KR20190036998A - Biomarkers for the diagnosis of vascular diseases using plasma metabolites - Google Patents

Abstract

The present invention relates to a kit for diagnosing a vascular disease, which measures a concentration of a diagnostic marker of a vascular disease included in plasma of a target object, and a method for providing diagnostic information of a vascular disease, including a method for measuring a concentration of the diagnostic marker of the vascular disease from blood collected from the target object.

Description

Technical Field [0001] The present invention relates to a biomarker for the diagnosis of vascular diseases using plasma metabolites,

The present invention includes a diagnostic marker for a vascular disease contained in plasma of a subject, a diagnostic kit for measuring vascular disease that measures the concentration of the diagnostic marker, and a method for measuring the concentration of the diagnostic marker of the vascular disease in the blood collected from the subject The present invention relates to a method for providing diagnosis information of a vascular disease.

Due to the development of modern society, modern people have a pattern of dietary intake that induces vascular diseases such as high fat / high carbohydrates, thereby increasing the incidence of vascular diseases related thereto.

In particular, cardiovascular disease (CVD) is the world's highest mortality. According to the World Health Organization (WHO), 23.6 million people are expected to die from cardiovascular disease by 2030. Myocardial infarction is the leading cause of sudden death in adults and continues to increase.

Cardiovascular disease is characterized by blockage of coronary blood flow, irregularity of oxygen supply to the heart muscle, and irreversible cardiac muscle cell damage. Coronary artery disease progresses to angina pectoris, which can be divided into stable angina pectoris and unstable angina. Stable formation of the lesion is predictive of recurrent and characteristic chest pain. Unlike stable angina pectoris, myocardial infarction ruptures vulnerable plaques and occurs as a symptom of sudden coronary thrombotic occlusion. Diagnosis of these vulnerable cirrhosis was made only by invasive methods such as using intravascular ultrasound (IVUS), and there is no biomarker that can be predicted and diagnosed before the onset so that early diagnosis is difficult .

Currently, the diagnosis of cardiovascular disease or heart failure is made by the use of GOT (glutamic oxaloacetic transaminase), LDH (lactate dehydrogenase), CK-MB (creatine kinase-MB), troponin I, troponin T, C- Although B-type natriuretic peptide (BNP) is used as a biomarker, the use of biomarkers for specific vascular diseases or metabolites found in blood as a diagnostic biomarker, which is caused by ingestion of high-fat / high- It is not.

Therefore, there is a demand for a technique for identifying a biomarker for a specific vascular disease caused by intake of a high-fat / high-carbohydrate food using a metabolite found in blood.

Accordingly, one object of the present invention is to provide a kit for the diagnosis of vascular diseases, which comprises a quantification device for measuring the concentration of a diagnostic marker of a vascular disease including a metabolite contained in plasma of a subject.

Yet another object of the present invention is to provide a method for detecting a diagnostic marker comprising: measuring the concentration of the diagnostic marker in blood taken from a subject; And comparing the concentration of the metabolite in the blood measured in the step with the concentration of the metabolite in the blood of the normal subject.

The present invention relates to a kit for diagnosing vascular diseases, comprising a quantification device for measuring the concentration of a diagnostic marker of a vascular disease including a plasma metabolite, and measuring the concentration of the diagnostic marker in the blood collected from the subject; And comparing the concentration of the metabolite in the blood measured in the step with the concentration of the metabolite of the normal individual blood.

Specifically, the present inventors conducted a human body test after consuming a test food or a reference food, and conducted metabolism analysis on 43 kinds of plasma metabolites. As a result, it was confirmed that 13 kinds of metabolites were significantly changed Thus, the present inventors have completed the present invention by confirming that the 13 selected metabolites can be used as biomarkers for diagnosing vascular diseases.

