Effects of rumen-protected creatine pyruvate on blood biochemical parameters and rumen fluid characteristics in transported beef cattle.

BACKGROUND: The fasting and stress associated with road transportation contributes to a lack of energy and a decline in the immune system of beef cattle. Therefore, it is essential for beef cattle to enhance energy reserves before transportation. Creatine pyruvate (CrPyr) is a new multifunctional nutrient that can provide both pyruvate and creatine, which are two intermediate products of energy metabolism. To investigate the effects of transport and rumen-protected (RP)-CrPyr on the blood biochemical parameters and rumen fluid characteristics of beef cattle, twenty male Simmental crossbred cattle (659 ± 16 kg) aged 18 months were randomly allocated to four groups (n = 5) using a 2 × 2 factorial arrangement with two RP-CrPyr supplemental levels (0 or 140 g/d) and two transport treatments (5 min or 12 h): T_CrPyr140, T_CrPyr0, NT_CrPyr140, and NT_CrPyr0. After feeding for 30 days, three cattle per treatment were slaughtered. RESULTS: Compared with nontransport, transport decreased the total antioxidant capacity, catalase activity, contents of IgA, interferon γ, interleukin-1ß (IL-1ß), and IL-6 in serum, and the amounts of total volatile fatty acids (TVFA), acetate, and butyrate in rumen (P < 0.05); increased the serum lipopolysaccharide (LPS) level, contents of rumen LPS and ammonia nitrogen (P < 0.05). RP-CrPyr supplementation decreased the levels of cortisol and LPS in serum and the butyrate concentration in the rumen of beef cattle compared with those in the unsupplemented groups (P < 0.05). RP-CrPyr and transport interaction had a significant effect on the contents of serum tumour necrosis factor-α, IL-6, LPS, ruminal pH, acetate content, and acetate/propionate (P < 0.05). In terms of ruminal bacterial composition, group T_CrPyr0 increased the Prevotella genus abundance compared with group NT_CrPyr0 (P < 0.05), while group T_CrPyr140 increased Firmicutes phylum abundance and decreased Bacteroidetes phylum and genus Prevotella abundance compared with group T_CrPyr0 (P < 0.05). Moreover, Bacteroidetes was positively correlated with serum LPS. CONCLUSIONS: These results indicated that dietary supplementation with RP-CrPyr might be beneficial to alleviate transport stress by decreasing serum cortisol and LPS levels and promoting the restoration of the rumen natural flora.

Dysregulated metabolism underpins Zika-virus-infection-associated impairment in fetal development.

Zika virus (ZIKV) is an Aedes-mosquito-borne flavivirus that causes debilitating congenital and developmental disorders. Improved understanding of ZIKV pathogenesis could assist efforts to fill the therapeutic and vaccine gap. We use several ZIKV strains, including a pair differing by a single phenylalanine-to-leucine substitution (M-F37L) in the membrane (M) protein, coupled with unbiased genomics to demarcate the border between attenuated and pathogenic infection. We identify infection-induced metabolic dysregulation as a minimal set of host alterations that differentiates attenuated from pathogenic ZIKV strains. Glycolytic rewiring results in impaired oxidative phosphorylation and mitochondrial dysfunction that trigger inflammation and apoptosis in pathogenic but not attenuated ZIKV strains. Critically, pyruvate supplementation prevents cell death, in vitro, and rescues fetal development in ZIKV-infected dams. Our findings thus demonstrate dysregulated metabolism as an underpinning of ZIKV pathogenicity and raise the potential of pyruvate supplementation in expectant women as a prophylaxis against congenital Zika syndrome.

Hyperpolarized 13C pyruvate magnetic resonance spectroscopy for in vivo metabolic phenotyping of rat HCC.

