氨基酸分析仪法批量测定小鼠皮质和海马中氨基酸类神经递质和牛磺酸的含量

Batch Determination of Amino Acid Neurotransmitters and Taurine in Mouse Cortex and Hippocampus by Amino Acid Analyzer

目的:建立一种简单、准确可靠、可批量检测小鼠皮质和海马内氨基酸类神经递质含量和牛磺酸含量的分析方法。方法:采用A300氨基酸分析仪,使用10%磺基水杨酸进行蛋白沉淀,锂盐试剂为流动相,茚三酮显色,检测波长为570 nm,对该方法的分离效果、线性范围、检出限及定量限、日内精密度和回收率、日间精密度进行测定;选择同日龄的SPF级KM小鼠分为雌雄两组,每组断头处死3只,每只均于冰上分离出皮质和海马,经匀浆、离心、蛋白沉淀、稀释、过膜等过程制成小鼠脑组织游离氨基酸样品,分别测定雌雄小鼠皮质和海马内天冬氨酸(aspartic acid,Asp)、谷氨酸(glutamic acid,Glu)、甘氨酸(glycine,Gly)、γ-氨基丁酸(gamma-aminobutyric acid,GABA)4种氨基酸类神经递质的含量和牛磺酸(taurine,Tau)的含量。结果:标准样品、雌雄小鼠皮质样品和海马样品中的5种目标氨基酸分离度好,在8~400μmol·L^-1的浓度范围内,线性良好(R^2≥0.999),检测限为1.29~1.86μmol·L^-1,定量限为4.29~6.20μmol·L^-1,日内精密度良好(标准样品相对标准偏差(relative standard deviation,RSD)<2.23%,脑组织样品RSD<2.76%),脑组织样品的加标回收率为91.52%~102.51%,采用绘制标样峰面积曲线的方法准确获取样品中的5种氨基酸含量,实验用雌雄小鼠之间未发现明显统计学差异(P>0.05)。结论:该方法简便、准确可靠,适用于小鼠皮质和海马组织中游离氨基酸含量的批量检测。

Objective:To establish a simple,accurate and reliable method for the batching detection of amino acid neurotransmitters and taurine in brain. Methods:The amino acids were detected by using A300 amino acid analyzer. The protein was precipitated by using 10% sulfosalicylic acid. Lithium salt reagent was used as mobile phase and ninhydrin was used as color reagent. The detection wavelength was 570 nm. The resolution,linear range,detection limit and quantitative limit,intraday precision and recovery,daytime precision of the method were determined. SPF-class KM mice at the same age were selected and divided into two groups:male and female. Three in each group were decapitated and executed,and each mouse was separated the cortex and hippocampus from the brain on ice. The free amino acid samples of mouse brain tissue were prepared by homogenization,centrifugation,protein precipitation,dilution and membrane transmembrane. The contents of aspartic acid(Asp),glutamic acid(Glu),glycine(Gly),gamma-aminobutyric acid(GABA)and Tau in cortex and hippocampus were determined by amino acid analyzer. Results: Five target amino acids in standard samples,cortical and hippocampal samples of male and female mice were well separated. Within the concentration range of 8~400 μmol·L^-1,the instrument had a good linear relationship(R^2≥0.999). Detection limit was 1.29~1.86 μmol·L^-1. Quantitative limit was 4.29~6.20 μmol·L^-1. The intraday relative standard deviation(RSD)of standard samples were less than 2.23%,the intraday RSD of brain tissue samples were less than 2.76% and the recovery of the spiked samples were in a range of 91.52%~102.51%. 5 kinds of amino acid content in the sample were accurately obtained by plotting the peak area curve of the standard sample. No statistically significant difference was found between the male and female mice(P>0.05). Conclusion:The method is simple,accurate,reliable and suitable for batching detection of free amino acid content in mouse cortex and hippocampus.