Red Blood Cells Membrane Permeability Research Articles

Correlation of Oxygen‐offloading rate from Red Blood Cells with Body Mass

The diffusion of O2 across red blood cell (RBC) membranes is critically important for the delivery of O2 to peripheral tissues. Although the traditional view had been that all O2 moves through RBC membranes by dissolving in and diffusing through membrane lipid, our recent work on murine RBCs indicates that most O2 traversing RBC membranes moves via protein channels (bioRxiv. doi:10.1101/2020.08.28.265066). In stopped-flow (SF) kinetics experiments, we use the absorbance spectrum of hemoglobin (Hb) to monitor the rate constant for O2 offloading from Hb (kHbO2). In RBCs from mice genetically deficient in both aquaporin-1 (AQP1) and the Rh complex (mainly RhAG), kHbO2 is 1/3 lower than in RBCs from wild-type (WT) mice. According to mathematical simulations, this 1/3 decrease in kHbO2 corresponds to a ~55% decrease in O2 permeability of RBC membranes (PM,O2). The combination of the double-knockout and pCMBS (nonspecific inhibitor of membrane proteins that is nonetheless excluded from the RBC interior) reduces PM,O2 by ~91%. Control experiments—hematology, RBC morphology, proteomics (abundance of individual proteins), lipidomics (abundance of individual membrane lipids)—rule out other potential explanations for our striking SF data. Our next challenge is to identify the membrane protein(s) responsible for the 91% – 55% = 36% of PM,O2 not accounted for by AQP1 and the Rh complex, as well as to establish the role of membrane lipids. Comparative physiology could be an important tool for achieving these goals. For each RBC sample, we correct the raw kHbO2 data for modest degrees of hemolysis that occur inside the SF reaction chamber. We assess this hemolysis using our novel SF assay, which is based on the release of carbonic anhydrase (CA) enzymes from RBCs. We find that, compared to RBCs from our lab-standard C57BL/6 WT mice, bovine RBCs have a significantly lower hemolysis (~2.5% vs. ~5%, N=3, p <0.01). More interestingly, we find a 71% lower CA activity in bovine RBCs compared to that in mouse RBCs (N=3, p <0.01). This observation could be important as we explore CO2 permeability in the near future. The kHbO2 is 41% lower for bovine RBCs than for RBCs from WT mice (N=3, p <0.01). Hematological analyses of our limited number of bovine samples do not yet show a significant difference from extensive murine samples in either mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC). A further evaluation of hematology, morphology, proteomics, lipidomics, and genomics (for deduced amino-acid sequences of proteins), will provide valuable insight to the contribution of specific membrane proteins and lipids to the relatively low kHbO2 of bovine RBCs. Our comparative analysis would benefit greatly from access to other mammals with a wide range of body mass (e.g., up to elephant size), from which we would use a small amount of blood (<1 ml) in the assays described above.

Computational analysis of nitric oxide biotransport in a microvessel influenced by red blood cells

It is pivotal that endothelium-dependent Nitric Oxide (NO) consumed by hemoglobin (Hb) inside red blood cells (RBCs) membrane, regulates the vascular tone. The whole processes of NO transport in vessel containing flowing RBCs is still not clear, such as NO production in endothelium, diffusion in plasma and consumption inside RBCs. In this work, the motion of RBCs in a microvessel is investigated by using immersed boundary lattice Boltzmann method (IB-LBM) first and the deformability of RBCs is expressed by using spring network model which is based on the minimum energy principle. Furthermore, the interaction between RBCs is considered. Based on the wall shear stress (WSS), NO production rate originated from endothelium was obtained by using a hyperbolic model. NO distribution inside the microvessel with multiple RBCs was computed by using immersed boundary finite difference method (IB-FDM). The result shows that a large (small) WSS exists at locations with a relatively wide(narrow) gap between the wall and cell. In terms of mass transfer, an increase of RBC membrane permeability leads to a decrease of NO concentration in the vessel and the surrounding endothelium significantly. In addition, with the increasing of hematocrit (Hct) value, NO concentration distribution in the whole vessel decreases both in the lumen and vascular wall. Finally, the thickness of RBCs-depleted layer gradually decreases with the weakened deformability of RBCs membrane, and the change degree of cell free layer (CFL) thickness decreases as the bending stiffness is relatively higher. Thus, when bending stiffness is higher, the NO concentration in vascular wall is reduced resulting from the thinner CFL.

