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Primaquine and Tafenoquine are the only approved drugs that can achieve a radical cure for Plasmodium vivax malaria but are contraindicated in patients who are deficient in glucose 6-phosphate dehydrogenase (G6PDd) due to risk of severe hemolysis from reactive oxygen species generated by redox cycling of drug metabolites. 5-hydroxyprimaquine and its quinoneimine cause robust redox cycling in red blood cells (RBCs) but are so labile as to not be detected in blood or urine. Rather, the quinoneimine is rapidly converted into primaquine-5,6-orthoquinone (5,6-POQ) that is then excreted in the urine. The extent to which 5,6-POQ contributes to hemolysis remains unclear, although some have suggested that it is a minor toxin that should be used predominantly as a surrogate to infer levels of 5-hydroxyprimaquine. In this report, we describe a novel humanized mouse model of the G6PD Mediterranean variant (hG6PD Med- ) that recapitulates the human biology of RBC age-dependent enzyme decay, as well as an isogenic matched control mouse with human nondeficient G6PD hG6PD ND In vitro challenge of RBCs with 5,6-POQ causes increased generation of superoxide and methemoglobin. Infusion of treated RBCs shows that 5,6-POQ selectively causes in vivo clearance of older hG6PD Med- RBCs. These findings support the hypothesis that 5,6-POQ directly induces hemolysis and challenges the notion that 5,6-POQ is an inactive metabolic waste product. Indeed, given the extreme lability of 5-hydroxyprimaquine and the relative stability of 5,6-POQ, these data raise the possibility that 5,6-POQ is a major hemolytic primaquine metabolite in vivo. SIGNIFICANCE STATEMENT: These findings demonstrate that 5,6-POQ, which has been considered an inert waste product of primaquine metabolism, directly induces ROS that cause clearance of older G6PDd RBCs. As 5,6-POQ is relatively stable compared with other active primaquine metabolites, these data support the hypothesis that 5,6-POQ is a major toxin in primaquine induced hemolysis. The findings herein also establish a new model of G6PDd and provide the first direct evidence, to our knowledge, that young G6PDd RBCs are resistant to primaquine-induced hemolysis., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Competing Interests: Competing interests statement:The authors declare no competing interest.

Mature red blood cells (RBCs) lack mitochondria and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine (HYPX) levels-and the genetic traits linked to them-were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes., Competing Interests: Declaration of interests A.D’A., K.C.H., and T.N. are founders of Omix Technologies Inc. and Altis Biosciences LLC. A.D’A., S.L.S., and T.N. are Scientific Advisory Board (SAB) members for Hemanext Inc. A.D’A. is an SAB member for Macopharma Inc. S.L.S. is an SAB member for Alcor, Inc.; a consultant for Team Conveyer Intellectual Properties; executive director for Worldwide Initiative for Rh Disease Eradication (WIRhE); and CEO for Ferrous Wheel Consultants, LLC. J.C.Z. is a founder of Svalinn Therapeutics., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mitapivat, a pyruvate kinase activator, shows great potential as a sickle cell disease (SCD)-modifying therapy. The safety and efficacy of mitapivat as a long-term maintenance therapy are currently being evaluated in two open-label studies. Here we applied a comprehensive multi-omics approach to investigate the impact of activating pyruvate kinase on red blood cells (RBC) from 15 SCD patients. HbSS patients were enrolled in one of the open-label, extended studies (NCT04610866). Leukodepleted RBC obtained from fresh whole blood at baseline, prior to drug initiation, and at longitudinal timepoints over the course of the study were processed for multi-omics through a stepwise extraction of metabolites, lipids and proteins. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBC. Mitapivat decreased 2,3-diphosphoglycerate levels, increased adenosine triphosphate levels, and improved hematologic and sickling parameters in patients with SCD. Agreement between omics measurements and clinical measurements confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of hemoglobin S oxygen affinity (p50) and sickling kinetics (t50) during treatment. Mitapivat markedly reduced levels of proteins of mitochondrial origin within 2 weeks of initiation of treatment, with minimal changes in reticulocyte counts. In the first 6 months of treatment there were also transient elevations of lysophosphatidylcholines and oxylipins with depletion of free fatty acids, suggestive of an effect on membrane lipid remodeling. Multi-omics analysis of RBC identified benefits for glycolysis, as well as activation of the Lands cycle.

