JP6198047B2 - Coronary artery disease test kit - Google Patents

Description

  The present specification relates to a test kit or the like for diagnosing coronary artery disease.

  Mortality from coronary artery disease is high worldwide, and there is a strong demand for early detection and prevention of coronary artery disease. Further, since coronary artery disease may require advanced medical treatment or may have long-term treatment, early detection is important in terms of medical economy.

  Coronary artery disease is a disease in which a chest pain attack occurs due to lack of oxygen in the myocardium. In coronary artery disease, the inside of a blood vessel is narrowed due to arteriosclerosis, etc., and the supply of blood to the myocardium is reduced. The balance is lost and ischemia occurs, and the myocardium becomes deficient in oxygen.

  There are two types of coronary artery disease: angina pectoris and myocardial infarction. Angina is mainly caused by chest pain due to transient myocardial ischemia, chest compression, etc. Myocardial infarction is caused by complete occlusion or significant stenosis of the coronary artery. It leads to myocardial necrosis.

  All of these coronary artery diseases have chest pain or chest compression as the main symptom, but diagnosis and identification of a stenotic site require myocardial scintigraphy, coronary artery CT, cardiac catheterization, and the like.

  Coronary artery disease is known to develop as a serious complication such as diabetes, chronic kidney disease, hyperlipidemia, and arteriosclerosis. As a method for examining the risk of coronary artery disease, a method based on a polymorphism in a specific gene is disclosed (Patent Document 1).

JP 2008-188016 A

  Various examination methods such as myocardial scintigraphy, coronary artery CT, and cardiac catheter examination performed for definitive diagnosis of coronary artery disease such as a chest pain attack are all meaningful examinations. However, there is a demerit that it is relatively invasive or only specific facilities can be used. Also, the inspection cost is high. For this reason, the tendency to be cautious about the inspection itself cannot be denied.

  In patients with primary diseases such as diabetes, early detection of coronary artery disease is extremely useful. However, genetic testing for predicting the risk of coronary artery disease has the disadvantage that it is expensive even if it is non-invasive. Moreover, in patients with primary diseases, regular monitoring for coronary artery disease, which is a major complication, is required rather than the risk of coronary artery disease.

  Furthermore, coronary artery disease is often in a serious state when a clear subjective symptom such as chest pain occurs, and periodic monitoring is considered effective even without a primary disease.

  To date, there is no simple test method that is advantageous for the diagnosis and early detection of coronary artery disease, and there is a strong demand for a test method for coronary artery disease that is simple and suitable for monitoring.

  The present specification provides a simple test kit and test method for coronary artery disease.

  The present inventors paid attention to prorenin. Prorenin is a precursor to renin, and its relationship with diabetic nephropathy and arteriosclerosis in diabetic patients has been speculated. However, blood prorenin and coronary artery disease have not been associated so far. The present inventors conducted examinations on various types of items regarding blood of patients with coronary artery disease, and unexpectedly obtained knowledge that plasma prorenin concentration and coronary artery disease can be correlated. This specification provides the following means based on this finding.

(1) A test kit for coronary artery disease, comprising one or more reagents for measuring prorenin.
(2) The kit according to (1), wherein the reagent contains an anti-prorenin antibody.
(3) A method for examining coronary artery disease,
A measurement process for measuring plasma prorenin,
A method comprising:
(4) Furthermore, an evaluation step for comparing the plasma prorenin amount with a preset threshold value,
(5) The method according to (4), wherein the threshold is set based on significant stenosis.
(6) The examination method according to any one of (3) to (5), wherein the coronary artery disease has a stenosis of 75% or more in coronary arteriography.
(7) The test method according to any one of (3) to (6), wherein the measurement step is a step of measuring the plasma prorenin by an immunoassay.
(8) A method for examining a stenosis of a coronary artery, comprising a measurement step of measuring plasma prorenin.

It is a figure which shows the blot regarding the plasma prorenin density | concentration about a non-coronary artery disease group and a coronary artery disease group.

