Science.gov

Sample records for 13c labeled samples

  1. Magic angle sample spinning sup 13 C nuclear magnetic resonance of isotopically labeled bacteriorhodopsin

    SciTech Connect

    Engelhard, M.; Hess, B.; Emeis, D.; Metz, G.; Kreutz, W.; Siebert, F. )

    1989-05-02

    Bacteriorhodopsin (bR), the light-driven proton pump protein from Halobacterium halobium, was biosynthetically labeled with (4-{sup 13}C)Asp. The incorporation yield was 48%. The magic angle sample spinning (MASS) {sup 13}C nuclear magnetic resonance (NMR) spectrum of this sample revealed six different peaks superimposed on a broad band of naturally abundant peptide-bond {sup 13}C. Two of the six carbonyl signals can be attributed to internal-protonated Asp carboxyl groups, one of which might be Asp115. An additional resonance at 110 ppm can be associated with the C-11 carbon of Trp, indicating an unusual biosynthetic pathway of this amino acid in Halobacterium halobium. Similar measurements performed on papain-treated purple membrane which lacks the C-terminal tail display two new intense signals at 178 and 178.9 ppm. If the same spectrum is taken without cross-polarization, these signals do not decrease or disappear. On the basis of their intensities and their chemical shifts, one can assign in addition to the C-terminal Asp four Asp residues facing the cytoplasmic phase. In native bR, at least two of these form a salt-bridge-like bond which also might include the C-terminal tail. These experiments not only provide data about the chemical environment of the Asp residues within the hydrophobic core of bacteriorhodopsin but also yield information about the interactions between surface components.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Variability of 13C-labeling in plant leaves.

    PubMed

    Nguyen Tu, Thanh Thuy; Biron, Philippe; Maseyk, Kadmiel; Richard, Patricia; Zeller, Bernd; Quénéa, Katell; Alexis, Marie; Bardoux, Gérard; Vaury, Véronique; Girardin, Cyril; Pouteau, Valérie; Billiou, Daniel; Bariac, Thierry

    2013-09-15

    Plant tissues artificially labeled with (13)C are increasingly used in environmental studies to unravel biogeochemical and ecophysiological processes. However, the variability of (13)C-content in labeled tissues has never been carefully investigated. Hence, this study aimed at documenting the variability of (13)C-content in artificially labeled leaves. European beech and Italian ryegrass were subjected to long-term (13)C-labeling in a controlled-environment growth chamber. The (13)C-content of the leaves obtained after several months labeling was determined by isotope ratio mass spectrometry. The (13)C-content of the labeled leaves exhibited inter- and intra-leaf variability much higher than those naturally occurring in unlabeled plants, which do not exceed a few per mil. This variability was correlated with labeling intensity: the isotope composition of leaves varied in ranges of ca 60‰ and 90‰ for experiments that led to average leaf (13)C-content of ca +15‰ and +450‰, respectively. The reported variability of isotope composition in (13)C-enriched leaves is critical, and should be taken into account in subsequent experimental investigations of environmental processes using (13)C-labeled plant tissues. Copyright © 2013 John Wiley & Sons, Ltd.

  7. 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.

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

    PubMed

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

    2014-08-01

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

  9. Metabolic flux analysis using 13C peptide label measurements

    USDA-ARS?s Scientific Manuscript database

    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...

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 13C isotope effects on 1H chemical shifts: NMR spectral analysis of 13C-labelled D-glucose and some 13C-labelled amino acids.

    PubMed

    Tiainen, Mika; Maaheimo, Hannu; Soininen, Pasi; Laatikainen, Reino

    2010-02-01

    The one- and two-bond (13)C isotope shifts, typically -1.5 to -2.5 ppb and -0.7 ppb respectively, in non-cyclic aliphatic systems and up to -4.4 ppb and -1.0 ppb in glucose cause effects that need to be taken into account in the adaptive NMR spectral library-based quantification of the isotopomer mixtures. In this work, NMR spectral analyses of some (13)C-labelled amino acids, D-glucose and other small compounds were performed in order to obtain rules for prediction of the (13)C isotope effects on (1)H chemical shifts. It is proposed that using the additivity rules, the isotope effects can be predicted with a sufficient accuracy for amino acid isotopomer applications. For glucose the effects were found strongly non-additive. The complete spectral analysis of fully (13)C-labelled D-glucose made it also possible to assign the exocyclic proton signals of the glucose. Copyright 2009 John Wiley & Sons, Ltd.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. Gram-scale synthesis and efficient purification of 13C-labeled levoglucosan from 13C glucose.

    PubMed

    Alexander, Lisa; Hoyt, Caroline; Michalczyk, Ryszard; Wu, Ruilian; Thorn, Dave L; Silks, L A Pete

    2013-01-01

    (13)C-Labeled levoglucosan has been synthesized and purified in good yield, and on the gram scale in one step from commercially available (13)C glucose. This one-step protocol uses 2-chloro-1,3-dimethylimidazolinium chloride that serves to selectively activate the anomeric carbon toward substitution reactions. The labeled glucose is then smoothly converted to the anhydroglucose. Purification is efficiently achieved on large scale by chromatography on silica gel. Published 2012. This article is a US Government work and is in the public domain in the USA.

  20. Metabolism of uniformly labeled (13)C-eicosapentaenoic acid and (13)C-arachidonic acid in young and old men.

    PubMed

    Léveillé, Pauline; Chouinard-Watkins, Raphaël; Windust, Anthony; Lawrence, Peter; Cunnane, Stephen C; Brenna, J Thomas; Plourde, Mélanie

    2017-08-01

    Background: Plasma eicosapentaenoic acid (EPA) and arachidonic acid (AA) concentrations increase with age.Objective: The aim of this study was to evaluate EPA and AA metabolism in young and old men by using uniformly labeled carbon-13 ((13)C) fatty acids.Design: Six young (∼25 y old) and 6 old (∼75 y old) healthy men were recruited. Each participant consumed a single oral dose of 35 mg (13)C-EPA and its metabolism was followed in the course of 14 d in the plasma and 28 d in the breath. After the washout period of ≥28 d, the same participants consumed a single oral dose of 50 mg (13)C-AA and its metabolism was followed for 28 d in plasma and breath.Results: There was a time × age interaction for (13)C-EPA (Ptime × age = 0.008), and the shape of the postprandial curves was different between young and old men. The (13)C-EPA plasma half-life was ∼2 d for both young and old men (P = 0.485). The percentage dose recovered of (13)C-EPA per hour as (13)CO2 and the cumulative β-oxidation of (13)C-EPA did not differ between young and old men. At 7 d, however, old men had a >2.2-fold higher plasma (13)C-DHA concentration synthesized from (13)C-EPA compared with young men (Page = 0.03). (13)C-AA metabolism was not different between young and old men. The (13)C-AA plasma half-life was ∼4.4 d in both young and old participants (P = 0.589).Conclusions: The metabolism of (13)C-AA was not modified by age, whereas (13)C-EPA metabolism was slightly but significantly different in old compared with young men. The higher plasma (13)C-DHA seen in old men may be a result of slower plasma DHA clearance with age. This trial was registered at clinicaltrials.gov as NCT02957188. © 2017 American Society for Nutrition.

  1. Biosynthesis of curdlan from culture media containing 13C-labeled glucose as the carbon source.

    PubMed

    Kai, A; Ishino, T; Arashida, T; Hatanaka, K; Akaike, T; Matsuzaki, K; Kaneko, Y; Mimura, T

    1993-02-24

    13C-Labeled curdlans were biosynthesized by Agrobacterium sp. (ATCC 31749) from culture media containing D-(1-13C)glucose, D-(6-13C)glucose, or D-(2-13C)glucose as the carbon source, and their structures were analyzed by 13C NMR spectroscopy. The labeling was mainly found in the original position, that is, C-1, C-6, or C-2, indicating direct polymerization of introduced glucose. In addition, C-3 in curdlan obtained from D-(1-13C)glucose, C-1 in curdlan obtained from D-(6-13C)glucose, and C-1 and C-3 in curdlan obtained from D-(2-13)glucose were labeled. From analysis of this labeling, the biosynthesis of curdlan was interpreted as involving five routes: (1) direct synthesis from glucose; (2) rearrangement (1-13C-->3-13C); and (3) isomerization (6-13C-->1-13C) of cleaved trioses by the Embden-Meyerhof pathway, followed by neogenesis of glucose and formation of curdlan; (4) from fructose 6-phosphate formed in the pentose cycle (2-13C-->1-13C, 3-13C); and (5) neogenesis of glucose from fragments produced in various pathways of glycolysis. The 13C-labeling at C-6 and C-2 in the starting glucoses is well preserved in the C-6 carbon and the C-1 to C-3 carbons, respectively, in the curdlan produced.

  2. 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

  3. Increased Resolution of Aromatic Cross Peaks Using Alternate 13C Labeling and TROSY

    PubMed Central

    Milbradt, Alexander G.; Arthanari, Haribabu; Takeuchi, Koh; Boeszoermenyi, Andras; Hagn, Franz; Wagner, Gerhard

    2016-01-01

    For typical globular proteins, contacts involving aromatic side chains would constitute the largest number of distance constraints that could be used to define the structure of proteins and protein complexes based on NOE contacts. However, the 1H NMR signals of aromatic side chains are often heavily overlapped, which hampers extensive use of aromatic NOE cross peaks. Some of this overlap can be overcome by recording 13C-dispersed NOESY spectra. However, the resolution in the carbon dimension is rather low due to the narrow dispersion of the carbon signals, large one-bond carbon-carbon (C-C) couplings, and line broadening due to chemical shift anisotropy (CSA). Although it has been noted that the CSA of aromatic carbons could be used in TROSY experiments for enhancing resolution, this has not been used much in practice because of complications arising from large aromatic one-bond C-C couplings, and 3D or 4D carbon dispersed NOESY are typically recorded at low resolution hampering straightforward peak assignments. Here we show that the aromatic TROSY effect can optimally be used when employing alternate 13C labeling using 2-13C glycerol, 2-13C pyruvate, or 3-13C pyruvate as carbon source. With the elimination of the strong one-bond C-C coupling, the TROSY effect can easily be exploited. We show that 1H-13C TROSY spectra of alternately 13C labeled samples can be recorded at high resolution, and we employ 3D NOESY aromatic-TROSY spectra to obtain valuable intramolecular and intermolecular cross peaks on a protein complex. PMID:25957757

  4. 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.

  5. 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

  6. 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.

  7. 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).

  8. 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

  9. 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.

  10. (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.

  11. 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

  12. 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.

  13. 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.

  14. Identification of degradation routes of metamitron in soil microcosms using (13)C-isotope labeling.

    PubMed

    Wang, Shizong; Miltner, Anja; Nowak, Karolina M

    2017-01-01

    Metamitron is one of the most commonly used herbicide in sugar beet and flower bulb cultures. Numerous laboratory and field studies on sorption and degradation of metamitron were performed. Detailed biodegradation studies in soil using (13)C-isotope labeling are still missing. Therefore, we aimed at providing a detailed turnover mass balance of (13)C6-metamitron in soil microcosms over 80 days. In the biotic system, metamitron mineralized rapidly, and (13)CO2 finally constituted 60% of the initial (13)C6-metamitron equivalents. In abiotic control experiments CO2 rose to only 7.4% of the initial (13)C6-metamitron equivalents. The (13)C label from (13)C6-metamitron was incorporated into microbial amino acids that were ultimately stabilized in the soil organic matter forming presumably harmless biogenic residues. Finally, (13)C label from (13)C6-metamitron was distributed between the (13)CO2 and the (13)C-biogenic residues indicating nearly complete biodegradation. The parallel increase of (13)C-alanine, (13)C-glutamate and (13)CO2 indicates that metamitron was initially biodegraded via the desamino-metamitron route suggesting its relevance in the growth metabolism. In later phases of biodegradation, the "Rhodococcus route" was indicated by the low (13)CO2 evolution and the high relevance of the pyruvate pathway, which aims at biomolecule synthesis and seems to be related to starvation. This is a first report on the detailed degradation route of metamitron in soil. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. 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.

  16. 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

  17. (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.

  18. Characterization of uniformly and atom-specifically 13C-labeled heparin and heparan sulfate polysaccharide precursors using 13C NMR spectroscopy and ESI mass spectrometry

    PubMed Central

    Nguyen, Thao K. N.; Tran, Vy M.; Victor, Xylophone V.; Skalicky, Jack J.; Kuberan, Balagurunathan

    2010-01-01

    The biological actions of heparin and heparan sulfate, two structurally related glycosaminoglycans, depend on the organization of the complex heparanome. Due to the structural complexity of the heparanome, the sequence of variably sulfonated uronic acid and glucosamine residues is usually characterized by the analysis of smaller oligosaccharide and disaccharide fragments. Even characterization of smaller heparin/heparan sulfate oligosaccharide or disaccharide fragments using simple 1D 1H NMR spectroscopy is often complicated by the extensive signal overlap. 13C NMR signals, on the other hand, overlap less and therefore, 13C NMR spectroscopy can greatly facilitate the structural elucidation of the complex heparanome and provide finer insights into the structural basis for biological functions. This is the first report of the preparation of anomeric carbon-specific 13C-labeled heparin/heparan sulfate precursors from the Escherichia coli K5 strain. Uniformly 13C- and 15N-labeled precursors were also produced and characterized by 13C NMR spectroscopy. Mass spectrometric analysis of enzymatically fragmented disaccharides revealed that anomeric carbon-specific labeling efforts resulted in a minor loss/scrambling of 13C in the precursor backbone, whereas uniform labeling efforts resulted in greater than 95% 13C isotope enrichment in the precursor backbone. These labeled precursors provided high-resolution NMR signals with great sensitivity and set the stage for studying the heparanome–proteome interactions. PMID:20832774

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 13C-labeled D-ribose: chemi-enzymic synthesis of various isotopomers.

    PubMed

    Serianni, A S; Bondo, P B

    1994-04-01

    Current interest in the use of heteronuclear multidimensional NMR methods to assess the structures, conformations and/or dynamics of oligonucleotides in solution has created an immediate need for nucleosides and their derivatives labeled in various ways with stable isotopes (13C, 2H, 15N and/or 17,18O). This short review focuses exclusively on chemienzymic methods to introduce one or more 13C labels into D-ribose, a precursor to ribo- and 2'-deoxyribonucleosides. It will be demonstrated that five convenient reactions, applied in specific sequences, provide access to 26 of the 32 13C-labeled isotopomers of D-ribose in acceptable yields. While not explicitly discussed herein, these same reactions, appropriately modified, can also be used to insert one or more 2H and/or 17,18O isotopes into this aldopentose.

  7. 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

  8. Bioaccumulation and Toxicity of (13)C-Skeleton Labeled Graphene Oxide in Wheat.

    PubMed

    Chen, Lingyun; Wang, Chenglong; Li, Hongliang; Qu, Xiulong; Yang, Sheng-Tao; Chang, Xue-Ling

    2017-09-05

    Graphene nanomaterials have many diverse applications, but are considered to be emerging environmental pollutants. Thus, their potential environmental risks and biosafety are receiving increased attention. Bioaccumulation and toxicity evaluations in plants are essential for biosafety assessment. In this study, (13)C-stable isotope labeling of the carbon skeleton of graphene oxide (GO) was applied to investigate the bioaccumulation and toxicity of GO in wheat. Bioaccumulation of GO was accurately quantified according to the (13)C/(12)C ratio. Wheat seedlings were exposed to (13)C-labeled GO at 1.0 mg/mL in nutrient solution for 15 d. (13)C-GO accumulated predominantly in the root with a content of 112 μg/g at day 15, hindered the development and growth of wheat plants, disrupted root structure and cellular ultrastructure, and promoted oxidative stress. The GO that accumulated in the root showed extremely limited translocation to the stem and leaves. During the experimental period, GO was excreted slowly from the root. GO inhibited the germination of wheat seeds at high concentrations (≥0.4 mg/mL). The mechanism of GO toxicity to wheat may be associated with oxidative stress induced by GO bioaccumulation, reflected by the changes of malondialdehyde concentration, catalase activity, and peroxidase activity. The results demonstrate that (13)C labeling is a promising method to investigate environmental impacts and fates of carbon nanomaterials in biological systems.

  9. 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. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  10. Synthesis of (6-(13)C)pyrimidine nucleotides as spin-labels for RNA dynamics.

    PubMed

    Wunderlich, Christoph H; Spitzer, Romana; Santner, Tobias; Fauster, Katja; Tollinger, Martin; Kreutz, Christoph

    2012-05-02

    We present a (13)C-based isotope labeling protocol for RNA. Using (6-(13)C)pyrimidine phosphoramidite building blocks, site-specific labels can be incorporated into a target RNA via chemical oligonucleotide solid-phase synthesis. This labeling scheme is particularly useful for studying milli- to microsecond dynamics via NMR spectroscopy, as an isolated spin system is a crucial prerequisite to apply Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion type experiments. We demonstrate the applicability for the characterization and detection of functional dynamics on various time scales by incorporating the (6-(13)C)uridine and -cytidine labels into biologically relevant RNAs. The refolding kinetics of a bistable terminator antiterminator segment involved in the gene regulation process controlled by the preQ(1) riboswitch class I was investigated. Using (13)C CPMG relaxation dispersion NMR spectroscopy, the milli- to microsecond dynamics of the HIV-1 transactivation response element RNA and the Varkud satellite stem loop V motif was addressed. © 2012 American Chemical Society

  11. 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

  12. The application of 13C-labeled tetramethylammonium hydroxide (13C-TMAH) thermochemolysis to study fungal degradation of wood

    Treesearch

    T.R. Filley; P.G. Hatcher; W.C. Shortle

    2000-01-01

    This paper presents the results from an assessment of the application of a new molecular analytical procedure, 13C-TMAH thermochemolysis, to study the chemical modification of lignin by white-rot and brown-rot fungi. This technique differs from other molecular chemolysis procedures (e.g. TMAH thermochemolysis and CuO alkaline oxidation) as it...

  13. 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%.

  14. 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.

  15. 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.

  16. Chiral Recognition by Dissolution DNP NMR Spectroscopy of (13)C-Labeled dl-Methionine.

    PubMed

    Monteagudo, Eva; Virgili, Albert; Parella, Teodor; Pérez-Trujillo, Míriam

    2017-05-02

    A method based on d-DNP NMR spectroscopy to study chiral recognition is described for the first time. The enantiodifferentiation of a racemic metabolite in a millimolar aqueous solution using a chiral solvating agent was performed. Hyperpolarized (13)C-labeled dl-methionine enantiomers were differently observed with a single-scan (13)C NMR experiment, while the chiral auxiliary at thermal equilibrium remained unobserved. The method developed entails a step forward in the chiral recognition of small molecules by NMR spectroscopy, opening new possibilities in situations where the sensitivity is limited, for example, when a low concentration of analyte is available or when the measurement of an insensitive nucleus, like (13)C, is required. The advantages and current limitations of the method, as well as future perspectives, are discussed.

  17. Observing 13C labelling kinetics in CO2 respired by a temperate grassland ecosystem.

    PubMed

    Gamnitzer, Ulrike; Schäufele, Rudi; Schnyder, Hans

    2009-10-01

    * The kinetic characteristics of the main sources of ecosystem respiration are quite unknown, partly because of methodological constraints. Here, we present a new open-top chamber (OTC) apparatus for continuous 13C/12C labelling and measurement of ecosystem CO2 fluxes, and report the tracer kinetics of nighttime respiration of a temperate grassland. * The apparatus includes four dynamic flow-through OTCs, a unit mixing CO2-free air with 13C-depleted CO2, and a CO2 analyser and an online isotope ratio mass spectrometer. * The concentration (367 +/- 6.5 micromol mol(-1)) and carbon isotopic composition, delta13C, (-46.9 +/- 0.4 per thousand) of CO2 in the OTCs were stable during photosynthesis as a result of high air through flux and minimal incursion through the buffered vent. Soil CO2 efflux was not affected by pressure effects during respiration measurements. The labelling kinetics of respiratory CO2 measured in the field agreed with that of excised soil + vegetation blocks measured in a laboratory-based system. The kinetics fitted a two-source system (r(2) = 0.97), with a rapidly labelled source (half-life 2.6 d) supplying 48% of respiration, and the other source (52%) releasing no tracer during 14 d of labelling. * Of the two sources supplying ecosystem respiration, one was closely connected to current photosynthesis (approximately autotrophic respiration) and the other was provided by decomposition of structural plant biomass (approximately heterotrophic respiration).

  18. 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

  19. 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.

  20. Fungal carbon sources in a pine forest: evidence from a 13C-labeled global change experiment

    Treesearch

    Erik A. Hobbie; Kirsten S. Hofmockel; Linda T.A. Van Diepen; Erik A. Lilleskov; Andrew P. Oiumette; Adrien C. Finzi

    2014-01-01

    We used natural abundance 13C:12C (δ13C) and 8 yr of labeling with 13C-depleted CO2 in a Pinus taeda Free Air CO2 Enrichment (FACE) experiment to investigate carbon sources of saprotrophic fungi, ectomycorrhizal...

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. A Comprehensive Metabolic Profile of Cultured Astrocytes Using Isotopic Transient Metabolic Flux Analysis and 13C-Labeled Glucose

    PubMed Central

    Amaral, Ana I.; Teixeira, Ana P.; Håkonsen, Bjørn I.; Sonnewald, Ursula; Alves, Paula M.

    2011-01-01

    Metabolic models have been used to elucidate important aspects of brain metabolism in recent years. This work applies for the first time the concept of isotopic transient 13C metabolic flux analysis (MFA) to estimate intracellular fluxes in primary cultures of astrocytes. This methodology comprehensively explores the information provided by 13C labeling time-courses of intracellular metabolites after administration of a 13C-labeled substrate. Cells were incubated with medium containing [1-13C]glucose for 24 h and samples of cell supernatant and extracts collected at different time points were then analyzed by mass spectrometry and/or high performance liquid chromatography. Metabolic fluxes were estimated by fitting a carbon labeling network model to isotopomer profiles experimentally determined. Both the fast isotopic equilibrium of glycolytic metabolite pools and the slow labeling dynamics of TCA cycle intermediates are described well by the model. The large pools of glutamate and aspartate which are linked to the TCA cycle via reversible aminotransferase reactions are likely to be responsible for the observed delay in equilibration of TCA cycle intermediates. Furthermore, it was estimated that 11% of the glucose taken up by astrocytes was diverted to the pentose phosphate pathway. In addition, considerable fluxes through pyruvate carboxylase [PC; PC/pyruvate dehydrogenase (PDH) ratio = 0.5], malic enzyme (5% of the total pyruvate production), and catabolism of branched-chained amino acids (contributing with ∼40% to total acetyl-CoA produced) confirmed the significance of these pathways to astrocytic metabolism. Consistent with the need of maintaining cytosolic redox potential, the fluxes through the malate–aspartate shuttle and the PDH pathway were comparable. Finally, the estimated glutamate/α-ketoglutarate exchange rate (∼0.7 μmol mg prot−1 h−1) was similar to the TCA cycle flux. In conclusion, this work demonstrates the potential of

  7. 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.

  8. 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.

  9. 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

  10. 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.

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

    PubMed

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

    1991-09-15

    The edible alga Spirulina platensis was uniformly labeled with 13C by growth in an atmosphere of pure 13CO2. 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 13C-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.

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

    PubMed Central

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

    1991-01-01

    The edible alga Spirulina platensis was uniformly labeled with 13C by growth in an atmosphere of pure 13CO2. 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 13C-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. Images PMID:11607211

  13. 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.

  14. Dynamic secondary ion mass spectrometry (SIMS) imaging of microbial populations utilizing 13C-labeled substrates in pure culture and in soil

    PubMed Central

    Pumphrey, Graham M.; Hanson, Buck T.; Chandra, Subhash; Madsen, Eugene L.

    2008-01-01

    Summary We demonstrate that dynamic secondary ion mass spectrometry (SIMS)-based ion microscopy can provide a means of measuring 13C assimilation into individual bacterial cells grown on 13C-labeled organic compounds in the laboratory and in field soil. We grew pure cultures of Pseudomonas putida NCIB 9816-4 in minimal media with known mixtures of 12C- and 13C-glucose and analyzed individual cells via SIMS imaging. Individual cells yielded signals of masses 12, 13, 24, 25, 26, and 27 as negative secondary ions indicating the presence of 12C−, 13C−, 24(12C2)−, 25(12C13C)−, 26(12C14N)−, and 27(13C14N)− ions, respectively. We verified that ratios of signals taken from the same cells only changed minimally during a ∼4.5-min period of primary O2+ beam sputtering by the dynamic SIMS instrument in microscope detection mode. There was a clear relationship between mass 27 and 26 signals in Psuedomonas putida cells grown in media containing varying proportions of 12C- to 13C-glucose: a standard curve was generated to predict 13C-enrichment in unknown samples. We then used two strains of Pseudomonas putida able to grow on either all or only a part of a mixture of 13C-labeled and unlabeled carbon sources to verify that differential 13C signals measured by SIMS were due to 13C assimilation into cell biomass. Finally, we made three key observations after applying SIMS ion microscopy to soil samples from a field experiment receiving 12C- or 13C-phenol: (i) cells enriched in 13C were heterogeneously distributed among soil populations; (ii) 13C-labeled cells were detected in soil that was dosed a single time with 13C-phenol; and (iii) in soil that received 12 doses of 13C-phenol, 27% of the cells in the total community were more than 90% 13C-labeled. PMID:18811644

  15. 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.

  16. 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

  17. 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

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

    2005-08-01

    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 (13)C CSA tensors were computed by quantum chemistry, using the gauge-including atomic-orbital approach within Hartree-Fock theory. Uniformly (13)C-labeled ergosterol was purified from Pichia pastoris cells grown on labeled methanol. After reconstitution into dimyristoylphosphatidylcholine lipids, the complete (1)H and (13)C 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 (13)C resonances (C(3) 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 degrees) with the inertial axis of the rigid four-ring system.

  18. Respiration of 13C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of 13C-labeled soil DNA.

    PubMed

    Padmanabhan, P; Padmanabhan, S; DeRito, C; Gray, A; Gannon, D; Snape, J R; Tsai, C S; Park, W; Jeon, C; Madsen, E L

    2003-03-01

    Our goal was to develop a field soil biodegradation assay using (13)C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived (13)C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of (13)C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for (13)CO(2) respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of (13)CO(2) emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF(6), that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired (13)CO(2). Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of (13)CO(2) released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of (13)C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with (13)C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, and Pedobacter spp. for glucose

  19. Effects of drought on C allocation and turnover in a Mediterranean shrub community - A 13C field labeling study

    NASA Astrophysics Data System (ADS)

    Gavrichkova, O.; Tian, J.; Spohn, M.; Guidolotti, G.; de Dato, G.; Liberati, D.; Pausch, J.; Brugnoli, E.; De Angelis, P.; Kuzyakov, Y.

    2012-04-01

    Predicting impacts of climate change on terrestrial ecosystem functioning is a big scientific challenge. Large-scale manipulation experiments may provide realistic estimates of the responses of biological processes to changes in their principal regulators such as temperature, CO2, and water availability. A field drought manipulation experiment has been established in a Mediterranean shrub community of Porto Conte (Sardegna, Italy) in 2002 as part of the INCREASE network. The INCREASE network aims at developing non-intrusive technologies for realistic climate change manipulations to study vulnerable shrubland ecosystems over Europe. In Porto Conte summer drought is extended by excluding precipitation with transparent roofs in order to mimic potential future changes in precipitation patterns. In October 2011 we performed a field 13C pulse labeling to explore effects of drought on carbon allocation and turnover in the shrub land of Porto Conte. For this purpose, Cistus monspeliensis, the dominant shrub species within the experimental site, was labeled in three plots subjected to extended summer droughts and in three control plots. Allocation of the tracer between various pools and fluxes in the plant-soil system was studied over a period of two weeks with an one day frequency. Aboveground carbon allocation and turnover was accessed by monitoring 13C content in shoots and in shoot-respired CO2. Belowground carbon allocation and turnover was explored by repeated determination of 13C label in roots, microbial biomass, and soil respired CO2. Two approaches of soil respired 13CO2 sampling were utilized and confronted here. Soil respiration and its δ13C were determined by Keeling plot approach. Additionally, cumulative amount of the soil respired CO2 and its isotopic signature were determined by trapping the evolved CO2 with soda traps. Conclusions on the sensitivity of C. monspeliensis to drought and its consequences for C cycling in shrub lands under extended summer

  20. Metabolic Response of Soil Microorganisms to Frost: A New Perspective from Position-specific 13C Labeling

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

    Cold adapted organisms and their biomolecules have received considerable attention in the last few decades, particularly in light of the perceived biotechnological potential. Mostly, these studies are based on pure isolated cultures from permafrost or permafrost samples with inherently adapted microbes. However, microbial activities in agricultural soils that are predominantly exposed to freeze conditions during winter in temperate ecosystems remain unclear. To analyze microbial metabolism at low soil temperatures, isotopomeres of position-specifically 13C labeled glucose were incubated at three temperature; 5 (control), -5 -20 oC. Soils were sampled after 1, 3 and 10 days (and after 30 days for samples at -20 °C). 13C was quantifed in CO2, bulk soil, microbial biomass and dissolved organic carbon (DOC). Highest 13C recovery in CO2 was obtained from C-1 position in control soil. Consequently, metabolic activity was dominated by pentose phosphate pathway at 5 °C. In contrast, metabolic behaviors switched towards a preferential respiration of the glucose C-4 position at -5 and -20 °C. High 13C recovery from C-4 position confirms previous studies suggesting that fermentation increases at subzero temperature. A 3-fold higher 13C recovery in microbial biomass at -5 °C than under control conditions points towards synthesis of intracellular antifreeze metabolites such as glycerol and ethanol and it is consistent with fermentative metabolism. A 5-fold higher 13C in bulk soil than microbial biomass at -20 °C does not reflect non-metabolized glucose because 13C recovery in DOC was less than 0.4% at day 1. Therefore, high 13C recovery in bulk soil at -20 °C was attributed to extracellular metabolites secreted to overcome frost. The shift in antifreeze mechanisms with temperature was brought about by shift in microbial community structure as indicated by incorporation into 13C into PLFA which was 2-fold higher in gram negative bacteria under control than frozen

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. A chemical synthesis of a multiply (13) C-labeled hexasaccharide: a high-mannose N-glycan fragment.

