阅读说明：本技术 冠状动脉疾病的诊断标记 (Diagnostic markers for coronary artery disease ) 是由 小野隆 滨田忠辉 紫垣修平 森田敦 于 2016-10-19 设计创作，主要内容包括：本发明涉及冠状动脉疾病的诊断。具体地,本发明涉及使用磷脂酰肌醇(36:5)或溶血磷脂酰肌醇(20:5)作为分子标记来诊断冠状动脉疾病。(The present invention relates to the diagnosis of coronary artery disease. In particular, the invention relates to the use of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) as molecular markers for the diagnosis of coronary artery disease.)

1. A method for detecting coronary artery disease, the method comprising the step of determining the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in a test sample derived from a subject, wherein the determined value is used as an indicator that the sample is derived from a human suffering from coronary artery disease.

2. The method of claim 1, wherein the fact that the determined value is less than a reference value is used as an indicator that the test sample is a sample derived from a person suffering from coronary artery disease.

3. The method of claim 2, wherein the reference value is an amount in a control sample derived from a human not suffering from coronary artery disease.

4. The method of any one of claims 1 to 3, wherein the test sample is blood.

5. A kit for detecting coronary artery disease, the kit comprising phosphatidylinositol (36:5) or lysophosphatidylinositol (20: 5).

6. A marker for detecting coronary artery disease, the marker consisting of phosphatidylinositol (36:5) or lysophosphatidylinositol (20: 5).

Technical Field

The present invention relates to the diagnosis of coronary artery disease. In detail, the present invention relates to the diagnosis of coronary artery disease using phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) as molecular markers.

Prior Art

In japan, heart disease is the second cause of death next to cancer (statistical data of the general of Internal affarts and Communications, statistical Bureau of japan), and most of them are coronary artery disease (ischemic heart disease). Coronary artery disease is a disease caused by poor blood flow into coronary arteries supplying blood to heart muscle and insufficient blood supply to heart muscle. Arteriosclerosis is a major cause of coronary artery disease. Stenosis within a blood vessel results in a decrease in the amount of blood supply and a disruption of blood flow to the heart muscle. The amount of oxygen supplied from the blood does not meet the oxygen demand required by the myocardium and creates a state of hypoxia. This leads to angina and myocardial infarction.

The main cause of arteriosclerosis is abnormal lipid metabolism including an increase in Low Density Lipoprotein (LDL) cholesterol. Thus, lipids that are altered with arteriosclerosis are potential candidates for biochemical markers of coronary artery disease. In particular, Low Density Lipoprotein (LDL) cholesterol is a marker and causative agent for diseases ranging from arteriosclerosis to coronary artery disease. However, sufficient reduction of Low Density Lipoprotein (LDL) cholesterol by administration of statins is not always able to prevent cardiovascular events (non-patent document 2). This does not indicate that Low Density Lipoprotein (LDL) cholesterol is an adequate marker or therapeutic target for coronary artery disease.

Regarding the marker of coronary artery disease, although there is a patent application (patent document 1) in which the amount of at least two lipid analytes in blood, such as phosphatidylinositol (36:1), is used as an index to diagnose coronary artery disease, a marker for diagnosing coronary artery disease by a more convenient evaluation method is not known.

Documents of the prior art

Patent document

Patent document 1: international publication No. 2011/063470

Non-patent document

Non-patent document 1: american Journal of Cardiology 2008, Vol.101, No. 8, supplement, pp.S 27-S35

Summary of The Invention

Problems to be solved by the invention

The present invention solves the problem of developing a convenient method for diagnosing arteriosclerosis.

Means for solving the problems

In view of the above problems, the inventors have made intensive studies and, as a result, succeeded in perfecting the present invention based on the following findings: coronary artery disease can be diagnosed by using phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in peripheral blood as a marker.

That is, the present invention relates to the following:

(1) a method for detecting coronary artery disease, the method comprising the step of determining the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in a test sample derived from a subject, wherein the determined value is used as an indicator that the sample is derived from a human suffering from coronary artery disease.

