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.

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.

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.

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.

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.

Hepatic metabolic response of Holstein cows in early and mid lactation is altered by nutrient supply and lipopolysaccharide in vitro.

The metabolic response of the liver during periods of inflammation is poorly understood. The objective of this study was to characterize the effects of nutrient supply and lipopolysaccharide (LPS) challenge on hepatic intermediate metabolism of early- and mid-lactation cows by employing gas chromatography-mass spectrometry with stable isotope tracer. Twelve multiparous Holstein-Friesian cows in early (n = 6; 12 ± 4.2 d in milk) and mid (n = 6; 115 ± 13.5 d in milk) lactation were used for this study. Liver biopsies were performed on all cows. Liver slices (40-60 mg) were incubated in a 37°C water bath for 2 h with either control (phosphate buffered saline), pyruvate (PYR; 1mM unlabeled pyruvate and 1mM [(13)C3]pyruvate), pyruvate + propionate (PYR+PRO; 1mM unlabeled pyruvate, 1mM [(13)C3]pyruvate, and 2mM sodium propionate), or pyruvate + AA (PYR+AA; 1mM unlabeled pyruvate, 1mM [(13)C3]pyruvate, and 2mM AA solution), and LPS (0.0 or 0.2 µg/mL) was added to flasks per treatment. Enrichment of isotopomers in metabolic equilibrium with Krebs cycle intermediates was assessed. Pyruvate fluxes and the enzymatic activity of pyruvate carboxylase (PC) versus pyruvate dehydrogenase (PDH) and phosphoenol pyruvate carboxykinase (PEPCK) were calculated. Media were analyzed for concentrations of tumor necrosis factor-α (TNF-α), glucose, and haptoglobin. Data were analyzed as randomized block (stage of lactation) design in a factorial arrangement of nutrient treatments by LPS dose. Challenge with LPS increased the mRNA abundance of TNF-α, haptoglobin, and serum amyloid A 2, and the concentration of TNF-α in media. Challenge with LPS increased mRNA abundance of PC but reduced the enrichment of (13)C1[M1] and (13)C2[M2]alanine and tended to reduce the enzymatic activity of PEPCK. Incubation with PYR+PRO and PYR+AA increased the flux of pyruvate to acetyl CoA. However, only PYR+PRO increased the enzymatic activity of PEPCK and PDH versus PC and decreased the mRNA abundance of PC. Cows in early lactation tended to receive a greater contribution of pyruvate to the oxaloacetate flux via the lower PDH versus PC activity and a higher mRNA abundance of PC than cows in mid lactation. Our results suggest that regardless of stage of lactation and nutrient supplement, hepatic gluconeogenesis was impaired during inflammation. Further research examining how various nutrients support liver function and improve the immunometabolic response of liver during inflammation is warranted.

Systemic pyruvate administration markedly reduces neuronal death and cognitive impairment in a rat model of Alzheimer's disease.

Alzheimer's disease (AD) is a major neurodegenerative disease of old age, characterized by progressive cognitive impairment, dementia and atrophy of the central nervous system. Amyloid-ß (Aß) oligomers are derived from proteolytic cleavage of amyloid precursor protein (APP) and recognized as the primary neurotoxic agents in AD. Pyruvate has a protective effect against Aß oligomer-induced neuronal cell death and inhibition of long-term potentiation (LTP) in hippocampal slice cultures, leading us to investigate the effect of systemic pyruvate administration in an intracerebroventricular Aß oligomer infusion model. We found that sodium pyruvate (500 mg/kg, intraperitoneally) improved neuron survival and sustained improvement in cognitive function as assessed by the Morris water maze. Pyuvate prevented the Aß oligomer-induced inhibition of LTP and protein phosphatase 2A (PP2A) activation. Pyruvate suppressed the Aß oligomer-induced poly[adenosine diphosphate (ADP) ribose] polymerase-1 (PARP-1) activity and ameliorated Aß oligomer-induced decrease of NAD(+) level. Moreover, pyuvate, but not lactate, decreased reactive oxygen species (ROS) accumulation in hippocampus of Aß1-42 oligomer-injection rat model. These results suggest that systemic pyruvate administration could significantly ameliorate Aß oligomer-induced spatial learning and memory impairment by the improvement of neuron survival and prevention of LTP inhibition, and the beneficial effect of pyruvate could be linked, at least in part, to the elimination of ROS accumulation, prevention of PP2A activation, amelioration of NAD(+) level and suppression of PARP-1 activity.

