Application of metabolic marker in pulmonary fibrosis diagnosis or prognosis evaluation

Abstract

The invention provides application of a metabolic marker in pulmonary fibrosis diagnosis or prognosis evaluation, and belongs to the technical field of biological medicine. Systematic analysis of metabonomics discovers that lipid metabolism of a patient suffering from pulmonary fibrosis is remarkably changed, downstream products Palmitoyl ethanolamide and 2-amino-1, 3, 4-octadecantool of lipid metabolism can be used as potential biomarkers for diagnosing pulmonary fibrosis, and objective quantitative indexes are provided for diagnosis and prognosis evaluation of pulmonary fibrosis. The invention proves that the Palmitoyl ethanolamide and the 2-amino-1, 3, 4-octadecantool are obviously related to the lung function injury degree and the concentration of the acute-stage blood inflammation marker of the pulmonary fibrosis patient, and the related characteristics prove that the Palmitoyl ethanolamide and the 2-amino-1, 3, 4-octadecantool can be used as the biomarker for evaluating the prognosis of the patient.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of metabolic markers in the diagnosis or prognosis evaluation of pulmonary fibrosis. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease with an insidious onset, a high misdiagnosis rate, and a median survival of only 2-4 years after diagnosis. Early symptoms lack specificity, while late-stage manifestations primarily include dry cough and progressive dyspnea. Improving the diagnosis rate and reducing misdiagnosis are crucial for the management of IPF. Currently, IPF is primarily diagnosed through a multidisciplinary approach based on chest HRCT findings, clinical symptoms, and pulmonary function tests, with invasive procedures such as lung biopsy required for definitive diagnosis. However, the diagnosis of IPF still lacks mature, quantifiable, objective, and sensitive clinical indicators. Therefore, systematic screening and identification of serum biomarkers specific for pulmonary fibrosis has significant clinical value for early screening and diagnosis of pulmonary fibrosis, establishing a dynamic monitoring system, and optimizing disease management strategies, thereby effectively delaying disease progression and improving patients' long-term quality of life.

[0003] During the pathogenesis of IPF, metabolic disorders such as protein metabolism, sugar metabolism, lipid metabolism, and fatty acid oxidation metabolism also occur. Among them, lipid metabolism disorders are particularly prominent. The abnormal accumulation of lipid metabolites in the lungs of IPF patients is an important factor in promoting the progression of IPF. However, there is still a gap in the current research on biomarkers for lipid metabolic reprogramming in IPF. Based on this, systematically exploring potential biomarkers in lipid metabolic reprogramming in IPF and using them as a basis for diagnosis and prognosis assessment of the disease has important clinical value. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of metabolic markers in the diagnosis or prognosis evaluation of pulmonary fibrosis, providing an effective means for idiopathic pulmonary fibrosis.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a kit for determining the level of metabolic markers in a sample for use in preparing a product for diagnosing pulmonary fibrosis or a product for evaluating the prognosis of pulmonary fibrosis. The metabolic markers include palmitoylethanolamide and 2-amino-1,3,4-octadecanetriol.

[0007] Preferably, the kit determines the level of the metabolite marker in a sample by mass spectrometry, chromatography, fluorescence assay, immunochemical analysis or nuclear magnetic resonance spectroscopy.

[0008] Preferably, the kit determines the level of the metabolite marker in a sample by mass spectrometry.

[0009] Preferably, the sample is serum.

[0010] Preferably, the idiopathic pulmonary fibrosis is primary pulmonary fibrosis.

[0011] The present invention also provides a kit for diagnosing pulmonary fibrosis or evaluating the prognosis of pulmonary fibrosis, wherein the kit comprises reagents for determining the level of metabolic markers in a sample, wherein the metabolic markers comprise palmitoylethanolamide and 2-amino-1,3,4-octadecantriol.

[0012] Preferably, the kit further comprises a pretreatment reagent for pretreating the sample.

[0013] The present invention also provides a system for diagnosing pulmonary fibrosis or evaluating the prognosis of pulmonary fibrosis, the system comprising a computing device for judging the risk of a subject suffering from idiopathic pulmonary fibrosis or evaluating the prognosis based on the levels of metabolic markers, wherein the metabolic markers include palmitoylethanolamide and 2-amino-1,3,4-octadecantriol.

