Žunić Gordana D, Spasić Slavica, Jelić-Ivanović Zorana
Institute for Medical Research, Military Medical Academy, Belgrade, Serbia.
Department of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia.
Methods Mol Biol. 2019;2030:315-326. doi: 10.1007/978-1-4939-9639-1_24.
Whole blood and/or plasma amino acids are useful for monitoring whole-body protein and amino acid metabolism in an organism under various physiological and pathophysiological conditions. Various methodological procedures are in use for their measurement in biological fluids. From the time when capillary electrophoresis was introduced as a technology offering rapid separation of various ionic and/or ionizable compounds with low sample and solvent consumption, there were many attempts to use it for the measurement of amino acids present in physiological fluids. As a rule, these methods require derivatization procedures for detection purposes.Here, we present two protocols for the analysis of free amino acids employing free zone capillary electrophoresis. Main advantage of both methods is an absence of any derivatization procedures that permits the analysis of free amino acid in physiological fluids. The method using direct detection and carrier electrolyte consisting of disodium monophosphate (10 mM at pH 2.90) permits determination of compounds that absorb in UV region (aromatic and sulfur containing amino acids, as well as some peptides such as carnosine, reduced, and oxidized glutathione). The other method use indirect absorbance detection, employing 8 mM p-amino salicylic acid buffered with sodium carbonate at pH 10.2 as running electrolyte. It permits quantification of 30 underivatized physiological amino acids and peptides. In our experience factorial design represents a useful tool for final optimization of the electrophoretic conditions if it is necessary.
全血和/或血浆氨基酸对于监测生物体在各种生理和病理生理条件下的全身蛋白质和氨基酸代谢很有用。目前有各种方法用于测量生物体液中的这些物质。自毛细管电泳作为一种能够快速分离各种离子型和/或可电离化合物且样品和溶剂消耗低的技术被引入以来，人们多次尝试用它来测量生理体液中的氨基酸。通常，这些方法需要衍生化程序用于检测目的。在此，我们介绍两种采用自由区毛细管电泳分析游离氨基酸的方法。这两种方法的主要优点是无需任何衍生化程序，从而能够分析生理体液中的游离氨基酸。使用直接检测且载体电解质由磷酸二氢钠（pH 2.90 时为 10 mM）组成的方法能够测定在紫外区域有吸收的化合物（芳香族和含硫氨基酸，以及一些肽如肌肽、还原型和氧化型谷胱甘肽）。另一种方法采用间接吸光度检测，使用 8 mM 对氨基水杨酸并用 pH 10.2 的碳酸钠缓冲作为运行电解质。它能够对 30 种未衍生化的生理氨基酸和肽进行定量。根据我们的经验，如果有必要，析因设计是最终优化电泳条件的有用工具。