(2) The method according to (1), wherein the fact that the determined value is smaller than the reference value is used as an index indicating that the test sample is a sample derived from a human suffering from coronary artery disease.

(3) The method according to (2), wherein the reference value is an amount in a control sample derived from a human not suffering from coronary artery disease.

(4) The method according to any one of (1) to (3), wherein the test sample is blood.

(5) The method according to any one of (1) to (4), wherein the determination is carried out using mass analysis or an antibody or antibody fragment that specifically binds to phosphatidylinositol (36:5) or lysophosphatidylinositol (20: 5).

(6) A kit for detecting coronary artery disease, the kit comprising phosphatidylinositol (36:5) or lysophosphatidylinositol (20: 5).

(7) A kit for detecting coronary artery disease comprising an antibody or antibody fragment that specifically binds to phosphatidylinositol (36:5) or lysophosphatidylinositol (20: 5).

(8) A marker for detecting coronary artery disease, the marker consisting of phosphatidylinositol (36:5) or lysophosphatidylinositol (20: 5).

(9) A method for treating coronary artery disease, by a coronary artery disease detected according to the method of any one of (1) to (5), and by one or more treatments selected from the group consisting of: pharmacotherapy, surgery, exercise therapy, and dietetic therapy.

Effects of the invention

According to the present invention, since, for example, phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) can be used alone as a marker, coronary artery disease can be conveniently diagnosed. The invention is further characterized by a reduction in stress in the subject, as can be determined using blood or serum.

Brief description of the drawings

FIG. 1 is a diagram showing an example of phosphatidylinositol (36: 5).

FIG. 2 is a diagram showing an example of lysophosphatidylinositol (20: 5).

FIG. 3 is a graph showing the relative values of the peripheral blood phosphatidylinositol (36:5) concentrations in the control group and the coronary artery disease group.

FIG. 4 shows the ROC curve for phosphatidylinositol (36: 5).

FIG. 5 is a graph showing the relative values of lysophosphatidylinositol (20:5) concentration in peripheral blood of the control group and the coronary artery disease group.

FIG. 6 shows the ROC curve for lysophosphatidylinositol (20: 5).

Examples of the invention

Terms used in the present specification are according to definitions generally used in the art, unless otherwise specified. Specific examples of the present invention are given below, but the present invention is not limited to these examples.

The present invention provides a method for determining whether a subject has coronary artery disease (is a human having coronary artery disease).

The method involves

A method for detecting coronary artery disease, the method comprising the step of determining the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in a test sample derived from a subject, wherein the determined value is used as an indicator that the sample is derived from a human suffering from coronary artery disease.

In addition, the present invention

Comprising a method for detecting coronary artery disease, the method comprising the step of determining the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in a test sample derived from a subject, wherein the fact that the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in the test sample is less than a reference value is used as an indicator that the test sample is a sample derived from a human having coronary artery disease.

Further, the present invention

Comprising a method for detecting coronary artery disease, the method comprising the step of determining the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in a test sample derived from a subject, wherein the fact that the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in the test sample is less than the amount of a control sample derived from a human not suffering from coronary artery disease is used as an indicator indicating that the test sample is a sample derived from a human suffering from coronary artery disease.

That is, a person suffering from coronary artery disease can be screened (diagnosed) by determining that a sample is derived from a person suffering from coronary artery disease using the above-described method.

A salient feature of the invention is the use of the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in a test sample derived from a subject as a molecular marker. The "test sample" is a sample collected from a subject including samples derived from urine, whole blood, plasma, serum or blood, and for convenience, a sample that can be prepared from peripheral blood is preferred. Peripheral blood may be collected from any site, but is typically collected from a vein of the subject. By collecting peripheral blood from the arm veins, the present invention can be conveniently carried out with reduced stress on the subject. Preferably, plasma is separated from the collected peripheral blood and the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in the plasma is determined. Phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) may be used alone as a marker for coronary artery disease or may be used in combination with other markers known to be capable of detecting coronary artery disease.

