Biomarker related to Parkinson's disease and application thereof

Abstract

The invention specifically relates to a kit for diagnosing whether a subject suffers from a neurodegenerative disease, especially Parkinson's disease (PD) or idiopathic tremor (ET), and application of a reagent for determining the level of a biomarker in a biological sample in preparation of the kit.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, virology and immunology, in particular the field of immunological diagnosis. The present application specifically relates to a kit for diagnosing whether a subject suffers from a neurodegenerative disease, in particular Parkinson's disease (PD) or essential tremor (ET), and the use of reagents for determining the level of biomarkers in a biological sample in the manufacture of a kit. BACKGROUND

[0002] The main pathological change of Parkinson's disease (PD) is the selective and progressive degeneration of dopamine (DA) neurons in the substantia nigra compacta of the midbrain. However, the specific pathogenesis of PD is still unclear. Currently, the diagnosis of PD is mainly based on the typical motor symptoms and signs, and there is a lack of precise and effective diagnostic biomarkers or indicators.

[0003] According to the recent standards of the International Movement Disorder Society, the course of PD is divided into preclinical and clinical PD. The preclinical stage includes the risk period and prodromal PD, during which no motor symptoms appear, and the core manifestations are non-motor symptoms such as constipation, olfactory reduction / loss, rapid eye movement sleep disorder (RBD), etc. The clinical PD is divided into early, middle and late stages according to Hoehn-Yahr classification, among which 1-2 stages are early PD, 2.5-3 stages are middle PD, and 4-5 stages are late PD. The diagnosis of PD is mainly based on typical clinical symptoms, but due to the lack of precision in current clinical diagnostic criteria, it often takes 3-5 years of follow-up to diagnose PD. In addition, essential tremor (ET) is a kind of idiopathic disease with tremor as the main manifestation, which is easily confused with early PD in clinical practice. Parkinson's disease often occurs in old age, and this period is also the peak age of essential tremor, resulting in many essential tremors being misdiagnosed as Parkinson's disease. Although typical Parkinson's disease has the characteristics of static tremor, muscle rigidity and motor retardation, it often lacks characteristic manifestations in the early stage of the disease, especially when it only has tremor at the onset, which often leads to misdiagnosis.

[0004] Therefore, it is necessary to find more effective diagnostic markers for PD patients, so that they can start intervention and treatment earlier, and better slow down the progression and control the symptoms of the disease. SUMMARY

[0005] The present application inventors obtained differential metabolites between primary Parkinson's disease (PD) patients, essential tremor (ET) patients and healthy subjects through analysis, and used the differential metabolites and combinations thereof to establish a prediction model. The model verified that these metabolites have strong discrimination ability for primary Parkinson's disease (PD) patients, essential tremor (ET) patients and healthy subjects. Therefore, the metabolites or combinations of metabolites of the present application are helpful for the diagnosis of PD or ET, and can simultaneously accurately distinguish PD patients, ET patients and healthy subjects.

[0006] Uses

[0007] In one aspect, the present application provides use of a reagent for determining a biomarker level in a biological sample in the preparation of a kit for diagnosing whether a subject has a neurodegenerative disease;

[0008] wherein the biomarker comprises any 1, 2, 3, 4, or 5 of biliverdin, erythritol, 11-dehydrothromboxane B2, neo-orientaline, adenosine monophosphate.

[0009] In certain embodiments, the biomarker is selected from biliverdin, erythritol, 11-dehydrothromboxane B2, neo-orientaline, adenosine monophosphate, or any combination thereof.

[0010] In certain embodiments, the neurodegenerative disease is selected from Parkinson's disease (PD), essential tremor (ET), Alzheimer's disease, or vascular dementia.

[0011] In certain embodiments, the kit is used to diagnose whether a subject has PD.

[0012] In certain embodiments, the kit is used to diagnose whether a subject has ET.

[0013] In certain embodiments, the kit is used to distinguish any two or all three of a subject with PD, a subject with ET, and a healthy subject.

[0014] In certain embodiments, the kit is used to distinguish a subject with PD and a subject with ET.

[0015] In certain embodiments, the kit is used to distinguish a subject with PD and a healthy subject.

[0016] In certain embodiments, the kit is used to distinguish a subject with ET and a healthy subject.

[0017] In certain embodiments, the kit is used to distinguish a subject with PD, a subject with ET, and a healthy subject.

[0018] In certain embodiments, the biomarker is 11-dehydrothrombix B2, propylthiosinigrone, adenosine monophosphate, biliverdin, or erythritol.

[0019] In certain embodiments, the biomarker is selected from any two of 11-dehydrothrombix B2, propylthiosinigrone, adenosine monophosphate, biliverdin, and erythritol.

[0020] In certain embodiments, the biomarker is selected from 11-dehydrothrombix B2 and propylthiosinigrone, 11-dehydrothrombix B2 and adenosine monophosphate, 11-dehydrothrombix B2 and biliverdin, 11-dehydrothrombix B2 and erythritol, propylthiosinigrone and adenosine monophosphate, propylthiosinigrone and biliverdin, propylthiosinigrone and erythritol, adenosine monophosphate and biliverdin, adenosine monophosphate and erythritol, or biliverdin and erythritol.

[0021] In certain embodiments, the biomarker is selected from any three of 11-dehydrothrombix B2, propylthiosinigrone, adenosine monophosphate, biliverdin, and erythritol.

[0022] In certain embodiments, the biomarker is selected from 11-dehydrothrombix B2, propylthiosinigrone, and adenosine monophosphate; 11-dehydrothrombix B2, propylthiosinigrone, and biliverdin; 11-dehydrothrombix B2, propylthiosinigrone, and erythritol; 11-dehydrothrombix B2, adenosine monophosphate, and biliverdin; 11-dehydrothrombix B2, adenosine monophosphate, and erythritol; or 11-dehydrothrombix B2, biliverdin, and erythritol.

[0023] In certain embodiments, the biomarker is selected from any four of 11-dehydrothrombix B2, propylthiosinigrone, adenosine monophosphate, biliverdin, and erythritol.

[0024] In certain embodiments, the biomarker is selected from 11-dehydrothrombix B2, propylthiosinigrone, adenosine monophosphate, and biliverdin; 11-dehydrothrombix B2, propylthiosinigrone, adenosine monophosphate, and erythritol; 11-dehydrothrombix B2, propylthiosinigrone, biliverdin, and erythritol; 11-dehydrothrombix B2, adenosine monophosphate, biliverdin, and erythritol; or propylthiosinigrone, adenosine monophosphate, biliverdin, and erythritol.

