一种基于超高效液相色谱-串联质谱法测定肝组织中N-乙酰谷氨酸合酶活性的新方法。
A novel UPLC-MS/MS based method to determine the activity of N-acetylglutamate synthase in liver tissue.
作者信息
Dercksen Marli, Duran Marinus, IJlst Lodewijk, Kulik Wim, Ruiter Jos P N, van Cruchten Arno, Tuchman Mendel, Wanders Ronald J A
机构信息
Laboratory Genetic Metabolic Diseases, Departments of Pediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Human Metabonomics, North-West University, Potchefstroom Campus, South Africa.
Laboratory Genetic Metabolic Diseases, Departments of Pediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
出版信息
Mol Genet Metab. 2016 Dec;119(4):307-310. doi: 10.1016/j.ymgme.2016.10.004. Epub 2016 Oct 13.
BACKGROUND
N-acetylglutamate synthase (NAGS) plays a key role in the removal of ammonia via the urea cycle by catalyzing the synthesis of N-acetylglutamate (NAG), the obligatory cofactor in the carbamyl phosphate synthetase 1 reaction. Enzymatic analysis of NAGS in liver homogenates has remained insensitive and inaccurate, which prompted the development of a novel method.
METHODS
UPLC-MS/MS was used in conjunction with stable isotope (N-acetylglutamic-2,3,3,4,4-d acid) dilution for the quantitative detection of NAG produced by the NAGS enzyme. The assay conditions were optimized using purified human NAGS and the optimized enzyme conditions were used to measure the activity in mouse liver homogenates.
RESULTS
A low signal-to-noise ratio in liver tissue samples was observed due to non-enzymatic formation of N-acetylglutamate and low specific activity, which interfered with quantitative analysis. Quenching of acetyl-CoA immediately after the incubation circumvented this analytical difficulty and allowed accurate and sensitive determination of mammalian NAGS activity. The specificity of the assay was validated by demonstrating a complete deficiency of NAGS in liver homogenates from Nags -/- mice.
CONCLUSION
The novel NAGS enzyme assay reported herein can be used for the diagnosis of inherited NAGS deficiency and may also be of value in the study of secondary hyperammonemia present in various inborn errors of metabolism as well as drug treatment.
背景
N - 乙酰谷氨酸合酶（NAGS）通过催化N - 乙酰谷氨酸（NAG）的合成，在经由尿素循环清除氨的过程中发挥关键作用，NAG是氨基甲酰磷酸合成酶1反应中必不可少的辅助因子。对肝脏匀浆中NAGS进行酶活性分析一直不够灵敏且不准确，这促使开发一种新方法。
方法
超高效液相色谱 - 串联质谱法（UPLC - MS/MS）结合稳定同位素（N - 乙酰谷氨酸 - 2,3,3,4,4 - d酸）稀释用于定量检测NAGS酶产生的NAG。使用纯化的人NAGS对测定条件进行优化，并将优化后的酶条件用于测量小鼠肝脏匀浆中的活性。
结果
由于N - 乙酰谷氨酸的非酶促形成和低比活性，在肝脏组织样品中观察到低信噪比，这干扰了定量分析。孵育后立即淬灭乙酰辅酶A克服了这一分析难题，并允许准确和灵敏地测定哺乳动物NAGS活性。通过证明Nags - / - 小鼠肝脏匀浆中NAGS完全缺乏，验证了该测定方法的特异性。
结论
本文报道的新型NAGS酶测定方法可用于诊断遗传性NAGS缺乏症，在研究各种先天性代谢缺陷以及药物治疗中出现的继发性高氨血症方面也可能具有价值。
Zotero 插件