Effect of laser on erythrocytes sedimentation rate and some hematological parameters

Background: This study was conducted in pursuit of gaining an understand effects of the low level laser irradiation on whole blood is very essential way in revealing the mechanisms of the action of laser radiation on biological tissues.Objective: The purpose of this study is to investigate the in vitroeffect of laser radiation on some hematological parameters and erythrocyte sedimentation rate.Subject and Method: Blood samples were obtained from 30 healthy volunteers, each sample was divided into four new samples, one of them was considered as control while the other three were exposed to three different laser doses. Thewavelength of532nm was used for irradiation with 4mm diameter beam spot on blood samples, with power density of 796.17mW/cm2. The irradiation times were 1.8, 3.7and 6.2 secand so the different doses of irradiation 1.5,3 and 5 J/cm2, respectively.Results: The erythrocyte sedimentation rate was measured after laser irradiation and compared with un-irradiated control sample. The results of this study showed thaterythrocyte sedimentation rate value increased significantly with a dose of 1.5J/cm² but not with the othertwo doses’ values in male persons but not in females. Moreover, the mean cell volume shows significant decrement post irradiation.Conclusion: Laser irradiation induces physical changes in red blood cell membrane permeability and blood viscosity and consequently alters erythrocyte sedimentation rate.

Vitamin C and Trolox decrease oxidative stress and hemolysis in cold-stored human red blood cells

ABSTRACTObjectives: To investigate the effects of sodium ascorbate (SA) (5–3125 μM) and a combination of SA and Trolox (25 and 125 μM) on oxidative changes generated in red blood cells (RBCs) followed by up to 20 days refrigerated storage.Methods: RBCs were isolated from CPD-preserved human blood. Percentage of hemolysis and extracellular activity of lactate dehydrogenase (LDH) were measured to assess the RBC membrane integrity. Lipid peroxidation (LPO), glutathione (GSH) and total antioxidant capacity (TAC) were quantified by thiobarbituric acid-reactive substances, Ellman’s reagent and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) -based assay, respectively.Results: SA failed to reduce the storage-induced hemolysis and RBC membrane permeability. Addition of SA resulted in a concentration-independent LPO inhibition and increased TAC. A combination of SA/Trolox supplemented to the RBC medium significantly inhibited hemolysis, LDH leakage, LPO, GSH depletion and enhanced TAC.Discussion: The effects of vitamin C action are closely concentration-dependent and may be modulated by a variety of compounds (e.g. Hb degradation products) released from RBCs during the prolonged storage, changing its properties from anti- to pro-oxidative. The two different class antioxidants (SA/Trolox) could possibly cooperate to be good potential RBC storage additives ensuring both antiradical and membrane stabilizing protection.

Carbamohydrazonothioate-based polymer-magnetic nanohybrids: Fabrication, characterization and bactericidal properties

Herein is presented fabrication of new efficient antibacterial polymer/gold/magnetic nanohybrids. These nanohybrids consist of a superparamagnetic nanoparticle core, which ensures their facile separation, purification and recyclability, and polymeric coating which exhibits bactericidal activity. They were prepared in an efficient four-step synthetic route involving the following: (1) magnetic nanoparticles fabrication by co-precipitation of Fe2+ and Fe3+ in the presence of ammonia solution; (2) gold shell formation; (3) immobilization of RAFT/MADIX (reversible addition-fragmentation transfer/macromolecular design via the interchange of xanthates) initiating agent on the surface of nanoparticles; and (4) surface-initiated RAFT/MADIX polymerizations of bactericidal monomers. Physicochemical properties of bare and functionalized magnetic nanoparticles were characterized by Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), magnetization measurements, transmission and scanning electron microscopies (TEM and SEM). Hemolytic and antibacterial activity of the obtained materials (monomers and polymer/gold/magnetic nanohybrids) against human red blood cells and Pseudomonas aeruginosa was determined. It was demonstrated that the tested agents do not affect red blood cells membrane permeability at the concentration range of 1–100μg/ml. Simultaneously, at this concentration, they effectively kill and restrict metabolic activity of planktonic P. aeruginosa as well as prevent its biofilm formation.