Phosphatidylinositol to be identified in the present invention is an acidic phospholipid having inositol at the polar group and is also abbreviated as PI for short. There are various molecular species with different fatty acid combinations at positions 1 and 2. For example, the annotation for phosphatidylinositol (38:4) is used in the following cases: wherein the ester-type bonded fatty acid has a total of 38 carbon atoms and a total of 4 double bonds at positions 1 and 2 based on molecular weight estimation assumed from the results of analysis of phosphatidylinositol molecular species by mass analysis.

As estimated based on molecular weights assumed from the analysis results by mass analysis, phosphatidylinositol to be determined in the present invention has ester-type bonded fatty acids at positions 1 and 2 (in which the number of carbon atoms is 36 in total and the number of double bonds is 5 in total), and thus it is phosphatidylinositol (36:5) (phosphatidylinositol has fatty acid residues at positions sn-1, 2 in which the number of carbon atoms is 36 in total and the number of double bonds is 5 in total).

FIG. 1 shows an example of phosphatidylinositol (36:5) to be identified in the present invention.

Lysophosphatidylinositol to be determined in the present invention is an acidic phospholipid having inositol at the polar group and is also abbreviated as LPI for short. There are various molecular species with different fatty acid contents. For example, the annotation for lysophosphatidylinositol (18:1) is used in the following cases: wherein the ester-type bonded fatty acid has a total of 18 carbon atoms and a total of 1 double bond estimated based on molecular weight assumed from the results of analysis of lysophosphatidylinositol molecular species by mass analysis.

As estimated based on the molecular weight assumed from the analysis result by mass analysis, lysophosphatidylinositol to be determined in the present invention has ester-type bonded fatty acids (in which the number of carbon atoms is 20 in total and the number of double bonds is 5 in total), and thus it is lysophosphatidylinositol (20:5) (lysophosphatidylinositol has fatty acid residues in which the number of carbon atoms is 20 in total and the number of double bonds is 5 in total).

FIG. 2 shows an example of lysophosphatidylinositol (20:5) to be determined in the present invention.

Examples of methods for determining the amount of phosphatidylinositol and lysophosphatidylinositol are mass analysis and immunoassays.

For example, mass analysis can be performed with reference to the following examples and Journal of Separation Science [ Journal of Separation Science ], 2012, volume 35, pages 1845-1853 or Analytical Biochemistry [ Analytical Biochemistry ], 2008, volume 375, pages 124-131.

The immunoassay may be a conventional immunoassay. Examples of conventional immunoassays are ELISA, RIA and western blot.

The type, derivative and kind of antibody used in the immunoassay are not particularly limited as long as the antibody has the ability to specifically bind to phosphatidylinositol (36:5) or lysophosphatidylinositol (20: 5). Monoclonal and polyclonal antibodies can be used, but monoclonal antibodies are preferred.

Antibodies can be prepared by conventional methods using phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) as the immunogen. Phosphatidylinositol (36:5) and lysophosphatidylinositol (20:5) are low molecular weight compounds, and when used alone as immunogens, the production of antibodies can be difficult. Thus, it is preferred to prepare compounds having a carrier protein. Typically, the mammal to be vaccinated with the immunogen is a rabbit, goat, sheep, rat, or the like. Immunization can be carried out by conventional methods, for example, by intravenous, intradermal, subcutaneous or intraperitoneal administration of the immunogen to a mammal. After immunization, antisera is collected from a mammal and polyclonal antibodies are prepared, or plasma cells (immune cells) are collected and monoclonal antibodies are prepared by a method described in, for example, "Molecular and Cellular Biology Basic Experimental Methods ]" (Nanedo Press, Takekazu Horie et al, 1994).

The antibody may be labeled with a label that can be detected and quantitatively determined. Radioactivity and fluorescence are known labels and can be selected appropriately. For example, biotinylated antibodies can be readily detected.

According to the method for detecting coronary artery disease of the present invention, the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in a test sample obtained from a subject is compared with a reference value, and when the amount is less than the reference value, the test sample is screened as a sample derived from a human having coronary artery disease.

