Characterization and comparison of the red blood cell membrane damage in severe human alpha- and beta-thalassemia.

The aim of the present work was to understand the pathophysiology of the severe human thalassemias as represented by beta-thalassemia intermedia and hemoglobin (Hb) H (alpha-thalassemia) disease. We have previously shown that the material properties of the red blood cell (RBC) and its membrane differ in severe alpha- and beta-thalassemia, and we now show that this difference is probably caused by accumulation of alpha-globin chains at the cytoskeleton in beta-thalassemia, whereas beta-globin chains are associated with the cytoskeleton in alpha-thalassemia. In both alpha- and beta-thalassemia, some of these globin chains have become oxidized as evidenced by loss of the free thiols. Furthermore, there is similar evidence of oxidation of protein 4.1 in beta-thalassemia, whereas beta-spectrin appears to be subject to oxidation in alpha-thalassemia. These observations support the idea that the association of partly oxidized globin chains with the cytoskeleton results in oxidation of adjacent skeletal proteins. The abnormality of protein 4.1 in beta-thalassemia is consistent with a prior observation, and is also in accord with the known importance of protein 4.1 in maintenance of membrane stability, a property that is abnormal in beta-thalassemic membranes.

Hereditary stomatocytosis: consistent association with an integral membrane protein deficiency.

We studied the RBC membrane proteins of four patients, including a mother and daughter, with hereditary stomatocytosis. One- and two-dimensional gel electrophoresis revealed that a 28 kDa integral protein, present in normal RBC membranes, was absent in all four patients. This abnormality, reported once previously (Lande et al, 1982), appears to be a characteristic feature of hereditary stomatocytosis, and may be related to the underlying permeability defect in this disorder.

Elevated fasting breath hydrogen and abnormal hydrogen breath tests in children with sickle cell disease: a preliminary report.

Hydrogen breath tests were performed in eight children with sickle cell disease (Hgb S-S), four of whom were growth retarded. Average base-line breath H2 values after an overnight fast were elevated (22.4 +/- 10.8 ppm; normal = 7.1 +/- 5.0). Breath H2 concentrations increased significantly above base line within 30-40 min after lactulose ingestion in the four growth-retarded children whereas a negligible rise was observed in the four with normal growth indices. Breath H2 production for each time interval in the first 120 min was greater in all Hgb S-S then in normal children (p less than 0.01 for each time interval measured before 60 min). The results indicate that children with sickle cell disease have intestinal abnormalities favoring excess production of H2 in the fasted state combined with early elevations in postlactulose breath H2 in those with growth retardation. The possible role of disordered gastrointestinal motility and/or anomalously distributed intestinal flora in growth retardation of children with Hgb S-S requires further investigation.

The incidence of painful crisis in homozygous sickle cell disease: correlation with red cell deformability.

To determine whether the vasoocclusive severity of homozygous sickle cell (SS) disease is influenced by cellular dehydration, we correlated the incidence of painful crisis with steady-state measurements of red cell hydration. Sixteen children with SS disease were followed for 3.3 to 8 years (mean, 6.8 years), and a single crisis rate was calculated for each patient. At the time of well visits, cellular hydration was assessed by measuring cell deformability, the percentage of red cells with a density greater than or equal to 1.1056 g/mL, and the percentage of irreversibly sickled cells (ISC). The incidence of painful crisis showed a strong positive correlation with Omax, a deformability measurement reflecting cellular hydration (r = .84, P less than .002), and with hemoglobin concentration (r = .59, P = .04). That is, higher crisis rates were observed in patients with less dehydrated, more deformable red cells and also in patients with higher hemoglobin concentrations. Furthermore, cell deformability and hemoglobin concentration were independent predictors of the incidence of painful crisis, which is consistent with separate effects of these two red cells parameters on vasoocclusive severity.

Bacteremia in sickle hemoglobinopathies.