Abstract: Recent large-scale multiomics studies suggest that genetic factors influence the chemical individuality of donated blood. To examine this concept, we performed metabolomics analyses of 643 blood units from volunteers who donated units of packed red blood cells (RBCs) on 2 separate occasions. These analyses identified carnitine metabolism as the most reproducible pathway across multiple donations from the same donor. We also measured l-carnitine and acyl-carnitines in 13 091 packed RBC units from donors in the Recipient Epidemiology and Donor Evaluation study. Genome-wide association studies against 879 000 polymorphisms identified critical genetic factors contributing to interdonor heterogeneity in end-of-storage carnitine levels, including common nonsynonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, and SLC16A9); carnitine synthesis (FLVCR1 and MTDH) and metabolism (CPT1A, CPT2, CRAT, and ACSS2), and carnitine-dependent repair of lipids oxidized by ALOX5. Significant associations between genetic polymorphisms on SLC22 transporters and carnitine pools in stored RBCs were validated in 525 Diversity Outbred mice. Donors carrying 2 alleles of the rs12210538 SLC22A16 single-nucleotide polymorphism exhibited the lowest l-carnitine levels, significant elevations of in vitro hemolysis, and the highest degree of vesiculation, accompanied by increases in lipid peroxidation markers. Separation of RBCs by age, via in vivo biotinylation in mice, and Percoll density gradients of human RBCs, showed age-dependent depletions of l-carnitine and acyl-carnitine pools, accompanied by progressive failure of the reacylation process after chemically induced membrane lipid damage. Supplementation of stored murine RBCs with l-carnitine boosted posttransfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Background: Red blood cell (RBC) storage promotes biochemical and morphological alterations, collectively referred to as storage lesions (SLs). Studies in humans have identified leukoreduction (LR) as a critical processing step that mitigates SLs. To date no study has evaluated the impact of LR on metabolic SLs in canine blood units using omics technologies., Objective: Compare the lipid and metabolic profiles of canine packed RBC (pRBC) units as a function of LR in fresh and stored refrigerated (up to 42 days) units., Animals: Packed RBC units were obtained from 8 donor dogs enrolled at 2 different Italian veterinary blood banks., Study Design and Methods: Observational study. A volume of 450 mL of whole blood was collected using Citrate-Phosphate-Dextrose-Saline-Adenine-Glucose-Mannitol (CPD-SAGM) transfusion bags with a LR filter to produce 2 pRBC units for each donor, without (nLR-pRBC) and with (LR-pRBC) LR. Units were stored in the blood bank at 4 ± 2°C. Sterile weekly samples were obtained from each unit for omics analyses., Results: A significant effect of LR on fresh and stored RBC metabolic phenotypes was observed. The nLR-pRBC were characterized by higher concentrations of free short and medium-chain fatty acids, carboxylic acids (pyruvate, lactate), and amino acids (arginine, cystine). The LR-pRBC had higher concentrations of glycolytic metabolites, high energy phosphate compounds (adenosine triphosphate [ATP]), and antioxidant metabolites (pentose phosphate, total glutathione)., Conclusion and Clinical Importance: Leukoreduction decreases the metabolic SLs of canine pRBC by preserving energy metabolism and preventing oxidative lesions., (© 2024 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Background: Understanding of the biochemical and morphological lesions associated with storage of equine blood is limited., Objective: To demonstrate the temporal sequences of lipid and metabolic profiles of equine fresh and stored (up to 42 days) and leukoreduced packed red blood cells (LR-pRBC) and non-leukoreduced packed RBC (nLR-pRBC)., Animals: Packed RBC units were obtained from 6 healthy blood donor horses enrolled in 2 blood banks., Methods: Observational study. Whole blood was collected from each donor using transfusion bags with a LR filter. Leukoreduction pRBC and nLR-pRBC units were obtained and stored at 4°C for up 42 days. Sterile weekly sampling was performed from each unit for analyses., Results: Red blood cells and supernatants progressively accumulated lactate products while high-energy phosphate compounds (adenosine triphosphate and 2,3-Diphosphoglycerate) declined. Hypoxanthine, xanthine, and free fatty acids accumulated in stored RBC and supernatants. These lesions were exacerbated in non-LR-pRBC., Conclusion and Clinical Importance: Leukoreduction has a beneficial effect on RBC energy and redox metabolism of equine pRBC and the onset and severity of the metabolic storage lesions RBC., (© 2024 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Our aim is to present an updated overview of the erythrocyte metabolism highlighting its richness and complexity. We have manually collected and connected the available biochemical pathways and integrated them into a functional metabolic map. The focus of this map is on the main biochemical pathways consisting of glycolysis, the pentose phosphate pathway, redox metabolism, oxygen metabolism, purine/nucleoside metabolism, and membrane transport. Other recently emerging pathways are also curated, like the methionine salvage pathway, the glyoxalase system, carnitine metabolism, and the lands cycle, as well as remnants of the carboxylic acid metabolism. An additional goal of this review is to present the dynamics of erythrocyte metabolism, providing key numbers used to perform basic quantitative analyses. By synthesizing experimental and computational data, we conclude that glycolysis, pentose phosphate pathway, and redox metabolism are the foundations of erythrocyte metabolism. Additionally, the erythrocyte can sense oxygen levels and oxidative stress adjusting its mechanics, metabolism, and function. In conclusion, fine-tuning of erythrocyte metabolism controls one of the most important biological processes, that is, oxygen loading, transport, and delivery., (© 2024 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Blood storage promotes the rapid depletion of red blood cell (RBC) high-energy adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG), which are critical regulators of erythrocyte physiology and function, as well as oxygen kinetics and posttransfusion survival. Sphingosine-1-phosphate (S1P) promotes fluxes through glycolysis. We hypothesized that S1P supplementation to stored RBC units would improve energy metabolism and posttransfusion recovery. We quantified S1P in 1929 samples (n = 643, storage days 10, 23, and 42) from the REDS RBC Omics study. We then supplemented human and murine RBCs from good storer (C57BL6/J) and poor storer strains (FVB) with S1P (1, 5, and 10 μM) before measurements of metabolism and posttransfusion recovery. Similar experiments were repeated for mice with genetic ablation of the S1P biosynthetic pathway (sphingosine kinase 1 [Sphk1] knockout [KO]). Sample analyses included metabolomics at steady state, tracing experiments with 1,2,3-13C3-glucose, proteomics, and analysis of end-of-storage posttransfusion recovery, under normoxic and hypoxic storage conditions. Storage promoted decreases in S1P levels, which were the highest in units donated by female or older donors. Supplementation of S1P to human and murine RBCs boosted the steady-state levels of glycolytic metabolites and glycolytic fluxes, ie the generation of ATP and DPG, at the expense of the pentose phosphate pathway. Lower posttransfusion recovery was observed upon S1P supplementation. All these phenomena were reversed in Sphk1 KO mice or with hypoxic storage. S1P is a positive regulator of energy metabolism and a negative regulator of antioxidant metabolism in stored RBCs, resulting in lower posttransfusion recoveries in murine models., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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- 13 C 3 -glucose, proteomics and end of storage post transfusion recovery., Results: Hypoxic storage improved post-transfusion recovery and energy metabolism, including increased steady state and 13 C 3 -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.

Blood donors with beta-thalassemia traits (βThal + ) have proven to be good "storers", since their stored RBCs are resistant to lysis and resilient against oxidative/proteotoxic stress. To examine the performance of these RBCs post-storage, stored βThal + and control RBCs were reconstituted in plasma donated from transfusion-dependent beta-thalassemic patients and healthy controls, and incubated for 24 h at body temperature. Several physiological parameters, including hemolysis, were evaluated. Moreover, labeled fresh/stored RBCs from the two groups were transfused in mice to assess 24 h recovery. All hemolysis metrics were better in the group of heterozygotes and distinguished them against controls in the plasma environment. The reconstituted βThal + samples also presented higher proteasome activity and fewer procoagulant extracellular vesicles. Transfusion to mice demonstrated that βThal + RBCs present a marginal trend for higher recovery, regardless of the recipient's immune background and the RBC storage age. According to correlation analysis, several of these advantageous post-storage characteristics are related to storage phenotypes, like the cytoskeleton composition, low cellular fragility, and enhanced membrane proteostasis that characterize stored βThal + RBCs. Overall, it seems that the intrinsic physiology of βThal + RBCs benefits them in conditions mimicking a recipient environment, and in the circulation of animal models; findings that warrant validation in clinical trials.

Blood banks around the world store blood components for several weeks ensuring its availability for transfusion medicine. Red blood cells (RBCs) are known to undergo compositional changes during storage, which may impact the cells' function and eventually the recipients' health. We extracted the RBC's cytoplasmic membrane (RBCcm) to study the effect of storage on the membranes' molecular structure and bending rigidity by a combination of X-ray diffraction (XRD), X-ray diffuse scattering (XDS) and coarse grained Molecular Dynamics (MD) simulations. Blood was stored in commercial blood bags for 2 and 5 weeks, respectively and compared to freshly drawn blood. Using mass spectrometry, we measured an increase of fatty acids together with a slight shift towards shorter tail lengths. We observe an increased fraction (6%) of liquid ordered (lo) domains in the RBCcms with storage time, and an increased lipid packing in these domains, leading to an increased membrane thickness and membrane order. The size of both, lo and liquid disordered (ld) lipid domains was found to decrease with increased storage time by up to 25%. XDS experiments reveal a storage dependent increase in the RBCcm's bending modulus κ by a factor of 2.8, from 1.9 kBT to 5.3 kBT. MD simulations were conducted in the absence of proteins. The results show that the membrane composition has a small contribution to the increased bending rigidity and suggests additional protein-driven mechanisms., Competing Interests: The authors have declared that no competing interests exist.

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice.

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of WT stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RBCs) that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency, Med(-), was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs, and deficient mice had increased hemolytic sequelae to oxidant stress. Nonerythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD-deficient and WT mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and advance our basic understanding of altered metabolism in this setting.

Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O2) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude. However, nothing is known about a role for erythrocyte ADORA2B in age-related functional decline. Here, we report that loss of murine erythrocyte-specific ADORA2B (eAdora2b-/-) accelerates early onset of age-related impairments in spatial learning, memory, and hearing ability. eAdora2b-/- mice display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus (HIP), cortex, or cochlea. Hypoxia sufficiently accelerates early onset of cognitive and cochlear functional decline and inflammatory response in eAdora2b-/- mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP-activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O2 delivery. Significantly, this finding led us to further discover that murine erythroblast ADORA2B and BPGM mRNA levels and erythrocyte BPGM activity are reduced during normal aging. Overall, we determined that erythrocyte ADORA2B-BPGM axis is a key component for anti-aging and anti-age-related functional decline., Competing Interests: The authors have declared that no competing interests exist.

