Restoration of Physiological Levels of Uric Acid and Ascorbic Acid Reroutes the Metabolism of Stored Red Blood Cells

Abstract

After blood donation, the red blood cells (RBCs) for transfusion are generally isolated by centrifugation and then filtrated and supplemented with additive solution. The consecutive changes of the extracellular environment participate to the occurrence of storage lesions. In this study, the hypothesis is that restoring physiological levels of uric and ascorbic acids (major plasmatic antioxidants) might correct metabolism defects and protect RBCs from the very beginning of the storage period, to maintain their quality. Leukoreduced CPD-SAGM RBC concentrates were supplemented with 416 µM uric acid and 114 µM ascorbic acid and stored during six weeks at 4 °C. Different markers, i.e., haematological parameters, metabolism, sensitivity to oxidative stress, morphology and haemolysis were analyzed. Quantitative metabolomic analysis of targeted intracellular metabolites demonstrated a direct modification of several metabolite levels following antioxidant supplementation. No significant differences were observed for the other markers. In conclusion, the results obtained show that uric and ascorbic acids supplementation partially prevented the metabolic shift triggered by plasma depletion that occurs during the RBC concentrate preparation. The treatment directly and indirectly sustains the antioxidant protective system of the stored RBCs.

Keywords: antioxidant; ascorbic acid; blood storage; metabolomics; red blood cell; transfusion; uric acid.

Figure A1

Evolution of haematological parameters and pH in red blood cell concentrates (RCCs) supplemented with uric and ascorbic acids (UA-AA) or control. (A) Haematological data: red blood cell (RBC) count, mean corpuscular volume (MCV) and size variation of the erythrocytes around the mean (SD-RDW) measured with Sysmex analyzer; (B) Evolution of the pH in the RCCs. Individual (symbols) and mean (lines) values for the four RCCs are presented ± standard deviation (shaded areas).

Figure A2

Effect of uric and ascorbic acids (UA-AA) supplementation on red blood cell (RBC) metabolism. Quantitative data of the time-course metabolomic analysis by HPLC-MS for the control RBCs stored in standard conditions (light grey lines) vs. the RBCs supplemented with UA-AA (dark grey lines). Individual (symbols) and mean (lines) values for the four RCCs are presented ± standard deviation (shaded areas). * p-value < 0.05 2way ANOVA.

Figure 1

Evolution of the morphology and haemolysis in the red blood cell concentrates (RCCs) Supplemented with uric and ascorbic acids (UA-AA) or control. Morphology analysis with digital holographic microscope (DHM): (A) Population analysis with standard deviation of the optical path difference parameter (SD-OPD); and (B) single-cell analysis with CellProfiler and CellProfiler Analyst (CPA); (C) percentage of small cells determined with the AMNIS imaging flow cytometer; (D) percentage of haemolysis in the blood bag determined using the Harboe spectrophotometric method. Individual (symbols) and mean (lines) values for the four RCCs are presented ± standard deviation (shaded areas).

Figure 2

Evolution of the antioxidant power (AOP) and sensitivity to oxidation in the red blood cell concentrates (RCCs) supplemented with uric and ascorbic acids (UA-AA) or control. (A) AOP in supernatant quantified by electrochemical pseudo-titration with the Edel device; (B) RBC sensitivity to oxidation under treatment with different concentration of hydrogen peroxide (H₂O₂) oxidant. The generation of reactive oxygen species (ROS) was reported using the 2′,7′-dichlorofluorescin diacetate (DCFH-DA) fluorescent dye. The higher the area under the curve (AUC), the more intracellular ROS generated. Individual (symbols) and mean (lines) values for the four RCCs are presented ± standard deviation (shaded areas). * p-value < 0.05 two-way ANOVA.

Figure 3

Evolution of the extracellular levels of glucose and lactate, and of the intracellular concentrations of ATP and 2,3-DPG in the red blood cell concentrates (RCCs) supplemented with uric and ascorbic acids (UA-AA) or control. The analyses were performed using commercial assay kits; (A) extracellular glucose level; (B) extracellular lactate level; (C) intracellular ATP level; (D) intracellular 2,3-DPG level. Individual (symbols) and mean (lines) values for the four RCCs are presented ± standard deviation (shaded areas).

Figure 4

Effect of uric and ascorbic acids (UA-AA) supplementation on red blood cell (RBC) metabolism—focus on the pentose phosphate pathway (PPP), and the purine and GMP metabolisms. The graphs present the results of time-course metabolomic analysis by mass spectrometry of several intracellular RBC metabolites. In the control RBCs, the metabolism seems to be pulled in the direction (path highlighted in red) of the non-oxidative PPP and toward the purine and GMP metabolism, probably as a result of the depletion of the intracellular UA pool. Individual (symbols) and mean (lines) values for the four RCCs are presented ± standard deviation (shaded areas). * p-value < 0.05 two-way ANOVA.

Figure 5

Effect of uric and ascorbic acids (UA-AA) supplementation on red blood cell (RBC) metabolism—focus on the anaerobic glycolysis, and the urea and remnants of the tricarboxylic acid cycle (TCA, or Krebs Cycle). The graphs present the results of time-course metabolomics analysis by mass spectrometry of several intracellular RBC metabolites. In the UA-AA-treated RBCs the glycolysis is favored (path highlighted in green). Mean values for the four RCCs are presented ± standard deviation (shaded areas). * p-value < 0.05 two-way ANOVA.

Figure 6

Effect of uric and ascorbic acids (UA-AA) supplementation on red blood cell (RBC) metabolism—focus on the S-adenosylmethionine cycle (SAM), and the reduced glutathione (GSH) synthesis and oxidized glutathione (GSSG) reduction pathways. The graphs present the results of time-course metabolomics analysis by mass spectrometry of several intracellular RBC metabolites. In the UA-AA-treated RBCs, the de novo synthesis of GSH seems to be favored (path highlighted in green). Mean values for the four RCCs are presented ± standard deviation (shaded areas). * p-value < 0.05 two-way ANOVA.