Thalassemic Red Blood Cells Research Articles

Oxidative Red Blood Cell Membrane Injury in the Pathophysiology of Severe Mouse β-Thalassemia

In severe human beta-thalassemia, the pathophysiology relates to accumulation of excess alpha-globin chains at the membrane. One hypothesis is that membrane-associated alpha-globin by virtue of it's iron or hemichromes produces oxidation of adjacent membrane proteins. The availability of a mouse model of severe beta-thalassemia, as well as a transgenic (thalassemic-sickle) mouse that expresses 12% of human beta s-chain, has allowed us to study the effect of graded accumulation of alpha-chains at the red blood cell (RBC) membrane on the clinical status of the animal and on the material properties of its RBCs. Proteins from control, beta-thalassemic, and transgenic mouse RBC membranes were analyzed for evidence of oxidation, as measured by thiol-disulfide exchange chromatography, which detects intramolecular sulfhydryl oxidation. Ratios of oxidized globin to protein 7 were calculated and increased amounts were seen in thalassemic mice as compared with control mice and transgenic mice. Furthermore, there were increased amounts of thiol-free protein 4.1 in the thalassemic mice, compared with very small amounts in the control mice and intermediate amounts in the transgenic mice. Membrane mechanical stability as assessed by ektacytometry showed that the thalassemic mouse RBCs were markedly unstable. Transgenic mouse RBCs showed intermediate levels of membrane instability compared with the controls. We propose that this oxidized globin, in conjunction with oxidized protein 4.1, accounts (at least in part) for membrane instability. A 12% increase in beta s-globin chain synthesis (by decreasing excess globin available) confers considerable protection against both oxidative damage and the consequent membrane instability.

Improvement of Mouse β-Thalassemia by Recombinant Human Erythropoietin

Abstract Homozygous beta thalassemic mice received 50 U (1,660 U/kg) of recombinant human erythropoietin (rhEpo) 5 days a week for 2 weeks. Hemoglobin increased from 9.2 +/- 0.6 g/dL to 10.5 +/- 0.4 g/dL (P = .002) and hematocrit increased from 29.2% +/- 0.9% to 34.1% +/- 1.9% (P = .0014). The beta minor/alpha globin chain synthesis ratio increased slightly but significantly between day -4 (0.75 +/- 0.07) and day 4 (0.81 +/- 0.04) (P = .01) and reached a minimum ratio (0.67 +/- 0.03) on day 15 (P = .001), being parallel to reticulocyte counts and to the incorporated trichloracetic acid (TCA)-insoluble radioactivity, therefore parallel to the erythropoietic output in thalassemic mice, as in normal mice. Erythrocyte defects were improved in beta thalassemic mice treated by rhEpo: membrane-associated alpha globin was significantly decreased (P less than .01), thiol group reactivity of ankyrin was significantly improved (P less than .05), spectrin alterations were reduced, and deformability of mouse thalassemic red blood cells was normalized. These results provide experimental criteria for modulating globin chain imbalance necessary for the therapy of human beta thalassemia intermedia, and suggest that rhEpo might be of interest to improve the red blood cell mass and reduce erythrocyte alterations in this disease.

Improvement of mouse beta-thalassemia by recombinant human erythropoietin

Homozygous beta thalassemic mice received 50 U (1,660 U/kg) of recombinant human erythropoietin (rhEpo) 5 days a week for 2 weeks. Hemoglobin increased from 9.2 +/- 0.6 g/dL to 10.5 +/- 0.4 g/dL (P = .002) and hematocrit increased from 29.2% +/- 0.9% to 34.1% +/- 1.9% (P = .0014). The beta minor/alpha globin chain synthesis ratio increased slightly but significantly between day -4 (0.75 +/- 0.07) and day 4 (0.81 +/- 0.04) (P = .01) and reached a minimum ratio (0.67 +/- 0.03) on day 15 (P = .001), being parallel to reticulocyte counts and to the incorporated trichloracetic acid (TCA)-insoluble radioactivity, therefore parallel to the erythropoietic output in thalassemic mice, as in normal mice. Erythrocyte defects were improved in beta thalassemic mice treated by rhEpo: membrane-associated alpha globin was significantly decreased (P less than .01), thiol group reactivity of ankyrin was significantly improved (P less than .05), spectrin alterations were reduced, and deformability of mouse thalassemic red blood cells was normalized. These results provide experimental criteria for modulating globin chain imbalance necessary for the therapy of human beta thalassemia intermedia, and suggest that rhEpo might be of interest to improve the red blood cell mass and reduce erythrocyte alterations in this disease.

