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Sample records for 13c label incorporation

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

    PubMed

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

    2008-08-01

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

  2. Incorporation of {sup 13}C-labeled intermediates into developing lignin revealed by analytical pyrolysis and CuO oxidation in combination with IRM-GC-MS

    SciTech Connect

    Eglinton, T.I.; Goni, M.A.; Boon, J.J.

    1995-12-31

    Tissue samples from Ginkgo shoots (Ginkgo biloba L.) and Rice grass (Oryzasitiva sp.) incubated in the presence of {sup 13}C-labeled substrates such as coniferin (postulated to be biosynthetic intermediates in lignin biosynthesis) were studied using thermal and chemical dissociation methods in combination with molecular-level isotopic measurements. The aim of the study was (1) to investigate dissociation mechanisms, and (2) to examine and quantify the proportions of labeled material incorporated within each sample. Isotopic analysis of specific dissociation products revealed the presence of the label in its original positions, and only within lignin-derived (phenolic) products. Moreover, the distribution and isotopic composition of the dissociation products strongly suggest an origin from newly-formed lignin. These results clearly indicate that there is no {open_quotes}scrambling{close_quotes} of carbon atoms as a result of the dissociation process, thereby lending support to this analytical approach. In addition, the data provide confidence in the selective labeling approach for elucidation of the structure and biosynthesis of lignin.

  3. Incorporation of 13C labelled root-shoot residues in soil in the presence of Lumbricus terrestris: An isotopic and molecular approach

    NASA Astrophysics Data System (ADS)

    Vidal, Alix; Alexis, Marie; Nguyen Tu, Thanh Tu; Anquetil, Christelle; Vaury, Véronique; Derenne, Sylvie; Quenea, Katell

    2016-04-01

    Litter from plant biomass deposited on soil surface can either be mineralized; releasing CO2 to the atmosphere, or transferred into the soil as organic compounds. Both pathways depend on biotic factors such as litter characteristics and the of soil organism activity. During the last decades, many studies have focused on the origin of organic matter, with a particular attention to the fate of root and shoot litter. It is generally admitted that roots decompose at a slower rate than shoots, resulting in a higher carbon sequestration in soil for compounds originating from roots. Earthworms play a central role in litter decomposition and carbon cycling, ingesting both organic and mineral compounds which are mixed, complexed and dejected in the form of casts at the soil surface or along earthworm burrows. The simultaneous impact of earthworms and root-shoot on soil carbon cycling is still poorly understood. This study aimed at (1) defining the rate of incorporation of root and shoot litter with or without earthworms and (2) characterizing the molecular composition of soil organic matter upon litter decomposition, after one year of experimentation. A mesocosm experiment was set up to follow the incorporation of 13C labelled Ryegrass root and shoot litter in the soil, in the presence of anecic earthworms (Lumbricus terrestris). Soil samples were collected at 0-20 and 40-60 cm, as well as surface casts, at the beginning and after 1, 2, 4, 8, 24 and 54 weeks of experiment. Organic carbon content and δ13C values were determined for all the samples with Elemental Analysis - Isotope Ratio Mass Spectrometry. Lipid-free soil and cast samples after 54 weeks of incubation were analyzed with Pyrolysis-Gas Chromatography-Mass Spectrometry. Pyrolysis products were grouped into six classes: polysaccharides, lignin derived compounds, phenols, N-compounds, aliphatic compounds and sterols. Each pyrolysis product was quantified thanks to its peak area, relative to the total area of the

  4. De novo biosynthesis of linoleic acid and its conversion to the hydrocarbon (Z,Z)-6,9-heptadecadiene in the astigmatid mite, Carpoglyphus lactis: incorporation experiments with 13C-labeled glucose.

    PubMed

    Shimizu, Nobuhiro; Naito, Michiya; Mori, Naoki; Kuwahara, Yasumasa

    2014-02-01

    De novo biosynthesis of linoleic acid (LA) and its conversion to (Z,Z)-6,9-heptadecadiene were examined in Carpoglyphus lactis (Acarina, Carpoglyphidae). Experiments involving (13)C-administration using [1-(13)C]-d-glucose revealed that (13)C atoms were incorporated into LA of total lipid extracted from the mite, resulting in labeling of all even-numbered carbons. This result demonstrated that LA was produced from (13)C-labeled acetyl-CoA, which is indicative of direct de novo biosynthesis. In these feeding experiments involving [1-(13)C]-D-glucose, (13)C atoms were also incorporated into (Z,Z)-6,9-heptadecadiene, which is one of the major secretory components in the mite. The labeling pattern of (Z,Z)-6,9-heptadecadiene at odd-numbered carbons agreed well with that of LA after loss of the carboxyl carbon. It was concluded that the mites could stably convert LA into (Z,Z)-6,9-heptadecadiene without the dietary requirement of this essential fatty acid.

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

    PubMed

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

    2016-10-12

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

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

    PubMed

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

    2014-08-01

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

  7. Metabolic flux analysis using 13C peptide label measurements

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Fontana, Carolina; Kovacs, Helena; Widmalm, Göran

    2014-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

  10. 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

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

    NASA Astrophysics Data System (ADS)

    Azurmendi, Hugo F.; Freedberg, Darón I.

    2013-03-01

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

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

    PubMed

    Azurmendi, Hugo F; Freedberg, Darón I

    2013-03-01

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

  13. Affordable uniform isotope labeling with (2)H, (13)C and (15)N in insect cells.

    PubMed

    Sitarska, Agnieszka; Skora, Lukasz; Klopp, Julia; Roest, Susan; Fernández, César; Shrestha, Binesh; Gossert, Alvar D

    2015-06-01

    For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80% can be achieved for (15)N and (13)C with yields comparable to expression in full media. For (2)H,(15)N and (2)H,(13)C,(15)N labeling, incorporation is only slightly lower with 75 and 73%, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins.

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

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

    PubMed

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

    2007-09-01

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

  16. Influence of 13C isotopic labeling location of 13C DNP of acetate using TEMPO free radical

    NASA Astrophysics Data System (ADS)

    Parish, Christopher; Niedbalski, Peter; Lumata, Lloyd

    2015-03-01

    Dynamic nuclear polarization (DNP) via the dissolution method enhances the liquid-state magnetic resonance (NMR or MRI) signals of insensitive nuclear spins by at least 10,000-fold. The basis for all these signal enhancements at room temperature is the polarization transfer from the electrons to nuclear spins at cryogenic temperature and high magnetic field. In this work, we have studied the influence of the location of 13C isotopic labeling on the DNP of sodium acetate at 3.35 T and 1.4 K using a wide ESR linewidth free radical 4-oxo-TEMPO. The carbonyl [1-13C]acetate spins produced a polarization level that is almost twice that of the methyl [2-13C]acetate spins. On the other hand, the polarization of the methyl 13C spins doubled to reach the level of [1-13C]acetate when the methyl group was deuterated. Meanwhile, the solid-state nuclear relaxation of these samples are the same and do not correlate with the polarization levels. These behavior implies that the nuclear relaxation for these samples is dominated by the contribution from the free radicals and the polarization levels can be explained by a thermodynamic picture of DNP.

  17. Structure of uniaxially aligned 13C labeled silk fibroin fibers with solid state 13C-NMR

    NASA Astrophysics Data System (ADS)

    Demura, Makoto; Yamazaki, Yasunobu; Asakura, Tetsuo; Ogawa, Katsuaki

    1998-01-01

    Carbon-13 isotopic labeling of B. mori silk fibroin was achieved biosynthetically with [1- 13C] glycine in order to determine the carbonyl bond orientation angle of glycine sites with the silk fibroin. Angular dependence of 13C solid state NMR spectra of uniaxially oriented silk fibroin fiber block sample due to the carbonyl 13C chemical shift anisotropy was simulated according to the chemical shift transformation with Euler angles, αF and βF, from principal axis system (PAS) to fiber axis system (FAS). The another Euler angles, αDCO and βDCO, for transformation from PAS to the molecular symmetry axis were determined from the [1- 13C] glycine sequence model compounds for the silk fibroin. By the combination of these Euler angles, the carbonyl bond orientation angle with respect to FAS of the [1- 13C] glycine sites of the silk fibroin was determined to be 90 ± 5°. This value is in agreement with the X-ray diffraction and our previous solid state NMR data of B. mori silk fibroin fiber (a typical β-pleated sheet) within experimental error.

  18. The use of 13-C-labelled polyaromatic hydrocarbons in soil bound residue formation

    SciTech Connect

    Richnow, H.H.; Seifert, R.; Hefter, J.

    1996-12-31

    The formation of non-extractable residues during biodegradation and humification processes in soils and sediments represent a major sink for organic contaminants. We studied the mode of incorporation of polycyclic aromatic hydrocarbons (PAM) and their metabolites into macromolecular organic matter during microbial degradation applying {sup 13}C-labelled compounds. Mineralization rates were determined by measuring the {sup 13}CO{sub 2} production. An incorporation of {sup 13}C-PAH-fragments into humic material could be traced by isotopic analysis of the bulk organic matter. Furthermore, selective chemical degradation reactions were applied to analyze the precise chemical structure of covalently bound {sup 13}C-labelled PAH fragments in soil humic substances. Structural assignments by GC-MS combined with isotope measurements on the bulk organic carbon and the molecular level (IRM-GC-MS) provide useful information on the fate of xenobiotics within the soil. The results are discussed in the context of long-term risk assessment of bioremediated soils.

  19. Synthesis and solid-state NMR structural characterization of 13C-labeled graphite oxide.

    PubMed

    Cai, Weiwei; Piner, Richard D; Stadermann, Frank J; Park, Sungjin; Shaibat, Medhat A; Ishii, Yoshitaka; Yang, Dongxing; Velamakanni, Aruna; An, Sung Jin; Stoller, Meryl; An, Jinho; Chen, Dongmin; Ruoff, Rodney S

    2008-09-26

    The detailed chemical structure of graphite oxide (GO), a layered material prepared from graphite almost 150 years ago and a precursor to chemically modified graphenes, has not been previously resolved because of the pseudo-random chemical functionalization of each layer, as well as variations in exact composition. Carbon-13 (13C) solid-state nuclear magnetic resonance (SSNMR) spectra of GO for natural abundance 13C have poor signal-to-noise ratios. Approximately 100% 13C-labeled graphite was made and converted to 13C-labeled GO, and 13C SSNMR was used to reveal details of the chemical bonding network, including the chemical groups and their connections. Carbon-13-labeled graphite can be used to prepare chemically modified graphenes for 13C SSNMR analysis with enhanced sensitivity and for fundamental studies of 13C-labeled graphite and graphene.

  20. Enhancing Phospholipid Fatty Acid Profiling of Soil Bacterial Communities via Substrate- Specific 13C-labelling

    NASA Astrophysics Data System (ADS)

    Evershed, R. P.; Maxfield, P. J.; Bingham, E. M.; Dildar, N.; Brennand, E. L.; Hornibrook, E.

    2008-12-01

    A range of culture-independent methods, has recently emerged to study environmental microorganisms in situ[1]. One such method is phospholipid fatty acid (PLFA) analysis, wherein these ubiquitous membrane lipids provide a powerful tool for the study of unculturable soil microorganisms. PLFA analyses have been used to investigate the impacts of a wide range of environmental factors on the soil microbial community. An acknowledged shortcoming of the PLFAs approach is the lack the chemotaxonoic specificity, which restricts the ability of the method to probe the activities of specific functional groups of the microbial community selectively. However, the selectivity of PLFAs analyses can be enhanced by incubating soils with 13C- labelled substrates followed by gas chromatography-combustion-isotope ratio mass spectrometry to reveal the specific PLFAs incorporating the 13C-label. The application of this approach will be demonstrated through our recent work on methanotrophic bacteria in soils. We applied this approach initially to mineral soils[2] and then extended chemotaxonomic assessments by using a combination of 13C-labelled PLFAs and hopanoids [3]. We have used this approach to explore the properties of high affinity methanotrophs in a range of environments, investigating the relationship between methane oxidation rates and the nature and magnitude of the methanotrophic community for the first time[4,5] More recently we extended the technique using a novel time series 13C-labelling of PLFAs[6] to estimate the rate and progression of 13C- label incorporation and turnover of methanotrophic populations. This modified approach has been used to investigate the impacts of various environmental variables, e.g. soil type, vegetation cover and land use, on the methanotrophic biomass[7.8]. The unique nature of the 13CH4 as a gaseous substate/carbon source means that can be readily introduced into soils via a specific subset of the soil microbial biomass, thereby offering many

  1. 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

  2. Hydrogen dynamics in soil organic matter as determined by 13C and 2H labeling experiments

    NASA Astrophysics Data System (ADS)

    Paul, Alexia; Hatté, Christine; Pastor, Lucie; Thiry, Yves; Siclet, Françoise; Balesdent, Jérôme

    2016-12-01

    Understanding hydrogen dynamics in soil organic matter is important to predict the fate of 3H in terrestrial environments. One way to determine hydrogen fate and to point out processes is to examine the isotopic signature of the element in soil. However, the non-exchangeable hydrogen isotopic signal in soil is complex and depends on the fate of organic compounds and microbial biosyntheses that incorporate water-derived hydrogen. To decipher this complex system and to understand the close link between hydrogen and carbon cycles, we followed labeled hydrogen and labeled carbon throughout near-natural soil incubations. We performed incubation experiments with three labeling conditions: 1 - 13C2H double-labeled molecules in the presence of 1H2O; 2 - 13C-labeled molecules in the presence of 2H2O; 3 - no molecule addition in the presence of 2H2O. The preservation of substrate-derived hydrogen after 1 year of incubation (ca. 5 % in most cases) was lower than the preservation of substrate-derived carbon (30 % in average). We highlighted that 70 % of the C-H bonds are broken during the degradation of the molecule, which permits the exchange with water hydrogen. Added molecules are used more for trophic resources. The isotopic composition of the non-exchangeable hydrogen was mainly driven by the incorporation of water hydrogen during microbial biosynthesis. It is linearly correlated with the amount of carbon that is degraded in the soil. The quantitative incorporation of water hydrogen in bulk material and lipids demonstrates that non-exchangeable hydrogen exists in both organic and mineral-bound forms. The proportion of the latter depends on soil type and minerals. This experiment quantified the processes affecting the isotopic composition of non-exchangeable hydrogen, and the results can be used to predict the fate of tritium in the ecosystem or the water deuterium signature in organic matter.

  3. Uniformly sup 13 C-labeled algal protein used to determine amino acid essentiality in vivo

    SciTech Connect

    Berthold, H.K.; Hachey, D.L.; Reeds, P.J.; Klein, P.D. ); Thomas, O.P. ); Hoeksema, S. )

    1991-09-15

    The edible alga Spirulina platensis was uniformly labeled with {sup 13}C by growth in an atmosphere of pure {sup 13}CO{sub 2}. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly {sup 13}C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo.

  4. Synthesis of isotopically labeled R- or S-[.sup.13C, .sup.2H] glycerols

    DOEpatents

    Martinez, Rodolfo A.; Unkefer, Clifford J.; Alvarez, Marc A.

    2008-01-22

    The present invention is directed to asymmetric chiral labeled glycerols including at least one chiral atom, from one to two .sup.13C atoms and from zero to four deuterium atoms bonded directly to a carbon atom, e.g., (2S) [1,2-.sup.13C.sub.2]glycerol and (2R) [1,2-.sup.13C.sub.2]glycerol, and to the use of such chiral glycerols in the preparation of labeled amino acids.

  5. 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.

  6. Preparation of 13C/15N-labeled oligomers using the polymerase chain reaction

    DOEpatents

    Chen, Xian; Gupta, Goutam; Bradbury, E. Morton

    2001-01-01

    Preparation of .sup.13 C/.sup.15 N-labeled DNA oligomers using the polymerase chain reaction (PCR). A PCR based method for uniform (.sup.13 C/.sup.15 N)-labeling of DNA duplexes is described. Multiple copies of a blunt-ended duplex are cloned into a plasmid, each copy containing the sequence of interest and restriction Hinc II sequences at both the 5' and 3' ends. PCR using bi-directional primers and uniformly .sup.13 C/.sup.15 N-labeled dNTP precursors generates labeled DNA duplexes containing multiple copies of the sequence of interest. Twenty-four cycles of PCR, followed by restriction and purification, gave the uniformly .sup.13 C/.sup.15 N-labeled duplex sequence with a 30% yield. Such labeled duplexes find significant applications in multinuclear magnetic resonance spectroscopy.

  7. Origin of acetaldehyde during milk fermentation using (13)C-labeled precursors.

    PubMed

    Ott, A; Germond, J E; Chaintreau, A

    2000-05-01

    Acetaldehyde formation by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus during fermentation of cow's milk was investigated using (13)C-labeled glucose, L-threonine, and pyruvate with a recent static-and-trapped-headspace technique that does not require derivatization of acetaldehyde prior to gas chromatography-mass spectrometry. Over 90% and almost 100% of acetaldehyde originated from glucose during fermentation by L. delbrueckii subsp. bulgaricus and S. thermophilus, respectively, taking into account both singly and doubly labeled acetaldehyde. As both microorganisms showed threonine aldolase activity and formed labeled acetaldehyde from (13)C-labeled threonine during the fermentation of milk, this amino acid should also contribute to the acetaldehyde produced.

  8. The use of 13C labeling to enhance the sensitivity of 13C solid-state CPMAS NMR to study polymorphism in low dose solid formulations.

    PubMed

    Booy, Kees-Jan; Wiegerinck, Peter; Vader, Jan; Kaspersen, Frans; Lambregts, Dorette; Vromans, Herman; Kellenbach, Edwin

    2005-02-01

    (13)C labeling was used to enhance the sensitivity of (13)C solid-state NMR to study the effect of tabletting on the polymorphism of a steroidal drug. The steroidal drug Org OD 14 was (13)C labeled and formulated into tablets containing only 0.5-2.5% active ingredient. The tablets were subsequently studied by solid-state (13)C CPMAS NMR. The crystalline form present in tablets could readily be analyzed in tablets. No change in crystalline form was observed as a result of formulation or in subsequent stability studies. Solid-state NMR in combination with (13)C labeling can, in suitable cases, be used as a strategy to study the effect of formulation on the polymorphism of low dose drugs.

  9. Economical synthesis of 13C-labeled opiates, cocaine derivatives and selected urinary metabolites by derivatization of the natural products.

    PubMed

    Karlsen, Morten; Liu, Huiling; Johansen, Jon Eigill; Hoff, Bård Helge

    2015-03-25

    The illegal use of opiates and cocaine is a challenge world-wide, but some derivatives are also valuable pharmaceuticals. Reference samples of the active ingredients and their metabolites are needed both for controlling administration in the clinic and to detect drugs of abuse. Especially, (13)C-labeled compounds are useful for identification and quantification purposes by mass spectroscopic techniques, potentially increasing accuracy by minimizing ion alteration/suppression effects. Thus, the synthesis of [acetyl-(13)C4]heroin, [acetyl-(13)C4-methyl-(13)C]heroin, [acetyl-(13)C2-methyl-(13)C]6-acetylmorphine, [N-methyl-(13)C-O-metyl-(13)C]codeine and phenyl-(13)C6-labeled derivatives of cocaine, benzoylecgonine, norcocaine and cocaethylene was undertaken to provide such reference materials. The synthetic work has focused on identifying (13)C atom-efficient routes towards these derivatives. Therefore, the (13)C-labeled opiates and cocaine derivatives were made from the corresponding natural products.

  10. 13C-labelled microdialysis studies of cerebral metabolism in TBI patients☆

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-06-16

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

  12. 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

  13. The biosynthetic pathway of curcuminoid in turmeric (Curcuma longa) as revealed by 13C-labeled precursors.

    PubMed

    Kita, Tomoko; Imai, Shinsuke; Sawada, Hiroshi; Kumagai, Hidehiko; Seto, Haruo

    2008-07-01

    In order to investigate the biosynthesis of curcuminoid in rhizomes of turmeric (Curcuma longa), we established an in vitro culture system of turmeric plants for feeding (13)C-labeled precursors. Analyses of labeled desmethoxycurcumin (DMC), an unsymmetrical curcuminoid, by (13)C-NMR, revealed that one molecule of acetic acid or malonic acid and two molecules of phenylalanine or phenylpropanoids, but not tyrosine, were incorporated into DMC. The incorporation efficiencies of the same precursors into DMC and curcumin were similar, and were in the order malonic acid > acetic acid, and cinnamic acid > p-coumaric acid > ferulic acid. These results suggest the possibility that the pathway to curcuminoids utilized two cinnamoyl CoAs and one malonyl CoA, and that hydroxy- and methoxy-functional groups on the aromatic rings were introduced after the formation of the curcuminoid skeleton.

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

    PubMed

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

    2015-03-01

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

  15. Synthesis and NMR studies of (13)C-labeled vitamin D metabolites.

    PubMed

    Okamura, William H; Zhu, Gui-Dong; Hill, David K; Thomas, Richard J; Ringe, Kerstin; Borchardt, Daniel B; Norman, Anthony W; Mueller, Leonard J

    2002-03-08

    Isotope-labeled drug molecules may be useful for probing by NMR spectroscopy the conformation of ligand associated with biological hosts such as membranes and proteins. Triple-labeled [7,9,19-(13)C(3)]-vitamin D(3) (56), its 25-hydroxylated and 1 alpha,25-dihydroxylated metabolites (58 and 68, respectively), and other labeled materials have been synthesized via coupling of [9-(13)C]-Grundmann's ketone 39 or its protected 25-hydroxy derivative 43 with labeled A ring enyne fragments 25 or 26. The labeled CD-ring fragment 39 was prepared by a sequence involving Grignard addition of [(13)C]-methylmagnesium iodide to Grundmann's enone 28, oxidative cleavage, functional group modifications leading to seco-iodide 38, and finally a kinetic enolate S(N)2 cycloalkylation. The C-7,19 double labeling of the A-ring enyne was achieved by the Corey-Fuchs/Wittig processes on keto aldehyde 11. By employing these labeled fragments in the Wilson-Mazur route, the C-7,9,19 triple-(13)C-labeled metabolites 56, 58, and 68 as well as other (13)C-labeled metabolites have been prepared. In an initial NMR investigation of one of the labeled metabolites prepared in this study, namely [7,9,19-(13)C(3)]-25-hydroxyvitamin D(3) (58), the three (13)C-labeled carbons of the otherwise water insoluble steroid could be clearly detected by (13)C NMR analysis at 0.1 mM in a mixture of CD(3)OD/D(2)O (60/40) or in aqueous dimethylcyclodextrin solution and at 2 mM in 20 mM sodium dodecyl sulfate (SDS) aqueous micellar solution. In the SDS micellar solution, a double half-filter NOESY experiment revealed that the distance between the H(19Z) and H(7) protons is significantly shorter than that of the corresponding distance calculated from the solid state (X-ray) structure of the free ligand. The NMR data in micelles reveals that 58 exists essentially completely in the alpha-conformer with the 3 beta-hydroxyl equatorially oriented, just as in the solid state. The shortened distance (H(19Z))-H(7)) in micellar

  16. Galacto-oligosaccharides have prebiotic activity in a dynamic in vitro colon model using a (13)C-labeling technique.

    PubMed

    Maathuis, Annet J H; van den Heuvel, Ellen G; Schoterman, Margriet H C; Venema, Koen

    2012-07-01

    Galacto-oligosaccharides (GOS) are considered to be prebiotic, although the contribution of specific members of the microbiota to GOS fermentation and the exact microbial metabolites that are produced upon GOS fermentation are largely unknown. We aimed to determine this using uniformly (13)C-labeled GOS. The normal (control) medium and unlabeled or (13)C-labeled GOS was added to a dynamic, validated, in vitro model of the large-intestine containing an adult-type microbiota. Liquid-chromatography MS was used to measure the incorporation of (13)C label into metabolites. 16S-rRNA stable isotope probing coupled to a phylogenetic micro-array was used to determine label incorporation in microbial biomass. The primary members within the complex microbiota that were directly involved in GOS fermentation were shown to be Bifidobacterium longum, B. bifidum, B. catenulatum, Lactobacillus gasseri, and L. salivarius, in line with the prebiotic effect of GOS, although some other species incorporated (13)C label also. GOS fermentation led to an increase in acetate (+49%) and lactate (+23%) compared with the control. Total organic acid production was 8.50 and 7.52 mmol/g of carbohydrate fed for the GOS and control experiments, respectively. At the same time, the cumulative production of putrefactive metabolites (branched-chain fatty acids and ammonia) was reduced by 55%. Cross-feeding of metabolites from primary GOS fermenters to other members of the microbiota was observed. Our findings support a prebiotic role for GOS and its potential to act as a synbiotic in combination with certain probiotic strains.

  17. 13C-NOESY-HSQC with Split Carbon Evolution for Increased Resolution with Uniformly Labeled Proteins

    NASA Astrophysics Data System (ADS)

    Baur, Matthias; Gemmecker, Gerd; Kessler, Horst

    1998-06-01

    Two new pulse sequences are presented for the recording of 2D13C-HSQC and 3D13C-NOESY-HSQC experiments, containing two consecutive carbon evolution periods. The two periods are separated by az-filter which creates a clean CxHz-quantum state for evolution in the second period. Each period is incremented (in anon-constant-time fashion) only to the extent that the defocusing of carbon inphase magnetization throughJ-coupling with neighboring carbons remains insignificant. Therefore,13C homonuclearJ-couplings are rendered ineffective, reducing the loss of signal and peak splitting commonly associated with long13C evolution times. The two periods are incremented according to a special acquisition protocol employing a13C-13C gradient echo to yield a data set analogous to one obtained by evolution over the added duration of both periods. The spectra recorded with the new technique on uniformly13C-labeled proteins at twice the evolution time of the standard13C-HSQC experiment display a nearly twofold enhancement of resolution in the carbon domain, while maintaining a good sensitivity even in the case of large proteins. Applied to the IIAManprotein ofE. coli(31 kDa), the13C-HSQC experiment recorded with a carbon evolution time of 2 × 8 ms showed a 36% decrease in linewidths compared to the standard13C-HSQC experiment, and theS/Nratio of representative cross-peaks was reduced to 40%. This reduction reflects mostly the typical loss of intensity observed when recording with an increased resolution. The13C-NOESY-HSQC experiment derived from the13C-HSQC experiment yielded additional NOE restraints between resonances which previously had been unresolved.

  18. Structural analysis of uniformly (13)C-labelled solids from selective angle measurements at rotational resonance.

    PubMed

    Patching, Simon G; Edwards, Rachel; Middleton, David A

    2009-08-01

    We demonstrate that individual H-C-C-H torsional angles in uniformly labelled organic solids can be estimated by selective excitation of (13)C double-quantum coherences under magic-angle spinning at rotational resonance. By adapting a straightforward one-dimensional experiment described earlier [T. Karlsson, M. Eden, H. Luhman, M.H. Levitt, J. Magn. Reson. 145 (2000) 95-107], a double-quantum filtered spectrum selective for Calpha and Cbeta of uniformly labelled L-[(13)C,(15)N]valine is obtained with 25% efficiency. The evolution of Calpha-Cbeta double-quantum coherence under the influence of the dipolar fields of bonded protons is monitored to provide a value of the Halpha-Calpha-Cbeta-Hbeta torsional angle that is consistent with the crystal structure. In addition, double-quantum filtration selective for C6 and C1' of uniformly labelled [(13)C,(15)N]uridine is achieved with 12% efficiency for a (13)C-(13)C distance of 2.5A, yielding a reliable estimate of the C6-H and C1'-H projection angle defining the relative orientations of the nucleoside pyrimidine and ribose rings. This procedure will be useful, in favourable cases, for structural analysis of fully labelled small molecules such as receptor ligands that are not readily synthesised with labels placed selectively at structurally diagnostic sites.

  19. Structural analysis of uniformly 13C-labelled solids from selective angle measurements at rotational resonance

    NASA Astrophysics Data System (ADS)

    Patching, Simon G.; Edwards, Rachel; Middleton, David A.

    2009-08-01

    We demonstrate that individual H-C-C-H torsional angles in uniformly labelled organic solids can be estimated by selective excitation of 13C double-quantum coherences under magic-angle spinning at rotational resonance. By adapting a straightforward one-dimensional experiment described earlier [T. Karlsson, M. Eden, H. Luhman, M.H. Levitt, J. Magn. Reson. 145 (2000) 95-107], a double-quantum filtered spectrum selective for Cα and Cβ of uniformly labelled L-[ 13C, 15N]valine is obtained with 25% efficiency. The evolution of Cα-Cβ double-quantum coherence under the influence of the dipolar fields of bonded protons is monitored to provide a value of the Hα-Cα-Cβ-Hβ torsional angle that is consistent with the crystal structure. In addition, double-quantum filtration selective for C6 and C1' of uniformly labelled [ 13C, 15N]uridine is achieved with 12% efficiency for a 13C- 13C distance of 2.5 Å, yielding a reliable estimate of the C6-H and C1'-H projection angle defining the relative orientations of the nucleoside pyrimidine and ribose rings. This procedure will be useful, in favourable cases, for structural analysis of fully labelled small molecules such as receptor ligands that are not readily synthesised with labels placed selectively at structurally diagnostic sites.

  20. 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

  1. 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

  2. The fate of (13)C-labelled and non-labelled inulin predisposed to large bowel fermentation in rats.

    PubMed

    Butts, Christine A; Paturi, Gunaranjan; Tavendale, Michael H; Hedderley, Duncan; Stoklosinski, Halina M; Herath, Thanuja D; Rosendale, Douglas; Roy, Nicole C; Monro, John A; Ansell, Juliet

    2016-04-01

    The fate of stable-isotope (13)C labelled and non-labelled inulin catabolism by the gut microbiota was assessed in a healthy rat model. Sprague-Dawley male rats were randomly assigned to diets containing either cellulose or inulin, and were fed these diets for 3 days. On day (d) 4, rats allocated to the inulin diet received (13)C-labelled inulin. The rats were then fed the respective non-labelled diets (cellulose or inulin) until sampling (d4, d5, d6, d7, d10 and d11). Post feeding of (13)C-labelled substrate, breath analysis showed that (13)C-inulin cleared from the host within a period of 36 hours. Faecal (13)C demonstrated the clearance of inulin from gut with a (13)C excess reaching maximum at 24 hours (d5) and then declining gradually. There were greater variations in caecal organic acid concentrations from d4 to d6, with higher concentrations of acetic, butyric and propionic acids observed in the rats fed inulin compared to those fed cellulose. Inulin influenced caecal microbial glycosidase activity, increased colon crypt depth, and decreased the faecal output and polysaccharide content compared to the cellulose diet. In summary, the presence of inulin in the diet positively influenced large bowel microbial fermentation.

  3. 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

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

    PubMed

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

    2014-11-01

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

  5. A method to trace root-respired CO2 using a 13C label

    NASA Astrophysics Data System (ADS)

    Cooperdock, S.; Breecker, D.; Litvak, M. E.

    2014-12-01

    In order to partition total soil respiration into root respiration and decomposition under ambient conditions in desert soils, the following method was developed using 13C-labeled CO2 in a modern juniper savannah in central New Mexico. The labeled CO2 was mixed with ambient air and pumped into a small (2.5 m diameter and 1.4 m tall) juniper tree canopy . 10 L of the 13CO2 was sufficient to generate a stream of air at 20 L/min for 1 hour with a CO2 concentration of 540 ppm and a δ13C value of approximately 35,000‰. Plastic tarpaulins were used as a wind block. The 13CO2 -labeled air was applied to the canopy during peak photosynthesis between 10 and 11 am on June 30 2014 during which canopy air CO2 was elevated by approximately 10 ppm over ambient and had δ13C values ranging from 50 to 1000 ‰. Over the next three days, gas and tissue samples were collected in order to trace the 13C label through the juniper tree. Leaf and root samples collected from the labeled tree and from several control trees were loaded into exetainer vials, flushed with CO2-free air and incubated in the dark for 5 hours in order to measure the carbon isotope composition of respired CO2. Samples of soil pore space gas were collected from wells under the labeled tree and a control tree and were transported to the laboratory in He-flushed exetainer vials. The δ13C values of CO2 in the soil gas samples and in the headspace of incubation vials were measured using an isotope ratio mass spectrometer. The δ13C values of foliar respiration were significantly higher than those of the control (by 3.6‰, p < 0.01) one and two days after labeling and δ13C values of root-respired CO2 were significantly higher (by 0.7‰, p = 0.01) than those of the control three days after labeling. In addition, δ13C values of soil respired CO2, determined from measurements of soil pore space CO2 at 50 cm three days after labeling, were significantly higher (by 0.7‰, p < 0.03)) for the labeled tree than control

  6. Linking autotrophic activity in environmental samples with specific bacterial taxa by detection of 13C-labelled fatty acids.

    PubMed

    Knief, Claudia; Altendorf, Karlheinz; Lipski, André

    2003-11-01

    A method for the detection of physiologically active autotrophic bacteria in complex microbial communities was developed based on labelling with the stable isotope 13C. Labelling of autotrophic nitrifying, sulphur-oxidizing and iron-oxidizing populations was performed in situ by incubation with NaH[13C]O3. Incorporated label into fatty acid methyl esters (FAMEs) was detected and quantified using gas chromatography-mass spectrometry in single ion monitoring mode. Before the analyses of different environmental samples, the protocol was evaluated in pure culture experiments. In different environmental samples a selective labelling of fatty acids demonstrated which microbial taxa were responsible for the respective chemolithoautotrophic activity. The most strongly labelled fatty acids of a sample from a sulphide treating biofilter from an animal rendering plant were cis-7-hexadecenoic acid (16:1 cis7) and 11-methyl hexadecanoic acid (16:0 11methyl), which are as-yet not known for any sulphide-oxidizing autotroph. The fatty acid labelling pattern of an experimental biotrickling filter sample supplied with dimethyl disulphide clearly indicated the presence and activity of sulphide-oxidizing bacteria of the genus Thiobacillus. For a third environmental sample from an acid mining lake sediment, the assignment of autotrophic activity to bacteria of the genus Leptospirillum but not to Acidithiobacillus could be made by this method, as the fatty acid patterns of these bacteria show clear differences.

  7. Isotopomer studies of gluconeogenesis and the Krebs cycle with 13C-labeled lactate.

    PubMed

    Katz, J; Wals, P; Lee, W N

    1993-12-05

    Fasted rats were intragastrically infused with either [2,3-13C]lactate or [1,2,3-13C]lactate. The infusate also contained 14C-labeled lactate and [3-3H]glucose. Glucose, alanine, glutamate, and glutamine were isolated from liver and blood. There was near complete equilibration of lactate and alanine, and the relative specific activities and relative enrichments were the same in blood and liver. Glucose was cleaved enzymatically to lactate. The compounds were examined by gas chromatography-mass spectroscopy. From the mass isotopomer spectra of the lactate, glutamate, and glutamine and their cleavage fragments the positional isotopomer composition of these compounds was obtained. The enrichment and isotopomer pattern in the lactate from cleaved glucose represents that in phosphoenolpyruvate (PEP). When [1,2,3-13C]lactate was infused the mass isotopomer spectrum of glutamates consisted only of compounds containing either one, two, or three 13C carbons per molecule (m1, m2, and m3). There was little 13C in C-4 and C-5 of glutamate. The rate of pyruvate decarboxylation is low, and 3-4% of the acetyl-CoA flux in the Krebs cycle is contributed by lactate carbon. The major isotopomers in lactate, alanine, and PEP were m3 and m2 with 13C in C-2 and C-3. The predominant isotopomer in PEP from [2,3-13C]lactate was m2 with 13C in C-2 and C-3. There was much more of m1 isotopomer with 13C in C-3 and C-2 than the m1 isotopomer with 13C in C-1. There was very little m3, the isotopomer with 13C in all three carbons. Most of the 13C in C-3 and C-4 of glucose and C-1 of glutamate was introduced via 13CO2 fixation. From the isotopomer distribution and the rate of glucose turnover we deduced, applying the analysis described in the "Appendix," the absolute rates of gluconeogenic pathways, recycling of PEP and the Cori cycle, and flux in the Krebs cycle. The flux from oxaloacetate (OAA)-->PEP was seven times that of OAA-->citrate, and about half of PEP was recycled to pyruvate via

  8. Retrobiosynthetic NMR studies with 13C-labeled glucose. Formation of gallic acid in plants and fungi.

    PubMed

    Werner, I; Bacher, A; Eisenreich, W

    1997-10-10

    The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina. Fungal cultures were grown with [1-13C]glucose or with a mixture of unlabeled glucose and [U-13C6]glucose. Young leaves of R. typhina were kept in an incubation chamber and were supplied with a solution containing a mixture of unlabeled glucose and [U-13C6]glucose via the leaf stem. Isotope distributions in isolated gallic acid and aromatic amino acids were analyzed by one-dimensional 1H and 13C NMR spectroscopy. A quantitative analysis of the complex isotopomer composition of metabolites was obtained by deconvolution of the 13C13C coupling multiplets using numerical simulation methods. This approach required the accurate analysis of heavy isotope chemical shift effects in a variety of different isotopomers and the analysis of long range 13C13C coupling constants. The resulting isotopomer patterns were interpreted using a retrobiosynthetic approach based on a comparison between the isotopomer patterns of gallic acid and tyrosine. The data show that both in the fungus and in the plant all carbon atoms of gallic acid are biosynthetically equivalent to carbon atoms of shikimate. Notably, the carboxylic group of gallic acid is derived from the carboxylic group of an early intermediate of the shikimate pathway and not from the side chain of phenylalanine or tyrosine. It follows that the committed precursor of gallic acid is an intermediate of the shikimate pathway prior to prephenate or arogenate, most probably 5-dehydroshikimate. A formation of gallic acid via phenylalanine, the lignin precursor, caffeic acid, or 3,4, 5-trihydroxycinnamic acid can be ruled out as major pathways in the fungus and in young leaves of R. typhina. The incorporation of uniformly 13C-labeled glucose followed by quantitative NMR analysis of isotopomer patterns is suggested as a general method for biosynthetic studies. As shown by the plant experiment, this

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

    PubMed

    Antoniewicz, Maciek R

    2015-12-01

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

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

    SciTech Connect

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

    2014-09-21

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

  11. Microbial transformations of free versus sorbed alanine analyzed by position-specific 13C and 14C labeling and 13C-PLFA analysis

    NASA Astrophysics Data System (ADS)

    Apostel, Carolin; Dippold, Michaela; Bore, Ezekiel; Kuzyakov, Yakov

    2015-04-01

    Sorption of charged or partially charged low molecular weight organic substances (LMWOS) to soil mineral surfaces delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil sciences, to compare the transformation mechanisms of sorbed and non-sorbed alanine in soil. Alanine as an amino acid links C- and N-cycles in soil and therefore is a model representative for the pool of LMWOS. To assess transformations of sorbed alanine, we combined position-specifically and uniformly 13C and 14C labeled alanine tracer solution with a loamy haplic luvisol that had previously been sterilized by γ-radiation. After shaking the mixtures, the supernatant was removed, as was all non-sorbed alanine by repeated shaking with millipore water. The labeled soil was added to non-sterilized soil from the same site. To compare the effect of sorption, soil labeled with the same position-specifically labeled tracers without previous sorption was prepared and incubated as well. We captured the respired CO2 and determined its 14C-activity at increasing time steps. The incorporation of 14C into microbial biomass was determined by CFE, and utilization of individual C positions by distinct microbial groups was evaluated by 13C-PLFA analysis. A dual peak in the respired CO2 revealed the influence of two sorption mechanisms. Microbial uptake and transformation of the sorbed alanine was 3 times slower compared to non-sorbed alanine. To compare the fate of individual C atoms independent of their concentration and pool size in soil, we introduced the divergence index (DI). The DI reveals the convergent or divergent behaviour of C from individual molecule positions during microbial utilization. The DI revealed, that alanines C-1 position was mainly oxidized to CO2, while its C-2 and C-3 were preferentially incorporated in microbial biomass and PLFAs. This indicates

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

    NASA Astrophysics Data System (ADS)

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

    2010-12-01

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

  13. Synthesis and applications of selectively {sup 13}C-labeled RNA

    SciTech Connect

    SantaLucia, J. Jr.; Shen, L.X.; Lewis, H.; Cai, Z.; Tinoci, I. Jr.

    1994-12-01

    Spectral overlap is a substantial problem in NMR studies of RNA molecules >30 nucleotides. To overcome this difficulty, we synthesized selectively {sup 13}C-labeled RNAs and adapted several isotope-edited two- and three-dimensional NMR experiments originally developed for protein studies. We optimized protocols for synthesis of multi-gram quantities of CTP, UTp, ATP, and GTP using a combination of synthetic organic and enzymatic methods. Uracil is prepared in 40 to 50% yield from {sup 13}C-cyanide in two steps. Using acetyl- tribenzoyl-ribose and standard chemistry uracil is then attached to the sugar (90% yield). The tribenzoyl-uridine intermediate is converted into uridine or cytidine quantitatively, depending on the deblocking protocol. Labeled purines are synthesized using simple pyrimidine precursors and reacting with {sup 13}C-formic acid (80% yield). Purine nucleosides are then synthesized using uridine phosphorylase and purine nucleoside phosphorylase. The nucleosides were converted to NMPs by treatment with POC1{sub 3} in triethylphosphate. We converted NMPs to NTPs by standard enzymatic methods. Selectively labeled RNAs were synthesized by run-off transcription using {sup 13}C-labeled NTPs. Several different strategies help solve over-lap problems in larger RNAs. Isotope-edited two-dimensional NMR experiments such as {omega}1-1/2 X-filtered NOESY simplify NMR spectra by dividing the normal NOESY spectrum into two subspectra-one involving NOEs from protons bound to {sup 12}C and one from protons bound to {sup 13}C. For example, we labeled A and U residues of a 34-nucleotide pseudoknot, and the {sup 12}C subspectrum of the 1/2 X-filtered NOESY contained NOEs only from G and C residues (along with adenine 2H); the {sup 13}C subspectrum contained NOEs only from A and U residues. Each subspectrum has less overlap than the NOESY of an unlabeled sample; the editing strategy allows each resonance to be identified by residue type (A, C, G, or U).

