Antigen perception in T cells by long-term Erk and NFAT signaling dynamics.

Immune system threat detection hinges on T cells' ability to perceive varying peptide-major histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs but diverge only over longer (9+ h) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception and establish a framework for understanding T cell responses under diverse contexts.

Metabolomics assessment reveals oxidative stress and altered energy production in the heart after ischemic acute kidney injury in mice.

Acute kidney injury (AKI) is a systemic disease associated with widespread effects on distant organs, including the heart. Normal cardiac function is dependent on constant ATP generation, and the preferred method of energy production is via oxidative phosphorylation. Following direct ischemic cardiac injury, the cardiac metabolome is characterized by inadequate oxidative phosphorylation, increased oxidative stress, and increased alternate energy utilization. We assessed the impact of ischemic AKI on the metabolomics profile in the heart. Ischemic AKI was induced by 22 minutes of renal pedicle clamping, and 124 metabolites were measured in the heart at 4 hours, 24 hours, and 7 days post-procedure. Forty-one percent of measured metabolites were affected, with the most prominent changes observed 24 hours post-AKI. The post-AKI cardiac metabolome was characterized by amino acid depletion, increased oxidative stress, and evidence of alternative energy production, including a shift to anaerobic forms of energy production. These metabolomic effects were associated with significant cardiac ATP depletion and with echocardiographic evidence of diastolic dysfunction. In the kidney, metabolomics analysis revealed shifts suggestive of energy depletion and oxidative stress, which were reflected systemically in the plasma. This is the first study to examine the cardiac metabolome after AKI, and demonstrates that effects of ischemic AKI on the heart are akin to the effects of direct ischemic cardiac injury.

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.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage.

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1-7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and - preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates.

Hydroxylamine Chemical Digestion for Insoluble Extracellular Matrix Characterization.

The extracellular matrix (ECM) is readily enriched by decellularizing tissues with nondenaturing detergents to solubilize and deplete the vast majority of cellular components. This approach has been used extensively to generate ECM scaffolds for regenerative medicine technologies and in 3D cell culture to model how the ECM contributes to disease progression. A highly enriched ECM fraction can then be generated using a strong chaotrope buffer that is compatible with downstream bottom-up proteomic analysis or 3D cell culture experiments after extensive dialysis. With most tissues, an insoluble pellet remains after chaotrope extraction that is rich in structural ECM components. Previously, we showed that this understudied fraction represented approximately 80% of total fibrillar collagen from the lung and other ECM fiber components that are known to be covalently cross-linked. Here, we present a hydroxylamine digestion approach for chaotrope-insoluble ECM analysis with comparison to an established CNBr method for matrisome characterization. Because ECM characteristics vary widely among tissues, we chose five tissues that represent unique and diverse ECM abundances, composition, and biomechanical properties. Hydroxylamine digestion is compatible with downstream proteomic workflows, yields high levels of ECM peptides from the insoluble ECM fraction, and reduces analytical variability when compared to CNBr digestion. Data are available via ProteomeXchange with identifier PXD006428.

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen.

Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼ 120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827.

Antigen perception in T cells by long-term Erk and NFAT signaling dynamics.

Immune system threat detection hinges on T cells' ability to perceive varying peptide major-histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs, but diverge only over longer (9+ hrs) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception, and establish a framework for understanding T cell responses under diverse contexts. SIGNIFICANCE STATEMENT: To counter diverse pathogens, T cells mount distinct responses to varying peptide-major histocompatibility complex ligands (pMHCs). They perceive the affinity of pMHCs for the T cell receptor (TCR), which reflects its foreignness, as well as pMHC abundance. By tracking signaling responses in single living cells to different pMHCs, we find that T cells can independently perceive pMHC affinity vs dose, and encode this information through the dynamics of Erk and NFAT signaling pathways downstream of the TCR. These dynamics are jointly decoded by gene regulatory mechanisms to produce pMHC-specific activation responses. Our work reveals how T cells can elicit tailored functional responses to diverse threats and how dysregulation of these responses may lead to immune pathologies.

