JP4398188B2 - Method for testing acute coronary syndrome and its pathology - Google Patents

Description

【００３０】
安定期冠動脈疾患群に対して、不安定狭心症群のプレβ1-HDL濃度は有意に高かった。また、プレβ1-HDL/HDL-C比およびプレβ1-HDL/アポA-Ｉ比の値も安定期冠動脈疾患群に対して、不安定狭心症群の方が有意に高かった。一方、従来公知の脂質マーカー（中性脂肪、総コレステロール、HDL-C、LDL-C、アポA-Ｉ、アポB）は、いずれの項目についても安定期冠動脈疾患群と不安定狭心症群で有意な差を認めなかった。
なお、健常者群（77例）のプレβ1-HDL濃度は27.6±7.0μg/mLであり、不安定狭心症群、安定期冠動脈疾患群に対して有意に低かった（ p < 0.0001 ）。
【００３１】
以上より、プレβ1-HDLが、ACSおよびその病態を検査する臨床化学的、特に脂質に関連する指標として有用であることがわかった。
【００３２】
（実施例２）
実施例１で得られたデータをもとに、プレβ1-HDL、プレβ1-HDL/HDL-C比およびプレβ1-HDL/アポA-Ｉ比のそれぞれについてROC曲線（receiver operating characteristic curve : 受診者動作特性曲線）を作成し、従来公知の脂質マーカーのROC曲線と比較した。
【００３３】
ROC曲線は、NCCLS（National committee for clinical laboratory standards）が、診断精度評価の標準的手法として採用している方法であり、検査の精度の評価や従来の検査と新しい検査の比較のため、カットオフ値を変えた場合の検査の感度と特異度との関係を示したグラフである。縦軸は診断的感度、つまり、実際の対象疾患罹患者のうちその検査で罹患者となる確率を示す。横軸は、1−特異度を示す。特異度とは、実際に罹患していない患者がその検査で罹患者にならない確率をいう。異なる検査の優劣を判定する場合は、この曲線がより左上方に位置するほど優れていることを示す。
【００３４】
安定期冠動脈疾患に対する不安定狭心症のROC曲線を各測定項目で画き診断の感度および特異度を比較した結果を図１に示した。
【００３５】
プレβ１-HDL、プレβ1-HDL/HDL-C比およびプレβ1-HDL/アポA-Ｉ比に関するROC曲線は、従来公知の脂質マーカーのROC曲線に比べ明瞭に左上方に位置しており、不安定狭心症群と安定期冠動脈疾患群の判別に有用であることがわかる。
【００３６】
（実施例３）
実施例１で得られたデータ中から、不安定狭心症群と安定期冠動脈疾患群それぞれについて、日本動脈硬化学会による「高脂血症治療ガイドライン」に示されている（１）血清総コレステロール濃度が220mg/dL未満、（２）HDL-コレステロール濃度が40mg/dL以上、（３）中性脂肪濃度が150mg/dL未満、を同時に満たすものを脂質マーカーの異常がないもの（正脂血症）として抽出し、プレβ1-HDL、プレβ1-HDL/HDL-C比およびプレβ1-HDL/アポA-Ｉ比を比較した。統計処理ソフト「Stat Mate III」（株式会社アトムス製）を用いてｔ検定を行った結果を含め表２に示した。
【００３７】
【表２】

【００３８】
安定期冠動脈疾患群に対して、不安定狭心症群のプレβ1-HDL濃度、プレβ1-HDL/HDL-C比およびプレβ1-HDL/アポA-Ｉ比は有意に高く、正脂血症の場合においても不安定狭心症群と安定期冠動脈疾患群を識別できることがわかる。
【００３９】
以上より、プレβ1-HDLは脂質に関連する指標でありながら、正脂血症のACSにおいても臨床化学的指標として有用であることがわかった。
【００４０】
（実施例４）
安定期冠動脈疾患（陳旧性心筋梗塞）に区分されていたが、その後に不安定狭心症の症状を呈した被検者について、不安定狭心症の症状を呈する前後のプレβ1-HDL濃度、プレβ1-HDL/HDL-C比およびプレβ1-HDL/アポA-Ｉ比を比較した。
【００４１】
安定期冠動脈疾患に区分されていた時点の血中プレβ1-HDL濃度は39.4μg/dLであったが、不安定狭心症に区分される症状を呈した後の血中プレβ1-HDL濃度は72.9μg/dLと著明に上昇していた。同様に、プレβ1-HDL/HDL-C比は、5.97から11.57へ、プレβ1-HDL/アポA-1比は 2.76から4.89へ、著明に上昇していた。
【００４２】
以上より、プレβ1-HDLが、ACSおよびその病態を検査する臨床化学的指標として、病態の変化や臨床症状の変化の把握や予知に有用であることがわかった。
【図面の簡単な説明】
【図１】安定期冠動脈疾患に対する不安定狭心症のROC曲線を各測定項目で画き診断の感度および特異度を比較した結果を示す図である。[0001]
[Field of the Invention]
The present invention relates to a method for examining acute coronary syndrome and its pathological condition.