  14. 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

  15. Biosynthesis, molecular structure, and domain architecture of potato suberin: a (13)C NMR study using isotopically labeled precursors.

    PubMed

    Yan, B; Stark, R E

    2000-08-01

    Although suberin in potato wound periderm is known to be a polyester containing long-chain fatty acids and phenolics embedded within the cell wall, many aspects of its molecular structure and polymer-polymer connectivities remain elusive. The present work combines biosynthetic incorporation of site-specifically (13)C-enriched acetates and phenylalanines with one- and two-dimensional solid-state (13)C NMR spectroscopic methods to monitor the developing suberin polymer. Exogenous acetate is found to be incorporated preferentially at the carboxyl end of the aliphatic carbon chains, suggesting addition during the later elongation steps of fatty acid synthesis. Carboxyl-labeled phenylalanine precursors provide evidence for the concurrent development of phenolic esters and of monolignols typical of lignin. Experiments with ring-labeled phenylalanine precursors demonstrate a predominance of sinapyl and guaiacyl structures among suberin's phenolic moieties. Finally, the analysis of spin-exchange (solid-state NOESY) NMR experiments in ring-labeled suberin indicates distances of no more than 0.5 nm between pairs of phenolic and oxymethine carbons, which are attributed to the aromatic-aliphatic polyester and the cell wall polysaccharide matrix, respectively. These results offer direct and detailed molecular information regarding the insoluble intermediates of suberin biosynthesis, indicate probable covalent linkages between moieties of its polyester and polysaccharide domains, and yield a clearer overall picture of this agriculturally important protective material.

  16. Asymmetry of (13)C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.

    PubMed

    Thakur, Chandar S; Dayie, T Kwaku

    2011-12-01

    Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of (13)C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-(13)C]-pyruvate affords ribonucleotides with site specific labeling at C5' (~95%) and C1' (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-(13)C]-glycerol for which the ribose ring is labeled in all but the C4' carbon position, leading to multiplet splitting of the C1', C2' and C3' carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.

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

    PubMed

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

    2016-11-01

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

  18. Pentose cycling and the distribution of 13C in trehalose during glucogenesis from 13C-labelled substrates in an insect.

    PubMed

    Thompson, S N; Scales, V M; Bochardt, D B

    1995-07-26

    Redistribution of 13C in trehalose (Tre) due to pentose cycling was observed in vivo in Manduca sexta during glucogenesis from [3-13C]alanine (Ala) and [2-13C]glycerol (Gly). The extent of cycling was affected by dietary composition. Larvae maintained on a low-carbohydrate diet (LCD) exhibited approximately 13% cycling, while those on a complete-balanced diet (CBD) or low-fat diet (LFD) displayed much higher rates of cycling. Significant incorporation of 13C via reversal of the non-oxidative phase was evident on all diets but was greatest on the CBD and LFD. In contrast to conclusions from previous studies with insects, the present results indicate that under normal conditions the pentose pathway is not the principal source of triose phosphates for oxidative catabolism during larval development.

  19. 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

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

    PubMed Central

    2011-01-01

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

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

    PubMed

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

    2016-09-01

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

  2. (13)C-Labeling the carbon-fixation pathway of a highly efficient artificial photosynthetic system.

    PubMed

    Liu, Chong; Nangle, Shannon N; Colón, Brendan C; Silver, Pamela A; Nocera, Daniel G

    2017-03-15

    Interfacing the CO2-fixing microorganism, Ralstonia eutropha, to the energy derived from hydrogen produced by water splitting is a viable approach to achieving renewable CO2 reduction at high efficiencies. We employ (13)C-labeling to report on the nature of CO2 reduction in the inorganic water splitting|R. eutropha hybrid system. Accumulated biomass in a reactor under a (13)C-enriched CO2 atmosphere may be sampled at different time points during CO2 reduction. Converting the sampled biomass into gaseous CO2 allows the (13)C/(12)C ratio to be determined by gas chromatography-mass spectrometry. After 2 hours of inoculation and the initiation of water splitting, the microbes adapted and began to convert CO2 into biomass. The observed time evolution of the (13)C/(12)C ratio in accumulated biomass is consistent with a Monod model for carbon fixation. Carbon dioxide produced by catabolism was found to be minimal. This rapid response of the bacteria to a hydrogen input and to subsequent CO2 reduction at high efficiency are beneficial to achieving artificial photosynthesis for the storage of renewable energy.

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

    PubMed Central

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

    2007-01-01

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

  4. Methanogenic capabilities of ANME-archaea deduced from (13) C-labelling approaches.

    PubMed

    Bertram, Sebastian; Blumenberg, Martin; Michaelis, Walter; Siegert, Michael; Krüger, Martin; Seifert, Richard

    2013-08-01

    Anaerobic methanotrophic archaea (ANME) are ubiquitous in marine sediments where sulfate dependent anaerobic oxidation of methane (AOM) occurs. Despite considerable progress in the understanding of AOM, physiological details are still widely unresolved. We investigated two distinct microbial mat samples from the Black Sea that were dominated by either ANME-1 or ANME-2. The (13) C lipid stable isotope probing (SIP) method using labelled substances, namely methane, bicarbonate, acetate, and methanol, was applied, and the substrate-dependent methanogenic capabilities were tested. Our data provide strong evidence for a versatile physiology of both, ANME-1 and ANME-2. Considerable methane production rates (MPRs) from CO2 -reduction were observed, particularly from ANME-2 dominated samples and in the presence of methane, which supports the hypothesis of a co-occurrence of methanotrophy and methanogenesis in the AOM systems (AOM/MPR up to 2:1). The experiments also revealed strong methylotrophic capabilities through (13) C-assimilation from labelled methanol, which was independent of the presence of methane. Additionally, high MPRs from methanol were detected in both of the mat samples. As demonstrated by the (13) C-uptake into lipids, ANME-1 was found to thrive also under methane free conditions. Finally, C35 -isoprenoid hydrocarbons were identified as new lipid biomarkers for ANME-1, most likely functioning as a hydrogen sink during methanogenesis.

  5. 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.

  6. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    PubMed

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

    2009-12-01

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

  9. Sparse (13)C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins.

    PubMed

    Liu, Jing; Liu, Chang; Fan, Ying; Munro, Rachel A; Ladizhansky, Vladimir; Brown, Leonid S; Wang, Shenlin

    2016-05-01

    We demonstrate a novel sparse (13)C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically (13)C labelled glycerol as carbon sources in the expression medium. This strategy improves the spectral resolution by 1.5 fold, displays site-specific labelling patterns, and has advantages for collecting long-range distance restraints for structure determination of large eukaryotic membrane proteins by solid-state NMR.

  10. Direct uptake of organic carbon by grass roots and allocation in leaves and phytoliths: 13C labeling evidence

    NASA Astrophysics Data System (ADS)

    Alexandre, A.; Balesdent, J.; Cazevieille, P.; Chevassus-Rosset, C.; Signoret, P.; Mazur, J.-C.; Harutyunyan, A.; Doelsch, E.; Basile-Doelsch, I.; Miche, H.; Santos, G. M.

    2015-12-01

    In the rhizosphere, the uptake of low molecular weight carbon (C) and nitrogen (N) by plant roots has been well documented. While organic N uptake relatively to total uptake is important, organic C uptake is supposed to be low relatively to the plant's C budget. Recently, radiocarbon analyses demonstrated that a fraction of C from the soil was occluded in amorphous silica micrometric particles that precipitate in plant cells (phytoliths). Here, we investigated whether and in which extent organic C absorbed by grass roots, under the form of either intact amino acids (AAs) or microbial metabolites, can feed the organic C occluded in phytoliths. For this purpose we added 13C- and 15N-labeled AAs to the silicon-rich hydroponic solution of the grass Festuca arundinacea. The experiment was designed to prevent C leakage from the labeled nutritive solution to the chamber atmosphere. After 14 days of growth, the 13C and 15N enrichments (13C-excess and 15N-excess) in the roots, stems and leaves, and phytoliths, as well as the 13C-excess in AAs extracted from roots and stems and leaves, were quantified relatively to a control experiment in which no labelled AAs were added. The net uptake of 13C derived from the labeled AAs supplied to the nutritive solution (AA-13C) by Festuca arundinacea represented 4.5 % of the total AA-13C supply. AA-13C fixed in the plant represented only 0.13 % of total C. However, the experimental conditions may have underestimated the extent of the process under natural and field conditions. Previous studies showed that 15N and 13C can be absorbed by the roots in several organic and inorganic forms. In the present experiment, the fact that phenylalanine and methionine, that were supplied in high amount to the nutritive solution, were more 13C-enriched than other AAs in the roots and stems and leaves strongly suggested that part of AA-13C was absorbed and translocated in its original AA form. The concentration of AA-13C represented only 0.15 % of the

  11. Partitioning CO2 effluxes from an Atlantic pine forest soil between endogenous soil organic matter and recently incorporated 13C-enriched plant material.

    PubMed

    Fernandez, Irene; Cabaneiro, Ana; González-Prieto, Serafín J

    2006-04-15

    Soil CO2 effluxes from recently added 13C-labeled phytomass versus endogenous soil organic matter (SOM) were studied in an acid soil from Atlantic pine forests (NW Spain). After several cultures to incorporate fresh 13C-enriched Lolium perenne to a Humic Cambisol with predominance of humus--Al over humus--Fe complexes, potential soil C mineralization was determined by laboratory aerobic incubation (84 days). Isotopic 13C analyses of SOM fractions were assessed to know in which organic compartments the 13C was preferentially incorporated. Although in the 13C-labeled soil the C mineralization coefficient totalized less than 3% of soil C, the 13C mineralization coefficient exceeded 14%, indicating a greater lability of the newly incorporated organic matter. Organic compounds coming from added phytomass showed a higher lability and contributed considerably to the total soil CO2 effluxes (52% of total soil CO2 evolved during the first decomposition stages and 27% at the end), even though added-C comprised less than 4% of total soil C. Good determination coefficients, when values of CO2--C released were fitted to a first-order double exponential kinetic model, support the existence of two C pools of different lability. Kinetic parameters obtained with this model indicated that phytomass addition augmented the biodegradability of the labile pool (instantaneous mineralization rate k increased from 0.07 d(-1) to 0.12 d(-1)) but diminished that of the recalcitrant pool (instantaneous mineralization rate h decreased from 2.7 x 10(-4) d(-1) to 1.6 x 10(-4) d(-1)). Consequently, the differentiation between both SOM pools increased, showing the importance of SOM quality on CO2 emissions from this kind of soil to the atmosphere.

  12. Detection of 2,4,6-Trinitrotoluene-Utilizing Anaerobic Bacteria by 15N and 13C Incorporation

    PubMed Central

    Gallagher, Erin M.; Young, Lily Y.; McGuinness, Lora M.; Kerkhof, Lee J.

    2010-01-01

    2,4,6-Trinitrotoluene (15N or 13C labeled) was added to Norfolk Harbor sediments to test whether anaerobic bacteria use TNT for growth. Stable-isotope probing (SIP)-terminal restriction fragment length polymorphism (TRFLP) detected peaks in the [15N]TNT cultures (60, 163, and 168 bp). The 60-bp peak was also present in the [13C]TNT cultures and was related to Lysobacter taiwanensis. PMID:20081008

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

    PubMed

    Niklas, Jens; Sandig, Volker; Heinzle, Elmar

    2011-12-01

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

  14. Origin of Epilachnapaenulata defensive alkaloids: incorporation of [1-13C]-sodium acetate and [methyl-2H3]-stearic acid.

    PubMed

    Camarano, S; González, A; Rossini, C

    2012-01-01

    Ladybird beetles produce a large number of defensive alkaloids. Previous studies suggest that the structural diversity of these endogenous alkaloids can be traced to a common biosynthetic route based on the condensation of several acetate units. In this study, adults of Epilachna paenulata, a phytophagous neotropical species, were fed on diet enriched with potential precursors (sodium acetate, fatty acids and the amino acids lysine and ornithine) labeled with stable isotopes ((13)C, (2)H and (15)N). Labeled acetate was incorporated into the structurally related homotropane and piperidine alkaloids. The later also showed incorporation of [methyl-(2)H3] stearic acid. Our results hence support a fatty acid pathway for the biosynthesis of E. paenulata alkaloids. To our knowledge, this is the first report on the incorporation of a labeled fatty acid into a defensive piperidine alkaloid in insects.

  15. Monitoring CO[subscript 2] Fixation Using GC-MS Detection of a [superscript 13]C-Label

    ERIC Educational Resources Information Center

    Hammond, Daniel G.; Bridgham, April; Reichert, Kara; Magers, Martin

    2010-01-01

    Much of our understanding of metabolic pathways has resulted from the use of chemical and isotopic labels. In this experiment, a heavy isotope of carbon, [superscript 13]C, is used to label the product of the well-known RuBisCO enzymatic reaction. This is a key reaction in photosynthesis that converts inorganic carbon to organic carbon; a process…

  16. Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling

    PubMed Central

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA – the combing, imaging by SIMS or CIS method – has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to 13C-labeling via the detection and quantification of the 13C 14N - recombinant ion and the use of the 13C: 12C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  17. Direct uptake of organically derived carbon by grass roots and allocation in leaves and phytoliths: 13C labeling evidence

    NASA Astrophysics Data System (ADS)

    Alexandre, Anne; Balesdent, Jérôme; Cazevieille, Patrick; Chevassus-Rosset, Claire; Signoret, Patrick; Mazur, Jean-Charles; Harutyunyan, Araks; Doelsch, Emmanuel; Basile-Doelsch, Isabelle; Miche, Hélène; Santos, Guaciara M.

    2016-03-01

    In the rhizosphere, the uptake of low-molecular-weight carbon (C) and nitrogen (N) by plant roots has been well documented. While organic N uptake relative to total uptake is important, organic C uptake is supposed to be low relative to the plant's C budget. Recently, radiocarbon analyses demonstrated that a fraction of C from the soil was occluded in amorphous silica micrometric particles that precipitate in plant cells (phytoliths). Here, we investigated whether and to what extent organically derived C absorbed by grass roots can feed the C occluded in phytoliths. For this purpose we added 13C- and 15N-labeled amino acids (AAs) to the silicon-rich hydroponic solution of the grass Festuca arundinacea. The experiment was designed to prevent C leakage from the labeled nutritive solution to the chamber atmosphere. After 14 days of growth, the 13C and 15N enrichments (13C excess and 15N excess) in the roots, stems and leaves as well as phytoliths were measured relative to a control experiment in which no labeled AAs were added. Additionally, the 13C excess was measured at the molecular level, in AAs extracted from roots and stems and leaves. The net uptake of labeled AA-derived 13C reached 4.5 % of the total AA 13C supply. The amount of AA-derived 13C fixed in the plant was minor but not nil (0.28 and 0.10 % of total C in roots and stems/leaves, respectively). Phenylalanine and methionine that were supplied in high amounts to the nutritive solution were more 13C-enriched than other AAs in the plant. This strongly suggested that part of AA-derived 13C was absorbed and translocated into the plant in its original AA form. In phytoliths, AA-derived 13C was detected. Its concentration was on the same order of magnitude as in bulk stems and leaves (0.15 % of the phytolith C). This finding strengthens the body of evidences showing that part of organic compounds occluded in phytoliths can be fed by C entering the plant through the roots. Although this experiment was done in

  18. {sup 13}C-enrichment at carbons 8 and 2 of uric acid after {sup 13}C-labeled folate dose in man

    SciTech Connect

    Baggott, Joseph E.; Gorman, Gregory S.; Morgan, Sarah L.; Tamura, Tsunenobu . E-mail: tamurat@uab.edu

    2007-09-21

    To evaluate folate-dependent carbon incorporation into the purine ring, we measured {sup 13}C-enrichment independently at C{sub 2} and C{sub 8} of urinary uric acid (the final catabolite of purines) in a healthy male after an independent oral dose of [6RS]-5-[{sup 13}C]-formyltetrahydrofolate ([6RS]-5-H{sup 13}CO-H{sub 4}folate) or 10-H{sup 13}CO-7,8-dihydrofolate (10-H{sup 13}CO-H{sub 2}folate). The C{sub 2} position was {sup 13}C-enriched more than C{sub 8} after [6RS]-5-H{sup 13}CO-H{sub 4}folate, and C{sub 2} was exclusively enriched after 10-H{sup 13}CO-H{sub 2}folate. The enrichment of C{sub 2} was greater from [6RS]-5-H{sup 13}CO-H{sub 4}folate than 10-H{sup 13}CO-H{sub 2}folate using equimolar bioactive doses. Our data suggest that formyl C of [6RS]-10-H{sup 13}CO-H{sub 4}folate was not equally utilized by glycinamide ribotide transformylase (enriches C{sub 8}) and aminoimidazolecarboxamide ribotide (AICAR) transformylase (enriches C{sub 2}), and the formyl C of 10-H{sup 13}CO-H{sub 2}folate was exclusively used by AICAR transformylase. 10-HCO-H{sub 2}folate may function in vivo as the predominant substrate for AICAR transformylase in humans.

  19. Stabilization of glucose-C in microbial cell membranes (PLFA) and cell walls (amino sugars) evaluated by 13C-labelling in a field experiment

    NASA Astrophysics Data System (ADS)

    Gunina, Anna; Kuzyakov, Yakov; Glaser, Bruno

    2015-04-01

    Microorganisms control carbon (C) cycle and strongly contribute to formation of soil organic matter. Strong differences in the turnover of microbial groups and cellular compounds complicate the assessment of their contribution to microbial food webs and C sequestration in soil in situ. The uptake and incorporation of 13C labeled glucose by microbial groups were traced during 50 days after the labeling under field conditions. 13C was analysed: i) in the cytosolic pool by chloroform fumigation extraction, ii) in cell membranes by phospholipid fatty acids (PLFA), iii) in cell walls by amino sugars, and iv) remaining in bulk soil. This allowed tracing C in microbial groups as well as cellular compounds. Mean residence times (MRT) of C in PLFA and the cytosol were 47 and 150 days, respectively. Such long cytosol MRT depends on its heterogeneous composition, which includes high and low molecular weight organics. Amino sugars were mainly originated from microbial residues and thus, observation periods higher than 1 year are required for estimation of their MRT. Relative 13C incorporation (13C portion in total pool C) was the highest for PLFAs (~1.5% at day 3), whereas 13C content of the cytosol and amino sugars was one and two orders of magnitude less, respectively. Relative 13C incorporation into amino sugars of living microorganisms showed only 0.57% on day 3. Therefore, the turnover of cell membrane components is two times faster than that of cell walls, even in living microorganisms. Both PLFAs and amino sugars showed that glucose C was preferentially used by bacteria. 13C incorporation into bacterial cell walls and membranes decreased with time, but increased or remained constant for fungi, reflecting faster turnover of bacteria than fungi. Consequently, bacteria contribute more to the decomposition of low molecular weight organics, whereas fungi consume bacterial products or necromass and contribute more to long-term C stabilisation. Thus, tracing of 13C in cellular

  20. 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.

  1. Determination of sup 13 C labeling pattern of citric acid cycle intermediates by gas chromatography-mass spectrometry

    SciTech Connect

    Di Donato, L.; Montgomery, J.A.; Des Rosiers, C.; David, F.; Garneau, M.; Brunengraber, H. )

    1990-02-26

    Investigations of the regulation of the citric acid cycle require determination of labeling patterns of cycle intermediates. These were assayed to date, using infusion of: (i) ({sup 14}C)tracer followed by chemical degradation of intermediates and (ii) ({sup 13}C)tracer followed by NMR analysis of intermediates. The authors developed a strategy to analyze by GC-MS the ({sup 13}C) labeling pattern of {mu}mole samples of citrate (CIT), isocitrate (ICIT), 2-ketoglutarate (2-KG), glutamate (GLU) and glutamine (GLN). These are enzymatically or chemically converted to 2-KG, ICIT, 4-aminobutyrate (GABA) and 2-hydroxyglutarate (2-OHG). GC-MS analyses of TMS or TBDMS derivatives of these compounds yield the enrichment of each carbon. The authors confirmed the identity of each fragment using the spectra of (1-{sup 13}C), (5-{sup 13}C), (2,3,3,4,4-{sup 2}H{sub 5})glutamate and (1-{sup 13}C), (1,4-{sup 13}C)GABA.

  2. Enzymatic (13)C labeling and multidimensional NMR analysis of miltiradiene synthesized by bifunctional diterpene cyclase in Selaginella moellendorffii.

    PubMed

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

    2011-12-16

    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-(13)C(6)]mevalonate, all carbons were labeled with (13)C stable isotope (>99%). The fully (13)C-labeled product was subjected to (13)C-(13)C 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

  3. Evidence of the photosynthetic origin of monoterpenes emitted by quercus ilex L. leaves by {sup 13}C labeling

    SciTech Connect

    Loreto, F.; Ciccioli, P.; Cecinato, A.; Brancaleoni, E. |

    1996-04-01

    The carbon of the four main monoterpenes emitted by Quercus ilex L. leaves was completely labeled with {sup 13}C after a 20-min feeding with 99% {sup 13}CO{sub 2}. This labeling time course is comparable with the labeling time course of isoprene, the terpenoid emitted by other Quercus species and synthesized in leaf chloroplasts. It is also comparable with that of phosphoglyceric acid. Our experiment therefore provides evidence that monoterpenes emitted by Q. ilex are formed photosynthesis intermediates and may share the same synthetic pathway with isoprene. By analyzing the rate and the distribution of labeling in the different fragments, we looked for evidence of differential carbon labeling in the {alpha}-pinene emitted. However, the labeling pattern was quite uniform in the different fragments, suggesting that the carbon skeleton of the emitted monoterpenes comes from a unique carbon source. 16 refs., 3 figs., 1 tab.

  4. Use of 13C-Labeled Substrates to Determine Relative Methane Production Rates in Hypersaline Microbial Communities

    NASA Astrophysics Data System (ADS)

    Kelley, C. A.; Bebout, B.; Chanton, J.

    2015-12-01

    Rates and pathways of methane production were determined from photosynthetic soft microbial mats and gypsum-encrusted endoevaporites collected in hypersaline environments from California, Mexico and Chile, as well as an organic-rich mud from a pond in the El Tatio volcanic fields, Chile. Samples (mud, homogenized soft mats and endoevaporites) were incubated anaerobically with deoxygenated site water, and the increase in methane concentration through time in the headspaces of the incubation vials was used to determine methane production rates. To ascertain the substrates used by the methanogens, 13C-labeled methylamines, methanol, dimethylsulfide, acetate or bicarbonate were added to the incubations (one substrate per vial) and the stable isotopic composition of the resulting methane was measured. The vials amended with 13C-labeled methylamines produced the most 13C-enriched methane, generally followed by the 13C-labeled methanol-amended vials. The stable isotope data and the methane production rates were used to determine first order rate constants for each of the substrates at each of the sites. Estimates of individual substrate use revealed that the methylamines produced 55 to 92% of the methane generated, while methanol was responsible for another 8 to 40%.

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

    NASA Astrophysics Data System (ADS)

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

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

  6. Fate of xylem-transported 11C- and 13C-labeled CO2 in leaves of poplar.

    PubMed

    Bloemen, Jasper; Bauweraerts, Ingvar; De Vos, Filip; Vanhove, Christian; Vandenberghe, Stefaan; Boeckx, Pascal; Steppe, Kathy

    2015-04-01

    In recent studies, assimilation of xylem-transported CO2 has gained considerable attention as a means of recycling respired CO2 in trees. However, we still lack a clear and detailed picture on the magnitude of xylem-transported CO2 assimilation, in particular within leaf tissues. To this end, detached poplar leaves (Populus × canadensis Moench 'Robusta') were allowed to take up a dissolved (13)CO2 label serving as a proxy of xylem-transported CO2 entering the leaf from the branch. The uptake rate of the (13)C was manipulated by altering the vapor pressure deficit (VPD) (0.84, 1.29 and 1.83 kPa). Highest tissue enrichments were observed under the highest VPD. Among tissues, highest enrichment was observed in the petiole and the veins, regardless of the VPD treatment. Analysis of non-labeled leaves showed that some (13)C diffused from the labeled leaves and was fixed in the mesophyll of the non-labeled leaves. However, (13)C leaf tissue enrichment analysis with elemental analysis coupled to isotope ratio mass spectrometry was limited in spatial resolution at the leaf tissue level. Therefore, (11)C-based CO2 labeling combined with positron autoradiography was used and showed a more detailed spatial distribution within a single tissue, in particular in secondary veins. Therefore, in addition to (13)C, (11) C-based autoradiography can be used to study the fate of xylem-transported CO2 at leaf level, allowing the acquisition of data at a yet unprecedented resolution.

  7. Carbon Transfer from the Host to Tuber melanosporum Mycorrhizas and Ascocarps Followed Using a 13C Pulse-Labeling Technique

    PubMed Central

    Le Tacon, François; Zeller, Bernd; Plain, Caroline; Hossann, Christian; Bréchet, Claude; Robin, Christophe

    2013-01-01

    Truffles ascocarps need carbon to grow, but it is not known whether this carbon comes directly from the tree (heterotrophy) or from soil organic matter (saprotrophy). The objective of this work was to investigate the heterotrophic side of the ascocarp nutrition by assessing the allocation of carbon by the host to Tuber melanosporum mycorrhizas and ascocarps. In 2010, a single hazel tree selected for its high truffle (Tuber melanosporum) production and situated in the west part of the Vosges, France, was labeled with 13CO2. The transfer of 13C from the leaves to the fine roots and T. melanosporum mycorrhizas was very slow compared with the results found in the literature for herbaceous plants or other tree species. The fine roots primarily acted as a carbon conduit; they accumulated little 13C and transferred it slowly to the mycorrhizas. The mycorrhizas first formed a carbon sink and accumulated 13C prior to ascocarp development. Then, the mycorrhizas transferred 13C to the ascocarps to provide constitutive carbon (1.7 mg of 13C per day). The ascocarps accumulated host carbon until reaching complete maturity, 200 days after the first labeling and 150 days after the second labeling event. This role of the Tuber ascocarps as a carbon sink occurred several months after the end of carbon assimilation by the host and at low temperature. This finding suggests that carbon allocated to the ascocarps during winter was provided by reserve compounds stored in the wood and hydrolyzed during a period of frost. Almost all of the constitutive carbon allocated to the truffles (1% of the total carbon assimilated by the tree during the growing season) came from the host. PMID:23741356

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-16

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

  10. Survival of free-living Acholeplasma in aerated pig manure slurry revealed by 13C-labeled bacterial biomass probing

    PubMed Central

    Hanajima, Dai; Aoyagi, Tomo; Hori, Tomoyuki

    2015-01-01

    Many studies have been performed on microbial community succession and/or predominant taxa during the composting process; however, the ecophysiological roles of microorganisms are not well understood because microbial community structures are highly diverse and dynamic. Bacteria are the most important contributors to the organic-waste decomposition process, while decayed bacterial cells can serve as readily digested substrates for other microbial populations. In this study, we investigated the active bacterial species responsible for the assimilation of dead bacterial cells and their components in aerated pig manure slurry by using 13C-labeled bacterial biomass probing. After 3 days of forced aeration, 13C-labeled and unlabeled dead Escherichia coli cell suspensions were added to the slurry. The suspensions contained 13C-labeled and unlabeled bacterial cell components, possibly including the cell wall and membrane, as well as intracellular materials. RNA extracted from each slurry sample 2 h after addition of E. coli suspension was density-resolved by isopycnic centrifugation and analyzed by terminal restriction fragment length polymorphism, followed by cloning and sequencing of bacterial 16S rRNA genes. In the heavy isotopically labeled RNA fraction, the predominant 13C-assimilating population was identified as belonging to the genus Acholeplasma, which was not detected in control heavy RNA. Acholeplasma spp. have limited biosynthetic capabilities and possess a wide variety of transporters, resulting in their metabolic dependence on external carbon and energy sources. The prevalence of Acholeplasma spp. was further confirmed in aerated pig manure slurry from four different pig farms by pyrosequencing of 16S rRNA genes; their relative abundance was ∼4.4%. Free-living Acholeplasma spp. had a competitive advantage for utilizing dead bacterial cells and their components more rapidly relative to other microbial populations, thus allowing the survival and prevalence

  11. Vibrational studies of {sup 13}C- and {sup 34}S-labelled bis(ethylenedithio)tetrathiafulvalene (ET) donor molecule

    SciTech Connect

    Ferraro, J.R.; Kini, A.M.; Williams, J.M.; Stout, P.

    1994-06-01

    FT-IR and FT-Raman studies of {sup 13}C- and {sup 34}S-labelled bis(ethylenedithio)-tetrathiafulvalene (BEDT-TTF or ET) electron-donor molecules were made and the results presented herein. Assignments for fundamental vibrations in ET were verified. Spectral data confirms that ET has no center-of-symmetry, and that the data can be reconciled by a D-type point group with only slight interactions occurring between the 4 molecules per unit cell.

  12. Vitamin K absorption and kinetics in human subjects after consumption of 13C-labelled phylloquinone from kale.

    PubMed

    Novotny, Janet A; Kurilich, Anne C; Britz, Steven J; Baer, David J; Clevidence, Beverly A

    2010-09-01

    The absorption and plasma disappearance of vitamin K were investigated by uniformly labelling phylloquinone in kale with carbon-13, and by feeding the kale to study subjects. Seven healthy volunteers ingested a single 400 g serving of kale with 30 g vegetable oil. The kale provided 156 nmol of phylloquinone. Serial plasma samples were collected and analysed for the appearance of 13C-phylloquinone by HPLC-MS. Six of the subjects showed significant amounts of labelled phylloquinone in plasma, though one subject's plasma was not consistently enriched above the detection limit, and this subject's baseline plasma phylloquinone level was the lowest in the group. After ingestion of the labelled kale, plasma 13C-phylloquinone concentration increased rapidly to a peak between 6 and 10 h, and then rapidly decreased. Average peak plasma concentration for the six subjects with detectable 13C-phylloquinone was 2.1 nmol/l. Plasma concentration-time data were analysed by compartmental modelling. Modelling results demonstrated a mean (n 6) bioavailability of phylloquinone from kale to be 4.7%. Plasma and tissue half-times for phylloquinone were found to be 8.8 and 215 h, respectively.

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

    PubMed Central

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

    2016-01-01

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

  14. Optimization of 13C dynamic nuclear polarization: isotopic labeling of free radicals

    NASA Astrophysics Data System (ADS)

    Niedbalski, Peter; Parish, Christopher; Kiswandi, Andhika; Lumata, Lloyd

    Dynamic nuclear polarization (DNP) is a physics technique that amplifies the nuclear magnetic resonance (NMR) signals by transferring the high polarization of the electrons to the nuclear spins. Thus, the choice of free radical is crucial in DNP as it can directly affect the NMR signal enhancement levels, typically on the order of several thousand-fold in the liquid-state. In this study, we have investigated the efficiency of four variants of the well-known 4-oxo-TEMPO radical (normal 4-oxo-TEMPO plus its 15N-enriched and/or perdeuterated variants) for use in DNP of an important metabolic tracer [1-13C]acetate. Though the variants have significant differences in electron paramagnetic resonance (EPR) spectra, we have found that changing the composition of the TEMPO radical through deuteration or 15N doping yields no significant difference in 13C DNP efficiency at 3.35 T and 1.2 K. On the other hand, deuteration of the solvent causes a significant increase of 13C polarization that is consistent over all the 4-oxo-TEMPO variants. These findings are consistent with the thermal mixing model of DNP. This work is supported by US Dept of Defense Award No. W81XWH-14-1-0048 and the Robert A. Welch Foundation Grant No. AT-1877.

  15. The effect of biochar amendment on the soil microbial community - PLFA analyses and 13C labeling results

    NASA Astrophysics Data System (ADS)

    Watzinger, A.; Feichtmair, S.; Rempt, F.; Anders, E.; Wimmer, B.; Kitzler, B.; Zechmeister-Boltenstern, S.; Horacek, M.; Zehetner, F.; Kloss, S.; Richoz, S.; Soja, G.

    2012-04-01

    The effects of biochar amendment on plant growth and on the chemical / physical soil characteristics are well explored but only few studies have investigated the impact on soil microorganisms. The response of the soil microbial community to biochar amendment was investigated by phospholipid fatty acid (PLFA) analysis in (i) a large scale pot experiment, (ii) a small scale pot experiment using 13C labeled biochar and (iii) an incubation study using 13C labeled biochar. In the large scale pot experiment, three different agricultural soils from Austria (Planosol, Cambisol, Chernozem) and four different types of biochar were investigated. In total, 25 treatments with 5 replicates each were set up and monitored over a year. The results from the pot experiments showed no significant influence of biochar amendment on the total microbial biomass in the first 100 days after biochar addition. However, discriminant analysis showed a distinction of biochar and control soils as well as a strong effect of the pyrolysis temperature on the microbial composition. The effect of biochar was dependent on the type of soil. In the Planosol, some PLFAs were affected positively, especially when adding biochar with a low pyrolysis temperature, in the first month. In the long term, microbial community composition altered. Growth of fungi and gram negative bacteria was enhanced. In the Chernozem, PLFAs from various microbial groups decreased in the long term. Variability in the incubation study was low. Consequently, many PLFAs were significantly affected by biochar amendment. Again, in the Planosol, gram negative bacteria, actinomycetes and, after 2 weeks, gram positive bacteria increased under biochar amendment whereas in the chernozem total microbial biomass and gram positive bacteria were negatively affected in the long term. The 13C labeling studies confirmed the low degradability of the biochar, i.e. no alteration of the content and the δ13C in the soil organic matter within 100 days

  16. 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.

  17. Atmospheric CO2 level affects plants' carbon use efficiency: insights from a 13C labeling experiment on sunflower stands

    NASA Astrophysics Data System (ADS)

    Gong, Xiaoying; Schäufele, Rudi; Schnyder, Hans

    2015-04-01

    The increase of atmospheric CO2 concentration has been shown to stimulate plant photosynthesis and (to a lesser extent) growth, thereby acting as a possible sink for the additional atmospheric CO2. However, this effect is dependent on the efficiency with which plants convert atmospheric carbon into biomass carbon, since a considerable proportion of assimilated carbon is returned to the atmosphere via plant respiration. As a core parameter for carbon cycling, carbon use efficiency of plants (CUE, the ratio of net primary production to gross primary production) quantifies the proportion of assimilated carbon that is incorporated into plant biomass. CUE has rarely been assessed based on measurements of complete carbon balance, due to methodological difficulties in measuring respiration rate of plants in light. Moreover, foliar respiration is known to be inhibited in light, thus foliar respiration rate is generally lower in light than in dark. However, this phenomenon, termed as inhibition of respiration in light (IRL), has rarely been assessed at the stand-scale and been incorporated into the calculation of CUE. Therefore, how CUE responses to atmospheric CO2 levels is still not clear. We studied CUE of sunflower stands grown at sub-ambient CO2 level (200 μmol mol-1) and elevated CO2 level (1000 μmol mol-1) using mesocosm-scale gas exchange facilities which enabled continuous measurements of 13CO2/12CO2 exchange. Appling steady-state 13C labeling, fluxes of respiration and photosynthesis in light were separated, and tracer kinetic in respiration was analyzed. This study provides the first data on CUE at a mesocosm-level including respiration in light in different CO2 environments. We found that CUE of sunflower was lower at an elevated CO2 level than at a sub-ambient CO2 level; and the ignorance of IRL lead to erroneous estimations of CUE. Variation in CUE at atmospheric CO2 levels was attributed to several mechanisms. In this study, CO2 enrichment i) affected the

  18. 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

  19. Reliable Identification of Cross-Linked Products in Protein Interaction Studies by 13C-Labeled p-Benzoylphenylalanine

    NASA Astrophysics Data System (ADS)

    Pettelkau, Jens; Ihling, Christian H.; Frohberg, Petra; van Werven, Lars; Jahn, Olaf; Sinz, Andrea

    2014-09-01

    We describe the use of the 13C-labeled artificial amino acid p-benzoyl-L-phenylalanine (Bpa) to improve the reliability of cross-linked product identification. Our strategy is exemplified for two protein-peptide complexes. These studies indicate that in many cases the identification of a cross-link without additional stable isotope labeling would result in an ambiguous assignment of cross-linked products. The use of a 13C-labeled photoreactive amino acid is considered to be preferred over the use of deuterated cross-linkers as retention time shifts in reversed phase chromatography can be ruled out. The observation of characteristic fragment ions additionally increases the reliability of cross-linked product assignment. Bpa possesses a broad reactivity towards different amino acids and the derived distance information allows mapping of spatially close amino acids and thus provides more solid structural information of proteins and protein complexes compared to the longer deuterated amine-reactive cross-linkers, which are commonly used for protein 3D-structure analysis and protein-protein interaction studies.

  20. High-throughput backbone resonance assignment of small 13C, 15N-labeled proteins by a triple-resonance experiment with four sequential connectivity pathways using chemical shift-dependent, apparent 1J ( 1H, 13C): HNCACB codedHAHB

    NASA Astrophysics Data System (ADS)

    Pegan, Scott; Kwiatkowski, Witek; Choe, Senyon; Riek, Roland

    2003-12-01

    The proposed three-dimensional triple-resonance experiment HNCACB codedHAHB correlates sequential 15N, 1H moieties via the chemical shifts of 13C α, 13C β, 1H α, and 1H β. The four sequential correlation pathways are achieved by the incorporation of the concept of chemical shift-coding [J. Biomol. NMR 25 (2003) 281] to the TROSY-HNCACB experiment. The monitored 1H α and 1H β chemical shifts are then coded in the line shape of the cross-peaks of 13C α, 13C β along the 13C dimension through an apparent residual scalar coupling, the size of which depends on the attached hydrogen chemical shift. The information of four sequential correlation pathways enables a rapid backbone assignment. The HNCACB codedHAHB experiment was applied to ˜85% labeled 13C, 15N-labeled amino-terminal fragment of Vaccinia virus DNA topoisomerase I comprising residues 1-77. After one day of measurement on a Bruker Avance 700 MHz spectrometer and 8 h of manual analysis of the spectrum 93% of the backbone assignment was achieved.

  1. Use of {sup 13}C NMR to assess the biodegradation of 1-{sup 13}C-labeled acenaphthene in the presence of creosote polynuclear hydrocarbons (PAHs) and naphthalene by mixed bacterial cultures

    SciTech Connect

    Selifonov, S.A.; Bortiatynski, J.M.; Nanny, M.A.; Hatcher, P.G.

    1996-10-01

    1-{sup 13}C-acenaphthene mixed with creosote PAH`s or naphthalene was incubated with bacterial strains known to degrade naphthalene, phenanthrene and acenaphthene. After incubation, the reaction mixtures were extracted with organic solvent, and the biodegradation products were identified by {sup 13}C NMR. An accumulation of intermediate degradation products was identified and attributed to the non-specific action of naphthalene catabolic pathways of the mixed bacterial cultures. An acenaphthene degrading strain, Pseudomonas sp. strain A2279 was added to the nixed bacterial cultures to minimize the formation of the observed dead-end products. The {sup 13}C NMR spectra obtained from the experiments in which strain A2279 was present clearly showed the complete biodegradation of 1-{sup 13}C-acenaphthene without the accumulation of {sup 13}C-labeled products. This set of experiments clearly demonstrates the utility of {sup 13}C NMR as an effective tool for the assessment of the biodegradation of PAH`s such as 1-{sup 13}C-acenaphthene by various microbial strains.

  2. Validating the Incorporation of 13C and 15N in a Shorebird That Consumes an Isotopically Distinct Chemosymbiotic Bivalve

    PubMed Central

    van Gils, Jan A.; Ahmedou Salem, Mohamed Vall

    2015-01-01

    The wealth of field studies using stable isotopes to make inferences about animal diets require controlled validation experiments to make proper interpretations. Despite several pleas in the literature for such experiments, validation studies are still lagging behind, notably in consumers dwelling in chemosynthesis-based ecosystems. In this paper we present such a validation experiment for the incorporation of 13C and 15N in the blood plasma of a medium-sized shorebird, the red knot (Calidris canutus canutus), consuming a chemosymbiotic lucinid bivalve (Loripes lucinalis). Because this bivalve forms a symbiosis with chemoautotrophic sulphide-oxidizing bacteria living inside its gill, the bivalve is isotopically distinct from ‘normal’ bivalves whose food has a photosynthetic basis. Here we experimentally tested the hypothesis that isotope discrimination and incorporation dynamics are different when consuming such chemosynthesis-based prey. The experiment showed that neither the isotopic discrimination factor, nor isotopic turnover time, differed between birds consuming the chemosymbiotic lucinid and a control group consuming a photosynthesis-based bivalve. This was true for 13C as well as for 15N. However, in both groups the 15N discrimination factor was much higher than expected, which probably had to do with the birds losing body mass over the course of the experiment. PMID:26458005

  3. High resolution 4D HPCH experiment for sequential assignment of (13)C-labeled RNAs via phosphodiester backbone.

    PubMed

    Saxena, Saurabh; Stanek, Jan; Cevec, Mirko; Plavec, Janez; Koźmiński, Wiktor

    2015-11-01

    The three-dimensional structure determination of RNAs by NMR spectroscopy requires sequential resonance assignment, often hampered by assignment ambiguities and limited dispersion of (1)H and (13)C chemical shifts, especially of C4'/H4'. Here we present a novel through-bond 4D HPCH NMR experiment involving phosphate backbone where C4'-H4' correlations are resolved along the (1)H3'-(31)P spectral planes. The experiment provides high peak resolution and effectively removes ambiguities encountered during assignments. Enhanced peak dispersion is provided by the inclusion of additional (31)P and (1)H3' dimensions and constant-time evolution of chemical shifts. High spectral resolution is obtained by using non-uniform sampling in three indirect dimensions. The experiment fully utilizes the isotopic (13)C-labeling with evolution of C4' carbons. Band selective (13)C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the C4'-C3' and C4'-C5' homonuclear couplings. Multiple quantum line narrowing is employed to minimize sensitivity loses. The 4D HPCH experiment is verified and successfully applied to a non-coding 34-nt RNA consisting typical structure elements and a 14-nt RNA hairpin capped by cUUCGg tetraloop.

