Effect of osmotic lysis and resealing on red cell structure and function

We have recently modified the dialysis tubing osmotic lysis and resealing method to examine the role of intracellular red blood cell (RBC) antioxidants. However, the potential effect of resealing on the RBC was not fully investigated. This study examined a number of cellular characteristics to determine the effects of physical lysis and resealing on the RBC. Following resealing, RBC exhibited normal morphology and at most only slight alterations in mean cell volume and mean cell hemoglobin concentration. RBC density distribution was significantly affected by resealing with increased populations of both light and dense cells, though the mean cell density was similar to that of control cells. Endogenous enzyme activities and adenosine triphosphate (ATP) concentration were unaffected by the resealing procedure. While reduced glutathione (GSH) concentration was decreased by 15%, RBC oxidant sensitivity was found to be unaltered. Cellular deformability of the resealed RBC was 80% to 90% that of the control cells. Membrane phospholipid and fatty acyl composition of the resealed RBC were unaffected when compared with matched control samples. Membrane transport, permeability, and Ca2(+)-mediated cellular vesiculation were minimally altered by resealing. Finally, entrapment of fluorescent compounds demonstrated that greater than 95% of the resealed RBC had incorporated exogenous agents. In summary, the osmotic lysis and resealing method described resulted in only minor changes in cellular characteristics while allowing for the efficient loading of compounds to which the RBC membrane is normally impermeable. Consequently, this method provides great potential for the selective modification of erythrocyte constituents in order to further define their roles within the RBC.