As used herein, the term "plasma metabolite" refers to a metabolite obtained from a liquid sample of blood origin. Preferably, the blood origin is whole blood, more preferably plasma. Preferably, the whole blood may be pretreated to detect the plasma metabolism. For example, filtration, distillation, extraction, separation, concentration, inactivation of interfering components, addition of reagents, and the like. In addition, the metabolites may include substances produced by metabolism and metabolism, biological enzymes, and substances generated by chemical metabolism by molecules.

As used herein, the term "increase in metabolite concentration compared to a normal subject" means that the concentration of the metabolite in the subject is significantly increased compared to the concentration of the metabolite in a normal adult without vascular disease Specifically, it means that the significance probability p value is less than 0.05, and more specifically, the p value is less than 0.01.

As used herein, the term "decrease in metabolite concentration compared to a normal subject" means that the concentration of the metabolite in the subject is significantly reduced compared to the concentration of the metabolite in a normal adult with no vascular disease Specifically, it means that the significance probability p value is less than 0.05, and more specifically, the p value is less than 0.01.

As used herein, the term "diagnosis" includes determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder (e.g., vascular disease) Determining the prognosis of an object that has suffered a particular disease or disorder, or therametrics (e.g., monitoring the status of an object to provide information about treatment efficacy).

As used herein, the term "patient " may be used in the sense of a human being as an object, unless otherwise stated.

Hereinafter, the present invention will be described in more detail.

In one aspect, the present invention relates to a method for the treatment of proline, pyrophosphate, oleanitrile, stearic acid, tryptophan, oleamide (including proline, pyrazole, oleamide, cholesterol, glycine, aspartic acid, 5-oxoproline, glucose, 9,12-octadecadienoic acid, acid and at least one metabolite selected from the group consisting of arachidonic acid. The present invention also relates to a kit for the diagnosis of vascular diseases, comprising a quantification device for measuring the concentration of a diagnostic marker of a vascular disease.

In one embodiment of the present invention, 112 high-fat / high-carbohydrate beverages were ingested for adult males and females, and then a control food or test food (a known mushroom extract known to be effective in treating vascular diseases) Six hours after the onset of the disease, blood samples were taken and plasma metabolites obtained from the blood samples were analyzed to identify thirteen kinds of plasma metabolites that can be used as biomarkers for vascular diseases.

In the above experiment, the group in which the high-fat / high-carbohydrate drink was consumed and the control food was consumed is referred to as a control group, and the group in which the test food is consumed is referred to as a test group.

The finally selected thirteen kinds of plasma metabolites are selected from the group consisting of proline, pyrophosphate, oleanitrile, stearic acid, tryptophan, , Oleamide, cholesterol, glycine, aspartic acid, 5-oxoproline, glucose, 9,12-octadecadienoic acid (9 , 12-octadecadienoic acid, and arachidonic acid.

At least one member selected from the group consisting of proline, pyrophosphate, oleanitrile, stearic acid, tryptophan, oleamide, and cholesterol is at least one selected from the group consisting of proline, pyrophosphate, oleanitrile, (5-oxoproline), glucose, 9,12 (5-oxoproline), and the like in the blood vessel disease patients. (9,12-octadecadienoic acid), and arachidonic acid is increased compared to a subject having a normal blood vessel disease level.

The subject may be a mammal including humans, primates such as monkeys, rodents such as rats and mice, or mammals other than humans, but is not limited thereto.

The vascular disease may be, but is not limited to, myocardial infarction, heart failure, arteriosclerosis, arrhythmia, atherosclerosis, hypertension, aneurysm, embolism, stroke, cerebral infarction, thrombosis, or angina.

The quantification device may be, but is not limited to, a nuclear magnetic resonance spectrometer ( ¹ H-NMR) or a chromatography / mass spectrometer.

In one embodiment of the present invention, the quantification device may be a chromatography / mass spectrometer.

The chromatography used in the present invention can be carried out by liquid-solid chromatography (LSC), paper chromatography (PC), thin-layer chromatography (TLC), gas- Gas-Solid Chromatography (GSC), Liquid-Liquid Chromatography (LLC), Foam Chromatography (FC), Emulsion Chromatography (EC) Including, but not limited to, gel filtration chromatography (GLC), ion chromatography (IC), Gel Filtration Chromatography (GFC), or Gel Permeation Chromatography All chromatographies for routine use can be used. Preferably, the chromatography used in the present invention is gas chromatography (GC).