Impaired Erythrocyte Methemoglobin Reduction in Sickle Cell Disease: Dependence of Methemoglobin Reduction on Reduced Nicotinamide Adenine Dinucleotide Content

Abstract We have examined aspects of methemoglobin (metHb) reduction in sickle and in thalassemic red blood cells (RBCs). NADH metHb reductase activity in sickle and thalassemic RBCs was significantly increased compared with normal RBCs. Because in vitro enzyme activity does not necessarily represent in vivo activity, we measured the rate of metHb reduction in intact RBCs. Intact thalassemic RBCs demonstrated a significantly increased rate of metHb reduction compared with normal RBCs. In contrast, intact sickle RBCs had a rate of metHb reduction that was similar to normal RBCs and significantly decreased relative to high reticulocyte RBCs of equivalent cell age. To determine the mechanism for the relative impairment of metHb reduction in sickle RBCs, we measured intraerythrocytic NADH, a cofactor in the metHb reduction reaction. Thalassemic RBCs had a significantly increased NADH content relative to normal RBCs. In contrast, sickle RBCs did not have an increase in NADH content. Furthermore, incubating normal RBCs under conditions that increase the NADH content resulted in an increased rate of metHb reduction. In contrast, conditions that decrease the NADH content in normal RBC resulted in a decreased rate of metHb reduction. These data and other results suggest that metHb reduction in intact RBCs is dependent on NADH content, and that the impaired metHb reduction rate in sickle RBCs may be a result of a lack of increase in NADH content. The dependence of metHb reduction on RBC NADH content and the ability to manipulate NADH content in vitro suggest a new strategy for decreasing oxidant damage to sickle RBCs in vivo.

Impaired erythrocyte methemoglobin reduction in sickle cell disease: dependence of methemoglobin reduction on reduced nicotinamide adenine dinucleotide content

We have examined aspects of methemoglobin (metHb) reduction in sickle and in thalassemic red blood cells (RBCs). NADH metHb reductase activity in sickle and thalassemic RBCs was significantly increased compared with normal RBCs. Because in vitro enzyme activity does not necessarily represent in vivo activity, we measured the rate of metHb reduction in intact RBCs. Intact thalassemic RBCs demonstrated a significantly increased rate of metHb reduction compared with normal RBCs. In contrast, intact sickle RBCs had a rate of metHb reduction that was similar to normal RBCs and significantly decreased relative to high reticulocyte RBCs of equivalent cell age. To determine the mechanism for the relative impairment of metHb reduction in sickle RBCs, we measured intraerythrocytic NADH, a cofactor in the metHb reduction reaction. Thalassemic RBCs had a significantly increased NADH content relative to normal RBCs. In contrast, sickle RBCs did not have an increase in NADH content. Furthermore, incubating normal RBCs under conditions that increase the NADH content resulted in an increased rate of metHb reduction. In contrast, conditions that decrease the NADH content in normal RBC resulted in a decreased rate of metHb reduction. These data and other results suggest that metHb reduction in intact RBCs is dependent on NADH content, and that the impaired metHb reduction rate in sickle RBCs may be a result of a lack of increase in NADH content. The dependence of metHb reduction on RBC NADH content and the ability to manipulate NADH content in vitro suggest a new strategy for decreasing oxidant damage to sickle RBCs in vivo.

Impaired antioxidant defense in hemoglobin E-containing erythrocytes: a mechanism protective against malaria?