[0025] In certain embodiments, the biomarker is 11-dehydrothrombix B2, propylthiosinigrone, adenosine monophosphate, biliverdin, and erythritol.

[0026] In certain embodiments, the 11-dehydrogenated thromboxane B2 has a CAS number of 67910-12-7. In certain embodiments, the neopetaside has a CAS number of 4567-33-3. In certain embodiments, the adenosine monophosphate has a CAS number of 61-19-8. In certain embodiments, the biliverdin has a CAS number of 114-25-0. In certain embodiments, the erythritol has a CAS number of 149-32-6.

[0027] In certain embodiments, the biological sample is obtained from whole blood, serum, plasma, or fecal matter of the subject.

[0028] In certain embodiments, the subject is a mammal, such as a human.

[0029] In certain embodiments, the kit further comprises a pre-treatment reagent or tool for pre-treating the biological sample.

[0030] In certain embodiments, the pre-treatment reagent or tool is used for extracting the biomarker in the biological sample.

[0031] In certain embodiments, the pre-treatment reagent or tool is selected from the group consisting of: methanol, 2-chloro-L-phenylalanine, a grinding tool (e.g., glass beads), a filtration membrane, or any combination thereof.

[0032] In certain embodiments, the reagent (e.g., the first, second, third, fourth, fifth reagent, or combination of reagents) determines the level of the biomarker in the biological sample by a method selected from the group consisting of: chromatography and / or mass spectrometry, fluorescence assay, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), and turbidimetry.

[0033] In certain embodiments, the reagent determines the level of the biomarker in the biological sample by spectroscopy, liquid or gas chromatography, mass spectrometry, liquid or gas chromatography coupled with mass spectrometry.

[0034] In certain embodiments, the kit further comprises a reagent and / or consumable for spectroscopy, a reagent and / or consumable for chromatography, a reagent and / or consumable for mass spectrometry, or any combination thereof.

[0035] In certain embodiments, the reagent and / or consumable for chromatography is selected from the group consisting of: a chromatography column, an aqueous acetonitrile solution, ammonium acetate, ammonium formate, formic acid, or any combination thereof.

[0036] In certain embodiments, the reagent and / or consumable for mass spectrometry is selected from the group consisting of: a mass spectrometry column, formic acid, acetonitrile, or any combination thereof.

[0037] Kit

[0038] In another aspect, the present application provides a kit for diagnosing whether a subject has a neurodegenerative disease, the kit comprising reagents for determining the level of a biomarker in a biological sample, the biomarker selected from biliverdin, erythritol, 11-dehydrothromboxane B2, neo-snakekin, adenosine monophosphate, or any combination thereof.

[0039] In certain embodiments, the neurodegenerative disease is selected from Parkinson's disease (PD), essential tremor (ET), Alzheimer's disease, or vascular dementia.

[0040] In certain embodiments, the kit is used to diagnose whether a subject has PD.

[0041] In certain embodiments, the kit is used to diagnose whether a subject has ET.

[0042] In certain embodiments, the kit is used to distinguish any two or all three of a subject having PD, a subject having ET, and a healthy subject.

[0043] In certain embodiments, the kit is used to distinguish a subject having PD and a subject having ET.

[0044] In certain embodiments, the kit is used to distinguish a subject having PD and a healthy subject.

[0045] In certain embodiments, the kit is used to distinguish a subject having ET and a healthy subject.

[0046] In certain embodiments, the kit is used to distinguish a subject having PD, a subject having ET, and a healthy subject.

[0047] In certain embodiments, the biomarker is 11-dehydrothromboxane B2, neo-snakekin, adenosine monophosphate, biliverdin, or erythritol.

[0048] In certain embodiments, the biomarker is selected from any two of 11-dehydrothromboxane B2, neo-snakekin, adenosine monophosphate, biliverdin, and erythritol.

[0049] In certain embodiments, the biomarker is selected from 11-dehydrothromboxane B2 and neo-snakekin, 11-dehydrothromboxane B2 and adenosine monophosphate, 11-dehydrothromboxane B2 and biliverdin, 11-dehydrothromboxane B2 and erythritol, neo-snakekin and adenosine monophosphate, neo-snakekin and biliverdin, neo-snakekin and erythritol, adenosine monophosphate and biliverdin, adenosine monophosphate and erythritol, or biliverdin and erythritol.

[0050] In certain embodiments, the biomarkers are selected from any three of 11-dehydrothromboxane B2, senkyunolide A, adenosine monophosphate, biliverdin, and erythritol.

[0051] In certain embodiments, the biomarkers are selected from 11-dehydrothromboxane B2, senkyunolide A, and adenosine monophosphate; 11-dehydrothromboxane B2, senkyunolide A, and biliverdin; 11-dehydrothromboxane B2, senkyunolide A, and erythritol; 11-dehydrothromboxane B2, adenosine monophosphate, and biliverdin; 11-dehydrothromboxane B2, adenosine monophosphate, and erythritol; or, 11-dehydrothromboxane B2, biliverdin, and erythritol.

[0052] In certain embodiments, the biomarkers are selected from any four of 11-dehydrothromboxane B2, senkyunolide A, adenosine monophosphate, biliverdin, and erythritol.

[0053] In certain embodiments, the biomarkers are selected from 11-dehydrothromboxane B2, senkyunolide A, adenosine monophosphate, and biliverdin; 11-dehydrothromboxane B2, senkyunolide A, adenosine monophosphate, and erythritol; 11-dehydrothromboxane B2, senkyunolide A, biliverdin, and erythritol; 11-dehydrothromboxane B2, adenosine monophosphate, biliverdin, and erythritol; or, senkyunolide A, adenosine monophosphate, biliverdin, and erythritol.

[0054] In certain embodiments, the biomarkers are 11-dehydrothromboxane B2, senkyunolide A, adenosine monophosphate, biliverdin, and erythritol.

[0055] In certain embodiments, the 11-dehydrothromboxane B2 has a CAS number of 67910-12-7. In certain embodiments, the senkyunolide A has a CAS number of 4567-33-3. In certain embodiments, the adenosine monophosphate has a CAS number of 61-19-8. In certain embodiments, the biliverdin has a CAS number of 114-25-0. In certain embodiments, the erythritol has a CAS number of 149-32-6.

[0056] In certain embodiments, the biological sample is obtained from a subject's whole blood, serum, plasma, or feces.

[0057] In certain embodiments, the subject is a mammal, e.g., a human.

[0058] In certain embodiments, the kit further comprises a pre-treatment reagent or means for pre-treating the biological sample.

[0059] In certain embodiments, the pre-treatment reagent or tool is used to extract the biomarker in the biological sample.