Background: Increases in the red blood cell (RBC) degree of fatty acid desaturation are reported in response to exercise, aging, or diseases associated with systemic oxidant stress. However, no studies have focused on the presence and activity of fatty acid desaturases (FADS) in the mature RBC., Study Design and Methods: Steady state metabolomics and isotope-labeled tracing experiments, immunofluorescence approaches, and pharmacological interventions were used to determine the degree of fatty acid unsaturation, FADS activity as a function of storage, oxidant stress, and G6PD deficiency in human and mouse RBCs., Results: In 250 blood units from the REDS III RBC Omics recalled donor population, we report a storage-dependent accumulation of free mono-, poly-(PUFAs), and highly unsaturated fatty acids (HUFAs), which occur at a faster rate than saturated fatty acid accumulation. Through a combination of immunofluorescence, pharmacological inhibition, tracing experiments with stable isotope-labeled fatty acids, and oxidant challenge with hydrogen peroxide, we demonstrate the presence and redox-sensitive activity of FADS2, FADS1, and FADS5 in the mature RBC. Increases in PUFAs and HUFAs in human and mouse RBCs correlate negatively with storage hemolysis and positively with posttransfusion recovery. Inhibition of these enzymes decreases accumulation of free PUFAs and HUFAs in stored RBCs, concomitant to increases in pyruvate/lactate ratios. Alterations of this ratio in G6PD deficient patients or units supplemented with pyruvate-rich rejuvenation solutions corresponded to decreased PUFA and HUFA accumulation., Conclusion: Fatty acid desaturases are present and active in mature RBCs. Their activity is sensitive to oxidant stress, storage duration, and alterations of the pyruvate/lactate ratio., (© 2021 AABB.)

Background: During storage, red blood cells (RBCs) undergo significant biochemical and morphologic changes, referred to collectively as the "storage lesion". It was hypothesized that these defects may arise from disrupted oxygen-based regulation of RBC energy metabolism, with resultant depowering of intrinsic antioxidant systems., Study Design and Methods: As a function of storage duration, the dynamic range in RBC metabolic response to three models of biochemical oxidant stress (methylene blue, hypoxanthine/xanthine oxidase, and diamide) was assessed, comparing glycolytic flux by NMR and UHPLC-MS methodologies. Blood was processed/stored under standard conditions (AS-1 additive solution) with leukoreduction. Over a 6-week period, RBC metabolic and antioxidant status were assessed at baseline and following exposure to the three biochemical oxidant models. Comparison was made of glycolytic flux ( 1 H-NMR tracking of [2- 13 C]-glucose and metabolomic phenotyping with [1,2,3- 13 C 3 ] glucose), reducing equivalent (NADPH/NADP + ) recycling, and thiol-based (GSH/GSSG) antioxidant status., Results: As a function of storage duration, we observed the following: (1) a reduction in baseline hexose monophosphate pathway (HMP) flux, the sole pathway responsible for the regeneration of the essential reducing equivalent NADPH; with (2) diminished stress-based dynamic range in both overall glycolytic as well as proportional HMP flux. In addition, progressive with storage duration, RBCs showed (3) constraint in reducing equivalent (NADPH) recycling capacity, (4) loss of thiol based (GSH) recycling capacity, and (5) dysregulation of metabolon assembly at the cytoplasmic domain of Band 3 membrane protein (cdB3)., Conclusion: Blood storage disturbs normal RBC metabolic control, depowering antioxidant capacity and enhancing vulnerability to oxidative injury., (© 2021 AABB.)

Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures ("exposome") may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell-Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine - though rarely detected in the blood donor population - has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could - through mechanisms involving sphingosine 1-phosphate-dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin - improve erythrocyte metabolism and storage quality.

Background: Obesity is a global pandemic characterized by multiple comorbidities, including cardiovascular and metabolic diseases. The aim of this study was to define the associations between blood donor body mass index (BMI) and RBC measurements of metabolic stress and hemolysis., Study Design and Methods: The associations between donor BMI (<25 kg/m 2 , normal weight; 25-29.9 kg/m 2 , overweight; and ≥30 kg/m 2 , obese) and hemolysis (storage, osmotic, and oxidative; n = 18 donors) or posttransfusion recovery (n = 14 donors) in immunodeficient mice were determined in stored leukocyte-reduced RBC units. Further evaluations were conducted using the National Heart, Lung, and Blood Institute RBC-Omics blood donor databases of hemolysis (n = 13 317) and metabolomics (n = 203)., Results: Evaluations in 18 donors revealed that BMI was significantly (P < 0.05) and positively associated with storage and osmotic hemolysis. A BMI of 30 kg/m 2 or greater was also associated with lower posttransfusion recovery in mice 10 minutes after transfusion (P = 0.026). Multivariable linear regression analyses in RBC-Omics revealed that BMI was a significant modifier for all hemolysis measurements, explaining 4.5%, 4.2%, and 0.2% of the variance in osmotic, oxidative, and storage hemolysis, respectively. In this cohort, obesity was positively associated (P < 0.001) with plasma ferritin (inflammation marker). Metabolomic analyses on RBCs from obese donors (44.1 ± 5.1 kg/m 2 ) had altered membrane lipid composition, dysregulation of antioxidant pathways (eg, increased oxidized lipids, methionine sulfoxide, and xanthine), and dysregulation of nitric oxide metabolism, as compared to RBCs from nonobese (20.5 ± 1.0 kg/m 2 ) donors., Conclusions: Obesity is associated with significant changes in RBC metabolism and increased susceptibility to hemolysis under routine storage of RBC units. The impact on transfusion efficacy warrants further evaluation., (© 2020 AABB.)

Here we describe the effects of a controlled, 30 min, high-intensity cycling test on blood rheology and the metabolic profiles of red blood cells (RBCs) and plasma from well-trained males. RBCs demonstrated decreased deformability and trended toward increased generation of microparticles after the test. Meanwhile, metabolomics and lipidomics highlighted oxidative stress and activation of membrane lipid remodeling mechanisms in order to cope with altered properties of circulation resulting from physical exertion during the cycling test. Of note, intermediates from coenzyme A (CoA) synthesis for conjugation to fatty acyl chains, in parallel with reversible conversion of carnitine and acylcarnitines, emerged as metabolites that significantly correlate with RBC deformability and the generation of microparticles during exercise. Taken together, we propose that RBC membrane remodeling and repair plays an active role in the physiologic response to exercise by altering RBC properties.

