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Sample records for 13c metabolic imaging

  1. Simultaneous imaging of 13C metabolism and 1H structure: technical considerations and potential applications.

    PubMed

    Gordon, Jeremy W; Fain, Sean B; Niles, David J; Ludwig, Kai D; Johnson, Kevin M; Peterson, Eric T

    2015-05-01

    Real-time imaging of (13)C metabolism in vivo has been enabled by recent advances in hyperpolarization. As a result of the inherently low natural abundance of endogenous (13)C nuclei, hyperpolarized (13)C images lack structural information that could be used to aid in motion detection and anatomical registration. Motion before or during the (13)C acquisition can therefore result in artifacts and misregistration that may obscure measures of metabolism. In this work, we demonstrate a method to simultaneously image both (1)H and (13)C nuclei using a dual-nucleus spectral-spatial radiofrequency excitation and a fully coincident readout for rapid multinuclear spectroscopic imaging. With the appropriate multinuclear hardware, and the means to simultaneously excite and receive on both channels, this technique is straightforward to implement requiring little to no increase in scan time. Phantom and in vivo experiments were performed with both Cartesian and spiral trajectories to validate and illustrate the utility of simultaneous acquisitions. Motion compensation of dynamic metabolic measurements acquired during free breathing was demonstrated using motion tracking derived from (1)H data. Simultaneous multinuclear imaging provides structural (1)H and metabolic (13)C images that are correlated both spatially and temporally, and are therefore amenable to joint (1)H and (13)C analysis and correction of structure-function images.

  2. Robust hyperpolarized (13)C metabolic imaging with selective non-excitation of pyruvate (SNEP).

    PubMed

    Chen, Way Cherng; Teo, Xing Qi; Lee, Man Ying; Radda, George K; Lee, Philip

    2015-08-01

    In vivo metabolic imaging using hyperpolarized [1-(13)C]pyruvate provides localized biochemical information and is particularly useful in detecting early disease changes, as well as monitoring disease progression and treatment response. However, a major limitation of hyperpolarized magnetization is its unrecoverable decay, due not only to T1 relaxation but also to radio-frequency (RF) excitation. RF excitation schemes used in metabolic imaging must therefore be able to utilize available hyperpolarized magnetization efficiently and robustly for the optimal detection of substrate and metabolite activities. In this work, a novel RF excitation scheme called selective non-excitation of pyruvate (SNEP) is presented. This excitation scheme involves the use of a spectral selective RF pulse to specifically exclude the excitation of [1-(13)C]pyruvate, while uniformly exciting the key metabolites of interest (namely [1-(13)C]lactate and [1-(13)C]alanine) and [1-(13)C]pyruvate-hydrate. By eliminating the loss of hyperpolarized [1-(13)C]pyruvate magnetization due to RF excitation, the signal from downstream metabolite pools is increased together with enhanced dynamic range. Simulation results, together with phantom measurements and in vivo experiments, demonstrated the improvement in signal-to-noise ratio (SNR) and the extension of the lifetime of the [1-(13)C]lactate and [1-(13)C]alanine pools when compared with conventional non-spectral selective (NS) excitation. SNEP has also been shown to perform comparably well with multi-band (MB) excitation, yet SNEP possesses distinct advantages, including ease of implementation, less stringent demands on gradient performance, increased robustness to frequency drifts and B0 inhomogeneity as well as easier quantification involving the use of [1-(13)C]pyruvate-hydrate as a proxy for the actual [1-(13)C] pyruvate signal. SNEP is therefore a promising alternative for robust hyperpolarized [1-(13)C]pyruvate metabolic imaging with high

  3. Hyperpolarized 13C dehydroascorbate as an endogenous redox sensor for in vivo metabolic imaging.

    PubMed

    Keshari, Kayvan R; Kurhanewicz, John; Bok, Robert; Larson, Peder E Z; Vigneron, Daniel B; Wilson, David M

    2011-11-15

    Reduction and oxidation (redox) chemistry is involved in both normal and abnormal cellular function, in processes as diverse as circadian rhythms and neurotransmission. Intracellular redox is maintained by coupled reactions involving NADPH, glutathione (GSH), and vitamin C, as well as their corresponding oxidized counterparts. In addition to functioning as enzyme cofactors, these reducing agents have a critical role in dealing with reactive oxygen species (ROS), the toxic products of oxidative metabolism seen as culprits in aging, neurodegenerative disease, and ischemia/ reperfusion injury. Despite this strong relationship between redox and human disease, methods to interrogate a redox pair in vivo are limited. Here we report the development of [1-(13)C] dehydroascorbate [DHA], the oxidized form of Vitamin C, as an endogenous redox sensor for in vivo imaging using hyperpolarized (13)C spectroscopy. In murine models, hyperpolarized [1-(13)C] DHA was rapidly converted to [1-(13)C] vitamin C within the liver, kidneys, and brain, as well as within tumor in a transgenic prostate cancer mouse. This result is consistent with what has been previously described for the DHA/Vitamin C redox pair, and points to a role for hyperpolarized [1-(13)C] DHA in characterizing the concentrations of key intracellular reducing agents, including GSH. More broadly, these findings suggest a prognostic role for this new redox sensor in determining vulnerability of both normal and abnormal tissues to ROS.

  4. Hyperpolarized [1,4-13C]-Diethylsuccinate: A Potential DNP Substrate for In Vivo Metabolic Imaging

    PubMed Central

    Billingsley, Kelvin L.; Josan, Sonal; Park, Jae Mo; Tee, Sui Seng; Spielman-Sun, Eleanor; Hurd, Ralph; Mayer, Dirk; Spielman, Daniel

    2014-01-01

    The tricarboxylic acid cycle (TCA) performs an essential role in the regulation of energy and metabolism, and deficiencies in this pathway are commonly correlated with various diseases. However, the development of non-invasive techniques for the assessment of the cycle in vivo has remained challenging. In this work, the applicability of a novel imaging agent, [1,4-13C]-diethylsuccinate, for hyperpolarized 13C metabolic imaging of the TCA cycle was explored. In vivo spectroscopic studies were conducted in conjunction with in vitro analyses to determine the metabolic fate of the imaging agent. Contrary to previous reports (Zacharias, N. M. et. al. J. Am. Chem. Soc. 2012, 134, 934-943), [13C]-labeled diethylsuccinate was primarily metabolized to succinate-derived products not originating from TCA cycle metabolism. These results illustrate potential issues of utilizing dialkyl ester analogs of TCA cycle intermediates as molecular probes for hyperpolarized 13C metabolic imaging. PMID:24421249

  5. 13C metabolic flux analysis.

    PubMed

    Wiechert, W

    2001-07-01

    Metabolic flux analysis using 13C-labeled substrates has become an important tool in metabolic engineering. It allows the detailed quantification of all intracellular fluxes in the central metabolism of a microorganism. The method has strongly evolved in recent years by the introduction of new experimental procedures, measurement techniques, and mathematical data evaluation methods. Many of these improvements require advanced skills in the application of nuclear magnetic resonance and mass spectrometry techniques on the one hand and computational and statistical experience on the other hand. This minireview summarizes these recent developments and sketches the major practical problems. An outlook to possible future developments concludes the text.

  6. Hyperpolarized [1-13C] glutamate: a metabolic imaging biomarker of IDH1 mutational status in glioma

    PubMed Central

    Chaumeil, Myriam M.; Larson, Peder E.Z.; Woods, Sarah M.; Cai, Larry; Eriksson, Pia; Robinson, Aaron E.; Lupo, Janine M.; Vigneron, Daniel B.; Nelson, Sarah J.; Pieper, Russell O.; Phillips, Joanna J.; Ronen, Sabrina M.

    2014-01-01

    Mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade glioma and secondary glioblastoma, represent an early pathogenic event, and are associated with epigenetically-driven modulations of metabolism. Of particular interest is the recently uncovered relationship between the IDH1 mutation and decreased activity of the branched-chain amino acid transaminase 1 (BCAT1) enzyme. Non-invasive imaging methods that can assess BCAT1 activity could therefore improve detection of mutant IDH1 tumors and aid in developing and monitoring new targeted therapies. BCAT1 catalyzes the transamination of branched-chain amino acids while converting α-ketoglutarate (α-KG) to glutamate. Our goal was to use 13C magnetic resonance spectroscopy to probe the conversion of hyperpolarized [1-13C] α-KG to hyperpolarized [1-13C] glutamate as a readout of BCAT1 activity. We investigated two isogenic glioblastoma lines that differed only in their IDH1 status, and performed experiments in live cells and in vivo in rat orthotopic tumors. Following injection of hyperpolarized [1-13C] α-KG, hyperpolarized [1-13C] glutamate production was detected both in cells and in vivo, and the level of hyperpolarized [1-13C] glutamate was significantly lower in mutant IDH1 cells and tumors compared to their IDH1-wild-type counterparts. Importantly however, in our cells the observed drop in hyperpolarized [1-13C] glutamate was likely mediated not only by a drop in BCAT1 activity, but also by reductions in aspartate transaminase and glutamate dehydrogenase activities, suggesting additional metabolic reprogramming at least in our model. Hyperpolarized [1-13C] glutamate could thus inform on multiple mutant IDH1-associated metabolic events that mediate reduced glutamate production. PMID:24876103

  7. Fast volumetric imaging of ethanol metabolism in rat liver with hyperpolarized [1-13C]-pyruvate

    PubMed Central

    Josan, Sonal; Spielman, Daniel; Yen, Yi-Fen; Hurd, Ralph; Pfefferbaum, Adolf; Mayer, Dirk

    2012-01-01

    Rapid, volumetric imaging of hyperpolarized 13C compounds allows the real time measurement of metabolic activity and can be useful in distinguishing between normal and diseased tissues. This work extends a fast 2D under-sampled spiral magnetic resonance spectroscopic imaging (MRSI) sequence to provide volumetric coverage, acquiring a 16×16×12 matrix with a nominal 5 mm isotropic resolution in 4.5 s. The rapid acquisition enables a high temporal resolution for dynamic imaging. This dynamic 3D MRSI method was used to investigate hyperpolarized [1-13C]-pyruvate metabolism modulated by the administration of ethanol in rat liver. A significant increase in the pyruvate to lactate conversion was observed in the liver due to the greater availability of NADH from ethanol metabolism. PMID:22331837

  8. Saturation-recovery metabolic-exchange rate imaging with hyperpolarized [1-13C] pyruvate using spectral-spatial excitation.

    PubMed

    Schulte, Rolf F; Sperl, Jonathan I; Weidl, Eliane; Menzel, Marion I; Janich, Martin A; Khegai, Oleksandr; Durst, Markus; Ardenkjaer-Larsen, Jan Henrik; Glaser, Steffen J; Haase, Axel; Schwaiger, Markus; Wiesinger, Florian

    2013-05-01

    Within the last decade hyperpolarized [1-13C] pyruvate chemical-shift imaging has demonstrated impressive potential for metabolic MR imaging for a wide range of applications in oncology, cardiology, and neurology. In this work, a highly efficient pulse sequence is described for time-resolved, multislice chemical shift imaging of the injected substrate and obtained downstream metabolites. Using spectral-spatial excitation in combination with single-shot spiral data acquisition, the overall encoding is evenly distributed between excitation and signal reception, allowing the encoding of one full two-dimensional metabolite image per excitation. The signal-to-noise ratio can be flexibly adjusted and optimized using lower flip angles for the pyruvate substrate and larger ones for the downstream metabolites. Selectively adjusting the excitation of the down-stream metabolites to 90° leads to a so-called "saturation-recovery" scheme with the detected signal content being determined by forward conversion of the available pyruvate. In case of repetitive excitations, the polarization is preserved using smaller flip angles for pyruvate. Metabolic exchange rates are determined spatially resolved from the metabolite images using a simplified two-site exchange model. This novel contrast is an important step toward more quantitative metabolic imaging. Goal of this work was to derive, analyze, and implement this "saturation-recovery metabolic exchange rate imaging" and demonstrate its capabilities in four rats bearing subcutaneous tumors.

  9. Hyperpolarized 13C Metabolic MRI of the Human Heart

    PubMed Central

    Lau, Justin Y.C.; Chen, Albert P.; Geraghty, Benjamin J.; Perks, William J.; Roifman, Idan; Wright, Graham A.; Connelly, Kim A.

    2016-01-01

    Rationale: Altered cardiac energetics is known to play an important role in the progression toward heart failure. A noninvasive method for imaging metabolic markers that could be used in longitudinal studies would be useful for understanding therapeutic approaches that target metabolism. Objective: To demonstrate the first hyperpolarized 13C metabolic magnetic resonance imaging of the human heart. Methods and Results: Four healthy subjects underwent conventional proton cardiac magnetic resonance imaging followed by 13C imaging and spectroscopic acquisition immediately after intravenous administration of a 0.1 mmol/kg dose of hyperpolarized [1-13C]pyruvate. All subjects tolerated the procedure well with no adverse effects reported ≤1 month post procedure. The [1-13C]pyruvate signal appeared within the chambers but not within the muscle. Imaging of the downstream metabolites showed 13C-bicarbonate signal mainly confined to the left ventricular myocardium, whereas the [1-13C]lactate signal appeared both within the chambers and in the myocardium. The mean 13C image signal:noise ratio was 115 for [1-13C]pyruvate, 56 for 13C-bicarbonate, and 53 for [1-13C]lactate. Conclusions: These results represent the first 13C images of the human heart. The appearance of 13C-bicarbonate signal after administration of hyperpolarized [1-13C]pyruvate was readily detected in this healthy cohort (n=4). This shows that assessment of pyruvate metabolism in vivo in humans is feasible using current technology. Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02648009. PMID:27635086

  10. BI-07HYPERPOLARIZED [1-13C] GLUTAMATE: A METABOLIC IMAGING BIOMARKER OF IDH1 MUTATIONAL STATUS IN GLIOMA

    PubMed Central

    Chaumeil, Myriam; Larson, Peder; Woods, Sarah; Cai, Larry; Eriksson, Pia; Robinson, Aaron; Lupo, Janine; Vigneron, Daniel; Nelson, Sarah; Pieper, Russell; Phillips, Joanna; Ronen, Sabrina

    2014-01-01

    Mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade glioma and secondary glioblastoma, represent an early pathogenic event and are being considered a promising therapeutic target. Consequently, non-invasive imaging methods are needed to monitor IDH1 status. Amongst these, we previously demonstrated the use of 13C MR spectroscopic imaging of hyperpolarized [1-13C] α-ketoglutarate (α-KG) to non-invasively assess IDH1 status through the detection of the conversion of hyperpolarized α-ketoglutarate to 2-hydroxyglutarate (2-HG) catalyzed by mutant IDH1. Importantly, in addition to its oncogenic role, IDH1 mutation is also associated with global modulations in metabolism. Interestingly, a study recently uncovered a relationship between presence of IDH1 mutation and decreased activity of the branched chained amino acid transaminase 1 (BCAT1) enzyme, which transaminates amino acids while converting α-KG to glutamate. Given this new study, we decided to expand on our previous findings and investigated the potential of hyperpolarized α-KG as an imaging probe to monitor BCAT1-driven α-KG-to-glutamate conversion and its modulation in the presence of IDH1 mutation. We investigated two isogenic glioblastoma lines that differed only in their IDH1 status, and performed experiments in live cells and in vivo in rat orthotopic tumors. Following injection of hyperpolarized α-KG, hyperpolarized glutamate production was detected both in cells and in vivo, and the level of hyperpolarized glutamate was significantly lower in mutant IDH1 cells and tumors compared to their IDH1-wild-type counterparts. Importantly however, the observed drop in hyperpolarized glutamate was likely mediated not only by a drop in BCAT1 activity, but also by reductions in aspartate transaminase and glutamate dehydrogenase activities, suggesting additional metabolic reprogramming at least in our model. Hyperpolarized glutamate could thus inform on multiple

  11. Multi-channel metabolic imaging, with SENSE reconstruction, of hyperpolarized [1- 13C] pyruvate in a live rat at 3.0 tesla on a clinical MR scanner

    NASA Astrophysics Data System (ADS)

    Tropp, James; Lupo, Janine M.; Chen, Albert; Calderon, Paul; McCune, Don; Grafendorfer, Thomas; Ozturk-Isik, Esin; Larson, Peder E. Z.; Hu, Simon; Yen, Yi-Fen; Robb, Fraser; Bok, Robert; Schulte, Rolf; Xu, Duan; Hurd, Ralph; Vigneron, Daniel; Nelson, Sarah

    2011-01-01

    We report metabolic images of 13C, following injection of a bolus of hyperpolarized [1-13C] pyruvate in a live rat. The data were acquired on a clinical scanner, using custom coils for volume transmission and array reception. Proton blocking of all carbon resonators enabled proton anatomic imaging with the system body coil, to allow for registration of anatomic and metabolic images, for which good correlation was achieved, with some anatomic features (kidney and heart) clearly visible in a carbon image, without reference to the corresponding proton image. Parallel imaging with sensitivity encoding was used to increase the spatial resolution in the SI direction of the rat. The signal to noise ratio in was in some instances unexpectedly high in the parallel images; variability of the polarization among different trials, plus partial volume effects, are noted as a possible cause of this.

  12. Imaging the time-integrated cerebral metabolic activity with subcellular resolution through nanometer-scale detection of biosynthetic products deriving from (13)C-glucose.

    PubMed

    Takado, Yuhei; Knott, Graham; Humbel, Bruno M; Masoodi, Mojgan; Escrig, Stéphane; Meibom, Anders; Comment, Arnaud

    2015-11-01

    Glucose is the primary source of energy for the brain but also an important source of building blocks for proteins, lipids, and nucleic acids. Little is known about the use of glucose for biosynthesis in tissues at the cellular level. We demonstrate that local cerebral metabolic activity can be mapped in mouse brain tissue by quantitatively imaging the biosynthetic products deriving from [U-(13)C]glucose metabolism using a combination of in situ electron microscopy and secondary ion mass-spectroscopy (NanoSIMS). Images of the (13)C-label incorporated into cerebral ultrastructure with ca. 100 nm resolution allowed us to determine the timescale on which the metabolic products of glucose are incorporated into different cells, their sub-compartments and organelles. These were mapped in astrocytes and neurons in the different layers of the motor cortex. We see evidence for high metabolic activity in neurons via the nucleus (13)C enrichment. We observe that in all the major cell compartments, such as e.g. nucleus and Golgi apparatus, neurons incorporate substantially higher concentrations of (13)C-label than astrocytes.

  13. Dynamic and High-Resolution Metabolic Imaging of Hyperpolarized [1-13C]-Pyruvate in the Rat Brain Using a High-Performance Gradient Insert

    PubMed Central

    Mayer, Dirk; Yen, Yi-Fen; Takahashi, Atsushi; Josan, Sonal; Tropp, James; Rutt, B.K.; Hurd, Ralph E.; Spielman, Daniel M.; Pfefferbaum, Adolf

    2010-01-01

    Fast chemical shift imaging techniques are advantageous in metabolic imaging of hyperpolarized compounds due to the limited duration of the signal amplification. At the same time, reducing the acquisition time in hyperpolarized imaging does not necessarily lead to the conventional penalty in signal-to-noise ratio that occurs in imaging at thermal equilibrium polarization levels. Here a high-performance gradient insert was used in combination with undersampled spiral chemical shift imaging to increase either the imaging speed or the spatial resolution of hyperpolarized 13C metabolic imaging on a clinical 3T MR scanner. Both a single-shot sequence with a total acquisition time of 125 ms and a 3-shot sequence with a nominal in-plane resolution of 1.5 mm were implemented. The k-space trajectories were measured and then used during image reconstruction. The technique was applied to metabolic imaging of the rat brain in vivo after the injection of hyperpolarized [1-13C]-pyruvate. Dynamic imaging afforded the measurement of region-of-interest-specific time courses of pyruvate and its metabolic products, while imaging at high spatial resolution was used to better characterize the spatial distribution of the metabolite signals. PMID:21500253

  14. (13)C-labeled biochemical probes for the study of cancer metabolism with dynamic nuclear polarization-enhanced magnetic resonance imaging.

    PubMed

    Salamanca-Cardona, Lucia; Keshari, Kayvan R

    2015-01-01

    In recent years, advances in metabolic imaging have become dependable tools for the diagnosis and treatment assessment in cancer. Dynamic nuclear polarization (DNP) has recently emerged as a promising technology in hyperpolarized (HP) magnetic resonance imaging (MRI) and has reached clinical relevance with the successful visualization of [1-(13)C] pyruvate as a molecular imaging probe in human prostate cancer. This review focuses on introducing representative compounds relevant to metabolism that are characteristic of cancer tissue: aerobic glycolysis and pyruvate metabolism, glutamine addiction and glutamine/glutamate metabolism, and the redox state and ascorbate/dehydroascorbate metabolism. In addition, a brief introduction of probes that can be used to trace necrosis, pH changes, and other pathways relevant to cancer is presented to demonstrate the potential that HP MRI has to revolutionize the use of molecular imaging for diagnosis and assessment of treatments in cancer.

  15. Application of Double Spin-Echo Spiral Chemical Shift Imaging to Rapid Metabolic Mapping of Hyperpolarized [1-13C]-Pyruvate

    PubMed Central

    Josan, Sonal; Yen, Yi-Fen; Hurd, Ralph; Pfefferbaum, Adolf; Spielman, Daniel; Mayer, Dirk

    2011-01-01

    Undersampled spiral CSI (spCSI) using a free induction decay (FID) acquisition allows sub-second metabolic imaging of hyperpolarized 13C. Phase correction of the FID acquisition can be difficult, especially with contributions from aliased out-of-phase peaks. This work extends the spCSI sequence by incorporating double spin-echo radiofrequency (RF) pulses to eliminate the need for phase correction and obtain high quality spectra in magnitude mode. The sequence also provides an added benefit of attenuating signal from flowing spins, which can otherwise contaminate signal in the organ of interest. The refocusing pulses can potentially lead to a loss of hyperpolarized magnetization in dynamic imaging due to flow of spins through the fringe field of the RF coil, where the refocusing pulses fail to provide complete refocusing. Care must be taken for dynamic imaging to ensure that the spins remain within the B1-homogeneous sensitive volume of the RF coil. PMID:21316280

  16. (13)C metabolic flux analysis of recombinant expression hosts.

    PubMed

    Young, Jamey D

    2014-12-01

    Identifying host cell metabolic phenotypes that promote high recombinant protein titer is a major goal of the biotech industry. (13)C metabolic flux analysis (MFA) provides a rigorous approach to quantify these metabolic phenotypes by applying isotope tracers to map the flow of carbon through intracellular metabolic pathways. Recent advances in tracer theory and measurements are enabling more information to be extracted from (13)C labeling experiments. Sustained development of publicly available software tools and standardization of experimental workflows is simultaneously encouraging increased adoption of (13)C MFA within the biotech research community. A number of recent (13)C MFA studies have identified increased citric acid cycle and pentose phosphate pathway fluxes as consistent markers of high recombinant protein expression, both in mammalian and microbial hosts. Further work is needed to determine whether redirecting flux into these pathways can effectively enhance protein titers while maintaining acceptable glycan profiles.

  17. /sup 13/C nuclear magnetic resonance studies of cardiac metabolism

    SciTech Connect

    Seeholzer, S.H.

    1985-01-01

    The last decade has witnessed the increasing use of Nuclear Magnetic Resonance (NMR) techniques for following the metabolic fate of compounds specifically labeled with /sup 13/C. The goals of the present study are: (1) to develop reliable quantitative procedures for measuring the /sup 13/C enrichment of specific carbon sites in compounds enriched by the metabolism of /sup 13/C-labeled substrates in rat heart, and (2) to use these quantitative measurements of fractional /sup 13/C enrichment within the context of a mathematical flux model describing the carbon flow through the TCA cycle and ancillary pathways, as a means for obtaining unknown flux parameters. Rat hearts have been perfused in vitro with various combinations of glucose, acetate, pyruvate, and propionate to achieve steady state flux conditions, followed by perfusion with the same substrates labeled with /sup 13/C in specific carbon sites. The hearts were frozen at different times after addition of /sup 13/C-labeled substrates and neutralized perchloric acid extracts were used to obtain high resolution proton-decoupled /sup 13/C NMR spectra at 90.55 MHz. The fractional /sup 13/C enrichment (F.E.) of individual carbon sites in different metabolites was calculated from the area of the resolved resonances after correction for saturation and nuclear Overhauser effects. These F.E. measurements by /sup 13/C NMR were validated by the analysis of /sup 13/C-/sup 1/H scalar coupling patterns observed in /sup 1/H NMR spectra of the extracted metabolites. The results obtained from perfusion of hearts glucose plus either (2-/sup 13/C) acetate or (3-/sup 13/C) pyruvate are similar to those obtained by previous investigators using /sup 14/C-labeled substrates.

  18. Metabolic flux analysis using 13C peptide label measurements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    13C metabolic flux analysis (MFA) has become the experimental method of choice to investigate cellular metabolism. MFA has established flux maps of central metabolism for dozens of microbes, cell cultures, and plant seeds. Steady-state MFA utilizes isotopic labeling measurements of amino acids obtai...

  19. EPR oxygen imaging and hyperpolarized 13C MRI of pyruvate metabolism as noninvasive biomarkers of tumor treatment response to a glycolysis inhibitor 3-bromopyruvate.

    PubMed

    Matsumoto, Shingo; Saito, Keita; Yasui, Hironobu; Morris, H Douglas; Munasinghe, Jeeva P; Lizak, Martin; Merkle, Hellmut; Ardenkjaer-Larsen, Jan Henrik; Choudhuri, Rajani; Devasahayam, Nallathamby; Subramanian, Sankaran; Koretsky, Alan P; Mitchell, James B; Krishna, Murali C

    2013-05-01

    The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia-sensitive drug. The small molecule 3-bromopyruvate blocks glycolysis pathway by inhibiting hypoxia inducible enzymes and enhanced cytotoxicity of 3-bromopyruvate under hypoxic conditions has been reported in vitro. However, the efficacy of 3-bromopyruvate was substantially attenuated in hypoxic tumor regions (pO2<10 mmHg) in vivo using squamous cell carcinoma (SCCVII)-bearing mouse model. Metabolic MRI studies using hyperpolarized 13C-labeled pyruvate showed that monocarboxylate transporter-1 is the major transporter for pyruvate and the analog 3-bromopyruvate in SCCVII tumor. The discrepant results between in vitro and in vivo data were attributed to biphasic oxygen dependent expression of monocarboxylate transporter-1 in vivo. Expression of monocarboxylate transporter-1 was enhanced in moderately hypoxic (8-15 mmHg) tumor regions but down regulated in severely hypoxic (<5 mmHg) tumor regions. These results emphasize the importance of noninvasive imaging biomarkers to confirm the action of hypoxia-activated drugs.

  20. 13C-based metabolic flux analysis: fundamentals and practice.

    PubMed

    Yang, Tae Hoon

    2013-01-01

    Isotope-based metabolic flux analysis is one of the emerging technologies applied to system level metabolic phenotype characterization in metabolic engineering. Among the developed approaches, (13)C-based metabolic flux analysis has been established as a standard tool and has been widely applied to quantitative pathway characterization of diverse biological systems. To implement (13)C-based metabolic flux analysis in practice, comprehending the underlying mathematical and computational modeling fundamentals is of importance along with carefully conducted experiments and analytical measurements. Such knowledge is also crucial when designing (13)C-labeling experiments and properly acquiring key data sets essential for in vivo flux analysis implementation. In this regard, the modeling fundamentals of (13)C-labeling systems and analytical data processing are the main topics we will deal with in this chapter. Along with this, the relevant numerical optimization techniques are addressed to help implementation of the entire computational procedures aiming at (13)C-based metabolic flux analysis in vivo.

  1. Towards hyperpolarized 13C-succinate imaging of brain cancer

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Pratip; Chekmenev, Eduard Y.; Perman, William H.; Harris, Kent C.; Lin, Alexander P.; Norton, Valerie A.; Tan, Chou T.; Ross, Brian D.; Weitekamp, Daniel P.

    2007-05-01

    We describe a novel 13C enriched precursor molecule, sodium 1- 13C acetylenedicarboxylate, which after hydrogenation by PASADENA (Parahydrogen and Synthesis Allows Dramatically Enhanced Nuclear Alignment) under controlled experimental conditions, becomes hyperpolarized 13C sodium succinate. Fast in vivo 3D FIESTA MR imaging demonstrated that, following carotid arterial injection, the hyperpolarized 13C-succinate appeared in the head and cerebral circulation of normal and tumor-bearing rats. At this time, no in vivo hyperpolarized signal has been localized to normal brain or brain tumor. On the other hand, ex vivo samples of brain harvested from rats bearing a 9L brain tumor, 1 h or more following in vivo carotid injection of hyperpolarized 13C sodium succinate, contained significant concentrations of the injected substrate, 13C sodium succinate, together with 13C maleate and succinate metabolites 1- 13C-glutamate, 5- 13C-glutamate, 1- 13C-glutamine and 5- 13C-glutamine. The 13C substrates and products were below the limits of NMR detection in ex vivo samples of normal brain consistent with an intact blood-brain barrier. These ex vivo results indicate that hyperpolarized 13C sodium succinate may become a useful tool for rapid in vivo identification of brain tumors, providing novel biomarkers in 13C MR spectral-spatial images.

  2. A scientific workflow framework for (13)C metabolic flux analysis.

    PubMed

    Dalman, Tolga; Wiechert, Wolfgang; Nöh, Katharina

    2016-08-20

    Metabolic flux analysis (MFA) with (13)C labeling data is a high-precision technique to quantify intracellular reaction rates (fluxes). One of the major challenges of (13)C MFA is the interactivity of the computational workflow according to which the fluxes are determined from the input data (metabolic network model, labeling data, and physiological rates). Here, the workflow assembly is inevitably determined by the scientist who has to consider interacting biological, experimental, and computational aspects. Decision-making is context dependent and requires expertise, rendering an automated evaluation process hardly possible. Here, we present a scientific workflow framework (SWF) for creating, executing, and controlling on demand (13)C MFA workflows. (13)C MFA-specific tools and libraries, such as the high-performance simulation toolbox 13CFLUX2, are wrapped as web services and thereby integrated into a service-oriented architecture. Besides workflow steering, the SWF features transparent provenance collection and enables full flexibility for ad hoc scripting solutions. To handle compute-intensive tasks, cloud computing is supported. We demonstrate how the challenges posed by (13)C MFA workflows can be solved with our approach on the basis of two proof-of-concept use cases.

  3. 13C metabolic flux analysis in complex systems.

    PubMed

    Zamboni, Nicola

    2011-02-01

    Experimental determination of in vivo metabolic rates by methods of (13)C metabolic flux analysis is a pivotal approach to unravel structure and regulation of metabolic networks, in particular with microorganisms grown in minimal media. However, the study of real-life and eukaryotic systems calls for the quantification of fluxes also in cellular compartments, rich media, cell-wide metabolic networks, dynamic systems or single cells. These scenarios drastically increase the complexity of the task, which is only partly dealt by existing approaches that rely on rigorous simulations of label propagation through metabolic networks and require multiple labeling experiments or a priori information on pathway inactivity to simplify the problem. Albeit qualitative and largely driven by human interpretation, statistical analysis of measured (13)C-patterns remains the exclusive alternative to comprehensively handle such complex systems. In the future, this practice will be complemented by novel modeling frameworks to assay particular fluxes within a network by stable isotopic tracer for targeted validation of well-defined hypotheses.

  4. 13C Tracer Studies of Metabolism in Mouse Tumor Xenografts

    PubMed Central

    Lane, Andrew N.; Yan, Jun; Fan, Teresa W-M.

    2015-01-01

    Mice are widely used for human tumor xenograft studies of cancer development and drug efficacy and toxicity. Stable isotope tracing coupled with metabolomic analysis is an emerging approach for assaying metabolic network activity. In mouse models there are several routes of tracer introduction, which have particular advantages and disadvantages that depend on the model and the questions addressed. This protocol describes the bolus i.v. route via repeated tail vein injections of solutions of stable isotope enriched tracers including 13C6-glucose and 13C5,15N2-glutamine. Repeated injections give higher enrichments and over longer labeling periods than a single bolus. Multiple injections of glutamine are necessary to achieve adequate enrichment in engrafted tumors. PMID:26693168

  5. An in Vivo 13C NMR Analysis of the Anaerobic Yeast Metabolism of 1-13C-Glucose

    NASA Astrophysics Data System (ADS)

    Giles, Brent J.; Matsche, Zenziwe; Egeland, Ryan D.; Reed, Ryan A.; Morioka, Scott S.; Taber, Richard L.

    1999-11-01

    A biochemistry laboratory experiment that studies the dynamics of the anaerobic yeast metabolism of 1-13C-D-glucose via NMR is described. Fleischmann's Active Dry yeast, under anaerobic conditions, produces primarily 2-13C-ethanol and some 1-13C-glycerol as end products. An experiment is described in which the yeast is subjected to osmotic shock from an increasing sodium chloride concentration. Under these conditions, the yeast increases the ratio of glycerol to ethanol. The experiment can be accomplished in a single laboratory period.

  6. 13C metabolic flux analysis at a genome-scale.

    PubMed

    Gopalakrishnan, Saratram; Maranas, Costas D

    2015-11-01

    Metabolic models used in 13C metabolic flux analysis generally include a limited number of reactions primarily from central metabolism. They typically omit degradation pathways, complete cofactor balances, and atom transition contributions for reactions outside central metabolism. This study addresses the impact on prediction fidelity of scaling-up mapping models to a genome-scale. The core mapping model employed in this study accounts for (75 reactions and 65 metabolites) primarily from central metabolism. The genome-scale metabolic mapping model (GSMM) (697 reaction and 595 metabolites) is constructed using as a basis the iAF1260 model upon eliminating reactions guaranteed not to carry flux based on growth and fermentation data for a minimal glucose growth medium. Labeling data for 17 amino acid fragments obtained from cells fed with glucose labeled at the second carbon was used to obtain fluxes and ranges. Metabolic fluxes and confidence intervals are estimated, for both core and genome-scale mapping models, by minimizing the sum of square of differences between predicted and experimentally measured labeling patterns using the EMU decomposition algorithm. Overall, we find that both topology and estimated values of the metabolic fluxes remain largely consistent between core and GSM model. Stepping up to a genome-scale mapping model leads to wider flux inference ranges for 20 key reactions present in the core model. The glycolysis flux range doubles due to the possibility of active gluconeogenesis, the TCA flux range expanded by 80% due to the availability of a bypass through arginine consistent with labeling data, and the transhydrogenase reaction flux was essentially unresolved due to the presence of as many as five routes for the inter-conversion of NADPH to NADH afforded by the genome-scale model. By globally accounting for ATP demands in the GSMM model the unused ATP decreased drastically with the lower bound matching the maintenance ATP requirement. A non

  7. Novel Imaging Contrast Methods for Hyperpolarized 13 C Magnetic Resonance Imaging

    NASA Astrophysics Data System (ADS)

    Reed, Galen Durant

    Magnetic resonance imaging using hyperpolarized 13C-labeled small molecules has emerged as an extremely powerful tool for the in vivo monitoring of perfusion and metabolism. This work presents methods for improved imaging, parameter mapping, and image contrast generation for in vivo hyperpolarized 13C MRI. Angiography using hyperpolarized urea was greatly improved with a highly T2-weighted acquisition in combination with 15N labeling of the urea amide groups. This is due to the fact that the T2 of [13C]urea is strongly limited by the scalar coupling to the neighboring quadrupolar 14N. The long in vivo T2 values of [13C, 15N2]urea were utilized for sub-millimeter projection angiography using a contrast agent that could be safely injected in concentrations of 10-100 mM while still tolerated in patients with renal insufficiency. This study also presented the first method for in vivo T2 mapping of hyperpolarized 13C compounds. The in vivo T2 of urea was short in the blood and long within the kidneys. This persistent signal component was isolated to the renal filtrate, thus enabling for the first time direct detection of an imaging contrast agent undergoing glomerular filtration. While highly T2-weighted acquisitions select for molecules with short rotational correlation times, high diffusion weighting selects for those with the long translational correlation times. A specialized spin-echo EPI sequence was developed in order to generate highly diffusion-weighted hyperpolarized 13C images on a clinical MRI system operating within clinical peak- RF and gradient amplitude constraints. Low power adiabatic spin echo pulses were developed in order to generate a sufficiently large refocused bandwidth while maintaining low nominal power. This diffusion weighted acquisition gave enhanced tumor contrast-to-noise ratio when imaging [1-13C]lactate after infusion of [1-13C]pyruvate. Finally, the first in-man hyperpolarized 13C MRI clinical trial is discussed.

  8. Kinetic modeling of hyperpolarized 13C 1-pyruvate metabolism in normal rats and TRAMP mice

    NASA Astrophysics Data System (ADS)

    Zierhut, Matthew L.; Yen, Yi-Fen; Chen, Albert P.; Bok, Robert; Albers, Mark J.; Zhang, Vickie; Tropp, Jim; Park, Ilwoo; Vigneron, Daniel B.; Kurhanewicz, John; Hurd, Ralph E.; Nelson, Sarah J.

    2010-01-01

    PurposeTo investigate metabolic exchange between 13C 1-pyruvate, 13C 1-lactate, and 13C 1-alanine in pre-clinical model systems using kinetic modeling of dynamic hyperpolarized 13C spectroscopic data and to examine the relationship between fitted parameters and dose-response. Materials and methodsDynamic 13C spectroscopy data were acquired in normal rats, wild type mice, and mice with transgenic prostate tumors (TRAMP) either within a single slice or using a one-dimensional echo-planar spectroscopic imaging (1D-EPSI) encoding technique. Rate constants were estimated by fitting a set of exponential equations to the dynamic data. Variations in fitted parameters were used to determine model robustness in 15 mm slices centered on normal rat kidneys. Parameter values were used to investigate differences in metabolism between and within TRAMP and wild type mice. ResultsThe kinetic model was shown here to be robust when fitting data from a rat given similar doses. In normal rats, Michaelis-Menten kinetics were able to describe the dose-response of the fitted exchange rate constants with a 13.65% and 16.75% scaled fitting error (SFE) for kpyr→lac and kpyr→ala, respectively. In TRAMP mice, kpyr→lac increased an average of 94% after up to 23 days of disease progression, whether the mice were untreated or treated with casodex. Parameters estimated from dynamic 13C 1D-EPSI data were able to differentiate anatomical structures within both wild type and TRAMP mice. ConclusionsThe metabolic parameters estimated using this approach may be useful for in vivo monitoring of tumor progression and treatment efficacy, as well as to distinguish between various tissues based on metabolic activity.

  9. Imaging pH with hyperpolarized 13C.

    PubMed

    Gallagher, Ferdia A; Kettunen, Mikko I; Brindle, Kevin M

    2011-10-01

    pH is a fundamental physiological parameter that is tightly controlled by endogenous buffers. The acid-base balance is altered in many disease states, such as inflammation, ischemia and cancer. Despite the importance of pH, there are currently no routine methods for imaging the spatial distribution of pH in humans. The enormous gain in sensitivity afforded by dynamic nuclear polarization (DNP) has provided a novel way in which to image tissue pH using MR, which has the potential to be translated into the clinic. This review explores the advantages and disadvantages of current pH imaging techniques and how they compare with DNP-based approaches for the measurement and imaging of pH with hyperpolarized (13)C. Intravenous injection of hyperpolarized (13)C-labeled bicarbonate results in the rapid production of hyperpolarized (13)CO(2) in the reaction catalyzed by carbonic anhydrase. As this reaction is close to equilibrium in the body and is pH dependent, the ratio of the (13)C signal intensities from H(13)CO(3)(-) and (13)CO(2), measured using MRS, can be used to calculate pH in vivo. The application of this technique to a murine tumor model demonstrated that it measured predominantly extracellular pH and could be mapped in the animal using spectroscopic imaging techniques. A second approach has been to use the production of hyperpolarized (13)CO(2) from hyperpolarized [1-(13)C]pyruvate to measure predominantly intracellular pH. In tissues with a high aerobic capacity, such as the heart, the hyperpolarized [1-(13)C]pyruvate undergoes rapid oxidative decarboxylation, catalyzed by intramitochondrial pyruvate dehydrogenase. Provided that there is sufficient carbonic anhydrase present to catalyze the rapid equilibration of the hyperpolarized (13)C label between CO(2) and bicarbonate, the ratio of their resonance intensities may again be used to estimate pH, which, in this case, is predominantly intracellular. As both pyruvate and bicarbonate are endogenous molecules they

  10. Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for (13)C metabolic flux analysis.

    PubMed

    Au, Jennifer; Choi, Jungik; Jones, Shawn W; Venkataramanan, Keerthi P; Antoniewicz, Maciek R

    2014-11-01

    In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and (13)C-metabolic flux analysis ((13)C-MFA). Here, cells were grown in parallel cultures with [1-(13)C]glucose and [U-(13)C]glucose as tracers and (13)C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of (13)C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for (13)C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased (13)C-flux measurements in C. acetobutylicum.

  11. Hyperpolarized 13C pyruvate mouse brain metabolism with absorptive-mode EPSI at 1 T

    NASA Astrophysics Data System (ADS)

    Miloushev, Vesselin Z.; Di Gialleonardo, Valentina; Salamanca-Cardona, Lucia; Correa, Fabian; Granlund, Kristin L.; Keshari, Kayvan R.

    2017-02-01

    The expected signal in echo-planar spectroscopic imaging experiments was explicitly modeled jointly in spatial and spectral dimensions. Using this as a basis, absorptive-mode type detection can be achieved by appropriate choice of spectral delays and post-processing techniques. We discuss the effects of gradient imperfections and demonstrate the implementation of this sequence at low field (1.05 T), with application to hyperpolarized [1-13C] pyruvate imaging of the mouse brain. The sequence achieves sufficient signal-to-noise to monitor the conversion of hyperpolarized [1-13C] pyruvate to lactate in the mouse brain. Hyperpolarized pyruvate imaging of mouse brain metabolism using an absorptive-mode EPSI sequence can be applied to more sophisticated murine disease and treatment models. The simple modifications presented in this work, which permit absorptive-mode detection, are directly translatable to human clinical imaging and generate improved absorptive-mode spectra without the need for refocusing pulses.

  12. Methods for metabolic evaluation of prostate cancer cells using proton and 13C HR-MAS spectroscopy and [3-13C] pyruvate as a metabolic substrate

    PubMed Central

    Levin, Yakir S.; Albers, Mark J.; Butler, Thomas N.; Spielman, Daniel; Peehl, Donna M.; Kurhanewicz, John

    2009-01-01

    Prostate cancer has been shown to undergo unique metabolic changes associated with neoplastic transformation, with associated changes in citrate, alanine, and lactate concentrations. 13C HR-MAS spectroscopy provides an opportunity to simultaneously investigate the metabolic pathways implicated in these changes by using 13C labeled substrates as metabolic probes. In this work, a method to reproducibly interrogate metabolism in prostate cancer cells in primary culture was developed using HR-MAS spectroscopy. Optimization of cell culture protocols, labeling parameters, harvesting, storage, and transfer was performed. Using [3-13C] pyruvate as a metabolic probe, 1H and 13C HR-MAS spectroscopy were used to quantify the net amount and fractional enrichment of several labeled metabolites that evolved in multiple cell samples from each of five different prostate cancers. Average enrichment across all cancers was 32.4±5.4% for [3-13C] alanine, 24.5±5.4% for [4-13C] glutamate, 9.1±2.5% for [3-13C] glutamate, 25.2±5.7% for [3-13C] aspartate, and 4.2±1.0% for [3-13C] lactate. Cell samples from the same parent population demonstrated reproducible fractional enrichments of alanine, glutamate, and aspartate to within 12%, 10%, and 10%, respectively. Furthermore, the cells produced a significant amount of [4-13C] glutamate, which supports the bioenergetic theory for prostate cancer. These methods will allow further characterization of metabolic properties of prostate cancer cells in the future. PMID:19780158

  13. Real-time cardiac metabolism assessed with hyperpolarized [1-13C]acetate in a large-animal model

    PubMed Central

    Flori, Alessandra; Liserani, Matteo; Frijia, Francesca; Giovannetti, Giulio; Lionetti, Vincenzo; Casieri, Valentina; Positano, Vincenzo; Aquaro, Giovanni Donato; Recchia, Fabio A.; Santarelli, Maria Filomena; Landini, Luigi; Ardenkjaer-Larsen, Jan Henrik; Menichetti, Luca

    2014-01-01

    Dissolution-Dynamic Nuclear Polarization (dissolution-DNP) for Magnetic Resonance (MR) Spectroscopic Imaging has recently emerged as a novel technique for non invasive studies of the metabolic fate of biomolecules in vivo. Since acetate is the most abundant extra- and intra-cellular short-chain fatty acid, we focused on [1-13C]acetate as a promising candidate for a chemical probe to study myocardial metabolism of beating heart. Dissolution-DNP procedure of Na[1-13C]acetate for in vivo cardiac applications with 3T MR scanner was optimized in pigs during bolus injection of doses up to 3 mmoles. The Na[1-13C]acetate formulation was characterized by a liquid-state polarization of 14.2% and T1Eff in vivo of 17.6 ± 1.7 s. In vivo Na[1-13C]acetate kinetic displayed a bimodal shape: [1-13C]acetyl carnitine (AcC) was detected in a slice covering the cardiac volume, and the signal of 13C-acetate and 13C-AcC was modeled using the total Area Under the Curve (AUC) for kinetic analysis. A good correlation was found between the ratio AUC(AcC)/AUC(acetate) and the apparent kinetic constant of metabolic conversion kAcC/r1 from [1-13C]acetate to [1-13C]AcC. Our study proved the feasibility and limitations of administration of large doses of hyperpolarized [1-13C]acetate with dissolution DNP to study by MR spectroscopy the myocardial conversion of [1-13C]acetate in [1-13C]acetyl-carnitine generated by acetyltransferase in healthy pigs. PMID:25201079

  14. In vivo investigation of cardiac metabolism in the rat using MRS of hyperpolarized [1-13C] and [2-13C]pyruvate.

    PubMed

    Josan, Sonal; Park, Jae Mo; Hurd, Ralph; Yen, Yi-Fen; Pfefferbaum, Adolf; Spielman, Daniel; Mayer, Dirk

    2013-12-01

    Hyperpolarized (13)C MRS allows the in vivo assessment of pyruvate dehydrogenase complex (PDC) flux, which converts pyruvate to acetyl-coenzyme A (acetyl-CoA). [1-(13)C]pyruvate has been used to measure changes in cardiac PDC flux, with demonstrated increase in (13)C-bicarbonate production after dichloroacetate (DCA) administration. With [1-(13)C]pyruvate, the (13)C label is released as (13 CO2 /(13)C-bicarbonate, and, hence, does not allow us to follow the fate of acetyl-CoA. Pyruvate labeled in the C2 position has been used to track the (13)C label into the TCA (tricarboxylic acid) cycle and measure [5-(13)C]glutamate as well as study changes in [1-(13)C]acetylcarnitine with DCA and dobutamine. This work investigates changes in the metabolic fate of acetyl-CoA in response to metabolic interventions of DCA-induced increased PDC flux in the fed and fasted state, and increased cardiac workload with dobutamine in vivo in rat heart at two different pyruvate doses. DCA led to a modest increase in the (13)C labeling of [5-(13)C]glutamate, and a considerable increase in [1-(13)C]acetylcarnitine and [1,3-(13)C]acetoacetate peaks. Dobutamine resulted in an increased labeling of [2-(13)C]lactate, [2-(13)C]alanine and [5-(13)C]glutamate. The change in glutamate with dobutamine was observed using a high pyruvate dose but not with a low dose. The relative changes in the different metabolic products provide information about the relationship between PDC-mediated oxidation of pyruvate and its subsequent incorporation into the TCA cycle compared with other metabolic pathways. Using a high dose of pyruvate may provide an improved ability to observe changes in glutamate.

  15. Effects of fasting on serial measurements of hyperpolarized [1-(13) C]pyruvate metabolism in tumors.

    PubMed

    Serrao, Eva M; Rodrigues, Tiago B; Gallagher, Ferdia A; Kettunen, Mikko I; Kennedy, Brett W C; Vowler, Sarah L; Burling, Keith A; Brindle, Kevin M

    2016-08-01

    Imaging of the metabolism of hyperpolarized [1-(13) C]pyruvate has shown considerable promise in preclinical studies in oncology, particularly for the assessment of early treatment response. The repeatability of measurements of (13) C label exchange between pyruvate and lactate was determined in a murine lymphoma model in fasted and non-fasted animals. The fasted state showed lower intra-individual variability, although the [1-(13) C]lactate/[1-(13) C]pyruvate signal ratio was significantly greater in fasted than in non-fasted mice, which may be explained by the higher tumor lactate concentrations in fasted animals. These results indicate that the fasted state may be preferable for the measurement of (13) C label exchange between pyruvate and lactate, as it reduces the variability and therefore should make it easier to detect the effects of therapy. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

  16. Effects of fasting on serial measurements of hyperpolarized [1‐13C]pyruvate metabolism in tumors

    PubMed Central

    Serrao, Eva M.; Rodrigues, Tiago B.; Gallagher, Ferdia A.; Kettunen, Mikko I.; Kennedy, Brett W. C.; Vowler, Sarah L.; Burling, Keith A.

    2016-01-01

    Imaging of the metabolism of hyperpolarized [1‐13C]pyruvate has shown considerable promise in preclinical studies in oncology, particularly for the assessment of early treatment response. The repeatability of measurements of 13C label exchange between pyruvate and lactate was determined in a murine lymphoma model in fasted and non‐fasted animals. The fasted state showed lower intra‐individual variability, although the [1‐13C]lactate/[1‐13C]pyruvate signal ratio was significantly greater in fasted than in non‐fasted mice, which may be explained by the higher tumor lactate concentrations in fasted animals. These results indicate that the fasted state may be preferable for the measurement of 13C label exchange between pyruvate and lactate, as it reduces the variability and therefore should make it easier to detect the effects of therapy. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd. PMID:27309986

  17. Propionate metabolism in the rat heart by 13C n.m.r. spectroscopy.

    PubMed Central

    Sherry, A D; Malloy, C R; Roby, R E; Rajagopal, A; Jeffrey, F M

    1988-01-01

    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. PMID:3178775

  18. Refined Analysis of Brain Energy Metabolism Using In Vivo Dynamic Enrichment of 13C Multiplets

    PubMed Central

    Dehghani M., Masoumeh; Duarte, João M. N.; Kunz, Nicolas; Gruetter, Rolf

    2016-01-01

    Carbon-13 nuclear magnetic resonance spectroscopy in combination with the infusion of 13C-labeled precursors is a unique approach to study in vivo brain energy metabolism. Incorporating the maximum information available from in vivo localized 13C spectra is of importance to get broader knowledge on cerebral metabolic pathways. Metabolic rates can be quantitatively determined from the rate of 13C incorporation into amino acid neurotransmitters such as glutamate and glutamine using suitable mathematical models. The time course of multiplets arising from 13C-13C coupling between adjacent carbon atoms was expected to provide additional information for metabolic modeling leading to potential improvements in the estimation of metabolic parameters. The aim of the present study was to extend two-compartment neuronal/glial modeling to include dynamics of 13C isotopomers available from fine structure multiplets in 13C spectra of glutamate and glutamine measured in vivo in rats brain at 14.1 T, termed bonded cumomer approach. Incorporating the labeling time courses of 13C multiplets of glutamate and glutamine resulted in elevated precision of the estimated fluxes in rat brain as well as reduced correlations between them. PMID:26969691

  19. Reproducibility study for free-breathing measurements of pyruvate metabolism using hyperpolarized (13) C in the heart.

    PubMed

    Lau, Angus Z; Chen, Albert P; Barry, Jennifer; Graham, John J; Dominguez-Viqueira, William; Ghugre, Nilesh R; Wright, Graham A; Cunningham, Charles H

    2013-04-01

    Spatially resolved images of hyperpolarized (13) C substrates and their downstream products provide insight into real-time metabolic processes occurring in vivo. Recently, hyperpolarized (13) C pyruvate has been used to characterize in vivo cardiac metabolism in the rat and pig, but accurate and reproducible measurements remain challenging due to the limited period available for imaging as well as physiological motion. In this article, time-resolved cardiac- and respiratory-gated images of [1-(13) C] pyruvate, [1-(13) C] lactate, and (13) C bicarbonate in the heart are acquired without the need for a breathhold. The robustness of these free-breathing measurements is demonstrated using the time-resolved data to produce a normalized metric of pyruvate dehydrogenase and lactate dehydrogenase activity in the heart. The values obtained are reproducible in a controlled metabolic state. In a 60-min ischemia/reperfusion model, significant differences in hyperpolarized bicarbonate and lactate, normalized using the left ventricular pyruvate signal, were detected between scans performed at baseline and 45 min after reperfusion. The sequence is anticipated to improve quantitative measurements of cardiac metabolism, leading to feasible validation studies using fewer subjects, and potentially improved diagnosis, serial monitoring, and treatment of cardiac disease in patients.

  20. Elucidating the role of copper in CHO cell energy metabolism using (13)C metabolic flux analysis.

    PubMed

    Nargund, Shilpa; Qiu, Jinshu; Goudar, Chetan T

    2015-01-01

    (13)C-metabolic flux analysis was used to understand copper deficiency-related restructuring of energy metabolism, which leads to excessive lactate production in recombinant protein-producing CHO cells. Stationary-phase labeling experiments with U-(13)C glucose were conducted on CHO cells grown under high and limiting copper in 3 L fed-batch bioreactors. The resultant labeling patterns of soluble metabolites were measured by GC-MS and used to estimate metabolic fluxes in the central carbon metabolism pathways using OpenFlux. Fluxes were evaluated 300 times from stoichiometrically feasible random guess values and their confidence intervals calculated by Monte Carlo simulations. Results from metabolic flux analysis exhibited significant carbon redistribution throughout the metabolic network in cells under Cu deficiency. Specifically, glycolytic fluxes increased (25%-79% relative to glucose uptake) whereas fluxes through the TCA and pentose phosphate pathway (PPP) were lower (15%-23% and 74%, respectively) compared with the Cu-containing condition. Furthermore, under Cu deficiency, 33% of the flux entering TCA via the pyruvate node was redirected to lactate and malate production. Based on these results, we hypothesize that Cu deficiency disrupts the electron transport chain causing ATP deficiency, redox imbalance, and oxidative stress, which in turn drive copper-deficient CHO cells to produce energy via aerobic glycolysis, which is associated with excessive lactate production, rather than the more efficient route of oxidative phosphorylation.

  1. Integrated 13C-metabolic flux analysis of 14 parallel labeling experiments in Escherichia coli.

    PubMed

    Crown, Scott B; Long, Christopher P; Antoniewicz, Maciek R

    2015-03-01

    The use of parallel labeling experiments for (13)C metabolic flux analysis ((13)C-MFA) has emerged in recent years as the new gold standard in fluxomics. The methodology has been termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. In this contribution, we have tested the limits of COMPLETE-MFA by demonstrating integrated analysis of 14 parallel labeling experiments with Escherichia coli. An effort on such a massive scale has never been attempted before. In addition to several widely used isotopic tracers such as [1,2-(13)C]glucose and mixtures of [1-(13)C]glucose and [U-(13)C]glucose, four novel tracers were applied in this study: [2,3-(13)C]glucose, [4,5,6-(13)C]glucose, [2,3,4,5,6-(13)C]glucose and a mixture of [1-(13)C]glucose and [4,5,6-(13)C]glucose. This allowed us for the first time to compare the performance of a large number of isotopic tracers. Overall, there was no single best tracer for the entire E. coli metabolic network model. Tracers that produced well-resolved fluxes in the upper part of metabolism (glycolysis and pentose phosphate pathways) showed poor performance for fluxes in the lower part of metabolism (TCA cycle and anaplerotic reactions), and vice versa. The best tracer for upper metabolism was 80% [1-(13)C]glucose+20% [U-(13)C]glucose, while [4,5,6-(13)C]glucose and [5-(13)C]glucose both produced optimal flux resolution in the lower part of metabolism. COMPLETE-MFA improved both flux precision and flux observability, i.e. more independent fluxes were resolved with smaller confidence intervals, especially exchange fluxes. Overall, this study demonstrates that COMPLETE-MFA is a powerful approach for improving flux measurements and that this methodology should be considered in future studies that require very high flux resolution.

  2. Parallel labeling experiments for pathway elucidation and (13)C metabolic flux analysis.

    PubMed

    Antoniewicz, Maciek R

    2015-12-01

    Metabolic pathway models provide the foundation for quantitative studies of cellular physiology through the measurement of intracellular metabolic fluxes. For model organisms metabolic models are well established, with many manually curated genome-scale model reconstructions, gene knockout studies and stable-isotope tracing studies. However, for non-model organisms a similar level of knowledge is often lacking. Compartmentation of cellular metabolism in eukaryotic systems also presents significant challenges for quantitative (13)C-metabolic flux analysis ((13)C-MFA). Recently, innovative (13)C-MFA approaches have been developed based on parallel labeling experiments, the use of multiple isotopic tracers and integrated data analysis, that allow more rigorous validation of pathway models and improved quantification of metabolic fluxes. Applications of these approaches open new research directions in metabolic engineering, biotechnology and medicine.

  3. Metabolism of hyperpolarized [1‐13C]pyruvate through alternate pathways in rat liver

    PubMed Central

    Moreno, Karlos X.; Wang, Jian‐Xiong; Fidelino, Leila; Merritt, Matthew E.; Sherry, A. Dean; Malloy, Craig R.

    2016-01-01

    The source of hyperpolarized (HP) [13C]bicarbonate in the liver during metabolism of HP [1‐13C]pyruvate is uncertain and likely changes with physiology. Multiple processes including decarboxylation through pyruvate dehydrogenase or pyruvate carboxylase followed by subsequent decarboxylation via phosphoenolpyruvate carboxykinase (gluconeogenesis) could play a role. Here we tested which metabolic fate of pyruvate contributed to the appearance of HP [13C]bicarbonate during metabolism of HP [1‐13C]pyruvate by the liver in rats after 21 h of fasting compared to rats with free access to food. The 13C NMR of HP [13C]bicarbonate was observed in the liver of fed rats, but not in fasted rats where pyruvate carboxylation and gluconeogenesis was active. To further explore the relative fluxes through pyruvate carboxylase versus pyruvate dehydrogenase in the liver under typical conditions of hyperpolarization studies, separate parallel experiments were performed with rats given non‐hyperpolarized [2,3‐13C]pyruvate. 13C NMR analysis of glutamate isolated from the liver of rats revealed that flux from injected pyruvate through pyruvate dehydrogenase was dominant under fed conditions whereas flux through pyruvate carboxylase dominated under fasted conditions. The NMR signal of HP [13C]bicarbonate does not parallel pyruvate carboxylase activity followed by subsequent decarboxylation reaction leading to glucose production. In the liver of healthy well‐fed rats, the appearance of HP [13C]bicarbonate exclusively reflects decarboxylation of HP [1‐13C]pyruvate via pyruvate dehydrogenase. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd. PMID:26836042

  4. Metabolism of hyperpolarized [1-(13)C]pyruvate through alternate pathways in rat liver.

    PubMed

    Jin, Eunsook S; Moreno, Karlos X; Wang, Jian-Xiong; Fidelino, Leila; Merritt, Matthew E; Sherry, A Dean; Malloy, Craig R

    2016-04-01

    The source of hyperpolarized (HP) [(13)C]bicarbonate in the liver during metabolism of HP [1-(13)C]pyruvate is uncertain and likely changes with physiology. Multiple processes including decarboxylation through pyruvate dehydrogenase or pyruvate carboxylase followed by subsequent decarboxylation via phosphoenolpyruvate carboxykinase (gluconeogenesis) could play a role. Here we tested which metabolic fate of pyruvate contributed to the appearance of HP [(13)C]bicarbonate during metabolism of HP [1-(13)C]pyruvate by the liver in rats after 21 h of fasting compared to rats with free access to food. The (13)C NMR of HP [(13)C]bicarbonate was observed in the liver of fed rats, but not in fasted rats where pyruvate carboxylation and gluconeogenesis was active. To further explore the relative fluxes through pyruvate carboxylase versus pyruvate dehydrogenase in the liver under typical conditions of hyperpolarization studies, separate parallel experiments were performed with rats given non-hyperpolarized [2,3-(13)C]pyruvate. (13)C NMR analysis of glutamate isolated from the liver of rats revealed that flux from injected pyruvate through pyruvate dehydrogenase was dominant under fed conditions whereas flux through pyruvate carboxylase dominated under fasted conditions. The NMR signal of HP [(13)C]bicarbonate does not parallel pyruvate carboxylase activity followed by subsequent decarboxylation reaction leading to glucose production. In the liver of healthy well-fed rats, the appearance of HP [(13)C]bicarbonate exclusively reflects decarboxylation of HP [1-(13)C]pyruvate via pyruvate dehydrogenase.

  5. In vivo single-shot 13C spectroscopic imaging of hyperpolarized metabolites by spatiotemporal encoding

    NASA Astrophysics Data System (ADS)

    Schmidt, Rita; Laustsen, Christoffer; Dumez, Jean-Nicolas; Kettunen, Mikko I.; Serrao, Eva M.; Marco-Rius, Irene; Brindle, Kevin M.; Ardenkjaer-Larsen, Jan Henrik; Frydman, Lucio

    2014-03-01

    Hyperpolarized metabolic imaging is a growing field that has provided a new tool for analyzing metabolism, particularly in cancer. Given the short life times of the hyperpolarized signal, fast and effective spectroscopic imaging methods compatible with dynamic metabolic characterizations are necessary. Several approaches have been customized for hyperpolarized 13C MRI, including CSI with a center-out k-space encoding, EPSI, and spectrally selective pulses in combination with spiral EPI acquisitions. Recent studies have described the potential of single-shot alternatives based on spatiotemporal encoding (SPEN) principles, to derive chemical-shift images within a sub-second period. By contrast to EPSI, SPEN does not require oscillating acquisition gradients to deliver chemical-shift information: its signal encodes both spatial as well as chemical shift information, at no extra cost in experimental complexity. SPEN MRI sequences with slice-selection and arbitrary excitation pulses can also be devised, endowing SPEN with the potential to deliver single-shot multi-slice chemical shift images, with a temporal resolution required for hyperpolarized dynamic metabolic imaging. The present work demonstrates this with initial in vivo results obtained from SPEN-based imaging of pyruvate and its metabolic products, after injection of hyperpolarized [1-13C]pyruvate. Multi-slice chemical-shift images of healthy rats were obtained at 4.7 T in the region of the kidney, and 4D (2D spatial, 1D spectral, 1D temporal) data sets were obtained at 7 T from a murine lymphoma tumor model.

  6. Determination of fructose metabolic pathways in normal and fructose-intolerant children: A sup 13 C NMR study using (U- sup 13 C)fructose

    SciTech Connect

    Gopher, A.; Lapidot, A. ); Vaisman, N. ); Mandel, H. )

    1990-07-01

    An inborn deficiency in the ability of aldolase B to split fructose 1-phosphate is found in humans with hereditary fructose intolerance (HFI). A stable isotope procedure to elucidate the mechanism of conversion of fructose to glucose in normal children and in HFI children has been developed. A constant infusion of D-(U-{sup 13}C)fructose was given nasogastrically to control and to HFI children. Hepatic fructose conversion to glucose was estimated by examination of {sup 13}C NMR spectra of plasma glucose. Significantly lower values ({approx}3-fold) for fructose conversion to glucose were obtained for the HFI patients as compared to the controls. A quantitative determination of the metabolic pathways of fructose conversion to glucose was derived from {sup 13}C NMR measurement of plasma ({sup 13}C)glucose isotopomer populations. The finding of isotopomer populations of three adjacent {sup 13}C atoms at glucose C-4 ({sup 13}C{sub 3}-{sup 13}C{sub 4}-{sup 13}C{sub 5}) suggests that there is a direct pathway from fructose, by-passing fructose-1-phosphate aldolase, to fructose 1,6-bisphosphate. The metabolism of fructose by fructose-1-phosphate aldolase activity accounts for only {approx}50% of the total amount of hepatic fructose conversion to glucose. In view of the marked decline by 67% in synthesis of glucose from fructose in HFI subjects found in this study, the extent of ({sup 13}C)glucose formation from a trace amount of (U-{sup 13}C)fructose infused into the patient can be used as a safe and noninvasive diagnostic test for inherent faulty fructose metabolism.

  7. 13C-labelled microdialysis studies of cerebral metabolism in TBI patients☆

    PubMed Central

    Carpenter, Keri L.H.; Jalloh, Ibrahim; Gallagher, Clare N.; Grice, Peter; Howe, Duncan J.; Mason, Andrew; Timofeev, Ivan; Helmy, Adel; Murphy, Michael P.; Menon, David K.; Kirkpatrick, Peter J.; Carpenter, T. Adrian; Sutherland, Garnette R.; Pickard, John D.; Hutchinson, Peter J.

    2014-01-01

    Human brain chemistry is incompletely understood and better methodologies are needed. Traumatic brain injury (TBI) causes metabolic perturbations, one result of which includes increased brain lactate levels. Attention has largely focussed on glycolysis, whereby glucose is converted to pyruvate and lactate, and is proposed to act as an energy source by feeding into neurons’ tricarboxylic acid (TCA) cycle, generating ATP. Also reportedly upregulated by TBI is the pentose phosphate pathway (PPP) that does not generate ATP but produces various molecules that are putatively neuroprotective, antioxidant and reparative, in addition to lactate among the end products. We have developed a novel combination of 13C-labelled cerebral microdialysis both to deliver 13C-labelled substrates into brains of TBI patients and recover the 13C-labelled metabolites, with high-resolution 13C NMR analysis of the microdialysates. This methodology has enabled us to achieve the first direct demonstration in humans that the brain can utilise lactate via the TCA cycle. We are currently using this methodology to make the first direct comparison of glycolysis and the PPP in human brain. In this article, we consider the application of 13C-labelled cerebral microdialysis for studying brain energy metabolism in patients. We set this methodology within the context of metabolic pathways in the brain, and 13C research modalities addressing them. PMID:24361470

  8. (13)C-labelled microdialysis studies of cerebral metabolism in TBI patients.

    PubMed

    Carpenter, Keri L H; Jalloh, Ibrahim; Gallagher, Clare N; Grice, Peter; Howe, Duncan J; Mason, Andrew; Timofeev, Ivan; Helmy, Adel; Murphy, Michael P; Menon, David K; Kirkpatrick, Peter J; Carpenter, T Adrian; Sutherland, Garnette R; Pickard, John D; Hutchinson, Peter J

    2014-06-16

    Human brain chemistry is incompletely understood and better methodologies are needed. Traumatic brain injury (TBI) causes metabolic perturbations, one result of which includes increased brain lactate levels. Attention has largely focussed on glycolysis, whereby glucose is converted to pyruvate and lactate, and is proposed to act as an energy source by feeding into neurons' tricarboxylic acid (TCA) cycle, generating ATP. Also reportedly upregulated by TBI is the pentose phosphate pathway (PPP) that does not generate ATP but produces various molecules that are putatively neuroprotective, antioxidant and reparative, in addition to lactate among the end products. We have developed a novel combination of (13)C-labelled cerebral microdialysis both to deliver (13)C-labelled substrates into brains of TBI patients and recover the (13)C-labelled metabolites, with high-resolution (13)C NMR analysis of the microdialysates. This methodology has enabled us to achieve the first direct demonstration in humans that the brain can utilise lactate via the TCA cycle. We are currently using this methodology to make the first direct comparison of glycolysis and the PPP in human brain. In this article, we consider the application of (13)C-labelled cerebral microdialysis for studying brain energy metabolism in patients. We set this methodology within the context of metabolic pathways in the brain, and (13)C research modalities addressing them.

  9. Profiling human gut bacterial metabolism and its kinetics using [U-13C]glucose and NMR.

    PubMed

    de Graaf, Albert A; Maathuis, Annet; de Waard, Pieter; Deutz, Nicolaas E P; Dijkema, Cor; de Vos, Willem M; Venema, Koen

    2010-01-01

    This study introduces a stable-isotope metabolic approach employing [U-(13)C]glucose that, as a novelty, allows selective profiling of the human intestinal microbial metabolic products of carbohydrate food components, as well as the measurement of the kinetics of their formation pathways, in a single experiment. A well-established, validated in vitro model of human intestinal fermentation was inoculated with standardized gastrointestinal microbiota from volunteers. After culture stabilization, [U-(13)C]glucose was added as an isotopically labeled metabolic precursor. System lumen and dialysate samples were taken at regular intervals. Metabolite concentrations and isotopic labeling were determined by NMR, GC, and enzymatic methods. The main microbial metabolites were lactate, acetate, butyrate, formate, ethanol, and glycerol. They together accounted for a (13)C recovery rate as high as 91.2%. Using an NMR chemical shift prediction approach, several minor products that showed (13)C incorporation were identified as organic acids, amino acids, and various alcohols. Using computer modeling of the (12)C contents and (13)C labeling kinetics, the metabolic fluxes in the gut microbial pathways for synthesis of lactate, formate, acetate, and butyrate were determined separately for glucose and unlabeled background substrates. This novel approach enables the study of the modulation of human intestinal function by single nutrients, providing a new rational basis for achieving control of the short-chain fatty acids profile by manipulating substrate and microbiota composition in a purposeful manner.

