The effect of ionic redistributions on the microwave dielectric response of cytosol water upon glucose uptake.

The sensitivity of cytosol water's microwave dielectric (MD) response to D-glucose uptake in Red Blood Cells (RBCs) allows the detailed study of cellular mechanisms as a function of controlled exposures to glucose and other related analytes like electrolytes. However, the underlying mechanism behind the sensitivity to glucose exposure remains a topic of debate. In this research, we utilize MDS within the frequency range of 0.5-40 GHz to explore how ionic redistributions within the cell impact the microwave dielectric characteristics associated with D-glucose uptake in RBC suspensions. Specifically, we compare glucose uptake in RBCs exposed to the physiological concentration of Ca2+ vs. Ca-free conditions. We also investigate the potential involvement of Na+/K+ redistribution in glucose-mediated dielectric response by studying RBCs treated with a specific Na+/K+ pump inhibitor, ouabain. We present some insights into the MD response of cytosol water when exposed to Ca2+ in the absence of D-glucose. The findings from this study confirm that ion-induced alterations in bound/bulk water balance do not affect the MD response of cytosol water during glucose uptake.

Hemoglobin Binding to the Red Blood Cell (RBC) Membrane Is Associated with Decreased Cell Deformability.

The deformability of red blood cells (RBCs), expressing their ability to change their shape as a function of flow-induced shear stress, allows them to optimize oxygen delivery to the tissues and minimize their resistance to flow, especially in microcirculation. During physiological aging and blood storage, or under external stimulations, RBCs undergo metabolic and structural alterations, one of which is hemoglobin (Hb) redistribution between the cytosol and the membrane. Consequently, part of the Hb may attach to the cell membrane, and although this process is reversible, the increase in membrane-bound Hb (MBHb) can affect the cell's mechanical properties and deformability in particular. In the present study, we examined the correlation between the MBHb levels, determined by mass spectroscopy, and the cell deformability, determined by image analysis. Six hemoglobin subunits were found attached to the RBC membranes. The cell deformability was negatively correlated with the level of four subunits, with a highly significant inter-correlation between them. These data suggest that the decrease in RBC deformability results from Hb redistribution between the cytosol and the cell membrane and the respective Hb interaction with the cell membrane.

Red Blood Cell Deformability Is Expressed by a Set of Interrelated Membrane Proteins.

Red blood cell (RBC) deformability, expressing their ability to change their shape, allows them to minimize their resistance to flow and optimize oxygen delivery to the tissues. RBC with reduced deformability may lead to increased vascular resistance, capillary occlusion, and impaired perfusion and oxygen delivery. A reduction in deformability, as occurs during RBC physiological aging and under blood storage, is implicated in the pathophysiology of diverse conditions with circulatory disorders and anemias. The change in RBC deformability is associated with metabolic and structural alterations, mostly uncharacterized. To bridge this gap, we analyzed the membrane protein levels, using mass spectroscopy, of RBC with varying deformability determined by image analysis. In total, 752 membrane proteins were identified. However, deformability was positively correlated with the level of only fourteen proteins, with a highly significant inter-correlation between them. These proteins are involved in membrane rafting and/or the membrane-cytoskeleton linkage. These findings suggest that the reduction of deformability is a programmed (not arbitrary) process of remodeling and shedding of membrane fragments, possibly mirroring the formation of extracellular vesicles. The highly significant inter-correlation between the deformability-expressing proteins infers that the cell deformability can be assessed by determining the level of a few, possibly one, of them.

The inhibition of glucose uptake to erythrocytes: microwave dielectric response.

Dielectric spectroscopy has been used in the study and development of non-invasive glucose monitoring (NIGM) sensors, including the range of microwave frequencies. Dielectric relaxation of red blood cell (RBC) cytosolic water in the microwave frequency band has been shown to be sensitive to variations in the glucose concentration of RBC suspensions. It has been hypothesized that this sensitivity stems from the utilization of D-glucose by RBCs. To verify this proposition, RBCs were pretreated with inhibitors of D-glucose uptake (cytochalasin B and forskolin). Then their suspensions were exposed to different D-glucose concentrations as measured by microwave dielectric spectroscopy (MDS) in the 500 MHz-40 GHz frequency band. After incubation of RBCs with either inhibitor, the dielectric response of water in the cytoplasm, and specifically its relaxation time, demonstrated minimal sensitivity to the change of D-glucose concentration in the medium. This result allows us to conclude that the sensitivity of MDS to glucose uptake is associated with variations in the balance of bulk and bound RBC cytosolic water due to intracellular D-glucose metabolism, verifying the correctness of the initial hypothesis. These findings represent a further argument to establish the dielectric response of water as a marker of glucose variation in RBCs.

