Acid pH induces formation of dense cells in sickle erythrocytes.

When erythrocytes from patients homozygous for hemoglobin S (SS) are swollen or exposed to pH less than 7.40, they lose K, Cl, and water through a volume and pH-dependent KCl cotransport system. We report that carbon monoxide-treated SS cells become progressively denser when incubated for eight to 12 hours in media with pH less than 7.40 (7.3 to 7.0) at constant cell 2,3-diphosphoglycerate (DPG). This phenomenon is maximal in fresh SS cells from the top and middle density fractions, and is absent in cells from the densest fraction. When AA cells are separated according to density, acid pH induces cell shrinkage in the least dense fraction of AA cells, which has considerable KCl cotransport, but produces no change in cell density in the densest fractions of AA erythrocyte, which have no KCl cotransport. These data suggest that dense cells can form in oxygenated SS erythrocytes when the KCl cotransport system is activated by acidification.

Hemoglobin Köln occurring in association with a beta zero thalassemia: hematologic and functional consequences.

Hemoglobin (Hb) Köln-beta zero thalassemia compound heterozygosity was discovered in a young Greek patient. This gave us the unique opportunity for studying the functional properties of this unstable high-oxygen affinity hemoglobin variant in red cells containing almost pure Hb Köln. The red cells of the proposita exhibit morphological alterations and hematologic indices corresponding to the presence of an unstable Hb and beta thalassemia. Globin chain synthesis confirmed the association with a beta zero thalassemia gene. Oxygen-binding curves for these cells were biphasic, indicating the presence of both heme-saturated and of approximately 20% of non-cooperative Hb Köln. The major component exhibits an increased oxygen affinity, reduced cooperativeness, and normal alkaline Bohr effect. The 35-year-old proposita is active, has not been splenectomized, and has not been transfused in several years.

Modeling sickle cell vasoocclusion in the rat leg: quantification of trapped sickle cells and correlation with 31P metabolic and 1H magnetic resonance imaging changes.

We have developed an animal model to elucidate the acute effects of perfusion abnormalities on muscle metabolism induced by different density-defined classes of erythrocytes isolated from sickle cell anemia patients. Technetium-99m (99mTc)-labeled, saline-washed normal (AA), homozygous sickle (SS), or high-density SS (SS4) erythrocytes were injected into the femoral artery of the rat and quantitative 99mTc imaging, 31P magnetic resonance spectroscopy by surface coil at 2 teslas, and 1H magnetic resonance imaging at 0.15 tesla were performed. Between 5 and 25 microliters of SS4 cells was trapped in the microcirculation of the thigh (or 1-6 x 10(7) cells per cubic centimeter of tissue). In contrast, fewer SS discocytes (SS2) or AA cells were trapped (an equivalent packed cell volume of less than 6.7 microliters and 0.3 microliters, respectively). After injection of SS4 cells an initial increase in inorganic phosphate was observed in the region of the thigh served by the femoral artery, intracellular pH decreased, and subsequently the proton relaxation time T1 reached a broad maximum at 18-28 hr. When T1 obtained at this time was plotted against the volume of cells trapped, an increase of T1 over the control value of 411 +/- 48 msec was found that was proportional to the number of cells trapped. We conclude that the densest SS cells are most effective at producing vasoocclusion. The extent of the change detected by 1H magnetic resonance imaging is dependent on the amount of cells trapped in the microcirculation and the magnitude of the initial increase of inorganic phosphate.

Clinical disorders of the red cell membrane skeleton.

The lipid bilayer of the adult red cell is supported on its inner surface by a complex arrangement of proteins known as the membrane skeleton. This filamentous network, a major component of which is a multifunctional protein called spectrin, has an essential role in determining the shape, structural integrity, and deformability of the red cell. A significant achievement of modern biochemistry and hematology has been the elucidation of the organization of the components of the membrane skeleton and their relationship to other membrane proteins and lipids. This article reviews current concepts of membrane skeleton structure and function and emphasizes recent advances which have been made in characterizing and classifying molecular defects of the skeleton which manifest clinically with changes in the shape and stability of the red cell. The pathobiology of hereditary skeletal defects associated with hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP) are comprehensively discussed. Secondary defects of the membrane skeleton occurring in glucose-6-phosphate dehydrogenase deficiency and sickle cell anemia are also briefly considered.

