WO2017107279A1 - 用于诊断冠状动脉粥样硬化的代谢标志物 - Google Patents

用于诊断冠状动脉粥样硬化的代谢标志物 Download PDF

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WO2017107279A1
WO2017107279A1 PCT/CN2016/071626 CN2016071626W WO2017107279A1 WO 2017107279 A1 WO2017107279 A1 WO 2017107279A1 CN 2016071626 W CN2016071626 W CN 2016071626W WO 2017107279 A1 WO2017107279 A1 WO 2017107279A1
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hydroxy
glycero
glycerol
phosphatidylethanolamine
metabolic
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齐炼文
赵艺静
范勇
朱伟
李萍
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
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

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  • the invention belongs to the field of biochemistry and relates to disease diagnosis markers, in particular to a group of metabolic markers for diagnosing coronary atherosclerosis.
  • 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.
  • plaque rupture leads to thrombosis, embolism, hemorrhage, partial or complete occlusion of the affected lumen, clinical manifestations of atherosclerotic vascular complications, the most common and most serious vascular complications in elderly patients are coronary heart disease (unstable Sexual angina and myocardial infarction are the main causes of coronary heart disease.
  • 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 atherosclerosis.
  • coronary angiography gold standard and clinical routine diagnosis of coronary heart disease, Shu Rongwen et al., Naval Medical Journal 2015 Issue 04.
  • coronary angiography can only find the degree of vascular stenosis, and it is also an interventional measurement method that requires interventional surgery and is expensive to diagnose.
  • the doctor also needs to make a final diagnosis based on the patient's electrocardiogram, cardiogram, treadmill exercise test, CT and other test 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 provides rapid and non-invasive analysis of body fluids such as blood and urine, and obtains metabolic markers that indicate various biochemical reactions through differences in metabolic profiles.
  • Currently commonly used 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.
  • the diagnosis of coronary atherosclerosis has not been performed using plasma metabolite levels.
  • the use of plasma metabolomics to find metabolic markers to diagnose coronary atherosclerosis is important for the rapid diagnosis of coronary atherosclerosis in early clinical stages.
  • a first object of the present invention is to provide a set of metabolic markers for diagnosing coronary atherosclerosis, which are simultaneously present in plasma and can be simultaneously analyzed and determined;
  • a second object is to provide a method capable of sensitively analyzing and detecting the metabolic marker;
  • a third object of the present invention is to provide an application of the metabolic marker in screening for a medicament for treating or relieving coronary atherosclerosis;
  • a fourth object of the present invention is to provide a detection kit based on the metabolic marker for diagnosing coronary atherosclerosis, improving diagnostic convenience, and promoting standardization of diagnostic methods; and a fifth object of the present invention is to provide the detection reagent The use of the cartridge in screening for drugs for treating or ameliorating coronary atherosclerosis.
  • a set of metabolic markers for the diagnosis of coronary atherosclerosis including one or more of the metabolic markers described below: 1-arachidonoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine, 1 - eicosenoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine, 1-oleoyl-sn-glycerol-3-phosphatidylcholine, 1-docosahexaenoyl-2-hydroxyl -sn-glycero-3-phosphatidylethanolamine, 1-docosapentaenoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine, 1-docosapentaenoyl-sn-glycerol-3 -phosphatidylcholine, 1-stearyl-sn-glycerol-3 phosphatidylcholine, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphatidy
  • any two of said metabolic markers are included.
  • any three of said metabolic markers are included.
  • the metabolic marker is derived from plasma.
  • a method of qualitatively or quantitatively analyzing said metabolic marker for characterizing or quantifying said metabolic marker by LC/MS and/or GC/MS is provided.
  • 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.
  • the metabolic markers can be used to screen for drugs that treat or alleviate coronary atherosclerosis.
