- 期刊
Conditions for Storage of Stroma-Free Hemoglobin Solutions for Fabricating Blood Substitutes
製造人工代血之血紅素保存條件之探討
摘要
以血紅素為基材之人工代血必須具備可大量生產及長期保存的特性，才能應付臨床上緊急情況下所需要的輸血問題。目前血紅素面臨無法長期保存的原因在於血紅素在有氧的情況下會自動氧化成變性血紅素(methemoglobin, metHb)，而失去攜氧功能。因此保存血紅素最大的目的就是抑制變性血紅素的生成。本研究分別就溫度、pH值、血紅素濃度、抗凍劑及冷凍回溫次數等不同保存條件下，探討對變性血紅素生成之影響。由本研究的實驗結果發現以兩水相萃取法得到的血紅素溶液純度相當高。當血紅素保存在4℃或-20℃條件下，變性血紅素含量僅能維持2個月不增加，爾後隨保存時間的增長，變性血紅素的含量明顯地有增加的趨勢。至於保存溶液之pH值的影響，我們發現當溶液pH值低於血紅素之等電點時，會改變血紅素上的胺基酸鏈帶電性，而影響彼此間的作用力，造成血紅素構形不穩定，而產生變性血紅素。同樣地，我們也發現當保存血紅素的濃度越高時，產生變性血紅素的含量也越高。此外，經過冷凍-回溫的過程，因為冰晶的形成及pH值的下降等因素，容易造成血紅素的析出及變性血紅素的生成，因此必須加入glucose或sucrose等抗凍劑，以減少對血紅素的傷害。我們也同時發現在了-80℃的保存條件下，即使未添加抗凍劑即能有良好的保存效果且至少可保存六個月以上。
並列摘要
The stroma-free hemoglobin (Hb) solutions used for fabricating blood substitutes must be stable for a long period of time during storage and be available when massive clinical transfusions are required. One of the major problems in the long-term storage of Hb is its susceptibility to autoxidation, and thus becomes denatured (methemoglobin, metHb) and loses its capability to bind oxygen reversibly. In the study, porcine stroma-free Hb solutions were prepared by the aqueous two-phase system. The effects of varying conditions (temperature, pH, Hb concentration, cryoprotectant, and frequency of freeze-and-thaw process) on the extent of metHb formation during storage of Hb solutions were investigated. The results demonstrated that the stroma-free Hb solution obtained by the aqueous two-phase system was highly pure. The stroma-free Hb solutions can be safely stored at 4℃ or -20℃ without denaturation only for 2 months. Afterward, deterioration of Hb started to occur as evidenced by the increase in the metHb content. The extent of metHb formation increased significantly when the pH value of the storage solution is lower than the isoelectric point of Hb. Additionally, the extent of metHb formation increased with increasing the Hb concentration. A higher frequency of freeze-and-thaw process induced a greater extent of metHb formation. However, addition of cryoprotectant in the storage solution may inhibit the extent of metHb formation. In contrast, the stroma-free Hb solution can be safely stored at -80℃ without formation of metHb for at least 6 months.