Biokinetic and dosimetric investigations of 14C-labeled substances in man using AMS

NASA Astrophysics Data System (ADS)

Mattsson, Sören; Gunnarsson, Mikael; Svegborn, Sigrid Leide; Nosslin, Bertil; Nilsson, Lars-Erik; Thorsson, Ola; Valind, Sven; Åberg, Magnus; Östberg, Henrik; Hellborg, Ragnar; Stenström, Kristina; Erlandsson, Bengt; Faarinen, Mikko; Kiisk, Madis; Magnusson, Carl-Erik; Persson, Per; Skog, Göran

2001-07-01

Up to now, radiation dose estimates from radiopharmaceuticals, labeled with pure β-emitting radionuclides, e.g., 14C or 3H have been very uncertain. Using accelerator mass spectrometry (AMS) we have derived new and improved data for 14C-triolein and 14C-urea and are currently running a program related to the biokinetics and dosimetry of 14C-glycocholic acid and 14C-xylose. The results of our investigations have made it possible to widen the indications for the clinical use of the 14C-urea test for Helicobacter pylori infection in children. The use of ultra-low activities, which is possible with AMS (down to 1/1000 of that used for liquid scintillation counting), has opened the possibility for metabolic investigations on children as well as on other sensitive patient groups like new-borns, and pregnant or breast-feeding women. Using the full potential of AMS, new 14C-labeled drugs could be tested on humans at a much earlier stage than today, avoiding uncertain extrapolations from animal models.

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

PubMed

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

2017-01-01

Metamitron is one of the most commonly used herbicide in sugar beet and flower bulb cultures. Numerous laboratory and field studies on sorption and degradation of metamitron were performed. Detailed biodegradation studies in soil using 13 C-isotope labeling are still missing. Therefore, we aimed at providing a detailed turnover mass balance of 13 C 6 -metamitron in soil microcosms over 80 days. In the biotic system, metamitron mineralized rapidly, and 13 CO 2 finally constituted 60% of the initial 13 C 6 -metamitron equivalents. In abiotic control experiments CO 2 rose to only 7.4% of the initial 13 C 6 -metamitron equivalents. The 13 C label from 13 C 6 -metamitron was incorporated into microbial amino acids that were ultimately stabilized in the soil organic matter forming presumably harmless biogenic residues. Finally, 13 C label from 13 C 6 -metamitron was distributed between the 13 CO 2 and the 13 C-biogenic residues indicating nearly complete biodegradation. The parallel increase of 13 C-alanine, 13 C-glutamate and 13 CO 2 indicates that metamitron was initially biodegraded via the desamino-metamitron route suggesting its relevance in the growth metabolism. In later phases of biodegradation, the "Rhodococcus route" was indicated by the low 13 CO 2 evolution and the high relevance of the pyruvate pathway, which aims at biomolecule synthesis and seems to be related to starvation. This is a first report on the detailed degradation route of metamitron in soil. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

Parish, Christopher; Niedbalski, Peter; Lumata, Lloyd

2015-03-01

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

Comprehensive and accurate tracking of carbon origin of LC-tandem mass spectrometry collisional fragments for 13C-MFA.

PubMed

Kappelmann, Jannick; Klein, Bianca; Geilenkirchen, Petra; Noack, Stephan

2017-03-01

In recent years the benefit of measuring positionally resolved 13 C-labeling enrichment from tandem mass spectrometry (MS/MS) collisional fragments for improved precision of 13 C-Metabolic Flux Analysis ( 13 C-MFA) has become evident. However, the usage of positional labeling information for 13 C-MFA faces two challenges: (1) The mass spectrometric acquisition of a large number of potentially interfering mass transitions may hamper accuracy and sensitivity. (2) The positional identity of carbon atoms of product ions needs to be known. The present contribution addresses the latter challenge by deducing the maximal positional labeling information contained in LC-ESI-MS/MS spectra of product anions of central metabolism as well as product cations of amino acids. For this purpose, we draw on accurate mass spectrometry, selectively labeled standards, and published fragmentation pathways to structurally annotate all dominant mass peaks of a large collection of metabolites, some of which with a complete fragmentation pathway. Compiling all available information, we arrive at the most detailed map of carbon atom fate of LC-ESI-MS/MS collisional fragments yet, comprising 170 intense and structurally annotated product ions with unique carbon origin from 76 precursor ions of 72 metabolites. Our 13 C-data proof that heuristic fragmentation rules often fail to yield correct fragment structures and we expose common pitfalls in the structural annotation of product ions. We show that the positionally resolved 13 C-label information contained in the product ions that we structurally annotated allows to infer the entire isotopomer distribution of several central metabolism intermediates, which is experimentally demonstrated for malate using quadrupole-time-of-flight MS technology. Finally, the inclusion of the label information from a subset of these fragments improves flux precision in a Corynebacterium glutamicum model of the central carbon metabolism.

Synthesis of empagliflozin, a novel and selective sodium-glucose co-transporter-2 inhibitor, labeled with carbon-14 and carbon-13.

PubMed

Hrapchak, Matt; Latli, Bachir; Wang, Xiao-Jun; Lee, Heewon; Campbell, Scot; Song, Jinhua J; Senanayake, Chris H

2014-10-01

Empagliflozin, (2S,3R,4R,5S,6R)-2-[4-chloro-3-[[4-[(3S)-oxolan-3-yl]oxyphenyl]methyl]phenyl]-6-(hydroxymethyl)oxane-3,4,5-triol was recently approved by the FDA for the treatment of chronic type 2 diabetes mellitus. Herein, we report the synthesis of carbon-13 and carbon-14 labeled empagliflozin. Carbon-13 labeled empagliflozin was prepared in five steps and in 34% overall chemical yield starting from the commercially available α-D-glucose-[(13)C6]. For the radiosynthesis, the carbon-14 atom was introduced in three different positions of the molecule. In the first synthesis, Carbon-14 D-(+)-gluconic acid δ-lactone was used to prepare specifically labeled empagliflozin in carbon-1 of the sugar moiety in four steps and in 19% overall radiochemical yield. Carbon-14 labeled empagliflozin with the radioactive atom in the benzylic position was obtained in eight steps and in 7% overall radiochemical yield. In the last synthesis carbon-14 uniformly labeled phenol was used to give [(14)C]empagliflozin in eight steps and in 18% overall radiochemical yield. In all these radiosyntheses, the specific activities of the final compounds were higher than 53 mCi/mmol, and the radiochemical purities were above 98.5%. Copyright © 2014 John Wiley & Sons, Ltd.

In vivo detection of 13C isotopomer turnover in the human brain by sequential infusion of 13C labeled substrates

NASA Astrophysics Data System (ADS)

Li, Shizhe; Zhang, Yan; Ferraris Araneta, Maria; Xiang, Yun; Johnson, Christopher; Innis, Robert B.; Shen, Jun

2012-05-01

This study demonstrates the feasibility of simultaneously detecting human brain metabolites labeled by two substrates infused in a sequential order. In vivo 13C spectra of carboxylic/amide carbons were acquired only during the infusion of the second substrate. This approach allowed dynamic detection of 13C labeling from two substrates with considerably different labeling patterns. [2-13C]glucose and [U-13C6]glucose were used to generate singlet and doublet signals of the same carboxylic/amide carbon atom, respectively. Because of the large one-bond 13C-13C homonuclear J coupling between a carboxylic/amide carbon and an aliphatic carbon (˜50 Hz), the singlet and doublet signals of the same carboxylic/amide carbon were well distinguished. The results demonstrated that different 13C isotopomer patterns could be simultaneously and distinctly measured in vivo in a clinical setting at 3 T.

Human metabolism and elimination of the anthocyanin, cyanidin-3-glucoside: a (13)C-tracer study.

PubMed

Czank, Charles; Cassidy, Aedín; Zhang, Qingzhi; Morrison, Douglas J; Preston, Tom; Kroon, Paul A; Botting, Nigel P; Kay, Colin D

2013-05-01

Evidence suggests that the consumption of anthocyanin-rich foods beneficially affects cardiovascular health; however, the absorption, distribution, metabolism, and elimination (ADME) of anthocyanin-rich foods are relatively unknown. We investigated the ADME of a (13)C5-labeled anthocyanin in humans. Eight male participants consumed 500 mg isotopically labeled cyanidin-3-glucoside (6,8,10,3',5'-(13)C5-C3G). Biological samples were collected over 48 h, and (13)C and (13)C-labeled metabolite concentrations were measured by using isotope-ratio mass spectrometry and liquid chromatography-tandem mass spectrometry. The mean ± SE percentage of (13)C recovered in urine, breath, and feces was 43.9 ± 25.9% (range: 15.1-99.3% across participants). The relative bioavailability was 12.38 ± 1.38% (5.37 ± 0.67% excreted in urine and 6.91 ± 1.59% in breath). Maximum rates of (13)C elimination were achieved 30 min after ingestion (32.53 ± 14.24 μg(13)C/h), whereas (13)C-labeled metabolites peaked (maximum serum concentration: 5.97 ± 2.14 μmol/L) at 10.25 ± 4.14 h. The half-life for (13)C-labeled metabolites ranged between 12.44 ± 4.22 and 51.62 ± 22.55 h. (13)C elimination was greatest between 0 and 1 h for urine (90.30 ± 15.28 μg/h), at 6 h for breath (132.87 ± 32.23 μg/h), and between 6 and 24 h for feces (557.28 ± 247.88 μg/h), whereas the highest concentrations of (13)C-labeled metabolites were identified in urine (10.77 ± 4.52 μmol/L) and fecal samples (43.16 ± 18.00 μmol/L) collected between 6 and 24 h. Metabolites were identified as degradation products, phenolic, hippuric, phenylacetic, and phenylpropenoic acids. Anthocyanins are more bioavailable than previously perceived, and their metabolites are present in the circulation for ≤48 h after ingestion. This trial was registered at clinicaltrials.gov as NCT01106729.

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

PubMed

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

2016-12-01

Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available 12/13 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.

49 CFR 172.411 - EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels..., 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label. (a) Except for size and color....5 and EXPLOSIVE 1.6 labels must be as follows: EXPLOSIVE 1.4: EC02MR91.016 EXPLOSIVE 1.5: EC02MR91...

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

DOEpatents

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

2001-01-01

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

49 CFR 172.411 - EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 2 2011-10-01 2011-10-01 false EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels..., EMERGENCY RESPONSE INFORMATION, TRAINING REQUIREMENTS, AND SECURITY PLANS Labeling § 172.411 EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label. (a) Except for size and color...

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

PubMed

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

2016-04-01

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

Determination of the Isotopic Enrichment of 13C- and 2H-Labeled Tracers of Glucose Using High-Resolution Mass Spectrometry: Application to Dual- and Triple-Tracer Studies.

PubMed

Trötzmüller, Martin; Triebl, Alexander; Ajsic, Amra; Hartler, Jürgen; Köfeler, Harald; Regittnig, Werner

2017-11-21

Multiple-tracer approaches for investigating glucose metabolism in humans usually involve the administration of stable and radioactive glucose tracers and the subsequent determination of tracer enrichments in sampled blood. When using conventional, low-resolution mass spectrometry (LRMS), the number of spectral interferences rises rapidly with the number of stable tracers employed. Thus, in LRMS, both computational effort and statistical uncertainties associated with the correction for spectral interferences limit the number of stable tracers that can be simultaneously employed (usually two). Here we show that these limitations can be overcome by applying high-resolution mass spectrometry (HRMS). The HRMS method presented is based on the use of an Orbitrap mass spectrometer operated at a mass resolution of 100 000 to allow electrospray-generated ions of the deprotonated glucose molecules to be monitored at their exact masses. The tracer enrichment determination in blood plasma is demonstrated for several triple combinations of 13 C- and 2 H-labeled glucose tracers (e.g., [1- 2 H 1 ]-, [6,6- 2 H 2 ]-, [1,6- 13 C 2 ]glucose). For each combination it is shown that ions arising from 2 H-labeled tracers are completely differentiated from those arising from 13 C-labeled tracers, thereby allowing the enrichment of a tracer to be simply calculated from the observed ion intensities using a standard curve with curve parameters unaffected by the presence of other tracers. For each tracer, the HRMS method exhibits low limits of detection and good repeatability in the tested 0.1-15.0% enrichment range. Additionally, due to short sample preparation and analysis times, the method is well-suited for high-throughput determination of multiple glucose tracer enrichments in plasma samples.

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2017-08-01

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

NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.

PubMed Central

Zimmer, D P; Crothers, D M

1995-01-01

A procedure for the enzymatic synthesis of uniformly 13C15N-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on 13C and 15N nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG.dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of 15N and 13C heteronuclear NMR experiments to isotopically labeled DNA is presented. Images Fig. 2 Fig. 3 Fig. 4 PMID:7724521

Structure of C 14 and B 14 from the C 14 , 15 ( d , He 3 ) B 13 , 14 reactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bedoor, S.; Wuosmaa, A. H.; Albers, M.

We have studied the C-14,C-15(d,He-3)B-13,B-14 proton-removing reactions in inverse kinematics. The (d,He-3) reaction probes the proton occupation of the target ground state, and also provides spectroscopic information about the final states in B-13,B-14. The experiments were performed using C-14,C-15 beams from the ATLAS accelerator at Argonne National Laboratory. The reaction products were analyzed with the HELIOS device. Angular distributions were obtained for transitions from both reactions. The C-14-beam data reveal transitions to excited states in B-13 that suggest configurations with protons outside the pi(0p(3/2)) orbital, and some possibility of proton cross-shell 0p-1s0d excitations, in the C-14 ground state. The C-15-beammore » data confirm the existence of a broad 2(-) excited state in B-14. The experimental data are compared to the results of shell-model calculations.« less

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

PubMed

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

2007-09-01

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

Selective detection of carbon-13, nitrogen-15, and deuterium labeled metabolites by capillary gas chromatography-chemical reaction interface/mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chace, D.H.; Abramson, F.P.

1989-12-15

We have applied a new chemical reaction interface/mass spectrometer technique (CRIMS) to the selective detection of 13C-, 15N-, and 2H-labeled phenytoin and its metabolites in urine following separation by capillary gas chromatography. The microwave-powered chemical reaction interface converts materials from their original forms into small molecules whose mass spectra serve to identify and quantify the nuclides that make up each analyte. The presence of each element is followed by monitoring the isotopic variants of CO2, NO, or H2 that are produced by the chemical reaction interface. Chromatograms showing only enriched 13C and 15N were produced by subtracting the abundance ofmore » naturally occurring isotopes from the observed M + 1 signal. A selective chromatogram of 2H (D) was obtained by measuring HD at m/z 3.0219 with a resolution of 2000. Metabolites representing less than 1.5% of the total labeled compounds could be identified in the chromatogram. Detection limits from urine of 380 pg/mL of a 15N-labeled metabolite, 7 ng/mL of a 13C-labeled metabolite, and 16 ng/mL of a deuterium labeled metabolite were determined at a signal to noise ratio of 2. Depending on the isotope examined, a linear dynamic range of 250-1000 was observed using CRIMS. To identify many of these labeled peaks (metabolites), the chromatographic analysis was repeated with the chemical reaction interface turned off and mass spectra obtained at the retention times found in the CRIMS experiment. CRIMS is a new analytical method that appears to be particularly useful for metabolism studies.« less

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

NASA Astrophysics Data System (ADS)

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

2013-04-01

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

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

NASA Astrophysics Data System (ADS)

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

2016-04-01

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

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

PubMed

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

2002-11-01

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 delta-, epsilon- and zeta-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.

AMS studies of the long-term turnover of 14C-labelled fat in man

NASA Astrophysics Data System (ADS)

Gunnarsson, M.; Mattsson, S.; Stenström, K.; Leide-Svegborn, S.; Erlandsson, B.; Faarinen, M.; Hellborg, R.; Kiisk, M.; Nilsson, L.-E.; Nosslin, B.; Persson, P.; Skog, G.; Åberg, M.

2000-10-01

To estimate the biokinetics of 14C-labelled fatty acids and the associated radiation absorbed dose to man, long-term retention of 14C from oral intake of glycerol tri[1- 14C]oleate (triolein) has been studied using accelerator mass spectrometry (AMS). As a complement to earlier reported data for three individuals, we present here results for one person from measurements up to 4.6 yr after administration, now also including 14C-levels in fat, muscle and bone. In this subject, a total of 44% of the administered activity was recovered in the exhaled air. Fasting increased the exhalation of 14C. The "excess" 14CO2 due to fasting had a half-life of about 400 d. AMS measurements on fat, muscle and bone biopsies taken from the same subject 4.5 yr after ingestion indicated that a small fraction of the administered activity was still present in fat. Also, bone tissue had a higher 14C specific activity than the current environmental level. No significantly increased level was found in the muscle sample.

Towards the measurement of the13C(d, p)14C cross section using AMS

NASA Astrophysics Data System (ADS)

Murillo-Morales, S.; Barrón-Palos, L.; Chávez, E.; Araujo-Escalona, V.

2017-07-01

A plan to study the total cross section for the13C(d, p)14C nuclear reaction has been developed for energies in the center-of-mass frame between 133 and 400 keV. The proposed experiment will use a deuterium beam (1-3 MeV of energy) from the Instituto de Física-UNAM 5.5 MV Van de Graaff accelerator and the produced14C will be afterwards measured by AMS technique in the LEMA-UNAM (HVEE 1 MV Tandetron). One of the main goals is to study the performance of the LEMA-UNAM facility in the cross section measurement in comparison with other data reported in the literature, measured by other techniques. In this work we present the current status of these studies. The relevance of the13C(d, p)14C reaction in the study of compound nucleus formation as well as in some astrophysics scenarios, and the importance of the development of the AMS technique to measure cross sections of nuclear reactions of astrophysical interest in Mixico are also discussed.

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

PubMed

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.

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

PubMed

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

2017-06-15

Metabolic flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography-high resolution mass spectrometry, without derivatized procedure, adapted for Metabolic flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic enrichments of free sugars compared to our previous methods [5, 6]. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolic fate and distribution of [{sup 14}C]1,3-dichloropropene in carrot, lettuce, radish, tomato, and wheat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barnkow, D.E.; Byrne, S.L.; Huskin, M.A.

1996-10-01

This study examined the nature of the radioactive residue in carrot, lettuce, radish, tomato, and wheat grown in soil treated with uniformly {sup 14}C-labeled cis- and trans-1,3-dichloropropene (1,3-D). Each crop was grown in soil treated at or above the maximum use rate. Carrot root and top, lettuce, radish root and top, tomato fruit and vine, and wheat forage, straw and grain were harvested, processed, and analyzed. The {sup 14}C residues were subjected to sequential extraction with diethyl ether and aqueous acetonitrile. The residues that remained were subjected to extraction, fractionation, and hydrolysis with buffer, enzymes, acid, alkali, and strong oxidizingmore » agents. Analyses of the solubilized residues demonstrated that no detectable parent, 1,3-D, or the putative metabolites 3-chloroallyl alcohol and 3-chloroacrylic acid were present. The components of the extractable residue included most major plant constituents (i.e., protein, pigments, organic acids, sucrose, cellulose, and lignin). Thus, natural incorporation of the {sup 14}C-label into natural plant biochemicals is demonstrated.« less

Synthesis of two potent glucocorticoid receptor agonists labeled with carbon-14 and stable isotopes.

PubMed

Latli, Bachir; Reeves, Jonathan T; Tan, Zhulin; Hrapchak, Matt; Song, Jinhua J; Busacca, Carl B; Senanayake, Chris H

2015-01-01

Two potent glucocorticoid receptor agonists were prepared labeled with carbon-14 and with stable isotopes to perform drug metabolism, pharmacokinetics, and bioanalytical studies. Carbon-14 labeled (1) was obtained from an enantiopure alkyne (5) via a Sonogashira coupling to a previously reported 5-amino-4-iodo-[2-(14)C]pyrimidine [(14)C]-(6), followed by a base-mediated cyclization (1) in 72% overall radiochemical yield. Carbon-14 labeled (2) was prepared in five steps employing a key benzoic acid intermediate [(14)C]-(13), which was synthesized in one pot from enolization of trifluoromethylketone (12), followed by bromine-magnesium exchange and then electrophile trapping reaction with [(14)C]-carbon dioxide. A chiral auxiliary (S)-1-(4-methoxyphenyl)ethylamine was then coupled to this acid to give [(14)C]-(15). Propargylation and separation of diastereoisomers by crystallizations gave the desired diastereomer [(14)C]-(17) in 34% yield. Sonogashira coupling to iodopyridine (10) followed by cyclization to the azaindole [(14)C]-(18) and finally removal of the chiral auxiliary gave [(14)C]-(2) in 7% overall yield. For stable isotope syntheses, [(13)C6]-(1) was obtained in three steps using [(13)C4]-(6) and trimethylsilylacetylene-[(13)C2] in 26% yield, while [(2)H5]-(2) was obtained by first preparing the iodopyridine [(2)H5]-(10) in five steps. Then, Sonogashira coupling to chiral alkyne (24) and cyclization gave [(2)H5]-(2) in 42% overall yield. Copyright © 2015 John Wiley & Sons, Ltd.

