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Sample records for 13c flux analysis

  1. 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.

  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. 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. PMID:26721184

  4. 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

  5. Novel biological insights through metabolomics and 13C-flux analysis.

    PubMed

    Zamboni, Nicola; Sauer, Uwe

    2009-10-01

    Metabolomics and (13)C-flux analysis have become instrumental for analyzing cellular metabolism and its regulation. Driven primarily by technical advances in mass spectrometry-based analytics, they provide unmatched readouts on metabolic state and activity. Functional genomics leverages metabolomics for the discovery of novel enzymes and unexpected secondary activities of annotated enzymes. (13)C-flux analyses are frequently used for empirical elucidation of pathways in poorly characterized species and for network-wide analysis of mechanisms that realize energy and redox balancing. Integration of metabolomics, (13)C-flux analysis and other data enable the condition-dependent characterization of regulatory circuits that ultimately govern the metabolic phenotype.

  6. Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells

    PubMed Central

    Metallo, Christian M.; Walther, Jason L.; Stephanopoulos, Gregory

    2009-01-01

    13C metabolic flux analysis (MFA) is the most comprehensive means of characterizing cellular metabolic states. Uniquely labeled isotopic tracers enable more focused analyses to probe specific reactions within the network. As a result, the choice of tracer largely determines the precision with which one can estimate metabolic fluxes, especially in complex mammalian systems that require multiple substrates. Here we have experimentally determined metabolic fluxes in a tumor cell line, successfully recapitulating the hallmarks of cancer cell metabolism. Using these data, we computationally evaluated specifically labeled 13C glucose and glutamine tracers for their ability to precisely and accurately estimate fluxes in central carbon metabolism. These methods enabled us to to identify the optimal tracer for analyzing individual fluxes, specific pathways, and central carbon metabolism as a whole. [1,2-13C2]glucose provided the most precise estimates for glycolysis, the pentose phosphate pathway, and the overall network. Tracers such as [2-13C]glucose and [3-13C]glucose also outperformed the more commonly used [1-13C]glucose. [U-13C5]glutamine emerged as the preferred isotopic tracer for analysis of the tricarboxylic acid (TCA) cycle. These results provide valuable, quantitative information on the performance of 13C-labeled substrates and can aid in the design of more informative MFA experiments in mammalian cell culture. PMID:19622376

  7. Metabolic networks in motion: 13C-based flux analysis.

    PubMed

    Sauer, Uwe

    2006-01-01

    Many properties of complex networks cannot be understood from monitoring the components--not even when comprehensively monitoring all protein or metabolite concentrations--unless such information is connected and integrated through mathematical models. The reason is that static component concentrations, albeit extremely informative, do not contain functional information per se. The functional behavior of a network emerges only through the nonlinear gene, protein, and metabolite interactions across multiple metabolic and regulatory layers. I argue here that intracellular reaction rates are the functional end points of these interactions in metabolic networks, hence are highly relevant for systems biology. Methods for experimental determination of metabolic fluxes differ fundamentally from component concentration measurements; that is, intracellular reaction rates cannot be detected directly, but must be estimated through computer model-based interpretation of stable isotope patterns in products of metabolism.

  8. 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

  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. 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. PMID:27626798

  11. OpenMebius: An Open Source Software for Isotopically Nonstationary 13C-Based Metabolic Flux Analysis

    PubMed Central

    Furusawa, Chikara

    2014-01-01

    The in vivo measurement of metabolic flux by 13C-based metabolic flux analysis (13C-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 13C-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 13C-MFA is conventionally performed under isotopically constant conditions, isotopically nonstationary 13C metabolic flux analysis (INST-13C-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-13C-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-13C-MFA. Confidence intervals determined by INST-13C-MFA were less than those determined by conventional methods, indicating the potential of INST-13C-MFA for precise metabolic flux analysis. OpenMebius is the open source software for the general application of INST-13C-MFA. PMID:25006579

  12. 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.

  13. 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

  14. 13CFLUX2—high-performance software suite for 13C-metabolic flux analysis

    PubMed Central

    Weitzel, Michael; Nöh, Katharina; Dalman, Tolga; Niedenführ, Sebastian; Stute, Birgit; Wiechert, Wolfgang

    2013-01-01

    Summary: 13C-based metabolic flux analysis (13C-MFA) is the state-of-the-art method to quantitatively determine in vivo metabolic reaction rates in microorganisms. 13CFLUX2 contains all tools for composing flexible computational 13C-MFA workflows to design and evaluate carbon labeling experiments. A specially developed XML language, FluxML, highly efficient data structures and simulation algorithms achieve a maximum of performance and effectiveness. Support of multicore CPUs, as well as compute clusters, enables scalable investigations. 13CFLUX2 outperforms existing tools in terms of universality, flexibility and built-in features. Therewith, 13CFLUX2 paves the way for next-generation high-resolution 13C-MFA applications on the large scale. Availability and implementation: 13CFLUX2 is implemented in C++ (ISO/IEC 14882 standard) with Java and Python add-ons to run under Linux/Unix. A demo version and binaries are available at www.13cflux.net. Contact: info@13cflux.net or k.noeh@fz-juelich.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23110970

  15. (13)C MRS and LC-MS Flux Analysis of Tumor Intermediary Metabolism.

    PubMed

    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 (13)C magnetic resonance spectroscopy ((13)C 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 (13)C 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

  16. 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

  17. 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/

  18. Mapping cancer cell metabolism with13C flux analysis: Recent progress and future challenges

    PubMed Central

    Duckwall, Casey Scott; Murphy, Taylor Athanasaw; Young, Jamey Dale

    2013-01-01

    The reprogramming of energy metabolism is emerging as an important molecular hallmark of cancer cells. Recent discoveries linking specific metabolic alterations to cancer development have strengthened the idea that altered metabolism is more than a side effect of malignant transformation, but may in fact be a functional driver of tumor growth and progression in some cancers. As a result, dysregulated metabolic pathways have become attractive targets for cancer therapeutics. This review highlights the application of13C metabolic flux analysis (MFA) to map the flow of carbon through intracellular biochemical pathways of cancer cells. We summarize several recent applications of MFA that have identified novel biosynthetic pathways involved in cancer cell proliferation and shed light on the role of specific oncogenes in regulating these pathways. Through such studies, it has become apparent that the metabolic phenotypes of cancer cells are not as homogeneous as once thought, but instead depend strongly on the molecular alterations and environmental factors at play in each case. PMID:23961260

  19. (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.

  20. (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.

  1. 13C metabolic flux analysis for larger scale cultivation using gas chromatography-combustion-isotope ratio mass spectrometry.

    PubMed

    Yuan, Yongbo; Yang, Tae Hoon; Heinzle, Elmar

    2010-07-01

    (13)C-based metabolic flux analysis ((13)CMFA) is limited to smaller scale experiments due to very high costs of labeled substrates. We measured (13)C enrichment in proteinogenic amino acid hydrolyzates using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) from a series of parallel batch cultivations of Corynebacterium glutamicum utilizing mixtures of natural glucose and [1-(13)C] glucose, containing 0%, 0.5%, 1%, 2%, and 10% [1-(13)C] glucose. Decreasing the [1-(13)C] glucose content, kinetic isotope effects played an increasing role but could be corrected. From the corrected (13)C enrichments in vivo fluxes in the central metabolism were determined by numerical optimization. The obtained flux distribution was very similar to those obtained from parallel labeling experiments using conventional high labeling GC-MS method and to published results. The GC-C-IRMS-based method involving low labeling degree of expensive tracer substrate, e.g. 1%, is well suited for larger laboratory and industrial pilot scale fermentations.

  2. 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.

  3. 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. PMID:26375179

  4. 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.

  5. 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.

  6. 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

  7. 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; Wu, Qingyu

    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

  8. 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; Hebbelmann, Inga; Rolletschek, Hardy; Schwender, Jorg

    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

  9. 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

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

    PubMed

    Ma, Fangfang; Jazmin, Lara J; Young, Jamey D; Allen, Doug K

    2014-11-25

    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 (13)C-labeling patterns of intracellular metabolites. However, experimental and computational difficulties have hindered its application to terrestrial plant systems. We performed in vivo isotopic labeling of Arabidopsis thaliana rosettes with (13)CO2 and estimated fluxes throughout leaf photosynthetic metabolism by INST-MFA. Plants grown at 200 µmol m(-2)s(-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). This study highlights the potential of (13)C INST-MFA to describe emergent flux phenotypes that respond to environmental conditions or plant physiology and cannot be obtained by other complementary approaches. PMID:25368168

  11. 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.

  12. Metabolic evolution and (13) C flux analysis of a succinate dehydrogenase deficient strain of Yarrowia lipolytica.

    PubMed

    Yuzbashev, Tigran V; Bondarenko, Pavel Yu; Sobolevskaya, Tatiana I; Yuzbasheva, Evgeniya Yu; Laptev, Ivan A; Kachala, Vadim V; Fedorov, Alexander S; Vybornaya, Tatiana V; Larina, Anna S; Sineoky, Sergey P

    2016-11-01

    Bio-based succinic acid production can redirect industrial chemistry processes from using limited hydrocarbons to renewable carbohydrates. A fermentation process that does not require pH-titrating agents will be advantageous to the industry. Previously, a Yarrowia lipolytica strain that was defective for succinate dehydrogenase was constructed and was found to accumulate up to 17.5 g L(-1) of succinic acid when grown on glycerol without buffering. Here, a derivative mutant was isolated that produced 40.5 g L(-1) of succinic acid in 36 h with a yield of 0.32 g g(-1) glycerol. A combination approach of induced mutagenesis and metabolic evolution allowed isolation of another derivative that could utilize glucose efficiently and accumulated 50.2 g L(-1) succinic acid in 54 h with a yield of 0.43 g g(-1) . The parent strain of these isolated mutants was used for [1,6-(13) C2 ]glucose assimilation analysis. At least 35% glucose was estimated to be utilized through the pentose phosphate pathway, while ≥84% succinic acid was formed through the oxidative branch of the tricarboxylic acid cycle. Biotechnol. Bioeng. 2016;113: 2425-2432. © 2016 Wiley Periodicals, Inc. PMID:27182846

  13. 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.

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

    PubMed

    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.

  15. 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

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

    PubMed

    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

  17. 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.

  18. 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.

  19. 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

  20. Serial 13C-based flux analysis of an L-phenylalanine-producing E. coli strain using the sensor reactor.

    PubMed

    Wahl, Aljoscha; El Massaoudi, Mohamed; Schipper, Dick; Wiechert, Wolfgang; Takors, Ralf

    2004-01-01

    With the aid of the recently developed Sensor reactor system, a series of three subsequent (13)C labeling experiments was performed mirroring the l-phenylalanine (l-Phe) production phase of a recombinant E. coli strain that was cultivated under industry-like conditions in a 300 L bioreactor. On the basis of the data from NMR labeling analysis, three subsequent flux patterns were successfully derived monitoring the l-Phe formation during an observation window from 14 to 23.3 h process time. Linear programming was performed to identify optimal flux patterns for l-Phe formation. Additionally, flux sensitivity analysis was used to identify the most promising metabolic engineering target. As a result, high rates of phosphoenolpyruvate (PEP) to pyruvate (PYR) conversion were identified as the most important reason for deterioration of the l-Phe/glucose yield from 20 to finally 11 mol %. Considering the characteristics of the enzyme kinetics involved, the working hypothesis was formulated that phosphoenolpyruvate synthase activity was increasingly hampered by rising oxaloacetate and 2-oxoglutarate concentrations, while at the same time pyruvate kinase activity arose due to activation by fructose 1,6-diphosphate. Hence, pps overexpression should be performed to optimize the existing production strain.

  1. 13C-flux Analysis Reveals NADPH-balancing Transhydrogenation Cycles in Stationary Phase of Nitrogen-starving Bacillus subtilis *

    PubMed Central

    Rühl, Martin; Le Coq, Dominique; Aymerich, Stéphane; Sauer, Uwe

    2012-01-01

    In their natural habitat, microorganisms are typically confronted with nutritional limitations that restrict growth and force them to persevere in a stationary phase. Despite the importance of this phase, little is known about the metabolic state(s) that sustains it. Here, we investigate metabolically active but non-growing Bacillus subtilis during nitrogen starvation. In the absence of biomass formation as the major NADPH sink, the intracellular flux distribution in these resting B. subtilis reveals a large apparent catabolic NADPH overproduction of 5.0 ± 0.6 mmol·g−1·h−1 that was partly caused by high pentose phosphate pathway fluxes. Combining transcriptome analysis, stationary 13C-flux analysis in metabolic deletion mutants, 2H-labeling experiments, and kinetic flux profiling, we demonstrate that about half of the catabolic excess NADPH is oxidized by two transhydrogenation cycles, i.e. isoenzyme pairs of dehydrogenases with different cofactor specificities that operate in reverse directions. These transhydrogenation cycles were constituted by the combined activities of the glyceraldehyde 3-phosphate dehydrogenases GapA/GapB and the malic enzymes MalS/YtsJ. At least an additional 6% of the overproduced NADPH is reoxidized by continuous cycling between ana- and catabolism of glutamate. Furthermore, in vitro enzyme data show that a not yet identified transhydrogenase could potentially reoxidize ∼20% of the overproduced NADPH. Overall, we demonstrate the interplay between several metabolic mechanisms that concertedly enable network-wide NADPH homeostasis under conditions of high catabolic NADPH production in the absence of cell growth in B. subtilis. PMID:22740702

  2. 13C-flux analysis reveals NADPH-balancing transhydrogenation cycles in stationary phase of nitrogen-starving Bacillus subtilis.

    PubMed

    Rühl, Martin; Le Coq, Dominique; Aymerich, Stéphane; Sauer, Uwe

    2012-08-10

    In their natural habitat, microorganisms are typically confronted with nutritional limitations that restrict growth and force them to persevere in a stationary phase. Despite the importance of this phase, little is known about the metabolic state(s) that sustains it. Here, we investigate metabolically active but non-growing Bacillus subtilis during nitrogen starvation. In the absence of biomass formation as the major NADPH sink, the intracellular flux distribution in these resting B. subtilis reveals a large apparent catabolic NADPH overproduction of 5.0 ± 0.6 mmol g(-1)h(-1) that was partly caused by high pentose phosphate pathway fluxes. Combining transcriptome analysis, stationary (13)C-flux analysis in metabolic deletion mutants, (2)H-labeling experiments, and kinetic flux profiling, we demonstrate that about half of the catabolic excess NADPH is oxidized by two transhydrogenation cycles, i.e. isoenzyme pairs of dehydrogenases with different cofactor specificities that operate in reverse directions. These transhydrogenation cycles were constituted by the combined activities of the glyceraldehyde 3-phosphate dehydrogenases GapA/GapB and the malic enzymes MalS/YtsJ. At least an additional 6% of the overproduced NADPH is reoxidized by continuous cycling between ana- and catabolism of glutamate. Furthermore, in vitro enzyme data show that a not yet identified transhydrogenase could potentially reoxidize ∼20% of the overproduced NADPH. Overall, we demonstrate the interplay between several metabolic mechanisms that concertedly enable network-wide NADPH homeostasis under conditions of high catabolic NADPH production in the absence of cell growth in B. subtilis.

  3. Characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative 13C flux analysis

    PubMed Central

    Frick, Oliver; Wittmann, Christoph

    2005-01-01

    Background One of the most fascinating properties of the biotechnologically important organism Saccharomyces cerevisiae is its ability to perform simultaneous respiration and fermentation at high growth rate even under fully aerobic conditions. In the present work, this Crabtree effect called phenomenon was investigated in detail by comparative 13C metabolic flux analysis of S. cerevisiae growing under purely oxidative, respiro-fermentative and predominantly fermentative conditions. Results The metabolic shift from oxidative to fermentative growth was accompanied by complex changes of carbon flux throughout the whole central metabolism. This involved a flux redirection from the pentose phosphate pathway (PPP) towards glycolysis, an increased flux through pyruvate carboxylase, the fermentative pathways and malic enzyme, a flux decrease through the TCA cycle, and a partial relocation of alanine biosynthesis from the mitochondrion to the cytosol. S. cerevisiae exhibited a by-pass of pyruvate dehydrogenase in all physiological regimes. During oxidative growth this by-pass was mainly provided via pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthase and transport of acetyl-CoA into the mitochondrion. During fermentative growth this route, however, was saturated due to limited enzyme capacity. Under these conditions the cells exhibited high carbon flux through a chain of reactions involving pyruvate carboxylase, the oxaloacetate transporter and malic enzyme. During purely oxidative growth the PPP alone was sufficient to completely supply NADPH for anabolism. During fermentation, it provided only 60 % of the required NADPH. Conclusion We conclude that, in order to overcome the limited capacity of pyruvate dehydrogenase, S. cerevisiae possesses different metabolic by-passes to channel carbon into the mitochondrion. This involves the conversion of cytosolic pyruvate either into acetyl CoA or oxaloacetate followed by intercompartmental transport of these

  4. 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.

  5. Quantitative Metabolomics and Instationary 13C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris.

    PubMed

    Jordà, Joel; Rojas, Hugo Cueto; Carnicer, Marc; Wahl, Aljoscha; Ferrer, Pau; Albiol, Joan

    2014-05-05

    Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13C metabolic flux analysis (INST 13C-MFA) using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol) on P. pastoris central carbon metabolism. Higher oxygen uptake and CO2 production rates and slightly reduced biomass yield suggest an increased energy demand for the producing strain. This observation is further confirmed by 13C-based metabolic flux analysis. In particular, the flux through the methanol oxidation pathway and the TCA cycle was increased in the Rol-producing strain compared to the reference strain. Next to changes in the flux distribution, significant variations in intracellular metabolite concentrations were observed. Most notably, the pools of trehalose, which is related to cellular stress response, and xylose, which is linked to methanol assimilation, were significantly increased in the recombinant strain.

  6. Quantitative Metabolomics and Instationary 13C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris

    PubMed Central

    Jordà, Joel; Cueto Rojas, Hugo; Carnicer, Marc; Wahl, Aljoscha; Ferrer, Pau; Albiol, Joan

    2014-01-01

    Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13C metabolic flux analysis (INST 13C-MFA) using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol) on P. pastoris central carbon metabolism. Higher oxygen uptake and CO2 production rates and slightly reduced biomass yield suggest an increased energy demand for the producing strain. This observation is further confirmed by 13C-based metabolic flux analysis. In particular, the flux through the methanol oxidation pathway and the TCA cycle was increased in the Rol-producing strain compared to the reference strain. Next to changes in the flux distribution, significant variations in intracellular metabolite concentrations were observed. Most notably, the pools of trehalose, which is related to cellular stress response, and xylose, which is linked to methanol assimilation, were significantly increased in the recombinant strain. PMID:24957027

  7. 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

  8. 13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

    PubMed Central

    Beste, Dany J. V.; Bonde, Bhushan; Hawkins, Nathaniel; Ward, Jane L.; Beale, Michael H.; Noack, Stephan; Nöh, Katharina; Kruger, Nicholas J.; Ratcliffe, R. George; McFadden, Johnjoe

    2011-01-01

    Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using 13C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with 13C labeled glycerol or sodium bicarbonate. Through measurements of the 13C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate – oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO2 into biomass. As the human host is abundant in CO2 this finding requires further investigation in vivo as CO2 fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using 13C-MFA. PMID:21814509

  9. 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.

  10. 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. PMID:25311863

  11. Metabolomic and 13C-Metabolic Flux Analysis of a Xylose-Consuming Saccharomyces cerevisiae Strain Expressing Xylose Isomerase

    PubMed Central

    Wasylenko, Thomas M.; Stephanopoulos, Gregory

    2016-01-01

    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 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. PMID:25311863

  12. Production process monitoring by serial mapping of microbial carbon flux distributions using a novel Sensor Reactor approach: II--(13)C-labeling-based metabolic flux analysis and L-lysine production.

    PubMed

    Drysch, A; El Massaoudi, M; Mack, C; Takors, R; de Graaf, A A; Sahm, H

    2003-04-01

    Corynebacterium glutamicum is intensively used for the industrial large-scale (fed-) batch production of amino acids, especially glutamate and lysine. However, metabolic flux analyses based on 13C-labeling experiments of this organism have hitherto been restricted to small-scale batch conditions and carbon-limited chemostat cultures, and are therefore of questionable relevance for industrial fermentations. To lever flux analysis to the industrial level, a novel Sensor Reactor approach was developed (El Massaoudi et al., Metab. Eng., submitted), in which a 300-L production reactor and a 1-L Sensor Reactor are run in parallel master/slave modus, thus enabling 13C-based metabolic flux analysis to generate a series of flux maps that document large-scale fermentation courses in detail. We describe the successful combination of this technology with nuclear magnetic resonance (NMR) analysis, metabolite balancing methods and a mathematical description of 13C-isotope labelings resulting in a powerful tool for quantitative pathway analysis during a batch fermentation. As a first application, 13C-based metabolic flux analysis was performed on exponentially growing, lysine-producing C. glutamicum MH20-22B during three phases of a pilot-scale batch fermentation. By studying the growth, (co-) substrate consumption and (by-) product formation, the similarity of the fermentations in production and Sensor Reactor was verified. Applying a generally applicable mathematical model, which included metabolite and carbon labeling balances for the analysis of proteinogenic amino acid 13C-isotopomer labeling data, the in vivo metabolic flux distribution was investigated during subsequent phases of exponential growth. It was shown for the first time that the in vivo reverse C(4)-decarboxylation flux at the anaplerotic node in C. glutamicum significantly decreased (70%) in parallel with threefold increased lysine formation during the investigated subsequent phases of exponential growth.

  13. 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.

  14. 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. PMID:27454589

  15. 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

  16. 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

  17. 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.

  18. Kinetic isotope effects significantly influence intracellular metabolite 13C labeling patterns and flux determination

    PubMed Central

    Wasylenko, Thomas M.; Stephanopoulos, Gregory

    2014-01-01

    Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from 13C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of 13C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the 13C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the 13C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in 13C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC–MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in 13C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions. PMID:23828762

  19. (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

  20. 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.

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

    PubMed Central

    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. PMID:25392417

  2. 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. PMID:26513365

  3. 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.

  4. 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

  5. Elucidation of intrinsic biosynthesis yields using 13C-based metabolism analysis

    PubMed Central

    2014-01-01

    This paper discusses the use of 13C-based metabolism analysis for the assessment of intrinsic product yields — the actual carbon contribution from a single carbon substrate to the final product via a specific biosynthesis route — in the following four cases. First, undefined nutrients (such as yeast extract) in fermentation may contribute significantly to product synthesis, which can be quantified through an isotopic dilution method. Second, product and biomass synthesis may be dependent on the co-metabolism of multiple-carbon sources. 13C labeling experiments can track the fate of each carbon substrate in the cell metabolism and identify which substrate plays a main role in product synthesis. Third, 13C labeling can validate and quantify the contribution of the engineered pathway (versus the native pathway) to the product synthesis. Fourth, the loss of catabolic energy due to cell maintenance (energy used for functions other than production of new cell components) and low P/O ratio (Phosphate/Oxygen Ratio) significantly reduces product yields. Therefore, 13C-metabolic flux analysis is needed to assess the influence of suboptimal energy metabolism on microbial productivity, and determine how ATP/NAD(P)H are partitioned among various cellular functions. Since product yield is a major determining factor in the commercialization of a microbial cell factory, we foresee that 13C-isotopic labeling experiments, even without performing extensive flux calculations, can play a valuable role in the development and verification of microbial cell factories. PMID:24642094

  6. Elucidation of intrinsic biosynthesis yields using 13C-based metabolism analysis.

    PubMed

    Varman, Arul M; He, Lian; You, Le; Hollinshead, Whitney; Tang, Yinjie J

    2014-03-19

    This paper discusses the use of 13C-based metabolism analysis for the assessment of intrinsic product yields - the actual carbon contribution from a single carbon substrate to the final product via a specific biosynthesis route - in the following four cases. First, undefined nutrients (such as yeast extract) in fermentation may contribute significantly to product synthesis, which can be quantified through an isotopic dilution method. Second, product and biomass synthesis may be dependent on the co-metabolism of multiple-carbon sources. 13C labeling experiments can track the fate of each carbon substrate in the cell metabolism and identify which substrate plays a main role in product synthesis. Third, 13C labeling can validate and quantify the contribution of the engineered pathway (versus the native pathway) to the product synthesis. Fourth, the loss of catabolic energy due to cell maintenance (energy used for functions other than production of new cell components) and low P/O ratio (Phosphate/Oxygen Ratio) significantly reduces product yields. Therefore, 13C-metabolic flux analysis is needed to assess the influence of suboptimal energy metabolism on microbial productivity, and determine how ATP/NAD(P)H are partitioned among various cellular functions. Since product yield is a major determining factor in the commercialization of a microbial cell factory, we foresee that 13C-isotopic labeling experiments, even without performing extensive flux calculations, can play a valuable role in the development and verification of microbial cell factories.

  7. 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

  8. Quantification of compartmented metabolic fluxes in developing soybean embryos by employing biosynthetically directed fractional (13)C labeling, two-dimensional [(13)C, (1)H] nuclear magnetic resonance, and comprehensive isotopomer balancing.

    PubMed

    Sriram, Ganesh; Fulton, D Bruce; Iyer, Vidya V; Peterson, Joan Marie; Zhou, Ruilian; Westgate, Mark E; Spalding, Martin H; Shanks, Jacqueline V

    2004-10-01

    Metabolic flux quantification in plants is instrumental in the detailed understanding of metabolism but is difficult to perform on a systemic level. Toward this aim, we report the development and application of a computer-aided metabolic flux analysis tool that enables the concurrent evaluation of fluxes in several primary metabolic pathways. Labeling experiments were performed by feeding a mixture of U-(13)C Suc, naturally abundant Suc, and Gln to developing soybean (Glycine max) embryos. Two-dimensional [(13)C, (1)H] NMR spectra of seed storage protein and starch hydrolysates were acquired and yielded a labeling data set consisting of 155 (13)C isotopomer abundances. We developed a computer program to automatically calculate fluxes from this data. This program accepts a user-defined metabolic network model and incorporates recent mathematical advances toward accurate and efficient flux evaluation. Fluxes were calculated and statistical analysis was performed to obtain sds. A high flux was found through the oxidative pentose phosphate pathway (19.99 +/- 4.39 micromol d(-1) cotyledon(-1), or 104.2 carbon mol +/- 23.0 carbon mol per 100 carbon mol of Suc uptake). Separate transketolase and transaldolase fluxes could be distinguished in the plastid and the cytosol, and those in the plastid were found to be at least 6-fold higher. The backflux from triose to hexose phosphate was also found to be substantial in the plastid (21.72 +/- 5.00 micromol d(-1) cotyledon(-1), or 113.2 carbon mol +/-26.0 carbon mol per 100 carbon mol of Suc uptake). Forward and backward directions of anaplerotic fluxes could be distinguished. The glyoxylate shunt flux was found to be negligible. Such a generic flux analysis tool can serve as a quantitative tool for metabolic studies and phenotype comparisons and can be extended to other plant systems.

  9. Constraining Methane Flux Estimates Using Atmospheric Observations of Methane and 1^3C in Methane

    NASA Astrophysics Data System (ADS)

    Mikaloff Fletcher, S. E.; Tans, P. P.; Miller, J. B.; Bruhwiler, L. M.

    2002-12-01

    Understanding the budget of methane is crucial to predicting climate change and managing earth's carbon reservoirs. Methane is responsible for approximately 15% of the anthropogenic greenhouse forcing and has a large impact on the oxidative capacity of Earth's atmosphere due to its reaction with hydroxyl radical. At present, many of the sources and sinks of methane are poorly understood due in part to the large spatial and temporal variability of the methane flux. Model simulations of methane mixing ratios using most process-based source estimates typically over-predict the latitudinal gradient of atmospheric methane relative to the observations; however, the specific source processes responsible for this discrepancy have not been identified definitively. The aim of this work is to use the isotopic signatures of the sources to attribute these discrepancies to a source process or group of source processes and create global and regional budget estimates that are in agreement with both the atmospheric observations of methane and 1^3C in methane. To this end, observations of isotopic ratios of 1^3C in methane and isotopic signatures of methane source processes are used in conjunction with an inverse model of the methane budget. Inverse modeling is a top-down approach which uses observations of trace gases in the atmosphere, an estimate of the spatial pattern of trace gas fluxes, and a model of atmospheric transport to estimate the sources and sinks. The atmospheric transport was represented by the TM3 three-dimensional transport model. The GLOBALVIEW 2001 methane observations were used along with flask measurements of 1^3C in methane at six of the CMDL-NOAA stations by INSTAAR. Initial results imply interesting differences from previous methane budget estimates. For example, the 1^3C isotope observations in methane call for an increase in southern hemisphere sources with a bacterial isotopic signature such as wetlands, rice paddies, termites, and ruminant animals. The

  10. 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.

  11. 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.

  12. 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. PMID:24771296

  13. Interaction between rhizosphere microorganisms and plant roots: 13C fluxes in the rhizosphere after pulse labeling

    NASA Astrophysics Data System (ADS)

    Yevdokimov, I. V.; Ruser, R.; Buegger, F.; Marx, M.; Munch, J. C.

    2007-07-01

    The input dynamics of labeled C into pools of soil organic matter and CO2 fluxes from soil were studied in a pot experiment with the pulse labeling of oats and corn under a 13CO2 atmosphere, and the contribution of the root and microbial respiration to the emission of CO2 from the soil was determined from the fluxes of labeled C in the microbial biomass and the evolved carbon dioxide. A considerable amount of 13C (up to 96% of the total amount of the label found in the rhizosphere soil) was incorporated into the biomass of the rhizosphere microorganisms. The diurnal fluctuations of the labeled C pools in the microbial biomass, dissolved organic carbon, and CO2 released in the rhizosphere of oats and corn were related to the day/night changes, i.e., to the on and off periods of the photosynthetic activity of the plants. The average contribution of the corn root respiration (70% of the total CO2 emission from the soil surface) was higher than that of the oats roots (44%), which was related to the lower incorporation of rhizodeposit carbon into the microbial biomass in the soil under the corn plants than in the soil under the oats plants.

  14. Variability of Root Exudate δ13C and Fluxes in Relation to Environmental Conditions and Plant Characteristics in a Bottomland Temperate Forest

    NASA Astrophysics Data System (ADS)

    Gougherty, S. W.; Bauer, J. E.; Pohlman, J.

    2015-12-01

    Plant root exudation of organic carbon (OC) is thought to be an important, yet poorly quantified and highly variable component of net primary productivity that influences soil biogeochemistry and ecology. In situ measurements of plant root OC exudation are relatively rare, and δ13C measurements of root exudates are generally lacking. Understanding both exudate fluxes and δ13C relative to other plant components, root characteristics and environmental parameters (e.g., vapor pressure deficit and soil moisture) will lead to better quantitative understanding of atmosphere—plant—soil linkages. We used a field based collection system to obtain root exudates from fine roots (diameter <5mm) over five sampling periods in a ~20 year old bottomland forest in central Ohio, USA. Exudates were analyzed for dissolved OC concentration and δ13C signatures. Exudate flux estimates were made at both the individual root level and also scaled to the entire sampling area. Preliminary data analysis suggests the mean root exudation rate was 26 µmol C g root -1 day-1 and when scaled to the 5600 m2 sampling area represents a mean flux of 4,200 µmol C m-2 day-1 from tree roots. The flux estimates presented here suggest root exudation may account for as much as 6% of net ecosystem production at the field location. Available data also suggests that exudate δ13C is enriched by 1-2 ‰ compared the root material from which exudates were collected. We will also assess the relationship between exudate, root and leaf δ13C, environmental parameters, and C fluxes at the site. If root exudation rate or δ13C varies as a function of environmental conditions this may suggest that heterotrophic remineralization of root exudates is one potential driver of correlations between soil δ13C-CO2 and environmental parameters.

  15. Approaches to studies on neuronal/glial relationships by 13C-MRS analysis.

    PubMed

    Taylor, A; McLean, M; Morris, P; Bachelard, H

    1996-01-01

    The use of different 13C-labelled precursors alone or in combination ([1-13C]glucose, [2-13C]glucose, [1-13C]acetate, [2-13C]acetate and [1,2-13C2]acetate) to study neuronal/glial metabolic relationships by MRS is discussed. Glutamine and citrate resonances represent glial metabolism if a combination of [1-13C]glucose + [2-13C]acetate is used, but only for short time periods. A combination of [2-13C]glucose + [2-13C]acetate will label -COO- groups from glucose and -CH2 groups from acetate, respectively, which distinguish well in theory. However, this approach is severely limited by the long T1S of -COO- groups and low S/N. Contributions of the anaplerotic pathway can be assessed using [2-13C]glucose, but again can be limited by the long T1S of -COO- groups. Labelling of glycerol-3-phosphate (believed to be produced in glia) from [1-13C]glucose is difficult to see under normal conditions but has proved useful in, e.g., hypoxia. We believe the most promising approach is the use of [1-13C] glucose with [1,2-13C2]acetate, by analysis of the multiplets ('isotopomers') of the amino acid resonances.

  16. Carbon fluxes to the soil in a mature temperate forest assessed by 13C isotope tracing.

    PubMed

    Steinmann, Katharina; Siegwolf, Rolf T W; Saurer, Matthias; Körner, Christian

    2004-11-01

    Photosynthetic carbon uptake and respiratory C release from soil are major components of the global carbon balance. The use of 13C depleted CO2)(delta13C = -30 per thousand) in a free air CO2 enrichment experiment in a mature deciduous forest permitted us to trace the carbon transfer from tree crowns to the rhizosphere of 100-120 years old trees. During the first season of CO2 enrichment the CO2 released from soil originated substantially from concurrent assimilation. The small contribution of recent carbon in fine roots suggests a much slower fine root turnover than is often assumed. 13C abundance in soil air correlated best with temperature data taken from 4 to 10 days before air sampling time and is thus rapidly available for root and rhizosphere respiration. The spatial variability of delta13C in soil air showed relationships to above ground tree types such as conifers versus broad-leaved trees. Considering the complexity and strong overlap of roots from different individuals in a forest, this finding opens an exciting new possibility of associating respiration with different species. What might be seen as signal noise does in fact contain valuable information on the spatial heterogeneity of tree-soil interaction.

  17. 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

  18. Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry.

    PubMed

    Heinzle, Elmar; Yuan, Yongbo; Kumar, Sathish; Wittmann, Christoph; Gehre, Matthias; Richnow, Hans-Herrmann; Wehrung, Patrick; Adam, Pierre; Albrecht, Pierre

    2008-09-15

    The applicability of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the quantification of 13C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the 13C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-13C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the 13C enrichment. The approach to correct for these effects in metabolic flux studies using delta13C measurement by GC-C-IRMS uses two parallel experiments applying substrate with natural abundance and 13C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of 13C in proteinogenic amino acids over the whole range of applied concentrations of [1-13C]glucose. The current findings indicate a great potential of GC-C-IRMS for labeling quantification in 13C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.

  19. Real-time measurements of CH4 and CO2 flux and del13C from a proglacial wetland in southwestern Greenland.

    NASA Astrophysics Data System (ADS)

    Stern, J. C.; White, J. R.; Pratt, L. M.; Thompson, H. A.

    2015-12-01

    Arctic permafrost environments represent a large repository of stored carbon that may be mobilized as global temperatures increase, providing a substrate for microbial CH4 production. Proglacial wetlands and lakes are important targets of study to better understand how rapidly changing landscapes affected by climate warming adapt their carbon cycling. Recent advances in portable laser spectrometry have enabled rapid in situ measurements of not only greenhouse gas fluxes, but also del13C compositions of these gases. Here we use a Picarro CH4 and CO2 isotope analyzer to continuously measure CH4 and CO2 flux in situ for comparison to static closed chamber measurements where samples are collected at discrete time intervals and returned to the laboratory for analysis. Real-time, in situ analysis also allowed simple light/dark experiments to be performed on chambers containing different vegetation. In addition, this instrument can be used to measure concentration and del13C of both dissolved CH4 and CO­­2 in lake waters when appropriate gas stripped methods are used. We present data for CH4 and CO2 flux and del13C of emitted and dissolved gases from permafrost-affected wetlands and lakes associated with proglacial landscapes in southwestern Greenland near the Russell Glacier.

  20. Flux-p: automating metabolic flux analysis.

    PubMed

    Ebert, Birgitta E; Lamprecht, Anna-Lena; Steffen, Bernhard; Blank, Lars M

    2012-11-12

    Quantitative knowledge of intracellular fluxes in metabolic networks is invaluable for inferring metabolic system behavior and the design principles of biological systems. However, intracellular reaction rates can not often be calculated directly but have to be estimated; for instance, via 13C-based metabolic flux analysis, a model-based interpretation of stable carbon isotope patterns in intermediates of metabolism. Existing software such as FiatFlux, OpenFLUX or 13CFLUX supports experts in this complex analysis, but requires several steps that have to be carried out manually, hence restricting the use of this software for data interpretation to a rather small number of experiments. In this paper, we present Flux-P as an approach to automate and standardize 13C-based metabolic flux analysis, using the Bio-jETI workflow framework. Exemplarily based on the FiatFlux software, it demonstrates how services can be created that carry out the different analysis steps autonomously and how these can subsequently be assembled into software workflows that perform automated, high-throughput intracellular flux analysis of high quality and reproducibility. Besides significant acceleration and standardization of the data analysis, the agile workflow-based realization supports flexible changes of the analysis workflows on the user level, making it easy to perform custom analyses.

  1. Direct analysis of δ13C and concentration of dissolved organic carbon (DOC) in environmental samples by TOC-IRMS

    NASA Astrophysics Data System (ADS)

    Kirkels, Frédérique; Cerli, Chiara; Federherr, Eugen; Kalbitz, Karsten

    2014-05-01

    Dissolved organic carbon (DOC) plays an important role in carbon cycling in terrestrial and aquatic systems. Stable isotope analysis (delta 13C) of DOC could provide valuable insights in its origin, fluxes and environmental fate. Precise and routine analysis of delta 13C and DOC concentration are therefore highly desirable. A promising, new system has been developed for this purpose, linking a high-temperature combustion TOC analyzer trough an interface with a continuous flow isotope ratio mass spectrometer (Elementar group, Hanau, Germany). This TOC-IRMS system enables simultaneous stable isotope (bulk delta 13C) and concentration analysis of DOC, with high oxidation efficiency by high-temperature combustion for complex mixtures as natural DOC. To give delta 13C analysis by TOC-IRMS the necessary impulse for broad-scale application, we present a detailed evaluation of its analytical performance for realistic and challenging conditions inclusive low DOC concentrations and environmental samples. High precision (standard deviation, SD predominantly < 0.15 permil) and accuracy (R2 = 0.9997, i.e. comparison TOC-IRMS and conventional EA-IRMS) were achieved by TOC-IRMS for a broad diversity of DOC solutions. This precision is comparable or even slightly better than that typically reported for EA-IRMS systems, and improves previous techniques for δ13C analysis of DOC. Simultaneously, very good precision was obtained for DOC concentration measurements. Assessment of natural abundance and slightly 13C enriched DOC, a wide range of concentrations (0.2-150 mgC/L) and injection volumes (0.05-3 ml), demonstrated good analytical performance with negligible memory effects, no concentration/volume effects and a wide linearity. Low DOC concentrations (< 2 mgC/L), were correctly analyzed without any pre-concentration. Moreover, TOC-IRMS was successfully applied to analyze DOC from diverse terrestrial, freshwater and marine environments (SD < 0.23 permil). In summary, the TOC

  2. A simple mathematical model and practical approach for evaluating citric acid cycle fluxes in perfused rat hearts by 13C-NMR and 1H-NMR spectroscopy.

    PubMed

    Tran-Dinh, S; Hoerter, J A; Mateo, P; Bouet, F; Herve, M

    1997-04-15

    We propose a simple mathematical model and a practical approach for evaluating the flux constant and the absolute value of flux in the citric acid cycle in perfused organs by 13C-NMR and 1H-NMR spectroscopy. We demonstrate that 13C-NMR glutamate spectra are independent of the relative sizes of the mitochondrial and cytosolic compartments and the exchange rates of glutamates, unless there is a difference in 13C chemical shifts of glutamate carbons between the two compartments. Wistar rat hearts (five beating and four KCl-arrested hearts) were aerobically perfused with 100% enriched [2-(13)C]acetate and the kinetics of glutamate carbon labeling from perchloric acid extracts were studied at various perfusion times. Under our experimental conditions, the citric acid cycle flux constant, which represents the fraction of glutamate in exchange with the citric acid cycle per unit time, is about 0.350 +/- 0.003 min(-1) for beating hearts and 0.0741 +/- 0.004 min(-1) for KCl-arrested hearts. The absolute values of the citric acid flux for beating hearts and for KCl-arrested hearts are 1.06 +/- 0.06 micromol x min(-1) x mg(-1) and 0.21 +/- 0.02 micromol x min(-1) x g(-1), respectively. The fraction of unlabeled acetate determined from the proton signal of the methyl group is small and essentially the same in beating and arrested hearts (7.4 +/- 1.7% and 8.8 +/- 2.1%, respectively). Thus, the large difference in the Glu C2/C4 between beating and arrested hearts is not due to the important contribution from anaplerotic sources in arrested hearts but simply to a substantial difference in citric acid cycle fluxes. Our model fits the experimental data well, indicating a fast exchange between 2-oxoglutarate and glutamate in the mitochondria of rat hearts. Analysis of the flux constant, calculated from the half-time of glutamate C4 labeling given in the literature, allows for a comparison of the citric acid flux for various working conditions in different animal species.

  3. 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

  4. Mathematical models for determining metabolic fluxes through the citric acid and the glyoxylate cycles in Saccharomyces cerevisiae by 13C-NMR spectroscopy.

    PubMed

    Tran-Dinh, S; Bouet, F; Huynh, Q T; Herve, M

    1996-12-15

    We propose, first, a practical method for studying the isotopic transformation of glutamate or any other metabolite isotopomers in the citric acid and the glyoxylate cycles; second, two mathematical models, one for evaluating the flux through the citric acid cycle and the other for evaluating the flux through the latter coupled to the glyoxylate cycle in yeast. These models are based on the analysis of 13C-NMR spectra of glutamate obtained from Saccharomyces cerevisiae, NCYC strain, fed with 100% enriched [2-13C]acetate. The population of each glutamate isotopomer, the change in intensity of each multiplet component or the enrichment of any glutamate carbon is expressed by a specific analytical equation from which the flux in the citric acid and the glyoxylate cycles can be deduced. The aerobic metabolism of 100% [2-13C]acetate in acetate-grown S. cerevisiae cells was studied as a function of time using 13C-NMR. 1H-NMR and biochemical techniques. The C1 and C6 doublet and singlet of labeled trehalose increase continuously with time indicating that there is no isotopic transformation between trehalose isotopomers even though the corresponding formation rates are different. By contrast, the glutamate C4 singlet increases then decreases with time. The C4 doublet, which is lower than the singlet for t < 60 min, increases continuously and becomes higher than the singlet for t > 90 min. A similar observation was made for the C2 resonance singlet and doublet. In addition, the glutamate C2 multiplet consists of only seven instead of nine peaks as in random labeling. These results agree well with our models and demonstrate that, in the presence of acetate, anaplerotic carbon sources involved in the synthesis of acetyl-CoA are negligible in yeast. The flux in the citric acid cycle was deduced from a plot of the C4 area versus incubation time, while the flux within the glyoxylate cycle was determined from the relative intensity of the glutamate C4 doublet and singlet. The

  5. 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

  6. 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

  7. 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

  8. Position-Specific Isotope Analysis of Xanthines: A (13)C Nuclear Magnetic Resonance Method to Determine the (13)C Intramolecular Composition at Natural Abundance.

    PubMed

    Diomande, Didier G; Martineau, Estelle; Gilbert, Alexis; Nun, Pierrick; Murata, Ariaki; Yamada, Keita; Watanabe, Naoharu; Tea, Illa; Robins, Richard J; Yoshida, Naohiro; Remaud, Gérald S

    2015-07-01

    The natural xanthines caffeine, theobromine, and theophylline are of major commercial importance as flavor constituents in coffee, cocoa, tea, and a number of other beverages. However, their exploitation for authenticity, a requirement in these commodities that have a large origin-based price-range, by the standard method of isotope ratio monitoring by mass spectrometry (irm-MS) is limited. We have now developed a methodology that overcomes this deficit that exploits the power of isotopic quantitative (13)C nuclear magnetic resonance (NMR) spectrometry combined with chemical modification of the xanthines to enable the determination of positional intramolecular (13)C/(12)C ratios (δ(13)Ci) with high precision. However, only caffeine is amenable to analysis: theobromine and theophylline present substantial difficulties due to their poor solubility. However, their N-methylation to caffeine makes spectral acquisition feasible. The method is confirmed as robust, with good repeatability of the δ(13)Ci values in caffeine appropriate for isotope fractionation measurements at natural abundance. It is shown that there is negligible isotope fractionation during the chemical N-methylation procedure. Thus, the method preserves the original positional δ(13)Ci values. The method has been applied to measure the position-specific variation of the (13)C/(12)C distribution in caffeine. Not only is a clear difference between caffeine isolated from different sources observed, but theobromine from cocoa is found to show a (13)C pattern distinct from that of caffeine. PMID:26067163

  9. A 13C labelling study on carbon fluxes in Arctic plankton communities under elevated CO2 levels

    NASA Astrophysics Data System (ADS)

    de Kluijver, A.; Soetaert, K.; Czerny, J.; Schulz, K. G.; Boxhammer, T.; Riebesell, U.; Middelburg, J. J.

    2013-03-01

    The effect of CO2 on carbon fluxes (production, consumption, and export) in Arctic plankton communities was investigated during the 2010 EPOCA (European project on Ocean Acidification) mesocosm study off Ny Ålesund, Svalbard. 13C labelled bicarbonate was added to nine mesocosms with a range in pCO2 (185 to 1420 μatm) to follow the transfer of carbon from dissolved inorganic carbon (DIC) into phytoplankton, bacterial and zooplankton consumers, and export. A nutrient-phytoplankton-zooplankton-detritus model amended with 13C dynamics was constructed and fitted to the data to quantify uptake rates and carbon fluxes in the plankton community. The plankton community structure was characteristic for a post-bloom situation and retention food web and showed high bacterial production (∼31% of primary production), high abundance of mixotrophic phytoplankton, low mesozooplankton grazing (∼6% of primary production) and low export (∼7% of primary production). Zooplankton grazing and export of detritus were sensitive to CO2: grazing decreased and export increased with increasing pCO2. Nutrient addition halfway through the experiment increased the export, but not the production rates. Although mixotrophs showed initially higher production rates with increasing CO2, the overall production of POC (particulate organic carbon) after nutrient addition decreased with increasing CO2. Interestingly, and contrary to the low nutrient situation, much more material settled down in the sediment traps at low CO2. The observed CO2 related effects potentially alter future organic carbon flows and export, with possible consequences for the efficiency of the biological pump.

  10. Comparing three methods of NEE-flux partitioning from the same grassland ecosystem: the 13C, 18O isotope approach and using simulated Ecosystem respiration

    NASA Astrophysics Data System (ADS)

    Siegwolf, R.; Bantelmann, E.; Saurer, M.; Eugster, W.; Buchmann, N.

    2007-12-01

    As a change in the global climate occurs with increasing temperatures, the Carbon exchange processes of terrestrial ecosystems will change as well. However, it is difficult to quantify the degree to what ecosystem respiration will change relative to the CO2 uptake by photosynthesis. To estimate the carbon sequestration potential of terrestrial vegetation cover it is essential to know both fluxes: ecosystem respiration and the carbon uptake by the vegetation cover. Therefore the net ecosystem exchange of CO2 (NEE) was measured with the eddy covariance method and separated into assimilation and respiration flux. We applied three different approaches, 1) the conventional method, applying the nighttime relationship between soil temperature and NEE for calculating the respiration flux during the day, 2) the use of stable carbon and 3) oxygen isotopes. We compared the results of the three partitioning exercises for a temperate grassland ecosystem in the pre-Alps of Switzerland for four days in June 2004. The assimilation flux derived with the conventional NEE partitioning approach, was best represented at low PAR and low temperatures, in the morning between 5 and 9 am. With increasing temperature and PAR the assimilation for the whole canopy was underestimated. For partitioning NEE via 18O approach, correlations of temperature and radiation with assimilation and respiration flux were significantly higher for the partitioning approach with 18O than for the 13C NEE partitioning. A sensitivity analysis showed the importance of an accurate determination of the equilibrium term θ between CO2 and leaf water δ18O for the NEE partitioning with 18O. For using 13C to partition NEE, the correct magnitude of the 13C fractionation and for the respiration term is essential. The analysis of the data showed that for low light and low morning temperatures the conventional method delivers reasonably good results. When the temperatures exceeded 21°C the isotope approach provided the

  11. Increasing Pyruvate Dehydrogenase Flux as a Treatment for Diabetic Cardiomyopathy: A Combined 13C Hyperpolarized Magnetic Resonance and Echocardiography Study

    PubMed Central

    Le Page, Lydia M.; Rider, Oliver J.; Lewis, Andrew J.; Ball, Vicky; Clarke, Kieran; Johansson, Edvin; Carr, Carolyn A.; Heather, Lisa C.; Tyler, Damian J.

    2015-01-01

    Although diabetic cardiomyopathy is widely recognised, there are no specific treatments available. Altered myocardial substrate selection has emerged as a candidate mechanism behind the development of cardiac dysfunction in diabetes. As pyruvate dehydrogenase (PDH) activity appears central to the balance of substrate utilisation, we aimed to investigate the relationship between PDH flux and myocardial function in a rodent model of type-II diabetes and to explore whether or not increasing PDH flux, with dichloroacetate, would restore the balance of substrate utilisation and improve cardiac function. All animals underwent in vivo hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopy and echocardiography to assess cardiac PDH flux and function respectively. Diabetic animals showed significantly higher blood glucose (10.8±0.7mM vs 8.4±0.5mM), lower PDH flux (0.005±0.001s−1 vs 0.017±0.002s−1) and significantly impaired diastolic function (E/E’ 12.2±0.8 vs 20±2) in keeping with early diabetic cardiomyopathy. Twenty-eight days treatment with dichloroacetate restored PDH flux to normal levels (0.018±0.002s−1), reversed diastolic dysfunction (E/E’ 14±1) and normalized blood glucose (7.5±0.7mM). Treatment of diabetes with dichloroacetate therefore restored the balance of myocardial substrate selection, reversed diastolic dysfunction and normalised blood glucose levels. This suggests that PDH modulation could be a novel therapy for the treatment and/or prevention of diabetic cardiomyopathy. PMID:25795215

  12. Characterizing biomass fast pyrolysis oils by 13C-NMR and chemometric analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several biomass fast pyrolysis oils were characterized by 13C and DEPT NMR analysis to determine chemical functional group compositions as related to their energy content. Pyrolysis oils were produced from a variety of feedstocks including energy crops, woods, animal wastes and oil seed presscakes,...

  13. Analysis of mutational lesions of acetate metabolism in Neurospora crassa by 13C nuclear magnetic resonance.

    PubMed Central

    Thomas, G H; Baxter, R L

    1987-01-01

    The adaptation of Neurospora crassa mycelium to growth on acetate as the sole carbon source was examined by using 13C nuclear magnetic resonance. Extracts were examined by nuclear magnetic resonance at various times after transfer of the mycelium from medium containing sucrose to medium containing [2-13C]acetate as the sole carbon source. The label was initially seen to enter the alanine, glutamate, and glutamine pools, and after 6 h 13C-enriched trehalose was evident, indicating that gluconeogenesis was occurring. Analysis of the isotopomer ratios in the alanine and glutamate-glutamine pools indicated that substantial glyoxylate cycle activity became evident between 2 and 4 h after transfer. Immediately after transfer of the mycelium to acetate medium, the alanine pool increased to about four times its previous level, only a small fraction of which was enriched with 13C. The quantity of 13C-enriched alanine remained almost constant between 2 and 7.5 h after the transfer, whereas the overall alanine pool decreased to its original level. The selective catabolism of the unenriched alanine leads us to suggest that the alanine pool is partitioned into two compartments during adaptation. Two acetate-nonutilizing mutants were also studied by this technique. An acu-3 strain, deficient for isocitrate lyase (EC 4.1.3.1) activity, showed metabolic changes consistent with this lesion. An acp strain, previously thought to be deficient in an inducible acetate permease, took up [2-13C]acetate but showed no evidence of glyoxylate cycle activity despite synthesizing the necessary enzymes; the lesion was therefore reinterpreted. PMID:2947898

  14. Rapid detection and characterization of surface CO2 leakage through the real-time measurement of δ13C signatures in CO2 flux from the ground

    NASA Astrophysics Data System (ADS)

    Krevor, S.; Perrin, J.; Esposito, A.; Rella, C.; Benson, S. M.

    2009-12-01

    A portable stable carbon isotope ratio analyzer for carbon dioxide, based on wavelength scanned cavity ringdown spectroscopy, has been used to detect and characterize an intentional leakage of CO2 from an underground pipeline at the ZERT experimental facility in Bozeman, Montana. Rapid (~1 hour) walking surveys of the entire 100m x 100m site were collected using this mobile, real-time instrument. The resulting concentration and 13C isotopic abundance maps were processed using simple yet powerful analysis techniques, permitting not only the identification of specific leakage locations, but providing the ability to distinguish petrogenic sources of CO2 from biogenic sources. At the site an approximately 100-meter horizontal well has been drilled below an alfalfa field at a depth between 1-3 meters below the surface. The well has perforations along the central 70 meters of the well. The overlying strata are highly permeable sand, silt, and topsoil. The flora consists generally of long grasses and was cut to a height of less than 6 inches before the start of the experiment. For 30 days starting July 15, 2009, CO2 was injected at a rate of 0.2 tonnes per day. The injection rate is designed to simulate leakage from a mature storage reservoir at an annual rate of between .001 and .01%. The isotopic composition of the gas from the tank is at δ13C signature of approximately -52‰, far more negative than either atmospheric (approx. -8‰) or CO2 from soil respiration (approx. -26‰) at the site. The CO2 isotopic and concentration measurements were taken with a Picarro WS-CRDS analyzer with 1/8” tubing connected to a sampling inlet. Simultaneous with CO2 concentrations (including 13C), position data was logged using a GPS receiver. Datapoints are taken around every second. The analyzer was powered using batteries and housed in a conventional garden cart. The surveys consisted of traverses of the site along the length of the pipeline and extending out 100 meters on either

  15. Strength and limits using 13C phospholipid fatty acid analysis in soil ecology

    NASA Astrophysics Data System (ADS)

    Watzinger, Andrea

    2016-04-01

    This presentation on microbial phospholipid biomarkers, their isotope analysis and their ability to reveal soil functions summarizes experiences gained by the author for more than 10 years. The amount and composition of phospholipid fatty acids (PLFAs) measured in environmental samples strongly depend on the methodology. To achieve comparable results the extraction, separation and methylation method must be kept constant. PLFAs patterns are sensitive to microbial community shifts even though the taxonomic resolution of PLFAs is low. The possibility to easily link lipid biomarkers with stable isotope techniques is identified as a major advantage when addressing soil functions. Measurement of PLFA isotopic ratios is sensitive and enables detecting isotopic fractionation. The difference between the carbon isotopic ratio of single PLFAs and their substrate (δ13C) can vary between -6 and +11‰. This difference derives from the fractionation during biosynthesis and from substrate inhomogeneity. Consequently, natural abundance studies are restricted to quantifying substrate uptake of the total microbial biomass. In contrast, artificial labelling enables quantifying carbon uptake into single PLFAs, but labelling success depends on homogeneous and undisturbed label application. Current developments in microbial ecology (e.g. 13C and 15N proteomics) and isotope techniques (online monitoring of CO2 isotope ratios) will likely improve soil functional interpretations in the future. 13C PLFA analysis will continue to contribute because it is affordable, sensitive and allows frequent sampling combined with the use of small amounts of 13C label.

  16. Effects of insulin on perfused liver from streptozotocin-diabetic and untreated rats: /sup 13/C NMR assay of pyruvate kinase flux

    SciTech Connect

    Cohen, S.M.