The in vivo assessment of tissue metabolism represents a novel strategy for the evaluation of oncologic disease. Hepatocellular carcinoma (HCC) is a high-prevalence, high-mortality tumor entity often discovered at a late stage. Recent evidence indicates that survival differences depend on metabolic alterations in tumor tissue, with particular focus on glucose metabolism and lactate production. Here, we present an in vivo imaging technique for metabolic tumor phenotyping in rat models of HCC. Endogenous HCC was induced in Wistar rats by oral diethyl-nitrosamine administration. Peak lactate-to-alanine signal ratios (L/A) were assessed with hyperpolarized magnetic resonance spectroscopic imaging (HPMRSI) after [1-13C]pyruvate injection. Cell lines were derived from a subset of primary tumors, re-implanted in nude rats, and assessed in vivo with dynamic hyperpolarized magnetic resonance spectroscopy (HPMRS) after [1-13C]pyruvate injection and kinetic modelling of pyruvate metabolism, taking into account systemic lactate production and recirculation. For ex vivo validation, enzyme activity and metabolite concentrations were spectroscopically quantified in cell and tumor tissue extracts. Mean peak L/A was higher in endogenous HCC compared to non-tumorous tissue. Dynamic HPMRS revealed higher pyruvate-to-lactate conversion rates (kpl) and lactate signal in subcutaneous tumors derived from high L/A tumor cells, consistent with ex vivo measurements of higher lactate dehydrogenase (LDH) levels in these cells. In conclusion, HPMRS and HPMRSI reveal distinct tumor phenotypes corresponding to differences in glycolytic metabolism in HCC tumor tissue.

Clinical Cardiovascular Applications of Hyperpolarized Magnetic Resonance.

Current cardiovascular magnetic resonance imaging techniques provide an exquisite assessment of the structure and function of the heart and great vessels, but their ability to assess the molecular processes that underpin changes in cardiac function in health and disease is limited by inherent insensitivity. Hyperpolarized magnetic resonance is a new technology which overcomes this limitation, generating molecular contrast agents with an improvement in magnetic resonance signal of up to five orders of magnitude. One key molecule, hyperpolarized [1-13C]pyruvate, shows particular promise for the assessment of cardiac energy metabolism and other fundamental biological processes in cardiovascular disease. This molecule has numerous potential applications of clinical relevance and has now been translated to human use in early clinical studies. This review outlines the principles of hyperpolarized magnetic resonance and key potential cardiovascular applications for this new technology. Finally, we provide an overview of the pipeline for forthcoming hyperpolarized agents and their potential applications in cardiovascular disease.

Transition to 37°C reveals importance of NADPH in mitigating oxidative stress in stored RBCs.

The RBC storage lesion is a multiparametric response that occurs during storage at 4°C, but its impact on transfused patients remains unclear. In studies of the RBC storage lesion, the temperature transition from cold storage to normal body temperature that occurs during transfusion has received limited attention. We hypothesized that multiple deleterious events might occur in this period of increasing temperature. We show dramatic alterations in several properties of therapeutic blood units stored at 4°C after warming them to normal body temperature (37°C), as well as febrile temperature (40°C). In particular, the intracellular content and redox state of NADP(H) were directly affected by post-storage incubation at 37°C, as well as by pro-oxidant storage conditions. Modulation of the NADPH-producing pentose phosphate pathway, but not the prevention of hemoglobin autoxidation by conversion of oxyhemoglobin to carboxyhemoglobin, provided protection against storage-induced alterations in RBCs, demonstrating the central role of NADPH in mitigating increased susceptibility of stored RBCs to oxidative stress. We propose that assessing RBC oxidative status after restoration of body temperature constitutes a sensitive method for detecting storage-related alterations that has the potential to improve the quality of stored RBCs for transfusion.

Quantitative myocardial first-pass cardiovascular magnetic resonance perfusion imaging using hyperpolarized [1-13C] pyruvate.