  4. Synthesis and biosynthesis of {sup 13}C-, {sup 15}N-labeled deoxynucleosides useful for biomolecular structural determinations

    SciTech Connect

    Ashburn, D.A.; Garcia, K.; Hanners, J.L.; Silks, L.A. III; Unkefer, C.J.

    1994-12-01

    Currently, there is a great emphasis on elucidating the structure, function, and dynamics of DNA. Much of the research involved in this study uses nuclear magnetic resonance (NMR) spectroscopy. Effective use of NMR spectroscopy for DNA molecules with mw > 10,000 requires stable isotope enrichment. We present strategies for site-specific isotopic labeling of the purine bases adenosine and guanosine and the biosynthesis of (U-{sup 13}C, {sup 15}N) DNA from methylotropic bacteria. With commercially available 6-chloropurine, an effective two-step route leads to 2{prime}-deoxy-(amino-{sup 15}N)adenosine (dA). The resulting d(amino-{sup 15}N)A is used in a series of reactions to synthesize 2{prime}-deoxy-(2-{sup 13}C,1,amino-{sup 15}N{sub 2})guanosine or any combination thereof. An improved biosynthesis of labeled DNA has been accomplished using Methylobacterium extorquens AS1. Each liter of growth medium contains 4 g of methanol to yield 1 g of lyophilized cells. As much as 200 mg of RNA per liter of culture has been obtained. We are currently developing large-scale isolation protocols. General synthetic pathways to oligomeric DNA will be presented.

  5. Molecular Investigation of the Short-term Sequestration of Natural Abundance 13C -labelled Cow Dung in the Surface Horizons of a Temperate Grassland Soil

    NASA Astrophysics Data System (ADS)

    Dungait, J.; Bol, R.; Evershed, R. P.

    2004-12-01

    An adequate understanding of the carbon (C) sequestration potential of grasslands requires that the quantity and residence times of C inputs be measured. Herbivore dung is largely comprised of plant cell wall material, a significant source of stable C in intensively grazed temperate grassland ecosystems that contributes to the soil carbon budget. Our work uses compound-specific isotope analysis to identify the pattern of input of dung-derived compounds from natural abundance 13C/-labelled cow dung into the surface horizons of a temperate grassland soil over one year. C4 dung (δ 13C \\-12.6 ‰ ) from maize fed cows was applied to a temperate grassland surface (δ 13C \\-29.95 ‰ ) at IGER-North Wyke (Devon, UK), and dung remains and soil cores beneath the treatments collected at ŧ = 7, 14, 28, 56, 112, 224 and 372 days. Bulk dung carbon present in the 0\\-1 cm and 1\\-5 cm surface horizons of a grassland soil over one year was estimated using Δ 13C between C4 dung and C3 dung, after Bol {\\et al.} (2000). The major biochemical components of dung were quantified using proximate forage fibre analyses, after Goering and Van Soest (1970) and identified using `wet' chemical and GC-MS methods. Plant cell wall polysaccharides and lignin were found to account for up to 67 {%} of dung dry matter. Hydrolysed polysaccharides were prepared as alditol acetates for analyses (after Docherty {\\et al.}, 2001), and a novel application of an off-line pyrolysis method applied to measure lignin-derived phenolic compounds (after Poole & van Bergen, 2002). This paper focuses on major events in the incorporation of dung carbon, estimated using natural abundance 13C&-slash;labelling technique. This revealed a major bulk input of dung carbon after a period of significant rainfall with a consequent decline in bulk soil δ 13C values until the end of the experiment (Dungait {\\et al.}, submitted). Findings will be presented revealing contribution of plant cell wall polysaccharides and

  6. 13C labeling analysis of sugars by high resolution-mass spectrometry for metabolic flux analysis.

    PubMed

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

    2017-02-14

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

  7. Regio-selective detection of dynamic structure of transmembrane alpha-helices as revealed from (13)C NMR spectra of [3-13C]Ala-labeled bacteriorhodopsin in the presence of Mn2+ ion.

    PubMed

    Tuzi, S; Hasegawa, J; Kawaminami, R; Naito, A; Saitô, H

    2001-07-01

    13C Nuclear magnetic resonance (NMR) spectra of [3-(13)C]Ala-labeled bacteriorhodopsin (bR) were edited to give rise to regio-selective signals from hydrophobic transmembrane alpha-helices by using NMR relaxation reagent, Mn(2+) ion. As a result of selective suppression of (13)C NMR signals from the surfaces in the presence of Mn(2+) ions, several (13)C NMR signals of Ala residues in the transmembrane alpha-helices were identified on the basis of site-directed mutagenesis without overlaps from (13)C NMR signals of residues located near the bilayer surfaces. The upper bound of the interatomic distances between (13)C nucleus in bR and Mn(2+) ions bound to the hydrophilic surface to cause suppressed peaks by the presence of Mn(2+) ion was estimated as 8.7 A to result in the signal broadening to 100 Hz and consistent with the data based on experimental finding. The Ala C(beta) (13)C NMR peaks corresponding to Ala-51, Ala-53, Ala-81, Ala-84, and Ala-215 located around the extracellular half of the proton channel and Ala-184 located at the kink in the helix F were successfully identified on the basis of (13)C NMR spectra of bR in the presence of Mn(2+) ion and site-directed replacement of Ala by Gly or Val. Utilizing these peaks as probes to observe local structure in the transmembrane alpha-helices, dynamic conformation of the extracellular half of bR at ambient temperature was examined, and the local structures of Ala-215 and 184 were compared with those elucidated at low temperature. Conformational changes in the transmembrane alpha-helices induced in D85N and E204Q and its long-range transmission from the proton release site to the site around the Schiff base in E204Q were also examined.

  8. Investigation of the degradation of 13C-labeled fungal biomass in soil - fate of carbon in a soil bioreactor system

    NASA Astrophysics Data System (ADS)

    Schweigert, Michael; Fester, Thomas; Miltner, Anja; Kaestner, Matthias

    2015-04-01

    Nutrient balances and degradation processes in boreal forests are mainly influenced by interactions of plant roots and ectomycorrhizal fungi. Plants benefit from nitrogen compounds provided by their symbiotic interaction partner. In return ectomycorrhiza are provided by large amounts of carbon from the plants which is used for the synthesis of hyphal networks in soil and for metabolic activity for nutrient uptake. Therefore, ectomycorrhizal fungi play a major role in ecosystems of boreal forests and are consequently an important sink for carbon by building large amount of mycelia. Recently, it has been shown that microbial biomass residues contribute significantly to soil organic matter formation. This suggests that also residues of ectomycorrhizal fungi may be an important source for soil organic matter formation in forest soils where these fungi are abundant. However, the fate of ectomycorrhizal biomass residues in soils is unknown. We therefore investigated the fate of ectomycorrhizal biomass in soil in a soil bioreactor system to quantify the contribution of this material to soil organic matter formation. As a model organism, we selected Laccaria bicolor, which was labelled by growing the fungus on 13C glucose. The stable isotope-labeled biomass was then homogenized and incubated in a podzol from a typical forest site in Central Germany. The fate of the labeled biomass was traced by analyzing the amount of 13C mineralized and the amount remaining in the soil. The fungal biomass carbon was mineralized rather rapidly during the first 50 days. Then the mineralization rate slowed down, but mineralization continued until the end of the experiment, when approximately 40% of the 13C was mineralized and 60% remained in soil. In addition, we analyzed biomolecules such as fatty acids to trace the incorporation of the L. bicolor-derived biomass carbon into other microorganisms and to identify potential primary consumers of fungal biomass. By these analyses, we found a

  9. Investigation of the degradation of 13C-labeled fungal biomass in soil - fate of carbon in a soil bioreactor system

    NASA Astrophysics Data System (ADS)

    Schweigert, Michael; Fester, Thomas; Miltner, Anja; Kästner, Matthias

    2014-05-01

    Nutrient balances and degradation processes in boreal forests are mainly influenced by interactions of plant roots and ectomycorrhizal fungi. Plants benefit from nitrogen compounds provided by their symbiotic interaction partner. In return ectomycorrhiza are provided by large amounts of carbon from the plants which is used for the synthesis of hyphal networks in soil and for metabolic activity for nutrient uptake. Therefore ectomycorrhizal fungi play a major role in ecosystems of boreal forests and are consequently an important sink for carbon by building large amounts of mycelia. Recently, it has been shown that microbial biomass residues contribute significantly to soil organic matter formation. This suggests that also residues of ectomycorrhizal fungi may be an important source for soil organic matter formation in forest soils where these fungi are abundant. However, the fate of ectomycorrhizal biomass residues in soils is unknown. We therefore investigated the fate of ectomycorrhizal biomass in soil in a bioreactor system to quantify the contribution of this material to soil organic matter formation. As a model organism, we selected Laccaria bicolor, which was labelled by growing the fungus on 13C glucose. The stable isotope-labeled biomass was then homogenized and incubated in a podzol from a typical forest site in Central Germany. The fate of the labeled biomass was traced by analyzing the amount of 13C mineralized and the amount remaining in the soil. The fungal biomass carbon was mineralized rather rapidly during the first 25 days. Then the mineralization rate slowed down, but mineralization continued until the end of the experiment, when approximately 40% of the 13C was mineralized and 60% remained in soil. In addition, we analyzed biomolecules such as fatty acids to trace the incorporation of the L. bicolor-derived biomass carbon into other microorganisms and to identify potential primary consumers of fungal biomass. By these analyses, we found a

  10. 13C-TRIPLY Labeled Ethyl Cyanide Submillimeterwave Study with Lille's Fast Scan Dds-Based Spectrometer

    NASA Astrophysics Data System (ADS)

    Pienkina, A.; Motiyenko, R. A.; Margulès, L.; Müller, Holger S. P.; Guillemin, J.-C.

    2016-06-01

    This study of the 13C-triply labeled species of ethyl cyanide (CH_3CH_2CN) follows our recent work on the three 13C-doubly-labeled that allowed their detection in the line survey recently obtained with ALMA (EMoCA). The detection of isotopologues could improve the knowledge of the astrochemistry. The other goal is to clean the surveys from the lines of known molecules in order to detect new ones, this is especially important for the abundant complex organic molecules like ethyl cyanide. As in the case of the doubly substitued species, no spectroscopic studies exist up to now for 13CH_313CH_213CN, the first predictions were thus obtained from scaled ab initio calculations. The spectra were recorded and analyzed up to 1 THz. More than 5500 lines were fitted with quantum numbers J and K_a up to 95 and 25 respectively. The spectra were obtained with the new version of the Lille's solid state spectrometers. This new version used Direct Digital Synthesizer in order to speed up acquisition time. We constructed a spectrometer covering a decade, from 150 to 1500 GHz, it scans the full range in 24 hours with high sensitivity and accuracy. This work was supported by the CNES and the Action sur Projets de l'INSU, PCMI. This work was also done under ANR-13-BS05-0008-02 IMOLABS Margules, L.; et al. 2015, 69th International Symposium on Molecular Spectroscopy, RI06 Belloche, A.; et al. 2014, Science, 345, 1584

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

    PubMed

    Christiansen, Torben; Christensen, Bjarke; Nielsen, Jens

    2002-04-01

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

  12. Ner protein of phage Mu: Assignments using {sup 13}C/{sup 15}N-labeled protein

    SciTech Connect

    Strzelecka, T.; Gronenborn, A.M.; Clore, G.M.

    1994-12-01

    The Ner protein is a small (74-amino acid) DNA-binding protein that regulates a switch between the lysogenic and lytic stages of phage Mu. It inhibits expression of the C repressor gene and down-regulates its own expression. Two-dimensional NMR experiments on uniformly {sup 15}N-labeled protein provided most of the backbone and some of the sidechain proton assignments. The secondary structure determination using two-dimensional NOESY experiments showed that Ner consists of five {alpha}-helices. However, because most of the sidechain protons could not be assigned, the full structure was not determined. Using uniformly {sup 13}C/{sup 15}N-labeled Ner and a set of three-dimensional experiments, we were able to assign all of the backbone and 98% of the sidechain protons. In particular, the CBCANH and CBCA(CO)NH experiments were used to sequentially assign the C{alpha} and C{beta} resonances; the HCCH-CTOCSY and HCCH-COSY were used to assign sidechain carbon and proton resonances.

  13. Incorporation of labeled small molecules into rubratoxin.

    PubMed

    Emeh, C O; Marth, E H

    1978-07-01

    A sterile glucose-mineral salts broth was inoculated with conidia of Penicillium rubrum P-13 and P-3290. Radiolabeled compounds were added to some cultures, these being incubated quiescently at 28 degrees C for 14 days. Other stationary cultures were grown for 21 days, received labeled compounds, and were then grown for 5 more days. The remaining cultures were inoculated with 72-h-old mycelial pellets, received labeled materials and were incubated with shaking for 60 h. Rubratoxin was resolved by thin-layer chromatography. Labeled [1(14)C]acetate, [1,5(14)C]citrate, [2(14)C]malonate, [1(14)C]glucose, [U14C]glucose or [1(14)C]hexanoate were incorporated into rubratoxins A and B by P. rubrum 3290 and into rubratoxin B by P. rubrum 13. Incorporation of [1(14)C]acetate and [2(14)C]malonate increased when exogenous unlabeled acetate, malonate, pyruvate, or phosphoenol-pyruvate was added. Acetate incorporation was influenced by cultural conditions, attaining maximum amounts in quiescent cultures which received labeled acetate after 21 days of incubation. Acetate incorporation in shake cultures was enhanced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and by unlabeled exogenous citrate.

  14. Identification of aquatically available carbon from algae through solution-state NMR of whole (13)C-labelled cells.

    PubMed

    Akhter, Mohammad; Dutta Majumdar, Rudraksha; Fortier-McGill, Blythe; Soong, Ronald; Liaghati-Mobarhan, Yalda; Simpson, Myrna; Arhonditsis, George; Schmidt, Sebastian; Heumann, Hermann; Simpson, André J

    2016-06-01

    Green algae and cyanobacteria are primary producers with profound impact on food web functioning. Both represent key carbon sources and sinks in the aquatic environment, helping modulate the dissolved organic matter balance and representing a potential biofuel source. Underlying the impact of algae and cyanobacteria on an ecosystem level is their molecular composition. Herein, intact (13)C-labelled whole cell suspensions of Chlamydomonas reinhardtii, Chlorella vulgaris and Synechocystis were studied using a variety of 1D and 2D (1)H/(13)C solution-state nuclear magnetic resonance (NMR) spectroscopic experiments. Solution-state NMR spectroscopy of whole cell suspensions is particularly relevant as it identifies species that are mobile (dissolved or dynamic gels), 'aquatically available' and directly contribute to the aquatic carbon pool upon lysis, death or become a readily available food source on consumption. In this study, a wide range of metabolites and structural components were identified within the whole cell suspensions. In addition, significant differences in the lipid/triacylglyceride (TAG) content of green algae and cyanobacteria were confirmed. Mobile species in algae are quite different from those in abundance in 'classic' dissolved organic matter (DOM) indicating that if algae are major contributors to DOM, considerable selective preservation of minor components (e.g. sterols) or biotransformation would have to occur. Identifying the metabolites and dissolved components within algal cells by NMR permits future studies of carbon transfer between species and through the food chain, whilst providing a foundation to better understand the role of algae in the formation of DOM and the sequestration/transformation of carbon in aquatic environments.

  15. Absorption and Distribution Kinetics of the 13C-Labeled Tomato Carotenoid Phytoene in Healthy Adults1234

    PubMed Central

    Riedl, Kenneth M; Rogers, Randy B; Grainger, Elizabeth M; Erdman, John W; Clinton, Steven K

    2016-01-01

    Background: Phytoene is a tomato carotenoid that may contribute to the apparent health benefits of tomato consumption. Although phytoene is a less prominent tomato carotenoid than lycopene, it is a major carotenoid in various human tissues. Phytoene distribution to plasma lipoproteins and tissues differs from lycopene, suggesting the kinetics of phytoene and lycopene differ. Objective: The objective of this study was to characterize the kinetic parameters of phytoene absorption, distribution, and excretion in adults, to better understand why biodistribution of phytoene differs from lycopene. Methods: Four adults (2 males, 2 females) maintained a controlled phytoene diet (1–5 mg/d) for 42 d. On day 14, each consumed 3.2 mg 13C-phytoene, produced using tomato cell suspension culture technology. Blood samples were collected at 0, 1–15, 17, 21, and 24 h and 2, 3, 4, 7, 10, 14, 17, 21, and 28 d after 13C-phytoene consumption. Plasma-unlabeled and plasma-labeled phytoene concentrations were determined using ultra-HPLC–quadrupole time-of-flight-mass spectrometry, and data were fit to a 7-compartment carotenoid kinetic model using WinSAAM 3.0.7 software. Results: Subjects were compliant with a controlled phytoene diet, consuming a mean ± SE of 2.5 ± 0.6 mg/d, resulting in a plasma unlabeled phytoene concentration of 71 ± 14 nmol/L. A maximal plasma 13C-phytoene concentration of 55.6 ± 5.9 nM was achieved 19.8 ± 9.2 h after consumption, and the plasma half-life was 2.3 ± 0.2 d. Compared with previous results for lycopene, phytoene bioavailability was nearly double at 58% ± 19%, the clearance rate from chylomicrons was slower, and the rates of deposition into and utilization by the slow turnover tissue compartment were nearly 3 times greater. Conclusions: Although only differing from lycopene by 4 double bonds, phytoene exhibits markedly different kinetic characteristics in human plasma, providing insight into metabolic processes contributing to phytoene

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2009-06-01

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

  18. Probing the origin of acetyl-CoA and oxaloacetate entering the citric acid cycle from the 13C labeling of citrate released by perfused rat hearts.

    PubMed

    Comte, B; Vincent, G; Bouchard, B; Des Rosiers, C

    1997-10-17

    We present a strategy for simultaneous assessment of the relative contributions of anaplerotic pyruvate carboxylation, pyruvate decarboxylation, and fatty acid oxidation to citrate formation in the perfused rat heart. This requires perfusing with a mix of 13C-substrates and determining the 13C labeling pattern of a single metabolite, citrate, by gas chromatography-mass spectrometry. The mass isotopomer distributions of the oxaloacetate and acetyl moieties of citrate allow calculation of the flux ratios: (pyruvate carboxylation)/(pyruvate decarboxylation), (pyruvate carboxylation)/(citrate synthesis), (pyruvate decarboxylation)/(citrate synthesis) (pyruvate carboxylation)/(fatty acid oxidation), and (pyruvate decarboxylation)/(fatty acid oxidation). Calculations, based on precursor-product relationship, are independent of pool size. The utility of our method was demonstrated for hearts perfused under normoxia with [U-13C3](lactate + pyruvate) and [1-13C]octanoate under steady-state conditions. Under these conditions, effluent and tissue citrate were similarly enriched in all 13C mass isotopomers. The use of effluent citrate instead of tissue citrate allows probing substrate fluxes through the various reactions non-invasively in the intact heart. The methodology should also be applicable to hearts perfused with other 13C-substrates, such as 1-13C-labeled long chain fatty acid, and under various conditions, provided that assumptions on which equations are developed are valid.

  19. 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.

  20. Does the time of the sampling matter in 13C pulse labeling and chasing experiments? A case study on beech seedlings

    NASA Astrophysics Data System (ADS)

    Gavrichkova, Olga; Thoms, Ronny; Muhr, Jan; Karlowsky, Stefan; Keitel, Claudia; Kayler, Zachary; Calfapietra, Carlo; Gessler, Arthur; Brugnoli, Enrico; Gleixner, Gerd

    2016-04-01

    13C pulse labeling and chasing is a valuable and very popular tool for determination of the fate and turnover rates of C in plant-soil systems. Continuous isoflux measurements became an accessible reality allowing to cover completely the diurnal variation in label assimilation and respiration fluxes. Label turnover in multiple pools, especially of those located belowground, is more often assessed instead by isolated day-time samplings. By increasing the sampling frequency of belowground compartments we aimed to catch the short-term diurnal variations in label allocation and to link these processes with label dynamics in the aboveground biomass. For these purposes we labeled 3-m height soil-grown European beech seedlings with 13C enriched CO2 and traced the flow of 13C within belowground plant-soil continuum. Continuous soil isoflux measurements were accompanied by a 3-h-frequency sampling of root and soil material during the first 48 h, followed by a daily sampling in the successive 5 days. The amount of label found in microbial biomass depended partially on the amount of roots in the sample. Microbial biomass C (MBC) and microbial respiration showed very strong correlation, suggesting the possibility to use one as a proxy of the other. MBC enrichment showed a clear diurnal pattern with night-time and early morning peaks. These peaks were similar in shape and shifted by one sampling when compared to root sugars enrichment. Soil respiration showed instead a single bell-shape peak in 13C, likely due to a sequence of peaks of root and microbial origin. 13C flow into soil microbial functional groups was assessed less frequently through phospholipid fatty acid analyses (PLFA). The microorganisms were separated into two distinct groups by the time of the appearance of the label in the single PLFAs. The first group was characterized by a fast appearance of the label and higher enrichment and was composed of Gram negative bacteria and saprotrophic fungi likely living in

  1. Carbon transfer, partitioning and residence time in the plant-soil system: a comparison of two 13C-CO2 labelling techniques

    NASA Astrophysics Data System (ADS)

    Studer, Mirjam S.; Siegwolf, Rolf T. W.; Schmidt, Michael W. I.; Abiven, Samuel

    2014-05-01

    13C-CO2 labelling is a powerful tool to study the carbon (C) dynamics in plant-soil systems, whereby various approaches have been applied, differing in the duration of label exposure, the applied label strength and the sampling intervals. We made a direct comparison of the two main 13C-CO2 labelling techniques - pulse and continuous labelling - and evaluated if different approaches yield the same results regarding the C transfer time, C partitioning and the C residence time in different plant-soil compartments. We conducted a pulse labelling (exposure to 99 atom% 13C-CO2 for three hours, traced for eight days) and a continuous labelling (exposure to 10 atom% 13C-CO2, traced for 14 days) on identical plant-soil systems (Populus deltoides x nigra, Cambisol soil) and under controlled environmental conditions. The plant-soil systems were destructively harvested at five sampling dates, and the soil CO2 efflux was sampled throughout the experiments. The 13C distribution into leaves, petioles, stems, cuttings, roots, soil, microbial biomass and soil respiration was analysed and wee applied exponential (pulse labelling) and logistic (continuous labelling) functions to model the C dynamics. Our results confirm that pulse labelling is best suited to assess the minimum C transfer time, while continuous labelling can be applied to assess the C transfer through a compartment, including short-term storage pools. Both experiments yielded the same C partitioning patterns at the specific sampling days, however, the time of sampling was crucial. For example the results of belowground C partitioning were consistent only after eight days of labelling. The C mean residence times estimated by the rate constant of the exponential and logistic function were largely different for the two techniques, mostly due to the strong model assumptions (e.g. steady state). Pulse and continuous labelling techniques are both well suited to assess C cycling. With pulse labelling, the dynamics of fresh

  2. Bioconversion of (13)C-labeled microalgal phytosterols to cholesterol by the Northern Bay scallop, Argopecten irradians irradians.

    PubMed

    Giner, José-Luis; Zhao, Hui; Dixon, Mark S; Wikfors, Gary H

    2016-02-01

    Bivalve mollusks lack de novo cholesterol biosynthesis capabilities and therefore rely upon dietary sources of sterols for rapid growth. Microalgae that constitute the main source of nutrition for suspension-feeding bivalves contain a diverse array of phytosterols, in most cases lacking cholesterol. Rapid growth of bivalves on microalgal diets with no cholesterol implies that some phytosterols can satisfy the dietary requirement for cholesterol through metabolic conversion to cholesterol, but such metabolic pathways have not been rigorously demonstrated. In the present study, stable isotope-labeled phytosterols were used to supplement a unialgal diet of Rhodomonas sp. and their biological transformation to cholesterol within scallop tissues was determined using (13)C-NMR spectroscopy. Scallops efficiently dealkylated ∆(5) C29 (24-ethyl) sterols to cholesterol, and the only C28 sterol that was dealkylated efficiently possessed the 24(28)-double bond. Non-metabolized dietary phytosterols accumulated in the soft tissues. Observed formation of ∆(5,7) sterols (provitamin D) from ∆(5) sterols may represent initiation of steroid hormone (possibly ecdysone) biosynthesis. These findings provide a key component necessary for formulation of nutritionally complete microalgal diets for hatchery production of seed for molluscan aquaculture.

  3. Interresidue carbonyl-carbonyl polarization transfer experiments in uniformly 13C, 15N-labeled peptides and proteins

    NASA Astrophysics Data System (ADS)

    Janik, Rafal; Ritz, Emily; Gravelle, Andrew; Shi, Lichi; Peng, Xiaohu; Ladizhansky, Vladimir

    2010-03-01

    In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl-carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly 13C, 15N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory. At spinning frequencies used in this study, 22 kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600 MHz. To match HORROR condition over all powder orientations, variable amplitude radio frequency (RF) fields are required, and efficient direct transfers on the order of 20-30% can be straightforwardly established. Two- and three-dimensional chemical shift correlation experiments establishing long-range interresidue connectivities (e.g., (N[i]-CO[i - 2])) are demonstrated on the model peptide N-acetyl-valine-leucine, and on the third immunoglobulin binding domain of protein G. Possible future developments are discussed.

  4. 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

  5. Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

    PubMed Central

    Thakur, Chandar S.; Sama, Jacob N.; Jackson, Melantha E.; Chen, Bin

    2010-01-01

    Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2016-05-10

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

  8. Changes in microbial structure and functional communities at different soil depths during 13C labelled root litter degradation

    NASA Astrophysics Data System (ADS)

    Sanaullah, Muhammad; Baumann, Karen; Chabbi, Abad; Dignac, Marie-France; Maron, Pierre-Alain; Kuzyakov, Yakov; Rumpel, Cornelia

    2014-05-01

    Soil organic matter turnover depends on substrate quality and microbial activity in soil but little is known about how addition of freshly added organic material modifies the diversity of soil microbial communities with in a soil profile. We took advantage of a decomposition experiment, which was carried out at different soil depths under field conditions and sampled litterbags with 13C-labelled wheat roots, incubated in subsoil horizons at 30, 60 and 90 cm depth for up to 36 months. The effect of root litter addition on microbial community structure, diversity and activity was studied by determining total microbial biomass, PLFA signatures, molecular tools (DNA genotyping and pyrosequencing of 16S and 18S rDNAs) and extracellular enzyme activities. Automated ribosomal intergenic spacer analysis (ARISA) was also carried out to determine the differences in microbial community structure. We found that with the addition of root litter, total microbial biomass as well as microbial community composition and structure changed at different soil depths and change was significantly higher at top 30cm soil layer. Moreover, in the topsoil, population of both gram-positive and gram-negative bacteria increased with root litter addition over time, while subsoil horizons were relatively dominated by fungal community. Extra-cellular enzyme activities confirmed relatively higher fungal community at subsoil horizons compared with surface soil layer with bacteria dominant microbial population. Bacterial-ARISA profiling illustrated that the addition of root litter enhanced the abundance of Actinobacteria and Proteobacteria, at all three soil depths. These bacteria correspond to copiotrophic attributes, which can preferentially consume of labile soil organic C pools. While disappearance of oligotrophic Acidobacteria confirmed the shifting of microbial communities due to the addition of readily available substrate. We concluded that root litter mixing altered microbial community

  9. Using 13C-labeled benzene and Raman gas spectroscopy to investigate respiration and biodegradation kinetics following soil contamination

    NASA Astrophysics Data System (ADS)

    Jochum, Tobias; Popp, Juergen; Frosch, Torsten

    2016-04-01

    Soil and groundwater contamination with benzene can cause serious environmental damages. However, many soil microorganisms are capable to adapt and known to strongly control the fate of organic contamination. Cavity enhanced Raman gas spectroscopy (CERS) was applied to investigate the short-term response of indigenous soil bacteria to a sudden surface contamination with benzene regarding the temporal variations of gas products and their exchange rates with the adjacent atmosphere. 13C-labeled benzene was spiked on a silty-loamy soil column (sampled from Hainich National Park, Germany) in order to track and separate the changes in heterotrophic soil respiration - involving 12CO2 and O2 - from the microbial process of benzene degradation, which ultimately forms 13CO2.1 The respiratory quotient (RQ) of 0.98 decreased significantly after the spiking and increased again within 33 hours to a value of 0.72. This coincided with maximum 13CO2 concentration rates (0.63 μ mol m-2 s-1), indicating highest benzene degradation at 33 hours after the spiking event. The diffusion of benzene in the headspace and the biodegradation into 13CO2 were simultaneously monitored and 12 days after the benzene spiking no measurable degradation was detected anymore.1 The RQ finally returned to a value of 0.96 demonstrating the reestablished aerobic respiration. In summary, this study shows the potential of combining Raman gas spectroscopy and stable isotopes to follow soil microbial biodegradation dynamics while simultaneously monitoring the underlying respiration behavior. Support by the Collaborative Research Center 1076 Aqua Diva is kindly acknowledged. We thank Beate Michalzik for soil analysis and discussion. 1. T. Jochum, B. Michalzik, A. Bachmann, J. Popp and T. Frosch, Analyst, 2015, 140, 3143-3149.

  10. 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

  11. 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.

  12. Spatial and temporal distribution of 13C labelled plant residues in soil aggregates and Lumbricus terrestris surface casts: A combination of Transmission Electron Microscopy and Nanoscale Secondary Ion Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Vidal, Alix; Remusat, Laurent; Watteau, Françoise; Derenne, Sylvie; Quenea, Katell

    2016-04-01

    Earthworms play a central role in litter decomposition, soil structuration and carbon cycling. They ingest both organic and mineral compounds which are mixed, complexed with mucus and dejected in form of casts at the soil surface and along burrows. Bulk isotopic or biochemical technics have often been used to study the incorporation of litter in soil and casts, but they could not reflect the complex interaction between soil, plant and microorganisms at the microscale. However, the heterogeneous distribution of organic carbon in soil structures induces contrasted microbial activity areas. Nano-scale secondary ion mass spectrometry (NanoSIMS), which is a high spatial resolution method providing elemental and isotopic maps of organic and mineral materials, has recently been applied in soil science (Herrmann et al., 2007; Vogel et al., 2014). The combination of Nano-scale secondary ion mass spectrometry (NanoSIMS) and Transmission Electron Microscopy (TEM) has proven its potential to investigate labelled residues incorporation in earthworm casts (Vidal et al., 2016). In line of this work, we studied the spatial and temporal distribution of plant residues in soil aggregates and earthworm surface casts. This study aimed to (1) identify the decomposition states of labelled plant residues incorporated at different time steps, in casts and soil, (2) identify the microorganisms implied in this decomposition (3) relate the organic matter states of decomposition with their 13C signature. A one year mesocosm experiment was set up to follow the incorporation of 13C labelled Ryegrass (Lolium multiflorum) litter in a soil in the presence of anecic earthworms (Lumbricus terrestris). Soil and surface cast samples were collected after 8 and 54 weeks, embedded in epoxy resin and cut into ultra-thin sections. Soil was fractionated and all and analyzed with TEM and NanoSIMS, obtaining secondary ion images of 12C, 16O, 12C14N, 13C14N and 28Si. The δ13C maps were obtained using the 13C14

  13. Straightforward preparation of labeled potassium cyanate by ozonation and application to the synthesis of [13C] or [14C]ureidocarboxylic acids.

    PubMed

    Loreau, Olivier; Marlière, Philippe

    2013-06-15

    The development of new efficient syntheses of labeled reagents is a great challenge. Avoidance of overcomplicated procedures, availability and cost of starting materials are important considerations in choosing the synthetic route. In this report, we describe a facile and rapid preparation of labeled cyanate by ozonation of cyanide, a basic precursor. The crude cyanate was used without purification for the synthesis of various [(13)C] or [(14)C]ureidocarboxylic acids (20-68% yield from potassium cyanide). According to these results, cyanide ozonation may prove to be a promising alternative to traditional preparations of labeled cyanate.

  14. Pre-malbrancheamide: Synthesis, Isotopic Labeling, Biosynthetic Incorporation, and Detection in Cultures of Malbranchea aurantiaca

    PubMed Central

    Ding, Yousong; Greshock, Thomas J.; Miller, Kenneth A.

    2009-01-01

    An advanced metabolite, named pre-malbrancheamide, involved in the biosynthesis of malbrancheamide (1) and malbrancheamide B (2) has been synthesized in double 13C-labeled form and was incorporated into the indole alkaloid 2 by Malbranchea aurantiaca. In addition, pre-malbrancheamide has been detected as a natural metabolite in cultures of M. aurantiaca. The biosynthetic implications of these experiments are discussed. PMID:18844365

  15. Streamlined pentafluorophenylpropyl column liquid chromatography-tandem quadrupole mass spectrometry and global (13)C-labeled internal standards improve performance for quantitative metabolomics in bacteria.

    PubMed

    Yang, Song; Sadilek, Martin; Lidstrom, Mary E

    2010-11-19

    Streamlined quantitative metabolomics in central metabolism of bacteria would be greatly facilitated by a high-efficiency liquid chromatography (LC) method in conjunction with accurate quantitation. To achieve this goal, a methodology for LC-tandem quadrupole mass spectrometry (LC-MS/MS) involving a pentafluorophenylpropyl (PFPP) column and culture-derived global (13)C-labeled internal standards (I.Ss.) has been developed and compared to hydrophilic interaction liquid chromatography (HILIC)-MS/MS and published combined two-dimensional gas chromatography and LC methods. All 50 tested metabolite standards from 5 classes (amino acids, carboxylic acids, nucleotides, acyl-CoAs and sugar phosphates) displayed good chromatographic separation and sensitivity on the PFPP column. In addition, many important critical pairs such as isomers/isobars (e.g. isoleucine/leucine, methylsuccinic acid/ethylmalonic acid and malonyl-CoA/3-hydroxybutyryl-CoA) and metabolites of similar structure (e.g. malate/fumarate) were resolved better on the PFPP than on the HILIC column. Compared to only one (13)C-labeled I.S., the addition of global (13)C-labeled I.Ss. improved quantitative linearity and accuracy. PFPP-MS/MS with global (13)C-labeled I.Ss. allowed the absolute quantitation of 42 metabolite pool sizes in Methylobacterium extorquens AM1. A comparison of metabolite level changes published previously for ethylamine (C2) versus succinate (C4) cultures of M. extorquens AM1 indicated a good consistency with the data obtained by PFPP-MS/MS, suggesting this single approach has the capability of providing comprehensive metabolite profiling similar to the combination of methods. The more accurate quantification obtained by this method forms a fundamental basis for flux measurements and can be used for metabolism modeling in bacteria in future studies.

  16. Evidence of polycyclic aromatic hydrocarbon biodegradation in a contaminated aquifer by combined application of in situ and laboratory microcosms using (13)C-labelled target compounds.

    PubMed

    Bahr, Arne; Fischer, Anko; Vogt, Carsten; Bombach, Petra

    2015-02-01

    The number of approaches to evaluate the biodegradation of polycyclic aromatic hydrocarbons (PAHs) within contaminated aquifers is limited. Here, we demonstrate the applicability of a novel method based on the combination of in situ and laboratory microcosms using (13)C-labelled PAHs as tracer compounds. The biodegradation of four PAHs (naphthalene, fluorene, phenanthrene, and acenaphthene) was investigated in an oxic aquifer at the site of a former gas plant. In situ biodegradation of naphthalene and fluorene was demonstrated using in situ microcosms (BACTRAP(®)s). BACTRAP(®)s amended with either [(13)C6]-naphthalene or [(13)C5/(13)C6]-fluorene (50:50) were incubated for a period of over two months in two groundwater wells located at the contaminant source and plume fringe, respectively. Amino acids extracted from BACTRAP(®)-grown cells showed significant (13)C-enrichments with (13)C-fractions of up to 30.4% for naphthalene and 3.8% for fluorene, thus providing evidence for the in situ biodegradation and assimilation of those PAHs at the field site. To quantify the mineralisation of PAHs, laboratory microcosms were set up with BACTRAP(®)-grown cells and groundwater. Naphthalene, fluorene, phenanthrene, or acenaphthene were added as (13)C-labelled substrates. (13)C-enrichment of the produced CO2 revealed mineralisation of between 5.9% and 19.7% for fluorene, between 11.1% and 35.1% for acenaphthene, between 14.2% and 33.1% for phenanthrene, and up to 37.0% for naphthalene over a period of 62 days. Observed PAH mineralisation rates ranged between 17 μg L(-1) d(-1) and 1639 μg L(-1) d(-1). The novel approach combining in situ and laboratory microcosms allowed a comprehensive evaluation of PAH biodegradation at the investigated field site, revealing the method's potential for the assessment of PAH degradation within contaminated aquifers.

  17. Development of an LC-MS/MS method for the determination of endogenous cortisol in hair using (13)C3-labeled cortisol as surrogate analyte.

    PubMed

    Binz, Tina M; Braun, Ueli; Baumgartner, Markus R; Kraemer, Thomas

    2016-10-15

    Hair cortisol levels are increasingly applied as a measure for stress in humans and mammals. Cortisol is an endogenous compound and is always present within the hair matrix. Therefore, "cortisol-free hair matrix" is a critical point for any analytical method to accurately quantify especially low cortisol levels. The aim of this project was to modify current methods used for hair cortisol analysis to more accurately determine low endogenous cortisol concentrations in hair. For that purpose, (13)C3-labeled cortisol, which is not naturally present in hair (above 13C natural abundance levels), was used for calibration and comparative validation applying cortisol versus (13)C3-labeled cortisol. Cortisol was extracted from 20mg hair (standard sample amount) applying an optimized single step extraction protocol. An LC-MS/MS method was developed for the quantitative analysis of cortisol using either cortisol or (13)C3-cortisol as calibrators and D7-cortisone as internal standard (IS). The two methods (cortisol/(13)C3-labeled cortisol) were validated in a concentration range up to 500pg/mg and showed good linearity for both analytes (cortisol: R(2)=0.9995; (13)C3-cortisol R(2)=0.9992). Slight differences were observed for limit of detection (LOD) (0.2pg/mg/0.1pg/mg) and limit of quantification (LOQ) (1pg/mg/0.5pg/mg). Precision was good with a maximum deviation of 8.8% and 10% for cortisol and (13)C3-cortisol respectively. Accuracy and matrix effects were good for both analytes except for the quality control (QC) low cortisol. QC low (2.5pg/mg) showed matrix effects (126.5%, RSD 35.5%) and accuracy showed a deviation of 26% when using cortisol to spike. These effects are likely to be caused by the unknown amount of endogenous cortisol in the different hair samples used to determine validation parameters like matrix effect, LOQ and accuracy. No matrix effects were observed for the high QC (400pg/mg) samples. Recovery was good with 92.7%/87.3% (RSD 9.9%/6.2%) for QC low and

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2009-12-01

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

  20. A Comparison between Radiolabeled Fluorodeoxyglucose Uptake and Hyperpolarized 13C-Labeled Pyruvate Utilization as Methods for Detecting Tumor Response to Treatment12

    PubMed Central

    Witney, Timothy H; Kettunen, Mikko I; Day, Samuel E; Hu, De-en; Neves, Andre A; Gallagher, Ferdia A; Fulton, Sandra M; Brindle, Kevin M

    2009-01-01

    Detection of early tumor responses to treatment can give an indication of clinical outcome. Positron emission tomography measurements of the uptake of the glucose analog, [18F] 2-fluoro-2-deoxy-d-glucose (FDG), have demonstrated their potential for detecting early treatment response in the clinic. We have shown recently that 13C magnetic resonance spectroscopy and spectroscopic imaging measurements of the uptake and conversion of hyperpolarized [1-13C]pyruvate into [1-13C]lactate can be used to detect treatment response in a murine lymphoma model. The present study compares these magnetic resonance measurements with changes in FDG uptake after chemotherapy. A decrease in FDG uptake was found to precede the decrease in flux of hyperpolarized 13C label between pyruvate and lactate, both in tumor cells in vitro and in tumors in vivo. However, the magnitude of the decrease in FDG uptake and the decrease in pyruvate to lactate flux was comparable at 24 hours after drug treatment. In cells, the decrease in FDG uptake was shown to correlate with changes in plasma membrane expression of the facilitative glucose transporters, whereas the decrease in pyruvate to lactate flux could be explained by an increase in poly(ADP-ribose) polymerase activity and subsequent depletion of the NAD(H) pool. These results show that measurement of flux between pyruvate and lactate may be an alternative to FDG-positron emission tomography for imaging tumor treatment response in the clinic. PMID:19484146

  1. Multidimensional High-Resolution Magic Angle Spinning and Solution-State NMR Characterization of 13C-labeled Plant Metabolites and Lignocellulose

    PubMed Central

    Mori, Tetsuya; Tsuboi, Yuuri; Ishida, Nobuhiro; Nishikubo, Nobuyuki; Demura, Taku; Kikuchi, Jun

    2015-01-01

    Lignocellulose, which includes mainly cellulose, hemicellulose, and lignin, is a potential resource for the production of chemicals and for other applications. For effective production of materials derived from biomass, it is important to characterize the metabolites and polymeric components of the biomass. Nuclear magnetic resonance (NMR) spectroscopy has been used to identify biomass components; however, the NMR spectra of metabolites and lignocellulose components are ambiguously assigned in many cases due to overlapping chemical shift peaks. Using our 13C-labeling technique in higher plants such as poplar samples, we demonstrated that overlapping peaks could be resolved by three-dimensional NMR experiments to more accurately assign chemical shifts compared with two-dimensional NMR measurements. Metabolites of the 13C-poplar were measured by high-resolution magic angle spinning NMR spectroscopy, which allows sample analysis without solvent extraction, while lignocellulose components of the 13C-poplar dissolved in dimethylsulfoxide/pyridine solvent were analyzed by solution-state NMR techniques. Using these methods, we were able to unambiguously assign chemical shifts of small and macromolecular components in 13C-poplar samples. Furthermore, using samples of less than 5 mg, we could differentiate between two kinds of genes that were overexpressed in poplar samples, which produced clearly modified plant cell wall components. PMID:26143886

  2. Amino acids as a nitrogen source in temperate upland grasslands: the use of dual labelled ((13)C, (15)N) glycine to test for direct uptake by dominant grasses.

