Inhibition of Glutathione Synthesis via Decreased Glucose Metabolism in Stored RBCs
Cellular Physiology and Biochemistry
Background/Aims: Although red blood cells (RBCs) transfusions can be lifesaving, they are not without risk. RBCs storage is associated with the abnormal metabolism of glutathione (GSH), which may increase the risk of the oxidative damage of RBCs after transfusion. The responsible mechanisms remain unknown. Methods: We determined the L-cysteine efflux and influx by evaluating the changes of free -SH concentrations in stored RBCs. The glutamate cysteine ligase (GCL) activities and protein content
... and protein content in stored RBCs was determined by fluorescence assay and western blotting. In addition, the glucose metabolism enzyme activity of RBCs was measured by spectrophotometric assay under in vitro incubation conditions. Results: We found that both L-cysteine transport and GCL activity significantly declined, thereby inducing the dysfunction of GSH synthesis during blood storage, which could be attenuated by ATP supplement and DTT treatment. In addition, the glycometabolic enzyme (G6PDH, HK, PK and LDH) activity significantly decreased after 6 weeks storage. Oxidant stress-induced dysfunction in glucose metabolism was the driving force for decreased GSH synthesis during storage. Conclusion: These experimental findings reflect an underlying molecular mechanism that oxidant stress induced glucose metabolism dysfunction contribute to decreased GSH synthesis in stored RBCs. Materials and Methods Collection and processing of blood Whole blood from 20 eligible young blood donors was collected. They underwent routine blood donation for this purpose. Blood donation and the entire preparation and storage of RBC units were performed according to international guidelines in the local blood bank centre. 450±50 mL of whole blood were collected and suspended in 100 mL of preservation solution salineadenine-glucose-mannitol (SAGM; 8.77 g/L NaCl, 0.169 g/L adenine, 9.00 g/L dextrose monohydrate [d-glucose], 5.25 g/L mannitol) and leukoreduced by filtration (final concentration, less than 10 6 white blood cells/RBC unit). After filtration, RBCs were stored for 7 weeks at 2 to 6°C. Units were sterilely sampled (15 mL per time point) at days 0, 7, 21, 42 by a sterile sampling device, after gentle mixing of the unit content by inversion for approximately 5 minutes. The study gained approval from the local ethics committee and informed consent was obtained from all subjects. All additive solutions (ASs) were made up in SAGM. When these various solutions were added to the RBCs suspended in SAGM they increased the volume in the packsby 2.5% and gave final concentrations of 1) SAGM only (control); 2) 5 mmol/L N-acetylcysteine (NAC), alanine and glycine; 3) 0.2U/ml HX 1.5mM XO (hypoxanthine/xanthine oxidase) to increase the oxidative load and 4) 1.0 mM dithiothreitol (DTT) to increase the antioxidant capacity. The solutions were added under sterile conditions and the RBCs were stored at 4°C. A 5mL sample was aseptically collected at Weeks 1, 3, and 6 of storage from each of the bags and pH was measured at 22°C using a laboratory pH meter.