    PubMed

    Zhang, Wenhui; Pan, Qingfeng; Serianni, Anthony S

    2016-12-01

    As covalent modifiers of proteins, high-mannose N-glycans are important in maintaining protein structure and function in vivo. The conformations of these glycans can be studied by nuclear magnetic resonance spectroscopy using spin-spin couplings (J-couplings; scalar couplings) and other nuclear magnetic resonance parameters that are sensitive to the geometries of their constituent glycosidic linkages and other mobile elements in their structures. These analyses often require (13) C-labeling at specific carbon atoms, especially when measurements of (13) C-(13) C J-couplings are of interest. The selection of particular (13) C isotopomers of a glycan depends on the type of question under scrutiny. A chemical synthesis of a mannose-containing hexasaccharide, α[1-(13) C]Man(1→2)α[1,2-(13) C2 ]Man(1→6)[α[1-(13) C]Man(1→2)α[1,2-(13) C2 ]Man(1→3)]α[1,2-(13) C2 ]Man(1→6)βManOCH3 , which is a nested fragment of the high-mannose N-glycans of human glycoproteins and contains eight (13) C-enriched carbon sites, is described in this report. The selected (13) C isotopomer was chosen to maximize the measurement of J-couplings sensitive to linkage conformations. This work demonstrates that chemical syntheses of multiply (13) C-labeled oligosaccharides are technically feasible and practical using present synthetic methods. The availability of this and other multiply (13) C-labeled mannose-containing oligosaccharides will promote future studies of their conformations in solution and in the bound state. Copyright © 2016 John Wiley & Sons, Ltd.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. Assessment of the production of 13C labeled compounds from phototrophic microalgae at laboratory scale.

    PubMed

    Acién Fernández, Francisco G; Alías, Celeste Brindley; García-Malea López, María C; Fernández Sevilla, José M; Ibáñez González, María J; Gómez, Rafael Núñez; Molina Grima, Emilio

    2003-07-01

    An integrated process for the indoor production of 13C labeled polyunsaturated fatty acids (PUFAs) from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor operating with recirculation of the exhaust gas using a low-pressure compressor. Oxygen accumulation in the system is avoided by bubbling the exhaust gas from the reactor in a sodium sulfite solution before returning to it. To achieve a high 13C enrichment in the biomass obtained, the culture medium is initially stripped of carbon, and labeled 13CO(2) is automatically injected on-demand during operation for pH control and carbon supply. The reactor was operated in both batch and semicontinuous modes. In semicontinuous mode, the reactor was operated at a dilution rate of 0.01 h(-1), resulting in a biomass productivity of 0.1 g l(-1) per day. The elemental analysis of the inlet and outlet flows of the reactor showed that 64.9% of carbon was turned into microalgal biomass, 34.9% remained in the supernatant mainly as inorganic compounds. Only 3.8% of injected carbon was effectively fixed as the target labeled product (EPA). Regarding the isotopic composition of fatty acids, results showed that fatty acids were not labeled in the same proportion, the higher the number of carbons the lower the percentage of 13C. Isotopic composition of EPA ranged from 36.5 to 53.5%, as a function of the methodology used (GC-MS, EA-IRMS or gas chromatography-combustion-isotope ratio mass spectrometry (GC-IRMS)). The low carbon uptake efficiency combined with the high cost of 13CO(2) make necessary to redefine the designed culture system to increase the efficiency of the conversion of 13CO(2) into the target product. Therefore, the possibility of removing 12C from the fresh medium, and recovering and recirculating the inorganic carbon in the supernatant and the organic carbon from the EPA depleted biomass was studied. The inorganic carbon of the fresh medium was removed by acidification and

  13. 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…

  14. 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…

  15. 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

  16. 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.

  17. Probing crystal packing of uniformly (13)C-enriched powder samples using homonuclear dipolar coupling measurements.

    PubMed

    Mollica, Giulia; Dekhil, Myriam; Ziarelli, Fabio; Thureau, Pierre; Viel, Stéphane

    2015-02-01

    The relationship between the crystal packing of powder samples and long-range (13)C-(13)C homonuclear dipolar couplings is presented and illustrated for the case of uniformly (13)C-enriched L-alanine and L-histidine·HCl·H2O. Dipolar coupling measurement is based on the partial reintroduction of dipolar interactions by spinning the sample slightly off-magic-angle, while the coupling of interest for a given spin pair is isolated with a frequency-selective pulse. A cost function is used to correlate the so-derived dipolar couplings to trial crystal structures of the samples under study. This procedure allowed for the investigation of the l-alanine space group and L-histidine·HCl·H2O space group and unit-cell parameters.

  18. High Resolution 13C MRI With Hyperpolarized Urea: In Vivo T2 Mapping and 15N Labeling Effects

    PubMed Central

    Reed, Galen D.; von Morze, Cornelius; Bok, Robert; Koelsch, Bertram L.; Van Criekinge, Mark; Smith, Kenneth J.; Shang, Hong; Larson, Peder E. Z.; Kurhanewicz, John; Vigneron, Daniel B.

    2014-01-01

    13C steady state free precession (SSFP) magnetic resonance imaging and effective spin-spin relaxation time (T2) mapping were performed using hyperpolarized [13C] urea and [13C, 15N2] urea injected intravenously in rats. 15N labeling gave large T2 increases both in solution and in vivo due to the elimination of a strong scalar relaxation pathway. The T2 increase was pronounced in the kidney, with [13C, 15N2] urea giving T2 values of 6.3±1.3 s in the cortex and medulla, and 11±2 s in the renal pelvis. The measured T2 in the aorta was 1.3±0.3 s. [13C] urea showed shortened T2 values in the kidney of 0.23±0.03 s compared to 0.28±0.03 s measured in the aorta. The enhanced T2 of [13C, 15N2] urea was utilized to generate large signal enhancement by SSFP acquisitions with flip angles approaching the fully refocused regime. Projection images at 0.94 mm in-plane resolution were acquired with both urea isotopes, with [13C, 15N2] urea giving a greater than four-fold increase in signal-to-noise ratio [13C] over urea. PMID:24235273

  19. 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

  20. Biochemical synthesis of uniformly (13)C-labeled diterpene hydrocarbons and their bioconversion to diterpenoid phytoalexins in planta.

    PubMed

    Ye, Zhongfeng; Nakagawa, Kazuya; Natsume, Masahiro; Nojiri, Hideaki; Kawaide, Hiroshi; Okada, Kazunori

    2017-06-01

    Phytocassanes and momilactones are the major diterpenoid phytoalexins inductively produced in rice as bioactive substances. Regardless of extensive studies on the biosynthetic pathways of these phytoalexins, bioconversion of diterpene hydrocarbons is not shown in planta. To elucidate the entire biosynthetic pathways of these phytoalexins, uniformly (13)C-labeled ent-cassadiene and syn-pimaradiene were enzymatically synthesized with structural verification by GC-MS and (13)C-NMR. Application of the (13)C-labeled substrates on rice leaves led to the detection of (13)C-labeled metabolites using LC-MS/MS. Further application of this method in the moss Hypnum plumaeforme and the nearest out-group of Oryza species Leersia perrieri, respectively, resulted in successful bioconversion of these labeled substrates into phytoalexins in these plants. These results demonstrate that genuine biosynthetic pathways from these diterpene hydrocarbons to the end product phytoalexins occur in these plants and that enzymatically synthesized [U-(13)C20] diterpene substrates are a powerful tool for chasing endogenous metabolites without dilution with naturally abundant unlabeled compounds.

  1. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  3. Probing RNA dynamics via longitudinal exchange and CPMG relaxation dispersion NMR spectroscopy using a sensitive 13C-methyl label.

    PubMed

    Kloiber, Karin; Spitzer, Romana; Tollinger, Martin; Konrat, Robert; Kreutz, Christoph

    2011-05-01

    The refolding kinetics of bistable RNA sequences were studied in unperturbed equilibrium via (13)C exchange NMR spectroscopy. For this purpose a straightforward labeling technique was elaborated using a 2'-(13)C-methoxy uridine modification, which was prepared by a two-step synthesis and introduced into RNA using standard protocols. Using (13)C longitudinal exchange NMR spectroscopy the refolding kinetics of a 20 nt bistable RNA were characterized at temperatures between 298 and 310K, yielding the enthalpy and entropy differences between the conformers at equilibrium and the activation energy of the refolding process. The kinetics of a more stable 32 nt bistable RNA could be analyzed by the same approach at elevated temperatures, i.e. at 314 and 316 K. Finally, the dynamics of a multi-stable RNA able to fold into two hairpin- and a pseudo-knotted conformation was studied by (13)C relaxation dispersion NMR spectroscopy.

  4. First airborne samples of a volcanic plume for δ13C of CO2 determinations

    NASA Astrophysics Data System (ADS)

    Fischer, Tobias P.; Lopez, Taryn M.

    2016-04-01

    Volcanic degassing is one of the main natural sources of CO2 to the atmosphere. Carbon isotopes of volcanic gases enable the determination of CO2 sources including mantle, organic or carbonate sediments, and atmosphere. Until recently, this work required sample collection from vents followed by laboratory analyses. Isotope ratio infrared analyzers now enable rapid analyses of plume δ13C-CO2, in situ and in real time. Here we report the first analyses of δ13C-CO2 from airborne samples. These data combined with plume samples from the vent area enable extrapolation to the volcanic source δ13C. We performed our experiment at the previously unsampled and remote Kanaga Volcano in the Western Aleutians. We find a δ13C source composition of -4.4‰, suggesting that CO2 from Kanaga is primarily sourced from the upper mantle with minimal contributions from subducted components. Our method is widely applicable to volcanoes where remote location or activity level precludes sampling using traditional methods.

  5. 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

  6. Assessing microbial utilization of free versus sorbed Alanine by using position-specific 13C labeling and 13C-PLFA analysis

    NASA Astrophysics Data System (ADS)

    Herschbach, Jennifer; Apostel, Carolin; Spielvogel, Sandra; Kuzyakov, Yakov; Dippold, Michaela

    2016-04-01

    Microbial utilization is a key transformation process of soil organic matter (SOM). Sorption of low molecular weight organic substances (LMWOS) to soil mineral surfaces blocks or delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil science, combined with 13C-phospholipid fatty acid (PLFA) analysis, to assess microbial utilization of sorbed and non-sorbed Alanine in soil. Alanine has various functional groups enabling different sorption mechanisms via its positive charge (e.g. to clay minerals by cation exchange), as well as via its negative charge (e.g. to iron oxides by ligand exchange). To assess changes in the transformation pathways caused by sorption, we added uniformly and position-specifically 13C and 14C labeled Alanine to the Ap of a loamy Luvisol in a short-term (10 days) incubation experiment. To allow for sorption of the tracer solution to an aliquot of this soil, microbial activity was minimized in this subsample by sterilizing the soil by γ-radiation. After shaking, the remaining solutions were filtered and the non-sorbed Alanine was removed with Millipore water and then added to non-sterilized soil. For the free Alanine treatment, solutions with Alanine of similar amount and isotopic composition were prepared, added to the soil and incubated as well. The respired CO2 was trapped in NaOH and its 14C-activity was determined at increasing times intervals. Microbial utilization of Alanine's individual C positions was evaluated in distinct microbial groups classified by 13C-PLFA analysis. Sorption to soil minerals delayed respiration to CO2 and reduced initial respiration rate by 80%. Irrespective of sorption, the highest amount was respired from the carboxylic position (C-1), whereas the amino-bound (C-2) and the methylic position (C-3) were preferentially incorporated into PLFA of microorganisms due to the

  7. 13C nuclear magnetic resonance and gas chromatography-mass spectrometry studies of carbon metabolism in the actinomycin D producer Streptomyces parvulus by use of 13C-labeled precursors.

    PubMed

    Inbar, L; Lapidot, A

    1991-12-01

    Fructose and glutamate metabolism was monitored in cell suspensions of streptomyces parvulus by 13C nuclear magnetic resonance. The experiments were performed for cells grown with various 13C sources in a growth medium containing D-[U-13C]fructose, L-[13C]glutamate, or L-[U-13C]aspartate and with nonlabeled precursors to compare intracellular pools in S. parvulus cells at different periods of the cell life cycle. The transport of fructose into the cells was biphasic in nature; during rapid transport, mannitol, fructose, and glucose 6-phosphate were accumulated intracellularly, whereas during the passive diffusion of fructose, the intracellular carbohydrate pool comprised mainly trehalose (1,1'-alpha-alpha-D-glucose). The regulation of fructokinase activity by the intracellular intermediates may play an important role in fructose catabolism in S. parvulus. Transaldolase activity in S. parvulus was determined from the 13C nuclear magnetic resonance labeling pattern of trehalose carbons obtained from cells grown in medium containing either L-[U-13C]aspartate or L-[U-13C]glutamate. Only carbons 4, 5, and 6 of the disaccharide were labeled. Isotopomer analysis of the trehalose carbons led us to conclude that the flux through the reverse glycolytic pathway, condensation of glyceraldehyde 3-phosphate with dihydroxyacetone phosphate, makes at best a minor contribution to the 13C-labeled glucose units observed in trehalose. The pentose pathway and transaldolase activity can explain the labeling pattern of 4,5,6-13C3 of trehalose. Moreover, the transfer of the 13C label of L-[U-13C]aspartate into the different isotopomers of trehalose C4, C5, and C6 by the transaldolase activity allowed us to calculate the relative fluxes from oxaloacetate via gluconeogenesis and through the tricarboxylic acid cycle. The ratio of the two fluxes is approximately 1. However, the main carbon source for trehalose synthesis in S. parvulus is fructose and not glutamate or aspartate. The 13C

  8. Surface dynamics of bacteriorhodopsin as revealed by (13)C NMR studies on [(13)C]Ala-labeled proteins: detection of millisecond or microsecond motions in interhelical loops and C-terminal alpha-helix.

    PubMed

    Yamaguchi, S; Tuzi, S; Yonebayashi, K; Naito, A; Needleman, R; Lanyi, J K; Saitô, H

    2001-03-01

    We have recorded (13)C NMR spectra of [2-(13)C]-, [1-(13)C]-, [3-(13)C],- and [1,2,3-(13)C(3)]Ala-labeled bacteriorhodopsin (bR), and its mutants, A196G, A160G, and A103C, by means of cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques, to reveal the conformation and dynamics of bR, with emphasis on the loop and C-terminus structures. The (13)C NMR signals of the loop (C-D, E-F, and F-G) regions were almost completely suppressed from [2-(13)C]-, [1-(13)C]Ala-, and [1-(13)C]Gly-labeled bR, due to the presence of conformational fluctuation with correlation times of 10(-4) s that interfered with the peak-narrowing by magic angle spinning. The observation of such suppressed peaks for specific residues provides a unique means of detecting intermediate frequency motions on the time scale of ms or micros in the surface loops of membrane proteins. Instead, the three well-resolved (13)C CP-MAS NMR signals of [2-(13)C]Ala-bR, at 50.38, 49.90, and 47.96 ppm, were ascribed to the C-terminal alpha-helix previously proposed from the data for [3-(13)C]Ala-bR: the former two peaks were assigned to Ala 232 and 238, in view of the results of successive proteolysis experiments, while the highest-field peak was ascribed to Ala 235 prior to Pro 236. Even such (13)C NMR signals were substantially broadened when (13)C NMR spectra of fully labeled [1,2,3-(13)C]Ala-bR were recorded, because the broadening and splitting of peaks due to the accelerated transverse relaxation rate caused by the increased number of relaxation pathways through a number of (13)C-(13)C homo-nuclear dipolar interactions and scalar J couplings, respectively, are dominant among (13)C-labeled nuclei. In addition, approximate correlation times for local conformational fluctuations of different domains, including the C-terminal tail, C-terminal alpha-helix, loops, and transmembrane alpha-helices, were estimated by measurement of the spin-lattice relaxation

  9. Unlocking the molecular structure of fungal melanin using 13C biosynthetic labeling and solid-state NMR.

    PubMed

    Tian, Shiying; Garcia-Rivera, Javier; Yan, Bin; Casadevall, Arturo; Stark, Ruth E

    2003-07-15

    Melanins are enigmatic pigments found in all biological kingdoms that are associated with a variety of functions, including microbial virulence. Despite being ubiquitous in nature, melanin pigments have long resisted atomic-level structural examination because of their insolubility and amorphous organization. Cryptococcus neoformans is a human pathogenic fungus that melanizes only when provided with exogenous substrate, thus offering a unique system for exploring questions related to melanin structure at the molecular level. We have exploited the requirement for exogenous substrate in melanin synthesis as well as the capabilities of high-resolution solid-state nuclear magnetic resonance (NMR) to establish the predominantly aliphatic composition of l-dopa melanin and to introduce (13)C labels that permit the identification of proximal carbons in the developing biopolymer. By swelling solid melanin samples in organic solvents and using two-dimensional heteronuclear NMR in conjunction with magic-angle spinning, we have identified chemical bonding patterns typical of alkane, alkene, alcohol, ketone, ester, and indole functional groups. These findings demonstrate the feasibility of a novel approach to determining the structure of melanin using metabolic labeling and NMR spectroscopy.

  10. NMR studies of bent DNA using {sup 13}C-enriched samples

    SciTech Connect

    Zimmer, D.P.; Crothers, D.M.

    1994-12-01

    Bending of the DNA double helix can be brought about by introducing runs of adenines (A-tracts) in phase with the helical repeat of the DNA. The requirements for bending of DNA by A-tracts are that the length of the A-tract be greater than 3 base pairs and that the A-tracts must be in phase with the helical repeat (every 10 or 11 bp). Other factors, such as the number of adenines in the run, flanking sequences, and whether the A-tracts are phased with respect to the 5{prime}A or the 3{prime}A, have effects upon the degree of bending as assayed by electrophoretic mobility on native polyacrylamide gels. There are a number of models for bending A-tract DNA. The junction-bending model postulates that the structure of A-tracts is similar to the fiber diffraction structure of poly A, in which there is a significant degree of base pair tilt with respect to the helix axis. In this model, bending occurs at the junction between the A-tract and the B-form helix to allow favorable stacking interactions to occur. The bend of the helix could arise as a result of some other perturbation of B-form DNA by A-tracts, such as propeller twist; bending also could be due to a combination of factors. Our goal is to find the structural features of A-tracts responsible for bending of the helix by performing NMR on oligonucleotides containing A-tracts to obtain higher resolution structural data. One of the problems encountered in NMR structure determination of nucleic acids and other macromolecules is the assignment of resonances to nuclei. This procedure can be greatly facilitated through the use of {sup 13}C-enriched nucleic acid samples. We are developing a technique for the enzymatic synthesis of labeled DNA for NMR. The technique we are developing is similar to RNA labeling techniques already in use. The technique involves growth of methylotrophic bacteria on {sup 13}CH{sub 3}OH.

  11. Nic1 inactivation enables stable isotope labeling with 13C615N4-arginine in Schizosaccharomyces pombe.

    PubMed

    Carpy, Alejandro; Patel, Avinash; Tay, Ye Dee; Hagan, Iain M; Macek, Boris

    2015-01-01

    Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, (13)C(6) (15)N(4)-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of (13)C(6) (15)N(4)-arginine is catabolized by arginase and urease activity to (15)N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni(2+)-dependent urease activity, through deletion of the sole Ni(2+) transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable (13)C(6) (15)N(4)-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. 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.

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

    NASA Technical Reports Server (NTRS)

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

    1982-01-01

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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. Carbon transfer from the host to Tuber melanosporum mycorrhizas and ascocarps followed using a 13C pulse-labeling technique.

    PubMed

    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 (13)CO2. The transfer of (13)C 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 (13)C and transferred it slowly to the mycorrhizas. The mycorrhizas first formed a carbon sink and accumulated (13)C prior to ascocarp development. Then, the mycorrhizas transferred (13)C to the ascocarps to provide constitutive carbon (1.7 mg of (13)C 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. Carrot solution culture bioproduction of uniformly labeled (13)C-lutein and in vivo dosing in non-human primates.

    PubMed

    Smith, Joshua W; Rogers, Randy B; Jeon, Sookyoung; Rubakhin, Stanislav S; Wang, Lin; Sweedler, Jonathan V; Neuringer, Martha; Kuchan, Matthew J; Erdman, John W

    2017-02-01

    Lutein is a xanthophyll abundant in nature and most commonly present in the human diet through consumption of leafy green vegetables. With zeaxanthin and meso-zeaxanthin, lutein is a component of the macular pigment of the retina, where it protects against photooxidation and age-related macular degeneration. Recent studies have suggested that lutein may positively impact cognition throughout the lifespan, but outside of the retina, the deposition, metabolism, and function(s) of lutein are poorly understood. Using a novel botanical cell culture system ( Daucus carota), the present study aimed to produce a stable isotope lutein tracer for use in future investigations of dietary lutein distribution and metabolism. Carrot cultivars were initiated into liquid solution culture, lutein production conditions optimized, and uniformly labeled (13)C-glucose was provided as the sole media carbon source for four serial growth cycles. Lutein yield was 2.58 ± 0.24 µg/g, and mass spectrometry confirmed high enrichment of (13)C: 64.9% of lutein was uniformly labeled and 100% of lutein was labeled on at least 37 of 40 possible carbons. Purification of carrot extracts yielded a lutein dose of 1.92 mg with 96.0 ± 0.60% purity. (13)C-lutein signals were detectable in hepatic extracts of an adult rhesus macaque monkey ( Macaca mulatta) dosed with (13)C-lutein, but not in hepatic samples collected from control animals. This novel botanical biofactory approach can be used to produce sufficient quantities of highly enriched and pure (13)C-lutein doses for use in tracer studies investigating lutein distribution, metabolism, and function.

  6. 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

  7. Extreme 13C depletion of CCl2F2 in firn air samples from NEEM, Greenland

    NASA Astrophysics Data System (ADS)

    Zuiderweg, A.; Holzinger, R.; Martinerie, P.; Schneider, R.; Kaiser, J.; Witrant, E.; Etheridge, D.; Rubino, M.; Petrenko, V.; Blunier, T.; Röckmann, T.

    2012-07-01

    A series of 12 high volume air samples collected from the S2 firn core during the North Greenland Eemian Ice Drilling (NEEM) 2009 campaign have been measured for mixing ratio and stable carbon isotope composition of the chlorofluorocarbon CFC-12 (CCl2F2). While the mixing ratio measurements compare favorably to other firn air studies, the isotope results show extreme 13C depletion at the deepest measurable depth (65 m), to values lower than δ13C = -80‰ vs. VPDB (the international stable carbon isotope scale), compared to present day surface tropospheric measurements near -40‰. Firn air modeling was used to interpret these measurements. Reconstructed atmospheric time series indicate even larger depletions (to -120‰) near 1950 AD, with subsequent rapid enrichment of the atmospheric reservoir of the compound to the present day value. Mass-balance calculations show that this change must have been caused by a large change in the isotopic composition of anthropogenic CFC-12 emissions, probably due to technological changes in the CFC production process over the last 80 yr. Propagating the mass-balance calculations into the future demonstrates that as emissions decrease to zero, isotopic fractionation by the stratospheric sinks will lead to continued 13C enrichment in atmospheric CFC-12.

  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. 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.

  10. Synthesis and incorporation of 13C-labeled DNA building blocks to probe structural dynamics of DNA by NMR

    PubMed Central

    Nußbaumer, Felix; Juen, Michael Andreas; Gasser, Catherina; Kremser, Johannes; Müller, Thomas; Tollinger, Martin

    2017-01-01

    Abstract We report the synthesis of atom-specifically 13C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-13C-modified pyrimidine and 8-13C purine DNA phosphoramidites were synthesized and incorporated into a polypurine tract DNA/RNA hybrid duplex to showcase the facile resonance assignment using site-specific labeling. We also addressed micro- to millisecond dynamics in the mini-cTAR DNA. This DNA is involved in the HIV replication cycle and our data points toward an exchange process in the lower stem of the hairpin that is up-regulated in the presence of the HIV-1 nucleocapsid protein 7. As another example, we picked a G-quadruplex that was earlier shown to exist in two folds. Using site-specific 8-13C-2′deoxyguanosine labeling we were able to verify the slow exchange between the two forms on the chemical shift time scale. In a real-time NMR experiment the re-equilibration of the fold distribution after a T-jump could be monitored yielding a rate of 0.012 min−1. Finally, we used 13C-ZZ-exchange spectroscopy to characterize the kinetics between two stacked X-conformers of a Holliday junction mimic. At 25°C, the refolding process was found to occur at a forward rate constant of 3.1 s−1 and with a backward rate constant of 10.6 s−1.

  11. 13C measurements on organic aerosol - ambient samples versus source studies

    NASA Astrophysics Data System (ADS)

    Dusek, Ulrike; Meusinger, Carl; Oyama, Beatriz; Ramon, Wichert; de Wilde, Peter A.; Holzinger, Rupert; Röckmann, Thomas

    2013-04-01

    The stable carbon isotopes 12C and 13C can be used to get information about sources and processing of organic aerosol (OA). We developed and tested a method to measure δ13C values of OA collected on filter samples in different volatility classes. These filter samples are introduced into an oven, where organic compounds are thermally desorbed in He at different temperatures. The compounds released at each temperature step are oxidized to CO2 using a platinum catalyst at 550 °C. The CO2 is then passed on to an isotope ratio mass spectrometer (IRMS) to measure δ13C ratios. With a similar setup the chemical composition at each temperature step can be determined using a Proton Transfer Reaction Time-of-Flight Mass Spectrometer (PTR-ToF-MS). System evaluation with controlled test compounds showed that organic compounds usually start evaporating from the filter when their melting point is reached. Isotopic fractionation occurs only, if one temperature step is within a few degrees of the melting point of the substance, so that the substance only partially evaporates. However, this effect should be limited in an ambient sample containing thousands of individual chemical compounds. We analysed aerosol samples collected in a tunnel in Brazil (vehicular emissions), laboratory generated secondary organic aerosol (SOA) from alpha-pinene ozonolysis, and ambient filter samples from a regional site in the Netherlands and an urban site in Belgium. First results indicate that SOA is more volatile than organic aerosol from ambient or tunnel filters. The δ13C ratios of SOA and vehicular emissions do not change strongly with oven temperature, i.e. the more refractory organic compounds have similar isotopic composition as the more volatile compounds. This is in contrast to ambient organic aerosol where the more volatile compounds evaporating below 200°C are depleted with respect to the refractory compounds. Possible reasons for this difference (mixture of sources vs the role of

  12. 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.

  13. 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

  14. Probing RNA dynamics via longitudinal exchange and CPMG relaxation dispersion NMR spectroscopy using a sensitive 13C-methyl label

    PubMed Central

    Kloiber, Karin; Spitzer, Romana; Tollinger, Martin; Konrat, Robert; Kreutz, Christoph

    2011-01-01

    The refolding kinetics of bistable RNA sequences were studied in unperturbed equilibrium via 13C exchange NMR spectroscopy. For this purpose a straightforward labeling technique was elaborated using a 2′-13C-methoxy uridine modification, which was prepared by a two-step synthesis and introduced into RNA using standard protocols. Using 13C longitudinal exchange NMR spectroscopy the refolding kinetics of a 20 nt bistable RNA were characterized at temperatures between 298 and 310 K, yielding the enthalpy and entropy differences between the conformers at equilibrium and the activation energy of the refolding process. The kinetics of a more stable 32 nt bistable RNA could be analyzed by the same approach at elevated temperatures, i.e. at 314 and 316 K. Finally, the dynamics of a multi-stable RNA able to fold into two hairpin- and a pseudo-knotted conformation was studied by 13C relaxation dispersion NMR spectroscopy. PMID:21252295

  15. The effects of isotope-labeled analogs on the LC-IDMS measurement by comparison of ESI responses and matrix effect of melamine, (13)C3-melamine, (13)C3+(15)N3-melamine, and (15)N3-melamine.

