Effect of a glycerol-containing hypotonic medium on erythrocyte phospholipid asymmetry and aminophospholipid transport during storage

Abstract

Previous studies from our laboratory have shown that under blood bank storage conditions red blood cell (RBC) ATP and lipid content were better maintained in a glycerol-containing hypotonic experimental additive solution (EAS 25) than in the conventional storage medium Adsola. The objective of this study was to determine the mechanism of the protective effect of EAS 25, by measuring transmembrane phospholipid asymmetry and the membrane integrity of stored RBCs. Split units of packed RBCs were stored in either EAS 25 or Adsol®. RBCs were analyzed after 0, 42, and 84 days and vesicles shed from stored RBCs were analyzed after 84 days of storage. Phospholipid asymmetry was measured by phospholipase A 2 digestion (RBCs) and activation of the prothrombinase complex (RBCs, vesicles). RBC membrane exhibited a significantly greater (P < 0.01) amount of phosphatidylethanolamine externalized after storage in Adsol® than in EAS 25 (44.3% ± 11.7 vs. 25.3% ± 5.7, respectively). Prothrombin converting activities in RBCs were significantly lower than in shed vesicles (P < 0.001) suggesting the presence of phosphatidylserine in the outer monolayer of vesicle, but not in RBC membranes. The rates of inwardly-directed aminophospholipid transport in RBCs decreased by ∼ 50% and glutathione levels decreased by ≈ 50% in both media. RBC cholesterol and phospholipid content of stored RBCs remained significantly greater (P > 0.01) in EAS 25 than in Adsole. The results indicate that despite comparable reduction in the rate of aminophospholipid transport and reduced GSH concentrations, RBC phospholipid asymmetry was better maintained during storage in EAS 25 than in Adsol®. The data suggest that glycerol in the hypotonic EAS helps preserve RBC lipid organization and membrane integrity during storage.