    PubMed

    Streeter, T C; Bol, R; Bardgett, R D

    2000-01-01

    It is becoming increasingly apparent that soil amino acids are a principal source of nitrogen (N) for certain plants, and especially those of N-limited environments. This study of temperate upland grasslands used glycine-2-(13)C-(15)N and ((15)NH4)(2)SO(4) labelling techniques to test the hypothesis that plant species which dominate 'unimproved' semi-natural grasslands (Festuca-Agrostis-Galium) are able to utilise amino acid N for growth, whereas those plants which dominate 'improved' grasslands (Lolium-Cynosurus), that receive regular applications of inorganic fertiliser, use inorganic N forms as their main N source. Data from field experiments confirmed that 'free' amino acids were more abundant in 'unimproved' than 'improved' grassland and that glycine was the dominant amino acid type (up to 42% of total). Secondly, the injection of representative amounts of glycine-2-(13)C-(15)N (4.76 and 42.86 mM) into intact soil cores from the two grassland types provided evidence of direct uptake of glycine by plants, with both (15)N and (13)C being detected in plant material of both grasslands. Finally, a microcosm experiment demonstrated no preferential uptake of amino acid N by the grasses which dominate the grassland types, namely Holcus lanatus, Festuca rubra, Agrostis capillaris from the 'unimproved' grassland, and Lolium perenne from the 'improved' grassland. Again, both (13)C and (15)N were detected in all grass species suggesting uptake of intact glycine by these plants.

  3. Evaluation of biodegradability of phenol and bisphenol A during mesophilic and thermophilic municipal solid waste anaerobic digestion using 13C-labeled contaminants.

    PubMed

    Limam, Intissar; Mezni, Mohamed; Guenne, Angéline; Madigou, Céline; Driss, Mohamed Ridha; Bouchez, Théodore; Mazéas, Laurent

    2013-01-01

    In this paper, the isotopic tracing using (13)C-labeled phenol and bisphenol A was used to study their biodegradation during anaerobic digestion of municipal solid waste. Microcosms were incubated anaerobically at 35 °C (mesophilic conditions) and 55 °C (thermophilic conditions) without steering. A continuous follow-up of the production of biogas (CH(4) and CO(2)), was carried out during 130 d until the establishment of stable methanogenesis. Then (13)C(12)-BPA, and (13)C(6)-phenol were injected in microcosms and the follow-up of their degradation was performed simultaneously by gas chromatography isotope-ratio mass spectrometry (GC-IRMS) and gas chromatography mass spectrometry (GC-MS). Moreover, Carbon-13 Nuclear Magnetic Resonance ((13)C-NMR) Spectroscopy is used in the identification of metabolites. This study proves that the mineralization of phenol to CO(2) and CH(4) occurs during anaerobic digestion both in mesophilic and thermophilic conditions with similar kinetics. In mesophilic condition phenol degradation occurs through the benzoic acid pathway. In thermophilic condition it was not possible to identify the complete metabolic pathway as only acetate was identified as metabolite. Our results suggest that mineralization of phenol under thermophilic condition is instantaneous explaining why metabolites are not observed as they do not accumulate. No biodegradation of BPA was observed.

  4. C4'/H4' selective, non-uniformly sampled 4D HC(P)CH experiment for sequential assignments of (13)C-labeled RNAs.

    PubMed

    Saxena, Saurabh; Stanek, Jan; Cevec, Mirko; Plavec, Janez; Koźmiński, Wiktor

    2014-11-01

    A through bond, C4'/H4' selective, "out and stay" type 4D HC(P)CH experiment is introduced which provides sequential connectivity via H4'(i)-C4'(i)-C4'(i-1)-H4'(i-1) correlations. The (31)P dimension (used in the conventional 3D HCP experiment) is replaced with evolution of better dispersed C4' dimension. The experiment fully utilizes (13)C-labeling of RNA by inclusion of two C4' evolution periods. An additional evolution of H4' is included to further enhance peak resolution. Band selective (13)C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the of C4'-C3' and C4'-C5' homonuclear couplings. For reasonable resolution, non-uniform sampling is employed in all indirect dimensions. To reduce sensitivity losses, multiple quantum coherences are preserved during shared-time evolution and coherence transfer delays. In the experiment the intra-nucleotide peaks are suppressed whereas inter-nucleotide peaks are enhanced to reduce the ambiguities. The performance of the experiment is verified on a fully (13)C, (15)N-labeled 34-nt hairpin RNA comprising typical structure elements.

  5. High-resolution direct infusion-based mass spectrometry in combination with whole 13C metabolome isotope labeling allows unambiguous assignment of chemical sum formulas.

    PubMed

    Giavalisco, Patrick; Hummel, Jan; Lisec, Jan; Inostroza, Alvaro Cuadros; Catchpole, Gareth; Willmitzer, Lothar

    2008-12-15

    A new strategy for direct infusion-based metabolite analysis employing a combination of high-resolution mass spectrometry and (13)C-isotope labeling of entire metabolomes is described. Differentially isotope labeled metabolite extracts from otherwise identically grown reference plants were prepared and infused into a Fourier transform ion cyclotron resonance mass spectrometer. The derived accurate mass lists from each extract were searched, using an in-house-developed database search tool, against a number of comprehensive metabolite databases. Comparison of the retrieved chemical formulas from both, the (12)C and (13)C samples, leads to two major advantages compared to nonisotope-based metabolite fingerprinting: first, removal of background contaminations from the result list, due to the (12)C/(13)C peak pairing principle and therefore positive identification of compounds of true biological origin; second, elimination of ambiguity in chemical formula assignment due to the same principle, leading to the clear association of one measured mass to only one chemical formula. Applying this combination of strategies to metabolite extracts of the model plant Arabidopsis thaliana therefore resulted in the reproducible identification of more than 1000 unambiguous chemical sum formulas of biological origin of which more than 80% have not been associated to Arabidopsis before.

  6. Timing and magnitude of C partitioning through a young loblolly pine (Pinus taeda L.) stand using 13C labeling and shade treatments

    DOE PAGES

    Warren, Jeffrey M.; Iversen, Colleen M.; Garten, Jr., Charles T.; ...

    2011-12-30

    The dynamics of rapid changes in carbon (C) partitioning within forest ecosystems are not well understood, which limits improvement of mechanistic models of C cycling. Our objective was to inform model processes by describing relationships between C partitioning and accessible environmental or physiological measurements, with a special emphasis on short-term C flux through a forest ecosystem. We exposed eight 7-year-old loblolly pine (Pinus taeda L.) trees to air enriched with 13CO2 and then implemented adjacent light shade (LS) and heavy shade (HS) treatments in order to manipulate C uptake and flux. The impacts of shading on photosynthesis, plant water potential,more » sap flow, basal area growth, root growth, and soil CO2 efflux rate (CER) were assessed for each tree over a three-week period. The progression of the 13C label was concurrently tracked from the atmosphere through foliage, phloem, roots, and surface soil CO2 efflux. The HS treatment significantly reduced C uptake, sap flow, stem growth and fine root standing crop, and resulted in greater residual soil water content to 1 m depth. Sap flow was strongly correlated with CER on the previous day, but not the current day, with no apparent treatment effect on the relationship. Although there were apparent reductions in new C flux belowground, the heavy shade treatment did not noticeably reduce the magnitude of belowground autotrophic and heterotrophic respiration based on surface soil CO2 efflux rate (CER), which was overwhelmingly driven by soil temperature and moisture. The 13C label was immediately detected in foliage on label day (half-life = 0.5 d), progressed through phloem by day 2 (half-life = 4.7 d), roots by day 2-4, and subsequently was evident as respiratory release from soil which peaked between days 3-6. The δ13C of soil CO2 efflux was strongly correlated with phloem 13C on the previous day, or two days earlier. While the 13C label was readily tracked through the ecosystem, the fate of root

  7. Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies

    NASA Astrophysics Data System (ADS)

    Fasshuber, Hannes Klaus; Demers, Jean-Philippe; Chevelkov, Veniamin; Giller, Karin; Becker, Stefan; Lange, Adam

    2015-03-01

    Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, α-ketoisovalerate and α-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are exclusively 13C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the type-three secretion system needle of Shigella flexneri, where 49 methyl-methyl and methyl-nitrogen distance restraints including 10 unambiguous long-range distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved.

  8. Quantitation of a spin polarization-induced nuclear Overhauser effect (SPINOE) between a hyperpolarized (13) C-labeled cell metabolite and water protons.

    PubMed

    Marco-Rius, Irene; Bohndiek, Sarah E; Kettunen, Mikko I; Larkin, Timothy J; Basharat, Meer; Seeley, Colm; Brindle, Kevin M

    2014-01-01

    The spin polarization-induced nuclear Overhauser effect (SPINOE) describes the enhancement of spin polarization of solvent nuclei by the hyperpolarized spins of a solute. In this communication we demonstrate that SPINOEs can be observed between [1,4-(13) C2 ]fumarate, hyperpolarized using the dissolution dynamic nuclear polarization technique, and solvent water protons. We derive a theoretical expression for the expected enhancement and demonstrate that this fits well with experimental measurements. Although the magnitude of the effect is relatively small (around 2% measured here), the SPINOE increases at lower field strengths, so that at clinically relevant magnetic fields (1.5-3 T) it may be possible to track the passage through the circulation of a bolus containing a hyperpolarized (13) C-labeled substrate through the increase in solvent water (1) H signal.

  9. Simultaneous quantification of labeled (2)H5-glycerol, (13)C6-glucose, and endogenous D-glucose in mouse plasma using liquid chromatography tandem mass spectrometry.

    PubMed

    Jahouh, Farid; Wang, Rong

    2015-11-01

    Monitoring the level of glucose and glycerol or their labeled derivatives in biological fluid for kinetic studies has always been challenging, especially in mice, because of the limited volume in addition to the complexity of plasma. For such application, we developed a simple, fast, and sensitive method for the simultaneous measurement of absolute concentrations of labeled (2)H5-glycerol and (13)C6-glucose as well as endogenous D-glucose using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In our study, 15.0 μL of mouse plasma was processed by a one-step protein precipitation, followed by LC-MS/MS analysis. The quantification of the analytes was carried out by monitoring the product ion scan of their corresponding deprotonated molecular ions and constructing the extracted ion fragmentogram by choosing a specific product ion for each analyte (equivalent to precursor ion to product ion transitions). The limit of detection (LOD) was evaluated to be 1.0 μM for both (2)H5-glycerol and (13)C6-glucose, and the limit of quantitation (LOQ) was observed to be 5.0 μM for both (2)H5-glycerol and (13)C6-glucose in diluted mice plasma that corresponds to 50 μM in plasma or 4.60 and 9.01 mg/dL of glycerol and glucose in plasma, respectively. The extraction recoveries are 81.9 % (CV = 8.1 %) for (2)H5-glycerol and 26.2 % (CV = 13.6 %) for (13)C6-glucose.

  10. HCN, A Triple-Resonance NMR Technique for Selective Observation of Histidine and Tryptophan Side Chains in 13C/ 15N-Labeled Proteins

    NASA Astrophysics Data System (ADS)

    Sudmeier, James L.; Ash, Elissa L.; Günther, Ulrich L.; Luo, Xuelian; Bullock, Peter A.; Bachovchin, William W.

    1996-12-01

    HCN, a new 3D NMR technique for stepwise coherence transfer from1H to13C to15N and reverse through direct spin couplings1JCHand1JCN, is presented as a method for detection and assignment of histidine and tryptophan side-chain1H,13C, and15N resonances in uniformly13C/15N-labeled proteins. Product-operator calculations of cross-peak volumes vs adjustable delay τ3were employed for determination of optimal τ3. For the phosphatidylinositol 3-kinase (PI3K SH3 domain, MW = 9.6 kD) at pH 6, H(C)N, the1H/15N projection, produced observable cross peaks within 20 min. and was completely selective for the single tryptophan and single histidine. The 3D HCN experiment yielded well-defined cross peaks in 20 h for the13C/15N-labeled origin-specific DNA binding domain from simian virus 40 T-antigen (T-ag-OBD131-259, MW = 15.4 kD) at pH 5.5. Resonances from all six histidines in T-ag-OBD were observed, and 11 of the 121H and13C chemical shifts and 10 of the 1215N chemical shifts were determined. The13C dimension proved essential in assignment of the multiply overlapping1H and15N resonances. From the spectra recorded at a single pH, three of the imidazoles were essentially neutral and the other three were partially protonated (22-37%). HCN yielded strong cross peaks after 18 h on a 2.0 mMsample of phenylmethanesulfonyl fluoride (PMSF)-inhibited α-lytic protease (MW = 19.8 kD) at pH 4.4. No spectra have been obtained, however, of native or boronic acid-inhibited α-lytic protease after 18 h at various temperatures ranging from 5 to 55°C, probably due to efficient relaxation of active-site imidazole1H and/or15N nuclei.

  11. HCN, a triple-resonance NMR technique for selective observation of histidine and tryptophan side chains in 13C/15N-labeled proteins.

    PubMed

    Sudmeier, J L; Ash, E L; Günther, U L; Luo, X; Bullock, P A; Bachovchin, W W

    1996-12-01

    HCN, a new 3D NMR technique for stepwise coherence transfer from 1H to 13C to 15N and reverse through direct spin couplings 1JCH and 1JCN, is presented as a method for detection and assignment of histidine and tryptophan side-chain 1H, 13C, and 15N resonances in uniformly 13C/15N-labeled proteins. Product-operator calculations of cross-peak volumes vs adjustable delay tau 3 were employed for determination of optimal tau 3. For the phosphatidylinositol 3-kinase (PI3K SH3 domain, MW = 9.6 kD) at pH 6, H(C)N, the 1H/15N projection, produced observable cross peaks within 20 min. and was completely selective for the single tryptophan and single histidine. The 3D HCN experiment yielded well-defined cross peaks in 20 h for the 13C/15N-labeled origin-specific DNA binding domain from simian virus 40 T-antigen (T-ag-OBD131-259, MW = 15.4 kD) at pH 5.5. Resonances from all six histidines in T-ag-OBD were observed, and 11 of the 12 1H and 13C chemical shifts and 10 of the 12 15N chemical shifts were determined. The 13C dimension proved essential in assignment of the multiply overlapping 1H and 15N resonances. From the spectra recorded at a single pH, three of the imidazoles were essentially neutral and the other three were partially protonated (22-37%). HCN yielded strong cross peaks after 18 h on a 2.0 mM sample of phenylmethanesulfonyl fluoride (PMSF)-inhibited alpha-lytic protease (MW = 19.8 kD) at pH 4.4. No spectra have been obtained, however, of native or boronic acid-inhibited alpha-lytic protease after 18 h at various temperatures ranging from 5 to 55 degrees C, probably due to efficient relaxation of active-site imidazole 1H and/or 15N nuclei.

  12. Balancing the (carbon) budget: Using linear inverse models to estimate carbon flows and mass-balance 13C:15N labelling experiments in low oxygen sediments.

    NASA Astrophysics Data System (ADS)

    Hunter, William Ross; Van Oevelen, Dick; Witte, Ursula

    2013-04-01

    Over 1 million km2 of seafloor experience permanent low-oxygen conditions within oxygen minimum zones (OMZs). OMZs are predicted to grow as a consequence of climate change, potentially affecting oceanic biogeochemical cycles. The Arabian Sea OMZ impinges upon the western Indian continental margin at bathyal depths (150 - 1500m) producing a strong depth dependent oxygen gradient at the sea floor. The influence of the OMZ upon the short term processing of organic matter by sediment ecosystems was investigated using in situ stable isotope pulse chase experiments. These deployed doses of 13C:15N labeled organic matter onto the sediment surface at four stations from across the OMZ (water depth 540 - 1100 m; [O2] = 0.35 - 15 μM). In order to prevent experimentally anoxia, the mesocosms were not sealed. 13C and 15N labels were traced into sediment, bacteria, fauna and 13C into sediment porewater DIC and DOC. However, the DIC and DOC flux to the water column could not be measured, limiting our capacity to obtain mass-balance for C in each experimental mesocosm. Linear Inverse Modeling (LIM) provides a method to obtain a mass-balanced model of carbon flow that integrates stable-isotope tracer data with community biomass and biogeochemical flux data from a range of sources. Here we present an adaptation of the LIM methodology used to investigate how ecosystem structure influenced carbon flow across the Indian margin OMZ. We demonstrate how oxygen conditions affect food-web complexity, affecting the linkages between the bacteria, foraminifera and metazoan fauna, and their contributions to benthic respiration. The food-web models demonstrate how changes in ecosystem complexity are associated with oxygen availability across the OMZ and allow us to obtain a complete carbon budget for the stationa where stable-isotope labelling experiments were conducted.

  13. Vitamin K absorption and kinetics in human subjects after consumption of 13C-labeled phylloquinone from kale

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The absorption and plasma elimination of vitamin K was investigated by uniformly labeling phylloquinone in kale with carbon-13 and feeding the kale to study subjects. Seven healthy volunteers ingested a single 400 g serving of kale with 30 g vegetable oil. The kale provided 156 nmol of phylloquino...

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

    PubMed

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

    2013-12-01

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

  15. Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry.

    PubMed

    Giavalisco, Patrick; Li, Yan; Matthes, Annemarie; Eckhardt, Aenne; Hubberten, Hans-Michael; Hesse, Holger; Segu, Shruthi; Hummel, Jan; Köhl, Karin; Willmitzer, Lothar

    2011-10-01

    The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.

  16. Metabolomic profiling of 13C-labelled cellulose digestion in a lower termite: insights into gut symbiont function

    PubMed Central

    Tokuda, Gaku; Tsuboi, Yuuri; Kihara, Kumiko; Saitou, Seikou; Moriya, Sigeharu; Lo, Nathan; Kikuchi, Jun

    2014-01-01

    Termites consume an estimated 3–7 billion tonnes of lignocellulose annually, a role in nature which is unique for a single order of invertebrates. Their food is digested with the help of microbial symbionts, a relationship that has been recognized for 200 years and actively researched for at least a century. Although DNA- and RNA-based approaches have greatly refined the details of the process and the identities of the participants, the allocation of roles in space and time remains unclear. To resolve this issue, a pioneer study is reported using metabolomics to chart the in situ catabolism of 13C-cellulose fed to the dampwood species Hodotermopsis sjostedti. The results confirm that the secretion of endogenous cellulases by the host may be significant to the digestive process and indicate that a major contribution by hindgut bacteria is phosphorolysis of cellodextrins or cellobiose. This study provides evidence that essential amino acid acquisition by termites occurs following the lysis of microbial tissue obtained via proctodaeal trophallaxis. PMID:25009054

  17. 13C- and 15N-Labeling Strategies Combined with Mass Spectrometry Comprehensively Quantify Phospholipid Dynamics in C. elegans

    PubMed Central

    Drechsler, Robin; Gafken, Philip R.; Olsen, Carissa Perez

    2015-01-01

    Membranes define cellular and organelle boundaries, a function that is critical to all living systems. Like other biomolecules, membrane lipids are dynamically maintained, but current methods are extremely limited for monitoring lipid dynamics in living animals. We developed novel strategies in C. elegans combining 13C and 15N stable isotopes with mass spectrometry to directly quantify the replenishment rates of the individual fatty acids and intact phospholipids of the membrane. Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations. In fact, our analysis shows that the majority of membrane lipids are replaced each day. Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids. Additionally, the compromised membrane maintenance as defined by LC-MS/MS with SCD RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals. Not only have these combined methodologies identified new facets of the impact of SCDs on the membrane, but they also have great potential to reveal many undiscovered regulators of phospholipid metabolism. PMID:26528916

  18. Human lactation: oxidation and maternal transfer of dietary (13)C-labelled α-linolenic acid into human milk.

    PubMed

    Demmelmair, Hans; Kuhn, Angelika; Dokoupil, Katharina; Hegele, Verena; Sauerwald, Thorsten; Koletzko, Berthold

    2016-06-01

    The origin of fatty acids in milk has not been elucidated in detail. We investigated the contribution of dietary α-linolenic acid (ALA) to human milk fat, its oxidation and endogenous conversion to long-chain polyunsaturated fatty acids. Ten lactating women were given (13)C-ALA orally, and breath and milk samples were collected for a five-day period, while dietary intakes were assessed. 37.5 ± 2.7 % (M ± SE) of the tracer was recovered in breath-CO2, and 7.3 ± 1.1 % was directly transferred into milk. About 0.25 % of the tracer was found in milk long-chain polyunsaturated fatty acids. Combining intake and milk data, we estimate that about 65 % of milk ALA is directly derived from maternal diet. Thus, the major portion of milk ALA is directly derived from the diet, but dietary ALA does not seem to contribute much as a precursor to milk n-3 long-chain polyunsaturated fatty acids within the studied time period.

  19. Suppression of glycine-15N incorporation into urinary uric acid by adenine-8-13C in normal and gouty subjects

    PubMed Central

    Seegmiller, J. Edwin; Klinenberg, James R.; Miller, John; Watts, R. W. E.

    1968-01-01

    Adenine inhibited the de novo synthesis of purines in both normal and gouty man as shown by inhibition of the incorporation of glycine-15N into urinary uric acid without altering the incorporation of glycine-15N into urinary creatinine. The diminished purine synthesis did not result in a diminution in the 24 hr excretion of uric acid. This observation was explainable in part by the prompt conversion of adenine to uric acid. In addition to this direct conversion, adenine-8-13C provided a slow and prolonged contribution to urinary uric acid. A feedback inhibition of purine synthesis by nucleotides derived from adenine provides the best interpretation of these results. PMID:5645862

  20. The Cyanide Ligands of [FeFe] Hydrogenase: Pulse EPR Studies of 13C and 15N-Labeled H-Cluster

    PubMed Central

    2015-01-01

    The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous l-tyrosine. Using selectively labeled tyrosine substrates, the cyanides were isotopically labeled via a recently developed in vitro maturation procedure allowing advanced electron paramagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic 13C hyperfine interactions for the two CN– ligands—a reporter of spin density on their respective coordinating iron ions—collapses from ≈5.8 for the Hox form of hydrogenase to <2 for the CO-inhibited form. Additionally, when the maturation was carried out using [15N]-tyrosine, no features previously ascribed to the nitrogen of the bridging dithiolate ligand were observed suggesting that this bridge is not sourced from tyrosine. PMID:25133957

  1. Redox dependent changes at the heme propionates in cytochrome c oxidase from Paracoccus denitrificans: direct evidence from FTIR difference spectroscopy in combination with heme propionate 13C labeling.

    PubMed

    Behr, J; Hellwig, P; Mäntele, W; Michel, H

    1998-05-19

    Specific isotope labeling at the carboxyl groups of the four heme propionates of cytochrome c oxidase from Paracoccus denitrificans was used in order to assign signals observed in electrochemically induced redox Fourier transform infrared (FTIR) difference spectra of this enzyme. For this purpose, the hemA gene of the P. denitrificans strain PD1222, coding for 5-aminolevulinate synthase, was deleted by partial replacement with a kanamycin resistance cartridge, resulting in a stable 5-aminolevulinic acid (ALA) auxotrophy. Normal growth of this deficient strain and cytochrome c oxidase yield comparable to that of P. dentrificans wild-type strain PD1222 could be obtained by supplementation with 0.1 mM ALA in the growth medium. Visible spectra and reduced-minus-oxidized FTIR spectra showed that the purified cytochrome c oxidase had spectral characteristics identical to those of the wild-type enzyme. The decrease of a negative signal at 1676 cm-1 in the reduced-minus-oxidized FTIR difference spectra of the 13C-labeled cytochrome c oxidase in comparison to those of the unlabeled protein allowed the assignment of this signal to a COOH vibration mode of at least one of the four heme propionates. Moreover, a negative band at approximately 1570 cm-1 shifted to smaller wavenumbers in the spectra of the 13C-labeled enzyme in comparison to the spectra of the unlabeled enzyme and was thus assigned to contributions from an antisymmetric COO- mode of one or more of the four heme propionates. Additionally, a positive signal at 1538 cm-1 shifted to approximately 1500 cm-1 in the spectra of the isotopically labeled protein and was therefore assigned to at least one antisymmetric COO- mode of the heme propionates. A negative signal at 1390 cm-1, which has been shifted to 1360 cm-1 in the spectra of the 13C-labeled enzyme, is due to a symmetric COO- mode from at least one heme propionate. These results suggest that at least two of the four heme propionates in cytochrome c oxidase

  2. Multidimensional solid-state NMR studies of the structure and dynamics of pectic polysaccharides in uniformly 13C-labeled Arabidopsis primary cell walls

    SciTech Connect

    Dick-Perez, Marilu; Wang, Tuo; Salazar, Andre; Zabotina, Olga A.; Hong, Mei

    2012-07-08

    Plant cell wall (CW) polysaccharides are responsible for the mechanical strength and growth of plant cells; however, the high-resolution structure and dynamics of the CW polysaccharides are still poorly understood because of the insoluble nature of these molecules. Here, we use 2D and 3D magic-angle-spinning (MAS) solid-state NMR (SSNMR) to investigate the structural role of pectins in the plant CW. Intact and partially depectinated primary CWs of Arabidopsis thaliana were uniformly labeled with 13C and their NMR spectra were compared. Recent 13C resonance assignment of the major polysaccharides in Arabidopsis thaliana CWs allowed us to determine the effects of depectination on the intermolecular packing and dynamics of the remaining wall polysaccharides. 2D and 3D correlation spectra show the suppression of pectin signals, confirming partial pectin removal by chelating agents and sodium carbonate. Importantly, higher cross peaks are observed in 2D and 3D 13C spectra of the depectinated CW, suggesting higher rigidity and denser packing of the remaining wall polysaccharides compared with the intact CW. 13C spin–lattice relaxation times and 1H rotating-frame spin–lattice relaxation times indicate that the polysaccharides are more rigid on both the nanosecond and microsecond timescales in the depectinated CW. Taken together, these results indicate that pectic polysaccharides are highly dynamic and endow the polysaccharide network of the primary CW with mobility and flexibility, which may be important for pectin functions. This study demonstrates the capability of multidimensional SSNMR to determine the intermolecular interactions and dynamic structures of complex plant materials under near-native conditions. Copyright © 2012 John Wiley & Sons, Ltd.

  3. Determining the in vivo regulation of cardiac pyruvate dehydrogenase based on label flux from hyperpolarised [1-13C]pyruvate.

    PubMed

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

    2011-10-01

    Pyruvate dehydrogenase (PDH) is a key regulator of cardiac substrate selection and is regulated by both pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation and feedback inhibition. The extent to which chronic upregulation of PDK protein levels, acutely increased PDK activity and acute feedback inhibition limit PDH flux remains unclear because existing in vitro assessment methods inherently disrupt the regulation of the enzyme complex. We have demonstrated previously that hyperpolarised (13)C-labelled metabolic tracers coupled with MRS can monitor flux through PDH in vivo. The aim of this study was to determine the relative contributions of acute and chronic changes in PDK and PDH activities to in vivo myocardial PDH flux. We examined both fed and fasted rats with either hyperpolarised [1-(13)C]pyruvate alone or hyperpolarised [1-(13)C]pyruvate co-infused with malate [to modulate mitochondrial nicotinamide adenine dinucleotide (NADH/NAD(+)) and acetyl-coenzyme A (acetyl-CoA)/CoA ratios, which alter both PDH activity and flux]. To confirm the metabolic fate of infused malate, we performed in vitro (1)H NMR spectroscopy on cardiac tissue extracts. We observed that, in fed rats, where PDH activity was high, the presence of malate increased PDH flux by 27%, whereas, in the fasted state, malate infusion had no effect on PDH flux. These observations suggest that pyruvate oxidation is limited by feedback inhibition from acetyl-CoA only when PDH activity is high. Therefore, in the case of PDH, and potentially other enzymes, hyperpolarised (13)C MRI can be used to assess noninvasively enzymatic regulation.

  4. Chemo-Enzymatic Synthesis of (13)C Labeled Complex N-Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry.

    PubMed

    Echeverria, Begoña; Etxebarria, Juan; Ruiz, Nerea; Hernandez, Álvaro; Calvo, Javier; Haberger, Markus; Reusch, Dietmar; Reichardt, Niels-Christian

    2015-11-17

    Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of (13)C labeled N-glycan libraries and provide an example for their use as internal standards in the profiling and absolute quantification of mAb glycans by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. A synthetic biantennary glycan precursor was (13)C-labeled on all four amino sugar residues and enzymatically derivatized to produce a library of 15 glycan isotopologues with a mass increment of 8 Da over the natural products. Asymmetrically elongated glycans were accessible by performing enzymatic reactions on partially protected UV-absorbing intermediates, subsequent fractionation by preparative HPLC, and final hydrogenation. Using a preformulated mixture of eight internal standards, we quantified the glycans in a monoclonal therapeutic antibody with excellent precision and speed.

  5. Quantitative twoplex glycan analysis using (12)C6 and (13)C6 stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry.

    PubMed

    Váradi, Csaba; Mittermayr, Stefan; Millán-Martín, Silvia; Bones, Jonathan

    2016-12-01

    Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available (12/13)C6 stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for (12)C6 'light' and (13)C6 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Synthesis of [13C4]-labeled ∆9-tetrahydrocannabinol and 11-nor-9-carboxy-∆9-tetrahydrocannabinol as internal standards for reducing ion suppressing/alteration effects in LC/MS-MS quantification.

    PubMed

    Karlsen, Morten; Liu, Huiling; Johansen, Jon Eigill; Hoff, Bård Helge

    2014-09-01

    (-)-∆9-Tetrahydrocannabinol is the principal psychoactive component of the cannabis plant and also the active ingredient in some prescribed drugs. To detect and control misuse and monitor administration in clinical settings, reference samples of the native drugs and their metabolites are needed. The accuracy of liquid chromatography/mass spectrometric quantification of drugs in biological samples depends among others on ion suppressing/alteration effects. Especially, 13C-labeled drug analogues are useful for minimzing such interferences. Thus, to provide internal standards for more accurate quantification and for identification purpose, synthesis of [13C4]-∆9-tetrahydro-cannabinol and [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was developed via [13C4]-olivetol. Starting from [13C4]-olivetol the synthesis of [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was shortened from three to two steps by employing nitromethane as a co-solvent in condensation with (+)-apoverbenone.

  8. In situ assessment of the velocity of carbon transfer by tracing 13 C in trunk CO2 efflux after pulse labelling: variations among tree species and seasons.

    PubMed

    Dannoura, Masako; Maillard, Pascale; Fresneau, Chantal; Plain, Caroline; Berveiller, Daniel; Gerant, Dominique; Chipeaux, Christophe; Bosc, Alexandre; Ngao, Jérôme; Damesin, Claire; Loustau, Denis; Epron, Daniel

    2011-04-01

    Phloem is the main pathway for transferring photosynthates belowground. In situ(13) C pulse labelling of trees 8-10 m tall was conducted in the field on 10 beech (Fagus sylvatica) trees, six sessile oak (Quercus petraea) trees and 10 maritime pine (Pinus pinaster) trees throughout the growing season. Respired (13) CO2 from trunks was tracked at different heights using tunable diode laser absorption spectrometry to determine time lags and the velocity of carbon transfer (V). The isotope composition of phloem extracts was measured on several occasions after labelling and used to estimate the rate constant of phloem sap outflux (kP ). Pulse labelling together with high-frequency measurement of the isotope composition of trunk CO2 efflux is a promising tool for studying phloem transport in the field. Seasonal variability in V was predicted in pine and oak by bivariate linear regressions with air temperature and soil water content. V differed among the three species consistently with known differences in phloem anatomy between broadleaf and coniferous trees. V increased with tree diameter in oak and beech, reflecting a nonlinear increase in volumetric flow with increasing bark cross-sectional area, which suggests changes in allocation pattern with tree diameter in broadleaf species. Discrepancies between V and kP indicate vertical changes in functional phloem properties.

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

    PubMed

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

    2015-02-01

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

  10. Efficient Measurement of 3JN,Cγ and 3JC‧,Cγ Coupling Constants of Aromatic Residues in 13C, 15N-Labeled Proteins

    NASA Astrophysics Data System (ADS)

    Löhr, Frank; Rüterjans, Heinz

    2000-09-01

    An NMR pulse sequence is proposed for the simultaneous determination of side chain χ1 torsion-angle related 3JN,Cγ and 3JC‧,Cγ couplings in aromatic amino acid spin systems. The method is of the quantitative J correlation type and takes advantage of attenuated 15N and 1H transverse relaxation by means of the TROSY principle. Unlike previously developed schemes for the measurement of either of the two coupling types, spectra contain internal reference peaks that are usually recorded in separate experiments. Therefore, the desired information is extracted from a single rather than four data sets. The new method is demonstrated with uniformly 13C/15N labeled Desulfovibrio vulgaris flavodoxin, which contains 14 aromatic out of 147 total amino acid residues.

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

    PubMed

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

    2014-03-15

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

  12. Use of 13C Labeled Carbon Tetrachloride to Demonstrate the Transformation to Carbon Dioxide under Anaerobic Conditions in a Continuous Flow Column

    NASA Astrophysics Data System (ADS)

    Semprini, L.; Azizian, M.

    2012-12-01

    The demonstration of transformation of chlorinated aliphatic compounds (CAHs) in the subsurface is a challenge, especially when the products are carbon dioxide (CO2) and chloride ion. The groundwater contaminant carbon tetrachloride (CT) is of particular interest since a broad range of transformation products can be potentially formed under anaerobic conditions. The ability to demonstrate the transformation of CT to CO2 as a non toxic endproduct, is also of great interest. Results will be presented from a continuous flow column study where 13C labeled CT was used to demonstrate its transformation to CO2. The column was packed with a quartz sand and bioaugmented the Evanite Culture (EV) that is capable of transforming tetrachloroethene (PCE) to ethene. The column was continously fed a synthetic groundwater that was amended with PCE (0.10 mM) and either formate (1.5 mM) or lactate (1.1 mM), which ferments to produce hydrogen (H2) as the ultimate electron donor. Earlier CT transformation studies with the column, in the absence of sulfate reduction, and with formate added as a donor found CT (0.015 mM) was over 98% transformed with about 20% converted to chloroform (CF) (0.003 mM) and with a transient detection of chloromethane (CM). Methane and carbon disulfide, as potential products, were not detected. Neither CT nor CF inhibited the reductive dehalogenation of PCE to ethene. A series of transient studies conducted after these initial CT transformation tests, but in the absence of CT, showed formate remained an effective substrate for maintaining sulfate reduction and PCE transformation. Lactate, which was effectively fermented prior to CT addition, was not effectively fermented, with propionate accumulating as a fermentation product. When lactate was added, PCE was mainly transformed to cis-dichloroethene (cis-DCE) and VC, and sulfate reduction did not occur. In order to restore effective lactate fermentation the column was then bioaugmented with an EV culture that

  13. [13C]-Specific labeling of 8-2' linked (-)-cis-blechnic, (-)-trans-blechnic and (-)-brainic acids in the fern Blechnum spicant

    NASA Technical Reports Server (NTRS)

    Davin, Laurence B.; Wang, Chang-Zeng; Helms, Gregory L.; Lewis, Norman G.

    2003-01-01

    In vivo administration experiments using stable (13C) and radio (14C) labeled precursors established that the optically active 8-2' linked lignans, (-)-cis-blechnic, (-)-trans-blechnic and (-)-trans-brainic acids, were directly derived from L-phenylalanine, cinnamate, and p-coumarate but not either from tyrosine or acetate. The radiochemical time course data suggest that the initial coupling product is (-)-cis-blechnic acid, which is then apparently converted into both (-)-trans-blechnic and (-)-trans-brainic acids in vivo. These findings provide additional evidence for vascular plant proteins engendering distinct but specific phenolic radical-radical coupling modes, i.e., for full control over phenylpropanoid coupling in vivo, whether stereoselective or regiospecific.

  14. Mineral fertilization did not affect decay of old lignin and SOC in a 13C-labeled arable soil over 36 years

    NASA Astrophysics Data System (ADS)

    Hofmann, A.; Heim, A.; Gioacchini, P.; Miltner, A.; Gehre, M.; Schmidt, M. W. I.

    2009-07-01

    Retardation of soil organic carbon (SOC) decay after nitrogen addition to litter or soil has been suggested in several recent studies and has been attributed to a retardation in lignin decay. With our study we tested the long-term effect of mineral fertilization (N+P) on the decay of the SOC component lignin in arable soil. To achieve this, we tracked 13C-labeled lignin and SOC in an arable soil that is part of a 36-year field experiment (conversion from C3 to C4 crops) with two mineral fertilization levels. We could show that fertilization neither retarded nor enhanced the decay of old SOC or lignin over a period of 36 years, proposing that decay of lignin was less sensitive to fertilization than previously suggested. However, for new, C4-derived lignin there were indications that decay might have been enhanced by the fertilization treatment, whereas decay of new SOC was unaffected.

  15. Intraseasonal carbon sequestration and allocation in larch trees growing on permafrost in Siberia after (13)C labeling (two seasons of 2013-2014 observation).

    PubMed

    Masyagina, Oxana; Prokushkin, Anatoly; Kirdyanov, Alexander; Artyukhov, Aleksey; Udalova, Tatiana; Senchenkov, Sergey; Rublev, Aleksey

    2016-12-01

    This research is an attempt to study seasonal translocation patterns of photoassimilated carbon within trees of one of the high latitudes widespread deciduous conifer species Larix gmelinii (Rupr. Rupr). For this purpose, we applied whole-tree labeling by (13)CO2, which is a powerful and effective tool for tracing newly developed assimilates translocation to tissues and organs of a tree. Experimental plot has been established in a mature 105-year-old larch stand located within the continuous permafrost area near Tura settlement (Central Siberia, 64°17'13″N, 100°11'55″E, 148 m a.s.l.). Measurements of seasonal photosynthetic activity and foliage parameters (i.e., leaf length, area, biomass, etc.), and sampling were arranged from early growing season (June 8, 2013; May 14, 2014) until yellowing and senescence of needles (September 17, 2013; September 14, 2014). Labeling by (13)C of the tree branch (June 2013, for 3 branch replicates in 3 different trees) and the whole tree was conducted at early (June 2014), middle (July 2014), and late (August 2013) phase of growing season (for different trees in 3 replicates each time) by three pulses [(CO2)max = 3000-4000 ppmv, (13)CO2 (30 % v/v)]. We found at least two different patterns of carbon translocation associated with larch CO2 assimilation depending on needle phenology. In early period of growing season (June), (13)C appearing in newly developed needles is a result of remobilized storage material use for growth purposes. Then approximately at the end of June, growth processes is switching to storage processes lasting to the end of growing season.

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

    PubMed Central

    2014-01-01

    Background Mapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation. Results Intracellular fluxes of the CHO-K1 cell line central carbon metabolism were successfully determined for a complex network using non-stationary 13C metabolic flux analysis. Mass isotopomers of extracellular metabolites were determined using [U-13C6] glucose as labeled substrate. Metabolic compartmentation and extracellular transport reversibility proved essential to successfully reproduce the dynamics of the labeling patterns. Alanine and pyruvate reversibility changed dynamically even if their net production fluxes remained constant. Cataplerotic fluxes of cytosolic phosphoenolpyruvate carboxykinase and mitochondrial malic enzyme and pyruvate carboxylase were successfully determined. Glycolytic pyruvate channeling to lactate was modeled by including a separate pyruvate pool. In the exponential growth phase, alanine, glycine and glutamate were excreted, and glutamine, aspartate, asparagine and serine were taken up; however, all these amino acids except asparagine were exchanged reversibly with the media. High fluxes were determined in the pentose phosphate pathway and the TCA cycle. The latter was fueled mainly by glucose but also by amino acid catabolism. Conclusions The CHO-K1 central metabolism in controlled batch culture proves to be robust. It has the main purpose to ensure fast growth on a mixture of substrates and also to mitigate oxidative stress. It achieves this by using compartmentation to control NADPH and NADH availability and by simultaneous synthesis and catabolism of amino acids. PMID:24773761

  17. Priming effect of 13C-labelled wheat straw in no-tillage soil under drying and wetting cycles in the Loess Plateau of China

    PubMed Central

    Liu, Enke; Wang, Jianbo; Zhang, Yanqing; Angers, Denis A.; Yan, Changrong; Oweis, Theib; He, Wenqing; Liu, Qin; Chen, Baoqing

    2015-01-01

    The objectives of this study were to determine the effects of drying and wetting (DW) cycles on soil organic carbon (SOC) mineralisation and on the priming effect (PE) induced by the addition of 13C-labelled wheat straw to long-term no-tillage (NT) and conventional-tillage (CT) soils. We observed that the SOC mineralisation rate in rewetted soils was greater than that in soils that were kept at constant water content. The proportion of CO2 derived from the straw declined dramatically during the first 10 days. The priming direction was first positive, and then became slightly negative. The PE was higher under DW cycles than under constant water content. There was no significant effect of the tillage system on the SOC mineralisation rate or PE. The data indicate that the DW cycles had a significant effect on the SOC mineralisation rate and on the PE, demonstrating a positive combined effect between wheat straw and moisture fluctuations. Further research is needed to study the role of microbial communities and C pools in affecting the SOC mineralisation response to DW cycles. PMID:26345303

  18. Two-dimensional IR spectroscopy and segmental 13C labeling reveals the domain structure of human γD-crystallin amyloid fibrils.

    PubMed

    Moran, Sean D; Woys, Ann Marie; Buchanan, Lauren E; Bixby, Eli; Decatur, Sean M; Zanni, Martin T

    2012-02-28

    The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly (13)C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins.