Background: Red blood cells (RBCs) derived from patients who receive testosterone replacement therapy (TRT) may be considered eligible for component production and transfusion. The aim of this study was to identify testosterone-dependent changes in RBC metabolism and to evaluate its impact on susceptibility to hemolysis during cold storage., Study Design and Methods: We characterized stored RBCs from two cohorts of TRT patients who were matched with control donors (no TRT) based upon sex, age, and ethnicity. We further evaluated the impact of testosterone deficiency (orchiectomy) on RBC metabolism in FVB/NJ mice. RBC metabolites were quantified by ultra-high-pressure liquid chromatography-mass spectrometry. RBC storage stability was determined in RBC units from TRT and controls by quantifying storage, osmotic, and oxidative hemolysis., Results: Orchiectomy in mice was associated with significant (P < 0.05) changes in RBC metabolism as compared with intact males including increased levels of acyl-carnitines, long-chain fatty acids (eg, docosapentaenoic acids), arginine, and dopamine. Stored RBCs from TRT patients exhibited higher levels of pentose phosphate pathway metabolites, glutathione, and oxidized purines (eg, hypoxanthine), suggestive of increased activation of antioxidant pathways in this group. Further analyses indicated significant changes in free fatty acids and acyl-carnitines in response to testosterone therapies. With regard to hemolysis, TRT was associated with enhanced susceptibility to osmotic hemolysis. Correlation analyses identified acyl-carnitines as significant modifiers of RBC predisposition to osmotic and oxidative hemolysis., Conclusions: These observations provide new insights into testosterone-mediated changes in RBC metabolome and biology that may impact the storage capacity and posttransfusion efficacy of RBCs from TRT donors., (© 2020 AABB.)

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, or mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading and metabolic rewiring toward the hexose monophosphate shunt, RBCs from COVID-19 patients may be less capable of responding to environmental variations in hemoglobin oxygen saturation/oxidant stress when traveling from the lungs to peripheral capillaries and vice versa.

Background: Characteristics of red blood cells (RBCs) are influenced by donor variability. This study assessed quality and metabolomic variables of RBC subpopulations of varied biologic age in red blood cell concentrates (RCCs) from male and female donors to evaluate their contribution to the storage lesion., Study Design and Methods: Red blood cell concentrates from healthy male (n = 6) and female (n = 4) donors were Percoll separated into less dense ("young", Y-RCCs) and dense ("old", O-RCCs) subpopulations, which were assessed weekly for 28 days for changes in hemolysis, mean cell volume (MCV), hemoglobin concentration (MCHC), hemoglobin autofluorescence (HGB), morphology index (MI), oxygen affinity (p50), rigidity, intracellular reactive oxygen species (ROS), calcium ([Ca 2+ ]), and mass spectrometry-based metabolomics., Results: Young RCCs having disc-to-discoid morphology showed higher MCV and MI, but lower MCHC, HGB, and rigidity than O-RCCs, having discoid-to-spheroid shape. By Day 14, Y-RCCs retained lower hemolysis and rigidity and higher p50 compared to O-RCCs. Donor sex analyses indicated that females had higher MCV, HGB, ROS, and [Ca 2+ ] and lower hemolysis than male RBCs, in addition to having a decreased rate of change in hemolysis by Day 28. Metabolic profiling indicated a significant sex-related signature across all groups with increased markers of high membrane lipid remodeling and antioxidant capacity in Y-RCCs, whereas O-RCCs had increased markers of oxidative stress and decreased coping capability., Conclusion: The structural, functional, and metabolic dissimilarities of Y-RCCs and O-RCCs from female and male donors demonstrate RCC heterogeneity, where RBCs from females contribute less to the storage lesion and age slower than males., (© 2020 AABB.)

Macaques are emerging as a critical animal model in transfusion medicine, because of their evolutionary similarity to humans and perceived utility in discovery and translational science. However, little is known about the metabolism of Rhesus macaque red blood cells (RBC) and how this compares to human RBC metabolism under standard blood banking conditions. Metabolomic and lipidomic analyses, and tracing experiments with [1,2,3- 13 C 3 ]glucose, were performed using fresh and stored RBC (sampled weekly until storage day 42) obtained from Rhesus macaques (n=20) and healthy human volunteers (n=21). These results were further validated with targeted quantification against stable isotope-labeled internal standards. Metabolomic analyses demonstrated inter-species differences in RBC metabolism independent of refrigerated storage. Although similar trends were observed throughout storage for several metabolic pathways, species- and sex-specific differences were also observed. The most notable differences were in glutathione and sulfur metabolites, purine and lipid oxidation metabolites, acylcarnitines, fatty acyl composition of several classes of lipids (including phosphatidylserines), glyoxylate pathway intermediates, and arginine and carboxylic acid metabolites. Species-specific dietary and environmental compounds were also detected. Overall, the results suggest an increased basal and refrigerator-storage-induced propensity for oxidant stress and lipid remodeling in Rhesus macaque RBC cells, as compared to human red cells. The overlap between Rhesus macaque and human RBC metabolic phenotypes suggests the potential utility of a translational model for simple RBC transfusions, although inter-species storage-dependent differences need to be considered when modeling complex disease states, such as transfusion in trauma/hemorrhagic shock models., (Copyright© 2020 Ferrata Storti Foundation.)

Rationale: Hypoxia promotes renal damage and progression of chronic kidney disease (CKD). The erythrocyte is the only cell type for oxygen (O 2 ) delivery. Sphingosine 1-phosphate (S1P)-a highly enriched biolipid in erythrocytes-is recently reported to be induced under high altitude in normal humans to enhance O 2 delivery. However, nothing is known about erythrocyte S1P in CKD., Objective: To investigate the function and metabolic basis of erythrocyte S1P in CKD with a goal to explore potential therapeutics., Methods and Results: Using erythrocyte-specific SphK1 (sphingosine kinase 1; the only enzyme to produce S1P in erythrocytes) knockout mice ( eSphK1 -/- ) in an experimental model of hypertensive CKD with Ang II (angiotensin II) infusion, we found severe renal hypoxia, hypertension, proteinuria, and fibrosis in Ang II-infused eSphk1 -/- mice compared with controls. Untargeted metabolomics profiling and in vivo U- 13 C 6 isotopically labeled glucose flux analysis revealed that SphK1 is required for channeling glucose metabolism toward glycolysis versus pentose phosphate pathway, resulting in enhanced erythroid-specific Rapoport-Luebering shunt in Ang II-infused mice. Mechanistically, increased erythrocyte S1P functioning intracellularly activates AMPK (AMP-activated protein kinase) 1α and BPGM (bisphosphoglycerate mutase) by reducing ceramide/S1P ratio and inhibiting PP2A (protein phosphatase 2A), leading to increased 2,3-bisphosphoglycerate (an erythrocyte-specific metabolite negatively regulating Hb [hemoglobin]-O 2 mice and prevented development of CKD in the control mice by inducing 2,3-bisphosphoglycerate production and thus enhancing renal oxygenation. Translational research validated mouse findings in erythrocytes of hypertensive CKD patients and cultured human erythrocytes.2 delivery to counteract kidney hypoxia and progression to CKD. Preclinical studies revealed that an AMPK agonist or a PP2A inhibitor rescued the severe CKD phenotype in Ang II-infused eSphK1 -/- mice and prevented development of CKD in the control mice by inducing 2,3-bisphosphoglycerate production and thus enhancing renal oxygenation. Translational research validated mouse findings in erythrocytes of hypertensive CKD patients and cultured human erythrocytes., Conclusions: Our study elucidates the beneficial role of eSphk1-S1P in hypertensive CKD by channeling glucose metabolism toward Rapoport-Luebering shunt and inducing 2,3-bisphosphoglycerate production and O 2 delivery via a PP2A-AMPK1α signaling pathway. These findings reveal the metabolic and molecular basis of erythrocyte S1P in CKD and new therapeutic avenues.

