This study was purposed to evaluate the effect of trehalose-loading on physiological and biochemistry properties of red blood cell (RBC) membrane. The samples were divided into the control group (RBC without trehalose loading) and the test group (RBC with trehalose loading). Osmotic fragility reaction was used to determine the osmotic fragility change of loaded RBC membrane in NaCl solution of different osmotic concentration. Flow cytometry and deformeter were used to assay the integrality and deformability of the RBC, respectively. The results showed that the NaCl solution osmotic concentrations were 160 mOsm and 121.4 mOsm, respectively when the haemolysis rate was 50% of the control group and the test group. Flow cytometry data demonstrated that incubation of RBC in a hypertonic trehalose solution resulted in a fraction of cells with different complexity that attached to little Annexin V-FITC, and that it could be removed by washing and resuspending the RBC in an iso-osmotic (300 mOsm PBS) medium. The deformability of the loaded RBC descend, the statistical difference was significant between control and test groups (P < 0.01). It is concluded that the membrane physiological and biochemistry stability and membrane integrality of RBC in a hyper osmotic pressure can be retained after trehalose loading.
Blood Preservation ; methods ; Cryopreservation ; methods ; Erythrocyte Membrane ; drug effects ; Erythrocytes ; drug effects ; Humans ; Osmotic Fragility ; drug effects ; Trehalose ; pharmacology

The effects of amphotericin B, an antifungal antibiotic, on erythrocyte volume and cation permeability were investigated by measuring the hematocrit, cell volume, cation content, fragility and osmotic behavior in rat erythrocytes, in vitro. 1. When erythrocytes were incubated in a Ringer solution containing amphotericin B (5-25 microgram/ml) the hematocrit and the cell volume increased, the effect being proportional to the concentration of the drug and the incubation time period. 2. Amphotericin B increased the Na content and decreased the K content of the erythrocyte. In normal Ringer solution (NaCl-Ringer)containing amphotericin B the magnitude of cellular Na gain was greater than that of K loss. Therefore, the total cellular cation content increased. On the other hand, when cells were incubated in the amphotericin B containing Ringer solution in which NaCl was replaced by Na2SO4 (Na2SO4-Ringer) the magnitude of cellular K loss exceeded that of cellular Na gain. Consequently, the total cellular cation content was reduced. 3. Amphotericin B increased cell volume (hematocrit) when erythrocytes were incubated in the Na2SO4-Ringer solution. 4. The fragility of erythrocytes increased when cells were preincubated in the amphotericin B containing normal Ringer solution, whereas it decreased in tile cells preincubated in the amphotericin B containing Na2SO4-Ringer solution. 5. The cell volume was linearly related to the reciprocal of medium osmolality(200 to 900 mOsm/kg H2O) in both NaCl-and Na2SO4-Ringer solutions, and the linearity was not altered by amphotericin B. The antibiotic did not change the slope of the correlation line (V vs. 1/OSM). It, however, increased the intercept of the line with the ordinate in normal Ringer solution and decreased that in the Na2SO4-Ringer solution. These results indicate that amphctericin B alters the cell volume by changing the permeability of Na and K across the membrane.
Amphotericin B/pharmacology* ; Animal ; Erythrocyte Volume/drug effects* ; Erythrocytes/analysis* ; In Vitro ; Osmotic Fragility/drug effects ; Potassium/blood* ; Rats ; Sodium/blood*

Though high concentration of glucose can benefit the survival of lyophilized human red blood cells, the high concentration of glucose can result in serious damage of red blood cells. This study was aimed to investigate the inhibitory effect of trehalose on damage of red blood cells induced by high concentration of glucose. After incubation with the high concentration of glucose buffer containing different concentrations of trehalose for three hours at 37 degrees C, the phosphatidylserine exposure and the osmotic fragility of cells were analyzed by flow cytometry and the lipid peroxidation of membrane was evaluated by TBA method. The results showed that the high concentration of glucose could lead to phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage which were dependent on the glucose concentrations and incubation temperature. However, trehalose could effectively prevent the phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage induced by high concentration glucose. With increase of the trehalose concentrations. As the trehalose concentration increases, the phosphatidylserine exposure, maloni-aldehyde concentration and cell debris rate decreased gradually. In conclusion, the high concentration of glucose can lead to phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage of red blood cells. However, trehalose can inhibit the damaging effects of high concentration of glucose on red blood cells, which may be useful for the application of sugars to lyophilization of red blood cells.
Blood Preservation ; methods ; Erythrocyte Membrane ; drug effects ; metabolism ; Erythrocytes ; drug effects ; Flow Cytometry ; Glucose ; adverse effects ; Humans ; Lipid Peroxidation ; drug effects ; Membrane Lipids ; metabolism ; Osmotic Fragility ; drug effects ; Phosphatidylserines ; pharmacology ; Trehalose ; pharmacology

The key points for better protection of trehalose in freeze-drying red blood cells (RBCs) are to resolve non-osmosis of trehalose to red blood cells and to make cytoplasmic trehalose to reach effective concentration. This study was aimed to investigate the regularity of loading RBCs with trehalose, screen out optimal loading condition and evaluate the effect of trehalose on physico-chemical parameters of RBCs during the period of loading. The cytoplasmic trehalose concentration in red blood cells, free hemoglobin and ATP level were determined at different incubation temperatures (4, 22 and 37 degrees C), different trehaolse concentrations (0, 200, 400, 600, 800 and 1000 mmol/L) and different incubation times (2, 4, 6, 8 and 10 hours), the cytoplasmic trehalose, free hemoglobin (FHb), hemoglobin (Hb) and mean corpuscular volume (MCV) in fresh RBCs and RBCs stored for 72 hours at 4 degrees C were compared, when loading condition was ensured. The results showed that with increase of incubation temperature, time and extracellular trehalose concentration, the loading of trehalose in RBCs also increased. Under the optimal loading condition, cytoplasmic trehalose concentration and free hemoglobin level of fresh RBCs and RBCs stored for 72 hours at 4 degrees C were 65.505 +/- 6.314 mmol/L, 66.2 +/- 5.002 mmol/L and 6.567 +/- 2.568 g/L, 16.168 +/- 3.922 g/L respectively. It is concluded that the most optimal condition of loading trehalose is that fresh RBCs incubate in 800 mmol/L trehalose solution for 8 hours at 37 degrees C. This condition can result in a efficient cytoplasmic trehalose concentration. The study provides an important basis for long-term preservation of RBCs.
Biological Transport, Active ; drug effects ; Blood Preservation ; methods ; Cryopreservation ; methods ; Cryoprotective Agents ; metabolism ; pharmacology ; Erythrocyte Membrane ; metabolism ; Erythrocytes ; Freeze Drying ; Humans ; Osmotic Fragility ; Temperature ; Time Factors ; Trehalose ; metabolism ; pharmacology