  10. A Peptide-Based Method for 13C Metabolic Flux Analysis in Microbial Communities

    PubMed Central

    Ghosh, Amit; Nilmeier, Jerome; Weaver, Daniel; Adams, Paul D.; Keasling, Jay D.; Mukhopadhyay, Aindrila; Petzold, Christopher J.; Martín, Héctor García

    2014-01-01

    The study of intracellular metabolic fluxes and inter-species metabolite exchange for microbial communities is of crucial importance to understand and predict their behaviour. The most authoritative method of measuring intracellular fluxes, 13C Metabolic Flux Analysis (13C MFA), uses the labeling pattern obtained from metabolites (typically amino acids) during 13C labeling experiments to derive intracellular fluxes. However, these metabolite labeling patterns cannot easily be obtained for each of the members of the community. Here we propose a new type of 13C MFA that infers fluxes based on peptide labeling, instead of amino acid labeling. The advantage of this method resides in the fact that the peptide sequence can be used to identify the microbial species it originates from and, simultaneously, the peptide labeling can be used to infer intracellular metabolic fluxes. Peptide identity and labeling patterns can be obtained in a high-throughput manner from modern proteomics techniques. We show that, using this method, it is theoretically possible to recover intracellular metabolic fluxes in the same way as through the standard amino acid based 13C MFA, and quantify the amount of information lost as a consequence of using peptides instead of amino acids. We show that by using a relatively small number of peptides we can counter this information loss. We computationally tested this method with a well-characterized simple microbial community consisting of two species. PMID:25188426

  11. Cardiac perfusion imaging using hyperpolarized 13c urea using flow sensitizing gradients

    PubMed Central

    Miller, Jack J.; Robson, Matthew D.; Tyler, Damian J.

    2015-01-01

    Purpose To demonstrate the feasibility of imaging the first passage of a bolus of hyperpolarized 13C urea through the rodent heart using flow‐sensitizing gradients to reduce signal from the blood pool. Methods A flow‐sensitizing bipolar gradient was optimized to reduce the bright signal within the cardiac chambers, enabling improved contrast of the agent within the tissue capillary bed. The gradient was incorporated into a dynamic golden angle spiral 13C imaging sequence. Healthy rats were scanned during rest (n = 3) and under adenosine stress‐induced hyperemia (n = 3). Results A two‐fold increase in myocardial perfusion relative to rest was detected during adenosine stress‐induced hyperemia, consistent with a myocardial perfusion reserve of two in rodents. Conclusion The new pulse sequence was used to obtain dynamic images of the first passage of hyperpolarized 13C urea in the rodent heart, without contamination from bright signal within the neighboring cardiac lumen. This probe of myocardial perfusion is expected to enable new hyperpolarized 13C studies in which the cardiac metabolism/perfusion mismatch can be identified. Magn Reson Med, 2015. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Magn Reson Med 75:1474–1483, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance. PMID:25991580

  12. A comparison of quantitative methods for clinical imaging with hyperpolarized (13)C-pyruvate.

    PubMed

    Daniels, Charlie J; McLean, Mary A; Schulte, Rolf F; Robb, Fraser J; Gill, Andrew B; McGlashan, Nicholas; Graves, Martin J; Schwaiger, Markus; Lomas, David J; Brindle, Kevin M; Gallagher, Ferdia A

    2016-04-01

    Dissolution dynamic nuclear polarization (DNP) enables the metabolism of hyperpolarized (13)C-labelled molecules, such as the conversion of [1-(13)C]pyruvate to [1-(13)C]lactate, to be dynamically and non-invasively imaged in tissue. Imaging of this exchange reaction in animal models has been shown to detect early treatment response and correlate with tumour grade. The first human DNP study has recently been completed, and, for widespread clinical translation, simple and reliable methods are necessary to accurately probe the reaction in patients. However, there is currently no consensus on the most appropriate method to quantify this exchange reaction. In this study, an in vitro system was used to compare several kinetic models, as well as simple model-free methods. Experiments were performed using a clinical hyperpolarizer, a human 3 T MR system, and spectroscopic imaging sequences. The quantitative methods were compared in vivo by using subcutaneous breast tumours in rats to examine the effect of pyruvate inflow. The two-way kinetic model was the most accurate method for characterizing the exchange reaction in vitro, and the incorporation of a Heaviside step inflow profile was best able to describe the in vivo data. The lactate time-to-peak and the lactate-to-pyruvate area under the curve ratio were simple model-free approaches that accurately represented the full reaction, with the time-to-peak method performing indistinguishably from the best kinetic model. Finally, extracting data from a single pixel was a robust and reliable surrogate of the whole region of interest. This work has identified appropriate quantitative methods for future work in the analysis of human hyperpolarized (13)C data.

  13. OpenMebius: an open source software for isotopically nonstationary 13C-based metabolic flux analysis.

    PubMed

    Kajihata, Shuichi; Furusawa, Chikara; Matsuda, Fumio; Shimizu, Hiroshi

    2014-01-01

    The in vivo measurement of metabolic flux by (13)C-based metabolic flux analysis ((13)C-MFA) provides valuable information regarding cell physiology. Bioinformatics tools have been developed to estimate metabolic flux distributions from the results of tracer isotopic labeling experiments using a (13)C-labeled carbon source. Metabolic flux is determined by nonlinear fitting of a metabolic model to the isotopic labeling enrichment of intracellular metabolites measured by mass spectrometry. Whereas (13)C-MFA is conventionally performed under isotopically constant conditions, isotopically nonstationary (13)C metabolic flux analysis (INST-(13)C-MFA) has recently been developed for flux analysis of cells with photosynthetic activity and cells at a quasi-steady metabolic state (e.g., primary cells or microorganisms under stationary phase). Here, the development of a novel open source software for INST-(13)C-MFA on the Windows platform is reported. OpenMebius (Open source software for Metabolic flux analysis) provides the function of autogenerating metabolic models for simulating isotopic labeling enrichment from a user-defined configuration worksheet. Analysis using simulated data demonstrated the applicability of OpenMebius for INST-(13)C-MFA. Confidence intervals determined by INST-(13)C-MFA were less than those determined by conventional methods, indicating the potential of INST-(13)C-MFA for precise metabolic flux analysis. OpenMebius is the open source software for the general application of INST-(13)C-MFA.

  14. HYPERPOLARIZED 13C MAGNETIC RESONANCE AND ITS USE IN METABOLIC ASSESSMENT OF CULTURED CELLS AND PERFUSED ORGANS

    PubMed Central

    Lumata, Lloyd; Yang, Chendong; Ragavan, Mukundan; Carpenter, Nicholas; DeBerardinis, Ralph J.; Merritt, Matthew E.

    2016-01-01

    Diseased tissue is often characterized by abnormalities in intermediary metabolism. Observing these alterations in situ may lead to an improved understanding of pathological processes and novel ways to monitor these processes non-invasively in human patients. Although 13C is a stable isotope safe for use in animal models of disease as well as human subjects, its utility as a metabolic tracer has largely been limited to ex vivo analyses employing analytical techniques like mass spectrometry or nuclear magnetic resonance spectroscopy. Neither of these techniques is suitable for non-invasive metabolic monitoring, and the low abundance and poor gyromagnetic ratio of conventional 13C make it a poor nucleus for imaging. However, the recent advent of hyperpolarization methods, particularly dynamic nuclear polarization (DNP), make it possible to enhance the spin polarization state of 13C by many orders of magnitude, resulting in a temporary amplification of the signal sufficient for monitoring kinetics of enzyme-catalyzed reactions in living tissue through magnetic resonance spectroscopy or magnetic resonance imaging. Here we review DNP techniques to monitor metabolism in cultured cells, perfused hearts, and perfused livers, focusing on our experiences with hyperpolarized [1-13C]pyruvate. We present detailed approaches to optimize the DNP procedure, streamline biological sample preparation, and maximize detection of specific metabolic activities. We also discuss practical aspects in the choice of metabolic substrates for hyperpolarization studies, and outline some of the current technical and conceptual challenges in the field, including efforts to use hyperpolarization to quantify metabolic rates in vivo. PMID:26358902

  15. Real-time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy.

    PubMed

    Schroeder, Marie A; Atherton, Helen J; Ball, Daniel R; Cole, Mark A; Heather, Lisa C; Griffin, Julian L; Clarke, Kieran; Radda, George K; Tyler, Damian J

    2009-08-01

    The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart disease. In this study, we measured Krebs cycle flux in real time in perfused rat hearts using hyperpolarized magnetic resonance spectroscopy (MRS). [2-(13)C]Pyruvate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of (13)C-labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of (13)C-labeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly elevated. These results showed that the control and fluxes of the Krebs cycle in heart disease can be studied using hyperpolarized [2-(13)C]pyruvate.

  16. Biosynthesis of highly enriched 13C-lycopene for human metabolic studies using repeated batch tomato cell culturing with 13C-glucose.

    PubMed

    Moran, Nancy Engelmann; Rogers, Randy B; Lu, Chi-Hua; Conlon, Lauren E; Lila, Mary Ann; Clinton, Steven K; Erdman, John W

    2013-08-15

    While putative disease-preventing lycopene metabolites are found in both tomato (Solanum lycopersicum) products and in their consumers, mammalian lycopene metabolism is poorly understood. Advances in tomato cell culturing techniques offer an economical tool for generation of highly-enriched (13)C-lycopene for human bioavailability and metabolism studies. To enhance the (13)C-enrichment and yields of labelled lycopene from the hp-1 tomato cell line, cultures were first grown in (13)C-glucose media for three serial batches and produced increasing proportions of uniformly labelled lycopene (14.3±1.2%, 39.6±0.5%, and 48.9±1.5%) with consistent yields (from 5.8 to 9 mg/L). An optimised 9-day-long (13)C-loading and 18-day-long labelling strategy developed based on glucose utilisation and lycopene yields, yielded (13)C-lycopene with 93% (13)C isotopic purity, and 55% of isotopomers were uniformly labelled. Furthermore, an optimised acetone and hexane extraction led to a fourfold increase in lycopene recovery from cultures compared to a standard extraction.

  17. Microbial metabolism in soil at low temperatures: Mechanisms unraveled by position-specific 13C labeling

    NASA Astrophysics Data System (ADS)

    Bore, Ezekiel

    2016-04-01

    Microbial transformation of organic substances in soil is the most important process of the C cycle. Most of the current studies base their information about transformation of organic substances on incubation studies under laboratory conditions and thus, we have a profound knowledge on SOM transformations at ambient temperatures. However, metabolic pathway activities at low temperature are not well understood, despite the fact that the processes are relevant for many soils globally and seasonally. To analyze microbial metabolism at low soil temperatures, isotopomeres of position-specifically 13C labeled glucose were incubated at three temperature; 5, -5 -20 oC. Soils were sampled after 1, 3 and 10 days and additionally after 30 days for samples at -20 °C. The 13C from individual molecule position was quantifed in respired CO2, bulk soil, extractable organic C and extractable microbial biomass by chloroform fumigation extraction (CFE) and cell membranes of microbial communities classified by 13C phospholipid fatty acid (PLFA) analysis. 13CO2 released showed a dominance of the flux from C-1 position at 5 °C. Consequently, at 5 °C, pentose phosphate pathway activity is a dominant metabolic pathway of glucose metabolization. In contrast to -5 °C and -20 oC, metabolic behaviors completely switched towards a preferential respiration of the glucose C-4 position. With decreasing temperature, microorganism strongly shifted towards metabolization of glucose via glycolysis which indicates a switch to cellular maintenance. High recoveries of 13C in extractable microbial biomass at -5 °C indicates optimal growth condition for the microorganisms. PLFA analysis showed high incorporation of 13C into Gram negative bacteria at 5 °C but decreased with temperature. Gram positive bacteria out-competed Gram negatives with decreasing temperature. This study revealed a remarkable microbial activity at temperatures below 0 °C, differing significantly from that at ambient

  18. Metabolic pathways for ketone body production. /sup 13/C NMR spectroscopy of rat liver in vivo using /sup 13/C-multilabeled fatty acids

    SciTech Connect

    Pahl-Wostl, C.; Seelig, J.

    1986-11-04

    The hormonal regulation of ketogenesis in the liver of living rat has been studied noninvasively with /sup 13/C nuclear magnetic resonance. The spatial selection for the liver was better than 90%, with extrahepatic adipose tissue contribution only a very small amount of signal. The metabolic activities of the liver were investigated by infusion of /sup 13/C-labeled butyrate in the jugular vein of the anesthetized rat. The rate of butyrate infusion was chosen to be close to the maximum oxidative capacity of the rat liver, and the /sup 13/C signal intensities were enhanced by using doubly labeled (1,3-/sup 13/C)butyrate as a substrate. Different /sup 13/C NMR spectra and hence different metabolites were observed depending on the hormonal state of the animal. The /sup 13/C NMR studies demonstrate that even when rate of acetyl-CoA production are high, the disposal of this compound is not identical in fasted and diabetic animals. This supports previous suggestions that the redox state of the mitochondrion represents the most important factor in regulation. For a given metabolic state of the animal, different signal intensities were obtained depending on whether butyrate was labeled at C-1, C-3, or C-1,3. From the ratios of incorporation of /sup 13/C label into the carbons of 3-hydroxybutyrate, it could be estimated that a large fraction of butyrate evaded ..beta..-oxidation to acetyl-CoA but was converted directly to acetoacetyl-CoA. /sup 13/C-labeled glucose could be detected in vivo in the liver of diabetic rats.

  19. Multi-objective experimental design for (13)C-based metabolic flux analysis.

    PubMed

    Bouvin, Jeroen; Cajot, Simon; D'Huys, Pieter-Jan; Ampofo-Asiama, Jerry; Anné, Jozef; Van Impe, Jan; Geeraerd, Annemie; Bernaerts, Kristel

    2015-10-01

    (13)C-based metabolic flux analysis is an excellent technique to resolve fluxes in the central carbon metabolism but costs can be significant when using specialized tracers. This work presents a framework for cost-effective design of (13)C-tracer experiments, illustrated on two different networks. Linear and non-linear optimal input mixtures are computed for networks for Streptomyces lividans and a carcinoma cell line. If only glucose tracers are considered as labeled substrate for a carcinoma cell line or S. lividans, the best parameter estimation accuracy is obtained by mixtures containing high amounts of 1,2-(13)C2 glucose combined with uniformly labeled glucose. Experimental designs are evaluated based on a linear (D-criterion) and non-linear approach (S-criterion). Both approaches generate almost the same input mixture, however, the linear approach is favored due to its low computational effort. The high amount of 1,2-(13)C2 glucose in the optimal designs coincides with a high experimental cost, which is further enhanced when labeling is introduced in glutamine and aspartate tracers. Multi-objective optimization gives the possibility to assess experimental quality and cost at the same time and can reveal excellent compromise experiments. For example, the combination of 100% 1,2-(13)C2 glucose with 100% position one labeled glutamine and the combination of 100% 1,2-(13)C2 glucose with 100% uniformly labeled glutamine perform equally well for the carcinoma cell line, but the first mixture offers a decrease in cost of $ 120 per ml-scale cell culture experiment. We demonstrated the validity of a multi-objective linear approach to perform optimal experimental designs for the non-linear problem of (13)C-metabolic flux analysis. Tools and a workflow are provided to perform multi-objective design. The effortless calculation of the D-criterion can be exploited to perform high-throughput screening of possible (13)C-tracers, while the illustrated benefit of multi

  20. Photobioreactor design for isotopic non-stationary 13C-metabolic flux analysis (INST 13C-MFA) under photoautotrophic conditions.

    PubMed

    Martzolff, Arnaud; Cahoreau, Edern; Cogne, Guillaume; Peyriga, Lindsay; Portais, Jean-Charles; Dechandol, Emmanuel; Le Grand, Fabienne; Massou, Stéphane; Gonçalves, Olivier; Pruvost, Jérémy; Legrand, Jack

    2012-12-01

    Adaptive metabolic behavior of photoautotrophic microorganisms toward genetic and environmental perturbations can be interpreted in a quantitative depiction of carbon flow through a biochemical reaction network using isotopic non-stationary (13) C-metabolic flux analysis (INST (13) C-MFA). To evaluate (13) C-metabolic flux maps for Chlamydomonas reinhardtii, an original experimental framework was designed allowing rapid, reliable collection of high-quality isotopomer data against time. It involved (i) a short-time (13) C labeling injection device based on mixing control in a torus-shaped photobioreactor with plug-flow hydrodynamics allowing a sudden step-change in the (13) C proportion in the substrate feed and (ii) a rapid sampling procedure using an automatic fast filtration method coupled to a manual rapid liquid nitrogen quenching step. (13) C-substrate labeling enrichment was controlled through the total dissolved inorganic carbon concentration in the pulsed solution. First results were obtained from steady-state continuous culture measurements allowing the characterization of the kinetics of label incorporation into light-limited growing cells cultivated in a photobioreactor operating at the maximal biomass productivity for an incident photon flux density of 200 µmol m(-2) s(-1). (13)C label incorporation was measured for 21 intracellular metabolites using IC-MS/MS in 58 samples collected across a labeling experiment duration of 7 min. The fastest labeling rate was observed for 2/3-phosphoglycerate with an apparent isotopic stationary state reached after 300 s. The labeling rate was consistent with the optimized mixing time of about 4.9 s inside the reactor and the shortest reliable sampling period assessed at 5 s.

  1. Monitoring electron donor metabolism under variable electron acceptor conditions using 13C-labeled lactate

    NASA Astrophysics Data System (ADS)

    Bill, M.; Conrad, M. E.; Yang, L.; Beller, H. R.; Brodie, E. L.

    2010-12-01

    Three sets of flow-through columns constructed with aquifer sediment from Hanford (WA) were used to study reduction of Cr(VI) to poorly soluble Cr(III) under denitrifying, sulfate-reducing/fermentative, and iron-reducing conditions with lactate as the electron donor. In order to understand the relationship between electron donors and biomarkers, and to determine the differences in carbon isotope fractionation resulting from different microbial metabolic processes, we monitored the variation in carbon isotopes in dissolved inorganic carbon (DIC), in total organic carbon (TOC), and in lactate, acetate and propionate. The greatest enrichment in 13C in columns was observed under denitrifying conditions. The δ13C of DIC increased by ~1750 to ~2000‰ fifteen days after supplementation of natural abundance lactate with a 13C-labeled lactate tracer (for an influent δ13C of ~2250‰ for the lactate) indicating almost complete oxidation of the electron donor. The denitrifying columns were among the most active columns and had the highest cell counts and the denitrification rate was highly correlated with Cr(VI) reduction rate. δ13C values of DIC ranged from ~540 to ~1170‰ for iron-reducing conditions. The lower enrichment in iron columns was related to the lower biological activity observed with lower yields of RNA and cell numbers in the column effluents. The carbon isotope shift in the sulfate-reducing ~198 to ~1960‰ for sulfate-reducing conditions reflecting the lower levels of the lactate in these columns. Additionally, in two of the sulfate columns, almost complete fermentation of the lactate occurred, producing acetate and propionate with the labeled carbon signature, but relatively smaller amounts of inorganic carbon. For all electron-accepting conditions, TOC yielded similar δ13C values as lactate stock solutions. Differences in C use efficiency, metabolic rate or metabolic pathway contributed to the differing TOC δ13C to DIC δ13C ratios between treatments

  2. Metabolism of [U-13C]glucose in Human Brain Tumors In Vivo

    PubMed Central

    Maher, Elizabeth A.; Marin-Valencia, Isaac; Bachoo, Robert M.; Mashimo, Tomoyuki; Raisanen, Jack; Hatanpaa, Kimmo J.; Jindal, Ashish; Jeffrey, F. Mark; Choi, Changho; Madden, Christopher; Mathews, Dana; Pascual, Juan M.; Mickey, Bruce E.; Malloy, Craig R.; DeBerardinis, Ralph J.

    2012-01-01

    Glioblastomas (GBMs) and brain metastases demonstrate avid uptake of 18fluoro-2-deoxyglucose (FDG) by positron emission tomography (PET) and display perturbations of intracellular metabolite pools by 1H magnetic resonance spectroscopy (MRS). These observations suggest that metabolic reprogramming contributes to brain tumor growth in vivo. The Warburg effect, excess metabolism of glucose to lactate in the presence of oxygen, is a hallmark of cancer cells in culture. FDG-positive tumors are assumed to metabolize glucose in a similar manner, with high rates of lactate formation compared to mitochondrial glucose oxidation, but few studies have specifically examined the metabolic fates of glucose in vivo. In particular, the capacity of human brain malignancies to oxidize glucose in the tricarboxylic acid cycle is unknown. Here we studied the metabolism of human brain tumors in situ. [U-13C]glucose was infused during surgical resection, and tumor samples were subsequently subjected to 13C NMR spectroscopy. Analysis of tumor metabolites revealed lactate production, as expected. We also determined that pyruvate dehydrogenase, turnover of the TCA cycle, anaplerosis and de novo glutamine and glycine synthesis contributed significantly to the ultimate disposition of glucose carbon. Surprisingly, less than 50% of the acetyl-CoA pool was derived from blood-borne glucose, suggesting that additional substrates contribute to tumor bioenergetics. This study illustrates a convenient approach that capitalizes on the high information content of 13C NMR spectroscopy and enables the analysis of intermediary metabolism in diverse malignancies growing in their native microenvironment. PMID:22419606

  3. Simultaneous steady-state and dynamic 13C NMR can differentiate alternative routes of pyruvate metabolism in living cancer cells.

    PubMed

    Yang, Chendong; Harrison, Crystal; Jin, Eunsook S; Chuang, David T; Sherry, A Dean; Malloy, Craig R; Merritt, Matthew E; DeBerardinis, Ralph J

    2014-02-28

    Metabolic reprogramming facilitates cancer cell growth, so quantitative metabolic flux measurements could produce useful biomarkers. However, current methods to analyze flux in vivo provide either a steady-state overview of relative activities (infusion of (13)C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized (13)C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined (13)C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-(13)C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-(13)C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[(13)C]O3(-) and [1-(13)C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-(13)C]pyruvate. Quantitation of hyperpolarized H[(13)C]O3(-) provided a single definitive metabolic rate, which was then used to convert relative rates derived from isotopomer analysis into quantitative fluxes. This revealed that H[(13)C]O3(-) appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1-(13)C]pyruvate metabolism, enhancing exchanges with [1-(13)C]lactate and suppressing H[(13)C]O3(-) formation. Furthermore, inhibiting Akt, an oncogenic kinase that stimulates glycolysis, reversed these effects, indicating that metabolism of pyruvate by both LDH and pyruvate dehydrogenase is subject to the acute effects of oncogenic signaling on glycolysis. The data suggest that combining (13)C isotopomer analyses and dynamic hyperpolarized (13)C spectroscopy may enable

  4. (13) C-metabolic flux analysis of human adenovirus infection: Implications for viral vector production.

    PubMed

    Carinhas, Nuno; Koshkin, Alexey; Pais, Daniel A M; Alves, Paula M; Teixeira, Ana P

    2017-01-01

    Adenoviruses are human pathogens increasingly used as gene therapy and vaccination vectors. However, their impact on cell metabolism is poorly characterized. We performed carbon labeling experiments with [1,2-(13) C]glucose or [U-(13) C]glutamine to evaluate metabolic alterations in the amniocyte-derived, E1-transformed 1G3 cell line during production of a human adenovirus type 5 vector (AdV5). Nonstationary (13) C-metabolic flux analysis revealed increased fluxes of glycolysis (17%) and markedly PPP (over fourfold) and cytosolic AcCoA formation (nearly twofold) following infection of growing cells. Interestingly, infection of growth-arrested cells increased overall carbon flow even more, including glutamine anaplerosis and TCA cycle activity (both over 1.5-fold), but was unable to stimulate the PPP and was associated with a steep drop in AdV5 replication (almost 80%). Our results underscore the importance of nucleic and fatty acid biosynthesis for adenovirus replication. Overall, we portray a metabolic blueprint of human adenovirus infection, highlighting similarities with other viruses and cancer, and suggest strategies to improve AdV5 production. Biotechnol. Bioeng. 2017;114: 195-207. © 2016 Wiley Periodicals, Inc.

  5. (13)C-metabolic flux analysis of co-cultures: A novel approach.

    PubMed

    Gebreselassie, Nikodimos A; Antoniewicz, Maciek R

    2015-09-01

    In this work, we present a novel approach for performing (13)C metabolic flux analysis ((13)C-MFA) of co-culture systems. We demonstrate for the first time that it is possible to determine metabolic flux distributions in multiple species simultaneously without the need for physical separation of cells or proteins, or overexpression of species-specific products. Instead, metabolic fluxes for each species in a co-culture are estimated directly from isotopic labeling of total biomass obtained using conventional mass spectrometry approaches such as GC-MS. In addition to determining metabolic fluxes, this approach estimates the relative population size of each species in a mixed culture and inter-species metabolite exchange. As such, it enables detailed studies of microbial communities including species dynamics and interactions between community members. The methodology is experimentally validated here using a co-culture of two E. coli knockout strains. Taken together, this work greatly extends the scope of (13)C-MFA to a large number of multi-cellular systems that are of significant importance in biotechnology and medicine.

  6. Concentric Rings K-Space Trajectory for Hyperpolarized 13C MR Spectroscopic Imaging

    PubMed Central

    Jiang, Wenwen; Lustig, Michael; Larson, Peder E.Z.

    2014-01-01

    Purpose To develop a robust and rapid imaging technique for hyperpolarized 13C MR Spectroscopic Imaging (MRSI) and investigate its performance. Methods A concentric rings readout trajectory with constant angular velocity is proposed for hyperpolarized 13C spectroscopic imaging and its properties are analyzed. Quantitative analyses of design tradeoffs are presented for several imaging scenarios. The first application of concentric rings on 13C phantoms and in vivo animal hyperpolarized 13C MRSI studies were performed to demonstrate the feasibility of the proposed method. Finally, a parallel imaging accelerated concentric rings study is presented. Results The concentric rings MRSI trajectory has the advantages of acquisition timesaving compared to echo-planar spectroscopic imaging (EPSI). It provides sufficient spectral bandwidth with relatively high SNR efficiency compared to EPSI and spiral techniques. Phantom and in vivo animal studies showed good image quality with half the scan time and reduced pulsatile flow artifacts compared to EPSI. Parallel imaging accelerated concentric rings showed advantages over Cartesian sampling in g-factor simulations and demonstrated aliasing-free image quality in a hyperpolarized 13C in vivo study. Conclusion The concentric rings trajectory is a robust and rapid imaging technique that fits very well with the speed, bandwidth, and resolution requirements of hyperpolarized 13C MRSI. PMID:25533653

  7. Metabolic network reconstruction, growth characterization and 13C-metabolic flux analysis of the extremophile Thermus thermophilus HB8.

    PubMed

    Swarup, Aditi; Lu, Jing; DeWoody, Kathleen C; Antoniewicz, Maciek R

    2014-07-01

    Thermus thermophilus is an extremely thermophilic bacterium with significant biotechnological potential. In this work, we have characterized aerobic growth characteristics of T. thermophilus HB8 at temperatures between 50 and 85°C, constructed a metabolic network model of its central carbon metabolism and validated the model using (13)C-metabolic flux analysis ((13)C-MFA). First, cells were grown in batch cultures in custom constructed mini-bioreactors at different temperatures to determine optimal growth conditions. The optimal temperature for T. thermophilus grown on defined medium with glucose was 81°C. The maximum growth rate was 0.25h(-1). Between 50 and 81°C the growth rate increased by 7-fold and the temperature dependence was described well by an Arrhenius model with an activation energy of 47kJ/mol. Next, we performed a (13)C-labeling experiment with [1,2-(13)C] glucose as the tracer and calculated intracellular metabolic fluxes using (13)C-MFA. The results provided support for the constructed network model and highlighted several interesting characteristics of T. thermophilus metabolism. We found that T. thermophilus largely uses glycolysis and TCA cycle to produce biosynthetic precursors, ATP and reducing equivalents needed for cells growth. Consistent with its proposed metabolic network model, we did not detect any oxidative pentose phosphate pathway flux or Entner-Doudoroff pathway activity. The biomass precursors erythrose-4-phosphate and ribose-5-phosphate were produced via the non-oxidative pentose phosphate pathway, and largely via transketolase, with little contribution from transaldolase. The high biomass yield on glucose that was measured experimentally was also confirmed independently by (13)C-MFA. The results presented here provide a solid foundation for future studies of T. thermophilus and its metabolic engineering applications.

  8. SUMOFLUX: A Generalized Method for Targeted 13C Metabolic Flux Ratio Analysis.

    PubMed

    Kogadeeva, Maria; Zamboni, Nicola

    2016-09-01

    Metabolic fluxes are a cornerstone of cellular physiology that emerge from a complex interplay of enzymes, carriers, and nutrients. The experimental assessment of in vivo intracellular fluxes using stable isotopic tracers is essential if we are to understand metabolic function and regulation. Flux estimation based on 13C or 2H labeling relies on complex simulation and iterative fitting; processes that necessitate a level of expertise that ordinarily preclude the non-expert user. To overcome this, we have developed SUMOFLUX, a methodology that is broadly applicable to the targeted analysis of 13C-metabolic fluxes. By combining surrogate modeling and machine learning, we trained a predictor to specialize in estimating flux ratios from measurable 13C-data. SUMOFLUX targets specific flux features individually, which makes it fast, user-friendly, applicable to experimental design and robust in terms of experimental noise and exchange flux magnitude. Collectively, we predict that SUMOFLUX's properties realistically pave the way to high-throughput flux analyses.

  9. SUMOFLUX: A Generalized Method for Targeted 13C Metabolic Flux Ratio Analysis

    PubMed Central

    Kogadeeva, Maria

    2016-01-01

    Metabolic fluxes are a cornerstone of cellular physiology that emerge from a complex interplay of enzymes, carriers, and nutrients. The experimental assessment of in vivo intracellular fluxes using stable isotopic tracers is essential if we are to understand metabolic function and regulation. Flux estimation based on 13C or 2H labeling relies on complex simulation and iterative fitting; processes that necessitate a level of expertise that ordinarily preclude the non-expert user. To overcome this, we have developed SUMOFLUX, a methodology that is broadly applicable to the targeted analysis of 13C-metabolic fluxes. By combining surrogate modeling and machine learning, we trained a predictor to specialize in estimating flux ratios from measurable 13C-data. SUMOFLUX targets specific flux features individually, which makes it fast, user-friendly, applicable to experimental design and robust in terms of experimental noise and exchange flux magnitude. Collectively, we predict that SUMOFLUX's properties realistically pave the way to high-throughput flux analyses. PMID:27626798

  10. Optimal tracers for parallel labeling experiments and (13)C metabolic flux analysis: A new precision and synergy scoring system.

    PubMed

    Crown, Scott B; Long, Christopher P; Antoniewicz, Maciek R

    2016-11-01

    (13)C-Metabolic flux analysis ((13)C-MFA) is a widely used approach in metabolic engineering for quantifying intracellular metabolic fluxes. The precision of fluxes determined by (13)C-MFA depends largely on the choice of isotopic tracers and the specific set of labeling measurements. A recent advance in the field is the use of parallel labeling experiments for improved flux precision and accuracy. However, as of today, no systemic methods exist for identifying optimal tracers for parallel labeling experiments. In this contribution, we have addressed this problem by introducing a new scoring system and evaluating thousands of different isotopic tracer schemes. Based on this extensive analysis we have identified optimal tracers for (13)C-MFA. The best single tracers were doubly (13)C-labeled glucose tracers, including [1,6-(13)C]glucose, [5,6-(13)C]glucose and [1,2-(13)C]glucose, which consistently produced the highest flux precision independent of the metabolic flux map (here, 100 random flux maps were evaluated). Moreover, we demonstrate that pure glucose tracers perform better overall than mixtures of glucose tracers. For parallel labeling experiments the optimal isotopic tracers were [1,6-(13)C]glucose and [1,2-(13)C]glucose. Combined analysis of [1,6-(13)C]glucose and [1,2-(13)C]glucose labeling data improved the flux precision score by nearly 20-fold compared to widely use tracer mixture 80% [1-(13)C]glucose +20% [U-(13)C]glucose.

  11. Metabolite channeling and compartmentation in the human cell line AGE1.HN determined by 13C labeling experiments and 13C metabolic flux analysis.

    PubMed

    Niklas, Jens; Sandig, Volker; Heinzle, Elmar

    2011-12-01

    This study focused on analyzing active pathways and the metabolic flux distribution in human neuronal AGE1.HN cells that is a desirable basis for a rational design and optimization of producing cell lines and production processes for biopharmaceuticals. (13)C-labeling experiments and (13)C metabolic flux analysis were conducted using glucose, glutamine, alanine and lactate tracers in parallel experiments. Connections between cytosolic and mitochondrial metabolite pools were verified, e.g., flux from TCA cycle metabolite (13)C to glycolytic metabolites. It was also found that lactate and alanine are produced from the same pyruvate pool and that consumed alanine is mainly directly metabolized and secreted as lactate. Activity of the pentose phosphate pathway was low being around 2.3% of the glucose uptake flux. This might be compensated in AGE1.HN by high mitochondrial malic enzyme flux producing NADPH. Mitochondrial pyruvate transport was almost zero. Instead pyruvate carbons were channeled via oxaloacetate into the TCA cycle which was mainly fed via α-ketoglutarate and oxaloacetate during the investigated phase. The data indicate that further optimization of this cell line should focus on the improved substrate usage which can be accomplished by an improved connectivity between glycolytic and mitochondrial pyruvate pools or by better control of the substrate uptake.

  12. HepatoDyn: A Dynamic Model of Hepatocyte Metabolism That Integrates 13C Isotopomer Data

    PubMed Central

    Foguet, Carles; Selivanov, Vitaly A.; Fanchon, Eric; Guinovart, Joan J.; de Atauri, Pedro; Cascante, Marta

    2016-01-01

    The liver performs many essential metabolic functions, which can be studied using computational models of hepatocytes. Here we present HepatoDyn, a highly detailed dynamic model of hepatocyte metabolism. HepatoDyn includes a large metabolic network, highly detailed kinetic laws, and is capable of dynamically simulating the redox and energy metabolism of hepatocytes. Furthermore, the model was coupled to the module for isotopic label propagation of the software package IsoDyn, allowing HepatoDyn to integrate data derived from 13C based experiments. As an example of dynamical simulations applied to hepatocytes, we studied the effects of high fructose concentrations on hepatocyte metabolism by integrating data from experiments in which rat hepatocytes were incubated with 20 mM glucose supplemented with either 3 mM or 20 mM fructose. These experiments showed that glycogen accumulation was significantly lower in hepatocytes incubated with medium supplemented with 20 mM fructose than in hepatocytes incubated with medium supplemented with 3 mM fructose. Through the integration of extracellular fluxes and 13C enrichment measurements, HepatoDyn predicted that this phenomenon can be attributed to a depletion of cytosolic ATP and phosphate induced by high fructose concentrations in the medium. PMID:27124774

  13. 13C MRS and LC–MS Flux Analysis of Tumor Intermediary Metabolism

    PubMed Central

    Shestov, Alexander A.; Lee, Seung-Cheol; Nath, Kavindra; Guo, Lili; Nelson, David S.; Roman, Jeffrey C.; Leeper, Dennis B.; Wasik, Mariusz A.; Blair, Ian A.; Glickson, Jerry D.

    2016-01-01

    We present the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism, including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, the TCA cycle as well as its anaplerotic pathways, pyruvate–malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. The model that is called Bonded Cumomer Analysis for application to 13C magnetic resonance spectroscopy (13C MRS) data and Fragmented Cumomer Analysis for mass spectrometric data is a refined and efficient form of isotopomer analysis that can readily be expanded to incorporate glycogen, phospholipid, and other pathways thereby encompassing all the key pathways of tumor intermediary metabolism. Validation was achieved by demonstrating agreement of experimental measurements of the metabolic rates of oxygen consumption, glucose consumption, lactate production, and glutamate pool size with independent measurements of these parameters in cultured human DB-1 melanoma cells. These cumomer models have been applied to studies of DB-1 melanoma and DLCL2 human diffuse large B-cell lymphoma cells in culture and as xenografts in nude mice at 9.4 T. The latter studies demonstrate the potential translation of these methods to in situ studies of human tumor metabolism by MRS with stable 13C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T). The melanoma studies indicate that this tumor line obtains 51% of its ATP by mitochondrial metabolism and 49% by glycolytic metabolism under both euglycemic (5 mM glucose) and hyperglycemic conditions (26 mM glucose). While a high level of glutamine uptake is detected corresponding to ~50% of TCA cycle flux under hyperglycemic conditions, and ~100% of TCA cycle flux under euglycemic conditions, glutaminolysis flux and its contributions to ATP synthesis were very small. Studies of human lymphoma cells demonstrated that inhibition of

  14. (13)C-metabolic flux analysis in S-adenosyl-L-methionine production by Saccharomyces cerevisiae.

    PubMed

    Hayakawa, Kenshi; Kajihata, Shuichi; Matsuda, Fumio; Shimizu, Hiroshi

    2015-11-01

    S-Adenosyl-L-methionine (SAM) is a major biological methyl group donor, and is used as a nutritional supplement and prescription drug. Yeast is used for the industrial production of SAM owing to its high intracellular SAM concentrations. To determine the regulation mechanisms responsible for such high SAM production, (13)C-metabolic flux analysis ((13)C-MFA) was conducted to compare the flux distributions in the central metabolism between Kyokai no. 6 (high SAM-producing) and S288C (control) strains. (13)C-MFA showed that the levels of tricarboxylic acid (TCA) cycle flux in SAM-overproducing strain were considerably increased compared to those in the S228C strain. Analysis of ATP balance also showed that a larger amount of excess ATP was produced in the Kyokai 6 strain because of increased oxidative phosphorylation. These results suggest that high SAM production in Kyokai 6 strains could be attributed to enhanced ATP regeneration with high TCA cycle fluxes and respiration activity. Thus, maintaining high respiration efficiency during cultivation is important for improving SAM production.

  15. Hybrid optimization for 13C metabolic flux analysis using systems parametrized by compactification

    PubMed Central

    Yang, Tae Hoon; Frick, Oliver; Heinzle, Elmar

    2008-01-01

    Background The importance and power of isotope-based metabolic flux analysis and its contribution to understanding the metabolic network is increasingly recognized. Its application is, however, still limited partly due to computational inefficiency. 13C metabolic flux analysis aims to compute in vivo metabolic fluxes in terms of metabolite balancing extended by carbon isotopomer balances and involves a nonlinear least-squares problem. To solve the problem more efficiently, improved numerical optimization techniques are necessary. Results For flux computation, we developed a gradient-based hybrid optimization algorithm. Here, independent flux variables were compactified into [0, 1)-ranged variables using a single transformation rule. The compactified parameters could be discriminated between non-identifiable and identifiable variables after model linearization. The developed hybrid algorithm was applied to the central metabolism of Bacillus subtilis with only succinate and glutamate as carbon sources. This creates difficulties caused by symmetry of succinate leading to limited introduction of 13C labeling information into the system. The algorithm was found to be superior to its parent algorithms and to global optimization methods both in accuracy and speed. The hybrid optimization with tolerance adjustment quickly converged to the minimum with close to zero deviation and exactly re-estimated flux variables. In the metabolic network studied, some fluxes were found to be either non-identifiable or nonlinearly correlated. The non-identifiable fluxes could correctly be predicted a priori using the model identification method applied, whereas the nonlinear flux correlation was revealed only by identification runs using different starting values a posteriori. Conclusion This fast, robust and accurate optimization method is useful for high-throughput metabolic flux analysis, a posteriori identification of possible parameter correlations, and also for Monte Carlo

  16. Probing metabolic processes of intact soil microbial communities using position-specific 13C-labeled glucose

    NASA Astrophysics Data System (ADS)

    Fairbanks, D. E.; Hungate, B. A.; KOCH, G. W.; Schwartz, E.; Dijkstra, P.

    2012-12-01

    Soils represent one of the largest carbon pools in the terrestrial biosphere and fluxes into or out of this pool may feedback to current climate change. Understanding the mechanisms behind microbial processes regulating C cycling, microbial turnover, and soil organic matter stabilization is hindered by our lack of understanding of the details of microbial physiology in soils. Position-specific 13C labeled metabolic tracers are proposed as a new way to probe microbial community energy production, biosynthesis, C use efficiency (the proportion of substrate incorporated into microbial biomass), and enables the determination of C fluxes through the various C metabolic pathways. We determined the 13CO2 production from microbial communities within a one hour time frame by adding six isotopomers (1-13C, 2-13C, 3-13C, 4-13C, 5-13C, 6-13C) of glucose in parallel incubations using a young volcanic soil (Pinyon-juniper wood, near Sunset Crater, Flagstaff, Arizona). We compared the measured rates of position-specific 13CO2 production with modeled results based on glucose (1-13C and U-13C) and pyruvate (1-13C and 2,3-13C) incubations. These labeling and modeling techniques may improve our ability to analyze the biochemistry and ecophysiology of intact soil microbial communities.

  17. Spinning sidebands from chemical shift anisotropy in 13C MAS imaging.

    PubMed

    Scheler, U; Blümich, B; Spiess, H W

    1993-07-01

    Solid state imaging by 13C MAS imaging is described. The spinning sidebands occurring at moderate spinning speeds, which disturb the images, can be suppressed by TOSS. For rigid solids the spatial resolution that can be achieved in this way is better than that of 1H images at the same spinning speed. Spatially resolved spectra with or without spinning sidebands can likewise be recorded providing information about the isotropic and the anisotropic chemical shifts which can be exploited for the study of structure, order and dynamics. The techniques are demonstrated on a phantom made with 13C-labelled glycine.

  18. 15N and 13C NMR Determination of Allantoin Metabolism in Developing Soybean Cotyledons 1

    PubMed Central

    Coker, George T.; Schaefer, Jacob

    1985-01-01

    The metabolism of allantoin by immature cotyledons of soybean (Glycine max L. cv Elf) grown in culture was investigated using solid state 13C and 15N nuclear magnetic resonance. All of the nitrogens of allantoin were incorporated into protein in a manner similar to that of each other and to the amide nitrogen of glutamine. The C-2 of allantoin was not incorporated into cellular material; presumably it was lost as CO2. About 50% of the C-5 of allantoin was incorporated into cellular material as a methylene carbon; the other 50% was presumably also lost as CO2. The 13C-15N bonds of [5-13C;1-15N] and [2-13C;1,3-15N]allantoin were broken prior to the incorporation of the nitrogens into protein. These data are consistent with allantoin's degradation to two molecules of urea and one two-carbon fragment. Cotyledons grown on allantoin as a source of nitrogen accumulated 21% of the nitrogen of cotyledons grown on glutamine. Only 50% of the nitrogen of the degraded allantoin was incorporated into the cotyledon as organic nitrogen; the other 50% was recovered as NH4+ in the media in which the cotyledons had been grown. The latter results suggests that the lower accumulation of nitrogen by cotyledons grown on allantoin was in part due to failure to assimilate NH4+ produced from allantoin. The seed coats had a higher activity of glutamine synthetase and a higher rate of allantoin degradation than cotyledons indicating that seed coats play an important role in the assimilation and degradation of allantoin. PMID:16663995

  19. Application of (13)C flux analysis to identify high-productivity CHO metabolic phenotypes.

    PubMed

    Templeton, Neil; Smith, Kevin D; McAtee-Pereira, Allison G; Dorai, Haimanti; Betenbaugh, Michael J; Lang, Steven E; Young, Jamey D

    2017-01-23

    Industrial bioprocesses place high demands on the energy metabolism of host cells to meet biosynthetic requirements for maximal protein expression. Identifying metabolic phenotypes that promote high expression is therefore a major goal of the biotech industry. We conducted a series of (13)C flux analysis studies to examine the metabolic response to IgG expression during early stationary phase of CHO cell cultures grown in 3L fed-batch bioreactors. We examined eight clones expressing four different IgGs and compared with three non-expressing host-cell controls. Some clones were genetically manipulated to be apoptosis-resistant by expressing Bcl-2Δ, which correlated with increased IgG production and elevated glucose metabolism. The metabolic phenotypes of the non-expressing, IgG-expressing, and Bcl-2Δ/IgG-expressing clones were fully segregated by hierarchical clustering analysis. Lactate consumption and citric acid cycle fluxes were most strongly associated with specific IgG productivity. These studies indicate that enhanced oxidative metabolism is a characteristic of high-producing CHO cell lines.

  20. Energy metabolism in the rat cortex under thiopental anaesthesia measured In Vivo by (13) C MRS.

    PubMed

    Sonnay, Sarah; Duarte, João M N; Just, Nathalie; Gruetter, Rolf

    2017-03-19

    Barbiturates, commonly used as general anaesthetics, depress neuronal activity and thus cerebral metabolism. Moreover, they are likely to disrupt the metabolic support of astrocytes to neurons, as well as the uptake of nutrients from circulation. By employing (13) C magnetic resonance spectroscopy (MRS) in vivo at high magnetic field, we characterized neuronal and astrocytic pathways of energy metabolism in the rat cortex under thiopental anaesthesia. The neuronal tricarboxylic acid (TCA) cycle rate was 0.46 ± 0.02 µmol/g/min, and the rate of the glutamate-glutamine cycle was 0.09 ± 0.02 µmol/g/min. In astrocytes, the TCA cycle rate was 0.16 ± 0.02 µmol/g/min, accounting for a quarter of whole brain glucose oxidation, pyruvate carboxylase rate was 0.02 ± 0.01 µmol/g/min, and glutamine synthetase was 0.12 ± 0.01 µmol/g/min. Relative to previous experiments under light α-chloralose anaesthesia, thiopental reduced oxidative metabolism in neurons and even more so in astrocytes. Interestingly, total oxidative metabolism in the cortex under thiopental anaesthesia surpassed the rate of pyruvate production by glycolysis, indicating substantial utilisation of substrates other than glucose, likely plasma lactate. © 2017 Wiley Periodicals, Inc.

  1. 13C and 15N natural isotope abundance reflects breast cancer cell metabolism

    NASA Astrophysics Data System (ADS)

    Tea, Illa; Martineau, Estelle; Antheaume, Ingrid; Lalande, Julie; Mauve, Caroline; Gilard, Francoise; Barillé-Nion, Sophie; Blackburn, Anneke C.; Tcherkez, Guillaume

    2016-09-01

    Breast cancer is the most common cancer in women worldwide. Despite the information provided by anatomopathological assessment and molecular markers (such as receptor expression ER, PR, HER2), breast cancer therapies and prognostics depend on the metabolic properties of tumor cells. However, metabolomics have not provided a robust and congruent biomarker yet, likely because individual metabolite contents are insufficient to encapsulate all of the alterations in metabolic fluxes. Here, we took advantage of natural 13C and 15N isotope abundance to show there are isotopic differences between healthy and cancer biopsy tissues or between healthy and malignant cultured cell lines. Isotope mass balance further suggests that these differences are mostly related to lipid metabolism, anaplerosis and urea cycle, three pathways known to be impacted in malignant cells. Our results demonstrate that the isotope signature is a good descriptor of metabolism since it integrates modifications in C partitioning and N excretion altogether. Our present study is thus a starting point to possible clinical applications such as patient screening and biopsy characterization in every cancer that is associated with metabolic changes.

  2. 13C and 15N natural isotope abundance reflects breast cancer cell metabolism

    PubMed Central

    Tea, Illa; Martineau, Estelle; Antheaume, Ingrid; Lalande, Julie; Mauve, Caroline; Gilard, Francoise; Barillé-Nion, Sophie; Blackburn, Anneke C.; Tcherkez, Guillaume

    2016-01-01

    Breast cancer is the most common cancer in women worldwide. Despite the information provided by anatomopathological assessment and molecular markers (such as receptor expression ER, PR, HER2), breast cancer therapies and prognostics depend on the metabolic properties of tumor cells. However, metabolomics have not provided a robust and congruent biomarker yet, likely because individual metabolite contents are insufficient to encapsulate all of the alterations in metabolic fluxes. Here, we took advantage of natural 13C and 15N isotope abundance to show there are isotopic differences between healthy and cancer biopsy tissues or between healthy and malignant cultured cell lines. Isotope mass balance further suggests that these differences are mostly related to lipid metabolism, anaplerosis and urea cycle, three pathways known to be impacted in malignant cells. Our results demonstrate that the isotope signature is a good descriptor of metabolism since it integrates modifications in C partitioning and N excretion altogether. Our present study is thus a starting point to possible clinical applications such as patient screening and biopsy characterization in every cancer that is associated with metabolic changes. PMID:27678172

  3. Metabolic Flux Elucidation for Large-Scale Models Using 13C Labeled Isotopes

    PubMed Central

    Suthers, Patrick F.; Burgard, Anthony P.; Dasika, Madhukar S.; Nowroozi, Farnaz; Van Dien, Stephen; Keasling, Jay D.; Maranas, Costas D.

    2007-01-01

    A key consideration in metabolic engineering is the determination of fluxes of the metabolites within the cell. This determination provides an unambiguous description of metabolism before and/or after engineering interventions. Here, we present a computational framework that combines a constraint-based modeling framework with isotopic label tracing on a large-scale. When cells are fed a growth substrate with certain carbon positions labeled with 13C, the distribution of this label in the intracellular metabolites can be calculated based on the known biochemistry of the participating pathways. Most labeling studies focus on skeletal representations of central metabolism and ignore many flux routes that could contribute to the observed isotopic labeling patterns. In contrast, our approach investigates the importance of carrying out isotopic labeling studies using a more comprehensive reaction network consisting of 350 fluxes and 184 metabolites in Escherichia coli including global metabolite balances on cofactors such as ATP, NADH, and NADPH. The proposed procedure is demonstrated on an E. coli strain engineered to produce amorphadiene, a precursor to the anti-malarial drug artemisinin. The cells were grown in continuous culture on glucose containing 20% [U-13C]glucose; the measurements are made using GC-MS performed on 13 amino acids extracted from the cells. We identify flux distributions for which the calculated labeling patterns agree well with the measurements alluding to the accuracy of the network reconstruction. Furthermore, we explore the robustness of the flux calculations to variability in the experimental MS measurements, as well as highlight the key experimental measurements necessary for flux determination. Finally, we discuss the effect of reducing the model, as well as shed light onto the customization of the developed computational framework to other systems. PMID:17632026

  4. (13)C-metabolic flux analysis of lipid accumulation in the oleaginous fungus Mucor circinelloides.

    PubMed

    Zhao, Lina; Zhang, Huaiyuan; Wang, Liping; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

    2015-12-01

    The oleaginous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with (13)C-glucose were performed with the growth of M. circinelloides and subsequent gas chromatography-mass spectrometric detection of (13)C-patterns in proteinogenic amino acids was carried out to identify the metabolic network topology and estimate intracellular fluxes. Our results showed that the high oleaginous strain WJ11 had higher flux of pentose phosphate pathway and malic enzyme, lower flux in tricarboxylic acid cycle, higher flux in glyoxylate cycle and ATP: citrate lyase, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis.

  5. Ursodeoxycholic acid treatment of hepatic steatosis: a (13)C NMR metabolic study.

    PubMed

    Nunes, Patrícia M; Jones, John G; Rolo, Anabela P; Palmeira, Carlos M M; Carvalho, Rui A

    2011-11-01

    Ursodeoxycholic acid (UDCA) is commonly used for the treatment of hepatobiliary disorders. In this study, we tested whether a 4-week treatment with this bile acid (12-15 mg/kg/day) could improve hepatic fatty acid oxidation in obese Zucker rats - a model for nonalcoholic fatty liver disease and steatosis. After 24 h of fasting, livers were perfused with physiological concentrations of [U-(13) C]nonesterified fatty acids and [3-(13) C]lactate/[3-(13) C]pyruvate. Steatosis was associated with abundant intracellular glucose, lactate, alanine and methionine, and low concentrations of choline and betaine. Steatotic livers also showed the highest output of glucose and lactate. Glucose and glycolytic products were mostly unlabeled, indicating active glycogenolysis and glycolysis after 24 h of fasting. UDCA treatment resulted in a general amelioration of liver metabolic abnormalities with a decrease in intracellular glucose and lactate, as well as their output. Hepatic betaine and methionine were also normalized after UDCA treatment, suggesting the amelioration of anti-oxidative defenses. Choline levels were not affected by the bile acid, which may indicate a deficient synthesis of very-low-density lipoproteins. The percentage contribution of [U-(13) C]nonesterified fatty acids to acetyl-coenzyme A entering the tricarboxylic acid (TCA) cycle was significantly lower in livers from Zucker obese rats relative to control rats: 23.1 ± 4.9% versus 44.1 ± 2.7% (p < 0.01). UDCA treatment did not alter significantly fatty acid oxidation in control rats, but improved significantly oxidation in Zucker obese rats to 46.0 ± 6.1% (p > 0.05), comparable with control group values. The TCA cycle activity subsequent to fatty acid oxidation was reduced in steatotic livers and improved when UDCA was administered (0.24 ± 0.04 versus 0.37 ± 0.05, p = 0.05). We further suggest that the mechanism of action of UDCA is either related to the activity of the

  6. Investigate the Metabolic Reprogramming of Saccharomyces cerevisiae for Enhanced Resistance to Mixed Fermentation Inhibitors via 13C Metabolic Flux Analysis

    PubMed Central

    Guo, Weihua; Chen, Yingying; Wei, Na; Feng, Xueyang

    2016-01-01

    The fermentation inhibitors from the pretreatment of lignocellulosic materials, e.g., acetic acid and furfural, are notorious due to their negative effects on the cell growth and chemical production. However, the metabolic reprogramming of the cells under these stress conditions, especially metabolic response for resistance to mixed inhibitors, has not been systematically investigated and remains mysterious. Therefore, in this study, 13C metabolic flux analysis (13C-MFA), a powerful tool to elucidate the intracellular carbon flux distributions, has been applied to two Saccharomyces cerevisiae strains with different tolerances to the inhibitors under acetic acid, furfural, and mixed (i.e., acetic acid and furfural) stress conditions to unravel the key metabolic responses. By analyzing the intracellular carbon fluxes as well as the energy and cofactor utilization under different conditions, we uncovered varied metabolic responses to different inhibitors. Under acetate stress, ATP and NADH production was slightly impaired, while NADPH tended towards overproduction. Under furfural stress, ATP and cofactors (including both NADH and NADPH) tended to be overproduced. However, under dual-stress condition, production of ATP and cofactors was severely impaired due to synergistic stress caused by the simultaneous addition of two fermentation inhibitors. Such phenomenon indicated the pivotal role of the energy and cofactor utilization in resisting the mixed inhibitors of acetic acid and furfural. Based on the discoveries, valuable insights are provided to improve the tolerance of S. cerevisiae strain and further enhance lignocellulosic fermentation. PMID:27532329

  7. Integration of a constraint-based metabolic model of Brassica napus developing seeds with (13)C-metabolic flux analysis.

    PubMed

    Hay, Jordan O; Shi, Hai; Heinzel, Nicolas; Hebbelmann, Inga; Rolletschek, Hardy; Schwender, Jorg

    2014-01-01

    The use of large-scale or genome-scale metabolic reconstructions for modeling and simulation of plant metabolism and integration of those models with large-scale omics and experimental flux data is becoming increasingly important in plant metabolic research. Here we report an updated version of bna572, a bottom-up reconstruction of oilseed rape (Brassica napus L.; Brassicaceae) developing seeds with emphasis on representation of biomass-component biosynthesis. New features include additional seed-relevant pathways for isoprenoid, sterol, phenylpropanoid, flavonoid, and choline biosynthesis. Being now based on standardized data formats and procedures for model reconstruction, bna572+ is available as a COBRA-compliant Systems Biology Markup Language (SBML) model and conforms to the Minimum Information Requested in the Annotation of Biochemical Models (MIRIAM) standards for annotation of external data resources. Bna572+ contains 966 genes, 671 reactions, and 666 metabolites distributed among 11 subcellular compartments. It is referenced to the Arabidopsis thaliana genome, with gene-protein-reaction (GPR) associations resolving subcellular localization. Detailed mass and charge balancing and confidence scoring were applied to all reactions. Using B. napus seed specific transcriptome data, expression was verified for 78% of bna572+ genes and 97% of reactions. Alongside bna572+ we also present a revised carbon centric model for (13)C-Metabolic Flux Analysis ((13)C-MFA) with all its reactions being referenced to bna572+ based on linear projections. By integration of flux ratio constraints obtained from (13)C-MFA and by elimination of infinite flux bounds around thermodynamically infeasible loops based on COBRA loopless methods, we demonstrate improvements in predictive power of Flux Variability Analysis (FVA). Using this combined approach we characterize the difference in metabolic flux of developing seeds of two B. napus genotypes contrasting in starch and oil content.

  8. Integration of a constraint-based metabolic model of Brassica napus developing seeds with 13C-metabolic flux analysis

    PubMed Central

    Hay, Jordan O.; Shi, Hai; Heinzel, Nicolas; Hebbelmann, Inga; Rolletschek, Hardy; Schwender, Jorg

    2014-01-01

    The use of large-scale or genome-scale metabolic reconstructions for modeling and simulation of plant metabolism and integration of those models with large-scale omics and experimental flux data is becoming increasingly important in plant metabolic research. Here we report an updated version of bna572, a bottom-up reconstruction of oilseed rape (Brassica napus L.; Brassicaceae) developing seeds with emphasis on representation of biomass-component biosynthesis. New features include additional seed-relevant pathways for isoprenoid, sterol, phenylpropanoid, flavonoid, and choline biosynthesis. Being now based on standardized data formats and procedures for model reconstruction, bna572+ is available as a COBRA-compliant Systems Biology Markup Language (SBML) model and conforms to the Minimum Information Requested in the Annotation of Biochemical Models (MIRIAM) standards for annotation of external data resources. Bna572+ contains 966 genes, 671 reactions, and 666 metabolites distributed among 11 subcellular compartments. It is referenced to the Arabidopsis thaliana genome, with gene-protein-reaction (GPR) associations resolving subcellular localization. Detailed mass and charge balancing and confidence scoring were applied to all reactions. Using B. napus seed specific transcriptome data, expression was verified for 78% of bna572+ genes and 97% of reactions. Alongside bna572+ we also present a revised carbon centric model for 13C-Metabolic Flux Analysis (13C-MFA) with all its reactions being referenced to bna572+ based on linear projections. By integration of flux ratio constraints obtained from 13C-MFA and by elimination of infinite flux bounds around thermodynamically infeasible loops based on COBRA loopless methods, we demonstrate improvements in predictive power of Flux Variability Analysis (FVA). Using this combined approach we characterize the difference in metabolic flux of developing seeds of two B. napus genotypes contrasting in starch and oil content. PMID

  9. Integration of a constraint-based metabolic model of Brassica napus developing seeds with 13C-metabolic flux analysis

    DOE PAGES

    Hay, Jordan O.; Shi, Hai; Heinzel, Nicolas; ...

    2014-12-19

    The use of large-scale or genome-scale metabolic reconstructions for modeling and simulation of plant metabolism and integration of those models with large-scale omics and experimental flux data is becoming increasingly important in plant metabolic research. Here we report an updated version of bna572, a bottom-up reconstruction of oilseed rape (Brassica napus L.; Brassicaceae) developing seeds with emphasis on representation of biomass-component biosynthesis. New features include additional seed-relevant pathways for isoprenoid, sterol, phenylpropanoid, flavonoid, and choline biosynthesis. Being now based on standardized data formats and procedures for model reconstruction, bna572+ is available as a COBRA-compliant Systems Biology Markup Language (SBML) modelmore » and conforms to the Minimum Information Requested in the Annotation of Biochemical Models (MIRIAM) standards for annotation of external data resources. Bna572+ contains 966 genes, 671 reactions, and 666 metabolites distributed among 11 subcellular compartments. It is referenced to the Arabidopsis thaliana genome, with gene-protein-reaction (GPR) associations resolving subcellular localization. Detailed mass and charge balancing and confidence scoring were applied to all reactions. Using B. napus seed specific transcriptome data, expression was verified for 78% of bna572+ genes and 97% of reactions. Alongside bna572+ we also present a revised carbon centric model for 13C-Metabolic Flux Analysis (13C-MFA) with all its reactions being referenced to bna572+ based on linear projections. By integration of flux ratio constraints obtained from 13C-MFA and by elimination of infinite flux bounds around thermodynamically infeasible loops based on COBRA loopless methods, we demonstrate improvements in predictive power of Flux Variability Analysis (FVA). In conclusion, using this combined approach we characterize the difference in metabolic flux of developing seeds of two B. napus genotypes contrasting in starch

  10. Mechanisms linking metabolism of Helicobacter pylori to (18)O and (13)C-isotopes of human breath CO2.

    PubMed

    Som, Suman; De, Anulekha; Banik, Gourab Dutta; Maity, Abhijit; Ghosh, Chiranjit; Pal, Mithun; Daschakraborty, Sunil B; Chaudhuri, Sujit; Jana, Subhra; Pradhan, Manik

    2015-06-03

    The gastric pathogen Helicobacter pylori utilize glucose during metabolism, but the underlying mechanisms linking to oxygen-18 ((18)O) and carbon-13 ((13)C)-isotopic fractionations of breath CO2 during glucose metabolism are poorly understood. Using the excretion dynamics of (18)O/(16)O and (13)C/(12)C-isotope ratios of breath CO2, we found that individuals with Helicobacter pylori infections exhibited significantly higher isotopic enrichments of (18)O in breath CO2 during the 2h-glucose metabolism regardless of the isotopic nature of the substrate, while no significant enrichments of (18)O in breath CO2 were manifested in individuals without the infections. In contrast, the (13)C-isotopic enrichments of breath CO2 were significantly higher in individuals with Helicobacter pylori compared to individuals without infections in response to (13)C-enriched glucose uptake, whereas a distinguishable change of breath (13)C/(12)C-isotope ratios was also evident when Helicobacter pylori utilize natural glucose. Moreover, monitoring the (18)O and (13)C-isotopic exchange in breath CO2 successfully diagnosed the eradications of Helicobacter pylori infections following a standard therapy. Our findings suggest that breath (12)C(18)O(16)O and (13)C(16)O(16)O can be used as potential molecular biomarkers to distinctively track the pathogenesis of Helicobacter pylori and also for eradication purposes and thus may open new perspectives into the pathogen's physiology along with isotope-specific non-invasive diagnosis of the infection.

  11. Effect of glyphosate on plant cell metabolism. 31P and 13C NMR studies.

    PubMed

    Gout, E; Bligny, R; Genix, P; Tissut, M; Douce, R

    1992-01-01

    The effect of glyphosate (N-phosphonomethyl glycine; the active ingredient of Roundup herbicide) on plant cells metabolism was analysed by 31P and 13C NMR using suspension-cultured sycamore (Acer pseudoplatanus L) cells. Cells were compressed in the NMR tube and perfused with an original arrangement enabling a tight control of the circulating nutrient medium. Addition of 1 mM glyphosate to the nutrient medium triggered the accumulation of shikimate (20-30 mumol g-1 cell wet weight within 50 h) and shikimate 3-phosphate (1-1.5 mumol g-1 cell wet weight within 50 h). From in vivo spectra it was demonstrated that these two compounds were accumulated in the cytoplasm where their concentrations reached potentially lethal levels. On the other hand, glyphosate present in the cytoplasmic compartment was extensively metabolized to yield aminomethylphosphonic acid which also accumulated in the cytoplasm. Finally, the results presented in this paper indicate that although the cell growth was stopped by glyphosate the cell respiration rates and the level of energy metabolism intermediates remained unchanged.

  12. High-throughput hyperpolarized 13C metabolic investigations using a multi-channel acquisition system

    NASA Astrophysics Data System (ADS)

    Lee, Jaehyuk; Ramirez, Marc S.; Walker, Christopher M.; Chen, Yunyun; Yi, Stacey; Sandulache, Vlad C.; Lai, Stephen Y.; Bankson, James A.

    2015-11-01

    Magnetic resonance imaging and spectroscopy of hyperpolarized (HP) compounds such as [1-13C]-pyruvate have shown tremendous potential for offering new insight into disease and response to therapy. New applications of this technology in clinical research and care will require extensive validation in cells and animal models, a process that may be limited by the high cost and modest throughput associated with dynamic nuclear polarization. Relatively wide spectral separation between [1-13C]-pyruvate and its chemical endpoints in vivo are conducive to simultaneous multi-sample measurements, even in the presence of a suboptimal global shim. Multi-channel acquisitions could conserve costs and accelerate experiments by allowing acquisition from multiple independent samples following a single dissolution. Unfortunately, many existing preclinical MRI systems are equipped with only a single channel for broadband acquisitions. In this work, we examine the feasibility of this concept using a broadband multi-channel digital receiver extension and detector arrays that allow concurrent measurement of dynamic spectroscopic data from ex vivo enzyme phantoms, in vitro anaplastic thyroid carcinoma cells, and in vivo in tumor-bearing mice. Throughput and the cost of consumables were improved by up to a factor of four. These preliminary results demonstrate the potential for efficient multi-sample studies employing hyperpolarized agents.

  13. (13)C metabolic flux analysis of the extremely thermophilic, fast growing, xylose-utilizing Geobacillus strain LC300.

    PubMed

    Cordova, Lauren T; Antoniewicz, Maciek R

    2016-01-01

    Thermophiles are increasingly used as versatile hosts in the biotechnology industry. One of the key advantages of thermophiles is the potential to achieve high rates of feedstock conversion at elevated temperatures. The recently isolated Geobacillus strain LC300 grows extremely fast on xylose, with a doubling time of less than 30 min. In the accompanying paper, the genome of Geobacillus LC300 was sequenced and annotated. In this work, we have experimentally validated the metabolic network model using parallel (13)C-labeling experiments and applied (13)C-metabolic flux analysis to quantify precise metabolic fluxes. Specifically, the complete set of singly labeled xylose tracers, [1-(13)C], [2-(13)C], [3-(13)C], [4-(13)C], and [5-(13)C]xylose, was used for the first time. Isotopic labeling of biomass amino acids was measured by gas chromatography mass spectrometry (GC-MS). Isotopic labeling of carbon dioxide in the off-gas was also measured by an on-line mass spectrometer. The (13)C-labeling data was then rigorously integrated for flux elucidation using the COMPLETE-MFA approach. The results provided important new insights into the metabolism of Geobacillus LC300, its efficient xylose utilization pathways, and the balance between carbon, redox and energy fluxes. The pentose phosphate pathway, glycolysis and TCA cycle were found to be highly active in Geobacillus LC300. The oxidative pentose phosphate pathway was also active and contributed significantly to NADPH production. No transhydrogenase activity was detected. Results from this work provide a solid foundation for future studies of this strain and its metabolic engineering and biotechnological applications.

  14. Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by 13C NMR Spectroscopy

    PubMed Central

    Ashworth, Dennis J.; Lee, Rino Y.; Adams, Douglas O.

    1987-01-01

    Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-13C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-13C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the 13C NMR spectra due to loss of the label as 13CO2. Uptake of [3-13C]pyruvate by the cells was considerably slower than with [2-13C]acetate; however, the labelling patterns were similar with the exception of increased [3-13C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-13C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine. PMID:16665721

  15. IsoDesign: a software for optimizing the design of 13C-metabolic flux analysis experiments.

    PubMed

    Millard, Pierre; Sokol, Serguei; Letisse, Fabien; Portais, Jean-Charles

    2014-01-01

    The growing demand for (13) C-metabolic flux analysis ((13) C-MFA) in the field of metabolic engineering and systems biology is driving the need to rationalize expensive and time-consuming (13) C-labeling experiments. Experimental design is a key step in improving both the number of fluxes that can be calculated from a set of isotopic data and the precision of flux values. We present IsoDesign, a software that enables these parameters to be maximized by optimizing the isotopic composition of the label input. It can be applied to (13) C-MFA investigations using a broad panel of analytical tools (MS, MS/MS, (1) H NMR, (13) C NMR, etc.) individually or in combination. It includes a visualization module to intuitively select the optimal label input depending on the biological question to be addressed. Applications of IsoDesign are described, with an example of the entire (13) C-MFA workflow from the experimental design to the flux map including important practical considerations. IsoDesign makes the experimental design of (13) C-MFA experiments more accessible to a wider biological community. IsoDesign is distributed under an open source license at http://metasys.insa-toulouse.fr/software/isodes/

  16. (13)C-metabolic flux analysis for mevalonate-producing strain of Escherichia coli.

    PubMed

    Wada, Keisuke; Toya, Yoshihiro; Banno, Satomi; Yoshikawa, Katsunori; Matsuda, Fumio; Shimizu, Hiroshi

    2017-02-01

    Mevalonate (MVA) is used to produce various useful products such as drugs, cosmetics and food additives. An MVA-producing strain of Escherichia coli (engineered) was constructed by introducing mvaES genes from Enterococcus faecalis. The engineered strain produced 1.84 mmol/gDCW/h yielding 22% (C-mol/C-mol) of MVA from glucose in the aerobic exponential growth phase. The mass balance analysis revealed that the MVA yield of the engineered strain was close to the upper limit at the biomass yield. Since MVA is synthesized from acetyl-CoA using NADPH as a cofactor, the production of MVA affects central metabolism in terms of carbon utilization and NADPH requirements. The reason for this highly efficient MVA production was investigated based on (13)C-metabolic flux analysis. The estimated flux distributions revealed that the fluxes of acetate formation and the TCA cycle in the engineered strain were lower than those in the control strain. Although the oxidative pentose phosphate pathway is considered as the NADPH generating pathway in E. coli, no difference of the flux was observed between the control and engineered strains. The production/consumption balance of NADPH suggested that additional requirement of NADPH for MVA synthesis was obtained from the transhydrogenase reaction in the engineered strain. Comparison between the measured flux distribution and the ideal values for MVA production proposes a strategy for further engineering to improve the MVA production in E. coli.

  17. Detecting response of rat C6 glioma tumors to radiotherapy using hyperpolarized [1- 13C]pyruvate and 13C magnetic resonance spectroscopic imaging.