[0060] In certain embodiments, the pre-treatment reagent or tool is selected from the group consisting of: methanol, 2-chloro-L-phenylalanine, a grinding tool (e.g., glass beads), a filtration membrane, or any combination thereof.

[0061] In certain embodiments, the reagent (e.g., first, second, third, fourth, fifth reagent or combination of reagents) determines the level of biomarker in the biological sample by: a chromatographic and / or mass spectrometric assay, a fluorescence assay, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), and nephelometry.

[0062] In certain embodiments, the reagent determines the level of biomarker in the biological sample by spectroscopy, liquid or gas chromatography, mass spectrometry, liquid or gas chromatography coupled with mass spectrometry.

[0063] In certain embodiments, the kit further comprises a reagent and / or consumable for spectroscopy, a reagent and / or consumable for chromatography, a reagent and / or consumable for mass spectrometry, or any combination thereof.

[0064] In certain embodiments, the reagent and / or consumable for chromatography is selected from the group consisting of a chromatographic column, an aqueous acetonitrile solution, ammonium acetate, ammonium formate, formic acid, or any combination thereof.

[0065] In certain embodiments, the reagent and / or consumable for mass spectrometry is selected from the group consisting of a mass spectrometry column, formic acid, acetonitrile, or any combination thereof.

[0066] Method

[0067] In another aspect, the present application provides a method for diagnosing whether a subject has Parkinson’s disease (PD) or essential tremor (ET), comprising:

[0068] (1) obtaining a biological sample comprising a biomarker from the subject;

[0069] (2) determining the level of the biomarker in the biological sample;

[0070] (3) diagnosing whether the subject has Parkinson’s disease (PD) or essential tremor (ET) based on the level of the biomarker;

[0071] wherein the biomarker is selected from the group consisting of biliverdin, erythritol, 11-dehydrochiroloban B2, neo-oxypeucedanin, adenosine monophosphate, or any combination thereof.

[0072] In certain embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, or feces.

[0073] In certain embodiments, in step (2), the level of the biomarker in the biological sample is determined by spectroscopy, liquid or gas chromatography, mass spectrometry, liquid or gas chromatography coupled to mass spectrometry.

[0074] In certain embodiments, in step (3), the subject is diagnosed as having Parkinson's disease (PD) or essential tremor (ET) by comparing the level of the biomarker to a reference value. In such embodiments, the reference value is the level or range of the biomarker in a biological sample obtained from a normal population. In certain embodiments, the level of the biomarker in the biological sample is determined by chromatography and / or mass spectrometry assays, fluorescence assays, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), and turbidimetry.

[0075] In certain embodiments, the biomarker is 11-dehydrothromboxane B2, neo- snailcine, adenosine monophosphate, biliverdin, or erythritol.

[0076] In certain embodiments, the biomarker is selected from any two of 11- dehydrothromboxane B2, neo-snailcine, adenosine monophosphate, biliverdin, and erythritol.

[0077] In certain embodiments, the biomarker is selected from 11-dehydrothromboxane B2 and neo-snailcine, 11-dehydrothromboxane B2 and adenosine monophosphate, 11-dehydrothromboxane B2 and biliverdin, 11-dehydrothromboxane B2 and erythritol, neo-snailcine and adenosine monophosphate, neo-snailcine and biliverdin, neo-snailcine and erythritol, adenosine monophosphate and biliverdin, adenosine monophosphate and erythritol, or biliverdin and erythritol.

[0078] In certain embodiments, the biomarker is selected from any three of 11- dehydrothromboxane B2, neo-snailcine, adenosine monophosphate, biliverdin, and erythritol.

[0079] In certain embodiments, the biomarker is selected from 11-dehydrothromboxane B2, neo-snailcine, and adenosine monophosphate; 11-dehydrothromboxane B2, neo-snailcine, and biliverdin; 11-dehydrothromboxane B2, neo-snailcine, and erythritol; 11-dehydrothromboxane B2, adenosine monophosphate, and biliverdin; 11-dehydrothromboxane B2, adenosine monophosphate, and erythritol; or 11-dehydrothromboxane B2, biliverdin, and erythritol.

[0080] In certain embodiments, the biomarkers are selected from any four of 11-dehydrothromboxane B2, neopetaside, adenosine monophosphate, biliverdin, and erythritol.

[0081] In certain embodiments, the biomarkers are selected from 11-dehydrothromboxane B2, neopetaside, adenosine monophosphate, and biliverdin; 11-dehydrothromboxane B2, neopetaside, adenosine monophosphate, and erythritol; 11-dehydrothromboxane B2, neopetaside, biliverdin, and erythritol; 11-dehydrothromboxane B2, adenosine monophosphate, biliverdin, and erythritol; or, neopetaside, adenosine monophosphate, biliverdin, and erythritol.

[0082] In certain embodiments, the biomarkers are 11-dehydrothromboxane B2, neopetaside, adenosine monophosphate, biliverdin, and erythritol.

[0083] In certain embodiments, the CAS number of 11-dehydrothromboxane B2 is 67910-12-7. In certain embodiments, the CAS number of neopetaside is 4567-33-3. In certain embodiments, the CAS number of adenosine monophosphate is 61-19-8. In certain embodiments, the CAS number of biliverdin is 114-25-0. In certain embodiments, the CAS number of erythritol is 149-32-6.

[0084] Terminology

[0085] As used herein, the term "neurodegenerative disease" is a group of progressive diseases characterized by the massive loss of specific neurons. It mainly includes Parkinson's disease (PD), essential tremor (ET), Alzheimer's disease (AD), vascular dementia, etc.

[0086] As used herein, the term "biomarker" refers to a biochemical indicator that can mark the changes of system, organ, tissue, cell and subcellular structure or function, or the changes that can be suffered, with very wide use. Biomarkers can be used for disease diagnosis, disease staging, or to evaluate the safety and effectiveness of new drugs or new therapies in the target population.

[0087] As used herein, the term "reference value" refers to a predetermined value for a biomarker, derived from the levels of the biomarker in a control sample (e.g., a biological sample obtained from a healthy population). Reference values ​​can be used as thresholds to distinguish between subjects who may be at risk of a disease and those who are not. Reference values ​​can be relative values, ranges with upper and lower limits, averages, medians, etc. Those skilled in the art can select appropriate control samples, determine, and obtain reference values ​​using methods disclosed in the prior art. Such methods can be found, for example, in Burtis CA et al., 2008, Chapter 14, section "statistical treatment of reference values," which is incorporated herein by reference in its entirety.

[0088] As used herein, the term “subject” includes, but is not limited to, various animals, particularly mammals such as humans.