Rapid radiosynthesis of [11C] and [14C]azelaic, suberic, and sebacic acids for in vivo mechanistic studies of systemic acquired resistance in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Best M.; Fowler J.; Best, M.

2011-11-25

A recent report that the aliphatic dicarboxylic acid, azelaic acid (1,9-nonanedioic acid) but not related acids, suberic acid (1,8-octanedioic acid) or sebacic (1,10-decanedioic acid) acid induces systemic acquired resistance to invading pathogens in plants stimulated the development of a rapid method for labeling these dicarboxylic acids with {sup 11}C and {sup 14}C for in vivo mechanistic studies in whole plants. {sup 11}C-labeling was performed by reaction of ammonium [{sup 11}C]cyanide with the corresponding bromonitrile precursor followed by hydrolysis with aqueous sodium hydroxide solution. Total synthesis time was 60 min. Median decay-corrected radiochemical yield for [{sup 11}C]azelaic acid was 40% relativemore » to trapped [{sup 11}C]cyanide, and specific activity was 15 GBq/{micro}mol. Yields for [{sup 11}C]suberic and sebacic acids were similar. The {sup 14}C-labeled version of azelaic acid was prepared from potassium [{sup 14}C]cyanide in 45% overall radiochemical yield. Radiolabeling procedures were verified using {sup 13}C-labeling coupled with {sup 13}C-NMR and liquid chromatography-mass spectrometry analysis. The {sup 11}C and {sup 14}C-labeled azelaic acid and related dicarboxylic acids are expected to be of value in understanding the mode-of-action, transport, and fate of this putative signaling molecule in plants.« less

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

NASA Astrophysics Data System (ADS)

Dippold, Michaela; Kuzyakov, Yakov

2015-04-01

Understanding the soil organic matter (SOM) dynamics is one of the most important challenges in soil science. Transformation of low molecular weight organic substances (LMWOS) is a key step in biogeochemical cycles because 1) all high molecular substances pass this stage during their decomposition and 2) only LMWOS will be taken up by microorganisms. Previous studies on LMWOS were focused on determining net fluxes through the LMWOS pool, but they rarely identified transformations. As LMWOS are the preferred C and energy source for microorganisms, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the biochemical pathways and its controlling factors. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools. Up to now, studies on LMWOS were nearly exclusively based on uniformly labeled organic substances i.e. all C atoms in the molecules were labeled with 13C or 14C. However, this classical approach did not allow the differentiation between use of intact initial substances in any process, or whether they were transformed to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of LMWOS and quantification of 13CO2 and 13C in bulk soil enabled following the basic metabolic pathways of soil microorganisms. However, only the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites allowed 1) tracing specific anabolic pathways in diverse microbial communities in soils and 2) identification of specific pathways of individual functional microbial groups. So, these are the prerequisites for soil fluxomics. Our studies combining position-specific labeled glucose with amino

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

PubMed Central

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

2015-01-01

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

A simple method to determine mineralization of (14) C-labeled compounds in soil.

PubMed

Myung, Kyung; Madary, Michael W; Satchivi, Norbert M

2014-06-01

Degradation of organic compounds in soil is often determined by measuring the decrease of the parent compound and analyzing the occurrence of its metabolites. However, determining carbon species as end products of parent compound dissipation requires using labeled materials that allow more accurate determination of the environmental fate of the compound of interest. The current conventional closed system widely used to monitor degradation of (14) C-labeled compounds in soil is complex and expensive and requires a specialized apparatus and facility. In the present study, the authors describe a simple system that facilitates measurement of mineralization of (14) C-labeled compounds applied to soil samples. In the system, soda lime pellets to trap mineralized (14) C-carbon species, including carbon dioxide, were placed in a cup, which was then inserted above the treated soil sample in a tube. Mineralization of [(14) C]2,4-D applied to soil samples in the simple system was compared with that in the conventional system. The simple system provided an equivalent detection of (14) C-carbon species mineralized from the parent compound. The results demonstrate that this cost- and space-effective simple system is suitable for examining degradation and mineralization of (14) C-labeled compounds in soil and could potentially be used to investigate their mineralization in other biological matrices. © 2014 SETAC.

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

DOEpatents

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

2008-01-22

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

Obtaining molecular and structural information from 13C-14N systems with 13C FIREMAT experiments.

PubMed

Strohmeier, Mark; Alderman, D W; Grant, David M

2002-04-01

The effect of dipolar coupling to 14N on 13C FIREMAT (five pi replicated magic angle turning) experiments is investigated. A method is developed for fitting the 13C FIREMAT FID employing the full theory to extract the 13C-14N dipolar and 13C chemical shift tensor information. The analysis requires prior knowledge of the electric field gradient (EFG) tensor at the 14N nucleus. In order to validate the method the analysis is done for the amino acids alpha-glycine, gamma-glycine, l-alanine, l-asparagine, and l-histidine on FIREMAT FIDs recorded at 13C frequencies of 50 and 100 MHz. The dipolar and chemical shift data obtained with this analysis are in very good agreement with the previous single-crystal 13C NMR results and neutron diffraction data on alpha-glycine, l-alanine, and l-asparagine. The values for gamma-glycine and l-histidine obtained with this new method are reported for the first time. The uncertainties in the EFG tensor on the resultant 13C chemical shift and dipolar tensor values are assessed. (c) 2002 Elsevier Science (USA).

In vivo stationary flux analysis by 13C labeling experiments.

PubMed

Wiechert, W; de Graaf, A A

1996-01-01

Stationary flux analysis is an invaluable tool for metabolic engineering. In the last years the metabolite balancing technique has become well established in the bioengineering community. On the other hand metabolic tracer experiments using 13C isotopes have long been used for intracellular flux determination. Only recently have both techniques been fully combined to form a considerably more powerful flux analysis method. This paper concentrates on modeling and data analysis for the evaluation of such stationary 13C labeling experiments. After reviewing recent experimental developments, the basic equations for modeling carbon labeling in metabolic systems, i.e. metabolite, carbon label and isotopomer balances, are introduced and discussed in some detail. Then the basics of flux estimation from measured extracellular fluxes combined with carbon labeling data are presented and, finally, this method is illustrated by using an example from C. glutamicum. The main emphasis is on the investigation of the extra information that can be obtained with tracer experiments compared with the metabolite balancing technique alone. As a principal result it is shown that the combined flux analysis method can dispense with some rather doubtful assumptions on energy balancing and that the forward and backward flux rates of bidirectional reaction steps can be simultaneously determined in certain situations. Finally, it is demonstrated that the variant of fractional isotopomer measurement is even more powerful than fractional labeling measurement but requires much higher numerical effort to solve the balance equations.

Metabolic flux analysis using 13C peptide label measurements

USDA-ARS?s Scientific Manuscript database

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

Pediatric microdose study of [(14)C]paracetamol to study drug metabolism using accelerated mass spectrometry: proof of concept.

PubMed

Mooij, Miriam G; van Duijn, Esther; Knibbe, Catherijne A J; Windhorst, Albert D; Hendrikse, N Harry; Vaes, Wouter H J; Spaans, Edwin; Fabriek, Babs O; Sandman, Hugo; Grossouw, Dimitri; Hanff, Lidwien M; Janssen, Paul J J M; Koch, Birgit C P; Tibboel, Dick; de Wildt, Saskia N

2014-11-01

Pediatric drug development is hampered by practical, ethical, and scientific challenges. Microdosing is a promising new method to obtain pharmacokinetic data in children with minimal burden and minimal risk. The use of a labeled oral microdose offers the added benefit to study intestinal and hepatic drug disposition in children already receiving an intravenous therapeutic drug dose for clinical reasons. The objective of this study was to present pilot data of an oral [(14)C]paracetamol [acetaminophen (AAP)] microdosing study as proof of concept to study developmental pharmacokinetics in children. In an open-label microdose pharmacokinetic pilot study, infants (0-6 years of age) received a single oral [(14)C]AAP microdose (3.3 ng/kg, 60 Bq/kg) in addition to intravenous therapeutic doses of AAP (15 mg/kg intravenous every 6 h). Blood samples were taken from an indwelling catheter. AAP blood concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and [(14)C]AAP and metabolites ([(14)C]AAP-Glu and [(14)C]AAP-4Sul) were measured by accelerator mass spectrometry. Ten infants (aged 0.1-83.1 months) were included; one was excluded as he vomited shortly after administration. In nine patients, [(14)C]AAP and metabolites in blood samples were detectable at expected concentrations: median (range) maximum concentration (C max) [(14)C]AAP 1.68 (0.75-4.76) ng/L, [(14)C]AAP-Glu 0.88 (0.34-1.55) ng/L, and [(14)C]AAP-4Sul 0.81 (0.29-2.10) ng/L. Dose-normalized oral [(14)C]AAP C max approached median intravenous average concentrations (C av): 8.41 mg/L (3.75-23.78 mg/L) and 8.87 mg/L (3.45-12.9 mg/L), respectively. We demonstrate the feasibility of using a [(14)C]labeled microdose to study AAP pharmacokinetics, including metabolite disposition, in young children.

Pharmacokinetic analysis of 14C-ursodiol in newborn infants using accelerator mass spectrometry.

PubMed

Gordi, Toufigh; Baillie, Rebecca; Vuong, Le T; Abidi, Saira; Dueker, Stephen; Vasquez, Herbert; Pegis, Priscilla; Hopper, Andrew O; Power, Gordon G; Blood, Arlin B

2014-09-01

Pharmacokinetic studies in the neonatal population are often limited by the small volume of blood that can be collected. The high sensitivity of (14) C-accelerator mass spectrometry (AMS) enables pharmacokinetic studies to be conducted with greatly reduced sample volumes. We demonstrated the utility of AMS in infants by studying the plasma pharmacokinetic behavior of nanogram doses of (14) C-ursodiol administered as a non-perturbing microdose or as a microtracer with therapeutic doses of non-labeled ursodiol in infants. Five non-cholestatic infants were administered 3 consecutive oral microdoses of (14) C-ursodiol: 8 ng (1.0 nCi), 26 ng (3.3 nCi), and 80 ng (10 nCi) 48 hours apart. Three additional infants with cholestasis were administered a single 80 ng (10.0 nCi) oral dose of (14) C-ursodiol together with a therapeutic dose of 40 mg/kg of non-labeled ursodiol. A pharmacokinetic model describing ursodiol concentrations was developed using nonlinear mixed-effects modeling. The pharmacokinetics of ursodiol in this pilot study were best described by a two-compartment model with first-order elimination. This study demonstrates the feasibility and utility of microdose and microtrace methodology in pediatric research. © 2014, The American College of Clinical Pharmacology.

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

PubMed Central

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

2011-01-01

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

Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance.

PubMed

Sugiki, Toshihiko; Furuita, Kyoko; Fujiwara, Toshimichi; Kojima, Chojiro

2018-06-20

Amino acid selective isotope labeling is an important nuclear magnetic resonance technique, especially for larger proteins, providing strong bases for the unambiguous resonance assignments and information concerning the structure, dynamics, and intermolecular interactions. Amino acid selective 15 N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. Carbonyl 13 C atoms experience less isotope scrambling than the main-chain 15 N atoms do. However, little is known about the side-chain 13 C atoms. Here, the 13 C scrambling profiles of the Cα and side-chain carbons were investigated for 15 N scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in 13 Cα and 13 C side-chain labeling than in 15 N labeling. We utilized this reduced scrambling-prone character of 13 C as a simple and efficient method for amino acid selective 13 C labeling using an Escherichia coli cold-shock expression system and high-cell density fermentation. Using this method, the 13 C labeling efficiency was >80% for Leu and Ile, ∼60% for Tyr and Phe, ∼50% for Thr, ∼40% for Val, and 30-40% for Ala. 1 H- 15 N heteronuclear single-quantum coherence signals of the 15 N scrambling-prone amino acid were also easily filtered using 15 N-{ 13 Cα} spin-echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.

Tracing phenolic biosynthesis in Vitis vinifera via in situ C-13 labeling and liquid chromatography-diode-array detector-mass spectrometer/mass spectrometer detection.

PubMed

Chassy, Alexander W; Adams, Douglas O; Laurie, V Felipe; Waterhouse, Andrew L

2012-10-17

Phenolic compounds in Vitis vinifera contribute important flavor, functionality, and health qualities to both table and wine grapes. The plant phenolic metabolic pathway has been well characterized, however many important questions remain regarding the influence of environmental conditions on pathway regulation. As a diagnostic for this pathway's regulation, we present a technique to incorporate a stable-isotopic tracer, L-phenyl-(13)C(6)-alanine (Phe(13)), into grape berries in situ and the accompanying high throughput analytical method based on LC-DAD-MS/MS to quantify and track the label into phenylalanine metabolites. Clusters of V. vinifera cv. Cabernet Sauvignon, either near the onset of ripening or 4 weeks later, were exposed to Phe(13) in the vineyard. Phe(13) was present in berries 9 days afterwards as well as labeled flavonols and anthocyanins, all of which possessed a molecular ion shift of 6 amu. However, nearly all the label was found in anthocyanins, indicating tight regulation of phenolic biosynthesis at this stage of maturity. This method provides a framework for examining the regulation of phenolic metabolism at different stages of maturity or under different environmental conditions. Additionally, this technique could serve as a tool to further probe the metabolism/catabolism of grape phenolics. Copyright © 2012 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2013-01-01

While putative disease-preventing lycopene metabolites are found in both tomato (Solanum lycopersicum) products and in their consumers, mammalian lycopene metabolism is poorly understood. Advances in tomato cell culturing techniques offer an economical tool for generation of highly-enriched 13C-lycopene for human bioavailability and metabolism studies. To enhance the 13C-enrichment and yields of labeled lycopene from the hp-1 tomato cell line, cultures were first grown in 13C-glucose media for three serial batches and produced increasing proportions of uniformly labeled lycopene (14.3 +/− 1.2 %, 39.6 +/− 0.5 %, and 48.9 +/− 1.5% with consistent yields (from 5.8 to 9 mg/L). An optimized 9-day-long 13C-loading and 18-day-long labeling strategy developed based on glucose utilization and lycopene yields, yielded 13C-lycopene with 93% 13C isotopic purity, and 55% of isotopomers were uniformly labeled. Furthermore, an optimized acetone and hexane extraction led to a four-fold increase in lycopene recovery from cultures compared to a standard extraction. PMID:23561155

Exploiting Uniformly 13C-Labeled Carbohydrates for Probing Carbohydrate-Protein Interactions by NMR Spectroscopy.

PubMed

Nestor, Gustav; Anderson, Taigh; Oscarson, Stefan; Gronenborn, Angela M

2017-05-03

NMR of a uniformly 13 C-labeled carbohydrate was used to elucidate the atomic details of a sugar-protein complex. The structure of the 13 C-labeled Manα(1-2)Manα(1-2)ManαOMe trisaccharide ligand, when bound to cyanovirin-N (CV-N), was characterized and revealed that in the complex the glycosidic linkage torsion angles between the two reducing-end mannoses are different from the free trisaccharide. Distances within the carbohydrate were employed for conformational analysis, and NOE-based distance mapping between sugar and protein revealed that Manα(1-2)Manα(1-2)ManαOMe is bound more intimately with its two reducing-end mannoses into the domain A binding site of CV-N than with the nonreducing end unit. Taking advantage of the 13 C spectral dispersion of 13 C-labeled carbohydrates in isotope-filtered experiments is a versatile means for a simultaneous mapping of the binding interactions on both, the carbohydrate and the protein.

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

PubMed

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

2017-05-01

In this paper, the effect of isotope-labeled analogs on the liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) measurement was evaluated based on the comparison research of electrospray ionization responses (ESI) and matrix effect of melamine, 13 C 3 -melamine, 13 C 3 + 15 N 3 -melamine, and 15 N 3 -melamine. The isotope-labeled melamines had similar ionization efficiency with melamine in the electrospray ionization source, but the intensity of corresponding quantitative fragment ions had distinctive differences. Based on the density functional theory at the B3LYP/6-311+G** level, this phenomenon was explained very well. The rare cleavage pathways of melamine, which just could be exactly identified by 15 N-labeled melamines, resulted in the difference of quantitative fragment ions between 15 N-labeled melamines and melamine. The interaction of ESI response between melamine and isotope-labeled melamines was investigated using MRM monitor mode. 15 N-labeled melamine had significant ion inter-suppression effect on melamine, while 13 C-labeled melamine had little influence on melamine. Finally, the influence of different isotope-labeled melamines on the LC-IDMS result was evaluated using the IDMS correction factor (θ). Taking the determination of melamine in milk powder as an example, the matrix effects of different isotope-labeled melamines and melamine had notable difference and the impact of this difference on the measurement results depended on the concentrations of analyte and matrix solution. It was worth noting that 15 N 3 -melamine exhibited significant ion suppression to melamine in matrix solution. The deviation of the results from IDMS method might reach 59% using 15 N 3 -melamine as internal standard in special matrix solution. Graphical Abstract The comparison of ESI responses of melamine, 13 C 3 -melamine, 13 C 3 + 15 N 3 -melamine and 15 N 3 -melamine.

Photosynthetic carbon metabolism in seagrasses C-labeling evidence for the c(3) pathway.

PubMed

Andrews, T J; Abel, K M

1979-04-01

The delta(13)C values of several seagrasses were considerably less negative than those of terrestrial C(3) plants and tended toward those of terrestrial C(4) plants. However, for Thalassia hemprichii (Ehrenb.) Aschers and Halophila spinulosa (R. Br.) Aschers, phosphoglycerate and other C(3) cycle intermediates predominated among the early labeled products of photosynthesis in (14)C-labeled seawater (more than 90% at the earliest times) and the labeling pattern at longer times was brought about by the operation of the C(3) pathway. Malate and aspartate together accounted for only a minor fraction of the total fixed label at all times and the kinetic data of this labeling were not at all consistent with these compounds being early intermediates in seagrass photosynthesis. Pulse-chase (14)C-labeling studies further substantiated these conclusions. Significant labeling of photorespiratory intermediates was observed in all experiments. The kinetics of total fixation of label during some steady-state and pulse-chase experiments suggested that there may be an intermediate pool of inorganic carbon of variable size closely associated with the leaves, either externally or internally. Such a pool may be one cause for the C(4)-like carbon isotope ratios of seagrasses.

Apportioning carbon sources of authigenic carbonate of extremely 13C-depleted foraminifera from the western North Pacific sediments: Implication from the coupled 13C and 14C isotopic mass balance approach

NASA Astrophysics Data System (ADS)

Uchida, M.; Ohkushi, K.; Ahagon, N.; Kimoto, K.; Inagaki, F.; Shibata, Y.

