Interior Of Red Blood Cells 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.

The significant blood resistance to lung nitric oxide transfer lies within the red cell

The lung nitric oxide (NO) diffusing capacity (DlNO) mainly reflects alveolar-capillary membrane conductance (Dm). However, blood resistance has been shown in vitro and in vivo. To explore whether this resistance lies in the plasma, the red blood cell (RBC) membrane, or in the RBC interior, we measured the NO diffusing capacity (Dno) in a membrane oxygenator circuit containing ∼1 liter of horse or human blood exposed to 14 parts per million NO under physiological conditions on 7 separate days. We compared results across a 1,000-fold change in extracellular diffusivity using dextrans, plasma, and physiological salt solution. We halved RBC surface area by comparing horse and human RBCs. We altered the diffusive resistance of the RBC interior by adding sodium nitrite converting oxyhemoglobin to methemoglobin. Neither increased viscosity nor reduced RBC size reduced Dno. Adding sodium nitrite increased methemoglobin and was associated with a steady fall in Dno (P < 0.001). Similar results were obtained at NO concentrations found in vivo. The RBC interior appears to be the site of the blood resistance.

A note on a bronchial tree with 23 levels of bifurcation

Our recent article ''Why do we have a bronchial tree with 23 levels of bifurcation?'' (1) came out in Heat and Mass Transfer in 2009, in which we proposed an analytical model for the lung architecture, namely, quasi-steady one- dimensional mass transfer model for the lung. In its introduction, we referred to the paper of Prof. Reis' group, namely, ''Constructal theory of flow architectures of the lungs'' (2) as one of the most important papers in this particular topic, explicitly stating, ''Others (i.e. Prof. Reis' group) were certainly successful to some extent in relating the number of the bifurcation levels to the best oxygen access to the alveolar sacs.'' We believe that Prof. Reis' group certainly deserves credit for explaining for the first time the reason why we have a bronchial tree with 23 levels of bifurcation on the basis of Bejan's Constructal law. In our paper, we mentioned the originality of our paper, stating, ''Here, we shall present a simple and yet rational model for the respiratory system, namely, a quasi-steady one-dimensional mass transfer model. The model describes the mass transfer between the airway inlet and the red blood cell interior. Using this mass transfer model, we shall show that there exists the optimal number of the bifurcation levels, namely, 23, that yields the minimum overall mass transfer resistance for the mass transport from the external air to the red blood cells.'' Although the conclusions drawn in the two papers, namely, the optimal number of the bifurcation levels being 23, are the same, the difference in the approaches taken in the two papers is quite clear. We proposed a quasi-steady one-dimensional mass transfer model to explain the reason why we have a bronchial tree with 23 levels of bifurcation, whereas they elegantly resorted to Bejan's Constructal law with some assumptions such as isothermal and adiabatic flows with all species having the same chemical potential. Naturally, the resulting equations giving the total num- ber of bifurcations differ substantially from each other. Our equation requires the total pressure difference for breathing and the alveolar sac effective diffusivity, whereas theirs requires quantities such as the gas constant, absolute tem- perature, the oxygen relative concentration at the entrance of the trachea and that in the external air. Despite of the substantial difference in appearance of the two equations, both equations give the same optimal number of the bifurcation levels. This, however, is not surprising, since all rational scientific approaches should lead to the same fact in nature.

Why do we have a bronchial tree with 23 levels of bifurcation?

A simple one-dimensional mass transfer model has been proposed for the oxygen transport through a bronchial tree to alveolar tissues as well as the carbon dioxide removal from the tissues in the human respiratory system. The proposed model mathematically describes the mass transfer between the airway inlet and the red blood cell interior in the pulmonary capillaries. The quasi steady one-dimensional analysis based on the model reveals that the bronchial tree is constructed such that it promotes the easiest access to the external air. Naturally, there exists the optimal number of the bifurcation levels, namely, 23, that yields the minimum overall mass transfer resistance for the mass transport from the external air to the red blood cells.

Bartonella Henselae Survives after the Storage Period of the Red Blood Cell Units.

Introduction: The genus Bartonella comprises a unique group of emerging, Gram-negative, facultative intracellular bacteria, which can cause Carrión's disease, trench fever, cat scratch disease and bacillary angiomatosis. Man is a reservoir host of Bartonella species. Ten to 15% of the populations in areas that are hyperendemic for Carrión's disease are asymptomatic carriers of B. bacilliformis. In addition, positive serologic tests for B. henselae were found in 2 to 6% of Americans, 48% of Swiss, 19% of Germans, and 13.7% of Brazilians. Blood donors can be asymptomatic carrier of Bartonella spp. and the risk for transmission by transfusion should be considered. The aim of this study was to demonstrate that B. henselae remains viable in red blood cell (RBC) units at the end of the storage period.Methods: Two RBC units from healthy blood donors were collected in CPDA1 and each one split into two portions via a sterile connecting device. One bag from each split unit received experimentally B. henselae (Adolpho Lutz Institute, Sao Paulo, Brazil) and the second one served as a control. A brain and heart infusion (BHI) suspension of B. henselae colonies (Houston 1, American Type Culture Collection, Rockville, MD, ATCC 49882T) was used to obtain equivalence with tube 10 of McFarland scale, which determined an initial suspension with approximately 3×109 colony-forming units (CFU)/mL. This bacterial suspension was inoculated (0,33mL of the initial suspension) in one split RBC unit using a Sampling Site Coupler. Thus approximately 5,5×106 CFU of B. henselae were obtained per mL of RBC concentrate. All split units were stored (4 ±2°C) for 35 days. Aliquots were collected weekly using, Sample Site Coupler for, culture in dish with chocolate agar (incubated at 37°C in environment with 5% CO2), samples for blood culture bottles Bact/Alert (BioMérieux, Inc, USA) and Karnovisky medium for future evaluation by transmission electron microscopy.Results: All dishes with chocolate agar culture medium from infected units (seeded on days D0, D7, D14, D21, D28 and D35) presented growth of exuberant and mucoid colonies, after the fourth day of incubation. The electron microscopy from these samples showed typical Gram-negative cell wall in the interior of red blood cells. Samples from infected units presented negative blood culture bottles. All cultures from control units had negative results.Discussion: Bartonella ssp. is fastidious bacteria. Incubation for a short period of time and in standard medium does not allow for B. henselae growth as observed in the bottle culture of this study. Cultivation in blood-enriched media and incubation in environment with high CO2 levels of infected smears resulted positive. In conclusion, this study has demonstrated that B. henselae remain viable in RBC units during the storage period, at 4±2°C. These data reinforce the possibility of infection by transfusion of blood units collected from asymptomatic blood donors. Receptors of blood transfusion many times are or may become immunocompromissed, with risk of developing severe forms of bartonelloses.

