Margaret R. Clark

Analysis of factors regulating erythrocyte deformability.

Using a laser diffraction technique, we have studied factors that influence the deformability of erythrocytes. Variations in suspending medium osmolality and applied shear stress were employed to isolate the individual contributions to whole cell deformability of internal viscosity, surface area-to-volume ratio, and viscoelastic properties of the membrane. An experimental system was devised in which normal cells were modified in vitro to induce specific alterations in each factor. Measurements of deformability as a function of medium osmolality showed characteristic behavior of the modified cells. Reduced surface area-to-volume ratio was detected by an exaggeration of the normal decrease in deformability as medium osmolality was decreased. In contrast, increased internal viscosity was detected by an increase in deformability as osmolality was decreased. Finally, decreased membrane flexibility was detected by reduced deformation at low shear stress. These methods of analysis were applied to cells from patients with hereditary spherocytosis, hereditary pyropoikilocytosis, and hemoglobin CC disease to define the basis of reduced deformability. Hereditary spherocytes showed the combined effects of reduced surface area and increased internal viscosity. Hereditary pyropoikilocytes revealed the effects of severely reduced surface area-to-volume ratio. Hemoglobin CC cells showed only the effects of high internal viscosity. An increase in the membrane shear modulus (decreased membrane deformability) was not evident in these disorders.

Deformability of oxygenated irreversibly sickled cells.

The deformability characteristics of isolated subpopulations of irreversibly sickled cells (ISC) have been studied in an ektacytometer. Analysis of laser diffraction patterns of well-oxygenated cells subjected to shear stress in solutions of varying osmolality has demonstrated a profound influence of mean corpuscular hemoglobin concentration and intracellular viscosity on the deformability of ISC. Virtually undeformable at 290 mosM, ISC became almost totally deformable at 130 mosM. In addition, when ISC membranes were loaded with normal hemoglobin at low concentration, they deformed easily in isotonic medium, as did resealed normal cell membranes. The restoration of deformability of ISC upon reduction of their hemoglobin concentration and internal viscosity to normal levels suggests that altered membrane properties are not the primary determinant of decreased deformability in these cells. Rather, cellular dehydration induced by previous sickling would appear to contribute in a major way to their abnormal rheological behavior.

The effect of malonyldialdehyde, a product of lipid peroxidation, on the deformability, dehydration and 51Cr‐survival of erythrocytes

Summary. Erythrocyte membrane lipid peroxidation has been reported to occur in various haemolytic anaemias. In the present study, treatment of human erythrocytes with malonyldialdehyde (MDA). a product of fatty acid peroxidation, induced membrane rigidity, cellular dehydration and reduced whole cell deformability. These effects of MDA were blocked by histamine and fluorescamine, which can act as alternate substrates for MDA. Additionally, reduced deformability of MDA‐treated rabbit cells was associated with shortened 51Cr survival in vivo. These findings suggest a biochemical basis for decreased survival of erythrocytes undergoing peroxidative damage of the membrane.

Concurrent sickle cell anemia and alpha-thalassemia. Effect on pathological properties of sickle erythrocytes.

The concurrence of sickle cell anemia and alpha-thalassemia results in less severe hemolytic anemia apparently as a result of reduced intraerythrocytic concentration of hemoglobin S and its retarded polymerization. We have evaluated the effect of alpha-globin gene number on several interrelated properties of sickle erythrocytes (RBC) that are expected to correlate with the hemolytic and rheologic consequences of sickle cell disease. The irreversibly sickled cell number, proportion of very dense sickle RBC, and diminished deformability of sickle RBC each varied directly with alpha-globin gene number. Sickle RBC density was a direct function of the mean corpuscular hemoglobin concentration (MCHC). Even in nonsickle RBC, alpha-globin gene number varied directly with RBC density. Despite differences in alpha-globin gene number, sickle RBC of the same density had the same degree of deformability and dehydration. These data indicate that the fundamental effect of alpha-thalassemia is to inhibit the generation of sickle RBC having high density and MCHC, and that the other beneficial effects of sickle RBC are secondary to this process. The less consistent effect on overall clinical severity reported for subjects with this concurrence may reflect an undefined detrimental effect of alpha-thalassemia, possibly on the whole blood viscosity or on sickle RBC membrane-mediated adherence phenomena.

Effects of Oxygen Inhalation on Endogenous Erythropoietin Kinetics, Erythropoiesis, and Properties of Blood Cells in Sickle-Cell Anemia

The role of oxygen therapy in sickle-cell anemia is not established, and its effects on erythropoiesis and on the rheologic properties of sickled erythrocytes are controversial. When three patients with sickle-cell anemia who were not in crisis or infected breathed oxygen at a rate of 5 liters per minute continuously through nasal prongs for five days, there was a rapid decline in erythropoietin levels that had initially been elevated, a delayed fall in the number of reticulocytes, and a fall in the number of irreversibly sickled cells, which, in two of the subjects, preceded the suppression of reticulocytosis. After cessation of oxygen therapy, erythropoietin levels and the number of irreversibly sickled cells increased promptly, followed by an increase in the number of reticulocytes. Calculated erythropoietin half-lives were 1.51 to 2.92 hours, and clearances were 43 to 84 ml per minute during oxygen administration. These are normal values. In two subjects, the number of irreversibly sickled cells rose to exceed base-line values after oxygen therapy was discontinued, and both subjects had acute painful episodes at this time. We conclude that in patients with sickle-cell anemia, substantial changes in erythropoiesis and in the rheologic properties of blood occur in association with oxygen inhalation and that when oxygen therapy is administered to such patients, it should be given intermittently rather than continuously.

