13C-Tracer Experiments in DIII-D Preliminary to Thermal Oxidation Experiments to Understand Tritium Recovery in DIII-D, JET, C-Mod, and MAST

International Nuclear Information System (INIS)

Stangeby, P.; Allen, S.; Bekris, N.; Brooks, N.; Christie, K.; Chrobak, C.; Coad, J.; Counsell, G.; Davis, J.; Elder, J.; Fenstermacher, M.; Groth, M.; Haasz, A.; Likonen, J.; Lipschultz, B.; McLean, A.; Philipps, V.; Porter, G.; Rudakov, D.; Shea, J.; Wampler, W.; Watkins, J.; West, W.; Whyte, D.

2006-01-01

Retention of tritium in carbon co-deposits is a serious concern for ITER. Developing a reliable in-situ removal method of the co-deposited tritium would allow the use of carbon plasma-facing components which have proven reliable in high heat flux conditions and compatible with high performance plasmas. Thermal oxidation is a potential solution, capable of reaching even hidden locations. It is necessary to establish the least severe conditions to achieve adequate tritium recovery, minimizing damage and reconditioning time. The first step in this multi-machine project is 13 C-tracer experiments in DIII-D, JET, C-Mod and MAST. In DIII-D and JET, 13 CH 4 has been (and in C-Mod and MAST, will be) injected toroidally symmetrically, facilitating quantification and interpretation of the results. Tiles have been removed, analyzed for 13 C content and will next be evaluated in a thermal oxidation test facility in Toronto with regard to the ability of different severities of oxidation exposure to remove the different types of (known and measured) 13 C co-deposit. Removal of D/T from B on Mo tiles from C-Mod will also be tested. OEDGE interpretive code analysis of the 13 C deposition patterns is used to generate the understanding needed to apply findings to ITER. First results are reported here for the 13 C injection experiments IN DIII-D

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

Science.gov (United States)

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.

Changes in Lignin and Polysaccharide Components in 13 Cultivars of Rice Straw following Dilute Acid Pretreatment as Studied by Solution-State 2D 1H-13C NMR

Science.gov (United States)

Teramura, Hiroshi; Sasaki, Kengo; Oshima, Tomoko; Aikawa, Shimpei; Matsuda, Fumio; Okamoto, Mami; Shirai, Tomokazu; Kawaguchi, Hideo; Ogino, Chiaki; Yamasaki, Masanori; Kikuchi, Jun; Kondo, Akihiko

2015-01-01

A renewable raw material, rice straw is pretreated for biorefinery usage. Solution-state two-dimensional (2D) 1H-13 C hetero-nuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy, was used to analyze 13 cultivars of rice straw before and after dilute acid pretreatment, to characterize general changes in the lignin and polysaccharide components. Intensities of most (15 of 16) peaks related to lignin aromatic regions, such as p-coumarate, guaiacyl, syringyl, p-hydroxyphenyl, and cinnamyl alcohol, and methoxyl, increased or remained unchanged after pretreatment. In contrast, intensities of most (11 of 13) peaks related to lignin aliphatic linkages or ferulate decreased. Decreased heterogeneity in the intensities of three peaks related to cellulose components in acid-insoluble residues resulted in similar glucose yield (0.45–0.59 g/g-dry biomass). Starch-derived components showed positive correlations (r = 0.71 to 0.96) with glucose, 5-hydroxymethylfurfural (5-HMF), and formate concentrations in the liquid hydrolysates, and negative correlations (r = –0.95 to –0.97) with xylose concentration and acid-insoluble residue yield. These results showed the fate of lignin and polysaccharide components by pretreatment, suggesting that lignin aromatic regions and cellulose components were retained in the acid insoluble residues and starch-derived components were transformed into glucose, 5-HMF, and formate in the liquid hydrolysate. PMID:26083431

Utilization of low molecular weight organics by soil microorganisms: combination of 13C-labelling with PLFA analysis

Science.gov (United States)

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

(13)C/(12)C isotope ratios of organic acids, glucose and fructose determined by HPLC-co-IRMS for lemon juices authenticity.

Science.gov (United States)

Guyon, Francois; Auberger, Pauline; Gaillard, Laetita; Loublanches, Caroline; Viateau, Maryse; Sabathié, Nathalie; Salagoïty, Marie-Hélène; Médina, Bernard

2014-03-01

High performance liquid chromatography linked to isotope ratio mass spectrometry via an interface allowing the chemical oxidation of organic matter (HPLC-co-IRMS) was used to simultaneously determine carbon 13 isotope ratio (δ(13)C) of organic acids, glucose and fructose in lime and lemon juices. Because of the significant difference between organic acids and sugars concentrations, the experimental protocol was optimised by applying a "current jump" to the IRMS device. The filament current is increased of 300μA during elution in order to enhance IRMS sensitivity. Then, analysis were performed on 35 lemon and lime fruits from various geographical origins and squeezed in the laboratory. An overall average δ(13)C values of -25.40±1.62‰, -23.83±1.82‰ and -25.67±1.72‰ is found for organic acids mixture mainly made up of citric acid, glucose and fructose, respectively. These authentic samples allowed the definition of a confidence domain to which have been confronted 30 commercial juices (24 "pure juices" and 6 coming from concentrate). Among these 30 samples, 10 present δ(13)C values outside the defined range revealing an added "C4" type organic acids or sugars, addition not specified on the label that is not in agreement with EU regulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Syntheses of DL-[2-13C]leucine and its use in the preparation of [3-DL-[2-13C]leucine]oxytocin and [8-DL-[2-13C]leucine]oxytocin

International Nuclear Information System (INIS)

Viswanatha, V.; Larsen, B.; Hruby, V.J.

1979-01-01

DL-[2- 13 C]Leucine was prepared by condensing the sodium salt of ethyl acetamido-[2- 13 C]cyanoacetate with isobutylbromide in hexamethylphosphoroustriamide followed by acid hydrolysis. N-BOC-DL-[2- 13 C]Leucine was prepared and incorporated into [8-DL-[2- 13 C]leucine]oxytocin by total synthesis. The 13 C-labeled hormone derivative [8-[2- 13 C]leucine]oxytocin was separated from its 8-position diastereoisomer by partition chromatography. The specifically 13 C-labeled peptide hormone diastereoisomeric analog [3-DL-[2- 13 C]leucine]oxytocin also was prepared by solid phase peptide synthesis. No suitable solvent system for partition chromatography separation of the latter diastereoisomeric peptide mixture could be found. However an excellent preparative separation of the diastereoisomers could be obtained by reverse phase high pressure liquid chromatography on a partisil 10 M9 ODS column using the solvent system 0.05 M ammonium acetate (pH 4.0), acetonitrile (81:19, v/v) to give pure [3-[2- 13 C]leucine]oxytocin and [3-D-[2- 13 C]leucine]oxytocin. An excellent separation of [8-[2- 13 C]leucine]oxytocin and the corresponding delata-D-leucine diastereoisomer derivative could also be accomplished by high pressure liquid chromatography. (author)

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

Science.gov (United States)

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

2015-10-01

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

Synthesis of deuterium labeled perillyl alcohol and dual C-13 and deuterium labeled perillic acid, major metabolites of d-limonene

International Nuclear Information System (INIS)

Chen, Haitao; Chan, K.C.

1997-01-01

Dual C-13 and deuterium labeled perillic acid, [(1,1-dideuterio-1- 13 C-2-methyl)ethenyl]-1-cyclohexene -1-carboxylic acid (6) and deuterated perillyl alcohol, [(2,2-dideuterio-1-methyl)ethenyl]-1-deuteriohydroxymethyl-1-cyclo -hexene (9) were synthesized from commercially available (4S)-(-)-perillaidehyde (1). Compound 1 was first protected with ethylene glycol to yield the ethylene ketal followed by oxidation with OsO 4 /NalO 4 to cleave the terminal double bond to afford the key intermediate ketone, 4-acetyl-1-cyclohexene-1-carboxaldehyde ethylene ketal (3). 3 was then converted to the labeled perillyl aldehyde by Wittig reaction with prepared Ph 3 P 13 CD 3 l or Ph 3 PCD 3 l. Followed by deprotection to give the labeled perillaldehydes, [(2,2-dideuterio-2- 13 C-1-methyl)ethenyl] -1-cyclohexene-1-carboxaldehyde-1-carboxaldehyde (5) or [(2,2-dideuterio-1-methyl)ethenyl] -1-cyclohexene-1-carboxaldehyde (8). 5 was further oxidized by freshly prepared Ag 2 O to give the desired compound 6. 8 was reduced by LiAID 4 to afford the desired compound 9. The same synthetic procedure may be adopted to synthesize the radioactive isotope labeled perillic acid and perilly alcohol. (author)

An in Situ NMR Study of the Mechanism for the Catalytic Conversion of Fructose to 5-Hydroxymethylfurfural and then to Levulinic Acid Using ¹³ C Labeled d -Fructose

Energy Technology Data Exchange (ETDEWEB)

Zhang, Jing [Department of Chemistry and Institute for Atom Efficient; Weitz, Eric [Department of Chemistry and Institute for Atom Efficient

2012-04-26

The pathways for the formation of 5-hydroxymethylfurfural (HMF) by dehydration of d-fructose and for the formation of levulinic acid and formic acid from HMF by rehydration were investigated by in situ13C and 1H NMR using both unlabeled and 13C-labeled fructose. Water or DMSO was used as the solvent with Amberlyst 70, PO43–/niobic acid, or sulfuric acid as catalysts. Only HMF is observed using NMR for fructose dehydration in DMSO with any of the three catalysts or without a catalyst. For each system, results with 13C-labeled fructose indicate that the first carbon (C-1) or sixth carbon (C-6) of fructose maps onto the corresponding carbons of HMF. For fructose dehydration in H2O with a PO43–/niobic acid catalyst, in addition to HMF, furfural was observed as a product. However, we show that furfural is not a reaction product deriving from HMF under our conditions. Rather our data indicate that there is a parallel reaction pathway open to fructose when the reaction takes place in H2O with a PO43–/niobic acid catalyst. The corresponding 13C-labeled results show that the first carbon in fructose maps onto the first carbon (aldehyde carbon) in furfural. Using 13C-enriched HMF formed from dehydration of 13C-labeled fructose in DMSO or H2O, we investigated the pathway for HMF rehydration to levulinic and formic acid. The data in different solvents and with different catalysts are consistent with a common mechanism for HMF rehydration, which results in the C-1 and C-6 carbon of HMF being transformed to the carbon of formic acid and methyl carbon (C-5) of levulinic acid, respectively.

Identifying inter-residue resonances in crowded 2D {sup 13}C-{sup 13}C chemical shift correlation spectra of membrane proteins by solid-state MAS NMR difference spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Miao Yimin; Cross, Timothy A. [Florida State University, Department of Chemistry and Biochemistry (United States); Fu Riqiang, E-mail: rfu@magnet.fsu.edu [National High Magnet Field Lab (United States)

2013-07-15

The feasibility of using difference spectroscopy, i.e. subtraction of two correlation spectra at different mixing times, for substantially enhanced resolution in crowded two-dimensional {sup 13}C-{sup 13}C chemical shift correlation spectra is presented. With the analyses of {sup 13}C-{sup 13}C spin diffusion in simple spin systems, difference spectroscopy is proposed to partially separate the spin diffusion resonances of relatively short intra-residue distances from the longer inter-residue distances, leading to a better identification of the inter-residue resonances. Here solid-state magic-angle-spinning NMR spectra of the full length M2 protein embedded in synthetic lipid bilayers have been used to illustrate the resolution enhancement in the difference spectra. The integral membrane M2 protein of Influenza A virus assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH and is essential for the viral lifecycle. Based on known amino acid resonance assignments from amino acid specific labeled samples of truncated M2 sequences or from time-consuming 3D experiments of uniformly labeled samples, some inter-residue resonances of the full length M2 protein can be identified in the difference spectra of uniformly {sup 13}C labeled protein that are consistent with the high resolution structure of the M2 (22-62) protein (Sharma et al., Science 330(6003):509-512, 2010)

Rapid measurement of 13C-enrichment of acetic, propionic and butyric acids in plasma with solid phase microextraction coupled to gas chromatography-mass spectrometry

International Nuclear Information System (INIS)

Moreau, N.M.; Delepee, R.; Maume, D.; Le Bizec, B.; Nguyen, P.G.; Champ, M.M.; Martin, L.J.; Dumon, H.J.

2004-01-01

An analytical procedure based on solid phase microextraction (SPME) has been developed to quantify [1- 13 C]-labelled short-chain fatty acids (SCFAs)--mainly acetic, propionic and butyric acids--in a small volume (120 μl) of deproteinised plasma (corresponding to 200 μl of raw plasma) by gas chromatography-mass spectrometry (GC-MS) analysis. Simultaneous SCFA extraction was optimal after 5 min using a 75 μm Carboxen/polydimethylsiloxane-coated fiber. The base peak of the three analytes has been characterised by middle-resolution mass spectrometry (R>6000). All these data allowed the specificity reinforcement of the measure. The validation of the method also considered the linearity and the repeatability of the [ 13 C]SCFA measurements by SPME-GC-MS. Results were linear in a range from 5 to 100 mol% of [ 13 C]SCFA enrichment and the method provided a good intra-day (R.S.D. 13 C]butyric acid) by cecal infusion before blood sampling in portal vein. Results of [1- 13 C]butyric acid enrichment showed an excellent correlation (r 2 =0.9832; n=30) with data obtained on the same samples using a previously published procedure based on diethyl extraction and derivatisation before GC-MS analyses. SPME coupled to GC-MS appears to be a powerful analytical tool for the direct isotopic measurements of low deproteinised plasma volume avoiding consequently preliminary treatment such as extraction or derivatisation. The presented method could be of great interest for real time [ 13 C]SCFA plasma determination of in metabolic in vivo studies in small animal models

13C-NMR assignment, structure, and dynamics of deoxyoligonucleotides

International Nuclear Information System (INIS)

Zanatta, N.; Borer, P.N.; Levy, G.C.

1986-01-01

The unique spectral properties of 13 C-NMR for studying nucleic acids and some of the important features of 13 C-NMR in oligonucleotide studies are demostrated. The main difficulty in studying oligonucleotides by 13 C-NMR and recent improvements in NMR instrumentation and advances in oligonucleotide synthesis are presented. The high resolution 13 C-NMR spectra, T 1 relaxation times and NOEs were measured for duplex of the self-complementary oligo-DNAs: d(CG) 3 and d(GGTATACC) are studied. The target of this study is to developed a systematic 13 C-NMR spectral assignment and to investigate the structure and dynamics of these two sequences by this techniques. (M.J.C.) [pt

Determination of the conformation of 2-hydroxy- and 2-aminobenzoic acid dimers using 13C NMR and density functional theory/natural bond order analysis: the central importance of the carboxylic acid carbon.

Science.gov (United States)

Burnette, Ronald R; Weinhold, Frank

2006-07-20

The 13C chemical shift for the carboxylic acid carbon provides a powerful diagnostic probe to determine the preferred isomeric dimer structures of benzoic acid derivatives undergoing intra- and intermolecular H-bonding in the gas, solution and crystalline phases. We have employed hybrid density functional calculations and natural bond orbital analysis to elucidate the electronic origins of the observed 13C shieldings and their relationship to isomeric stability. We find that delocalizing interactions from the carbonyl oxygen lone pairs (nO) into vicinal carbon-oxygen and carbon-carbon antibonds (sigmaCO*,sigmaCC*) make critical contributions to the 13C shieldings, and these nO --> sigmaCO*, nO --> sigmaCC* interactions are in turn sensitive to the intramolecular interactions that dictate dimer structure and stability. The carboxyl carbon atom can thus serve as a useful detector of subtle structural and conformational features in this pharmacologically important class of carboxylic acid interactions.

3D Hyperpolarized C-13 EPI with Calibrationless Parallel Imaging

DEFF Research Database (Denmark)

Gordon, Jeremy W.; Hansen, Rie Beck; Shin, Peter J.

2018-01-01

With the translation of metabolic MRI with hyperpolarized 13C agents into the clinic, imaging approaches will require large volumetric FOVs to support clinical applications. Parallel imaging techniques will be crucial to increasing volumetric scan coverage while minimizing RF requirements and tem...... strategies to accelerate and undersample hyperpolarized 13C data using 3D blipped EPI acquisitions and multichannel receive coils, and demonstrated its application in a human study of [1-13C]pyruvate metabolism....

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

Science.gov (United States)

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 C 6 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 C 6 'light' and 13 C 6 '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.

What is hiding behind ontogenic d13C variations in mollusk shells: New insights from scallops.

Science.gov (United States)

Chauvaud, L.; Lorrain, A.; Gillikin, D. P.; Thebault, J.; Paulet, Y.; Strand, O.; Blamart, D.; Guarini, J.; Clavier, J.

2008-12-01

We examined d13Ccalcite variations along scallop shells (Pecten maximus) sampled in Norway, France and Spain. Time series of shell calcite d13C show a consistent pattern of decreasing d13C with age. This almost linear d13C trend reflects an increasing contribution of metabolic CO2 to skeletal carbonate throughout ontogeny. We have removed this ontogenic trend to try to extract other information from our shell calcite d13C dataset. Scallops from the Bay of Brest (western Brittany, France) were then used to interpret the data as many environmental parameters were available for this site. d13Ccalcite variations were compared to d13C of dissolved inorganic carbon (DIC) and Chl a. The detrended calcite d13C profiles seem to follow a seasonal pattern, but surprisingly are inversely related to the d13C DIC and chlorophyll a concentrations measured within the water column. Theses results suggest that shell d13C variations are not controlled by isotopic variation of DIC. Since scallops eat phytoplankton and microphytobenthos cells, and, as a consequence respire organic mater largely depleted in 13C, we therefore suggest that in mollusk suspension feeders the shell d13Ccalcite might still be used to track the annual number of phytoplankton blooms when d13C values of calcite are detrended. We must consider this trend as a potential biological filter hiding precious environmental records.

Investigation of α-cluster states in 13C via the (6Li,d) reaction

CERN Document Server

Rodrigues, M R D; Horodynski-Matsushigue, L B; Cunsolo, A; Cappuzzello, F; Duarte, J L M; Rodrigues, C L; Ukita, G M; Souza, M A; Miyake, H

2010-01-01

The 9Be(6Li,d)13C reaction was used to investigate possible α-cluster states in 13C. The reaction was measured at 25.5 MeV incident energy, employing the São Paulo Pelletron-Enge-Spectrograph facility and the nuclear emulsion detection technique. Ten out of sixteen known levels of 13C, up to 11 MeV of excitation, were observed and, due to the much improved energy resolution of 50 keV, at least three doublets could be resolved. This work presents a preliminary analysis of five of the most intensely populated states, also in comparison with the results of former transfer studies.

Biosynthesis of the sesquiterpene germacrene D in Solidago canadensis: 13C and (2)H labeling studies.

Science.gov (United States)

Steliopoulos, Panagiotis; Wüst, Matthias; Adam, Klaus-Peter; Mosandl, Armin

2002-05-01

The biogenetic origin of the isoprenoid building blocks of the sesquiterpene germacrene D was studied in Solidago canadensis. Feeding experiments were carried out with 1-[5,5-D(2)]deoxy-D-xylulose-5-phosphate (D(2)-DOXP), [5-13C]mevalonolactone (13C-MVL) and [1-13C]-D-glucose. The hydrodistillate of a cut shoot fed with D(2)-DOXP was investigated by enantio-MDGC-MS and the volatile fraction of a shoot supplied with 13C-MVL was examined by GC-C-IRMS. The incorporation of [1-13C]-D-glucose was analyzed by quantitative 13C NMR spectroscopy after isolation of germacrene D from the essential oil. Our labeling studies revealed that the biosynthesis of the C-15 skeleton of sesquiterpene germacrene D in Solidago canadensis proceeds predominantly via the methylerythritol phosphate pathway.

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

Energy Technology Data Exchange (ETDEWEB)

Martinez, Rodolfo A. (Santa Fe, NM), Unkefer; Clifford J. (Los Alamos, NM), Alvarez; Marc, A [Santa Fe, NM

2012-06-12

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

Analysis of epoxyeicosatrienoic acids by chiral liquid chromatography/electron capture atmospheric pressure chemical ionization mass spectrometry using [13C]-analog internal standards

Science.gov (United States)

Mesaros, Clementina; Lee, Seon Hwa; Blair, Ian A.

2012-01-01

The metabolism of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) is thought to be mediated primarily by the cytochromes P450 (P450s) from the 2 family (2C9, 2C19, 2D6, and 2J2). In contrast, P450s of the 4 family are primarily involved in omega oxidation of AA (4A11 and 4A22). The ability to determine enantioselective formation of the regioisomeric EETs is important in order to establish their potential biological activities and to asses which P450 isoforms are involved in their formation. It has been extremely difficult to analyze individual EET enantiomers in biological fluids because they are present in only trace amounts and they are extremely difficult to separate from each other. In addition, the deuterium-labeled internal standards that are commonly used for stable isotope dilution liquid chromatography/mass spectrometry (LC/MS) analyses have different LC retention times when compared with the corresponding protium forms. Therefore, quantification by LC/MS-based methodology can be compromised by differential suppression of ionization of the closely eluting isomers. We report the preparation of [13C20]-EET analog internal standards and the use of a validated high-sensitivity chiral LC/electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the trace analysis of endogenous EETs as their pentafluorobenzyl (PFB) ester derivatives. The assay was then used to show the exquisite enantioselectivity of P4502C19-, P4502D6-, P4501A1-, and P4501B1-mediated conversion of AA into EETs and to quantify the enantioselective formation of EETs produced by AA metabolism in a mouse epithelial hepatoma (Hepa) cell line. PMID:20972997

13C Metabolic Flux Analysis for systematic metabolic engineering of S. cerevisiae for overproduction of fatty acids.

Directory of Open Access Journals (Sweden)

Amit Ghosh

2016-10-01

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

Computational analysis of KRAS mutations: implications for different effects on the KRAS p.G12D and p.G13D mutations.

Directory of Open Access Journals (Sweden)

Chih-Chieh Chen

Full Text Available BACKGROUND: The issue of whether patients diagnosed with metastatic colorectal cancer who harbor KRAS codon 13 mutations could benefit from the addition of anti-epidermal growth factor receptor therapy remains under debate. The aim of the current study was to perform computational analysis to investigate the structural implications of the underlying mutations caused by c.38G>A (p.G13D on protein conformation. METHODS: Molecular dynamics (MD simulations were performed to understand the plausible structural and dynamical implications caused by c.35G>A (p.G12D and c.38G>A (p.G13D. The potential of mean force (PMF simulations were carried out to determine the free energy profiles of the binding processes of GTP interacting with wild-type (WT KRAS and its mutants (MT. RESULTS: Using MD simulations, we observed that the root mean square deviation (RMSD increased as a function of time for the MT c.35G>A (p.G12D and MT c.38G>A (p.G13D when compared with the WT. We also observed that the GTP-binding pocket in the c.35G>A (p.G12D mutant is more open than that of the WT and the c.38G>A (p.G13D proteins. Intriguingly, the analysis of atomic fluctuations and free energy profiles revealed that the mutation of c.35G>A (p.G12D may induce additional fluctuations in the sensitive sites (P-loop, switch I and II regions. Such fluctuations may promote instability in these protein regions and hamper GTP binding. CONCLUSIONS: Taken together with the results obtained from MD and PMF simulations, the present findings implicate fluctuations at the sensitive sites (P-loop, switch I and II regions. Our findings revealed that KRAS mutations in codon 13 have similar behavior as KRAS WT. To gain a better insight into why patients with metastatic colorectal cancer (mCRC and the KRAS c.38G>A (p.G13D mutation appear to benefit from anti-EGFR therapy, the role of the KRAS c.38G>A (p.G13D mutation in mCRC needs to be further investigated.

d13C Signatures of Flora, Macrofauna and Sediment of a Mangrove ...

African Journals Online (AJOL)

Mangrove organic carbon (d13C±SD = - 27.97±0.25) was found to be an important component of the sediment organic matter except at the marine fringe zone (d13C±SD = - 17.26±0.83). The areal distribution of d13C values show that the marine fringe zone was rich in organic carbon of marine origin. The terrestrial fringe ...

Testing compound-specific δ13C of amino acids in mussels as a new approach to determine the average 13C values of primary production in littoral ecosystems

Science.gov (United States)

Vokhshoori, N. L.; Larsen, T.; McCarthy, M.

2012-12-01

Compound-specific isotope analysis of amino acids (CSI-AA) is a technique used to decouple trophic enrichment patterns from source changes at the base of the food web. With this new emerging tool, it is possible to precisely determine both trophic position and δ15N or δ13C source values in higher feeding organisms. While most work to date has focused on nitrogen (N) isotopic values, early work has suggested that δ13C CSI-AA has great potential as a new tracer both to a record δ13C values of primary production (unaltered by trophic transfers), and also to "fingerprint" specific carbon source organisms. Since essential amino acids (EAA) cannot be made de novo in metazoans but must be obtained from diet, the δ13C value of the primary producer is preserved through the food web. Therefore, the δ13C values of EAAs act as a unique signature of different primary producers and can be used to fingerprint the dominant carbon (C) source driving primary production at the base of the food web. In littoral ecosystems, such as the California Upwelling System (CUS), the likely dominant C sources of suspended particulate organic matter (POM) pool are kelp, upwelling phytoplankton or estuarine phytoplankton. While bulk isotopes of C and N are used extensively to resolve relative consumer hierarchy or shifting diet in a food web, we found that the δ13C bulk values in mussels cannot distinguish exact source in littoral ecosystems. Here we show 15 sites within the CUS, between Cape Blanco, OR and La Jolla, CA where mussels were sampled and analyzed for both bulk δ13C and CSI-AA. We found no latitudinal trends, but rather average bulk δ13C values for the entire coastal record were highly consistent (-15.7 ± 0.9‰). The bulk record would suggest either nutrient provisioning from kelp or upwelled phytoplankton, but 13C-AA fingerprinting confines these two sources to upwelling. This suggests that mussels are recording integrated coastal phytoplankton values, with the enriched �

CACA-TOCSY with alternate 13C-12C labeling: a 13Cα direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification

International Nuclear Information System (INIS)

Takeuchi, Koh; Frueh, Dominique P.; Sun, Zhen-Yu J.; Hiller, Sebastian; Wagner, Gerhard

2010-01-01

We present a 13 C direct detection CACA-TOCSY experiment for samples with alternate 13 C- 12 C labeling. It provides inter-residue correlations between 13 C α resonances of residue i and adjacent C α s at positions i - 1 and i + 1. Furthermore, longer mixing times yield correlations to C α nuclei separated by more than one residue. The experiment also provides C α -to-sidechain correlations, some amino acid type identifications and estimates for ψ dihedral angles. The power of the experiment derives from the alternate 13 C- 12 C labeling with [1,3- 13 C] glycerol or [2- 13 C] glycerol, which allows utilizing the small scalar 3 J CC couplings that are masked by strong 1 J CC couplings in uniformly 13 C labeled samples.

Non-stationary (13)C-metabolic flux ratio analysis.

Science.gov (United States)

Hörl, Manuel; Schnidder, Julian; Sauer, Uwe; Zamboni, Nicola

2013-12-01

(13)C-metabolic flux analysis ((13)C-MFA) has become a key method for metabolic engineering and systems biology. In the most common methodology, fluxes are calculated by global isotopomer balancing and iterative fitting to stationary (13)C-labeling data. This approach requires a closed carbon balance, long-lasting metabolic steady state, and the detection of (13)C-patterns in a large number of metabolites. These restrictions mostly reduced the application of (13)C-MFA to the central carbon metabolism of well-studied model organisms grown in minimal media with a single carbon source. Here we introduce non-stationary (13)C-metabolic flux ratio analysis as a novel method for (13)C-MFA to allow estimating local, relative fluxes from ultra-short (13)C-labeling experiments and without the need for global isotopomer balancing. The approach relies on the acquisition of non-stationary (13)C-labeling data exclusively for metabolites in the proximity of a node of converging fluxes and a local parameter estimation with a system of ordinary differential equations. We developed a generalized workflow that takes into account reaction types and the availability of mass spectrometric data on molecular ions or fragments for data processing, modeling, parameter and error estimation. We demonstrated the approach by analyzing three key nodes of converging fluxes in central metabolism of Bacillus subtilis. We obtained flux estimates that are in agreement with published results obtained from steady state experiments, but reduced the duration of the necessary (13)C-labeling experiment to less than a minute. These results show that our strategy enables to formally estimate relative pathway fluxes on extremely short time scale, neglecting cellular carbon balancing. Hence this approach paves the road to targeted (13)C-MFA in dynamic systems with multiple carbon sources and towards rich media. © 2013 Wiley Periodicals, Inc.

CACA-TOCSY with alternate 13C-12C labeling: a 13Calpha direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification.

Science.gov (United States)

Takeuchi, Koh; Frueh, Dominique P; Sun, Zhen-Yu J; Hiller, Sebastian; Wagner, Gerhard

2010-05-01

We present a (13)C direct detection CACA-TOCSY experiment for samples with alternate (13)C-(12)C labeling. It provides inter-residue correlations between (13)C(alpha) resonances of residue i and adjacent C(alpha)s at positions i - 1 and i + 1. Furthermore, longer mixing times yield correlations to C(alpha) nuclei separated by more than one residue. The experiment also provides C(alpha)-to-sidechain correlations, some amino acid type identifications and estimates for psi dihedral angles. The power of the experiment derives from the alternate (13)C-(12)C labeling with [1,3-(13)C] glycerol or [2-(13)C] glycerol, which allows utilizing the small scalar (3)J(CC) couplings that are masked by strong (1)J(CC) couplings in uniformly (13)C labeled samples.

Spectrally edited 2D 13Csbnd 13C NMR spectra without diagonal ridge for characterizing 13C-enriched low-temperature carbon materials

Science.gov (United States)

Johnson, Robert L.; Anderson, Jason M.; Shanks, Brent H.; Fang, Xiaowen; Hong, Mei; Schmidt-Rohr, Klaus

2013-09-01

Two robust combinations of spectral editing techniques with 2D 13Csbnd 13C NMR have been developed for characterizing the aromatic components of 13C-enriched low-temperature carbon materials. One method (exchange with protonated and nonprotonated spectral editing, EXPANSE) selects cross peaks of protonated and nearby nonprotonated carbons, while the other technique, dipolar-dephased double-quantum/single-quantum (DQ/SQ) NMR, selects signals of bonded nonprotonated carbons. Both spectra are free of a diagonal ridge, which has many advantages: Cross peaks on the diagonal or of small intensity can be detected, and residual spinning sidebands or truncation artifacts associated with the diagonal ridge are avoided. In the DQ/SQ experiment, dipolar dephasing of the double-quantum coherence removes protonated-carbon signals; this approach also eliminates the need for high-power proton decoupling. The initial magnetization is generated with minimal fluctuation by combining direct polarization, cross polarization, and equilibration by 13C spin diffusion. The dipolar dephased DQ/SQ spectrum shows signals from all linkages between aromatic rings, including a distinctive peak from polycondensed aromatics. In EXPANSE NMR, signals of protonated carbons are selected in the first spectral dimension by short cross polarization combined with dipolar dephasing difference. This removes ambiguities of peak assignment to overlapping signals of nonprotonated and protonated aromatic carbons, e.g. near 125 ppm. Spin diffusion is enhanced by dipolar-assisted rotational resonance. Before detection, Csbnd H dipolar dephasing by gated decoupling is applied, which selects signals of nonprotonated carbons. Thus, only cross peaks due to magnetization originating from protonated C and ending on nearby nonprotonated C are retained. Combined with the chemical shifts deduced from the cross-peak position, this double spectral editing defines the bonding environment of aromatic, COO, and Cdbnd O carbons

Study of the (d,α) reactions on the nuclei 10B, 11B, 12C, and 13C and the reaction 13C(p,α)10B and their microscopic and semicroscopic analysis

International Nuclear Information System (INIS)

Abd el-Kariem, S.E.S.

1984-01-01

In the framework of a systematic analysis of many-particle transfer reactions on light nuclei in the present thesis the two-particle transfer reactions of the type (d,α) on the nucleus 10 B at Esub(d) = 16 MeV and on the nuclei 11 B, 12 C and 13 C at Esub(d) = 24 MeV as well as the three-particle transfer reaction 13 C(p,α) 10 B at eight incident energies between 16 and 45 MeV have been studied. In the case of the residual nuclei 10 B and 11 B transitions up to an excitation energy Esub(x) approx.= 7.5 respectively approx.= 9,0 MeV, in the case of the residual nuclei 8 Be and 9 Be transitions up to Esub(x) approx.= 17 respectively 2.5 MeV were evaluated. Under the assumption that the studied reactions behave as direct one-stage transfer processes the measurement results were analyzed in the framework of the DWBA theory in zero-range approximation. The parameters for the optical potentials used in the DWBA calculations were taken from literature and partly modified by fitting to the angular distributions of the reactions studied here. Microscopic and semimicroscopic calculations were performed. In the semimicroscopic calculations the spectroscopic amplitudes calculated microscopically or in SU(3) approximation were used together with a cluster form factor, in the other case with a microscopically calculated form factor. For the residual nucleus for some higher excited states results on spin, parity, and isospin could be partly obtained, partly confirmed. (orig./HSI) [de

Routing of Fatty Acids from Fresh Grass to Milk Restricts the Validation of Feeding Information Obtained by Measuring (13)C in Milk.

Science.gov (United States)

Auerswald, Karl; Schäufele, Rudi; Bellof, Gerhard

2015-12-09

Dairy production systems vary widely in their feeding and livestock-keeping regimens. Both are well-known to affect milk quality and consumer perceptions. Stable isotope analysis has been suggested as an easy-to-apply tool to validate a claimed feeding regimen. Although it is unambiguous that feeding influences the carbon isotope composition (δ(13)C) in milk, it is not clear whether a reported feeding regimen can be verified by measuring δ(13)C in milk without sampling and analyzing the feed. We obtained 671 milk samples from 40 farms distributed over Central Europe to measure δ(13)C and fatty acid composition. Feeding protocols by the farmers in combination with a model based on δ(13)C feed values from the literature were used to predict δ(13)C in feed and subsequently in milk. The model considered dietary contributions of C3 and C4 plants, contribution of concentrates, altitude, seasonal variation in (12/13)CO2, Suess's effect, and diet-milk discrimination. Predicted and measured δ(13)C in milk correlated closely (r(2) = 0.93). Analyzing milk for δ(13)C allowed validation of a reported C4 component with an error of information. However, the error was not random but varied seasonally and correlated with the seasonal variation in long-chain fatty acids. This indicated a bypass of long-chain fatty acids from fresh grass to milk.

Incorporation of deuterium-labeled trans- and cis-13-octadecenoic acids in human plasma lipids

International Nuclear Information System (INIS)

Emken, E.A.; Adlof, R.O.; Rohwedder, W.K.; Gulley, R.M.

