Distribution of erythrocyte in a model vessel exposed to inhomogeneous magnetic fields

In order to investigate magnetic field effects on blood flow, changes in the flow of erythrocytes in a model branched vessel were observed in an inhomogeneous magnetic field. The magnetic field was applied perpendicular to the straight vessel before branching. When the suspension containing paramagnetic erythrocytes with high spin methemoglobin or deoxygenated hemoglobin flowed in the model vessel, the erythrocytes were attracted towards the stronger magnetic field (i.e. to the side branch) and an excess flow of erythrocytes to the side branch was detected. This excess flow of erythrocytes to the side branch was the highest at a hematocrit of about 5% for the suspension containing erythrocytes with high spin methemoglobin. In the case of mixed suspensions containing erythrocytes with high spin methemoglobin and oxygenated erythrocytes, the excess flow of erythrocytes to the side branch reached its maximum at the "partial hematocrit" for the paramagnetic erythrocyte of around 5% and remained nearly constant with a further increase of the "partial hematocrit." The effect of magnetic field decreased as the flow velocity increased. These results are explained with the paramagnetism of erythrocytes and with the assumption of a hydrodynamic interaction among erythrocytes which are pulled in the direction of the magnetic field. It is suggested that a strong inhomogeneous magnetic field is not totally negligible to the blood circulation.     

Analysis of the distribution of flowing erythrocytes in a model vessel under an inhomogeneous magnetic field

Changes in the distribution of flowing erythrocytes in a straight cylinder were studied under an inhomogeneous magnetic field. The magnetic field was applied perpendicular to a cylinder, which had a 90° side vessel at the end (oriented towards the magnetic field) to detect changes in the erythrocyte distribution within the cylinder. (1) The attraction of paramagnetic erythrocytes by the magnetic field was demonstrated by an increase in the concentration (or number) of erythrocytes drawn into the side vessel. The flow of diamagnetic, oxygenated erythrocytes was unaffected. (2) The degree of attraction of the paramagnetic erythrocytes was proportional to ``(magnetic susceptibility)' and to ``(magnetic flux density) × (magnetic field gradient)' up to 10 T²/m, but it saturated at high magnetic field. The onset of the saturation depended on the magnetic susceptibility of the erythrocytes. (3) The degree of attraction depended on the hematocrit of the flowing erythrocyte suspension, with a maximum value at a low hematocrit. These phenomena are explained on the basis of the balance between the paramagnetic attractive force of the magnetic field and the collision rate between erythrocytes. Received: 2 May 1996 / Accepted: 1 July 1996     

High gradient magnetic separation of erythrocytes

The high gradient magnetic separation technique has been applied to separate paramagnetic erythrocytes from a cell suspension that also contained diamagnetic cells. Paramagnetism was induced in the red blood cells by oxidizing the iron atoms in the cell hemoglobin to the ferric state (methemoglobin). Diamagnetic cells were either untreated erythrocytes, containing oxyferrohemoglobin, or leukocytes in a suspension of mouse spleen cells. Cell suspensions were passed through a column containing 40 micron diameter stainless steel wire in a high magnetic field (33 kG). The paramagnetic cells were retained on the surface of the wire while the diamagnetic cells passed through. Elution of the paramagnetic cells was accomplished by removing the column from the magnet, in effect turning off the field.     

Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.     

