Travis Nemkov

Routine storage of red blood cell (RBC) units in additive solution‐3: a comprehensive investigation of the RBC metabolome

In most countries, red blood cells (RBCs) can be stored up to 42 days before transfusion. However, observational studies have suggested that storage duration might be associated with increased morbidity and mortality. While clinical trials are under way, impaired metabolism has been documented in RBCs stored in several additive solutions (ASs). Here we hypothesize that, despite reported beneficial effects, storage in AS‐3 results in metabolic impairment weeks before the end of the unit shelf life.

Metabolomics in transfusion medicine

Biochemical investigations on the regulatory mechanisms of red blood cell (RBC) and platelet (PLT) metabolism have fostered a century of advances in the field of transfusion medicine. Owing to these advances, storage of RBCs and PLT concentrates has become a lifesaving practice in clinical and military settings. There, however, remains room for improvement, especially with regard to the introduction of novel storage and/or rejuvenation solutions, alternative cell processing strategies (e.g., pathogen inactivation technologies), and quality testing (e.g., evaluation of novel containers with alternative plasticizers). Recent advancements in mass spectrometry–based metabolomics and systems biology, the bioinformatics integration of omics data, promise to speed up the design and testing of innovative storage strategies developed to improve the quality, safety, and effectiveness of blood products.

Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days. Proteomics analyses revealed a storage-dependent oxidation of GAPDH at functional Cys152, 156, 247, and His179. Activity loss by oxidation occurred with increasing storage duration and was progressively irreversible. Irreversibly oxidized GAPDH accumulated in stored erythrocyte membranes and supernatants through storage day 42. By combining state-of-the-art ultra-high-pressure liquid chromatography-mass spectrometry metabolic flux analysis with redox and switch-tag proteomics, we identify for the first time ex vivo functionally relevant reversible and irreversible (sulfinic acid; Cys to dehydroalanine) oxidations of GAPDH without exogenous supplementation of excess pro-oxidant compounds in clinically relevant blood products. Oxidative and metabolic lesions, exacerbated by storage under hyperoxic conditions, were ameliorated by hypoxic storage. Storage-dependent reversible oxidation of GAPDH represents a mechanistic adaptation in stored erythrocytes to promote PPP activation and generate reducing equivalents. Removal of irreversibly oxidized, functionally compromised GAPDH identifies enhanced vesiculation as a self-protective mechanism in ex vivo aging erythrocytes.

Red blood cell storage in additive solution-7 preserves energy and redox metabolism: a metabolomics approach.

Storage and transfusion of red blood cells (RBCs) has a huge medical and economic impact. Routine storage practices can be ameliorated through the implementation of novel additive solutions (ASs) that tackle the accumulation of biochemical and morphologic lesion during routine cold liquid storage in the blood bank, such as the recently introduced alkaline solution AS‐7. Here we hypothesize that AS‐7 might exert its beneficial effects through metabolic modulation during routine storage.

Beneficial role of erythrocyte adenosine A2B receptor-mediated AMP-activated protein kinase activation in high-altitude hypoxia

Background: High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia. Methods: Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m. Results: This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O2 releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O2 release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O2 release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O2 release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation. Conclusions: Together, human and mouse studies reveal novel mechanisms of hypoxia adaptation and potential therapeutic approaches for counteracting hypoxia-induced tissue damage.

Biomarkers defining the metabolic age of red blood cells during cold storage

Metabolomic investigations of packed red blood cells (RBCs) stored under refrigerated conditions in saline adenine glucose mannitol (SAGM) additives have revealed the presence of 3 distinct metabolic phases, occurring on days 0-10, 10-18, and after day 18 of storage. Here we used receiving operating characteristics curve analysis to identify biomarkers that can differentiate between the 3 metabolic states. We first recruited 24 donors and analyzed 308 samples coming from RBC concentrates stored in SAGM and additive solution 3. We found that 8 extracellular compounds (lactic acid, nicotinamide, 5-oxoproline, xanthine, hypoxanthine, glucose, malic acid, and adenine) form the basis for an accurate classification/regression model and are able to differentiate among the metabolic phases. This model was then validated by analyzing an additional 49 samples obtained by preparing 7 new RBC concentrates in SAGM. Despite the technical variability associated with RBC processing strategies, verification of these markers was independently confirmed in 2 separate laboratories with different analytical setups and different sample sets. The 8 compounds proposed here highly correlate with the metabolic age of packed RBCs, and can be prospectively validated as biomarkers of the RBC metabolic lesion.