1983-01-01

The absorption and distribution of deuterated trans- and cis-13-octadecenoic acid (13t-18:1 and 13c-18:1) in plasma lipids were compared to deuterated cis-9-octadecenoic acid (9c-18:1) in two young adult male subjects. A mixture of triglycerides was fed in a multiple-labeled experiment where each triglyceride contained a fatty acid labeled with a different number of deuterium atoms. Analysis of human plasma lipids by mass spectroscopy allowed the distribution of the two 13-octadecenoic acid isomers to be directly compared to cis-9-octadecenoic acid. Plasma lipids selectively excluded both the 13t-18:1 and 13c-18:1 isomers relative to 9c-18:1 in all neutral and phospholipid fractions. Discrimination against incorporation of the 13t-18:1 isomer into plasma cholesteryl ester and 2-acyl phosphatidylcholine was nearly absolute. The 1-acyl phosphatidylcholine fraction exhibited a large positive selectivity for the 13t-18:1 isomer. Differences in the relative distribution of the trans and cis 13-18:1 isomers vs. 9c-18:1 in the various lipoprotein lipid classes were found. Analysis of the chylomicron triglyceride component of the plasma lipids indicated all three fatty acids were equally well absorbed

Pt.3. Carbon-13 fractionation in the decomposition of formic acid initiated by phosphoric anhydride. 13C fractionation in the decomposition of HCOOH initiated by P2O5

International Nuclear Information System (INIS)

Zielinski, M.; Zielinska, A.

1998-01-01

13 C isotope effects in the decarbonylation of formic acid of natural isotopic composition initiated by phosphorus pentoxide have been studied in a large temperature range (-5 o C) - (+90 o C). The 13 C fractionation in the carbon monooxide production at -5 o C increased from a low value of 1.2% characteristic of the first fractions of consecutively controlled portions of carbon monooxide to higher values of 13 C KIE observed in the decarbonylation of pure formic acid at corresponding temperatures. The temperature and time dependences of the measured 13 C fractionation are functions of the relative number of milimoles of formic acid and the dehydrating phosphoric anhydride, P 2 O 5 . The addition of metaphosphoric acid reagent to unreacted formic acid containing H 3 PO 4 significantly increased the 13 C fractionation in subsequent decarbonylations at 70.4 o C but to a slightly less degree than expected ( 13 C KIE = 1.0503 instead 1.0535). The addition of metaphosphoric acid reagent to formic acid saturated with NaCl results in the experimental 13 C fractionation of the value of 1.0534 very close to the theoretical one. An explanation of the low values of 13 C KIE in the initial stages of HCOOH/P 2 O 5 decarbonylations has been presented. (author)

Neutron orbital radii in {sup 13} C; Radios orbitales neutronicos en {sup 13} C

Energy Technology Data Exchange (ETDEWEB)

Aguilera R, E.F.; Murillo, G.; Ramirez, J.J.; Avila, O.L. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

1988-01-15

In this work its were carried out experimental measurements of the reaction {sup 12}C(d,p) {sup 13}C at low energy. Preliminary results of a DWBA analysis of the data are presented, and the possibility of using this reaction to obtain the orbital radius of the transferred neutron is investigated. (Author)

Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations

International Nuclear Information System (INIS)

Hong, M.; Jakes, K.

1999-01-01

The selective and extensive 13C labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported. The novel 13C labeling approach takes advantage of the amino acid biosynthetic pathways in bacteria and suppresses the synthesis of the amino acid products of the citric acid cycle. The selectivity and extensiveness of labeling significantly simplify the solid-state NMR spectra, reduce line broadening, and should permit the simultaneous measurement of multiple structural constraints. We show the assignment of most 13C resonances to specific amino acid types based on the characteristic chemical shifts, the 13C labeling pattern, and the amino acid composition of the protein. The assignment is partly confirmed by a 2D homonuclear double-quantum-filter experiment under magic-angle spinning. The high sensitivity and spectral resolution attained with this 13C-labeling protocol, which is termed TEASE for ten-amino acid selective and extensive labeling, are demonstrated

NMR structure analysis of uniformly 13C-labeled carbohydrates.

Science.gov (United States)

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.

CACA-TOCSY with alternate {sup 13}C-{sup 12}C labeling: a {sup 13}C{sup {alpha}} direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification

Energy Technology Data Exchange (ETDEWEB)

Takeuchi, Koh [National Institute of Advanced Industrial Science and Technology (AIST), Biomedicinal Information Research Center (BIRC) (Japan); Frueh, Dominique P.; Sun, Zhen-Yu J.; Hiller, Sebastian; Wagner, Gerhard, E-mail: gerhard_wagner@hms.harvard.ed [Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology (United States)

2010-05-15

We present a {sup 13}C direct detection CACA-TOCSY experiment for samples with alternate {sup 13}C-{sup 12}C labeling. It provides inter-residue correlations between {sup 13}C{sup {alpha}} resonances of residue i and adjacent C{sup {alpha}s} at positions i - 1 and i + 1. Furthermore, longer mixing times yield correlations to C{sup {alpha}} nuclei separated by more than one residue. The experiment also provides C{sup {alpha}}-to-side chain correlations, some amino acid type identifications and estimates for {psi} dihedral angles. The power of the experiment derives from the alternate {sup 13}C-{sup 12}C labeling with [1,3-{sup 13}C] glycerol or [2-{sup 13}C] glycerol, which allows utilizing the small scalar {sup 3}J{sub CC} couplings that are masked by strong {sup 1}J{sub CC} couplings in uniformly {sup 13}C labeled samples.

Asymmetric synthesis including enzymatic catalysis of 11C and 13N labelled amino acids

International Nuclear Information System (INIS)

Langstrom, B.; Antonio, G.; Bjurling, P.; Fasth, K.J.; Westerberg, G.; Watanabe, Y.

1993-01-01

Use of asymmetric synthesis in production of 11 C- and 13 N-labelled amino acids has been shown to be a useful approach in order to prepare amino acids routinely for PET-studies. Such PET-studies are focused either on problems related to amino acid transport, protein synthesis rate or the turnover of neurotransmitters from amino acids. The paper discusses matters regarding synthetic strategies and techniques involving production of precursors, labelled intermediates and main reaction sequences. In synthesis using the short-lived β + -emitters like 11 C and 13 N with T 1/2 of 20.3 and 10.0 min respectively, many special aspects have to be considered. The use of enzymes as catalysts has shown to be a useful tool in such preparations. The design of the labelled amino acids especially considering the stereochemistry, the position of the label will be addressed since these points are important both with regard to the application of the labelled amino acids as well as to the synthesis itself. In this presentation of the synthesis of labelled amino acids these various aspects are discussed

The cerebral metabolism of amino acids and related metabolites as studied by 13C and 14C labelling

International Nuclear Information System (INIS)

Hassel, B.

1995-11-01

The present investigations show the feasibility of analyzing the cerebral metabolism of amino acids and related metabolites by 13 C-and 14 C-labelling using labelled acetate and glucose as markers for glial and neuronal metabolism, respectively. Using [ 13 C[acetate, it was shown that glial cells export ∼60% of their TCA cycle intermediates, mostly as glutamine, and that this glutamine is used by neurons partly as an energy reserve, and partly it is converted directly to glutamate and GABA. Using [ 13 C[glucose, the glial process or pyruvate carboxylation was shown to compensate fully for the loss of glutamine. The mechanism of action of two neurotoxins, fluorocitrate and 3-nitropropionate was elucidated. The latter toxin was shown to inhibit the TCA cycle of GABAergic neurons selectively. Formation of pyruvate and lactate from glial TCA cycle intermediates was demonstrated in vivo. This pathway may be important for glial inactivation of transmitter glutamate and GABA. The results illustrate glianeuronal interactions, and they suggest the applicability of 13 CNMR spectroscopy to the detailed study of the cerebral metabolism of amino acids in the intact, unanesthetized human brain. 174 refs

Quantitative importance of the pentose phosphate pathway determined by incorporation of 13C from [2-13C]- and [3-13C]glucose into TCA cycle intermediates and neurotransmitter amino acids in functionally intact neurons.

Science.gov (United States)

Brekke, Eva M F; Walls, Anne B; Schousboe, Arne; Waagepetersen, Helle S; Sonnewald, Ursula

2012-09-01

The brain is highly susceptible to oxidative injury, and the pentose phosphate pathway (PPP) has been shown to be affected by pathological conditions, such as Alzheimer's disease and traumatic brain injury. While this pathway has been investigated in the intact brain and in astrocytes, little is known about the PPP in neurons. The activity of the PPP was quantified in cultured cerebral cortical and cerebellar neurons after incubation in the presence of [2-(13)C]glucose or [3-(13)C]glucose. The activity of the PPP was several fold lower than glycolysis in both types of neurons. While metabolism of (13)C-labeled glucose via the PPP does not appear to contribute to the production of releasable lactate, it contributes to labeling of tricarboxylic acid (TCA) cycle intermediates and related amino acids. Based on glutamate isotopomers, it was calculated that PPP activity accounts for ~6% of glucose metabolism in cortical neurons and ~4% in cerebellar neurons. This is the first demonstration that pyruvate generated from glucose via the PPP contributes to the synthesis of acetyl CoA for oxidation in the TCA cycle. Moreover, the fact that (13)C labeling from glucose is incorporated into glutamate proves that both the oxidative and the nonoxidative stages of the PPP are active in neurons.

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

Science.gov (United States)

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.

Synthesis of selectively 13C-labelled benzoic acid for nuclear magnetic resonance spectroscopic measurement of glycine conjugation activity

International Nuclear Information System (INIS)

Akira, Kazuki; Hasegawa, Hiroshi; Baba, Shigeo

1995-01-01

The synthesis of [4- 13 C]benzoic acid (BA) labelled in a single protonated carbon, for use as a probe to measure glycine conjugation activity by nuclear magnetic resonance (NMR) spectroscopy, has been reported. The labelled compound was prepared by a seven-step synthetic scheme on a relatively small scale using [2- 13 C] acetone as the source of label in overall yield of 16%. The usefulness of [4- 13 C]BA was demonstrated by the NMR spectroscopic monitoring of urinary excretion of [4- 13 C]hippuric acid in the rat administered with the labelled BA. (Author)

Styrylpyrylium Salts: 1H and 13C NMR High-Resolution Spectroscopy (1D and 2D

Directory of Open Access Journals (Sweden)

Jean Claude W. Ouédraogo

2010-01-01

Full Text Available 1H and 13C NMR high-resolution spectroscopy (1D and 2D (1H, 1H-COSY, HSQC, HMBC for four styrylpyrylium perchlorates were carried out and signal attributions are reported. Chemical shifts observed on 13C NMR spectra for the styrylpyrylium salts were compared with net atomic charge for carbon obtained by AM1 semiempirical calculations. The position of the styryl group present low effect on chemical shifts for carbon atoms, while the presence of methyl group led to the unshielding of the substituted carbon.

Fourier spectroscopy of the 12C2, 13C2, and 12C13C (0-0) swan bands

International Nuclear Information System (INIS)

Amiot, C.

1983-01-01

The (0-0) band of the C 2 Swan electronic system d 3 Pi/sub g/→a 3 Pi/sub u/ has been recorded by Fourier spectroscopy. The three isotopes species 12 C 2 , 13 C 2 , and 12 C 13 C were investigated. The observed wavenumbers were reduced to molecular parameters using a nonlinear least-square fitting procedure. Well-known perturbations at N' = 47 and N' = 51 again observed in the e 12 C 2 d 3 Pi/sub g/ (v = 0) level. Perturbations of the same kind are present in the 13 C 2 spectrum at N' = 34 and N' = 44,48,52. The 12 C 13 C spectrum exhibits in the observed spectral range a unique perturbation for N' = 41

Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis.

Science.gov (United States)

Sumida, Yosuke; Iwai, Sachio; Nishiya, Yoshiaki; Kumagai, Shinya; Yamada, Toshihide; Azuma, Masayuki

2018-03-01

d-Amino acids are important building blocks for various compounds, such as pharmaceuticals and agrochemicals. A more cost-effective enzymatic method for d-amino acid production is needed in the industry. We improved a one-pot enzymatic method for d-amino acid production by the dynamic kinetic resolution of N-succinyl amino acids using two enzymes: d-succinylase (DSA) from Cupriavidus sp. P4-10-C, which hydrolyzes N-succinyl-d-amino acids enantioselectively to their corresponding d-amino acid, and N-succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503. In this study, DSA and NSAR were purified and their properties were investigated. The optimum temperature of DSA was 50°C and it was stable up to 55°C. The optimum pH of DSA and NSAR was around 7.5. In d-phenylalanine production, the optical purity of product was improved to 91.6% ee from the examination about enzyme concentration. Moreover, 100 mM N-succinyl-dl-tryptophan was converted to d-tryptophan at 81.8% yield with 94.7% ee. This enzymatic method could be useful for the industrial production of various d-amino acids. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A new method to reconstruct fish diet and movement patterns from δ 13 C values in otolith amino acids

KAUST Repository

McMahon, Kelton W.; Fogel, Marilyn L.; Johnson, Beverly J.; Houghton, Leah A.; Thorrold, Simon R.; Gillanders, Bronwyn

2011-01-01

Fish ecologists have used geochemical values in otoliths to examine habitat use, migration, and population connectivity for decades. However, it remains difficult to determine an unambiguous dietary δ 13C signature from bulk analysis of otolith. Studies to date have focused on the aragonite component of otoliths with less attention paid to the organic fraction. We describe the application of compound-specific stable isotope analysis (SIA) to analyze amino acid (AA) δ 13C values from small amounts (<1 mg) of otolith powder. We examined δ 13C values of otolith and muscle AAs from a reef-associated snapper (Lutjanus ehrenbergii (Peters, 1869)) collected along a carbon isotope gradient (isoscape) from seagrass beds to coral reefs. Carbon isotope values in otolith and muscle samples were highly correlated within and among coastal habitats. Moreover, δ 13C values of otolith AAs provided a purely dietary record that avoided dilution from dissolved inorganic carbon. Otolith AAs served as a robust tracer of δ 13C values at the base of the food web, making compound-specific SIA a powerful tool for dietary reconstructions and tracking the movement of fishes across isoscapes.

A new method to reconstruct fish diet and movement patterns from δ 13 C values in otolith amino acids

KAUST Repository

McMahon, Kelton W.

2011-08-01

Fish ecologists have used geochemical values in otoliths to examine habitat use, migration, and population connectivity for decades. However, it remains difficult to determine an unambiguous dietary δ 13C signature from bulk analysis of otolith. Studies to date have focused on the aragonite component of otoliths with less attention paid to the organic fraction. We describe the application of compound-specific stable isotope analysis (SIA) to analyze amino acid (AA) δ 13C values from small amounts (<1 mg) of otolith powder. We examined δ 13C values of otolith and muscle AAs from a reef-associated snapper (Lutjanus ehrenbergii (Peters, 1869)) collected along a carbon isotope gradient (isoscape) from seagrass beds to coral reefs. Carbon isotope values in otolith and muscle samples were highly correlated within and among coastal habitats. Moreover, δ 13C values of otolith AAs provided a purely dietary record that avoided dilution from dissolved inorganic carbon. Otolith AAs served as a robust tracer of δ 13C values at the base of the food web, making compound-specific SIA a powerful tool for dietary reconstructions and tracking the movement of fishes across isoscapes.

Fate of free amino acids in paddy and upland soils by using 13C and 15N tracer techniques

International Nuclear Information System (INIS)

Yamamuro, Shigekazu; Ueno, Hideto; Takahashi, Shigeru

1999-01-01

Direct and indirect (=through decomposition) uptakes of free amino acids (FAA) by rice and tomato plants were investigated by using 13 C- and 15 N-labeled aspartic and glutamic acids, serine, leusine and ammonium as tracers. 1) One week after the surface application of amino acid-N or NH 4 -N to paddy soil, the amounts of ammonium remaining in the soil, assimilated ammonium, denitrificated ammonium and amounts taken up by plants were similar. 2) From 5.5 to 7.7% of the FAA applied was absorbed directly by rice plants, and from 42.5 to 47.2% of that was indirectly absorbed as ammonium after decomposition. It is suggested that the FAA degraded to ammonium around 2 or 3 d and the 1- 13 C absorption rates of the FAA (RCH(NH 2 ) 13 COOH) were high in proportion to the number of carbon atoms of the R side-chain. 3) The absorption rate of N derived from the FAA by tomato plants was lower than that by rice plants, namely, from 0.4 to 1.9% in direct-uptake and from 16.0 to 29.8% in indirect-uptake. Percentage of direct-uptake of the FAA in upland soil was much lower than that in the paddy field. (author)

13C/12C ratios in human urine concrementes

International Nuclear Information System (INIS)

Hoefs, J.; Armbruster, T.

1978-01-01

Oxalate, uric acid, and phosphate stones have been analyzed for their carbon isotope composition. The oxalate stones show delta 13 C values between -17.0 and -19.5 pro mille, the uric acid stones between -14.9 and -19.4 pro mille, and the phosphate stones between -13.0 and -23.9 pro mille. It is proposed that endogenic rather than exogenic sources are responsible for the 13 C/ 12 C ratios of the stones. The isotopic composition of the phosphate stones seems to be influenced primarily by bacterial activity. (orig.) [de

Metabolic characteristics of 13-cis-retinoic acid (isotretinoin) and anti-tumour activity of the 13-cis-retinoic acid metabolite 4-oxo-13-cis-retinoic acid in neuroblastoma.

Science.gov (United States)

Sonawane, Poonam; Cho, Hwang Eui; Tagde, Ashujit; Verlekar, Dattesh; Yu, Alice L; Reynolds, C Patrick; Kang, Min H

2014-12-01

Isotretinoin (13-cis-retinoic acid; 13-cRA) is a differentiation inducer used to treat minimal residual disease after myeloablative therapy for high-risk neuroblastoma. However, more than 40% of children develop recurrent disease during or after 13-cRA treatment. The plasma concentrations of 13-cRA in earlier studies were considered subtherapeutic while 4-oxo-13-cis-RA (4-oxo-13-cRA), a metabolite of 13-cRA considered by some investigators as inactive, were greater than threefold higher than 13-cRA. We sought to define the metabolic pathways of 13-cRA and investigated the anti-tumour activity of its major metabolite, 4-oxo-13-cRA. Effects of 13-cRA and 4-oxo-13-cRA on human neuroblastoma cell lines were assessed by DIMSCAN and flow cytometry for cell proliferation, MYCN down-regulation by reverse transcription PCR and immunoblotting, and neurite outgrowth by confocal microscopy. 13-cRA metabolism was determined using tandem MS in human liver microsomes and in patient samples. Six major metabolites of 13-cRA were identified in patient samples. Of these, 4-oxo-13-cRA was the most abundant, and 4-oxo-13-cRA glucuronide was also detected at a higher level in patients. CYP3A4 was shown to play a major role in catalysing 13-cRA to 4-oxo-13-cRA. In human neuroblastoma cell lines, 4-oxo-13-cRA and 13-cRA were equi-effective at inducing neurite outgrowth, inhibiting proliferation, decreasing MYCN mRNA and protein, and increasing the expression of retinoic acid receptor-β mRNA and protein levels. We showed that 4-oxo-13-cRA is as active as 13-cRA against neuroblastoma cell lines. Plasma levels of both 13-cRA and 4-oxo-13-cRA should be evaluated in pharmacokinetic studies of isotretinoin in neuroblastoma. © 2014 The British Pharmacological Society.

Synthesis of 1-13C-1-indanone and 2-13C-1,2,3,4-tetrahydroquinoline

International Nuclear Information System (INIS)

Pickering, R.E.; Wysocki, M.A.; Eisenbraun, E.J.

1985-01-01

The synthesis of 2- 13 C-1,2,3,4-tetrahydroquinoline (5) via 1- 13 C-3-phenylpropanoic acid (1), 1- 13 C-1-indanone (2), 1- 13 C-1-indanone hydrazone (3) and 2- 13 C-3,4-dihydro-2(1H)-quinolinone (4) proceeded in 78, 96, 95, 79, and 85% individual yields respectively for 1, 2, 3, 4, 5 and 61% overall yield of the latter from 1. (author)

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

Science.gov (United States)

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

2015-11-01

S-Adenosyl-L-methionine (SAM) is a major biological methyl group donor, and is used as a nutritional supplement and prescription drug. Yeast is used for the industrial production of SAM owing to its high intracellular SAM concentrations. To determine the regulation mechanisms responsible for such high SAM production, (13)C-metabolic flux analysis ((13)C-MFA) was conducted to compare the flux distributions in the central metabolism between Kyokai no. 6 (high SAM-producing) and S288C (control) strains. (13)C-MFA showed that the levels of tricarboxylic acid (TCA) cycle flux in SAM-overproducing strain were considerably increased compared to those in the S228C strain. Analysis of ATP balance also showed that a larger amount of excess ATP was produced in the Kyokai 6 strain because of increased oxidative phosphorylation. These results suggest that high SAM production in Kyokai 6 strains could be attributed to enhanced ATP regeneration with high TCA cycle fluxes and respiration activity. Thus, maintaining high respiration efficiency during cultivation is important for improving SAM production. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

D-seco-Vitamin D analogs having reversed configurations at C-13 and C-14: Synthesis, docking studies and biological evaluation.

Science.gov (United States)

Szybinski, Marcin; Sokolowska, Katarzyna; Sicinski, Rafal R; Plum, Lori A; DeLuca, Hector F

2017-10-01

Prompted by results of molecular modeling performed on the seco-d-ring-vitamins D, we turned our attention to such analogs, having reversed configurations at C-13 and C-14, as the next goals of our studies on the structure-activity relationship for vitamin D compounds. First, we developed an efficient total synthesis of the "upper" C/seco-d-ring fragment with a 7-carbon side chain. Then, we coupled it with A-ring fragments using Sonogashira or Wittig-Horner protocol, providing the targeted D-seco analogs of 1α,25-dihydroxyvitamin D 3 and 1α,25-dihydroxy-19-norvitamin D 3 possessing a vinyl substituent at C-14 and a double bond between C-17 and C-20. The affinities of the synthesized vitamin D analogs to the full-length recombinant rat VDR were examined, as well as their differentiating and transcriptional activities. In these in vitro tests, they were significantly less active compared to 1α,25-(OH) 2 D 3 . Moreover, it was established that the analogs tested in vivo in rats showed no calcemic potency. Copyright © 2016 Elsevier Ltd. All rights reserved.

Determination of the anomeric specificity of the Escherichia coli CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase by 13C NMR spectroscopy

International Nuclear Information System (INIS)

Kohlbrenner, W.E.; Fesik, S.W.

1985-01-01

[99%, 1- 13 C]- and [90%, 2- 13 C]3-deoxy-D-manno-octulosonic acid (KDO) were prepared enzymatically and used to determine the anomeric specificity of the CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase (CMP-KDO synthetase) by 13 C NMR spectroscopy. Addition of CMP-KDO synthetase to reaction mixtures containing either 1- 13 C- or 2- 13 C-labeled KDO resulted in rapid CMP-KDO formation which was accompanied by a substantial decrease in the 13 C-enriched resonances of the beta-pyranose form of KDO relative to the resonances of other KDO species in solution, demonstrating that the beta-pyranose is the preferred substrate. Concomitant with the production of CMP-KDO was the appearance of peaks at 174.3 and 101.4 ppm when [1- 13 C]- and [2- 13 C]KDO, respectively, were used as substrates. The correspondence of these resonances to the enriched carbons in CMP-KDO was confirmed by the expected 3-bond (3JP,C-1 = 6.9 Hz) and 2-bond coupling (2JP,C-2 = 8.3 Hz) between the labeled carbons and the ketosidically linked phosphoryl group. A large coupling (3J = 5.7 Hz) was observed in proton-coupled spectra of CMP-[1- 13 C]KDO between carbon 1 and the axial proton at carbon 3 of KDO. The magnitude of this coupling constant supports a diaxial relationship between these two groups and, along with chemical shift data, indicates that KDO retains the beta-configuration when linked in CMP-KDO

Structural characterization of supramolecular assemblies by {sup 13}C spin dilution and 3D solid-state NMR

Energy Technology Data Exchange (ETDEWEB)

Habenstein, Birgit; Loquet, Antoine; Giller, Karin; Becker, Stefan; Lange, Adam, E-mail: adla@nmr.mpibpc.mpg.de [Max Planck Institute for Biophysical Chemistry, Department of NMR-based Structural Biology (Germany)

2013-01-15

{sup 13}C spin diluted protein samples can be produced using [1-{sup 13}C] and [2-{sup 13}C]-glucose (Glc) carbon sources in the bacterial growth medium. The {sup 13}C spin dilution results in favorable {sup 13}C spectral resolution and polarization transfer behavior. We recently reported the combined use of [1-{sup 13}C]- and [2-{sup 13}C]-Glc labeling to facilitate the structural analysis of insoluble and non-crystalline biological systems by solid-state NMR (ssNMR), including sequential assignment, detection of long-range contacts and structure determination of macromolecular assemblies. In solution NMR the beneficial properties of sparsely labeled samples using [2-{sup 13}C]-glycerol ({sup 13}C labeled C{alpha} sites on a {sup 12}C diluted background) have recently been exploited to provide a bi-directional assignment method (Takeuchi et al. in J Biomol NMR 49(1):17-26, 2011 ). Inspired by this approach and our own recent results using [2-{sup 13}C]-Glc as carbon sources for the simplification of ssNMR spectra, we present a strategy for a bi-directional sequential assignment of solid-state NMR resonances and additionally the detection of long-range contacts using the combination of {sup 13}C spin dilution and 3D NMR spectroscopy. We illustrate our results with the sequential assignment and the collection of distance restraints on an insoluble and non-crystalline supramolecular assembly, the Salmonella typhimurium type III secretion system needle.

Comparison of bolus versus fractionated oral applications of [13C]-linoleic acid in humans.

Science.gov (United States)

Demmelmair, H; Iser, B; Rauh-Pfeiffer, A; Koletzko, B

1999-07-01

The endogenous conversion of linoleic acid into long-chain polyunsaturated fatty acids is of potential importance for meeting substrate requirements, particularly in young infants. After application of [13C]-linoleic acid, we estimated its conversion to dihomo-gamma-linolenic and arachidonic acids from only two blood samples. Oral tracer doses were given to five healthy adults as a single bolus. In four subjects the tracer was given in nine equal portions over 3 days. Concentration and 13C content of fatty acids from serum phospholipids were analysed by gas chromatography combustion isotope ratio-mass spectrometry. Areas under the tracer-concentration curves were calculated, and fractional transfer and turnover rates estimated from compartmental models. The median fractional turnover of linoleic acid was 93.7% per day (interquartile range 25.3) in the bolus group and 80. 0% per day (6.3) in the fraction group (NS). Fractional conversion of linoleic to dihomo-gamma-linolenic acid was 1.5% (0.9) vs. 2.1% (0.7) (bolus vs. fraction, P /= 0.94, P < 0.05) with the ratio of areas under the curve. Using areas under the curve overestimates the conversion, because different residence times are not considered. Estimation of conversion intensity appears possible with only one blood sample obtained after tracer application.

The simultaneous biosynthesis and uptake of amino acids by Lactococcus lactis studied by C-13-labeling experiments

DEFF Research Database (Denmark)

Jensen, N.B.S.; Christensen, B.; Nielsen, Jette

2002-01-01

Uniformly C-13 labeled glucose was fed to a lactic acid bacterium growing on a defined medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with C-13 disclosing a substantial de novo...... biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange flux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium....

Benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives as multiple inhibitors of bacterial Mur ligases (MurC-MurF).

Science.gov (United States)

Perdih, Andrej; Hrast, Martina; Barreteau, Hélène; Gobec, Stanislav; Wolber, Gerhard; Solmajer, Tom

2014-08-01

Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of the bacterial Mur ligase family-MurC, MurD, MurE and MurF-are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Park's nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC-MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC-MurF enzymes in biochemical inhibition assays and molecules 10-14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC-MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d-Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

International Nuclear Information System (INIS)

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

1999-01-01

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

Synthesis of edatrexate (2-13C-glutamate)

International Nuclear Information System (INIS)

DeGraw, J.I.; Colwell, W.T.; Jue, Thomas

1997-01-01

The experimental antitumor drug Edatrexate, labeled with 99% 13 C at the 2-position of the glutamate acid group was required for 13 C-magnetic resonance spectroscopy studies in biological media. Coupling of 2,4-diamino-4-deoxy-10-ethyl-10-deazapteroic acid with diethyl L-2- 13 C-glutamate as promoted by BOP reagent afforded Edatrexate (2- 13 C-glu) diethyl ester in 60% yield following purification by column chromatography. Saponification by aqueous NaOH in 2-methoxyethanol gave the target molecule in 44% yield or 26% overall. (author)

Fatty acid biomarkers: validation of food web and trophic markers using C-13-labelled fatty acids in juvenile sandeel ( Ammodytes tobianus )

DEFF Research Database (Denmark)

Dalsgaard, Anne Johanne Tang; St. John, Michael

2004-01-01

A key issue in marine science is parameterizing trophic interactions in marine food webs, thereby developing an understanding of the importance of top-down and bottom-up controls on populations of key trophic players. This study validates the utility of fatty acid food web and trophic markers usi......), respectively. Lack of temporal trends in nonlabelled fatty acids confirmed the conservative incorporation of labelled fatty acids by the fish.......A key issue in marine science is parameterizing trophic interactions in marine food webs, thereby developing an understanding of the importance of top-down and bottom-up controls on populations of key trophic players. This study validates the utility of fatty acid food web and trophic markers using...... C-13-labelled fatty acids to verify the conservative incorporation of fatty acid tracers by juvenile sandeel (Ammodytes tobianus) and assess their uptake, clearance, and metabolic turnover rates. Juvenile sandeel were fed for 16 days in the laboratory on a formulated diet enriched in (13)C16...

A facile synthesis of δ-aminolevulinic acid (ALA) regio-selectively labeled with 13C and direct observation of enzymatic transformation from ALA to porphobilinogen (PBG)

International Nuclear Information System (INIS)

Kurumaya, Katsuyuki; Okazaki, Takeo; Seido, Nobuo; Akasaka, Yuzuru; Kawajiri, Yoshiki; Kajiwara, Masahiro; Kondo, Masao

1989-01-01

δ-Aminolevulinic acid (ALA), labeled with 13 C at position 1, 2, 3, 4, or 5, was synthesized from 13 C-labeled glycine, Meldrum's acid, or bromoacetate. The latter compounds were prepared from 13 C-sodium acetate or 13 C-acetic acid. Enzymatic transformation from ALA to porphobilinogen (PBG) was directly observed by 13 C-NMR. (author)

The cerebral metabolism of amino acids and related metabolites as studied by {sup 13}C and {sup 14}C labelling

Energy Technology Data Exchange (ETDEWEB)

Hassel, B

1995-11-01

The present investigations show the feasibility of analyzing the cerebral metabolism of amino acids and related metabolites by {sup 13}C-and {sup 14}C-labelling using labelled acetate and glucose as markers for glial and neuronal metabolism, respectively. Using [{sup 13}C]acetate, it was shown that glial cells export {approx}60% of their TCA cycle intermediates, mostly as glutamine, and that this glutamine is used by neurons partly as an energy reserve, and partly it is converted directly to glutamate and GABA. Using [{sup 13}C]glucose, the glial process or pyruvate carboxylation was shown to compensate fully for the loss of glutamine. The mechanism of action of two neurotoxins, fluorocitrate and 3-nitropropionate was elucidated. The latter toxin was shown to inhibit the TCA cycle of GABAergic neurons selectively. Formation of pyruvate and lactate from glial TCA cycle intermediates was demonstrated in vivo. This pathway may be important for glial inactivation of transmitter glutamate and GABA. The results illustrate glianeuronal interactions, and they suggest the applicability of {sup 13}CNMR spectroscopy to the detailed study of the cerebral metabolism of amino acids in the intact, unanesthetized human brain. 174 refs.

Climate signal in d13C of wood lignin methoxyl groups from high-elevation alpine larch trees

Science.gov (United States)

Reichelmann, Dana; Greule, Markus; Treydte, Kerstin; Keppler, Frank; Esper, Jan

2015-04-01

Tree-rings of high alpine larch trees (Larix decidua) were investigated by a recently established method that measures d13C values of the wood lignin methoxyl groups (Greule et al. 2009). The resulting d13C time series were tested for their potential as a climate proxy. For this 37 larch trees were sampled at the tree line near Simplon Village (Southern Switzerland). They were analysed for their tree-ring width (TRW) and from five individuals d13C of the wood lignin methoxyl groups (d13Cmethoxyl) were measured at annual resolution from 1971-2009 and at pentadal resolution from 1747-2009. The d13Cmethoxyl chronologies were corrected for the anthropogenic change in atmospheric CO2 concentration and its decreasing d13C value. Further, the physiological response of the trees to these atmospheric changes was corrected using the flexible correction factor approach of Treydte et al. (2009), which minimise the residuals with the target climate data. This approach results in the highest so far reported correction factors of 0.032 - 0.036‰/ppmv CO2, which are explained by a low water-use efficiency of deciduous larch. The climate response of the new d13Cmethoxyl proxy shows a significant correlation of 0.75 for the annually and 0.87 for the pentadally resolved data with June to August temperatures. TRW shows also significant correlations with June to August temperatures, but they are lower than the correlations observed for the d13Cmethoxyl chronologies. These results indicate the potential of d13Cmethoxyl chronologies as a summer temperature proxy from high-elevation alpine trees with an even stronger signal than reported from earlier published tree-ring width and maximum latewood density temperature reconstructions. References: Greule, M., Mosandl, A., Hamilton, J.T.G., Keppler, F., 2009. A simple rapid method to precisely determine 13C/12C ratios of plant methoxyl groups. Rapid Communications in Mass Spectrometry, 23(11): 1710-1714. Treydte, K.S., Frank, D.C., Saurer, M

Practical use of chemical shift databases for protein solid-state NMR: 2D chemical shift maps and amino-acid assignment with secondary-structure information

Energy Technology Data Exchange (ETDEWEB)

Fritzsching, K. J.; Yang, Y.; Schmidt-Rohr, K.; Hong Mei, E-mail: mhong@iastate.edu [Iowa State University, Department of Chemistry (United States)

2013-06-15

We introduce a Python-based program that utilizes the large database of {sup 13}C and {sup 15}N chemical shifts in the Biological Magnetic Resonance Bank to rapidly predict the amino acid type and secondary structure from correlated chemical shifts. The program, called PACSYlite Unified Query (PLUQ), is designed to help assign peaks obtained from 2D {sup 13}C-{sup 13}C, {sup 15}N-{sup 13}C, or 3D {sup 15}N-{sup 13}C-{sup 13}C magic-angle-spinning correlation spectra. We show secondary-structure specific 2D {sup 13}C-{sup 13}C correlation maps of all twenty amino acids, constructed from a chemical shift database of 262,209 residues. The maps reveal interesting conformation-dependent chemical shift distributions and facilitate searching of correlation peaks during amino-acid type assignment. Based on these correlations, PLUQ outputs the most likely amino acid types and the associated secondary structures from inputs of experimental chemical shifts. We test the assignment accuracy using four high-quality protein structures. Based on only the C{alpha} and C{beta} chemical shifts, the highest-ranked PLUQ assignments were 40-60 % correct in both the amino-acid type and the secondary structure. For three input chemical shifts (CO-C{alpha}-C{beta} or N-C{alpha}-C{beta}), the first-ranked assignments were correct for 60 % of the residues, while within the top three predictions, the correct assignments were found for 80 % of the residues. PLUQ and the chemical shift maps are expected to be useful at the first stage of sequential assignment, for combination with automated sequential assignment programs, and for highly disordered proteins for which secondary structure analysis is the main goal of structure determination.