Magnetic behavior of human erythrocytes at different hemoglobin states

The effect of a static magnetic field on human erythrocytes at different hemoglobin states (normal, oxidized and reduced hemoglobin) was investigated. Three different blood samples, normal, iron deficiency anemic and beta thalassemia minor, were studied. Measurements of the magnetization curves of the erythrocytes for all blood samples in all states showed diamagnetic behavior; however, oxidation was found to enhance this behavior. These measurements have also shown that the normal and iron deficiency samples in the reduced states exhibit a less diamagnetic response in comparison with the normal state. This result indicates that the reduction process gave rise to a paramagnetic component of the magnetization. Analysis of the measured paramagnetic behavior, using a Brillouin function, gave an effective magnetic moment of 8 muB per reduced hemoglobin molecule for both normal and anemic samples. This result shows that both anemic and normal blood have similar magnetic behavior and the only difference is the number of hemoglobin molecules per erythrocyte. For the beta thalassemia minor blood sample, magnetic measurements showed that both the normal and reduced states have almost the same diamagnetic behavior. However, this diamagnetic response is less than that for the normal state of the iron deficiency anemic sample. This result may indicate a low oxygen intake for the blood in the normal state for the beta thalassemia minor blood. All magnetic measurements were made using a vibrating sample magnetometer using field steps of 0.001 T from 1 T to -1 T.     

Red blood cell magnetophoresis

The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 x 10(-6) mm(3) s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 x 10(-6) mm(3) s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from -0.2 x 10(-6) mm(3) s/kg to +0.30 x 10(-6) mm(3) s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules.     

Orientation of glutaraldehyde-fixed erythrocytes in strong static magnetic fields

In a uniform static magnetic field up to 8 Telsa, glutaraldehyde-fixed erythrocytes showed an orientation in which their disk plane was perpendicular to the magnetic field. The paramagnetism of membrane-bound hemoglobin was thought to contribute significantly to this orientation. The observation of magnetic orientation is directed toward understanding the fundamental microstructural aspects of the erythrocyte. © 1996 Wiley-Liss, Inc.     

Hemoglobin degradation, lipid peroxidation, and inhibition of Na+/K(+)-ATPase in rat erythrocytes exposed to acrylonitrile

The effect of acrylonitrile (VCN) on erythrocyte lipid metabolism was investigated in vitro in metabolically active red cells from male Sprague-Dawley rats containing three types of hemoglobins: oxyhemoglobin, methemoglobin, and carbon monoxyhemoglobin. VCN at the concentration of 10 mM rapidly depleted erythrocyte glutathione (GSH) (75% of control) and induced lipid peroxidation (274% of control). Degradation of oxy- and methemoglobin was directly proportional to the extent of lipid peroxidation (r = 0.89). Addition of glucose to the incubation medium decreased hemoglobin degradation while it slightly increased VCN-induced lipid peroxidation. The highest amount of lipid peroxidation occurred in erythrocytes containing carbon monoxyhemoglobin and glucose. In the isolated red cell membranes incubated with 10 mM VCN, the lipid peroxidation was 400% of controls. VCN (25 mM) noncompetitively inhibited erythrocyte membrane Na+/K(+)-ATPase activity and the degree of inhibition was inversely proportional to the reaction temperature (r = -0.88). These findings indicate that the VCN induced hemoglobin degradation and lipid peroxidation are two extremes of a spectrum of oxidative damage in red cells leading to a change in physical state of membrane structure causing inhibition of adenosine triphosphate (ATPase) activity.     

Damage to erythrocytes caused by the interaction of nitrite-ions with hemoglobin

The formation of two hemoglobin forms (methemoglobin and nitrite methemoglobin) in native human erythrocytes in the presence of sodium nitrite in suspension was shown. In normal erythrocytes, the interaction of intracellular oxyhemoglobin with nitrite ions results in the formation of methemoglobin, whereas in metabolically exhausted erythrocytes, this leads predominantly to the formation of nitrite methemoglobin. The nitrite methemoglobin reacts with hydrogen peroxide to form reactive intermediates (e.g. peroxynitrous acid) and the products of hemoglobin destruction. During the storage of erythrocyte suspensions containing methemoglobin and modified nitrite methemoglobin, differences in the forms of erythrocytes and the degree of their hemolysis were revealed. It is assumed that the formation of methemoglobin leads to the destruction of erythrocytes.     