[0014] Preferably, the system further includes any one or more of the following:

[0015] (1) a detection device for detecting the level of the metabolic marker;

[0016] (2) a reference device for receiving the metabolic marker levels output by the detection device and performing comparisons based on known grouping information of a healthy control group and an idiopathic pulmonary fibrosis patient group;

[0017] (3) An analysis device for comparing the metabolic marker levels of the subject with known grouping information and outputting a risk or prognosis assessment result.

[0018] The present invention also provides the use of a metabolic marker composition in constructing a computational model for diagnosing pulmonary fibrosis or evaluating the prognosis of pulmonary fibrosis, wherein the metabolic marker composition comprises palmitoylethanolamide and 2-amino-1,3,4-octadecantriol.

[0019] Beneficial effects of the present invention:

[0020] Through systematic metabolomics analysis, the present invention discovered significant changes in lipid metabolism in patients with pulmonary fibrosis. The downstream products of lipid metabolism, Palmitoyl ethanolamide and 2-amino-1,3,4-octadecanetriol, can serve as potential biomarkers for diagnosing pulmonary fibrosis, providing objective quantitative indicators for the diagnosis and prognosis of pulmonary fibrosis. The present invention confirmed that Palmitoyl ethanolamide and 2-amino-1,3,4-octadecanetriol were significantly correlated with the degree of lung function damage and the concentrations of acute blood inflammatory markers in patients with pulmonary fibrosis. This correlation confirmed their potential as biomarkers for assessing patient prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a flow chart of the experiment of the present invention;

[0022] Figure 2 This is the lipid metabolism profile and differential metabolite screening results of patients with pulmonary fibrosis;

[0023] Figure 3 This figure shows the comparison of the levels of Palmitoyl ethanolamide and 2-amino-1,3,4-octadecanetriol in the serum of patients with pulmonary fibrosis and non-patients;

[0024] Figure 4 This is a graph showing the correlation between Palmitoyl ethanolamide and lung function and serum inflammatory markers in patients with pulmonary fibrosis;

[0025] Figure 5 This figure shows the correlation between 2-amino-1,3,4-octadecanetriol and lung function and serum inflammatory markers in patients with pulmonary fibrosis. DETAILED DESCRIPTION

[0026] The present invention provides a kit for determining the level of metabolic markers in a sample for use in preparing a product for diagnosing pulmonary fibrosis or a product for evaluating the prognosis of pulmonary fibrosis. The metabolic markers include palmitoylethanolamide and 2-amino-1,3,4-octadecanetriol.

[0027] In the present invention, preferably, the kit determines the level of the metabolite marker in the sample by the following methods: mass spectrometry, chromatography, fluorescence measurement, immunochemical analysis, or nuclear magnetic resonance spectroscopy. Further preferably, the mass spectrometry method may include the following steps: taking 1 mL of sample, lyophilizing it in a lyophilizer, adding 100 μL of 80% methanol in water, vortexing, standing in an ice bath for 5 minutes, and centrifuging at 15,000 g and 4°C for 15 minutes; taking a certain amount of supernatant and diluting it with mass spectrometry-grade water to a methanol content of 53%, centrifuging it at 15,000 g and 4°C for 15 minutes, collecting the supernatant, and injecting it into LC-MS for analysis. Using CD3.1 data processing software, the chromatographic peaks detected in the sample are integrated, and background ions are removed using a blank sample. The raw quantitative results are normalized using the total peak area. Metabolites with a coefficient of variation (CV) of less than 30% in the QC sample are retained, and finally, metabolite identification and relative quantification results are obtained. All samples were grouped and data were compared between groups. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to investigate intrinsic metabolic variation and data quality in metabolic profiling. The OPLS-DA model's VIP (Variable Importance in the Projection) value, combined with the p-value from an independent-samples T-test, was used to identify differentially expressed metabolites. The threshold for screening differential metabolites was: |log2FC| ≥ 1 & OPLS-DA_VIP ≥ 1 & P-value ≤ 0.05. The measured metabolites were annotated using the KEGGPATHWAY database.