Degeneration of spinal motor neurons by chronic AMPA-induced excitotoxicity in vivo and protection by energy substrates.

INTRODUCTION: Several data suggest that excitotoxicity due to excessive glutamatergic neurotransmission may be an important factor in the mechanisms of motor neuron (MN) death occurring in amyotrophic lateral sclerosis (ALS). We have previously shown that the overactivation of the Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptor type, through the continuous infusion of AMPA in the lumbar spinal cord of adult rats during several days, results in progressive rear limb paralysis and bilateral MN degeneration. Because it has been shown that energy failure and oxidative stress are involved in MN degeneration, in both ALS and experimental models of spinal MN degeneration, including excitotoxicity, in this work we tested the protective effect of the energy substrates pyruvate and ß-hydroxybutyrate (ßHB) and the antioxidants glutathione ethyl ester (GEE) and ascorbate in this chronic AMPA-induced neurodegeneration. RESULTS: AMPA infusion induced remarkable progressive motor deficits, assessed by two motor tasks, that by day seven reach bilateral rear limb paralysis. These effects correlate with the death of >80% of lumbar spinal MNs in the infused and the neighbor spinal cord segments, as well as with notable astrogliosis in the ventral horns, detected by glial fibrillary acidic protein immunohistochemistry. Co-infusion with pyruvate or ßHB notably prevented the motor deficits and paralysis, decreased MN loss to <25% and completely prevented the induction of astrogliosis. In contrast, the antioxidants tested were ineffective regarding all parameters analyzed. CONCLUSIONS: Chronic progressive excitotoxicity due to AMPA receptors overactivation results in MN death and astrogliosis, with consequent motor deficits and paralysis. Because of the notable protection against these effects exerted by pyruvate and ßHB, which are well established mitochondrial energy substrates, we conclude that deficits in mitochondrial energy metabolism are an important factor in the mechanisms of this slowly developed excitotoxic MN death, while the lack of protective effect of the antioxidants indicates that oxidative stress seems to be less significant factor. Because excitotoxicity may be involved in MN degeneration in ALS, these findings suggest possible preventive or therapeutic strategies for the disease.

[Quantitative cellular metabolism can be estimated by hyperpolarized magnetic resonance]. / Hyperpolariseret magnetisk resonans kan in vivo kvantificere cellulær metabolism.

A new MR methodology, hyperpolarized MR spectroscopy (MRS), enhances the MRS signals by more than a factor 10,000, enabling fast and unique insight into in situ metabolic processes. So far the method has provided new and exciting metabolic details in prostate, heart, brain, kidney and liver examinations. In diseases characterized by abnormal metabolic profiles, such as in diabetes and tumour tissue, hyperpolarized MRS provides highly detailed spatial information as well as quantitative estimates of individual steps in glycolysis and real-time amino acid metabolism.

Rapid sequential injections of hyperpolarized [1-¹³C]pyruvate in vivo using a sub-kelvin, multi-sample DNP polarizer.