We analyzed 178 episodes of bacteremia that occurred during 13,771 patient-years of follow-up of 3451 patients with sickle hemoglobinopathies. Age-specific incidence rates of bacteremia were calculated for patients with sickle cell anemia (SS) and sickle cell-hemoglobin C (SC) disease. The incidence rate was highest among children with SS and SC younger than age 2 years. Children with SC showed an abrupt decrease after age 2 years, whereas children with SS had a gradual decline in rate from 2 to 6 years of age. The predominant pathogen in patients younger than 6 years was Streptococcus pneumoniae (66%); gram-negative organisms were responsible for 50% of bacteremias in patients 6 years and older. Urinary tract infection was present during 73% of Escherichia coli bacteremias, and 77% of Salmonella bacteremias were associated with osteomyelitis. In contrast, no focus of infection was present in 52% of pneumococcal bacteremias. The incidence of pneumococcal bacteremia in children with SS younger than age 3 years was 6.1 events/100 patient-years; the case fatality rate for pneumococcal sepsis in this age group was 24%. No hematologic or demographic variables were associated with occurrence of pneumococcal bacteremia in young children. Retrospective analysis of pneumococcal bacteremia suggests that the prophylactic use of penicillin may decrease the incidence in children younger than 3 years of age.

Haemolytic anaemia associated with increased cation permeability.

Volume regulation in the red cell depends on the balance between the osmotic swelling pressure contributed by the polyvalent anions 2,3-DPG and haemoglobin, and the counterbalancing osmotic pressure provided by extracellular Na+. Although changes in 2,3-DPG have been identified in some cases, increases in the electrodiffusional leak of Na+ and K+ are most likely responsible for the disorders of volume regulation leading to haemolytic anaemia. The current challenge is to identify the membrane proteins and/or lipids which play a key role in passive transport of the monovalent cations.

Two-dimensional electrophoretic analysis of human erythrocyte cylindrin.

Cylindrin, a macromolecule isolated from the human erythrocyte, and the band 7 proteins of the erythrocyte membrane were analyzed by one- and two-dimensional electrophoresis. Cylindrin was recovered from both the cytosol and cell membranes of hypotonically lysed erythrocytes, and its identity was confirmed by electrophoresis and transmission electron microscopy. Cylindrin from either source produced eight bands on one-dimensional SDS gels, and seventeen spots on two-dimensional gels, revealing a more complex composition than previously reported. It is unlikely that this complexity was due to proteolysis, since preparations of cylindrin with various protease inhibitors gave the same electrophoretic patterns. Mixing experiments showed that the polypeptide subunits of the cylindrin complex are distinct from the band 7 proteins of the erythrocyte membrane. This finding failed to support a role for the cylindrin macromolecule in the permeability disorders of the erythrocyte membrane associated with a missing band 7 protein.

Influence of pH on the rheologic and antisickling effects of dimethyl adipimidate.

Dimethyl adipimidate, a bifunctional imidoester, is an effective inhibitor of sickling in vitro. We determined the reaction conditions necessary to inhibit sickling without impairing cell deformability. Although imidoesters are routinely used at alkaline pH, we found that reaction of red blood cells with dimethyl adipimidate at pH 8.4 shortened the survival of rat red blood cells and impaired the deformability of human red blood cells. These adverse effects were eliminated by changing the pH of the reaction from 8.4 to 7.4. Furthermore, treatment of sickle cells with dimethyl adipimidate at pH 7.4 inhibited sickling, as demonstrated by dose-dependent reductions in the percentage of sickle forms, viscosity, and potassium efflux. Dimethyl adipimidate appeared to inhibit sickling by several mechanisms: namely, it increased deoxygenated sickle hemoglobin solubility, oxygen affinity, and cell hydration. We conclude that dimethyl adipimidate at pH 7.4 can inhibit sickling without impairing the rheologic properties of the red blood cell.

Prolongation of sickle cell survival by dimethyl adipimidate is compromised by immune sensitization.

The effect of dimethyl adipimidate (DMA), an amino-reactive crosslinking reagent with demonstrated antisickling properties in vitro, on the survival of 51Cr-labeled autologous sickle cells was evaluated in five adult males with sickle cell anemia. The survival of cells pretreated with 5 mmol/L DMA (pH 7.4), normal (t1/2 28-33 days) in four subjects and near-normal (t1/2 20 days) in the fifth, was considerably longer than that usually observed in sickle cell disease. In fact, the effect of DMA on the survival of sickle cells in vivo equals or exceeds that of any other agent tested to date. In three subjects, the survival of a second infusion of DMA-treated red cells was much shorter (t1/2 1.8, 3, 4.5 days) than in the initial study. An antibody was detected in the serum of these subjects that was directed to DMA-treated red cells. Modification of the immunogenicity of treated cells will be required before further consideration of DMA for use in the therapy of sickle cell anemia.

