Biomarker, analysis method and application of biomarker in COPD

Abstract

The invention discloses a biomarker, an analysis method and application, can be effectively used for disease course monitoring and prognosis evaluation of chronic obstructive pulmonary disease, is high in sensitivity, rapid and convenient, and accurate and reliable in result, can be used for monitoring patients with mild, moderate, severe and extremely severe chronic obstructive pulmonary disease for multiple times in a stable phase and an acute exacerbation phase, and provides 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 biochemistry, and in particular to a biomarker, an analysis method of this biomarker, and the application of this biomarker in the monitoring of chronic obstructive pulmonary disease. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a common and preventable and treatable disease, characterized by persistent respiratory symptoms and airflow limitation, usually caused by abnormal inflammation of the airways and / or alveoli due to exposure to harmful particles or gases. It is reported that in 2016, COPD has risen to the 3rd leading cause of death globally and the 7th in the global disease economic burden, and it is still on the rise. The number of COPD patients aged ≥20 years in China is about 100 million, and it has become the most common chronic disease on a par with hypertension and diabetes, constituting a major disease burden. The situation of acute exacerbation of COPD in China is not optimistic. 65% of the patients have experienced acute exacerbation, and frequent moderate acute exacerbations or even one severe acute exacerbation will even increase the death risk of COPD patients.

[0003] Currently, the "gold standard" for the diagnosis of COPD is that the ratio of the forced expiratory volume in the first second / forced vital capacity (FEV1 / FVC%) < 70% after the patient inhales a bronchodilator is the boundary for determining the existence of persistent airflow limitation. If other known etiologies or airflow limitation diseases with characteristic pathological manifestations can be excluded at the same time, then the diagnosis of COPD can be clearly made. However, epidemiological studies have shown that the examination rate of lung function in COPD patients is less than 10%, showing a significant phenomenon of underdiagnosis, which not only limits the opportunity for patients to obtain timely and effective treatment, but also exacerbates the negative impact of COPD on personal health and social economy.

[0004] For high-risk populations and diagnosed patients with COPD, enhancing the accessibility and equality of early screening, standardized diagnosis and treatment, and long-term follow-up management helps to relieve disease symptoms, delay disease progression, and improve the quality of life. Developing biomarkers and kits for COPD to achieve precise diagnosis and stratified management of the disease is of great significance for improving treatment effects and reducing medical costs. 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 the prediction, diagnosis, risk stratification, monitoring, and prognosis of COPD, with high sensitivity, fast and convenient, and accurate and reliable results.

[0006] The technical solution of the present invention is: This biomarker comprises at least one of the following substances: ceramide sphingolipid d18:1 / 16:0, ceramide sphingolipid d18:0 / 18:0, ceramide sphingolipid d18:1 / 18:0, hexosylated sphingolipid d16:1 / 24:0, phosphorylated sphingolipid d18:1 / 14:1, ceramide sphingolipid d18:1 / 24:0, ceramide sphingolipid d18:2 / 16:0, hexosylated sphingolipid d18:2 / 22:0, ceramide sphingolipid d18:1 / 22:0, hexosylated sphingolipid d18:1 / 18:0, phosphorylated sphingolipid d18:1 / 22:0, dihexosylated sphingolipid d18:2 / 16:0, ceramide sphingolipid d18:2 / 24:1, ceramide sphingolipid d18:1 / 24:2, lactosylated sphingolipid d18:1 / 22:0, lactosylated sphingolipid d18:1 / 24:1, hexosylated sphingolipid d18:1 / 24:0, hexosylated sphingolipid d18:1 / 22:1, ceramide sphingolipid d18:2 / 20:0, sphingosine, phosphorylated sphingolipid d18:1 / 16:0, ceramide sphingolipid d18:1 / 26:0, hexosylated sphingolipid d18:1 / 24:1, hexosylated sphingolipid d18:1 / 16:0, sphingosine-1-phosphate, hexosylated sphingolipid d18:2 / 24:0, hexosylated sphingolipid d16:1 / 20:0, ceramide sphingolipid d18:1 / 24:1.