Red Cell Transport Abnormalities As Predictors of the Development of Renal Disease in Children with Sickle Cell Disease

Background:Patients with sickle cell disease (SCD) have the abnormal haemoglobin (Hb) HbS in their red blood cells (RBCs). The complications of SCD may be extensive but are markedly variable between patients. Renal damage (sometimes called sickle cell nephropathy, SCN) is a common finding in about a third of children with SCD, a significant number of whom will proceed to renal failure. As yet, it is not possible to predict which children will develop SCN and who would therefore benefit from earlier and aggressive management. Various biomarkers have been investigated but none have proved efficacious. Changes in RBC membrane permeability are closely linked with polymerisation of HbS and, as such, may represent an important early event in pathogenesis.We postulated that certain RBC characteristics (sickling, K+ transport, PS exposure), which may be inherited independently of the HbS mutation, may correlate with renal pathology, but more importantly, may be identifiable in advance of damage, thereby providing prognostic markers to inform patient management.Participants and Methods:Children (>4 years old) with sickle cell anaemia (SCA) were recruited from the paediatric haematology clinic at King’s College Hospital in South London, UK. Patients who had received blood transfusion in the preceding four months or who were on medications known to directly alter RBC cation transport (e.g. dipyridamole and Ca2+ channel blockers) were excluded. Patients on hydroxyurea were included. All patients were in steady state, with no acute symptoms requiring treatment for at least seven days. Clinical and laboratory parameters were collected prospectively at routine clinical attendance. Details of enuresis were collected using specifically developed proforma and patients were divided into two groups: those who stopped wetting their bed before the age of five years and those who were still enuretic after five years of age. RBC permeability was assessed using 86Rb+ as a congener for K+ to measure the activity of main cation transport systems involved in RBC dehydration. The activity of RBC transport pathways and percentage of sickling was measured at 100mmHg, 35mmHg, 15mmHg and 0mmHg. Statistical analysis was conducted using IBM-SPSS, New York.Results: 112 patients of HbSS genotype (SCA) consented to take part in the study. Their clinical profile is summarised Table 1. The percentage of children with enuresis, hypertension and significant albuminuria is shown in Table 2. There was significant correlation between activity of Gardos channel at O2 tensions of 35mmHg, 15mmHg and 0mmHg and enuresis after the age of five years. Psickle activity also showed significant correlation but only in fully deoxygenated RBCs (O2 of 0mmHg.) Table 3.ConclusionIn conclusion, the present findings emphasise the significance of a thorough appreciation of RBC permeability to the pathogenesis of SCD. Whilst preliminary, these findings are exciting because they show that the activity of a particular transport pathways, abnormally elevated in SCA patients, are associated with enuresis. These findings suggest that the early identification of increased Gardos and Psickle activity may identify children who would benefit from early treatment for nocturnal enuresis. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

New insights on hereditary erythrocyte membrane defects.

After the first proposed model of the red blood cell membrane skeleton 36 years ago, several additional proteins have been discovered during the intervening years, and their relationship with the pathogenesis of the related disorders have been somewhat defined. The knowledge of erythrocyte membrane structure is important because it represents the model for spectrin-based membrane skeletons in all cells and because defects in its structure underlie multiple hemolytic anemias. This review summarizes the main features of erythrocyte membrane disorders, dividing them into structural and altered permeability defects, focusing particularly on the most recent advances. New proteins involved in alterations of the red blood cell membrane permeability were recently described. The mechanoreceptor PIEZO1 is the largest ion channel identified to date, the fundamental regulator of erythrocyte volume homeostasis. Missense, gain-of-function mutations in the PIEZO1 gene have been identified in several families as causative of dehydrated hereditary stomatocytosis or xerocytosis. Similarly, the KCNN4 gene, codifying the so called Gardos channel, has been recently identified as a second causative gene of hereditary xerocytosis. Finally, ABCB6 missense mutations were identified in different pedigrees of familial pseudohyperkalemia. New genomic technologies have improved the quality and reduced the time of diagnosis of these diseases. Moreover, they are essential for the identification of the new causative genes. However, many questions remain to solve, and are currently objects of intensive studies.