    1987-01-27

    The effects of insulin in vitro on perfused liver from streptozotocin-diabetic rats and their untreated littermates during gluconeogenesis from either (3-/sup 13/C)alanine + ethanol or (2-/sup 13/C)pyruvate + NH/sub 4/Cl + ethanol were studied by /sup 13/C NMR. A /sup 13/C NMR determination of the rate of pyruvate kinase flux under steady-state conditions of active gluconeogenesis was developed; this assay includes a check on the reuse of recycled pyruvate. The preparations studied provided gradations of pyruvate kinase flux within the confines of the assay's requirement of active gluconeogenesis. By this determination, the rate of pyruvate kinase flux was 0.74 +/- 0.04 of the gluconeogenic rate in liver from 24-h-fasted controls; in liver from 12-h fasted controls, relative pyruvate kinase flux increased to 1.0 +/- 0.2. In diabetic liver, this flux was undetectable by the authors NMR method. Insulin's hepatic influence in vitro was greatest in the streptozotocin model of type 1 diabetes: upon treatment of diabetic liver with 7 nM insulin in vitro, a partial reversal of many of the differences noted between diabetic and control liver was demonstrated by /sup 13/C NMR. A major effect of insulin in vitro upon diabetic liver was the induction of a large increase in the rate of pyruvate kinase flux, bringing relative and absolute fluxes up to the levels measured in 24-h-fasted controls. By way of comparison, the effects of ischemia on diabetic liver were studied by /sup 13/C NMR to test whether changes in allosteric effectors under these conditions could also increase pyruvate kinase flux. A large increase in this activity was demonstrated in ischemic diabetic liver.

  17. 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.

  18. A 13C labelling study on carbon fluxes in Arctic plankton communities under elevated CO2 levels

    NASA Astrophysics Data System (ADS)

    de Kluijver, A.; Soetaert, K.; Czerny, J.; Schulz, K. G.; Boxhammer, T.; Riebesell, U.; Middelburg, J. J.

    2012-07-01

    The effect of CO2 on carbon fluxes in Arctic plankton communities was investigated during the 2010 EPOCA mesocosm study in Ny Ålesund, Svalbard. Nine mesocosms were set up with initial pCO2 levels ranging from 185 to 1420 μatm for 5 weeks. 13C labelled bicarbonate was added at the start of the experiment to follow the transfer of carbon from dissolved inorganic carbon (DIC) into phytoplankton, bacteria, total particulate organic carbon (POC), zooplankton, and settling particles. Polar lipid derived fatty acids (PLFA) were used to trace carbon dynamics of phytoplankton and bacteria and allowed distinction of two groups of phytoplankton: phyto I (autotrophs) and phyto II (mixotrophs). Nutrients were added on day 13. A nutrient-phytoplankton-zooplankton-detritus model amended with 13C dynamics was constructed and fitted to the data to quantify uptake rates and carbon fluxes in the plankton community during the phase prior to nutrient addition (phase 1, days 0-12). During the first 12 days, a phytoplankton bloom developed that was characterized by high growth rates (0.87 days-1) for phyto I and lower growth rates (0.18 days-1) for phyto II. A large part of the carbon fixed by phytoplankton (~31%) was transferred to bacteria, while mesozooplankton grazed only ~6% of the production. After 6 days, the bloom collapsed and part of the organic matter subsequently settled into the sediment traps. The sedimentation losses of detritus in phase 1 were low (0.008 days-1) and overall export was only ~7% of production. Zooplankton grazing and detritus sinking losses prior to nutrient addition were sensitive to CO2: grazing decreased with increasing CO2, while sinking increased. Phytoplankton production increased again after nutrient addition on day 13. Although phyto II showed initially higher growth rates with increasing CO2 (days 14-22), the overall production of POC after nutrient addition (phase 2, days 14-29) decreased with increasing CO2. Significant sedimentation occurred

  19. 13C solid-state NMR chemical shift anisotropy analysis of the anomeric carbon in carbohydrates.

    PubMed

    Chen, Ying-Ying; Luo, Shun-Yuan; Hung, Shang-Cheng; Chan, Sunney I; Tzou, Der-Lii M

    2005-03-21

    (13)C NMR solid-state structural analysis of the anomeric center in carbohydrates was performed on six monosaccharides: glucose (Glc), mannose (Man), galactose (Gal), galactosamine hydrochloride (GalN), glucosamine hydrochloride (GlcN), and N-acetyl-glucosamine (GlcNAc). In the 1D (13)C cross-polarization/magic-angle spinning (CP/MAS) spectrum, the anomeric center C-1 of these carbohydrates revealed two well resolved resonances shifted by 3-5ppm, which were readily assigned to the anomeric alpha and beta forms. From this experiment, we also extracted the (13)C chemical shift anisotropy (CSA) tensor elements of the two forms from their spinning sideband intensities, respectively. It was found out that the chemical shift tensor for the alpha anomer was more axially symmetrical than that of the beta form. A strong linear correlation was obtained when the ratio of the axial asymmetry of the (13)C chemical shift tensors of the two anomeric forms was plotted in a semilogarithmic plot against the relative population of the two anomers. Finally, we applied REDOR spectroscopy to discern whether or not there were any differences in the sugar ring conformation between the anomers. Identical two-bond distances of 2.57A (2.48A) were deduced for both the alpha and beta forms in GlcNAc (GlcN), suggesting that the two anomers have essentially identical sugar ring scaffolds in these sugars. In light of these REDOR distance measurements and the strong correlation observed between the ratio of the axial asymmetry parameters of the (13)C chemical shift tensors and the relative population between the two anomeric forms, we concluded that the anomeric effect arises principally from interaction of the electron charge clouds between the C-1-O-5 and the C-1-O-1 bonds in these monosaccharides.

  20. 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. PMID:12009795

  1. Analysis of defect structure in silicon. Characterization of samples from UCP ingot 5848-13C

    NASA Technical Reports Server (NTRS)

    Natesh, R.; Guyer, T.; Stringfellow, G. B.

    1982-01-01

    Statistically significant quantitative structural imperfection measurements were made on samples from ubiquitous crystalline process (UCP) Ingot 5848 - 13 C. Important trends were noticed between the measured data, cell efficiency, and diffusion length. Grain boundary substructure appears to have an important effect on the conversion efficiency of solar cells from Semix material. Quantitative microscopy measurements give statistically significant information compared to other microanalytical techniques. A surface preparation technique to obtain proper contrast of structural defects suitable for QTM analysis was perfected.

  2. Analysis of Hydroperoxides in Solid Polyethylene by MAS (13)C NMR and EPR

    SciTech Connect

    ASSINK,ROGER A.; CELINA,MATHIAS C.; DUNBAR,TIMOTHY D.; ALAM,TODD M.; CLOUGH,ROGER LEE; GILLEN,KENNETH T.

    1999-11-19

    {sup 13}C-enriched polyethylene was subjected to {gamma}-irradiation in the presence of air at 25 and 80 C for total doses ranging from 71 to 355 kGy. Significant quantities of hydroperoxides were detected in the 25 C irradiated sample by {sup 13}C magic angle spinning NMR spectroscopy. This method of detection was performed on the solid polymer and required no chemical derivatization or addition of solvent. The chemical stability and subsequent products of the hydroperoxide species were studied by annealing the irradiated samples in air at temperatures ranging from 22 to 110 C. A time-temperature superposition analysis provided an activation energy of 108 kJ/mol for the hydroperoxide decomposition process. The primary products of hydroperoxide decomposition were ketones and secondary alcohols with lesser amounts of acids and esters. EPR measurements suggest that the reactive hydroperoxide species reside in the amorphous phase of polyethylene, consistent with degradation occurring in the amorphous phase.

  3. Phenyl galactopyranosides - 13C CPMAS NMR and conformational analysis using genetic algorithm

    NASA Astrophysics Data System (ADS)

    Wałejko, Piotr; Paradowska, Katarzyna; Bukowicki, Jarosław; Witkowski, Stanisław; Wawer, Iwona

    2015-08-01

    Structural analyses of four compounds (phenyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (1), phenyl β-D-galactopyranoside (2), phenyl 2,3,4,6-tetra-O-acetyl-α-D-galactopyranoside (3) and phenyl α-D-galactopyranoside (4)) have been performed using solid-state 13C MAS NMR spectroscopy and theoretical methods. Conformational analysis involved grid search and genetic algorithm (GAAGS). Low-energy conformers found by GAAGS were further optimized by DFT and chemical shifts were calculated using GIAO/DFT approach. 13C CPMAS NMR chemical shift of carbon C2 is indicative of the glycoside torsional angle. Separated or merged resonances of C2 and C6 suggest free rotation of phenyl ring in the solid phase.

  4. Fecal /sup 13/C analysis for the detection and quantitation of intestinal malabsorption

    SciTech Connect

    Schoeller, D.A.; Klein, P.D.; MacLean, W.C. Jr.; Watkins, J.B.; Van Santen, E.

    1981-03-01

    The use of /sup 14/CO/sub 2/ breath tests and fecal analyses for the detection and quantitation of intestinal malabsorption has been extensively documented in adult subjects. The use of stable isotopes has extended the range of breath test applications to include pediatric and obstetric subjects. Here we report a fecal /sup 13/C analysis that can be used in conjunction with /sup 13/CO/sub 2/ breath tests. Twenty-four-hour fecal samples were collected before and after the administration of a labeled substrate. The samples were homogenized and combusted to CO/sub 2/, and the /sup 13/C abundance was determined by high-precision, differential isotope ratio mass spectrometry. The isotopic variation between successive 24 hr fecal samples was 0.6 per thousand (0.0006 atom percent). This variation limited the sensitivity of the fecal analysis to 13 ..mu..mol of /sup 13/C label per mole of fecal carbon. Simultaneous cholyglycine /sup 13/CO/sub 2/ breath tests and fecal assays were performed in five children. One child with bacterial overgrowth had an abnormal breath test and a normal fecal test. Of three children with ileal dysfunction, only one had an abnormal breath test, whereas the fecal test was abnormal in all three. Both the breath test and fecal test were abnormal for a child who had undergone an ileal resection. Both tests were normal for a child with ulcerative colitis.

  5. High-resolution FTIR analysis and rotational constants for the ν12 band of ethylene-1-13C (13C12CH4)

    NASA Astrophysics Data System (ADS)

    Gabona, M. G.; Tan, T. L.; Woo, J. Q.

    2014-11-01

    The Fourier transform infrared (FTIR) absorption spectrum of the ν12 fundamental band of ethylene-1-13C (or 13C12CH4) was recorded in the frequency range of 1350-1510 cm-1 with an unapodized resolution of 0.0063 cm-1. The upper state (ν12 = 1) and ground state rotational constants derived in the present analysis cover a wide wavenumber range and high J and Ka (J = 41 and Ka = 14). By assigning and fitting 1602 infrared transitions using Watson's A-reduced Hamiltonian in the Ir representation, upper state (ν12 = 1) constants consisting of three rotational, five quartic and two sextic constants were more accurately determined. The root-mean-square deviation of the fit was 0.00030 cm-1. Ground state rotational constants were also improved from the fit of 808 ground state combination differences (GSCDs) with a root-mean-square deviation of 0.00032 cm-1. The unperturbed A-type ν12 band is centered at 1439.34612(2) cm-1. The inertial defect Δ of 0.05381(8) μÅ2 for the ground state has been derived using the ground state rotational constants obtained from this work.

  6. 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.

  7. 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

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

    PubMed

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

    2015-07-15

    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. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]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.

  9. Systematic R -matrix analysis of the 13C(p ,γ )14N capture reaction

    NASA Astrophysics Data System (ADS)

    Chakraborty, Suprita; deBoer, Richard; Mukherjee, Avijit; Roy, Subinit

    2015-04-01

    Background: The proton capture reaction 13C(p ,γ )14N is an important reaction in the CNO cycle during hydrogen burning in stars with mass greater than the mass of the Sun. It also occurs in astrophysical sites such as red giant stars: the asymptotic giant branch (AGB) stars. The low energy astrophysical S factor of this reaction is dominated by a resonance state at an excitation energy of around 8.06 MeV (Jπ=1-,T =1 ) in 14N. The other significant contributions come from the low energy tail of the broad resonance with Jπ=0-,T =1 at an excitation of 8.78 MeV and the direct capture process. Purpose: Measurements of the low energy astrophysical S factor of the radiative capture reaction 13C(p ,γ )14N reported extrapolated values of S (0 ) that differ by about 30 % . Subsequent R -matrix analysis and potential model calculations also yielded significantly different values for S (0 ) . The present work intends to look into the discrepancy through a detailed R -matrix analysis with emphasis on the associated uncertainties. Method: A systematic reanalysis of the available decay data following the capture to the Jπ=1-,T =1 resonance state of 14N around 8.06 MeV excitation had been performed within the framework of the R -matrix method. A simultaneous analysis of the 13C(p ,p0 ) data, measured over a similar energy range, was carried out with the capture data. The data for the ground state decay of the broad resonance state (Jπ=0-,T =1 ) around 8.78 MeV excitations was included as well. The external capture model along with the background poles to simulate the internal capture contribution were used to estimate the direct capture contribution. The asymptotic normalization constants (ANCs) for all states were extracted from the capture data. The multichannel, multilevel R -matrix code azure2 was used for the calculation. Results: The values of the astrophysical S factor at zero relative energy, resulting from the present analysis, are found to be consistent within the

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

    PubMed

    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

    2016-03-01

    A network model for the determination of tumor metabolic fluxes from (13)C 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-(13)C2]glucose under normoxic conditions at 37 °C and monitored by (13)C 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

  11. Microbial utilization of sugars in soil assessed by position-specific labeling and compound-specific 13C-PLFA-analysis

    NASA Astrophysics Data System (ADS)

    Apostel, Carolin; Dippold, Michaela; Glaser, Bruno; Kuzyakov, Yakov

    2014-05-01

    For the transformation of low molecular weight organic substances (LMWOS) in soil, which is an important process in the turnover of organic matter, microbial utilization is one of the most important processes. Position-specific labeling combined with compound-specific 13C-PLFA-analysis allows a closer look on the mechanisms of LMWOS transformation in soil. We assessed short- (3 and 10 days) and long-term (half year) transformations of monosaccharides by adding position-specifically 13C labeled glucose and ribose to soil in a field experiment conducted on an agriculturally used luvisol located in north-western Bavaria. We quantified the microbial utilization of the different functional groups by 13C-analysis of microbial biomass with the chloroform-fumigation-extraction method (CFE). 13C-PLFA analysis enabled us to distinguish individual microbial groups and compare their C-utilization. Preferential degradation of glucoses C-3 and C-4 respectively C-1 position enabled differentiation between the two main hexose metabolic pathways - glycolysis and the pentose phosphate pathway. Microbial groups revealed different incorporation of specific C positions into their PLFA. The highest incorporation was reached by the prokaryotic gram- negative groups. The application of position-specifically labeled substances, coupled with compound-specific 13C-PLFA analysis opens a new way to investigate the microbial transformations of LMWOS in soil. Observing single C atoms and their utilization by specific microbial groups allow conclusions about the mechanisms and kinetics of microbial utilization and interaction between these groups and therefore will improve our understanding of soil carbon fluxes.

  12. Seasonal and inter-annual variability in 13C composition of ecosystem carbon fluxes in the U.S. Southern Great Plains

    SciTech Connect

    Torn, M.S.; Biraud, S.; Still, C.J.; Riley, W.J.; Berry, J.A.

    2010-09-22

    The {delta}{sup 13}C signature of terrestrial carbon fluxes ({delta}{sub bio}) provides an important constraint for inverse models of CO{sub 2} sources and sinks, insight into vegetation physiology, C{sub 3} and C{sub 4} vegetation productivity, and ecosystem carbon residence times. From 2002-2009, we measured atmospheric CO{sub 2} concentration and {delta}{sup 13}C-CO{sub 2} at four heights (2 to 60 m) in the U.S. Southern Great Plains (SGP) and computed {delta}{sub bio} weekly. This region has a fine-scale mix of crops (primarily C{sub 3} winter wheat) and C{sub 4} pasture grasses. {delta}{sub bio} had a large and consistent seasonal cycle of 6-8{per_thousand}. Ensemble monthly mean {delta}{sub bio} ranged from -25.8 {+-} 0.4{per_thousand} ({+-}SE) in March to -20.1 {+-} 0.4{per_thousand} in July. Thus, C{sub 3} vegetation contributed about 80% of ecosystem fluxes in winter-spring and 50% in summer-fall. In contrast, prairie-soil {delta}{sub 13}C values were about -15{per_thousand}, indicating that historically the region was dominated by C{sub 4} vegetation and had more positive {delta}{sub bio} values. Based on a land-surface model, isofluxes ({delta}{sub bio} x NEE) in this region have large seasonal amplitude because {delta}{sub bio} and net ecosystem exchange (NEE) covary. Interannual variability in isoflux was driven by variability in NEE. The large seasonal amplitude in {delta}{sub bio} and isoflux imply that carbon inverse analyses require accurate estimates of land cover and temporally resolved {sup 13}CO{sub 2} and CO{sub 2} fluxes.

  13. Mathematical modelling of the citric acid cycle for the analysis of glutamine isotopomers from cerebellar astrocytes incubated with [1(-13)C]glucose.

    PubMed

    Merle, M; Martin, M; Villégier, A; Canioni, P

    1996-08-01

    A mathematical model of the citric acid cycle devoted to the analysis of 13C-NMR data was developed for determining the relative flux of molecules through the anaplerotic versus oxidative pathways and the relative pyruvate carboxylase versus pyruvate dehydrogenase activities. Different variants of the model were considered depending on the reversibility of the conversion of fumarate into malate and oxaloacetate. The model also included the possibility of orientation-conserved transfer of the four-carbon citric acid cycle intermediates, leading to conversion of succinyl-CoA C1 into either malate C1 or C4. It was used to analyse NMR data from glutamine isotopomers produced by cerebellar astrocytes incubated with [1-13C]glucose. Partial cycling (39%) between oxaloacetate and fumarate was evident from the analysis. Application of the model to glutamate isotopomers from granule cells incubated with [1-13C]glucose [Martin, M.. Portais, J.C.. Labouesse. J., Canioni. P, & Merle, M. (1993) Eur. J. Biochem. 217, 617-625] indicated that total cycling of oxaloacetate into fumarate was, in this case, required to get the best fit. The results emphasized some important differences in carbon metabolism between cerebellar astrocytes and granule cells concerning the sources of carbon fuelling the citric acid cycle and the carbon fluxes on different pathways.

  14. Food webs in Mongolian grasslands: the analysis of 13C and 15N natural abundances.

    PubMed

    Kohzu, Ayato; Iwata, T; Kato, M; Nishikawa, J; Wada, Eitaro; Amartuvshin, N; Namkhaidorj, B; Fujita, N

    2009-09-01

    Overgrazing often lowers species richness and productivity of grassland communities. For Mongolian grassland ecosystems, a lack of detailed information about food-web structures makes it difficult to predict the effects of overgrazing on species diversity and community composition. We analysed the delta13C and delta15N signatures of herbaceous plants, arthropods (grouped by feeding habit), wild and domestic mammals, and humans in central Mongolia to understand the predominant food-web pathways in this grassland ecosystem. The delta13C and delta15N values of mammals showed little variation within species, but varied considerably with slope position for arthropods. The apparent isotopic discrimination between body tissue and hair of mammals was estimated as 2.0 per thousand for delta13C and 2.1 per thousand for delta15N, which was large enough to cause overestimation of the trophic level of mammals if not taken into account when using hair samples to measure isotopic enrichment.

  15. Quantitative Analysis of Metabolic Mixtures by 2D 13C-Constant-Time TOCSY NMR Spectroscopy

    PubMed Central

    Bingol, Kerem; Zhang, Fengli; Bruschweiler-Li, Lei; Brüschweiler, Rafael

    2013-01-01

    An increasing number of organisms can be fully 13C-labeled, which has the advantage that their metabolomes can be studied by high-resolution 2D NMR 13C–13C constant-time (CT) TOCSY experiments. Individual metabolites can be identified via database searching or, in the case of novel compounds, through the reconstruction of their backbone-carbon topology. Determination of quantitative metabolite concentrations is another key task. Because significant peak overlaps in 1D NMR spectra prevents straightforward quantification through 1D peak integrals, we demonstrate here the direct use of 13C–13C CT-TOCSY spectra for metabolite quantification. This is accomplished through the quantum-mechanical treatment of the TOCSY magnetization transfer at short and long mixing times or by the use of analytical approximations, which are solely based on the knowledge of the carbon-backbone topologies. The methods are demonstrated for carbohydrate and amino-acid mixtures. PMID:23773204

  16. 13C-n.m.r. analysis of some sulphate derivatives of chitosan.

    PubMed

    Hirano, S; Hasegawa, M; Kinugawa, J

    1991-10-01

    Positions of substitution with sulphate in three water-soluble sulphated derivatives of chitosan were analysed by 13C n.m.r. The structures of N-acetylchitosan 3,6-O-disulphate, sodium chitosan N-, 6-O-disulphate, and sodium chitosan 6-O-monosulphate were confirmed.

  17. Compound specific 13C- and 18O-isotope analysis of organic aerosols

    NASA Astrophysics Data System (ADS)

    Blees, Jan; Saurer, Matthias; Siegwolf, Rolf T. W.; Dommen, Josef; Baltensperger, Urs

    2014-05-01

    . Elements other than carbon may provide valuable additional information. Here we report on the development of methods for the analysis of stable carbon and oxygen isotope ratios of organic compounds in aerosols, through GC-combustion-irMS and GC-pyrolysis-irMS. We apply these analyses to environmental aerosol samples and samples of smog-chamber experiments, with the aim of identifying isotopic signatures of sources and pathways. We will pay special attention to derivatisation techniques - notably alternatives to the often-used trimethylsilyl derivatives in GC-pyrolysis-irMS for δ18O analysis - and to compound separation and identification. We present initial data of combined δ13C and δ18O studies on (secondary) organic aerosol samples, and their added value for source apportionment studies.

  18. Double cross polarization /sup 13/C-NMR experiment in solid fossil fuel structure analysis

    SciTech Connect

    Hagaman, E.W.; Woody, M.C.

    1988-01-01

    The Double Cross Polarization /sup 13/C-MAS/NMR experiment has been used to derive a new operational classification of solid fossil fuels based on chemical reactivity. The method requires labeling reactive sites in the organic matrix with a magnetically active isotope not present in the precursor material, and using the local, isolated dipole-dipole interaction between this nucleus and nearby /sup 13/C nuclei to detect via cross polarization the carbon centers in the vicinity of the label. The technique is a marriage of chemistry and spectroscopy and the information content of the DCP spectra is defined by both partners. /sup 1/H-/sup 13/C-/sup 31/P DCP/MAS /sup 13/C-NMR spectroscopy has been used to statistically describe phenolic ortho-substitution patterns of coals via their aryl phosphinate or phosphate derivatives. In these applications of DCP NMR the new, detailed structure and/or reactivity information is realized by detection of carbon resonances one or more bonds removed from the reaction center, but in a volume element of intramolecular dimensions. To the extent that intermolecular contributions to the spectrum are detected, and not recognized as such, the structure/reactivity correlation is weakened. Direct substitution of phosphorus on the aromatic rings in the organic matrix of the coal is not readily accomplished. This environment potentially can be labeled with fluorine in a selective fashion using newly developed reagents. The possibility of determining the changes in average ring substitution patterns as a function of chemical treatment or coal diagenesis emerges. Recent developments in the field of DCP /sup 13/C NMR are presented.

  19. Analysis of states in {sup 13}C populated in {sup 9}Be + {sup 4}He resonant scattering

    SciTech Connect

    Freer, M.; Ashwood, N. I.; Curtis, N.; Kokalova, Tz.; Wheldon, C.; Di Pietro, A.; Figuera, P.; Fisichella, M.; Scuderi, V.; Torresi, D.; Grassi, L.; Jelavic Malenica, D.; Koncul, M.; Mijatovic, T.; Prepolec, L.; Skukan, N.; Soic, N.; Szilner, S.; Tokic, V.; Milin, M.

    2011-09-15

    Measurements of {sup 9}Be + {alpha} resonant scattering have been performed using the thick-target approach with a {sup 4}He gas volume and a large-area silicon strip detector. {sup 9}Be beam energies in the range 12 to 21.4 MeV were used to measure the {sup 13}C excitation energy spectrum between 13.2 and 16.2 MeV. An R-matrix analysis has been performed to characterize the spins and widths of {sup 13}C resonances, some of which have been proposed to be associated with a 3{alpha}+n molecular band.

  20. Transmembrane Exchange of Hyperpolarized 13C-Urea in Human Erythrocytes: Subminute Timescale Kinetic Analysis

    PubMed Central

    Pagès, Guilhem; Puckeridge, Max; Liangfeng, Guo; Tan, Yee Ling; Jacob, Chacko; Garland, Marc; Kuchel, Philip W.

    2013-01-01

    The rate of exchange of urea across the membranes of human erythrocytes (red blood cells) was quantified on the 1-s to 2-min timescale. 13C-urea was hyperpolarized and subjected to rapid dissolution and the previously reported (partial) resolution of 13C NMR resonances from the molecules inside and outside red blood cells in suspensions was observed. This enabled a stopped-flow type of experiment to measure the (initially) zero-trans transport of urea with sequential single-pulse 13C NMR spectra, every second for up to ∼2 min. Data were analyzed using Bayesian reasoning and a Markov chain Monte Carlo method with a set of simultaneous nonlinear differential equations that described nuclear magnetic relaxation combined with transmembrane exchange. Our results contribute to quantitative understanding of urea-exchange kinetics in the whole body; and the methodological approach is likely to be applicable to other cellular systems and tissues in vivo. PMID:24209840

  1. Food webs in Mongolian grasslands: the analysis of 13C and 15N natural abundances.

    PubMed

    Kohzu, Ayato; Iwata, T; Kato, M; Nishikawa, J; Wada, Eitaro; Amartuvshin, N; Namkhaidorj, B; Fujita, N

    2009-09-01

    Overgrazing often lowers species richness and productivity of grassland communities. For Mongolian grassland ecosystems, a lack of detailed information about food-web structures makes it difficult to predict the effects of overgrazing on species diversity and community composition. We analysed the delta13C and delta15N signatures of herbaceous plants, arthropods (grouped by feeding habit), wild and domestic mammals, and humans in central Mongolia to understand the predominant food-web pathways in this grassland ecosystem. The delta13C and delta15N values of mammals showed little variation within species, but varied considerably with slope position for arthropods. The apparent isotopic discrimination between body tissue and hair of mammals was estimated as 2.0 per thousand for delta13C and 2.1 per thousand for delta15N, which was large enough to cause overestimation of the trophic level of mammals if not taken into account when using hair samples to measure isotopic enrichment. PMID:19507080

  2. Degradation pathways of dissolved carbon in landfill leachate traced with compound-specific (13)C analysis of DOC.

    PubMed

    Mohammadzadeh, Hossein; Clark, Ian

    2008-09-01

    The isotopic compositions of carbon compounds in landfill leachate provide insights into the biodegradation pathways that dominate the different stages of waste decomposition. In this study, the carbon geochemistry of different carbon pools, environmental stable isotopes and compound-specific isotope analysis (CSIA) of leachate dissolved organic carbon (DOC) fractions and gases show distinctions in leachate biogeochemistry and methane production between the young area of active waste emplacement and the old area of historical emplacement at the Trail Road Landfill (TRL). The active area leachate has low DOC concentrations (<200 mg l(-1)) dominated by fulvic acid (FA=160 mg l(-1)), and produces CH(4) dominantly by CO(2) reduction (D- excess=20.6 per thousand). Leachate generated in the area of older waste has high DOC (>4770 mg l(-1)) dominated by FA (4482 mg l(-1)) and simple fatty acids (acetic=1008 mg l(-1) and propionic=608 mg l(-1)), and produces CH(4) by the acetate fermentation pathway (D- excess=9.8 per thousand). CSIA shows an advanced degradation and a progressive accumulation of (13)C of fatty acids in leachate from the older area. The enriched (13)C value of FA (-20 and-26 per thousand for the older and active parts, respectively,) and of low molecular weight DOC (-8 and-27 per thousand) as well as of the bulk DOC (-21 and-25 per thousand) shows more advanced degradation in the older part of the landfill, which is consistent with the shift in the humic/FA ratios (0.05 and 0.18). The (13)C enrichment of acetate (-12 per thousand) above the (13)C of DOC (-21 per thousand) and of propionic acid (-19 per thousand), in older leachate, suggests that this acetate has not evolved from the simple degradation of larger organic molecules, but by homoacetogenesis from the enriched dissolved inorganic carbon (DIC) pool (8 per thousand) and H(2,) which produce a more enriched (13)C of acetate. In contrast, the (13)C of the minor acetate in the active area (-17 per

  3. /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.

  4. Statistical signal analysis of the Phanerozoic ð13C curve: implications for Earth system evolution

    NASA Astrophysics Data System (ADS)

    Bachan, A.; Kump, L. R.; Payne, J.; Saltzman, M.; Thomas, E.

    2014-12-01

    In recent years, vast amounts of carbon isotopic data have been collected allowing the construction of the Phanerozoic δ13C curve in unprecedented detail. Our dataset comprises 8143 points spanning the last 541 m.y., with a mean spacing of 66 k.y. The average δ13C of Phanerozoic carbonate is 1 ‰ ± 2 ‰, in accordance with the canonical values measured in the past. However, the record also shows numerous, highly resolved, large (± 6 ‰) excursions whose magnitude declines through time, especially going into the late Mesozoic and Cenozoic. When the magnitude - distribution of the excursions is tabulated we find that it follows a power law: plotting the min-max differences vs. number of bins in which a particular value occurs reveals that the data fall on a semilogarithmic line with a slope of -0.23 and R2 = 0.99. The result is insensitive to outliers: smoothing the data with lowess, spline, Savitzky-Golay, and Butterworth filters yields similar results. The continuity from small variation to large perturbations, both positive and negative, suggests that, despite the numerous proposed causes for individual carbon isotopic evens, there is likely an underlying mechanism which governs the magnitude of δ13C response to perturbations. We suggest that a mechanism acting to amplify carbon cycle perturbations is the key to explaining the power-law distribution, and identify the anoxia-productivity feedback as the most likely candidate. Establishment of sulfidic conditions is accompanied by increased release of phosphate to the water column, which allows for further productivity, and thus acts as a destabilizing, positive, feedback. This feedback would act to increase carbon cycle swings irrespective of their proximal trigger. The decline in frequency of anoxic-sulfidic bottom waters in the world's oceans, and potential disappearance in the Late Mesozoic-Cenozoic, may account for a reduction in the Earth system's gain and increase in its resilience.

  5. 13C solid-state NMR analysis of heterogeneous structure of beeswax in native state

    NASA Astrophysics Data System (ADS)

    Kameda, Tsunenori

    2005-12-01

    I investigated the molecular structure of natural wax from Japanese bees (Apis cerana japonica) in its native state (neither purified nor recrystallized) by 13C and 1H solid-state NMR. Two strong 13C peaks at 32.9 and 34.0 ppm were attributed to signals from internal-chain methylene carbons [int-(CH2)] in two types of crystal form. The peak at 32.9 ppm was assigned to an orthorhombic crystal form, and that at 34.0 ppm was assigned to a triclinic or monoclinic form. In both crystalline regions, bi-exponential decay of 13C spin-lattice relaxation [T1(C)] for the crystalline peaks due to chain diffusion was observed. 1H spin-lattice relaxation [T1(H)] values for protons of the CH3 group and for int-(CH2) in the crystalline and amorphous regions were identical; this was interpreted as being due to averaging of the T1(H) relaxation rates via spin diffusion. In contrast, although the T_{{1}_{\\rho}}(H) decay curves for protons of the CH3 group and for int-(CH2) in the amorphous and orthorhombic forms were almost identical, those of the triclinic or monoclinic forms were different. This unhomogeneous character of T_{{1}_{\\rho}}(H) was interpreted as resulting from differences in the molecular composition of each crystal form. Moreover, two components with long and short 1H spin-spin relaxation [T2(H)] values, arising from the mobile and rigid phases, respectively, were observed at above about -30 °C.

  6. Stable isotope (13C, 15N and 34S) analysis of the hair of modern humans and their domestic animals.

    PubMed

    Bol, Roland; Pflieger, Christian

    2002-01-01

    Relationships between dietary status and recent migration were examined by delta(13)C, delta(15)N and delta(34)S analysis of hair samples from 43 modern humans living in a rural community in SW England. The isotopic content of 38 'local' hair samples was compared with that of five recently arrived individuals (from Canada, Chile, Germany and the USA). Hair samples from domestic animals (i.e. mainly cats, dogs, cows and horses) were analysed to examine the difference in delta(13)C, delta(15)N and delta(34)S values between herbivores and carnivores. Generally, modern human hair data from the triple stable isotope (delta(13)C, delta(15)N and delta(34)S) provided enough information to confirm the dietary status and origin of the individual subjects. The dietary intake was generally reflected in the animal hair delta(15)N and delta(13)C values, i.e. highest in the carnivores (cats). However, a non-local origin of food sources given to domesticated omnivores (i.e. dogs) was suggested by their hair delta(34)S values.

  7. A computer-assisted structural analysis of regular polysaccharides on the basis of 13C-n.m.r. data.

    PubMed

    Lipkind, G M; Shashkov, A S; Knirel, Y A; Vinogradov, E V; Kochetkov, N K

    1988-04-01

    A computerised approach to the structural analysis of unbranched regular polysaccharides is described, which is based on an evaluation of the 13C-n.m.r. spectra for all possible primary structures within the additive scheme starting from the chemical shifts of the 13C resonances of the constituent monosaccharides and the average values of the glycosylation effects. The analysis reveals a structure (or structures), the evaluated spectrum of which resembles most closely that observed. The approach has been verified by using a series of bacterial polysaccharides of known structure and, in combination with methylation analysis data, for the determination of the presently unknown structures of the O-specific polysaccharides from Salmonella arizonae O59 and O63, and Proteus hauseri O19.

  8. Continuous Flow - Cavity RingDown Spectroscopy Using a Novel Universal Interface for High-Precision Bulk 13C Analysis

    NASA Astrophysics Data System (ADS)

    Saad, Nabil; Richman, Bruce

    2010-05-01

    We have developed the world's first optical spectroscopy-based system for bulk stable isotope analysis of 13C. The system is based on a novel universal interface, named LIAISON, capable of coupling to almost any CO2-generating sample preparation front-end ranging from an elemental analyzer to any dissolved carbon analysis module, which are of significant use in geochemical, ecological and food authentication studies. In one specific application, we have coupled LIAISON to an elemental analyzer (EA) and to a cavity ring-down spectrometer (CRDS) for 13C isotopic analysis of adulterated honey samples. Another application was developed to analyze dissolved inorganic carbon in water samples. LIAISON is suited for handling a high-throughput sample analysis process by running three different gas handling operations in parallel: Admitting combustion gas from the EA into a first gas bellows, analyzing the previous sample collected into a second gas bellows with CRDS, and flushing and purging a third gas bellows in preparation for the upcoming sample collection operation. The sample-to-sample analysis time is 10 minutes and the operation is completely automated for the whole front-end auto-sampler tray capacity, requiring no operator intervention. The CRDS data are collected, tabulated and saved into an output text file. The memory effect between the USGS L-Glutamic acid standard at natural abundance and the moderately enriched USGS L-Glutamic acid standard is excluded by the selection of the adequate number and duration of flush and purge cycles of the gas sample bags. The system's proven accuracy was cross-checked with EA-IRMS and its achieved precision was typically less than 0.2 permil, including the 13C-enriched tested samples. The LIAISON-CRDS system presented here provides a fully automated solution for 13C bulk stable isotope analysis with unprecedented ease-of-use and possible field portability and application with the availability of a compact front-end. In

  9. Isotope ratio mass spectrometry: delta13C and delta15 N analysis for tracing the origin of illicit drugs.

    PubMed

    Galimov, E M; Sevastyanov, V S; Kulbachevskaya, E V; Golyavin, A A

    2005-01-01

    Gas chromatography/combustion/mass spectrometry (GC-C-MS) and elemental analysis/mass spectrometry (EA-MS) techniques are proposed to estimate delta(13)C and delta(15)N values in heroin, morphine, cocaine and hemp leaves, for the purposes of tracing the geographical origins of seized drugs. The values of isotope ratios for pure drugs and drugs with impurities were compared. It was demonstrated that large samples (up to 3 x 10(-6) g C) were combusted completely, so that the results obtained were valid. The data are considered to be an essential supplement to a wide-scale database designed specifically for the delta(13)C and delta(15)N values of drugs. The potential forensic and academic significance of the results is discussed.

  10. LASER BIOLOGY AND MEDICINE: Laser analysis of the 13C/12C isotope ratio in CO2 in exhaled air

    NASA Astrophysics Data System (ADS)

    Stepanov, E. V.

    2002-11-01

    Tunable diode lasers (TDLs) are applied to the diagnostics of gastroenterological diseases using respiratory tests and preparations enriched with the stable 13C isotope. This method of the analysis of the 13C/12C isotope ratio in CO2 in exhaled air is based on the selective measurement of the resonance absorption at the vibrational — rotational structure of 12CO2 and 13CO2. The CO2 transmission spectra in the region of 4.35 μm were measured with a PbEuSe double-heterostructure TDL. The accuracy of carbon isotope ratio measurements in CO2 of exhaled air performed with the TDL was ~0.5%. The data of clinical tests of the developed laser-based analyser are presented.

  11. Selective {sup 2}H and {sup 13}C labeling in NMR analysis of solution protein structure and dynamics

    SciTech Connect

    LeMaster, D.M.

    1994-12-01

    Preparation of samples bearing combined isotope enrichment patterns has played a central role in the recent advances in NMR analysis of proteins in solution. In particular, uniform {sup 13}C, {sup 15}N enrichment has made it possible to apply heteronuclear multidimensional correlation experiments for the mainchain assignments of proteins larger than 30 KDa. In contrast, selective labeling approaches can offer advantages in terms of the directedness of the information provided, such as chirality and residue type assignments, as well as through enhancements in resolution and sensitivity that result from editing the spectral complexity, the relaxation pathways and the scalar coupling networks. In addition, the combination of selective {sup 13}C and {sup 2}H enrichment can greatly facilitate the determination of heteronuclear relaxation behavior.

  12. 13C-NMR analysis of Aspergillus mutants disturbed in pyruvate metabolism.

    PubMed

    Dijkema, C; Visser, J

    1987-12-10

    The metabolic consequences of two defects in pyruvate metabolism of the hyphal fungus Aspergillus nidulans have been investigated by natural abundance 13C-NMR spectroscopy. A pyruvate dehydrogenase complex (pdh) mutant, grown on acetate, accumulates alanine upon starvation which is derived from mannitol reserves. The L-alanine level increases further upon incubation with the non-permissive substrate D-glucose. L-Glutamate is absent from these spectra as it is required both for the transamination of pyruvate and as a reaction on an impaired energy metabolism in such a pdh-deficient strain. A pyruvate carboxylase (pyc) mutant, grown upon acetate, only starts to accumulate alanine after a long incubation period with D-glucose, due to the long-lasting presence of phosphoenolpyruvate carboxykinase and malic enzyme, which are both induced by growth on acetate. When this strain is grown on D-fructose and L-glutamate, alanine also accumulates within 3 h upon transfer to D-glucose.

  13. Compositional differences among Chinese soy sauce types studied by (13)C NMR spectroscopy coupled with multivariate statistical analysis.

    PubMed

    Kamal, Ghulam Mustafa; Wang, Xiaohua; Bin Yuan; Wang, Jie; Sun, Peng; Zhang, Xu; Liu, Maili

    2016-09-01

    Soy sauce a well known seasoning all over the world, especially in Asia, is available in global market in a wide range of types based on its purpose and the processing methods. Its composition varies with respect to the fermentation processes and addition of additives, preservatives and flavor enhancers. A comprehensive (1)H NMR based study regarding the metabonomic variations of soy sauce to differentiate among different types of soy sauce available on the global market has been limited due to the complexity of the mixture. In present study, (13)C NMR spectroscopy coupled with multivariate statistical data analysis like principle component analysis (PCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) was applied to investigate metabonomic variations among different types of soy sauce, namely super light, super dark, red cooking and mushroom soy sauce. The main additives in soy sauce like glutamate, sucrose and glucose were easily distinguished and quantified using (13)C NMR spectroscopy which were otherwise difficult to be assigned and quantified due to serious signal overlaps in (1)H NMR spectra. The significantly higher concentration of sucrose in dark, red cooking and mushroom flavored soy sauce can directly be linked to the addition of caramel in soy sauce. Similarly, significantly higher level of glutamate in super light as compared to super dark and mushroom flavored soy sauce may come from the addition of monosodium glutamate. The study highlights the potentiality of (13)C NMR based metabonomics coupled with multivariate statistical data analysis in differentiating between the types of soy sauce on the basis of level of additives, raw materials and fermentation procedures. PMID:27343582

  14. Large-Scale 13C flux profiling reveals conservation of the Entner-Doudoroff pathway as a glycolytic strategy among marine bacteria that use glucose.

    PubMed

    Klingner, Arne; Bartsch, Annekathrin; Dogs, Marco; Wagner-Döbler, Irene; Jahn, Dieter; Simon, Meinhard; Brinkhoff, Thorsten; Becker, Judith; Wittmann, Christoph

    2015-04-01

    Marine bacteria form one of the largest living surfaces on Earth, and their metabolic activity is of fundamental importance for global nutrient cycling. Here, we explored the largely unknown intracellular pathways in 25 microbes representing different classes of marine bacteria that use glucose: Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia of the Bacteriodetes phylum. We used (13)C isotope experiments to infer metabolic fluxes through their carbon core pathways. Notably, 90% of all strains studied use the Entner-Doudoroff (ED) pathway for glucose catabolism, whereas only 10% rely on the Embden-Meyerhof-Parnas (EMP) pathway. This result differed dramatically from the terrestrial model strains studied, which preferentially used the EMP pathway yielding high levels of ATP. Strains using the ED pathway exhibited a more robust resistance against the oxidative stress typically found in this environment. An important feature contributing to the preferential use of the ED pathway in the oceans could therefore be enhanced supply of NADPH through this pathway. The marine bacteria studied did not specifically rely on a distinct anaplerotic route, but the carboxylation of phosphoenolpyruvate (PEP) or pyruvate for fueling of the tricarboxylic acid (TCA) cycle was evenly distributed. The marine isolates studied belong to clades that dominate the uptake of glucose, a major carbon source for bacteria in seawater. Therefore, the ED pathway may play a significant role in the cycling of mono- and polysaccharides by bacterial communities in marine ecosystems.

  15. Large-Scale 13C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine Bacteria That Use Glucose

    PubMed Central

    Klingner, Arne; Bartsch, Annekathrin; Dogs, Marco; Wagner-Döbler, Irene; Jahn, Dieter; Simon, Meinhard; Brinkhoff, Thorsten; Becker, Judith

    2015-01-01

    Marine bacteria form one of the largest living surfaces on Earth, and their metabolic activity is of fundamental importance for global nutrient cycling. Here, we explored the largely unknown intracellular pathways in 25 microbes representing different classes of marine bacteria that use glucose: Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia of the Bacteriodetes phylum. We used 13C isotope experiments to infer metabolic fluxes through their carbon core pathways. Notably, 90% of all strains studied use the Entner-Doudoroff (ED) pathway for glucose catabolism, whereas only 10% rely on the Embden-Meyerhof-Parnas (EMP) pathway. This result differed dramatically from the terrestrial model strains studied, which preferentially used the EMP pathway yielding high levels of ATP. Strains using the ED pathway exhibited a more robust resistance against the oxidative stress typically found in this environment. An important feature contributing to the preferential use of the ED pathway in the oceans could therefore be enhanced supply of NADPH through this pathway. The marine bacteria studied did not specifically rely on a distinct anaplerotic route, but the carboxylation of phosphoenolpyruvate (PEP) or pyruvate for fueling of the tricarboxylic acid (TCA) cycle was evenly distributed. The marine isolates studied belong to clades that dominate the uptake of glucose, a major carbon source for bacteria in seawater. Therefore, the ED pathway may play a significant role in the cycling of mono- and polysaccharides by bacterial communities in marine ecosystems. PMID:25616803

  16. Conditional flux analysis and stable isotopes

    NASA Astrophysics Data System (ADS)

    Zeeman, M. J.; Knohl, A.; Sturm, P.; Buchmann, N. C.; Thomas, C. K.

    2009-12-01

    We propose to investigate to what extend conditional flux analysis can benefit from the addition of stable isotope information. Stable isotopes have been recognized for their potential as process tracer, and could add an extra dimension to the conditional flux concept, which aims at directly quantifying component fluxes and identifying their sources. Differences in 13C abundance in carbon dioxide can be used to distinguish assimilation or respiration sources, whereas the 18O abundance expresses differences in water exchange, for instance between canopy and soil. Lending to recent advances in measurement technology, stable isotopes can now be measured at high temporal resolutions (10Hz) required for commonly applied micrometeorological methods such as the eddy-covariance technique, or related conditional flux methods. We will present current ideas on how the conditional flux method, as recently proposed and evaluated by Thomas et al. (2008), Scanlon & Sahu (2008), to perform daytime flux partitioning at the ecosystem level, can be refined by stable isotope analysis (13C and 18O) of carbon dioxide as additional dimension for identification of fluxes.

  17. /sup 13/C NMR spectroscopy in the analysis of conjugate metabolites in the bile of fish exposed to petroleum

    SciTech Connect

    Hellou, J.; Banoub, J.H.; Payne, J.F.

    1986-01-01

    The first natural abundance /sup 13/C NMR investigation of a complex mixture of conjugate metabolites obtained from the gall bladder bile of fish exposed to hydrocarbons is presented. Cunners were exposed to water accommodated No. 2 fuel oil containing about 68% saturates and 22% aromatics. Spectral analysis indicated that the hydrocarbon derivatives were present predominantly as ..beta..-glucuronides, with the oxygen at carbon-1 of glucuronic acid preferentially attached to an aliphatic carbon. The conjugate metabolites were enriched in aromatic-type carbons when compared to the fuel oil or the aromatic fraction of oil.

  18. Triterpenes in the hexane extract of leaves of Olea europaea L.: analysis using 13C-NMR spectroscopy.

    PubMed

    Duquesnoy, Emilie; Castola, Vincent; Casanova, Joseph

    2007-01-01

    Two neutral triterpenes and a triterpene acid were identified and quantified directly, in the absence of any purification steps, in a precipitate obtained during the industrial extraction of the leaves of Olea europaea L. using 13C-NMR spectroscopy (spectrometer operating at 4.7 T equipped with a 10 mm probe). The method was optimised in order to reduce the duration of analysis with a routine NMR spectrometer. Together with long-chain linear compounds, erythrodiol, uvaol and oleanolic acid accounted for 27.3, 18.3 and 12.5% of the precipitate, respectively.

  19. Ab initio study of {sup 13}C NMR chemical shifts for the chromophores of rhodopsin and bacteriorhodopsin. 2. Comprehensive analysis of the {sup 13}C chemical shifts of protonated all-trans-retinylidene Schiff base

    SciTech Connect

    Sakurai, Minoru; Wada, Mitsuhito; Inoue, Yoshio; Tamura, Yusuke; Watanabe, Yoichi

    1996-02-01

    Theoretical analysis was performed for the {sup 13}C chemical shifts of the retinal chromophore in bacteriorhodopsin (bR) by means of ab initio NMR shielding calculation, based on the localized orbital/ local origin method. In order to comprehensively investigate the correlation between the {sup 13}C chemical shieldings of the unsaturated carbons and physicochemical perturbations relating to the spectral tuning of bacteriorhodopsin, the following three factors are taken into account in the present calculation: (1) change in strength of the hydrogen bonding between protonated retinylidene Schiff base and its counterion, (2) conformational changes about single bonds of the conjugated chain, and (3) electrostatic interactions between the Schiff base and electric dipoles. On the basis of these calculations, we successfully find a molecular model for which the shielding calculation almost completely reproduces the observed chemical shift data for the chromophore of bR. 47 refs., 13 figs.

  20. Real-time analysis of δ13C- and δD-CH4 by high precision laser spectroscopy

    NASA Astrophysics Data System (ADS)

    Eyer, Simon; Emmenegger, Lukas; Tuzson, Béla; Fischer, Hubertus; Mohn, Joachim

    2014-05-01

    analysis of CH4 isotopologues. The infrared radiation emitted by the two cw-QC laser sources are combined and coupled into a 0.5 L astigmatic multipass absorption cell with an optical path length of 76 m. An Allan variance minimum of the isotope ratio time-series of 0.1 o for δ13C-CH4 and 0.3 o for δD-CH4 has been achieved using 300 s integration time. First experiments of the developed analytical technique demonstrate its potential with respect to field-applicability and temporal resolving power. References: [1] WMO, Greenhouse Gas Bulletin No. 9, 2013, WMO GAW, pp. 4. [2] H. Fischer, M. Behrens, M. Bock, U. Richter, J. Schmitt, L. Loulergue, J. Chappellaz, R. Spahni, T. Blunier, M. Leuenberger and T. F. Stocker, Nature, 2008, 452, 864-867. [3] J. Mohn, B. Tuzson, A. Manninen, N. Yoshida, S. Toyoda, W. A. Brand, and L. Emmenegger, Atmos. Meas. Tech., 2012, 5, 1601-1609. [4] Tuzson, B., Hiller, R. V., Zeyer, K., Eugster, W., Neftel, A., Ammann, C., and L. Emmenegger, Atmos. Meas. Tech., 2010, 3,1519-1531.

  1. Retrospective characterization of ontogenetic shifts in killer whale diets via δ13C and δ15N analysis of teeth

    USGS Publications Warehouse

    Newsome, Seth D.; Etnier, Michael A.; Monson, Daniel H.; Fogel, Marilyn L.

    2009-01-01

    Metabolically inert, accretionary structures such as the dentin growth layers in teeth provide a life history record of individual diet with near-annual resolution. We constructed ontogenetic ??13C and ??15N profiles by analyzing tooth dentin growth layers from 13 individual killer whales Orcinus orca collected in the eastern northeast Pacific Ocean between 1961 and 2003. The individuals sampled were 6 to 52 yr old, representing 2 ecotypes-resident and transient - collected across ???25?? of latitude. The average isotopic values of transient individuals (n = 10) are consistent with a reliance on mammalian prey, while the average isotopic values of residents (n = 3) are consistent with piscivory. Regardless of ecotype, most individuals show a decrease in ??15N values of ???2.5% through the first 3 yr of life, roughly equivalent to a decrease of one trophic level. We interpret this as evidence of gradual weaning, after which, ontogenetic shifts in isotopic values are highly variable. A few individuals (n = 2) maintained relatively stable ??15N and ??13C values throughout the remainder of their lives, whereas ??15N values of most (n = 11) increased by ???1.5%, suggestive of an ontogenetic increase in trophic level. Significant differences in mean ??13C and ??15N values among transients collected off California suggest that individuality in prey preferences may be prevalent within this ecotype. Our approach provides retrospective individual life history and dietary information that cannot be obtained through traditional field observations of free-ranging and elusive species such as killer whales, including unique historic ecological information that pre-dates modern studies. By providing insights into individual diet composition, stable isotope analysis of teeth and/or bones may be the only means of evaluating a number of hypothesized historical dietary shifts in killer whales of the northeast Pacific Ocean. ?? Inter-Research 2009.

  2. Retrospective characterization of ontogenetic shifts in killer whale diets via δ13C and δ15N analysis of teeth

    USGS Publications Warehouse

    Newsome, Seth D.; Etnier, Michael A.; Monson, Daniel H.; Fogel, Marilyn L.

    2009-01-01

    Metabolically inert, accretionary structures such as the dentin growth layers in teeth provide a life history record of individual diet with near-annual resolution. We constructed ontogenetic δ13C and δ15N profiles by analyzing tooth dentin growth layers from 13 individual killer whales Orcinus orca collected in the eastern northeast Pacific Ocean between 1961 and 2003. The individuals sampled were 6 to 52 yr old, representing 2 ecotypes—resident and transient—collected across ~25° of latitude. The average isotopic values of transient individuals (n = 10) are consistent with a reliance on mammalian prey, while the average isotopic values of residents (n = 3) are consistent with piscivory. Regardless of ecotype, most individuals show a decrease in δ15N values of ~2.5‰ through the first 3 yr of life, roughly equivalent to a decrease of one trophic level. We interpret this as evidence of gradual weaning, after which, ontogenetic shifts in isotopic values are highly variable. A few individuals (n = 2) maintained relatively stable δ15N and δ13C values throughout the remainder of their lives, whereas δ15N values of most (n = 11) increased by ~1.5‰, suggestive of an ontogenetic increase in trophic level. Significant differences in mean δ13C and δ15N values among transients collected off California suggest that individuality in prey preferences may be prevalent within this ecotype. Our approach provides retrospective individual life history and dietary information that cannot be obtained through traditional field observations of free-ranging and elusive species such as killer whales, including unique historic ecological information that pre-dates modern studies. By providing insights into individual diet composition, stable isotope analysis of teeth and/or bones may be the only means of evaluating a number of hypothesized historical dietary shifts in killer whales of the northeast Pacific Ocean

  3. A Capillary Absorption Spectrometer for Stable Carbon Isotope Ratio (13C/12C) Analysis in Very Small Samples

    SciTech Connect

    Kelly, James F.; Sams, Robert L.; Blake, Thomas A.; Newburn, Matthew K.; Moran, James J.; Alexander, M. L.; Kreuzer, Helen W.

    2012-02-06

    A capillary absorption spectrometer (CAS) suitable for IR laser isotope analysis of small CO{sub 2} samples is presented. The system employs a continuous-wave (cw) quantum cascade laser to study nearly adjacent rovibrational transitions of different isotopologues of CO{sub 2} near 2307 cm{sup -1} (4.34 {mu}m). This initial CAS system can achieve relative isotopic precision of about 10 ppm {sup 13}C, or {approx}1{per_thousand} (per mil in delta notation relative to Vienna Pee Dee Belemnite) with 20-100 picomoles of entrained sample within the hollow waveguide for CO{sub 2} concentrations {approx}400 to 750 ppm. Isotopic analyses of such gas fills in a 1-mm ID hollow waveguide of 0.8 m overall physical path length can be carried out down to {approx}2 Torr. Overall {sup 13}C/{sup 12}C ratios can be calibrated to {approx}2{per_thousand} accuracy with diluted CO{sub 2} standards. A novel, low-cost method to reduce cw-fringing noise resulting from multipath distortions in the hollow waveguide is presented, which allows weak absorbance features to be studied at the few ppm level (peak-to-rms) after 1,000 scans are co-added in {approx}10 sec. The CAS is meant to work directly with converted CO{sub 2} samples from a Laser Ablation-Catalytic-Combustion (LA CC) micro-sampler to provide {sup 13}C/{sup 12}C ratios of small biological isolates with spatial resolutions {approx}50 {mu}m.

  4. Application of multivariate image analysis in QSPR study of 13C chemical shifts of naphthalene derivatives: a comparative study.