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

    PubMed

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

    2016-08-10

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

  20. Direct incorporation of fatty acids into microbial phospholipids in soils: Position-specific labeling tells the story

    NASA Astrophysics Data System (ADS)

    Dippold, Michaela A.; Kuzyakov, Yakov

    2016-02-01

    Fatty acids have been used as plant and microbial biomarkers, and knowledge about their transformation pathways in soils and sediments is crucial for interpreting fatty acid signatures, especially because the formation, recycling and decomposition processes are concurrent. We analyzed the incorporation of free fatty acids into microbial fatty acids in soil by coupling position-specific 13C labeling with compound-specific 13C analysis. Position-specifically and uniformly 13C labeled palmitate were applied in an agricultural Luvisol. Pathways of fatty acids were traced by analyzing microbial utilization of 13C from individual molecule positions of palmitate and their incorporation into phospholipid fatty acids (PLFA). The fate of palmitate 13C in the soil was characterized by the main pathways of microbial fatty acid metabolism: Odd positions (C-1) were preferentially oxidized to CO2 in the citric acid cycle, whereas even positions (C-2) were preferentially incorporated into microbial biomass. This pattern is a result of palmitate cleavage to acetyl-CoA and its further use in the main pathways of C metabolism. We observed a direct, intact incorporation of more than 4% of the added palmitate into the PLFA of microbial cell membranes, indicating the important role of palmitate as direct precursor for microbial fatty acids. Palmitate 13C was incorporated into PLFA as intact alkyl chain, i.e. the C backbone of palmitate was not cleaved, but palmitate was incorporated either intact or modified (e.g. desaturated, elongated or branched) according to the fatty acid demand of the microbial community. These modifications of the incorporated palmitate increased with time. Future PLFA studies must therefore consider the recycling of existing plant and microbial-derived fatty acids. This study demonstrates the intact uptake and recycling of free fatty acids such as palmitate in soils, as well as the high turnover and transformation of cellular PLFA. Knowledge about the intact

  1. Tracking the incorporation of 15N from labeled beech litter into mineral-organic associations

    NASA Astrophysics Data System (ADS)

    Kleber, M.; Hatton, P.; Derrien, D.; Lajtha, K.; Zeller, B.

    2008-12-01

    Nitrogen containing organic compounds are thought to have a role in the complex web of processes that control the turnover time of soil organic matter. The sequential density fractionation technique is increasingly used for the purpose of investigating the association of organic materials with the mineral matrix. Organic materials in the denser fractions (>2.0 kg L-1) typically show 13C NMR signals indicative of carbohydrate and aliphatic structures, an absence of lignin and tannin structures and a narrow C:N ratio, suggesting a microbial origin of organic matter in these fractions. Here we take advantage of a labeling experiment conducted at two different sites in Germany and in France to investigate the incorporation of organic nitrogen into physical fractions of increasing density, representing a proximity gradient to mineral surfaces. 15N labeled beech litter was applied to two acidic forest topsoils 8 and 12 years ago. Although there are differences in the distribution patterns between the two soils, and the majority of the organic nitrogen was recovered in fractions representing organic matter of plant origin and not bound to the mineral matrix, our data clearly show that after a decade, significant amounts of the nitrogen had been incorporated in mineral-organic fractions of supposedly slow turnover. It remains to be shown to which extent the N in the densest fractions was incorporated by soil microbiota and associated with mineral surfaces in organic form or adsorbed to mineral surfaces in inorganic form (NH4+).

  2. Multi-isotope labelling of organic matter by diffusion of 2H/18O-H2O vapour and 13C-CO2 into the leaves and its distribution within the plant

    NASA Astrophysics Data System (ADS)

    Studer, M. S.; Siegwolf, R. T. W.; Leuenberger, M.; Abiven, S.

    2015-03-01

    Isotope labelling is a powerful tool to study elemental cycling within terrestrial ecosystems. Here we describe a new multi-isotope technique to label organic matter (OM). We exposed poplars (Populus deltoides × nigra) for 14 days to an atmosphere enriched in 13CO2 and depleted in 2H218O. After 1 week, the water-soluble leaf OM (δ13C = 1346 ± 162‰) and the leaf water were strongly labelled (δ18O = -63 ± 8, δ2H = -156 ± 15‰). The leaf water isotopic composition was between the atmospheric and stem water, indicating a considerable back-diffusion of vapour into the leaves (58-69%) in the opposite direction to the net transpiration flow. The atomic ratios of the labels recovered (18O/13C, 2H/13C) were 2-4 times higher in leaves than in the stems and roots. This could be an indication of the synthesis of more condensed compounds in roots and stems (e.g. lignin vs. cellulose) or might be the result of O and H exchange and fractionation processes during phloem transport and biosynthesis. We demonstrate that the three major OM elements (C, O, H) can be labelled and traced simultaneously within the plant. This approach could be of interdisciplinary interest in the fields of plant physiology, palaeoclimatic reconstruction or soil science.

  3. Medium- and long-chain triglycerides labeled with 13C: a comparison of oxidation after oral or parenteral administration in humans.

    PubMed

    Metges, C C; Wolfram, G

    1991-01-01

    The special physical properties of medium-chain triglycerides (MCT) result in some substantial differences in their metabolism compared to that of long-chain triglycerides (LCT). Administering MCT is of importance in enteral nutrition of patients with disturbances of fat digestion or lipoprotein lipase deficiency. Their use in parenteral nutrition is also of interest. The purpose of this study was to compare the rate of conversion of MCT and LCT to CO2 after parenteral or oral administration in humans. At 1-wk intervals, a liquid formula diet (418 kJ/h for 8 h) was given to five healthy volunteers following an overnight fast. Two hours after starting this, they were given either 100 mg [13C]trioctanoate or [13C]trioleate orally or parenterally. Excess 13C in breath carbon dioxide was analyzed by mass-spectrometry, and oxidation rates over 7.5 h were calculated. Oxidation rates for [13C]trioctanoate were on the average 34.7% after enteral and 31.0% after parenteral administration, and for [13C]trioleate, 25.3 and 24.9%, respectively (p less than 0.05, trioctanoate vs. trioleate). The results show that the oxidation of trioctanoate in healthy humans is greater both after oral and parenteral administration and increases more rapidly than that of [13C]trioleate.

  4. The Semiquinone at the Qi Site of the bc1 Complex Explored Using HYSCORE Spectroscopy and Specific Isotopic Labeling of Ubiquinone in Rhodobacter sphaeroides via 13C Methionine and Construction of a Methionine Auxotroph

    PubMed Central

    2015-01-01

    Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe 13C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group 13C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of 13C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly. PMID:25184535

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

    PubMed

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

    1998-08-01

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

  6. Production of Hydrolysable Tannin-Like Structures During the Microbial Demethylation of lignin: An Assessment Using13C-Labeled Tetramethylammonium Hydroxide Thermochemolysis.

    NASA Astrophysics Data System (ADS)

    Filley, T.; Blanchette, R.; Nierop, K.; Gamblin, D.

    2003-12-01

    Phenolic compounds in soils are important mediators of microbial activity, metal mobility, soil redox, and soil organic matter building processes. Direct tannin input and the microbial decomposition of lignin in litter and soil are important contributors to this pool of phenols. The ability to accurately assess the relative differences in lignin decay (which are initiated by demethylation and side chain oxidation) among synapyl, coniferyl, and p-coumaryl components of detrital lignin requires the ability to determine microbial demethylation within the complex soil residues. Differentiating between hydrolysable tannins and contributions from advanced lignin decay can be problematic for many of the most common molecular techniques such as alkaline CuO oxidation, pyrolysis GC, and tetramethylammonium hydroxide thermochemolysis because of either the masking effects of derivatizing agents, oxidative damage to ortho-phenols or low volatility of lignin monomers. In this study we investigate lignin demethylation and polyhydroxyl-aromatic production in BC and C horizons of sandy forest soils dominated by oak, the A horizon from a red spruce forest, and controlled microbial inoculation studies of woody tissue using in-line 13C-labeled tetramethylammonium hydroxide thermochemolysis. Both white-rot and brown-rot decay resulted in syringyl demethylation, with the latter exhibiting more aggressive demethylation chemistry, while coniferyl monomer demethylation was essentially restricted to brown-rot decay. In a typical brown-rot sequence demethylation of syringyl components occurs more rapidly than coniferyl units within the same tissue and lower molecular weight fragments are likewise more demethylated than lignin monomers containing the full glycerol side chain. Demethylation of both methoxyl groups in the syringyl monomer is evident in soil horizons as well as laboratory inoculations. The latter may suggest demethylation after lignin depolymerization. Low molecular weight

  7. 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

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

    PubMed

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

    2012-12-01

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

  9. Synthesis of 3H, 13C,2H3,15N and 14C-labelled SCH 466036, a histamine 3 receptor antagonist.

    PubMed

    Hesk, D; Borges, S; Dumpit, R; Hendershot, S; Koharski, D; Lavey, C; McNamara, P; Voronin, K

    2015-02-01

    The synthesis of [(3)H]SCH 466036, [Me-(3)H3]SCH 466036, [(13)C,(2)H3,(15)N]SCH 466036 and [(14)C]SCH 466036 is described. [(3)H]SCH 466036 was prepared in two steps via Raney Ni-catalysed exchange with tritiated water. [Me-(3)H3]SCH 466036 was prepared in a single step from [(3)H]methyl iodide in 45% yield. [(13)C,(2)H3,(15)N]SCH 466036 was prepared in two steps from [(15)N]hydroxylamine and [(13)C,(2)H3]methyl iodide with an overall yield of 16%. [(14)C]SCH 466036 was prepared in seven steps from [(14)C]potassium cyanide in an overall yield of 13%.

  10. Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.

    PubMed

    Hefke, Frederik; Bagaria, Anurag; Reckel, Sina; Ullrich, Sandra Johanna; Dötsch, Volker; Glaubitz, Clemens; Güntert, Peter

    2011-02-01

    We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273-6279 (1982)), types of amino acids are labeled with (13)C or/and (15)N such that cross peaks between (13)CO(i - 1) and (15)NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with (13)C and the second with (15)N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B(2)R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.

  11. Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [13C,15N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

    PubMed Central

    2014-01-01

    Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure–activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [13C,15N]-N-acetylglucosamine (GlcNAc). UDP-[13C,15N]GlcNAc was synthesized enzymatically in a one-pot reaction from [13C]glucose and [15N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[13C,15N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [13C,15N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein

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

    PubMed

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

    2015-06-02

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

  13. Proteome Analysis using Selective Incorporation of Isotopically Labeled Amino Acids

    SciTech Connect

    Veenstra, Timothy D.; Martinovic, Suzana; Anderson, Gordon A.; Pasa-Tolic, Liljiana; Smith, Richard D.

    2000-01-01

    A method is described for identifying intact proteins from genomic databases using a combination of accurate molecular mass measurements and partial amino acid content. An initial demonstration was conducted for proteins isolated from Escherichia coli (E. coli) using a multiple auxotrophic strain of K12. Proteins were extracted from the organism grown in natural isotopic abundance minimal medium and also minimal medium containing isotopically labeled leucine (Leu-D10), were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled Leu residue has no effect on the CIEF separation of the protein, therefore both versions of the protein are observed within the same FTICR spectrum. The difference in the molecular mass of the natural isotopic abundance and Leu-D10 isotopically labeled proteins is used to determine the number of Leu residues present in that particular protein. Knowledge of the molecular mass and number of Leu residues present can be used to unambiguously identify the intact protein. Preliminary results show the efficacy of using this method to unambiguously identify proteins isolated from E. coli.

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

    SciTech Connect

    Pahl-Wostl, C.; Seelig, J.

    1986-11-04

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

  15. Folate is absorbed across the human colon: evidence by using enteric-coated caplets containing 13C-labeled [6S]-5-formyltetrahydrofolate1, 2, 3, 4

    PubMed Central

    Lakoff, Alanna; Fazili, Zia; Aufreiter, Susanne; Pfeiffer, Christine M; Connolly, Bairbie; Gregory, Jesse F; Pencharz, Paul B; O’Connor, Deborah L

    2016-01-01

    Background Folate intakes that do not meet or greatly exceed requirements may be associated with negative health outcomes. A better understanding of contributors that influence the input side will help establish dietary guidance that ensures health benefits without associated risks. Colonic microbiota produce large quantities of folate, and [13C5]5-formyltetrahydrofolate infused during colonoscopy is absorbed. However, it is unclear if significant quantities of folate are absorbed in an intact microbiome. Objective We determined whether and how much of a physiologic dose of [13C5]5-formyltetrahydrofolate delivered in a pH-sensitive enteric caplet to an intact colonic microbiome is absorbed. Design Healthy adults ingested a specially designed pH-sensitive acrylic copolymer–coated barium sulfate caplet that contained 855 nmol (400 μg) [13C5]5-formyltetrahydrofolate. After a washout period ≥4 wk, subjects received an intravenous injection of the same compound (214 nmol). Serially collected blood samples before and after each test dose were analyzed by using a microbiological assay and liquid chromatography–tandem mass spectrometry. Results Caplet disintegration in the colon was observed by fluoroscopic imaging for 6 subjects with a mean (±SD) complete disintegration time of 284 ± 155 min. The mean (±SEM) rate of appearance of [13C5]5-methyltetrahydrofolate in plasma was 0.33 ± 0.09 (caplet) and 5.8 ± 1.2 (intravenous) nmol/h. Likely because of the significant time in the colon, the mean apparent absorption across the colon was 46%. Conclusions Folate is absorbed across the colon in humans with an undisturbed microbiome. This finding and previous observations of the size of the colonic depot of folate and its potential for manipulation by diet (eg, dietary fiber, oligosaccharides, and probiotics) suggest that an individual’s dietary folate requirement may differ depending on the consumption of dietary constituents that affect the size and composition of

  16. 13C metabolic flux analysis.

    PubMed

    Wiechert, W

    2001-07-01

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

  17. 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.

  18. 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.

  19. In Situ 13C and 23Na Magic Angle Spinning NMR Investigation of Supercritical CO2 Incorporation in Smectite-Natural Organic Matter Composites

    SciTech Connect

    Bowers, Geoffrey M.; Hoyt, David W.; Burton, Sarah D.; Ferguson, Brennan O.; Varga, Tamas; Kirkpatrick, Robert J.

    2014-01-29

    This paper presents an in situ NMR study of clay-natural organic polymer systems (a hectoritehumic acid [HA] composite) under CO2 storage reservoir conditions (90 bars CO2 pressure, 50°C). The 13C and 23Na NMR data show that supercritical CO2 interacts more strongly with the composite than with the base clay and does not react to form other C-containing species over several days at elevated CO2. With and without organic matter, the data suggest that CO2 enters the interlayer space of Na-hectorite equilibrated at 43% relative humidity. The presence of supercritical CO2 also leads to increased 23Na signal intensity, reduced line width at half height, increased basal width, more rapid 23Na T1 relaxation rates, and a shift to more positive resonance frequencies. Larger changes are observed for the hectorite-HA composite than for the base clay. In light of recently reported MD simulations of other polymer-Na-smectite composites, we interpret the observed changes as an increase in the rate of Na+ site hopping in the presence of supercritical CO2, the presence of potential new Na+ sorption sites when the humic acid is present, and perhaps an accompanying increase in the number of Na+ ions actively involved in site hopping. The results suggest that the presence of organic material either in clay interlayers or on external particle surfaces can significantly affect the behavior of supercritical CO2 and the mobility of metal ions in reservoir rocks.

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

    PubMed

    Salamanca-Cardona, Lucia; Keshari, Kayvan R

    2015-01-01

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

  1. 13 C solid-state NMR study of the 13 C-labeled peptide, (E)8 GGLGGQGAG(A)6 GGAGQGGYGG as a model for the local structure of Nephila clavipes dragline silk (MaSp1) before and after spinning.

    PubMed

    Yazawa, Koji; Yamaguchi, Erika; Knight, David; Asakura, Tetsuo

    2012-06-01

    We prepared the water soluble model peptide, (E)(8) GGLGGQGAG(A)(6) GGAGQGGYGG, to throw light on the local structure of spidroin 1 (MaSpl) protein in spider dragline silk of Nephila clavipes before and after spinning. Solution (13) C NMR showed that the conformation of the peptide in aqueous solution was essentially random coil. Solid-state NMR was used to follow conformation-dependent (13) C chemical shifts in (13) C selectively labeled versions of the peptide. The peptide lyophilized from an aqueous solution at neutral pH (hereafter referred to as "without acid treatment)"was used to mimic the state of the spidroin stored in the spider's silk gland while the peptide precipitated from the acidic solution ("with acid treatment") was used to simulate the role of acid treatment in inducing conformation change in the natural spinning process. In without acid treatment, the fraction of random coil conformation was lowest in the N-terminal region (residues 15-18) when compared with the C-terminus. The conformational change produced by the acid treatment occurred in the sequence, G(15) AG(A)(6) GGAG(27), interposed between pairs of Gly residues pairs, Gly(12,13), and Gly(29,30). The acid treated peptide showed a remarkable decrease in the fraction of random coil conformation from A(20) to A(23) in the poly-Ala region when compared with the peptide without acid treatment. These observations taken together suggest that the peptide can be used as a model for studying the localization of the conformation change in spider silk fibroin in the natural spinning and the role of acid treatment in this process.

  2. Dynamic nuclear polarization-enhanced 13C NMR spectroscopy of static biological solids

    PubMed Central

    Potapov, Alexey; Yau, Wai-Ming; Tycko, Robert

    2013-01-01

    We explore the possibility of using dynamic nuclear polarization (DNP) to enhance signals in structural studies of biological solids by solid state NMR without sample spinning. Specifically, we use 2D 13C-13C exchange spectroscopy to probe the peptide backbone torsion angles (ϕ,ψ) in a series of selectively 13C-labeled 40-residue β-amyloid (Aβ1–40) samples, in both fibrillar and non-fibrillar states. Experiments are carried out at 9.39 T and 8 K, using a static double-resonance NMR probe and low-power microwave irradiation at 264 GHz. In frozen solutions of Aβ1–40 fibrils doped with DOTOPA-TEMPO, we observe DNP signal enhancement factors of 16–21. We show that the orientation- and frequency-dependent spin polarization exchange between sequential backbone carbonyl 13C labels can be simulated accurately using a simple expression for the exchange rate, after experimentally determined homogeneous 13C lineshapes are incorporated in the simulations. The experimental 2D 13C-13C exchange spectra place constraints on the ϕ and ψ angles between the two carbonyl labels. Although the data are not sufficient to determine ϕ and ψ uniquely, the data do provide non-trivial constraints that could be included in structure calculations. With DNP at low temperatures, 2D 13C-13C exchange spectra can be obtained from a 3.5 mg sample of Aβ1–40 fibrils in 4 hr or less, despite the broad 13C chemical shift anisotropy line shapes that are observed in static samples. PMID:23562665

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

    PubMed

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

    2014-03-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1987-01-01

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

  7. 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.

  8. 13C pulse-labeling assessment of the community structure of active fungi in the rhizosphere of a genetically starch-modified potato (Solanum tuberosum) cultivar and its parental isoline.

    PubMed

    Hannula, S E; Boschker, H T S; de Boer, W; van Veen, J A

    2012-05-01

    • The aim of this study was to gain understanding of the carbon flow from the roots of a genetically modified (GM) amylopectin-accumulating potato (Solanum tuberosum) cultivar and its parental isoline to the soil fungal community using stable isotope probing (SIP). • The microbes receiving (13)C from the plant were assessed through RNA/phospholipid fatty acid analysis with stable isotope probing (PLFA-SIP) at three time-points (1, 5 and 12 d after the start of labeling). The communities of Ascomycota, Basidiomycota and Glomeromycota were analysed separately with RT-qPCR and terminal restriction fragment length polymorphism (T-RFLP). • Ascomycetes and glomeromycetes received carbon from the plant as early as 1 and 5 d after labeling, while basidiomycetes were slower in accumulating the labeled carbon. The rate of carbon allocation in the GM variety differed from that in its parental variety, thereby affecting soil fungal communities. • We conclude that both saprotrophic and mycorrhizal fungi rapidly metabolize organic substrates flowing from the root into the rhizosphere, that there are large differences in utilization of root-derived compounds at a lower phylogenetic level within investigated fungal phyla, and that active communities in the rhizosphere differ between the GM plant and its parental cultivar through effects of differential carbon flow from the plant.

  9. An automated GCxGC-TOF-MS protocol for batch-wise extraction and alignment of mass isotopomer matrixes from differential 13C-labelling experiments: a case study for photoautotrophic-mixotrophic grown Chlamydomonas reinhardtii cells.

    PubMed

    Kempa, Stefan; Hummel, Jan; Schwemmer, Thorsten; Pietzke, Matthias; Strehmel, Nadine; Wienkoop, Stefanie; Kopka, Joachim; Weckwerth, Wolfram

    2009-02-01

    Two dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-TOF-MS) is a promising technique to overcome limits of complex metabolome analysis using one dimensional GC-TOF-MS. Especially at the stage of data export and data mining, however, convenient procedures to cope with the complexity of GCxGC-TOF-MS data are still in development. Here, we present a high sample throughput protocol exploiting first and second retention index for spectral library search and subsequent construction of a high dimensional data matrix useful for statistical analysis. The method was applied to the analysis of (13)C-labelling experiments in the unicellular green alga Chlamydomonas reinhardtii. We developed a rapid sampling and extraction procedure for Chlamydomonas reinhardtii laboratory strain (CC503), a cell wall deficient mutant. By testing all published quenching protocols we observed dramatic metabolite leakage rates for certain metabolites. To circumvent metabolite leakage, samples were directly quenched and analyzed without separation of the medium. The growth medium was adapted to this rapid sampling protocol to avoid interference with GCxGC-TOF-MS analysis. To analyse batches of samples a new software tool, MetMax, was implemented which extracts the isotopomer matrix from stable isotope labelling experiments together with the first and second retention index (RI1 and RI2). To exploit RI1 and RI2 for metabolite identification we used the Golm metabolome database (GMD [1] with RI1/RI2-reference spectra and new search algorithms. Using those techniques we analysed the dynamics of (13)CO(2) and (13)C-acetate uptake in Chlamydomonas reinhardtii cells in two different steady states namely photoautotroph and mixotroph growth conditions.

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

    DOEpatents

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

    2004-03-30

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

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

    PubMed

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

    2010-01-01

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

  12. /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.

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

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash; Lorey, Daniel R., II

    2007-02-01

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

  14. Linear ion trap MS(n) of enzymatically synthesized 13C-labeled fructans revealing differentiating fragmentation patterns of β (1-2) and β (1-6) fructans and providing a tool for oligosaccharide identification in complex mixtures.

    PubMed

    Harrison, Scott; Xue, Hong; Lane, Geoff; Villas-Boas, Silas; Rasmussen, Susanne

    2012-02-07

    Fructans are polymeric carbohydrates, which play important roles as plant reserve carbohydrates and stress protectants, and are beneficial for human health and animal production. Fructans are formed by the addition of β-d-fructofuranosyl units to sucrose, leading to very complex mixtures of 1-kestose based inulins, 6-kestose linked levans, and 6G-kestose derived neoseries inulins and levans in cool season grasses such as Lolium perenne. The identification of isomeric fructan oligomers in chromatographic analysis of crude plant extracts is often hampered by the lack of authentic standards, and unambiguous peak assignment usually requires time-consuming analyses of purified fructan oligomers. We have developed a LC-MS(n) method for the separation and detection of fructan isomers and present here evidence for specific MS(n) fragmentation patterns associated with β 1-2 (inulins) and β 2-6 (levans) fructans. LC-MS(n) analysis of (13)C labeled fructan oligomers produced by L. perenne fructosyltransferases expressed in yeast has enabled us to account for the observed fragmentation patterns in terms of preferential cleavage of the glycosidic bond between O- and fructose C2 in both inulins and levans and to differentiate reducing-end from nonreducing end cross ring cleavages in levans. We propose that higher order MS fragmentation patterns can be used to distinguish between the two major classes of fructan, i.e., inulins and levans, without the need for authentic standards.

  15. Uniform {sup 15}N- and {sup 15}N/{sup 13}C-labeling of proteins in mammalian cells and solution structure of the amino terminal fragment of u-PA

    SciTech Connect

    Hansen, A.P.; Petros, A.M.; Meadows, R.P.; Mazar, A.P.; Nettesheim, D.G.; Pederson, T.M.; Fesik, S.W.

    1994-12-01

    Urokinase-type plasminogen activator (u-PA) is a 54-kDa glycoprotein that catalyzes the conversion of plasminogen to plasmin, a broad-specificity protease responsible for the degradation of fibrin clots and extracellular matrix components. The u-PA protein consists of three individual modules: a growth factor domain (GFD), a kringle, and a serine protease domain. The amino terminal fragment (ATF) includes the GFD-responsible for u-PA binding to its receptor-and the kringle domains. This protein was expressed and uniformly {sup 15}N-and {sup 15}N/{sup 13}C-labeled in mammalian cells by methods that will be described. In addition, we present the three-dimensional structure of ATF that was derived from 1299 NOE-derived distance restraints along with the {phi} angle and hydrogen bonding restraints. Although the individual domains in the structures were highly converged, the two domains are structurally independent. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor domain of u-PA and other homologous proteins were observed in the region that has been implicated in binding the urokinase receptor. These results may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.

  16. 13C Tracking after 13CO2 Supply Revealed Diurnal Patterns of Wood Formation in Aspen1

    PubMed Central

    Mahboubi, Amir; Linden, Pernilla; Moritz, Thomas

    2015-01-01

    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 13CO2 labeling system to study carbon transport and incorporation to developing wood of hybrid aspen (Populus tremula × tremuloides). Tracking of 13C 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 13C incorporation to lignin and cell wall carbohydrates. No 13C 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 13C labeling method for the analysis of wood formation and secondary growth in trees. PMID:25931520

  17. INCORPORATION OF LABELED NITRIC OXIDE INTO RESPIRATORY TRACT LINING FLUIDS AND BLOOD PLASMA DURING LUNG INFLAMMATION

    EPA Science Inventory

    Incorporation of labeled nitric oxide (N18O) into respiratory tract lining fluids and blood plasma during lung inflammation. Slade, R., Norwood, J., Crissman, K., McKee, J., Hatch, G. PTB, ETD, NHEERL, ORD, USEPA, Res. Tri. Pk., NC

    Our earlier studies have demonstrated t...

  18. 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.

  19. Photoaffinity labeling of Ras converting enzyme using peptide substrates that incorporate benzoylphenylalanine (Bpa) residues: improved labeling and structural implications.

    PubMed

    Kyro, Kelly; Manandhar, Surya P; Mullen, Daniel; Schmidt, Walter K; Distefano, Mark D

    2011-12-15

    Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide analogues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photoaffinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near the Rce1p active site. By incorporating the photoactive group via p-benzoyl-l-phenylalanine (Bpa) residues directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that residues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor design.

  20. Photoaffinity Labeling of Ras Converting Enzyme using Peptide Substrates that Incorporate Benzoylphenylalanine (Bpa) Residues: Improved Labeling and Structural Implications

    PubMed Central

    Kyro, Kelly; Manandhar, Surya P.; Mullen, Daniel; Schmidt, Walter K.; Distefano, Mark D.

    2012-01-01

    Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide analogues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photoaffinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near the Rce1p active site. By incorporating the photoactive group via p-benzoyl-L-phenylalanine (Bpa) residues directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that residues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor design. PMID:22079863

  1. 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.

  2. Targeted 13C enrichment of lipid and protein pools in the body reveals circadian changes in oxidative fuel mixture during prolonged fasting: a case study using Japanese quail.

    PubMed

    McCue, Marshall D; Amaya, James A; Yang, Alice S; Erhardt, Erik B; Wolf, Blair O; Hanson, David T

    2013-12-01

    Many animals undergo extended periods of fasting. During these fasts, animals oxidize a ratio of macronutrients dependent on the nutritional, energetic, and hydric requirements of the fasting period. In this study, we use Japanese quail (Coturnix coturnix japonica), a bird with natural intermediate fasting periods, to examine macronutrient use during a 6d fast. We raised groups of quail on isotopically labeled materials ((13)C-1-leucine, (13)C-U-glucose, or (13)C-1-palmitic acid) with the intent of labeling specific macronutrient/tissue pools in each treatment, and then traced their use as fuels by measuring the δ(13)C values of breath CO2. Based on changes in δ(13)C values during the fast, it appears that the carbohydrate label,(13)C-U-glucose, was largely incorporated into the lipid pool and thus breath samples ultimately reflected lipid use rather than carbohydrate use. In the lipid treatment, the (13)C-1-palmitic acid faithfully labeled the lipid pool and was reflected in the kinetics δ(13)C values in breath CO2 during the fast. Endogenous lipid oxidation peaked after 24h of fasting and remained constantly elevated thereafter. The protein label,(13)C-1-leucine, showed clear diurnal periods of protein sparing and degradation, with maximal rates of protein oxidation occurring at night and the lowest rates occurring during the day time. This stable isotope tracer method provides a noninvasive approach to study the nutrient dynamics of fasting animals and should provide new insights into how different types of animals use specific nutrient pools during fasting and possibly other non-steady physiological states.

  3. A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the [2-13C]Glucose labeling scheme

    NASA Astrophysics Data System (ADS)

    Lv, Guohua; Faßhuber, Hannes Klaus; Loquet, Antoine; Demers, Jean-Philippe; Vijayan, Vinesh; Giller, Karin; Becker, Stefan; Lange, Adam

    2013-03-01

    The unambiguous stereospecific assignment of the prochiral methyl groups in Val and Leu plays an important role in the structural investigation of proteins by NMR. Here, we present a straightforward method for their stereospecific solid-state NMR assignment based on [2-13C]Glucose ([2-13C]Glc) as the sole carbon source during protein expression. The approach is fundamentally based on the stereo-selective biosynthetic pathway of Val and Leu, and the co-presence of [2-13C]pyruvate produced mainly by glycolysis and [3-13C]/[1,3-13C]pyruvate most probably formed through scrambling in the pentose phosphate pathway. As a consequence, the isotope spin pairs 13Cβ-13Cγ2 and 13Cα-13Cγ1 in Val, and 13Cγ-13Cδ2 and 13Cβ-13Cδ1 in Leu are obtained. The approach is successfully demonstrated with the stereospecific assignment of the methyl groups of Val and Leu of type 3 secretion system PrgI needles and microcrystalline ubiquitin.

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

    PubMed

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

    2007-08-15

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

  5. 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.

  6. Palmitic acid-labeled lipids selectively incorporated into platelet cytoskeleton during aggregation

    SciTech Connect

    Packham, M.A.; Guccione, M.A.; Bryant, N.L.; Livne, A. )

    1990-07-01

    Previous experiments showed that during the early stages (20-30 seconds) of aggregation induced by adenosine diphosphate (ADP, 2 microM) or thrombin (0.1 U/mL) of rabbit or human platelets prelabeled with (3H)palmitic acid, labeled lipid became associated with the cytoskeleton isolated after lysis with 1% Triton X-100, 5 mM EGTA (ethylene glycol-bis-(beta-aminoethyl ether))-N,N,N',N'-tetra-acetic acid. The association appeared to be related to the number of sites of contact and was independent of the release of granule contents. We have now investigated the nature of the labeled lipids by thin-layer and column chromatography and found differences between the distribution of the label in intact platelets (both stimulated and unstimulated) and the isolated cytoskeletons. In both species, and with either ADP or thrombin as aggregating agent, 70-85% of the label in both intact platelets and in the cytoskeletons was in phospholipids. The distribution of label among the phospholipids in the cytoskeletons was similar to that in intact platelets except that the percentage of label in phosphatidylcholine was significantly higher in the cytoskeletons of human platelets than in the intact platelets, and the percentage of label in phosphatidylserine/phosphatidylinositol was significantly lower in the cytoskeletons of rabbit platelets and thrombin-aggregated human platelets than in intact platelets. The cytoskeletons contained a lower percentage of label in triacylglycerol, diacylglycerol, and cholesterol ester than the intact platelets. Contrary to a report in the literature, we found no evidence for the incorporation of diacylglycerol and palmitic acid into the cytoskeleton.

  7. Macrolide biosynthesis. 7. Incorporation of polyketide chain elongation intermediates into methymycin

    SciTech Connect

    Cane, D.E.; Lambalot, R.H.; Prabhakaran, P.C.; Ott, W.R. )

    1993-01-27

    Administration of [1-[sup 13]C]propionate to cultures of Streptomyces venezuelae SC 2366 gave methymycin (1), which was shown by [sup 13]C NMR analysis to be labeled at the predicted sites, C-1, C-3, C-5, C-9, and C-11. Similarly, incorporation of [1,2-[sup 13]C]acetate gave methymycin labeled at C-7 and C-8. A series of presumptive intermediates of polyketide chain elongation was also successfully incorporated. Thus, feeding of (2S,3R)-[2,3-[sup 13]C[sub 2

  8. Glucogenesis in an insect, Manduca sexta L., estimated from the 13C isotopomer distribution in trehalose synthesized from [1,3-13C2]glycerol.

    PubMed

    Thompson, S N

    1997-07-19

    Glucogenesis from [3-13C]alanine and [1,3-13C2]glycerol was demonstrated in the insect Manduca sexta by examining the 13C enrichment of trehalose, a non-reducing disaccharide of glucose synthesized in the insect fat body and released into the blood or hemolymph. In insects maintained on a low carbohydrate diet, trehalose synthesized from [3-13C]alanine was selectively enriched at C1 and C6, and C2 and C5. The 13C-labelling pattern indicated the carboxylation of [3-13C]pyruvate, formed by transamination of the [3-13C]alanine followed by randomization of the label at the fumarate step of the tricarboxylic acid cycle and glucose synthesis via the gluconeogenic pathway. 13C enrichment of trehalose was absent in similarly maintained insect larvae administered 3-mercaptopicolinic acid, an inhibitor of hepatic phosphoenolpyruvate carboxykinase. Insects on the low carbohydrate diet also synthesized trehalose from [1,3-13C2]glycerol. 13C multiplets were observed in trehalose C3 and C4 demonstrating the synthesis of three 13C enriched glucose isotopomers from the 13C-labelled glycerol. The relative contributions of 13C-labelled glycerol and unlabelled 3 carbon substrates to the synthesis of the 13C enriched trehalose isotopomers were determined from the multiplet structure at C3, and calculation of minimal rates of glucogenesis were based on the 13C enrichment of C4. The C4/C3 13C enrichment ratio in trehalose synthesized from [1,3-13C2]glycerol was close to unity, and total glucogenesis was calculated after estimation of the expected contribution of unlabelled trehalose synthesis from 3 carbon substrates by comparison of the ratio of unlabelled and labelled contributions to the 13C enriched trehalose isotopomers with the 13C enrichment of [1,3-13C2]glycerol-3-phosphate. The estimated total rates of glucogenesis varied from 0.33 to 2.80 micromol glucose/g fresh weight/h. The blood sugar level of M. sexta was also highly variable. Although the potential importance of

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

    PubMed

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

    2003-07-02

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

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

    PubMed

    Young, Jamey D

    2014-12-01

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

  11. Variations in labeling protocol influence incorporation, distribution and retention of iron oxide nanoparticles into human umbilical vein endothelial cells.

    PubMed

    van Tiel, Sandra T; Wielopolski, Piotr A; Houston, Gavin C; Krestin, Gabriel P; Bernsen, Monique R

    2010-01-01

    Various studies have shown that various cell types can be labeled with iron oxide particles and visualized by magnetic resonance imaging (MRI). However, reported protocols for cell labeling show a large variation in terms of labeling dose and incubation time. It is therefore not clear how different labeling protocols may influence labeling efficiency. Systematic assessment of the effects of various labeling protocols on labeling efficiency of human umbilical vein endothelial cells (HUVEC) using two different types of iron oxide nanoparticles, i.e. super paramagnetic iron oxide particles (SPIOs) and microparticles of iron oxide (MPIOs), demonstrated that probe concentration, incubation time and particle characteristics all influence the efficiency of label incorporation, label distribution, label retention and cell behavior. For SPIO the optimal labeling protocol consisted of a dose of 12.5 µg iron/2 ml/9.5 cm(2) and an incubation time of 24 h, resulting in an average iron load of 12.0 pg iron/per cell (uptake efficiency of 9.6%). At 4 h many SPIOs are seen sticking to the outside of the cell instead of being taken up by the cell. For MPIO optimal labeling was obtained with a dose of 50 µg iron/2 ml/9.5 cm(2). Incubation time was of less importance since most of the particles were already incorporated within 4 h with a 100% labeling efficiency, resulting in an intracellular iron load of 626 pg/cell. MPIO were taken up more efficiently than SPIO and were also better tolerated. HUVEC could be exposed to and contain higher amounts of iron without causing significant cell death, even though MPIO had a much more pronounced effect on cell appearance. Using optimal labeling conditions as found for HUVEC on other cell lines, we observed that different cell types react differently to identical labeling conditions. Consequently, for each cell type separately an optimal protocol has to be established.

  12. Dynamics of PAHs and derived organic compounds in a soil-plant mesocosm spiked with (13)C-phenanthrene.

    PubMed

    Cennerazzo, Johanne; de Junet, Alexis; Audinot, Jean-Nicolas; Leyval, Corinne

    2017-02-01

    Polycyclic Aromatic Hydrocarbons (PAHs) are ubiquitous and persistent soil pollutants. Their fate and the influence of the plant rhizosphere on their dynamics has been extensively studied, but studies mainly focused on their dissipation rate. We conducted a plant-soil mesocosm experiment to study the fate and distribution of PAHs or derived compounds in the extractable fraction, the residual soil, the shoot biomass and the root biomass. The experiment was conducted for 21 days using ryegrass and a forest soil spiked with (13)C-labeled phenanthrene (PHE), using combined IRMS and NanoSIMS for analyses. Almost 90% of the initial extractable PHE content was dissipated within 3 weeks, but no rhizospheric effect was highlighted on PHE dissipation. More than 40% of (13)C-PHE was still in the soil at the end of the experiment, but not as PHE or PAH-derived compounds. Therefore it was under the form of new compounds (metabolites) and/or had been incorporated into the microbial biomass. About 0.36% of the initial (13)C-PHE was recovered in the root and shoot tissues, representing similar (13)C enrichment (E(13)C) as in the soil (E(13)C ≈ 0.04 at.%). Using NanoSIMS, (13)C was also localized at the microscale in the roots and their close environment. Global (13)C enrichment confirmed the results obtained by IRMS. Some hotspots of (13)C enrichment were found, with a high (32)S/(12)C(14)N ratio. Comparing the ratios, sizes and shapes of these hotspots suggested that they could be bacteria.

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

    results suggest that soil microorganisms primarily consume substrates that exhibit constant δ13C values throughout the soil profile, like root litter or dissolved organic carbon from litter leachates or root exudates that percolates through the soil column. δ13C values of PLFA produced by both fungi and bacteria, in contrast to the group specific PLFA, strongly increased with depth and were tightly correlated to F:B ratios (R2 > 0.84), which decreased with depth. Because group-specific PLFA did not exhibit increased δ13C with depth, the increase observed in the general biomarker δ13C values, associated with the aggregated microbial community, was not the consequence of microbial incorporation of more 13C enriched SOC at greater depth. Rather, the increase in community δ13C reflects a shift in community structure towards more 13C enriched bacteria with depth. Our results indicate that, higher δ13C values associated with microbial biomass at a greater depth likely contributes to the increase in δ13CSOC with depth via more 13C enriched contributions from necromass to SOC.

  14. 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.

  15. 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.

  16. A study on the biosynthesis of hygrophorone B(12) in the mushroom Hygrophorus abieticola reveals an unexpected labelling pattern in the cyclopentenone moiety.

    PubMed

    Otto, Alexander; Porzel, Andrea; Schmidt, Jürgen; Wessjohann, Ludger; Arnold, Norbert

    2015-10-01

    The hitherto unknown natural formation of hygrophorones, antibacterial and antifungal cyclopentenone derivatives from mushrooms, was investigated for hygrophorone B(12) in Hygrophorus abieticola Krieglst. ex Gröger & Bresinsky by feeding experiments in the field using (13)C labelled samples of D-glucose and sodium acetate. The incorporation of (13)C isotopes was extensively studied using 1D and 2D NMR spectroscopy as well as ESI-HRMS analyses. In the experiment with [U-(13)C6]-glucose, six different (13)C2 labelled isotopomers were observed in the 2D INADEQUATE spectrum due to incorporation of [1,2-(13)C2]-acetyl-CoA. This labelling pattern demonstrated that hygrophorone B(12) is derived from a fatty acid-polyketide route instead of a 1,4-α-D-glucan derived anhydrofructose pathway. The experiment with [2-(13)C]-acetate revealed an unexpected incorporation pattern in the cyclopentenone functionality of hygrophorone B(12). Four single-labelled isotopomers, in particular [1-(13)C]-, [2-(13)C]-, [3-(13)C]-, and [4-(13)C]-hygrophorone B(12), were detected that showed only half enrichment in comparison to the respective labelled alkyl side chain carbons. This labelling pattern indicates the formation of a symmetrical intermediate during hygrophorone B(12) biosynthesis. Based on these observations, a biogenetic route via a 4-oxo fatty acid and a chrysotrione B homologue is discussed.

  17. 13C nuclear magnetic resonance data of lanosterol derivatives—Profiling the steric topology of the steroid skeleton via substituent effects on its 13C NMR

    NASA Astrophysics Data System (ADS)

    Dias, Jerry Ray; Gao, Hongwu

    2009-12-01

    The 13C NMR spectra of over 24 tetracyclic triterpenoid derivatives have been structurally analyzed. The 13C NMR chemical shifts allow one to probe the steric topology of the rigid steroid skeleton and inductive effects of its substituents. Use of deuterium labeling in chemical shift assignment and B-ring aromatic terpenoids are also featured.

  18. Measurement of δ13C values of soil amino acids by GC-C-IRMS using trimethylsilylation: a critical assessment.

    PubMed

    Rubino, Mauro; Milin, Sylvie; D'Onofrio, Antonio; Signoret, Patrick; Hatté, Christine; Balesdent, Jérôme

    2014-01-01

    In this study, we evaluated trimethylsilyl (TMS) derivatives as derivatization reagents for the compound-specific stable carbon isotope analysis of soil amino acids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). We used non-proteinogenic amino acids to show that the extraction-derivatization-analysis procedure provides a reliable method to measure δ(13)C values of amino acids extracted from soil. However, we found a number of drawbacks that significantly increase the final total uncertainty. These include the following: production of multiple peaks for each amino acid, identified as di-, tri- and tetra-TMS derivatives; a number of TMS-carbon (TMS-C) atoms added lower than the stoichiometric one, possibly due to incomplete combustion; different TMS-C δ(13)C for di-, tri- and tetra-TMS derivatives. For soil samples, only four amino acids (leucine, valine, threonine and serine) provide reliable δ(13)C values with a total average uncertainty of 1.3 ‰. We conclude that trimethylsilyl derivatives are only suitable for determining the (13)C incorporation in amino acids within experiments using (13)C-labelled tracers but cannot be applied for amino acids with natural carbon isotope abundance until the drawbacks described here are overcome and the measured total uncertainty significantly decreased.

