Changes of phosphatidylserine distribution in human red blood cells during the process of loading sugars

The plasma membrane of red blood cells permits sugars to be loaded into the cytoplasm simply by incubation in a suitable buffer solution containing the sugar. This may provide some hope for the freeze-drying of human red blood cells. However, the effect of the loading process on red blood cells has not been fully investigated. The exposure of phosphatidylserine (PS) on the surface of the cell can be recognized by macrophages and result in shortened circulation in vivo. This study evaluates the effects of the concentration, the incubation time, and the temperature of exposure of human red blood cells to extracellular trehalose or glucose. Exposure of PS was demonstrated by annexin V labeling. It was shown that the efficiency of loading of glucose was significantly greater than that of trehalose. The loading efficiency of both sugars increased with increase in extracellular sugar concentration, prolongation of incubation time, and increase of incubation temperature. The percentages of cells with exposed PS and of damaged cells were dependent on the extracellular sugar concentration, the incubation time, and the temperature. With an extracellular glucose concentration of 0.8M, the percentage of cells with exposed PS was more than 80% and significantly higher than that of red blood cells loaded with trehalose (approximate 20%, P<0.01). As the incubation time was prolonged, the percentage of PS exposure and of damaged cells also increased. After incubation for 5h, the percentage of red cells with exposed PS following loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (40%, P<0.01). In addition, the incubation temperature had a major effect on PS exposure. The percentage of cells with PS exposure and the proportion of damaged cells increased with increase of incubation temperature. At 37 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (P<0.01). However, when the temperature was below 25 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose or trehalose were both less than 10%. In conclusion, the loading efficiency for glucose was higher than that for trehalose, but the lesser effect of trehalose on exposure of PS suggests that it can maintain the asymmetrical distribution of membrane phospholipids and the intracellular trehalose can increase the osmotic tolerance of cells.