Nancy E. Witowski

Metabolomics classifies phase of care and identifies risk for mortality in a porcine model of multiple injuries and hemorrhagic shock.

BACKGROUND Early recognition and intervention in hemorrhagic shock is essential to improved outcomes. However, the lack of robust diagnostic tools readily available to identify patients in the field inhibits the ability to provide timely intervention. Therefore, the development of a reliable prognostic indicator, such as a serum biomarker or a metabolic profile, has significant potential to improve far-forward trauma care. In this study, we used metabolomics as a tool to identify a metabolic state associated with the hemorrhagic shock and outcome in our porcine model of multiple injuries, shock, and resuscitation. METHODS Proton nuclear magnetic resonance spectroscopy was used to evaluate serum metabolites from 23 animals that underwent multiple injuries, controlled hemorrhage, and 20 hours of a standard resuscitation protocol. Serum samples were collected from the animals at baseline (before hemorrhage), at shock (after 45 minutes of shock), and at 8 hours of full resuscitation. RESULTS We were able to demonstrate shifts in the metabolome throughout different time points and construct a metabolic profile associated with mortality using partial least squares discriminate analysis. The metabolites most responsible for the classification of hemorrhagic shock in our model serve as markers for ischemia, changes in energy production, and cellular damage. Hemorrhagic shock was characterized by marked increases in tricarboxylic acid cycle intermediates, glycolytic-gluconeogenic by-products, purine-pyrimidine catabolism, and fatty acid oxidation. CONCLUSION The results of this study demonstrate the potential for metabolomics as a tool to classify the metabolic flux, to identify relevant biochemical pathways, and to identify clinically useful biomarkers.

Liver Metabolomic Changes Identify Biochemical Pathways in Hemorrhagic Shock

BACKGROUND Despite ongoing advances in treatment, thousands of patients still die annually from complications due to hemorrhagic shock, a condition causing dramatic physiologic and metabolic changes as cells switch to anaerobic metabolism in response to oxygen deprivation. As the shift from aerobic to anaerobic metabolism occurs in the peripheral tissues during shock, the liver must increase production of endogenous glucose as well as process excess lactate produced in the periphery. This places the liver at the center of metabolic regulation in the body during hemorrhagic shock. Therefore, we hypothesized that liver tissue from pigs during an in vivo model of hemorrhagic shock (n = 6) would reflect resultant metabolic changes. MATERIALS AND METHODS The in vivo model of shock consisted of 45 min of shock followed by 8 h of hypotensive resuscitation (80 mmHg) and subsequent normotensive resuscitation (90 mmHg) ending 48 h after the shock period. Control groups of pigs (n = 3) (1) shock with no resuscitation, and (2) only anesthesia and instrumentation, also were included. Metabolic changes within the liver after shock and during resuscitation were investigated using both proton ((1)H) and phosphorous ((31)P) nuclear magnetic resonance (NMR) spectroscopy. RESULTS Concentrations of glycerylphosphorylcholine (GPC) and glycerylphosphorylethanolamine (GPE) were significantly lower at 8 h after shock, with recovery to baseline by 23 and 48 h after shock. Uridine diphosphate-glucose (UDP-glucose), and phosphoenolpyruvate (PEP) were elevated 23 h after shock. CONCLUSIONS These results indicate that (1)H and (31)P NMR spectroscopy can be used to identify differences in liver metabolites in an in vivo model of hemorrhagic shock, indicating that metabolomic analysis can be used to elucidate biochemical events occurring during this complex disease process.

Urine Metabolomics in Hemorrhagic Shock: Normalization of Urine in the Face of Changing Intravascular Fluid Volume and Perturbations in Metabolism

There are many ways to normalize biofluid metabolomics data to account for changes in dilution, all of which have been thoroughly examined in model systems. Here, urine metabolomics data was examined under relevant physiological conditions obtained from a porcine model of hemorrhagic shock and resuscitation. This includes highly variable intravascular fluid volume and urine output coupled with large perturbations in the abundance of endogenous metabolites. Seven different normalization techniques and raw data were evaluated to determine an appropriate normalization technique in this setting, including spectral post-processing methods and physiological measures of concentration. Relationships between normalization constants for each urine sample were examined, as well as relationships between urinary and serum creatinine concentrations. Principal components analysis was used to examine clustering of metabolomics data. The set of normalization constants associated with each sample were reflective of urine concentration, with a trend toward concentration decreases during late resuscitation timepoints. Urinary creatinine normalized to urine output was most reflective of serum creatinine levels. Principal components analysis showed that urine samples clustered according to experimental timepoint for all normalization methods examined. Little separation was seen in raw data. Urine output-normalized data stands out from the six other normalization methods studied because it is reflective of renal clearance and should be used when comparing urine and serum metabolomics data.