    PubMed

    Li, Xiu Qin; Zhang, Qing He; Yang, Zong; Li, Hong Mei; Huang, Dong Feng

    2017-05-01

    In this paper, the effect of isotope-labeled analogs on the liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) measurement was evaluated based on the comparison research of electrospray ionization responses (ESI) and matrix effect of melamine, (13)C3-melamine, (13)C3+(15)N3-melamine, and (15)N3-melamine. The isotope-labeled melamines had similar ionization efficiency with melamine in the electrospray ionization source, but the intensity of corresponding quantitative fragment ions had distinctive differences. Based on the density functional theory at the B3LYP/6-311+G** level, this phenomenon was explained very well. The rare cleavage pathways of melamine, which just could be exactly identified by (15)N-labeled melamines, resulted in the difference of quantitative fragment ions between (15)N-labeled melamines and melamine. The interaction of ESI response between melamine and isotope-labeled melamines was investigated using MRM monitor mode. (15)N-labeled melamine had significant ion inter-suppression effect on melamine, while (13)C-labeled melamine had little influence on melamine. Finally, the influence of different isotope-labeled melamines on the LC-IDMS result was evaluated using the IDMS correction factor (θ). Taking the determination of melamine in milk powder as an example, the matrix effects of different isotope-labeled melamines and melamine had notable difference and the impact of this difference on the measurement results depended on the concentrations of analyte and matrix solution. It was worth noting that (15)N3-melamine exhibited significant ion suppression to melamine in matrix solution. The deviation of the results from IDMS method might reach 59% using (15)N3-melamine as internal standard in special matrix solution. Graphical Abstract The comparison of ESI responses of melamine, (13)C3-melamine, (13)C3+(15)N3-melamine and (15)N3-melamine.

  16. 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

  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. Non-targeted determination of (13)C-labeling in the Methylobacterium extorquens AM1 metabolome using the two-dimensional mass cluster method and principal component analysis.

    PubMed

    Reaser, Brooke C; Yang, Song; Fitz, Brian D; Parsons, Brendon A; Lidstrom, Mary E; Synovec, Robert E

    2016-02-05

    A novel analytical workflow is presented for the analysis of time-dependent (13)C-labeling of the metabolites in the methylotrophic bacterium Methylobacterium extorquens AM1 using gas chromatography time-of-flight mass spectrometry (GC-TOFMS). Using (13)C-methanol as the substrate in a time course experiment, the method provides an accurate determination of the number of carbons converted to the stable isotope. The method also extracts a quantitative isotopic dilution time course profile for (13)C uptake of each metabolite labeled that could in principle be used to obtain metabolic flux rates. The analytical challenges encountered require novel analytical platforms and chemometric techniques. GC-TOFMS offers advanced separation of mixtures, identification of individual components, and high data density for the application of advanced chemometrics. This workflow combines both novel and traditional chemometric techniques, including the recently reported two-dimensional mass cluster plot method (2D m/z cluster plot method) as well as principal component analysis (PCA). The 2D m/z cluster plot method effectively indexed all metabolites present in the sample and deconvoluted metabolites at ultra-low chromatographic resolution (RS≈0.04). Using the pure mass spectra extracted, two PCA models were created. Firstly, PCA was used on the first and last time points of the time course experiment to determine and quantify the extent of (13)C uptake. Secondly, PCA modeled the full time course in order to quantitatively extract the time course profile for each metabolite. The 2D m/z cluster plot method found 152 analytes (metabolites and reagent peaks), with 54 pure analytes, and 98 were convoluted, with 65 of the 98 requiring mathematical deconvolution. Of the 152 analytes surveyed, 83 were metabolites determined by the PCA model to have incorporated (13)C while 69 were determined to be either metabolites or reagent peaks that remained unlabeled.

  19. 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

  20. Quantification of peptide m/z distributions from 13C-labeled cultures with high-resolution mass spectrometry.

    PubMed

    Allen, Doug K; Goldford, Joshua; Gierse, James K; Mandy, Dominic; Diepenbrock, Christine; Libourel, Igor G L

    2014-02-04

    Isotopic labeling studies of primary metabolism frequently utilize GC/MS to quantify (13)C in protein-hydrolyzed amino acids. During processing some amino acids are degraded, which reduces the size of the measurement set. The advent of high-resolution mass spectrometers provides a tool to assess molecular masses of peptides with great precision and accuracy and computationally infer information about labeling in amino acids. Amino acids that are isotopically labeled during metabolism result in labeled peptides that contain spatial and temporal information that is associated with the biosynthetic origin of the protein. The quantification of isotopic labeling in peptides can therefore provide an assessment of amino acid metabolism that is specific to subcellular, cellular, or temporal conditions. A high-resolution orbital trap was used to quantify isotope labeling in peptides that were obtained from unlabeled and isotopically labeled soybean embryos and Escherichia coli cultures. Standard deviations were determined by estimating the multinomial variance associated with each element of the m/z distribution. Using the estimated variance, quantification of the m/z distribution across multiple scans was achieved by a nonlinear fitting approach. Observed m/z distributions of uniformly labeled E. coli peptides indicated no significant differences between observed and simulated m/z distributions. Alternatively, amino acid m/z distributions obtained from GC/MS were convolved to simulate peptide m/z distributions but resulted in distinct profiles due to the production of protein prior to isotopic labeling. The results indicate that peptide mass isotopologue measurements faithfully represent mass distributions, are suitable for quantification of isotope-labeling-based studies, and provide additional information over existing methods.

  1. 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.

  2. 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.

  3. 13C NMR and isotopic (δ13C) investigations on modern vegetation samples: a tool to understand the soil organic matter degradation dynamics and preferences

    NASA Astrophysics Data System (ADS)

    Rakshit, Subhadeep; Sanyal, Prasanta; Vardhan Gaur, Harsh

    2015-04-01

    Soil organic carbon, one of the largest reservoirs of carbon, is a heterogeneous mixture of organic compounds with dominant contribution derived from decomposition of plants in various stages. Although general ideas about the processes and mechanisms of soil organic matter (SOM) degradation have been developed, a very few study has linked the SOM with its parent material. In this study we aim to generate reference data set of functional groups from modern vegetation samples (C3 and C4plants) to better understand the degradation dynamics and preferences. The carbon functional groups from modern vegetation samples (eight C3 and nine C4 plants collected from Mohanpur, Nadia, West Bengal, India) were examined by solid state 13C CPMAS NMR spectroscopy. Additionally, isotopic investigations (δ13C) has also been carried out on the modern vegetation samples to understand the relationship of bulk isotopic values to the concentration of functional groups. The major functional groups (alkyl C, O-alkyl C, aromatic C, carbonyl C and aldehyde/ketone) of modern vegetation samples form 16%, 65%, 5%, 14% and 1% respectively in C3 plants. Considerable differences has been observed for C4 plants with average values of alkyl C, O-alkyl C, aromatic C, carbonyl C and aldehyde/ketone are 8%, 83%, 3%, 5% and 1% respectively. The concentration of functional groups from the modern vegetational samples can be considered as reference scale to compare with the 13C NMR data derived from the different soil horizons to understand the SOM degradation dynamics. The δ13CV PDB values of modern vegetation samples plotted against the individual concentration of functional groups shows significant correlation in C4 plants, whereas a lack in correlation has been observed for C3 plants. We assume this difference in relationship of δ13CV PDB values with functional groups of C3 and C4plants can be due to the differences in photosynthesis pathways, the fractionation of CO2 and accumulation of the products

  4. Extreme 13C depletion of CCl2F2 in firn air samples from NEEM, Greenland

    NASA Astrophysics Data System (ADS)

    Zuiderweg, A.; Holzinger, R.; Martinerie, P.; Schneider, R.; Kaiser, J.; Witrant, E.; Etheridge, D.; Petrenko, V.; Blunier, T.; Röckmann, T.

    2013-01-01

    A series of 12 high volume air samples collected from the S2 firn core during the North Greenland Eemian Ice Drilling (NEEM) 2009 campaign have been measured for mixing ratio and stable carbon isotope composition of the chlorofluorocarbon CFC-12 (CCl2F2). While the mixing ratio measurements compare favorably to other firn air studies, the isotope results show extreme 13C depletion at the deepest measurable depth (65 m), to values lower than δ13C = -80‰ vs. VPDB (the international stable carbon isotope scale), compared to present day surface tropospheric measurements near -40‰. Firn air modeling was used to interpret these measurements. Reconstructed atmospheric time series indicate even larger depletions (to -120‰) near 1950 AD, with subsequent rapid enrichment of the atmospheric reservoir of the compound to the present day value. Mass-balance calculations show that this change is likely to have been caused by a large change in the isotopic composition of anthropogenic CFC-12 emissions, probably due to technological advances in the CFC production process over the last 80 yr, though direct evidence is lacking.

  5. Biosynthesis of pyrroloquinoline quinone. 1. Identification of biosynthetic precursors using /sup 13/C labeling and NMR spectroscopy

    SciTech Connect

    Houck, D.R.; Hanners, J.L.; Unkefer, C.J.

    1988-09-28

    The biosynthesis of pyrroloquinoline quinone (PQQ) in the methylotropic bacterium methylobacterium AM1 has been investigated using /sup 13/C-labelling of the products and NMR spectroscopy. The data indicated that the quinoline portion of PQQ is formed by a novel condensation of N-1, C-2, -3, and -4 of glutamate with a symmetrical six-carbon ring derived from the shikimate pathway. It is postulated that tyrosine is the shikimate-derived percursor, since pyrrole could be formed by the internal cyclization of the amino acid backbone. 18 references, 2 figures, 2 tables.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. HNCA-TOCSY-CANH experiments with alternate 13C-12C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

    PubMed Central

    Takeuchi, Koh; Gal, Maayan; Takahashi, Hideo; Shimada, Ichio

    2011-01-01

    Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak 3JCαCα coupling. These pulse sequences, which resemble recently described 13C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in 1H2O, and use 1H excitation and detection. These experiments require alternate 13C-12C labeling together with perdeuteration, which allows utilizing the small 3JCαCα scalar coupling that is otherwise masked by the stronger 1JCC couplings in uniformly 13C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the 13Cα of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i−1, i + 1 and i−2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOC-SY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the 15N-1H spin pair of residue i to adjacent amide protons and nitrogens at positions i−2, i−1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments. PMID:21110064

  11. 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.

  12. Preparative chromatography for specific δ13C isotopic analysis of individual carbohydrates in environmental samples

    NASA Astrophysics Data System (ADS)

    Nouara, Amel; Panagiotopoulos, Christos; Balesdent, Jérôme; Sempéré, Richard

    2017-04-01

    Carbohydrates are among the most abundant organic molecules on the Earth and are present in all geochemical systems. Despite their high abundance in the environment, very few studies assessed their origin using molecular carbohydrate isotopic analyses. In contrast with bulk stable isotope analysis (BSIA), which gives the isotopic signature of the entire sample without any specification about its chemical composition, compound specific 13C isotopic analysis of individual sugars (CSIA) offers valuable information about the origin of single molecules. Previous investigations used gas or liquid chromatography coupled with isotope ratio mass spectroscopy (GC-IRMS; HPLC-IRMS) for CSIA of sugars however the former requires δ13C corrections due to the carbon added to the sugar (derivatization) while the later does not provide always adequate separations among monosaccharides. Here we used cation preparative chromatography (Ca2+, Pb2+ and Na+) with refractive index detection in order to produce pure monosaccharide targets for subsequent EA-IRMS analyses. Milli-Q water was used as eluant at a flow rate 0.6 ml min-1. In general, three successive purifications (Ca2+, Pb2+, Ca2+) were sufficient to produce pure compounds. Pure monosaccharides were compared with authentic monosaccharide standards using 1H NMR and/or mass spectroscopy. The detection limit of our technique was about 1µM/sugar with a precision of 10% (n=6). Blanks run with Milli-Q water after three successive purifications resulted in carbon content of 0.13 to 2.77 µgC per collected sugar. These values are much lower than the minimum required amount (5 µgC) of the EA-IRSMS system with a precision of ± 0.35 ‰. Application of our method to environmental samples resulted in δ13C values of glucose, fructose, and levoglucosan in the range of -24 to -26 ‰ (PM10 atmospheric particles), and -15‰ to -22 ‰ for arabinose, glucose, and xylose (marine high molecular dissolved organic matter). These results fall in

  13. 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

  14. Respiratory Carbon Metabolism following Illumination in Intact French Bean Leaves Using 13C/12C Isotope Labeling1

    PubMed Central

    Nogués, Salvador; Tcherkez, Guillaume; Cornic, Gabriel; Ghashghaie, Jaleh

    2004-01-01

    The origin of the carbon atoms in the CO2 respired by French bean (Phaseolus vulgaris) leaves in the dark has been studied using 13C/12C isotopes as tracers. The stable isotope labeling was achieved through a technical device that uses an open gas-exchange system coupled online to an elemental analyzer and linked to an isotope ratio mass spectrometer. The isotopic analysis of the CO2 respired in the dark after a light period revealed that the CO2 was labeled, but the labeling level decreased progressively as the dark period increased. The pattern of disappearance depended on the amount of carbon fixed during the labeling and indicated that there were several pools of respiratory metabolites with distinct turnover rates. We demonstrate that the carbon recently assimilated during photosynthesis accounts for less than 50% of the carbon in the CO2 lost by dark respiration and that the proportion is not influenced by leaf starvation in darkness before the labeling. Therefore, most of the carbon released by dark respiration after illumination does not come from new photosynthates. PMID:15377781

  15. Respiratory carbon metabolism following illumination in intact French bean leaves using (13)C/(12)C isotope labeling.

    PubMed

    Nogués, Salvador; Tcherkez, Guillaume; Cornic, Gabriel; Ghashghaie, Jaleh

    2004-10-01

    The origin of the carbon atoms in the CO(2) respired by French bean (Phaseolus vulgaris) leaves in the dark has been studied using (13)C/(12)C isotopes as tracers. The stable isotope labeling was achieved through a technical device that uses an open gas-exchange system coupled online to an elemental analyzer and linked to an isotope ratio mass spectrometer. The isotopic analysis of the CO(2) respired in the dark after a light period revealed that the CO(2) was labeled, but the labeling level decreased progressively as the dark period increased. The pattern of disappearance depended on the amount of carbon fixed during the labeling and indicated that there were several pools of respiratory metabolites with distinct turnover rates. We demonstrate that the carbon recently assimilated during photosynthesis accounts for less than 50% of the carbon in the CO(2) lost by dark respiration and that the proportion is not influenced by leaf starvation in darkness before the labeling. Therefore, most of the carbon released by dark respiration after illumination does not come from new photosynthates.

  16. PICquant: A Quantitative Platform to Measure Differential Peptide Abundance Using Dual-Isotopic Labeling with 12C6- and 13C6-Phenylisocyanate

    PubMed Central

    Lyons, Charles E.; Victor, Ken G.; Moshnikov, Sergey A.; Bachmann, Lorin M.; Baras, Alexander S.; Dettmann, Kathleen M.; Cross, Janet V.; Templeton, Dennis J.

    2011-01-01

    We have developed a complete system for the isotopic labeling, fractionation, and automated quantification of differentially expressed peptides that significantly facilitates candidate biomarker discovery. We describe a new stable mass tagging reagent pair, 12C6- and 13C6-phenylisocyanate (PIC), that offers significant advantages over currently available tags. Peptides are labeled predominantly at their amino termini and exhibit elution profiles that are independent of label isotope. Importantly, PIC-labeled peptides have unique neutral-mass losses upon CID fragmentation that enable charge state and label isotope identification and, thereby, decouple the sequence identification from the quantification of candidate biomarkers. To exploit these properties, we have coupled peptide fractionation protocols with a Thermo LTQ-XL LC-MS2 data acquisition strategy and a suite of automated spectrum analysis software that identifies quantitative differences between labeled samples. This approach, dubbed the PICquant platform, is independent of protein sequence identification and excludes unlabeled peptides that otherwise confound biomarker discovery. Application of the PICquant platform to a set of complex clinical samples showed that the system allows rapid identification of peptides that are differentially expressed between control and patient groups. PMID:21192683

  17. (13)C glucose labelling studies using 2D NMR are a useful tool for determining ex vivo whole organ metabolism during hypothermic machine perfusion of kidneys.

    PubMed

    Nath, Jay; Smith, Tom; Hollis, Alex; Ebbs, Sam; Canbilen, Sefa W; Tennant, Daniel A; Ready, Andrew R; Ludwig, Christian

    2016-01-01

    The aim of this study is to determine the feasibility of using nuclear magnetic resonance (NMR) tracer studies ((13)C-enriched glucose) to detect ex vivo de novo metabolism in the perfusion fluid and cortical tissue of porcine kidneys during hypothermic machine perfusion (HMP). Porcine kidneys (n = 6) were subjected to 24 h of HMP using the Organ Recovery Systems LifePort Kidney perfusion device. Glucose, uniformly enriched with the stable isotope (13)C ([U-(13)C] glucose), was incorporated into KPS-1-like perfusion fluid at a concentration of 10 mM. Analysis of perfusate was performed using both 1D (1)H and 2D (1)H,(13)C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The metabolic activity was then studied by quantifying the proportion of key metabolites containing (13)C in both perfusate and tissue samples. There was significant enrichment of (13)C in a number of central metabolites present in both the perfusate and tissue extracts and was most pronounced for lactate and alanine. The total amount of enriched lactate (per sample) in perfusion fluid increased during HMP (31.1 ± 12.2 nmol at 6 h vs 93.4 ± 25.6 nmol at 24 h p < 0.01). The total amount of enriched alanine increased in a similar fashion (1.73 ± 0.89 nmol at 6 h vs 6.80 ± 2.56 nmol at 24 h p < 0.05). In addition, small amounts of enriched acetate and glutamic acid were evident in some samples. This study conclusively demonstrates that de novo metabolism occurs during HMP and highlights active metabolic pathways in this hypothermic, hypoxic environment. Whilst the majority of the (13)C-enriched glucose is metabolised into glycolytic endpoint metabolites such as lactate, the presence of non-glycolytic pathway derivatives suggests that metabolism during HMP is more complex than previously thought. Isotopic labelled ex vivo organ perfusion studies using 2D NMR are feasible and informative.

  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. 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].

  20. An efficient procedure for assignment of the proton, carbon and nitrogen resonances in 13C/15N labeled nucleic acids.

    PubMed

    Nikonowicz, E P; Pardi, A

    1993-08-20

    An efficient method is presented for the assignment of the proton, carbon, and nitrogen resonances in the NMR spectra of isotopically labeled nucleic acids. The assignment strategy starts by identifying all protons and carbons belonging to the same sugar ring through application of a set of 2D or 3D heteronuclear HCCH NMR experiments. Next the individual sugar rings are connected to their corresponding bases through intra-residue 1H-1H nuclear Overhauser effects (NOEs) observed in a 3D (1H, 13C, 1H) NOESY-HMQC experiment. Sequential NOE connectivities observed in this experiment are then used to assign each residue in the nucleotide sequence. The imino protons and nitrogens, and the cytidine amino protons and nitrogens, are assigned by 2D (15N, 1H) HMQC and 3D (1H, 15N, 1H) NOESY-HMQC experiments in H2O. This assignment procedure is illustrated on the 99% 13C/15N labeled RNA duplex r(GGCGCUUGCGUC)2. The application of these multi-dimensional heteronuclear magnetic resonance experiments enormously simplifies the resonance assignment of nucleic acids and allows assignment of many more protons, carbons and nitrogens than was possible using standard techniques on unlabeled molecules. Since a larger percentage of the protons can now be assigned by these experiments, much more NMR structural information can be obtained which will significantly extend the size limit for solution structure determinations of RNAs.

  1. 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.

  2. 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.

  3. Assimilation of xylem-transported 13C-labelled CO2 in leaves and branches of sycamore (Platanus occidentalis L.).

    PubMed

    McGuire, M A; Marshall, J D; Teskey, R O

    2009-01-01

    Previous reports have shown that CO(2) dissolved in xylem sap in tree stems can move upward in the transpiration stream. To determine the fate of this dissolved CO(2), the internal transport of respired CO(2) at high concentration from the bole of the tree was simulated by allowing detached young branches of sycamore (Platanus occidentalis L.) to transpire water enriched with a known quantity of (13)CO(2) in sunlight. Simultaneously, leaf net photosynthesis and CO(2) efflux from woody tissue were measured. Branch and leaf tissues were subsequently analysed for (13)C content to determine the quantity of transported (13)CO(2) label that was fixed. Treatment branches assimilated an average of 35% (SE=2.4) of the (13)CO(2) label taken up in the treatment water. The majority was fixed in the woody tissue of the branches, with smaller amounts fixed in the leaves and petioles. Overall, the fixation of internally transported (13)CO(2) label by woody tissues averaged 6% of the assimilation of CO(2) from the atmosphere by the leaves. Woody tissue assimilation rates calculated from measurements of (13)C differed from rates calculated from measurements of CO(2) efflux in the lower branch but not in the upper branch. The results of this study showed unequivocally that CO(2) transported in xylem sap can be fixed in photosynthetic cells in the leaves and branches of sycamore trees and provided evidence that recycling of xylem-transported CO(2) may be an important means by which trees reduce the carbon cost of respiration.

  4. 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.

  5. Capillary Absorption Spectrometer for 13C Isotopic Composition of Pico to Subpico Molar Sample Quantities

    NASA Astrophysics Data System (ADS)

    Moran, J.; Kelly, J.; Sams, R.; Newburn, M.; Kreuzer, H.; Alexander, M.

    2011-12-01

    Quick incorporation of IR spectroscopy based isotope measurements into cutting edge research in biogeochemical cycling attests to the advantages of a spectroscopy versus mass spectrometry method for making some 13C measurements. The simple principles of optical spectroscopy allow field portability and provide a more robust general platform for isotope measurements. We present results with a new capillary absorption spectrometer (CAS) with the capability of reducing the sample size required for high precision isotopic measurements to the picomolar level and potentially the sub-picomolar level. This work was motivated by the minute sample size requirements for laser ablation isotopic studies of carbon cycling in microbial communities but has potential to be a valuable tool in other areas of biological and geological research. The CAS instrument utilizes a capillary waveguide as a sample chamber for interrogating CO2 via near IR laser absorption spectroscopy. The capillary's small volume (~ 0.5 mL) combined with propagation and interaction of the laser mode with the entire sample reduces sample size requirements to a fraction of that accessible with commercially available IR absorption including those with multi-pass or ring-down cavity systems. Using a continuous quantum cascade laser system to probe nearly adjacent rovibrational transitions of different isotopologues of CO2 near 2307 cm-1 permits sample measurement at low analyte pressures (as low as 2 Torr) for further sensitivity improvement. A novel method to reduce cw-fringing noise in the hollow waveguide is presented, which allows weak absorbance features to be studied at the few ppm level after averaging 1,000 scans in 10 seconds. Detection limits down to the 20 picomoles have been observed, a concentration of approximately 400 ppm at 2 Torr in the waveguide with precision and accuracy at or better than 1 %. Improvements in detection and signal averaging electronics and laser power and mode quality are

  6. 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

  7. Biomineralization of 1,4-dioxane in Pure Culture, Microcosm, and Column Studies Using 13C Labeling

    NASA Astrophysics Data System (ADS)

    Rolston, H. M.; Azizian, M.; Hyman, M. R.; Semprini, L.

    2016-12-01

    1,4-dioxane (1,4-D), a probable human carcinogen at low (< 1ppb) concentrations, is a widely occurring groundwater contaminant due to its use as a stabilizer for chlorinated solvents. Aerobic cometabolism, the use of a primary substrate to induce the production of microbial enzymes capable of transforming contaminants into innocuous products, is a promising in-situ treatment strategy for 1,4-D because it has the potential to mineralize trace 1,4-D concentrations to carbon dioxide (CO2). Previous work has confirmed the bacterium Rhodococcus rhodochrous (strain ATCC 21198) will transform 1,4-D when grown on isobutane. In this study, 13C uniformly labeled 1,4-D was used to determine the extent to which strain ATCC 21198 biomineralizes 1,4-D to CO2. Batch experiments have been conducted with pure culture 21198 and in microcosms constructed with aquifer sediments. The rate of resting cell transformation of 1,4-D by ATCC 21198 was over 100 times faster than the rate of CO2 accumulation, indicating the presence of intermediates that were slowly mineralized to CO2 . In microcosms, the use of isobutane as a primary substrate effectively stimulated the native microbial community to transform 1,4-D. Microcosms were also bioaugmented with ATCC 21198. After an initial lag and subsequent additions of isobutane, transformation rates in the native microcosms approached those of the bioaugmented microcosms. Cometabolically active microbes survived several periods of starvation in all microcosms, and nutrient amendment allowed for sustained transformation rates. 13C labeled 1,4-D is currently being used to determine the rates and extents of biomineralization in the microcosms. Column studies are also being conducted to evaluate cometabolism and biominerazation potential of isobutane as a biostimulant and 21198 for bioaugmentation under geochemical and flow conditions more representative of in-situ bioremediation.

  8. The interactions with solvent, heat stability, and 13C-labelling of alamethicin, an ion-channel-forming peptide.

    PubMed

    Yee, A A; Marat, K; O'Neil, J D

    1997-01-15

    The peptide alamethicin was labelled with 13C and 15N by growing the fungus Trichoderma viride in a medium containing [U-13C] glucose and K15NO3. Spin-echo difference spectroscopy showed that 13C was incorporated to a level of about 50% and 15N to about 98%. Incorporation of 13C into the peptide provided residue-specific probes of the interactions with solvent and heat stability of this ion-channel-forming peptide. All of the carbonyl carbons and the alpha-carbons of the alpha-aminoisobutyric acid [Ala(Me)] residues of alamethicin in methanol were assigned using two-dimensional and three-dimensional heteronuclear correlation experiments. Measurements of 1JC'N revealed hydrogen bonding with solvent at residues 1 and 19 at the ends of the peptide and at Gly11 in the middle. The data also support the thesis [see Juranic, N., Ilich, P. K. & Macara, S. (1995) J. Am. Chem. Soc. 117, 405-410 that intramolecular hydrogen bonds in proteins and peptides are weaker than hydrogen bonds to solvent. The sensitivity of alamethicin carbonyl and proton chemical shifts to perturbation by dimethyl sulfoxide correlates well with the calculated solvent accessibilities of the carbonyls in the crystal structures and reveals residues in the middle of the peptide and at the C-terminus which interact with solvent. Taken together with the 1JC'N measurements, the data support a model in which hydrogen bonding to solvent at the Gly11/Leu12 amide could provide a site of hydration in the interior of the alamethicin channel structure. The temperature dependencies of the carbonyl chemical shifts support the suggestion that the peptide is flexible in the regions where solvent interacts with the backbone of the peptide. The linear temperature dependence of the carbonyl chemical shifts and molar ellipticity indicate that, due to steric constraints at the Ala(Me) residues, the peptide folding/unfolding transition is non-cooperative and that the peptide is remarkably heat stable.

  9. 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.

  10. 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.

  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. 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.

  14. hNCOcanH pulse sequence and a robust protocol for rapid and unambiguous assignment of backbone ((1)H(N), (15)N and (13)C') resonances in (15)N/(13)C-labeled proteins.

    PubMed

    Kumar, Dinesh; Hosur, Ramakrishna V

    2011-09-01

    A three-dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in (15)N/(13)C-labeled proteins. The experiment has been derived by a simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147]; t2 evolution is used to frequency label (13)C' rather than (15)N (similar trick has also been used in the design of hNCAnH pulse sequence from hNcaNH [Frueh et al., JACS, 131 (2009) 12880-12881]). The modification results in a spectrum equivalent to HNCO, but in addition to inter-residue correlation peaks (i.e. Hi , Ci-1), the spectrum also contains additional intra-residue correlation peaks (i.e. Hi-1 , Ci-1) in the direct proton dimension which has maximum resolution. This is the main strength of the experiment and thus, even a small difference in amide (1) H chemical shifts (5-6 Hz) can be used for establishing a sequential connectivity. This experiment in combination with the HNN experiment described previously [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] leads to a more robust assignment protocol for backbone resonances ((1) H(N) , (15)N) than could be derived from the combination of HNN and HN(C)N experiments [Bhavesh et al., Biochemistry, 40 (2001) 14727-14735]. Further, this new protocol enables assignment of (13)C' resonances as well. We believe that the experiment and the protocol presented here will be of immense value for structural-and functional-proteomics research by NMR. Performance of this experiment has been demonstrated using (13)C/(15)N labeled ubiquitin. Copyright © 2011 John Wiley & Sons, Ltd.

  15. Identification of metabolites of honokiol in rat urine using 13C stable isotope labeling and liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

    PubMed

    Liu, Juan; Tang, Minghai; Lai, Huijun; Dong, Yinfeng; Xie, Caifeng; Ye, Haoyu; Ma, Liang; Qiu, Neng; Li, Yanfang; Cai, Lulu; Chen, Lijuan

    2013-06-21

    A general approach based on stable isotope labeling and UPLC/Q-TOF-MS analysis of in vivo novel metabolites of honokiol has been developed in our study. In this method, urine samples were collected after intravenous administration of mixture of regular and [(13)C6]-labeled honokiol at 1:1 ratio to healthy rats. The metabolites could be easily recognized by the determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to that between isotope-labeled and non-isotope-labeled honokiol. A total of 51 metabolites were detected, 37 of which were tentatively identified based on mass accuracy (<5 ppm). Among them, 33 of honokiol metabolites were first reported with 5 metabolites belonging to phase I and other 32 metabolites belonging to phase II metabolites. Our results highlighted that the main phase I metabolic pathways of honokiol in rats were oxidation, and the phase II metabolic pathways were sulfation, glucuronidation, acetylation as well as amino acids conjugation. This was the first research focused on the biotransformation of honokiol in rats, and the identification of these metabolites might provide us essential information for further pharmacological and clinical studies of honokiol. Copyright © 2013 Elsevier B.V. All rights reserved.