BACKGROUND: The feasibility of absolute myocardial blood flow quantification and suitability of hyperpolarized [1-13C] pyruvate as contrast agent for first-pass cardiovascular magnetic resonance (CMR) perfusion measurements are investigated with simulations and demonstrated in vivo in a swine model. METHODS: A versatile simulation framework for hyperpolarized CMR subject to physical, physiological and technical constraints was developed and applied to investigate experimental conditions for accurate perfusion CMR with hyperpolarized [1-13C] pyruvate. Absolute and semi-quantitative perfusion indices were analyzed with respect to experimental parameter variations and different signal-to-noise ratio (SNR) levels. Absolute myocardial blood flow quantification was implemented with an iterative deconvolution approach based on Fermi functions. To demonstrate in vivo feasibility, velocity-selective excitation with an echo-planar imaging readout was used to acquire dynamic myocardial stress perfusion images in four healthy swine. Arterial input functions were extracted from an additional image slice with conventional excitation that was acquired within the same heartbeat. RESULTS: Simulations suggest that obtainable SNR and B0 inhomogeneity in vivo are sufficient for the determination of absolute and semi-quantitative perfusion with ≤25% error. It is shown that for expected metabolic conversion rates, metabolic conversion of pyruvate can be neglected over the short duration of acquisition in first-pass perfusion CMR. In vivo measurements suggest that absolute myocardial blood flow quantification using hyperpolarized [1-13C] pyruvate is feasible with an intra-myocardial variability comparable to semi-quantitative perfusion indices. CONCLUSION: The feasibility of quantitative hyperpolarized first-pass perfusion CMR using [1-13C] pyruvate has been investigated in simulations and demonstrated in swine. Using an approved and metabolically active compound is envisioned to increase the value of hyperpolarized perfusion CMR in patients.

Cafeteria Diet Feeding in Young Rats Leads to Hepatic Steatosis and Increased Gluconeogenesis under Fatty Acids and Glucagon Influence.

Gluconeogenesis overstimulation due to hepatic insulin resistance is the best-known mechanism behind elevated glycemia in obese subjects with hepatic steatosis. This suggests that glucose production in fatty livers may differ from that of healthy livers, also in response to other gluconeogenic determinant factors, such as the type of substrate and modulators. Thus, the aim of this study was to investigate the effects of these factors on hepatic gluconeogenesis in cafeteria diet-induced obese adult rats submitted to a cafeteria diet at a young age. The livers of the cafeteria group exhibited higher gluconeogenesis rates when glycerol was the substrate, but lower rates were found when lactate and pyruvate were the substrates. Stearate or glucagon caused higher stimulations in gluconeogenesis in cafeteria group livers, irrespective of the gluconeogenic substrates. An increased mitochondrial NADH/NAD⁺ ratio and a reduced rate of 14CO2 production from [14C] fatty acids suggested restriction of the citric acid cycle. The higher glycogen and lipid levels were possibly the cause for the reduced cellular and vascular spaces found in cafeteria group livers, likely contributing to oxygen consumption restriction. In conclusion, specific substrates and gluconeogenic modulators contribute to a higher stimulation of gluconeogenesis in livers from the cafeteria group.

Pyruvate-enriched resuscitation for shock.

This commentary addresses the recent retraction of an article which reported favorable outcomes in septic patients treated with intravenous pyruvate. The retracted report was cited in the authors' recent minireview on the cellular mechanisms and clinical application of pyruvate to improve cardiac performance. Because the retracted article reports pyruvate-enhanced resuscitation of critically ill patients, the authors wish to inform the readership, especially critical care providers, that this particular clinical application of pyruvate is not now supported by robust evidence. After discussing the retraction's implications for the clinical application of pyruvate-enriched resuscitation for sepsis, this commentary summarizes the extensive preclinical evidence of the efficacy and mechanisms of pyruvate resuscitation in animal models of hemorrhagic and septic shock, which argues for renewed clinical investigation of pyruvate-enriched resuscitation. Impact statement This commentary addresses the recent retraction of a clinical report of significant benefits of intravenous pyruvate resuscitation in septic patients, including sharply lowered mortality and decreased circulating pro-inflammatory cytokines, which was cited in the authors' minireview in Experimental Biology and Medicine. The potential implications of the retraction, and the extensive preclinical evidence supporting the use of pyruvate-enriched resuscitation for shock states, are summarized and discussed.

Pyruvate in reduced osmolarity oral rehydration salt corrected lactic acidosis in sever scald rats.