    PubMed

    Day, Sam E; Kettunen, Mikko I; Cherukuri, Murali Krishna; Mitchell, James B; Lizak, Martin J; Morris, H Douglas; Matsumoto, Shingo; Koretsky, Alan P; Brindle, Kevin M

    2011-02-01

    We show here that hyperpolarized [1-(13) C]pyruvate can be used to detect treatment response in a glioma tumor model; a tumor type where detection of response with (18) fluoro-2-deoxyglucose, using positron emission tomography, is limited by the high background signals from normal brain tissue. (13) C chemical shift images acquired following intravenous injection of hyperpolarized [1-(13) C]pyruvate into rats with implanted C6 gliomas showed significant labeling of lactate within the tumors but comparatively low levels in surrounding brain.Labeled pyruvate was observed at high levels in blood vessels above the brain and from other major vessels elsewhere but was detected at only low levels in tumor and brain.The ratio of hyperpolarized (13) C label in tumor lactate compared to the maximum pyruvate signal in the blood vessels was decreased from 0.38 ± 0.16 to 0.23 ± 0.13, (a reduction of 34%) by 72 h following whole brain irradiation with 15 Gy.

  18. The aspartate metabolism pathway is differentiable in human hepatocellular carcinoma: transcriptomics and (13) C-isotope based metabolomics.

    PubMed

    Darpolor, Moses M; Basu, Sankha S; Worth, Andrew; Nelson, David S; Clarke-Katzenberg, Regina H; Glickson, Jerry D; Kaplan, David E; Blair, Ian A

    2014-04-01

    Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than a year following diagnosis. Although bioinformatic analyses have indicated differentially expressed genes and cancer related mutations in HCC, integrated genetic and metabolic pathway analyses remain to be investigated. Herein, gene (i.e. messenger RNA, mRNA) enrichment analysis was performed to delineate significant alterations of metabolic pathways in HCC. The objective of this study was to investigate the pathway of aspartate metabolism in HCC of humans. Coupled with transcriptomic (i.e. mRNA) and NMR based metabolomics of human tissue extracts, we utilized liquid chromatography mass spectrometry based metabolomics analysis of stable [U-(13) C6 ]glucose metabolism or [U-(13) C5 ,(15) N2 ]glutamine metabolism of HCC cell culture. Our results indicated that aspartate metabolism is a significant and differentiable metabolic pathway of HCC compared with non-tumor liver (p value < 0.0001). In addition, branched-chain amino acid metabolism (p value < 0.0001) and tricarboxylic acid metabolism (p value < 0.0001) are significant and differentiable. Statistical analysis of measurable NMR metabolites indicated that at least two of the group means were significantly different for the metabolites alanine (p value = 0.0013), succinate (p value = 0.0001), lactate (p value = 0.0114), glycerophosphoethanolamine (p value = 0.015), and inorganic phosphate (p value = 0.0001). However, (13) C isotopic enrichment analysis of these metabolites revealed less than 50% isotopic enrichment with either stable [U-(13) C6 ]glucose metabolism or [U-(13) C5 ,(15) N2 ]glutamine. This may indicate the differential account of total metabolite pool versus de novo metabolites from a (13) C labeled substrate. The ultimate translation of these findings will be to determine putative enzyme activity via

  19. In vivo and in vitro liver cancer metabolism observed with hyperpolarized [5-13C]glutamine

    NASA Astrophysics Data System (ADS)

    Cabella, C.; Karlsson, M.; Canapè, C.; Catanzaro, G.; Colombo Serra, S.; Miragoli, L.; Poggi, L.; Uggeri, F.; Venturi, L.; Jensen, P. R.; Lerche, M. H.; Tedoldi, F.

    2013-07-01

    Glutamine metabolism is, with its many links to oncogene expression, considered a crucial step in cancer metabolism and it is thereby a key target for alteration in cancer development. In particular, strong correlations have been reported between oncogene expression and expression and activity of the enzyme glutaminase. This mitochondrial enzyme, which is responsible for the deamidation of glutamine to form glutamate, is overexpressed in many tumour tissues. In animal models, glutaminase expression is correlated with tumour growth rate and it is readily possible to limit tumour growth by suppression of glutaminase activity. In principle, hyperpolarized 13C MR spectroscopy can provide insight to glutamine metabolism and should hence be a valuable tool to study changes in glutaminase activity as tumours progress. However, no such successful in vivo studies have been reported, even though several good biological models have been tested. This may, at least partly, be due to problems in preparing glutamine for hyperpolarization. This paper reports a new and improved preparation of hyperpolarized [5-13C]glutamine, which provides a highly sensitive 13C MR marker. With this preparation of hyperpolarized [5-13C]glutamine, glutaminase activity in vivo in a rat liver tumour was investigated. Moreover, this marker was also used to measure response to drug treatment in vitro in cancer cells. These examples of [5-13C]glutamine used in tumour models warrant the new preparation to allow metabolic studies with this conditionally essential amino acid.

  20. Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by (13)C metabolic flux analysis.

    PubMed

    Gonzalez, Jacqueline E; Long, Christopher P; Antoniewicz, Maciek R

    2017-01-01

    Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no (13)C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in (13)C metabolic flux analysis ((13)C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated (13)C-MFA using the optimal tracers [1,2-(13)C]glucose, [1,6-(13)C]glucose, [1,2-(13)C]xylose and [5-(13)C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U-(13)C]glucose and [U-(13)C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO2 originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH2, NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology.

  1. Evolution of E. coli on [U-13C]Glucose Reveals a Negligible Isotopic Influence on Metabolism and Physiology

    PubMed Central

    Sandberg, Troy E.; Long, Christopher P.; Gonzalez, Jacqueline E.; Feist, Adam M.; Antoniewicz, Maciek R.; Palsson, Bernhard O.

    2016-01-01

    13C-Metabolic flux analysis (13C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia coli were adaptively evolved for ~1000 generations on uniformly labeled 13C-glucose, a commonly used isotope for 13C-MFA. Phenotypic characterization of these evolved strains revealed ~40% increases in growth rate, with no significant difference in fitness when grown on either labeled (13C) or unlabeled (12C) glucose. The evolved strains displayed decreased biomass yields, increased glucose and oxygen uptake, and increased acetate production, mimicking what is observed after adaptive evolution on unlabeled glucose. Furthermore, full genome re-sequencing revealed that the key genetic changes underlying these phenotypic alterations were essentially the same as those acquired during adaptive evolution on unlabeled glucose. Additionally, glucose competition experiments demonstrated that the wild-type exhibits no isotopic preference for unlabeled glucose, and the evolved strains have no preference for labeled glucose. Overall, the results of this study indicate that there are no significant differences between 12C and 13C-glucose as a carbon source for E. coli growth. PMID:26964043

  2. Central metabolic responses to the overproduction of fatty acids in Escherichia coli based on 13C-metabolic flux analysis.

    PubMed

    He, Lian; Xiao, Yi; Gebreselassie, Nikodimos; Zhang, Fuzhong; Antoniewiez, Maciek R; Tang, Yinjie J; Peng, Lifeng

    2014-03-01

    We engineered a fatty acid overproducing Escherichia coli strain through overexpressing tesA (“pull”) and fadR (“push”) and knocking out fadE (“block”). This “pull-push-block” strategy yielded 0.17 g of fatty acids (C12–C18) per gram of glucose (equivalent to 48% of the maximum theoretical yield) in batch cultures during the exponential growth phase under aerobic conditions. Metabolic fluxes were determined for the engineered E. coli and its control strain using tracer ([1,2-13C]glucose) experiments and 13C-metabolic flux analysis. Cofactor (NADPH) and energy (ATP) balances were also investigated for both strains based on estimated fluxes. Compared to the control strain, fatty acid overproduction led to significant metabolic responses in the central metabolism: (1) Acetic acid secretion flux decreased 10-fold; (2) Pentose phosphate pathway and Entner–Doudoroff pathway fluxes increased 1.5- and 2.0-fold, respectively; (3) Biomass synthesis flux was reduced 1.9-fold; (4) Anaplerotic phosphoenolpyruvate carboxylation flux decreased 1.7-fold; (5) Transhydrogenation flux converting NADH to NADPH increased by 1.7-fold. Real-time quantitative RT-PCR analysis revealed the engineered strain increased the transcription levels of pntA (encoding the membrane-bound transhydrogenase) by 2.1-fold and udhA (encoding the soluble transhydrogenase) by 1.4-fold, which is in agreement with the increased transhydrogenation flux. Cofactor and energy balances analyses showed that the fatty acid overproducing E. coli consumed significantly higher cellular maintenance energy than the control strain. We discussed the strategies to future strain development and process improvements for fatty acid production in E. coli.

  3. Metabolic flux distribution analysis by 13C-tracer experiments using the Markov chain-Monte Carlo method.

    PubMed

    Yang, J; Wongsa, S; Kadirkamanathan, V; Billings, S A; Wright, P C

    2005-12-01

    Metabolic flux analysis using 13C-tracer experiments is an important tool in metabolic engineering since intracellular fluxes are non-measurable quantities in vivo. Current metabolic flux analysis approaches are fully based on stoichiometric constraints and carbon atom balances, where the over-determined system is iteratively solved by a parameter estimation approach. However, the unavoidable measurement noises involved in the fractional enrichment data obtained by 13C-enrichment experiment and the possible existence of unknown pathways prevent a simple parameter estimation method for intracellular flux quantification. The MCMC (Markov chain-Monte Carlo) method, which obtains intracellular flux distributions through delicately constructed Markov chains, is shown to be an effective approach for deep understanding of the intracellular metabolic network. Its application is illustrated through the simulation of an example metabolic network.

  4. Development and testing of hyperpolarized 13C MR calibrationless parallel imaging

    PubMed Central

    Feng, Yesu; Gordon, Jeremy W.; Shin, Peter J.; von Morze, Cornelius; Lustig, Michael; Larson, Peder E.Z.; Ohliger, Michael A.; Carvajal, Lucas; Tropp, James; Pauly, John M.; Vigneron, Daniel B.

    2016-01-01

    A calibrationless parallel imaging technique developed previously for 1H MRI was modified and tested for hyperpolarized 13C MRI for applications requiring large FOV and high spatial resolution. The technique was demonstrated with both retrospective and prospective under-sampled data acquired in phantom and in vivo rat studies. A 2-fold acceleration was achieved using a 2D symmetric EPI readout equipped with random blips on the phase encode dimension. Reconstructed images showed excellent qualitative agreement with fully sampled data. Further acceleration can be achieved using acquisition schemes that incorporate multi-dimensional under-sampling. PMID:26679288

  5. Mechanisms linking metabolism of Helicobacter pylori to 18O and 13C-isotopes of human breath CO2

    PubMed Central

    Som, Suman; De, Anulekha; Banik, Gourab Dutta; Maity, Abhijit; Ghosh, Chiranjit; Pal, Mithun; Daschakraborty, Sunil B.; Chaudhuri, Sujit; Jana, Subhra; Pradhan, Manik

    2015-01-01

    The gastric pathogen Helicobacter pylori utilize glucose during metabolism, but the underlying mechanisms linking to oxygen-18 (18O) and carbon-13 (13C)-isotopic fractionations of breath CO2 during glucose metabolism are poorly understood. Using the excretion dynamics of 18O/16O and 13C/12C-isotope ratios of breath CO2, we found that individuals with Helicobacter pylori infections exhibited significantly higher isotopic enrichments of 18O in breath CO2 during the 2h-glucose metabolism regardless of the isotopic nature of the substrate, while no significant enrichments of 18O in breath CO2 were manifested in individuals without the infections. In contrast, the 13C-isotopic enrichments of breath CO2 were significantly higher in individuals with Helicobacter pylori compared to individuals without infections in response to 13C-enriched glucose uptake, whereas a distinguishable change of breath 13C/12C-isotope ratios was also evident when Helicobacter pylori utilize natural glucose. Moreover, monitoring the 18O and 13C-isotopic exchange in breath CO2 successfully diagnosed the eradications of Helicobacter pylori infections following a standard therapy. Our findings suggest that breath 12C18O16O and 13C16O16O can be used as potential molecular biomarkers to distinctively track the pathogenesis of Helicobacter pylori and also for eradication purposes and thus may open new perspectives into the pathogen’s physiology along with isotope-specific non-invasive diagnosis of the infection. PMID:26039789

  6. Fast Volumetric Spatial-Spectral MR Imaging of Hyperpolarized 13C-Labeled Compounds using Multiple Echo 3D bSSFP

    PubMed Central

    Perman, William H.; Bhattacharya, Pratip; Leupold, Jochen; Lin, Alexander P.; Harris, Kent C.; Norton, Valerie A.; Hovener, Jan B.; Ross, Brian D.

    2010-01-01

    PURPOSE The goal of this work was to develop a fast 3D chemical shift imaging technique for the non-invasive measurement of hyperpolarized 13C-labeled substrates and metabolic products at low concentration. MATERIALS AND METHODS Multiple echo 3D balanced steady state MR imaging (ME-3DbSSFP) was performed in vitro on a syringe containing hyperpolarized [1,3,3-2H3; 1-13C]2-hydroxyethylpropionate (HEP) adjacent to a 13C-enriched acetate phantom, and in vivo on a rat before and after IV injection of hyperpolarized HEP at 1.5 T. Chemical shift images of the hyperpolarized HEP were derived from the multiple echo data by Fourier transformation along the echoes on a voxel by voxel basis for each slice of the 3D data set. RESULTS ME-3DbSSFP imaging was able to provide chemical shift images of hyperpolarized HEP in vivo, and in a rat with isotropic 7 mm spatial resolution, 93 Hz spectral resolution and 16 second temporal resolution for a period greater than 45 seconds. CONCLUSION Multiple echo 3D bSSFP imaging can provide chemical shift images of hyperpolarized 13C-labeled compounds in vivo with relatively high spatial resolution and moderate spectral resolution. The increased signal-to-noise ratio (SNR) of this 3D technique will enable the detection of hyperpolarized 13C-labeled metabolites at lower concentrations as compared to a 2D technique. PMID:20171034

  7. Alterations in Cytosolic and Mitochondrial [U-13C]Glucose Metabolism in a Chronic Epilepsy Mouse Model

    PubMed Central

    Carrasco-Pozo, Catalina

    2017-01-01

    Abstract Temporal lobe epilepsy is a common form of adult epilepsy and shows high resistance to treatment. Increasing evidence has suggested that metabolic dysfunction contributes to the development of seizures, with previous studies indicating impairments in brain glucose metabolism. Here we aim to elucidate which pathways involved in glucose metabolism are impaired, by tracing the hippocampal metabolism of injected [U-13C]glucose (i.p.) during the chronic stage of the pilocarpine-status epilepticus mouse model of epilepsy. The enrichment of 13C in the intermediates of glycolysis and the TCA cycle were quantified in hippocampal extracts using liquid chromatography–tandem mass spectroscopy, along with the measurement of the activities of enzymes in each pathway. We show that there is reduced incorporation of 13C in the intermediates of glycolysis, with the percentage enrichment of all downstream intermediates being highly correlated with those of glucose 6-phosphate. Furthermore, the activities of all enzymes in this pathway including hexokinase and phosphofructokinase were unaltered, suggesting that glucose uptake is reduced in this model without further impairments in glycolysis itself. The key findings were 33% and 55% losses in the activities of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, respectively, along with reduced 13C enrichment in TCA cycle intermediates. This lower 13C enrichment is best explained in part by the reduced enrichment in glycolytic intermediates, whereas the reduction of key TCA cycle enzyme activity indicates that TCA cycling is also impaired in the hippocampal formation. Together, these data suggest that multitarget approaches may be necessary to restore metabolism in the epileptic brain. PMID:28303258

  8. Alterations in Cytosolic and Mitochondrial [U-(13)C]Glucose Metabolism in a Chronic Epilepsy Mouse Model.

    PubMed

    McDonald, Tanya S; Carrasco-Pozo, Catalina; Hodson, Mark P; Borges, Karin

    2017-01-01

    Temporal lobe epilepsy is a common form of adult epilepsy and shows high resistance to treatment. Increasing evidence has suggested that metabolic dysfunction contributes to the development of seizures, with previous studies indicating impairments in brain glucose metabolism. Here we aim to elucidate which pathways involved in glucose metabolism are impaired, by tracing the hippocampal metabolism of injected [U-(13)C]glucose (i.p.) during the chronic stage of the pilocarpine-status epilepticus mouse model of epilepsy. The enrichment of (13)C in the intermediates of glycolysis and the TCA cycle were quantified in hippocampal extracts using liquid chromatography-tandem mass spectroscopy, along with the measurement of the activities of enzymes in each pathway. We show that there is reduced incorporation of (13)C in the intermediates of glycolysis, with the percentage enrichment of all downstream intermediates being highly correlated with those of glucose 6-phosphate. Furthermore, the activities of all enzymes in this pathway including hexokinase and phosphofructokinase were unaltered, suggesting that glucose uptake is reduced in this model without further impairments in glycolysis itself. The key findings were 33% and 55% losses in the activities of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, respectively, along with reduced (13)C enrichment in TCA cycle intermediates. This lower (13)C enrichment is best explained in part by the reduced enrichment in glycolytic intermediates, whereas the reduction of key TCA cycle enzyme activity indicates that TCA cycling is also impaired in the hippocampal formation. Together, these data suggest that multitarget approaches may be necessary to restore metabolism in the epileptic brain.

  9. (13)C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids.

    PubMed

    Ghosh, Amit; Ando, David; Gin, Jennifer; Runguphan, Weerawat; Denby, Charles; Wang, George; Baidoo, Edward E K; Shymansky, Chris; Keasling, Jay D; García Martín, Héctor

    2016-01-01

    Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined (13)C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. These genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%.

  10. 13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

    PubMed Central

    Ghosh, Amit; Ando, David; Gin, Jennifer; Runguphan, Weerawat; Denby, Charles; Wang, George; Baidoo, Edward E. K.; Shymansky, Chris; Keasling, Jay D.; García Martín, Héctor

    2016-01-01

    Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. These genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%. PMID:27761435

  11. Energy contribution of octanoate to intact rat brain metabolism measured by 13C nuclear magnetic resonance spectroscopy.

    PubMed

    Ebert, Douglas; Haller, Ronald G; Walton, Marlei E

    2003-07-02

    Glucose is the dominant oxidative fuel for brain, but studies have indicated that fatty acids are used by brain as well. We postulated that fatty acid oxidation in brain could contribute significantly to overall energy usage and account for non-glucose-derived energy production. [2,4,6,8-13C4]octanoate oxidation in intact rats was determined by nuclear magnetic resonance spectroscopy. We found that oxidation of 13C-octanoate in brain is avid and contributes approximately 20% to total brain oxidative energy production. Labeling patterns of glutamate and glutamine were distinct, and analysis of these metabolites indicated compartmentalized oxidation of octanoate in brain. Examination of liver and blood spectra revealed that label from 13C-octanoate was incorporated into glucose and ketones, which enabled calculation of its overall energy contribution to brain metabolism: glucose (predominantly unlabeled) and 13C-labeled octanoate can account for the entire oxidative metabolism of brain. Additionally, flux through anaplerotic pathways relative to tricarboxylic acid cycle flux (Y) was calculated to be 0.08 +/- 0.039 in brain, indicating that anaplerotic flux is significant and should be considered when assessing brain metabolism. Y was associated with the glutamine synthesis compartment, consistent with the view that anaplerotic flux occurs primarily in astrocytes.

  12. Cutting the Gordian Knot: Identifiability of anaplerotic reactions in Corynebacterium glutamicum by means of (13) C-metabolic flux analysis.

    PubMed

    Kappelmann, Jannick; Wiechert, Wolfgang; Noack, Stephan

    2016-03-01

    Corynebacterium glutamicum is the major workhorse for the microbial production of several amino and organic acids. As long as these derive from tricarboxylic acid cycle intermediates, the activity of anaplerotic reactions is pivotal for a high biosynthetic yield. To determine single anaplerotic activities (13) C-Metabolic Flux Analysis ((13) C-MFA) has been extensively used for C. glutamicum, however with different network topologies, inconsistent or poorly determined anaplerotic reaction rates. Therefore, in this study we set out to investigate whether a focused isotopomer model of the anaplerotic node can at all admit a unique solution for all fluxes. By analyzing different scenarios of active anaplerotic reactions, we show in full generality that for C. glutamicum only certain anaplerotic deletion mutants allow to uniquely determine the anaplerotic fluxes from (13) C-isotopomer data. We stress that the result of this analysis for different assumptions on active enzymes is directly transferable to other compartment-free organisms. Our results demonstrate that there exist biologically relevant metabolic network topologies for which the flux distribution cannot be inferred by classical (13) C-MFA.

  13. Metabolic phenotyping of the diseased rat heart using 13C-substrates and ex vivo perfusion in the working mode.

    PubMed

    Vincent, Geneviève; Khairallah, Maya; Bouchard, Bertrand; Des Rosiers, Christine

    2003-01-01

    The objective of the present study was to compare energy substrate fluxes through metabolic pathways leading to mitochondrial citrate synthesis and release in normal and diseased rat hearts using 13C-substrates and mass isotopomer analysis by gas chromatography-mass spectrometry (GCMS). This study was prompted by our previous finding of a modulated citrate release by perfused rat hearts and by the possibility that a dysregulated myocardial citrate release represents a specific chronic alteration of energy metabolism in cardiac patients. The 15-week-old spontaneously hypertensive rat (SHR) was chosen as our animal model of disease and the Wistar-Kyoto (WKY) rat as its matched control. Ex vivo work-performing hearts were perfused with a semi-recirculating buffer containing physiological concentrations of unlabeled (glucose) and 13C-labeled ([U-13C3](lactate + pyruvate) and/or [1-(13)C]oleate) substrates. In parallel to the continuous monitoring of indices of the heart's functional and physiological status, the following metabolic parameters were documented: (i) citrate release rates and citric acid cycle intermediate tissue levels, (ii) the contribution of fatty acids as well as pyruvate decarboxylation and carboxylation to citrate synthesis, and (iii) lactate and pyruvate uptake and efflux rates. Working hearts from both rat species showed a similar percent contribution of carbohydrates for citrate synthesis through decarboxylation (70%) and carboxylation (10%). SHR hearts showed the following metabolic alterations: a higher citrate release rate, which was associated with a parallel increase in its tissue level, a lower contribution of oleate beta-oxidation to citrate synthesis, and an accelerated efflux rate of unlabeled lactate from glycolysis. These metabolic changes were not explained by differences in myocardial oxygen consumption, cardiac performance or efficiency, nor correlated with indices of tissue necrosis or ischemia. This study demonstrates how the

  14. Genome-based metabolic mapping and 13C flux analysis reveal systematic properties of an oleaginous microalga Chlorella protothecoides.

    PubMed

    Wu, Chao; Xiong, Wei; Dai, Junbiao; Wu, Qingyu

    2015-02-01

    Integrated and genome-based flux balance analysis, metabolomics, and (13)C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass and corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary (13)C metabolic flux analysis as a complementing strategy to flux balance analysis. The result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Taken together, the metabolic network modeling assisted by experimental metabolomics and (13)C

  15. Genome-Based Metabolic Mapping and 13C Flux Analysis Reveal Systematic Properties of an Oleaginous Microalga Chlorella protothecoides

    DOE PAGES

    Wu, Chao; Xiong, Wei; Dai, Junbiao; ...

    2014-12-15

    We report that integrated and genome-based flux balance analysis, metabolomics, and 13C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass andmore » corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary 13C metabolic flux analysis as a complementing strategy to flux balance analysis. We found that the result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Lastly, taken together, the metabolic network modeling assisted

  16. 13 C-metabolic flux analysis in heterologous cellulase production by Bacillus subtilis genome-reduced strain.

    PubMed

    Toya, Yoshihiro; Hirasawa, Takashi; Morimoto, Takuya; Masuda, Kenta; Kageyama, Yasushi; Ozaki, Katsuya; Ogasawara, Naotake; Shimizu, Hiroshi

    2014-06-10

    The great potential of Bacillus subtilis to produce biomaterials would be further enhanced by the development of strains with deletions of non-essential genomic regions. Here, using stationary (13)C-metabolic flux analysis ((13)C-MFA), we investigated the metabolism during cellulase production by the genome-reduced B. subtilis strain MGB874. We transformed MGB874 and wild-type strains with the heterologous cellulase gene, and cultured these on a synthetic medium containing glucose as carbon source. The addition of glutamate and the genome reduction enhanced cellulase production, which led us to use (13)C-MFA to assess the effects of glutamate addition and gene deletions on metabolism. We found that there was a significant increase in the flux in the pentose phosphate (PP) pathway, whereas the fluxes of reactions from acetyl-CoA to α-ketoglutarate were repressed in the presence of glutamate. We hypothesize that the increase in the PP pathway flux was caused by the decrease of citrate synthase flux through the accumulation of glycolytic intermediates. Excess NADPH produced by the PP pathway may affect the increase in cellulase production. Furthermore, the fluxes on glycolysis and the acetate formation of the cellulase-producing wild-type strain were significantly larger than that of the cellulase-producing MGB874 strain when the strains were cultured with glucose and glutamate.

  17. Use of hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate to probe the effects of the anticancer agent dichloroacetate on mitochondrial metabolism in vivo in the normal rat.

    PubMed

    Hu, Simon; Yoshihara, Hikari A I; Bok, Robert; Zhou, Jenny; Zhu, Minhua; Kurhanewicz, John; Vigneron, Daniel B

    2012-12-01

    Development of hyperpolarized technology utilizing dynamic nuclear polarization has enabled the measurement of (13)C metabolism in vivo at very high signal-to-noise ratio (SNR). In vivo mitochondrial metabolism can, in principle, be monitored with pyruvate, which is catalyzed to acetyl-CoA via pyruvate dehydrogenase (PDH). The purpose of this work was to determine whether the compound sodium dichloroacetate (DCA) could aid the study of mitochondrial metabolism with hyperpolarized pyruvate. DCA stimulates PDH by inhibiting its inhibitor, pyruvate dehydrogenase kinase. In this work, hyperpolarized [1-(13)C]pyruvate and [2-(13)C]pyruvate were used to probe mitochondrial metabolism in normal rats. Increased conversion to bicarbonate (+181±69%, P=.025) was measured when [1-(13)C]pyruvate was injected after DCA administration, and increased glutamate (+74±23%, P=.004), acetoacetate (+504±281%, P=.009) and acetylcarnitine (+377±157%, P=.003) were detected when [2-(13)C]pyruvate was used.

  18. Carbon Metabolism of Soil microorganisms at Low Temperatures: Position-Specific 13C Labeled Glucose Reveals the Story

    NASA Astrophysics Data System (ADS)

    Apostel, C.; Bore, E. K.; Halicki, S.; Kuzyakov, Y.; Dippold, M.

    2015-12-01

    Metabolic pathway activities at low temperature are not well understood, despite the fact that the processes are relevant for many soils globally and seasonally. To analyze soil metabolism at low temperature, isotopomeres of position-specifically 13C labeled glucose were applied at three temperature levels; +5, -5 -20 oC. In additon, one sterilization treatment with sodium azide at +5 oC was also performed. Soils were incubated for 1, 3 and 10 days while soil samples at -20 oC were additionally sampled after 30 days. The 13C from individual molecule position in respired CO2 was quantifed. Incorporation of 13C in bulk soil, extractable microbial biomass by chloroform fumigation extraction (CFE) and cell membranes of different microbial communities classified by 13C phospholipid fatty acid analysis (PLFA) was carried out. Our 13CO2 data showed a dominance of C-1 respiration at +5 °C for treatments with and without sodium azide, but total respiration for sodium azide inhibited treatments increased by 14%. In contrast, at -5 and -20 oC metabolic behavior showed intermingling of preferential respiration of the glucose C-4 and C-1 positions. Therefore, at +5 °C, pentose phosphate pathway activity is a dominant metabolic pathway used by microorganisms to metabolize glucose. The respiration increase due to NaN3 inhibition was attributed to endoenzymes released from dead organisms that are stabilized at the soil matrix and have access to suitable substrate and co-factors to permit their funtions. Our PLFA analysis showed that incorporation of glucose 13C was higher in Gram negative bacteria than other microbial groups as they are most competitive for LMWOS. Only a limited amount of microbial groups maintained their glucose utilizing activity at -5 and -20 °C and they strongly shifted towards a metabolization of glucose via both glycolysis and pentose phosphate pathways indicating both growth and cellular maintenance. This study revealed a remarkable microbial acitivity

  19. Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells*

    PubMed Central

    Shestov, Alexander A.; Mancuso, Anthony; Lee, Seung-Cheol; Guo, Lili; Nelson, David S.; Roman, Jeffrey C.; Henry, Pierre-Gilles; Leeper, Dennis B.; Blair, Ian A.; Glickson, Jerry D.

    2016-01-01

    A network model for the determination of tumor metabolic fluxes from 13C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-13C2]glucose under normoxic conditions at 37 °C and monitored by 13C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism. PMID:26703469

  20. Acute porcine renal metabolic effect of endogastric soft drink administration assessed with hyperpolarized [1‐13c]pyruvate

    PubMed Central

    Hansen, Esben Søvsø Szocska; Kjærgaard, Uffe; Bertelsen, Lotte Bonde; Ringgaard, Steffen; Stødkilde‐Jørgensen, Hans

    2015-01-01

    Purpose Our aim was to determine the quantitative reproducibility of metabolic breakdown products in the kidney following intravenous injection of hyperpolarized [1‐13C]pyruvate and secondly to investigate the metabolic effect on the pyruvate metabolism of oral sucrose load using dissolution dynamic nuclear polarization. By this technique, metabolic alterations in several different metabolic related diseases and their metabolic treatment responses can be accessed. Methods In four healthy pigs the lactate‐to‐pyruvate, alanine‐to‐pyruvate and bicarbonate‐to‐pyruvate ratio was measured following administration of regular cola and consecutive injections of hyperpolarized [1‐13C]pyruvate four times within an hour. Results The overall lactate‐to‐pyruvate metabolic profile changed significantly over one hour following an acute sucrose load leading to a significant rise in blood glucose. Conclusion The reproducibility of hyperpolarized magnetic resonance spectroscopy in the healthy pig kidney demonstrated a repeatability of more than 94% for all metabolites and, furthermore, that the pyruvate to lactate conversion and the blood glucose level is elevated following endogastric sucrose administration. Magn Reson Med 74:558–563, 2015. © 2015 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. PMID:26014387

  1. 13C metabolic flux analysis in Clostridium acetobutylicum during growth on L-arabinose

    NASA Astrophysics Data System (ADS)

    Hurley, Margaret; Sund, Christian; Liu, Sanchao; Germane, Katherine; Servinsky, Matthew; Gerlach, Elliot

    2015-03-01

    Clostridium acetobutylicum's metabolic pathways have been studied for decades due to its metabolic diversity and industrial value, yet many details of its metabolism are continuing to emerge. To elucidate the role of xylulose-5-P/fructose-6-P phosphoketolase (XFP), and the recently discovered Pentose Phosphate Pathway (PKP) in C. acetobutylicum, experimental and computational metabolic isotope analysis was performed under growth on glucose, xylose, and arabinose. Results indicate that PKP utilization increased with increasing xylose concentration and this trend was further pronounced during growth on arabinose. This was confirmed by mutation of the gene encoding XFP, which almost completely abolished flux through the PKP during growth on arabinose and resulted in decreased acetate:butyrate ratios. We discuss these experimental and computational results here, and the implications for our understanding of sugar metabolism in C. acetobutylicum.

  2. 13C labeling analysis of sugars by high resolution-mass spectrometry for metabolic flux analysis.

    PubMed

    Acket, Sébastien; Degournay, Anthony; Merlier, Franck; Thomasset, Brigitte

    2017-02-14

    Metabolic flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography-high resolution mass spectrometry, without derivatized procedure, adapted for Metabolic flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic enrichments of free sugars compared to our previous methods [5, 6].

  3. Tracing metabolic pathways of lipid biosynthesis in ectomycorrhizal fungi from position-specific 13C-labelling in glucose.

    PubMed

    Scandellari, Francesca; Hobbie, Erik A; Ouimette, Andrew P; Stucker, Valerie K

    2009-12-01

    Six position-specific (13)C-labelled isotopomers of glucose were supplied to the ectomycorrhizal fungi Suillus pungens and Tricholoma flavovirens. From the resulting distribution of (13)C among fungal PLFAs, the overall order and contribution of each glucose atom to fatty acid (13)C enrichment was: C6 (approximately 31%) > C5 (approximately 25%) > C1 (approximately 18%) > C2 (approximately 18%) > C3 (approximately 8%) > C4 (approximately 1%). These data were used to parameterize a metabolic model of the relative fluxes from glucose degradation to lipid synthesis. Our data revealed that a higher amount of carbon is directed to glycolysis than to the oxidative pentose phosphate pathway (60% and 40% respectively) and that a significant part flows through these pathways more than once (73%) due to the reversibility of some glycolysis reactions. Surprisingly, 95% of carbon cycled through glyoxylate prior to incorporation into lipids, possibly to consume the excess of acetyl-CoA produced during fatty acid turnover. Our approach provides a rigorous framework for analysing lipid biosynthesis in fungi. In addition, this approach could ultimately improve the interpretation of isotopic patterns at natural abundance in field studies.

  4. Pathway analysis using (13) C-glycerol and other carbon tracers reveals a bipartite metabolism of Legionella pneumophila.

    PubMed

    Häuslein, Ina; Manske, Christian; Goebel, Werner; Eisenreich, Wolfgang; Hilbi, Hubert

    2016-04-01

    Amino acids represent the prime carbon and energy source for Legionella pneumophila, a facultative intracellular pathogen, which can cause a life-threatening pneumonia termed Legionnaires' disease. Genome, transcriptome and proteome studies indicate that L. pneumophila also utilizes carbon substrates other than amino acids. We show here that glycerol promotes intracellular replication of L. pneumophila in amoeba or macrophages (but not extracellular growth) dependent on glycerol-3-phosphate dehydrogenase, GlpD. An L. pneumophila mutant strain lacking glpD was outcompeted by wild-type bacteria upon co-infection of amoeba, indicating an important role of glycerol during infection. Isotopologue profiling studies using (13) C-labelled substrates were performed in a novel minimal defined medium, MDM, comprising essential amino acids, proline and phenylalanine. In MDM, L. pneumophila utilized (13) C-labelled glycerol or glucose predominantly for gluconeogenesis and the pentose phosphate pathway, while the amino acid serine was used for energy generation via the citrate cycle. Similar results were obtained for L. pneumophila growing intracellularly in amoeba fed with (13) C-labelled glycerol, glucose or serine. Collectively, these results reveal a bipartite metabolism of L. pneumophila, where glycerol and carbohydrates like glucose are mainly fed into anabolic processes, while serine serves as major energy supply.

  5. Measuring and modeling C flux rates through the central metabolic pathways in microbial communities using position-specific 13C-labeled tracers

    NASA Astrophysics Data System (ADS)

    Dijkstra, P.; van Groenigen, K.; Hagerty, S.; Salpas, E.; Fairbanks, D. E.; Hungate, B. A.; KOCH, G. W.; Schwartz, E.

    2012-12-01

    The production of energy and metabolic precursors occurs in well-known processes such as glycolysis and Krebs cycle. We use position-specific 13C-labeled metabolic tracers, combined with models of microbial metabolic organization, to analyze the response of microbial community energy production, biosynthesis, and C use efficiency (CUE) in soils, decomposing litter, and aquatic communities. The method consists of adding position-specific 13C -labeled metabolic tracers to parallel soil incubations, in this case 1-13C and 2,3-13C pyruvate and 1-13C and U-13C glucose. The measurement of CO2 released from the labeled tracers is used to calculate the C flux rates through the various metabolic pathways. A simplified metabolic model consisting of 23 reactions is solved using results of the metabolic tracer experiments and assumptions of microbial precursor demand. This new method enables direct estimation of fundamental aspects of microbial energy production, CUE, and soil organic matter formation in relatively undisturbed microbial communities. We will present results showing the range of metabolic patterns observed in these communities and discuss results from testing metabolic models.

  6. A Marked Gradient in δ13C Values of Clams Mercenaria mercenaria Across a Marine Embayment May Reflect Variations in Ecosystem metabolism

    EPA Science Inventory

    Although stable isotopes of organic carbon (δ13C) are typically used as indicators of terrestrial, intertidal, and offshore organic carbon sources to coastal ecosystems, there is evidence that δ13C values are also sensitive to in situ ecosystem metabolism. To investigate this phe...

  7. Monitoring tumor response of prostate cancer to radiation therapy by multi-parametric 1H and hyperpolarized 13C magnetic resonance imaging

    NASA Astrophysics Data System (ADS)

    Zhang, Vickie Yi

    Radiation therapy is one of the most common curative therapies for patients with localized prostate cancer, but despite excellent success rates, a significant number of patients suffer post- treatment cancer recurrence. The accurate characterization of early tumor response remains a major challenge for the clinical management of these patients. Multi-parametric MRI/1H MR spectroscopy imaging (MRSI) has been shown to increase the diagnostic performance in evaluating the effectiveness of radiation therapy. 1H MRSI can detect altered metabolic profiles in cancerous tissue. In this project, the concentrations of prostate metabolites from snap-frozen biopsies of recurrent cancer after failed radiation therapy were correlated with histopathological findings to identify quantitative biomarkers that predict for residual aggressive versus indolent cancer. The total choline to creatine ratio was significantly higher in recurrent aggressive versus indolent cancer, suggesting that use of a higher threshold tCho/Cr ratio in future in vivo 1H MRSI studies could improve the selection and therapeutic planning for patients after failed radiation therapy. Varying radiation doses may cause a diverse effect on prostate cancer micro-environment and metabolism, which could hold the key to improving treatment protocols for individual patients. The recent development and clinical translation of hyperpolarized 13C MRI have provided the ability to monitor both changes in the tumor micro-environment and its metabolism using a multi-probe approach, [1-13C]pyruvate and 13C urea, combined with 1H Multi-parametric MRI. In this thesis, hyperpolarized 13C MRI, 1H dynamic contrast enhancement, and diffusion weighted imaging were used to identify early radiation dose response in a transgenic prostate cancer model. Hyperpolarized pyruvate to lactate metabolism significantly decreased in a dose dependent fashion by 1 day after radiation therapy, prior to any changes observed using 1H DCE and diffusion

  8. (13)C Metabolic Flux Analysis of acetate conversion to lipids by Yarrowia lipolytica.

    PubMed

    Liu, Nian; Qiao, Kangjian; Stephanopoulos, Gregory

    2016-11-01

    Volatile fatty acids (VFAs) are an inexpensive and renewable carbon source that can be generated from gas fermentation and anaerobic digestion of fermentable wastes. The oleaginous yeast Yarrowia lipolytica is a promising biocatalyst that can utilize VFAs and convert them into triacylglycerides (TAGs). However, currently there is limited knowledge on the metabolism of Y. lipolytica when cultured on VFAs. To develop a better understanding, we used acetate as the sole carbon source to culture two strains, a control strain and a previously engineered strain for lipid overaccumulation. For both strains, metabolism during the growth phase and lipid production phase were investigated by metabolic flux analysis using two parallel sodium acetate tracers. The resolved flux distributions demonstrate that the glyoxylate shunt pathway is constantly active and the flux through gluconeogenesis varies depending on strain and phase. In particular, by regulating the activities of malate transport and pyruvate kinase, the cells divert only a portion of the glyoxylate shunt flux required to satisfy the needs for anaplerotic reactions and NADPH production through gluconeogenesis and the oxidative pentose phosphate pathway (PPP). Excess flux flows back to the tricarboxylic acid (TCA) cycle for energy production. As with the case of glucose as the substrate, the primary source for lipogenic NADPH is derived from the oxidative PPP.

  9. Isotopically nonstationary 13C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation

    DOE PAGES

    Ma, Fangfang; Jazmin, Lara J.; Young, Jamey D.; ...

    2014-11-03

    Improving plant productivity is an important aim for metabolic engineering. There are few comprehensive methods that quantitatively describe leaf metabolism, although such information would be valuable for increasing photosynthetic capacity, enhancing biomass production, and rerouting carbon flux toward desirable end products. Isotopically nonstationary metabolic flux analysis (INST-MFA) has been previously applied to map carbon fluxes in photoautotrophic bacteria, which involves model-based regression of transient 13C-labeling patterns of intracellular metabolites. However, experimental and computational difficulties have hindered its application to terrestrial plant systems. Here, we performed in vivo isotopic labeling of Arabidopsis thaliana rosettes with 13CO2 and estimated fluxes throughout leafmore » photosynthetic metabolism by INST-MFA. Plants grown at 200 µmol m$-$2s$-$1 light were compared with plants acclimated for 9 d at an irradiance of 500 µmol∙m$-$2∙s$-$1. Approximately 1,400 independent mass isotopomer measurements obtained from analysis of 37 metabolite fragment ions were regressed to estimate 136 total fluxes (54 free fluxes) under each condition. The results provide a comprehensive description of changes in carbon partitioning and overall photosynthetic flux after long-term developmental acclimation of leaves to high light. Despite a doubling in the carboxylation rate, the photorespiratory flux increased from 17 to 28% of net CO2 assimilation with high-light acclimation (Vc/Vo: 3.5:1 vs. 2.3:1, respectively). In conclusion, this study highlights the potential of 13C INST-MFA to describe emergent flux phenotypes that respond to environmental conditions or plant physiology and cannot be obtained by other complementary approaches.« less

  10. Dual resonant birdcage coils for 1H detected 13C microscopic imaging at 11.7 T.

    PubMed

    Hudson, A M; Köckenberger, W; Bowtell, R W

    2000-06-01

    Liquid state, rotating frame cross polarisation experiments are very sensitive to RF field inhomogeneity. In this work, we present an easily fabricated, co-resident high- and low-pass linear birdcage resonator, optimised to perform liquid state rotating frame polarisation transfer at 1H and 13C frequencies. Both the RF fields have been experimentally mapped, and used to validate the spatial signal dependence of a proton detected, 13C image. The predicted performance was then confirmed using PRAWN-based, cyclic J-cross polarisation (CYCLCROP) imaging. A novel variant of a B(1)-field mapping approach is also presented, using the signal enhancement of the CYCLCROP sequence to generate proton detected, 13C field maps.

  11. Metabolic network analysis of Bacillus clausii on minimal and semirich medium using (13)C-labeled glucose.

    PubMed

    Christiansen, Torben; Christensen, Bjarke; Nielsen, Jens

    2002-04-01

    Using (13)C-labeled glucose fed to the facultative alkalophilic Bacillus clausii producing the alkaline serine protease Savinase, the intracellular fluxes were quantified in continuous cultivation and in batch cultivation on a minimal medium. The flux through the pentose phosphate pathway was found to increase with increasing specific growth rate but at a much lower level than previously reported for Bacillus subtilis. Two futile cycles in the pyruvate metabolism were included in the metabolic network. A substantial flux in the futile cycle involving malic enzyme was estimated, whereas only a very small or zero flux through PEP carboxykinase was estimated, indicating that the latter enzyme was not active during growth on glucose. The uptake of the amino acids in a semirich medium containing 15 of the 20 amino acids normally present in proteins was estimated using fully labeled glucose in batch cultivations. It was found that leucine, isoleucine, and phenylalanine were taken up from the medium and not synthesized de novo from glucose. In contrast, serine and threonine were completely synthesized from other metabolites and not taken up from the medium. Valine, proline, and lysine were partly taken up from the medium and partly synthesized from glucose. The metabolic network analysis was extended to include analysis of growth on the semirich medium containing amino acids, and the metabolic flux distribution on this medium was estimated and compared with growth on minimal medium.

  12. Investigation of metabolic changes in STZ-induced diabetic rats with hyperpolarized [1-13C]acetate

    PubMed Central

    Koellisch, Ulrich; Laustsen, Christoffer; Nørlinger, Thomas S; Østergaard, Jakob Appel; Flyvbjerg, Allan; Gringeri, Concetta V; Menzel, Marion I; Schulte, Rolf F; Haase, Axel; Stødkilde-Jørgensen, Hans

    2015-01-01

    In the metabolism of acetate several enzymes are involved, which play an important role in free fatty acid oxidation. Fatty acid metabolism is altered in diabetes patients and therefore acetate might serve as a marker for pathological changes in the fuel selection of cells, as these changes occur in diabetes patients. Acetylcarnitine is a metabolic product of acetate, which enables its transport into the mitochondria for energy production. This study investigates whether the ratio of acetylcarnitine to acetate, measured by noninvasive hyperpolarized [1-13C]acetate magnetic resonance spectroscopy, could serve as a marker for myocardial, hepatic, and renal metabolic changes in rats with Streptozotocin (STZ)-induced diabetes in vivo. We demonstrate that the conversion of acetate to acetylcarnitine could be detected and quantified in all three organs of interest. More interestingly, we found that the hyperpolarized acetylcarnitine to acetate ratio was independent of blood glucose levels and prolonged hyperglycemia following diabetes induction in a type-1 diabetes model. PMID:26272734

  13. Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe--a quantitative approach using 13C-based metabolic flux analysis.

    PubMed

    Klein, Tobias; Lange, Sabrina; Wilhelm, Nadine; Bureik, Matthias; Yang, Tae-Hoon; Heinzle, Elmar; Schneider, Konstantin

    2014-01-01

    Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying (13)C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.

  14. Flux analysis of central metabolic pathways in the Fe(III)-reducing organism Geobacter metallireducens via 13C isotopiclabeling

    SciTech Connect

    Tang, Yinjie J.; Chakraborty, Romy; Martin, Hector Garcia; Chu,Jeannie; Hazen, Terry C.; Keasling, Jay D.

    2007-08-13

    We analyzed the carbon fluxes in the central metabolism ofGeobacter metallireducens strain GS-15 using 13C isotopomer modeling.Acetate labeled in the 1st or 2nd position was the sole carbon source,and Fe-NTA was the sole terminal electron acceptor. The measured labeledacetate uptake rate was 21 mmol/gdw/h in the exponential growth phase.The resulting isotope labeling pattern of amino acids allowed an accuratedetermination of the in vivo global metabolic reaction rates (fluxes)through the central metabolic pathways using a computational isotopomermodel. The model indicated that over 90 percent of the acetate wascompletely oxidized to CO2 via a complete tricarboxylic acid (TCA) cyclewhile reducing iron. Pyruvate carboxylase and phosphoenolpyruvatecarboxykinase were present under these conditions, but enzymes in theglyoxylate shunt and malic enzyme were absent. Gluconeogenesis and thepentose phosphate pathway were mainly employed for biosynthesis andaccounted for less than 3 percent of total carbon consumption. The modelalso indicated surprisingly high reversibility in the reaction betweenoxoglutarate and succinate. This step operates close to the thermodynamicequilibrium possibly because succinate is synthesized via a transferasereaction, and its product, acetyl-CoA, inhibits the conversion ofoxoglutarate to succinate. These findings enable a better understandingof the relationship between genome annotation and extant metabolicpathways in G. metallireducens.

  15. In vivo measurement of ethanol metabolism in the rat liver using magnetic resonance spectroscopy of hyperpolarized [1-13C]pyruvate

    PubMed Central

    Spielman, Daniel M.; Mayer, Dirk; Yen, Yi-Fen; Tropp, James; Hurd, Ralph E.; Pfefferbaum, Adolf

    2009-01-01

    [1-13C]pyruvate is readily polarizable substrate that has been the subject of numerous magnetic resonance spectroscopy (MRS) studies of in vivo metabolism. In this work, 13C-MRS of hyperpolarized [1-13C]pyruvate is used to interrogate a metabolic pathway involved in neither aerobic nor anaerobic metabolism. In particular, ethanol consumption leads to altered liver metabolism, which when excessive is associated with adverse medical conditions including fatty liver disease, hepatitis, cirrhosis, and cancer. Here we present a method for noninvasively monitoring this important process in vivo. Following the bolus injection of hyperpolarized [1-13C]pyruvate, we demonstrate a significantly increased rat liver lactate production rate with the co-administration of ethanol (P = 0.0016 unpaired t-test). The affect is attributable to increased liver nicotinamide adenine dinucleotide (NADH) associated with ethanol metabolism in combination with NADH's role as a coenzyme in pyruvate to lactate conversion. Beyond studies of liver metabolism, this novel in vivo assay of changes in NADH levels makes hyperpolarized [1-13C]pyruvate a potentially viable substrate for studying the multiple in vivo metabolic pathways that use NADH (or NAD+) as a coenzyme, thus broadening the range of applications that have been discussed in the literature to date. PMID:19526498

  16. Reduced mitochondrial malate dehydrogenase activity has a strong effect on photorespiratory metabolism as revealed by 13C labelling

    PubMed Central

    Lindén, Pernilla; Keech, Olivier; Stenlund, Hans; Gardeström, Per; Moritz, Thomas

    2016-01-01

    Mitochondrial malate dehydrogenase (mMDH) catalyses the interconversion of malate and oxaloacetate (OAA) in the tricarboxylic acid (TCA) cycle. Its activity is important for redox control of the mitochondrial matrix, through which it may participate in regulation of TCA cycle turnover. In Arabidopsis, there are two isoforms of mMDH. Here, we investigated to which extent the lack of the major isoform, mMDH1 accounting for about 60% of the activity, affected leaf metabolism. In air, rosettes of mmdh1 plants were only slightly smaller than wild type plants although the fresh weight was decreased by about 50%. In low CO2 the difference was much bigger, with mutant plants accumulating only 14% of fresh weight as compared to wild type. To investigate the metabolic background to the differences in growth, we developed a 13CO2 labelling method, using a custom-built chamber that enabled simultaneous treatment of sets of plants under controlled conditions. The metabolic profiles were analysed by gas- and liquid- chromatography coupled to mass spectrometry to investigate the metabolic adjustments between wild type and mmdh1. The genotypes responded similarly to high CO2 treatment both with respect to metabolite pools and 13C incorporation during a 2-h treatment. However, under low CO2 several metabolites differed between the two genotypes and, interestingly most of these were closely associated with photorespiration. We found that while the glycine/serine ratio increased, a concomitant altered glutamine/glutamate/α-ketoglutarate relation occurred. Taken together, our results indicate that adequate mMDH activity is essential to shuttle reductants out from the mitochondria to support the photorespiratory flux, and strengthen the idea that photorespiration is tightly intertwined with peripheral metabolic reactions. PMID:26889011

  17. Structure and Metabolic-Flow Analysis of Molecular Complexity in a (13) C-Labeled Tree by 2D and 3D NMR.

    PubMed

    Komatsu, Takanori; Ohishi, Risa; Shino, Amiu; Kikuchi, Jun

    2016-05-10

    Improved signal identification for biological small molecules (BSMs) in a mixture was demonstrated by using multidimensional NMR on samples from (13) C-enriched Rhododendron japonicum (59.5 atom%) cultivated in air containing (13) C-labeled carbon dioxide for 14 weeks. The resonance assignment of 386 carbon atoms and 380 hydrogen atoms in the mixture was achieved. 42 BSMs, including eight that were unlisted in the spectral databases, were identified. Comparisons between the experimental values and the (13) C chemical shift values calculated by density functional theory supported the identifications of unlisted BSMs. Tracing the (13) C/(12) C ratio by multidimensional NMR spectra revealed faster and slower turnover ratios of BSMs involved in central metabolism and those categorized as secondary metabolites, respectively. The identification of BSMs and subsequent flow analysis provided insight into the metabolic systems of the plant.

  18. Use of Position-Specific 13C Isotopomers to Examine Central Carbon Metabolism in the Thermophile 'Thermoflexus hugenholtzii'

    NASA Astrophysics Data System (ADS)

    Thomas, S.; Tamadonfar, K. O.; Dijkstra, P.; Dodsworth, J. A.; Hedlund, B. P.

    2013-12-01

    'Thermoflexus hugenholtzii' is a member of a newly discovered class of Chloroflexi. It is the dominant microorganism in certain hot springs; however, very little is known about its physiology, and it is unable to grow on defined media. In order to examine central carbon metabolism in 'T. hugenholtzii', the genome was annotated for genes encoding enzymes for central carbon metabolism, revealing complete pathways for glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway (PPP). Isotope experiments were conducted to test predicted activities by adding position-specific carbon-13 (13C)-labeled metabolites of glucose, pyruvate, acetate, TCA metabolites, and amino acids and measuring the production of 13CO2 during exponential growth. Use of these metabolites demonstrated broad heterotrophic activity of 'T. hugenholtzii,' despite its inability to grow on defined media. Use of glucose-U demonstrated an active glycolytic pathway and pyruvate-1 demonstrated the functioning of the pyruvate oxidation pathway after glycolysis. Use of the TCA cycle intermediates citrate and succinate demonstrated an active TCA cycle. Production of CO2 from alanine and cysteine demonstrated oxidation of amino acids. However, lack of activity on glucose-1 failed to reveal an active PPP suggesting 'T. hugenholtzii' may rely on exogenous sources of pentoses for nucleic acid biosynthesis.

  19. Effective Estimation of Dynamic Metabolic Fluxes Using 13C Labeling and Piecewise Affine Approximation: From Theory to Practical Applicability

    PubMed Central

    Schumacher, Robin; Wahl, S. Aljoscha

    2015-01-01

    The design of microbial production processes relies on rational choices for metabolic engineering of the production host and the process conditions. These require a systematic and quantitative understanding of cellular regulation. Therefore, a novel method for dynamic flux identification using quantitative metabolomics and 13C labeling to identify piecewise-affine (PWA) flux functions has been described recently. Obtaining flux estimates nevertheless still required frequent manual reinitalization to obtain a good reproduction of the experimental data and, moreover, did not optimize on all observables simultaneously (metabolites and isotopomer concentrations). In our contribution we focus on measures to achieve faster and robust dynamic flux estimation which leads to a high dimensional parameter estimation problem. Specifically, we address the following challenges within the PWA problem formulation: (1) Fast selection of sufficient domains for the PWA flux functions, (2) Control of over-fitting in the concentration space using shape-prescriptive modeling and (3) robust and efficient implementation of the parameter estimation using the hybrid implicit filtering algorithm. With the improvements we significantly speed up the convergence by efficiently exploiting that the optimization problem is partly linear. This allows application to larger-scale metabolic networks and demonstrates that the proposed approach is not purely theoretical, but also applicable in practice. PMID:26690237

  20. 13C metabolic flux analysis shows that resistin impairs the metabolic response to insulin in L6E9 myotubes

    PubMed Central

    2014-01-01

    Background It has been suggested that the adipokine resistin links obesity and insulin resistance, although how resistin acts on muscle metabolism is controversial. We aimed to quantitatively analyse the effects of resistin on the glucose metabolic flux profile and on insulin response in L6E9 myotubes at the metabolic level using a tracer-based metabolomic approach and our in-house developed software, Isodyn. Results Resistin significantly increased glucose uptake and glycolysis, altering pyruvate utilisation by the cell. In the presence of resistin, insulin only slightly increased glucose uptake and glycolysis, and did not alter the flux profile around pyruvate induced by resistin. Resistin prevented the increase in gene expression in pyruvate dehydrogenase-E1 and the sharp decrease in gene expression in cytosolic phosphoenolpyruvate carboxykinase-1 induced by insulin. Conclusions These data suggest that resistin impairs the metabolic activation of insulin. This impairment cannot be explained by the activity of a single enzyme, but instead due to reorganisation of the whole metabolic flux distribution. PMID:25217974

  1. Evidence for transketolase-like TKTL1 flux in CHO cells based on parallel labeling experiments and (13)C-metabolic flux analysis.

    PubMed

    Ahn, Woo Suk; Crown, Scott B; Antoniewicz, Maciek R

    2016-09-01

    The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. It provides precursors for the biosynthesis of nucleotides and contributes to the production of reducing power in the form of NADPH. It has been hypothesized that mammalian cells may contain a hidden reaction in PPP catalyzed by transketolase-like protein 1 (TKTL1) that is closely related to the classical transketolase enzyme; however, until now there has been no direct experimental evidence for this reaction. In this work, we have applied state-of-the-art techniques in (13)C metabolic flux analysis ((13)C-MFA) based on parallel labeling experiments and integrated flux fitting to estimate the TKTL1 flux in CHO cells. We identified a set of three parallel labeling experiments with [1-(13)C]glucose+[4,5,6-(13)C]glucose, [2-(13)C]glucose+[4,5,6-(13)C]glucose, and [3-(13)C]glucose+[4,5,6-(13)C]glucose and developed a new method to measure (13)C-labeling of fructose 6-phosphate by GC-MS that allows intuitive interpretation of mass isotopomer distributions to determine key fluxes in the model, including glycolysis, oxidative PPP, non-oxidative PPP, and the TKTL1 flux. Using these tracers we detected a significant TKTL1 flux in CHO cells at the stationary phase. The flux results suggest that the main function of oxidative PPP in CHO cells at the stationary phase is to fuel the TKTL1 reaction. Overall, this study demonstrates for the first time that carbon atoms can be lost in the PPP, by means other than the oxidative PPP, and that this loss of carbon atoms is consistent with the hypothesized TKTL1 reaction in mammalian cells.

  2. Comparative 13C metabolic flux analysis of pyruvate dehydrogenase complex-deficient, L-valine-producing Corynebacterium glutamicum.

    PubMed

    Bartek, Tobias; Blombach, Bastian; Lang, Siegmund; Eikmanns, Bernhard J; Wiechert, Wolfgang; Oldiges, Marco; Nöh, Katharina; Noack, Stephan

    2011-09-01

    L-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by (13)C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an L-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated for L-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP.

  3. C13C4.5/Spinster, an evolutionarily conserved protein that regulates fertility in C. elegans through a lysosome-mediated lipid metabolism process.

    PubMed

    Han, Mei; Chang, Hao; Zhang, Peng; Chen, Tao; Zhao, Yanhua; Zhang, Yongdeng; Liu, Pingsheng; Xu, Tao; Xu, Pingyong

    2013-05-01

    Lipid droplets, which are conserved across almost all species, are cytoplasmic organelles used to store neutral lipids. Identification of lipid droplet regulators will be conducive to resolving obesity and other fat-associated diseases. In this paper, we selected 11 candidates that might be associated with lipid metabolism in Caenorhabditis elegans. Using a BODIPY 493/503-based flow cytometry screen, 6 negative and 3 positive regulators of fat content were identified. We selected one negative regulator of lipid content, C13C4.5, for future study. C13C4.5 was mainly expressed in the worm intestine. We found that this gene was important for maintaining the metabolism of lipid droplets. Biochemical results revealed that 50% of triacylglycerol (TAG) was lost in C13C4.5 knockout worms. Stimulated Raman scattering (SRS) signals in C13C4.5 mutants showed only 49.6% of the fat content in the proximal intestinal region and 86.3% in the distal intestinal region compared with wild type animals. The mean values of lipid droplet size and intensity in C13C4.5 knockout animals were found to be significantly decreased compared with those in wild type worms. The LMP-1-labeled membrane structures in worm intestines were also enlarged in C13C4.5 mutant animals. Finally, fertility defects were found in C13C4.5(ok2087) mutants. Taken together, these results indicate that C13C4.5 may regulate the fertility of C. elegans by changing the size and fat content of lipid droplets by interfering with lysosomal morphology and function.

  4. A method for (13)C-labeling of metabolic carbohydrates within French bean leaves (Phaseolus vulgaris L.) for decomposition studies in soils.

    PubMed

    Girardin, Cyril; Rasse, Daniel P; Biron, Philippe; Ghashghaie, Jaleh; Chenu, Claire

    2009-06-01

    The molecular composition of plant residues is suspected to largely govern the fate of their constitutive carbon (C) in soils. Labile compounds, such as metabolic carbohydrates, are affected differently from recalcitrant and structural compounds by soil-C stabilisation mechanisms. Producing (13)C-enriched plant residues with specifically labeled fractions would help us to investigate the fate in soils of the constitutive C of these compounds. The objective of the present research was to test (13)C pulse chase labeling as a method for specifically enriching the metabolic carbohydrate components of plant residues, i.e. soluble sugars and starch. Bean plants were exposed to a (13)CO(2)-enriched atmosphere for 0.5, 1, 2, 3 and 21 h. The major soluble sugars were then determined on water-soluble extracts, and starch on HCl-hydrolysable extracts. The results show a quick differential labeling between water-soluble and water-insoluble compounds. For both groups, (13)C-labeling increased linearly with time. The difference in delta(13)C signature between water-soluble and insoluble fractions was 7 per thousand after 0.5 h and 70 per thousand after 21 h. However, this clear isotopic contrast masked a substantial labeling variability within each fraction. By contrast, metabolic carbohydrates on the one hand (i.e. soluble sugars + starch) and other fractions (essentially cell wall components) on the other hand displayed quite homogeneous signatures within fractions, and a significant difference in labeling between fractions: delta(13)C = 414 +/- 3.7 per thousand and 56 +/- 5.5 per thousand, respectively. Thus, the technique generates labeled plant residues displaying contrasting (13)C-isotopic signatures between metabolic carbohydrates and other compounds, with homogenous signatures within each group. Metabolic carbohydrates being labile compounds, our findings suggest that the technique is particularly appropriate for investigating the effect of compound lability on the long

  5. Simultaneous investigation of cardiac pyruvate dehydrogenase flux, Krebs cycle metabolism and pH, using hyperpolarized [1,2-(13)C2]pyruvate in vivo.

    PubMed

    Chen, Albert P; Hurd, Ralph E; Schroeder, Marie A; Lau, Angus Z; Gu, Yi-ping; Lam, Wilfred W; Barry, Jennifer; Tropp, James; Cunningham, Charles H

    2012-02-01

    (13)C MR spectroscopy studies performed on hearts ex vivo and in vivo following perfusion of prepolarized [1-(13)C]pyruvate have shown that changes in pyruvate dehydrogenase (PDH) flux may be monitored non-invasively. However, to allow investigation of Krebs cycle metabolism, the (13)C label must be placed on the C2 position of pyruvate. Thus, the utilization of either C1 or C2 labeled prepolarized pyruvate as a tracer can only afford a partial view of cardiac pyruvate metabolism in health and disease. If the prepolarized pyruvate molecules were labeled at both C1 and C2 positions, then it would be possible to observe the downstream metabolites that were the results of both PDH flux ((13)CO(2) and H(13)CO(3)(-)) and Krebs cycle flux ([5-(13)C]glutamate) with a single dose of the agent. Cardiac pH could also be monitored in the same experiment, but adequate SNR of the (13)CO(2) resonance may be difficult to obtain in vivo. Using an interleaved selective RF pulse acquisition scheme to improve (13)CO(2) detection, the feasibility of using dual-labeled hyperpolarized [1,2-(13)C(2)]pyruvate as a substrate for dynamic cardiac metabolic MRS studies to allow simultaneous investigation of PDH flux, Krebs cycle flux and pH, was demonstrated in vivo.

  6. Rapid adaptation of rat brain and liver metabolism to a ketogenic diet: an integrated study using (1)H- and (13)C-NMR spectroscopy.

    PubMed

    Roy, Maggie; Beauvieux, Marie-Christine; Naulin, Jérôme; El Hamrani, Dounia; Gallis, Jean-Louis; Cunnane, Stephen C; Bouzier-Sore, Anne-Karine

    2015-07-01

    The ketogenic diet (KD) is an effective alternative treatment for refractory epilepsy in children, but the mechanisms by which it reduces seizures are poorly understood. To investigate how the KD modifies brain metabolism, we infused control (CT) and 7-day KD rats with either [1-(13)C]glucose (Glc) or [2,4-(13)C2]β-hydroxybutyrate (β-HB). Specific enrichments of amino acids (AAs) measured by (1)H- and (13)C-NMR in total brain perchloric acid extracts were similar between CT and KD rats after [1-(13)C]Glc infusion whereas they were higher in KD rats after [2,4-(13)C2]β-HB infusion. This suggests better metabolic efficiency of ketone body utilization on the KD. The relative rapid metabolic adaptation to the KD included (1) 11%-higher brain γ-amino butyric acid (GABA)/glutamate (Glu) ratio versus CT, (2) liver accumulation of the ketogenic branched-chain AAs (BCAAs) leucine (Leu) and isoleucine (ILeu), which were never detected in CT, and (3) higher brain Leu and ILeu contents. Since Glu and GABA are excitatory and inhibitory neurotransmitters, respectively, higher brain GABA/Glu ratio could contribute to the mechanism by which the KD reduces seizures in epilepsy. Increased BCAA on the KD may also contribute to better seizure control.

  7. Rapid adaptation of rat brain and liver metabolism to a ketogenic diet: an integrated study using 1H- and 13C-NMR spectroscopy

    PubMed Central

    Roy, Maggie; Beauvieux, Marie-Christine; Naulin, Jérôme; El Hamrani, Dounia; Gallis, Jean-Louis; Cunnane, Stephen C; Bouzier-Sore, Anne-Karine

    2015-01-01

    The ketogenic diet (KD) is an effective alternative treatment for refractory epilepsy in children, but the mechanisms by which it reduces seizures are poorly understood. To investigate how the KD modifies brain metabolism, we infused control (CT) and 7-day KD rats with either [1-13C]glucose (Glc) or [2,4-13C2]β-hydroxybutyrate (β-HB). Specific enrichments of amino acids (AAs) measured by 1H- and 13C-NMR in total brain perchloric acid extracts were similar between CT and KD rats after [1-13C]Glc infusion whereas they were higher in KD rats after [2,4-13C2]β-HB infusion. This suggests better metabolic efficiency of ketone body utilization on the KD. The relative rapid metabolic adaptation to the KD included (1) 11%-higher brain γ-amino butyric acid (GABA)/glutamate (Glu) ratio versus CT, (2) liver accumulation of the ketogenic branched-chain AAs (BCAAs) leucine (Leu) and isoleucine (ILeu), which were never detected in CT, and (3) higher brain Leu and ILeu contents. Since Glu and GABA are excitatory and inhibitory neurotransmitters, respectively, higher brain GABA/Glu ratio could contribute to the mechanism by which the KD reduces seizures in epilepsy. Increased BCAA on the KD may also contribute to better seizure control. PMID:25785828

  8. Non-stationary 13C metabolic flux analysis of Chinese hamster ovary cells in batch culture using extracellular labeling highlights metabolic reversibility and compartmentation

    PubMed Central

    2014-01-01

    Background Mapping 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. Results Intracellular 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. Conclusions The 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. PMID:24773761

  9. Probing pyruvate metabolism in normal and mutant fibroblast cell lines using 13C-labeled mass isotopomer analysis and mass spectrometry.

    PubMed

    Riazi, Roya; Khairallah, Maya; Cameron, Jessie M; Pencharz, Paul B; Des Rosiers, Christine; Robinson, Brian H

    2009-12-01

    Fibroblast cell lines are frequently used to diagnose genetic mitochondrial defects in children. The effect of enzyme deficiency on overall flux rate through metabolic pathways is, however, not generally considered. We have transposed an experimental paradigm that was developed for isolated perfused organs using (13)C-labeled substrates and (13)C-isotopomer analysis to probe pyruvate mitochondrial metabolism in cultured human fibroblast cell lines with normal or genetically mutant pyruvate decarboxylation (PDC) or carboxylation (PC) activity. Cells were incubated with 1mM [U-(13)C]pyruvate, and the (13)C-molar percent enrichment (MPE) of intracellular pyruvate, citrate, malate (as a surrogate of oxaloacetate) and aspartate was assessed by mass spectrometry. We estimated various flux ratios relevant to metabolic pathways involved in energy production, namely pyruvate formation, PDC, PC, and citrate recycling in the citric acid cycle (CAC). In all cell lines, exogenous pyruvate was predominately decarboxylated (PC/PDC ratios 0.01-0.3). PC-deficient cell lines displayed an expected negligible contribution of PC flux to oxaloacetate formation for citrate synthesis (PC/CS), which was associated with a greater contribution of PDC to acetyl-CoA formation (PDC/CS), and greater recycling of (13)C-labeled citrate into the CAC. In PDH-deficient cell lines, metabolic flux alterations were most apparent in cells with more than 50% reduction in enzyme activity. This led to an unexpected lower PC/CS flux ratio, while the PDC/CS flux ratio was unchanged. These data illustrate the usefulness of this approach in identifying unexpected metabolic consequences of genetic defects related to pyruvate metabolism.

  10. Simultaneous hyperpolarized 13C-pyruvate MRI and 18F-FDG-PET in cancer (hyperPET): feasibility of a new imaging concept using a clinical PET/MRI scanner

    PubMed Central

    Gutte, Henrik; Hansen, Adam E; Henriksen, Sarah T; Johannesen, Helle H; Ardenkjaer-Larsen, Jan; Vignaud, Alexandre; Hansen, Anders E; Børresen, Betina; Klausen, Thomas L; Wittekind, Anne-Mette N; Gillings, Nic; Kristensen, Annemarie T; Clemmensen, Andreas; Højgaard, Liselotte; Kjær, Andreas

    2015-01-01

    In this paper we demonstrate, for the first time, the feasibility of a new imaging concept - combined hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging (MRSI) and 18F-FDG-PET imaging. This procedure was performed in a clinical PET/MRI scanner with a canine cancer patient. We have named this concept hyper PET. Intravenous injection of the hyperpolarized 13C-pyruvate results in an increase of 13C-lactate, 13C-alanine and 13C-CO2 (13C-HCO3) resonance peaks relative to the tissue, disease and the metabolic state probed. Accordingly, with dynamic nuclear polarization (DNP) and use of 13C-pyruvate it is now possible to directly study the Warburg Effect through the rate of conversion of 13C-pyruvate to 13C-lactate. In this study, we combined it with 18F-FDG-PET that studies uptake of glucose in the cells. A canine cancer patient with a histology verified local recurrence of a liposarcoma on the right forepaw was imaged using a combined PET/MR clinical scanner. PET was performed as a single-bed, 10 min acquisition, 107 min post injection of 310 MBq 18F-FDG. 13C-chemical shift imaging (CSI) was performed just after FDG-PET and 30 s post injection of 23 mL hyperpolarized 13C-pyruvate. Peak heights of 13C-pyruvate and 13C-lactate were quantified using a general linear model. Anatomic 1H-MRI included axial and coronal T1 vibe, coronal T2-tse and axial T1-tse with fat saturation following gadolinium injection. In the tumor we found clearly increased 13C-lactate production, which also corresponded to high 18F-FDG uptake on PET. This is in agreement with the fact that glycolysis and production of lactate are increased in tumor cells compared to normal cells. Yet, most interestingly, also in the muscle of the forepaw of the dog high 18F-FDG uptake was observed. This was due to activity in these muscles prior to anesthesia, which was not accompanied by a similarly high 13C-lactate production. Accordingly, this clearly demonstrates how the Warburg Effect directly

  11. Simultaneous hyperpolarized (13)C-pyruvate MRI and (18)F-FDG-PET in cancer (hyperPET): feasibility of a new imaging concept using a clinical PET/MRI scanner.

