Effect of excess alpha-hemoglobin chains on cellular and membrane oxidation in model beta-thalassemic erythrocytes. M D Scott, … , Y Beuzard, B H Lubin J Clin Invest. 1993;91(4):1706-1712. https://doi.org/10.1172/JCI116380. Research Article While red cells from individuals with beta thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess alpha-hemoglobin chains and the observed oxidant damage. To investigate the oxidative effects of unpaired alpha-hemoglobin chains, purified alpha-hemoglobin chains were entrapped within normal erythrocytes. These "model" beta-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 alpha-hemoglobin chain autoxidation and appearance of membrane- bound globin. In addition, alpha-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 alpha- hemoglobin chain-loaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001) in the alpha- 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 alpha-hemoglobin chains. In summary, entrapment of purified alpha-hemoglobin chains within normal erythrocytes significantly enhanced cellular oxidant stress […] Find the latest version: https://jci.me/116380/pdf Effect of Excess a-Hemoglobin Chains on Cellular and Membrane Oxidation in Model f,-Thalassemic Erythrocytes Mark D. Scott,* Jeroen J. M. van den Berg,* Tanya Repka,t Philippe Rouyer-Fessard,' Robert P. Hebbel,t Yves Beuzard,' and Bertram H. Lubin* *Children's Hospital Oakland Research Institute, Oakland, California 94609; tUniversity ofMinnesota, Minneapolis, Minnesota 55455; and §Unite de Recherche en Genetique Moleculaire et en Hematologie, Institut de la Sante et de la Recherche Medicale U.91 et Centre National de la Recherche Scientifique UA 607, H6pital Henri Mondor, 94010 Crteil, France Abstract absent, and a pool of unpaired a-hemoglobin chains is ob- served in erythroid precursors and, to a lesser extent, peripheral While red cells from individuals with ,8 thalassemias are char- blood erythrocytes. It is believed that as a consequence of these acterized by evidence of elevated in vivo oxidation, it has not excess a-hemoglobin chains, ineffective erythropoiesis and been possible to directly examine the relationship between ex- shortened erythrocyte survival is observed (3, 4). cess a-hemoglobin chains and the observed oxidant damage. To While no single mechanism is likely to account for the com- investigate the oxidative effects of unpaired a-hemoglobin plex pathophysiology of the fl-thalassemic erythrocyte, oxida- chains, purified a-hemoglobin chains were entrapped within tion of cellular components has been implicated as an impor- normal erythrocytes. These "model" ,8-thalassemic cells gener- tant factor. This hypothesis is supported by the observation ated significantly (P < 0.001 ) greater amounts of methemoglo- that isolated a-hemoglobin chains directly generate reactive bin and intracellular hydrogen peroxide than did control cells. oxygen species (5, 6). In addition, because ofthe inherent insta- This resulted in significant time-dependent decreases in the bility of a-hemoglobin chains, damage to the red cell by oxida- protein concentrations and reduced thiol content of spectrin and tive means may be further potentiated by the heme, or heme- ankyrin. These abnormalities correlated with the rate of a-he- derived iron, released from the excess a-hemoglobin chains moglobin chain autoxidation and appearance of membrane- (7-9). Indeed, elevated oxidant generation and heme release in bound globin. In addition, a-hemoglobin chain loading resulted thalassemic erythrocytes may be directly responsible for the in a direct decrease (38.5%) in catalase activity. In the absence decreased reactive sulfhydryl content of membrane proteins of exogenous oxidants, membrane peroxidation and vitamin E ( 10-13 ). Previous studies have demonstrated that the in vitro levels were unaltered. However, when challenged with an exter- oxidant sensitivity of hemoglobin E erythrocytes ( 14) (a fl-thal- nal oxidant, lipid peroxidation and vitamin E oxidation were assemia-like disease) and a-hemoglobin chain loaded ( 15) red significantly (P < 0.001 ) enhanced in the a-hemoglobin chain- blood cells to exogenous oxidants correlated with the amount loaded cells. Membrane bound heme and iron were also signifi- of membrane-bound hemoglobin or heme/iron and was am- cantly elevated (P < 0.001 ) in the