BACKGROUND: A novel pyruvate-based oral rehydration salt (Pyr-ORS) was demonstrated of superiority over bicarbonate- or citrate-based one to preserve organ function and correct lactic acidosis in rehydration of lethal shock in animals. This study further compared these effects between low-osmolar Pyr-ORS and equimolar citrate-based counterpart. METHODS: Eighty rats, using a fatal burn shock model, were randomized into four groups (two subgroups per group: n = 10): the sham group (group SR), Pyr-ORS group (group PR), WHO-ORS III group (group CR), and no rehydration group. ORS was delivered by manual gavage during 24 h following burns. Oral administration consisted of half of counted volume in the initial 8 h plus the rest in the later 16 h. Systemic hemodynamics, visceral organ surface blood flow, organ function, and metabolic acidosis were determined at 8 h and 24 h after burn. Another set of rats with identical surgical procedures without tests was observed for survival. RESULTS: Survival was markedly improved in the groups PR and CR; the former showed a higher survival rate than the latter at 24 h (40% versus 20%, P < 0.05). Systemic hemodynamics, visceral blood flow, and function of heart, liver, and kidney were greatly restored in group PR, compared with group CR (all P < 0.05). Hypoxic lactic acidosis was efficiently reversed in group PR, instead of group CR, (pH 7.36 versus 7.11, base excess 2.1 versus -9.1 mmol/L, lactate 4.28 versus 8.18 mmol/L; all P < 0.05) at 24 h after injury. CONCLUSIONS: Pyruvate was advantageous over citrate in low-osmolar ORS for protection of organs and survival; pyruvate, but not citrate, in the ORS corrected hypoxic lactic acidosis in rats subjected to lethal burn shock in 24 h.

Pyruvic acid prevents Cu2+/Zn2+-induced neurotoxicity by suppressing mitochondrial injury.

Zinc (Zn) is known as a co-factor for over 300 metalloproteins or enzymes, and has essential roles in many physiological functions. However, excessively high Zn concentrations are induced in pathological conditions such as interruption of blood flow in stroke or transient global ischemia-induced neuronal cell death. Furthermore, we recently found that copper (Cu2+) significantly exacerbates Zn2+ neurotoxicity in mouse hypothalamic neuronal cells, suggesting that Zn2+ interaction with Cu2+ is important for the development of neurological disease. Meanwhile, organic acids such as pyruvic acid and citric acid are reported to prevent neuronal cell death induced by various stresses. Thus, in this study, we focused on organic acids and searched for compounds that inhibit Cu2+/Zn2+-induced neurotoxicity. Initially, we examined the protective effect of various organic acids on Cu2+/Zn2+-induced neurotoxicity, and found that pyruvic acid clearly suppresses Cu2+/Zn2+-induced neurotoxicity in GT1-7 cells. Next, we examined the protective mechanisms of pyruvic acid against Cu2+/Zn2+-induced neurotoxicity. Specifically, we examined the possibilities that pyruvic acid chelates Cu2+ and Zn2+ or suppresses the ER stress response, but found that neither was suppressed by pyruvic acid treatment. In contrast, pyruvic acid significantly suppressed cytochrome c release into cytoplasm, an index of mitochondrial injury, in a dose-dependent manner. These results suggest that pyruvic acid prevents Cu2+/Zn2+-induced neuronal cell death by suppressing mitochondrial injury. Based on our results, we assume that pyruvic acid may be therapeutically beneficial for neurological diseases involving neuronal cell death such as vascular dementia.

Pyruvate enhancement of cardiac performance: Cellular mechanisms and clinical application.