Background: Taurine is an antioxidant that is abundant in some common energy drinks. Here we hypothesized that the antioxidant activity of taurine in red blood cells (RBCs) could be leveraged to counteract storage-induced oxidant stress., Study Design and Methods: Metabolomics analyses were performed on plasma and RBCs from healthy volunteers (n = 4) at baseline and after consumption of a whole can of a common, taurine-rich (1000 mg/serving) energy drink. Reductionistic studies were also performed by incubating human RBCs with taurine ex vivo (unlabeled or 13 C 15 N-labeled) at increasing doses (0, 100, 500, and 1000 μmol/L) at 37°C for up to 16 hours, with and without oxidant stress challenge with hydrogen peroxide (0.1% or 0.5%). Finally, we stored human and murine RBCs under blood bank conditions in additives supplemented with 500 μmol/L taurine, before metabolomics and posttransfusion recovery studies., Results: Consumption of energy drinks increased plasma and RBC levels of taurine, which was paralleled by increases in glycolysis and glutathione (GSH) metabolism in the RBC. These observations were recapitulated ex vivo after incubation with taurine and hydrogen peroxide. Taurine levels in the RBCs from the REDS-III RBC-Omics donor biobank were directly proportional to the total levels of GSH and glutathionylated metabolites and inversely correlated to oxidative hemolysis measurements. Storage of human RBCs in the presence of taurine improved energy and redox markers of storage quality and increased posttransfusion recoveries in FVB mice., Conclusion: Taurine modulates RBC antioxidant metabolism in vivo and ex vivo, an observation of potential relevance to transfusion medicine., (© 2020 AABB.)

Background: Cigarette smoking is a frequent habit across blood donors (approx. 13% of the donor population), that could compound biologic factors and exacerbate oxidant stress to stored red blood cells (RBCs)., Study Design and Methods: As part of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, a total of 599 samples were sterilely drawn from RBC units stored under blood bank conditions at Storage Days 10, 23, and 42 days, before testing for hemolysis parameters and metabolomics. Quantitative measurements of nicotine and its metabolites cotinine and cotinine oxide were performed against deuterium-labeled internal standards., Results: Donors whose blood cotinine levels exceeded 10 ng/mL (14% of the tested donors) were characterized by higher levels of early glycolytic intermediates, pentose phosphate pathway metabolites, and pyruvate-to-lactate ratios, all markers of increased basal oxidant stress. Consistently, increased glutathionylation of oxidized triose sugars and lipid aldehydes was observed in RBCs donated by nicotine-exposed donors, which were also characterized by increased fatty acid desaturation, purine salvage, and methionine oxidation and consumption via pathways involved in oxidative stress-triggered protein damage-repair mechanisms., Conclusion: RBCs from donors with high levels of nicotine exposure are characterized by increases in basal oxidant stress and decreases in osmotic hemolysis. These findings indicate the need for future clinical studies aimed at addressing the impact of smoking and other sources of nicotine (e.g., nicotine patches, snuff, vaping, secondhand tobacco smoke) on RBC storage quality and transfusion efficacy., (© 2020 AABB.)

Background: Red blood cell (RBC) storage in the blood bank is associated with the progressive accumulation of oxidant stress. While the mature erythrocyte is well equipped to cope with such stress, recreative habits like alcohol consumption may further exacerbate the basal level of oxidant stress and contribute to the progress of the storage lesion., Study Design and Methods: RBC levels of ethyl glucuronide, a marker of alcohol consumption, were measured via ultra-high-pressure liquid chromatography coupled with high-resolution mass spectrometry. Analyses were performed on 599 samples from the recalled donor population at Storage Days 10, 23, and 42 (n = 250), as part of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study. This cohort consisted of the 5th and 95th percentile of donors with extreme hemolytic propensity out of the original cohort of 13,403 subjects enrolled in the REDS-III RBC Omics study. Ehtyl glucuronide levels were thus correlated to global metabolomics and lipidomics analyses and RBC hemolytic propensity., Results: Ethyl glucuronide levels were positively associated with oxidant stress markers, including glutathione consumption and turnover, methionine oxidation, S-adenosylhomocysteine accumulation, purine oxidation, and transamination markers. Decreases in glycolysis and energy metabolism, the pentose phosphate pathway and ascorbate system were observed in those subjects with the highest levels of ethyl glucuronide, though hemolysis values were comparable between groups., Conclusion: Though preliminary, this study is suggestive that markers of alcohol consumption are associated with increases in oxidant stress and decreases in energy metabolism with no significant impact on hemolytic parameters in stored RBCs from healthy donor volunteers., (© 2020 AABB.)

Background: Coffee consumption is extremely common in the United States. Coffee is rich with caffeine, a psychoactive, purinergic antagonist of adenosine receptors, which regulate red blood cell energy and redox metabolism. Since red blood cell (purine) metabolism is a critical component to the red cell storage lesion, here we set out to investigate whether caffeine levels correlated with alterations of energy and redox metabolism in stored red blood cells., Study Design and Methods: We measured the levels of caffeine and its main metabolites in 599 samples from the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study via ultra-high-pressure-liquid chromatography coupled to high-resolution mass spectrometry and correlated them to global metabolomic and lipidomic analyses of RBCs stored for 10, 23, and 42 days., Results: Caffeine levels positively correlated with increased levels of the main red cell antioxidant, glutathione, and its metabolic intermediates in glutathione-dependent detoxification pathways of oxidized lipids and sugar aldehydes. Caffeine levels were positively correlated with transamination products and substrates, tryptophan, and indole metabolites. Expectedly, since caffeine and its metabolites belong to the family of xanthine purines, all xanthine metabolites were significantly increased in the subjects with the highest levels of caffeine. However, high-energy phosphate compounds ATP and DPG were not affected by caffeine levels, despite decreases in glucose oxidation products-both via glycolysis and the pentose phosphate pathway., Conclusion: Though preliminary, this study is suggestive of a beneficial correlation between the caffeine levels and improved antioxidant capacity of stored red cells., (© 2020 AABB.)

Insufficient O2 supply is frequently associated with fetal growth restriction (FGR), a leading cause of perinatal mortality and morbidity. Although the erythrocyte is the most abundant and only cell type to deliver O2 in our body, its function and regulatory mechanism in FGR remain unknown. Here, we report that genetic ablation of mouse erythrocyte equilibrative nucleoside transporter 1 (eENT1) in dams, but not placentas or fetuses, results in FGR. Unbiased high-throughput metabolic profiling coupled with in vitro and in vivo flux analyses with isotopically labeled tracers led us to discover that maternal eENT1-dependent adenosine uptake is critical in activating AMPK by controlling the AMP/ATP ratio and its downstream target, bisphosphoglycerate mutase (BPGM); in turn, BPGM mediates 2,3-BPG production, which enhances O2 delivery to maintain placental oxygenation. Mechanistically and functionally, we revealed that genetic ablation of maternal eENT1 increases placental HIF-1α; preferentially reduces placental large neutral aa transporter 1 (LAT1) expression, activity, and aa supply; and induces FGR. Translationally, we revealed that elevated HIF-1α directly reduces LAT1 gene expression in cultured human trophoblasts. We demonstrate the importance and molecular insight of maternal eENT1 in fetal growth and open up potentially new diagnostic and therapeutic possibilities for FGR.