2005-12-01

Recently, Uchida et al. (G-cubed, 2004) and Ohkushi et al. (G-cubed, 2005) interprete /delta 13C variations of planktonic and benthic foraminifera found in Last Glacial sediments in off Shimokita Peninsula and Tokachi as evidence for periodic releases of methane, arising from the dissociation of methane hydrate, and its subsequent oxidation in bottom- and/or surface-water environments. According to recent observations of anomalous bottom-simulating reflections, northwest Pacific marginal sediments around Japan main islands bear large abundances of methane hydrate. In this study, analyzed piston cores (42° 21.42' N, 144° 13.36' E) at a water depth 1066-m was retrieved from the off Tokachi continental slope in the Oyashio current region, where recently is found to bear immense amounts of methane hydrate. The piston core covered past 22 ka with high-resolution. Here we showed that carbon isotope signals indicated that planktonic and benthic foraminifera in several glacial sediment layers in the core were highly depleted in13 C; both the planktonic and benthic foraminiferal /delta 13C values ranged from about -10/permil to -2/permil. Most foraminiferal tests in these horizons were brown as a result of postdepositional alteration. Foraminiferal oxygen isotopes fluctuated abnormally in the glacial sediment layers, showing small (about 0.5/permil) positive shifts relative to normal glacial values. We attributed the positive shifts to authigenic carbonate formation in the foraminiferal tests. In order to decipher the relation between foraminifera carbon isotopic signal and methane release from the seafloor, we have apportioned carbon sources (methane from methane hydrate or not) of foraminiferal carbon isotopic anomalies using dual mass balance isotopic model (14C/ 12C and 13C/ 12C). It has been suggested that sulfate-dependent anaerobic methane oxidation (AOM) dominates carbon oxidation and attendant authigenic carbonate precipitation to foraminifera. To this assumption

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

PubMed

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

2016-11-01

13 C-Metabolic flux analysis ( 13 C-MFA) is a widely used approach in metabolic engineering for quantifying intracellular metabolic fluxes. The precision of fluxes determined by 13 C-MFA depends largely on the choice of isotopic tracers and the specific set of labeling measurements. A recent advance in the field is the use of parallel labeling experiments for improved flux precision and accuracy. However, as of today, no systemic methods exist for identifying optimal tracers for parallel labeling experiments. In this contribution, we have addressed this problem by introducing a new scoring system and evaluating thousands of different isotopic tracer schemes. Based on this extensive analysis we have identified optimal tracers for 13 C-MFA. The best single tracers were doubly 13 C-labeled glucose tracers, including [1,6- 13 C]glucose, [5,6- 13 C]glucose and [1,2- 13 C]glucose, which consistently produced the highest flux precision independent of the metabolic flux map (here, 100 random flux maps were evaluated). Moreover, we demonstrate that pure glucose tracers perform better overall than mixtures of glucose tracers. For parallel labeling experiments the optimal isotopic tracers were [1,6- 13 C]glucose and [1,2- 13 C]glucose. Combined analysis of [1,6- 13 C]glucose and [1,2- 13 C]glucose labeling data improved the flux precision score by nearly 20-fold compared to widely use tracer mixture 80% [1- 13 C]glucose +20% [U- 13 C]glucose. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Dynamic analysis of CO₂ labeling and cell respiration using membrane-inlet mass spectrometry.

PubMed

Yang, Tae Hoon

2014-01-01

Here, we introduce a mass spectrometry-based analytical method and relevant technical details for dynamic cell respiration and CO2 labeling analysis. Such measurements can be utilized as additional information and constraints for model-based (13)C metabolic flux analysis. Dissolved dynamics of oxygen consumption and CO2 mass isotopomer evolution from (13)C-labeled tracer substrates through different cellular processes can be precisely measured on-line using a miniaturized reactor system equipped with a membrane-inlet mass spectrometer. The corresponding specific rates of physiologically relevant gases and CO2 mass isotopomers can be quantified within a short-term range based on the liquid-phase dynamics of dissolved fermentation gases.

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

NASA Astrophysics Data System (ADS)

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

2012-12-01

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

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

PubMed

Knief, Claudia; Altendorf, Karlheinz; Lipski, André

2003-11-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Dien, Stephen J.

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

Formation of glutamine from (/sup 13/N)ammonia, (/sup 13/N)dinitrogen, and (/sup 14/C)glutamate by heterocysts isolated from Anabaena cylindrica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thomas, J.; Meeks, J.C.; Wolk, C.P.

A method is described for the isolation of metabolically active heterocysts from Anabaena cylindrica. These isolated heterocysts accounted for up to 34% of the acetylene-reducing activity of whole filaments and had a specific activity of up to 1,560 nmol of C/sub 2/H/sub 4/ formed per mg of heterocyst chlorophyll per min. Activity of glutamine synthetase was coupled to activity of nitrogenase in isolated heterocysts as shown by acetylene-inhibitable formation of (/sup 13/N)NH/sub 3/ and of amide-labeled (/sup 13/N)glutamine from (/sup 13/N)N/sub 2/. A method is also described for the production of 6-mCi amounts of (/sup 13/N)NH/sub 3/. Isolated heterocysts formedmore » (/sup 13/N)glutamine from (/sup 13/N)NH/sub 3/ and glutamate, and (/sup 14/C)glutamine from NH/sub 3/ and (/sup 14/C)glutamate, in the presence of magnesium adenosine 5'-triphosphate. Methionine sulfoximine strongly inhibited these syntheses. Glutamate synthase is, after nitrogenase and glutamine synthetase, the third sequential enzyme involved in the assimilation of N/sub 2/ by intact filaments. However, the kinetics of solubilization of the activity of glutamate synthase during cavitation of suspensions of A. cylindrica indicated that very little, if any, of the activity of that enzyme was located in heterocysts. Concordantly, isolated heterocysts failed to form substantial amounts of radioactive glutamate from either (/sup 13/N)glutamine or ..cap alpha..-(/sup 14/C)ketoglutarate in the presence of other substrates and cofactors of the glutamate synthase reaction. However, they formed (/sup 14/C)glutamate rapidly from ..cap alpha..-(/sup 14/C)ketoglutarate by aminotransferase reactions, with various amino acids as the nitrogen donor. The implications of these findings with regard to the identities of the substances moving between heterocysts and vegetative cells are discussed.« less

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

PubMed

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.

The synthesis of a tritium, carbon-14, and stable isotope-labeled cathepsin C inhibitors.

PubMed

Allen, Paul; Bragg, Ryan A; Caffrey, Moya; Ericsson, Cecilia; Hickey, Michael J; Kingston, Lee P; Elmore, Charles S

2017-02-01

As part of a medicinal chemistry program aimed at developing a highly potent and selective cathepsin C inhibitor, tritium, carbon-14, and stable isotope-labeled materials were required. The synthesis of tritium-labeled methanesulfonate 5 was achieved via catalytic tritiolysis of a chloro precursor, albeit at a low radiochemical purity of 67%. Tritium-labeled AZD5248 was prepared via a 3-stage synthesis, utilizing amide-directed hydrogen isotope exchange. Carbon-14 and stable isotope-labeled AZD5248 were successfully prepared through modifications of the medicinal chemistry synthetic route, enabling the use of available labeled intermediates. Copyright © 2016 John Wiley & Sons, Ltd.

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

NASA Astrophysics Data System (ADS)

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

2012-12-01

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

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

Treesearch

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

2014-01-01

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

[Quantifying rice (Oryza sativa L.) photo-assimilated carbon input into soil organic carbon pools following continuous 14C labeling].

PubMed

Nie, San-An; Zhou, Ping; Ge, Ti-Da; Tong, Cheng-Li; Xiao, He-Ai; Wu, Jin-Shui; Zhang, Yang-Zhu

2012-04-01

The microcosm experiment was carried out to quantify the input and distribution of photo-assimilated C into soil C pools by using a 14C continuous labeling technique. Destructive samplings of rice (Oryza sativa) were conducted after labeling for 80 days. The allocation of 14C-labeled photosynthates in plants and soil C pools such as dissolved organic C (DOC) and microbial biomass C (MBC) in rice-planted soil were examined over the 14C labeling span. The amounts of rice shoot and root biomass C was ranged from 1.86 to 5.60 g x pot(-1), 0.46 to 0.78 g x pot(-1) in different tested paddy soils after labeling for 80 days, respectively. The amount of 14C in the soil organic C (14C-SOC) was also dependent on the soils, ranged from 114.3 to 348.2 mg x kg(-1), accounting for 5.09% to 6.62% of the rice biomass 14C, respectively. The amounts of 14C in the dissolved organic C (14C-DOC) and in the microbial biomass C(14C-MBC), as proportions of 14C-SOC, were 2.21%-3.54% and 9.72% -17.2%, respectively. The 14C-DOC, 14C-MBC, and 14C-SOC as proportions of total DOC, MBC, and SOC, respectively, were 6.72% -14.64%, 1.70% -7.67%, and 0.73% -1.99%, respectively. Moreover, the distribution and transformation of root-derived C had a greater influence on the dynamics of DOC and MBC than on the dynamics of SOC. Further studies are required to ascertain the functional significance of soil microorganisms (such as C-sequestering bacteria and photosynthetic bacteria) in the paddy system.

Mechanism studies of the conversion of 13C-labeled n-butane on zeolite H-ZSM-5 by using 13C magic angle spinning NMR spectroscopy and GC-MS analysis.

PubMed

Luzgin, Mikhail V; Stepanov, Alexander G; Arzumanov, Sergei S; Rogov, Vladimir A; Parmon, Valentin N; Wang, Wei; Hunger, Michael; Freude, Dieter

2005-12-23

By using 13C MAS NMR spectroscopy (MAS = magic angle spinning), the conversion of selectively 13C-labeled n-butane on zeolite H-ZSM-5 at 430-470 K has been demonstrated to proceed through two pathways: 1) scrambling of the selective 13C-label in the n-butane molecule, and 2) oligomerization-cracking and conjunct polymerization. The latter processes (2) produce isobutane and propane simultaneously with alkyl-substituted cyclopentenyl cations and condensed aromatic compounds. In situ 13C MAS NMR and complementary ex situ GC-MS data provided evidence for a monomolecular mechanism of the 13C-label scrambling, whereas both isobutane and propane are formed through intermolecular pathways. According to 13C MAS NMR kinetic measurements, both pathways proceed with nearly the same activation energies (E(a) = 75 kJ mol(-1) for the scrambling and 71 kJ mol(-1) for isobutane and propane formation). This can be rationalized by considering the intermolecular hydride transfer between a primarily initiated carbenium ion and n-butane as being the rate-determining stage of the n-butane conversion on zeolite H-ZSM-5.

13C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicillium chrysogenum

PubMed Central

Kleijn, Roelco J.; van Winden, Wouter A.; Ras, Cor; van Gulik, Walter M.; Schipper, Dick; Heijnen, Joseph J.

2006-01-01

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h−1 yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio. PMID:16820467

The recovery of 13C-labeled oleic acid in rat lymph after administration of long chain triacylglycerols or specific structured triacylglycerols.

PubMed

Vistisen, Bodil; Mu, Huiling; Høy, Carl-Erik

2006-09-01

Consumption of specific structured triacylglycerols, MLM (M = medium chain fatty acid, L = long chain fatty acid), delivers fast energy and long chain fatty acids to the organism. The purpose of the present study was to compare lymphatic absorption of (13)C-labeled MLM and (13)C-labeled LLL in rats. Stable isotope labeling enables the separation of the endogenous and exogenous fatty acids. Lymph was collected during 24 h following administration of MLM or LLL. Lymph fatty acid composition and (13)C-enrichment were determined and quantified by gas chromatography combustion isotope ratio mass spectrometry. The recovery of 18:1n-9 was higher after administration of LLL compared with MLM (58.1% +/- 7.4% and 29.1% +/- 3.9%, respectively, P < 0.001). This may be due to a higher chylomicron formation stimulated by a higher amount of long chain fatty acids in the intestine after LLL compared with MLM administration. This was confirmed by the tendencies of higher lymphatic transport of endogenous fatty acids. The study revealed a higher lymphatic recovery of the administered long chain fatty acids after LLL compared with MLM consumption.

Determination of Multiple φ-Torsion Angles in Proteins by Selective and Extensive 13C Labeling and Two-Dimensional Solid-State NMR

NASA Astrophysics Data System (ADS)

Hong, Mei

1999-08-01

We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive 13C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective 13C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-13C]glucose preferentially labels the ends of the side chains, while [2-13C]glycerol labels the Cα of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles φ simultaneously, using an isotropic-anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively 13C labeled protein were performed using 15N-13C 2D correlation spectroscopy. From the time dependence of the 15N-13C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective 13C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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

2004-12-01

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

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

PubMed Central

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

2011-01-01

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

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

PubMed

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

2011-06-15

average δ(13)C (-14.8) than teeth from subjects raised in Japan (-13.5), Middle East and North Africa (-12.7) and South America (-10.9). In summary, isotopic analysis of carbon in enamel from a single tooth can give a good estimate of the year of birth of an individual and also provide information about the geographical origin of the individual. This strategy can assist police and forensic authorities when attempting to solve unidentified homicide cases and may facilitate the identification work associated with mass disasters. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

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

PubMed Central

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

2010-01-01

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

Synthesis of 14C-labeled perfluorooctanoic and perfluorodecanoic acids; Purification of perfluorodecanoic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reich, I.L.; Reich, H.J.; Menahan, L.A.

1987-01-01

Perfluorooctanoic and -decanoic acids are representative of a series of perfluorinated acids that have been used for a variety of industrial purposes primarily due to their surfactant properties. The toxicity of these compounds is being investigated in a number of laboratories. 14C-labeled materials would be useful in these studies but are not commercially available. Johncock prepared unlabeled PFOA in low yield by carbonation of the unstable perfluoroheptyllithium at -90 degrees Centigrade. We anticipated several problems in applying this procedure to the synthesis of the 14C-labeled material. Johncock's procedure was run on a fairly large scale (10 mmol) with excess CO2.

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

PubMed Central

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

Metabolism of C14-labeled phenylalanine and tyrosine in malaria-infected Culex-females (in German)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maier, W.A.; Nassif-Makki, H.

1975-01-01

Culex females are fed on C14-phenylalanine or C14-tyrosine in sugar solution. Autoradiographic studies on homogenated females 1 or 4 days after feeding, show that the labeled amino acids are metabolized on the first day and are not detectable on the fourth day. After increase of the amino acid concentration by saturation of the sugar solution with the unlabeled amino acid, the labeled acid and its metabolites are visible over a longer period of time. Phenylalanine is metabolized to tyrosine and at least four other substances. Radioactivity on the starting point of the chromatogram can be interpreted as incorporation of tyrosinemore » into proteins. After infection with Plasmodium cathemerium, and feeding of C14-phenylalanine C14-tyrosine is demonstrable over a longer period. (orig.)« less

In-Situ 13C-Labeling of Microbial Phospholipid Fatty Acids: Tracing Substrate Assimilation in a Petroleum-Contaminated Aquifer

NASA Astrophysics Data System (ADS)

Pombo, S. A.; Schroth, M. H.; Pelz, O.; Zeyer, J.

2001-12-01

Stable isotope analysis of phospholipid-derived fatty acids (PLFA) is a novel tool to trace assimilation of organic carbon in microbial communities. The 13C-labeling of biomarker fatty acids allows the identification of specific microbial populations involved in the metabolism of particular substrates, supplemented in 13C-labeled form. The goal of this study was to investigate the feasibility of 13C-labeling of PLFA and produced dissolved inorganic carbon (DIC) in a petroleum hydrocarbon (PHC)-contaminated aquifer during an in-situ experiment. To this end, we performed a single-well "push-pull" test in a monitoring well located in the denitrifying zone of a PHC-contaminated aquifer in Studen, Switzerland. During the experiment, we injected 500 L of site groundwater that was amended with 13C-labeled acetate (50% [2-13C]) and nitrate as reactants, and bromide as conservative tracer. Following the injection, we extracted a total of 1000 L of test solution/groundwater mixture after 4, 23 and 46 h from the same location. Concentrations of anions were measured in samples collected during the extraction. From these data, we computed first order rate coefficients for consumption of acetate (0.70 +/- 0.05 1/d) and nitrate (0.63 +/- 0.08 1/d). In addition, we extracted and identified PLFA, and measured \\delta13C values of PLFA and DIC. After only 4 h of incubation, we detected 13C-enrichment of certain PLFA in suspended biomass of extracted groundwater. After 46 h, we measured enrichments of up to 5000 per mil in certain PLFA (e.g. 16:1ω 7c), and up to 1500 per mil in the produced DIC. Our results demonstrate the feasibility of in-situ 13C-labeling of PLFA and DIC using push-pull tests to determine microbial activities in-situ in a natural ecosystem.

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

PubMed

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

Protein N- and C-Termini Identification Using Mass Spectrometry and Isotopic Labeling

USDA-ARS?s Scientific Manuscript database

A new method for protein N- and C-terminal analysis using mass spectrometry is introduced. A novel stable isotopic labeling scheme has been developed to identify terminal peptides generated from an enzyme digestion for the determination of both N- and C-termini of the protein. This method works dire...

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

NASA Astrophysics Data System (ADS)

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

2015-12-01

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

Determination of δ13C, δ15N, or δ34S by isotope-ratio-monitoring mass spectrometry using an elemental analyzer

USGS Publications Warehouse

Johnson, Craig A.; Stricker, Craig A.; Gulbransen, Cayce A.; Emmons, Matthew P.

2018-02-14

This report describes procedures used in the Geology, Geophysics, and Geochemistry Science Center of the U.S. Geological Survey in Denver, Colorado, to determine the stable-isotope ratios 13C/12C, 15N/14N, and 34S/32S in solid materials. The procedures use elemental analyzers connected directly to gas-source isotope-ratio mass spectrometers. A different elemental–analyzer–mass-spectrometer system is used for 13C/12C and 15N/14N than is used for 34S/32S to accommodate differences in reagents, catalysts, and instrument settings.

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

PubMed

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.

Comparative Study of 14C-Labeled Purified Protein Derivative from Various Mycobacteria

PubMed Central

Landi, S.; Held, H. R.; Tseng, M. C.

1970-01-01

Biologically active 14C-labeled purified protein derivative (14C-PPD) has been prepared from the culture filtrates of seven species of mycobacteria, namely Mycobacterium tuberculosis Johnston strain (PPD), M. bovis BCG (PPD-BCG), M. avium (PPD-A), M. kansasii (PPD-Y), M. intracellulare (PPD-B), M. scrofulaceum (PPD-G), and M. fortuitum (PPD-F). These mycobacteria were grown in a culture medium containing a mixture of 14C-labeled amino acids. The yield and specific radioactivity of the PPD, of the nucleic acid, of the bacterial cells, and of the CO2 developed during growth have been determined for each of the seven species of mycobacteria. Although the yields of 14C-PPD antigens differed greatly for the different species of mycobacteria tested, their specific radioactivities were similar. The 14C-PPD antigens have been used as a means to measure their adsorption to glass. When glass ampoules containing dilute solutions (0.001 mg of PPD per ml) of these PPD antigens (PPD, PPD-BCG, PPD-A, PPD-Y, PPD-G, PPD-B, and PPD-F) were stored for 12 months at 5 C, it was found that they all adsorbed equally well to glass surfaces. In fact, regardless of the origin of the PPD, a loss due to adsorption of about 90% occurred during the first month of storage, and thereafter the PPD content remained practically constant for the rest of the duration of the storage period. The addition of 0.0005% Tween 80 to the PPD solutions effectively reduced the adsorption to glass of most PPD antigens. However, adsorption of PPD-BCG was not quite so effectively prevented, even when the Tween 80 concentration was increased from 0.0005 to 0.0005%. Images PMID:5485082

Recent Developments in Carbonylation Chemistry Using [13 C]CO, [11 C]CO and [14 C]CO.

PubMed

Nielsen, Dennis U; Neumann, Karoline T; Lindhardt, Anders T; Skrydstrup, Troels

2018-06-01

Carbon monoxide represents the most important C1-building block for the chemical industry, both for the production of bulk and fine chemicals, but also for synthetic fuels. Yet, its toxicity and subsequently its cautious handling has limited its applications in medicinal chemistry research and in particular for the synthesis of pharmaceutically relevant molecules. Recent years have nevertheless witnessed a considerable headway on the development of carbon monoxide surrogates and reactor systems, which provide an ideal setting for performing carbonylation chemistry with stoichiometric and sub-stoichiometric carbon monoxide. Such set-ups are particularly ideal for the introduction of isotope labels such as carbon-11, carbon-13 and carbon-14 into bioactive compounds. This review summarizes this growing field and examines the large number of carbonylation reactions that can be exploited for the introduction of a carbon isotope. This article is protected by copyright. All rights reserved.

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

PubMed

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

2015-07-01

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

Fate of 14C-labeled dissolved organic matter in paddy and upland soils in responding to moisture.