Computational analysis of nitric oxide biotransport to red blood cell in the presence of free hemoglobin and NO donor

Red blood cells (RBCs) modulate nitric oxide (NO) bioavailability in the vasculature. Extracellular free hemoglobin (Hb) in the vascular lumen can cause NO bioavailability related complications seen in pathological conditions such as pancreatitis, sickle cell disease and malaria. In addition, the role of extracellular free Hb has been critical to estimate kinetic and transport properties of NO–RBCs interactions in ‘competition experiments’. We recently reported a strong dependence of NO transport on RBC membrane permeability and hematocrit. NO donors combined with anti-inflammatory drugs are an emergent treatment for diseases like cancer, cardiovascular complications and wound healing. However, the role of RBCs in transport NO from NO donors is not clearly understood. To understand the significance of extracellular free Hb in pathophysiology on NO availability and estimation of the NO-RBC interactions, we developed a computational model to simulate NO biotransport to the RBC in the presence of extracellular free Hb. Using this model, we studied the effect of hematocrit, RBC membrane permeability and NO donors on NO–RBC interactions in the presence and absence of extracellular free Hb. The plasma NO concentration gradients and average plasma NO concentrations changed minimally with increase in extracellular free Hb concentrations at the higher hematocrit as compared to those at the lower hematocrit irrespective of the NO delivery method, indicating that the presence of extracellular free Hb affects the NO transport only at a low hematocrit. We also observed that NO concentrations increased with NO donor concentrations in the absence as well as in the presence of extracellular free Hb. In addition, NO donor supplementation may increase NO availability in the plasma in the event of loss of endothelium-derived NO activity.

A moderate protective effect of quercetin against γ-irradiation- and storage-induced oxidative damage in red blood cells for transfusion

Purpose: To investigate the extent of γ-irradiation-induced oxidative membrane damage and antioxidant activity of quercetin in long-term, cold stored (4°C) acid-citrate-dextrose- preserved human red blood cells (RBC).Materials and methods: The extracellular activity of lactate dehydrogenase (LDH) was measured to assess RBC membrane integrity. Lipid peroxidation and reduced glutathione (GSH) levels were quantified by thiobarbituric acid-reactive substances (TBARS) and Ellman's reagent, respectively.Results: During storage of non-irradiated RBC (up 21 days) the LDH activity in the supernatant increased with time. In contrast to a low dose of ionizing radiation (30 Gy), irradiation at higher, but still clinically relevant doses, of 40–50 Gy resulted in elevation of the post-storage extracellular LDH activity. Quercetin (2–50 μM) dissolved in dimethyl sulfoxide (DMSO) significantly increased the LDH release in the irradiated and non-irradiated RBC, reflecting an increase of RBC membrane permeability. In the presence of ethanol as a solvent quercetin protected RBC against storage-induced oxidative damage – it inhibited the LDH release, GSH depletion, and lipid peroxidation.Conclusion: The level of protection offered by quercetin against the radiation- and storage-induced oxidative damage to RBC does not seem to be sufficient to warrant its application as an additive for conservation purposes. The findings indicate that the solvent can modulate a response of RBC to water-insoluble antioxidants changing their properties from anti-oxidative to pro-oxidative.

Mechanism of faster NO scavenging by older stored red blood cells

The blood storage lesion involves morphological and biochemical changes of red blood cells (RBCs) that occur during storage. These include conversion of the biconcave disc morphology to a spherical one, decreased mean corpuscular hemoglobin concentration, varied mean corpuscular volume, reduced integrity of the erythrocyte membrane with formation of microparticles, and increased cell-free hemoglobin. We studied the extent that older stored red blood cells scavenge nitric oxide (NO) faster than fresher stored red blood cells. Using electron paramagnetic resonance spectroscopy and stopped-flow absorption spectroscopy to measure the rate of NO uptake and reaction with hemoglobin in red cells, we found that older stored red blood cells scavenge NO about 1.8 times faster than fresher ones. Based on these experimental data, we simulated NO scavenging by fresher or older stored red blood cells with a biconcave or spherical geometry, respectively, in order to explore the mechanism of NO scavenging related to changes that occur during blood storage. We found that red blood cells with a spherical geometry scavenges NO about 2 times slower than ones with a biconcave geometry, and a smaller RBC hemoglobin concentration or volume increases NO scavenging by red blood cells. Our simulations demonstrate that even the most extreme possible changes in mean corpuscular hemoglobin concentration and mean corpuscular volume that favor increased NO scavenging are insufficient to account for what is observed experimentally. Therefore, RBC membrane permeability must increase during storage and we find that the permeability is likely to increase between 5 and 70 fold. Simulations using a two-dimensional blood vessel show that even a 5-fold increase in membrane permeability to NO can reduce NO bioavailability at the smooth muscle. BackgroundTransfusion of older stored blood may be harmful. ResultsOlder stored red blood cells scavenge nitric oxide more than fresher cells. ConclusionAs stored red blood cells age, structural and biochemical changes occur that lead to faster scavenging. SignificanceIncreased nitric oxide scavenging by red blood cells as a function of storage age contributes to deleterious effects upon transfusion.

