Cerebral metabolism of acetate and glucose studied by 13C-n.m.r. spectroscopy. A technique for investigating metabolic compartmentation in the brain.
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ABSTRACT:
SUBMITTER: Badar-Goffer RS
PROVIDER: S-EPMC1131106 | biostudies-other | 1990 Feb
REPOSITORIES: biostudies-other
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Project description:Incorporation of 13C label from either [1-13C]glucose to glutamate C-4 and lactate C-3 or from [2-13C]acetate to glutamate C-4 was monitored in situ in a superfused brain slice preparation by using 1H-detected/13C-edited (1H/13C) n.m.r. spectroscopy. The fractional enrichments of both metabolites were determined by this means in both brain slices and acid extracts of the preparations in order to assess their 1H-n.m.r. detectabilities. The 1H/13C satellite resonances from glutamate C-4 and lactate C-3 in brain tissue were followed from 4 min onwards in the presence of 5 mM [1-13C]glucose. Fractional enrichment of glutamate C-4 in the slice preparations was higher than in their acid extracts throughout the incubation of 100 min; at 30 min the enrichment was 15.9 +/- 0.6% in the slice preparations and 10.6 +/- 0.9% in extracts and at 100 min 24.5 +/- 1.7% compared with 19.7 +/- 0.4%, respectively. In contrast, lactate C-3 reached a steady-state fractional enrichment of approx. 43% by 15 min and there was no difference between the values determined in the slice preparations and the acid extracts. There was a significant difference between the glutamate C-4 fractional enrichments in the brain slices (7.4 +/- 0.6%) and extracts (5.1 +/- 0.3%) after 60 min of incubation with [2-13C]acetate. Thus 13C label from both glucose and exogenous acetate enters a pool of glutamate that is more amenable to 1H n.m.r. detection than total acid-extracted brain biochemical glutamate, whereas lactate is labelled with full 1H n.m.r. visibility. The results are discussed in the light of the biochemical factors that affect glutamate 1H-n.m.r. susceptibility and thus its n.m.r. visibility.
| S-EPMC1137991 | biostudies-other
Project description:11C-acetate is a positron emission tomography (PET) tracer of oxidative metabolism, whereas hyperpolarized 13C-acetate can be used in magnetic resonance imaging (MRI) for investigating specific metabolic processes. The aims of this study were to examine if the kinetic formalism of 11C-acetate PET in the kidneys is comparable to that of 13C-acetate MRI, and to compare the dynamic metabolic information of hyperpolarized 13C-acetate MRI with that obtained with 11C-acetate PET. Rats were examined with dynamic hyperpolarized 13C-acetate MRI or 11C-acetate PET before and after intravenous injection of furosemide, a loop diuretic known to alter both the hemodynamics and oxygen consumption in the kidney. The metabolic clearance rates (MCR) were estimated and compared between the two modalities experimentally in vivo and in simulations. There was a clear dependency on the mean transit time and MCR for both 13C-acetate and 11C-acetate following furosemide administration, while no dependencies on the apparent renal perfusion were observed. This study demonstrated that hyperpolarized 13C-acetate MRI is feasible for measurements of the intrarenal energetic demand via the MCR, and that the quantitative measures are correlated with those measured by 11C-acetate PET, even though the temporal window is more than 30 times longer with 11C-acetate.
| S-EPMC5700138 | biostudies-literature
Project description:Acetate is a well-known astrocyte-specific substrate that has been used extensively to probe astrocytic function in vitro and in vivo. Analysis of amino acid turnover curves from (13)C-acetate has been limited mainly for estimation of first-order rate constants from exponential fitting or calculation of relative rates from steady-state (13)C enrichments. In this study, we used (1)H-[(13)C]-Nuclear Magnetic Resonance spectroscopy with intravenous infusion of [2-(13)C]acetate-Na(+) in vivo to measure the cerebral kinetics of acetate transport and utilization in anesthetized rats. Kinetics were assessed using a two-compartment (neuron/astrocyte) analysis of the (13)C turnover curves of glutamate-C4 and glutamine-C4 from [2-(13)C]acetate-Na(+), brain acetate levels, and the dependence of steady-state glutamine-C4 enrichment on blood acetate levels. The steady-state enrichment of glutamine-C4 increased with blood acetate concentration until 90% of plateau for plasma acetate of 4 to 5 mmol/L. Analysis assuming reversible, symmetric Michaelis-Menten kinetics for transport yielded 27+/-2 mmol/L and 1.3+/-0.3 micromol/g/min for K(t) and T(max), respectively, and for utilization, 0.17+/-0.24 mmol/L and 0.14+/-0.02 micromol/g/min for K(M_util) and V(max_util), respectively. The distribution space for acetate was only 0.32+/-0.12 mL/g, indicative of a large excluded volume. The astrocytic and neuronal tricarboxylic acid cycle fluxes were 0.37+/-0.03 micromol/g/min and 1.41+/-0.11 micromol/g/min, respectively; astrocytes thus comprised approximately 21%+/-3% of total oxidative metabolism.
