Metabolic marker, kit and application of metabolic marker in auxiliary diagnosis of schizophrenia

Abstract

The invention provides a group of metabolic markers, a kit and application of the metabolic markers in auxiliary diagnosis of schizophrenia. The metabolic markers comprise arachidonic acid, palmitamide and lysophosphatidylcholine (LPC) (17: 0 / 0: 0). By utilizing the metabolic marker, auxiliary diagnosis of schizophrenia can be rapidly and accurately completed in the early stage of disease attack.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and to the application of metabolic markers in disease diagnosis, and in particular to metabolic markers, kits and their application in assisting the diagnosis of schizophrenia. Background Art

[0002] Schizophrenia is one of the serious diseases that cause mental disability and one of the top ten causes of disability in the world, which has brought a heavy burden to society, patients and their families. The current global prevalence of schizophrenia is 0.32%, and the number of patients worldwide announced by the WHO in 2022 is 24 million. Schizophrenia is manifested by hallucinations, delusions, disordered speech and behavior, emotional indifference, cognitive impairment, and occupational and social dysfunction that are detached from reality. The probability of early death in schizophrenia patients is 2-3 times higher than that of the general population, which is related to a higher risk of suicide and an increased possibility of concurrent physical illness.

[0003] Therefore, early identification of schizophrenia is crucial. However, the current diagnosis of schizophrenia still relies on the doctor's subjective assessment of its clinical symptoms. There is no clear laboratory test and a lack of reliable physiological, biochemical and pathological indicators. Biomarkers can help clinicians diagnose schizophrenia faster and more timely. Ideal biomarkers should have the characteristics of easy detection, simple operation, high specificity and high sensitivity. In recent years, molecular markers for the diagnosis of schizophrenia based on plasma and serum have gradually gained attention in the diagnosis of schizophrenia, but these markers are mostly concentrated at the gene and protein levels, with insufficient sensitivity and specificity, and cannot meet clinical needs.

[0004] Therefore, how to provide a marker that can identify schizophrenia at an early stage with good accuracy and sensitivity is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a group of metabolic markers, which are used for auxiliary diagnosis of schizophrenia.

[0006] The first object of the present invention is to provide a group of metabolic markers, wherein the metabolic markers include arachidonic acid, palmitamide and lysophosphatidylcholine LPC (17:0 / 0:0).

[0007] The metabolic markers of the present invention also include lysophosphatidylethanolamine LysoPE (0:0 / 20:4).

[0008] The second aspect of the present invention also provides the use of a group of metabolite markers in the auxiliary diagnosis of schizophrenia, which includes using the above-mentioned metabolite markers for auxiliary diagnosis of schizophrenia.

[0009] The application of the metabolic markers of the present invention in assisting the diagnosis of schizophrenia, wherein the area under the curve of arachidonic acid is 0.778;

[0010] The area under the curve of palmitamide is 0.788;

[0011] The area under the curve of the lysophosphatidylcholine LPC (17:0 / 0:0) was 0.787;

[0012] The area under the curve of the lysophosphatidylethanolamine LysoPE (0:0 / 20:4) is 0.756.

[0013] The third aspect of the present invention also provides a kit for auxiliary diagnosis of schizophrenia, which comprises the above-mentioned metabolic marker.

[0014] In the kit of the present invention, the internal standard solution of the kit is a methanol solution containing ketoprofen and L-2-chlorophenylalanine.

[0015] In the kit of the present invention, the concentration of ketoprofen in the methanol solution is 500 ng / mL.

[0016] In the kit of the present invention, the concentration of L-2-chlorophenylalanine in the methanol solution is 50 ng / mL.

[0017] Beneficial effects:

[0018] The metabolic markers, kit and use thereof in the auxiliary diagnosis of schizophrenia described in the present invention provide a group of metabolic markers including arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0) and phosphatidylethanolamine LysoPE (0:0 / 20:4). The above-mentioned metabolic markers can be used to quickly and accurately complete the auxiliary diagnosis of schizophrenia in the early stage of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a principal component analysis (PCA) score diagram of the positive ion mode of schizophrenia patients (Schizophrenia, SP) vs. healthy control group (Control, C) using the metabolic markers described in the present invention;

[0020] Figure 2 It is a principal component analysis (PCA) score diagram of the negative ion pattern of schizophrenia patients (Schizophrenia, SP) vs. healthy control group (Control, C) using the metabolic markers described in the present invention;