  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. The influence of preservation method and time on the delta13C value of dissolved inorganic carbon in water samples.

    PubMed

    Taipale, Sami J; Sonninen, Eloni

    2009-08-30

    The precise delta(13)C value of dissolved inorganic carbon (DIC) is important for various types of ecological studies. Without a preservation agent, microbial degradation of organic compounds continues in water samples and the delta(13)C value of DIC will become more depleted with time. HgCl(2) or acidification is often used to prevent microbial activity in water samples collected for carbon isotope ratio analyses of DIC. Mercury compounds are toxic and result in waste disposal problems. Other inhibiting agents or preservation methods are therefore needed. Two possible solutions are to use copper sulphate (CuSO(4)) as a preservative agent or to acidify water samples with phosphoric acid (H(3)PO(4)) within 12 mL measurement Exetainers (septum-capped vials). We prepared a set of lake water samples in three types of vials: glass vials with silicone/PTFE septa, high-density polyethylene vials (HD-PE, scintillation vials) and Exetainers (12 mL) with butyl rubber septa. Samples in glass and PE vials were preserved with and without CuSO(4), whereas lake water was injected into the Exetainer and acidified with H(3)PO(4). Isotope ratios were measured in two laboratories over 6 months. The delta(13)C values of DIC systematically increased with storage time for samples preserved in glass and PE vials with and without CuSO(4). A strong correlation between a decrease of CO(2) concentration and an increase in DIC delta(13)C values was found. The delta(13)C values and DIC concentrations were stable for 6 months in acidified samples stored in Exetainers with butyl rubber septa. Therefore, we conclude that the best method for up to 6 months of storage is to inject samples in the field into butyl rubber septum capped Exetainers containing H(3)PO(4), thereby avoiding the use of preservatives. 2009 John Wiley & Sons, Ltd.

  1. 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.

  2. Root and nodule growth in Pisum sativum L. in relation to photosynthesis: analysis using 13C-labelling.

    PubMed

    Voisin, A S; Salon, C; Jeudy, C; Warembourg, F R

    2003-10-01

    The effect of the nitrogen source (gaseous nitrogen, N2, or nitrate ions, NO3-) on the use of carbon (C) for root and nodule growth of pea (Pisum sativum L.) was investigated using 13C-labelling of assimilated CO2 at various stages of growth. Nitrate supply and growing conditions (sowing dates, air CO2 concentration) were varied to alter photosynthetic rates. Nodules are the sink with the highest demand for C in both the vegetative and flowering stages, growing at the expense of shoot and root in the vegetative stage, but only at the expense of roots at flowering. Until flowering, the addition of C into root and nodule biomass was linearly related to pre-existing biomass, thus determining net sink strengths which decreased with root and nodule age. Nodule growth patterns did not depend on the N source, whereas root growth was increased by nitrate when nodule biomass was low. At seed filling, the increase in C of biomass of the root system was no longer related to pre-existing biomass and C was preferentially diverted to roots of plants assimilating nitrate, or to nodules for plants fixing N2.

  3. 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.

  4. Weak coupling between magnetically inequivalent spins: The deceptively simple, complicated spectrum of a 13C-labeled trimethylated amine

    NASA Astrophysics Data System (ADS)

    le Paige, Ulric B.; Smits, Bauke; 't Hart, Peter; Lefeber, Fons; Martin, Nathaniel I.; van Ingen, Hugo

    2017-05-01

    Magnetic inequivalence of nuclear spins is well known to cause additional splittings that complicate spectral analysis. Here, we present an extraordinary case of magnetic inequivalence, manifested in the 13-spin system of a 13C,15N-labeled trimethylated amine. All methyl group protons are chemically equivalent due to the molecular symmetry, but not all are magnetically equivalent as they have different 1JCH and 3JCH couplings. In general, spectra of such a large spin system can be expected to be extremely complicated by the presence of hundreds if not thousands of extra lines, caused by the strong coupling between inequivalent nuclei. Surprisingly, the 1H spectrum presented consists of very few lines, in a pattern of the utmost simplicity. Using sub-spectral analysis we show that this is due to weak coupling between the magnetically inequivalent nuclei, as a consequence of the particular combination of coupling constants. We find that the 4JHH geminal methyl coupling constant is 0.43 Hz and 2JCC is ∼0 Hz. In addition, we demonstrate that homo-decoupling can be used to transform the spin system to a set of fully equivalent spins, resulting in disappearance of 4JHH-splittings. We believe this curious case is a highly instructive example of magnetic inequivalence. The spectra may be considered deceptively simple, as fewer lines are observed than one would anticipate. At the same time, the spectra are deceptively complicated, as they can very well be approximated by intuitive reasoning.

  5. 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

  6. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. 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

  8. 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

  9. 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.

  10. Cost-effective production of 13C, 15N stable isotope-labelled biomass from phototrophic microalgae for various biotechnological applications.

    PubMed

    Acién Fernández, F G; Fernández Sevilla, J M; Egorova-Zachernyuk, T A; Molina Grima, E

    2005-12-01

    The present study outlines a process for the cost-effective production of 13C/15N-labelled biomass of microalgae on a commercial scale. The core of the process is a bubble column photobioreactor with exhaust gas recirculation by means of a low-pressure compressor. To avoid accumulation of dissolved oxygen in the culture, the exhaust gas is bubbled through a sodium sulphite solution prior to its return to the reactor. The engineered system can be used for the production of 13C, 15N, and 13C-15N stable isotope-labelled biomass as required. To produce 13C-labelled biomass, 13CO2 is injected on demand for pH control and carbon supply, whereas for 15N-labelled biomass Na15NO3 is supplied as nitrogen source at the stochiometric concentration. The reactor is operated in semicontinuous mode at different biomass concentrations, yielding a maximum mean biomass productivity of 0.3 gL(-1) day(-1). In order to maximize the uptake efficiency of the labelled substrates, the inorganic carbon is recovered from the supernatant by acidification/desorption processes, while the nitrate is delivered at stochiometric concentration and the harvesting of biomass is performed when the 15NO3- is depleted. In these conditions, elemental analysis of both biomass and supernatant shows that 89.2% of the injected carbon is assimilated into the biomass and 6.9% remains in the supernatant. Based on elemental analysis, 97.8% of the supplied nitrogen is assimilated into the biomass and 1.3% remains in the supernatant. Stable isotope-labelling enrichment has been analysed by GC-MS results showing that the biomass is highly labelled. All the fatty acids are labelled; more than 96% of the carbon present in these fatty acids is 13C. The engineered system was stably operated for 3 months, producing over 160 g of 13C and/or 15N-labelled biomass. The engineered bioreactor can be applied for the labelling of various microalgae.

  11. The effect of sample hydration on 13C CPMAS NMR spectra of fulvic acids

    USGS Publications Warehouse

    Hatcher, P.G.; Wilson, M.A.

    1991-01-01

    Three fulvic acids, two of which have been well studied by a number of other groups (Armadale and Suwannee river fulvic acids) have been examined by high resolution solid-state 13C-NMR techniques to delineate the effect of absorbed water. Two main effects of absorbed water were observed: (1) changes in spin lattice relaxation times in the rotating frame and cross polarization times and (2) total loss of signal so that some fulvic acid is effectively in solution. These results suggest that discrepancies in the literature concerning observed relative signal intensities from different structural groups are due to absorbed water and emphasize the necessity for proper precautionary drying before spectroscopic analysis. ?? 1991.

  12. 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

  13. Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously.

    PubMed

    Blank, Lars M; Desphande, Rahul R; Schmid, Andreas; Hayen, Heiko

    2012-06-01

    Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly (13)C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously--i.e., (13)C and (15)N--in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with (13)C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon-nitrogen co-metabolism by administering both (13)C-labeled glucose and (15)N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes.

  14. Investigating CH4 production in an oxic plant-soil system -a new approach combining isotopic labelling (13C) and inhibitors

    NASA Astrophysics Data System (ADS)

    Lenhart, Katharina; Keppler, Frank

    2017-04-01

    Typically, aerated soil are net sinks of atmospheric methane (CH4), being highest in native ecosystems (pristine forests > managed forests > grasslands > crop fields). However, this does not exclude a simultaneous endogenic CH4 production in the plant-soil system, which cannot be detected simply via CH4 flux measurements. Methanogenic archaea producing CH4 under anoxic conditions were thought to be the only biotic source of CH4 in the soil. However, until recently a non-archaeal pathway of CH4 formation is known where CH4 is produced under oxic conditions in plants (Keppler et al. 2006) and fungi (Lenhart et al. 2012). Additionally, abiotic formation of CH4 from soil organic matter was reported (Jugold et al. 2012) and may be ubiquitous in terrestrial ecosystems. The major goal of this project was to determine soil endogenic CH4 sources and to estimate their contribution to the endogenic CH4 production. Especially the effect of plants and fungi on soil CH4 production was investigated. Therefore, a series of experiments was carried out on field fresh soil collected in a grassland and a forest ecosystem under controlled laboratory conditions. By combining selective inhibitors and 13C labelling, CH4 production rates of several CH4 sources were quantified. The major difficulty was to detect the comparatively small flux of CH4 production against the background of the high CH4 consumption rates due to methanotrophic bacteria. Therefore, we supplemented bare soil and soil with vegetation with selective inhibitors and 13C labelled substrates in a closed chamber system. In a first step, CH4 production was determined by the inhibition of CH4 oxidizing bacteria with Difluoromethane (DFM, 2ml l-1). In the following, a 13C labelled substrate (either CO2, Acetate, or Methionine -S-CH3 labelled) was added in combination with a specific inhibitor -either for archaeal methanogenesis (Bromoethanesulfonate), bacteria (Streptomycin), or fungi (Captan, Cycloheximide). Gas samples were

  15. Soil Fertilization Leads to a Decline in Between-Samples Variability of Microbial Community δ13C Profiles in a Grassland Fertilization Experiment

    PubMed Central

    Veresoglou, Stavros D.; Thornton, Barry; Menexes, George; Mamolos, Andreas P.; Veresoglou, Demetrios S.

    2012-01-01

    Gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) was used to measure the 13C/12C ratios of PLFAs at natural abundance levels from a temperate grassland nitrogen (N) and phosphorus (P) factorial fertilization experiment in northern Greece. In each plot two rhizosphere samples were derived centred around individual Agrostis capillaris and Prunella vulgaris plants. It was hypothesized that the isotopic signal of microbes that preferentially feed on recalcitrant litter such as fungi would be modified by fertilization more strongly than that of opportunistic microbes using labile C. Microbial community δ13C was affected by both P and N fertilization regime and plant species identity. However, we have been unable to detect significant nutrient effects on individual groups of microbes when analyzed separately in contrast to our original hypothesis. Intra-treatment variability, as evaluated from Hartley’s Fmax tests in the five first PCA components axes as well as the size of the convex hulls in PCA scoreplots and Mahalanobis distances, was considerably higher in the non-fertilized controls. Moreover, a significant relationship was established between the change in PLFA abundances and their respective changes in δ13C for the aggregate of samples and those simultaneously fertilized with N and P. We conclude that use of compound specific isotope analysis in the absence of labelling represents a valuable and overlooked tool in obtaining an insight of microbial community functioning. PMID:22962602

  16. Synthesis of /sup 13/C-labeled standards for use in coal liquefaction studies. II. Dissolving metal reactions applied to naphthalenes and indoles: effect of sonication

    SciTech Connect

    Pickering, R.E.

    1986-01-01

    High yield syntheses of /sup 13/C-labeled standards for later use in coal liquefaction studies are described. An alternate route for the synthesis of 1-cyclohexanone-1-/sup 13/C is discussed. Naphthalene and 2,3-dimethylnaphthalene were reduced with metal-amine solutions. The effect of sonication on product distribution was investigated. The effect of different amine solvent and different metals also was studied. A series of indoles were reduced with lithium and ethylenediamine to study the effect of sonication. To aid identification of some reduction products, independent syntheses were conducted. Naphthalenol-1-/sup 13/C, phenol-1-/sup 13/C, 1-indanone-1-/sup 13/C, 3,4-dihydro-2(1H)-quinolinone-2-/sup 13/C, 1,2,3,4-tetrahydroquinoline-2-/sup 13/C and carbazole-9a-/sup 13/C were synthesized in good yield. The carbonation of 1,5-pentanedimagnesium bromide was shown to be a viable alternative route for the synthesis of 1-cyclohexanone-1-/sup 13/C. Sonication was found to be an efficient agitation method for the reductive dimerization or the reductive amination of naphthalene and 2,3-dimethylnaphthalene. The product distribution was found to be dependent on the selection of the amine solvent. Sodium was shown to be a better reagent for reductive dimerization than potassium, lithium, calcium, or magnesium. The synthesis of 6,6',7,7'-tetramethyl-,1'-binaphthyl, 6,6',7,7'-tetramethyl-1,2'-binaphthyl, 6,6',7,7'-tetramethyl-2,2'-binaphthyl and some of their hydro-derivatives are described. The reduction of indoles with lithium-ethylenediamine was found to be influenced by sonication which significant increased the yield of the 4,5,6,7-tetrahydro-derivative. N-Substituted indoles were shown to undergo reductive cleavage with lithium-ethylenediamine while being treated with ultrasound.

  17. 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

  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. 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.

  20. Kinetics of labelling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labelled isotopes.

    PubMed

    Roessner-Tunali, Ute; Liu, JunLi; Leisse, Andrea; Balbo, Ilse; Perez-Melis, Alicia; Willmitzer, Lothar; Fernie, Alisdair R

    2004-08-01

    Metabolic pathways of primary metabolism of discs isolated from potato tubers were evaluated by the use of a gas chromatography-mass spectrometry (GC-MS) method generated specifically for this purpose. After testing several possible methods including chemical ionization, it was decided for reasons of sensitivity, reproducibility and speed to use electron impact ionization-based GC-MS analysis. The specific labelling and label accumulation of over 30 metabolites including a broad number of sugars, organic and amino acids was analysed following the incubation of tuber discs in [U-(13)C]glucose. The reproducibility of this method was similar to that found for other GC-MS-based analyses and comparison of flux estimates from this method with those obtained from parallel, yet less comprehensive, radiolabel experiments revealed close agreement. Therefore, the novel method allows quantitatively evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate), chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabelled substrates. As a first experiment the GC-MS method has been applied to compare the metabolism of wild type and well-characterized transgenic potato tubers exhibiting an enhanced sucrose mobilization. The fact that this method is able to rapidly yield further comprehensive information into primary metabolism illustrates its power as a further phenotyping tool for the analysis of plant metabolism.

  1. A capillary absorption spectrometer for stable carbon isotope ratio (13C/12C) analysis in very small samples

    NASA Astrophysics Data System (ADS)

    Kelly, J. F.; Sams, R. L.; Blake, T. A.; Newburn, M.; Moran, J.; Alexander, M. L.; Kreuzer, H.

    2012-02-01

    A capillary absorption spectrometer (CAS) suitable for IR laser isotope analysis of small CO2 samples is presented. The system employs a continuous-wave (cw) quantum cascade laser to study nearly adjacent rovibrational transitions of different isotopologues of CO2 near 2307 cm-1 (4.34 μm). This initial CAS system can achieve relative isotopic precision of about 10 ppm 13C, or ˜1‰ (per mil in delta notation relative to Vienna Pee Dee Belemnite) with 20-100 picomoles of entrained sample within the hollow waveguide for CO2 concentrations ˜400-750 ppm. Isotopic analyses of such gas fills in a 1-mm ID hollow waveguide of 0.8 m overall physical path length can be carried out down to ˜2 Torr. Overall 13C/12C ratios can be calibrated to ˜2‰ accuracy with diluted CO2 standards. A novel, low-cost method to reduce cw-fringing noise resulting from multipath distortions in the hollow waveguide is presented, which allows weak absorbance features to be studied at the few ppm level (peak-to-rms) after 1000 scans are co-added in ˜10 s. The CAS is meant to work directly with converted CO2 samples from a laser ablation-catalytic combustion micro-sampler to provide 13C/12C ratios of small biological isolates currently operating with spatial resolutions ˜50 μm.

  2. 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

  3. 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.

  4. Organic vs. conventional grassland management: do (15)N and (13)C isotopic signatures of hay and soil samples differ?

    PubMed

    Klaus, Valentin H; Hölzel, Norbert; Prati, Daniel; Schmitt, Barbara; Schöning, Ingo; Schrumpf, Marion; Fischer, Markus; Kleinebecker, Till

    2013-01-01

    Distinguishing organic and conventional products is a major issue of food security and authenticity. Previous studies successfully used stable isotopes to separate organic and conventional products, but up to now, this approach was not tested for organic grassland hay and soil. Moreover, isotopic abundances could be a powerful tool to elucidate differences in ecosystem functioning and driving mechanisms of element cycling in organic and conventional management systems. Here, we studied the δ(15)N and δ(13)C isotopic composition of soil and hay samples of 21 organic and 34 conventional grasslands in two German regions. We also used Δδ(15)N (δ(15)N plant - δ(15)N soil) to characterize nitrogen dynamics. In order to detect temporal trends, isotopic abundances in organic grasslands were related to the time since certification. Furthermore, discriminant analysis was used to test whether the respective management type can be deduced from observed isotopic abundances. Isotopic analyses revealed no significant differences in δ(13)C in hay and δ(15)N in both soil and hay between management types, but showed that δ(13)C abundances were significantly lower in soil of organic compared to conventional grasslands. Δδ(15)N values implied that management types did not substantially differ in nitrogen cycling. Only δ(13)C in soil and hay showed significant negative relationships with the time since certification. Thus, our result suggest that organic grasslands suffered less from drought stress compared to conventional grasslands most likely due to a benefit of higher plant species richness, as previously shown by manipulative biodiversity experiments. Finally, it was possible to correctly classify about two third of the samples according to their management using isotopic abundances in soil and hay. However, as more than half of the organic samples were incorrectly classified, we infer that more research is needed to improve this approach before it can be efficiently

  5. Organic vs. Conventional Grassland Management: Do 15N and 13C Isotopic Signatures of Hay and Soil Samples Differ?

    PubMed Central

    Klaus, Valentin H.; Hölzel, Norbert; Prati, Daniel; Schmitt, Barbara; Schöning, Ingo; Schrumpf, Marion; Fischer, Markus; Kleinebecker, Till

    2013-01-01

    Distinguishing organic and conventional products is a major issue of food security and authenticity. Previous studies successfully used stable isotopes to separate organic and conventional products, but up to now, this approach was not tested for organic grassland hay and soil. Moreover, isotopic abundances could be a powerful tool to elucidate differences in ecosystem functioning and driving mechanisms of element cycling in organic and conventional management systems. Here, we studied the δ15N and δ13C isotopic composition of soil and hay samples of 21 organic and 34 conventional grasslands in two German regions. We also used Δδ15N (δ15N plant - δ15N soil) to characterize nitrogen dynamics. In order to detect temporal trends, isotopic abundances in organic grasslands were related to the time since certification. Furthermore, discriminant analysis was used to test whether the respective management type can be deduced from observed isotopic abundances. Isotopic analyses revealed no significant differences in δ13C in hay and δ15N in both soil and hay between management types, but showed that δ13C abundances were significantly lower in soil of organic compared to conventional grasslands. Δδ15N values implied that management types did not substantially differ in nitrogen cycling. Only δ13C in soil and hay showed significant negative relationships with the time since certification. Thus, our result suggest that organic grasslands suffered less from drought stress compared to conventional grasslands most likely due to a benefit of higher plant species richness, as previously shown by manipulative biodiversity experiments. Finally, it was possible to correctly classify about two third of the samples according to their management using isotopic abundances in soil and hay. However, as more than half of the organic samples were incorrectly classified, we infer that more research is needed to improve this approach before it can be efficiently used in practice

  6. Importance of bacterivory and preferential selection toward diatoms in larvae of Crepidula fornicata (L.) assessed by a dual stable isotope (13C, 15N) labeling approach

    NASA Astrophysics Data System (ADS)

    Leroy, Fanny; Riera, Pascal; Jeanthon, Christian; Edmond, Frédérique; Leroux, Cédric; Comtet, Thierry

    2012-05-01

    In Europe, the gastropod Crepidula fornicata is an invasive species characterized by a long reproductive period (from February to November). Thus, its larvae are exposed to variations in available food sources (in terms of quantity and quality). We aimed to investigate if bacteria could contribute to larval food both in presence or absence of phytoplankton, and to compare these results to seasonal variations of bacteria and phytoplankton abundances at a coastal site in the English Channel. First, ingestion of fluorescent beads of 0.5 to 2 μm diameter, showed that larvae were able to ingest particles of typical bacterial size. Then we used a dual stable isotope labeling approach which consisted in labeling a bacterial pelagic community with 15N and a diatom (Chaetoceros gracilis) culture with 13C, and supplying larvae with 15N-labeled bacteria, 13C-labeled diatoms, and both labeled sources. This technique has, to our knowledge, never been applied to invertebrate larvae. After 24 h of experiment, larvae were significantly enriched in all treatments: + 21.5‰ (∆δ13C) when supplied with diatoms, + 1364‰ (∆δ15N) when supplied with bacteria, and + 24‰ (∆δ13C) and + 135‰ (∆δ15N) when supplied with the two mixed sources. These results indicated that bacteria can contribute to the larval nutrition in C. fornicata, even in the presence of phytoplankton. Our results however suggested that larvae of C. fornicata preferentially used diatoms and showed that the supply of free bacteria did not alter the uptake of diatoms. Considering the seasonal variations of bacteria and phytoplankton abundances at the study site, these results suggested that bacteria may constitute a complementary resource for the larvae of C. fornicata when phytoplankton is abundant and may become a substitute resource when phytoplankton is less available. This approach offers promising perspectives to trace food sources and assess nitrogen and carbon fluxes between planktotrophic larvae

  7. Identifying sources of methane sampled in the Arctic using δ13C in CH4 and Lagrangian particle dispersion modelling.

    NASA Astrophysics Data System (ADS)

    Cain, Michelle; France, James; Pyle, John; Warwick, Nicola; Fisher, Rebecca; Lowry, Dave; Allen, Grant; O'Shea, Sebastian; Illingworth, Samuel; Jones, Ben; Gallagher, Martin; Welpott, Axel; Muller, Jennifer; Bauguitte, Stephane; George, Charles; Hayman, Garry; Manning, Alistair; Myhre, Catherine Lund; Lanoisellé, Mathias; Nisbet, Euan

    2016-04-01

    An airmass of enhanced methane was sampled during a research flight at ~600 m to ~2000 m altitude between the North coast of Norway and Svalbard on 21 July 2012. The largest source of methane in the summertime Arctic is wetland emissions. Did this enhancement in methane come from wetland emissions? The airmass was identified through continuous methane measurements using a Los Gatos fast greenhouse gas analyser on board the UK's BAe-146 Atmospheric Research Aircraft (ARA) as part of the MAMM (Methane in the Arctic: Measurements and Modelling) campaign. A Lagrangian particle dispersion model (the UK Met Office's NAME model) was run backwards to identify potential methane source regions. This was combined with a methane emission inventory to create "pseudo observations" to compare with the aircraft observations. This modelling was used to constrain the δ13C CH4 wetland source signature (where δ13C CH4 is the ratio of 13C to 12C in methane), resulting in a most likely signature of -73‰ (±4‰7‰). The NAME back trajectories suggest a methane source region of north-western Russian wetlands, and -73‰ is consistent with in situ measurements of wetland methane at similar latitudes in Scandinavia. This analysis has allowed us to study emissions from remote regions for which we do not have in situ observations, giving us an extra tool in the determination of the isotopic source variation of global methane emissions.

  8. Combining asymmetric 13C-labeling and isotopic filter/edit NOESY: a novel strategy for rapid and logical RNA resonance assignment.

    PubMed

    LeBlanc, Regan M; Longhini, Andrew P; Le Grice, Stuart F J; Johnson, Bruce A; Dayie, Theodore K

    2017-09-19

    Although ∼98% of the human genomic output is transcribed as non-protein coding RNA, <2% of the protein data bank structures comprise RNA. This huge structural disparity stems from combined difficulties of crystallizing RNA for X-ray crystallography along with extensive chemical shift overlap and broadened linewidths associated with NMR of RNA. While half of the deposited RNA structures in the PDB were solved by NMR methods, the usefulness of NMR is still limited by the high cost of sample preparation and challenges of resonance assignment. Here we propose a novel strategy for resonance assignment that combines new strategic 13C labeling technologies with filter/edit type NOESY experiments to greatly reduce spectral complexity and crowding. This new strategy allowed us to assign important non-exchangeable resonances of proton and carbon (1', 2', 2, 5, 6 and 8) nuclei using only one sample and <24 h of NMR instrument time for a 27 nt model RNA. The method was further extended to assigning a 6 nt bulge from a 61 nt viral RNA element justifying its use for a wide range RNA chemical shift resonance assignment problems. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. 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.

  10. 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.

  11. Stability of (13) C-Urea Breath Test Samples Over Time in the Diagnosis of Helicobacter pylori.

    PubMed

    Perets, Tsachi Tsadok; Shporn, Einav; Boltin, Doron; Dickman, Ram; Niv, Yaron

    2016-05-01

    The accuracy and repeatability of breath test in the diagnosis of Helicobacter pylori infection have not been adequately investigated. Although it has been shown that storage for long periods does not affect the analysis results, no data are available on the effect of repetitive testing. In this study, our aim was to evaluate the repeatability of the analyses of breath samples at room temperature. A total of 202 positive breath samples were collected in duplicates, before and after administration of 75 mg (13) C- urea dissolved in 100 ml of orange juice. Breath test results were expressed as delta (13) CO2 . The cut-off value was 3.5 parts per thousand. Each sample was analyzed in a mass spectrometer 7, 14, 21, and 28 days after collection. The accuracy calculation was based on the comparison of the delta (13) CO2 obtained in the three consecutive weeks following the first test run to the delta (13) CO2 obtained in the first test run. Two hundred (99%), 197 (97.52%), and 196 (97%) of the 202 samples tested positive in the second, third, and fourth test runs, respectively. The accuracy of the delta (13) CO2 was 98.6%, 99.2%, and 96.7% in the three consecutive runs, respectively. Short-term storage of 1 month does not affect sample stability or the results of (13) C-urea breath tests in up to three consecutive repeats. © 2015 Wiley Periodicals, Inc.

  12. Carbon isotopic composition (δ(13)C and (14)C activity) of plant samples in the vicinity of the Slovene nuclear power plant.

    PubMed

    Sturm, Martina; Vreča, Polona; Krajcar Bronić, Ines

    2012-08-01

    δ(13)C values of various plants (apples, wheat, and maize) collected in the vicinity of the Krško Nuclear Power Plant (Slovenia) during 2008 and 2009 were determined. By measuring dried samples and their carbonized counterparts we showed that no significant isotopic fractionation occurs during the carbonization phase of the sample preparation process in the laboratory. The measured δ(13)C values of the plants were used for δ(13)C correction of their measured (14)C activities.

  13. Using Isotope Ratio Infrared Spectrometer to determine δ13C and δ18O of carbonate samples

    NASA Astrophysics Data System (ADS)

    Smajgl, Danijela; Stöbener, Nils; Mandic, Magda

    2017-04-01

    The isotopic composition of calcifying organisms is a key tool for reconstruction past seawater temperature and water chemistry. Therefore stable carbon and oxygen isotopes (δ13C and δ18O) in carbonates have been widely used for reconstruction of paleoenvironments. Precise and accurate determination of isotopic composition of carbon (13C) and oxygen (18O) from carbonate sample with proper referencing and data evaluation algorithm presents a challenge for scientists. Mass spectrometry was the only widely used technique for this kind of analysis, but recent advances make laser based spectroscopy a viable alternative. The Thermo Scientific Delta Ray Isotope Ratio Infrared Spectrometer (IRIS) analyzer with the Universal Reference Interface (URI) Connect is one of those alternatives and with TELEDYNE Cetac ASX-7100 autosampler extends the traditional offerings with a system of high precision and throughput of samples. To establish precision and accuracy of measurements and also to develop optimal sample preparation method for measurements with Delta Ray IRIS and URI Connect, IAEA reference materials were used. Preparation is similar to a Gas Bench II method. Carbonate material is added into the vials, flushed with CO2 free synthetic air and acidified with few droplets of 104% H3PO4. Sample amount used for analysis can be as low as 200 μg. Samples are measured after acidification and equilibration time of one hour at 70°C. The CO2 gas generated by reaction is flushed into the variable volume inside the URI Connect through the Nafion based built-in water trap. For this step, carrier gas (CO2 free air) is used to flush the gas from the vial into the variable volume with a maximum volume of 100 ml. A small amount of the sample is then used for automatic concentration determination present in the variable volume. The Thermo Scientific Qtegra Software automatically adjusts any additional dilution of the sample to achieve the desired concentration (usually 400 ppm) in the

  14. 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. Published by Elsevier B.V.

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

    SciTech Connect

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

    2012-02-06

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

  16. 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

  17. 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.

