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 oxid ative 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 protec t membrane damage in the banked RBCs following an oxidant challenge. RBCs s tored at 1-6 degreesC for 0, 42 and 84 days in a conventional additive solu tion (Adsol(R)) were subjected to oxidative stress using ferric/ascorbic ac id (Fe/ASC) before and after enriching them with GSH or catalase using a hy potonic lysis-isoosmotic resealing procedure. This lysis-resealing procedur e in the presence of GSH/catalase raised intracellular GSH and catalase con centrations 4-6 fold, yet produced only a small reduction in mean cell volu me (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration s (MCHC). Indicators of oxidative stress and membrane integrity were measur ed, including acetylcholinesterase (AChE) activity, GSH concentration, phos phatidylserine (PS) externalization (prothrombin-converting activity) and t ransmembrane lipid movements (C-14-lyso phosphatidylcholine flip-flop and P S transport). GSH-enrichment protected AChE activity in fresh (0 day) and s tored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respec tively, compared with not-enriched controls. Following oxidative stress, th e rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3 % in 42-day stored cells. Phosphatidylserine exposure, as me asured 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 (similar to 50 % ), whereas transport rates declined by up to 75 % in stored RBCs when chall enged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid f lip-flop and the increase in prothrombinase activity, but offered no protec tion against oxidative damage of PS transport. In contrast to these effects , catalase-enrichment failed to protect GSH levels and AChE activity upon o xidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblo tting with antifluorescein antibodies. Significant oxidation of membrane pr oteins was confirmed by a greater loss of thiols in stored RBCs than in fre sh RBCs. These results demonstrate that it may be possible to prevent stora ge-mediated loss of AChE, increased lipid flip-flop, and increased PS expos ure, by maintaining or increasing GSH levels of banked RBCs.