    PubMed

    Garkani-Nejad, Zahra; Poshteh-Shirani, Marziyeh

    2010-11-15

    A new implemented QSPR method, whose descriptors achieved from bidimensional images, was applied for predicting (13)C NMR chemical shifts of 25 mono substituted naphthalenes. The resulted descriptors were subjected to principal component analysis (PCA) and the most significant principal components (PCs) were extracted. MIA-QSPR (multivariate image analysis applied to quantitative structure-property relationship) modeling was done by means of principal component regression (PCR) and principal component-artificial neural network (PC-ANN) methods. Eigen value ranking (EV) and correlation ranking (CR) were used here to select the most relevant set of PCs as inputs for PCR and PC-ANN modeling methods. The results supported that the correlation ranking-principal component-artificial neural network (CR-PC-ANN) model could predict the (13)C NMR chemical shifts of all 10 carbon atoms in mono substituted naphthalenes with R(2) ≥ 0.922 for training set, R(2) ≥ 0.963 for validation set and R(2) ≥ 0.936 for the test set. Comparison of the results with other existing factor selection method revealed that less accurate results were obtained by the eigen value ranking procedure. PMID:21035668

  5. Stable isotope analysis (δ (13)C and δ (15)N) of soil nematodes from four feeding groups.

    PubMed

    Melody, Carol; Griffiths, Bryan; Dyckmans, Jens; Schmidt, Olaf

    2016-01-01

    Soil nematode feeding groups are a long-established trophic categorisation largely based on morphology and are used in ecological indices to monitor and analyse the biological state of soils. Stable isotope ratio analysis ((13)C/(12)C and (15)N/(14)N, expressed as δ (13)C and δ (15)N) has provided verification of, and novel insights into, the feeding ecology of soil animals such as earthworms and mites. However, isotopic studies of soil nematodes have been limited to date as conventional stable isotope ratio analysis needs impractically large numbers of nematodes (up to 1,000) to achieve required minimum sample weights (typically >100 µg C and N). Here, micro-sample near-conventional elemental analysis-isotopic ratio mass spectrometry (μEA-IRMS) of C and N using microgram samples (typically 20 µg dry weight), was employed to compare the trophic position of selected soil nematode taxa from four feeding groups: predators (Anatonchus and Mononchus), bacterial feeders (Plectus and Rhabditis), omnivores (Aporcelaimidae and Qudsianematidae) and plant feeder (Rotylenchus). Free-living nematodes were collected from conventionally and organically managed arable soils. As few as 15 nematodes, for omnivores and predators, were sufficient to reach the 20 µg dry weight target. There was no significant difference in δ (15)N (p = 0.290) or δ (13)C (p = 0.706) between conventional and organic agronomic treatments but, within treatments, there was a significant difference in N and C stable isotope ratios between the plant feeder, Rotylenchus (δ (15)N = 1.08 to 3.22 mUr‰, δ (13)C = -29.58 to -27.87 mUr) and all other groups. There was an average difference of 9.62 mUr in δ (15)N between the plant feeder and the predator group (δ (15)N = 9.89 to 12.79 mUr, δ (13)C = -27.04 to -25.51 mUr). Isotopic niche widths were calculated as Bayesian derived standard ellipse areas and were smallest for the plant feeder (1.37 mUr(2)) and the predators (1.73 mUr(2)), but largest for

  6. Stable isotope analysis (δ (13)C and δ (15)N) of soil nematodes from four feeding groups.

    PubMed

    Melody, Carol; Griffiths, Bryan; Dyckmans, Jens; Schmidt, Olaf

    2016-01-01

    Soil nematode feeding groups are a long-established trophic categorisation largely based on morphology and are used in ecological indices to monitor and analyse the biological state of soils. Stable isotope ratio analysis ((13)C/(12)C and (15)N/(14)N, expressed as δ (13)C and δ (15)N) has provided verification of, and novel insights into, the feeding ecology of soil animals such as earthworms and mites. However, isotopic studies of soil nematodes have been limited to date as conventional stable isotope ratio analysis needs impractically large numbers of nematodes (up to 1,000) to achieve required minimum sample weights (typically >100 µg C and N). Here, micro-sample near-conventional elemental analysis-isotopic ratio mass spectrometry (μEA-IRMS) of C and N using microgram samples (typically 20 µg dry weight), was employed to compare the trophic position of selected soil nematode taxa from four feeding groups: predators (Anatonchus and Mononchus), bacterial feeders (Plectus and Rhabditis), omnivores (Aporcelaimidae and Qudsianematidae) and plant feeder (Rotylenchus). Free-living nematodes were collected from conventionally and organically managed arable soils. As few as 15 nematodes, for omnivores and predators, were sufficient to reach the 20 µg dry weight target. There was no significant difference in δ (15)N (p = 0.290) or δ (13)C (p = 0.706) between conventional and organic agronomic treatments but, within treatments, there was a significant difference in N and C stable isotope ratios between the plant feeder, Rotylenchus (δ (15)N = 1.08 to 3.22 mUr‰, δ (13)C = -29.58 to -27.87 mUr) and all other groups. There was an average difference of 9.62 mUr in δ (15)N between the plant feeder and the predator group (δ (15)N = 9.89 to 12.79 mUr, δ (13)C = -27.04 to -25.51 mUr). Isotopic niche widths were calculated as Bayesian derived standard ellipse areas and were smallest for the plant feeder (1.37 mUr(2)) and the predators (1.73 mUr(2)), but largest for

  7. Stable isotope analysis13C and δ15N) of soil nematodes from four feeding groups

    PubMed Central

    Griffiths, Bryan; Dyckmans, Jens; Schmidt, Olaf

    2016-01-01

    Soil nematode feeding groups are a long-established trophic categorisation largely based on morphology and are used in ecological indices to monitor and analyse the biological state of soils. Stable isotope ratio analysis (13C/12C and 15N/14N, expressed as δ13C and δ15N) has provided verification of, and novel insights into, the feeding ecology of soil animals such as earthworms and mites. However, isotopic studies of soil nematodes have been limited to date as conventional stable isotope ratio analysis needs impractically large numbers of nematodes (up to 1,000) to achieve required minimum sample weights (typically >100 µg C and N). Here, micro-sample near-conventional elemental analysis–isotopic ratio mass spectrometry (μEA–IRMS) of C and N using microgram samples (typically 20 µg dry weight), was employed to compare the trophic position of selected soil nematode taxa from four feeding groups: predators (Anatonchus and Mononchus), bacterial feeders (Plectus and Rhabditis), omnivores (Aporcelaimidae and Qudsianematidae) and plant feeder (Rotylenchus). Free-living nematodes were collected from conventionally and organically managed arable soils. As few as 15 nematodes, for omnivores and predators, were sufficient to reach the 20 µg dry weight target. There was no significant difference in δ15N (p = 0.290) or δ13C (p = 0.706) between conventional and organic agronomic treatments but, within treatments, there was a significant difference in N and C stable isotope ratios between the plant feeder, Rotylenchus (δ15N = 1.08 to 3.22 mUr‰, δ13C = –29.58 to –27.87 mUr) and all other groups. There was an average difference of 9.62 mUr in δ15N between the plant feeder and the predator group (δ15N = 9.89 to 12.79 mUr, δ13C = –27.04 to –25.51 mUr). Isotopic niche widths were calculated as Bayesian derived standard ellipse areas and were smallest for the plant feeder (1.37 mUr2) and the predators (1.73 mUr2), but largest for omnivores (3.83 mUr2

  8. Stable isotope analysis13C and δ15N) of soil nematodes from four feeding groups

    PubMed Central

    Griffiths, Bryan; Dyckmans, Jens; Schmidt, Olaf

    2016-01-01

    Soil nematode feeding groups are a long-established trophic categorisation largely based on morphology and are used in ecological indices to monitor and analyse the biological state of soils. Stable isotope ratio analysis (13C/12C and 15N/14N, expressed as δ13C and δ15N) has provided verification of, and novel insights into, the feeding ecology of soil animals such as earthworms and mites. However, isotopic studies of soil nematodes have been limited to date as conventional stable isotope ratio analysis needs impractically large numbers of nematodes (up to 1,000) to achieve required minimum sample weights (typically >100 µg C and N). Here, micro-sample near-conventional elemental analysis–isotopic ratio mass spectrometry (μEA–IRMS) of C and N using microgram samples (typically 20 µg dry weight), was employed to compare the trophic position of selected soil nematode taxa from four feeding groups: predators (Anatonchus and Mononchus), bacterial feeders (Plectus and Rhabditis), omnivores (Aporcelaimidae and Qudsianematidae) and plant feeder (Rotylenchus). Free-living nematodes were collected from conventionally and organically managed arable soils. As few as 15 nematodes, for omnivores and predators, were sufficient to reach the 20 µg dry weight target. There was no significant difference in δ15N (p = 0.290) or δ13C (p = 0.706) between conventional and organic agronomic treatments but, within treatments, there was a significant difference in N and C stable isotope ratios between the plant feeder, Rotylenchus (δ15N = 1.08 to 3.22 mUr‰, δ13C = –29.58 to –27.87 mUr) and all other groups. There was an average difference of 9.62 mUr in δ15N between the plant feeder and the predator group (δ15N = 9.89 to 12.79 mUr, δ13C = –27.04 to –25.51 mUr). Isotopic niche widths were calculated as Bayesian derived standard ellipse areas and were smallest for the plant feeder (1.37 mUr2) and the predators (1.73 mUr2), but largest for omnivores (3.83 mUr2

  9. Description and Preliminary Results From an On-Line Method of Analysis of δ 13C and δ 18O of CO2

    NASA Astrophysics Data System (ADS)

    Pierce, D. L.; Bissell, S.; Giles, L.; Wright, A.; van Haren, J.; Yakir, D.; Berry, J. A.; Lin, G.

    2003-12-01

    At the Biosphere 2 Center in Oracle, Arizona we have developed a continuous flow analysis method to measure in "real-time" the concentration, 13C/12C, and 18O/16O of CO2 in air. CO2 isotope analysis in air samples is crucial for the partitions of the net ecosystem flux into its net assimilation and respiration components. Previous analysis techniques depend on having many glass flasks and transporting the flask samples to the lab for analysis. Our technique transports gas using 1/4 inch tubing from various sampling points in Biosphere 2 Center to an on-site laboratory, which houses the on-line discrimination stable isotope mass spectrometer (O.L.D.S.I.M.S.). OLDSIMS is a system designed to combine a Licor 6262 gas analyzer (Lincoln, NE) for instant CO2 concentration values, a SIRA (Stable Isotope Ratio Analyzer, VG-Micromass, UK), and a Process Control computer for peripheral system control and coordination with the SIRA. The stable isotope and concentration analysis of one sample as well as a know reference gas currently takes OLDSIMS 12.4 minutes to run. Results from a diurnal run of a standard CO2 tank are: [CO2] = 998.7 +/- 0.45 ppm, δ 13C = -3.34 +/- 0.098 ‰ , δ 18O = -11.75 +/- 0.108 ‰ vs PDB. During our presentation we will show this and other data collected at the Biosphere 2 Center tropical rainforest biome to demonstrate the capabilities of this "real-time" technique.

  10. Analysis of 14C and 13C in teeth provides precise birth dating and clues to geographical origin

    PubMed Central

    K, Alkass; BA, Buchholz; H, Druid; KL, Spalding

    2011-01-01

    The identification of human bodies in situations when there are no clues as to the person’s identity from circumstantial data, poses a difficult problem to investigators. The determination of age and sex of the body can be crucial in order to limit the search to individuals that are a possible match. We analyzed the proportion of bomb pulse derived carbon-14 (14C) incorporated in the enamel of teeth from individuals from different geographical locations. The ‘bomb pulse’ refers to a significant increase in 14C levels in the atmosphere caused by above ground test detonations of nuclear weapons during the cold war (1955-1963). By comparing 14C levels in enamel with 14C atmospheric levels systematically recorded over time, high precision birth dating of modern biological material is possible. Above ground nuclear bomb testing was largely restricted to a couple of locations in the northern hemisphere, producing differences in atmospheric 14C levels at various geographical regions, particularly in the early phase. Therefore, we examined the precision of 14C birth dating of enamel as a function of time of formation and geographical location. We also investigated the use of the stable isotope 13C as an indicator of geographical origin of an individual. Dental enamel was isolated from 95 teeth extracted from 84 individuals to study the precision of the 14C method along the bomb spike. For teeth formed before 1955 (N = 17), all but one tooth showed negative Δ14C values. Analysis of enamel from teeth formed during the rising part of the bomb-spike (1955-1963, N = 12) and after the peak (>1963, N = 66) resulted in an average absolute date of birth estimation error of 1.9 ±1.4 and 1.3 ± 1.0 years, respectively. Geographical location of an individual had no adverse effect on the precision of year of birth estimation using radiocarbon dating. In 46 teeth, measurement of 13C was also performed. Scandinavian teeth showed a substantially greater depression in average δ13C

  11. Analysis of 14C and 13C in teeth provides precise birth dating and clues to geographical origin.

    PubMed

    Alkass, K; Buchholz, B A; Druid, H; Spalding, K L

    2011-06-15

    The identification of human bodies in situations when there are no clues as to the person's identity from circumstantial data, poses a difficult problem to the investigators. The determination of age and sex of the body can be crucial in order to limit the search to individuals that are a possible match. We analyzed the proportion of bomb pulse derived carbon-14 ((14)C) incorporated in the enamel of teeth from individuals from different geographical locations. The 'bomb pulse' refers to a significant increase in (14)C levels in the atmosphere caused by above ground test detonations of nuclear weapons during the cold war (1955-1963). By comparing (14)C levels in enamel with (14)C atmospheric levels systematically recorded over time, high precision birth dating of modern biological material is possible. Above ground nuclear bomb testing was largely restricted to a couple of locations in the northern hemisphere, producing differences in atmospheric (14)C levels at various geographical regions, particularly in the early phase. Therefore, we examined the precision of (14)C birth dating of enamel as a function of time of formation and geographical location. We also investigated the use of the stable isotope (13)C as an indicator of geographical origin of an individual. Dental enamel was isolated from 95 teeth extracted from 84 individuals to study the precision of the (14)C method along the bomb spike. For teeth formed before 1955 (N=17), all but one tooth showed negative Δ(14)C values. Analysis of enamel from teeth formed during the rising part of the bomb-spike (1955-1963, N=12) and after the peak (>1963, N=66) resulted in an average absolute date of birth estimation error of 1.9±1.4 and 1.3±1.0 years, respectively. Geographical location of an individual had no adverse effect on the precision of year of birth estimation using radiocarbon dating. In 46 teeth, measurement of (13)C was also performed. Scandinavian teeth showed a substantially greater depression in

  12. Analysis of 14C and 13C in teeth provides precise birth dating and clues to geographical origin.

    PubMed

    Alkass, K; Buchholz, B A; Druid, H; Spalding, K L

    2011-06-15

    The identification of human bodies in situations when there are no clues as to the person's identity from circumstantial data, poses a difficult problem to the investigators. The determination of age and sex of the body can be crucial in order to limit the search to individuals that are a possible match. We analyzed the proportion of bomb pulse derived carbon-14 ((14)C) incorporated in the enamel of teeth from individuals from different geographical locations. The 'bomb pulse' refers to a significant increase in (14)C levels in the atmosphere caused by above ground test detonations of nuclear weapons during the cold war (1955-1963). By comparing (14)C levels in enamel with (14)C atmospheric levels systematically recorded over time, high precision birth dating of modern biological material is possible. Above ground nuclear bomb testing was largely restricted to a couple of locations in the northern hemisphere, producing differences in atmospheric (14)C levels at various geographical regions, particularly in the early phase. Therefore, we examined the precision of (14)C birth dating of enamel as a function of time of formation and geographical location. We also investigated the use of the stable isotope (13)C as an indicator of geographical origin of an individual. Dental enamel was isolated from 95 teeth extracted from 84 individuals to study the precision of the (14)C method along the bomb spike. For teeth formed before 1955 (N=17), all but one tooth showed negative Δ(14)C values. Analysis of enamel from teeth formed during the rising part of the bomb-spike (1955-1963, N=12) and after the peak (>1963, N=66) resulted in an average absolute date of birth estimation error of 1.9±1.4 and 1.3±1.0 years, respectively. Geographical location of an individual had no adverse effect on the precision of year of birth estimation using radiocarbon dating. In 46 teeth, measurement of (13)C was also performed. Scandinavian teeth showed a substantially greater depression in

  13. Mathematical model for evaluating the Krebs cycle flux with non-constant glutamate-pool size by 13C-NMR spectroscopy. Evidence for the existence of two types of Krebs cycles in cells.

    PubMed

    Tran-Dinh, S; Beganton, F; Nguyen, T T; Bouet, F; Herve, M

    1996-12-01

    A practical method using matrix operations is proposed for studying the isotopic transformation of glutamate, or any other metabolite isotopomers, in the Krebs cycle. Two mathematical models were constructed for evaluating the Krebs cycle flux where the enrichment of [2-13C]acetyl-CoA is not 100% and the total glutamate concentration remains constant or varies during incubation. A comparative study of [1-13C]glucose metabolism was subsequently carried out using Saccharomyces cerevisiae cells from two different strains (ATCC-9763 and NCYC-239) by 13C-NMR spectroscopy and biochemical techniques. The results show that there are two types of Krebs cycles in cells. The first is represented by the ATCC cells which contain a small amount of 2-oxoglutarate dehydrogenase and hence the flux in the Krebs cycle is negligible. With [1-13C]glucose as a carbon source, the 13C-NMR spectra of glutamate exhibit the C2 and C4 resonances that are almost equivalent and much greater than that of the C3. Labeled metabolites derived from [1-13C]glucose enter the Krebs cycle at two points: oxaloacetate and citrate. The second cell type is represented by NCYC-239. The C2 and C3 areas are equivalent and smaller than the C4 resonance. The results suggest that labeled metabolites enter the Krebs cycle only at the citrate level via acetyl-CoA, 2-oxoglutarate dehydrogenase is present but pyruvate carboxylase is virtually absent or inactivated. When both are incubated with glucose, the total concentration of glutamate was found to decrease with the incubation time. The fraction of glutamate in isotopic exchange with the Krebs cycle in NCYC-239 cells is about 2.6% and the reduction in glutamate concentration is about 0.5%/min. Using our model, with a variable glutamate pool size, good agreement between the theoretical and experimental data is obtained.

  14. Carbon isotope analysis in urea at high 13C-abundances using the 13/12CO2-breath test device FANci2.

    PubMed

    Schmidt, G

    2002-09-01

    The increasing application of 13C-labelled urea in medicine requires simple and reasonable methods for measuring highly enriched C in urea. The combination: ultimate organic analysis--mass spectrometry so far prescribed is complicated and expensive. For medical diagnosis, however, isotope selective nondispersive infrared spectrometers (NDIRS) have been available for many years. One of these tools is FANci2 which is very reasonable and easily to be operated. By means of such devices also urea highly enriched in 13C can be analysed, provided that the samples are first diluted with a defined amount of urea of natural isotopic composition and then transformed into carbon dioxide by means of urease. The relative abundance of 13C in this carbon dioxide, measured by nondispersive infrared spectrometry, is then a measure of the 13C abundance in the initial urea sample. Comparison of results of such measurements with those attained by mass spectrometry proves that this procedure is feasible and yields precis results.

  15. [FTIR and 13C NMR Analysis of Dissolved Organic Matter (DOM) in the Treatment Process of Tannery Wastewater].

    PubMed

    Fan, Chun-hui; Zhang, Ying-chao; Tang, Ze-heng; Wang, Jia-hong

    2015-05-01

    Nowadays, the wastewater quantity discharged yearly from tannery industry is around 0. 2 billion t in China. The contaminants of tannery wastewater include macromolecular organic matters, such as grease, fur scraps and collagen, and the alkaline wastewater appears to be of high content of salt and COD. The quality of tannery wastewater is monitored strictly among all kinds of industry wastewater. In the treatment process of tannery wastewater, the quality of inlet and outlet water is generally analyzed. In fact, the transformation behavior of contaminants should be additionally checked to optimize the treatment conditions. Dissolved organic matter (DOM) is commonly existed in water-bodies and helpful to understand the physicochemical characteristics, while the related work should be further studied on tannery wastewater. The approaches of elemental analysis, thermal gravimetric analysis (TG), Fourier infrared spectroscopy (FTIR) and 13C nuclear magnetic resonance (13C NMR) were used to reveal the characteristics of DOM in the treatment process of tannery wastewater. The results showed the carbon content of DOM samples increased gradually, atomic ratios of H/C increased firstly and then decreased, indicating the organic matters were decomposed into chain structures firstly, finally forming the component hard to degraded. The pyrolysis process of DOM mainly proceeded in the regions of 110~530 °C (aliphatic compound, protein, etc. ) and 530~800 °C (aromatic ring, single bond of C-C, etc. ). The functional groups of DOM included -OH, -NH2, C=O and so on, and the aromatic substances were detected, shown from FTIR figures, in the later period of the reaction, caused by the metabolism effect of micro-organism. The content of alkoxy-C increased to the maximum in the second biochemical pond, and the minimum content of aromatic-C appeared in the second biochemical pond, suggesting the transformation behavior of carbon functional groups. The investigation on DOM in tannery

  16. Analysis of the Fusarium mycotoxin moniliformin in cereal samples using 13C2-moniliformin and high-resolution mass spectrometry.

    PubMed

    von Bargen, Katharina Walburga; Lohrey, Lilia; Cramer, Benedikt; Humpf, Hans-Ulrich

    2012-04-11

    Moniliformin is a mycotoxin produced by fungi of the Fusarium genus and occurs as a contaminant of different cereals worldwide. This study describes the first application of isotopically labeled (13)C(2)-moniliformin for the analysis of moniliformin in cereals. Moniliformin is a small and ionic molecule that forms only a single sensitive fragment ion in the collision cell of a tandem mass spectrometer. Therefore, the methods described in the literature for this kind of instrument observe only a single mass transition and show a relatively poor sensitivity. The use of high-resolution mass spectrometry was described to be a suitable alternative technique for the detection of this compound and was therefore applied in this study. The developed method is based on the use of strong anion exchange columns for cleanup prior to HPLC analysis and has a recovery rate of 75.3%, a limit of detection (LOD) of 0.7 μg/kg, and a limit of quantitation (LOQ) of 2.5 μg/kg. Twenty-three different cereal samples were analyzed for their moniliformin content. Twenty of them showed positive results with levels up to 126 ± 12.2 μg/kg.

  17. Detection of adulteration in honey samples added various sugar syrups with 13C/12C isotope ratio analysis method.

    PubMed

    Tosun, Murat

    2013-06-01

    Honey can be adulterated in various ways. One of the adulteration methods is the addition of different sugar syrups during or after honey production. Starch-based sugar syrups, high fructose corn syrup (HFCS), glucose syrup (GS) and saccharose syrups (SS), which are produced from beet or canes, can be used for adulterating honey. In this study, adulterated honey samples were prepared with the addition of HFCS, GS and SS (beet sugar) at a ratio of 0%, 10%, 20%, 40% and 50% by weight. (13)C/(12)C analysis was conducted on these adulterated honey samples using an isotope ratio mass spectrometer in combination with an elemental analyser (EA-IRMS). As a result, adulteration using C(4) sugar syrups (HFCS and GS) could be detected to a certain extent while adulteration of honey using C(3) sugar syrups (beet sugar) could not be detected. Adulteration by using SS (beet sugar) still has a serious detection problem, especially in countries in which beet is used in manufacturing sugar. For this reason, practice and analysis methods are needed to meet this deficit and to detect the adulterations precisely in the studies that will be conducted. PMID:23411291

  18. 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

  19. Simultaneous Steady-state and Dynamic 13C NMR Can Differentiate Alternative Routes of Pyruvate Metabolism in Living Cancer Cells*

    PubMed Central

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

    2014-01-01

    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 13C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized 13C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined 13C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-13C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-13C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[13C]O3− and [1-13C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-13C]pyruvate. Quantitation of hyperpolarized H[13C]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[13C]O3− appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1-13C]pyruvate metabolism, enhancing exchanges with [1-13C]lactate and suppressing H[13C]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 13C isotopomer analyses and dynamic hyperpolarized 13C spectroscopy may enable quantitative flux measurements in

  20. Glycation Isotopic Labeling with 13C-Reducing Sugars for Quantitative Analysis of Glycated Proteins in Human Plasma*

    PubMed Central

    Priego-Capote, Feliciano; Scherl, Alexander; Müller, Markus; Waridel, Patrice; Lisacek, Frédérique; Sanchez, Jean-Charles

    2010-01-01

    Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [13C6]glucose incubation to evaluate the native glycated proteins labeled with [12C6]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive

  1. Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii*

    PubMed Central

    Sugai, Yoshinori; Ueno, Yohei; Hayashi, Ken-ichiro; Oogami, Shingo; Toyomasu, Tomonobu; Matsumoto, Sadamu; Natsume, Masahiro; Nozaki, Hiroshi; Kawaide, Hiroshi

    2011-01-01

    Diterpenes show diverse chemical structures and various physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) are involved in hormone and defense compound biosyntheses in bryophytes and gymnosperms, respectively. The BDTCs catalyze the successive two-step type-B (protonation-initiated cyclization) and type-A (ionization-initiated cyclization) reactions of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes with the majority being uncharacterized. The cDNA from S. moellendorffii encoding a BDTC-like enzyme, miltiradiene synthase (SmMDS), was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with a molecular mass of 272 Da. Mutation in the type-B active motif of SmMDS abolished the cyclase activity, whereas (+)-copalyl diphosphate, the reaction intermediate from the conversion of GGDP to the hydrocarbon product, rescued the cyclase activity of the mutant to form a diterpene hydrocarbon. Another mutant lacking type-A activity accumulated copalyl diphosphate as the reaction intermediate. When the diterpene hydrocarbon was enzymatically synthesized from [U-13C6]mevalonate, all carbons were labeled with 13C stable isotope (>99%). The fully 13C-labeled product was subjected to 13C-13C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multidimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a putative biosynthetic precursor of tanshinone identified from the Chinese medicinal herb Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii. In this study, we demonstrate that one-dimensional and

  2. Analysis of dynamics and mechanism of ligand binding to Artocarpus integrifolia agglutinin. A 13C and 19F NMR study.

    PubMed

    Krishna Sastry, M V; Swamy, M J; Surolia, A

    1988-10-15

    Binding of 13C-labeled N-acetylgalactosamine (13C-GalNAc) and N-trifluoroacetylgalactosamine (19F-GalNAc) to Artocarpus integrifolia agglutinin has been studied using 13C and 19F nuclear magnetic resonance spectroscopy, respectively. Binding of these saccharides resulted in broadening of the resonances, and no change in chemical shift was observed, suggesting that the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc experience a magnetically equivalent environment in the lectin combining site. The alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc were found to be in slow exchange between free and protein bound states. Binding of 13C-GalNAc was studied as a function of temperature. From the temperature dependence of the line broadening, the thermodynamic and kinetic parameters were evaluated. The association rate constants obtained for the alpha-anomers of 13C-GalNAc and 19F-GalNAc (k+1 = 1.01 x 10(5) M-1.s-1 and 0.698 x 10(5) M-1.s-1, respectively) are in close agreement with those obtained for the corresponding beta-anomers (k+1 = 0.95 x 10(5) M-1.s-1 and 0.65 x 10(5) M-1.s-1, respectively), suggesting that the two anomers bind to the lectin by a similar mechanism. In addition these values are several orders of magnitude slower than those obtained for diffusion controlled processes. The dissociation rate constants obtained are 49.9, 56.9, 42, and 43 s-1, respectively, for the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc. A two-step mechanism has been proposed for the interaction of 13C-GalNAc and 19F-GalNAc with A. integrifolia lectin in view of the slow association rates and high activation entropies. The thermodynamic parameters obtained for the association and dissociation reactions suggest that the binding process is entropically favored and that there is a small enthalpic contribution.

  3. Radiative Flux Analysis

    DOE Data Explorer

    Long, Chuck [NOAA

    2008-05-14

    The Radiative Flux Analysis is a technique for using surface broadband radiation measurements for detecting periods of clear (i.e. cloudless) skies, and using the detected clear-sky data to fit functions which are then used to produce continuous clear-sky estimates. The clear-sky estimates and measurements are then used in various ways to infer cloud macrophysical properties.

  4. Can 13C stable isotope analysis uncover essential amino acid provisioning by termite-associated gut microbes?

    PubMed Central

    Jones, Susan C.; Sabree, Zakee L.

    2015-01-01

    Gut-associated microbes of insects are postulated to provide a variety of nutritional functions including provisioning essential amino acids (EAAs). Demonstrations of EAA provisioning in insect-gut microbial systems, nonetheless, are scant. In this study, we investigated whether the eastern subterranean termite Reticulitermes flavipes sourced EAAs from its gut-associated microbiota. δ13CEAA data from termite carcass, termite gut filtrate and dietary (wood) samples were determined following 13C stable isotope analysis. Termite carcass samples (−27.0 ± 0.4‰, mean ± s.e.) were significantly different from termite gut filtrate samples (−27.53 ± 0.5‰), but not the wood diet (−26.0 ± 0.5‰) (F(2,64) = 6, P < 0.0052). δ13CEAA-offsets between termite samples and diet suggested possible non-dietary EAA input. Predictive modeling identified gut-associated bacteria and fungi, respectively as potential major and minor sources of EAAs in both termite carcass and gut filtrate samples, based on δ13CEAA data of four and three EAAs from representative bacteria, fungi and plant data. The wood diet, however, was classified as fungal rather than plant in origin by the model. This is attributed to fungal infestation of the wood diet in the termite colony. This lowers the confidence with which gut microbes (bacteria and fungi) can be attributed with being the source of EAA input to the termite host. Despite this limitation, this study provides tentative data in support of hypothesized EAA provisioning by gut microbes, and also a baseline/framework upon which further work can be carried out to definitively verify this function. PMID:26336647

  5. Additional Value of CH₄ Measurement in a Combined (13)C/H₂ Lactose Malabsorption Breath Test: A Retrospective Analysis.

    PubMed

    Houben, Els; De Preter, Vicky; Billen, Jaak; Van Ranst, Marc; Verbeke, Kristin

    2015-09-07

    The lactose hydrogen breath test is a commonly used, non-invasive method for the detection of lactose malabsorption and is based on an abnormal increase in breath hydrogen (H₂) excretion after an oral dose of lactose. We use a combined (13)C/H₂ lactose breath test that measures breath (13)CO₂ as a measure of lactose digestion in addition to H₂ and that has a better sensitivity and specificity than the standard test. The present retrospective study evaluated the results of 1051 (13)C/H₂ lactose breath tests to assess the impact on the diagnostic accuracy of measuring breath CH₄ in addition to H₂ and (13)CO₂. Based on the (13)C/H₂ breath test, 314 patients were diagnosed with lactase deficiency, 138 with lactose malabsorption or small bowel bacterial overgrowth (SIBO), and 599 with normal lactose digestion. Additional measurement of CH₄ further improved the accuracy of the test as 16% subjects with normal lactose digestion and no H₂-excretion were found to excrete CH₄. These subjects should have been classified as subjects with lactose malabsorption or SIBO. In conclusion, measuring CH₄-concentrations has an added value to the (13)C/H₂ breath test to identify methanogenic subjects with lactose malabsorption or SIBO.

  6. Additional Value of CH₄ Measurement in a Combined (13)C/H₂ Lactose Malabsorption Breath Test: A Retrospective Analysis.

    PubMed

    Houben, Els; De Preter, Vicky; Billen, Jaak; Van Ranst, Marc; Verbeke, Kristin

    2015-09-01

    The lactose hydrogen breath test is a commonly used, non-invasive method for the detection of lactose malabsorption and is based on an abnormal increase in breath hydrogen (H₂) excretion after an oral dose of lactose. We use a combined (13)C/H₂ lactose breath test that measures breath (13)CO₂ as a measure of lactose digestion in addition to H₂ and that has a better sensitivity and specificity than the standard test. The present retrospective study evaluated the results of 1051 (13)C/H₂ lactose breath tests to assess the impact on the diagnostic accuracy of measuring breath CH₄ in addition to H₂ and (13)CO₂. Based on the (13)C/H₂ breath test, 314 patients were diagnosed with lactase deficiency, 138 with lactose malabsorption or small bowel bacterial overgrowth (SIBO), and 599 with normal lactose digestion. Additional measurement of CH₄ further improved the accuracy of the test as 16% subjects with normal lactose digestion and no H₂-excretion were found to excrete CH₄. These subjects should have been classified as subjects with lactose malabsorption or SIBO. In conclusion, measuring CH₄-concentrations has an added value to the (13)C/H₂ breath test to identify methanogenic subjects with lactose malabsorption or SIBO. PMID:26371034

  7. 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

  8. 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

  9. Constraining {sup 13}C amounts in AGB stars through isotopic analysis of trace elements in presolar SiC.

    SciTech Connect

    Barzyk, J. G.; Savina, M. R.; Davis, A. M.; Gallino, R.; Gyngard, F.; Amari, S.; Zinner, E.; Pelliln, M. J.; Lewis, R. S.; Clayton, R. N.; Materials Science Division; Univ. Chicago; Chicago Ctr Cosmochem.; Universita di Torino; Washington Univ.

    2007-07-01

    Analyses of the isotopic compositions of multiple elements (Mo, Zr, and Ba) in individual mainstream presolar SiC grains were done by resonant ionization mass spectrometry (RIMS). While most heavy element compositions were consistent with model predictions for the slow neutron capture process (s-process) in low-mass (1.5-3 M{sub {circle_dot}}) asymptotic giant branch stars of solar metallicity when viewed on single-element three-isotope plots, grains with compositions deviating from model predictions were identified on multi-element plots. These grains have compositions that cannot result from any neutron capture process but can be explained by contamination in some elements with solar system material. Previous work in which only one heavy element per grain was examined has been unable to identify contaminated grains. The multi-element analyses of this study detected contaminated grains which were subsequently eliminated from consideration. The uncontaminated grains form a data set with a greatly reduced spread on the three-isotope plots of each element measured, corresponding to a smaller range of {sup 13}C pocket efficiencies in parent AGB stars. Furthermore, due to this reduced spread, the nature of the stellar starting material, previously interpreted as having solar isotopic composition, is uncertain. The constraint on {sup 13}C pocket efficiencies in parent stars of these grains may help uncover the mechanism responsible for formation of {sup 13}C, the primary neutron source for s-process nucleosynthesis in low-mass stars.

  10. 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. PMID:26691313

  11. An Oral Load of [13C3]Glycerol and Blood NMR Analysis Detect Fatty Acid Esterification, Pentose Phosphate Pathway, and Glycerol Metabolism through the Tricarboxylic Acid Cycle in Human Liver.

    PubMed

    Jin, Eunsook S; Sherry, A Dean; Malloy, Craig R

    2016-09-01

    Drugs and other interventions for high impact hepatic diseases often target biochemical pathways such as gluconeogenesis, lipogenesis, or the metabolic response to oxidative stress. However, traditional liver function tests do not provide quantitative data about these pathways. In this study, we developed a simple method to evaluate these processes by NMR analysis of plasma metabolites. Healthy subjects ingested [U-(13)C3]glycerol, and blood was drawn at multiple times. Each subject completed three visits under differing nutritional states. High resolution (13)C NMR spectra of plasma triacylglycerols and glucose provided new insights into a number of hepatic processes including fatty acid esterification, the pentose phosphate pathway, and gluconeogenesis through the tricarboxylic acid cycle. Fasting stimulated pentose phosphate pathway activity and metabolism of [U-(13)C3]glycerol in the tricarboxylic acid cycle prior to gluconeogenesis or glyceroneogenesis. Fatty acid esterification was transient in the fasted state but continuous under fed conditions. We conclude that a simple NMR analysis of blood metabolites provides an important biomarker of pentose phosphate pathway activity, triacylglycerol synthesis, and flux through anaplerotic pathways in mitochondria of human liver.

  12. An Oral Load of [13C3]Glycerol and Blood NMR Analysis Detect Fatty Acid Esterification, Pentose Phosphate Pathway, and Glycerol Metabolism through the Tricarboxylic Acid Cycle in Human Liver.

    PubMed

    Jin, Eunsook S; Sherry, A Dean; Malloy, Craig R

    2016-09-01

    Drugs and other interventions for high impact hepatic diseases often target biochemical pathways such as gluconeogenesis, lipogenesis, or the metabolic response to oxidative stress. However, traditional liver function tests do not provide quantitative data about these pathways. In this study, we developed a simple method to evaluate these processes by NMR analysis of plasma metabolites. Healthy subjects ingested [U-(13)C3]glycerol, and blood was drawn at multiple times. Each subject completed three visits under differing nutritional states. High resolution (13)C NMR spectra of plasma triacylglycerols and glucose provided new insights into a number of hepatic processes including fatty acid esterification, the pentose phosphate pathway, and gluconeogenesis through the tricarboxylic acid cycle. Fasting stimulated pentose phosphate pathway activity and metabolism of [U-(13)C3]glycerol in the tricarboxylic acid cycle prior to gluconeogenesis or glyceroneogenesis. Fatty acid esterification was transient in the fasted state but continuous under fed conditions. We conclude that a simple NMR analysis of blood metabolites provides an important biomarker of pentose phosphate pathway activity, triacylglycerol synthesis, and flux through anaplerotic pathways in mitochondria of human liver. PMID:27432878

  13. Enhanced forensic discrimination of pollutants by position-specific isotope analysis using isotope ratio monitoring by (13)C nuclear magnetic resonance spectrometry.

    PubMed

    Julien, Maxime; Nun, Pierrick; Höhener, Patrick; Parinet, Julien; Robins, Richard J; Remaud, Gérald S

    2016-01-15

    In forensic environmental investigations the main issue concerns the inference of the original source of the pollutant for determining the liable party. Isotope measurements in geochemistry, combined with complimentary techniques for contaminant identification, have contributed significantly to source determination at polluted sites. In this work we have determined the intramolecular (13)C profiles of several molecules well-known as pollutants. By giving additional analytical parameters, position-specific isotope analysis performed by isotope ratio monitoring by (13)C nuclear magnetic resonance (irm-(13)C NMR) spectrometry gives new information to help in answering the major question: what is the origin of the detected contaminant? We have shown that isotope profiling of the core of a molecule reveals both the raw materials and the process used in its manufacture. It also can reveal processes occurring between the contamination site 'source' and the sampling site. Thus, irm-(13)C NMR is shown to be a very good complement to compound-specific isotope analysis currently performed by mass spectrometry for assessing polluted sites involving substantial spills of pollutant.

  14. Enhanced forensic discrimination of pollutants by position-specific isotope analysis using isotope ratio monitoring by (13)C nuclear magnetic resonance spectrometry.

    PubMed

    Julien, Maxime; Nun, Pierrick; Höhener, Patrick; Parinet, Julien; Robins, Richard J; Remaud, Gérald S

    2016-01-15

    In forensic environmental investigations the main issue concerns the inference of the original source of the pollutant for determining the liable party. Isotope measurements in geochemistry, combined with complimentary techniques for contaminant identification, have contributed significantly to source determination at polluted sites. In this work we have determined the intramolecular (13)C profiles of several molecules well-known as pollutants. By giving additional analytical parameters, position-specific isotope analysis performed by isotope ratio monitoring by (13)C nuclear magnetic resonance (irm-(13)C NMR) spectrometry gives new information to help in answering the major question: what is the origin of the detected contaminant? We have shown that isotope profiling of the core of a molecule reveals both the raw materials and the process used in its manufacture. It also can reveal processes occurring between the contamination site 'source' and the sampling site. Thus, irm-(13)C NMR is shown to be a very good complement to compound-specific isotope analysis currently performed by mass spectrometry for assessing polluted sites involving substantial spills of pollutant. PMID:26592622

  15. Measurement of 13C and 15N isotope labeling by gas chromatography/combustion/isotope ratio mass spectrometry to study amino acid fluxes in a plant-microbe symbiotic association.

    PubMed

    Molero, Gemma; Aranjuelo, Iker; Teixidor, Pilar; Araus, José Luis; Nogués, Salvador

    2011-03-15

    We have developed a method based on a double labeling with stable isotopes and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses to study amino acid exchange in a symbiotic plant-microbe association. Isotopic precision was studied for 21 standards including 15 amino acid derivatives, three N-protected amino acid methyl esters, three amines and one international standard. High correlations were observed between the δ(13)C and δ(15)N values obtained by GC/C/IRMS and those obtained by an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (R(2) = 0.9868 and 0.9992, respectively). The mean precision measured was 0.04‰ for δ(13)C and 0.28‰ for δ(15)N (n = 15). This method was applied in vivo to the symbiotic relationship between alfalfa (Medicago sativa L.) and N(2)-fixing bacteria. Plants were simultaneously labeled over 10 days with (13)C-depleted CO(2) ((12)CO(2)), which was assimilated through photosynthesis by leaves, and (15)N(2) fixed via nodules. Subsequently, the C and N isotope compositions (i.e. δ(13)C and δ(15)N) of free amino acids were analyzed in leaves and nodules by GC/C/IRMS. The method revealed the pattern of C and N exchange between leaves and nodules, highlighting that γ-aminobutanoic acid and glycine may represent an important form of C transport from leaves to the nodules. The results confirmed the validity, reliability and accuracy of the method for assessing C and N fluxes between plants and symbiotic bacteria and support the use of this technique in a broad range of metabolic and fluxomic studies.

  16. Phase behavior and 13C NMR spectroscopic analysis of the mixed methane + ethane + propane hydrates in mesoporous silica gels.

    PubMed

    Lee, Seungmin; Cha, Inuk; Seo, Yongwon

    2010-11-25

    In this study, the phase behavior and quantitative determination of hydrate composition and cage occupancy for the mixed CH(4) + C(2)H(6) + C(3)H(8) hydrates were closely investigated through the experimental measurement of three-phase hydrate (H)-water-rich liquid (L(W))-vapor (V) equilibria and (13)C NMR spectra. To examine the effect of pore size and salinity, we measured hydrate phase equilibria for the quaternary CH(4) (90%) + C(2)H(6) (7%) + C(3)H(8) (3%) + water mixtures in silica gel pores of nominal diameters of 6.0, 15.0, and 30.0 nm and for the quinary CH(4) (90%) + C(2)H(6) (7%) + C(3)H(8) (3%) + NaCl + water mixtures of two different NaCl concentrations (3 and 10 wt %) in silica gel pores of a nominal 30.0 nm diameter. The value of hydrate-water interfacial tension for the CH(4) (90%) + C(2)H(6) (7%) + C(3)H(8) (3%) hydrate was found to be 47 ± 4 mJ/m(2) from the relation of the dissociation temperature depression with the pore size of silica gels at a given pressure. At a specified temperature, three-phase H-L(W)-V equilibrium curves of pore hydrates were shifted to higher pressure regions depending on pore sizes and NaCl concentrations. From the cage-dependent (13)C NMR chemical shifts of enclathrated guest molecules, the mixed CH(4) (90%) + C(2)H(6) (7%) + C(3)H(8) (3%) gas hydrate was confirmed to be structure II. The cage occupancies of each guest molecule and the hydration number of the mixed gas hydrates were also estimated from the (13)C NMR spectra.

  17. δ18O and δ13C Analysis in Tree Rings of Pterocarpus angolensis Growing in Zimbabwe

    NASA Astrophysics Data System (ADS)

    McLeran, K.; Schoof, J. T.; Lefticariu, L.; Therrell, M.

    2015-12-01

    Instrumental weather records in southern Africa are largely limited to the last 100 years and documentary weather-related data are rare prior to the 1800s, hindering our understanding of the natural and/or anthropogenic factors that influence climate variability over this region. Measuring stable isotopes ratios (commonly 13C/12C and 18O/16O) in tree rings can provide a good proxy for extending climate data beyond the instrumental record. The objective of this study is to characterize historical variations in the climatology underlying extreme climatic events in Zimbabwe using instrumental climate records (precipitation and temperature) and a multi-proxy approach (ring width, δ18O, and δ13C) for dendroclimatic proxy reconstructions. A 90-year (1900-1990) δ18O and δ13C tree ring record using four Pterocarpus angolensis samples is being developed and compared to tree ring width, monthly, seasonal, and annual precipitation totals, meteoric water δ18O values, and mean monthly and seasonal temperature. Preliminary results indicate significant correlations between the average δ18O record and the previous year December precipitation totals (r=0.41, p<0.0001), current year January precipitation totals (r=0.45, p<0.0001), and combined total precipitation for the previous year November and December and current year January (r=0.57, p<0.0001). Furthermore, we find that the δ18O values are strongly influenced by maximum temperature during the previous year December (r=0.39, p=0.0001) and current year January (r=0.40, p=0.0001), and average maximum temperature during the months of the previous year December and current year January and February (r=0.47, p<0.001). We thus present one of the first studies to integrate a multi-proxy approach to investigate historical climate variability in southern Africa using ring widths, and tree ring δ18O and δ13C values of trees growing in Zimbabwe.

  18. Expression, purification, and mass spectrometric analysis of 15N, 13C-labeled RGD-hirudin, expressed in Pichia pastoris, for NMR studies.

    PubMed

    Huang, Yinong; Zhang, Yanling; Wu, Yi; Wang, Jue; Liu, Xingang; Dai, Linsen; Wang, Longsheng; Yu, Min; Mo, Wei

    2012-01-01

    A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed (15)N, (13)C-labeled RGD-hirudin in Pichia pastoris in a fermenter. The protein was subsequently purified to yield sufficient quantities for structural and functional studies. The purified protein was characterized by HPLC and MALDI-TOF mass spectroscopy. Analysis revealed that the protein was pure and uniformly labeled with (15)N and (13)C. A bioassay showed that the anti-thrombin activity and the anti-platelet aggregation ability of the labeled protein were the same as those of unlabeled RGD-hirudin. Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone (15)N, (13)C and (1)H resonance assignments of the r-RGD-Hirudin. The (15)N-(1)H HSQC spectrum of uniformly (15)N, (13)C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the (15)N-(l)H correlation spectrum, and by selected planes of the CBCA(CO)NH, CBCANH, and HNCO experiments. These results provide a basis for further studies on the structure-function relationship of RGD-hirudin with thrombin and platelets. PMID:22879918

  19. Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

    PubMed Central

    Berditsch, Marina; Afonin, Sergii; Steineker, Anna; Orel, Nataliia; Jakovkin, Igor; Weber, Christian

    2015-01-01

    Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly 13C/15N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive 13C/15N-labeled amino acids. The most cost-effective production of 13C/15N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% 13C-glycerol and 0.5% 15N-ammonium sulfate, supplemented with only 0.025% of 13C/15N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state. PMID:25795666

  20. Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis.

    PubMed

    Berditsch, Marina; Afonin, Sergii; Steineker, Anna; Orel, Nataliia; Jakovkin, Igor; Weber, Christian; Ulrich, Anne S

    2015-06-01

    Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly (13)C/(15)N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive (13)C/(15)N-labeled amino acids. The most cost-effective production of (13)C/(15)N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% (13)C-glycerol and 0.5% (15)N-ammonium sulfate, supplemented with only 0.025% of (13)C/(15)N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state.

  1. FTIR and NDIR spectroscopies as valuable alternatives to IRMS spectrometry for the δ(13)C analysis of food.

    PubMed

    Pironti, Concetta; Proto, Antonio; Camin, Federica; Cucciniello, Raffaele; Zarrella, Ilaria; Motta, Oriana

    2016-11-01

    The (13)C/(12)C carbon isotope ratio is a chemical parameter with many important applications in several scientific area and the technique of choice currently used for the δ(13)C determination is the isotope ratio mass spectrometry (IRMS). This latter is highly accurate (0.1‰) and sensitive (up to 0.01‰), but at the same time expensive and complex. The objective of this work was to assess the reliability of FTIR and NDIRS techniques for the measurement of carbon stable isotope ratio of food sample, in comparison to IRMS. IRMS, NDIRS and FTIR were used to analyze samples of food, such as oil, durum, cocoa, pasta and sugar, in order to determine the natural abundance isotopic ratio of carbon in a parallel way. The results were comparable, showing a close relationship among the three techniques. The main advantage in using FTIR and NDIRS is related to their cheapness and easy-to-operate in comparison to IRMS. PMID:27591614

  2. Recalcitrance and structural analysis by water-only flowthrough pretreatment of (13)C enriched corn stover stem.

    PubMed

    Foston, Marcus; Trajano, Heather L; Samuel, Reichel; Wyman, Charles E; He, Jian; Ragauskas, Arthur J

    2015-12-01

    This study presents high temperature water-only continuous flowthrough pretreatment coupled with nuclear magnetic resonance (NMR) as a promising analytical tool to examine the plant cell wall, to understand its recalcitrance (i.e., cell wall resistance to deconstruction), and to probe the chemistry occurring during batch pretreatment of biomass. (13)C-enriched corn stover stems were pretreated at 170°C for 60min with a hot-water flow rate of 20mL/min to control fractionation of the cell wall. This approach helped elucidate the nature of plant cell wall chemical recalcitrance and biomass pretreatment chemistry by tracking cell wall fragmentation as a function of time. Fractions of the reactor effluent were collected in a time-resolved fashion and characterized by various NMR techniques to determine the degree and sequence of fragments released, as well as, the chemical composition, molecular structure, and relative molecular weight of those released fragments.

  3. 13C Pathway Analysis for the Role of Formate in Electricity Generation by Shewanella Oneidensis MR-1 Using Lactate in Microbial Fuel Cells

    PubMed Central

    Luo, Shuai; Guo, Weihua; H. Nealson, Kenneth; Feng, Xueyang; He, Zhen

    2016-01-01

    Microbial fuel cell (MFC) is a promising technology for direct electricity generation from organics by microorganisms. The type of electron donors fed into MFCs affects the electrical performance, and mechanistic understanding of such effects is important to optimize the MFC performance. In this study, we used a model organism in MFCs, Shewanella oneidensis MR-1, and 13C pathway analysis to investigate the role of formate in electricity generation and the related microbial metabolism. Our results indicated a synergistic effect of formate and lactate on electricity generation, and extra formate addition on the original lactate resulted in more electrical output than using formate or lactate as a sole electron donor. Based on the 13C tracer analysis, we discovered decoupled cell growth and electricity generation in S. oneidensis MR-1 during co-utilization of lactate and formate (i.e., while the lactate was mainly metabolized to support the cell growth, the formate was oxidized to release electrons for higher electricity generation). To our best knowledge, this is the first time that 13C tracer analysis was applied to study microbial metabolism in MFCs and it was demonstrated to be a valuable tool to understand the metabolic pathways affected by electron donors in the selected electrochemically-active microorganisms. PMID:26868848

  4. Flux analysis in plant metabolic networks: increasing throughput and coverage.

    PubMed

    Junker, Björn H

    2014-04-01

    Quantitative information about metabolic networks has been mainly obtained at the level of metabolite contents, transcript abundance, and enzyme activities. However, the active process of metabolism is represented by the flow of matter through the pathways. These metabolic fluxes can be predicted by Flux Balance Analysis or determined experimentally by (13)C-Metabolic Flux Analysis. These relatively complicated and time-consuming methods have recently seen significant improvements at the level of coverage and throughput. Metabolic models have developed from single cell models into whole-organism dynamic models. Advances in lab automation and data handling have significantly increased the throughput of flux measurements. This review summarizes advances to increase coverage and throughput of metabolic flux analysis in plants.

  5. 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

  6. Combined analysis by GC (RI), GC-MS and 13C NMR of the supercritical fluid extract of Abies alba twigs.

    PubMed

    Duquesnoy, Emilie; Marongiu, Bruno; Castola, Vincent; Piras, Alessandra; Porcedda, Silvia; Casanova, Joseph

    2010-12-01

    Two samples (leaves and twigs) of Abies alba Miller from Corsica were extracted using supercritical CO2 and their chemical compositions were compared with those of the essential oils obtained from the same batch of plant material. In total 45 components were identified using combined analysis by GC (RI), GC-MS and 13C NMR. It was observed that the contents of monoterpenes (mainly represented by limonene, alpha-pinene and camphene) were significantly lower in the supercritical fluid extract (SFE) than in the essential oil (EO). Conversely, the proportions of sesquiterpenes were much higher in CO2 extracts than in essential oils (around 30% vs 4%). Cis-abienol, a diterpene alcohol, was identified only in SFE, and the proportions of this constituent (7.5% and 17.3%) were determined using quantitative 13C NMR since it was under estimated using the standard conditions of GC. PMID:21299139

  7. Combined analysis by GC (RI), GC-MS and 13C NMR of the supercritical fluid extract of Abies alba twigs.

    PubMed

    Duquesnoy, Emilie; Marongiu, Bruno; Castola, Vincent; Piras, Alessandra; Porcedda, Silvia; Casanova, Joseph

    2010-12-01

    Two samples (leaves and twigs) of Abies alba Miller from Corsica were extracted using supercritical CO2 and their chemical compositions were compared with those of the essential oils obtained from the same batch of plant material. In total 45 components were identified using combined analysis by GC (RI), GC-MS and 13C NMR. It was observed that the contents of monoterpenes (mainly represented by limonene, alpha-pinene and camphene) were significantly lower in the supercritical fluid extract (SFE) than in the essential oil (EO). Conversely, the proportions of sesquiterpenes were much higher in CO2 extracts than in essential oils (around 30% vs 4%). Cis-abienol, a diterpene alcohol, was identified only in SFE, and the proportions of this constituent (7.5% and 17.3%) were determined using quantitative 13C NMR since it was under estimated using the standard conditions of GC.

  8. Chemical composition of the essential oil from Corsican Mentha aquatica--combined analysis by GC(RI), GC-MS and 13C NMR spectroscopy.

    PubMed

    Sutour, Sylvain; Tomi, Félix; Bradesi, Pascale; Casanova, Joseph

    2011-10-01

    The essential oil (EO) of M. aquatica L. growing wild in Corsica was isolated by dry vapor distillation and submitted to combined analysis by column chromatography over silica gel, GC(RI), GC-MS and 13C NMR spectroscopy. The composition was dominated byoxygenated monoterpenes and characterized by the occurrence of menthofuran (50.7%) as the major component. In parallel, seven laboratory-distilled oil samples isolated from individual plants collected in Corsica were analyzed by GC(RI) and 13C NMR spectroscopy. Onlyquantitative differences were observed between the samples. Beside the usual terpenes, various p-menthane lactones (mintlactone, isomintlactone, hydroxymintlactone, menthofurolactone and epimenthofurolactone) have been identified in all the oil samples.

  9. Real-time analysis of δ13C- and δD-CH4 in ambient air with laser spectroscopy: method development and first intercomparison results

    NASA Astrophysics Data System (ADS)

    Eyer, S.; Tuzson, B.; Popa, M. E.; van der Veen, C.; Röckmann, T.; Rothe, M.; Brand, W. A.; Fisher, R.; Lowry, D.; Nisbet, E. G.; Brennwald, M. S.; Harris, E.; Zellweger, C.; Emmenegger, L.; Fischer, H.; Mohn, J.

    2015-08-01

    In situ and simultaneous measurement of the three most abundant isotopologues of methane using mid-infrared laser absorption spectroscopy is demonstrated. A field-deployable, autonomous platform is realized by coupling a compact quantum cascade laser absorption spectrometer (QCLAS) to a preconcentration unit, called TRace gas EXtractor (TREX). This unit enhances CH4 mole fractions by a factor of up to 500 above ambient levels and quantitatively separates interfering trace gases such as N2O and CO2. The analytical precision of the QCLAS isotope measurement on the preconcentrated (750 ppm, parts-per-million, μmole/mole) methane is 0.1 and 0.5 ‰ for δ13C- and δD-CH4 at 10 min averaging time. Based on replicate measurements of compressed air during a two-week intercomparison campaign, the repeatability of the TREX-QCLAS was determined to be 0.19 and 1.9 ‰ for δ13C and δD-CH4, respectively. In this intercomparison campaign the new in situ technique is compared to isotope-ratio mass-spectrometry (IRMS) based on glass flask and bag sampling and real time CH4 isotope analysis by two commercially available laser spectrometers. Both laser-based analyzers were limited to methane mole fraction and δ13C-CH4 analysis, and only one of them, a cavity ring down spectrometer, was capable to deliver meaningful data for the isotopic composition. After correcting for scale offsets, the average difference between TREX-QCLAS data and bag/flask sampling-IRMS values are within the extended WMO compatibility goals of 0.2 and 5 ‰ for δ13C- and δD-CH4, respectively. Thus, the intercomparison also reveals the need for reference air samples with accurately determined isotopic composition of CH4 to further improve the interlaboratory compatibility.

  10. Bomb-pulse 14C analysis combined with 13C and 15N measurements in blood serum from residents of Malmö, Sweden.

    PubMed

    Georgiadou, Elisavet; Stenström, Kristina Eriksson; Uvo, Cintia Bertacchi; Nilsson, Peter; Skog, Göran; Mattsson, Sören

    2013-05-01

    The (14)C content of 60 human blood serum samples from residents of Malmö (Sweden) in 1978, obtained from a biobank, has been measured to estimate the accuracy of (14)C bomb-pulse dating. The difference between the date estimated using the Calibomb software and sampling date varied between -3 ± 0.4 and +0.2 ± 0.5 years. The average age deviation of all samples was -1.5 ± 0.7 years, with the delay between production and consumption of foodstuffs being probably the dominating cause. The potential influence of food habits on the (14)C date has been evaluated using stable isotope δ(13)C and δ(15)N analysis and information about the dietary habits of the investigated individuals. Although the group consisting of lacto-ovo vegetarians and vegans (pooled group) was not completely separated from the omnivores in a stable isotopic trophic level diagram, this analysis proved to add valuable information on probable dietary habits. The age deviation of the sampling date from the respective Calibomb date was found strongly correlated with the δ(13)C values, probably due to influence from marine diet components. For the omnivore individuals, there were indications of seasonal effects on δ(13)C and the age deviation. No significant correlation was found between the age deviation and the δ(15)N values of any dietary group. No influence of sex or year of birth was found on neither the (14)C nor the δ(13)C and δ(15)N values of the serum samples. The data were also divided into two groups (omnivores and pooled group), based on the level of δ(15)N in the samples. The consumption of high δ(15)N-valued fish and birds can be responsible for this clustering.

  11. Recalcitrance and structural analysis by water-only flowthrough pretreatment of 13C enriched corn stover stem

    DOE PAGES

    Foston, Marcus B.; Trajanob, Heather L.; Samuel, Reichel; Wyman, Charles E.; He, Jian; Ragauskas, Arthur J.