Cardiac contractile function is adenosine-5'-triphosphate (ATP)-intensive, and the myocardium's high demand for oxygen and energy substrates leaves it acutely vulnerable to interruptions in its blood supply. The myriad cardioprotective properties of the natural intermediary metabolite pyruvate make it a potentially powerful intervention against the complex injury cascade ignited by myocardial ischemia-reperfusion. A readily oxidized metabolic substrate, pyruvate augments myocardial free energy of ATP hydrolysis to a greater extent than the physiological fuels glucose, lactate and fatty acids, particularly when it is provided at supra-physiological plasma concentrations. Pyruvate also exerts antioxidant effects by detoxifying reactive oxygen and nitrogen intermediates, and by increasing nicotinamide adenine dinucleotide phosphate reduced form (NADPH) production to maintain glutathione redox state. These enhancements of free energy and antioxidant defenses combine to augment sarcoplasmic reticular Ca2+ release and re-uptake central to cardiac mechanical performance and to restore ß-adrenergic signaling of ischemically stunned myocardium. By minimizing Ca2+ mismanagement and oxidative stress, pyruvate suppresses inflammation in post-ischemic myocardium. Thus, pyruvate administration stabilized cardiac performance, augmented free energy of ATP hydrolysis and glutathione redox systems, and/or quelled inflammation in a porcine model of cardiopulmonary bypass, a canine model of cardiac arrest-resuscitation, and a caprine model of hypovolemia and hindlimb ischemia-reperfusion. Pyruvate's myriad benefits in preclinical models provide the mechanistic framework for its clinical application as metabolic support for myocardium at risk. Phase one trials have demonstrated pyruvate's safety and efficacy for intravenous resuscitation for septic shock, intracoronary infusion for heart failure and as a component of cardioplegia for cardiopulmonary bypass. The favorable outcomes of these trials, which argue for expanded, phase three investigations of pyruvate therapy, mirror findings in isolated, perfused hearts, underscoring the pivotal role of preclinical research in identifying clinical interventions for cardiovascular diseases. Impact statement This article reviews pyruvate's cardioprotective properties as an energy-yielding metabolic fuel, antioxidant and anti-inflammatory agent in mammalian myocardium. Preclinical research has shown these properties make pyruvate a powerful intervention to curb the complex injury cascade ignited by ischemia and reperfusion. In ischemically stunned isolated hearts and in large mammal models of cardiopulmonary bypass, cardiac arrest-resuscitation and hypovolemia, intracoronary pyruvate supports recovery of myocardial contractile function, intracellular Ca2+ homeostasis and free energy of ATP hydrolysis, and its antioxidant actions restore ß-adrenergic signaling and suppress inflammation. The first clinical trials of pyruvate for cardiopulmonary bypass, fluid resuscitation and intracoronary intervention for congestive heart failure have been reported. Receiver operating characteristic analyses show remarkable concordance between pyruvate's beneficial functional and metabolic effects in isolated, perfused hearts and in patients recovering from cardiopulmonary bypass in which they received pyruvate- vs. L-lactate-fortified cardioplegia. This research exemplifies the translation of mechanism-oriented preclinical studies to clinical application and outcomes.

Effect of Glycine, Pyruvate, and Resveratrol on the Regeneration Process of Postischemic Intestinal Mucosa.

BACKGROUND: Intestinal ischemia is often caused by a malperfusion of the upper mesenteric artery. Since the intestinal mucosa is one of the most rapidly proliferating organs in human body, this tissue can partly regenerate itself after the onset of ischemia and reperfusion (I/R). Therefore, we investigated whether glycine, sodium pyruvate, and resveratrol can either support or potentially harm regeneration when applied therapeutically after reperfusion injury. METHODS: I/R of the small intestine was initiated by occluding and reopening the upper mesenteric artery in rats. After 60 min of ischemia and 300 min of reperfusion, glycine, sodium pyruvate, or resveratrol was administered intravenously. Small intestine regeneration was analyzed regarding tissue damage, activity of saccharase, and Ki-67 positive cells. Additionally, systemic parameters and metabolic ones were obtained at selected periods. RESULTS: Resveratrol failed in improving the outcome after I/R, while glycine showed a partial beneficial effect. Sodium pyruvate ameliorated metabolic acidosis, diminished histopathologic tissue injury, and increased cell proliferation in the small intestine. CONCLUSION: While glycine could improve in part regeneration but not proliferation, sodium pyruvate seems to be a possible therapeutic agent to facilitate proliferation and to support mucosal regeneration after I/R injury to the small intestine.

In ovo feeding of creatine pyruvate alters energy reserves, satellite cell mitotic activity and myogenic gene expression of breast muscle in embryos and neonatal broilers.

