α-Synuclein转基因小鼠内源性儿茶酚异喹啉的高效液相色谱-串联质谱检测

Determination of Endogenous Catecholamine Isoquinolines inα-Synuclein Transgenic Mice by HPLC-MS/MS

建立内源性儿茶酚异喹啉类物质1-甲基-6,7-二羟基-1,2,3,4-四氢异喹啉(Salsolinol,Sal)、6,7-二羟基-1,2-二甲基-1,2,3,4-四氢异喹啉(N-methyl-Sal,NMSal)的高效液相色谱-串联质谱(HPLC-MS/MS)定量检测方法,研究α-Synuclein过表达对Sal、NMSal脑内水平的影响。实验优化了色谱及质谱条件,建立基于多反应监测(MRM)技术的Sal和NMSal的高灵敏度、高选择性、高重复性分析方法,并应用此方法对过表达人源野生型(WT)、突变型(A53T)α-Synuclein蛋白的转基因小鼠脑中Sal、NMSal含量进行测定,发现WT、A53T转基因小鼠脑内Sal、NMSal的含量与对照组相比均显著升高。结果表明:Sal、NMSal分别在6.10pmol/L^6.25nmol/L和97.70pmol/L^100.00nmol/L范围呈线性(相关系数r≥0.9995),日内、日间相对标准偏差(RSD)均小于15.0%,回收率分别为86.5%~90.7%和81.0%~91.4%,Sal、NMSal的检出限分别为1.53pmol/L和24.4pmol/L,定量限分别为6.10pmol/L和97.7pmol/L。α-Synuclein过表达会导致内源性神经毒素Sal、NMSal水平的升高,可能与帕金森病发病机制有关。

As a main component of Lewy bodies（LBs）,which represent the pathology characteristic of Parkinson＇sdisease（PD） ,a-synuclein can aggregate in the cell and participate in the onset of PD if it overexpresses. In orderto investigate the effect of a-synuclein aggregation on endogenous catecholamine isoquinolines-salsolinol（Sal） andN-methyl-salsolinol（NMSal）-in human A53T mutant and wild type a-synuclein transgenic mice, a sensitive,specific and stable analytical method based on high performance liquid chromatography-tandem massspectrometry（HPLC-MS/MS） working in multiple reaction monitoring （MRM） mode was established byoptimization of LC and MS condition. A pentafluorophenylpropyl stationary phase and an ammonium formatemobile phase of pH=4.0 were employed in the method. The MRM transition was m/z180.1--117.1 for Sal,rez194. 1---145.1 for NMSal and rez212.1-107.0 for isoproterenol as internal standard. The method realizedwide linear range （over 3 orders of magnitude）, high precision （RSD 15 %） and good recovery （81. 0%-91.4%）. The limits of detection were 1.53 pmol/L and 24.4 pmol/L,and the limits of quantification were 6.10pmol/L and 97.7 pmol/L for Sal and NMSal, respectively. The determination results of Sal and NMSal in thebrain of human a-synuclein transgenic mice showed that the concentration of Sal and NMSal in A53T mutanttype（Sal：671.7-210.6 pg/mg of tissue,NMSal.256.4-31.8 pg/mg of tissue） and wild type（Sal：564. 8.74-148.4 pg/mg of tissue, NMSal. 221. 1 q-16. 6 pg/mg of tissue） transgenic mice both increased significantlycompared to that of control（Sal. 370. 9-101.5 pg/mg of tissue, NMSal： 200. 84-16.4 pg/mg of tissue）. Itindicates that, abnormal aggregation of a -synuclein can elevate the level of endogenous neurotoxin Sal andNMSal, which may involve in the pathogenesis of PD.