Blood Storage Solutions Effecting Advanced Glycated End Products (AGEs) N‐CEL and N‐CML on Red Blood Cell Membranes

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Blood transfusions are critical, often lifesaving, procedures that result in approximately 10.5 million transfusions per year in the United States. Donated red blood cells (RBCs) is stored for a maximum of 42 days in FDA approved storage solutions. These storage solutions contain high levels of glucose to promote the longevity of the cells donated. However, AS‐1 storage solution contains 111 mM glucose, which is about 20 times higher than normal blood concentrations (5‐6 mM). Hyperglycemia is often associated with diabetes (7‐9 mM glucose), and prolonged hyperglycemic conditions are known to cause various blood flow‐related complications (retinopathy, neuropathy, nephropathy, cardiovascular disease). There is evidence of adverse RBCs changes in hyperglycemic conditions during the time over which storage would occur. Specifically, increased glycation is linked to increased amounts of advanced glycated end products (AGEs).AGEs are formed from the glycation process, as higher glucose conditions produce a spontaneous non‐enzymatic reaction of free reducing sugars with free amino groups that form Amadori products, which undergo irreversible changes that lead to the formation of AGEs. These compounds promote oxidative stress and inflammation and have been linked to various chronic diseases, often linked with diabetes. RBCs release the necessary ATP for proper blood circulation, and there is previous evidence showing the high glucose storage decreases ATP release over 36 days. Also, there is a consistent ATP levels for the AS‐1N (5 mM glucose) storage solution over 36 days which are higher than the hyperglycemic storage solution.Decreased ATP release during storage may be linked to hyperglycemic storage conditions increasing AGE biomarkers on the RBC membrane. Two of these AGE adducts of carboxymethyl lysine (N‐CEL and N‐CML) are used to quantify glycation on the RBC membrane by studying adduct concentration changes during storage. Two solutions were prepared (AS‐1 and AS‐1N) and used for storage for 36 days, with aliquots of RBCs removed every 4‐7 days for analysis. The amount of irreversible glycation products, thiols, and total lysine groups were determined to evaluate the amount of modified species over time. LC‐MS was used to quantify these modified N‐CEL and N‐CML adducts on the RBCs using internal standards. Overall, our results probe the hyperglycemic conditions on RBCs and examine the effects of increased glycation on cellular release of components from these stored cells.