Improved preservation of human red blood cells by lyophilization

The lyophilization of human red blood cells has important implications for blood transfusion in clinical medicine. In this study, sugars, human serum albumin, polyvinylpyrrolidone, and dimethyl sulfoxide were used as protective reagents for the lyophilization of red blood cells. Freezing temperature, shelf temperature, and the rehydration conditions were optimized. The results showed that extracellular disaccharides, especially trehalose, did not increase the recovery of hemoglobin. However, when the concentration of human serum albumin was higher than 25%, it had a considerable protective effect on the recovery of lyophilized red blood cells; the cellular hemoglobin recovery was over 70%, which was significantly higher than that in the group without human serum albumin (P<0.01). As the concentration of polyvinylpyrrolidone was increased, the extent of vitrification also increased. But when the concentration of polyvinylpyrrolidone was over 40%, the resulting concentration of free hemoglobin was over 1g/L, which was significantly higher than that with 40% (P<0.01). When lyophilization was carried out after freezing at different temperatures, the recovery of cells and hemoglobin was 70-80% and there were no significant differences among the five groups. When the shelf temperature was higher than -30 degrees C, the samples were partly collapsed, but when the shelf temperature was lower than -30 degrees C, the recovery of cells in the -40 and -45 degrees C groups was significantly higher than in the -30 and -35 degrees C groups (P<0.05). The recovery of cells and hemoglobin after lyophilization and rehydration in solutions containing low concentrations of polymers was over 80%, which is significantly higher than the other groups (P<0.01). In addition, when the temperature was higher than 25 degrees C, the concentration of free hemoglobin was significantly lower than it was at 4 degrees C (P<0.01). In conclusion, our study showed the lyophilization of red blood cells is feasible. Disaccharides have no protective effect on lyophilized cells when they are only extracellular and extensive vitrification may be not beneficial. Although the recovery of cells after lyophilization and rehydration by our method was over 70%, the ultrastructure of the cells may be compromised and some hemolysis does still exist. Further research is required.     

Phospholipid vesicles increase the survival of freeze-dried human red blood cells

In a previous report [Z. T?r?k, G. Satpathy, M. Banerjee, R. Bali, E. Little, R. Novaes, H. Van Ly, D. Dwyre, A. Kheirolomoom, F. Tablin, J.H. Crowe, N.M. Tsvetkova, Preservation of trehalose loaded red blood cells by lyophilization, Cell Preservation Technol. 3 (2005) 96-111.], we presented a method for preserving human red blood cells (RBCs) by loading them with trehalose and then freeze-drying. We have now improved that method, based on the discovery that addition of phospholipid vesicles to the lyophilization buffer substantially reduces hemolysis of freeze-dried RBCs after rehydration. The surviving cells synthesize 2,3-DPG, have low levels of methemoglobin, and have preserved morphology. Among the lipid species we studied, unsaturated PCs were found to be most effective in suppressing hemoglobin leakage. RBC-vesicle interactions depend on vesicle size and structure; unilamellar liposomes with average diameter of less than 300 nm were more effective in reducing the hemolysis than multilamellar vesicles. Trehalose loaded RBCs demonstrated high survival and low levels of methemoglobin during 10 weeks of storage at 4 degrees C in the dry state when lyophilized in the presence of liposomes.     

Temperature dependence of the survival of human erythrocytes frozen slowly in various concentrations of glycerol.

One widely accepted explanation of injury from slow freezing is that damage results when the concentration of electrolyte reaches a critical level in partly frozen solutions during freezing. We have conducted experiments on human red cells to further test this hypothesis. Cells were suspended in phosphate-buffered saline containing 0-3 M glycerol, held for 30 min at 20 degrees C to permit solute permeation, and frozen at 0.5 or 1.7 degrees C/min to various temperatures between -2 and -100 degrees C. Upon reaching the desired minimum temperature, the samples were warmed at rates ranging from 1 to 550 degrees C/min and the percent hemolysis was determined. The results for a cooling rate of 1.7 degrees C/min indicate the following: (a) Between 0.5 and 1.85 M glycerol, the temperature yielding 50% hemolysis (LT50) drops slowly from -18 to -35 degrees C. (b) The LT50's over this range of concentrations are relatively independent of warming rate. (c) With glycerol concentrations of 1.95 and 2.0 M, the LT50 drops abruptly to -60 degrees C and to below -100 degrees C, respectively, and becomes dependent on warming rate. The LT50 is lower with slow warming at 1 degree C/min than with rapid. With still higher concentrations (2.5 and 3.0 M), there is no LT50, i.e., more than 50% of the cells survive freezing to-100 degrees C. Results for cooling at 0.5 degrees C/min in 2 M glycerol were similar except that the LT50s were some 10-20 degrees C higher. A companion paper (Rall et al., Biophys. J. 23:101-120, 1978) examines the relation between survival and the concentrations of salts produced during freezing.     