PubMed

Chen, Xiangbi; Wang, Aihua; Li, Yang; Hu, Lening; Zheng, Hua; He, Xunyang; Ge, Tida; Wu, Jinshui; Kuzyakov, Yakov; Su, Yirong

2014-08-01

Soil organic matter (SOM) content in paddy soils is higher than that in upland soils in tropical and subtropical China. The dissolved organic matter (DOM) concentration, however, is lower in paddy soils. We hypothesize that soil moisture strongly controls the fate of DOM, and thereby leads to differences between the two agricultural soils under contrasting management regimens. A 100-day incubation experiment was conducted to trace the fate and biodegradability of DOM in paddy and upland soils under three moisture levels: 45%, 75%, and 105% of the water holding capacity (WHC). (14)C labeled DOM, extracted from the (14)C labeled rice plant material, was incubated in paddy and upland soils, and the mineralization to (14)CO2 and incorporation into microbial biomass were analyzed. Labile and refractory components of the initial (14)C labeled DOM and their respective half-lives were calculated by a double exponential model. During incubation, the mineralization of the initial (14)C labeled DOM in the paddy soils was more affected by moisture than in the upland soils. The amount of (14)C incorporated into the microbial biomass (2.4-11.0% of the initial DOM-(14)C activity) was less affected by moisture in the paddy soils than in the upland soils. At any of the moisture levels, 1) the mineralization of DOM to (14)CO2 within 100 days was 1.2-2.1-fold higher in the paddy soils (41.9-60.0% of the initial DOM-(14)C activity) than in the upland soils (28.7-35.7%), 2) (14)C activity remaining in solution was significantly lower in the paddy soils than in the upland soils, and 3) (14)C activity remaining in the same agricultural soil solution was not significantly different among the three moisture levels after 20 days. Therefore, moisture strongly controls DOM fate, but moisture was not the key factor in determining the lower DOM in the paddy soils than in the upland soils. The UV absorbance of DOM at 280 nm indicates less aromaticity of DOM from the paddy soils than from the

Tissue distribution and depuration kinetics of waterborne 14C-labeled light PAHs in mummichog (Fundulus heteroclitus).

PubMed

Valdez Domingos, F X; Oliveira Ribeiro, C A; Pelletier, É; Rouleau, C

2011-04-01

Light polycyclic aromatic hydrocarbons (PAHs) of petrogenic origin are commonly found in estuaries and coastal areas. Though they are known to be toxic to fish, little is known about their uptake and tissue distribution. This paper reports on the results of a study on uptake, elimination, and tissue distribution of three waterborne 14C-labeled PAHs in the mummichog, Fundulus heteroclitus, using whole-body autoradiography. After a 24 h exposure to 1 μCi·L(-1) of 14C-naphthalene, 14C-1-naphthol, and 14C-phenanthrene, fish were transferred to clean water and tissue distribution examined after 0, 1, 3, 7, 14, and 21 days of depuration. All compounds were readily accumulated by fish and were also rapidly eliminated (t0.5 range=1.1 to 3.0 days). Most of the radioactivity in naphthalene- and phenanthrene-treated fish was found in gall bladder≫liver>intestinal lumen. In naphthol-exposed fish, an important labeling of some brain areas was observed. Brain of naphthalene-exposed fish was also labeled after 24 h depuration, indicating that exposure to naphthalene may result in metabolite accumulation in the brain. This is the first study showing that naphthalene, naphthol, and/or unidentified metabolite(s) can accumulate in brain tissues, which may impair normal brain function.

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

PubMed

Antoniewicz, Maciek R

2015-12-01

Metabolic pathway models provide the foundation for quantitative studies of cellular physiology through the measurement of intracellular metabolic fluxes. For model organisms metabolic models are well established, with many manually curated genome-scale model reconstructions, gene knockout studies and stable-isotope tracing studies. However, for non-model organisms a similar level of knowledge is often lacking. Compartmentation of cellular metabolism in eukaryotic systems also presents significant challenges for quantitative (13)C-metabolic flux analysis ((13)C-MFA). Recently, innovative (13)C-MFA approaches have been developed based on parallel labeling experiments, the use of multiple isotopic tracers and integrated data analysis, that allow more rigorous validation of pathway models and improved quantification of metabolic fluxes. Applications of these approaches open new research directions in metabolic engineering, biotechnology and medicine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bacterial Production of Site Specific 13C Labeled Phenylalanine and Methodology for High Level Incorporation into Bacterially Expressed Recombinant Proteins

PubMed Central

Ramaraju, Bhargavi; McFeeters, Hana; Vogler, Bernhard; McFeeters, Robert L.

2016-01-01

Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific 13C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct 13C chemical shifts and multiple magnetically equivalent 1H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with 13C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated 1H-13C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study. PMID:28028744

A double-tracer technique to characterize absorption, distribution, metabolism and excretion (ADME) of [14C]-basimglurant and absolute bioavailability after oral administration and concomitant intravenous microdose administration of [13C6]-labeled basimglurant in humans.

PubMed

Guerini, Elena; Schadt, Simone; Greig, Gerard; Haas, Ruth; Husser, Christophe; Zell, Manfred; Funk, Christoph; Hartung, Thomas; Gloge, Andreas; Mallalieu, Navita L

2017-02-01

1. The emerging technique of employing intravenous microdose administration of an isotope tracer concomitantly with an [ 14 C]-labeled oral dose was used to characterize the disposition and absolute bioavailability of a novel metabotropic glutamate 5 (mGlu5) receptor antagonist under clinical development for major depressive disorder (MDD). 2. Six healthy volunteers received a single 1 mg [ 12 C/ 14 C]-basimglurant (2.22 MBq) oral dose and a concomitant i.v. tracer dose of 100 μg of [ 13 C 6 ]-basimglurant. Concentrations of [ 12 C]-basimglurant and the stable isotope [ 13 C 6 ]-basimglurant were determined in plasma by a specific LC/MS-MS method. Total [ 14 C] radioactivity was determined in whole blood, plasma, urine and feces by liquid scintillation counting. Metabolic profiling was conducted in plasma, urine, blood cell pellet and feces samples. 3. The mean absolute bioavailability after oral administration (F) of basimglurant was ∼67% (range 45.7-77.7%). The major route of [ 14 C]-radioactivity excretion, primarily in form of metabolites, was in urine (mean recovery 73.4%), with the remainder excreted in feces (mean recovery 26.5%). The median t max for [ 12 C]-basimglurant after the oral administration was 0.71 h (range 0.58-1.00) and the mean terminal half-life was 77.2 ± 38.5 h. Terminal half-life for the [ 14 C]-basimglurant was 178 h indicating presence of metabolites with a longer terminal half-life. Five metabolites were identified with M1-Glucuronide as major and the others in trace amounts. There was minimal binding of drug to RBCs. IV pharmacokinetics was characterized with a mean ± SD CL of 11.8 ± 7.4 mL/h and a Vss of 677 ± 229 L. 4. The double-tracer technique used in this study allowed to simultaneously characterize the absolute bioavailability and disposition characteristics of the new oral molecular entity in a single study.

Implications of new measurements of O-16 + p + C-12,13, N-14,15 for the abundances of C, N isotopes at the cosmic ray source

NASA Technical Reports Server (NTRS)

Guzik, T. G.; Wefel, J. P.; Crawford, H. J.; Greiner, D. E.; Lindstrom, P. J.; Schimmerling, W.; Symons, T. J. M.

1985-01-01

The fragmentation of a 225 MeV/n O-16 beam was investigated at the Bevalac. Preliminary cross sections for mass = 13, 14, 15 fragments are used to constrain the nuclear excitation functions employed in galactic propagation calculations. Comparison to cosmic ray isotonic data at low energies shows that in the cosmic ray source C-13/C approximately 2% and N-14/0=3-6%. No source abundance of N-15 is required with the current experimental results.

Microbial degradation of [C14C]polystyrene and 1,3-diphenylbutane.

PubMed

Sielicki, M; Focht, D D; Martin, J P

1978-07-01

Microbial degradation of [beta-14C]polystyrene and 1,3-diphenylbutane, a compound structurally representing the smallest repeating unit of styrene (dimer), was investigated in soil and liquid enrichment cultures. Degradation rates in soil, as determined by 14CO2 evolution from applied [14C]polystyrene, varied from 1.5 to 3.0% for a 4-month period. Although relatively low, these percentages were 15 to 30 times greater than values previously reported. Enrichment cultures, containing 1,3-diphenylbutane as the only carbon souce, were used to determine the mechanisms of microbial oxidation of the polymer chain ends. Metabolism of 1,3-diphenylbutane appeared to involve the attack by a monooxygenease to form 2-phenyl-4-hydroxyphenylbutane followed by a further oxidation and subsequent fission of the benzene ring to yield 4-phenylvaleric acid and an unidentified 5-carbon fragment via the classic meta-fission pathway. Phenylacetic acid was probably formed from 4-phenylvaleric acid by subsequent beta-oxidation of the side chain, methyl-oxidation and decarboxylation. An initial examination of the population of microorganisms in the diphenylbutane enrichment cultures indicated that these oxidative reactions are carried out by common soil microorganism of the genera Bacillus, Pseudomonas, Micrococcus, and Nocardia.

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

PubMed Central

2011-01-01

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

7Li(15N, 14C)8Be reaction at 81 MeV and 14C + 8Be interaction versus that of 13C + 8Be

NASA Astrophysics Data System (ADS)

Rudchik, A. T.; Rudchik, A. A.; Muravynets, L. M.; Kemper, K. W.; Rusek, K.; Koshchy, E. I.; Piasecki, E.; Trzcinska, A.; Pirnak, Val. M.; Ponkratenko, O. A.; Strojek, I.; Stolarz, A.; Plujko, V. A.; Sakuta, S. B.; Siudak, R.; Ilyin, A. P.; Stepanenko, Yu. M.; Shyrma, Yu. O.; Uleshchenko, V. V.

2018-03-01

Angular distributions of the 7Li(15N, 14C)8Be reaction were measured at the energy Elab(15N) = 81 MeV. Data for transfer to the ground and first two excited states in 8Be were acquired as well as to the 14C ground and excited states. The reaction data were analyzed within the coupled-reaction-channels (CRC) method. The required 15N + 7Li entrance channel potential was taken from the 15N + 7Li elastic scattering. The 14C + 8Be potential was found by fitting Woods-Saxon form potentials to those generated by double folded real and imaginary potentials in the region of interaction. These generated potentials were then used in the CRC calculations. Proton transfer dominants this reaction, including to the excited states of 8Be. The reaction dependence on the exit channel potential was examined by using the 13C + 8Be potential previously deduced from the 9Be(12C, 13C)8Be reaction and 14C + 8Be from the 13C(9Be, 8Be)14C reaction.

An alternative and robust synthesis of [(13) C4 ]Baraclude® (entecavir).

PubMed

Easter, John A; Burrell, Richard C; Bonacorsi, Samuel J

2013-10-01

Stable isotope-labeled [(13) C4 ]entecavir (1) was prepared in 11 steps. Commercially available [(13) C]guanidine hydrochloride and diethyl[1,2,3-(13) C3 ]malonate were condensed to yield 2-amino[2,4,5,6-(13) C4 ]pyrimidine-4,6-diol (8). This was converted to the desired purine (7) in five steps. Introduction of the chiral epoxide was followed by subsequent deprotection to give [(13) C4 ]entecavir (1), in an overall yield of 5.7% from labeled precursors. The chemical purity of the title compound was determined to be >99% by HPLC. The isotopic distribution was determined by mass spectrometry to be 282[M + 4], 98.4%; 281[M + 3], 1.6%; and 278[M + 0], <0.1%. Copyright © 2013 John Wiley & Sons, Ltd.

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

ERIC Educational Resources Information Center

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

2010-01-01

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

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

PubMed

Warren, J M; Iversen, C M; Garten, C T; Norby, R J; Childs, J; Brice, D; Evans, R M; Gu, L; Thornton, P; Weston, D J

2012-06-01

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

Two Phase 1, Open-Label, Mass Balance Studies to Determine the Pharmacokinetics of 14 C-Labeled Isavuconazonium Sulfate in Healthy Male Volunteers.

PubMed

Townsend, Robert; Kato, Kota; Hale, Christine; Kowalski, Donna; Lademacher, Christopher; Yamazaki, Takao; Akhtar, Shahzad; Desai, Amit

2018-02-01

Isavuconazonium sulfate is the water-soluble prodrug of the active triazole isavuconazole. Two phase 1 studies were conducted to identify the metabolic profile and mass balance of isavuconazole and BAL8728 (inactive cleavage product). Seven subjects in study 1 (isavuconazole mass balance) received a single oral dose of [cyano- 14 C]isavuconazonium sulfate corresponding to 200 mg isavuconazole. Six subjects in study 2 (BAL8728 mass balance) received a single intravenous dose of [pyridinylmethyl- 14 C]isavuconazonium sulfate corresponding to 75 mg BAL8728. Pharmacokinetic parameters of radioactivity in whole blood and plasma and of isavuconazole and BAL8728 in plasma were assessed. Radioactivity ratio of blood/plasma, percentage of dose, and cumulative percentage of radioactive dose recovered in urine and feces for isavuconazole and BAL8728 were assessed. Metabolic profiling was carried out by high-performance liquid chromatography and mass spectrometry. Mean plasma isavuconazole pharmacokinetic parameters included apparent clearance (2.3 ± 0.7 L/h), apparent volume of distribution (301.8 ± 105.7 L), and terminal elimination half-life (99.9 ± 44.6 hours). In study 1, isavuconazole-derived radioactivity was recovered approximately equally in urine and feces (46.1% and 45.5%, respectively). In study 2, BAL8728-derived radioactivity was predominantly recovered in urine (96.0%). Isavuconazole (study 1) and M4 (cleavage metabolite of BAL8728; study 2) were the predominant circulating components of radioactivity in plasma. © 2017 The Authors. Clinical Pharmacology in Drug Development Published by Wiley Periodicals, Inc. on behalf of The American College of Clinical Pharmacology.

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

NASA Astrophysics Data System (ADS)

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.

Dual carbon isotopes (14C and 13C) and optical properties of WSOC and HULIS-C during winter in Guangzhou, China.

PubMed

Liu, Junwen; Mo, Yangzhi; Ding, Ping; Li, Jun; Shen, Chengde; Zhang, Gan

2018-08-15

Water-soluble brown carbon (ws-BrC) exerts an important influence on climate change, but its emission sources and optical properties remain poorly understood. In this study, we isolated two ws-BrC proxies, water-soluble organic carbon (WSOC) and humic-like substance carbon (HULIS-C), from particulate matter collected in Guangzhou, China, during December 2012 for the measurement of dual carbon isotopes ( 14 C and 13 C) and light absorption. The mass absorption efficiencies of WSOC and HULIS-C at 365nm were 0.81±0.16 and 1.33±0.21m 2 g -1 C, respectively. The 14 C results showed that two-thirds of WSOC and HULIS-C were derived from non-fossil sources (e.g., biomass burning and biogenic emission), and the remaining third was derived from fossil sources. The δ 13 C values of WSOC and HULIS-C were -23.7±1.2‰ and -24.2±0.9‰, respectively, underlining the limited influences of C4 plants and natural gas on ws-BrC. Fitting the data to a multiple linear regression, we further concluded that approximately 80% and 10% of the light absorption at 365nm was due to non-fossil and fossil carbon, respectively. Non-fossil sources of ws-BrC, such as the burning of agricultural residue, were responsible for the light absorption recorded in Guangzhou. Copyright © 2018 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

Bore, Ezekiel

2016-04-01

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

TRITIUM-LABELED COMPOUNDS VII. ISOTOPE EFFECTS IN THE OXIDATION OF d- MANNITOLS-C$sup 14$ AND d-MANNITOLS-t TO d-FRUCTOSES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sniegoski, L.T.; Frush, H.L.; Isbell, H.S.

1961-09-01

D-Mannitols, labeled either with carbon-14 at C1, C2, or C3, or with tritium attached to C1, C2, or C3, were prepared. After oxidation by Acetobacter suboxydans, the distribution of radioactivity in each of the resulting labeled D- fructoses was determined. Labeled D-mannitol is unique among the hexitols in that it may be oxidized by A. suboxydans in either the labeled or the unlabeled part of the molecule. Except in the oxidation of D-mannitol-2-t, the competing reactions result in the formation of a mixture of D-fructoses, each having radioactivity in one of two different positions. Hence, the isotope effect, k*/ k,more » (where k* and k are, respectively, the rate constants for oxidation in the labeled and in the unlabeled part of the labeled emannitol molecule) is the ratio of the activities at the two positions of the product, D-fructose. The following isotope effects were found for the bacterial oxidation of labeled D-mannitols: for D-mannitol-2-C/sup 14/, k*/k = 0.93; for Dmannitol-2-t, k*/k = 0.23; and for D-mannitol-3-t, k*/k = 0.70. For D-mannitols labeled at other positions, no isotope effect was detected, since k*/k was unity. The large isotope effect for D-mannitol-2-t is indicative of rupture of the C2-H bond in the rate determining process. It is suggested that the secondary isotope effect for tritium at C3 indicates hyperconjugation of the C3 hydrogen atom in the activated enzyme-- substrate complex; the lack of such effect for tritium at C1 may be due to unfavorable steric conditions for hyperconjugation of the C1 hydrogen atoms in the complex. The following substances were prepared and their isotopic distributions determined: D-fructose1,6-C/sup 14/ and D-fructose-1,6-t (from 1- labeled D-mannitols); D-fructose-2,5-C/sup 14/ and D-fructose-5-t (from 2-labeled e-mannitols); and D-fructose-3,4-C/sup 14/ and D-fructose-3,4-t (from 3-labeled D- mannitols). A procedure, employing D-fructose-1,6-C/sup 14/ as an internal standard, was devised for the

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

DOE PAGES

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

2011-12-30

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

Comparison of Accuracy Between 13C- and 14C-Urea Breath Testing: Is an Indeterminate-Results Category Still Needed?

PubMed

Charest, Mathieu; Bélair, Marc-André

2017-06-01

Helicobacter pylori infection is the leading cause of peptic ulcer disease. The purpose of this study was, first, to assess the difference in the distribution of negative versus positive results between the older 14 C-urea breath test and the newer 13 C-urea breath test and, second, to determine whether use of an indeterminate-results category is still meaningful and what type of results should trigger repeated testing. Methods: A retrospective survey was performed of all consecutive patients referred to our service for urea breath testing. We analyzed 562 patients who had undergone testing with 14 C-urea and 454 patients who had undergone testing with 13 C-urea. Results: In comparison with the wide distribution of negative 14 C results, negative 13 C results were distributed farther from the cutoff and were grouped more tightly around the mean negative value. Distribution analysis of the negative results for 13 C testing, compared with those for 14 C testing, revealed a statistically significant difference between the two. Within the 13 C group, only 1 patient could have been classified as having indeterminate results using the same indeterminate zone as was used for the 14 C group. This is significantly less frequent than what was found for the 14 C group. Discussion: Borderline-negative results do occur with 13 C-urea breath testing, although less frequently than with 14 C-urea breath testing, and we will be carefully monitoring differences falling between 3.0 and 3.5 %Δ. 13 C-urea breath testing is safe and simple for the patient and, in most cases, provides clearer positive or negative results for the clinician. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

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

PubMed

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

2014-01-01

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

Comparison of different mass spectrometry techniques in the measurement of L-[ring-13C6]phenylalanine incorporation into mixed muscle proteins

PubMed Central

Zabielski, Piotr; Ford, G. Charles; Persson, X. Mai; Jaleel, Abdul; Dewey, Jerry D.; Nair, K Sreekumaran

2013-01-01

Precise measurement of low enrichment of stable isotope labeled amino-acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 hour intravenous infusion of L-[ring-13C6]phenylalanine and a bolus dose of L-[ring-13C6]phenylalanine in a mouse were utilized. Liquid Chromatography tandem mass spectrometry (LC/MS/MS), Gas Chromatography tandem mass spectrometry (GC/MS/MS) and Gas Chromatography/Mass spectrometry (GC/MS) were compared to the Gas Chromatography-Combustion-Isotope Ratio mass spectrometry (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring13C6]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 Molar Percent excess (MPE). As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R2 = 0.9962 and R2 = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra-assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter-assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS respectively. The muscle sample sizes required to obtain these results were 8μg, 0.8μg, 3μg and 3μg for GC/C/IRMS, LC/MS/MS, GC/MS/MS, and GC/MS respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L-[ring-13C6]phenylalanine tracer enrichment in low abundance and in small quantity samples. PMID:23378099

Effects of muffin processing on fumonisins from 14C-labeled toxins produced in cultured corn kernels.