[0021] Figure 3It is an orthogonal partial least squares discriminant analysis (OPLS-DA) score diagram of the metabolic markers of the present invention for schizophrenia patients (Schizophrenia, SP) vs. healthy control group (Control, C) in positive ion mode;

[0022] Figure 4 It is an orthogonal partial least squares discriminant analysis (OPLS-DA) score diagram of the metabolic markers of the present invention for negative ion patterns of schizophrenia patients (Schizophrenia, SP) vs. healthy control group (Control, C);

[0023] Figure 5 It is a permutation test diagram of the orthogonal partial least squares discriminant analysis OPLS-DA model of the metabolic markers of the present invention for schizophrenia patients (Schizophrenia, SP) vs. healthy control group (Control, C) in positive ion mode;

[0024] Figure 6 It is a permutation test diagram of the orthogonal partial least squares discriminant analysis OPLS-DA model of the metabolic markers of the present invention for the negative ion pattern of schizophrenia patients (Schizophrenia, SP) vs. healthy control group (Control, C);

[0025] Figure 7 The receiver operating characteristic curve (ROC) analysis of the metabolic marker arachidonic acid described in the present invention alone for diagnosing schizophrenia in the training set;

[0026] Figure 8 The receiver operating characteristic curve (ROC) analysis of the metabolite marker palmitamide described in the present invention alone for diagnosing schizophrenia in the training set;

[0027] Fig. 9 The receiver operating characteristic curve (ROC) analysis of the metabolic marker lysophosphatidylcholine LPC (17:0 / 0:0) described in the present invention for the diagnosis of schizophrenia in the training set alone;

[0028] Fig.10 The receiver operating characteristic curve (ROC) analysis of the metabolic marker lysophosphatidylethanolamine LysoPE (0:0 / 20:4) described in the present invention for the diagnosis of schizophrenia in the training set alone;

[0029] Fig.11 The receiver operating characteristic curve (ROC) analysis of the diagnosis of schizophrenia in the training set by combining the four metabolic markers of arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0), and lysophosphatidylethanolamine LysoPE (0:0 / 20:4) described in the present invention;

[0030] Fig.12 The receiver operating characteristic curve (ROC) analysis of the metabolic marker arachidonic acid described in the present invention alone for the diagnosis of schizophrenia in the test set;

[0031] Fig.13 The receiver operating characteristic curve (ROC) analysis of the metabolite marker palmitamide described in the present invention for diagnosing schizophrenia in the test set alone;

[0032] Fig.14 The receiver operating characteristic curve (ROC) analysis of the metabolite marker lysophosphatidylcholine LPC (17:0 / 0:0) described in the present invention for schizophrenia diagnosis in the test set alone;

[0033] Fig.15 The receiver operating characteristic curve (ROC) analysis of the metabolite marker lysophosphatidylethanolamine LysoPE (0:0 / 20:4) described in the present invention for diagnosing schizophrenia in the test set alone;

[0034] Fig.16 The receiver operating characteristic curve (ROC) analysis of the diagnosis of schizophrenia in the test set was performed by combining the four metabolite markers of arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0), and lysophosphatidylethanolamine LysoPE (0:0 / 20:4) described in the present invention;

[0035] Fig.17 This is a verification graph of the diagnostic accuracy of the four metabolic markers of the present invention, namely, arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0), and lysophosphatidylethanolamine LysoPE (0:0 / 20:4), in the training set for schizophrenia patients when used in combination;

[0036] Fig.18 This is a verification graph of the diagnostic accuracy of schizophrenia patients in a test set using the combined use of the four metabolic markers of arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0), and lysophosphatidylethanolamine LysoPE (0:0 / 20:4) described in the present invention. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art described in the present invention are generally understood. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or verification of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] As used herein, "include", "including", "have", "contain", etc. are all open terms, which means including but not limited to. As used herein, "and / or" includes any or all combinations of the things mentioned. Unless otherwise specified, % refers to mass volume percentage.

[0041] like Figures 1 to 18 As shown, the present invention provides a group of metabolic markers for auxiliary diagnosis of schizophrenia, wherein the metabolic markers include arachidonic acid, palmitamide and lysophosphatidylcholine LPC (17:0 / 0:0).

[0042] In certain embodiments, the metabolite markers are used for auxiliary diagnosis of schizophrenia, and the metabolite markers include arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0), and lysophosphatidylethanolamine LysoPE (0:0 / 20:4).