Further characterization of the effects of alpha-1-acid glycoprotein on the passage of human erythrocytes through micropores

Effects of human alpha-1-acid glycoprotein (AG) on the passage of human red blood cell(s) (RBC) through membrane filters with micropores were examined in vitro. RBCs, with a mean major diameter of 7.2 micron, that had been suspended at 1% in physiological phosphate-buffered saline (PBS), were filtered through membrane filters of various pore diameters under positive pressure. The percentages of cells that passed through the micropores and of cells hemolyzed during filtration were determined. RBCs suspended in PBS did not pass through micropores that had an average pore diameter of 3 micron; instead hemolysis took place. Neither temperature nor applied pressure affected cell passage; but when AG at 0.1 mg/ml or above was added to an RBC-suspension, it promoted cell passage through the 3 micron micropores and reduced the degree of hemolysis. The effects of AG were dose dependent up to a concentration of 0.5 mg/ml. The addition of AG to an RBC-suspension that contained 90% human serum had the same additive effects. Washing AG-treated RBCs with normal saline produced a marked decrease in cell passage through the 3 micron pores. Fluorescence antibody staining revealed that the exogenous AG was localized on the membrane surface of the RBCs. Our results suggest that the AG bound to the surface of the RBCs acts as a lubricant between the RBCs and the wall of the micropore; this would facilitate RBC-passage through the micropores.     

Antioxidant Status and Susceptibility of Sickle Erythrocytes to Oxidative and Osmotic Stress

The purpose of this study was to determine if differences in antioxidant status between the red blood cells (RBCs) of sickle cell anemia (SCA) patients and controls are responsible for the differential responses to oxidative and osmotic stress-induced hemolysis. Susceptibility to hemolysis was examined by incubating oxygenated and deoxygenated RBCs at 37°C with 73 mM 2,2' azobis (2-amidinopropane) HC1 (AAPH), a peroxyl radical generator, for up to 3.5 hours. The ability of RBCs to maintain membrane integrity under osmotic stress was determined over a range of diluted saline-phosphate buffer. Sickled RBCs showed a lesser degree of AAPH-induced hemolysis than control groups and were more resistant to osmotic stress-induced hemolysis. SCA patients had higher levels of RBC vitamin E and RBC lipids, but lower RBC GSH, plasma lipids and plasma carotenes than those of the hospital controls. No significant differences were observed in the levels of retinol, vitamin C, vitamin E, MDA and conjugated dienes in plasma, or the levels of MDA and conjugated dienes in RBCs. The results obtained suggest that the differences in antioxidant status between sickled RBCs and controls do not appear to be responsible for their different susceptibility to oxidative or osmotic stress-induced hemolysis observed.     

Deformability and stability of erythrocytes in high-frequency electric fields down to subzero temperatures.

