ZOOMICS : comparative metabolomics of red blood cells from dogs, cows, horses and donkeys during refrigerated storage for up to 42 days

Abstract

Background: The use of omics technologies in human transfusion medicine has improved our understanding of the red blood cell (RBC) storage lesion(s). Despite significant progress towards understanding the storage lesion(s) of human RBCs, a comparison of basal and post-storage RBC metabolism across multiple species using omics technologies has not yet been reported, and is the focus of this study.

Materials and methods: Blood was collected in a standard bag system (CPD-SAG-Mannitol) from dogs (n=8), horses, bovines, and donkeys (n=6). All bags were stored at 4°C for up to 42 days (i.e., the end of the shelf life in Italian veterinary clinics) and sampled weekly for metabolomics analyses. In addition, data comparisons to our ongoing Zoomics project are included to compare this study's results with those of non-human primates and humans.

Results: Significant interspecies differences in RBC metabolism were observed at baseline, at the time of donation, with bovine showing significantly higher levels of metabolites in the tryptophan/kynurenine pathway; dogs showing elevated levels of high-energy compounds (especially adenosine triphosphate and S-adenosyl-methionine) and equine (donkey and horse) RBCs showing almost overlapping phenotypes, with the highest levels of free branched chain amino acids, glycolytic metabolites (including 2,3-diphosphoglycerate), higher total glutathione pools, and elevated metabolites of the folate pathway compared to the other species. Strikingly, previously described metabolic markers of the storage lesion(s) in humans followed similar trends across all species, though the rate of accumulation/depletion of metabolites in energy and redox metabolism varied by species, with equine blood showing the lowest degree of storage lesion(s).

Discussion: These results interrogate RBC metabolism across a range of mammalian species and improve our understanding of both human and veterinary blood storage and transfusion.

Figure 1

Interspecies differences in red blood cell metabolism across dogs, cows, donkeys and horses Metabolomics analyses were performed on freshly drawn red blood cells from dogs (n=8), cows, donkeys and horses (n=6 for each one of the three species. (A) Significant baseline interspecies differences were observed in the metabolomes of fresh red blood cells, as determined by principal component analysis (B) and hierarchical clustering analysis of the top 50 metabolites by ANOVA (C).

Figure 2

Significant interspecies differences in red blood cell glycolysis, pentose phosphate pathway and glutathione metabolism Fresh red blood cells from cows (red), dogs (green), donkeys (dark blue) and horses (light blue) significantly differ in baseline levels of metabolites involved in central energy and redox metabolism. Asterisks indicate significance based on ANOVA with post-hoc multiple column comparisons (ns: not significant; * p <0.05; ** p < 0.01; *** p < 0.001).

Figure 3

Significant interspecies differences in stored red blood cell metabolism Leukofiltered red blood cells from cows (red), dogs (green), donkeys (dark blue) and horses (light blue) were stored in CPD-SAGM for 42 days (A). Sterile weekly sampling generated samples for metabolomics analyses via UHPLC-MS. Results indicate a significant impact of storage duration and species on red blood cell (B) and supernatant (C) metabolites, as determined by principal component analysis and hierarchical clustering analysis of significant metabolites by repeated measure ANOVA (D).

Figure 4

Comparative analysis of the storage lesion in cows, dogs, horses and donkeys vs previously published data from human and non-human primates Metabolomics analyses from the present study were compared against published literature in humans and non-human primates, baboons and rhesus macaques,, (A). In B, hierarchical clustering of red cell metabolomics data from the present and previous studies,,, as a function of storage and species.

Figure 5

Interspecies differences in the red blood cell storage lesion to glycolysis (A), the pentose phosphate pathway and glutathione pools (B), based on data from the present study (cows, dogs, donkeys and horses) and previous studies (humans, baboons, rhesus macaques) of the Zoomics project,, Line plots indicate median (solid line) + interquartile range (lighter, dotted lines) for top significant metabolites by Two-way ANOVA across all four species. Line plots are color coded according to the scheme in the bottom right corner of the figure.

Figure 6

Interspecies differences in the red blood cell storage lesion to purine deamination and oxidation, methionine and arginine metabolism, based on data from the present study (cows, dogs, donkeys and horses) and previous studies (humans, baboons, rhesus macaques) of the Zoomics project,, Line plots indicate median (solid line) + interquartile range (lighter, dotted lines) for top significant metabolites by Two-way ANOVA across all four species. Line plots are color coded according to the scheme in the bottom right corner of the figure.