Unit-to-unit variability in the deformability of red blood cells.

BACKGROUND: In blood banking practice, the storage duration is used as the primary criterion for inventory management, and usually, the packed red blood cells (PRBC) units are supplied primarily according to first-in-first-out (FIFO) principle. However, the actual functionality of individual PRBC units is mostly ignored. One of the main features of the RBCs not accounted for under this approach is the deformability of the red cells, i.e., their ability to affect the recipients' blood flow. The objective of the study was to analyze unit-to-unit variability in the deformability of PRBCs during their cold storage. METHODS: RBC samples were obtained from twenty leukoreduced PRBC units, stored in SAGM. The deformability of cells was monitored from the day of donation throughout 42 days. RBC deformability was determined using the computerized cell flow-properties analyzer (CFA) based on cell elongation under a shear stress of 3.0 Pa, expressed by the elongation-ratio (ER). The image analysis determines the ER for each cell and provides the ER distribution in the population of 3000-6000 cells. RESULTS: The deformability of freshly-collected RBCs exhibited marked variability already on the day of donation. We also found that the aging curve of PRBC deformability varies significantly among donors. SIGNIFICANCE: The present study has demonstrated that storage duration is only one of the factors, and seemingly not even the major one, affecting the PRBCs functionality. Therefore, the FIFO approach is not sufficient for assessing the potential transfusion outcome, and the PRBC functionality should be determined explicitly for each unit.

Preparation of packed red blood cell units in the blood bank: Alteration in red blood cell deformability.

BACKGROUND: Donated blood is stored in the blood bank as packed red blood cell units. In the process of packed cells preparation, the red blood cells (RBCs) are subjectedto high level of shear stress, which can induce alterations in their properties. In the present study, we examined the effect of packed RBCs preparation (which included leuko-filtration) on red cell deformability. METHODS: Blood samples were collected from 25 healthy donors and from corresponding units of packed RBCs. The portion of undeformable cells (%UDFC) was determined for each sample. RESULTS: The median value of %UDFC was equal to 6.75 ± 0.70 %, for freshly-donated RBCs, and to 6.36 ± 0.51 %, for packed cells. Wherein, %UDFC may increase or decrease following packed cells preparation, depending upon the initial portion of undeformable cells. CONCLUSION: Likely, exposure of RBCs to high shear stress, during packed cells preparation, induces opposing effects: (a) removal/destruction of rigid (undeformable) cells, thereby reducing their total amount (i.e., decreasing the %UDFC) on the one hand, and (b) mechanical damage to the cell membrane and subsequent reduction of the cell deformability (thereby increasing the %UDFC) on the other. As a consequence, the final impact of packed cells preparation is primarily determined by the initial state of erythrocytes in the blood of the donor.

Oxygenation state of hemoglobin defines dynamics of water molecules in its vicinity.

This study focuses on assessing the possible impact of changes in hemoglobin (Hb) oxygenation on the state of water in its hydration shell as it contributes to red blood cell deformability. Microwave Dielectric Spectroscopy (MDS) was used to monitor the changes in interactions between water molecules and Hb, the number of water molecules in the protein hydration shell, and the dynamics of pre-protein water in response to the transition of Hb from the tense (T) to the relaxed (R) state, and vice versa. Measurements were performed for Hb solutions of different concentrations (5 g/dl-30 g/dl) in phosphate-buffered saline buffer. Cole-Cole parameters of the main water relaxation peak in terms of interactions of water molecules (dipole-dipole/ionic dipole) during the oxygenation-deoxygenation cycle were used to analyze the obtained data. The water mobility-represented by α as a function of ln τ-differed dramatically between the R (oxygenated) state and the T (deoxygenated) state of Hb at physiologically relevant concentrations (30 g/dl-35 g/dl or 4.5 mM-5.5 mM). At these concentrations, oxygenated hemoglobin was characterized by substantially lower mobility of water in the hydration shell, measured as an increase in relaxation time, compared to deoxyhemoglobin. This change indicated an increase in red blood cell cytosolic viscosity when cells were oxygenated and a decrease in viscosity upon deoxygenation. Information provided by MDS on the intraerythrocytic water state of intact red blood cells reflects its interaction with all of the cytosolic components, making these measurements powerful predictors of the changes in the rheological properties of red blood cells, regardless of the cause.

