不同工艺人血白蛋白与地西泮结合功能的比较研究

Comparison of binding function of different albumin production to diazepam

目的研究比较不同生产工艺制备的人血白蛋白(HSA)与地西泮的结合功能。方法在0.1mol/L的地西泮储备液中加入PBS缓冲液,并调整地西泮的终浓度分别为5.0、10.0、15.0、20.0、25.0μmol/L,用可见-紫外分光光度计于波长254nm处测定各样品的吸光度(A)值并绘制标准曲线。取预处理后的各HSA样品(0.615mmol/L),分别加入终浓度为0.1、0.2、0.3、0.4、0.5mmol/L的地西泮储备液,采用超滤离心法测定超滤透析液中游离地西泮浓度,据此参照公式计算在地西泮浓度为0.1mmol/L时HSA与地西泮的结合率,以及各HSA样品与地西泮之间的结合常数。结果标准曲线显示地西泮浓度与A254值呈线性关系。各HSA样品与地西泮结合率的检测结果显示,HSA与地西泮的结合率最高为98.18%,最低为97.47%,不同生产工艺制备的样品间比较,HSA与地西泮的结合功能差异均无统计学意义。各HSA样品结合常数范围在6×104～10×104mol-1之间,国内不同生产工艺制备的各HSA样品的结合常数平均值约为9.00×104mol-1,与国外HSA样品(8.91×104mol-1)比较差异无统计学意义,其中采用Cohn’s改良法(离心)制备的样品结合常数平均值最低(7.78×104mol-1),采用Cohn’s经典法(压滤)制备的样品结合常数平均值最高(9.57×104mol-1),生产工艺对于HSA的结合常数没有明显影响。结论不同生产工艺制备的HSA与地西泮的结合功能基本一致。

Objective To compare the binding function of human albumins （HSA） produced by different processes to a small molecule drug, diazepam. Methods PBS buffer were added to a stock solution of diazepam （0.1 mol/L） to prepare different concentrations of diazepam （5.0, 10.0, 15.0, 20.0, and 25.0 μmol/L respectively）. Then, the absorbance （A） of the dilutions was detected at a wavelength of 254 nm by using a UV-vis spectrophotometer, and the standard curve was plotted. To test the binding function of HSA, pretreated HSA samples （0.615 mmol/L） were mixed with different concentrations of diazepam solutions （0.1, 0.2, 0.3, 0.4, and 0.5 mmol/L）. The mixtures were then centrifugated with a ultrafilter, and the concentration of the unbound diazepam in the filtrate was determined to calculate the percentage of HSA-diazepam. The association constants （Ka） between HSA and diazepam, and the binding rate at a diazepam concentration of 0. 1mmol/L were determined with a reference formula. Results The standard curve indicates a linear relationship between the concentration of diazepam and the absorbance （A254）. The percentage of HSA-diazepam （0.1 mmol/L） ranged from 97.47% to 98.18%. No significant difference was found in the binding function of HSA obtained by different processes to diazepam. The average Ka of HSA ranged from 6 ×10^4mol^-1 to 10 ×10^4mol^-1. No significant difference was detected in the mean Ka between domestic and imported HSA （9.00 ×10^4mol^-1 vs. 8.91 ×10^4mol^-1）. In the HSA employed in this study, the mean Ka of the sample produced using Cohn＇s modified （centrifugation） method was the lowest （7.78 ×10^4mol^-1）, while that of the HSA obtained by Cohn＇s traditional method （pressure filtration） was the highest （9.57 ×10^4mol^-1）. No significant effect was found in the Ka of HSA obtained by different processes. Conclusion Human albumins produced by different processes have similar binding functions with diazepam.