  19. The First in Vivo Observation of 13C- 15N Coupling in Mammalian Brain

    NASA Astrophysics Data System (ADS)

    Kanamori, Keiko; Ross, Brian D.

    2001-12-01

    [5-13C,15N]Glutamine, with 1J(13C-15N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by 13C MRS at 4.7 T. The brain [5-13C]glutamine peak consisted of the doublet from [5-13C,15N]glutamine and the center [5-13C,14N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-13C,15N]glutamine was monitored in vivo with a time resolution of 20-35 min. This [5-13C,15N]glutamine was formed by glial uptake of released neurotransmitter [5-13C]glutamate and its reaction with 15NH3 catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively13C-enriched by intravenous [2,5-13C]glucose infusion to 13C-label whole-brain glutamate C5, followed by [12C]glucose infusion to chase 13C from the small and rapidly turning-over glial glutamate pool, leaving 13C mainly in the neurotransmitter [5-13C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-13C,15N]glutamine arises from a coupling between 13C of neuronal origin and 15N of glial origin. Measurement of the rate of brain [5-13C,15N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    1988-01-01

    High-resolution 13C n.m.r. spectroscopy has been used to examine propionate metabolism in the perfused rat heart. A number of tricarboxylic acid (TCA) cycle intermediates are observable by 13C n.m.r. in hearts perfused with mixtures of pyruvate and propionate. When the enriched 13C-labelled nucleus originates with pyruvate, the resonances of the intermediates appear as multiplets due to formation of multiply-enriched 13C-labelled isotopomers, whereas when the 13C-labelled nucleus originates with propionate, these same intermediates appear as singlets in the 13C spectrum since entry of propionate into the TCA cycle occurs via succinyl-CoA. An analysis of the isotopomer populations in hearts perfused with [3-13C]pyruvate plus unlabelled propionate indicates that about 27% of the total pyruvate pool available to the heart is derived directly from unlabelled propionate. This was substantiated by perfusing a heart for 2 h with [3-13C]propionate as the only available exogenous substrate. Under these conditions, all of the propionate consumed by the heart, as measured by conventional chemical analysis, ultimately entered the oxidative pathway as [2-13C] or [3-13C]pyruvate. This is consistent with entry of propionate into the TCA cycle intermediate pools as succinyl-CoA and concomitant disposal of malate to pyruvate via the malic enzyme. 13C resonances arising from enriched methylmalonate and propionylcarnitine are also detected in hearts perfused with [3-13C] or [1-13C]propionate which suggests that 13C n.m.r. may be useful as a non-invasive probe in vivo of metabolic abnormalities involving the propionate pathway, such as methylmalonic aciduria or propionic acidaemia. PMID:3178775

  2. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

    PubMed Central

    Evans, Eric G.B.; Millhauser, Glenn L.

    2016-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain “K1,” and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron–electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite

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

    PubMed

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

    2015-11-01

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

  4. Local deposition of 13C tracer in the JET MKII-HD divertor

    NASA Astrophysics Data System (ADS)

    Likonen, Jari; Airila, M. I.; Coad, J. P.; Hakola, A.; Koivuranta, S.; Ahonen, E.; Alves, E.; Barradas, N.; Widdowson, A.; Rubel, M.; Brezinsek, S.; Groth, M.; JET-EFDA Contributors

    2013-07-01

    Migration and deposition of 13C have been investigated at JET by injecting 13C-labelled methane at the outer divertor base at the end of the 2009 campaign. The 13C deposition profile was measured with enhanced proton scattering (EPS) and secondary ion mass spectrometry (SIMS) techniques. A strong toroidal deposition band for 13C was observed experimentally on each of the analysed four outer divertor floor tiles. In addition, 13C was also found on the vertical edge of load bearing tile (LBT) and at the bottom of the LBT tile facing the puffing hole. Local 13C migration in the vicinity of the injection location was modelled by the ERO code. The ERO simulations also produced the strong toroidal 13C deposition band but there is strong deposition also on the vertical edge of the LBT tile and elsewhere on the horizontal part of the outer divertor floor tile.

  5. 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%.

  6. Metallic nanocrystallites-incorporated ordered mesoporous carbon as labels for a sensitive simultaneous multianalyte electrochemical immunoassay.

    PubMed

    Fang, Yishan; Huang, Xinjian; Zeng, Qiang; Wang, Lishi

    2015-11-15

    This work reports on a facile, novel multianalyte electrochemical immunoassay for simultaneous detection of a-fetoprotein (AFP) and human epidermal growth factor receptor type-2 (HER-2) using metal-containing nanomaterials confined in the ordered mesoporous carbon matrix (OMC-M) as labels. Well-dispersed uniform metallic nanocrystallites incorporated OMC materials were fabricated through a simple, economical, and green preparative strategy toward phenolic resol as a carbon source and metal nitrate as metal sources. The large amount of metallic nanocrystallites loading on the OMC nanomaterials, greatly amplified the detection signals, and the good biocompatibility of carbon nanotubes-chitosan retained excellent stability for the sandwich-type immunoassay. Under optimal experimental conditions, the proposed immunoassay exhibited high sensitivity and selectivity for the detection of analytes, providing a better linear response range from 0.001 to 150 ng/mL for AFP and for HER-2, with a lower limit of detectionof 0.6p g/mL and 0.35 pg/mL (S/N=3), respectively. The immunosensor exhibited convenience, low cost, rapidity, good specificity, acceptable stability and reproducibility. Moreover, satisfactory results were obtained for the determination of AFP and HER-2 in real human serum samples, indicating that the developed immunoassay has the potential to find application in clinical detection of AFP and HER-2 and other tumor markers as an alternative approach.

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

    PubMed

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

    2016-04-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2013-08-15

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

  10. NMR 13C-isotopic enrichment experiments to study carbon-partitioning into organic solutes in the red alga Grateloupia doryphora.

    PubMed

    Simon-Colin, Christelle; Kervarec, Nelly; Pichon, Roger; Deslandes, Eric

    2004-01-01

    The red alga Grateloupia doryphora Montagne (Howe) (Cryptonemiales, Halymeniaceae) was used as a model to investigate the effects of changes in seawater salinity on the intracellular low-molecular-weight organic compounds. Carbon-partitioning into major organic solutes was followed by 13C nuclear magnetic resonance (NMR) spectroscopy on living algae incubated in NaH13CO3-enriched seawater, and by high resolution 1H and 13C NMR experiments performed on 13C-enriched algal extracts. NMR and high performance liquid chromatography (HPLC) analyses both demonstrated that floridoside level was the most affected by changes in salinity: it rose under the hypersaline treatment and decreased under hyposaline one. Moreover, at low salinity, the high labeling of floridoside (45.3% 13C-enrichment for C1) together with its low concentrations both provided evidence of great increase in the de novo biosynthesis and turnover rate. Our experiments also demonstrated a high incorporation of photosynthetic carbon into amino acids, especially glutamate, under hypoosmotic conditions. On the other hand, isethionic acid and N-methyl-methionine sulfoxide were only partly labeled, which indicates they do not directly derive from carbon photoassimilation. In algae exposed to high salinity, elevated concentrations of floridoside coupled to a low labeling (9.4%) were observed. These results suggest that hyperosmotic conditions stimulated floridoside biosynthesis from endogen storage products rather than from carbon assimilation through photosynthesis.

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

    PubMed

    Wan, Qianfen; Wang, Yulan; Tang, Huiru

    2017-02-21

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

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

    PubMed

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

    2015-03-01

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

  13. NOTE The effect of 13C enrichment in the glassing matrix on dynamic nuclear polarization of [1-13C]pyruvate

    NASA Astrophysics Data System (ADS)

    Lumata, Lloyd; Kovacs, Zoltan; Malloy, Craig; Sherry, A. Dean; Merritt, Matthew

    2011-03-01

    Dimethyl sulfoxide (DMSO) can effectively form a glassy matrix necessary for dynamic nuclear polarization (DNP) experiments. We tested the effects of 13C enrichment in DMSO on DNP of [1-13C]pyruvate doped with trityl radical OX063Me. We found that the polarization build-up time τ of pyruvate in 13C-labeled DMSO glassing solution is twice as fast as the unenriched DMSO while the nuclear magnetic resonance enhancement was unchanged. This indicates that 13C-13C spin diffusion is a limiting factor in the kinetics of DNP in this system, but it has a minimal effect on the absolute value of polarization achievable for the target.

  14. The effect of 13C enrichment in the glassing matrix on dynamic nuclear polarization of [1-13C]pyruvate

    NASA Astrophysics Data System (ADS)

    Lumata, Lloyd; Kovacs, Zoltan; Malloy, Craig; Sherry, A. Dean; Merritt, Matthew

    2011-03-01

    Dimethyl sulfoxide (DMSO) can effectively form a glassy matrix necessary for dynamic nuclear polarization (DNP) experiments. We tested the effects of 13C enrichment in DMSO on DNP of [1-13C]pyruvate doped with trityl radical OX063Me. We found that the polarization build-up time τ of pyruvate in 13C-labeled DMSO glassing solution is twice as fast as the unenriched DMSO while the nuclear magnetic resonance enhancement was unchanged. This indicates that 13C-13C spin diffusion is a limiting factor in the kinetics of DNP in this system, but it has a minimal effect on the absolute value of polarization achievable for the target.

  15. 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.

  16. 1H–13C hetero-nuclear dipole–dipole couplings of methyl groups in stationary and magic angle spinning solid-state NMR experiments of peptides and proteins

    PubMed Central

    Wu, Chin H.; Das, Bibhuti B.; Opella, Stanley J.

    2010-01-01

    13C NMR of isotopically labeled methyl groups has the potential to combine spectroscopic simplicity with ease of labeling for protein NMR studies. However, in most high resolution separated local field experiments, such as polarization inversion spin exchange at the magic angle (PISEMA), that are used to measure 1H–13C hetero-nuclear dipolar couplings, the four-spin system of the methyl group presents complications. In this study, the properties of the 1H–13C hetero-nuclear dipolar interactions of 13C-labeled methyl groups are revealed through solid-state NMR experiments on a range of samples, including single crystals, stationary powders, and magic angle spinning of powders, of 13C3 labeled alanine alone and incorporated into a protein. The spectral simplifications resulting from proton detected local field (PDLF) experiments are shown to enhance resolution and simplify the interpretation of results on single crystals, magnetically aligned samples, and powders. The complementarity of stationary sample and magic angle spinning (MAS) measurements of dipolar couplings is demonstrated by applying polarization inversion spin exchange at the magic angle and magic angle spinning (PISEMAMAS) to unoriented samples. PMID:19896874

  17. 13C NMR of tunnelling methyl groups

    NASA Astrophysics Data System (ADS)

    Detken, A.

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

  18. 13C Tracer Studies of Metabolism in Mouse Tumor Xenografts

    PubMed Central

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

    2015-01-01

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

  19. Synthesis of [1-.sup.13C]pyruvic acid], [2-.sup.13C]pyruvic acid], [3-.sup.13C]pyruvic acid] and combinations thereof

    DOEpatents

    Martinez, Rodolfo A. , Unkefer; Clifford J. , Alvarez; Marc, A [Santa Fe, NM

    2012-06-12

    The present invention is directed to the labeled compounds, ##STR00001## wherein C* is each either .sup.13C and .sup.12C where at least one C* is .sup.13C, each hydrogen of the methylene group is hydrogen or deuterium, the methyl group includes either zero or three deuterium atoms, Q is sulfide, sulfinyl, or sulfone, Z is an aryl group such as 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, or a phenyl group ##STR00002## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each independently either hydrogen, a C.sub.1-C.sub.4 lower alkyl, a halogen, and an amino group such as NH.sub.2, NHR and NRR' where R and R' are each independently either a C.sub.1-C.sub.4 lower alkyl, a phenyl, and an alkoxy group, and the methyl group can include either zero or three deuterium atoms. The present invention is also directed to the labeled compounds ##STR00003##

  20. Incorporation of probabilistic seismic phase labels into a Bayesian multiple-event seismic locator

    SciTech Connect

    Myers, S; Johannesson, G; Hanley, W

    2008-01-17

    We add probabilistic phase labels to the multiple-event joint probability function of Myers et al., 2007 that formerly included event locations, travel-time corrections, and arrival-time measurement precision. Prior information on any of the multiple-event parameters may be used. The phase-label model includes a null label that captures phases not belonging to the collection of phases under consideration. Using the Markov-Chain Monte Carlo method, samples are drawn from the multiple-event joint probability function to infer the posteriori distribution that is consistent with priors and the arrival-time data set. Using this approach phase-label error can be accessed and phase-label error is propagated to all other multiple-event parameters. We test the method using a ground-truth data set of nuclear explosions at the Nevada Test Site. We find that posteriori phase labels agree with the meticulously analyzed data set in more than 97% of instances and the results are robust even when the input phase-label information is discarded. Only when a large percentage of the arrival-time data are corrupted does prior phase label information improve resolution of multiple-event parameters. Simultaneous modeling of the entire multiple-event system results in accurate posteriori probability regions for each multiple-event parameter.

  1. 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

  2. Imaging pH with hyperpolarized 13C.

    PubMed

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

    2011-10-01

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

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

    PubMed

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

    2013-11-01

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

  4. In vitro incorporation of label from (. gamma. /sup 32/P) ATP into isocitrate lyase of Escherichia coli

    SciTech Connect

    Robertson, F.; Reeves, H.C.

    1986-05-01

    A partially purified sonic extract of an E. coli mutant, constitutive for the glyoxylate by-pass enzymes, was incubated with (..gamma../sup 32/P) ATP in 50 mM MOPS buffer at pH 7.5 containing 2 mM 2-mercaptoethanol, 10 mM MgCl/sub 2/ and 10% glycerol at room temperature for 1 h. Incubation was continued for another hour following the addition of unlabeled ATP. The assay was terminated by the addition of 10 mM EDTA. The assay mixture was then analyzed, by several electrophoretic techniques and subsequent autoradiography, to determine which of the proteins in the extract had incorporated label. Isoelectric focusing was performed at pH 3-10 and pH 4-4.5. Specific enzyme staining of IEF gels revealed that active isocitrate lyase (ICL) co-migrated with a protein band which also was radioactive. Immuno blots of these IEF gels, using antibody raised in rabbits against ICL, also demonstrated incorporation of label. SDS-PAGE autoradiograms displayed a labeled protein which co-migrated with purified E. coli ICL, which has a subunit M/sub r/ of 48,000. The mixture was also analyzed by 2-D PAGE which further demonstrated that a labeled protein and authentic purified ICl co-migrate.

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

    PubMed

    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 (13)C, 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) (13)C. 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 (13)C-based approaches. For samples at natural abundance, we have developed a workflow to obtain (13)C-(13)C and (13)C-(1)H statistical correlations using 1D (13)C and (1)H NMR spectra. For samples that can be isotopically labeled, we describe another NMR approach to obtain direct (13)C-(13)C 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 (13)C 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.

  6. Bacteria and fungi respond differently to multifactorial climate change in a temperate heathland, traced with 13C-glycine and FACE CO2.

    PubMed

    Andresen, Louise C; Dungait, Jennifer A J; Bol, Roland; Selsted, Merete B; Ambus, Per; Michelsen, Anders

    2014-01-01

    It is vital to understand responses of soil microorganisms to predicted climate changes, as these directly control soil carbon (C) dynamics. The rate of turnover of soil organic carbon is mediated by soil microorganisms whose activity may be affected by climate change. After one year of multifactorial climate change treatments, at an undisturbed temperate heathland, soil microbial community dynamics were investigated by injection of a very small concentration (5.12 µg C g(-1) soil) of (13)C-labeled glycine ((13)C2, 99 atom %) to soils in situ. Plots were treated with elevated temperature (+1°C, T), summer drought (D) and elevated atmospheric carbon dioxide (510 ppm [CO2]), as well as combined treatments (TD, TCO2, DCO2 and TDCO2). The (13)C enrichment of respired CO2 and of phospholipid fatty acids (PLFAs) was determined after 24 h. (13)C-glycine incorporation into the biomarker PLFAs for specific microbial groups (Gram positive bacteria, Gram negative bacteria, actinobacteria and fungi) was quantified using gas chromatography-combustion-stable isotope ratio mass spectrometry (GC-C-IRMS). Gram positive bacteria opportunistically utilized the freshly added glycine substrate, i.e. incorporated (13)C in all treatments, whereas fungi had minor or no glycine derived (13)C-enrichment, hence slowly reacting to a new substrate. The effects of elevated CO2 did suggest increased direct incorporation of glycine in microbial biomass, in particular in G(+) bacteria, in an ecosystem subjected to elevated CO2. Warming decreased the concentration of PLFAs in general. The FACE CO2 was (13)C-depleted (δ(13)C = 12.2‰) compared to ambient (δ(13)C = ∼-8‰), and this enabled observation of the integrated longer term responses of soil microorganisms to the FACE over one year. All together, the bacterial (and not fungal) utilization of glycine indicates substrate preference and resource partitioning in the microbial community, and therefore suggests a diversified response pattern

  7. 13C NMR of Nephila clavipes major ampullate silk gland.

    PubMed

    Hijirida, D H; Do, K G; Michal, C; Wong, S; Zax, D; Jelinski, L W

    1996-12-01

    The major ampullate glands of the spider Nephila clavipes contain approximately 0.2 microliter each of a highly concentrated (approximately 50%) solution of silk fibroin. Therefore, the reservoir of silk in these glands presents an ideal opportunity to observe prefolded conformations of a protein in its native state. To this end, the structure and conformation of major ampullate gland silk fibroin within the glands of the spider N. clavipes were examined by 13C NMR spectroscopy. These results were compared to those from silk protein first drawn from the spinneret and then denatured. The 13C NMR chemical shifts, along with infrared and circular dichroism data, suggest that the silk fibroin in the glands exists in dynamically averaged helical conformations. Furthermore, there is no evidence of proline residues in U-(13)C-D-glucose-labeled silk. This transient prefolded "molten fibril" state may correspond to the silk I form found in Bombyx mori silk. There is no evidence of the final beta-sheet structure in the ampullate gland silk fibroin before final silk processing. However, the conformation of silk in the glands appears to be in a highly metastable state, as plasticization with water produces the beta-sheet structure. Therefore, the ducts connecting the ampullate glands to the spinnerets play a larger role in silk processing than previously thought.

  8. EPR assessment of protein sites for incorporation of Gd(III) MRI contrast labels

    PubMed Central

    Lagerstedt, Jens O.; Petrlova, Jitka; Hilt, Silvia; Marek, Antonin; Chung, Youngran; Sriram, Renuka; Budamagunta, Madhu S.; Desreux, Jean F.; Thonon, David; Jue, Thomas; Smirnov, Alex I.; Voss, John C.

    2013-01-01

    We have engineered Apolipoprotein A-I (apoA-I), a major protein constituent of high-density lipoprotein (HDL), to contain DOTA-chelated Gd(III) as an MRI contrast agent for the purpose of imaging reconstituted HDL (rHDL) biodistribution, metabolism, and regulation in vivo. This protein contrast agent was obtained by reacting the thiol-reactive Gd[MTS-ADO3A] label with Cys engineered at four distinct positions (52, 55, 76 and 80) in apoA-I. MRI of infused mice previously showed that the Gd-labeled apoA-I migrates to both the liver and the kidney, the organs responsible for HDL catabolism; however, the contrast properties of apoA-I are superior when the ADO3A moiety is located at position 55, compared to the protein labeled at positions 52, 76 or 80. It is shown here that continuous wave X-band (9 GHz) EPR spectroscopy is capable of detecting differences in the Gd(III) signal when comparing the labeled protein in the lipid-free to the rHDL state. Furthermore, the values of NMR relaxivity obtained for labeled variants in both the lipid-free and rHDL states correlate to the product of the X-band Gd(III) spectral width and the collision frequency between a nitroxide spin label and a polar relaxation agent. Consistent with its superior relaxivity measured by NMR, the rHDL-associated apoA-I containing the Gd[MTS-ADO3A] probe attached to position 55 displays favorable dynamic and water accessibility properties as determined by X-band EPR. While room temperature EPR requires >1 mM Gd(III)-labeled and only >10 μM nitroxide-labeled protein to resolve the spectrum, the volume requirement is exceptionally low (~5μL). Thus, X-band EPR provides a practical assessment for the suitability of imaging candidates containing the site-directed ADO3A contrast probe. PMID:23606429

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

    PubMed

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

    1990-01-01

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

  10. 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.

  11. Transfer of (13) C between paired Douglas-fir seedlings reveals plant kinship effects and uptake of exudates by ectomycorrhizas.

    PubMed

    Pickles, Brian J; Wilhelm, Roland; Asay, Amanda K; Hahn, Aria S; Simard, Suzanne W; Mohn, William W

    2017-04-01

    Processes governing the fixation, partitioning, and mineralization of carbon in soils are under increasing scrutiny as we develop a more comprehensive understanding of global carbon cycling. Here we examined fixation by Douglas-fir seedlings and transfer to associated ectomycorrhizal fungi, soil microbes, and full-sibling or nonsibling neighbouring seedlings. Stable isotope probing with 99% (13) C-CO2 was applied to trace (13) C-labelled photosynthate throughout plants, fungi, and soil microbes in an experiment designed to assess the effect of relatedness on (13) C transfer between plant pairs. The fixation and transfer of the (13) C label to plant, fungal, and soil microbial tissue was examined in biomass and phospholipid fatty acids. After a 6 d chase period, c. 26.8% of the (13) C remaining in the system was translocated below ground. Enrichment was proportionally greatest in ectomycorrhizal biomass. The presence of mesh barriers (0.5 or 35 μm) between seedlings did not restrict (13) C transfer. Fungi were the primary recipients of (13) C-labelled photosynthate throughout the system, representing 60-70% of total (13) C-enriched phospholipids. Full-sibling pairs exhibited significantly greater (13) C transfer to recipient roots in two of four Douglas-fir families, representing three- and fourfold increases (+ c. 4 μg excess (13) C) compared with nonsibling pairs. The existence of a root/mycorrhizal exudation-hyphal uptake pathway was supported.

  12. 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.

  13. 13C metabolic flux analysis in complex systems.

    PubMed

    Zamboni, Nicola

    2011-02-01

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

  14. Specific Cellular Incorporation of a Pyrene-Labelled Cholesterol: Lipoprotein-Mediated Delivery toward Ordered Intracellular Membranes

    PubMed Central

    Gaibelet, Gérald; Azalbert, Vincent; Bertrand-Michel, Justine; Hamdi, Safouane; Collet, Xavier; Orlowski, Stéphane

    2015-01-01

    In the aim of testing tools for tracing cell trafficking of exogenous cholesterol, two fluorescent derivatives of cholesterol, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), with distinctive chemico-physical characteristics, have been compared for their cell incorporation properties, using two cell models differently handling cholesterol, with two incorporation routes. In the Caco-2 cell model, the cholesterol probes were delivered in bile salt micelles, as a model of intestinal absorption. The two probes displayed contrasting behaviors for cell uptake characteristics, cell staining, and efflux kinetics. In particular, Pyr-met-Chol cell incorporation involved SR-BI, while that of NBD-Chol appeared purely passive. In the PC-3 cell model, which overexpresses lipoprotein receptors, the cholesterol probes were delivered via the serum components, as a model of systemic delivery. We showed that Pyr-met-Chol-labelled purified LDL or HDL were able to specifically deliver Pyr-met-Chol to the PC-3 cells, while NBD-Chol incorporation was independent of lipoproteins. Observations by fluorescence microscopy evidenced that, while NBD-Chol readily stained the cytosolic lipid droplets, Pyr-met-Chol labelling led to the intense staining of intracellular structures of membranous nature, in agreement with the absence of detectable esterification of Pyr-met-Chol. A 48 h incubation of PC-3 cells with either Pyr-met-Chol-labelled LDL or HDL gave same staining patterns, mainly colocalizing with Lamp1, caveolin-1 and CD63. These data indicated convergent trafficking downwards their respective receptors, LDL-R and SR-BI, toward the cholesterol-rich internal membrane compartments, late endosomes and multivesicular bodies. Interestingly, Pyr-met-Chol staining of these structures exhibited a high excimer fluorescence emission, revealing their ordered membrane environment, and indicating that Pyr-met-Chol behaves as a fair cholesterol tracer

  15. 13C MRS studies of neuroenergetics and neurotransmitter cycling in humans.

    PubMed

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

    2011-10-01

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

  16. Rapid aquatic toxicity assay utilizing labeled thymidine incorporation in sea urchin embryos

    SciTech Connect

    Jackim, E.; Nacci, D.

    1984-01-01

    Aquatic toxicity was evaluated in the sea urchin embryo (Arbacea punctulata) by the inhibition of tritiated thymidine incorporation after a brief exposure to toxic chemicals. Arbacia is a useful surrogate species for assay: well-studied, easily cultured and fertile virtually year round. The simplicity and speed of this test system lends itself to screening large numbers of compounds, mixtures or water samples.

  17. 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

  18. Gastrointestinal handling of [1-13C]palmitic acid in healthy controls and patients with cystic fibrosis

    PubMed Central

    Murphy, J.; Jones, A.; Stolinski, M.; Wootton, S.

    1997-01-01

    Accepted 2 January 1997
 AIM—To examine the gastrointestinal handling of [1-13C]palmitic acid given as the free acid by measuring the excretion of 13C label in stool in 16 healthy children and 11 patients with cystic fibrosis on their habitual enzyme replacement treatment.
METHODS—After an overnight fast, each child ingested 10 mg/kg body weight [1-13C]palmitic acid with a standardised test meal of low natural 13C abundance. A stool sample was collected before the test and all stools were collected thereafter for a period of up to five days. The total enrichment of 13C in stool and the species bearing the 13C label was measured using isotope ratio mass spectrometry.
RESULTS—The proportion of administered 13C label excreted in stool was 24.0% (range 10.7-64.9%) in healthy children and only 4.4% (range 1.2-11.6%) in cystic fibrosis patients. The enrichment of 13C in stool was primarily restricted to the species consumed by the subjects (that is as palmitic acid).
CONCLUSION—There does not appear to be a specific defect in the absorption of [1-13C]palmitic acid in patients with cystic fibrosis. The reasons why cystic fibrosis patients appear to absorb more of this saturated fatty acid than healthy children is not clear and requires further investigation.

 PMID:9196358

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

    PubMed

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

    2011-02-01

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

  20. [Determination of 13C enrichment in soil amino acid enantiomers by gas chromatogram/mass spectrometry].

    PubMed

    He, Hong-Bo; Zhang, Wei; Ding, Xue-Li; Bai, Zhen; Liu, Ning; Zhang, Xu-Dong

    2008-06-01

    The transformation and renewal of amino acid enantiomers is of significance in indicating the turnover mechanism of soil organic matter. In this paper, a method of gas chromatogram/mass spectrometry combined with U-13 C-glucose incubation was developed to determine the 13C enrichment in soil amino acid enantiomers, which could effectively differentiate the original and the newly synthesized amino acids in soil matrix. The added U-13 C-glucose was utilized rapidly to structure the amino acid carbon skeleton, and the change of relative abundance of isotope ions could be determined by mass spectrometry. The direct incorporation of U-13 C glucose was estimated by the intensity increase of m/z (F + n) to F (F was parent fragment, and n was the carbon number in the fragment), while the total isotope incorporation from the added 13C could be calculated according to the abundance ratio increment summation from m/z (Fa + 1) through (Fa + T) (Fa was the fragment containing all original skeleton carbons, and T was the carbon number in the amino acid molecule). The 13C enrichment in the target compound was expressed as atom percentage excess (APE), and that of D-amino acid needed to be corrected by the coefficient of hydrolysis-induced racemization. The 13C enrichment reflected the carbon turnover velocity of individual amino acid enantiomers, and was powerful to investigate the dynamics of soil amino acids.

  1. Optoacoustic 13C-breath test analyzer

    NASA Astrophysics Data System (ADS)

    Harde, Hermann; Helmrich, Günther; Wolff, Marcus

    2010-02-01

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

  2. 13C Metabolomics: NMR and IROA for Unknown Identification

    PubMed Central

    Clendinen, Chaevien S.; Stupp, Gregory S.; Wang, Bing; Garrett, Timothy J.; Edison, Arthur S.

    2016-01-01

    Abstract: Background Isotopic Ratio Outlier Analysis (IROA) is an untargeted metabolomics method that uses stable isotopic labeling and LC-HRMS for identification and relative quantification of metabolites in a biological sample under varying experimental conditions. Objective We demonstrate a method using high-sensitivity 13C NMR to identify an unknown metabolite isolated from fractionated material from an IROA LC-HRMS experiment. Methods IROA samples from the nematode Caenorhabditis elegans were fractionated using LC-HRMS using 5 repeated injections and collecting 30 sec fractions. These were concentrated and analyzed by 13C NMR. Results We isotopically labeled samples of C. elegans and collected 2 adjacent LC fractions. By HRMS, one contained at least 2 known metabolites, phenylalanine and inosine, and the other contained tryptophan and an unknown feature with a monoisotopic mass of m/z 380.0742 [M+H]+. With NMR, we were able to easily verify the known compounds, and we then identified the spin system networks responsible for the unknown resonances. After searching the BMRB database and comparing the molecular formula from LC-HRMS, we determined that the fragments were a modified anthranilate and a glucose modified by a phosphate. We then performed quantum chemical NMR chemical shift calculations to determine the most likely isomer, which was 3’-O-phospho-β-D-glucopyranosyl-anthranilate. This compound had previously been found in the same organism, validating our approach. Conclusion We were able to dereplicate previously known metabolites and identify a metabolite that was not in databases by matching resonances to NMR databases and using chemical shift calculations to determine the correct isomer. This approach is efficient and can be used to identify unknown compounds of interest using the same material used for IROA. PMID:28090435

  3. 13C-isotope analyses reveal that chemolithoautotrophic Gamma- and Epsilonproteobacteria feed a microbial food web in a pelagic redoxcline of the central Baltic Sea.

    PubMed

    Glaubitz, Sabine; Lueders, Tillmann; Abraham, Wolf-Rainer; Jost, Günter; Jürgens, Klaus; Labrenz, Matthias

    2009-02-01

    Marine pelagic redoxclines are zones of high dark CO(2) fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO(2) fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO(2) incorporation in (13)C-bicarbonate pulse experiments. The incorporation of (13)C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in (13)C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO(2)-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the (13)C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO(2)-fixing Proteobacteria within this habitat.

  4. 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

  5. Growth activity in human septal cartilage: age-dependent incorporation of labeled sulfate in different anatomic locations

    SciTech Connect

    Vetter, U.; Pirsig, W.; Heinze, E.

    1983-02-01

    Growth activity in different areas of human septal cartilage was measured by the in vitro incorporation of /sup 35/S-labeled NaSO/sub 4/ into chondroitin sulfate. Septal cartilage without perichondrium was obtained during rhinoplasty from 36 patients aged 6 to 35 years. It could be shown that the anterior free end of the septum displays high growth activity in all age groups. The supra-premaxillary area displayed its highest growth activity during prepuberty, showing thereafter a continuous decline during puberty and adulthood. A similar age-dependent pattern in growth activity was found in the caudal prolongation of the septal cartilage. No age-dependent variations could be detected in the posterior area of the septal cartilage.

  6. Confirmation of Fructans biosynthesized in vitro from [1-13C]glucose in asparagus tissues using MALDI-TOF MS and ESI-MS.

    PubMed

    Suzuki, Takashi; Maeda, Tomoo; Grant, Suzanne; Grant, Gordon; Sporns, Peter

    2013-05-15

    Accumulation of Fructans was confirmed in asparagus tissues that had been cultured for 2 days on media supplemented with glucose. It is very common that Fructans are biosynthesized from sucrose. We hypothesized however that Fructans could also be biosynthesized from glucose. Stem tissues of in vitro-cultured asparagus were subcultured for 72 h on a medium containing 0.5M of [1-(13)C]glucose. A medium containing 0.5M of normal ((12)C) glucose was used as control. Carbohydrates were extracted from the tissues and analyzed using HPLC, MALDI-TOF MS and ESI-MS. HPLC results indicated that the accumulation of short-chain Fructans was similar in both (13)C-labelled and control samples. Short-chain Fructans of DP=3-7 were detected using MALDI-TOF MS. The molecular mass of each oligomer in the (13)C-labelled sample was higher than the mass of the natural sample by 1 m/z unit per sugar moiety. The results of ESI-MS on the HPLC fractions of neokestose and 1-kestose showed that these oligomers (DP=3) were biosynthesized from exogenous glucose added to the medium. We conclude that not only exogenous sucrose but glucose can induce Fructan biosynthesis; fructans of both inulin type and inulin neoseries are also biosynthesized from glucose accumulated in asparagus tissues; the glucose molecules (or its metabolic products) were incorporated into Fructans as structural monomers.

  7. 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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2007-05-01

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

  11. Incorporation mechanisms of a branched nonylphenol isomer in soil-derived organo-clay complexes during a 180-day experiment.

    PubMed

    Riefer, Patrick; Klausmeyer, Timm; Adams, Alina; Schmidt, Burkhard; Schäffer, Andreas; Schwarzbauer, Jan

    2013-07-02

    The incorporation process of a defined (13)C- and (14)C-labeled nonylphenol isomer (4-(3,5-dimethylhept-3-yl)phenol) into soil-derived organo-clay complexes was investigated. Isolated organo-clay complexes were separated into humic subfractions. Noninvasive ((13)C-CP/MAS NMR) and invasive methods (sequential chemical degradation, pyrolysis) were applied to obtain detailed information about the mode of incorporation, chemical structure, and change of the incorporation character of nonextractable residues in course of incubation. (13)C-CP/MAS NMR measurements of humic acids revealed an increasing incorporation of phenolic compounds during the experimental time which was referred to residues of the introduced (13)C-labeled NP isomer. Detailed investigations by means of sequential chemical degradation indicated a predominant incorporation of nonextractable NP isomer residues via reversible ester (amide) bonds. In course of time, the amount of releasable compounds decreased, pointing to altering processes which affected the mode of incorporation. BBr3-treatment, RuO4 oxidation, and thermochemolysis released only low portions of nonextractable radioactivity giving evidence of strongly incorporated residues. With the comprehensive application of complementary methods (e.g., humic matter fractionation, (13)C-CP/MAS NMR, sequential chemical degradation) it was possible to provide a comparatively detailed insight into the incorporation behavior of the applied NP isomer.

  12. 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...

  13. Deposition of 13C tracer in the JET MkII-HD divertor

    NASA Astrophysics Data System (ADS)

    Likonen, J.; Airila, M.; Alves, E.; Barradas, N.; Brezinsek, S.; Coad, J. P.; Devaux, S.; Groth, M.; Grünhagen, S.; Hakola, A.; Jachmich, S.; Koivuranta, S.; Makkonen, T.; Rubel, M.; Strachan, J.; Stamp, M.; Widdowson, A.; EFDA contributors, JET-

    2011-12-01

    Migration of 13C has been investigated at JET by injecting 13C-labelled methane at the outer divertor base at the end of the 2009 campaign. The 13C deposition profiles on carbon fibre composite divertor tiles were measured by secondary ion mass spectrometry and Rutherford backscattering techniques. 13C was mainly deposited near the puffing location on the outer divertor base tiles. High amounts of 13C were also found at the outer vertical target: at the bottom of the lower and at the top of the upper plates. Thirty-three percent of puffed 13CH4 was instantly pumped out by the divertor cryopump, which is close to the pump duct entrance. Global 13C transport in the torus was modelled by the EDGE2D/EIRENE and DIVIMP codes, and local 13C migration in the vicinity of the injection location by the ERO code. The DIVIMP and EDGE2D simulations show strong prompt deposition of 13C directly adjacent to the injection point as well as in the far scrape-off layer (SOL) along both the inner and outer divertor targets. In addition, the measured 13C deposition along the outer divertor wall tiles is qualitatively reproduced. However, EDGE2D and DIVIMP do not predict any deposition along the divertor surfaces facing the private plasma on the inner floor tile and inboard of the outer strike point on tile 5. The ERO calculations also indicate that most of the deposition occurs close to the injection location on the vertical face of the LBSRP tile and the horizontal part of tile 6.

  14. 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.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2010-05-01

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

  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. In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies.

    PubMed

    Kanje, Sara; Hober, Sophia

    2015-04-01

    Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site.

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

    PubMed

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

    1993-07-01

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

  20. 13C-methionine breath tests for mitochondrial liver function assessment.

    PubMed

    Candelli, M; Miele, L; Armuzzi, A; Nista, E C; Pignataro, G; Fini, L; Cazzato, I A; Zocco, M A; Bartolozzi, F; Gasbarrini, G; Grieco, A; Gasbarrini, A

    2008-01-01

    13C-methionine breath test has been proposed as a non-invasive tool for the assessment of human hepatic mithocondrial function. Two methionine breath labeled with 13C in differents point of his molecular structure have been used for breath test analisys. Aim of this study was to compare two differently 13C-labeled methionines in the evaluation of mitochondrial oxidation in basal conditions and after an acute oxidative stress. 15 healthy male subjects (mean age 30.5 +/- 3.1) received [methyl-13C]-methionine dissolved in water. Breath samples were taken at baseline and and 10, 20, 30, 45, 60, 75, 90, 105 and 120 minutes after the ingestion of the labeled substrate. Forthy-eight hours later, subjects underwent the same test 30 minutes after ethanol ingestion (0,3 g/kg of body weight). Seven-day later, subjects underwent breath test using (L-methionine-1-13COOH) as substrate, in basal condition and after ethanol ingestion. At basal condition, the cumulative percentage of 13CO2 recovered in breath during the test period (%cum-dose) was higher using L-methionine-1-13COOH than [methyl-13C]-methionine (10.25 +/- 1.0 vs 4.07 +/- 0.8; p < 0.01). After ethanol ingestion, % cum dose was significantly decreased at 60 and 120 minutes with both methionines (120 min: 10.25 +/- 1.0 vs 5.03% +/- 1.8; < 0.01 and 4.07 +/- 0.8 vs 2.16% +/- 0.9; p < 0.01, respectively). However, %cum-dose during L-methionine-1-13C-breath test was significantly lower than that observed during methyl-13C-methionine breath test (120 minutes: 5.03% +/- 1.8 vs 2.16% +/- 0.9; p < 0.01). In conclusion, breath test based on L-methionine-1-13COOH seems to show a greater reliability when compared to [methyl-13C]-methionine to assess mitochondrial function because a larger amount of labeled carbon that reaches the Krebs' cicle.

  1. (13) C dynamic nuclear polarization using isotopically enriched 4-oxo-TEMPO free radicals.

    PubMed

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

    2016-12-01

    The nitroxide-based free radical 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) is a widely used polarizing agent in NMR signal amplification via dissolution dynamic nuclear polarization (DNP). In this study, we have thoroughly investigated the effects of (15) N and/or (2) H isotopic labeling of 4-oxo-TEMPO free radical on (13) C DNP of 3 M [1-(13) C] sodium acetate samples in 1 : 1 v/v glycerol : water at 3.35 T and 1.2 K. Four variants of this free radical were used for (13) C DNP: 4-oxo-TEMPO, 4-oxo-TEMPO-(15) N, 4-oxo-TEMPO-d16 and 4-oxo-TEMPO-(15) N,d16 . Our results indicate that, despite the striking differences seen in the electron spin resonance (ESR) spectral features, the (13) C DNP efficiency of these (15) N and/or (2) H-enriched 4-oxo-TEMPO free radicals are relatively the same compared with (13) C DNP performance of the regular 4-oxo-TEMPO. Furthermore, when fully deuterated glassing solvents were used, the (13) C DNP signals of these samples all doubled in the same manner, and the (13) C polarization buildup was faster by a factor of 2 for all samples. The data here suggest that the hyperfine coupling contributions of these isotopically enriched 4-oxo-TEMPO free radicals have negligible effects on the (13) C DNP efficiency at 3.35 T and 1.2 K. These results are discussed in light of the spin temperature model of DNP. Copyright © 2016 John Wiley & Sons, Ltd.

  2. 13C magnetic resonance spectroscopy measurements with hyperpolarized [1‐13C] pyruvate can be used to detect the expression of transgenic pyruvate decarboxylase activity in vivo

    PubMed Central

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

    2015-01-01

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

  3. (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.

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

    PubMed Central

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

    2016-01-01

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

  5. Synthesis of [.sup.13C] and [.sup.2H] substituted methacrylic acid, [.sup.13C] and [.sup.2H] substituted methyl methacrylate and/or related compounds

    DOEpatents

    Alvarez, Marc A.; Martinez, Rodolfo A.; Unkefer, Clifford J.

    2008-01-22

    The present invention is directed to labeled compounds of the formulae ##STR00001## wherein Q is selected from the group consisting of --S--, --S(.dbd.O)--, and --S(.dbd.O).sub.2--, Z is selected from the group consisting 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 selected from the group consisting of hydrogen, a C.sub.1-C.sub.4 lower alkyl, a halogen, and an amino group selected from the group consisting of NH.sub.2, NHR and NRR' where R and R' are each independently selected from the group consisting of a C.sub.1-C.sub.4 lower alkyl, an aryl, and an alkoxy group, and X is selected from the group consisting of hydrogen, a C.sub.1-C.sub.4 lower alkyl group, and a fully-deuterated C.sub.1-C.sub.4 lower alkyl group. The present invention is also directed to a process of preparing labeled compounds, e.g., process of preparing [.sup.13C]methacrylic acid by reacting a (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13CH.sub.2)-- aryl sulfone precursor with .sup.13CHI to form a (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13C(.sup.13CH.sub.3).sub.2)-- aryl sulfone intermediate, and, reacting the (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13C(.sup.13CH.sub.3).sub.2)-- aryl sulfone intermediate with sodium hydroxide, followed by acid to form [.sup.13C]methacrylic acid. The present invention is further directed to a process of preparing [.sup.2H.sub.8]methyl methacrylate by reacting a (HOOC--C(C.sup.2H.sub.3).sub.2-- aryl sulfinyl intermediate with CD.sub.3I to form a (.sup.2H.sub.3COOC--C(C.sup.2H.sub.3).sub.2)-- aryl sulfinyl intermediate, and heating the(.sup.2H.sub.3COOC--C(C.sup.2H.sub.3).sub.2)-- aryl sulfinyl intermediate at temperatures and for time sufficient to form [.sup.2H.sub.8]methyl methacrylate.

