Supplementation with uric and ascorbic acid protects stored red blood cells through enhancement of non-enzymatic antioxidant activity and metabolic rewiring

Abstract

Redox imbalance and oxidative stress have emerged as generative causes of the structural and functional degradation of red blood cells (RBC) that happens during their hypothermic storage at blood banks. The aim of the present study was to examine whether the antioxidant enhancement of stored RBC units following uric (UA) and/or ascorbic acid (AA) supplementation can improve their storability as well as post-transfusion phenotypes and recovery by using in vitro and animal models, respectively. For this purpose, 34 leukoreduced CPD/SAGM RBC units were aseptically split in 4 satellite units each. UA, AA or their mixture were added in the three of them, while the fourth was used as control. Hemolysis as well as redox and metabolic parameters were studied in RBC units throughout storage. The addition of antioxidants maintained the quality parameters of stored RBCs, (e.g., hemolysis, calcium homeostasis) and furthermore, shielded them against oxidative defects by boosting extracellular and intracellular (e.g., reduced glutathione; GSH) antioxidant powers. Higher levels of GSH seemed to be obtained through distinct metabolic rewiring in the modified units: methionine-cysteine metabolism in UA samples and glutamine production in the other two groups. Oxidatively-induced hemolysis, reactive oxygen species accumulation and membrane lipid peroxidation were lower in all modifications compared to controls. Moreover, denatured/oxidized Hb binding to the membrane was minor, especially in the AA and mix treatments during middle storage. The treated RBC were able to cope against pro-oxidant triggers when found in a recipient mimicking environment in vitro, and retain control levels of 24h recovery in mice circulation. The currently presented study provides (a) a detailed picture of the effect of UA/AA administration upon stored RBCs and (b) insight into the differential metabolic rewiring when distinct antioxidant "enhancers" are used.

Keywords: Antioxidant supplementation; Ascorbic acid; Glutathione metabolism; Oxidative stress; RBC storage lesion; Uric acid.

Graphical abstract

Fig. 1

Antioxidants supplementation effects upon qualitative parameters of stored red blood cells. Hemolysis parameters (A), pH (B), calcium accumulation along with calpain-1 recruitment to the membrane (C) and extracellular antioxidant capacity (D) during storage of red blood cells under standard conditions or upon supplementation with uric acid (UA) and/or ascorbic acid (AA). Representative immunoblots are shown (n = 6). 4.1R protein was used as internal loading control. Data are presented as mean ± SD. C: control samples. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Antioxidants supplementation effects upon the oxidative stress of stored red blood cells. Membrane lipid oxidation and protein carbonylation (A), membrane-bound hemichromes and hemoglobin (Hb) oligomers (B), oxidatively-induced hemolysis (C) and intracellular ROS accumulation (D) during storage of red blood cells under standard conditions or upon supplementation with uric acid (UA) and/or ascorbic acid (AA). Representative immunoblots are shown (n = 6). Band 3 and 4.1R proteins were used as internal loading controls for membrane samples, while stomatin served the same role in extracellular vesicles. Blue rectangular: statistically significant reduction (p<0.05). Data are presented as mean ± SD. C: control samples. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Antioxidants supplementation effects upon the proteasomal activity and extracellular vesicle's (EVs) parameters of stored red blood cells. Proteasomal activities in the cytosol (A) and at the membrane (B), and EV protein concentration, procoagulant activity and specific proteins (C) during storage of red blood cells under standard conditions or upon supplementation with uric acid (UA) and/or ascorbic acid (AA). Representative immunoblots are shown (n = 6). Stomatin was used as internal loading control. Data are presented as mean ± SD. C: control samples; Prdx2: peroxiredoxin-2; Casp-3: caspase-3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Antioxidants supplementation effects upon glucose metabolism. Fold change of selected metabolites during storage (x axis: storage days) after red blood cell supplementation with uric acid (UA) and/or ascorbic acid (AA). Y axis: fold change versus control. Transparent gray bands' thickness is representative of the mean ± SD of control samples. Highlighted pathways: significant divergence in specific supplementations (color defined). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Antioxidants supplementation effects upon methionine and glutathione (GSH) metabolism. Fold change of selected metabolites during storage (x axis: storage days) after red blood cell supplementation with uric acid (UA) and/or ascorbic acid (AA). Y axis: fold change versus control. Transparent gray bands' thickness is representative of the mean ± SD of control samples. GSSG: glutathione disulfide. Highlighted pathways: significant divergence in specific supplementations (color defined). Red highlight: final product of both pathways shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Antioxidants supplementation effects upon purine metabolism. Fold change of selected metabolites during storage (x axis: storage days) after red blood cell supplementation with uric acid (UA) and/or ascorbic acid (AA). Y axis: fold change versus control. Transparent gray bands' thickness is representative of the mean ± SD of control samples. Highlighted pathways: significant divergence in specific supplementations (color defined). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Post-storage physiology and post-transfusion recovery of stored red blood cells supplemented with antioxidants. (A) Hemolysis and redox parameters after 24h incubation in conditions mimicking a transfusion recipient environment (n=10 for each group). (B) Representative immunoblots of membrane-bound IgGs in early and late storage (n=6 for each group; 4.1R protein: internal loading control). Blue rectangular: statistically significant reduction (p<0.05). Data are presented as mean ± SD. (C) 24h RBC recovery post-transfusion in a xenobiotic animal model (n=6 for each group; X: mean). C: control, UA: uric acid, AA: ascorbic acid. (*) p<0.05 control vs. all treatments, (m) p<0.05 mix vs. control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)