We investigated the effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on energy reserves, satellite cell mitotic activity (SCMA) and myogenic gene expression in breast muscle of embryos and neonatal broilers. A total of 960 eggs were randomly allocated into three treatments: 1) non-injected control group, 2) saline group injected with 0.6 mL of physiological saline (0.75%), and 3) CrPyr group injected with 0.6 mL of physiological saline (0.75%) containing 12 mg CrPyr/egg at 17.5 d of incubation. After hatching, a total of 120 male chicks were randomly assigned to each treatment group, with eight replicate sets per group. Selected chicks had body BW close to the average of their pooled group. Our results showed that the total and relative breast muscle weights of broilers subjected to CrPyr treatment were higher than those in the control and saline groups on 19 d of incubation (19 E), the day of hatch, 3 and 7 d post-hatch (P < 0.05). The myofiber diameter and cross-sectional area of individuals in the CrPyr group were higher than those in other treatments on 3 and 7 d post-hatch (P < 0.05). Moreover, IOF of CrPyr increased (P < 0.05) creatine concentrations on 19 E, the day of hatch and 3 d post-hatch, the same treatment increased phosphocreatine concentrations on 19 E. Broilers in the CrPyr group showed higher expression of myogenic differentiation 1 (MyoD) (P < 0.05), myogenin and paired box 7 (Pax7), as well as higher index of SCMA on 3 d post-hatch. However, myostatin mRNA expression in CrPyr-treated broilers was down-regulated on 3 d post-hatch (P < 0.05). These results indicated that IOF of CrPyr increased energy reserves of embryos and SCMA of broilers on 3 d post-hatch, which led to enhanced muscle growth in the late embryos and neonatal broilers. Additionally, IOF of CrPyr increased the activity of satellite cells possibly through up-regulating MyoD, myogenin, and Pax7 mRNA expression and down-regulating myostatin mRNA expression.

Identification of a High-Affinity Pyruvate Receptor in Escherichia coli.

Two-component systems are crucial for signal perception and modulation of bacterial behavior. Nevertheless, to date, very few ligands have been identified that directly interact with histidine kinases. The histidine kinase/response regulator system YehU/YehT of Escherichia coli is part of a nutrient-sensing network. Here we demonstrate that this system senses the onset of nutrient limitation in amino acid rich media and responds to extracellular pyruvate. Binding of radiolabeled pyruvate was found for full-length YehU in right-side-out membrane vesicles as well as for a truncated, membrane-integrated variant, confirming that YehU is a high-affinity receptor for extracellular pyruvate. Therefore we propose to rename YehU/YehT as BtsS/BtsR, after "Brenztraubensäure", the name given to pyruvic acid when it was first synthesized. The function of BtsS/BtsR was also assessed in a clinically relevant uropathogenic E. coli strain. Quantitative transcriptional analysis revealed BtsS/BtsR importance during acute and chronic urinary-tract infections.

Featured Article: Pyruvate preserves antiglycation defenses in porcine brain after cardiac arrest.

Cardiac arrest (CA) and cardiocerebral resuscitation (CCR)-induced ischemia-reperfusion imposes oxidative and carbonyl stress that injures the brain. The ischemic shift to anaerobic glycolysis, combined with oxyradical inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), provokes excessive formation of the powerful glycating agent, methylglyoxal. The glyoxalase (GLO) system, comprising the enzymes glyoxalase 1 (GLO1) and GLO2, utilizes reduced glutathione (GSH) supplied by glutathione reductase (GR) to detoxify methylglyoxal resulting in reduced protein glycation. Pyruvate, a natural antioxidant that augments GSH redox status, could sustain the GLO system in the face of ischemia-reperfusion. This study assessed the impact of CA-CCR on the cerebral GLO system and pyruvate's ability to preserve this neuroprotective system following CA. Domestic swine were subjected to 10 min CA, 4 min closed-chest CCR, defibrillation and 4 h recovery, or to a non-CA sham protocol. Sodium pyruvate or NaCl control was infused (0.1 mmol/kg/min, intravenous) throughout CCR and the first 60 min recovery. Protein glycation, GLO1 content, and activities of GLO1, GR, and GAPDH were analyzed in frontal cortex biopsied at 4 h recovery. CA-CCR produced marked protein glycation which was attenuated by pyruvate treatment. GLO1, GR, and GAPDH activities fell by 86, 55, and 30%, respectively, after CA-CCR with NaCl infusion. Pyruvate prevented inactivation of all three enzymes. CA-CCR sharply lowered GLO1 monomer content with commensurate formation of higher molecular weight immunoreactivity; pyruvate preserved GLO1 monomers. Thus, ischemia-reperfusion imposed by CA-CCR disabled the brain's antiglycation defenses. Pyruvate preserved these enzyme systems that protect the brain from glycation stress. Impact statement Recent studies have demonstrated a pivotal role of protein glycation in brain injury. Methylglyoxal, a by-product of glycolysis and a powerful glycating agent in brain, is detoxified by the glutathione-catalyzed glyoxalase (GLO) system, but the impact of cardiac arrest (CA) and cardiocerebral resuscitation (CCR) on the brain's antiglycation defenses is unknown. This study in a swine model of CA and CCR demonstrated for the first time that the intense cerebral ischemia-reperfusion imposed by CA-resuscitation disabled glyoxalase-1 and glutathione reductase (GR), the source of glutathione for methylglyoxal detoxification. Moreover, intravenous administration of pyruvate, a redox-active intermediary metabolite and antioxidant in brain, prevented inactivation of glyoxalase-1 and GR and blunted protein glycation in cerebral cortex. These findings in a large mammal are first evidence of GLO inactivation and the resultant cerebral protein glycation after CA-resuscitation, and identify novel actions of pyruvate to minimize protein glycation in postischemic brain.

