冻干红细胞残余水的测定及其分布研究

The determination of residual water and its distribution in freeze-dried red blood cells

目的建立并比较冻干红细胞残余水含量测定方法,探讨冻干红细胞残余水的分布情况。方法一次性冻干等量(500μL)的含保护剂(主要成分12%甘油)的红细胞、无保护剂的红细胞,含保护剂(同前)的血影细胞、无保护剂的血影细胞及保护剂,用热重分析法及Karl-Fisher滴定法结合卡式炉法测定其残余水含量,同时用含水量固定的标准品(水分含量4.9%-5.2%)做检验,每组样品重复测量6次;分析各组之间的差异,比较冻干血影细胞及冻干红细胞残余水含量。结果热重分析法测定残余水含量(%):冻干红细胞、无保护剂冻干红细胞、冻干血影细胞、无保护剂冻干血影细胞、单纯冻干保护剂、标准品分别为19.01±2.18、4.60±0.78、18.95±1.89、4.87±1.01、16.12±1.04、5.28±0.16;冻干保护剂及含保护剂冻干红细胞的热重(TG)曲线走势接近,无保护剂冻干红细胞与前二者曲线走势差异明显;Karl-Fisher法测定残余水含量(%):冻干红细胞、无保护剂冻干红细胞、冻干血影细胞、无保护剂冻干血影细胞、单纯冻干保护剂、标准品分别为3.21±0.23、1.22±0.09、3.16±0.26、1.25±0.07、2.63±0.41、5.14±0.13。结论热重分析法测定含保护剂的冻干品残余水含量时的结果偏高,Karl-Fisher滴定法结合卡式炉法能够准确反映冻干红细胞的残余水含量;初步判断冻干红细胞残余水主要分布于细胞膜。

Objective To investigate the distribution of residual water in lyophilized erythrocytes and the effect of two different methods to determine the lyophilized red blood cell＇s residual water contents in order to provide basic data for further study. Methods Both RBCs and erythrocyte ghosts contained protective agent. Erythrocyte ghosts and pure protective agent were divided into five groups, and then underwent freeze-drying by freeze dryer under the same process at the same time. The residual water content were determined by thermogravimetric analysis and Karl Fisher titration. Water content in the standard group （4. 9%-5.2%） was also determined by those two methods as the sixth group. Each determination was repeated six times, while the results were statistically analyzed. Results The residual water contents of each group determined by thermogravimetric analysis were （ 19. 01±2. 18）%, （4. 60±0. 78）%, （ 18.95±1.89）%, （4. 87±1.01）%, （ 16. 12±1.04）%, （ 5.39±0. 20） %. The TG curve of freeze-dried protective agent was close to the TG curve of freeze-dried RBCs which contained the protective agent. Those two curves showed discrepancy to the TG curves of freeze-dried RBCs without protective agent. The residual water contents of each group determined by Karl Fisher titration were （ 3.21±0. 23） %, （ 1. 22±0. 09） %, （ 3.16±0. 26） %, （1.25±0.07） %, （ 16. 12± 1.04） %, （ 5.14±0. 13） %. Conclusion The result of two methods were reliable while water standard served as control. The Karl Fisher titration can determine the residual water content of freezingdried RBCs accurately. The residual water of freezing-dried RBCs is mainly distributed in the cell membrane.