Effect of Storage on Levels of Nitric Oxide Derivatives in Blood Components.
Authors: Rizzatti Fabiola G. , Stroncek David , Qazi Melissa , Sibmooh Nathawut , Piknova Barbora , Schechter Alan N. 
Published: October 2019

Abstract
The important role of erythrocytes in nitric oxide (NO) physiology changed the traditional view of the red blood cells (RBC) as only a carrier of oxygen and carbon monoxide. Nitrite is a primary oxidative NO metabolite and is considered a major intravascular storage pool for NO. In the vascular system, erythrocytes are the major storage sites of nitrite, which can be activated to NO by deoxyhemoglobin, but also are responsible for its rapid destruction after reaction with oxyhemoglobin.The purpose of this study was to quantify the nitric oxide metabolites, nitrite and nitrate, in red blood cells (RBCs) stored as packed cells or whole blood and to evaluate their levels with the time of storage. Whole blood, leukoreduced, and non-leukoreduced packed RBCs were obtained from healthy volunteer donors and were stored in polyvinyl chloride (PVC) bags to up to 42 days at 4°C. Sequential aliquots were taken from the bags using a liquid transfer set to maintain sterile conditions. Nitrite and nitrate were measured in the whole blood and in RBC components using reductive gas phase chemiluminescence. Nitrite concentrations decreased during the storage in the three blood components analyzed. The nitrite concentration in RBCs before storage was 202±45 nM, but fell rapidly upon storage. In the leukoreduced RBCs, nitrite levels were 81±36 nM on day 1 and 51±8 nM on day 42. The concentration of nitrate remained stable during blood storage, 30±14 uM on day 1 and 33±5 uM on day 42 of storage. The pH decreased slightly in all three blood components during storage, from pH 6.7±0.05 on day 1 to 6.5±0 on day 42. The blood pO2 before storage was 40.5±1.5 and increased to 251±4 mmHg on day 42, presumably due to the diffusion of oxygen from the room air. In control experiments, PVC bags were filled with normal saline used for medical purposes and stored up to 42 days at 4°C in room air; nitrite concentrations gradually increased while nitrate values remained stable. Similar results were observed for nitrite and nitrate concentrations in the non-leukoreduced RBCs and whole blood. Both cells and saline controls maintained in an argon chamber at 4°C for 42 days showed decreased levels of nitrite when compared to the bags stored in room air under the same temperature. Our results show that nitrite levels fall in hemocomponents during blood bank storage, nitrate remains stable, while pH decreases and pO2 increases. The decrease in nitrite levels could be explained either by its reaction with oxyhemoglobin, resulting in nitrate and methemoglobin, or with deoxyhemoglobin. The diffusion of oxides of nitrogen gases through the PVC bags could in part explain why nitrite levels do not completely disappear in the RBCs stored for transfusion, under standard transfusion medicine conditions. As erythrocytes may contribute to the control of blood flow and oxygen delivery through reduction of nitrite to NO under hypoxic conditions, our findings may provide insight into the vasodynamic effects of blood transfusion. These measurements of NO derivatives may have implication for transfusion therapy, explaining some adverse effects of RBC transfusion and/or optimizing the preservation of stored hemocomponents.