  18. Measurements of Atmospheric Methane and 13C/12C of Atmospheric Methane from Flask Air Samples (1999)

    DOE Data Explorer

    Quay, Paul; Stutsman, Johnny

    1999-01-01

    This database offers precise measurements of atmospheric methane and 13C/12C in atmospheric methane from flask air samples collected at eight sites worldwide and aboard NOAA cruises in the Pacific Ocean. The eight sites include Olympic Peninsula, Washington; Cape Grim, Tasmania; Fraserdale, Ontario; Marshall Islands; Baring Head, New Zealand; Mauna Loa, Hawaii; Point Barrow, Alaska; and American Samoa. The measurements span the period 1988 to mid-1996. These data are useful for global methane budget analyses and for determining the atmospheric isotopic composition of methane. All isotopic measurements have been corrected for standard drift.

  19. 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

  20. Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in (15)N-(13)C-ILV methyl labeled proteins.

    PubMed

    Frueh, Dominique P; Leed, Alison; Arthanari, Haribabu; Koglin, Alexander; Walsh, Christopher T; Wagner, Gerhard

    2009-11-01

    We present a time-shared 3D HSQC-NOESY experiment that enables one to simultaneously record (13)C- and (15)N-dispersed spectra in Ile, Leu and Val (ILV) methyl-labeled samples. This experiment is designed to delineate the two spectra which would otherwise overlap with one another when acquired together. These spectra display nOe correlations in the detected proton dimension, i.e. with maximum resolution. This is in contrast to NOESY-HSQC types of experiments that provide cross-peaks in the indirect dimension with low resolution due to limits in experimental time. The technique is particularly advantageous at high field where even longer experimental times would be required for comparable resolution in NOESY-HSQC experiments. The method is demonstrated at 900 MHz and at 750 MHz on 37 and 31 kDa proteins, respectively. The resolution and time saving provided in this experiment was crucial for solving the structures of these two proteins.

  1. Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in 15N-13C-ILV methyl labeled proteins

    PubMed Central

    Frueh, Dominique P.; Leed, Alison; Arthanari, Haribabu; Koglin, Alexander; Walsh, Christopher T.; Wagner, Gerhard

    2009-01-01

    We present a time-shared 3D HSQC-NOESY experiment that enables one to simultaneously record 13C- and 15N-dispersed spectra in Ile, Leu and Val (ILV) methyl-labeled samples. This experiment is designed to delineate the two spectra which would otherwise overlap with one another when acquired together. These spectra display nOe correlations in the detected proton dimension, i.e. with maximum resolution. This is in contrast to NOESY-HSQC types of experiments that provide cross-peaks in the indirect dimension with low resolution due to limits in experimental time. The technique is particularly advantageous at high field where even longer experimental times would be required for comparable resolution in NOESY-HSQC experiments. The method is demonstrated at 900 MHz and at 750 MHz on 37 kDa and 31 kDa proteins, respectively. The resolution and time saving provided in this experiment was crucial for solving the structures of these two proteins. PMID:19728110

  2. 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.

  3. Correlation between the synthetic origin of methamphetamine samples and their 15N and 13C stable isotope ratios.

    PubMed

    Billault, Isabelle; Courant, Frédérique; Pasquereau, Léo; Derrien, Solène; Robins, Richard J; Naulet, Norbert

    2007-06-12

    The active ingredient of ecstasy, N-methyl-3,4-methyldioxyphenylisopropylamine (MDMA) can be manufactured by a number of easy routes from simple precursors. We have synthesised 45 samples of MDMA following the five most common routes using N-precursors from 12 different origins and three different precursors for the aromatic moiety. The 13C and 15N contents of both the precursors and the MDMA samples derived therefrom were measured by isotope ratio mass spectrometry coupled to an elemental analyser (EA-IRMS). We show that within-pathway correlation between the 15N content of the precursor and that of the derived MDMA can be strong but that no general pattern of correlation can be defined. Rather, it is evident that the delta15N values of MDMA are strongly influenced by a combination of the delta15N values of the source of nitrogen used, the route by which the MDMA is synthesised, and the experimental conditions employed. Multivariate analysis (PCA) based on the delta15N values of the synthetic MDMA and of the delta15N and delta13C values of the N-precursors leads to good discrimination between the majority of the reaction conditions tested.

  4. 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.

  5. 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.

  6. 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

  7. Biosynthesis of pyrrolnitrin. Incorporation of 13C, 15N double-labelled D- and L-tryptophan.

    PubMed

    Zhou, P; Mocek, U; Siesel, B; Floss, H G

    1992-01-01

    Experiments on the incorporation of D- and L-[alanine-3-13C,2-15N]tryptophan into the antibiotic pyrrolnitrin in Pseudomonas aureofaciens confirmed earlier conclusions about the conversion of L-tryptophan into pyrrolnitrin. They also demonstrated that a fraction of the D isomer is incorporated without breakage of the 15N-carbon bond, consistent with the operation of a second pathway from D-tryptophan to pyrrolnitrin. Cell-free experiments confirmed the conversion of 3-(o-aminophenyl)pyrrole into aminopyrrolnitrin but failed to detect enzymatic oxidation of the latter to pyrrolnitrin.

  8. Metabolic Pathway for Propionate Utilization by Phosphorus-Accumulating Organisms in Activated Sludge: 13C Labeling and In Vivo Nuclear Magnetic Resonance

    PubMed Central

    Lemos, Paulo C.; Serafim, Luísa S.; Santos, Margarida M.; Reis, Maria A. M.; Santos, Helena

    2003-01-01

    In vivo 13C and 31P nuclear magnetic resonance techniques were used to study propionate metabolism by activated sludge in enhanced biological phosphorus removal systems. The fate of label supplied in [3-13C]propionate was monitored in living cells subjected to anaerobic/aerobic cycles. During the anaerobic phase, propionate was converted to polyhydroxyalkanoates (PHA) with the following monomer composition: hydroxyvalerate, 74.2%; hydroxymethylvalerate, 16.9%; hydroxymethylbutyrate, 8.6%; and hydroxybutyrate, 0.3%. The isotopic enrichment in the different carbon atoms of hydroxyvalerate (HV) produced during the first anaerobic stage was determined: HV5, 59%; HV4, 5.0%; HV3, 1.1%; HV2, 3.5%; and HV1, 2.8%. A large proportion of the supplied label ended up on carbon C-5 of HV, directly derived from the pool of propionyl-coenzyme A (CoA), which is primarily labeled on C-3; useful information on the nature of operating metabolic pathways was provided by the extent of labeling on C-1, C-2, and C-4. The labeling pattern on C-1 and C-2 was explained by the conversion of propionyl-CoA to acetyl-CoA via succinyl-CoA and the left branch of the tricarboxylic acid cycle, which involves scrambling of label between the inner carbons of succinate. This constitutes solid evidence for the operation of succinate dehydrogenase under anaerobic conditions. The labeling in HV4 is explained by backflux from succinate to propionyl-CoA. The involvement of glycogen in the metabolism of propionate was also demonstrated; moreover, it was shown that the acetyl moiety to the synthesis of PHA was derived preferentially from glycogen. According to the proposed metabolic scheme, the decarboxylation of pyruvate is coupled to the production of hydrogen, and the missing reducing equivalents should be derived from a source other than glycogen metabolism. PMID:12514001

  9. In vivo, large-scale preparation of uniformly (15)N- and site-specifically (13)C-labeled homogeneous, recombinant RNA for NMR studies.

    PubMed

    Le, My T; Brown, Rachel E; Simon, Anne E; Dayie, T Kwaku

    2015-01-01

    Knowledge of how ribonucleic acid (RNA) structures fold to form intricate, three-dimensional structures has provided fundamental insights into understanding the biological functions of RNA. Nuclear magnetic resonance (NMR) spectroscopy is a particularly useful high-resolution technique to investigate the dynamic structure of RNA. Effective study of RNA by NMR requires enrichment with isotopes of (13)C or (15)N or both. Here, we present a method to produce milligram quantities of uniformly (15)N- and site-specifically (13)C-labeled RNAs using wild-type K12 and mutant tktA Escherichia coli in combination with a tRNA-scaffold approach. The method includes a double selection protocol to obtain an E. coli clone with consistently high expression of the recombinant tRNA-scaffold. We also present protocols for the purification of the tRNA-scaffold from a total cellular RNA extract and the excision of the RNA of interest from the tRNA-scaffold using DNAzymes. Finally, we showcase NMR applications to demonstrate the benefit of using in vivo site-specifically (13)C-labeled RNA. © 2015 Elsevier Inc. All rights reserved.

  10. Amino-acid selective experiments on uniformly 13C and 15N labeled proteins by MAS NMR: Filtering of lysines and arginines.

    PubMed

    Jehle, Stefan; Rehbein, Kristina; Diehl, Anne; van Rossum, Barth-Jan

    2006-12-01

    Amino-acid selective magic-angle spinning (MAS) NMR experiments can aid the assignment of ambiguous cross-peaks in crowded spectra of solid proteins. In particular for larger proteins, data analysis can be hindered by severe resonance overlap. In such cases, filtering techniques may provide a good alternative to site-specific spin-labeling to obtain unambiguous assignments that can serve as starting points in the assignment procedure. In this paper we present a simple pulse sequence that allows selective excitation of arginine and lysine residues. To achieve this, we make use of a combination of specific cross-polarization for selective excitation [M. Baldus, A.T. Petkova, J. Herzfeld, R.G. Griffin, Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems, Mol. Phys. 95 (1998) 1197-1207.] and spin diffusion for transfer along the amino-acid side-chain. The selectivity of the filter is demonstrated with the excitation of lysine and arginine side-chain resonances in a uniformly 13C and 15N labeled protein preparation of the alpha-spectrin SH3 domain. It is shown that the filter can be applied as a building block in a 13C-13C lysine-only correlation experiment.

  11. 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

  12. Deuteration of Hyperpolarized (13) C-Labeled Zymonic Acid Enables Sensitivity-Enhanced Dynamic MRI of pH.

    PubMed

    Hundshammer, Christian; Düwel, Stephan; Köcher, Simone S; Gersch, Malte; Feuerecker, Benedikt; Scheurer, Christoph; Haase, Axel; Glaser, Steffen J; Schwaiger, Markus; Schilling, Franz

    2017-09-20

    Aberrant pH is characteristic of many pathologies such as ischemia, inflammation or cancer. Therefore, a non-invasive and spatially resolved pH determination is valuable for disease diagnosis, characterization of response to treatment and the design of pH-sensitive drug-delivery systems. We recently introduced hyperpolarized [1,5-(13) C2 ]zymonic acid (ZA) as a novel MRI probe of extracellular pH utilizing dissolution dynamic polarization (DNP) for a more than 10000-fold signal enhancement of the MRI signal. Here we present a strategy to enhance the sensitivity of this approach by deuteration of ZA yielding [1,5-(13) C2 , 3,6,6,6-D4 ]zymonic acid (ZAd ), which prolongs the liquid state spin lattice relaxation time (T1 ) by up to 39 % in vitro. Measurements with ZA and ZAd on subcutaneous MAT B III adenocarcinoma in rats show that deuteration increases the signal-to-noise ratio (SNR) by up to 46 % in vivo. Furthermore, we demonstrate a proof of concept for real-time imaging of dynamic pH changes in vitro using ZAd , potentially allowing for the characterization of rapid acidification/basification processes in vivo. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Incorporation of 13C labeled Pinus ponderosa needle and fine root litter into soil organic matter measured by Py-GC/MS-C-IRMS

    NASA Astrophysics Data System (ADS)

    Mambelli, S.; Gleixner, G.; Dawson, T. E.; Bird, J. A.; Torn, M. S.

    2006-12-01

    Developing effective strategies for enhancing C storage in soils requires understanding the influence of plant C quality. In turn, plant C quality impacts the decay continuum between plant residue and humified, stable SOM. This remains one of the least understood aspects of soil biogeochemistry. We investigated the initial phase of incorporation of 13C labeled Pinus ponderosa needle and fine root litter into SOM. The two litter types were placed in separate microcosms in the A horizon in a temperate conifer soil. Curie-point pyrolysis-gas chromatography coupled with on-line mass spectrometry and isotope ratio mass spectrometry (Py-GC/MS-C- IRMS) were used to determine the identity and the 13C enrichment of pyrolysis products (fragments of carbohydrates, lignin, proteins and lipids). We compared the two initial litter types, needles and fine roots, to samples of the bulk soil (A horizon, < 2mm) and soil humin fraction (from chemical solubility) obtained from each microcosm 1.5y after litter addition. Pyrolysis of plant material and SOM produced 56 suitable products for isotopic analysis; of them, 15 occurred in both the litter and bulk soil, 7 in both the litter and the humin fraction and 9 in both bulk soil and the humin fraction. The pyrolysis products found in common in the plant and soil were related either to polysaccharides or were non-specific and could have originated from various precursors. The data suggest that the majority of plant inputs, both from needles or fine roots, were degraded very rapidly. In the humin fraction, the most recalcitrant pool of C in soil, with a measured turnover time of 260y (this soil), only products from the fragmentation of polysaccharides and alkyl-benzene compounds were found. Comparisons of the enrichment normalized by input level suggest little difference between the incorporation of C from needles versus fine roots into SOM. The most enriched fragments in the humin fraction were products from polysaccharides degradation

  14. 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.

  15. 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. © 2014 The Authors. Contrast Media & Molecular Imaging published by John Wiley and Sons, Ltd.

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

    PubMed Central

    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 [1,4-13C2]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 13C-labeled substrate through the increase in solvent water 1H signal. © 2014 The Authors. Contrast Media & Molecular Imaging published by John Wiley and Sons, Ltd. PMID:24523064

  17. Long-distance effects of site-directed mutations on backbone conformation in bacteriorhodopsin from solid state NMR of [1-13C]Val-labeled proteins.

    PubMed Central

    Tanio, M; Inoue, S; Yokota, K; Seki, T; Tuzi, S; Needleman, R; Lanyi, J K; Naito, A; Saitô, H

    1999-01-01

    We have recorded 13C cross-polarization-magic angle spinning and dipolar decoupled-magic angle spinning NMR spectra of [1-13C]Val-labeled wild-type bacteriorhodopsin (bR), and the V49A, V199A, T46V, T46V/V49A, D96N, and D85N mutants, in order to study conformational changes of the backbone caused by site-directed mutations along the extracellular surface and the cytoplasmic half channel. On the basis of spectral changes in the V49A and V199A mutants, and upon specific cleavage by chymotrypsin, we assigned the three well-resolved 13C signals observed at 172.93, 172.00, and 171. 11 ppm to [1-13C]Val 69, Val 49, and Val 199, respectively. The local conformations of the backbone at these residues are revealed by the conformation-dependent 13C chemical shifts. We find that at the ambient temperature of these measurements Val 69 is not in a beta-sheet, in spite of previous observations by electron microscopy and x-ray diffraction at cryogenic temperatures, but in a flexible turn structure that undergoes conformational fluctuation. Results with the T46V mutant suggest that there is a long-distance effect on backbone conformation between Thr 46 and Val 49. From the spectra of the D85N and E204Q mutants there also appears to be coupling between Val 49 and Asp 85 and between Asp 85 and Glu 204, respectively. In addition, the T2 measurement indicates conformational interaction between Asp 96 and extracellular surface. The protonation of Asp 85 in the photocycle therefore might induce changes in conformation or dynamics, or both, throughout the protein, from the extracellular surface to the side chain of Asp 96. PMID:10388769

  18. Dynamics of amino acid redistribution in the carnivorous Venus flytrap (Dionaea muscipula) after digestion of (13) C/(15) N-labelled prey.

    PubMed

    Kruse, J; Gao, P; Eibelmeier, M; Alfarraj, S; Rennenberg, H

    2017-07-20

    Amino acids represent an important component in the diet of the Venus flytrap (Dionaea muscipula), and supply plants with much needed nitrogen resources upon capture of insect prey. Little is known about the significance of prey-derived carbon backbones of amino acids for the success of Dionaea's carnivorous life-style. The present study aimed at characterizing the metabolic fate of (15) N and (13) C in amino acids acquired from double-labeled insect powder. We tracked changes in plant amino acid pools and their δ(13) C- and δ(15) N-signatures over a period of five weeks after feeding, as affected by contrasting feeding intensity and tissue type (i.e., fed and non-fed traps and attached petioles of Dionaea). Isotope signatures (i.e., δ(13) C and δ(15) N) of plant amino acid pools were strongly correlated, explaining 60% of observed variation. Residual variation was related to contrasting effects of tissue type, feeding intensity and elapsed time since feeding. Synthesis of nitrogen-rich transport compounds (i.e., amides) during peak time of prey digestion increased (15) N- relative to (13) C- abundances in amino acid pools. After completion of prey digestion, (13) C in amino acid pools was progressively exchanged for newly fixed (12) C. The latter process was most evident for non-fed traps and attached petioles of plants that had received ample insect powder. We argue that prey-derived amino acids contribute to respiratory energy gain and loss of (13) CO2 during conversion into transport compounds (i.e., 2 days after feeding), and that amino-nitrogen helps boost photosynthetic carbon gain later on (i.e., 5 weeks after feeding). © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. Sample Pesticide Label for Label Review Training

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  4. 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

  5. NMR studies of the stability, protonation States, and tautomerism of (13)C- AND (15)N-labeled aldimines of the coenzyme pyridoxal 5'-phosphate in water.

    PubMed

    Chan-Huot, Monique; Sharif, Shasad; Tolstoy, Peter M; Toney, Michael D; Limbach, Hans-Heinrich

    2010-12-28

    We have measured the pH-dependent (1)H, (13)C, and (15)N NMR spectra of pyridoxal 5'-phosphate ((13)C(2)-PLP) mixed with equal amounts of either doubly (15)N-labeled diaminopropane, (15)N(α)-labeled l-lysine, or (15)N(ε)-labeled l-lysine as model systems for various intermediates of the transimination reaction in PLP-dependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of single- and double-headed aldimines (Schiff bases) with the added diamines is observed, and their (13)C and (15)N NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, the NMR parameters of the geminal diamine formed with diaminopropane at high pH are measured. However, no geminal diamine is formed with l-lysine. In contrast to the aldimine formed with the ε-amino group of lysine, the aldimine formed with the α-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR data, both the mole fractions of the different PLP species and up to 6 different protonation states including their pK(a) values were obtained. Furthermore, the data show that all Schiff bases are subject to a proton tautomerism along the intramolecular OHN hydrogen bond, where the zwitterionic form is favored before deprotonation occurs at high pH. This observation, as well as the observation that around pH 7 the different PLP species are present in comparable amounts, sheds new light on the mechanism of the transimination reaction.

  6. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  8. Efficient, scalable and economical preparation of tris(deuterium)- and 13C-labelled N-methyl-N-nitroso-p-toluenesulfonamide (Diazald®) and their conversion to labelled diazomethane.

    PubMed

    Shields, Samuel W J; Manthorpe, Jeffrey M

    2014-10-01

    A method for the preparation of multi-gramme quantities of N-methyl-d3-N-nitroso-p-toluenesulfonamide (Diazald-d3) and N-methyl-(13)C-N-nitroso-p-toluenesulfonamide (Diazald-(13)C) and their conversion to diazomethane-d2 and diazomethane-(13) C, respectively, is presented. This approach uses robust and reliable chemistry, and critically, employs readily commercially available and inexpensive methanol as the label source. Several reactions of labelled diazomethane are also reported, including alkene cyclopropanation, phenol methylation and α-diazoketone formation, as well as deuterium scrambling in the preparation of diazomethane-d2 and subsequent methyl esterification of benzoic acid. Copyright © 2014 John Wiley & Sons, Ltd.

  9. New Multidimensional Editing Experiments for Measurement of Amide Deuterium Isotope Effects on C βChemical Shifts in 13C, 15N-Labeled Proteins

    NASA Astrophysics Data System (ADS)

    Meissner, Axel; Sørensen, Ole Winneche

    1998-12-01

    Novel multidimensional NMR pulse sequences for measurement of the three- and four-bond amide deuterium isotope effect on the chemical shifts of13Cβin proteins are presented. The sequences result in editing into two subspectra of a heteronuclear triple resonance spectrum {ω(N), ω(Cβ), ω(Hα)} according to there being a deuterium or a proton attached to15N for the pertinent correlations. The new experiments are demonstrated by an application to the first module of the13C,15N-labeled protein RAP 18-112 (N-terminal module of α2-macroglobulin receptor associated protein).

  10. 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

  11. 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

  12. Norlittorine and norhyoscyamine identified as products of littorine and hyoscyamine metabolism by (13)C-labeling in Datura innoxia hairy roots.

    PubMed

    Al Balkhi, Mohamad Houssam; Schiltz, Séverine; Lesur, David; Lanoue, Arnaud; Wadouachi, Anne; Boitel-Conti, Michèle

    2012-02-01

    The presence of two compounds, norlittorine and norhyoscyamine, has been reported in leaves and roots of Datura innoxia; however their metabolic origin in the tropane alkaloid pathway has remained unknown. Precise knowledge of this pathway is a necessary pre-requisite to optimize the production of hyoscyamine and scopolamine in D. innoxia hairy root cultures. The exact structure of norlittorine and norhyoscyamine was confirmed by LC-MS/MS and NMR analyses. Isotopic labeling experiments, using [1-(13)C]-phenylalanine, [1'-(13)C]-littorine and [1'-(13)C]-hyoscyamine, combined with elicitor treatments, using methyl jasmonate, coronalon and 1-aminocyclopropane-1-carboxylic acid, were used to investigate the metabolic origin of the N-demethylated tropane alkaloids. The results suggest that norlittorine and norhyoscyamine are induced under stress conditions by conversion of littorine and hyoscyamine. We propose the N-demethylation of tropane alkaloids as a mechanism to detoxify cells in overproducing conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Development of Decarboxylative Cyanation Reactions for C-13/C-14 Carboxylic Acid Labeling Using an Electrophilic Cyanating Reagent.

    PubMed

    Song, Fengbin; Salter, Rhys; Chen, Lu

    2017-04-07

    Degradation-reconstruction approaches for isotope labeling synthesis have been known for their remarkable efficiency, but applications are scarce due to some fundamental limitations of the chemistries developed to date. The decarboxylative cyanation reaction, as a degradation-reconstruction approach, is especially useful in rapid carboxylic acid carbon isotope labeling, however development toward its application as a widespread technique has stalled at the early stages due to numerous limitations which include somewhat narrow applicability. Employing the electrophilic cyanating reagent N-cyano-N-phenyl-p-toluenesulfonamide (NCTS) as the cyano source, efficient decarboxylative cyanation chemistry has been developed for aryl and alkyl carboxylic acids respectively with two rationally designed reaction pathways. The reactions provided good yields of nitrile products from carboxylic acids, with complete retention of isotopic purity from the [(13)CN]-NCTS used. The reaction conditions are relatively mild requiring no oxidant and no excess toxic heavy metal and the reagent [(13/14)CN]-NCTS is a stable, easy-to-handle crystalline solid that can be prepared quickly and effectively from the readily available [(13/14)C]-KCN. The following work describes this novel and efficient method for alkyl and aryl carboxylic acid isotopic labeling using a single reagent.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed

    Tosun, Murat

    2013-06-01

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

  2. Measurement of compound-specific carbon isotope ratios (δ(13) C values) via direct injection of whole crude oil samples.

    PubMed

    Barrie, Craig D; Taylor, Kyle W R; Zumberge, John

    2016-04-15

    Stable isotope analysis is a powerful tool in understanding the generation, history and correlation of hydrocarbons. Compound-specific δ(13) C measurements of oils allow detailed comparison of individual compound groupings; however, most studies of these sample materials separate and isolate individual fractions based on the chemistries of particular compound groups, potentially losing considerable valuable isotopic data. Even if all fractions are analyzed, this represents a large increase in the data-processing burden, effectively multiplying data evaluation time and effort by the number of fractions produced. Gas chromatography/isotope ratio mass spectrometry (GC/IRMS) of untreated, whole crude oils allows the immediate collection of a larger suite of valuable isotopic data for these studies. Untreated ('neat', undiluted), whole crude oils were directly injected and measured on a GC/IRMS system, using split (40:1) injections and a 50 m HP-PONA column. The GC method, 97 min in duration, was designed to maximize baseline separation of target analyte peaks, while an additional oxygen flow was admitted into the combustion reactor to maximize the lifetime of the combustion chemicals. The method and setup utilized allow the measurement of a much greater range of the n-alkanes (n-C4 to n-C25+ ) than traditional methods, while also retaining important cycloalkane, aromatic and isoprenoid peaks within the same analysis. Carbon isotope (δ(13) C) evaluation of these additional compound classes reveals trends in maturity and origins which are not identifiable when exclusively assessing the traditional n-alkane package (>n-C12 ). The described setup and method open up new possibilities for assessing the origins and histories of crude oil samples. The data generated for the whole oil n-alkanes by this method is equivalent to that reported for isolated n-alkane studies, while also providing valuable additional data on many other important compounds. The end result of this

  3. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  4. Nuclear magnetic resonance study of interaction of ligands with Streptococcus faecium dihydrofolate reductase labeled with (. gamma. -/sup 13/C)tryptophan

    SciTech Connect

    London, R.E.; Groff, J.P.; Cocco, L.; Blakley, R.L.

    1982-01-01

    Dihydrofolate reductase from Streptococcus faecium has been labeled with (..gamma..-/sup 13/C)tryptophan. We have determined changes occurring in the chemical shifts and line widths of the four resonances of the /sup 13/C NMR spectrum of the labeled enzyme, due to its interaction with various ligands. These include the coenzyme, NPDPH and related nucleotides, folate and its polyglutamate derivatives, and many inhibitors including methotrexate and trimethoprim. In addition, paramagnetic relaxation effects produced by a bound spin-labeled analogue of 2'-phosphoadenosine-5'-diphosphoribose on the tryptophan C/sup ..gamma../ carbons have been measured. Distances calculated from the relaxation data have been compared with corresponding distances in the crystallographic model of the NADPH-methotrexate ternary complex of Lactobacillus casei reductase. The paramagnetic relaxation data indicate that the two downfield resonances (1 and 2) correspond to tryptophans (W/sub A/ and W/sub B/) that are more remote from the catalytic site, and from the crystallographic model these are seen to be Trp-115 and Trp-160. The upfield resonances (3 and 4) that show broadening due to chemical exchange correspond to closer residues (W/sub C/ and W/sub D/), and these are identified with Trp-6 and Trp-22. However, the relaxation data do not permit specific assignments within the nearer and farther pairs. Although resonance 3, which is split due to chemical exchange, was formerly assigned to Trp-6, data obtained for the enzyme in the presence of various ligands are better interpreted if resonance 3 is assigned to Trp-22, which is located on a loop that joins elements of secondary structure and forms one side of the ligand-binding cavity.

  5. Biogenic Volatile Organic Compound and Respiratory CO2 Emissions after 13C-Labeling: Online Tracing of C Translocation Dynamics in Poplar Plants

    PubMed Central

    Ghirardo, Andrea; Gutknecht, Jessica; Zimmer, Ina; Brüggemann, Nicolas; Schnitzler, Jörg-Peter

    2011-01-01

    Background Globally plants are the primary sink of atmospheric CO2, but are also the major contributor of a large spectrum of atmospheric reactive hydrocarbons such as terpenes (e.g. isoprene) and other biogenic volatile organic compounds (BVOC). The prediction of plant carbon (C) uptake and atmospheric oxidation capacity are crucial to define the trajectory and consequences of global environmental changes. To achieve this, the biosynthesis of BVOC and the dynamics of C allocation and translocation in both plants and ecosystems are important. Methodology We combined tunable diode laser absorption spectrometry (TDLAS) and proton transfer reaction mass spectrometry (PTR-MS) for studying isoprene biosynthesis and following C fluxes within grey poplar (Populus x canescens) saplings. This was achieved by feeding either 13CO2 to leaves or 13C-glucose to shoots via xylem uptake. The translocation of 13CO2 from the source to other plant parts could be traced by 13C-labeled isoprene and respiratory 13CO2 emission. Principal Finding In intact plants, assimilated 13CO2 was rapidly translocated via the phloem to the roots within 1 hour, with an average phloem transport velocity of 20.3±2.5 cm h−1. 13C label was stored in the roots and partially reallocated to the plants' apical part one day after labeling, particularly in the absence of photosynthesis. The daily C loss as BVOC ranged between 1.6% in mature leaves and 7.0% in young leaves. Non-isoprene BVOC accounted under light conditions for half of the BVOC C loss in young leaves and one-third in mature leaves. The C loss as isoprene originated mainly (76–78%) from recently fixed CO2, to a minor extent from xylem-transported sugars (7–11%) and from photosynthetic intermediates with slower turnover rates (8–11%). Conclusion We quantified the plants' C loss as respiratory CO2 and BVOC emissions, allowing in tandem with metabolic analysis to deepen our understanding of ecosystem C flux. PMID:21387007

  6. 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

  7. 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

  8. Biogenic volatile organic compound and respiratory CO2 emissions after 13C-labeling: online tracing of C translocation dynamics in poplar plants.