The development of hyperpolarized technology utilizing dynamic nuclear polarization (DNP) has enabled the rapid measurement of (13)C metabolism in vivo with very high SNR. However, with traditional DNP equipment, consecutive injections of a hyperpolarized compound in an animal have been subject to a practical minimum time between injections governed by the polarization build-up time, which is on the order of an hour for [1-(13)C]pyruvate. This has precluded the monitoring of metabolic changes occurring on a faster time scale. In this study, we demonstrated the ability to acquire in vivo dynamic magnetic resonance spectroscopy (MRS) and 3D magnetic resonance spectroscopic imaging (MRSI) data in normal rats with a 5 min interval between injections of hyperpolarized [1-(13)C]pyruvate using a prototype, sub-Kelvin dynamic nuclear polarizer with the capability to simultaneously polarize up to 4 samples and dissolve them in rapid succession. There were minimal perturbations in the hyperpolarized spectra as a result of the multiple injections, suggesting that such an approach would not confound the investigation of metabolism occurring on this time scale. As an initial demonstration of the application of this technology and approach for monitoring rapid changes in metabolism as a result of a physiological intervention, we investigated the pharmacodynamics of the anti-cancer agent dichloroacetate (DCA), collecting hyperpolarized data before administration of DCA, 1 min after administration, and 6 min after administration. Dramatic increases in (13)C-bicarbonate were detected just 1 min (as well as 6 min) after DCA administration.

Dietary zinc reduction, pyruvate supplementation, or zinc transporter 5 knockout attenuates ß-cell death in nonobese diabetic mice, islets, and insulinoma cells.

Pancreatic zinc (Zn(2+)) concentrations are linked to diabetes and pancreatic dysfunction, but Zn(2+) is also required for insulin processing and packaging. Zn(2+) released with insulin increases ß-cell pancreatic death after streptozotocin toxin exposure in vitro and in vivo. Triosephosphate accumulation, caused by NAD(+) loss and glycolytic enzyme dysfunction, occur in type-1 diabetics (T1DM) and animal models. We previously showed these mechanisms are also involved in Zn(2+) neurotoxicity and are attenuated by nicotinamide- or pyruvate-induced restoration of NAD(+) concentrations, Zn(2+) restriction, or inhibition of Sir2 proteins. We tested the hypothesis that similar Zn(2+)- and NAD(+)-mediated mechanisms are involved in ß-cell toxicity in models of ongoing T1DM using mouse insulinoma cells, islets, and nonobese diabetic (NOD) mice. Zn(2+), streptozotocin, and cytokines caused NAD(+) loss and death in insulinoma cells and islets, which were attenuated by Zn(2+) restriction, pyruvate, nicotinamide, NAD(+), and inhibitors of Sir2 proteins. We measured diabetes incidence and mortality in NOD mice and demonstrated that pyruvate supplementation, or genetic or dietary Zn(2+) reduction, attenuated these measures. T-lymphocyte infiltration, punctate Zn(2+) staining, and ß-cell loss increased with time in islets of NOD mice. Dietary Zn(2+) restriction or Zn(2+) transporter 5 knockout reduced pancreatic Zn(2+) staining and increased ß-cell mass, glucose homeostasis, and survival in NOD mice, whereas Zn(2+) supplementation had the opposite effects. Pancreatic Zn(2+) reduction or NAD(+) restoration (pyruvate or nicotinamide supplementation) are suggested as novel targets for attenuating T1DM.

Combined inhibitory effects of pyruvate and low temperature on postovulatory aging of mouse oocytes.

This study tested the hypothesis that oocyte aging could be prevented for a longer time by reducing the culture temperature while supplementing the culture medium with more pyruvate. Newly ovulated mouse oocytes were cultured at various temperatures for various times in HCZB medium (Kimura and Yanagimachi, Biol Reprod 1995; 52:709-720) containing various concentrations of pyruvate before examining for aging parameters and developmental potential. The increase in susceptibility to activating stimuli was efficiently prevented when oocytes were cultured in HCZB with 10.27 mM pyruvate at 37°C for 6 h, 25°C for 24 h, 15°C for 96 h, and 5°C for 48 h. Satisfactory blastocyst development of both parthenotes and fertilized zygotes was achieved after oocyte culture in HCZB containing 10.27 mM pyruvate at 37°C for 6 h, 25°C for 24 h, 15°C for 36 h, and 5°C for 24 h. Transfer of two-cell embryos or blastocysts showed no difference between newly ovulated control oocytes and oocytes cultured at 15°C for 36 h in either term pregnancy, live young per pregnant recipient, live young/transferred embryos, or birth weight of young. Oocytes with impaired developmental potential after culture at 15°C for 96 h and at 5°C for 48 h showed unrecoverable decreases in the content of glutathione, the glutathione/oxidized glutathione ratio, the BCL2 content, and in the numbers of oocytes with normal spindles and cortical granule distribution, suggesting induction of oxidative stress, which caused oocyte apoptosis and cytoskeleton alterations by downregulating BCL2. Because oocytes cultured at 15°C for 36 h were activated or fertilized after a 6-h recovery culture, aging of ovulated mouse oocytes has been successfully prevented for 42 h without impairing their developmental potential.