Carbohydrate fed state alters the metabolomic response to hemorrhagic shock and resuscitation in liver

Hemorrhagic shock with injury results in alterations of the metabolic state of an organism, which contribute to organ dysfunction and death. Previous investigations have explored the effects of carbohydrate prefeed in murine models but few in clinically relevant large animal models. We performed carbohydrate prefeed in pigs undergoing simulated polytrauma and hemorrhagic shock with resuscitation to determine if carbohydrate prefeeding if the metabolic response to shock is dependent on fed state. Sixty-four Yorkshire pigs were divided into two experimental groups: fasted (32) and prefed (32). Experimental animals were subjected to a standardized hemorrhagic shock protocol, including pulmonary contusion and liver crush injury. To determine molecular alterations in response to trauma as a result of prefeeding, liver biopsies were obtained at set timepoints throughout the procedure. Fifty-one metabolites were profiled for each sample via proton nuclear magnetic resonance spectroscopy (1H NMR). Partial-Least Squared Discriminant Analysis (PLS-DA) was used to examine clustering of the data with respect to fed state. Cross-validated models separated the fed from fasted animals. Metabolites contributing to the separation have known relationships to alternate carbon energy sources, amino acid metabolism, oxidative stress response, and membrane maintenance. In conclusion, metabolomic techniques allowed identification of an alternate response to shock and resuscitation, dependent upon fed state, through the use of metabolomics.

A Four-Compartment Metabolomics Analysis of the Liver, Muscle, Serum, and Urine Response to Polytrauma with Hemorrhagic Shock following Carbohydrate Prefeed

Objective Hemorrhagic shock accompanied by injury represents a major physiologic stress. Fasted animals are often used to study hemorrhagic shock (with injury). A fasted state is not guaranteed in the general human population. The objective of this study was to determine if fed animals would exhibit a different metabolic profile in response to hemorrhagic shock with trauma when compared to fasted animals. Methods Proton (1H) NMR spectroscopy was used to determine concentrations of metabolites from four different compartments (liver, muscle, serum, urine) taken at defined time points throughout shock/injury and resuscitation. PLS-DA was performed and VIP lists established for baseline, shock and resuscitation (10 metabolites for each compartment at each time interval) on metabolomics data from surviving animals. Results Fed status prior to the occurrence of hemorrhagic shock with injury alters the metabolic course of this trauma and potentially affects mortality. The death rate for CPF animals is higher than FS animals (47 vs 28%). The majority of deaths occur post-resuscitation suggesting reperfusion injury. The metabolomics response to shock reflects priorities evident at baseline. FS animals raise the baseline degree of proteolysis to provide additional amino acids for energy production while CPF animals rely on both glucose and, to a lesser extent, amino acids. During early resuscitation levels of metabolites associated with energy production drop, suggesting diminished demand. Conclusions Feeding status prior to the occurrence of hemorrhagic shock with injury alters the metabolic course of this trauma and potentially affects mortality. The response to shock reflects metabolic priorities at baseline.

Prolonged induced hypothermia in hemorrhagic shock is associated with decreased muscle metabolism: a nuclear magnetic resonance-based metabolomics study.

ABSTRACT Hemorrhagic shock is a leading cause of trauma-related death in war and is associated with significant alterations in metabolism. Using archived serum samples from a previous study, the purpose of this work was to identify metabolic changes associated with induced hypothermia in a porcine model of hemorrhagic shock. Twelve Yorkshire pigs underwent a standardized hemorrhagic shock and resuscitation protocol to simulate battlefield injury with prolonged evacuation to definitive care in cold environments. Animals were randomized to receive either hypothermic (33°C) or normothermic (39°C) limited resuscitation for 8 h, followed by standard resuscitation. Proton nuclear magnetic resonance spectroscopy was used to evaluate serum metabolites from these animals at intervals throughout the hypothermic resuscitation period. Animals in the hypothermic group had a significantly higher survival rate (P = 0.02) than normothermic animals. Using random forest analysis, a difference in metabolic response between hypothermic and normothermic animals was identified. Hypothermic resuscitation was characterized by decreased concentrations of several muscle-related metabolites including taurine, creatine, creatinine, and amino acids. This study suggests that a decrease in muscle metabolism as a result of induced hypothermia is associated with improved survival.