Do birds of like feather flock together? The use of a novel 13C+D combustion isotope analyzer to track bird migration

Science.gov (United States)

Saad, N.

2011-12-01

Information on spring migration routes, geographic linkages among winter, spring, and breeding locations, and potential geographic effects on arrival body condition of a variety of avian species is so far little known. Stable isotope ratios are exquisitely sensitive to the biochemistry of living organisms and the nutrients available to them. Isotope ratios provide detailed knowledge useful in a variety of fields, including birds migration, through a combination of stable-isotope measurements of carbon (13C/12C) and hydrogen (D/H) isotopes of flight feathers and breast feathers of tissues representing different periods of dietary integration and body composition analyses. Associations among specific geographic areas, habitat use, and arrival condition can be elucidated through the measurement of these dual isotopes. We report here on the development of a novel laser spectroscopy based system for the simultaneous analysis of the stable isotope ratios of carbon (13C/12C) and hydrogen (D/H) that is robust, easy-to-use, and is the first stable isotope ratio analysis system to combine the measurement of 13C/12C and D/H in one simple analysis from a bulk organic sample with an application to bird migration. The system comprises a combustion module to convert the organic sample into CO2 and H2O and a Cavity Ring-Down Spectrometer (CRDS) that analyzes the combustion species inside an optical cavity based on the molecular absorption of individual isotopomers. This CRDS uses dual lasers to target the four isotpomers of interest: 12CO2, 13CO2, H2O and HDO. The system delivers a typical precision of 0.1 permil for δ13C and 1.5 permil for δD that parallels that achieved by IRMS, but with an unprecedented simplicity that allows ecologists to leverage the science and elucidate the avian migration patterns.

delta 13C analyses of vegetable oil fatty acid components, determined by gas chromatography--combustion--isotope ratio mass spectrometry, after saponification or regiospecific hydrolysis.

Science.gov (United States)

Woodbury, S E; Evershed, R P; Rossell, J B

1998-05-01

The delta 13C values of the major fatty acids of several different commercially important vegetable oils were measured by gas chromatography--combustion--isotope ratio mass spectrometry. The delta 13C values obtained were found to fall into two distinct groups, representing the C3 and C4 plants classes from which the oils were derived. The delta 13C values of the oils were measured by continuous flow elemental isotope ratio mass spectrometry and were found to be similar to their fatty acids, with slight differences between individual fatty acids. Investigations were then made into the influence on the delta 13C values of fatty acids of the position occupied on the glycerol backbone. Pancreatic lipase was employed to selectively hydrolyse fatty acids from the 1- and 3-positions with the progress of the reaction being followed by high-temperature gas chromatography in order to determine the optimum incubation time. The 2-monoacylglycerols were then isolated by thin-layer chromatography and fatty acid methyl esters prepared. The delta 13C values obtained indicate that fatty acids from any position on the glycerol backbone are isotopically identical. Thus, whilst quantification of fatty acid composition at the 2-position and measurement of delta 13C values of oils and their major fatty acids are useful criteria in edible oil purity assessment, measurement of delta 13C values of fatty acids from the 2-position does not assist with oil purity assignments.

A facile approach for the synthesis of C13-C24 fragments of maltepolides A, C and D.

Science.gov (United States)

Rao, P Sankara; Srihari, P

2016-10-12

A linear, chiron approach for the synthesis of C13-C24 fragments of cytostatic maltepolides A, C and D consisting of a tetrahydrofuran subunit and a chiral alkenyl/alkyl substituent is achieved from (+)-diethyl l-tartrate. The other chiral stereocenters were generated by employing key reactions such as Crimmins aldol, alkynylation and CeCl 3 ·7H 2 O mediated Luche reduction reactions.

Oxidation of linoleic and palmitic acid in pre-hibernating and hibernating common noctule bats revealed by 13C breath testing.

Science.gov (United States)

Rosner, Elisabeth; Voigt, Christian C

2018-02-19

Mammals fuel hibernation by oxidizing saturated and unsaturated fatty acids from triacylglycerols in adipocytes, yet the relative importance of these two categories as an oxidative fuel may change during hibernation. We studied the selective use of fatty acids as an oxidative fuel in noctule bats ( Nyctalus noctula ). Pre-hibernating noctule bats that were fed 13 C-enriched linoleic acid (LA) showed 12 times higher tracer oxidation rates compared with conspecifics fed 13 C-enriched palmitic acid (PA). After this experiment, we supplemented the diet of bats with the same fatty acids on five subsequent days to enrich their fat depots with the respective tracer. We then compared the excess 13 C enrichment (excess atom percentage, APE) in breath of bats for torpor and arousal events during early and late hibernation. We observed higher APE values in breath of bats fed 13 C-enriched LA than in bats fed 13 C-enriched PA for both states (torpor and arousal), and also for both periods. Thus, hibernating bats selectively oxidized endogenous LA instead of PA, probably because of faster transportation rates of polyunsaturated fatty acids compared with saturated fatty acids. We did not observe changes in APE values in the breath of torpid animals between early and late hibernation. Skin temperature of torpid animals increased by 0.7°C between early and late hibernation in bats fed PA, whereas it decreased by -0.8°C in bats fed LA, highlighting that endogenous LA may fulfil two functions when available in excess: serving as an oxidative fuel and supporting cell membrane functionality. © 2018. Published by The Company of Biologists Ltd.

Synthesis and characterization of "1"3C_3-tristearin

International Nuclear Information System (INIS)

Wu Hangyu; Lin Lin; Li Lei; Chen Dazhou

2011-01-01

A highly efficient synthesis of "1"3C_3 labeled triglycerides of stearic acids from "1"3C_3-glycerol and stearic acids, by immobilized lipase-catalyzed in solvent-free medium was described. The structure of the product were characterized by fourier transform infrared spectrum (FT-IR), nuclear magnetic resonance (NMR), mass spectra (MS). The results showed that triglycerides of stearic acids contained three "1"3C atoms. The isotope abundance of "1"3C_3-tristearin was more than 99% and the yield was 80% of "1"3C_3-tristearin through calculation. Chemical purity (> 98%) was obtained by differential scanning calorimetry (DSC). (authors)

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

DEFF Research Database (Denmark)

Ghosh, Amit; Ando, David; Gin, Jennifer

2016-01-01

Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models...

Characterization of two minor saponins from Cordia piauhiensis by 1H and 13C NMR spectroscopy.

Science.gov (United States)

Santos, Renata P; Silveira, Edilberto R; Lemos, Telma Leda G; Viana, Francisco Arnaldo; Braz-Filho, Raimundo; Pessoa, Otília Deusdênia L

2005-06-01

A careful NMR analysis with full assignment of the 1H and 13C spectral data for two minor saponins isolated from stems of Cordia piauhiensis is reported. These saponins were isolated by high-performance liquid chromatography and characterized as 3beta-O-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl]pomolic acid 28-O-[beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranosyl] ester (1) and 3beta-O-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl]oleanolic acid 28-O-[beta-D-xylopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranosyl] ester (2). Their structures were established using a combination of 1D and 2D (1H, 1H-COSY, TOCSY, NOESY, gs-HMQC and gs-HMBC) NMR techniques, electrospray ionization mass spectrometry and chemical evidence. Copyright 2005 John Wiley & Sons, Ltd.

Tricarboxylic acid cycle activity measured by 13C magnetic resonance spectroscopy in rats subjected to the kaolin model of obstructed hydrocephalus

DEFF Research Database (Denmark)

Melø, Torun M; Håberg, Asta K; Risa, Øystein

2011-01-01

in the amounts of glutamate, alanine and taurine. In addition, the concentration of the neuronal marker N-acetyl aspartate was decreased. (13)C Labelling of most amino acids derived from [1,6-(13)C]glucose was unchanged 2 weeks after hydrocephalus induction. The only indication of astrocyte impairment......Evaluating early changes in cerebral metabolism in hydrocephalus can help in the decision making and the timing of surgical intervention. This study was aimed at examining the tricarboxylic acid (TCA) cycle rate and (13)C label incorporation into neurotransmitter amino acids and other compounds 2...

Creatinine and creatininium cation in water solution. Tautomerism and quantitative interpretation of the solution acidity effect on 1H, 13C and 1:4N NMR chemical shifts

International Nuclear Information System (INIS)

Kotsyubynskyy, D.; Molchanov, S.; Gryff-Keller, A.

2004-01-01

1 H, 13 C and 1 :4N NMR chemical shifts for creatinine in water solution of various acidity have been measured. Analysis of these data enabled determination of the acidity constant of creatininium cation and the chemical shifts of the neutral and protonated forms of creatinine. Molecular energies and carbon and nitrogen magnetic shielding constants for various tautomeric structures of the investigated species have been calculated using the quantum chemistry method GIAO DFT B3LYP/6-311++G(2d,p). Compilation of the available experimental and theoretical results has provided additional information on the problem of tautomerism of this important biological molecule. (author)

Noninvasive brain metabolism measurement using carbon-13 magnetic resonance spectroscopy ({sup 13}C-MRS); Tanso 13 jiki kyomei spectroscopy ({sup 13}C-MRS) ni yoru mushinshuteki notaisha keisoku

Energy Technology Data Exchange (ETDEWEB)

Okamoto, K.; Tsukada, Y. [Toshiba Corp., Tokyo (Japan)

1998-10-10

Carbon-13 magnetic resonance spectroscopy ({sup 13}C-MRS) and research and development efforts for brain metabolism measurement are described. Brain metabolism is a process characterized in that it not only extracts energy by disintegrating grape sugar that is the practically sole source of energy into H2O, CO2, etc., but also vigorously synthesizes amino acids that perform important functions in neural transmission, such as glutamic acid, glutamine, and {gamma}-amino acid. MRS is a technique that utilizes the magnetic resonance, which is generated when an atomic nucleus with a spin is placed in a magnetic field, for the isolation and identification of chemicals in a living body through examining the delicate difference in the magnetic resonance frequencies of the nuclei under observation. Since the signals from {sup 13}C are low in intensity as compared with those from other nuclides, a method was contrived around 1980, which observes {sup 1}H combined with {sup 13}C in grape sugar and amino acids, named the HSQC (heteronuclear single quantum coherence) method. The author et al., combining gradient magnetic pulses with HSQC, actually measure Homo sapiens brain metabolism using {sup 13}C-MRS, and now believe that the technology will be put to practical application. 7 refs., 10 figs., 1 tab.

Stereoselective synthesis of L-[4-13C]carnitine

International Nuclear Information System (INIS)

Unkefer, C.J.; Ehler, D.S.

1991-01-01

The stereoselective synthesis of L-[4- 13 C]carnitine was achieved in 5 steps. The label was introduced from K 13 CN into an easily separated diastereomeric pair of 3-deoxy-D-[1- 13 C]aldohexoses. Reductive amination of the labeled aldohexose yielded the corresponding D-1-(dimethylamino)[1- 13 C]alditol which was oxidized in two steps and alkylated with iodomethane to yield L-[4- 13 C]carnitine. The stereochemical integrity at C-2 of the 3-deoxy-D-[1- 13 C]glucose precursor was maintained throughout the synthesis of L-[4- 13 C]carnitine. (author)

Relative bioavailability of 13C5-folic acid in pectin-coated folate fortified rice in humans using stable isotope techniques.

Science.gov (United States)

de Ambrosis, A; Vishnumohan, S; Paterson, J; Haber, P; Arcot, J

2017-01-01

The aim of the study was to measure the relative bioavailability of labeled pteroylglutamic acid (13C5-PteGlu) from a pectin-coated fortified rice in vivo to measure any effect of the edible coating on folic acid bioavailability. Healthy volunteers (N=26) aged 18-39 years received three test meals in three randomized short-term cross-over trials: Trial 1: aqueous 400 μg 13C5-PteGlu, Trial 2: 200 g cooked white rice+400 μg 13C5-PteGlu,Trial 3: 200 g fortified cooked white rice with pectin-coated premix containing 400 μg 13C5-PteGlu. Blood samples were drawn at 0,1,2,5 and 8 h postprandial. The concentration of 13C5-5 methyl-tetrahydrofolate appearing in plasma was quantified using high performance liquid chromatography-mass spectrometry (MS)/MS. For 24 h before baseline estimation and during the area under the curve (AUC) study, the subjects were placed on a low folate diet (∼100 μg/day). The relative bioavailability of the folic acid following Trial 3 was measured by comparing the 13C5-5 methyl-tetrahydrofuran (THF) AUC with Trials 1 and 2. The bioavailability of folic acid in a pectin-coated rice premix was 68.7% (range 47-105) and 86.5% (range 65-115) in uncoated fortified rice relative to aqueous folic acid. This study is the first demonstration of the bioavailability of folate in pectin-coated fortified rice in humans.

Carbon (14C,13C) and hydrogen (D) isotope measurements on atmospheric methane and its sources (sinks)

International Nuclear Information System (INIS)

Levin, I.; Doerr, H.

1991-07-01

Concentrations and isotope ratios ( 13 C/ 12 C and D/H) of atmospheric methane and methane from other sources were determined by gas chromatography and mass spectroscopy in specially prepared samples. The results were used for assessing the atmospheric methane inventory. (BBR) [de

Pyrolysis compound specific isotopic analysis (δ13C and δD Py-CSIA) of soil organic matter size fractions under four vegetation covers.

Science.gov (United States)

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

2015-04-01

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

A facile synthesis of. delta. -aminolevulinic acid (ALA) regio-selectively labeled with sup 13 C and direct observation of enzymatic transformation from ALA to porphobilinogen (PBG)

Energy Technology Data Exchange (ETDEWEB)

Kurumaya, Katsuyuki; Okazaki, Takeo; Seido, Nobuo; Akasaka, Yuzuru; Kawajiri, Yoshiki; Kajiwara, Masahiro (Meiji College of Pharmacy, Tokyo (Japan)); Kondo, Masao (Institute of Public Health, Tokyo (Japan))

1989-02-01

{delta}-Aminolevulinic acid (ALA), labeled with {sup 13}C at position 1, 2, 3, 4, or 5, was synthesized from {sup 13}C-labeled glycine, Meldrum's acid, or bromoacetate. The latter compounds were prepared from {sup 13}C-sodium acetate or {sup 13}C-acetic acid. Enzymatic transformation from ALA to porphobilinogen (PBG) was directly observed by {sup 13}C-NMR. (author).

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

Science.gov (United States)

Qi, Haiping; Coplen, Tyler B.; Geilmann, Heike; Brand, Willi A.; Böhlke, J.K.

2003-01-01

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

Whole-core analysis by 13C NMR

International Nuclear Information System (INIS)

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

1991-01-01

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

Study for the charge symmetric systems, 12C+13N and 12C+13C with the orthogonalized coupled-reaction-channel method

International Nuclear Information System (INIS)

Imanishi, B.; Denisov, V.; Motobayashi, T.

1996-10-01

The charge-symmetric scattering systems, 12 C+ 13 N and 12 C+ 13 C have been investigated by using the orthogonalized coupled-reaction-channel (OCRC) method with the basis functions of the elastic, inelastic and transfer channels defined by the single-particle states, 1p1/2, 2s1/2, 1d5/2 and 1d3/2 of the valence nucleon in 13 N or 13 C. The data of the elastic scattering of 13 N on 12 C measured by Lienard et al. have been explained consistently with the data of the elastic and inelastic scattering of the 12 C+ 13 C system. The CRC effects both on the above systems are very strong, although those on the 12 C+ 13 N system are fairly weaker than the 12 C+ 13 C system. The role of the highly excited single-particle states 1d3/2 is particularly important in the formation of a specific CRC scheme, i.e., the formation of the covalent molecules due to the hybridization caused by the mixing of the different parity single-particle states. The fusion cross sections of the 12 C+ 13 C system at energies below the Coulomb barrier are strongly enhanced as a result of the strong CRC effects as compared with those of the 12 C+ 12 C system, while in 12 C+ 13 N system the enhancement of the sub-barrier fusion has not been observed. The above absorption mechanism for the 12 C+ 13 C system explains the lack of the molecular-resonance phenomena observed in the 12 C+ 12 C system. We check the effects of the dipole (E1) transition of the valence nucleon in 13 N (and also in 13 C) due to the core-core Coulomb interaction in the scattering at sub-barrier energies. The effects are not appreciable. (author)

Measurement of {sup 1}H-{sup 15}N and {sup 1}H-{sup 13}C residual dipolar couplings in nucleic acids from TROSY intensities

Energy Technology Data Exchange (ETDEWEB)

Ying Jinfa [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Wang Jinbu [National Cancer Institute, National Institutes of Health, Structural Biophysics Laboratory (United States); Grishaev, Alex [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Yu Ping; Wang Yunxing [National Cancer Institute, National Institutes of Health, Structural Biophysics Laboratory (United States); Bax, Ad, E-mail: bax@nih.gov [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

2011-09-15

Analogous to the recently introduced ARTSY method for measurement of one-bond {sup 1}H-{sup 15}N residual dipolar couplings (RDCs) in large perdeuterated proteins, we introduce methods for measurement of base {sup 13}C-{sup 1}H and {sup 15}N-{sup 1}H RDCs in protonated nucleic acids. Measurements are based on quantitative analysis of intensities in {sup 1}H-{sup 15}N and {sup 13}C-{sup 1}H TROSY-HSQC spectra, and are illustrated for a 71-nucleotide adenine riboswitch. Results compare favorably with those of conventional frequency-based measurements in terms of completeness and convenience of use. The ARTSY method derives the size of the coupling from the ratio of intensities observed in two TROSY-HSQC spectra recorded with different dephasing delays, thereby minimizing potential resonance overlap problems. Precision of the RDC measurements is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC reference spectrum, and is approximately given by 30/(S/N) Hz for {sup 15}N-{sup 1}H and 65/(S/N) Hz for {sup 13}C-{sup 1}H. The signal-to-noise ratio of both {sup 1}H-{sup 15}N and {sup 1}H-{sup 13}C spectra greatly benefits when water magnetization during the experiments is not perturbed, such that rapid magnetization transfer from bulk water to the nucleic acid, mediated by rapid amino and hydroxyl hydrogen exchange coupled with {sup 1}H-{sup 1}H NOE transfer, allows for fast repetition of the experiment. RDCs in the mutated helix 1 of the riboswitch are compatible with nucleotide-specifically modeled, idealized A-form geometry and a static orientation relative to the helix 2/3 pair, which differs by ca 6 Degree-Sign relative to the X-ray structure of the native riboswitch.

/sup 13/C(p vector,d)/sup 12/C and /sup 208/Pb(p vector,d)/sup 207/Pb reactions at 123 MeV

Energy Technology Data Exchange (ETDEWEB)

Kraushaar, J J; Shepard, J R [Colorado Univ., Boulder (USA). Nuclear Physics Lab.; Miller, D W; Jacobs, W W; Jones, W P; Devins, D W [Indiana Univ., Bloomington (USA). Dept. of Physics

1983-02-01

Cross-section and analyzing power angular distributions have been measured for /sup 13/C(p vector,d) and /sup 208/Pb(p vector, d) at 123 MeV to the strong low-lying residual states in both final nuclei. The data have been compared with the results of both zero- and exact-finite-range distorted wave calculations and some serious discrepancies were noted for the analyzing powers. For the case of the /sup 13/C calculations, marked improvement in the description of the data was achieved with the use of a damping factor in the nuclear interior.

1H and 13C NMR spectral data of new saponins from Cordia piauhiensis.

Science.gov (United States)

Santos, Renata P; Silveira, Edilberto R; Uchôa, Daniel Esdras de A; Pessoa, Otília Deusdênia L; Viana, Francisco Arnaldo; Braz-Filho, Raimundo

2007-08-01

Two new bidesmoside triterpenoid saponins were isolated from stems of Cordia piauhiensis. Their structures, characterized as 3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl pomolic acid 28-O-beta-D-glucopyranosyl ester (1) and 3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranosyl ester (2), were unequivocally established after extensive NMR (1H, 13C, DEPT 135 degrees, COSY, HSQC, HMBC, TOCSY, and NOESY) studies. Copyright 2007 John Wiley & Sons, Ltd.

The Indicator Amino Acid Oxidation Method with the Use of l-[1-13C]Leucine Suggests a Higher than Currently Recommended Protein Requirement in Children with Phenylketonuria.

Science.gov (United States)

Turki, Abrar; Ueda, Keiko; Cheng, Barbara; Giezen, Alette; Salvarinova, Ramona; Stockler-Ipsiroglu, Sylvia; Elango, Rajavel

2017-02-01

Phenylketonuria is characterized by mutations in the Phe hydroxylase gene that leads to the accumulation of Phe in plasma and the brain. The standard of care for phenylketonuria is nutritional management with dietary restriction of Phe and the provision of sufficient protein and energy for growth and health maintenance. The protein requirement in children with phenylketonuria is empirically determined based upon phenylketonuria nutritional guidelines that are adjusted individually in response to biochemical markers and growth. We determined dietary protein requirements in children with phenylketonuria with the use of the indicator amino acid oxidation (IAAO) technique, with l-[1- 13 C]Leu as the indicator amino acid. Four children (2 males; 2 females) aged 9-18 y with phenylketonuria [mild hyperphenylalanemia (mHPA); 6-10 mg/dL (360-600 μmol/L)] were recruited to participate in ≥7 separate test protein intakes (range: 0.2-3.2 g ⋅ kg -1 ⋅ d -1 ) with the IAAO protocol with the use of l-[1- 13 C]Leu followed by the collection of breath and urine samples over 8 h. The diets were isocaloric and provided energy at 1.7 times the resting energy expenditure. Protein was provided as a crystalline amino acid mixture based on an egg protein pattern, except Phe and Leu, which were maintained at a constant across intakes. Protein requirement was determined with the use of a 2-phase linear-regression crossover analysis of the rate of l-[1- 13 C]Leu tracer oxidation. The mean protein requirement was determined to be 1.85 g ⋅ kg -1 ⋅ d -1 (R 2 = 0.66; 95% CI: 1.37, 2.33). This result is substantially higher than the 2014 phenylketonuria recommendations (1.14-1.33 g ⋅ kg -1 ⋅ d -1 ; based on 120-140% above the current RDA for age). To our knowledge, this is the first study to directly define a quantitative requirement for protein intake in children with mHPA and indicates that current protein recommendations in children with phenylketonuria may be insufficient. This

Dereplication of depsides from the lichen Pseudevernia furfuracea by centrifugal partition chromatography combined to 13C nuclear magnetic resonance pattern recognition

International Nuclear Information System (INIS)

Oettl, Sarah K.; Hubert, Jane; Nuzillard, Jean-Marc; Stuppner, Hermann; Renault, Jean-Hugues; Rollinger, Judith M.

2014-01-01

Highlights: • The major depsides of a lichen extract were directly identified within mixtures. • The initial extract was rapidly fractionated by CPC in the pH-zone refining mode. • Hierarchical clustering of 13 C NMR signals resulted in the identification of depside molecular skeletons. • 13 C chemical shift clusters were assigned to structures using a 13 C NMR database. • Six depsides were unambiguously identified by this approach. - Abstract: Lichens produce a diversity of secondary metabolites, among them depsides comprised of two or more hydroxybenzoic acid units linked by ester, ether, or C-C-bonds. During classic solid support-based purification processes, depsides are often hydrolyzed and in many cases time, consuming procedures result only in the isolation of decomposition products. In an attempt to avoid extensive purification steps while maintaining metabolite structure integrity, we propose an alternative method to identify the major depsides of a lichen crude extract (Pseudevernia furfuracea var. ceratea (Ach.) D. Hawksw., Parmeliaceae) directly within mixtures. Exploiting the acidic character of depsides and differences in polarity, the extract was fractionated by centrifugal partition chromatography in the pH-zone refining mode resulting in twelve simplified mixtures of depsides. After 13 C nuclear magnetic resonance analysis of the produced fractions, the major molecular structures were directly identified within the fraction series by using a recently developed pattern recognition method, which combines spectral data alignment and hierarchical clustering analysis. The obtained clusters of 13 C chemical shifts were assigned to their corresponding molecular structures with the help of an in-house 13 C NMR chemical shift database, resulting in six unambiguously identified compounds, namely methyl β-orcinolcarboxylate (1), atranorin (2), 5-chloroatranorin (3), olivetol carboxylic acid (4), olivetoric acid (5), and olivetonide (6)

Practical use of chemical shift databases for protein solid-state NMR: 2D chemical shift maps and amino-acid assignment with secondary-structure information

International Nuclear Information System (INIS)

Fritzsching, K. J.; Yang, Y.; Schmidt-Rohr, K.; Hong Mei

2013-01-01

We introduce a Python-based program that utilizes the large database of 13 C and 15 N chemical shifts in the Biological Magnetic Resonance Bank to rapidly predict the amino acid type and secondary structure from correlated chemical shifts. The program, called PACSYlite Unified Query (PLUQ), is designed to help assign peaks obtained from 2D 13 C– 13 C, 15 N– 13 C, or 3D 15 N– 13 C– 13 C magic-angle-spinning correlation spectra. We show secondary-structure specific 2D 13 C– 13 C correlation maps of all twenty amino acids, constructed from a chemical shift database of 262,209 residues. The maps reveal interesting conformation-dependent chemical shift distributions and facilitate searching of correlation peaks during amino-acid type assignment. Based on these correlations, PLUQ outputs the most likely amino acid types and the associated secondary structures from inputs of experimental chemical shifts. We test the assignment accuracy using four high-quality protein structures. Based on only the Cα and Cβ chemical shifts, the highest-ranked PLUQ assignments were 40–60 % correct in both the amino-acid type and the secondary structure. For three input chemical shifts (CO–Cα–Cβ or N–Cα–Cβ), the first-ranked assignments were correct for 60 % of the residues, while within the top three predictions, the correct assignments were found for 80 % of the residues. PLUQ and the chemical shift maps are expected to be useful at the first stage of sequential assignment, for combination with automated sequential assignment programs, and for highly disordered proteins for which secondary structure analysis is the main goal of structure determination.

Deuterium isotope effects on 13C and 15N chemical shifts of intramolecularly hydrogen-bonded enaminocarbonyl derivatives of Meldrum’s and Tetronic acid

Science.gov (United States)

Ullah, Saif; Zhang, Wei; Hansen, Poul Erik

2010-07-01

Secondary deuterium isotope effects on 13C and 15N nuclear shieldings in a series of cyclic enamino-diesters and enamino-esters and acyclic enaminones and enamino-esters have been examined and analysed using NMR and DFT (B3LYP/6-31G(d,p)) methods. One-dimensional and two-dimensional NMR spectra of enaminocarbonyl and their deuterated analogues were recorded in CDCl 3 and CD 2Cl 2 at variable temperatures and assigned. 1JNH coupling constants for the derivatives of Meldrum's and tetronic acids reveal that they exist at the NH-form. It was demonstrated that deuterium isotope effects, for the hydrogen bonded compounds, due to the deuterium substitution at the nitrogen nucleus lead to large one-bond isotope effects at nitrogen, 1Δ 15N(D), and two-bond isotope effects on carbon nuclei, 2ΔC(ND), respectively. A linear correlations exist between 2ΔC(ND) and 1Δ 15N(D) whereas the correlation with δNH is divided into two. A good agreement between the experimentally observed 2ΔC(ND) and calculated dσ 13C/dR NH was obtained. A very good correlation between calculated NH bond lengths and observed NH chemical shifts is found. The observed isotope effects are shown to depend strongly on Resonance Assisted Hydrogen bonding.

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

Science.gov (United States)

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

2015-04-01

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

Preparation of 15N-13C-fulminic acid

International Nuclear Information System (INIS)

Wilmes, R.; Winnewisser, M.

1993-01-01

The precursor for the title compound was prepared in a three-step synthesis. The 13 C-label was incorporated in the first step employing 2- 13 C-ethyl acetate and the 15 N-label in the last step, using 15 N-sodium nitrite. Upon pyrolysis the precursor forms three fragments, one of them being the title compound. (Author)

Technique development of 3D dynamic CS-EPSI for hyperpolarized 13 C pyruvate MR molecular imaging of human prostate cancer.

Science.gov (United States)

Chen, Hsin-Yu; Larson, Peder E Z; Gordon, Jeremy W; Bok, Robert A; Ferrone, Marcus; van Criekinge, Mark; Carvajal, Lucas; Cao, Peng; Pauly, John M; Kerr, Adam B; Park, Ilwoo; Slater, James B; Nelson, Sarah J; Munster, Pamela N; Aggarwal, Rahul; Kurhanewicz, John; Vigneron, Daniel B

2018-03-25

The purpose of this study was to develop a new 3D dynamic carbon-13 compressed sensing echoplanar spectroscopic imaging (EPSI) MR sequence and test it in phantoms, animal models, and then in prostate cancer patients to image the metabolic conversion of hyperpolarized [1- 13 C]pyruvate to [1- 13 C]lactate with whole gland coverage at high spatial and temporal resolution. A 3D dynamic compressed sensing (CS)-EPSI sequence with spectral-spatial excitation was designed to meet the required spatial coverage, time and spatial resolution, and RF limitations of the 3T MR scanner for its clinical translation for prostate cancer patient imaging. After phantom testing, animal studies were performed in rats and transgenic mice with prostate cancers. For patient studies, a GE SPINlab polarizer (GE Healthcare, Waukesha, WI) was used to produce hyperpolarized sterile GMP [1- 13 C]pyruvate. 3D dynamic 13 C CS-EPSI data were acquired starting 5 s after injection throughout the gland with a spatial resolution of 0.5 cm 3 , 18 time frames, 2-s temporal resolution, and 36 s total acquisition time. Through preclinical testing, the 3D CS-EPSI sequence developed in this project was shown to provide the desired spectral, temporal, and spatial 5D HP 13 C MR data. In human studies, the 3D dynamic HP CS-EPSI approach provided first-ever simultaneously volumetric and dynamic images of the LDH-catalyzed conversion of [1- 13 C]pyruvate to [1- 13 C]lactate in a biopsy-proven prostate cancer patient with full gland coverage. The results demonstrate the feasibility to characterize prostate cancer metabolism in animals, and now patients using this new 3D dynamic HP MR technique to measure k PL , the kinetic rate constant of [1- 13 C]pyruvate to [1- 13 C]lactate conversion. © 2018 International Society for Magnetic Resonance in Medicine.

EDGE2D Simulations of JET 13C Migration Experiments

International Nuclear Information System (INIS)

Strachan, J.D.; Coad, J.P.; Corrigan, G.; Matthews, G.F.; Spence, J.

2004-01-01

Material migration has received renewed interest due to tritium retention associated with carbon transport to remote vessel locations. Those results influence the desirability of carbon usage on ITER. Subsequently, additional experiments have been performed, including tracer experiments attempting to identify material migration from specific locations. In this paper, EDGE2D models a well-diagnosed JET 13 C tracer migration experiment. The role of SOL flows upon the migration patterns is identified

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

Science.gov (United States)

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

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

Science.gov (United States)

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

2015-12-01

The oleaginous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with (13)C-glucose were performed with the growth of M. circinelloides and subsequent gas chromatography-mass spectrometric detection of (13)C-patterns in proteinogenic amino acids was carried out to identify the metabolic network topology and estimate intracellular fluxes. Our results showed that the high oleaginous strain WJ11 had higher flux of pentose phosphate pathway and malic enzyme, lower flux in tricarboxylic acid cycle, higher flux in glyoxylate cycle and ATP: citrate lyase, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Dereplication of depsides from the lichen Pseudevernia furfuracea by centrifugal partition chromatography combined to {sup 13}C nuclear magnetic resonance pattern recognition

Energy Technology Data Exchange (ETDEWEB)

Oettl, Sarah K. [Institute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80–82, 6020 Innsbruck (Austria); Hubert, Jane, E-mail: jane.hubert@univ-reims.fr [Institut de Chimie Moléculaire de Reims (UMR CNRS 7312), SFR CAP' sANTE, UFR de Pharmacie, Université de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2 (France); Nuzillard, Jean-Marc [Institut de Chimie Moléculaire de Reims (UMR CNRS 7312), SFR CAP' sANTE, UFR de Pharmacie, Université de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2 (France); Stuppner, Hermann [Institute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80–82, 6020 Innsbruck (Austria); Renault, Jean-Hugues [Institut de Chimie Moléculaire de Reims (UMR CNRS 7312), SFR CAP' sANTE, UFR de Pharmacie, Université de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2 (France); Rollinger, Judith M. [Institute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80–82, 6020 Innsbruck (Austria)

2014-10-10

Highlights: • The major depsides of a lichen extract were directly identified within mixtures. • The initial extract was rapidly fractionated by CPC in the pH-zone refining mode. • Hierarchical clustering of {sup 13}C NMR signals resulted in the identification of depside molecular skeletons. • {sup 13}C chemical shift clusters were assigned to structures using a {sup 13}C NMR database. • Six depsides were unambiguously identified by this approach. - Abstract: Lichens produce a diversity of secondary metabolites, among them depsides comprised of two or more hydroxybenzoic acid units linked by ester, ether, or C-C-bonds. During classic solid support-based purification processes, depsides are often hydrolyzed and in many cases time, consuming procedures result only in the isolation of decomposition products. In an attempt to avoid extensive purification steps while maintaining metabolite structure integrity, we propose an alternative method to identify the major depsides of a lichen crude extract (Pseudevernia furfuracea var. ceratea (Ach.) D. Hawksw., Parmeliaceae) directly within mixtures. Exploiting the acidic character of depsides and differences in polarity, the extract was fractionated by centrifugal partition chromatography in the pH-zone refining mode resulting in twelve simplified mixtures of depsides. After {sup 13}C nuclear magnetic resonance analysis of the produced fractions, the major molecular structures were directly identified within the fraction series by using a recently developed pattern recognition method, which combines spectral data alignment and hierarchical clustering analysis. The obtained clusters of {sup 13}C chemical shifts were assigned to their corresponding molecular structures with the help of an in-house {sup 13}C NMR chemical shift database, resulting in six unambiguously identified compounds, namely methyl β-orcinolcarboxylate (1), atranorin (2), 5-chloroatranorin (3), olivetol carboxylic acid (4), olivetoric acid (5

Simultaneous determination of wine sugars, glycerol and organic acids 13C/12C isotopic ratio by ion chromatography-co-IRMS

Directory of Open Access Journals (Sweden)

Guyon Francois

2014-01-01

Full Text Available Ion chromatography (IC isotope ratio mass spectrometry (IRMS coupling is possible using a liquid interface allowing chemical oxidation (co of organic compounds. Synthetic solutions containing a mixture of glycerol, sugars (sucrose, glucose and fructose and organic acids (gluconic, lactic, malic, tartaric, oxalic, fumaric, citric and isocitric were elaborated to estimate analytical applications of two different IC systems. It appears that the use of carbonated solution in the eluting phase is a perturbation for 13C isotope measurements as it creates a δ13C deviation from the expected values. A sample of authentic sweet wine was analyzed by IC-co-IRMS using KOH gradient. Ratios of isotopic, ratios of organic acids and glycerol was found to be, in average, equal 1.01 ± 0.04 that is in accordance with our previous results.