[0089] As used herein, the term "metabolite" or "metabolic product" refers to a substance produced during chemical or physical processes in the human body. It includes any chemical or biochemical product of metabolic processes, such as any compound produced through the processing, cleavage, or consumption of biomolecules. Examples of such molecules include, but are not limited to: acids and related compounds; mono, di, and tricarboxylic acids (saturated, unsaturated, aliphatic and cyclic, aryl, alkylaryl); aldehydes, keto acids; lactone forms; gibberellins; abscisic acid; alcohols, polyols, derivatives, and related compounds; ethanol, benzyl alcohol, methanol; propylene glycol, glycerol, phytol; inositol, furfuryl alcohol, menthol; aldehydes, ketones, quinones, derivatives, and related compounds; acetaldehyde, butyraldehyde, benzaldehyde, acrolein, furfural, glyoxal; acetone, butanone; anthraquinones; carbohydrates; monosaccharides, disaccharides, trisaccharides; alkaloids, amines, and other bases; pyridines (including nicotinic acid and nicotinamide); pyrimidines (including cytosine and thymine). The following are permitted compounds: purines (including guanine, adenine, xanthine / hypoxanthine, and kinetin); pyrrole; quinoline (including isoquinoline); morphinan, tropane, cinchonans, nucleotides, oligonucleotides, derivatives, and related compounds; guanosine, cytosine, adenosine, thymidine, and inosine; amino acids, oligopeptides, derivatives, and related compounds; esters; phenols and related compounds; heterocyclic compounds and derivatives; pyrrole, tetrapyrrole; flavonoids; indole; lipids (including fatty acids and triglycerides), derivatives, and related compounds; carotenoids, phytopenic acid, and sterols, isoprene-like compounds, including terpenes; and modified forms of the above molecules. In some embodiments, the metabolite is a product of the metabolism of an endogenous substance. In some embodiments, the metabolite is a product of the metabolism of an exogenous substance. In some embodiments, the metabolite is a product of the metabolism of both endogenous and exogenous substances.

[0090] Advantages of the invention

[0091] The biomarkers provided by the present application have a relatively accurate distinguishing ability for primary Parkinson's disease (PD) patients, essential tremor (ET) patients and healthy subjects. Therefore, the biomarkers or combinations of biomarkers of the present application can be used for the diagnosis of PD or ET, and can simultaneously accurately distinguish PD patients, ET patients and healthy subjects.

[0092] In addition, the levels of the above-mentioned biomarkers can be detected by collecting the blood or feces of the subject. Compared with traditional lumbar puncture detection, the detection of the biomarkers of the present application has less trauma and lower risk, thereby realizing rapid, convenient, non-invasive and non-radiation auxiliary diagnosis of PD or ET. BRIEF DESCRIPTION OF DRAWINGS

[0093] Figure 1 The difference of 11-dehydrothromboxane B2 between PD, ET and HC is shown.

[0094] Figure 2A The diagnostic results of 11-dehydrothromboxane B2 in distinguishing PD patients from HC healthy controls are shown.

[0095] Figure 2B The diagnostic results of 11-dehydrothromboxane B2 in distinguishing PD patients from ET patients are shown.

[0096] Figure 3 The difference of Neocnidilide between PD, ET and HC is shown.

[0097] Figure 4A The diagnostic results of Neocnidilide in distinguishing ET patients from HC healthy controls are shown.

[0098] Figure 4B The diagnostic results of Neocnidilide in distinguishing ET patients from PD patients are shown.

[0099] Figure 5 The difference of Adenosine monophosphate between PD, ET and HC is shown.

[0100] Figure 6 The diagnostic results of Adenosine monophosphate in distinguishing PD patients from ET patients are shown.

[0101] Figure 7 The diagnostic results of the combination of 11-dehydrothromboxane B2, Neocnidilide and Adenosine monophosphate in distinguishing PD patients from HC\ET population are shown.

[0102] Figure 8Diagnostic results showing the discrimination between PD vs HC\ET population after combination of 11-dehydrothromboxane B2, oualane and adenosine monophosphate.

[0103] Figure 9 Diagnostic results showing the discrimination between HC vs PD\ET population after combination of 11-dehydrothromboxane B2, oualane and adenosine monophosphate.

[0104] Figure 10 Diagnostic results showing the discrimination between PD and HC healthy controls for biliverdin.

[0105] Figure 11 Diagnostic results showing the discrimination between PD and HC healthy controls for biliverdin.

[0106] Figure 12 Diagnostic results showing the discrimination between ET and HC healthy controls for biliverdin.

[0107] Figure 13 Diagnostic results showing the discrimination between PD and ET patients for erythritol.

[0108] Figure 14A Diagnostic results showing the discrimination between PD and HC healthy controls for erythritol.

[0109] Figure 14B Diagnostic results showing the discrimination between PD and HC healthy controls for erythritol.

[0110] Figure 15 Diagnostic results showing the discrimination between PD vs HC\ET population for biliverdin and erythritol.

[0111] Figure 16 Diagnostic results showing the discrimination between ET vs HC\PD population for biliverdin and erythritol.

[0112] Figure 17 Diagnostic accuracy showing the discrimination between HC vs ET\PD population for biliverdin and erythritol.

[0113] Figure 18 Diagnostic results showing the discrimination between PD vs HC\ET for adenosine monophosphate and biliverdin.

[0114] Figure 19 Diagnostic results showing the discrimination between ET vs PD\HC for adenosine monophosphate and biliverdin.

[0115] Figure 20 Diagnostic results showing the discrimination between HC vs PD\ET for adenosine monophosphate and biliverdin. DETAILED DESCRIPTION

[0116] Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are for illustrative purposes only and should not be construed as limiting the scope of the present application. If the specific conditions are not specified in the examples, they are carried out under conventional conditions or according to the manufacturer's recommendations. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be obtained commercially.

[0117] Example 1 : Sample collection

[0118] Ten patients with primary Parkinson's disease (PD), ten patients with essential tremor (ET) and ten HC healthy controls were included in this application in 2021-2022 in the Department of Neurology of Zhejiang Second Hospital. All PD patients were diagnosed by at least one experienced neurologist according to the UK Brain Bank criteria diagnostic criteria and had not undergone anti-Parkinson's disease drug treatment before enrollment, and all HCs had no positive signs of nervous system. Those who meet one or more of the following conditions are excluded: 1. Past or present IBD history or other serious digestive diseases; 2. Active systemic inflammatory diseases such as tuberculosis, rheumatoid arthritis, etc.; 3. History of serious mental illness such as schizophrenia, major depression, etc.; 4. Other neurodegenerative diseases or secondary PD syndrome, PD superposition syndrome; 5. Severe organic diseases such as cancer, coronary heart disease, myocardial infarction and stroke, etc. The research protocol of this study conforms to the Declaration of Helsinki and has been approved by the Ethics Committee of Zhejiang Second Hospital. All subjects are fully aware of the process and program of this study and have voluntarily signed the informed consent form. All subjects received detailed demographic data collection and clinical evaluation. All PD patients received detailed clinical scale assessment of disease severity and cognitive, emotional and other states. In addition, all subjects were surveyed by non-motor symptom questionnaire to assess the frequency of Parkinson's disease-related non-motor symptoms.