  6. Extreme (13)C depletion of carbonates formed during oxidation of biogenic methane in fractured granite.

    PubMed

    Drake, Henrik; Åström, Mats E; Heim, Christine; Broman, Curt; Åström, Jan; Whitehouse, Martin; Ivarsson, Magnus; Siljeström, Sandra; Sjövall, Peter

    2015-05-07

    Precipitation of exceptionally 13C-depleted authigenic carbonate is a result of, and thus a tracer for, sulphate-dependent anaerobic methane oxidation, particularly in marine sediments. Although these carbonates typically are less depleted in 13C than in the source methane, because of incorporation of C also from other sources, they are far more depleted in 13C13C as light as -69‰ V-PDB) than in carbonates formed where no methane is involved. Here we show that oxidation of biogenic methane in carbon-poor deep groundwater in fractured granitoid rocks has resulted in fracture-wall precipitation of the most extremely 13C-depleted carbonates ever reported, δ13C down to -125‰ V-PDB. A microbial consortium of sulphate reducers and methane oxidizers has been involved, as revealed by biomarker signatures in the carbonates and S-isotope compositions of co-genetic sulphide. Methane formed at shallow depths has been oxidized at several hundred metres depth at the transition to a deep-seated sulphate-rich saline water. This process is so far an unrecognized terrestrial sink of methane.

  7. Extreme 13C depletion of carbonates formed during oxidation of biogenic methane in fractured granite

    PubMed Central

    Drake, Henrik; Åström, Mats E.; Heim, Christine; Broman, Curt; Åström, Jan; Whitehouse, Martin; Ivarsson, Magnus; Siljeström, Sandra; Sjövall, Peter

    2015-01-01

    Precipitation of exceptionally 13C-depleted authigenic carbonate is a result of, and thus a tracer for, sulphate-dependent anaerobic methane oxidation, particularly in marine sediments. Although these carbonates typically are less depleted in 13C than in the source methane, because of incorporation of C also from other sources, they are far more depleted in 13C13C as light as −69‰ V-PDB) than in carbonates formed where no methane is involved. Here we show that oxidation of biogenic methane in carbon-poor deep groundwater in fractured granitoid rocks has resulted in fracture-wall precipitation of the most extremely 13C-depleted carbonates ever reported, δ13C down to −125‰ V-PDB. A microbial consortium of sulphate reducers and methane oxidizers has been involved, as revealed by biomarker signatures in the carbonates and S-isotope compositions of co-genetic sulphide. Methane formed at shallow depths has been oxidized at several hundred metres depth at the transition to a deep-seated sulphate-rich saline water. This process is so far an unrecognized terrestrial sink of methane. PMID:25948095

  8. Extreme 13C depletion of carbonates formed during oxidation of biogenic methane in fractured granite

    NASA Astrophysics Data System (ADS)

    Drake, Henrik; Åström, Mats E.; Heim, Christine; Broman, Curt; Åström, Jan; Whitehouse, Martin; Ivarsson, Magnus; Siljeström, Sandra; Sjövall, Peter

    2015-05-01

    Precipitation of exceptionally 13C-depleted authigenic carbonate is a result of, and thus a tracer for, sulphate-dependent anaerobic methane oxidation, particularly in marine sediments. Although these carbonates typically are less depleted in 13C than in the source methane, because of incorporation of C also from other sources, they are far more depleted in 13C13C as light as -69‰ V-PDB) than in carbonates formed where no methane is involved. Here we show that oxidation of biogenic methane in carbon-poor deep groundwater in fractured granitoid rocks has resulted in fracture-wall precipitation of the most extremely 13C-depleted carbonates ever reported, δ13C down to -125‰ V-PDB. A microbial consortium of sulphate reducers and methane oxidizers has been involved, as revealed by biomarker signatures in the carbonates and S-isotope compositions of co-genetic sulphide. Methane formed at shallow depths has been oxidized at several hundred metres depth at the transition to a deep-seated sulphate-rich saline water. This process is so far an unrecognized terrestrial sink of methane.

  9. NMR analyses of the cold cataract. III. /sup 13/C acrylamide studies

    SciTech Connect

    Lerman, S.; Megaw, J.M.; Moran, M.N.

    1985-10-01

    /sup 13/C-enriched acrylamide was employed to further delineate the action of this compound in preventing the cold cataract phenomenon when it is incorporated (in vitro) into young human and rabbit lenses. The extent of acrylamide incorporation, in the dark and with concurrent UV exposure, was monitored by /sup 13/C NMR spectroscopy. These studies provide further evidence that UV exposure causes permanent acrylamide photobinding within the lens. In such lenses, the gamma crystallin fraction of the soluble lens proteins is affected to the greatest extent. It appears to become aggregated and/or combined with the alpha and beta fractions resulting in an apparent loss of most of the gamma monomers. There is also an age-related effect with respect to the amount of acrylamide that can be incorporated into the lens. The decrease in acrylamide incorporation with age directly parallels the age-related decline in gamma crystallin levels.

  10. GFT projection NMR for efficient (1)H/ (13)C sugar spin system identification in nucleic acids.

    PubMed

    Atreya, Hanudatta S; Sathyamoorthy, Bharathwaj; Jaipuria, Garima; Beaumont, Victor; Varani, Gabriele; Szyperski, Thomas

    2012-12-01

    A newly implemented G-matrix Fourier transform (GFT) (4,3)D HC(C)CH experiment is presented in conjunction with (4,3)D HCCH to efficiently identify (1)H/(13)C sugar spin systems in (13)C labeled nucleic acids. This experiment enables rapid collection of highly resolved relay 4D HC(C)CH spectral information, that is, shift correlations of (13)C-(1)H groups separated by two carbon bonds. For RNA, (4,3)D HC(C)CH takes advantage of the comparably favorable 1'- and 3'-CH signal dispersion for complete spin system identification including 5'-CH. The (4,3)D HC(C)CH/HCCH based strategy is exemplified for the 30-nucleotide 3'-untranslated region of the pre-mRNA of human U1A protein.

  11. 13C Tracers for Glucose Degrading Pathway Discrimination in Gluconobacter oxydans 621H

    PubMed Central

    Ostermann, Steffen; Richhardt, Janine; Bringer, Stephanie; Bott, Michael; Wiechert, Wolfgang; Oldiges, Marco

    2015-01-01

    Gluconobacter oxydans 621H is used as an industrial production organism due to its exceptional ability to incompletely oxidize a great variety of carbohydrates in the periplasm. With glucose as the carbon source, up to 90% of the initial concentration is oxidized periplasmatically to gluconate and ketogluconates. Growth on glucose is biphasic and intracellular sugar catabolism proceeds via the Entner–Doudoroff pathway (EDP) and the pentose phosphate pathway (PPP). Here we studied the in vivo contributions of the two pathways to glucose catabolism on a microtiter scale. In our approach we applied specifically 13C labeled glucose, whereby a labeling pattern in alanine was generated intracellularly. This method revealed a dynamic growth phase-dependent pathway activity with increased activity of EDP in the first and PPP in the second growth phase, respectively. Evidence for a growth phase-independent decarboxylation-carboxylation cycle around the pyruvate node was obtained from 13C fragmentation patterns of alanine. For the first time, down-scaled microtiter plate cultivation together with 13C-labeled substrate was applied for G. oxydans to elucidate pathway operation, exhibiting reasonable labeling costs and allowing for sufficient replicate experiments. PMID:26404385

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

    PubMed Central

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

    2015-01-01

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

  13. Hyperpolarized 13C Metabolic MRI of the Human Heart

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Reed, Galen Durant

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

  15. Incorporating functionalized polyethylene glycol lipids into reprecipitated conjugated polymer nanoparticles for bioconjugation and targeted labeling of cells

    NASA Astrophysics Data System (ADS)

    Kandel, Prakash K.; Fernando, Lawrence P.; Ackroyd, P. Christine; Christensen, Kenneth A.

    2011-03-01

    We report a simple and rapid method to prepare extremely bright, functionalized, stable, and biocompatible conjugated polymer nanoparticles incorporating functionalized polyethylene glycol (PEG) lipids by reprecipitation. These nanoparticles retain the fundamental spectroscopic properties of conjugated polymer nanoparticles prepared without PEG lipid, but demonstrate greater hydrophilicity and quantum yield compared to unmodified conjugated polymer nanoparticles. The sizes of these nanoparticles, as determined by TEM, were 21-26 nm. Notably, these nanoparticles were prepared with several PEG lipid functional end groups, including biotin and carboxy moieties that can be easily conjugated to biomolecules. We have demonstrated the availability of these end groups for functionalization using the interaction of biotin PEG lipid conjugated polymer nanoparticles with streptavidin. Biotinylated PEG lipid conjugated polymer nanoparticles bound streptavidin-linked magnetic beads, while carboxy and methoxy PEG lipid modified nanoparticles did not. Similarly, biotinylated PEG lipid conjugated polymer nanoparticles bound streptavidin-coated glass slides and could be visualized as diffraction-limited spots, while nanoparticles without PEG lipid or with non-biotin PEG lipid end groups were not bound. To demonstrate that nanoparticle functionalization could be used for targeted labelling of specific cellular proteins, biotinylated PEG lipid conjugated polymer nanoparticles were bound to biotinylated anti-CD16/32 antibodies on J774A.1 cell surface receptors, using streptavidin as a linker. This work represents the first demonstration of targeted delivery of conjugated polymer nanoparticles and demonstrates the utility of these new nanoparticles for fluorescence based imaging and sensing.We report a simple and rapid method to prepare extremely bright, functionalized, stable, and biocompatible conjugated polymer nanoparticles incorporating functionalized polyethylene glycol (PEG

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

    PubMed Central

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

    2016-01-01

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

  17. Dynamic nuclear polarization-enhanced 1H–13C double resonance NMR in static samples below 20 K

    PubMed Central

    Potapov, Alexey; Thurber, Kent R.; Yau, Wai-Ming; Tycko, Robert

    2012-01-01

    We demonstrate the feasibility of one-dimensional and two-dimensional 1H–13C double resonance NMR experiments with dynamic nuclear polarization (DNP) at 9.4 T and temperatures below 20 K, including both 1H–13C cross-polarization and 1H decoupling, and discuss the effects of polarizing agent type, polarizing agent concentration, temperature, and solvent deuteration. We describe a two-channel low-temperature DNP/NMR probe, capable of carrying the radio-frequency power load required for 1H–13C cross-polarization and high-power proton decoupling. Experiments at 8 K and 16 K reveal a significant T2 relaxation of 13C, induced by electron spin flips. Carr–Purcell experiments and numerical simulations of Carr–Purcell dephasing curves allow us to determine the effective correlation time of electron flips under our experimental conditions. The dependence of the DNP signal enhancement on electron spin concentration shows a maximum near 80 mM. Although no significant difference in the absolute DNP enhancements for triradical (DOTOPA-TEMPO) and biradical (TOTAPOL) dopants was found, the triradical produced greater DNP build-up rates, which are advantageous for DNP experiments. Additionally the feasibility of structural measurements on 13C-labeled biomolecules was demonstrated with a two-dimensional 13C–13C exchange spectrum of selectively 13C-labeled β-amyloid fibrils. PMID:22743540

  18. Methylation patterns of aquatic humic substances determined by 13C NMR spectroscopy

    USGS Publications Warehouse

    Thorn, K.A.; Steelink, C.; Wershaw, R. L.

    1987-01-01

    13C NMR spectroscopy is used to examine the hydroxyl group functionality of a series of humic and fulvic acids from different aquatic environments. Samples first are methylated with 13C-labeled diazomethane. The NMR spectra of the diazomethylated samples allow one to distinguish between methyl esters of carboxylic acids, methyl ethers of phenolic hydroxyls, and methyl ethers of phenolic hydroxyls adjacent to two substituents. Samples are then permethylated with 13C-labeled methyl iodide/NaH. 13C NMR spectra of permethylated samples show that a significant fraction of the hydroxyl groups is not methylated with diazomethane alone. In these spectra methyl ethers of carbohydrate and aliphatic hydroxyls overlap with methyl ethers of phenolic hydroxyls. Side reactions of the methyltion procedure including carbon methylation in the CH3I/NaH procedure, are also examined. Humic and fulvic acids from bog, swamp, groundwater, and lake waters showssome differences in their distribution of hydroxyl groups, mainly in the concentrations of phenolic hydroxyls, which may be attributed to their different biogeochemical origins. ?? 1987.

  19. Increased incorporation of an infused labelled amino acid into plasma proteins as a means of assessing the severity of injury or activity of disease in surgical patients

    SciTech Connect

    Clague, M.B.; Carmichael, M.J.; Keir, M.J.; Rogaly, E.; Wright, P.D.; Johnston, I.D.

    1982-07-01

    Following injury there is increased synthesis of several plasma proteins. During a constant rate infusion of 1-(1-/sup 14/C) leucine some of the labeled amino acid became incorporated into plasma proteins, the amount being readily determined after precipitation with perchloric acid. This value was correlated with the clinical state of the patient to see if an index of severity of injury could be derived. Before operation 6.7 +/- 0.5% (mean +/- SD, n . 16) of the infused labeled amino acid was incorporated in asymptomatic patients, irrespective of their dietary intakes, while in the presence of symptomatology this value rose, correlating with tumor staging in patients with colorectal neoplasia (r . 0.988, p . 0.001). It returned to normal following tumor resection, but was elevated in the presence of recurrence or residual disease. After operation it also rose, being maximal in the early postoperative period and following the more major surgical procedures, the increase correlating with the change in cortisol level in patients following cholecystectomy (r . 0.84, p less than 0.01; n . 8). The pattern of distribution of the label among the plasma proteins did not change with increased incorporation. The increase in incorporation of 1-(1-14C) leucine into plasma proteins would appear to offer a good index for severity of injury of activity of a disease state.

  20. (Bio)degradation of glyphosate in water-sediment microcosms - A stable isotope co-labeling approach.

    PubMed

    Wang, Shizong; Seiwert, Bettina; Kästner, Matthias; Miltner, Anja; Schäffer, Andreas; Reemtsma, Thorsten; Yang, Qi; Nowak, Karolina M

    2016-08-01

    Glyphosate and its metabolite aminomethylphosphonic acid (AMPA) are frequently detected in water and sediments. Up to date, there are no comprehensive studies on the fate of glyphosate in water-sediment microcosms according to OECD 308 guideline. Stable isotope co-labeled (13)C3(15)N-glyphosate was used to determine the turnover mass balance, formation of metabolites, and formation of residues over a period of 80 days. In the water-sediment system, 56% of the initial (13)C3-glyphosate equivalents was ultimately mineralized, whereas the mineralization in the water system (without sediment) was low, reaching only 2% of (13)C-glyphosate equivalents. This finding demonstrates the key role of sediments in its degradation. Glyphosate was detected below detection limit in the water compartment on day 40, but could still be detected in the sediments, ultimately reaching 5% of (13)C3(15)N-glyphosate equivalents. A rapid increase in (13)C(15)N-AMPA was noted after 10 days, and these transformation products ultimately constituted 26% of the (13)C3-glyphosate equivalents and 79% of the (15)N-glyphosate equivalents. In total, 10% of the (13)C label and 12% of the (15)N label were incorporated into amino acids, indicating no risk bearing biogenic residue formation from (13)C3(15)N-glyphosate. Initially, glyphosate was biodegraded via the sarcosine pathway related to microbial growth, as shown by co-labeled (13)C(15)N-glycine and biogenic residue formation. Later, degradation via AMPA dominated under starvation conditions, as shown by the contents of (13)C-glycine. The presented data provide the first evidence of the speciation of the non-extractable residues as well as the utilization of glyphosate as a carbon and nitrogen source in the water-sediment system. This study also highlights the contribution of both the sarcosine and the AMPA degradation pathways under these conditions.

  1. 13C NMR Metabolomics: Applications at Natural Abundance

    PubMed Central

    2015-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  3. Incorporation of fluorescein conjugated function-spacer-lipid constructs into the red blood cell membrane facilitates detection of labeled cells for the duration of ex-vivo storage.

    PubMed

    Ki, Katrina K; Flower, Robert L; Faddy, Helen M; Dean, Melinda M

    2016-02-01

    The contribution of ex-vivo storage duration of packed red blood cells (PRBC) to patient outcomes and transfusion-related immunomodulation (TRIM) remains a broadly debated area in transfusion medicine. Kode™ Technology with fluorescein conjugated function-spacer-lipid (FSL-FLRO4) constructs is a tool that can aid in-vitro visualization and tracking of red blood cells (RBC) during routine storage. FSL-FLRO4 is incorporated into the RBC membrane without altering cell function. In this study, we explore the suitability of this technology to label clinical grade PRBC and to determine if the label would be retained during ex-vivo storage. Firstly, to confirm feasibility and assess the limit of detection of FSL-FLRO4 on PRBC at date of expiry (42 days post-collection), we tracked the binding of FSL-FLRO4 on PRBC at weekly intervals during routine storage. Over the time course, all cells remained labelled with FSL-FLRO4, although a decrease in the intensity of labelling was observed (P<0.0001). We then further investigated differences in FSL-FLRO4 labelling during RBC storage by labelling separated light-young and dense-old RBC from the same PRBC unit. There were no differences in the capacity of FSL-FLRO4 to label these different RBC subsets. Together, these data demonstrate that FSL-FLRO4 is a suitable reagent for labelling PRBC at any point during routine storage. This technology will facilitate the development of immunoassays and transfusion models focused on addressing the mechanisms involved in TRIM.

  4. Hyperpolarized 13C MR Markers of Renal Tumor Aggressiveness

    DTIC Science & Technology

    2013-10-01

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

  5. 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...

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

    NASA Astrophysics Data System (ADS)

    Baggs, E. M.

    2005-12-01

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

  7. A 13C mass isotopomer study of anaplerotic pyruvate carboxylation in perfused rat hearts.

    PubMed

    Comte, B; Vincent, G; Bouchard, B; Jetté, M; Cordeau, S; Rosiers, C D

    1997-10-17

    Anaplerotic pyruvate carboxylation was examined in hearts perfused with physiological concentrations of glucose, [U-13C3]lactate, and [U-13C3]pyruvate. Also, a fatty acid, [1-13C]octanoate, or ketone bodies were added at concentrations providing acetyl-CoA at a rate resulting in either low or substantial pyruvate decarboxylation. Relative contributions of pyruvate and fatty acids to citrate synthesis were determined from the 13C labeling pattern of effluent citrate by gas chromatography-mass spectrometry (see companion article, Comte, B., Vincent, G., Bouchard, B., and Des Rosiers, C. (1997) J. Biol. Chem. 272, 26117-26124). Precision on flux measurements of anaplerotic pyruvate carboxylation depended on the mix of substrates supplied to the heart. Anaplerotic fluxes were precisely determined under conditions where acetyl-CoA was predominantly supplied by beta-oxidation, as it occurred with 0.2 or 1 mM octanoate. Then, anaplerotic pyruvate carboxylation provided 3-8% of the OAA moiety of citrate and was modulated by concentrations of lactate and pyruvate in the physiological range. Also, the contribution of pyruvate to citrate formation through carboxylation was equal to or greater than through decarboxylation. Furthermore, 13C labeling data on tissue citric acid cycle intermediates and pyruvate suggest that (i) anaplerosis occurs also at succinate and (ii) cataplerotic malate decarboxylation is low. Rather, the presence of citrate in the effluent perfusate of hearts perfused with physiological concentrations of glucose, lactate, and pyruvate and concentrations of octanoate leading to maximal oxidative rates suggests a cataplerotic citrate efflux from mitochondria to cytosol. Taken altogether, our data raise the possibility of a link between pyruvate carboxylation and mitochondrial citrate efflux. In view of the proposed feedback regulation of glycolysis by cytosolic citrate, such a link would support a role of anaplerosis and cataplerosis in metabolic signal

  8. 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.

  9. Identification of biomass utilizing bacteria in a carbon-depleted glacier forefield soil by the use of 13C DNA stable isotope probing.

    PubMed

    Zumsteg, Anita; Schmutz, Stefan; Frey, Beat

    2013-06-01

    As Alpine glaciers are retreating rapidly, bare soils with low organic C and N contents are becoming exposed. Carbon availability is a key factor regulating microbial diversity and ecosystem functioning in these soils. The aim of this study was to investigate how bacterial activity, community structure and composition are influenced by organic carbon availability. Bare soils were supplied with (13)C-labelled fungal (Penicillium sp.) and green algal (Chlorella sp.) biomass and the CO2 evolution and its δ(13)C signature were monitored up to 60 days. These organisms have previously been isolated near the glacier terminus. DNA stable isotope probing followed by T-RFLP profiling and sequencing of 16S rRNA genes was employed to identify consumers able to assimilate carbon from these biomass amendments. Higher respiration and higher bacterial activity indicated a more efficient utilization of algal cells than fungal cells. Flavobacterium sp. predominantly incorporated fungal-derived C, whereas the algal-derived C was mainly incorporated by Acidobacteria and Proteobacteria. This study emphasizes the important role of both fungal and algal biomass in increasing the carbon pool in recently deglaciated bare soils, as only 20% of the added C was respired as CO2, and the rest, we presume, remained in the soil.

  10. 15N and 13C NMR Determination of Allantoin Metabolism in Developing Soybean Cotyledons 1

    PubMed Central

    Coker, George T.; Schaefer, Jacob

    1985-01-01

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

  11. 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

  12. Detection of intracellular lactate with localized diffusion { 1H- 13C}-spectroscopy in rat glioma in vivo

    NASA Astrophysics Data System (ADS)

    Pfeuffer, Josef; Lin, Joseph C.; DelaBarre, Lance; Ugurbil, Kamil; Garwood, Michael

    2005-11-01

    The aim of this study was to compare the diffusion characteristic of lactate and alanine in a brain tumor model to that of normal brain metabolites known to be mainly intracellular such as N-acetylaspartate or creatine. The diffusion of 13C-labeled metabolites was measured in vivo with localized NMR spectroscopy at 9.4 T (400 MHz) using a previously described localization and editing pulse sequence known as ACED-STEAM ('adiabatic carbon editing and decoupling'). 13C-labeled glucose was administered and the apparent diffusion coefficients of the glycolytic products, { 1H- 13C}-lactate and { 1H- 13C}-alanine, were determined in rat intracerebral 9L glioma. To obtain insights into { 1H- 13C}-lactate compartmentation (intra- versus extracellular), the pulse sequence used very large diffusion weighting (50 ms/μm 2). Multi-exponential diffusion attenuation of the lactate metabolite signals was observed. The persistence of a lactate signal at very large diffusion weighting provided direct experimental evidence of significant intracellular lactate concentration. To investigate the spatial distribution of lactate and other metabolites, 1H spectroscopic images were also acquired. Lactate and choline-containing compounds were consistently elevated in tumor tissue, but not in necrotic regions and surrounding normal-appearing brain. Overall, these findings suggest that lactate is mainly associated with tumor tissue and that within the time-frame of these experiments at least some of the glycolytic product ([ 13C] lactate) originates from an intracellular compartment.

  13. Resolving Confounding Enrichment Kinetics Due to Overlapping Resonance Signals From 13C-Enriched Long Chain Fatty Acid Oxidation and Uptake Within Intact Hearts

    PubMed Central

    O'Donnell, J. Michael; Fasano, Matthew J.; Lewandowski, E. Douglas

    2014-01-01

    Purpose Long chain fatty acid (LCFA) oxidation measurements in the intact heart from 13C-NMR rely on detection of 13C-enriched glutamate. However, progressive increases in overlapping resonance signal from LCFA can confound detection of the glutamate 4-carbon (GLU-C4) signal. We evaluated alternative 13C labeling for exogenous LCFA and developed a simple scheme to distinguish kinetics of LCFA uptake and storage from oxidation. Methods Sequential 13C-NMR spectra were acquired from isolated rat hearts perfused with 13C LCFA and glucose. Spectra were evaluated from hearts supplied: U 13C LCFA, [2,4,6,8,10,12,14,16-13C8] palmitate, [2,4,6,8,10,12,14,16,18-13C9] oleate, [4,6,8,10,12,14,16-13C7] palmitate, or [4,6,8,10,12, 14,16,18-13C8] oleate. Results 13C signal reflected the progressive enrichment at 34.6 ppm from GLU-C4, confounded by additional signal with distinct kinetics attributed to 13C-enriched LCFA 2-carbon (34.0 ppm). Excluding 13C at the 2-carbon of both palmitate and oleate eliminated signal overlap and enabled detection of the exponential enrichment of GLU-C4 for assessing LCFA oxidation. Conclusion Eliminating enrichment at the 2-carbon of 13C LCFA resolved confounding kinetics between GLU-C4 and LCFA 2-carbon signals. With this enrichment scheme, oxidation of LCFA, the primary fuel for cardiac ATP synthesis, can now be more consistently examined in whole organs with dynamic mode, proton-decoupled 13C-NMR. PMID:25199499

  14. Study of Urban environmental quality through Isotopes δ13C

    NASA Astrophysics Data System (ADS)

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

    2012-04-01

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

  15. Modeling the contribution of individual proteins to mixed skeletal muscle protein synthetic rates over increasing periods of label incorporation.

    PubMed

    Miller, Benjamin F; Wolff, Christopher A; Peelor, Fredrick F; Shipman, Patrick D; Hamilton, Karyn L

    2015-03-15

    Advances in stable isotope approaches, primarily the use of deuterium oxide ((2)H2O), allow for long-term measurements of protein synthesis, as well as the contribution of individual proteins to tissue measured protein synthesis rates. Here, we determined the influence of individual protein synthetic rates, individual protein content, and time of isotopic labeling on the measured synthesis rate of skeletal muscle proteins. To this end, we developed a mathematical model, applied the model to an established data set collected in vivo, and, to experimentally test the impact of different isotopic labeling periods, used (2)H2O to measure protein synthesis in cultured myotubes over periods of 2, 4, and 7 days. We first demonstrated the influence of both relative protein content and individual protein synthesis rates on measured synthesis rates over time. When expanded to include 286 individual proteins, the model closely approximated protein synthetic rates measured in vivo. The model revealed a 29% difference in measured synthesis rates from the slowest period of measurement (20 min) to the longest period of measurement (6 wk). In support of these findings, culturing of C2C12 myotubes with isotopic labeling periods of 2, 4, or 7 days revealed up to a doubling of the measured synthesis rate in the shorter labeling period compared with the longer period of labeling. From our model, we conclude that a 4-wk period of labeling is ideal for considering all proteins in a mixed-tissue fraction, while minimizing the slowing effect of fully turned-over proteins. In addition, we advocate that careful consideration must be paid to the period of isotopic labeling when comparing mixed protein synthetic rates between studies.

  16. Single-step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides: Detection of apoptosis and bromodeoxyuridine incorporation

    SciTech Connect

    Xun Li; Traganos, F.; Melamed, M.R.; Darzynkiewicz, Z.

    1995-06-01

    The methods of in situ labeling of DNA strand breaks have been used to identify apoptotic cells and/or DNA replicating cells. While discrimination of apoptotic cells is based on the inherent presence of numerous DNA strand breaks in their chromatin, DNA proliferating cells can be discriminated by the selective DNA strand break induction by photolysis (SBIP) methodology at the sites that contain incorporated bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). In both instances, DNA strand breaks are labeled with biotin- or digoxygenin-conjugated deoxynucleotides, preferably in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase; fluorescein tagged avidin (streptavidin) or digoxygenin antibody is used in the second step of the reaction. In the present study, DNA strand break labeling was simplified by using directly labeled deoxynucleotides, in a single-step reaction. Apoptotic cells in HL-60 cultures treated with camptothecin or in primary cultures of non-Hodgkin`s lymphoma cells treated with prednisolone were easily identified utilizing BODIPY-conjugated dUTP (B-dUTP). The single-step procedure, requiring fewer centrifugation steps, resulted in less cell loss compared to the two-step cell labeling technique. The morphology of cells subjected to SBIP was excellent, allowing visualization of distinct DNA replication points. Because, unlike the immunocytochemical methods used to detect BrdUrd incorporation, the SBIP methodology does not require DNA denaturation by heat or acid, nuclear proteins are expected to remain undenatured in situ, allowing one to study colocalization of various constituents, detected immunocytochemically, at the DNA replication points. 30 refs., 7 figs.

  17. {sup 13}C chemical shift anisotropies for carbonate ions in cement minerals and the use of {sup 13}C, {sup 27}Al and {sup 29}Si MAS NMR in studies of Portland cement including limestone additions

    SciTech Connect

    Sevelsted, Tine F.; Herfort, Duncan

    2013-10-15

    {sup 13}C isotropic chemical shifts and chemical shift anisotropy parameters have been determined for a number of inorganic carbonates relevant in cement chemistry from slow-speed {sup 13}C MAS or {sup 13}C({sup 1}H) CP/MAS NMR spectra (9.4 T or 14.1 T) for {sup 13}C in natural abundance. The variation in the {sup 13}C chemical shift parameters is relatively small, raising some doubts that different carbonate species in Portland cement-based materials may not be sufficiently resolved in {sup 13}C MAS NMR spectra. However, it is shown that by combining {sup 13}C MAS and {sup 13}C({sup 1}H) CP/MAS NMR carbonate anions in anhydrous and hydrated phases can be distinguished, thereby providing valuable information about the reactivity of limestone in cement blends. This is illustrated for three cement pastes prepared from an ordinary Portland cement, including 0, 16, and 25 wt.% limestone, and following the hydration for up to one year. For these blends {sup 29}Si MAS NMR reveals that the limestone filler accelerates the hydration for alite and also results in a smaller fraction of tetrahedrally coordinated Al incorporated in the C-S-H phase. The latter result is more clearly observed in {sup 27}Al MAS NMR spectra of the cement–limestone blends and suggests that dissolved aluminate species in the cement–limestone blends readily react with carbonate ions from the limestone filler, forming calcium monocarboaluminate hydrate. -- Highlights: •{sup 13}C chemical shift anisotropies for inorganic carbonates from {sup 13}C MAS NMR. •Narrow {sup 13}C NMR chemical shift range (163–171 ppm) for inorganic carbonates. •Anhydrous and hydrated carbonate species by {sup 13}C MAS and {sup 13}C({sup 1}H) CP/MAS NMR. •Limestone accelerates the hydration for alite in Portland – limestone cements. •Limestone reduces the amount of aluminium incorporated in the C-S-H phase.

  18. Random isotopolog libraries for protein perturbation studies. 13C NMR studies on lumazine protein of Photobacterium leiognathi.

    PubMed

    Illarionov, Boris; Lee, Chan Yong; Bacher, Adelbert; Fischer, Markus; Eisenreich, Wolfgang

    2005-11-25

    [graph: see text] Lumazine proteins of luminescent bacteria are paralogs of riboflavin synthase which are devoid of catalytic activity but bind the riboflavin synthase substrate, 6,7-dimethyl-8-ribityllumazine, with high affinity and are believed to serve as optical transponders for bioluminescence emission. Lumazine protein of Photobacterium leiognathi was expressed in a recombinant Escherichia coli host and was reconstituted with mixtures (random libraries) of 13C-labeled isotopologs of 6,7-dimethyl-8-ribityllumazine or riboflavin that had been prepared by biotransformation of [U-(13)C6]-, [1-(13)C1]-, [2-(13)C1]-, and [3-(13)C1]glucose. 13C NMR analysis of the protein/ligand complexes afforded the assignments of the 13C NMR chemical shifts for all carbon atoms of the protein-bound ligands by isotopolog abundance editing. The carbon atoms of the ribityl groups of both ligands studied were shifted up to 6 ppm upon binding to the protein. Chemical shift modulation of the side chain and chromophore carbon atoms due to protein/ligand interaction is discussed on the basis of the sequence similarity between lumazine protein and riboflavin synthase.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2017-03-08

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

  1. /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.

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

    PubMed

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

    2014-01-01

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

  3. 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

  4. 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.

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

    NASA Astrophysics Data System (ADS)

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

    1999-11-01

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

  6. Measuring the 13C content of soil-respired CO2 using a novel open chamber system.

    PubMed

    Midwood, Andrew J; Thornton, Barry; Millard, Pete

    2008-07-01

    Carbon dioxide respired by soils comes from both autotrophic and heterotrophic respiration. 13C has proved useful in differentiating between these two sources, but requires the collection and analysis of CO2 efflux from the soil. We have developed a novel, open chamber system which allows for the accurate and precise quantification of the delta13C of soil-respired CO2. The chamber was tested using online analyses, by configuring a GasBench II and continuous flow isotope ratio mass spectrometer, to measure the delta13C of the chamber air every 120 s. CO2 of known delta13C value was passed through a column of sand and, using the chamber, the CO2 concentration stabilized rapidly, but 60 min was required before the delta13C value was stable and identical to the cylinder gas (-33.3 per thousand). Changing the chamber CO2 concentration between 200 and 900 micromol.mol(-1) did not affect the measured delta13C of the efflux. Measuring the delta13C of the CO2 efflux from soil cores in the laboratory gave a spread of +/-2 per thousand, attributed to heterogeneity in the soil organic matter and roots. Lateral air movement through dry sand led to a change in the delta13C of the surface efflux of up to 8 per thousand. The chamber was used to measure small transient changes (+/-2 per thousand) in the delta13C of soil-respired CO2 from a peaty podzol after gradual heating from 12 to 35 degrees C over 12 h. Finally, soil-respired CO2 was partitioned in a labelling study and the contribution of autotrophic and heterotrophic respiration to the total efflux determined. Potential applications for the chamber in the study of soil respiration are discussed.

  7. sup 18 F-labeled insulin: A prosthetic group methodology for incorporation of a positron emitter into peptides and proteins

    SciTech Connect

    Shai, Y.; Kirk, K.L.; Channing, M.A.; Dunn, B.B.; Lesniak, M.A.; Eastman, R.C.; Finn, R.D.; Roth, J.; Jacobson, K.A. )

    1989-05-30

    In the present study we synthesize {sup 18}F-labeled insulin of high specific radioactivity. A new prosthetic group methodology, in which ({sup 18}F)fluoride displaces a bromide group of 4-(bromomethyl)-benzoylamine intermediates, was used. The 4-(fluoromethyl)benzoyl product was chemically stable. {sup 18}F-Labeled insulin retains the essential biological properties of native insulin, as measured in vitro by binding to insulin receptors on human cells and stimulation of glucose metabolism in rat adipocytes. The overall process can be carried out speedily to yield a product of sufficient purity to permit in vivo studies. The method appears to be applicable to a wide variety of peptides.

  8. Turnover of microbial groups and cell components in soil: 13C analysis of cellular biomarkers

    NASA Astrophysics Data System (ADS)

    Gunina, Anna; Dippold, Michaela; Glaser, Bruno; Kuzyakov, Yakov

    2017-01-01

    Microorganisms regulate the carbon (C) cycle in soil, controlling the utilization and recycling of organic substances. To reveal the contribution of particular microbial groups to C utilization and turnover within the microbial cells, the fate of 13C-labelled glucose was studied under field conditions. Glucose-derived 13C was traced in cytosol, amino sugars and phospholipid fatty acid (PLFA) pools at intervals of 3, 10 and 50 days after glucose addition into the soil. 13C enrichment in PLFAs ( ˜ 1.5 % of PLFA C at day 3) was an order of magnitude greater than in cytosol, showing the importance of cell membranes for initial C utilization. The 13C enrichment in amino sugars of living microorganisms at day 3 accounted for 0.57 % of total C pool; as a result, we infer that the replacement of C in cell wall components is 3 times slower than that of cell membranes. The C turnover time in the cytosol (150 days) was 3 times longer than in PLFAs (47 days). Consequently, even though the cytosol pool has the fastest processing rates compared to other cellular compartments, intensive recycling of components here leads to a long C turnover time. Both PLFA and amino-sugar profiles indicated that bacteria dominated in glucose utilization. 13C enrichment decreased with time for bacterial cell membrane components, but it remained constant or even increased for filamentous microorganisms. 13C enrichment of muramic acid was the 3.5 times greater than for galactosamine, showing a more rapid turnover of bacterial cell wall components compared to fungal. Thus, bacteria utilize a greater proportion of low-molecular-weight organic substances, whereas filamentous microorganisms are responsible for further C transformations. Thus, tracing 13C in cellular compounds with contrasting turnover rates elucidated the role of microbial groups and their cellular compartments in C utilization and recycling in soil. The results also reflect that microbial C turnover is not restricted to the death or

  9. Unraveling the 13C NMR chemical shifts in single-walled carbon nanotubes: dependence on diameter and electronic structure.

    PubMed

    Engtrakul, Chaiwat; Irurzun, Veronica M; Gjersing, Erica L; Holt, Josh M; Larsen, Brian A; Resasco, Daniel E; Blackburn, Jeffrey L

    2012-03-14

    The atomic specificity afforded by nuclear magnetic resonance (NMR) spectroscopy could enable detailed mechanistic information about single-walled carbon nanotube (SWCNT) functionalization as well as the noncovalent molecular interactions that dictate ground-state charge transfer and separation by electronic structure and diameter. However, to date, the polydispersity present in as-synthesized SWCNT populations has obscured the dependence of the SWCNT (13)C chemical shift on intrinsic parameters such as diameter and electronic structure, meaning that no information is gleaned for specific SWCNTs with unique chiral indices. In this article, we utilize a combination of (13)C labeling and density gradient ultracentrifugation (DGU) to produce an array of (13)C-labeled SWCNT populations with varying diameter, electronic structure, and chiral angle. We find that the SWCNT isotropic (13)C chemical shift decreases systematically with increasing diameter for semiconducting SWCNTs, in agreement with recent theoretical predictions that have heretofore gone unaddressed. Furthermore, we find that the (13)C chemical shifts for small diameter metallic and semiconducting SWCNTs differ significantly, and that the full-width of the isotropic peak for metallic SWCNTs is much larger than that of semiconducting nanotubes, irrespective of diameter.

  10. 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.

  11. Spectral density mapping at multiple magnetic fields suitable for 13C NMR relaxation studies

    NASA Astrophysics Data System (ADS)

    Kadeřávek, Pavel; Zapletal, Vojtěch; Fiala, Radovan; Srb, Pavel; Padrta, Petr; Přecechtělová, Jana Pavlíková; Šoltésová, Mária; Kowalewski, Jozef; Widmalm, Göran; Chmelík, Josef; Sklenář, Vladimír; Žídek, Lukáš

    2016-05-01

    Standard spectral density mapping protocols, well suited for the analysis of 15N relaxation rates, introduce significant systematic errors when applied to 13C relaxation data, especially if the dynamics is dominated by motions with short correlation times (small molecules, dynamic residues of macromolecules). A possibility to improve the accuracy by employing cross-correlated relaxation rates and on measurements taken at several magnetic fields has been examined. A suite of protocols for analyzing such data has been developed and their performance tested. Applicability of the proposed protocols is documented in two case studies, spectral density mapping of a uniformly labeled RNA hairpin and of a selectively labeled disaccharide exhibiting highly anisotropic tumbling. Combination of auto- and cross-correlated relaxation data acquired at three magnetic fields was applied in the former case in order to separate effects of fast motions and conformational or chemical exchange. An approach using auto-correlated relaxation rates acquired at five magnetic fields, applicable to anisotropically moving molecules, was used in the latter case. The results were compared with a more advanced analysis of data obtained by interpolation of auto-correlated relaxation rates measured at seven magnetic fields, and with the spectral density mapping of cross-correlated relaxation rates. The results showed that sufficiently accurate values of auto- and cross-correlated spectral density functions at zero and 13C frequencies can be obtained from data acquired at three magnetic fields for uniformly 13C -labeled molecules with a moderate anisotropy of the rotational diffusion tensor. Analysis of auto-correlated relaxation rates at five magnetic fields represents an alternative for molecules undergoing highly anisotropic motions.

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

    PubMed

    Winter, Klaus

    1979-01-01

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

  13. {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.

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

    PubMed

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

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

    PubMed

    Gebreselassie, Nikodimos A; Antoniewicz, Maciek R

    2015-09-01

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

  17. 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.

  18. Dynamic nuclear polarization of carbonyl and methyl 13C spins in acetate using trityl OX063

    NASA Astrophysics Data System (ADS)

    Niedbalski, Peter; Parish, Christopher; Lumata, Lloyd

    2015-03-01

    Hyperpolarization via dissolution dynamic nuclear polarization (DNP) is a physics technique that amplifies the magnetic resonance signals by several thousand-fold for biomedical NMR spectroscopy and imaging (MRI). Herein we have investigated the effect of carbon-13 isotopic location on the DNP of acetate (one of the biomolecules commonly used for hyperpolarization) at 3.35 T and 1.4 K using a narrow ESR linewidth free radical trityl OX063. We have found that the carbonyl 13C spins yielded about twice the polarization produced in methyl 13C spins. Deuteration of the methyl group, beneficial in the liquid-state, did not produce an improvement in the polarization level at cryogenic conditions. Concurrently, the solid-state nuclear relaxation of these samples correlate with the polarization levels achieved. These results suggest that the location of the 13C isotopic labeling in acetate has a direct impact on the solid-state polarization achieved and is mainly governed by the nuclear relaxation leakage factor.

  19. 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

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

    SciTech Connect

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

    1982-02-10

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

  1. Quantitative 13C NMR characterization of fast pyrolysis oils

    DOE PAGES

    Happs, Renee M.; Lisa, Kristina; Ferrell, III, Jack R.

