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首页> 外文期刊>RSC Advances >Determination of total, free, and reduced homocysteine and related aminothiols in uremic patients undergoing hemodialysis by precolumn derivatization HPLC with fluorescence detection
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Determination of total, free, and reduced homocysteine and related aminothiols in uremic patients undergoing hemodialysis by precolumn derivatization HPLC with fluorescence detection

机译：柱前衍生HPLC-荧光检测法测定尿毒症患者血液透析中总半胱氨酸，游离半胱氨酸和还原性高半胱氨酸及相关氨基硫醇的含量

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摘要

Homocysteine (Hcy) and its metabolically related aminothiols cysteine (Cys), cysteinylglycine (CysGly), and glutathione (GSH) play important roles in the pathogenesis of uremia. Most of these aminothiols exist in plasma as disulphide and mixed disulphide forms. A new, simple, sensitive and reliable precolumn derivatization HPLC method, with fluorescence detection, for the determination of four kinds of aminothiols, including total, free and reduced fractions, in plasma simultaneously was established and validated. After a simple derivatization using N-(1-pyrenyl) maleimide (NPM), the assay was performed using a C-18 column with gradient elution. The method used for the total aminothiol determinations was linear in the range of 2.00-80.0, 10.0-1500, 1.00-120, and 3.00-240 mu mol L-1 for GSH, Cys, Hcy, and CysGly, respectively. The method used for the reduced aminothiols was linear in the range of 0.10-8.00, 1.25-50.0, 1.25-50.0, and 0.01-4.00 mu mol L-1 for GSH, Cys, Hcy, and CysGly, respectively. The method was successfully applied to the analysis of plasma from uremic patients (n = 29) and healthy subjects (n = 28). The results showed that the concentrations of the total, free and reduced forms of Hcy, Cys and CysGly were higher, while the concentrations of the three forms of GSH were lower (p < 0.05) in uremic patients than in the healthy controls. The concentrations of the three forms of Hcy and Cys were significantly decreased, but the concentrations of the three fractions of GSH and CysGly had no significant change, during hemodialysis. In conclusion, the developed method is simple, fast, accurate, and suitable for clinical measurements.
机译：同型半胱氨酸（Hcy）及其代谢相关的氨基硫半胱氨酸（Cys），半胱氨酰甘氨酸（CysGly）和谷胱甘肽（GSH）在尿毒症的发病机理中起重要作用。这些氨基硫醇中的大多数以二硫化物和混合的二硫化物形式存在于血浆中。建立并验证了一种新的，简便，灵敏和可靠的柱前衍生HPLC方法，该方法具有荧光检测功能，可同时测定血浆中的四种氨基硫醇，包括总级分，游离级分和还原级分。使用N-（1-吡啶基）马来酰亚胺（NPM）进行简单衍生化后，使用带有梯度洗脱的C-18色谱柱进行测定。对于GSH，Cys，Hcy和CysGly，用于总氨基硫醇测定的方法分别在2.00-80.0、10.0-1500、1.00-120和3.00-240μmolL-1范围内呈线性。对于GSH，Cys，Hcy和CysGly，用于还原的氨基硫醇的方法分别在0.10-8.00、1.25-50.0、1.25-50.0和0.01-4.00μmolL-1范围内呈线性。该方法已成功应用于尿毒症患者（n = 29）和健康受试者（n = 28）的血浆分析。结果显示，尿毒症患者的Hcy，Cys和CysGly的总，游离和还原形式的浓度均较高，而尿毒症患者中三种形式的GSH的浓度均低于健康对照组（p <0.05）。在血液透析期间，三种形式的Hcy和Cys的浓度均显着降低，但GSH和CysGly的三种馏分的浓度没有明显变化。综上所述，该方法简便，快速，准确，适用于临床测量。

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