  The disclosure of the present specification relates to a test kit and a test method for coronary artery disease. According to the test kit and the test method disclosed in the present specification, the possibility of coronary artery disease can be predicted with high accuracy with minimal invasiveness by simply collecting blood. In addition, the possibility of coronary artery disease can be predicted at low cost. If the presence or absence of significant stenosis can be detected without coronary angiography, treatment for coronary artery disease can be started more easily and early. Therefore, the inspection kit and the inspection method according to the present disclosure can easily and easily detect coronary artery disease that has been mainly performed by invasive, advanced, and high-cost inspection, and is greatly useful for early detection and prevention of coronary artery disease. Can contribute. It is also meaningful for regular monitoring of coronary artery disease, which is a serious complication of primary diseases such as diabetes, chronic kidney disease, hyperlipidemia, and arteriosclerosis. From the above, the methods and kits disclosed in this specification are extremely useful for primary screening of coronary artery disease. Hereinafter, various embodiments related to the disclosure of this specification will be described in detail.

  In this specification, coronary artery disease means that the blood vessels are stenotic regardless of the cause and the supply of blood to the myocardium is reduced, or the blood flow is interrupted and the myocardium becomes ischemic and the myocardium is deficient in oxygen. And the resulting heart condition. Therefore, it includes angina pectoris due to myocardial ischemia, myocardial infarction, heart failure, arrhythmia, ventricular fibrillation and the like.

  In the present specification, coronary artery disease means a case where the coronary artery has significant stenosis. Here, the term “significant stenosis” generally means that 75% or more of the stenosis (75% or more of the original lumen diameter is narrowed in the coronary artery by the coronary angiography (the lumen is the original lumen diameter). The state in which the diameter has become 25% or less) is observed. Of the coronary artery, the main trunk of the left coronary artery is assumed to have significant stenosis when the stenosis is 50% or more.

  In general, when there is significant stenosis, it is treated as a coronary artery disease. When significant stenosis does not occur, it is generally not a treatment target, and mainly improves daily life if necessary.

  In the present specification, coronary artery disease mainly means coronary artery disease in mammals including humans. Examples of animals other than humans include domestic animals and pets such as dogs, cats, rabbits, horses, cows, monkeys, and the like.

  From the above, the present inspection method and inspection kit can be implemented not only as a coronary artery disease inspection kit and method but also as a coronary artery stenosis inspection kit and method.

(Test method for coronary artery disease)
The test method disclosed in the present specification can include a measurement step of measuring plasma prorenin.

  Prorenin is an inactive precursor of active renin involved in blood pressure regulation and homeostasis. Renin is an enzyme that cleaves angiotensinogen and converts it into angiotensin I (this conversion rate is called renin activity). Prorenin has a renin skeleton having a three-dimensional structure having a cleft (groove) having an enzyme active center, and further has a prorenin prosegment of 43 amino acids covering the enzyme active center. For this reason, prorenin cannot be converted to angiotensin I because the prosegment part blocks the interaction with the active center of angiotensinogen. In plasma, prorenin is stable. It is known that when a prorenin prosegment is reacted with solubilized trypsin at about 4 ° C. for a short time, it is converted to renin and exhibits renin activity (for example, Clin Chem 2009; 55: 876-877).

  The measurement method is not particularly limited as long as plasma prorenin can be measured. In addition, prorenin itself can be measured by renin enzyme activity or by an immunoassay.

  A method for measuring prorenin based on enzyme activity (renin activity) is known (for example, Clin Chem 2009; 55: 876-877). When prorenin is measured by renin enzyme activity, angiotensinogen as a substrate and a reagent for evaluating the conversion to angiotensin I can be used. Such reagents are known (such as Clin Chem 2009 described above).

  The plasma prorenin concentration can be calculated from the total renin concentration in plasma-plasma renin concentration. The total renin concentration in plasma can be obtained by renin enzyme activity (based on angiotensin I production) by prorenin activated by renin and trypsin in plasma. The renin concentration in plasma can be obtained by renin enzyme activity (based on the amount of angiotensin I produced) by renin in plasma and open structure prorenin (Clin Chem 2009 and the like described above).

  Prorenin itself can also be measured by immunoassay. Although it does not specifically limit as an immunoassay, ELISA method etc. are mentioned. In the case of the immunoassay, a prorenin-specific antibody can be used as a reagent for measuring prorenin (Japanese Patent Laid-Open No. 10-279600). Such antibodies are also commercially available (Prorenine Elisa Assay kit, Innovative Research Inc.). Other reagents and reagents used in the final detection method in immunoassay are well known to those skilled in the art, and those skilled in the art can measure plasma prorenin as appropriate if antibodies are available.