[0028] In the present invention, the chromatography method may comprise the following steps:

[0029] After freeze-drying and concentration, 0.5-2 mL of sample was extracted with 80-90% aqueous methanol (volume 50-200 μL). The sample was vortexed for 1-3 minutes, placed in an ice bath for 5-10 minutes, and centrifuged at 10,000-15,000 g and 2-8°C for 10-20 minutes. The supernatant was filtered through a 0.22 μm organic filter membrane and separated using a reversed-phase column (C18 or C8, particle size 1.7-5 μm, column length 50-150 mm). The mobile phases consisted of aqueous phase (A) containing 0.1-0.3% formic acid and acetonitrile / methanol (B). The gradient elution rate was 0.2-0.6 mL / min, the column temperature was 30-40°C, and the detector wavelength was set according to the UV absorption characteristics of the target metabolite (e.g., 210-280 nm). Quantification was performed by standard retention time matching and peak area normalization. After data collection, internal standard method or total peak area normalization was combined to screen stable metabolites with CV < 30%.

[0030] Note: The mobile phase gradient needs to be adjusted according to the polarity of the target metabolites to avoid overloading the chromatographic column; regular column efficiency testing (theoretical plate number > 5000) is required to ensure the separation degree.

[0031] In the present invention, the fluorescence assay may include the following steps:

[0032] After lyophilization, serum samples were extracted for metabolites using a polar solvent (e.g., a methanol / acetonitrile / water mixture, 70-90% by volume). The samples were vortexed for 1-5 minutes, placed on ice for 5-15 minutes, and centrifuged (8000-12000g, 4-10°C, 10-15 minutes). The supernatant was then collected. A specific fluorescent probe (e.g., dansyl chloride or an o-phthalaldehyde derivative, 10-50 μM) was added and incubated in the dark for 10-30 minutes (temperature 25-37°C). The excitation and emission wavelengths were determined based on the characteristics of the probe-metabolite complex (e.g., Ex 340-380 nm, Em 420-480 nm). Relative fluorescence intensity was measured by spectrofluorometry and quantified using a standard curve. Data were analyzed after blank subtraction and internal standard correction.

[0033] Note: Optimize probe concentration and incubation time to avoid fluorescence quenching; strictly control the reaction pH (e.g., 7.0-8.5) to ensure derivatization efficiency.

[0034] In the present invention, the immunochemical analysis may include the following steps:

[0035] Using enzyme-linked immunosorbent assay (ELISA), a specific antibody (e.g., anti-Palmitoyl ethanolamide monoclonal antibody, coating concentration 1-5 μg / mL) is immobilized on a 96-well plate and incubated at 4°C for 12-24 hours, followed by blocking (PBS containing 1-5% BSA). A serial dilution of the sample (10-100-fold dilution) and the standard are added and incubated at room temperature for 1-2 hours. After washing, an enzyme-labeled secondary antibody (e.g., HRP-labeled antibody, 1000-5000-fold dilution) is added and incubated for 30-60 minutes. A chromogenic substrate (e.g., TMB) is added for 5-15 minutes. After termination, the absorbance at 450 nm is measured, and the metabolite concentration is calculated using a four-parameter standard curve fit.

[0036] Note: Antibody cross-reactivity needs to be verified (cross-reactivity rate <5%); samples need to avoid repeated freezing and thawing to prevent metabolite degradation.

[0037] In the present invention, the nuclear magnetic resonance spectroscopy may include the following steps:

[0038] Dissolve the lyophilized sample in a deuterated solvent (e.g., D2O / CD3OD mixture, 50-200 μL), vortex for 1-3 min, centrifuge (10,000-15,000 g, 4°C) for 5-10 min, and collect the supernatant into an NMR tube; set up a one-dimensional 1 H-NMR parameters (spectral width 10-12 ppm, sampling times 32-128 times, relaxation delay 1-3 s) were used, and the water peak was suppressed using the presaturation method. The characteristic peaks of metabolites were matched by chemical shift (such as δ0.5-9.0 ppm), and attributed in combination with a standard database (such as HMDB). The target peak area was integrated and normalized to the total metabolite signal or internal standard (such as TSP) for semi-quantitative analysis.

[0039] Note: Samples must be deproteinized (e.g., acetonitrile precipitation) to improve spectral resolution; metabolite concentrations should be >10 μM to ensure a signal-to-noise ratio (S / N > 3).