A multisample dissolution dynamic nuclear polarization system for serial injections in small animals.

PURPOSE: Several in vivo applications of dissolution dynamic nuclear polarization (DNP) require rapid successive injections of hyperpolarized substrates. Here we present the design and performance of a custom-built multisample dissolution DNP setup for small animal research. METHODS: The DNP setup consists of a commercial wide-bore magnet charged to 3.35 T, a cryostat, a 94-GHz microwave source, and a custom-built skeleton that accommodates four identical sample sticks. Each sample stick features a dissolver locked into the skeleton port and a lifter, which permits moving the sample cup out of the liquid helium bath for dissolution. RESULTS: The dissolution of the first sample was triggered after 2 hours of polarization buildup during single-shot operation of the cryostat. Thereafter, a time window of 75-90 min was available to dissolve the remaining three polarized samples. The average liquid state polarization over all four sticks was measured as 18.7% ± 2.3% for [1-13C] pyruvate 30 s after dissolution. In vivo applicability of the setup using serial injections of [1-13C] pyruvate to study cardiac metabolism in rats revealed good reproducibility. CONCLUSION: The proposed four-sample DNP insert provides reproducible liquid state polarization of [1-13C] pyruvate and allows for rapid repeat injections in small animals. Magn Reson Med 77:904-910, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Pyruvate treatment attenuates cerebral metabolic depression and neuronal loss after experimental traumatic brain injury.

Experimental traumatic brain injury (TBI) is known to produce an acute increase in cerebral glucose utilization, followed rapidly by a generalized cerebral metabolic depression. The current studies determined effects of single or multiple treatments with sodium pyruvate (SP; 1000mg/kg, i.p.) or ethyl pyruvate (EP; 40mg/kg, i.p.) on cerebral glucose metabolism and neuronal injury in rats with unilateral controlled cortical impact (CCI) injury. In Experiment 1 a single treatment was given immediately after CCI. SP significantly improved glucose metabolism in 3 of 13 brain regions while EP improved metabolism in 7 regions compared to saline-treated controls at 24h post-injury. Both SP and EP produced equivalent and significant reductions in dead/dying neurons in cortex and hippocampus at 24h post-CCI. In Experiment 2 SP or EP were administered immediately (time 0) and at 1, 3 and 6h post-CCI. Multiple SP treatments also significantly attenuated TBI-induced reductions in cerebral glucose metabolism (in 4 brain regions) 24h post-CCI, as did multiple injections of EP (in 4 regions). The four pyruvate treatments produced significant neuroprotection in cortex and hippocampus 1day after CCI, similar to that found with a single SP or EP treatment. Thus, early administration of pyruvate compounds enhanced cerebral glucose metabolism and neuronal survival, with 40mg/kg of EP being as effective as 1000mg/kg of SP, and multiple treatments within 6h of injury did not improve upon outcomes seen following a single treatment.

Pyruvate alleviates lipid peroxidation and multiple-organ dysfunction in rats with hemorrhagic shock.