CACA-TOCSY with alternate 13C–12C labeling: a 13Cα direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification

Science.gov (United States)

Takeuchi, Koh; Frueh, Dominique P.; Sun, Zhen-Yu J.; Hiller, Sebastian

2010-01-01

We present a 13C direct detection CACA-TOCSY experiment for samples with alternate 13C–12C labeling. It provides inter-residue correlations between 13Cα resonances of residue i and adjacent Cαs at positions i − 1 and i + 1. Furthermore, longer mixing times yield correlations to Cα nuclei separated by more than one residue. The experiment also provides Cα-to-sidechain correlations, some amino acid type identifications and estimates for ψ dihedral angles. The power of the experiment derives from the alternate 13C–12C labeling with [1,3-13C] glycerol or [2-13C] glycerol, which allows utilizing the small scalar 3JCC couplings that are masked by strong 1JCC couplings in uniformly 13C labeled samples. PMID:20383561

Combined chemometric analysis of (1)H NMR, (13)C NMR and stable isotope data to differentiate organic and conventional milk.

Science.gov (United States)

Erich, Sarah; Schill, Sandra; Annweiler, Eva; Waiblinger, Hans-Ulrich; Kuballa, Thomas; Lachenmeier, Dirk W; Monakhova, Yulia B

2015-12-01

The increased sales of organically produced food create a strong need for analytical methods, which could authenticate organic and conventional products. Combined chemometric analysis of (1)H NMR-, (13)C NMR-spectroscopy data, stable-isotope data (IRMS) and α-linolenic acid content (gas chromatography) was used to differentiate organic and conventional milk. In total 85 raw, pasteurized and ultra-heat treated (UHT) milk samples (52 organic and 33 conventional) were collected between August 2013 and May 2014. The carbon isotope ratios of milk protein and milk fat as well as the α-linolenic acid content of these samples were determined. Additionally, the milk fat was analyzed by (1)H and (13)C NMR spectroscopy. The chemometric analysis of combined data (IRMS, GC, NMR) resulted in more precise authentication of German raw and retail milk with a considerably increased classification rate of 95% compared to 81% for NMR and 90% for IRMS using linear discriminate analysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Synthesis of the mevalonic acid labelled with "1"4C, "1"3C and "3H

International Nuclear Information System (INIS)

Rousseau, Bernard

1982-01-01

This thesis describes five new methods of synthesis of the (R,S) mevalonic acid adapted to the labelling with "1"4C and "1"3C in positions 4,5 or 5 or 3', or with tritium in position 3'. Three of them use the tri-oxa-2,4,10 adamantyl group as masked carboxyl function. The two others take benefit from the regioselectivity of the bis-hydro-boration of terminal acetylenics by the 9-borabicyclo [3-3-1]nonane. The acylation of the bis-trimethylsilyl lithiomalonate, and the chemistry of dithiannes are also involved. Acetylene and methyl iodide labelled with isotopes are used as cheap base products [fr

Quantitative analysis of deuterium using the isotopic effect on quaternary {sup 13}C NMR chemical shifts

Energy Technology Data Exchange (ETDEWEB)

Darwish, Tamim A., E-mail: tamim.darwish@ansto.gov.au [National Deuteration Facility, Australian Nuclear Science and Technology Organisation, Locked Bag 21, Kirrawee DC, NSW 2232 (Australia); Yepuri, Nageshwar Rao; Holden, Peter J. [National Deuteration Facility, Australian Nuclear Science and Technology Organisation, Locked Bag 21, Kirrawee DC, NSW 2232 (Australia); James, Michael [Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168 (Australia)

2016-07-13

Quantitative analysis of specifically deuterated compounds can be achieved by a number of conventional methods, such as mass spectroscopy, or by quantifying the residual {sup 1}H NMR signals compared to signals from internal standards. However, site specific quantification using these methods becomes challenging when dealing with non-specifically or randomly deuterated compounds that are produced by metal catalyzed hydrothermal reactions in D{sub 2}O, one of the most convenient deuteration methods. In this study, deuterium-induced NMR isotope shifts of quaternary {sup 13}C resonances neighboring deuterated sites have been utilized to quantify the degree of isotope labeling of molecular sites in non-specifically deuterated molecules. By probing {sup 13}C NMR signals while decoupling both proton and deuterium nuclei, it is possible to resolve {sup 13}C resonances of the different isotopologues based on the isotopic shifts and the degree of deuteration of the carbon atoms. We demonstrate that in different isotopologues, the same quaternary carbon, neighboring partially deuterated carbon atoms, are affected to an equal extent by relaxation. Decoupling both nuclei ({sup 1}H, {sup 2}H) resolves closely separated quaternary {sup 13}C signals of the different isotopologues, and allows their accurate integration and quantification under short relaxation delays (D1 = 1 s) and hence fast accumulative spectral acquisition. We have performed a number of approaches to quantify the deuterium content at different specific sites to demonstrate a convenient and generic analysis method for use in randomly deuterated molecules, or in cases of specifically deuterated molecules where back-exchange processes may take place during work up. - Graphical abstract: The relative intensities of quaternary {sup 13}C {"1H,"2H} resonances are equal despite the different relaxation delays, allowing the relative abundance of the different deuterated isotopologues to be calculated using NMR fast

13C metabolic flux analysis: optimal design of isotopic labeling experiments.

Science.gov (United States)

Antoniewicz, Maciek R

2013-12-01

Measuring fluxes by 13C metabolic flux analysis (13C-MFA) has become a key activity in chemical and pharmaceutical biotechnology. Optimal design of isotopic labeling experiments is of central importance to 13C-MFA as it determines the precision with which fluxes can be estimated. Traditional methods for selecting isotopic tracers and labeling measurements did not fully utilize the power of 13C-MFA. Recently, new approaches were developed for optimal design of isotopic labeling experiments based on parallel labeling experiments and algorithms for rational selection of tracers. In addition, advanced isotopic labeling measurements were developed based on tandem mass spectrometry. Combined, these approaches can dramatically improve the quality of 13C-MFA results with important applications in metabolic engineering and biotechnology. Copyright © 2013 Elsevier Ltd. All rights reserved.

Correlation between the 12C+12C, 12C+13C, and 13C+13C fusion cross sections

Science.gov (United States)

Notani, M.; Esbensen, H.; Fang, X.; Bucher, B.; Davies, P.; Jiang, C. L.; Lamm, L.; Lin, C. J.; Ma, C.; Martin, E.; Rehm, K. E.; Tan, W. P.; Thomas, S.; Tang, X. D.; Brown, E.

2012-01-01

The fusion cross section for 12C+13C has been measured down to Ec.m.=2.6 MeV, at which the cross section is of the order of 20 nb. By comparing the cross sections for the three carbon isotope systems, 12C+12C, 12C+13C, and 13C+13C, it is found that the cross sections for 12C+13C and 13C+13C provide an upper limit for the fusion cross section of 12C+12C over a wide energy range. After calibrating the effective nuclear potential for 12C+12C using the 12C+13C and 13C+13C fusion cross sections, it is found that a coupled-channels calculation with the ingoing wave boundary condition (IWBC) is capable of predicting the major peak cross sections in 12C+12C. A qualitative explanation for this upper limit is provided by the Nogami-Imanishi model and by level density differences among the compound nuclei. It is found that the strong resonance found at 2.14 MeV in 12C+12C exceeds this upper limit by a factor of more than 20. The preliminary result from the most recent measurement shows a much smaller cross section at this energy, which agrees with our predicted upper limit.

Lactic acid-producing yeast cells having nonfunctional L- or D-lactate:ferricytochrome C oxidoreductase cells

Science.gov (United States)

Miller, Matthew [Boston, MA; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Highland Ranch, CO; Hause, Benjamin Matthew [Currie, MN; Van Hoek, Pim [Camarillo, CA; Dundon, Catherine Asleson [Minneapolis, MN

2012-03-20

Yeast cells having an exogenous lactate dehydrogenase gene ae modified by reducing L- or D-lactate:ferricytochrome c oxidoreductase activity in the cell. This leads to reduced consumption of lactate by the cell and can increase overall lactate yields in a fermentation process. Cells having the reduced L- or D-lactate:ferricytochrome c oxidoreductase activity can be screened for by resistance to organic acids such as lactic or glycolic acid.

Solid-state NMR detection of 14N-13C dipolar couplings between amino acid side groups provides constraints on amyloid fibril architecture.

Science.gov (United States)

Middleton, David A

2011-02-01

Solid-state nuclear magnetic resonance (SSNMR) is a powerful technique for the structural analysis of amyloid fibrils. With suitable isotope labelling patterns, SSNMR can provide constraints on the secondary structure, alignment and registration of β-strands within amyloid fibrils and identify the tertiary and quaternary contacts defining the packing of the β-sheet layers. Detection of (14)N-(13)C dipolar couplings may provide potentially useful additional structural constraints on β-sheet packing within amyloid fibrils but has not until now been exploited for this purpose. Here a frequency-selective, transfer of population in double resonance SSNMR experiment is used to detect a weak (14)N-(13)C dipolar coupling in amyloid-like fibrils of the peptide H(2)N-SNNFGAILSS-COOH, which was uniformly (13)C and (15)N labelled across the four C-terminal amino acids. The (14)N-(13)C interatomic distance between leucine and asparagine side groups is constrained between 2.4 and 3.8 Å, which allows current structural models of the β-spine arrangement within the fibrils to be refined. This procedure could be useful for the general structural analysis of other proteins in condensed phases and environments, such as biological membranes. Copyright © 2011 John Wiley & Sons, Ltd.

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

Science.gov (United States)

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

2014-05-01

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

13C-NMR chemical shift databases as a quick tool to evaluate structural models of humic substances

DEFF Research Database (Denmark)

Nyrop Albers, Christian; Hansen, Poul Erik

2010-01-01

Models for humic and fulvic acids are discussed based on 13C liquid state NMR spectra combined with results from elemental analysis and titration studies. The analysis of NMR spectra is based on a full reconstruction of the NMR spectrum done with help of 13C-NMR data bases by adding up chemical...... side missing structural elements in the models can be suggested. A number of proposed structures for humic and fulvic acids are discussed based on the above analysis....

Ageing processes and soil microbial community effects on the biodegradation of soil 13C-2,4-D nonextractable residues

International Nuclear Information System (INIS)

Lerch, T.Z.; Dignac, M.-F.; Nunan, N.; Barriuso, E.; Mariotti, A.

2009-01-01

The biodegradation of nonextractable residues (NER) of pesticides in soil is still poorly understood. The aim of this study was to evaluate the influence of NER ageing and fresh soil addition on the microbial communities responsible for their mineralisation. Soil containing either 15 or 90-day-old NER of 13 C-2,4-D (NER15 and NER90, respectively) was incubated for 90 days with or without fresh soil. The addition of fresh soil had no effect on the mineralisation of NER90 or of SOM, but increased the extent and rate of NER15 mineralisation. The analyses of 13 C-enriched FAME (fatty acids methyl esters) profiles showed that the fresh soil amendment only influenced the amount and structure of microbial populations responsible for the biodegradation of NER15. By coupling biological and chemical analyses, we gained some insight into the nature and the biodegradability of pesticide NER. - Ageing processes influence the NER mineralisation rate and the microbial population involved.

OEDGE modeling of the DIII-D double null (CH4)-C-13 puffing experiment

International Nuclear Information System (INIS)

Elder, J.D.; Wampler, W.R.; McLean, A.G.; Stangeby, P.C.; Allen, S.L.; Bray, B.D.; Brooks, N.H.; Leonard, A.W.; Unterberg, Ezekial A.; Watkins, J.G.

2011-01-01

Unbalanced double null ELMy H-mode configurations in DIII-D are used to simulate the situation in ITER high triangularity, burning plasma magnetic equilibria, where the second X-point lies close to the top of the vacuum vessel, creating a secondary divertor region at the upper blanket modules. The measured plasma conditions in the outer secondary divertor closely duplicated those projected for ITER. (CH4)-C-13 was injected into the secondary outer divertor to simulate sputtering there. The majority of the C-13 found was in the secondary outer divertor. This material migration pattern is radically different than that observed for main wall (CH4)-C-13 injections into single null configurations where the deposition is primarily at the inner divertor. The implications for tritium codeposition resulting from sputtering at the secondary divertor in ITER are significant since release of tritium from Be co-deposits at the main wall bake temperature for ITER, 240 degrees C, is incomplete. The principal features of the measured C-13 deposition pattern have been replicated by the OEDGE interpretive code.

Site-selective 13C labeling of proteins using erythrose

International Nuclear Information System (INIS)

Weininger, Ulrich

2017-01-01

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with 13 C and/or 1 H, which is achieved in the most general way by using site-selectively 13 C-enriched glucose (1- and 2- 13 C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively 13 C-enriched erythrose (1-, 2-, 3- and 4- 13 C) as a suitable precursor for 13 C labeled aromatic side chains. We quantify 13 C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the 13 C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated 13 C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective 13 C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.

5D {sup 13}C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion

Energy Technology Data Exchange (ETDEWEB)

Novacek, Jiri [Masaryk University, Faculty of Science, NCBR, and CEITEC (Czech Republic); Zawadzka-Kazimierczuk, Anna [University of Warsaw, Faculty of Chemistry (Poland); Papouskova, Veronika; Zidek, Lukas, E-mail: lzidek@chemi.muni.cz [Masaryk University, Faculty of Science, NCBR, and CEITEC (Czech Republic); Sanderova, Hana; Krasny, Libor [Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Molecular Genetics of Bacteria and Department of Bacteriology (Czech Republic); Kozminski, Wiktor [University of Warsaw, Faculty of Chemistry (Poland); Sklenar, Vladimir [Masaryk University, Faculty of Science, NCBR, and CEITEC (Czech Republic)

2011-05-15

Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of {sup 13}C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.

Carbon-13 kinetic isotope effects in the decarbonylation of lactic acid of natural isotopic composition in phosphoric acid medium

International Nuclear Information System (INIS)

Zielinski, M.; Czarnota, G.; Papiernik-Zielinska, H.; Kasprzyk, G.; Gumolka, L.; Staedter, W.

1993-01-01

The 13 C kinetic isotope effect fractionation in the decarbonylation of lactic acid (LA) of natural isotopic composition by concentrated phosphoric acids (PA) and by 85% H 3 PO 4 has been studied in the temperature interval of 60-150 deg C. The values of the 13 C (1) isotope effects in the decarbonylation of lactic acid in 100% H 3 PO 4 , in pyrophosphoric acid and in more concentrated phosphoric acids are intermediate between the values calculated assuming that the C (1)- OH bond is broken in the rate-controlling step of dehydration and those calculated for rupture of the carbon-carbon bond in the transition state. In the temperature interval of 90-130 deg C the experimental 13 C fractionation factors determined in concentrated PA approach quite closely the 13 C fractionation corresponding to C (2)- C (1) bond scission. The 13 C (1) kinetic isotope effects in the decarbonylation of LA in 85% orthophosphoric acid in the temperature range of 110-150 deg C coincide with the 13 C isotope effects calculated assuming that the frequency corresponding to the C (1) -OH vibration is lost in the transition state of decarbonylation. A change of the mechanism of decarbonylation of LA in going from concentrated PA medium to 85% H 3 PO 4 has been suggested. A possible secondary 18 O and a primary 18 O kinetic isotope effect in decarbonylation of lactic acid in phosphoric acids media have been discussed, too. (author) 21 refs.; 3 tabs

Two-Scale 13C Metabolic Flux Analysis for Metabolic Engineering.

Science.gov (United States)

Ando, David; Garcia Martin, Hector

2018-01-01

Accelerating the Design-Build-Test-Learn (DBTL) cycle in synthetic biology is critical to achieving rapid and facile bioengineering of organisms for the production of, e.g., biofuels and other chemicals. The Learn phase involves using data obtained from the Test phase to inform the next Design phase. As part of the Learn phase, mathematical models of metabolic fluxes give a mechanistic level of comprehension to cellular metabolism, isolating the principle drivers of metabolic behavior from the peripheral ones, and directing future experimental designs and engineering methodologies. Furthermore, the measurement of intracellular metabolic fluxes is specifically noteworthy as providing a rapid and easy-to-understand picture of how carbon and energy flow throughout the cell. Here, we present a detailed guide to performing metabolic flux analysis in the Learn phase of the DBTL cycle, where we show how one can take the isotope labeling data from a 13 C labeling experiment and immediately turn it into a determination of cellular fluxes that points in the direction of genetic engineering strategies that will advance the metabolic engineering process.For our modeling purposes we use the Joint BioEnergy Institute (JBEI) Quantitative Metabolic Modeling (jQMM) library, which provides an open-source, python-based framework for modeling internal metabolic fluxes and making actionable predictions on how to modify cellular metabolism for specific bioengineering goals. It presents a complete toolbox for performing different types of flux analysis such as Flux Balance Analysis, 13 C Metabolic Flux Analysis, and it introduces the capability to use 13 C labeling experimental data to constrain comprehensive genome-scale models through a technique called two-scale 13 C Metabolic Flux Analysis (2S- 13 C MFA) [1]. In addition to several other capabilities, the jQMM is also able to predict the effects of knockouts using the MoMA and ROOM methodologies. The use of the jQMM library is

Mechanistic studies of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

International Nuclear Information System (INIS)

Dotson, G.D.; Woodard, R.W.

1994-01-01

The enzyme 3-deOXY-D-manno-octulosonic acid 8-phosphate synthase (KDO 8-P synthase) catalyses the condensation of arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give the unique eight-carbon acidic sugar 3-deoxy-D-nianno-octulosonic acid 8-phosphate (KDO 8-P) found only in gram-negative bacteria and required for lipid A maturation and cellular growth. The E. coli gene kdsA that encodes KDO 8-P synthase has been amplified by standard PCR methodologies. The synthetic gene, subcloned into the expression vector pT7-7 was used to infect E. coli BL 21 (DE 3). Purification of crude supernatant from this transformant on Q Sepharose yields >200 mg of near-homogeneous KDO 8-P synthase per liter of cell culture. To explore the mechanism of KDO 8-P synthase, we prepared (E)- and (Z)-(3 2 H)PEP, (2- 13 C)PEP, and (2- 13 C, 18 O)PEP chemically from the appropriately labeled 3-bromopyruvates by reaction with trimethylphosphite under Perkow reaction conditions. Our 1 H-NMR analysis of the stereochemistry at C3 of the KDO 8-Ps, obtained by separate incubation of (E)- and (Z)-(3- 2 H)PEP with A 5-P in the presence of KDO 8-P synthase, demonstrated that the reaction is stereospecific with respect to both the C3 of PEP and the C1 carbonyl of A 5-P. (Z)-(3- 2 H)PEP gave predominantly (3S)-(3 2 H)KDO 8-P and (E)-(3- 2 H)PEP gave predominantly (3R)-(3 2 H)KDO-8P, which indicates condensation of the si face of PEP upon the re face of A 5-P-an orientation analogous to that seen with the similar aldehyde Iyase DAH 7-P synthase. The fate of the enolic oxygen of (2- 13 C, 18 O)PEP, during the course of the KDO 8-P synthase-catalyzed reaction as monitored by both 13 C- and 31 P-NMR spectroscopy demonstrated that the inorganic phosphate (Pi) and not the KDO 8-P contained the 18 O

Mechanistic studies of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

Energy Technology Data Exchange (ETDEWEB)

Dotson, G.D.; Woodard, R.W. [Univ. of Michigan, Ann Arbor, MI (United States)

1994-12-01

The enzyme 3-deOXY-D-manno-octulosonic acid 8-phosphate synthase (KDO 8-P synthase) catalyses the condensation of arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give the unique eight-carbon acidic sugar 3-deoxy-D-nianno-octulosonic acid 8-phosphate (KDO 8-P) found only in gram-negative bacteria and required for lipid A maturation and cellular growth. The E. coli gene kdsA that encodes KDO 8-P synthase has been amplified by standard PCR methodologies. The synthetic gene, subcloned into the expression vector pT7-7 was used to infect E. coli BL 21 (DE 3). Purification of crude supernatant from this transformant on Q Sepharose yields >200 mg of near-homogeneous KDO 8-P synthase per liter of cell culture. To explore the mechanism of KDO 8-P synthase, we prepared (E)- and (Z)-(3{sup 2}H)PEP, (2-{sup 13}C)PEP, and (2-{sup 13}C,{sup 18}O)PEP chemically from the appropriately labeled 3-bromopyruvates by reaction with trimethylphosphite under Perkow reaction conditions. Our {sup 1}H-NMR analysis of the stereochemistry at C3 of the KDO 8-Ps, obtained by separate incubation of (E)- and (Z)-(3-{sup 2}H)PEP with A 5-P in the presence of KDO 8-P synthase, demonstrated that the reaction is stereospecific with respect to both the C3 of PEP and the C1 carbonyl of A 5-P. (Z)-(3-{sup 2}H)PEP gave predominantly (3S)-(3{sup 2}H)KDO 8-P and (E)-(3-{sup 2}H)PEP gave predominantly (3R)-(3{sup 2}H)KDO-8P, which indicates condensation of the si face of PEP upon the re face of A 5-P-an orientation analogous to that seen with the similar aldehyde Iyase DAH 7-P synthase. The fate of the enolic oxygen of (2-{sup 13}C, {sup 18}O)PEP, during the course of the KDO 8-P synthase-catalyzed reaction as monitored by both {sup 13}C- and {sup 31}P-NMR spectroscopy demonstrated that the inorganic phosphate (Pi) and not the KDO 8-P contained the {sup 18}O.

Relaxation-compensated difference spin diffusion NMR for detecting 13C–13C long-range correlations in proteins and polysaccharides

International Nuclear Information System (INIS)

Wang, Tuo; Williams, Jonathan K.; Schmidt-Rohr, Klaus; Hong, Mei

2015-01-01

The measurement of long-range distances remains a challenge in solid-state NMR structure determination of biological macromolecules. In 2D and 3D correlation spectra of uniformly 13 C-labeled biomolecules, inter-residue, inter-segmental, and intermolecular 13 C– 13 C cross peaks that provide important long-range distance constraints for three-dimensional structures often overlap with short-range cross peaks that only reflect the covalent structure of the molecule. It is therefore desirable to develop new approaches to obtain spectra containing only long-range cross peaks. Here we show that a relaxation-compensated modification of the commonly used 2D 1 H-driven spin diffusion (PDSD) experiment allows the clean detection of such long-range cross peaks. By adding a z-filter to keep the total z-period of the experiment constant, we compensate for 13 C T 1 relaxation. As a result, the difference spectrum between a long- and a scaled short-mixing time spectrum show only long-range correlation signals. We show that one- and two-bond cross peaks equalize within a few tens of milliseconds. Within ∼200 ms, the intensity equilibrates within an amino acid residue and a monosaccharide to a value that reflects the number of spins in the local network. With T 1 relaxation compensation, at longer mixing times, inter-residue and inter-segmental cross peaks increase in intensity whereas intra-segmental cross-peak intensities remain unchanged relative to each other and can all be subtracted out. Without relaxation compensation, the difference 2D spectra exhibit both negative and positive intensities due to heterogeneous T 1 relaxation in most biomolecules, which can cause peak cancellation. We demonstrate this relaxation-compensated difference PDSD approach on amino acids, monosaccharides, a crystalline model peptide, a membrane-bound peptide and a plant cell wall sample. The resulting difference spectra yield clean multi-bond, inter-residue and intermolecular correlation peaks

An inproved synthesis of 24- 13C-labeled bile acids using formyl esters and a modified lead tetraacetate procedure

International Nuclear Information System (INIS)

Tserng, K.Y.; Klein, P.D.

1977-01-01

An improved synthesis of 24- 13 C-labeled bile acids has been achieved using formyl derivatives of bile acids and a modified lead tetraacetate procedure. The formylated bile acids were degraded by lead tetraacetate and lithium chloride to formylated 23-chloronorcholanes in 72 to 83% yield. Formylated 23-chloronorcholanes were converted to nitriles in dimethylformamide, which were then hydrolyzed to obtain C-24 labeled bile acids in yield of 80 to 90% of labeled sodium cyanide used. This method results in a higher yield and a purer product with less manipulation than previously reported procedures for synthesis of labeled bile acids

Metabolic Characterization of Acutely Isolated Hippocampal and Cerebral Cortical Slices Using [U-13C]Glucose and [1,2-13C]Acetate as Substrates.

Science.gov (United States)

McNair, Laura F; Kornfelt, Rasmus; Walls, Anne B; Andersen, Jens V; Aldana, Blanca I; Nissen, Jakob D; Schousboe, Arne; Waagepetersen, Helle S

2017-03-01

Brain slice preparations from rats, mice and guinea pigs have served as important tools for studies of neurotransmission and metabolism. While hippocampal slices routinely have been used for electrophysiology studies, metabolic processes have mostly been studied in cerebral cortical slices. Few comparative characterization studies exist for acute hippocampal and cerebral cortical slices, hence, the aim of the current study was to characterize and compare glucose and acetate metabolism in these slice preparations in a newly established incubation design. Cerebral cortical and hippocampal slices prepared from 16 to 18-week-old mice were incubated for 15-90 min with unlabeled glucose in combination with [U- 13 C]glucose or [1,2- 13 C]acetate. Our newly developed incubation apparatus allows accurate control of temperature and is designed to avoid evaporation of the incubation medium. Subsequent to incubation, slices were extracted and extracts analyzed for 13 C-labeling (%) and total amino acid contents (µmol/mg protein) using gas chromatography-mass spectrometry and high performance liquid chromatography, respectively. Release of lactate from the slices was quantified by analysis of the incubation media. Based on the measured 13 C-labeling (%), total amino acid contents and relative activity of metabolic enzymes/pathways, we conclude that the slice preparations in the current incubation apparatus exhibited a high degree of metabolic integrity. Comparison of 13 C-labeling observed with [U- 13 C]glucose in slices from cerebral cortex and hippocampus revealed no significant regional differences regarding glycolytic or total TCA cycle activities. On the contrary, results from the incubations with [1,2- 13 C]acetate suggest a higher capacity of the astrocytic TCA cycle in hippocampus compared to cerebral cortex. Finally, we propose a new approach for assessing compartmentation of metabolite pools between astrocytes and neurons using 13 C-labeling (%) data obtained from

Chloromonilinic Acids C and D, Phytotoxic Tetrasubstituted 3-Chromanonacrylic Acids Isolated from Cochliobolus australiensis with Potential Herbicidal Activity against Buffelgrass (Cenchrus ciliaris).

Science.gov (United States)

Masi, Marco; Meyer, Susan; Clement, Suzette; Pescitelli, Gennaro; Cimmino, Alessio; Cristofaro, Massimo; Evidente, Antonio

2017-10-27

The fungal pathogen Cochliobolus australiensis isolated from infected leaves of the invasive weed buffelgrass (Pennisetum ciliare) was grown in vitro to evaluate its ability to produce phytotoxic metabolites that could potentially be used as natural herbicides against this weed. Two new tetrasubstituted 3-chromanonacrylic acids, named chloromonilinic acids C (1) and D (2), were isolated from the liquid cultures of C. australiensis, together with the known chloromonilinic acid B. Chloromonilinic acids C and D were characterized by spectroscopic and chemical methods as (E)-3-chloro-3-[(5-hydroxy-3-(1-hydroxy-2-methoxy-2-oxoethyl)-7-methyl-4-oxo-4H-chromen-2-yl)]acrylic acid and (Z)-3-chloro-3-[(5-hydroxy-3-(2-methoxy-2-oxoethyl)-7-methyl-4-oxo-4H-chromen-2-yl)]acrylic acid, respectively. The stereochemistry of chloromonilinic acids C and D was determined using a combination of spectroscopic and computational methods, including electronic circular dichroism. The fungus produced these compounds in two different liquid media together with cochliotoxin, radicinin, radicinol, and their 3-epimers. The radicinin-related compounds were also produced when the fungus was grown in wheat seed solid culture, but chloromonilinic acids were not found in the solid culture organic extract. All three chloromonilinic acids were toxic to buffelgrass in a seedling elongation bioassay, with significantly delayed germination and dramatically reduced radicle growth, especially at a concentration of 5 × 10 -3 M.

Synthesis of new derivatives of 1-(3-aminophenyl-4-benzoyl-5-phenyl-1H-pyrazole-3-carboxylic acid

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RAHMI KASIMOGULLAR

2010-12-01

Full Text Available 1-(3-Aminophenyl-4-benzoyl-5-phenyl-1H-pyrazole-3-carboxylic acid (1 was synthesized according to the literature. 2-(3-Aminophenyl-2,6-dihydro-3,4-diphenyl-7H-pyrazolo[3,4-d]pyridazin-7-one (5 was obtained by the cyclocondensation reaction of 1 with hydrazine hydrate. New pyrazole derivatives of compounds 1 and 5 were synthesized by their reaction with β-diketones, β-ketoesters, β-naphthol, phenol and various other reagents. The structures of the synthesized compounds were characterized by 1H-NMR, 13C-NMR, IR and mass spectroscopy, as well as elemental analysis.

Authentication of the origin of vanillin using quantitative natural abundance 13C NMR.

Science.gov (United States)

Tenailleau, Eve J; Lancelin, Pierre; Robins, Richard J; Akoka, Serge

2004-12-29

The use of 13C isotopic distribution as an efficient means to determine the origin of vanillin has been substantiated. Using quantitative 13C NMR, the 13C/12C ratios at all eight carbon positions can be exploited. On a set of 21 samples of vanillin from five different origins, complete discrimination can be achieved. It is shown that, for many purposes, a rapid analysis in which only five sites are used is sufficient. However, improved discrimination using all eight sites is preferable to differentiate between different methods of production from natural ferulic acid or between natural and lignin-derived vanillin on the basis of the 13C/12C ratios characteristic of different origins. The C1 and C8 positions are demonstrated to be the most significant sites for discrimination using principle component analysis. However, aromatic carbon positions make an essential contribution, notably in differentiating between natural and lignin-derived vanillin.

13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses.

Science.gov (United States)

Azizan, Kamalrul Azlan; Ressom, Habtom W; Mendoza, Eduardo R; Baharum, Syarul Nataqain

2017-01-01

Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13 C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis ( r ) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis' central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) in L. lactis , in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis . Overall, the

13C based proteinogenic amino acid (PAA and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP pathway in response to agitation and temperature related stresses

Directory of Open Access Journals (Sweden)

Kamalrul Azlan Azizan

2017-07-01

Full Text Available Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C and agitation (with and without agitation at 150 rpm. Collectively, the concentrations of proteinogenic amino acids (PAAs and free fatty acids (FAAs were compared, and Pearson correlation analysis (r was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA. Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis’ central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA from pyruvate (PYR reaction in all conditions suggested the activation of pyruvate carboxylate (pycA in L. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis. Overall

13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses

Science.gov (United States)

2017-01-01

Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis (r) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis’ central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) in L. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis. Overall, the

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

International Nuclear Information System (INIS)

Pahl-Wostl, C.; Seelig, J.

1986-01-01

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

Expression, purification, crystallization and preliminary X-ray analysis of the d-alanyl carrier protein DltC from Staphylococcus epidermidis

International Nuclear Information System (INIS)

Huang, Chi-Hung; Kao, Chao-Hung; Yang, Chia-Shin; Chang, Chi-Huang; Chen, Sheng-Chia; Kuan, Shu-Min; Su, Yen-Chao; Huang, Yu-Han; Chang, Ming-Chung; Chen, Yeh

2012-01-01

The S. epidermidis carrier protein DltC has been crystallized in order to elucidate the functional role of DltC in the alanylation of lipoteichoic acids in bacteria. The d-alanyl lipoteichoic acids (d-alanyl LTAs) present in the cell walls of Gram-positive bacteria play crucial roles in autolysis, cation homeostasis and biofilm formation. The alanylation of LTAs requires the d-alanyl carrier protein DltC to transfer d-Ala onto a membrane-associated LTA. Here, DltC from Staphylococcus epidermidis (SeDltC) was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.83 Å and belonged to space group P2, with unit-cell parameters a = 66.26, b = 53.28, c = 88.05 Å, β = 98.22°. The results give a preliminary crystallographic analysis of SeDltC and shed light on the functional role of DltC in the alanylation of LTAs

Quantitation of alpha-linolenic acid elongation to eicosapentaenoic and docosahexaenoic acid as affected by the ratio of n6/n3 fatty acids

Directory of Open Access Journals (Sweden)

Somoza Veronika

2009-02-01

Full Text Available Abstract Background Conversion of linoleic acid (LA and alpha-linolenic acid (ALA to their higher chain homologues in humans depends on the ratio of ingested n6 and n3 fatty acids. Design and methods In order to determine the most effective ratio with regard to the conversion of ALA to eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA, human hepatoma cells were incubated with varying ratios of [13C] labeled linoleic acid ([13C]LA- and alpha-linolenic acid ([13C]ALA-methylesters. Regulative cellular signal transduction pathways involved were studied by determinations of transcript levels of the genes encoding delta-5 desaturase (D5D and delta-6 desaturase (D6D, peroxisome proliferator-activated receptor alpha (PPARα and sterol regulatory element binding protein 1c (SREBP-1c. Mitogen-activated protein kinase kinase 1 (MEK1 and mitogen-activated protein kinase kinase kinase 1 (MEKK1 were also examined. Results Maximum conversion was observed in cells incubated with the mixture of [13C]LA/[13C]ALA at a ratio of 1:1, where 0.7% and 17% of the recovered [13C]ALA was converted to DHA and EPA, respectively. Furthermore, differential regulation of enzymes involved in the conversion at the transcript level, dependent on the ratio of administered n6 to n3 fatty acids in human hepatocytes was demonstrated. Conclusion Formation of EPA and DHA was highest at an administered LA/ALA ratio of 1:1, although gene expression of PPARα, SREBP-1c and D5D involved in ALA elongation were higher in the presence of ALA solely. Also, our findings suggest that a diet-induced enhancement of the cell membrane content of highly unsaturated fatty acids is only possible up to a certain level.

Enhancing the understanding of earthworm feeding behaviour via the use of fatty acid delta13C values determined by gas chromatography-combustion-isotope ratio mass spectrometry.

Science.gov (United States)

Dungait, Jennifer A J; Briones, Maria J I; Bol, Roland; Evershed, Richard P

2008-06-01

Litter-dwelling (epigeic) Lumbricus rubellus and soil-dwelling (endogeic) Allolobophora chlorotica earthworms were observed aggregating under C(3) (delta(13)C = -31.3 per thousand; delta(15)N = 10.7 per thousand) and C(4) (delta(13)C = -12.6 per thousand; delta(15)N = 7.5 per thousand) synthetic dung pats applied to a temperate grassland (delta(13)C = -30.3 per thousand; delta(15)N = 5.7 per thousand) in an experiment carried out for 372 days. Bulk delta(13)C values of earthworms collected from beneath either C(3) or C(4) dung after 28, 56, 112 and 372 days demonstrated that (i) L. rubellus beneath C(4) dung were significantly (13)C-enriched after 56 days (delta(13)C = -23.8 per thousand) and 112 days (delta(13)C = -22.4 per thousand) compared with those from C(3) dung treatments (56 days, delta(13)C = -26.5 per thousand; 112 days, delta(13)C = -27.0 per thousand), and (ii) A. chlorotica were 2.1 per thousand (13)C-enriched (delta(13)C = -24.2 per thousand) relative to those from C(3) dung (delta(13)C = -26.3 per thousand) treatments after 372 days. Bulk delta(15)N values did not suggest significant uptake of dung N by either species beneath C(3) or C(4) dung, but showed that the endogeic species (total mean delta(15)N = 3.3 per thousand) had higher delta(15)N values than the epigeic species (total mean delta(15)N = 5.4 per thousand). Although the two species exhibited similar fatty acid profiles, individual fatty acid delta(13)C values revealed extensive routing of dietary C into body tissue of L. rubellus, but minor incorporation into A. chlorotica. In particular, the direct incorporation of microbial biomarker fatty acids (iC(17:0), aC(17:0)) from (13)C-labelled dung in situ, the routing of dung C into de novo synthesised compounds (iC(20:4)(omega)(6),C(20:5)(omega)(3), and the assimilation of essential fatty acids ((C(18:1)(omega)(9), C(18:1)(omega(7), C(18:2)(omega(6), C(18:3)(omega)(3)) derived from dung, were determined. John Wiley & Sons, Ltd

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

Science.gov (United States)

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    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 therefore should be studied further to clarify the fate of carbon in the human body. In addition to excreta, data for serum and blood cell samples were also collected from the subjects to examine the metabolism of 13 C in human body.

