Effect of Storage on Levels of Nitric Oxide Derivatives in Blood Components.
Authors: Rizzatti Fabiola G. , Stroncek David , Sibmooh Nathawut , Schechter Alan N. 
Published: April 2020

Abstract
The traditional view of the red blood cell (RBC) as only a carrier of oxygen and carbon monoxide has been changed by the current understanding of the erythrocyte’s role in nitric oxide (NO) physiology. 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 NO metabolites, nitrite and nitrate, in whole blood and packed red blood cells and to evaluate their correlations with the time of storage. Whole blood, leukoreduced, and non-leukoreduced packed RBCs were obtained from 3 donors and were stored in polyvinyl chloride (PVC) bags 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. We found that both nitrite and nitrate decreased during the storage in the three blood components analyzed. The nitrite concentration measured in whole blood was 180± 18 before storage, 45±4 on day 1 and 43±5 nM on day 42. The nitrite concentration in RBCs was 225±51 before storage. In the leukoreduced RBCs, 75±12 on day 1 and 57±9 nM on day 42. In the non-leukoreduced RBCs, 68±10 on day 1 and 38±13 nM on day 42. The concentration of nitrate in whole blood was 50±7 before storage, 36±9 on day 1 and 27±3 uM on day 42. The concentration of nitrate in RBCs before storage was 43±5 uM. In the leukoreduced RBCs, 30±14 on day 1 and 33±5 uM on day 42. In the non-leukoreduced RBCs, 20±3 on day 1 and 23±3 uM on day 42. The whole blood pH after phlebotomy was 7.4±0.025 and decreased from 7±0.005 on day 1 to 6.5±0 on day 42 of storage. In leukoreduced RBC bags, the pH was 6.7±0.05 on day 1 and 6.5±0 on day 42 of storage. Similar values of pH were measured in the non-leukoreduced RBC bags. The blood pO2 before storage was 40.5±1.5 and increased to 251±4 mmHg in the leukoreduced RBC bag on day 42 of storage. Similar results were found for the non-leukoreduced RBCs and whole blood. In control experiments, a PVC bag was filled with saline and stored up to 42 days at 4°C. Nitrite and nitrate concentrations increased during saline storage. Our results showed that nitrite and nitrate levels fall in hemocomponents during blood bank storage, 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. Surprisingly nitrate levels did not increase during storage as one might expect from consumption of NO and nitrite. We are now investigating mechanisms for the possible reduction of nitrate under these conditions. The physiology of NO may have implication for transfusion medicine, ranging from the adverse effects of RBC transfusion to optimizing the preservation of stored hemocomponents. In addition, as erythrocytes may contribute to the control of blood flow and oxygen delivery through reduction of nitrite to NO under hypoxic and acid conditions, our findings may provide insight into the vasodynamic effects of blood transfusion and transfusion-related disorders.