PubMed

Avantaggiato, Giuseppina; De La Campa, Regina; Miller, J David; Visconti, Angelo

2003-10-01

The persistence of fumonisins during cooking is known to be affected by several factors, including thermal degradation and the presence of various ingredients in corn-based food recipes that can react with the toxin. A method for the production of corn kernels containing 14C-fumonisins was developed. The corn kernels were colonized by Fusarium verticillioides MRC 826 and supplemented with 1,2-14C-sodium acetate. The specific activity of 14C-FB1 produced made the study of its fate in cornmeal muffins possible. The double-extraction acetonitrile-water-methanol/immunoaffinity column/o-phthaldialdehyde high-performance liquid chromatography (HPLC) method was used to determine FB1 levels in cornmeal muffins. Reductions in FB1 levels in muffins spiked with 14C-labeled and unlabeled FB1 (43 and 48%, respectively) were similar, indicating that the extraction method was efficient and consistent with previous reports. However, with the labeled corn kernel material, recovery levels based on the 14C counts for the eluate from an immunoaffinity column were much higher (90%). This finding indicates that some fumonisin-related compounds other than FB1 that were present in the cornmeal were recognized by the antibodies but not by the HPLC method.

Relative quantitation of glycosylation variants by stable isotope labeling of enzymatically released N-glycans using [12C]/[13C] aniline and ZIC-HILIC-ESI-TOF-MS.

PubMed

Giménez, Estela; Sanz-Nebot, Victòria; Rizzi, Andreas

2013-09-01

Glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline was used for relative quantitation of N-glycans. In a first step, the labeling method by reductive amination was optimized for this reagent. It could be demonstrated that selecting aniline as limiting reactant and using the reductant in excess is critical for achieving high derivatization yields (over 95 %) and good reproducibility (relative standard deviations ∼1-5 % for major and ∼5-10 % for minor N-glycans). In a second step, zwitterionic-hydrophilic interaction liquid chromatography in capillary columns coupled to electrospray mass spectrometry with time-of-flight analyzer (μZIC-HILIC-ESI-TOF-MS) was applied for the analysis of labeled N-glycans released from intact glycoproteins. Ovalbumin, bovine α1-acid-glycoprotein and bovine fetuin were used as test glycoproteins to establish and evaluate the methodology. Excellent separation of isomeric N-glycans and reproducible quantitation via the extracted ion chromatograms indicate a great potential of the proposed methodology for glycoproteomic analysis and for reliable relative quantitation of glycosylation variants in biological samples.

[Ozone effects on soil microbial community of rice investigated by 13C isotope labeling].

PubMed

Chen, Zhan; Wang, Xiao-Ke; Shang, He

2014-10-01

This study was initiated to explore the effects of dynamic ozone (O3) exposure on soil microbial biomass and phospholipid fatty acids (PLFAs) under potted rice. A pulse-chase labeling experiment was designed to expose potted rice with 13CO2 for 6 h after one and two months, the rice were fumigated by elevated O3 concentration with an 8 h mean of 110 nL · L(-1) (O3). The allocation of the assimilated 13C to soil microorganisms was estimated by analyzing the 13C profile of microbial phospholipid fatty acids (PLFAs). After one month O3 exposure, the soil microbial biomass carbon was not affected, while the 13C-microbial biomass was significantly decreased with elevated O3. Both the total and 13C microbial biomass carbon was remarkably lower than that of control treatment after two months O3 exposure. Principal components analysis of 13C-PLFA data showed that elevated O3 significantly changed soil microbial structure after two month exposures, while there was no difference of 13C-PLFA structure between control and elevated O3 treatments after one month exposure. Δδ13C per hundred thousand of individual PLFA was significantly affected by O3 after both one and two month exposures. Only did ozone change the relative abundance of individual 13C-PLFA (13C%) of bacterial fatty acids after one month exposure, while after two month exposures, the 13C% of fungal and actinomycetic fatty acids were markedly changed by elevated O3.

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

PubMed

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

2014-11-01

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

[13C] GC-C-IRMS analysis of methylboronic acid derivatives of glucose from liver glycogen after the ingestion of [13C] labeled tracers in rats.

PubMed

Luengo, Catherine; Azzout-Marniche, Dalila; Fromentin, Claire; Piedcoq, Julien; Lemosquet, Sophie; Tomé, Daniel; Gaudichon, Claire

2009-11-01

We developed a complete method to measure low [(13)C] enrichments in glycogen. Fourteen rats were fed a control diet. Six of them also ingested either [U-(13)C] glucose (n=2) or a mixture of 20 [U-(13)C] amino acids (n=4). Hepatic glycogen was extracted, digested to glucose and purified on anion-cation exchange resins. After the optimization of methylboronic acid derivatization using GC-MS, [(13)C] enrichment of extracted glucose was measured by GC-C-IRMS. The accuracy was addressed by measuring the enrichment excess of a calibration curve, which observed values were in good agreement with the expected values (R=0.9979). Corrected delta values were -15.6+/-1.6 delta(13)C (per thousand) for control rats (n=8) and increased to -5 to 8 delta(13)C (per thousand) per thousand and 12-14 delta(13)C (per thousand) per thousand after the ingestion of [U-(13)C] amino acids or [U-(13)C] glucose as oral tracers, respectively. The method enabled the determination of dietary substrate transfer into glycogen. The sequestration of dietary glucose in liver glycogen 4 h after the meal was 35% of the ingested dose whereas the transfer of carbon skeletons from amino acids was only 0.25 to 1%.

Production of isotopically labeled standards from a uniformly labeled precursor for quantitative volatile metabolomic studies.

PubMed

Gómez-Cortés, Pilar; Brenna, J Thomas; Sacks, Gavin L

2012-06-19

Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-α-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve

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

NASA Astrophysics Data System (ADS)

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

2008-12-01

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

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

NASA Technical Reports Server (NTRS)

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

2003-01-01

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

Buscopan labeled with carbon-14 and deuterium.

PubMed

Latli, Bachir; Stiasni, Michael; Hrapchak, Matt; Li, Zhibin; Grinberg, Nelu; Lee, Heewon; Busacca, Carl A; Senanayake, Chris H

2016-11-01

Hyosine butyl bromide, the active ingredient in Buscopan, is an anticholinergic and antimuscarinic drug used to treat pain and discomfort caused by abdominal cramps. A straightforward synthesis of carbon-14- and deuterium-labeled Buscopan was developed using scopolamine, n-butyl-1- 14 C bromide, and n-butyl- 2 H 9 bromide, respectively. In a second carbon-14 synthesis, the radioactive carbon was incorporated in the tropic acid moiety to follow its metabolism. Herein, we describe the detailed preparations of carbon-14- and deuterium-labeled Buscopan. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolite pools and carbon flow during C4 photosynthesis in maize: 13CO2 labeling kinetics and cell type fractionation.

PubMed

Arrivault, Stéphanie; Obata, Toshihiro; Szecówka, Marek; Mengin, Virginie; Guenther, Manuela; Hoehne, Melanie; Fernie, Alisdair R; Stitt, Mark

2017-01-01

Worldwide efforts to engineer C 4 photosynthesis into C 3 crops require a deep understanding of how this complex pathway operates. CO 2 is incorporated into four-carbon metabolites in the mesophyll, which move to the bundle sheath where they are decarboxylated to concentrate CO 2 around RuBisCO. We performed dynamic 13 CO 2 labeling in maize to analyze C flow in C 4 photosynthesis. The overall labeling kinetics reflected the topology of C 4 photosynthesis. Analyses of cell-specific labeling patterns after fractionation to enrich bundle sheath and mesophyll cells revealed concentration gradients to drive intercellular diffusion of malate, but not pyruvate, in the major CO 2 -concentrating shuttle. They also revealed intercellular concentration gradients of aspartate, alanine, and phosphenolpyruvate to drive a second phosphoenolpyruvate carboxykinase (PEPCK)-type shuttle, which carries 10-14% of the carbon into the bundle sheath. Gradients also exist to drive intercellular exchange of 3-phosphoglycerate and triose-phosphate. There is rapid carbon exchange between the Calvin-Benson cycle and the CO 2 -concentrating shuttle, equivalent to ~10% of carbon gain. In contrast, very little C leaks from the large pools of metabolites in the C concentration shuttle into respiratory metabolism. We postulate that the presence of multiple shuttles, alongside carbon transfer between them and the Calvin-Benson cycle, confers great flexibility in C 4 photosynthesis. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Fate of 14C-terpolymer (methylmethacrylate-14C, 2-hydroxyethylmethacrylate, butylacrylate) nanoparticles after peroral administration to rats.

PubMed

Kukan, M; Bezek, S; Koprda, V; Labský, J; Kálal, J; Bauerová, K; Trnovec, T

1989-05-01

The fate of 14C-terpolymer (methylmethacrylate-14C, 2-hydroxyethylmethacrylate, butylacrylate) nanoparticles was studied in male Wistar rats after peroral administration. These nanoparticles may reach systemic circulation as evidenced by the plasma 14C level, excretion of the label in the urine, as well as organ label deposition. It was found that at least 2% of the dose of 14C was absorbed from the gastrointestinal tract. As expected, the radioactive nanoparticles were excreted predominantly via the feces. The amount of the label in the gastrointestinal tract, liver, and carcasses fell below the limit of detection on day seven after administration. However in the spleen and lung some slight radioactivity persisted after 7 d of experiment.

Measuring transfer of 14C-PCB from maternal diet to milk in a goat model using an accelerator mass spectrometer (AMS)

NASA Astrophysics Data System (ADS)

Janle, E.; Sojka, J.; Jackson, G. S.; Lachcik, P.; Einstien, J. A.; Santerre, C. R.

2007-06-01

Environmental pollutants pose a substantial risk to nursing infants. Many of these toxicants (i.e. PCBs, PBDEs, mercury) are passed from the maternal diet to the nursing infant in breast milk. Determining the toxicokinetics has been difficult to measure due to ethical limitations. Since extremely small amounts of 14C can be measured using Accelerator Mass Spectrometry (AMS), a goat model was used to establish a minimum oral dose of 14C-labeled PCB (2,2‧,4,4‧,5,5‧-hexachlorobiphenyl-UL-14C) that could be given to a lactating animal and traced into the milk. An oral dose of 66 nCi/kg body weight (1.84 μg PCB/kg bw) was administered. Plasma and milk samples were collected for 2 months after dosing. The concentration of 14C label reached a peak value of 1.71 ng/ml PCB equivalents in the milk on day 2 and then declined to about 135 pg/ml PCB equivalents in the milk at 3 weeks. A second goat was administered a smaller dose (22 nCi/kg bw; 616 ng PCB/kg bw). A peak concentration of 485 pg PCB equivalents/ml milk occurred at 3 days and declined to 77.6 pg PCB equivalents/ml milk by 3 weeks. Our results indicated that an even lower dosage of labeled-PCB could be used due to the extreme sensitivity of AMS measurement. Extrapolating from current data it is estimated that the dose could be reduced by a factor of 20 (31 ng PCB/kg bw; 1.1 nCi/kg bw) and still be detectable after 2 months. Thus, the potential exists for developing protocols for studying toxicokinetics in humans using radiologically- and toxicologically-benign doses of labeled environmental toxicants.

Differentiation of the Stereochemistry and Anomeric Configuration for 1-3 Linked Disaccharides Via Tandem Mass Spectrometry and 18O-labeling

NASA Astrophysics Data System (ADS)

Konda, Chiharu; Bendiak, Brad; Xia, Yu

2012-02-01

Collision-induced dissociation (CID) of deprotonated hexose-containing disaccharides ( m/z 341) with 1-2, 1-4, and 1-6 linkages yields product ions at m/z 221, which have been identified as glycosyl-glycolaldehyde anions. From disaccharides with these linkages, CID of m/z 221 ions produces distinct fragmentation patterns that enable the stereochemistries and anomeric configurations of the non-reducing sugar units to be determined. However, only trace quantities of m/z 221 ions can be generated for 1-3 linkages in Paul or linear ion traps, preventing further CID analysis. Here we demonstrate that high intensities of m/z 221 ions can be built up in the linear ion trap (Q3) from beam-type CID of a series of 1-3 linked disaccharides conducted on a triple quadrupole/linear ion trap mass spectrometer. 18O-labeling at the carbonyl position of the reducing sugar allowed mass-discrimination of the "sidedness" of dissociation events to either side of the glycosidic linkage. Under relatively low energy beam-type CID and ion trap CID, an m/z 223 product ion containing 18O predominated. It was a structural isomer that fragmented quite differently than the glycosyl-glycolaldehydes and did not provide structural information about the non-reducing sugar. Under higher collision energy beam-type CID conditions, the formation of m/z 221 ions, which have the glycosyl-glycolaldehyde structures, were favored. Characteristic fragmentation patterns were observed for each m/z 221 ion from higher energy beam-type CID of 1-3 linked disaccharides and the stereochemistry of the non-reducing sugar, together with the anomeric configuration, were successfully identified both with and without 18O-labeling of the reducing sugar carbonyl group.

Distribution and biomarkers of carbon-14-labeled fullerene C60 ([(14) C(U)]C60 ) in female rats and mice for up to 30 days after intravenous exposure.

PubMed

Sumner, Susan C J; Snyder, Rodney W; Wingard, Christopher; Mortensen, Ninell P; Holland, Nathan A; Shannahan, Jonathan H; Dhungana, Suraj; Pathmasiri, Wimal; Han, Li; Lewin, Anita H; Fennell, Timothy R

2015-12-01

A comprehensive distribution study was conducted in female rats and mice exposed to a suspension of uniformly carbon-14-labeled C60 ([(14) C(U)]C60 ). Rodents were administered [(14) C(U)]C60 (~0.9 mg kg(-1) body weight) or 5% polyvinylpyrrolidone-saline vehicle alone via a single tail vein injection. Tissues were collected at 1 h and 1, 7, 14 and 30 days after administration. A separate group of rodents received five daily injections of suspensions of either [(14) C(U)]C60 or vehicle with tissue collection 14 days post exposure. Radioactivity was detected in over 20 tissues at all time points. The highest concentration of radioactivity in rodents at each time point was in liver, lungs and spleen. Elimination of [(14) C(U)]C60 was < 2% in urine and feces at any 24 h time points. [(14) C(U)]C60 and [(14) C(U)]C60 -retinol were detected in liver of rats and together accounted for ~99% and ~56% of the total recovered at 1 and 30 days postexposure, respectively. The blood radioactivity at 1 h after [(14) C(U)]C60 exposure was fourfold higher in rats than in mice; blood radioactivity was still in circulation at 30 days post [(14) C(U)]C60 exposure in both species (<1%). Levels of oxidative stress markers increased by 5 days after exposure and remained elevated, while levels of inflammation markers initially increased and then returned to control values. The level of cardiovascular marker von Willebrand factor, increased in rats, but remained at control levels in mice. This study demonstrates that [(14) C(U)]C60 is retained in female rodents with little elimination by 30 days after i.v. exposure, and leads to systemic oxidative stress. Copyright © 2015 John Wiley & Sons, Ltd.

Disposition of the 14C-labeled phosphorothioate oligonucleotide ISIS 2105 after intravenous administration to rats.

PubMed

Cossum, P A; Sasmor, H; Dellinger, D; Truong, L; Cummins, L; Owens, S R; Markham, P M; Shea, J P; Crooke, S

1993-12-01

5'-TTGCTTCCATCTTCCTCGTC-3' (ISIS 2105) is a phosphorothioate oligodeoxynucleotide currently being evaluated as an intralesional antiviral drug for the treatment of genital warts that are caused by the human papillomavirus. ISIS 2105, labeled with 14C (at the carbon-2 position of thymine) was administered as a single i.v. injection (3.6 mg/kg) to female Sprague-Dawley rats to assess the disposition of the drug. After i.v. administration of [14C]2105, blood radioactivity disappeared in a multiexponential manner with the half-lives of the phases equal to 0.4, 1.9, 7.1 and 5.1 hr. The initial volume of distribution was 22 ml and the postdistribution volume of distribution was 1076 ml, which indicated an extensive distribution of radioactivity. The apparent blood clearance was 14.7 ml/hr. The radioactivity in the expired air accounted for 51% of the administered dose over the 10-day period. Urinary and fecal radioactivity accounted for 15% and 5% of the administered dose, respectively. The major sites of radioactivity uptake were the liver (up to 22.6% of the dose), kidneys (renal cortex, up to 14% of the dose), bone marrow (up to 14% of the dose), skin (up to 13% of the dose) and skeletal muscle (up to 9% of the dose). Other tissues contained approximately 1% or less of the dose. The overall recovery of radioactivity 10 days postdosing was 95.1 +/- 7.5% (mean +/- S.D.) of the administered single dose. The radioactivity in the blood was almost completely in the plasma during the course of the study. In the plasma, the radioactivity was extensively bound to proteins, as assessed by size-exclusion high-performance liquid chromatography (HPLC), in samples up to 8 hr postdosing. Retention data on size-exclusion HPLC and in vitro incubations using purified proteins suggested that the plasma proteins that bound [14C]2105 were albumin and alpha 2-macroglobulin. The complex formed between the plasma proteins and [14C]2105-derived radioactivity was dissociated on anion

Analysis of 14C-bearing compounds released by the corrosion of irradiated steel using accelerator mass spectrometry.

PubMed

Cvetković, B Z; Salazar, G; Kunz, D; Szidat, S; Wieland, E

2018-06-25

The combination of ion chromatography (IC) with accelerator mass spectrometry (AMS) was developed to determine the speciation of 14C-(radiocarbon) bearing organic compounds in the femto to pico molar concentration range. The development of this compound-specific radiocarbon analysis (CSRA) of carboxylic acids is reported and the application of the method on a leaching solution from neutron-irradiated steel is demonstrated. The background and the dynamic range of the AMS-based method were quantified. On using 14C-labelled standards, the measurements demonstrate the repeatability of the analytical method and the reproducible recovery of the main target carboxylic acids (i.e., acetate, formate, malonate, and oxalate). The detection limit was determined to be in the mid fmol 14C per L level while the dynamic range of the analytical method covers three orders of magnitude from the low fmol to the mid pmol 14C per L level. Cross contamination was found to be negligible during IC fractionation and was accounted for during eluate processing and 14C detection by AMS. The 14C-bearing carboxylates released from an irradiated steel nut into an alkaline leaching solution were analysed using the CSRA-based analytical method with the aim to check the applicability of the approach and develop appropriate sample preparation. The concentrations of 14C-bearing formate and acetate, the main organic corrosion products, were at a low pmol 14C per L level for convenient dimensions of the alkaline leaching experiment which demonstrates that compound-specific 14C AMS is an extremely sensitive analytical method for analysing 14C-bearing compounds. The content of total organic 14C in solution (TO14C) determined by the direct measurement of an aliquot of the leaching solution agrees well with the sum of the 14C concentrations of the individual carboxylates within the uncertainty of the data. Furthermore, the TO14C content is in good agreement with the calculated value using the corrosion rate

A rapid phospholipase A2 bioassay using 14C-oleate-labelled E. coli bacterias.

PubMed

Meyer, T; von Wichert, P; Weins, D

1989-02-01

Two methods of phospholipase A2 determination using 14C-labelled E. coli bacterias as substrate were compared. One method works with a filter membrane for separation of cleaved 14C-oleate from remaining phospholipids, the other uses the well-known thin-layer chromatography for lipid analysis. Some features of human serum phospholipase A2 regarding pH and Ca2+ dependency were investigated. Possible sources of errors were discussed. It was shown that either method can differentiate between normal and pathologically elevated phospholipase A2 levels, but that the filter method is superior in terms of sensitivity and workload.

Evidence for Biomass Burning from 14C and 13C/12C Measurements at T-0 and T-1 during MILAGRO.

NASA Astrophysics Data System (ADS)

Gaffney, J. S.; Marley, N. A.; Tackett, M. J.; Sturchio, N. C.; Heraty, L. J.; Martinez, N.; Hardy, K.; Guilderson, T.