[0119] Fecal and blood samples of the above 30 subjects were collected for subsequent analysis.

[0120] Example 2: Extraction and analysis of metabolites in fecal samples

[0121] Sample preparation

[0122] Metabolites in the fecal samples of the subjects were extracted by the following steps:

[0123] 1. Accurately weigh an appropriate amount of sample in a 2 mL centrifuge tube, add 600 μL of methanol (containing 2-chloro-L-phenylalanine 4 ppm), store at -20°C, vortex for 30 s;

[0124] 2. Add 100 mg of glass beads and put them into a tissue grinder, grind at 60 Hz for 90 s;

[0125] 3. Room temperature ultrasonic 10 min;

[0126] 4. 12,000 rpm 4℃ centrifugal 10 min, take the supernatant through 0.22 μm membrane filter, add the filtrate to the detection bottle, used for LC-MS detection.

[0127] Chromatography conditions

[0128] Thermo Vanquish (Thermo Fisher Scientific, USA) ultra-high performance liquid system, using ACQUITY HSST3 (2.1 × 150 mm, 1.8 μm) (Waters, Milford, MA, USA) chromatographic column, 0.25 mL / min flow rate, column temperature 40℃, injection volume 2 μL. Positive ion mode, mobile phase is 0.1% formic acid acetonitrile (C) and 0.1% formic acid water (D), gradient elution program: 0-1 min, 2% C; 1-9 min, 2%-50% C; 9-12 min, 50%-98% C; 12-13.5 min, 98% C; 13.5-14 min, 98%-2% C; 14-20 min, 2% C. Negative ion mode, mobile phase is acetonitrile (A) and 5 mM ammonium formate water (B), gradient elution program: 0-1 min, 2% A; 1-9 min, 2%-50% A; 9-12 min, 50%-98% A; 12-13.5 min, 98% A; 13.5-14 min, 98%-2% A; 14-17 min, 2% A.

[0129] Mass spectrometry conditions

[0130] Thermo Orbitrap Exploris 120 mass spectrometry detector (Thermo Fisher Scientific, USA), electrospray ion source (ESI), positive and negative ion modes respectively collect data. Positive ion spray voltage is 3.50 kV, negative ion spray voltage is -2.50 kV, sheath gas 30 arb, auxiliary gas 10 arb. Capillary temperature 325℃, with resolution 60000 for primary full scan, primary ion scan range m / z 100-1000, and use HCD for secondary fragmentation, collision voltage 30%, secondary resolution 15000, collect signals before 4 ions for fragmentation, while using dynamic exclusion to remove unnecessary MS / MS information.

[0131] Analysis of differential metabolites between PD, ET, HC groups

[0132] Through the above experiments, a total of 124 differentially expressed metabolites were identified between the PD vs HC groups, 56 between the ET vs HC groups, and 60 between the PD vs ET groups. Furthermore, among these differentially expressed metabolites, we identified several particularly prominent metabolites and conducted follow-up studies.

[0133] Example 3: Validation of metabolites in fecal samples

[0134] 11 -dehydrothromboxane B2

[0135] The method described in Example 2 was used to detect the difference in levels of the metabolite 11-dehydro-thromboxane B2 between PD and HC. The results showed a significant intergroup difference in 11-dehydro-thromboxane B2 levels between PD and HC. Figure 1 Even after adjusting for the gender and age of the participants, the differences remained significant (p). PD vs HC =0.046).

[0136] Furthermore, the receiver operating characteristic (ROC) curve was used to verify the discriminant ability of 11-dehydro-thromboxane B2 for PD vs HC. A logistic regression model was constructed with 11-dehydro-thromboxane B2 level as the independent variable and PD / HC grouping as the dependent variable. Based on the predicted results of the logistic regression model for PD / HC grouping and the actual PD / HC grouping, ROC curves were plotted, and the area under the curve (AUC value) was calculated. The analysis results showed that the AUC value of the ROC curve reached 0.90 (…). Figure 2A This study confirmed that 11-dehydro-thromboxane B2 has a good ability to distinguish between PD and HC.

[0137] Furthermore, the receiver operating characteristic (ROC) curve was used to verify the discriminant ability of 11-dehydro-thromboxane B2 for PD vs ET. A logistic regression model was constructed with 11-dehydro-thromboxane B2 level as the independent variable and PD / HC grouping as the dependent variable. Based on the predicted results of the logistic regression model for PD / ET grouping and the actual PD / ET grouping, ROC curves were plotted, and the area under the curve (AUC value) was calculated. The analysis results showed that the AUC value of the ROC curve reached 0.85 (…). Figure 2B), confirming that 11-Dehydro-thromboxane B2 has certain ability to distinguish ET vs PD.

[0138] Novel secoisolaricretic acid

[0139] The difference of Neocnidilide level between ET patients vs HC healthy controls was detected by the method of Example 2, and the results showed that Neocnidilide has significant difference between ET vs HC, ET vs PD ( Figure 3 ). Even after correction for the gender, age of the subjects, the difference is still significant (p ET vs HC = 0.038, p ET vsPD = 0.035).

[0140] Further, the ability of Neocnidilide to distinguish ET vs HC was verified by the receiver operating characteristic (ROC) curve. The logistic regression model was constructed with the level of Neocnidilide as the independent variable, and the ET\HC grouping as the dependent variable. According to the prediction results of the logistic regression model for PD\HC grouping and the true grouping of ET\HC, the ROC curve was drawn, and the area under the curve (ACU value) was calculated. The experimental results showed that the AUC value of the ROC curve reached 0.87 ( Figure 4A ), confirming that Neocnidilide has good ability to distinguish ET vs HC.

[0141] Further, the ability of Neocnidilide to distinguish ET vs PD was verified by the receiver operating characteristic (ROC) curve. The logistic regression model was constructed with the level of Neocnidilide as the independent variable, and the ET\PD grouping as the dependent variable. According to the prediction results of the logistic regression model for ET\PD grouping and the true grouping of ET\PD, the ROC curve was drawn, and the area under the curve (ACU value) was calculated. The experimental results showed that the AUC value of the ROC curve reached 0.83 ( Figure 4B ), confirming that Neocnidilide has good ability to distinguish ET vs PD.

