结核性胸膜炎特异代谢物的质谱检测新技术研究及其诊断价值评估

Tuberculous pleurisy is the main type of extra-pulmonary tuberculosis. However, it is very difficult to make a definite diagnosis of the disease. Rapid development of the Mass Spectrometry techniques provided a new platform for finding new disease specific metabolites. In the previous study we analyzed serum metabolites utilizing GC/TOF-MS. We found that several basic metabolic pathways in active pulmonary tuberculosis patients altered, and also found a specific metabolite that was associated with pathologic damages. However, the diagnostic sensitivity and specificity are not satisfying. To dig deep in searching tuberculous pleurisy specific metabolites, this study will bring in higher resolution and higher throughput LC/MS-MS technique, a novel spectrum analysis method and an autonomous Mycobacterium tuberculosis lipid compound database. Combining targeted and non-targeted metabolomic detection and analysis methods, we will search for human endogenous metabolites specifically associated with tuberculous pleurisy, and analyze the relevant metabolic pathways. We will also search for lipid compounds unique to Mycobacterium tuberculosis utilizing lipid metabolomic detection methods and the autonomous database. Then a diagnostic model based on detection of specific metabolites will be constructed and sensitivity and specificity of the model will be evaluated. This study will provide new theoretical evidences for getting in-depth knowledge of metabolic mechanisms of the tuberculous pleurisy. Detection of novel metabolic biomarkers will lay the theoretical and methodological foundation for rapid diagnosis of tuberculous pleurisy.

结核性胸膜炎是最常见的肺外结核类型，但其确诊存在很大困难。近年质谱技术发展迅速，为筛选疾病特异代谢物提供了新技术平台。前期我们利用GC/TOF-MS技术检测了肺结核患者及健康人血清代谢物，发现与发病状态及病理损伤相关的特异代谢物，但其诊断敏感性和特异性有待提高。本研究将利用更高分辨率、更高通量的LC/MS-MS技术，结合新质谱峰分析方法和自主建立的结核分枝杆菌脂类化合物数据库，深入研究能鉴别诊断结核性胸膜炎和恶性胸膜炎的特异代谢物。结合靶向和非靶向代谢组学检测方法，寻找与结核性胸膜炎发病特异相关的人体内源代谢物，分析其相关代谢通路。同时，利用脂类代谢组学检测方法和自主建立的数据库，寻找结核分枝杆菌来源的特异脂类化合物。最后建立基于代谢物检测的诊断模型，评估其敏感性和特异性。本研究将为深入了解结核性胸膜炎代谢机理变化提供新的理论证据，并为结核性胸膜炎的快速诊断奠定新的理论方法学基础。

结核性胸膜炎是最常见的肺外结核类型，但由于胸腔积液中病原菌含量非常少，确诊很困难。临床上结核病及恶性肿瘤引发的胸腔积液最为常见，也是鉴别诊断的难点。本研究采用组合式准靶向代谢组学检测分析方法探索了结核性胸腔积液及恶性胸腔积液的主要代谢通路及相关代谢物变化。首先，我们用准靶向代谢组学策略进行检测，筛选出差异代谢物，再纳入新病例进行了验证。结果显示，结核性胸腔积液与恶性胸腔积液相比，氨基酸类、三羧酸循环中间产物以及游离脂肪酸类有显著下降，但是乙酰基肉碱类及胆汁酸类显著上升。这些代谢物及相关代谢途径变化提示，结核性胸膜炎患者相比恶性胸腔积液患者具有更高的能量损耗。此外，我们发现犬尿氨酸（kynurenine）代谢途径中的多种代谢物变化显著。其中该代谢途径限速反应的底物色氨酸显著降低，产物犬尿氨酸显著升高，且犬尿氨酸下游多种代谢物也显著升高。犬尿氨酸：色氨酸丰度比值作为诊断指标对结核性与恶性胸腔积液进行鉴别，敏感性达到92.7%，特异性达到86.1%。该比值联合常见的生化指标ADA，诊断敏感性和特异性达到97.6%和91.3%。犬尿氨酸、色氨酸以及这两种代谢物丰度比值在正离子模式、负离子模式下均显示出相一致的变化趋势，同时在不同批次收集的样品中均显示出非常稳定的变化趋势。由于犬尿氨酸：色氨酸丰度比值是相对值，不需要进行单个化合物绝对定量，因此操作更为简单、快捷，成本更为低廉，具有良好的临床应用价值。此外，我们还探索了能否从结核病患者胸腔积液中检测结核分枝杆菌特异代谢物或生物大分子。在总代谢物及脂类代谢物中我们均未能找到结核分枝杆菌特异的化合物，这也进一步说明结核分枝杆菌菌量在胸腔积液中非常低。但是在胸腔积液的游离DNA中，我们检测到了结核分枝杆菌特异的IS6110序列。通过检测结核分枝杆菌特异游离DNA，诊断敏感性能达到75%，特异性达到100%。对于提高结核性胸膜炎的确诊率有很重要的临床意义。