a-hemoglobin chain-loaded plified by increasing the amount ofmembrane-bound hemoglo- cells and lipid peroxidation could be partially inhibited by en- bin. Similarly, in vivo fl-thalassemic red cells show evidence of trapment of an iron chelator. In contrast, chemical inhibition of elevated oxidative damage, as well as enhanced sensitivity to cellular catalase activity enhanced the detrimental effects of xenobiotic oxidants ( 16-18). entrapped a-hemoglobin chains. In summary, entrapment of However, while many erythrocyte abnormalities in fi thalas- purified a-hemoglobin chains within normal erythrocytes signif- semia have been related to the presence ofincreased a-hemoglo- icantly enhanced cellular oxidant stress and resulted in patho- bin chains, it has not been possible to determine whether these logical changes characteristic of thalassemic cells in vivo. This changes arise directly from the presence of these chains or to model provides a means by which the pathophysiological ef- other factors present during erythropoiesis. Since the most dam- fects of excess a-hemoglobin chains can be examined. (J. Clin. aged cells are destroyed within the bone marrow, and are there- Invest. 1993. 91:1706-1712.) Key words: catalase * vitamin E. fore not available for analysis, we have directly determined the glutathione. iron * heme effects of unbound a-hemoglobin chains on erythrocyte oxida- tion by entrapping purified a-hemoglobin chains within nor- Introduction mal erythrocytes by reversible osmotic lysis ( 19-21 ). As previ- The f thalassemias arise as a consequence of decreased or ab- ously shown, osmotic lysis and resealing results in resealed sent synthesis of the d-hemoglobin chains ( 1, 2). As a result of erythrocytes exhibiting normal morphology, hemoglobin con- the altered :-hemoglobin synthesis, the concentration of the centration, volume, ATP concentration, oxidant sensitivity, a2f2-hemoglobin tetramer (Hb A) is substantially reduced, or and deformability while allowing for the efficient entrapment of exogenous compounds. The effect of entrapped a-hemoglo- bin chains on oxidant generation, antioxidant status, hemoglo- Address correspondence and reprint requests to Mark D. Scott, Ph.D., bin oxidation, and membrane peroxidation in normal erythro- Children's Hospital Oakland Research Institute, 747 52nd St., Oak- cytes was then assessed. land, CA 94609. Received for publication 8 October 1991 and in revised form 18 Methods September 1992. After informed consent, red blood cells (RBC)' were collected in hepa- rinized tubes from laboratory volunteers at Children's Hospital (Oak- J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 1. Abbreviations -used in this paper: DFO-Dex, dextran derivative of 0021-9738/93/04/1706/07 $2.00 deferoxamine; IOMs, inside-out membranes; NEM, N-ethylmalemide; Volume 91, April 1993, 1706-1712 RBC, red blood cells. 1706 Scott, van den Berg, Repka, Rouyer-Fessard, Hebbel, Beuzard, and Lubin land, CA). Blood samples were processed immediately or stored at 4VC removal, by washing, of heme/Fe simply entrapped within the mem- for a maximum of 24 h before utilization. All biochemicals, unless brane ghosts. Furthermore, studies in sickle cells have demonstrated a otherwise noted, were obtained from Sigma Chemical Co. (St. Louis, close correlation between IOM-bound heme and Fe and susceptibility MO). Hemoglobin A (aAfl2) and the heme-containing a and fl subunits to membrane oxidation (40). To further investigate the effects of were prepared as previously described (22-24). In some experiments, membrane bound heme and Fe on the membrane's susceptibility to radiolabeled a-hemoglobin chains were used to quantitate their intra- iron-mediated oxidants, a high molecular mass(70 kD) dextran deriva- cellular entrapment (21 ). tive of deferoxamine (DFO-Dex) (41, 42) was entrapped within the Osmotic lysis and resealing was done by the method of Scott et al. control and a-hemoglobin chain-loaded cells. As previously shown, (19-21 ). After resealing, cells were washed at 4VC with isotonic saline DFO-Dex has virtually identical iron-binding characteristics to defer- until the supernatant was clear (five to seven times). Unless otherwise oxamine but, unlike DFO, is unable to cross the membrane and does indicated, the results presented in this study used RBC resealed in the not mediate methemoglobin