BACKGROUNDGlucose-6-phosphate dehydrogenase (G6PD) deficiency decreases the ability of red blood cells (RBCs) to withstand oxidative stress. Refrigerated storage of RBCs induces oxidative stress. We hypothesized that G6PD-deficient donor RBCs would have inferior storage quality for transfusion as compared with G6PD-normal RBCs.METHODSMale volunteers were screened for G6PD deficiency; 27 control and 10 G6PD-deficient volunteers each donated 1 RBC unit. After 42 days of refrigerated storage, autologous 51-chromium 24-hour posttransfusion RBC recovery (PTR) studies were performed. Metabolomics analyses of these RBC units were also performed.RESULTSThe mean 24-hour PTR for G6PD-deficient subjects was 78.5% ± 8.4% (mean ± SD), which was significantly lower than that for G6PD-normal RBCs (85.3% ± 3.2%; P = 0.0009). None of the G6PD-normal volunteers (0/27) and 3 G6PD-deficient volunteers (3/10) had PTR results below 75%, a key FDA acceptability criterion for stored donor RBCs. As expected, fresh G6PD-deficient RBCs demonstrated defects in the oxidative phase of the pentose phosphate pathway. During refrigerated storage, G6PD-deficient RBCs demonstrated increased glycolysis, impaired glutathione homeostasis, and increased purine oxidation, as compared with G6PD-normal RBCs. In addition, there were significant correlations between PTR and specific metabolites in these pathways.CONCLUSIONBased on current FDA criteria, RBCs from G6PD-deficient donors would not meet the requirements for storage quality. Metabolomics assessment identified markers of PTR and G6PD deficiency (e.g., pyruvate/lactate ratios), along with potential compensatory pathways that could be leveraged to ameliorate the metabolic needs of G6PD-deficient RBCs.TRIAL REGISTRATIONClinicalTrials.gov NCT04081272.FUNDINGThe Harold Amos Medical Faculty Development Program, Robert Wood Johnson Foundation grant 71590, the National Blood Foundation, NIH grant UL1 TR000040, the Webb-Waring Early Career Award 2017 by the Boettcher Foundation, and National Heart, Lung, and Blood Institute grants R01HL14644 and R01HL148151.

Purpose of Review: The erythrocyte is the most abundant cell type in our body, acting as both a carrier/deliverer and sensor of oxygen (O2). Erythrocyte O2 delivery capacity is finely regulated by sophisticated metabolic control. In recent years, unbiased and robust human metabolomics screening and mouse genetic studies have advanced erythroid research revealing the differential role of erythrocyte hypoxic metabolic reprogramming in normal individuals at high altitudes and patients facing hypoxia, such as sickle cell disease (SCD) and chronic kidney disease (CKD). Here we summarize recent progress and highlight potential therapeutic possibilities., Recent Findings: Initial studies showed that elevated soluble CD73 (sCD73, converts AMP to adenosine) results in increased circulating adenosine that activates the A2B adenosine receptor (ADORA2B). Signaling through this axis is co-operatively strengthened by erythrocyte-specific synthesis of sphingosine-1-phosphate (S1P). Ultimately, these mechanisms promote the generation of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific allosteric modulator that decreases haemoglobin--O2-binding affinity, and thus, induces deoxygenated sickle Hb (deoxyHbS), deoxyHbS polymerization, sickling, chronic inflammation and tissue damage in SCD. Similar to SCD, plasma adenosine and erythrocyte S1P are elevated in humans ascending to high altitude. At high altitude, these two metabolites are beneficial to induce erythrocyte metabolic reprogramming and the synthesis of 2,3-BPG, and thus, increase O2 delivery to counteract hypoxic tissue damage. Follow-up studies showed that erythrocyte equilibrative nucleoside transporter 1 (eENT1) is a key purinergic cellular component controlling plasma adenosine in humans at high altitude and mice under hypoxia and underlies the quicker and higher elevation of plasma adenosine upon re-ascent because of prior hypoxia-induced degradation of eENT1. More recent studies demonstrated the beneficial role of erythrocyte ADORA2B-mediated 2,3-BPG production in CKD., Summary: Taken together, these findings revealed the differential role of erythrocyte hypoxic metabolic reprogramming in normal humans at high altitude and patients with CKD vs. SCD patients and immediately suggest differential and precision therapies to counteract hypoxia among these groups.

Background: Blood transfusion is a lifesaving intervention for millions of recipients worldwide every year. Storing blood makes this possible but also promotes a series of alterations to the metabolism of the stored erythrocyte. It is unclear whether the metabolic storage lesion is correlated with clinically relevant outcomes and whether strategies aimed at improving the metabolic quality of stored units, such as hypoxic storage, ultimately improve performance in the transfused recipient., Study Design and Methods: Twelve healthy donor volunteers were recruited in a two-arm cross-sectional study, in which each subject donated 2 units to be stored under standard (normoxic) or hypoxic conditions (Hemanext technology). End-of-storage measurements of hemolysis and autologous posttransfusion recovery (PTR) were correlated to metabolomics measurements at Days 0, 21, and 42., Results: Hypoxic red blood cells (RBCs) showed superior PTR and comparable hemolysis to donor-paired standard units. Hypoxic storage improved energy and redox metabolism (glycolysis and 2,3-diphosphoglycerate), improved glutathione and methionine homeostasis, decreased purine oxidation and membrane lipid remodeling (free fatty acid levels, unsaturation and hydroxylation, acyl-carnitines). Intra- and extracellular metabolites in these pathways (including some dietary purines) showed significant correlations with PTR and hemolysis, though the degree of correlation was influenced by sulfur dioxide (SO 2 ) levels., Conclusion: Hypoxic storage improves energy and redox metabolism of stored RBCs, which results in improved posttransfusion recoveries in healthy autologous recipients-a Food and Drug Administration gold standard of stored blood quality. In addition, we identified candidate metabolic predictors of PTR for RBCs stored under standard and hypoxic conditions., (© 2020 AABB.)

Background: In response to stress, anucleate red blood cells (RBCs) can undergo a process of atypical cell death characterised by intracellular Ca 2+ accumulation and phosphatidylserine (PS) externalisation. Here we studied alterations in RBC metabolism, a critical contributor to their capacity to survive environmental challenges, during this process., Materials and Methods: Metabolomics analyses of RBCs and supernatants, using ultra-high-pressure liquid chromatography coupled to mass spectrometry, were performed after in vitro exposure of RBCs to different pathophysiological cell stressors, including starvation, extracellular hypertonicity, hyperthermia, and supraphysiological ionic stress. Cell death was examined by flow cytometry., Results: Our data show that artificially enhancing RBC cytosolic Ca 2+ influx significantly enhanced purine oxidation and strongly affected cellular bioenergetics by reducing glycolysis. Depleting extracellular Ca 2+ curtailed starvation-induced cell death, an effect paralleled by the activation of compensatory pathways such as the pentose phosphate pathway, carboxylic acid metabolism, increased pyruvate to lactate ratios (methemoglobin reductase activation), one-carbon metabolism (protein-damage repair) and glutathione synthesis; RBCs exposed to hypertonic shock displayed a similar metabolic profile. Furthermore, cell stress promoted lipid remodelling as reflected by the levels of free fatty acids, acyl-carnitines and CoA precursors. Notably, RBC PS exposure, independently of the stressor, showed significant correlation with the levels of free fatty acids, glutamate, cystine, spermidine, tryptophan, 5-oxoproline, lactate, and hypoxanthine., Discussion: In conclusion, different cell death-inducing pathophysiological stressors, encountered in various clinical conditions, result in differential RBC metabolic phenotypes, only partly explained by intracellular Ca 2+ levels and ATP availability.

