Browsing by Subject "Pyruvate"
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Item EXAMINING PYRUVATE S ANTI-INFLAMMATORY ACTIONS IN BRAIN FOLLOWING CARDIAC ARREST(2013-04-12) Nguyen, AnhPurpose: Approximately 0.5 million Americans suffer cardiac arrest each year. Cardiac arrest is usually lethal, and survivors often face severe neurological disability due to irreversible brain injury inflicted by global ischemia/reperfusion. Brain ischemia and reperfusion activates matrix metalloproteinases (MMPs) that disrupt the blood-brain barrier (BBB), enabling inflammatory cells to invade the brain parenchyma. We have shown that pyruvate, a metabolic intermediate and antioxidant, prevents death of hippocampal and cerebellar neurons and preserves neurobehavioral function in dogs after cardiac arrest. However, pyruvate's neuroprotective mechanism is elusive. We are testing the hypothesis that pyruvate preserves BBB integrity following cardiac arrest by suppressing MMPs and evoking expression of protective proteins hypoxia-inducible factor (HIF)-1ɑ and heat shock protein (Hsp)-70. Methods: Yorkshire swine (30-40 kg) were subjected to cardiac arrest-resuscitation or non-arrest sham protocols. Ventricular fibrillation was induced by a train of electric impulses transmitted to the right ventricle via a pacing wire. Precordial compressions were given at 6-10 min arrest, and then sinus rhythm was restored with defibrillatory transthoracic countershocks. NaCl or Na-pyruvate was infused iv at the rate of 0.1 mmol/kg/min during chest compressions and the first 60 min post-defibrillation. Cerebral cortex was snap-frozen in liquid nitrogen for immunoblot or fixed in 4% paraformaldehyde for immunohistochemistry (IHC) at 4 h after defibrillation. HIF-1ɑ and Hsp-70 contents were assessed by immunoblots and IHC. Four groups were studied: cardiac arrest + NaCl (n = 9) or Na-pyruvate (n = 7), or sham + NaCl (n = 5) or Na-pyruvate (n = 5). Results: Although immunoblots did not reveal appreciable differences in temporal cortex HIF-1ɑ content among the groups, IHC revealed increased HIF-1ɑ content in pyruvate-treated compared to NaCl-treated sham and cardiac arrest groups. Hsp-70 content also was similar among the groups, suggesting that this cytoprotective protein was not augmented within 3 h after 60 min pyruvate treatment. Conclusions: Current studies are examining neurobehavioral function and brain proteins over 7 days post-arrest. MMP activities in brain are being measured by gel zymography. Future experiments will determine plasma concentrations of neurofilament light chain, which, when elevated up to 7 days post-cardiac arrest, is associated with poor neurological outcomes.Item INTRAVENOUS PYRUVATE FOR CARDIAC ARREST DOES NOT CAUSE PERSISTENT HYPERNATREMIA(2013-04-12) Cherry, BrandonPurpose: Intravenous infusion of sodium pyruvate can protect internal organs from ischemia-reperfusion imposed by cardiac arrest and resuscitation, but may produce hypernatremia due to the sodium load. The purpose of this study was to examine the effects of Na-pyruvate infusion on plasma pyruvate, bicarbonate and sodium concentrations in order to test the hypothesis that pyruvate infusion does not cause persistent hypernatremia. Methods: Swine were subjected to 6 min cardiac arrest, 4 min closed-chest CPR, defibrillation and 4 h recovery. Na-pyruvate (n=7) or NaCl control (n=9) were infused iv (0.1 mmol/kg/min) during CPR and the first 60 min recovery. Results: Pyruvate infusion produced a sustained increase in plasma bicarbonate concentration (44.2 ± 1.2 mM in pyruvate-treated vs. 27.5 ± 2.6 mM in NaCl-treated group at 3 hours post-treatment; P<0.05), which may potentially offset post-arrest acidemia. Although pyruvate and NaCl infusions similarly increased plasma sodium concentrations (146 ± 2 mM and 148 ± l mM, respectively), the hypernatremia resolved to pre-arrest concentrations by 3 h post-pyruvate (140 ± 1 mM), but persisted 3 h post-NaCl (147 ± 2 mM; P<0.05). Conclusions: This study confirms the hypothesis that pyruvate administration after cardiac arrest did not cause persistent hypernatremia.Item PYRUVATE DIMERIZATION BY DIVALENT METAL CATIONS(2013-04-12) Barnes, DavidPurpose: Pyruvate enriched cardioplegia has been shown to reduce cardiopulmonary bypass induced inflammation and oxidative stress. Cardioplegia solutions are stored in the hospital pharmacy at room temperature. It has been argued that pyruvate is unstable in aqueous solutions containing divalent metal cations and undergoes an aldol condensation reaction to form parapyruvate. This substance is a potential inhibitor