Inter-donor variability in deformability of red blood cells in blood units.

OBJECTIVE: This study aimed to examine the donor-to-donor variability in the deformability of red blood cells (RBCs) from freshly collected blood donations (F-RBC) and packed RBCs. BACKGROUND: Packed RBCs are supplied for transfusion by the first-in-first-out (FIFO) criterion, assuming that their quality is the same for packed RBCs with equal storage duration. To challenge this notion, we determined the deformability of F-RBC and packed RBCs stored for different durations. METHODS: Three RBC groups were employed: A. 79 samples of F-RBC; B. 76 samples of packed RBC units, randomly used for transfusion at different storage durations; and C. 65 samples of outdated packed RBCs stored for 35 to 37 days. All packed RBC units were non-leukofiltrated and stored in Citrate-phosphate-dextrose solution with adenine (CPDA-1). RBC deformability was determined using a computerised cell-flow properties analyser, which monitors the shape change of cells directly visualised in a narrow-gap flow chamber and provides the cells' deformability distribution in a large RBC population. RESULTS: The F-RBC deformability exhibited a wide-range inter-donor variability. The cold storage of packed RBCs exerted a mild reduction of deformability, which became significant, compared to the initial inter-donor variability, only after 3 weeks of storage. CONCLUSION: Packed RBCs are generally supplied for transfusion by the FIFO criterion based on the assumption that the storage duration is a key factor of RBC quality. This study demonstrates that the deformability of red blood cells is significantly different in donors, and substantial variability persists throughout the entire process of their storage. Therefore, the FIFO criterion is not sufficient for assessing the RBC deformability, which should, therefore, be specifically characterised for each unit.

Erythrocyte survival is controlled by microRNA-142.

Hematopoietic-specific microRNA-142 is a critical regulator of various blood cell lineages, but its role in erythrocytes is unexplored. Herein, we characterize the impact of microRNA-142 on erythrocyte physiology and molecular cell biology, using a mouse loss-of-function allele. We report that microRNA-142 is required for maintaining the typical erythrocyte biconcave shape and structural resilience, for the normal metabolism of reactive oxygen species, and for overall lifespan. microRNA-142 further controls ACTIN filament homeostasis and membrane skeleton organization. The analyses presented reveal previously unappreciated functions of microRNA-142 and contribute to an emerging view of small RNAs as key players in erythropoiesis. Finally, the work herein demonstrates how a housekeeping network of cytoskeletal regulators can be reshaped by a single micro-RNA denominator in a cell type specific manner.

Biophysical and Biochemical Markers of Red Blood Cell Fragility.

BACKGROUND: Red blood cells (RBCs) undergo a natural aging process occurring in the blood circulation throughout the RBC lifespan or during routine cold storage in the blood bank. The aging of RBCs is associated with the elevation of mechanical fragility (MF) or osmotic fragility (OF) of RBCs, which can lead to cell lysis. The present study was undertaken to identify RBC properties that characterize their susceptibility to destruction under osmotic/mechanical stress. METHODS: RBCs were isolated from freshly donated blood or units of packed RBCs (PRBCs) and suspended in albumin-supplemented phosphate-buffered saline (PBS). In addition, PRBCs were separated by filtration through a microsphere column into two fractions: enriched with rigid (R-fraction) and deformable (D-fraction) cells. The RBCs were subjected to determination of deformability, MF and OF, moreover, the level of cell surface phosphatidylserine (PS) and the stomatin level in isolated RBC membranes were measured. RESULTS: In the RBC population, the cells that were susceptible to mechanical and osmotic stress were characterized by low deformability and increased level of surface PS. The OF/MF was higher in the R-fraction than in the D-fraction. Stomatin was depleted in destroyed cells and in the R-fraction. CONCLUSION: RBC deformability, the levels of surface PS, and membrane stomatin can be used as markers of RBC fragility.