2007-12-01

Both stable carbon isotopic and radiocarbon characterizations of aerosols can yield important information regarding the sources of carbonaceous aerosols in urban and regional environments. Biomass derived materials are labeled due to their recent photochemical activity in radiocarbon and vary depending upon the photochemical pathway (either C-4 or C-3) in stable carbon-13 content. C-4 being enriched over C-3. During the MILAGRO campaign, quartz filter samples were taken at 12 hour intervals from 5 am to 5 pm (day) and from 5 pm to 5 am (night) during the month of March 2006. These samples were taken at the two super-sites, T-0 (Instituto Mexicano de Petroleo in Mexico City) and T-1 (Universidad Technologica de Tecamac, State of Mexico). The total carbon content was analyzed for stable carbon isotopic composition as well as for radiocarbon. Stable isotope mass spectroscopy was used to determine the carbon-13 to carbon-12 isotopic ratios on carbon dioxide. The carbon dioxide was then converted to graphite for analysis by accelerator mass spectrometry at the Center for Accelerator Mass Spectrometry at Lawrence Livermore National Laboratory. Results are presented for the carbon-13 content relative to the PDB standard and radiocarbon is given relative to recent carbon. The results for total radiocarbon content show that the carbonaceous aerosol content in Mexico City has more than half of the carbon coming from biomass derived sources. These can include inflow of biomass burning aerosols into the T-0 site as well as the input from local burning of biofuels and trash containing biomass derived materials (paper, boxes, etc.). Data also indicate that at the T-1 site biomass burning of C-4 grasses appears to be significant in that the carbon-13 values observed are enriched. Also at T-1 the radiocarbon levels are also found to be slightly higher indicating regional biomass burning as a significant contributor to aerosol carbon in the 0.1 to 1.0 micron size fraction. Some day

PiTS-1: Carbon Partitioning in Loblolly Pine after 13C Labeling and Shade Treatments

DOE Data Explorer

Warren, J. M.; Iversen, C. M.; Garten, Jr., C. T.; Norby, R. J.; Childs, J.; Brice, D.; Evans, R. M.; Gu, L.; Thornton, P.; Weston, D. J.

2013-01-01

The PiTS task was established with the objective of improving the C partitioning routines in existing ecosystem models by exploring mechanistic model representations of partitioning tested against field observations. We used short-term field manipulations of C flow, through 13CO2 labeling, canopy shading and stem girdling, to dramatically alter C partitioning, and resultant data are being used to test model representation of C partitioning processes in the Community Land Model (CLM4 or CLM4.5).

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

PubMed

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

2016-12-01

This research is an attempt to study seasonal translocation patterns of photoassimilated carbon within trees of one of the high latitudes widespread deciduous conifer species Larix gmelinii (Rupr. Rupr). For this purpose, we applied whole-tree labeling by 13 CO 2 , which is a powerful and effective tool for tracing newly developed assimilates translocation to tissues and organs of a tree. Experimental plot has been established in a mature 105-year-old larch stand located within the continuous permafrost area near Tura settlement (Central Siberia, 64°17'13″N, 100°11'55″E, 148 m a.s.l.). Measurements of seasonal photosynthetic activity and foliage parameters (i.e., leaf length, area, biomass, etc.), and sampling were arranged from early growing season (June 8, 2013; May 14, 2014) until yellowing and senescence of needles (September 17, 2013; September 14, 2014). Labeling by 13 C of the tree branch (June 2013, for 3 branch replicates in 3 different trees) and the whole tree was conducted at early (June 2014), middle (July 2014), and late (August 2013) phase of growing season (for different trees in 3 replicates each time) by three pulses [(CO 2 )max = 3000-4000 ppmv, 13 CO 2 (30 % v/v)]. We found at least two different patterns of carbon translocation associated with larch CO 2 assimilation depending on needle phenology. In early period of growing season (June), 13 C appearing in newly developed needles is a result of remobilized storage material use for growth purposes. Then approximately at the end of June, growth processes is switching to storage processes lasting to the end of growing season.

Imaging of pH in vivo using hyperpolarized 13C-labelled zymonic acid

PubMed Central

Düwel, Stephan; Hundshammer, Christian; Gersch, Malte; Feuerecker, Benedikt; Steiger, Katja; Buck, Achim; Walch, Axel; Haase, Axel; Glaser, Steffen J.; Schwaiger, Markus; Schilling, Franz

2017-01-01

Natural pH regulatory mechanisms can be overruled during several pathologies such as cancer, inflammation and ischaemia, leading to local pH changes in the human body. Here we demonstrate that 13C-labelled zymonic acid (ZA) can be used as hyperpolarized magnetic resonance pH imaging sensor. ZA is synthesized from [1-13C]pyruvic acid and its 13C resonance frequencies shift up to 3.0 p.p.m. per pH unit in the physiological pH range. The long lifetime of the hyperpolarized signal enhancement enables monitoring of pH, independent of concentration, temperature, ionic strength and protein concentration. We show in vivo pH maps within rat kidneys and subcutaneously inoculated tumours derived from a mammary adenocarcinoma cell line and characterize ZA as non-toxic compound predominantly present in the extracellular space. We suggest that ZA represents a reliable and non-invasive extracellular imaging sensor to localize and quantify pH, with the potential to improve understanding, diagnosis and therapy of diseases characterized by aberrant acid-base balance. PMID:28492229

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loreto, F.; Ciccioli, P.; Cecinato, A.

1996-04-01

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

Earth's magnetic field enabled scalar coupling relaxation of 13C nuclei bound to fast-relaxing quadrupolar 14N in amide groups.

PubMed

Chiavazza, Enrico; Kubala, Eugen; Gringeri, Concetta V; Düwel, Stephan; Durst, Markus; Schulte, Rolf F; Menzel, Marion I

2013-02-01

Scalar coupling relaxation, which is usually only associated with closely resonant nuclei (e.g., (79)Br-(13)C), can be a very effective relaxation mechanism. While working on hyperpolarized [5-(13)C]glutamine, fast liquid-state polarization decay during transfer to the MRI scanner was observed. This behavior could hypothetically be explained by substantial T(1) shortening due to a scalar coupling contribution (type II) to the relaxation caused by the fast-relaxing quadrupolar (14)N adjacent to the (13)C nucleus in the amide group. This contribution is only effective in low magnetic fields (i.e., less than 800 μT) and prevents the use of molecules bearing the (13)C-amide group as hyperpolarized MRS/MRI probes. In the present work, this hypothesis is explored both theoretically and experimentally. The results show that high hyperpolarization levels can be retained using either a (15)N-labeled amide or by applying a magnetic field during transfer of the sample from the polarizer to the MRI scanner. Copyright © 2012 Elsevier Inc. All rights reserved.

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

DOEpatents

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

2004-03-30

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

Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent.

PubMed

Kim, Eun-Mi; Joung, Min-Hee; Lee, Chang-Moon; Jeong, Hwan-Jeong; Lim, Seok Tae; Sohn, Myung-Hee; Kim, Dong Wook

2010-07-15

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the 'click-to-chelate' protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12, [(99m)Tc(CO)(3)]13, and [(99m)Tc(CO)(3)]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)(3)]12, [Re(CO)(3)]13, and [Re(CO)(3)]14, were shown to have high binding affinities (0.13 microM, 0.06 microM, and 0.16 microM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12-14) to c-Met receptor positive U87MG cells. 2010 Elsevier Ltd. All rights reserved.

Localization of 14C-labeled 2% lidocaine hydrochloride after intraosseous anesthesia in the rabbit.

PubMed

Goto, Takashi; Mamiya, Hideki; Ichinohe, Tatsuya; Kaneko, Yuzuru

2011-10-01

The purpose of this study was to investigate the tissue distribution of lidocaine hydrochloride in mandibular bone marrow after intraosseous anesthesia (IOA) in rabbits. We used macroautoradiography to examine the tissue distribution of a (14)C-labeled 2% lidocaine hydrochloride solution containing 1:80,000 epinephrine ((14)C-lidocaine). Under general anesthesia, (14)C-lidocaine was injected intraosseously or paraperiosteally. After IOA, animals were divided into three groups and observed at 1 (IOA-1), 5 (IOA-5), and 10 minutes (IOA-10) after injection. After infiltration anesthesia (IA), animals were observed at 1 minute after injection. The accumulation of (14)C-lidocaine was observed around the injection site in both the IA and the IOA groups. Paraperiosteally injected (14)C-lidocaine diffused to the surrounding tissues such as the lip, whereas IOA showed concentrated accumulation around the root apex throughout the experiment. The distribution area was significantly smaller in the IOA-1 group than in the IA group. The distribution area in the IOA-5 group was larger than those in the IOA-1 and IOA-10 groups. The accumulation of (14)C-lidocaine injected by IOA in rabbits was concentrated around the root apex. These results may explain the rapid onset time of IOA. Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Analytical Validation of Accelerator Mass Spectrometry for Pharmaceutical Development: the Measurement of Carbon-14 Isotope Ratio.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keck, B D; Ognibene, T; Vogel, J S

2010-02-05

Accelerator mass spectrometry (AMS) is an isotope based measurement technology that utilizes carbon-14 labeled compounds in the pharmaceutical development process to measure compounds at very low concentrations, empowers microdosing as an investigational tool, and extends the utility of {sup 14}C labeled compounds to dramatically lower levels. It is a form of isotope ratio mass spectrometry that can provide either measurements of total compound equivalents or, when coupled to separation technology such as chromatography, quantitation of specific compounds. The properties of AMS as a measurement technique are investigated here, and the parameters of method validation are shown. AMS, independent of anymore » separation technique to which it may be coupled, is shown to be accurate, linear, precise, and robust. As the sensitivity and universality of AMS is constantly being explored and expanded, this work underpins many areas of pharmaceutical development including drug metabolism as well as absorption, distribution and excretion of pharmaceutical compounds as a fundamental step in drug development. The validation parameters for pharmaceutical analyses were examined for the accelerator mass spectrometry measurement of {sup 14}C/C ratio, independent of chemical separation procedures. The isotope ratio measurement was specific (owing to the {sup 14}C label), stable across samples storage conditions for at least one year, linear over 4 orders of magnitude with an analytical range from one tenth Modern to at least 2000 Modern (instrument specific). Further, accuracy was excellent between 1 and 3 percent while precision expressed as coefficient of variation is between 1 and 6% determined primarily by radiocarbon content and the time spent analyzing a sample. Sensitivity, expressed as LOD and LLOQ was 1 and 10 attomoles of carbon-14 (which can be expressed as compound equivalents) and for a typical small molecule labeled at 10% incorporated with {sup 14}C corresponds to 30

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

NASA Astrophysics Data System (ADS)

Azurmendi, Hugo F.; Freedberg, Darón I.

2013-03-01

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

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

PubMed

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

2014-03-15

As fecal excretion is one of important routes of elimination of drugs and their metabolites, it is indispensable to investigate the metabolites in feces for more comprehensive information on biotransformation in vivo. In this study, a sensitive and reliable approach based on ultra-performance liquid chromatography/quadrupole-time-of-flight-mass spectrometry (UHPLC-Q-TOF-MS) was applied to characterize the metabolic profile of honokiol in rat feces after the administration of an equimolar mixture of honokiol and [(13)C6]-labeled honokiol. Totally 42 metabolites were discovered and tentatively identified in rat feces samples, 26 metabolites were first reported, including two novel classes of metabolites, methylated and dimeric metabolites of honokiol. Moreover, this study provided basic comparative data on the metabolites in rat plasma, feces and urine, which gave better understanding of the metabolic fate of honokiol in vivo. Copyright © 2014 Elsevier B.V. All rights reserved.

Distribution and biomarker of carbon-14 labeled fullerene C60 ([(14) C(U)]C60 ) in pregnant and lactating rats and their offspring after maternal intravenous exposure.

PubMed

Snyder, Rodney W; Fennell, Timothy R; Wingard, Christopher J; Mortensen, Ninell P; Holland, Nathan A; Shannahan, Jonathan H; Pathmasiri, Wimal; Lewin, Anita H; Sumner, Susan C J

2015-12-01

A comprehensive distribution study was conducted in pregnant and lactating rats exposed to a suspension of uniformly carbon-14 labeled C60 ([(14) C(U)]C60 ). Rats were administered [(14) C(U)]C60 (~0.2 mg [(14) C(U)]C60 kg(-1) body weight) or 5% polyvinylpyrrolidone (PVP)-saline vehicle via a single tail vein injection. Pregnant rats were injected on gestation day (GD) 11 (terminated with fetuses after either 24 h or 8 days), GD15 (terminated after 24 h or 4 days), or GD18 (terminated after 24 h). Lactating rats were injected on postnatal day 8 and terminated after 24 h, 3 or 11 days. The distribution of radioactivity in pregnant dams was influenced by both the state of pregnancy and time of termination after exposure. The percentage of recovered radioactivity in pregnant and lactating rats was highest in the liver and lungs. Radioactivity was quantitated in over 20 tissues. Radioactivity was found in the placenta and in fetuses of pregnant dams, and in the milk of lactating rats and in pups. Elimination of radioactivity was < 2% in urine and feces at each time point. Radioactivity remained in blood circulation up to 11 days after [(14) C(U)]C60 exposure. Biomarkers of inflammation, cardiovascular injury and oxidative stress were measured to study the biological impacts of [(14) C(U)]C60 exposure. Oxidative stress was elevated in female pups of exposed dams. Metabolomics analysis of urine showed that [(14) C(U)]C60 exposure to pregnant rats impacted the pathways of vitamin B, regulation of lipid and sugar metabolism and aminoacyl-tRNA biosynthesis. This study demonstrated that [(14) C(U)]C60 crosses the placenta at all stages of pregnancy examined, and is transferred to pups via milk. Copyright © 2015 John Wiley & Sons, Ltd.

[2,4-(13)C]β-hydroxybutyrate metabolism in astrocytes and C6 glioblastoma cells.

PubMed

Eloqayli, Haytham; Melø, Torun M; Haukvik, Anne; Sonnewald, Ursula

2011-08-01

This study was undertaken to determine if the ketogenic diet could be useful for glioblastoma patients. The hypothesis tested was whether glioblastoma cells can metabolize ketone bodies. Cerebellar astrocytes and C6 glioblastoma cells were incubated in glutamine and serum free medium containing [2,4-(13)C]β-hydroxybutyrate (BHB) with and without glucose. Furthermore, C6 cells were incubated with [1-(13)C]glucose in the presence and absence of BHB. Cell extracts were analyzed by mass spectrometry and media by (1)H magnetic resonance spectroscopy and HPLC. Using [2,4-(13)C]BHB and [1-(13)C]glucose it could be shown that C6 cells, in analogy to astrocytes, had efficient mitochondrial activity, evidenced by (13)C labeling of glutamate, glutamine and aspartate. However, in the presence of glucose, astrocytes were able to produce and release glutamine, whereas this was not accomplished by the C6 cells, suggesting lack of anaplerosis in the latter. We hypothesize that glioblastoma cells kill neurons by not supplying the necessary glutamine, and by releasing glutamate.

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

NASA Astrophysics Data System (ADS)

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

2012-04-01

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

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

NASA Astrophysics Data System (ADS)

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

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

Preparation of [13C3]-melamine and [13C3]-cyanuric acid and their application to the analysis of melamine and cyanuric acid in meat and pet food using liquid chromatography-tandem mass spectrometry.

PubMed

Varelis, P; Jeskelis, R

2008-10-01

For the determination of melamine and cyanuric acid the labelled internal standards [(13)C(3)]-melamine and [(13)C(3)]-cyanuric acid were synthesized using the common substrate [(13)C(3)]-cyanuric chloride by reaction with ammonia and acidified water, respectively. Standards with excellent isotopic and chemical purities were obtained in acceptable yields. These compounds were used to develop an isotope dilution liquid chromatography/mass spectrometry (LC/MS) method to determine melamine and cyanuric acid in catfish, pork, chicken, and pet food. The method involved extraction into aqueous methanol, liquid-liquid extraction and ion exchange solid phase clean-up, with normal phase high-performance liquid chromatography (HPLC) in the so-called hydrophilic interaction mode. The method had a limit of detection (LOD) of 10 microg kg(-1) for both melamine and cyanuric acid in the four foods with a percentage coefficient of variation (CV) of less than 10%. The recovery of the method at this level was in the range of 87-110% and 96-110% for melamine and cyanuric acid, respectively.

Intracavity optogalvanic spectroscopy. An analytical technique for 14C analysis with subattomole sensitivity.

PubMed

Murnick, Daniel E; Dogru, Ozgur; Ilkmen, Erhan

2008-07-01

We show a new ultrasensitive laser-based analytical technique, intracavity optogalvanic spectroscopy, allowing extremely high sensitivity for detection of (14)C-labeled carbon dioxide. Capable of replacing large accelerator mass spectrometers, the technique quantifies attomoles of (14)C in submicrogram samples. Based on the specificity of narrow laser resonances coupled with the sensitivity provided by standing waves in an optical cavity and detection via impedance variations, limits of detection near 10(-15) (14)C/(12)C ratios are obtained. Using a 15-W (14)CO2 laser, a linear calibration with samples from 10(-15) to >1.5 x 10(-12) in (14)C/(12)C ratios, as determined by accelerator mass spectrometry, is demonstrated. Possible applications include microdosing studies in drug development, individualized subtherapeutic tests of drug metabolism, carbon dating and real time monitoring of atmospheric radiocarbon. The method can also be applied to detection of other trace entities.

Large Variations of Atmospheric 14C Associated With Dansgaard-Oeschger Cycles 10- 13

NASA Astrophysics Data System (ADS)

Weyhenmeyer, C. E.; Burns, S. J.; Fleitmann, D.; Mangini, A.; Matter, A.; Guilderson, T.; Reimer, P. J.

2006-12-01

A 1.7 m long stalagmite from Moomi Cave, Socotra Island in the Indian Ocean provides a continuous, high- resolution record of climate change between 53 and 41 kyr BP. In the northern high-latitude regions, this time period is characterized by several rapid climate change events, corresponding to Dansgaard-Oeschger (D/O) cycles 10-13. It has been suggested that these D/O cycles may be global events but high-resolution data from the low-latitude regions are scarce. As a result, the driving and feedback mechanisms of these rapid changes remain poorly understood. The presented stalagmite data of U/Th, stable isotopes (del 18O, del 13C) and radiocarbon (14C) provide unique information regarding the nature and timing of rapid climate changes in the tropics. A depth-age model for the Moomi Cave stalagmite was developed from 25 high-precision U/Th measurements, providing a solid chronology for this record. Oxygen isotope measurements of the stalagmite calcite reveal several large variations that are believed to reflect changes in the amount of precipitation, rather than temperature. A comparison to the Greenland Ice Core records shows a remarkable similarity to D/O cycles 10- 13 with warmer periods in the high-latitude regions being associated with increased precipitation in the tropics and vice versa. The stalagmite radiocarbon (14C) values from over 100 individual measurements reveal an almost identical cyclic pattern, tracing all four D/O cycles. Assuming no changes in the carbonate chemistry of the precipitating fluid, the radiocarbon values of the stalagmite calcite directly reflect changes in global atmospheric 14C concentrations. There are three possible explanations for these cyclic variations of 14C values: 1) changes in the carbonate chemistry of the drip water resulting in changes of the dead carbon fraction (DCF); 2) changes in the solar activity and/or Earth's magnetic field resulting in direct variations of atmospheric 14C concentrations; and 3) changes in

Integration of parallel 13 C-labeling experiments and in silico pathway analysis for enhanced production of ascomycin.

PubMed

Qi, Haishan; Lv, Mengmeng; Song, Kejing; Wen, Jianping

2017-05-01

Herein, the hyper-producing strain for ascomycin was engineered based on 13 C-labeling experiments and elementary flux modes analysis (EFMA). First, the metabolism of non-model organism Streptomyces hygroscopicus var. ascomyceticus SA68 was investigated and an updated network model was reconstructed using 13 C- metabolic flux analysis. Based on the precise model, EFMA was further employed to predict genetic targets for higher ascomycin production. Chorismatase (FkbO) and pyruvate carboxylase (Pyc) were predicted as the promising overexpression and deletion targets, respectively. The corresponding mutant TD-FkbO and TD-ΔPyc exhibited the consistency effects between model prediction and experimental results. Finally, the combined genetic manipulations were performed, achieving a high-yield ascomycin engineering strain TD-ΔPyc-FkbO with production up to 610 mg/L, 84.8% improvement compared with the parent strain SA68. These results manifested that the integration of 13 C-labeling experiments and in silico pathway analysis could serve as a promising concept to enhance ascomycin production, as well as other valuable products. Biotechnol. Bioeng. 2017;114: 1036-1044. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Synthesis of carbon-14 and stable isotope labeled Avagacestat: a novel gamma secretase inhibitor for the treatment of Alzheimer's disease.