Contribution of membrane permeability and unstirred layer diffusion to nitric oxide–red blood cell interaction

Nitric oxide (NO) consumption by red blood cell (RBC) hemoglobin (Hb) in vasculature is critical in regulating the vascular tone. The paradox of NO production at endothelium in close proximity of an effective NO scavenger Hb in RBCs is mitigated by lower NO consumption by RBCs compared to that of free Hb due to transport resistances including membrane resistance, extra- and intra-cellular resistances for NO biotransport to the RBC. Relative contribution of each transport resistance on NO–RBC interactions is still not clear. We developed a mathematical model of NO transport to a single RBC to quantify the contributions from individual transport barriers by analyzing the effect of RBC membrane permeability (Pm), hematocrit (Hct) and NO–Hb reaction rate constants on NO–RBC interactions. Our results indicated that intracellular diffusion of NO was not a rate limiting step for NO–RBC interactions. The extracellular diffusion contributed 70–90% of total transport resistance for Pm>1cms−1 whereas membrane resistance accounts for 50–75% of total transport resistance for Pm<0.1cms−1. We propose a narrow Pm range of 0.21–0.44cms−1 for 10–45% Hct, respectively, below which membrane resistance is more significant and above which extracellular diffusion is a dominating transport resistance for NO–RBC interactions.

Role of membrane permeability and extracellular diffusion in nitric oxide transport to the red blood cells

Nitric oxide (NO) availability for vasodilation in vasculature is dependent on its consumption by red blood cell (RBC) hemoglobin. Extracellular diffusion in the plasma, cell free layer near the vessel wall and the RBC membrane permeability (Pm) reportedly lower NO consumption by RBCs. To understand the contribution of RBC membrane permeability and extracellular diffusion from the plasma layer surrounding the RBC towards NO uptake by RBCs, we developed a mathematical model simulating NO transport to a single RBC. Simulations were performed for Pm values ranging from 0.0415–40 cm/s and hematocrit (Hct) ranging from 1–45% and NO concentrations at the RBC membrane (CNOm inside and CNOm outside) were estimated. NO concentration gradients in the plasma layer increased with Pm. CNOm inside increased while CNOm outside decreased with Pm. At Pm =40 cm/s, CNOm inside and CNOm outside were similar to each other and both increased with Hct, indicating that extracellular diffusional resistance provides a significant transport barrier for NO at higher RBC membrane permeability. At Pm = 0.0415 cm/s, estimated CNOm outside were much higher than CNOm inside and both were independent of Hct for Hct ≥ 15%, suggesting that at lower RBC membrane permeability, for Hct ≥ 15%, RBC permeability is a major barrier for resistance for NO transport. Insights from this study may prove useful in understanding interaction of NO with RBC. Supported by AHA 0530050N &amp; R15 HL087287 grants.

Taking Charge: Feeding Malaria via Anion Channels

The human malaria parasite Plasmodium falciparum increases red blood cell membrane permeability during infection to allow for import of nutrients and other solutes. Nguitragool etal. (2011) have now identified parasite-encoded CLAG3 proteins as key components of the import channel located on the erythrocyte membrane.

Effect of Membrane Permeability and Extracellular Diffusion on NO uptake by Erythrocytes

Quantification of nitric oxide (NO)‐red blood cells (RBC) reaction metabolites is necessary to evaluate the extent of diffusional resistances for nitric oxide transport in vascular lumen. Delivery of physiologically relevant concentration of nitric oxide and RBC‐hemoglobin are a critical factor in quantification of in vitro NO‐RBC reaction. In this study, we developed a flask bioreactor which delivers gaseous NO to the RBCs and studied the effects of hematocrit (hct) and NO concentrations on NO‐RBC interactions. We developed a computational model of NO transport to single RBCs using experimental data to analyze the effect of RBC membrane permeability on steady state NO concentrations in the bulk of liquid surrounding the RBCs and at the RBC membrane. We observed that the steady state NO concentrations in the bulk of liquid and at the RBC membrane 1) decreased with increase in RBC membrane permeability for a given equilibrium NO concentration and hct; 2) increased with increase available NO concentrations at a given RBC membrane permeability; 3) decreased with increase in hct for a given RBC membrane permeability. Our results indicate that the extracellular diffusion and RBC membrane permeability are significant diffusional barriers for NO transport to the RBCs and the RBC NO membrane permeability should be in the range of 0.0415‐ 0.4 cm/s. Supported by: AHA 0530050N, NIH R01 HL084337 &amp; R15 HL087287.

