Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia.
Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia.
Biochemistry (Mosc). 2022 Apr;87(4):356-365. doi: 10.1134/S0006297922040058.
2-Oxoacids are involved in a number of important metabolic processes and can be used as biomarkers in some human diseases. A new optimized method for quantification of 2,4-dinitrophenylhydrazine derivatives of 2-oxoacids using high-performance liquid chromatography was developed based on available techniques for quantification of 2-oxoacids in mammalian brain. The use of the 2,4-dinitrophenylhydrazine derivatives of 2-oxoacids was shown to be more advantageous in comparison with the previously used phenylhydrazine derivatives, due to a high chemical stability of the former. Here, we determined the concentrations of pyruvate, glyoxylate, 2-oxoglutarate, 2-oxomalonate, and 4-methylthio-2-oxobutyrate in the methanol/acetic acid extracts of the rat brain using the developed method, as well discussed the procedures for the sample preparation in analysis of mammalian brain extracts. The validation parameters of the method demonstrated that the quantification limits for each of the analyzed of 2-oxoacids was 2 nmol/mg tissue. The developed method facilitates identification of subtle changes in the tissue and cellular content of 2-oxoacids as (patho)physiological biomarkers of metabolism in mammalian tissues.
2- 氧代酸参与许多重要的代谢过程，可作为某些人类疾病的生物标志物。基于已有的哺乳动物脑组织中 2- 氧代酸定量技术，开发了一种新的优化方法，用于高效液相色谱法定量 2- 氧代酸的 2,4- 二硝基苯腙衍生物。与以前使用的苯肼衍生物相比，2- 氧代酸的 2,4- 二硝基苯腙衍生物的使用具有更高的化学稳定性，因此更为有利。在这里，我们使用所开发的方法确定了大鼠脑组织甲醇/乙酸提取物中丙酮酸、乙醛酸、2- 酮戊二酸、2- 酮戊二酸和 4- 甲基巯基-2- 氧代丁酸的浓度，并讨论了分析哺乳动物脑组织提取物时的样品制备程序。该方法的验证参数表明，分析的每种 2- 氧代酸的定量限为 2 nmol/mg 组织。该方法有助于识别组织和细胞中 2- 氧代酸含量的细微变化，作为哺乳动物组织代谢的（病理）生理生物标志物。