[0002]
[Prior art]
Inclusive of unstable angina, acute myocardial infarction, and sudden cardiac death, it is called acute coronary syndrome (ACS). ACS refers to the above three ischemic heart diseases as plaque (plaque: atherosclerotic lesion) rupture, then sudden thrombus formation at the rupture site, and complete or incomplete occlusion of coronary artery due to thrombus formation As a result of continuous onset, unstable angina pectoris caused by temporary occlusion or severe stenosis in the coronary artery lumen due to thrombus, and acute myocardial infarction resulting from complete occlusion The idea is that sudden cardiac death occurs (Non-Patent Documents 1, 2, etc.). In clinical practice, about half of cases of acute myocardial infarction have angina symptoms observed within a few weeks before the onset, so unstable angina is at high risk of progression to myocardial infarction and sudden cardiac death, It is understood to be a precursor stage leading to the onset of acute myocardial infarction.
[0003]
Conventionally, coronary stenosis gradually progressed due to thickening of the intima associated with the occurrence of plaque as the onset mechanism of ischemic heart disease, but acute myocardial infarction is Since lesions of 50% or less occur in 68% (Non-patent Document 2), the plaque state rather than the degree of stenosis, that is, a plaque that causes ACS even if it is not highly stenotic ( The presence of unstable plaques) and thrombus formation due to plaque rupture have been considered to be important as a pathogenic mechanism.
[0004]
Unstable plaques have a higher proportion of lipid cores, mainly cholesterol esters, and contain more macrophages and foam cells, but less collagen and smooth muscle cells than stable plaques. These contents are covered with a thin fibrous film, and are thought to be easily broken by damage to the vascular endothelium, stress on the blood vessel wall, inflammation, and the like.
[0005]
ACS and its pathological examination methods include subjective methods such as chest pain, as well as physical methods such as angiography, angioscopy, and MRI, but are related to established clinical chemistry indicators, particularly lipids No indicators have been found. This gives information on the presence of ACS and its pathology, for example, vulnerable plaque, and clinical chemistry indicators that can be used to understand and predict changes in the pathology and clinical symptoms in the near future (for example, the onset of myocardial infarction) In particular, there has been a demand for an index related to lipids and an inspection method thereof.