High-frequency electric fields can be used to induce deformation of red blood cells. In the temperature domain T = 0 degrees to -15 degrees C (supercooled suspension) and for 25 degrees C this paper examines for human erythrocytes (discocytes, young cell population suspended in a low ionic strength solution with conductivity sigma(25 degrees) = 154 microS/cm) in a sinusoidal electric field (nu = 1 MHz, E0 = 0-18 kV/cm) the following properties and effects as a function of field strength and temperature: 1) viscoelastic response, 2) (shear) deformation (steady-state value obtained from the viscoelastic response time), 3) stability (by experimentally observed breakdown of cell polarization and hemolysis), 4) electrical membrane breakdown and field-induced hemolysis (theoretical calculations for ellipsoidal particles), and 5) mechanical hemolysis. The items 2-4 were also examined for the frequency nu = 100 kHz and for a nonionic solution of very low conductivity (sigma(25 degrees) = 10 microS/cm) to support our interpretations of the results for 1 MHz. Below 0 degrees C with decreasing temperature the viscoelastic response time tau(res)(T) for the cells to reach steady-state deformation values d(infinity,E) increases and the deformation d(infinity,E)(T) decreases strongly. Both effects are especially high for low field strengths. The longest response time of approximately 30 s was obtained for -15 degrees C and small deformations. For 1 MHz the cells can be highly elongated up to 2.3 times their initial diameter a0 for 25 degrees and 0 degrees C, 2.1a0 for -10 degrees C and still 1.95a0 for -15 degrees C. For T > or = 0 degrees C the deformation is limited by hemolysis of the cells, which sets in for E0(lysis)(25 degrees) approximately 8 kV/cm and E0(lysis)(0 degrees) approximately 14 kV/cm. These values are approximately three times higher than the corresponding calculated critical field strengths for electrically induced pore formation. Nevertheless, the observed depolarization and hemolysis of the cells is provoked by electrical membrane breakdown rather than by mechanical forces due to the high deformation. For the nonionic solution, where no electrical breakdown is expected in the whole range for E0, the cells can indeed be deformed to even higher values with a low hemolytic rate. Below 0 degrees C we observe no hemolysis at all, not even for the frequency 100 kHz, where the cells hemolyze at 25 degrees C for the much lower field strength E0(lysis) approximately 2.5 kV/cm. Obviously, pore formation and growth are weak for subzero temperatures.     

海藻糖载入红细胞及其冷冻干燥的实验研究

冷冻干燥保存是长期保存人体红细胞的理想方案之一。冻干保护剂海藻糖渗入细胞内后,对细胞膜和细胞内物质有保护作用,其中的一个作用是增加细胞质的浓度,使冻干过程容易形成稳定的玻璃态。应用高渗法处理红细胞,通过考察胞内海藻糖含量、红细胞冻干后的存活率、腺苷三磷酸酶(ATPase)、超氧化物歧化酶(SOD)活力以及细胞形态变化,研究胞内海藻糖含量对红细胞冻干后活性的影响。结果显示:海藻糖对红细胞冻干具有明显的保护作用,随胞内海藻糖浓度升高,其保护性能逐渐增强;43.8mmol/L的胞内海藻糖浓度对红细胞保护最好,细胞存活率达到53.6%,形态保持良好,ATP和SOD活力均在正常的范围内。     

Protective effects of flavonols and their glycosides against free radical-induced oxidative hemolysis of red blood cells

The in vitro oxidative hemolysis of human red blood cells (RBCs) was used as a model to study the free radical-induced damage of biological membranes and the protective effect of flavonols and their glycosides (FOHs), i.e., myricetin (MY), quercetin (Q), morin (MO), kaempferol (K), rutin (R), quercetin galactopyranoside (QG), quercetin rhamnopyranoside (QR), and kaempferol glucopyranoside (KG). The hemolysis of RBCs was induced by a water-soluble free radical initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). It was found that addition of AAPH at 37 degrees C to the suspension of RBCs caused fast hemolysis after a short period of inhibition period, and addition of FOHs significantly suppressed the hemolysis. The FOHs (MY, Q, R, QG and QR) which bears an ortho-dihydroxyl functionality showed much more effective anti-hemolysis activity than that of the other FOHs (MO, K and KG) bearing no such functionality.     

Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes

Abstract

Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol^-1. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.     

The effect of initial tonicity on freeze/thaw injury to human red cells suspended in solutions of sodium chloride