Hematologic and systemic metabolic alterations due to Mediterranean class II G6PD deficiency in 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.

Order by chance: origins and benefits of stochasticity in immune cell fate control.

To protect against diverse challenges, the immune system must continuously generate an arsenal of specialized cell types, each of which can mount a myriad of effector responses upon detection of potential threats. To do so, it must generate multiple differentiated cell populations with defined sizes and proportions, often from rare starting precursor cells. Here, we discuss the emerging view that inherently probabilistic mechanisms, involving rare, rate-limiting regulatory events in single cells, control fate decisions and population sizes and fractions during immune development and function. We first review growing evidence that key fate control points are gated by stochastic signaling and gene regulatory events that occur infrequently over decision-making timescales, such that initially homogeneous cells can adopt variable outcomes in response to uniform signals. We next discuss how such stochastic control can provide functional capabilities that are harder to achieve with deterministic control strategies, and may be central to robust immune system function.

Selective organ ischaemia/reperfusion identifies liver as the key driver of the post-injury plasma metabolome derangements.

BACKGROUND: Understanding the molecular mechanisms in perturbation of the metabolome following ischaemia and reperfusion is critical in developing novel therapeutic strategies to prevent the sequelae of post-injury shock. While the metabolic substrates fueling these alterations have been defined, the relative contribution of specific organs to the systemic metabolic reprogramming secondary to ischaemic or haemorrhagic hypoxia remains unclear. MATERIALS AND METHODS: A porcine model of selected organ ischaemia was employed to investigate the relative contribution of liver, kidney, spleen and small bowel ischaemia/reperfusion to the plasma metabolic phenotype, as gleaned through ultra-high performance liquid chromatography-mass spectrometry-based metabolomics. RESULTS: Liver ischaemia/reperfusion promotes glycaemia, with increases in circulating carboxylic acid anions and purine oxidation metabolites, suggesting that this organ is the dominant contributor to the accumulation of these metabolites in response to ischaemic hypoxia. Succinate, in particular, accumulates selectively in response to the hepatic ischemia, with levels 6.5 times spleen, 8.2 times small bowel, and 6 times renal levels. Similar trends, but lower fold-change increase in comparison to baseline values, were observed upon ischaemia/reperfusion of kidney, spleen and small bowel. DISCUSSION: These observations suggest that the liver may play a critical role in mediating the accumulation of the same metabolites in response to haemorrhagic hypoxia, especially with respect to succinate, a metabolite that has been increasingly implicated in the coagulopathy and pro-inflammatory sequelae of ischaemic and haemorrhagic shock.

All animals are equal but some animals are more equal than others: Plasma lactate and succinate in hemorrhagic shock-A comparison in rodents, swine, nonhuman primates, and injured patients.

BACKGROUND: Plasma levels of lactate and succinate are predictors of mortality in critically injured patients in military and civilian settings. In relative terms, these metabolic derangements have been recapitulated in rodent, swine, and nonhuman primate models of severe hemorrhage. However, no direct absolute quantitative comparison has been evaluated across these species. METHODS: Ultra-high pressure liquid chromatography-mass spectrometry with stable isotope standards was used to determine absolute concentrations of baseline and postshock levels of lactate and succinate in rats, pigs, macaques, and injured patients. RESULTS: Baseline levels of lactate and succinate were most comparable to humans in macaques, followed by pigs and rats. Baseline levels of lactate in pigs and baseline and postshock levels of lactate and succinate in rats were significantly higher than those measured in macaques and humans. Postshock levels of lactate and succinate in pigs and macaques, respectively, were directly comparable to measurements in critically injured patients. CONCLUSION: Acknowledging the caveats associated with the variable degrees of shock in the clinical cohort, our data indicate that larger mammals represent a better model than rodents when investigating metabolic derangements secondary to severe hemorrhage.