  • a test kit for diagnosing coronary atherosclerosis 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.
  • test kit for screening drugs for treating or ameliorating coronary atherosclerosis.
  • the metabolic marker provided by the present invention can accurately diagnose coronary atherosclerosis.
  • the area value AUC under the ROC curve 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 diagnosing coronary atherosclerosis alone (compared with healthy people), and the AUC is above 0.7; when combined, the AUC is closer to 1 than the individual, and the diagnostic effect is more Good; when combined with eleven, the AUC is closest to 1, and the diagnosis 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 metabolic markers provided by the present invention can effectively diagnose whether the condition of coronary atherosclerosis is treated or alleviated, and can be used for screening drugs for treating or relieving coronary atherosclerosis.
  • the detection kit provided by the invention can be used for diagnosing coronary atherosclerosis, improving the convenience of diagnosis, and promoting standardization of diagnostic methods.
  • the kit based on the metabolic marker provided by the present invention can effectively diagnose whether coronary atherosclerosis is treated or relieved, and can be used for screening for a drug for treating or relieving coronary atherosclerosis.
  • Example 1 Screening and Characterization of Plasma Differential Metabolites Between Patients with Coronary Atherosclerosis and Healthy People
  • peripheral venous blood plasma was collected from 480 patients with coronary atherosclerosis and 350 healthy subjects from September 2010 to June 2015 in Jiangsu Provincial People's Hospital. All patients or healthy persons were confirmed by coronary angiography. The age and gender of healthy people match those of coronary atherosclerosis. All patients with coronary atherosclerosis and healthy people have normal cardiopulmonary 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; standard products include 1-arachidonoyl-2 -hydroxy-sn-glycero-3-phosphatidylethanolamine, 1-eicosyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine, 1-oleoyl-sn-glycerol-3-phosphatidylcholine , 1-docosahexaenoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine, 1-docosapentaenoyl-2
  • 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.
  • a total of 11 differential metabolites were identified by screening: 1-arachidonoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine, 1-eicocarbyl-2-hydroxy-sn-glycerol-3 -phosphatidylethanolamine, 1-oleoyl-sn-glycero-3-phosphatidylcholine, 1-docosahexaenoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine, 1-22 Cefenoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine, 1-docosapentaenoyl-sn-glycero-3-phosphatidylcholine, 1-stearoyl-sn-glycerol- 3 phosphatidylcholine, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine, 1-palmitoyl-2-hydroxy-sn-glycero-3
  • Example 2 Construction of ROC curves Comparison of 11 differential metabolites to diagnose the ability of patients with coronary atherosclerosis and healthy people
  • the AUC for single and any 2 to 10 joint use for diagnosis is shown in Tables 1-3.
  • Example 3 Application in screening drugs for treating or relieving coronary atherosclerosis
  • the plasma of 155 patients with coronary atherosclerosis before and after drug treatment showed that the patient's condition gradually eased with the increase of medication treatment.
  • the 11 metabolic markers provided by the present invention are in plasma. The level gradually became higher in healthy people, and there was no significant difference in plasma levels between healthy and healthy people after complete cure (P>0.05). This result is consistent with the results of coronary angiography. Therefore, the metabolic markers provided by the present invention can be used for screening treatment or alleviation. Coronary atherosclerosis drugs.
  • 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;
  • test kit it is not necessary to completely contain the above-mentioned 11 metabolic markers, and only a few of them can be used in combination. These standards can be packaged individually or in a mixture.
  • the kit is designed based on the metabolic markers provided by the present invention and can be used to screen for drugs for treating or ameliorating coronary atherosclerosis.
  • the present invention effectively overcomes the deficiencies in the prior art and has a high industrial utilization value.

