WO2017128162A1 - 诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物 - Google Patents

诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物 Download PDF

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WO2017128162A1
WO2017128162A1 PCT/CN2016/072440 CN2016072440W WO2017128162A1 WO 2017128162 A1 WO2017128162 A1 WO 2017128162A1 CN 2016072440 W CN2016072440 W CN 2016072440W WO 2017128162 A1 WO2017128162 A1 WO 2017128162A1
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stable angina
coronary atherosclerosis
metabolic
metabolic marker
angina pectoris
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French (fr)
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齐炼文
朱伟
秦勇
陈彦
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齐炼文
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the invention belongs to the field of biochemistry and relates to a metabolic marker for diagnosis and classification of coronary heart disease, and particularly relates to a group of metabolic markers for diagnosing coronary atherosclerosis and stable angina pectoris.
  • Coronary heart disease also known as ischemic heart disease, involves atherosclerosis in the arteries supplying myocardial blood, that is, coronary atherosclerotic lesions cause vascular stenosis or plaque formation or even rupture, complete blockage, resulting in myocardial ischemia, hypoxia Or necrosis leads to ischemic heart disease, which causes a series of serious cardiovascular events such as clinical angina and myocardial infarction.
  • Coronary heart disease is the main killer of human health. It has the characteristics of high incidence, high disability rate, high recurrence rate, high mortality rate and many complications. It has become a major disease threatening the health of our people.
  • coronary heart disease is divided into chronic stable coronary heart disease (ie, stable angina) and acute coronary syndrome; acute coronary syndrome is further divided into unstable angina and acute myocardial infarction.
  • Coronary heart disease from scratch, from light to heavy usually includes the following stages of development: normal coronary artery ⁇ coronary atherosclerosis ⁇ stable angina pectoris ⁇ unstable angina pectoris ⁇ acute myocardial infarction.
  • Coronary atherosclerosis is the main cause of coronary heart disease and is an early condition of coronary heart disease. Coronary atherosclerosis is a common progressive arterial disease. The lesions mainly involve moderately sized myometrial arteries, arterial intima lipid deposits, smooth muscle cell hyperplasia, and the formation of localized plaques that can harden the arterial wall. The rupture of the plaque leads to thrombosis, embolism, hemorrhage, partial or complete occlusion of the affected lumen, and clinical manifestations of atherosclerotic vascular complications. Early atherosclerotic lesions can occur before the age of 10 years. The disease causes arterial stenosis to take 20 to 30 years. It has no clinical symptoms in the early stage and is difficult to be discovered and valued. Therefore, it can be effective for early prevention and diagnosis of coronary atherosclerosis. To prevent the occurrence of coronary heart disease.
  • Coronary angiography can accurately determine the degree of coronary artery stenosis, and is the gold standard for the diagnosis of coronary heart disease [Comparative study of coronary angiography gold standard and clinical routine diagnosis of coronary heart disease, Shu Rongwen et al., Naval Medical Journal 2015 04] .
  • coronary angiography can only find the degree of vascular stenosis, and it is also an interventional measurement method, requiring interventional surgery, and the diagnosis is expensive.
  • the doctor also needs to make a final diagnosis based on the patient's electrocardiogram, cardiogram, treadmill exercise test, CT and other examination results.
  • Metabolomics is a science that studies the whole body of endogenous metabolites and their changes with internal and external factors. It is an important part of systems biology. It can perform rapid and non-invasive analysis of body fluids such as blood and urine. The difference in the spectrum gives a metabolic marker that can indicate various biochemical reactions.
  • analytical techniques include nuclear magnetic resonance (NMR), mass spectrometry (LC-MS/GC-MS), and the like.
  • LC-MS/GC-MS has the characteristics of low sample preparation, high sensitivity, wide dynamic range, etc. It can be used to detect metabolites with large differences in concentration in samples, and thus become more and more applied in the study of metabolomics. platform.
  • Plasma analysis is a commonly used disease diagnosis method in clinical practice and is widely used because of its advantages of simplicity, rapidity, economy, and relatively non-invasiveness.
  • a first object of the present invention is to provide a set of metabolic markers for diagnosing coronary atherosclerosis and stable angina pectoris, which are simultaneously present in plasma and can be simultaneously analyzed and determined.
  • a second object of the present invention is to provide a method capable of sensitively detecting and detecting the metabolic marker;
  • a third object of the present invention is to provide a detection kit based on the metabolic marker for diagnosing a coronary artery Atherosclerosis and stable angina pectoris improve diagnostic convenience and promote standardization of diagnostic methods.
