In vivo detection of brain Krebs cycle intermediate by hyperpolarized magnetic resonance.

The Krebs (or tricarboxylic acid (TCA)) cycle has a central role in the regulation of brain energy regulation and metabolism, yet brain TCA cycle intermediates have never been directly detected in vivo. This study reports the first direct in vivo observation of a TCA cycle intermediate in intact brain, namely, 2-oxoglutarate, a key biomolecule connecting metabolism to neuronal activity. Our observation reveals important information about in vivo biochemical processes hitherto considered undetectable. In particular, it provides direct evidence that transport across the inner mitochondria membrane is rate limiting in the brain. The hyperpolarized magnetic resonance protocol designed for this study opens the way to direct and real-time studies of TCA cycle kinetics.

Soybean (Glycine max. L.) and bacteroid glyoxylate cycle activities during nodular senescence.

Soybean (Glycine max. L.) nodular senescence results in the dismantling of the peribacteroid membrane (PBM) and in an increase of soybean isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2) mRNA and protein levels. This suggests that in senescing soybean nodular cells, the specific glyoxylate cycle enzyme activities might be induced to reallocate carbon obtained from the PBM degradation. In order to evaluate as well the carbon metabolism of the nitrogen-fixing Bradyrhizobium japonicum endosymbiotic bacteroids during nodular senescence, their glyoxylate cycle activities were also investigated. To this end, partial DNA sequences were isolated from their icl and ms genes, but the corresponding mRNAs were not detected in the microorganisms. It was also observed that the bacteroid ICL and MS activities were negligible during nodular senescence. This suggests that glyoxylate cycle activities are not reinitiated in the bacteroids under these physiological conditions. In case the microorganisms nevertheless feed on the PBM degradation products, this might occur via the citric acid cycle exclusively.

Soybean (Glycine max. L.) and bacteroid glyoxylate cycle activities during nodular senescence

Soybean (Glycine max. L.) nodular senescence results in the dismantling of the peribacteroid membrane (PBM) and in an increase of soybean isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2) mRNA and protein levels. This suggests that in senescing soybean nodular cells, the specific glyoxylate cycle enzyme activities might be induced to reallocate carbon obtained from the PBM degradation. In order to evaluate as well the carbon metabolism of the nitrogen-fixing Bradyrhizobium japonicum endosymbiotic bacteroids during nodular senescence, their glyoxylate cycle activities were also investigated. To this end, partial DNA sequences were isolated from their icl and ms genes, but the corresponding mRNAs were not detected in the microorganisms. It was also observed that the bacteroid ICL and MS activities were negligible during nodular senescence. This suggests that glyoxylate cycle activities are not reinitiated in the bacteroids under these physiological conditions. In case the microorganisms nevertheless feed on the PBM degradation products, this might occur via the citric acid cycle exclusively.

Direct noninvasive estimation of myocardial tricarboxylic acid cycle flux in vivo using hyperpolarized (13)C magnetic resonance.

BACKGROUND: The heart relies on continuous energy production and imbalances herein impair cardiac function directly. The tricarboxylic acid (TCA) cycle is the primary means of energy generation in the healthy myocardium, but direct noninvasive quantification of metabolic fluxes is challenging due to the low concentration of most metabolites. Hyperpolarized (13)C magnetic resonance spectroscopy (MRS) provides the opportunity to measure cellular metabolism in real time in vivo. The aim of this work was to noninvasively measure myocardial TCA cycle flux (VTCA) in vivo within a single minute. METHODS AND RESULTS: Hyperpolarized [1-(13)C]acetate was administered at different concentrations in healthy rats. (13)C incorporation into [1-(13)C]acetylcarnitine and the TCA cycle intermediate [5-(13)C]citrate was dynamically detected in vivo with a time resolution of 3s. Different kinetic models were established and evaluated to determine the metabolic fluxes by simultaneously fitting the evolution of the (13)C labeling in acetate, acetylcarnitine, and citrate. VTCA was estimated to be 6.7±1.7μmol·g(-1)·min(-1) (dry weight), and was best estimated with a model using only the labeling in citrate and acetylcarnitine, independent of the precursor. The TCA cycle rate was not linear with the citrate-to-acetate metabolite ratio, and could thus not be quantified using a ratiometric approach. The (13)C signal evolution of citrate, i.e. citrate formation was independent of the amount of injected acetate, while the (13)C signal evolution of acetylcarnitine revealed a dose dependency with the injected acetate. The (13)C labeling of citrate did not correlate to that of acetylcarnitine, leading to the hypothesis that acetylcarnitine formation is not an indication of mitochondrial TCA cycle activity in the heart. CONCLUSIONS: Hyperpolarized [1-(13)C]acetate is a metabolic probe independent of pyruvate dehydrogenase (PDH) activity. It allows the direct estimation of VTCA in vivo, which was shown to be neither dependent on the administered acetate dose nor on the (13)C labeling of acetylcarnitine. Dynamic (13)C MRS coupled to the injection of hyperpolarized [1-(13)C]acetate can enable the measurement of metabolic changes during impaired heart function.