    PubMed

    Gutte, Henrik; Hansen, Adam E; Henriksen, Sarah T; Johannesen, Helle H; Ardenkjaer-Larsen, Jan; Vignaud, Alexandre; Hansen, Anders E; Børresen, Betina; Klausen, Thomas L; Wittekind, Anne-Mette N; Gillings, Nic; Kristensen, Annemarie T; Clemmensen, Andreas; Højgaard, Liselotte; Kjær, Andreas

    2015-01-01

    In this paper we demonstrate, for the first time, the feasibility of a new imaging concept - combined hyperpolarized (13)C-pyruvate magnetic resonance spectroscopic imaging (MRSI) and (18)F-FDG-PET imaging. This procedure was performed in a clinical PET/MRI scanner with a canine cancer patient. We have named this concept hyper PET. Intravenous injection of the hyperpolarized (13)C-pyruvate results in an increase of (13)C-lactate, (13)C-alanine and (13)C-CO2 ((13)C-HCO3) resonance peaks relative to the tissue, disease and the metabolic state probed. Accordingly, with dynamic nuclear polarization (DNP) and use of (13)C-pyruvate it is now possible to directly study the Warburg Effect through the rate of conversion of (13)C-pyruvate to (13)C-lactate. In this study, we combined it with (18)F-FDG-PET that studies uptake of glucose in the cells. A canine cancer patient with a histology verified local recurrence of a liposarcoma on the right forepaw was imaged using a combined PET/MR clinical scanner. PET was performed as a single-bed, 10 min acquisition, 107 min post injection of 310 MBq (18)F-FDG. (13)C-chemical shift imaging (CSI) was performed just after FDG-PET and 30 s post injection of 23 mL hyperpolarized (13)C-pyruvate. Peak heights of (13)C-pyruvate and (13)C-lactate were quantified using a general linear model. Anatomic (1)H-MRI included axial and coronal T1 vibe, coronal T2-tse and axial T1-tse with fat saturation following gadolinium injection. In the tumor we found clearly increased (13)C-lactate production, which also corresponded to high (18)F-FDG uptake on PET. This is in agreement with the fact that glycolysis and production of lactate are increased in tumor cells compared to normal cells. Yet, most interestingly, also in the muscle of the forepaw of the dog high (18)F-FDG uptake was observed. This was due to activity in these muscles prior to anesthesia, which was not accompanied by a similarly high (13)C-lactate production. Accordingly, this clearly

  12. Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate.

    PubMed

    Gallagher, Ferdia A; Kettunen, Mikko I; Day, Sam E; Hu, De-En; Ardenkjaer-Larsen, Jan Henrik; Zandt, René in 't; Jensen, Pernille R; Karlsson, Magnus; Golman, Klaes; Lerche, Mathilde H; Brindle, Kevin M

    2008-06-12

    As alterations in tissue pH underlie many pathological processes, the capability to image tissue pH in the clinic could offer new ways of detecting disease and response to treatment. Dynamic nuclear polarization is an emerging technique for substantially increasing the sensitivity of magnetic resonance imaging experiments. Here we show that tissue pH can be imaged in vivo from the ratio of the signal intensities of hyperpolarized bicarbonate (H(13)CO(3)(-)) and (13)CO(2) following intravenous injection of hyperpolarized H(13)CO(3)(-). The technique was demonstrated in a mouse tumour model, which showed that the average tumour interstitial pH was significantly lower than the surrounding tissue. Given that bicarbonate is an endogenous molecule that can be infused in relatively high concentrations into patients, we propose that this technique could be used clinically to image pathological processes that are associated with alterations in tissue pH, such as cancer, ischaemia and inflammation.

  13. Exo-MFA - A 13C metabolic flux analysis framework to dissect tumor microenvironment-secreted exosome contributions towards cancer cell metabolism.

    PubMed

    Achreja, Abhinav; Zhao, Hongyun; Yang, Lifeng; Yun, Tae Hyun; Marini, Juan; Nagrath, Deepak

    2017-01-11

    Dissecting the pleiotropic roles of tumor micro-environment (TME) on cancer progression has been brought to the foreground of research on cancer pathology. Extracellular vesicles such as exosomes, transport proteins, lipids, and nucleic acids, to mediate intercellular communication between TME components and have emerged as candidates for anti-cancer therapy. We previously reported that cancer-associated fibroblast (CAF) derived exosomes (CDEs) contain metabolites in their cargo that are utilized by cancer cells for central carbon metabolism and promote cancer growth. However, the metabolic fluxes involved in donor cells towards packaging of metabolites in extracellular vesicles and exosome-mediated metabolite flux upregulation in recipient cells are still not known. Here, we have developed a novel empirical and computational technique, exosome-mediated metabolic flux analysis (Exo-MFA) to quantify flow of cargo from source cells to recipient cells via vesicular transport. Our algorithm, which is based on (13)C metabolic flux analysis, successfully predicts packaging fluxes to metabolite cargo in CAFs, dynamic changes in rate of exosome internalization by cancer cells, and flux of cargo release over time. We find that cancer cells internalize exosomes rapidly leading to depletion of extracellular exosomes within 24h. However, metabolite cargo significantly alters intracellular metabolism over the course of 24h by regulating glycolysis pathway fluxes via lactate supply. Furthermore, it can supply up to 35% of the TCA cycle fluxes by providing TCA intermediates and glutamine. Our algorithm will help gain insight into (i) metabolic interactions in multicellular systems (ii) biogenesis of extracellular vesicles and their differential packaging of cargo under changing environments, and (iii) regulation of cancer cell metabolism by its microenvironment.

  14. Impacts of high β-galactosidase expression on central metabolism of recombinant Pichia pastoris GS115 using glucose as sole carbon source via (13)C metabolic flux analysis.

    PubMed

    Nie, Yongsheng; Huang, Mingzhi; Lu, Junjie; Qian, Jiangchao; Lin, Weilu; Chu, Ju; Zhuang, Yingping; Zhang, Siliang

    2014-10-10

    The yeast Pichia pastoris GS115 is a widely used microbial cell factory for the production of heterologous protein. In order to reveal the impacts of high heterologous protein expression on the central metabolism of Pichia pastoris GS115 using glucose as sole carbon source, we engineered a high β-galactosidase expression strain P. pastoris G1HL and a low expression control strain P. pastoris GHL through controlling the initiation strength of constitutive promoter pGAP. The carbon flux distributions in these two strains were quantified via (13)C metabolic flux analysis. Compared to the control strain, G1HL showed a lower growth rate, a higher flux through glycolysis pathway, a higher flux through pentose phosphate pathway, and a lower flux through by-products secretion pathway. The metabolic flux redistribution in G1HL was thought to compensate the increased redox cofactors and energy demands caused by the high protein expression. Although the fluxes through Krebs cycle in two engineered strains were almost the same, they were significantly lower than those in wild strain. The enhanced expression of β-galactosidase by glutamate supplementation demonstrated the potential of P. pastoris GS115 to catabolize more carbon through the Krebs cycle for even higher protein expression. In conclusion, our work indicates that P. pastoris GS115 can readjusts the central metabolism for higher heterologous protein expression and provides strategies for strain development or process optimization for enhancing production of heterologous protein.

  15. Genome-Based Metabolic Mapping and 13C Flux Analysis Reveal Systematic Properties of an Oleaginous Microalga Chlorella protothecoides1[OPEN

    PubMed Central

    Wu, Chao; Xiong, Wei; Dai, Junbiao; Wu, Qingyu

    2015-01-01

    Integrated and genome-based flux balance analysis, metabolomics, and 13C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass and corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary 13C metabolic flux analysis as a complementing strategy to flux balance analysis. The result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Taken together, the metabolic network modeling assisted by experimental metabolomics and 13C labeling

  16. Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

    PubMed Central

    Soong, Jennifer L; Reuss, Dan; Pinney, Colin; Boyack, Ty; Haddix, Michelle L; Stewart, Catherine E; Cotrufo, M. Francesca

    2014-01-01

    Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or 2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components. Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components

  17. Design and operation of a continuous 13C and 15N labeling chamber for uniform or differential, metabolic and structural, plant isotope labeling.

    PubMed

    Soong, Jennifer L; Reuss, Dan; Pinney, Colin; Boyack, Ty; Haddix, Michelle L; Stewart, Catherine E; Cotrufo, M Francesca

    2014-01-16

    Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as (13)C with (15)N, (18)O or (2)H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation(1-4). From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage(5-7). The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing (13)C and (15)N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components. Here, we present the construction and operation of a continuous (13)C and (15)N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%(13)C and 6.7 atom%(15)N uniform plant label, or material that is differentially labeled by up to 1.29 atom%(13)C and 0.56 atom%(15)N in its metabolic and structural components (hot water extractable and hot water

  18. 13C-Tracer and Gas Chromatography-Mass Spectrometry Analyses Reveal Metabolic Flux Distribution in the Oleaginous Microalga Chlorella protothecoides1[C][W][OA

    PubMed Central

    Xiong, Wei; Liu, Lixia; Wu, Chao; Yang, Chen; Wu, Qingyu

    2010-01-01

    The green alga Chlorella protothecoides has received considerable attention because it accumulates neutral triacylglycerols, commonly regarded as an ideal feedstock for biodiesel production. In order to gain a better understanding of its metabolism, tracer experiments with [U-13C]/[1-13C]glucose were performed with heterotrophic growth of C. protothecoides for identifying the metabolic network topology and estimating intracellular fluxes. Gas chromatography-mass spectrometry analysis tracked the labeling patterns of protein-bound amino acids, revealing a metabolic network consisting of the glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle with inactive glyoxylate shunt. Evidence of phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme activity was also obtained. It was demonstrated that the relative activity of the pentose phosphate pathway to glycolysis under nitrogen-limited environment increased, reflecting excess NADPH requirements for lipid biosynthesis. Although the growth rate and cellular oil content were significantly altered in response to nitrogen limitation, global flux distribution of C. protothecoides remained stable, exhibiting the rigidity of central carbon metabolism. In conclusion, quantitative knowledge on the metabolic flux distribution of oleaginous alga obtained in this study may be of value in designing strategies for metabolic engineering of desirable bioproducts. PMID:20720172

  19. Isotopically nonstationary 13C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improving plant productivity is an important aim for metabolic engineering. There are few comprehensive methods that quantitatively describe the primary metabolism of leaves, though such information would be valuable for improving photosynthetic capacity, increasing biomass production, and rerouting...

  20. Measuring changes in substrate utilization in the myocardium in response to fasting using hyperpolarized [1-13C]butyrate and [1-13C]pyruvate

    PubMed Central

    Bastiaansen, Jessica A. M.; Merritt, Matthew E.; Comment, Arnaud

    2016-01-01

    Cardiac dysfunction is often associated with a shift in substrate preference for ATP production. Hyperpolarized (HP) 13C magnetic resonance spectroscopy (MRS) has the unique ability to detect real-time metabolic changes in vivo due to its high sensitivity and specificity. Here a protocol using HP [1-13C]pyruvate and [1-13C]butyrate is used to measure carbohydrate versus fatty acid metabolism in vivo. Metabolic changes in fed and fasted Sprague Dawley rats (n = 36) were studied at 9.4 T after tail vein injections. Pyruvate and butyrate competed for acetyl-CoA production, as evidenced by significant changes in [13C]bicarbonate (−48%), [1-13C]acetylcarnitine (+113%), and [5-13C]glutamate (−63%), following fasting. Butyrate uptake was unaffected by fasting, as indicated by [1-13C]butyrylcarnitine. Mitochondrial pseudoketogenesis facilitated the labeling of the ketone bodies [1-13C]acetoacetate and [1-13C]β-hydroxybutyryate, without evidence of true ketogenesis. HP [1-13C]acetoacetate was increased in fasting (250%) but decreased during pyruvate co-injection (−82%). Combining HP 13C technology and co-administration of separate imaging agents enables noninvasive and simultaneous monitoring of both fatty acid and carbohydrate oxidation. This protocol illustrates a novel method for assessing metabolic flux through different enzymatic pathways simultaneously and enables mechanistic studies of the changing myocardial energetics often associated with disease. PMID:27150735

  1. Quantification of 13C pyruvate and 13C lactate in dog blood by reversed-phase liquid chromatography-electrospray ionization mass spectrometry after derivatization with 3-nitrophenylhydrazine.

    PubMed

    Uran, Steinar; Landmark, Kristin Eitrem; Hjellum, Gro; Skotland, Tore

    2007-08-15

    Injection of hyperpolarized (13)C-labelled pyruvate ((13)C pyruvate) is under evaluation as an agent for medical metabolic imaging by measuring formation of (13)C lactate using magnetic resonance spectroscopy of the (13)C nuclei. A quantitative method for analysis of these (13)C-labelled substances in dog blood was needed as part of the development of this agent and we here describe a liquid chromatography-mass spectrometry method for that purpose. Immediately after blood collection, the blood proteins were precipitated using methanol added internal standard ([U-(13)C]pyruvate and [U-(13)C]lactate). Prior to analysis, the compounds were derivatized using 3-nitrophenylhydrazine. Following separation on a Supelco Discovery HS C18 column, (13)C pyruvate and (13)C lactate were detected using negative electrospray ionization mass spectrometry. Calibration standards (4.5-4500 microM (13)C pyruvate and 9-9000 microM (13)C lactate) and added internal standard were used to make the calibration curves, which were fitted to a non-linear equation y=a+bx+cx(2) and weighted with a weighting factor of 1/y(2). The analytical lower limit of quantification of (13)C pyruvate and (13)C lactate was 4.5 and 9 microM, respectively. The total precision of the method was below 9.2% for (13)C pyruvate and below 5.8% for (13)C lactate. The accuracy of the method showed a relative error less than 2.4% for (13)C pyruvate and less than 6.3% for (13)C lactate. The recoveries were in the range 93-115% for (13)C pyruvate and 70-111% for (13)C lactate. Both substances were stable in protein-free supernatant when stored for up to 3 weeks in a -20 degrees C freezer, during three freeze/thaw cycles, and when stored in an autosampler for at least 30 h.

  2. Metabolic Fate of the Carboxyl Groups of Malate and Pyruvate and their Influence on δ13C of Leaf-Respired CO2 during Light Enhanced Dark Respiration

    PubMed Central

    Lehmann, Marco M.; Wegener, Frederik; Barthel, Matti; Maurino, Veronica G.; Siegwolf, Rolf T. W.; Buchmann, Nina; Werner, Christiane; Werner, Roland A.

    2016-01-01

    The enhanced CO2 release of illuminated leaves transferred into darkness, termed “light enhanced dark respiration (LEDR)”, is often associated with an increase in the carbon isotope ratio of the respired CO2 (δ13CLEDR). The latter has been hypothesized to result from different respiratory substrates and decarboxylation reactions in various metabolic pathways, which are poorly understood so far. To provide a better insight into the underlying metabolic processes of δ13CLEDR, we fed position-specific 13C-labeled malate and pyruvate via the xylem stream to leaves of species with high and low δ13CLEDR values (Halimium halimifolium and Oxalis triangularis, respectively). During respective label application, we determined label-derived leaf 13CO2 respiration using laser spectroscopy and the 13C allocation to metabolic fractions during light–dark transitions. Our results clearly show that both carboxyl groups (C-1 and C-4 position) of malate similarly influence respiration and metabolic fractions in both species, indicating possible isotope randomization of the carboxyl groups of malate by the fumarase reaction. While C-2 position of pyruvate was only weakly respired, the species-specific difference in natural δ13CLEDR patterns were best reflected by the 13CO2 respiration patterns of the C-1 position of pyruvate. Furthermore, 13C label from malate and pyruvate were mainly allocated to amino and organic acid fractions in both species and only little to sugar and lipid fractions. In summary, our results suggest that respiration of both carboxyl groups of malate (via fumarase) by tricarboxylic acid cycle reactions or by NAD-malic enzyme influences δ13CLEDR. The latter supplies the pyruvate dehydrogenase reaction, which in turn determines natural δ13CLEDR pattern by releasing the C-1 position of pyruvate. PMID:27375626

  3. Solid-state /sup 13/C nuclear magnetic resonance spectroscopy of simultaneously metabolized acetate and phenol in a soil Pseudomonas sp

    SciTech Connect

    Heiman, A.S.; Copper, W.T.

    1987-01-01

    An investigation was made of the concentration-dependent primary and secondary substrate relationships in the simultaneous metabolism of the ubiquitous pollutant phenol and the naturally occurring substrate acetate by a Pseudomonas sp. soil isolate capable of utilizing either substance as a sole source of carbon and energy. In addition to conventional analytical techniques, solid-state /sup 13/C nuclear magnetic resonance spectroscopy was used to follow the cellular distribution of (1-/sup 13/C)acetate in the presence of unlabeled phenol. These results suggest that, when phenol is present as the primary substrate, acetate is preferentially shuttled into fatty acyl chain synthesis, whereas phenol carbon is funnelled into the tricarboxylic acid cycle. Thus, simultaneous use of a xenobiotic compound and a natural substrate apparently does occur, and the relative concentrations of the two substrates do influence the rate and manner in which the compounds are utilized. These results also demonstrate the unique advantage of using solid-state nuclear magnetic resonance techniques combined with /sup 13/C labeling of specific sites in substrates when doing microbial degradation studies. In this work, the entire cellular biomass was examined directly without extensive extraction, fractionation, or isolation of subcellular units; thus, there is no uncertainty about chemical alteration of substrate metabolites as a result of these often harsh treatments.

  4. How Closely Do the δ13C Values of Crassulacean Acid Metabolism Plants Reflect the Proportion of CO2 Fixed during Day and Night?1

    PubMed Central

    Winter, Klaus; Holtum, Joseph A.M.

    2002-01-01

    The extent to which Crassulacean acid metabolism (CAM) plant δ13C values provide an index of the proportions of CO2 fixed during daytime and nighttime was assessed. Shoots of seven CAM species (Aloe vera, Hylocereus monocanthus, Kalanchoe beharensis, Kalanchoe daigremontiana, Kalanchoe pinnata, Vanilla pauciflora, and Xerosicyos danguyi) and two C3 species (teak [Tectona grandis] and Clusia sp.) were grown in a cuvette, and net CO2 exchange was monitored for up to 51 d. In species exhibiting net dark CO2 fixation, between 14% and 73.3% of the carbon gain occurred in the dark. δ13C values of tissues formed inside the cuvette ranged between −28.7‰ and −11.6‰, and correlated linearly with the percentages of carbon gained in the light and in the dark. The δ13C values for new biomass obtained solely during the dark and light were estimated as −8.7‰ and −26.9‰, respectively. For each 10% contribution of dark CO2 fixation integrated over the entire experiment, the δ13C content of the tissue was, thus, approximately 1.8‰ less negative. Extrapolation of the observations to plants previously surveyed under natural conditions suggests that the most commonly expressed version of CAM in the field, “the typical CAM plant,” involves plants that gain about 71% to 77% of their carbon by dark fixation, and that the isotopic signals of plants that obtain one-third or less of their carbon in the dark may be confused with C3 plants when identified on the basis of carbon isotope content alone. PMID:12177497

  5. How closely do the delta(13)C values of Crassulacean Acid metabolism plants reflect the proportion of CO(2) fixed during day and night?

    PubMed

    Winter, Klaus; Holtum, Joseph A M

    2002-08-01

    The extent to which Crassulacean acid metabolism (CAM) plant delta(13)C values provide an index of the proportions of CO(2) fixed during daytime and nighttime was assessed. Shoots of seven CAM species (Aloe vera, Hylocereus monocanthus, Kalanchoe beharensis, Kalanchoe daigremontiana, Kalanchoe pinnata, Vanilla pauciflora, and Xerosicyos danguyi) and two C(3) species (teak [Tectona grandis] and Clusia sp.) were grown in a cuvette, and net CO(2) exchange was monitored for up to 51 d. In species exhibiting net dark CO(2) fixation, between 14% and 73.3% of the carbon gain occurred in the dark. delta(13)C values of tissues formed inside the cuvette ranged between -28.7 per thousand and -11.6 per thousand, and correlated linearly with the percentages of carbon gained in the light and in the dark. The delta(13)C values for new biomass obtained solely during the dark and light were estimated as -8.7 per thousand and -26.9 per thousand, respectively. For each 10% contribution of dark CO(2) fixation integrated over the entire experiment, the delta(13)C content of the tissue was, thus, approximately 1.8 per thousand less negative. Extrapolation of the observations to plants previously surveyed under natural conditions suggests that the most commonly expressed version of CAM in the field, "the typical CAM plant," involves plants that gain about 71% to 77% of their carbon by dark fixation, and that the isotopic signals of plants that obtain one-third or less of their carbon in the dark may be confused with C(3) plants when identified on the basis of carbon isotope content alone.

  6. GC-MS/MS survey of collision-induced dissociation of tert-butyldimethylsilyl-derivatized amino acids and its application to (13)C-metabolic flux analysis of Escherichia coli central metabolism.

    PubMed

    Okahashi, Nobuyuki; Kawana, Shuichi; Iida, Junko; Shimizu, Hiroshi; Matsuda, Fumio

    2016-09-01

    Stable isotope labeling experiments using mass spectrometry have been employed to investigate carbon flow levels (metabolic flux) in mammalian, plant, and microbial cells. To achieve a more precise (13)C-metabolic flux analysis ((13)C-MFA), novel fragmentations of tert-butyldimethylsilyl (TBDMS)-amino acids were investigated by gas chromatography-tandem mass spectrometry (GC-MS/MS). The product ion scan analyses of 15 TBDMS-amino acids revealed 24 novel fragment ions. The amino acid-derived carbons included in the five fragment ions were identified by the analyses of (13)C-labeled authentic standards. The identification of the fragment ion at m/z 170 indicated that the isotopic abundance of S-methyl carbon in methionine could be determined from the cleavage of C5 in the precursor of [M-159](+) (m/z 218). It was also confirmed that the precision of (13)C-MFA in Escherichia coli central carbon metabolism could be improved by introducing (13)C-labeling data derived from novel fragmentations. Graphical Abstract Novel collision-induced dissociation fragmentations of tert-butyldimethylsilyl amino acids were investigated and identified by GC-MS/MS.

  7. Computational analysis of storage synthesis in developing Brassica napus L. (oilseed rape) embryos: Flux variability analysis in relation to 13C-metabolic flux analysis

    SciTech Connect

    Hay, J.; Schwender, J.

    2011-08-01

    Plant oils are an important renewable resource, and seed oil content is a key agronomical trait that is in part controlled by the metabolic processes within developing seeds. A large-scale model of cellular metabolism in developing embryos of Brassica napus (bna572) was used to predict biomass formation and to analyze metabolic steady states by flux variability analysis under different physiological conditions. Predicted flux patterns are highly correlated with results from prior 13C metabolic flux analysis of B. napus developing embryos. Minor differences from the experimental results arose because bna572 always selected only one sugar and one nitrogen source from the available alternatives, and failed to predict the use of the oxidative pentose phosphate pathway. Flux variability, indicative of alternative optimal solutions, revealed alternative pathways that can provide pyruvate and NADPH to plastidic fatty acid synthesis. The nutritional values of different medium substrates were compared based on the overall carbon conversion efficiency (CCE) for the biosynthesis of biomass. Although bna572 has a functional nitrogen assimilation pathway via glutamate synthase, the simulations predict an unexpected role of glycine decarboxylase operating in the direction of NH4+ assimilation. Analysis of the light-dependent improvement of carbon economy predicted two metabolic phases. At very low light levels small reductions in CO2 efflux can be attributed to enzymes of the tricarboxylic acid cycle (oxoglutarate dehydrogenase, isocitrate dehydrogenase) and glycine decarboxylase. At higher light levels relevant to the 13C flux studies, ribulose-1,5-bisphosphate carboxylase activity is predicted to account fully for the light-dependent changes in carbon balance.

  8. Metabolic flux analysis of Arthrobacter sp. CGMCC 3584 for cAMP production based on 13C tracer experiments and gas chromatography-mass spectrometry.

    PubMed

    Niu, Huanqing; Chen, Yong; Yao, Shiwei; Liu, Lixia; Yang, Chen; Li, Bingbing; Liu, Dong; Xie, Jingjing; Chen, Xiaochun; Wu, Jinglan; Ying, Hanjie

    2013-12-01

    Arthrobacter sp. CGMCC 3584 are able to produce cAMP from glucose by the purine synthesis pathway via de novo or salvage biosynthesis. In order to gain an improved understanding of its metabolism, (13)C-labeling experiment and gas chromatography-mass spectrometry (GC-MS) analysis were employed to determine the metabolic network structure and estimate the intracellular fluxes. GC-MS analysis helps to reflect the activity of the intracellular pathways and reactions. The metabolic network mainly contains glycolytic and pentose phosphate pathways, the tricarboxylic acid cycle, and the inactive glyoxylate shunt. Hypoxanthine as a precursor of cAMP and sodium fluoride as an inhibitor of glycolysis were found to increase the cAMP production, as well as the flux through the PP pathway. The effects of adding hypoxanthine and sodium fluoride are discussed based on the enzyme assays and metabolic flux analysis. In conclusion, our results provide quantitative insights into how cells manipulate the metabolic network under different culture conditions and this may be of value in metabolic regulation for desirable production.

  9. Characterization of metabolic profile of honokiol in rat feces using liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and (13)C stable isotope labeling.

    PubMed

    Dong, Yinfeng; Tang, Minghai; Song, Hang; Li, Rong; Wang, Chunyu; Ye, Haoyu; Qiu, Neng; Zhang, Yongkui; Chen, Lijuan; Wei, Yuquan

    2014-03-15

    As fecal excretion is one of important routes of elimination of drugs and their metabolites, it is indispensable to investigate the metabolites in feces for more comprehensive information on biotransformation in vivo. In this study, a sensitive and reliable approach based on ultra-performance liquid chromatography/quadrupole-time-of-flight-mass spectrometry (UHPLC-Q-TOF-MS) was applied to characterize the metabolic profile of honokiol in rat feces after the administration of an equimolar mixture of honokiol and [(13)C6]-labeled honokiol. Totally 42 metabolites were discovered and tentatively identified in rat feces samples, 26 metabolites were first reported, including two novel classes of metabolites, methylated and dimeric metabolites of honokiol. Moreover, this study provided basic comparative data on the metabolites in rat plasma, feces and urine, which gave better understanding of the metabolic fate of honokiol in vivo.

  10. (13)C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway.

    PubMed

    Gebril, Hoda M; Avula, Bharathi; Wang, Yan-Hong; Khan, Ikhlas A; Jekabsons, Mika B

    2016-02-01

    Glycolysis, mitochondrial substrate oxidation, and the pentose phosphate pathway (PPP) are critical for neuronal bioenergetics and oxidation-reduction homeostasis, but quantitating their fluxes remains challenging, especially when processes such as hexose phosphate (i.e., glucose/fructose-6-phosphate) recycling in the PPP are considered. A hexose phosphate recycling model was developed which exploited the rates of glucose consumption, lactate production, and mitochondrial respiration to infer fluxes through the major glucose consuming pathways of adherent cerebellar granule neurons by replicating [(13)C]lactate labeling from metabolism of [1,2-(13)C2]glucose. Flux calculations were predicated on a steady-state system with reactions having known stoichiometries and carbon atom transitions. Non-oxidative PPP activity and consequent hexose phosphate recycling, as well as pyruvate production by cytoplasmic malic enzyme, were optimized by the model and found to account for 28 ± 2% and 7.7 ± 0.2% of hexose phosphate and pyruvate labeling, respectively. From the resulting fluxes, 52 ± 6% of glucose was metabolized by glycolysis, compared to 19 ± 2% by the combined oxidative/non-oxidative pentose cycle that allows for hexose phosphate recycling, and 29 ± 8% by the combined oxidative PPP/de novo nucleotide synthesis reactions. By extension, 62 ± 6% of glucose was converted to pyruvate, the metabolism of which resulted in 16 ± 1% of glucose oxidized by mitochondria and 46 ± 6% exported as lactate. The results indicate a surprisingly high proportion of glucose utilized by the pentose cycle and the reactions synthesizing nucleotides, and exported as lactate. While the in vitro conditions to which the neurons were exposed (high glucose, no lactate or other exogenous substrates) limit extrapolating these results to the in vivo state, the approach provides a means of assessing a number of metabolic fluxes within the context of hexose phosphate recycling in the PPP from a

  11. NMR ({sup 1}H and {sup 13}C) based signatures of abnormal choline metabolism in oral squamous cell carcinoma with no prominent Warburg effect

    SciTech Connect

    Bag, Swarnendu; Banerjee, Deb Ranjan; Basak, Amit; Das, Amit Kumar; Pal, Mousumi; Banerjee, Rita; Paul, Ranjan Rashmi; Chatterjee, Jyotirmoy

    2015-04-17

    At functional levels, besides genes and proteins, changes in metabolome profiles are instructive for a biological system in health and disease including malignancy. It is understood that metabolomic alterations in association with proteomic and transcriptomic aberrations are very fundamental to unravel malignant micro-ambient criticality and oral cancer is no exception. Hence deciphering intricate dimensions of oral cancer metabolism may be contributory both for integrated appreciation of its pathogenesis and to identify any critical but yet unexplored dimension of this malignancy with high mortality rate. Although several methods do exist, NMR provides higher analytical precision in identification of cancer metabolomic signature. Present study explored abnormal signatures in choline metabolism in oral squamous cell carcinoma (OSCC) using {sup 1}H and {sup 13}C NMR analysis of serum. It has demonstrated down-regulation of choline with concomitant up-regulation of its break-down product in the form of trimethylamine N-oxide in OSCC compared to normal counterpart. Further, no significant change in lactate profile in OSCC possibly indicated that well-known Warburg effect was not a prominent phenomenon in such malignancy. Amongst other important metabolites, malonate has shown up-regulation but D-glucose, saturated fatty acids, acetate and threonine did not show any significant change. Analyzing these metabolomic findings present study proposed trimethyl amine N-oxide and malonate as important metabolic signature for oral cancer with no prominent Warburg effect. - Highlights: • NMR ({sup 1}H and {sup 13}C) study of Oral Squamous cell Carcinoma Serum. • Abnormal Choline metabolomic signatures. • Up-regulation of Trimethylamine N-oxide. • Unchanged lactate profile indicates no prominent Warburg effect. • Proposed alternative glucose metabolism path through up-regulation of malonate.

  12. Impacts of proline on the central metabolism of an industrial erythromycin-producing strain Saccharopolyspora erythraea via (13)C labeling experiments.

    PubMed

    Hong, Ming; Huang, Mingzhi; Chu, Ju; Zhuang, Yingping; Zhang, Siliang

    2016-08-10

    Saccharopolyspora erythraea E3 is an important industrial strain for erythromycin production and knowledge on its metabolism is limited. In the present work, (13)C labeling experiments were conducted to characterize the metabolism of S. erythraea E3. We found that S. erythraea E3 was difficult to grow on minimal medium with glucose as sole carbon source and the addition of proline remarkably improved the cell growth. The activity of EMP pathway was very low and ED pathway was alternatively the main glucose utilization pathway. The addition of proline resulted in remarkable changes in the fluxes of central metabolism. The fluxes in PP pathway, in TCA cycle and in ED pathway were 90% higher, 64% and 31% lower on Glc/Pro than on Glc, respectively. The maintenance energy on Glc/Pro was 58.4% lower than that on Glc. The energy charge was lower on Glc than on Glc/Pro, indicating that the cells on Glc suffered from energy burden. This study elucidates the impacts of proline on the central metabolism of S. erythraea and deepens the understanding of its metabolism.

  13. Early estrogen-induced metabolic changes and their inhibition by actinomycin D and cycloheximide in human breast cancer cells: sup 31 P and sup 13 C NMR studies

    SciTech Connect

    Neeman, M.; Degani, H. )

    1989-07-01

    Metabolic changes following estrogen stimulation and the inhibition of these changes in the presence of actinomycin D and cycloheximide were monitored continuously in perfused human breast cancer T47D clone 11 cells with {sup 31}P and {sup 13}C NMR techniques. The experiments were performed by estrogen rescue of tamoxifen-treated cells. Immediately after perfusion with estrogen-containing medium, a continuous enhancement in the rates of glucose consumption, lactate production by glycolysis, and glutamate synthesis by the Krebs cycle occurred with a persistent 2-fold increase at 4 hr. Pretreatment with either actinomycin D or cycloheximide, at concentrations known to inhibit mRNA and protein synthesis, respectively, and simultaneous treatment with estrogen and each inhibitor prevented the estrogen-induced changes in glucose metabolism. This suggested that the observed estrogen stimulation required synthesis of mRNA and protein. These inhibitors also modulated several metabolic activities that were not related to estrogen stimulation. The observed changes in the in vivo kinetics of glucose metabolism may provide a means for the early detection of the response of human breast cancer cells to estrogen versus tamoxifen treatment.

  14. 13C-metabolic flux ratio and novel carbon path analyses confirmed that Trichoderma reesei uses primarily the respirative pathway also on the preferred carbon source glucose

    PubMed Central

    Jouhten, Paula; Pitkänen, Esa; Pakula, Tiina; Saloheimo, Markku; Penttilä, Merja; Maaheimo, Hannu

    2009-01-01

    Background The filamentous fungus Trichoderma reesei is an important host organism for industrial enzyme production. It is adapted to nutrient poor environments where it is capable of producing large amounts of hydrolytic enzymes. In its natural environment T. reesei is expected to benefit from high energy yield from utilization of respirative metabolic pathway. However, T. reesei lacks metabolic pathway reconstructions and the utilization of the respirative pathway has not been investigated on the level of in vivo fluxes. Results The biosynthetic pathways of amino acids in T. reesei supported by genome-level evidence were reconstructed with computational carbon path analysis. The pathway reconstructions were a prerequisite for analysis of in vivo fluxes. The distribution of in vivo fluxes in both wild type strain and cre1, a key regulator of carbon catabolite repression, deletion strain were quantitatively studied by performing 13C-labeling on both repressive carbon source glucose and non-repressive carbon source sorbitol. In addition, the 13C-labeling on sorbitol was performed both in the presence and absence of sophorose that induces the expression of cellulase genes. Carbon path analyses and the 13C-labeling patterns of proteinogenic amino acids indicated high similarity between biosynthetic pathways of amino acids in T. reesei and yeast Saccharomyces cerevisiae. In contrast to S. cerevisiae, however, mitochondrial rather than cytosolic biosynthesis of Asp was observed under all studied conditions. The relative anaplerotic flux to the TCA cycle was low and thus characteristic to respiratory metabolism in both strains and independent of the carbon source. Only minor differences were observed in the flux distributions of the wild type and cre1 deletion strain. Furthermore, the induction of the hydrolytic gene expression did not show altered flux distributions and did not affect the relative amino acid requirements or relative anabolic and respirative activities

  15. Metabolomic and (13)C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase.

    PubMed

    Wasylenko, Thomas M; Stephanopoulos, Gregory

    2015-03-01

    Over the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis.

  16. Metabolic Flux Analysis of Lipid Biosynthesis in the Yeast Yarrowia lipolytica Using 13C-Labled Glucose and Gas Chromatography-Mass Spectrometry.

    PubMed

    Zhang, Huaiyuan; Wu, Chao; Wu, Qingyu; Dai, Junbiao; Song, Yuanda

    2016-01-01

    The oleaginous yeast Yarrowia lipolytica has considerable potential for producing single cell oil, which can be converted to biodiesel, a sustainable alternative to fossil fuels. However, extensive fundamental and engineering efforts must be carried out before commercialized production become cost-effective. Therefore, in this study, metabolic flux analysis of Y. lipolytica was performed using 13C-labeled glucose as a sole carbon source in nitrogen sufficient and insufficient media. The nitrogen limited medium inhibited cell growth while promoting lipid accumulation (from 8.7% of their biomass to 14.3%). Metabolic flux analysis showed that flux through the pentose phosphate pathway was not significantly regulated by nitrogen concentration, suggesting that NADPH generation is not the limiting factor for lipid accumulation in Y. lipolytica. Furthermore, metabolic flux through malic enzyme was undetectable, confirming its non-regulatory role in lipid accumulation in this yeast. Nitrogen limitation significantly increased flux through ATP:citrate lyase (ACL), implying that ACL plays a key role in providing acetyl-CoA for lipid accumulation in Y. lipolytica.

  17. Metabolic Flux Analysis of Lipid Biosynthesis in the Yeast Yarrowia lipolytica Using 13C-Labled Glucose and Gas Chromatography-Mass Spectrometry

    PubMed Central

    Zhang, Huaiyuan; Wu, Chao; Wu, Qingyu; Dai, Junbiao; Song, Yuanda

    2016-01-01

    The oleaginous yeast Yarrowia lipolytica has considerable potential for producing single cell oil, which can be converted to biodiesel, a sustainable alternative to fossil fuels. However, extensive fundamental and engineering efforts must be carried out before commercialized production become cost-effective. Therefore, in this study, metabolic flux analysis of Y. lipolytica was performed using 13C-labeled glucose as a sole carbon source in nitrogen sufficient and insufficient media. The nitrogen limited medium inhibited cell growth while promoting lipid accumulation (from 8.7% of their biomass to 14.3%). Metabolic flux analysis showed that flux through the pentose phosphate pathway was not significantly regulated by nitrogen concentration, suggesting that NADPH generation is not the limiting factor for lipid accumulation in Y. lipolytica. Furthermore, metabolic flux through malic enzyme was undetectable, confirming its non-regulatory role in lipid accumulation in this yeast. Nitrogen limitation significantly increased flux through ATP:citrate lyase (ACL), implying that ACL plays a key role in providing acetyl-CoA for lipid accumulation in Y. lipolytica. PMID:27454589

  18. Investigation of Metabolism of Exogenous Glucose at the Early Stage and Onset of Diabetes Mellitus in Otsuka Long-Evans Tokushima Fatty Rats Using [1, 2, 3-13C]Glucose Breath Tests

    PubMed Central

    Kijima, Sho; Tanaka, Hideki

    2016-01-01

    This study aimed to evaluate changes in glucose metabolism at the early stage and onset of diabetes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Specifically, after the oral administration of [1, 2, 3-13C]glucose, the levels of exhaled 13CO2, which most likely originated from pyruvate decarboxylation and tricarboxylic acid, were measured. Eight OLETF rats and eight control rats (Long-Evans Tokushima Otsuka [LETO]) were administered 13C-glucose. Three types of 13C-glucose breath tests were performed thrice in each period at 2-week intervals. [3-13C]glucose results in a 13C isotope at position 1 in the pyruvate molecule, which provides 13CO2. The 13C at carbons 1 and 2 of glucose is converted to 13C at carbons 2 and 1 of acetate, respectively, which produce 13CO2. Based on metabolic differences of the labeled sites, glucose metabolism was evaluated using the results of three breath tests. The increase in 13CO2 excretion in OLETF rats was delayed in all three breath tests compared to that in control rats, suggesting that OLETF rats had a lower glucose metabolism than control rats. In addition, overall glucose metabolism increased with age in both groups. The utilization of [2-13C]glucose was suppressed in OLETF rats at 6–12 weeks of age, but they showed higher [3-13C]glucose oxidation than control rats at 22–25 weeks of age. In the [1-13C]glucose breath test, no significant differences in the area under the curve until 180 minutes (AUC180) were observed between OLETF and LETO rats of any age. Glucose metabolism kinetics were different between the age groups and two groups of rats; however, these differences were not significant based on the overall AUC180 of [1-13C]glucose. We conclude that breath 13CO2 excretion is reduced in OLETF rats at the primary stage of prediabetes, indicating differences in glucose oxidation kinetics between OLETF and LETO rats. PMID:27483133

  19. Investigation of Metabolism of Exogenous Glucose at the Early Stage and Onset of Diabetes Mellitus in Otsuka Long-Evans Tokushima Fatty Rats Using [1, 2, 3-13C]Glucose Breath Tests.

    PubMed

    Kawagoe, Naoyuki; Kano, Osamu; Kijima, Sho; Tanaka, Hideki; Takayanagi, Masaaki; Urita, Yoshihisa

    2016-01-01

    This study aimed to evaluate changes in glucose metabolism at the early stage and onset of diabetes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Specifically, after the oral administration of [1, 2, 3-13C]glucose, the levels of exhaled 13CO2, which most likely originated from pyruvate decarboxylation and tricarboxylic acid, were measured. Eight OLETF rats and eight control rats (Long-Evans Tokushima Otsuka [LETO]) were administered 13C-glucose. Three types of 13C-glucose breath tests were performed thrice in each period at 2-week intervals. [3-13C]glucose results in a 13C isotope at position 1 in the pyruvate molecule, which provides 13CO2. The 13C at carbons 1 and 2 of glucose is converted to 13C at carbons 2 and 1 of acetate, respectively, which produce 13CO2. Based on metabolic differences of the labeled sites, glucose metabolism was evaluated using the results of three breath tests. The increase in 13CO2 excretion in OLETF rats was delayed in all three breath tests compared to that in control rats, suggesting that OLETF rats had a lower glucose metabolism than control rats. In addition, overall glucose metabolism increased with age in both groups. The utilization of [2-13C]glucose was suppressed in OLETF rats at 6-12 weeks of age, but they showed higher [3-13C]glucose oxidation than control rats at 22-25 weeks of age. In the [1-13C]glucose breath test, no significant differences in the area under the curve until 180 minutes (AUC180) were observed between OLETF and LETO rats of any age. Glucose metabolism kinetics were different between the age groups and two groups of rats; however, these differences were not significant based on the overall AUC180 of [1-13C]glucose. We conclude that breath 13CO2 excretion is reduced in OLETF rats at the primary stage of prediabetes, indicating differences in glucose oxidation kinetics between OLETF and LETO rats.

  20. Synergy between 13C-metabolic flux analysis and flux balance analysis for understanding metabolic adaption to anaerobiosis in e. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome-based Flux Balance Analysis (FBA, constraints based flux analysis) and steady state isotopic-labeling-based Metabolic Flux Analysis (MFA) are complimentary approaches to predicting and measuring the operation and regulation of metabolic networks. Here a genome-derived model of E. coli metabol...

  1. Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through 13C-based metabolic flux analysis.

    PubMed

    Papini, Marta; Nookaew, Intawat; Siewers, Verena; Nielsen, Jens

    2012-08-01

    Several bacterial species and filamentous fungi utilize the phosphoketolase pathway (PHK) for glucose dissimilation as an alternative to the Embden-Meyerhof-Parnas pathway. In Aspergillus nidulans, the utilization of this metabolic pathway leads to increased carbon flow towards acetate and acetyl CoA. In the first step of the PHK, the pentose phosphate pathway intermediate xylulose-5-phosphate is converted into acetylphosphate and glyceraldehyde-3-phosphate through the action of xylulose-5-phosphate phosphoketolase, and successively acetylphosphate is converted into acetate by the action of acetate kinase. In the present work, we describe a metabolic engineering strategy used to express the fungal genes of the phosphoketolase pathway in Saccharomyces cerevisiae and the effects of the expression of this recombinant route in yeast. The phenotype of the engineered yeast strain MP003 was studied during batch and chemostat cultivations, showing a reduced biomass yield and an increased acetate yield during batch cultures. To establish whether the observed effects in the recombinant strain MP003 were due directly or indirectly to the expression of the phosphoketolase pathway, we resolved the intracellular flux distribution based on (13)C labeling during chemostat cultivations. From flux analysis it is possible to conclude that yeast is able to use the recombinant pathway. Our work indicates that the utilization of the phosphoketolase pathway does not interfere with glucose assimilation through the Embden-Meyerhof-Parnas pathway and that the expression of this route can contribute to increase the acetyl CoA supply, therefore holding potential for future metabolic engineering strategies having acetyl CoA as precursor for the biosynthesis of industrially relevant compounds.

  2. Flux response of glycolysis and storage metabolism during rapid feast/famine conditions in Penicillium chrysogenum using dynamic (13)C labeling.

    PubMed

    de Jonge, Lodewijk; Buijs, Nicolaas A A; Heijnen, Joseph J; van Gulik, Walter M; Abate, Alessandro; Wahl, S Aljoscha

    2014-03-01

    The scale-up of fermentation processes frequently leads to a reduced productivity compared to small-scale screening experiments. Large-scale mixing limitations that lead to gradients in substrate and oxygen availability could influence the microorganism performance. Here, the impact of substrate gradients on a penicillin G producing Penicillium chrysogenum cultivation was analyzed using an intermittent glucose feeding regime. The intermittent feeding led to fluctuations in the extracellular glucose concentration between 400 μM down to 6.5 μM at the end of the cycle. The intracellular metabolite concentrations responded strongly and showed up to 100-fold changes. The intracellular flux changes were estimated on the basis of dynamic (13) C mass isotopomer measurements during three cycles of feast and famine using a novel hybrid modeling approach. The flux estimations indicated a high turnover of internal and external storage metabolites in P. chrysogenum under feast/famine conditions. The synthesis and degradation of storage requires cellular energy (ATP and UTP) in competition with other cellular functions including product formation. Especially, 38% of the incoming glucose was recycled once in storage metabolism. This result indicated that storage turnover is increased under dynamic cultivation conditions and contributes to the observed decrease in productivity compared to reference steady-state conditions.

  3. Uncoupling Protein 2 (UCP2) Function in the Brain as Revealed by the Cerebral Metabolism of (1-(13)C)-Glucose.

    PubMed

    Contreras, Laura; Rial, Eduardo; Cerdan, Sebastian; Satrustegui, Jorgina

    2017-01-01

    The mitochondrial aspartate/glutamate transporter Aralar/AGC1/Slc25a12 is critically involved in brain aspartate synthesis, and AGC1 deficiency results in a drastic fall of brain aspartate levels in humans and mice. It has recently been described that the uncoupling protein UCP2 transports four carbon metabolites including aspartate. Since UCP2 is expressed in several brain cell types and AGC1 is mainly neuronal, we set to test whether UCP2 could be a mitochondrial aspartate carrier in the brain glial compartment. The study of the cerebral metabolism of (1-(13)C)-glucose in vivo in wild type and UCP2-knockout mice showed no differences in C3 or C2 labeling of aspartate, suggesting that UCP2 does not function as a mitochondrial aspartate carrier in brain. However, surprisingly, a clear decrease (of about 30-35 %) in the fractional enrichment of glutamate, glutamine and GABA was observed in the brains of UCP2-KO mice which was not associated with differences in either glucose or lactate enrichments. The results suggest that the dilution in the labeling of glutamate and its downstream metabolites could originate from the uptake of an unlabeled substrate that could not leave the matrix via UCP2 becoming trapped in the matrix. Understanding the nature of the unlabeled substrate and its precursor(s) as alternative substrates to glucose is of interest in the context of neurological diseases associated with UCP2.

  4. Quantitative role of splanchnic region in leucine metabolism: L-(1-13C,15N)leucine and substrate balance studies

    SciTech Connect

    Yu, Y.M.; Wagner, D.A.; Tredget, E.E.; Walaszewski, J.A.; Burke, J.F.; Young, V.R. )

    1990-07-01

    The role of the splanchnic region (Sp) in whole body leucine metabolism was assessed in six chronically catheterized fasting mongrel dogs and in eight dogs during constant enteral feeding of a complete amino acid solution (0.24 g.kg-1.h-1). We used primed continuous intravenous infusions of L-(1-13C,15N)leucine and L-(1-14C)leucine and measurements of arteriovenous isotope and leucine balance across the gut, liver, and Sp. In the fasted condition, 3.5% of arterial leucine supply was oxidized in the Sp, accounting for 13% of total body leucine oxidation, with 10% by liver. With amino acid feeding (1) leucine carbon and nitrogen fluxes and oxidation were increased (P less than 0.01) at the whole body level; (2) the percent of whole body leucine oxidation occurring in the Sp and liver increased (P less than 0.01) to 41 and 27%, respectively; (3) fractional metabolic utilization of leucine delivered to the Sp was reduced (P less than 0.01) from 47 to 35%; (4) the deamination rate of leucine in the gut was increased (P less than 0.05), along with an increased reamination rate of alpha-ketoisocaproic acid in the Sp (P less than 0.05). These findings reveal that the Sp accounts for a small fraction of whole body leucine oxidation during the fasting condition, but it plays a quantitatively important role in total body leucine oxidation during amino acid feeding; the gut and liver play cooperative roles in controlling leucine supply to peripheral tissues.

  5. Mass spectrometry-based microassay of 2H and 13C plasma glucose labeling to quantify liver metabolic fluxes in vivo

    PubMed Central

    Hasenour, Clinton M.; Wall, Martha L.; Ridley, D. Emerson; Hughey, Curtis C.; James, Freyja D.; Wasserman, David H.

    2015-01-01

    Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [13C3]propionate, [2H2]water, and [6,6-2H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations. PMID:25991647

  6. Imaging metabolic syndrome

    PubMed Central

    Han, Weiping; Chuang, Kai-Hsiang; Chang, Young-Tae; Olivo, Malini; Velan, S Sendhil; Bhakoo, Kishore; Townsend, David; Radda, George K

    2010-01-01

    Metabolic syndrome is a fast growing public health burden for almost all the developed countries and many developing nations. Despite intense efforts from both biomedical and clinical scientists, many fundamental questions regarding its aetiology and development remain unclear, partly due to the lack of suitable imaging technologies to visualize lipid composition and distribution, insulin secretion, β-cell mass and functions in vivo. Such technologies would not only impact on our understanding of the complexity of metabolic disorders such as obesity and diabetes, but also aid in their diagnosis, drug development and assessment of treatment efficacy. In this article we discuss and propose several strategies for visualization of physiological and pathological changes that affect pancreas and adipose tissue as a result of the development of metabolic diseases. PMID:20533426

  7. Comparison of Glutamate Turnover in Nerve Terminals and Brain Tissue During [1,6-(13)C2]Glucose Metabolism in Anesthetized Rats.

    PubMed

    Patel, Anant B; Lai, James C K; Chowdhury, Golam I M; Rothman, Douglas L; Behar, Kevin L

    2017-01-01

    The (13)C turnover of neurotransmitter amino acids (glutamate, GABA and aspartate) were determined from extracts of forebrain nerve terminals and brain homogenate, and fronto-parietal cortex from anesthetized rats undergoing timed infusions of [1,6-(13)C2]glucose or [2-(13)C]acetate. Nerve terminal (13)C fractional labeling of glutamate and aspartate was lower than those in whole cortical tissue at all times measured (up to 120 min), suggesting either the presence of a constant dilution flux from an unlabeled substrate or an unlabeled (effectively non-communicating on the measurement timescale) glutamate pool in the nerve terminals. Half times of (13)C labeling from [1,6-(13)C2]glucose, as estimated by least squares exponential fitting to the time course data, were longer for nerve terminals (GluC4, 21.8 min; GABAC2 21.0 min) compared to cortical tissue (GluC4, 12.4 min; GABAC2, 14.5 min), except for AspC3, which was similar (26.5 vs. 27.0 min). The slower turnover of glutamate in the nerve terminals (but not GABA) compared to the cortex may reflect selective effects of anesthesia on activity-dependent glucose use, which might be more pronounced in the terminals. The (13)C labeling ratio for glutamate-C4 from [2-(13)C]acetate over that of (13)C-glucose was twice as large in nerve terminals compared to cortex, suggesting that astroglial glutamine under the (13)C glucose infusion was the likely source of much of the nerve terminal dilution. The net replenishment of most of the nerve terminal amino acid pools occurs directly via trafficking of astroglial glutamine.

  8. An Alternative Scheme for 13C Chemical-Shift Imaging via Inverse Detection of Protons through an MILS (or Inverse SLIM) Technique

    PubMed

    Lee; Tzou; Chiou; Yeung

    1998-01-01

    An alternative scheme in acquiring 13C spectroscopic images using inverse detection via polarization transfer through protons was proposed and experimentally verified by a phantom using a heteronuclear multiple-quantum coherence technique. This scheme has some features that, in special circumstances, can be exploited to one's advantages. These features are: (1) signal enhancement, a feat realizable under favorable conditions; (2) spectroscopic encoding via constant time; and (3) improvement of the time efficiency of the constant-time method via optimization by singular-value decomposition analysis. Features (1) and (2) can, in some cases, yield better sensitivity than the conventional 3DFT technique without sacrificing the most important attribute of 13C, the enormity of the chemical shifts. Such is the case because spectroscopic images acquired by the technique proposed are based on the chemical shifts of carbon, in contrast to the other more prevalent inverse-detection schemes. Feature (2) can also offer higher spatial resolution by shifting the emphasis of signal encoding from a spectral-resolution-first viewpoint to one that favors spatial resolution. Feature (3) was adopted from a method previously known as MILS (metabolite imaging of lines in a spectrum), or the inverse of SLIM, an economical scheme originally proposed by Hu et al. (1988, Magn. Reson. Med. 8, 314) for obtaining a compartmentalized NMR spectrum. Copyright 1998 Academic Press. Copyright 1998 Academic Press

  9. The Effect of Molecular Conformation on the Accuracy of Theoretical (1)H and (13)C Chemical Shifts Calculated by Ab Initio Methods for Metabolic Mixture Analysis.

    PubMed

    Chikayama, Eisuke; Shimbo, Yudai; Komatsu, Keiko; Kikuchi, Jun

    2016-04-14

    NMR spectroscopy is a powerful method for analyzing metabolic mixtures. The information obtained from an NMR spectrum is in the form of physical parameters, such as chemical shifts, and construction of databases for many metabolites will be useful for data interpretation. To increase the accuracy of theoretical chemical shifts for development of a database for a variety of metabolites, the effects of sets of conformations (structural ensembles) and the levels of theory on computations of theoretical chemical shifts were systematically investigated for a set of 29 small molecules in the present study. For each of the 29 compounds, 101 structures were generated by classical molecular dynamics at 298.15 K, and then theoretical chemical shifts for 164 (1)H and 123 (13)C atoms were calculated by ab initio quantum chemical methods. Six levels of theory were used by pairing Hartree-Fock, B3LYP (density functional theory), or second order Møller-Plesset perturbation with 6-31G or aug-cc-pVDZ basis set. The six average fluctuations in the (1)H chemical shift were ±0.63, ± 0.59, ± 0.70, ± 0.62, ± 0.75, and ±0.66 ppm for the structural ensembles, and the six average errors were ±0.34, ± 0.27, ± 0.32, ± 0.25, ± 0.32, and ±0.25 ppm. The results showed that chemical shift fluctuations with changes in the conformation because of molecular motion were larger than the differences between computed and experimental chemical shifts for all six levels of theory. In conclusion, selection of an appropriate structural ensemble should be performed before theoretical chemical shift calculations for development of an accurate database for a variety of metabolites.

  10. Impact of carbon metabolism on 13C signatures of cyanobacteria and green non-sulfur-like bacteria inhabiting a microbial mat from an alkaline siliceous hot spring in Yellowstone National Park (USA).

    PubMed

    van der Meer, Marcel T J; Schouten, Stefan; Damsté, Jaap S Sinninghe; Ward, David M

    2007-02-01

    Alkaline siliceous hot spring microbial mats in Yellowstone National Park are composed of two dominant phototropic groups, cyanobacteria and green non-sulfur-like bacteria (GNSLB). While cyanobacteria are thought to cross-feed low-molecular-weight organic compounds to support photoheterotrophic metabolism in GNSLB, it is unclear how this could lead to the heavier stable carbon isotopic signatures in GNSLB lipids compared with cyanobacterial lipids found in previous studies. The two groups of phototrophs were separated using percoll density gradient centrifugation and subsequent lipid and stable carbon isotopic analysis revealed that we obtained fractions with a approximately 60-fold enrichment in cyanobacterial and an approximately twofold enrichment in GNSLB biomass, respectively, compared with the mat itself. This technique was used to study the diel cycling and 13C content of the glucose pools in and the uptake of 13C-bicarbonate by the cyanobacteria and GNSLB, as well as the transfer of incorporated 13C from cyanobacteria to GNSLB. The results show that cyanobacteria have the highest bicarbonate uptake rates and accumulate glucose during the afternoon in full light conditions. In contrast, GNSLB have relatively higher bicarbonate uptake rates compared with cyanobacteria in the morning at low light levels. During the night GNSLB take up carbon that is likely derived through fermentation of cyanobacterial glucose enriched in 13C. The assimilation of 13C-enriched cyanobacterial carbon may thus lead to enriched 13C-contents of GNSLB cell components.

  11. Tracing bacterial metabolism using multi-nuclear (1H, 2H, and 13C) Solid State NMR: Realizing an Idea Initiated by James Scott

    NASA Astrophysics Data System (ADS)

    Cody, G.; Fogel, M. L.; Jin, K.; Griffen, P.; Steele, A.; Wang, Y.

    2011-12-01

    Approximately 6 years ago, while at the Geophysical Laboratory, James Scott became interested in the application of Solid State Nuclear Magnetic Resonance Spectroscopy to study bacterial metabolism. As often happens, other experiments intervened and the NMR experiments were not pursued. We have revisited Jame's question and find that using a multi-nuclear approach (1H, 2H, and 13C Solid State NMR) on laboratory cell culture has some distinct advantages. Our experiments involved batch cultures of E. coli (MG1655) harvested at stationary phase. In all experiments the growth medium consisted of MOPS medium for enterobacteria, where the substrate is glucose. In one set of experiments, 10 % of the water was D2O; in another 10 % of the glucose was per-deuterated. The control experiment used both water and glucose at natural isotopic abundance. A kill control of dead E. coli immersed in pure D2O for an extended period exhibited no deuterium incorporation. In both deuterium enriched experiments, considerable incorporation of deuterium into E. coli's biomolecular constituents was detected via 2H Solid State NMR. In the case of the D2O enriched experiment, 58 % of the incorporated deuterium is observed in a sharp peak at a frequency of 0.31 ppm, consistent with D incorporation in the cell membrane lipids, the remainder is observed in a broad peak at a higher frequency (centered at 5.4 ppm, but spanning out to beyond 10 ppm) that is consistent with D incorporation into predominantly DNA and RNA. In the case of the D-glucose experiments, 61 % of the deuterium is observed in a sharp resonance peak at 0.34 ppm, also consistent with D incorporation into membrane lipids, the remainder of the D is observed at a broad resonance peak centered at 4.3 ppm, consistent with D enrichment in glycogen. Deuterium abundance in the E. coli cells grown in 10 % D2O is nearly 2X greater than that grown with 10 % D-glucose. Very subtle differences are observed in both the 1H and 13C solid

  12. Gas Chromatography-Quadrupole Time-of-Flight Mass Spectrometry-Based Determination of Isotopologue and Tandem Mass Isotopomer Fractions of Primary Metabolites for (13)C-Metabolic Flux Analysis.

    PubMed

    Mairinger, Teresa; Steiger, Matthias; Nocon, Justyna; Mattanovich, Diethard; Koellensperger, Gunda; Hann, Stephan

    2015-12-01

    For the first time an analytical work flow based on accurate mass gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOFMS) with chemical ionization for analysis providing a comprehensive picture of (13)C distribution along the primary metabolism is elaborated. The method provides a powerful new toolbox for (13)C-based metabolic flux analysis, which is an emerging strategy in metabolic engineering. In this field, stable isotope tracer experiments based on, for example, (13)C are central for providing characteristic patterns of labeled metabolites, which in turn give insights into the regulation of metabolic pathway kinetics. The new method enables the analysis of isotopologue fractions of 42 free intracellular metabolites within biotechnological samples, while tandem mass isotopomer information is also accessible for a large number of analytes. Hence, the method outperforms previous approaches in terms of metabolite coverage, while also providing rich isotopomer information for a significant number of key metabolites. Moreover, the established work flow includes novel evaluation routines correcting for isotope interference of naturally distributed elements, which is crucial following derivatization of metabolites. Method validation in terms of trueness, precision, and limits of detection was performed, showing excellent analytical figures of merit with an overall maximum bias of 5.8%, very high precision for isotopologue and tandem mass isotopomer fractions representing >10% of total abundance, and absolute limits of detection in the femtomole range. The suitability of the developed method is demonstrated on a flux experiment of Pichia pastoris employing two different tracers, i.e., 1,6(13)C2-glucose and uniformly labeled (13)C-glucose.

  13. The pharmacokinetics and metabolism of 14C/13C-labeled ortho-phenylphenol formation following dermal application to human volunteers.

    PubMed

    Timchalk, C; Selim, S; Sangha, G; Bartels, M J

    1998-08-01

    1. The pharmacokinetics and metabolism of uniformly labeled 14C/13C-ortho-phenylphenol (OPP) were followed in six human male volunteers given a single 8 h dermal dose of 6 microg OPP/kg body weight formulated as a 0.4% (w/v) solution in isopropyl alcohol. The application site was covered with a non-occlusive dome allowing free movement of air, but preventing the loss of radioactivity due to physical contact. At 8 h post-exposure the non-occlusive dome was removed, the dose site was wiped with isopropyl alcohol containing swabs and the skin surface repeatedly stripped with tape. Blood specimens, urine, and feces were collected from each volunteer over a 5 day post-exposure period and were analyzed for radioactivity and metabolites (urine only). 2. Following dermal application, peak plasma levels of radioactivity were obtained within 4 h post-exposure and rapidly declined with virtually all of the absorbed dose rapidly excreted into the urine within 24 h post-exposure. A one-compartment pharmacokinetic model was used to describe the time-course of OPP absorption and clearance in male human volunteers. Approximately 43% of the dermally applied dose was absorbed through the skin with an average absorption half-life of 10 h. Once absorbed the renal clearance of OPP was rapid with an average half-life of 0.8 h. The rate limiting step for renal clearance was the relatively slower rate of dermal absorption; therefore the pharmacokinetics of OPP in humans was described by a 'flip-flop' single compartment model. Overall, the pharmacokinetics were similar between individuals, and the model parameters were in excellent agreement with the experimental data. 3. Approximately 73% of the total urinary radioactivity was accounted for as free OPP, OPP-sulfate and OPP-glucuronide conjugates. The sulfate conjugate was the major metabolite (approximately 69%). Therefore, total urinary OPP equivalents (acid-labile conjugates+free OPP) can be used to estimate the systemically absorbed

  14. Design and operation of a continuous 13C and 15N labeling chamber for uniform or differential, metabolic and structural, plant tissue isotope labeling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tracing heavy stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O o...

  15. An automated growth enclosure for metabolic labeling of Arabidopsis thaliana with 13C-carbon dioxide - an in vivo labeling system for proteomics and metabolomics research

    PubMed Central

    2011-01-01

    Background Labeling whole Arabidopsis (Arabidopsis thaliana) plants to high enrichment with 13C for proteomics and metabolomics applications would facilitate experimental approaches not possible by conventional methods. Such a system would use the plant's native capacity for carbon fixation to ubiquitously incorporate 13C from 13CO2 gas. Because of the high cost of 13CO2 it is critical that the design conserve the labeled gas. Results A fully enclosed automated plant growth enclosure has been designed and assembled where the system simultaneously monitors humidity, temperature, pressure and 13CO2 concentration with continuous adjustment of humidity, pressure and 13CO2 levels controlled by a computer running LabView software. The enclosure is mounted on a movable cart for mobility among growth environments. Arabidopsis was grown in the enclosure for up to 8 weeks and obtained on average >95 atom% enrichment for small metabolites, such as amino acids and >91 atom% for large metabolites, including proteins and peptides. Conclusion The capability of this labeling system for isotope dilution experiments was demonstrated by evaluation of amino acid turnover using GC-MS as well as protein turnover using LC-MS/MS. Because this 'open source' Arabidopsis 13C-labeling growth environment was built using readily available materials and software, it can be adapted easily to accommodate many different experimental designs. PMID:21310072

  16. Application of Metabolic 13C Labeling in Conjunction with High-Field Nuclear Magnetic Resonance Spectroscopy for Comparative Conformational Analysis of High Mannose-Type Oligosaccharides

    PubMed Central

    Kamiya, Yukiko; Yanagi, Kotaro; Kitajima, Toshihiko; Yamaguchi, Takumi; Chiba, Yasunori; Kato, Koichi

    2013-01-01

    High mannose-type oligosaccharides are enzymatically trimmed in the endoplasmic reticulum, resulting in various processing intermediates with exposed glycotopes that are recognized by a series of lectins involved in glycoprotein fate determination in cells. Although recent crystallographic data have provided the structural basis for the carbohydrate recognition of intracellular lectins, atomic information of dynamic oligosaccharide conformations is essential for a quantitative understanding of the energetics of carbohydrate–lectin interactions. Carbohydrate NMR spectroscopy is useful for characterizing such conformational dynamics, but often hampered by poor spectral resolution and lack of recombinant techniques required to produce homogeneous glycoforms. To overcome these difficulties, we have recently developed a methodology for the preparation of a homogeneous high mannose-type oligosaccharide with 13C labeling using a genetically engineered yeast strain. We herein successfully extended this method to result in the overexpression of 13C-labeled Man9GlcNAc2 (M9) with a newly engineered yeast strain with the deletion of four genes involved in N-glycan processing. This enabled high-field NMR analyses of 13C-labeled M9 in comparison with its processing product lacking the terminal mannose residue ManD2. Long-range NOE data indicated that the outer branches interact with the core in both glycoforms, and such foldback conformations are enhanced upon the removal of ManD2. The observed conformational variabilities might be significantly associated with lectins and glycan-trimming enzymes. PMID:24970159

  17. Comparative absorption of [13C]glucose and [13C]lactose by premature infants.

    PubMed

    Murray, R D; Boutton, T W; Klein, P D; Gilbert, M; Paule, C L; MacLean, W C

    1990-01-01

    Oxidation of orally administered [13C]glucose and [13C]lactose and fecal recovery of malabsorbed substrates were determined in two groups of premature infants. Eighteen studies were performed with six infants at Johns Hopkins Hospital (JHH); 24 studies were performed with nine infants at Columbus Children's Hospital (CCH). The two groups differed in that JHH infants had shorter gestations but were older when studied. Fecal 13C loss after [13C]glucose administration did not differ between the two groups. Compared with glucose, the metabolism of lactose appeared to involve more malabsorption and colonic fermentation in JHH infants than in CCH infants and resulted in higher fecal losses of substrate carbon. Maturation appeared to involve increased proximal intestinal absorption and greater retention of absorbed carbohydrate. Simultaneous absorption of substrate from the small and large intestine may limit the usefulness of breath tests for 13C in the premature infant.

  18. Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids.

    PubMed

    Jordà, Joel; de Jesus, Sérgio S; Peltier, Solenne; Ferrer, Pau; Albiol, Joan

    2014-01-25

    The yeast Pichia pastoris has emerged as one of the most promising yeast cell factories for the production of heterologous proteins. The readily available genetic tools and the ease of high-cell density cultivations using methanol or glycerol/methanol mixtures are among the key factors for this development. Previous studies have shown that the use of mixed feeds of glycerol and methanol seem to alleviate the metabolic burden derived from protein production, allowing for higher specific and volumetric process productivities. However, initial studies of glycerol/methanol co-metabolism in P. pastoris by classical metabolic flux analyses using (13)C-derived Metabolic Flux Ratio (METAFoR) constraints were hampered by the reduced labelling information obtained when using C3:C1 substrate mixtures in relation to the conventional C6 substrate, that is, glucose. In this study, carbon flux distributions through the central metabolic pathways in glycerol/methanol co-assimilation conditions have been further characterised using biosynthetically directed fractional (13)C labelling. In particular, metabolic flux distributions were obtained under 3 different glycerol/methanol ratios and growth rates by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids using the software tool (13)CFlux2. Specifically, cells were grown aerobically in chemostat cultures fed with 80:20, 60:40 and 40:60 (w:w) glycerol/methanol mixtures at two dilutions rates (0.05 hour(-1) and 0.16 hour(-1)), allowing to obtain additional data (biomass composition and extracellular fluxes) to complement pre-existing datasets. The performed (13)C-MFA reveals a significant redistribution of carbon fluxes in the central carbon metabolism as a result of the shift in the dilution rate, while the ratio of carbon sources has a lower impact on carbon flux distribution in cells growing at the same dilution rate. At low growth rate, the percentage of methanol directly dissimilated to CO2 ranges

  19. Profiling sulfation/epimerization pattern of full-length heparan sulfate by NMR following cell culture 13C-glucose metabolic labeling.

    PubMed

    Pegeot, Mathieu; Sadir, Rabia; Eriksson, Inger; Kjellen, Lena; Simorre, Jean-Pierre; Gans, Pierre; Lortat-Jacob, Hugues

    2015-02-01

    Through its ability to interact with proteins, heparan sulfate (HS) fulfills a large variety of functions. Protein binding depends on the level of HS sulfation and epimerization which are cell specific and dynamically regulated. Characterization of this molecule, however, has been restricted to oligosaccharide fragments available in large amount for structural investigation or to sulfate distribution through compositional analysis. Here we developed a (1)H-(13)C 2D NMR-based approach, directly performed on HS isolated from (13)C-labeled cells. By integrating the peak volumes measured at different chemical shifts, this non-destructive analysis allows us to determine both the sulfation and the iduronic/glucuronic profiles of the polysaccharide. Applied to wild-type and N-deacetylase/N-sulfotransferase-deficient fibroblasts as well as to epithelial cells differentiation, it also gives insights into the functional relationships existing between HS biosynthetic enzymes. This approach should be of significant interest to better understand HS changes that occur through physiologic regulations or during pathological development.

