The Metabolopathy of Tissue Injury, Hemorrhagic Shock, and Resuscitation in a Rat Model.

INTRODUCTION: The metabolic consequences of trauma induce significant clinical pathology. In this study, we evaluate the independent, metabolic contributions of tissue injury (TI) and combined tissue injury and hemorrhagic shock (TI/HS) using mass spectrometry (MS) metabolomics in a controlled animal model of critical injury. METHODS: Sprague-Dawley rats (n = 14) underwent TI alone or TI/HS, followed by resuscitation with normal saline and shed blood. Plasma was collected (baseline, post-laparotomy, post-HS, post-resuscitation) for ultra-high pressure liquid chromatography MS-metabolomics. Repeated-measures ANOVA with Tukey multiple column comparison test compared the fold change of metabolite concentration among the animal groups at corresponding time points. RESULTS: Four hundred forty metabolites were identified. TI alone did not change the metabolite levels versus baseline. TI/HS induced changes in metabolites from glycolysis, the tricarboxylic acid cycle, the pentose phosphate, fatty acid and glutathione homeostasis pathways, sulfur metabolism, and urea cycle versus TI alone. Following resuscitation many metabolites normalized to TI alone levels, including lactate, most tri-carboxylic acid metabolites, most urea cycle metabolites, glutathione disulfide, and some metabolites from both the pentose phosphate pathway and sulfur metabolism. CONCLUSIONS: Significant changes occur immediately following TI/HS versus TI alone. These metabolic changes are not explained by dilution as a number of metabolites remained unchanged or even increased following resuscitation. The differential metabolic changes resulting from TI alone and TI/HS provide foundation for future investigations severe injury in humans, where TI and HS are often concurrent. This investigation provides a foundation to evaluate metabolic-related outcomes and design-targeted resuscitation strategies.

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.

Red blood cells in hemorrhagic shock: a critical role for glutaminolysis in fueling alanine transamination in rats.

Red blood cells (RBCs) are the most abundant host cell in the human body and play a critical role in oxygen transport and systemic metabolic homeostasis. Hypoxic metabolic reprogramming of RBCs in response to high-altitude hypoxia or anaerobic storage in the blood bank has been extensively described. However, little is known about the RBC metabolism following hemorrhagic shock (HS), the most common preventable cause of death in trauma, the global leading cause of total life-years lost. Metabolomics analyses were performed through ultra-high pressure liquid chromatography-mass spectrometry on RBCs from Sprague-Dawley rats undergoing HS (mean arterial pressure [MAP], <30 mm Hg) in comparison with sham rats (MAP, >80 mm Hg). Steady-state measurements were accompanied by metabolic flux analysis upon tracing of in vivo-injected 13C15N-glutamine or inhibition of glutaminolysis using the anticancer drug CB-839. RBC metabolic phenotypes recapitulated the systemic metabolic reprogramming observed in plasma from the same rodent model. Results indicate that shock RBCs rely on glutamine to fuel glutathione (GSH) synthesis and pyruvate transamination, whereas abrogation of glutaminolysis conferred early mortality and exacerbated lactic acidosis and systemic accumulation of succinate, a predictor of mortality in the military and civilian critically ill populations. Glutamine is here identified as an essential amine group donor in HS RBCs, plasma, liver, and lungs, providing additional rationale for the central role glutaminolysis plays in metabolic reprogramming and survival following severe hemorrhage.

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.