The mass spectrometer can be a conventional mass spectrometer without any particular limitation, and can be specifically exemplified by MALDI-TOF MS, Q-TOF MS and LTQ-Orbitrap MS.

Preferably, the mass spectrometer used in the present invention can be a conventional mass spectrometer without any particular limitation, but specifically, for example, MALDI-TOF MS, Q-TOF MS and LTQ-Orbitrap MS, May be a TOF MS.

At least one member selected from the group consisting of proline, pyrophosphate, oleanitrile, stearic acid, tryptophan, oleamide, and cholesterol is at least one selected from the group consisting of proline, pyrophosphate, oleanitrile, The present invention can be characterized by diagnosing the subject as a vascular disease when the concentration in the vascular disease patient is decreased compared with that of the normal subject and the glycine, aspartic acid, 5-oxoproline (5 at least one selected from the group consisting of -oxoproline, glucose, 9,12-octadecadienoic acid, and arachidonic acid, The subject is diagnosed as a vascular disease.

In another aspect, the present invention relates to a method of treating a blood sample collected from a subject with proline, pyrophosphate, oleanitrile, stearic acid, tryptophan, Cholesterol, glycine, aspartic acid, 5-oxoproline, glucose, 9,12-octadecadienoic acid (9, 12) measuring the concentration of at least one metabolite selected from the group consisting of salicylic acid, salicylic acid, -octadecadienoic acid, and arachidonic acid; And

And comparing the concentration of the metabolite in the blood measured in the step with the concentration of the metabolite in the blood of the normal control group.

At least one member selected from the group consisting of proline, pyrophosphate, oleanitrile, stearic acid, tryptophan, oleamide, and cholesterol is at least one selected from the group consisting of proline, pyrophosphate, oleanitrile, The subject can be diagnosed as a vascular disease when the concentration of the blood vessel disease is lower than that of a normal subject and the subject can be diagnosed as glycine, aspartic acid, 5-oxoproline, One or more selected from the group consisting of glucose, 9,12-octadecadienoic acid, and arachidonic acid has been found to be increased in the blood vessel disease compared to the normal subjects State, the subject can be diagnosed as a vascular disease.

The diagnostic kit for the vascular disease may be equally applied to a method for providing diagnostic information of a vascular disease.

The present invention relates to a kit for diagnosing a vascular disease which measures the concentration of a diagnostic marker of a vascular disease contained in a plasma of a subject and a method for measuring the concentration of the diagnostic marker of the vascular disease in the blood collected from the subject The present invention relates to a method for providing diagnostic information, and is useful for easily diagnosing vascular disease using a metabolite present in plasma of a subject.

FIG. 1 is a graph showing the results of multivariate analysis of plasma metabolism changes in vascular diseases due to high fat / high carbohydrate intake using the OPLS-DA method according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of analysis of 43 plasma metabolites in a test group different from the control group on OPLS-DA analysis according to an embodiment of the present invention, with 16 metabolisms having variable importance plots (VIP) The results are shown in Fig.
FIG. 3 illustrates hierarchical analysis of 16 metabolites having variable importance plots (VIP) values selected according to an embodiment of the present invention, ) Were prepared, and the results of the final selection of 13 metabolites showing statistically significant differences in the test group as compared with the control group are shown.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1. Plasma Metabolism  Vascular disease bio Marker  Selection

1-1. Blood sampling after ingestion of food known to be effective in treating vascular disease

In order to select blood vessel disease biomarkers using plasma metabolism, 112 healthy men and women aged 30 to 65 were recruited from Ewha Womans University Mokdong Hospital (Seoul, Korea), and 112 subjects were randomly selected Control and test groups were divided into two groups: high-fat / high-carbohydrate drinks (palm oil, dextrose, protein powder, Nutricia) , 600 mg of control food (dextrin) for the control group, 600 mg of the known mushroom extract known to be effective for the treatment of vascular diseases for the test group, and rest for 6 hours so that the metabolic change occurs After the venous blood was obtained from 112 subjects, they were collected in a sterilized plastic test tube which had been treated with EDTA as an anticoagulant, and immediately centrifuged Performed to obtained the plasma metabolite samples, all samples obtained above were stored in -80 ℃ until analysis.