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Abstract

一组用于诊断冠状动脉粥样硬化的代谢标志物，包括1-花生四烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十碳烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-油酰-sn-甘油-3-磷脂酰胆碱、1-二十二碳六烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-sn-甘油-3-磷脂酰胆碱、1-硬脂酰-sn-甘油-3磷脂酰胆碱、1-油酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-sn-甘油-3-磷脂酰胆碱和1-十八碳酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺中的一种或多种。该代谢标志物能精确诊断区分冠状动脉粥样硬化患者与健康人，精确度高，灵敏度和特异性强。

Description

用于诊断冠状动脉粥样硬化的代谢标志物 技术领域
本发明属于生物化学领域，涉及疾病诊断标志物，具体涉及一组用于诊断冠状动脉粥样硬化的代谢标志物。
背景技术
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，老年患者中最常见和最严重的血管并发症是冠心病(不稳定性心绞痛和心肌梗塞)，是冠心病的主要病因。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，是诊断冠状动脉粥样硬化的金标准[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]。作为冠状动脉粥样硬化诊断的金标准，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，对冠状动脉粥样硬化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，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状动脉粥样硬化进行诊断。应用血浆代谢组学寻找代谢标志物以诊断冠状动脉粥样硬化对于临床早期快速确诊冠状动脉粥样硬化具有重要意义。
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冠状动脉粥样硬化的代谢标志物，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所述代谢标志物在筛选治疗或缓解冠状动脉粥样硬化的药物中的应用；本发明的第四目的在于提供一种基于所述代谢标志物的检测试剂盒，用于诊断冠状动脉粥样硬化，提高诊断便利性，促进诊断方法标准化；本发明的第五目的在于提供所述检测试剂盒在筛选治疗或缓解冠状动脉粥样硬化的药物中的应用。
上述目的是通过如下技术方案得以实现的：
一组用于诊断冠状动脉粥样硬化的代谢标志物，包括一个或多个如下所述的代谢标志物：1-花生四烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十碳烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-油酰-sn-甘油-3-磷脂酰胆碱、1-二十二碳六烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-sn-甘油-3-磷脂酰胆碱、1-硬脂酰-sn-甘油-3磷脂酰胆碱、1-油酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-sn-甘油-3-磷脂酰胆碱和1-十八碳酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺。
进一步地，包括任意两个所述的代谢标志物。
进一步地，包括任意三个所述的代谢标志物。
进一步地，包括任意四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代谢标志物的方法，通过液质联用和/或气质联用对所述的代谢标志物定性或定量。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。
冠状动脉粥样硬化治疗或缓解后，会在代谢标志物的表达水平上有所体现。因此，所述的代谢标志物可以用于筛选治疗或缓解冠状动脉粥样硬化的药物。
一种用于诊断冠状动脉粥样硬化的检测试剂盒，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。
所述的检测试剂盒在筛选治疗或缓解冠状动脉粥样硬化的药物中的应用。
本发明的优点：
(1)本发明提供的代谢标志物能精确诊断冠状动脉粥样硬化。ROC曲线评价方法中，ROC曲线下的面积值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冠状动脉粥样硬化时(与健康人比较)，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；十一个联合应用时，AUC最接近于1，诊断效果最好。
(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)本发明提供的代谢标志物可以有效诊断冠状动脉粥样硬化的病情是否得到治疗或缓解，可以用于筛选治疗或缓解冠状动脉粥样硬化的药物。
(4)本发明提供的检测试剂盒可以用于诊断冠状动脉粥样硬化，提高诊断便利性，促进诊断方法标准化。
(5)本发明提供的基于所述代谢标志物的试剂盒可以有效诊断冠状动脉粥样硬化是否得到治疗或缓解，可以用于筛选治疗或缓解冠状动脉粥样硬化的药物。
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与健康人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相匹配。所有冠状动脉粥样硬化患者和健康人均有正常的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1-花生四烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十碳烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-油酰-sn-甘油-3-磷脂酰胆碱、1-二十二碳六烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、 1-二十二碳五烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-sn-甘油-3-磷脂酰胆碱、1-硬脂酰-sn-甘油-3磷脂酰胆碱、1-油酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-sn-甘油-3-磷脂酰胆碱和1-十八碳酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺，购于美国Sigma-Aldrich。
3、血浆差异代谢物的表征
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检测表征
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。
3.2GC-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检测表征
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与健康人之间代谢轮廓贡献较大(VIP＞1.0且p＜0.01)的差异代谢物。
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
共筛选表征11个差异代谢产物，分别为：1-花生四烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十碳烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-油酰-sn-甘油-3-磷脂酰胆碱、1-二十二碳六烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-sn-甘油-3-磷脂酰胆碱、1-硬脂酰-sn-甘油-3磷脂酰胆碱、1-油酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-sn-甘油-3-磷脂酰胆碱和1-十八碳酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺。
