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2263562 
Journal Article 
Abstract 
Nitric oxide storage lesion in stored blood 
Donadee, C; Raat, NJH; Meyer, EM; Kelley, E; Wang, X; Qu, L; Donnenberg, AD; Lee, J; Kim-Shapiro, DB; Gladwin, MT 
2010 
Yes 
American Journal of Respiratory and Critical Care Medicine
ISSN: 1073-449X
EISSN: 1535-4970 
181 
A6343 
English 
WOS:000208771004702 
is part of a larger document 3452678 Proceedings of the American Thoracic Society 2010 International Conference, May 14-19, 2010, New Orleans
Rationale: While blood storage is associated with increased risk of cardiovascular events and multi-organ failure, especially in physiologically compromised patient populations receiving multiple units of > 2 weeks aged blood, the fundamental mechanisms underlying the “storage lesion” in blood remain uncertain. This study examines the hypothesis that the storage lesion is associated hemolysis-dependent nitric oxide (NO) dysregulation, characterized by an increase in markers of red blood cell destruction/free hemoglobin (Hb) production and consequent NO and arginine catabolism. Central to this hypothesis is the understanding that hemolysis releases low concentrations of hemoglobin into plasma which reacts rapidly with NO to form bio-inert nitrate, and also releases other mediators into plasma from the red blood cell, such as arginase 1 and adenosine deaminase, which catabolize arginine and adenosine.

Methods: We performed multiple assays to evaluate the degree of hemolysis in RBCs stored over time and the effect on NO signaling, including free Hb by ELISA and spectroscopy, Arginase-1 by ELISA, the ability of stored blood plasma to consume NO, and quantification of microvesicle formation by flow cytometry. We evaluated the effect of storage on mechanical fragility of RBCs over time to evaluate propensity of packed red blood cells to hemolyze during infusion and in the circulation after transfusion.

Results: Arginase-1 levels were 3000 ng/mL in 1 week old plasma vs 100,000 ng/mL at 4 weeks. Plasma free Hb and NO consumption were 10µM at 1 week and 75µM at 4 weeks of storage. The plasma hemoglobin remained in the ferrous redox state capable of stoichiometric scavenging reactions with NO. Consistent with this the PRBC plasma heme levels directly correlated with the concentration of NO consumed (R= 0.9655; P<0.0001). In the RBC fragility assay free Hb increased from 193µM ± in blood stored for 2 days to 333µM ± 51 (mean±SEM) at 4 weeks of storage. Microvesicles formation increased from as low as 0.68% of events at 2 days to as high as 11.48% of
events at 39 days of storage.

Conclusions: Red cell hemolysis and mechanical fragility increase with time during RBC storage, producing ferrous hemoglobin, arginase I, and red cell microvesicles. These factors directly inhibit NO in vitro suggesting that such a process might increase cardiovascular risk after transfusion of aged blood. This study provides new evidence that the storage lesion in banked blood is due to increased RBC destruction over time leading to increased NO consumption and consequent vascular dysfunction. 
American Thoracic Society 2010 International Conference 
New Orleans, LA 
May 14-19, 2010