Prehemolytic effects of hydrogen peroxide and t-butylhydroperoxide on selected red cell properties.

To provide further understanding of how oxidative damage affects red cell membrane function, the effects of low levels of two different types of oxidants on selected red cell properties have been studied. Hydrogen peroxide (H2O2), an example of a water soluble oxidant, and t-butylhydroperoxide (tBHP), a hydrophobic hydroperoxide, were compared with respect to their effects on membrane permeability, membrane mechanical properties and binding of autologous serum antibodies to the cell surface. Whereas H2O2 treatment resulted in a dose-dependent increase in membrane permeability to potassium that was evident after one hour of oxidant exposure, cells treated with tBHP at doses up to 5 mumol/ml cells showed no immediate change in cation permeability. H2O2 also caused a marked decrease in membrane deformability, whereas tBHP-treated cells showed minimal loss of deformability. However, tBHP treatment did result in a dose-dependent increase in the susceptibility of the membrane to fragmentation under high shear stress. With exclusion of treated samples that bound excess rabbit anti-spectrin antibody, indicating exposure of intracellular components, neither agent promoted the binding of autologous serum antibody in amounts comparable to that found in vivo on high density or some pathologic red cells. Taken together, the results suggest that tBHP and H2O2 cause damage to human red cells by distinct oxidative mechanisms which do not lead directly to substantive generation of binding sites for autologous serum antibodies.

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.

Specificity of autologous antibody binding to phenylhydrazine-treated human erythrocytes: implications for models of red blood cell aging.

A fundamental difficulty in the study of red cell senescence has been that of isolating a population of cells that have been aged in vivo. It has been proposed that the induction of Heinz body formation through the use of oxidizing agents, including phenylhydrazine, can provide a model for elucidating mechanisms of normal red cell aging. In an effort to evaluate the applicability of this model, we examined the nature of autologous antibody binding to phenylhydrazine-treated cells. Using both radioisotopic and immunofluorescent probes, we confirmed the previous observation of increased immunoglobulin G (IgG) binding to phenylhydrazine-treated cells. However, further analysis of the cell population indicated that the majority of the IgG bound to cells that had suffered severe membrane lesions, allowing access to intracellular consituents. Elution and analysis of the bound autologous IgG confirmed that it showed specificity for some of these intracellular constituents as well as for the surface epitopes available on intact cells. We conclude that phenylhydrazine treatment is a problematic model for study of red blood cell aging mechanisms, because the binding of autologous IgG appears to be limited to a small population of severely damaged cells.

Improved isolation of normal human reticulocytes via exploitation of chloride-dependent potassium transport.

Studies on normal human reticulocytes have been limited by a lack of methods for effective reticulocyte enrichment. This study shows a convenient new approach for selective enrichment of reticulocytes from normal blood samples. We have developed a modified arabinogalactan density gradient that contains high potassium levels, approximating the internal cation composition of red blood cells (RBC). The low-density populations from this gradient are enriched in reticulocytes, and the highly selected lowest density fraction shows a much higher reticulocyte enrichment than that obtained with high sodium chloride arabinogalactan density gradients, or other previously reported density gradient methods. We found that this improved isolation is caused by suppression of potassium loss and reticulocyte dehydration via chloride (KCI) cotransport. When the low-density fraction of RBC from a high-potassium gradient was subsequently incubated in high sodium chloride medium and reseparated on a sodium chloride density gradient, the reticulocytes dehydrated and were recovered in high-density fractions. The highest-density fractions from this secondary gradient yield 95% to 99% reticulocytes. We anticipate that this method will benefit investigators who require reticulocyte enriched populations for a wide variety of applications.

Ektacytometric measurement of sickle cell deformability as a continuous function of oxygen tension.

In an effort to study the rheologic effects of small amounts of hemoglobin S (HbS) polymer in sickle red cells, we have used the ektacytometer, a laser diffraction couette viscometer, to measure sickle cell deformability as a function of oxygen tension. Sickle cell populations of defined intracellular hemoglobin concentration (MCHC) were isolated using Stractan density gradients and were resuspended in buffered polyvinylpyrrolidone solutions for deformability measurements. Using a gas-porous, hollow fiber gas exchange system to establish a linear gradient in oxygen tension, deformability was measured over a pO2 range of 76 to 0 mm Hg. Parallel spectroscopic determinations of oxygen saturation permitted determination of cell deformability as a function of oxygen saturation for each discrete MCHC population. From these measurements the level of oxygen saturation at which a loss in cell deformability was first detected could be defined. Then, using the data of Noguchi and Schecter, the amount of polymerized HbS in the cells at that defined level of oxygen saturation was estimated. The results of this analysis suggested that the quantity of polymer that caused a detectable loss in cell deformability increased with increasing MCHC. In addition, for MCHC above 30 g/dL, this represented a substantial fraction of the total HbS in the cell.

Comparison of serum anti-band 3 and anti-Gal antibody binding to density-separated human red blood cells.

This study examines the quantitative relationship between two natural serum antibodies, anti-band 3 and anti-alpha-galactosyl (anti-Gal), in their capacity to bind to human red blood cell (RBC) populations separated on density gradients. The question was approached in two ways. First, we determined the extent of rebinding of affinity-purified human serum antibodies to RBCs that had been stripped of in situ antibody. Second, we eluted the in situ bound antibody at low pH from density-separated RBCs and determined the proportion of total eluted antibody that bound specifically to erythrocyte band 3 or to a Gal-alpha-(1,3)-Gal structure. Our results show that high-density human RBCs bind increased amounts of both antibodies. Anti-Gal rebinding was specific, because it was saturable and occurred in the presence of serum IgG depleted of anti-Gal. Binding assays using control natural autoantibodies directed against antigens not found on the RBC surface showed that high-density RBCs also bind increased amounts of these antibodies as compared with low-density RBCs. However, the extent of this binding is substantially lower than that of anti-band 3 and anti-Gal. Binding studies using the lectins Bandeiraea Simplicifolia (alpha-galactosyl specific) and Arachis Hypogaea (peanut agglutinin, beta-galactosyl specific) indicated that only the alpha-galactosyl sites are exposed on high density RBCs, and not the beta-galactosyl structure characteristic of T antigen. Antibody that is eluted at low pH from high density RBCs contains a 5.0% to 18.0% component that binds to band 3 protein, and a 9.1% to 39.0% component that recognizes the alpha-galactosyl structure. Together, the two antibodies appear to constitute an average of 35% (range 17.2% to 57.4%) of the in situ bound antibody from high-density human RBCs.

Host-associated iron transfer factor in normal humans and patients with transfusion siderosis.

A low molecular weight iron-binding substance that promotes bacterial growth in vitro by increasing iron availability was identified in human blood and urine. Partial purification and physical characterization indicate that this factor is similar to the host-associated iron transfer factor (HAITF) previously isolated from mammalian tissue. HAITF was found to be significantly elevated in the blood of patients with thalassemia who have transfusional siderosis. The level of HAITF in the blood of these patients was also found to correlate with that of serum iron and serum glutamic-oxaloacetic transaminase (SGOT) but not with that of serum ferritin. Thus, elevated blood levels of HAITF may explain the increased susceptibility to infection seen in patients with iron overload. Its physiologic role, however, may involve the transport of iron within cells.