Soil carbon inventories and d 13C along a moisture gradient in Botswana

NARCIS (Netherlands)

Bird, M.I.; Veenendaal, E.M.; Lloyd, J.

2004-01-01

We present a study of soil organic carbon (SOC) inventories and d 13C values for 625 soil cores collected from well-drained, coarse-textured soils in eight areas along a 1000 km moisture gradient from Southern Botswana, north into southern Zambia. The spatial distribution of trees and grass in the

The hallucinogen d-lysergic acid diethylamide (d-LSD) induces the immediate-early gene c-Fos in rat forebrain.

Science.gov (United States)

Frankel, Paul S; Cunningham, Kathryn A

2002-12-27

The hallucinogen d-lysergic acid diethylamide (d-LSD) evokes dramatic somatic and psychological effects. In order to analyze the neural activation induced by this unique psychoactive drug, we tested the hypothesis that expression of the immediate-early gene product c-Fos is induced in specific regions of the rat forebrain by a relatively low, behaviorally active, dose of d-LSD (0.16 mg/kg, i.p.); c-Fos protein expression was assessed at 30 min, and 1, 2 and 4 h following d-LSD injection. A time- and region-dependent expression of c-Fos was observed with a significant increase (PLSD administration. These data demonstrate a unique pattern of c-Fos expression in the rat forebrain following a relatively low dose of d-LSD and suggest that activation of these forebrain regions contributes to the unique behavioral effects of d-LSD. Copyright 2002 Elsevier Science B.V.

A new organic reference material, l-glutamic acid, USGS41a, for δ(13) C and δ(15) N measurements - a replacement for USGS41.

Science.gov (United States)

Qi, Haiping; Coplen, Tyler B; Mroczkowski, Stanley J; Brand, Willi A; Brandes, Lauren; Geilmann, Heike; Schimmelmann, Arndt

2016-04-15

The widely used l-glutamic acid isotopic reference material USGS41, enriched in both (13) C and (15) N, is nearly exhausted. A new material, USGS41a, has been prepared as a replacement for USGS41. USGS41a was prepared by dissolving analytical grade l-glutamic acid enriched in (13) C and (15) N together with l-glutamic acid of normal isotopic composition. The δ(13) C and δ(15) N values of USGS41a were directly or indirectly normalized with the international reference materials NBS 19 calcium carbonate (δ(13) CVPDB = +1.95 mUr, where milliurey = 0.001 = 1 ‰), LSVEC lithium carbonate (δ(13) CVPDB = -46.6 mUr), and IAEA-N-1 ammonium sulfate (δ(15) NAir = +0.43 mUr) and USGS32 potassium nitrate (δ(15) N = +180 mUr exactly) by on-line combustion, continuous-flow isotope-ratio mass spectrometry, and off-line dual-inlet isotope-ratio mass spectrometry. USGS41a is isotopically homogeneous; the reproducibility of δ(13) C and δ(15) N is better than 0.07 mUr and 0.09 mUr, respectively, in 200-μg amounts. It has a δ(13) C value of +36.55 mUr relative to VPDB and a δ(15) N value of +47.55 mUr relative to N2 in air. USGS41 was found to be hydroscopic, probably due to the presence of pyroglutamic acid. Experimental results indicate that the chemical purity of USGS41a is substantially better than that of USGS41. The new isotopic reference material USGS41a can be used with USGS40 (having a δ(13) CVPDB value of -26.39 mUr and a δ(15) NAir value of -4.52 mUr) for (i) analyzing local laboratory isotopic reference materials, and (ii) quantifying drift with time, mass-dependent isotopic fractionation, and isotope-ratio-scale contraction for isotopic analysis of biological and organic materials. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

Measurement of the natural variation of 13C/12C isotope ratio in organic samples

International Nuclear Information System (INIS)

Ducatti, C.

1977-01-01

The isotopic ratio analysis for 13 C/ 12 C by mass spectrometry using a 'Working standard' allows the study of 13 C natural variation in organic material, with a total analytical error of less than 0,2%. Equations were derived in order to determine 13 C/ 12 C and 18 O/ 16 O ratios related to the 'working standard' CENA-std and to the international standard PDB. Isotope ratio values obtained with samples prepared in two different combustion apparatus were compared; also the values obtained preparing samples by acid decomposition of carbonaceous materials were compared with the values obtained in different international laboratories. Utilizing the methodology proposed, several leaves collected at different heights of different vegetal species, found 'inside' and 'outside' of the Ducke Forest Reserve, located in the Amazon region, are analysed. It is found that the 13 C natural variation depends upon metabolic process and environmental factors, both being factors which may be qualified as parcial influences on the CO 2 cycle in the forest. (author) [pt

Synthesis of /sup 14/C-labeled felbamate from phenylacetic-(methylene-/sup 14/C) acid

Energy Technology Data Exchange (ETDEWEB)

Choi, Y.M.; Kucharczyk, N.; Sofia, R.D.

1986-05-01

A synthetic procedure for the preparation of 2-phenyl-1,3-propanediol-2-/sup 14/C dicarbamate, starting with phenylacetic-(methylene-/sup 14/C) acid was developed. The procedure from phenylacetic acid to 2-phenyl-1,3-propanediol dicarbamate has four steps via phenylmalonic acid and 2-phenyl-1,3-propanediol. The overall yield of all four steps was 28%.

N-13 labeled amino acids: biodistribution, metabolism and dosimetric considerations

International Nuclear Information System (INIS)

Rosenspire, K.C.; Gelbard, A.S.

1986-01-01

With the growing interest in metabolic imaging and with the increasing number of cyclotron/PET facilities, more studies are being performed in animal and humans using short-lived positron-emitting radionuclides. Amino acids labeled either with N-13 or C-11 are one group of compounds being used to study in vivo regional organ (i.e., brain and heart) or tumor metabolism. Of the studies previously reported using C-11 or N-13 labeled amino acids (methionine, alanine, valine, glutamate, glutamine and tryptophan), imaging was restricted mainly to the organ or tissue of interest with little information obtained about the whole-bode distribution of the label. Such data are important for studying interorgan transport of amino acids and for determining accurate dosimetric measurements after intravenous injection of labeled amino acids. The goals of the authors study were to compare the distribution of several N-13 L-amino acids and N-13 ammonia in tumor-bearing mice and to determine the metabolic fate of the label in vivo. The following amino acids were enzymatically labeled using N-13 ammonia: glutamine, glutamate, methionine, α-aminobutyric acid, valine and leucine. 30 references, 2 figures, 14 tables

Experimental and DFT evaluation of the 1H and 13C NMR chemical shifts for calix[4]arenes

Science.gov (United States)

Guzzo, Rodrigo N.; Rezende, Michelle Jakeline Cunha; Kartnaller, Vinicius; Carneiro, José Walkimar de M.; Stoyanov, Stanislav R.; Costa, Leonardo Moreira da

2018-04-01

The density functional theory is employed to determine the efficiency of 11 exchange-correlation (XC) functionals to compute the 1H and 13C NMR chemical shifts of p-tert-butylcalix[4]arene (ptcx4, R1 = C(CH3)3) and congeners using the 6-31G(d,p) basis set. The statistical analysis shows that B3LYP, B3PW91 and PBE1PBE are the best XC functionals for the calculation of 1H chemical shifts. Moreover, the best results for the 13C chemical shifts are obtained using the LC-WPBE, M06-2X and wB97X-D functionals. The performance of these XC functionals is tested for three other calix[4]arenes: p-sulfonic acid calix[4]arene (sfxcx4 - R1 = SO3H), p-nitro-calix[4]arene (ncx4, R1 = NO2) and calix[4]arene (cx4 - R1 = H). For 1H chemical shifts B3LYP, B3PW91 and PBE1PBE yield similar results, although B3PW91 shows more consistency in the calculated error for the different structures. For 13C NMR chemical shifts, the XC functional that stood out as best is LC-WPBE. Indeed, the three functionals selected for each of 1H and 13C show good accuracy and can be used in future studies involving the prediction of 1H and 13C chemical shifts for this type of compounds.

13C-nuclear magnetic resonance studies of the biosynthesis of 5-aminolevolinic acid destined for chlorophyll formation in dark-grown Scenedesmus Obliquus

International Nuclear Information System (INIS)

Oh-hama, Tamiko; Seto, Harvo; Miyachi, Shigetoh

1985-01-01

The 13 C-nuclear magnetic resonance (NMR) spectra of chlorophyll-α-formed in dark-grown Scenedesmus Obliquus (Turp.) Kutzing in the presence of (1- 13 C) glutamate, (2- 13 C) and (1- 13 C) glycine showed that the 13 C of glutamate was specifically incorporated into the eight-carbon atoms in the tetrapyrrole macrocyles derived from C-5 of 5-aminolevolinic acid (ALA), while the C-2 of glycine was only incorporated into the methyl carbon of the methoxycarbonyl group attached to the isolcyclic ring of chlorophyll a formed in the presence of (1- 13 C)-glycine. These labelling patterns provide evidence for the operation of the C 5 -pathway and against the operation of the ALA synthase pathway for chlorophyll formation in darkness. (author)

(13)C MR spectroscopy study of lactate as substrate for rat brain.

Science.gov (United States)

Qu, H; Håberg, A; Haraldseth, O; Unsgård, G; Sonnewald, U

2000-01-01

In order to address the question whether lactate in blood can serve as a precursor for cerebral metabolites, fully awake rats were injected intravenously with [U-(13)C]lactate or [U-(13)C]glucose followed 15 min later by decapitation. Incorporation of label from [U-(13)C]glucose was seen mainly in glutamate, GABA, glutamine, aspartate, alanine and lactate. More label was found in glutamate than glutamine, underscoring the predominantly neuronal metabolism of pyruvate from [U-(13)C]glucose. It was estimated that the neuronal metabolism of acetyl CoA from glucose accounts for at least 66% and the glial for no more than 34% of the total glucose consumption. When [U-(13)C]lactate was the precursor, label incorporation was similar to that observed from [U-(13)C]glucose, but much reduced. Plasma analysis revealed the presence of approximately equal amounts of [1,2,3-(13)C]- and [1,2-(13)C]glucose, showing gluconeogenesis from [U-(13)C]lactate. It was thus possible that the labeling seen in the cerebral amino acids originated from labeled glucose, not [U-(13)C]lactate. However, the presence of significantly more label in [U-(13)C]- than in [2,3-(13)C]alanine demonstrated that [U-(13)C]lactate did indeed cross the blood-brain barrier, and was metabolized further in the brain. Furthermore, contributions from pyruvate carboxylase (glial enzyme) were detectable in glutamine, glutamate and GABA, and were comparatively more pronounced in the glucose group. This indicated that relatively more pyruvate from lactate than glucose was metabolized in neurons. Surprisingly, the same amount of lactate was synthesized via the tricarboxylic acid cycle in both groups, indicating transfer of neurotransmitters from the neuronal to the astrocytic compartment, as previous studies have shown that this lactate is synthesized primarily in astrocytes. Taking into consideration that astrocytes take up glutamate more avidly than GABA, it is conceivable that neuronal lactate metabolism was more

Automated no-carrier-added synthesis of [1-11C]-labeled D- and L-enantiomers of lactic acid

International Nuclear Information System (INIS)

Drandarov, Konstantin; Schubiger, P. August; Westera, Gerrit

2006-01-01

The first purely chemical method for automated no-carrier-added synthesis of [1- 11 C]-labeled D(R)- and L(S)-2-hydroxypropanoic acid (lactic acid) was developed for experimental neurophysiology studies and position emission tomography (PET) diagnosis. Starting from sodium 1-hydroxyethanesulfonate and [ 11 C]HCN (trapped as [ 11 C]KCN) the intermediate DL-(R,S)-[1- 11 C]-2-hydroxypropanenitrile was prepared. Its rapid acid hydrolysis gave DL-(R,S)-[1- 11 C]lactic acid, which was isolated by preparative reversed phase HPLC and automatically injected on a second preparative C 18 HPLC column coated with a chiral selector, where both [1- 11 C]lactic acid enantiomers were separated by chiral ligand-exchange chromatography. Two novel chiral selectors for HPLC enantiomeric separation of α-hydroxy acids, namely D(R)- or L(S)-2-amino-3-methyl-3-(5-phenylpentylsulfanyl)-butanoic acid were utilized for the preparative HPLC separation of the [1- 11 C]lactic acid enantiomers. The preparation of the selectors and the coating procedure for the manufacturing of the preparative chiral HPLC columns are described. A highly efficient trap for [ 11 C]HCN is presented. The whole radiosynthesis is automated, takes about 45 min and leads to more than 80% decay corrected overall radiochemical yield of each enantiomer (up to 2.5 GBq) with over 99% radiochemical, chemical and enantiomeric purity. The specific activity at the end of the synthesis is about 400 GBq/μmol

(1 H, 13 C and 31 P) NMR of phosphonic acid derivatives

International Nuclear Information System (INIS)

Campos, Valdevino; Costa, Valentim E. Uberti

1991-01-01

In the last years the development of phosphates analogues in the medical and agricultural pesticides has being very expressive. 1 H, 13 C and mainly 31 P NMR are used for stereochemical and conformational analysis, and reactivity studies on the compounds resulting from those chemical processes

Comparison of liquid chromatography-isotope ratio mass spectrometry (LC/IRMS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) for the determination of collagen amino acid δ13C values for palaeodietary and palaeoecological reconstruction.

Science.gov (United States)

Dunn, Philip J H; Honch, Noah V; Evershed, Richard P

2011-10-30

Results are presented of a comparison of the amino acid (AA) δ(13)C values obtained by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) and liquid chromatography-isotope ratio mass spectrometry (LC/IRMS). Although the primary focus was the compound-specific stable carbon isotope analysis of bone collagen AAs, because of its growing application for palaeodietary and palaeoecological reconstruction, the results are relevant to any field where AA δ(13)C values are required. We compare LC/IRMS with the most up-to-date GC/C/IRMS method using N-acetyl methyl ester (NACME) AA derivatives. This comparison involves the analysis of standard AAs and hydrolysates of archaeological human bone collagen, which have been previously investigated as N-trifluoroacetyl isopropyl esters (TFA/IP). It was observed that, although GC/C/IRMS analyses required less sample, LC/IRMS permitted the analysis of a wider range of AAs, particularly those not amenable to GC analysis (e.g. arginine). Accordingly, reconstructed bulk δ(13)C values based on LC/IRMS-derived δ(13)C values were closer to the EA/IRMS-derived δ(13)C values than those based on GC/C/IRMS values. The analytical errors for LC/IRMS AA δ(13)C values were lower than GC/C/IRMS determinations. Inconsistencies in the δ(13)C values of the TFA/IP derivatives compared with the NACME- and LC/IRMS-derived δ(13)C values suggest inherent problems with the use of TFA/IP derivatives, resulting from: (i) inefficient sample combustion, and/or (ii) differences in the intra-molecular distribution of δ(13)C values between AAs, which are manifested by incomplete combustion. Close similarities between the NACME AA δ(13)C values and the LC/IRMS-derived δ(13)C values suggest that the TFA/IP derivatives should be abandoned for the natural abundance determinations of AA δ(13)C values. Copyright © 2011 John Wiley & Sons, Ltd.

Identification of rice Os4BGlu13 as a β-glucosidase which hydrolyzes gibberellin A4 1-O-β-d-glucosyl ester, in addition to tuberonic acid glucoside and salicylic acid derivative glucosides.

Science.gov (United States)

Hua, Yanling; Ekkhara, Watsamon; Sansenya, Sompong; Srisomsap, Chantragan; Roytrakul, Sittiruk; Saburi, Wataru; Takeda, Ryosuke; Matsuura, Hideyuki; Mori, Haruhide; Ketudat Cairns, James R

2015-10-01

Gibberellin 1-O-β-d-glucose ester hydrolysis activity has been detected in rice seedling extracts, but no enzyme responsible for this activity has ever been purified and identified. Therefore, gibberellin A4 glucosyl ester (GA4-GE) β-d-glucosidase activity was purified from ten-day rice seedling stems and leaves. The family 1 glycoside hydrolase Os4BGlu13 was identified in the final purification fraction. The Os4BGlu13 cDNA was amplified from rice seedlings and expressed as an N-terminal thioredoxin-tagged fusion protein in Escherichia coli. The purified recombinant Os4BGlu13 protein (rOs4BGlu13) had an optimum pH of 4.5, for hydrolysis of p-nitrophenyl β-d-glucopyranoside (pNPGlc), which was the best substrate identified, with a kcat/Km of 637 mM(-1) s(-1). rOs4BGlu13 hydrolyzed helicin best among natural glycosides tested (kcat/Km of 74.4 mM(-1) s(-1)). Os4BGlu13 was previously designated tuberonic acid glucoside (TAG) β-glucosidase (TAGG), and here the kcat/Km of rOsBGlu13 for TAG was 6.68 mM(-1) s(-1), while that for GA4-GE was 3.63 mM(-1) s(-1) and for salicylic acid glucoside (SAG) is 0.88 mM(-1) s(-1). rOs4BGlu13 also hydrolyzed oligosaccharides, with preference for short β-(1 → 3)-linked over β-(1 → 4)-linked glucooligosaccharides. The enzymatic data suggests that Os4BGlu13 may contribute to TAG, SAG, oligosaccharide and GA4-GE hydrolysis in the rice plant, although helicin or a similar compound may be its primary target. Copyright © 2015 Elsevier Inc. All rights reserved.

Metabolic regulation in Streptomyces parvulus during actinomycin D synthesis, studied with 13C- and 15N-labeled precursors by 13C and 15N nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry

International Nuclear Information System (INIS)

Inbar, L.; Lapidot, A.

1988-01-01

Recent studies have suggested that the onset of synthesis of actinomycin D in Streptomyces is due to a release from L-glutamate catabolic repression. In the present investigation we showed that S. parvulus has the capacity to maintain high levels of intracellular glutamate during the synthesis of actinomycin D. The results seem contradictory, since actinomycin D synthesis cannot start before a release from L-glutamate catabolic repression, but a relatively high intracellular pool of glutamate is needed for the synthesis of actinomycin D. Utilizing different labeled precursors, D-[U- 13 C]fructose and 13 C- and 15 N-labeled L-glutamate, and nuclear magnetic resonance techniques, we showed that carbon atoms of an intracellular glutamate pool of S. parvulus were not derived biosynthetically from the culture medium glutamte source but rather from D-fructose catabolism. A new intracellular pyrimidine derivative whose nitrogen and carbon skeletons were derived from exogenous L-glutamate was obtained as the main glutamate metabolite. Another new pyrimidine derivative that had a significantly reduced intracellular mobility and that was derived from D-fructose catabolism was identified in the cell extracts of S. parvulus during actinomycin D synthesis. These pyrimidine derivatives may serve as a nitrogen store for actinomycin D synthesis. In the present study, the N-trimethyl group of a choline derivative was observed by 13 C nuclear magnetic resonance spectroscopy in growing S. parvulus cells. The choline group, as well as the N-methyl groups of sarcosine, N-methyl-valine, and the methyl groups of an actinomycin D chromophore, arose from D-fructose catabolism. The 13 C enrichments found in the peptide moieties of actinomycin D were in accordance with a mechanism of actinomycin D synthesis from L-glutamate and D-fructose

2D 1H -13C Heteronuclear Shift Correlation Of 2a - Hydroxy Aiantolactone From Pulicaria Undulata C.A. Mey

Directory of Open Access Journals (Sweden)

A. Rustaiyan

1992-07-01

Full Text Available We have reported recently the isolation and characterization of several sesquiterpene lactones from Pulicaria undulata (1."nThe lactones were isolated from an Et20 - Petrol (1:3 fraction by different chromatographic techniques including HPLC (RP 8, MeOH - H2O, 13:7."nIn this way three eudesmanolides 1 - 3, a guaianolide 4, a nor -guaianolide 5, as well as the pseudoguaianolide 6 and the xanthanolide 7 were isolated. One of the eudesmanolides (2a - hydroxy aiantolactone, 1, was present as the main component."nSuch lactones being known as biologically active substances, we have decided to describe for the first time a detailed interpretation of proton, 1H -NMR, 13C - NMR and 2D lH -13C - heteronuclear shift correlation spectra of 2a - hydroxy aiantolactone. The stereochemistry of C - 2 , C - 7 and C - 8 was determined by the NOESY experiments, H - 7 and H - 8 are in the a configuration and H - 2 is in the b configuration.

Stereoselective synthesis of the Halaven C14-C26 fragment from D-quinic acid: crystallization-induced diastereoselective transformation of an α-methyl nitrile.

Science.gov (United States)

Belanger, Francis; Chase, Charles E; Endo, Atsushi; Fang, Francis G; Li, Jing; Mathieu, Steven R; Wilcoxen, Annie Z; Zhang, Huiming

2015-04-20

Crystallization-induced diastereoselective transformation (CIDT) of an α-methyl nitrile completes an entirely non-chromatographic synthesis of the halichondrin B C14-C26 stereochemical array. The requisite α-methyl nitrile substrate is derived from D-quinic acid through a series of substrate-controlled stereoselective reactions via a number of crystalline intermediates that benefit from a rigid polycyclic template. Therefore, all four stereogenic centers in the Halaven C14-C26 fragment were derived from the single chiral source D-quinic acid. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Validation of 13C-acetic acid breath test by measuring effects of loperamide, morphine, mosapride, and itopride on gastric emptying in mice.

Science.gov (United States)

Matsumoto, Kenjiro; Kimura, Hiroshi; Tashima, Kimihito; Uchida, Masayuki; Horie, Syunji

2008-10-01

Several methods are used to evaluate gastric motility in rodents, but they all have technical limitations. Recent technical developments enable a convenient method to evaluate gastric motility. The (13)C-acetic acid breath test in rodents is a non-invasive and repeatable method that can be used without physical restraints. The present study aimed to validate the (13)C-acetic acid breath test by measuring the effects of loperamide, morphine, mosapride, and itopride on gastric emptying in mice. Loperamide (1-10 mg/kg) and morphine (1.25-10 mg/kg) slowed gastric emptying and decreased the maximum concentration (C(max)) and area under the curve (AUC(90 min)) value in a dose-dependent manner. Mosapride (0.2-5 mg/kg) accelerated gastric emptying and increased C(max) value. Mosapride (20 mg/kg) did not accelerate gastric emptying on the (13)C-breath test. Itopride (30 mg/kg, per os) significantly accelerated gastric emptying compared with the vehicle group. In a comparison with the conventional phenol red test, there was a correlation between the C(max) value of breath test and gastric emptying (%) of phenol red tests in treatment with loperamide or mosapride. These results indicate that the (13)C-acetic acid breath test is an accurate, noninvasive, and simple method for monitoring gastric emptying in mice. This method is useful to assess the effect of drugs and gut function pharmacologically.

The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4.

Science.gov (United States)

Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

2016-02-03

Organic acids are essential to fruit flavor. The vacuolar H(+) transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties 'Ordinary Ponkan (OPK)' and an early maturing mutant 'Zaoshu Ponkan (ZPK)'. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis.

Chemical constituents of Ottonia corcovadensis Miq. from Amazon forest: 1H and 13C chemical shift assignments

International Nuclear Information System (INIS)

Facundo, Valdir A.; Morais, Selene M.; Braz Filho, Raimundo

2004-01-01

In an ethanolic extract of leaves of Ottonia corcovadensis (Piperaceae) were identified sixteen terpenoids of essential oil and the three flavonoids 3',4',5,5',7-penta methoxyflavone (1), 3',4',5,7-tetra methoxyflavone (2) and 5-hydroxy-3',4',5',7-tetra methoxyflavone (3) and cafeic acid (4). Two amides (5 and 6) were isolated from an ethanolic extract of the roots. The structures were established by spectral analysis, meanly NMR (1D and 2D) and mass spectra. Extensive NMR analysis was also used to complete 1 H and 13 C chemical shift assignments of the flavonoids and amides. The components of the essential oil were identified by computer library search, retention indices and visual interpretation of mass spectra. (author)

Site-selective {sup 13}C labeling of proteins using erythrose

Energy Technology Data Exchange (ETDEWEB)

Weininger, Ulrich, E-mail: ulrich.weininger@physik.uni-halle.de [Lund University, Department of Biophysical Chemistry, Center for Molecular Protein Science (Sweden)

2017-03-15

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with {sup 13}C and/or {sup 1}H, which is achieved in the most general way by using site-selectively {sup 13}C-enriched glucose (1- and 2-{sup 13}C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively {sup 13}C-enriched erythrose (1-, 2-, 3- and 4-{sup 13}C) as a suitable precursor for {sup 13}C labeled aromatic side chains. We quantify {sup 13}C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the {sup 13}C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated {sup 13}C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective {sup 13}C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.

Relaxation-compensated difference spin diffusion NMR for detecting {sup 13}C–{sup 13}C long-range correlations in proteins and polysaccharides

Energy Technology Data Exchange (ETDEWEB)

Wang, Tuo; Williams, Jonathan K. [Massachusetts Institute of Technology, Department of Chemistry (United States); Schmidt-Rohr, Klaus [Brandeis University, Department of Chemistry (United States); Hong, Mei, E-mail: meihong@mit.edu [Massachusetts Institute of Technology, Department of Chemistry (United States)

2015-02-15

The measurement of long-range distances remains a challenge in solid-state NMR structure determination of biological macromolecules. In 2D and 3D correlation spectra of uniformly {sup 13}C-labeled biomolecules, inter-residue, inter-segmental, and intermolecular {sup 13}C–{sup 13}C cross peaks that provide important long-range distance constraints for three-dimensional structures often overlap with short-range cross peaks that only reflect the covalent structure of the molecule. It is therefore desirable to develop new approaches to obtain spectra containing only long-range cross peaks. Here we show that a relaxation-compensated modification of the commonly used 2D {sup 1}H-driven spin diffusion (PDSD) experiment allows the clean detection of such long-range cross peaks. By adding a z-filter to keep the total z-period of the experiment constant, we compensate for {sup 13}C T{sub 1} relaxation. As a result, the difference spectrum between a long- and a scaled short-mixing time spectrum show only long-range correlation signals. We show that one- and two-bond cross peaks equalize within a few tens of milliseconds. Within ∼200 ms, the intensity equilibrates within an amino acid residue and a monosaccharide to a value that reflects the number of spins in the local network. With T{sub 1} relaxation compensation, at longer mixing times, inter-residue and inter-segmental cross peaks increase in intensity whereas intra-segmental cross-peak intensities remain unchanged relative to each other and can all be subtracted out. Without relaxation compensation, the difference 2D spectra exhibit both negative and positive intensities due to heterogeneous T{sub 1} relaxation in most biomolecules, which can cause peak cancellation. We demonstrate this relaxation-compensated difference PDSD approach on amino acids, monosaccharides, a crystalline model peptide, a membrane-bound peptide and a plant cell wall sample. The resulting difference spectra yield clean multi-bond, inter

Conditions to obtain precise and true measurements of the intramolecular {sup 13}C distribution in organic molecules by isotopic {sup 13}C nuclear magnetic resonance spectrometry

Energy Technology Data Exchange (ETDEWEB)

Bayle, Kevin [EBSI Team, Interdisciplinary Chemistry: Synthesis, Analysis, Modelling (CEISAM), University of Nantes-CNRS UMR 6230, 2 Rue de la Houssinière, BP 92208, F-44322, Nantes Cedex 3 (France); Gilbert, Alexis [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Earth–Life Science Institute, Tokyo Institute of Technology, Meguro, Tokyo 152-8551 (Japan); Julien, Maxime [EBSI Team, Interdisciplinary Chemistry: Synthesis, Analysis, Modelling (CEISAM), University of Nantes-CNRS UMR 6230, 2 Rue de la Houssinière, BP 92208, F-44322, Nantes Cedex 3 (France); Yamada, Keita [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Silvestre, Virginie; Robins, Richard J.; Akoka, Serge [EBSI Team, Interdisciplinary Chemistry: Synthesis, Analysis, Modelling (CEISAM), University of Nantes-CNRS UMR 6230, 2 Rue de la Houssinière, BP 92208, F-44322, Nantes Cedex 3 (France); Yoshida, Naohiro [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Earth–Life Science Institute, Tokyo Institute of Technology, Meguro, Tokyo 152-8551 (Japan); Remaud, Gérald S., E-mail: gerald.remaud@univ-nantes.fr [EBSI Team, Interdisciplinary Chemistry: Synthesis, Analysis, Modelling (CEISAM), University of Nantes-CNRS UMR 6230, 2 Rue de la Houssinière, BP 92208, F-44322, Nantes Cedex 3 (France)

2014-10-10

Highlights: • Evaluation of the trueness and precision criteria of isotopic {sup 13}C NMR spectrometry. • Use of bi-labelled [1,2-{sup 13}C{sub 2}]acetic acid to determine the performance of the instrumental response. • Inter-calibration of the {sup 13}C intramolecular composition of acetic acid using the technique GC-Py–irm-MS. - Abstract: Intramolecular {sup 13}C composition gives access to new information on the (bio) synthetic history of a given molecule. Isotopic {sup 13}C NMR spectrometry provides a general tool for measuring the position-specific {sup 13}C content. As an emerging technique, some aspects of its performance are not yet fully delineated. This paper reports on (i) the conditions required to obtain satisfactory trueness and precision for the determination of the internal {sup 13}C distribution, and (ii) an approach to determining the “absolute” position-specific {sup 13}C content. In relation to (i), a precision of <1% can be obtained whatever the molecule on any spectrometer, once quantitative conditions are met, in particular appropriate proton decoupling efficiency. This performance is a prerequisite to the measurement of isotope fractionation either on the transformed or residual compound when a chemical reaction or process is being studied. The study of the trueness has revealed that the response of the spectrometer depends on the {sup 13}C frequency range of the studied molecule, i.e. the chemical shift range. The “absolute value” and, therefore, the trueness of the {sup 13}C NMR measurements has been assessed on acetic acid and by comparison to the results obtained on the fragments from COOH and CH{sub 3} by isotopic mass spectrometry coupled to a pyrolysis device (GC-Py–irm-MS), this technique being the reference method for acetic acid. Of the two NMR spectrometers used in this work, one gave values that corresponded to those obtained by GC-Py–irm-MS (thus, the “true” value) while the other showed a bias, which was

A new organic reference material, L-glutamic acid, USGS41a, for δ13C and δ15N measurements − a replacement for USGS41

Science.gov (United States)

Qi, Haiping; Coplen, Tyler B.; Mroczkowski, Stanley J.; Brand, Willi A.; Brandes, Lauren; Geilmann, Heike; Schimmelmann, Arndt

2016-01-01

RationaleThe widely used l-glutamic acid isotopic reference material USGS41, enriched in both 13C and 15N, is nearly exhausted. A new material, USGS41a, has been prepared as a replacement for USGS41.MethodsUSGS41a was prepared by dissolving analytical grade l-glutamic acid enriched in 13C and 15N together with l-glutamic acid of normal isotopic composition. The δ13C and δ15N values of USGS41a were directly or indirectly normalized with the international reference materials NBS 19 calcium carbonate (δ13CVPDB = +1.95 mUr, where milliurey = 0.001 = 1 ‰), LSVEC lithium carbonate (δ13CVPDB = −46.6 mUr), and IAEA-N-1 ammonium sulfate (δ15NAir = +0.43 mUr) and USGS32 potassium nitrate (δ15N = +180 mUr exactly) by on-line combustion, continuous-flow isotope-ratio mass spectrometry, and off-line dual-inlet isotope-ratio mass spectrometry.ResultsUSGS41a is isotopically homogeneous; the reproducibility of δ13C and δ15N is better than 0.07 mUr and 0.09 mUr, respectively, in 200-μg amounts. It has a δ13C value of +36.55 mUr relative to VPDB and a δ15N value of +47.55 mUr relative to N2 in air. USGS41 was found to be hydroscopic, probably due to the presence of pyroglutamic acid. Experimental results indicate that the chemical purity of USGS41a is substantially better than that of USGS41.ConclusionsThe new isotopic reference material USGS41a can be used with USGS40 (having a δ13CVPDB value of −26.39 mUr and a δ15NAir value of −4.52 mUr) for (i) analyzing local laboratory isotopic reference materials, and (ii) quantifying drift with time, mass-dependent isotopic fractionation, and isotope-ratio-scale contraction for isotopic analysis of biological and organic materials. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

Inhibitors of the alpha-ketoglutarate dehydrogenase complex alter [1-13C]glucose and [U-13C]glutamate metabolism in cerebellar granule neurons.

Science.gov (United States)

Santos, Sónia Sá; Gibson, Gary E; Cooper, Arthur J L; Denton, Travis T; Thompson, Charles M; Bunik, Victoria I; Alves, Paula M; Sonnewald, Ursula

2006-02-15

Diminished activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), an important component of the tricarboxylic acid (TCA) cycle, occurs in several neurological diseases. The effect of specific KGDHC inhibitors [phosphonoethyl ester of succinyl phosphonate (PESP) and the carboxy ethyl ester of succinyl phosphonate (CESP)] on [1-13C]glucose and [U-13C]glutamate metabolism in intact cerebellar granule neurons was investigated. Both inhibitors decreased formation of [4-13C]glutamate from [1-13C]glucose, a reduction in label in glutamate derived from [1-13C]glucose/[U-13C]glutamate through a second turn of the TCA cycle and a decline in the amounts of gamma-aminobutyric acid (GABA), aspartate, and alanine. PESP decreased formation of [U-13C]aspartate and total glutathione, whereas CESP decreased concentrations of valine and leucine. The findings are consistent with decreased KGDHC activity; increased alpha-ketoglutarate formation; increased transamination of alpha-ketoglutarate with valine, leucine, and GABA; and new equilibrium position of the aspartate aminotransferase reaction. Overall, the findings also suggest that some carbon derived from alpha-ketoglutarate may bypass the block in the TCA cycle at KGDHC by means of the GABA shunt and/or conversion of valine to succinate. The results suggest the potential of succinyl phosphonate esters for modeling the biochemical and pathophysiological consequences of reduced KGDHC activity in brain diseases.

13C, 1H spin-spin coupling constants. Pt. 4

International Nuclear Information System (INIS)

Aydin, R.; Guenther, H.

1979-01-01

One-bond, geminal, and vicinal 13 C, 1 H coupling constants have been determined for adamantane using α-and β-[D]adamantane and the relation sup(n)J( 13 C, 1 H)=6,5144sup(n)J( 13 C, 2 H) for the conversion of the measured sup(n)J( 13 C, 2 H) values. It is shown that the magnitude of 3 Jsub(trans) is strongly influenced by the substitution pattern. Relative H,D isotope effects for 13 C chemical shifts are given. (orig.) [de

Structure of Selected Derivates of p-Hydroxy Cynamonic Acid According to 13C CP MAS NMR and DFT Calculation

International Nuclear Information System (INIS)

Pisklak, D.M.; Wawer, I.; Tkaczyk, M.