    2015-08-28

    Here, this study presents high temperature water-only continuous flowthrough pretreatment coupled with nuclear magnetic resonance (NMR) as a promising analytical tool to examine the plant cell wall, to understand its recalcitrance (i.e., cell wall resistance to deconstruction), and to probe the chemistry occurring during batch pretreatment of biomass. 13C-enriched corn stover stems were pretreated at 170 °C for 60 min with a hot-water flow rate of 20 mL/min to control fractionation of the cell wall. This approach helped elucidate the nature of plant cell wall chemical recalcitrance and biomass pretreatment chemistry by tracking cell wall fragmentation as a function ofmore » time. Fractions of the reactor effluent were collected in a time-resolved fashion and characterized by various NMR techniques to determine the degree and sequence of fragments released, as well as, the chemical composition, molecular structure, and relative molecular weight of those released fragments.« less

  12. Compartmentation of glycogen metabolism revealed from 13C isotopologue distributions

    PubMed Central

    2011-01-01

    Background Stable isotope tracers are used to assess metabolic flux profiles in living cells. The existing methods of measurement average out the isotopic isomer distribution in metabolites throughout the cell, whereas the knowledge of compartmental organization of analyzed pathways is crucial for the evaluation of true fluxes. That is why we accepted a challenge to create a software tool that allows deciphering the compartmentation of metabolites based on the analysis of average isotopic isomer distribution. Results The software Isodyn, which simulates the dynamics of isotopic isomer distribution in central metabolic pathways, was supplemented by algorithms facilitating the transition between various analyzed metabolic schemes, and by the tools for model discrimination. It simulated 13C isotope distributions in glucose, lactate, glutamate and glycogen, measured by mass spectrometry after incubation of hepatocytes in the presence of only labeled glucose or glucose and lactate together (with label either in glucose or lactate). The simulations assumed either a single intracellular hexose phosphate pool, or also channeling of hexose phosphates resulting in a different isotopic composition of glycogen. Model discrimination test was applied to check the consistency of both models with experimental data. Metabolic flux profiles, evaluated with the accepted model that assumes channeling, revealed the range of changes in metabolic fluxes in liver cells. Conclusions The analysis of compartmentation of metabolic networks based on the measured 13C distribution was included in Isodyn as a routine procedure. The advantage of this implementation is that, being a part of evaluation of metabolic fluxes, it does not require additional experiments to study metabolic compartmentation. The analysis of experimental data revealed that the distribution of measured 13C-labeled glucose metabolites is inconsistent with the idea of perfect mixing of hexose phosphates in cytosol. In contrast

  13. Composition and chemical variability of the leaf oil from Corsican Juniperus thurifera. Integrated analysis by GC(RI), GC-MS and 13C NMR.

    PubMed

    Ottavioli, Josephine; Casanova, Joseph; Bighelli, Ange

    2010-12-01

    The composition of 16 samples of leaf oil from Corsican Juniperus thurifera was investigated by integrated techniques, GC, GC-MS and 13C NMR. K-means partitioning and PCA analysis of the data allowed the definition of a main group (14 samples) dominated by limonene (mean = 52.2%, SD = 6.4) and alpha-pinene (mean = 7.2%, SD = 3.8). Limonene and beta-elemol (up to 19.7%) were identified as the major components of two atypic samples.

  14. Experimental design principles for isotopically instationary 13C labeling experiments.

    PubMed

    Nöh, Katharina; Wiechert, Wolfgang

    2006-06-01

    13C metabolic flux analysis (MFA) is a well-established tool in Metabolic Engineering that found numerous applications in recent years. However, one strong limitation of the current method is the requirement of an-at least approximate-isotopic stationary state at sampling time. This requirement leads to a principle lower limit for the duration of a 13C labeling experiment. A new methodological development is based on repeated sampling during the instationary transient of the 13C labeling dynamics. The statistical and computational treatment of such instationary experiments is a completely new terrain. The computational effort is very high because large differential equations have to be solved and, moreover, the intracellular pool sizes play a significant role. For this reason, the present contribution works out principles and strategies for the experimental design of instationary experiments based on a simple example network. Hereby, the potential of isotopically instationary experiments is investigated in detail. Various statistical results on instationary flux identifiability are presented and possible pitfalls of experimental design are discussed. Finally, a framework for almost optimal experimental design of isotopically instationary experiments is proposed which provides a practical guideline for the analysis of large-scale networks.

  15. Food web structure of the epibenthic and infaunal invertebrates on the Catalan slope (NW Mediterranean): Evidence from δ 13C and δ 15N analysis

    NASA Astrophysics Data System (ADS)

    Fanelli, E.; Papiol, V.; Cartes, J. E.; Rumolo, P.; Brunet, C.; Sprovieri, M.

    2011-01-01

    , together with macrophyte remains. The stronger δ 13C-δ 15N correlations found in February and April suggest that during the period of water column homogeneization (winter-spring), the benthic community was sustained by phytodetritus inputs originating from the peak of surface primary production in February. Conversely, weaker δ 13C-δ 15N correlations were observed during the period of water column stratification (beginning in June-July), suggesting that the benthic community in this period was sustained, with a delay of ca. 2/3 months, by multiple carbon sources including continental inputs from river discharge (with the maxima in April-May). Thus both advective and vertical fluxes seem to be food sources for benthos on the Catalonian slope. Pigments in the guts of key species were generally degraded, and only the active suspension feeder A. longicallus ingested fresh chlorophyll during periods of high primary production at the surface (February and April 2007).

  16. Amino acid δ13C analysis shows flexibility in the routing of dietary protein and lipids to the tissue of an omnivore.

    PubMed

    Newsome, Seth D; Wolf, Nathan; Peters, Jacob; Fogel, Marilyn L

    2014-11-01

    Stable-isotope analysis (SIA) has revolutionized animal ecology by providing time-integrated estimates of the use of resources and/or habitats. SIA is based on the premise that the isotopic composition of a consumer's tissues originates from its food, but is offset by trophic-discrimination (enrichment) factors controlled by metabolic processes associated with the assimilation of nutrients and the biosynthesis of tissues. Laboratory preparation protocols dictate that tissues both of consumers and of their potential prey be lipid-extracted prior to analysis, because (1) lipids have carbon isotope (δ(13)C) values that are lower by approximately 3-8‰ than associated proteins and (2) amino acids in consumers' proteinaceous tissues are assumed to be completely routed from dietary protein. In contrast, models of stable-isotope mixing assume that dietary macromolecules are broken into their elemental constituents from which non-essential amino acids are resynthesized to build tissues. Here, we show that carbon from non-protein dietary macromolecules, namely lipids, was used to synthesize muscle tissue in an omnivorous rodent (Mus musculus). We traced the influence of dietary lipids on the synthesis of consumers' tissues by inversely varying the dietary proportion of C4-based lipids and C3-based protein while keeping carbohydrate content constant in four dietary treatments, and analyzing the δ(13)C values of amino acids in mouse muscle after 4 months of feeding. The influence of dietary lipids on non-essential amino acids varied as function of biosynthetic pathway. The source of carbon in ketogenic amino acids synthesized through the Krebs cycle was highly sensitive to dietary lipid content, with significant increases of approximately 2-4‰ in Glutamate and Aspartate δ(13)C values from the 5% to 15% dietary lipid treatment. Glucogenic amino acids (Glycine and Serine) were less sensitive to dietary lipid, but increased by approximately 3-4‰ from the 25% to 40% lipid

  17. Real-time analysis of δ13C- and δD-CH4 in ambient air with laser spectroscopy: Method development and intercomparison

    NASA Astrophysics Data System (ADS)

    Harris, E. J.; Eyer, S.; Mohn, J.; Röckmann, T.; Popa, E.; Lowry, D.; Nisbet, E. G.; Fisher, R. E.; Brennwald, M. S.; Fischer, H.; Emmenegger, L.; Tuzson, B.; Zellweger, C.

    2015-12-01

    Methane (CH4) is the second most important anthropogenically emitted greenhouse gas after carbon dioxide (CO2). Its mole fraction has increased from around 722 ppb in pre-industrial times to 1824 ppb in 2013 and the anthropogenic fraction is estimated to be 60 % of the total emissions. A promising approach to improve the understanding of the CH4 budget is the use of isotopologues to distinguish between various CH4 source processes. In the presented study in situ and simultaneous measurement of the three most abundant isotopologues of methane using mid-infrared laser absorption spectroscopy is demonstrated. A field-deployable, autonomous platform is realized by coupling a compact quantum cascade laser absorption spectrometer (QCLAS) to a preconcentration unit, called TRace gas EXtractor (TREX). This unit enhances CH4 mole fractions by a factor of up to 500 above ambient levels and quantitatively separates interfering trace gases such as N2O and CO2. The analytical precision of the QCLAS isotope measurement on the preconcentrated (750 ppm, parts-per-million, mmole/mole) methane is 0.1‰ and 0.5 ‰ for δ13C- and δD-CH4 at 10 min averaging time. [1] Based on replicate measurements of compressed air during a two-week intercomparison campaign, the repeatability of the TREX-QCLAS was determined to be 0.19 ‰ and 1.9 ‰ for δ13C and δD-CH4, respectively. In this intercomparison campaign the new in situ technique is compared to IRMS based on glass flask and bag sampling and real time CH4 isotope analysis by two commercially available laser spectrometers (Figure). Both laser-based analyzers were limited to methane mole fraction and δ13C-CH4 analysis, and only one of them, a cavity ring down spectrometer, was capable to deliver meaningful data for the isotopic composition. After correcting for scale offsets, the average difference between TREX-QCLAS data and bag/flask sampling-IRMS values are within the extended WMO compatibility goals of 0.2 and 5 ‰ for δ13C- and

  18. Glucuronoxylomannan of Cryptococcus neoformans serotype B: structural analysis by gas-liquid chromatography-mass spectrometry and 13C-nuclear magnetic resonance spectroscopy.

    PubMed

    Turner, S H; Cherniak, R

    1991-04-01

    The major extracellular polysaccharide (glucuronoxylomannan, GXM) from six strains of Cryptococcus neoformans serotype B was characterized by gas-liquid chromatography (g.l.c.), g.l.c.-mass spectrometry (g.l.c.-m.s.), and nuclear magnetic resonance (n.m.r.) spectroscopy. Ultrasonic irradiation (u.i.) was used to reduce the mol.wt. of native GXM from 9.75 x 10(5) to 1.15 x 10(5) without apparent change in its composition (GXM-S). The Xylp:Manp:GlcpA molar ratio of the GXM and GXM-S from the six strains of C. neoformans serotype B is approximately 3.5:3.0:0.6. GXM-S was O-deacetylated (GXM-D) by treatment with NH4OH. The 13C-n.m.r. analysis of GXM-D gave spectra that served as characteristic fingerprints of the structure and also facilitated the assignment of the anomeric carbon resonances to specific structural moieties present in GXM-D. The GXM-D from each serotype B strain was found to be similar by 13C-n.m.r. spectroscopy. The structure contains a linear (1----3)-alpha-D-Manp backbone substituted with 2-O-beta-GlcpA and 2-O-beta-Xylp. beta-Xylp is also O-4 linked to the Manp substituted with GlcpA. In addition, a model for the disposition of the Xylp and GlcpA side chain substituents along the mannopyranan backbone is proposed, based upon results from the combination of g.l.c.-m.s. and 13C-n.m.r. spectroscopy. PMID:1773425

  19. Provenancing Archaeological Wool Textiles from Medieval Northern Europe by Light Stable Isotope Analysis13C, δ15N, δ2H)

    PubMed Central

    von Holstein, Isabella C. C.; Walton Rogers, Penelope; Craig, Oliver E.; Penkman, Kirsty E. H.; Newton, Jason; Collins, Matthew J.

    2016-01-01

    We investigate the origin of archaeological wool textiles preserved by anoxic waterlogging from seven medieval archaeological deposits in north-western Europe (c. 700–1600 AD), using geospatial patterning in carbon (δ13C), nitrogen (δ15N) and non-exchangeable hydrogen (δ2H) composition of modern and ancient sheep proteins. δ13C, δ15N and δ2H values from archaeological wool keratin (n = 83) and bone collagen (n = 59) from four sites were interpreted with reference to the composition of modern sheep wool from the same regions. The isotopic composition of wool and bone collagen samples clustered strongly by settlement; inter-regional relationships were largely parallel in modern and ancient samples, though landscape change was also significant. Degradation in archaeological wool samples, examined by elemental and amino acid composition, was greater in samples from Iceland (Reykholt) than in samples from north-east England (York, Newcastle) or northern Germany (Hessens). A nominal assignment approach was used to classify textiles into local/non-local at each site, based on maximal estimates of isotopic variability in modern sheep wool. Light element stable isotope analysis provided new insights into the origins of wool textiles, and demonstrates that isotopic provenancing of keratin preserved in anoxic waterlogged contexts is feasible. We also demonstrate the utility of δ2H analysis to understand the location of origin of archaeological protein samples. PMID:27764106

  20. Carbon sequestration and estimated carbon credit values as measured using 13C labelling and analysis by means of an optical breath test analyser.

    PubMed

    Hood, R C; Khan, M; Haque, A; Khadir, M; Bonetto, J P; Syamsul, R; Mayr, L; Heiling, M

    2004-05-01

    Recent developments in optical systems (isotope-selective non-dispersive infrared spectrometry) for breath testing have provided a robust, low-cost option for undertaking (13)C analysis. Although these systems were initially developed for breath testing for Helicobacter pylori, they have an enormous potential as a soil science research tool. The relatively low cost of the equipment, US$15,000-25,000, is within the research budgets of most institutes or universities. The simplicity of the mechanisms and optical nature mean that the equipment requires relatively low maintenance and minimal training. Thus methods were developed to prepare soil and plant materials for analysis using the breath test analyser. Results that compare conventional mass spectrometric methods with the breath test analyser will be presented. In combination with simple (13)C-plant-labeling techniques it is possible to devise methods for estimating carbon sequestration under different agronomic management practices within a short time frame. This enables assessment of the carbon credit value of a particular agronomic practice, which can in turn be used by policy makers for decision-making purposes. For global understanding of the effect of agricultural practices on the carbon cycle, data are required from a range of cropping systems and agro-ecological zones. The method and the approach described will enable collection of hard data within a reasonable time.

  1. Validation of pentaacetylaldononitrile derivative for dual 2H gas chromatography/mass spectrometry and 13C gas chromatography/combustion/isotope ratio mass spectrometry analysis of glucose.

    PubMed

    Sauvinet, Valérie; Gabert, Laure; Qin, Du; Louche-Pélissier, Corinne; Laville, Martine; Désage, Michel

    2009-12-01

    A reference method to accurately define kinetics in response to the ingestion of glucose in terms of total, exogenous and endogenous glucose is to use stable-isotope-labelled compounds such as 2H and 13C glucose followed by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis. The use of the usual pentaacetyl (5Ac) derivative generates difficulties in obtaining accurate and reproducible results due to the two chromatographic peaks for the syn and anti isomers, and to the isotopic effect occurring during acetylation. Therefore, the pentaacetylaldononitrile derivative (Aldo) was validated for both isotopes, and compared with the 5Ac derivative. A correction factor including carbon atom dilution (stoichiometric equation) and the kinetic isotopic effect (KIE) was determined. Analytical validation results for the 2H GC/MS and 13C GC/C/IRMS measurements produced acceptable results with both derivatives. When 2H enrichments of plasma samples were < or = 1 mol % excess (MPE), the repeatability (RSD(Aldo Intra assay and Intra day) <0.94%, RSD(5Ac Intra assay and Intra day) <3.29%), accuracy (Aldo <3.4%, 5Ac <29.0%), and stability of the derivatized samples were significantly better when the Aldo derivatives of the plasma samples were used (p < 0.05). When the glucose kinetics were assessed in nine human subjects, after glucose ingestion, the plasma glucose 2H enrichments were identical with both derivatives, whereas the 13C enrichments needed a correction factor to fit together. Due to KIE variation, this correction factor was not constant and had to be calculated for each batch of analyses, to obtain satisfactory results. Mean quantities of exogenous glucose exhibit marked difference (20.9 +/- 1.3g (5Ac) vs. 26.7 +/- 2.5g (Aldo)) when calculated with stoichiometric correction, but fit perfectly when calculated after application of the correction factor (22.1 +/- 1.3g (5Ac) vs. 22.9 +/- 1.9g

  2. Feeding and migration habits of white shark Carcharodon carcharias (Lamniformes: Lamnidae) from Isla Guadalupe inferred by analysis of stable isotopes delta15N and delta13C.

    PubMed

    Jaime-Rivera, Mario; Caraveo-Patiño, Javier; Hoyos-Padilla, Mauricio; Galván-Magaña, Felipe

    2014-06-01

    Stable isotope composition of marine top predator's tissues provides insight information of its trophic ecology and migratory behavior. Previous reports have shown that dermal tissues could record longer patterns of hunting and movement. Based on this, the aim of this study was to describe the feeding and migratory habits of the white shark from Isla Guadalupe, using stable isotopic analysis of dermis. We considered a small subset of many possible prey taxa that the sharks could have eaten throughout their migration: pinnipeds, squid and tuna. We grouped the data in five focal areas: Gulf of California, Coast of California, Isla Guadalupe, SOFA and Hawaii. We performed a Bayesian mixing model to study the trophic ecology of this top predator. Average isotopic values for dermis tissue of white shark were delta13C (-14.5 per thousand) and delta15N (19.1 per thousand). Corrected white shark dermal mean values to resemble muscle were delta13C (-16.6 per thousand) and delta15N (21.2 per thousand). Mixing model data from dermis showed predation in offshore areas such the SOFA and a main importance of pinnipeds as prey of the white shark in Isla Guadalupe. PMID:25102646

  3. Determination of methanogenic pathways through carbon isotope (δ13C) analysis for the two-stage anaerobic digestion of high-solids substrates.

    PubMed

    Gehring, Tito; Klang, Johanna; Niedermayr, Andrea; Berzio, Stephan; Immenhauser, Adrian; Klocke, Michael; Wichern, Marc; Lübken, Manfred

    2015-04-01

    This study used carbon isotope (δ(13)C)-based calculations to quantify the specific methanogenic pathways in a two-stage experimental biogas plant composed of three thermophilic leach bed reactors (51-56 °C) followed by a mesophilic (36.5 °C) anaerobic filter. Despite the continuous dominance of the acetoclastic Methanosaeta in the anaerobic filter, the methane (CH4) fraction derived from carbon dioxide reduction (CO2), fmc, varied significantly over the investigation period of 200 days. At organic loading rates (OLRs) below 6.0 gCOD L(-1) d(-1), the average fmc value was 33%, whereas at higher OLRs, with a maximum level of 17.0 gCOD L(-1) d(-1), the fmc values reached 47%. The experiments allowed for a clear differentiation of the isotope fractionation related to the formation and consumption of acetate in both stages of the plant. Our data indicate constant carbon isotope fractionation for acetate formation at different OLRs within the thermophilic leach bed reactors as well as a negligible contribution of homoacetogenesis. These results present the first quantification of methanogenic pathway (fmc values) dynamics for a continually operated mesophilic bioreactor and highlight the enormous potential of δ(13)C analysis for a more comprehensive understanding of the anaerobic degradation processes in CH4-producing biogas plants. PMID:25741999

  4. Structural study of humic acids during composting of activated sludge-green waste: elemental analysis, FTIR and 13C NMR.

    PubMed

    Amir, Soumia; Jouraiphy, Abdelmajid; Meddich, Abdelilah; El Gharous, Mohamed; Winterton, Peter; Hafidi, Mohamed

    2010-05-15

    The humic acids extracted from a compost of activated sludge at different stages of maturity were characterized by various chemical techniques. Elemental analysis showed the reduction of H, and the H/C and C/N ratios and an increase in the proportion of N and S. At the end of composting C% and O% presented the same values as initially, although they increased in the intermediate stage. Based on the ratios of FTIR absorbance it was shown that the end product was enriched in etherified and peptidic compounds absorbing at 1384, 1034 and 1544 cm(-1). The alkyl and other N-rich and oxidized recalcitrant structures compose the new humic polymers produced during composting. In principal components analysis, the first axis, PC1: 49.75% considers the variability between structures in decomposition from the other parameters that concern the stable new humic polymers formed after composting. PC2 (40.5%) shows a negative correlation between (aromatic carbon, FA level) and (aliphatic carbon, HA level) during composting. PMID:20106591

  5. On-site analysis of d13C- and dD-CH4 by laser spectroscopy for the allocation of source processes

    NASA Astrophysics Data System (ADS)

    Eyer, Simon; Tuzson, Béla; Popa, Elena; van der Veen, Carina; Röckmann, Thomas; Brand, Willi A.; Fisher, Rebecca; Lowry, David; Nisbet, Euan G.; Brennwald, Matthias S.; Harris, Eliza; Emmenegger, Lukas; Fischer, Hubertus; Mohn, Joachim

    2015-04-01

    Analysis of the most abundant methane isotopologues 12CH4, 13CH4 and 12CH3D can be used to disentangle source/sink processes (Fischer et al. 2008) and to develop target oriented reduction strategies. Isotopic analysis of CH4 is accomplished by isotope-ratio mass-spectrometry (IRMS) and more recently by mid-infrared laser spectroscopy. For high precision measurements in ambient air, however, both techniques rely on preconcentration of the target gas (Eyer et al. 2014). We developed a field-deployable analyser for real-time, on-site analysis of CH4 isotopologues which is based on a dual quantum cascade laser absorption spectrometer (QCLAS) in combination with an innovative preconcentration technique named trace gas extractor (TREX). The core part of the 19 ″ rack-mounted preconcentration unit is a highly efficient adsorbent trap attached to the cold end of a Stirling cooler. The system achieves preconcentration factors >500. For fast desorption and optimal heat management, the trap is decoupled from the cooler during desorption. The QCLAS has been developed based on a previously described instrument (Tuzson 2010). It comprises two cw-QC laser sources combined and coupled into an astigmatic multipass absorption cell with 76 m optical path. The developed technique reaches an unsurpassed precision of 0.1‰ for d13C-CH4 and <0.5‰ for dD-CH4 at 600 s spectral averaging. The potential of the new analytical system for field applications has been shown in June 2014, where the system has achieved an overall repeatability of 0.19‰ for d13C and 1.7‰ for dD-CH4 for repeated target gas measurements. Compatibility of TREX - QCLAS with flask sampling - IRMS for analysis of ambient CH4 fulfilled the extended WMO/GAW compatibility goals of 0.2‰ for d13C-CH4 and 5‰ for dD-CH4. References: Fischer, H., Behrens, M., Bock, M., Richter, U., Schmitt, J., Loulergue, L., Chappellaz, J., Spahni, R., Blunier, T., Leuenberger, M., Stocker, T. F. (2008) Nature 452: 864-867. Eyer, S

  6. Soil Organic Matter Characterization by 13C-NMR and Thermal Analysis in Deep Tropical Soil Profiles from the Luquillo Critical Zone Observatory

    NASA Astrophysics Data System (ADS)

    Plante, A. F.; Hockaday, W. C.

    2015-12-01

    Tropical forest soils store large quantities of carbon (C) as soil organic matter (SOM), a substantial proportion of which is stored deep (> 30 cm) in the soil profile. Characterization of tropical SOM remains difficult, in part due to the analytical challenges associated high iron and low C concentrations. In this study, we combined solid-state 13C nuclear magnetic resonance (NMR) spectroscopy with analytical thermal analysis (differential scanning calorimetry, DSC; evolved CO2 gas analysis, CO2-EGA) to explore patterns in SOM composition in deep soil profiles from two contrasting soil types at the Luquillo Critical Zone Observatory (LCZO) in northeast Puerto Rico. Prior to 13C NMR, soils were repeatedly demineralized with hydrofluoric acid (HF) to remove paramagnetic compounds and concentrate organic matter. Given the scant information on tropical subsoil OM, we also sought to evaluate the effect of HF acid treatments on tropical subsoil SOM. HF treatments effectively enriched sample C and removed paramagnetic compounds, allowing us to obtain high-quality NMR spectra for low-C subsoils. C:N ratios before and after HF treatment were nearly identical (mean = 16.6 ± 0.8), suggesting that the SOM pool was not substantially fractionated, though C recoveries were low and variable. Thermal analyses confirmed the loss of a substantial fraction of the soil mineral matrix, however, retention of several endothermic regions in post-HF Inceptisol soils indicated that not all minerals were completely solubilized. In addition, important differences in the DSC and CO2-EGA thermograms were observed in comparing samples before versus after HF treatments. These results suggest that the organo-mineral associations were substantially altered, though it is not immediately clear the degree to which alterations in chemical composition versus binding association have changed. In addition to these qualitative changes, quantitative interpretations of 13C-NMR results from low-C and high

  7. Pyrolysis compound specific isotopic analysis13C and δD Py-CSIA) of soil organic matter size fractions under four vegetation covers.

    NASA Astrophysics Data System (ADS)

    Jiménez-Morillo, Nicasio T.; González-Vila, Francisco J.; Almendros, Gonzalo; De la Rosa, José M.; González-Pérez, José A.

    2015-04-01

    A chemical characterization of soil organic matter (SOM) under different ground cover from a Mediterranean climate (Doñana National Park, Andalusia, Spain) is approached using bulk δ15N, δ13C, δ18O and δD isotopic analysis (C/TC-IRMS) and δ13C and δD pyrolysis compound specific isotopic analysis (Py-CSIA: Py-GC-C/TC-IRMS). Soil samples were collected in sandy soils, Arenosols (WRB 2006) from the Doñana National Park (SW Spain) under different vegetation cover: cork oak (Quercus suber, QS), eagle fern (Pteridium aquilinum, PA), pine (Pinus pinea, PP) and rockrose (Halimium halimifolium, HH). Two size fractions; coarse (C: 1-2 mm) and fine (F: <0.05 mm) were studied from each soil. A complete conventional analytical pyrolysis (Py-GC/MS) of these samples have been studied in detail (Jiménez-Morillo et al., 2015). Bulk isotopic analysis of stable light elements (δ15N, δ13C, δ18O and δD) revealed particular isotopic signatures showing differences related with the main vegetation cover and the different soil size fraction. All samples had a carbon isotopic signature between -26 and -29 ‰, which indicated that the organic matter in the two fractions of each soil sample derived from C3-type plants. The bulk δD isotopic signature in whole soil sample indicate a lower deuterium fractionation occurs in SOM under arboreal than under no-arboreal vegetation, this can be caused by the occurrence of a higher water evaporation rate under bush vegetation and/or to differences due to leaf morphology as previously described (Leaney et al., 1985). A δ15N vs. δ18O chart may provide some clues about N origin in the soil and particularly about the original source of nitrates (Kendall et al., 1996). In in all sample and size fractions our values are in the chart area corresponding to NO3 in precipitation, with lighter δ18O (c. 20 ‰) values compatible with fertilizers may be from adjacent crops. In addition we were able to assign δ13C and δD values for a number of

  8. Pyrolysis compound specific isotopic analysis13C and δD Py-CSIA) of soil organic matter size fractions under four vegetation covers.

    NASA Astrophysics Data System (ADS)

    Jiménez-Morillo, Nicasio T.; González-Vila, Francisco J.; Almendros, Gonzalo; De la Rosa, José M.; González-Pérez, José A.

    2015-04-01

    A chemical characterization of soil organic matter (SOM) under different ground cover from a Mediterranean climate (Doñana National Park, Andalusia, Spain) is approached using bulk δ15N, δ13C, δ18O and δD isotopic analysis (C/TC-IRMS) and δ13C and δD pyrolysis compound specific isotopic analysis (Py-CSIA: Py-GC-C/TC-IRMS). Soil samples were collected in sandy soils, Arenosols (WRB 2006) from the Doñana National Park (SW Spain) under different vegetation cover: cork oak (Quercus suber, QS), eagle fern (Pteridium aquilinum, PA), pine (Pinus pinea, PP) and rockrose (Halimium halimifolium, HH). Two size fractions; coarse (C: 1-2 mm) and fine (F: <0.05 mm) were studied from each soil. A complete conventional analytical pyrolysis (Py-GC/MS) of these samples have been studied in detail (Jiménez-Morillo et al., 2015). Bulk isotopic analysis of stable light elements (δ15N, δ13C, δ18O and δD) revealed particular isotopic signatures showing differences related with the main vegetation cover and the different soil size fraction. All samples had a carbon isotopic signature between -26 and -29 ‰, which indicated that the organic matter in the two fractions of each soil sample derived from C3-type plants. The bulk δD isotopic signature in whole soil sample indicate a lower deuterium fractionation occurs in SOM under arboreal than under no-arboreal vegetation, this can be caused by the occurrence of a higher water evaporation rate under bush vegetation and/or to differences due to leaf morphology as previously described (Leaney et al., 1985). A δ15N vs. δ18O chart may provide some clues about N origin in the soil and particularly about the original source of nitrates (Kendall et al., 1996). In in all sample and size fractions our values are in the chart area corresponding to NO3 in precipitation, with lighter δ18O (c. 20 ‰) values compatible with fertilizers may be from adjacent crops. In addition we were able to assign δ13C and δD values for a number of

  9. Comparison among Different Gilthead Sea Bream (Sparus aurata) Farming Systems: Activity of Intestinal and Hepatic Enzymes and 13C-NMR Analysis of Lipids

    PubMed Central

    Coco, Laura Del; Papadia, Paride; Pascali, Sandra A. De; Bressani, Giorgia; Storelli, Carlo; Zonno, Vincenzo; Fanizzi, Francesco Paolo

    2009-01-01

    In order to evaluate differences in general health and nutritional values of gilthead sea bream (Sparus aurata), the effects of semi-intensive, land-based tanks and sea-cages intensive rearing systems were investigated, and results compared with captured wild fish. The physiological state was determined by measuring the activity of three different intestinal digestive enzymes: alkaline phosphatase (ALP), leucine aminopeptidase (LAP) and maltase; and the activity of the hepatic ALP. Also, the hepatic content in protein, cholesterol, and lipid were assessed. 13C-NMR analysis for qualitative and quantitative characterization of the lipid fraction extracted from fish muscles for semi-intensive and land based tanks intensive systems was performed. The lipid fraction composition showed small but significant differences in the monounsaturated/saturated fatty acid ratio, with the semi-intensive characterized by higher monounsaturated and lower saturated fatty acid content with respect to land based tanks intensive rearing system. PMID:22253985

  10. Application of Spectroscopic Techniques (FT-IR, 13C NMR) to the analysis of humic substances in volcanic soils along an environmental gradient (Tenerife, Canary Islands, Spain)

    NASA Astrophysics Data System (ADS)

    Rodriguez Rodriguez, Antonio; María Armas Herrera, Cecilia; González Pérez, José Antonio; González-Vila, Francisco Javier; Arbelo Rodríguez, Carmen Dolores; Mora Hernández, Juan Luis; Polvillo Polo, Oliva

    2010-05-01

    Andosols and andic soils are considered as efficient C-sinks in terms of C sequestration. These soils are usually developed from volcanic materials, and are characterized by a predominance of short-range ordered minerals like allophanes, imogolite and other Fe and Al oxyhydroxides. Such materials occur commonly associated with organic compounds, thus generating highly stable organo-mineral complexes and leading to the accumulation of a high amount of organic carbon. Spectroscopic methods like FT-IR and 13C NMR are suitable for the analysis of the chemical structure of soil humic substances, and allow identifying distinct functional groups and protein, lipids, lignin, carbohydrate-derived fragments. In this work we study the structural features of four soils developed on Pleistocene basaltic lavae in Tenerife (Canary Island, Spain), distributed along an altitudinal climatic gradient. The soil sequence comprises soils with different degree of geochemical evolution and andic character, including a mineral ‘Hypersalic Solonchak' (Tabaibal de Rasca), a slightly vitric ‘Luvic Phaeozem' (Los Frailes), a degraded and shallow ‘Endoleptic, fulvic, silandic Andosol' (Siete Lomas), and a well-developed and deep ‘Fulvic, silandic, Andosol' (Ravelo). Samples of the raw soil and humic and fulvic acids isolated from the surface horizons were analyzed. The results show a low content of organic carbon in the mineral soil, the inherited humin predominating, and a very high content of humic and fulvic acids in Andosols. The FT-IR and 13C NMR spectra of the raw soil samples show a low resolution, related to interferences from mineral complexes signals, particularly in soils with lower organic carbon content. 13C NMR shows a predominance of O-alkyl carbon (derived of carbohydrates) in andic soils, whereas O-alkyl and aromatic fractions are most evident in the mineral soil. The humic acids spectra are characterized by a dominance of alkyl and aromatic fractions with a high degree

  11. Adaptation of continuous-flow cavity ring-down spectroscopy for batch analysis of δ13C of CO2 and comparison with isotope ratio mass spectrometry.

    PubMed

    Berryman, E M; Marshall, J D; Rahn, T; Cook, S P; Litvak, M

    2011-08-30

    Measurements of δ(13)C in CO(2) have traditionally relied on samples stored in sealed vessels and subsequently analyzed using magnetic sector isotope ratio mass spectrometry (IRMS), an accurate but expensive and high-maintenance analytical method. Recent developments in optical spectroscopy have yielded instruments that can measure δ(13)CO(2) in continuous streams of air with precision and accuracy approaching those of IRMS, but at a fraction of the cost. However, continuous sampling is unsuited for certain applications, creating a need for conversion of these instruments for batch operation. Here, we present a flask (syringe) adaptor that allows the collection and storage of small aliquots (20-30 mL air) for injection into the cavity ring-down spectroscopy (CRDS) instrument. We demonstrate that the adaptor's precision is similar to that of traditional IRMS (standard deviation of 0.3‰ for 385 ppm CO(2) standard gas). In addition, the concentration precision (±0.3% of sample concentration) was higher for CRDS than for IRMS (±7% of sample concentration). Using the adaptor in conjunction with CRDS, we sampled soil chambers and found that soil-respired δ(13)C varied between two different locations in a piñon-juniper woodland. In a second experiment, we found no significant discrimination between the respiration of a small beetle (~5 mm) and its diet. Our work shows that the CRDS system is flexible enough to be used for the analysis of batch samples as well as for continuous sampling. This flexibility broadens the range of applications for which CRDS has the potential to replace magnetic sector IRMS.

  12. Differences in CH4 oxidation and pathways of production between rice cultivars deduced from measurements of CH4 flux and δ13C of CH4 and CO2

    NASA Astrophysics Data System (ADS)

    Bilek, Rebecca S.; Tyler, Stanley C.; Sass, Ronald L.; Fisher, Frank M.

    We report measurements of CH4 flux and δ13C and δD values of emitted CH4 and sediment CH4 and CO2 during the 1995 rice growing season in Beaumont, Texas. Four rice plant cultivars, Lemont, Mars, Cypress, and Della, and an unplanted plot were studied to provide possible explanations for the differences in CH4 emissions between cultivars. Using the measured isotope values, along with data of CH4 and CO2 concentrations and other ecosystem data, we determined differences between cultivars in the processes of oxidation and production throughout the growing season. For instance, rhizospheric CH4 oxidation increased as the season progressed in both Mars and Lemont cultivars. Late in the season, however, 71+/-10% of CH4 produced in the Mars plot was oxidized compared to only 39+/-10% in the Lemont plot. The contribution of acetate fermentation to methanogenesis at specific times during the season was calculated using measured isotopic values and assuming identical isotopic fractionation factors in methanogenic pathways for the cultivars. In these calculations a range of values for the contribution to CH4 production from acetate fermentation and CO2 reduction with H2 was estimated by considering different fractionation factors for the methanogenic CO2 reduction pathway and the possibility of a 10% contribution to CH4 production from acetate produced by homoacetogenesis. In general, a steady increase in the CH4 portion produced by acetate fermentation was noted in the Lemont cultivar, while an increase followed by a decrease near the end of the season was observed for the Mars cultivar.

  13. Proton-detected 3D 1H/13C/1H correlation experiment for structural analysis in rigid solids under ultrafast-MAS above 60 kHz

    NASA Astrophysics Data System (ADS)

    Zhang, Rongchun; Nishiyama, Yusuke; Ramamoorthy, Ayyalusamy

    2015-10-01

    A proton-detected 3D 1H/13C/1H chemical shift correlation experiment is proposed for the assignment of chemical shift resonances, identification of 13C-1H connectivities, and proximities of 13C-1H and 1H-1H nuclei under ultrafast magic-angle-spinning (ultrafast-MAS) conditions. Ultrafast-MAS is used to suppress all anisotropic interactions including 1H-1H dipolar couplings, while the finite-pulse radio frequency driven dipolar recoupling (fp-RFDR) pulse sequence is used to recouple dipolar couplings among protons and the insensitive nuclei enhanced by polarization transfer technique is used to transfer magnetization between heteronuclear spins. The 3D experiment eliminates signals from non-carbon-bonded protons and non-proton-bonded carbons to enhance spectral resolution. The 2D (F1/F3) 1H/1H and 2D 13C/1H (F2/F3) chemical shift correlation spectra extracted from the 3D spectrum enable the identification of 1H-1H proximity and 13C-1H connectivity. In addition, the 2D (F1/F2) 1H/13C chemical shift correlation spectrum, incorporated with proton magnetization exchange via the fp-RFDR recoupling of 1H-1H dipolar couplings, enables the measurement of proximities between 13C and even the remote non-carbon-bonded protons. The 3D experiment also gives three-spin proximities of 1H-1H-13C chains. Experimental results obtained from powder samples of L-alanine and L-histidine ṡ H2O ṡ HCl demonstrate the efficiency of the 3D experiment.

  14. Proton-detected 3D (1)H/(13)C/(1)H correlation experiment for structural analysis in rigid solids under ultrafast-MAS above 60 kHz.

    PubMed

    Zhang, Rongchun; Nishiyama, Yusuke; Ramamoorthy, Ayyalusamy

    2015-10-28

    A proton-detected 3D (1)H/(13)C/(1)H chemical shift correlation experiment is proposed for the assignment of chemical shift resonances, identification of (13)C-(1)H connectivities, and proximities of (13)C-(1)H and (1)H-(1)H nuclei under ultrafast magic-angle-spinning (ultrafast-MAS) conditions. Ultrafast-MAS is used to suppress all anisotropic interactions including (1)H-(1)H dipolar couplings, while the finite-pulse radio frequency driven dipolar recoupling (fp-RFDR) pulse sequence is used to recouple dipolar couplings among protons and the insensitive nuclei enhanced by polarization transfer technique is used to transfer magnetization between heteronuclear spins. The 3D experiment eliminates signals from non-carbon-bonded protons and non-proton-bonded carbons to enhance spectral resolution. The 2D (F1/F3) (1)H/(1)H and 2D (13)C/(1)H (F2/F3) chemical shift correlation spectra extracted from the 3D spectrum enable the identification of (1)H-(1)H proximity and (13)C-(1)H connectivity. In addition, the 2D (F1/F2) (1)H/(13)C chemical shift correlation spectrum, incorporated with proton magnetization exchange via the fp-RFDR recoupling of (1)H-(1)H dipolar couplings, enables the measurement of proximities between (13)C and even the remote non-carbon-bonded protons. The 3D experiment also gives three-spin proximities of (1)H-(1)H-(13)C chains. Experimental results obtained from powder samples of L-alanine and L-histidine ⋅ H2O ⋅ HCl demonstrate the efficiency of the 3D experiment.

  15. Effects of cooking on the cell walls (dietary fiber) of 'Scarlet Warren' winter squash ( Cucurbita maxima ) studied by polysaccharide linkage analysis and solid-state (13)C NMR.

    PubMed

    Ratnayake, R M Sunil; Sims, Ian M; Newman, Roger H; Melton, Laurence D

    2011-07-13

    Cell wall polysaccharides of 'Scarlet Warren' winter squash ( Cucurbita maxima ) were investigated before and after thermal processing. Linkage analysis of polysaccharides was done by gas chromatography coupled to mass spectrometry (GC-MS). The linkage analysis showed the cell wall polysaccharide compositions of raw and cooked squash were similar. The total pectic polysaccharides (galacturonan, rhamnogalacturonan, arabinan, and arabinogalactan) contents of the cell walls of both raw and cooked squash were 39 mol %. The amounts of pectic polysaccharides and xyloglucan in the cell walls of squash showed little alteration on heating. The cellulose content of the raw and cooked cell walls was relatively high at 47 mol %, whereas the xyloglucan content was low at 4 mol %. Solid-state (13)C nuclear magnetic resonance (NMR) spectroscopy techniques were used to examine the molecular motion of the polysaccharides in the cell walls. The mobility of highly flexible galactan depends on the water content of the sample, but no difference was seen between raw and cooked samples. Likewise, the mobility of semimobile pectic polysaccharides was apparently unaltered by cooking. No change was detected in the rigid cellulose microfibrils on cooking.

  16. Quantitative analysis of cereulide toxin from Bacillus cereus in rice and pasta using synthetic cereulide standard and 13C6-cereulide standard - a short validation study.

    PubMed

    Zuberovic Muratovic, Aida; Tröger, Rikard; Granelli, Kristina; Hellenäs, Karl-Erik

    2014-12-11

    A single laboratory validation study of a rapid and sensitive quantitative method for the analysis of cereulide toxin produced by Bacillus cereus using ultra high performance liquid chromatography-electrospray-tandem mass spectrometry is presented. The analysis of this cyclic peptide toxin was validated for pasta and rice samples using a newly presented synthetic cereulide peptide standard, together with 13C6-cereulide that previously have not been commercially available. The use of cereulide standard was also compared to the most frequently used surrogate standard, the antibiotic valinomycin. The performance of the method was evaluated by analyzing spiked sample pools from different types of rice and pasta, as well as 21 individual rice and pasta samples from differently prepared meals. Inoculation of samples with three cereulide toxin-producing strains of Bacillus cereus was finally used to mimic naturally contaminated foods. The quantification range of the method was 1-500 ng/g (R2 = 0.999) and the limits of detection and quantification were 0.1 and 1 ng/g, respectively. The precision varied from 3% to 7% relative standard deviation and the trueness from -2% to +6% relative bias at different concentration levels in cooked rice and pasta.

  17. 13C NMR and fluorescence analysis of tryptophan dynamics in wild-type and two single-Trp variants of Escherichia coli thioredoxin.

    PubMed Central

    Kemple, M D; Yuan, P; Nollet, K E; Fuchs, J A; Silva, N; Prendergast, F G

    1994-01-01

    The rotational motion of tryptophan side chains in oxidized and reduced wild-type (WT) Escherichia coli thioredoxin and in two single-tryptophan variants of E. coli thioredoxin was studied in solution in the temperature range 20-50 degrees C from 13C-NMR relaxation rate measurements at 75.4 and 125.7 MHz and at 20 degrees C from steady-state and time-resolved trp fluorescence anisotropy measurements. Tryptophan enriched with 13C at the delta 1 and epsilon 3 sites of the indole ring was incorporated into WT thioredoxin and into two single-trp mutants, W31F and W28F, in which trp-28 or trp-31 of WT thioredoxin was replaced, respectively, with phenylalanine. The NMR relaxation data were interpreted using the Lipari and Szabo "model-free" approach (G. Lipari and A. Szabo. 1982. J. Amer. Chem. Soc. 104:4546-4559) with trp steady-state anisotropy data included for the variants at 20 degrees C. Values for the correlation time for the overall rotational motion (tau m) from NMR of oxidized and reduced WT thioredoxin at 35 degrees C agree well with those given by Stone et al. (Stone, M. J., K. Chandrasekhar, A. Holmgren, P. E. Wright, and H. J. Dyson. 1993. Biochemistry. 32:426-435) from 15N NMR relaxation rates, and the dependence of tau m on viscosity and temperature was in accord with the Stokes-Einstein relationship. Order parameters (S2) near 1 were obtained for the trp side chains in the WT proteins even at 50 degrees C. A slight increase in the amplitude of motion (decrease in S2) of trp-31, which is near the protein surface, but not of trp-28, which is partially buried in the protein matrix, was observed in reduced relative to oxidized WT thioredoxin. For trp-28 in W31F, order parameters near 1 (S2 > or = 0.8) at 20 degrees C were found, whereas trp-31 in W28F yielded the smallest order parameters (S2 approximately 0.6) of any of the cases. Analysis of time-resolved anisotropy decays in W28F and W31F yielded S2 values in good agreement with NMR, but gave tau m values

  18. 13C/12C and 15N/14N Isotope Analysis to Characterize Natural Degradation of Atrazine: Evidence from Parent and Daughter Compound Values

    NASA Astrophysics Data System (ADS)

    Elsner, Martin; Meyer, Armin

    2013-04-01

    The mobile and still herbicidal metabolites desethylatrazine (DEA) and desisopropylatrazine (DIA) are frequently detected together with its parent compound atrazine (Atz) in the aquatic environment. Interpretation of their transformation state is often difficult with current methods, which are mainly measuring concentrations. Alternatively, compound specific isotope analyses (CSIA) has become a novel tool to detect degradation processes of contaminants in groundwater. The aim of our study was to investigate on the lab scale 13C/12C and 15N/14N isotope trends in parent and daughter compounds associated with different degradation scenarios of atrazine likely to occur in the environment. Thus atrazine was dealkylated with (i) permanganate and (ii) the bacterium Rhodococcus sp. NI86/21. In both transformations, 13C/12C ratios of atrazine increased strongly (epsilon carbon/permanganate = -4.6 ± 0.6 ‰ and epsilon carbon/Rhodoccoccus = -3.8 ± 0.2 ‰) whereas nitrogen isotope fractionation was small. 13C/12C ratios of DEA showed the following trends. (i) When DEA was formed as only product (Atz + permanganate) 13C/12C remained constant, close to the initial value of Atz. (ii) When DEA was formed together with deisopropylatrazine (biodegradation of Atz) 13C/12C increased, but only within 2‰. (iii) When DEA and DIA was further biodegraded, 13C/12C increased for both metabolites up to 9‰. Thus strong enrichment of 13C/12C in the metabolites in comparison to Atz can give strong testimony for further breakdown of the metabolite.

  19. Combined analysis of the essential oil of Chenopodium ambrosioides by GC, GC-MS and 13C-NMR spectroscopy: quantitative determination of ascaridole, a heat-sensitive compound.

    PubMed

    Cavalli, Jean-François; Tomi, Félix; Bernardini, Antoine-François; Casanova, Joseph

    2004-01-01

    A commercial sample of the essential oil of Chenopodium ambrosioides L. from Madagascar was analysed by GC, GC-MS and 13C-NMR. By GC analysis, the major constituents were found to be ascaridole (1) (41.8%), isoascaridole (2) (18.1%), p-cymene (16.2%), alpha-terpinene (9.7%) and limonene (3.8%). However, ascaridole undergoes a partial thermal isomerisation to 2 and hence the amount of 1 is under-estimated by GC analysis. The actual contents of 1 and 2 (55.3 and 4.6%, respectively) were obtained following combined analysis of the sample by GC and 13C-NMR. Several hydroxy- and polyhydroxy-menthanes were identified by 13C-NMR.

  20. 13C18O in Earth's Atmosphere: a New Proxy for Constraining CO Budget

    NASA Astrophysics Data System (ADS)

    Guo, W.; Eiler, J. M.

    2005-12-01

    Despite its low average mixing ratio (70-100 ppbv), carbon monoxide plays an important role in atmospheric chemistry. It is the major sink of OH radicals, and thus strongly influences the oxidizing capacity of the atmosphere, and the lifetimes of many other atmospheric trace gases (e.g., methane, NHMCs and HCFCs). At present, the budget of atmospheric CO is constrained by its mixing ratio, δ13C, δ18O, δ17O values, inventory analysis and inverse modeling. However, the major sources of CO (CH4-oxidation, NMHC-oxidation, biomass burning, anthropogenic emissions and the ocean) vary in flux and isotopic composition, and some overlap one another in isotopic composition. Therefore, its atmospheric budget cannot be rigorously defined by inversion of the existing isotopic and concentration records. Here we introduce measurements of the abundance anomaly of the 13C18O isotopologue of carbon monoxide as an additional constraint on its atmospheric budget. We define the 13C18O anomaly as the deviation of its actual abundance from its expected statistical abundance,Δ13C18O=(([13C18O]actual/[12C16O]actual)/([13C18O]stati stical /[12C16O]statistical)-1)×1000. Abundances of 13C18O are measured by quantitatively oxidizing CO to CO2 over the Schutze reagent, and then measuring mass 47 (mainly 13C18O16O) in the product CO2, which is proportional to the abundance of 13C18O in the starting CO. External precision of Δ13C18O for repeated measurements of pure CO averages 0.03‰(one standard deviation). We expect Δ13C18O in atmospheric carbon monoxide to be sensitive to: mixing between CO of different isotopic compositions, thermodynamic fractionations, diffusion, and kinetic isotope effects accompanying chemical reactions. We have investigated the thermodynamic fractionation of Δ13C18O by performing measurements on carbon monoxide samples catalytically equilibrated at high temperatures (300-1000°C). Measured Δ13C18O values, ranging from ~0.08‰ to ~0.47‰, vary as a function

  1. Stable Isotope (δ13C, δ15N, δ34S) Analysis and Satellite Telemetry Depict the Complexity of Gray Wolf (Canis lupus) Diets in Southwest Alaska

    NASA Astrophysics Data System (ADS)

    Stanek, A.; Watts, D. E.; Cohn, B. R.; Spencer, P.; Mangipane, B.; Welker, J. M.

    2010-12-01

    Throughout Alaska, gray wolves (Canis lupus) are a top predator of large ungulates. While they primarily rely on ungulates such as moose (Alces alces) and caribou (Rangifer tarandus) as food, they are opportunistic and use alternative resources. The variation and supplemental protein sources in wolf diet has not been studied extensively on live animals currently using the landscape. With large seasonal influxes of Pacific salmon (Oncorhynchus sp.) into Alaska, terrestrial carnivore use of marine species is of particular interest. Using stable isotope (δ13C, δ15N, δ34S) analysis of wolf guard hair and blood, this study aims to determine the proportion of marine derived nutrients (MDN) in the diet of wolf packs within and surrounding Lake Clark National Park and Preserve and Alaska Peninsula and Becharof National Wildlife Refuges in Southwest Alaska. Satellite telemetry from the animals sampled facilitates quantification of landscape use patterns in correspondence with isotopic traits. Wolf pack territories within and surrounding the Lake Clark region appear to vary in spatial extent and in availability of MDN, such as salmon. Initial analysis shows that two packs with smaller home ranges, centrally located around areas with greater salmon availability, have enriched δ15N values compared to packs that have larger home ranges not centralized around salmon spawning waters. This pattern of isotopic enrichment is found in red blood cells, blood serum and hair, representing diets over different time scales. The enrichment in both blood and hair indicates a sustained use of MDN over the previous six to nine months. In the Lake Clark region, simple mixing model estimates suggest that up to 30% of wolf pack diets may be from marine sources. In contrast, packs with larger home ranges and less access to salmon have stable isotope values representative of a terrestrial diet.

  2. Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by 13C-/12C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Guo, Kevin; Bamforth, Fiona; Li, Liang

    2011-02-01

    Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by 13C-dansyl and 12C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner.

  3. Metabolic flux analysis of Cyanothece sp. ATCC 51142 under mixotrophic conditions.

    PubMed

    Alagesan, Swathi; Gaudana, Sandeep B; Sinha, Avinash; Wangikar, Pramod P

    2013-11-01

    Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several "proof of principle" studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by (13)C metabolic flux analysis ((13)C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report (13)C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of (12)C and (13)C from unlabeled CO2 and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism's preferred mode under nitrogen-fixing conditions. The (13)C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism's distinct metabolic features under nitrogen-fixing and -non-fixing conditions.

  4. Conformational analysis, spectroscopic study (FT-IR, FT-Raman, UV, 1H and 13C NMR), molecular orbital energy and NLO properties of 5-iodosalicylic acid.

    PubMed

    Karaca, Caglar; Atac, Ahmet; Karabacak, Mehmet

    2015-02-01

    In this study, 5-iodosalicylic acid (5-ISA, C7H5IO3) is structurally characterized by FT-IR, FT-Raman, NMR and UV spectroscopies. There are eight conformers, Cn, n=1-8 for this molecule therefore the molecular geometry for these eight conformers in the ground state are calculated by using the ab-initio density functional theory (DFT) B3LYP method approach with the aug-cc-pVDZ-PP basis set for iodine and the aug-cc-pVDZ basis set for the other elements. The computational results identified that the most stable conformer of 5-ISA is the C1 form. The vibrational spectra are calculated DFT method invoking the same basis sets and fundamental vibrations are assigned on the basis of the total energy distribution (TED) of the vibrational modes, calculated with scaled quantum mechanics (SQM) method with PQS program. Total density of state (TDOS) and partial density of state (PDOS) and also overlap population density of state (COOP or OPDOS) diagrams analysis for C1 conformer were calculated using the same method. The energy and oscillator strength are calculated by time-dependent density functional theory (TD-DFT) results complement with the experimental findings. Besides, charge transfer occurring in the molecule between HOMO and LUMO energies, frontier energy gap, molecular electrostatic potential (MEP) are calculated and presented. The NMR chemical shifts ((1)H and (13)C) spectra are recorded and calculated using the gauge independent atomic orbital (GIAO) method. Mulliken atomic charges of the title molecule are also calculated, interpreted and compared with salicylic acid. The optimized bond lengths, bond angles and calculated NMR and UV, vibrational wavenumbers showed the best agreement with the experimental results. PMID:25448933

  5. Conformational analysis, spectroscopic study (FT-IR, FT-Raman, UV, 1H and 13C NMR), molecular orbital energy and NLO properties of 5-iodosalicylic acid.

    PubMed

    Karaca, Caglar; Atac, Ahmet; Karabacak, Mehmet

    2015-02-01

    In this study, 5-iodosalicylic acid (5-ISA, C7H5IO3) is structurally characterized by FT-IR, FT-Raman, NMR and UV spectroscopies. There are eight conformers, Cn, n=1-8 for this molecule therefore the molecular geometry for these eight conformers in the ground state are calculated by using the ab-initio density functional theory (DFT) B3LYP method approach with the aug-cc-pVDZ-PP basis set for iodine and the aug-cc-pVDZ basis set for the other elements. The computational results identified that the most stable conformer of 5-ISA is the C1 form. The vibrational spectra are calculated DFT method invoking the same basis sets and fundamental vibrations are assigned on the basis of the total energy distribution (TED) of the vibrational modes, calculated with scaled quantum mechanics (SQM) method with PQS program. Total density of state (TDOS) and partial density of state (PDOS) and also overlap population density of state (COOP or OPDOS) diagrams analysis for C1 conformer were calculated using the same method. The energy and oscillator strength are calculated by time-dependent density functional theory (TD-DFT) results complement with the experimental findings. Besides, charge transfer occurring in the molecule between HOMO and LUMO energies, frontier energy gap, molecular electrostatic potential (MEP) are calculated and presented. The NMR chemical shifts ((1)H and (13)C) spectra are recorded and calculated using the gauge independent atomic orbital (GIAO) method. Mulliken atomic charges of the title molecule are also calculated, interpreted and compared with salicylic acid. The optimized bond lengths, bond angles and calculated NMR and UV, vibrational wavenumbers showed the best agreement with the experimental results.

  6. A conifer-friendly high-throughput α-cellulose extraction method for δ13C and δ18O stable isotope ratio analysis

    NASA Astrophysics Data System (ADS)

    Lin, W.; Noormets, A.; domec, J.; King, J. S.; Sun, G.; McNulty, S.

    2012-12-01

    Wood stable isotope ratios (δ13C and δ18O) offer insight to water source and plant water use efficiency (WUE), which in turn provide a glimpse to potential plant responses to changing climate, particularly rainfall patterns. The synthetic pathways of cell wall deposition in wood rings differ in their discrimination ratios between the light and heavy isotopes, and α-cellulose is broadly seen as the best indicator of plant water status due to its local and temporal fixation and to its high abundance within the wood. To use the effects of recent severe droughts on the WUE of loblolly pine (Pinus taeda) throughout Southeastern USA as a harbinger of future changes, an effort has been undertaken to sample the entire range of the species and to sample the isotopic composition in a consistent manner. To be able to accommodate the large number of samples required by this analysis, we have developed a new high-throughput method for α-cellulose extraction, which is the rate-limiting step in such an endeavor. Although an entire family of methods has been developed and perform well, their throughput in a typical research lab setting is limited to 16-75 samples per week with intensive labor input. The resin exclusion step in conifersis is particularly time-consuming. We have combined the recent advances of α-cellulose extraction in plant ecology and wood science, including a high-throughput extraction device developed in the Potsdam Dendro Lab and a simple chemical-based resin exclusion method. By transferring the entire extraction process to a multiport-based system allows throughputs of up to several hundred samples in two weeks, while minimizing labor requirements to 2-3 days per batch of samples.