[0007] The analysis method of the biomarker comprises the following steps:

[0008] (1) Sample preparation and pretreatment: Collect the plasma sample of the subject, precisely pipette 50 μL of the plasma sample, add 200 μL of methanol solution to precipitate proteins, vortex for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and take the supernatant;

[0009] (2) Perform chromatographic detection: Use a reverse-phase high-performance liquid chromatography column as the analytical chromatographic column; select the column length to be 5 - 250 mm, mobile phase A is an aqueous solution, mobile phase B is a methanol solution, the 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%

[0010] formic acid and 0 - 10 mmol / L ammonium acetate; the flow rate is 0.1 - 1.0 mL / min; the column temperature of the chromatographic column is 4 - 50 °C; the injection volume is 1 - 20 μL;

[0011] (3) Mass spectrometry detection: An electrospray ionization source was used, in positive ion detection mode, with a nebulizing gas flow rate of 1 - 5 L / min, a heating gas flow rate of 5 - 20 L / min, an interface temperature of 250 - 400 °C, a desolvation temperature of 500 - 600 °C, a DL temperature of 200 - 300 °C, and a drying gas flow rate of 5 - 20 L / min. The multiple reaction monitoring (MRM) mode was used for quantitative analysis of the target sphingolipid metabolites;

[0012] (4) Sphingolipid metabolite analysis: Considering the results of hypothesis testing and ROC analysis of sphingolipid metabolites between groups, biomarkers related to chronic obstructive pulmonary disease were screened.

[0013] The biomarkers and analysis method of the present invention can be effectively used for the course monitoring and prognosis evaluation of chronic obstructive pulmonary disease, with high sensitivity, rapidity, convenience, and accurate and reliable results. They can be used for multiple monitoring of patients with mild, moderate, severe, and very severe chronic obstructive pulmonary disease during the stable period and acute exacerbation period, providing a basis for clinical decision-making, and at the same time providing a certain foundation for subsequent basic research and clinical research, with great application and research value.

[0014] The application of the biomarker is also provided for the monitoring and prognosis evaluation of chronic obstructive pulmonary disease.

[0015] The application of the biomarker is also provided for the monitoring and prognosis evaluation of chronic bronchitis and emphysema.

[0016] The application of the biomarker is also provided for a chip, test strip, or kit for the monitoring of chronic obstructive pulmonary disease, to obtain the level of the biomarker in a biological sample of the test subject and to determine the disease status of chronic obstructive pulmonary disease in the test subject. Description of the Drawings

[0017] Figure 1 Shown is a flow chart of biomarker-assisted chronic obstructive pulmonary disease process management.

[0018] Figure 2 Shown is an MRM diagram of 44 sphingolipid metabolites.

[0019] Figure 3 Shown is a bar chart comparing the between-group differences in the levels of sphingolipid metabolites in the plasma of patients with chronic obstructive pulmonary disease (COPD) and healthy subjects (HC).

[0020] Figure 4 Shown is an ROC diagram comparing the between-group differences in the levels of sphingolipid metabolites in the plasma of patients with chronic obstructive pulmonary disease (COPD) and healthy subjects (HC).

[0021] Figure 5 Shown is a bar chart comparing the between-group differences in the levels of sphingolipid metabolites in the plasma of patients with acute exacerbation of chronic obstructive pulmonary disease (A) and patients with stable chronic obstructive pulmonary disease (S).

[0022] Figure 6 ROC curve showing the comparison of the levels of sphingolipid metabolites in plasma between patients with acute exacerbation of chronic obstructive pulmonary disease (A) and patients with stable chronic obstructive pulmonary disease (S).

[0023] Figure 7 Bar chart showing the comparison of the levels of sphingolipid metabolites in plasma between patients with acute exacerbation of chronic obstructive pulmonary disease (A) and healthy subjects (HC).

[0024] Figure 8 ROC curve showing the comparison of the levels of sphingolipid metabolites in plasma between patients with acute exacerbation of chronic obstructive pulmonary disease (A) and healthy subjects (HC).

[0025] Figure 9 Bar chart showing the comparison of the levels of sphingolipid metabolites in plasma between patients with stable chronic obstructive pulmonary disease (S) and healthy subjects (HC).

[0026] Figure 10 ROC curve showing the comparison of the levels of sphingolipid metabolites in plasma between patients with stable chronic obstructive pulmonary disease (S) and healthy subjects (HC).