PubMed

Burrell, Richard C; Easter, John A; Cassidy, Michael P; Gillman, Kevin W; Olson, Richard E; Bonacorsi, Samuel J

2014-08-01

Bristol-Myers Squibb and others are developing drugs that target novel mechanisms to combat Alzheimer's disease. γ-Secretase inhibitors are one class of potential therapies that have received considerable attention. (R)-2-(4-Chloro-N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)phenylsulfonamido)-5,5,5-trifluoropentanamide (Avagacestat) is a γ-secretase-inhibiting drug that has been investigated by Bristol-Myers Squibb in preclinical and clinical studies. An important step in the development process was the synthesis of a carbon-14-labeled analog for use in a human absorption, distribution, metabolism, and excretion study and a stable isotope labeled analog for use as a standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. Carbon-14 labeled Avagacestat was synthesized in seven steps in a 33% overall yield from carbon-14 labeled potassium cyanide. A total of 5.95 mCi was prepared with a specific activity of 0.81 μCi/mg and a radiochemical purity of 99.9%. (13) C6 -Labeled Avagacestat was synthesized in three steps in a 15% overall yield from 4-chloro[(13) C6 ]aniline. A total of 585 mg was prepared with a ultraviolet purity of 99.9%. Copyright © 2014 John Wiley & Sons, Ltd.

14C and delta13C characteristics of organic matter and carbonate in saltmarsh sediments from south west Scotland.

PubMed

MacKenzie, A B; Cook, G T; Barth, J; Gulliver, P; McDonald, P

2004-05-01

The distribution of contaminant radionuclides from the Sellafield nuclear fuel reprocessing plant was used to establish chronologies for three saltmarsh sediment cores from south west Scotland. delta(13)C and (14)C analyses indicated that the cores provided a useful archive record of variations in input of organic matter and carbonate. The results imply that prior to major releases of contaminant (14)C from Sellafield, the (14)C specific activity of organic matter in Irish Sea offshore sediments was about 24 Bq kg(-1) C, while that of the carbonate component was below the limit of detection. These results provide baseline data for modelling the uptake of contaminant (14)C by the Irish Sea sediment system. The study confirmed that small(13)C analyses provide a sensitive means of apportioning the origin of saltmarsh organic matter between C(3) terrigenous plants, C(4) terrigenous plants and suspended particulate marine organic matter. For the <2 mm fraction of sediment, a clear pattern of decreasing marine organic input was observed in response to increasing elevation of the marsh surface as a result of sediment accumulation. Bulk sediment, including detrital vegetation, had a dominant input from terrigenous plants. The combined use of delta(13)C and (14)C data revealed that organic matter in the marine organic component of the <2 mm fraction of contemporary surface sediments of the saltmarshes is dominated by recycled old organic material.

Chlorophyll a specific Δ14C, δ13C and δ15N values in stream periphyton: implications for aquatic food web studies

NASA Astrophysics Data System (ADS)

Ishikawa, N. F.; Yamane, M.; Suga, H.; Ogawa, N. O.; Yokoyama, Y.; Ohkouchi, N.

2015-07-01

We determined the isotopic composition of chlorophyll a in periphytic algae attached to a streambed substrate (periphyton). The samples were collected from a stream flowing on limestone bedrock in the Seri River, central Japan. Stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) and natural radiocarbon abundances (Δ14C) were measured in chlorophyll a (δ13Cchl, δ15Nchl and Δ14Cchl) and bulk (δ13Cbulk, δ15Nbulk and Δ14Cbulk) for periphyton, pure aquatic primary producer (Cladophora sp.) and terrestrial primary producer (Quercus glauca). Periphyton δ13Cbulk and δ13Cchl values did not necessarily correspond to δ13Cbulk for an algal-grazing specialist (Mayfly larva, Epeorus latifolium), suggesting that periphyton δ13C values do not faithfully trace carbon transfer between primary producers and primary consumers. Periphyton Δ14Cchl values (-258 ‰ in April and -190 ‰ in October) were slightly lower than Δ14Cbulk values (-228 ‰ in April and -179 ‰ in October), but were close to the Δ14C value for dissolved inorganic carbon (DIC) (-217 ± 31 ‰), which is a mixture of weathered carbonates (Δ14C = -1000 ‰) and dissolved atmospheric CO2 (Δ14C approximately +30 ‰ in 2013). Δ14Cchl values were also close to Δ14Cbulk for E. latifolium (-215 ‰ in April and -199 ‰ in October) and Cladophora sp. (-210 ‰), whereas the Δ14Cbulk value for Q. glauca (+27 ‰) was closer to Δ14C for atmospheric CO2. Although the bulk isotopic composition of periphyton is recognised as a surrogate for the photosynthetic algal community, natural periphyton is a mixture of aquatic and terrestrial organic materials. Our results indicate that the bulk periphyton matrix at the study site consists of 89 to 95 % algal carbon (derived from 14C-depleted DIC) and 5 to 11 % terrestrial organic carbon (derived from 14C-enriched atmospheric CO2).

Natural isotope correction of MS/MS measurements for metabolomics and (13)C fluxomics.

PubMed

Niedenführ, Sebastian; ten Pierick, Angela; van Dam, Patricia T N; Suarez-Mendez, Camilo A; Nöh, Katharina; Wahl, S Aljoscha

2016-05-01

Fluxomics and metabolomics are crucial tools for metabolic engineering and biomedical analysis to determine the in vivo cellular state. Especially, the application of (13)C isotopes allows comprehensive insights into the functional operation of cellular metabolism. Compared to single MS, tandem mass spectrometry (MS/MS) provides more detailed and accurate measurements of the metabolite enrichment patterns (tandem mass isotopomers), increasing the accuracy of metabolite concentration measurements and metabolic flux estimation. MS-type data from isotope labeling experiments is biased by naturally occurring stable isotopes (C, H, N, O, etc.). In particular, GC-MS(/MS) requires derivatization for the usually non-volatile intracellular metabolites introducing additional natural isotopes leading to measurements that do not directly represent the carbon labeling distribution. To make full use of LC- and GC-MS/MS mass isotopomer measurements, the influence of natural isotopes has to be eliminated (corrected). Our correction approach is analyzed for the two most common applications; (13)C fluxomics and isotope dilution mass spectrometry (IDMS) based metabolomics. Natural isotopes can have an impact on the calculated flux distribution which strongly depends on the substrate labeling and the actual flux distribution. Second, we show that in IDMS based metabolomics natural isotopes lead to underestimated concentrations that can and should be corrected with a nonlinear calibration. Our simulations indicate that the correction for natural abundance in isotope based fluxomics and quantitative metabolomics is essential for correct data interpretation. © 2015 Wiley Periodicals, Inc.

Synthesis of 2H- and 13C-substituted dithanes

DOEpatents

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

2003-01-01

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

Synthesis Of 2h- And 13c-Substituted Dithanes

DOEpatents

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

2004-05-04

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

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

NASA Astrophysics Data System (ADS)

Gunina, Anna; Kuzyakov, Yakov; Glaser, Bruno

2015-04-01

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

Coral skeletal carbon isotopes (δ13C and Δ14C) record the delivery of terrestrial carbon to the coastal waters of Puerto Rico

USGS Publications Warehouse

Moyer, R.P.; Grottoli, A.G.

2011-01-01

Tropical small mountainous rivers deliver a poorly quantified, but potentially significant, amount of carbon to the world's oceans. However, few historical records of land-ocean carbon transfer exist for any region on Earth. Corals have the potential to provide such records, because they draw on dissolved inorganic carbon (DIC) for calcification. In temperate systems, the stable- (δ13C) and radiocarbon (Δ14C) isotopes of coastal DIC are influenced by the δ13C and Δ14C of the DIC transported from adjacent rivers. A similar pattern should exist in tropical coastal DIC and hence coral skeletons. Here, δ13C and Δ14C measurements were made in a 56-year-old Montastraea faveolata coral growing ~1 km from the mouth of the Rio Fajardo in eastern Puerto Rico. Additionally, the δ13C and Δ14C values of the DIC of the Rio Fajardo and its adjacent coastal waters were measured during two wet and dry seasons. Three major findings were observed: (1) synchronous depletions of both δ13C and Δ14C in the coral skeleton are annually coherent with the timing of peak river discharge, (2) riverine DIC was always more depleted in δ13C and Δ14C than seawater DIC, and (3) the correlation of δ13C and Δ14C was the same in both coral skeleton and the DIC of the river and coastal waters. These results indicate that coral skeletal δ13C and Δ14C are recording the delivery of riverine DIC to the coastal ocean. Thus, coral records could be used to develop proxies of historical land-ocean carbon flux for many tropical regions. Such information could be invaluable for understanding the role of tropical land-ocean carbon flux in the context of land-use change and global climate change.

Coral skeletal carbon isotopes (δ13C and Δ14C) record the delivery of terrestrial carbon to the coastal waters of Puerto Rico

USGS Publications Warehouse

Moyer, R.P.; Grottoli, A.G.

2011-01-01

Tropical small mountainous rivers deliver a poorly quantified, but potentially significant, amount of carbon to the world's oceans. However, few historical records of land-ocean carbon transfer exist for any region on Earth. Corals have the potential to provide such records, because they draw on dissolved inorganic carbon (DIC) for calcification. In temperate systems, the stable- (??13C) and radiocarbon (??14C) isotopes of coastal DIC are influenced by the ??13C and ??14C of the DIC transported from adjacent rivers. A similar pattern should exist in tropical coastal DIC and hence coral skeletons. Here, ??13C and ??14C measurements were made in a 56-year-old Montastraea faveolata coral growing ~1 km from the mouth of the Rio Fajardo in eastern Puerto Rico. Additionally, the ??13C and ??14C values of the DIC of the Rio Fajardo and its adjacent coastal waters were measured during two wet and dry seasons. Three major findings were observed: (1) synchronous depletions of both ??13C and ??14C in the coral skeleton are annually coherent with the timing of peak river discharge, (2) riverine DIC was always more depleted in ??13C and ??14C than seawater DIC, and (3) the correlation of ??13C and ??14C was the same in both coral skeleton and the DIC of the river and coastal waters. These results indicate that coral skeletal ??13C and ??14C are recording the delivery of riverine DIC to the coastal ocean. Thus, coral records could be used to develop proxies of historical land-ocean carbon flux for many tropical regions. Such information could be invaluable for understanding the role of tropical land-ocean carbon flux in the context of land-use change and global climate change. ?? 2011 United States Geological Survey.

Source apportionment of carbonaceous aerosol in Sao Paulo using 13C and 14C measurements

NASA Astrophysics Data System (ADS)

Oyama, Beatriz; Andrade, Maria de Fatima; Holzinger, Rupert; Röckmann, Thomas; Meijer, Harro A. J.; Dusek, Ulrike

2016-04-01

The Metropolitan Area of Sao Paulo is affected by high aerosol concentrations, which contain a large fraction of organic material. Up to date, not much is known about the composition and origin of the organic aerosol in this city. We present the first source apportionment of the carbonaceous aerosol fraction in Sao Paulo, using stable (13C) and radioactive carbon isotopes (14C). 14C provides a clear-cut distinction between fossil sources, which contain no 14C, and contemporary sources such as biofuels, biomass burning, or biogenic sources, which contain a typical contemporary 14C/12C ratio. 13C can be used to distinguish C3 plants, such as maize and sugarcane, from C4 plants. This can help to identify a possible impact of sugarcane field burning in the rural areas of Sao Paulo State on the aerosol carbon in the city. In the first part of the study, we compare two tunnel studies: Tunnel 1 is frequented only by light duty vehicles, which run mainly on mixtures of gasoline with ethanol (gasohol, 25% ethanol and 85% gasoline) or hydrated ethanol (5% water and 95% ethanol). Tunnel 2 contains a significant fraction of heavy-duty diesel vehicles, and therefore the fraction of biofuels in the average fleet is lower. Comparison of 14C in organic and elemental carbon (OC and EC) shows that in both tunnels there is no significant contribution of biofuels to EC. Combusting ethanol-gasoline fuels in a vehicle engine does apparently not result in significant EC formation from ethanol. Biofuels contribute around 45% to OC in Tunnel 1 an only 20% in Tunnel 2, reflecting a strong impact of diesel vehicles in Tunnel 2. In the second part of the study we conduct a source apportionment of ambient aerosol carbon collected in a field study during winter (July-August) 2012. Ambient EC has two main sources, vehicular emissions and biomass burning. We estimate a contribution of vehicular sources to EC of roughly 90% during weekdays and 80% during weekends, using the 14C values measured in

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

PubMed

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

2015-02-01

The number of approaches to evaluate the biodegradation of polycyclic aromatic hydrocarbons (PAHs) within contaminated aquifers is limited. Here, we demonstrate the applicability of a novel method based on the combination of in situ and laboratory microcosms using (13)C-labelled PAHs as tracer compounds. The biodegradation of four PAHs (naphthalene, fluorene, phenanthrene, and acenaphthene) was investigated in an oxic aquifer at the site of a former gas plant. In situ biodegradation of naphthalene and fluorene was demonstrated using in situ microcosms (BACTRAP(®)s). BACTRAP(®)s amended with either [(13)C6]-naphthalene or [(13)C5/(13)C6]-fluorene (50:50) were incubated for a period of over two months in two groundwater wells located at the contaminant source and plume fringe, respectively. Amino acids extracted from BACTRAP(®)-grown cells showed significant (13)C-enrichments with (13)C-fractions of up to 30.4% for naphthalene and 3.8% for fluorene, thus providing evidence for the in situ biodegradation and assimilation of those PAHs at the field site. To quantify the mineralisation of PAHs, laboratory microcosms were set up with BACTRAP(®)-grown cells and groundwater. Naphthalene, fluorene, phenanthrene, or acenaphthene were added as (13)C-labelled substrates. (13)C-enrichment of the produced CO2 revealed mineralisation of between 5.9% and 19.7% for fluorene, between 11.1% and 35.1% for acenaphthene, between 14.2% and 33.1% for phenanthrene, and up to 37.0% for naphthalene over a period of 62 days. Observed PAH mineralisation rates ranged between 17 μg L(-1) d(-1) and 1639 μg L(-1) d(-1). The novel approach combining in situ and laboratory microcosms allowed a comprehensive evaluation of PAH biodegradation at the investigated field site, revealing the method's potential for the assessment of PAH degradation within contaminated aquifers. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2015-04-01

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

Chlorophyll a-specific Δ14C, δ13C and δ15N values in stream periphyton: implications for aquatic food web studies

NASA Astrophysics Data System (ADS)

Ishikawa, N. F.; Yamane, M.; Suga, H.; Ogawa, N. O.; Yokoyama, Y.; Ohkouchi, N.

2015-11-01

Periphytic algae attached to a streambed substrate (periphyton) are an important primary producer in stream ecosystems. We determined the isotopic composition of chlorophyll a in periphyton collected from a stream flowing on limestone bedrock in the Seri River, central Japan. Stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) and natural radiocarbon abundances (Δ14C) were measured in chlorophyll a (δ13Cchl, δ15Nchl and Δ14Cchl) and bulk (δ13Cbulk, δ15Nbulk and Δ14Cbulk) for periphyton, a pure aquatic primary producer (Cladophora sp.) and a terrestrial primary producer (Quercus glauca). Periphyton δ13Cbulk and δ13Cchl values did not necessarily correspond to δ13Cbulk for an algal-grazing specialist (Epeorus latifolium). Periphyton Δ14Cchl values (-258 ‰ in April and -190 ‰ in October) were slightly lower than Δ14Cbulk values (-228 ‰ in April and -179 ‰ in October) but were close to the Δ14C value for dissolved inorganic carbon (DIC; -217 ± 31 ‰), which is a mixture of weathered carbonates (Δ14C = -1000 ‰), CO2 derived from aquatic and terrestrial organic matters (variable Δ14C) and dissolved atmospheric CO2 (Δ14C approximately +30 ‰ in 2013). Δ14Cchl values were also close to Δ14Cbulk for E. latifolium (-215 ‰ in April and -199 ‰ in October) and Cladophora sp. (-210 ‰), whereas the Δ14Cbulk value for Q. glauca (+27 ‰) was closer to Δ14C for atmospheric CO2. Although the bulk isotopic composition of periphyton is recognised as a surrogate for the photosynthetic algal community, natural periphyton is a mixture of aquatic and terrestrial organic materials. Our results indicate that the bulk periphyton matrix at the study site consists of 89 to 95 % algal carbon (derived from 14C-depleted DIC) and 5 to 11 % terrestrial organic carbon (derived from 14C-enriched atmospheric CO2).

Palladium-catalyzed double carbonylation using near stoichiometric carbon monoxide: expedient access to substituted 13C2-labeled phenethylamines.

PubMed

Nielsen, Dennis U; Neumann, Karoline; Taaning, Rolf H; Lindhardt, Anders T; Modvig, Amalie; Skrydstrup, Troels

2012-07-20

A novel and general approach for (13)C(2)- and (2)H-labeled phenethylamine derivatives has been developed, based on a highly convergent single-step assembly of the carbon skeleton. The efficient incorporation of two carbon-13 isotopes into phenethylamines was accomplished using a palladium-catalyzed double carbonylation of aryl iodides with near stoichiometric carbon monoxide.

The curved 14C vs. δ13C relationship in dissolved inorganic carbon: A useful tool for groundwater age- and geochemical interpretations

USGS Publications Warehouse

Han, Liang-Feng; Plummer, Niel; Aggarwal, Pradeep

2014-01-01

Determination of the 14C content of dissolved inorganic carbon (DIC) is useful for dating of groundwater. However, in addition to radioactive decay, the 14C content in DIC (14CDIC) can be affected by many geochemical and physical processes and numerous models have been proposed to refine radiocarbon ages of DIC in groundwater systems. Changes in the δ13C content of DIC (δ13CDIC) often can be used to deduce the processes that affect the carbon isotopic composition of DIC and the 14C value during the chemical evolution of groundwater. This paper shows that a curved relationship of 14CDIC vs. δ13CDIC will be observed for groundwater systems if (1) the change in δ13C value in DIC is caused by a first-order or pseudo-first-order process, e.g. isotopic exchange between DIC and solid carbonate, (2) the reaction/process progresses with the ageing of the groundwater, i.e. with decay of 14C in DIC, and (3) the magnitude of the rate of change in δ13C of DIC is comparable with that of 14C decay. In this paper, we use a lumped parameter method to derive a model based on the curved relationship between 14CDICand δ13CDIC. The derived model, if used for isotopic exchange between DIC and solid carbonate, is identical to that derived by Gonfiantini and Zuppi (2003). The curved relationship of 14CDIC vs. δ13CDIC can be applied to interpret the age of the DIC in groundwater. Results of age calculations using the method discussed in this paper are compared with those obtained by using other methods that calculate the age of DIC based on adjusted initial radiocarbon values for individual samples. This paper shows that in addition to groundwater age interpretation, the lumped parameter method presented here also provides a useful tool for geochemical interpretations, e.g. estimation of apparent rates of geochemical reactions and revealing the complexity of the geochemical environment.

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

NASA Astrophysics Data System (ADS)

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

2006-12-01

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

The impact of carbon-13 and deuterium on relative quantification of proteins using stable isotope diethyl labeling.

PubMed

Koehler, Christian J; Arntzen, Magnus Ø; Thiede, Bernd

2015-05-15

Stable isotopic labeling techniques are useful for quantitative proteomics. A cost-effective and convenient method for diethylation by reductive amination was established. The impact using either carbon-13 or deuterium on quantification accuracy and precision was investigated using diethylation. We established an effective approach for stable isotope labeling by diethylation of amino groups of peptides. The approach was validated using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nanospray liquid chromatography/electrospray ionization (nanoLC/ESI)-ion trap/orbitrap for mass spectrometric analysis as well as MaxQuant for quantitative data analysis. Reaction conditions with low reagent costs, high yields and minor side reactions were established for diethylation. Furthermore, we showed that diethylation can be applied to up to sixplex labeling. For duplex experiments, we compared diethylation in the analysis of the proteome of HeLa cells using acetaldehyde-(13) C(2)/(12) C(2) and acetaldehyde-(2) H(4)/(1) H(4). Equal numbers of proteins could be identified and quantified; however, (13) C(4)/(12) C(4) -diethylation revealed a lower variance of quantitative peptide ratios within proteins resulting in a higher precision of quantified proteins and less falsely regulated proteins. The results were compared with dimethylation showing minor effects because of the lower number of deuteriums. The described approach for diethylation of primary amines is a cost-effective and accurate method for up to sixplex relative quantification of proteomes. (13) C(4)/(12) C(4) -diethylation enables duplex quantification based on chemical labeling without using deuterium which reduces identification of false-negatives and increases the quality of the quantification results. Copyright © 2015 John Wiley & Sons, Ltd.

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

PubMed Central

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

2014-01-01

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

Absorption, Distribution, Metabolism, and Excretion of [14C]-Volixibat in Healthy Men: Phase 1 Open-Label Study.