  7. Conformational analysis, spectroscopic study (FT-IR, FT-Raman, UV, 1H and 13C NMR), molecular orbital energy and NLO properties of 5-iodosalicylic acid

    NASA Astrophysics Data System (ADS)

    Karaca, Caglar; Atac, Ahmet; Karabacak, Mehmet

    2015-02-01

    In this study, 5-iodosalicylic acid (5-ISA, C7H5IO3) is structurally characterized by FT-IR, FT-Raman, NMR and UV spectroscopies. There are eight conformers, Cn, n = 1-8 for this molecule therefore the molecular geometry for these eight conformers in the ground state are calculated by using the ab-initio density functional theory (DFT) B3LYP method approach with the aug-cc-pVDZ-PP basis set for iodine and the aug-cc-pVDZ basis set for the other elements. The computational results identified that the most stable conformer of 5-ISA is the C1 form. The vibrational spectra are calculated DFT method invoking the same basis sets and fundamental vibrations are assigned on the basis of the total energy distribution (TED) of the vibrational modes, calculated with scaled quantum mechanics (SQM) method with PQS program. Total density of state (TDOS) and partial density of state (PDOS) and also overlap population density of state (COOP or OPDOS) diagrams analysis for C1 conformer were calculated using the same method. The energy and oscillator strength are calculated by time-dependent density functional theory (TD-DFT) results complement with the experimental findings. Besides, charge transfer occurring in the molecule between HOMO and LUMO energies, frontier energy gap, molecular electrostatic potential (MEP) are calculated and presented. The NMR chemical shifts (1H and 13C) spectra are recorded and calculated using the gauge independent atomic orbital (GIAO) method. Mulliken atomic charges of the title molecule are also calculated, interpreted and compared with salicylic acid. The optimized bond lengths, bond angles and calculated NMR and UV, vibrational wavenumbers showed the best agreement with the experimental results.

  8. Solid state 13C NMR analysis of shales and coals from Laramide Basins. Final report, March 1, 1995--March 31, 1996

    SciTech Connect

    Miknis, F.P.; Jiao, Z.S.; Zhao, Hanqing; Surdam, R.C.

    1998-12-31

    This Western Research Institute (WRI) jointly sponsored research (JSR) project augmented and complemented research conducted by the University of Wyoming Institute For Energy Research for the Gas Research Institute. The project, {open_quotes}A New Innovative Exploitation Strategy for Gas Accumulations Within Pressure Compartments,{close_quotes} was a continuation of a project funded by the GRI Pressure Compartmentalization Program that began in 1990. That project, {open_quotes}Analysis of Pressure Chambers and Seals in the Powder River Basin, Wyoming and Montana,{close_quotes} characterized a new class of hydrocarbon traps, the discovery of which can provide an impetus to revitalize the domestic petroleum industry. In support of the UW Institute For Energy Research`s program on pressure compartmentalization, solid-state {sup 13}C NMR measurements were made on sets of shales and coals from different Laramide basins in North America. NMR measurements were made on samples taken from different formations and depths of burial in the Alberta, Bighorn, Denver, San Juan, Washakie, and Wind River basins. The carbon aromaticity determined by NMR was shown to increase with depth of burial and increased maturation. In general, the NMR data were in agreement with other maturational indicators, such as vitrinite reflectance, illite/smectite ratio, and production indices. NMR measurements were also obtained on residues from hydrous pyrolysis experiments on Almond and Lance Formation coals from the Washakie Basin. These data were used in conjunction with mass and elemental balance data to obtain information about the extent of carbon aromatization that occurs during artificial maturation. The data indicated that 41 and 50% of the original aliphatic carbon in the Almond and Lance coals, respectively, aromatized during hydrous pyrolysis.

  9. Optimized [1-13C]glucose infusion protocol for 13C magnetic resonance spectroscopy at 3 Tesla of human brain glucose metabolism under euglycemic and hypoglycemic conditions

    PubMed Central

    van de Ven, Kim C.C.; van der Graaf, Marinette; Tack, Cees J.J.; Klomp, Dennis W.J.; Heerschap, Arend; de Galan, Bastiaan E.

    2009-01-01

    The effect of insulin-induced hypoglycemia on cerebral glucose metabolism is largely unknown. 13C MRS is a unique tool to study cerebral glucose metabolism, but the concurrent requirement for [1-13C]glucose administration limits its use under hypoglycemic conditions. To facilitate 13C MRS data analysis we designed separate [1-13C]glucose infusion protocols for hyperinsulinemic euglycemic and hypoglycemic clamps in such a way that plasma isotopic enrichment of glucose was stable and comparable under both glycemic conditions. 13C MR spectra were acquired with optimized 13C MRS measurement techniques to obtain high quality 13C MR spectra with these protocols. PMID:19913052

  10. Additional Value of CH4 Measurement in a Combined 13C/H2 Lactose Malabsorption Breath Test: A Retrospective Analysis

    PubMed Central

    Houben, Els; De Preter, Vicky; Billen, Jaak; Van Ranst, Marc; Verbeke, Kristin

    2015-01-01

    The lactose hydrogen breath test is a commonly used, non-invasive method for the detection of lactose malabsorption and is based on an abnormal increase in breath hydrogen (H2) excretion after an oral dose of lactose. We use a combined 13C/H2 lactose breath test that measures breath 13CO2 as a measure of lactose digestion in addition to H2 and that has a better sensitivity and specificity than the standard test. The present retrospective study evaluated the results of 1051 13C/H2 lactose breath tests to assess the impact on the diagnostic accuracy of measuring breath CH4 in addition to H2 and 13CO2. Based on the 13C/H2 breath test, 314 patients were diagnosed with lactase deficiency, 138 with lactose malabsorption or small bowel bacterial overgrowth (SIBO), and 599 with normal lactose digestion. Additional measurement of CH4 further improved the accuracy of the test as 16% subjects with normal lactose digestion and no H2-excretion were found to excrete CH4. These subjects should have been classified as subjects with lactose malabsorption or SIBO. In conclusion, measuring CH4-concentrations has an added value to the 13C/H2 breath test to identify methanogenic subjects with lactose malabsorption or SIBO. PMID:26371034

  11. Achieving Metabolic Flux Analysis for S. cerevisiae at a Genome-Scale: Challenges, Requirements, and Considerations

    PubMed Central

    Gopalakrishnan, Saratram; Maranas, Costas D.

    2015-01-01

    Recent advances in 13C-Metabolic flux analysis (13C-MFA) have increased its capability to accurately resolve fluxes using a genome-scale model with narrow confidence intervals without pre-judging the activity or inactivity of alternate metabolic pathways. However, the necessary precautions, computational challenges, and minimum data requirements for successful analysis remain poorly established. This review aims to establish the necessary guidelines for performing 13C-MFA at the genome-scale for a compartmentalized eukaryotic system such as yeast in terms of model and data requirements, while addressing key issues such as statistical analysis and network complexity. We describe the various approaches used to simplify the genome-scale model in the absence of sufficient experimental flux measurements, the availability and generation of reaction atom mapping information, and the experimental flux and metabolite labeling distribution measurements to ensure statistical validity of the obtained flux distribution. Organism-specific challenges such as the impact of compartmentalization of metabolism, variability of biomass composition, and the cell-cycle dependence of metabolism are discussed. Identification of errors arising from incorrect gene annotation and suggested alternate routes using MFA are also highlighted. PMID:26393660

  12. Flux balance analysis accounting for metabolite dilution.

    PubMed

    Benyamini, Tomer; Folger, Ori; Ruppin, Eytan; Shlomi, Tomer

    2010-01-01

    Flux balance analysis is a common method for predicting steady-state flux distributions within metabolic networks, accounting for the growth demand for the synthesis of a predefined set of essential biomass precursors. Ignoring the growth demand for the synthesis of intermediate metabolites required for balancing their dilution leads flux balance analysis to false predictions in some cases. Here, we present metabolite dilution flux balance analysis, which addresses this problem, resulting in improved metabolic phenotype predictions. PMID:20398381

  13. Flux balance analysis accounting for metabolite dilution.

    PubMed

    Benyamini, Tomer; Folger, Ori; Ruppin, Eytan; Shlomi, Tomer

    2010-01-01

    Flux balance analysis is a common method for predicting steady-state flux distributions within metabolic networks, accounting for the growth demand for the synthesis of a predefined set of essential biomass precursors. Ignoring the growth demand for the synthesis of intermediate metabolites required for balancing their dilution leads flux balance analysis to false predictions in some cases. Here, we present metabolite dilution flux balance analysis, which addresses this problem, resulting in improved metabolic phenotype predictions.

  14. Rotational analysis of the Ångström system (B1Σ+-A1Π) in the rare 13C17O isotopologue

    NASA Astrophysics Data System (ADS)

    Hakalla, Rafał; Zachwieja, Mirosław

    2012-02-01

    Although yet unobserved in the very rare 13C17O isotopologue, the Ångström system (B1Σ+-A1Π) was obtained under high resolution as an emission spectrum using a conventional spectroscopic technique. The emission from the discharge was observed with a plane grating spectrograph and recorded by a photomultiplier tube. In total, 192 transition wave numbers belonging to two bands (0-1 and 0-2) were precisely measured and rotationally analyzed. This method allowed us to determine the merged rotational constants B0 = 1.8131941(58) cm-1 and D0 = 5.5620(46) × 10-6 cm-1 and the individual molecular constant B1 = 1.471059(47) cm-1, D1 = 5.315(64) × 10-6 cm-1, B2 = 1.451762(13) cm-1, D2 = 7.812(16) × 10-6 cm-1 for the as yet unanalyzed 13C17O molecule B1Σ+ and A1Π states, respectively. The band origins σ of the Ångström system in the 13C17O molecule were also calculated. Numerous rotational perturbations observed in the A1Π state in 13C17O molecule have been identified and analyzed in detail. The suspected candidates responsible for these irregularities were indicated by means of a graph of the rovibronic levels of the neighboring states based on the estimated term value in the 13C17O molecule. The values of these perturbations have also been defined for both the e- and f-parity of the A1Π(v = 1 and 2) state. Simultaneously, the B1Σ+ state was observed to be quite regular up to the observed Jmax level.

  15. Constrained Allocation Flux Balance Analysis.

    PubMed

    Mori, Matteo; Hwa, Terence; Martin, Olivier C; De Martino, Andrea; Marinari, Enzo

    2016-06-01

    New experimental results on bacterial growth inspire a novel top-down approach to study cell metabolism, combining mass balance and proteomic constraints to extend and complement Flux Balance Analysis. We introduce here Constrained Allocation Flux Balance Analysis, CAFBA, in which the biosynthetic costs associated to growth are accounted for in an effective way through a single additional genome-wide constraint. Its roots lie in the experimentally observed pattern of proteome allocation for metabolic functions, allowing to bridge regulation and metabolism in a transparent way under the principle of growth-rate maximization. We provide a simple method to solve CAFBA efficiently and propose an "ensemble averaging" procedure to account for unknown protein costs. Applying this approach to modeling E. coli metabolism, we find that, as the growth rate increases, CAFBA solutions cross over from respiratory, growth-yield maximizing states (preferred at slow growth) to fermentative states with carbon overflow (preferred at fast growth). In addition, CAFBA allows for quantitatively accurate predictions on the rate of acetate excretion and growth yield based on only 3 parameters determined by empirical growth laws.

  16. Constrained Allocation Flux Balance Analysis.

    PubMed

    Mori, Matteo; Hwa, Terence; Martin, Olivier C; De Martino, Andrea; Marinari, Enzo

    2016-06-01

    New experimental results on bacterial growth inspire a novel top-down approach to study cell metabolism, combining mass balance and proteomic constraints to extend and complement Flux Balance Analysis. We introduce here Constrained Allocation Flux Balance Analysis, CAFBA, in which the biosynthetic costs associated to growth are accounted for in an effective way through a single additional genome-wide constraint. Its roots lie in the experimentally observed pattern of proteome allocation for metabolic functions, allowing to bridge regulation and metabolism in a transparent way under the principle of growth-rate maximization. We provide a simple method to solve CAFBA efficiently and propose an "ensemble averaging" procedure to account for unknown protein costs. Applying this approach to modeling E. coli metabolism, we find that, as the growth rate increases, CAFBA solutions cross over from respiratory, growth-yield maximizing states (preferred at slow growth) to fermentative states with carbon overflow (preferred at fast growth). In addition, CAFBA allows for quantitatively accurate predictions on the rate of acetate excretion and growth yield based on only 3 parameters determined by empirical growth laws. PMID:27355325

  17. Constrained Allocation Flux Balance Analysis

    PubMed Central

    Mori, Matteo; Hwa, Terence; Martin, Olivier C.

    2016-01-01

    New experimental results on bacterial growth inspire a novel top-down approach to study cell metabolism, combining mass balance and proteomic constraints to extend and complement Flux Balance Analysis. We introduce here Constrained Allocation Flux Balance Analysis, CAFBA, in which the biosynthetic costs associated to growth are accounted for in an effective way through a single additional genome-wide constraint. Its roots lie in the experimentally observed pattern of proteome allocation for metabolic functions, allowing to bridge regulation and metabolism in a transparent way under the principle of growth-rate maximization. We provide a simple method to solve CAFBA efficiently and propose an “ensemble averaging” procedure to account for unknown protein costs. Applying this approach to modeling E. coli metabolism, we find that, as the growth rate increases, CAFBA solutions cross over from respiratory, growth-yield maximizing states (preferred at slow growth) to fermentative states with carbon overflow (preferred at fast growth). In addition, CAFBA allows for quantitatively accurate predictions on the rate of acetate excretion and growth yield based on only 3 parameters determined by empirical growth laws. PMID:27355325

  18. 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...

  19. Localized in vivo13C NMR spectroscopy of the brain

    PubMed Central

    Gruetter, Rolf; Adriany, Gregor; Choi, In-Young; Henry, Pierre-Gilles; Lei, Hongxia; Öz, Gülin

    2006-01-01

    Localized 13C NMR spectroscopy provides a new investigative tool for studying cerebral metabolism. The application of 13C NMR spectroscopy to living intact humans and animals presents the investigator with a number of unique challenges. This review provides in the first part a tutorial insight into the ingredients required for achieving a successful implementation of localized 13C NMR spectroscopy. The difficulties in establishing 13C NMR are the need for decoupling of the one-bond 13C–1H heteronuclear J coupling, the large chemical shift range, the low sensitivity and the need for localization of the signals. The methodological consequences of these technical problems are discussed, particularly with respect to (a) RF front-end considerations, (b) localization methods, (c) the low sensitivity, and (d) quantification methods. Lastly, some achievements of in vivo localized 13C NMR spectroscopy of the brain are reviewed, such as: (a) the measurement of brain glutamine synthesis and the feasibility of quantifying glutamatergic action in the brain; (b) the demonstration of significant anaplerotic fluxes in the brain; (c) the demonstration of a highly regulated malate-aspartate shuttle in brain energy metabolism and isotope flux; (d) quantification of neuronal and glial energy metabolism; and (e) brain glycogen metabolism in hypoglycemia in rats and humans. We conclude that the unique and novel insights provided by 13C NMR spectroscopy have opened many new research areas that are likely to improve the understanding of brain carbohydrate metabolism in health and disease. PMID:14679498

  20. Short-term natural δ13C variations in pools and fluxes in a beech forest: the transfer of isotopic signal from recent photosynthates to soil respired CO2

    NASA Astrophysics Data System (ADS)

    Gavrichkova, O.; Proietti, S.; Moscatello, S.; Portarena, S.; Battistelli, A.; Matteucci, G.; Brugnoli, E.

    2011-03-01

    The fate of photosynthetic products within the plant-soil continuum determines how long the reduced carbon resides within the ecosystem and when it returns back to the atmosphere in the form of respiratory CO2. We have tested the possibility of measuring natural variation in δ13C to disentangle potential times needed to transfer carbohydrates produced by photosynthesis down to roots and, in general, to belowground up to its further release in the form of soil respiration into the atmosphere in a beech (Fagus sylvatica) forest. For these purposes we have measured the variation in stable carbon and oxygen isotope compositions in plant material and in soil respired CO2 every three hours for three consequent days. Possible steps and different signs of post-photosynthetic fractionation during carbon translocation were also identified. A 12 h-periodicity was observed for variation in δ13C in soluble sugars in the top crown leaves and it can be explained by starch day/night dynamics in synthesis and breakdown and by stomatal limitations under elevated vapour pressure deficits. Photosynthetic products were transported down the trunk and mixed with older carbon pools, therefore causing the dampening of the δ13C signal variation. The strongest periodicity of 24 h was found in δ13C in soil respiration indicating changes in root contribution to the total CO2 efflux. Nevertheless, it was possible to identify the speed of carbon translocation through the plant-soil continuum. A period of 24 h was needed to transfer the C assimilated by photosynthesis from the top crown leaves to the tree trunk at breast height and additional 3 h for further respiration of that C by roots and soil microorganisms and its to subsequent diffusion back to the atmosphere.

  1. High-resolution FTIR spectroscopic analysis of the ν11 and ν2 + ν7 bands of 13C2D4

    NASA Astrophysics Data System (ADS)

    Gabona, M. G.; Tan, T. L.

    2016-06-01

    The FTIR spectrum of the ν11 band of 13C2D4 was recorded at a resolution of 0.0063 cm-1 in the 2130-2250 cm-1 region. This band was perturbed by the unobserved ν2 + ν7 band. By fitting 862 infrared transitions for the ν11 band with a rms deviation of 0.0024 cm-1 using a Watson's A-reduced Hamiltonian in the Ir representation including a Coriolis coupling constant, the rovibrational constants for the ν11 = 1 state and three rotational constants for the ν2 = ν7 = 1 state of 13C2D4 were derived for the first time. The band centers of ν11 and ν2 + ν7 were determined to be 2193.75982(25) cm-1 and 2184.613(11) cm-1 respectively.

  2. Tequila authenticity assessment by headspace SPME-HRGC-IRMS analysis of 13C/12C and 18O/16O ratios of ethanol.

    PubMed

    Aguilar-Cisneros, Blanca O; López, Mercedes G; Richling, Elke; Heckel, Frank; Schreier, Peter

    2002-12-18

    By use of headspace SPME sampling and a PLOT column, on-line capillary gas chromatography-isotope ratio mass spectrometry was employed in the combustion (C) and the pyrolysis (P) modes (HRGC-C/P-IRMS) to determine the delta(13)C(VPDB) and delta(18)O(VSMOW) values of ethanol in authentic (n = 14) and commercial tequila samples (n = 15) as well as a number of other spirits (n = 23). Whereas with delta(13)C(VPDB) values ranging from -12.1 to -13.2 per thousand and from -12.5 to -14.8 per thousand similar variations were found for 100% agave and mixed tequilas, respectively, the delta(18)O(VSMOW) data differed slightly within these categories: ranges from +22.1 to +22.8 per thousand and +20.8 to +21.7 per thousand were determined for both the authentic 100% agave and mixed products, respectively. The data recorded for commercial tequilas were less homogeneous; delta(13)C(VPDB) data from -10.6 to -13.9 per thousand and delta(18)O(VSMOW) values from +15.5 to +22.7 per thousand were determined in tequilas of both categories. Owing to overlapping data, attempts to differentiate between white, rested, and aged tequilas within each of the two categories failed. In addition, discrimination of tequila samples from other spirits by means of delta(13)C(VPDB) and delta(18)O(VSMOW) data of ethanol was restricted to the products originating from C(3) as well as C(4)/CAM raw materials.

  3. Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis.

    PubMed

    Dauner, M; Bailey, J E; Sauer, U

    2001-09-01

    Fluxes in central carbon metabolism of a genetically engineered, riboflavin-producing Bacillus subtilis strain were investigated in glucose-limited chemostat cultures at low (0.11 h(-1)) and high (0.44 h(-1)) dilution rates. Using a mixture of 10% [U-(13)C] and 90% glucose labeled at natural abundance, (13)C-labeling experiments were carried out to provide additional information for metabolic flux balancing. The resulting labeling pattern in the proteinogenic amino acids were analyzed by two-dimensional [(13)C, (1)H] nuclear magnetic resonance (NMR) spectroscopy. To account rigorously for all available data from these experiments, we developed a comprehensive isotopomer model of B. subtilis central metabolism. Using this model, intracellular carbon net and exchange fluxes were estimated on the basis of validated physiological data and biomass composition in combination with 2D NMR data from 45 individual carbon atom spectra in the amino acids. Glucose catabolism proceeded primarily via glycolysis but pentose phosphate pathway fluxes increased with increasing growth rate. Moreover, significant back fluxes from the TCA cycle to the lower part of glycolysis via the gluconeogenic PEP carboxykinase were detected. The malic enzyme reaction, in contrast, was found to be inactive. A thorough statistical analysis was performed to prove the reliability of the isotopomer balance model and the obtained results. Specifically, a chi(2) test was applied to validate the model and the chi-square criterion was used to explore the sensitivity of model predictions to the experimental data.

  4. Carbon 13-Metabolic Flux Analysis derived constraint-based metabolic modelling of Clostridium acetobutylicum in stressed chemostat conditions.

    PubMed

    Wallenius, Janne; Maaheimo, Hannu; Eerikäinen, Tero

    2016-11-01

    The metabolism of butanol producing bacteria Clostridium acetobutylicum was studied in chemostat with glucose limited conditions, butanol stimulus, and as a reference cultivation. COnstraint-Based Reconstruction and Analysis (COBRA) was applied using additional constraints from (13)C Metabolic Flux Analysis ((13)C-MFA) and experimental measurement results. A model consisting of 451 metabolites and 604 reactions was utilized in flux balance analysis (FBA). The stringency of the flux spaces considering different optimization objectives, i.e. growth rate maximization, ATP maintenance, and NADH/NADPH formation, for flux variance analysis (FVA) was studied in the different modelled conditions. Also a previously uncharacterized exopolysaccharide (EPS) produced by C. acetobutylicum was characterized on monosaccharide level. The major monosaccharide components of the EPS were 40n-% rhamnose, 34n-% glucose, 13n-% mannose, 10n-% galactose, and 2n-% arabinose. The EPS was studied to have butanol adsorbing property, 70(butanol)mg(EPS)g(-1) at 37°C. PMID:27501035

  5. Short-term natural δ13C and δ18O variations in pools and fluxes in a beech forest: the transfer of isotopic signal from recent photosynthates to soil respired CO2

    NASA Astrophysics Data System (ADS)

    Gavrichkova, O.; Proietti, S.; Moscatello, S.; Portarena, S.; Battistelli, A.; Matteucci, G.; Brugnoli, E.

    2011-10-01

    The fate of photosynthetic products within the plant-soil continuum determines how long the reduced carbon resides within the ecosystem and when it returns back to the atmosphere in the form of respiratory CO2. We have tested the possibility of measuring natural variation in δ13C and δ18O to disentangle the potential times needed to transfer carbohydrates produced by photosynthesis down to trunk, roots and, in general, to belowground up to its further release in the form of soil respiration into the atmosphere in a beech (Fagus sylvatica) forest. We have measured the variation in stable carbon and oxygen isotope compositions in plant material and in soil respired CO2 every three hours for three consecutive days. Possible steps and different signs of post-photosynthetic fractionation during carbon translocation were also identified. A 12 h-periodicity was observed for variation in δ13C in soluble sugars in the top crown leaves and it can be explained by starch day/night dynamics in synthesis and breakdown and by stomatal limitations under elevated vapour pressure deficits. Photosynthetic products were transported down the trunk and mixed with older carbon pools, therefore causing the dampening of the δ13C signal variation. The strongest periodicity of 24 h was found in δ13C in soil respiration indicating changes in root contribution to the total CO2 efflux. Other non-biological causes like diffusion fractionation and advection induced by gas withdrawn from the measurement chamber complicate data interpretation on this step of C transfer path. Nevertheless, it was possible to identify the speed of carbohydrates' translocation from the point of assimilation to the trunk breast height because leaf-imprinted enrichment of δ18O in soluble sugars was less modified along the downward transport and was well related to environmental parameters potentially linked to stomatal conductance. The speed of carbohydrates translocation from the site of assimilation to the trunk

  6. 1H and 13C NMR Chemical Shift Assignments and Conformational Analysis for the Two Diastereomers of the Vitamin K Epoxide Reductase Inhibitor Brodifacoum

    SciTech Connect

    Cort, John R.; Cho, Herman M.

    2009-10-01

    Proton and 13C NMR chemical shift assignments and 1H-1H scalar couplings for the two diastereomers of the vitamin K epoxide reductase (VKOR) inhibitor brodifacoum have been determined from acetone solutions containing both diastereomers. Data were obtained from homo- and heteronuclear correlation spectra acquired at 1H frequencies of 750 and 900 MHz over a 268-303 K temperature range. Conformations inferred from scalar coupling and 1-D NOE measurements exhibit large differences between the diastereomers. Pacific Northwest National Laboratory is operated by Battelle for the US Department of Energy.

  7. Mechanism of Au(III) reduction by chitosan: comprehensive study with 13C and 1H NMR analysis of chitosan degradation products.

    PubMed

    Pestov, Alexander; Nazirov, Alexander; Modin, Evgeny; Mironenko, Alexander; Bratskaya, Svetlana

    2015-03-01

    The mechanism of Au(III) reduction by chitosan has been proposed on the basis of comprehensive study of kinetics of Au(III) reduction and chitosan chain degradation using UV-vis spectroscopy and viscosimetry, and identification of reaction products using colloid titration and (13)C, (1)H NMR spectroscopy. We have shown that formation of gold nanoparticles in H[AuCl4]/chitosan solutions starts with hydrolysis of chitosan catalyzed by Au(III). The products of chitosan hydrolysis rather than chitosan itself act as the main reducing species. According to (13)C and (1)H NMR spectroscopy data, chitosan/Au(0) composites contain chitosan with reduced molecular weight and acetylation degree, whereas water-soluble by-products consist of chitosan oligomers with higher acetylation degree, derivatives of glucosamine acids, and formate ion. Chitosan degradation has significantly contributed to the decrease of its efficiency as a gold nanoparticles stabilizer. The gold particle size increased from 6.9 nm to 16.2 nm, when Au(III)/chitosan molar ratio changed from 1:80 to 1:10.

  8. Spatial distribution of PAH concentrations and stable isotope signatures (δ13C, δ15N) in mosses from three European areas--characterization by multivariate analysis.

    PubMed

    Foan, L; Leblond, S; Thöni, L; Raynaud, C; Santamaría, J M; Sebilo, M; Simon, V

    2014-01-01

    Polycyclic aromatic hydrocarbon (PAH) concentrations and N, C stable isotope signatures were determined in mosses Hypnum cupressiforme Hedw. from 61 sites of 3 European regions: Île-de-France (France); Navarra (Spain); the Swiss Plateau and Basel area (Switzerland). Total PAH concentrations of 100-700 ng g(-1), as well as δ(13)C values of -32 to -29‰ and δ(15)N values of -11 to -3‰ were measured. Pearson correlation tests revealed opposite trends between high molecular weight PAH (4-6 aromatic rings) content and δ(13)C values. Partial Least Square regressions explained the very significant correlations (r > 0.91, p < 0.001) between high molecular weight PAH concentrations by local urban land use (<10 km) and environmental factors such as elevation and pluviometry. Finally, specific correlations between heavy metal and PAH concentrations were attributed to industrial emissions in Switzerland and road traffic emissions in Spain.

  9. Using Headspace Equilibration to Measure the d13C of Soil-Respired CO2

    NASA Astrophysics Data System (ADS)

    Robertson, M. A.; Powers, E.; Marshall, J.

    2007-12-01

    Soil respiration is an important component of the global carbon cycle and can account for as much as 70% of ecosystem respiration. Soil gas flux measurements have been combined with stable isotope analysis to examine ecosystem properties and processes such as water-use efficiency and the role of above ground weather in controlling soil respiration. However, current methods of measuring the δ13C of soil-respired CO2 are either inherently inaccurate or time-consuming and tedious. An alternative method of obtaining this value offers a potential solution to these problems. In this method, plastic chambers are fitted with rubber septa to allow for sample collection, then inverted and partially buried in soil. The chamber headspace is allowed to come to equilibrium with soil air. In this study we tested the viability of this method by examining whether frequent resampling of respiration chambers affected δ13C measurements, whether headspace CO2 concentration and δ13C values approached equilibrium asymptotically, and whether simulated and actual diel temperature cycles affected estimates of δ13C. All experiments were conducted on respiration chambers inverted in potting soil and placed in a Conviron growth chamber, with the exception of one field test that was conducted on respiration chambers installed in a Northern Idaho experimental forest. Samples were collected with a syringe and stored in glass vials for analysis by a ratioing mass spectrometer. We found that resampling respiration chambers as frequently as every 10 minutes had no significant effect on final δ13C values, that both chamber CO2 concentrations and δ13C values exhibited an asymptotic approach to equilibrium, and that the equilibrium value was offset from the initial flux by the amount we expected, approximately 4 ‰. However, we also found that diel temperature variation affected both headspace CO2 concentration and δ13C in the lab and in the field. We concluded that if this method is used in

  10. Flux and reflux: metabolite reflux in plant suspension cells and its implications for isotope-assisted metabolic flux analysis.

    PubMed

    Nargund, Shilpa; Misra, Ashish; Zhang, Xiaofeng; Coleman, Gary D; Sriram, Ganesh

    2014-06-01

    Isotope-assisted metabolic flux analysis (MFA) is a powerful methodology to quantify intracellular fluxes via isotope labeling experiments (ILEs). In batch cultures, which are often convenient, inexpensive or inevitable especially for eukaryotic systems, MFA is complicated by the presence of the initially present biomass. This unlabeled biomass may either mix with the newly synthesized labeled biomass or reflux into the metabolic network, thus masking the true labeling patterns in the newly synthesized biomass. Here, we report a detailed investigation of such metabolite reflux in cell suspensions of the tree poplar. In ILEs supplying 28% or 98% U-(13)C glucose as the sole organic carbon source, biomass components exhibited lower (13)C enrichments than the supplied glucose as well as anomalous isotopomers not explainable by simple mixing of the initial and newly synthesized biomass. These anomalous labeling patterns were most prominent in a 98% U-(13)C glucose ILE. By comparing the performance of light- and dark-grown cells as well as by analyzing the isotope labeling patterns in aspartic and glutamic acids, we eliminated photosynthetic or anaplerotic fixation of extracellular (12)CO2 as explanations for the anomalous labeling patterns. We further investigated four different metabolic models for interpreting the labeling patterns and evaluating fluxes: (i) a carbon source (glucose) dilution model, (ii) an isotopomer correction model with uniform dilution for all amino acids, (iii) an isotopomer correction model with variable dilution for different amino acids, and (iv) a comprehensive metabolite reflux model. Of these, the metabolite reflux model provided a substantially better fit for the observed labeling patterns (sum of squared residues: 538) than the other three models whose sum of squared residues were (i) 4626, (ii) 4983, and (iii) 1748, respectively. We compared fluxes determined using the metabolite reflux model to those determined using an independent

  11. 2H NMR and 13C-IRMS analyses of acetic acid from vinegar, 18O-IRMS analysis of water in vinegar: international collaborative study report.

    PubMed

    Thomas, Freddy; Jamin, Eric

    2009-09-01

    An international collaborative study of isotopic methods applied to control the authenticity of vinegar was organized in order to support the recognition of these procedures as official methods. The determination of the 2H/1H ratio of the methyl site of acetic acid by SNIF-NMR (site-specific natural isotopic fractionation-nuclear magnetic resonance) and the determination of the 13C/12C ratio, by IRMS (isotope ratio mass spectrometry) provide complementary information to characterize the botanical origin of acetic acid and to detect adulterations of vinegar using synthetic acetic acid. Both methods use the same initial steps to recover pure acetic acid from vinegar. In the case of wine vinegar, the determination of the 18O/16O ratio of water by IRMS allows to differentiate wine vinegar from vinegars made from dried grapes. The same set of vinegar samples was used to validate these three determinations. The precision parameters of the method for measuring delta13C (carbon isotopic deviation) were found to be similar to the values previously obtained for similar methods applied to wine ethanol or sugars extracted from fruit juices: the average repeatability (r) was 0.45 per thousand, and the average reproducibility (R) was 0.91 per thousand. As expected from previous in-house study of the uncertainties, the precision parameters of the method for measuring the 2H/1H ratio of the methyl site were found to be slightly higher than the values previously obtained for similar methods applied to wine ethanol or fermentation ethanol in fruit juices: the average repeatability was 1.34 ppm, and the average reproducibility was 1.62 ppm. This precision is still significantly smaller than the differences between various acetic acid sources (delta13C and delta18O) and allows a satisfactory discrimination of vinegar types. The precision parameters of the method for measuring delta18O were found to be similar to the values previously obtained for other methods applied to wine and

  12. Kinetic analysis of reactions of Si-based epoxy resins by near-infrared spectroscopy, 13C NMR and soft-hard modelling.

    PubMed

    Garrido, Mariano; Larrechi, Maria Soledad; Rius, F Xavier; Mercado, Luis Adolfo; Galià, Marina

    2007-02-01

    Soft- and hard-modelling strategy was applied to near-infrared spectroscopy data obtained from monitoring the reaction between glycidyloxydimethylphenyl silane, a silicon-based epoxy monomer, and aniline. On the basis of the pure soft-modelling approach and previous chemical knowledge, a kinetic model for the reaction was proposed. Then, multivariate curve resolution-alternating least squares optimization was carried out under a hard constraint, that compels the concentration profiles to fulfil the proposed kinetic model at each iteration of the optimization process. In this way, the concentration profiles of each species and the corresponding kinetic rate constants of the reaction, unpublished until now, were obtained. The results obtained were contrasted with 13C NMR. The joint interval test of slope and intercept for detecting bias was not significant (alpha=5%).

  13. Synthesis of Isotopically Labeled (13)C3-Simazine and Development of a Simultaneous UPLC-MS/MS Method for the Analysis of Simazine in Soil.

    PubMed

    Song, Yan; Guo, Yangzhen; Zhang, Xia; Yang, Yue; Chen, Shuo; She, Gaimei; She, Dongmei

    2016-01-14

    The isotope dilution mass spectrometry (IDMS) is a highly efficient method for tackling the ion suppression in complex matrix by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), but a lack of commercial internal standards is a limiting factor for these analyses. Herein, an economical and efficient strategy for the synthesis of (13)C3-simazine via a three-step procedure was developed. The isotope-labeled internal standard was used for determination of simazine residue in soil samples. The quantitation method has a limit of detection of 0.015 μg/kg and quantitation of 0.08 μg/kg. The inter-day and intra-day precision of the method were below 4.6%. Recovery values were ranged between 92.9% and 99.2%. All the samples obtained from six provinces in China contained from 1 to 62 μg/kg of simazine.

  14. 13C-1H dipolar-driven 13C-13C recoupling without 13C rf irradiation in nuclear magnetic resonance of rotating solids

    NASA Astrophysics Data System (ADS)

    Takegoshi, K.; Nakamura, Shinji; Terao, Takehiko

    2003-02-01

    Two recently proposed 13C-13C recoupling methods under magic angle spinning (MAS), resonant interference recoupling (RIR), and 13C-1H dipolar-assisted rotational resonance (DARR), are examined on a common theoretical foundation using the average Hamiltonian theory. In both methods, a rf field is applied on not 13C but 1H to recouple the 13C-1H dipolar interactions, and spectral overlap necessary to conserve energy for 13C-13C polarization transfer is achieved by the 13C-1H dipolar line broadening. While DARR employs time-independent 13C-1H interactions recoupled by suitable rf irradiation to 1H spins, RIR uses time-dependent 13C-1H interactions modulated appropriately by 1H rf irradiation. There are two distinct cases where 13C-1H line broadening realizes 13C-13C spectral overlap. For a pair of a carbonyl or aromatic carbon and an aliphatic carbon, spectral overlap can be achieved between one of the spinning sidebands of the former 13C resonance and the 13C-1H dipolar powder pattern of the latter. On the other hand for a pair of spins with a small chemical shift difference, the two center bands are overlapped with each other due to 13C-1H dipolar broadening. For the former, we show that both RIR and DARR occur in the first order, while for the latter, DARR recoupling is appreciable for time-independent 13C-1H interactions. We refer to the former DARR as the first-order DARR recoupling and the latter as the second-order DARR. Experimentally, we examined the following 13C-1H recoupling methods for DARR: 1H CW irradiation fulfilling a rotary-resonance condition or a modulatory-resonance condition, and 1H π pulses applied synchronously to MAS. For RIR, the FSLG-m2m¯m sequence is applied to 1H. Several one-dimensional DARR and RIR experiments were done for N-acetyl[1,2-13C, 15N] DL-valine, and [2,3-13C] L-alanine. It was found that the polarization transfer rate for RIR is larger than that for DARR except for fast spinning, while the rate for DARR is less sensitive to

  15. Rapid analysis of 13C in plant-wax n-alkanes for reconstruction of terrestrial vegetation signals from aquatic sediments

    NASA Astrophysics Data System (ADS)

    McDuffee, Kelsey E.; Eglinton, Timothy I.; Sessions, Alex L.; Sylva, Sean; Wagner, Thomas; Hayes, John M.

    2004-10-01

    Long-chain, odd-carbon-numbered C25 to C35n-alkanes are characteristic components of epicuticular waxes produced by terrestrial higher plants. They are delivered to aquatic systems via eolian and fluvial transport and are preserved in underlying sediments. The isotopic compositions of these products can serve as records of past vegetation. We have developed a rapid method for stable carbon isotopic analyses of total plant-wax n-alkanes using a novel, moving-wire system coupled to an isotope-ratio mass spectrometer (MW-irMS). The n-alkane fractions are prepared from sediment samples by (1) saponification and extraction with organic solvents, (2) chromatographic separation using silica gel, (3) isolation of straight-chain carbon skeletons using a zeolite molecular sieve, and (4) oxidation and removal of unsaturated hydrocarbons with RuO4. Short-chain n-alkanes of nonvascular plant origin (13C values obtained by MW-irMS did not differ significantly from weighted averages of individual n-alkane δ13C values obtained by irmGC-MS. Isotopic variations in compound-class n-alkane fractions from a latitudinal transect of core-top sediments from the Southwest African margin (3°N-28°S) were congruent with those measured by compound-specific isotopic analyses of plant-wax n-alkanes. The amplitude of the variations was smaller, indicating contributions from non-plant-wax hydrocarbons, but the measurements revealed variations in carbon isotopic composition that are consistent with vegetation zones on the adjacent continent.

  16. Rapid analysis of 13C in plant-wax n-alkanes for reconstruction of terrestrial vegetation signals from aquatic sediments

    NASA Astrophysics Data System (ADS)

    McDuffee, Kelsey E.; Eglinton, Timothy I.; Sessions, Alex L.; Sylva, Sean; Wagner, Thomas; Hayes, John M.

    2004-10-01

    Long-chain, odd-carbon-numbered C25 to C35 n-alkanes are characteristic components of epicuticular waxes produced by terrestrial higher plants. They are delivered to aquatic systems via eolian and fluvial transport and are preserved in underlying sediments. The isotopic compositions of these products can serve as records of past vegetation. We have developed a rapid method for stable carbon isotopic analyses of total plant-wax n-alkanes using a novel, moving-wire system coupled to an isotope-ratio mass spectrometer (MW-irMS). The n-alkane fractions are prepared from sediment samples by (1) saponification and extraction with organic solvents, (2) chromatographic separation using silica gel, (3) isolation of straight-chain carbon skeletons using a zeolite molecular sieve, and (4) oxidation and removal of unsaturated hydrocarbons with RuO4. Short-chain n-alkanes of nonvascular plant origin (13C values obtained by MW-irMS did not differ significantly from weighted averages of individual n-alkane δ13C values obtained by irmGC-MS. Isotopic variations in compound-class n-alkane fractions from a latitudinal transect of core-top sediments from the Southwest African margin (3°N-28°S) were congruent with those measured by compound-specific isotopic analyses of plant-wax n-alkanes. The amplitude of the variations was smaller, indicating contributions from non-plant-wax hydrocarbons, but the measurements revealed variations in carbon isotopic composition that are consistent with vegetation zones on the adjacent continent.

  17. 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.

  18. 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

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

    PubMed

    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) (13)C 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-(13)C]pyruvate and [1-(13)C]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 [(13)C]bicarbonate (-48%), [1-(13)C]acetylcarnitine (+113%), and [5-(13)C]glutamate (-63%), following fasting. Butyrate uptake was unaffected by fasting, as indicated by [1-(13)C]butyrylcarnitine. Mitochondrial pseudoketogenesis facilitated the labeling of the ketone bodies [1-(13)C]acetoacetate and [1-(13)C]β-hydroxybutyryate, without evidence of true ketogenesis. HP [1-(13)C]acetoacetate was increased in fasting (250%) but decreased during pyruvate co-injection (-82%). Combining HP (13)C 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

  20. 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.

  1. Biosynthetic uniform 13C,15N-labelling of zervamicin IIB. Complete 13C and 15N NMR assignment.

    PubMed

    Ovchinnikova, Tatyana V; Shenkarev, Zakhar O; Yakimenko, Zoya A; Svishcheva, Natalia V; Tagaev, Andrey A; Skladnev, Dmitry A; Arseniev, Alexander S

    2003-01-01

    Zervamicin IIB is a member of the alpha-aminoisobutyric acid containing peptaibol antibiotics. A new procedure for the biosynthetic preparation of the uniformly 13C- and 15N-enriched peptaibol is described This compound was isolated from the biomass of the fungus-producer Emericellopsis salmosynnemata strain 336 IMI 58330 obtained upon cultivation in the totally 13C, 15N-labelled complete medium. To prepare such a medium the autolysed biomass and the exopolysaccharides of the obligate methylotrophic bacterium Methylobacillus flagellatus KT were used. This microorganism was grown in totally 13C, 15N-labelled minimal medium containing 13C-methanol and 15N-ammonium chloride as the only carbon and nitrogen sources. Preliminary NMR spectroscopic analysis indicated a high extent of isotope incorporation (> 90%) and led to the complete 13C- and 15N-NMR assignment including the stereospecific assignment of Aib residues methyl groups. The observed pattern of the structurally important secondary chemical shifts of 1H(alpha), 13C=O and 13C(alpha) agrees well with the previously determined structure of zervamicin IIB in methanol solution. PMID:14658801

  2. 1H, 13C and 15N resonance assignments and secondary structure analysis of CmPI-II, a serine protease inhibitor isolated from marine snail Cenchritis muricatus.

    PubMed

    Cabrera-Muñoz, Aymara; Rojas, Laritza; Alonso-del-Rivero Antigua, Maday; Pires, José Ricardo

    2016-04-01

    A protease inhibitor (CmPI-II) (UNIPROT: IPK2_CENMR) from the marine mollusc Cenchritis muricatus, has been isolated and characterized. It is the first member of a new group (group 3) of non-classical Kazal-type inhibitors. CmPI-II is a tight-binding inhibitor of serine proteases: trypsin, human neutrophil elastase (HNE), subtilisin A and pancreatic elastase. This specificity is exceptional in the members of Kazal-type inhibitor family. Several models of three-dimensional structure of CmPI-II have been constructed by homology with other inhibitors of the family but its structure has not yet been solved experimentally. Here we report the (1)H, (15)N and (13)C chemical shift assignments of CmPI-II as basis for NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data have identified three β-strands β1: residues 14-19, β2: 23-35 and β3: 43-45 and one helix α1: 28-37 arranged in the sequential order β1-β2-α1-β3. These secondary structure elements suggest that CmPI-II adopts the typical scaffold of a Kazal-type inhibitor. PMID:26547437

  3. Precise ro-vibrational analysis of molecular bands forbidden in absorption: The ν8 +ν10 band of 13C2H4

    NASA Astrophysics Data System (ADS)

    Ulenikov, O. N.; Gromova, O. V.; Bekhtereva, E. S.; Kashirina, N. V.; Maul, C.; Bauerecker, S.

    2015-10-01

    The high resolution spectra of the 13C2H4 molecule was recorded with a Bruker IFS 120 Fourier transform spectrometer and theoretically analyzed in the 1650 - 1800cm-1 region of the ν8 +ν10 band which is forbidden in absorption. About 1200 experimental transitions with the maximum values of quantum numbers Jmax. = 34 and Kamax. = 17 were assigned to the ν8 +ν10 band. On that basis the 516 high accuracy ro-vibrational energies of the (v8=v10=1) vibrational state, as well as energy levels with J ≤ 2 of the (v4 =v8 = 1) and (v7 =v8 = 1) vibrational states, were determined which then were used as input data in the weighted fit of spectroscopic parameters of the Hamiltonian (strong local resonance interactions of the ν8 +ν10 band with the bands ν4 +ν8 and ν7 +ν8 have been taken into account). A set of 34 vibrational, rotational, centrifugal distortion, and resonance interaction parameters was obtained from the fit. These parameters reproduce positions of about 1200 experimentally recorded and assigned transitions with the rms error drms = 0.00018cm-1 (blended and very weak transitions are not taken into account in that case).

  4. Stability and biodegradability of organic matter from Arctic soils of Western Siberia: Insights from 13C-NMR spectroscopy and elemental analysis

    NASA Astrophysics Data System (ADS)

    Ejarque, Elisabet; Abakumov, Evgeny

    2016-04-01

    Arctic soils contain large amounts of organic matter which, globally, exceed the amount of carbon stored in vegetation biomass and in the atmosphere. Recent studies emphasize the potential sensitivity for this soil organic matter (SOM) to be mineralised when faced with increasing ambient temperatures. In order to better refine the predictions about the response of SOM to climate warming, there is a need to increase the spatial coverage of empirical data on SOM quantity and quality in the Arctic area. This study provides, for the first time, a characterisation of SOM from the Gydan Peninsula in the Yamal Region, Western Siberia, Russia. On the one hand, soil humic acids and their humification state were characterised by measuring the elemental composition and diversity of functional groups using solid-state 13C-NMR spectroscopy. Also, the total mineralisable carbon was measured. Our results show that there is a predominance of aliphatic carbon structures, with a distribution of functional groups that has a minimal variation both regionally and within soil depth. Such vertical homogeneity and low level of aromaticity reflects the accumulation in soil of lowly decomposed organic matter due to cold temperatures. Mineralisation rates were found to be independent of SOM quality, and to be mainly explained solely by the total carbon content. Overall, our results provide further evidence on the sensitivity that the soils of Western Siberia may have to increasing ambient temperatures and highlight the important role that this region can play in the global carbon balance under the effects of climate warming.

  5. Solid-state and solution /sup 13/C NMR in the conformational analysis of methadone-hydrochloride and related narcotic analgesics

    SciTech Connect

    Sumner, S.C.J.

    1986-01-01

    Solid state and solution /sup 13/C NMR have been used to study the conformations of the racemic mixtures and single enantiomers of methadone hydrochloride, alpha and beta methadol hydrochloride, and alpha and beta acetylmethadol hydrochloride. The NMR spectra acquired for the compounds as solids, and in polar and nonpolar solvents are compared, in order to determine the conformation of the molecules in solution. To determine the reliability of assigning solution conformations by comparing solution and solid state chemical shift data, three bond coupling constants measured in solution are compared with those calculated from X-ray data. The conformations of the racemic mixture and plus enantiomer of methadone hydrochloride have been shown to be very similar in the solid state, where minor differences in conformation can be seen by comparing NMR spectra obtained for the solids. Also shown is that the molecules of methadone hydrochloride have conformations in polar and in nonpolar solvents which are very similar to the conformation of the molecules in the solid state.

  6. Analysis of mercerization process based on the intensity change of deconvoluted resonances of (13)C CP/MAS NMR: Cellulose mercerized under cooling and non-cooling conditions.

    PubMed

    Miura, Kento; Nakano, Takato

    2015-08-01

    The area intensity change of C1, C4, and C6 in spectrum obtained by (13)C CP/MAS NMR and the mutual relationship between their changes were examined for cellulose samples treated with various concentrations of aqueous NaOH solutions under non-cooling and cooling conditions. The area intensity of C1-up and C6-down changed cooperatively with that of C4-down which corresponds to the crystallinity of samples: "-up" and "-down" are the up- and down- field component in a splitting peak of NMR spectrum, respectively. The intensity change of C1-up starts to decrease with decreasing in that of C4-down after that of C6-down is almost complete. These changes were more clearly observed for samples treated under cooling condition. It can be suggested that their characteristic change relates closely to the change in conformation of cellulose chains by induced decrystallization and the subsequent crystallization of cellulose II, and presumed that their changes at microscopic level relate to the macroscopic morphological changes such as contraction along the length of cellulose chains and recovery along the length. PMID:26042706

  7. INCA: a computational platform for isotopically non-stationary metabolic flux analysis

    PubMed Central

    2014-01-01

    Summary: 13C flux analysis studies have become an essential component of metabolic engineering research. The scope of these studies has gradually expanded to include both isotopically steady-state and transient labeling experiments, the latter of which are uniquely applicable to photosynthetic organisms and slow-to-label mammalian cell cultures. Isotopomer network compartmental analysis (INCA) is the first publicly available software package that can perform both steady-state metabolic flux analysis and isotopically non-stationary metabolic flux analysis. The software provides a framework for comprehensive analysis of metabolic networks using mass balances and elementary metabolite unit balances. The generation of balance equations and their computational solution is completely automated and can be performed on networks of arbitrary complexity. Availability and implementation: MATLAB p-code files are freely available for non-commercial use and can be downloaded at http://mfa.vueinnovations.com. Commercial licenses are also available. Contact: j.d.young@vanderbilt.edu PMID:24413674

  8. ¹³C-based metabolic flux analysis in yeast: the Pichia pastoris case.

    PubMed

    Ferrer, Pau; Albiol, Joan

    2014-01-01

    Metabolic flux analysis based on tracing patterns of stable isotopes, particularly (13)C, comprises a set of methodologies to experimentally quantify intracellular biochemical reaction rates, i.e., to measure carbon flux distributions through a metabolic network. This allows quantifying the response of a metabolic network to an environmental or genetic perturbation (i.e., the metabolic phenotype). Here, we describe a protocol based on growing yeast on a (13)C-labelled substrate and subsequent NMR detection of (13)C-patterns in proteinogenic amino acids. To calculate metabolic fluxes, we describe two complementary mathematical approaches using available software; namely, an approach based on the estimation of local ratios in network nodes, and a method based on a global iterative fitting approach. Furthermore, we consider specificities of these protocols for their application to the yeast Pichia pastoris growing on multicarbon substrates other than glucose (glycerol), as well as the case when methanol is used as co-substrate in combination with glucose or glycerol.

  9. Analyzing sites of OH radical attack (ring vs. side chain) in oxidation of substituted benzenes via dual stable isotope analysis (δ(13)C and δ(2)H).

    PubMed

    Zhang, Ning; Geronimo, Inacrist; Paneth, Piotr; Schindelka, Janine; Schaefer, Thomas; Herrmann, Hartmut; Vogt, Carsten; Richnow, Hans H

    2016-01-15

    OH radicals generated by the photolysis of H2O2 can degrade aromatic contaminants by either attacking the aromatic ring to form phenolic products or oxidizing the substituent. We characterized these competing pathways by analyzing the carbon and hydrogen isotope fractionation (εC and εH) of various substituted benzenes. For benzene and halobenzenes that only undergo ring addition, low values of εC (-0.7‰ to -1.0‰) were observed compared with theoretical values (-7.2‰ to -8‰), possibly owing to masking effect caused by pre-equilibrium between the substrate and OH radical preceding the rate-limiting step. In contrast, the addition of OH radicals to nitrobenzene ring showed a higher εC (-3.9‰), probably due to the lower reactivity. Xylene isomers, anisole, aniline, N,N-dimethylaniline, and benzonitrile yielded normal εH values (-2.8‰ to -29‰) indicating the occurrence of side-chain reactions, in contrast to the inverse εH (11.7‰ to 30‰) observed for ring addition due to an sp(2) to sp(3) hybridization change at the reacting carbon. Inverse εH values for toluene (14‰) and ethylbenzene (30‰) were observed despite the formation of side-chain oxidation products, suggesting that ring addition has a larger contribution to isotope fractionation. Dual element isotope slopes (∆δ(2)H/∆δ(13)C) therefore allow identification of significant degradation pathways of aromatic compounds by photochemically induced OH radicals. Issues that should be addressed in future studies include quantitative determination of the contribution of each competing pathway to the observed isotope fractionation and characterization of physical processes preceding the reaction that could affect isotope fractionation.

  10. 1H, 13C and 15N resonance assignments and secondary structure analysis of translation initiation factor 1 from Pseudomonas aeruginosa

    PubMed Central

    Bernal, Alejandra; Hu, Yanmei; Palmer, Stephanie O.; Silva, Aaron; Bullard, James; Zhang, Yonghong

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of infection in humans. P. aeruginosa can acquire resistance against multiple groups of antimicrobial agents, including β-lactams, aminoglycosides and fluoroquinolones, and multidrug resistance is increasing in this organism which makes treatment of the infections difficult and expensive. This has led to the unmet need for discovery of new compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Translation initiation factor 1 from P. aeruginosa (Pa-IF1) is the smallest of the three initiation factors that acts to establish the 30S initiation complex to initiate translation during protein biosynthesis, and its structure is unknown. Here we report the 1H, 13C and 15N chemical shift assignments of Pa-IF1 as the basis for NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data have identified five β-strands with an unusually extended β-strand at the C-terminal end of the protein and one short α-helix arranged in the sequential order β1–β2–β3–α1–β4–β5. This is further supported by 15N–{1H} hetero NOEs. These secondary structure elements suggest the Pa-IF1 adopts the typical β-barrel structure and is composed of an oligomer-binding motif. PMID:26983940

  11. 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.

  12. 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%.

  13. 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

  14. In vivo13C spectroscopy in the rat brain using hyperpolarized [1- 13C]pyruvate and [2- 13C]pyruvate

    NASA Astrophysics Data System (ADS)

    Marjańska, Małgorzata; Iltis, Isabelle; Shestov, Alexander A.; Deelchand, Dinesh K.; Nelson, Christopher; Uğurbil, Kâmil; Henry, Pierre-Gilles

    2010-10-01

    The low sensitivity of 13C spectroscopy can be enhanced using dynamic nuclear polarization. Detection of hyperpolarized [1- 13C]pyruvate and its metabolic products has been reported in kidney, liver, and muscle. In this work, the feasibility of measuring 13C signals of hyperpolarized 13C metabolic products in the rat brain in vivo following the injection of hyperpolarized [1- 13C]pyruvate and [2- 13C]pyruvate is investigated. Injection of [2- 13C]pyruvate led to the detection of [2- 13C]lactate, but no other downstream metabolites such as TCA cycle intermediates were detected. Injection of [1- 13C]pyruvate enabled the detection of both [1- 13C]lactate and [ 13C]bicarbonate. A metabolic model was used to fit the hyperpolarized 13C time courses obtained during infusion of [1- 13C]pyruvate and to determine the values of VPDH and VLDH.

  15. Determination of de novo synthesized amino acids in cellular proteins revisited by 13C NMR spectroscopy.

    PubMed

    Flögel, U; Willker, W; Leibfritz, D

    1997-04-01

    13C nuclear magnetic resonance spectroscopy was used to determine the absolute amounts to de novo synthesized amino acids in both the perchloric acid extracts and the hydrolyzed protein fractions of F98 glioma cells incubated for 2 h with 5 mmol/l [U-13C]glucose. 13C NMR spectra of the hydrolyzed protein fraction revealed a marked incorporation of 13C-labelled alanine, aspartate and glutamate into the proteins of F98 cells within the incubation period. Additionally, small amounts of 13C-labelled glycine, proline and serine could unambiguously be identified in the protein fraction. Astonishingly, approximately equal amounts of 13C-labelled glutamate and aspartate were incorporated into the cellular proteins, although the cytosolic steady-state concentration of aspartate was below 13C NMR detectability. Hypertonic stress decreased the incorporation of 13C-labelled amino acids into the total protein, albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. On the other hand, hypotonic stress increased the amount of 13C-labelled proline incorporated into the cellular proteins even though the cytosolic concentration of 13C-labelled proline was largely decreased. Apparently, hypoosmotic conditions stimulate the synthesis of proteins or peptides with a high proline content. The results show that already after 2 h of incubation with [U-13C]glucose there is a pronounced flux of 13C label into the cellular proteins, which is usually disregarded if cytosolic fluids are examined only. This means that calculations of metabolic fluxes based on 13C NMR spectroscopic data obtained from perchloric acid extracts of cells or tissues and also from in vivo measurements consider only the labelled 'NMR visible' cytosolic metabolites, which may have to be corrected for fast label flowing off into other compartments.

  16. Quantum mechanical study of the structure and spectroscopic (FT-IR, FT-Raman, 13C, 1H and UV), NBO and HOMO-LUMO analysis of 2-quinoxaline carboxylic acid

    NASA Astrophysics Data System (ADS)

    Prabavathi, N.; Nilufer, A.; Krishnakumar, V.

