CN114544806A - 血清肉豆蔻酸作为哮喘诊断标志物的用途 - Google Patents
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Abstract
本发明公开了血清肉豆蔻酸作为哮喘诊断标志物的用途。本发明通过Q Exactive Orbitrap LC‑MS对实验组血清的非靶向代谢组学进行分析,发现了肉豆蔻酸等12种哮喘相关血清核心代谢物。其中,哮喘患者血清肉豆蔻酸明显高于健康者,差异有统计学意义,且血清肉豆蔻酸对于诊断哮喘具有较高的敏感性,并且通过验证组对上述结果进行了验证。本发明提供的血清肉豆蔻酸作为哮喘诊断标志物具有较高的敏感性和特异性,且更为简便,因此,血清肉豆蔻酸对于哮喘的诊断具有良好的应用前景。
Description
技术领域
本发明涉及血清肉豆蔻酸作为哮喘诊断标志物的用途,属于检测试剂技术领域。
背景技术
支气管哮喘(bronchial asthma)简称哮喘,是一种以气道炎症和气道高反应性为特征的慢性呼吸系统疾病,其主要临床表现为反复发作的喘息、气短、胸闷、咳嗽等呼吸道症状。哮喘是全球最常见的慢性疾病之一,全球哮喘患病率在不同国家约为1%~18%,在中国约为4.2%,全球哮喘患者总数已超过3亿人,严重威胁人类健康。2021年全球哮喘防治倡议(Global Initiative for Asthma,GINA)中关于哮喘疾病的诊断,仍是基于临床表现和肺功能等辅助检查结果,当前哮喘的诊断没有金标准,也没有单一检测方法可以诊断(或排除)哮喘。因此发现一个新的高特异性和敏感性的生物标记物对于哮喘的病情的评估和治疗有着十分重要的作用。
代谢组学是组学研究方法之一,旨在探索生物样本中代谢物的总体特征及其相互作用。代谢组学的研究方法分为非靶向性代谢组学以及靶向性代谢组学。非靶向性代谢组学测量所提取样本中所存在的最广泛的代谢物范围,具有高选择性和高敏感性。靶向性代谢组学通过测量样本中一组预先确定的代谢物的浓度,在具有先验信息的基础上,可针对分析样本中特定的代谢物和代谢途径进行分析,将两者结合,能够为分析验证提供更好的数据。代谢物处于生物系统中生化活动的终端,因此反映的是已经发生的生物学事件。基因表达和环境因素的变化对生物系统所产生的影响都可在代谢物水平上得到最终的表型体现。代谢组学可对生物体所有脂肪酸和氨基酸等代谢物进行全面而准确的检查和分析,能够阐述疾病发病机制及相关分子通路,识别新的生物标志物,能预测疾病进展和指导个性化治疗。因此,将代谢数据和临床特征结合能提高其预测能力。肉豆蔻酸(Myristoleicacid)又称为十四烷酸,是一种饱和脂肪酸,现已发现哮喘患者血清肉豆蔻酸水平明显高于健康者,但是目前还没有关于将血清肉豆蔻酸用于诊断哮喘的相关报道。
发明内容
本发明所要解决的技术问题是:现有技术中的标记物并不能方便准确地诊断哮喘,即现有的哮喘诊断标志物存在敏感性和特异性不高等问题。
为了解决上述技术问题,本发明提供了检测血清肉豆蔻酸的试剂在制备用于诊断哮喘的试剂或试剂盒中的应用。
优选地,所述血清肉豆蔻酸的诊断阈值为286.94nmol/L。
本发明还提供了一种用于诊断哮喘的试剂或试剂盒,所述的试剂或试剂盒包括用于检测血清肉豆蔻酸的试剂。
优选地,所述血清肉豆蔻酸的诊断阈值为286.94nmol/L。
与现有技术相比,本发明的有益效果在于:
本发明提供了血清肉豆蔻酸作为哮喘诊断标志物的用途,血清肉豆蔻酸作为哮喘诊断标志物具有较高的敏感性和特异性,且更为简便,因此,血清肉豆蔻酸对于哮喘的诊断具有良好的应用前景。
附图说明
图1为试验组人群基于Q Exactive Orbitrap LC-MS系统检测的血清肉豆蔻酸水平;图中****表示经过统计学分析,两组间具有显著性差异,P<0.0001;
图2为试验组人群基于Q Exactive Orbitrap LC-MS系统检测的血清肉豆蔻酸水平拟合的ROC曲线结果;
图3为验证组人群基于UHPLC-MRM-MS/MS系统检测的血清肉豆蔻酸水平;图中**表示经过统计学分析,两组间具有显著性差异,P<0.01;
图4为验证组人群基于UHPLC-MRM-MS/MS系统检测的肉豆蔻酸水平拟合的ROC曲线结果。
具体实施方式
为使本发明更明显易懂,兹以优选实施例,并配合附图作详细说明如下。
以下实施例中,试验组哮喘患者和健康受试者血清收集:纳入标准为以全球哮喘倡议(GINA)指南诊断为哮喘102例患者,其中男性55例,女性47例;对照组18例,均为健康成年人。采集哮喘患者及健康人空腹的静脉血,在采血前禁食12小时血样收集后立即于3000rpm离心10分钟,分离血清,将分离出的血清样品置于已标记好的试管中,在超低温冰箱中低于-80℃冷冻保存,为了减少代谢组学分析中的批次效应,这批所有样品都在同一时间进行非靶向性代谢组学Q Exactive Orbitrap LC-MS系统检测。作为验证组,选取另一组哮喘患者30例和健康受试者15例,按照上述方法继续收集、保存和制备血清样品,在同一时间对所有样品进行靶向性代谢组学UHPLC-MS/MS分析测定。
以下实施例中,非靶向性代谢组学Q Exactive Orbitrap LC-MS系统血清中代谢物的分析检测方法为:
在持续至少12小时的夜间禁食后采集试验组人群的血样。然后,将所有血清样本储存在-80℃下,直至处理完毕。为了减少代谢组学分析中的批次效应,所有样品均同时进行分析,通过Q Exactive Orbitrap LC-MS/MS系统对血清的非靶向代谢组学进行分析。简言之,在50μL样品中加入200μL提取液(乙腈:甲醇=1:1,含有同位素标记的内标混合物)。然后,将样品旋转30秒,在冰水浴中超声处理10分钟,并在-40℃下培养1小时以沉淀蛋白质。在4℃下以12000rpm离心15分钟后,将所得上清液行进一步分析。质量控制(QC)样品是通过从所有样品中混合等量的上清液制备的。同时,实验中包括14个质控样品。LC-MS/MS分析使用UHPLC系统(Vanquish,Thermo Fisher Scientific)进行,该系统带有UPLC色谱柱(2.1mm×100mm,1.7μm),与Q Exactive HFX质谱仪(Orbitrap MS,Thermo)耦合。流动相由A:水(25mmol/L醋酸铵和25mmol/L氨水,pH=9.75)和B:乙腈组成。洗脱梯度设置为:0~0.5min,95%B;0.5~7.0min,95%~65%B;7.0~8.0min,65%~40%B;8.0~9.0min,40%B;9.0~9.1min,40%~95%B;9.1~12.0min,95%B,柱温30℃。自动取样器温度为4℃,进样量为3μL。QE HFX质谱仪能够在采集软件(Xcalibur,Thermo)的控制下,以信息相关采集(IDA)模式采集MS/MS光谱。在此模式下,采集软件持续评估全扫描MS光谱。ESI源条件设置如下:鞘气流速50arb,辅助气流速10arb,毛细管温度320℃,全MS分辨率60000,MS/MS分辨率7500,归一化碰撞能量(NCE)模式下的碰撞能量为10/30/60,离子源喷射电压:为3.5kV(正)或-3.2kV(负)。
以下实施例中,靶向性代谢组学UHPLC-MS/MS系统检测血清肉豆蔻酸的方法为:
通过UHPLC-MS/MS分析测定验证组人群血清中肉豆蔻酸水平。血清样本的采集、保存和制备如上所述。血清样品解冻后,将100μL等份样品精确转移至Eppendorf管中。在加入400μL萃取溶液(乙腈-甲醇,1:1)后,将样品旋转30秒,并在冰水浴中超声15分钟。接下来,对样品进行培养和离心。然后,将70μL等分清液转移至自动取样器小瓶中进行UHPLC-MS/MS分析。对于标准溶液制备,将每种标准物质溶解或稀释至10mmol/L的最终浓度。使用Agilent 1290Infinity II series(Agilent Technologies)超高效液相色谱仪,通过Waters ACQUITY UPLC BEH Amide(100×2.1mm,1.7μm,Waters)液相色谱柱对目标化合物进行色谱分离,AJS-ESI离子源的Agilent 6460三重四极杆质谱仪,以多反应监测(MRM)模式进行质谱分析。然后,通过Agilent MassHunter Work Station Software(B.08.00,Agilent Technologies)来完成MRM数据采集和处理。随后,对校准溶液进行UPLC-MRM-MS/MS分析,以建立每个代谢物的校准曲线。最后,测定血清中肉豆蔻酸浓度。
实施例
本实施例提供了血清肉豆蔻酸作为哮喘诊断标志物的用途:
将收集的哮喘患者和健康受试者(试验组)血清,用Q Exactive Orbitrap LC-MS对血清的非靶向代谢组学进行分析,发现了肉豆蔻酸等12种哮喘相关血清核心代谢物。哮喘患者血清肉豆蔻酸(17629808.23±20999189.42)明显高于健康者(6940110.31±7041734.38),差异有统计学意义(P<0.0001),如图1所示,且血清肉豆蔻酸对于诊断哮喘具有较高的敏感性(82.35%)和特异性(77.78%),ROC曲线的AUC为0.801,结果如图2所示。为了进一步评估血清肉豆蔻酸对于哮喘诊断的价值,又纳入了另外一组哮喘患者和健康受试者作为验证组,并采用靶向性代谢组学UHPLC-MRM-MS/MS法检测验证组患者血清肉豆蔻酸的具体水平,结果显示哮喘患者血清肉豆蔻酸水平(514.142±319.878nmol/L)明显高于健康者(263.653±167.058nmol/L),差异有统计学意义(P<0.01),如图3所示,当取286.94nmol/L作为诊断阈值(Cut-off value)时,诊断哮喘的敏感性为83.33%,特异性为73.33%,ROC曲线的AUC为0.789,如图4所示。
上述实施例仅为本发明的优选实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (4)
1.检测血清肉豆蔻酸的试剂在制备用于诊断哮喘的试剂或试剂盒中的应用。
2.如权利要求1所述的应用,其特征在于,所述血清肉豆蔻酸的诊断阈值为286.94nmol/L。
3.一种用于诊断哮喘的试剂或试剂盒,其特征在于,所述的试剂或试剂盒包括用于检测血清肉豆蔻酸的试剂。
4.如权利要求3所述的用于诊断哮喘的试剂或试剂盒,其特征在于,所述血清肉豆蔻酸的诊断阈值为286.94nmol/L。
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