  20. 13C-nuclear magnetic resonance spectroscopy studies of hepatic glucose metabolism in normal subjects and subjects with insulin-dependent diabetes mellitus.

    PubMed Central

    Cline, G W; Rothman, D L; Magnusson, I; Katz, L D; Shulman, G I

    1994-01-01

    To determine the effect of insulin-dependent diabetes mellitus (IDDM) on rates and pathways of hepatic glycogen synthesis, as well as flux through hepatic pyruvate dehydrogenase, we used 13C-nuclear magnetic resonance spectroscopy to monitor the peak intensity of the C1 resonance of the glucosyl units of hepatic glycogen, in combination with acetaminophen to sample the hepatic UDP-glucose pool and phenylacetate to sample the hepatic glutamine pool, during a hyperglycemic-hyperinsulinemic clamp using [1-13C]-glucose. Five subjects with poorly controlled IDDM and six age-weight-matched control subjects were clamped at a mean plasma glucose concentration of approximately 9 mM and mean plasma insulin concentrations approximately 400 pM for 5 h. Rates of hepatic glycogen synthesis were similar in both groups (approximately 0.43 +/- 0.09 mumol/ml liver min). However, flux through the indirect pathway of glycogen synthesis (3 carbon units-->-->glycogen) was increased by approximately 50% (P < 0.05), whereas the relative contribution of pyruvate oxidation to TCA cycle flux was decreased by approximately 30% (P < 0.05) in the IDDM subjects compared to the control subjects. These studies demonstrate that patients with poorly controlled insulin-dependent diabetes mellitus have augmented hepatic gluconeogenesis and relative decreased rates of hepatic pyruvate oxidation. These abnormalities are not immediately reversed by normalizing intraportal concentrations of glucose, insulin, and glucagon and may contribute to postprandial hyperglycemia. PMID:7989593

  1. Videofluorometer for imaging tissue metabolism

    NASA Astrophysics Data System (ADS)

    Kelly, Jeffrey J.; Rorvik, Dawn A.; Richmond, Keith N.; Barlow, Clyde H.

    1989-11-01

    A videofluorometer is described that directly acquires digital metabolic images of reduced nicotinamide adenine dinucleotide (NADH) fluorescence in tissue. NADH fluorescence provides an intrinsic indicator of the state of tissue mitochondrial oxidative metabolism. The device combines a computer-controlled fluorescence excitation system with digital image acquisition to quantify tissue bioenergetics in both spatial and time domains. Localized ischemia following coronary artery ligation in a perfused rat heart (model for a coronary artery occlusion heart attack) is used as an example to demonstrate the capabilities of the system. This videofluorometer permits monitoring changes in physiological state of organs and tissue without interfering with tissue metabolism. The digital nature of the acquired image allows detailed analysis of physiological features and their time dependence.

  2. CeCaFDB: a curated database for the documentation, visualization and comparative analysis of central carbon metabolic flux distributions explored by 13C-fluxomics.

    PubMed

    Zhang, Zhengdong; Shen, Tie; Rui, Bin; Zhou, Wenwei; Zhou, Xiangfei; Shang, Chuanyu; Xin, Chenwei; Liu, Xiaoguang; Li, Gang; Jiang, Jiansi; Li, Chao; Li, Ruiyuan; Han, Mengshu; You, Shanping; Yu, Guojun; Yi, Yin; Wen, Han; Liu, Zhijie; Xie, Xiaoyao

    2015-01-01

    The Central Carbon Metabolic Flux Database (CeCaFDB, available at http://www.cecafdb.org) is a manually curated, multipurpose and open-access database for the documentation, visualization and comparative analysis of the quantitative flux results of central carbon metabolism among microbes and animal cells. It encompasses records for more than 500 flux distributions among 36 organisms and includes information regarding the genotype, culture medium, growth conditions and other specific information gathered from hundreds of journal articles. In addition to its comprehensive literature-derived data, the CeCaFDB supports a common text search function among the data and interactive visualization of the curated flux distributions with compartmentation information based on the Cytoscape Web API, which facilitates data interpretation. The CeCaFDB offers four modules to calculate a similarity score or to perform an alignment between the flux distributions. One of the modules was built using an inter programming algorithm for flux distribution alignment that was specifically designed for this study. Based on these modules, the CeCaFDB also supports an extensive flux distribution comparison function among the curated data. The CeCaFDB is strenuously designed to address the broad demands of biochemists, metabolic engineers, systems biologists and members of the -omics community.

  3. Calculation of total meal d13C from individual food d13C.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Variations in the isotopic signature of carbon in biological samples can be used to distinguish dietary patterns and monitor shifts in metabolism. But for these variations to have meaning, the isotopic signature of the diet must be known. We sought to determine if knowledge of the 13C isotopic abund...

  4. The use of dynamic nuclear polarization (13)C-pyruvate MRS in cancer.

    PubMed

    Gutte, Henrik; Hansen, Adam Espe; Johannesen, Helle Hjorth; Clemmensen, Andreas Ettrup; Ardenkjær-Larsen, Jan Henrik; Nielsen, Carsten Haagen; Kjær, Andreas

    2015-01-01

    In recent years there has been an immense development of new targeted anti-cancer drugs. For practicing precision medicine, a sensitive method imaging for non-invasive, assessment of early treatment response and for assisting in developing new drugs is warranted. Magnetic Resonance Spectroscopy (MRS) is a potent technique for non-invasive in vivo investigation of tissue chemistry and cellular metabolism. Hyperpolarization by Dynamic Nuclear Polarization (DNP) is capable of creating solutions of molecules with polarized nuclear spins in a range of biological molecules and has enabled the real-time investigation of in vivo metabolism. The development of this new method has been demonstrated to enhance the nuclear polarization more than 10,000-fold, thereby significantly increasing the sensitivity of the MRS with a spatial resolution to the millimeters and a temporal resolution at the subsecond range. Furthermore, the method enables measuring kinetics of conversion of substrates into cell metabolites and can be integrated with anatomical proton magnetic resonance imaging (MRI). Many nuclei and substrates have been hyperpolarized using the DNP method. Currently, the most widely used compound is (13)C-pyruvate due to favoring technicalities. Intravenous injection of the hyperpolarized (13)C-pyruvate results in appearance of (13)C-lactate, (13)C-alanine and (13)C-bicarbonate resonance peaks depending on the tissue, disease and the metabolic state probed. In cancer, the lactate level is increased due to increased glycolysis. The use of DNP enhanced (13)C-pyruvate has in preclinical studies shown to be a sensitive method for detecting cancer and for assessment of early treatment response in a variety of cancers. Recently, a first-in-man 31-patient study was conducted with the primary objective to assess the safety of hyperpolarized (13)C-pyruvate in healthy subjects and prostate cancer patients. The study showed an elevated (13)C-lactate/(13)C-pyruvate ratio in regions of

  5. The use of dynamic nuclear polarization 13C-pyruvate MRS in cancer

    PubMed Central

    Gutte, Henrik; Hansen, Adam Espe; Johannesen, Helle Hjorth; Clemmensen, Andreas Ettrup; Ardenkjær-Larsen, Jan Henrik; Nielsen, Carsten Haagen; Kjær, Andreas

    2015-01-01

    In recent years there has been an immense development of new targeted anti-cancer drugs. For practicing precision medicine, a sensitive method imaging for non-invasive, assessment of early treatment response and for assisting in developing new drugs is warranted. Magnetic Resonance Spectroscopy (MRS) is a potent technique for non-invasive in vivo investigation of tissue chemistry and cellular metabolism. Hyperpolarization by Dynamic Nuclear Polarization (DNP) is capable of creating solutions of molecules with polarized nuclear spins in a range of biological molecules and has enabled the real-time investigation of in vivo metabolism. The development of this new method has been demonstrated to enhance the nuclear polarization more than 10,000-fold, thereby significantly increasing the sensitivity of the MRS with a spatial resolution to the millimeters and a temporal resolution at the subsecond range. Furthermore, the method enables measuring kinetics of conversion of substrates into cell metabolites and can be integrated with anatomical proton magnetic resonance imaging (MRI). Many nuclei and substrates have been hyperpolarized using the DNP method. Currently, the most widely used compound is 13C-pyruvate due to favoring technicalities. Intravenous injection of the hyperpolarized 13C-pyruvate results in appearance of 13C-lactate, 13C-alanine and 13C-bicarbonate resonance peaks depending on the tissue, disease and the metabolic state probed. In cancer, the lactate level is increased due to increased glycolysis. The use of DNP enhanced 13C-pyruvate has in preclinical studies shown to be a sensitive method for detecting cancer and for assessment of early treatment response in a variety of cancers. Recently, a first-in-man 31-patient study was conducted with the primary objective to assess the safety of hyperpolarized 13C-pyruvate in healthy subjects and prostate cancer patients. The study showed an elevated 13C-lactate/13C-pyruvate ratio in regions of biopsy

  6. Hyperpolarized 13C MR Markers of Renal Tumor Aggressiveness

    DTIC Science & Technology

    2013-10-01

    reliably distinguish renal cancer aggressiveness for optimal triage of therapies . Hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI...reliably distinguish renal cancer aggressiveness for optimal triage of therapies . Hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) is... cancer and normal tissues were obtained from nephrectomy specimens and sliced using Krumdieck slicer. With a precision gauge micrometer, the slice

  7. MRI with hyperpolarised [1-13C]pyruvate detects advanced pancreatic preneoplasia prior to invasive disease in a mouse model

    PubMed Central

    Serrao, Eva M; Kettunen, Mikko I; Rodrigues, Tiago B; Dzien, Piotr; Wright, Alan J; Gopinathan, Aarthi; Gallagher, Ferdia A; Lewis, David Y; Frese, Kristopher K; Almeida, Jaime; Howat, William J; Tuveson, David A; Brindle, Kevin M

    2016-01-01

    Objectives Pancreatic cancer (PCa) is treatable by surgery when detected at an early stage. Non-invasive imaging methods able to detect both established tumours and their precursor lesions are needed to select patients for surgery. We investigated here whether pancreatic preneoplasia could be detected prior to the development of invasive cancers in genetically engineered mouse models of PCa using metabolic imaging. Design The concentrations of alanine and lactate and the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were measured in extracts prepared from the pancreas of animals at different stages of disease progression; from pancreatitis, through tissue with predominantly low-grade and then high-grade pancreatic intraepithelial neoplasia and then tumour. 13C magnetic resonance spectroscopic imaging (13C-MRSI) was used to measure non-invasively changes in 13C labelling of alanine and lactate with disease progression, following injection of hyperpolarised [1-13C]pyruvate. Results Progressive decreases in the alanine/lactate concentration ratio and ALT/LDH activity ratio with disease progression were accompanied by a corresponding decrease in the [1-13C]alanine/[1-13C]lactate signal ratio observed in 13C-MRSI images of the pancreas. Conclusions Metabolic imaging with hyperpolarised [1-13C]pyruvate enables detection and monitoring of the progression of PCa precursor lesions. Translation of this MRI technique to the clinic has the potential to improve the management of patients at high risk of developing PCa. PMID:26347531

  8. In search of the mechanisms behind soil carbon metabolism of a Douglas fir forest in complex terrain using naturally abundant 13C

    NASA Astrophysics Data System (ADS)

    Kayler, Z. E.; Sulzman, E. W.; Barnard, H. R.; Kennedy, A.; Phillips, C.; Mix, A.; Bond, B. J.

    2008-12-01

    Soil is well known for being highly variable, spatially and temporally, in moisture, texture, nutrients, carbon content and organisms. The magnitude of variation in soil characteristics represented in a study is, in part, determined by the choice in site location. Choosing sites that are topographically flat reduces variability due to environmental gradients, variability that is amplified in sites of complex terrain. We measured soil respiration, an integrative measure of ecosystem biological and physical processes, and its isotopic signature (δ13CR-s) to accomplish two goals: 1. Explore how gradients in temperature and moisture within a steeply sloped watershed affect the flux and isotopic signature of soil CO2 2. Deconvolve the isotopic signature of soil respiration into autotrophic and heterotrophic sources using a multi-source mixing model constrained by samples of soil organic matter and water soluble extracts of leaf foliage. Our site is located in a steep catchment within the central Cascades of Oregon (HJ Andrews LTER) where we made respiration measurements in plots established along side a sensor transect that continuously measures soil moisture and temperature; air relative humidity and temperature; and tree transpiration. There was a distinct difference in soil metabolism between the south and north aspects in the watershed. Temperature-corrected basal respiration of the south facing slope was 1 μmol m-2s-1 greater than the north facing slope. There was also a difference in isotopic signature between the two slopes that could be as great as 2 per mil depending on the period within the growing season. The strength of the correlation between environmental variables and soil carbon flux was non-uniform across the catchment. There was, however, a strong positive correlation between soil flux with recent transpiration rates (0 to 3 days prior) as well as with transpiration rates that occurred up to 9 days previously. This pattern was especially prevalent

  9. Single shot three‐dimensional pulse sequence for hyperpolarized 13C MRI

    PubMed Central

    Wang, Jiazheng; Wright, Alan J.; Hu, De‐en; Hesketh, Richard

    2016-01-01

    Purpose Metabolic imaging with hyperpolarized 13C‐labeled cell substrates is a promising technique for imaging tissue metabolism in vivo. However, the transient nature of the hyperpolarization, and its depletion following excitation, limits the imaging time and the number of excitation pulses that can be used. We describe here a single‐shot three‐dimensional (3D) imaging sequence and demonstrate its capability to generate 13C MR images in tumor‐bearing mice injected with hyperpolarized [1‐13C]pyruvate. Methods The pulse sequence acquires a stack‐of‐spirals at two spin echoes after a single excitation pulse and encodes the kz‐dimension in an interleaved manner to enhance robustness to B0 inhomogeneity. Spectral‐spatial pulses are used to acquire dynamic 3D images from selected hyperpolarized 13C‐labeled metabolites. Results A nominal spatial/temporal resolution of 1.25 × 1.25 × 2.5 mm3 × 2 s was achieved in tumor images of hyperpolarized [1‐13C]pyruvate and [1‐13C]lactate acquired in vivo. Higher resolution in the z‐direction, with a different k‐space trajectory, was demonstrated in measurements on a thermally polarized [1‐13C]lactate phantom. Conclusion The pulse sequence is capable of imaging hyperpolarized 13C‐labeled substrates at relatively high spatial and temporal resolutions and is robust to moderate system imperfections. Magn Reson Med 77:740–752, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PMID:26916384

  10. Hyperpolarized (13)C-lactate to (13)C-bicarbonate ratio as a biomarker for monitoring the acute response of anti-vascular endothelial growth factor (anti-VEGF) treatment.

    PubMed

    Park, Jae Mo; Spielman, Daniel M; Josan, Sonal; Jang, Taichang; Merchant, Milton; Hurd, Ralph E; Mayer, Dirk; Recht, Lawrence D

    2016-05-01

    Hyperpolarized [1-(13)C]pyruvate MRS provides a unique imaging opportunity to study the reaction kinetics and enzyme activities of in vivo metabolism because of its favorable imaging characteristics and critical position in the cellular metabolic pathway, where it can either be reduced to lactate (reflecting glycolysis) or converted to acetyl-coenzyme A and bicarbonate (reflecting oxidative phosphorylation). Cancer tissue metabolism is altered in such a way as to result in a relative preponderance of glycolysis relative to oxidative phosphorylation (i.e. Warburg effect). Although there is a strong theoretical basis for presuming that readjustment of the metabolic balance towards normal could alter tumor growth, a robust noninvasive in vivo tool with which to measure the balance between these two metabolic processes has yet to be developed. Until recently, hyperpolarized (13)C-pyruvate imaging studies had focused solely on [1-(13)C]lactate production because of its strong signal. However, without a concomitant measure of pyruvate entry into the mitochondria, the lactate signal provides no information on the balance between the glycolytic and oxidative metabolic pathways. Consistent measurement of (13)C-bicarbonate in cancer tissue, which does provide such information, has proven difficult, however. In this study, we report the reliable measurement of (13)C-bicarbonate production in both the healthy brain and a highly glycolytic experimental glioblastoma model using an optimized (13)C MRS imaging protocol. With the capacity to obtain signal in all tumors, we also confirm for the first time that the ratio of (13)C-lactate to (13)C-bicarbonate provides a more robust metric relative to (13)C-lactate for the assessment of the metabolic effects of anti-angiogenic therapy. Our data suggest a potential application of this ratio as an early biomarker to assess therapeutic effectiveness. Furthermore, although further study is needed, the results suggest that anti

  11. SIMS ion microscopy imaging of boronophenylalanine (BPA) and 13C15N-labeled phenylalanine in human glioblastoma cells: Relevance of subcellular scale observations to BPA-mediated boron neutron capture therapy of cancer

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash; Lorey, Daniel R., II

    2007-02-01

    p-Boronophenylalanine (BPA) is a clinically approved boron neutron capture therapy (BNCT) agent currently being used in clinical trials of glioblastoma multiforme, melanoma and liver metastases. Secondary ion mass spectrometry (SIMS) observations from the Cornell SIMS Laboratory provided support for using a 6 h infusion of BPA, instead of a 2 h infusion, for achieving higher levels of boron in brain tumor cells. These observations were clinically implemented in Phase II experimental trials of glioblastoma multiforme in Sweden. However, the mechanisms for higher BPA accumulation with longer infusions have remained unknown. In this work, by using 13C15N-labeled phenylalanine and T98G human glioblastoma cells, comparisons between the 10B-delivery of BPA and the accumulation of labeled phenylalanine after 2 and 6 h treatments were made with a Cameca IMS-3f SIMS ion microscope at 500 nm spatial resolution in fast frozen, freeze-fractured, freeze-dried cells. Due to the presence of the Na-K-ATPase in the plasma membrane of most mammalian cells, the cells maintain an approximately 10/1 ratio of K/Na in the intracellular milieu. Therefore, the quantitative imaging of these highly diffusible species in the identical cell in which the boron or labeled amino acid was imaged provides a rule-of-thumb criterion for validation of SIMS observations and the reliability of the cryogenic sampling. The labeled phenylalanine was detected at mass 28, as the 28(13C15N)- molecular ion. Correlative analysis with optical and confocal laser scanning microscopy revealed that fractured freeze-dried glioblastoma cells contained well-preserved ultrastructural details with three discernible subcellular regions: a nucleus or multiple nuclei, a mitochondria-rich perinuclear cytoplasmic region and the remaining cytoplasm. SIMS analysis revealed that the overall cellular signals of both 10B from BPA and 28CN- from labeled phenylalanine increased approximately 1.6-fold between the 2 and 6 h exposures

  12. Real-time Detection of Hepatic Gluconeogenic and Glycogenolytic States Using Hyperpolarized [2-13C]Dihydroxyacetone*

    PubMed Central

    Moreno, Karlos X.; Satapati, Santhosh; DeBerardinis, Ralph J.; Burgess, Shawn C.; Malloy, Craig R.; Merritt, Matthew E.

    2014-01-01

    Glycogenolysis and gluconeogenesis are sensitive to nutritional state, and the net direction of flux is controlled by multiple enzymatic steps. This delicate balance in the liver is disrupted by a variety of pathological states including cancer and diabetes mellitus. Hyperpolarized carbon-13 magnetic resonance is a new metabolic imaging technique that can probe intermediary metabolism nondestructively. There are currently no methods to rapidly distinguish livers in a gluconeogenic from glycogenolytic state. Here we use the gluconeogenic precursor dihydroxyacetone (DHA) to deliver hyperpolarized carbon-13 to the perfused mouse liver. DHA enters gluconeogenesis at the level of the trioses. Perfusion conditions were designed to establish either a gluconeogenic or a glycogenolytic state. Unexpectedly, we found that [2-13C]DHA was metabolized within a few seconds to the common intermediates and end products of both glycolysis and gluconeogenesis under both conditions, including [2,5-13C]glucose, [2-13C]glycerol 3-phosphate, [2-13C]phosphoenolpyruvate (PEP), [2-13C]pyruvate, [2-13C]alanine, and [2-13C]lactate. [2-13C]Phosphoenolpyruvate, a key branch point in gluconeogenesis and glycolysis, was monitored in functioning tissue for the first time. Observation of [2-13C]PEP was not anticipated as the free energy difference between PEP and pyruvate is large. Pyruvate kinase is the only regulatory step of the common glycolytic-gluconeogenic pathway that appears to exert significant control over the kinetics of any metabolites of DHA. A ratio of glycolytic to gluconeogenic products distinguished the gluconeogenic from glycogenolytic state in these functioning livers. PMID:25352600

  13. Real-time detection of hepatic gluconeogenic and glycogenolytic states using hyperpolarized [2-13C]dihydroxyacetone.

    PubMed

    Moreno, Karlos X; Satapati, Santhosh; DeBerardinis, Ralph J; Burgess, Shawn C; Malloy, Craig R; Merritt, Matthew E

    2014-12-26

    Glycogenolysis and gluconeogenesis are sensitive to nutritional state, and the net direction of flux is controlled by multiple enzymatic steps. This delicate balance in the liver is disrupted by a variety of pathological states including cancer and diabetes mellitus. Hyperpolarized carbon-13 magnetic resonance is a new metabolic imaging technique that can probe intermediary metabolism nondestructively. There are currently no methods to rapidly distinguish livers in a gluconeogenic from glycogenolytic state. Here we use the gluconeogenic precursor dihydroxyacetone (DHA) to deliver hyperpolarized carbon-13 to the perfused mouse liver. DHA enters gluconeogenesis at the level of the trioses. Perfusion conditions were designed to establish either a gluconeogenic or a glycogenolytic state. Unexpectedly, we found that [2-(13)C]DHA was metabolized within a few seconds to the common intermediates and end products of both glycolysis and gluconeogenesis under both conditions, including [2,5-(13)C]glucose, [2-(13)C]glycerol 3-phosphate, [2-(13)C]phosphoenolpyruvate (PEP), [2-(13)C]pyruvate, [2-(13)C]alanine, and [2-(13)C]lactate. [2-(13)C]Phosphoenolpyruvate, a key branch point in gluconeogenesis and glycolysis, was monitored in functioning tissue for the first time. Observation of [2-(13)C]PEP was not anticipated as the free energy difference between PEP and pyruvate is large. Pyruvate kinase is the only regulatory step of the common glycolytic-gluconeogenic pathway that appears to exert significant control over the kinetics of any metabolites of DHA. A ratio of glycolytic to gluconeogenic products distinguished the gluconeogenic from glycogenolytic state in these functioning livers.

  14. Synthesis of exemestane labelled with (13)C.

    PubMed

    Fontana, Erminia; Pignatti, Alberto; Giribone, Danilo; Di Salle, Enrico

    2008-08-01

    The synthesis of exemestane Aromasin, an irreversible steroidal aromatase inhibitor, specifically labelled with (13)C is reported. The preparation of [(13)C(3)]exemestane was achieved according to an eight-step procedure starting from the commercially available testosterone.

  15. Detection of inflammatory cell function using 13C magnetic resonance spectroscopy of hyperpolarized [6-13C]-arginine

    PubMed Central

    Najac, Chloé; Chaumeil, Myriam M.; Kohanbash, Gary; Guglielmetti, Caroline; Gordon, Jeremy W.; Okada, Hideho; Ronen, Sabrina M.

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) are highly prevalent inflammatory cells that play a key role in tumor development and are considered therapeutic targets. MDSCs promote tumor growth by blocking T-cell-mediated anti-tumoral immune response through depletion of arginine that is essential for T-cell proliferation. To deplete arginine, MDSCs express high levels of arginase, which catalyzes the breakdown of arginine into urea and ornithine. Here, we developed a new hyperpolarized 13C probe, [6-13C]-arginine, to image arginase activity. We show that [6-13C]-arginine can be hyperpolarized, and hyperpolarized [13C]-urea production from [6-13C]-arginine is linearly correlated with arginase concentration in vitro. Furthermore we show that we can detect a statistically significant increase in hyperpolarized [13C]-urea production in MDSCs when compared to control bone marrow cells. This increase was associated with an increase in intracellular arginase concentration detected using a spectrophotometric assay. Hyperpolarized [6-13C]-arginine could therefore serve to image tumoral MDSC function and more broadly M2-like macrophages. PMID:27507680

  16. δ(13)C values of some succulent plants from Madagascar.

    PubMed

    Winter, Klaus

    1979-01-01

    δ(13)C values were determined in 20 succulents from Madagascar. The values were indicative of Crassulacean Acid Metabolism in 10 species of the Didiereaceae, 4 species of the Euphorbiaceae, 2 species of the Crassulaceae and 1 species of the Cucurbitaceae. The Didiereaceae and Euphorbiaceae studied are major components of a high biomass xerophytic flora in the semi-arid southwest and south of Madagascar. Three species of the Euphorbiaceae with succulent stems and non-succulent leaves, which were cultivated outdoors in the Tananarive Botanic Garden, showed C3 like δ(13)C values for both leaves and stems. δ(13)C values of leaf and stem material from a similar species, collected in the south of Madagascar, indicated Crassulacean Acid Metabolism.

  17. Whole-core analysis by sup 13 C NMR

    SciTech Connect

    Vinegar, H.J.; Tutunjian, P.N. ); Edelstein, W.A.; Roemer, P.B. )

    1991-06-01

    This paper reports on a whole-core nuclear magnetic resonance (NMR) system that was used to obtain natural abundance {sup 13}C spectra. The system enables rapid, nondestructive measurements of bulk volume of movable oil, aliphatic/aromatic ratio, oil viscosity, and organic vs. carbonate carbon. {sup 13}C NMR can be used in cores where the {sup 1}H NMR spectrum is too broad to resolve oil and water resonances separately. A 5 1/4-in. {sup 13}C/{sup 1}H NMR coil was installed on a General Electric (GE) CSI-2T NMR imager/spectrometer. With a 4-in.-OD whole core, good {sup 13}C signal/noise ratio (SNR) is obtained within minutes, while {sup 1}H spectra are obtained in seconds. NMR measurements have been made of the {sup 13}C and {sup 1}H density of crude oils with a wide range of API gravities. For light- and medium-gravity oils, the {sup 13}C and {sup 1}H signal per unit volume is constant within about 3.5%. For heavy crudes, the {sup 13}C and {sup 1}H density measured by NMR is reduced by the shortening of spin-spin relaxation time. {sup 13}C and {sup 1}H NMR spin-lattice relaxation times were measured on a suite of Cannon viscosity standards, crude oils (4 to 60{degrees} API), and alkanes (C{sub 5} through C{sub 16}) with viscosities at 77{degrees}F ranging from 0.5 cp to 2.5 {times} 10{sup 7} cp. The {sup 13}C and {sup 1}H relaxation times show a similar correlation with viscosity from which oil viscosity can be estimated accurately for viscosities up to 100 cp. The {sup 13}C surface relaxation rate for oils on water-wet rocks is very low. Nonproton decoupled {sup 13}C NMR is shown to be insensitive to kerogen; thus, {sup 13}C NMR measures only the movable hydrocarbon content of the cores. In carbonates, the {sup 13}C spectrum also contains a carbonate powder pattern useful in quantifying inorganic carbon and distinguishing organic from carbonate carbon.

  18. Optical metabolic imaging of live tissue cultures

    NASA Astrophysics Data System (ADS)

    Walsh, Alex J.; Cook, Rebecca S.; Arteaga, Carlos L.; Skala, Melissa C.

    2013-02-01

    The fluorescence properties, both intensity and fluorescence lifetime, of NADH and FAD, two coenzymes of metabolism, are sensitive, high resolution measures of cellular metabolism. However, often in vivo measurements of tissue are not feasible. In this study, we investigate the stability over time of two-photon auto-fluorescence imaging of NADH and FAD in live-cultured tissues. Our results demonstrate that cultured tissues remain viable for at least several days post excision. Furthermore, the optical redox ratio, NADH fluorescence lifetime, and FAD fluorescence lifetime do not significantly change in the cultured tissues over time. With these findings, we demonstrate the potential of sustained tissue culture techniques for optical metabolic imaging.

  19. (13)C NMR Metabolomics: INADEQUATE Network Analysis.

    PubMed

    Clendinen, Chaevien S; Pasquel, Christian; Ajredini, Ramadan; Edison, Arthur S

    2015-06-02

    The many advantages of (13)C NMR are often overshadowed by its intrinsically low sensitivity. Given that carbon makes up the backbone of most biologically relevant molecules, (13)C NMR offers a straightforward measurement of these compounds. Two-dimensional (13)C-(13)C correlation experiments like INADEQUATE (incredible natural abundance double quantum transfer experiment) are ideal for the structural elucidation of natural products and have great but untapped potential for metabolomics analysis. We demonstrate a new and semiautomated approach called INETA (INADEQUATE network analysis) for the untargeted analysis of INADEQUATE data sets using an in silico INADEQUATE database. We demonstrate this approach using isotopically labeled Caenorhabditis elegans mixtures.

  20. New guidelines for δ13C measurements

    USGS Publications Warehouse

    Coplen, Tyler B.; Brand, Willi A.; Gehre, Matthias; Groning, Manfred; Meijer, Harro A. J.; Toman, Blaza; Verkouteren, R. Michael

    2006-01-01

    Consistency of δ13C measurements can be improved 39−47% by anchoring the δ13C scale with two isotopic reference materials differing substantially in 13C/12C. It is recommended thatδ13C values of both organic and inorganic materials be measured and expressed relative to VPDB (Vienna Peedee belemnite) on a scale normalized by assigning consensus values of −46.6‰ to L-SVEC lithium carbonate and +1.95‰ to NBS 19 calcium carbonate. Uncertainties of other reference material values on this scale are improved by factors up to two or more, and the values of some have been notably shifted:  the δ13C of NBS 22 oil is −30.03%.

  1. Enhancing the [13C]bicarbonate signal in cardiac hyperpolarized [1-13C]pyruvate MRS studies by infusion of glucose, insulin and potassium.

    PubMed

    Lauritzen, Mette Hauge; Laustsen, Christoffer; Butt, Sadia Asghar; Magnusson, Peter; Søgaard, Lise Vejby; Ardenkjær-Larsen, Jan Henrik; Åkeson, Per

    2013-11-01

    A change in myocardial metabolism is a known effect of several diseases. MRS with hyperpolarized (13)C-labelled pyruvate is a technique capable of detecting changes in myocardial pyruvate metabolism, and has proven to be useful for the evaluation of myocardial ischaemia in vivo. However, during fasting, the myocardial glucose oxidation is low and the fatty acid oxidation (β-oxidation) is high, which complicates the interpretation of pyruvate metabolism with the technique. The aim of this study was to investigate whether the infusion of glucose, insulin and potassium (GIK) could increase the myocardial glucose oxidation in the citric acid cycle, reflected as an increase in the [(13)C]bicarbonate signal in cardiac hyperpolarized [1-(13)C]pyruvate MRS measurements in fasted rats. Two groups of rats were infused with two different doses of GIK and investigated by MRS after injection of hyperpolarized [1-(13)C]pyruvate. No [(13)C]bicarbonate signal could be detected in the fasted state. However, a significant increase in the [(13)C]bicarbonate signal was observed by the infusion of a high dose of GIK. This study demonstrates that a high [(13)C]bicarbonate signal can be achieved by GIK infusion in fasted rats. The increased [(13)C]bicarbonate signal indicates an increased flux of pyruvate through the pyruvate dehydrogenase enzyme complex and an increase in myocardial glucose oxidation through the citric acid cycle.

  2. Novel Approaches to Imaging Tumor Metabolism.

    PubMed

    Tee, Sui-Seng; Keshari, Kayvan R

    2015-01-01

    The field of metabolism research has made a dramatic resurgence in recent years, fueled by a newfound appreciation of the interactions between metabolites and phenotype. Metabolic substrates and their products can be biomarkers of a wide range of pathologies, including cancer, but our understanding of their in vivo interactions and pathways has been hindered by the robustness of noninvasive imaging approaches. The past 3 decades have been flushed with the development of new techniques for the study of metabolism in vivo. These methods include nuclear-based, predominantly positron emission tomography and magnetic resonance imaging, many of which have been translated to the clinic. The purpose of this review was to introduce both long-standing imaging strategies as well as novel approaches to the study of perturbed metabolic pathways in the setting of carcinogenesis. This will involve descriptions of nuclear probes labeled with C and F as well C for study using hyperpolarized magnetic resonance imaging. Highlighting both advantages and disadvantages of each approach, the aim of this summary was to provide the reader with a framework for interrogation of metabolic aberrations in their system of interest.

  3. 13C NMR of tunnelling methyl groups

    NASA Astrophysics Data System (ADS)

    Detken, A.

    The dipolar interactions between the protons and the central 13C nucleus of a 13CH3 group are used to study rotational tunnelling and incoherent dynamics of such groups in molecular solids. Single-crystal 13C NMR spectra are derived for arbitrary values of the tunnel frequency upsilon t. Similarities to ESR and 2H NMR are pointed out. The method is applied to three different materials. In the hydroquinone/acetonitrile clathrate, the unique features in the 13C NMR spectra which arise from tunnelling with a tunnel frequency that is much larger than the dipolar coupling between the methyl protons and the 13C nucleus are demonstrated, and the effects of incoherent dynamics are studied. The broadening of the 13C resonances is related to the width of the quasi-elastic line in neutron scattering. Selective magnetization transfer experiments for studying slow incoherent dynamics are proposed. For the strongly hindered methyl groups of L-alanine, an upper limit for upsilon is derived from the 13C NMR spectrum. In aspirinTM (acetylsalicylic acid), incoherent reorientations dominate the spectra down to the lowest temperatures studied; their rate apparently increases with decreasing temperature below 25K.

  4. Optical metabolic imaging for monitoring tracheal health

    NASA Astrophysics Data System (ADS)

    Sharick, Joe T.; Gil, Daniel A.; Choma, Michael A.; Skala, Melissa C.

    2016-04-01

    The health of the tracheal mucosa and submucosa is a vital yet poorly understood component of critical care medicine, and a minimally-invasive method is needed to monitor tracheal health in patients. Of particular interest are the ciliated cells of the tracheal epithelium that move mucus away from the lungs and prevent respiratory infection. Optical metabolic imaging (OMI) allows cellular-level measurement of metabolism, and is a compelling method for assessing tracheal health because ciliary motor proteins require ATP to function. In this pilot study, we apply multiphoton imaging of the fluorescence intensities and lifetimes of metabolic co-enzymes NAD(P)H and FAD to the mucosa and submucosa of ex vivo mouse trachea. We demonstrate the feasibility and potential diagnostic utility of these measurements for assessing tracheal health and pathophysiology at the single-cell level.

  5. Towards imaging metabolic pathways in tissues.

    PubMed

    Dekker, Tim J A; Jones, Emrys A; Corver, Willem E; van Zeijl, René J M; Deelder, André M; Tollenaar, Rob A E M; Mesker, Wilma E; Morreau, Hans; McDonnell, Liam A

    2015-03-01

    Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging using 9-aminoacridine as the matrix leads to the detection of low mass metabolites and lipids directly from cancer tissues. These included lactate and pyruvate for studying the Warburg effect, as well as succinate and fumarate, metabolites whose accumulation is associated with specific syndromes. By using the pathway information present in the human metabolome database, it was possible to identify regions within tumor tissue samples with distinct metabolic signatures that were consistent with known tumor biology. We present a data analysis workflow for assessing metabolic pathways in their histopathological context.

  6. Galactose oxidation using (13)C in healthy and galactosemic children.

    PubMed

    Resende-Campanholi, D R; Porta, G; Ferrioli, E; Pfrimer, K; Ciampo, L A Del; Junior, J S Camelo

    2015-03-01

    Galactosemia is an inborn error of galactose metabolism that occurs mainly as the outcome of galactose-1-phosphate uridyltransferase (GALT) deficiency. The ability to assess galactose oxidation following administration of a galactose-labeled isotope (1-(13)C-galactose) allows the determination of galactose metabolism in a practical manner. We aimed to assess the level of galactose oxidation in both healthy and galactosemic Brazilian children. Twenty-one healthy children and seven children with galactosemia ranging from 1 to 7 years of age were studied. A breath test was used to quantitate (13)CO2 enrichment in exhaled air before and at 30, 60, and 120 min after the oral administration of 7 mg/kg of an aqueous solution of 1-(13)C-galactose to all children. The molar ratios of (13)CO2 and (12)CO2 were quantified by the mass/charge ratio (m/z) of stable isotopes in each air sample by gas-isotope-ratio mass spectrometry. In sick children, the cumulative percentage of (13)C from labeled galactose (CUMPCD) in the exhaled air ranged from 0.03% at 30 min to 1.67% at 120 min. In contrast, healthy subjects showed a much broader range in CUMPCD, with values from 0.4% at 30 min to 5.58% at 120 min. The study found a significant difference in galactose oxidation between children with and without galactosemia, demonstrating that the breath test is useful in discriminating children with GALT deficiencies.

  7. Optical hyperpolarization of 13C nuclear spins in nanodiamond ensembles

    NASA Astrophysics Data System (ADS)

    Chen, Q.; Schwarz, I.; Jelezko, F.; Retzker, A.; Plenio, M. B.

    2015-11-01

    Dynamical nuclear polarization holds the key for orders of magnitude enhancements of nuclear magnetic resonance signals which, in turn, would enable a wide range of novel applications in biomedical sciences. However, current implementations of DNP require cryogenic temperatures and long times for achieving high polarization. Here we propose and analyze in detail protocols that can achieve rapid hyperpolarization of 13C nuclear spins in randomly oriented ensembles of nanodiamonds at room temperature. Our protocols exploit a combination of optical polarization of electron spins in nitrogen-vacancy centers and the transfer of this polarization to 13C nuclei by means of microwave control to overcome the severe challenges that are posed by the random orientation of the nanodiamonds and their nitrogen-vacancy centers. Specifically, these random orientations result in exceedingly large energy variations of the electron spin levels that render the polarization and coherent control of the nitrogen-vacancy center electron spins as well as the control of their coherent interaction with the surrounding 13C nuclear spins highly inefficient. We address these challenges by a combination of an off-resonant microwave double resonance scheme in conjunction with a realization of the integrated solid effect which, together with adiabatic rotations of external magnetic fields or rotations of nanodiamonds, leads to a protocol that achieves high levels of hyperpolarization of the entire nuclear-spin bath in a randomly oriented ensemble of nanodiamonds even at room temperature. This hyperpolarization together with the long nuclear-spin polarization lifetimes in nanodiamonds and the relatively high density of 13C nuclei has the potential to result in a major signal enhancement in 13C nuclear magnetic resonance imaging and suggests functionalized and hyperpolarized nanodiamonds as a unique probe for molecular imaging both in vitro and in vivo.

  8. Application of 13C-labeling and 13C-13C COSY NMR experiments in the structure determination of a microbial natural product.

    PubMed

    Kwon, Yun; Park, Sunghyouk; Shin, Jongheon; Oh, Dong-Chan

    2014-08-01

    The elucidation of the structures of complex natural products bearing many quaternary carbons remains challenging, even in this advanced spectroscopic era. (13)C-(13)C COSY NMR spectroscopy shows direct couplings between (13)C and (13)C, which comprise the backbone of a natural product. Thus, this type of experiment is particularly useful for natural products bearing consecutive quaternary carbons. However, the low sensitivity of (13)C-based NMR experiments, due to the low natural abundance of the (13)C nucleus, is problematic when applying these techniques. Our efforts in the (13)C labeling of a microbial natural product, cyclopiazonic acid (1), by feeding (13)C-labeled glucose to the fungal culture, enabled us to acquire (13)C-(13)C COSY NMR spectra on a milligram scale that clearly show the carbon backbone of the compound. This is the first application of (13)C-(13)C COSY NMR experiments for a natural product. The results suggest that (13)C-(13)C COSY NMR spectroscopy can be routinely used for the structure determination of microbial natural products by (13)C-enrichment of a compound with (13)C-glucose.

  9. A 13C NMR spectrometric method for the determination of intramolecular δ13C values in fructose from plant sucrose samples.

    PubMed

    Gilbert, Alexis; Silvestre, Virginie; Robins, Richard J; Tcherkez, Guillaume; Remaud, Gérald S

    2011-07-01

    Recent developments in (13) C NMR spectrometry have allowed the determination of intramolecular (13) C/(12) C ratios with high precision. However, the analysis of carbohydrates requires their derivatization to constrain the anomeric carbon. Fructose has proved to be particularly problematic because of a byproduct occurring during derivatization and the complexity of the NMR spectrum of the derivative. Here, we describe a method to determine the intramolecular (13) C/(12) C ratios in fructose by (13) C NMR analysis of the acetyl-isopropylidene derivative. We have applied this method to measure the intramolecular (13) C/(12) C distribution in the fructosyl moiety of sucrose and have compared this with that in the glucosyl moiety. Three prominent features stand out. First, in sucrose from both C(3) and C(4) plants, the C-1 and C-2 positions of the glucosyl and fructosyl moieties are markedly different. Second, these positions in C(3) and C(4) plants show a similar profile. Third, the glucosyl and fructosyl moieties of sucrose from Crassulacean acid metabolism (CAM) metabolism have a different profile. These contrasting values can be interpreted as a result of the isotopic selectivity of enzymes that break or make covalent bonds in glucose metabolism, whereas the distinctive (13) C pattern in CAM sucrose probably indicates a substantial contribution of gluconeogenesis to glucose synthesis.

  10. Synthesis Of [2h, 13c] And [2h3, 13c]Methyl Aryl Sulfides

    DOEpatents

    Martinez, Rodolfo A.; Alvarez, Marc A.; Silks, III, Louis A.; Unkefer, Clifford J.

    2004-03-30

    The present invention is directed to labeled compounds, [.sup.2 H.sub.1, .sup.13 C], [.sup.2 H.sub.2, .sup.13 C] and [.sup.2 H.sub.3, .sup.13 C]methyl aryl sulfides wherein the .sup.13 C methyl group attached to the sulfur of the sulfide includes exactly one, two or three deuterium atoms and the aryl group is selected from the group consisting of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure ##STR1## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are each independently, hydrogen, a C.sub.1 -C.sub.4 lower alkyl, a halogen, an amino group from the group consisting of NH.sub.2, NHR and NRR' where R and R' are each a C.sub.1 -C.sub.4 lower alkyl, a phenyl, or an alkoxy group. The present invention is also directed to processes of preparing [.sup.2 H.sub.1, .sup.13 C], [.sup.2 H.sub.2,.sup.13 C] and [.sup.2 H.sub.3, .sup.13 C]methyl aryl sulfides wherein the .sup.13 C methyl group attached to the sulfur of the sulfide includes exactly one, two or three deuterium atoms. The present invention is also directed to the labeled compounds of [.sup.2 H.sub.1, .sup.13 C]methyl iodide and [.sup.2 H.sub.2, .sup.13 C]methyl iodide.

  11. Mapping cerebral glutamate 13C turnover and oxygen consumption by in vivo NMR.

    PubMed

    Hyder, Fahmeed; Brown, Peter; Nixon, Terennce W; Behar, Kevin L

    2003-01-01

    Regional rates of 13C incorporation from glucose to glutamate were detected in anesthetized rat brain in vivo at 7T with high temporal and spatial resolution using NMR method ICED PEPSI (in vivo carbon edited detection with proton echo planar spectroscopic imaging). Time courses of regional glutamate 13C turnover were fitted by a metabolic model to obtain regional tri-carboxylic acid (TCA) cycle flux and cerebral metabolic rate of oxygen consumption (CMRO2) in each voxel (8 microL) of rat cortex. CMRO2 maps obtained for rats under either alpha-chloralose or morphine anesthesia revealed average cortical values of 1.5 +/- 0.2 (n = 3) and 3.2 +/- 0.3 (n = 4) mumol/g/min, respectively. These values of CMRO2 are in good agreement with previous cortical measurements with coarser spatial resolution. The heterogeneity within each map, which depicted predominantly gray and white matter differences, was significantly greater under morphine (higher cortical activity) than under-alpha-chloralose (lower cortical activity) anesthesia. The regional variations in the basal awake state, which are expected to be even greater, should be considered to avoid partial-volume artifacts in functional activation studies of awake subjects.

  12. 13C MRS studies of neuroenergetics and neurotransmitter cycling in humans.

    PubMed

    Rothman, Douglas L; De Feyter, Henk M; de Graaf, Robin A; Mason, Graeme F; Behar, Kevin L

    2011-10-01

    In the last 25 years, (13)C MRS has been established as the only noninvasive method for the measurement of glutamate neurotransmission and cell-specific neuroenergetics. Although technically and experimentally challenging, (13)C MRS has already provided important new information on the relationship between neuroenergetics and neuronal function, the energy cost of brain function, the high neuronal activity in the resting brain state and how neuroenergetics and neurotransmitter cycling are altered in neurological and psychiatric disease. In this article, the current state of (13)C MRS as it is applied to the study of neuroenergetics and neurotransmitter cycling in humans is reviewed. The focus is predominantly on recent findings in humans regarding metabolic pathways, applications to clinical research and the technical status of the method. Results from in vivo (13)C MRS studies in animals are discussed from the standpoint of the validation of MRS measurements of neuroenergetics and neurotransmitter cycling, and where they have helped to identify key questions to address in human research. Controversies concerning the relationship between neuroenergetics and neurotransmitter cycling and factors having an impact on the accurate determination of fluxes through mathematical modeling are addressed. We further touch upon different (13)C-labeled substrates used to study brain metabolism, before reviewing a number of human brain diseases investigated using (13)C MRS. Future technological developments are discussed that will help to overcome the limitations of (13)C MRS, with special attention given to recent developments in hyperpolarized (13)C MRS.

  13. Natural (13) C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern.

    PubMed

    Lamade, Emmanuelle; Tcherkez, Guillaume; Darlan, Nuzul Hijri; Rodrigues, Rosario Lobato; Fresneau, Chantal; Mauve, Caroline; Lamothe-Sibold, Marlène; Sketriené, Diana; Ghashghaie, Jaleh

    2016-01-01

    Oil palm has now become one of the most important crops, palm oil representing nearly 25% of global plant oil consumption. Many studies have thus addressed oil palm ecophysiology and photosynthesis-based models of carbon allocation have been used. However, there is a lack of experimental data on carbon fixation and redistribution within palm trees, and important C-sinks have not been fully characterized yet. Here, we carried out extensive measurement of natural (13) C-abundance (δ(13) C) in oil palm tissues, including fruits at different maturation stages. We find a (13) C-enrichment in heterotrophic organs compared to mature leaves, with roots being the most (13) C-enriched. The δ(13) C in fruits decreased during maturation, reflecting the accumulation in (13) C-depleted lipids. We further used observed δ(13) C values to compute plausible carbon fluxes using a steady-state model of (13) C-distribution including metabolic isotope effects ((12) v/(13) v). The results suggest that fruits represent a major respiratory loss (≈39% of total tree respiration) and that sink organs such as fruits are fed by sucrose from leaves. That is, glucose appears to be a quantitatively important compound in palm tissues, but computations indicate that it is involved in dynamic starch metabolism rather that C-exchange between organs.

  14. Accurate determinations of one-bond 13C-13C couplings in 13C-labeled carbohydrates

    NASA Astrophysics Data System (ADS)

    Azurmendi, Hugo F.; Freedberg, Darón I.

    2013-03-01

    Carbon plays a central role in the molecular architecture of carbohydrates, yet the availability of accurate methods for 1DCC determination has not been sufficiently explored, despite the importance that such data could play in structural studies of oligo- and polysaccharides. Existing methods require fitting intensity ratios of cross- to diagonal-peaks as a function of the constant-time (CT) in CT-COSY experiments, while other methods utilize measurement of peak separation. The former strategies suffer from complications due to peak overlap, primarily in regions close to the diagonal, while the latter strategies are negatively impacted by the common occurrence of strong coupling in sugars, which requires a reliable assessment of their influence in the context of RDC determination. We detail a 13C-13C CT-COSY method that combines a variation in the CT processed with diagonal filtering to yield 1JCC and RDCs. The strategy, which relies solely on cross-peak intensity modulation, is inspired in the cross-peak nulling method used for JHH determinations, but adapted and extended to applications where, like in sugars, large one-bond 13C-13C couplings coexist with relatively small long-range couplings. Because diagonal peaks are not utilized, overlap problems are greatly alleviated. Thus, one-bond couplings can be determined from different cross-peaks as either active or passive coupling. This results in increased accuracy when more than one determination is available, and in more opportunities to measure a specific coupling in the presence of severe overlap. In addition, we evaluate the influence of strong couplings on the determination of RDCs by computer simulations. We show that individual scalar couplings are notably affected by the presence of strong couplings but, at least for the simple cases studied, the obtained RDC values for use in structural calculations were not, because the errors introduced by strong couplings for the isotropic and oriented phases are very

  15. Accurate determinations of one-bond 13C-13C couplings in 13C-labeled carbohydrates.

    PubMed

    Azurmendi, Hugo F; Freedberg, Darón I

    2013-03-01

    Carbon plays a central role in the molecular architecture of carbohydrates, yet the availability of accurate methods for (1)D(CC) determination has not been sufficiently explored, despite the importance that such data could play in structural studies of oligo- and polysaccharides. Existing methods require fitting intensity ratios of cross- to diagonal-peaks as a function of the constant-time (CT) in CT-COSY experiments, while other methods utilize measurement of peak separation. The former strategies suffer from complications due to peak overlap, primarily in regions close to the diagonal, while the latter strategies are negatively impacted by the common occurrence of strong coupling in sugars, which requires a reliable assessment of their influence in the context of RDC determination. We detail a (13)C-(13)C CT-COSY method that combines a variation in the CT processed with diagonal filtering to yield (1)J(CC) and RDCs. The strategy, which relies solely on cross-peak intensity modulation, is inspired in the cross-peak nulling method used for J(HH) determinations, but adapted and extended to applications where, like in sugars, large one-bond (13)C-(13)C couplings coexist with relatively small long-range couplings. Because diagonal peaks are not utilized, overlap problems are greatly alleviated. Thus, one-bond couplings can be determined from different cross-peaks as either active or passive coupling. This results in increased accuracy when more than one determination is available, and in more opportunities to measure a specific coupling in the presence of severe overlap. In addition, we evaluate the influence of strong couplings on the determination of RDCs by computer simulations. We show that individual scalar couplings are notably affected by the presence of strong couplings but, at least for the simple cases studied, the obtained RDC values for use in structural calculations were not, because the errors introduced by strong couplings for the isotropic and

  16. Multi-modality imaging to assess metabolic response to dichloroacetate treatment in tumor models

    PubMed Central

    Neveu, Marie-Aline; Preter, Géraldine De; Joudiou, Nicolas; Bol, Anne; Brender, Jeffery R.; Saito, Keita; Kishimoto, Shun; Grégoire, Vincent; Jordan, Bénédicte F.; Krishna, Murali C.; Feron, Olivier; Gallez, Bernard

    2016-01-01

    Reverting glycolytic metabolism is an attractive strategy for cancer therapy as upregulated glycolysis is a hallmark in various cancers. Dichloroacetate (DCA), long used to treat lactic acidosis in various pathologies, has emerged as a promising anti-cancer drug. By inhibiting the pyruvate dehydrogenase kinase, DCA reactivates the mitochondrial function and decreases the glycolytic flux in tumor cells resulting in cell cycle arrest and apoptosis. We recently documented that DCA was able to induce a metabolic switch preferentially in glycolytic cancer cells, leading to a more oxidative phenotype and decreasing proliferation, while oxidative cells remained less sensitive to DCA treatment. To evaluate the relevance of this observation in vivo, the aim of the present study was to characterize the effect of DCA in glycolytic MDA-MB-231 tumors and in oxidative SiHa tumors using advanced pharmacodynamic metabolic biomarkers. Oxygen consumption, studied by 17O magnetic resonance spectroscopy, glucose uptake, evaluated by 18F-FDG PET and pyruvate transformation into lactate, measured using hyperpolarized 13C-magnetic resonance spectroscopy, were monitored before and 24 hours after DCA treatment in tumor bearing mice. In both tumor models, no clear metabolic shift was observed. Surprisingly, all these imaging parameters concur to the conclusion that both glycolytic tumors and oxidative tumors presented a similar response to DCA. These results highlight a major discordance in metabolic cancer cell bioenergetics between in vitro and in vivo setups, indicating critical role of the local microenvironment in tumor metabolic behaviors. PMID:28082726

  17. Investigation of Carbohydrate Metabolism and Transport in Castor Bean Seedlings by Cyclic JCross Polarization Imaging and Spectroscopy

    NASA Astrophysics Data System (ADS)

    Heidenreich, M.; Köckenberger, W.; Kimmich, R.; Chandrakumar, N.; Bowtell, R.

    1998-05-01

    NMR experiments using13C-labeled compounds offer the possibility of noninvasive monitoring of carbohydrate transport and metabolism in living plants, but are usually hampered by the low sensitivity of the13C nucleus. The problem of low sensitivity can be overcome by using the cyclicJcross polarization (CYCLCROP) technique, which allows the indirect detection of13C nuclei coupled to1H nuclei with the high NMR sensitivity of protons. We report here on methods for imaging and spectroscopy based on the CYCLCROP technique, and their use in the firstin vivoNMR study of carbohydrate transport and metabolism in castor bean seedlings (Ricinus communis L.). Comprehensive acquisition strategies for the various NMR methods are given, including the procedure for setting up the experiments. In addition, a full analysis of the effect of relaxation on the signals generated from smallJ-coupled spin systems by the CYCLCROP sequence is given, and the high sensitivity of the sequence is demonstrated. In thein vivostudy of six-day-old castor bean seedlings, we were able to measure the uptake of labeled hexoses, supplied in solution to the cotyledons, and their conversion to sucrose, as well as the transport of this sucrose in the vascular bundles. Images of the actual distribution of labeled sucrose in the hypocotyl of the seedling have also been obtained. The resulting data show some evidence for a preferential incorporation of labeled fructose in the process of sucrose synthesis, which decreases with the time of incubation.

  18. A 13C-NMR study of exopolysaccharide synthesis in Rhizobium meliloti Su47 strain

    NASA Astrophysics Data System (ADS)

    Tavernier, P.; Portais, J.-C.; Besson, I.; Courtois, J.; Courtois, B.; Barbotin, J.-N.

    1998-02-01

    Metabolic pathways implied in the synthesis of succinoglycan produced by the Su47 strain of R. meliloti were evaluated by 13C-NMR spectroscopy after incubation with [1{-}13C] or [2{-}13C] glucose. The biosynthesis of this polymer by R. meliloti from glucose occurred by a direct polymerisation of the introduced glucose and by the pentose phosphate pathway. Les voies métaboliques impliquées dans la synthèse du succinoglycane produit par la souche Su47 de R. meliloti ont été évaluées par la spectroscopie de RMN du carbone 13 après incubation des cellules avec du [1{-}13C] ou [2{-}13C] glucose. La biosynthèse de ce polymère à partir du glucose se produit par polymérisation directe du glucose et par la voie des pentoses phosphate.

  19. A Method to Constrain Genome-Scale Models with 13C Labeling Data

    PubMed Central

    García Martín, Héctor; Kumar, Vinay Satish; Weaver, Daniel; Ghosh, Amit; Chubukov, Victor; Mukhopadhyay, Aindrila; Arkin, Adam; Keasling, Jay D.

    2015-01-01

    Current limitations in quantitatively predicting biological behavior hinder our efforts to engineer biological systems to produce biofuels and other desired chemicals. Here, we present a new method for calculating metabolic fluxes, key targets in metabolic engineering, that incorporates data from 13C labeling experiments and genome-scale models. The data from 13C labeling experiments provide strong flux constraints that eliminate the need to assume an evolutionary optimization principle such as the growth rate optimization assumption used in Flux Balance Analysis (FBA). This effective constraining is achieved by making the simple but biologically relevant assumption that flux flows from core to peripheral metabolism and does not flow back. The new method is significantly more robust than FBA with respect to errors in genome-scale model reconstruction. Furthermore, it can provide a comprehensive picture of metabolite balancing and predictions for unmeasured extracellular fluxes as constrained by 13C labeling data. A comparison shows that the results of this new method are similar to those found through 13C Metabolic Flux Analysis (13C MFA) for central carbon metabolism but, additionally, it provides flux estimates for peripheral metabolism. The extra validation gained by matching 48 relative labeling measurements is used to identify where and why several existing COnstraint Based Reconstruction and Analysis (COBRA) flux prediction algorithms fail. We demonstrate how to use this knowledge to refine these methods and improve their predictive capabilities. This method provides a reliable base upon which to improve the design of biological systems. PMID:26379153

  20. Clinical NOE 13C MRS for neuropsychiatric disorders of the frontal lobe

    NASA Astrophysics Data System (ADS)

    Sailasuta, Napapon; Robertson, Larry W.; Harris, Kent C.; Gropman, Andrea L.; Allen, Peter S.; Ross, Brian D.

    2008-12-01

    In this communication, a scheme is described whereby in vivo 13C MRS can safely be performed in the frontal lobe, a human brain region hitherto precluded on grounds of SAR, but important in being the seat of impaired cognitive function in many neuropsychiatric and developmental disorders. By combining two well known features of 13C NMR—the use of low power NOE and the focus on 13C carbon atoms which are only minimally coupled to protons, we are able to overcome the obstacle of SAR and develop means of monitoring the 13C fluxes of critically important metabolic pathways in frontal brain structures of normal volunteers and patients. Using a combination of low-power WALTZ decoupling, variants of random noise for nuclear overhauser effect enhancement it was possible to reduce power deposition to 20% of the advised maximum specific absorption rate (SAR). In model solutions 13C signal enhancement achieved with this scheme were comparable to that obtained with WALTZ-4. In human brain, the low power procedure effectively determined glutamine, glutamate and bicarbonate in the posterior parietal brain after [1- 13C] glucose infusion. The same 13C enriched metabolites were defined in frontal brain of human volunteers after administration of [1- 13C] acetate, a recognized probe of glial metabolism. Time courses of incorporation of 13C into cerebral glutamate, glutamine and bicarbonate were constructed. The results suggest efficacy for measurement of in vivo cerebral metabolic rates of the glutamate-glutamine and tricarboxylic acid cycles in 20 min MR scans in previously inaccessible brain regions in humans at 1.5T. We predict these will be clinically useful biomarkers in many human neuropsychiatric and genetic conditions.

  1. Optoacoustic 13C-breath test analyzer

    NASA Astrophysics Data System (ADS)

    Harde, Hermann; Helmrich, Günther; Wolff, Marcus

    2010-02-01

    The composition and concentration of exhaled volatile gases reflects the physical ability of a patient. Therefore, a breath analysis allows to recognize an infectious disease in an organ or even to identify a tumor. One of the most prominent breath tests is the 13C-urea-breath test, applied to ascertain the presence of the bacterium helicobacter pylori in the stomach wall as an indication of a gastric ulcer. In this contribution we present a new optical analyzer that employs a compact and simple set-up based on photoacoustic spectroscopy. It consists of two identical photoacoustic cells containing two breath samples, one taken before and one after capturing an isotope-marked substrate, where the most common isotope 12C is replaced to a large extent by 13C. The analyzer measures simultaneously the relative CO2 isotopologue concentrations in both samples by exciting the molecules on specially selected absorption lines with a semiconductor laser operating at a wavelength of 2.744 μm. For a reliable diagnosis changes of the 13CO2 concentration of 1% in the exhaled breath have to be detected at a concentration level of this isotope in the breath of about 500 ppm.

  2. A roadmap for interpreting 13C metabolite labeling patterns from cells

    PubMed Central

    Buescher, Joerg M.; Antoniewicz, Maciek R.; Boros, Laszlo G.; Burgess, Shawn C.; Brunengraber, Henri; Clish, Clary B.; DeBerardinis, Ralph J.; Feron, Olivier; Frezza, Christian; Ghesquiere, Bart; Gottlieb, Eyal; Hiller, Karsten; Jones, Russell G.; Kamphorst, Jurre J.; Kibbey, Richard G.; Kimmelman, Alec C.; Locasale, Jason W.; Lunt, Sophia Y.; Maddocks, Oliver D. K.; Malloy, Craig; Metallo, Christian M.; Meuillet, Emmanuelle J.; Munger, Joshua; Nöh, Katharina; Rabinowitz, Joshua D.; Ralser, Markus; Sauer, Uwe; Stephanopoulos, Gregory; St-Pierre, Julie; Tennant, Daniel A.; Wittmann, Christoph; Vander Heiden, Matthew G.; Vazquez, Alexei; Vousden, Karen; Young, Jamey D.; Zamboni, Nicola; Fendt, Sarah-Maria

    2015-01-01

    Measuring intracellular metabolism has increasingly led to important insights in biomedical research. 13C tracer analysis, although less information-rich than quantitative 13C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting 13C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments. PMID:25731751

  3. Metabolic Imaging of Glutamine in Cancer.

    PubMed

    Zhu, Lin; Ploessl, Karl; Zhou, Rong; Mankoff, David; Kung, Hank F

    2017-04-01

    Glucose and glutamine are the most abundant nutrients for producing energy and building blocks in normal and tumor cells. Increased glycolysis in tumors, the Warburg Effect, is the basis for (18)F-FDG PET imaging. Cancer cells can also be genetically reprogrammed to use glutamine. 5-(11)C-(2S)-glutamine and (18)F-(2S,4R)4-fluoroglutamine may be useful complementary tools to measure changes in tumor metabolism. In glioma patients, the tracer (18)F-(2S,4R)4-fluoroglutamine showed tumor-to-background contrast different from that of (18)F-FDG and differences in uptake in glioma patients with clinical progression of disease versus stable disease (tumor-to-brain ratio > 3.7 in clinically active glioma tumors, minimal or no specific uptake in clinically stable tumors). These preliminary results suggest that (18)F-(2S,4R)4-fluoroglutamine PET may be a new tool for probing in vivo metabolism of glutamine in cancer patients and for guiding glutamine-targeted therapeutics. Further studies of uptake mechanism, and comparison of kinetics for (18)F-(2S,4R)4-fluoroglutamine versus the (11)C-labeled native glutamine, will be important and enlightening.

  4. Biokinetics of (13)C in the human body after oral administration of (13)C-labeled glucose as an index for the biokinetics of (14)C.

    PubMed

    Masuda, Tsuyoshi; Tako, Yasuhiro; Matsushita, Kensaku; Takeda, Hiroshi; Endo, Masahiro; Nakamura, Yuji; Hisamatsu, Shun'ichi

    2016-09-01

    The retention of (13)C in the human body after oral administration of (13)C-labeled glucose was studied in three healthy volunteer subjects to estimate the 50 year cumulative body burden for (13)C as an index of the committed dose of the radioisotope (14)C. After administration of (13)C-labeled glucose, the volunteers ingested controlled diets with a fixed number of calories for 112 d. Samples of breath and urine were collected up to 112 d after administration. Samples of feces were collected up to 14 d after administration. Hair samples were obtained at 119 d after administration and analyzed as a representative index of the rate of excretion of organic (13)C via pathways such as skin cell exfoliation and mucus secretion. All samples were analyzed for (13)C/(12)C atomic ratio to determine the rate of excretion via each pathway. We then constructed a metabolic model with a total of four pathways (breath, urine, feces, and other) comprising seven compartments. We determined the values of the biokinetic parameters in the model by using the obtained excretion data. From 74% to 94% of the (13)C administered was excreted in breath, whereas  <2% was excreted in urine and feces. In the other pathway, the excretion rate constant in the compartment with the longest residence time stretched to hundreds of days but the rate constant for each subject was not statistically significant (P value  >  0.1). In addition, the dataset for one of the three subjects was markedly different from those of the other two. When we estimated the 50 year cumulative body burden for (13)C by using our model and we included non-statistically significant parameters, a considerable cumulative body burden was found in the compartments excreting to the other pathway. Although our results on the cumulative body burden of (13)C from orally administered carbon as glucose were inconclusive, we found that the compartments excreting to the other pathway had a markedly long residence time and

  5. In vivo dynamic turnover of cerebral 13C isotopomers from [U- 13C]glucose

    NASA Astrophysics Data System (ADS)

    Xu, Su; Shen, Jun

    2006-10-01

    An INEPT-based 13C MRS method and a cost-effective and widely available 11.7 Tesla 89-mm bore vertical magnet were used to detect dynamic 13C isotopomer turnover from intravenously infused [U- 13C]glucose in a 211 μL voxel located in the adult rat brain. The INEPT-based 1H → 13C polarization transfer method is mostly adiabatic and therefore minimizes signal loss due to B 1 inhomogeneity of the surface coils used. High quality and reproducible data were acquired as a result of combined use of outer volume suppression, ISIS, and the single-shot three-dimensional localization scheme built in the INEPT pulse sequence. Isotopomer patterns of both glutamate C4 at 34.00 ppm and glutamine C4 at 31.38 ppm are dominated first by a doublet originated from labeling at C4 and C5 but not at C3 (with 1JC4C5 = 51 Hz) and then by a quartet originated from labeling at C3, C4, and C5 (with 1JC3C4 = 35 Hz). A lag in the transition of glutamine C4 pattern from doublet-dominance to quartet dominance as compared to glutamate C4 was observed, which provides an independent verification of the precursor-product relationship between neuronal glutamate and glial glutamine and a significant intercompartmental cerebral glutamate-glutamine cycle between neurons and glial cells.

  6. Synthesis of 2H- and 13C-substituted dithanes

    DOEpatents

    Martinez, Rodolfo A.; Alvarez, Marc A.; Silks, III, Louis A.; Unkefer, Clifford J.

    2003-01-01

    The present invention is directed to labeled compounds, [2-.sup.13 C]dithiane wherein the .sup.13 C atom is directly bonded to one or two deuterium atoms. The present invention is also directed to processes of preparing [2-.sup.13 C]dithiane wherein the .sup.13 C atom is directly bonded to one or two deuterium atoms. The present invention is also directed to labeled compounds, e.g., [.sup.2 H.sub.1-2, .sup.13 C]methanol (arylthio)-, acetates wherein the .sup.13 C atom is directly bonded to exactly one or two deuterium atoms.

  7. Synthesis Of 2h- And 13c-Substituted Dithanes

    DOEpatents

    Martinez, Rodolfo A.; Alvarez, Marc A.; Silks, III, Louis A.; Unkefer, Clifford J.

    2004-05-04

    The present invention is directed to labeled compounds, [2-.sup.13 C]dithane wherein the .sup.13 C atom is directly bonded to one or two deuterium atoms. The present invention is also directed to processes of preparing [2-.sup.13 C]dithane wherein the .sup.13 C atom is directly bonded to one or two deuterium atoms. The present invention is also directed to labeled compounds, e.g., [.sup.2 H.sub.1-2, .sup.13 C]methanol (arylthio)-, acetates wherein the .sup.13 C atom is directly bonded to exactly one or two deuterium atoms.

  8. Hypothalamic metabolic compartmentation during appetite regulation as revealed by magnetic resonance imaging and spectroscopy methods

    PubMed Central

    Lizarbe, Blanca; Benitez, Ania; Peláez Brioso, Gerardo A.; Sánchez-Montañés, Manuel; López-Larrubia, Pilar; Ballesteros, Paloma; Cerdán, Sebastián

    2013-01-01

    We review the role of neuroglial compartmentation and transcellular neurotransmitter cycling during hypothalamic appetite regulation as detected by Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) methods. We address first the neurochemical basis of neuroendocrine regulation in the hypothalamus and the orexigenic and anorexigenic feed-back loops that control appetite. Then we examine the main MRI and MRS strategies that have been used to investigate appetite regulation. Manganese-enhanced magnetic resonance imaging (MEMRI), Blood oxygenation level-dependent contrast (BOLD), and Diffusion-weighted magnetic resonance imaging (DWI) have revealed Mn2+ accumulations, augmented oxygen consumptions, and astrocytic swelling in the hypothalamus under fasting conditions, respectively. High field 1H magnetic resonance in vivo, showed increased hypothalamic myo-inositol concentrations as compared to other cerebral structures. 1H and 13C high resolution magic angle spinning (HRMAS) revealed increased neuroglial oxidative and glycolytic metabolism, as well as increased hypothalamic glutamatergic and GABAergic neurotransmissions under orexigenic stimulation. We propose here an integrative interpretation of all these findings suggesting that the neuroendocrine regulation of appetite is supported by important ionic and metabolic transcellular fluxes which begin at the tripartite orexigenic clefts and become extended spatially in the hypothalamus through astrocytic networks becoming eventually MRI and MRS detectable. PMID:23781199

  9. Accurate measurements of 13C-13C distances in uniformly 13C-labeled proteins using multi-dimensional four-oscillating field solid-state NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Straasø, Lasse Arnt; Nielsen, Jakob Toudahl; Bjerring, Morten; Khaneja, Navin; Nielsen, Niels Chr.

    2014-09-01

    Application of sets of 13C-13C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important 13C-13C distances in uniformly 13C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl (13C') and aliphatic (13Caliphatic) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys. 130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly 13C,15N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of 13C'-13Caliphatic distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform 13C,15N-labeling on the FGAIL fragment.

  10. Quantitative 13C traces of glucose fate in hepatitis B virus infected hepatocytes.

    PubMed

    Wan, Qianfen; Wang, Yulan; Tang, Huiru

    2017-02-21

    Quantitative characterization of 13C-labeled metabolites is an important part of the stable isotope tracing method widely used in metabolic flux analysis. Due to long relaxation time and low sensitivity of 13C nuclei, direct measurement of 13C labeled metabolites using one dimensional 13C NMR often fails to meet the demand of metabolomics studies especially with large number of samples and metabolites having low abundance. Although HSQC-based 2D NMR methods have improved sensitivity with inversion detection, they are time-consuming thus unsuitable for high-throughput absolute quantification of 13C-labeled metabolites. In this study, we developed a method for absolute quantification of 13C labeled metabolites using naturally abundant TSP as a reference with the first increment of HMQC pulse sequence, taking polarization transfer efficiencies into consideration. We validated this method using a mixture of 13C-labeled alanine, methionine, glucose and formic acid together with a mixture of alanine, lactate, glycine, uridine, cytosine, and hypoxanthine having natural 13C abundance with known concentrations. We subsequently applied this method to analyze the flux of glucose in HepG2 cells infected with hepatitis B virus (HBV). The results showed that HBV infection increased the cellular uptake of glucose, stimulated glycolysis and enhanced the pentose phosphate and hexosamine pathways for biosynthesis of RNA and DNA and nucleotide sugars to facilitate HBV replication. This method saves experimental time and provides a possibility for absolute quantitative tracking of the 13C labeled metabolites for high throughput studies.