Background: Resuscitation from hemorrhagic shock (HS) by blood transfusion restores oxygen (O2) delivery and provides hemodynamic stability. Current regulations allow red blood cells (RBCs) to be stored and used for up to 42 days. During storage, RBCs undergo many structural and functional changes. These storage lesions have been associated with adverse events and increased mortality after transfusion, increasing the need for improved RBC storage protocols. This study evaluates the efficacy of anaerobically stored RBCs to resuscitate rats from severe HS compared with conventionally stored RBCs., Methods and Results: Rat RBCs were stored under anaerobic, anaerobic/hypercapnic, or conventional conditions for a period of 3 weeks. Hemorrhage was induced by controlled bleeding, shock was maintained for 30 min, and RBCs were transfused to restore and maintain blood pressure near the prhemorrhage level. All storage conditions met current regulatory 24-h posttransfusion recovery requirements. Transfusion of anaerobically stored RBCs required significantly less RBC volume to restore and maintain hemodynamics. Anaerobic or anaerobic/hypercapnic RBCs restored hemodynamics better than conventionally stored RBCs. Resuscitation with conventionally stored RBCs impaired indices of left ventricular cardiac function, increased hypoxic tissue staining and inflammatory markers, and affected organ function compared with anaerobically stored RBCs., Conclusions: Resuscitation from HS via transfusion of anaerobically stored RBCs recovered cardiac function, restored hemodynamic stability, and improved outcomes.

Red blood cells (RBCs) are the most numerous cell type in the body and serve a vital purpose of delivering oxygen to essentially all tissues. In addition to the central role of RBCs in health and disease, RBC storage is a requirement for the >90 million units of RBC transfusions given to millions of recipients each year, worldwide. It is well known that there is genetic donor-to-donor variability in how human RBCs store, rendering blood a nonstandardized therapeutic with a wide range of biological properties from unit to unit, by the time it is transfused. As with humans, genetic variation exists in how murine RBCs, from different strains of mice, store and perform after transfusion. The genetic mechanisms for variation, in humans and mice, both remain obscure. Combining advanced metabolomics, genetics, and molecular and cellular biology approaches, we identify genetic variation in six-transmembrane epithelial antigen of prostate 3 (Steap3) expression as a critical and previously unrecognized mechanism of oxidative damage of RBCs during storage. Increased levels of Steap3 result in degradation of cellular membrane through lipid peroxidation, leading to failure of RBC homeostasis and hemolysis/clearance of RBCs. This article is the first report of a role of Steap3 in mature RBCs; it defines a new mechanism of redox biology in RBCs with a substantial effect upon RBC function and provides a novel mechanistic determinant of genetic variation of RBC storage., (© 2019 by The American Society of Hematology.)

Background: The chromium-51-labeled posttransfusion recovery (PTR) study has been the gold-standard test for assessing red blood cell (RBC) quality. Despite guiding RBC storage development for decades, it has several potential sources for error., Methods: Four healthy adult volunteers each donated an autologous, leukoreduced RBC unit, aliquots were radiolabeled with technetium-99m after 1 and 6 weeks of storage, and then infused. Subjects were imaged by single-photon-emission computed tomography immediately and 4 hours after infusion. Additionally, from subjects described in a previously published study, adenosine triphosphate levels in transfusates infused into 52 healthy volunteers randomized to a single autologous, leukoreduced, RBC transfusion after 1, 2, 3, 4, 5, or 6 weeks of storage were correlated with PTR and laboratory parameters of hemolysis., Results: Evidence from one subject imaged after infusion of technetium-99m-labeled RBCs suggests that, in some individuals, RBCs may be temporarily sequestered in the liver and spleen immediately following transfusion and then subsequently released back into circulation; this could be one source of error leading to PTR results that may not accurately predict the true quantity of RBCs cleared by intra- and/or extravascular hemolysis. Indeed, adenosine triphosphate levels in the transfusates correlated more robustly with measures of extravascular hemolysis in vivo (e.g., serum iron, indirect bilirubin, non-transferrin-bound iron) than with PTR results or measures of intravascular hemolysis (e.g., plasma free hemoglobin)., Conclusions: Sources of measurement error are inherent in the chromium-51 PTR method. Transfusion of an entire unlabeled RBC unit, followed by quantifying extravascular hemolysis markers, may more accurately measure true posttransfusion RBC recovery., (© 2019 AABB.)

Red blood cells (RBCs) have been historically regarded as a critical model to investigate cellular and oxidant stress biology. First of all, they are constantly exposed to oxidant stress, as their main function is to transport and deliver oxygen to tissues. Second, they are devoid of de novo protein synthesis capacity, which prevents RBCs from replacing irreversibly oxidised proteins with newly synthesised ones. As such, RBCs have evolved to (i) protect themselves from oxidant stress, in order to prevent oxidant damage from reactive species; (ii) repair oxidatively damaged proteins, through mechanisms that involve glutathione and one-carbon metabolism; (iii) destroy irreversibly oxidised proteins through proteasomal or protease-dependent degradation; and (iv) sacrifice membrane portions through mechanism of vesiculation. In this brief review we will summarize these processes and their relevance to RBC redox biology (within the context of blood storage), with a focus on how polymorphisms in RBC antioxidant responses could contribute to explaining the heterogeneity in the progression and severity of the RBC storage lesion that can be observed across the healthy donor population.

Mature red blood cells (RBCs) not only account for ~83% of the total host cells in the human body, but they are also exposed to all body tissues during their circulation in the bloodstream. In addition, RBCs are devoid of de novo protein synthesis capacity and, as such, they represent a perfect model to investigate system-wide alterations of cellular metabolism in the context of aging and age-related oxidant stress without the confounding factor of gene expression. In the present study, we employed ultra-high-pressure liquid chromatography coupled with mass spectrometry (UHPLC-MS)-based metabolomics and proteomics to investigate RBC metabolism across age in male mice (6, 15, and 25 months old). We report that RBCs from aging mice face a progressive decline in the capacity to cope with oxidant stress through the glutathione/NADPH-dependent antioxidant systems. Oxidant stress to tryptophan and purines was accompanied by declines in late glycolysis and methyl-group donors, a potential compensatory mechanism to repair oxidatively damaged proteins. Moreover, heterochronic parabiosis experiments demonstrated that the young environment only partially rescued the alterations in one-carbon metabolism in old mice, although it had minimal to no impact on glutathione homeostasis, the pentose phosphate pathway, and oxidation of purines and tryptophan, which were instead aggravated in old heterochronic parabionts.

Red blood cell (RBC) storage in the blood bank is characterized by the progressive loss of metabolic regulation, a phenomenon that targets energy and antioxidant metabolism; While the progression of the storage lesion is inevitable, the rate at which this phenomenon occurs varies from donor to donor; Red blood cells from some donors at the end of storage are metabolically superior to RBCs from other donors at the beginning of storage, suggesting that the age of blood alone may not be a sufficiently accurate predictor of stored blood quality; and Animal studies and large-scale omics screening in blood donors have helped identify mechanistic contributors to the metabolic heterogeneity of stored blood units., (© 2019 AABB.)