[0027] Figure 11 Structural formula of the metabolite involved in this specification. Detailed implementation manners

[0028] This biomarker includes at least one of the following substances: ceramide sphingolipid d18:1 / 16:0, ceramide sphingolipid d18:0 / 18:0, ceramide sphingolipid d18:1 / 18:0, hexosylceramide d16:1 / 24:0, phosphorylated sphingolipid d18:1 / 14:1, ceramide sphingolipid d18:1 / 24:0, ceramide sphingolipid d18:2 / 16:0, hexosylceramide d18:2 / 22:0, ceramide sphingolipid d18:1 / 22:0, hexosylceramide d18:1 / 18:0, phosphorylated sphingolipid d18:1 / 22:0, dihexosylceramide d18:2 / 16:0, ceramide sphingolipid d18:2 / 24:1, ceramide sphingolipid d18:1 / 24:2, lactosylceramide d18:1 / 22:0, lactosylceramide d18:1 / 24:1, hexosylceramide d18:1 / 24:0, hexosylceramide d18:1 / 22:1, ceramide sphingolipid d18:2 / 20:0, sphingosine, phosphorylated sphingolipid d18:1 / 16:0, ceramide sphingolipid d18:1 / 26:0, hexosylceramide d18:1 / 24:1, hexosylceramide d18:1 / 16:0, sphingosine-1-phosphate, hexosylceramide d18:2 / 24:0, hexosylceramide d16:1 / 20:0, ceramide sphingolipid d18:1 / 24:1.

[0029] A method for analyzing biomarkers, comprising the following steps:

[0030] (1) Sample preparation and pretreatment: Collect plasma samples from subjects, precisely pipette 50 μL of plasma samples, add 200 μL of methanol solution to precipitate proteins, vortex for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and take the supernatant;

[0031] (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%

[0032] formic acid and 0 - 10 mmol / L ammonium acetate; the flow rate is 0.1 - 1.0 mL / min; the column temperature is

[0033] 4 - 50 °C; the injection volume is 1 - 20 μL;

[0034] (3) Perform mass spectrometry 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 target sphingolipid metabolites;

[0035] (4) Sphingolipid metabolite analysis: Comprehensively consider the hypothesis testing of sphingolipid metabolites between groups and

[0036] the results of ROC analysis to screen biomarkers related to chronic obstructive pulmonary disease.

[0037] The biomarkers and analysis method of the present invention can be effectively used for the course monitoring and prognosis evaluation of chronic obstructive pulmonary disease, with high sensitivity, fast and convenient, accurate and reliable results. It can monitor mild, moderate, severe, and very severe chronic obstructive pulmonary disease patients multiple times during the stable period and acute exacerbation period, provide a basis for clinical decision-making, and at the same time provide a certain basis for subsequent basic research and clinical research, having great application and research value.

[0038] Preferably, in the step (2), the column length of the reverse-phase high-performance liquid chromatography column is 30 - 200 mm, 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 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 column temperature of the chromatographic column is 10 - 50 °C, and the injection volume is 1 - 10 μL.

[0039] Preferably, in the step (2), the column length of the reverse-phase high-performance liquid chromatography column is 30 - 150 mm, the filler is octadecylsilyl-bonded silica gel, the analysis time is 5 - 18 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 of the chromatographic column is 25 - 50 °C, and the injection volume is 5 μL.

[0040] Preferably, in the step (2), the reverse-phase high-performance liquid chromatography column is Agilent Poroshell 120 EC-C18, the analysis time is 18 min, the elution program is gradient elution from 70% of mobile phase B to 100% of mobile phase B, the flow rate is 0.3 mL / min, and the column temperature of the chromatographic column is 40 °C.

[0041] The application of the biomarker is also provided for the monitoring and prognosis evaluation of chronic obstructive pulmonary disease.

[0042] The application of the biomarker is also provided for the monitoring and prognosis evaluation of chronic bronchitis and emphysema.

[0043] The application of the biomarker is also provided for a chip, test strip or kit for monitoring chronic obstructive pulmonary disease, to obtain the biomarker level in the biological sample of the test subject and to judge the disease condition of chronic obstructive pulmonary disease in the test subject.

[0044] Preferably, the biological sample is plasma, serum, whole blood, whole blood dried blood spot, dried plasma spot, urine, sputum, bronchoalveolar lavage fluid, exhaled breath condensate or lung tissue sample.

[0045] Preferably, the pretreatment method of the biological sample before detection is as follows: aspirate 15 - 50 μL of human whole blood or plasma, drop it onto a dried blood spot collection card, and 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 sample to a solid-phase extraction cartridge with the function of removing phospholipids / proteins, and elute it with an organic reagent containing 5% - 50% water.

[0046] The following is a detailed description of the embodiments of the present invention.

[0047] Example 1: Screening of Biomarkers for the Diagnosis of Chronic Obstructive Pulmonary Disease Based on Hypothesis Testing and ROC Analysis

[0048] 1 Sample Source and Grouping Information

[0049] After approval by the Ethics Committee of China-Japan Friendship Hospital, plasma samples from 80 patients with chronic obstructive pulmonary disease and plasma samples from 20 healthy subjects were collected. All participants were from China-Japan Friendship Hospital and were clinically diagnosed with chronic obstructive pulmonary disease, excluding other chronic pulmonary diseases (including interstitial lung disease). The plasma was collected in the early morning on an empty stomach. All samples were stored in a -80 °C refrigerator for later use.