    The FTIR and FT-Raman spectra of 2-quinoxaline carboxylic acid (2-QCA) has been recorded in the region 4000-450 and 4000-100 cm-1, respectively. The conformational analysis, optimized geometry, frequency and intensity of the vibrational bands of 2-QCA were obtained by the density functional theory (DFT) with complete relaxation in the potential energy surface using 6-31G(d) basis set. The harmonic vibrational frequencies were calculated and the scaled values have been compared with experimental FTIR and FT-Raman spectra. The observed and the calculated frequencies are found to be in good agreement. The 1H and13C NMR spectra have been recorded and 1H and 13C nuclear magnetic resonance chemical shifts of the molecule were also calculated using the gauge independent atomic orbital (GIAO) method and their respective linear correlations were obtained. The theoretical UV-visible spectrum of the compound using CIS method and the electronic properties, such as HOMO and LUMO energies, were performed by time-dependent DFT (TD-DFT) approach. The calculated HOMO and LUMO energies show that charge transfer occurs within molecule. The Mulliken charges, the values of electric dipole moment (μ) of the molecule were computed using DFT calculations. The change in electron density (ED) in the σ* antibonding orbitals and stabilization energies E(2) have been calculated by natural bond (NBO) analysis to give clear evidence of stabilization originating in the hyper conjugation of hydrogen-bonded interactions.

  17. Measurement of Small One-Bond Proton-Carbon Residual Dipolar Coupling Constants in Partially Oriented 13C Natural Abundance Oligosaccharide Samples: Analysis of Heteronuclear 1JCH-Modulated Spectra with the BIRD Inversion Pulse

    NASA Astrophysics Data System (ADS)

    Pham, Tran Nghia; Liptaj, Tibor; Bromek, Krystyna; Uhrín, Dušan

    2002-08-01

    Two 2D J-modulated HSQC-based experiments were designed for precise determination of small residual dipolar one-bond carbon-proton coupling constants in 13C natural abundance carbohydrates. Crucial to the precision of a few hundredths of Hz achieved by these methods was the use of long modulation intervals and BIRD pulses, which acted as semiselective inversion pulses. The BIRD pulses eliminated effective evolution of all but 1JCH couplings, resulting in signal modulation that can be described by simple modulation functions. A thorough analysis of such modulation functions for a typical four-spin carbohydrate spin system was performed for both experiments. The results showed that the evolution of the 1H- 1H and long-range 1H- 13C couplings during the BIRD pulses did not necessitate the introduction of more complicated modulation functions. The effects of pulse imperfections were also inspected. While weakly coupled spin systems can be analyzed by simple fitting of cross peak intensities, in strongly coupled spin systems the evolution of the density matrix needs to be considered in order to analyse data accurately. However, if strong coupling effects are modest the errors in coupling constants determined by the "weak coupling" analysis are of similar magnitudes in oriented and isotropic samples and are partially cancelled during dipolar coupling calculation. Simple criteria have been established as to when the strong coupling treatment needs to be invoked.

  18. Quantum mechanical study and spectroscopic (FT-IR, FT-Raman, 13C, 1H) study, first order hyperpolarizability, NBO analysis, HOMO and LUMO analysis of 2-acetoxybenzoic acid by density functional methods

    NASA Astrophysics Data System (ADS)

    Bhavani, K.; Renuga, S.; Muthu, S.; Sankara narayanan, K.

    2015-02-01

    In this work, colorless crystals of 2-acetoxybenzoic acid were grown by slow evaporation method and the FT-IR and FT-Raman spectra of the sample were recorded in the region 4000-500 cm-1 and 4000-100 cm-1 respectively. Molecular structure is optimized with the help of density functional theory method (B3LYP) with 6-31+G(d,p), 6-311++G(d,p) basis sets. Stability of the molecule arising from hyperconjugation and charge delocalization is confirmed by the natural bond orbital analysis (NBO). The results show that electron density (ED) in the σ∗ antibonding orbitals and E(2) energies confirms the occurrence of intramolecular charge transfer (ICT) within the molecule. The assignments of the vibrational spectra have been carried out with the help of normal coordinate analysis following the scaled quantum mechanical force field (SQMFF) methodology. The results of the calculations were applied to simulated spectra of the title compound, which show excellent agreement with observed spectra. The 1H and 13C nuclear magnetic resonance (NMR) chemical shifts of the molecule were calculated by GIAO method. Mulliken population analysis on atomic charges is also calculated. The calculated HOMO and LUMO energy gap shows that charge transfer occurs within the molecule.

  19. An estimation of Central Iberian Peninsula atmospheric δ13C and water δD in the Upper Cretaceous using pyrolysis compound specific isotopic analysis (Py-CSIA) of a fossil conifer.

    NASA Astrophysics Data System (ADS)

    González-Pérez, José A.; Jiménez-Morillo, Nicasio T.; De la Rosa, José M.; Almendros, Gonzalo; González-Vila, Francisco J.

    2015-04-01

    Frenelopsis is a frequently found genus of the Cretaceous floras adapted to dry, saline and in general to environmental conditions marked by severe water stress [1]. Stable isotope analysis of fossil organic materials can be used to infer palaeoenvironmental variables helpful to reconstruct plant paleohabitats [2]. In this study stable isotope analysis of organic fossil remains (FR) and humic fractions (FA, HA and humin) of Frenelopsis oligiostomata are studied in bulk (C, H, O, N IRMS) and in specific compounds released by pyrolysis (C, H, Py-CSIA). Well preserved F. oligiostomata fossils were handpicked from a limestone included in compacted marls from Upper Cretaceous (Senonian c. 72 Mya) in Guadalix de la Sierra (Madrid, Spain) [3]. The fossils were decarbonated with 6M HCl. Humic substances were extracted from finely ground fossil remains (FR) by successive treatments with 0.1M Na4P2O7 + NaOH [4]. The extract was acidified resulting into insoluble HA and soluble FA fractions. The HA and FA were purified as in [5] and [6] respectively. Bulk stable isotopic analysis13C, δD, δ18O, δ15N IRMS) was done in an elemental micro-analyser coupled to a continuous flow Delta V Advantage isotope ratio mass spectrometer (IRMS). Pyrolysis compound specific isotopic analysis Py-CSIA (δ13C, δD): was done by coupling a double-shot pyrolyzer to a chromatograph connected to an IRMS. Structural features of specific peaks were inferred by comparing/matching mass spectra from conventional Py-GC/MS (data not shown) with Py-GC/IRMS chromatograms obtained using the same chromatographic conditions. Bulk C isotopic signature found for FR (-20.5±0.02 ‰) was in accordance with previous studies [2, 7-9]. This heavy isotopic δ13C signature indicates a depleted stomatal conductance and paleoenvironmental growth conditions of water and salt stress. This is in line with the morphological and depositional characteristics [3] confirming that F. oligostomata was adapted to highly xeric

  20. Synthesis and NMR Spectral Analysis of Amine Heterocycles: The Effect of Asymmetry on the [superscript 1]H and [superscript 13]C NMR Spectra of N,O-Acetals

    ERIC Educational Resources Information Center

    Saba, Shahrokh; Ciaccio, James A.; Espinal, Jennifer; Aman, Courtney E.

    2007-01-01

    The stereochemical investigation is conducted to give students the combined experience of chemical synthesis of amines and N-heterocycles and structural stereochemical analysis using NMR spectroscopy. Students are introduced to the concept of topicity-stereochemical relationships between ligands within a molecule by synthesizing N,O-acetals.

  1. Dual element ((15)N/(14)N, (13)C/(12)C) isotope analysis of glyphosate and AMPA by derivatization-gas chromatography isotope ratio mass spectrometry (GC/IRMS) combined with LC/IRMS.

    PubMed

    Mogusu, Emmanuel O; Wolbert, J Benjamin; Kujawinski, Dorothea M; Jochmann, Maik A; Elsner, Martin

    2015-07-01

    To assess sources and degradation of the herbicide glyphosate [N-(phosphonomethyl) glycine] and its metabolite AMPA (aminomethylphosphonic acid), concentration measurements are often inconclusive and even (13)C/(12)C analysis alone may give limited information. To advance isotope ratio analysis of an additional element, we present compound-specific (15)N/(14)N analysis of glyphosate and AMPA by a two step derivatization in combination with gas chromatography/isotope ratio mass spectrometry (GC/IRMS). The N-H group was derivatized with isopropyl chloroformate (iso-PCF), and remaining acidic groups were subsequently methylated with trimethylsilyldiazomethane (TMSD). Iso-PCF treatment at pH <10 gave too low (15)N/(14)N ratios indicating an incomplete derivatization; in contrast, too high (15)N/(14)N ratios at pH >10 indicated decomposition of the derivative. At pH 10, and with an excess of iso-PCF by 10-24, greatest yields and accurate (15)N/(14)N ratios were obtained (deviation from elemental analyzer-IRMS: -0.2 ± 0.9% for glyphosate; -0.4 ± 0.7% for AMPA). Limits for accurate δ(15)N analysis of glyphosate and AMPA were 150 and 250 ng injected, respectively. A combination of δ(15)N and δ(13)C analysis by liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) (1) enabled an improved distinction of commercial glyphosate products and (2) showed that glyphosate isotope values during degradation by MnO2 clearly fell outside the commercial product range. This highlights the potential of combined carbon and nitrogen isotopes analysis to trace sources and degradation of glyphosate.

  2. Determining the Local Abundance of Martian Methane and its 13-C/l2-C and D/H Isotopic Ratios for Comparison with Related Gas and Soil Analysis on the 2011 Mars Science Laboratory (MSL) Mission

    NASA Technical Reports Server (NTRS)

    Webster, Christopher R.; Mahaffy, Paul R.

    2011-01-01

    Understanding the origin of Martian methane will require numerous complementary measurements from both in situ and remote sensing investigations and laboratory work to correlate planetary surface geophysics with atmospheric dynamics and chemistry. Three instruments (Quadrupole Mass Spectrometer (QMS), Gas Chromatograph (GC) and Tunable Laser Spectrometer (TLS)) with sophisticated sample handling and processing capability make up the Sample Analysis at Mars (SAM) analytical chemistry suite on NASA s 2011 Mars Science Laboratory (MSL) Mission. Leveraging off the SAM sample and gas processing capability that includes methane enrichment, TLS has unprecedented sensitivity for measuring absolute methane (parts-per-trillion), water, and carbon dioxide abundances in both the Martian atmosphere and evolved from heated soil samples. In concert with a wide variety of associated trace gases (e.g. SO2, H2S, NH3, higher hydrocarbons, organics, etc.) and other isotope ratios measured by SAM, TLS will focus on determining the absolute abundances of methane, water and carbon dioxide, and their isotope ratios: 13C/12C and D/H in methane; 13C/12C and 18O/17O/16O in carbon dioxide; and 18O/17O/16O and D/H in water. Measurements near the MSL landing site will be correlated with satellite (Mars Express, Mars 2016) and ground-based observations.

  3. Development of qualitative and quantitative analysis methods in pharmaceutical application with new selective signal excitation methods for 13 C solid-state nuclear magnetic resonance using 1 H T1rho relaxation time.

    PubMed

    Nasu, Mamiko; Nemoto, Takayuki; Mimura, Hisashi; Sako, Kazuhiro

    2013-01-01

    Most pharmaceutical drug substances and excipients in formulations exist in a crystalline or amorphous form, and an understanding of their state during manufacture and storage is critically important, particularly in formulated products. Carbon 13 solid-state nuclear magnetic resonance (NMR) spectroscopy is useful for studying the chemical and physical state of pharmaceutical solids in a formulated product. We developed two new selective signal excitation methods in (13) C solid-state NMR to extract the spectrum of a target component from such a mixture. These methods were based on equalization of the proton relaxation time in a single domain via rapid intraproton spin diffusion and the difference in proton spin-lattice relaxation time in the rotating frame ((1) H T1rho) of individual components in the mixture. Introduction of simple pulse sequences to one-dimensional experiments reduced data acquisition time and increased flexibility. We then demonstrated these methods in a commercially available drug and in a mixture of two saccharides, in which the (13) C signals of the target components were selectively excited, and showed them to be applicable to the quantitative analysis of individual components in solid mixtures, such as formulated products, polymorphic mixtures, or mixtures of crystalline and amorphous phases. PMID:23147444

  4. A Column Experiment To Determine Black Shale Degradation And Colonization By Means of δ13C and 14C Analysis Of Phospholipid Fatty Acids And DNA Extraction

    NASA Astrophysics Data System (ADS)

    Seifert, A.; Gleixner, G.

    2008-12-01

    We investigated the degradation of black shale organic matter by microbial communities. We inoculated two columns respectively, with the fungi Schizophyllum commune, the gram-positive bacterium Pseudomonas putida and the gram-negative bacteria Streptomyces griseus and Streptomyces chartreusis. These microorganisms are known to degrade a wide variety of organic macromolecules. Additionally, we had two sets of control columns. To one set the same nutrient solution was added as to the inoculated columns and to the other set only sterile deionised water was supplied. All columns contained 1.5 kg of freshly crushed not autoclaved black shale material with a particle size of 0.63-2 mm. The columns were incubated at 28° C and 60% humidity in the dark. The aim was to investigate, which microorganisms live on black shales and if these microorganisms are able to degrade ancient organic matter. We used compound specific stable isotope measurement techniques and compound specific 14C-dating methods. After 183 days PLFAs were extracted from the columns to investigate the microbial community, furthermore we extracted on one hand total-DNA of column material and on the other hand DNA from pure cultures isolates which grew on Kinks-agar B, Starch-casein-nitrate-agar (SCN) and on complete-yeast-medium-agar (CYM). According to the PLFA analysis bacteria dominated in the columns, whereas in pure cultures more fungi were isolated. A principal component analysis revealed differences between the columns in accordance with the inoculation, but it seems that the inoculated microorganisms were replaced by the natural population. For AMS measurements palmitic acid (C 16:0) was re-isolated from total-PLFA-extract with a preparative fraction collector (PFC). Preliminary results of the study revealed that microorganisms are able to degrade black shale material and that PLFA analysis are useful methods to be combined with analysis of stable isotope and 14C measurements to study microbial

  5. Recurrence Analysis of Eddy Covariance Fluxes

    NASA Astrophysics Data System (ADS)

    Lange, Holger; Flach, Milan; Foken, Thomas; Hauhs, Michael

    2015-04-01

    The eddy covariance (EC) method is one key method to quantify fluxes in biogeochemical cycles in general, and carbon and energy transport across the vegetation-atmosphere boundary layer in particular. EC data from the worldwide net of flux towers (Fluxnet) have also been used to validate biogeochemical models. The high resolution data are usually obtained at 20 Hz sampling rate but are affected by missing values and other restrictions. In this contribution, we investigate the nonlinear dynamics of EC fluxes using Recurrence Analysis (RA). High resolution data from the site DE-Bay (Waldstein-Weidenbrunnen) and fluxes calculated at half-hourly resolution from eight locations (part of the La Thuile dataset) provide a set of very long time series to analyze. After careful quality assessment and Fluxnet standard gapfilling pretreatment, we calculate properties and indicators of the recurrent structure based both on Recurrence Plots as well as Recurrence Networks. Time series of RA measures obtained from windows moving along the time axis are presented. Their interpretation is guided by three different questions: (1) Is RA able to discern periods where the (atmospheric) conditions are particularly suitable to obtain reliable EC fluxes? (2) Is RA capable to detect dynamical transitions (different behavior) beyond those obvious from visual inspection? (3) Does RA contribute to an understanding of the nonlinear synchronization between EC fluxes and atmospheric parameters, which is crucial for both improving carbon flux models as well for reliable interpolation of gaps? (4) Is RA able to recommend an optimal time resolution for measuring EC data and for analyzing EC fluxes? (5) Is it possible to detect non-trivial periodicities with a global RA? We will demonstrate that the answers to all five questions is affirmative, and that RA provides insights into EC dynamics not easily obtained otherwise.

  6. {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.

  7. 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.

  8. 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. PMID:26479486

  9. Determination of 13C/ 12C ratios with (d, p) nuclear reactions

    NASA Astrophysics Data System (ADS)

    Wang, Y. Q.; Zhang, J.; Tesmer, J. R.; Li, Y. H.; Greco, R.; Grim, G. P.; Obst, A. W.; Rundberg, R. S.; Wilhelmy, J. B.

    2010-06-01

    Stable isotope ratios such as 13C/ 12C play an important role in many applications including environment and energy research. Since many surface analysis techniques are plagued with unavoidable hydrocarbon contamination issues during analysis, it is highly desirable that 13C and 12C isotopes be measured simultaneously especially in specimens with a minute amount of 13C, in order to reliably determine 13C/ 12C ratios. In this paper, we report that deuterium induced proton particle reactions, 13C(d, p) 14C and 12C(d, p) 13C, provide a convenient and reliable approach for 13C/ 12C ratio determination. Optimizations on experimental considerations and potential interferences from other common light isotopes are discussed as well as results from the application of this technique to diagnose the performance of a target debris collection in an inertial confinement fusion (ICF) experiment.

  10. Crystal structure and thermodynamic analysis of diagnostic mAb 106.3 complexed with BNP 5-13 (C10A)

    SciTech Connect

    Longenecker, Kenton L.; Ruan, Qiaoqiao; Fry, Elizabeth H.; Saldana, Sylvia C.; Brophy, Susan E.; Richardson, Paul L.; Tetin, Sergey Y.

    2010-09-02

    B-type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention in human physiology. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using the high-affinity monoclonal IgG1 antibody 106.3, which binds an epitope spanning residues 5-13 of the mature bioactive peptide. To understand the structural basis of this molecular recognition, we crystallized the Fab fragment complexed with the peptide epitope and determined the three-dimensional structure by X-ray diffraction to 2.1 {angstrom} resolution. The structure reveals the detailed interactions that five of the complementarity-determining regions make with the partially folded peptide. Thermodynamic measurements using fluorescence spectroscopy suggest that the interaction is enthalpy driven, with an overall change in free energy of binding, {Delta}G = -54 kJ/mol, at room temperature. The parameters are interpreted on the basis of the structural information. The kinetics of binding suggest a diffusion-limited mechanism, whereby the peptide easily adopts a bound conformation upon interaction with the antibody. Moreover, comparative analysis with alanine-scanning results of the epitope explains the basis of selectivity for BNP over other related natriuretic peptides.

  11. Analysis of the 1-v" Progression of the 3A Band System (c(3)Pi-a(3)Pi) in the (13)C(16)O Molecule Spectrum.

    PubMed

    Hakalla

    2000-12-01

    The 1-0, 1-1, and 1-2 bands of the 3A system of (13)CO (c(3)Pi-a(3)Pi) have been recorded for the first time, in the form of discharge emission spectra photographed at high resolution. Preliminary rotational analysis suggests that the v = 1 level of the c(3)Pi state is perturbed in a fashion similar to the v = 0 level, previously observed by Dabrowski et al. (1987. I. Dabrowski, M. Vervolet, and D. C. Wang, Can. J. Phys. 65, 1171-1177). Rotational combination differences from the 1720 measured lines have been used to obtain rotational constants for the v = 0-2 levels of the a(3)Pi state, using the Hamiltonian of Brown et al. (1979. J. M. Brown, E. A. Calbourn, J. K. G. Watson, and F. D. Wayne, J. Mol. Spectrosc. 74, 294-318). and the least squares formalism of Curl and Dane (1988. J. Mol. Spectrosc. 128, 406-412), as modified by Watson (1989. J. Mol. Spectrosc. 138, 302-308). Term values for the c(3)Pi, v = 1 level have then been obtained from the line frequencies, and a set of effective rotational constants for the c(3)Pi (v = 1) level of (13)CO has been derived. Copyright 2000 Academic Press.

  12. Conformational Analysis, Thermal Rearrangement, and EI-MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol by (13)C-Labeling Experiments.

    PubMed

    Rabe, Patrick; Barra, Lena; Rinkel, Jan; Riclea, Ramona; Citron, Christian A; Klapschinski, Tim A; Janusko, Aron; Dickschat, Jeroen S

    2015-11-01

    An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen ((13)C1)FPP isotopomers (FPP=farnesyl diphosphate) and ((13)C15)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including (13)C, (13)C COSY experiments. The ((13)C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product. PMID:26361082

  13. Conformational Analysis, Thermal Rearrangement, and EI-MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol by (13)C-Labeling Experiments.

    PubMed

    Rabe, Patrick; Barra, Lena; Rinkel, Jan; Riclea, Ramona; Citron, Christian A; Klapschinski, Tim A; Janusko, Aron; Dickschat, Jeroen S

    2015-11-01

    An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen ((13)C1)FPP isotopomers (FPP=farnesyl diphosphate) and ((13)C15)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including (13)C, (13)C COSY experiments. The ((13)C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product.

  14. 'Wave' analysis of Venus ionospheric flux ropes

    NASA Technical Reports Server (NTRS)

    Luhmann, J. G.

    1990-01-01

    The behavior of the general field fluctuations observed by the Pioneer Venus Orbiter magnetometer in the Venusian ionosphere is approached via the application of wave-analysis techniques to their compressional amplitude, ellipticity, and polarization. The most marked change in these properties occurs within about 15 deg of the terminator; ellipticity and compressional power plummet, implying that flux ropes either drastically change in character there or are confined to the dayside hemisphere, within about 75 percent of the subsolar point. Attention is given to a heuristic model which attempts to unify dayside flux-rope and 'terminator wave' interpretations.

  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. Feasibility of Multianimal Hyperpolarized 13C MRS

    PubMed Central

    Ramirez, Marc S.; Lee, Jaehyuk; Walker, Christopher M.; Chen, Yunyun; Kingsley, Charles V.; De La Cerda, Jorge; Maldonado, Kiersten L.; Lai, Stephen Y.; Bankson, James A.

    2014-01-01

    Purpose There is great potential for real-time investigation of metabolism with MRS and hyperpolarized (HP) 13C agents. Unfortunately, HP technology has high associated costs and efficiency limitations that may constrain in vivo studies involving many animals. To improve the throughput of preclinical investigations, we evaluate the feasibility of performing HP MRS on multiple animals simultaneously. Methods Simulations helped assess the viability of a dual-coil strategy for spatially-localized multivolume MRS.A dual-mouse system was assembled and characterized based on bench- and scanner-based experiments. Enzyme phantoms mixed with HP [1-13C] pyruvate emulated real-time metabolism and offered a controlled mechanism for evaluating system performance. Finally, a normal mouse and a mouse bearing a subcutaneous xenograft of colon cancer were simultaneously scanned in vivo using an agent containing HP [1-13C] pyruvate. Results Geometric separation/rotation, active decoupling, and use of low input impedance preamplifiers permitted an encode-by-channel approach for spatially-localized MRS. A pre-calibrated shim allowed straightforward metabolite differentiation in enzyme phantom and in vivo experiments at 7 T, with performance similar to conventional acquisitions. Conclusion The initial feasibility of multi-animal HP 13C MRS was established. Throughput scales with the number of simultaneously-scanned animals, demonstrating the potential for significant improvements in study efficiency. PMID:24903532

  17. Life history of the individuals buried in the St. Benedict Cemetery (Prague, 15th-18th centuries): insights from (14)C dating and stable isotope (δ(13)C, δ(15)N, δ(18)O) analysis.

    PubMed

    Salesse, Kevin; Dufour, Élise; Castex, Dominique; Velemínský, Petr; Santos, Frédéric; Kuchařová, Hedvika; Jun, Libor; Brůžek, Jaroslav

    2013-06-01

    Funerary practices and bioarchaeological (sex and age) data suggest that a mortality crisis linked to an epidemic episode occurred during the fifth phase of the St. Benedict cemetery in Prague (Czech Republic). To identify this mass mortality episode, we reconstructed individual life histories (dietary and mobility factors), assessed the population's biological homogeneity, and proposed a new chronology through stable isotope analysis (δ(13)C, δ(18)O and δ(15)N) and direct radiocarbon dating. Stable isotope analysis was conducted on the bone and tooth enamel (collagen and carbonate) of 19 individuals from three multiple graves (MG) and 12 individuals from individual graves (IG). The δ(15)N values of collagen and the difference between the δ(13)C values of collagen and bone carbonate could indicate that the IG individuals had a richer protein diet than the MG individuals or different food resources. The human bone and enamel carbonate and δ(18)O values suggest that the majority of individuals from MG and all individuals from IG spent most of their lives outside of the Bohemian region. Variations in δ(18)O values also indicate that all individuals experienced residential mobility during their lives. The stable isotope results, biological (age and sex) data and eight (14)C dates clearly differentiate the MG and IG groups. The present work provides evidence for the reuse of the St. Benedict cemetery to bury soldiers despite the funeral protest ban (1635 AD). The Siege of Prague (1742 AD) by French-Bavarian-Saxon armies is identified as the cause of the St. Benedict mass mortality event.

  18. Life history of the individuals buried in the St. Benedict Cemetery (Prague, 15th-18th centuries): insights from (14)C dating and stable isotope (δ(13)C, δ(15)N, δ(18)O) analysis.

    PubMed

    Salesse, Kevin; Dufour, Élise; Castex, Dominique; Velemínský, Petr; Santos, Frédéric; Kuchařová, Hedvika; Jun, Libor; Brůžek, Jaroslav

    2013-06-01

    Funerary practices and bioarchaeological (sex and age) data suggest that a mortality crisis linked to an epidemic episode occurred during the fifth phase of the St. Benedict cemetery in Prague (Czech Republic). To identify this mass mortality episode, we reconstructed individual life histories (dietary and mobility factors), assessed the population's biological homogeneity, and proposed a new chronology through stable isotope analysis (δ(13)C, δ(18)O and δ(15)N) and direct radiocarbon dating. Stable isotope analysis was conducted on the bone and tooth enamel (collagen and carbonate) of 19 individuals from three multiple graves (MG) and 12 individuals from individual graves (IG). The δ(15)N values of collagen and the difference between the δ(13)C values of collagen and bone carbonate could indicate that the IG individuals had a richer protein diet than the MG individuals or different food resources. The human bone and enamel carbonate and δ(18)O values suggest that the majority of individuals from MG and all individuals from IG spent most of their lives outside of the Bohemian region. Variations in δ(18)O values also indicate that all individuals experienced residential mobility during their lives. The stable isotope results, biological (age and sex) data and eight (14)C dates clearly differentiate the MG and IG groups. The present work provides evidence for the reuse of the St. Benedict cemetery to bury soldiers despite the funeral protest ban (1635 AD). The Siege of Prague (1742 AD) by French-Bavarian-Saxon armies is identified as the cause of the St. Benedict mass mortality event. PMID:23588853

  19. Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle.

    PubMed

    Alves, Tiago C; Pongratz, Rebecca L; Zhao, Xiaojian; Yarborough, Orlando; Sereda, Sam; Shirihai, Orian; Cline, Gary W; Mason, Graeme; Kibbey, Richard G

    2015-11-01

    Mass isotopomer multi-ordinate spectral analysis (MIMOSA) is a step-wise flux analysis platform to measure discrete glycolytic and mitochondrial metabolic rates. Importantly, direct citrate synthesis rates were obtained by deconvolving the mass spectra generated from [U-(13)C6]-D-glucose labeling for position-specific enrichments of mitochondrial acetyl-CoA, oxaloacetate, and citrate. Comprehensive steady-state and dynamic analyses of key metabolic rates (pyruvate dehydrogenase, β-oxidation, pyruvate carboxylase, isocitrate dehydrogenase, and PEP/pyruvate cycling) were calculated from the position-specific transfer of (13)C from sequential precursors to their products. Important limitations of previous techniques were identified. In INS-1 cells, citrate synthase rates correlated with both insulin secretion and oxygen consumption. Pyruvate carboxylase rates were substantially lower than previously reported but showed the highest fold change in response to glucose stimulation. In conclusion, MIMOSA measures key metabolic rates from the precursor/product position-specific transfer of (13)C-label between metabolites and has broad applicability to any glucose-oxidizing cell. PMID:26411341

  20. Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle.

    PubMed

    Alves, Tiago C; Pongratz, Rebecca L; Zhao, Xiaojian; Yarborough, Orlando; Sereda, Sam; Shirihai, Orian; Cline, Gary W; Mason, Graeme; Kibbey, Richard G

    2015-11-01

    Mass isotopomer multi-ordinate spectral analysis (MIMOSA) is a step-wise flux analysis platform to measure discrete glycolytic and mitochondrial metabolic rates. Importantly, direct citrate synthesis rates were obtained by deconvolving the mass spectra generated from [U-(13)C6]-D-glucose labeling for position-specific enrichments of mitochondrial acetyl-CoA, oxaloacetate, and citrate. Comprehensive steady-state and dynamic analyses of key metabolic rates (pyruvate dehydrogenase, β-oxidation, pyruvate carboxylase, isocitrate dehydrogenase, and PEP/pyruvate cycling) were calculated from the position-specific transfer of (13)C from sequential precursors to their products. Important limitations of previous techniques were identified. In INS-1 cells, citrate synthase rates correlated with both insulin secretion and oxygen consumption. Pyruvate carboxylase rates were substantially lower than previously reported but showed the highest fold change in response to glucose stimulation. In conclusion, MIMOSA measures key metabolic rates from the precursor/product position-specific transfer of (13)C-label between metabolites and has broad applicability to any glucose-oxidizing cell.

  1. Monomeric and dimeric structures analysis and spectroscopic characterization of 3,5-difluorophenylboronic acid with experimental (FT-IR, FT-Raman, 1H and 13C NMR, UV) techniques and quantum chemical calculations

    NASA Astrophysics Data System (ADS)

    Karabacak, Mehmet; Kose, Etem; Atac, Ahmet; Asiri, Abdullah M.; Kurt, Mustafa

    2014-01-01

    The spectroscopic properties of 3,5-difluorophenylboronic acid (3,5-DFPBA, C6H3F2B(OH)2) were investigated by FT-IR, FT-Raman UV-Vis, 1H and 13C NMR spectroscopic techniques. FT-IR (4000-400 cm-1) and FT-Raman spectra (3500-10 cm-1) in the solid phase and 1H and 13C NMR spectra in DMSO solution were recorded. The UV spectra that dissolved in ethanol and water were recorded in the range of 200-400 nm for each solution. The structural and spectroscopic data of the molecule have been obtained for possible three conformers from DFT (B3LYP) with 6-311++G(d,p) basis set calculations. The geometry of the molecule was fully optimized, vibrational spectra were calculated and fundamental vibrations were assigned on the basis of the total energy distribution (TED) of the vibrational modes, calculated with scaled quantum mechanics (SQM) method and PQS program. Hydrogen-bonded dimer of title molecule, optimized by counterpoise correction, was also studied B3LYP at the 6-311++G(d,p) level and the effects of molecular association through O-H⋯O hydrogen bonding have been discussed. 1H and 13C NMR chemical shifts were calculated by using the gauge-invariant atomic orbital (GIAO) method. The electronic properties, such as excitation energies, oscillator strength, wavelengths, HOMO and LUMO energies, were performed by time-dependent density functional theory (TD-DFT) results complements with the experimental findings. Total and partial density of state (TDOS and PDOS) and also overlap population density of state (OPDOS) diagrams analysis were presented. The effects due to the substitutions of boric acid group and halogen were investigated. The results of the calculations were applied to simulate spectra of the title compound, which show excellent agreement with observed spectra. Besides, frontier molecular orbitals (FMO), molecular electrostatic potential (MEP), nonlinear optical properties (NLO) and thermodynamic features were performed.

  2. 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.

  3. ¹³C-based metabolic flux analysis of Saccharomyces cerevisiae with a reduced Crabtree effect.

    PubMed

    Kajihata, Shuichi; Matsuda, Fumio; Yoshimi, Mika; Hayakawa, Kenshi; Furusawa, Chikara; Kanda, Akihisa; Shimizu, Hiroshi

    2015-08-01

    Saccharomyces cerevisiae shows a Crabtree effect that produces ethanol in a high glucose concentration even under fully aerobic condition. For efficient production of cake yeast or compressed yeast for baking, ethanol by-production is not desired since glucose limited chemostat or fed-batch cultivations are performed to suppress the Crabtree effect. In this study, the (13)C-based metabolic flux analysis ((13)C-MFA) was performed for the S288C derived S. cerevisiae strain to characterize a metabolic state under the reduced Crabtree effect. S. cerevisiae cells were cultured at a low dilution rate (0.1 h(-1)) under the glucose-limited chemostat condition. The estimated metabolic flux distribution showed that the acetyl-CoA in mitochondria was mainly produced from pyruvate by pyruvate dehydrogenase (PDH) reaction and that the level of the metabolic flux through the pentose phosphate pathway was much higher than that of the Embden-Meyerhof-Parnas pathway, which contributes to high biomass yield at low dilution rate by supplying NADPH required for cell growth.

  4. ¹³C-based metabolic flux analysis of recombinant Pichia pastoris.

    PubMed

    Ferrer, Pau; Albiol, Joan

    2014-01-01

    Overexpression of a foreign protein may negatively affect several cell growth parameters, as well as cause cellular stress. Central (or core) metabolism plays a crucial role since it supplies energy, reduction equivalents, and precursor molecules for the recombinant product, cell's maintenance, and growth needs. However, the number of quantitative physiology studies of the impact of recombinant protein production on the central metabolic pathways of yeast cell factories has been traditionally rather limited, thereby hampering the application of rational strain engineering strategies targeting central metabolism.The development and application of quantitative physiology and modelling tools and methodologies is allowing for a systems-level understanding of the effect of bioprocess parameters such as growth rate, temperature, oxygen availability, and substrate(s) choice on metabolism, and its subsequent interactions with recombinant protein synthesis, folding, and secretion.Here, we review the recent developments and applications of (13)C-based metabolic flux analysis ((13)C-MFA) of Pichia pastoris and the gained understanding of the metabolic behavior of this yeast in recombinant protein production bioprocesses. We also discuss the potential of multilevel studies integrating (13)C-MFA with other omics analyses, as well as future perspectives on the metabolic modelling approaches to study and design metabolic engineering strategies for improved protein production.

  5. {sup 13}C-{sup 1}H and {sup 13}C-{sup 13}C spin couplings in [2`-{sup 13}C]2`-deoxyribonucleosides: Correlations with molecular structure

    SciTech Connect

    Bandyopadhyay, T.; Stripe, W.A.; Carmichael, I.; Serianni, A.S.; Wu, J.

    1997-02-19

    2`-Deoxyribonucleosides (2`-deoxyadenosine (1), 2`-depoxycytidine (2), thymidine (3)) singly enriched with {sup 13}C at C2` have been prepared and used to obtain one-, two-, and three-bond {sup 13}C-{sup 1}H and {sup 13}C-{sup 13}C spin-coupling constants involving C2`. Spin couplings in 1-3 involving C1` and C2`are also compared to corresponding values in ribonucleosides in order to assess the effects of nucleoside structure and conformation on J values within the furanose ring. {sup 1}J{sub C2`,H2`R} and {sup 1}J{sub C2`,H2`S} in 1-3 and {sup 1}J{sub C2`,H2`} in ribonucleosides depend on C-H bond orientation: {sup 1}J{sub C1`,H1`} in 1-3 and in ribonucleosides exhibits a similar dependence. The latter couplings appear to be essentially unaffected by N-glycoside torsion. {sup 1}J{sub CC} values depend on the number and distribution of electronegative substituents on the C-C fragment. A modified projection curve is proposed to aid in the interpretation of {sup 2}J{sub C2`,H1`} values; the presence of N substitution at C1` caused a shift to more negative couplings relative to the O-substituted analog. In contrast, {sup 2}J{sub C1`,H2`} is essentially unaffected by the same change in the electronegative substituent at C1`. {sup 2}J{sub CC} values within the furanose ring are determined buy two coupling pathways; in one case (i.e., {sup 2}J{sub C1`,C3`}), the observed coupling is shown to be the algebraic sum of the two couplings arising from each pathway. 41 refs., 4 figs., 2 tabs.

  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. The Late Pleistocene history of surface water δ13C in the Sulu Sea: Possible relationship to Pacific Deepwater δ13C changes

    NASA Astrophysics Data System (ADS)

    Linsley, Braddock K.; Dunbar, Robert B.

    1994-04-01

    A reconstruction of late Pleistocene surface water carbon isotopic (δ13C) variability is presented from Ocean Drilling Program (ODP) site 769 in the Sulu Sea in the western tropical Pacific. The Sulu Sea is a shallowly silled back arc basin with a maximum sill depth of 420 m. Site 769 was drilled on a bathymetric high in 3643 m of water and has average late Pleistocene sedimentation rates of 8.5 cm/kyr. The oxygen isotope record (δ18O) of Globigerinoides ruber at site 769 shows a strong correlation with the SPECMAP stacked δ18O record, attesting to the continuity of sediment archive at the site. Surface δ13C displays consistent glacial-interglacial variability which averages ˜0.9‰ and has varied from 0.75 to 1.1‰ over the last 800 kyr. Comparison to surface water δ13C records in the South China Sea and western tropical Pacific suggests that the glacial-interglacial surface δ13C variability is regional in scale. Planktonic δ13C data from ODP site 677 in the eastern Pacific is also coherent with the site 769. Additionally, we have found that the site 769 surface δ13C record is coherent at periods of 100 and 41 kyr with deepwater δ13C records from the Pacific. The highest correlation occurs with the deep eastern Pacific, where benthic δ13C data from cores RC13-110 and ODP site 677 closely match the Sulu Sea surface water record. We evaluate several possible controls of surface water δ13C in the Sulu Sea that may explain the coherent timing with Pacific deepwater δ13C records. These include variations in terrestrial organic matter flux to the basin, the upwelling of subsurface water and productivity changes, and the influx of western Pacific intermediate water to the Sulu Sea. Our preferred explanation involves a region of upper intermediate water upwelling in the far western Pacific which has been shown to outgas CO2 from subsurface waters into surface waters. Upwelling also occurs in the area of Panama Basin site 677. These equatorial upwelling zones

  8. Biomimetic oxidative treatment of spruce wood studied by pyrolysis-molecular beam mass spectrometry coupled with multivariate analysis and 13C-labeled tetramethylammonium hydroxide thermochemolysis: implications for fungal degradation of wood.

    PubMed

    Arantes, Valdeir; Qian, Yuhui; Kelley, Stephen S; Milagres, Adriane M F; Filley, Timothy R; Jellison, Jody; Goodell, Barry

    2009-11-01

    In this work, pyrolysis-molecular beam mass spectrometry analysis coupled with principal components analysis and (13)C-labeled tetramethylammonium hydroxide thermochemolysis were used to study lignin oxidation, depolymerization, and demethylation of spruce wood treated by biomimetic oxidative systems. Neat Fenton and chelator-mediated Fenton reaction (CMFR) systems as well as cellulosic enzyme treatments were used to mimic the nonenzymatic process involved in wood brown-rot biodegradation. The results suggest that compared with enzymatic processes, Fenton-based treatment more readily opens the structure of the lignocellulosic matrix, freeing cellulose fibrils from the matrix. The results demonstrate that, under the current treatment conditions, Fenton and CMFR treatment cause limited demethoxylation of lignin in the insoluble wood residue. However, analysis of a water-extractable fraction revealed considerable soluble lignin residue structures that had undergone side chain oxidation as well as demethoxylation upon CMFR treatment. This research has implications for our understanding of nonenzymatic degradation of wood and the diffusion of CMFR agents in the wood cell wall during fungal degradation processes.

  9. Measuring (13)C-(2)D dipolar couplings with a universal REDOR dephasing curve

    PubMed

    Gullion

    2000-09-01

    A (13)C-observe REDOR experiment is described which allows (13)C-(2)D dipolar couplings to be obtained by a universal dipolar dephasing curve. Previous (13)C-observe REDOR experiments on (13)C-(2)D spin pairs generally relied on numerical simulations to obtain the dipolar coupling. The REDOR experiment described in this article is based on a deuterium composite pulse, and the data analysis eliminates the need for numerical simulations and is the same as the traditional REDOR analysis performed on pairs of spin-12 nuclei. Copyright 2000 Academic Press. PMID:10968975

  10. Proton-detected 3D {sup 1}H/{sup 13}C/{sup 1}H correlation experiment for structural analysis in rigid solids under ultrafast-MAS above 60 kHz

    SciTech Connect

    Zhang, Rongchun; Ramamoorthy, Ayyalusamy; Nishiyama, Yusuke

    2015-10-28

    A proton-detected 3D {sup 1}H/{sup 13}C/{sup 1}H chemical shift correlation experiment is proposed for the assignment of chemical shift resonances, identification of {sup 13}C-{sup 1}H connectivities, and proximities of {sup 13}C-{sup 1}H and {sup 1}H-{sup 1}H nuclei under ultrafast magic-angle-spinning (ultrafast-MAS) conditions. Ultrafast-MAS is used to suppress all anisotropic interactions including {sup 1}H-{sup 1}H dipolar couplings, while the finite-pulse radio frequency driven dipolar recoupling (fp-RFDR) pulse sequence is used to recouple dipolar couplings among protons and the insensitive nuclei enhanced by polarization transfer technique is used to transfer magnetization between heteronuclear spins. The 3D experiment eliminates signals from non-carbon-bonded protons and non-proton-bonded carbons to enhance spectral resolution. The 2D (F1/F3) {sup 1}H/{sup 1}H and 2D {sup 13}C/{sup 1}H (F2/F3) chemical shift correlation spectra extracted from the 3D spectrum enable the identification of {sup 1}H-{sup 1}H proximity and {sup 13}C-{sup 1}H connectivity. In addition, the 2D (F1/F2) {sup 1}H/{sup 13}C chemical shift correlation spectrum, incorporated with proton magnetization exchange via the fp-RFDR recoupling of {sup 1}H-{sup 1}H dipolar couplings, enables the measurement of proximities between {sup 13}C and even the remote non-carbon-bonded protons. The 3D experiment also gives three-spin proximities of {sup 1}H-{sup 1}H-{sup 13}C chains. Experimental results obtained from powder samples of L-alanine and L-histidine ⋅ H{sub 2}O ⋅ HCl demonstrate the efficiency of the 3D experiment.

  11. 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. PMID:26228944

  12. 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.

  13. Lithium metaborate flux in silicate analysis

    USGS Publications Warehouse

    Ingamells, C.O.

    1970-01-01

    Lithium metaborate is an effective flux for silicates and other rock-forming minerals. The glass resulting from fusion is mechanically strong, reasonably nonhygroscopic, and is readily soluble in dilute acids. These characteristics lead to its use in X-ray spectrography and in methods which require whole-rock solutions, such as atomic absorption and emission spectrometry. Difficulties have been encountered in the use of such techniques : a high-quality reagent has been difficult to obtain ; fusion conditions must be rather closely controlled; graphite crucibles used in the fusions need special treatment. Methods for overcoming these difficulties are outlined. Selected procedures for various instrumental methods of analysis are described. ?? 1970.

  14. Large-scale synthesis of isotopically labeled 13C2-tenuazonic acid and development of a rapid HPLC-MS/MS method for the analysis of tenuazonic acid in tomato and pepper products.

    PubMed

    Lohrey, Lilia; Marschik, Stefanie; Cramer, Benedikt; Humpf, Hans-Ulrich

    2013-01-01

    Tenuazonic acid is a fungal secondary metabolite that is produced by a number of Alternaria species and is therefore a natural contaminant of food and feed samples. This paper describes a new strategy for the efficient and economical large-scale synthesis of the isotopically labeled internal standard (13)C(2)-tenuazonic acid via a three-step procedure. Furthermore, a new reliable and quick method based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) cleanup is presented for the determination of tenuazonic acid in food and feed samples utilizing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) by application of the stable isotope dilution analysis. This new method has a limit of detection (LOD) of 0.86 μg/kg and a limit of quantitation (LOQ) of 2.89 μg/kg. In total 26 tomato samples and 4 bell pepper samples from the German market were analyzed. Tenuazonic acid was found in each sample with levels from 3 to 2330 μg/kg.

  15. Large-scale synthesis of isotopically labeled 13C2-tenuazonic acid and development of a rapid HPLC-MS/MS method for the analysis of tenuazonic acid in tomato and pepper products.

    PubMed

    Lohrey, Lilia; Marschik, Stefanie; Cramer, Benedikt; Humpf, Hans-Ulrich

    2013-01-01

    Tenuazonic acid is a fungal secondary metabolite that is produced by a number of Alternaria species and is therefore a natural contaminant of food and feed samples. This paper describes a new strategy for the efficient and economical large-scale synthesis of the isotopically labeled internal standard (13)C(2)-tenuazonic acid via a three-step procedure. Furthermore, a new reliable and quick method based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) cleanup is presented for the determination of tenuazonic acid in food and feed samples utilizing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) by application of the stable isotope dilution analysis. This new method has a limit of detection (LOD) of 0.86 μg/kg and a limit of quantitation (LOQ) of 2.89 μg/kg. In total 26 tomato samples and 4 bell pepper samples from the German market were analyzed. Tenuazonic acid was found in each sample with levels from 3 to 2330 μg/kg. PMID:23230907

  16. Analysis of the Rotational Structure in the High-Resolution Infrared Spectrum of trans-Hexatriene-1-13C1; a Semiexperimental Equilibrium Structure for the C6 Backbone for trans-Hexatriene

    SciTech Connect

    Craig, Norman C.; Tian, Hengfeng; Blake, Thomas A.

    2012-03-29

    trans-Hexatriene-1-13C1 (tHTE-1-13C1) has been synthesized, and its high-resolution (0.0015 cm-1) infrared spectrum has been recorded. The rotational structure in the C-type bands for v26 at 1011 cm-1 and v30 at 894 cm-1 has been analyzed. To the 1458 ground state combination differences from these bands, ground state rotational constants were fitted to a Watson-type Hamiltonian to give A0 = 0.8728202(9), B0 = 0.0435868(4), and C0 = 0.0415314(2) cm-1. Upper state rotational constants for the v30 band were also fitted. Predictions of the ground state rotational constants for t-HTE-1-13C1 from a B3LYP/cc-pVTZ model with scale factors based on the normal species were in excellent agreement with observations. Similar good agreement was found between predicted and observed ground state rotational constants for the three 13C1 isotopologues of cis-hexatriene (cHTE), as determined from microwave spectroscopy. Equilibrium rotational constants for tHTE and its three 13C1 isotopologues, of which two were predicted, were used to find a semiexperimental equilibrium structure for the C6 backbone of tHTE. This structure shows increased structural effects of pi-electron delocalization in comparison with butadiene.

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

    PubMed Central

    Duarte, João M. N.; Lanz, Bernard; Gruetter, Rolf

    2011-01-01

    Cerebral metabolism is compartmentalized between neurons and glia. Although glial glycolysis is thought to largely sustain the energetic requirements of neurotransmission while oxidative metabolism takes place mainly in neurons, this hypothesis is matter of debate. The compartmentalization of cerebral metabolic fluxes can be determined by 13C nuclear magnetic resonance (NMR) spectroscopy upon infusion of 13C-enriched compounds, especially glucose. Rats under light α-chloralose anesthesia were infused with [1,6-13C]glucose and 13C enrichment in the brain metabolites was measured by 13C NMR spectroscopy with high sensitivity and spectral resolution at 14.1 T. This allowed determining 13C enrichment curves of amino acid carbons with high reproducibility and to reliably estimate cerebral metabolic fluxes (mean error of 8%). We further found that TCA cycle intermediates are not required for flux determination in mathematical models of brain metabolism. Neuronal tricarboxylic acid cycle rate (VTCA) and neurotransmission rate (VNT) were 0.45 ± 0.01 and 0.11 ± 0.01 μmol/g/min, respectively. Glial VTCA was found to be 38 ± 3% of total cerebral oxidative metabolism, accounting for more than half of neuronal oxidative metabolism. Furthermore, glial anaplerotic pyruvate carboxylation rate (VPC) was 0.069 ± 0.004 μmol/g/min, i.e., 25 ± 1% of the glial TCA cycle rate. These results support a role of glial cells as active partners of neurons during synaptic transmission beyond glycolytic metabolism. PMID:21713114

  18. 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

  19. A method for simultaneous echo planar imaging of hyperpolarized 13C pyruvate and 13C lactate

    NASA Astrophysics Data System (ADS)

    Reed, Galen D.; Larson, Peder E. Z.; von Morze, Cornelius; Bok, Robert; Lustig, Michael; Kerr, Adam B.; Pauly, John M.; Kurhanewicz, John; Vigneron, Daniel B.

    2012-04-01

    A rapid echo planar imaging sequence for dynamic imaging of [1-13C] lactate and [1-13C] pyruvate simultaneously was developed. Frequency-based separation of these metabolites was achieved by spatial shifting in the phase-encoded direction with the appropriate choice of echo spacing. Suppression of the pyruvate-hydrate and alanine resonances is achieved through an optimized spectral-spatial RF waveform. Signal sampling efficiency as a function of pyruvate and lactate excitation angle was simulated using two site exchange models. Dynamic imaging is demonstrated in a transgenic mouse model, and phantom validations of the RF pulse frequency selectivity were performed.

  20. Ground-fire effects on the composition of dissolved and total organic matter in forest floor and soil solutions from Scots pine forests in Germany: new insights from solid state 13C NMR analysis

    NASA Astrophysics Data System (ADS)

    Näthe, Kerstin; Michalzik, Beate; Levia, Delphis; Steffens, Markus

    2016-04-01

    Fires represent an ecosystem disturbance and are recognized to seriously pertubate the nutrient budgets of forested ecosystems. While the effects of fires on chemical, biological, and physical soil properties have been intensively studied, especially in Mediterranean areas and North America, few investigations examined the effects of fire-induced alterations in the water-bound fluxes and the chemical composition of dissolved and particulate organic carbon and nitrogen (DOC, POC, DN, PN). The exclusion of the particulate organic matter fraction (0.45 μm < POM < 500 μm) potentially results in misleading inferences and budgeting gaps when studying the effects of fires on nutrient and energy fluxes. To our best knowledge, this is the first known study to present fire-induced changes on the composition of dissolved and total organic matter (DOM, TOM) in forest floor (FF) and soil solutions (A, B horizon) from Scots pine forests in Germany. In relation to control sites, we test the effects of low-severity fires on: (1) the composition of DOM and TOM in forest floor and soil solutions; and (2) the translocated amount of particulate in relation to DOC and DN into the subsoil. The project aims to uncover the mechanisms of water-bound organic matter transport along an ecosystem profile and its compositional changes following a fire disturbance. Forest floor and soil solutions were fortnightly sampled from March to December 2014 on fire-manipulated and control plots in a Scots pine forest in Central Germany. Shortly after the experimental duff fire in April 2014 pooled solutions samples were taken for solid-state 13C NMR spectroscopy to characterize DOM (filtered solution < 0.8μm pore size) and TOM in unfiltered solutions. Independent from fire manipulation, the composition of TOM was generally less aromatic (aromaticity index [%] according to Hatcher et al., 1981) with values between 18 (FF) - 25% (B horizon) than the DOM fraction with 23 (FF) - 27% (B horizon). For DOM

  1. High-resolution proton NMR studies of intracellular metabolites in yeast using 13C decoupling

    NASA Astrophysics Data System (ADS)

    Sillerud, Laurel O.; Alger, Jeffry R.; Shulman, Robert G.

    The resolution and specificity of 1H NMR in studies of yeast cellular metabolism were increased by feeding a 13C-labeled substrate and observing 1H difference spectra in the presence and absence of 13C decoupling fields. [2- 13C]Acetate was utilized as a respiratory substrate in an aerobic suspension of Saccharomyces cerevisiae. The broad cellular background proton resonances are removed by the technique, leaving only signals from the protons of the substrate, or its metabolites, that are coupled to 13C. Spectra of the yeast suspension after acetate feeding show the disappearance of label from the acetate pool and the subsequent appearance of 13C in glutamate C 3 and C 4 and in aspartate C 3. These results are in accord with the known fluxes of metabolites. Selective single-frequency 13C decoupling was used to provide assignments for the difference signals. The limitations on single-frequency decoupling coming from finite decoupling fields are investigated. The technique shows a potential for application in a wide variety of systems where the resolution of the 13C spectrum may be combined with the sensitivity for proton detection to observe metabolites that have been previously unobservable.

  2. 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.

  3. 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.

  4. 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...

  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. Comparison and analysis of objective functions in flux balance analysis.

    PubMed

    García Sánchez, Carlos Eduardo; Torres Sáez, Rodrigo Gonzalo

    2014-01-01

    Flux balance analysis (FBA) is currently one of the most important and used techniques for estimation of metabolic reaction rates (fluxes). This mathematical approach utilizes an optimization criterion in order to select a distribution of fluxes from the feasible space delimited by the metabolic reactions and some restrictions imposed over them, assuming that cellular metabolism is in steady state. Therefore, the obtained flux distribution depends on the specific objective function used. Multiple studies have been aimed to compare distinct objective functions at given conditions, in order to determine which of those functions produces values of fluxes closer to real data when used as objective in the FBA; in other words, what is the best objective function for modeling cell metabolism at a determined environmental condition. However, these comparative studies have been designed in very dissimilar ways, and in general, several factors that can change the ideal objective function in a cellular condition have not been adequately considered. Additionally, most of them have used only one dataset for representing one condition of cell growth, and different measuring techniques have been used. For these reasons, a rigorous study on the effect of factors such as the quantity of used data, the number and type of fluxes utilized as input data, and the selected classification of growth conditions, are required in order to obtain useful conclusions for these comparative studies, allowing limiting clearly the application range on any of those results. PMID:25044958

  7. Comparison and analysis of objective functions in flux balance analysis.

    PubMed

    García Sánchez, Carlos Eduardo; Torres Sáez, Rodrigo Gonzalo

    2014-01-01

    Flux balance analysis (FBA) is currently one of the most important and used techniques for estimation of metabolic reaction rates (fluxes). This mathematical approach utilizes an optimization criterion in order to select a distribution of fluxes from the feasible space delimited by the metabolic reactions and some restrictions imposed over them, assuming that cellular metabolism is in steady state. Therefore, the obtained flux distribution depends on the specific objective function used. Multiple studies have been aimed to compare distinct objective functions at given conditions, in order to determine which of those functions produces values of fluxes closer to real data when used as objective in the FBA; in other words, what is the best objective function for modeling cell metabolism at a determined environmental condition. However, these comparative studies have been designed in very dissimilar ways, and in general, several factors that can change the ideal objective function in a cellular condition have not been adequately considered. Additionally, most of them have used only one dataset for representing one condition of cell growth, and different measuring techniques have been used. For these reasons, a rigorous study on the effect of factors such as the quantity of used data, the number and type of fluxes utilized as input data, and the selected classification of growth conditions, are required in order to obtain useful conclusions for these comparative studies, allowing limiting clearly the application range on any of those results.

  8. Dipolar-coupling-mediated total correlation spectroscopy in solid-state 13C NMR: Selection of individual 13C- 13C dipolar interactions

    NASA Astrophysics Data System (ADS)

    Spano, Justin; Wi, Sungsool

    2010-06-01

    Herein is described a useful approach in solid-state NMR, for selecting homonuclear 13C- 13C spin pairs in a multiple- 13C homonuclear dipolar coupled spin system. This method builds upon the zero-quantum (ZQ) dipolar recoupling method introduced by Levitt and coworkers (Marin-Montesinos et al., 2006 [30]) by extending the originally introduced one-dimensional (1D) experiment into a two-dimensional (2D) method with selective irradiation scheme, while moving the 13C- 13C mixing scheme from the transverse to the longitudinal mode, together with a dramatic improvement in the proton decoupling efficiency. Selective spin-pair recoupling experiments incorporating Gaussian and cosine-modulated Gaussian pulses for inverting specific spins were performed, demonstrating the ability to detect informative, simplified/individualized, long-range 13C- 13C homonuclear dipolar coupling interactions more accurately by removing less informative, stronger, short-range 13C- 13C interactions from 2D correlation spectra. The capability of this new approach was demonstrated experimentally on uniformly 13C-labeled Glutamine and a tripeptide sample, GAL.

  9. Study of conformations and hydrogen bonds in the configurational isomers of pyrrole-2-carbaldehyde oxime by 1H, 13C and 15N NMR spectroscopy combined with MP2 and DFT calculations and NBO analysis.

    PubMed

    Afonin, Andrei V; Ushakov, Igor A; Pavlov, Dmitry V; Ivanov, Andrei V; Mikhaleva, Al'bina I

    2010-09-01

    The (1)H, (13)C and (15)N NMR studies have shown that the E and Z isomers of pyrrole-2-carbaldehyde oxime adopt preferable conformation with the syn orientation of the oxime group with respect to the pyrrole ring. The syn conformation of E and Z isomers of pyrrole-2-carbaldehyde oxime is stabilized by the N-H...N and N-H...O intramolecular hydrogen bonds, respectively. The N-H...N hydrogen bond in the E isomer causes the high-frequency shift of the bridge proton signal by about 1 ppm and increase the (1)J(N, H) coupling by approximately 3 Hz. The bridge proton shows further deshielding and higher increase of the (1)J(N, H) coupling constant due to the strengthening of the N-H...O hydrogen bond in the Z isomer. The MP2 calculations indicate that the syn conformation of E and Z isomers is by approximately 3.5 kcal/mol energetically less favorable than the anti conformation. The calculations of (1)H shielding and (1)J(N, H) coupling in the syn and anti conformations allow the contribution to these constants from the N-H...N and N-H...O hydrogen bondings to be estimated. The NBO analysis suggests that the N-H...N hydrogen bond in the E isomer is a pure electrostatic interaction while the charge transfer from the oxygen lone pair to the antibonding orbital of the N-H bond through the N-H...O hydrogen bond occurs in the Z isomer. PMID:20623827

  10. Sediment features, macrozoobenthic assemblages and trophic relationships (delta13C and delta15N analysis) following a dystrophic event with anoxia and sulphide development in the Santa Giusta lagoon (western Sardinia, Italy).