[0043] The second aspect of the present invention also provides the use of a group of markers in assisting the diagnosis of schizophrenia, which includes the use of the above-mentioned metabolic markers.

[0044] In the above scheme, the application of the metabolic markers in assisting the diagnosis of schizophrenia, wherein:

[0045] The area under the curve of arachidonic acid is 0.778;

[0046] The area under the curve of palmitamide is 0.788;

[0047] The area under the curve of the lysophosphatidylcholine LPC (17:0 / 0:0) was 0.787;

[0048] The area under the curve of the lysophosphatidylethanolamine LysoPE (0:0 / 20:4) is 0.756.

[0049] The third aspect of the present invention also provides a kit for auxiliary diagnosis of schizophrenia, which comprises the above-mentioned metabolic marker.

[0050] In the above scheme, in the kit, the internal standard solution of the kit is a methanol solution containing ketoprofen and L-2-chlorophenylalanine.

[0051] In the above scheme, in the kit, the concentration of ketoprofen in the methanol solution is 500 ng / mL.

[0052] In the above scheme, in the kit, the concentration of L-2-chlorophenylalanine in the methanol solution is 50 ng / mL.

[0053] Example

[0054] In order to verify that the metabolic markers described in the present invention can quickly and accurately assist in the diagnosis of schizophrenia in the early stages of schizophrenia, the following experiment was conducted.

[0055] 1. Experimental instruments and reagents

[0056] The U1timate 3000 ultra-high performance liquid chromatography system (Dionex, USA) was connected in series with a Q-Exactive quadrupole-electrostatic field orbitrap high-resolution mass spectrometer (Thermo Fisher Scientific, USA). The chromatographic column used was ACQUITY HSS T3 1.8 μm (2.1×100 mm) (Waters, Milford, MA, USA); Heraeus Fresco 17 centrifuge (Thermo Fisher Scientific).

[0057] HPLC grade methanol, acetonitrile, and formic acid were purchased from Thermo Fisher, USA; L-2-chlorophenylalanine and ketoprofen standards were purchased from J&K Chemical (Beijing, China); Wahaha purified water was used in the experiments;

[0058] 2. Experimental Methods

[0059] 1. Experimental samples

[0060] After obtaining the patient's consent, hospitalized patients who met the diagnostic criteria for schizophrenia in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) were included, and a total of 116 schizophrenia patients admitted to the inpatient department of Zhumadian Second People's Hospital from May 2023 to August 2023 were collected. In addition, the physical examination department collected 110 healthy volunteers as a normal control group.

[0061] From the above 226 samples, 88 healthy samples and 93 schizophrenia samples were randomly selected to form a training set, which was used to find the differential metabolites between schizophrenia patients and healthy people and build a diagnostic model;

[0062] The remaining 22 healthy subjects samples and 23 schizophrenia patient samples constituted the test set to verify the ability of differential metabolites as metabolic markers for diagnosing schizophrenia.

[0063] All blood samples were collected from patients in the early morning when they were fasting. 3 mL of venous blood was collected from each subject and placed in an EDTA anticoagulant blood collection tube. After collection, the blood was centrifuged at 3000 r / min for 10 min at 4°C, and the supernatant plasma was aspirated and immediately stored in a -80°C refrigerator after packaging.

[0064] 2. Preparation of plasma samples

[0065] 2.1 After taking out the plasma sample, place it on ice to thaw. After vortex mixing, pipette 50 μL of plasma sample into a 1.5 mL centrifuge tube, add 150 μL of methanol solution containing internal standard (containing ketoprofen 500 ng / mL, L-2-chlorophenylalanine 50 ng / mL), vortex for 30 seconds to mix, centrifuge at 13000 rpm for 10 min (4°C), and pipette the supernatant into a sample vial.

[0066] 2.2 Take 10 μL of each plasma sample and vortex mix, and perform QC sample pretreatment in the same way as above.

[0067] In order to ensure the reliability of the data, QC sample analysis was interspersed in the process of all sample metabolomics data collection. Before sample analysis, 5 QC samples were tested continuously. After the instrument was stable, sample analysis began, and QC solution was tested every 5 samples.

