Departamento de Biología Celular y Molecular, CUCBA, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico.
Neurochem Res. 2012 Jul;37(7):1457-64. doi: 10.1007/s11064-012-0736-y. Epub 2012 Mar 8.
Microdialysis coupled to HPLC is the preferred method for quantification of glutamate (Glu) concentrations, both in normal and pathological conditions. However, HPLC is a time consuming technique that suffers from poor temporal resolution. Here we describe an alternative method to measure glutamate concentrations in small-volume dialysis samples by quantifying hydrogen peroxide released by glutamate oxidase using the Amplex Red method. This system permits continuous automatic sample collection and the detection of a fluorescent reaction product, resorufin, which provides a measure of the glutamate concentration. Quantification can be carried out in small microdialysis samples to allow a temporal resolution of 60 s. Both in vitro and in vivo tests showed that this method was reproducible and reliable, detecting Glu along a linear scale. To validate the proposed method, extracellular Glu concentrations in the rat brain were measured and correlated with electrophysiological activity prior, during and after seizure induction with 4-aminopyridine. This method may be adapted to monitor other biologically active compounds, including acetylcholine and glucose, as well as other compounds that generate hydrogen peroxide as a reaction product and may be used as an alternative to other neurochemical methods.
微透析与 HPLC 耦联是定量分析谷氨酸（Glu）浓度的首选方法，无论是在正常还是病理条件下。然而，HPLC 是一种耗时的技术，其时间分辨率较差。在这里，我们描述了一种替代方法，通过使用 Amplex Red 方法定量谷氨酸氧化酶释放的过氧化氢来测量小体积透析样品中的谷氨酸浓度。该系统允许连续自动采集样品，并检测荧光反应产物 Resorufin，这提供了谷氨酸浓度的测量值。可以对小的微透析样品进行定量，以实现 60 秒的时间分辨率。体外和体内测试均表明，该方法具有可重复性和可靠性，能够在线性范围内检测 Glu。为了验证所提出的方法，在使用 4-氨基吡啶诱导癫痫发作之前、期间和之后，测量了大鼠脑中的细胞外 Glu 浓度，并与电生理活性相关联。该方法可以适应于监测其他生物活性化合物，包括乙酰胆碱和葡萄糖，以及其他作为反应产物产生过氧化氢的化合物，并且可以作为其他神经化学方法的替代方法。
