Amarnath Kapil, Amarnath Venkataraman, Amarnath Kalyani, Valentine Holly L, Valentine William M
Department of Pathology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Talanta. 2003 Aug 29;60(6):1229-38. doi: 10.1016/S0039-9140(03)00232-7.
We present a highly selective and sensitive method for the determination of cysteine (Cys) and related aminothiols that play important roles in health and disease. The key step in the analysis is treatment with 1,1'-thiocarbonyldiimidazole (TCDI) that rapidly and quantitatively reacts with both the amino and thiol groups to form stable cyclic dithiocarbamates with intense UV absorption. Cys, homocysteine (hCys), and cysteinylglycine in plasma (75 microl), urine (100 microl), or cerebrospinal fluid (100-500 microl) were determined by separating and measuring their cyclic derivatives by a high performance liquid chromatograph (HPLC) connected to a UV detector. The chromatograms obtained using TCDI contained fewer and better-resolved peaks than those produced by less selective reagents used previously. Using chemically similar 2-methylcysteine as the internal standard, high repeatability (variation of less than 5%) and adequate sensitivity to detect small increments (10-20%) in the concentrations of cysteinylglycine and hCys were achieved. The HPLC method can also be modified to measure d-penicillamine (greater than 0.8 muM) in plasma (50 microl) providing a potential method to monitor plasma levels of this drug in patients.
我们提出了一种高选择性和高灵敏度的方法，用于测定在健康和疾病中起重要作用的半胱氨酸（Cys）及相关氨基硫醇。分析的关键步骤是用1,1'-硫代羰基二咪唑（TCDI）处理，它能与氨基和硫醇基团快速定量反应，形成具有强烈紫外吸收的稳定环状二硫代氨基甲酸盐。通过连接紫外检测器的高效液相色谱仪（HPLC）分离并测量血浆（75微升）、尿液（100微升）或脑脊液（100 - 500微升）中的Cys、同型半胱氨酸（hCys）和半胱氨酰甘氨酸的环状衍生物来进行测定。与先前使用的选择性较差的试剂相比，使用TCDI获得的色谱图峰数更少且分离效果更好。以化学性质相似的2-甲基半胱氨酸作为内标，实现了高重复性（变异小于5%）以及对检测半胱氨酰甘氨酸和hCys浓度小幅度增加（10 - 20%）有足够的灵敏度。该HPLC方法还可进行改进，用于测量血浆（50微升）中的d-青霉胺（大于0.8微摩尔），为监测患者体内该药物的血浆水平提供了一种潜在方法。
