Energy-related Metabolites Research Articles

The effects of levosimendan on brain metabolism during initial recovery from global transient ischaemia/hypoxia

BackroundNeuroprotective strategies after cardiopulmonary resuscitation are currently the focus of experimental and clinical research. Levosimendan has been proposed as a promising drug candidate because of its cardioprotective properties, improved haemodynamic effects in vivo and reduced traumatic brain injury in vitro. The effects of levosimendan on brain metabolism during and after ischaemia/hypoxia are unknown.MethodsTransient cerebral ischaemia/hypoxia was induced in 30 male Wistar rats by bilateral common carotid artery clamping for 15 min and concomitant ventilation with 6% O2 during general anaesthesia with urethane. After 10 min of global ischaemia/hypoxia, the rats were treated with an i.v. bolus of 24 μg kg-1 levosimendan followed by a continuous infusion of 0.2 μg kg-1 min-1. The changes in the energy-related metabolites lactate, the lactate/pyruvate ratio, glucose and glutamate were monitored by microdialysis. In addition, the effects on global haemodynamics, cerebral perfusion and autoregulation, oedema and expression of proinflammatory genes in the neocortex were assessed.ResultsLevosimendan reduced blood pressure during initial reperfusion (72 ± 14 vs. 109 ± 2 mmHg, p = 0.03) and delayed flow maximum by 5 minutes (p = 0.002). Whereas no effects on time course of lactate, glucose, pyruvate and glutamate concentrations in the dialysate could be observed, the lactate/pyruvate ratio during initial reperfusion (144 ± 31 vs. 77 ± 8, p = 0.017) and the glutamate release during 90 minutes of reperfusion (75 ± 19 vs. 24 ± 28 μmol·L-1) were higher in the levosimendan group. The increased expression of IL-6, IL-1ß TNFα and ICAM-1, extend of cerebral edema and cerebral autoregulation was not influenced by levosimendan.ConclusionAlthough levosimendan has neuroprotective actions in vitro and on the spinal cord in vivo and has been shown to cross the blood–brain barrier, the present results showed that levosimendan did not reduce the initial neuronal injury after transient ischaemia/hypoxia.

Fluctuations in energy-related metabolites during the peri-parturition period in Lori-Bakhtiari ewes

This study describes the fluctuations in serum energy-related metabolites during a period of 2 weeks before, to 2 weeks after parturition in Lori-Bakhtiari ewes. The effect of parity was also studied. Blood profiles were determined in 60 healthy pregnant ewes with single (n=30) and twin (n=30) lambings. Blood was collected from each ewe on days 14 and 7 prepartum, and days 7 and 14 postpartum to determine the serum non-esterified fatty acid (NEFA), β-hydroxybutyrate (BHBA), glucose, cholesterol, blood urea nitrogen (BUN) and calcium (Ca) levels. The age of the ewes had no significant effect on the energy metabolism indicators. Serum NEFA, BHBA, glucose, BUN and calcium concentrations recorded peak levels 7 days before parturition. However, NEFA and BHBA recorded significant changes (P<0.05) during the peri-parturition period. All metabolites changed significantly in ewes carrying twin-bearing ewes, compared to single-bearing ewes. Serum BHBA concentrations recorded positive correlations with the serum NEFA (P<0.01) and cholesterol (P<0.05), while blood glucose had negative correlations (P<0.01) with NEFA, BHBA and Ca. Blood NEFA and BHBA recorded positive correlations (P<0.05) with the BUN levels and negative correlations (P<0.05) with Ca. The results showed that blood NEFA and BHBA levels are sensitive indicators of the energy balance during the peri-parturition period in ewes.

Visualization of Spatiotemporal Energy Dynamics of Hippocampal Neurons by Mass Spectrometry during a Kainate-Induced Seizure

We report the use of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry combined with capillary electrophoresis (CE) mass spectrometry to visualize energy metabolism in the mouse hippocampus by imaging energy-related metabolites. We show the distribution patterns of ATP, ADP, and AMP in the hippocampus as well as changes in their amounts and distribution patterns in a murine model of limbic, kainate-induced seizure. As an acute response to kainate administration, we found massive and moderate reductions in ATP and ADP levels, respectively, but no significant changes in AMP levels—especially in cells of the CA3 layer. The results suggest the existence of CA3 neuron-selective energy metabolism at the anhydride bonds of ATP and ADP in the hippocampal neurons during seizure. In addition, metabolome analysis of energy synthesis pathways indicates accelerated glycolysis and possibly TCA cycle activity during seizure, presumably due to the depletion of ATP. Consistent with this result, the observed energy depletion significantly recovered up to 180 min after kainate administration. However, the recovery rate was remarkably low in part of the data-pixel population in the CA3 cell layer region, which likely reflects acute and CA3-selective neural death. Taken together, the present approach successfully revealed the spatiotemporal energy metabolism of the mouse hippocampus at a cellular resolution—both quantitatively and qualitatively. We aim to further elucidate various metabolic processes in the neural system.