  • a set of metabolic markers for the diagnosis of coronary atherosclerosis and stable angina including one or more of the following metabolic markers: phosphocholine, palmitoylethanolamine, phytosphingosine, phosphatidylcholine , ethyl chenodeoxycholic acid, lysophosphatidylcholine (16:0), lysophosphatidylcholine (18:2), and phosphatidylinositol (20:4/0:0).
  • Lysophosphatidylcholine (16:0), 1-palmitoyl-glycerol-3-phosphatidylcholine.
  • Lysophosphatidylcholine (18:2), 1- linoleoyl-glycerol-3-phosphatidylcholine.
  • Phosphatidylinositol (20:4/0:0), 1-arachidontylglycerol phosphatidylinositol.
  • the set of metabolic markers for diagnosing coronary atherosclerosis and stable angina includes any of the three described metabolic markers.
  • the set of metabolic markers for diagnosing coronary atherosclerosis and stable angina includes any of the four described metabolic markers.
  • the set of metabolic markers for diagnosing coronary atherosclerosis and stable angina includes any of the five described metabolic markers.
  • the set of metabolic markers for diagnosing coronary atherosclerosis and stable angina comprises any of the six described metabolic markers.
  • the set of metabolic markers for diagnosing coronary atherosclerosis and stable angina comprises all eight of said metabolic markers.
  • the metabolic marker is derived from plasma.
  • a qualitative or quantitative method for diagnosing a metabolic marker for distinguishing between coronary atherosclerosis and stable angina pectoris is: qualitatively characterizing said metabolic marker by LC/MS and/or GC/MS Or quantitative analysis.
  • the liquid and temperament detection limits are low and the sensitivity is high, and the metabolic markers in the biological samples can be sensitively analyzed and quantified.
  • a kit for the diagnosis of coronary atherosclerosis and stable angina pectoris comprising a standard of said metabolic marker, said standard being a chemical monomer or mixture of each metabolic marker.
  • Standards can be used to quickly and accurately characterize and quantify metabolic markers in biological samples.
  • the kit facilitates standardization of testing and improves ease of detection and reproducibility.
  • the detection kit further comprises a solvent that dissolves the standard and/or a solvent that extracts the metabolic marker.
  • the metabolic markers provided by the present invention can accurately diagnose and distinguish between coronary atherosclerosis and stable angina pectoris.
  • the area under the ROC curve AUC is greater than 0.5, and the closer to 1, the better the diagnostic effect.
  • AUC has lower accuracy from 0.5 to 0.7
  • AUC has a certain accuracy from 0.7 to 0.9
  • AUC has higher accuracy at above 0.9. It has been verified that the metabolic markers provided by the present invention are used for the diagnosis of coronary atherosclerosis and stable angina pectoris, and the AUC is above 0.7; when combined, the AUC is closer to 1 than the individual, and the diagnosis is better. When eight joint applications, the AUC is closest to 1, and the diagnostic distinction is best.
  • the method for analyzing and detecting the metabolic marker provided by the present invention has high sensitivity and accurate and reliable results.
  • the detection kit provided by the invention can be used for diagnosing coronary atherosclerosis and stable angina pectoris, improving the convenience of diagnosis, and promoting standardization of diagnostic methods.
  • Example 1 Screening and Characterization of Plasma Differential Metabolites Between Patients with Coronary Atherosclerosis and Stable Angina Pectoris
  • the peripheral blood plasma of 480 patients with coronary atherosclerosis, 280 patients with stable angina pectoris and 350 healthy people from September 2010 to June 2015 in Jiangsu Provincial People's Hospital were collected. All patients or health Per capita was confirmed by coronary angiography. The age and gender of healthy people matched with patients with coronary atherosclerosis and stable angina. All patients with coronary atherosclerosis, stable angina pectoris and healthy people have normal heart, lung, liver and kidney and hematopoietic function.
  • the blood collection time is in the early morning fasting state.
  • Acetonitrile and formic acid were purchased from ROE Company of the United States; chromatographic grade methanol and chloroform were purchased from Jiangsu Hanbang Technology Co., Ltd.; methoxyamine and N-methyl-N-(trimethylsilane)trifluoroacetamide (containing 1% trimethylchlorosilane) purchased from Sigma-Aldrich, USA; deionized water was prepared by Millipore's MIlli-Q ultrapure water system; standards include phosphorylcholine, palmitoylethanolamine, Phytosphingosine, phosphatidylcholine, ethyl chenodeoxycholic acid, lysophosphatidylcholine (16:0), lysophosphatidylcholine (18:2) and phosphatidylinositol (20:4/0) :0), purchased from Sigma-Aldrich, USA.