    PubMed

    Magni, P; Rajagopal, S; van der Velde, G; Fenzi, G; Kassenberg, J; Vizzini, S; Mazzola, A; Giordani, G

    2008-01-01

    Macrozoobenthic assemblages and stable carbon (delta(13)C) and nitrogen (delta(15)N) isotope values of various primary producers (macroalgae and angiosperms) and consumers (macroinvertebrate filter/suspension feeders, deposit feeders, detritivores/omnivores and carnivores and fishes) were studied in the Santa Giusta lagoon (Sardinia, Italy) before (spring) and after (autumn) a dystrophic event which occurred in the summer of 2004. A few days after the dystrophy, the physico-chemical characteristics of sediments and macrozoobenthic assemblages were also investigated. In the latter occasion, high total organic carbon (3.9%) and organic matter (15.9%) contents of surface sediments went together with peaks in acid-volatile sulphide concentrations. Certain immediate effects were quite extreme, such as the drastic reduction in macrozoobenthos and the massive fish kill in August 2004. Among the macrozoobenthos, there were few individuals of chironomid larvae and Capitella cf. capitata left. However, by October, chironomid larvae were numerous, indicating a lack of predators (e.g. fish) and competitors. In addition, some bivalve species and polychaetes which were absent, or present in small numbers before the event, became relatively numerous. The results are discussed based on a knowledge of the sulphide tolerance of these species. Stable isotope analysis clearly showed that the basal level of the food web for most consumers consisted mainly of macroalgae and sedimentary organic matter, and that the values before and after the dystrophic event were not significantly different from one another. This indicates that the relations among different trophic levels were quickly restored following the dystrophic event.

  11. Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism

    PubMed Central

    Sauer, Uwe; Lasko, Daniel R.; Fiaux, Jocelyne; Hochuli, Michel; Glaser, Ralf; Szyperski, Thomas; Wüthrich, Kurt; Bailey, James E.

    1999-01-01

    The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional 13C-1H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as

  12. 13C-DEPLETED MICROBIAL LIPIDS INDICATE SEASONAL METHANOTROPHIC ACTIVITY IN SHALLOW ESTUARINE SEDIMENTS

    EPA Science Inventory

    Compound specific isotope analysis was combined with phospholipid fatty acid (PLFA) analysis to identify methanotrophic activity in members of the sedimentary microbial community in the Altamaha and Savannah River estuaries in Georgia. 13C-depleted PLFAs indicate methane utilizat...

  13. Analysis of the rotational structure in the high-resolution infrared spectrum of trans-hexatriene-1-13C1: a semiexperimental equilibrium structure for the C6 backbone of trans-hexatriene.

    PubMed

    Craig, Norman C; Tian, Hengfeng; Blake, Thomas A

    2012-03-29

    trans-Hexatriene-1-(13)C(1) (tHTE-1-(13)C(1)) has been synthesized, and its high-resolution (0.0015 cm(-1)) infrared spectrum has been recorded. The rotational structure in the C-type bands for ν(26) at 1011 cm(-1) and ν(30) at 894 cm(-1) has been analyzed. To the 1458 ground state combination differences from these bands, ground state rotational constants were fitted to a Watson-type Hamiltonian to give A(0) = 0.8728202(9), B(0) = 0.0435868(4), and C(0) = 0.0415314(2) cm(-1). Upper state rotational constants for the ν(30) band were also fitted. Predictions of the ground state rotational constants for tHTE-1-(13)C(1) from a B3LYP/cc-pVTZ model with scale factors based on the normal species were in excellent agreement with observations. Similar good agreement was found between predicted and observed ground state rotational constants for the three (13)C(1) isotopologues of cis-hexatriene, as determined from microwave spectroscopy. Equilibrium rotational constants for tHTE and its three (13)C(1) isotopologues, of which two were predicted, were used to find a semiexperimental equilibrium structure for the C(6) backbone of tHTE. This structure shows increased structural effects of π-electron delocalization in comparison with butadiene and some differences from the cis isomer of HTE. Structures predicted with the MP2/cc-pVTZ model are also compared.

  14. Flux Coupling Analysis of Genome-Scale Metabolic Network Reconstructions

    PubMed Central

    Burgard, Anthony P.; Nikolaev, Evgeni V.; Schilling, Christophe H.; Maranas, Costas D.

    2004-01-01

    In this paper, we introduce the Flux Coupling Finder (FCF) framework for elucidating the topological and flux connectivity features of genome-scale metabolic networks. The framework is demonstrated on genome-scale metabolic reconstructions of Helicobacter pylori, Escherichia coli, and Saccharomyces cerevisiae. The analysis allows one to determine whether any two metabolic fluxes, v1 and v2, are (1) directionally coupled, if a non-zero flux for v1 implies a non-zero flux for v2 but not necessarily the reverse; (2) partially coupled, if a non-zero flux for v1 implies a non-zero, though variable, flux for v2 and vice versa; or (3) fully coupled, if a non-zero flux for v1 implies not only a non-zero but also a fixed flux for v2 and vice versa. Flux coupling analysis also enables the global identification of blocked reactions, which are all reactions incapable of carrying flux under a certain condition; equivalent knockouts, defined as the set of all possible reactions whose deletion forces the flux through a particular reaction to zero; and sets of affected reactions denoting all reactions whose fluxes are forced to zero if a particular reaction is deleted. The FCF approach thus provides a novel and versatile tool for aiding metabolic reconstructions and guiding genetic manipulations. PMID:14718379

  15. 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.

  16. /sup 13/C nuclear magnetic resonance study of the complexation of calcium by taurine

    SciTech Connect

    Irving, C.S.; Hammer, B.E.; Danyluk, S.S.; Klein, P.D.

    1980-01-01

    /sup 13/C Nuclear magnetic resonance chemical shifts, /sup 1/J/sub c-c/ scalar coupling constants, spin-lattice relaxation times, and nuclear Overhauser effects were determined for taurine-(1, 2 /sup 13/C) and a taurine-(1 /sup 13/C) and taurine-(2 /sup 13/C) mixture in the presence and absence of calcium. Comparison of taurine titration shifts to values for related compounds reveals some unusual electronic properties of the taurine molecule. Stability constants of 1:1 calcium complexes with taurine zwitterions and anions, as well as their /sup 13/C chemical shifts, were obtained by least squares analysis of titration curves measured in the presence of calcium. The stability constants of calcium-taurine complexes were significantly lower than previous values and led to estimates that only approximately one percent of intracellular calcium of mammalian myocardial cells would exist in a taurine complex.

  17. 1H-13C/1H-15N Heteronuclear Dipolar Recoupling by R-Symmetry Sequences Under Fast Magic Angle Spinning for Dynamics Analysis of Biological and Organic Solids

    PubMed Central

    Hou, Guangjin; Byeon, In-Ja L.; Ahn, Jinwoo; Gronenborn, Angela M.; Polenova, Tatyana

    2011-01-01

    Fast magic angle spinning (MAS) NMR spectroscopy is becoming increasingly important in structural and dynamics studies of biological systems and inorganic materials. Superior spectral resolution due to the efficient averaging of the dipolar couplings can be attained at MAS frequencies of 40 kHz and higher with appropriate decoupling techniques, while proton detection gives rise to significant sensitivity gains, therefore making fast MAS conditions advantageous across the board compared with the conventional slow- and moderate-MAS approaches. At the same time, many of the dipolar recoupling approaches that currently constitute the basis for structural and dynamics studies of solid materials and that are designed for MAS frequencies of 20 kHz and below, fail above 30 kHz. In this report, we present an approach for 1H-13C/1H-15N heteronuclear dipolar recoupling under fast MAS conditions using R-type symmetry sequences, which is suitable even for fully protonated systems. A series of rotor-synchronized R-type symmetry pulse schemes are explored for the determination of structure and dynamics in biological and organic systems. The investigations of the performance of the various RNnv-symmetry sequences at the MAS frequency of 40 kHz experimentally and by numerical simulations on [U-13C,15N]-alanine and [U-13C,15N]-N-acetyl-valine, revealed excellent performance for sequences with high symmetry number ratio (N/2n > 2.5). Further applications of this approach are presented for two proteins, sparsely 13C/uniformly 15N enriched CAP-Gly domain of dynactin and U-13C,15N-Tyr enriched C-terminal domain of HIV-1 CA protein. 2D and 3D R1632-based DIPSHIFT experiments carried out at the MAS frequency of 40 kHz, yielded site-specific 1H-13C/1H-15N heteronuclear dipolar coupling constants for CAP-Gly and CTD CA, reporting on the dynamic behavior of these proteins on time scales of nano- to microseconds. The R-symmetry based dipolar recoupling under fast MAS is expected to find

  18. Multi-isotope labelling (13C, 18O, 2H) for studying organic matter cycling within plant-soil systems

    NASA Astrophysics Data System (ADS)

    Studer, M. S.; Abiven, S.; Schmidt, M. W. I.; Siegwolf, R. T. W.

    2012-04-01

    Carbon cycling has become of major interest for the understanding and mitigation of global climatic change. Terrestrial ecosystems have a large carbon sequestration potential, but many processes and fluxes of organic matter (OM) cycling within the plant-soil system are not yet well understood [1]. The dynamics of OM cycling within the plant soil-system are determined by environmental parameters, as well as chemical quality of OM input. A well-known technique to study OM dynamics is to label OM inputs with stable isotopes (e.g 13C). Changes in OM quality in the plant and in the soil can be assessed by compound specific isotopic analysis [2]. These techniques give a precise insight of the OM composition, but are laborious and expensive. Here we suggest a new multi-isotope labelling technique using stable 13C in combination with stable 18O and 2H isotopes, which provides information on OM quality by simple bulk material analysis. The method is based on the creation of an isotopic van Krevelen diagram, which is used to describe different compound groups by plotting the atomic ratios of O/C vs. H/C [3]. We could show that new assimilates can be labelled with 13C, 18O and 2H by adding the stable isotopes (continuously) in the gaseous phase (CO2 and water vapour) to the plants atmosphere. The label has been traced within the bulk material of different compartments of the plant-soil system (e.g. leaves, stems, roots, bulk soil). Our first results showed that after 2, 8 and 14 days of labelling the 18O/13C(new) ratio was notably different in leaf, stem and root tissue (0.0024, 0.0011 and 0.0007, respectively), suggesting a change in OM quality towards more C-rich compounds. d2H analysis will follow and an isotopic van Krevelen diagram will be produced (18O/13C(new) vs. 2H/13C(new)) to describe the changes in OM quality. The new multi-isotope labelling approach represent a powerful tool to address open questions in plant and soil research such as the allocation of organic

  19. 13C isotopic fractionation during biodegradation of agricultural wastes.

    PubMed

    Chalk, Phillip M; Inácio, Caio T; Urquiaga, Segundo; Chen, Deli

    2015-01-01

    Significant differences in δ(13)C signatures occur within and between plant tissues and their constituent biochemical entities, and also within and between heterotrophic bacteria and fungi and their metabolic products. Furthermore, (13)C isotopic fractionation occurs during the biodegradation of organic molecules as seen in the substrate, respired CO(2) and the microbial biomass, which could be related to substrate composition and/or microbial metabolism. The (13)C isotopic fractionation observed during the decomposition of a single defined C substrate appears to be due to the intra-molecular heterogeneity in (13)C in the substrate and to (13)C isotopic fractionation during microbial metabolism. Very limited data suggest that the latter may be quantitatively more important than the former. Studies with defined fungi in culture media have highlighted the complexities associated with the interpretation of the observed patterns of (13)C isotopic fractionation when a single defined C source is added to the culture medium which itself contains one or more C sources. Techniques involving (13)C enrichment or paired treatments involving an equivalent C(3)- and C(4)-derived substrate have been devised to overcome the problem of background C in the culture medium and (13)C isotopic fractionation during metabolism. Studies with complex substrates have shown an initial (13)C depletion phase in respired CO(2) followed by a (13)C enrichment phase which may or may not be followed by another (13)C depletion phase. Basic studies involving an integrated approach are required to gain a new insight into (13)C isotopic fractionation during organic residue decomposition, by simultaneous measurements of δ(13)C in all C moieties. New analytical tools to measure real-time changes in δ(13)CO(2) and the intra-molecular δ(13)C distribution within plant biochemical entities offer new opportunities for unravelling the complex interactions between substrate and microbial metabolism with

  20. Catabolism of glucose and lactose in Bifidobacterium animalis subsp. lactis, studied by 13C Nuclear Magnetic Resonance.

    PubMed

    González-Rodríguez, Irene; Gaspar, Paula; Sánchez, Borja; Gueimonde, Miguel; Margolles, Abelardo; Neves, Ana Rute

    2013-12-01

    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 (13)C nuclear magnetic resonance (NMR) spectroscopy. The metabolism of [1-(13)C]glucose was characterized in cells grown in glucose as the sole carbon source. Moreover, the metabolism of lactose specifically labeled with (13)C 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 (13)C on carbon 1, 2, or 3 in cells grown in glucose or lactose specifically labeled in carbon 1 of the glucose moiety ([1-(13)Cglucose]lactose), lactose specifically labeled in carbon 1 of the galactose moiety ([1-(13)Cgalactose]lactose), and [1-(13)C]glucose in lactose-grown cells were determined in cell extracts by (13)C 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

  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. Analysis of commercial proanthocyanidins. Part 4: solid state (13)C NMR as a tool for in situ analysis of proanthocyanidin tannins, in heartwood and bark of quebracho and acacia, and related species.

    PubMed

    Reid, David G; Bonnet, Susan L; Kemp, Gabre; van der Westhuizen, Jan H

    2013-10-01

    (13)C NMR is an effective method of characterizing proanthocyanidin (PAC) tannins in quebracho (Schinopsis lorentzii) heartwood and black wattle (Acacia mearnsii) bark, before and after commercial extraction. The B-rings of the constituent flavan-3-ols, catechols (quebracho) or pyrogallols (wattle), are recognized in unprocessed source materials by "marker" signals at ca. 118 or 105ppm, respectively. NMR allows the minimum extraction efficiency to be calculated; ca. 30%, and ca. 80%, for quebracho heartwood and black wattle bark, respectively. NMR can also identify PAC tannin (predominantly robinetinidin), and compare tannin content, in bark from other acacia species; tannin content decreases in the order A. mearnsii, Acacia pycnantha (87% of A. mearnsii), Acacia dealbata and Acacia decurrens (each 74%) and Acacia karroo (30%). Heartwood from an underexploited PAC tannin source, Searsia lancea, taxonomically close to quebracho, shows abundant profisetinidin and catechin PACs. NMR offers the advantage of being applicable to source materials in their native state, and has potential applications in optimizing extraction processes, identification of tannin sources, and characterization of tannin content in cultivar yield improvement programmes. PMID:23838626

  3. Analysis of commercial proanthocyanidins. Part 4: solid state (13)C NMR as a tool for in situ analysis of proanthocyanidin tannins, in heartwood and bark of quebracho and acacia, and related species.

    PubMed

    Reid, David G; Bonnet, Susan L; Kemp, Gabre; van der Westhuizen, Jan H

    2013-10-01

    (13)C NMR is an effective method of characterizing proanthocyanidin (PAC) tannins in quebracho (Schinopsis lorentzii) heartwood and black wattle (Acacia mearnsii) bark, before and after commercial extraction. The B-rings of the constituent flavan-3-ols, catechols (quebracho) or pyrogallols (wattle), are recognized in unprocessed source materials by "marker" signals at ca. 118 or 105ppm, respectively. NMR allows the minimum extraction efficiency to be calculated; ca. 30%, and ca. 80%, for quebracho heartwood and black wattle bark, respectively. NMR can also identify PAC tannin (predominantly robinetinidin), and compare tannin content, in bark from other acacia species; tannin content decreases in the order A. mearnsii, Acacia pycnantha (87% of A. mearnsii), Acacia dealbata and Acacia decurrens (each 74%) and Acacia karroo (30%). Heartwood from an underexploited PAC tannin source, Searsia lancea, taxonomically close to quebracho, shows abundant profisetinidin and catechin PACs. NMR offers the advantage of being applicable to source materials in their native state, and has potential applications in optimizing extraction processes, identification of tannin sources, and characterization of tannin content in cultivar yield improvement programmes.

  4. Sources and transfers of particulate organic matter in a tropical reservoir (Petit Saut, French Guiana): a multi-tracers analysis using δ13C, C/N ratio and pigments

    NASA Astrophysics Data System (ADS)

    de Junet, A.; Abril, G.; Guérin, F.; Billy, I.; de Wit, R.

    2005-08-01

    Carbon cycling and organic matter transfers in the tropical Sinnamary river system (French Guiana), including a mid-stream reservoir (Petit Saut) and its estuary on the Atlantic coast, were studied during the dry season by analyzing the organic carbon isotopic ratios (δ13C-OC), C/N ratios and pigments contents of suspended matter, sediments, sediments traps and epiphytic and epilithic biofilms. In the River upstream as well as in surface sediments at the entrance of the reservoir and at the littoral zone of the reservoir, particulate organic matter (POM) was in majority of terrestrial origin, with a δ13C-C/N signature close to the one of soil and litter collected in the surrounding forest and with high OC/total pigments ratios. High concentrations of Pheopigments a and b in these surface sediments showed that this terrestrial POM, either carried by the river and eolian transport or present in the soil before flooding, undergoes intense degradation. Deeper in the sediment, the δ13C profile showed a decreasing trend with depth typical of what is found in soils, showing that the flooded soil still remains present at the reservoir bottom 10 years after flooding. At the center of the reservoir, POM in the water column, in sediment traps and in surface sediments was in majority of aquatic origin with low C/N and OC/total pigments ratios. In the oxic epilimnion at 3 m depth, Chl a, Chl b and Lutein showed the predominance of Chlorophyceae to the phytoplankton community. At this depth, a C/N ratio of 21 suggests a large contribution of transparent exopolymeric particles to the bulk POM, which, in addition, was 13C-depleted due to a significant contribution of methanotrophic bacteria. At 7 m depth, below the oxicline, high concentrations of BChl d and occasionally BChl c revealed the presence of anoxygenic phototrophic bacteria, namely Chlorobiaceae. In the sediment traps, Chl a, Chl b, Lutein and BChl c and BChl d confirmed the contribution of plankton to the

  5. Anaerobic Methane Oxidation in Soils - revealed using 13C-labelled methane tracers

    NASA Astrophysics Data System (ADS)

    Riekie, G. J.; Baggs, E. M.; Killham, K. S.; Smith, J. U.

    2008-12-01

    In marine sediments, anaerobic methane oxidation is a significant biogeochemical process limiting methane flux from ocean to atmosphere. To date, evidence for anaerobic methane oxidation in terrestrial environments has proved elusive, and its significance is uncertain. In this study, an isotope dilution method specifically designed to detect the process of anaerobic methane oxidation in methanogenic wetland soils is applied. Methane emissions of soils from three contrasting permanently waterlogged sites in Scotland are investigated in strictly anoxic microcosms to which 13C- labelled methane is added, and changes in the concentration and 12C/13C isotope ratios of methane and carbon dioxide are subsequently measured and used to calculate separate the separate components of the methane flux. The method used takes into account the 13C-methane associated with methanogenesis, and the amount of methane dissolved in the soil. The calculations make no prior assumptions about the kinetics of methane production or oxidation. The results indicate that methane oxidation can take place in anoxic soil environments. The clearest evidence for anaerobic methane oxidation is provided by soils from a minerotrophic fen site (pH 6.0) in Bin Forest underlain by ultra-basic and serpentine till. In the fresh soil anoxic microcosms, net consumption methane was observed, and the amount of headspace 13C-CO2 increased at a greater rate than the 12+13C-CO2, further proof of methane oxidation. A net increase in methane was measured in microcosms of soil from Murder Moss, an alkaline site, pH 6.5, with a strong calcareous influence. However, the 13C-CH4 data provided evidence of methane oxidation, both in the disappearance of C- CH4 and appearance of smaller quantities of 13C-CO2. The least alkaline (pH 5.5) microcosms, of Gateside Farm soil - a granitic till - exhibited net methanogenesis and the changes in 13C-CH4 and 13C-CO2 here followed the pattern expected if no methane is consumed

  6. sup 13 C-enriched ribonucleosides: Synthesis and application of sup 13 C- sup 1 H and sup 13 C- sup 13 C spin-coupling constants to assess furanose and N-glycoside bond conformations

    SciTech Connect

    Kline, P.C.; Serianni, A.S. )

    1990-09-26

    Adenosine (1), cytidine (2), guanosine (3), and uridine (4) have been prepared chemically with {sup 13}C enrichment (99 atom %) at C1{prime} and C2{prime} of the ribose ring. Reliable synthetic protocols have been developed to permit access to millimole quantities of labeled ribonucleosides required for structural studies of stable isotopically labeled oligonucleotides and for in vivo metabolism studies. High-resolution {sup 1}H and {sup 13}C NMR spectra of the enriched ribonucleosides have been obtained, and {sup 13}C-{sup 13}C and {sup 13}C-{sup 1}H spin-coupling constants have been measured for pathways within the {beta}-D-ribofuranose ring and across the N-glycoside bond. Related couplings were determined in methyl {alpha}- and {beta}-D-riboruanosides (5,6), and in two conformationally constrained nucleosides, 2,2{prime}-anhydro-(1-{beta}-D-arabinofuranosyl)uracil (7) and 2{prime},3{prime}-O-isopropylidene-2,5{prime}-O-cyclouridine (8). The latter data were used to construct a crude Karplus curve for the {sup 13}C-C-N-{sup 13}C coupling pathway across the N-glycoside bond in 1-4. {sup 1}H-{sup 1}H, {sup 13}C-{sup 1}H, and {sup 13}C-{sup 13}C coupling data are used to evaluate current models describing the conformational dynamics of 1-4 in aqueous solution.

  7. Leaf and twig delta13C during growth in relation to biochemical composition and respired CO2.

    PubMed

    Eglin, Thomas; Fresneau, Chantal; Lelarge-Trouverie, Caroline; Francois, Christophe; Damesin, Claire

    2009-06-01

    In deciduous trees, the delta(13)C values of leaves are known to diverge during growth from those of woody organs. The main purpose of this study is to determine whether the divergence in delta(13)C between leaves and current-year twigs of Fagus sylvatica (L.) is influenced by changes (i) in the relative contents of organic matter fractions and (ii) in the delta(13)C of respired CO(2). The delta(13)C values of bulk matter, extractive-free matter, lignin, holocellulose, starch, soluble sugars, water-soluble fraction and respired CO(2), as well as their relative contents in bulk matter were determined. The delta(13)C values of biochemical fractions and respired CO(2) showed very similar temporal variations for both leaves and twigs. Variations in bulk matter delta(13)C during growth were, therefore, poorly explained by changes in biochemical composition or in respiratory fractionation and were attributed to the transition from (13)C-enriched reserves (mainly starch) to (13)C-depleted new photoassimilates. The divergence between leaves and twigs was related to higher values of soluble sugar delta(13)C in twigs. However, the difference between lignin and holocellulose delta(13)C varied during growth. This phenomenon was attributed to the delay between holocellulose and lignin deposition. These results may have implications for analysis of organic matter delta(13)C in trees and forest ecosystems.

  8. 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.

  9. High-Resolution Stimulated Raman Spectroscopy and Analysis of ν_2 and ν_3 Bands of of 13C_2H_4 Using the D2h Top Data System

    NASA Astrophysics Data System (ADS)

    Alkadrou, Abdulsamee; Rotger, Maud; Bermejo, Dionisio; Domenech, Jose Luis; Boudon, Vincent

    2016-06-01

    High resolution stimulated Raman spectra of 13C_2H_4 in the regions of the ν_2 and ν_3 Raman active modes have been recorded at at two temperatures (145 and 296 K) based on the quasi continuous-wave (cw) stimulated Raman spectrometer at Instituto de Estructura de la Materia (CSIC) in Madrid. A tensorial formalism adapted to X2Y4 planar asymmetric tops with D2h symmetry has been developed in Dijon and a program suite called D2hTDS (now part of the XTDS/SPVIEW spectroscopic software was proposed to calculate their high-resolution spectra. The effective Hamiltonian operator, involving a polyad structure, and transition moment (dipole moment and polarizability) operators can be systematically expanded to carry out global analyses of many rovibrational bands. A total of 103 and 51 lines corresponding to ν_2 and ν_3 Raman active modes have been assigned and fitted in frequency with a global root mean square deviation of 0.54 × 10-3 cm-1 and 0.36 × 10-3 cm-1, respectively. The figures below shows the stimulated Raman spectrum of the ν_2 and ν_3 bands of 13C_2H_4, compared to the simulation at 296 K. Raballand W, Rotger M, Boudon V, Loëte M. J Mol Spectrosc 2003;217:239-48. Wenger Ch, Boudon V, Rotger M, Champion JP, Sanzharov M. J Mol Spectrosc 2008;251:102-13.

  10. New study of the astrophysical reaction 13C(a,n)16O via the 13C(7Li,t)17O transfer reaction

    NASA Astrophysics Data System (ADS)

    Pellegriti, Maria Grazia; Hammache, F.; Roussel, P.; Audouin, L.; Beaumel, D.; Fortier, S.; Gaudefroy, L.; Kiener, J.; Lefebvre-Schujl, A.; Stanoiu, M.; Tatischeff, V.; Vilmay, M.

    PoS(NIC-IX)161 , , [1] , L. Gaudefroy[2] , J. Kiener[3] , A. Lefebvre-Schuhl[3] , M. Stanoiu[4] , V. The cross section of the 13 C(α,n)16 O reaction is a key ingredient for the comprehension of the s-process (slow neutron captures) in stars. This reaction is considered as the main neutron source for the s-process in low-mass Asymptotic Giant Branch (AGB) stars (1-3 solar mass) [1, 2, 3]. At the α-13 C energies of astrophysical interest (Ecm around 190 keV, corresponding to a tem- perature of 108 K) the contribution of the 17 O α-decay subthreshold resonance at 6.356 MeV to the 13 C(α,n)16 O cross section should be taken into account. The effect of this resonance is controversial after the different analyses of the Kubono et al. measurement [4] of the 6.356 MeV α-spectroscopic factor (Sα ) via the transfer reaction 13 C(6 Li,d)17 O . In order to further investigate the contribution of the 6.356 MeV resonance to the 13 C(α,n)16 O cross section, we performed a new measurement of its Sα factor via a different α-transfer reac- tion, namely the 13 C(7 Li,t)17 O reaction. The experiment was performed at the Orsay Tandem by using a 7 Li beam of 28 and 34 MeV on a 13C target. The angular distribution for the transfer dif- ferential cross section was measured by detecting the tritons at the focal plane of the SPLITPOLE spectrometer. The analysis procedure used in order to extract the yield of the 6.356 MeV level will be described. Preliminary results of the angular distribution will be shown.

  11. Late Quaternary Biomass Changes from 13C Measurements in a Highland Peatbog from Equatorial Africa (Burundi)

    NASA Astrophysics Data System (ADS)

    Aucour, Anne-Marie; Hillaire-Marcel, Claude; Bonnefille, Raymonde

    1994-03-01

    Stable carbon isotope ratios of total organic matter were measured in two cores collected from the Kashiru peatbog in Burundi, Equatorial Africa. The record, which spans at least the last 40,000 yr, documents the C 3-C 4 biomass balance in the organic sediment. Among the major modern peat formers, most plants are C 3 species and are characterized by δ 13C values of -25.5 ± 2.3% (vs PDB). The C 4 plants, which are characterized by higher δ 13C values (-11.3 ± 0.7%) belong to the Gramineae ( Miscanthidium sp.) and Cyperaceae families ( Cyperus latifolius, C. papyrus, Pycreus nigricans). In the fossil record, δ 13C values of total organic matter vary between -28 and -15% in response to the relative fluxes of C 3 and C 4 plants. Before 30,000 yr B.P., low δ 13C values (-23.5 ± 1.1%) match high arboreal pollen contents. From 30,000 to 15,000 yr B.P., higher δ 13C values (-17.6 ± 1.1%) correspond to a significant increase in percentages of grass pollen. During this episode, a short and sharp shift toward lighter carbon isotopic compositions at 21,000 yr B.P. is synchronous with higher input of arboreal pollen. From 15,000 to 12,000 yr B.P., the 13C content decreases (δ 13C = -22.9 ± 1.4%). This shift, which cannot be explained by an increase in the arboreal vegetation, could be explained by the spreading of C 3 Gramineae or C 3 Cyperaceae. The interval from 12,000 to 7000 yr B.P. is poorly documented in these cores due to much lower organic matter accumulation. Low δ 13C values (δ 13C = -25.2 ± 1.3%) are observed from 7000 to 5000 yr B.P., when the pollen data show development of C 3 mountain forest. The Late Holocene is characterized by a mixed C 3-C 4 organic matter accumulation (δ 13C = -20.9 ± 1.6%). This study depicts a change in the dominant photosynthetic pathway among the herbaceous components, notably at the glacial-interglacial transition, when C 3 plants were favored by increased water supply and/or higher atmospheric CO 2 concentration.

  12. 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.

  13. 13C/12C ratios in CO2 extracted from Antarctic ice

    NASA Astrophysics Data System (ADS)

    Friedli, H.; Moor, E.; Oeschger, H.; Siegenthaler, U.; Stauffer, B.

    Air is extracted from bubbles of polar ice samples by a dry extraction method, and the 13C/12C ratio is measured on CO2 separated from the air. Ice samples of typically 700 g are crushed at ca. -20°C, the evolving air is trapped cryogenically, and CO2 is frozen out from this air for mass-spectrometric isotope analysis. First δ13C and δ 18O results of CO2 from Antarctic ice cores are presented, and δ13C is discussed in relation to atmospheric CO2 variations. δ13C of 400 to 800 year old ice is ca. 1.1 %o higher than the 1980 atmospheric value, which agrees well with model-based estimations. The measurement of three ca. 50,000 yr old samples yielded astonishingly low values, but contamination cannot be excluded.

  14. 13C Magic angle spinning NMR analysis and quantum chemical modeling of the bathochromic shift of astaxanthin in alpha-crustacyanin, the blue carotenoprotein complex in the carapace of the lobster Homarus gammarus.

    PubMed

    Weesie, R J; Jansen, F J; Merlin, J C; Lugtenburg, J; Britton, G; de Groot, H J

    1997-06-17

    Selective isotope enrichment, 13C magic angle spinning (MAS) NMR, and semiempirical quantum chemical modeling, have been used to analyze ligand-protein interactions associated with the bathochromic shift of astaxanthin in alpha-crustacyanin, the blue carotenoprotein complex from the carapace of the lobster Homarus gammarus. Spectra of alpha-crustacyanin were obtained after reconstitution with astaxanthins labeled with 13C at positions 4,4', 12,12', 13,13', or 20,20'. The data reveal substantial downfield shifts of 4.9 and 7.0 ppm at positions 12 and 12' in the complex, respectively. In contrast, at the 13 and 13' positions, small upfield shifts of 1.9 ppm were observed upon binding to the protein. These data are in line with previously obtained results for positions 14,14' (3.9 and 6.8 ppm downfield) and 15,15' (0.6 ppm upfield) and confirm the unequal perturbation of both halves after binding of the chromophore. However, these results also show that the main perturbation is of symmetrical origin, since the chemical shift differences exhibit a similar pattern in both halves of the astaxanthin molecule. A small downfield shift of 2.4 ppm was detected for the 4 and 4' positions. Finally, the 20,20' methyl groups are shifted 0.4 ppm upfield by the protein. The full data set provides convincing evidence that charge polarization is of importance for the bathochromic shift. The NMR shifts are compared with calculated charge densities for astaxanthin subjected to variations in protonation states of the ring-functional groups, as models of ligand-protein interactions. Taking into account the color shift and other available optical data, the current model for the mechanisms of interaction with the protein was refined. The results point toward a mechanism in which the astaxanthin is charged and subject to strong electrostatic polarizations originating from both keto groups, most likely a double protonation. PMID:9200677

  15. Fossil chironomid d13C as a new proxy for past methanogenic contribution to benthic food-webs in lakes?

    NASA Astrophysics Data System (ADS)

    van Hardenbroek, M.; Heiri, O. M.; Grey, J.; Bodelier, P. L. E.; Lotter, A. F.

    2009-04-01

    Lake sediments are an important source of atmospheric methane. Methanogenic archaea in lake sediments produce 13C-depleted methane that is partly released to the water column and the atmosphere. Another part is utilized by methane oxidizing bacteria (MOB) that are an important food source for deposit-feeding chironomid larvae (Diptera: Chironomidae). If methane-derived carbon is a significant component of the chironomid diet this will lead to strongly negative d13C in the tissue and exoskeleton of chironomid larvae. Chironomid cuticles, especially the strongly sclerotized head capsules, are well preserved as fossils in lake sediments. If the relationship between modern methane fluxes in lakes and chironomid d13C can be established this would therefore provide an approach for estimating past methane fluxes based on d13C of fossil chironomid remains. Using culturing experiments we show that the stable carbon isotope signature of MOB and other food sources can be traced in chironomid muscle tissue as well as in the fossilizing exoskeleton. In addition we measured d13C in chironomid larval head capsules and other invertebrate remains from a range of surface and downcore sediment samples. Small intra-specific variability (-27.1 ± 0.08 permille) was measured in replicate samples of chironomid head capsules of Corynocera ambigua (n=7). d13C of chironomid head capsules from a several different taxa ranged from -28.0 to -25.8 permille, but in some instances we observed d13C values as low as -36.9 to -31.5 permille, suggesting that carbon from MOB can be successfully traced in fossil and subfossil chironomid remains. Our results demonstrate that the stable carbon isotope signature of MOB is incorporated into chironomid head capsules. Future research will focus on quantifying the relationship between methane fluxes, MOB, and head capsule d13C in order to reconstruct past methane fluxes based on the lake sediment record.

  16. 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

    along the canopy to trunk continuum. We further hypothesize that pronounced depletion along the basipetal transport pathway in A. longifolia (more than 6 per mil from leaf water soluble organic matter to trunk phloem sap) may be due to high stem photosynthesis rates in this green-barked legume. Regardless of these fractionation effects, phloem sap d13C correlated well with environmental parameters driving photosynthesis (photosynthetic photon flux density, soil moisture, vapor pressure deficit) for both species indicating that phloem sap d13C is a good integrative tracer of changes in canopy-level carbon discrimination once species-specific differences in post-photosynthetic fractionation are accounted for. Furthermore, we illustrate that combining sap flow estimated canopy stomatal conductance (gs) with measurements of phloem sap d13C (adjusted for post-photosynthetic fractionation) has significant potential as a relatively non-intensive method for estimating canopy-level carbon assimilation rates in field studies.

  17. Regioselective Syntheses of [13C]4-Labelled Sodium 1-Carboxy-2-(2-ethylhexyloxycarbonyl)ethanesulfonate and Sodium 2-Carboxy-1-(2-ethylhexyloxycarbonyl)ethanesulfonate from [13C]4-Maleic Anhydride

    PubMed Central

    Barsamian, Adam L.; Perkins, Matt J.; Field, Jennifer A.; Blakemore, Paul R.

    2014-01-01

    The entitled monohydrolysis products, also known as α- and β-ethylhexyl sulfosuccinate ('EHSS'), of the surfactant diisooctyl sulfosuccinate ('DOSS') were synthesized in stable isotope labelled form from [13C]4-maleic anhydride. Sodium [13C]4-1-carboxy-2-(2-ethylhexyloxycarbonyl)ethanesulfonate (α-EHSS) was prepared by the method of Larpent by reaction of 2-ethylhexan-1-ol with [13C]4-maleic anhydride followed by regioselective conjugate addition of sodium bisulfite to the resulting monoester (38% overall yield). The regiochemical outcome of bisulfite addition was confirmed by a combination of 13C/13C (INADEQUATE) and 1H/13C (HMBC) NMR spectral correlation experiments. Sodium [13C]4-2-carboxy-1-(2-ethylhexyloxycarbonyl)ethanesulfonate (β-EHSS) was prepared in four steps by reaction of 4-methoxybenzyl alcohol (PMBOH) with [13C]4-maleic anhydride, regioselective sodium bisulfite addition, DCC mediated esterification with 2-ethylhexan-1-ol, and PMB ester deprotection with trifluoroacetic acid (13% overall yield). The regiochemical outcome of the second synthesis was confirmed by a combination of 1JCC scalar coupling constant analysis and 1H/13C (HMBC) NMR spectral correlation. The materials prepared are required as internal standards for the LC-MS/MS trace analysis of the degradation products of DOSS, the anionic surfactant found in Corexit, the oil dispersant used during emergency response efforts connected to the Deepwater Horizon oil spill of April 2010. PMID:24700711

  18. Coupling aboveground and belowground activities using short term fluctuations in 13C composition of soil respiration

    NASA Astrophysics Data System (ADS)

    Epron, D.; Parent, F.; Grossiord, C.; Plain, C.; Longdoz, B.; Granier, A.

    2011-12-01

    There is a growing amount of evidence that belowground processes in forest ecosystems are tightly coupled to aboveground activities. Soil CO2 efflux, the largest flux of CO2 to the atmosphere, is dominated by root respiration and by respiration of microorganisms that find the carbohydrates required to fulfil their energetic costs in the rhizosphere. A close coupling between aboveground photosynthetic activity and soil CO2 efflux is therefore expected. The isotopic signature of photosynthates varies with time because photosynthetic carbon isotope discrimination is dynamically controlled by environmental factors. This temporal variation of δ13C of photosynthate is thought to be transferred along the tree-soil continuum and it will be retrieved in soil CO2 efflux after a time lag that reflects the velocity of carbon transport from canopy to belowground. However, isotopic signature of soil CO2 efflux is not solely affected by photosynthetic carbon discrimination, bur also by post photosynthetic fractionation, and especially by fractionation processes affecting CO2 during the transport from soil layers to surface. Tunable diode laser spectrometry is a useful tool to quantify short-term variation in δ13C of soil CO2 efflux and of CO2 in the soil atmosphere. We set up hydrophobic tubes to measure the vertical profile of soil CO2 concentration and its δ13C composition in a temperate beech forest, and we monitored simultaneously δ13C of trunk and soil CO2 efflux, δ13C of phloem exudate and δ13C of leaf sugars. We evidenced that temporal changes in δ13C of soil CO2 and soil CO2 efflux reflected changes in environmental conditions that affect photosynthetic discrimination and that soil CO2 was 4.4% enriched compared to soil CO2 efflux according to diffusion fractionation. However, this close coupling can be disrupted when advective transport of CO2 took place. We also reported evidences that temporal variations in the isotopic composition of soil CO2 efflux reflect

  19. Software applications toward quantitative metabolic flux analysis and modeling.

    PubMed

    Dandekar, Thomas; Fieselmann, Astrid; Majeed, Saman; Ahmed, Zeeshan

    2014-01-01

    Metabolites and their pathways are central for adaptation and survival. Metabolic modeling elucidates in silico all the possible flux pathways (flux balance analysis, FBA) and predicts the actual fluxes under a given situation, further refinement of these models is possible by including experimental isotopologue data. In this review, we initially introduce the key theoretical concepts and different analysis steps in the modeling process before comparing flux calculation and metabolite analysis programs such as C13, BioOpt, COBRA toolbox, Metatool, efmtool, FiatFlux, ReMatch, VANTED, iMAT and YANA. Their respective strengths and limitations are discussed and compared to alternative software. While data analysis of metabolites, calculation of metabolic fluxes, pathways and their condition-specific changes are all possible, we highlight the considerations that need to be taken into account before deciding on a specific software. Current challenges in the field include the computation of large-scale networks (in elementary mode analysis), regulatory interactions and detailed kinetics, and these are discussed in the light of powerful new approaches.

  20. Tracing the biosynthetic source of essential amino acids in marine turtles using delta13C fingerprints.

    PubMed

    Arthur, Karen E; Kelez, Shaleyla; Larsen, Thomas; Choy, C Anela; Popp, Brian N

    2014-05-01

    Plants, bacteria, and fungi produce essential amino acids (EAAs) with distinctive patterns of delta13C values that can be used as naturally occurring fingerprints of biosynthetic origin of EAAs in a food web. Because animals cannot synthesize EAAs and must obtain them from food, their tissues reflect delta13C(EAA) patterns found in diet, but it is not known how microbes responsible for hindgut fermentation in some herbivores influence the delta13C values of EAAs in their hosts' tissues. We examined whether distinctive delta13C fingerprints of hindgut flora are evident in the tissues of green turtles (Chelonia mydas), which are known to be facultative hindgut fermenters. We determined delta13C(EAA) values in tissues of green turtles foraging herbivorously in neritic habitats of Hawaii and compared them with those from green, olive ridley, and loggerhead turtles foraging carnivorously in oceanic environments of the central and southeast Pacific Ocean. Results of multivariate statistical analysis revealed two distinct groups that could be distinguished based on unique delta13C(EAA) patterns. A three-end-member predictive linear discriminant model indicated that delta13C(EAA) fingerprints existed in the tissues of carnivorous turtles that resembled patterns found in microalgae, which form the base of an oceanic food web, whereas herbivorous turtles derive EAAs from a bacterial or seagrass source. This study demonstrates the capacity for delta13C fingerprinting to establish the biosynthetic origin of EAAs in higher consumers, and that marine turtles foraging on macroalgal diets appear to receive nutritional supplementation from bacterial symbionts in their digestive system. PMID:25000760

  1. Tracing the biosynthetic source of essential amino acids in marine turtles using delta13C fingerprints.

    PubMed

    Arthur, Karen E; Kelez, Shaleyla; Larsen, Thomas; Choy, C Anela; Popp, Brian N

    2014-05-01

    Plants, bacteria, and fungi produce essential amino acids (EAAs) with distinctive patterns of delta13C values that can be used as naturally occurring fingerprints of biosynthetic origin of EAAs in a food web. Because animals cannot synthesize EAAs and must obtain them from food, their tissues reflect delta13C(EAA) patterns found in diet, but it is not known how microbes responsible for hindgut fermentation in some herbivores influence the delta13C values of EAAs in their hosts' tissues. We examined whether distinctive delta13C fingerprints of hindgut flora are evident in the tissues of green turtles (Chelonia mydas), which are known to be facultative hindgut fermenters. We determined delta13C(EAA) values in tissues of green turtles foraging herbivorously in neritic habitats of Hawaii and compared them with those from green, olive ridley, and loggerhead turtles foraging carnivorously in oceanic environments of the central and southeast Pacific Ocean. Results of multivariate statistical analysis revealed two distinct groups that could be distinguished based on unique delta13C(EAA) patterns. A three-end-member predictive linear discriminant model indicated that delta13C(EAA) fingerprints existed in the tissues of carnivorous turtles that resembled patterns found in microalgae, which form the base of an oceanic food web, whereas herbivorous turtles derive EAAs from a bacterial or seagrass source. This study demonstrates the capacity for delta13C fingerprinting to establish the biosynthetic origin of EAAs in higher consumers, and that marine turtles foraging on macroalgal diets appear to receive nutritional supplementation from bacterial symbionts in their digestive system.

  2. 13C editing of glutamate in human brain using J-refocused coherence transfer spectroscopy at 4.1 T.

    PubMed

    Pan, J W; Mason, G F; Vaughan, J T; Chu, W J; Zhang, Y; Hetherington, H P

    1997-03-01

    The method of single quantum 13C editing is analyzed and implemented with water suppressed J-refocused coherence transfer spectroscopy. Analysis of the 13C inversion pulse demonstrates that it is optimally placed into the second echo of the J-refocused sequence. We have used this method to acquire 13C-edited spectra of glutamate from phantoms and in vivo. The turnover of 13C4-labeled glutamate in human brain in vivo was observed in parasagittal gray matter using a volume head coil at 4.1 T with a time resolution of 5.3 min.

  3. A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

    PubMed Central

    2011-01-01

    Background Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of 13C isotopologue data for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) synthesized from [U-13C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes. Results We utilized a stable isotope resolved metabolomics (SIRM) approach to determine the timecourse of 13C incorporation from [U-13C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. 13C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS) was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional 13C enrichment in these subunits against experimental data

  4. Sensitivity and specificity of an abbreviated 13C-mixed triglyceride breath test for measurement of pancreatic exocrine function

    PubMed Central

    Meier, Viola; Wolfram, Kristina U; Rosien, Ulrich; Layer, Peter

    2014-01-01

    Background A modified 13C-mixed triglyceride breath test (13C -MTGT) detects moderate pancreatic exocrine insufficiency noninvasively and reliably, but it requires prolonged breath sampling (6 hours (hr)). Objective We aimed to investigate whether 13C -MTGT can be abbreviated, to optimize clinical usability. Methods We analyzed the 13C-MTGT of 200 consecutive patients, retrospectively. Cumulative 1–5 hr 13C-exhalation values were compared with the standard parameter (6-hr cumulative 13C-exhalation). We determined the sensitivity and specificity of shortened breath sampling periods, by comparison with the normal values from 10 healthy volunteers, whom also underwent a secretin test to quantitate pancreatic secretion. Moreover, we evaluated the influence of gastric emptying (GE), using a 13C-octanoic acid breath test in a subset (N = 117). Results The 1–5 hr cumulative 13C-exhalation tests correlated highly and significantly with the standard parameter (p < 0.0001). Sensitivity for detection of impaired lipolysis was high (≥77%), but the specificity was low (≥38%) for the early measurements. Both parameters were high after 4 hrs (88% and 94%, respectively) and 5 hrs (98% and 91%, respectively). Multivariate linear correlation analysis confirmed that GE strongly influenced early postprandial 13C-exhalation during the 13C-MTGT. Conclusion Shortening of the 13C -MTGT from 6 to 4 hrs of duration was associated with similar diagnostic accuracy, yet increased clinical usability. The influence of GE on early postprandial results of the 13C-MTGT precluded further abbreviation of the test. PMID:25083286

  5. Determination of the Orientation and Dynamics of Ergosterol in Model Membranes Using Uniform 13C Labeling and Dynamically Averaged 13C Chemical Shift Anisotropies as Experimental Restraints

    PubMed Central

    Soubias, O.; Jolibois, F.; Massou, S.; Milon, A.; Réat, V.

    2005-01-01

    A new strategy was established to determine the average orientation and dynamics of ergosterol in dimyristoylphosphatidylcholine model membranes. It is based on the analysis of chemical shift anisotropies (CSAs) averaged by the molecular dynamics. Static 13C CSA tensors were computed by quantum chemistry, using the gauge-including atomic-orbital approach within Hartree-Fock theory. Uniformly 13C-labeled ergosterol was purified from Pichia pastoris cells grown on labeled methanol. After reconstitution into dimyristoylphosphatidylcholine lipids, the complete 1H and 13C assignment of ergosterol's resonances was performed using a combination of magic-angle spinning two-dimensional experiments. Dynamically averaged CSAs were determined by standard side-band intensity analysis for isolated 13C resonances (C3 and ethylenic carbons) and by off-magic-angle spinning experiments for other carbons. A set of 18 constraints was thus obtained, from which the sterol's molecular order parameter and average orientation could be precisely defined. The validity of using computed CSAs in this strategy was verified on cholesterol model systems. This new method allowed us to quantify ergosterol's dynamics at three molar ratios: 16 mol % (Ld phase), 30 mol % (Lo phase), and 23 mol % (mixed phases). Contrary to cholesterol, ergosterol's molecular diffusion axis makes an important angle (14°) with the inertial axis of the rigid four-ring system. PMID:15923221

  6. Analysis of metabolic flux phenotypes for two Arabidopsis mutants with severe impairment in seed storage lipid synthesis

    SciTech Connect

    Lonien, J.; Schwender, J.

    2009-11-01

    Major storage reserves of Arabidopsis (Arabidopsis thaliana) seeds are triacylglycerols (seed oils) and proteins. Seed oil content is severely reduced for the regulatory mutant wrinkled1 (wri1-1; At3g54320) and for a double mutant in two isoforms of plastidic pyruvate kinase (pkp{beta}{sub 1}pkp{alpha}; At5g52920 and At3g22960). Both already biochemically well-characterized mutants were now studied by {sup 13}C metabolic flux analysis of cultured developing embryos based on comparison with their respective genetic wild-type backgrounds. For both mutations, in seeds as well as in cultured embryos, the oil fraction was strongly reduced while the fractions of proteins and free metabolites increased. Flux analysis in cultured embryos revealed changes in nutrient uptakes and fluxes into biomass as well as an increase in tricarboxylic acid cycle activity for both mutations. While in both wild types plastidic pyruvate kinase (PK{sub p}) provides most of the pyruvate for plastidic fatty acid synthesis, the flux through PK{sub p} is reduced in pkp{beta}{sub 1}pkp{alpha} by 43% of the wild-type value. In wri1-1, PK{sub p} flux is even more reduced (by 82%), although the genes PKp{beta}{sub 1} and PKp{alpha} are still expressed. Along a common paradigm of metabolic control theory, it is hypothesized that a large reduction in PK{sub p} enzyme activity in pkp{beta}{sub 1}pkp{alpha} has less effect on PK{sub p} flux than multiple smaller reductions in glycolytic enzymes in wri1-1. In addition, only in the wri1-1 mutant is the large reduction in PK{sub p} flux compensated in part by an increased import of cytosolic pyruvate and by plastidic malic enzyme. No such limited compensatory bypass could be observed in pkp{beta}{sub 1}pkp{alpha}.

  7. 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

  8. In vivo quantification of neuro-glial metabolism and glial glutamate concentration using 1H-[13C] MRS at 14.1T.

    PubMed

    Lanz, Bernard; Xin, Lijing; Millet, Philippe; Gruetter, Rolf

    2014-01-01

    Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo (13) C NMR spectroscopy coupled with the infusion labeled glial-specific substrate, such as acetate. In this study, we infused alpha-chloralose anesthetized rats with [2-(13) C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using (1) H-[(13) C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two-compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (Vg ) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (VNT ) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (VPC ) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (Vx (g) and Vx (n) ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations. In this (13) C-acetate labeling study, we propose a refined two-compartment analysis of brain energy metabolism based on (13) C turnover curves of acetate, glutamate and glutamine measured with state of the art in vivo dynamic MRS at high magnetic field in rats, enabling a deeper understanding of the specific role of glial cells in brain oxidative metabolism. In addition, the robustness of the metabolic fluxes determination relative to MRS data quality was carefully studied. PMID:24117599

  9. Hyperpolarized [1,(13)C]pyruvate in lysed human erythrocytes: effects of co-substrate supply on reaction time courses.

    PubMed

    Pagès, Guilhem; Tan, Yee Ling; Kuchel, Philip W

    2014-10-01

    Hyperpolarized [1,(13)C]pyruvate was injected rapidly into haemolysates in which hydrolysis of nicotinamide adenine dinucleotide (phosphate) (NAD(P))/NAD(P)H had been inhibited with nicotinamide. Haemolysates provide a stable glycolytic system in which membrane permeability is not a flux-controlling step, and they enable the concentration of NADH to be adjusted experimentally while keeping the rest of the sample with the same composition as that of the cytoplasm of the cell (albeit diluted twofold at the time of injection of the [1,(13)C]pyruvate). We showed that the maximum amplitude of the (13)C NMR signal from the [1,(13)C]L-lactate, produced from [1,(13)C]pyruvate, and the time at which it occurred was dependent on NADH concentration, as predicted by enzyme-kinetic analysis. The main feature of such curves was dictated by the immediacy of the supply of the co-substrate of lactate dehydrogenase (LDH, EC 1.1.1.27), and we posit that this also pertains in vivo in various tissues including neoplasms. By constructing an appropriate mathematical model and by using a Markov-chain Monte Carlo approach, we fitted experimental data to estimate LDH and NADH concentrations. Experiments carried out with only endogenous NADH present enabled the estimation of its effective concentration in human RBCs; the ability to make this estimate is a special feature of the rapid-dissolution dynamic nuclear polarization method. We found an endogenous NADH concentration in human RBCs two to four times higher than previously reported. PMID:25111006

  10. Structure of B sub 13 C sub 2

    SciTech Connect

    Bylander, D.M.; Kleinman, L. )

    1991-01-15

    By comparing calculated lattice constants with x-ray data as well as by comparison of calculated free energies, we find that the correct structure of B{sub 13}C{sub 2} is B{sub 12}(CBC) rather than B{sub 11}C(BBC), as had been suggested. We also show that B{sub 12}C{sub 3} is stable against 13B{sub 12}C{sub 3}{r arrow}12B{sub 13}C{sub 2}+15C as is B{sub 13}C{sub 2} against 3B{sub 13}C{sub 2}{r arrow}2B{sub 12}C{sub 3}+15B.

  11. 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.

  12. Dihydroflavanonols from Cedrus deodara, A (13)C NMR study.

    PubMed

    Agrawal, P K; Agarwal, S K; Rastogi, R P; Osterdahal, B G

    1981-09-01

    High resolution (13)C NMR study of taxifolin, cedeodarin, cedrin and their methyl ethers allowed unambiguous placement of the Me in 5,7-dihydroxyflavanonol nucleus, besides providing other valuable information on the substitution pattern in the molecule.

  13. Late-Quaternary variation in C3 and C4 grass abundance in southeastern Australia as inferred from δ13C analysis: Assessing the roles of climate, pCO2, and fire

    NASA Astrophysics Data System (ADS)

    Nelson, David M.; Urban, Michael A.; Kershaw, A. Peter; Hu, Feng Sheng

    2016-05-01

    Climate, atmospheric pCO2, and fire all may exert major influences on the relative abundance of C3 and C4 grasses in the present-day vegetation. However, the relative role of these factors in driving variation in C3 and C4 grass abundances in the paleorecord is uncertain, and C4 abundance is often interpreted narrowly as a proxy indicator of aridity or pCO2. We measured δ13C values of individual grains of grass (Poaceae) pollen in the sediments of two sites in southeastern Australia to assess changes in the proportions of C3 and C4 grasses during the past 25,000 years. These data were compared with shifts in pCO2, temperature, moisture balance, and fire to assess how these factors were related to long-term variation of C4 grass abundance during the late Quaternary. At Caledonia Fen, a high-elevation site in the Snowy Mountains, C4 grass abundance decreased from an average of 66% during the glacial period to 11% during the Holocene, primarily in response to increased pCO2 and temperature. In contrast, this pattern did not exist in low-elevation savannah woodlands around Tower Hill Northwest Crater, where C4 grass abundance instead varied in response to shifts in regional aridity. Fire did not appear to have strongly influenced the proportions of C3 and C4 grasses on the landscape at millennial timescales at either site. These patterns are similar to those of a recent study in East Africa, suggesting that elevation-related climatic differences influence how the abundance of C3 and C4 grasses responds to shifts in climate and pCO2. These results caution against using C4 plant abundance as a proxy indicator of either climate or pCO2 without an adequate understanding of key controlling factors.

  14. Pulsed polarization transfer for 13C NMR in solids

    NASA Astrophysics Data System (ADS)

    Bax, Ad; Szeverenyi, Nikolaus M.; Maciel, Gary E.

    A new pulsed polarization transfer experiment method is described for the polarization of 13C spins in a solid by magnetization transfer from protons. The method is directly analogous to the INEPT sequence for liquids introduced by Freeman and Morris. As polarization is transferred in PPT between individual 1H 13C pairs, rather than between spin reservoirs, different opportunities exist for structurally selective experiments. Results on p-diethoxybenzene and coronene are presented.

  15. Stable isotopic signatures (δ13C, δD) of methane from European landfill sites

    NASA Astrophysics Data System (ADS)

    Bergamaschi, P.; Lubina, C.; KöNigstedt, R.; Fischer, H.; Veltkamp, A. C.; Zwaagstra, O.

