蛋白结合类尿毒症毒素蛋白结合率方法测定

Determination of plasma protein binding rate of protein binding uremic toxins

目的建立蛋白结合类尿毒症毒素蛋白结合率的检测方法,测定慢性肾脏病（chronic kidney disease,CKD）患者血清马尿酸（hippuric acid,HA）、硫酸吲哚酚（indoxyl sulfate,IS）、硫酸对甲酚（p-cresyl sulfate,PCS）及3-羧基-4-甲基-5-丙基-2-呋喃丙酸（3-carboxy-4-methyl-5-propyl-2-furan-propionic acid,CMPF）4种蛋白结合类尿毒症毒素的蛋白结合率。方法通过对方法的专属性、线性关系、精密度等考察,验证超滤法联合高效液相色谱-串联质谱法（high performance liquid chromatagraphy-tandem mass spectrometry,HPLC-MS/MS）测定蛋白结合类尿毒症毒素的游离浓度的可行性,并采用该方法测定CKD患者血清HA、IS、PCS、CMPF的蛋白结合率。结果内源性干扰物质在待测物的出峰位置基本不干扰HA、IS、PCS、CMPF的测定;本方法在10-1 000 ng/ml（IS、PCS、CMPF）、50-5 000 ng/ml（HA）范围内线性良好;日内精密度为0.84%,日间精密度为1.18%;超滤膜回收率均〉95%;健康对照组HA、IS、PCS及CMPF的蛋白结合率为（83.44±3.04）%、（98.73±0.55）%、（98.42±0.95）%、（96.66±2.65）%;CKD 3-5期HA、IS、PCS及CMPF的蛋白结合率依次为3期（79.72±3.82）%、（99.18±0.45）%、（98.64±0.64）%、（95.89±3.52）%;4期（71.09±7.66）%、（98.24±0.95）%、（98.26±0.73）%、（91.82±5.10）%;5期（61.22±7.62）%、（97.41±1.10）%、（96.85±1.51）%、（97.51±1.52）%。结论所建立的方法灵敏度高,专属性和重复性好,操作简单,能够满足定量分析测试要求。HA蛋白结合率均〉60%,与HSA之间具有中等强度结合,IS、PCS及CMPF的蛋白结合率均〉90%,与HSA之间具有高强度结合。

Objective To detect the serum albumin binding rate of protein binding uremic toxins, and then detect the serum albumin binding rate of hippuric acid （HA）, indoxyl sulfate （IS）, p-cresyl sulfate （PCS） and 3-carboxy-4-methyl-5-propyl-2-furan-propionic acid （CMPF） in chronic kidney disease （CKD） patients. Methods This method was inspected through specificity, linearity and precision, validation the feasibility of ultrafiltration method which combined with high performance liquid chromatagraphy-tandem mass spectrometry （HPLC-MS/MS） to detect free concentration of protein-bound uremic toxins, and to detect the serum albumin binding rate of HA, IS, PCS and CMPF in CKD patients. Result Endogenous interfering substances had little interference in the peak position of HA, IS, PCS, and CMPF when they were detected. The liner was good with 10-1 000 ng/ml of IS, PCS CMPF and 50-5 000 ng/ml of HA; days of precision was 0.84%, while daytime precision was 1.18%; ultrafiltration membrane recovery was above 95%; the protein binding rates of HA, IS, PCS and CMPF in healthy controls were （83.44±3.04）%, （98.73±0.55）%, （98.42±0.95）%, （96.66±2.65）%, which were （79.72±3.82）%, （99.18±0.45）%, （98.64±0.64）%, （95.89±3.52）% in CKD stage 3, and （71.09±7.66）%, （98.24±0.95）%, （98.26±0.73）%, （91.82±5.10）% in CKD stage 4, and （61.22±7.62）%, （97.41±1.10）%, （96.855±1.51）%, （97.515±1.52）% in CKD stage 5, respectively. Conclusion The method which has high sensitivity, good specificity and reproduction can satisfy the requirement of quantitive analysis. The protein conjugation rate of HA was more 60%, which was middle degree with human serum albumin （HSA） while the binding of IS, PCS and CMPF were more than 90%, which had high degree with HSA