of a critical step in the TCA cycle, oxoglutarate dehydrogenase, and therefore may be toxic. Our pilot study is testing whether pyruvate remains stable in solution in the presence of a metal catalyst. Methods: Aqueous solutions of 5 mM pyruvate were prepared in 0.5 M Tris and aliquoted into test tubes. Zn2+ (ZnCl2) was then added to the test tubes at 0, 0.1, 0.3 and 1.0 mM concentrations. The solution was buffered to pH 4 to prevent precipitation of the Zn2+ which occurred at neutral pH. The solutions were stored in the dark at 20 and 4 ℃ to assess the effect of temperature on the aldol condensation. Absorbance of ultraviolet light (315 nm wavelength) by the pyruvate-Zn solution was measured by spectrophotometry at 0 and 10 days to detect formation of parapyruvate, which absorbs light at that wavelength. Results: The changes in absorbance of the solutions kept at 20 ℃ were as follows: 0 mM Zn2+ -0.002, 0.1 mM Zn2+ 0.007, 0.3 mM Zn2+ -0.011, 1.0 mM Zn2+ -0.0195. The corresponding absorbance changes in the solutions at 0 ℃ for 10 days were as follows: 0 mM Zn2+ -0.009, 0.1 mM Zn2+ 0.0015, 0.3 mM Zn2+ -0.005, 1.0 mM Zn2+ -0.0105. Thus, at both temperatures, only the highest Zn2+ concentration, 1 mM, produced a detectable, albeit modest, change in absorbance at 315 nm, and that change was in the direction opposite that which would indicate pyruvate dimerization. Conclusions: Over the course of 10 days, no appreciable pyruvate dimerization could be detected by spectrophotometry. Thus, pyruvate appeared to be stable in aqueous solution, even over 10 days at 20 ℃. To confirm these results, we are currently measuring pyruvate concentrations at 0, 1, 3, 7 and 14 days by a spectrophotometric assay at 340 nm utilizing the lactate dehydrogenase reaction, in which pyruvate reduction to lactate consumes NADH. This method will enable us to verify that the apparent absence of parapyruvate is accompanied by a stable concentration of pyruvate over time. Future studies will test the impact of other divalent metals, including Mg2+, an important component of many cardioplegia formulations.Item Pyruvate-Enhanced Fluid Resuscitation for Hemorrhagic Shock and Hindlimb Ischemia(2009-05-01) Flaherty, Devin C.; Mallet, Robert T.Traumatic blood loss often necessitates the use of resuscitative fluids to replenish blood volume and stabilize blood pressure. The use of tourniquets to achieve hemostasis imposes ischemia-reperfusion on wounded limbs after release. Resuscitation with the physiological antioxidant and natural intermediary metabolite pyruvate may abrogate reperfusion injury of muscle by scavenging oxyradicals and stabilizing cytoprotective proteins. This study was designed to determine the effects of pyruvate in the setting of hemorrhagic shock with resuscitation and hindlimb ischemia-reperfusion. All experiments were conducted on isoflurane-anesthetized male goats. A controlled hemorrhage was performed to lower mean arterial pressure (MAP) to c. 50 mmHg, then the right femoral artery and vein were occluded for 90 min. Lactate Ringer’s (LR) or pyruvate Ringer’s (PR) was infused intravenously (10 ml/min) for 90 min, from 30 min occlusion until 30 min after reperfusion. At 4 h reperfusion, the right gastrocnemius muscle and left ventricular myocardium were biopsied and flash-frozen for analyses of metabolites, enzymes, pro- and anti-apoptotic proteins and markers of oxidative and inflammatory stress. During the first phase of experimentation we hypothesized that controlled resuscitation with PR vs. LR more effectively stabilizes MAP and attenuates myocardial inflammation post-resuscitation. MAP (mmHg) was increased in PR (59 ± 4) vs. LR (47 ± 3) resuscitated goats (p During the second phase of experimentation, we hypothesized that PR resuscitation would protect ischemic hindlimb muscle in the setting of hemorrhagic shock and limb reperfusion. Lactate dehydrogenase and creatine kinase activities fell by 36 and 20%, respectively in LR-resuscitated vs. sham muscle (p We conclude that 1) Systemic hypotension and hindlimb ischemia-reperfusion with conventional LR treatment imposed pro-oxidative and pro-inflammatory stress both systemically and locally, thus preventing stabilization of MAP during recovery and initiating apoptotic mechanisms in the hindlimb musculature; 2) Pyruvate-fortified Ringer’s effectively stabilized hemodynamics and dampened systemic inflammation after hemorrhagic shock with resuscitation and hindlimb ischemia-reperfusion; 3) PR-fortified resuscitation blunted oxidative and inflammatory stress within the ischemic hindlimb and suppressed pro-apoptotic