Citrate- vs. acetate-based dialysate in bicarbonate haemodialysis: consequences on haemodynamics, coagulation, acid-base status, and electrolytes.

BACKGROUND: A concentrate for bicarbonate haemodialysis acidified with citrate instead of acetate has been marketed in recent years. The small amount of citrate used (one-fifth of the concentration adopted in regional anticoagulation) protects against intradialyser clotting while minimally affecting the calcium concentration. The aim of this study was to compare the impact of citrate- and acetate-based dialysates on systemic haemodynamics, coagulation, acid-base status, calcium balance and dialysis efficiency. METHODS: In 25 patients who underwent a total of 375 dialysis sessions, an acetate dialysate (A) was compared with a citrate dialysate with (C+) or without (C) calcium supplementation (0.25 mmol/L) in a randomised single-blind cross-over study. Systemic haemodynamics were evaluated using pulse-wave analysis. Coagulation, acid-base status, calcium balance and dialysis efficiency were assessed using standard biochemical markers. RESULTS: Patients receiving the citrate dialysate had significantly lower systolic blood pressure (BP) (-4.3 mmHg, p < 0.01) and peripheral resistances (PR) (-51 dyne.sec.cm-5, p < 0.001) while stroke volume was not increased. In hypertensive patients there was a substantial reduction in BP (-7.8 mmHg, p < 0.01). With the C+ dialysate the BP gap was less pronounced but the reduction in PR was even greater (-226 dyne.sec.cm-5, p < 0.001). Analyses of the fluctuations in PR and of subjective tolerance suggested improved haemodynamic stability with the citrate dialysate. Furthermore, an increase in pre-dialysis bicarbonate and a decrease in pre-dialysis BUN, post-dialysis phosphate and ionised calcium were noted. Systemic coagulation activation was not influenced by citrate. CONCLUSION: The positive impact on dialysis efficiency, acid-base status and haemodynamics, as well as the subjective tolerance, together indicate that citrate dialysate can significantly contribute to improving haemodialysis in selected patients.

High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements.

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart (125 mmx4 mm I.D.) Nucleosil 100-5 microm C18AB cartridge column, using a gradient elution of MeOH-buffer pH 3.9 1:99-->15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate-citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H3PO4 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2+/-2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (ClR) and systemic (Cls) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney ClR/ClS calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73+/-0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87+/-0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.

Compartmentalized Cerebral Metabolism of [1,6-(13)C]Glucose Determined by in vivo (13)C NMR Spectroscopy at 14.1 T.

Cerebral metabolism is compartmentalized between neurons and glia. Although glial glycolysis is thought to largely sustain the energetic requirements of neurotransmission while oxidative metabolism takes place mainly in neurons, this hypothesis is matter of debate. The compartmentalization of cerebral metabolic fluxes can be determined by (13)C nuclear magnetic resonance (NMR) spectroscopy upon infusion of (13)C-enriched compounds, especially glucose. Rats under light α-chloralose anesthesia were infused with [1,6-(13)C]glucose and (13)C enrichment in the brain metabolites was measured by (13)C NMR spectroscopy with high sensitivity and spectral resolution at 14.1 T. This allowed determining (13)C enrichment curves of amino acid carbons with high reproducibility and to reliably estimate cerebral metabolic fluxes (mean error of 8%). We further found that TCA cycle intermediates are not required for flux determination in mathematical models of brain metabolism. Neuronal tricarboxylic acid cycle rate (V(TCA)) and neurotransmission rate (V(NT)) were 0.45 ± 0.01 and 0.11 ± 0.01 μmol/g/min, respectively. Glial V(TCA) was found to be 38 ± 3% of total cerebral oxidative metabolism, accounting for more than half of neuronal oxidative metabolism. Furthermore, glial anaplerotic pyruvate carboxylation rate (V(PC)) was 0.069 ± 0.004 μmol/g/min, i.e., 25 ± 1% of the glial TCA cycle rate. These results support a role of glial cells as active partners of neurons during synaptic transmission beyond glycolytic metabolism.