[0142] Adenosine monophosphate

[0143] The levels of Adenosine monophosphate (AMP) were detected by the method of Example 2 to detect the difference between PD patients and ET patients, and the results showed that Adenosine monophosphate (AMP) had significant group difference between PD and ET Figure 5 ). Even after correction for the gender and age of the subjects, the difference was still significant (p PD vs ET = 0.025).

[0144] Further, the discriminant ability of Adenosine monophosphate (AMP) for PD vs ET was verified by the receiver operating characteristic (ROC) curve. The Adenosine monophosphate (AMP) level was used as the independent variable, and the ET\PD grouping was used as the dependent variable to construct a logistic regression model. According to the prediction results of the logistic regression model for PD\PD grouping and the true grouping of ET\PD, the ROC curve was drawn, and the area under the curve (ACU value) was calculated. The experimental results showed that the AUC value of the ROC curve reached 0.87 Figure 6 ), confirming that Adenosine monophosphate has good discriminant ability for PD vs ET.

[0145] 11 -dehydrothromboxane B2, novel secoisolaricretic acid, adenosine monophosphate in combination

[0146] The same mass spectrometry method as in Example 1 was used to detect the levels of three metabolites in the feces of PD patients vs ET patients. According to the levels of metabolites 11-Dehydro-thromboxane B2, Neocnidilide, and Adenosine monophosphate (AMP), a prediction model was established to distinguish PD vs HC\ET, ET vs PD\HC, and HC vs PD\ET: 11-Dehydro-thromboxane B2, Neocnidilide, and Adenosine monophosphate (AMP) levels were used as three independent variables, and PD vs HC\ET, ET vs PD\HC, and HC vs PD\ET groupings were used as dependent variables to construct a multiple logistic regression model. According to the prediction results of the multiple logistic regression model for each grouping and the true grouping, the ROC curve was drawn, and the area under the curve (ACU value) was calculated.

[0147] The experimental results show that the combination of 11-dehydro-thromboxane B2, neocnidilide, and adenosine monophosphate (AMP) has good discrimination ability for PD vs HC\ET, ET vs PD\HC, and HC vs PD\ET, and the AUC values of the ROC curves are 0.87 Figure 7 ), 0.93 Figure 8 ), and 0.92 Figure 9 ), respectively.

[0148] Example 4: Extraction and analysis of metabolites in blood samples

[0149] Sample preparation

[0150] The metabolites in the blood samples of the subjects are extracted by the following steps:

[0151] 1. Thaw the experimental samples at 4°C, and vortex the samples for 1 min after thawing to mix them evenly;

[0152] 2. Accurately transfer an appropriate amount of the sample into a 2 mL centrifuge tube;

[0153] 3. Add 400 μL of methanol solution (stored at -20°C) and vortex for 1 min;

[0154] 4. Centrifuge at 12,000 rpm at 4°C for 10 min, take all the supernatant, and transfer it to a new 2 mL centrifuge tube, and concentrate and dry it;

[0155] 5. Accurately add 150 μL of 2-chloro-L-phenylalanine (4 ppm) solution prepared with 80% methanol water (stored at 4°C) to reconstitute the sample, take the supernatant, filter it through a 0.22 μm membrane, and add the filtrate to a detection bottle for LC-MS detection.

[0156] Chromatography conditions

[0157] Thermo Vanquish (Thermo Fisher Scientific, USA) ultra-high performance liquid system, using ACQUITY HSST3 (2.1 x 150 mm, 1.8 μm) (Waters, Milford, MA, USA) column, 0.25 mL / min flow rate, 40 °C column temperature, 2 μL injection volume. Positive ion mode, mobile phase of 0.1% formic acid in acetonitrile (C) and 0.1% formic acid in water (D), gradient elution program: 0-1 min, 2% C; 1-9 min, 2%-50% C; 9-12 min, 50%-98% C; 12-13.5 min, 98% C; 13.5-14 min, 98%-2% C; 14-20 min, 2% C. Negative ion mode, mobile phase of acetonitrile (A) and 5 mM ammonium formate in water (B), gradient elution program: 0-1 min, 2% A; 1-9 min, 2%-50% A; 9-12 min, 50%-98% A; 12-13.5 min, 98% A; 13.5-14 min, 98%-2% A; 14-17 min, 2% A.

[0158] Mass spectrometry conditions

[0159] Thermo Orbitrap Exploris 120 mass spectrometer detector (Thermo Fisher Scientific, USA), electrospray ion source (ESI), positive and negative ion modes were used to collect data respectively. The positive ion spray voltage was 3.50 kV, and the negative ion spray voltage was -2.50 kV, the sheath gas was 30 arb, and the auxiliary gas was 10 arb. The capillary temperature was 325 °C, the first level full scan was performed at a resolution of 60000, the first level ion scan range was m / z 100-1000, and the HCD was used for secondary fragmentation, the collision voltage was 30%, the secondary resolution was 15000, and the signals of the first 4 ions were collected for fragmentation, and dynamic exclusion was used to remove unnecessary MS / MS information.

[0160] Differential metabolites between PD, ET, HC groups

[0161] By analyzing, 120 PD vs HC group difference metabolites, 110 ET vs HC group difference metabolites, and 34 PD vs ET group difference metabolites were found. Further, among these difference metabolites, several metabolites were found to be particularly prominent and were subjected to subsequent research.

[0162] Example 5: Validation of metabolites in blood samples

[0163] Biliverdin

[0164] The levels of metabolite Biliverdin were detected between different groups by the method of Example 4, and the results showed that it had significant group difference between PD vs HC, ET vs HC( Figure 10 ). The difference was still significant (p PD vs HC = 0.024, p ET vs HC = 0.040) even after correction for the gender and age of the subjects.

[0165] Further, the discriminant ability of Biliverdin for PD vs HC and ET vs HC was verified by the receiver operating characteristic (ROC) curve. The logistic regression model was constructed with the level of Biliverdin as the independent variable and the PD\HC, ET\HC grouping as the dependent variable. According to the prediction results of the logistic regression model on the grouping and the true grouping, the ROC curve was drawn, and the area under the curve (ACU value) was calculated. The experimental results showed that the AUC value of the ROC curve reached 0.96( Figure 11 ) and 0.94( Figure 12 ) respectively, confirming that Biliverdin had good discriminant ability for PD vs HC and ET vs HC.

[0166] Erythritol

[0167] The levels of metabolite Erythritol were detected between different groups by the method of Example 4, and the results showed that it had significant group difference between PD vs ET( Figure 13 ). The difference was still significant (p PD vs ET = 0.035) even after correction for the gender and age of the subjects.