Hypertonic cryohemolysis of pathologic red blood cells.

Human erythrocytes suspended in hypertonic solutions undergo hemolysis when the temperature of the suspension is changed from 37 degrees C toward 0-4 degrees C. It has been suggested that the hypertonic environment causes some proteins of the skeletal network to be changed in such a way that their normal adaptation to temperature changes is prevented, thus resulting in cryohemolysis. In the present study, we compared the cryohemolysis of some pathologic red blood cells in hypertonic sucrose and NaCl to normal cells. Erythrocytes of hereditary spherocytosis (HS) were found to be significantly more fragile than all others in hypertonic sucrose, while they behaved normally in hypertonic NaCl. In contrast, erythrocytes of thalassemic patients showed decreased susceptibility to cryohemolysis, both in hypertonic sucrose and in NaCl. Autoimmune hemolytic anemia samples behaved like normal samples, both in NaCl and in sucrose. The erythrocytes of congenital dyserythropoietic anemia-type II patients showed two types of cryohemolysis; one pattern was similar to that of HS, and the other one presented normal levels in sucrose and reduced levels in NaCl. The different patterns of cryohemolysis described for the pathologic cells are thought to reflect different lesions in the membranes of the erythrocytes of the various hemolytic disorders. It is hoped that studying the cryohemolysis of abnormal red cells may contribute some illumination as to molecular interactions in intact cells in health and in disease.

pH effects on red cell deformability.

The effect of pH on erythrocyte deformability was determined in the ektacytometer. In isotonic solutions, cell deformability was essentially unaltered at pH values from 6.44 to 7.7, despite the known cell volume changes that occur over this pH range. Analysis by osmotic scan ektacytometry showed that erythrocyte deformability is homeostatically maintained constant during acidosis and alkalosis because of a balance between the countervailing effects of changes in intracellular viscosity and in the surface to volume ratio. This balance is perturbed in the sickle erythrocyte, so that acidosis improves deformability.

31P NMR study of erythrocytes from a patient with hereditary pyrimidine-5'-nucleotidase deficiency.

The composition of phosphate metabolites and the intracellular pH in erythrocytes from a patient with hereditary pyrimidine-5'-nucleotidase deficiency were examined using 31P NMR spectroscopy. Several resonances were identified in spectra from intact cells and from extracts. The 2,3-bisphosphoglycerate line intensities were normal but the NTP resonances were about twice normal due to the presence of millimolar quantities of pyrimidine phosphates. Several intense resonances were also observed in the diphosphodiester region of the spectrum. One compound contributing to these lines has been identified as cytidine diphosphocholine. The resonances of NTPs were in a position indicating that the additional triphosphates were also bound by Mg2+. Direct measurement shows that there is a nearly proportional increase in total cell Mg2+ in the patient's cells, in agreement with the interpretation of the spectra. The intracellular pH was about 0.2 unit lower in the patient's erythrocytes. This lower pH is due to the elevation in intracellular fixed negative charges and the shift in permeable anions consequent to the Donnan equilibrium. We suggest that the lower intracellular pH may explain the lower oxygen affinity of these cells in the presence of otherwise normal 2,3-bisphosphoglycerate levels and the increased Mg2+ triphosphates level, because the Mg2+ form of NTPs is known not to alter the oxygen affinity of hemoglobin under physiologic conditions. Furthermore, the lower intracellular pH can also explain the abnormalities in glycolytic intermediates observed for these cells.

Effects of isovolumetric venesection on blood viscosity, on red cell deformability, and on arterial flow velocity in chronic respiratory failure with secondary polycythemia.

Normovolemic hemodilution produced by isovolumetric venesection was done in 8 patients with polycythemia and respiratory failure secondary to chronic obstructive pulmonary disease. The 'deformability index' was studied by means of a filtration technique. The blood flow velocity in ascending aorta and right pulmonary artery was also studied by means of ultrasonic Echo-Doppler technique. The procedure showed in all patients an increase of oxygen tension and a decrease of carbonic gas tension in arterial blood. At the same time it was possible to demonstrate an improvement of red cell deformability and an increase of arterial flow velocity. Isovolumetric venesection is an important procedure in the management of patients with polycythemia secondary to respiratory failure.

Viability and function of stored sickle erythrocytes.

