高效液相色谱-质谱法对冠心病患者血清的脂质组学研究及其临床意义

HPLC-MS Study on Lipidomics for Coronary Artery Disease Patients’Serum and Its Significance in Clinical Medicine

采用高效液相色谱-质谱法(HPLC-MS)对冠状动脉慢性完全堵塞(CTO)患者在不同患病阶段的血清试样进行了脂质组学研究。试验采集了CTO疑似人群、CTO患者及CTO患者经皮冠状动脉介入治疗(PCI)手术后24h和72h的血清,分别采用氯仿和体积比为25∶75的乙酸乙酯-异辛烷混合溶液提取脂质,离心分离后,上清液用于Shotgun法(MS)和多反应监测(MRM)法(HPLC-MS)分析。样本直接进样电喷雾(ESI)质谱分析,用于Shotgun法进行广谱的脂质组学分析鉴定。通过Analyst 1.6软件提取质谱数据,并通过生物信息学工具"Lipid MS Predict"确认脂质分子,总共得到1504种脂质分子,并利用MRM法确认其中有临床意义的631个脂质分子。通过生物信息学分析,得到对于CTO具有诊断价值的包括胆固醇酯CE 16∶1和溶血磷脂酰乙醇胺LPE 18∶1在内的9个标志性脂质分子。结合临床回访结果和脂质组学研究数据证实,CTO患者手术治疗后的预后情况和磷脂酸、磷脂酰胆碱、胆固醇酯等3类脂质的含量变化有关。确定的脂质标志物应用于临床诊断早期冠心病和评估手术治疗后的风险,具有显著而明确的临床医学价值。

HPLC-MS was established to study the lipidomics of serum samples for the patients with chronic complete coronary occlusion(CTO)at the different stages of disease.Patients′serum of suspected CTO,CTO,and CTO after 24 hand 72 hof percutaneous coronary intervention(PCI)surgery were collected respectively in the test.The lipid in samples was extracted with chloroform and ethyl acetate-isooctane with volume ratio of 25∶75,respectively.After centrifugation,the supernatant was taken for Shotgun method(MS)and multiple reaction monitoring(MRM)method(HPLC-MS).The supernatant sample was injected directly to electrospray(ESI)mass spectrometry for a broad-lipidomics analysis and identification by Shotgun method.The MS data was indexed and analyzed by software Analyst 1.6,and the lipid molecules were confirmed by the bioinformatics tool"Lipid MS Predict".A total of 1504 lipid molecules were identified,and among them,631 lipid molecules with clinical significance were confirmed by using the MRM method.Through bioinformatics analysis,9 lipid molecules with significance in CTO diagnosis at early stage were ascertained as the biomarker,including cholesterol ester(CE)16∶1 and lysophosphatidylethanolamine(LPE)18∶1.Feedback from the clinical return visit as well as research data confirmed that the postoperative risk prognosis of CTO patients was correlated positively with the increase of content of 3 lipid molecules including phosphatidic acid,phosphatidylcholine,and cholesterol ester.The lipid biomarkers found in the study can be applied to the clinical diagnosis of coronary heart disease at early stage and assessment of the risk after surgery,with definite significance and clear clinical medical value.