Transient decrease in tumor oxygenation after intravenous administration of pyruvate.

MRI using hyperpolarized (13) C-labeled pyruvate is a promising tool to biochemically profile tumors and monitor their response to therapy. This technique requires injection of pyruvate into tumor-bearing animals. Pyruvate is an endogenous entity but the influence of exogenously injected bolus doses of pyruvate on tumor microenvironment is not well understood. In this study, the effect of injecting a bolus of pyruvate on tumor oxygen status was investigated. EPR oxygen imaging revealed that the partial pressure of oxygen (pO(2)) in squamous cell carcinoma implanted in mice decreased significantly 30 min after [1-(13) C]pyruvate injection, but recovered to preinjection levels after 5 h. Dynamic contrast-enhanced-MRI studies showed that, at the dose of pyruvate used, no changes in tumor perfusion were noticed. Immunohistochemical analysis of hypoxic marker pimonidazole independently verified that the squamous cell carcinoma tumor transiently became more hypoxic by pyruvate injection. Efficacy of radiotherapy was suppressed when X-irradiation was delivered during the period of pyruvate-induced transient hypoxia. These results suggest importance of taking into account the transient decrease in tumor pO(2) after pyruvate injection in hyperpolarized (13) C MRI, because tumor oxygen status is an important factor in determining outcomes of therapies.

Delayed sodium pyruvate treatment improves working memory following experimental traumatic brain injury.

Prior work indicates that cerebral glycolysis is impaired following traumatic brain injury (TBI) and that pyruvate treatment acutely after TBI can improve cerebral metabolism and is neuroprotective. Since extracellular levels of glucose decrease during periods of increased cognitive demand and exogenous glucose improves cognitive performance, we hypothesized that pyruvate treatment prior to testing could ameliorate cognitive deficits in rats with TBI. Based on pre-surgical spatial alternation performance in a 4-arm plus-maze, adult male rats were randomized to receive either sham injury or unilateral (left) cortical contusion injury (CCI). On days 4, 9 and 14 after surgery animals received an intraperitoneal injection of either vehicle (Sham-Veh, n=6; CCI-Veh, n=7) or 1000 mg/kg of sodium pyruvate (CCI-SP, n=7). One hour after each injection rats were retested for spatial alternation performance. Animals in the CCI-SP group showed no significant working memory deficits in the spatial alternation task compared to Sham-Veh controls. The percent four/five alternation scores for CCI-Veh rats were significantly decreased from Sham-Veh scores on days 4 and 9 (p<0.01) and from CCI-SP scores on days 4, 9 and 14 (p<0.05). Measures of cortical contusion volume, regional cerebral metabolic rates of glucose and regional cytochrome oxidase activity at day 15 post-injury did not differ between CCI-SP and CCI-Veh groups. These results show that spatial alternation testing can reliably detect temporal deficits and recovery of working memory after TBI and that delayed pyruvate treatment can ameliorate TBI-induced cognitive impairments.

Detection of tumor response to a vascular disrupting agent by hyperpolarized 13C magnetic resonance spectroscopy.