Variations of energy related biochemical metabolites during periparturition period in fat-tailed Baloochi breed sheep.

Negative energy balance in pregnant ewes during last two month of gestation could result in fatal pregnancy toxemia and affects ewe and lamb health. The understanding of variations of energy related metabolites during this critical period may cast light upon to a better management of the situation. This study describes the variations of serum energy related biochemical metabolites at a period of 45 days before to 21 days after parturition in Baloochi breed ewes. Blood from the jugular vein was collected from each sheep at days 45, 30, 20, 10, 1 prior to parturition, and days 4, 7, 13, 19 and 21 post-partum to determine total protein, albumin, urea , cholesterol, glucose, triglyceride, aspartate aminotransferase (AST), -Hydroxybutyrate (BHBA) and non-esterified fatty acid (NEFA). NEFA and BHBA concentrations reached peak levels at parturition. However, AST activity had highest levels at the first and second week after lambing. AST activity significantly correlated to energy metabolism indicators. This enzyme had positive significant correlations with NEFA and BHBA and negative correlations with cholesterol and triglyceride. NEFA concentrations had a positive correlation with BHBA and negative correlation with glucose and cholesterol. At pre-partum period NEFA correlation with BHBA and AST were stronger than post-partum. This finding indicates that NEFA testing at pre-parturition days is a reliable predictor of fat mobilization and energy status of pre-parturition period in sheep. Urea and total protein concentrations reached lowest levels at parturition and then gradually increased afterwards.

Lactate-to-Pyruvate Ratio as a Marker of Propofol Infusion Syndrome After Subarachnoid Hemorrhage

Propofol infusion syndrome (PRIS) is a rare but frequently fatal condition. It is characterized by cardiovascular collapse and metabolic derangement due to propofol exposure. The pathophysiology of PRIS is poorly understood, and its study has previously been limited to animal models and clinical observations. We present the first in vivo brain biochemical data in a patient with PRIS. We report the case of a 37-year-old woman with PRIS following aneurysmal subarachnoid hemorrhage who was monitored by cerebral microdialysis (CMD). A CMD catheter was inserted into the brain and provided near real-time monitoring of brain energy-related metabolites, including lactate and pyruvate, during the time period surrounding the diagnosis of PRIS. We recorded propofol exposure, clinical manifestations, and relevant laboratory measurements. CMD revealed a temporal association between propofol exposure and the cerebral lactate-to-pyruvate ratio (LPR). The LPR increased linearly after propofol was restarted following an off period, and the LPR decreased linearly after propofol was discontinued. Serum lactate correlated with clinical worsening after the onset of PRIS, whereas cerebral LPR correlated with propofol exposure. Cerebral LPR may be a sensitive marker of PRIS. Increases in LPR following propofol exposure should alert clinicians to the possibility of PRIS and might prompt early discontinuation of propofol thereby avoiding fatal complications.

Seasonal Changes in Energy-related Blood Metabolites and Mineral Profiles of Nguni and Crossbred Cattle on Communal Rangelands in the Eastern Cape, South Africa