[0050] 2 Experimental Instruments and Materials

[0051] 2.1 Experimental Instruments: Shimadzu LC-MS 8060NX Liquid Chromatography-Mass Spectrometry System.

[0052] 2.2 Experimental Materials: Methanol (mass spectrometry grade, batch number: 226015) was purchased from Fisher Company, chloroform (chromatography grade, batch number: 015167) was purchased from Beijing Maida Technology Co., Ltd., isopropanol (mass spectrometry grade, batch number: 224989) was purchased from Fisher Company, ammonium acetate (mass spectrometry grade, batch number: M KCJ6670) was purchased from Sigma Company, formic acid (mass spectrometry grade, batch number: 214911) was purchased from Sigma Company, and purified water (batch number / Lot#: 111305BS) was purchased from Hangzhou Wahaha Company.

[0053] 3 Experimental Methods

[0054] 3.1 Analytical Methods

[0055] 3.1.1 Chromatographic Conditions

[0056] Chromatographic column: Agilent Poroshell 120EC-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 1, injection volume: 5 μL, column temperature: 40 °C.

[0057] Table 1 Mobile Phase Gradient Elution Program

[0058]

[0059] 3.1.2 Mass Spectrometry Conditions

[0060] Ion source: ESI; Detection mode: positive ion mode; Ion pairs and related parameter settings are shown in Table 3; Interface: IonFocus, 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.

[0061] Forty-four sphingolipid metabolites were predicted using the multiple reaction monitoring (MRM) mode. The retention times and related mass spectrometry parameters of each sphingolipid metabolite are shown in Table 2. The MRM chromatograms of glycosphingolipid metabolites in the plasma of patients with chronic obstructive pulmonary disease are shown in Figure 2 .

[0062] 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 d18:1 / 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), Phosphorylated sphingolipids d18:0 / 16:0: CerP(d18:0 / 16:0), Phosphorylated sphingolipids d18:1 / 12:0: CerP(d18:1 / 12:0), Phosphorylated sphingolipids d18:1 / 14:0: CerP(d18:1 / 14:0), Phosphorylated sphingolipids d18:1 / 14:1: CerP(d18:1 / 14:1), Phosphorylated sphingolipids d18:1 / 16:0: CerP(d18:1 / 16:0), Phosphorylated sphingolipids d18:1 / 20:0: CerP(d18:1 / 20:0), Phosphorylated sphingolipids d18:1 / 22:0: CerP(d18:1 / 22:0), Dihexosylceramide d18:2 / 16:0: Hex2Cer(d18:2 / 16:0), Hexosylceramide d16:1 / 20:0: HexCer(d16:1 / 20:0), Hexosylceramide d16:1 / 24:0: HexCer(d16:1 / 24:0),Hexosylceramide d18:1 / 12:0: HexCer(d18:1 / 12:0), Hexosylceramide d18:1 / 16:0: HexCer(d18:1 / 16:0), Hexosylceramide d18:1 / 18:0: HexCer(d18:1 / 18:0), Hexosylceramide d18:1 / 20:0: HexCer(d18:1 / 20:0), 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 d18:2 / 22:0: HexCer(d18:2 / 22:0), Hexosylceramide d18:2 / 24:0: HexCer(d18:2 / 24:0), Lactosylceramide d18:1 / 20:0: LacCer(d18:1 / 20:0), Lactosylceramide d18:1 / 22:0: LacCer(d18:1 / 22:0), Lactosylceramide d18:1 / 24:1: LacCer(d18:1 / 24:1), Dihydrosphingosine: Sphinganine, Sphingosine: Sphingosine, Sphingosine 1-phosphate: Sphingosine1-phosphate.,

[0063] Table 2 Retention times and related mass spectrometry parameters of ceramide-based sphingolipid metabolites

[0064]

[0065]

[0066]

[0067] 3.3 Sample pretreatment method

[0068] Precisely pipette 50 μL of plasma, add 200 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.

