Target metabolomic analysis of Red Blood Cells transfusion bags: role of hypoxanthine in neutrophil activation.

Red Blood Cells (RBCs) stored in blood bank conditions, undergo biological/biochemical changes collectively referred as "storage lesions". The RBCs bags metabolic profile was evaluated during storage (up to 42 days) by 1H-NMR spectroscopy [1]. More than 30 metabolites were identified: we observed a significant increase of hypoxanthine (HX) concentration in aged RBCs units [2]. This intermediate of purine pathway is a pro-oxidant molecule: its oxidation by xanthine oxidase (XO) produces reactive oxygen species (ROS, O2•- and H2O2) as by-products. ROS play a central role in inflammation, acting as direct mediators and/or being involved in neutrophil activation and downstream inflammatory response mediated by cytokines and chemokines. In vitro studies on neutrophil priming/activation, induced by RBCs storage medium, attributed this effect to the presence of bioactive substances, such as lipids [3]. The search of other mediators is still an open question. Since the role of HX was not previously investigated, our aim is to discern its role on neutrophil activation. The effect of 42 days old RBCs supernatants and HX (+/- XO in both conditions) on isolated neutrophils in culture was assessed by flow cytometry and ELISA assays. An increase of the intracellular cytokine TNFα and a release of chemokine IL-8 in the extra-cellular space were observed. Both effects were absent in presence of allopurinol, XO inhibitor, thus demonstrating the role played by HX on neutrophil activation. References [1] Pertinhez, T.A., Berni, P., Casali, E., Lindner, L., Spisni, A., Baricchi, R. Blood Transfus. 12, 548 (2014) [2] Casali, E., Berni, P., Spisni, A., Baricchi, R., Pertinhez, T.A. Blood Transfus. 14, 555 (2016) [3] Silliman, C.C., Moore, E.E., Kelher, M.R., Khan, S.Y., Gellar, L., Elzi, D. J. Transf. 51, 2549 (2011)