Hemoglobin degradation lipid peroxidation,and inhibition of na+/k+-atpase in rat erythrocytes exposed to acrylonitrile

The effect of acrylonitrile (VCN) on erythrocyte lipid metabolism was investigated in vitro in metabolically active red cells from male Sprague-Dawley rats containing three types of hemoglobins: oxyhemoglobin, methemoglobin, and carbon monoxyhemoglobin. VCN at the concentration of 10 mM rapidly depleted erythrocyte glutathione (GSH) (75% of control) and induced lipid peroxidation (274% of control). Degradation of oxy- and methemoglobin was directly proportional to the extent of lipid peroxidation (r = 0.89). Addition of glucose to the incubation medium decreased hemoglobin degradation while it slightly increased VCN-induced lipid peroxidation. The highest amount of lipid peroxidation occurred in erythrocytes containing carbon monoxyhemoglobin and glucose. In the isolated red cell membranes incubated with 10 mM VCN, the lipid peroxidation was 400% of controls. VCN (25 mM) noncompetitively inhibited erythrocyte membrane Na⁺/K⁺-ATPase activity and the degree of inhibition was inversely proportional to the reaction temperature (r = ?0.88). These findings indicate that the VCN induced hemoglobin degradation and lipid peroxidation are two extremes of a spectrum of oxidative damage in red cells leading to a change in physical state of membrane structure causing inhibition of adenosine triphosphatase (ATPase) activity.     

Identification of hemoglobins within individual rhesus monkey (Macaca mulatta) erythrocytes.

Rabbit and goat antibodies to monkey adult and fetal hemoglobin were prepared and purified to apparent monospecificity. After conjugation with fluorescein isothiocyanate, the antibodies were employed to identify the hemoglobin types within individual cells in peripheral erythrocyte smears. The percentage of neonatal monkey erythrocytes containing fetal hemoglobin was found to decrease with time. The existence of adult or fetal hemoglobin in the erythrocytes appeared to be mutually exclusive.     

Osmolality dependence of erythrocyte sedimentation and aggregation in a strong magnetic field

In order to specify the major determinant of the magnetic enhancement of erythrocyte sedimentation observed previously, the dependence of erythrocyte sedimentation rate (ESR) on osmolality was measured under a strong magnetic field. Even at hypotonic osmolality, an increase in ESR due to aggregation was observed in plasma solution as compared with that without aggregation in saline solution. However, the magnetic field did not enhance ESR at hypotonic osmolality, when the cell shape was an isotropic sphere (spherocyte). Thus, we narrowed our search to a mechanism that would explain the enhanced ESR found specifically in anisotropic erythrocytes. It was concluded that the major determinant can only work for anisotropic erythrocytes and is a magnetic field-induced increase in an intermembrane adhesive area due to magnetic orientation of anisotropic erythrocytes.     

Interaction of liposomes with erythrocytes in the process of their lysis]

Interaction of phosphatidyl choline liposomes with erythrocytes during spontaneous lysis of the latter has been studied. It is shown that hemoglobin which is released during the lysis of erythrocytes is found by liposomes which in their turn are absorbed on the external erythrocyte surface. In this case the binding of hemoglobin by liposomes takes place with a greater speed than its release during erythrocyte lysis and is accompanied by a change in its conformation. Possibilities of the microcalorimetry methods for studying the interaction of liposomes with erythrocytes under the conditions mentioned above are considered.     

Erythrocyte membrane antigen expression during friend cell differentiation: Analysis of two non-inducible variants