The objective of the current study was to determine seasonal changes in glucose, cholesterol, non-esterified fatty acids (NEFA), serum inorganic phosphorous (SIP), calcium, magnesium and iron concentrations in Nguni and crossbred cattle on the sweet and sour rangelands of the Eastern Cape Province of South Africa. Body weights, body condition scores (BCS) and serum concentrations of energy-related metabolites and mineral profiles were determined in late cool-dry, hot-dry, hot-wet, post-rainy and early cool-dry seasons in 100 cattle raised on communal rangelands from August 2007 to May 2008. Nguni cattle had lower (p<0.05) and higher (p<0.05) serum concentrations of glucose in the hot-wet and post-rainy seasons, respectively, compared to crossbreds in the same seasons. Serum cholesterol and NEFA concentrations in Nguni were lower (p<0.05) than in the crossbreds. Nguni and crossbred cattle had higher (p<0.05) serum NEFA concentrations on the sweet rangeland during the late cool-dry season than on sour rangeland. Nguni cattle had higher (p<0.05) SIP concentration in the hot-wet season than the crossbreds. Generally, both breeds had lowest SIP concentration during the hot-wet season on the sour rangeland. The lowest magnesium and highest iron concentrations were observed in the hot-wet and post-rainy seasons, respectively, compared to other seasons. Cattle on the sour rangeland had lower (p<0.05) iron concentrations than those on the sweet rangeland. It was concluded that Nguni cattle had lower cholesterol and NEFA, and higher SIP concentrations in the hot-wet season than crossbreds and energy deficits mostly occurred during the late cool-dry season on the sweet rangeland.

Highlights of This Issue

Inhibition of DNA polymerase β (Polβ) enhances the cytotoxicity of chemotherapeutic DNA-damaging agents. To define the mechanism of cell death resulting from Polβ inhibition, Tang and colleagues depleted human cells of Polβ and demonstrate that unrepaired base excision repair (BER) intermediates elicit hyperactivation of poly(ADP) ribose synthesis causing cell death via depletion of NAD+ and other energy-related metabolites. This study implicates a linkage between BER, cell survival, and cellular bioenergetics after exposure to chemotherapeutic alkylating agents and has significant implications for therapeutic development of DNA repair and NAD+ biosynthesis inhibitors.The CXCR4 and CXCR7 receptors that bind prometastatic α-chemokine SDF- 1 were cloned and their activities were analyzed in a model of less metastatic human embryonal-rhabdomyosarcoma cell line (RD) and more metastatic alveolar-like rhabdomyosarcoma (RD cells transduced with PAX3-FKHR fusion gene). While the expression of CXCR4 required NRF-1 binding site and was additionally upregulated by direct interaction of NRF-1 with PAX3-FKHR, CXCR7 promoter activity required a proximal NF-κB binding motif. Moreover, while hypoxia enhanced expression of both receptors on RD cells, it inhibited CXCR7 expression in RD/PAX3-FKHR cells. Thus, the change in CXCR4/CXCR7 ratio by PAX3-FKHR or hypoxia modulates rhabdomyosarcoma metastasis.Thyroid hormone receptors (TR) play important roles in normal physiology and development, whereas dominant-negative TR mutants are associated with certain neoplasia, including human hepatocellular carcinoma (HCC). Chan and Privalsky report that the dominant-negative properties of two HCC-TR mutants map to lesions at lysine 74, an allosteric sensor that, in wild-type TRs, couples DNA binding to transcriptional regulation. These results reveal a mutational hot spot by which TRs can acquire dominant-negative activity, and help improve our understanding of how wild-type and mutant nuclear receptors recognize DNA so as to confer gene repression or activation.Because the activation of STAT3 mediates proliferation and metastasis of numerous human cancers, including multiple myleoma, inhibitors of this pathway are highly desirable. Sandur and colleagues report the identification of a compound plumbagin from an Ayurvedic plant Chitrak (Plumbago zeylanica) that can suppress the activation of STAT3 through the induction of expression of a protein tyrosine phosphatase that specifically dephosphorylates STAT3. Additionally, plumbagin inhibits the activation of upstream kinases c-Src, JAK1, and JAK2. This kind of promiscuous mechanism of action of plumbagin is highly desirable for its potential for treatment of cancer.

Extracellular Metabolite Biomarkers of Bortezomib Resistance in Multiple Myeloma Indicate Involvement of Unexpected Metabolic Pathways.