  • Extraction solvent optimization by response surface method The extraction of rich metabolites in plasma by different solvents (acetonitrile, methanol, ethanol, chloroform, water) was investigated by the number of peaks and total peak area in mass spectrometry ESI+ and ESI-detection modes. Set efficiency. The experimentally measured data was subjected to multivariate analysis, and the importance of the variable to the model response was reflected by the importance importance of the PLS model. The acetonitrile, methanol, ethanol, chloroform and water have the highest VIP values of 1.503, 0.802, 0.651, 0.688 and 0.987. The extraction efficiency of acetonitrile is the highest, so acetonitrile is selected as the extraction solvent for plasma samples.
  • Sample processing Take 100 ⁇ L of plasma in a 1.5 mL centrifuge tube, add 400 ⁇ L of acetonitrile, vortex for 30 seconds, mix, centrifuge at 13000 rpm ⁇ 10 min (4 ° C), take 200 ⁇ L of supernatant in a 1.5 mL centrifuge tube, and use nitrogen at room temperature. The mixture was blown dry, and the resulting residue was dissolved in 300 ⁇ L of a 20% aqueous acetonitrile solution.
  • the chromatographic separation was performed by ultra performance liquid chromatography (UPLC, Agilent 1290, USA).
  • the column was a Waters BEH C18 column (100 mm x 2.1 mm, 1.7 ⁇ m), the column temperature was 25 ° C, the injection chamber temperature was room temperature, and the injection volume was 2 ⁇ L.
  • the positive and negative ion mode mobile phase compositions are all A with a volume concentration of 0.1% formic acid aqueous solution and B with a volume concentration of 0.1% formic acid acetonitrile solution.
  • Mass spectrometry was performed using a quadrupole-time-of-flight mass spectrometer (Agilent 6530Q-TOF/MS, USA).
  • ESI electrospray ionization source
  • dry gas flow rate is 7L/min
  • dry gas temperature is 300 °C
  • dry gas and cone gas are high purity nitrogen
  • ion source temperature is 100 °C
  • positive ion and In negative ion mode the capillary voltage is 3000V and the collision voltage is 100V.
  • the data acquisition is performed three times per second in full scan mode.
  • the scanning mass range is m/z 100-1000 Daltons.
  • GC-Q/MS conditions American Agilent 7890B-5977A gas chromatography-mass spectrometer.
  • Column HP-5MS capillary column (30.0m ⁇ 0.25mm, capillary thickness 0.25 ⁇ m); carrier gas is high purity helium, flow rate 1.0mL / min; injection volume 2 ⁇ L; programmed temperature: 80 ° C constant temperature 2min, 80 ° C - 300 ° C (5 ° C / min) constant temperature 6 min; no split, injection temperature 300 ° C; interface temperature 300 ° C; ion source temperature 200 ° C; electron energy 50 eV; solvent delay 3 min; using full scan mode, scanning mass range: m / z 30-600 Daltons.
  • the expression levels of the above 8 differential metabolites in the plasma of patients with stable angina were down-regulated.
  • the quantitative expression of the above-mentioned differential metabolites in the plasma of patients with stable angina pectoris was 0.7-0.8 times lower than that of patients with coronary atherosclerosis. It can be seen that the above eight differential metabolites have different expression levels in the plasma of patients with stable angina pectoris and coronary atherosclerosis, and can be used for the diagnosis of coronary atherosclerosis and stable angina pectoris.
  • Example 2 Construction of ROC curves to verify the ability of 8 differential metabolites to diagnose coronary atherosclerosis and stable angina pectoris
  • the receiver operating curve (ROC) method was used to verify the differential expression levels of plasma metabolites in patients with stable angina and coronary atherosclerosis for the diagnosis of stable angina patients and patients with coronary atherosclerosis.
  • the results showed that phosphorylcholine, palmitoylethanolamine, phytosphingosine, phosphatidylcholine, ethyl chenodeoxycholic acid, lysophosphatidylcholine (16:0), lysophosphatidylcholine (18:2)
  • the eight differential metabolites of phosphatidylinositol (20:4/0:0) alone have a strong ability to diagnose patients with stable angina and coronary atherosclerosis, and the area under the ROC curve (AUC) is greater than 0.7, has clinical diagnostic significance; when used in combination, as the number of joints increases, the AUC is further increased, the highest in all 8 combinations, the AUC is 0.985, and the sensitivity and specificity are 97.4 at the best cutoff
  • Table 1 Single differential metabolite diagnosis distinguishes patients with coronary atherosclerosis and patients with stable angina
  • Table 3 any three to seven differential metabolites combined diagnosis to distinguish patients with coronary atherosclerosis and stable angina pectoris
  • these 8 differential metabolites can be used as metabolic markers for the diagnosis of coronary atherosclerosis and stable angina.