    PubMed

    Ghirardo, Andrea; Gutknecht, Jessica; Zimmer, Ina; Brüggemann, Nicolas; Schnitzler, Jörg-Peter

    2011-02-28

    Globally plants are the primary sink of atmospheric CO(2), but are also the major contributor of a large spectrum of atmospheric reactive hydrocarbons such as terpenes (e.g. isoprene) and other biogenic volatile organic compounds (BVOC). The prediction of plant carbon (C) uptake and atmospheric oxidation capacity are crucial to define the trajectory and consequences of global environmental changes. To achieve this, the biosynthesis of BVOC and the dynamics of C allocation and translocation in both plants and ecosystems are important. We combined tunable diode laser absorption spectrometry (TDLAS) and proton transfer reaction mass spectrometry (PTR-MS) for studying isoprene biosynthesis and following C fluxes within grey poplar (Populus x canescens) saplings. This was achieved by feeding either (13)CO(2) to leaves or (13)C-glucose to shoots via xylem uptake. The translocation of (13)CO(2) from the source to other plant parts could be traced by (13)C-labeled isoprene and respiratory (13)CO(2) emission. In intact plants, assimilated (13)CO(2) was rapidly translocated via the phloem to the roots within 1 hour, with an average phloem transport velocity of 20.3±2.5 cm h(-1). (13)C label was stored in the roots and partially reallocated to the plants' apical part one day after labeling, particularly in the absence of photosynthesis. The daily C loss as BVOC ranged between 1.6% in mature leaves and 7.0% in young leaves. Non-isoprene BVOC accounted under light conditions for half of the BVOC C loss in young leaves and one-third in mature leaves. The C loss as isoprene originated mainly (76-78%) from recently fixed CO(2), to a minor extent from xylem-transported sugars (7-11%) and from photosynthetic intermediates with slower turnover rates (8-11%). We quantified the plants' C loss as respiratory CO(2) and BVOC emissions, allowing in tandem with metabolic analysis to deepen our understanding of ecosystem C flux.

  9. Influence of 13C-enriched Foliage Respired CO2 on 13C of Ecosystem-respired CO2 Estimated From Mid-canopy CO2 Sampled During Several Hours After Sunset

    NASA Astrophysics Data System (ADS)

    Mortazavi, B.; Chanton, J.; Smith, M.

    2005-12-01

    Nighttime CO2 concentration and 13C isotopic ratio were measured in the summer and fall of 2004 by two approaches at a Biosphere Atmosphere Stable Isotope Network (BASIN) network site in Florida, USA. The site is a naturally regenerated pine ecosystem dominated by longleaf and slash pine. The first approach consisted in making measurements at multiple heights within the canopy from ground level to top of the canopy just prior to dawn. The second approach consisted in making measurements at mid- canopy at different times from 1 hour after sunset till 2 am. For each method a Keeling plot was used to estimate the isotopic composition of respired CO2 (dCr). dCr values determined with the time-series were enriched relative to those determined from multiple heights just prior to dawn. The 13C of slash pine foliage respired CO2 (dCf) was also measured at temperatures ranging from 6.6 to 30 °C. dCf was enriched initially after dark by as much as 9° and then declined, reaching constant values after a few hours. Maximum enrichment in dCf was measured at the lowest temperature and the decline in dCf towards constant values took longer at lower temperatures. The influence of 13C enriched and temporally variable nighttime foliage respired CO2 accounts for the enriched time-series dCr. A model constrained by field and experimental data was used to determine the impact of the non-constancy of dCf on the linearity and intercept of dCr Keeling plots determined from CO2 samples collected during several hours at mid-canopy height. For the months examined, results indicate that (i) Keeling plots determined from mid-canopy heights were linear despite the addition of temporally variable dCf (range: 5 °) and (ii) the enrichment in the Keeling intercepts determined from samples collected during several hours at mid-canopy compared to those determined from multiple heights were consistent with the addition of 13C enriched foliage respired CO2. A common approach that specifies the time

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. [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.

  15. 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

  16. Optimization of automated gas sample collection and isotope ratio mass spectrometric analysis of delta(13)C of CO(2) in air.

    PubMed

    Zeeman, Matthias J; Werner, Roland A; Eugster, Werner; Siegwolf, Rolf T W; Wehrle, Günther; Mohn, Joachim; Buchmann, Nina

    2008-12-01

    The application of (13)C/(12)C in ecosystem-scale tracer models for CO(2) in air requires accurate measurements of the mixing ratios and stable isotope ratios of CO(2). To increase measurement reliability and data intercomparability, as well as to shorten analysis times, we have improved an existing field sampling setup with portable air sampling units and developed a laboratory setup for the analysis of the delta(13)C of CO(2) in air by isotope ratio mass spectrometry (IRMS). The changes consist of (a) optimization of sample and standard gas flow paths, (b) additional software configuration, and (c) automation of liquid nitrogen refilling for the cryogenic trap. We achieved a precision better than 0.1 per thousand and an accuracy of 0.11 +/- 0.04 per thousand for the measurement of delta(13)C of CO(2) in air and unattended operation of measurement sequences up to 12 h.

  17. Determination of the mutual orientation of the 15N and 13C NMR chemical shift tensors of 13- 15N double labeled model peptides for silk fibroin from the dipolar-coupled powder patterns

    NASA Astrophysics Data System (ADS)

    Asakura, Tetsuo; Yamazaki, Yasunobu; Seng, Koo Wey; Demura, Makoto

    1998-05-01

    The 15N and 13C chemical shift tensors, and the orientation of the principal axis system relative to the molecular symmetry axes were determined for 15N and 13C carbonyl carbon sites of 13C 15N double labeled model peptides for Bombyx mori silk fibroin, that is, Boc-[1- 13C]Ala[ 15N]Gly-OMe, Boc-[1- 13C]Ala[ 15N]GlyAlaGly-OPac, Boc-AlaGly[1- 13C]Ala[ 15N]GlyAlaGly-OPac, Boc-[1- 13C]Gly[ 15N]AlaGlyAla-OPac, Boc-GlyAla[1- 13C]Gly[ 15N]AlaGlyAla-OPac and Boc-[1- 13C]Gly[ 15N]ValGlyAla-OPac, where Boc is t-butoxycarbonyl, OMe is methyl ester, OPac is phenacyl ester, Ala is alanine, Gly is glycine and Val is valine. From the comparisons of the 15N chemical shift tensors and the orientations of the principal axis system relative to the molecular symmetry axes among three compounds having [1- 13C]Ala[ 15N]Gly units, it is concluded that the intermolecular interactions such as hydrogen bonding are different between Boc-[1- 13C]Ala[ 15N]Gly-OMe and two compounds, Boc-[1- 13C]Ala[ 15N]GlyAlaGly-OPac and Boc-AlaGly[1- 13C]Ala[ 15N]GlyAlaGly-OPac although the latter two compounds have similar structures. A similar conclusion has also been obtained from the 13C chemical shift tensors of these compounds.

  18. 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.

  19. Assignments of the Pfr-Pr FTIR difference spectrum of cyanobacterial phytochrome Cph1 using 15N and 13C isotopically labeled phycocyanobilin chromophore.

    PubMed

    van Thor, Jasper J; Fisher, Nicholas; Rich, Peter R

    2005-11-03

    The reversible red and far-red light-induced transitions of cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 were investigated by Fourier transform infrared (FTIR) difference spectroscopy. High-quality light-induced Pfr-Pr difference FTIR spectra were recorded for the 58 kDa N-terminal domain of Cph1 by repetitive photochemical cycling and signal averaging. The Pfr-Pr difference spectra in H(2)O and D(2)O were very similar to those previously reported for full-length 85 kDa Cph1.(1) Published assignments were extended by analysis of the effects of (13)C and (15)N isotope substitutions at selected sites in the phycocyanobilin chromophore and by (15)N global labeling of the protein. The Pfr-Pr difference spectra were dominated by an amide I peak/trough at 1653 cm(-1)(+)/1631 cm(-1)(-) and a smaller amide II band at 1554 cm(-1). Labeling effects allowed specific chromophore assignments for the C(1)=O (1736 cm(-1)(-)/1724 cm(-1)(+)) and C(19)=O (1704 cm(-1)(-)) carbonyl vibrations, C=C vibrations at 1589 cm(-1)(+), and bands at 1537(-), 1512(+), 1491(-), 1163(+), 1151(-), 1134(+), 1109(-), and 1072(-) cm(-1) that must involve chromophore C-N bonds. A variety of additional changes were insensitive to isotope labeling of the chromophore. Effects of (15)N labeling of the protein were used to tentatively assign some of these to specific amino acid changes. Those insensitive to (15)N labeling included a protonated aspartic or glutamic acid at 1734 cm(-1)(-)/1722 cm(-1)(+) and a cysteine at 2575 cm(-1)(+)/2557 cm(-1)(-). Bands sensitive to (15)N protein labeling at 1487 cm(-1)(+)/1502 cm(-1)(-) might arise from trytophan and bands at 1261 cm(-1)(+)/1244 cm(-1)(-) and 1107 cm(-1)(-)/1095 cm(-1)(+) might arise from a histidine environment or protonation change. These assignments are discussed in light of the 15Z-E photoisomerization model of phototransformation and the associated protein conformational changes.

  20. δ13C of volatile organic compounds (VOCS) in airborne samples by thermal desorption-gas chromatography-isotope ratio-mass spectrometry (TD-GC-IR-MS)

    NASA Astrophysics Data System (ADS)

    Turner, Nicole; Jones, Mark; Grice, Kliti; Dawson, Daniel; Ioppolo-Armanios, Marisa; Fisher, Steven J.

    This paper is a preliminary investigation into the use of a thermal desorption-gas chromatography-isotope ratio mass spectrometry (TD-GC-IR-MS) method to determine stable carbon isotopic compositions ( δ13C) of low molecular-weight volatile organic compounds (VOCs) in airborne samples (e.g. industrial and car exhaust emissions) as a means of differentiating their sources in the environment. A TD-GC-IR-MS method for obtaining δ13C of VOCs (benzene, toluene, chlorobenzene, ethylbenzene, m-xylene and propylbenzene) in air samples has been optimised, and is proven to be both reproducible and linear. The δ13C of the VOC standards was found to be comparable (within analytical error) to that obtained from direct GC-IR-MS analysis. This novel method of VOC analysis is valuable in environmental and forensic investigations.

  1. 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.

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

    PubMed

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

    2014-11-01

    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 [(13)C5]5-formyltetrahydrofolate infused during colonoscopy is absorbed. However, it is unclear if significant quantities of folate are absorbed in an intact microbiome. We determined whether and how much of a physiologic dose of [(13)C5]5-formyltetrahydrofolate delivered in a pH-sensitive enteric caplet to an intact colonic microbiome is absorbed. Healthy adults ingested a specially designed pH-sensitive acrylic copolymer-coated barium sulfate caplet that contained 855 nmol (400 μg) [(13)C5]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. 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 [(13)C5]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%. 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 their gastrointestinal microbiota. In

  3. 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

  4. Influence of an intervention targeting a reduction in sugary beverage intake on the δ13C sugar intake biomarker in a predominantly obese, health-disparate sample.

    PubMed

    Davy, Brenda M; Jahren, A Hope; Hedrick, Valisa E; You, Wen; Zoellner, Jamie M

    2017-01-01

    Controversy exists surrounding the health effects of added sugar (AS) and sugar-sweetened beverage (SSB) intakes, primarily due to a reliance on self-reported dietary intake. The purpose of the current investigation was to determine if a 6-month intervention targeting reduced SSB intake would impact δ13C AS intake biomarker values. A randomized controlled intervention trial. At baseline and at 6 months, participants underwent assessments of anthropometrics and dietary intake. Fasting fingerstick blood samples were obtained and analysed for δ13C value using natural abundance stable isotope MS. Statistical analysis included descriptive statistics, correlational analyses and multilevel mixed-effects linear regression analysis using an intention-to-treat approach. Rural Southwest Virginia, USA. Adults aged ≥18 years who consumed ≥200 kcal SSB/d (≥837 kJ/d) were randomly assigned to either the intervention (n 155) or a matched-contact group (n 146). Participants (mean age 42·1 (sd 13·4) years) were primarily female and overweight (21·5 %) or obese (57·0 %). A significant group by time difference in δ13C value was detected (P<0·001), with mean (sd) δ13C value decreasing in the intervention group (pre: -18·92 (0·65) ‰, post: -18·97 (0·65) ‰) and no change in the comparison group (pre: -18·94 (0·72) ‰, post: -18·92 (0·73) ‰). Significant group differences in weight and BMI change were also detected. Changes in biomarker δ13C values were consistent with changes in self-reported AS and SSB intakes. The δ13C sugar intake biomarker assessed using fingerstick blood samples shows promise as an objective indicator of AS and SSB intakes which could be feasibly included in community-based research trials.

  5. 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.

  6. Application of a nanoEA-IRMS system for δ13C measurement of biomineral-bound organics in samples of diatom opal with nanomolar quantities of C

    NASA Astrophysics Data System (ADS)

    Méndez-Vicente, Ana; María Mejía-Ramírez, Luz; Stoll, Heather

    2013-04-01

    We describe the isotopic measurement of δ13C in very small samples of diatom opal (nanomolar quantities of C) both from fossil sediments and cultures. We use a nano-EA system composed of a combustion elemental analyzer (EA3000 series, Eurovector), with standard 18 mm diameter quartz oxidation-reduction reactors and an ash removal device that aids in removal of uncombusted opal and ensures a long reactor lifetime. This is coupled to a custom designed trapping and cromatography system (Nano-CF, Nu Instruments Ltd.) which cryogenically removes CO2 generated by sample combustion and introduces the gas into a low-flow helium carrier stream to the mass spectrometer (Nu Perspective IRMS instrument, Nu Instruments Ltd.). This technique allows for an important reduction in the minimum sample requirements for analysis compared to a typical EA, however the need to reduce the contribution of the blank to the measured values becomes all the more critical. Blank from the capsules can be minimized through specific protocols including cleaning with solvents and reducing the size of the capsule by cutting it to a smaller size, attaining blanks as low as 13.75±2.15 nmol C. Under these conditions we can accurately measure both standards and diatom reference materials in the range of 100 to 330 nmol C, with a precision of 2σ < 1 ‰. The measured δ13C is independent of sample size in this range for standards or samples with δ13C < -11 ‰, which is the compositional range expected for natural diatom samples. Furthermore, no memory effect is observed in samples with an isotopic δ13C value differing by > 10 ‰ analysed in sequence. Applied to measure biomineral-bound organics in cleaned diatom samples from sediments, the low sample size requirements of this technique allows us to analyse multiple size fractions within one sample, and explore isotopic fractionation patterns between them. We have analysed samples from sediments of both centric and pennate diatoms typically in the

  7. 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

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

    PubMed Central

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

    2012-01-01

    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 13C 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. PMID:22328156

  9. 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.

  10. 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.

  11. Towards an inhalative 13C breath test method.

    PubMed

    Krumbiegel, P; Rolle-Kampczyk, U; Liebergeld, P; Herbarth, O; Köbrich, R

    2002-06-01

    Customary 13CO2 breath tests--and also 15N urine tests--always start with an oral administration of a test substrate. The test person swallows a stable isotope labelled diagnostic agent. This technique has been used to study several pathophysiological changes in gastrointestinal organs. However, to study pathophysiological changes of the bronchial and lung epithelium, the inhalative administration of a stable isotope labelled agent appeared more suitable to us. [1-13C]Hexadecanol and [1-13C]glucose were chosen. Inhaled [1-13C]hexadecanol did not yield 13CO2 in the exhaled air, but [1-13C]glucose did. To study the practicability of the [1-13C]glucose method and the reproducibility of the results, 18 inhalation tests were performed with healthy subjects. In 6 self-tests, the optimum inhalative dose of [13C]glucose was determined to be 205 mg. Using the APS aerosol provocation system with the nebulizer 'Medic Aid' (Erich Jaeger Würzburg), a 25% aqueous solution was inhaled. Then, breath samples were collected at 15 min. intervals and analysed for 13CO2. 75-120 min after the end of inhalation a well-reproducible maximum delta13C value of 6%o over baseline (DOB) was detected for 12 healthy probands. Speculating that the pulmonary resorption of the [13C]glucose is the rate-limiting step of elimination, decompensations in the epithelium ought to be reflected in changed [1-13C]glucose resorption rates and changed 13CO2 output. Therefore, we speculate that the inhalation of suitable 13C-labelled substrates will pave the way for a new group of 13CO2 breath tests aiding investigations of specific pathophysiological changes in the pulmonary tract, such as inflammations of certain sections and decompensations of cell functions.

  12. A novel LCMSMS method for quantitative measurement of short-chain fatty acids in human stool derivatized with (12)C- and (13)C-labelled aniline.

    PubMed

    Chan, James Chun Yip; Kioh, Dorinda Yan Qin; Yap, Gaik Chin; Lee, Bee Wah; Chan, Eric Chun Yong

    2017-05-10

    A novel liquid chromatography tandem mass spectrometry (LCMSMS) method for the quantitative measurement of gut microbial-derived short-chain fatty acids (SCFAs) in human infant stool has been developed and validated. Baseline chromatographic resolution was achieved for 12 SCFAs (acetic, butyric, caproic, 2,2-dimethylbutyric, 2-ethylbutyric, isobutyric, isovaleric, 2-methylbutyric, 4-methylvaleric, propionic, pivalic and valeric acids) within an analysis time of 15min. A novel sequential derivatization of endogenous and spiked SCFAs in stool via (12)C- and (13)C-aniline respectively, facilitated the accurate quantitation of (12)C-aniline derivatized endogenous SCFAs based on calibration of exogenously (13)C-derivatized SCFAs. Optimized quenching of derivatization agents prior to LCMSMS analysis further reduced to negligible levels the confounding chromatographic peak due to in-line derivatization of unquenched aniline with residual acetic acid present within the LCMS system. The effect of residual acetic acid, a common LCMS modifier, in analysis of SCFAs has not been addressed in previous SCFA assays. For the first time, a total of 9 SCFAs (acetic, butyric, caproic, isobutyric, isovaleric, 2-methylbutyric, 4-methylvaleric, propionic and valeric acids) were detected and quantitated in 107 healthy infant stool samples. The abundance and diversity of SCFAs in infant stool vary temporally from 3 weeks onwards and stabilize towards the end of 12 months. This in turn reflects the maturation of infant SCFA-producing gut microbiota community. In summary, this novel method is applicable to future studies that investigate the biological roles of SCFAs in paediatric health and diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. 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.

  14. {sup 13}C-METHYL FORMATE: OBSERVATIONS OF A SAMPLE OF HIGH-MASS STAR-FORMING REGIONS INCLUDING ORION-KL AND SPECTROSCOPIC CHARACTERIZATION

    SciTech Connect

    Favre, Cécile; Bergin, Edwin A.; Crockett, Nathan R.; Neill, Justin L.; Carvajal, Miguel; Field, David; Jørgensen, Jes K.; Bisschop, Suzanne E.; Brouillet, Nathalie; Despois, Didier; Baudry, Alain; Kleiner, Isabelle; Margulès, Laurent; Huet, Thérèse R.; Demaison, Jean E-mail: miguel.carvajal@dfa.uhu.es

    2015-01-01

    We have surveyed a sample of massive star-forming regions located over a range of distances from the Galactic center for methyl formate, HCOOCH{sub 3}, and its isotopologues H{sup 13}COOCH{sub 3} and HCOO{sup 13}CH{sub 3}. The observations were carried out with the APEX telescope in the frequency range 283.4-287.4 GHz. Based on the APEX observations, we report tentative detections of the {sup 13}C-methyl formate isotopologue HCOO{sup 13}CH{sub 3} toward the following four massive star-forming regions: Sgr B2(N-LMH), NGC 6334 IRS 1, W51 e2, and G19.61-0.23. In addition, we have used the 1 mm ALMA science verification observations of Orion-KL and confirm the detection of the {sup 13}C-methyl formate species in Orion-KL and image its spatial distribution. Our analysis shows that the {sup 12}C/{sup 13}C isotope ratio in methyl formate toward the Orion-KL Compact Ridge and Hot Core-SW components (68.4 ± 10.1 and 71.4 ± 7.8, respectively) are, for both the {sup 13}C-methyl formate isotopologues, commensurate with the average {sup 12}C/{sup 13}C ratio of CO derived toward Orion-KL. Likewise, regarding the other sources, our results are consistent with the {sup 12}C/{sup 13}C in CO. We also report the spectroscopic characterization, which includes a complete partition function, of the complex H{sup 13}COOCH{sub 3} and HCOO{sup 13}CH{sub 3} species. New spectroscopic data for both isotopomers H{sup 13}COOCH{sub 3} and HCOO{sup 13}CH{sub 3}, presented in this study, have made it possible to measure this fundamentally important isotope ratio in a large organic molecule for the first time.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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%.

  1. 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

  2. A gas chromatography/combustion/isotope ratio mass spectrometry system for high-precision delta13C measurements of atmospheric methane extracted from ice core samples.

    PubMed

    Behrens, Melanie; Schmitt, Jochen; Richter, Klaus-Uwe; Bock, Michael; Richter, Ulrike C; Levin, Ingeborg; Fischer, Hubertus

    2008-10-01

    Past atmospheric composition can be reconstructed by the analysis of air enclosures in polar ice cores which archive ancient air in decadal to centennial resolution. Due to the different carbon isotopic signatures of different methane sources high-precision measurements of delta13CH4 in ice cores provide clues about the global methane cycle in the past. We developed a highly automated (continuous-flow) gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) technique for ice core samples of approximately 200 g. The methane is melt-extracted using a purge-and-trap method, then separated from the main air constituents, combusted and measured as CO2 by a conventional isotope ratio mass spectrometer. One CO2 working standard, one CH4 and two air reference gases are used to identify potential sources of isotope fractionation within the entire sample preparation process and to enhance the stability, reproducibility and accuracy of the measurement. After correction for gravitational fractionation, pre-industrial air samples from Greenland ice (1831 +/- 40 years) show a delta13C(VPDB) of -49.54 +/- 0.13 per thousand and Antarctic samples (1530 +/- 25 years) show a delta13C(VPDB) of -48.00 +/- 0.12 per thousand in good agreement with published data.

  3. Using Isotope Ratio Infrared Spectrometer to determine δ13C of CaCO3 carbonate and DIC samples and δ18O of water

    NASA Astrophysics Data System (ADS)

    Mandic, M.; Stöbener, N.; Mandic, L.; Smajgl, D.; Jost, H. J. H.

    2016-12-01

    Precise and accurate determination of isotopic composition of carbon (13C) and oxygen (18O) from carbonate or DIC sample with proper referencing and data evaluation algorithm presents a challenge for scientists. Mass spectrometry was the only widely used technique for this kind of analysis, but recent advances make laser based isotope ratio infrared spectroscopy (IRIS) a viable alternative. To analyze discrete samples, the Universal Reference Interface (URI) Connect was developed. CO2 free syntethic air is used to flush out the contents of a sample container into a variable volume. If necessary, the sample is further diluted before entering the analysis chamber. Reference gas measurements are automatically performed at the same concentration as sample measurements to compensate for instrument drifts and non linearity. The URI Connect can handle about 100 samples per day from an autosampler, or samples can be injected one at a time through a septum on the front of the instrument. Gas samples collected in flasks, bags, syringes, or vials can be analyzed. The system only needs the equivalent of about 80µg - or 40µL - of pure CO2 gas to complete an analysis. Due to it's small weight and robustness, sample analysis can be performed in the field, e.g. aboard a research vessel. To demonstrate the performance, a test experiment with 1% CO2 in 12 ml vials was performed. We achieved an internal precision of better than 0.07‰ and 0.1‰ for δ13C and δ18O, respectively. Analyses with sample amounts as low as 200 μg of carbonate can also be performed reliably with IRIS. We present measurements of three international reference materials, and one of them treated as an unknown. Five samples each of approximately 1mg each were acidified using a few droplets of 43% H3PO4 and left for equilibration overnight at 25°C. The standard deviation was less than 0.1‰ δ13C and the accuracy <0.01‰ As another example of head space analysis in 12 ml vials, we determined δ18O of

  4. UV-laser-based microscopic dissection of tree rings - a novel sampling tool for δ(13) C and δ(18) O studies.

    PubMed

    Schollaen, Karina; Heinrich, Ingo; Helle, Gerhard

    2014-02-01

    UV-laser-based microscopic systems were utilized to dissect and sample organic tissue for stable isotope measurements from thin wood cross-sections. We tested UV-laser-based microscopic tissue dissection in practice for high-resolution isotopic analyses (δ(13) C/δ(18) O) on thin cross-sections from different tree species. The method allows serial isolation of tissue of any shape and from millimetre down to micrometre scales. On-screen pre-defined areas of interest were automatically dissected and collected for mass spectrometric analysis. Three examples of high-resolution isotopic analyses revealed that: in comparison to δ(13) C of xylem cells, woody ray parenchyma of deciduous trees have the same year-to-year variability, but reveal offsets that are opposite in sign depending on whether wholewood or cellulose is considered; high-resolution tree-ring δ(18) O profiles of Indonesian teak reflect monsoonal rainfall patterns and are sensitive to rainfall extremes caused by ENSO; and seasonal moisture signals in intra-tree-ring δ(18) O of white pine are weighted by nonlinear intra-annual growth dynamics. The applications demonstrate that the use of UV-laser-based microscopic dissection allows for sampling plant tissue at ultrahigh resolution and unprecedented precision. This new technique facilitates sampling for stable isotope analysis of anatomical plant traits like combined tree eco-physiological, wood anatomical and dendroclimatological studies.

  5. 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

  6. A study of 15N- 15N and 15N- 13C spin couplings in some 15N labeled mesoionic 1-oxa and 1-thia-2,3,4-triazoles

    NASA Astrophysics Data System (ADS)

    Jaźwiński, J.; Staszewska, O.; Stefaniak, L.; Webb, G. A.

    1996-03-01

    15N- 15N and 15N- 13C spin-spin couplings are reported for seven 15N labeled 1-oxa and 1-thia-2,3,4-triazoles and three sydnonimines. For the former class of compounds the spin-spin coupling data show a close similarity between the N2N3 and N3N4 bonds which had not previously been suspected from chemical shift measurements.

  7. Wanted: dead or alive? Isotopic analysis (δ13C and δ15N) of Pygoscelis penguin chick tissues supports opportunistic sampling.

    PubMed

    Vasil, Christopher A; Polito, Michael J; Patterson, William P; Emslie, Steven D

    2012-03-15

    Physiological stress and starvation have been shown to affect δ(13)C and δ(15)N isotope values and, given that animals often die from starvation, the cause of death may be an important factor to consider in stable isotope analyses of opportunistically collected samples. We addressed this issue by comparing tissue stable isotope values of living and deceased Adélie (Pygoscelis adeliae) and Chinstrap Penguin (P. antarctica) chicks collected from the same respective populations. No significant difference was found between living and deceased penguin chick feather, down, and toenail isotope values and both groups displayed similar isotopic trends between tissue types. In addition, similar relationships were observed between both species and across several seasons. Furthermore, sub-dermal adiposity and cause of death (starvation and/or predation) had no significant effect on the δ(13)C and δ(15)N values. Our findings suggest that tissues from deceased penguins can be isotopically representative of tissues obtained from the living population, despite the cause of death, and support the use of opportunistic sampling in stable isotope analyses. Copyright © 2012 John Wiley & Sons, Ltd.

  8. (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.

  9. Energy expenditure of grazing cows and cows fed grass indoors as determined by the 13C bicarbonate dilution technique using an automatic blood sampling system.

    PubMed

    Kaufmann, L D; Münger, A; Rérat, M; Junghans, P; Görs, S; Metges, C C; Dohme-Meier, F

    2011-04-01

    The objectives of the study were to assess the 13C bicarbonate dilution technique using an automatic blood sampling system and to use this technique to estimate energy expenditure (EE) based on the CO2 production of 14 lactating Holstein cows on pasture or in a freestall barn. The effects of physical activity and eating behavior on EE were also assessed. Cows were exposed to each feeding system in a crossover design with two 14-d experimental periods, each consisting of an adaptation period and a 7-d data collection period. Cows either grazed on pasture or had ad libitum access, in the freestall barn, to grass cut daily from the same paddock. All cows were supplemented with a cereal-based concentrate. The EE of each cow was determined from 0700 to 1300 h on 1 d of each collection period. Blood samples for the 13C bicarbonate dilution technique were taken either manually in the barn or using an automatic blood sampling system on pasture. Eating pattern and physical activity were recorded from 0700 to 1300 h using a behavior recorder and an activity meter, respectively. Milk yield was recorded daily. Individual feed intake was estimated using the alkane double-indicator technique. Two preceding experiments confirmed that the sampling technique (manual or automatic) and the following storage of the blood samples (frozen directly after withdrawal or first cooled on ice and then frozen 6 h later) had no effect on 13CO2 enrichment in the extracted blood CO2 or on the subsequent calculation of CO2 production. During the 6-h measurement period, the EE of cows on pasture was higher than that of cows in the freestall barn. Daily feed intake and milk production were not affected by the feeding treatment. Grazing cows spent more time walking and less time standing and lying than did cows fed indoors. Time spent eating was greater and time spent ruminating was lower for cows on pasture compared with grass-fed cows in the barn. In conclusion, the 13C bicarbonate dilution

  10. Synthesis and application of (13)C-labeled 2-acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI), an immunosuppressant observed in caramel food colorings.