[0006]
[Patent Document 1]
JP 2001-066634 A [Patent Document 2]
Japanese Patent Application No. 2002-181102 [Patent Document 3]
JP 2000-239300 A [Non-Patent Document 1]
Fuster, V., et al; N. Engl. J. Med., 326, 242-250 (1992)
[Non-Patent Document 2]
Falk, E., et al; Circulation, 92, 657-671 (1995)
[Non-Patent Document 3]
Sviridov, D. & Nestel, P.; Atherosclerosis, 161, 245-254 (2002)
[Non-Patent Document 4]
Miida, T., et al; Clin. Chem., 42, 1992-1995 (1996)
[Non-Patent Document 5]
O'connor, PJ., Et al; Anal. Biochem., 251, 234-240 (1997)
[Non-Patent Document 6]
Nanjee, MN. & Brinton, EA.; Clin. Chem., 46, 207-223 (2000)
[Non-Patent Document 7]
Miyazaki, O., et al; J. Lipid Res., 41, 2083-2088 (2000)
[0007]
[Problems to be solved by the invention]
The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide an ACS and its pathological examination method using clinical chemistry indicators, particularly lipid-related indicators.
[0008]
[Means for Solving the Problems]
The present inventors provide information on the presence of ACS and its pathology, for example, the presence of vulnerable plaque, and can grasp and predict changes in the near future and clinical symptoms (for example, the onset of myocardial infarction). As a result of searching and examining chemical indices, particularly indices related to lipids, it was found that pre-β1-HDL can be used to solve the above problems, and the present invention was completed.
[0009]
That is, the present invention provides an ACS comprising measuring pre-β1-HDL in a blood sample and a method for examining the disease state thereof.
[0010]
Pre-β1-HDL is one of the subfractions of high-density lipoprotein (HDL) that plays a central role in the reverse cholesterol transport system that extracts excess cholesterol from tissues and returns it to the liver, and is involved in the early stages of cholesterol withdrawal It has been speculated that blood levels increase in coronary heart disease (CHD), hypertriglyceridemia, and hypercholesterolemia. Non-patent document 3).
[0011]
In addition, by incubating blood samples collected from subjects over time and measuring pre-β1-HDL behavior in the samples over time, abnormalities in lipid metabolism or diseases caused by abnormal lipid metabolism can be detected. A detection method has also been reported (Patent Document 1).
[0012]
Furthermore, the present inventors measured the pre-β1-HDL in a blood sample, so that a subject known in the art cannot find a known lipid marker such as HDL-cholesterol, metabolic risk factors such as diabetes and hypertension. A method for inspecting morbidity of arteriosclerosis has been invented and a patent application has been filed (Patent Document 2).
[0013]
In this way, pre-β1-HDL has been reported or found several findings related to hyperlipidemia and arteriosclerosis, but it gives information on ACS and its pathology, changes in clinical conditions and clinical symptoms in the near future It was not known at all that it would be a clinical chemistry index that could grasp and predict changes in the body.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the blood sample collected from the subject may be whole blood, plasma, or serum. When using plasma, EDTA or sodium citrate can be used as an anticoagulant. After blood collection, it is preferably kept under ice cooling, and when plasma or serum is obtained, it is preferably centrifuged under cooling.
[0015]
Pre-β1-HDL in blood samples is known to vary in concentration due to storage and freezing and thawing. Pre-β1-HDL is stabilized by adding monosaccharides, disaccharides, or glycerol to blood samples and coexisting them. It can be made.
[0016]
Pre-β1-HDL is measured by two-dimensional electrophoresis followed by non-denaturing two-dimensional electrophoresis (Non-Patent Document 4) in which Western blotting is performed using a radiolabeled anti-apo A-1 antibody. Ultrafiltration isotope dilution method (Non-patent Document 5) utilizing mixing of a certain amount of β1-HDL with a sample and measuring the radioactivity in the ultrafiltered filtrate, using two connected columns Gel filtration method (Non-patent Document 6) for measuring apo A-1 in fractions corresponding to pre-β1-HDL fractionated by gel filtration, using anti-pre-β1-HDL monoclonal antibody and anti-apo A-1 antibody A known measurement method such as a sandwich ELISA method (Non-patent Document 7) can be used. Of these measurement methods, the ELISA method is preferable because of its excellent reproducibility and simplicity.
