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Molecular BioSystems Accepted Manuscript This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/molecularbiosystems Page 1 of 40 Molecular BioSystems Metabolomic Analysis of Survival in Carbohydrate Pre-fed Pigs Subjected to Shock and Polytrauma Nancy E Witowski 1, Elizabeth R Lusczek 1, Charles E Determan Jr 1, Daniel R Lexcen 1, Kristine E Mulier 1, Andrea Wolf 1, Beverly G Ostrowski 2, Greg J Beilman 1 1 Department of Surgery, University of Minnesota, Minneapolis, MN 2 Minnesota NMR Center, University of Minnesota, Minneapolis, MN Abstract: 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 Manuscript 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 Accepted 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 BioSystems animals. Table of contents: Metabolomics data from four compartments in porcine polytrauma suggest differences in carbohydrate processing between survivors and non-survivors even before injury. 1. Introduction Molecular Hemorrhagic shock is the pathophysiologic state that accompanies extensive blood loss and remains a dominant factor in battlefield morbidity and mortality 1. The response to trauma is 1 Molecular BioSystems Page 2 of 40 complex 2, with several predictive models of outcome and severity of injury studied 3-5. One such predictor is pre-trauma state. Unsurprisingly, pre-existing disease negatively impacts outcomes in trauma patients 6. Alternatively, pre-injury state may be leveraged to improve outcomes. Studies in rodents report higher survival rates associated with carbohydrate feeding prior to the induction of hemorrhagic shock 7-9. We have previously reported that the metabolic as well as the physiologic profiles in pre-fed animals differed from that of fasted animals in a porcine model of hemorrhagic shock with injury and resuscitation 10-13 . In this study, both fasted and pre-fed pigs were allowed water ad libitum during an overnight fast. The pre-fed group then received an oral bolus of carbohydrate solution one hour prior to surgery. Contrary to previous reports in rodent models 7-9, the death rate for pre-fed animals in our study was nearly double that of fasted animals (15/32 or 47% vs 9/32 or 28%) 10 . Manuscript In an attempt to explain the unexpected death rate for pre-fed animals, we further analyzed the metabolic and physiologic profiles of pre-fed non-survivors and pre-fed survivors from our original experiment 10-13 . We hypothesized that non-survivors would exhibit profiles that differed from survivors. In this report we present the metabolic and physiologic profiles associated with outcome in the pre-fed group across 4 compartments (liver, muscle, serum, and urine) at specific time intervals: pre-shock, shock, and resuscitation. As hypothesized we found that pre-fed pigs Accepted that died as a result of hemorrhage and trauma showed differences in their metabolic and physiologic profiles in all time intervals. Based on these 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. 2. Methods 2.1 Animal Preparation and Hemorrhagic Shock Protocol BioSystems The experimental protocol was approved by the University of Minnesota Institutional Animal Care and Use Committee and was conducted in accordance with established guidelines for the treatment of laboratory animals. A modification of our well-established model of porcine hemorrhagic shock 14, 15 was used as described in previous work 10, 12, 13 (Fig. 1 ). In short, for the pre-fed group, 32 Yorkshire pigs (Manthei Hog Farm, LLC, Elk River, MN) weighing between 15-20 kg were fasted overnight but allowed free access to water for 12 hours prior to the execution of the protocol. Animals were given an oral carbohydrate solution (Karo Light ®, Molecular 7cc/kg, diluted with an equal volume of water) 60 minutes prior to the administration of initial anesthesia. [ 1H NMR of Karo Light ® indicated it is a mixture of mono- and di-saccharides (glucose, fructose, maltose, sucrose)]. 2 Page 3 of 40 Molecular BioSystems Animals were anesthetized, intubated and ventilated to maintain a PaO2 of 70-120 torr and a PaCO 2 of 35-45 torr (SERVO Ventilator 900C, Siemens, Malvern, PA). After surgical preparation, animals were allowed to stabilize until plasma lactate levels reach a value of 2.0 mmol/L or less, at which time Baseline (B) measurements were taken. Pigs were exposed to a blunt percussive chest injury, hemorrhage by withdrawal of blood from the inferior vena cava to a systolic blood pressure <60mm Hg, and a liver crush injury using a Holcomb clamp 16 . 45 minutes after the start of shock induction, animals were resuscitated with boluses of lactated Ringer’s solution during the first hour, and boluses of lactated Ringer’s solution and shed blood during the next 20 hours of resuscitation (for the detailed resuscitation protocol see Colling et al. 10 ). Pigs were extubated after 20 hours of full resuscitation. 48 hours after the end of resuscitation, animals were re-intubated for endpoint sample harvesting and then Manuscript euthanized with intravenous Beuthanasia D (1 ml/10kg). Physiologic and oxygen parameters were analyzed as described in Colling et al 10 . 2.2 Sampling for Metabolomic Analysis Serum, urine and tissue (muscle and liver) samples were collected at Baseline (B), 45 minutes after the beginning of the shock period (S45) and two hours after the advent of full resuscitation Accepted (FR2). Samples were stored at -80 °C until analysis. 2.3 Nuclear Magnetic Resonance Spectroscopy (NMR) 2.3.1 NMR Spectroscopy of Tissue Samples. Muscle and liver samples were processed for NMR analysis according to established protocol 17 . Frozen biopsy specimens were pulverized to a fine powder using a liquid nitrogen-chilled mortar and pestle. Ice-cold perchloric acid (6%, 5 mL/g wet weight) was added to the pulverized tissue (100-150 mg wet weight), and the sample vortexed for 30 seconds. Following a 10 minute incubation on ice, samples were centrifuged (14,000 rpm, 4° C), the pH of the supernatants adjusted to between 7 and 8 with 2M K 2CO 3 and BioSystems the neutralized samples incubated on ice for 30 min. The KClO 4 precipitate was removed by centrifugation. The resulting supernatant was lyophilized (LABCONCO Freezone 6 plus freeze- dryer, Kansas City, MO) and stored at _80 °C until NMR analysis. Lyophilized samples were reconstituted in 500 µl D 2O (99.9%; Sigma-Aldrich, St. Louis, MO); 50 µl of 3 mM 2,2-dimethyl-2- silapentane-5-sulfonate (DSS, Cambridge Labs) was added to serve as a lock signal and chemical shift reference (δ =0.0). Solution pH was adjusted with DCl Molecular and NaOD to 7.4. The final volume was brought to 600 µL with D 2O making the solution 0.25 mM in DSS and the sample was transferred to a 5mm NMR tube (Wilmad, Vineland, NJ). 3 Molecular BioSystems Page 4 of 40 1H NMR data acquisition was performed at 600 MHz using a Varian spectrometer with a 5mm HCN triple resonance probe at 25 °C. Muscle and liver spectra were generated from 128 scans with a basic 1H acquisition protocol consisting of a 45° tip angle, a relaxation delay of 1 sec and an acquisition time of 7 sec.