| S-EPMC2879471 | biostudies-literature
Project description:In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.
| S-EPMC5226623 | biostudies-literature
Project description:BackgroundStable isotope tracers are used to assess metabolic flux profiles in living cells. The existing methods of measurement average out the isotopic isomer distribution in metabolites throughout the cell, whereas the knowledge of compartmental organization of analyzed pathways is crucial for the evaluation of true fluxes. That is why we accepted a challenge to create a software tool that allows deciphering the compartmentation of metabolites based on the analysis of average isotopic isomer distribution.ResultsThe software Isodyn, which simulates the dynamics of isotopic isomer distribution in central metabolic pathways, was supplemented by algorithms facilitating the transition between various analyzed metabolic schemes, and by the tools for model discrimination. It simulated 13C isotope distributions in glucose, lactate, glutamate and glycogen, measured by mass spectrometry after incubation of hepatocytes in the presence of only labeled glucose or glucose and lactate together (with label either in glucose or lactate). The simulations assumed either a single intracellular hexose phosphate pool, or also channeling of hexose phosphates resulting in a different isotopic composition of glycogen. Model discrimination test was applied to check the consistency of both models with experimental data. Metabolic flux profiles, evaluated with the accepted model that assumes channeling, revealed the range of changes in metabolic fluxes in liver cells.ConclusionsThe analysis of compartmentation of metabolic networks based on the measured 13C distribution was included in Isodyn as a routine procedure. The advantage of this implementation is that, being a part of evaluation of metabolic fluxes, it does not require additional experiments to study metabolic compartmentation. The analysis of experimental data revealed that the distribution of measured 13C-labeled glucose metabolites is inconsistent with the idea of perfect mixing of hexose phosphates in cytosol. In contrast, the observed distribution indicates the presence of a separate pool of hexose phosphates that is channeled towards glycogen synthesis.
| S-EPMC3292525 | biostudies-literature
Project description:High-resolution 13C n.m.r. spectroscopy has been used to examine propionate metabolism in the perfused rat heart. A number of tricarboxylic acid (TCA) cycle intermediates are observable by 13C n.m.r. in hearts perfused with mixtures of pyruvate and propionate. When the enriched 13C-labelled nucleus originates with pyruvate, the resonances of the intermediates appear as multiplets due to formation of multiply-enriched 13C-labelled isotopomers, whereas when the 13C-labelled nucleus originates with propionate, these same intermediates appear as singlets in the 13C spectrum since entry of propionate into the TCA cycle occurs via succinyl-CoA. An analysis of the isotopomer populations in hearts perfused with [3-13C]pyruvate plus unlabelled propionate indicates that about 27% of the total pyruvate pool available to the heart is derived directly from unlabelled propionate. This was substantiated by perfusing a heart for 2 h with [3-13C]propionate as the only available exogenous substrate. Under these conditions, all of the propionate consumed by the heart, as measured by conventional chemical analysis, ultimately entered the oxidative pathway as [2-13C] or [3-13C]pyruvate. This is consistent with entry of propionate into the TCA cycle intermediate pools as succinyl-CoA and concomitant disposal of malate to pyruvate via the malic enzyme. 13C resonances arising from enriched methylmalonate and propionylcarnitine are also detected in hearts perfused with [3-13C] or [1-13C]propionate which suggests that 13C n.m.r. may be useful as a non-invasive probe in vivo of metabolic abnormalities involving the propionate pathway, such as methylmalonic aciduria or propionic acidaemia.
| S-EPMC1135119 | biostudies-other
Project description:BackgroundMapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation.ResultsIntracellular fluxes of the CHO-K1 cell line central carbon metabolism were successfully determined for a complex network using non-stationary 13C metabolic flux analysis. Mass isotopomers of extracellular metabolites were determined using [U-13C6] glucose as labeled substrate. Metabolic compartmentation and extracellular transport reversibility proved essential to successfully reproduce the dynamics of the labeling patterns. Alanine and pyruvate reversibility changed dynamically even if their net production fluxes remained constant. Cataplerotic fluxes of cytosolic phosphoenolpyruvate carboxykinase and mitochondrial malic enzyme and pyruvate carboxylase were successfully determined. Glycolytic pyruvate channeling to lactate was modeled by including a separate pyruvate pool. In the exponential growth phase, alanine, glycine and glutamate were excreted, and glutamine, aspartate, asparagine and serine were taken up; however, all these amino acids except asparagine were exchanged reversibly with the media. High fluxes were determined in the pentose phosphate pathway and the TCA cycle. The latter was fueled mainly by glucose but also by amino acid catabolism.ConclusionsThe CHO-K1 central metabolism in controlled batch culture proves to be robust. It has the main purpose to ensure fast growth on a mixture of substrates and also to mitigate oxidative stress. It achieves this by using compartmentation to control NADPH and NADH availability and by simultaneous synthesis and catabolism of amino acids.