Erythrocyte Glut1 Triggers Dehydroascorbic Acid Uptake in Mammals Unable to Synthesize Vitamin C

Of all cells, human erythrocytes express the highest level of the Glut1 glucose transporter. However, the regulation and function of Glut1 during erythropoiesis are not known. Here, we report that glucose transport actually decreases during human erythropoiesis despite a >3-log increase in Glut1 transcripts. In contrast, Glut1-mediated transport of L-dehydroascorbic acid (DHA), an oxidized form of ascorbic acid (AA), is dramatically enhanced. We identified stomatin, an integral erythrocyte membrane protein, as regulating the switch from glucose to DHA transport. Notably though, we found that erythrocyte Glut1 and associated DHA uptake are unique traits of humans and the few other mammals that have lost the ability to synthesize AA from glucose. Accordingly, we show that mice, a species capable of synthesizing AA, express Glut4 but not Glut1 in mature erythrocytes. Thus, erythrocyte-specific coexpression of Glut1 with stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize vitamin C.

Nitric oxide red blood cell membrane permeability at high and low oxygen tension

Red blood cell (RBC) encapsulated hemoglobin in the blood scavenges nitric oxide (NO) much more slowly than cell-free hemoglobin would. Part of this reduced NO scavenging has been attributed to an intrinsic membrane barrier to diffusion of NO through the RBC membrane. Published values for the permeability of RBCs to NO vary over several orders of magnitude. Recently, the rate that RBCs scavenge NO has been shown to depend on the hematocrit (percentage volume of RBCs) and oxygen tension. The difference in rate constants was hypothesized to be due to oxygen modulation of the RBC membrane permeability, but also could have been due to the difference in bimolecular rate constants for the reaction of NO and oxygenated vs deoxygenated hemoglobin. Here, we model NO scavenging by RBCs under previously published experimental conditions. A finite-element based computer program model is constrained by published values for the reaction rates of NO with oxygenated and deoxygenated hemoglobin as well as RBC NO scavenging rates. We find that the permeability of RBCs to NO under oxygenated conditions is between 4400 and 5100 μm s −1 while the permeability under deoxygenated conditions is greater than 64,000 μm s −1. The permeability changes by a factor of 10 or more upon oxygenation of anoxic RBCs. These results may have important implications with respect to NO import or export in physiology.

Computational Modeling of Nitric Oxide Bioavailability in Hemolytic Anemias.

Intravascular hemoglobin (Hb) scavenges nitric oxide (NO), which decreases the availability of endothelial-derived NO, thereby reducing smooth muscle relaxation. Under normal conditions, there are various mechanisms that limit consumption of NO by Hb in vivo includinga physical barrier to NO diffusion across the red blood cell (RBC) membrane,an unstirred layer creating a concentration gradient in NO outside the RBC, andand a cell-free zone between the endothelium where NO is made and the location of the RBCs.However, in hemolytic conditions, these mechanisms can be compromised, leading to reduced levels of NO reaching the smooth muscle cells to cause vasodilation. Although it is generally accepted that cell-free Hb scavenges NO more effectively than Hb encapsulated in the red blood cell (RBC), some do not believe that the low concentrations of cell-free Hb that are present in hemolytic conditions, such as sickle cell disease, can impact NO bioavailability in the presence of the large amounts of RBC encapsulated Hb (about 10 mM in heme) present in vivo. We have performed computational modeling looking at the effects of cell-free Hb on NO bioavailability within blood vessels. We have found that concentrations of cell-free Hb as low as one micromolar significantly reduces the availability of NO. Thus, 0.01% hemolysis has a significant affect on steady state NO levels. Extravasation of cell-free Hb into the interstitial space of as low as one micromolar further reduces the bioavailability of NO. At low hematocrit values, cell-free Hb scavenging of NO was found to be more efficient than at high hematocrit values. We have also found that the effect of RBC membrane permeability diminishes at cell-free Hb concentrations of only four micromolar and above. These theoretical results support experimental evidence indicating that cell-free Hb contributes to the pathology of hemolytic diseases. Supported by NIH grant RG0489.