[0069] 3.4 Data analysis

[0070] The Shimadzu Labsolution series workstation was used to perform peak extraction and integration of the target sphingolipid metabolites, and the peak areas of the target analytes were used for subsequent data processing. In this study, the sensitivity and specificity of sphingolipid metabolites for the diagnosis, staging, and grading of COPD were comprehensively investigated through receiver operating characteristic curve (ROC curve) analysis. In large-scale data analysis, an area under the ROC curve greater than 0.7 was considered to indicate good diagnostic ability of the indicator; in the case of progressive significance p < 0.05, an area under the ROC curve greater than 0.65 was also considered to indicate good diagnostic ability of the indicator, and it was defined as a candidate biomarker. Both the ROC curve and the independent samples t-test were calculated and plotted using the Graphpad Prism 10.2.3 statistical analysis software.

[0071] 4. Analysis of experimental results

[0072] The results of sphingolipid metabolites in the plasma of COPD patients and healthy subjects were analyzed, and the t-test and ROC analysis results of sphingolipid metabolites between groups were comprehensively considered to screen for biomarkers for COPD diagnosis.

[0073] The between-group differences in the levels of sphingolipid metabolites in the plasma of COPD patients and healthy subjects are shown in Table 3 and Figure 3 and 4, where Cer(d18:0 / 18:0) (AUC = 0.6758, p = 0.0255), Cer(d18:1 / 16:0) (AUC = 0.7831, p < 0.0001), Cer(d18:1 / 18:0) (AUC = 0.6734, p = 0.0239), Cer(d18:1 / 22:0) (AUC = 0.6829, p = 0.0169), Cer(d18:1 / 24:0) (AUC = 0.7263, p = 0.0002), Cer(d18:1 / 24:1) (AUC = 0.6764, p = 0.0142), Cer(d18:1 / 24:2) (AUC = 0.7353, p = 0.0027), Cer(d18:1 / 26:0) (AUC = 0.7349, p = 0.0002), Cer(d18:2 / 16:0) (AUC = 0.7300, p = 0.0036), Cer(d18:2 / 20:0) (AUC = 0.6950, p = 0.0060), Cer(d18:2 / 24:1) (AUC = 0.6880, p = 0.0168), CerP(d18:1 / 14:1) (AUC = 0.6588, p = 0.0269), CerP(d18:1 / 16:0) (AUC = 0.6750, p = 0.0082), CerP(d18:1 / 22:0) (AUC = 0.7729, p = 0.0024), Hex2Cer(d18:2 / 16:0) (AUC = 0.8446, p = 0.0014), HexCer(d16:1 / 20:0) (AUC = 0.7031, p = 0.0089), HexCer(d16:1 / 24:0) (AUC = 0.6601, p = 0.0184), HexCer(d18:1 / 16:0) (AUC = 0.7840, p = 0.0013), HexCer(d18:1 / 18:0) (AUC = 0.8344, p = 0.0002), HexCer(d18:1 / 22:1) (AUC = 0.7300, p = 0.0002), HexCer(d18:1 / 24:0) (AUC = 0.6675, p = 0.0142), HexCer(d18:1 / 24:1) (AUC = 0.6992, p = 0.0331), HexCer(d18:2 / 22:0) (AUC = 0.7218, p < 0.0001), HexCer(d18:2 / 24:0) (AUC = 0.7119, p = 0.0111), LacCer(d18:1 / 22:0) (AUC = 0.7077, p = 0.0014), LacCer(d18:1 / 24:1) (AUC = 0.7160, p = 0.0129), Sphingosine (AUC = 0.7463, p = 0.0058), Sphingosine 1 - phosphate (AUC = 0.6518, p = 0.0038) have good diagnostic discrimination ability and can be used for the auxiliary diagnosis and management of COPD.

[0074] Table 3 Comparison of between - group differences in sphingolipid metabolite levels in plasma of COPD patients and healthy subjects

[0075]

[0076]

[0077]

[0078] Example 2: Screening of markers for distinguishing acute - phase COPD and stable - phase COPD according to hypothesis testing and ROC analysis

[0079] 1 Sample source and grouping information

[0080] After approval by the Ethics Committee of China - Japan Friendship Hospital, plasma samples from 45 COPD (acute exacerbation phase) patients and 20 healthy subjects were collected. All participants were from China - Japan Friendship Hospital, clinically diagnosed with COPD, and other chronic pulmonary diseases (including interstitial lung 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.

[0081] 2 Experimental instruments and materials: The same as in Example 1.

[0082] 3 Experimental methods: The same as in Example 1. The correspondence between compound names and abbreviations is the same as in Example 1.

[0083] 4. Analysis of experimental results

[0084] The results of sphingolipid metabolites in plasma of acute - phase COPD patients and stable - phase COPD patients were analyzed. Considering the t - test and ROC analysis results of sphingolipid metabolites between groups comprehensively, markers for distinguishing acute - phase COPD and stable - phase COPD were screened.