Insights into neuronal cell metabolism using NMR spectroscopy: uridyl diphosphate N-acetyl-glucosamine as a unique metabolic marker.

Making the switch: Compounds 1 and 2 are used as metabolic markers for NMR detection. When neuronal cells switch to a glycolytic state, an uneven distribution of (13) C in the N-acetyl group results, thus giving a mixture of the metabolites 1 and 2. It is therefore possible to monitor flux through different metabolic pathways, such as glycolysis, the tricarboxylic acid cycle, and the hexosamine biosynthetic pathway, using a single molecule.

Cloning and characterization of Helicobacter pylori succinyl CoA:acetoacetate CoA-transferase, a novel prokaryotic member of the CoA-transferase family.

Sequencing of a fragment of Helicobacter pylori genome led to the identification of two open reading frames showing striking homology with Coenzyme A (CoA) transferases, enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The genes were present in all H. pylori strains tested by polymerase chain reaction or slot blotting but not in Campylobacter jejuni. Genes for the putative A and B subunits of H. pylori CoA-transferase were introduced into the bacterial expression vector pKK223-3 and expressed in Escherichia coli JM105 cells. Amino acid sequence comparisons, combined with measurements of enzyme activities using different CoA donors and acceptors, identified the H. pylori CoA-transferase as a succinyl CoA:acetoacetate CoA-transferase. This activity was consistently observed in different H. pylori strains. Antibodies raised against either recombinant A or B subunits recognized two distinct subunits of Mr approximately 26,000 and 24, 000 that are both necessary for H. pylori CoA-transferase function. The lack of alpha-ketoglutarate dehydrogenase and of succinyl CoA synthetase activities indicates that the generation of succinyl CoA is not mediated by the tricarboxylic acid cycle in H. pylori. We postulate the existence of an alternative pathway where the CoA-transferase is essential for energy metabolism.

Quelles applications raisonnables pour l'anticoagulation régionale au citrate en épuration extrarénale [What reasonable applications for regional citrate anticoagulation in renal replacement therapy?].

Regional citrate anticoagulation of the extracorporeal circuits (CRA) experienced considerable growth over the past decade. This development is partly explained by the significant progresses made in the field of bioengineering. These allow a secure administration of citrate, while an increasing availability of ionized calcium measurement at the bedside allows reactivity in monitoring the treatment. An increasing severity of the medical condition of patients requiring blood purification treatment gives more contrast to the profile of patient who may benefit from a CRA. If some methods of renal replacement therapy are well suited to this mode of anticoagulation, others are, to date, only at the stage of development and are applied under close medical supervision.

Amyloid-beta aggregates cause alterations of astrocytic metabolic phenotype: impact on neuronal viability.

Amyloid-beta (Abeta) peptides play a key role in the pathogenesis of Alzheimer's disease and exert various toxic effects on neurons; however, relatively little is known about their influence on glial cells. Astrocytes play a pivotal role in brain homeostasis, contributing to the regulation of local energy metabolism and oxidative stress defense, two aspects of importance for neuronal viability and function. In the present study, we explored the effects of Abeta peptides on glucose metabolism in cultured astrocytes. Following Abeta(25-35) exposure, we observed an increase in glucose uptake and its various metabolic fates, i.e., glycolysis (coupled to lactate release), tricarboxylic acid cycle, pentose phosphate pathway, and incorporation into glycogen. Abeta increased hydrogen peroxide production as well as glutathione release into the extracellular space without affecting intracellular glutathione content. A causal link between the effects of Abeta on glucose metabolism and its aggregation and internalization into astrocytes through binding to members of the class A scavenger receptor family could be demonstrated. Using astrocyte-neuron cocultures, we observed that the overall modifications of astrocyte metabolism induced by Abeta impair neuronal viability. The effects of the Abeta(25-35) fragment were reproduced by Abeta(1-42) but not by Abeta(1-40). Finally, the phosphoinositide 3-kinase (PI3-kinase) pathway appears to be crucial in these events since both the changes in glucose utilization and the decrease in neuronal viability are prevented by LY294002, a PI3-kinase inhibitor. This set of observations indicates that Abeta aggregation and internalization into astrocytes profoundly alter their metabolic phenotype with deleterious consequences for neuronal viability.