Nuclear spin hyperpolarization can dramatically increase the sensitivity of the (13)C magnetic resonance experiment, allowing dynamic measurements of the metabolism of hyperpolarized (13)C-labeled substrates in vivo. Here, we report a preclinical study of the response of lymphoma tumors to the vascular disrupting agent (VDA), combretastatin-A4-phosphate (CA4P), as detected by measuring changes in tumor metabolism of hyperpolarized [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate. These measurements were compared with dynamic contrast agent-enhanced magnetic resonance imaging (DCE-MRI) measurements of tumor vascular function and diffusion-weighted MRI (DW-MRI) measurements of the tumor cell necrosis that resulted from subsequent loss of tumor perfusion. The rate constant describing flux of hyperpolarized (13)C label between [1-(13)C]pyruvate and lactate was decreased by 34% within 6 hours of CA4P treatment and remained at this lower level at 24 hours. The rate constant describing production of labeled malate from hyperpolarized [1,4-(13)C(2)]fumarate increased 1.6-fold and 2.5-fold at 6 and 24 hours after treatment, respectively, and correlated with the degree of necrosis detected in histologic sections. Although DCE-MRI measurements showed a substantial reduction in perfusion at 6 hours after treatment, which had recovered by 24 hours, DW-MRI showed no change in the apparent diffusion coefficient of tumor water at 6 hours after treatment, although there was a 32% increase at 24 hours (P < 0.02) when regions of extensive necrosis were observed by histology. Measurements of hyperpolarized [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate metabolism may provide, therefore, a more sustained and sensitive indicator of response to a VDA than DCE-MRI or DW-MRI, respectively.

Multi-compound polarization by DNP allows simultaneous assessment of multiple enzymatic activities in vivo.

Methods for the simultaneous polarization of multiple 13C-enriched metabolites were developed to probe several enzymatic pathways and other physiologic properties in vivo, using a single intravenous bolus. A new method for polarization of 13C sodium bicarbonate suitable for use in patients was developed, and the co-polarization of 13C sodium bicarbonate and [1-(13)C] pyruvate in the same sample was achieved, resulting in high solution-state polarizations (15.7% and 17.6%, respectively) and long spin-lattice relaxation times (T1) (46.7 s and 47.7 s respectively at 3 T). Consistent with chemical shift anisotropy dominating the T1 relaxation of carbonyls, T1 values for 13C bicarbonate and [1-(13)C] pyruvate were even longer at 3 T (49.7s and 67.3s, respectively). Co-polarized 13C bicarbonate and [1-(13)C] pyruvate were injected into normal mice and a murine prostate tumor model at 3T. Rapid equilibration of injected hyperpolarized 13C sodium bicarbonate with 13C CO2 allowed calculation of pH on a voxel by voxel basis, and simultaneous assessment of pyruvate metabolism with cellular uptake and conversion of [1-(13)C] pyruvate to its metabolic products. Initial studies in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model demonstrated higher levels of hyperpolarized lactate and lower pH within tumor, relative to surrounding benign tissues and to the abdominal viscera of normal controls. There was no significant difference observed in the tumor lactate/pyruvate ratio obtained after the injection of co-polarized 13C bicarbonate and [1-(13)C] pyruvate or polarized [1-(13)C] pyruvate alone. The technique was extended to polarize four 13C labelled substrates potentially providing information on pH, metabolism, necrosis and perfusion, namely [1-(13)C]pyruvic acid, 13C sodium bicarbonate, [1,4-(13)C]fumaric acid, and 13C urea with high levels of solution polarization (17.5%, 10.3%, 15.6% and 11.6%, respectively) and spin-lattice relaxation values similar to those recorded for the individual metabolites. These studies demonstrated the feasibility of simultaneously measuring in vivo pH and tumor metabolism using nontoxic, endogenous species, and the potential to extend the multi-polarization approach to include up to four hyperpolarized probes providing multiple metabolic and physiologic measures in a single MR acquisition.

Lactate and adenosine triphosphate in the extender enhance the cryosurvival of rat epididymal sperm.