  • a detection kit is prepared based on the metabolic marker provided by the present invention, and the kit includes the following components:
  • Plasma metabolite extraction solvent 100% acetonitrile and 20% acetonitrile aqueous solution (for UPLC-Q/TOF-MS sample preparation); a ratio of 2.5:1:1 methanol, a mixed solution of chloroform and water, methoxyamine pyridine and N-methyl-N-trimethylsilyltrifluoroacetamide (for GC-Q/MS sample preparation); UPLC-Q/TOF-MS screening characterization, 20% aqueous acetonitrile can be used as a dissolution standard Solvent; GC-Q/MS screening characterization, using a plasma metabolite extraction solvent to prepare a standard solution by sample preparation;
  • the kit is designed based on the metabolic markers provided by the present invention and can be used to diagnose patients with coronary atherosclerosis and stable angina.
  • the present invention effectively overcomes the deficiencies in the prior art and has a high industrial utilization value.

Abstract

诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，包括磷酸胆碱、棕榈酰乙醇胺、植物鞘氨醇、磷脂酰胆碱、乙基鹅去氧胆酸、溶血磷脂酰胆碱（16:0）、溶血磷脂酰胆碱（18:2）和磷脂酰肌醇（20:4/0:0）中的一个或多个。单个用于诊断区分稳定型心绞痛患者和冠状动脉粥样硬化患者时，ROC曲线下面积（AUC）均大于0.7，具有临床诊断意义；联合用于诊断时，随着联合个数的增加，AUC进一步提高，8个全部联合时最高，AUC达0.985，在最佳cutoff值下，灵敏度和特异性分别为97.4％和98.0％。这些代谢标志物能准确地诊断区分冠状动脉粥样硬化和稳定型心绞痛，准确性高，灵敏度和特异性强。

Description

诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物 技术领域
本发明属于生物化学领域，涉及冠心病诊断分型的代谢标志物，具体涉及一组用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物。
背景技术
冠心病亦称为缺血性心脏病，涉及供应心肌血液的动脉发生粥样硬化，即冠状动脉粥样硬化病变引起血管腔狭窄或斑块形成甚至破裂、完全堵塞，造成心肌缺血、缺氧或坏死而导致心脏缺血性疾病，从而引起临床上心绞痛、心肌梗死等一系列严重的心血管事件。冠心病是人类健康的主要杀手，具发病率高、致残率高、复发率高、病死率高、并发症多等特点，已经成为威胁我国人民健康的主要疾病。
目前，基于病理生理机制，将冠心病分为慢性稳定性冠心病(即稳定型心绞痛)和急性冠脉综合征；急性冠脉综合征又进一步分为不稳定型心绞痛和急性心肌梗死。冠心病从无至有、从轻至重通常包括如下发展阶段：冠状动脉正常→冠状动脉粥样硬化→稳定型心绞痛→不稳定型心绞痛→急性心肌梗死。
冠状动脉粥样硬化是冠心病的主要病因，是冠心病的早期病症。冠状动脉粥样硬化是一种常见的进行性动脉疾病，病变主要累及中等大小的肌层动脉，动脉内膜脂质沉积，平滑肌细胞增生，形成局限性斑块，可使动脉壁变硬，严重时斑块破裂导致血栓、栓塞、出血，受累管腔部分或完全闭塞，临床表现为动脉粥样硬化血管并发症的发生。动脉粥样硬化早期病变可发生在10岁之前，病变引起动脉狭窄需经历20至30年时间，早期无临床症状，不易被发现和重视，因此对冠状动脉粥样硬化的早期预防和诊断可以有效地防止冠心病的发生。