[0017]
In the present invention, the examination of ACS and its pathological condition means that the subject suffers from unstable angina pectoris and acute myocardial infarction with the desired accuracy even when there is no subjective symptom or laboratory test result. It means that it can be estimated that there is unstable plaque. Furthermore, it also means that it is possible to grasp and predict changes in the pathology and clinical symptoms in the near future (for example, the onset of myocardial infarction).
[0018]
ACS and its pathological examination can be performed by setting a certain standard for the amount of pre-β1-HDL in the blood sample, and comparing and evaluating the measured value of the blood sample with the standard. Examples of how to set the standard include a 95th percentile value often used in the field of clinical testing and a method of setting from desired testing accuracy using an ROC curve described later.
[0019]
In addition to the method of performing the pre-β1-HDL measurement value alone, the evaluation may be performed by associating the measurement value of the pre-β1-HDL with another index, for example, the measurement value of a known lipid marker. Examples of lipid markers associated here include HDL-cholesterol or apoA-I. In the present specification, associating the abundance with the abundance means obtaining information that could not be obtained with pre-β1-HDL alone using a calculation formula.
[0020]
The method for associating the abundance with the abundance is to divide the abundance of pre-β1-HDL by the abundance of a known lipid marker and use the calculated ratio (ratio of the two abundances) as a new index. Etc. can be illustrated. Thereby, the accuracy of the inspection can be improved. Moreover, it is possible to improve the accuracy by appropriately combining with measured values of substances other than HDL-cholesterol and apoA-I (which may be an index other than lipid), and these are naturally included in the present invention. .
[0021]
Pre-β1-HDL is currently accumulating knowledge on clinical significance and physiological roles, and it is considered that international reference materials and pricing methods will be proposed after this application. However, even in such a case, it goes without saying that the present invention can be implemented without hindrance by appropriately converting the standard substances described in this specification.
[0022]
【The invention's effect】
According to the present invention, it is possible to examine the clinical chemistry index established so far, in particular, ACS and its pathology for which an index related to lipid did not exist, using the index related to lipid, and the failure of plaque It is useful for the diagnosis, prevention and treatment of ACS, which is a concept that captures a series of pathological conditions beginning with, and for understanding and predicting changes in pathological conditions and clinical symptoms.
[0023]
【Example】
EXAMPLES Next, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.
[0024]
(Measurement of pre-β1-HDL)
(1) Preparation of specimen Whole blood collected from the cubital vein containing EDTA or a serum separating agent-containing blood collection tube during early morning fasting was centrifuged at 3000 rpm for 15 minutes under cooling to obtain EDTA plasma and serum, respectively. A 50% sucrose aqueous solution was added to the obtained EDTA-containing plasma and serum in a volume 20 times that of the plasma or serum, and frozen until stored.
[0025]
(2) ELISA reagent described in JP 2000-239300 A (Patent Document 3) “(1) Human apolipoprotein AI present in HDL having a molecular weight of 150,000 or less and having no human apolipoprotein A-II and ( 2) Monoclonal antibody 55201 that specifically reacts with human apolipoprotein AI not bound to lipid (deposited with FERM BP-6938 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (original deposit date 1998) Nov. 17, 1) ELISA reagent using a monoclonal antibody produced by hybridoma 55201). Purified human apoprotein AI was used as the standard for converting the measured absorbance into the pre-β1-HDL concentration (μg / mL). Purified human apo AI was prepared from human plasma by affinity chromatography using an anti-apo AI antibody-coupled Sepharose column and gel filtration method, and bovine serum albumin was used as a calibration substance to determine the protein amount by quantification by BCA method.
[0026]
(Measurement of lipid markers)
Neutral fat, total cholesterol, HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C) are enzymatic methods, Apo AI and Apo B are reagents by immunoturbidimetry (all made by Daiichi Chemical) Measured using.
[0027]
(Criteria for grouping subjects)
1. Unstable angina group Followed the American Heart Association (AHA) classification of angina (1975). In other words, seizures start within 3 weeks, final seizures occur within 1 week, and there is no electrocardiogram change or serum enzyme elevation indicating acute myocardial infarction, and any of the following three criteria Unstable angina pectoris.