Functional and metabolic characteristics of fresh and three-week stored erythrocytes from patients with sickle cell anemia were compared. The storage-related changes in ATP, 2,3-DPG, and P50 in sickle erythrocytes were similar to those in control (HbA) red blood cells. After storage in CPD, sickle erythrocytes maintained significantly higher levels of 2,3-DPG (mean 2.20 +/- 0.73 mM/ml RBC) than did control cells (mean 0.36 +/- 0.13 mM/ml RBC). The posttransfusion recovery and survival of stored SS erythrocytes in autologous recipients and in an animal test system were at least as good as those before storage. Tolerance of the storage lesion by sickle erythrocytes is probably related to their young mean cell age. These results also suggest that the option of autotransfusion should be explored for selected patients with sickle cell disease in special clinical settings.

Sickle-cell hemoglobin: fall in osmotic pressure upon deoxygenation.

Macromolecules such as hemoglobin exert both kinetic and matrix effects on osmotic pressure. The kinetic osmotic pressure of sickle-cell hemoglobin is lost upon deoxygenation at physiological erythrocyte concentrations. The non-kinetic or matrix component of osmotic pressure remains relatively unchanged. Loss of thermal-osmotic activity during deoxygenation occurs throughout a hemoglobin concentration range between 2.5 and 35 g/100 ml. Deoxygenation of sickle-cell hemoglobin causes aggregation such that the matrix effect is unchanged but the kinetic (van't Hoff) effect nearly vanishes. A loss of intracellular osmotic pressure during deoxygenation could dehydrate the erythrocyte sufficiently to promote more rapid sickle-cell hemoglobin aggregation. Subsequently, complete gelation of these aggregates could cause additional water loss and thrust the sickled cell into an irreversible cycle. The osmotic pressure of normal hemoglobin does not change appreciably during deoxygenation and is essentially the same as the osmotic pressure of oxygenated sickle-cell hemoglobin.

Changes in shape of erythrocytes in rabbits on atherogenic diet and onion extracts.

The effects of administration of cholesterol alone and of cholesterol and fresh whole onion extract, on the shape of erythrocytes, were studied in albino rabbits. In animals on a cholesterol-enriched diet, the erythrocytes changed shape and showed an increased tendency to aggregate, whereas they retained their normal appearance in animals on cholesterol and onion extract, and did not differ from those of normal control rabbits.

Influence of red cell water content on the morphology of sickling.

The response of sickle cells with varying water content to alterations in oxygen tension has been studied. Cells that were severely dehydrated while sickled retained the characteristic sickled morphology even after prolonged reoxygenation. When the cell water content was increased by reduction of the suspending medium osmolality, the cells unsickled. Cells that were dehydrated before deoxygenation were unable to assume the spiculated morphology typical of sicked cells. This was true both for high mean cell hemoglobin concentration (MCHC) discoid sickle cells and for irreversibly sickled cells. When such cells were resuspended in hypotonic medium before deoxygenation, they sickled with the characteristic morphology of sickle cells with normal MCHC. The morphological behavior of Ca-loaded sickled cells as well as irreversibly sickled cells showed a major influence of increased hemoglobin concentration and extremely high internal viscosity. Constraint on cell morphology by putative membrane rigidity was not observed.

Adhesion of normal and sickle erythrocytes to endothelial monolayer cultures.

Experiments were carried out to test the hypothesis that the differences between the surfaces of erythrocytes from normal and sickle cell patients are reflected in the degree of attachment to the capillary lining. An assay was used that measured the number of 51Cr-labeled erythrocytes (normal or sickle) attaching to a monolayer of endothelium cultured from calf aortas. Under these conditions, erythrocytes from sickle cell patients adhered better to the endothelium than did those from normal patients. The results suggested that the enhanced adhesion of the sickle cells to the endothelium may be partially responsible for the increased blockage of capillaries that produce the symptoms in sickle cell anemia.

Effects of oxygen tension and pH on the ultrasonic absorption properties of sickle cells.

The ultrasonic absorption of sickle cells was compared with the morphological changes in these cells are produced by varying oxygen tensions and pH. A decrease in oxygen tension caused increased sickling and an increase in the ultrasonic absorption of the blood. An increase in pH reduced the ultrasonic absorption and decreased sickling. These findings indicate that ultrasonic absorption can detect changes in the aggregation state of sickle cell hemoglobin in situ within the red cell and that the method may be used to monitor the degree of sickling under various environmental conditions.