冠状动脉造影可以对冠状动脉的狭窄程度作出准确的判断，是诊断冠心病的金标准[冠状动脉造影金标准与临床常规诊断冠心病差异性比较研究，舒荣文等，海军医学杂志2015年04期]。作为冠心病诊断的金标准，冠脉造影只能发现血管狭窄程度，而且它还是一种介入测量手段，需要介入手术，且诊断昂贵。另一方面，医生还需要依据对患者的心电图、心动图、平板运动试验、CT等检查结果作出最终诊断，由于医生主观性判断、病人叙述不清等情况的出现，对冠心病的诊断仍存在较大的误诊和漏诊，这对病人的预后影响非常大。为了改善患者的生活质量，降低患者受到的生命威胁，我们亟需发展一种具有诊断率高、经济、无侵入性、操作简便等特性的诊断方法。
代谢组学是研究生物体内源性代谢物质的整体及其随内因和外因变化的科学，是系统生物学的一个重要组成部分。它可以对体液如血液和尿液进行快速及无侵入性的分析，通过代 谢谱的差异获得可以指示各种生化反应的代谢标志物。目前常用的分析技术包括核磁共振(NMR)、质谱(LC-MS/GC-MS)等。LC-MS/GC-MS具有对样本制备要求低、灵敏度高、动态范围宽等特点，可用于检测样本中浓度相差大的代谢物，因而成为代谢组学的研究中应用越来越多的技术平台。血浆分析是临床上常用的一种疾病诊断方法，因其简便、快速、经济且相对无创的优点而被广泛采用。
目前尚未有人使用血浆代谢物水平对冠心病进行诊断分型。应用血浆代谢组学寻找冠状动脉正常人与冠状动脉粥样硬化患者及不同分型冠心病患者血浆代谢物的水平差异对于临床早期快速确诊冠心病并进行分型具有重要意义。
发明内容
为了克服现有技术的不足，本发明第一目的在于提供一组用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，该组代谢标志物同时存在于血浆中，可同时分析测定；本发明的第二目的在于提供一种能够灵敏地分析检测所述代谢标志物的方法；本发明的第三目的在于提供一种基于所述代谢标志物的检测试剂盒，用于诊断区分冠状动脉粥样硬化和稳定型心绞痛，提高诊断便利性，促进诊断方法标准化。
上述目的是通过如下技术方案得以实现的：
一组用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，包括一个或多个如下所述的代谢标志物：磷酸胆碱、棕榈酰乙醇胺、植物鞘氨醇、磷脂酰胆碱、乙基鹅去氧胆酸、溶血磷脂酰胆碱(16:0)、溶血磷脂酰胆碱(18:2)和磷脂酰肌醇(20:4/0:0)。
溶血磷脂酰胆碱(16:0)，即1-棕榈酰-甘油-3-磷脂酰胆碱。
溶血磷脂酰胆碱(18:2)，即1-亚油酰-甘油-3-磷脂酰胆碱。
磷脂酰肌醇(20:4/0:0)，即1-花生四烯酰甘油磷脂酰肌醇。
进一步地，所述的一组用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，包括任意三个所述的代谢标志物。
进一步地，所述的一组用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，包括任意四个所述的代谢标志物。
进一步地，所述的一组用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，包括任意五个所述的代谢标志物。
进一步地，所述的一组用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，包括任意六个所述的代谢标志物。
进一步地，所述的一组用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，包括全部八个所述的代谢标志物。
进一步地，所述的代谢标志物来源于血浆。
一种定性或定量分析所述的用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物的方法为：采用液质联用和/或气质联用对所述的代谢标志物进行定性或定量分析。液质和气质检测限低、灵敏度高，能灵敏地分析检测生物样本中的代谢标志物并对其定量。
一种用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的检测试剂盒，包括所述的代谢标志物的标准品，所述标准品为各代谢标志物的化学单体或混合物。使用标准品可以对生物样本中的代谢标志物进行快速准确地定性、定量分析。试剂盒有助于实现检测标准化，提高检测便利性和重现性。