[0040] In the present invention, preferably, the sample is serum. The serum is preferably processed by the following method: after the serum is thawed on ice at 4°C, 10ul of serum is added to 150ul of ice methanol, vortexed for 30s, 500ul of chloroform is added and shaken for 20min, then 125ul of ultrapure water is added, vortexed, centrifuged at 4°C and 12000rpm, 600ul of the upper layer of liquid is collected, concentrated by nitrogen blowdown, and re-dissolved in 100ul (acetonitrile isopropanol water 65:35:5), shaken, vortexed, centrifuged at 4°C and 12000rpm for 10 minutes, and the supernatant is collected for sampling.

[0041] In the present invention, preferably, the pulmonary fibrosis is primary pulmonary fibrosis. The present invention also provides a kit for diagnosing pulmonary fibrosis or assessing the prognosis of pulmonary fibrosis, the kit comprising reagents for determining the levels of metabolic markers in a sample, the metabolic markers comprising palmitoylethanolamide and 2-amino-1,3,4-octadecantriol. Preferably, the kit also comprises a pretreatment reagent for pretreating the sample.

[0042] The present invention also provides a system for diagnosing pulmonary fibrosis or evaluating the prognosis of pulmonary fibrosis, the system comprising a computing device for judging the risk of a subject suffering from idiopathic pulmonary fibrosis or evaluating the prognosis based on the levels of metabolic markers, wherein the metabolic markers include palmitoylethanolamide and 2-amino-1,3,4-octadecantriol.

[0043] Preferably, the system further comprises any one or more of the following: (1) a detection device for detecting the level of the metabolite marker; (2) a reference device for receiving the metabolite marker level output by the detection device and comparing it based on known grouping information of a healthy control group and an idiopathic pulmonary fibrosis patient group; (3) an analysis device for comparing the metabolite marker level of the subject with the known grouping information and outputting a risk or prognosis assessment result.

[0044] The present invention also provides the use of a metabolic marker composition in constructing a computational model for diagnosing pulmonary fibrosis or evaluating the prognosis of pulmonary fibrosis, wherein the metabolic marker composition comprises palmitoylethanolamide and 2-amino-1,3,4-octadecantriol.

[0045] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example

[0047] Serum sample processing and data analysis for non-targeted metabolic testing

[0048] Take 1 mL of sample and freeze-dry it in a freeze dryer. Add 100 μL of 80% methanol aqueous solution, vortex and shake, let it stand in an ice bath for 5 minutes, and centrifuge it at 15,000g and 4°C for 15 minutes. Take a certain amount of supernatant and dilute it with mass spectrometry-grade water to a methanol content of 53%. Centrifuge it at 15,000g and 4°C for 15 minutes, collect the supernatant, and inject it into LC-MS for analysis.

[0049] Using CD3.1 data processing software, chromatographic peaks detected in the samples were integrated, and background ions were removed using blank samples. The raw quantitative results were normalized using total peak area. Metabolites with a coefficient of variation (CV) of less than 30% in the QC samples were retained. Metabolite identification and relative quantification were then determined. All samples were grouped, and intergroup comparisons were performed. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to investigate intrinsic metabolic variation and data quality in metabolic profiling. The VIP (Variable Importance in the Projection) value of the OPLS-DA model was used in combination with the p-value of the independent sample T-test to identify differentially expressed metabolites. The threshold for screening differential metabolites was: |log2FC| ≥ 1 & OPLS-DA_VIP ≥ 1 & P-value ≤ 0.05. The measured metabolites were annotated using the KEGG PATHWAY database.

[0050] After the serum was melted on ice at 4°C, 10ul serum was added to 150ul ice methanol, vortexed for 30s, 500ul chloroform was added and shaken for 20min, then 125ul ultrapure water was added, vortexed, and centrifuged at 4°C and 12000rpm. 600ul of the upper liquid was collected, concentrated by nitrogen blow-through, and re-dissolved in 100ul (acetonitrile, isopropanol, and water 65:35:5), shaken, vortexed, and centrifuged at 4°C and 12000rpm for 10 minutes. The supernatant was collected and injected.