Arctic ground squirrels provide a unique model to investigate metabolic responses to hibernation in mammals. During winter months these rodents are exposed to severe hypothermia, prolonged fasting, and hypoxemia. In the light of their role in oxygen transport/off-loading and owing to the absence of nuclei and organelles (and thus de novo protein synthesis capacity), mature red blood cells have evolved metabolic programs to counteract physiological or pathological hypoxemia. However, red blood cell metabolism in hibernation has not yet been investigated. Here we employed targeted and untargeted metabolomics approaches to investigate erythrocyte metabolism during entrance to torpor to arousal, with a high resolution of the intermediate time points. We report that torpor and arousal promote metabolism through glycolysis and pentose phosphate pathway, respectively, consistent with previous models of oxygen-dependent metabolic modulation in mature erythrocytes. Erythrocytes from hibernating squirrels showed up to 100-fold lower levels of biomarkers of reperfusion injury, such as the pro-inflammatory dicarboxylate succinate. Altered tryptophan metabolism during torpor was here correlated to the accumulation of potentially neurotoxic catabolites kynurenine, quinolinate, and picolinate. Arousal was accompanied by alterations of sulfur metabolism, including sudden spikes in a metabolite putatively identified as thiorphan (level 1 confidence)-a potent inhibitor of several metalloproteases that play a crucial role in nociception and inflammatory complication to reperfusion secondary to ischemia or hemorrhage. Preliminary studies in rats showed that intravenous injection of thiorphan prior to resuscitation mitigates metabolic and cytokine markers of reperfusion injury, etiological contributors to inflammatory complications after shock.

Cold storage of blood for 5 to 6 weeks has been shown to impair endothelial function after transfusion and has been associated with measures of end-organ dysfunction. Although the products of hemolysis, such as cell-free plasma hemoglobin, arginase, heme, and iron, in part mediate these effects, a complete analysis of transfused metabolites that may affect organ function has not been evaluated to date. Blood stored for either 5 or 42 days was collected from 18 healthy autologous volunteers, prior to and after autologous transfusion into the forearm circulation, followed by metabolomics analyses. Significant metabolic changes were observed in the plasma levels of hemolytic markers, oxidized purines, plasticizers, and oxidized lipids in recipients of blood stored for 42 days, compared with 5 days. Notably, transfusion of day 42 red blood cells (RBCs) increased circulating levels of plasticizers (diethylhexyl phthalate and derivatives) by up to 18-fold. Similarly, transfusion of day 42 blood significantly increased circulating levels of proinflammatory oxylipins, including prostaglandins, hydroxyeicosatrienoic acids (HETEs), and dihydroxyoctadecenoic acids. Oxylipins were the most significantly increasing metabolites (for 9-HETE: up to ∼41-fold, P = 3.7e-06) in day 42 supernatants. Measurements of arginine metabolism confirmed an increase in arginase activity at the expense of nitric oxide synthesis capacity in the bloodstream of recipients of day 42 blood, which correlated with measurements of hemodynamics. Metabolic changes in stored RBC supernatants impact the plasma metabolome of healthy transfusion recipients, with observed increases in plasticizers, as well as vasoactive, pro-oxidative, proinflammatory, and immunomodulatory metabolites after 42 days of storage., (© 2019 by The American Society of Hematology.)

Red blood cells (RBCs) are a specialised organ that enabled the evolution of multicellular organisms by supplying a sufficient quantity of oxygen to cells that cannot obtain oxygen directly from ambient air via diffusion, thereby fueling oxidative phosphorylation for highly efficient energy production. RBCs have evolved to optimally serve this purpose by packing high concentrations of haemoglobin in their cytosol and shedding nuclei and other organelles. During their circulatory lifetimes in humans of approximately 120 days, RBCs are poised to transport oxygen by metabolic/redox enzymes until they accumulate damage and are promptly removed by the reticuloendothelial system. These elaborate evolutionary adaptions, however, are no longer effective when RBCs are removed from the circulation and stored hypothermically in blood banks, where they develop storage-induced damages ("storage lesions") that accumulate over the shelf life of stored RBCs. This review attempts to provide a comprehensive view of the literature on the subject of RBC storage lesions and their purported clinical consequences by incorporating the recent exponential growth in available data obtained from "omics" technologies in addition to that published in more traditional literature. To summarise this vast amount of information, the subject is organised in figures with four panels: i) root causes; ii) RBC storage lesions; iii) physiological effects; and iv) reported outcomes. The driving forces for the development of the storage lesions can be roughly classified into two root causes: i) metabolite accumulation/depletion, the target of various interventions (additive solutions) developed since the inception of blood banking; and ii) oxidative damages, which have been reported for decades but not addressed systemically until recently. Downstream physiological consequences of these storage lesions, derived mainly by in vitro studies, are described, and further potential links to clinical consequences are discussed. Interventions to postpone the onset and mitigate the extent of the storage lesion development are briefly reviewed. In addition, we briefly discuss the results from recent randomised controlled trials on the age of stored blood and clinical outcomes of transfusion.

Background: Biological and technical variability has been increasingly appreciated as a key factor impacting red blood cell (RBC) storability and, potentially, transfusion outcomes. Here, we performed metabolomics analyses to investigate the impact of factors other than storage duration on the metabolic phenotypes of stored RBC in a multicenter study., Study Design and Methods: Within the framework of the REDS-III (Recipient Epidemiology and Donor Evaluation Study-III) RBC-Omics study, 13,403 donors were enrolled from four blood centers across the United States and tested for the propensity of their RBCs to hemolyze after 42 days of storage. Extreme hemolyzers were recalled and donated a second unit of blood. Units were stored for 10, 23, and 42 days prior to sample acquisition for metabolomics analyses., Results: Unsupervised analyses of metabolomics data from 599 selected samples revealed a strong impact (14.2% of variance) of storage duration on metabolic phenotypes of RBCs. The blood center collecting and processing the units explained an additional 12.2% of the total variance, a difference primarily attributable to the storage additive (additive solution 1 vs. additive solution 3) used in the different hubs. Samples stored in mannitol-free/citrate-loaded AS-3 were characterized by elevated levels of high-energy compounds, improved glycolysis, and glutathione homeostasis. Increased methionine metabolism and activation of the transsulfuration pathway was noted in samples processed in the center using additive solution 1., Conclusion: Blood processing impacts the metabolic heterogeneity of stored RBCs from the largest multicenter metabolomics study in transfusion medicine to date. Studies are needed to understand if these metabolic differences influenced by processing/storage strategies impact the effectiveness of transfusions clinically., (© 2018 AABB.)

Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter and sample analysis, we developed a research-specific RBC cryopreservation protocol and assessed its impact on data fidelity for key biochemical and physiological assays. RBCs drawn from healthy volunteers were aliquotted for immediate analysis or following glycerol-based cryopreservation, thawing, and deglycerolization. RBC phenotype was assessed by (1) scanning electron microscopy (SEM) imaging and standard morphometric RBC indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge with superoxide thermal source; SOTS-1), as well as in vivo quantification (following human to mouse RBC xenotransfusion) of (9) blood oxygenation content mapping and flow dynamics (velocity and adhesion). Our revised glycerolization protocol (40% v/v final) resulted in >98.5% RBC recovery following freezing (-80°C) and thawing (37°C), with no difference compared to the standard reported method (40% w/v final). Full deglycerolization (>99.9% glycerol removal) of 40% v/v final samples resulted in total cumulative lysis of ~8%, compared to ~12-15% with the standard method. The post cryopreservation/deglycerolization RBC phenotype was indistinguishable from that for fresh RBCs with regard to physical RBC parameters (morphology, volume, and density), osmotic fragility, deformability, endothelial adhesivity, O2 affinity, vasoregulation, metabolomics, and flow dynamics. These results indicate that RBC cryopreservation/deglycerolization in 40% v/v glycerol final does not significantly impact RBC phenotype (compared to fresh cells)., Competing Interests: The authors have declared that no competing interests exist.