Deformability of transfused red blood cells is a potent determinant of transfusion-induced change in recipient's blood flow.

OBJECTIVE: There is a growing concern regarding the risks in the transfusion of PRBC, as numerous studies have reported negative transfusion outcomes, including reduced blood perfusion. In search of this phenomenon's mechanism, the effect of PRBC deformability, a major determinant of blood flow, on transfusion outcome was explored. METHODS: The effect of PRBC deformability was examined by the transfusion-induced change in recipients' ∆SBF, in ß-TM patients, who are routinely treated with lifelong frequent transfusions. SBF was determined using a laser Doppler imager. RESULTS: ∆SBF was examined vs PRBC deformability, the transfusion-induced increase in ∆Hct and the recipients' SBF before transfusion (SBFB ). ∆SBF elevated with increasing PRBC deformability, with a highly significant dependence, while its elevation with ∆Hct was much less significant. ∆SBF was inversely proportional to the SBFB . CONCLUSIONS: This study provides, for the first time in humans, direct evidence that the deformability of transfused PRBC is a potent effector of transfusion outcome. Currently, PRBC are supplied primarily by the first-in-first-out criteria, while their functionality is ignored. The testing of PRBC hemodynamic quality would introduce a new paradigm into blood banking, which would contribute substantially to improving transfusion therapy.

Diabetic foot disease is associated with reduced erythrocyte deformability.

The pathogenesis of diabetic foot disease is multifactorial and encompasses microvascular and macrovascular pathologies. Abnormal blood rheology may also play a part in its development. Using a cell flow analyser (CFA), we examined the association between erythrocyte deformability and diabetic foot disease. Erythrocytes from diabetic patients with no known microvascular complications (n = 11) and patients suffering from a diabetic foot ulcer (n = 11) were isolated and their average elongation ratio (ER) as well as the ER distribution curve were measured. Average ER was decreased in the diabetic foot patients compared with the patients with diabetes and no complications (1·64 ± 0·07 versus 1·71 ± 0·1; P = 0·036). A significant rise in the percentage of minimally deformable red blood cells RBCs in diabetic foot patients compared with the patients with no complications was observed (37·89% ± 8·12% versus 30·61% ± 10·17%; P = 0·039) accompanied by a significant decrease in the percentage of highly deformable RBCs (12·47% ± 4·43% versus 17·49% ± 8·17% P = 0·046). Reduced erythrocyte deformability may slow capillary flow in the microvasculature and prolong wound healing in diabetic foot patients. Conversely, it may be the low-grade inflammatory state imposed by diabetic foot disease that reduces erythrocyte deformability. Further study of the rheological changes associated with diabetic foot disease may enhance our understanding of its pathogenesis and aid in the study of novel therapeutic approaches.

Storage-induced damage to red blood cell mechanical properties can be only partially reversed by rejuvenation.

BACKGROUND: The storage of red blood cells (RBC) is associated with impairment of their properties that can induce a circulatory risk to recipients. In a preceding study (2009), we reported that post-storage rejuvenation (RJ) of stored RBC (St-RBC) efficiently reduced the storage-induced RBC/endothelial cell interaction, while only partially reversing the level of intracellular Ca(2+), reactive oxygen species, and surface phosphatidylserine. In the present study, we examined the RJ effectiveness in repairing St-RBC mechanical properties. METHODS: RBC, stored in CPDA-1 without pre-storage leukoreduction, were subjected to post-storage RJ, and the deformability, osmotic fragility (OF), and mechanical fragility (MF) of the rejuvenated St-RBC (St-RBCRj) were compared to those of untreated St-RBC and of freshly-collected RBC (F-RBC). RESULTS: 5-week storage considerably increased OF and MF, and reduced the deformability of St-RBC. All alterations were only partially (40-70%) reversed by RJ, depending on the extent of the damage: the greater the damage, the lesser the relative effect of RJ. CONCLUSION: The findings of the present and preceding studies suggest that different St-RBC properties are differentially reversed by RJ, implying that some of the changes occur during storage and are irreversible.

Resuscitation with aged blood exacerbates liver injury in a hemorrhagic rat model.