[0085] The comparison of between - group differences in sphingolipid metabolite levels in plasma of acute - phase COPD patients and stable - phase COPD patients is shown in Table 4 and Figure 5 、 6, where Cer(d18:0 / 18:0) (AUC = 0.7514, p = 0.0003), Cer(d18:0 / 20:0) (AUC = 0.9184, <0.0001), Cer(d18:1 / 16:0) (AUC = 0.6686, p = 0.0072), Cer(d18:1 / 18:0) (AUC = 0.6919, p = 0.0009), CerP(d18:1 / 12:0) (AUC = 0.6778, p = 0.0036), LacCer(d18:1 / 20:0) (AUC = 0.7086, p = 0.0035) have good diagnostic discrimination ability and can be used for the auxiliary diagnosis and management of chronic obstructive pulmonary disease.

[0086] Table 4 Comparison of between-group differences in sphingolipid metabolite levels in plasma of patients with acute exacerbation of chronic obstructive pulmonary disease and patients with stable chronic obstructive pulmonary disease

[0087]

[0088]

[0089]

[0090] Example 3: Screening of markers for distinguishing healthy subjects from patients with stable chronic obstructive pulmonary disease based on hypothesis testing and ROC analysis

[0091] 1 Sample source and grouping information

[0092] After approval by the Ethics Committee of China-Japan Friendship Hospital, plasma samples from 35 patients with chronic obstructive pulmonary disease (stable stage) and plasma samples from 20 healthy subjects were collected. All participants were from China-Japan Friendship Hospital and were clinically diagnosed with chronic obstructive pulmonary disease, excluding other chronic pulmonary diseases (including interstitial lung disease). The plasma was collected in the early morning on an empty stomach. All samples were stored in a -80°C refrigerator for later use.

[0093] 2 Experimental instruments and materials: The same as in Example 1.

[0094] 3 Experimental methods: The same as in Example 1. The correspondence between compound names and abbreviations is the same as in Example 1.

[0095] 4. Analysis of experimental results

[0096] The results of sphingolipid metabolites in plasma of healthy subjects and patients with stable chronic obstructive pulmonary disease were analyzed. Considering the t-test and ROC analysis results of sphingolipid metabolites between groups, biomarkers for distinguishing healthy subjects from patients with stable chronic obstructive pulmonary disease were screened.

[0097] The comparison of between-group differences in sphingolipid metabolite levels in plasma of healthy subjects and patients with stable chronic obstructive pulmonary disease is shown in Table 5 andFigure 7 , 8 , among which Cer(d18:1 / 16:0) (AUC = 0.7200, p = 0.0028), Cer(d18:1 / 22:0) (AUC = 0.6735, p = 0.0311), Cer(d18:1 / 24:0) (AUC = 0.7106, p = 0.0047), Cer(d18:1 / 24:2) (AUC = 0.7100, p = 0.0131), Cer(d18:1 / 26:0) (AUC = 0.7219, p = 0.0025), Cer(d18:2 / 16:0) (AUC = 0.7343, p = 0.0089), Cer(d18:2 / 20:0) (AUC = 0.6544, p = 0.0425), Cer(d18:2 / 24:1) (AUC = 0.6618, p = 0.0463), CerP(d18:1 / 22:0) (AUC = 0.8000, p = 0.0031), Hex2Cer(d18:2 / 16:0) (AUC = 0.8673, p = 0.0012), HexCer(d18:1 / 16:0) (AUC = 0.8095, p = 0.0012), HexCer(d18:1 / 18:0) (AUC = 0.8018, p = 0.0010), HexCer(d18:1 / 22:1) (AUC = 0.6571, p = 0.0327), HexCer(d18:1 / 24:0) (AUC = 0.6786, p = 0.0253), HexCer(d18:1 / 24:1) (AUC = 0.7121, p = 0.0337), HexCer(d18:2 / 22:1) (AUC = 0.6571, p = 0.0327), HexCer(d18:1 / 24:0) (AUC = 0.6786, p = 0.0253), HexCer(d18:1 / 24:1) (AUC = 0.7121, p = 0.0337), HexCer(d18:2 / 22:0) (AUC = 0.6614, p = 0.0246), HexCer(d18:2 / 24:0) (AUC = 0.7105, p = 0.0193), LacCer(d18:1 / 22:0) (AUC = 0.7324, p = 0.0007), LacCer(d18:1 / 24:1) (AUC = 0.7317, p = 0.0136), Sphingosine (AUC = 0.7265, p = 0.0082) have good diagnostic discrimination ability and can be used for the auxiliary diagnosis and management of chronic obstructive pulmonary disease.