进一步地，所述检测试剂盒还包括溶解所述标准品的溶剂和/或提取富集所述代谢标志物的溶剂。
本发明的优点：
(1)本发明提供的代谢标志物能准确地诊断区分冠状动脉粥样硬化和稳定型心绞痛。ROC曲线评价方法中，ROC曲线下面积AUC在大于0.5的情况下，越接近于1，说明诊断效果越好。AUC在0.5～0.7时有较低准确性，AUC在0.7～0.9时有一定准确性，AUC在0.9以上时有较高准确性。经验证，本发明提供的代谢标志物单个用于诊断区分冠状动脉粥样硬化和稳定型心绞痛时，AUC均在0.7以上；多个联合应用时，AUC比单个更接近于1，诊断效果更好；八个联合应用时，AUC最接近于1，诊断区分效果最好。
(2)本发明提供的分析检测所述代谢标志物的方法灵敏度高，结果准确可靠。
(3)本发明提供的检测试剂盒可以用于诊断区分冠状动脉粥样硬化和稳定型心绞痛，提高诊断便利性，促进诊断方法标准化。
具体实施方式
下面结合实施例进一步说明本发明实质性内容。使用的仪器或试剂未做详细说明的均为常规仪器和试剂；未具体描述的试验操作方法均为本领域普通技术人员公知的常规操作方法。
实施例1：冠状动脉粥样硬化患者和稳定型心绞痛患者之间血浆差异代谢物的筛选表征
一、对象和方法
1、标本来源
在取得患者同意后，收集江苏省人民医院2010年9月～2015年6月480例冠状动脉粥样硬化患者、280例稳定型心绞痛患者和350例健康人的外周静脉血血浆，所有患者或健康人均经冠脉造影证实。健康人的年龄、性别与冠状动脉粥样硬化患者、稳定型心绞痛患者相匹配。所有冠状动脉粥样硬化患者、稳定型心绞痛患者和健康人均有正常心肺肝肾及造血功能。
采血时间均为清晨空腹状态。
2、主要试剂
乙腈及甲酸(UPLC纯)购于美国ROE公司；色谱级别甲醇和氯仿购于江苏汉邦科技有限公司；氯化甲氧胺及N-甲基-N-(三甲基硅烷)三氟乙酰胺(含1％三甲基氯硅烷)购于美国Sigma-Aldrich公司；去离子水由美国密理博(Millipore)公司的MIlli-Q超纯水系统制备；标准品包括磷酸胆碱、棕榈酰乙醇胺、植物鞘氨醇、磷脂酰胆碱、乙基鹅去氧胆酸、溶血磷脂酰胆碱(16:0)、溶血磷脂酰胆碱(18:2)和磷脂酰肌醇(20:4/0:0)，购于美国Sigma-Aldrich。
3、血浆差异代谢物的筛选表征
3.1 UPLC-Q/TOF-MS筛选表征
3.1.1样品制备
利用响应面法进行提取溶剂优化：以质谱ESI+和ESI-检测模式下的峰个数和总峰面积为因素考察不同溶剂(乙腈、甲醇、乙醇、氯仿、水)对血浆中代谢物的提取富集效率。将实验测得数据进行多变量分析，利用PLS模型中重要性因子(variable importance to projection，VIP值)反映变量对模型响应的重要性。乙腈、甲醇、乙醇、氯仿、水的VIP值依次为1.503，0.802，0.651，0.688和0.987，乙腈的提取效率最高，故选择乙腈作为血浆样本的提取溶剂。
样品处理：取100μL血浆于1.5mL离心管中，加入400μL乙腈，涡旋30秒后混匀，13000rpm×10min离心(4℃)，取200μL上清于1.5mL离心管中，在室温下用氮吹仪吹干，所得残渣用300μL的20％乙腈水溶液溶解，即得。
3.1.2试验条件及参数
UPLC-Q/TOF-MS条件：
色谱分离采用超高效液相色谱(UPLC，Agilent 1290，USA)。色谱柱为Waters BEH C18柱(100mm×2.1mm，1.7μm)，柱温25℃，进样室温度为室温，进样量2μL。正、负离子模式流动相组成均是A为体积浓度0.1％甲酸水溶液，B为体积浓度0.1％甲酸乙腈溶液。梯度洗脱条件：0～1min为0～30％B相，2min内B相线性增加到60％，3～8min线性变化至90％B相，然后在8～9min线性增加至100％B相并保持1min；流速0.3mL/min，柱后流出液不经分流直接导入质谱系统检测。
质谱分析采用四级杆-飞行时间质谱(Agilent 6530Q-TOF/MS，USA)。以电喷雾离子源(ESI)正、负离子模式检测；干燥气流速为7L/min，干燥气温度为300℃，干燥气和锥孔气均为高纯氮气；离子源温度100℃，正离子和负离子模式下毛细管电压均为3000V，碰撞电压为100V；采用全扫描模式每秒进行三次数据采集，扫描质量范围：m/z 100-1000道尔顿。
3.2 GC-Q/MS筛选表征
3.2.1样品制备