  11. sup 13 C and sup 18 O isotopic disequilibrium in biological carbonates: I. Patterns

    SciTech Connect

    McConnaughey, T. )

    1989-01-01

    Biological carbonates frequently precipitate out of {sup 18}O and {sup 13}C equilibrium with ambient waters. Two patterns of isotopic disequilibrium are particularly common. Kinetic disequilibria, so designated because they apparently result from kinetic isotope effects during CO{sub 2} hydration and hydroxylation, involve simultaneous depletions of {sup 18}O and {sup 13}C as large as 4{per thousand} and 10 to 15{per thousand}, respectively. Rapid skeletogenesis favors strong kinetic effects, and approximately linear correlations between skeletal {delta}{sup 18}O and {delta}{sup 13}C are common in carbonates showing mainly the kinetic pattern. Metabolic effects involve additional positive or negative modulation of skeletal {delta}{sup 13}C, reflecting changes in the {delta}{sup 13}C of dissolved inorganic carbon, caused mainly by photosynthesis and respiration. Kinetic isotope disequilibria tend to be fairly consistent in rapidly growing parts of photosynthetic corals, and time dependent isotopic variations therefore reflect changes in environmental conditions. {delta}{sup 18}O variations from Galapagos corals yields meaningful clues regarding seawater temperature, while {delta}{sup 13}C variations reflect changes in photosynthesis, modulated by cloudiness.

  12. T(2) relaxation times of (13)C metabolites in a rat hepatocellular carcinoma model measured in vivo using (13)C-MRS of hyperpolarized [1-(13)C]pyruvate.

    PubMed

    Yen, Yi-Fen; Le Roux, Patrick; Mayer, Dirk; King, Randy; Spielman, Daniel; Tropp, James; Butts Pauly, Kim; Pfefferbaum, Adolf; Vasanawala, Shreyas; Hurd, Ralph

    2010-05-01

    A single-voxel Carr-Purcell-Meibloom-Gill sequence was developed to measure localized T(2) relaxation times of (13)C-labeled metabolites in vivo for the first time. Following hyperpolarized [1-(13)C]pyruvate injections, pyruvate and its metabolic products, alanine and lactate, were observed in the liver of five rats with hepatocellular carcinoma and five healthy control rats. The T(2) relaxation times of alanine and lactate were both significantly longer in HCC tumors than in normal livers (p < 0.002). The HCC tumors also showed significantly higher alanine signal relative to the total (13)C signal than normal livers (p < 0.006). The intra- and inter-subject variations of the alanine T(2) relaxation time were 11% and 13%, respectively. The intra- and inter-subject variations of the lactate T(2) relaxation time were 6% and 7%, respectively. The intra-subject variability of alanine to total carbon ratio was 16% and the inter-subject variability 28%. The intra-subject variability of lactate to total carbon ratio was 14% and the inter-subject variability 20%. The study results show that the signal level and relaxivity of [1-(13)C]alanine may be promising biomarkers for HCC tumors. Its diagnostic values in HCC staging and treatment monitoring are yet to be explored.

  13. Multi-year estimates of plant and ecosystem 13C discrimination at AmeriFlux sites

    NASA Astrophysics Data System (ADS)

    Dang, X.; Lai, C.; Hollinger, D. Y.; Bush, S.; Randerson, J. T.; Law, B. E.; Schauer, A. J.; Ehleringer, J.

    2011-12-01

    We estimated plant and ecosystem 13C discrimination continuously at 8 AmeriFlux sites (Howland Forest, Harvard Forest, Wind River Forest, Rannells Prairie, Freeman Ranch, Chestnut Ridge, Metolius, and Marys River fir) over 8 years (2002-2009). We used an observation-based approach from weekly measurements of eddy covariance CO2 fluxes and their 13C/12C ratios to estimate photosynthetic 13C discrimination (△A) and respiration (δ13CR) on seasonal and interannual time scales. The coordinated, systematic flask sampling across the AmeriFlux subnetwork were used for cross-site synthesis of monthly flux estimates [Dang et al. Combining tower mixing ratio and community model data to estimate regional-scale net ecosystem carbon exchange by boundary layer inversion over 4 flux towers in the U.S.A., Journal of Geophysical Research-Biogeosciences, in press]. Here, we evaluated environmental factors that also influenced temporal variability in △A and δ13CR from daily to interannual time scales, comparing atmospheric 13C/12C measurements, leaf and needle organic matter, and tree ring cellulose. Across these major biomes that dominate the continent, we show differential ecophysiological responses to environmental stresses, among which water availability appeared to be a dominant factor. Our decadal measurement period provided robust estimates of atmospheric 13C discrimination by terrestrial ecosystems, but also suggest regions where enhanced monitoring efforts are required (e.g., 13C/12C emission from fire and urban metabolism; increased temporal resolution of 13C measurements in stress-sensitive ecosystems) to make atmospheric 13C/12C measurements an effective constraint for continental-scale assessments of the terrestrial carbon cycle.

  14. The effect of chemical processing on the δ 13C value of plant tissue

    NASA Astrophysics Data System (ADS)

    Van de Water, Peter K.

    2002-04-01

    The effect of standard processing techniques on the δ 13C value of plant tissue was tested using species representing the three photosynthetic pathways, including angiosperms and gymnosperms within the C 3 taxonomic division. The species include Cowania mexicana (C 3 angiosperm), Juniperus osteosperma (C 3 gymnosperm), Opuntia spp. (crassulacean acid metabolism [CAM] angiosperm), and Atriplex canescens (C 4 angiosperm). Each species is represented by 5 plants collected at two different sites, for a total of 10 samples. The samples were processed to whole plant tissue, holocellulose, α-cellulose, and nitrocellulose. An additional process was added with the discovery of residual Ca-oxalate crystals in holocellulose samples. Both C 3 species show δ 13C values becoming 13C enriched with increased processing. The CAM representative shows the opposite trend, with 13C depletion during the progression of treatments. The greatest range of values and most inconsistent trends occur in the C 4 representative. Removal of the Ca-oxalate fraction resulted in different mean weight percentages and δ 13C values among the species. Calculated δ 13C values of the Ca-oxalate crystals show depletion from the tissue values in the two C 3 species and enrichment in the C 4 and CAM representatives. The C. mexicana samples show the greatest change between the tissue and Ca-oxalates (7.3‰) but the least mean weight percentage (11%), whereas A. canescens shows the greatest overall change, with a -2.8‰ isotopic shift and over 48% mean weight percentage. Variability within the samples undergoing each treatment remained relatively unchanged even with increased cellulose purity. This paper provides estimates of isotopic offsets necessary to correct from one treatment to another. Significant differences in δ 13C among different treatments confirm the need to state the tissue fraction analyzed when reporting δ 13C results.

  15. Synthesis Of [2h, 13c]M [2h2m 13c], And [2h3,, 13c] Methyl Aryl Sulfones And Sulfoxides

    DOEpatents

    Martinez, Rodolfo A.; Alvarez, Marc A.; Silks, III, Louis A.; Unkefer, Clifford J.; Schmidt, Jurgen G.

    2004-07-20

    The present invention is directed to labeled compounds, [.sup.2 H.sub.1, .sup.13 C], [.sup.2 H.sub.2, .sup.13 C] and [.sup.2 H.sub.3, .sup.13 C]methyl aryl sulfones and [.sup.2 H.sub.1, .sup.13 C], [.sup.2 H.sub.2, .sup.13 C] and [.sup.2 H.sub.3, .sup.13 C]methyl aryl sulfoxides, wherein the .sup.13 C methyl group attached to the sulfur of the sulfone or sulfoxide includes exactly one, two or three deuterium atoms and the aryl group is selected from the group consisting of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure: ##STR1## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each independently, hydrogen, a C.sub.1 -C.sub.4 lower alkyl, a halogen, an amino group from the group consisting of NH.sub.2, NHR and NRR' where R and R' are each a C.sub.1 -C.sub.4 lower alkyl, a phenyl, or an alkoxy group. The present invention is also directed to processes of preparing methyl aryl sulfones and methyl aryl sulfoxides.

  16. 13C magnetic resonance spectroscopy measurements with hyperpolarized [1‐13C] pyruvate can be used to detect the expression of transgenic pyruvate decarboxylase activity in vivo

    PubMed Central

    Dzien, Piotr; Tee, Sui‐Seng; Kettunen, Mikko I.; Lyons, Scott K.; Larkin, Timothy J.; Timm, Kerstin N.; Hu, De‐En; Wright, Alan; Rodrigues, Tiago B.; Serrao, Eva M.; Marco‐Rius, Irene; Mannion, Elizabeth; D'Santos, Paula; Kennedy, Brett W. C.

    2015-01-01

    Purpose Dissolution dynamic nuclear polarization can increase the sensitivity of the 13C magnetic resonance spectroscopy experiment by at least four orders of magnitude and offers a novel approach to the development of MRI gene reporters based on enzymes that metabolize 13C‐labeled tracers. We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC 4.1.1.1), which catalyzes the decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide. Methods Pyruvate decarboxylase from Zymomonas mobilis (zmPDC) and a mutant that lacked enzyme activity were expressed using an inducible promoter in human embryonic kidney (HEK293T) cells. Enzyme activity was measured in the cells and in xenografts derived from the cells using 13C MRS measurements of the conversion of hyperpolarized [1‐13C] pyruvate to H13 CO3–. Results Induction of zmPDC expression in the cells and in the xenografts derived from them resulted in an approximately two‐fold increase in the H13 CO3–/[1‐13C] pyruvate signal ratio following intravenous injection of hyperpolarized [1‐13C] pyruvate. Conclusion We have demonstrated the feasibility of using zmPDC as an in vivo reporter gene for use with hyperpolarized 13C MRS. Magn Reson Med 76:391–401, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PMID:26388418

  17. 13C NMR Metabolomics: Applications at Natural Abundance

    PubMed Central

    2015-01-01

    13C NMR has many advantages for a metabolomics study, including a large spectral dispersion, narrow singlets at natural abundance, and a direct measure of the backbone structures of metabolites. However, it has not had widespread use because of its relatively low sensitivity compounded by low natural abundance. Here we demonstrate the utility of high-quality 13C NMR spectra obtained using a custom 13C-optimized probe on metabolomic mixtures. A workflow was developed to use statistical correlations between replicate 1D 13C and 1H spectra, leading to composite spin systems that can be used to search publicly available databases for compound identification. This was developed using synthetic mixtures and then applied to two biological samples, Drosophila melanogaster extracts and mouse serum. Using the synthetic mixtures we were able to obtain useful 13C–13C statistical correlations from metabolites with as little as 60 nmol of material. The lower limit of 13C NMR detection under our experimental conditions is approximately 40 nmol, slightly lower than the requirement for statistical analysis. The 13C and 1H data together led to 15 matches in the database compared to just 7 using 1H alone, and the 13C correlated peak lists had far fewer false positives than the 1H generated lists. In addition, the 13C 1D data provided improved metabolite identification and separation of biologically distinct groups using multivariate statistical analysis in the D. melanogaster extracts and mouse serum. PMID:25140385

  18. SU-E-QI-11: Measurement of Renal Pyruvate-To-Lactate Exchange with Hyperpolarized 13C MRI

    SciTech Connect

    Adamson, E; Johnson, K; Fain, S; Gordon, J

    2014-06-15

    Purpose: Previous work [1] modeling the metabolic flux between hyperpolarized [1-13C]pyruvate and [1-13C]lactate in magnetic resonance spectroscopic imaging (MRSI) experiments failed to account for vascular signal artifacts. Here, we investigate a method to minimize the vascular signal and its impact on the fidelity of metabolic modeling. Methods: MRSI was simulated for renal metabolism in MATLAB both with and without bipolar gradients. The resulting data were fit to a two-site exchange model [1], and the effects of vascular partial volume artifacts on kinetic modeling were assessed. Bipolar gradients were then incorporated into a gradient echo sequence to validate the simulations experimentally. The degree of diffusion weighting (b = 32 s/mm{sup 2}) was determined empirically from 1H imaging of murine renal vascular signal. The method was then tested in vivo using MRSI with bipolar gradients following injection of hyperpolarized [1-{sup 13}C]pyruvate (∼80 mM at 20% polarization). Results: In simulations, vascular signal contaminated the renal metabolic signal at resolutions as high as 2 × 2 mm{sup 2} due to partial volume effects. The apparent exchange rate from pyruvate to lactate (k{sub p}) was underestimated in the presence of these artifacts due to contaminating pyruvate signal. Incorporation of bipolar gradients suppressed vascular signal and improved the accuracy of kp estimation. Experimentally, the in vivo results supported the ability of bipolar gradients to suppress vascular signal. The in vivo exchange rate increased, as predicted in simulations, from k{sub p} = 0.012 s-{sup 1} to k{sub p} = 0.020-{sup 1} after vascular signal suppression. Conclusion: We have demonstrated the limited accuracy of the two-site exchange model in the presence of vascular partial volume artifacts. The addition of bipolar gradients suppressed vascular signal and improved model accuracy in simulations. Bipolar gradients largely affected kp estimation in vivo. Currently

  19. Conditions for 13C NMR Detection of 2-Hydroxyglutarate in Tissue Extracts from IDH-Mutated Gliomas

    PubMed Central

    Pichumani, Kumar; Mashimo, Tomoyuki; Baek, Hyeon-Man; Ratnakar, James; Mickey, Bruce; DeBerardinis, Ralph J.; Maher, Elizabeth A.; Bachoo, Robert M.; Malloy, Craig R.; Kovacs, Zoltan

    2015-01-01

    13C NMR spectroscopy of extracts from patient tumor samples provides rich information about metabolism. However, in IDH-mutant gliomas 13C labeling is obscured in glutamate and glutamine by the oncometabolite, 2-hydroxyglutaric acid (2HG), prompting development of a simple method to resolve the metabolites. J-coupled multiplets in 2HG were similar to glutamate and glutamine and could be clearly resolved at pH 6. A cryogenically-cooled 13C probe but not J-resolved heteronuclear single quantum coherence spectroscopy significantly improved detection of 2HG. These methods enable the monitoring of 13C-13C spin-spin couplings in 2HG expressing IDH mutant gliomas. PMID:25908561

  20. Identification of VPS13C as a Galectin-12-Binding Protein That Regulates Galectin-12 Protein Stability and Adipogenesis

    PubMed Central

    Yang, Ri-Yao; Xue, Huiting; Yu, Lan; Velayos-Baeza, Antonio; Monaco, Anthony P.; Liu, Fu-Tong

    2016-01-01

    Galectin-12, a member of the galectin family of β-galactoside-binding animal lectins, is preferentially expressed in adipocytes and required for adipocyte differentiation in vitro. This protein was recently found to regulate lipolysis, whole body adiposity, and glucose homeostasis in vivo. Here we identify VPS13C, a member of the VPS13 family of vacuolar protein sorting-associated proteins highly conserved throughout eukaryotic evolution, as a major galectin-12-binding protein. VPS13C is upregulated during adipocyte differentiation, and is required for galectin-12 protein stability. Knockdown of Vps13c markedly reduces the steady-state levels of galectin-12 by promoting its degradation through primarily the lysosomal pathway, and impairs adipocyte differentiation. Our studies also suggest that VPS13C may have a broader role in protein quality control. The regulation of galectin-12 stability by VPS13C could potentially be exploited for therapeutic intervention of obesity and related metabolic diseases. PMID:27073999

  1. Functional groups identified by solid state 13C NMR spectroscopy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Animal manure is generally high in organic matter intensity so it is well suitable for 13C nuclear magnetic resonance (NMR) analysis. Solid-state 13C NMR techniques used in characterizing organic matter and its components include, but are not limited to, cross-polarization /magic angle spinning (CP...

  2. Neuroprotective effects of caffeine in MPTP model of Parkinson's disease: A (13)C NMR study.

    PubMed

    Bagga, Puneet; Chugani, Anup N; Patel, Anant B

    2016-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of nigrostriatal dopaminergic neurons with an accompanying neuroinflammation leading to loss of dopamine in the basal ganglia. Caffeine, a well-known A2A receptor antagonist is reported to slow down the neuroinflammation caused by activated microglia and reduce the extracellular glutamate in the brain. In this study, we have evaluated the neuroprotective effect of caffeine in the MPTP model of PD by monitoring the region specific cerebral energy metabolism. Adult C57BL6 mice were treated with caffeine (30 mg/kg, i.p.) 30 min prior to MPTP (25 mg/kg, i.p.) administration for 8 days. The paw grip strength of mice was assessed in order to evaluate the motor function after various treatments. For metabolic studies, mice were infused with [1,6-(13)C2]glucose, and (13)C labeling of amino acids was monitored using ex vivo(1)H-[(13)C]-NMR spectroscopy. The paw grip strength was found to be reduced following the MPTP treatment. The caffeine pretreatment showed significant protection against the reduction of paw grip strength in MPTP treated mice. The levels of GABA and myo-inositol were found to be elevated in the striatum of MPTP treated mice. The (13)C labeling of GluC4, GABAC2 and GlnC4 from [1,6-(13)C2]glucose was decreased in the cerebral cortex, striatum, olfactory bulb, thalamus and cerebellum suggesting impaired glutamatergic and GABAergic neuronal activity and neurotransmission of the MPTP treated mice. Most interestingly, the pretreatment of caffeine maintained the (13)C labeling of amino acids to the control values in cortical, olfactory bulb and cerebellum regions while it partially retained in striatal and thalamic regions in MPTP treated mice. The pretreatment of caffeine provides a partial neuro-protection against severe striatal degeneration in the MPTP model of PD.

  3. Metabolic imaging of patients with cardiomyopathy

    SciTech Connect

    Geltman, E.M. )

    1991-09-01

    The cardiomyopathies comprise a diverse group of illnesses that can be characterized functionally by several techniques. However, the delineation of derangements of regional perfusion and metabolism have been accomplished only relatively recently with positron emission tomography (PET). Regional myocardial accumulation and clearance of 11C-palmitate, the primary myocardial substrate under most conditions, demonstrate marked spatial heterogeneity when studied under fasting conditions or with glucose loading. PET with 11C-palmitate permits the noninvasive differentiation of patients with nonischemic from ischemic dilated cardiomyopathy, since patients with ischemic cardiomyopathy demonstrate large zones of intensely depressed accumulation of 11C-palmitate, probably reflecting prior infarction. Patients with hypertrophic cardiomyopathy and Duchenne's muscular dystrophy demonstrate relatively unique patterns of myocardial abnormalities of perfusion and metabolism. The availability of new tracers and techniques for the evaluation of myocardial metabolism (11C-acetate), perfusion (H2(15)O), and autonomic tone (11-C-hydroxyephedrine) should facilitate further understanding of the pathogenesis of the cardiomyopathies.

  4. Production and NMR signal optimization of hyperpolarized 13C-labeled amino acids

    NASA Astrophysics Data System (ADS)

    Parish, Christopher; Niedbalski, Peter; Ferguson, Sarah; Kiswandhi, Andhika; Lumata, Lloyd

    Amino acids are targeted nutrients for consumption by cancers to sustain their rapid growth and proliferation. 13C-enriched amino acids are important metabolic tracers for cancer diagnostics using nuclear magnetic resonance (NMR) spectroscopy. Despite this diagnostic potential, 13C NMR of amino acids however is hampered by the inherently low NMR sensitivity of the 13C nuclei. In this work, we have employed a physics technique known as dynamic nuclear polarization (DNP) to enhance the NMR signals of 13C-enriched amino acids. DNP works by transferring the high polarization of electrons to the nuclear spins via microwave irradiation at low temperature and high magnetic field. Using a fast dissolution method in which the frozen polarized samples are dissolved rapidly with superheated water, injectable solutions of 13C-amino acids with highly enhanced NMR signals (by at least 5,000-fold) were produced at room temperature. Factors that affect the NMR signal enhancement levels such as the choice of free radical polarizing agents and sample preparation will be discussed along with the thermal mixing physics model of DNP. The authors would like to acknowledge the support by US Dept of Defense Award No. W81XWH-14-1-0048 and Robert A. Welch Foundation Grant No. AT-1877.

  5. A cellular perspective on brain energy metabolism and functional imaging.

    PubMed

    Magistretti, Pierre J; Allaman, Igor

    2015-05-20

    The energy demands of the brain are high: they account for at least 20% of the body's energy consumption. Evolutionary studies indicate that the emergence of higher cognitive functions in humans is associated with an increased glucose utilization and expression of energy metabolism genes. Functional brain imaging techniques such as fMRI and PET, which are widely used in human neuroscience studies, detect signals that monitor energy delivery and use in register with neuronal activity. Recent technological advances in metabolic studies with cellular resolution have afforded decisive insights into the understanding of the cellular and molecular bases of the coupling between neuronal activity and energy metabolism and point at a key role of neuron-astrocyte metabolic interactions. This article reviews some of the most salient features emerging from recent studies and aims at providing an integration of brain energy metabolism across resolution scales.

  6. Linking Biogeochemistry to Microbial Diversity Using New 13C Approaches

    NASA Astrophysics Data System (ADS)

    Baggs, E. M.

    2005-12-01

    The use of 13C enables us to overcome uncertainties associated with soil C processes and to assess the links between species diversity and ecosystem function. Recent advances in stable isotope techniques enable determination of process rates, for example CH4 oxidation by direct measurement of 13C-CH4 and 13C-CO2. This overcomes uncertainties associated with reliance on changes in net CH4 emission, which may have compromised some earlier studies as both methanogenesis and CH4 oxidation may occur simultaneously in soil, providing significant advances in our understanding of the process of CH4 oxidation. These stable isotope techniques can be combined with molecular techniques (analysis of gene expression, stable isotope probing (SIP)) to relate the measured process to the microbial populations responsible. Here we will give a synthesis of results from experiments in which we applied 13C-CH4 to accurately determine CH4 oxidation rates in soils, and also present results of 13C-SIP from which we can identify the key players in the microbial population that are using the applied 13C substrate. With the 13C-CH4 technique we were able to provide direct evidence of inhibition of CH4 oxidation following fertiliser application (50-300 kg N ha-1) that was less under elevated pCO2, and evidence for anaerobic CH4 oxidation occurring in soil at 75% soil water filled pore space that would not have been apparent from changes in net CH4 emissions. 13C-SIP both through plants (using 13C-CO2) and directly into soil (using 13C-methane and -organic substrates) has revealed how key players in C utilisation vary under different soil conditions, for example, under improved and unimproved grasslands.

  7. Distinct fungal and bacterial δ13C signatures can drive the increase in soil δ13C with depth

    NASA Astrophysics Data System (ADS)

    Kohl, Lukas; Laganièrea, Jérôme; Edwards, Kate A.; Billings, Sharon A.; Morrill, Penny L.; Van Biesen, Geert; Ziegler, Susan E.

    2015-04-01

    Soil microbial biomass is a key precursor of soil organic carbon (SOC), and the enrichment in 13C during SOC diagenesis has been purported to be driven by increasing proportions of microbially derived SOC. Yet, little is known about how the δ13C of soil microbial biomass - and by extension the δ13C of microbial inputs to SOC - vary in space, time, or with the composition of the microbial community. Phospholipid fatty acids (PLFA) can be analyzed to measure the variation of the natural abundance δ13C values of both individual groups of microorganisms and the microbial community as a whole. Here, we show how variations of δ13CPLFA within the soil profile provides insight into C fluxes in undisturbed soils and demonstrate that distinct δ13C of fungal and bacterial biomass and their relative abundance can drive the increase of bulk δ13CSOC with depth. We studied the variation in natural abundance δ13C signatures of PLFA in podzolic soil profiles from mesic boreal forests in Atlantic Canada. Samples from the organic horizons (L,F,H) and the mineral (B; top 10 cm) horizons were analyzed for δ13C values of PLFA specific to fungi, G+ bacteria, or G- bacteria as proxies for the δ13C of the biomass of these groups, and for δ13C values of PLFA produced by a wide range of microorganisms (e.g. 16:0) as a proxy for the δ13C value of microbial biomass as a whole. Results were compared to fungi:bacteria ratios (F:B) and bulk δ13CSOC values. The δ13C values of group-specific PLFA were driven by differences among source organisms, with fungal PLFA consistently depleted (2.1 to 6.4‰) relative to and G+ and G- bacterial PLFA in the same sample. All group-specific PLFA, however, exhibited nearly constant δ13C values throughout the soil profile, apparently unaffected by the over 2.8‰ increase in δ13CSOC with depth from the L to B horizons. This indicates that bulk SOC poorly represents the substrates actually consumed by soil microorganisms in situ. Instead, our

  8. NMR structure analysis of uniformly 13C-labeled carbohydrates.

    PubMed

    Fontana, Carolina; Kovacs, Helena; Widmalm, Göran

    2014-06-01

    In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of (13)C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly (13)C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-(13)C)-sucrose, 342 Da] and one compound of medium molecular weight ((13)C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The (13)C resonances are traced using (13)C-(13)C correlations from homonuclear experiments, such as (H)CC-CT-COSY, (H)CC-NOESY, CC-CT-TOCSY and/or virtually decoupled (H)CC-TOCSY. Based on the assignment of the (13)C resonances, the (1)H chemical shifts are derived in a straightforward manner using one-bond (1)H-(13)C correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J CC splitting of the (13)C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either (13)C or (1)H detected experiments, namely CC-CT-COSY, band-selective (H)CC-TOCSY, HC-CT-HSQC-NOESY or long-range HC-CT-HSQC. However, due to the short T2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the (1)H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the (13)C-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with (15)N at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and (13)C-detected (H)CACO spectra.

  9. Specific 13C functional pathways as diagnostic targets in gastroenterology breath-tests: tricks for a correct interpretation.

    PubMed

    Pizzoferrato, M; Del Zompo, F; Mangiola, F; Lopetuso, L R; Petito, V; Cammarota, G; Gasbarrini, A; Scaldaferri, F

    2013-01-01

    Breath tests are non-invasive, non-radioactive, safe, simple and effective tests able to determine significant metabolic alterations due to specific diseases or lack of specific enzymes. Carbon isotope (13)C, the stable-non radioactive isotope of carbon, is the most used substrate in breath testing, in which (13)C/(12)C ratio is measured and expressed as a delta value, a differences between readings and a fixed standard. (13)C/(12)C ratio is measured with isotope ratio mass spectrometry or non-dispersive isotope-selective infrared spectrometer and generally there is a good agreement between these techniques in the isotope ratio estimation. (13)C/(12)C ratio can be expressed as static measurement (like delta over baseline in urea breath test) or as dynamic measurement as percent dose recovery, but more dosages are necessary. (13)C Breath-tests are involved in many fields of interest within gastroenterology, such as detection of Helicobacter pylori infection, study of gastric emptying, assessment of liver and exocrine pancreatic functions, determination of oro-caecal transit time, evaluation of absorption and to a lesser extend detection of bacterial overgrowth. The use of every single test in a clinical setting is vary depending on accuracy and substrate costs. This review is meant to present (13)C the meaning of (13)C/(12)C ratio and static and dynamic measure and, finally, the instruments dedicated to its use in gastroenterology. A brief presentation of (13)C breath tests in gastroenterology is also provided.

  10. Metabolic positron emission tomography imaging of cancer: Pairing lipid metabolism with glycolysis

    PubMed Central

    Kwee, Sandi A; Lim, John

    2016-01-01

    The limitations of fluorine-18 fluorodeoxy-D-glucose (FDG) in detecting some cancers has prompted a longstanding search for other positron emission tomography (PET) tracers to complement the imaging of glycolysis in oncology, with much attention paid to lipogenesis based on observations that the production of various lipid and lipid-containing compounds is increased in most cancers. Radiolabeled analogs of choline and acetate have now been used as oncologic PET probes for over a decade, showing convincingly improved detection sensitivity over FDG for certain cancers. However, neither choline nor acetate have been thoroughly validated as lipogenic biomarkers, and while acetyl-CoA produced from acetate is used in de-novo lipogenesis to synthesize fatty acids, acetate is also consumed by various other synthetic and metabolic pathways, with recent experimental observations challenging the assumption that lipogenesis is its predominant role in all cancers. Since tumors detected by acetate PET are also frequently detected by choline PET, imaging of choline metabolism might serve as an alternative albeit indirect marker of lipogenesis, particularly if the fatty acids produced in cancer cells are mainly destined for membrane synthesis through incorporation into phosphatidylcholines. Aerobic glycolysis may or may not coincide with changes in lipid metabolism, resulting in combinatorial metabolic phenotypes that may have different prognostic or therapeutic implications. Consequently, PET imaging using dual metabolic tracers, in addition to being diagnostically superior to imaging with individual tracers, could eventually play a greater role in supporting precision medicine, as efforts to develop small-molecule metabolic pathway inhibitors are coming to fruition. To prepare for this advent, clinical and translational studies of metabolic PET tracers must go beyond simply estimating tracer diagnostic utility, and aim to uncover potential therapeutic avenues associated with

  11. Mapping carbon fate during bleaching in a model cnidarian symbiosis: the application of (13) C metabolomics.

    PubMed

    Hillyer, Katie E; Dias, Daniel A; Lutz, Adrian; Roessner, Ute; Davy, Simon K

    2017-03-08

    Coral bleaching is a major threat to the persistence of coral reefs. Yet we lack detailed knowledge of the metabolic interactions that determine symbiosis function and bleaching-induced change. We mapped autotrophic carbon fate within the free metabolite pools of both partners of a model cnidarian-dinoflagellate symbiosis (Aiptasia-Symbiodinium) during exposure to thermal stress via the stable isotope tracer ((13) C bicarbonate), coupled to GC-MS. Symbiont photodamage and pronounced bleaching coincided with substantial increases in the turnover of non(13) C-labelled pools in the dinoflagellate (lipid and starch store catabolism). However, (13) C enrichment of multiple compounds associated with ongoing carbon fixation and de novo biosynthesis pathways was maintained (glucose, fatty acid and lipogenesis intermediates). Minimal change was also observed in host pools of (13) C-enriched glucose (a major symbiont-derived mobile product). However, host pathways downstream showed altered carbon fate and/or pool composition, with accumulation of compatible solutes and nonenzymic antioxidant precursors. In hospite symbionts continue to provide mobile products to the host, but at a significant cost to themselves, necessitating the mobilization of energy stores. These data highlight the need to further elucidate the role of metabolic interactions between symbiotic partners, during the process of thermal acclimation and coral bleaching.

  12. On the use of phloem sap δ13C to estimate canopy carbon discrimination

    NASA Astrophysics Data System (ADS)

    Rascher, Katherine; Máguas, Cristina; Werner, Christiane

    2010-05-01

    Although the carbon stable isotope composition (d13C) of bulk leaf material is a good integrative parameter of photosynthetic discrimination and can be used as a reliable ecological index of plant functioning; it is not a good tracer of short-term changes in photosynthetic discrimination. In contrast, d13C of phloem sap is potentially useful as an indicator of short-term changes in canopy photosynthetic discrimination. However, recent research indicates that d13C signatures may be substantially altered by metabolic processes downstream of initial leaf-level carbon fixation (e.g. post-photosynthetic fractionation). Accordingly, before phloem sap d13C can be used as a proxy for canopy level carbon discrimination an understanding of factors influencing the degree and magnitude of post-photosynthetic fractionation and how these vary between species is of paramount importance. In this study, we measured the d13C signature along the basipetal transport pathway in two co-occurring tree species in the field - an understory invasive exotic legume, Acacia longifolia, and a native pine, Pinus pinaster. We measured d13C of bulk leaf and leaf water soluble organic matter (WSOM), phloem sap sampled at two points along the plant axis and leaf and root dark respiration. In general, species differences in photosynthetic discrimination resulted in more enriched d13C values in the water-conserving P. pinaster relative to the water-spending A. longifolia. Post-photosynthetic fractionation led to differences in d13C of carbon pools along the plant axis with progressively more depleted d13C from the canopy to the trunk (~6.5 per mil depletion in A. longifolia and ~0.8per mil depletion in P. pinaster). Leaf and root respiration, d13C, were consistently enriched relative to putative substrates. We hypothesize that the pronounced enrichment of leaf respired CO2 relative to leaf WSOM may have left behind relatively depleted carbon to be loaded into the phloem resulting in d13C depletion

  13. Metabolic Imaging in Prostate Cancer: Where We Are

    PubMed Central

    Testa, Claudia; Pultrone, Cristian; Manners, David Neil; Schiavina, Riccardo; Lodi, Raffaele

    2016-01-01

    In recent years, the development of diagnostic methods based on metabolic imaging has been aimed at improving diagnosis of prostate cancer (PCa) and perhaps at improving therapy. Molecular imaging methods can detect specific biological processes that are different when detected within cancer cells relative to those taking place in surrounding normal tissues. Many methods are sensitive to tissue metabolism; among them, positron emission tomography (PET) and magnetic resonance spectroscopic imaging (MRSI) are widely used in clinical practice and clinical research. There is a rich literature that establishes the role of these metabolic imaging techniques as valid tools for the diagnosis, staging, and monitoring of PCa. Until recently, European guidelines for PCa detection still considered both MRSI/MRI and PET/CT to be under evaluation, even though they had demonstrated their value in the staging of high risk PCa, and in the restaging of patients presenting elevated prostatic-specific antigen levels following radical treatment of PCa, respectively. Very recently, advanced methods for metabolic imaging have been proposed in the literature: multiparametric MRI (mpMRI), hyperpolarized MRSI, PET/CT with the use of new tracers and finally PET/MRI. Their detection capabilities are currently under evaluation, as is the feasibility of using such techniques in clinical studies. PMID:27882307

  14. Study of Urban environmental quality through Isotopes δ13C

    NASA Astrophysics Data System (ADS)

    González-Sosa, E.; Mastachi-Loza, C.; Becerril-Piña, R.; Ramos-Salinas, N. M.

    2012-04-01

    Usually, trees with similar pH values on their bark develop epiphytes of similar species, the acidity to be a factor for growth. The aim of the study was evaluate the air quality through isotope δ13C in order to define the levels of environmental quality in the city of Queretaro, Mexico. In this work were collected at least 4 epiphytes positioned in trees of the species Prosopis Laevigata at 25 sites of Queretaro City. The samples were analyzed for trace elements with an inductively coupled plasma atomic emission spectroscopy (ICP). The collecting took place during dry period, in May and early rain June 2011 period, and on four sectors to identify the spatial distribution of pollution, using isotopic analysis of concentration of δ 13C. According with the results there are significant differences among the species in each of the sampled areas. The 5 February Avenue presented greater diversity and richness of δ13C, followed by those who were surveyed in the proximity of the UAQ and finally in the middle-east area. An average value of δ13C-17.92%, followed by those surveyed in the vicinity of the UAQ that correspond to sector I and II with an concentration of δ13C-17.55% and δ13C-17.22%, and finally the samples collected in trees scattered in the East-Sector II and IV with a value of δ13C-17.02% and δ13C-15.62%, respectively. Also were observed differences between the dry and wet period. It is likely that these results of δ 13C in moist period reflect the drag of the isotopes due to rain events that could mark a trend in the dilution of this element, however there is a trend in terms of abundance and composition of finding more impact in those species sampled in dry period, in May and early June 2011.

  15. Application of 13C Nuclear Magnetic Resonance To Elucidate the Unexpected Biosynthesis of Erythritol by Leuconostoc oenos

    PubMed Central

    Veiga-Da-Cunha, Maria; Firme, Paula; Romão, M. Vitória San; Santos, Helena

    1992-01-01

    Natural-abundance 13C nuclear magnetic resonance (13C-NMR) revealed the production of erythritol and glycerol by nongrowing cells of Leuconostoc oenos metabolizing glucose. The ratio of erythritol to glycerol was strongly influenced by the aeration conditions of the medium. The elucidation of the metabolic pathway responsible for erythritol production was achieved by 13C-NMR and 1H-NMR spectroscopy using specifically 13C-labelled d-glucose. The 1H-NMR spectrum of the cell supernatant resulting from the metabolism of [2-13C]glucose showed that only 75% of the glucose supplied was metabolized heterofermentatively and that the remaining 25% was channelled to the production of erythritol. The synthesis of this polyol resulted from the reduction of the C-4 moiety of the intermediate fructose 6-phosphate. Oxygen has an inhibitory effect on the production of erythritol by L. oenos. Preaeration of a suspension of nongrowing cells of L. oenos resulted in 30% less erythritol and in 70% more glycerol formed during the anaerobic metabolism of glucose. The anaerobic production of erythritol from glucose was also found in growing cultures of L. oenos, although to a smaller extent. PMID:16348738

  16. Hyperpolarized 13C allows a direct measure of flux through a single enzyme-catalyzed step by NMR

    PubMed Central

    Merritt, Matthew E.; Harrison, Crystal; Storey, Charles; Jeffrey, F. Mark; Sherry, A. Dean; Malloy, Craig R.

    2007-01-01

    13C NMR is a powerful tool for monitoring metabolic fluxes in vivo. The recent availability of automated dynamic nuclear polarization equipment for hyperpolarizing 13C nuclei now offers the potential to measure metabolic fluxes through select enzyme-catalyzed steps with substantially improved sensitivity. Here, we investigated the metabolism of hyperpolarized [1-13C1]pyruvate in a widely used model for physiology and pharmacology, the perfused rat heart. Dissolved 13CO2, the immediate product of the first step of the reaction catalyzed by pyruvate dehydrogenase, was observed with a temporal resolution of ≈1 s along with H13CO3−, the hydrated form of 13CO2 generated catalytically by carbonic anhydrase. In hearts presented with the medium-chain fatty acid octanoate in addition to hyperpolarized [1-13C1]pyruvate, production of 13CO2 and H13CO3− was suppressed by ≈90%, whereas the signal from [1-13C1]lactate was enhanced. In separate experiments, it was shown that O2 consumption and tricarboxylic acid (TCA) cycle flux were unchanged in the presence of added octanoate. Thus, the rate of appearance of 13CO2 and H13CO3− from [1-13C1]pyruvate does not reflect production of CO2 in the TCA cycle but rather reflects flux through pyruvate dehydrogenase exclusively. PMID:18056642

  17. Gamma images in benign and metabolic bone diseases: volume 1

    SciTech Connect

    Sy, W.M.

    1981-01-01

    Volume 1 of ''Gamma images in benign and metabolic bone diseases'' comprises chapters devoted to: general remarks and considerations, radiopharmaceuticals, Paget disease, osteomyelitis, trauma, benign bone tumors, chronic renal dialysis, acute renal failure, osteomalacia and rickets, and osteoporosis. Although published in 1981, the most recent references in the book were 1978 and most are 1977 or earlier. One of the strongest aspects of the volume are tables which categorize diseases, pathophysiology of disease, and image abnormalities. (JMT)

  18. Molecular Imaging of Metabolic Reprograming in Mutant IDH Cells.

    PubMed

    Viswanath, Pavithra; Chaumeil, Myriam M; Ronen, Sabrina M

    2016-01-01

    Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG). Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG). In turn, 2-HG, which has been termed an "oncometabolite," inhibits key α-KG-dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprograming that extends beyond 2-HG production, and this reprograming often differs from what has been previously reported in other cancer types. In this review, we will discuss in detail what is known to date about the metabolic reprograming of mutant IDH cells, and how this reprograming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells, and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

  19. Molecular imaging and therapy targeting copper metabolism in hepatocellular carcinoma.

    PubMed

    Wachsmann, Jason; Peng, Fangyu

    2016-01-07

    Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Significant efforts have been devoted to identify new biomarkers for molecular imaging and targeted therapy of HCC. Copper is a nutritional metal required for the function of numerous enzymatic molecules in the metabolic pathways of human cells. Emerging evidence suggests that copper plays a role in cell proliferation and angiogenesis. Increased accumulation of copper ions was detected in tissue samples of HCC and many other cancers in humans. Altered copper metabolism is a new biomarker for molecular cancer imaging with position emission tomography (PET) using radioactive copper as a tracer. It has been reported that extrahepatic mouse hepatoma or HCC xenografts can be localized with PET using copper-64 chloride as a tracer, suggesting that copper metabolism is a new biomarker for the detection of HCC metastasis in areas of low physiological copper uptake. In addition to copper modulation therapy with copper chelators, short-interference RNA specific for human copper transporter 1 (hCtr1) may be used to suppress growth of HCC by blocking increased copper uptake mediated by hCtr1. Furthermore, altered copper metabolism is a promising target for radionuclide therapy of HCC using therapeutic copper radionuclides. Copper metabolism has potential as a new theranostic biomarker for molecular imaging as well as targeted therapy of HCC.

  20. Molecular imaging and therapy targeting copper metabolism in hepatocellular carcinoma

    PubMed Central

    Wachsmann, Jason; Peng, Fangyu

    2016-01-01

    Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Significant efforts have been devoted to identify new biomarkers for molecular imaging and targeted therapy of HCC. Copper is a nutritional metal required for the function of numerous enzymatic molecules in the metabolic pathways of human cells. Emerging evidence suggests that copper plays a role in cell proliferation and angiogenesis. Increased accumulation of copper ions was detected in tissue samples of HCC and many other cancers in humans. Altered copper metabolism is a new biomarker for molecular cancer imaging with position emission tomography (PET) using radioactive copper as a tracer. It has been reported that extrahepatic mouse hepatoma or HCC xenografts can be localized with PET using copper-64 chloride as a tracer, suggesting that copper metabolism is a new biomarker for the detection of HCC metastasis in areas of low physiological copper uptake. In addition to copper modulation therapy with copper chelators, short-interference RNA specific for human copper transporter 1 (hCtr1) may be used to suppress growth of HCC by blocking increased copper uptake mediated by hCtr1. Furthermore, altered copper metabolism is a promising target for radionuclide therapy of HCC using therapeutic copper radionuclides. Copper metabolism has potential as a new theranostic biomarker for molecular imaging as well as targeted therapy of HCC. PMID:26755872

  1. Geochemical Approach to Archaeal Ecology: δ13C of GDGTs

    NASA Astrophysics Data System (ADS)

    Lichtin, S.; Warren, C.; Pearson, A.; Pagani, M.

    2015-12-01

    Over the last decade and a half, glycerol dialkyl glycerol tetraethers (GDGTs) have increasingly been used to reconstruct environmental temperatures; proxies like TEX86 that correlate the relative abundance of these archaeal cell membrane lipids to sea surface temperature are omnipresent in paleoclimatology literature. While it has become common to make claims about past temperatures using GDGTs, our present understanding of the organisms that synthesize the compounds is still quite limited. The generally accepted theory states that microorganisms like the Thaumarchaeota modify the structure of membrane lipids to increase intermolecular interactions, strengthening the membrane at higher temperatures. Yet to date, culture experiments have been largely restricted to a single species, Nitrosopumilus maritimes, and recent studies on oceanic archaeal rRNA have revealed that these biomarkers are produced in diverse, heterogeneous, and site-specific communities. This brings up questions as to whether different subclasses of GDGTs, and all subsequent proxies, represent adaptation within a single organismal group or a shift in community composition. To investigate whether GDGTs with different chain structures, from the simple isoprenoidal GDGT-0 to Crenarchaeol with its many cyclopentane groups, are sourced from archaea with similar or disparate metabolic pathways—and if that information is inherited in GDGTs trapped in marine sediments—this study examines the stable carbon isotope values (δ13C) of GDGTs extracted from the uppermost meters of sediment in the Orca Basin, Gulf of Mexico, using spooling-wire microcombustion isotope-ratio mass spectrometer (SWiM-IRMS), tackling a fundamental assumption of the TEX86 proxy that influences the way we perceive the veracity of existing temperature records.

  2. Metabolic PET Imaging in Cancer Detection and Therapy Response

    PubMed Central

    Zhu, Aizhi; Lee, Daniel; Shim, Hyunsuk

    2010-01-01

    Positron emission tomography (PET) is a noninvasive imaging technique that provides a functional or metabolic assessment of normal tissue or disease conditions. 18F-fluorodeoxyglucose PET imaging (FDG-PET) is widely used clinically for tumor imaging due to increased glucose metabolism in most types of tumors, and has been shown to improve the diagnosis and subsequent treatment of cancers. In this chapter, we review its use in cancer diagnosis, staging, restaging, and assessment of response to treatment. In addition, other metabolic PET imaging agents in research or clinical trial stages are discussed, including amino acid analogs based on increased protein synthesis, and choline, which is based on increased membrane lipid synthesis. Amino acid analogs and choline are more specific to tumor cells than FDG, so they play an important role in differentiating cancers from benign conditions and in the diagnosis of cancers with low FDG uptake or high background FDG uptake. For decades, researchers have shown that tumors have altered metabolic profiles and display elevated uptake of glucose, amino acids, and lipids, which can be used for cancer diagnosis and monitoring of the therapeutic response with excellent signal-to-noise ratios. PMID:21362516

  3. Constraining 3-PG with a new δ13C submodel: a test using the δ13C of tree rings.

    PubMed

    Wei, Liang; Marshall, John D; Link, Timothy E; Kavanagh, Kathleen L; DU, Enhao; Pangle, Robert E; Gag, Peter J; Ubierna, Nerea

    2014-01-01

    A semi-mechanistic forest growth model, 3-PG (Physiological Principles Predicting Growth), was extended to calculate δ(13)C in tree rings. The δ(13)C estimates were based on the model's existing description of carbon assimilation and canopy conductance. The model was tested in two ~80-year-old natural stands of Abies grandis (grand fir) in northern Idaho. We used as many independent measurements as possible to parameterize the model. Measured parameters included quantum yield, specific leaf area, soil water content and litterfall rate. Predictions were compared with measurements of transpiration by sap flux, stem biomass, tree diameter growth, leaf area index and δ(13)C. Sensitivity analysis showed that the model's predictions of δ(13)C were sensitive to key parameters controlling carbon assimilation and canopy conductance, which would have allowed it to fail had the model been parameterized or programmed incorrectly. Instead, the simulated δ(13)C of tree rings was no different from measurements (P > 0.05). The δ(13)C submodel provides a convenient means of constraining parameter space and avoiding model artefacts. This δ(13)C test may be applied to any forest growth model that includes realistic simulations of carbon assimilation and transpiration.

  4. Synthesis of Site-Specifically (13)C Labeled Linoleic Acids.

    PubMed

    Offenbacher, Adam R; Zhu, Hui; Klinman, Judith P

    2016-10-12

    Soybean lipoxygenase-1 (SLO-1) catalyzes the C-H abstraction from the reactive carbon (C-11) in linoleic acid as the first and rate-determining step in the formation of alkylhydroperoxides. While previous labeling strategies have focused on deuterium labeling to ascertain the primary and secondary kinetic isotope effects for this reaction, there is an emerging interest and need for selectively enriched (13)C isotopologues. In this report, we present synthetic strategies for site-specific (13)C labeled linoleic acid substrates. We take advantage of a Corey-Fuchs formyl to terminal (13)C-labeled alkyne conversion, using (13)CBr4 as the labeling source, to reduce the number of steps from a previous fatty acid (13)C synthetic labeling approach. The labeled linoleic acid substrates are useful as nuclear tunneling markers and for extracting active site geometries of the enzyme-substrate complex in lipoxygenase.

  5. Anomalous 13C enrichment in modern marine organic carbon

    USGS Publications Warehouse

    Arthur, M.A.; Dean, W.E.; Claypool, G.E.

    1985-01-01

    Marine organic carbon is heavier isotopically (13C enriched) than most land-plant or terrestrial organic C1. Accordingly, ??13C values of organic C in modern marine sediments are routinely interpreted in terms of the relative proportions of marine and terrestrial sources of the preserved organic matter2,3. When independent geochemical techniques are used to evaluate the source of organic matter in Cretaceous or older rocks, those rocks containing mostly marine organic C are found typically to have lighter (more-negative) ??13C values than rocks containing mostly terrestrial organic C. Here we conclude that marine photosynthesis in mid-Cretaceous and earlier oceans generally resulted in a greater fractionation of C isotopes and produced organic C having lighter ??13C values. Modern marine photosynthesis may be occurring under unusual geological conditions (higher oceanic primary production rates, lower PCO2) that limit dissolved CO2 availability and minimize carbon isotope fractionation4. ?? 1985 Nature Publishing Group.

  6. Biosynthetic production of universally (13)C-labelled polyunsaturated fatty acids as reference materials for natural health product research.

    PubMed

    Le, Phuong Mai; Fraser, Catherine; Gardner, Graeme; Liang, Wei-Wan; Kralovec, Jaroslav A; Cunnane, Stephen C; Windust, Anthony J

    2007-09-01

    Long-chain polyunsaturated fatty acids (LCPUFA) including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have become important natural health products with numerous proven benefits related to brain function and cardiovascular health. Not only are omega-3 fatty acids available in a plethora of dietary supplements, but they are also increasingly being incorporated as triglycerides into conventional foods, including bread, milk, yoghurt and confectionaries. Recently, transgenic oil seed crops and livestock have been developed that enhance omega-3 fatty acid content. This diverse array of matrices presents a difficult analytical challenge and is compounded further by samples generated through clinical research. Stable isotope (13)C-labelled LCPUFA standards offer many advantages as research tools because they may be distinguished from their naturally abundant counterparts by mass spectrometry and directly incorporated as internal standards into analytical procedures. Further, (13)C-labelled LCPUFAs are safe to use as metabolic tracers to study uptake and metabolism in humans. Currently, (13)C-labelled LCPUFAs are expensive, available in limited supply and not in triglyceride form. To resolve these issues, marine heterotrophic microorganisms are being isolated and screened for LCPUFA production with a view to the efficient biosynthetic production of U-(13)C-labelled fatty acids using U-(13)C glucose as a carbon source. Of 37 isolates obtained, most were thraustochytrids, and either DHA or omega-6 docosapentaenoic acid (22:5n-6) were produced as the major LCPUFA. The marine protist Hyalochlorella marina was identified as a novel source of EPA and omega-3 docosapentaenoic acid (22:5n-3). As proof of principle, gram-level production of (13)C-labelled DHA has been achieved with high chemical purity ( >99%) and high (13)C incorporation levels (>90%), as confirmed by NMR and MS analyses. Finally, U-(13)C-DHA was enzymatically re-esterified to

  7. Assessing Oxidative Stress in Tumors by Measuring the Rate of Hyperpolarized [1-13C]Dehydroascorbic Acid Reduction Using 13C Magnetic Resonance Spectroscopy*

    PubMed Central

    Timm, Kerstin N.; Hu, De-En; Williams, Michael; Wright, Alan J.; Kettunen, Mikko I.; Kennedy, Brett W. C.; Larkin, Timothy J.; Dzien, Piotr; Marco-Rius, Irene; Bohndiek, Sarah E.; Brindle, Kevin M.

    2017-01-01

    Rapid cancer cell proliferation promotes the production of reducing equivalents, which counteract the effects of relatively high levels of reactive oxygen species. Reactive oxygen species levels increase in response to chemotherapy and cell death, whereas an increase in antioxidant capacity can confer resistance to chemotherapy and is associated with an aggressive tumor phenotype. The pentose phosphate pathway is a major site of NADPH production in the cell, which is used to maintain the main intracellular antioxidant, glutathione, in its reduced state. Previous studies have shown that the rate of hyperpolarized [1-13C]dehydroascorbic acid (DHA) reduction, which can be measured in vivo using non-invasive 13C magnetic resonance spectroscopic imaging, is increased in tumors and that this is correlated with the levels of reduced glutathione. We show here that the rate of hyperpolarized [1-13C]DHA reduction is increased in tumors that have been oxidatively prestressed by depleting the glutathione pool by buthionine sulfoximine treatment. This increase was associated with a corresponding increase in pentose phosphate pathway flux, assessed using 13C-labeled glucose, and an increase in glutaredoxin activity, which catalyzes the glutathione-dependent reduction of DHA. These results show that the rate of DHA reduction depends not only on the level of reduced glutathione, but also on the rate of NADPH production, contradicting the conclusions of some previous studies. Hyperpolarized [1-13C]DHA can be used, therefore, to assess the capacity of tumor cells to resist oxidative stress in vivo. However, DHA administration resulted in transient respiratory arrest and cardiac depression, which may prevent translation to the clinic. PMID:27994059

  8. Metabolic Imaging Biomarkers of Postradiotherapy Xerostomia

    SciTech Connect

    Cannon, Blake; Schwartz, David L.; Dong Lei

    2012-08-01

    Purpose: Xerostomia is a major complication of head and neck radiotherapy (RT). Available xerostomia measures remain flawed. [{sup 18}F]fluorodeoxyglucose-labeled positron emission tomography-computed tomography (FDG-PET-CT) is routinely used for staging and response assessment of head and neck cancer. We investigated quantitative measurement of parotid gland FDG uptake as a potential biomarker for post-RT xerostomia. Methods and Materials: Ninety-eight locally advanced head and neck cancer patients receiving definitive RT underwent baseline and post-RT FDG-PET-CT on a prospective imaging trial. A separate validation cohort of 14 patients underwent identical imaging while prospectively enrolled in a second trial collecting sialometry and patient-reported outcomes. Radiation dose and pre- and post-RT standard uptake values (SUVs) for all voxels contained within parotid gland ROI were deformably registered. Results: Average whole-gland or voxel-by-voxel models incorporating parotid D{sub Met} (defined as the pretreatment parotid SUV weighted by dose) accurately predicted posttreatment changes in parotid FDG uptake (e.g., fractional parotid SUV). Fractional loss of parotid FDG uptake closely paralleled early parotid toxicity defined by posttreatment salivary output (p < 0.01) and Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer xerostomia scores (p < 0.01). Conclusions: In this pilot series, loss of parotid FDG uptake was strongly associated with acute clinical post-RT parotid toxicity. D{sub Met} may potentially be used to guide function-sparing treatment planning. Prospective validation of FDG-PET-CT as a convenient, quantifiable imaging biomarker of parotid function is warranted and ongoing.

  9. /sup 13/C nuclear magnetic resonance studies of the biosynthesis by Microbacterium ammoniaphilum of L-glutamate selectively enriched with carbon-13

    SciTech Connect

    Walker, T.E.; Han, C.H.; Kollman, V.H.; London, R.E.; Matwiyoff, N.A.

    1982-02-10

    /sup 13/C NMR of isotopically enriched metabolites has been used to study the metabolism of Microbacterium ammoniaphilum, a bacterium which excretes large quantities of L-glutamic acid into the medium. Biosynthesis from 90% (1-/sup 13/C) glucose results in relatively high specificity of the label, with (2,4-/sup 13/C/sub 2/) glutamate as the major product. The predominant biosynthetic pathway for synthesis of glutamate from glucose was determined to be the Embden Meyerhof glycolytic pathway followed by P-enolpyruvate carboxylase and the first third of the Krebs cycle. Different metabolic pathways are associated with different correlations in the enrichment of the carbons, reflected in the spectrum as different /sup 13/C-/sup 13/C scalar multiplet intensities. Hence, intensity and /sup 13/C-/sup 13/C multiplet analysis allows quantitation of the pathways involved. Although blockage of the Krebs cycle at the ..cap alpha..-ketoglutarate dehydrogenase step is the basis for the accumulation of glutamate, significant Krebs cycle activity was found in glucose grown cells, and extensive Krebs cycle activity in cells metabolizing (1-/sup 13/C) acetate. In addition to the observation of the expected metabolites, the disaccharide ..cap alpha..,..cap alpha..-trehalose and ..cap alpha..,..beta..-glucosylamine were identified from the /sup 13/C NMR spectra.

  10. Computational Platform for Flux Analysis Using 13C-Label Tracing- Phase I SBIR Final Report

    SciTech Connect

    Van Dien, Stephen J.

    2005-04-12

    Isotopic label tracing is a powerful experimental technique that can be combined with metabolic models to quantify metabolic fluxes in an organism under a particular set of growth conditions. In this work we constructed a genome-scale metabolic model of Methylobacterium extorquens, a facultative methylotroph with potential application in the production of useful chemicals from methanol. A series of labeling experiments were performed using 13C-methanol, and the resulting distribution of labeled carbon in the proteinogenic amino acids was determined by mass spectrometry. Algorithms were developed to analyze this data in context of the metabolic model, yielding flux distributions for wild-type and several engineered strains of M. extorquens. These fluxes were compared to those predicted by model simulation alone, and also integrated with microarray data to give an improved understanding of the metabolic physiology of this organism.

  11. Longitudinal optical imaging of tumor metabolism and hemodynamics

    NASA Astrophysics Data System (ADS)

    Skala, Melissa C.; Fontanella, Andrew; Lan, Lan; Izatt, Joseph A.; Dewhirst, Mark W.

    2010-01-01

    An important feature of tumor hypoxia is its temporal instability, or ``cycling hypoxia.'' The primary consequence of cycling hypoxia is increased tumor aggressiveness and treatment resistance beyond that of chronic hypoxia. Longitudinal imaging of tumor metabolic demand, hemoglobin oxygen saturation, and blood flow would provide valuable insight into the mechanisms and distribution of cycling hypoxia in tumors. Fluorescence imaging of metabolic demand via the optical redox ratio (fluorescence intensity of FAD/NADH), absorption microscopy of hemoglobin oxygen saturation, and Doppler optical coherence tomography of vessel morphology and blood flow are combined to noninvasively monitor changes in oxygen supply and demand in the mouse dorsal skin fold window chamber tumor model (human squamous cell carcinoma) every 6 h for 36 h. Biomarkers for metabolic demand, blood oxygenation, and blood flow are all found to significantly change with time (p<0.05). These variations in oxygen supply and demand are superimposed on a significant (p<0.05) decline in metabolic demand with distance from the nearest vessel in tumors (this gradient was not observed in normal tissues). Significant (p<0.05), but weak (r<=0.5) correlations are found between the hemoglobin oxygen saturation, blood flow, and redox ratio. These results indicate that cycling hypoxia depends on both oxygen supply and demand, and that noninvasive optical imaging could be a valuable tool to study therapeutic strategies to mitigate cycling hypoxia, thus increasing the effectiveness of radiation and chemotherapy.

  12. Increased tricarboxylic acid cycle flux in rat brain during forepaw stimulation detected with 1H[13C]NMR.

    PubMed Central

    Hyder, F; Chase, J R; Behar, K L; Mason, G F; Siddeek, M; Rothman, D L; Shulman, R G

    1996-01-01

    NMR spectroscopy was used to test recent proposals that the additional energy required for brain activation is provided through nonoxidative glycolysis. Using localized NMR spectroscopic methods, the rate of C4-glutamate isotopic turnover from infused [1-(13)C]glucose was measured in the somatosensory cortex of rat brain both at rest and during forepaw stimulation. Analysis of the glutamate turnover data using a mathematical model of cerebral glucose metabolism showed that the tricarboxylic acid cycle flux [(V(TCA)] increased from 0.49 +/- 0.03 at rest to 1.48 +/- 0.82 micromol/g/min during stimulation (P < 0.01). The minimum fraction of C4-glutamate derived from C1-glucose was approximately 75%, and this fraction was found in both the resting and stimulated rats. Hence, the percentage increase in oxidative cerebral metabolic rate of glucose use (CMRglc) equals the percentage increases in V(TCA) and cerebral metabolic rate of oxygen consumption (CMRO2). Comparison with previous work for the same rat model, which measured total CMRglc [Ueki, M., Linn, F. & Hossman, K. A. (1988) J. Cereb. Blood Flow Metab. 8, 486-4941, indicates that oxidative CMRglc supplies the majority of energy during sustained brain activation. Images Fig. 2 PMID:8755523

  13. 13C and 1H Nuclear Magnetic Resonance Study of Glycogen Futile Cycling in Strains of the Genus Fibrobacter

    PubMed Central

    Matheron, Christelle; Delort, Anne-Marie; Gaudet, Geneviève; Forano, Evelyne; Liptaj, Tibor

    1998-01-01

    We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. 13C nuclear magnetic resonance (NMR) analysis of [1-13C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by 13C NMR by following the simultaneous metabolism of labeled [13C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH2 of succinate and the CH3 of acetate when the resting cells were metabolizing [1-13C]glucose and unlabeled glycogen were precisely quantified by using 13C-filtered spin-echo difference 1H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics. PMID:12033219

  14. Constraining riverine δ13C-DIC using Late Cretaceous and Early Paleogene freshwater bivalve mollusks (Unionoidea) form Montana

    NASA Astrophysics Data System (ADS)

    Gillikin, D. P.; Goodwin, D. H.; Davidson, M.; Hartman, J.

    2014-12-01

    Interpretation of carbon isotope variation in freshwater unionoid mollusk shells (δ13CSHELL) is not straightforward because of the variable contributions of metabolic (i.e., food) and dissolved inorganic carbon (DIC). Bivalve shells typically contain between 0 and 50% metabolic carbon (CM), which has a δ13C value close to the animal's food source. In marine systems, the food source (usually phytoplankton) has a δ13C value typically around -20 ‰ and a d13CDIC value around ~0 ‰. In freshwater systems, these numbers can vary considerably, with food sources ranging from -35 to -10 ‰. Typically, δ13C-DIC values range between -25 to 0‰ and are dependent on numerous factors; carbonate weathering and equilibrium with the atmosphere typically leading to high values and respiration of organic matter and oxidation of methane leading to lower values. Therefore, δ13C-DIC values reflect numerous processes occurring in the watershed. Nevertheless, here we suggest δ13CSHELL values can constrain the lower bounds of riverine δ13C-DIC values, despite the influence of CM. The metabolic end-member δ13C value is typically lower than the DIC end member and consequently will lead to higher calculated δ13C-DIC when using δ13CSHELL values. Therefore, if the CM fraction is set to 0 %, δ13CSHELL values will provide the lowest possible riverine δ13C-DIC values (after accounting for fractionation). Applying this method to modern shells from waters with known δ13C-DIC values (ranging from -3.2 to -12.8 ‰) results in calculated δ13C-DIC values from -6.0 to -12.4 ‰, which is close to measured DIC data from the waters in which the mussel grew. This can then in turn be applied to well-preserved fossil shells. Freshwater unionoid shells from the uppermost Cretaceous Hell Creek Formation and the lower Paleogene Fort Union Formation are exceptionally well preserved. Applying this method to these shells results in δ13C-DIC values ranging from -6 to -11‰, which is consistent

  15. Synthesis and applications of {sup 13}C glycerol

    SciTech Connect

    Stocking, E.; Khalsa, O.; Martinez, R.A.; Silks, L.A. III

    1994-12-01

    Due in part to the use of labeled glycerol for the {sup 13}C enrichment of biomolecules, we are currently developing new synthetic routes to various isotopomers of glycerol. Judging from our experience, traditional methods of glycerol synthesis are not easily adapted for isotopic enrichment and/or have poor overall yields (12 to 15%). Furthermore, the use of glycerol for enrichment can be prohibitively expensive and its availability is limited by the level of demand. We are presently developing a short de novo synthesis of glycerol from carbon dioxide ({approximately}53% overall yield for four steps) and are examining the feasibility of synthesizing site-specific {sup 13}C-labeled glycerol and dihydroxyacetone (DHA) from labeled methanol and carbon dioxide. One application of {sup 13}C glycerol we have examined is enzymatic conversion of glycerol to glyceraldehyde-3-monophosphate or dihydroxyacetone monophosphate (DHAP) with yields ranging from 25 to 50% (as determined by NMR spectroscopy). We are also pursuing the chemical conversion of {sup 13}C-labeled DHA to DHAP. We are especially interested in {sup 13}C-labeled DHAP because we are investigating its use as a chemo-enzymatic precursor for both labeled 2-deoxyribose and 2-deoxyribonucleic acids.

  16. Impact of Ho3+-doping on 13C dynamic nuclear polarization using trityl OX063 free radical

    PubMed Central

    Kiswandhi, Andhika; Niedbalski, Peter; Parish, Christopher; Kaur, Pavanjeet; Martins, André; Fidelino, Leila; Khemtong, Chalermchai; Song, Likai; Sherry, A. Dean

    2016-01-01

    We have investigated the effects of Ho-DOTA doping on the dynamic nuclear polarization (DNP) of [1-13C] sodium acetate using trityl OX063 free radical at 3.35 T and 1.2 K. Our results indicate that addition of 2 mM Ho-DOTA on 3 M [1-13C] sodium acetate sample in 1:1 v/v glycerol:water with 15 mM trityl OX063 improves the DNP-enhanced 13C solid-state nuclear polarization by a factor of around 2.7-fold. Similar to the Gd3+ doping effect on 13C DNP, the locations of the positive and negative 13C maximum polarization peaks in the 13C microwave DNP sweep are shifted towards each other with the addition of Ho-DOTA on the DNP sample. W-band electron spin resonance (ESR) studies have revealed that while the shape and linewidth of the trityl OX063 ESR spectrum was not affected by Ho3+-doping, the electron spin-lattice relaxation time T1 of trityl OX063 was prominently reduced at cryogenic temperatures. The reduction of trityl OX063 electron T1 by Ho-doping is linked to the 13C DNP improvement in light of the thermodynamic picture of DNP. Moreover, the presence of Ho-DOTA in the dissolution liquid at room temperature has negligible reduction effect on liquid-state 13C T1, in contrast to Gd3+-doping which drastically reduces the 13C T1. The results here suggest that Ho3+-doping is advantageous over Gd3+ in terms of preservation of hyperpolarized state—an important aspect to consider for in vitro and in vivo NMR or imaging (MRI) experiments where a considerable preparation time is needed to administer the hyperpolarized 13C liquid. PMID:27424954

  17. Imaging enzymes at work: metabolic mapping by enzyme histochemistry.

    PubMed

    Van Noorden, Cornelis J F

    2010-06-01

    For the understanding of functions of proteins in biological and pathological processes, reporter molecules such as fluorescent proteins have become indispensable tools for visualizing the location of these proteins in intact animals, tissues, and cells. For enzymes, imaging their activity also provides information on their function or functions, which does not necessarily correlate with their location. Metabolic mapping enables imaging of activity of enzymes. The enzyme under study forms a reaction product that is fluorescent or colored by conversion of either a fluorogenic or chromogenic substrate or a fluorescent substrate with different spectral characteristics. Most chromogenic staining methods were developed in the latter half of the twentieth century but still find new applications in modern cell biology and pathology. Fluorescence methods have rapidly evolved during the last decade. This review critically evaluates the methods that are available at present for metabolic mapping in living animals, unfixed cryostat sections of tissues, and living cells, and refers to protocols of the methods of choice.

  18. Metabolic Imaging of Head and Neck Cancer Organoids

    PubMed Central

    Shah, Amy T.; Heaster, Tiffany M.; Skala, Melissa C.

    2017-01-01

    Head and neck cancer patients suffer from toxicities, morbidities, and mortalities, and these ailments could be minimized through improved therapies. Drug discovery is a long, expensive, and complex process, so optimized assays can improve the success rate of drug candidates. This study applies optical imaging of cell metabolism to three-dimensional in vitro cultures of head and neck cancer grown from primary tumor tissue (organoids). This technique is advantageous because it measures cell metabolism using intrinsic fluorescence from NAD(P)H and FAD on a single cell level for a three-dimensional in vitro model. Head and neck cancer organoids are characterized alone and after treatment with standard therapies, including an antibody therapy, a chemotherapy, and combination therapy. Additionally, organoid cellular heterogeneity is analyzed quantitatively and qualitatively. Gold standard measures of treatment response, including cell proliferation, cell death, and in vivo tumor volume, validate therapeutic efficacy for each treatment group in a parallel study. Results indicate that optical metabolic imaging is sensitive to therapeutic response in organoids after 1 day of treatment (p<0.05) and resolves cell subpopulations with distinct metabolic phenotypes. Ultimately, this platform could provide a sensitive high-throughput assay to streamline the drug discovery process for head and neck cancer. PMID:28099487

  19. {sup 13}C relaxation in an RNA hairpin

    SciTech Connect

    King, G.C. |; Akratos, C.; Xi, Z.; Michnica, M.J.

    1994-12-01

    This initial survey of {sup 13}C relaxation in the {triangle}TAR RNA element has generated a number of interesting results that should prove generally useful for future studies. The most readily comparable study in the literature monitored {sup 13}C relaxation of the methyl groups from unusual bases in tRNA{sup Phe}. The study, which used T{sub 1} and NOE data only, reported order parameters for the methyl group axis that ranged between 0.51 and 0.97-a range similar to that observed here. However, they reported a breakdown of the standard order parameter analysis at higher (118-MHz {sup 13}C) frequencies, which should serve to emphasize the need for a thorough exploration of suitable motional models.

