反相高效液相色谱荧光法测定大鼠脑组织中氨基酸类神经递质  

Determination of the Amino Acids in Rat Brain by High-performance Liquid Chromatography and Fluorescence Detection*

作　　者：吴强恩[1] 郑力行[1] 谢芳[1] 吴同俊[1] 阮国洪[1] 周志俊[1] 

机构地区：[1]复旦大学公共卫生学院劳动卫生教研室-公共卫生安全国家教育部重点实验室,上海200032

出　　处：《复旦学报（医学版）》2005年第3期355-358,共4页Fudan University Journal of Medical Sciences

基　　金：国家 973项目 (2 0 0 2CB5 12 90 5 );上海市曙光计划资助项目

摘　　要：目的　建立并改良反相高效液相色谱法,测定大鼠不同脑组织分区氨基酸类神经递质含量。方法　改良使用K2 CO3 缓冲溶液作为稀释液,反相高效液相色谱洗脱,高丝氨酸作为内标,邻苯二甲醛柱前衍生和荧光检测器检测大鼠大脑皮质、海马、纹状体和小脑中4种氨基酸类神经递质:天冬氨酸(Asp)、谷氨酸(Glu)、甘氨酸(Gly)和γ氨基丁酸(GABA)的含量。结果　4种氨基酸在18min内洗脱完全,分离效果良好;在0 .5～4 0 μmol/L浓度范围有较好的线性关系,其相关系数不低于0 .9997;4种氨基酸天内精密度范围为0 .70 %～4 .0 1% ,天间精密度为2 .78%～9.5 8% ;Asp、Glu和GABA的最低检测限为0 .0 1μmol/L ,而Gly为0 .0 2 μmol/L ;海马组织4种氨基酸绝对回收率不低于89.39%。结论　改良后的反相高效液相色谱荧光法灵敏度高、重复性好,能有效分离检测大鼠脑组织分区中氨基酸类神经递质。Purpose To establish and improve the meth od of determining the amino acid neurotransmitters in different rat brain tissues homogenates by isocratic reversed high-performance liquid chromatography (HPLC) . Methods Standards and the samples were prepared by K 2 CO 3 solution,the HPLC system with fluorescence detection (FL) was used to exam ine amino aci d derivatives,including aspartate,glutamate,glycine and GABA amino acid neurot ransmitters in 4 different rat brain regions (cortex, hippocampus,striatum and cerebellum) formed by a reaction with o-phthalaldehyde (OPA) in the presenc e of the internal standard homose-rine. Results Precise separation of the five amino acids was achieved using isocratic elution within 18 minutes.A good linear response was ob tained for the 4 standards over the range of 0.5～40 μmol/L with cor relation coefficients not less than 0.999 7.For these amino acids,good precision s were obtained between 0.70%～4.01% for the intra-day precisions (RSD,%) a nd 2.78%～9.58% for the inter-day precisions.The detection limits were in t he range of 0.01 μmol/L (aspartate,glutamate and GABA)～0.02 μmol/L (glycine ).The absolute recoveries was not less than 89.39% in the hippocampus. Conclusions It was a more reliable and sensitive method to measure the amino acid neurotransmitters in rat brain sections by the revise d high-performance liquid chromatography with a reaction with o-phthalaldehyde (OPA) in the presence of the dilution K 2CO 3.

关 键 词：高效液相色谱 氨基酸类神经递质 脑组织 大鼠 

分 类 号：Q592.5[生物学—生物化学] R322.8[医药卫生—人体解剖和组织胚胎学]