毛细管区带电泳检测糖尿病肾病变患者血液内红细胞中的谷胱甘肽

Determination of Glutathione in Red Blood Cells of Diabetic Nephropathy Patient with Capillary Zone Electrophoresis

采用高效毛细管区带电泳法对糖尿病肾病变患者血液内红细胞中参与氧化应激的氧化型谷胱甘肽(GSSG)和还原型谷胱甘肽(GSH)进行了测定。对影响分离的条件(缓冲液pH、缓冲液浓度、分离电压和毛细管温度)进行了优化,使用非涂层的毛细管(21cm×75μmi.d.)和20mmol/LpH6 86的磷酸缓冲液,在25kV,30℃和200nm条件下,可在3min内同时对血红细胞中GSSG和GSH定量分析。方法具有良好的重现性(迁移时间和峰面积的相对标准偏差均小于2 0%)、较高的灵敏度(GSH:3 0μmol/L,GSSG:1 5μmol/L)和较宽的线性范围(GSH:3 0～750 0μmol/L,GSSG:1 5～600 0μmol/L),完全可用于临床上对患者体内氧化应激状况的监测。

A new capillary zone electrophoretic method for the determination of both the reduced (GSH) and oxidized (GSSG) forms of glutathione was optimized in terms of buffer pH, buffer concentration, separation voltage and capillary temperature. GSH and GSSG, which are related to oxidative stress and several diseases, in red blood cells of diabetic nephropathy (DN) patients could be baseline separated within 3 min using a fusedsilica capillary (21 cm×75 μm i.d.) and a 20 mmol/L phosphate buffer (pH 686) with a separation voltage of 25 kV at 30 ℃, and a detection wavelength of 200 nm. The red blood cells were obtained by centrifugation of the DN plasma samples at 3?000 r/min for 5 min and deproteinized with trichloroacetic acid. After centrifugation at 10?000 r/min for 10 min, the supernatant fluid was injected into the capillary directly. The method was applied to detect the clinical samples without derivatization. The linear ranges were 30-7500 μmol/L and 15-6000 μmol/L and the limits of detection were 30 and 15 μmol/L for GSH and GSSG, respectively. The proposed method also has a high reproducibility (RSDs of both migration times and peak areas were lower than 2%) and has been successfully applied to the determination of GSH and GSSG in the clinical samples and monitoring of the status of the oxidized stress in patients' plasma.