[0098] Comparison of between-group differences in the levels of sphingolipid metabolites in plasma of healthy subjects and patients with stable chronic obstructive pulmonary disease

[0099]

[0100]

[0101]

[0102] Example 4: Screening of biomarkers for distinguishing healthy subjects from patients in the acute stage of chronic obstructive pulmonary disease based on hypothesis testing and ROC analysis

[0103] 1 Sample source and grouping information

[0104] After approval by the Ethics Committee of China-Japan Friendship Hospital, plasma samples from 45 patients with chronic obstructive pulmonary disease (in the acute exacerbation stage) and plasma samples from 20 healthy subjects were collected. All participants were from China-Japan Friendship Hospital and were clinically diagnosed with chronic obstructive pulmonary disease, excluding other chronic pulmonary diseases (including interstitial lung disease). The plasma was collected in the early morning on an empty stomach. All samples were stored in a -80 °C refrigerator for later use.

[0105] 2 Experimental instruments and materials: The same as in Example 1.

[0106] 3 Experimental methods: The same as in Example 1. The correspondence between compound names and abbreviations is the same as in Example 1.

[0107] 4. Analysis of experimental results

[0108] The results of sphingolipid metabolites in plasma of healthy subjects and patients in the acute stage of chronic obstructive pulmonary disease were analyzed. Considering the t-test and ROC analysis results of sphingolipid metabolites between groups, biomarkers for distinguishing healthy subjects from patients in the acute stage of chronic obstructive pulmonary disease were screened.

[0109] Comparison of between-group differences in the levels of sphingolipid metabolites in plasma of healthy subjects and patients in the acute stage of chronic obstructive pulmonary disease is shown in Table 6 and Figure 9 、 10, where Cer(d18:0 / 18:0) (AUC = 0.7877, p = 0.0028), Cer(d18:0 / 20:0) (AUC = 0.8774, p < 0.0001), Cer(d18:0 / 22:0) (AUC = 0.7090, p = 0.0041), Cer(d18:1 / 16:0) (AUC = 0.8322, p < 0.0001), Cer(d18:1 / 18:0) (AUC = 0.7500, p = 0.0024), Cer(d18:1 / 22:0) (AUC = 0.6506, p = 0.0322), Cer(d18:1 / 24:0) (AUC = 0.7378, p = 0.0012), Cer(d18:1 / 24:1) (AUC = 0.6654, p = 0.0182), Cer(d18:1 / 24:2) (AUC = 0.7667, p = 0.0013), Cer(d18:1 / 26:0) (AUC = 0.7311, p = 0.0048), Cer(d18:2 / 16:0) (AUC = 0.7267, p = 0.0041), Cer(d18:2 / 20:0) (AUC = 0.7189, p = 0.0019), Cer(d18:2 / 24:1) (AUC = 0.6815, p = 0.0213), CerP(d18:1 / 14:1) (AUC = 0.6767, p = 0.0171), CerP(d18:1 / 16:0) (AUC = 0.6944, p = 0.0144), CerP(d18:1 / 22:0) (AUC = 0.7519, p = 0.0044), Hex2Cer(d18:2 / 16:0) (AUC = 0.8270, p = 0.0024), HexCer(d16:1 / 20:0) (AUC = 1, p < 0.0001), HexCer(d16:1 / 24:0) (AUC = 0.6922, p = 0.0205), HexCer(d18:1 / 16:0) (AUC = 0.7642, p = 0.0034), HexCer(d18:1 / 18:0) (AUC = 0.8597, p = 0.0001), HexCer(d18:1 / 22:0) (AUC = 0.6633, p = 0.0070), HexCer(d18:1 / 22:1) (AUC = 0.7867, p < 0.0001), HexCer(d18:1 / 24:0) (AUC = 0.6589, p = 0.0305), HexCer(d18:1 / 24:1) (AUC = 0.7029, p = 0.0390), HexCer(d18:2 / 22:0) (AUC = 0.7545, p < 0.0001), HexCer(d18:2 / 24:0) (AUC = 0.7129, p = 0.0165), LacCer(d18:1 / 22:0) (AUC = 0.6886, p = 0.0193), LacCer(d18:1 / 24:1) (AUC = 0.7037, p = 0.0210), Sphinganine (AUC = 0.6683, p = 0.0067), Sphingosine (AUC = 0.7625, p = 0.0059), Sphingosine 1-phosphate (AUC = 0.6635, p = 0.0050) have good diagnostic discrimination ability and can be used for the auxiliary diagnosis and management of chronic obstructive pulmonary disease.