| S-EPMC4022241 | biostudies-literature
Project description:BackgroundTo engineer Saccharomyces cerevisiae for efficient xylose utilization, a fungal pathway consisting of xylose reductase, xylitol dehydrogenase, and xylulose kinase is often introduced to the host strain. Despite extensive in vitro studies on the xylose pathway, the intracellular metabolism rewiring in response to the heterologous xylose pathway remains largely unknown. In this study, we applied 13C metabolic flux analysis and stoichiometric modeling to systemically investigate the flux distributions in a series of xylose utilizing S. cerevisiae strains.ResultsAs revealed by 13C metabolic flux analysis, the oxidative pentose phosphate pathway was actively used for producing NADPH required by the fungal xylose pathway during xylose utilization of recombinant S. cerevisiae strains. The TCA cycle activity was found to be tightly correlated with the requirements of maintenance energy and biomass yield. Based on in silico simulations of metabolic fluxes, reducing the cell maintenance energy was found crucial to achieve the optimal xylose-based ethanol production. The stoichiometric modeling also suggested that both the cofactor-imbalanced and cofactor-balanced pathways could lead to optimal ethanol production, by flexibly adjusting the metabolic fluxes in futile cycle. However, compared to the cofactor-imbalanced pathway, the cofactor-balanced xylose pathway can lead to optimal ethanol production in a wider range of fermentation conditions.ConclusionsBy applying 13C-MFA and in silico flux balance analysis to a series of recombinant xylose-utilizing S. cerevisiae strains, this work brings new knowledge about xylose utilization in two aspects. First, the interplays between the fungal xylose pathway and the native host metabolism were uncovered. Specifically, we found that the high cell maintenance energy was one of the key factors involved in xylose utilization. Potential strategies to reduce the cell maintenance energy, such as adding exogenous nutrients and evolutionary adaptation, were suggested based on the in vivo and in silico flux analysis in this study. In addition, the impacts of cofactor balance issues on xylose utilization were systemically investigated. The futile pathways were identified as the key factor to adapt to different degrees of cofactor imbalances and suggested as the targets for further engineering to tackle cofactor-balance issues.
| S-EPMC3842631 | biostudies-literature
Project description:The arrangement of isoprene units in pig liver dolichol-18, -19 and -20 was determined by 1H- and 13C-n.m.r. spectroscopies. The alignment of trans and cis isoprene units was found to be in the order: dimethylallyl unit, two trans units, a sequence of 14-16 cis units, and a saturated isoprene unit terminated with a hydroxyl group, which verified the presumed chemical structure of dolichol. The absence of geometric isomers was confirmed. A slight amount of impurity was detected in each reversed-phase h.p.l.c. fraction of dolichol obtained by a conventional method. Detailed assignments of the 13C-n.m.r. spectrum were given for these dolichols by using model compounds and INEPT (insensitive nuclei enhanced by polarization transfer) measurement. The chemical structure of synthetic dolichol-19, which was prepared by the addition of a saturated isoprene unit to the polyprenol-18 isolated from Ginkgo biloba, was confirmed to be identical with that of pig liver dolichol-19.
| S-EPMC1147880 | biostudies-other
Project description:The biosynthesis of alginate by a mucoid strain of Pseudomonas aeruginosa, isolated from a cystic-fibrosis patient, was monitored by using 13C-n.m.r. spectroscopy of bacterial cultures incubated with 1-13C- or 2-13C-enriched fructose. When 1-13C- or 2-13C-enriched fructose was used as the precursor of alginate, enrichment with 13C in the constituent uronic acid monomers of the polysaccharide could only be detected in C-1 or C-2 respectively, indicating that alginate is synthesized in Ps. aeruginosa directly from fructose, with the hexose molecule being retained intact; this rules out the involvement of C3 intermediates, which occurs when glucose is the alginate precursor. The absence of detectable poly-L-gluluronate block sequences from the alginate of Ps. aeruginosa was confirmed, and it was shown that there is no modification of the arrangement of the constituent uronic acids between polymerization to form alginate and the appearance of the mature alginate in the extracellular medium. The 13C-n.m.r. data also provided independent evidence for acetylation on D-mannuronate residues and for the ratio of D-mannuronate to L-guluronate residues in newly synthesized alginate, which had previously been determined only for material secreted from bacteria into the extracellular medium.
| S-EPMC1131336 | biostudies-other