[0051] Clinical data and correlation analysis

[0052] Various lung function indicators of patients were collected, such as total lung capacity (TLC), vital capacity (VC), forced vital capacity (FVC), diffusing capacity of the lung for carbon monoxide (DLCO), peak expiratory flow (PEF), and functional residual capacity (FRC). Laboratory parameters were collected from all patients, including complete blood count (WBC), neutrophil percentage (NEUT%), lymphocyte percentage (LYMPH%), hemoglobin (HB), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). Coagulation parameters, including prothrombin time (PT), prothrombin time activity (PTA), fibrinogen (Fib), activated partial thromboplastin time (APTT), thrombin time (TT), international normalized ratio (INR), and D-dimer, were also collected. GraphPad Prism software was used for statistical analysis. The Kolmogorov-Smirnov test was used to assess the normality of the distribution of continuous variables. Categorical variables were compared between groups using the chi-square test, and continuous variables were compared using the independent t-test (for normally distributed data) or the Mann-Whitney U test (for non-normally distributed data), depending on the data distribution. Correlations between the two biomarkers and clinical variables were assessed using Spearman and Pearson correlations, depending on the variable distribution.

[0053] Figure 1 The experimental results of the present invention are shown in Figures 2-4 , Figure 2It showed that patients with pulmonary fibrosis had dysregulated lipid metabolism and that the downstream products Palmitoyl ethanolamide and 2-amino-1,3,4-octadecanetriol were differentially expressed metabolites; Figure 3 It was shown that Palmitoyl ethanolamide and 2-amino-1,3,4-octadecanetriol were significantly elevated in the serum of patients with pulmonary fibrosis; Figure 4 It showed that Palmitoyl ethanolamide was significantly correlated with lung function and serum inflammatory markers in patients with pulmonary fibrosis; Figure 5 The results showed that 2-amino-1,3,4-octadecanetriol was significantly correlated with lung function and serum inflammatory markers in patients with pulmonary fibrosis.

[0054] As demonstrated in the examples above, the present invention successfully screened and identified two key differential metabolites: palmitoyl ethanolamide and 2-amino-1,3,4-octadecanetriol. These metabolites were significantly upregulated and strongly correlated with indicators of lung function impairment and inflammatory markers. KEGG pathway annotation further revealed the central role of lipid metabolism reprogramming in the disease process. This present invention provides a novel potential biomarker combination for pulmonary fibrosis, offering a methodological reference for understanding the pathological mechanisms of related diseases and developing precise diagnostic and treatment strategies.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a kit for determining the level of a metabolic marker in a sample in the preparation of a product for diagnosing pulmonary fibrosis or a product for evaluating the prognosis of pulmonary fibrosis, characterized in that: The metabolic markers include palmitoylethanolamide and 2-amino-1,3,4-octadecantriol.

2. The use according to claim 1, characterized in that The kit determines the level of the metabolite marker in a sample by mass spectrometry, chromatography, fluorescence measurement, immunochemical analysis or nuclear magnetic resonance spectroscopy.

3. The use according to claim 2, characterized in that The kit determines the level of the metabolite marker in a sample by mass spectrometry.

4. The use according to claim 1, characterized in that The sample is serum.

5. The use according to any one of claims 1 to 4, characterized in that: The pulmonary fibrosis is primary pulmonary fibrosis.

6. A kit for diagnosing pulmonary fibrosis or assessing the prognosis of pulmonary fibrosis, the kit comprising reagents for determining the level of metabolic markers in a sample, characterized in that: The metabolic markers include palmitoylethanolamide and 2-amino-1,3,4-octadecantriol.

7. The kit according to claim 6, characterized in that The kit also includes a pretreatment reagent for pretreating the sample.

8. A system for diagnosing pulmonary fibrosis or evaluating the prognosis of pulmonary fibrosis, characterized in that: The system includes a computing device for judging the risk of a subject suffering from pulmonary fibrosis or evaluating the prognosis according to the levels of metabolic markers, wherein the metabolic markers include palmitoylethanolamide and 2-amino-1,3,4-octadecantriol.

9. The system according to claim 8, characterized in that The system also includes any one or more of the following: (1) a detection device for detecting the level of the metabolic marker; (2) a reference device for receiving the metabolic marker levels output by the detection device and performing comparisons based on known grouping information of the healthy control group and the pulmonary fibrosis patient group; (3) An analysis device for comparing the metabolic marker levels of the subject with known grouping information and outputting a risk or prognosis assessment result.

10. Use of a metabolic marker composition in constructing a computational model for diagnosing pulmonary fibrosis or evaluating the prognosis of pulmonary fibrosis, characterized in that: The metabolic marker composition includes palmitoylethanolamide and 2-amino-1,3,4-octadecantriol.

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