2005-01-01

Derivatives of p-hydroxy cynamonic acid are widely occurring in fruits, vegetables, tea and coffee. They exhibit strong antioxidant activity due to the presence of phenolic group. Epidemiological, biological and biochemical data support health beneficial role of this group of compounds and anticarcinogenic, antimutagenic and antiinflamatory effects have been reported. The most common caffeic acid contributes significantly to the total polyphenol intake and has been suggested to play a role in the apparent association between the regular consumption of polyphenol-rich food and beverages, and the prevetion of inflammatory and proliferative diseases. 13 C MAS NMR spectra were recorded on a BRUKER DSX 400 spectrometer at 400,13. Powder samples were spun in a 4 mm rotor at 10 kHz ( 13 C). Signals were assigned:- By comparison with solution spectra; - Using dipolar dephasing and variable contact time experiments; - Confirmed by DFT calculations of shielding constants. The differences in chemical shifts between solution and solid state spectra are due to the formation of intramolecular and intermolecular hydrogen bonds, including C-OH...OC within cyclic dimers. (author)

Oxidation of C18 Hydroxy-Polyunsaturated Fatty Acids to Epoxide or Ketone by Catalase-Related Hemoproteins Activated with Iodosylbenzene.

Science.gov (United States)

Teder, Tarvi; Boeglin, William E; Brash, Alan R

2017-07-01

Small catalase-related hemoproteins with a facility to react with fatty acid hydroperoxides were examined for their potential mono-oxygenase activity when activated using iodosylbenzene. The proteins tested were a Fusarium graminearum 41 kD catalase hemoprotein (Fg-cat, gene FGSG_02217), a Pseudomonas fluorescens Pfl01 catalase (37.5 kD, accession number WP_011333788.1), and a Mycobacterium avium ssp. paratuberculosis 33 kD catalase (gene MAP-2744c). 13-Hydroxy-octadecenoic acids (which are normally unreactive) were selected as substrates because these enzymes react specifically with the corresponding 13S-hydroperoxides (Pakhomova et al. 18:2559-2568, 5; Teder et al. 1862:706-715, 14). In the presence of iodosylbenzene Fg-cat converted 13S-hydroxy-fatty acids to two products: the 15,16-double bond of 13S-hydroxy α-linolenic acid was oxidized stereospecifically to the 15S,16R-cis-epoxide or the 13-hydroxyl was oxidized to the 13-ketone. Products were identified by UV, HPLC, LC-MS, NMR and by comparison with authentic standards prepared for this study. The Pfl01-cat displayed similar activity. MAP-2744c oxidized 13S-hydroxy-linoleic acid to the 13-ketone, and epoxidized the double bonds to form the 9,10-epoxy-13-hydroxy, 11,12-epoxy-13-hydroxy, and 9,10-epoxy-13-keto derivatives; equivalent transformations occurred with 9S-hydroxy-linoleic acid as substrate. In parallel incubations in the presence of iodosylbenzene, human catalase displayed no activity towards 13S-hydroxy-linoleic acid, as expected from the highly restricted access to its active site. The results indicated that with suitable transformation to Compound I, monooxygenase activity can be demonstrated by these catalase-related hemoproteins with tyrosine as the proximal heme ligand.

Stereochemistry of C18 monounsaturated cork suberin acids determined by spectroscopic techniques including (1) H-NMR multiplet analysis of olefinic protons.

Science.gov (United States)

Santos, Sara; Graça, José

2014-01-01

Suberin is a biopolyester responsible for the protection of secondary plant tissues, and yet its molecular structure remains unknown. The C18:1 ω-hydroxyacid and the C18:1 α,ω-diacid are major monomers in the suberin structure, but the configuration of the double bond remains to be elucidated. To unequivocally define the configuration of the C18:1 suberin acids. Pure C18:1 ω-hydroxyacid and C18:1 α,ω-diacid, isolated from cork suberin, and two structurally very close C18:1 model compounds of known stereochemistry, methyl oleate and methyl elaidate, were analysed by NMR spectroscopy, Fourier transform infrared (FTIR) and Raman spectroscopy, and GC-MS. The GC-MS analysis showed that both acids were present in cork suberin as only one geometric isomer. The analysis of dimethyloxazoline (DMOX) and picolinyl derivatives proved the double bond position to be at C-9. The FTIR spectra were concordant with a cis-configuration for both suberin acids, but their unambiguous stereochemical assignment came from the NMR analysis: (i) the chemical shifts of the allylic (13) C carbons were shielded comparatively to the trans model compound, and (ii) the complex multiplets of the olefinic protons could be simulated only with (3) JHH and long-range (4) JHH coupling constants typical of a cis geometry. The two C18:1 suberin acids in cork are (Z)-18-hydroxyoctadec-9-enoic acid and (Z)-octadec-9-enedoic acid. Copyright © 2013 John Wiley & Sons, Ltd.

In Folio Respiratory Fluxomics Revealed by 13C Isotopic Labeling and H/D Isotope Effects Highlight the Non-cyclic Nature of the Tricarboxylic Acid 'Cycle' in Illuminated Leaves

International Nuclear Information System (INIS)

Tcherkez, G.; Mahe, A.; Gauthier, P.; Hodges, M.; Tcherkez, G.; Mauve, C.; Cornic, G.; Gout, E.; Bligny, R.

2009-01-01

While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, 13 C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA 'cycle' does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation. (authors)

In-phantom dosimetry using the 13C(d,n)14N reaction for BNCT (boron neutron capture therapy)

International Nuclear Information System (INIS)

Burlon, Alejandro; Kreiner, Andres J.; White, S.; Blackburn, B.; Gierga, David; Yanch, Jacquelyn C.

2000-01-01

The use of the 13 C(d,n) 14 N reaction at E d =1.5 MeV for accelerator-based boron neutron capture therapy is investigated. The 13 C(d,n) 14 N reaction presents the advantages of carbon as a target material and its large cross section. The deuteron beam was produced by a tandem accelerator at MIT's Laboratory for Accelerator Beam Applications. The resulting neutron spectra were evaluated in terms of RBE-dose rates at different depths inside a water-filled brain phantom using a heavy water moderator and lead reflector assembly. All results were simulated using the code MCNP. (author)

Syntheses of {gamma}-aminobutyric-1-{sup 14}C and of {alpha}-aminoadipic-6-{sup 14}C acid from methoxy-3 chloropropyl-magnesium and marked carbon dioxide; Syntheses de l'acide {gamma}-aminobutyrique{sup 14}C-1 et de l'acide {alpha}-aminoadipique {sup 14}C-6 a partir de methoxy-3 chloropropylmagnesium et d'anhydride carbonique marque

Energy Technology Data Exchange (ETDEWEB)

Liem, Phung Nhu [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires, Departement des radioelements, Service des molecules marquees

1967-04-01

Carbonation of {gamma}-methoxypropyl-magnesium chloride by CO{sub 2} gives {gamma}-methoxy-butyric carboxylic-{sup 14}C acid with a yield of about 95 per cent. When the latter is treated successively with anhydrous HBr and with diazomethane, methyl carboxylic {gamma}-bromobutyrate-{sup 14}C is formed. This in turn gives {gamma}-amino-butyric carboxylic-{sup 14}C acid with an overall yield of 66 per cent with respect to Ba{sup 14}CO{sub 3}, when it is condensed with potassium phthalimide and hydrolyzed by acid. By reacting methyl-{gamma}-bromobutyrate-{sup 14}C with the sodium derivative of ethyl cyanacetamido-acetate in ethanol, followed by an acid hydrolysis, {alpha}-aminoadipic-6-{sup 14}C acid is obtained with an overall yield of 46 per cent with respect to Ba{sup 14}CO{sub 3}. (author) [French] La carbonatation du chlorure de {gamma}-methoxypropylmagnesium par {sup 14}CO{sub 2} donne l'acide {gamma}-methoxybutyrique carboxyle {sup 14}C avec un rendement d'environ 95 pour cent. Ce dernier traite successivement par HBr anhydre et par le diazomethane conduit au {gamma}-bromobutyrate de methyle carboxyle {sup 14}C. Celui-ci condense avec le phtalimide de potassium suivi d'une hydrolyse acide fournit l'acide {gamma}-aminobutyrique carboxyle {sup 14}C avec un rendement global de 66 pour cent par rapport a Ba{sup 14}CO{sub 3}. L'action du {gamma}-bromobutyrate de methyle {sup 14}C sur le derive sode du cyanacetamidoacetate d'ethyle dans l'ethanol suivie d'hydrolyse acide donne l'acide {alpha}-aminoadipique {sup 14}C-6 avec un rendement global de 46 pour cent par rapport a Ba{sup 14}CO{sub 3}. (auteur)

13C NMR Chemical Shifts of the Triclinic and Monoclinic Crystal forms of Valinomycin

International Nuclear Information System (INIS)

Kameda, Tsunenori; McGeorge, Gary; Orendt, Anita M.; Grant, David M.

2004-01-01

Two different crystalline polymorphs of valinomycin, the triclinic and monoclinic forms, have been studied by high resolution, solid state 13 C CP-MAS NMR spectroscopy. Although the two polymorphs of the crystal are remarkably similar, there are distinct differences in the isotropic chemical shifts between the two spectra. For the triclinic form, the carbon chemical shift tensor components for the alpha carbons adjacent to oxygen in the lactic acid and hydroxyisovaleric acid residues and the ester carbonyls of the valine residue were obtained using the FIREMAT experiment. From the measured components, it was found that the behavior of the isotropic chemical shift, δ iso , for valine residue ester carbonyl carbons is predominately influenced by the intermediate component, δ 22 . Additionally it was found that the smallest shift component, δ 33 , for the L-lactic acid (L-Lac) and D-α-hydroxyisovaleric acid (D-Hyi) C α -O carbon was significantly displaced depending upon the nature of individual amino acid residues, and it is the δ 33 component that governs the behavior of δ iso in these alpha carbons

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

Science.gov (United States)

Webster, Christopher R.; Mahaffy, Paul R.

2011-01-01

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

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

Science.gov (United States)

Thomas, Freddy; Jamin, Eric

2009-09-01

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

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

International Nuclear Information System (INIS)

Thomas, Freddy; Jamin, Eric

2009-01-01

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

Synthesis and antimicrobial activities of new higher amino acid Schiff base derivatives of 6-aminopenicillanic acid and 7-aminocephalosporanic acid

Science.gov (United States)

Özdemir (nee Güngör), Özlem; Gürkan, Perihan; Özçelik, Berrin; Oyardı, Özlem

2016-02-01

Novel β-lactam derivatives (1c-3c) (1d-3d) were produced by using 6-aminopenicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA) and the higher amino acid Schiff bases. The synthesized compounds were characterized by elemental analysis, IR, 1H/13C NMR and UV-vis spectra. Antibacterial activities of all the higher amino acid Schiff bases (1a-3a) (1b-3b) and β-lactam derivatives were screened against three gram negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii RSKK 02026), three gram positive bacteria (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 07005, Bacillus subtilis ATCC 6633) and their drug-resistant isolates by using broth microdilution method. Two fungi (Candida albicans and Candida krusei) were used for antifungal activity.

Determination of 13C isotopic enrichment of valine and threonine by GC-C-IRMS after formation of the N(O,S)-ethoxycarbonyl ethyl ester derivatives of the amino acids.

Science.gov (United States)

Godin, Jean-Philippe; Faure, Magali; Breuille, Denis; Hopfgartner, Gérard; Fay, Laurent-Bernard

2007-06-01

We describe a new method of assessing, in a single run, (13)C isotopic enrichment of both Val and Thr by gas chromatography-combustion-isotope-ratio mass spectrometry (GC-C-IRMS). This method characterised by a rapid one-step derivatisation procedure performed at room temperature to form the N(O,S)-ethoxycarbonyl ethyl ester derivatives, and a polar column for GC. The suitability of this method for Val and Thr in in-vivo samples (mucosal hydrolysate) was demonstrated by studying protein metabolism with two tracers ((13)C-valine or (13)C-threonine). The intra-day and inter-day repeatability were both assessed either with standards or with in-vivo samples at natural abundance and at low (13)C isotopic enrichment. For inter-day repeatability CVs were between 0.8 and 1.5% at natural abundance and lower than 5.5% at 0.112 and 0.190 atom% enrichment for Val and Thr, respectively. Overall isotopic precision was studied for eleven standard amino acid derivatives (those of Val, Ala, Leu, Iso, Gly, Pro, Asp, Thr, Ser, Met, and Phe) and was assessed at 0.32 per thousand. The (13)C isotopic measurement was then extended to the other amino acids (Ala, Val, Leu, Iso, Gly, Pro, Thr, and Phe) at natural abundance for in-vivo samples. The isotopic precision was better than 0.002 atom% per amino acid (for n = 4 rats). This analytical method was finally applied to an animal study to measure Thr utilization in protein synthesis.

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

International Nuclear Information System (INIS)

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

2011-01-01

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak 3 J(C α C α ) coupling. These pulse sequences, which resemble recently described 13 C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in 1 H 2 O, and use 1 H excitation and detection. These experiments require alternate 13 C- 12 C labeling together with perdeuteration, which allows utilizing the small 3 J(C α C α ) scalar coupling that is otherwise masked by the stronger 1 J CC couplings in uniformly 13 C 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 13 (C α ) 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-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the 15 N- 1 H 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.

Preparation, isolation and identification of non-conjugated C18:2 fatty acid isomers.

Science.gov (United States)

Fardin-Kia, Ali Reza

2016-12-01

Non-conjugated geometric/positional isomers of linoleic acid (c9,c12-18:2) are often present in processed foods and oils. The following work presents a simple addition/elimination reaction for preparation of non-conjugated 18:2 fatty acid isomers. A mixture containing positional and geometric isomers of C18:2 fatty acids was produced by addition of hydrobromic acid to the fatty acid double bonds, followed by its elimination with a strong sterically hindered base. Pure 8,12-, 8,13-, 9,12-, and 9,13-18:2 fatty acid methyl esters were isolated from the synthetic mixture by a combination of sub-ambient RP-HPLC and Ag + -HPLC. The determination of the double bond position was achieved by GC-MS using picolinyl esters derivatives. The determination of the fatty acid double bond geometric configuration was obtained by partial hydrogenation of the isolated isomer with hydrazine, followed by the GC-FID analysis. Published by Elsevier Ireland Ltd.

Synthesis of two hyaluronic-acid-related oligosaccharide 4-methoxyphenyl glycosides having a β-D-glucuronic acid residue at the reducing end

NARCIS (Netherlands)

Vliegenthart, J.F.G.; Halkes, K.M.; Slaghek, T.M.; Hyppönen, T.K.; Kamerling, J.P.

1999-01-01

Synthesis of two hyaluronic-acid-related oligosaccharides, the 4-methoxyphenyl beta-glycosides of beta-D-GlcpA-(1->3)-beta-D-GlcpNAc-(1->4)-D-GlcpA and beta-D-GlcpA-(1->3)-beta-D-GlcpNAc-(1->4)-beta-D-GlcpA-(1->3)-beta-D-GlcpNAc-(1->4)-D-GlcpA, is described. D-Glucopyranosyluronic acid residues were

Analysis of neuron–astrocyte metabolic cooperation in the brain of db/db mice with cognitive decline using 13C NMR spectroscopy

Science.gov (United States)

Zheng, Hong; Zheng, Yongquan; Wang, Dan; Cai, Aimin; Lin, Qiuting; Zhao, Liangcai; Chen, Minjiang; Deng, Mingjie; Ye, Xinjian

2016-01-01

Type 2 diabetes has been linked to cognitive impairment, but its potential metabolic mechanism is still unclear. The present study aimed to explore neuron–astrocyte metabolic cooperation in the brain of diabetic (db/db, BKS.Cg-m+/+ Leprdb/J) mice with cognitive decline using 13C NMR technique in combination with intravenous [2-13C]-acetate and [3-13C]-lactate infusions. We found that the 13C-enrichment from [2-13C]-acetate into tricarboxylic acid cycle intermediate, succinate, was significantly decreased in db/db mice with cognitive decline compared with wild-type (WT, C57BLKS/J) mice, while an opposite result was obtained after [3-13C]-lactate infusion. Relative to WT mice, db/db mice with cognitive decline had significantly lower 13C labeling percentages in neurotransmitters including glutamine, glutamate, and γ-aminobutyric acid after [2-13C]-acetate infusion. However, [3-13C]-lactate resulted in increased 13C-enrichments in neurotransmitters in db/db mice with cognitive decline. This may indicate that the disturbance of neurotransmitter metabolism occurred during the development of cognitive decline. In addition, a reduction in 13C-labeling of lactate and an increase in gluconeogenesis were found from both labeled infusions in db/db mice with cognitive decline. Therefore, our results suggest that the development of cognitive decline in type 2 diabetes may be implicated to an unbalanced metabolism in neuron–astrocyte cooperation and an enhancement of gluconeogenesis. PMID:26762505

Analysis of neuron-astrocyte metabolic cooperation in the brain of db/db mice with cognitive decline using 13C NMR spectroscopy.

Science.gov (United States)

Zheng, Hong; Zheng, Yongquan; Wang, Dan; Cai, Aimin; Lin, Qiuting; Zhao, Liangcai; Chen, Minjiang; Deng, Mingjie; Ye, Xinjian; Gao, Hongchang

2017-01-01

Type 2 diabetes has been linked to cognitive impairment, but its potential metabolic mechanism is still unclear. The present study aimed to explore neuron-astrocyte metabolic cooperation in the brain of diabetic (db/db, BKS.Cg-m +/+ Leprdb/J) mice with cognitive decline using 13 C NMR technique in combination with intravenous [2- 13 C]-acetate and [3- 13 C]-lactate infusions. We found that the 13 C-enrichment from [2- 13 C]-acetate into tricarboxylic acid cycle intermediate, succinate, was significantly decreased in db/db mice with cognitive decline compared with wild-type (WT, C57BLKS/J) mice, while an opposite result was obtained after [3- 13 C]-lactate infusion. Relative to WT mice, db/db mice with cognitive decline had significantly lower 13 C labeling percentages in neurotransmitters including glutamine, glutamate, and γ-aminobutyric acid after [2- 13 C]-acetate infusion. However, [3- 13 C]-lactate resulted in increased 13 C-enrichments in neurotransmitters in db/db mice with cognitive decline. This may indicate that the disturbance of neurotransmitter metabolism occurred during the development of cognitive decline. In addition, a reduction in 13 C-labeling of lactate and an increase in gluconeogenesis were found from both labeled infusions in db/db mice with cognitive decline. Therefore, our results suggest that the development of cognitive decline in type 2 diabetes may be implicated to an unbalanced metabolism in neuron-astrocyte cooperation and an enhancement of gluconeogenesis. © The Author(s) 2016.

Automated no-carrier-added synthesis of [1-{sup 11}C]-labeled D- and L-enantiomers of lactic acid

Energy Technology Data Exchange (ETDEWEB)

Drandarov, Konstantin [Center for Radiopharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, University Hospital Zuerich, CH-8091 Zurich (Switzerland); Paul Scherrer Institute, CH-5232 Villigen (Switzerland); Schubiger, P. August [Center for Radiopharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, University Hospital Zuerich, CH-8091 Zurich (Switzerland); Paul Scherrer Institute, CH-5232 Villigen (Switzerland); Westera, Gerrit [Center for Radiopharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, University Hospital Zuerich, CH-8091 Zurich (Switzerland) and Paul Scherrer Institute, CH-5232 Villigen (Switzerland)]. E-mail: gerrit.westera@usz.ch

2006-12-15

The first purely chemical method for automated no-carrier-added synthesis of [1-{sup 11}C]-labeled D(R)- and L(S)-2-hydroxypropanoic acid (lactic acid) was developed for experimental neurophysiology studies and position emission tomography (PET) diagnosis. Starting from sodium 1-hydroxyethanesulfonate and [{sup 11}C]HCN (trapped as [{sup 11}C]KCN) the intermediate DL-(R,S)-[1-{sup 11}C]-2-hydroxypropanenitrile was prepared. Its rapid acid hydrolysis gave DL-(R,S)-[1-{sup 11}C]lactic acid, which was isolated by preparative reversed phase HPLC and automatically injected on a second preparative C{sub 18} HPLC column coated with a chiral selector, where both [1-{sup 11}C]lactic acid enantiomers were separated by chiral ligand-exchange chromatography. Two novel chiral selectors for HPLC enantiomeric separation of {alpha}-hydroxy acids, namely D(R)- or L(S)-2-amino-3-methyl-3-(5-phenylpentylsulfanyl)-butanoic acid were utilized for the preparative HPLC separation of the [1-{sup 11}C]lactic acid enantiomers. The preparation of the selectors and the coating procedure for the manufacturing of the preparative chiral HPLC columns are described. A highly efficient trap for [{sup 11}C]HCN is presented. The whole radiosynthesis is automated, takes about 45 min and leads to more than 80% decay corrected overall radiochemical yield of each enantiomer (up to 2.5 GBq) with over 99% radiochemical, chemical and enantiomeric purity. The specific activity at the end of the synthesis is about 400 GBq/{mu}mol.

Contribution of diet to δ15N, δ13C, and Δ14C values in the Pacific rat (Rattus exulans)

International Nuclear Information System (INIS)

Beavan, N.R.

2001-01-01

This thesis is the outcome of a research project undertaken to determine how diet can affect the reliability of rat bone (especially that of Rattus exulans) for accurate radiocarbon analysis. It addresses questions about the reliability of R. exulans bone for radiocarbon dating with respect to the survival of bone in archaeological and natural burial sites, laboratory processing methods, and the extent to which diet influences radiocarbon ages of bone. I have shown that R. exulans bone can return reliable radiocarbon dates, based on bone survival and the efficacy of chemical treatment to remove burial contamination. In three dietary investigations, stable isotope (δ 13 C and δ 15 N) and radiocarbon ( 14 C) analyses of modern populations and archaeological specimens also indicated that Rattus exulans and other omnivorous species can have radiocarbon values influenced by a diet linked to 14 C - depleted reservoirs. Moreover, depleted carbon reservoir effects are localised and variable in their magnitude. Work on modern populations also provided an improved means of isotopic analysis in an ecological study, where bomb-generated radiocarbon (Δ 14 C) signatures in the natural environment were used as for other 'tracer' studies, in conjunction with stable isotopes (δ 13 C and δ 15 N). Results indicated that the changing atmospheric 14 C signal fixed into the biota in a given year by photosynthesis is transferred from plants through trophic levels to end-members, including rats. The additional isotopic 'clue' about diet given by radiocarbon analysis offered a better understanding of the variation in diets of R. exulans in different habitats. Variation in the isotopic signal among individuals supported other observations that diets of scavenging rats are opportunistic and associated with faunal availability in different habitats. To better understand diet-induced radiocarbon variations, I also examined the carbon contribution to bone protein from essential amino acids

Analysis of neuron–astrocyte metabolic cooperation in the brain of db/db mice with cognitive decline using 13C NMR spectroscopy

OpenAIRE

Zheng, Hong; Zheng, Yongquan; Wang, Dan; Cai, Aimin; Lin, Qiuting; Zhao, Liangcai; Chen, Minjiang; Deng, Mingjie; Ye, Xinjian; Gao, Hongchang

2016-01-01

Type 2 diabetes has been linked to cognitive impairment, but its potential metabolic mechanism is still unclear. The present study aimed to explore neuron–astrocyte metabolic cooperation in the brain of diabetic (db/db, BKS.Cg-m+/+ Leprdb/J) mice with cognitive decline using 13C NMR technique in combination with intravenous [2-13C]-acetate and [3-13C]-lactate infusions. We found that the 13C-enrichment from [2-13C]-acetate into tricarboxylic acid cycle intermediate, succinate, was significant...

Electric dipole moment of 13C

Science.gov (United States)

Yamanaka, Nodoka; Yamada, Taiichi; Hiyama, Emiko; Funaki, Yasuro

2017-06-01

We calculate for the first time the electric dipole moment (EDM) of 13C generated by the isovector charge conjugation-parity (CP)-odd pion exchange nuclear force in the α -cluster model, which describes well the structures of low-lying states of the 13C nucleus. The linear dependence of the EDM of 13C on the neutron EDM and the isovector CP-odd nuclear coupling is found to be d13C=-0.33 dn-0.0020 G¯π(1 ) . The linear enhancement factor of the CP-odd nuclear coupling is smaller than that of the deuteron, due to the difference of the structure between the 1 /21- state and the opposite-parity (1 /2+ ) states. We clarify the role of the structure played in the enhancement of the EDM. This result provides good guiding principles to search for other nuclei with large enhancement factor. We also mention the role of the EDM of 13C in determining the new physics beyond the standard model.

In Folio Respiratory Fluxomics Revealed by {sup 13}C Isotopic Labeling and H/D Isotope Effects Highlight the Non-cyclic Nature of the Tricarboxylic Acid 'Cycle' in Illuminated Leaves

Energy Technology Data Exchange (ETDEWEB)

Tcherkez, G; Mahe, A; Gauthier, P; Hodges, M [Institut de Biotechnologie des Plantes, Plateforme Metabolisme-Metabolome IFR87, Batiment 630, Universite Paris-Sud 11, 91405 Orsay cedex (France); Tcherkez, G; Mauve, C; Cornic, G [Laboratoire d' Ecophysiologie Vegetale, Ecologie Systematique Evolution (G.C.), Batiment 630, Universite Paris-Sud 11, 91405 Orsay cedex (France); Gout, E; Bligny, R [Laboratoire de Physiologie Cellulaire Vegetale, Commissariat a l' Energie Atomique-Grenoble, 38054 Grenoble cedex 9 (France)

2009-07-01

While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, {sup 13}C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA 'cycle' does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation. (authors)

Sensitivity enhancement of 13C nuclei in 2D J-resolved NMR spectroscopy using a recycled-flow system

International Nuclear Information System (INIS)

Ha, S.T.K.; Lee, R.W.K.; Wilkins, C.L.

1987-01-01

Recycled-flow nuclear magnetic resonance for sensitivity enhancement in 1/2 spin nuclei has been reported previously, achieving several-fold signal enhancement. The success of the method depends upon premagnetization of nuclei prior to flowing into the detector region, obviating the need for delays following data acquisition to allow spin-lattice relaxation and reduce experiment time. The actual gains of sensitivity enhancement for 13 C- 1 H 2D J-resolved NMR using a recycled-flow method are evaluated. Possible enhancements for two types of J-resolved measurements, namely, one-bond 13 C- 1 H and long range J-resolved spectroscopy, are estimated using a simple Carr-Purcell spin-echo approach to quantify the 13 C signals. The pulse sequence is simply 90 0 -t /sub 1/2/-180 0 -t/sub 1/2/-AT-t/sub d/, where t/sub 1/2/ is half the evolution time, AT is the acquisition time, and t/sub d/ the experiment repetition time. In a static 2D NMR experiment, t/sub d/ usually must be the same order of the longest spin-lattice relaxation time (T 1 ) of nuclei. Quantitative measurements using a recycled-flow system indicate t/dub d/ can be reduced to a fraction of T 1 ; hence significant time savings can be achieved. Time-savings of between 2 and 25 can be anticipated for 2D spectroscopy under flow measurement conditions used in the present study. Other types of 2D NMR spectroscopy (autocorrelation and double quantum NMR) are discussed

Synthesis of O- and C-glycosides derived from β-(1,3)-D-glucans.

Science.gov (United States)

Marca, Eduardo; Valero-Gonzalez, Jessika; Delso, Ignacio; Tejero, Tomás; Hurtado-Guerrero, Ramon; Merino, Pedro

2013-12-15

A series of β-(1,3)-d-glucans have been synthesized incorporating structural variations specifically on the reducing end of the oligomers. Both O- and C-glucosides derived from di- and trisaccharides have been obtained in good overall yields and with complete selectivity. Whereas the O-glycosides were obtained via a classical Koenigs-Knorr glycosylation, the corresponding C-glycosides were obtained through allylation of the anomeric carbon and further cross-metathesis reaction. Finally, the compounds were evaluated against two glycosidases and two endo-glucanases and no inhibitory activity was observed. Copyright © 2013 Elsevier Ltd. All rights reserved.

Synthesis and applications of 13C glycerol

International Nuclear Information System (INIS)

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

1994-01-01

The authors are currently developing new synthetic routes to the various isotopomers of glycerol. Labeled glycerol is useful for 13 C enrichment of biomolecules. However, traditional methods of glycerol synthesis are not easily adapted for isotopic enrichment or have poor overall yields (12-15%). In addition, the use of glycerol for enrichment can be prohibitively expensive and its availability depends on the level of demand. The authors have developed a short de novo synthesis of [U- 13 C]glycerol from carbon dioxide (∼53% overall yield for four steps) and are currently examining the feasibility of synthesizing site-specific 13 C labeled glycerol and dihydroxyacetone (DHA) from methanol and carbon dioxide. The authors have examined the enzymatic conversion of [U- 13 C]glycerol to glyceraldehyde-3-monophosphate or dihydroxyacetone monophosphate (DHAP) with yields ranging from 25-50% (as determined by NMR spectroscopy). The authors are also pursuing the chemical conversion of 13 C labeled DHA to DHAP and the results are presented. Labeled DHAP is a possible enzymatic precursor for both labeled 2-deoxyribose and 2-deoxyribonucleic acids

Gap Analysis Bulletin No. 13

Science.gov (United States)

2005-12-01

we would like to web developer; gather comments from GAP researchers and data users. We are * facilitate collaboration among GAP projects by...N.Y. Research Grant #012/01 A. 42 Gap Analysis Bulletin No. 13, December 2005 Ga pAnalysis Smith, S. D., W. A. Brown, C. R. Smith, and M. E. Richmond... GAP will be focusing activities have greatly reduced the habitat available to support on the enduring features of the Great Lakes basin. Influences

The 12C/ 13C isotopic ratio in Titan hydrocarbons from Cassini/CIRS infrared spectra

Science.gov (United States)

Nixon, C. A.; Achterberg, R. K.; Vinatier, S.; Bézard, B.; Coustenis, A.; Irwin, P. G. J.; Teanby, N. A.; de Kok, R.; Romani, P. N.; Jennings, D. E.; Bjoraker, G. L.; Flasar, F. M.

2008-06-01

We have analyzed infrared spectra of Titan recorded by the Cassini Composite Infrared Spectrometer (CIRS) to measure the isotopic ratio 12C/ 13C in each of three chemical species in Titan's stratosphere: CH 4, C 2H 2 and C 2H 6. This is the first measurement of 12C/ 13C in any C 2 molecule on Titan, and the first measurement of 12CH 4/ 13CH 4 (non-deuterated) on Titan by remote sensing. Our spectra cover five widely-spaced latitudes, 65° S to 71° N and we have searched for both latitude variability of 12C/ 13C within a given species, and also for differences between the 12C/ 13C in the three gases. For CH 4 alone, we find C12/C13=76.6±2.7 (1- σ), essentially in agreement with the 12CH 4/ 13CH 4 measured by the Huygens Gas Chromatograph/Mass Spectrometer instrument (GCMS) [Niemann, H.B., and 17 colleagues, 2005. Nature 438, 779-784]: 82.3±1.0, and also with measured values in H 13CN and 13CH 3D by CIRS at lower precision [Bézard, B., Nixon, C., Kleiner, I., Jennings, D., 2007. Icarus 191, 397-400; Vinatier, S., Bézard, B., Nixon, C., 2007. Icarus 191, 712-721]. For the C 2 species, we find C12/C13=84.8±3.2 in C 2H 2 and 89.8±7.3 in C 2H 6, a possible trend of increasingly value with molecular mass, although these values are both compatible with the Huygens GCMS value to within error bars. There are no convincing trends in latitude. Combining all fifteen measurements, we obtain a value of C12/C13=80.8±2.0, also compatible with GCMS. Therefore, the evidence is mounting that 12C/ 13C is some 8% lower on Titan than on the Earth (88.9, inorganic standard), and lower than typical for the outer planets ( 88±7 [Sada, P.V., McCabe, G.H., Bjoraker, G.L., Jennings, D.E., Reuter, D.C., 1996. Astrophys. J. 472, 903-907]). There is no current model for this enrichment, and we discuss several mechanisms that may be at work.

Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins

International Nuclear Information System (INIS)

Jacob, Jaison; Louis, John M.; Nesheiwat, Issa; Torchia, Dennis A.

2002-01-01

Analysis of 2D [ 13 C, 1 H]-HSQC spectra of biosynthetic fractionally 13 C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional 13 C labeling yields aromatic rings in which some of the 13 C- 13 C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the δ-, ε- and ζ-carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the 13 C constant-time period in 2D [ 13 C, 1 H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic 13 C CSA and 13 C- 1 H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution 13 C constant-time spectra with good sensitivity

SOIL RESPIRED D13C SIGNATURES REFLECT ROOT EXUDATE OR ROOT TURNOVER SIGNATURES IN AN ELEVATED CO2 AND OZONE MESOCOSM EXPERIMENT

Science.gov (United States)

Bulk tissue and root and soil respired d13C signatures were measured throughout the soil profile in a Ponderosa Pine mesocosm experiment exposed to ambient and elevated CO2 concentrations. For the ambient treatment, root (0-1mm, 1-2mm, and >2mm) and soil d13C signatures were ?24...

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

Science.gov (United States)

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

2015-07-01

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

OpenFLUX: efficient modelling software for 13C-based metabolic flux analysis

Directory of Open Access Journals (Sweden)

Nielsen Lars K

2009-05-01

Full Text Available Abstract Background The quantitative analysis of metabolic fluxes, i.e., in vivo activities of intracellular enzymes and pathways, provides key information on biological systems in systems biology and metabolic engineering. It is based on a comprehensive approach combining (i tracer cultivation on 13C substrates, (ii 13C labelling analysis by mass spectrometry and (iii mathematical modelling for experimental design, data processing, flux calculation and statistics. Whereas the cultivation and the analytical part is fairly advanced, a lack of appropriate modelling software solutions for all modelling aspects in flux studies is limiting the application of metabolic flux analysis. Results We have developed OpenFLUX as a user friendly, yet flexible software application for small and large scale 13C metabolic flux analysis. The application is based on the new Elementary Metabolite Unit (EMU framework, significantly enhancing computation speed for flux calculation. From simple notation of metabolic reaction networks defined in a spreadsheet, the OpenFLUX parser automatically generates MATLAB-readable metabolite and isotopomer balances, thus strongly facilitating model creation. The model can be used to perform experimental design, parameter estimation and sensitivity analysis either using the built-in gradient-based search or Monte Carlo algorithms or in user-defined algorithms. Exemplified for a microbial flux study with 71 reactions, 8 free flux parameters and mass isotopomer distribution of 10 metabolites, OpenFLUX allowed to automatically compile the EMU-based model from an Excel file containing metabolic reactions and carbon transfer mechanisms, showing it's user-friendliness. It reliably reproduced the published data and optimum flux distributions for the network under study were found quickly ( Conclusion We have developed a fast, accurate application to perform steady-state 13C metabolic flux analysis. OpenFLUX will strongly facilitate and

Studies of the intermediary metabolism in cultured cells of the insect Spodoptera frugiperda using 13C- or 15N-labelled tracers

Directory of Open Access Journals (Sweden)

Bacher Adelbert

2005-11-01

Full Text Available Abstract Background Insect cells can serve as host systems for the recombinant expression of eukaryotic proteins. Using this platform, the controlled expression of 15N/13C labelled proteins requires the analysis of incorporation paths and rates of isotope-labelled precursors present in the medium into amino acids. For this purpose, Spodoptera frugiperda cells were grown in a complex medium containing [U-13C6]glucose. In a second experiment, cultures of S. frugiperda were grown in the presence of 15N-phenylalanine. Results Quantitative NMR analysis showed incorporation of the proffered [U-13C6]glucose into the ribose moiety of ribonucleosides (40 – 45% and into the amino acids, alanine (41%, glutamic acid/glutamine (C-4 and C-5, 30% and aspartate/asparagine (15%. Other amino acids and the purine ring of nucleosides were not formed from exogenous glucose in significant amounts (> 5%. Prior to the incorporation into protein the proffered 15N-phenylalanine lost about 70% of its label by transamination and the labelled compound was not converted into tyrosine to a significant extent. Conclusion Growth of S. frugiperda cells in the presence of [U-13C6]glucose is conducive to the fractional labelling of ribonucleosides, alanine, glutamic acid/glutamine and aspartic acid/asparagine. The isotopolog compositions of the ribonucleosides and of alanine indicate considerable recycling of carbohydrate intermediates in the reductive branch of the pentose phosphate pathway. The incorporation of 15N-labelled amino acids may be hampered by loss of the 15N-label by transamination.