Abstract 1839Poster Board I-865 Introduction and Objectives:Bortezomib (BZ) is a chemotherapeutic agent approved for the treatment of multiple myeloma (MM). BZ acts through proteasome inhibition, inducing significant ER stress and ideally resulting in cell death. Unfortunately, nearly 20% of MM patients are primarily resistant to BZ treatment and responses to BZ are difficult to predict based on the currently available clinical, cytogenetic and genomic biomarkers. Our function hypothesis is that extracellular metabolites have a greater potential to be found in circulating biofluids as biomarkers. As a result we used a metabolite ‘footprinting' approach in cell growth media to examine the metabolic consequences of BZ treatment using eight human MM cell lines, three of which have been determined to be less sensitive to BZ treatment than the others with a 10 fold difference in their IC50 at 24 hours (5 nM vs 50 nM). Our aims were 1) to establish whether analysis of growth media was suitable for monitoring metabolic changes and 2) to determine specific biopatterns of BZ resistance. Methods:Eight MM cell lines (MM1S, MM1R, INA6, U266, RPMI8266, OPM2, KMS11 and PCL1) were cultured under standard conditions without (control group, 10% FBS) or with bortezomib added (10nM). Media samples were taken for metabolic analysis at 6 and 24 hours for a total of 32 media profiles. Metabolite profiling was accomplished using gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). GC-MS data was analysed using AMDIS (NIST), and NMR data using Chenomx NMR Suite. Significant metabolites were identified using multivariate regression analysis by supervised projection methods (two-way orthogonal partial least squares discriminant analysis, O2PLS-DA) using SIMCA-P (Umetrics). Results:An average of 756 chemical or metabolite components per sample were profiled, which was reduced to a subset of 116 unique features that were shared in at least 75% of samples. An initial O2PLS-DA model was successfully built from the GC-MS feature set using both growth time (p=0.03) and BZ status (p=6.9e-13) in the Y-matrix. Figure 1 shows a scores plot, where each point represents a single sample, and the position is calculated as a combination of the underlying metabolite concentrations. Changes in cell growth were consistent with the known uptake of carbohydrate substrates and elimination of various amino acids and waste products such as lactate. The remarkable metabolic difference between BZ-treated and untreated cells resulted from reduced energy-related metabolites such as citric acid cycle intermediates and sugars, with a concomitant increase in selected amino acids. Intriguingly, the BZ-insensitive cell cultures exhibit overall metabolic phenotypes much more similar to the BZ-sensitive cultures than to the untreated group, with the exception of a single sample after 6 hours (denoted with an asterisk in Figure 1) which showed an averaged profile. To further probe the phenomenon of BZ resistance, the treated group was analyzed independently, with the 37 most influential components providing discriminating ability between the BZ-insensitive and BZ-sensitive cells (p= 0.04) in an OPLS-DA model. Conclusions:We conclude that metabolite footprinting is a reliable and robust method for monitoring metabolic events for both cell growth and BZ treatment. Furthermore, BZ-insensitivity is accompanied by a notable shift from carbohydrate metabolism to fatty acid metabolism, while the overall metabolic phenotype remains very similar in both BZ-sensitive and insensitive strains in the presence of the drug. This result suggests that BZ function remains largely intact in both sensitive and insensitive cell lines, and resistance is conferred through alternate mechanisms with measureable metabolic endpoints. Success in measuring extracellular metabolites also supports the notion of serum-accessible biomarkers or biopatterns of BZ resistance. The unique genetic instability underlying each cell line may provide a further avenue for characterizing resistance mechanisms along with analysis of various intracellular components. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Effects of anion supplementation to low-potassium prepartum diets on macromineral status and performance of periparturient dairy cows

Data from multiparous Holstein cows (n=43) were used to determine whether supplementation of anions to low-potassium (K) prepartum diets would improve periparturient energy and macromineral status and affect performance during the postpartum period. Beginning 21 d before expected parturition, cows were fed a control diet (1.29% K; +10mEq/100g; n=21) or a low dietary cation-anion difference (DCAD) diet (1.29% K; −15mEq/100g; n=22) with anions provided through a combination of sulfate from calcium sulfate dihydrate (0.40% S total ration) and chloride (1.17% Cl total ration) from SoyChlor 16–7 (West Central, Ralston, IA). All cows were fed the same postpartum diet from parturition through 63 d postpartum. Feeding anions decreased overall urine pH (8.17 vs. 6.70) during the prepartum period. Overall, peripartum concentrations of plasma Ca, P, and Mg were similar between treatments; however, concentrations of plasma Ca tended to be increased during the first 24h postcalving in cows fed the low DCAD diet. Overall, concentrations of plasma P tended to be increased by feeding the anionic diet prepartum; this effect was more pronounced during the immediate peripartal period. Anionic supplementation did not affect incidence of clinical (<5mg/dL) and subclinical (5 to 8mg/dL) hypocalcemia, clinical hypophosphatemia (<2mg/dL), or clinical (<1.1mg/dL) and subclinical (1.1 to 1.8mg/dL) hypomagnesemia. Nevertheless, subclinical hypophosphatemia (2 to 4mg/dL) tended to be decreased at 16h postcalving and was decreased at d 2 postpartum for cows fed the anionic diet prepartum. Anion supplementation decreased prepartum dry matter intake (15.6 vs. 14.4kg/d), but did not affect postpartum dry matter intake (22.4 vs. 23.0kg/d), milk yield (46.5 vs. 46.1kg/d), or content and yield of milk fat and true protein. Plasma concentrations of energy-related metabolites (glucose, nonesterified fatty acids, β-hydroxybutyrate) were similar for both groups during the prepartum and postpartum periods. Glucose rate of appearance was determined by continuous infusion of 6,6-dideuterated glucose in a subset of cows between 6 and 10 d prepartum (control, n=12; low DCAD, n=9) and 7 and 10 d postpartum (control, n=9; low DCAD, n=8) periods. Glucose rate of appearance was not affected by treatment during the prepartum or postpartum periods. Overall, anion supplementation of low K diets improved P status during the early postpartum period, but did not affect aspects of energy metabolism or periparturient performance.