    2016-10-20

    Quantitative 13C NMR analysis of model catalytic fast pyrolysis (CFP) oils following literature procedures showed poor agreement for aromatic hydrocarbons between NMR measured concentrations and actual composition. Furthermore, modifying integration regions based on DEPT analysis for aromatic carbons resulted in better agreement. Solvent effects were also investigated for hydrotreated CFP oil.

  2. Modeling of the 2007 JET ^13C migration experiments

    NASA Astrophysics Data System (ADS)

    Strachan, J. D.; Likonen, J.; Hakola, A.; Coad, J. P.; Widdowson, A.; Koivuranta, S.; Hole, D. E.; Rubel, M.

    2010-11-01

    Using the last run day of the 2007 JET experimental campaign, ^13CH4 was introduced repeatedly from the vessel top into a single plasma type (H-mode, Ip= 1.6 MA, Bt= 1.6 T). Similar experiments were performed in 2001 (vessel top into L-Mode) and 2004 (outer divertor into H-Mode). Divertor and wall tiles were removed and been analysed using secondary ion mass spectrometry (SIMS) and Rutherford backscattering (RBS) to determine the ^13C migration. ^13C was observed to migrate both to the inner (largest deposit), outer divertor (less) , and the floor tiles (least). This paper reports the EDGE2D/NIMBUS based modelling of the carbon migration. The emphasis is on the comparison of the 2007 results with the 2001 results where both injections were from the machine top but ELMs were present in 2007 but not present in 2001. The ELMs seemed to cause more ^13C re-erosion near the inner strike point. Also of interest is the difference in the Private Flux Region deposits where the changes in divertor geometry between 2004 and 2007 caused differences in the deposits. In 2007, the tilting of the load bearing tile caused regions of the PFR to be shadowed from the inner strike point which were not shadowed in 2004, indicating ^13C neutrals originated from the OSP.

  3. Complete 1H and 13C spectral assignment of floridoside.

    PubMed

    Simon-Colin, Christelle; Kervarec, Nelly; Pichon, Roger; Deslandes, Eric

    2002-02-11

    Floridoside (2-O-alpha-D-galactopyranosylglycerol) was extracted from the red marine alga Rhodymenia palmata, and purified by ion-exchange chromatography: 1D and 2D NMR spectroscopy experiments were used to unambiguously assign the complete 1H and 13C spectra.

  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. Quantitative analysis of deuterium using the isotopic effect on quaternary (13)C NMR chemical shifts.

    PubMed

    Darwish, Tamim A; Yepuri, Nageshwar Rao; Holden, Peter J; James, Michael

    2016-07-13

    Quantitative analysis of specifically deuterated compounds can be achieved by a number of conventional methods, such as mass spectroscopy, or by quantifying the residual (1)H NMR signals compared to signals from internal standards. However, site specific quantification using these methods becomes challenging when dealing with non-specifically or randomly deuterated compounds that are produced by metal catalyzed hydrothermal reactions in D2O, one of the most convenient deuteration methods. In this study, deuterium-induced NMR isotope shifts of quaternary (13)C resonances neighboring deuterated sites have been utilized to quantify the degree of isotope labeling of molecular sites in non-specifically deuterated molecules. By probing (13)C NMR signals while decoupling both proton and deuterium nuclei, it is possible to resolve (13)C resonances of the different isotopologues based on the isotopic shifts and the degree of deuteration of the carbon atoms. We demonstrate that in different isotopologues, the same quaternary carbon, neighboring partially deuterated carbon atoms, are affected to an equal extent by relaxation. Decoupling both nuclei ((1)H, (2)H) resolves closely separated quaternary (13)C signals of the different isotopologues, and allows their accurate integration and quantification under short relaxation delays (D1 = 1 s) and hence fast accumulative spectral acquisition. We have performed a number of approaches to quantify the deuterium content at different specific sites to demonstrate a convenient and generic analysis method for use in randomly deuterated molecules, or in cases of specifically deuterated molecules where back-exchange processes may take place during work up.

  7. 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.

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

    PubMed

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

    2015-01-01

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

  9. Target-controlled formation of silver nanoclusters in abasic site-incorporated duplex DNA for label-free fluorescence detection of theophylline

    NASA Astrophysics Data System (ADS)

    Park, Ki Soo; Oh, Seung Soo; Soh, H. Tom; Park, Hyun Gyu

    2014-08-01

    A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to the cytosine nucleobase is prevented. Consequently, fluorescent silver nanoclusters are not formed causing a significant reduction of the fluorescence signal. By employing this new sensor, theophylline can be highly selectively detected at a concentration as low as 1.8 μM. Finally, the diagnostic capability and practical application of this sensor were demonstrated by its use in detecting theophylline in human blood serum.A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to

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

    PubMed Central

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

    2016-01-01

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

  11. Ontogenetic shift in crayfish δ(13)C as a measure of land-water ecotonal coupling.

    PubMed

    France, Robert

    1996-07-01

    Although ontogenetic changes in the carbon isotope ratios of marine fauna have been well studied those of freshwater organisms have not. As a result, we may have a less than adequate assessment of the incorporation of allochthonous detritus into freshwater foodwebs. This study found a δ(13)C range of 9‰ for crayfish (Orconectes virilis) from oligotrophic Canadian Shield lakes. Much (60-83%) of this variability was explained by body size. A simple isotopic mixing model suggests that by their third year of life, crayfish in these lakes rely more substantially upon terrestrial detritus than epilithic algae for energy.

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

    PubMed Central

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

    1992-01-01

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

  13. Area per lipid and cholesterol interactions in membranes from separated local-field (13)C NMR spectroscopy.

    PubMed

    Leftin, Avigdor; Molugu, Trivikram R; Job, Constantin; Beyer, Klaus; Brown, Michael F

    2014-11-18

    Investigations of lipid membranes using NMR spectroscopy generally require isotopic labeling, often precluding structural studies of complex lipid systems. Solid-state (13)C magic-angle spinning NMR spectroscopy at natural isotopic abundance gives site-specific structural information that can aid in the characterization of complex biomembranes. Using the separated local-field experiment DROSS, we resolved (13)C-(1)H residual dipolar couplings that were interpreted with a statistical mean-torque model. Liquid-disordered and liquid-ordered phases were characterized according to membrane thickness and average cross-sectional area per lipid. Knowledge of such structural parameters is vital for molecular dynamics simulations, and provides information about the balance of forces in membrane lipid bilayers. Experiments were conducted with both phosphatidylcholine (dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC)) and egg-yolk sphingomyelin (EYSM) lipids, and allowed us to extract segmental order parameters from the (13)C-(1)H residual dipolar couplings. Order parameters were used to calculate membrane structural quantities, including the area per lipid and bilayer thickness. Relative to POPC, EYSM is more ordered in the ld phase and experiences less structural perturbation upon adding 50% cholesterol to form the lo phase. The loss of configurational entropy is smaller for EYSM than for POPC, thus favoring its interaction with cholesterol in raftlike lipid systems. Our studies show that solid-state (13)C NMR spectroscopy is applicable to investigations of complex lipids and makes it possible to obtain structural parameters for biomembrane systems where isotope labeling may be prohibitive.

  14. Short-term d13C changes in cultivated soils from Mexico

    NASA Astrophysics Data System (ADS)

    Lounejeva, E.; Etchevers, J.; Morales Puente, P.; Cienfuegos Alvarado, E.; Sedov, S.; Solleiro, E.; Hidalgo, C.

    2007-05-01

    (generally less than -20%o). The common feature for the soils under the Traditional and Traditional improved treatments was a C3 enrichment of the superficial SOM component compared to the underlying layers as a consequence of the dominance of the cultivated C3 species. A similar but more accentuated negative shift is also observed in the SOM from the forest soil (non-cultivated soil d13C -25.2), so the interpretation is uncertain. In the Traditional Organic treatment a clear and perceptible increment of d13C in the SOM carbon signature was observed. This was attributed mainly to the fact that cows manure may contain a lot of C4 coming from feedstuff rich in corn grain that is provided to the animals during grass shortage periods . However, the maize crop introduced in the rotation during the 3rd year had no major effect on the tepetates carbon isotopic signature. The stable isotopic carbon data corresponding to a short period (4 years) of observation in uniformly managed soil ecosystems showed that d13C changed due to the quality of the residues (relative abundance of C3/C4 species) incorporated to the SOC, but this memory is susceptible to undergo changes in the short term and could be rapidly reversed as a consequence of crop management.

  15. 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

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

    PubMed Central

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

    2015-01-01

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

  17. A novel proteomic approach for specific identification of tyrosine kinase substrates using [13C]tyrosine.

    PubMed

    Ibarrola, Nieves; Molina, Henrik; Iwahori, Akiko; Pandey, Akhilesh

    2004-04-16

    Proteomic studies to find substrates of tyrosine kinases generally rely on identification of protein bands that are "pulled down" by antiphosphotyrosine antibodies from ligand-stimulated samples. One can obtain erroneous results from such experiments because of two major reasons. First, some proteins might be basally phosphorylated on tyrosine residues in the absence of ligand stimulation. Second, proteins can bind non-specifically to the antibodies or the affinity matrix. Induction of phosphorylation of proteins by ligand must therefore be confirmed by a different approach, which is not always feasible. We have developed a novel proteomic approach to identify substrates of tyrosine kinases in signaling pathways studies based on in vivo labeling of proteins with "light" (12C-labeled) or "heavy" (13C-labeled) tyrosine. This stable isotope labeling in cell culture method enables the unequivocal identification of tyrosine kinase substrates, as peptides derived from true substrates give rise to a unique signature in a mass spectrometry experiment. By using this approach, from a single experiment, we have successfully identified several known substrates of insulin signaling pathway and a novel substrate, polymerase I and transcript release factor, a protein that is implicated in the control of RNA metabolism and regulation of type I collagen promoters. This approach is amenable to high throughput global studies as it simplifies the specific identification of substrates of tyrosine kinases as well as serine/threonine kinases using mass spectrometry.

  18. Millimeter and submillimeter wave spectra of 13C methylamine

    NASA Astrophysics Data System (ADS)

    Motiyenko, R. A.; Margulès, L.; Ilyushin, V. V.; Smirnov, I. A.; Alekseev, E. A.; Halfen, D. T.; Ziurys, L. M.

    2016-03-01

    Context. Methylamine (CH3NH2) is a light molecule of astrophysical interest, which has an intensive rotational spectrum that extends in the submillimeter wave range and far beyond, even at temperatures characteristic for the interstellar medium. It is likely for 13C isotopologue of methylamine to be identified in astronomical surveys, but there is no information available for the 13CH3NH2 millimeter and submillimeter wave spectra. Aims: In this context, to provide reliable predictions of 13CH3NH2 spectrum in millimeter and submillimeter wave ranges, we have studied rotational spectra of the 13C methylamine isotopologue in the frequency range from 48 to 945 GHz. Methods: The spectrum of 13C methylamine was recorded using conventional absorption spectrometers. The analysis of the rotational spectrum of 13C methylamine in the ground vibrational state was performed on the basis of the group-theoretical high-barrier tunneling Hamiltonian that was developed for methylamine. The available multiple observations of the parent methylamine species toward Sgr B2(N) at 1, 2, and 3 mm using the Submillimeter Telescope and the 12 m antenna of the Arizona Radio Observatory were used to make a search for interstellar 13CH3NH2. Results: In the recorded spectra, we have assigned 2721 rotational transitions that belong to the ground vibrational state of the 13CH3NH2. These measurements were fitted to the Hamiltonian model that uses 75 parameters to achieve an overall weighted rms deviation of 0.73. On the basis of these spectroscopic results, predictions of transition frequencies in the frequency range up to 950 GHz with J ≤ 50 and Ka ≤ 20 are presented. The search for interstellar 13C methylamine in available observational data was not successful and therefore only an upper limit of 6.5 × 1014 cm-2 can be derived for the column density of 13CH3NH2 toward Sgr B2(N), assuming the same source size, temperature, linewidth, and systemic velocity as for parent methylamine isotopic

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

    PubMed Central

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

    2016-01-01

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

  20. 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.

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

    PubMed

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

    2014-04-01

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

  2. 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.

  3. Carbon assimilation and turnover in grassland vegetation using an in situ (13)CO(2) pulse labelling system.

    PubMed

    Ostle, N; Ineson, P; Benham, D; Sleep, D

    2000-01-01

    A mobile laboratory was developed to administer a controlled flow of (13)C labelled CO(2) at ambient concentrations ( approximately 350 ppm) in the field. The stable isotope delivery (SID) system consists of an isotope-mixing unit with flow control to a series of 12 independent labelling chambers. In-line CPU controlled infrared gas analysers allow automated measurement of chamber CO(2) concentrations and gas flow management. A preliminary experiment was established on an upland pasture located at the NERC Soil Biodiversity experimental site, Sourhope, UK, in August 1999. The objective of this investigation was to determine the magnitude of pulse-derived C incorporation into a typical upland plant community. To achieve this, the SID system was deployed to pulse-label vegetation with CO(2) enriched with (13)C (50 atom %) at ambient concentrations ( approximately 350 ppm) on two consecutive days in August 1999. Samples of headspace CO(2), shoot and root were taken on four occasions over a period of 28 days after (13)C labelling. These materials were then prepared for (13)C/(12)C ratio determination by continuous-flow/combustion/isotope ratio mass spectrometry (CF-C-IRMS). Results showed that pulse derived CO(2)-C was assimilated at a rate of 128 +/- 32 microg g shoot-C hour(-1). Dynamic samplings showed that pulse-derived (13)C concentrations in the labelled plant tissues declined by 77.4 +/- 6% after 48 hours. The rapid decline in (13)C concentrations in plant matter was the result of C loss from the plant in the form of respired CO(2) and root exudates, and dilution by subsequent unlabelled C assimilates. This novel system offers considerable potential for in situ tracer investigations.

  4. The 13C nuclear magnetic resonance in graphite intercalation compounds

    NASA Technical Reports Server (NTRS)

    Tsang, T.; Resing, H. A.

    1985-01-01

    The (13)C NMR chemical shifts of graphite intercalation compounds were calculated. For acceptor types, the shifts come mainly from the paramagnetic (Ramsey) intra-atomic terms. They are related to the gross features of the two-dimensional band structures. The calculated anisotropy is about -140 ppm and is independent of the finer details such as charge transfer. For donor types, the carbon 2p pi orbitals are spin-polarized because of mixing with metal conduction electrons, thus there is an additional dipolar contribution which may be correlated with the electronic specific heat. The general agreement with experimental data is satisfactory.

  5. Oxygen atom transfer and oxidative water incorporation in cuboidal Mn3MO(n) complexes based on synthetic, isotopic labeling, and computational studies.

    PubMed

    Kanady, Jacob S; Mendoza-Cortes, Jose L; Tsui, Emily Y; Nielsen, Robert J; Goddard, William A; Agapie, Theodor

    2013-01-23

    The oxygen-evolving complex (OEC) of photosystem II contains a Mn(4)CaO(n) catalytic site, in which reactivity of bridging oxidos is fundamental to OEC function. We synthesized structurally relevant cuboidal Mn(3)MO(n) complexes (M = Mn, Ca, Sc; n = 3,4) to enable mechanistic studies of reactivity and incorporation of μ(3)-oxido moieties. We found that Mn(IV)(3)CaO(4) and Mn(IV)(3)ScO(4) were unreactive toward trimethylphosphine (PMe(3)). In contrast, our Mn(III)(2)Mn(IV)(2)O(4) cubane reacts with this phosphine within minutes to generate a novel Mn(III)(4)O(3) partial cubane plus Me(3)PO. We used quantum mechanics to investigate the reaction paths for oxygen atom transfer to phosphine from Mn(III)(2)Mn(IV)(2)O(4) and Mn(IV)(3)CaO(4). We found that the most favorable reaction path leads to partial detachment of the CH(3)COO(-) ligand, which is energetically feasible only when Mn(III) is present. Experimentally, the lability of metal-bound acetates is greatest for Mn(III)(2)Mn(IV)(2)O(4). These results indicate that even with a strong oxygen atom acceptor, such as PMe(3), the oxygen atom transfer chemistry from Mn(3)MO(4) cubanes is controlled by ligand lability, with the Mn(IV)(3)CaO(4) OEC model being unreactive. The oxidative oxide incorporation into the partial cubane, Mn(III)(4)O(3), was observed experimentally upon treatment with water, base, and oxidizing equivalents. (18)O-labeling experiments provided mechanistic insight into the position of incorporation in the partial cubane structure, consistent with mechanisms involving migration of oxide moieties within the cluster but not consistent with selective incorporation at the site available in the starting species. These results support recent proposals for the mechanism of the OEC, involving oxido migration between distinct positions within the cluster.

  6. Regulatory issues on breath tests and updates of recent advances on [13C]-breath tests.

    PubMed

    Modak, Anil S

    2013-09-01

    Over the last decade non invasive diagnostic phenotype [(13)C]-breath tests as well as tests using endogenous volatile organic compounds (VOCs) in breath have been researched extensively. However, only three breath tests have been approved by the FDA over the last 15 years. Despite the potential benefits of these companion diagnostic tests (CDx) for evaluation of drug metabolizing enzyme activities and standalone diagnostic tests for disease diagnosis to personalize medicine, the clinical and commercial development of breath tests will need to overcome a number of regulatory, financial and scientific hurdles prior to their acceptance into routine clinical practice. The regulatory agencies (FDA and EMEA) need to adapt and harmonize their approval process for companion diagnostic tests as well as standalone diagnostic breath tests for personalized medicine. The Center for Devices and Radiological Health has deemed any breath test that involves a labeled (13)C substrate/drug and a device requires a Pre Market Approval (PMA), which is analogous to an approved New Drug Application. A PMA is in effect, a private license granted to the applicant for marketing a particular medical device. Any breath test with endogenous VOCs along with a device can be approved via the 510(k) application. A number of (13)C breath tests with clinical applications have been researched recently and results have been published in reputed journals. Diagnostic companies will need to invest the necessary financial resources to develop and get regulatory approval for diagnostic breath tests capable of identifying responders/non responders for FDA approved drugs with narrow therapeutic indices (personalized medicine) or for evaluating the activity of drug metabolizing P450 polymorphic enzymes or for diagnosing diseases at an early stage or for monitoring the efficacy of medications. The financial success of these diagnostic breath tests will then depend entirely on how the test is marketed to

  7. Tracking the flow of bacterially derived 13C and 15N through soil faunal feeding channels.

    PubMed

    Crotty, F V; Blackshaw, R P; Murray, P J

    2011-06-15

    The soil food web has been referred to as a 'black box', a 'poor man's tropical rainforest' and an 'enigma', due to its opacity, diversity and the limited insight into feeding specificity. Here we investigate the flow of C and N through the soil food web as a way to gain understanding of the feeding interactions occurring. A bacterium, Pseudomonas lurida, was introduced to soil cores from two different habitats, a grassland and a woodland with the same soil type, enriched to 99 atom% in (13)C and (15)N, to trace the flow of bacterial C and N through the soil food web. Throughout the experiment the soil remained enriched in (13)C and (15)N. Almost all the invertebrates tested gained C and N enrichment indicative of the labelled bacteria, implying that bacterial feeding is a common mechanism within the soil. Only three groups were significantly enriched in both (13)C and (15)N in both habitats. These were Collembola (Entomobryomorpha), Acari (Oribatida), and Nematoda, indicating that these organisms are consuming the most bacteria within both systems. When the invertebrates were grouped into hypothesised trophic levels, those considered secondary decomposers were gaining the most enrichment across all invertebrates tested. This enrichment was also high in the micro-predators within the soil, implying that their main food source was the secondary decomposers, particularly the Collembola. Using an enriched bacterium to track the trophic transfer between organisms within the soil food web is a novel way of empirically showing that interactions are occurring, which normally cannot be seen.

  8. Acetate and bicarbonate assimilation and metabolite formation in Chlamydomonas reinhardtii: a 13C-NMR study.

    PubMed

    Singh, Himanshu; Shukla, Manish R; Chary, Kandala V R; Rao, Basuthkar J

    2014-01-01

    Cellular metabolite analyses by (13)C-NMR showed that C. reinhardtii cells assimilate acetate at a faster rate in heterotrophy than in mixotrophy. While heterotrophic cells produced bicarbonate and CO2aq, mixotrophy cells produced bicarbonate alone as predominant metabolite. Experiments with singly (13)C-labelled acetate ((13)CH(3)-COOH or CH(3)-(13)COOH) supported that both the (13)C nuclei give rise to bicarbonate and CO2(aq). The observed metabolite(s) upon further incubation led to the production of starch and triacylglycerol (TAG) in mixotrophy, whereas in heterotrophy the TAG production was minimal with substantial accumulation of glycerol and starch. Prolonged incubation up to eight days, without the addition of fresh acetate, led to an increased TAG production at the expense of bicarbonate, akin to that of nitrogen-starvation. However, such TAG production was substantially high in mixotrophy as compared to that in heterotrophy. Addition of mitochondrial un-coupler blocked the formation of bicarbonate and CO2(aq) in heterotrophic cells, even though acetate uptake ensued. Addition of PSII-inhibitor to mixotrophic cells resulted in partial conversion of bicarbonate into CO2(aq), which were found to be in equilibrium. In an independent experiment, we have monitored assimilation of bicarbonate via photoautotrophy and found that the cells indeed produce starch and TAG at a much faster rate as compared to that in mixotrophy and heterotrophy. Further, we noticed that the accumulation of starch is relatively more as compared to TAG. Based on these observations, we suggest that acetate assimilation in C. reinhardtii does not directly lead to TAG formation but via bicarbonate/CO2(aq) pathways. Photoautotrophic mode is found to be the best growth condition for the production of starch and TAG and starch in C. reinhardtii.

  9. 13C and 1H Nuclear Magnetic Resonance Study of Glycogen Futile Cycling in Strains of the Genus Fibrobacter

    PubMed Central

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

    1998-01-01

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

  10. 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.

  11. Production and application of high quality stable isotope-labeled human immunoglobulin G1 for mass spectrometry analysis.

    PubMed

    Phillip, Amsler; Thierry, Wolf; Christian, Lanshoeft; Anja, Bettighofer; Jochen, Eisfeld; Thomas, Moenius; Claudia, Probst; Coralie, Etter; Olivier, Heudi

    2016-12-12

    Here, we describe the production of stable isotope-labeled human immunoglobulin G1 ([(13) C]-hIgG1) using [(13) C]-L-lysine/arginine-labeled hIgG1. The fermentation process was run in shake flasks containing labeled arginine and lysinethat were incorporated into the produced recombinant hIgG1. The [(13) C]-hIgG1 was purified, and label incorporation was determined to be >99% at all lysine and arginine moieties. Sequence coverage was confirmed by peptide mapping. [(13) C]-hIgG1 was then used as an internal standard (IS) for the development of a liquid chromatography-tandem mass spectrometry method applicable to the quantitative analysis of all human types of hIgG1 in rat serum. Four conserved peptides, namely, GPSVFPLAPSSK, TTPPVLDSDGSFFLYSK, VVSVLTVLHQDWLNGK, and FNWYVDGVEVHNAK, originating from different parts of the fraction crystallizable region of hIgG1, were used for quantitation of hIgG1 in rat serum. The calibration curves with a coefficient of determination (r(2) ) between 0.9950 and 0.9962 resulting from the peak area ratio of each peptide to its respective labeled IS were reproducible. A mean bias within ±20.0% of the nominal values and a precision of ≤20.0 % were obtained for the calibration standards and quality control samples for each peptide. [(13) C]-hIgG1 was shown as a suitable IS for quantitative hIgG1 analysis in preclinical species by LC-MS/MS.

  12. Solubilization and localization of weakly polar lipids in unsonicated egg phosphatidylcholine: A sup 13 C MAS NMR study

    SciTech Connect

    Hamilton, J.A. ); Fujito, D.T.; Hammer, C.F. )

    1991-03-19

    The weakly polar lipids cholesteryl ester, triacylglycerol, and diacylglycerol incorporate to a limited extent into the lamellar structure of small unilamellar vesicles. The localization of the carbonyl group(s) at the aqueous interface was detected by ({sup 13}C)carbonyl chemical shift changes relative to the neat unhydrated lipid. This study uses {sup 13}C NMR to investigate the interactions of thes lipids with unsonicated (multilamellar) phosphatidylcholine, a model system for cellular membranes and surfaces of emulsion particles with low curvature. Magic angle spinning reduced the broad lines of the unsonicated dispersions to narrow lines comparable to those from sonicated dispersions. ({sup 13}C)Carbonyl chemical shifts revealed incorporation of the three lipids into the lamellar structure of the unsonicated phospholipids and a partial hydration of the carbonyl groups similar to that observed in small vesicles. Other properties of interfacial weakly polar lipids in multilayers were similar to those in small unilamellar bilayers. There is thus a general tendency of weakly polar lipids to incorparate at least to a small extent into the lamellar structure of phospholipids and take on interfacial properties that are distinct from their bulk-phase properties. This pool of surface-located lipid is likely to be directly involved in enzymatyic transformations and protein-mediated transport. The {sup 13}C magic angle spinning NMR method may be generally useful for determining the orientation of molecules in model membranes.

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  15. Dynamic pictures of membrane proteins in two-dimensional crystal, lipid bilayer and detergent as revealed by site-directed solid-state 13C NMR.

    PubMed

    Saitô, Hazime

    2004-11-01

    We have compared site-directed 13C solid-state NMR spectra of [3-13C]Ala- and/or [1-13C]Val-labeled membrane proteins, including bacteriorhodopsin (bR), pharaonis phoborhodopin (ppR), its cognate transducer (pHtrII) and Escherichia coli diacylglycerol kinase (DGK), in two-dimensional (2D) crystal, lipid bilayers, and detergent. Restricted fluctuation motions of these membrane proteins due to oligomerization of bR by specific protein-protein interactions in the 2D crystalline lattice or protein complex between ppR and pHtrII provide the most favorable environment to yield well-resolved, fully visible 13C NMR signals for [3-13C]Ala-labeled proteins. In contrast, several signals from such membrane proteins were broadened or lost owing to interference of inherent fluctuation frequencies (10(4)-10(5)Hz) with frequency of either proton decoupling or magic angle spinning, if their 13C NMR spectra were recorded as a monomer in lipid bilayers at ambient temperature. The presence of such protein dynamics is essential for the respective proteins to achieve their own biological functions. Finally, spectral broadening found for bR and DGK in detergents were discussed.

  16. 15N and13C NMR investigation of hydroxylamine-derivatized humic substances

    USGS Publications Warehouse

    Thorn, K.A.; Arterburn, J.B.; Mikita, M.A.

    1992-01-01

    Five fulvic and humic acid samples of diverse origins were derivatized with 15N-labeled hydroxylamine and analyzed by liquid-phase 15N NMR spectrometry. The 15N NMR spectra indicated that hydroxylamine reacted similarly with all samples and could discriminate among carbonyl functional groups. Oximes were the major derivatives; resonances attributable to hydroxamic acids, the reaction products of hydroxylamine with esters, and resonances attributable to the tautomeric equilibrium position between the nitrosophenol and monoxime derivatives of quinones, the first direct spectroscopic evidence for quinones, also were evident. The 15N NMR spectra also suggested the presence of nitriles, oxazoles, oxazolines, isocyanides, amides, and lactams, which may all be explained in terms of Beckmann reactions of the initial oxime derivatives. INEPT and ACOUSTIC 15N NMR spectra provided complementary information on the derivatized samples. 13C NMR spectra of derivatized samples indicated that the ketone/quinone functionality is incompletely derivatized with hydroxylamine. ?? 1991 American Chemical Society.

  17. Absorption and distribution kinetics of the 13C-labeled tomato carotenoid phytoene in healthy adults

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytoene is a tomato carotenoid which may contribute to the apparent health benefits of tomato consumption. While phytoene is a less prominent tomato carotenoid than lycopene, it is a major carotenoid in various human tissues. Phytoene distribution to plasma lipoproteins and tissues differs from lyc...

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

    PubMed

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

    2008-06-12

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

  19. In vivo measurement of aldehyde dehydrogenase-2 activity in rat liver ethanol model using dynamic MRSI of hyperpolarized [1-(13) C]pyruvate.

    PubMed

    Josan, Sonal; Xu, Tao; Yen, Yi-Fen; Hurd, Ralph; Ferreira, Julio; Chen, Che-Hong; Mochly-Rosen, Daria; Pfefferbaum, Adolf; Mayer, Dirk; Spielman, Daniel

    2013-06-01

    To date, measurements of the activity of aldehyde dehydrogenase-2 (ALDH2), a critical mitochondrial enzyme for the elimination of certain cytotoxic aldehydes in the body and a promising target for drug development, have been largely limited to in vitro methods. Recent advancements in MRS of hyperpolarized (13) C-labeled substrates have provided a method to detect and image in vivo metabolic pathways with signal-to-noise ratio gains greater than 10 000-fold over conventional MRS techniques. However aldehydes, because of their toxicity and short T1 relaxation times, are generally poor targets for such (13) C-labeled studies. In this work, we show that dynamic MRSI of hyperpolarized [1-(13) C]pyruvate and its conversion to [1-(13) C]lactate can provide an indirect in vivo measurement of ALDH2 activity via the concentration of NADH (nicotinamide adenine dinucleotide, reduced form), a co-factor common to both the reduction of pyruvate to lactate and the oxidation of acetaldehyde to acetate. Results from a rat liver ethanol model (n = 9) show that changes in (13) C-lactate labeling following the bolus injection of hyperpolarized pyruvate are highly correlated with changes in ALDH2 activity (R(2) = 0.76).

  20. High-field 13C NMR spectroscopy of tissue in Vivo. A double-resonance surface-coil probe

    NASA Astrophysics Data System (ADS)

    Reo, Nicholas V.; Ewy, Coleen S.; Siegfried, Barry A.; Ackerman, Joseph J. H.

    A double-resonance surface-coil NMR probe is described for performance of high-field (8.5 T) proton decoupled carbon-13 experiments with tissue in vivo. The probe may be accommodated in standard, 89 mm i.d. clear bore, commercial spectrometers and is suitable for studies utilizing small laboratory animals such as mice, hamsters, and rats. A coaxial coil design is employed (10 mm diameter 13C coil, 20 mm diameter 1H coil) which provides ca. 40 dB attenuation between the 13C observe and 1H decouple channels. The inherent efficiency of the surface-coil configuration provides a sensitivity comparable to a commercial probe of the same nominal dimension (10 mm Helmholtz coil) and assures adequate decoupling in conductive samples with ca. 3-5 W power. In the absence of 13C isotopic enrichment, NMR spectra of rat leg, liver, and brain in vivo provide signalto-noise sufficient for 10 min time resolution. Administration of 100 mg of 90% 13C-labeled glucose into a peripheral vein of a ca. 300 g rat resulted in a liver glucose resonance which could be monitored with good signal-to-noise and 3 min time resolution.

  1. Following Suberization in Potato Wound Periderm by Histochemical and Solid-State 13C Nuclear Magnetic Resonance Methods.

    PubMed

    Stark, R. E.; Sohn, W.; Pacchiano Jr, R. A.; Al-Bashir, M.; Garbow, J. R.

    1994-02-01

    The time course of suberization in wound periderm from potato (Solanum tuberosum L.) has been monitored by histochemical and high-resolution solid-state nuclear magnetic resonance (NMR) methods. Light microscopy conducted after selective staining of the lipid and double-bonded constituents shows that suberin is deposited at the outermost intact cell-wall surface during the first 7 d of wound healing; suberization forms a barrier to tissue infiltration at later times. Cross polarization-magic angle spinning 13C NMR spectra demonstrate the deposition of a polyester containing all major suberin functional groups after just 4 d of wound healing. Initially the suberin includes a large proportion of aromatic groups and fairly short aliphatic chains, but the spectral data demonstrate the growing dominance of long-chain species during the period 7 to 14 d after wounding. The results of preliminary 13C-labeling experiments with sodium [2-13C]acetate and DL-[1-13C]phenylalanine provide an excellent prospectus for future NMR-based studies of suberin biosynthesis.

  2. Improved ground state and ν12 = 1 state rovibrational constants for ethylene-13C2 (13C2H4)

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

    The Fourier transform infrared (FTIR) absorption spectrum of the ν12 fundamental band of ethylene-13C2 (13C2H4) was recorded in the frequency range of 1350-1550 cm-1 with unapodized resolution of 0.0063 cm-1. Improved upper state (ν12 = 1) rovibrational constants consisting of three rotational, five quartic and five sextic constants were derived by assigning and fitting 1731 infrared transitions using Watson's A-reduced Hamiltonian in the Ir representation. The root-mean-square deviation of the fit was 0.00030 cm-1. More higher-order upper state (ν12 = 1) constant were derived in the present analysis than previously reported. Improved ground state rovibrational constants were also determined from the fit of 1104 ground state combination differences (GSCD) with a root-mean-square deviation of 0.00029 cm-1. The A-type ν12 band centered at 1436.65409 ± 0.00002 cm-1 has a calculated inertial defect Δ12 is 0.242896 ± 0.000007 μÅ2. No indications of perturbation were found in the analysis of the band.

  3. A Proof of Concept Study to Detect Urease Producing Bacteria in Lungs Using Aerosolized 13C-Urea

    PubMed Central

    Timmins, Graham; Davies, Lea; Heynekamp, Theresa; Harkins, Michelle; Sharp, Zachary D.; Kelly, H. William

    2016-01-01

    This is a “proof of concept” study to determine whether inhalation of 13C-urea can be safely used to detect the presence of urease producing bacteria in the airways of patients with cystic fibrosis (CF) by detecting 13CO2 in breath. This was a prospective, 2-part, open label, single-center, single-arm, single-administration, dose-escalation investigational device exemption trial. First, the safety of 20 and 50 mg inhaled 13C-urea was evaluated in 6 healthy adult participants. Then, 3 adult CF participants colonized with Pseudomonas aeruginosa were enrolled for each dose of inhaled 13C-urea. The safety of inhaled 13C-urea was assessed by spirometry and physical examination. 13C-urea was administered using a jet nebulizer, followed by serial spirometry (10 min and 30 min post inhalation) and collection of exhaled breath at 5, 10, and 15 min post inhalation. There was no clinical significant change in any of the spirometry values compared to baseline in healthy participants and CF patients. Mean of 13CO2/12CO2 delta over baseline (DOB) values in CF participants at 5, 10, and 15 min post inhalation was as follows: 20 mg dose 4‰ (2.2‰–4.9‰), 1‰ (1.0‰–1.4‰), and 1‰ (0.4‰–1.5‰); 50 mg dose: 10‰ (6.2‰–14.5‰), 3‰ (2.1‰–4.3‰), and 1.5‰ (0.6‰–2.3‰). Inhaled 13C-urea for detection of urease producing bacteria was safe, and preliminary data suggest that 13CO2/12CO2 DOB values may be higher in CF patients with P. aeruginosa at 5–10 min after inhalation of 13C-urea. A future direction is to investigate use of inhaled 13C-urea in young children who have difficulty producing sputum for culturing. PMID:27458537

  4. The Influence of Plant Litter on Soil Water Repellency: Insight from 13C NMR Spectroscopy.

    PubMed

    Cesarano, Gaspare; Incerti, Guido; Bonanomi, Giuliano

    2016-01-01

    Soil water repellency (SWR, i.e. reduced affinity for water owing to the presence of organic hydrophobic coatings on soil particles) has relevant hydrological implications because low rates of infiltration enhance water runoff, and untargeted diffusion of fertilizers and pesticides. Previous studies investigated the occurrence of SWR in ecosystems with different vegetation cover but did not clarify its relationships with litter biochemical quality. Here, we investigated the capability of different plant litter types to induce SWR by using fresh and decomposed leaf materials from 12 species, to amend a model sandy soil over a year-long microcosm experiment. Water repellency, measured by the Molarity of an Ethanol Droplet (MED) test, was tested for the effects of litter species and age, and compared with litter quality assessed by 13C-CPMAS NMR in solid state and elemental chemical parameters. All litter types were highly water repellent, with MED values of 18% or higher. In contrast, when litter was incorporated into the soil, only undecomposed materials induced SWR, but with a large variability of onset and peak dynamics among litter types. Surprisingly, SWR induced by litter addition was unrelated to the aliphatic fraction of litter. In contrast, lignin-poor but labile C-rich litter, as defined by O-alkyl C and N-alkyl and methoxyl C of 13C-CPMAS NMR spectral regions, respectively, induced a stronger SWR. This study suggests that biochemical quality of plant litter is a major controlling factor of SWR and, by defining litter quality with 13C-CPMAS NMR, our results provide a significant novel contribution towards a full understanding of the relationships between plant litter biochemistry and SWR.

  5. The Influence of Plant Litter on Soil Water Repellency: Insight from 13C NMR Spectroscopy

    PubMed Central

    Cesarano, Gaspare; Incerti, Guido; Bonanomi, Giuliano

    2016-01-01

    Soil water repellency (SWR, i.e. reduced affinity for water owing to the presence of organic hydrophobic coatings on soil particles) has relevant hydrological implications because low rates of infiltration enhance water runoff, and untargeted diffusion of fertilizers and pesticides. Previous studies investigated the occurrence of SWR in ecosystems with different vegetation cover but did not clarify its relationships with litter biochemical quality. Here, we investigated the capability of different plant litter types to induce SWR by using fresh and decomposed leaf materials from 12 species, to amend a model sandy soil over a year-long microcosm experiment. Water repellency, measured by the Molarity of an Ethanol Droplet (MED) test, was tested for the effects of litter species and age, and compared with litter quality assessed by 13C-CPMAS NMR in solid state and elemental chemical parameters. All litter types were highly water repellent, with MED values of 18% or higher. In contrast, when litter was incorporated into the soil, only undecomposed materials induced SWR, but with a large variability of onset and peak dynamics among litter types. Surprisingly, SWR induced by litter addition was unrelated to the aliphatic fraction of litter. In contrast, lignin-poor but labile C-rich litter, as defined by O-alkyl C and N-alkyl and methoxyl C of 13C-CPMAS NMR spectral regions, respectively, induced a stronger SWR. This study suggests that biochemical quality of plant litter is a major controlling factor of SWR and, by defining litter quality with 13C-CPMAS NMR, our results provide a significant novel contribution towards a full understanding of the relationships between plant litter biochemistry and SWR. PMID:27022916

  6. Millimeter and submillimeter wave spectra of 13C-glycolaldehydes

    NASA Astrophysics Data System (ADS)

    Haykal, I.; Motiyenko, R. A.; Margulès, L.; Huet, T. R.

    2013-01-01

    Context. Glycolaldehyde (CH2OHCHO) is the simplest sugar and an important intermediate in the path toward forming more complex biologically relevant molecules. Astronomical surveys of interstellar molecules, such as those available with the very sensitive ALMA telescope, require preliminary laboratory investigations of the microwave and submillimeter-wave spectra of molecular species including new isotopologs - to identify these in the interstellar media. Aims: To achieve the detection of the 13C isotopologs of glycolaldehyde in the interstellar medium, their rotational spectra in the millimeter and submillimeter-wave regions were studied. Methods: The spectra of 13CH2OHCHO and CH2OH13CHO were recorded in the 150-945 GHz spectral range in the laboratory using a solid-state submillimeter-wave spectrometer in Lille. The observed line frequencies were measured with an accuracy of 30 kHz up to 700 GHz and of 50 kHz above 700 GHz. We analyzed the spectra with a standard Watson Hamiltonian. Results: About 10 000 new lines were identified for each isotopolog. The spectroscopic parameters were determined for the ground- and the three lowest vibrational states up to 945 and 630 GHz. Previous microwave assignments of 13CH2OHCHO were not confirmed. Conclusions: The provided line-lists and sets of molecular parameters meet the needs for a first astrophysical search of 13C-glycolaldehydes. Full Tables 3 and 4 are only available at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/549/A96

  7. Differential allocation of carbon in mosses and grasses governs ecosystem sequestration: a 13C tracer study in the high Arctic.

    PubMed

    Woodin, S J; van der Wal, R; Sommerkorn, M; Gornall, J L

    2009-12-01

    *This study investigates the influence of vegetation composition on carbon (C) sequestration in a moss-dominated ecosystem in the Arctic. *A (13)C labelling study in an arctic wet meadow was used to trace assimilate into C pools of differing recalcitrance within grasses and mosses and to determine the retention of C by these plant groups. *Moss retained 70% of assimilated (13)C over the month following labelling, which represented half the growing season. By contrast, the vascular plants, comprising mostly grasses, retained only 40%. The mechanism underlying this was that moss allocated 80% of the (13)C to recalcitrant C pools, a much higher proportion than in grasses (56%). *This method enabled elucidation of a plant trait that will influence decomposition and hence persistence of assimilated C in the ecosystem. We predict that moss-dominated vegetation will retain sequestered C more strongly than a grass-dominated community. Given the strong environmental drivers that are causing a shift from moss to grass dominance, this is likely to result in a reduction in future ecosystem C sink strength.

  8. Positional Enrichment by Proton Analysis (PEPA): A One-Dimensional (1) H-NMR Approach for (13) C Stable Isotope Tracer Studies in Metabolomics.

    PubMed

    Vinaixa, Maria; Rodríguez, Miguel A; Aivio, Suvi; Capellades, Jordi; Gómez, Josep; Canyellas, Nicolau; Stracker, Travis H; Yanes, Oscar

    2017-03-20

    A novel metabolomics approach for NMR-based stable isotope tracer studies called PEPA is presented, and its performance validated using human cancer cells. PEPA detects the position of carbon label in isotopically enriched metabolites and quantifies fractional enrichment by indirect determination of (13) C-satellite peaks using 1D-(1) H-NMR spectra. In comparison with (13) C-NMR, TOCSY and HSQC, PEPA improves sensitivity, accelerates the elucidation of (13) C positions in labeled metabolites and the quantification of the percentage of stable isotope enrichment. Altogether, PEPA provides a novel framework for extending the high-throughput of (1) H-NMR metabolic profiling to stable isotope tracing in metabolomics, facilitating and complementing the information derived from 2D-NMR experiments and expanding the range of isotopically enriched metabolites detected in cellular extracts.

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

    PubMed

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

    2003-01-01

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