液相层析串联质谱法检测脑脊液食欲素诊断1型发作性睡病的初步探索  

Detection of CSF hypocretin-1 on the narcolepsy type 1 patients by LC-MS/MS:A primary observation

作　　者：赵显超 任佳封 程金湘 吴境宇 王荔月 张潇羽 郑威 李华 刘爽 汪宇佳 程凯汀 汤媛媛 宿长军 ZHAO Xianchao;REN Jiafeng;CHENG Jinxiang(Department of Neurology,Tangdu Hospital of Air Force Military Medical University,Xi’an 710038,China)

机构地区：[1]空军军医大学唐都医院神经内科,陕西西安710038 [2]广州金域医学检验中心有限公司,广东广州510145 [3]上海百趣代谢组学技术研究中心,上海201815 [4]广州医科大学金域检验学院,广东广州510145

出　　处：《中风与神经疾病杂志》2025年第3期195-199,F0002,共6页Journal of Apoplexy and Nervous Diseases

基　　金：陕西省重点产业创新链项目(2022ZDLSF03-07);广州市2023年度市校(院)企联合资助专题(2023A03J0558)。

摘　　要：目的1型发作性睡病(NT1)伴有脑脊液食欲素(Hcrt-1)的降低。目前国内检测Hcrt-1的方法多是利用进口试剂进行的放射免疫法测定(RIA),但此方法只能在有限的放射性同位素安全认证的实验室开展,而且受限于试剂中同位素的衰减、抗体依赖的方法学、缓慢与繁琐的步骤和较长的反应时间。本研究的目的在于探索出一种非放射性且非抗体依赖的脑脊液食欲素快速检测方法。方法利用液相层析串联质谱法(LC-MS/MS)技术对临床确诊的14例NT1患者和10例非发作性睡病患者的脑脊液进行了反复测试,包括基本的方法学建立和优化。主要步骤是将加入非放射性同位素内标的脑脊液经过固相萃取后进行质谱信号捕获和定量分析,并与相应的RIA结果进行比较。结果LC-MS/MS方法简单快速,且在5~2500 pg/ml范围内显示良好的线性关系和重复性。虽然这种绝对定量的检测方法显示与RIA结果有数值方面的差异和不一致性,但它足以区分NT1和非发作性睡病对照组且不存在灰区。结论LC-MS/MS是一种更便利、快速、安全、可靠的可用于NT1诊断的检测方法。Objective Narcolepsy type 1(NT1)is known to be associated with low levels of hypocretin⁃1(Hcrt⁃1)in cerebrospinal fluid(CSF).The standard method for Hcrt⁃1 measurement is radioimmunoassay(RIA)with imported reagents,but this antibody⁃dependent method is limited to radiation safety⁃certified lab,gradual radioactivity degradation,and slow turn⁃around time.The purpose of this study is to explore a non⁃radioactive,faster,and antibody independent domestic method in China for Hcrt⁃1 detection.Methods Repeated testing of cerebrospinal fluid from 14 clinically diagnosed NT1 patients and 10 non⁃narcolepsy patients was performed using liquid chromatography⁃tandem mass spectrometry(LC⁃MS/MS)technology,including the establishment and optimization of fundamental methodological procedures.The main steps involved the addition of non⁃radioactive isotope⁃labeled internal standards to the cerebrospinal fluid,followed by solid⁃phase extraction,mass spectrometry signal acquisition,and quantitative analysis.The results were then com⁃pared with the corresponding radioimmunoassay(RIA)findings.Results The LC⁃MS/MS method showed faster speed,and good linearity across a wider range of synthesized standard(5~2500 pg/ml),and good repeatability.Although this absolute⁃quantitation⁃based LC⁃MS/MS method and RIA method have different reading values in Hcrt⁃1 quantitation,they both can segregate NT1 group from non⁃NT1 group well.Conclusion Although larger cohorts are needed to set up a standard method in China,LC⁃MS/MS method is proved to be an easier,safer,faster,and possibly more accurate method for Hcrt⁃1 quantitation and detection for NT1 diagnosis.

关 键 词：发作性睡病 脑脊液食欲素 液相层析串联质谱法 放射免疫法 

分 类 号：R338.63[医药卫生—人体生理学] R741.04[医药卫生—基础医学]