Effect of yeast culture on milk production and metabolic and reproductive performance of early lactation dairy cows

BackgroundThe main objective of this study was to estimate the effect of supplementation with Saccaromyces cerevisiae (SC) (Yea-Sacc® 1026) on milk production, metabolic parameters and the resumption of ovarian activity in early lactation dairy cows.MethodsThe experiment was conducted during 2005/2006 in a commercial tied-house farm with an average of 200 milking Estonian Holstein Friesian cows. The late pregnant multiparous cows (n = 46) were randomly divided into two groups; one group received 10 g yeast culture from two weeks before to 14 weeks after calving. The groups were fed a total mixed ration with silages and concentrates. Milk recording data and blood samples for plasma metabolites were taken. Resumption of luteal activity was determined using milk progesterone (P4) measurements. Uterine bacteriology and ovarian ultrasonography (US) were performed and body condition scores (BCS) and clinical disease occurrences were recorded. For analysis, the statistical software Stata 9.2 and R were used to compute Cox proportional hazard and linear mixed models.ResultsThe average milk production per cow did not differ between the groups (32.7 ± 6.4 vs 30.7 ± 5.3 kg/day in the SC and control groups respectively), but the production of milk fat (P < 0.001) and milk protein (P < 0.001) were higher in the SC group. There was no effect of treatment on BCS. The analysis of energy-related metabolites in early lactation showed no significant differences between the groups. In both groups higher levels of β-hydroxybutyrate (BHB) appeared from days 14 to 28 after parturition and the concentration of non-esterfied fatty acid (NEFA) was higher from days 1–7 post partum (PP). According to US and P4 results, all cows in both groups ovulated during the experimental period. The resumption of ovarian activity (first ovulations) and time required for elimination of bacteria from the uterus did not differ between the groups.ConclusionSupplementation with SC had an effect on milk protein and fat production, but did not influence the milk yield. No effects on PP metabolic status, bacterial elimination from the uterus nor the resumption of ovarian activity were found.

Metabolic profiling of a Schistosoma mansoni infection in mouse tissues using magic angle spinning-nuclear magnetic resonance spectroscopy

In order to enhance our understanding of physiological and pathological consequences of a patent Schistosoma mansoni infection in the mouse, we examined the metabolic responses of different tissue samples recovered from the host animal using a metabolic profiling strategy. Ten female NMRI mice were infected with ∼80 S. mansoni cercariae each, and 10 uninfected age- and sex-matched animals served as controls. At day 74 post infection (p.i.), mice were killed and jejunum, ileum, colon, liver, spleen and kidney samples were removed. We employed 1H magic angle spinning-nuclear magnetic resonance spectroscopy to generate tissue-specific metabolic profiles. The spectral data were analyzed using multivariate modelling methods including an orthogonal signal corrected-projection to latent structure analysis and hierarchical principal component analysis to assess the differences and/or similarities in metabolic responses between infected and non-infected control mice. Most tissues obtained from S. mansoni-infected mice were characterized by high levels of amino acids, such as leucine, isoleucine, lysine, glutamine and asparagine. High levels of membrane phospholipid metabolites, including glycerophosphoryl choline and phosphoryl choline were found in the ileum, colon, liver and spleen of infected mice. Additionally, low levels of energy-related metabolites, including lipids, glucose and glycogen were observed in ileum, spleen and liver samples of infected mice. Energy-related metabolites in the jejunum, liver and renal medulla were found to be positively correlated with S. mansoni worm burden upon dissection. These findings show that a patent S. mansoni infection causes clear disruption of metabolism in a range of tissues at a molecular level, which can be interpreted in relation to the previously reported signature in a biofluid (i.e. urine), giving further evidence of the global effect of the infection.