Omics markers of the red cell storage lesion and metabolic linkage.

The introduction of omics technologies in the field of Transfusion Medicine has significantly advanced our understanding of the red cell storage lesion. While the clinical relevance of such a lesion is still a matter of debate, quantitative and redox proteomics approaches, as well quantitative metabolic flux analysis and metabolic tracing experiments promise to revolutionise our understanding of the role of blood processing strategies, inform the design and testing of novel additives or technologies (such as pathogen reduction), and evaluate the clinical relevance of donor and recipient biological variability with respect to red cell storability and transfusion outcomes. By reviewing existing literature in this rapidly expanding research endeavour, we highlight for the first time a correlation between metabolic markers of the red cell storage age and protein markers of haemolysis. Finally, we introduce the concept of metabolic linkage, i.e. the appreciation of a network of highly correlated small molecule metabolites which results from biochemical constraints of erythrocyte metabolic enzyme activities. For the foreseeable future, red cell studies will advance Transfusion Medicine and haematology by addressing the alteration of metabolic linkage phenotypes in response to stimuli, including, but not limited to, storage additives, enzymopathies (e.g. glucose 6-phosphate dehydrogenase deficiency), hypoxia, sepsis or haemorrhage.

Plasma succinate is a predictor of mortality in critically injured patients.

BACKGROUND: Trauma is the leading cause of mortality under the age of 40 years. Recent observations on metabolic reprogramming during hypoxia and ischemia indicate that hypoxic mitochondrial uncoupling promotes the generation of succinate, which in turn mediates reperfusion injury and inflammatory sequelae upon reoxygenation. Plasma levels of succinate significantly increase in response to trauma and hemorrhage in experimental models and clinical samples, suggesting that succinate may represent a candidate marker of systemic perfusion in trauma. METHODS: Quantitative mass spectrometry-based metabolomics was used to quantify succinate and lactate in 595 plasma samples from severely injured patients enrolled at the Denver Health Medical Center, a Level I trauma center in Denver, Colorado. RESULTS: A total of 95 severely injured patients were sampled for up to 10 time points (595 total samples), from field blood to 7 days postinjury. Results indicate that plasma levels of succinate increased up to 25.9-fold in deceased patients versus the median of the surviving patients (p = 2.75e-100; receiver operating characteristic area under the curve, 0.911). On the other hand, only 2.4-fold changes increases in lactate were observed (p = 5.8e-21; area under the curve, 0.874). CONCLUSION: Succinate represents a uniquely sensitive biomarker of postshock metabolic derangement and may be an important mediator of sequelae. LEVEL OF EVIDENCE: Prognostic study, level III.

Hemorrhagic shock and tissue injury drive distinct plasma metabolome derangements in swine.

BACKGROUND: Tissue injury and hemorrhagic shock induce significant systemic metabolic reprogramming in animal models and critically injured patients. Recent expansions of the classic concepts of metabolomic aberrations in tissue injury and hemorrhage opened the way for novel resuscitative interventions based on the observed abnormal metabolic demands. We hypothesize that metabolic demands and resulting metabolic signatures in pig plasma will vary in response to isolated or combined tissue injury and hemorrhagic shock. METHODS: A total of 20 pigs underwent either isolated tissue injury, hemorrhagic shock, or combined tissue injury and hemorrhagic shock referenced to a sham protocol (n = 5/group). Plasma samples were analyzed by UHPLC-MS. RESULTS: Hemorrhagic shock promoted a hypermetabolic state. Tissue injury alone dampened metabolic responses in comparison to sham and hemorrhagic shock, and attenuated the hypermetabolic state triggered by shock with respect to energy metabolism (glycolysis, glutaminolysis, and Krebs cycle). Tissue injury and hemorrhagic shock had a more pronounced effect on nitrogen metabolism (arginine, polyamines, and purine metabolism) than hemorrhagic shock alone. CONCLUSION: Isolated or combined tissue injury and hemorrhagic shock result in distinct plasma metabolic signatures. These findings indicate that optimized resuscitative interventions in critically ill patients are possible based on identifying the severity of tissue injury and hemorrhage.