OBJECTIVE: Pyruvate can reduce lipid peroxidation, which plays a critical role in organ injury, in various models. However, it is not fully understood if this inhibition occurs in resuscitation of hemorrhagic shock (HS). This study examines effects of pyruvate Ringer solution (PR) in this respect in rats. METHODS: Rats, subjected to 45% blood loss, were randomly allocated to the 3 groups (n = 18): HS with no fluid resuscitation (group NR), HS resuscitated with lactated Ringer solution (LR) (group LR), and HS resuscitated with PR (group PR). Mean arterial pressure, plasma levels of thiobarbituric acid reactive substances (TBARS), and superoxide dismutase were measured at various time points until 360 minutes after hemorrhage. Visceral organs were harvested at the end for evaluations of the TBARS, antioxidant enzyme, and tissue water content. Other 54 rats with identical procedures without sampling were documented for 24-hour survival rates (n = 18) after fluid resuscitation. RESULTS: Pyruvate Ringer solution significantly increased mean arterial pressure and decreased blood TBARS levels after lethal HS. It also reduced TBARS concentrations and glutathione peroxidase activities but significantly enhanced glutathione reductase activities in most organs and greatly improved the ratios of reduced glutathione over oxidized glutathione in various organs in group PR, compared to group LR. Furthermore, PR significantly improved various organ function and water contents relative to LR. Group PR showed a more than 2-fold higher 24-hour survival rate of group LR. CONCLUSIONS: Pyruvate Ringer solution alleviated organ edema and injury and prompted survival partially through inhibition of lipid peroxidation in various organs in severe HS rats.

Ethanol, ethyl and sodium pyruvate decrease the inflammatory responses of human lung epithelial cells via Akt and NF-κB in vitro but have a low impact on hepatocellular cells.

Increases in pro-inflammatory cytokine levels and tissue-infiltrating leukocytes have been closely linked to increased systemic and local inflammation, which result in organ injury. Previously, we demonstrated the beneficial and hepatoprotective anti-inflammatory effects of acute ethanol (EtOH) ingestion in an in vivo model of acute inflammation. Due to its undesirable side-effects, however, EtOH does not represent a therapeutic option for treatment of acute inflammation. Therefore, in this study, we compared the effects of acute EtOH exposure with ethyl pyruvate (EtP) as an alternative anti-inflammatory drug in an in vitro model of hepatic and pulmonary inflammation. Human hepatocellular carcinoma cells Huh7 and alveolar epithelial cells A549 were stimulated with either interleukin (IL) IL-1ß (1 ng/ml, 24 h) or tumor necrosis factor (TNF) (10 ng/ml, 4 h), and then treated with EtP (2.5-10 mM), sodium pyruvate (NaP, 10 mM) or EtOH (85-170 mM) for 1 h. IL-6 or IL-8 release from Huh7 or A549 cells, respectively, was measured by an enzyme­linked immunosorbent assay. Neutrophil adhesion to cell monolayers and CD54 expression were also analyzed. Bcl-2 and Bax gene expression was determined by RT-qPCR, and western blot analysis was performed to determine the mechanisms involved. Treating A549 cells with either EtOH or EtP significantly reduced the IL-1ß- or TNF­induced IL-8 release, whereas treating Huh7 cells did not significantly alter IL-6 release. Similarly, neutrophil adhesion to stimulated A549 cells was significantly reduced by EtOH or EtP, whereas for Huh7 cells the tendency for reduced neutrophil adhesion rates by EtOH or EtP was not significant. CD54 expression was noticeably reduced in A549 cells, but this was not the case in Huh7 cells after treatment. The Bax/Bcl-2 ratio was dose­dependently decreased by EtOH and by high-dose EtP in A549 cells, indicating a reduction in apoptosis, whereas this effect was not observed in Huh7 cells. The underlying mechanisms involve reduced phosphorylation of Akt and nuclear factor-κB (NF-κB) p65. We noted that as with EtP, EtOH reduced the inflammatory response in lung epithelial cells under acute inflammatory conditions. However, due to the low impact which EtP and EtOH had on the hepatocellular cells, our data suggest that both substances exerted different effects depending on the cellular entity. The possible underlying mechanisms involved the downregulation of Akt and the transcription factor NF-κB, but further research on this subject is required.