Evaluation of Milk Enzymes and Electrolytes, Plasma Metabolites, and Oxidative Status in Twin Cows Milked in an Automatic Milking System or Twice Daily in a Conventional Milking Parlor

The aim of this paper was to evaluate the effects of automatic milking (AM) on milk enzymes and minerals related to mammary epithelial integrity in comparison with twice-daily conventional milking (CM). One cow from each of 6 pairs of twins was assigned to be milked with AM or with CM throughout first lactation. Milk production was recorded and milk samples were collected at 4, 11, 18, 25, 32, and 39 wk of lactation (WOL) to determine fat and protein content, somatic cell count, pH, plasminogen (pl) and plasmin (Pl) activities, Na, K, and Cl. Body condition score was monitored; blood samples were collected to determine energy-related metabolites in the first third of lactation (14 WOL), and plasma oxidative status throughout lactation. Overall mean and standard deviation of milking frequency (MF) in AM were 2.69 and 0.88, respectively. Milk production, fat and protein contents, and somatic cell count did not differ between milking systems. The pl and pl+Pl activities were lesser in AM than in CM. Milk pH was greater in AM than in CM. Milk Na, K, Na/K ratio, and Cl did not differ across the whole lactation. Milk pH had a positive correlation with milk Pl activity (r=0.41), Na (r=0.37), and Cl (r=0.40) concentration, and negative correlation with the log10 of pl/Pl ratio (r=−0.47). The milk Na/K ratio had a positive correlation (r=0.55) with milk Pl activity. Milking system (MS) did not seem to affect mammary epithelial permeability. The differences in enzymatic (proteolytic) activity due to the MS, probably related to daily MF, lead one to suppose that the quality of the protein fraction for the cheese-making process was preserved better with AM than with CM, even if differences in pH might negatively interfere. No difference was detected in BCS, and in plasma concentration of triglycerides and nonesterified fatty acids, whereas plasma cholesterol concentration during the first 10 WOL was lesser in AM than CM. Oxidative status, measured by plasma reactive oxygen metabolites and thiol groups, did not differ between MS throughout the whole lactation. These results suggest that early lactation of AM primiparous cows may give rise to crucial situations: for milk production, when a low MF may impair further mammary cell proliferation; for milk quality, if an irregular MF, with prolonged milking intervals, leads to an increased milk pH with increased conversion of pl to Pl.

Metabonomics evaluation of urine from rats given acute and chronic doses of acetaminophen using NMR and UPLC/MS

Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously. The principal component analysis (PCA) of NMR or UPLC/MS spectra showed that metabolic changes observed in both acute and chronic dosing of acetaminophen were similar. Histopathology and clinical chemistry studies were performed and correlated well with the PCA analysis and magnitude of metabolite changes. Depletion of antioxidants (e.g. ferulic acid), trigonelline, S-adenosyl- l-methionine, and energy-related metabolites indicated that oxidative stress was caused by acute and chronic acetaminophen administration. Similar patterns of metabolic changes in response to acute or chronic dosing suggest similar detoxification and recovery mechanisms following APAP administration.

AMPK and ACC phosphorylation: Effect of leptin, muscle fibre type and obesity

Leptin stimulates fatty acid oxidation via the phosphorylation of AMPK (AMP-activated protein kinase) and ACC (acetyl-CoA carboxylase). Obesity is associated with resistance to the effects of leptin. We determined the action of leptin on AMPKalpha and ACCbeta phosphorylation and lipid metabolism in soleus (SOL) and extensor digitorum longus (EDL) muscles from lean and obese Wistar rats after 1 and 100 nM leptin. Both leptin doses stimulated phosphorylation of AMPKalpha and ACCbeta (P<or=0.05) only in EDL muscles from lean animals. Malonyl-CoA levels were decreased in EDL muscles from lean animals after 1 and 100 nM leptin and significantly after 100 nM leptin in obese animals (P<or=0.05). Long-chain fatty acyl-CoA concentrations were decreased in EDL muscles from both phenotypes after 100 nM leptin. AMPK activation by leptin occurred independently of energy-related metabolites. These data demonstrate that the leptin effect on AMPKalpha and ACCbeta is muscle fibre type dependent and fails in diet-induced obesity.