Sphingosine-1-phosphate promotes erythrocyte glycolysis and oxygen release for adaptation to high-altitude hypoxia

Sphingosine-1-phosphate (S1P) is a bioactive signalling lipid highly enriched in mature erythrocytes, with unknown functions pertaining to erythrocyte physiology. Here by employing nonbiased high-throughput metabolomic profiling, we show that erythrocyte S1P levels rapidly increase in 21 healthy lowland volunteers at 5,260 m altitude on day 1 and continue increasing to 16 days with concurrently elevated erythrocyte sphingonisne kinase 1 (Sphk1) activity and haemoglobin (Hb) oxygen (O2) release capacity. Mouse genetic studies show that elevated erythrocyte Sphk1-induced S1P protects against tissue hypoxia by inducing O2 release. Mechanistically, we show that intracellular S1P promotes deoxygenated Hb anchoring to the membrane, enhances the release of membrane-bound glycolytic enzymes to the cytosol, induces glycolysis and thus the production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific glycolytic intermediate, which facilitates O2 release. Altogether, we reveal S1P as an intracellular hypoxia-responsive biolipid promoting erythrocyte glycolysis, O2 delivery and thus new therapeutic opportunities to counteract tissue hypoxia.

A three-minute method for high-throughput quantitative metabolomics and quantitative tracing experiments of central carbon and nitrogen pathways

RATIONALE The implementation of mass spectrometry (MS)-based metabolomics is advancing many areas of biomedical research. The time associated with traditional chromatographic methods for resolving metabolites prior to mass analysis has limited the potential to perform large-scale, highly powered metabolomics studies and clinical applications. METHODS Here we describe a three-minute method for the rapid profiling of central metabolic pathways through UHPLC/MS, tracing experiments in vitro and in vivo, and targeted quantification of compounds of interest using spiked in heavy labeled standards. RESULTS This method has shown to be linear, reproducible, selective, sensitive, and robust for the semi-targeted analysis of central carbon and nitrogen metabolism. Isotopically labeled internal standards are used for absolute quantitation of steady-state metabolite levels and de novo synthesized metabolites in tracing studies. We further propose exploratory applications to biofluids, cell and tissue extracts derived from relevant biomedical/clinical samples. CONCLUSIONS While limited to the analysis of central carbon and nitrogen metabolism, this method enables the analysis of hundreds of samples per day derived from diverse biological matrices. This approach makes it possible to analyze samples from large patient populations for translational research, personalized medicine, and clinical metabolomics applications. Copyright

Citrate metabolism in red blood cells stored in additive solution-3

Red blood cells (RBCs) are thought to have a relatively simple metabolic network compared to other human cell types. Recent proteomics reports challenge the notion that RBCs are mere hemoglobin carriers with limited metabolic activity. Expanding our understanding of RBC metabolism has key implications in many biomedical areas, including transfusion medicine.

Early hemorrhage triggers metabolic responses that build up during prolonged shock

Metabolic staging after trauma/hemorrhagic shock is a key driver of acidosis and directly relates to hypothermia and coagulopathy. Metabolic responses to trauma/hemorrhagic shock have been assayed through classic biochemical approaches or NMR, thereby lacking a comprehensive overview of the dynamic metabolic changes occurring after shock. Sprague-Dawley rats underwent progressive hemorrhage and shock. Baseline and postshock blood was collected, and late hyperfibrinolysis was assessed (LY30 >3%) in all of the tested rats. Extreme and intermediate time points were collected to assay the dynamic changes of the plasma metabolome via ultra-high performance liquid chromatography-mass spectrometry. Sham controls were used to determine whether metabolic changes could be primarily attributable to anesthesia and supine positioning. Early hemorrhage-triggered metabolic changes that built up progressively and became significant during sustained hemorrhagic shock. Metabolic phenotypes either resulted in immediate hypercatabolism, or late hypercatabolism, preceded by metabolic deregulation during early hemorrhage in a subset of rats. Hemorrhagic shock consistently promoted hyperglycemia, glycolysis, Krebs cycle, fatty acid, amino acid, and nitrogen metabolism (urate and polyamines), and impaired redox homeostasis. Early dynamic changes of the plasma metabolome are triggered by hemorrhage in rats. Future studies will determine whether metabolic subphenotypes observed in rats might be consistently observed in humans and pave the way for tailored resuscitative strategies.