Sanne de Bruin

156 Chapter 6 As a result, ATP levels were significantly higher and less glucosewas present in PAGGGM stored RBCs, resulting in increased levels of 7 metabolites of the glycolysis including hexose phosphate (including unresolved isomers glucose 1 or 6-phosphate and fruc- tose 6-phosphate with the chromatographic conditions adopted in this study), fruc- tose-1,6-biphosphate, glyceraldehyde-3-phosphate, diphosphoglycerate (including isomers 1,3 and 2,3-DPG), phosphoglycerate, phosphoenolpyruvate and lactate. Sev- eral differences remained shortly after transfusion. Glucose-phosphate was higher 10 minutes after transfusion in the PAGGGM stored RBCs but was similar after 6 hours. DPG, phosphoglycerate and phosphoenolpyruvate were significantly higher up to six hours after transfusion. In SAGM RBCs, these levels increased after transfusion, while in PAGGGMRBCs a decrease was observed. Consequently, one day after transfusion, no differences were found between SAGM and PAGGGM stored RBCs that were still circulat- ing at 24h. Also, no differences were found in AMP, ADP and ATP levels after transfusion. Redox metabolism in PAGGGM RBCs In addition to the metabolomics analysis, G6PD activity was also assessed using a flow cytometric assay. PAGGGMstored RBCs showed an increased PPP flux prior to transfusion indicated by higher levels of 6-phosphogluconate levels in the metabolomics analysis (Figure 3) and increased G6PD activity as determined by our flow cytometric assay (Figure 6). Immediately after transfusion, the differences between 6-phosphogluconate levels and G6PD activity were corrected. On the other hand, ribose phosphate – the final product of the PPP – was significantly higher in transfused PAGGGM RBCs at 10 min, 6h and 1d after transfusion (Figure 3). Sedoheptulose phosphate levels were higher 10 minutes after transfusion in PAGGGM stored RBCs, while prior to transfusion higher levels were found in SAGM stored RBCs. Glutathione disulfide (GSSG) levels were significantly higher in PAGGGM RBCs prior to transfusion. After transfusion these differences rapidly disappeared. Reduced glutathione (GSH) and the breakdown product of GSH, 5-oxoprolin was not significantly different on any of the time point prior or after transfusion. Purine metabolism in PAGGGM RBCs Hypoxanthine is a deaminated purine, which is a product of ATP, AMP and adenosine metabolism. In PAGGGM RBCs hypoxanthine levels were significantly higher compared to 35 days stored SAGM RBCs. This difference remained present up to 6 hours after transfusion in the restored RBCs (Figure 4). Hypoxanthine can be converted via xan- thine to urate with hydrogen peroxide as by-product. Xanthine was significantly higher in PAGGGM prior to transfusion but not in the RBC that were recovered after transfu- sion. In contrast, urate levels were similar in SAGM and PAGGGM RBCs before and after transfusion.