Effect of Storage on Levels of Nitric Oxide Derivatives in Blood Components

In light of recent papers stressing the importance of decreased levels of SNO-hemoglobin (SNO-Hb) to the pronounced nature of deleterious effects of transfusion of stored red blood cells (RBCs), there has been an increased interest in the practice of blood storage. Dejam et al. (Blood, 2005) previously demonstrated the critical nature of RBCs in NO physiology: they serve as the major intravascular stores of nitrite, which is eventually converted to NO, an important player in vasoregulation. The purpose of this study is to quantify the NO metabolites, nitrite and nitrate, in three blood components and evaluate their levels over time of storage. Blood obtained from 6 healthy volunteer donors was split into whole blood, leukoreduced, and non-leukoreduced packed RBCs and stored in polyvinyl chloride (PVC) bags for 42 days at 4°C. PVC bags were maintained in either room air or an argon chamber. Nitrite, nitrate, and SNO-Hb/nitrosyl-hemoglobin (HbNO) were measured using reductive gas-phase chemiluminescence. In all blood components, the nitrite and nitrate were detected in higher concentrations in RBCs than in the extracellular fluid compartment. Mean nitrite value immediately before storage was 152±13nM, but fell rapidly upon storage. Nitrite levels continued to decrease with storage time, while nitrate levels remained constant for the duration of storage. In the leukoreduced blood product, nitrite levels were 75±8nM on day 1 and 50±9nM by day 42; the concentration of nitrate in the leukoreduced blood product was 34±3uM on day 1 and 34±4uM on day 42. The nitrite levels in non-leukoreduced blood product were 76±12nM on day 1 and 37±7 by day 42; the nitrate concentration in the non-leukoreduced blood product was 35±3uM on day 1 and 32±0.4uM on day 42. In whole blood, nitrite levels were 64±11nM on day 1 and 44±9nM by day 42; the nitrate concentration was 47±2uM on day 1 and 43±6uM on day 42. SNO-Hb levels were very low in fresh blood and virtually undetectable after one day of storage. Interestingly, nitrite levels never reached zero. Enzyme inhibitors—L-NAME (nitric oxide synthase inhibitor), acetazolamide (carbonic anhydrase inhibitor), and oxypurinol (xanthine oxidase inhibitor)—did not lower nitrite levels enough to explain the remaining nitrite present in the PVC bags after 42 days. pH decreased slightly, while pO₂ increased in all three components during storage; this is likely due to the diffusion of oxygen from room air into the PVC bags. Control experiments with saline showed an increase in nitrite levels, while nitrate levels remained stable over 42 days. When stored in an argon chamber, both blood and saline samples showed relatively lower nitrite levels than their room air counterparts. Thus, during blood bank storage, nitrite levels decrease in blood while nitrate levels remain stable. The diffusion of nitrogenous gases may explain why nitrite does not completely disappear under standard storage conditions. Our results suggest that most of the NO pathway is initially retained, but greater changes occur with prolonged storage. These measurements of NO derivatives may have implications for transfusion therapy, explaining some of the adverse effects seen with RBC transfusion and providing a foundation for enhancing blood preservation through improvement of storage practices.