In the measurement step, plasma prorenin is quantitatively measured. That is, it is determined as the amount of prorenin for a predetermined amount of plasma. Whether quantification of plasma prorenin is based on enzyme activity or immunoassay, those skilled in the art can measure plasma prorenin by appropriately setting conditions. Plasma is prepared from blood by a known method. If a known method capable of measuring prorenin is applied, plasma prorenin can be measured by preparing plasma from a whole blood sample or by collecting collected plasma.

  According to this test method, early detection of coronary artery disease, detection of coronary artery stenosis based on the amount of plasma prorenin obtained and its change, that is, progression or improvement of coronary artery stenosis, monitoring of coronary artery disease is possible Become. Since this measurement method can be carried out simply and promptly, it is possible to detect coronary artery disease and to take measures against coronary artery disease regardless of the equipment and location.

(Evaluation process)
The inspection method can further include an evaluation step. By performing the evaluation process, the basis for diagnosis of coronary artery disease can be obtained with higher accuracy. The evaluation step is preferably an evaluation step in which the plasma prorenin amount is compared with a preset threshold value. By contrasting with the set threshold value, a highly accurate diagnosis basis can be obtained.

  The threshold value can be obtained by measuring the plasma prorenin level for a sample (blood or plasma) collected from a patient with a confirmed diagnosis of coronary artery disease. A definitive diagnosis of coronary artery disease can be obtained by an already established diagnostic method such as myocardial scintigraphy, coronary CT, or cardiac catheterization.

  In addition, as described above, as a coronary artery disease, it is preferable to set a threshold value for the plasma prorenin amount according to the degree of stenosis by coronary angiography. For example, a threshold value corresponding to significant stenosis (generally 75% or more stenosis) obtained by definitive diagnosis by coronary angiography can be set. For example, the threshold value may be set based on a stenosis of 70% or more. Further, the threshold value may be set based on a stenosis of 75% or more. A threshold value may be set based on a stenosis of 80% or more. Furthermore, the threshold value may be set based on a stenosis of 85% or more. Furthermore, the threshold value may be set based on 90% or more stenosis. Preferably, the threshold is set based on the lowest 75% of significant stenosis. This is because 75% stenosis is a stage where treatment is required and there is a case where there is still no subjective symptom, and detection by examination of coronary artery disease is a significant stage. The threshold value can be set based on a positive predictive value or the like.

  As shown in the examples described later, for example, the threshold value of the plasma prorenin amount can be 1000 pg / ml. When the threshold is exceeded or exceeded, stenosis is often a coronary artery disease of 75% or more. In this case, generally the positive predictive value can be 80% or more. More preferably, the threshold is 1100 pg / ml. When the threshold is exceeded or exceeded, the positive predictive value of coronary artery disease with stenosis of 75% or more is generally 90% or more.

(Inspection kit)
The test kit disclosed in the present specification is a test kit for coronary artery disease. The test kit can include one or more reagents for measuring prorenin. A reagent for measuring prorenin can be appropriately selected by those skilled in the art depending on the measurement method. In the case of the enzyme activity method, it is angiotensinogen or the like as a substrate for renin, and in the case of the immunoassay method, an anti-prorenin antibody or the like can be mentioned.

  Hereinafter, the disclosure of the present specification will be described with specific examples, but the disclosure of the present specification is not limited thereto.

  Among patients undergoing coronary angiography for definitive diagnosis of coronary artery disease complaining of chest pain or chest discomfort, patients with irregular angina or myocardial infarction in the last 3 months, severe heart failure (NYHA: IV And / or 85 patient groups excluding left ventricular ejection fraction were examined for plasma prorenin and other biochemical items. In addition, the coronary angiography on which the coronary angiography was examined was also diagnosed as coronary artery disease when the coronary artery had a stenosis of 75% or more. 59 people). A summary of the patient group is shown in Table 1. Of the patients, 24 were orally administered angiotensin converting enzyme inhibitors (ACEIs) or angiotensin II receptor inhibitors (ARBs), 30 were orally administered calcium channel inhibitors, and were orally administered β inhibitors. There were 12 people.