  20. A 13C mass isotopomer study of anaplerotic pyruvate carboxylation in perfused rat hearts.

    PubMed

    Comte, B; Vincent, G; Bouchard, B; Jetté, M; Cordeau, S; Rosiers, C D

    1997-10-17

    Anaplerotic pyruvate carboxylation was examined in hearts perfused with physiological concentrations of glucose, [U-13C3]lactate, and [U-13C3]pyruvate. Also, a fatty acid, [1-13C]octanoate, or ketone bodies were added at concentrations providing acetyl-CoA at a rate resulting in either low or substantial pyruvate decarboxylation. Relative contributions of pyruvate and fatty acids to citrate synthesis were determined from the 13C labeling pattern of effluent citrate by gas chromatography-mass spectrometry (see companion article, Comte, B., Vincent, G., Bouchard, B., and Des Rosiers, C. (1997) J. Biol. Chem. 272, 26117-26124). Precision on flux measurements of anaplerotic pyruvate carboxylation depended on the mix of substrates supplied to the heart. Anaplerotic fluxes were precisely determined under conditions where acetyl-CoA was predominantly supplied by beta-oxidation, as it occurred with 0.2 or 1 mM octanoate. Then, anaplerotic pyruvate carboxylation provided 3-8% of the OAA moiety of citrate and was modulated by concentrations of lactate and pyruvate in the physiological range. Also, the contribution of pyruvate to citrate formation through carboxylation was equal to or greater than through decarboxylation. Furthermore, 13C labeling data on tissue citric acid cycle intermediates and pyruvate suggest that (i) anaplerosis occurs also at succinate and (ii) cataplerotic malate decarboxylation is low. Rather, the presence of citrate in the effluent perfusate of hearts perfused with physiological concentrations of glucose, lactate, and pyruvate and concentrations of octanoate leading to maximal oxidative rates suggests a cataplerotic citrate efflux from mitochondria to cytosol. Taken altogether, our data raise the possibility of a link between pyruvate carboxylation and mitochondrial citrate efflux. In view of the proposed feedback regulation of glycolysis by cytosolic citrate, such a link would support a role of anaplerosis and cataplerosis in metabolic signal

  1. Influence of 13C isotopic labeling location of 13C DNP of acetate using TEMPO free radical

    NASA Astrophysics Data System (ADS)

    Parish, Christopher; Niedbalski, Peter; Lumata, Lloyd

    2015-03-01

    Dynamic nuclear polarization (DNP) via the dissolution method enhances the liquid-state magnetic resonance (NMR or MRI) signals of insensitive nuclear spins by at least 10,000-fold. The basis for all these signal enhancements at room temperature is the polarization transfer from the electrons to nuclear spins at cryogenic temperature and high magnetic field. In this work, we have studied the influence of the location of 13C isotopic labeling on the DNP of sodium acetate at 3.35 T and 1.4 K using a wide ESR linewidth free radical 4-oxo-TEMPO. The carbonyl [1-13C]acetate spins produced a polarization level that is almost twice that of the methyl [2-13C]acetate spins. On the other hand, the polarization of the methyl 13C spins doubled to reach the level of [1-13C]acetate when the methyl group was deuterated. Meanwhile, the solid-state nuclear relaxation of these samples are the same and do not correlate with the polarization levels. These behavior implies that the nuclear relaxation for these samples is dominated by the contribution from the free radicals and the polarization levels can be explained by a thermodynamic picture of DNP.

  2. Structure of uniaxially aligned 13C labeled silk fibroin fibers with solid state 13C-NMR

    NASA Astrophysics Data System (ADS)

    Demura, Makoto; Yamazaki, Yasunobu; Asakura, Tetsuo; Ogawa, Katsuaki

    1998-01-01

    Carbon-13 isotopic labeling of B. mori silk fibroin was achieved biosynthetically with [1- 13C] glycine in order to determine the carbonyl bond orientation angle of glycine sites with the silk fibroin. Angular dependence of 13C solid state NMR spectra of uniaxially oriented silk fibroin fiber block sample due to the carbonyl 13C chemical shift anisotropy was simulated according to the chemical shift transformation with Euler angles, αF and βF, from principal axis system (PAS) to fiber axis system (FAS). The another Euler angles, αDCO and βDCO, for transformation from PAS to the molecular symmetry axis were determined from the [1- 13C] glycine sequence model compounds for the silk fibroin. By the combination of these Euler angles, the carbonyl bond orientation angle with respect to FAS of the [1- 13C] glycine sites of the silk fibroin was determined to be 90 ± 5°. This value is in agreement with the X-ray diffraction and our previous solid state NMR data of B. mori silk fibroin fiber (a typical β-pleated sheet) within experimental error.

  3. Time-gated FLIM microscope for corneal metabolic imaging

    NASA Astrophysics Data System (ADS)

    Silva, Susana F.; Batista, Ana; Domingues, José Paulo; Quadrado, Maria João.; Morgado, António Miguel

    2016-03-01

    Detecting corneal cells metabolic alterations may prove a valuable tool in the early diagnosis of corneal diseases. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent metabolic co-factors that allow the assessment of metabolic changes through non-invasive optical methods. These co-factors exhibit double-exponential fluorescence decays, with well-separated short and lifetime components, which are related to their protein-bound and free-states. Corneal metabolism can be assessed by measuring the relative contributions of these two components. For that purpose, we have developed a wide-field time-gated fluorescence lifetime microscope based on structured illumination and one-photon excitation to record FAD lifetime images from corneas. NADH imaging was not considered as its UV excitation peak is regarded as not safe for in vivo measurements. The microscope relies on a pulsed blue diode laser (λ=443 nm) as excitation source, an ultra-high speed gated image intensifier coupled to a CCD camera to acquire fluorescence signals and a Digital Micromirror Device (DMD) to implement the Structured Illumination technique. The system has a lateral resolution better than 2.4 μm, a field of view of 160 per 120 μm and an optical sectioning of 6.91 +/- 0.45 μm when used with a 40x, 0.75 NA, Water Immersion Objective. With this setup we were able to measure FAD contributions from ex-vivo chicken corneas collected from a local slaughterhouse..

  4. Bolus tracking for improved metabolic imaging of hyperpolarised compounds

    NASA Astrophysics Data System (ADS)

    Durst, Markus; Koellisch, Ulrich; Gringeri, Concetta; Janich, Martin A.; Rancan, Giaime; Frank, Annette; Wiesinger, Florian; Menzel, Marion I.; Haase, Axel; Schulte, Rolf F.

    2014-06-01

    Dynamic nuclear polarisation has enabled real-time metabolic imaging of pyruvate and its metabolites. Conventional imaging sequences rely on predefined settings and do not account for intersubject variations in biological parameters such as perfusion. We present a fully automatic real-time bolus tracking sequence for hyperpolarised substrates which starts the imaging acquisition at a defined point on the bolus curve. This reduces artefacts due to signal change and allows for a more efficient use of hyperpolarised magnetisation. For single time point imaging methods, bolus tracking enables a more reliable and consistent quantification of metabolic activity. An RF excitation with a small flip angle is used to obtain slice-selective pyruvate tracking information in rats. Moreover, in combination with a copolarised urea and pyruvate injection, spectrally selective tracking on urea allows obtaining localised bolus tracking information without depleting the pyruvate signal. Particularly with regard to clinical application, the bolus tracking technique could provide an important step towards a routine assessment protocol which removes operator dependencies and ensures comparable results.

  5. Quantitative 13C NMR characterization of fast pyrolysis oils

    DOE PAGES

    Happs, Renee M.; Lisa, Kristina; Ferrell, III, Jack R.

    2016-10-20

    Quantitative 13C NMR analysis of model catalytic fast pyrolysis (CFP) oils following literature procedures showed poor agreement for aromatic hydrocarbons between NMR measured concentrations and actual composition. Furthermore, modifying integration regions based on DEPT analysis for aromatic carbons resulted in better agreement. Solvent effects were also investigated for hydrotreated CFP oil.

  6. Modeling of the 2007 JET ^13C migration experiments

    NASA Astrophysics Data System (ADS)

    Strachan, J. D.; Likonen, J.; Hakola, A.; Coad, J. P.; Widdowson, A.; Koivuranta, S.; Hole, D. E.; Rubel, M.

    2010-11-01

    Using the last run day of the 2007 JET experimental campaign, ^13CH4 was introduced repeatedly from the vessel top into a single plasma type (H-mode, Ip= 1.6 MA, Bt= 1.6 T). Similar experiments were performed in 2001 (vessel top into L-Mode) and 2004 (outer divertor into H-Mode). Divertor and wall tiles were removed and been analysed using secondary ion mass spectrometry (SIMS) and Rutherford backscattering (RBS) to determine the ^13C migration. ^13C was observed to migrate both to the inner (largest deposit), outer divertor (less) , and the floor tiles (least). This paper reports the EDGE2D/NIMBUS based modelling of the carbon migration. The emphasis is on the comparison of the 2007 results with the 2001 results where both injections were from the machine top but ELMs were present in 2007 but not present in 2001. The ELMs seemed to cause more ^13C re-erosion near the inner strike point. Also of interest is the difference in the Private Flux Region deposits where the changes in divertor geometry between 2004 and 2007 caused differences in the deposits. In 2007, the tilting of the load bearing tile caused regions of the PFR to be shadowed from the inner strike point which were not shadowed in 2004, indicating ^13C neutrals originated from the OSP.

  7. Complete 1H and 13C spectral assignment of floridoside.

    PubMed

    Simon-Colin, Christelle; Kervarec, Nelly; Pichon, Roger; Deslandes, Eric

    2002-02-11

    Floridoside (2-O-alpha-D-galactopyranosylglycerol) was extracted from the red marine alga Rhodymenia palmata, and purified by ion-exchange chromatography: 1D and 2D NMR spectroscopy experiments were used to unambiguously assign the complete 1H and 13C spectra.

  8. Does the Shuram δ13C excursion record Ediacaran oxygenation?

    NASA Astrophysics Data System (ADS)

    Husson, J. M.; Maloof, A. C.; Schoene, B.; Higgins, J. A.

    2013-12-01

    The most negative carbon isotope excursion in Earth history is found in carbonate rocks of the Ediacaran Period (635-542 Ma). Known colloquially as the the 'Shuram' excursion, workers have long noted its tantalizing, broad concordance with the rise of abundant macro-scale fossils in the rock record, variously interpreted as animals, giant protists, macro-algae and lichen, and known as the 'Ediacaran Biota.' Thus, the Shuram excursion has been interpreted by many in the context of a dramatically changing redox state of the Ediacaran oceans - e.g., a result of methane cycling in a low O2 atmosphere, the final destruction of a large pool of recalcitrant dissolved organic carbon (DOC), and the step-wise oxidation of the Ediacaran oceans. More recently, diagenetic interpretations of the Shuram excursion - e.g. sedimentary in-growth of very δ13C depleted authigenic carbonates, meteoric alteration of Ediacaran carbonates, late-stage burial diagenesis - have challenged the various Ediacaran redox models. A rigorous geologic context is required to discriminate between these explanatory models, and determine whether the Shuram excursion can be used to evaluate terminal Neoproterozoic oxygenation. Here, we present chemo-stratigraphic data (δ13C, δ18O, δ44/42Ca and redox sensitive trace element abundances) from 12 measured sections of the Ediacaran-aged Wonoka Formation (Fm.) of South Australia that require a syn-depositional age for the extraordinary range of δ13C values (-12 to +4‰) observed in the formation. In some locations, the Wonoka Fm. is ~700 meters (m) of mixed shelf limestones and siliclastics that record the full 16 ‰ δ13C excursion in a remarkably consistent fashion across 100s of square kilometers of basin area. Fabric-altering diagenesis, where present, occurs at the sub-meter vertical scale, only results in sub-permil offsets in δ13C and cannot be used to explain the full δ13C excursion. In other places, the Wonoka Fm. is host to deep (1 km

  9. Recent Advances in Metabolic Profiling And Imaging of Prostate Cancer

    PubMed Central

    Thapar, Roopa; Titus, Mark A

    2015-01-01

    Cancer is a metabolic disease. Cancer cells, being highly proliferative, show significant alterations in metabolic pathways such as glycolysis, respiration, the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, lipid metabolism, and amino acid metabolism. Metabolites like peptides, nucleotides, products of glycolysis, the TCA cycle, fatty acids, and steroids can be an important read out of disease when characterized in biological samples such as tissues and body fluids like urine, serum, etc. The cancer metabolome has been studied since the 1960s by analytical techniques such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Current research is focused on the identification and validation of biomarkers in the cancer metabolome that can stratify high-risk patients and distinguish between benign and advanced metastatic forms of the disease. In this review, we discuss the current state of prostate cancer metabolomics, the biomarkers that show promise in distinguishing indolent from aggressive forms of the disease, the strengths and limitations of the analytical techniques being employed, and future applications of metabolomics in diagnostic imaging and personalized medicine of prostate cancer. PMID:25632377

  10. Monitoring CO[subscript 2] Fixation Using GC-MS Detection of a [superscript 13]C-Label

    ERIC Educational Resources Information Center

    Hammond, Daniel G.; Bridgham, April; Reichert, Kara; Magers, Martin

    2010-01-01

    Much of our understanding of metabolic pathways has resulted from the use of chemical and isotopic labels. In this experiment, a heavy isotope of carbon, [superscript 13]C, is used to label the product of the well-known RuBisCO enzymatic reaction. This is a key reaction in photosynthesis that converts inorganic carbon to organic carbon; a process…

  11. Synthesis of [1-.sup.13C]pyruvic acid], [2-.sup.13C]pyruvic acid], [3-.sup.13C]pyruvic acid] and combinations thereof

    DOEpatents

    Martinez, Rodolfo A. , Unkefer; Clifford J. , Alvarez; Marc, A [Santa Fe, NM

    2012-06-12

    The present invention is directed to the labeled compounds, ##STR00001## wherein C* is each either .sup.13C and .sup.12C where at least one C* is .sup.13C, each hydrogen of the methylene group is hydrogen or deuterium, the methyl group includes either zero or three deuterium atoms, Q is sulfide, sulfinyl, or sulfone, Z is an aryl group such as 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, or a phenyl group ##STR00002## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each independently either hydrogen, a C.sub.1-C.sub.4 lower alkyl, a halogen, and an amino group such as NH.sub.2, NHR and NRR' where R and R' are each independently either a C.sub.1-C.sub.4 lower alkyl, a phenyl, and an alkoxy group, and the methyl group can include either zero or three deuterium atoms. The present invention is also directed to the labeled compounds ##STR00003##

  12. Synthesis of [1-.sup.13C]pyruvic acid], [2-.sup.13C]pyruvic acid], [3-.sup.13C]pyruvic acid] and combinations thereof

    DOEpatents

    Martinez, Rodolfo A.; Unkefer, Clifford J.; Alvarez, Marc A.

    2009-09-01

    The present invention is directed to labeled compounds, of the formulae ##STR00001## wherein C* is each independently selected from the group consisting of .sup.13C and .sup.12C with the proviso that at least one C* is .sup.13C, each hydrogen of the methylene group can independently be either hydrogen or deuterium, the methyl group includes either zero or three deuterium atoms, Q is from the group of sulfide, sulfinyl, and sulfone, Z is an aryl group from the group of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure ##STR00002## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each independently from the group of hydrogen, a C.sub.1-C.sub.4 lower alkyl, a halogen, and an amino group from the group of NH.sub.2, NHR and NRR' where R and R' are each independently from the group of a C.sub.1-C.sub.4 lower alkyl, a phenyl, and an alkoxy group, and the methyl group can include either zero or three deuterium atoms.

  13. Natural isotope correction of MS/MS measurements for metabolomics and (13)C fluxomics.

    PubMed

    Niedenführ, Sebastian; ten Pierick, Angela; van Dam, Patricia T N; Suarez-Mendez, Camilo A; Nöh, Katharina; Wahl, S Aljoscha

    2016-05-01

    Fluxomics and metabolomics are crucial tools for metabolic engineering and biomedical analysis to determine the in vivo cellular state. Especially, the application of (13)C isotopes allows comprehensive insights into the functional operation of cellular metabolism. Compared to single MS, tandem mass spectrometry (MS/MS) provides more detailed and accurate measurements of the metabolite enrichment patterns (tandem mass isotopomers), increasing the accuracy of metabolite concentration measurements and metabolic flux estimation. MS-type data from isotope labeling experiments is biased by naturally occurring stable isotopes (C, H, N, O, etc.). In particular, GC-MS(/MS) requires derivatization for the usually non-volatile intracellular metabolites introducing additional natural isotopes leading to measurements that do not directly represent the carbon labeling distribution. To make full use of LC- and GC-MS/MS mass isotopomer measurements, the influence of natural isotopes has to be eliminated (corrected). Our correction approach is analyzed for the two most common applications; (13)C fluxomics and isotope dilution mass spectrometry (IDMS) based metabolomics. Natural isotopes can have an impact on the calculated flux distribution which strongly depends on the substrate labeling and the actual flux distribution. Second, we show that in IDMS based metabolomics natural isotopes lead to underestimated concentrations that can and should be corrected with a nonlinear calibration. Our simulations indicate that the correction for natural abundance in isotope based fluxomics and quantitative metabolomics is essential for correct data interpretation.

  14. Temperature-mediated changes in microbial carbon use efficiency and 13C discrimination

    NASA Astrophysics Data System (ADS)

    Lehmeier, C. A.; Ballantyne, F., IV; Min, K.; Billings, S. A.

    2015-10-01

    Understanding how carbon dioxide (CO2) flux from soils feeds back to climate warming depends in part on our ability to quantify the efficiency with which microorganisms convert soil organic carbon (C) into either biomass or CO2. Quantifying ecosystem-level respiratory CO2 losses often also requires assumptions about stable C isotope fractionations associated with the microbial transformation of soil organic substrates. However, the diversity of organic substrates' δ13C and the challenges of measuring microbial C use efficiency (CUE) in soils fundamentally limit our ability to project soil, and thus ecosystem, C budgets in a warming climate. Here, we quantify the effect of temperature on C fluxes during metabolic transformations of cellobiose, a common microbial substrate, by a cosmopolitan soil microorganism growing at a constant rate. Specific respiration rate increased by 250 % between 13 and 26.5 °C, decreasing CUE from 77 to 56 %. Specific respiration rate was positively correlated with an increase in respiratory 13C discrimination from 4.4 to 6.7 ‰ across the same temperature range. This first demonstration of a direct link between temperature, microbial CUE and associated isotope fluxes provides a critical step towards understanding δ13C of respired CO2 at multiple scales, and towards a framework for predicting future soil C fluxes.

  15. Temperature-mediated changes in microbial carbon use efficiency and 13C discrimination

    NASA Astrophysics Data System (ADS)

    Lehmeier, Christoph A.; Ballantyne, Ford, IV; Min, Kyungjin; Billings, Sharon A.

    2016-06-01

    Understanding how carbon dioxide (CO2) flux from ecosystems feeds back to climate warming depends in part on our ability to quantify the efficiency with which microorganisms convert organic carbon (C) into either biomass or CO2. Quantifying ecosystem-level respiratory CO2 losses often also requires assumptions about stable C isotope fractionations associated with the microbial transformation of organic substrates. However, the diversity of organic substrates' δ13C and the challenges of measuring microbial C use efficiency (CUE) in their natural environment fundamentally limit our ability to project ecosystem C budgets in a warming climate. Here, we quantify the effect of temperature on C fluxes during metabolic transformations of cellobiose, a common microbial substrate, by a cosmopolitan microorganism growing at a constant rate. Biomass C specific respiration rate increased by 250 % between 13 and 26.5 °C, decreasing CUE from 77 to 56 %. Biomass C specific respiration rate was positively correlated with an increase in respiratory 13C discrimination from 4.4 to 6.7 ‰ across the same temperature range. This first demonstration of a direct link between temperature, microbial CUE, and associated isotope fluxes provides a critical step towards understanding δ13C of respired CO2 at multiple scales, and towards a framework for predicting future ecosystem C fluxes.

  16. Optimization of 13C dynamic nuclear polarization: isotopic labeling of free radicals

    NASA Astrophysics Data System (ADS)

    Niedbalski, Peter; Parish, Christopher; Kiswandi, Andhika; Lumata, Lloyd

    Dynamic nuclear polarization (DNP) is a physics technique that amplifies the nuclear magnetic resonance (NMR) signals by transferring the high polarization of the electrons to the nuclear spins. Thus, the choice of free radical is crucial in DNP as it can directly affect the NMR signal enhancement levels, typically on the order of several thousand-fold in the liquid-state. In this study, we have investigated the efficiency of four variants of the well-known 4-oxo-TEMPO radical (normal 4-oxo-TEMPO plus its 15N-enriched and/or perdeuterated variants) for use in DNP of an important metabolic tracer [1-13C]acetate. Though the variants have significant differences in electron paramagnetic resonance (EPR) spectra, we have found that changing the composition of the TEMPO radical through deuteration or 15N doping yields no significant difference in 13C DNP efficiency at 3.35 T and 1.2 K. On the other hand, deuteration of the solvent causes a significant increase of 13C polarization that is consistent over all the 4-oxo-TEMPO variants. These findings are consistent with the thermal mixing model of DNP. This work is supported by US Dept of Defense Award No. W81XWH-14-1-0048 and the Robert A. Welch Foundation Grant No. AT-1877.

  17. Cardiovascular Metabolic Imaging: Physiologic and Biochemical Dynamics In Vivo

    PubMed Central

    McMillin-Wood, Jeanie B.; Bassingthwaighte, James B.

    2010-01-01

    Fifty-eight investigators from the fields of biochemistry, physiology, cardiology, nuclear medicine, and physics met to discuss the development of metabolic imaging techniques for application to cardiovascular and pulmonary studies in health and disease. The workshop was sponsored by the Division of Heart and Vascular Diseases, National Heart, Lung, and Blood Institute and was held on September 16 to 18 in Bethesda, Maryland, in facilities provided by the American College of Cardiology. This report summarizes the presentations and discussions and presents recommendations for future studies. PMID:3902281

  18. Cardiovascular Metabolic Imaging: Physiologic and Biochemical Dynamics In Vivo

    PubMed Central

    Bassingthwaighte, James B.; McMillin-Wood, Jeanie B.; Brown, Truman R.; Budinger, Thomas F.; Ingwall, Joanne S.; Rovetto, Michael J.; Schelbert, Heinrich R.; McCallum, Zena

    2010-01-01

    Fifty-eight investigators from the fields of biochemistry, physiology, cardiology, nuclear medicine, and physics met to discuss the development of metabolic imaging techniques for application to cardiovascular and pulmonary studies in health and disease. The workshop was sponsored by the Division of Heart and Vascular Diseases, National Heart, Lung, and Blood Institute and was held on September 16 to 18 in Bethesda, Maryland, in facilities provided by the American College of Cardiology. This report summarizes the presentations and discussions and presents recommendations for future studies. PMID:3876891

  19. 13C NMR of Nephila clavipes major ampullate silk gland.

    PubMed

    Hijirida, D H; Do, K G; Michal, C; Wong, S; Zax, D; Jelinski, L W

    1996-12-01

    The major ampullate glands of the spider Nephila clavipes contain approximately 0.2 microliter each of a highly concentrated (approximately 50%) solution of silk fibroin. Therefore, the reservoir of silk in these glands presents an ideal opportunity to observe prefolded conformations of a protein in its native state. To this end, the structure and conformation of major ampullate gland silk fibroin within the glands of the spider N. clavipes were examined by 13C NMR spectroscopy. These results were compared to those from silk protein first drawn from the spinneret and then denatured. The 13C NMR chemical shifts, along with infrared and circular dichroism data, suggest that the silk fibroin in the glands exists in dynamically averaged helical conformations. Furthermore, there is no evidence of proline residues in U-(13)C-D-glucose-labeled silk. This transient prefolded "molten fibril" state may correspond to the silk I form found in Bombyx mori silk. There is no evidence of the final beta-sheet structure in the ampullate gland silk fibroin before final silk processing. However, the conformation of silk in the glands appears to be in a highly metastable state, as plasticization with water produces the beta-sheet structure. Therefore, the ducts connecting the ampullate glands to the spinnerets play a larger role in silk processing than previously thought.

  20. Construction and 13C NMR signal-amplification efficiency of a dynamic nuclear polarizer at 6.4 T and 1.4 K

    NASA Astrophysics Data System (ADS)

    Kiswandhi, Andhika; Niedbalski, Peter; Parish, Christopher; Ferguson, Sarah; Taylor, David; McDonald, George; Lumata, Lloyd

    Dissolution dynamic nuclear polarization (DNP) is a rapidly emerging technique in biomedical and metabolic imaging since it amplifies the liquid-state nuclear magnetic resonance (NMR) and imaging (MRI) signals by >10,000-fold. Originally used in nuclear scattering experiments, DNP works by creating a non-Boltzmann nuclear spin distribution by transferring the high electron (γ = 28,000 MHz/T) thermal polarization to the nuclear spins via microwave irradiation of the sample at high magnetic field and low temperature. A dissolution device is used to rapidly dissolve the frozen sample and consequently produces an injectable ``hyperpolarized'' liquid at physiologically-tolerable temperature. Here we report the construction and performance evaluation of a dissolution DNP hyperpolarizer at 6.4 T and 1.4 K using a continuous-flow cryostat. The solid and liquid-state 13C NMR signal enhancement levels of 13C acetate samples doped with trityl OX063 and 4-oxo-TEMPO free radicals will be discussed and compared with the results from the 3.35 T commercial hyperpolarizer. This work is supported by US Dept of Defense Award No. W81XWH-14-1-0048 and Robert A. Welch Foundation Grant No. AT-1877.

  1. Catabolism of Glucose and Lactose in Bifidobacterium animalis subsp. lactis, Studied by 13C Nuclear Magnetic Resonance

    PubMed Central

    González-Rodríguez, Irene; Gaspar, Paula; Sánchez, Borja; Gueimonde, Miguel; Neves, Ana Rute

    2013-01-01

    Bifidobacteria are widely used as probiotics in several commercial products; however, to date there is little knowledge about their carbohydrate metabolic pathways. In this work, we studied the metabolism of glucose and lactose in the widely used probiotic strain Bifidobacterium animalis subsp. lactis BB-12 by in vivo 13C nuclear magnetic resonance (NMR) spectroscopy. The metabolism of [1-13C]glucose was characterized in cells grown in glucose as the sole carbon source. Moreover, the metabolism of lactose specifically labeled with 13C on carbon 1 of the glucose or the galactose moiety was determined in suspensions of cells grown in lactose. These experiments allowed the quantification of some intermediate and end products of the metabolic pathways, as well as determination of the consumption rate of carbon sources. Additionally, the labeling patterns in metabolites derived from the metabolism of glucose specifically labeled with 13C on carbon 1, 2, or 3 in cells grown in glucose or lactose specifically labeled in carbon 1 of the glucose moiety ([1-13Cglucose]lactose), lactose specifically labeled in carbon 1 of the galactose moiety ([1-13Cgalactose]lactose), and [1-13C]glucose in lactose-grown cells were determined in cell extracts by 13C NMR. The NMR analysis showed that the recovery of carbon was fully compatible with the fructose 6-phosphate, or bifid, shunt. The activity of lactate dehydrogenase, acetate kinase, fructose 6-phosphate phosphoketolase, and pyruvate formate lyase differed significantly between glucose and lactose cultures. The transcriptional analysis of several putative glucose and lactose transporters showed a significant induction of Balat_0475 in the presence of lactose, suggesting a role for this protein as a lactose permease. This report provides the first in vivo experimental evidence of the metabolic flux distribution in the catabolic pathway of glucose and lactose in bifidobacteria and shows that the bifid shunt is the only pathway

  2. Millimeter and submillimeter wave spectra of 13C methylamine

    NASA Astrophysics Data System (ADS)

    Motiyenko, R. A.; Margulès, L.; Ilyushin, V. V.; Smirnov, I. A.; Alekseev, E. A.; Halfen, D. T.; Ziurys, L. M.

    2016-03-01

    Context. Methylamine (CH3NH2) is a light molecule of astrophysical interest, which has an intensive rotational spectrum that extends in the submillimeter wave range and far beyond, even at temperatures characteristic for the interstellar medium. It is likely for 13C isotopologue of methylamine to be identified in astronomical surveys, but there is no information available for the 13CH3NH2 millimeter and submillimeter wave spectra. Aims: In this context, to provide reliable predictions of 13CH3NH2 spectrum in millimeter and submillimeter wave ranges, we have studied rotational spectra of the 13C methylamine isotopologue in the frequency range from 48 to 945 GHz. Methods: The spectrum of 13C methylamine was recorded using conventional absorption spectrometers. The analysis of the rotational spectrum of 13C methylamine in the ground vibrational state was performed on the basis of the group-theoretical high-barrier tunneling Hamiltonian that was developed for methylamine. The available multiple observations of the parent methylamine species toward Sgr B2(N) at 1, 2, and 3 mm using the Submillimeter Telescope and the 12 m antenna of the Arizona Radio Observatory were used to make a search for interstellar 13CH3NH2. Results: In the recorded spectra, we have assigned 2721 rotational transitions that belong to the ground vibrational state of the 13CH3NH2. These measurements were fitted to the Hamiltonian model that uses 75 parameters to achieve an overall weighted rms deviation of 0.73. On the basis of these spectroscopic results, predictions of transition frequencies in the frequency range up to 950 GHz with J ≤ 50 and Ka ≤ 20 are presented. The search for interstellar 13C methylamine in available observational data was not successful and therefore only an upper limit of 6.5 × 1014 cm-2 can be derived for the column density of 13CH3NH2 toward Sgr B2(N), assuming the same source size, temperature, linewidth, and systemic velocity as for parent methylamine isotopic

  3. Detection of intracellular lactate with localized diffusion { 1H- 13C}-spectroscopy in rat glioma in vivo

    NASA Astrophysics Data System (ADS)

    Pfeuffer, Josef; Lin, Joseph C.; DelaBarre, Lance; Ugurbil, Kamil; Garwood, Michael

    2005-11-01

    The aim of this study was to compare the diffusion characteristic of lactate and alanine in a brain tumor model to that of normal brain metabolites known to be mainly intracellular such as N-acetylaspartate or creatine. The diffusion of 13C-labeled metabolites was measured in vivo with localized NMR spectroscopy at 9.4 T (400 MHz) using a previously described localization and editing pulse sequence known as ACED-STEAM ('adiabatic carbon editing and decoupling'). 13C-labeled glucose was administered and the apparent diffusion coefficients of the glycolytic products, { 1H- 13C}-lactate and { 1H- 13C}-alanine, were determined in rat intracerebral 9L glioma. To obtain insights into { 1H- 13C}-lactate compartmentation (intra- versus extracellular), the pulse sequence used very large diffusion weighting (50 ms/μm 2). Multi-exponential diffusion attenuation of the lactate metabolite signals was observed. The persistence of a lactate signal at very large diffusion weighting provided direct experimental evidence of significant intracellular lactate concentration. To investigate the spatial distribution of lactate and other metabolites, 1H spectroscopic images were also acquired. Lactate and choline-containing compounds were consistently elevated in tumor tissue, but not in necrotic regions and surrounding normal-appearing brain. Overall, these findings suggest that lactate is mainly associated with tumor tissue and that within the time-frame of these experiments at least some of the glycolytic product ([ 13C] lactate) originates from an intracellular compartment.

  4. Development of imaging methods to assess adiposity and metabolism.

    PubMed

    Heymsfield, S B

    2008-12-01

    Body composition studies were first recorded around the time of the renaissance, and advances by the mid-twentieth century facilitated growth in the study of physiology, metabolism and pathological states. The field developed during this early period around the 'two-compartment' molecular level model that partitions body weight into fat and fat-free mass. Limited use was also made of X-rays as a means of estimating fat-layer thickness, but the revolutionary advance was brought about by the introduction of three-dimensional images provided by computed tomography (CT) in the mid 1970s, followed soon thereafter by magnetic resonance imaging (MRI). Complete in vivo reconstruction of all major anatomic body compartments and tissues became possible, thus providing major new research opportunities. This imaging revolution has continued to advance with further methodology refinements including functional MRI, diffusion tensor imaging and combined methods such as positron emission tomography+CT or MRI. The scientific advances made possible by these new and innovative methods continue to unfold today and hold enormous promise for the future of obesity research.

  5. Regulatory issues on breath tests and updates of recent advances on [13C]-breath tests.

    PubMed

    Modak, Anil S

    2013-09-01

    Over the last decade non invasive diagnostic phenotype [(13)C]-breath tests as well as tests using endogenous volatile organic compounds (VOCs) in breath have been researched extensively. However, only three breath tests have been approved by the FDA over the last 15 years. Despite the potential benefits of these companion diagnostic tests (CDx) for evaluation of drug metabolizing enzyme activities and standalone diagnostic tests for disease diagnosis to personalize medicine, the clinical and commercial development of breath tests will need to overcome a number of regulatory, financial and scientific hurdles prior to their acceptance into routine clinical practice. The regulatory agencies (FDA and EMEA) need to adapt and harmonize their approval process for companion diagnostic tests as well as standalone diagnostic breath tests for personalized medicine. The Center for Devices and Radiological Health has deemed any breath test that involves a labeled (13)C substrate/drug and a device requires a Pre Market Approval (PMA), which is analogous to an approved New Drug Application. A PMA is in effect, a private license granted to the applicant for marketing a particular medical device. Any breath test with endogenous VOCs along with a device can be approved via the 510(k) application. A number of (13)C breath tests with clinical applications have been researched recently and results have been published in reputed journals. Diagnostic companies will need to invest the necessary financial resources to develop and get regulatory approval for diagnostic breath tests capable of identifying responders/non responders for FDA approved drugs with narrow therapeutic indices (personalized medicine) or for evaluating the activity of drug metabolizing P450 polymorphic enzymes or for diagnosing diseases at an early stage or for monitoring the efficacy of medications. The financial success of these diagnostic breath tests will then depend entirely on how the test is marketed to

  6. Comparing Soil and Bison δ13C to Field Estimates of C4 Plant Abundances in North America

    NASA Astrophysics Data System (ADS)

    Griffith, D.; Still, C. J.; Cotton, J.; Powell, B.

    2015-12-01

    Stable carbon isotope data (i.e., δ13C) from soils and herbivore tissue are commonly used as a proxy for the relative abundance of C4 and C3 plants at a site. These data are also increasingly used to represent other climatologically relevant properties of vegetated environments, such as productivity, aridity, water use efficiency, and tree cover. The δ13C values of soils and herbivore tissues are generally assumed to resemble their source vegetation, after accounting for diverse fractionation processes during litter decomposition and tissue metabolism and turnover. However, δ13C values have rarely been compared to source vegetation at a regional to continental scale. As a result, the quality of δ13C as a proxy has not been thoroughly evaluated, and the importance of modifying factors have not been assessed at biogeographically relevant scales. To address some of these issues, we combined three multi-source datasets from North America: herbaceous C4 plant abundances from thousands of vegetation plots, hundreds of soil δ13C measurements, and hundreds of bison collagen, hair, and enamel δ13C data (tissues with different turnover rates). These datasets were resampled to common grid for comparison. A stronger relationship with C4 vegetation existed for bison as compared to soil δ13C. To determine which factors might explain deviations in the vegetation plot and isotopic data, we used statistical models to quantify the influence of soil variables, mean annual precipitation and temperature, tree cover, presence of invasives, and fire frequency in conjunction with plot-based C4 abundance data. Our bison model was only improved by the addition of invasives. In contrast, our soil model was significantly improved when accounting for tree cover (C3 vegetation and shade), precipitation, various soil parameters, and invasive grasses, suggesting that soils are more likely to be biased from source vegetation in ways that could influence interpretation as a proxy at broad

  7. Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling

    PubMed Central

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA – the combing, imaging by SIMS or CIS method – has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to 13C-labeling via the detection and quantification of the 13C 14N - recombinant ion and the use of the 13C: 12C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  8. Advanced Imaging Approaches to Characterize Stromal and Metabolic Changes in In Vivo Mammary Tumor Models

    DTIC Science & Technology

    2015-02-01

    Optical imaging , metabolism, tumor microenvironment, NADH, FAD, intravital imaging , collagen, metastasis 3.Overall Project Summary Our preliminary...Keely, KW Eliceiri. Novel Intravital Imaging Approaches to Characterize Collagen Alignment in Defined Mammary Tumor Models. Microscopy and...fixturing for intravital FLIM imaging through a rodent mammary imaging window. Stage is raised to accommodate tall 20xW objective. 14     Figure

  9. The 13C nuclear magnetic resonance in graphite intercalation compounds

    NASA Technical Reports Server (NTRS)

    Tsang, T.; Resing, H. A.

    1985-01-01

    The (13)C NMR chemical shifts of graphite intercalation compounds were calculated. For acceptor types, the shifts come mainly from the paramagnetic (Ramsey) intra-atomic terms. They are related to the gross features of the two-dimensional band structures. The calculated anisotropy is about -140 ppm and is independent of the finer details such as charge transfer. For donor types, the carbon 2p pi orbitals are spin-polarized because of mixing with metal conduction electrons, thus there is an additional dipolar contribution which may be correlated with the electronic specific heat. The general agreement with experimental data is satisfactory.

  10. Dynamic nuclear polarization of carbonyl and methyl 13C spins in acetate using trityl OX063

    NASA Astrophysics Data System (ADS)

    Niedbalski, Peter; Parish, Christopher; Lumata, Lloyd

    2015-03-01

    Hyperpolarization via dissolution dynamic nuclear polarization (DNP) is a physics technique that amplifies the magnetic resonance signals by several thousand-fold for biomedical NMR spectroscopy and imaging (MRI). Herein we have investigated the effect of carbon-13 isotopic location on the DNP of acetate (one of the biomolecules commonly used for hyperpolarization) at 3.35 T and 1.4 K using a narrow ESR linewidth free radical trityl OX063. We have found that the carbonyl 13C spins yielded about twice the polarization produced in methyl 13C spins. Deuteration of the methyl group, beneficial in the liquid-state, did not produce an improvement in the polarization level at cryogenic conditions. Concurrently, the solid-state nuclear relaxation of these samples correlate with the polarization levels achieved. These results suggest that the location of the 13C isotopic labeling in acetate has a direct impact on the solid-state polarization achieved and is mainly governed by the nuclear relaxation leakage factor.

  11. Metabolic alterations in bladder cancer: applications for cancer imaging.

    PubMed

    Whyard, Terry; Waltzer, Wayne C; Waltzer, Douglas; Romanov, Victor

    2016-02-01

    Treatment planning, outcome and prognosis are strongly related to the adequate tumor staging for bladder cancer (BC). Unfortunately, a large discrepancy exists between the preoperative clinical and final pathologic staging. Therefore, an advanced imaging-based technique is crucial for adequate staging. Although Magnetic Resonance Imaging (MRI) is currently the best in vivo imaging technique for BC staging because of its excellent soft-tissue contrast and absence of ionizing radiation it lacks cancer-specificity. Tumor-specific positron emission tomography (PET), which is based on the Warburg effect (preferential uptake of glucose by cancer cells), exploits the radioactively-labeled glucose analogs, i.e., FDG. Although FDG-PET is highly cancer specific, it lacks resolution and contrast quality comparable with MRI. Chemical Exchange Saturation Transfer (CEST) MRI enables the detection of low concentrations of metabolites containing protons. BC is an attractive target for glucose CEST MRI because, in addition to the typical systemic administration, glucose might also be directly applied into the bladder to reduce toxicity-related complications. As a first stage of the development of a contrast-specific BC imaging technique we have studied glucose uptake by bladder epithelial cells and have observed that glucose is, indeed, consumed by BC cells with higher intensity than by non-transformed urothelial cells. This effect might be partly explained by increased expression of glucose transporters GLUT1 and GLUT3 in transformed cells as compared to normal urothelium. We also detected higher lactate production by BC cells which is another cancer-specific manifestation of the Warburg effect. In addition, we have observed other metabolic alterations in BC cells as compared to non-transformed cells: in particular, increased pyruvate synthesis. When glucose was substituted by glutamine in culture media, preferential uptake of glutamine by BC cells was observed. The preferential

  12. Swift Acetate Glial Assay (SAGA): An accelerated human 13C MRS brain exam for clinical diagnostic use

    NASA Astrophysics Data System (ADS)

    Sailasuta, Napapon; Tran, Thao T.; Harris, Kent C.; Ross, B. D.

    2010-12-01

    We demonstrate a robust procedure for the quantitative characterization of glial metabolism in human brain. In the past, the slope of the uptake and production of enriched label at steady state were used to determine metabolic rates, requiring the patient to be in the magnet for 120-160 min. In the present method, 13C cerebral metabolite profiles were acquired at steady state alone on a routine clinical MR scanner in 25.6 min. Results obtained from the new short method (SAGA) were comparable to those achieved in a conventional, long method and effective for determination of glial metabolic rate in posterior-parietal and frontal brain regions.

  13. Glucogenesis in an insect, Manduca sexta L., estimated from the 13C isotopomer distribution in trehalose synthesized from [1,3-13C2]glycerol.

    PubMed

    Thompson, S N

    1997-07-19

    Glucogenesis from [3-13C]alanine and [1,3-13C2]glycerol was demonstrated in the insect Manduca sexta by examining the 13C enrichment of trehalose, a non-reducing disaccharide of glucose synthesized in the insect fat body and released into the blood or hemolymph. In insects maintained on a low carbohydrate diet, trehalose synthesized from [3-13C]alanine was selectively enriched at C1 and C6, and C2 and C5. The 13C-labelling pattern indicated the carboxylation of [3-13C]pyruvate, formed by transamination of the [3-13C]alanine followed by randomization of the label at the fumarate step of the tricarboxylic acid cycle and glucose synthesis via the gluconeogenic pathway. 13C enrichment of trehalose was absent in similarly maintained insect larvae administered 3-mercaptopicolinic acid, an inhibitor of hepatic phosphoenolpyruvate carboxykinase. Insects on the low carbohydrate diet also synthesized trehalose from [1,3-13C2]glycerol. 13C multiplets were observed in trehalose C3 and C4 demonstrating the synthesis of three 13C enriched glucose isotopomers from the 13C-labelled glycerol. The relative contributions of 13C-labelled glycerol and unlabelled 3 carbon substrates to the synthesis of the 13C enriched trehalose isotopomers were determined from the multiplet structure at C3, and calculation of minimal rates of glucogenesis were based on the 13C enrichment of C4. The C4/C3 13C enrichment ratio in trehalose synthesized from [1,3-13C2]glycerol was close to unity, and total glucogenesis was calculated after estimation of the expected contribution of unlabelled trehalose synthesis from 3 carbon substrates by comparison of the ratio of unlabelled and labelled contributions to the 13C enriched trehalose isotopomers with the 13C enrichment of [1,3-13C2]glycerol-3-phosphate. The estimated total rates of glucogenesis varied from 0.33 to 2.80 micromol glucose/g fresh weight/h. The blood sugar level of M. sexta was also highly variable. Although the potential importance of

  14. Evaluating North Sea carbon sources using radiogenic (224Ra and 228Ra) and stable carbon isotope (DI13C) tracers

    NASA Astrophysics Data System (ADS)

    Burt, William; Thomas, Helmuth; Hagens, Mathilde; Brenner, Heiko; Paetsch, Johannes; Clargo, Nikki

    2015-04-01

    In the North Sea, much uncertainty still exists regarding the role of boundary fluxes (e.g. benthic input from sediments or lateral inputs from the coastline) in the overall biogeochemical cycling of the system. The stable carbon isotope signature of dissolved inorganic carbon (δ13C-DIC) is a common tool for following transformations of carbon in the water column and identifying carbon sources and sinks. Here, analyses of the first basin-wide observations of δ13C-DIC reveal that a balance between biological production and respiration, as well as a freshwater input near the European continental coast, predominantly control surface distributions in the North Sea. A strong relationship between the biological component of DIC (DICbio) and δ13C-DIC is then used to quantify the metabolic DIC flux associated with changes in the carbon isotopic signature. Correlations are also found between δ13C-DIC and naturally-occurring Radium isotopes (224Ra and 228Ra), which have well-identified sources from the seafloor and coastal boundaries. The relationship between δ13C-DIC and the longer-lived 228Ra isotope (half-life = 5.8 years) is used to derive a metabolic DIC flux from the European continental coastline. 228Ra is also shown to be a highly effective tracer of North Sea total alkalinity (TA) compared to the more conventional use of salinity as a tracer. Coastal alkalinity inputs are calculated using relationships with 228Ra, and ratios of DIC and TA suggest denitrification as the main metabolic pathway for the formation of these coastal inputs. Finally, coastal TA inputs are translated into inputs of protons to quantify their impact on the buffering capacity of the Southern North Sea.

  15. Interkingdom metabolic transformations captured by microbial imaging mass spectrometry

    PubMed Central

    Moree, Wilna J.; Phelan, Vanessa V.; Wu, Cheng-Hsuan; Bandeira, Nuno; Cornett, Dale S.; Duggan, Brendan M.; Dorrestein, Pieter C.

    2012-01-01

    In polymicrobial infections, microbes can interact with both the host immune system and one another through direct contact or the secretion of metabolites, affecting disease progression and treatment options. The thick mucus in the lungs of patients with cystic fibrosis is highly susceptible to polymicrobial infections by opportunistic pathogens, including the bacterium Pseudomonas aeruginosa and the fungus Aspergillus fumigatus. Unravelling the hidden molecular interactions within such polymicrobial communities and their metabolic exchange processes will require effective enabling technologies applied to model systems. In the present study, MALDI-TOF and MALDI-FT-ICR imaging mass spectrometry (MALDI-IMS) combined with MS/MS networking were used to provide insight into the interkingdom interaction between P. aeruginosa and A. fumigatus at the molecular level. The combination of these technologies enabled the visualization and identification of metabolites secreted by these microorganisms grown on agar. A complex molecular interplay was revealed involving suppression, increased production, and biotransformation of a range of metabolites. Of particular interest is the observation that P. aeruginosa phenazine metabolites were converted by A. fumigatus into other chemical entities with alternative properties, including enhanced toxicities and the ability to induce fungal siderophores. This work highlights the capabilities of MALDI-IMS and MS/MS network analysis to study interkingdom interactions and provides insight into the complex nature of polymicrobial metabolic exchange and biotransformations. PMID:22869730

  16. Influence of different proton pump inhibitors on activity of cytochrome P450 assessed by [(13)C]-aminopyrine breath test.

    PubMed

    Kodaira, Chise; Uchida, Shinya; Yamade, Mihoko; Nishino, Masafumi; Ikuma, Mutsuhiro; Namiki, Noriyuki; Sugimoto, Mitsushige; Watanabe, Hiroshi; Hishida, Akira; Furuta, Takahisa

    2012-03-01

    Aminopyrine is metabolized by cytochrome P450 (CYP) in the liver. The investigators evaluated influences of different PPIs on CYP activity as assessed by the [(13)C]-aminopyrine breath test ([(13)C]-ABT). Subjects were 15 healthy volunteers with different CYP2C19 status (5 rapid metabolizers [RMs], 5 intermediate metabolizers [IMs], and 5 poor metabolizers [PMs]). Breath samples were collected before and every 15 to 30 minutes for 3 hours after oral ingestion of [(13)C]-aminopyrine 100 mg on day 8 of each of the following regimens: control; omeprazole 20 mg and 80 mg, lansoprazole 30 mg, and rabeprazole 20 mg. Changes in carbon isotope ratios in carbon dioxide ((13)CO(2)/(12)CO(2)) in breath samples were measured by infrared spectrometry and expressed as delta-over-baseline (DOB) ratios (‰). Mean areas under the curve of DOB from 0 to 3 h (AUC(0-3h) of DOB) were significantly decreased by omeprazole 20 mg and lansoprazole 30 mg but not by rabeprazole 20 mg. Conversely, higher PPI dose (ie, omeprazole 80 mg) seemed to further decrease AUC(0-3h) of DOB in RMs but increased it in PMs. Omeprazole and lansoprazole at the standard doses inhibit CYP activity but rabeprazole does not, whereas high-dose omeprazole seems to induce CYPs.

  17. Improved ground state and ν12 = 1 state rovibrational constants for ethylene-13C2 (13C2H4)

    NASA Astrophysics Data System (ADS)

    Gabona, M. G.; Tan, T. L.

    2014-05-01

    The Fourier transform infrared (FTIR) absorption spectrum of the ν12 fundamental band of ethylene-13C2 (13C2H4) was recorded in the frequency range of 1350-1550 cm-1 with unapodized resolution of 0.0063 cm-1. Improved upper state (ν12 = 1) rovibrational constants consisting of three rotational, five quartic and five sextic constants were derived by assigning and fitting 1731 infrared transitions using Watson's A-reduced Hamiltonian in the Ir representation. The root-mean-square deviation of the fit was 0.00030 cm-1. More higher-order upper state (ν12 = 1) constant were derived in the present analysis than previously reported. Improved ground state rovibrational constants were also determined from the fit of 1104 ground state combination differences (GSCD) with a root-mean-square deviation of 0.00029 cm-1. The A-type ν12 band centered at 1436.65409 ± 0.00002 cm-1 has a calculated inertial defect Δ12 is 0.242896 ± 0.000007 μÅ2. No indications of perturbation were found in the analysis of the band.

  18. 13C Metabolomics: NMR and IROA for Unknown Identification

    PubMed Central

    Clendinen, Chaevien S.; Stupp, Gregory S.; Wang, Bing; Garrett, Timothy J.; Edison, Arthur S.

    2016-01-01

    Abstract: Background Isotopic Ratio Outlier Analysis (IROA) is an untargeted metabolomics method that uses stable isotopic labeling and LC-HRMS for identification and relative quantification of metabolites in a biological sample under varying experimental conditions. Objective We demonstrate a method using high-sensitivity 13C NMR to identify an unknown metabolite isolated from fractionated material from an IROA LC-HRMS experiment. Methods IROA samples from the nematode Caenorhabditis elegans were fractionated using LC-HRMS using 5 repeated injections and collecting 30 sec fractions. These were concentrated and analyzed by 13C NMR. Results We isotopically labeled samples of C. elegans and collected 2 adjacent LC fractions. By HRMS, one contained at least 2 known metabolites, phenylalanine and inosine, and the other contained tryptophan and an unknown feature with a monoisotopic mass of m/z 380.0742 [M+H]+. With NMR, we were able to easily verify the known compounds, and we then identified the spin system networks responsible for the unknown resonances. After searching the BMRB database and comparing the molecular formula from LC-HRMS, we determined that the fragments were a modified anthranilate and a glucose modified by a phosphate. We then performed quantum chemical NMR chemical shift calculations to determine the most likely isomer, which was 3’-O-phospho-β-D-glucopyranosyl-anthranilate. This compound had previously been found in the same organism, validating our approach. Conclusion We were able to dereplicate previously known metabolites and identify a metabolite that was not in databases by matching resonances to NMR databases and using chemical shift calculations to determine the correct isomer. This approach is efficient and can be used to identify unknown compounds of interest using the same material used for IROA. PMID:28090435

  19. Millimeter and submillimeter wave spectra of 13C-glycolaldehydes

    NASA Astrophysics Data System (ADS)

    Haykal, I.; Motiyenko, R. A.; Margulès, L.; Huet, T. R.

    2013-01-01

    Context. Glycolaldehyde (CH2OHCHO) is the simplest sugar and an important intermediate in the path toward forming more complex biologically relevant molecules. Astronomical surveys of interstellar molecules, such as those available with the very sensitive ALMA telescope, require preliminary laboratory investigations of the microwave and submillimeter-wave spectra of molecular species including new isotopologs - to identify these in the interstellar media. Aims: To achieve the detection of the 13C isotopologs of glycolaldehyde in the interstellar medium, their rotational spectra in the millimeter and submillimeter-wave regions were studied. Methods: The spectra of 13CH2OHCHO and CH2OH13CHO were recorded in the 150-945 GHz spectral range in the laboratory using a solid-state submillimeter-wave spectrometer in Lille. The observed line frequencies were measured with an accuracy of 30 kHz up to 700 GHz and of 50 kHz above 700 GHz. We analyzed the spectra with a standard Watson Hamiltonian. Results: About 10 000 new lines were identified for each isotopolog. The spectroscopic parameters were determined for the ground- and the three lowest vibrational states up to 945 and 630 GHz. Previous microwave assignments of 13CH2OHCHO were not confirmed. Conclusions: The provided line-lists and sets of molecular parameters meet the needs for a first astrophysical search of 13C-glycolaldehydes. Full Tables 3 and 4 are only available at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/549/A96

  20. Quantitative imaging of subcellular metabolism with stable isotopes and multi-isotope imaging mass spectrometry.

    PubMed

    Steinhauser, Matthew L; Lechene, Claude P

    2013-01-01

    Multi-isotope imaging mass spectrometry (MIMS) is the quantitative imaging of stable isotope labels in cells with a new type of secondary ion mass spectrometer (NanoSIMS). The power of the methodology is attributable to (i) the immense advantage of using non-toxic stable isotope labels, (ii) high resolution imaging that approaches the resolution of usual transmission electron microscopy and (iii) the precise quantification of label down to 1 part-per-million and spanning several orders of magnitude. Here we review the basic elements of MIMS and describe new applications of MIMS to the quantitative study of metabolic processes including protein and nucleic acid synthesis in model organisms ranging from microbes to humans.

  1. Nitrogen control of 13C enrichment in heterotrophic organs relative to leaves in a landscape-building desert plant species

    NASA Astrophysics Data System (ADS)

    Zhang, J.; Gu, L.; Bao, F.; Cao, Y.; Hao, Y.; He, J.; Li, J.; Li, Y.; Ren, Y.; Wang, F.; Wu, R.; Yao, B.; Zhao, Y.; Lin, G.; Wu, B.; Lu, Q.; Meng, P.

    2015-01-01

    A longstanding puzzle in isotope studies of C3 plant species is that heterotrophic plant organs (e.g., stems, roots, seeds, and fruits) tend to be enriched in 13C compared to the autotrophic organ (leaves) that provides them with photosynthate. Our inability to explain this puzzle suggests key deficiencies in understanding post-photosynthetic metabolic processes. It also limits the effectiveness of applications of stable carbon isotope analyses in a variety of scientific disciplines ranging from plant physiology to global carbon cycle studies. To gain insight into this puzzle, we excavated whole plant architectures of Nitraria tangutorum Bobrov, a C3 species that has an exceptional capability of fixing sands and building sand dunes, in two deserts in northwestern China. We systematically and simultaneously measured carbon isotope ratios and nitrogen and phosphorous contents of different parts of the excavated plants. We also determined the seasonal variations in leaf carbon isotope ratios on nearby intact plants of N. tangutorum. We found, for the first time, that higher nitrogen contents in heterotrophic organs were significantly correlated with increased heterotrophic 13C enrichment compared to leaves. However, phosphorous contents had no effect on the enrichment. In addition, new leaves had carbon isotope ratios similar to roots but were progressively depleted in 13C as they matured. We concluded that a nitrogen-mediated process, hypothesized to be the refixation of respiratory CO2 by phosphoenolpyruvate (PEP) carboxylase, was responsible for the differences in 13C enrichment among different heterotrophic organs, while processes such as fractionating foliar metabolism and preferentially loading into phloem of 13C-enriched sugars may contribute to the overall autotrophic-heterotrophic difference in carbon isotope compositions.

  2. The First in Vivo Observation of 13C- 15N Coupling in Mammalian Brain

    NASA Astrophysics Data System (ADS)

    Kanamori, Keiko; Ross, Brian D.

    2001-12-01

    [5-13C,15N]Glutamine, with 1J(13C-15N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by 13C MRS at 4.7 T. The brain [5-13C]glutamine peak consisted of the doublet from [5-13C,15N]glutamine and the center [5-13C,14N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-13C,15N]glutamine was monitored in vivo with a time resolution of 20-35 min. This [5-13C,15N]glutamine was formed by glial uptake of released neurotransmitter [5-13C]glutamate and its reaction with 15NH3 catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively13C-enriched by intravenous [2,5-13C]glucose infusion to 13C-label whole-brain glutamate C5, followed by [12C]glucose infusion to chase 13C from the small and rapidly turning-over glial glutamate pool, leaving 13C mainly in the neurotransmitter [5-13C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-13C,15N]glutamine arises from a coupling between 13C of neuronal origin and 15N of glial origin. Measurement of the rate of brain [5-13C,15N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.

  3. 13C and 31P NMR for the diagnosis of muscular phosphorylase-kinase deficiency

    NASA Astrophysics Data System (ADS)

    Jehenson, P.; Duboc, D.; Laforet, P.; Eymard, B.; Lombès, A.; Fardeau, M.; Brunet, P.; Syrota, A.

    1998-02-01

    To further develop and specify the range of medical applications of in vivo NMR spectroscopy for the study of myopathies, it is ncessary to study the largest number of well characterized cases. We here report on the 31P and 13C NMR study of a purely muscular form of phosphorylase-kinase (PK) deficiency. Abnormalities were observed that agree with and increase our pathophysiological knowledge, in particular on the activation of phosphorylase and PK. Also, the abnormalities are different from those found in other clinically similar metabolic myopathies and could be used for the differential diagnosis. Afin de continuer à développer et préciser les applications médicales de la spectroscopie RMN in vivo, il faut étudier le plus grand nombre possible de cas bien caractérisés. Nous avons étudié ici une forme purement musculaire de déficit en phosphorylase-kinase (PK) par RMN du phosphore 31 et du carbone 13. Les altérations observées sont en accord avec et augmentent nos connaissances physiopathologiques, par exemple concernant l'activation de la phosphorylase et PK. Par ailleurs, la combinaison d'altérations observées en 31P et 13C est différente de celle retrouvée dans d'autres myopathies métaboliques cliniquement semblables et pourrait être utilisée pour le diagnostic différentiel.

  4. Uniformly sup 13 C-labeled algal protein used to determine amino acid essentiality in vivo

    SciTech Connect

    Berthold, H.K.; Hachey, D.L.; Reeds, P.J.; Klein, P.D. ); Thomas, O.P. ); Hoeksema, S. )

    1991-09-15

    The edible alga Spirulina platensis was uniformly labeled with {sup 13}C by growth in an atmosphere of pure {sup 13}CO{sub 2}. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly {sup 13}C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo.

  5. Reaction cross sections for. nu. sup 13 C r arrow e sup minus sup 13 N and. nu. sup 13 C r arrow. nu. prime sup 13 C sup * for low energy neutrinos

    SciTech Connect

    Fukugita, M. ); Kohyama, Y.; Kubodera, K.; Kuramoto, T. )

    1990-04-01

    Cross sections for {nu}+{sup 13}C reactions are calculated both for charged- and neutral-current reactions in order to estimate the efficiency of a {sup 13}C target as a solar neutrino detector. The relevant transition matrix elements are obtained using the semiphenomenological effective-operator approach for {ital p}-shell nuclei.

  6. 13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum.

    PubMed

    Kleijn, Roelco J; van Winden, Wouter A; Ras, Cor; van Gulik, Walter M; Schipper, Dick; Heijnen, Joseph J

    2006-07-01

    In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h(-1) yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.

  7. 13C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites

    PubMed Central

    McCullough, Christopher R.; Pullela, Phani Kumar; Im, Sang-Choul; Waskell, Lucy

    2012-01-01

    The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a 13CH3-reporter attached. This 13C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site. PMID:19199046

  8. Novel Peak Assignments of in Vivo 13C MRS in Human Brain at 1.5 T

    NASA Astrophysics Data System (ADS)

    Blüml, Stefan; Hwang, Jong-Hee; Moreno, Angel; Ross, Brian D.

    2000-04-01

    13C MRS studies at natural abundance and after intravenous 1-13C glucose infusion were performed on a 1.5-T clinical scanner in four subjects. Localization to the occipital cortex was achieved by a surface coil. In natural abundance spectra glucose C3β,5β, myo-inositol, glutamate C1,2,5, glutamine C1,2,5, N-acetyl-aspartate C1-4,Cdbnd O, creatine CH2, CH3, and CCdbnd N, taurine C2,3, bicarbonate HCO-3 were identified. After glucose infusion 13C enrichment of glucose C1α,1β, glutamate C1-4, glutamine C1-4, aspartate C2,3, N-acetyl-aspartate C2,3, lactate C3, alanine C3, and HCO-3 were observed. The observation of 13C enrichment of resonances resonating at >150 ppm is an extension of previously published studies and will provide a more precise determination of metabolic rates and substrate decarboxylation in human brain.

  9. Regional, seasonal and interspecific variation in 15N and 13C in sympatric mouse lemurs

    NASA Astrophysics Data System (ADS)

    Rakotondranary, S. Jacques; Struck, Ulrich; Knoblauch, Christian; Ganzhorn, Jörg U.

    2011-11-01

    Madagascar provides some of the rare examples where two or more primate species of the same genus and with seemingly identical niche requirements occur in sympatry. If congeneric primate species co-occur in other parts of the world, they differ in size in a way that is consistent with Hutchinson's rule for coexisting species, or they occupy different ecological niches. In some areas of Madagascar, mouse lemurs do not follow these "rules" and thus seem to violate one of the principles of community ecology. In order to understand the mechanisms that allow coexistence of sympatric congeneric species without obvious niche differentiation, we studied food composition of two identical sized omnivorous mouse lemur species, Microcebus griseorufus and M. murinus with the help of stable isotope analyses ( δ 15N and δ 13C). The two species are closely related sister species. During the rich season, when food seems abundant, the two species do not differ in their nitrogen isotope composition, indicating that the two species occupy the same trophic level. But they differ in their δ 13C values, indicating that M. griseorufus feeds more on C4 and CAM (Crassulacean-acid-metabolism) plants than M. murinus. During the lean season, M. murinus has lower δ 15N values, indicating that the two species feed at different trophic levels during times of food shortage. Hybrids between the two species showed intermediate food composition. The results reflect subtle differences in foraging or metabolic adaptations that are difficult to quantify by traditional observations but that represent possibilities to allow coexistence of species.

  10. PASADENA hyperpolarization of 13C biomolecules: equipment design and installation

    PubMed Central

    Hövener, Jan-Bernd; Chekmenev, Eduard Y.; Harris, Kent C.; Perman, William H.; Robertson, Larry W.; Bhattacharya, Pratip

    2009-01-01

    Object The PASADENA method has achieved hyperpolarization of 16–20% (exceeding 40,000-fold signal enhancement at 4.7 T), in liquid samples of biological molecules relevant to in vivo MRI and MRS. However, there exists no commercial apparatus to perform this experiment conveniently and reproducibly on the routine basis necessary for translation of PASADENA to questions of biomedical importance. The present paper describes equipment designed for rapid production of six to eight liquid samples per hour with high reproducibility of hyperpolarization. Materials and methods Drawing on an earlier, but unpublished, prototype, we provide diagrams of a delivery circuit, a laminar-flow reaction chamber within a low field NMR contained in a compact, movable housing. Assembly instructions are provided from which a computer driven, semiautomated PASADENA polarizer can be constructed. Results Together with an available parahydrogen generator, the polarizer, which can be operated by a single investigator, completes one cycle of hyperpolarization each 52 s. Evidence of efficacy is presented. In contrast to competing, commercially available devices for dynamic nuclear polarization which characteristically require 90 min per cycle, PASADENA provides a low-cost alternative for high throughput. Conclusions This equipment is suited to investigators who have an established small animal NMR and wish to explore the potential of heteronuclear (13C and 15N) MRI, MRS, which harnesses the enormous sensitivity gain offered by hyperpolarization. PMID:19067008

  11. New optical analyzer for 13C-breath test

    NASA Astrophysics Data System (ADS)

    Harde, Hermann; Dressler, Matthias; Helmrich, Günther; Wolff, Marcus; Groninga, Hinrich

    2008-04-01

    Medical breath tests are well established diagnostic tools, predominantly for gastroenterological inspections, but also for many other examinations. Since the composition and concentration of exhaled volatile gases reflect the physical condition of a patient, a breath analysis allows one to recognize an infectious disease in an organ or even to identify a tumor. One of the most prominent breath tests is the 13C-urea-breath test, applied to ascertain the presence of the bacterium helicobacter pylori in the stomach wall as an indication of a gastric ulcer. In this contribution we present a new optical analyzer that is based on photoacoustic spectroscopy and uses a DFB diode laser at 2.744 μm. The concentration ratio of the CO II isotopologues is determined by measuring the absorption on a 13CO II line in comparison to a 12CO II line. In the specially selected spectral range the lines have similar strengths, although the concentrations differ by a factor of 90. Therefore, the signals are well comparable. Due to an excellent signal-noise-ratio isotope variations of less than 1% can be resolved as required for the breath test.

  12. Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging.

    PubMed

    Neves, André A; Wainman, Yéléna A; Wright, Alan; Kettunen, Mikko I; Rodrigues, Tiago B; McGuire, Sarah; Hu, De-En; Bulat, Flaviu; Geninatti Crich, Simonetta; Stöckmann, Henning; Leeper, Finian J; Brindle, Kevin M

    2016-01-22

    Glycosylation is a ubiquitous post-translational modification, present in over 50 % of the proteins in the human genome,1 with important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases,2 including cancer.3 We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal MRI probe. Significant N-azidoacetylgalactosamine dependent T1 contrast was observed in vivo two hours after probe administration. Tumor, kidney, and liver showed significant contrast, and several other tissues, including the pancreas, spleen, heart, and intestines, showed a very high contrast (>10-fold). This approach has the potential to enable the rapid and non-invasive magnetic resonance imaging of glycosylated tissues in vivo in preclinical models of disease.

  13. Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging.

    PubMed

    Neves, André A; Wainman, Yéléna A; Wright, Alan; Kettunen, Mikko I; Rodrigues, Tiago B; McGuire, Sarah; Hu, De-En; Bulat, Flaviu; Geninatti Crich, Simonetta; Stöckmann, Henning; Leeper, Finian J; Brindle, Kevin M

    2016-01-22

    Glycosylation is a ubiquitous post-translational modification, present in over 50% of the proteins in the human genome, with important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases, including cancer. We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal MRI probe. Significant N-azidoacetylgalactosamine dependent T1  contrast was observed in vivo two hours after probe administration. Tumor, kidney, and liver showed significant contrast, and several other tissues, including the pancreas, spleen, heart, and intestines, showed a very high contrast (>10-fold). This approach has the potential to enable the rapid and non-invasive magnetic resonance imaging of glycosylated tissues in vivo in preclinical models of disease.

  14. Citrate and Sugar Cofermentation in Leuconostoc oenos, a (sup13)C Nuclear Magnetic Resonance Study

    PubMed Central

    Ramos, A.; Santos, H.

    1996-01-01

    (sup13)C nuclear magnetic resonance spectroscopy was used to investigate citrate-glucose cometabolism in nongrowing cell suspensions of the wine lactic acid bacterium Leuconostoc oenos. The use of isotopically enriched substrates allowed us to identify and quantify in the end products the carbon atoms derived from each of the substrates supplied; furthermore, it was possible to differentiate between products derived from the metabolism of endogenous carbon reserves and those derived from external substrates. Citrate-sugar cometabolism was also monitored in dilute cell suspensions for comparison with the nuclear magnetic resonance results. A clear metabolic shift of the end products from glucose metabolism was observed when citrate was provided along with glucose: ethanol was replaced by acetate, and 2,3-butanediol was produced. Reciprocally, the production of lactate and 2,3-butanediol from citrate was increased in the presence of glucose. When citrate was cometabolized with glucose, a 10-fold reduction in the intracellular concentration of glucose-6-phosphate was observed, a result in line with the observed citrate-induced stimulation of glucose consumption. The presence of citrate provided additional pathways for NADP(sup+) regeneration and allowed the diversion of sugar carbon to reactions in which ATP was synthesized. The increased growth rates and maximal biomass yields of L. oenos growing on citrate-glucose mixtures resulted from increased ATP synthesis both by substrate-level phosphorylation and by a chemiosmotic mechanism. PMID:16535363

  15. Program for PET image alignment: Effects on calculated differences in cerebral metabolic rates for glucose

    SciTech Connect

    Phillips, R.L.; London, E.D.; Links, J.M.; Cascella, N.G. )

    1990-12-01

    A program was developed to align positron emission tomography images from multiple studies on the same subject. The program allowed alignment of two images with a fineness of one-tenth the width of a pixel. The indications and effects of misalignment were assessed in eight subjects from a placebo-controlled double-blind crossover study on the effects of cocaine on regional cerebral metabolic rates for glucose. Visual examination of a difference image provided a sensitive and accurate tool for assessing image alignment. Image alignment within 2.8 mm was essential to reduce variability of measured cerebral metabolic rates for glucose. Misalignment by this amount introduced errors on the order of 20% in the computed metabolic rate for glucose. These errors propagate to the difference between metabolic rates for a subject measured in basal versus perturbed states.

  16. Accurate measurements of {sup 13}C-{sup 13}C distances in uniformly {sup 13}C-labeled proteins using multi-dimensional four-oscillating field solid-state NMR spectroscopy

    SciTech Connect

    Straasø, Lasse Arnt; Nielsen, Jakob Toudahl; Bjerring, Morten; Nielsen, Niels Chr.; Khaneja, Navin

    2014-09-21

    Application of sets of {sup 13}C-{sup 13}C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important {sup 13}C-{sup 13}C distances in uniformly {sup 13}C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl ({sup 13}C′) and aliphatic ({sup 13}C{sub aliphatic}) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys. 130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly {sup 13}C,{sup 15}N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of {sup 13}C′-{sup 13}C{sub aliphatic} distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform {sup 13}C,{sup 15}N-labeling on the FGAIL fragment.

  17. 40 CFR 721.6505 - Polymers of C13C15 oxoalcohol ethoxolates.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Polymers of C13C15 oxoalcohol... Specific Chemical Substances § 721.6505 Polymers of C13C15 oxoalcohol ethoxolates. (a) Chemical substance... polymers of C13C15 oxoalcohol ethoxolates (PMNs P-96-950/951) are subject to reporting under this...

  18. 40 CFR 721.6505 - Polymers of C13C15 oxoalcohol ethoxolates.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Polymers of C13C15 oxoalcohol... Specific Chemical Substances § 721.6505 Polymers of C13C15 oxoalcohol ethoxolates. (a) Chemical substance... polymers of C13C15 oxoalcohol ethoxolates (PMNs P-96-950/951) are subject to reporting under this...

  19. 40 CFR 721.6505 - Polymers of C13C15 oxoalcohol ethoxolates.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Polymers of C13C15 oxoalcohol... Specific Chemical Substances § 721.6505 Polymers of C13C15 oxoalcohol ethoxolates. (a) Chemical substance... polymers of C13C15 oxoalcohol ethoxolates (PMNs P-96-950/951) are subject to reporting under this...

  20. 40 CFR 721.6505 - Polymers of C13C15 oxoalcohol ethoxolates.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Polymers of C13C15 oxoalcohol... Specific Chemical Substances § 721.6505 Polymers of C13C15 oxoalcohol ethoxolates. (a) Chemical substance... polymers of C13C15 oxoalcohol ethoxolates (PMNs P-96-950/951) are subject to reporting under this...