Human red blood cells, suspended in solutions of sodium chloride, have been frozen to temperatures between -2 and -14 degrees C and thawed, and the extent of hemolysis was measured. In parallel experiments, red cells were exposed to similar cycles of change in the composition of the suspending solution, but by dialysis at 21 degrees C. The tonicity of the saline in which the cells were initially suspended was varied between 0.6x isotonic and 4x isotonic; some samples from each experimental treatment were returned to isotonic saline before hemolysis was measured. It was found that the tonicity of the saline used to suspend the cells for the main body of the experiment affected the amount of hemolysis measured: raising the tonicity from 0.6x to 1x to 2x reduced hemolysis, both in the freezing and in the dialysis experiments, whereas raising the tonicity further to 4x reversed that trend. There was little difference between the freeze/thaw and the dialysis treatments for the cells suspended in 1x or 2x saline, whether or not the cells were returned to isotonic conditions. However, the cells suspended in 0.6x saline showed greater damage from freezing and thawing than from the comparable change in the composition of the solution, whether or not they were returned to isotonic conditions. Cells that were suspended in 4x saline and exposed to changes in salt concentration by dialysis showed less hemolysis when they were assayed in the 4x solution than cells that had received the comparable freezing/thaw treatment, but when the experiment included a return to isotonicity, the two treatments gave similar results. Returning the cells to isotonic saline had a negligible affect on the cells in 0.6x and 1x saline, but caused considerable hemolysis in the 2x and 4x samples, more so after dialysis than after freezing and thawing. We conclude that cells suspended in 0.6x and 4x saline behave differently from cells suspended in 1x and 2x saline and hence that cells suspended in a range of solutions of differing initial tonicity should not be treated as a homogeneous population. We argue that an effect of the unfrozen fraction of water (U) cannot be distinguished, within the framework of these freeze/thaw experiments alone, from an effect of initial tonicity, and that the biphasic nature of the correlation between haemolysis and U makes a causal connection improbable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thermal shock hemolysis in human red cells. I. The effects of temperature, time, and osmotic stress

Thermal shock is a form of hemolysis which occurs in human red cells exposed to greater than a critical level of osmotic stress of 1.4 Osm and subsequently cooled from above about 12 degrees C to below that temperature. Higher concentrations and higher cooling rates each increase the amount of hemolysis, within limits. Incubation for varying periods in hypertonic solutions and varying temperatures of incubation affect the amount of thermal shock. The effect of cooling rate on thermal shock is independent of the period of exposure to hypertonic solutions. Thermal shock is not the cause of freezing injury in human red cells, at least above -10 degrees C.     

Effect of phosphoenolpyruvate on metabolic and morphological recovery of red cells after prolonged liquid storage and subsequent freezing in glycerol medium.

The present study was designed to determine the effects of (i) phosphoenolpyruvate (PEP) treatment of red blood cells (RBCs) previously cold stored for a prolonged period in a liquid medium and (ii) the freezing of these treated cells in glycerol. RBCs stored for 21 days at 4 degrees C were incubated for 30 min at 37 degrees C with rejuvenant solution containing 50 mM PEP, 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, and 1 mM adenine, pH 6.0, and then frozen at -80 degrees C for 4 weeks. Red cell recovery as frozen and thawed red cells (FTRCs) after deglycerolization was increased to 80 +/- 4% compared to 43 +/- 9% in units without rejuvenation; the percentage of PEP-treated FTRCs was similar to the percentage of FTRCs recovered from fresh RBCs within 5 days after donation. Incubation of RBCs with PEP solution restored ATP and 2,3-DPG to levels seen in fresh RBCs, and also facilitated transformation of crenated RBCs to discocytes. These results indicate that maximum recovery of viable RBCs can be attained when FTRCs are processed from cells stored in the frozen state after they had been rejuvenated with PEP even after prolonged liquid storage.     

Role of membrane thermotropic properties on hypotonic hemolysis and hypertonic cryohemolysis of human red blood cells

The hypothesis of a correlation between the effects of temperature on red blood cells hypotonic hemolysis and hypertonic cryohemolysis and two thermotropic structural transitions evidenced by EPR studies has been tested. Hypertonic cryohemolysis of red blood cells shows critical temperatures at 7 degrees C and 19 degrees C. In hypotonic solution, the osmotic resistance increases near 10 degrees C and levels off above 20 degrees C. EPR studies of red blood cell membrane of a 16-dinyloxyl stearic acid spin label show, in the 0-50 degrees C range, the presence of three thermotropic transitions at 8, 20, and 40 degrees C. Treatments of red blood cells with acidic or alkaline pH, glutaraldehyde, and chlorpromazine abolish hypertonic cryohemolysis and reduce the effect of temperature on hypotonic hemolysis. 16-Dinyloxyl stearic acid spectra of red blood cells treated with glutaraldehyde and chlorpromazine show the disappearance of the 8 degrees C transition. Both the 8 degrees C and the 20 degrees C transitions were abolished by acidic pH treatment. The correlation between the temperature dependence of red blood cell lysis and thermotropic breaks might be indicative of the presence of structural transitions producing areas of mismatching between differently ordered membrane components where the osmotic resistance is decreased.     

Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes

Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol?1. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.     

Trifluoperazine inhibits Sendai virus-induced hemolysis

Sendai virus-induced hemolysis, a manifestation of virus-red cell fusion, is inhibited by exposure of the virus to 50 microM and higher concentrations of trifluoperazine. Trifluoperazine does not disrupt the virus, since trifluoperazine-treated virus with no hemolytic activity sediments slightly faster than untreated virus on sucrose density gradients and contains viral proteins in proportions characteristic of untreated virus. Trifluoperazine affects the fusion protein to a greater extent than the hemagglutinin, since trifluoperazine-treated virus with no hemolytic activity is as active or nearly as active in agglutinating red cells. The partition coefficient of trifluoperazine between the virus membrane and buffer is lower at 4 degrees C than, but the same at 37 degrees C, as that between the red cell membrane and buffer. Nevertheless, virus-independent red cell lysis and inactivation of virus-mediated hemolysis occur when the red cell and viral membranes, respectively, contain similar concentrations of trifluoperazine. Furthermore, 13-28% more trifluoperazine is necessary to achieve either effect at 4 degrees C or at 25 degrees C than at 37 degrees C. Changes in the surface activity of trifluoperazine do not explain these results, insofar as the critical micellar concentration of (0.75 mM) and maximal reduction in surface tension by (40 dyn/cm) trifluoperazine are the same at 25 degrees C and 37 degrees C. The fluorescence of viral tryptophan decreases by approx. 25% when viral hemolysis is inactivated by trifluoperazine, by trypsin treatment or by heating at 100 degrees C for 5 min.     

Temperature transition of human hemoglobin at body temperature: effects of calcium

We studied the effects of calcium ion concentration on the temperature dependence of rheological behavior of human red blood cells (RBCs) and concentrated hemoglobin solutions. Our previous study (G. M. Artmann, C. Kelemen, D. Porst, G. Büldt, and S. Chien, 1998, Biophys. J., 75:3179-3183) showed a critical temperature (Tc) of 36.4 +/- 0.3 degrees C at which the RBCs underwent a transition from non-passage to passage through 1.3 microm micropipettes in response to an aspiration pressure of -2.3 kPa. An increase in intracellular Ca2+ concentration by using the ionophore A23187 reduced the passability of intact RBCs through small micropipettes above T(c); the micropipette diameter needed for >90% passage increased to 1.7 microm. Viscometry of concentrated hemoglobin solutions (45 and 50 g/dl) showed a sudden viscosity transition at 36 +/- 1 degrees C (Tc(eta)) at all calcium concentrations investigated. Below Tc(eta), the viscosity value of the concentrated hemoglobin solution at 1.8 mM Ca(2+) was higher than that at other concentrations (0.2 microM, 9 mM, and 18 mM). Above Tc(eta), the viscosity was almost Ca2+ independent. At 1.8 mM Ca2+ and 36 +/- 1 degrees C, the activation energy calculated from the viscometry data showed a strong dependence on the hemoglobin concentration. We propose that the transition of rheological behavior is attributable to a high-to-low viscosity transition mediated by a partial release of the hemoglobin-bound water.     

Rapid removal of glycerol from frozen‐thawed red blood cells

The storage of red blood cells (RBCs) in a refrigerated state allows a shelf life of a few weeks, whereas RBCs frozen in 40% glycerol have a shelf life of 10 years. Despite the clear logistical advantages of frozen blood, it is not widely used in transfusion medicine. One of the main reasons is that existing post‐thaw washing methods to remove glycerol are prohibitively time consuming, requiring about an hour to remove glycerol from a single unit of blood. In this study, we have investigated the potential for more rapid removal of glycerol. Using published biophysical data for human RBCs, we mathematically optimized a three‐step deglycerolization process, yielding a procedure that was less than 32 s long. This procedure was found to yield 70% hemolysis, a value that was much higher than expected. Consequently, we systematically evaluated three‐step deglycerolization procedures, varying the solution composition and equilibration time in each step. Our best results consisted of less than 20% hemolysis for a deglycerolization time of 3 min, and it is expected that even further improvements could be made with a more thorough optimization and more reliable biophysical data. Our results demonstrate the potential for significantly reducing the deglycerolization time compared with existing methods. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:609–620, 2013     