(1) A seizure is the first episode or has recurred after 6 months of asymptomatic symptoms (first onset angina).
(2) Those with stable exertion angina are those in which the frequency, duration, and intensity of chest pain attacks have increased, and the effects of nitroglycerin have been attenuated (exacerbation type).
(3) A seizure that develops even at rest and persists for more than 15 minutes, is not improved by nitroglycerin, or is accompanied by a transient ECG change (initial resting angina).
2. Angina pectoris that appeared in the stable coronary artery disease group with an almost constant effort, and has no symptoms as listed in unstable angina (stable effort angina) or after the onset of myocardial infarction 1 More than a month (old-aged myocardial infarction) without subsequent myocardial ischemic attack and no progression of coronary angiography.
[0028]
Example 1
Plasma pre-β1-HDL levels and lipid markers in the unstable angina group (35 cases) and stable coronary artery disease group (79 cases) were measured. About the obtained measured value, t test was performed using statistical processing software "Stat Mate III" (made by Atoms Co., Ltd.). The results are shown in Table 1. Note BMI Oyo BiOsamu Chijimiki blood pressure as basic information for the measurement it was possible examples, shown in Table 1 together also diastolic blood pressure.
[0029]
[Table 1]

[0030]
The pre-β1-HDL concentration in the unstable angina group was significantly higher than that in the stable coronary artery disease group. The pre-β1-HDL / HDL-C ratio and the pre-β1-HDL / apo A-I ratio were also significantly higher in the unstable angina group than in the stable coronary artery disease group. On the other hand, conventionally known lipid markers (neutral fat, total cholesterol, HDL-C, LDL-C, apoA-I, apoB) are stable coronary artery disease group and unstable angina group for all items. There was no significant difference.
The pre-β1-HDL concentration in the healthy group (77 cases) was 27.6 ± 7.0 μg / mL, which was significantly lower than the unstable angina group and the stable coronary artery disease group (p <0.0001).
[0031]
From the above, it was found that pre-β1-HDL is useful as an index related to clinical chemistry, particularly lipids, for examining ACS and its pathological conditions.
[0032]
(Example 2)
Based on the data obtained in Example 1, each of the pre-β1-HDL, pre-β1-HDL / HDL-C ratio, and pre-β1-HDL / apoA-I ratio is ROC curve (receiver operating characteristic curve) Operating characteristic curve) was prepared and compared with the ROC curve of a conventionally known lipid marker.
[0033]
The ROC curve is a method adopted by the NCCLS (National Committee for Clinical Laboratory Standards) as a standard method for evaluating diagnostic accuracy, and is used to evaluate the accuracy of tests and to compare conventional tests with new tests. It is the graph which showed the relationship between the sensitivity of a test | inspection at the time of changing a value, and specificity. The vertical axis shows the diagnostic sensitivity, that is, the probability of being affected by the test among the actual affected patients. The horizontal axis indicates 1-specificity. Specificity refers to the probability that a patient who is not actually affected will not be affected by the test. When judging the superiority or inferiority of different examinations, it indicates that the curve is more excellent as it is located at the upper left.
[0034]
The ROC curve of unstable angina for stable coronary artery disease was plotted for each measurement item, and the results of comparing the sensitivity and specificity of the diagnosis are shown in FIG.
[0035]
The ROC curves for pre-β1-HDL, pre-β1-HDL / HDL-C ratio and pre-β1-HDL / apoA-I ratio are clearly located at the upper left compared to the ROC curves of conventionally known lipid markers, It can be seen that it is useful for discriminating between unstable angina and stable coronary artery disease groups.