Separate Mechanisms of Deformability Loss in ATP-depleted and Ca-loaded Erythrocytes

Membrane rigidity has been widely accepted as the dominant cause of reduced deformability both of ATP-depleted erythrocytes and erythrocytes containing excess calcium (Ca). However, recent studies have shown normal membrane deformability in ATP-depleted erythrocytes. In addition, Ca accumulation causes massive ion and water loss, and it has been shown that extensive dehydration causes an increase in intracellular viscosity with attendant loss of whole cell deformability. To obtain a detailed understanding of the processes accompanying ATP depletion and/or Ca accumulation that limit cell deformability, we have used a viscodiffractometric method to identify the cellular factors contributing to reduced whole cell deformability. Analysis of the influence of the suspending medium osmolality on deformability showed the presence of two independent processes. One was a Ca-independent reduction in cell surface area/volume ratio, resulting from the spheroechinocyte formation that follows total ATP consumption. The other was a Ca-dependent increase in intracellular viscosity resulting from a Ca-induced loss of intracellular potassium and water. This deformability loss due to increased intracellular viscosity was found for cells depleted of ATP in the presence of Ca and in cells treated with Ca and A23187 without prior depletion. Ionophore-treated cells at high Ca concentration (>500 muM) formed spheroechinocytes with reduced surface area and a further loss of whole cell deformability. The rate of deformability loss associated with Ca-induced spheroechinocytosis was much more rapid than that associated with ATP-depletion-induced spheroechinocytosis, suggesting different mechanisms for the morphologic changes. No major effects of altered membrane elasticity on the reduced deformability of either ATP-depleted or Ca-loaded cells were observed.

Monovalent cation transport in irreversibly sickled cells.

Using discontinuous density gradients of Stractan II, we have separated sickle cell blood into discrete subpopulations of reticulocytes, mature discoid cells, and irreversibly sickled cells (ISCs). We have measured active and passive fluxes of monovalent cations in mature discoid cells, ISCs, and normal control cells, also separated upon density gradients. These measurements revealed a decreased active cation transport in ISC-rich populations. However, parallel measurements of Na, K-ATPase activity showed normal ouabain-sensitive ATPase activity in ISCs. Passive permeability to external Rb was also normal in ISCs. The observation of depressed pump activity in intact ISCs, contrasted with normal ATPase activity in ISC membranes, suggests the presence of factors in the intact cell which inhibit the active transport of Na and K in ISCs.

Study on the dehydrating effect of the red cell Na+/K+-pump in nystatin-treated cells with varying Na+ and water contents

Using the antibiotic Nystatin, we have developed a systematic method for the preparation of red blood cells with independently selected levels of intracellular Na+ concentrations and water content. Such cells provided an experimental model to study the effect of Na+/K+ pump stimulation on red cell water content. Even in initially dehydrated cells, stimulation of the Na+/K+ pump by elevated intracellular Na+ caused subsequent further loss of cell water. Cell water loss was reflected in decreased monovalent cation content per unit mass of hemoglobin and by a shift in the density distribution of the cell populations to higher densities on discontinuous Stractan gradients. We conclude that the 3 Na+out : 2 K+in stoichiometry of the Na+/K+ pump results in a net desalting effect with increased pump activity. Under the conditions of these experiments, the cell appears to have no effective mechanism to compensate for a net loss of ions and water.

Hydration of sickle cells using the sodium ionophore Monensin: a model for therapy.

Mean cell hemoglobin concentration (MCHC) is thought to have an important influence in sickle cell disease, both through the strong dependence of sickling rates on hemoglobin S concentration, and through the profoundly limiting effect of high MCHC on the rheologic competence of oxygenated, irreversibly sickled cells (ISC). Recent studies have tested the ability of antidiuretic hormone to reduce sickle cell MCHC by reducing plasma sodium (Na) and osmolality. An alternative means of reducing MCHC is to elevate intracellular cation content, rather than to depress extracellular cation concentration. In an effort to do this, we have treated sickle cells with Monensin, an antibiotic that selectively enhances membrane Na permeability. At submicromolar concentrations, Monensin substantially reduced the MCHC of whole sickle blood and isolated ISC, causing an improvement in cell deformability. Monensins effectiveness in producing a controlled increase in erythrocyte water content suggests that agents that selectively increase membrane Na permeability could be therapeutically useful.

AUR memorial Award. Induced alignment of flowing sickle erythrocytes in a magnetic field. A preliminary report.

Deoxygenated sickle erythrocytes in static suspension align perpendicular to a magnetic field. To assess the importance of this observation to MRI of sickle-cell disease, an in vitro flow apparatus was devised and the orientation of sickle erythrocytes flowing through a 0.38 T magnetic field was investigated. We showed a significant perpendicular alignment of fully deoxygenated sickle erythrocytes flowing at 3 to 4 mm/minute (P less than .001). These results suggest that deoxygenated erythrocytes in a sickle-cell patient could orient perpendicular to a magnetic field, and therefore that MRI of such patients could possibly result in worsening of vaso-occlusive complications. Further studies are needed to assess the possible hazards of MRI of sickle-cell disease, especially at high field strengths.