Erythrocyte membrane antigens have been detected on induced Friend erythroleukemic cells with a rabbit antiserum raised against mouse erythrocyte membranes. The antibody specificities of this antiserum have been quantitatively analyzed using a cellular radioimmunoassay. After absorption with thymocytes, the rabbit anti-erythrocyte membrane serum bound to dimethylsulfoxide (DMSO)-induced Friend erythroleukemic cells and to mouse erythrocytes but not to uninduced Friend cells or thymocytes. Reciprocal inhibition studies demonstrated that, following complete thymocyte absorption, the antiserum detected similar antigenic specificities, termed erythrocyte membrane antigens (EMA), on both mature erythrocytes and induced Friend cells. The expression of these erythrocyte membrane antigens was also induced on Friend cells by other agents, such as ouabain and dimethylacetamide (DMA). In contrast, exogenous hematin, which did not induce hemoglobin synthesis in the Friend cell clones used in this study, also did not induce erythrocyte membrane antigen expression. Two independently derived variant clones which do not produce hemoglobin in reponse to DMSO were analyzed for their ability to produce erythrocyte membrane antigens in response to various inducers of Friend cell differentiation. Clone TG-13 is not inducible by DMSO or hematin but is weakly induced by DMA for both hemoglobin production and erythrocyte membrane antigen expression. Another variant clone, M18, was also analyzed. This clone does not synthesize detectable hemoglobin when grown in either DMSO or hematin alone, but undergoes extensive hemoglobin synthesis when grown in medium containing both DMSO and hematin. M18 does, however, express erythrocyte membrane antigens when grown in DMSO alone: the presence of hematin and DMSO together in the growth medium does not enhance expression of these antigens. Thus M18 appears to be defective for hemoglobin inducibility, and this defect can be overcome by exogenous hematin; however, the expression of erythrocyte membrane antigens is not affected by this block in hemoglobin synthesis. The results with the variant clones are discussed in terms of a program for Friend cell differentiation in which the induction of hemoglobin synthesis and erythrocyte membrane antigen expression are under both co-ordinate and separate controls.     

Spin-label detection of hemoglobin-membrane interaction at physiological pH

The interaction between hemoglobin and the cytoplasmic surface of human erythrocyte membranes at physiological pH was studied by monitoring the electron paramagnetic resonance (EPR) signal of spin-labeled membrane ghosts in hemoglobin solutions of various concentrations. The EPR spectra indicate the existence of a significant hemoglobin-membrane interaction which exhibits a substantial hemoglobin concentration dependence over the concentration range 0-12 mg/mL. An equilibrium binding model yields a hemoglobin-membrane dissociation constant, Kd, on the order of 10(-4) M, at and above physiological pH; the interaction is classified as very low-affinity binding. The interaction increases significantly when the pH is decreased. Half-saturation of the binding sites occurs at a ratio of about 10(8) hemoglobins per cell.     

High-resolution NMR studies of transmembrane cation transport in uremic patients

Cation transport in erythrocytes of some uremic patients is impaired. Most studies have focused on the defect of the erythrocyte Na+/K+ pump in these diseased states. Herein, this cation transport defect was studied by using nuclear magnetic resonance spectroscopy (NMR) which is a non-invasive method permitting study on living erythrocytes. Firstly, we verified that the Na+ transport defect in uremic erythrocytes was not due to non-specific causes such as membrane alteration or a modification of the intracellular metabolism. The proton relaxation data, determined using a paramagnetic doping method, are consistent with a lack of erythrocytic membrane damage in uremic patients. Also, 31P-NMR results showed that in our experimental conditions, uremic and normal erythrocytes exhibit similar variations of ATP level over time. Lastly, the use of anionic paramagnetic shift reagent in 23Na-NMR revealed a defect in the Na+/K+ pump of erythrocytes from uremic patients with high Nain concentration. This defect seems to be due to a reduced number of pump units and to the presence of an endogenous inhibitor in uremic plasma.     