Efflux of cholesterol and phospholipids derived from the haemoglobin-lipid adduct in human red blood cells into plasma

Objective: The interior of red blood cells (RBCs) contains a variable amount of cholesterol and phospholipids bound to haemoglobin (Hb). This current study was devised to determine if this pool of lipids (termed Hb-Ch) was available for exchange with plasma lipoproteins. Design and methods: We studied the in vitro efflux of lipids from human RBCs into fasting plasma in men with either low (control group) or high Hb-Ch (study group). Results: When plasma was incubated with a two-fold excess of autologous RBCs the plasma cholesterol level increased due to a decrease in the level of cholesterol from the RBC membrane (in the control group) and due to a decrease in the level of cholesterol both from the RBC membrane and the Hb-Ch fraction (in the study group). The loss of Hb-Ch-derived phospholipids during lipid efflux was roughly equal to that of Hb-Ch-derived cholesterol. The loss of RBC cholesterol into plasma high-density lipoproteins (HDL) was more pronounced in our study group and correlated with the loss of cholesterol from Hb-Ch. Conclusion: The Hb-Ch adduct significantly contributes to the lipid efflux from RBCs into plasma. The majority of cholesterol released from Hb-Ch appears in the plasma HDL fraction suggesting that Hb-Ch may play a role in reverse cholesterol transport in vivo.

The detection of intracellular bluetongue virus particles within ovine erythrocytes

1. 1. We report here a simplified method for detecting viruses and other antigenic agents in red blood cells (RBCs). Using a nonionic detergent to prepare cytoskeletons, the interior of RBCs can be scanned rapidly using immunoelectron microscopy. 2. 2. In this study, RBCs from bluetongue (BLU) virus-infected sheep were adsorbed directly onto Formvar-coated, gold electron microscope grids. 3. 3. Cytoskeletons were prepared and then probed using a monoclonal antibody to VP 7, a structural BLU-virus protein and Protein-A gold. 4. 4. Of the ca 32,000 RBCs that were examined from BLU virus-infected sheep, 34 (0.106%) contained labelled BLU virus particles. 5. 5. No labelled particles were observed in any of ca 8000 RBCs taken prior to BLU virus inoculation of sheep. 6. 6. If the antigenic BLU virus particles (which may be viral cores) are in fact infectious, this method of sequestration of virus within RBCs could contribute to the prolonged viremia typical of this arboviral disease, which is known to occur concurrently with circulating neutralizing antibody.

The effect of diabetes on blood flow properties.

Blood flow is a complex process combining fluid shearing in both plasma and the interior of red blood cells with elastic deformation of blood's solid elements. The red cell membrane is the major solid in blood, but platelets and white cells contribute solid behavior as well. Changes in blood's flow properties are often hidden by blood's ability to change its pattern of response. Capillary viscometry can be used to examine serum, plasma, and hemoglobin solutions directly, but rotational viscometry, where regularity of fluid shearing can be controlled to a narrow shear rate range, is required to study blood flow effectively. The most striking diabetic blood flow abnormality is best seen in time-based rotational viscometer studies that demonstrate the pattern of development of shear stress as flow becomes established. In such studies blood demonstrates both viscoelastic and thixotropic behavior; in diabetes blood's thixotropy is substantially increased. The diabetic pattern appears to be produced by a combination of reduced erythrocyte deformability and increased erythrocyte aggregation due to plasma protein changes. The plasma protein changes observed in diabetes are linked to the development of glucose intolerance but they are not specific to diabetes. The combined increase in aggregation and resistance to deformation of red blood cells produces blood flow abnormalities that can be detected primarily at low shear rate. Plasma protein changes and alteration in blood viscosity are absent from children with diabetes, while such changes tend to be associated with diabetic microangiopathy in adults. In some in vivo conditions, the effects on blood flow that can be linked to erythrocyte deformability are magnified out of proportion to measurements made in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of tricomplex fixation on lung tissue

The effect of tricomplex fixation on lungs fixed through vascular perfusion or through instillation of fixatives into the airways has been studied. To test the specificity of electron dense precipitates for phospholipids, some preparations were treated with tricomplex fixatives after lipid extraction with chloroform—methanol in a Soxhlet apparatus. The same experiment was repeated with red blood cells taken from the inferior vena cava of rats. It is concluded that precipitates reported in lung tissue after tricomplex flocculation cannot be regarded as specific for surfactant phospholipids, since they appear in various locations of several cell types including the interior of red blood cells; their occurrence is furthermore not influenced by lipid extraction prior to the application of tricomplex fixatives.