Development of automatic system for the determination of biogenic amines in brain by microdialysis-HPLC with fluorescence detection

荧光微透析高效液相色谱法测定脑生物胺自动系统的研制

• 批准号: 08672493
• 负责人: YAMAGUCHI Masatoshi
• 金额: $ 1.41万
• 依托单位: Fukuoka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08672493/
• 关键词: bioactive amines; serotonin; norepinephrine; brain; microdialysis; liquid chromatography; benzylamine; fluorescence detection; マイクロダイアリシス; HPLC; カテコールアミン; インドールアミン; カチコールアミン

Biogenic amines such as serotonin and norepinephrine are well known as neurotransmitters in the control and regulation of many brain functions, and have been strongly implicated in several pathological conditions such as aggressive and predatory behavior, migraine, depression and carcinoid syndrome. Moreover, various drugs such as antigepressants act on the central serotonergic syustem. It is very importnt to measure time-dependent changes of serotonin and/or norepinephrine levels for the investigations of the relationships between the amine levels and various pharmacentical activities. The brain microdialysis sampling technique has been successfully introduced into in vivo studies of the neurotransmitters. Microdialysis can be applied to the continuous measurement of the neurotransmitters of a freely moving animals.In this study, we have developed a microbore high-performance liquid chromatographic method based on precolumn fluorescence derivatization with benzylamine for the determination of the amines in microdialysate from rat brain. The method is based on the fluorescence derivatization of the amines by reaction with benzylamine in the presence of potassium hexacyanoferrate (III). A microdialysis probe was implanted in the rat brain, and continuously perfused at 2.0mul/min with Ringer solution. The microdialysate, collected every 5 min, was added with a reagent solution composed of CAPS buffer (pH 12.0), benzylamine, potassium hexacyanoferrate (III) and methanol. The derivatization reaction was completed by standing at room temperature for 5 min. The benzylamine derivatives of the amines could be separated within 15 minon a reverserd-phase microbore column with isocratic elution. The detection limits of the amines are 30-100 amol for a 5mul injection. The effects of increased potassium ion concentration in the Ringer solution and a single intraperitoneal injection of methanphetamine on the amine levels in microdialysate were examined.

生物胺如5-羟色胺和去甲肾上腺素是众所周知的在许多脑功能的控制和调节中的神经递质，并且已经强烈地涉及若干病理状况，如攻击性和掠夺性行为、偏头痛、抑郁症和类癌综合征。此外，各种药物如抗抑郁药作用于中枢神经系统。测定5-羟色胺和/或去甲肾上腺素水平的时间依赖性变化对于研究胺水平与各种药理活性之间的关系是非常重要的。脑微透析取样技术已成功地应用于神经递质的在体研究。微透析可用于自由活动动物体内神经递质的连续测定，本文建立了一种基于苄胺柱前荧光衍生化的大鼠脑微透析液中胺类化合物的微柱高效液相色谱测定方法。该方法是基于在六氰合铁（III）酸钾存在下，通过与苄胺反应而使胺的荧光衍生化。将微透析探针植入大鼠脑内，用林格液以2.0 μ l/min持续灌注。每5分钟收集一次的微透析液中加入由CAPS缓冲液（pH 12.0）、苄胺、六氰高铁酸钾（III）和甲醇组成的试剂溶液。衍生化反应在室温下静置5 min即可完成。在反相微孔柱上等度洗脱，可在15 min内分离胺的苄胺衍生物。5 μ l进样，胺的检出限为30-100阿莫尔。研究了增加林格溶液中钾离子浓度和单次腹腔注射甲非他明对微透析液中胺水平的影响。