The interaction of phenyldichloroarsine with erythrocytes

The purpose of the study was to identify binding sites of organic arsenic in the erythrocyte and to explain species differences in binding. Washed erythrocytes were exposed to graded concentrations of [U-14C]phenyldichloroarsine (PDA) in phosphate-buffered saline containing 0.1% glucose and 0.1% bovine serum albumin. At low PDA concentrations, all cells bound the arsenical rapidly (within 10 min) and quantitatively. Human, pig, hamster, guinea pig, and mouse erythrocytes approached saturation at 0.02-0.3 mumol PDA/10(9) cells, depending on the species. Saturation points correlated well with each respective species' erythrocyte glutathione content. In contrast, rat erythrocytes showed no sign of saturation at PDA loads as high as 3.0 mumol/10(9) cells. Hemolysates of PDA-treated erythrocytes were subjected to Sephadex G-75 gel filtration chromatography. 14C from rat hemolysate was distributed between the hemoglobin and small molecular weight (glutathione-containing) fractions. In all other species, the 14C eluted almost exclusively with the glutathione-containing fractions. In equilibrium dialysis experiments, human hemoglobin did not bind PDA, whereas rat hemoglobin bound 2 PDA/mol with Kd approximately 5 microM. In conclusion, glutathione is the principal binding site of phenyldichloroarsine in erythrocytes. In most species, the arsenical does not bind to hemoglobin, even though it has free (titratable) sulfhydryls considerably in excess of the glutathione concentration. In rat erythrocytes, phenlydichloroarsine binds both to glutathione and to hemoglobin. Arsenical binding by rat hemoglobin is presumably due to the unique location of the extra titratable cysteine in that protein.     

Differential monooxygenase-like activity of fetal and adult erythrocytes

The monooxygenase-like activity of human erythrocytes was measured by monitoring the rate of para-hydroxylation of aniline. Erythrocytes from umbilical cord blood samples were found to be 3–5 times more active than erythrocytes from adult peripheral venous blood samples. This result may be attributed to an intrinsic difference in the reactivity of the particular form of hemoglobin which predominates in each of these erythrocyte types. Thus, the fetal hemoglobin isolated and purified from the cord blood displayed 2–6 times more activity than purified adult hemoglobin when each was tested in reconstituted aniline hydroxylation systems containing NADPH.     

Genetic resistance to malaria, oxidative stress and hemoglobin oxidation

I describe a model which posits the molecular basis of some malaria-resistance genes in the interaction between oxidized hemoglobin and membrane components. The model is supported by a considerable body of evidence which indicates that erythrocytes of genetically protected individuals (carriers of sickle cell trait, alpha- and beta-thalassemia, and G6PD deficiency) are susceptible to the increase of oxidation of hemoglobin following H2O2 release in the host cell by Plasmodium falciparum. I suggest that the irreversible interaction between oxidized hemoglobin and the red cell membrane could trigger mechanisms that: (i) reduce invasion of erythrocytes by the falciparum parasite; (ii) impair parasite survival and development within the cell; (iii) accelerate infected erythrocyte clearance by phagocytosis.     

Differential time‐dependent volumetric and surface area changes and delayed induction of new permeation pathways in P. falciparum‐infected hemoglobinopathic erythrocytes

During intraerythrocytic development, Plasmodium falciparum increases the ion permeability of the erythrocyte plasma membrane to an extent that jeopardizes the osmotic stability of the host cell. A previously formulated numeric model has suggested that the parasite prevents premature rupture of the host cell by consuming hemoglobin (Hb) in excess of its own anabolic needs. Here, we have tested the colloid‐osmotic model on the grounds of time‐resolved experimental measurements on cell surface area and volume. We have further verified whether the colloid‐osmotic model can predict time‐dependent volumetric changes when parasites are grown in erythrocytes containing the hemoglobin variants S or C. A good agreement between model‐predicted and empirical data on both infected erythrocyte and intracellular parasite volume was found for parasitized HbAA and HbAC erythrocytes. However, a delayed induction of the new permeation pathways needed to be taken into consideration for the latter case. For parasitized HbAS erythrocyte, volumes diverged from model predictions, and infected erythrocytes showed excessive vesiculation during the replication cycle. We conclude that the colloid‐osmotic model provides a plausible and experimentally supported explanation of the volume expansion and osmotic stability of P. falciparum‐infected erythrocytes. The contribution of vesiculation to the malaria‐protective function of hemoglobin S is discussed.