[0168] Further, the discriminant ability of Erythritol for PD vs ET was verified by the receiver operating characteristic (ROC) curve. The logistic regression model was constructed with the level of Erythritol as the independent variable and the PD\ET grouping as the dependent variable. According to the prediction results of the logistic regression model on the grouping and the true grouping, the ROC curve was drawn, and the area under the curve (ACU value) was calculated. The experimental results showed that the AUC value of the ROC curve reached 0.94( Figure 14A ), confirming that Erythritol had good discriminant ability for PD vs ET.

[0169] Further, the ability of Erythritol to distinguish PD vs HC was verified by receiver operating characteristic (ROC) curve. A logistic regression model was constructed with the level of Erythritol as independent variable and PD\HC grouping as dependent variable. According to the prediction results of the logistic regression model on the grouping and the real grouping, the ROC curve was drawn and the area under the curve (ACU value) was calculated. The experimental results showed that the AUC value of the ROC curve reached 0.94 Figure 14B ), which confirmed that Erythritol had good ability to distinguish PD vs HC.

[0170] Biliverdin and erythritol in combination

[0171] The same mass spectrometry method as in Example 4 was used to detect the levels of two metabolites in the blood of PD patients vs ET patients. According to the levels of metabolites Biliverdin and Erythritol, a prediction model was established to distinguish PD vs HC\ET, ET vs PD\HC and HC vs PD\ET: a multiple logistic regression model was constructed with the levels of Biliverdin and Erythritol as two independent variables and PD vs HC\ET, ET vs PD\HC and HC vs PD\ET grouping as dependent variables, respectively. According to the prediction results of the multiple logistic regression model on each grouping and the real grouping, the ROC curve was drawn and the area under the curve (ACU value) was calculated.

[0172] The experimental results showed that the combination of Biliverdin and Erythritol exhibited good distinguishing ability for PD vs HC\ET, ET vs PD\HC and HC vs PD\ET, and the AUC values of the ROC curve reached 0.93 Figure 15 ), 0.79 Figure 16 ), 0.97 Figure 17 ), respectively.

[0173] Example 6: Validation of metabolites in fecal and blood samples

[0174] Adenosine monophosphate and biliverdin in combination

[0175] The levels of fecal metabolite Adenosine monophosphate (AMP) and blood metabolite Biliverdin were detected by the methods of Examples 3 and 4 to determine the differences between different groups.

[0176] According to the levels of metabolites Adenosine monophosphate (AMP) and Biliverdin, a prediction model was established to distinguish PD vs HC\ET, ET vs PD\HC and HC vs PD\ET: With the levels of Adenosine monophosphate (AMP) and Biliverdin as two independent variables, and PD vs HC\ET, ET vs PD\HC, and HC vs PD\ET groupings as dependent variables, a multiple logistic regression model was constructed. According to the prediction results of each grouping and the true grouping of the multiple logistic regression model, the ROC curve was drawn respectively, and the area under the curve (ACU value) was calculated.

[0177] The experimental results show that the combination of Adenosine monophosphate (AMP) and Biliverdin exhibits good discrimination ability for PD vs HC\ET, ET vs PD\HC, and HC vs PD\ET, and the AUC values of the ROC curve are 0.84 Figure 18 ), 0.83 Figure 19 ), and 0.95 Figure 20 ), respectively.

[0178] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details in light of the all teachings disclosed herein, and such changes are intended to be within the scope of the present application. The full scope of the application is given by the appended claims and any equivalents thereof.

Claims

1. Use of reagents for determining the level of biomarkers in biological samples in the preparation of a kit for diagnosing whether a subject has a neurodegenerative disease; in, The biomarkers include any one, two, three, four, or five of the following: biliverdin, erythritol, 11-dehydrothrombin B2, neo-ostrichol, and adenosine monophosphate. Preferably, the biomarker is selected from biliverdin, erythritol, 11-dehydrothrombin B2, neo-ostrichol, adenosine monophosphate, or any combination thereof.

2. The use as described in claim 1, wherein, The neurodegenerative diseases mentioned are selected from Parkinson's disease (PD), essential tremor (ET), Alzheimer's disease, or vascular dementia; Preferably, the kit is used to diagnose whether a subject has PD; Preferably, the kit is used to diagnose whether a subject has ET; Preferably, the kit is used to distinguish any two or all three of the following: subjects with PD, subjects with ET, and healthy subjects. Preferably, the kit has one or more features selected from the following: (1) The kit is used to distinguish between subjects with PD and subjects with ET; (2) The kit is used to distinguish between subjects with PD and healthy subjects; (3) The kit is used to distinguish between subjects with ET and healthy subjects; (4) The kit is used to distinguish between subjects with PD, subjects with ET and healthy subjects.

3. The use as described in claim 1 or 2, wherein, The kit has one or more of the following characteristics: (1) The biomarkers are 11-dehydrothromethane B2, neo-spatholoben, adenosine monophosphate, biliverdin or erythritol. (2) The biomarker is selected from any two of 11-dehydrothrombin B2, neo-spatholoben, adenosine monophosphate, biliverdin, and erythritol; Preferably, the biomarker is selected from 11-dehydrothromethane B2 and neoostrichone, 11-dehydrothromethane B2 and adenosine monophosphate, 11-dehydrothromethane B2 and biliverdin, 11-dehydrothromethane B2 and erythritol, neoostrichone and adenosine monophosphate, neoostrichone and biliverdin, neoostrichone and erythritol, adenosine monophosphate and biliverdin, adenosine monophosphate and erythritol, or biliverdin and erythritol; (3) The biomarker is selected from any three of 11-dehydrothromethane B2, neo-spatholoben, adenosine monophosphate, biliverdin, and erythritol; Preferably, the biomarker is selected from 11-dehydrothromethane B2, neoostrichone, and adenosine monophosphate; 11-dehydrothromethane B2, neoostrichone, and biliverdin; 11-dehydrothromethane B2, neoostrichone, and erythritol; 11-dehydrothromethane B2, adenosine monophosphate, and biliverdin; 11-dehydrothromethane B2, adenosine monophosphate, and erythritol; or 11-dehydrothromethane B2, biliverdin, and erythritol. (4) The biomarker is selected from any four of 11-dehydrothrombin B2, neo-spatholoben, adenosine monophosphate, biliverdin, and erythritol; Preferably, the biomarker is selected from 11-dehydrothromethane B2, neoostrichol, adenosine monophosphate, and biliverdin; 11-dehydrothromethane B2, neoostrichol, adenosine monophosphate, and erythritol; 11-dehydrothromethane B2, neoostrichol, biliverdin, and erythritol; 11-dehydrothromethane B2, adenosine monophosphate, biliverdin, and erythritol; or neoostrichol, adenosine monophosphate, biliverdin, and erythritol. (5) The biomarkers are 11-dehydrothrombin B2, neo-spatholoben, adenosine monophosphate, biliverdin and erythritol.