[0110] Table 6 Comparison of between-group differences in sphingolipid metabolite levels in plasma of healthy subjects and patients with acute exacerbation of chronic obstructive pulmonary disease

[0111]

[0112]

[0113] The above are only the preferred embodiments of the present invention, and do not impose any formal limitations on the present invention. Any simple modifications, equivalent changes and modifications 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 in that: At least one of the following substances: ceramide sphingolipid d18:1 / 16:0, ceramide sphingolipid d18:0 / 18:0, ceramide sphingolipid d18:1 / 18:0, hexosylated sphingolipid d16:1 / 24:0, phosphorylated sphingolipid d18:1 / 14:1, ceramide sphingolipid d18:1 / 24:0, ceramide sphingolipid d18:2 / 16:0, hexosylated sphingolipid d18:2 / 22:0, ceramide sphingolipid d18:1 / 22:0, hexosylated sphingolipid d18:1 / 18:0, phosphorylated sphingolipid d18:1 / 22:0, dihexosylated sphingolipid d18:2 / 16:0, ceramide sphingolipid d18:2 / 24:1, ceramide sphingolipid d18:1 / 24:2, lactosylated sphingolipid d18:1 / 22:0, lactosylated sphingolipid d18:1 / 24:1, hexosylated sphingolipid d18:1 / 24:0, hexosylated sphingolipid d18:1 / 22:1, ceramide sphingolipid d18:2 / 20:0, sphingosine, phosphorylated sphingolipid d18:1 / 16:0, ceramide sphingolipid d18:1 / 26:0, hexosylated sphingolipid d18:1 / 24:1, hexosylated sphingolipid d18:1 / 16:0, sphingosine-1-phosphate, hexosylated sphingolipid d18:2 / 24:0, hexosylated sphingolipid d16:1 / 20:0, ceramide sphingolipid d18:1 / 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 the plasma sample of the subject, precisely pipette 50 μL of the plasma sample, add 200 μL of methanol solution to precipitate proteins, vortex for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes, and take the supernatant; (2) Perform chromatographic detection: Use a reversed-phase high-performance liquid chromatography column as the analytical chromatographic column; select the column length of 5 - 250 mm, mobile phase A is an aqueous solution, mobile phase B is a methanol solution, the 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 ion 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 hypothesis test and ROC analysis results of sphingolipid metabolites between groups to screen the biomarkers related to chronic obstructive pulmonary disease.

3. The method for analyzing a biomarker according to claim 2, wherein: In step (2), the column length of the reversed-phase high-performance liquid chromatography column is 30 - 200 mm, 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 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 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 step (2), the column length of the reversed-phase high-performance liquid chromatography column is 30 - 150 mm, with octadecylsilyl-bonded silica gel as the packing material, the analysis time is 5 - 18 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 is 25 - 50 °C, and the injection volume is 5 μL.

5. The biomarker analysis method according to claim 4, characterized in that: In step (2), the reversed-phase high-performance liquid chromatography column is Agilent Poroshell 120 EC-C18, the analysis time is 18 min, the elution program is gradient elution from 70% of mobile phase B to 100% of mobile phase B, the flow rate is 0.3 mL / min, and the column temperature is 40 °C.

6. Use of the biomarker according to claim 1, characterized in that: For the monitoring and prognosis evaluation of chronic obstructive pulmonary disease.

7. Use of the biomarker according to claim 1, characterized in that: For the monitoring and prognosis evaluation of chronic bronchitis and emphysema.

8. The use of the biomarker according to claim 1, characterized in that: A chip, test strip or kit for monitoring chronic obstructive pulmonary disease, which obtains the biomarker level in the biological sample of the test subject and is used to judge the disease status of chronic obstructive pulmonary disease in the test subject.

9. Use of the biomarker according to claim 8, characterized in that: The biological sample is plasma, serum, whole blood, whole blood dried blood spot, dried plasma spot, urine, sputum, bronchoalveolar lavage fluid, exhaled breath condensate or lung tissue sample.

10. Use of the biomarker according to claim 8, characterized in that: The pretreatment method of the biological sample before detection is as follows: aspirate 15 - 50 μL of human whole blood or plasma, drop it onto a dried blood spot collection card, and 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 sample to a solid-phase extraction small column with the function of removing phospholipids / proteins, and elute it with an organic reagent containing 5% - 50% water.

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