Effect of excess alpha-hemoglobin chains on cellular and membrane oxidation in model beta-thalassemic erythrocytes.

Abstract

While red cells from individuals with ,8 thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess a-hemoglobin chains and the observed oxidant damage. To investigate the oxidative effects of unpaired a-hemoglobin chains, purified a-hemoglobin chains were entrapped within normal erythrocytes. These "model" ,8-thalassemic cells generated significantly (P < 0.001 ) greater amounts of methemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of a-hemoglobin chain autoxidation and appearance of membranebound globin. In addition, a-hemoglobin chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P < 0.001 ) enhanced in the a-hemoglobin chainloaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001 ) in the a-hemoglobin chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped a-hemoglobin chains. In summary, entrapment of purified a-hemoglobin chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess a-hemoglobin chains can be examined. (J. Clin.

Microfluidic analysis of cellular deformability of normal and oxidatively damaged red blood cells

“…However, over time the damage becomes more apparent. Indeed, the oxidant and challenge dosage (50 mM PMS) used in this study was specifically chosen as previous studies have demonstrated that the oxidant treated RBC exhibited similar characteristics to sickle and thalassemic cells [15,17,24]. In support of this finding, the PMS-treated murine cells demonstrated very similar in vivo circulation to that of model ß thalassemia murine RBC [3,17,49].…”

“…Survival of fluorescently labeled control and PMS-oxidized RBC was monitored by analyzing the percent of fluorescently labeled RBC by flow cytometry (FACSCalibur Flow Cytometer, BD Biosciences, San Jose, CA) [45,46]. To better correlate PMS-treatment with a known hematological abnormality, model murine ß thalassemic cells were prepared via osmotic lysis and resealing as previously described [3,17,48,49]. Model ß thalassemic murine RBC are produced via the entrapment of purified human alpha-hemoglobin chains within normal murine RBC.…”

“…Model ß thalassemic murine RBC are produced via the entrapment of purified human alpha-hemoglobin chains within normal murine RBC. Human model ß-thalassemic cells have previously been shown to exhibit structural and function changes very similar to patient derived samples [3,17,49]. The model murine ß thalassemic cells were labeled with PKH-26 and in vivo survival was followed as above.…”

“…Altered cellular deformability is a component of multiple clinically significant red cell abnormalities and hemoglobinopathies such as Sickle Cell Disease (SCD) or bThalassemia [3,[16][17][18][19][20][21][22][23]. In SCD, the inter-chain hydrophobic interactions and polymerization of the deoxygenated hemoglobin impairs deformability as well as promotes microocclusions via vasculature adhesion and blockage [18].…”

“…In b-Thalassemia, the unpaired and unstable a-hemoglobin chains generate reactive oxygen species damaging cytoskeletal proteins and oxidizing lipids and also tightly adhere to the membrane leading to a reduction in cellular deformability. The loss of deformability is further compounded by the decreased surface area to volume ratio of the microcytic red cells [3,17,22]. Experimentally, the aforementioned red cell damage can be modeled by exposing them to oxidants such as phenazine methosulphate (PMS) [3,15,17,24,25].…”