Missing band 7 membrane protein in two patients with high Na, low K erythrocytes.

We investigated the erythrocyte membrane proteins of two patients with congenital hemolytic anemia due to increased permeability of the erythrocyte membrane to Na and K (hereditary stomatocytosis and cryohydrocytosis). One-dimensional sodium dodecyl sulfate (SDS) gel electrophoresis resolved the band 7 erythrocyte membrane proteins into three components with approximate molecular weights of 30,000, 28,000, and 26,000. The 28,000-dalton component was decreased in both patients with permeability disorders. Two-dimensional electrophoresis (nonequilibrium pH gradient electrophoresis in the first dimension combined with SDS gel electrophoresis in the first dimension combined with SDS gel electrophoresis in the second dimension) resolved the 28,000-dalton component from normal erythrocyte membranes into two proteins with different isoelectric points, designated 22 x 8 and 60 x 8. In the patients with hereditary stomatocytosis and cryohydrocytosis, 22 x 8 was completely absent, whereas 60 x 8 was detected as usual. In contrast, all the band 7 proteins (including 22 x 8) were invariably present in a survey of normal subjects and reticulocytosis controls. The unique finding of a missing band 7 protein in the patients with hereditary stomatocytosis and cryohydrocytosis raises the possibility that the absence of this protein is responsible for the increased Na and K permeability in these disorders.

Concurrent sickle-cell anemia and alpha-thalassemia: effect on severity of anemia.

We studied 47 patients with sickle-cell anemia to determine the effect of alpha-thalassemia on the severity of their hemolytic anemia. We diagnosed alpha-thalassemia objectively by using alpha-globin-gene mapping to detect alpha-globin-gene deletions, studying 25 subjects with the normal four alpha-globin-genes, 18 with three, and four with two. The mean hemoglobin, hematocrit, and absolute reticulocyte levels (+/- S.D.) were 7.9 +/- 0.9 g per deciliter (4.9 +/- 0.6 mmol per liter), 22.9 +/- 2.9 per cent, and 501,000 +/- 126,000 per cubic millimeter, respectively, in the non-thalassemic group; 9.8 +/- 1.6 g per deciliter (6.1 +/- 1.0 mmol per liter), 29.0 +/- 5.0 per cent, and 361,000 +/- 51,000 per cubic millimeter in the group with three alpha-globin genes; and 9.2 +/- 1.0 g per deciliter (5.7 +/- 0.6 mmol per liter), 27.5 +/- 3.0 per cent, and 100,000 +/- 15,000 per cubic millimeter in the group with two alpha-globin genes. Deletion of alpha-globin genes was also accompanied by a decreased mean corpuscular hemoglobin concentration (MCHC) in post-reticulocyte erythrocytes and by increased hemoglobin F levels. The decreased intraerythrocytic hemoglobin S concentration and elevated hemoglobin F levels associated with alpha-thalassemia appear to diminish the degree of hemolytic anemia found in sickle-cell disease.

Membrane effects of imidoesters in hereditary stomatocytosis.

The marked increase in cation (Na+, K+) permeability that results in swollen, cup-shaped red cells in the hereditary stomatocytosis syndrome can be corrected in vitro with a bifunctional crosslinking reagent, dimethyl adipimidate (DMA). 45Ca influx in intact RBC, 45Ca efflux in red ghosts, and 45Ca retention in red ghosts are normal and not influenced by DMA. Endocytosis in resealed red ghosts is strikingly impaired but becomes normal if cells are first treated with 2 mM DMA. Protein kinase mediated phosphorylation of membrane proteins by AT32P--only 20--40% of normal control values in both shortterm (5 min) and more extended (60 min) incubations--is not improved by DMA. After reaction of 14C-DMA with stomatocytes, radiolabel is found associated with phosphatidyl serine and phosphatidyl ethanolamine and is also widely distributed among membrane proteins. Cation permeability of stomatocytes in corrected at DMA concentrations (1 mM) that result in barely detectable crosslinking of aminophospholipids or proteins, suggesting that either crosslinking of a minor component present in only small quantities or intramolecular (rather than intermolecular) crosslinking is responsible for the permeability effects. DMA, whose maximal crosslinking dimension is 7.3--9 A, is the most effective bifunctional imidoester of those tested. Shorter (dimethyl malonimidate) or longer (dimethylsuberimidate) reagents are either less effective than DMA or totally without effect.