结果：与健康人相比，上述11个差异代谢产物在冠脉粥样硬化患者血浆中的表达水平均下调。通过标准品定量检测，与健康人相比，上述差异代谢产物在冠脉粥样硬化病人血浆中的表达水平下调0.7～0.8倍。
实施例2：构建ROC曲线比较11个差异代谢产物诊断区分冠状动脉粥样硬化患者与健康人的能力
采用受试者工作曲线(ROC)法进行验证，通过冠状动脉粥样硬化患者与健康人血浆中 11个差异代谢产物的表达水平判断其用于诊断冠状动脉粥样硬化的能力。结果表明，1-花生四烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十碳烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-油酰-sn-甘油-3-磷脂酰胆碱、1-二十二碳六烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-sn-甘油-3-磷脂酰胆碱、1-硬脂酰-sn-甘油-3磷脂酰胆碱、1-油酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-sn-甘油-3-磷脂酰胆碱和1-十八碳酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺这11个差异代谢产物单个用于诊断区分冠状动脉粥样硬化患者与健康人的能力较强，ROC曲线下面积(AUC)均大于0.7，具有临床诊断意义；这11个差异代谢产物联合用于诊断时，AUC进一步提高，11个联合起来诊断冠状动脉粥样硬化的AUC达0.995，在最佳cutoff值下，灵敏度和特异性分别为99.1％和99.8％。
单个及任意2～10个联合用于诊断时的AUC见表1～3。
表1单个差异代谢产物用于诊断冠状动脉粥样硬化的AUC
单个差异代谢物 AUC 灵敏度 特异性
1-花生四烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺 0.864 85.9％ 87.5％
1-二十碳烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺 0.845 83.5％ 83.1％
1-油酰-sn-甘油-3-磷脂酰胆碱 0.818 82.2％ 82.3％
1-二十二碳六烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺 0.762 76.0％ 78.9％
1-二十二碳五烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺 0.735 72.8％ 75.4％
1-二十二碳五烯酰-sn-甘油-3-磷脂酰胆碱 0.724 72.5％ 74.3％
1-硬脂酰-sn-甘油-3磷脂酰胆碱 0.721 72.4％ 73.0％
1-油酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺 0.711 72.1％ 71.9％
1-棕榈酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺 0.709 71.0％ 71.7％
1-棕榈酰-sn-甘油-3-磷脂酰胆碱 0.706 70.5％ 71.4％
1-十八碳酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺 0.701 70.2％ 70.3％
表2两个差异代谢产物联合用于诊断冠状动脉粥样硬化的AUC
Figure PCTCN2016071626-appb-000001
Figure PCTCN2016071626-appb-000002
表3任意三至十个差异代谢产物联合用于诊断冠状动脉粥样硬化的AUC
联合个数 AUC 灵敏度 特异性
三个 ≥0.909 ≥90.8％ ≥93.5％
四个 ≥0.919 ≥91.1％ ≥94.3％
五个 ≥0.931 ≥91.5％ ≥95.7％
六个 ≥0.935 ≥92.7％ ≥96.9％
七个 ≥0.954 ≥93.3％ ≥97.7％
八个 ≥0.968 ≥94.6％ ≥98.5％
九个 ≥0.978 ≥95.5％ ≥99.0％
十个 ≥0.982 ≥97.9％ ≥99.3％
从表1可以看出，这11个差异代谢产物单个用于诊断区分冠状动脉粥样硬化患者与健康人的能力较强，AUC均大于0.7，灵敏度高、特异性强，具有临床诊断意义；从表2可以看出，这11个差异代谢产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、特异性强，具有临床诊断意义；从表3可以看出，用这11个差异代谢产物中的3～10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11个差异代谢产物可以作为诊断冠状动脉粥样硬化的代谢标志物。
实施例3：在筛选治疗或缓解冠状动脉粥样硬化的药物中的应用
对155例冠状动脉粥样硬化患者药物治疗前和药物治疗后的血浆进行检测，结果表明：随着药物治疗疗程的增加，患者病情逐步缓解，本发明提供的11个代谢标志物在血浆中的水平逐渐趋于健康人的水平，且完全治愈后与健康人的血浆水平无显著性差异(P＞0.05)。该结果与冠脉造影的检测结果一致。因此，可以利用本发明提供的代谢标志物筛选治疗或缓解 冠状动脉粥样硬化的药物。
实施例4：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1-花生四烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十碳烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-油酰-sn-甘油-3-磷脂酰胆碱、1-二十二碳六烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-sn-甘油-3-磷脂酰胆碱、1-硬脂酰-sn-甘油-3磷脂酰胆碱、1-油酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-sn-甘油-3-磷脂酰胆碱和1-十八碳酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺，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11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，可以用于筛选治疗或缓解冠状动脉粥样硬化的药物。
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. 一组用于诊断冠状动脉粥样硬化的代谢标志物，其特征在于包括一个或多个如下所述的代谢标志物：1-花生四烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十碳烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-油酰-sn-甘油-3-磷脂酰胆碱、1-二十二碳六烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-二十二碳五烯酰-sn-甘油-3-磷脂酰胆碱、1-硬脂酰-sn-甘油-3磷脂酰胆碱、1-油酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺、1-棕榈酰-sn-甘油-3-磷脂酰胆碱和1-十八碳酰-2-羟基-sn-甘油-3-磷脂酰乙醇胺。
  2. 如权利要求1所述的代谢标志物，其特征在于：包括任意两个所述的代谢标志物。
  3. 如权利要求1所述的代谢标志物，其特征在于：包括任意三个所述的代谢标志物。
  4. 如权利要求1所述的代谢标志物，其特征在于：包括任意四个所述的代谢标志物。
  5. 如权利要求1～4任一所述的代谢标志物，其特征在于：该代谢标志物来源于血浆。
  6. 一种定性或定量分析权利要求1～4任一所述的代谢标志物的方法，其特征在于：通过液质联用和/或气质联用对所述的代谢标志物定性或定量。
  7. 权利要求1～4任一所述的代谢标志物在筛选治疗或缓解冠状动脉粥样硬化的药物中的应用。
  8. 一种用于诊断冠状动脉粥样硬化的检测试剂盒，其特征在于：包括权利要求1～4任一所述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。
  9. 如权利要求8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。
  10. 权利要求8或9所述的检测试剂盒在筛选治疗或缓解冠状动脉粥样硬化的药物中的应用。
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