In this study we attempt t o establish the consequence of in vitro hydrogen peroxide (H,O,)-induced membrane damage as manifested by spectrin-hemoglobin (Sp-Hb) complex formation and decreased red blood cell (RBC) deformability t o in vivo RBC survival in baboons. After exposure t o 135 t o 581 pmol/L H, O, and reduction with dithiothreitol (DTE), baboon RBCs were infused into the animal, and the fraction of cells remaining in circulation after 24 hours and the lifespan of surviving cells were quantitated. In a dosedependent fashion, a positive correlation was observed between in vitro membrane alterations and the 24-hour in vivo survival. While 12% of the control cells were removed from circulation in 24 hours, 23% were removed after treatment with 339 pmol/L H , O , , and 36% following exposure t o 581 pmol/L H, O, .Pretreatment with carbon monoxide before exposure with H, O, increased the survival of oxidized RBCs. RBCs not removed from circulation in the first 24 hours had a normal lifespan. Moreover, by selectively isolating biotin-labeled, peroxide-treated cells XTENSIVE BIOCHEMICAL, biophysical, and immu-

In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta- subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.