取200μL血浆于1.5mL离心管中，加入50μL 1mg/mL的2-异丙基苹果酸溶液内标，涡旋20秒混匀，加入400μL甲醇、氯仿和水的混合溶液(比例为2.5∶1∶1)，然后在70℃的金属浴上振摇30min(1200rpm)，16000g×5min离心(4℃)，取500μL上清于1.5mL离心管中，加入500μL蒸馏水，涡旋混匀，然后16000g×5min离心(4℃)，取500μL上清于1.5mL离心管中，在室温下用氮吹仪吹干，所得残渣用80μL的甲氧胺吡啶溶液溶解，在50℃条件下肟化8h，加入60μL N-甲基-N-三甲基硅基三氟乙酰胺，在70℃条件下衍生化2h，即得。
3.2.2试验条件及参数
GC-Q/MS条件：美国Agilent 7890B-5977A气相色谱-质谱联用仪。色谱柱HP-5MS毛细管柱(30.0m×0.25mm，毛细管厚度0.25μm)；载气为高纯氦气，流速1.0mL/min；进样量2μL；程序升温：80℃恒温2min，80℃-300℃(5℃/min)恒温6min；不分流，进样温度300℃；接口温度300℃；离子源温度200℃；电子能量50eV；溶剂延迟3min；采用全扫描模式，扫描质量范围：m/z 30-600道尔顿。
4、数据处理和分析
将UPLC-Q/TOF-MS和GC-Q/MS得到的数据导入SIMCA软件(version 13.0.2，Umetrics)进行多元统计分析。通过建立OPLS-DA(正交偏最小二乘法-判别分析)模型，寻找冠状动脉粥样硬化患者和稳定型心绞痛患者之间代谢轮廓贡献较大(VIP＞1.0且p＜0.01)的代谢物。
通过HMDB(http：//www.hmdb.ca/)和Metline(http：//metlin.scripps.edu/)等数据库进行物质结构的检索，利用数据库中提供的精确分子量和质谱所得的MS/MS图谱初步鉴定上述差异代谢物的结构。最终通过购买标准品，用标准品的分子量、色谱保留时间和相应的多级MS裂解谱比对，确证差异代谢物的结构。
二、结果
筛选表征出8个差异代谢物，分别为：磷酸胆碱、棕榈酰乙醇胺、植物鞘氨醇、磷脂酰胆碱、乙基鹅去氧胆酸、溶血磷脂酰胆碱(16:0)、溶血磷脂酰胆碱(18:2)和磷脂酰肌醇(20:4/0:0)。
与冠状动脉硬化患者相比，上述8个差异代谢物在稳定型心绞痛患者血浆中的表达水平均下调。通过标准品定量检测，与冠状动脉硬化患者相比，上述差异代谢物在稳定型心绞痛患者血浆中的表达水平下调0.7～0.8倍。由此可见，上述8个差异代谢物在稳定型心绞痛患者和冠状动脉硬化患者血浆中的表达水平明显不同，可以用于诊断区分冠状动脉粥样硬化和稳定型心绞痛。
实施例2：构建ROC曲线验证8个差异代谢物用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的能力
采用受试者工作曲线(ROC)法进行验证，通过稳定型心绞痛患者和冠状动脉粥样硬化患者血浆中差异代谢物的表达水平判断其用于诊断区分稳定型心绞痛患者和冠状动脉粥样硬化患者的能力。结果表明，磷酸胆碱、棕榈酰乙醇胺、植物鞘氨醇、磷脂酰胆碱、乙基鹅去氧胆酸、溶血磷脂酰胆碱(16:0)、溶血磷脂酰胆碱(18:2)和磷脂酰肌醇(20:4/0:0)这8个差异代谢物单个用于诊断区分稳定型心绞痛患者和冠状动脉粥样硬化患者的能力较强，ROC曲线下面积(AUC)均大于0.7，具有临床诊断意义；联合用于诊断时，随着联合个数的增加，AUC进一步提高，8个全部联合时最高，AUC达0.985，在最佳cutoff值下，灵敏度和特异性分别为97.4％和98.0％。单个及任意2～7个联合诊断结果见表1～3。
表1单个差异代谢物诊断区分冠状动脉粥样硬化患者和稳定型心绞痛患者
单个差异代谢物 AUC 灵敏度 特异性
磷酸胆碱 0.867 86.6％ 87.8％
棕榈酰乙醇胺 0.854 83.3％ 84.5％
植物鞘氨醇 0.832 81.1％ 82.3％
磷脂酰胆碱 0.795 77.4％ 78.6％
乙基鹅去氧胆酸 0.779 75.8％ 77.0％
溶血磷脂酰胆碱(16:0) 0.758 73.7％ 74.9％
溶血磷脂酰胆碱(18:2) 0.733 71.2％ 72.4％
磷脂酰肌醇(20:4/0:0) 0.711 70.1％ 71.3％
表2两个差异代谢物联合诊断区分冠状动脉粥样硬化患者和稳定型心绞痛患者
Figure PCTCN2016072440-appb-000001
表3任意三～七个差异代谢物联合诊断区分冠状动脉粥样硬化患者和稳定型心绞痛患者
联合个数 AUC 灵敏度 特异性
三个 ≥0.907 ≥90.8％ ≥90.2％
四个 ≥0.919 ≥91.3％ ≥92.0％
五个 ≥0.923 ≥93.6％ ≥93.4％
六个 ≥0.946 ≥95.2％ ≥94.8％