  Measurement of plasma prorenin and renin was performed as follows. Blood was collected from the median cubital vein before fasting breakfast on the day of cardiac catheterization. The blood was immediately placed on ice, centrifuged at 3000 g for 10 minutes at 4 ° C., and then immediately stored at −80 ° C. until analysis. Plasma prorenin was performed using an ELISA kit (LINCO Research Inc., USA). Plasma BNP was measured by immunoradiometric assay (Shinoria BNP RIA kit; Shionogi, Japan). In addition, HbAlc, total cholesterol, LDL-cholesterol, HDL-cholesterol and estimated glomerular filtration rate (eGFR) were also measured by conventional methods.

  The results for the 86 patient group are shown in Tables 1 and 2. The test results obtained by classifying the plasma prorenin concentration of 1100 pg / ml as a cut-off value in the coronary artery disease group are shown in Table 3, and a blot of plasma prorenin concentration, non-coronary artery disease group, and coronary artery disease group is shown in FIG.

  As shown in Table 1, regarding the hemodynamics, hyperlipidemia and left ventricular ejection fraction were significantly different (P value = 0.001) between the non-coronary artery disease group and the coronary artery disease group. Moreover, as shown in Table 2, there was no significant difference between the non-coronary artery disease group and the coronary artery disease group regarding the plasma prorenin concentration. Similarly, the plasma renin activity was not significantly different between the non-coronary artery disease group and the coronary artery disease group.

  Throughout all patient groups, plasma prorenin concentration had a weak correlation with renin concentration (r = 0.35, p = 0.001) and also had a weak correlation with renin activity (r = 0.34, p = 0.001). However, there was no correlation between prorenin and aldosterone, brain natriuretic peptide, left ventricular ejection fraction and estimated glomerular filtration rate.

  On the other hand, as shown in Table 3, when the coronary artery disease group was classified using a plasma prorenin concentration of 1100 pg / ml as a cut-off value, there was a significant difference between the plasma prorenin concentration and the plasma renin concentration. Moreover, as shown in FIG. 1, the ratio of the coronary artery disease group increased as the plasma prorenin concentration increased.

  When the plasma prorenin concentration was 1100 pg / ml or more, the positive predictive value was 94% (15/16), and the negative predictive value was 36%. When the plasma renin activity was cut off at 0.8 ng / ml / hr, the positive predictive value was 82% and the negative predictive value was 35%.

  Based on the above, plasma prorenin concentration enables early detection of coronary artery disease, and plasma prorenin concentration is an indicator of the degree of progression to coronary artery disease and the degree of improvement of coronary artery disease. It was found to be effective for early detection, prevention and treatment of coronary artery disease.

Claims (8)

A test kit for coronary artery disease,
Comprising one or more reagents for measuring plasma prorenin concentration based on an immunoassay or a renin enzyme activity assay;
A kit for detecting significant stenosis of a coronary artery on coronary angiography based on the plasma prorenin concentration .   The kit according to claim 1, wherein the reagent comprises an anti-prorenin antibody. A test method for coronary artery disease,
A measuring step for measuring plasma prorenin concentration ,
With
It examines the coronary artery disease based on a threshold associated with a significant narrowing of the coronary arteries on the plasma prorenin concentration and coronary angiography methods. The test method according to claim 3, wherein the threshold is set based on a plasma prorenin concentration of a coronary artery disease patient having a stenosis of 75% or more in a coronary artery on coronary angiography . The test method according to claim 3, wherein the threshold is set based on a plasma prorenin concentration of a coronary artery disease patient having a stenosis of 50% or more in the left coronary artery main trunk on coronary angiography . The test method according to claim 3 , wherein the threshold value is 1100 pg / ml as a plasma prorenin concentration . The test | inspection method in any one of Claims 3-6 used for monitoring of a coronary artery disease . A method for examining coronary artery stenosis in coronary artery disease ,
A measuring step for measuring plasma prorenin concentration ,
With
It examines the stenotic coronary artery based on a threshold associated with a significant narrowing of the coronary arteries on the plasma prorenin concentration and coronary angiography methods.