Swartz K J, Matson W R, MacGarvey U, Ryan E A, Beal M F
Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115.
Anal Biochem. 1990 Mar;185(2):363-76. doi: 10.1016/0003-2697(90)90309-w.
Kynurenic acid is a broad-spectrum excitatory amino acid (EAA) receptor antagonist which is present in the mammalian central nervous system. We describe a method for the measurement of kynurenic acid using isocratic reverse-phase high-performance liquid chromatography (HPLC) with fluorometric detection enhanced by Zn2+ as a postcolumn reagent. The method requires no prior sample preparation procedures other than extraction with 0.1 M HClO4. The reliability of the primary fluorometric method was verified by comparing measurements of tissue concentrations of kynurenic acid in human cerebral cortex and putamen using three different methods of separation with fluorometric detection, as well as four methods utilizing HPLC with coulometric electrode array system (CEAS) detection. All seven methods produced comparable results. The concentration of kynurenic acid in human cerebral cortex was 2.07 +/- 0.61 pmol/mg protein, and in human putamen, 3.38 +/- 0.81 pmol/mg protein. Kynurenic acid was also found to be present in human cerebrospinal fluid (CSF) at a concentration of 5.09 +/- 1.04 nM. The regional distribution of kynurenic acid in the rat brain was examined. Kynurenic acid concentrations were highest in brainstem (149.6 fmol/mg protein) and olfactory bulb (103.9 fmol/mg protein) and lowest in thalamus (26.0 fmol/mg protein). There were no significant postmortem changes in kynurenic acid concentrations in cerebral cortex, hippocampus, and striatum at intervals ranging from 0 to 24 h. Perfusion of the cerebral vasculature with normal saline prior to sacrifice did not significantly alter kynurenic acid content in rat hippocampus, cerebral cortex, or striatum. The analytical methods described are the most sensitive (10-30 fmol injection-1) and specific (utilizing both excitation and emissions properties and electrochemical reaction potentials, respectively) methods for determining kynurenic acid in brain tissue extracts and CSF. These methods should prove useful in examining whether kynurenic acid modulates EAA-mediated neurotransmission under physiologic conditions, as well as in determining the role of kynurenic acid in excitotoxic neuronal death.
犬尿喹啉酸是一种存在于哺乳动物中枢神经系统中的广谱兴奋性氨基酸（EAA）受体拮抗剂。我们描述了一种使用等度反相高效液相色谱（HPLC）结合锌离子作为柱后试剂增强荧光检测来测定犬尿喹啉酸的方法。该方法除了用0.1 M高氯酸提取外，无需任何样品前处理程序。通过比较使用三种不同荧光检测分离方法以及四种使用带库仑电极阵列系统（CEAS）检测的HPLC方法来测量人脑皮质和壳核中犬尿喹啉酸的组织浓度，验证了主要荧光法的可靠性。所有七种方法都得出了可比的结果。人脑皮质中犬尿喹啉酸的浓度为2.07±0.61 pmol/mg蛋白质，人壳核中为3.38±0.81 pmol/mg蛋白质。还发现人脑脊液（CSF）中存在犬尿喹啉酸，浓度为5.09±1.04 nM。研究了犬尿喹啉酸在大鼠脑中的区域分布。犬尿喹啉酸浓度在脑干（149.6 fmol/mg蛋白质）和嗅球（103.9 fmol/mg蛋白质）中最高，在丘脑（26.0 fmol/mg蛋白质）中最低。在0至24小时的间隔内，脑皮质、海马体和纹状体中犬尿喹啉酸浓度没有显著的死后变化。处死前用生理盐水灌注脑血管并未显著改变大鼠海马体、脑皮质或纹状体中犬尿喹啉酸的含量。所描述的分析方法是用于测定脑组织提取物和脑脊液中犬尿喹啉酸的最灵敏（10 - 30 fmol进样量-1）和特异（分别利用激发和发射特性以及电化学反应电位）的方法。这些方法在研究犬尿喹啉酸在生理条件下是否调节EAA介导的神经传递以及确定犬尿喹啉酸在兴奋性毒性神经元死亡中的作用方面应会证明是有用的。