Red blood cell (RBC) antioxidant defense was investigated in eight individuals with hemoglobin E (Six EE and two E-B(+) thalassemia) and compared to that in six individuals with thalassemia and ten normal subjects. Individuals with hemoglobin E had increased incubated Heinz body formation (68% +/- 18%; p less than 0.001) compared to normal and thalassemic RBC (10% +/- 2% and 11% +/- 5%, respectively). Stimulated pentose phosphate shunt activity was increased in the thalassemic and decreased in the hemoglobin E RBC as compared to normal. The 2,3-diphosphoglycerate (DPG) content of the EE RBC was increased to 5.59 +/- 0.69 mumol/ml RBC as compared to normal (4.51 +/- 0.77; p less than 0.001). In the EE RBC, there was a direct correlation between Heinz body formation and DPG content (r = 0.73). Ascorbic and dehydroascorbic acid (0.1 and 1.0 mM) were able to decrease the degree of Heinz body formation in the hemoglobin E RBC. Ascorbic acid (0.1 mM) prolonged the response of the pentose shunt. Thus impaired antioxidant defense may account for the persistence of the hemoglobin E gene in areas where malaria is endemic. Oxidant medications should be used with caution in individuals of Southeast Asian origin.

Quantitative changes of red blood cell shapes in relation to clinical features in beta-thalassemia/HbE disease.

Quantitative analysis of red blood cell (RBC) shapes was performed in 79 patients with beta-thalassemia/HbE disease, using scanning electron microscopy (SEM). Most patients had many abnormal RBC shapes, namely: torocytes, codocytes, dacryocytes, keratocytes, and schizocytes with a reduction in the number of normally shaped red blood cells (discocytes). Splenectomy, larger splenic sizes, and increasing anemia all were associated with a further reduction in the number of discocytes, reflecting increasing numbers of abnormally shaped RBC. The number of torocytes was increased with anemia; a larger spleen was associated with a decrease and splenectomy with an increase in the number of torocytes, indicating that they are destroyed by the spleen. The numbers of codocytes and echinocytes were singularly associated with splenectomy. Most likely, almost all of the echinocytes are destroyed by the spleen. The red blood cell types that occur as a result of RBC fragmentation, namely, dacryocytes, keratocytes, and schizocytes, were remarkably associated with the splenic status; larger splenic size was associated with an increased number of these cells, while splenectomy was associated with a noticeable reduction in their numbers. This finding confirms the role of the spleen in the fragmentation of thalassemic RBC. An increasing degree of anemia did not affect the number of codocytes and echinocytes, but was associated with a mild increase in the number of fragmented RBC and a significant increase in the number of torocytes.

Microwave dielectric properties of normal and homozygous beta-thalassemic erythrocytes

Recently, the dielectric properties of haemoglobin's suspending medium inside human erythrocytes have been studied in the frequency range from 0.1 to 250 MHz (Jenin and Schwan Biophys. J., vol.30, p.235, 1980). It was found that in this frequency range, the erythrocyte water content exhibits rotational mobilities similar to those in pure liquid and the interior of erythrocytes has electrical properties consistent with those of a standard haemoglobin solution. In particular, dielectric and conductometric measurements have shown no evidence of an interaction between the cell membrane and the adjacent water. To verify these conclusions by direct observations, the authors present measurements on the dielectric properties of erythrocyte suspensions, carried out at a frequency of 10.0 GHz in the temperature interval from 5 to 45 degrees C. They also present a comparative analysis between the dielectric properties of homozygous beta -thalassemic red blood cells and normal cells.

Demonstration of a Natural Antigalactosyl IgG Antibody on Thalassemic Red Blood Cells

A modified antiglobulin test, based on the high affinity between the Fc portion of the red blood cell (RBC) bound IgG and the Fc receptor on the myeloid cell K-562, was utilized for demonstration of immunoglobulins (Ig) on thalassemic RBC. Ig was found on the RBC of 73 out of 80 patients with thalassemia. The immunoglobulins on the thalassemic RBC belonged to the IgG subclass and were autoreactive. Elution studies utilizing various carbohydrates, or by thermal stripping, indicated that at least part of the IgG molecules found on the thalassemic RBC were specifically reactive with terminal galactosyl residues on the RBC membrane. IgG antibodies with similar reactivity were also demonstrated in normal human serum. These natural antigalactosyl IgG antibodies from normal sera could bind to IgG-depleted thalassemic RBC. Thalassemic RBC and normal senescent RBC were previously found to contain reduced amounts of membrane sialic acid (SA). It is suggested that the antigalactosyl IgG antibodies interact with newly exposed galactosyl residues underlying the sialic acid units. Such interaction may lead to the shortened lifespan of thalassemic RBC and may result in sequestration of senescent normal RBC by the reticuloendothelial system.