signaling. These investigations demonstrate the anti-oxidative and anti-inflammatory effects of pyruvate in a system exposed to hemorrhagic shock with fluid resuscitation, as well as identify the cytoprotection pyruvate affords tissue experiencing ischemia-reperfusion.Item The Effects of Pyruvate on Oxidative Stress and Myocardial Energetics During Cardioplegic Arrest and Reperfusion(2005-10-01) Knott, E. Marty; Robert T. Mallet; James Caffrey; Jerry SimeckaKnott, E. Marty, The Effects of Pyruvate on Oxidative Stress and Myocardial Energetics During Cardioplegic Arrest and Reperfusion Doctor of Philosophy (Integrative Physiology), October 2005, 118 pp, 1 tables, 17 figures, references, 130 titles. Cardioplegic arrest for bypass surgery imposes global ischemia on the myocardium generating oxyradicals which contribute to post surgical cardiac dysfunction. Early clinical trials have demonstrated that pyruvate-fortified cardioplegia reduces myocardial energy and improves mechanical recovery in patients undergoing elective cardiopulmonary bypass for coronary artery bypass grafting. This study was designed to determine the effects of the natural carbohydrate, pyruvate, on oxidative stress, myocardial energy state, and activities of myocardial metabolic enzymes during and immediately following cardiopulmonary bypass. In the first set of experiments, in situ swine hearts were arrested for 60 min with a 4:1 mixture of blood and crystalloid cardioplegia solution containing 188 mM glucose alone (control) or with additional 23.8 mM lactate or 23.8 mM pyruvate, then reperfused for 3 min with cardioplegia-free blood. Glutathione redox state (GSH/GSSG) and phosphocreatine phosphorylation potential were determine from measurements of myocardial metabolites in left ventricular heart tissue snap frozen at 45 min arrest and 3 min reperfusion. Coronary sinus 8-isoprostane indexed oxidative stress. Pyruvate-fortified cardioplegia decreased oxidative stress, lowering 8-isoporstane content accumulate during arrest and reperfusion. Phosphorylation potential was maintained in all groups during arrest but fell upon reperfusion in the control and lactate and cardioplegia groups. Use of pyruvate cardioplegia during arrest prevented the decline in phosphorylation potential during reperfusion. Pyruvate cardioplegia doubled GSH/GSSG during arrest as compared to lactate, but GSH/GSSG fell during reperfusion all 3 groups. Pyruvate proved to be an effective antioxidant and energy yielding fuel in the setting of carioplegic arrest and reperfusion. From these data, we hypothesized that pyruvate would protect oxidant-sensitive enzymes from inactivation. To test this hypothesis, in situ swine hearts were arrested for 60 min with control cardioplegia and reperfused for 3 min with cardioplegia-free blood alone or with co-infusion c. 12 mM pyruvate. Activities of oxidant-sensitive enzymes, 8-isoprostane content, and energy and antioxidant metabolites were measured in left ventricular myocardium snap-frozen at 45 min arrest and 3 min reperfusion. At 3 min reperfusion, glutathione redox state fell by 70% while 8-isoprostane content increased 75%. Pyruvate administration during reperfusion suppressed oxidative stress, maintained glutathione redox state, and enhanced phosphocreatine phosphorylation potential. Aconitase and glucose 6-phosphate dehydrogenase activities fell during arrest; creatine kinase and phosphofructokinase were inactivated upon reperfusion. Pyruvate protected creatine kinase and reactivated aconitase, which are at least partially mitochondrial enzymes, but did not modify the cytosolic enzymes glucose 6-phosphofructokinase. We conclude that 1) pyruvate-fortified cardioplegia and administration of pyruvate during early perfusion increase the antioxidant state of the heart and reduce oxidative stress occurring as a result of cardioplegic arrest and reperfusion; 2) pyruvate bolsters the myocardial energy state during early reperfusion when administered during cardioplegic arrest or during reperfusion; 3) Cardioplegic arrest and reperfusion inactivated several key metabolic enzymes. Pyruvate administration during reperfusion, the period of most intense oxidative stress, increases the activity of two mitochondrial enzymes during early reperfusion when compared to control. These investigations provide likely mechanisms for the ability of pyruvate-fortified cardioplegia to reduce myocardial injury and improve post-surgical cardiac performance in patients undergoing CPB. More research must be done to solidify pyruvate’s role as a cardioprotective intervention during CPB.