    1998-04-01

    The stable isotopic signatures (δ13C, δD) of CH4 from four German and Dutch landfill sites have been characterized using different techniques for isotope analysis (tunable diode laser absorption spectroscopy and isotope ratio mass spectrometry). Samples taken directly from the gas collection systems show fairly uniform, biogenic δ13C-δD isotopic signatures [δ13C = (-59.0±2.2)‰ VPDB (n = 104); δD = (-304±10)‰ VSMOW (n = 46)]. In contrast, emission samples taken with static chambers on soil-covered landfill areas exhibit a considerable δ13C-δD variability, mainly due to the influence of aerobic bacterial CH4 oxidation, which occurs when the biogas CH4 encounters atmospheric oxygen available in the uppermost region of the cover soil. Soil gas samples from the landfill covers clearly show the progressive isotopic enrichment within the aerobic regions of the soil. Isotope fractionation factors due to CH4 oxidation were determined to be α(δ13C) = 1.008±0.004 and α(δD) = 1.039±0.026. On average, about 80% (70-97%) of CH4 is oxidized during the transport through cover soils, while no significant CH4 oxidation was found in uncovered areas consisting of freshly dumped waste. Area-integrated δ13C values of total emissions were derived from upwind-downwind measurements around the landfill and show very little temporal and site-to-site variation (δ13C = (-55.4±1.4)‰ VPDB (n = 13; four different landfills)). CH4 budgets were established for two landfill sites, indicating that projected CH4 surface emissions from uncovered and covered areas are significantly lower compared to total CH4 production (for a landfill without gas collection) or compared to the difference between CH4 production and recovery (for a landfill with a gas collection system). For these two landfill sites the overall fraction of CH4 oxidation is estimated to be 46 and 39% (53%) of total CH4 production (minus recovery). Furthermore, the δ13C balance (comparing the δ13C values of the

  16. Abundance anomaly of the 13C species of CCH

    NASA Astrophysics Data System (ADS)

    Sakai, N.; Saruwatari, O.; Sakai, T.; Takano, S.; Yamamoto, S.

    2010-03-01

    Aims: We have observed the N = 1-0 lines of CCH and its 13C isotopic species toward a cold dark cloud, TMC-1 and a star-forming region, L1527, to investigate the 13C abundances and formation pathways of CCH. Methods: The observations have been carried out with the IRAM 30 m telescope. Results: We have successfully detected the lines of 13CCH and C13CH toward the both sources and found a significant intensity difference between the two 13C isotopic species. The [C13CH] /[13CCH] abundance ratios are 1.6 ± 0.4 (3σ) and 1.6 ± 0.1 (3σ) for TMC-1 and L1527, respectively. The abundance difference between C13CH and 13CCH means that the two carbon atoms of CCH are not equivalent in the formation pathway. On the other hand, the [CCH]/[C13CH] and [CCH]/[13CCH] ratios are evaluated to be larger than 170 and 250 toward TMC-1, and to be larger than 80 and 135 toward L1527, respectively. Therefore, both of the 13C species are significantly diluted in comparison with the interstellar 12C/13C ratio of 60. The dilution is discussed in terms of a behavior of 13C in molecular clouds.

  17. Measurement of soil carbon oxidation state and oxidative ratio by 13C nuclear magnetic resonance

    USGS Publications Warehouse

    Hockaday, W.C.; Masiello, C.A.; Randerson, J.T.; Smernik, R.J.; Baldock, J.A.; Chadwick, O.A.; Harden, J.W.

    2009-01-01

    The oxidative ratio (OR) of the net ecosystem carbon balance is the ratio of net O2 and CO2 fluxes resulting from photosynthesis, respiration, decomposition, and other lateral and vertical carbon flows. The OR of the terrestrial biosphere must be well characterized to accurately estimate the terrestrial CO2 sink using atmospheric measurements of changing O2 and CO2 levels. To estimate the OR of the terrestrial biosphere, measurements are needed of changes in the OR of aboveground and belowground carbon pools associated with decadal timescale disturbances (e.g., land use change and fire). The OR of aboveground pools can be measured using conventional approaches including elemental analysis. However, measuring the OR of soil carbon pools is technically challenging, and few soil OR data are available. In this paper we test three solid-state nuclear magnetic resonance (NMR) techniques for measuring soil OR, all based on measurements of the closely related parameter, organic carbon oxidation state (Cox). Two of the three techniques make use of a molecular mixing model which converts NMR spectra into concentrations of a standard suite of biological molecules of known C ox. The third technique assigns Cox values to each peak in the NMR spectrum. We assess error associated with each technique using pure chemical compounds and plant biomass standards whose Cox and OR values can be directly measured by elemental analyses. The most accurate technique, direct polarization solid-state 13C NMR with the molecular mixing model, agrees with elemental analyses to ??0.036 Cox units (??0.009 OR units). Using this technique, we show a large natural variability in soil Cox and OR values. Soil Cox values have a mean of -0.26 and a range from -0.45 to 0.30, corresponding to OR values of 1.08 ?? 0.06 and a range from 0.96 to 1.22. We also estimate the OR of the carbon flux from a boreal forest fire. Analysis of soils from nearby intact soil profiles imply that soil carbon losses associated

  18. An overview of methods using 13C for improved compound identification in metabolomics and natural products

    PubMed Central

    Clendinen, Chaevien S.; Stupp, Gregory S.; Ajredini, Ramadan; Lee-McMullen, Brittany; Beecher, Chris; Edison, Arthur S.

    2015-01-01

    Compound identification is a major bottleneck in metabolomics studies. In nuclear magnetic resonance (NMR) investigations, resonance overlap often hinders unambiguous database matching or de novo compound identification. In liquid chromatography-mass spectrometry (LC-MS), discriminating between biological signals and background artifacts and reliable determination of molecular formulae are not always straightforward. We have designed and implemented several NMR and LC-MS approaches that utilize 13C, either enriched or at natural abundance, in metabolomics applications. For LC-MS applications, we describe a technique called isotopic ratio outlier analysis (IROA), which utilizes samples that are isotopically labeled with 5% (test) and 95% (control) 13C. This labeling strategy leads to characteristic isotopic patterns that allow the differentiation of biological signals from artifacts and yield the exact number of carbons, significantly reducing possible molecular formulae. The relative abundance between the test and control samples for every IROA feature can be determined simply by integrating the peaks that arise from the 5 and 95% channels. For NMR applications, we describe two 13C-based approaches. For samples at natural abundance, we have developed a workflow to obtain 13C–13C and 13C–1H statistical correlations using 1D 13C and 1H NMR spectra. For samples that can be isotopically labeled, we describe another NMR approach to obtain direct 13C–13C spectroscopic correlations. These methods both provide extensive information about the carbon framework of compounds in the mixture for either database matching or de novo compound identification. We also discuss strategies in which 13C NMR can be used to identify unknown compounds from IROA experiments. By combining technologies with the same samples, we can identify important biomarkers and corresponding metabolites of interest. PMID:26379677

  19. Transport and Deposition of 13c From Methane Injection into Detached H-Mode Plasmas in DIII-D

    SciTech Connect

    Wampler, W R; McLean, A G; Allen, S L; Brooks, N H; Elder, J D; Fenstermacher, M E; Groth, M; Stangeby, P C; West, W P; Whyte, D G

    2006-06-01

    Experiments are described which examine the transport and deposition of carbon entering the main plasma scrape-off layer in DIII-D. {sup 13}CH{sub 4} was injected from a toroidally symmetric source into the crown of lower single-null detached ELMy H-mode plasmas. {sup 13}C deposition, mapped by nuclear reaction analysis of tiles, was high at the inner divertor but absent at the outer divertor, as found previously for low density L-mode plasmas. This asymmetry indicates that ionized carbon is swept towards the inner divertor by a fast flow in the scrape-off layer. In the private flux region between inner and outer strike points, carbon deposition was low for L-mode but high for the H-mode plasmas. OEDGE modeling reproduces observed deposition patterns and indicates that neutral carbon dominates deposition in the divertor from detached H-mode plasmas.

  20. Anthropogenic and solar forcing in δ13C time pattern of coralline sponges.

    PubMed

    Madonia, Paolo; Reitner, Joachim

    2014-01-01

    We present the results of a re-analysis of a previously published carbon isotope data-set related to coralline sponges in the Caribbean Sea. The original interpretation led to the discrimination between a pre-industrial period, with a signal controlled by solar-induced climatic variations, followed by the industrial era, characterized by a progressive δ(13)C negative shift due to the massive anthropogenic carbon emissions. Our re-analysis allowed to extract from the raw isotopic data evidence of a solar forcing still visible during the industrial era, with a particular reference to the 88-year Gleissberg periods. These signals are related to slope changes in both the δ(13)C versus time and the δ(13)C versus carbon emission curves.

  1. Inelastic pion scattering by /sup 13/C at low energies

    SciTech Connect

    Mitchell, J.H.

    1987-03-01

    Angular distributions for inelastically scattered pions were obtained for several states in /sup 13/C at an incident energy of 65 MeV. The data include results from both ..pi../sup +/ and ..pi../sup -/ measurements. In addition, ..pi../sup -/ measurements were made at T/sub ..pi../ = 50 MeV at one angle to give a two point fixed-q excitation function. The data are compared to theory and the data of others. As might be expected, medium corrections are shown to be considerably more important at low energies than at resonance. This is true for inelastic transitions of multipolarity 0,2 and 3. Parameters derived from an analysis of elastic pion scattering and SCX data also provide an adequate description of the inelastic transitions. The charge asymmetry in the cross sections for the 9/2/sup +/ state that was seen at resonance persists at these energies. This result is consistent with an impulse approximation treatment of the spin-flip amplitude. This is true even though the incoming energy of the pions is far below the range where the validity of an impulse treatment is expected. 65 refs., 45 figs.

  2. 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.

  3. Two new organic reference materials for δ13C and δ15N measurements and a new value for the δ13C of NBS 22 oil

    USGS Publications Warehouse

    Qi, Haiping; Coplen, Tyler B.; Geilmann, Heike; Brand, Willi A.; Bohlke, John Karl

    2003-01-01

    Analytical grade L-glutamic acid is chemically stable and has a C/N mole ratio of 5, which is close to that of many of natural biological materials, such as blood and animal tissue. Two L-glutamic acid reference materials with substantially different 13C and 15N abundances have been prepared for use as organic reference materials for C and N isotopic measurements. USGS40 is analytical grade L-glutamic acid and has a δ13C value of −26.24‰ relative to VPDB and a δ15N value of −4.52‰ relative to N2 in air. USGS41 was prepared by dissolving analytical grade L-glutamic acid with L-glutamic acid enriched in 13C and 15N. USGS41 has a δ13C value of +37.76‰ and a δ15N value of +47.57‰. The δ13C and δ15N values of both materials were measured against the international reference materials NBS 19 calcium carbonate (δ13C = +1.95‰), L-SVEC lithium carbonate (δ13C = −46.48‰), IAEA-N-1 ammonium sulfate (δ15N = 0.43‰), and USGS32 potassium nitrate (δ15N = 180‰) by on-line combustion continuous-flow and off-line dual-inlet isotope-ratio mass spectrometry. Both USGS40 and USGS41 are isotopically homogeneous; reproducibility of δ13C is better than 0.13‰, and that of δ15N is better than 0.13‰ in 100-μg amounts. These two isotopic reference materials can be used for (i) calibrating local laboratory reference materials, and (ii) quantifying drift with time, mass-dependent fractionations, and isotope-ratio-scale contraction in the isotopic analysis of various biological materials. Isotopic results presented in this paper yield a δ13C value for NBS 22 oil of −29.91‰, in contrast to the commonly accepted value of −29.78‰ for which off-line blank corrections probably have not been quantified satisfactorily.

  4. Steady-state flux ratio analysis: application to biological transport.

    PubMed

    Wagg, J; Chapman, J B

    1995-05-01

    A thermodynamically constrained method of flux ratio analysis, based upon a previously developed thermodynamic rate ratio equation has been developed. In this paper it is demonstrated that, for a complex reaction, application of the thermodynamic rate ratio equation may provide a useful tool for the interpretation of unidirectional flux data thought to be mediated by the reaction, provided that: (i) a clearly defined mechanism for the reaction has been proposed; (ii) a set of partial reaction components may be defined for the reaction from the proposed mechanism, with the rate ratio of at least one of these components being amenable to experimental measurement. This paper defines the conditions for which the rate ratio of a component reaction may be measured, and illustrates the basic principles underlying this approach to flux ratio analysis by direct application to a number of hypothetical mechanisms for biological transport phenomena.

  5. Application of 13C isotope labeling using liquid chromatography mass spectrometry (LC-MS) to determining phosphate-containing metabolic incorporation

    PubMed Central

    Bhowmik, Salil Kumar; Putluri, Vasanta; Kommagani, Ramakrishna; Konde, Sai Aparna; Lydon, John P.; Sreekumar, Arun; Putluri, Nagireddy

    2016-01-01

    Here, we describe an approach wherein negative electrospray ionization mass spectrometry has used to understand the relative flux through phosphate containing metabolic intermediates associated with central carbon metabolism after administering cells with 13C-labeled substrates. The method was applied to examine the 13C incorporation through glycolysis in T47D breast cancer cells and showed reduction of glycolytic relative flux upon treatment with 2-Deoxyglucose. PMID:24338880

  6. Origin differentiation of heroin sample and its acetylating agent with (13)C isotope ratio mass spectrometry.

    PubMed

    Zhang, Daming; Sun, Wei; Yuan, Zengping; Ju, Huangxian; Shi, Xuejun; Wang, Chonghu

    2005-01-01

    A novel method for deducing the origins of heroin and the reagent used for acetylation was established based on delta(13)C determinations of heroin and its hydrolysate, morphine, using gas chromatography (13)C isotope ratio mass spectrometry (GC-C-IRMS). The alkaline and acid hydrolysis conditions of heroin were optimized. Both yield and purity of morphine produced could meet the requirement for a GC-C-IRMS analysis. Using (2-diethylaminoethyl-2,2- diphenylvalerate) as internal standard the determinations of heroin and morphine contents were performed with a GC method in a linear range of 0.2 to 2.0 mg ml(1) that was required to gain the isotope ratio results. The hydrolysis and synthesis of heroin did not change the delta(13)C value of morphine. The precision for delta(13)C detection of both heroin and morphine was sufficient for origin differentiation of heroin samples. The information about the origins of acetylation reagents could be deduced from the difference of delta(13)C values between heroin and morphine. The results for origin differentiation of 10 heroin samples grouped into different regions and their acetylating agents were satisfactory.

  7. An Analysis of Moisture Fluxes into the Gulf of California

    NASA Technical Reports Server (NTRS)

    Wu, Man-Li C.; Schubert, Siegfried D.; Suarez, Max J.; Huang, Norden E.

    2009-01-01

    This study examines the nature of episodes of enhanced warm-season moisture flux into the Gulf of California. Both spatial structure and primary time scales of the fluxes are examined using the 40-yr ECMWF Re-Analysis data for the period 1980-2001. The analysis approach consists of a compositing technique that is keyed on the low-level moisture fluxes into the Gulf of California. The results show that the fluxes have a rich spectrum of temporal variability, with periods of enhanced transport over the gulf linked to African easterly waves on subweekly (3-8 day) time scales, the Madden-Julian oscillation (MJO) at intraseasonal time scales (20-90 day), and intermediate (10-15 day) time-scale disturbances that appear to originate primarily in the Caribbean Sea-western Atlantic Ocean. In the case of the MJO, enhanced low-level westerlies and large-scale rising motion provide an environment that favors large-scale cyclonic development near the west coast of Central America that, over the course of about 2 weeks, expands northward along the coast eventually reaching the mouth of the Gulf of California where it acts to enhance the southerly moisture flux in that region. On a larger scale, the development includes a northward shift in the eastern Pacific ITCZ, enhanced precipitation over much of Mexico and the southwestern United States, and enhanced southerly/southeasterly fluxes from the Gulf of Mexico into Mexico and the southwestern and central United States. In the case of the easterly waves, the systems that reach Mexico appear to redevelop/reorganize on the Pacific coast and then move rapidly to the northwest to contribute to the moisture flux into the Gulf of California. The most intense fluxes into the gulf on these time scales appear to be synchronized with a midlatitude short-wave trough over the U.S. West Coast and enhanced low-level southerly fluxes over the U.S. Great Plains. The intermediate (10-15 day) time-scale systems have zonal wavelengths roughly twice

  8. Transport and imaging of brute-force (13)C hyperpolarization.

    PubMed

    Hirsch, Matthew L; Smith, Bryce A; Mattingly, Mark; Goloshevsky, Artem G; Rosay, Melanie; Kempf, James G

    2015-12-01

    We demonstrate transport of hyperpolarized frozen 1-(13)C pyruvic acid from its site of production to a nearby facility, where a time series of (13)C images was acquired from the aqueous dissolution product. Transportability is tied to the hyperpolarization (HP) method we employ, which omits radical electron species used in other approaches that would otherwise relax away the HP before reaching the imaging center. In particular, we attained (13)C HP by 'brute-force', i.e., using only low temperature and high-field (e.g., T<∼2K and B∼14T) to pre-polarize protons to a large Boltzmann value (∼0.4% (1)H polarization). After polarizing the neat, frozen sample, ejection quickly (<1s) passed it through a low field (B<100G) to establish the (1)H pre-polarization spin temperature on (13)C via the process known as low-field thermal mixing (yielding ∼0.1% (13)C polarization). By avoiding polarization agents (a.k.a. relaxation agents) that are needed to hyperpolarize by the competing method of dissolution dynamic nuclear polarization (d-DNP), the (13)C relaxation time was sufficient to transport the sample for ∼10min before finally dissolving in warm water and obtaining a (13)C image of the hyperpolarized, dilute, aqueous product (∼0.01% (13)C polarization, a >100-fold gain over thermal signals in the 1T scanner). An annealing step, prior to polarizing the sample, was also key for increasing T1∼30-fold during transport. In that time, HP was maintained using only modest cryogenics and field (T∼60K and B=1.3T), for T1((13)C) near 5min. Much greater time and distance (with much smaller losses) may be covered using more-complete annealing and only slight improvements on transport conditions (e.g., yielding T1∼5h at 30K, 2T), whereas even intercity transfer is possible (T1>20h) at reasonable conditions of 6K and 2T. Finally, it is possible to increase the overall enhancement near d-DNP levels (i.e., 10(2)-fold more) by polarizing below 100mK, where nanoparticle

  9. Transport and imaging of brute-force 13C hyperpolarization

    NASA Astrophysics Data System (ADS)

    Hirsch, Matthew L.; Smith, Bryce A.; Mattingly, Mark; Goloshevsky, Artem G.; Rosay, Melanie; Kempf, James G.

    2015-12-01

    We demonstrate transport of hyperpolarized frozen 1-13C pyruvic acid from its site of production to a nearby facility, where a time series of 13C images was acquired from the aqueous dissolution product. Transportability is tied to the hyperpolarization (HP) method we employ, which omits radical electron species used in other approaches that would otherwise relax away the HP before reaching the imaging center. In particular, we attained 13C HP by 'brute-force', i.e., using only low temperature and high-field (e.g., T < ∼2 K and B ∼ 14 T) to pre-polarize protons to a large Boltzmann value (∼0.4% 1H polarization). After polarizing the neat, frozen sample, ejection quickly (<1 s) passed it through a low field (B < 100 G) to establish the 1H pre-polarization spin temperature on 13C via the process known as low-field thermal mixing (yielding ∼0.1% 13C polarization). By avoiding polarization agents (a.k.a. relaxation agents) that are needed to hyperpolarize by the competing method of dissolution dynamic nuclear polarization (d-DNP), the 13C relaxation time was sufficient to transport the sample for ∼10 min before finally dissolving in warm water and obtaining a 13C image of the hyperpolarized, dilute, aqueous product (∼0.01% 13C polarization, a >100-fold gain over thermal signals in the 1 T scanner). An annealing step, prior to polarizing the sample, was also key for increasing T1 ∼ 30-fold during transport. In that time, HP was maintained using only modest cryogenics and field (T ∼ 60 K and B = 1.3 T), for T1(13C) near 5 min. Much greater time and distance (with much smaller losses) may be covered using more-complete annealing and only slight improvements on transport conditions (e.g., yielding T1 ∼ 5 h at 30 K, 2 T), whereas even intercity transfer is possible (T1 > 20 h) at reasonable conditions of 6 K and 2 T. Finally, it is possible to increase the overall enhancement near d-DNP levels (i.e., 102-fold more) by polarizing below 100 mK, where

  10. A 13C-NMR study of azacryptand complexes.

    PubMed

    Wild, Aljoscha A C; Fennell, Kevin; Morgan, Grace G; Hewage, Chandralal M; Malthouse, J Paul G

    2014-09-28

    An azacryptand has been solubilised in aqueous media containing 50% (v/v) dimethyl sulphoxide. (13)C-NMR has been used to determine how the azacryptand is affected by zinc binding at pH 10. Using (13)C-NMR and (13)C-enriched bicarbonate we have been able to observe the formation of 4 different carbamate derivatives of the azacryptand at pH 10. The azacryptand was shown to solubilise zinc or cadmium at alkaline pHs. Two moles of zinc are bound per mole of azacryptand and this complex binds 1 mole of carbonate. By replacing the zinc with cadmium-113 we have shown that the (13)C-NMR signal of the (13)C-enriched carbon of the bound carbonate is split into two triplets at 2.2 °C. This shows that two cadmium complexes are formed and in each of these complexes the carbonate group is bound by two magnetically equivalent metal ions. It also demonstrates that these cadmium complexes are not in fast exchange. From temperature studies we show that in the zinc complexes both complexes are in fast exchange with each other but are in slow exchange with free bicarbonate. HOESY is used to determine the position of the carbonate carbon in the complex. The solution and crystal structures of the zinc-carbonate-azacryptand complexes are compared. PMID:25091182

  11. 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.

  12. Correlation of 13C /12C and 34S /32S secular variations

    NASA Astrophysics Data System (ADS)

    Veizer, Ján; Holser, William T.; Wilgus, Cheryl K.

    1980-04-01

    Statistical evaluation of 3056 δ13C measurements in carbonate rocks and fossils shows that they record a 2‰ 13C depletion from the late Proterozoic to the early Paleozoic, a 2.5‰ enrichment to the Permian, and a 1.5‰ depletion to the Cenozoic. These variations, not controlled primarily by facies or alteration phenomena, correlate negatively with the δ34S sulfate secular trend, as confirmed by collation of 1083 δ34S measurements. The correlation suggests that the biologically mediated redox fluxes of the C and S cycles have been approximately balanced through this long span of geological time, generally levelling available oxygen. Such a redox system is consistent with the controlling mechanism proposed by GARRELS and PERRY (1974). Consequently, the sedimentary reservoirs of C organic as well as S bacteriological'have varied through geological time.

  13. Direct observation of glycogen synthesis in human muscle with sup 13 C NMR

    SciTech Connect

    Jue, T.; Rothman, D.L.; Shulman, G.I.; Tavitian, B.A.; DeFronzo, R.A.; Shulman, R.G. )

    1989-06-01

    On the basis of previous indirect measurements, skeletal muscle has been implicated as the major site of glucose uptake and it has been suggested that muscle glycogen formation is the dominant pathway. However, direct measurements of the rates of glycogen synthesis have not been possible by previous techniques. The authors have developed {sup 13}C NMR methods to measure directly the rate of human muscle glycogen formation from infused, isotopically labeled (1-{sup 13}C)glucose. They show that under conditions of imposed hyperglycemia and hyperinsulinemia, a majority of the infused glucose was converted to muscle glycogen in a normal man. This directly shows that muscle is the major site of glucose disposal under these conditions, and provides quantitation of the glucose flux to muscle glycogen.

  14. 1H and 13C NMR assignments for the cyanine dyes SYBR Safe and thiazole orange.

    PubMed

    Evenson, William E; Boden, Lauren M; Muzikar, Katy A; O'Leary, Daniel J

    2012-12-01

    Analysis of (1)H and (13)C NMR and mass spectral data for the fluorescent nucleic acid stain SYBR Safe indicates that it contains a cyanine-based cationic core structure identical to thiazole orange. The difference between these two compounds is the type of N-substitution on the quinolinium ring system (SYBR Safe, n-Pr; thiazole orange, Me). The (1)H and (13)C NMR resonances for both compounds were assigned on the basis of one- and two-dimensional (COSY, ROESY, HSQC, and HMBC) experiments. The preferred conformation of these compounds was computed by ab initio methods and found to be consistent with the NMR data.

  15. The Ratio of Acetate-to-Glucose Oxidation in Astrocytes from a Single 13C NMR Spectrum of Cerebral Cortex

    PubMed Central

    Marin-Valencia, Isaac; Hooshyar, M. Ali; Pichumani, Kumar; Sherry, A. Dean; Malloy, Craig R.

    2014-01-01

    The 13C labeling patterns in glutamate and glutamine from brain tissue are quite different after infusion of a mixture of 13C-enriched glucose and acetate. Two processes contribute to this observation, oxidation of acetate by astrocytes but not neurons, and preferential incorporation of α-ketoglutarate into glutamate in neurons, and incorporation of α-ketoglutarate into glutamine in astrocytes. The acetate:glucose ratio, introduced previously for analysis of a single 13C NMR spectrum, provides a useful index of acetate and glucose oxidation in the brain tissue. However, quantitation of relative substrate oxidation at the cell compartment level has not been reported. A simple mathematical method is presented to quantify the ratio of acetate to glucose oxidation in astrocytes, based on the standard assumption that neurons do not oxidize acetate. Mice were infused with [1,2-13C]acetate and [1,6-13C]glucose, and proton decoupled 13C NMR spectra of cortex extracts were acquired. A fit of those spectra to the model indicated that 13C-labeled acetate and glucose contributed approximately equally to acetyl-CoA (0.96) in astrocytes. Since this method relies on a single 13C NMR spectrum, it can be readily applied to multiple physiologic and pathologic conditions. PMID:25231025

  16. Diploptene δ13C values from contemporary thermokarst lake sediments show complex spatial variation

    NASA Astrophysics Data System (ADS)

    Davies, Kimberley L.; Pancost, Richard D.; Edwards, Mary E.; Anthony, Katey M. Walter; Langdon, Peter G.; Chaves Torres, Lidia

    2016-05-01

    Cryospheric changes in northern high latitudes are linked to significant greenhouse gas flux to the atmosphere, for example, methane that originates from organic matter decomposition in thermokarst lakes. The set of pathways that link methane production in sediments, via oxidation in the lake system, to the flux of residual methane to the atmosphere is complex and exhibits temporal and spatial variation. The isotopic signal of bacterial biomarkers (hopanoids, e.g. diploptene) in sediments has been used to identify contemporary ocean-floor methane seeps and, in the geological record, periods of enhanced methane production (e.g. the PETM). The biomarker approach could potentially be used to assess temporal changes in lake emissions through the Holocene via the sedimentary biomarker record. However, there are no data on the consistency of the signal of isotopic depletion in relation to source or on the amount of noise (unexplained variation) in biomarker values from modern lake sediments. We assessed methane oxidation as represented by the isotopic signal of biomarkers from methane oxidising bacteria (MOB) in multiple surface sediment samples in three distinct areas known to emit varying levels of methane in two shallow Alaskan thermokarst lakes. Diploptene was present and had δ13C values lower than -38 ‰ in all sediments analysed, suggesting methane oxidation was widespread. However, there was considerable variation in δ13C values within each area. The most 13C-depleted diploptene was found in an area of high methane ebullition in Ace Lake (diploptene δ13C values between -68.2 and -50.1 ‰). In contrast, significantly higher diploptene δ13C values (between -42.9 and -38.8 ‰) were found in an area of methane ebullition in Smith Lake. δ13C values of diploptene between -56.8 and -46.9 ‰ were found in the centre of Smith Lake, where ebullition rates are low but diffusive methane efflux occurs. The small-scale heterogeneity of the samples may reflect patchy

  17. Structural analysis of airborne flux estimates over a region

    NASA Technical Reports Server (NTRS)

    Caramori, Paulo; Schuepp, Peter; Desjardins, Raymond; Macpherson, Ian

    1994-01-01

    Aircraft-based observations of turbulence fields of velocity, moisture, and temperature are used to study coherent turbulent structures that dominate turbulent transfer of moisture and heat above three different eco-systems. Flux traces are defragmented, to reconstruct the presumed full size (along the sampled transect) of these structures, and flux traces are simplified by elimination of those that contribute negligibly to the flux estimate. Structures are analyzed in terms of size, spatial distribution, and contribution to the flux, in the four 'quadrant' modes of eddy-covariance transfer (excess up/down and deficit up/down). The effect of nonlinear detrending of moisture and temperature data on this 'structural analysis,' over surfaces with heterogeneous surface wetness, is also examined. Results over grassland, wetland, and moist and dry agricultural land, show that nonlinear detrending may provide a more physically realistic description of structures. Significant differences are observed between structure size and associated relative flux contribution, between moist and dry areas, with smaller structures playing a more important role over the moist areas. Structure size generally increases with height, as spatial reorganization from smaller structures into larger ones takes place. This coincides with a gradual loss of surface 'signature' (position and clustering of plumes above localized source areas). The data are expected to provide a basis for an eventual statistical description of boundary-layer transfer events , and help to interpret the link between boundary-layer transfer and hydrological surface conditions.

  18. Measuring doubly 13C-substituted ethane by mass spectrometry

    NASA Astrophysics Data System (ADS)

    Clog, M.; Ling, C.; Eiler, J. M.

    2012-12-01

    Ethane (C2H6) is present in non-negligible amounts in most natural gas reservoirs and is used to produce ethylene for petrochemical industries. It is one of the by-products of lipid metabolism and is the arguably simplest molecule that can manifest multiple 13C substitutions. There are several plausible controls on the relative abundances of 13C2H6 in natural gases: thermodynamically controlled homogeneous isotope exchange reactions analogous to those behind carbonate clumped isotope thermometry; inheritance from larger biomolecules that under thermal degradation to produce natural gas; mixing of natural gases that differ markedly in bulk isotopic composition; or combinations of these and/or other, less expected fractionations. There is little basis for predicting which of these will dominate in natural samples. Here, we focus on an analytical techniques that will provide the avenue for exploring these phenomena. The method is based on high-resolution gas source isotope ratio mass spectrometry, using the Thermo 253-Ultra (a new prototype mass spectrometer). This instrument achieves the mass resolution (M/Δ M) up to 27,000, permitting separation of the isobaric interferences of potential contaminants and isotopologues of an analtye or its fragments which share a cardinal mass. We present techniques to analyze several isotopologues of molecular and fragment ions of C2H6. The critical isobaric separations for our purposes include: discrimination of 13C2H6 from 13C12CDH5 at mass 32 and separation of the 13CH3 fragment from 12CH4 at mass 16, both requiring at least a mass resolution of 20000 to make an adequate measurement. Other obvious interferences are either cleanly separated (e.g., O2, O) or accounted for by peak-stripping (CH3OH on mass 32 and NH2 on mass 16). We focus on a set of measurements which constrain: the doubly-substituted isotopologue, 13C2H6, and the 13CH3/12CH3 ratio of the methyl fragment, which constrains the bulk δ 13C. Similar methods can be

  19. Reaction rate of the 13C(α,n)16O neutron source using the ANC of the -3 keV resonance measured with the THM

    NASA Astrophysics Data System (ADS)

    La Cognata, M.; Spitaleri, C.; Trippella, O.; Kiss, G. G.; Rogachev, G. V.; Mukhamedzhanov, A. M.; Avila, M.; Guardo, G. L.; Koshchiy, E.; Kuchera, A.; Lamia, L.; Puglia, S. M. R.; Romano, S.; Santiago, D.; Spartà, R.

    2016-01-01

    The s-process is responsible of the synthesis of most of the nuclei in the mass range 90 ≤ A ≤ 208. It consists in a series of neutron capture reactions on seed nuclei followed by β-decays, since the neutron accretion rate is slower than the β-decay rate. Such small neutron flux is supplied by the 13C(α,n)16O reaction. It is active inside the helium-burning shell of asymptotic giant branch stars, at temperatures < 108 K, corresponding to an energy interval of 140-230 keV. In this region, the astrophysical S (E)-factor is dominated by the -3 keV sub-threshold resonance due to the 6.356 MeV level in 17O. In this work, we have applied the Trojan Horse Method (THM) to the 13C(6Li,n16O)d quasi-free reaction to extract the 6.356 MeV level resonance parameters, in particular the asymptotic normalization coefficient . A preliminary analysis of a partial data set has lead to , slightly larger than the values in the literature. However, the deduced 13C(α, n)16O reaction rate is in agreement with most results in the literature at ˜ 108 K, with enhanced accuracy thanks to our innovative approach merging together ANC and THM.

  20. Identification of natural metabolites in mixture: a pattern recognition strategy based on (13)C NMR.

    PubMed

    Hubert, Jane; Nuzillard, Jean-Marc; Purson, Sylvain; Hamzaoui, Mahmoud; Borie, Nicolas; Reynaud, Romain; Renault, Jean-Hugues

    2014-03-18

    Because of their highly complex metabolite profile, the chemical characterization of bioactive natural extracts usually requires time-consuming multistep purification procedures to achieve the structural elucidation of pure individual metabolites. The aim of the present work was to develop a dereplication strategy for the identification of natural metabolites directly within mixtures. Exploiting the polarity range of metabolites, the principle was to rapidly fractionate a multigram quantity of a crude extract by centrifugal partition extraction (CPE). The obtained fractions of simplified chemical composition were subsequently analyzed by (13)C NMR. After automatic collection and alignment of (13)C signals across spectra, hierarchical clustering analysis (HCA) was performed for pattern recognition. As a result, strong correlations between (13)C signals of a single structure within the mixtures of the fraction series were visualized as chemical shift clusters. Each cluster was finally assigned to a molecular structure with the help of a locally built (13)C NMR chemical shift database. The proof of principle of this strategy was achieved on a simple model mixture of commercially available plant secondary metabolites and then applied to a bark extract of the African tree Anogeissus leiocarpus Guill. & Perr. (Combretaceae). Starting from 5 g of this genuine extract, the fraction series was generated by CPE in only 95 min. (13)C NMR analyses of all fractions followed by pattern recognition of (13)C chemical shifts resulted in the unambiguous identification of seven major compounds, namely, sericoside, trachelosperogenin E, ellagic acid, an epimer mixture of (+)-gallocatechin and (-)-epigallocatechin, 3,3'-di-O-methylellagic acid 4'-O-xylopyranoside, and 3,4,3'-tri-O-methylflavellagic acid 4'-O-glucopyranoside. PMID:24555703

  1. Prognostic value of 13C-phenylalanine breath test on predicting survival in patients with chronic liver failure

    PubMed Central

    Gallardo-Wong, I; Morán, S; Rodríguez-Leal, G; Castañeda-Romero, B; Mera, R; Poo, J; Uribe, M; Dehesa, M

    2007-01-01

    AIM: To evaluate the prognostic value of percentage of 13C-phenylalanine oxidation (13C-PheOx) obtained by 13C-phenylalanine breath test (13C-PheBT) on the survival of patients with chronic liver failure. METHODS: The hepatic function was determined by standard liver blood tests and the percentage of 13C-PheOx in 118 chronic liver failure patients. The follow-up period was of 64 mo. Survival analysis was performed by the Kaplan-Meier method and variables that were significant (P < 0.10) in univariate analysis and subsequently introduced in a multivariate analysis according to the hazard model proposed by Cox. RESULTS: Forty-one patients died due to progressive liver failure during the follow-up period. The probability of survival at 12, 24, 36, 48 and 64 mo was 0.88, 0.78, 0.66, 0.57 and 0.19, respectively. Multivariate analysis demonstrated that Child-Pugh classes, age, creatinine and the percentage of 13C-PheOx (HR 0.338, 95% CI: 0.150-0.762, P = 0.009) were independent predictors of survival. When Child-Pugh classes were replaced by all the parameters of the score, only albumin, bilirubin, creatinine, age and the percentage of 13C-PheOx (HR 0.449, 95% CI: 0.206-0.979, P = 0.034) were found to be independent predictors of survival. CONCLUSION: Percentage of 13C-PheOx obtained by 13C-PheBT is a strong predictor of survival in patients with chronic liver disease. PMID:17729409

  2. Neutron Backgrounds: 13C({alpha}, n) etc

    SciTech Connect

    Mitsui, Tadao

    2005-09-08

    13C({alpha}, n) reaction is the main neutron source in an underground large-volume liquid-scintillator detector KamLAND. {alpha} sources, targets, cross sections, and neutron transport are studied to estimate the backgrounds of v-bare signal.

  3. Spectral editing for in vivo 13C magnetic resonance spectroscopy

    NASA Astrophysics Data System (ADS)

    Xiang, Yun; Shen, Jun

    2012-01-01

    In vivo detection of carboxylic/amide carbons is a promising technique for studying cerebral metabolism and neurotransmission due to the very low RF power required for proton decoupling. In the carboxylic/amide region, however, there is severe spectral overlap between acetate C1 and glutamate C5, complicating studies that use acetate as an astroglia-specific substrate. There are no known in vivo MRS techniques that can spectrally resolve acetate C1 and glutamate C5 singlets. In this study, we propose to spectrally separate acetate C1 and glutamate C5 by a two-step J-editing technique after introducing homonuclear 13C- 13C scalar coupling between carboxylic/amide carbons and aliphatic carbons. By infusing [1,2- 13C 2]acetate instead of [1- 13C]acetate the acetate doublet can be spectrally edited because of the large separation between acetate C2 and glutamate C4 in the aliphatic region. This technique can be applied to studying acetate transport and metabolism in brain in the carboxylic/amide region without spectral interference.

  4. 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

  5. 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.

  6. Molecular structure, spectroscopic (FTIR, FT-Raman, 13C and 1H NMR, UV), polarizability and first-order hyperpolarizability, HOMO-LUMO analysis of 2,4-difluoroacetophenone

    NASA Astrophysics Data System (ADS)

    Jeyavijayan, S.

    2015-02-01

    The FTIR and FT-Raman spectra of 2,4-difluoroacetophenone (DFAP) have been recorded in the regions 4000-400 cm-1 and 3500-50 cm-1, respectively. Utilizing the observed FTIR and FT-Raman data, a complete vibrational assignment and analysis of the fundamental modes of the compound were carried out. The optimum molecular geometry, harmonic vibrational frequencies, infrared intensities and Raman scattering activities, were calculated by density functional theory (DFT/B3LYP) method with 6-31+G(d,p) and 6-311++G(d,p) basis sets. The difference between the observed and scaled wavenumber values of most of the fundamentals is very small. A detailed interpretation of the infrared and Raman spectra of DFAP is also reported based on total energy distribution (TED). Stability of the molecule arising from hyperconjugative interactions, charge delocalization have been analyzed using natural bond orbital (NBO) analysis. The MEP map shows the negative potential sites are on oxygen atom as well as the positive potential sites are around the hydrogen atoms. The UV-Vis spectral analysis of DFAP has also been done which confirms the charge transfer of DFAP. The chemical shifts of H atoms and C atoms were calculated using NMR analysis. Furthermore, the polarizability, the first hyperpolarizability and total dipole moment of the molecule have been calculated.

  7. Testing compound-specific δ13C of amino acids in mussels as a new approach to determine the average 13C values of primary production in littoral ecosystems

    NASA Astrophysics Data System (ADS)

    Vokhshoori, N. L.; Larsen, T.; McCarthy, M.

    2012-12-01

    Compound-specific isotope analysis of amino acids (CSI-AA) is a technique used to decouple trophic enrichment patterns from source changes at the base of the food web. With this new emerging tool, it is possible to precisely determine both trophic position and δ15N or δ13C source values in higher feeding organisms. While most work to date has focused on nitrogen (N) isotopic values, early work has suggested that δ13C CSI-AA has great potential as a new tracer both to a record δ13C values of primary production (unaltered by trophic transfers), and also to "fingerprint" specific carbon source organisms. Since essential amino acids (EAA) cannot be made de novo in metazoans but must be obtained from diet, the δ13C value of the primary producer is preserved through the food web. Therefore, the δ13C values of EAAs act as a unique signature of different primary producers and can be used to fingerprint the dominant carbon (C) source driving primary production at the base of the food web. In littoral ecosystems, such as the California Upwelling System (CUS), the likely dominant C sources of suspended particulate organic matter (POM) pool are kelp, upwelling phytoplankton or estuarine phytoplankton. While bulk isotopes of C and N are used extensively to resolve relative consumer hierarchy or shifting diet in a food web, we found that the δ13C bulk values in mussels cannot distinguish exact source in littoral ecosystems. Here we show 15 sites within the CUS, between Cape Blanco, OR and La Jolla, CA where mussels were sampled and analyzed for both bulk δ13C and CSI-AA. We found no latitudinal trends, but rather average bulk δ13C values for the entire coastal record were highly consistent (-15.7 ± 0.9‰). The bulk record would suggest either nutrient provisioning from kelp or upwelled phytoplankton, but 13C-AA fingerprinting confines these two sources to upwelling. This suggests that mussels are recording integrated coastal phytoplankton values, with the enriched

  8. 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.

  9. 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.

    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##

  10. Regional flux analysis of longitudinal atrophy in Alzheimer's disease.

    PubMed

    Lorenzi, Marco; Ayache, Nicholas; Xavier, Pennec

    2012-01-01

    The longitudinal analysis of the brain morphology in Alzheimer's disease(AD) is fundamental for understanding and quantifying the dynamics of the pathology. This study provides a new measure of the brain longitudinal changes based on the Helmholtz decomposition of deformation fields. We used the scalar pressure map associated to the irrotational component in order to identify a consistent group-wise set of areas of maximal volume change. The atrophy was then quantified in these areas for each subject by the probabilistic integration of the flux of the longitudinal deformations across the boundaries. The presented framework unifies voxel-based and regional approaches, and robustly describes the longitudinal atrophy at group level as a spatial process governed by consistently defined regions. Our experiments showed that the resulting regional flux analysis is able to detect the differential atrophy patterns across populations, and leads to precise and statistically powered quantifications of the longitudinal changes in AD, even in mild/premorbid cases.

  11. Pre-treatment Effects on Coral Skeletal δ 13C and δ 18O

    NASA Astrophysics Data System (ADS)

    Grottoli, A. G.; Gibb, O.; Wellington, G. M.

    2003-12-01

    Pre-treatment protocols for coral skeletal stable carbon (δ 13C) and oxygen (δ 18O) isotope analyses include no treatment, bleach (NaOH), hydrogen peroxide (H2O2), or vacuum roasting prior to analysis. Such pre-treatments are used to remove organic material prior to isotopic analyses. Researchers that do not pre-treat samples argue that such treatments result in non-linear shifts in coral skeletal δ 13C and δ 18O thus increasing the analytical error in the δ 13C and δ 18O values. Vacuum roasting does cause isotopic shifts and is no longer practiced. However, both no pre-treatment and pre-treatment (with either NaOH or H2O2) coral δ 13C and δ 18O values continue to be published in the literature. In all previous studies of the effects of NaOH and H2O2 pre-treatments on coral δ 13C and δ 18O, the samples sizes were typically small and the exact time interval being sampled and compared was not specifically controlled. Here, we evaluated the effects of NaOH and H2O2 pre-treatments on coral skeletal δ 13C and δ 18O in Pavona clavus and Pavona gigantea from Panama, and Porites compressa from Hawaii. In Panama, at least five coral fragments from five different colonies of each species were stained on November 1978 and April 1979 then collected in November 1979. In Hawaii, at least five coral fragments from five different colonies at 1.7 and 7 m depths were stained on 1 September and 21 November 1996 then collected 2 March 1997. For each fragment, a bulk skeletal sample was extracted representing the entire growth interval between the two stain lines yielding at least 24 mg of material. Sampling between the stain lines ensured that all of the fragments from a given site and species were sampled over the same time interval and avoided any potential contamination from the tissue layer. Eight milligram subsamples from each fragment were subjected to 24 hours of the following treatments: NaOH, H2O2, Milli-Q filtered water (control), or no pre-treatment (control

  12. The Late Devonian Frasnian-Famennian (F/F) biotic crisis: Insights from δ13C carb, δ13C org and 87Sr / 86Sr isotopic systematics

    NASA Astrophysics Data System (ADS)

    Chen, Daizhao; Qing, Hairuo; Li, Renwei

    2005-06-01

    A severe biotic crisis occurred during the Late Devonian Frasnian-Famennian (F/F) transition (± 367 Myr). Here we present δ13C carb, δ13C org and 87Sr / 86Sr isotopic systematics, from identical samples of two sections across F/F boundary in South China, which directly demonstrate large and frequent climatic fluctuations (˜200 kyr) from warming to cooling during the F/F transition. These climate fluctuations are interpreted to have been induced initially by increased volcanic outgassing, and subsequent enhanced chemical weathering linked to the rapid expansion of vascular plants on land, which would have increased riverine delivery to oceans and primary bioproductivity, and subsequent burial of organic matter, thereby resulting in climate cooling. Such large and frequent climatic fluctuations, together with volcanic-induced increases in nutrient (e.g., biolimiting Fe), toxin (sulfide) and anoxic water supply, and subsequent enhanced riverine fluxes and microbial bloom, were likely responsible for the stepwise faunal demise of F/F biotic crisis.

  13. Analysis of metabolic pathways and fluxes in a newly discovered thermophilic and ethanol-tolerant Geobacillus strain.

    PubMed

    Tang, Yinjie J; Sapra, Rajat; Joyner, Dominique; Hazen, Terry C; Myers, Samuel; Reichmuth, David; Blanch, Harvey; Keasling, Jay D

    2009-04-01

    A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and is tolerant to high ethanol concentrations (10%, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications. Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner-Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions (including the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not be accurately determined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 100 units (g DCW)(-1) h(-1)) through the TCA cycle and oxidative pentose phosphate pathway were reduced from 64 +/- 3 to 25 +/- 2 and from 30 +/- 2 to 19 +/- 2, respectively. The carbon flux under micro-aerobic growth was directed to ethanol, L-lactate (> 99% optical purity), acetate, and formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38 +/- 0.07 mol mol(-1) glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG reduces the maximum ethanol yield by approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production.

  14. Analysis of Metabolic Pathways and Fluxes in a Newly Discovered Thermophilic and Ethanol-Tolerant Geobacillus Strain

    SciTech Connect

    Tang, Yinjie J.; Sapra, Rajat; Joyner, Dominique; Hazen, Terry C.; Myers, Samuel; Reichmuth, David; Blanch, Harvey; Keasling, Jay D.

    2009-01-20

    A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and istolerant to high ethanol concentrations (10percent, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications. Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner?Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions (including the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not be accuratelydetermined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 100 units (g DCW)-1 h-1) through the TCA cycle and oxidative pentose phosphate pathway were reduced from 64+-3 to 25+-2 and from 30+-2 to 19+-2, respectively. The carbon flux under micro-aerobic growth was directed formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38+-0.07 mol mol-1 glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG reduces the maximum ethanol yieldby approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production.

  15. Reflections on the application of 13C-MRS to research on brain metabolism.

    PubMed

    Morris, Peter; Bachelard, Herman

    2003-01-01

    The power of (13)C-MRS lies in its unique chemical specificity, enabling detection and quantification of metabolic intermediates which would not be so readily monitored using conventional radiochemical techniques. Examples from animal studies, by examination of tissue extracts from the whole brain, brain slices and cultured cells, include observation of intermediates such as citrate and triose phosphates which have yielded novel information on neuronal/glial relationships. The use of (13)C-labelled acetate as a specific precursor for glial metabolism provided evidence in support of the view that some of the GABA produced in the brain is derived from glial glutamine. Such studies have also provided direct evidence on the contribution of anaplerotic pathways to intermediary metabolism. Analogous studies are now being performed on the human brain, where (13)C-acetate is used to quantitate the overall contribution of glial cells to intermediary metabolism, and use of (13)C-glucose enables direct calculation of rates of flux through the TCA (F(TCA)) and of the glutamate-glutamine cycle (F(CYC)), leading to the conclusion that the rate of glial recycling of glutamate accounts for some 50% of F(TCA). The rate of 0.74 micromol min(-1) g(-1) for F(TCA) is compatible with PET rates of CMRglc of 0.3-0.4 micromol min(-1) g(-1) (since each glucose molecule yields two molecules of pyruvate entering the TCA). Our brain activation studies showed a 60% increase in F(TCA), which is very similar to the increases in CBF and in CMRglc observed in PET activation studies.

  16. Direct Monitoring of γ-Glutamyl Transpeptidase Activity In Vivo Using a Hyperpolarized (13) C-Labeled Molecular Probe.

    PubMed

    Nishihara, Tatsuya; Yoshihara, Hikari A I; Nonaka, Hiroshi; Takakusagi, Yoichi; Hyodo, Fuminori; Ichikawa, Kazuhiro; Can, Emine; Bastiaansen, Jessica A M; Takado, Yuhei; Comment, Arnaud; Sando, Shinsuke

    2016-08-26

    The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for in vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2 O at 9.4 T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin. PMID:27483206

  17. Spatio-temporal variability of Δ13C in tree-rings of Aleppo pine

    NASA Astrophysics Data System (ADS)

    del Castillo, Jorge; Ferrio, Juan Pedro; Voltas, Jordi

    2014-05-01

    Aim: To study the spatiotemporal variability of Δ13C using a tree-ring network of Aleppo pine (Pinus halepensis Mill.) in the eastern part of the Iberian Peninsula. In this study, we tried to understand some of the environmental drivers behind changes in Δ13C as well as to decide the most optimal sites to infer paleoclimatic information using such variables. We also try to understand key physiological aspects of P. halepensis. Methods: In order to do that, we have collected biannual Δ13C time series (1950-1998) together with mean annual precipitation (MAP), tree-ring width (TRW) and remote sensing (NDVI) data, for 7 different locations along a precipitation gradient. We assessed how correlations between variables changed along that gradient. In addition to that, we have also looked at how that precipitation gradient changed along the years and thus its relationships with the Δ13C at the spatial level, giving us an idea whether changes in MAP at each site could affect the relationship between these two variables. Results: We found that a log model better explains the relationship between Δ13C and MAP and that it reaches a saturation point at values above 800 mm of MAP. Similarly, we found that, in the drier sites, correlations between Δ13C and precipitation were higher than in wetter ones. In addition, the coefficient of variation (CV) of Δ13C was a good indicator of the correlation between Δ13C and MAP. Similarly, the mean and the CV of TRW and summer NDVI were good indicators of the level of such correlation between Δ13C and MAP. On the other hand, the inter-site analysis of the data suggested that during dry years exists a stronger relationship between Δ13C and precipitation than in wet years. Discussion: Our results pointed out that the threshold for water limitation for Aleppo pine was around MAP=800 mm, an amount that might be sufficient for the tree to grow during most of the growing season without altering its water use efficiency (WUE) by closing

  18. Structural analysis of airborne flux estimates over a region

    SciTech Connect

    Caramori, P.; Schuepp, P. ); Desjardins, R. ); MacPherson, I. )

    1994-05-01

    Aircraft-based observations of turbulence fields of velocity, moisture, and temperature are used to study coherent turbulent structures that dominate turbulent transfer of moisture and heat above three different ecosystems. Flux traces are defragmented, to reconstruct the presumed full size (along the sampled transect) of these structures, and flux traces are simplified by elimination of those that contribute negligibly to the flux estimates. Structures are analyzed in terms of size, spatial distribution, and contribution to the flux, in the four [open quotes]quadrant[close quotes] modes of eddy-covariance transfer (excess up/down and deficit up/down). The effect of nonlinear detrending of moisture and temperature data on this [open quotes]structural analysis,[close quotes] over surfaces with heterogeneous surface wetness, is also examined. Results over grassland, wetland, and moist and dry agricultural land, show that nonlinear detrending may provide a more physically realistic description of structures. Significant difference are observed between structure size and associated relative flux contribution, between moist and dry areas, with smaller structures playing a more important role over the moist areas. Structure size generally increases with height, as spatial reorganization from smaller structures into larger ones takes place. This coincides with a gradual loss of surface [open quotes]signature[close quotes] (position and clustering of plumes above localized source area). The data are expected to provide a basis for an eventual statistical description of boundary-layer transfer events, and help to interpret the link between boundary-layer transfer and hydrological surface conditions. 48 refs., 15 figs. 2 tabs.

  19. /sup 1/H and /sup 13/C spin-lattice relaxation in gaseous benzene

    SciTech Connect

    Folkendt, M.M.; Weiss-Lopez, B.E.; True, N.S.

    1988-08-25

    The nuclear spin-lattice relaxation time, T/sub 1/, measured for benzene protons at densities between 0.81 and 54.4 mol/m/sup 3/ (15 and 980 Torr) at 381 K exhibits a characteristic nonlinear density dependence. Analysis of the density-dependent T/sub 1/ data yields a spin-rotation coupling constant, C/sub eff/, of /vert bar/182.6 (0.4)/vert bar/ Hz and an angular momentum reorientation cross section, sigma, of 131 (1) /Angstrom//sup 2/. The /sup 13/C spin-lattice relaxation time of singly labeled /sup 13/C benzene is a linear function of density over the density range 1.07-75.12 mol/m/sup 3/ (20-1330 Torr). /sup 13/C T/sub 1/ values are shorter than /sup 1/H T/sub 1/ values by a factor of ca. 100 at comparable densities. The nuclear Overhauser enhancement factor, /eta/, is 0.0 /plus minus/ 0.02 at densities between 11 and 85.3 mol/m/sup 3/ (200 and 1500 Torr), demonstrating that dipole-dipole relaxation is relatively inefficient in this region. The spin-rotation coupling constant, C/sub eff/, for /sup 13/C nuclei in benzene is estimated to be /vert bar/1602 (68)/vert bar/ Hz.

  20. 13C Tracking after 13CO2 Supply Revealed Diurnal Patterns of Wood Formation in Aspen.

    PubMed

    Mahboubi, Amir; Linden, Pernilla; Hedenström, Mattias; Moritz, Thomas; Niittylä, Totte

    2015-06-01

    Wood of trees is formed from carbon assimilated in the photosynthetic tissues. Determining the temporal dynamics of carbon assimilation, subsequent transport into developing wood, and incorporation to cell walls would further our understanding of wood formation in particular and tree growth in general. To investigate these questions, we designed a (13)CO2 labeling system to study carbon transport and incorporation to developing wood of hybrid aspen (Populus tremula × tremuloides). Tracking of (13)C incorporation to wood over a time course using nuclear magnetic resonance spectroscopy revealed diurnal patterns in wood cell wall biosynthesis. The dark period had a differential effect on (13)C incorporation to lignin and cell wall carbohydrates. No (13)C was incorporated into aromatic amino acids of cell wall proteins in the dark, suggesting that cell wall protein biosynthesis ceased during the night. The results show previously unrecognized temporal patterns in wood cell wall biosynthesis, suggest diurnal cycle as a possible cue in the regulation of carbon incorporation to wood, and establish a unique (13)C labeling method for the analysis of wood formation and secondary growth in trees.

  1. Quantitative identification of metastable magnesium carbonate minerals by solid-state 13C NMR spectroscopy.

    PubMed

    Moore, Jeremy K; Surface, J Andrew; Brenner, Allison; Wang, Louis S; Skemer, Philip; Conradi, Mark S; Hayes, Sophia E

    2015-01-01

    In the conversion of CO2 to mineral carbonates for the permanent geosequestration of CO2, there are multiple magnesium carbonate phases that are potential reaction products. Solid-state (13)C NMR is demonstrated as an effective tool for distinguishing magnesium carbonate phases and quantitatively characterizing magnesium carbonate mixtures. Several of these mineral phases include magnesite, hydromagnesite, dypingite, and nesquehonite, which differ in composition by the number of waters of hydration or the number of crystallographic hydroxyl groups. These carbonates often form in mixtures with nearly overlapping (13)C NMR resonances which makes their identification and analysis difficult. In this study, these phases have been investigated with solid-state (13)C NMR spectroscopy, including both static and magic-angle spinning (MAS) experiments. Static spectra yield chemical shift anisotropy (CSA) lineshapes that are indicative of the site-symmetry variations of the carbon environments. MAS spectra yield isotropic chemical shifts for each crystallographically inequivalent carbon and spin-lattice relaxation times, T1, yield characteristic information that assist in species discrimination. These detailed parameters, and the combination of static and MAS analyses, can aid investigations of mixed carbonates by (13)C NMR.

  2. 13C NMR spectra of pyridine chalcone analogs

    NASA Astrophysics Data System (ADS)

    Jovanović, B. Ž.; Mišić-Vuković, M.; Marinković, A. D.; Csanádi, J.

    1999-05-01

    13C NMR spectra of two series of pyridine chalcone analogs were determined in deuterated dimethylsulphoxide (DMSO-d 6). It was established that these compounds were in more stable E-configurations except for the 4-pyridalacetophenone which was in Z-configuration. On the basis of the Hammett correlations of 13C NMR chemical shifts of the ethylenic bond carbon atoms and the σ values for the pyridine "aza" groups, the polarization of ethylenic bonds were discussed. It was established that the opposite effect of the pyridine substituents at the electronic density distribution in pyridalacetophenone and cinnamoylpyridines depends on its direct bonding to the ethylenic carbon or the transmission electronic effects via the carbonyl group, respectively.

  3. Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and (13)c NMR analysis.

    PubMed

    Gilbert, M; Brisson, J R; Karwaski, M F; Michniewicz, J; Cunningham, A M; Wu, Y; Young, N M; Wakarchuk, W W

    2000-02-11

    We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an alpha-2, 3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis lo