[0036]
(Example 3)
From the data obtained in Example 1, each of the unstable angina group and the stable coronary artery disease group is shown in the “Guideline for Treatment of Hyperlipidemia” by the Japanese Atherosclerosis Society (1) Serum total cholesterol Concentration of less than 220 mg / dL, (2) HDL-cholesterol concentration of 40 mg / dL or more, and (3) triglyceride concentration of less than 150 mg / dL, with no abnormal lipid marker (hyperlipidemia) ) And pre-β1-HDL, pre-β1-HDL / HDL-C ratio and pre-β1-HDL / apoA-I ratio were compared. The results of t-test using statistical processing software “Stat Mate III” (manufactured by Atoms Co., Ltd.) are shown in Table 2.
[0037]
[Table 2]

[0038]
Compared to stable coronary artery disease group, pre-β1-HDL concentration, pre-β1-HDL / HDL-C ratio and pre-β1-HDL / apoA-I ratio in unstable angina group were significantly higher It can be seen that an unstable angina group and a stable coronary artery disease group can be distinguished even in the case of symptom.
[0039]
From the above, it was found that pre-β1-HDL is useful as a clinical chemistry index in ACS of normolipidemia although it is an index related to lipid.
[0040]
Example 4
For subjects who had been classified as stable coronary artery disease (old myocardial infarction) but subsequently presented with unstable angina, pre-β1-HDL before and after presenting symptoms of unstable angina Concentration, pre-β1-HDL / HDL-C ratio and pre-β1-HDL / apoA-I ratio were compared.
[0041]
The blood pre-β1-HDL concentration at the time of being classified as stable coronary artery disease was 39.4 μg / dL, but the blood pre-β1-HDL concentration after showing symptoms classified as unstable angina Was significantly increased to 72.9 μg / dL. Similarly, the pre-β1-HDL / HDL-C ratio increased significantly from 5.97 to 11.57, and the pre-β1-HDL / Apo A-1 ratio increased from 2.76 to 4.89.
[0042]
From the above, it was found that pre-β1-HDL is useful for grasping and predicting changes in pathological conditions and clinical symptoms as clinical chemistry indicators for examining ACS and its pathological conditions.
[Brief description of the drawings]
FIG. 1 is a diagram showing the results of comparing the sensitivity and specificity of diagnostic imaging by plotting ROC curves of unstable angina against stable coronary artery disease for each measurement item.

Claims (10)

  A method for examining acute coronary syndrome and its pathological condition, comprising measuring pre-β1-HDL in a blood sample.   Measure the abundance of pre-β1-HDL in the blood sample and the abundance of an index selected from the group consisting of neutral fat, total cholesterol, HDL-cholesterol, LDL-cholesterol, apoA-I, and apoB, A method for examining acute coronary syndrome and its pathological condition, comprising determining the ratio of   A method for examining acute coronary syndrome and its pathological condition, comprising measuring the abundance of pre-β1-HDL and abundance of HDL-cholesterol or apoA-I in a blood sample and determining a ratio thereof.   The method for examining acute coronary syndrome and its disease state according to any one of claims 1 to 3, wherein the acute coronary syndrome is unstable angina.   The method for examining acute coronary syndrome and its disease state according to any one of claims 1 to 3, wherein the acute coronary syndrome is a stage before acute myocardial infarction.   The method for examining acute coronary syndrome and its disease state according to any one of claims 1 to 5, wherein the blood sample is obtained from a subject who has chest pain or who has had chest pain in the past.   The method for examining acute coronary syndrome and its disease state according to any one of claims 1 to 5, wherein the blood sample is obtained from a subject with unstable angina. The test method according to any one of claims 1 to 5, wherein the blood sample is obtained from a hyperlipidemic subject.   A blood sample was obtained from a subject having (1) a total cholesterol concentration of less than 220 mg / dL, (2) an HDL-cholesterol concentration of 40 mg / dL or more, and (3) a neutral fat concentration of less than 150 mg / dL. The method for examining acute coronary syndrome and its pathological condition according to claim 8. The method for examining acute coronary syndrome and its disease state according to any one of claims 1 to 9, wherein the disease state of acute coronary syndrome is the presence of unstable plaque.

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