Hypoxic storage of murine red blood cells improves energy metabolism and post-transfusion recoveries

Abstract

Background: The Red blood cell (RBC) storage lesion results in decreased circulation and function of transfused RBCs. Elevated oxidant stress and impaired energy metabolism are a hallmark of the storage lesion in both human and murine RBCs. Although human studies don't suffer concerns that findings may not translate, they do suffer from genetic and environmental variability amongst subjects. Murine models can control for genetics, environment, and much interventional experimentation can be carried out in mice that is neither technically feasible nor ethical in humans. However, murine models are only useful to the extent that they have similar biology to humans. Hypoxic storage has been shown to mitigate the storage lesion in human RBCs, but has not been investigated in mice.

Materials and methods: RBCs from a C57BL6/J mouse strain were stored under normoxic (untreated) or hypoxic conditions (SO2 ~ 26%) for 1h, 7 and 12 days. Samples were tested for metabolomics at steady state, tracing experiments with 1,2,3-¹³C₃-glucose, proteomics and end of storage post transfusion recovery.

Results: Hypoxic storage improved post-transfusion recovery and energy metabolism, including increased steady state and ¹³C₃-labeled metabolites from glycolysis, high energy purines (adenosine triphosphate) and 2,3-diphospholgycerate. Hypoxic storage promoted glutaminolysis, increased glutathione pools, and was accompanied by elevation in the levels of free fatty acids and acyl-carnitines.

Discussion: This study isolates hypoxia, as a single independent variable, and shows similar effects as seen in human studies. These findings also demonstrate the translatability of murine models for hypoxic RBC storage and provide a pre-clinical platform for ongoing study.

Figure 1

Post-transfusion recovery and omis of normoxic and hypoxic murine RBCs Hypoxic storage of murine RBCs improves post-transfusion recovery (A) and has a significant effect on the RBC metabolome and proteome. RBCs were stored for 1h, 7 or 12 days under normoxic (shades of blue) or hypoxic (shades of red) conditions. At storage day 12, post-transfusion recovery studies showed a significant increase (p<0.001) in PTR in mouse RBCs stored under hypoxia. Metabolomics analyses showed a significant impact of storage duration and normoxic vs hypoxic storage on mouse RBCs, as gleaned by unsupervised hierarchical clustering analysis of the top 75 metabolites by repeated measures ANOVA (B). Similarly, storage impacted the RBC proteome (C). Bar plots with superimposed dot plots (n = 3) are shown for vinculine and peptidyl-prolyl cis-trans isomerase (PPIA), two of the most significantly impacted proteins in this analysis (mean + standard deviation). Paired t-test between normoxic or hypoxic samples from the same mice are shown for each time point (Welsch τ-test; ns: not significant; *p<0.05).

Figure 2

Impact of normoxic or hypoxic storage on RBC glycolysis and high-energy purinemetabolism RBCs were stored for 1h, 7 or 12 days under normoxic (shades of blue) or hypoxic (shades of red) conditions. Bar plots with superimposed dot plots (n = 3) are shown for selected metabolites in this pathway (mean + standard deviation). Paired t-test between normoxic or hypoxic samples from the same mice are shown for each time point (Welsch τ-test; ns: not significant; *p<0.05).

Figure 3

Glucose tracing experiments confirm up-regulation of glycolysis at the expense of PPP in murine RBCs under hypoxic storage Metabolic tracing experiments were performed by incubating murine RBCs for 1h, 7 or 12 days under normoxic (standard) or hypoxic conditions in presence of 1,2,3-13C3-glucose. Results indicate comparable levels of glucose depletion, but a significantly higher accumulation of labeled glycolytic intermediates (e.g., fructose bisphosphate – FBP) and byproducts (lactate) in hypoxic RBCs (A). Determination of fluxes through the Embden-Meyerhof-Parnas (EMP) glycolytic pathway and the pentose phosphate pathway (PPP – B) can be performed through determination of the levels of lactate isotopologues +2 (from the PPP) and +3 (derived from the EMP – C). In keeping with previous reports in human RBCs, murine hypoxic RBCs have lower levels of PPP-derived lactate +2 and higher levels of EMP derived lactate +3, consistent with increased glycolytic fluxes in hypoxic RBCs.

Figure 4

Impact of normoxic or hypoxic storage on RBC pentose phosphate pathway and glutathione homeostasis RBCs were stored for 1h, 7 or 12 days under normoxic (shades of blue) or hypoxic (shades of red) conditions. Bar plots with superimposed dot plots (n=3) are shown for selected metabolites in this pathway (mean + standard deviation). Paired t-test between normoxic or hypoxic samples from the same mice are shown for each time point (Welsch τ-test; ns: not significant; *p<0.05).

Figure 5

Impact of normoxic or hypoxic storage on RBC carboxylic acid metabolism To be noted that an incomplete, non-canonical carboxylic acid cycle is present in mature RBCs and here represented as the standard Krebs cycle for simplicity. RBCs were stored for 1h, 7 or 12 days under normoxic (shades of blue) or hypoxic (shades of red) conditions. Bar plots with superimposed dot plots (n = 3) are shown for selected metabolites in this pathway (mean + standard deviation). Paired t-test between normoxic or hypoxic samples from the same mice are shown for each time point (Welsch τ-test; ns: not significant; *p<0.05).

Figure 6

A impact of normoxic or hypoxic storage on RBC purine deamination and carboxylic acid metabolism RBCs were stored for 1h, 7 or 12 days under normoxic (shades of blue) or hypoxic (shades of red) conditions. B Impact of normoxic or hypoxic storage on RBC free fatty acids and acyl-carnitine metabolism. RBCs were stored for 1h, 7 or 12 days under normoxic (shades of blue) or hypoxic (shades of red) conditions. Bar plots with superimposed dot plots (n = 3) are shown for selected metabolites in this pathway (mean + standard deviation). Paired t-test between normoxic or hypoxic samples from the same mice are shown for each time point (Welsch τ-test; ns: not significant; *p<0.05).