基于同位素稀释质谱技术的多发性骨髓瘤相关血清代谢物测定及应用研究

Multiple myeloma (MM) is the second most frequent hematologic malignancy, and its pathogenesis is complicated. Recent observations have consistently found that several species of serum amino acids, carnitine and acylcarnitines, lysophosphatidylcholines, sphingolipids and fatty acids are closely related to the development of MM. It is highly desirable a precise approach for simultaneous measurement of all metabolites and then they need to be validated on external independent populations for further clinical confirmation and application. Analytical methods require separate sample preparations and analyses and respective measurement of species of these compounds. So it is urgent to carry out the study on the simultaneously accurate determination of all metabolites. We have developed a method for quantification of 27 of serum analytes by isotope dilution liquid chromatography tandem mass spectrometry. Serum concentrations of metabolites were analyzed and significant differences were observed between the MM patients and a matched control group. Based on our previous studies, this current project aims to develop a simple, precise and specific isotope dilution mass spectrometry to detect all metabolites for MM. With our developed methods, the levels of these biomarkers will be determined in serum of control subjects and MM patients at the time of diagnosis, before and after achieving partial remission and achieving complete remission and finally their relationships will be analyzed. This project will help to develop a method for simultaneous quantification of different classes of metabolites in serum, to find an efficient tool in MM research and to provide potentially useful new biomarkers for auxiliary diagnosis and evaluation of therapeutic efficacy of MM.

多发性骨髓瘤（MM）发病率占血液系统肿瘤第二位，发病机制复杂。研究发现血清氨基酸类、肉毒碱类、溶血卵磷脂类、鞘脂类和脂肪酸类代谢物水平与MM发生发展关系密切，而进一步临床确认及应用需要精准定量所有代谢物并进行人群验证。目前每类代谢物测定的方式各不相同，缺乏同时准确可靠检测所有物质的简便方法。项目组前期建立了同时测定其中27种血清代谢物的同位素稀释液相色谱串联质谱法，并发现在MM患者和对照组间部分代谢物水平有显著差异。申请人拟在前期工作基础上纳入其余待测物，建立简便、精密、特异的同时检测所有MM相关代谢物的方法，并在人群中确证这些标志物与MM发病、未达到部分缓解、部分缓解和完全缓解的关系。预期该研究方法可以搭建血清样本中多类型代谢物同时准确检测的平台，为MM代谢物的研究提供有应用价值的技术手段，为辅助MM诊断和治疗效果评价提供潜在有效的代谢标志物。

多发性骨髓瘤（MM）是恶性浆细胞克隆性疾病，起病隐匿，发病率在血液系统恶性肿瘤中居第二位，目前临床上无法治愈，早期鉴定其主要改变生物学指标并及时进行有效干预，是准确诊断、疗效评估及延长生存期的重要手段。研究发现血清氨基酸类、肉毒碱类、溶血卵磷脂类和鞘脂类代谢物水平与MM发生发展关系密切，而进一步临床确认及应用需要精准定量所有代谢物并进行人群验证。我们首先用标准溶液建立各代谢物液相色谱分离和质谱测定的条件。用含0.1%甲酸的异丙醇提取血清各目标代谢物，氮气下吹干，用流动相重组后进行LC-MS/MS分析。液相色谱分离采用HILIC色谱柱，流动相为75%甲醇（0.1%甲酸，3mM甲酸铵），柱温25℃，进样2μl。在API 5500质谱，电喷雾离子源（ESI）多反应监测模式下，所有代谢物在15分钟内有效分离。同时，用同位素内标法定量，评价方法的线性、检测限、定量限、基质效应和不精密度。方法的相对回收率在80.12%-109.33%。平均批内不精密度在5%以内，总不精密度大多在10%以内，检测限和定量限都小于血清最低浓度。收集160名MM患者和184名对照组人群，记录年龄、性别等基本信息，同时测定白蛋白（ALB），球蛋白（GLB），乳酸脱氢酶（LDH），血肌酐（Cr），血钙（Ca2+），血红蛋白（Hb）和血小板（PLT）等MM相关诊断指标，分析各代谢物与传统诊断指标、与MM的关系。结果发现，氨基酸类代谢物中，MM组Phe、Glu水平显著高于对照组，Gln和Trp水平显著低于对照组；饱和的LPC水平显著高于对照组，不饱和的LPC显著低于对照组；MM患者组所有的肉毒碱类代谢物均低于对照组血清水平。多元logistic回归分析发现性别，ALB和Hb与MM显着相关，通过公式“MM指数=-（-18×gender-3×Alb-Hb）/10”建立了MM的诊断模型。MM组和对照组的MM指数分别为22（20-25）和30（29-31），两组之间有显着差异（P<0.001）。研究的完成有助于为日后开展这些代谢物血清水平与健康和MM发生、发展关系的流行病学研究奠定方法学基础。