Structure-dependent and receptor-independent increase in osmotic fragility of rat erythrocytes by short-chain fatty acids

We examined short-chain fatty acids (SCFAs) with 1 (C1) to 5 (C5) carbon atoms for osmotic fragility (OF) in isolated red blood cells (RBCs) in rats. The RBCs were used as prototypical plasma membrane model. The dense packed RBC was incubated in a phosphate-NaCl buffer solution containing each SCFA at 0 to 100 mM. The RBC suspensions were transferred into the OF test tubes containing NaCl from 0.2 to 0.9%. The hemoglobin concentration was determined and the EC50 in hemolysis was calculated. The OF in RBCs was dose-dependently increased by exposure to SCFAs, except for C1, with an increasing number of carbon atoms. Branched-chain fatty acids (isomers of C4 and C5) have a smaller effect on OF than straight-chain fatty acids (C4 and C5). The SCFA-induced increases in OF were not affected by pretreatment of RBCs with trypsin. The response of the RBC membrane to SCFAs depends on their concentration, carbon chain length and chain structure (straight or branched). The SCFAs probably disturb the lipid bilayer of the RBC membrane and result in a decrease in osmotic resistance. The plasma membrane in rat RBCs could respond to the structure of the SCFAs in detail by using the OF as an indicator.     

Structure-dependent and receptor-independent increase in osmotic fragility of rat erythrocytes by short-chain fatty acids

We examined short-chain fatty acids (SCFAs) with 1 (C1) to 5 (C5) carbon atoms for osmotic fragility (OF) in isolated red blood cells (RBCs) in rats. The RBCs were used as prototypical plasma membrane model. The dense packed RBC was incubated in a phosphate-NaCl buffer solution containing each SCFA at 0 to 100 mM. The RBC suspensions were transferred into the OF test tubes containing NaCl from 0.2 to 0.9%. The hemoglobin concentration was determined and the EC50 in hemolysis was calculated. The OF in RBCs was dose-dependently increased by exposure to SCFAs, except for C1, with an increasing number of carbon atoms. Branched-chain fatty acids (isomers of C4 and C5) have a smaller effect on OF than straight-chain fatty acids (C4 and C5). The SCFA-induced increases in OF were not affected by pretreatment of RBCs with trypsin. The response of the RBC membrane to SCFAs depends on their concentration, carbon chain length and chain structure (straight or branched). The SCFAs probably disturb the lipid bilayer of the RBC membrane and result in a decrease in osmotic resistance. The plasma membrane in rat RBCs could respond to the structure of the SCFAs in detail by using the OF as an indicator.     

Loading red blood cells with trehalose: a step towards biostabilization

A method for freeze-drying red blood cells (RBCs) while maintaining a high degree of viability has important implications in blood transfusion and clinical medicine. The disaccharide trehalose, found in animals capable of surviving dehydration can aid in this process. As a first step toward RBC preservation, we present a method for loading RBCs with trehalose. The method is based on the thermal properties of the RBC plasma membranes and provides efficient uptake of the sugar at 37 degrees C in a time span of 7 h. The data show that RBCs can be loaded with trehalose from the extracellular medium through a combination of osmotic imbalance and the phospholipid phase transition, resulting in intracellular trehalose concentrations of about 40 mM. During the loading period, the levels of ATP and 2,3-DPG are maintained close to the levels of fresh RBCs. Increasing the membrane fluidity through the use of a benzyl alcohol results in a higher concentration of intracellular trehalose, suggesting the importance of the membrane physical state for the uptake of the sugar. Osmotic fragility data show that trehalose exerts osmotic protection on RBCs. Flow cytometry data demonstrate that incubation of RBCs in a hypertonic trehalose solution results in a fraction of cells with different complexity and that it can be removed by washing and resuspending the RBCs in an iso-osmotic medium. The data provide an important first step in long-term preservation of RBCs.