.2 Preparation method of internal standard working solution: The same as in Example 1.

[0074] 3.3 Sample pretreatment method: The same as in Example 1.

[0075] 3.4 Data analysis: The same as in Example 1.

[0076] 4. Analysis of experimental results The measurement results of ceramide sphingolipid metabolites in plasma samples of patients with controlled asthma and severe asthma were analyzed. Considering the t-tests of ceramide sphingolipid metabolites between groups, the comparison of fold changes, and the results of ROC analysis, biomarkers for the process management of severe asthma were screened. The inter-group differences in the levels of ceramide sphingolipid metabolites in the plasma of patients with controlled asthma and severe asthma are shown in Table 8 below and Appendix Figure 8 、 Figure 9 , Cer(d18:0 / 18:0) (AUC = 0.7884, p < 0.0001), Cer(d18:0 / 20:0) (AUC = 0.7902, p < 0.0001), Cer(d18:0 / 22:1) (AUC = 0.7016, p < 0.0001), Cer(d18:1 / 14:0) (AUC = 0.7695, p < 0.0001), Cer(d18:1 / 16:0) (AUC = 0.8649, p < 0.0001), Cer(d18:1 / 18:0) (AUC = 0.8104, p < 0.0001), Cer(d18:1 / 20:0) (AUC = 0.8601, p < 0.0001), Cer(d18:1 / 22:0) (AUC = 0.7041, p = 0.0001), Cer(d18:1 / 22:1) (AUC = 0.8543, p < 0.0001), Cer(d18:1 / 24:1) (AUC = 0.8633, p < 0.0001), Cer(d18:1 / 24:2) (AUC = 0.9751, p < 0.0001), Cer(d18:1 / 26:0) (AUC = 0.7277, p < 0.0001), Cer(d18:1 / 26:1) (AUC = 0.9343, p < 0.0001), Cer(d18:2 / 16:0) (AUC = 0.8329, p < 0.0001), Cer(d18:2 / 20:0) (AUC = 0.8170, p < 0.0001), Cer(d18:2 / 22:0) (AUC = 0.7203, p = 0.0006), Cer(d18:2 / 24:1) (AUC = 0.8252, p < 0.0001) have good diagnostic discrimination ability and can be used for the process management of asthma.

[0077] Table 8 Comparison of inter-group differences in the levels of ceramide sphingolipid metabolites in the plasma of patients with controlled asthma and severe asthma

[0078] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A biomarker characterized by: At least one of the following substances is included: hexosylated sphingolipid d18:1 / 12:0, hexosylated sphingolipid d18:1 / 14:0, hexosylated sphingolipid d18:1 / 16:0, hexosylated sphingolipid d18:1 / 20:0, hexosylated sphingolipid d18:1 / 22:0, hexosylated sphingolipid d18:1 / 24:0, hexosylated sphingolipid d18:1 / 24:1, hexosylated sphingolipid d18:1 / 26:1, hexosylated sphingolipid d18:2 / 16:0, hexosylated sphingolipid lipid d18:2 / 20:0, hexosylated sphingolipid d18:2 / 22:0, hexosylated sphingolipid d18:2 / 24:0, lactosylated sphingolipid d18:1 / 12:0, lactosylated sphingolipid d18:1 / 16:0, lactosylated sphingolipid d18:1 / 18:0, lactosylated sphingolipid d18:1 / 20:0, lactosylated sphingolipid d18:1 / 24:1, lactosylated sphingolipid d18:1 / 26:0, dihexosylated sphingolipid d18:2 / 16:0, phosphorylated sphingolipid d18:1 / 12:0, phosphorylated sphingolipids d18:1 / 14:0, phosphorylated sphingolipids d18:1 / 16:0, phosphorylated sphingolipids d18:1 / 18:0, phosphorylated sphingolipids d18:1 / 20:0, phosphorylated sphingolipids d18:1 / 22:0, phosphorylated sphingolipids d18:1 / 26:0, ceramide sphingolipids d18:0 / 18:0, ceramide sphingolipids d18:0 / 20:0, ceramide sphingolipids d18:0 / 22:1, ceramide sphingolipids d18 :1 / 14:0, ceramide sphingolipids d18:1 / 22:0, ceramide sphingolipids d18:1 / 22:1, ceramide sphingolipids d18:1 / 24:2, ceramide sphingolipids d18:1 / 26:0, ceramide sphingolipids d18:1 / 26:1, ceramide sphingolipids d18:2 / 16:0, ceramide sphingolipids d18:2 / 20:0, ceramide sphingolipids d18:2 / 22:0, ceramide sphingolipids d18:2 / 24:

1.