Brain Metabolism During a Decrease in Cerebral Perfusion Pressure Caused by an Elevated Intracranial Pressure in the Porcine Neocortex

Cranial hypertension coincides with a reduction in cerebral blood flow as well as in oxygen delivery and influences outcome. In this study, we monitored changes in energy-related metabolites in the porcine cortex during an increase of intracranial pressure (ICP) and to determine the level at which damage occurs. Male domestic pigs (32-40 kg) were anesthetized, mechanically ventilated, and randomly assigned to either the experimental (n = 6) or control groups (n = 5). A microdialysis probe (CMA 70) was inserted into the cortex to measure extracellular dialysate concentrations of lactate, pyruvate, glucose, glutamate, and glycerol. Every hour an increase of 10 mm Hg in ICP was preformed in the experimental group by infusion of artificial cerebrospinal fluid into the ventricular system of the brain until a maximum ICP of 50 mm Hg was reached. We demonstrated a significant increase of lactate and glycerol compared with control at ICP values > or =30 mm Hg and cerebral perfusion pressure (CPP) below 50 mm Hg. The increase of ICP to > or =40 mm Hg in conjunction with a reduction in CPP below 40 mm Hg led to a significant increase in the lactate/pyruvate-ratio and glutamate, as well as a decrease of glucose in relation to control. Our data strongly suggest that, during a defined ICP increase, lower CPP values may be tolerable until severe damage occurs. Borderline ICP and CPP values of 30 and 40 mm Hg, respectively, could be advised.

Variations of energy-related biochemical metabolites during transition period in dairy cows

This study describes the variation of serum biochemical metabolites during transition period in dairy cows. Blood samples were obtained on days 22 and 8 prepartum and days 7 and 21 postpartum (pp), to determine total protein, albumin, blood urea nitrogen, cholesterol, magnesium, inorganic phosphorus, calcium, glucose, triglyceride, aspartate aminotransferase (AST), β-hydroxybutyrate (BHBA), and non-esterified fatty acid (NEFA). NEFA concentrations during the pre-fresh period had a positive correlation with triglyceride, BHBA, and AST during post-calving days. This finding indicates that NEFA testing during the last days of a dry period is a reliable predictor of fat mobilization and energy status during this transition period. Serum glucose showed no strong correlations with other energy-related metabolites. AST was significantly correlated to triglyceride, BHBA, NEFA, cholesterol, and glucose at day 8 pp. AST also showed significant correlation with triglyceride concentrations at days −8 and +8 relating to parturition. In addition, AST was significantly correlated to BHBA and NEFA at day 21 pp.

Inhibition of Nitric Oxide Synthase with 7-Nitroindazole does not Modify Early Metabolic Recovery Following Focal Cerebral Ischemia in Rats

Nitric oxide has been strongly implicated in the development of tissue infarction in response to focal cerebral ischemia. Nitric oxide and its derivatives can inhibit components of the electron transport chain, providing a likely target for these substances in ischemic and post-ischemic brain. Lactate content is increased during post-ischemic reperfusion in tissue destined to become infarcted, consistent with impairment of mitochondrial respiration. To investigate the possible involvement of nitric oxide in generating these changes, we have tested the effect of 7-nitroindazole, a nitric oxide synthase (NOS) inhibitor, on the content of lactate and other metabolites during early reperfusion following temporary focal ischemia. This treatment inhibited total NOS by approximately 50%. However, the treatment did not significantly affect the marked increases in lactate in post-ischemic brain nor did it alter the recovery of other energy-related metabolites. These findings indicate that inhibition of oxidative metabolism is probably not the primary site of the deleterious effects of nitric oxide and derivatives during early post-ischemic reperfusion.