NEUROPATHOPHYSIOLOGICAL MECHANISMS IN GLUTARIC ACIDURIA TYPE I: 3-HYDROXYGLUTARIC ACID LEADS TO AMMONIA INCREASE AND NON-APOPTOTIC CELL DEATH

A 3D in vitro model of rat organotypic brain cell cultures in aggregates was used to investigate neurotoxicity mechanisms in glutaric aciduria type I (GA-I). 1 mM glutarate (GA) or 3-hydroxyglutarate (3OHGA) were repeatedly added to the culture media at two different time points. In cultures treated with 3OHGA, we observed an increase in lactate in the medium, pointing to a possible inhibition of Krebs cycle and respiratory chain. We further observed that 3OHGA and to a lesser extend GA induced an increase in ammonia production with concomitant decrease of glutamine concentrations, which may suggest an inhibition of the astrocytic enzyme glutamine synthetase. These previously unreported findings may uncover a pathogenic mechanism in this disease which has deleterious effects on early stages of brain development. By immunohistochemistry we showed that 3OHGA increased non-apoptotic cell death. On the cellular level, 3OHGA and to a lesser extend GA led to cell swelling and loss of astrocytic fibers whereas a loss of oligodendrocytes was only observed for 3OHGA. We conclude that 3OHGAwas the most toxic metabolite in our model for GA-I. 3OHGA induced deleterious effects on glial cells, an increase of ammonia production, and resulted in accentuated cell death of non-apoptotic origin.

A phosphorylation cycle shapes gradients of the DYRK family kinase Pom1 at the plasma membrane.

Concentration gradients regulate many cell biological and developmental processes. In rod-shaped fission yeast cells, polar cortical gradients of the DYRK family kinase Pom1 couple cell length with mitotic commitment by inhibiting a mitotic inducer positioned at midcell. However, how Pom1 gradients are established is unknown. Here, we show that Tea4, which is normally deposited at cell tips by microtubules, is both necessary and, upon ectopic cortical localization, sufficient to recruit Pom1 to the cell cortex. Pom1 then moves laterally at the plasma membrane, which it binds through a basic region exhibiting direct lipid interaction. Pom1 autophosphorylates in this region to lower lipid affinity and promote membrane release. Tea4 triggers Pom1 plasma membrane association by promoting its dephosphorylation through the protein phosphatase 1 Dis2. We propose that local dephosphorylation induces Pom1 membrane association and nucleates a gradient shaped by the opposing actions of lateral diffusion and autophosphorylation-dependent membrane detachment.

Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans.

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ∆foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ∆scdA∆echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ∆scdA∆echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.

In vitro prevention of the emergence of daptomycin resistance in Staphylococcus aureus and enterococci following combination with amoxicillin/clavulanic acid or ampicillin.

Daptomycin is bactericidal against meticillin-resistant Staphylococcus aureus (MRSA), glycopeptide-intermediate-resistant S. aureus (GISA) and vancomycin-susceptible and -resistant enterococci. However, selection for daptomycin-resistant derivatives has occasionally been reported during therapy in humans. Here we evaluate whether selection for daptomycin-resistant S. aureus or enterococci could be prevented in vitro by combining daptomycin with amoxicillin/clavulanic acid, ampicillin, gentamicin or rifampicin. Six strains of S. aureus (four MRSA and two GISA) and four strains of enterococci (two Enterococcus faecalis and two Enterococcus faecium) were serially exposed in broth to two-fold stepwise increasing concentrations of daptomycin alone or in combination with a fixed concentration [0.25x minimum inhibitory concentration (MIC)] of either of the second agents. The daptomycin MIC was examined after each cycle. Exposure to daptomycin alone gradually selected for S. aureus and enterococci with an increased MIC. Gentamicin did not prevent the emergence of daptomycin-resistant bacteria. Rifampicin was also unable to prevent daptomycin resistance, although resistance was slightly delayed. In contrast, amoxicillin/clavulanic acid or ampicillin prevented or greatly delayed the selection of daptomycin-resistant mutants in S. aureus and enterococci, respectively. Addition of amoxicillin/clavulanic acid or ampicillin to daptomycin prevents, or greatly delays, daptomycin resistance in vitro. Future studies in animal models are needed to predict the utility of these combinations in humans.