4. The use according to any one of claims 1-3, wherein, The biological samples were obtained from the subject's whole blood, serum, plasma, or feces; Preferably, the subject is a mammal, such as a human; Preferably, the kit further comprises pretreatment reagents or tools for pretreating biological samples; Preferably, the pretreatment reagent or tool is used to extract biomarkers from the biological sample; Preferably, the pretreatment reagent or tool is selected from: methanol, 2-chloro-L-phenylalanine, grinding tools (e.g., glass beads), filter membranes, or any combination thereof.

5. The use according to any one of claims 1-4, wherein, The reagents (e.g., the first, second, third, fourth, fifth reagents or a combination of reagents) are used to determine the levels of biomarkers in the biological samples by the following methods: chromatographic and / or mass spectrometry, fluorescence assay, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near-infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), and turbidimetry. Preferably, the reagent is used to determine the level of biomarkers in the biological sample by spectroscopy, liquid or gas chromatography, mass spectrometry, or liquid or gas chromatography coupled with mass spectrometry. Preferably, the kit further includes reagents and / or consumables for spectroscopy, reagents and / or consumables for chromatography, reagents and / or consumables for mass spectrometry, or any combination thereof; Preferably, the reagents and / or consumables used for chromatography are selected from chromatographic columns, aqueous acetonitrile solutions, ammonium acetate, ammonium formate, formic acid, or any combination thereof; Preferably, the reagents and / or consumables used for mass spectrometry are selected from mass spectrometry columns, formic acid, acetonitrile, or any combination thereof.

6. A kit for diagnosing whether a subject has a neurodegenerative disease, the kit comprising reagents for determining the level of a biomarker in a biological sample, said biomarker being selected from biliverdin, erythritol, 11-dehydrothrombin B2, neo-ostrichol, adenosine monophosphate, or any combination thereof.

7. The kit according to claim 6, wherein, The neurodegenerative diseases mentioned are selected from Parkinson's disease (PD), essential tremor (ET), Alzheimer's disease, or vascular dementia; Preferably, the kit is used to diagnose whether a subject has PD; Preferably, the kit is used to diagnose whether a subject has ET; Preferably, the kit is used to distinguish any two or all three of the following: subjects with PD, subjects with ET, and healthy subjects. Preferably, the kit has one or more features selected from the following: (1) The kit is used to distinguish between subjects with PD and subjects with ET; (2) The kit is used to distinguish between subjects with PD and healthy subjects; (3) The kit is used to distinguish between subjects with ET and healthy subjects; (4) The kit is used to distinguish between subjects with PD, subjects with ET and healthy subjects.

8. The kit according to claim 6 or 7, wherein, The kit has one or more of the following characteristics: (1) The biomarkers are 11-dehydrothromethane B2, neo-spatholoben, adenosine monophosphate, biliverdin or erythritol. (2) The biomarker is selected from any two of 11-dehydrothrombin B2, neo-spatholoben, adenosine monophosphate, biliverdin, and erythritol; Preferably, the biomarker is selected from 11-dehydrothromethane B2 and neoostrichone, 11-dehydrothromethane B2 and adenosine monophosphate, 11-dehydrothromethane B2 and biliverdin, 11-dehydrothromethane B2 and erythritol, neoostrichone and adenosine monophosphate, neoostrichone and biliverdin, neoostrichone and erythritol, adenosine monophosphate and biliverdin, adenosine monophosphate and erythritol, or biliverdin and erythritol; (3) The biomarker is selected from any three of 11-dehydrothromethane B2, neo-spatholoben, adenosine monophosphate, biliverdin, and erythritol; Preferably, the biomarker is selected from 11-dehydrothromethane B2, neoostrichone, and adenosine monophosphate; 11-dehydrothromethane B2, neoostrichone, and biliverdin; 11-dehydrothromethane B2, neoostrichone, and erythritol; 11-dehydrothromethane B2, adenosine monophosphate, and biliverdin; 11-dehydrothromethane B2, adenosine monophosphate, and erythritol; or 11-dehydrothromethane B2, biliverdin, and erythritol. (4) The biomarker is selected from any four of 11-dehydrothrombin B2, neo-spatholoben, adenosine monophosphate, biliverdin, and erythritol; Preferably, the biomarker is selected from 11-dehydrothromethane B2, neoostrichol, adenosine monophosphate, and biliverdin; 11-dehydrothromethane B2, neoostrichol, adenosine monophosphate, and erythritol; 11-dehydrothromethane B2, neoostrichol, biliverdin, and erythritol; 11-dehydrothromethane B2, adenosine monophosphate, biliverdin, and erythritol; or neoostrichol, adenosine monophosphate, biliverdin, and erythritol. (5) The biomarkers are 11-dehydrothrombin B2, neo-spatholoben, adenosine monophosphate, biliverdin and erythritol.

9. The kit according to any one of claims 6-8, wherein, The biological samples were obtained from the subject's whole blood, serum, plasma, or feces; Preferably, the subject is a mammal, such as a human; Preferably, the kit further comprises pretreatment reagents or tools for pretreating biological samples; Preferably, the pretreatment reagent or tool is used to extract biomarkers from the biological sample; Preferably, the pretreatment reagent or tool is selected from: methanol, 2-chloro-L-phenylalanine, grinding tools (e.g., glass beads), filter membranes, or any combination thereof.

10. The kit according to any one of claims 6-9, wherein, The reagents (e.g., the first, second, third, fourth, fifth reagents or a combination of reagents) are used to determine the levels of biomarkers in the biological samples by the following methods: chromatographic and / or mass spectrometry, fluorescence assay, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near-infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), and turbidimetry. Preferably, the reagent is used to determine the level of biomarkers in the biological sample by spectroscopy, liquid or gas chromatography, mass spectrometry, or liquid or gas chromatography coupled with mass spectrometry. Preferably, the kit further includes reagents and / or consumables for spectroscopy, reagents and / or consumables for chromatography, reagents and / or consumables for mass spectrometry, or any combination thereof; Preferably, the reagents and / or consumables used for chromatography are selected from chromatographic columns, aqueous acetonitrile solutions, ammonium acetate, ammonium formate, formic acid, or any combination thereof; Preferably, the reagents and / or consumables used for mass spectrometry are selected from mass spectrometry columns, formic acid, acetonitrile, or any combination thereof.

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