    PubMed

    Elsinghorst, Paul W; Raters, Marion; Dingel, Anna; Fischer, Jochen; Matissek, Reinhard

    2013-08-07

    2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is a minor toxic contaminant observed in caramel food colorings and was shown to exert immunosuppressant activity when fed to rodents. Because of this toxicity, maximum levels of THI in caramel food colorings have been defined by international and European authorities. Several reports of THI analysis using external standardization have been published for liquid foods such as beers and soft drinks. However, no suitable internal standard has yet been described allowing THI analysis in more complex samples. In this paper we describe the preparation of a labeled [(13)C6]THI analogue and its application for the successful validation of the first stable isotope dilution assay (SIDA) of THI in caramel food colorings. A brief survey of THI levels in commercially available caramel class III (E 150c) and IV (E 150d) food colorings is also included, corroborating that THI occurs only in caramel class III food colorings.

  11. 19 CFR 12.22 - Labels; samples.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 1 2013-04-01 2013-04-01 false Labels; samples. 12.22 Section 12.22 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY SPECIAL CLASSES OF MERCHANDISE Viruses, Serums, Toxins, Antitoxins, and Analogous Products for the...

  12. 19 CFR 12.22 - Labels; samples.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Labels; samples. 12.22 Section 12.22 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY SPECIAL CLASSES OF MERCHANDISE Viruses, Serums, Toxins, Antitoxins, and Analogous Products for the...

  13. 19 CFR 12.22 - Labels; samples.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Labels; samples. 12.22 Section 12.22 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY SPECIAL CLASSES OF MERCHANDISE Viruses, Serums, Toxins, Antitoxins, and Analogous Products for the...

  14. 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.

  15. A New Substrate Cycle in Plants. Evidence for a High Glucose-Phosphate-to-Glucose Turnover from in Vivo Steady-State and Pulse-Labeling Experiments with [13C]Glucose and [14C]Glucose1

    PubMed Central

    Alonso, Ana Paula; Vigeolas, Hélène; Raymond, Philippe; Rolin, Dominique; Dieuaide-Noubhani, Martine

    2005-01-01

    Substrate (futile) cycling involving carbohydrate turnover has been widely reported in plant tissues, although its extent, mechanisms, and functions are not well known. In this study, two complementary approaches, short and steady-state labeling experiments, were used to analyze glucose metabolism in maize (Zea mays) root tips. Unidirectional rates of synthesis for storage compounds (starch, Suc, and cell wall polysaccharides) were determined by short labeling experiments using [U-14C]glucose and compared with net synthesis fluxes to determine the rate of glucose production from these storage compounds. Steady-state labeling with [1-13C]glucose and [U-13C]glucose showed that the redistribution of label between carbon C-1 and C-6 in glucose is close to that in cytosolic hexose-P. These results indicate a high resynthesis flux of glucose from hexose-P that is not accounted for by glucose recycling from storage compounds, thus suggesting the occurrence of a direct glucose-P-to-glucose conversion. An enzyme assay confirmed the presence of substantial glucose-6-phosphatase activity in maize root tips. This new glucose-P-to-glucose cycle was shown to consume around 40% of the ATP generated in the cell, whereas Suc cycling consumes at most 3% to 6% of the ATP produced. The rate of glucose-P cycling differs by a factor of 3 between a maize W22 line and the hybrid maize cv Dea, and is significantly decreased by a carbohydrate starvation pretreatment. PMID:16024683

  16. Measurements of CH4 Mixing Ratio and D/H and 13C/12C Isotope Ratios in Atmospheric Samples from Continental United States

    NASA Astrophysics Data System (ADS)

    Tyler, S. C.; Ajie, H. O.; Gotoh, A. A.; Rice, A. L.

    2002-12-01

    We report δD and δ13C measurements of atmospheric CH4 from air samples collected bi-weekly from fixed surface sites in the United States. Our fixed surface sites are located at the mid-continental site Niwot Ridge, CO (41°N, 105°W) and a Pacific coastal northern hemispheric site receiving strong westerlies, Montaña de Oro, CA (35°N, 121°W). Data from multiyear bi-weekly sampling provide information relating seasonal cycling of CH4 sources and sinks in background air, provide a record of long term trends in CH4 mixing and isotope ratio related to atmospheric CH4 loading, and may reveal information on regional sources of CH4. Measurements of δD-CH4 average -93+/-3‰ (versus V-SMOW) at Niwot Ridge from 1999 through 2001 and -97+/-4‰ at Montaña de Oro from 2000 through 2001. Annual seasonal cycles observed at both sites are 6-7‰ , exhibiting strong anti-correlation with CH4 mixing ratio seasonality that appears to be largely sink-driven (i.e. through reaction with OH). More extensive time series in δ13C-CH4 average -47.2+/-0.1‰ (versus V-PDB) at Niwot Ridge from 1995 through 2001 and -47.3+/-0.2‰ at Montaña de Oro from 1996 through 2001. In contrast to δD-CH4, seasonality in δ13C-CH4 has a relatively poor anti-correlation with seasonality in CH4 mixing ratio, indicating significant source-driven influence. We discuss the implications of these measurements on the atmospheric CH4 budget and detail the precision and accuracy of our data with respect international reference standards. It is our intention to have these data archived and available for modeling and advanced calculations by other atmospheric researchers in the near future.

  17. 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.

  18. Methane mole fraction and δ13C above and below the trade wind inversion at Ascension Island in air sampled by aerial robotics

    NASA Astrophysics Data System (ADS)

    Brownlow, R.; Lowry, D.; Thomas, R. M.; Fisher, R. E.; France, J. L.; Cain, M.; Richardson, T. S.; Greatwood, C.; Freer, J.; Pyle, J. A.; MacKenzie, A. R.; Nisbet, E. G.

    2016-11-01

    Ascension Island is a remote South Atlantic equatorial site, ideal for monitoring tropical background CH4. In September 2014 and July 2015, octocopters were used to collect air samples in Tedlar bags from different heights above and below the well-defined Trade Wind Inversion (TWI), sampling a maximum altitude of 2700 m above mean sea level. Sampling captured both remote air in the marine boundary layer below the TWI and also air masses above the TWI that had been lofted by convective systems in the African tropics. Air above the TWI was characterized by higher CH4, but no distinct shift in δ13C was observed compared to the air below. Back trajectories indicate that lofted CH4 emissions from Southern Hemisphere Africa have bulk δ13CCH4 signatures similar to background, suggesting mixed emissions from wetlands, agriculture, and biomass burning. The campaigns illustrate the usefulness of unmanned aerial system sampling and Ascension's value for atmospheric measurement in an understudied region.

  19. 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.

  20. 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.

  1. (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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. Nutritional status, fecal elastase-1, and 13C-labeled mixed triglyceride breath test in the long-term after pancreaticoduodenectomy.

    PubMed

    Muniz, Cinara Knychala; dos Santos, José Sebastião; Pfrimer, Karina; Ferrioli, Eduardo; Kemp, Rafael; Marchini, Júlio Sérgio; Cunha, Selma Freire

    2014-04-01

    This study aimed to compare the body composition, dietary intake and serum levels of vitamins and minerals, and exocrine pancreatic function in patients late after pancreaticoduodenectomy (PD) and healthy subjects. Fifteen patients (PD group) who had undergone PD over 1 year before the study and 15 health volunteers (control group) were included in the study. All volunteers underwent dietary intake evaluation, body composition, laboratory data, exocrine pancreatic function by elastase-1, and carbon (C )-labeled triglycerides in breath tests. The PD group subjects also underwent upper gastrointestinal endoscopy and small intestinal bacterial overgrowth analysis. Nutrient intake was adequate, and there were no differences in body mass index and mineral serum levels between the groups. The PD group showed lower serum levels of retinol, α-tocopherol, and ascorbic acid. Small intestinal bacterial overgrowth occurred in 39% of the patients. Fecal elastase-1 was lower in the PD group. The PD group had a higher C peak time; the cumulative label C recovery in 7 hours was similar in both groups. Fecal elastase-1 decreased, and the excretion of C in breath was similar to healthy controls. Although the data point toward an adaptation in the absorptive capacity of fats, A, C, and E hypovitaminosis indicate that some absorptive insufficiency persists late after PD.

  10. Carbonation of C–S–H and C–A–S–H samples studied by {sup 13}C, {sup 27}Al and {sup 29}Si MAS NMR spectroscopy

    SciTech Connect

    Sevelsted, Tine F.; Skibsted, Jørgen

    2015-05-15

    Synthesized calcium silicate hydrate (C–S–H) samples with Ca/Si ratios of 0.66, 1.0, and 1.5 have been exposed to atmospheric CO{sub 2} at room temperature and high relative humidity and studied after one to 12 weeks. {sup 29}Si NMR reveals that the decomposition of C–S–H caused by carbonation involves two steps and that the decomposition rate decreases with increasing Ca/Si ratio. The first step is a gradual decalcification of the C–S–H where calcium is removed from the interlayer and defect sites in the silicate chains until Ca/Si = 0.67 is reached, ideally corresponding to infinite silicate chains. In the seconds step, calcium from the principal layers is consumed, resulting in the final decomposition of the C–S–H and the formation of an amorphous silica phase composed of Q{sup 3} and Q{sup 4} silicate tetrahedra. The amount of solid carbonates and of carbonate ions in a hydrous environment increases with increasing Ca/Si ratio for the C–S–H, as shown by {sup 13}C NMR. For C–A–S–H samples with Ca/Si = 1.0 and 1.5, {sup 27}Al NMR demonstrates that all aluminium sites associated with the C–S–H are consumed during the carbonation reactions and incorporated mainly as tetrahedral Al(–OSi){sub 4} units in the amorphous silica phase. A small amount of penta-coordinated Al sites has also been identified in the silica phase.

  11. Coupling a high-temperature catalytic oxidation total organic carbon analyzer to an isotope ratio mass spectrometer to measure natural-abundance delta13C-dissolved organic carbon in marine and freshwater samples.

    PubMed

    Panetta, Robert J; Ibrahim, Mina; Gélinas, Yves

    2008-07-01

    The stable isotope composition of dissolved organic carbon (delta(13)C-DOC) provides powerful information toward understanding carbon sources and cycling, but analytical limitations have precluded its routine measurement in natural samples. Recent interfacing of wet oxidation-based dissolved organic carbon analyzers and isotope ratio mass spectrometers has simplified the measurement of delta(13)C-DOC in freshwaters, but the analysis of salty estuarine/marine samples still proves difficult. Here we describe the coupling of the more widespread high-temperature catalytic oxidation-based total organic carbon analyzer to an isotope ratio mass spectrometer (HTC-IRMS) through cryogenic trapping of analyte gases exiting the HTC analyzer for routine analysis of delta(13)C-DOC in aquatic and marine samples. Targeted elimination of major sources of background CO2 originating from the HTC analyzer allows for the routine measurement of samples over the natural range of DOC concentrations (from 40 microM to over 2000 microM), and salinities (<0.1-36 g/kg). Because consensus reference natural samples for delta(13)C-DOC do not exist, method validation was carried out with water-soluble stable isotope standards as well as previously measured natural samples (IAEA sucrose, Suwannee River Fulvic Acids, Deep Sargasso Sea consensus reference material, and St. Lawrence River water) and result in excellent delta(13)C-DOC accuracy (+/-0.2 per thousand) and precision (+/-0.3 per thousand).

  12. 16 CFR 301.33 - Labeling of samples.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... REGULATIONS UNDER FUR PRODUCTS LABELING ACT Regulations § 301.33 Labeling of samples. Where samples of furs or fur products subject to the act are used to promote or effect sales of fur products, said samples, as well as the fur products purchased therefrom, shall be labeled to show the information required...

  13. 16 CFR 301.33 - Labeling of samples.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... REGULATIONS UNDER FUR PRODUCTS LABELING ACT Regulations § 301.33 Labeling of samples. Where samples of furs or fur products subject to the act are used to promote or effect sales of fur products, said samples, as well as the fur products purchased therefrom, shall be labeled to show the information required...

  14. 16 CFR 301.33 - Labeling of samples.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... REGULATIONS UNDER FUR PRODUCTS LABELING ACT Regulations § 301.33 Labeling of samples. Where samples of furs or fur products subject to the act are used to promote or effect sales of fur products, said samples, as well as the fur products purchased therefrom, shall be labeled to show the information required...

  15. 16 CFR 301.33 - Labeling of samples.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... REGULATIONS UNDER FUR PRODUCTS LABELING ACT Regulations § 301.33 Labeling of samples. Where samples of furs or fur products subject to the act are used to promote or effect sales of fur products, said samples, as well as the fur products purchased therefrom, shall be labeled to show the information required...

  16. 16 CFR 301.33 - Labeling of samples.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... REGULATIONS UNDER FUR PRODUCTS LABELING ACT Regulations § 301.33 Labeling of samples. Where samples of furs or fur products subject to the act are used to promote or effect sales of fur products, said samples, as well as the fur products purchased therefrom, shall be labeled to show the information required...

  17. Four-dimensional sup 13 C/ sup 13 C-edited nuclear Overhauser Enhancement Spectroscopy of a protein in solution: Application to interleukin 1. beta

    SciTech Connect

    Clore, G.M.; Kay, L.E.; Bax, A.; Gronenborn, A.M. )

    1991-01-01

    A four-dimensional {sup 13}C/{sup 13}C-edited NOESY experiment is described which dramatically improves the resolution of protein NMR spectra and enables the straightforward assignment of nuclear Overhauser effects involving aliphatic and/or aromatic protons in larger proteins. The experiment is demonstrated for uniformly (>95{percent}) {sup 13}C-labeled interleukin 1{beta}, a protein of 153 residues and 17.4 kDa, which plays a key role in the immune response. NOEs between aliphatic and/or aromatic protons are first spread out into a third dimension by the {sup 13}C chemical shift of the carbon atom attached to the originating proton and subsequently into a fourth dimension by the {sup 13}C chemical shift of the carbon atom attached to the destination proton. Thus, each NOE cross peak is labeled by four chemical shifts. By this means, ambiguities in the assignment of NOEs that arise from chemical shift overlap and degeneracy are completely removed. Further, NOEs between protons with the same chemical shifts can readily be detected providing their attached carbon atoms have different {sup 13}C chemical shifts. The design of the pulse sequence requires special care to minimize the level of artifacts arising from undesired coherence transfer pathways, and in particular those associated with diagonal peaks which correspond to magnetization that has not been transferred from one proton to another. The 4D {sup 13}C/{sup 13}C-edited NOESY experiment is characterized by high sensitivity as the through-bond transfer steps involve the large {sup 1}J{sub CH} (130 Hz) couplings, and it is possible to obtain high-quality spectra on 1-2 mM samples of {sup 13}C-labeled protein in as little as 3 days. This experiment should open up the application of protein structure determination by NMR to a large number of medium-sized proteins (150-300 residues) of biological interest.

  18. Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of 13C-valine isotopic ratios in complex biological samples.

    PubMed

    Godin, Jean-Philippe; Breuillé, Denis; Obled, Christiane; Papet, Isabelle; Schierbeek, Henk; Hopfgartner, Gérard; Fay, Laurent-Bernard

    2008-10-01

    On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision (13)C isotopic enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure (13)C isotopic enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(delta(13)C) reported with this method was found to be below 1 per thousand . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (delta(13)C= -12.3 to 150.8 per thousand), the calculated root-mean-square (rms) of SD was 0.38 and 0.46 per thousand and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (delta(13)C) and absolute (atom%, APE and MPE) isotopic enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic enrichment of protein-bound (13)C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p>0.05). The results of this work indicate that

  19. 1H-13C HSQC NMR spectroscopy for estimating procyanidin/prodelphinidin and cis/trans flavan-3-ol ratios of condensed tannin samples: correlation with thiolysis

    USDA-ARS?s Scientific Manuscript database

    Studies with a diverse array of 22 condensed tannin (CT) fractions from 9 plant species demonstrated that procyanidin/prodelphinidin (PC/PD) and cis/trans flavan-3-ol ratios can be appraised by 1H-13C HSQC NMR. The method was developed from fractions containing 44 to ~100% CT, PC/PD ratios ranging f...

  20. 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. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  1. 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.

  2. 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

  3. Automated simultaneous measurement of the δ(13) C and δ(2) H values of methane and the δ(13) C and δ(18) O values of carbon dioxide in flask air samples using a new multi cryo-trap/gas chromatography/isotope ratio mass spectrometry system.

    PubMed

    Brand, Willi A; Rothe, Michael; Sperlich, Peter; Strube, Martin; Wendeberg, Magnus

    2016-07-15

    The isotopic composition of greenhouse gases helps to constrain global budgets and to study sink and source processes. We present a new system for high-precision stable isotope measurements of carbon, hydrogen and oxygen in atmospheric methane and carbon dioxide. The design is intended for analyzing flask air samples from existing sampling programs without the need for extra sample air for methane analysis. CO2 and CH4 isotopes are measured simultaneously using two isotope ratio mass spectrometers, one for the analysis of δ(13) C and δ(18) O values and the second one for δ(2) H values. The inlet carousel delivers air from 16 sample positions (glass flasks 1-5 L and high-pressure cylinders). Three 10-port valves take aliquots from the sample stream. CH4 from 100-mL air aliquots is preconcentrated in 0.8-mL sample loops using a new cryo-trap system. A precisely calibrated working reference air is used in parallel with the sample according to the Principle of Identical Treatment. It takes about 36 hours for a fully calibrated analysis of a complete carousel including extractions of four working reference and one quality control reference air. Long-term precision values, as obtained from the quality control reference gas since 2012, account for 0.04 ‰ (δ(13) C values of CO2 ), 0.07 ‰ (δ(18) O values of CO2 ), 0.11 ‰ (δ(13) C values of CH4 ) and 1.0 ‰ (δ(2) H values of CH4 ). Within a single day, the system exhibits a typical methane δ(13) C standard deviation (1σ) of 0.06 ‰ for 10 repeated measurements. The system has been in routine operation at the MPI-BGC since 2012. Consistency of the data and compatibility with results from other laboratories at a high precision level are of utmost importance. A high sample throughput and reliability of operation are important achievements of the presented system to cope with the large number of air samples to be analyzed. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  4. /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.

  5. 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.

  6. 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

  7. 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.

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

    PubMed Central

    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.

    2009-01-01

    We describe a novel 13C enriched precursor molecule, sodium 1-13C acetylenedicarboxylate, which after hydrogenation by PASADE-NA (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. PMID:17303454

  9. 16 CFR Appendix L to Part 305 - Sample Labels

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 16 Commercial Practices 1 2013-01-01 2013-01-01 false Sample Labels L Appendix L to Part 305... UNDER THE ENERGY POLICY AND CONSERVATION ACT (âAPPLIANCE LABELING RULEâ) Pt. 305, App. L Appendix L to.... At 76 FR 79058, Dec. 21, 2011, appendix L was amended by redesignating samples 10, 11, 12, and icon...

  10. 16 CFR Appendix L to Part 305 - Sample Labels

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 1 2014-01-01 2014-01-01 false Sample Labels L Appendix L to Part 305... RULEâ) Pt. 305, App. L Appendix L to Part 305—Sample Labels Link to a correction published at 78 FR... affecting appendix L, see the List of CFR Sections Affected, which appears in the Finding Aids section of...

  11. 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.

  12. 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.

  13. Leaf functional response to increasing atmospheric CO(2) concentrations over the last century in two northern Amazonian tree species: a historical δ(13) C and δ(18) O approach using herbarium samples.

    PubMed

    Bonal, Damien; Ponton, Stéphane; Le Thiec, Didier; Richard, Béatrice; Ningre, Nathalie; Hérault, Bruno; Ogée, Jérôme; Gonzalez, Sophie; Pignal, Marc; Sabatier, Daniel; Guehl, Jean-Marc

    2011-08-01

    We assessed the extent of recent environmental changes on leaf morphological (stomatal density, stomatal surface, leaf mass per unit area) and physiological traits (carbon isotope composition, δ(13)C(leaf) , and discrimination, Δ(13)C(leaf) , oxygen isotope composition, δ(18)O(leaf) ) of two tropical rainforest species (Dicorynia guianensis; Humiria balsamifera) that are abundant in the Guiana shield (Northern Amazonia). Leaf samples were collected in different international herbariums to cover a 200 year time-period (1790-2004) and the whole Guiana shield. Using models describing carbon and oxygen isotope fractionations during photosynthesis, different scenarios of change in intercellular CO(2) concentrations inside the leaf (C(i)), stomatal conductance (g), and photosynthesis (A) were tested in order to understand leaf physiological response to increasing air CO(2) concentrations (C(a)). Our results confirmed that both species displayed physiological response to changing C(a) . For both species, we observed a decrease of about 1.7‰ in δ(13)C(leaf) since 1950, without significant change in Δ(13)C(leaf) and leaf morphological traits. Furthermore, there was no clear change in δ(18)O(leaf) for Humiria over this period. Our simulation approach revealed that an increase in A, rather than a decrease in g, explained the observed trends for these tropical rainforest species, allowing them to maintain a constant ratio of C(i)/C(a) .

  14. Matching isotopic distributions from metabolically labeled samples.

    PubMed

    McIlwain, Sean; Page, David; Huttlin, Edward L; Sussman, Michael R

    2008-07-01

    In recent years stable isotopic labeling has become a standard approach for quantitative proteomic analyses. Among the many available isotopic labeling strategies, metabolic labeling is attractive for the excellent internal control it provides. However, analysis of data from metabolic labeling experiments can be complicated because the spacing between labeled and unlabeled forms of each peptide depends on its sequence, and is thus variable from analyte to analyte. As a result, one generally needs to know the sequence of a peptide to identify its matching isotopic distributions in an automated fashion. In some experimental situations it would be necessary or desirable to match pairs of labeled and unlabeled peaks from peptides of unknown sequence. This article addresses this largely overlooked problem in the analysis of quantitative mass spectrometry data by presenting an algorithm that not only identifies isotopic distributions within a mass spectrum, but also annotates matches between natural abundance light isotopic distributions and their metabolically labeled counterparts. This algorithm is designed in two stages: first we annotate the isotopic peaks using a modified version of the IDM algorithm described last year; then we use a probabilistic classifier that is supplemented by dynamic programming to find the metabolically labeled matched isotopic pairs. Such a method is needed for high-throughput quantitative proteomic metabolomic experiments measured via mass spectrometry. The primary result of this article is that the dynamic programming approach performs well given perfect isotopic distribution annotations. Our algorithm achieves a true positive rate of 99% and a false positive rate of 1% using perfect isotopic distribution annotations. When the isotopic distributions are annotated given 'expert' selected peaks, the same algorithm gets a true positive rate of 77% and a false positive rate of 1%. Finally, when annotating using machine selected peaks, which

  15. Matching isotopic distributions from metabolically labeled samples

    PubMed Central

    McIlwain, Sean; Page, David; Huttlin, Edward L.; Sussman, Michael R.

    2008-01-01

    Motivation: In recent years stable isotopic labeling has become a standard approach for quantitative proteomic analyses. Among the many available isotopic labeling strategies, metabolic labeling is attractive for the excellent internal control it provides. However, analysis of data from metabolic labeling experiments can be complicated because the spacing between labeled and unlabeled forms of each peptide depends on its sequence, and is thus variable from analyte to analyte. As a result, one generally needs to know the sequence of a peptide to identify its matching isotopic distributions in an automated fashion. In some experimental situations it would be necessary or desirable to match pairs of labeled and unlabeled peaks from peptides of unknown sequence. This article addresses this largely overlooked problem in the analysis of quantitative mass spectrometry data by presenting an algorithm that not only identifies isotopic distributions within a mass spectrum, but also annotates matches between natural abundance light isotopic distributions and their metabolically labeled counterparts. This algorithm is designed in two stages: first we annotate the isotopic peaks using a modified version of the IDM algorithm described last year; then we use a probabilistic classifier that is supplemented by dynamic programming to find the metabolically labeled matched isotopic pairs. Such a method is needed for high-throughput quantitative proteomic metabolomic experiments measured via mass spectrometry. Results: The primary result of this article is that the dynamic programming approach performs well given perfect isotopic distribution annotations. Our algorithm achieves a true positive rate of 99% and a false positive rate of 1% using perfect isotopic distribution annotations. When the isotopic distributions are annotated given ‘expert’ selected peaks, the same algorithm gets a true positive rate of 77% and a false positive rate of 1%. Finally, when annotating using

  16. A new set-up for simultaneous high-precision measurements of CO2, δ13C-CO2 and δ18O-CO2 on small ice core samples

    NASA Astrophysics Data System (ADS)

    Jenk, Theo Manuel; Rubino, Mauro; Etheridge, David; Ciobanu, Viorela Gabriela; Blunier, Thomas

    2016-08-01

    Palaeoatmospheric records of carbon dioxide and its stable carbon isotope composition (δ13C) obtained from polar ice cores provide important constraints on the natural variability of the carbon cycle. However, the measurements are both analytically challenging and time-consuming; thus only data exist from a limited number of sampling sites and time periods. Additional analytical resources with high analytical precision and throughput are thus desirable to extend the existing datasets. Moreover, consistent measurements derived by independent laboratories and a variety of analytical systems help to further increase confidence in the global CO2 palaeo-reconstructions. Here, we describe our new set-up for simultaneous measurements of atmospheric CO2 mixing ratios and atmospheric δ13C and δ18O-CO2 in air extracted from ice core samples. The centrepiece of the system is a newly designed needle cracker for the mechanical release of air entrapped in ice core samples of 8-13 g operated at -45 °C. The small sample size allows for high resolution and replicate sampling schemes. In our method, CO2 is cryogenically and chromatographically separated from the bulk air and its isotopic composition subsequently determined by continuous flow isotope ratio mass spectrometry (IRMS). In combination with thermal conductivity measurement of the bulk air, the CO2 mixing ratio is calculated. The analytical precision determined from standard air sample measurements over ice is ±1.9 ppm for CO2 and ±0.09 ‰ for δ13C. In a laboratory intercomparison study with CSIRO (Aspendale, Australia), good agreement between CO2 and δ13C results is found for Law Dome ice core samples. Replicate analysis of these samples resulted in a pooled standard deviation of 2.0 ppm for CO2 and 0.11 ‰ for δ13C. These numbers are good, though they are rather conservative estimates of the overall analytical precision achieved for single ice sample measurements. Facilitated by the small sample requirement

  17. 16 CFR Appendix L to Part 305 - Sample Labels

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Sample Labels L Appendix L to Part 305... UNDER THE ENERGY POLICY AND CONSERVATION ACT (âAPPLIANCE LABELING RULEâ) Pt. 305, App. L Appendix L to... Editorial Note: For Federal Register citations affecting appendix L, see the List of CFR Sections Affected...

  18. 16 CFR Appendix L to Part 305 - Sample Labels

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 1 2012-01-01 2012-01-01 false Sample Labels L Appendix L to Part 305... UNDER THE ENERGY POLICY AND CONSERVATION ACT (âAPPLIANCE LABELING RULEâ) Pt. 305, App. L Appendix L to.... For Federal Register citations affecting appendix L, see the List of CFR Sections Affected, which...

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

    NASA Astrophysics Data System (ADS)

    Watzinger, Andrea

    2016-04-01

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

  20. 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.

  1. Solid-state {sup 19}F and {sup 13}C NMR of room temperature fluorinated graphite and samples thermally treated under fluorine: Low-field and high-resolution studies

    SciTech Connect

    Giraudet, J.; Dubois, M.; Guerin, K.; Pinheiro, J.P.; Hamwi, A.; Stone, W.E.E.; Pirotte, P.; Masin, F. . E-mail: fmasin@ulb.ac.be

    2005-04-15

    Room temperature graphite fluorides consisting of raw material and samples post-treated in pure fluorine atmosphere in the temperature range 100-500 deg. C have been studied by solid-state NMR. Several NMR approaches have been used, both high and low-field {sup 19}F, {sup 19}F MAS and {sup 13}C MAS with {sup 19}F to {sup 13}C cross polarization. The modifications, in the graphitic lattice, of the catalytic iodine fluorides products have been examined. A transformation of the C-F bond character from semi-ionic to covalent has been found to occur at a post-treatment temperature close to 400 deg. C. It is shown that covalency increases with temperature.

  2. Small Cab-like proteins retard degradation of photosystem II-associated chlorophyll in Synechocystis sp. PCC 6803: kinetic analysis of pigment labeling with 15N and 13C.

    PubMed

    Vavilin, Dmitrii; Yao, Danny; Vermaas, Wim

    2007-12-28

    Isotope (Na(15)NO(3), ((15)NH(4))SO(4) or [(13)C]glucose) labeling was used to analyze chlorophyll synthesis and degradation rates in a set of Synechocystis mutants that lacked single or multiple small Cab-like proteins (SCPs), as well as photosystem I or II. When all five small Cab-like proteins were inactivated in the wild-type background, chlorophyll stability was not affected unless the scpABCDE(-) strain was grown at a moderately high light intensity of 100-300 micromol photons m(-2) s(-1). However, the half-life time of chlorophyll was 5-fold shorter in the photosystem I-less/scpABCDE(-) strain than in the photosystem I-less strain even when grown at low light intensity (~3 micromol photons m(-2) s(-1)) (32 +/- 5 and 161 +/- 25 h, respectively). In other photosystem I-less mutants that lacked one to four of the scp genes the chlorophyll lifetime was in between these two values, with the chlorophyll lifetime generally decreasing with an increasing number of inactivated scps. In contrast, the chlorophyll biosynthesis rate was only marginally affected by inactivation of scps except when all five scp genes were deleted. Small Cab-like protein deficiency did not significantly affect photoinhibition or turnover of photosystem II-associated beta-carotene. It is concluded that SCPs do not alter the stability of functional photosystem II complexes but retard the degradation of photosystem II-associated chlorophyll, consistent with the proposed involvement of SCPs in photosystem II re-assembly or/and repair processes by temporarily binding chlorophyll while photosystem II protein components are being replaced.