Brain metabolism and extracellular space diffusion parameters during and after transient global hypoxia in the rat cortex

Hypoxia results in both reversible and irreversible changes in the brain extracellular space (ECS). This study utilized microdialysis to monitor changes in the energy-related metabolites lactate, pyruvate, glucose and glutamate in the rat cortex before, during and after 30-min transient global hypoxia, induced in anesthetized rats by reducing inspired oxygen to 6% O 2 in nitrogen. Changes in metabolite levels were compared with ECS diffusion parameters calculated from diffusion curves of tetramethylammonium applied by iontophoresis. Significant increases in lactate concentration and the lactate/pyruvate ratio, as well as decreased glucose levels, were found in the cortex immediately after the induction of hypoxia. Following recovery to ventilation with air, extracellular lactate and glucose levels and the lactate/pyruvate ratio returned to control levels within 40, 20 and 30 min, respectively. Glutamate levels started to increase 20–30 min after the onset of hypoxia and returned to prehypoxic values within 30–40 min of reoxygenation. The ECS volume fraction α decreased by about 5% from 0.18 ± 0.01 during the first 20–25 min of hypoxia; after 25 min α dropped a further 22% to 0.14 ± 0.01. Within 10 min of reoxygenation, α returned to control values, then increased to 0.20 ± 0.01 and remained at this level until the end of the experiment. The observed 22% decrease in α markedly influences dialysate levels measured during hypoxia. In our study, the complete posthypoxic recovery of cortical metabolite levels and ECS diffusion properties suggests that metabolic enzymes and related cellular components (e.g., mitochondria) may tolerate prolonged hypoxic periods and recover to prehypoxic values.

Changes in diffusion parameters, energy-related metabolites and glutamate in the rat cortex after transient hypoxia/ischemia

It has been shown that global anoxia leads to dramatic changes in the diffusion properties of the extracellular space (ECS). In this study, we investigated how changes in ECS volume and geometry in the rat somatosensory cortex during and after transient hypoxia/ischemia correlate with extracellular concentrations of energy-related metabolites and glutamate. Adult male Wistar rats ( n = 12) were anesthetized and subjected to hypoxia/ischemia for 30 min (ventilation with 10% oxygen and unilateral carotid artery occlusion). The ECS diffusion parameters, volume fraction and tortuosity, were determined from concentration–time profiles of tetramethylammonium applied by iontophoresis. Concentrations of lactate, glucose, pyruvate and glutamate in the extracellular fluid (ECF) were monitored by microdialysis ( n = 9). During hypoxia/ischemia, the ECS volume fraction decreased from initial values of 0.19 ± 0.03 (mean ± S.E.M.) to 0.07 ± 0.01 and tortuosity increased from 1.57 ± 0.01 to 1.88 ± 0.03. During reperfusion the volume fraction returned to control values within 20 min and then increased to 0.23 ± 0.01, while tortuosity only returned to original values (1.53 ± 0.06). The concentrations of lactate and glutamate, and the lactate/pyruvate ratio, substantially increased during hypoxia/ischemia, followed by continuous recovery during reperfusion. The glucose concentration decreased rapidly during hypoxia/ischemia with a subsequent return to control values within 20 min of reperfusion. We conclude that transient hypoxia/ischemia causes similar changes in ECS diffusion parameters as does global anoxia and that the time course of the reduction in ECS volume fraction correlates with the increase of extracellular concentration of glutamate. The decrease in the ECS volume fraction can therefore contribute to an increased accumulation of toxic metabolites, which may aggravate functional deficits and lead to damage of the central nervous system (CNS).

Welfare Assessment Based on Metabolic and Endocrine Aspects in Primiparous Cows Milked in a Parlor or with an Automatic Milking System

An automatic milking system (AMS) was compared with a traditional milking parlor (MP) to evaluate metabolic and psycho-physiological aspects of animal welfare. Twenty Italian Friesian heifers were allocated to 2 groups of 10 cows each after calving and maintained in the same free-stall barn. The first group was milked twice daily in a MP; the second group was milked in a single box AMS. Feed and diet characteristics were analyzed. Health status and body condition score (BCS) were evaluated in each cow. Blood samples were obtained from −14 to 154 d in milk (DIM) to determine metabolic profile and basal concentrations of cortisol in plasma. Data collected from 10 cows per group were processed. No significant difference was detected in milk yield, BCS, and energy-related metabolites (glucose, nonesterified fatty acids, β-hydroxybutyrate, and triglycerides) from cows in MP or in AMS during the first 22 wk of lactation. These results, jointly with the absence of significant differences in plasma metabolites related to protein metabolism, mineral metabolism, and liver function during the first 22 wk of lactation, indicates that cows in AMS did not suffer metabolically. Greater basal concentrations of plasma cortisol in AMS cows, even if absolute values were considered to be in an acceptable range, might indicate chronic stress in these primiparous cows. Further research is necessary to confirm this hypothesis.