Demonstration of a natural antigalactosyl IgG antibody on thalassemic red blood cells

A modified antiglobulin test, based on the high affinity between the Fc portion of the red blood cell (RBC) bound IgG and the Fc receptor on the myeloid cell K-562, was utilized for demonstration of immunoglobulins (Ig) on thalassemic RBC. Ig was found on the RBC of 73 out of 80 patients with thalassemia. The immunoglobulins on the thalassemic RBC belonged to the IgG subclass and were autoreactive. Elution studies utilizing various carbohydrates, or by thermal stripping, indicated that at least part of the IgG molecules found on the thalassemic RBC were specifically reactive with terminal galactosyl residues on the RBC membrane. IgG antibodies with similar reactivity were also demonstrated in normal human serum. These natural antigalactosyl IgG antibodies from normal sera could bind to IgG- depleted thalassemic RBC. Thalassemic RBC and normal senescent RBC were previously found to contain reduced amounts of membrane sialic acid (SA). It is suggested that the antigalactosyl IgG antibodies interact with newly exposed galactosyl residues underlying the sialic acid units. Such interaction may lead to the shortened lifespan of thalassemic RBC and may result in sequestration of senescent normal RBC by the reticuloendothelial system.

Phagocytosis of Nucleated and Mature β Thalassaemic Red Blood Cells by Mouse Macro‐phages in Vitro

Physiological or experimental decrease in sialic acid (SA) content on the red blood cell (RBC) membrane is believed to play an important role in the recognition of these cells by macrophages. Since there is a 20-30% decrease in the SA content on the membrane of thalassaemic RBC, the interaction between macrophages and these RBC was studied in vitro. Using mouse peritoneal macrophages, it was found that these macrophages 'recognize' and phagocytize thalassaemic RBC while RBC from normal donors are hardly phagocytized. The average level of phagocytosis of thalassaemic RBC from splenectomized patients was found to be 22-fold higher than that of RBC from normal donors. The phagocytized cells consisted of both mature and nucleated RBC. Mouse peritoneal macrophages seem to be a useful in vitro system for the study of the accelerated sequestration and shortened life span of thalassaemic RBC.

“Lysosomal” enzyme activities in red blood cells of normal individuals and patients with homozygous β-thalassaemia

Four hydrolases, β-galactosidase, β-glucuronidase, β- N-acetylglucosaminidase and acid phosphatase were examined in red blood cells (RBC) of normal donors and patients with homozygous ß-thalassaemia. Highly sensitive fluorimetric substrates were used to determine the specific activities of these enzymes. In order to avoid contamination by lysosomal activities derived from white blood cells (WBC), the mature RBC were separated from other blood elements by cellulose chromatography. The hydrolase activities in normal RBC were detected only in their plasma membranes and were found to be considerably lower than in WBC or platelets. In thalassaemic RBC, hydrolase activities were present in both plasma membranes and in the soluble fraction. The normoblast fraction contributed most of the hydrolase activity found in these preparations, suggesting the presence of lysosomal particles in thalassaemic RBC. No differences in the enzymatic activities were found when purified membranes of mature RBC from thalassemic and normal preparations were compared. The origin and roles of these hydrolytic enzymes in normal and thalassaemic RBC membranes are not known.

Increased Permeability of Erythrocyte Membrane in Thalassemia

Abstract Increased sodium efflux is demonstrated in vitro in thalassemic red blood cells (and in those of patients with some other hemolytic conditions). This increase, in the presence of either normal or augmented sodium accumulation, must reflect an increased sodium leak into the cells. In vivo, the intracellular content of sodium and potassium is nearly normal. In vitro, however, thalassemia major RBC incubated for 24 hr undergo increased sodium gain, with somewhat more potassium loss. The resultant loss of cations presumably leads to intracellular dehydration. The converse obtains in hereditary spherocytosis, insofar as a sodium gain greater than the potassium loss leads to accumulation of water. It is concluded that increased permeability of the erythrocyte membrane often reflects an underlying hemolytic process. It is postulated that inability to maintain a normal composition of cations and water plays a part in the pathogenesis of hemolysis in thalassemia.