The term "reference value" is defined as the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in a control sample, e.g. derived from a human not suffering from coronary artery disease. The amount in the control sample is preferably an average of a plurality of samples. The amount in the control sample may be determined simultaneously with the test sample, or the cut-off value may be set by a predetermined determination.

Phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in samples derived from persons with coronary artery disease are much less than phosphatidylinositol or lysophosphatidylinositol in control samples derived from persons without coronary artery disease. Therefore, in the method for detecting coronary artery disease of the present invention, when the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) or the calculated value based on the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in the test sample is significantly smaller than the amount of the above-mentioned "control sample", or is smaller than the preset cutoff value, it can be said that the test sample is a sample derived from a human suffering from coronary artery disease with a high possibility.

Note that the "cutoff value" in the present invention is defined as a value for distinguishing a person having coronary artery disease from a person not having coronary artery disease by comparing phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) for detecting coronary artery disease obtained by the above-mentioned method, or a calculated value based on the amount of phosphatidylinositol (36:5) or lysophosphatidylinositol (20: 5). Although the method for setting the cutoff value is not particularly limited, the cutoff value may be set using an ROC curve as in the following example.

When a cutoff value is used to detect coronary artery disease, the cutoff value varies according to changes in the method of determining the marker. Therefore, it is preferable to set a target marker, a cutoff value and perform measurement based on the cutoff value in advance before determining the definite values of no coronary artery disease and coronary artery disease. According to an example of the present invention, when a marker is determined using serum of a subject, coronary artery disease can be detected by referring to the numbers obtained in the example of the present invention.

Note that when 2 or more determined values, for example, compare values of a test sample to values of a control sample, the comparison can use one or more statistical analyses (e.g., t-test, Welch t-test, Wilcoxon rank sum test, analysis of variance (ANOVA), recursive decomposition, or random forest).

The present invention provides a kit for detecting coronary artery disease comprising phosphatidylinositol (36:5) or lysophosphatidylinositol (20: 5). The present invention further provides a kit characterized by containing an antibody specifically recognizing phosphatidylinositol (36:5) or lysophosphatidylinositol (20: 5). These kits can be used in the method for detecting coronary artery disease of the present invention.

In addition, these kits typically further comprise one or more structural elements necessary for the assay. The structural element may be a reference standard, a reagent (diluent, buffer, etc.), a container and/or a device.

Furthermore, the present invention makes it possible to treat a person diagnosed with coronary artery disease by diagnosing with phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) by the above-mentioned method. In particular, the present invention is a method of treating coronary artery disease comprising

A step of detecting coronary artery disease, wherein in the above-mentioned method for detecting coronary artery disease, the fact that the amount in the test sample is smaller than the amount of the control sample derived from a human not suffering from coronary artery disease is used as an index indicating that the test sample is derived from a human suffering from coronary artery disease, and

a step of treating a subject in which coronary artery disease has been detected by one or more treatments selected from the group consisting of: pharmacotherapy, surgery, exercise therapy, and dietetic therapy.

That is, the present invention includes a method for treating a human suffering from coronary artery disease by:

(a) determining the concentration of phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) in a test sample derived from the subject,

(b) using the determined value as an indicator for detecting coronary artery disease, the indicator indicating that the sample originates from a person suffering from coronary artery disease, an

(c) Treating a subject in which coronary artery disease has been detected by one or more treatments selected from the group consisting of: pharmacotherapy, surgery, exercise therapy, and dietetic therapy.

Examples of drug therapies are nitric acid drugs, beta blockers, calcium antagonists, antiplatelet drugs (aspirin, etc.) and cholesterol lowering drugs.

Examples of surgery are Coronary Artery Bypass Grafting (CABG), balloon angioplasty, and stent implantation.

The invention will now be described in further detail and more specifically using examples, to which the invention is not limited.

Example 1

Diagnosis of coronary artery disease using phosphatidylinositol (36:5) in peripheral blood as a molecular marker

To investigate the performance of phosphatidylinositol (36:5) as a novel marker of coronary artery disease, plasma phosphatidylinositol (36:5) concentrations were compared in 20 persons in the control group and 20 persons in the coronary artery disease group.

