An on-column derivatization method for the determination of homocysteine-thiolactone and protein N-linked homocysteine
An on-column derivatization method for the determination of homocysteine-thiolactone and protein N-linked homocysteine
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DOI：
10.1007/s00726-010-0521-7
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发表时间：
2011-06-01
期刊：
影响因子：
3.5
通讯作者：
Jakubowski, Hieronim
中科院分区：
文献类型：
--
作者：
Glowacki, Rafal;Bald, Edward;Jakubowski, Hieronim
同型半胱氨酸(Hcy)是通过硫酯Hcy-内酯与蛋白质赖氨酸残基的epsilon-氨基反应合成N-Hcy-蛋白质。这种反应损害和改变了蛋白质的功能，并与动脉粥样硬化性血栓疾病有关。在这里，我们描述了基于邻苯二甲醛柱上衍生化和荧光检测的新的高效液相色谱分析方法，用于测定同型半胱氨酸-硫内酯、蛋白质N-连接的同型半胱氨酸和同型半胱氨酸。柱上衍生化产生窄的峰，允许快速运行(3-5分钟)，并便于直接从血浆蛋白质的酸解产物中测定N-连接的同型半胱氨酸。用人体尿液和血浆样本验证了这些检测方法的实用性。
Homocysteine (Hcy) is incorporated into protein via a reaction of the thioester Hcy-thiolactone with epsilon-amino group of a protein lysine residue generating N-Hcy-protein. This reaction impairs and alters protein's function and has been implicated in atherothrombotic disease. Here, we describe new high-performance liquid chromatography assays for the determination of Hcy-thiolactone, protein N-linked Hcy, and Hcy based on an on-column derivatization with o-phthaldialdehyde and fluorescence detection. The on-column derivatization generates narrow peaks, which allows fast run times (3-5 min) and facilitates determination of N-linked Hcy directly from acid hydrolysates of plasma protein. Utility of these assays was demonstrated with human urine and plasma samples.