1-2. plasma Metabolism  analysis

Analysis using the gas chromatography-time of flight mass spectrometry (GC-TOF-MS) was carried out in the following manner to analyze the plasma metabolite samples obtained in Example 1-1.

Specifically, the plasma metabolite sample obtained in Example 1-1 was dissolved in 1,000 uL of methanol (JTBaker, 9093-68) supplemented with 10 uL of 2-chloro-1-phenylalanine (Sigma-Aldrich, 47766) And homogenized for 10 minutes using Mixermill (SPEXSamplePrep, 8000M). After homogenization, the supernatant was sonicated for 1 minute and stored at 4 ° C for 1 hour. The supernatant was centrifuged at 13,500 xg for 10 minutes at 4 ° C After separation, it was filtered using 0.2 μm polytetrafluoroethylene (PTFE) (Sigma-Aldrich, P0325), dried and oximidated with 50 μL methoxyamine hydrochloride (Sigma-Aldrich, 226904) for 30 min. 50 μL of N-methyl-N-trimethylsilyl-trifluoroacetamide (Sigma-Aldrich, 69482) was added to the reaction mixture and reacted at 37 for 30 min. The reaction product was analyzed by GC-TOF-MS.

(Id, 30 m x 0.25 mm, 0.25 μm particle size; Restek Corp., Bellefonte, Calif.) Using an Agilent 7890 gas chromatograph system (Agilent Technologies, Palo Alto, CA, USA) , PA, USA), and helium was flowed constantly at 1.5 mL / min according to the protocol provided by the manufacturer and 1 μL of the reactant sample was injected. The temperatures of the front inlet and the transfer line were 250 ° C. and 240 ° C. , Electron ionization was performed at -70 eV, and data were collected in the range of 50-1000 m / z.

1-3. Statistical analysis

To analyze the results of plasma metabolite GC-TOF-MS analysis in Example 1-2, peak values of each sample were analyzed using SIMCA-P software version 14.1 (Umetrics, Umea, Sweden) (Fig. 1), and metabolites with a variable importance plots (VIP) value of 1.0 or higher were selected as metabolites that contribute to the difference between the control and test groups (Fig. 2), and a hierarchical analysis was conducted on the selected peaks to generate a heat map (Fig. 3). The significant difference between the metabolites in the two groups was that the student's t -test (p <0.05).

Experimental Results 1. Plasma from blood vessel diseases due to high fat / high carbohydrate intake Metabolite  change

1-1. OPLS -DA Analytical  Difference

The results of the multivariate analysis of the plasma metabolism in the vascular disease according to the ingestion of the high fat / high carbohydrate of Example 1-3 using the OPLS-DA method are shown in FIG.

1, a clear multivariate analysis was obtained between the control group and the test group. As a result, it was found that the metabolism of the 43 metabolites in the test group consuming the food, .

1-2. 16 kinds of plasma using VIP parameters Metabolite  Selection

Of the 43 plasma metabolites in the test group that differed from the control group in the OPLS-DA analysis, VIP value was more than 1 through variable importance plots (VIP), which significantly contributed to the difference between the two groups It can be regarded as a metabolite. As shown in FIG. 2, 43 kinds of metabolites were listed in VIP order, and 16 kinds of metabolites having VIP value of 1 or more were selected.

1-3. 13 kinds of plasma Metabolite  Selection

As shown in FIG. 3, a hierarchy analysis is performed on 16 metabolites having a variable importance plots (VIP) value of 1 or more to generate a heat map And 13 kinds of metabolites showing a statistically significant difference in the test group were finally selected compared with the control group.