  1. 40 CFR 721.6505 - Polymers of C13C15 oxoalcohol ethoxolates.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Polymers of C13C15 oxoalcohol... Specific Chemical Substances § 721.6505 Polymers of C13C15 oxoalcohol ethoxolates. (a) Chemical substance... polymers of C13C15 oxoalcohol ethoxolates (PMNs P-96-950/951) are subject to reporting under this...

  2. 13C nuclear magnetic resonance data of lanosterol derivatives—Profiling the steric topology of the steroid skeleton via substituent effects on its 13C NMR

    NASA Astrophysics Data System (ADS)

    Dias, Jerry Ray; Gao, Hongwu

    2009-12-01

    The 13C NMR spectra of over 24 tetracyclic triterpenoid derivatives have been structurally analyzed. The 13C NMR chemical shifts allow one to probe the steric topology of the rigid steroid skeleton and inductive effects of its substituents. Use of deuterium labeling in chemical shift assignment and B-ring aromatic terpenoids are also featured.

  3. Uranyl nitrate inhibits lactate gluconeogenesis in isolated human and mouse renal proximal tubules: A {sup 13}C-NMR study

    SciTech Connect

    Renault, Sophie; Faiz, Hassan; Gadet, Rudy; Ferrier, Bernard; Martin, Guy; Baverel, Gabriel; Conjard-Duplany, Agnes

    2010-01-01

    As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl nitrate in isolated human and mouse kidney cortex tubules metabolizing the physiological substrate lactate. In the millimolar range, uranyl nitrate reduced lactate removal and gluconeogenesis and the cellular ATP level in a dose-dependent fashion. After incubation in phosphate-free Krebs-Henseleit medium with 5 mM L-[1-{sup 13}C]-, or L-[2-{sup 13}C]-, or L-[3-{sup 13}C]lactate, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. In the presence of 3 mM uranyl nitrate, glucose production and the intracellular ATP content were significantly reduced in both human and mouse tubules. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism revealed an inhibition of fluxes through lactate dehydrogenase and the gluconeogenic enzymes in the presence of 3 mM uranyl nitrate; in human and mouse tubules, fluxes were lowered by 20% and 14% (lactate dehydrogenase), 27% and 32% (pyruvate carboxylase), 35% and 36% (phosphoenolpyruvate carboxykinase), and 39% and 45% (glucose-6-phosphatase), respectively. These results indicate that natural uranium is an inhibitor of renal lactate gluconeogenesis in both humans and mice.

  4. Impact of Gd3+ doping and glassing solvent deuteration on 13C DNP at 5 Tesla

    NASA Astrophysics Data System (ADS)

    Kiswandhi, Andhika; Lama, Bimala; Niedbalski, Peter; Goderya, Mudrekh; Long, Joanna; Lumata, Lloyd

    Dynamic nuclear polarization (DNP) is a technique which can be used to amplify signals in nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) by several thousand-fold. The most commonly available DNP system typically operates at the W-band field or 3.35 T, at which it has been shown that 13C NMR signal can be enhanced by deuteration and Gd3+ doping. In this work, we have investigated the applicability of these procedures at 5 T. Our results indicate that the deuteration of the glassing matrix still yields an enhancement of 13C DNP when 4-oxo-TEMPO free radical is used. The effect is attributed to the lower heat load of the deuterons compared to protons. An addition of a trace amount of Gd3+ gives a modest enhancement of the signal when trityl OX063 is used, albeit with a less pronounced relative enhancement compared to the results obtained at 3.35 T. The results suggest that the enhancement obtained via Gd3+ doping may become saturated at higher field. These results will be discussed using a thermodynamic model of DNP. This work is supported by US Dept of Defense Award No. W81XWH-14-1-0048 and Robert A. Welch Foundation Grant No. AT-1877.

  5. NOTE The effect of 13C enrichment in the glassing matrix on dynamic nuclear polarization of [1-13C]pyruvate

    NASA Astrophysics Data System (ADS)

    Lumata, Lloyd; Kovacs, Zoltan; Malloy, Craig; Sherry, A. Dean; Merritt, Matthew

    2011-03-01

    Dimethyl sulfoxide (DMSO) can effectively form a glassy matrix necessary for dynamic nuclear polarization (DNP) experiments. We tested the effects of 13C enrichment in DMSO on DNP of [1-13C]pyruvate doped with trityl radical OX063Me. We found that the polarization build-up time τ of pyruvate in 13C-labeled DMSO glassing solution is twice as fast as the unenriched DMSO while the nuclear magnetic resonance enhancement was unchanged. This indicates that 13C-13C spin diffusion is a limiting factor in the kinetics of DNP in this system, but it has a minimal effect on the absolute value of polarization achievable for the target.

  6. The effect of 13C enrichment in the glassing matrix on dynamic nuclear polarization of [1-13C]pyruvate

    NASA Astrophysics Data System (ADS)

    Lumata, Lloyd; Kovacs, Zoltan; Malloy, Craig; Sherry, A. Dean; Merritt, Matthew

    2011-03-01

    Dimethyl sulfoxide (DMSO) can effectively form a glassy matrix necessary for dynamic nuclear polarization (DNP) experiments. We tested the effects of 13C enrichment in DMSO on DNP of [1-13C]pyruvate doped with trityl radical OX063Me. We found that the polarization build-up time τ of pyruvate in 13C-labeled DMSO glassing solution is twice as fast as the unenriched DMSO while the nuclear magnetic resonance enhancement was unchanged. This indicates that 13C-13C spin diffusion is a limiting factor in the kinetics of DNP in this system, but it has a minimal effect on the absolute value of polarization achievable for the target.

  7. The use of 13C labeling to enhance the sensitivity of 13C solid-state CPMAS NMR to study polymorphism in low dose solid formulations.

    PubMed

    Booy, Kees-Jan; Wiegerinck, Peter; Vader, Jan; Kaspersen, Frans; Lambregts, Dorette; Vromans, Herman; Kellenbach, Edwin

    2005-02-01

    (13)C labeling was used to enhance the sensitivity of (13)C solid-state NMR to study the effect of tabletting on the polymorphism of a steroidal drug. The steroidal drug Org OD 14 was (13)C labeled and formulated into tablets containing only 0.5-2.5% active ingredient. The tablets were subsequently studied by solid-state (13)C CPMAS NMR. The crystalline form present in tablets could readily be analyzed in tablets. No change in crystalline form was observed as a result of formulation or in subsequent stability studies. Solid-state NMR in combination with (13)C labeling can, in suitable cases, be used as a strategy to study the effect of formulation on the polymorphism of low dose drugs.

  8. Using Position-Specific 13C and 14C Labeling and 13C-PLFA Analysis to Assess Microbial Transformations of Free Versus Sorbed Alanine

    NASA Astrophysics Data System (ADS)

    Apostel, C.; Herschbach, J.; Bore, E. K.; Kuzyakov, Y.; Dippold, M. A.

    2015-12-01

    Sorption of charged or partially charged low molecular weight organic substances (LMWOS) to soil mineral surfaces delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil sciences, to compare the transformation mechanisms of sorbed and non-sorbed alanine in soil. Alanine as an amino acid links C- and N-cycles in soil and therefore is a model substance for the pool of LMWOS. To assess transformations of sorbed alanine, we added position-specific and uniformly 13C and 14C labeled alanine tracer to soil that had previously been sterilized by γ-radiation. The labeled soil was added to non-sterilized soil from the same site and incubated. Soil labeled with the same tracers without previous sorption was prepared and incubated as well. We captured the respired CO2 and determined its 14C-activity at increasing time intervals. The incorporation of 14C into microbial biomass was determined by chloroform fumigation extraction (CFE), and utilization of individual C positions by distinct microbial groups was evaluated by 13C-phospholipid fatty acid analysis (PLFA). A dual peak in the respired CO2 revealed two sorption mechanisms. To compare the fate of individual C atoms independent of their concentration and pool size in soil, we applied the divergence index (DI). The DI reveals the convergent or divergent behavior of C from individual molecule positions during microbial utilization. Alanine C-1 position was mainly oxidized to CO2, while its C-2 and C-3 were preferentially incorporated in microbial biomass and PLFA. This indicates that sorption by the COOH group does not protect this group from preferential oxidation. Microbial metabolism was determinative for the preferential oxidation of individual molecule positions. The use of position-specific labeling revealed mechanisms and kinetics of microbial utilization of sorbed and non

  9. Methanogenesis produces strong 13C enrichment in stromatolites of Lagoa Salgada, Brazil: a modern analogue for Palaeo-/Neoproterozoic stromatolites?

    PubMed

    Birgel, D; Meister, P; Lundberg, R; Horath, T D; Bontognali, T R R; Bahniuk, A M; de Rezende, C E; Vasconcelos, C; McKenzie, J A

    2015-05-01

    Holocene stromatolites characterized by unusually positive inorganic δ(13) CPDB values (i.e. up to +16‰) are present in Lagoa Salgada, a seasonally brackish to hypersaline lagoon near Rio de Janeiro (Brazil). Such positive values cannot be explained by phototrophic fixation of CO2 alone, and they suggest that methanogenesis was a dominating process during the growth of the stromatolites. Indeed, up to 5 mm methane was measured in the porewater. The archaeal membrane lipid archaeol showing δ(13) C values between -15 and 0‰ suggests that archaea are present and producing methane in the modern lagoon sediment. Moreover, (13) C-depleted hopanoids diplopterol and 3β-methylated C32 17β(H),21β(H)-hopanoic acid (both -40‰) are preserved in lagoon sediments and are most likely derived from aerobic methanotrophic bacteria thriving in the methane-enriched water column. Loss of isotopically light methane through the water column would explain the residual (13) C-enriched pool of dissolved inorganic carbon from where the carbonate constituting the stromatolites precipitated. The predominance of methanogenic archaea in the lagoon is most likely a result of sulphate limitation, suppressing the activity of sulphate-reducing bacteria under brackish conditions in a seasonally humid tropical environment. Indeed, sulphate-reduction activity is very low in the modern sediments. In absence of an efficient carbonate-inducing metabolic process, we propose that stromatolite formation in Lagoa Salgada was abiotically induced, while the (13) C-enriched organic and inorganic carbon pools are due to methanogenesis. Unusually, (13) C-enriched stromatolitic deposits also appear in the geological record of prolonged periods in the Palaeo- and Neoproterozoic. Lagoa Salgada represents a possible modern analogue to conditions that may have been widespread in the Proterozoic, at times when low sulphate concentrations in sea water allowed methanogens to prevail over sulphate

  10. Comparison of Kinetic Models for Analysis of Pyruvate-to-Lactate Exchange by Hyperpolarized 13C NMR

    PubMed Central

    Harrison, Crystal; Yang, Chendong; Jindal, Ashish; DeBerardinis, Ralph J.; Hooshyar, M.A.; Merritt, Matthew; Sherry, A. Dean; Malloy, Craig R.

    2012-01-01

    The activity of specific enzyme-catalyzed reactions may be detected in vivo by 13C NMR of hyperpolarized (HP) substrates. The signal from HP substrates and products, acquired over time, have been fit to a number of different mathematical models to determine fluxes, but these models have not been critically compared. In this study, two-pool and three-pool first-order models were constructed to measure flux through lactate dehydrogenase in isolated glioblastoma cells by NMR detection of lactate and pyruvate following addition of hyperpolarized [1-13C]pyruvate. Mass spectrometry (MS) was used to independently monitor 13C enrichment in intra- and extracellular lactate. Six models were evaluated using time dependent pyruvate C2 and lactate C1 HP NMR data acquired by use of selective excitation pulses plus 13C enrichment data from intracellular and extracellular lactate measured by MS. A three-pool bi-directional model provided the most accurate description of pyruvate metabolism in these cells. With computed values for the T1 of pyruvate and lactate as well as the effect of pulsing, the initial flux through lactate dehydrogenase (LDH) was well-determined by both the two-pool bidirectional and unidirectional models when only HP data was available. The three-pool model was necessary to fit the combined data from both MS and HP, but the simpler two-pool exchange model was sufficient to determine the 13C lactate concentration when the lactate appearance was measured only by HP. PMID:22451442

  11. Assessing microbial utilization of free versus sorbed Alanine by using position-specific 13C labeling and 13C-PLFA analysis

    NASA Astrophysics Data System (ADS)

    Herschbach, Jennifer; Apostel, Carolin; Spielvogel, Sandra; Kuzyakov, Yakov; Dippold, Michaela

    2016-04-01

    Microbial utilization is a key transformation process of soil organic matter (SOM). Sorption of low molecular weight organic substances (LMWOS) to soil mineral surfaces blocks or delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil science, combined with 13C-phospholipid fatty acid (PLFA) analysis, to assess microbial utilization of sorbed and non-sorbed Alanine in soil. Alanine has various functional groups enabling different sorption mechanisms via its positive charge (e.g. to clay minerals by cation exchange), as well as via its negative charge (e.g. to iron oxides by ligand exchange). To assess changes in the transformation pathways caused by sorption, we added uniformly and position-specifically 13C and 14C labeled Alanine to the Ap of a loamy Luvisol in a short-term (10 days) incubation experiment. To allow for sorption of the tracer solution to an aliquot of this soil, microbial activity was minimized in this subsample by sterilizing the soil by γ-radiation. After shaking, the remaining solutions were filtered and the non-sorbed Alanine was removed with Millipore water and then added to non-sterilized soil. For the free Alanine treatment, solutions with Alanine of similar amount and isotopic composition were prepared, added to the soil and incubated as well. The respired CO2 was trapped in NaOH and its 14C-activity was determined at increasing times intervals. Microbial utilization of Alanine's individual C positions was evaluated in distinct microbial groups classified by 13C-PLFA analysis. Sorption to soil minerals delayed respiration to CO2 and reduced initial respiration rate by 80%. Irrespective of sorption, the highest amount was respired from the carboxylic position (C-1), whereas the amino-bound (C-2) and the methylic position (C-3) were preferentially incorporated into PLFA of microorganisms due to the

  12. Imaging collagen remodeling and sensing transplanted autologous fibroblast metabolism in mouse dermis using multimode nonlinear optical imaging

    NASA Astrophysics Data System (ADS)

    Zhuo, Shuangmu; Chen, Jianxin; Cao, Ning; Jiang, Xingshan; Xie, Shusen; Xiong, Shuyuan

    2008-06-01

    Collagen remodeling and transplanted autologous fibroblast metabolic states in mouse dermis after cellular injection are investigated using multimode nonlinear optical imaging. Our findings show that the technique can image the progress of collagen remodeling in mouse dermis. It can also image transplanted autologous fibroblasts in their collagen matrix environment in the dermis, because of metabolic activity. It was also found that the approach can provide two-photon ratiometric redox fluorometry based on autologous fibroblast fluorescence from reduced nicotinamide adenine dinucleotide coenzyme and oxidized flavoproteins for sensing the autologous fibroblast metabolic state. These results show that the multimode nonlinear optical imaging technique may have potential in a clinical setting as an in vivo diagnostic and monitoring system for cellular therapy in plastic surgery.

  13. Multimodal optical imaging system for in vivo investigation of cerebral oxygen delivery and energy metabolism

    PubMed Central

    Yaseen, Mohammad A.; Srinivasan, Vivek J.; Gorczynska, Iwona; Fujimoto, James G.; Boas, David A.; Sakadžić, Sava

    2015-01-01

    Improving our understanding of brain function requires novel tools to observe multiple physiological parameters with high resolution in vivo. We have developed a multimodal imaging system for investigating multiple facets of cerebral blood flow and metabolism in small animals. The system was custom designed and features multiple optical imaging capabilities, including 2-photon and confocal lifetime microscopy, optical coherence tomography, laser speckle imaging, and optical intrinsic signal imaging. Here, we provide details of the system’s design and present in vivo observations of multiple metrics of cerebral oxygen delivery and energy metabolism, including oxygen partial pressure, microvascular blood flow, and NADH autofluorescence. PMID:26713212

  14. Synthesis and solid-state NMR structural characterization of 13C-labeled graphite oxide.

    PubMed

    Cai, Weiwei; Piner, Richard D; Stadermann, Frank J; Park, Sungjin; Shaibat, Medhat A; Ishii, Yoshitaka; Yang, Dongxing; Velamakanni, Aruna; An, Sung Jin; Stoller, Meryl; An, Jinho; Chen, Dongmin; Ruoff, Rodney S

    2008-09-26

    The detailed chemical structure of graphite oxide (GO), a layered material prepared from graphite almost 150 years ago and a precursor to chemically modified graphenes, has not been previously resolved because of the pseudo-random chemical functionalization of each layer, as well as variations in exact composition. Carbon-13 (13C) solid-state nuclear magnetic resonance (SSNMR) spectra of GO for natural abundance 13C have poor signal-to-noise ratios. Approximately 100% 13C-labeled graphite was made and converted to 13C-labeled GO, and 13C SSNMR was used to reveal details of the chemical bonding network, including the chemical groups and their connections. Carbon-13-labeled graphite can be used to prepare chemically modified graphenes for 13C SSNMR analysis with enhanced sensitivity and for fundamental studies of 13C-labeled graphite and graphene.

  15. Local deposition of 13C tracer in the JET MKII-HD divertor

    NASA Astrophysics Data System (ADS)

    Likonen, Jari; Airila, M. I.; Coad, J. P.; Hakola, A.; Koivuranta, S.; Ahonen, E.; Alves, E.; Barradas, N.; Widdowson, A.; Rubel, M.; Brezinsek, S.; Groth, M.; JET-EFDA Contributors

    2013-07-01

    Migration and deposition of 13C have been investigated at JET by injecting 13C-labelled methane at the outer divertor base at the end of the 2009 campaign. The 13C deposition profile was measured with enhanced proton scattering (EPS) and secondary ion mass spectrometry (SIMS) techniques. A strong toroidal deposition band for 13C was observed experimentally on each of the analysed four outer divertor floor tiles. In addition, 13C was also found on the vertical edge of load bearing tile (LBT) and at the bottom of the LBT tile facing the puffing hole. Local 13C migration in the vicinity of the injection location was modelled by the ERO code. The ERO simulations also produced the strong toroidal 13C deposition band but there is strong deposition also on the vertical edge of the LBT tile and elsewhere on the horizontal part of the outer divertor floor tile.

  16. Metabolic brain imaging correlated with clinical features of brain tumors

    SciTech Connect

    Alavi, J.; Alavi, A.; Dann, R.; Kushner, M.; Chawluk, J.; Powlis, W.; Reivich, M.

    1985-05-01

    Nineteen adults with brain tumors have been studied with positron emission tomography utilizing FDG. Fourteen had biopsy proven cerebral malignant glioma, one each had meningioma, hemangiopericytoma, primitive neuroectodermal tumor (PNET), two had unbiopsied lesions, and one patient had an area of biopsy proven radiation necrosis. Three different patterns of glucose metabolism are observed: marked increase in metabolism at the site of the known tumor in (10 high grade gliomas and the PNET), lower than normal metabolism at the tumor (in 1 grade II glioma, 3 grade III gliomas, 2 unbiopsied low density nonenhancing lesions, and the meningioma), no abnormality (1 enhancing glioma, the hemangiopericytoma and the radiation necrosis.) The metabolic rate of the tumor or the surrounding brain did not appear to be correlated with the history of previous irradiation or chemotherapy. Decreased metabolism was frequently observed in the rest of the affected hemisphere and in the contralateral cerebellum. Tumors of high grade or with enhancing CT characteristics were more likely to show increased metabolism. Among the patients with proven gliomas, survival after PETT scan tended to be longer for those with low metabolic activity tumors than for those with highly active tumors. The authors conclude that PETT may help to predict the malignant potential of tumors, and may add useful clinical information to the CT scan.

  17. Characterization of uniformly and atom-specifically 13C-labeled heparin and heparan sulfate polysaccharide precursors using 13C NMR spectroscopy and ESI mass spectrometry

    PubMed Central

    Nguyen, Thao K. N.; Tran, Vy M.; Victor, Xylophone V.; Skalicky, Jack J.; Kuberan, Balagurunathan

    2010-01-01

    The biological actions of heparin and heparan sulfate, two structurally related glycosaminoglycans, depend on the organization of the complex heparanome. Due to the structural complexity of the heparanome, the sequence of variably sulfonated uronic acid and glucosamine residues is usually characterized by the analysis of smaller oligosaccharide and disaccharide fragments. Even characterization of smaller heparin/heparan sulfate oligosaccharide or disaccharide fragments using simple 1D 1H NMR spectroscopy is often complicated by the extensive signal overlap. 13C NMR signals, on the other hand, overlap less and therefore, 13C NMR spectroscopy can greatly facilitate the structural elucidation of the complex heparanome and provide finer insights into the structural basis for biological functions. This is the first report of the preparation of anomeric carbon-specific 13C-labeled heparin/heparan sulfate precursors from the Escherichia coli K5 strain. Uniformly 13C- and 15N-labeled precursors were also produced and characterized by 13C NMR spectroscopy. Mass spectrometric analysis of enzymatically fragmented disaccharides revealed that anomeric carbon-specific labeling efforts resulted in a minor loss/scrambling of 13C in the precursor backbone, whereas uniform labeling efforts resulted in greater than 95% 13C isotope enrichment in the precursor backbone. These labeled precursors provided high-resolution NMR signals with great sensitivity and set the stage for studying the heparanome–proteome interactions. PMID:20832774

  18. 13C MRS of Human Brain at 7 Tesla Using [2-13C]Glucose Infusion and Low Power Broadband Stochastic Proton Decoupling

    PubMed Central

    Li, Shizhe; An, Li; Yu, Shao; Araneta, Maria Ferraris; Johnson, Christopher S.; Wang, Shumin; Shen, Jun

    2015-01-01

    Purpose 13C magnetic resonance spectroscopy (MRS) of human brain at 7 Tesla (T) may pose patient safety issues due to high RF power deposition for proton decoupling. The purpose of present work is to study the feasibility of in vivo 13C MRS of human brain at 7 T using broadband low RF power proton decoupling. Methods Carboxylic/amide 13C MRS of human brain by broadband stochastic proton decoupling was demonstrated on a 7 T scanner. RF safety was evaluated using the finite-difference time-domain method. 13C signal enhancement by nuclear Overhauser effect (NOE) and proton decoupling was evaluated in both phantoms and in vivo. Results At 7 T, the peak amplitude of carboxylic/amide 13C signals was increased by a factor of greater than 4 due to the combined effects of NOE and proton decoupling. The 7 T 13C MRS technique used decoupling power and average transmit power of less than 35 W and 3.6 W, respectively. Conclusion In vivo 13C MRS studies of human brain can be performed at 7 T well below the RF safety threshold by detecting carboxylic/amide carbons with broadband stochastic proton decoupling. PMID:25917936

  19. Helicobacter pylori infection in type 1 diabetes children and adolescents using 13C urea breath test.

    PubMed

    Chobot, Agata; Bak-Drabik, Katarzyna; Skała-Zamorowska, Eliza; Krzywicka, Agnieszka; Kwiecień, Jarosław; Polańska, Joanna

    2014-01-01

    There is a 10-30% prevalence of HP infection in the general pediatric population in Poland. This study aimed to determine its prevalence in T1DM children in Upper Silesia, Poland and estimate its influence on metabolic control of patients. We studied 149 (82 female) children with T1DM (duration > 12 months, mean HbA1c) and 298 (164 female) age-matched controls. In all cases height and weight z-scores and Cole's index were assessed. In T1DM patients additionally glycated hemoglobin A1c and T1DM duration were analyzed. Presence of HP infection was determined using 13C-isotope-labeled urea breath test (UBT) (fasting and 30 min after ingestion 75 mg of 13C urea). HP infection was present in 17 (11.4%) T1DM patients and in 49 (16.4%) controls (p > 0.05). T1DM patients presented higher values of anthropometric parameters than healthy controls (weight SDS 0.25[-0.46 divided by 0.84] vs. -0.25 [-1.06 divided by 0.26], height SDS 0.09 [-0.60 divided by 0.69] vs. -0.31[-1.17 divided by 0.48] and Cole's index 103% [93 divided by 111%] vs. 97% [86 divided by 106%]; for all p < 0001). Within both groups--T1DM children and controls--no differences regarding sex, age and any of the anthropometric parameters were determined. T1DM duration and HbA1c showed no relation to prevalence of HP infection. Prevalence of HP infection in pediatric T1DM patients is similar to that of healthy peers and shows no relation to glycemic control.

  20. Real-time magnetic resonance imaging and quantification of lipoprotein metabolism in vivo using nanocrystals.

    PubMed

    Bruns, Oliver T; Ittrich, Harald; Peldschus, Kersten; Kaul, Michael G; Tromsdorf, Ulrich I; Lauterwasser, Joachim; Nikolic, Marija S; Mollwitz, Birgit; Merkel, Martin; Bigall, Nadja C; Sapra, Sameer; Reimer, Rudolph; Hohenberg, Heinz; Weller, Horst; Eychmüller, Alexander; Adam, Gerhard; Beisiegel, Ulrike; Heeren, Joerg

    2009-03-01

    Semiconductor quantum dots and superparamagnetic iron oxide nanocrystals have physical properties that are well suited for biomedical imaging. Previously, we have shown that iron oxide nanocrystals embedded within the lipid core of micelles show optimized characteristics for quantitative imaging. Here, we embed quantum dots and superparamagnetic iron oxide nanocrystals in the core of lipoproteins--micelles that transport lipids and other hydrophobic substances in the blood--and show that it is possible to image and quantify the kinetics of lipoprotein metabolism in vivo using fluorescence and dynamic magnetic resonance imaging. The lipoproteins were taken up by liver cells in wild-type mice and displayed defective clearance in knock-out mice lacking a lipoprotein receptor or its ligand, indicating that the nanocrystals did not influence the specificity of the metabolic process. Using this strategy it is possible to study the clearance of lipoproteins in metabolic disorders and to improve the contrast in clinical imaging.

  1. Real-time magnetic resonance imaging and quantification of lipoprotein metabolism in vivo using nanocrystals

    NASA Astrophysics Data System (ADS)

    Bruns, Oliver T.; Ittrich, Harald; Peldschus, Kersten; Kaul, Michael G.; Tromsdorf, Ulrich I.; Lauterwasser, Joachim; Nikolic, Marija S.; Mollwitz, Birgit; Merkel, Martin; Bigall, Nadja C.; Sapra, Sameer; Reimer, Rudolph; Hohenberg, Heinz; Weller, Horst; Eychmüller, Alexander; Adam, Gerhard; Beisiegel, Ulrike; Heeren, Joerg

    2009-03-01

    Semiconductor quantum dots and superparamagnetic iron oxide nanocrystals have physical properties that are well suited for biomedical imaging. Previously, we have shown that iron oxide nanocrystals embedded within the lipid core of micelles show optimized characteristics for quantitative imaging. Here, we embed quantum dots and superparamagnetic iron oxide nanocrystals in the core of lipoproteins-micelles that transport lipids and other hydrophobic substances in the blood-and show that it is possible to image and quantify the kinetics of lipoprotein metabolism in vivo using fluorescence and dynamic magnetic resonance imaging. The lipoproteins were taken up by liver cells in wild-type mice and displayed defective clearance in knock-out mice lacking a lipoprotein receptor or its ligand, indicating that the nanocrystals did not influence the specificity of the metabolic process. Using this strategy it is possible to study the clearance of lipoproteins in metabolic disorders and to improve the contrast in clinical imaging.

  2. Utilization of low molecular weight organics by soil microorganisms: combination of 13C-labelling with PLFA analysis

    NASA Astrophysics Data System (ADS)

    Gunina, Anna; Dippold, Michaela; Kuzyakov, Yakov

    2014-05-01

    Microbial metabolisation is the main transformation pathway of low molecular weight organic substances (LMWOS), but detailed knowledge concerning the fate of LMWOS in soils is strongly limited. Considering that various LMWOS classes enter biochemical cycles at different steps, we hypothesise that the percentage of their LMWOS-Carbon (C) used for microbial biomass (MB) production and consequently medium-term stabilisation in soil is different. We traced the three main groups of LMWOS: amino acids, sugars and carboxylic acids, by uniformly labelled 13C-alanine, -glutamate, -glucose, -ribose, -acetate and -palmitate. Incorporation of 13C from these LMWOS into MB (fumigation-extraction method) and into phospholipid fatty acids (PLFAs) (Bligh-Dyer extraction, purification and GC-C-IRMS measurement) was investigated under field conditions 3 d and 10 d after LMWOS application. The activity of microbial utilization of LMWOS for cell membrane construction was estimated by replacement of PLFA-C with 13C. Decomposition of LMWOS-C comprised 20-65% of the total label, whereas incorporation of 13C into MB amounted to 20-50% of initially applied 13C on day three and was reduced to 5-30% on day 10. Incorporation of 13C-labelled LMWOS into MB followed the trend sugars > carboxylic acids > amino acids. Differences in microbial utilisation between LMWOS were observed mainly at day 10. Thus, instead of initial rapid uptake, further metabolism within microbial cells accounts for the individual fate of C from different LMWOS in soils. Incorporation of 13C from each LMWOS into each PLFA occurred, which reflects the ubiquitous ability of all functional microbial groups for LMWOS utilization. The preferential incorporation of palmitate can be attributed to its role as a direct precursor for many fatty acids (FAs) and PLFA formation. Higher incorporation of alanine and glucose compared to glutamate, ribose and acetate reflect the preferential use of glycolysis-derived substances in the FAs

  3. Medium- and long-chain triglycerides labeled with 13C: a comparison of oxidation after oral or parenteral administration in humans.

    PubMed

    Metges, C C; Wolfram, G

    1991-01-01

    The special physical properties of medium-chain triglycerides (MCT) result in some substantial differences in their metabolism compared to that of long-chain triglycerides (LCT). Administering MCT is of importance in enteral nutrition of patients with disturbances of fat digestion or lipoprotein lipase deficiency. Their use in parenteral nutrition is also of interest. The purpose of this study was to compare the rate of conversion of MCT and LCT to CO2 after parenteral or oral administration in humans. At 1-wk intervals, a liquid formula diet (418 kJ/h for 8 h) was given to five healthy volunteers following an overnight fast. Two hours after starting this, they were given either 100 mg [13C]trioctanoate or [13C]trioleate orally or parenterally. Excess 13C in breath carbon dioxide was analyzed by mass-spectrometry, and oxidation rates over 7.5 h were calculated. Oxidation rates for [13C]trioctanoate were on the average 34.7% after enteral and 31.0% after parenteral administration, and for [13C]trioleate, 25.3 and 24.9%, respectively (p less than 0.05, trioctanoate vs. trioleate). The results show that the oxidation of trioctanoate in healthy humans is greater both after oral and parenteral administration and increases more rapidly than that of [13C]trioleate.

  4. Imaging Sensitivity of Quiescent Cancer Cells to Metabolic Perturbations in Bone Marrow Spheroids

    PubMed Central

    Cavnar, Stephen P.; Xiao, Annie; Gibbons, Anne E.; Rickelmann, Andrew D.; Neely, Taylor; Luker, Kathryn E.; Takayama, Shuichi; Luker, Gary D.

    2016-01-01

    Malignant cells from breast cancer and other common cancers such as prostate and melanoma may persist in bone marrow as quiescent, non-dividing cells that remain viable for years or even decades before resuming proliferation to cause recurrent disease. This phenomenon, referred to clinically as tumor dormancy, poses tremendous challenges to curing patients with breast cancer. Quiescent tumor cells resist chemotherapy drugs that predominantly target proliferating cells, limiting success of neo-adjuvant and adjuvant therapies. We recently developed a 3D spheroid model of quiescent breast cancer cells in bone marrow for mechanistic and drug testing studies. We combined this model with optical imaging methods for label-free detection of cells preferentially utilizing glycolysis versus oxidative metabolism to investigate the metabolic state of co-culture spheroids with different bone marrow stromal and breast cancer cells. Through imaging and biochemical assays, we identified different metabolic states of bone marrow stromal cells that control metabolic status and flexibilities of co-cultured breast cancer cells. We tested metabolic stresses and targeted inhibition of specific metabolic pathways to identify approaches to preferentially eliminate quiescent breast cancer cells from bone marrow environments. These studies establish an integrated imaging approach to analyze metabolism in complex tissue environments to identify new metabolically-targeted cancer therapies. PMID:27478871

  5. Functional imaging using the retinal function imager: direct imaging of blood velocity, achieving fluorescein angiography-like images without any contrast agent, qualitative oximetry, and functional metabolic signals.

    PubMed

    Izhaky, David; Nelson, Darin A; Burgansky-Eliash, Zvia; Grinvald, Amiram

    2009-07-01

    The Retinal Function Imager (RFI; Optical Imaging, Rehovot, Israel) is a unique, noninvasive multiparameter functional imaging instrument that directly measures hemodynamic parameters such as retinal blood-flow velocity, oximetric state, and metabolic responses to photic activation. In addition, it allows capillary perfusion mapping without any contrast agent. These parameters of retinal function are degraded by retinal abnormalities. This review delineates the development of these parameters and demonstrates their clinical applicability for noninvasive detection of retinal function in several modalities. The results suggest multiple clinical applications for early diagnosis of retinal diseases and possible critical guidance of their treatment.

  6. Bioorthogonal chemical imaging of metabolic activities in live mammalian hippocampal tissues with stimulated Raman scattering

    PubMed Central

    Hu, Fanghao; Lamprecht, Michael R.; Wei, Lu; Morrison, Barclay; Min, Wei

    2016-01-01

    Brain is an immensely complex system displaying dynamic and heterogeneous metabolic activities. Visualizing cellular metabolism of nucleic acids, proteins, and lipids in brain with chemical specificity has been a long-standing challenge. Recent development in metabolic labeling of small biomolecules allows the study of these metabolisms at the global level. However, these techniques generally require nonphysiological sample preparation for either destructive mass spectrometry imaging or secondary labeling with relatively bulky fluorescent labels. In this study, we have demonstrated bioorthogonal chemical imaging of DNA, RNA, protein and lipid metabolism in live rat brain hippocampal tissues by coupling stimulated Raman scattering microscopy with integrated deuterium and alkyne labeling. Heterogeneous metabolic incorporations for different molecular species and neurogenesis with newly-incorporated DNA were observed in the dentate gyrus of hippocampus at the single cell level. We further applied this platform to study metabolic responses to traumatic brain injury in hippocampal slice cultures, and observed marked upregulation of protein and lipid metabolism particularly in the hilus region of the hippocampus within days of mechanical injury. Thus, our method paves the way for the study of complex metabolic profiles in live brain tissue under both physiological and pathological conditions with single-cell resolution and minimal perturbation. PMID:28000773

  7. Bioorthogonal chemical imaging of metabolic activities in live mammalian hippocampal tissues with stimulated Raman scattering

    NASA Astrophysics Data System (ADS)

    Hu, Fanghao; Lamprecht, Michael R.; Wei, Lu; Morrison, Barclay; Min, Wei

    2016-12-01

    Brain is an immensely complex system displaying dynamic and heterogeneous metabolic activities. Visualizing cellular metabolism of nucleic acids, proteins, and lipids in brain with chemical specificity has been a long-standing challenge. Recent development in metabolic labeling of small biomolecules allows the study of these metabolisms at the global level. However, these techniques generally require nonphysiological sample preparation for either destructive mass spectrometry imaging or secondary labeling with relatively bulky fluorescent labels. In this study, we have demonstrated bioorthogonal chemical imaging of DNA, RNA, protein and lipid metabolism in live rat brain hippocampal tissues by coupling stimulated Raman scattering microscopy with integrated deuterium and alkyne labeling. Heterogeneous metabolic incorporations for different molecular species and neurogenesis with newly-incorporated DNA were observed in the dentate gyrus of hippocampus at the single cell level. We further applied this platform to study metabolic responses to traumatic brain injury in hippocampal slice cultures, and observed marked upregulation of protein and lipid metabolism particularly in the hilus region of the hippocampus within days of mechanical injury. Thus, our method paves the way for the study of complex metabolic profiles in live brain tissue under both physiological and pathological conditions with single-cell resolution and minimal perturbation.

  8. Pentose cycling and the distribution of 13C in trehalose during glucogenesis from 13C-labelled substrates in an insect.

    PubMed

    Thompson, S N; Scales, V M; Bochardt, D B

    1995-07-26

    Redistribution of 13C in trehalose (Tre) due to pentose cycling was observed in vivo in Manduca sexta during glucogenesis from [3-13C]alanine (Ala) and [2-13C]glycerol (Gly). The extent of cycling was affected by dietary composition. Larvae maintained on a low-carbohydrate diet (LCD) exhibited approximately 13% cycling, while those on a complete-balanced diet (CBD) or low-fat diet (LFD) displayed much higher rates of cycling. Significant incorporation of 13C via reversal of the non-oxidative phase was evident on all diets but was greatest on the CBD and LFD. In contrast to conclusions from previous studies with insects, the present results indicate that under normal conditions the pentose pathway is not the principal source of triose phosphates for oxidative catabolism during larval development.

  9. Isotopic 13C NMR spectrometry to assess counterfeiting of active pharmaceutical ingredients: site-specific 13C content of aspirin and paracetamol.

    PubMed

    Silvestre, Virginie; Mboula, Vanessa Maroga; Jouitteau, Catherine; Akoka, Serge; Robins, Richard J; Remaud, Gérald S

    2009-10-15

    Isotope profiling is a well-established technique to obtain information about the chemical history of a given compound. However, the current methodology using IRMS can only determine the global (13)C content, leading to the loss of much valuable data. The development of quantitative isotopic (13)C NMR spectrometry at natural abundance enables the measurement of the (13)C content of each carbon within a molecule, thus giving simultaneous access to a number of isotopic parameters. When it is applied to active pharmaceutical ingredients, each manufactured batch can be characterized better than by IRMS. Here, quantitative isotopic (13)C NMR is shown to be a very promising and effective tool for assessing the counterfeiting of medicines, as exemplified by an analysis of aspirin (acetylsalicylic acid) and paracetamol (acetaminophen) samples collected from pharmacies in different countries. It is proposed as an essential complement to (2)H NMR and IRMS.

  10. Hyperpolarized 13C MR Markers of Renal Tumor Aggressiveness

    DTIC Science & Technology

    2014-10-01

    lactate in the media. We observed three fold higher rate of lactate secreted into the media by the ccRCC tissue compared to the normal kidney (figure...imaging sequences with higher spatial resolution as well as sensitivity to generate contrast between the mouse kidney and the tumor grafts. This will...signal in the tumor grafts compared to the high background signal arising from the mouse kidney , several optimizations were explored, such as diffusion

  11. Site-specific 13C content by quantitative isotopic 13C nuclear magnetic resonance spectrometry: a pilot inter-laboratory study.

    PubMed

    Chaintreau, Alain; Fieber, Wolfgang; Sommer, Horst; Gilbert, Alexis; Yamada, Keita; Yoshida, Naohiro; Pagelot, Alain; Moskau, Detlef; Moreno, Aitor; Schleucher, Jürgen; Reniero, Fabiano; Holland, Margaret; Guillou, Claude; Silvestre, Virginie; Akoka, Serge; Remaud, Gérald S

    2013-07-25

    Isotopic (13)C NMR spectrometry, which is able to measure intra-molecular (13)C composition, is of emerging demand because of the new information provided by the (13)C site-specific content of a given molecule. A systematic evaluation of instrumental behaviour is of importance to envisage isotopic (13)C NMR as a routine tool. This paper describes the first collaborative study of intra-molecular (13)C composition by NMR. The main goals of the ring test were to establish intra- and inter-variability of the spectrometer response. Eight instruments with different configuration were retained for the exercise on the basis of a qualification test. Reproducibility at the natural abundance of isotopic (13)C NMR was then assessed on vanillin from three different origins associated with specific δ (13)Ci profiles. The standard deviation was, on average, between 0.9 and 1.2‰ for intra-variability. The highest standard deviation for inter-variability was 2.1‰. This is significantly higher than the internal precision but could be considered good in respect of a first ring test on a new analytical method. The standard deviation of δ (13)Ci in vanillin was not homogeneous over the eight carbons, with no trend either for the carbon position or for the configuration of the spectrometer. However, since the repeatability for each instrument was satisfactory, correction factors for each carbon in vanillin could be calculated to harmonize the results.

  12. [The use of the [13C]/[12C] ratio for the assay of the microbial oxidation of hydrocarbons].

    PubMed

    Ziakun, A M; Kosheleva, I A; Zakharchenko, V N; Kudriavtseva, A I; Peshenko, V A; Filonov, A E; Boronin, A M

    2003-01-01

    The study deals with a comparative analysis of the relative abundances of the carbon isotopes 12C and 13C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope composition of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (delta 13C = -44.6 +/- 0.2@1000) were characterized by the isotope effects delta 13CCO2 = -50.2 +/- 0.4@1000, delta 13Cbiom = -46.6 +/- 0.4@1000 and delta 13Cexo = -41.5 +/- 0.4@1000, respectively. The isotope composition of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (delta 13C = -21 +/- 0.4@1000) were characterized by the isotope effects delta 13CCO2 = -24.1 +/- 0.4@1000, delta 13Cbiom = -19.2 +/- 0.4@1000 and delta 13Cexo = -19.1 +/- 0.4@1000, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the enviroment is discussed.

  13. Quantifying the Contribution of Grape Hexoses to Wine Volatiles by High-Precision [U13C]-Glucose Tracer Studies

    PubMed Central

    Nisbet, Mark A.; Tobias, Herbert J.; Brenna, J. Thomas; Sacks, Gavin L.; Mansfield, Anna Katharine

    2016-01-01

    Many fermentation volatiles important to wine aroma potentially arise from yeast metabolism of hexose sugars, but assessing the relative importance of these pathways is challenging due to high endogenous hexose substrate concentrations. To overcome this problem, gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) was used to measure high-precision 13C/12C isotope ratios of volatiles in wines produced from juices spiked with tracer levels (0.01–1 APE) of uniformly labeled [U-13C]-glucose. The contribution of hexose to individual volatiles was determined from the degree of 13C enrichment. As expected, straight-chain fatty acids and their corresponding ethyl esters were derived almost exclusively from hexoses. Most fusel alcohols and their acetate esters were also majority hexose-derived, indicating the importance of anabolic pathways for their formation. Only two compounds were not derived primarily from hexoses (hexanol and isobutyric acid). This approach can be extended to other food systems or substrates for studying precursor–product relationships. PMID:24960193

  14. The influence of diet on the δ 13C of shell carbon in the pulmonate snail Helix aspersa

    NASA Astrophysics Data System (ADS)

    Stott, Lowell D.

    2002-02-01

    The influence of diet and atmospheric CO 2 on the carbon isotope composition of shell aragonite and shell-bound organic carbon in the pulmonate snail Helix aspersa raised in the laboratory was investigated. Three separate groups of snails were raised on romaine lettuce (C3 plant, δ 13C=-25.8‰), corn (C4 plant, δ 13C=-10.5‰), and sour orange ( 12C-enriched C3 plant, δ 13C=-39.1‰). The isotopic composition of body tissues closely tracked the isotopic composition of the snail diet as demonstrated previously. However, the isotopic composition of the acid insoluble organic matrix extracted from the aragonite shells does not track diet in all groups. In snails that were fed corn the isotopic composition of the organic matrix was more negative than the body by as much as 5‰ whereas the matrix was approximately 1‰ heavier than the body tissues in snails fed a diet of C3 plant material. These results indicate that isotopic composition of the organic matrix carbon cannot be used as an isotopic substrate for paleodietary reconstructions without first determining the source of the carbon and any associated fractionations. The isotopic composition of the shell aragonite is offset from the body tissues by 12.3‰ in each of the culture groups. This offset was not influenced by the consumption of carbonate and is not attributable to the diffusion of atmospheric CO 2 into the hemolymph. The carbon isotopic composition of shell aragonite is best explained in terms of equilibrium fractionations associated with exchange between metabolic CO 2 and HCO 3 in the hemolymph and the fractionation associated with carbonate precipitation. These results differ from previous studies, based primarily on samples collected in the field, that have suggested atmospheric carbon dioxide contributes significantly to the shell δ 13C. The culture results indicate that the δ 13C of aragonite is a good recorder of the isotopic composition of the snail body tissue, and therefore a better

  15. 13cRA regulates the differentiation of antler chondrocytes through targeting Runx3.

    PubMed

    Zhang, Hong-Liang; Cao, Hang; Yang, Zhan-Qing; Geng, Shuang; Wang, Kai; Yu, Hai-Fan; Guo, Bin; Yue, Zhan-Peng

    2017-03-01

    Although 13cRA is involved in the regulation of cellular proliferation and differentiation, its physiological roles in chondrocyte proliferation and differentiation still remain unknown. Here, we showed that 13cRA could induce the proliferation of sika deer antler chondrocytes and expression of Ccnd3 and Cdk6. Administration of 13cRA to antler chondrocytes resulted in an obvious increase in the expression of chondrocyte marker Col II and hypertrophic chondrocyte marker Col X. Silencing of Crabp2 expression by specific siRNA could prevent the 13cRA-induced up-regulation of Col X, whereas overexpression of Crabp2 showed the opposite effects. Further study found that Crabp2 mediated the regulation of 13cRA on the expression of Runx3 which was highly expressed in the antler cartilage and inhibited the differentiation of antler chondrocytes. Moreover, attenuation of Runx3 expression greatly raised 13cRA-induced chondrocyte differentiation. Simultaneously, 13cRA could stimulate the expression of Cyp26a1 and Cyp26b1 in the antler chondrocytes. Inhibition of Cyp26a1 and/or Cyp26b1 reinforced the effects of 13cRA on the expression of Col X and Runx3, while overexpression of Cyp26b1 rendered the antler chondrocytes hyposensitive to 13cRA. Collectively, 13cRA may play an important role in the differentiation of antler chondrocytes through targeting Runx3. Crabp2 enhances the effects of 13cRA on chondrocyte differentiation, while Cyp26a1 and Cyp26b1 weaken the sensitivity of antler chondrocytes to 13cRA.

  16. Synthesis of isotopically labeled R- or S-[.sup.13C, .sup.2H] glycerols

    DOEpatents

    Martinez, Rodolfo A.; Unkefer, Clifford J.; Alvarez, Marc A.

    2008-01-22

    The present invention is directed to asymmetric chiral labeled glycerols including at least one chiral atom, from one to two .sup.13C atoms and from zero to four deuterium atoms bonded directly to a carbon atom, e.g., (2S) [1,2-.sup.13C.sub.2]glycerol and (2R) [1,2-.sup.13C.sub.2]glycerol, and to the use of such chiral glycerols in the preparation of labeled amino acids.

  17. 13C-NOESY-HSQC with Split Carbon Evolution for Increased Resolution with Uniformly Labeled Proteins

    NASA Astrophysics Data System (ADS)

    Baur, Matthias; Gemmecker, Gerd; Kessler, Horst

    1998-06-01

    Two new pulse sequences are presented for the recording of 2D13C-HSQC and 3D13C-NOESY-HSQC experiments, containing two consecutive carbon evolution periods. The two periods are separated by az-filter which creates a clean CxHz-quantum state for evolution in the second period. Each period is incremented (in anon-constant-time fashion) only to the extent that the defocusing of carbon inphase magnetization throughJ-coupling with neighboring carbons remains insignificant. Therefore,13C homonuclearJ-couplings are rendered ineffective, reducing the loss of signal and peak splitting commonly associated with long13C evolution times. The two periods are incremented according to a special acquisition protocol employing a13C-13C gradient echo to yield a data set analogous to one obtained by evolution over the added duration of both periods. The spectra recorded with the new technique on uniformly13C-labeled proteins at twice the evolution time of the standard13C-HSQC experiment display a nearly twofold enhancement of resolution in the carbon domain, while maintaining a good sensitivity even in the case of large proteins. Applied to the IIAManprotein ofE. coli(31 kDa), the13C-HSQC experiment recorded with a carbon evolution time of 2 × 8 ms showed a 36% decrease in linewidths compared to the standard13C-HSQC experiment, and theS/Nratio of representative cross-peaks was reduced to 40%. This reduction reflects mostly the typical loss of intensity observed when recording with an increased resolution. The13C-NOESY-HSQC experiment derived from the13C-HSQC experiment yielded additional NOE restraints between resonances which previously had been unresolved.

  18. Lowest bending mode of 13C-substituted C3 and an experimentally derived structure

    NASA Astrophysics Data System (ADS)

    Breier, Alexander A.; Büchling, Thomas; Schnierer, Rico; Lutter, Volker; Fuchs, Guido W.; Yamada, Koichi M. T.; Mookerjea, Bhaswati; Stutzki, Jürgen; Giesen, Thomas F.

    2016-12-01

    The ν2 lowest bending mode of linear C3 and of all its 13C-substituted isotopologues was recorded using a terahertz-supersonic jet spectrometer in combination with a laser ablation source. Sixty-five ro-vibrational transitions between 1.8 and 1.9 THz have been assigned to linear 12C12C12C, 12C12C13, 12C13C12C, 13C13C12C, 13C12C13C, and 13C13C13. For each isotopologue, molecular parameters were obtained and the C-C-bond length was derived experimentally. All results are in excellent agreement with recent ab initio calculations [B. Schröder and P. Sebald, J. Chem. Phys. 144, 044307 (2016)]. The new measurements explain why the interstellar search for singly substituted 12C12C13C has failed so far. A spectral line list with recommended transition frequencies based on global data fits is given to foster future interstellar detections.

  19. Survival of free-living Acholeplasma in aerated pig manure slurry revealed by 13C-labeled bacterial biomass probing

    PubMed Central

    Hanajima, Dai; Aoyagi, Tomo; Hori, Tomoyuki

    2015-01-01

    Many studies have been performed on microbial community succession and/or predominant taxa during the composting process; however, the ecophysiological roles of microorganisms are not well understood because microbial community structures are highly diverse and dynamic. Bacteria are the most important contributors to the organic-waste decomposition process, while decayed bacterial cells can serve as readily digested substrates for other microbial populations. In this study, we investigated the active bacterial species responsible for the assimilation of dead bacterial cells and their components in aerated pig manure slurry by using 13C-labeled bacterial biomass probing. After 3 days of forced aeration, 13C-labeled and unlabeled dead Escherichia coli cell suspensions were added to the slurry. The suspensions contained 13C-labeled and unlabeled bacterial cell components, possibly including the cell wall and membrane, as well as intracellular materials. RNA extracted from each slurry sample 2 h after addition of E. coli suspension was density-resolved by isopycnic centrifugation and analyzed by terminal restriction fragment length polymorphism, followed by cloning and sequencing of bacterial 16S rRNA genes. In the heavy isotopically labeled RNA fraction, the predominant 13C-assimilating population was identified as belonging to the genus Acholeplasma, which was not detected in control heavy RNA. Acholeplasma spp. have limited biosynthetic capabilities and possess a wide variety of transporters, resulting in their metabolic dependence on external carbon and energy sources. The prevalence of Acholeplasma spp. was further confirmed in aerated pig manure slurry from four different pig farms by pyrosequencing of 16S rRNA genes; their relative abundance was ∼4.4%. Free-living Acholeplasma spp. had a competitive advantage for utilizing dead bacterial cells and their components more rapidly relative to other microbial populations, thus allowing the survival and prevalence

  20. Nitrogen control of 13C enrichment in heterotrophic organs relative to leaves in a landscape-building desert plant species

    NASA Astrophysics Data System (ADS)

    Zhang, J.; Gu, L.; Bao, F.; Cao, Y.; Hao, Y.; He, J.; Li, J.; Li, Y.; Ren, Y.; Wang, F.; Wu, R.; Yao, B.; Zhao, Y.; Lin, G.; Wu, B.; Lu, Q.; Meng, P.

    2014-09-01

    A longstanding puzzle in isotope studies of C3 plant species is that heterotrophic plant organs (e.g., stems, roots, seeds, and fruits) tend to be enriched in 13C compared to the autotrophic organ (leaves) that provides them with photosynthate. Our inability to explain this puzzle suggests key deficiencies in understanding post-photosynthetic metabolic processes. It also limits the effectiveness of applications of stable carbon isotope analyses in a variety of scientific disciplines ranging from plant physiology to global carbon cycle studies. To gain insight into this puzzle, we excavated whole plant architectures of Nitraria tangutorum Bobrov, a C3 species that has an exceptional capability of fixing sands and building sand dunes, in two deserts in northwestern China. We systematically and simultaneously measured carbon isotope ratios and nitrogen and phosphorous contents of different parts of the excavated plants. We also determined the seasonal variations in leaf carbon isotope ratios on nearby intact plants of N. tangutorum. We found, for the first time, that higher nitrogen contents in heterotrophic organs were significantly correlated with increased heterotrophic 13C enrichment compared to leaves. However, phosphorous contents had no effect on the enrichment. In addition, new leaves had carbon isotope ratios similar to roots but were progressively depleted in 13C as they matured. We concluded that a nitrogen-mediated process, probably the refixation of respiratory CO2 by phosphoenolpyruvate (PEP) carboxylase, was responsible for the differences in 13C enrichment among different heterotrophic organs while processes within leaves or during phloem loading may contribute to the overall autotrophic - heterotrophic difference in carbon isotope compositions.

  1. Nitrogen control of 13C enrichment in heterotrophic organs relative to leaves in a landscape-building desert plant species

    DOE PAGES

    Zhang, J.; Gu, L.; Bao, F.; ...

    2014-09-10

    A longstanding puzzle in isotope studies of C3 plant species is that heterotrophic plant organs (e.g., stems, roots, seeds, and fruits) tend to be enriched in 13C compared to the autotrophic organ (leaves) that provides them with photosynthate. Our inability to explain this puzzle suggests key deficiencies in understanding post-photosynthetic metabolic processes. It also limits the effectiveness of applications of stable carbon isotope analyses in a variety of scientific disciplines ranging from plant physiology to global carbon cycle studies. To gain insight into this puzzle, we excavated whole plant architectures of Nitraria tangutorum Bobrov, a C3 species that has anmore » exceptional capability of fixing sands and building sand dunes, in two deserts in northwestern China. We systematically and simultaneously measured carbon isotope ratios and nitrogen and phosphorous contents of different parts of the excavated plants. We also determined the seasonal variations in leaf carbon isotope ratios on nearby intact plants of N. tangutorum. We found, for the first time, that higher nitrogen contents in heterotrophic organs were significantly correlated with increased heterotrophic 13C enrichment compared to leaves. However, phosphorous contents had no effect on the enrichment. In addition, new leaves had carbon isotope ratios similar to roots but were progressively depleted in 13C as they matured. We concluded that a nitrogen-mediated process, probably the refixation of respiratory CO2 by phosphoenolpyruvate (PEP) carboxylase, was responsible for the differences in 13C enrichment among different heterotrophic organs while processes within leaves or during phloem loading may contribute to the overall autotrophic – heterotrophic difference in carbon isotope compositions.« less

  2. Microbial transformations of free versus sorbed alanine analyzed by position-specific 13C and 14C labeling and 13C-PLFA analysis

    NASA Astrophysics Data System (ADS)

    Apostel, Carolin; Dippold, Michaela; Bore, Ezekiel; Kuzyakov, Yakov

    2015-04-01

    Sorption of charged or partially charged low molecular weight organic substances (LMWOS) to soil mineral surfaces delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil sciences, to compare the transformation mechanisms of sorbed and non-sorbed alanine in soil. Alanine as an amino acid links C- and N-cycles in soil and therefore is a model representative for the pool of LMWOS. To assess transformations of sorbed alanine, we combined position-specifically and uniformly 13C and 14C labeled alanine tracer solution with a loamy haplic luvisol that had previously been sterilized by γ-radiation. After shaking the mixtures, the supernatant was removed, as was all non-sorbed alanine by repeated shaking with millipore water. The labeled soil was added to non-sterilized soil from the same site. To compare the effect of sorption, soil labeled with the same position-specifically labeled tracers without previous sorption was prepared and incubated as well. We captured the respired CO2 and determined its 14C-activity at increasing time steps. The incorporation of 14C into microbial biomass was determined by CFE, and utilization of individual C positions by distinct microbial groups was evaluated by 13C-PLFA analysis. A dual peak in the respired CO2 revealed the influence of two sorption mechanisms. Microbial uptake and transformation of the sorbed alanine was 3 times slower compared to non-sorbed alanine. To compare the fate of individual C atoms independent of their concentration and pool size in soil, we introduced the divergence index (DI). The DI reveals the convergent or divergent behaviour of C from individual molecule positions during microbial utilization. The DI revealed, that alanines C-1 position was mainly oxidized to CO2, while its C-2 and C-3 were preferentially incorporated in microbial biomass and PLFAs. This indicates

  3. The Nature of Carbonate and Organic δ13C Covariance Through Geological Time

    NASA Astrophysics Data System (ADS)

    Oehlert, A. M.; Swart, P. K.

    2014-12-01

    Significant evolutionary, climatic, and oceanographic events in Earth history are often accompanied by excursions in the carbon isotope composition (δ13C) of marine carbonates and co-occurring sedimentary organic material. The observation of synchronous excursions in the δ13C values of marine carbonates and coeval organic matter is commonly thought to prove that the deposit has not been altered by diagenesis, and that the variations in the δ13C records are the result of a significant change in global carbon cycling. Furthermore, this model suggests that the covariance of carbonate and organic δ13C records is driven only by changes in the δ13C value of the dissolved inorganic carbon in the surface waters of the ocean. However, recent work suggests that there may be at least two alternate models for generating covariance between carbonate and organic δ13C values in the geologic record. One of the models invokes sea-level driven syndepositional mixing between isotopically distinct sources of carbonate and organic material to produce positive covariance between carbonate and organic δ13C values. The second model suggests that post-depositional alteration to the carbonate δ13C values during meteoric diagenesis, in concert with concurrent contributions of terrestrial organic material during subaerial exposure, can also produce co-occurring negative excursions with tightly covariant δ13C records. In contrast to earlier interpretations of covariant δ13C values, these models suggest that both syndepositional and post-depositional factors can significantly influence the relationship between carbonate and organic δ13C values in a variety of depositional environments. The implications for reconstructions of ancient global carbon cycle events will be explored within the context of these three models, and their relative importance throughout geologic time will be discussed.

  4. Preliminary studies of a canine 13C-aminopyrine demethylation blood test.

    PubMed Central

    Moeller, E M; Steiner, J M; Williams, D A; Klein, P D

    2001-01-01

    The objectives of this study were to determine whether a 13C-aminopyrine demethylation blood test is technically feasible in clinically healthy dogs, whether oral administration of 13C-aminopyrine causes a detectable increase in percent dose/min (PCD) of 13C administered as 13C-aminopyrine and recovered in gas extracted from blood, and whether gas extraction efficiency has an impact on PCD. A dose of 2 mg/kg body weight of 13C-aminopyrine dissolved in deionized water was administered orally to 6 clinically healthy dogs. Blood samples were taken from each dog 0, 30, 60, and 120 min after administration of the 13C-aminopyrine. Carbon dioxide was extracted from blood samples by addition of acid and analyzed by fractional mass spectrometry. None of the 6 dogs showed any side effects after 13C-aminopyrine administration. All 6 dogs showed a measurable increase of the PCD in gas samples extracted from blood samples at 30 min, 60 min, and 120 min after 13C-aminopyrine administration. Coefficients of variation between the triplicate samples were statistically significantly higher for the %CO2, a measure of extraction efficiency, than for PCD values (P < 0.0001). The 13C-aminopyrine demethylation blood test described here is technically feasible. Oral administration of 13C-aminopyrine did not lead to gross side effects in the 6 dogs. Clinically healthy dogs show a measurable increase of PCD in gas extracted from blood samples after oral administration of 13C-aminopyrine. Efficiency of CO2 extraction from blood samples does not have an impact on PCD determined from these blood samples. This test may prove useful to evaluate hepatic function in dogs. PMID:11227194

  5. Preserved pontine glucose metabolism in Alzheimer disease: A reference region for functional brain image (PET) analysis

    SciTech Connect

    Minoshima, Satoshi; Frey, K.A.; Foster, N.L.; Kuhl, D.W.

    1995-07-01

    Our goal was to examine regional preservation of energy metabolism in Alzheimer disease (AD) and to evaluate effects of PET data normalization to reference regions. Regional metabolic rates in the pons, thalamus, putamen, sensorimotor cortex, visual cortex, and cerebellum (reference regions) were determined stereotaxically and examined in 37 patients with probable AD and 22 normal controls based on quantitative {sup 18}FDG-PET measurements. Following normalization of metabolic rates of the parietotemporal association cortex and whole brain to each reference region, distinctions of the two groups were assessed. The pons showed the best preservation of glucose metabolism in AD. Other reference regions showed relatively preserved metabolism compared with the parietotemporal association cortex and whole brain, but had significant metabolic reduction. Data normalization to the pons not only enhanced statistical significance of metabolic reduction in the parietotemporal association cortex, but also preserved the presence of global cerebral metabolic reduction indicated in analysis of the quantitative data. Energy metabolism in the pons in probable AD is well preserved. The pons is a reliable reference for data normalization and will enhance diagnostic accuracy and efficiency of quantitative and nonquantitative functional brain imaging. 39 refs., 2 figs., 3 tabs.

  6. A novel proteomic approach for specific identification of tyrosine kinase substrates using [13C]tyrosine.

    PubMed

    Ibarrola, Nieves; Molina, Henrik; Iwahori, Akiko; Pandey, Akhilesh

    2004-04-16

    Proteomic studies to find substrates of tyrosine kinases generally rely on identification of protein bands that are "pulled down" by antiphosphotyrosine antibodies from ligand-stimulated samples. One can obtain erroneous results from such experiments because of two major reasons. First, some proteins might be basally phosphorylated on tyrosine residues in the absence of ligand stimulation. Second, proteins can bind non-specifically to the antibodies or the affinity matrix. Induction of phosphorylation of proteins by ligand must therefore be confirmed by a different approach, which is not always feasible. We have developed a novel proteomic approach to identify substrates of tyrosine kinases in signaling pathways studies based on in vivo labeling of proteins with "light" (12C-labeled) or "heavy" (13C-labeled) tyrosine. This stable isotope labeling in cell culture method enables the unequivocal identification of tyrosine kinase substrates, as peptides derived from true substrates give rise to a unique signature in a mass spectrometry experiment. By using this approach, from a single experiment, we have successfully identified several known substrates of insulin signaling pathway and a novel substrate, polymerase I and transcript release factor, a protein that is implicated in the control of RNA metabolism and regulation of type I collagen promoters. This approach is amenable to high throughput global studies as it simplifies the specific identification of substrates of tyrosine kinases as well as serine/threonine kinases using mass spectrometry.

  7. Analogy between mission critical detection in distributed systems and 13C isotope separation column

    NASA Astrophysics Data System (ADS)

    Boca, Maria L.; Secara, Mihai

    2015-02-01

    Carbon represents the fourth most abundant chemical element in the world, having two stable and one radioactive isotope. The 13 Carbon isotopes, with a natural abundance of 1.1%, plays an important role in numerous applications, such as the study of human metabolism changes, molecular structure studies, non-invasive respiratory tests, Alzheimer tests, air pollution and global warming effects on plants [2]. Distributed systems are increasingly being applied in critical real-time applications and their complexity forces programmers to use design methods which guarantee correctness and increase the maintainability of the products. Objectoriented methodologies are widely used to cope with complexity in any kind of system, but most of them lack a formal foundation to allow the analysis and verification of designs, which is one of the main requirements for dealing with concurrent and reactive systems. This research is intended to make an analogy between two tips of industrial processes, one 13C Isotope Separation Column and other one distributed systems. We try to highlight detection of "mission critical "situations for this two processes and show with one is more critical and needs deeply supervisyon [1], [3].

  8. Environmental controls over methanol production, emission, and δ13C values from Lycopersicon esculentum

    NASA Astrophysics Data System (ADS)

    Oikawa, P.; Giebel, B. M.; Mak, J. E.; Riemer, D. D.; Swart, P. K.; Lerdau, M.

    2009-12-01

    Phytogenic methanol is the dominant source of methanol to the atmosphere, where it is the second most abundant organic compound. Beyond methanol’s role in atmospheric chemistry, it is an indicator of plant function and is linked to plant wound response. Methanol emissions are considered to be a by-product of cell wall expansion and, more specifically, the demethylation of pectin by pectin methylesterase (PME) in cell walls. Production of methanol was investigated in mature and immature tomato Lycopersicon esculentum via measurement of methanol flux, foliar PME activity, and methanol extraction from leaf, root, and stem tissues. δ13C values for mature and immature methanol emissions were also measured using a GC-IRMS system. Environmental control over methanol production and emission was studied by changing temperature and light while holding stomatal conductance constant. As seen previously, mature leaf methanol emissions were significantly less than immature emissions. Surprisingly, preliminary results suggest mature leaf methanol production to be similar to immature leaves, indicating an enhanced metabolic sink for methanol in mature leaves. These data enhance our understanding of methanol production, a term which is not well constrained in current methanol flux models.

  9. Positional Enrichment by Proton Analysis (PEPA): A One-Dimensional (1) H-NMR Approach for (13) C Stable Isotope Tracer Studies in Metabolomics.

    PubMed

    Vinaixa, Maria; Rodríguez, Miguel A; Aivio, Suvi; Capellades, Jordi; Gómez, Josep; Canyellas, Nicolau; Stracker, Travis H; Yanes, Oscar

    2017-03-20

    A novel metabolomics approach for NMR-based stable isotope tracer studies called PEPA is presented, and its performance validated using human cancer cells. PEPA detects the position of carbon label in isotopically enriched metabolites and quantifies fractional enrichment by indirect determination of (13) C-satellite peaks using 1D-(1) H-NMR spectra. In comparison with (13) C-NMR, TOCSY and HSQC, PEPA improves sensitivity, accelerates the elucidation of (13) C positions in labeled metabolites and the quantification of the percentage of stable isotope enrichment. Altogether, PEPA provides a novel framework for extending the high-throughput of (1) H-NMR metabolic profiling to stable isotope tracing in metabolomics, facilitating and complementing the information derived from 2D-NMR experiments and expanding the range of isotopically enriched metabolites detected in cellular extracts.

  10. Economical synthesis of 13C-labeled opiates, cocaine derivatives and selected urinary metabolites by derivatization of the natural products.

    PubMed

    Karlsen, Morten; Liu, Huiling; Johansen, Jon Eigill; Hoff, Bård Helge

    2015-03-25

    The illegal use of opiates and cocaine is a challenge world-wide, but some derivatives are also valuable pharmaceuticals. Reference samples of the active ingredients and their metabolites are needed both for controlling administration in the clinic and to detect drugs of abuse. Especially, (13)C-labeled compounds are useful for identification and quantification purposes by mass spectroscopic techniques, potentially increasing accuracy by minimizing ion alteration/suppression effects. Thus, the synthesis of [acetyl-(13)C4]heroin, [acetyl-(13)C4-methyl-(13)C]heroin, [acetyl-(13)C2-methyl-(13)C]6-acetylmorphine, [N-methyl-(13)C-O-metyl-(13)C]codeine and phenyl-(13)C6-labeled derivatives of cocaine, benzoylecgonine, norcocaine and cocaethylene was undertaken to provide such reference materials. The synthetic work has focused on identifying (13)C atom-efficient routes towards these derivatives. Therefore, the (13)C-labeled opiates and cocaine derivatives were made from the corresponding natural products.

  11. Combining position-specific 13C labeling with compound-specific isotope analysis: first steps towards soil fluxomics

    NASA Astrophysics Data System (ADS)

    Dippold, Michaela; Kuzyakov, Yakov

    2015-04-01

    Understanding the soil organic matter (SOM) dynamics is one of the most important challenges in soil science. Transformation of low molecular weight organic substances (LMWOS) is a key step in biogeochemical cycles because 1) all high molecular substances pass this stage during their decomposition and 2) only LMWOS will be taken up by microorganisms. Previous studies on LMWOS were focused on determining net fluxes through the LMWOS pool, but they rarely identified transformations. As LMWOS are the preferred C and energy source for microorganisms, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the biochemical pathways and its controlling factors. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools. Up to now, studies on LMWOS were nearly exclusively based on uniformly labeled organic substances i.e. all C atoms in the molecules were labeled with 13C or 14C. However, this classical approach did not allow the differentiation between use of intact initial substances in any process, or whether they were transformed to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of LMWOS and quantification of 13CO2 and 13C in bulk soil enabled following the basic metabolic pathways of soil microorganisms. However, only the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites allowed 1) tracing specific anabolic pathways in diverse microbial communities in soils and 2) identification of specific pathways of individual functional microbial groups. So, these are the prerequisites for soil fluxomics. Our studies combining position-specific labeled glucose with amino

  12. [In vivo imaging for biodistribution and metabolism evaluations of new chemical entities].

    PubMed

    Corot, C; Idée, J-M; Raynaud, J-S; Salazar, J-F; Catoen, S

    2012-01-01

    Due to numerous technical developments, in vivo imaging is suitable for pharmacokinetic and metabolism studies of new chemical entities as well as for evaluating their pharmacological or biological effects. MRI, nuclear medicine, X-Ray, ultrasound and optical imaging are available for both clinical and experimental imaging with even higher performance. For all these imaging modalities, diagnostic agents are useful to improve contrast and specificity. Specific targeting of biological events is addressed by molecular imaging. From a pharmacodynamic perspective, radiolabeling of a new chemical entity allows in vivo visualization quantitative measure of its biodistribution, its elimination and its specific molecular binding. Non-invasive imaging methods are useful for longitudinal investigations of biological changes. Based on nanotechnologies, specificity of drug delivery can be monitored by imaging. New developments in hybrid imaging technologies as well as multimodal contrast agents reinforce in vivo experimental and clinical proof of mechanism of new chemical entities.

  13. Protonation of carbon single-walled nanotubes studied using 13