The subject was a coronary artery disease patient and plasma samples derived from the patient provided by Proteogenex were used. The control group was an adult who did not suffer from coronary artery disease. Experiments were performed using human tissues and genes of Shionogi & Company (Shionogi corporation) after obtaining approval from the ethical research review board. Blood was collected with written consent and frozen in aliquots at-80 ℃.

The concentration of phosphatidylinositol (36:5) in the peripheral blood of the subject was quantitatively determined by liquid chromatography tandem mass spectrometry (hereinafter LC/MS).

The specific LC/MS/MS system used by the inventors to determine the concentration of phosphatidylinositol (36:5) in peripheral blood is described below.

After adding the internal standard solution to the plasma derived from each subject, an organic solvent was added and deproteinization treatment was performed, the product was stirred and centrifuged, and the supernatant was recovered. The supernatant was dried with nitrogen and then redissolved with an organic solvent. Phosphatidylinositol in blood was quantitatively determined by LC/MS/MS using reconstituted solution as the assay solution (36: 5). Nexera (Shimadzu Corporation [ Shimadzu Corporation, Japan ]) -AP15000(AB SCIEX) was used as the LC/MS/MS system. Blood components were separated and eluted under reverse phase conditions and then detected by electrospray ionization in negative ion mode and MRM (multiple reaction monitoring). The peak area and internal standard of phosphatidylinositol (36:5) were analyzed by an analyst (AB SCIEX) and the area ratio was calculated. Phosphatidylinositol in plasma was quantitatively determined using, as a relative value, a value obtained by dividing the peak area ratio of each subject by the average of the peak area ratios of the control group (36: 5).

FIG. 3 summarizes the relative values of the amount of phosphatidylinositol (36:5) in plasma in the control group and the coronary artery disease group. The relative value of phosphatidylinositol (36:5) in the plasma of the control group was 100.

The concentration of phosphatidylinositol (36:5) in the coronary artery disease group was about 79% lower than that in the control group, and a significant difference was observed (P < 0.0001).

Example 2

FIG. 4 summarizes the results of the ROC analysis of the results obtained in example 1. The area under the curve (AUC) was 0.963.

According to the results of fig. 4, the cutoff value was 43% when coronary artery disease was diagnosed using the relative value of phosphatidylinositol (36:5) concentration, and a concentration at or below the cutoff value could be diagnosed as possible coronary artery disease.

Example 3

Diagnosis of coronary artery disease using lysophosphatidylinositol (20:5) in peripheral blood as a molecular marker

To investigate the performance of lysophosphatidylinositol (20:5) as a novel marker of coronary artery disease, the plasma concentrations of lysophosphatidylinositol (20:5) in 20 persons in the control group and 20 persons in the coronary artery disease group were compared by the same method as in example 1.

FIG. 5 summarizes the relative values of the amounts of lysophosphatidylinositol (20:5) in plasma of the control group and the coronary artery disease group. The relative value of lysophosphatidylinositol (20:5) in the plasma of the control group was 100.

The lysophosphatidylinositol (20:5) concentration in the coronary artery disease group was about 69% lower than that in the control group, and a significant difference was observed (P < 0.0001).

Example 4

Figure 6 summarizes the results of the ROC analysis of the results obtained in example 3. The area under the curve (AUC) was 0.897.

According to the results of fig. 6, the cutoff value was 44% when coronary artery disease was diagnosed using the relative value of the lysophosphatidylinositol (20:5) concentration, and the concentration at or below the cutoff value could be diagnosed as possible coronary artery disease.

As described above, it was confirmed that phosphatidylinositol (36:5) or lysophosphatidylinositol (20:5) can be quantitatively determined in peripheral blood and can be used as a molecular marker for coronary artery disease.

Industrial applicability

The invention can be used in the field of pharmacy, in particular in the field of pharmacy for the in vitro diagnosis of coronary artery diseases.