Glutathione loading prevents free radical injury in red blood cells after storage

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity^1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1–6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4–6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (¹⁴C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (～ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.