Preinjury Fed State Alters the Physiologic Response in a Porcine Model of Hemorrhagic Shock and Polytrauma.

ABSTRACT Introduction: Hemorrhagic shock and injury lead to dramatic changes in metabolic demands and continue to be a leading cause of death. We hypothesized that altering the preinjury metabolic state with a carbohydrate load prior to injury would affect subsequent metabolic responses to injury and lead to improved survival. Methods: Sixty-four pigs were randomized to fasted (F) or carbohydrate prefeeding (CPF) groups and fasted 12 h prior to experiment. The CPF pigs received an oral carbohydrate load 1 h prior to anesthesia. All pigs underwent a standardized injury/hemorrhagic shock protocol. Physiologic parameters and laboratory values were obtained at set time points. Results: Carbohydrate prefeeding did not convey a survival benefit; instead, CPF animals had greater mortality rates (47% vs. 28%; P = 0.153; log-rank [Mantel-Cox]). Carbohydrate prefeeding animals also had higher rates of acute lung injury (odds ratio, 4.23; 95% confidence interval, 1.1–16.3) and altered oxygen utilization. Prior to shock and throughout resuscitation, CPF animals had significantly higher serum glucose levels than did the F animals. Conclusions: Carbohydrate prefeeding did not provide a survival benefit to swine subjected to hemorrhagic shock and polytrauma. Carbohydrate prefeeding led to significantly different metabolic profile than in fasted animals, and prefeeding led to a greater incidence of lung injury, increased multiorgan dysfunction, and altered oxygen utilization.

Fed state prior to hemorrhagic shock and polytrauma in a porcine model results in altered liver transcriptomic response.

Hemorrhagic shock is a leading cause of trauma-related mortality in both civilian and military settings. Resuscitation often results in reperfusion injury and survivors are susceptible to developing multiple organ failure (MOF). The impact of fed state on the overall response to shock and resuscitation has been explored in some murine models but few clinically relevant large animal models. We have previously used metabolomics to establish that the fed state results in a different metabolic response in the porcine liver following hemorrhagic shock and resuscitation. In this study, we used our clinically relevant model of hemorrhagic shock and polytrauma and the Illumina HiSeq platform to determine if the liver transcriptomic response is also altered with respect to fed state. Functional analysis of the response to shock and resuscitation confirmed several typical responses including carbohydrate metabolism, cytokine inflammation, decreased cholesterol synthesis, and apoptosis. Our findings also suggest that the fasting state, relative to a carbohydrate prefed state, displays decreased carbohydrate metabolism, increased cytoskeleton reorganization and decreased inflammation in response to hemorrhagic shock and reperfusion. Evidence suggests that this is a consequence of a shrunken, catabolic state of the liver cells which provides an anti-inflammatory condition that partially mitigates hepatocellar damage.

Metabolomic analysis of survival in carbohydrate pre-fed pigs subjected to shock and polytrauma

Hemorrhagic shock, a result of extensive blood loss, is a dominant factor in battlefield morbidity and mortality. Early rodent studies in hemorrhagic shock reported carbohydrate feeding prior to the induction of hemorrhagic shock decreased mortality. When repeated in our laboratory with a porcine model, carbohydrate pre-feed resulted in a 60% increase in death rate following hemorrhagic shock with trauma when compared to fasted animals (15/32 or 47% vs. 9/32 or 28%). In an attempt to explain the unexpected death rate for pre-fed animals, we further investigated the metabolic profiles of pre-fed non-survivors (n = 15) across 4 compartments (liver, muscle, serum, and urine) at specific time intervals (pre-shock, shock, and resuscitation) and compared them to pre-fed survivors (n = 17). As hypothesized, pre-fed pigs that died as a result of hemorrhage and trauma showed differences in their metabolic and physiologic profiles at all time intervals and in all compartments when compared to pre-fed survivors. Our data suggest that, although all animals were subjected to the same shock and trauma protocol, non-survivors exhibited altered carbohydrate processing as early as the pre-shock sampling point. This was evident in (for example) the higher levels of ATP and markers of greater anabolic activity in the muscle at the pre-shock time point. Based on the metabolic findings, we propose two mechanisms that connect pre-fed status to a higher death rate: (1) animals that die are more susceptible to opening of the mitochondrial permeability transition pore, a major factor in ischemia/reperfusion injury; and (2) loss of fasting-associated survival mechanisms in pre-fed animals.