Combining position-specific 13C labeling with compound-specific isotope analysis: first steps towards soil fluxomics

Science.gov (United States)

Dippold, Michaela; Kuzyakov, Yakov

2015-04-01

sugar 13C analysis showed that oxidizing catabolic pathways and anabolic pathways, i.e. building-up new cellular compounds, occurred in soils simultaneously. This involved an intensive C recycling within the microorganisms that was observed not only for cytosolic compounds but also for cell wall polymers. Fungal metabolism and fluxes were slower than bacterial intracellular C recycling and turnover. Furthermore, position-specific labeling of glutamate and subsequent 13C analysis of microbial phospholipid fatty acids (PLFA) revealed starvation pathways, which were only active in specific microbial groups in soils. These studies revealed that position-specific labeling enables the reconstruction of metabolic pathways of LMWOS within diverse microbial communities in complex media such as soil. Processes occurring simultaneously in soil i.e. 1) within individual, reversible metabolic pathways and 2) in various microbial groups could be traced by position-specific labeling in soils in situ. Tracing these pathways and understanding their regulating factors are crucial for soil C fluxomics, the extremely complex network of transformations towards mineralization versus the formation of microbial biomass compounds. Quantitative models to assess microbial group specific metabolic networks can be generated and parameterized by this approach. The submolecular knowledge of transformation steps and biochemical pathways in soils and their regulating factors is essential for understanding C cycling and long-term C storage in soils.

Mono terpenes characterization by {sup 1} H and {sup 13} C-1 NMR; Caracterizacao de monoterpenos por RMN - {sup 1} H e de {sup 13} C-1

Energy Technology Data Exchange (ETDEWEB)

Araujo, Martha T. de [Universidade Federal Fluminense, Niteroi, RJ (Brazil). Dept. de Fisico-Quimica; Silveira, Carmen L.P. [Universidade Federal Fluminense, Niteroi, RJ (Brazil). Dept. de Quimica Organica; Mcchesney, James D [Mississippi Univ., University, MS (United States). Research Inst. of Pharmaceutical Sciences

1992-12-31

Artemisinine, a new lactone sesquiterpene containing one peroxide binding, is the main anti malarial agent obtained from the Artemisia annua L. Viewing to obtain a simple synthetic route for artemisinic acid preparation, which is the key intermediary for total synthesis of this type of anti malarial agent, R-carvone has been chosen as starting material. The S-carvone was used as model for reaction optimization and preparation of derivatives to be used for NMR studies. The main objective of this work is the signalling of the {sup 13} C and {sup 1} H NMR spectra, using the 2 D-COSY and 2 D-Hector spectra 4 refs., 3 figs., 1 tab

In Folio Respiratory Fluxomics Revealed by 13C Isotopic Labeling and H/D Isotope Effects Highlight the Noncyclic Nature of the Tricarboxylic Acid “Cycle” in Illuminated Leaves1[W

Science.gov (United States)

Tcherkez, Guillaume; Mahé, Aline; Gauthier, Paul; Mauve, Caroline; Gout, Elizabeth; Bligny, Richard; Cornic, Gabriel; Hodges, Michael

2009-01-01

While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, 13C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA “cycle” does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation. PMID:19675152

{sup 15}N and {sup 13}C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated, selectively [{sup 1}H,{sup 13}C]-labeled proteins

Energy Technology Data Exchange (ETDEWEB)

Rossi, Paolo, E-mail: rossip@umn.edu; Xia, Youlin; Khanra, Nandish; Veglia, Gianluigi, E-mail: vegli001@umn.edu; Kalodimos, Charalampos G., E-mail: ckalodim@umn.edu [University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics (United States)

2016-12-15

The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply ‘SOFAST-HMQC’ to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and {sup 15}N, methyl labeled samples in H{sub 2}O. The experiments benefit from a combination of selective T{sub 1} relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear {sup 15}N,{sup 13}C-edited, or with diagonal-free {sup 13}C aromatic/methyl-resolved 3D-SOFAST-HMQC–NOESY–HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY–HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination.

In vivo 13C MRS in the mouse brain at 14.1 Tesla and metabolic flux quantification under infusion of [1,6-13C2]glucose.

Science.gov (United States)

Lai, Marta; Lanz, Bernard; Poitry-Yamate, Carole; Romero, Jackeline F; Berset, Corina M; Cudalbu, Cristina; Gruetter, Rolf

2017-01-01

In vivo 13 C magnetic resonance spectroscopy (MRS) enables the investigation of cerebral metabolic compartmentation while, e.g. infusing 13 C-labeled glucose. Metabolic flux analysis of 13 C turnover previously yielded quantitative information of glutamate and glutamine metabolism in humans and rats, while the application to in vivo mouse brain remains exceedingly challenging. In the present study, 13 C direct detection at 14.1 T provided highly resolved in vivo spectra of the mouse brain while infusing [1,6- 13 C 2 ]glucose for up to 5 h. 13 C incorporation to glutamate and glutamine C4, C3, and C2 and aspartate C3 were detected dynamically and fitted to a two-compartment model: flux estimation of neuron-glial metabolism included tricarboxylic acid cycle (TCA) flux in astrocytes (V g  = 0.16 ± 0.03 µmol/g/min) and neurons (V TCA n  = 0.56 ± 0.03 µmol/g/min), pyruvate carboxylase activity (V PC  = 0.041 ± 0.003 µmol/g/min) and neurotransmission rate (V NT  = 0.084 ± 0.008 µmol/g/min), resulting in a cerebral metabolic rate of glucose (CMR glc ) of 0.38 ± 0.02 µmol/g/min, in excellent agreement with that determined with concomitant 18 F-fluorodeoxyglucose positron emission tomography ( 18 FDG PET).We conclude that modeling of neuron-glial metabolism in vivo is accessible in the mouse brain from 13 C direct detection with an unprecedented spatial resolution under [1,6- 13 C 2 ]glucose infusion.

Synthesis and applications of 13C glycerol

International Nuclear Information System (INIS)

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

1994-01-01

Due in part to the use of labeled glycerol for the 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 (∼53% overall yield for four steps) and are examining the feasibility of synthesizing site-specific 13 C-labeled glycerol and dihydroxyacetone (DHA) from labeled methanol and carbon dioxide. One application of 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 13 C-labeled DHA to DHAP. We are especially interested in 13 C-labeled DHAP because we are investigating its use as a chemo-enzymatic precursor for both labeled 2-deoxyribose and 2-deoxyribonucleic acids

A method for differentiating between vinegar produced by fermentation and vinegar made from synthetic acetic acid based on determination of 13C/12C-isotope ratio by mass spectrometry

International Nuclear Information System (INIS)

Schmid, E.R.; Fogy, I.

1978-01-01

The 13 C/ 12 C-isotope ratio is characteristic for vinegar of fermentation and synthetic origin respectively and used for their differentiation. The acetic acid was isolated from the vinegar as calcium acetate, the calcium acetate was pyrolysed to CaCO 3 and the CO 2 was released from the CaCO 3 with H 3 PO 4 . The CO 2 was measured in a mass spectrometer with double collector. The difference in the 13 C- content between the two varieties of vinegar is 5 0 / 00 ; the accuracy of the measurement is between 0,5 0 / 00 and 1 0 / 00 . Therefore, addition of synthetic acetic acid in excess of 15-20% to fermentation vinegar can be detected by this method. (orig.) [de

Structural characteristics of marine sedimentary humic acids by CP/MAS sup(13)C NMR spectroscopy

Digital Repository Service at National Institute of Oceanography (India)

Sardessai, S.; Wahidullah, S.

quino- nes or semiquinones which exhibit 13C resonance at around 195 ppm. Free radicals of semiquinone type have been reported from the aquatic environment [2]. Spectra of all the four samples show absorption at 195 ppm. The total acidity of marine..., significant resonance at 55 ppm (8.9) from aromatic methoxy carbon suggests that phenol is present mostly as methyl ether and not as free phenol. The signal at 55 ppm is also attributed to aliphatic carbon adjacent to the amino functional group as in amino...

Integrated multidimensional and comprehensive 2D GC analysis of fatty acid methyl esters.

Science.gov (United States)

Zeng, Annie Xu; Chin, Sung-Tong; Marriott, Philip J

2013-03-01

Fatty acid methyl ester (FAME) profiling in complex fish oil and milk fat samples was studied using integrated comprehensive 2D GC (GC × GC) and multidimensional GC (MDGC). Using GC × GC, FAME compounds--cis- and trans-isomers, and essential fatty acid isomers--ranging from C18 to C22 in fish oil and C18 in milk fat were clearly displayed in contour plot format according to structural properties and patterns, further identified based on authentic standards. Incompletely resolved regions were subjected to MDGC, with Cn (n = 18, 20) zones transferred to a (2)D column. Elution behavior of C18 FAME on various (2)D column phases (ionic liquids IL111, IL100, IL76, and modified PEG) was evaluated. Individual isolated Cn zones demonstrated about four-fold increased peak capacities. The IL100 provided superior separation, good peak shape, and utilization of elution space. For milk fat-derived FAME, the (2)D chromatogram revealed at least three peaks corresponding to C18:1, more than six peaks for cis/trans-C18:2 isomers, and two peaks for C18:3. More than 17 peaks were obtained for the C20 region of fish oil-derived FAMEs using MDGC, compared with ten peaks using GC × GC. The MDGC strategy is useful for improved FAME isomer separation and confirmation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of natural metabolites in mixture: a pattern recognition strategy based on (13)C NMR.

Science.gov (United States)

Hubert, Jane; Nuzillard, Jean-Marc; Purson, Sylvain; Hamzaoui, Mahmoud; Borie, Nicolas; Reynaud, Romain; Renault, Jean-Hugues

2014-03-18

Because of their highly complex metabolite profile, the chemical characterization of bioactive natural extracts usually requires time-consuming multistep purification procedures to achieve the structural elucidation of pure individual metabolites. The aim of the present work was to develop a dereplication strategy for the identification of natural metabolites directly within mixtures. Exploiting the polarity range of metabolites, the principle was to rapidly fractionate a multigram quantity of a crude extract by centrifugal partition extraction (CPE). The obtained fractions of simplified chemical composition were subsequently analyzed by (13)C NMR. After automatic collection and alignment of (13)C signals across spectra, hierarchical clustering analysis (HCA) was performed for pattern recognition. As a result, strong correlations between (13)C signals of a single structure within the mixtures of the fraction series were visualized as chemical shift clusters. Each cluster was finally assigned to a molecular structure with the help of a locally built (13)C NMR chemical shift database. The proof of principle of this strategy was achieved on a simple model mixture of commercially available plant secondary metabolites and then applied to a bark extract of the African tree Anogeissus leiocarpus Guill. & Perr. (Combretaceae). Starting from 5 g of this genuine extract, the fraction series was generated by CPE in only 95 min. (13)C NMR analyses of all fractions followed by pattern recognition of (13)C chemical shifts resulted in the unambiguous identification of seven major compounds, namely, sericoside, trachelosperogenin E, ellagic acid, an epimer mixture of (+)-gallocatechin and (-)-epigallocatechin, 3,3'-di-O-methylellagic acid 4'-O-xylopyranoside, and 3,4,3'-tri-O-methylflavellagic acid 4'-O-glucopyranoside.

Intramolecular 13C analysis of tree rings provides multiple plant ecophysiology signals covering decades.

Science.gov (United States)

Wieloch, Thomas; Ehlers, Ina; Yu, Jun; Frank, David; Grabner, Michael; Gessler, Arthur; Schleucher, Jürgen

2018-03-22

Measurements of carbon isotope contents of plant organic matter provide important information in diverse fields such as plant breeding, ecophysiology, biogeochemistry and paleoclimatology. They are currently based on 13 C/ 12 C ratios of specific, whole metabolites, but we show here that intramolecular ratios provide higher resolution information. In the glucose units of tree-ring cellulose of 12 tree species, we detected large differences in 13 C/ 12 C ratios (>10‰) among carbon atoms, which provide isotopically distinct inputs to major global C pools, including wood and soil organic matter. Thus, considering position-specific differences can improve characterisation of soil-to-atmosphere carbon fluxes and soil metabolism. In a Pinus nigra tree-ring archive formed from 1961 to 1995, we found novel 13 C signals, and show that intramolecular analysis enables more comprehensive and precise signal extraction from tree rings, and thus higher resolution reconstruction of plants' responses to climate change. Moreover, we propose an ecophysiological mechanism for the introduction of a 13 C signal, which links an environmental shift to the triggered metabolic shift and its intramolecular 13 C signature. In conclusion, intramolecular 13 C analyses can provide valuable new information about long-term metabolic dynamics for numerous applications.

PET tomographic 2-D and/or 3-D imaging of nitrogen-13, derived from 13N-amino acids, in the heart, pancreas, liver

International Nuclear Information System (INIS)

Myers, W.G.; Bigler, R.E.; Gelbard, A.S.; Benua, R.S.; Graham, M.C.; Laughlin, J.S.

1982-01-01

The loci of accumulations of Nitrogen-13, derived from enzymatically-synthesized, intravenously-injected, natural L-forms of (N-13)-L-Glutamate, (N-13)-L-Leucine, (N-13)-L-Valine, or (N-13)-L-Methionine, were determined in situ in several organs after a large meal. The images were made with the first commercial version of the MGH ''positron'' camera. When operated in the transverse, coronal, 2-D mode, high concentrations of N-13 were found consistently in the Heart and Pancreas, and lesser amounts in the Liver, after (N-13)-Glutamate or (N-13)-L-Leucine. Little accumulation occurred in the Heart after (N-13)-L-Methionine or (N-13)-L-Valine. High concentrations of N-13 were found in the Pancreas after the (N-13)-L-Methionine or (N-13)-L-Valine. When the PCT-4200 instrument was operated in the 3-D orthogonal mode, following intravenous (N-13)-L-Glutamate, five transverse 1.0-cm-thick slice images were made at 1.4-cm sequential spacings, both of the Heart and of the Pancreas. The computer simultaneously collected the data for reconstrutions subsequently of all ten image slices through both the Heart and the Pancreas

Analysis of dissolved organic carbon concentration and 13C isotopic signature by TOC-IRMS - assessment of analytical performance

Science.gov (United States)

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

2013-04-01

Stable carbon isotopes provide a powerful tool to assess carbon pools and their dynamics. Dissolved organic carbon (DOC) has been recognized to play an important role in ecosystem functioning and carbon cycling and has therefore gained increased research interest. However, direct measurement of 13C isotopic signature of carbon in the dissolved phase is technically challenging particularly using high temperature combustion. Until recently, mainly custom-made systems existed which were modified for coupling of TOC instruments with IRMS for simultaneous assessment of C content and isotopic signature. The variety of coupled systems showed differences in their analytical performances. For analysis of DOC high temperature combustion is recognized as best performing method, owing to its high efficiency of conversion to CO2 also for highly refractory components (e.g. humic, fulvic acids) present in DOC and soil extracts. Therefore, we tested high temperature combustion TOC coupled to IRMS (developed by Elementar Group) for bulk measurements of DOC concentration and 13C signature. The instruments are coupled via an Interface to exchange the carrier gas from O2 to He and to concentrate the derived CO2 for the isotope measurement. Analytical performance of the system was assessed for a variety of organic compounds characterized by different stability and complexity, including humic acid and DOM. We tested injection volumes between 0.2-3 ml, thereby enabling measurement of broad concentration ranges. With an injection volume of 0.5 ml (n=3, preceded by 1 discarded injection), DOC and 13C signatures for concentrations between 5-150 mg C/L were analyzed with high precision (standard deviation (SD) predominantly TOC-IRMS in comparison with other systems capable of determining C concentration and isotopic signatures. We recognize the advantages of this system providing: - High sample throughput, short measurement time (15 minutes), flexible sample volume - Easy maintenance

The structure of teichoic acid from Bacillus subtilis var. niger WM as determined by 13C nuclear-magnetic-resonance spectroscopy

International Nuclear Information System (INIS)

De Boer, W.R.; Kruyssen, F.J.; Wouters, J.T.M.; Kruk, C.

1976-01-01

The walls of Bacillus subtilis var. niger WM, grown in a Mg 2+ -limited chemostat culture (carbon source glucose, dilution rate = 0.2 h -1 , 37 0 C, pH 7) contained 45% (w/w) teichoic acid, a polymer composed of glycerol, phosphate and glucose in the molar ratio 1.00 : 1.00 : 0.88. Alkaline hydrolysis of this teichoic acid yielded 1-O-β-glucosylglycerol phosphate (together with small amounts of glycerol phosphate), and 13 C nuclear magnetic resonance spectra of this hydrolysis product, and its derivative after alkaline phosphatase treatment, confirmed that the monomeric unit was 1-O-β-glucosylglycerol-3-phosphate. Assignment of the resonances in the spectrum of undegraded teichoic acid revealed that the polymer was a poly[(2,3)glycerol phosphate], glucosidically substituted on C-1 of glycerol with β-glucose. (orig.) [de

In Folio Respiratory Fluxomics Revealed by {sup 13}C Isotopic Labeling and H/D Isotope Effects Highlight the Non-cyclic Nature of the Tricarboxylic Acid 'Cycle' in Illuminated Leaves

Energy Technology Data Exchange (ETDEWEB)

Tcherkez, G.; Mahe, A.; Gauthier, P.; Hodges, M. [Institut de Biotechnologie des Plantes, Plateforme Metabolisme-Metabolome IFR87, Batiment 630, Universite Paris-Sud 11, 91405 Orsay cedex (France); Tcherkez, G.; Mauve, C.; Cornic, G. [Laboratoire d' Ecophysiologie Vegetale, Ecologie Systematique Evolution (G.C.), Batiment 630, Universite Paris-Sud 11, 91405 Orsay cedex (France); Gout, E.; Bligny, R. [Laboratoire de Physiologie Cellulaire Vegetale, Commissariat a l' Energie Atomique-Grenoble, 38054 Grenoble cedex 9 (France)

2009-07-01

While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, {sup 13}C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA 'cycle' does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation. (authors)

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

Energy Technology Data Exchange (ETDEWEB)

Takeuchi, Koh; Gal, Maayan [Harvard Medical School, Department of Biochemistry and Molecular Pharmacology (United States); Takahashi, Hideo; Shimada, Ichio [National Institute of Advanced Industrial Science and Technology, Biomedicinal Information Research Center (Japan); Wagner, Gerhard, E-mail: gerhard_wagner@hms.harvard.edu [Harvard Medical School, Department of Biochemistry and Molecular Pharmacology (United States)

2011-01-15

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak {sup 3}J(C{sub {alpha}}C{sub {alpha}}) coupling. These pulse sequences, which resemble recently described {sup 13}C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in {sup 1}H{sub 2}O, and use {sup 1}H excitation and detection. These experiments require alternate {sup 13}C-{sup 12}C labeling together with perdeuteration, which allows utilizing the small {sup 3}J(C{sub {alpha}}C{sub {alpha}}) scalar coupling that is otherwise masked by the stronger {sup 1}J{sub CC} couplings in uniformly {sup 13}C 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 {sup 13}(C{sub {alpha}}) 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-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the {sup 15}N-{sup 1}H 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.

Constraining genome-scale models to represent the bow tie structure of metabolism for 13C metabolic flux analysis

DEFF Research Database (Denmark)

Backman, Tyler W.H.; Ando, David; Singh, Jahnavi

2018-01-01

for a minimum of fluxes into core metabolism to satisfy these experimental constraints. Together, these methods accelerate and automate the identification of a biologically reasonable set of core reactions for use with 13C MFA or 2S- 13C MFA, as well as provide for a substantially lower set of flux bounds......Determination of internal metabolic fluxes is crucial for fundamental and applied biology because they map how carbon and electrons flow through metabolism to enable cell function. 13C Metabolic Flux Analysis (13C MFA) and Two-Scale 13C Metabolic Flux Analysis (2S-13C MFA) are two techniques used...

Gas chromatography/isotope ratio mass spectrometry: analysis of methanol, ethanol and acetic acid by direct injection of aqueous alcoholic and acetic acid samples.

Science.gov (United States)

Ai, Guomin; Sun, Tong; Dong, Xiuzhu

2014-08-15

Methanol, ethanol, and acetic acid are not easily extracted from aqueous samples and are susceptible to isotope fractionation in gas chromatography/isotope ratio mass spectrometry (GC/IRMS) analysis. Developing a direct dilution GC/IRMS method for aqueous samples, by adjusting the sample concentrations in common solvents to be similar to each other and using a fixed GC split ratio, is very convenient and important because any linearity effects caused by amount-dependent isotope fractionation can be avoided. The suitability of acetonitrile and acetone solvents for the GC/IRMS analysis of pure methanol, ethanol and acetic acid, and commercial liquor and vinegar samples was evaluated using n-hexane and water as control solvents. All the solvents including water were separated from the analyte on a HP-INNOWAX column and were diverted away from the combustion interface. The influence of liquor matrix on the ethanol GC/IRMS analyses was evaluated by adding pure ethanol to liquor samples. Acetonitrile and acetone gave similar δ(13) C values for pure ethanol and pure acetic acid to those obtained in water and n-hexane, and also gave similar δ(13) C values of ethanol in liquor and acetic acid in white vinegar to that obtained in water. For methanol analysis, acetonitrile and refined acetone gave similar δ(13) C values to that obtained in water, but n-hexane was not a suitable solvent. In addition, isotopic fractionation caused by solvent and solute interactions was observed. We recommend using acetonitrile for the GC/IRMS analysis of aqueous alcoholic samples, and acetone for the analysis of aqueous acetic acid samples. This direct dilution method can provide high accurate and precise GC/IRMS analysis of the relative changes in δ(13) C values of methanol, ethanol, and acetic acid. Copyright © 2014 John Wiley & Sons, Ltd.

Stable carbon isotope analysis (δ13C values) of polybrominated diphenyl ethers and their UV-transformation products

International Nuclear Information System (INIS)

Rosenfelder, Natalie; Bendig, Paul; Vetter, Walter

2011-01-01

Polybrominated diphenyl ethers (PBDEs) are frequently detected in food and environmental samples. We used compound specific isotope analysis to determine the δ 13 C values of individual PBDEs in two technical mixtures. Within the same technical product (DE-71 or DE-79), BDE congeners were the more depleted in 13 C the higher brominated they were. In contrast, the products of light-induced hydrodebromination of BDE 47 and technical DE-79 were more enriched in 13 C because of more stable bonds between 13 C and bromine. As a result, the δ 13 C values of the irradiated solution progressed diametrically compared to those of the technical synthesis. The ratio of the δ 13 C values of BDE 47 to BDE 99 and of BDE 99 to BDE 153 are thus suggested as indicators to distinguish native technical products from transformation products. Ratios 1) is typical of transformation products. - Highlights: → δ 13 C values of PBDEs were determined by means of compound specific isotope analysis. → PBDEs in technical mixtures were the more depleted in 13 C the higher they were brominated. → Solutions of individual PBDEs and technical PBDE mixtures were irradiated by UV light. → δ 13 C values of irradiated PBDEs and technical PBDEs progressed diametrically. → Ratios of the δ 13 C values were used to distinguish native from transformed PBDEs. - Diametrically progressing δ 13 C values in technical mixtures and UV-transformation products of DE-79 may be useful for source appointment of PBDEs in environmental samples

Conventional high-performance liquid chromatography versus derivative spectrophotometry for the determination of 1,3,6-pyrenetrisulfonic acid trisodium salt and 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt in the color additive D&C Green No. 8 (Pyranine).

Science.gov (United States)

Jitian, Simion; White, Samuel R; Yang, H-H Wendy; Weisz, Adrian

2014-01-10

Specifications in the U.S. Code of Federal Regulations for the color additive D&C Green No. 8 (Colour Index No. 59040) limit the levels of the subsidiary colors 1,3,6-pyrenetrisulfonic acid trisodium salt (P3S) and 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (P4S). The present paper describes a comparative study of two possible methods to replace the currently used multi-step TLC/spectrophotometry method of separating and quantifying the minor components P3S and P4S in G8. One of the new approaches uses conventional high-performance liquid chromatography (HPLC) and the other, derivative spectrophotometry. While the derivative spectrophotometric method was shown to be inadequate for the analysis of minor components overwhelmed by components of much higher concentration, the HPLC method was proven highly effective. The closely related, very polar compounds P3S and P4S were separated by the new HPLC method in less than 4 min using a conventional HPLC instrument. P3S and P4S were quantified by using five-point calibration curves with data points that ranged from 0.45 to 7.63% and from 0.13 to 1.82%, by weight, for P3S and P4S, respectively. The HPLC method was applied to the analysis of test portions from 20 batches of D&C Green No. 8 submitted to the U.S. Food and Drug Administration for certification. Published by Elsevier B.V.

Quantitative amino acid profiling and stable isotopically labeled amino acid tracer enrichment used for in vivo human systemic and tissue kinetics measurements

DEFF Research Database (Denmark)

Bornø, Andreas; van Hall, Gerrit

2014-01-01

An important area within clinical functional metabolomics is in vivo amino acid metabolism and protein turnover measurements for which accurate amino acid concentrations and stable isotopically labeled amino acid enrichments are mandatory not the least when tissue metabolomics is determined....... The present study describes a new sensitive liquid chromatography tandem mass-spectrometry method quantifying 20 amino acids and their tracer(s) ([ring-(13)C6]/D5Phenylalanine) in human plasma and skeletal muscle specimens. Before analysis amino acids were extracted and purified via deprotonization....../ion exchange, derivatized using a phenylisothiocyanate reagent and each amino acid was quantitated with its own stable isotopically labeled internal standard (uniformly labeled-(13)C/(15)N). The method was validated according to general recommendations for chromatographic analytical methods. The calibration...

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

Science.gov (United States)

Kogadeeva, Maria

2016-01-01

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

FluxPyt: a Python-based free and open-source software for 13C-metabolic flux analyses.

Science.gov (United States)

Desai, Trunil S; Srivastava, Shireesh

2018-01-01

13 C-Metabolic flux analysis (MFA) is a powerful approach to estimate intracellular reaction rates which could be used in strain analysis and design. Processing and analysis of labeling data for calculation of fluxes and associated statistics is an essential part of MFA. However, various software currently available for data analysis employ proprietary platforms and thus limit accessibility. We developed FluxPyt, a Python-based truly open-source software package for conducting stationary 13 C-MFA data analysis. The software is based on the efficient elementary metabolite unit framework. The standard deviations in the calculated fluxes are estimated using the Monte-Carlo analysis. FluxPyt also automatically creates flux maps based on a template for visualization of the MFA results. The flux distributions calculated by FluxPyt for two separate models: a small tricarboxylic acid cycle model and a larger Corynebacterium glutamicum model, were found to be in good agreement with those calculated by a previously published software. FluxPyt was tested in Microsoft™ Windows 7 and 10, as well as in Linux Mint 18.2. The availability of a free and open 13 C-MFA software that works in various operating systems will enable more researchers to perform 13 C-MFA and to further modify and develop the package.

Estimation of glucose carbon recycling in children with glycogen storage disease: A 13C NMR study using [U-13C]glucose

International Nuclear Information System (INIS)

Kalderon, B.; Korman, S.H.; Gutman, A.; Lapidot, A.

1989-01-01

A stable isotope procedure to estimate hepatic glucose carbon recycling and thereby elucidate the mechanism by which glucose is produced in patients lacking glucose 6-phosphatase is described. A total of 10 studies was performed in children with glycogen storage disease type I (GSD-I) and type III (GSD-III) and control subjects. A primed dose-constant nasogastric infusion of D-[U- 13 C]glucose or an infusion diluted with nonlabeled glucose solution was administered following different periods of fasting. Hepatic glucose carbon recycling was estimated from 13 C NMR spectra. The values obtained for GSD-I patients coincided with the standard [U- 13 C]glucose dilution curve. These results indicate that the plasma glucose of GSD-I subjects comprises only a mixture of 99% 13 C-enriched D-[U- 13 C]glucose and unlabeled glucose but lacks any recycled glucose. Significantly different glucose carbon recycling values were obtained for two GSD-III patients in comparison to GSD-I patients. The results eliminate a mechanism for glucose production in GSD-I children involving gluconeogenesis. However, glucose release by amylo-1,6-glucosidase activity would result in endogenous glucose production of non- 13 C-labeled and nonrecycled glucose carbon, as was found in this study. In GSD-III patients gluconeogenesis is suggested as the major route for endogenous glucose synthesis. The contribution of the triose-phosphate pathway in these patients has been determined

Selective release of D and 13C from insoluble organic matter of the Murchison meteorite by impact shock

Science.gov (United States)

Mimura, Koichi; Okamoto, Michioki; Sugitani, Kenichiro; Hashimoto, Shigemasa

2007-03-01

We performed shock-recovery experiments on insoluble organic matter (IOM) purified from the Murchison meteorite, and determined the abundances and isotope ratios of hydrogen and carbon in the shocked IOM sample. We also performed shock experiments on type III kerogen and compared the results of these experiments with the experimental results regarding IOM.The shock selectively released D and 13C from the IOM, while it preferably released H and 12C from the kerogen. The release of these elements from IOM cannot be explained in terms of the isotope effect, whereas their release from kerogen can be explained by this effect. The selective release of heavier isotopes from IOM would be due to its structure, in which D and 13C-enriched parts are present as an inhomogeneity and are weakly attached to the main network. Shock gave rise to a high release of D even at a lower degree of dehydrogenation compared with the stepwise heating of IOM. This effective release of D is probably an inherent result of shock, in which a dynamic high-pressure and high-temperature condition prevails. Thus, shock would effectively control the hydrogen isotope behavior of extraterrestrial organic matter during the evolution of the solar nebula.

Accurate determination of 13C/12C in CO2 of past atmospheres from 13C/12C in tree rings by removal of climatic interferences. Final technical progress report

International Nuclear Information System (INIS)

Long, A.; Leavitt, S.W.

1982-07-01

Analysis of delta 13 C in tree rings offers the potential for reconstructing changes in delta 13 C of atmospheric CO 2 , which combined with 14 C trends will be important in evaluating the past and present role of the biosphere as a net carbon dioxide source or sink. One problem with such tree-ring delta 13 C records is that in addition to changes in atmospheric delta 13 C, climatic factors may influence the isotopic composition of the rings. This study examines delta 13 C in cellulose of five-year intervals of juniper tree rings. The sampling sites were near weather stations for which climate data are readily available. delta 13 C vs winter climate relationships were derived from those sites not under the influence of local pollution sources. These relationships facilitated development of a delta 13 C record from which climate effects have been factored out. The resulting 1930-1979 delta 13 C reconstruction shows a pronounced delta 13 C decrease from 1930 to 1960 and then a post-1960 flattening of the curve. We interpret this trend as representing an increasing role of biospheric CO 2 sinks relative to sources, although other interpretations may be possible within the limits of precision of data analysis. Research with this technique is continuing with pinyon pine and dendroclimatological reconstructions to verify these results and to develop a climate-free delta 13 C chronology back to pre-industrial time

Fatty acid synthesis in Xylella fastidiosa: correlations between genome studies, 13C NMR data, and molecular models

International Nuclear Information System (INIS)

Osiro, Denise; Muniz, Joao Renato C.; Coleta Filho, Helvecio Della; Alves de Sousa, Alessandra; Machado, Marcos Antonio; Garratt, Richard C.; Colnago, Luiz Alberto

2004-01-01

Xylella fastidiosa was the first plant pathogen to have its complete genome sequence elucidated. Routine database analyses suggested that two enzymes essential for fatty acid synthesis were missing, one of these is the holo-acyl-carrier-protein synthase. However, here we demonstrate, using 13 C NMR spectroscopy, that X. fastidiosa is indeed able to synthesize fatty acids from acetate via an apparently conventional metabolic pathway. We further identify a gene product HetI, an alternative phosphopantetheinyl transferase, which we propose to fill the missing link. Homology modeling of HetI shows conservation of the Coenzyme A binding site suggesting it to be an active enzyme and reveals several interesting structural features when compared with the surfactin synthase-activating enzyme, on which the model was built. These include a simplified topology due to N- and C-terminal deletions and the observation of a novel serine ladder

Synthesis and applications of {sup 13}C glycerol

Energy Technology Data Exchange (ETDEWEB)

Stocking, E.; Khalsa, O.; Martinez, R.A.; Silks, L.A. III [Los Alamos National Laboratory, NM (United States)

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.

Structural Characterization of Febuxostat/l-Pyroglutamic Acid Cocrystal Using Solid-State 13C-NMR and Investigational Study of Its Water Solubility

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Ji-Hun An

2017-12-01

Full Text Available Febuxostat (FB is a poorly water-soluble drug that belongs to BCS class II. The drug is employed for the treatment of inflammatory disease arthritis urica (gout, and the free base, FB form-A, is most preferred for drug formulation. In order to achieve a goal of improving the water solubility of FB form-A, this study was carried out using the cocrystallization technique called the liquid-assisted grinding method to produce FB cocrystals. Here, five amino acids containing amine (NH, oxygen (O, and hydroxyl (OH functional groups, and possessing difference of pKa less than 3 with FB, were selected as coformers. Then, solvents including methanol, ethanol, isopropyl alcohol, n-hexane, dichloromethane, and acetone were used for the cocrystal screening. As a result, a cocrystal was obtained when acetone and l-pyroglutamic acid (PG of 0.5 eq. were employed as solvent and coformer, respectively. The ratio of 2:1, which is the ratio of FB to PG within FB-PG cocrystal, was predicted by means of solid-state CP/MAS 13C-NMR, solution-state NMR (1H, 13C, and 2D and FT-IR. Moreover, Powder X-ray Diffraction (PXRD, Differential Scanning Calorimetry (DSC, and Thermogravimetric Analysis (TGA were used to investigate the characteristics of FB-PG cocrystal. In addition, comparative solubility tests between FB-PG cocrystal and FB form-A were conducted in deionized water and under simulated gastrointestinal pH (1.2, 4, and 6.8 conditions. The result revealed that FB-PG cocrystal has a solubility of four-fold higher than FB form-A in deionized water and two-fold and five-fold greater than FB form-A at simulated gastrointestinal pH 1.2 and pH 4, respectively. Besides, solubilities of FB-PG cocrystal and FB form-A at pH 6.8 were similar to the results measured in deionized water. Therefore, it is postulated that FB-PG cocrystal has a potential overcoming the limitations related to the low aqueous solubility of FB form-A. Accordingly, FB-PG cocrystal is suggested as an

Acinetobacter baumannii K13 and K73 capsular polysaccharides differ only in K-unit side branches of novel non-2-ulosonic acids: di-N-acetylated forms of either acinetaminic acid or 8-epiacinetaminic acid.

