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Impact of donor biology on stored RBC metabolism
form a biomarker analysis of the whole population versus the seven subjects carrying G6PD A- SNP. This analysis highlighted pyruvate (G6PD deficient if pyruvate <0.546; AUC: 0.95; P-value =4.59 e-33), pyruvate/lactate ratios and dopamine (whose metabolism is NADPH-dependent) and glyceraldehyde 3-phosphate (whose metabolism is NADH-dependent) as the top markers of G6PD deficiency in this study (Figure 6D-E).
Putative G6PD deficient donors were indeed character- ized by significant increases in oxidative hemolysis and decreases in GSH (Figure 7). Even though G6PD deficien- cy was inferred from the SNP detected in the GWAS data, metabolomics analyses confirmed that RBC from the donors bearing those SNP were indeed characterized by an ablation of the PPP and complete depletion of the total glutathione pool, including GSH but also its oxidized form, GSH-adducts (Figure 7A). By limiting the availabili- ty of reduced GSH and NADPH, G6PD deficiency nega- tively impacts recycling of peroxidized lipids through the glyoxalase pathway and aldehyde dehydrogenase 1 (ALDH1)-dependent steps (Figure 7D). Significantly, decreases were indeed observed in the levels of metabo- lites that from pathways requiring either/both NADPH and GSH for the detoxification of oxidized lipids (Figure 7D), including lactaldehyde, lactoyl-glutathione, glu- tathionyl- dihydroxynonane (GSDHN).
Discussion
The mature erythrocyte has evolved to maximize its capacity to carry and deliver oxygen to tissues. As such, RBC are packed with ~270 million copies of hemoglobin per cell and ~66% of the total iron in the human body. When in the presence of oxygen (e.g., oxygen saturation rises above 90% just after 3 weeks of storage owing to the rapid consumption of DPG22), iron can trigger Fenton reactions that generate reactive oxygen species. These phenomena are largely impacted by post-donation hemo- globin-oxygen saturation, which has been noted to vary widely across healthy blood donors.13,21 While RBC have also evolved strategies to cope with such oxidant stress, the propensity of RBC to hemolyze following oxidative injury has recently been noted to depend on donor demo- graphics, including donor sex, age and race-ethnicity.33 Understanding the etiological contributors to such donor- dependent variance might shed light not just on RBC stor- age biology, but also contribute significantly to our under- standing of those pathologies associated with RBC hemolysis due to exacerbated oxidant stress (e.g., sickle cell disease, b-thalassemia, pulmonary hypertension, Down syndrome, inflammaging39). In the present study, we sought to investigate the metabolic underpinnings of RBC susceptibility to oxidant stress-induced hemolysis at the end of storage.
This study challenges the dogma that the “age of blood” is the main factor impacting RBC storability and that the progressive accumulation of the storage lesion inevitably results in poor quality blood across all donors by the end of the shelf-life of the stored unit. While donor biology, including sex, age and ethnicity, had been previ- ously identified as determinants of RBC susceptibility to hemolysis following oxidant insults,33 by leveraging state of the art metabolomics and lipidomics tools, we report for the first time that these observations could be
explained by metabolic factors in the RBC from these donors. Specifically, donors who are less susceptible to oxidant hemolysis (e.g., female donors, donors over 60 years of age and donors of Asian/South Asian race-ethnic- ity, and leucoreduction RBC components preserved in AS-3) are characterized by higher levels of reduced GSH – independently of the storage duration – higher activa- tion of the PPP and improved preservation of the protein damage-repair mechanisms that rely on methionine metabolism (and intertwined arginine metabolism).
Caution is advised in the interpretation of the present- ed results, in that (i) it is unclear whether oxidative hemolysis is predictive of clinically-relevant outcomes and (ii) RBC that are less susceptible to oxidant hemolysis may on the other hand be more susceptible to lysis fol- lowing mechanical and osmotic insults. In this view, the biomarkers and biological correlates to oxidative hemoly- sis identified here are relevant and potentially translatable to studies pertaining oxidant injury, though they may be less relevant for studies on RBC function under mechani- cal or osmotic stress, such as for example in the capillaries and in the kidney. Future studies will investigate metabol- ic correlates and potential mechanistic determinants to these phenotypes. However, prior work has highlighted the clinical relevance of conditions that are associated with increased oxidative hemolysis, such as a causative link between G6PD deficiency and decreases in end of storage autologous post-transfusion recovery in healthy volunteers30 and post-transfusion circulation in sickle cell recipients.29
Previous studies had shown that RBC from G6PD defi- cient donors (the most common enzymopathy in humans, affecting ~10% of the donor population in some metropolitan areas26) are naturally incapable of coping with oxidant stress owing to the ablation of the NADPH- generating PPP. As a result, RBC from G6PD deficient donors favor energy metabolism over antioxidant path- ways, which protects them from morphological changes but makes them more susceptible to oxidant insults upon storage.28 This observation has potential clinical implica- tions, in that transfusion of blood from G6PD deficient donors into recipients suffering from elevated oxidative stress (e.g., sickle cell recipients40) may be suboptimal and could potentially promote untoward transfusion conse- quences or, at least, decrease the dose-efficacy of transfu- sion therapies. In the present study, by combining metabolomics and genomics data from the RBC-Omics study (Page et al. under review and Guo et al.41), we iden- tified a key role of antioxidant systems that rely on reduc- ing equivalents such as GSH and NADPH to cope with oxidant stress – such as G6PD deficiency. While this result was largerly expected, the present study advanced our understanding of G6PD deficiency by providing genetic and metabolomic metabolic markers (e.g., pyru- vate/lactate ratios, absolute concentration of pyruvate) that complement spectrophotometric assays of the enzy- matic activity of G6PD.42 Some markers like pyruvate to lactate ratio were previously associated with increased activation of NADH-dependent methemoglobin reduc- tase.28 As such, since glyceraldehyde 3-phosphate metab- olism is dependent on NAD+/NADH ratios, the two markers observed here are metabolically linked. On the other hand, dopamine metabolism is NADPH-dependent – suggesting a role for monoamine oxidase activity in the context of RBC metabolism and storability as a function
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