[0068] 3. Detection methods of metabolites in plasma

[0069] Chromatographic conditions Chromatographic separation was performed by ultra-high performance liquid chromatography (UPLC, Waters Ultimate 3000, USA), the chromatographic column was ACQUITY UPLC BEH C18 (50×2.1 mm, 1.7 μm), and the column temperature was 40°C. The mobile phase composition was acetonitrile in phase A and 0.1% formic acid in water in phase B. Gradient elution conditions:

[0070] 0~0.5min,5%A;

[0071] 0.5~1.0min,5%~60%A;

[0072] 0.5~1.0min,5%~60%A;

[0073] 1.0~7.0min, 60%~80%A;

[0074] 7.0~9.0min,80%~100%A;

[0075] 9.0~11.0min,100%A;

[0076] 11.0~13.0min,5%A;

[0077] The flow rate was 0.2 mL / min, and the post-column effluent was directly fed into the mass spectrometer for detection without splitting.

[0078] Mass spectrometry conditions Mass spectrometry analysis was performed using a Q Exactive quadrupole-electrostatic field orbital trap high-resolution mass spectrometer, and the ion source was a heatable electrospray ionization source (HESI). The auxiliary gas temperature was 300℃, the ion source temperature was 350℃, the capillary temperature was 320℃, the auxiliary gas flow rate was 10uL / min, the mass spectrometry analysis scanning mode was full scan / ddms2, the primary mass spectrometry resolution was 70000, the secondary mass spectrometry resolution was 17500, and the mass-to-charge ratio window width was set to 8; the collision energy gradient was 20eV, 40eV and 60eV; the scanning range in positive and negative ion modes was 80.00-1200.00m / z, and the spray voltage and sheath gas flow rate were 3.50kV, 40μL / min and 2.80kV, 38μL / min, respectively. All samples were randomly injected.

[0079] 4. Data processing and analysis

[0080] All data were collected and processed by Thermo Xcalibur software (version 3.0, Thermo Scientific, USA), and then Compound Discovery software (version 3.0, Thermo Scientific, USA) was used for peak correction, peak matching and peak alignment information extraction. The specific parameters are as follows: the retention time (RT) width is set to 0.1min, the mass width is 5ppm, and the intensity threshold for filtering the ion peak is 1,000,000 in both positive and negative ion modes. The data set was imported into the multivariate statistical analysis software SIMCA (version 14.1, Umetrics, Sweden) for orthogonal partial least squares discriminant analysis (OPLS-DA), as well as 200 permutation tests to assess the risk of model overfitting. By establishing an OPLS-DA model, the variable weight importance ranking (VIP value) was obtained, and the metabolites with VIP>1 were subjected to an independent sample t-test to screen out statistically significant (P<0.05) differential metabolites, and the primary accurate m / z, secondary fragment ion map, retention time (RT) and other information collected by mass spectrometry were compared with the database for identification. The databases include ChemSpider and MassList with primary mass spectrometry information; mzCloud with primary and secondary mass spectrometry information, the laboratory's self-built database mzVault, the Human Metabolome database (HMBD, http: / / hmdb.ca / ), MassBank (https: / / massbank.eu / MassBank / ), and the PubChem compound database (https: / / pubchem.ncbi.nlm.nih.gov / ). Finally, the compound structure was confirmed by comparison with the standard.

[0081] SPSS software (version 26.0, SPSS, Chicago, Illinois) was used to screen out factors with significant influence as independent variables through stepwise regression analysis, and the optimal regression equation was established to find potential biomarkers for distinguishing schizophrenia patients from healthy people. The diagnostic accuracy (Area Under Curve, AUC), sensitivity (Sensitivity) and specificity (specificity) of potential biomarkers in the training set and test set were evaluated by combining binary logistic regression and receiver operating characteristic (ROC) curve analysis. The coordinate value taken when calculating the maximum value of the Youden index through ROC curve analysis was the cut-off value, and the test set samples were predicted to evaluate the predictive ability of the metabolic marker group for schizophrenia.

[0082] 3. Experimental Results

[0083] Through principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), such as Figures 1 to 3 As shown, Figures 1 to 6 A represents the positive ion mode and B represents the negative ion mode. It can be seen that in the training set, the schizophrenia group (Schizophrenia, SP) and the normal control group (Control, C) composed of healthy people can be clearly separated and clustered separately, indicating that there are significant differences in the endogenous metabolites of schizophrenia patients and healthy people. The model evaluation parameters of OPLS-DA show that the model has good explanatory and predictive abilities. The response ranking test (RPT) method was used to perform permutation verification after 200 modelings, which confirmed that the model was stable and reliable without overfitting.