Mass Spectrometry-Based Bottom-Up Proteomics: Sample Preparation, LC-MS/MS Analysis, and Database Query Strategies.

Recent technological advances in mass spectrometry (MS) have made possible the investigation and quantification of complex mixtures of biomolecules. The exceptional sensitivity and resolving power of today's mass spectrometers allow for the detection of proteins and peptides at low femtomole quantities; however, these attributes demand high sample purity to minimize artifacts and achieve the highest degree of biomolecule identification. Tissue preparation for proteomic studies is particularly challenging due to their heterogeneity in cell type, presence of insoluble biomaterials, and wide diversity of biomolecules. The workflow described herein details sample preparation from tissues through protein extraction, proteolysis, and purification to generate peptides for MS analysis. Increased peptide resolution and a corresponding increase in protein identification is accomplished using polarity-based fractionation (C18 resin) at the peptide level. Additionally, approaches to instrument set up, including the use of nanoscale liquid chromatography and quadrupole Orbitrap MS, along with database searching, are described. © 2016 by John Wiley & Sons, Inc.

Data on how several physiological parameters of stored red blood cells are similar in glucose 6-phosphate dehydrogenase deficient and sufficient donors.

This article contains data on the variation in several physiological parameters of red blood cells (RBCs) donated by eligible glucose-6-phosphate dehydrogenase (G6PD) deficient donors during storage in standard blood bank conditions compared to control, G6PD sufficient (G6PD(+)) cells. Intracellular reactive oxygen species (ROS) generation, cell fragility and membrane exovesiculation were measured in RBCs throughout the storage period, with or without stimulation by oxidants, supplementation of N-acetylcysteine and energy depletion, following incubation of stored cells for 24 h at 37 °C. Apart from cell characteristics, the total or uric acid-dependent antioxidant capacity of the supernatant in addition to extracellular potassium concentration was determined in RBC units. Finally, procoagulant activity and protein carbonylation levels were measured in the microparticles population. Further information can be found in "Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red blood cells" [1].

Plasma First Resuscitation Reduces Lactate Acidosis, Enhances Redox Homeostasis, Amino Acid and Purine Catabolism in a Rat Model of Profound Hemorrhagic Shock.

The use of aggressive crystalloid resuscitation to treat hypoxemia, hypovolemia, and nutrient deprivation promoted by massive blood loss may lead to the development of the blood vicious cycle of acidosis, hypothermia, and coagulopathy and, utterly, death. Metabolic acidosis is one of the many metabolic derangements triggered by severe trauma/hemorrhagic shock, also including enhanced proteolysis, lipid mobilization, as well as traumatic diabetes. Appreciation of the metabolic benefit of plasma first resuscitation is an important concept. Plasma resuscitation has been shown to correct hyperfibrinolysis secondary to severe hemorrhage better than normal saline. Here, we hypothesize that plasma first resuscitation corrects metabolic derangements promoted by severe hemorrhage better than resuscitation with normal saline. Ultra-high-performance liquid chromatography-mass spectrometry-based metabolomics analyses were performed to screen plasma metabolic profiles upon shock and resuscitation with either platelet-free plasma or normal saline in a rat model of severe hemorrhage. Of the 251 metabolites that were monitored, 101 were significantly different in plasma versus normal saline resuscitated rats. Plasma resuscitation corrected lactate acidosis by promoting glutamine/amino acid catabolism and purine salvage reactions. Plasma first resuscitation may benefit critically injured trauma patients by relieving the lactate burden and promoting other non-clinically measured metabolic changes. In the light of our results, we propose that plasma resuscitation may promote fueling of mitochondrial metabolism, through the enhancement of glutaminolysis/amino acid catabolism and purine salvage reactions. The treatment of trauma patients in hemorrhagic shock with plasma first resuscitation is likely not only to improve coagulation, but also to promote substrate-specific metabolic corrections.

Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red blood cells.

Storage of packed red blood cells (RBCs) is associated with progressive accumulation of lesions, mostly triggered by energy and oxidative stresses, which potentially compromise the effectiveness of the transfusion therapy. Concerns arise as to whether glucose 6-phosphate dehydrogenase deficient subjects (G6PD(-)), ~5% of the population in the Mediterranean area, should be accepted as routine donors in the light of the increased oxidative stress their RBCs suffer from. To address this question, we first performed morphology (scanning electron microscopy), physiology and omics (proteomics and metabolomics) analyses on stored RBCs from healthy or G6PD(-) donors. We then used an in vitro model of transfusion to simulate transfusion outcomes involving G6PD(-) donors or recipients, by reconstituting G6PD(-) stored or fresh blood with fresh or stored blood from healthy volunteers, respectively, at body temperature. We found that G6PD(-) cells store well in relation to energy, calcium and morphology related parameters, though at the expenses of a compromised anti-oxidant system. Additional stimuli, mimicking post-transfusion conditions (37°C, reconstitution with fresh healthy blood, incubation with oxidants) promoted hemolysis and oxidative lesions in stored G6PD(-) cells in comparison to controls. On the other hand, stored healthy RBC units showed better oxidative parameters and lower removal signaling when reconstituted with G6PD(-) fresh blood compared to control. Although the measured parameters of stored RBCs from the G6PD deficient donors appeared to be acceptable, the results from the in vitro model of transfusion suggest that G6PD(-) RBCs could be more susceptible to hemolysis and oxidative stresses post-transfusion. On the other hand, their chronic exposure to oxidative stress might make them good recipients, as they better tolerate exposure to oxidatively damaged long stored healthy RBCs.

Glutamine metabolism drives succinate accumulation in plasma and the lung during hemorrhagic shock.

BACKGROUND: Metabolomic investigations have consistently reported succinate accumulation in plasma after critical injury. Succinate receptors have been identified on numerous tissues, and succinate has been directly implicated in postischemic inflammation, organ dysfunction, platelet activation, and the generation of reactive oxygen species, which may potentiate morbidity and mortality risk to patients. Metabolic flux (heavy-isotope labeling) studies demonstrate that glycolysis is not the primary source of increased plasma succinate during protracted shock. Glutamine is an alternative parent substrate for ATP generation during anaerobic conditions, a biochemical mechanism that ultimately supports cellular survival but produces succinate as a catabolite. We hypothesize that succinate accumulation during hemorrhagic shock is driven by glutaminolysis. METHODS: Sprague-Dawley rats were subjected to hemorrhagic shock for 45 minutes (shock, n = 8) and compared with normotensive shams (sham, n = 8). At 15 minutes, animals received intravenous injection of C5-N2-glutamine solution (iLG). Blood, brain, heart, lung, and liver tissues were harvested at defined time points. Labeling distribution in samples was determined by ultrahigh-pressure liquid chromatography-mass spectrometry metabolomic analysis. Repeated-measures analysis of variance with Tukey comparison determined significance of relative fold change in metabolite level from baseline. RESULTS: Hemorrhagic shock instigated succinate accumulation in plasma and lungs tissues (8.5- vs. 1.1-fold increase plasma succinate level from baseline, shock vs. sham, p = 0.001; 3.2-fold higher succinate level in lung tissue, shock vs. sham, p = 0.006). Metabolomic analysis identified labeled glutamine and labeled succinate in plasma (p = 0.002) and lung tissue (p = 0.013), confirming glutamine as the parent substrate. Kinetic analyses in shams showed constant total levels of all metabolites without significant change due to iLG. CONCLUSION: Glutamine metabolism contributes to increased succinate concentration in plasma during hemorrhagic shock. The glutaminolytic pathway is implicated as a therapeutic target to prevent the contribution of succinate accumulation in plasma and the lung-to-postshock pathogenesis.