Stephen B. Shohet

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.

Congenital Hemolytic Anemia with High Sodium, Low Potassium Red Cells

Abstract Evaluation of the erythrocytes of a child with hemolytic anemia of unknown cause showed that the intracellular concentrations of sodium and potassium were 100 mEq per liter of cells and 40...

Erythrocyte Membrane Lipid Reorganization during the Sickling Process

Summary. In order to study possible alterations in membrane lipids during sickling, we have measured the difference in susceptibility to lipid peroxidation, binding of trinitrobenzenesulfonic acid (TNBS) to aminophospholipids, and fatty acid uptake in cells containing sickle haemoglobin under aerobic and anaerobic conditions. We have also examined TNBS binding in irreversibly sickled cells in an attempt to evaluate the permanent effects of any such alterations.

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.

Deformability of isolated red blood cell membranes.

We have used a laser diffraction method (ektacytometry) to directly measure the membrane component of red cell deformability, without contributions from either cell geometry or internal viscosity. This technique was validated by subjecting resealed erythrocyte ghosts to manipulations previously shown to increase the membrane shear modulus. Heating above 45 degrees C, pH greater than 9.0 and less than 5.0, and micromolar concentrations of the cross-linking agents, glutaraldehyde and diamide, all reduced the deformability of resealed erythrocyte ghosts. We have applied this assay to the study of reduced cellular deformability of calcium-loaded red cells, and have shown that, for physiological concentrations of calcium, the effect of calcium on the physical properties of the membrane may be negligible when compared to its effect of promoting cell dehydration and subsequent increased cytoplasmic viscosity.

Calcium potentiates the peroxidation of erythrocyte membrane lipids

To explore the possible role of intracellular calcium in membrane lipid peroxidation, we subjected red cells to conditions designed to increase intracellular calcium levels and then measured lipid peroxidation after exposure to a peroxidant threat. Human erythrocytes were pretreated for 3 h with either very high levels of CaCl2, or with low levels in the presence of the ionophore A23187. The erythrocytes were subsequently exposed to a peroxide-generating system consisting of xanthine and xanthine oxidase, or H2O2 for 1 h at 37 degrees C. As measured by a malonyldialdehyde assay, the calcium-treated cell showed up to a 2-fold increase in lipid peroxidation in comparison to untreated cells. In experiments with the ionophore, calcium concentration-dependent effects were detected at levels as low as 10 microM and were maximal at 50 microM. A significant loss of phosphatidylserine and phosphatidylethanolamine was observed in calcium- and peroxide-treated erythrocytes. This potentiation of membrane lipid peroxidation and lipid loss could be prevented by either lipid antioxidants or EGTA. The present study shows that pretreatment of erythrocytes with calcium increases their sensitivity to lipid peroxidation. This suggests that increased calcium concentration may be a factor in the potentiation of membrane lipid peroxidation of erythrocytes known to have increased calcium levels such as sickled and senescent red cells.

Effect of L-carnitine and acetyl-L-carnitibe on the human erythrocyte membrane stability and deformability

In this study we examined the effect of carnitine and acetylcarnitine on the human erythrocyte membrane stability and membrane deformability. Since erythrocyte membranes are impermeable to these compounds, we resealed erythrocyte ghosts in the presence of different concentrations of carnitine or acetylcarnitine. Resealed ghosts can be adequately studied in their cellular deformability and membrane stability properties by means of ektacytometry. Both carnitine and acetylcarnitine alter the membrane stability but not membrane deformability of the red cell membrane. Resealed ghosts containing 20, 50, 150, and 300 microM carnitine had 1.1, 1.6, 0.9, and 0.7 times the normal stability. While resealed ghosts containing 20, 50, 150, and 300 microM acetylcarnitine had 1.1, 1.5, 1.3, and 1.2 times the normal stability. Such changes were found to be reversible. We also conducted SDS PAGE of cytoskeletal membrane proteins from membrane fragments and residual membranes produced during membrane stability analysis, and unsheared resealed membranes in those samples where we observed an increase or a decrease of membrane stability. No changes in the cytoskeletal membrane proteins were noticed, even when the samples, prior SDS PAGE analysis, were treated with or without dithiothreitol. In addition, fluorescence steady state anisotropy of DPH in the erythrocyte membrane treated with carnitine or acetylcarnitine shows no modification of the lipid order parameter. Our results would suggest that both carnitine and its acetyl-ester, at physiological concentrations, may increase membrane stability in mature erythrocytes, most likely via a specific interaction with one or more cytoskeletal proteins, and that this effect would manifest when the erythrocytes are subjected to high shear stress.

Energy Metabolism in Human Erythrocytes: I. EFFECTS OF SODIUM FLUORIDE

Exposure of red cells to fluoride produces a variety of metabolic alterations, most of which are based upon the secondary effects of enolase inhibition, which reduces pyruvate synthesis and interferes with the regeneration of diphosphopyridine nucleotide (NAD). Adenosine triphosphate (ATP) is consumed in the hexokinase and phosphofructokinase reactions but is not regenerated since the deficiency of NAD limits glyceraldehyde phosphate dehydrogenase. ATP depletion in the presence of fluoride and calcium induces a massive loss of cations and water. Of the other known sites of ATP utilization, membrane-bound ATPase is inhibited by fluoride, but the incorporation of fatty acids into membrane phospholipids is unaffected until ATP is depleted. The addition of methylene blue to fluoride-treated red cells regenerates NAD, permitting triose oxidation and the generation of 3-phosphoglycerate and 2,3-diphosphoglycerate. Enolase inhibition is then partially overcome by mass action, and sufficient glycolysis proceeds to maintain the concentration of ATP. This in turn prevents the massive cation and water loss, and permits membrane phospholipid renewal to proceed. Membrane ATPase activity is not restored by the oxidant so that normal cation leakage remains unopposed by cation pumping in red cells exposed to the combination of fluoride and methylene blue.