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Kenyon, Johanna J; Kasimova, Anastasiya A; Notaro, Anna; Arbatsky, Nikolay P; Speciale, Immacolata; Shashkov, Alexander S; De Castro, Cristina; Hall, Ruth M; Knirel, Yuriy A

2017-11-27

Structures of capsular polysaccharides of Acinetobacter baumannii isolates carrying KL13 and KL73 gene clusters were established. The closely related KL73 and KL13 gene clusters differ only by one gene in the module responsible for synthesis of the non-2-ulosonic acids. The K13 and K73 polysaccharides differ only in a single side-chain sugar, which is either 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-altro- or -d-glycero-l-altro-non-2-ulosonic acid [di-N-acetylated forms of acinetaminic acid (Aci5Ac7Ac) or 8-epiacinetaminic acid (8eAci5Ac7Ac), respectively]. The KL13 also is closely related to the KL12 gene cluster, which contains a different wzy gene encoding the K unit polymerase. Accordingly, the otherwise near identical K units are linked differently via an α-d-FucpNAc-(1 → 4)-d-Galp linkage in K13 and K73 or an α-d-FucpNAc-(1 → 3)-d-GalpNAc linkage in K12. This finding confirms the predicted substrate of the ItrB3 initiating transferase as d-FucpNAc. Glycosyltransferases predicted to catalyse the linkage of d-Galp or d-GalpNAc to l-FucpNAc in the growing K13 and K73 or K12 units, respectively, differ by only two amino acids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis of output trends from Varian 2100C/D and 600C/D accelerators

International Nuclear Information System (INIS)

Grattan, M W D; Hounsell, A R

2011-01-01

Analysis of Varian linear accelerator output trends is reported. Two groups, consisting of four matched Varian 2100C/D and four matched Varian 600C/D accelerators, with different designs of monitor chamber, have been investigated and the data acquired from regular calibrated ion chamber/electrometer measurements of the output performance of the eight accelerators analysed. The trend of machine output with time, having removed the effect of adjusting the monitor chamber response, was compared on a monthly and annual basis for monitor chambers with ages ranging between 1 year and 7 years. The results indicate that the response is generally consistent within each set of accelerators with different monitor chamber designs. Those used in a Varian 600C/D machine result in a reduction in measured output over time, with an average monthly reduction of 0.35 ± 0.09% over the course of the first 4 years of use. The chambers used in a 2100C/D accelerator result in an increase in measured output over time, with an average monthly increase of 0.26 ± 0.09% over the course of the first 4 years of use. The output increase then reduces towards the end of this period of time, with the average monthly change falling to -0.03 ± 0.02% for the following 3 years. The output response trend was similar for all clinical energies used on the 2100C/D accelerators--6, 15 MV x-ray beams, and 4, 6, 9, 12, 16 and 20 MeV electron beams. By tracking these changes it has been possible to predict the response over time to allow appropriate adjustments in monitor chamber response to maintain a measured accelerator output within tolerance and give confidence in performance. It has also provided data to indicate the need for planned preventative intervention and indicate if the monitor chamber response is behaving as expected. (note)

Analyzing power measurements for the /sup 13/C(p vector,d)/sup 12/C reaction at 200 and 400 MeV

Energy Technology Data Exchange (ETDEWEB)

Liljestrand, R P; Cameron, J M; Hutcheon, D A; MacDonald, R; McDonald, W J; Miller, C A; Olsen, W C [Alberta Univ., Edmonton (Canada); Kraushaar, J J; Shepard, J R [Colorado Univ., Boulder (USA). Nuclear Physics Lab.; Rogers, J G [British Columbia Univ., Vancouver (Canada). TRIUMF Facility

1981-02-26

Cross sections and analyzing powers for the /sup 13/C(p vector,d) reaction have been measured at 200 and 400 MeV to the O/sup +/, ground state and 2/sup +/, 4.44 MeV state of /sup 12/C. While the cross sections are rather structureless, DWBA calculations in exact finite range account well for both the magnitude and shape of the angular distributions. On the other hand, the measured analyzing powers are in serious disagreement with the DWBA calculations.

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

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Winter, Klaus; Holtum, Joseph A.M.

2002-01-01

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

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

Science.gov (United States)

Rascher, Katherine; Máguas, Cristina; Werner, Christiane

2010-05-01

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

Racemization of aspartic acid and phenylalanine in the sweetener aspartame at 100 degrees C.

Science.gov (United States)

Boehm, M F; Bada, J L

1984-01-01

The racemization half-lives (i.e., the time required to reach a D/L = 0.33) at pH 6.8 for aspartic acid and phenylalanine in the sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester) were determined to be 13 and 23 hours, respectively, at 100 degrees C. Racemization at this pH does not occur in aspartame but rather in its diketopiperazine decomposition product. Our results indicate that the use of aspartame to sweeten neutral pH foods and beverages that are then heated at elevated temperature could generate D-aspartic acid and D-phenylalanine. The nutritive consequences of these D-amino acids in the human diet are not well established, and thus aspartame should probably not be used as a sweetener when the exposure of neutral pH foods and beverages to elevated temperatures is required. At pH 4, a typical pH of most foods and beverages that might be sweetened with aspartame, the half-lives are 47 hours for aspartic acid and 1200 hours for phenylalanine at 100 degrees C. Racemization at pH 4 takes place in aspartame itself. Although the racemization rates at pH 4 are slow and no appreciable racemization of aspartic acid and phenylalanine should occur during the normal use of aspartame, some food and beverage components could conceivably act as catalysts. Additional studies are required to evaluate whether the use of aspartame as a sugar substitute might not in turn result in an increased human consumption of D-aspartic acid and D-phenylalanine. PMID:6591191

Analysis of 13C-mixed triacylglycerol in stool by bulk (EA-IRMS) and compound specific (GC/MS) methods.

Science.gov (United States)

Slater, C; Ling, S C; Preston, T; Weaver, L T

2002-06-01

This paper was presented in poster form at the 17th International Congress of Nutrition, August 27-31, Vienna, Austria (Annals of Nutrition & Metabolism 2001; 45(Suppl.1):349). Some of the data were also presented in poster form at the British Society of Gastroenterology Meeting, March 18-21, Glasgow, UK (Gut 2001; 48(Suppl.1):A91). The 13C-mixed triacylglycerol (MTG) breath test is used to measure intraluminal fat digestion. In normal digestion, 20-40% of the ingested 13C label is recovered in breath CO2. We aimed to identify the proportions of ingested label excreted in stool, as well as breath following ingestion of 13C-MTG by children with impaired exocrine pancreatic function and healthy controls. 13C enrichment of breath samples was measured by continuous flow isotope ratio mass spectrometry (IRMS) and cumulative percent dose recovered (cPDR) in 10 h was calculated. Total 13C of a faecal fat extract from each stool was measured by elemental analyser-IRMS, and 13C enrichment and concentration of the TBDMS derivative of octanoic acid was measured by GC/MS after hydrolysis of the fat extract. Stool 5-day cPDR was calculated. Mean breath cPDR was 35%. Mean cPDR in stool by combustion-IRMS and GC/ MS, respectively, was 0.8% and 1.0%. Therefore, the remaining 64% of the 13C label must remain in the body and variability in breath cPDR is due to postabsorptive rather than predigestive factors.

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

Science.gov (United States)

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

2014-01-01

The growing demand for (13) C-metabolic flux analysis ((13) C-MFA) in the field of metabolic engineering and systems biology is driving the need to rationalize expensive and time-consuming (13) C-labeling experiments. Experimental design is a key step in improving both the number of fluxes that can be calculated from a set of isotopic data and the precision of flux values. We present IsoDesign, a software that enables these parameters to be maximized by optimizing the isotopic composition of the label input. It can be applied to (13) C-MFA investigations using a broad panel of analytical tools (MS, MS/MS, (1) H NMR, (13) C NMR, etc.) individually or in combination. It includes a visualization module to intuitively select the optimal label input depending on the biological question to be addressed. Applications of IsoDesign are described, with an example of the entire (13) C-MFA workflow from the experimental design to the flux map including important practical considerations. IsoDesign makes the experimental design of (13) C-MFA experiments more accessible to a wider biological community. IsoDesign is distributed under an open source license at http://metasys.insa-toulouse.fr/software/isodes/ © 2013 Wiley Periodicals, Inc.

Structure of 14C via elastic and inelastic neutron scattering from 13C: Measurement, R-matrix analysis, and shell model calculations

International Nuclear Information System (INIS)

Resler, D.A.

1987-03-01

The specific purpose of this work is to provide a better understanding of the 14 C level structure; the general purpose is to provide the details for using shell model calculations in R-matrix analyses. Using the TOF facilities of the Ohio University Accelerator Laboratory, the elastic and first 3 inelastic differential scattering cross sections for 13 C + n were measured at 69 energies for 4.5 ≤ E/sub n/ ≤ 11 MeV. A multiple scattering code was developed which provided a simulation of the experimental scattering process allowing accurate corrections to the small inelastic data. The integrated 13 C(n,α) 10 Be cross section is estimated. The sequential 2n-decay of 14 C states populated by 13 C + n was observed. A shell model code was developed. Normal and nonnormal parity calculations were made for the lithium isotopes using a new two-body interaction. The results for 5 Li predict the 2s/sub 1/2/ and 1d/sub 5/2/ single-particle states to be located below the 3/2 + state. Similar calculations were made for 13 C, 13 N, and 14 C. Results for 13 C and 13 N show for E/sub x/ 7 Li and 14 C, 2 h-barω calculations were done. Shell model calculations generated the R-matrix parameters for the elastic and first 3 inelastic channels of 13 C + n. After adjusting some energies, the predicted structure generally agrees with experiment for E/sub n/ 13 C + n data were refit to replace R 0 background terms by more realistic broad states and to get better agreement with model calculations. R-matrix fitting of the full data set produced new 14 C level information. For E/sub n/ > 4 MeV (E/sub x/ > 12 MeV), 5 states are given definite J/sup π/ assignments; 3, tentative assignments. 122 refs., 91 figs., 30 tabs

Distribuciones simuladas de valores de d13c de colágeno humano: implicancias para los estudios paleodietarios

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Barrientos, Gustavo

2005-01-01

Full Text Available El objetivo de este trabajo es presentar los primeros resultados derivados de la aplicación de un programa de simulación de distribuciones de valores de δ13C de colágeno humano (DeltaCarb130.0, desarrollado por los autores. DeltaCarb130.0 es un programa simple que simula la composición isotópica (δ13C del colágeno de huesos humanos obtenida a través de una dieta de composición variable, constituida por tres ingestas diarias, durante un período de 1825 días (5 años. La composición de la dieta y los valores de δ13C de cada recurso dietario (vegetales C3 y C4, carne de herbívoros consumidores de C3 y C4, de mamíferos marinos, de peces y moluscos se obtienen, en cada etapa de la simulación, a través de un proceso aleatorio a partir de datos de abundancia relativa y rangos de valores de δ13C de cada tipo de recurso. Se analizarán estadísticamente las distribuciones de valores de δ13C obtenidas a partir de n corridas del programa para cada combinación dietaria, con la finalidad de obtener medidas de tendencia central y de dispersión asociadas a cada tipo de dieta y calcular la probabilidad de obtener valores "outliers", un rasgo frecuente en el estudio de casos arqueológicos.

SRS-sensor 13C/12C isotops measurements for detecting Helicobacter Pylori

Science.gov (United States)

Grishkanich, Aleksandr; Chubchenko, Yan; Elizarov, Valentin; Zhevlakov, Aleksandr; Konopelko, Leonid

2018-02-01

We developed SRS-sensor 13C/12C isotops measurements detecting Helicobacter Pylori for medical diagnostics of human health. Measuring of absolute 13C/12C isotope amount ratios allows to explore the topical problems of the modern world, alcoholic beverages and tobacco, medical diagnostics of human health. SRS method is used to measure the ratio of carbon isotopes in the exhaled carbon dioxide, which is used to diagnose the human infection of Helicobacter pylori and the influence of the Helicobacter pylori bacterium on the occurrence of gastritis, gastric and duodenal ulcers. A method for the analysis of human infection with Helicobacter pylori was developed on the basis of measurements of the ratio of 13C / 12C carbon isotopes in human exhaled air with a high level of measurement accuracy. The article reviews the work in the field of provision comparability of absolute 13C/12C isotope amount ratios in the environment and food. The analysis of the technical and metrological characteristics of traditional and perspective instruments for measuring isotope ratios is presented. The provision of comparability of absolute 13C/12C isotope amount ratios is carried by gravimetrically prepared reference standards. The key features and emerging issues are discussed.

Abundance and δ13C values of fatty acids in lacustrine surface sediments: Relationships with in-lake methane concentrations

Science.gov (United States)

Stötter, Tabea; Bastviken, David; Bodelier, Paul L. E.; van Hardenbroek, Maarten; Rinta, Päivi; Schilder, Jos; Schubert, Carsten J.; Heiri, Oliver

2018-07-01

Proxy-indicators in lake sediments provide the only approach by which the dynamics of in-lake methane cycling can be examined on multi-decadal to centennial time scales. This information is necessary to constrain how lacustrine methane production, oxidation and emissions are expected to respond to global change drivers. Several of the available proxies for reconstructing methane cycle changes of lakes rely on interpreting past changes in the abundance or relevance of methane oxidizing bacteria (MOB), either directly (e.g. via analysis of bacterial lipids) or indirectly (e.g. via reconstructions of the past relevance of MOB in invertebrate diet). However, only limited information is available about the extent to which, at the ecosystem scale, variations in abundance and availability of MOB reflect past changes in in-lake methane concentrations. We present a study examining the abundances of fatty acids (FAs), particularly of 13C-depleted FAs known to be produced by MOB, relative to methane concentrations in 29 small European lakes. 39 surface sediment samples were obtained from these lakes and FA abundances were compared with methane concentrations measured at the lake surface, 10 cm above the sediments and 10 cm within the sediments. Three of the FAs in the surface sediment samples, C16:1ω7c, C16:1ω5c/t, and C18:1ω7c were characterized by lower δ13C values than the remaining FAs. We show that abundances of these FAs, relative to other short-chain FAs produced in lake ecosystems, are related with sedimentary MOB concentrations assessed by quantitative polymerase chain reaction (qPCR). We observed positive relationships between methane concentrations and relative abundances of C16:1ω7c, C16:1ω5c/t, and C18:1ω7c and the sum of these FAs. For the full dataset these relationships were relatively weak (Spearman's rank correlation (rs) of 0.34-0.43) and not significant if corrected for multiple testing. However, noticeably stronger and statistically significant

Cloning, Phylogenetic Analysis and 3D Modeling of a Putative Lysosomal Acid Lipase from the Camel, Camelus dromedarius

Directory of Open Access Journals (Sweden)

Farid Shokry Ataya

2012-08-01

Full Text Available Acid lipase belongs to a family of enzymes that is mainly present in lysosomes of different organs and the stomach. It is characterized by its capacity to withstand acidic conditions while maintaining high lipolytic activity. We cloned for the first time the full coding sequence of camel’s lysosomal acid lipase, cLIPA using RT-PCR technique (Genbank accession numbers JF803951 and AEG75815, for the nucleotide and aminoacid sequences respectively. The cDNA sequencing revealed an open reading frame of 1,197 nucleotides that encodes a protein of 399 aminoacids which was similar to that from other related mammalian species. Bioinformatic analysis was used to determine the aminoacid sequence, 3D structure and phylogeny of cLIPA. Bioinformatics analysis suggested the molecular weight of the translated protein to be 45.57 kDa, which could be decreased to 43.16 kDa after the removal of a signal peptide comprising the first 21 aminoacids. The deduced cLIPA sequences exhibited high identity with Equus caballus (86%, Numascus leucogenys (85%, Homo sapiens (84%, Sus scrofa (84%, Bos taurus (82% and Ovis aries (81%. cLIPA shows high aminoacid sequence identity with human and dog-gastric lipases (58%, and 59% respectively which makes it relevant to build a 3D structure model for cLIPA. The comparison confirms the presence of the catalytic triad and the oxyanion hole in cLIPA. Phylogenetic analysis revealed that camel cLIPA is grouped with monkey, human, pig, cow and goat. The level of expression of cLIPA in five camel tissues was examined using Real Time-PCR. The highest level of cLIPA transcript was found in the camel testis (162%, followed by spleen (129%, liver (100%, kidney (20.5% and lung (17.4%.

Carbon-13 and oxygen-18 isotope effects in the decarboxylation of nicotinic acid of natural isotopic composition

International Nuclear Information System (INIS)

Zielinski, M.; Zielinska, A.; Papiernik-Zielinska, H.; McKenzie, J.A.; Bernasconi, S.; Paul, H.

1998-01-01

Carbon-13 and oxygen-18 isotope effects in the decarboxylation of nicotinic acid of natural isotopic composition above and below its melting temperature have been studied and compared with the primary (PKIE) and secondary kinetic isotope effects (SKIE) of 13 C and 18 O, respectively, in the decarboxylation of other heterocyclic acids. The temperature dependence of the secondary oxygen-18 isotope effects is negative in the total 221-255 deg C temperature interval investigated initially. The 13 C KIE measured above melting point of N.A. (temperature interval 235-270 deg C) are located in the range 1.007-1.009. Below melting point of nicotinic acid the 13 C KIE are larger and reveal the negative temperature dependence ( 13 C KIE decreases with decreasing the reaction temperature from 1.013/at 230 deg C to 1.0114/at 221 deg C). A discussion of the above isotopic results is presented. (author)

[The importance of C-terminal aspartic acid residue (D141) to the antirestriction activity of the ArdB (R64) protein].

Science.gov (United States)

Kudryavtseva, A A; Osetrova, M S; Livinyuk, V Ya; Manukhov, I V; Zavilgelsky, G B

2017-01-01

Antirestriction proteins of the ArdB/KlcA family are specific inhibitors of restriction (endonuclease) activity of type-I restriction/modification enzymes. The effect of conserved amino acid residues on the antirestriction activity of the ArdB protein encoded by the transmissible R64 (IncI1) plasmid has been investigated. An analysis of the amino acid sequences of ArdB homologues demonstrated the presence of four groups of conserved residues ((1) R16, E32, and W51; (2) Y46 and G48; (3) S81, D83 and E132, and (4) N77, L(I)140, and D141) on the surface of the protein globule. Amino acid residues of the fourth group showed a unique localization pattern with the terminal residue protruding beyond the globule surface. The replacement of two conserved amino acids (D141 and N77) located in the close vicinity of each other on the globule surface showed that the C-terminal D141 is essential for the antirestriction activity of ArdB. The deletion of this residue, as well as replacement by a hydrophobic threonine residue (D141T), completely abolished the antirestriction activity of ArdB. The synonymous replacement of D141 by a glutamic acid residue (D141E) caused an approximately 30-fold decrease of the antirestriction activity of ArdB, and the point mutation N77A caused an approximately 20-fold decrease in activity. The residues D141 and N77 located on the surface of the protein globule are presumably essential for the formation of a contact between ArdB and a currently unknown factor that modulates the activity of type-I restriction/modification enzymes.

Determination of fructose metabolic pathways in normal and fructose-intolerant children: A 13C NMR study using [U-13C]fructose

International Nuclear Information System (INIS)

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

1990-01-01

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

IMPROVED LINE DATA FOR THE SWAN SYSTEM 12C13C ISOTOPOLOGUE

International Nuclear Information System (INIS)

Ram, Ram S.; Brooke, James S. A.; Bernath, Peter F.; Sneden, Christopher; Lucatello, Sara

2014-01-01

We present new, accurate predictions for rotational line positions, excitation energies, and transition probabilities of the 12 C 13 C isotopologue Swan d 3 Π-a 3 Π system 0-0, 0–1, 0–2, 1–0, 1–1, 1–2, 2–0, 2–1, and 2–2 vibrational bands. The line positions and energy levels were predicted through new analyses of published laboratory data for the 12 C 13 C lines. Transition probabilities were derived from recent computations of transition dipole moments and related quantities. The 12 C 13 C line data were combined with similar data for 12 C 2, reported in a companion paper, and applied to produce synthetic spectra of carbon-rich metal-poor stars that have strong C 2 Swan bands. The matches between synthesized and observed spectra were used to estimate band head positions for a few of the 12 C 13 C vibrational bands and to verify that the new computed line data match observed spectra. The much weaker C 2 lines of the bright red giant Arcturus were also synthesized in the band head regions

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture

Science.gov (United States)

Bernhart, Eva; Kollroser, Manfred; Rechberger, Gerald; Reicher, Helga; Heinemann, Akos; Schratl, Petra; Hallström, Seth; Wintersperger, Andrea; Nusshold, Christoph; DeVaney, Trevor; Zorn-Pauly, Klaus; Malli, Roland; Graier, Wolfgang; Malle, Ernst; Sattler, Wolfgang

2014-01-01

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA1, LPA2, and LPA3 on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS. PMID:19899077

The role of leucine in isoprenoid metabolism. Incorporation of [3-13C]leucine and of [2-3H,4-14C]-β,β-dimethyl-acrylic acid into phytosterols by tissue cultures of Andrographis paniculata

International Nuclear Information System (INIS)

Anastasis, P.; Freer, I.; Overton, K.; Rycroft, D.; Singh, S.B.

1985-01-01

[3- 13 C]Leucine is incorporated into phytosterols by tissue cultures of Andrographis paniculata by breakdown to acetyl-CoA and its subsequent incorporation via (3S)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) and mevalonic acid; [2- 3 H,4- 14 C]-β,β-dimethylacrylic acid also is not incorporated intact. (author)

Low Mass MS/MS Fragments of Protonated Amino Acids Used for Distinction of Their 13C- Isotopomers in Metabolic Studies

Science.gov (United States)

Ma, Xin; Dagan, Shai; Somogyi, Árpád; Wysocki, Vicki H.; Scaraffia, Patricia Y.

2013-04-01

Glu, Gln, Pro, and Ala are the main amino acids involved in ammonia detoxification in mosquitoes. In order to develop a tandem mass spectrometry method (MS2) to monitor each carbon of the above isotopically-labeled 13C-amino acids for metabolic studies, the compositions and origins of atoms in fragments of the protonated amino acid should be first elucidated. Thus, various electrospray (ESI)-based MS2 tools were employed to study the fragmentation of these unlabeled and isotopically-labeled amino acids and better understand their dissociation pathways. A broad range of fragments, including previously-undescribed low m/z fragments was revealed. The formulae of the fragments (from m/z 130 down to m/z 27) were confirmed by their accurate masses. The structures and conformations of the larger fragments of Glu were also explored by ion mobility mass spectrometry (IM-MS) and gas-phase hydrogen/deuterium exchange (HDX) experiments. It was found that some low m/z fragments ( m/z 27-30) are common to Glu, Gln, Pro, and Ala. The origins of carbons in these small fragments are discussed and additional collision induced dissociation (CID) MS2 fragmentation pathways are proposed for them. It was also found that small fragments (≤ m/z 84) of protonated, methylated Glu, and methylated Gln are the same as those of the underivatized Glu and Gln. Taken together, the new approach of utilizing low m/z fragments can be applied to distinguish, identify, and quantify 13C-amino acids labeled at various positions, either in the backbone or side chain.

{sup 2}H NMR and {sup 13}C-IRMS analyses of acetic acid from vinegar, {sup 18}O-IRMS analysis of water in vinegar: International collaborative study report

Energy Technology Data Exchange (ETDEWEB)

Thomas, Freddy [Eurofins Scientific Analytics, BP42301, 44323 Nantes (France); Jamin, Eric [Eurofins Scientific Analytics, BP42301, 44323 Nantes (France)

2009-09-01

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

Evaluation of auto-assessment method for C-D analysis based on support vector machine

International Nuclear Information System (INIS)

Takei, Takaaki; Ikeda, Mitsuru; Imai, Kuniharu; Kamihira, Hiroaki; Kishimoto, Tomonari; Goto, Hiroya

2010-01-01

Contrast-Detail (C-D) analysis is one of the visual quality assessment methods in medical imaging, and many auto-assessment methods for C-D analysis have been developed in recent years. However, for the auto-assessment method for C-D analysis, the effects of nonlinear image processing are not clear. So, we have made an auto-assessment method for C-D analysis using a support vector machine (SVM), and have evaluated its performance for the images processed with a noise reduction method. The feature indexes used in the SVM were the normalized cross correlation (NCC) coefficient on each signal between the noise-free and noised image, the contrast to noise ratio (CNR) on each signal, the radius of each signal, and the Student's t-test statistic for the mean difference between the signal and background pixel values. The results showed that the auto-assessment method for C-D analysis by using Student's t-test statistic agreed well with the visual assessment for the non-processed images, but disagreed for the images processed with the noise reduction method. Our results also showed that the auto-assessment method for C-D analysis by the SVM made of NCC and CNR agreed well with the visual assessment for the non-processed and noise-reduced images. Therefore, the auto-assessment method for C-D analysis by the SVM will be expected to have the robustness for the non-linear image processing. (author)

13C and 31P NMR study of gluconeogenesis: utilization of 13C-labeled substrates by perfused liver from streptozotocin-diabetic and untreated rats

International Nuclear Information System (INIS)

Cohen, S.M.

1987-01-01

The metabolism of 13 C-labeled substrates was followed by 13 C and 31 P NMR in perfused liver from the streptozotocin-treated rat model of insulin-dependent diabetes. Comparison was made with perfused liver from untreated littermates, fasted either 24 or 12 h. The major routes of pyruvate metabolism were followed by a 13 C NMR approach that provided for the determination of the metabolic fate of several substances simultaneously. The rate of gluconeogenesis was 2-4-fold greater and β-hydroxybutyrate production was 50% greater in liver from the chronically diabetic rats as compared with the control groups. Large differences in the distribution of 13 C label in hepatic alanine were measured between diabetic and control groups. The biosyntheses of 13 C-labeled glutathione and N-carbamoylaspartate were monitored in time-resolved 13 C NMR spectra of perfused liver. Assignments for the resonances of glutathione and N-carbamoylaspartate were made with the aid of 13 C NMR studies of perchloric acid extracts of the freeze-clamped livers. 13 C NMR spectroscopy of the perfusates provided a convenient, rapid assay of the rate of oxidation of [2- 13 C]ethanol, the hepatic output of [2- 13 ]acetaldehyde, and the accumulation of [2- 13 C]acetate in the perfusate. By 31 P NMR spectroscopy, carbamoyl phosphate was measured in all diabetic livers and an unusual P,P'-diesterified pyrophosphate was observed in one-fourth of the diabetic livers examined. Neither of these phosphorylated metabolites was detected in control liver. Both 13 C and 31 P NMR were useful in defining changes in hepatic metabolism in experimental diabetes

Adulteration of honey : relation between microscopic analysis and delta C-13 measurements

NARCIS (Netherlands)

Kerkvliet, JD; Meijer, HAJ

2000-01-01

Upon routine microscopic analysis of some honey samples, parenchyma cells, single rings of ring vessels and epidermal cells are found. These cells originate from the sugar cane stem. We investigated whether there was a relation between these plant fragments and the delta C-13 value of honey. 17

Laboratory spectra of C-13 ethane

Science.gov (United States)

Kurtz, Joe; Reuter, Dennis C.; Jennings, Donald E.; Hillman, John J.

1991-01-01

The laboratory infrared spectrum of C-13 monosubstituted ethane has been obtained at high resolution (0.0025/cm) using the McMath Fourier transform spectrometer at Kitt Peak National Observatory in May 1990. A preliminary analysis of the nu12 rQ0 branch (substituted species) suggests that its intensity is 1.15 + or - 0.05 times stronger than the equivalent nu9 branch in the normal (C-12)2H6 species. This result leads to a correction of a previously published estimate for the C-12/C-13 ratio in the atmosphere of Jupiter from about 94 to about 106.

Synthesis of 13C-labeled vitamin E and interaction between vitamin E and phospholipid in liposome

International Nuclear Information System (INIS)

urano, S.; Matsuo, M.

1986-01-01

Vitamin E with a 13 C-labeled isoprenoid side chain, [4'a- 13 C], [6'- 13 C], [8'a- 13 C] and [12'a and 13'- 13 C]α-tocopherols were synthesized. These compounds were incorporated into three kinds of lecithin liposomes from dipalmitoyl phosphatidyl cholin, egg lecithin and rat liver lecithin, of which arachidonic acid contents are 0, 2.6 and 19.0%, respectively. T 1 values, which were measured by NMR for the labeled carbons, indicate that the segmental motion tends to increase with the increase of the distance from the chroman ring. This tendency is not affected with the arachidonic acid contents of phospholipids. This result can not be explained by Lucy's hypothesis. 1 figure; 1 table

Method for differentiating between vinegar produced by fermentation and vinegar made from synthetic acetic acid based on determination of the /sup 13/C//sup 12/C-isotope ratio by mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

Schmid, E R; Fogy, I [Vienna Univ. (Austria). Inst. fuer Analytische Chemie; Schwartz, P [International Atomic Energy Agency, Vienna (Austria)

1978-02-01

The /sup 13/C//sup 12/C-isotope ratio is characteristic for vinegar of fermentation and synthetic origin respectively and used for their differentiation. The acetic acid was isolated from the vinegar as calcium acetate, the calcium acetate was pyrolysed to CaCO/sub 3/ and the CO/sub 2/ was released from the CaCO/sub 3/ with H/sub 3/PO/sub 4/. The CO/sub 2/ was measured in a mass spectrometer with double collector. The difference in the /sup 13/C- content between the two varieties of vinegar is 5/sup 0///sub 00/; the accuracy of the measurement is between 0,5/sup 0///sub 00/ and 1/sup 0///sub 00/. Therefore, addition of synthetic acetic acid in excess of 15 to 20% to fermentation vinegar can be detected by this method.

13C NMR study of effects of fasting and diabetes on the metabolism of pyruvate in the tricarboxylic acid cycle and of the utilization of pyruvate and ethanol in lipogenesis in perfused rat liver

International Nuclear Information System (INIS)

Cohen, S.M.

1987-01-01

13 C NMR has been used to study the competition of pyruvate dehydrogenase with pyruvate carboxylase for entry of pyruvate into the tricarboxylic acid (TCA) cycle in perfused liver from streptozotocin-diabetic and normal donor rats. The relative proportion of pyruvate entering the TCA cycle by these two routes was estimated from the 13 C enrichments at the individual carbons of glutamate when [3- 13 C]alanine was the only exogenous substrate present. In this way, the proportion of pyruvate entering by the pyruvate dehydrogenase route relative to the pyruvate carboxylase route was determined to be 1:1.2 +/- 0.1 in liver from fed controls, 1:7.7 +/- 2 in liver from 24-fasted controls, and 1:2.6 +/- 0.3 in diabetic liver. Pursuant to this observation that conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) was greatest in perfused liver from fed controls, the incorporation of 13 C label into fatty acids was monitored in this liver preparation. With the exception of the repeating methylene carbons, fatty acyl carbons labeled by [1- 13 C]acetyl-CoA (from [2- 13 C]pyruvate) gave rise to resonances distinguishable on the basis of chemical shift from those observed when label was introduced by [3- 13 C]alanine plus [2- 13 C]ethanol, which are converted to [2- 13 C]acetyl-CoA. Thus, measurement of 13 C enrichment at several specific sites in the fatty acyl chains in time-resolved spectra of perfused liver offers a novel way of monitoring the kinetics of the biosynthesis of fatty acids. In addition to obtaining the rate of lipogenesis, it was possible to distinguish the contributions of chain elongation from those of the de novo synthesis pathway and to estimate the average chain length of the 13 C-labeled fatty acids produced

Characterization of Chlamydia MurC-Ddl, a fusion protein exhibiting D-alanyl-D-alanine ligase activity involved in peptidoglycan synthesis and D-cycloserine sensitivity.

Science.gov (United States)

McCoy, Andrea J; Maurelli, Anthony T

2005-07-01

Recent characterization of chlamydial genes encoding functional peptidoglycan (PG)-synthesis proteins suggests that the Chlamydiaceae possess the ability to synthesize PG yet biochemical evidence for the synthesis of PG has yet to be demonstrated. The presence of D-amino acids in PG is a hallmark of bacteria. Chlamydiaceae do not appear to encode amino acid racemases however, a D-alanyl-D-alanine (D-Ala-D-Ala) ligase homologue (Ddl) is encoded in the genome. Thus, we undertook a genetics-based approach to demonstrate and characterize the D-Ala-D-Ala ligase activity of chlamydial Ddl, a protein encoded as a fusion with MurC. The full-length murC-ddl fusion gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible ara promoter and transformed into a D-Ala-D-Ala ligase auxotroph of Escherichia coli possessing deletions of both the ddlA and ddlB genes. Viability of the E. coliDeltaddlADeltaddlB mutant in the absence of exogenous D-Ala-D-Ala dipeptide became dependent on the expression of the chlamydial murC-ddl thus demonstrating functional ligase activity. Domain mapping of the full-length fusion protein and site-directed mutagenesis of the MurC domain revealed that the structure of the full fusion protein but not MurC enzymatic activity was required for ligase activity in vivo. Recombinant MurC-Ddl exhibited substrate specificity for D-Ala. Chlamydia growth is inhibited by D-cycloserine (DCS) and in vitro analysis provided evidence for the chlamydial MurC-Ddl as the target for DCS sensitivity. In vivo sensitivity to DCS could be reversed by addition of exogenous D-Ala and D-Ala-D-Ala. Together, these findings further support our hypothesis that PG is synthesized by members of the Chlamydiaceae family and suggest that D-amino acids, specifically D-Ala, are present in chlamydial PG.

The transformation and fate of sub-Arctic microphytobenthos carbon revealed through 13C-labeling

DEFF Research Database (Denmark)

Oakes, Joanne M.; Rysgaard, Søren; Glud, Ronnie N.

2016-01-01

Microphytobenthos (MPB) at higher latitudes has been poorly studied. This study used pulse-chase C-13-labeling to investigate the production, processing, and fate of MPB-derived carbon (MPB-C) in sub-Arctic intertidal sediments over 31 d. Gross primary production (2.1 mmolCm(-2)h(-1)+/- 0.4 mmol...... accounted for49.8% of this C-13. The C-13 content of sediment organic carbon declined over time, but>31% of the C-13 fixed within the first tidal cycle remained after 31 d, suggesting that sub-Arctic MPB may contribute to coastal carbon retention during the productive season. Over 21 d, 10.6% of the fixed C......-13 was removed via DIC fluxes and 0.3% via DOC fluxes from inundated sediment, and 0.6% as CO2 from exposed sediment. The greatest loss of C-13 (38.2%) was via unmeasured pathways, including resuspension and/or removal by mobile consumers. The rates of MPB-C production and the relative importance...

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

Science.gov (United States)

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

2014-05-01

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