OBJECTIVE: Blood loss and transfusion are frequent among patients undergoing liver surgery. Concerns have been raised about the safety and efficacy of transfusing stored blood. The influence of transfusing fresh vs. stored blood on the liver has not been studied to date. We tested the hypothesis that transfusion of stored, but not fresh blood, adversely affects liver outcome in vivo following acute hemorrhage. Additionally, possible mechanisms linking adverse liver outcome with increased storage duration were evaluated. DESIGN: Prospective, controlled, animal study. SETTING: University research laboratory. SUBJECTS: Adult male Sprague-Dawley rats INTERVENTIONS: Anesthetized rats were randomized to control, hemorrhagic and shock group (acute bleeding; HSG), or hemorrhagic and blood resuscitation groups (BR) (with fresh blood [BR-d0], blood stored for 4 [BR-d4] or 7 [BR-d7] days, or packed RBCs stored for 7 days [packed RBC-d7]). MEASUREMENTS AND MAIN RESULTS: Administration of blood or packed RBC stored for 7 days exacerbated liver injury as reflected by liver necrosis and enhanced apoptosis (p < 0.001). Functional MRI analysis of the liver demonstrated significant improvement in liver perfusion with fresh blood (% change in functional MRI signal intensity due to hyperoxia was 16% ± 3% in BR-d0 vs. 4% ± 3% in hemorrhagic group, p < 0.001) but not with stored blood (12% ± 2% and 9% ± 5% for BR-d4 and BR-d7, respectively). Analysis of stored blood showed reduction in RBC deformability at 7 days of storage, reflecting a five-fold increase in the number of undeformable cells. CONCLUSION: Liver injury is exacerbated by the transfusion of stored blood, primarily due to the change in the rheological properties of RBC. This data call for clinical studies in patients undergoing liver resection or transplantation.

The Impact of Ca2+ on Intracellular Distribution of Hemoglobin in Human Erythrocytes.

The membrane-bound hemoglobin (Hb) fraction impacts red blood cell (RBC) rheology and metabolism. Therefore, Hb-RBC membrane interactions are precisely controlled. For instance, the signaling function of membrane-bound deoxy-Hb and the structure of the docking sites in the cytosolic domain of the anion exchanger 1 (AE-1) protein are well documented; however, much less is known about the interaction of Hb variants with the erythrocyte's membrane. Here, we identified factors other than O2 availability that control Hb abundance in the membrane-bound fraction and the possible variant-specific binding selectivity of Hb to the membrane. We show that depletion of extracellular Ca2+ by chelators, or its omission from the extracellular medium, leads to membrane-bound Hb release into the cytosol. The removal of extracellular Ca2+ further triggers the redistribution of HbA0 and HbA2 variants between the membrane and the cytosol in favor of membrane-bound HbA2. Both effects are reversible and are no longer observed upon reintroduction of Ca2+ into the extracellular medium. Fluctuations of cytosolic Ca2+ also impact the pre-membrane Hb pool, resulting in the massive transfer of Hb to the cellular cytosol. We hypothesize that AE-1 is the specific membrane target and discuss the physiological outcomes and possible clinical implications of the Ca2+ regulation of the intracellular Hb distribution.

Biochemical and Biophysical Properties of Red Blood Cells in Disease.

Red blood cells (RBCs, erythrocytes) are highly specialized cells devoted to the transport of respiratory gases [...].

Hemolytic Activity of Nanoparticles as a Marker of Their Hemocompatibility.

The potential use of nanomaterials in medicine offers opportunities for novel therapeutic approaches to treating complex disorders. For that reason, a new branch of science, named nanotoxicology, which aims to study the dangerous effects of nanomaterials on human health and on the environment, has recently emerged. However, the toxicity and risk associated with nanomaterials are unclear or not completely understood. The development of an adequate experimental strategy for assessing the toxicity of nanomaterials may include a rapid/express method that will reliably, quickly, and cheaply make an initial assessment. One possibility is the characterization of the hemocompatibility of nanomaterials, which includes their hemolytic activity as a marker. In this review, we consider various factors affecting the hemolytic activity of nanomaterials and draw the reader's attention to the fact that the formation of a protein corona around a nanoparticle can significantly change its interaction with the red cell. This leads us to suggest that the nanomaterial hemolytic activity in the buffer does not reflect the situation in the blood plasma. As a recommendation, we propose studying the hemocompatibility of nanomaterials under more physiologically relevant conditions, in the presence of plasma proteins in the medium and under mechanical stress.