Comparison of X‐ray vs. gamma irradiation of CPDA‐1 red cells

Transfusion-associated graft-versus-host disease (TA-GVHD) is a serious, potential complication of blood transfusion that is essentially prevented by blood product irradiation. Blood product irradiation is currently performed using gamma irradiation. X-ray irradiation is an alternative that has certain advantages: an X-ray irradiator is less expensive and does not have a radioactive source. However, the biochemical effects of X-ray irradiation on red blood cells (RBCs) have not been well characterized. The primary purpose of our study was to compare the effects of X-ray irradiation with gamma irradiation on RBC membrane permeability. A secondary purpose was to verify that X-ray irradiation has the same effect on lymphocytes as gamma irradiation. Ten RBC units were split into two portions; five units of each were then irradiated with either gamma or X-ray at a dose of 35 Gy or 25 Gy, respectively. Free plasma haemoglobin and extracellular potassium levels were measured in each unit over time. Another unit was divided into three parts for lymphocyte studies; one part was not irradiated as a control and the other two received 25 Gy of irradiation via X-ray or gamma irradiation, respectively. Minimally increased free plasma haemoglobin was found only in X-ray-irradiated units after 25 Gy compared to gamma-irradiated units. However, extracellular potassium levels were not significantly different in these groups. Extracellular potassium levels were slightly higher only in gamma irradiated units at 35 Gy. X-ray and gamma irradiation were equivalent in affecting lymphocyte function. Small differences in RBC membrane permeability are found between gamma-irradiated and X-ray-irradiated units. However, these differences are not likely to be clinically important.

Effect of 50 Hz, 0.2 mT magnetic fields on RBC properties and heart functions of albino rats.

In this work the effect of sinusoidal 50 Hz, 0.2 mT magnetic fields on the red blood cells (RBCs) and heart functions of Albino rats were investigated. Twenty-four male Albino rats were equally divided into four groups, A, B, C, and D. Animals from groups B were continuously exposed to the magnetic field for 15 days; and groups C and D, for 30 days. Group A was used as control. Animals from group D were kept after exposure to the magnetic field for a period of 45 days for delayed effect studies. The osmotic fragility and shape of RBCs' membrane and hemoglobin (Hb) structure tests were carried out for all groups. The dielectric relaxation of Hb molecules was measured in the frequency range of 0.1-10 MHz and the dielectric increment (Deltaepsilon), relaxation time (tau), molecular radius (r), and Cole-Cole parameter (alpha) were calculated for all groups. The ECG was measured for all animals before and after exposure to the magnetic field. The results indicated that exposure of the animals to 50 Hz, 0.2 mT magnetic fields resulted in the decrease of RBCs membrane elasticity and permeability and changes in the molecular structure of Hb. The ECG of the exposed animals was considerably altered. The data also indicated that there was no sign of repair in the newly generated RBCs structure and the ECG after removing the animals from the magnetic field, which indicates that the blood generating system was severely injured. The injuries in the heart of the animals were attributed to the loss of some physiological functions of the RBCs as a result of exposures of the rats to the magnetic field.

Erythrocyte Consumption of Nitric Oxide: Competition Experiment and Model Analysis

It is generally believed that the erythrocyte membrane is highly permeable to nitric oxide (NO). To prevent NO from freely entering and being scavenged by the red blood cell (RBC), it has been suggested that NO consumption is limited by the mass transfer resistance of the diffusion layer adjacent to the erythrocyte membrane. Recently, we (Vaughn et al. (2000). J. Biol. Chem. 275, 2342) presented an experimental technique that overcomes experimental diffusional limitations and showed that RBCs also possess a mechanism to slow nitric oxide uptake. Here, we present a mathematical analysis of this technique by modeling the NO uptake of a single cell. We obtain additional data (n = 33, total) by use of the competition experiment and, through application of the model, show that either the RBC membrane permeability to NO or the intracellular reaction rate between NO and hemoglobin (Hb) is at least 2000-fold lower than previously thought. As a result, RBCs react with NO at a rate three orders of magnitude slower than free oxyHb. This phenomena may play an important role in NO bioavailability.