We evaluated the cryosurvival of rat epididymal sperm preserved in raffinose-modified Krebs-Ringer bicarbonate-egg yolk extender supplemented with various energy-yielding substrates (glucose, pyruvate, lactate, and ATP) and assessed the effect on sperm oxygen consumption. The incubation of sperm at 37 degrees C for 10 min in lactate-free extender decreased sperm motility and oxygen consumption before and after thawing compared with those of sperm in glucose- and pyruvate-free mediums. We then focused on the effect of supplementing the extender with lactate (0, 10.79, 21.58, 32.37, and 43.16 mM) and found that sperm frozen and thawed in extender supplemented with 32.37 mM lactate exhibited the highest motility. When we supplemented extender containing 32.37 mM lactate with ATP (0, 0.92, 1.85, 3.70, and 5.55 mM), sperm frozen and thawed in the extender supplemented with 1.85 mM ATP exhibited considerably higher motility and viability than those of sperm frozen and thawed in ATP-free extender. These results provide the first evidence that supplementation of the raffinose-modified Krebs-Ringer bicarbonate-egg yolk extender with 32.37 mM lactate and 1.85 mM ATP increases of number of motile sperm before freezing and enhances the cryosurvival of rat sperm. These supplements to the extender may enhance sperm cryosurvival by improving the metabolic capacity of sperm before freezing.

Characterization and evaluation of pigment distribution and response to therapy in melasma using in vivo reflectance confocal microscopy: a preliminary study.

BACKGROUND: Melasma is a frequent skin disorder characterized by the appearance of abnormal pigment (melanin) deposits in different layers of the skin. Melasma has been classified into epidermal, dermal and mixed types using Wood's lamp, and the type and extent of the pigment deposits determine the type and invasiveness of the treatment. AIMS: The aims of this study were to carry out a preliminary evaluation of the effective usefulness of reflectance confocal microscopy (RCM) in pigment distribution definition and subsequent re-classification of melasma types. Moreover, RCM therapeutical follow-up efficiency to combination therapy with pyruvic acid and hydroquinone was also tested. MATERIALS AND METHODS: A small group (n=15) of patients previously diagnosed with facial melasma were selected and their pigment distribution was evaluated by RCM. In seven of these patients therapeutical follow-up was performed. RESULTS: The results of the study suggest that RCM is more accurate than techniques previously used in the diagnosis of melasma, thus providing precise information on the location and extent of pigment deposits. DISCUSSION AND CONCLUSION: The non-invasive nature of this technique suggests that RCM may be a suitable tool for treatment monitoring, providing additional information not only on the evolution of the disorder but also on the possible occurrence of therapeutical side or adverse effects.

Effect of speed of rewarming and administration of anti-inflammatory or anti-oxidant agents on acute lung injury in an intestinal ischemia model treated with therapeutic hypothermia.

AIM OF THE STUDY: Acute lung injury (ALI) develops in various clinical situations and is associated with high morbidity and mortality and therapeutic hypothermia (HT) has been studied to attenuate the ALI. However, the optimal method of rewarming has not been determined. We determined the effect of speed of rewarming and the administration of anti-inflammatory or anti-oxidant agents on ALI in an intestinal ischemia and reperfusion (I/R) model treated with HT. MATERIALS AND METHODS: A Sprague-Dawley rat model of intestine ischemia and reperfusion was used. Two parallel animal experiments were conducted. In the survival study, rats (n=5 per group) underwent normothermic intestinal ischemia (60min, 36-38 degrees C) and then randomized into 7 groups with reperfusion: normothermia (NT), HT without rewarming (30-32 degrees C, HT), 2h HT+rewarming for 1h (RW1), 2h HT+rewarming for 2h (RW2), RW1+N-acetyl cysteine (RW-NAC), RW1+ethylpyruvate (RW-EP), and RW1+dexamethasone (RW+Dexa). In the second experiment, we investigated the histological and biochemical effects on the lung 4h after reperfusion (n=8 per group). RESULTS: The survival rate was lowest after NT. The HT, RW2, and RW-Dexa groups survived longer than the RW1, RW-NAC, and RW-EP groups. ALI scores were lower in the HT, RW2, and RW-Dexa groups than RW1. Lung malondialdehyde content was also lower in these groups. Interleukin (IL)-6 was significantly higher in the RW1 group. Inducible NO synthase gene expression in lung was lower in the HT, RW2, and RW-Dexa than RW1, and serum NO was lower in the RW2 and RW-Dexa than RW1. CONCLUSION: Gradual rewarming and administration of dexamethasone improved survival and attenuated ALI after intestinal I/R injury treated with HT in rats.