2. The biomarker analysis method according to claim 1, characterized in that: It includes the following steps: (1) Sample preparation and pretreatment: Collect plasma samples from asthma patients in the control period and plasma samples from patients with severe asthma, prepare internal standard working solution, accurately pipette 50 μL of plasma sample, add 10 μL of internal standard working solution, add 140 μL of methanol solution to precipitate protein, vortex for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and collect the supernatant; (2) Chromatographic detection: A reversed-phase high performance liquid chromatography column is used as the analytical column; the column length is 5-250 mm, the mobile phase A is an aqueous solution, the mobile phase B is a methanol solution, the mobile phase ratio is: A+B=100%, the analysis time is 5-50 min, the elution program is a gradient elution from 0-80% mobile phase B to 60-100% mobile phase B; the mobile phase additives are 0.1%-1% formic acid and 0-10mmol / L ammonium acetate; the flow rate is 0.1-1.0 mL / min; the column temperature is 4-50 °C; the injection volume is 1-20 μL; (3) Perform mass spectrometry detection: use an electrospray ion source, positive ion detection mode, nebulizer gas flow rate of 1-5 L / min, heating gas flow rate of 5-20 L / min, interface temperature of 250-400 °C, desolvation temperature of 500-600 °C, DL temperature of 200-300 °C, drying gas flow rate of 5-20 L / min, and use multiple reaction monitoring (MRM) mode to quantitatively analyze the target sphingolipid metabolites; (4) Sphingolipid metabolite analysis: The process management biomarkers for severe asthma were screened by comprehensively considering the t-test, change ratio comparison and ROC analysis results of sphingolipid metabolites among the groups.

3. The biomarker analysis method according to claim 2, characterized in that: In the step (2), the reverse phase high performance liquid chromatography column preferably has a column length of 30-200 mm, mobile phase A is an aqueous solution, mobile phase B is a methanol solution, the mobile phase ratio is: A+B=100%, the analysis time is 5-20 min, the elution program is a gradient elution from 0-80% mobile phase B to 80-100% mobile phase B; the mobile phase additives are 0.1%-0.5% formic acid and 0-1 mmol / L ammonium acetate, the flow rate is 0.1-0.8 mL / min, the chromatographic column temperature is: 10-50 °C, and the injection volume is 1-10 μL.

4. The biomarker analysis method according to claim 3, characterized in that: In the step (2), the reverse phase high performance liquid chromatography column preferably has a column length of 30-150 mm, uses octadecylsilane bonded silica gel as filler, mobile phase A is an aqueous solution, mobile phase B is a methanol solution, the mobile phase ratio is: A+B=100%, the analysis time is 5-15 min, the elution program is a gradient elution from 50-80% mobile phase B to 80-100% mobile phase B, the mobile phase additives are 0.1% formic acid and 1 mmol / L ammonium acetate, the flow rate is 0.2-0.4 mL / min, the column temperature is: 25-50 °C, and the injection volume is 5 μL.

5. The biomarker analysis method according to claim 4, characterized in that: In the step (2), the reversed-phase HPLC column is Agilent Poroshell 120 EC-C18, the mobile phase A is an aqueous solution, the mobile phase B is a methanol solution, the mobile phase ratio is: A+B=100%, the analysis time is 15 min, the elution program is a gradient elution from 70% mobile phase B to 100% mobile phase B, the mobile phase additives are 0.1% formic acid and 1 mmol / L ammonium acetate, the flow rate is 0.3 mL / min, the column temperature is: 40°C, and the injection volume is 5 μL.

6. The use of the biomarker according to claim 1, characterized in that: Used for prediction, diagnosis, risk stratification, monitoring and prognosis of bronchial asthma.

7. The use of the biomarker according to claim 1, characterized in that: Used for the prediction, diagnosis, risk stratification, monitoring and prognosis of cough variant asthma, chest tightness variant asthma, latent asthma and drug-induced asthma.

8. The use of the biomarker according to claim 1, characterized in that: Chips, test strips or kits used for asthma management obtain the levels of sphingolipid biomarkers in the biological samples of the test subjects and use them to determine the disease status of asthma in the test subjects, thereby achieving the goal of chronic asthma management.

9. The use of the biomarker according to claim 8, characterized in that: The biological sample is plasma, serum, whole blood, dried whole blood spots, dried plasma spots, urine, sputum, or breath condensate.

10. The use of the biomarker according to claim 8, characterized in that: The pretreatment method of biological samples before detection is as follows: draw 15-50 μL of human whole blood or plasma and drop it onto a dry blood spot collection card, dry it at room temperature for 1-4 hours to obtain a dry blood spot or dry plasma spot sample; use a puncher to take one or more samples with a diameter of 3-8 mm from the dry blood spot; add the dry blood spot or dry plasma spot sample to a solid phase extraction column with phospholipid / protein removal function, and elute it with an organic reagent containing 5%-50% water.

Images