七个 ≥0.968 ≥96.3％ ≥96.5％
从表1可以看出，这8个差异代谢物单个用于诊断区分冠状动脉粥样硬化患者和稳定型心绞痛患者的能力较强，AUC均大于0.7，灵敏度较高、特异性较强，具有临床诊断意义；从表2可以看出，这8个差异代谢物两两联合用于诊断时，AUC比单个用于诊断时更高，灵敏度高、特异性较高，具有临床诊断意义；从表3可以看出，用这8个差异代谢物中的3～7个联合用于诊断时，AUC进一步提高，灵敏度高、特异性强，具有临床诊断意义。
因此，这8个差异代谢物可以作为用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物。
实施例3：检测试剂盒的制备
基于本发明提供的代谢标志物制备了检测试剂盒，该试剂盒包括如下成分：
代谢标志物的标准品：包括磷酸胆碱、棕榈酰乙醇胺、植物鞘氨醇、磷脂酰胆碱、乙基鹅去氧胆酸、溶血磷脂酰胆碱(16:0)、溶血磷脂酰胆碱(18:2)和磷脂酰肌醇(20:4/0:0)，各标准品分别封装；
血浆代谢物提取溶剂：100％乙腈和20％乙腈水溶液(用于UPLC-Q/TOF-MS样品制备)；比例为2.5∶1∶1的甲醇、氯仿和水的混合溶液、甲氧胺吡啶和N-甲基-N-三甲基硅基三氟乙酰胺(用于GC-Q/MS样品制备)；UPLC-Q/TOF-MS筛选表征中，20％乙腈水溶液可以用作溶解标准品的溶剂；GC-Q/MS筛选表征中，用血浆代谢物提取溶剂按样品制备的方法制备标准品溶液；
内标：2-异丙基苹果酸。
当然，设计检测试剂盒时，并不需要完全包含上述8个代谢标志物的标准品，可以仅使用其中几个进行组合。这些标准品可以单独封装，也可以制成混合物封装。
该试剂盒是基于本发明提供的代谢标志物而设计的，可以用于诊断区分冠状动脉粥样硬化患者和稳定型心绞痛患者。
综上所述，本发明有效克服了现有技术中的不足，且具高度产业利用价值。
上述实施例的作用在于说明本发明的实质性内容，但并不以此限定本发明的保护范围。 本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和保护范围。

Claims (10)

  1. 一组用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，其特征在于，包括一个或多个如下所述的代谢标志物：磷酸胆碱、棕榈酰乙醇胺、植物鞘氨醇、磷脂酰胆碱、乙基鹅去氧胆酸、溶血磷脂酰胆碱(16:0)、溶血磷脂酰胆碱(18:2)和磷脂酰肌醇(20:4/0:0)。
  2. 根据权利要求1所述的用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，其特征在于：包括任意三个所述的代谢标志物。
  3. 根据权利要求1所述的用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，其特征在于：包括任意四个所述的代谢标志物。
  4. 根据权利要求1所述的用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，其特征在于：包括任意五个所述的代谢标志物。
  5. 根据权利要求1所述的用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，其特征在于：包括任意六个所述的代谢标志物。
  6. 根据权利要求1所述的用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，其特征在于：包括全部八个所述的代谢标志物。
  7. 根据权利要求1～6任一所述的用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物，其特征在于：所述代谢标志物来源于血浆。
  8. 一种定性或定量分析权利要求1～6任一所述的用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的代谢标志物的方法，其特征在于：采用液质联用和/或气质联用对所述的代谢标志物进行定性或定量分析。
  9. 一种用于诊断区分冠状动脉粥样硬化和稳定型心绞痛的检测试剂盒，其特征在于：包括权利要求1～6任一所述的代谢标志物的标准品，所述标准品为各代谢标志物的化学单体或混合物。
  10. 根据权利要求9所述的检测试剂盒，其特征在于：还包括溶解所述标准品的溶剂和/或提取富集所述代谢标志物的溶剂。
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