[0084] Differential metabolites were screened out with VIP>1.0 and p<0.05 as the standard, and four metabolic markers were further screened by SPSS stepwise regression analysis, namely, arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0), and lysophosphatidylethanolamine LysoPE (0:0 / 20:4), and the results are shown in Table 1. Compared with the normal control group, the expression levels of arachidonic acid and lysophosphatidylcholine LPC (17:0 / 0:0) in the plasma of schizophrenia patients were significantly downregulated, and the expression levels of palmitamide and lysophosphatidylethanolamine LysoPE (0:0 / 20:4) in the plasma of schizophrenia patients were significantly upregulated, which can be used as potential diagnostic markers for schizophrenia.

[0085] Table 1 Plasma metabolic markers in patients with schizophrenia

[0086]

[0087] The diagnostic accuracy of the above four metabolic markers for schizophrenia was further tested by drawing ROC curves. The results showed that: Figure 7-11 As shown in the figure, in the training set, when arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0), and lysophosphatidylethanolamine LysoPE (0:0 / 20:4) were used alone for the diagnosis of schizophrenia, the areas under the ROC curve (AUC) were 0.778, 0.788, 0.787, and 0.756, respectively. When the four markers were used in combination for diagnosis, the AUC reached 0.942 (sensitivity 87.1%, specificity 90.9%). Figure 12-16 As shown in the figure, in the test set, when arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0), and lysophosphatidylethanolamine LysoPE (0:0 / 20:4) were used individually for the diagnosis of schizophrenia, the areas under the ROC curve (AUC) were 0.681, 0.915, 0.674, and 0.776, respectively. When the four markers were used in combination for diagnosis, the AUC reached 0.972 (sensitivity 91.3%, specificity 95.5%).

[0088] In the ROC curve evaluation method, when the area under the ROC curve (AUC) is greater than 0.5, the closer it is to 1, the better the diagnostic effect. AUC has lower accuracy when it is between 0.5 and 0.7, has certain accuracy when it is between 0.7 and 0.9, and has higher accuracy when it is above 0.9. It has been verified that when the four metabolites of arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0), and lysophosphatidylethanolamine LysoPE (0:0 / 20:4) are used in combination, the AUC is as high as 0.9 or above, with high diagnostic accuracy.

[0089] In the training set, the optimal cut-off value (Cut-off value) of arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0), and lysophosphatidylethanolamine LysoPE (0:0 / 20:4) was obtained for joint diagnosis based on the best sensitivity and specificity. The sample prediction using this cut-off value showed that the prediction accuracy of the metabolic marker group composed of these four metabolites for the training set was 94.2%, and the prediction accuracy for the test set was 97.2%. The results are as follows Figures 17-18 shown.

[0090] In conclusion, arachidonic acid, palmitamide, lysophosphatidylcholine LPC (17:0 / 0:0) and lysophosphatidylethanolamine LysoPE (0:0 / 20:4) can be used in combination to diagnose schizophrenia with high diagnostic accuracy.

[0091] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

Claims

1. A group of metabolic markers for use in the auxiliary diagnosis of schizophrenia, characterized in that: The metabolic markers include arachidonic acid, palmitamide and lysophosphatidylcholine LPC (17:0 / 0:0).

2. The metabolic marker according to claim 1, characterized in that Also included is lysophosphatidylethanolamine LysoPE (0:0 / 20:4).

3. Application of a group of markers in assisting the diagnosis of schizophrenia, characterized in that: The method comprises using the metabolic marker as described in any one of claims 1 to 2.

4. Use of a group of markers according to claim 3 in assisting the diagnosis of schizophrenia, characterized in that: The area under the curve of arachidonic acid is 0.778; The area under the curve of palmitamide is 0.788; The area under the curve of the lysophosphatidylcholine LPC (17:0 / 0:0) was 0.787; The area under the curve of the lysophosphatidylethanolamine LysoPE (0:0 / 20:4) is 0.

756.

5. A kit for auxiliary diagnosis of schizophrenia, characterized in that: The method comprises the metabolic marker according to any one of claims 1 to 2.

6. The kit according to claim 5, characterized in that The internal standard solution of the kit is a methanol solution containing ketoprofen and L-2-chlorophenylalanine.

7. The kit according to claim 6, characterized in that The concentration of the ketoprofen in the methanol solution is 500 ng / mL.

8. The kit according to claim 6, characterized in that The concentration of L-2-chlorophenylalanine in the methanol solution is 50 ng / mL.

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