  3. An automated GC-C-GC-IRMS setup to measure palaeoatmospheric δ13C-CH4, δ15N-N2O and δ18O-N2O in one ice core sample

    NASA Astrophysics Data System (ADS)

    Sperlich, P.; Buizert, C.; Jenk, T. M.; Sapart, C. J.; Prokopiou, M.; Röckmann, T.; Blunier, T.

    2013-08-01

    Air bubbles in ice core samples represent the only opportunity to study the mixing ratio and isotopic variability of palaeoatmospheric CH4 and N2O. The highest possible precision in isotope measurements is required to maximize the resolving power for CH4 and N2O sink and source reconstructions. We present a new setup to measure δ13C-CH4, δ15N-N2O and δ18O-N2O isotope ratios in one ice core sample and with one single IRMS instrument, with a precision of 0.09, 0.6 and 0.7‰, respectively, as determined on 0.6-1.6 nmol CH4 and 0.25-0.6 nmol N2O. The isotope ratios are referenced to the VPDB scale (δ13C-CH4), the N2-air scale (δ15N-N2O) and the VSMOW scale (δ18O-N2O). Ice core samples of 200-500 g are melted while the air is constantly extracted to minimize gas dissolution. A helium carrier gas flow transports the sample through the analytical system. We introduce a new gold catalyst to oxidize CO to CO2 in the air sample. CH4 and N2O are then separated from N2, O2, Ar and CO2 before they get pre-concentrated and separated by gas chromatography. A combustion unit is required for δ13C-CH4 analysis, which is equipped with a constant oxygen supply as well as a post-combustion trap and a post-combustion GC column (GC-C-GC-IRMS). The post-combustion trap and the second GC column in the GC-C-GC-IRMS combination prevent Kr and N2O interferences during the isotopic analysis of CH4-derived CO2. These steps increase the time for δ13C-CH4 measurements, which is used to measure δ15N-N2O and δ18O-N2O first and then δ13C-CH4. The analytical time is adjusted to ensure stable conditions in the ion source before each sample gas enters the IRMS, thereby improving the precision achieved for measurements of CH4 and N2O on the same IRMS. The precision of our measurements is comparable to or better than that of recently published systems. Our setup is calibrated by analysing multiple reference gases that were injected over bubble-free ice samples. We show that our measurements

  4. 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

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

    NASA Astrophysics Data System (ADS)

    Xu, Su; Shen, Jun

    2006-10-01

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

  6. (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.

  7. 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.

  8. Galactose oxidation using 13C in healthy and galactosemic children

    PubMed Central

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

    2015-01-01

    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 (1-13C-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 13CO2 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 1-13C-galactose to all children. The molar ratios of 13CO2 and 12CO2 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 13C 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. PMID:25608239

  9. 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.

  10. Effects of dietary alpha-linolenic acid on the conversion and oxidation of 13C-alpha-linolenic acid.

    PubMed

    Vermunt, S H; Mensink, R P; Simonis, M M; Hornstra, G

    2000-02-01

    The effects of a diet rich in alpha-linolenic acid vs. one rich in oleic acid on the oxidation of uniformly labeled 13C-alpha-linolenic acid and its conversion into longer-chain polyunsaturates (LCP) were investigated in vivo in healthy human subjects. Volunteers received a diet rich in oleic acid (n = 5) or a diet rich in alpha-linolenic acid (n = 7; 8.3 g/d) for 6 wk before and during the study. After 6 wk, subjects were given 45 mg of 13C-alpha-linolenic acid dissolved in olive oil. Blood samples were collected at t = 0, 5, 11, 24, 96, and 336 h. Breath was sampled and CO2 production was measured each hour for the first 12 h. The mean (+/- SEM) maximal absolute amount of 13C-eicosapentaenoic acid (EPA) in plasma total lipids was 0.04 +/- 0.01 mg in the alpha-linolenic acid group, which was significantly lower (P = 0.01) than the amount of 0.12 +/- 0.03 mg 13C-EPA in the oleic acid group. Amounts of 13C-docosapentaenoic acid (DPA) and 13C-docosahexaenoic acid (DHA) tended to be lower as well. The mean proportion of labeled alpha-linolenic acid (ALA) recovered as 13CO2 in breath after 12 h was 20.4% in the ALA and 15.7% in the oleic acid group, which was not significantly different (P = 0.12). The cumulative recovery of 13C from 13C-ALA in breath during the first 12 h was negatively correlated with the maximal amounts of plasma 13C-EPA (r = -0.58, P = 0.047) and 13C-DPA (r = -0.63, P = 0.027), but not of 13C-DHA (r = -0.49, P = 0.108). In conclusion, conversion of 13C-ALA into its LCP may be decreased on diets rich in ALA, while oxidation of 13C-ALA is negatively correlated with its conversion into LCP. In a few pilot samples, low 13C enrichments of n-3 LCP were observed in a diet rich in EPA/DHA as compared to oleic acid.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Visual detection of melamine in milk samples based on label-free and labeled gold nanoparticles.

    PubMed

    Huang, Hui; Li, Li; Zhou, Guohua; Liu, Zhihong; Ma, Qiao; Feng, Yuqi; Zeng, Guoping; Tinnefeld, Philip; He, Zhike

    2011-08-15

    Melamine that can cause serious damage to the organs of animal or human beings was found to bind to polythymine via hydrogen bonding. With this novel discovery, colorimetric detection of melamine based on label-free and labeled gold nanoparticles was developed, respectively. Both of the methods revealed good selectivity for melamine over other components that may exist in milk and good anti-influence ability. The raw milk samples were pretreated according to the National standard method combined with a solid phase extraction monolithic column. The accurate quantification of melamine as low as 41.7 nM and 46.5 nM was obtained, respectively. It also guarantees fast and reliable readout with naked eyes, making visual detection possible. Further comparison between label-free and labeled based methods was discussed in this paper. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. 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

  17. 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.

  18. 16 CFR 1702.13 - Labeling and packaging samples.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Labeling and packaging samples. 1702.13 Section 1702.13 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION POISON PREVENTION PACKAGING ACT OF 1970 REGULATIONS PETITIONS FOR EXEMPTIONS FROM POISON PREVENTION PACKAGING ACT REQUIREMENTS;...

  19. 16 CFR 1702.13 - Labeling and packaging samples.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 2 2012-01-01 2012-01-01 false Labeling and packaging samples. 1702.13 Section 1702.13 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION POISON PREVENTION PACKAGING ACT OF 1970 REGULATIONS PETITIONS FOR EXEMPTIONS FROM POISON PREVENTION PACKAGING ACT REQUIREMENTS;...

  20. 16 CFR 1702.13 - Labeling and packaging samples.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 2 2011-01-01 2011-01-01 false Labeling and packaging samples. 1702.13 Section 1702.13 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION POISON PREVENTION PACKAGING ACT OF 1970 REGULATIONS PETITIONS FOR EXEMPTIONS FROM POISON PREVENTION PACKAGING ACT REQUIREMENTS;...

  1. 16 CFR 1702.13 - Labeling and packaging samples.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 16 Commercial Practices 2 2013-01-01 2013-01-01 false Labeling and packaging samples. 1702.13 Section 1702.13 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION POISON PREVENTION PACKAGING ACT OF 1970 REGULATIONS PETITIONS FOR EXEMPTIONS FROM POISON PREVENTION PACKAGING ACT REQUIREMENTS;...

  2. 16 CFR 1702.13 - Labeling and packaging samples.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 2 2014-01-01 2014-01-01 false Labeling and packaging samples. 1702.13 Section 1702.13 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION POISON PREVENTION PACKAGING ACT OF 1970 REGULATIONS PETITIONS FOR EXEMPTIONS FROM POISON PREVENTION PACKAGING ACT REQUIREMENTS;...

  3. Automated determination of the stable carbon isotopic composition (δ13C) of total dissolved inorganic carbon (DIC) and total nonpurgeable dissolved organic carbon (DOC) in aqueous samples: RSIL lab codes 1851 and 1852

    USGS Publications Warehouse

    Révész, Kinga M.; Doctor, Daniel H.

    2014-01-01

    The purposes of the Reston Stable Isotope Laboratory (RSIL) lab codes 1851 and 1852 are to determine the total carbon mass and the ratio of the stable isotopes of carbon (δ13C) for total dissolved inorganic carbon (DIC, lab code 1851) and total nonpurgeable dissolved organic carbon (DOC, lab code 1852) in aqueous samples. The analysis procedure is automated according to a method that utilizes a total carbon analyzer as a peripheral sample preparation device for analysis of carbon dioxide (CO2) gas by a continuous-flow isotope ratio mass spectrometer (CF-IRMS). The carbon analyzer produces CO2 and determines the carbon mass in parts per million (ppm) of DIC and DOC in each sample separately, and the CF-IRMS determines the carbon isotope ratio of the produced CO2. This configuration provides a fully automated analysis of total carbon mass and δ13C with no operator intervention, additional sample preparation, or other manual analysis. To determine the DIC, the carbon analyzer transfers a specified sample volume to a heated (70 °C) reaction vessel with a preprogrammed volume of 10% phosphoric acid (H3PO4), which allows the carbonate and bicarbonate species in the sample to dissociate to CO2. The CO2 from the reacted sample is subsequently purged with a flow of helium gas that sweeps the CO2 through an infrared CO2 detector and quantifies the CO2. The CO2 is then carried through a high-temperature (650 °C) scrubber reactor, a series of water traps, and ultimately to the inlet of the mass spectrometer. For the analysis of total dissolved organic carbon, the carbon analyzer performs a second step on the sample in the heated reaction vessel during which a preprogrammed volume of sodium persulfate (Na2S2O8) is added, and the hydroxyl radicals oxidize the organics to CO2. Samples containing 2 ppm to 30,000 ppm of carbon are analyzed. The precision of the carbon isotope analysis is within 0.3 per mill for DIC, and within 0.5 per mill for DOC.

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

    USDA-ARS?s Scientific Manuscript database

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

  5. 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

  6. 2D DIGE saturation labeling for minute sample amounts.

    PubMed

    Arnold, Georg J; Fröhlich, Thomas

    2012-01-01

    The 2D DIGE technique, based on fluorophores covalently linked to amino acid side chain residues and the concept of an internal standard, has significantly improved reproducibility, sensitivity, and the dynamic range of protein quantification. In saturation DIGE, sulfhydryl groups of cysteines are labeled with cyanine dyes to completion, providing a so far unraveled sensitivity for protein detection and quantification in 2D gel-based proteomic experiments. Only a few micrograms of protein per 2D gel facilitate the analysis of about 2,000 analytes from complex mammalian cell or tissue samples. As a consequence, 2D saturation DIGE is the method of choice when only minute sample amounts are available for quantitative proteome analysis at the level of proteins rather than peptides. Since very low amounts of samples have to be handled in a reproducible manner, saturation DIGE-based proteomic experiments are technically demanding. Moreover, successful saturation DIGE approaches require a strict adherence to adequate reaction conditions at each step. This chapter is dedicated to colleagues already experienced in 2D PAGE protein separation and intends to support the establishment of this ultrasensitive technique in proteomic workgroups. We provide basic guidelines for the experimental design and discuss crucial aspects concerning labeling chemistry, sample preparation, and pitfalls caused by labeling artifacts. A detailed step-by-step protocol comprises all aspects from initial sample preparation to image analysis and statistical evaluation. Furthermore, we describe the generation of preparative saturation DIGE gels necessary for mass spectrometry-based spot identification.

  7. 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. © 2013 Elsevier Inc. All rights reserved.

  8. 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

  9. (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.

  10. Simultaneous determination of stable isotopic compositions of nitrous oxide (δ15N and δ18O of N2O) and methane (δ13C of CH4) in nanomolar quantities from a single water sample

    NASA Astrophysics Data System (ADS)

    Hirota, A.; Tsunogai, U.; Komatsu, D. D.; Nakagawa, F.

    2010-12-01

    The stable isotopic compositions of nitrous oxide (δ15N of N2O and δ18O of N2O, respectively) and methane (δ13C of CH4) have provided us with some interesting geochemical insights. We have developed a rapid, sensitive, and automated analytical system to simultaneously determine the concentrations and stable isotopic compositions of nanomolar quantities of N2O and CH4 in the environmental water, by combining continuous-flow isotope-ratio mass spectrometry and a He-sparging system to extract and purify the dissolved gases. Our system, which is composed of a sparging bottle, a chemical trap, four cold traps and a capillary gas chromatograph that use ultra-pure helium as the carrier gas, achieves complete extraction of N2O and CH4 in a water sample and separation among N2O, CH4, and the other component gases. The flow path subsequent to gas chromatograph was periodically changed to pass the gases through the combustion furnace to convert CH4 and the other hydrocarbons into CO2, or to bypass the combustion furnace for the direct introduction of eluted N2O into the mass spectrometer, for determining the stable isotopic compositions through monitoring m/z = 44, 45, and 46, on the bases of CO2+ and N2O+, respectively. The analytical system can be operated automatically with sequential software programmed on a personal computer. The analytical precisions (the standard deviation of a single measurement) were better than 0.2‰ for δ15N of N2O and 0.3‰ for δ18O of N2O, in the case of more than 6.7 nmol N2O injection and better than 1.4‰ for δ15N of N2O and 2.6‰ for δ18O of N2O, in the case of more than 0.2 nmol N2O injection, respectively. Simultaneously, the analytical precisions were better than 0.07‰ for δ13C of CH4, in the case of more than 5.5 nmol CH4 infection and better than 2.1‰ for δ13C of CH4, when more than 0.024 nmol CH4 injection. In this manner, we can simultaneously determine stable isotopic compositions of a 120 mL water sample having

  11. Notes on the origin of copromacrinite based on nitrogen functionalities and δ13C and δ15N determined on samples from the Peach Orchard coal bed, southern Magoffin County, Kentucky

    USGS Publications Warehouse

    Valentim, Bruno; Algarra, Manuel; Guedes, Alexandra; Ruppert, Leslie F.; Hower, James C.

    2016-01-01

    The study of Peach Orchard coal samples using reflected-light microscopy, isotopic composition, and nitrogen-forms analyses revealed that the macrinite-rich sample contains macrinite with coprolitic features (e.g. oxidation rind, mix of undigested palynomorphs, frequent and randomly located funginite, agglutination pulp of semifusinite reflectance, internal lack of bedding fabric, and suggestion of structures resulting from intestines and stomach walls), more pyrrolic-N (~ 16%), and lower δ13C (~ 2‰ VPDB) and δ15N (~ 4‰ Air) values than the vitrinite and semifusinite + fusinite rich samples. These findings suggest that the maceral macrinite has multiple origins based on petrography and measurable chemical differences between the macrinite, vitrinite, and semifusinite + fusinite fractions within the coal. Assuming that copromacrinite observed is an excretion then the anomalies observed may result from the symbiotic relations between the macrofauna (e.g. cockroaches) and microbiota during the digestive processes, and the nitrogen balance mechanisms inside macrofauna body.

  12. Quantification of isotope-labeled and unlabeled folates and folate catabolites in urine samples by stable isotope dilution assay.

    PubMed

    Büttner, Barbara E; Ohrvik, Veronica E; Köhler, Peter; Witthöft, Cornelia M; Rychlik, Michael

    2013-01-01

    Dual-label stable isotope dilution assays for the simultaneous quantification of isotopologic folates in clinical samples offer the perspective for differentiating between unlabeled folates from endogenous body pools and administered [13C5]-labeled folates from a test dose when performing bioavailability trials. In contrast to intact folates, this methodology could hitherto not be applied to the quantification of the folate catabolites, p-aminobenzoyl glutamate and p-acetamidobenzoyl glutamate. In this study, [2H4]-p-aminobenzoyl glutamate, [2H4]-p-acetamidobenzoyl glutamate, and unlabeled p-acetamidobenzoyl glutamate were synthesized. The synthesis of the [2H4]-labeled compounds started at unlabeled p-aminobenzoic acid. For the formation of p-acetamidobenzoyl glutamate, p-aminobenzoyl glutamate was acetylated. The new substances were applied successfully in stable isotope dilution assays for the simultaneous quantification of the [13C5]-labeled and unlabeled folate catabolites, p-aminobenzoyl glutamate and p-acetamidobenzoyl glutamate, along with the predominant folate vitamers in urine. The assays were based on clean-up by strong anion exchange followed by liquid chromatography-tandem mass spectrometry detection. Assay sensitivity was sufficient to detect the folate catabolites in physiologic concentrations. The limit of detection was below 0.4 and 0.3 nmol/100 g for p-aminobenzoyl glutamate isotopologues and p-acetamidobenzoyl glutamate isotopologues in urine, respectively. The successful synthesis of [2H4]-p-aminobenzoyl glutamate, [2H4]-p-acetamidobenzoyl glutamate, and unlabeled p-acetamidobenzoyl glutamate and the implementation of these substances in stable isotope dilution assays allows dual-label designs that provide a more detailed insight into human folate metabolism.

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

    SciTech Connect

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

    1981-03-01

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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  19. 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.

  20. Evaluation of ¹³C- and ²H-labeled internal standards for the determination of amphetamines in biological samples, by reversed-phase ultra-high performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Berg, Thomas; Karlsen, Morten; Oiestad, Ase Marit Leere; Johansen, Jon Eigill; Liu, Huiling; Strand, Dag Helge

    2014-05-30

    Stable isotope-labeled internal standards (SIL-ISs) are often used when applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze for legal and illegal drugs. ISs labeled with (13)C, (15)N, and (18)O are expected to behave more closely to their corresponding unlabeled analytes, compared with that of the more classically used (2)H-labeled ISs. This study has investigated the behavior of amphetamine, (2)H3-, (2)H5, (2)H6-, (2)H8-, (2)H11-, and (13)C6-labeled amphetamine, during sample preparation by liquid-liquid extraction and LC-MS/MS analyses. None or only minor differences in liquid-liquid extraction recoveries of amphetamine and the SIL-ISs were observed. The chromatographic resolution between amphetamine and the (2)H-labeled amphetamines increased with the number of (2)H-substitutes. For chromatographic studies we also included seven additional (13)C6-amphetamines and their analytes. All the (13)C6-labeled ISs were co-eluting with their analytes, both when a basic and when an acidic mobile phase were used. MS/MS analyses of amphetamine and its SIL-ISs showed that the ISs with the highest number of (2)H-substitutes required more energy for fragmentation in the collision cell compared with that of the ISs with a lower number. The findings, in this study, support those of previous studies, showing that (13)C-labeled ISs are superior to (2)H-labeled ISs, for analytical purposes.

  1. Kinetics of 13C-DHA before and during fish-oil supplementation in healthy older individuals.

    PubMed

    Plourde, Mélanie; Chouinard-Watkins, Raphaël; Rioux-Perreault, Christine; Fortier, Mélanie; Dang, Marie Thuy Mai; Allard, Marie-Julie; Tremblay-Mercier, Jennifer; Zhang, Ying; Lawrence, Peter; Vohl, Marie-Claude; Perron, Patrice; Lorrain, Dominique; Brenna, J Thomas; Cunnane, Stephen C

    2014-07-01

    Docosahexaenoic acid (DHA) kinetics appear to change with intake, which is an effect that we studied in an older population by using uniformly carbon-13-labeled DHA ((13)C-DHA). We evaluated the influence of a fish-oil supplement over 5 mo on the kinetics of (13)C-DHA in older persons. Thirty-four healthy, cognitively normal participants (12 men, 22 women) aged between 52 and 90 y were recruited. Two identical kinetic studies were performed, each with the use of a single oral dose of 40 mg (13)C-DHA. The first kinetic study was performed before participants started taking a 5-mo supplementation that provided 1.4 g DHA/d plus 1.8 g eicosapentaenoic acid (EPA)/d (baseline); the second study was performed during the final month of supplementation (supplement). In both kinetic studies, blood and breath samples were collected ≤8 h and weekly over 4 wk to analyze (13)C enrichment. The time × supplement interaction for (13)C-DHA in the plasma was not significant, but there were separate time and supplement effects (P < 0.0001). The area under the curve for plasma (13)C-DHA was 60% lower while subjects were taking the supplement than at baseline (P < 0.0001). The uniformly carbon-13-labeled EPA concentration was 2.6 times as high 1 d posttracer while patients were taking the supplement as it was at baseline. The mean (±SEM) plasma (13)C-DHA half-life was 4.5 ± 0.4 d at baseline compared with 3.0 ± 0.2 d while taking the supplement (P < 0.0001). Compared with baseline, the mean whole-body half-life was 61% lower while subjects were taking the supplement. The loss of (13)C-DHA through β-oxidation to carbon dioxide labeled with carbon-13 increased from 0.085% of dose/h at baseline to 0.208% of dose/h while subjects were taking the supplement. In older persons, a supplement of 3.2 g EPA + DHA/d increased β-oxidation of (13)C-DHA and shortened the plasma (13)C-DHA half-life. Therefore, when circulating concentrations of EPA and DHA are increased, more DHA is available

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

    NASA Astrophysics Data System (ADS)

    Kanamori, Keiko; Ross, Brian D.

    2001-12-01

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

  3. 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

  4. Study of molecular interactions with 13C DNP-NMR

    NASA Astrophysics Data System (ADS)

    Lerche, Mathilde H.; Meier, Sebastian; Jensen, Pernille R.; Baumann, Herbert; Petersen, Bent O.; Karlsson, Magnus; Duus, Jens Ø.; Ardenkjær-Larsen, Jan H.

    2010-03-01

    NMR spectroscopy is an established, versatile technique for the detection of molecular interactions, even when these interactions are weak. Signal enhancement by several orders of magnitude through dynamic nuclear polarization alleviates several practical limitations of NMR-based interaction studies. This enhanced non-equilibrium polarization contributes sensitivity for the detection of molecular interactions in a single NMR transient. We show that direct 13C NMR ligand binding studies at natural isotopic abundance of 13C gets feasible in this way. Resultant screens are easy to interpret and can be performed at 13C concentrations below μM. In addition to such ligand-detected studies of molecular interaction, ligand binding can be assessed and quantified with enzymatic assays that employ hyperpolarized substrates at varying enzyme inhibitor concentrations. The physical labeling of nuclear spins by hyperpolarization thus provides the opportunity to devise fast novel in vitro experiments with low material requirement and without the need for synthetic modifications of target or ligands.

  5. 9 CFR 327.12 - Foreign canned or packaged products bearing trade labels; sampling and inspection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... bearing trade labels; sampling and inspection. 327.12 Section 327.12 Animals and Animal Products FOOD....12 Foreign canned or packaged products bearing trade labels; sampling and inspection. (a) Samples of foreign canned or packaged products bearing on their immediate containers trade labels which have not been...

  6. 9 CFR 327.12 - Foreign canned or packaged products bearing trade labels; sampling and inspection.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... bearing trade labels; sampling and inspection. 327.12 Section 327.12 Animals and Animal Products FOOD....12 Foreign canned or packaged products bearing trade labels; sampling and inspection. (a) Samples of foreign canned or packaged products bearing on their immediate containers trade labels which have not been...

  7. 9 CFR 327.12 - Foreign canned or packaged products bearing trade labels; sampling and inspection.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... bearing trade labels; sampling and inspection. 327.12 Section 327.12 Animals and Animal Products FOOD....12 Foreign canned or packaged products bearing trade labels; sampling and inspection. (a) Samples of foreign canned or packaged products bearing on their immediate containers trade labels which have not...

  8. Measurement of (13) C turnover into glutamate and glutamine pools in brain tumor patients.

    PubMed

    Pichumani, Kumar; Mashimo, Tomoyuki; Vemireddy, Vamsidhara; Ijare, Omkar B; Mickey, Bruce E; Malloy, Craig R; Marin-Valencia, Isaac; Baskin, David S; Bachoo, Robert M; Maher, Elizabeth A

    2017-09-30

    Malignant brain tumors are known to utilize acetate as an alternate carbon source in the citric acid cycle for their bioenergetics. (13) C NMR based isotopomer analysis has been used to measure turnover of (13) C-acetate carbons into glutamate and glutamine pools in tumors. Plasma from the patients infused with [1,2-(13) C]acetate further revealed the presence of (13) C isotopomers of glutamine, glucose and lactate in the circulation that were generated due to metabolism of [1,2-(13) C]acetate by peripheral organs. In the tumor cells, [4-(13) C] and [3,4-(13) C] glutamate and glutamine isotopomers were generated from blood-borne (13) C labeled glucose and lactate which were formed due to [1,2-(13) C[acetate metabolism of peripheral tissues. [4,5-(13) C] and [3,4,5-(13) C] glutamate and glutamine isotopomers were produced from [1,2-(13) C]acetyl-CoA that were derived from direct oxidation of [1,2-(13) C]acetate in the tumor This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  9. Enantioselective syntheses of alpha-Fmoc-Pbf-[2-(13)C]-L-arginine and Fmoc-[1,3-(13)C2]-L-proline and Incorporation into the neurotensin receptor 1 ligand, NT(8-13).

    PubMed

    Song, Chuanjun; Tapaneeyakorn, Satita; Murphy, Annabel C; Butts, Craig; Watts, Anthony; Willis, Christine L

    2009-12-04

    Enantioselective syntheses of selectively labeled, orthogonally protected [2-(13)C]-L-arginine and [1,3-(13)C(2)]-L-proline are described from the commercially available precursors [2-(13)C]bromoacetic acid and potassium [(13)C]cyanide. Interestingly the enhanced signal assigned to C-2 in the (13)C NMR spectrum of alpha-Fmoc-Pbf-[2-(13)C]-L-arginine was very broad at room temperature. The two Fmoc-labeled amino acids were used to prepare [2-(13)C]-Arg9 and [1,3-(13)C(2)]-Pro10 labeled ligand (NT(8-13)) by manual Fmoc-SPSS.

  10. 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

  11. Probing site-specific 13C/15N-isotope enrichment of spider silk with liquid-state NMR spectroscopy.

    PubMed

    Shi, Xiangyan; Yarger, Jeffery L; Holland, Gregory P

    2013-05-01

    Solid-state nuclear magnetic resonance (NMR) has been extensively used to elucidate spider silk protein structure and dynamics. In many of these studies, site-specific isotope enrichment is critical for designing particular NMR methods for silk structure determination. The commonly used isotope analysis techniques, isotope-ratio mass spectroscopy and liquid/gas chromatography-mass spectroscopy, are typically not capable of providing the site-specific isotope information for many systems because an appropriate sample derivatization method is not available. In contrast, NMR does not require any sample derivatization or separation prior to analysis. In this article, conventional liquid-state (1)H NMR was implemented to evaluate incorporation of (13)C/(15)N-labeled amino acids in hydrolyzed spider dragline silk. To determine site-specific (13)C and (15)N isotope enrichments, an analysis method was developed to fit the (1)H-(13)C and (1)H-(15)N J-splitting (J CH and J NH) (1)H NMR peak patterns of hydrolyzed silk fiber. This is demonstrated for Nephila clavipes spiders, where [U-(13)C3,(15)N]-Ala and [1-(13)C,(15)N]-Gly were dissolved in their water supplies. Overall, contents for Ala and Gly isotopomers are extracted for these silk samples. The current methodology can be applied to many fields where site-specific tracking of isotopes is of interest.

  12. Simultaneous measurement of 13C- and 15N-isotopic enrichments of threonine by mass spectrometry.

    PubMed

    Godin, Jean-Philippe; Mermoud, Anne-France; Rémond, Didier; Faure, Magali; Breuille, Denis; Williamson, Gary; Peré-Trepat, Emma; Ramadan, Ziad; Fay, Laurent-Bernard; Kochhar, Sunil

    2009-04-01

    Under conditions of high isotopic dilution, e.g. in a tracer study, the ability to determine accurately and quantitatively small variations in isotopic enrichments of differently labelled chemical compounds (e.g. (13)C and (15)N in threonine) in a single run by gas chromatography/mass spectrometry (GC/MS) is desirable but remains a technological challenge. Here, we report a new, rapid and simple GC/MS method for simultaneously measuring the isotopic enrichments of doubly labelled threonine ([U(13)C] and (15)N) with isotopic enrichment lower than 1.5 Molar Percent Excess (MPE). The long-term reproducibility measured was around 0.09 MPE for both tracers (throughout a 6 week period). The intra-day repeatability was lower than 0.05 and 0.06 MPE for [U(13)C]-Thr and (15)N-Thr, respectively. To calculate both isotopic enrichments, two modes of calculations were used: one based on work by Rosenblatt et al. in 1992 and the other one using a matrix approach. Both methods gave similar results (ANOVA, P >0.05) with close precision for each mode of calculation. The GC/MS method was then used to investigate the differential utilization of threonine in different organs according to its route of administration in minipigs after administration of both tracers. In plasma samples, the lowest isotopic enrichment measured between two successive time points was at 0.01 and 0.02 MPE for [U(13)C]-Thr and (15)N-Thr, respectively. Moreover, the accuracy of GC/MS (13)C-isotopic enrichment measured was valida