The 13 selected metabolites are shown in Table 1. In the control group showing vascular diseases, the metabolites of 7 kinds of metabolites (proline, pyrophosphate, oleanitrile, stearic acid, tryptophan, oleamide, cholesterol) , 6 metabolites (glycine, aspartic acid, 5-oxoproline, glucose, 9,12-octadecadienoic acid, arachidonic acid).

From the above results, it was confirmed that the 13 selected metabolites were biomarkers capable of diagnosing vascular disease due to high fat / high carbohydrate intake.

Metabolism P-value Arachidonic acid 0.0180 Aspartic acid 0.0030 Cholesterol <0.0001 Glucose 0.0147 Glycine 0.0139 Oleamide <0.0001 Oleanitrile <0.0001 Proline 0.0156 Pyrophosphate 0.0013 Stearic acid 0.0002 Tryptophan 0.0015 5-oxoproline 0.0363 9,12-octadecadienoic acid 0.0085

Claims (12)

A proline, a pyrophosphate, an oleanitrile, a stearic acid, a tryptophan, an oleamide, a cholesterol, a cholesterol, and the like contained in plasma of a subject, Glycine, aspartic acid, 5-oxoproline, glucose, 9,12-octadecadienoic acid, and arachidonic acid. wherein the concentration of the at least one metabolite selected from the group consisting of glucose, ascorbic acid, ascorbic acid and ascorbic acid is measured. The diagnostic kit according to claim 1, wherein the quantification device is a nuclear magnetic resonance spectroscopic analyzer ( ¹ H-NMR) or a chromatography / mass spectrometer. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises the proline, pyrophosphate, oleanitrile, stearic acid, tryptophan, oleamide, and cholesterol. Wherein the subject is diagnosed as a vascular disease when the concentration of one or more selected from the group is lower than that of a normal subject. The method of claim 1, wherein the glycine, aspartic acid, 5-oxoproline, glucose, 9,12-octadecadienoic acid ), And arachidonic acid is diagnosed as a vascular disease when the concentration of the at least one selected from the group consisting of arachidonic acid is increased compared to that of a normal subject with vascular disease. The diagnostic kit according to claim 1, wherein the vascular disease is myocardial infarction, heart failure, arteriosclerosis, arrhythmia, atherosclerosis, hypertension, aneurysm, embolism, stroke, cerebral infarction, thrombosis, or angina pectoris. The diagnostic kit of claim 1, wherein the subject is a mammalian animal, including a human. In the blood taken from the subject, proline, pyrophosphate, oleanitrile, stearic acid, tryptophan, oleamide, cholesterol, , Glycine, aspartic acid, 5-oxoproline, glucose, 9,12-octadecadienoic acid, and arachidonic acid ( arachidonic acid) in a measuring device; And
And comparing the concentration of the metabolite in the blood measured in the step with the concentration of the metabolite in the normal individual blood. 8. The method according to claim 7, wherein the quantification device is a nuclear magnetic resonance spectroscopic analyzer ( ¹ H-NMR) or a chromatography / mass spectrometer. 8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition comprises the proline, pyrophosphate, oleanitrile, stearic acid, tryptophan, oleamide, and cholesterol. Wherein the subject is diagnosed as a vascular disease when the concentration of the at least one selected from the group is lower than that of the subject having normal vascular disease. The method of claim 7, wherein the glycine, aspartic acid, 5-oxoproline, glucose, 9,12-octadecadienoic acid ) And arachidonic acid is diagnosed as a vascular disease when the concentration of the at least one selected from the group consisting of arachidonic acid, How to provide information. The method according to claim 7, wherein the vascular disease is diagnosed as myocardial infarction, heart failure, arteriosclerosis, arrhythmia, atherosclerosis, hypertension, aneurysm, embolism, stroke, cerebral infarction, thrombosis, or angina. 8. The method according to claim 7, wherein the object is a mammalian animal including human.

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