PubMed

Siebers, Nicholas; Palmer, Melissa; Silberg, Debra G; Jennings, Lee; Bliss, Caleb; Martin, Patrick T

2018-02-01

Volixibat is a potent inhibitor of the apical sodium-dependent bile acid transporter in development for the treatment of nonalcoholic steatohepatitis. This phase 1, open-label study investigated the absorption, distribution, metabolism, and excretion of [ 14 C]-volixibat in heathy men. Eligible men (n = 8) aged 18-50 years (body mass index 18.0-30.0 kg/m 2 ; weight >50 kg) received a single oral dose of [ 14 C]-volixibat 50 mg containing ~5.95 µCi radioactivity. The primary objectives were to assess the pharmacokinetics of [ 14 C]-volixibat and to determine the total radioactivity in whole blood, plasma, urine, and feces at pre-selected time points over 6 days. The secondary objectives were to characterize metabolites and to assess the safety and tolerability. Low concentrations of volixibat (range 0-0.179 ng/mL) were detected in plasma up to 8 h following administration; the pharmacokinetic parameters could not be calculated. No radioactivity was observed in plasma or whole blood. The percentage (mean ± standard deviation) of total radioactivity in urine was 0.01 ± 0.007%. The vast majority (92.3 ± 5.25%) of volixibat was recovered in feces (69.2 ± 33.1% within 24 h). Unchanged volixibat was the only radioactive component detected in feces. Adverse events were mild in severity and mostly gastrointestinal. Changes in laboratory values were not clinically meaningful. Following oral administration, [ 14 C]-volixibat was excreted unchanged from the parent compound almost exclusively via fecal excretion, indicating that the drug is minimally absorbed. Consistent with other studies, adverse events were primarily gastrointestinal in nature. ClinicalTrials.gov identifier NCT02571192.

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

NASA Astrophysics Data System (ADS)

Xu, Su; Shen, Jun

2006-10-01

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

Bio-Carbon Accounting for Bio-Oil Co-Processing: 14C and 13C/ 12C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mora, Claudia I.; Li, Zhenghua; Vance, Zachary

This is a powerpoint presentation on bio-carbon accounting for bio-oil co-processing. Because of the overlapping range in the stable C isotope compositions of fossil oils and biooils from C3-type feedstocks, it is widely thought that stable isotopes are not useful to track renewable carbon during co-production. In contrast, our study demonstrates the utility of stable isotopes to: • capture a record of renewable carbon allocation between FCC products of co-processing • record changes in carbon apportionments due to changes in reactor or feed temperature Stable isotope trends as a function of percent bio-oil in the feed are more pronounced whenmore » the δ 13C of the bio-oil endmember differs greatly from the VGO (i.e., it has a C4 biomass source–corn stover, switch grass, Miscanthus, sugarcane– versus a C3 biomass source– pine, wheat, rice, potato), but trends on the latter case are significant for endmember differences of just a few permil. The correlation between measured 14C and δ 13C may be useful as an alternative to carbon accounting, but the relationship must first be established for different bio-oil sources.« less

Incorporation of sup 14 C from ( sup 14 C)phenylalanine into condensed tannin of sorghum grain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reddy, V.; Butler, L.G.

A procedure is described for obtaining condensed tannin from sorghum (Sorghum bicolor (L.) Moench) seeds metabolically labeled from ({sup 14}C)phenylalanine. The ({sup 14}C)tannin should be useful in determining the metabolic fate of dietary condensed tannin.

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

NASA Astrophysics Data System (ADS)

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

2016-06-01

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

Accelerator mass spectrometry analysis of 14C-oxaliplatin concentrations in biological samples and 14C contents in biological samples and antineoplastic agents

NASA Astrophysics Data System (ADS)

Toyoguchi, Teiko; Kobayashi, Takeshi; Konno, Noboru; Shiraishi, Tadashi; Kato, Kazuhiro; Tokanai, Fuyuki

2015-10-01

Accelerator mass spectrometry (AMS) is expected to play an important role in microdose trials. In this study, we measured the 14C concentration in 14C-oxaliplatin-spiked serum, urine and supernatant of fecal homogenate samples in our Yamagata University (YU) - AMS system. The calibration curves of 14C concentration in serum, urine and supernatant of fecal homogenate were linear (the correlation coefficients were ⩾0.9893), and the precision and accuracy was within the acceptance criteria. To examine a 14C content of water in three vacuum blood collection tubes and a syringe were measured. 14C was not detected from water in these devices. The mean 14C content in urine samples of 6 healthy Japanese volunteers was 0.144 dpm/mL, and the intra-day fluctuation of 14C content in urine from a volunteer was little. The antineoplastic agents are administered to the patients in combination. Then, 14C contents of the antineoplastic agents were quantitated. 14C contents were different among 10 antineoplastic agents; 14C contents of paclitaxel injection and docetaxel hydrate injection were higher than those of the other injections. These results indicate that our quantitation method using YU-AMS system is suited for microdosing studies and that measurement of baseline and co-administered drugs might be necessary for the studies in low concentrations.

High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cocuron, Jean-Christophe; Tsogtbaatar, Enkhtuul; Alonso, Ana P.

Accurate assessment of mass isotopomer distributions (MIDs) of intracellular metabolites, such as free amino acids (AAs), is crucial for quantifying in vivo fluxes. To date, the majority of studies that measured AA MIDs have relied on the analysis of proteinogenic rather than free AAs by: i) GC–MS, which involved cumbersome process of derivatization, or ii) NMR, which requires large quantities of biological sample. In this work, the development and validation of a high-throughput LC–MS/MS method allowing the quantification of the levels and labeling of free AAs is described. Sensitivity in the order of the femtomol was achieved using multiple reactionmore » monitoring mode (MRM). The MIDs of all free AAs were assessed without the need of derivatization, and were validated (except for Trp) on a mixture of unlabeled AA standards. Finally, this method was applied to the determination of the 13C-labeling abundance in free AAs extracted from maize embryos cultured with 13C-glutamine or 13C-glucose. Although Cys was below the limit of detection in these biological samples, the MIDs of a total of 18 free AAs were successfully determined. Due to the increased application of tandem mass spectrometry for 13C-Metabolic Flux Analysis, this novel method will enable the assessment of more complete and accurate labeling information of intracellular AAs, and therefore a better definition of the fluxes.« less

High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry

DOE PAGES

Cocuron, Jean-Christophe; Tsogtbaatar, Enkhtuul; Alonso, Ana P.

2017-02-16

Accurate assessment of mass isotopomer distributions (MIDs) of intracellular metabolites, such as free amino acids (AAs), is crucial for quantifying in vivo fluxes. To date, the majority of studies that measured AA MIDs have relied on the analysis of proteinogenic rather than free AAs by: i) GC–MS, which involved cumbersome process of derivatization, or ii) NMR, which requires large quantities of biological sample. In this work, the development and validation of a high-throughput LC–MS/MS method allowing the quantification of the levels and labeling of free AAs is described. Sensitivity in the order of the femtomol was achieved using multiple reactionmore » monitoring mode (MRM). The MIDs of all free AAs were assessed without the need of derivatization, and were validated (except for Trp) on a mixture of unlabeled AA standards. Finally, this method was applied to the determination of the 13C-labeling abundance in free AAs extracted from maize embryos cultured with 13C-glutamine or 13C-glucose. Although Cys was below the limit of detection in these biological samples, the MIDs of a total of 18 free AAs were successfully determined. Due to the increased application of tandem mass spectrometry for 13C-Metabolic Flux Analysis, this novel method will enable the assessment of more complete and accurate labeling information of intracellular AAs, and therefore a better definition of the fluxes.« less

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

DOEpatents

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

2012-06-12

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

Positron emitting nuclides and their synthetic incorporation in radiopharmaceuticals. [Labeled with /sup 11/C, /sup 13/N, and /sup 18/F

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowler, J.S.

/sup 11/C, /sup 13/N, and /sup 15/O has potential applicability to the study of metabolism in humans. Problems in the synthesis of radiopharmaceuticals labeled with /sup 11/C, /sup 13/N, and /sup 18/F are described: quality control, radiation exposure, carboxylic acids, glucose, amines, amino acids, nitrosources, fluoroethanol. 54 references. (DLC)

13C-13C rotational resonance in a transmembrane peptide: A comparison of the fluid and gel phases

NASA Astrophysics Data System (ADS)

Langlais, Denis B.; Hodges, Robert S.; Davis, James H.

1999-05-01

A comparative study of two doubly 13C labeled amphiphilic transmembrane peptides was undertaken to determine the potential of rotational resonance for measuring internuclear distances through the direct dipolar coupling in the presence of motion. The two peptides, having the sequence acetyl-K2-G-L16-K2-A-amide, differed only in the position of 13C labels. The first peptide, [1-13C]leu11:[α-13C]leu12, had labels on adjacent residues, at the carbonyl of leu11 and the α carbon of leu12. The second, [1-13C]leu8:[α-13\\|C]leu11, was labeled on consecutive turns of the α-helical peptide. The internuclear distance between labeled positions of the first peptide, which for an ideal α helix has a value of 2.48 Å, is relatively independent of internal flexibility or peptide conformational change. The dipolar coupling between these two nuclei is sensitive to motional averaging by molecular reorientation, however, making this peptide ideal for investigating these motions. The internuclear distance between labels on the second peptide has an expected static ideal α-helix value of 4.6 Å, but this is sensitive to internal flexibility. In addition, the dipolar coupling between these two nuclei is much weaker because of their larger separation, making this peptide a much more difficult test of the rotational resonance technique. The dipolar couplings between the labeled nuclei of these two peptides were measured by rotational resonance in the dry peptide powders and in multilamellar dispersions with dimyristoylphosphatidylcholine in the gel phase, at -10 °C, and in the fluid phase, at 40 °C. The results for the peptide having adjacent labels can be readily interpreted in terms of a simple model for the peptide motion. The results for the second peptide show that, in the fluid phase, the motionally averaged dipolar coupling is too small to be measured by rotational resonance. Rotational resonance, rotational echo double resonance, and related techniques can be used to

Combination of liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry with 13C-labeling for chemical assignment of sulfur-containing metabolites in onion bulbs.

PubMed

Nakabayashi, Ryo; Sawada, Yuji; Yamada, Yutaka; Suzuki, Makoto; Hirai, Masami Yokota; Sakurai, Tetsuya; Saito, Kazuki

2013-02-05

Phytochemicals containing heteroatoms (N, O, S, and halogens) often have biological activities that are beneficial to humans. Although targeted profiling methods for such phytochemicals are expected to contribute to rapid chemical assignments, thus making phytochemical genomics and crop breeding much more efficient, there are few profiling methods for the metabolites. Here, as an ultrahigh performance approach, we propose a practical profiling method for S-containing metabolites (S-omics) using onions (Allium cepa) as a representative species and (12)C- and (13)C-based mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses by liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry (LC-FTICR-MS). Use of the ultrahigh quality data from FTICR-MS enabled simplifying the previous methods to determine specific elemental compositions. MS analysis with a resolution of >250,000 full width at half-maximum and a mass accuracy of <1 ppm can distinguish S-containing monoisotopic ions from other ions on the basis of the natural abundance of (32)S and (34)S and the mass differences among the S isotopes. Comprehensive peak picking using the theoretical mass difference (1.99579 Da) between (32)S-containing monoisotopic ions and their (34)S-substituted counterparts led to the assignment of 67 S-containing monoisotopic ions from the (12)C-based MS spectra, which contained 4693 chromatographic ions. The unambiguous elemental composition of 22 ions was identified through comparative analysis of the (12)C- and (13)C-based MS spectra. Finally, of these, six ions were found to be derived from S-alk(en)ylcysteine sulfoxides and glutathione derivatives. This S-atom-driven approach afforded an efficient chemical assignment of S-containing metabolites, suggesting its potential application for screening not only S but also other heteroatom-containing metabolites in MS-based metabolomics.

Easy Extraction Method To Evaluate δ13C Vanillin by Liquid Chromatography-Isotopic Ratio Mass Spectrometry in Chocolate Bars and Chocolate Snack Foods.

PubMed

Bononi, Monica; Quaglia, Giancarlo; Tateo, Fernando

2015-05-20

An easy extraction method that permits the use of a liquid chromatography-isotopic ratio mass spectrometry (LC-IRMS) system to evaluate δ(13)C of vanillin in chocolate products and industrial flavorings is presented. The method applies the determination of stable isotopes of carbon to discriminate between natural vanillin from vanilla beans and vanillin from other sources (mixtures from beans, synthesis, or biotechnology). A series of 13 chocolate bars and chocolate snack foods available on the Italian market and 8 vanilla flavorings derived from industrial quality control processes were analyzed. Only 30% of products considered in this work that declared "vanilla" on the label showed data that permitted the declaration "vanilla" according to European Union (EU) Regulation 1334/2008. All samples not citing "vanilla" or "natural flavoring" on the label gave the correct declaration. The extraction method is presented with data useful for statistical evaluation.

Application of isotope labeling experiments and (13)C flux analysis to enable rational pathway engineering.

PubMed

McAtee, Allison G; Jazmin, Lara J; Young, Jamey D

2015-12-01

Isotope labeling experiments (ILEs) and (13)C flux analysis provide actionable information for metabolic engineers to identify knockout, overexpression, and/or media optimization targets. ILEs have been used in both academic and industrial labs to increase product formation, discover novel metabolic functions in previously uncharacterized organisms, and enhance the metabolic efficiency of host cell factories. This review highlights specific examples of how ILEs have been used in conjunction with enzyme or metabolic engineering to elucidate host cell metabolism and improve product titer, rate, or yield in a directed manner. We discuss recent progress and future opportunities involving the use of ILEs and (13)C flux analysis to characterize non-model host organisms and to identify and subsequently eliminate wasteful byproduct pathways or metabolic bottlenecks. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of alcohol consumption on the liver detoxication capacity as measured by [13C2]aminopyrine and L-[1-13C]phenylalanine breath tests.

PubMed

Wutzke, Klaus D; Wigger, Marianne

2009-09-01

The aim of this study was to investigate the hepatic microsomal and cytosolic functions by using the 13CO2 breath test in healthy subjects either before or after consumption of red wine. Twelve adults received [13C2]aminopyrine and L-[1-13C]phenylalanine together with a standardised dinner. Expired air samples were taken over 6 h. After a wash-out period, the subjects consumed 0.4 ml ethanol per kg per day together with dinner over a 7.5-day period on average. Thereafter, 13C-tracer administration was repeated under identical conditions. The 13CO2 enrichments were measured by isotope ratio mass spectrometry. The mean cumulative percentage 13C-dose recovery after administration of [13C2]aminopyrine and L-[1-13C]phenylalanine either without or with red wine consumption amounted to 17.0+/-4.4 vs. 14.7+/-3.1% (p=0.170) and 14.0+/-2.8 vs. 11.5+/-3.9% (p=0.084), respectively. Moderate alcohol consumption does not induce significant short-term changes of the microsomal and the cytosolic function of the human liver in healthy subjects.

Differentiation of Pigment in Eggs Using Carbon ((13)C/(12)C) and Nitrogen ((15)N/(14)N) Stable Isotopes.

PubMed

Sun, Feng M; Shi, Guang Y; Wang, Hui W

2016-07-01

Consumers prefer natural and healthy food, but artificial pigments are often abused in egg products. The study aimed at differentiating the origin of pigments in eggs by applying the technique of carbon ((13)C/(12)C) and nitrogen ((15)N/(14)N) stable isotope analysis. Five hundred sixty laying hens were randomly distributed into 14 treatments, which were divided into four groups: maize, carophyll red pigment, carophyll yellow pigment, and a mixture of carophyll red and yellow pigments. Eggs were collected and pretreated to determe the values of the Roche Yolk Color Fan (RCF), δ(13)C, and δ(15)N. With increasing maize content, the RCF and δ(13)C values of yolks increased. Moreover, the RCF values in the three pigment groups were significantly influenced by the artificial colors, but δ(13)C values were not significantly different, regardless of the existence of pigment. The δ(15)N values in all treatments did not vary as regularly as the carbon stable isotope. A strong positive correlation was found between RCF and δ(13)C in the maize group, but no such correlation was be observed in the pigment groups. It is concluded that carbon stable isotope ratio analysis (δ(13)C) of the yolk can be used to differentiate the origin of the pigment added to eggs.

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms*

PubMed Central

Nakajima, Kazuki; Ito, Emi; Ohtsubo, Kazuaki; Shirato, Ken; Takamiya, Rina; Kitazume, Shinobu; Angata, Takashi; Taniguchi, Naoyuki

2013-01-01

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using 13C6-glucose and 13C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with 13C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a 13C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using 13C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism. PMID:23720760

16 CFR 300.14 - Substitute label requirement.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Substitute label requirement. 300.14 Section 300.14 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.14 Substitute label...

Tracing photosynthetic carbon in leaves with nanoSIMS after 13CO2 labelling

NASA Astrophysics Data System (ADS)

Dannoura, Masako; Takeuchi, Miyuki; Kominami, Yuji; Takanashi, Satoru; Kenichi, Yoshimura; Ataka, Mioko

2015-04-01

To understand the carbon allocation of the tree and forest ecosystem, it is important to consider the residence time of carbon in different pools at suitable time scales. For example the carbon used for respiration will stay a few minutes to a few days in the tree, the carbon used for storage or structure of leaves will stay months to years, and the carbon used for wood structure, it will stay over the whole lifespan of the tree. The leaves are the entrance of carbon in trees where it can be used for foliage growth and maintenance or exported to the other organs or the other forest ecosystem compartments. Tracing carbon isotope using NanoSIMS technique is one of useful methods to estimate where and how long the carbon stay in the tree organs. In this study, 13CO2 pulse labelling were conducted and 13C was measured by IRMS to see the amount of C remaining in the leaves with time.NanoSIMS was used to localize where the labelled C remained within the leaf tissue. Twice labelling were done on branches of Quercus serrata at FFPRI(Forest and Forest Products research Institute) in Kyoto, Japan. The first labelling was in 30 April 2012 when the leaves start flushing and the second one was in 29 May 2012 when the leaves were completely deployed. For both labelling experiment, one branch was selected and covered with transparent plastic bag. CO2 concentration was recorded with IRGA and air temperature inside the chamber was monitored. Then 13CO2 was injected into the bag, and after 1 hour, the bag was removed and the branch was again exposed to ambient air. Leaves were collected before and 10-12 times after labelling and their isotope compositions were measured by IRMS. The leaf collected just after labelling and 6 days after labelling were used for NanoSIMS observation. Samples for nanoSIMS were preserved in glutaraldehyde and then embed in epoxy resin. The sliced sample were placed on the silicon wafer and observed by NanoSIMS 50L(Cameca, France). The 13C was highest just

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

NASA Astrophysics Data System (ADS)

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

2016-12-01

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

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry.

PubMed

Lechene, Claude; Hillion, Francois; McMahon, Greg; Benson, Douglas; Kleinfeld, Alan M; Kampf, J Patrick; Distel, Daniel; Luyten, Yvette; Bonventre, Joseph; Hentschel, Dirk; Park, Kwon Moo; Ito, Susumu; Schwartz, Martin; Benichou, Gilles; Slodzian, Georges

2006-01-01

Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse. MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.

Specific coupling between the 13-keto carbonyl and chlorin skeletal vibrational modes of synthetic 13 1- 18O-(un)labelled metallochlorophyll derivatives

NASA Astrophysics Data System (ADS)

Morishita, Hidetada; Tamiaki, Hitoshi

2009-03-01

Metal complexes of methyl 13 1- 18O-labelled pyropheophorbide- a1-M- 18O (M = Zn, Cu and Ni) were prepared by metallation of the 18O-labelled free base ( 1- 18O) and 18O-labelling of unlabelled nickel complex ( 1-Ni). The FT-IR spectra of 1-Zn and 1-Zn- 18O in CH 2Cl 2 showed that the 13-keto carbonyl stretching vibration mode moved to about a 30-cm -1 lower wavenumber by 18O-labelling of the 13 1-oxo moiety. In 1-Cu- 18O and 1-Ni- 18O, the 13-C dbnd 18O stretching modes were close to the highest-energy wavenumber mode of chlorin skeletal C-C/C-N vibrations at around 1650 cm -1 and they were coupled in CH 2Cl 2 to give two split IR bands (Fermi resonance). A similar coupling was observed in the resonance Raman scattering of 1-Ni- 18O in the solid state. The hydrogen-bonded 13-C dbnd 16O vibration mode of 1-Ni similarly coupled with the skeletal C-C/C-N mode in CCl 4 containing 1% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, while such a coupling was not observed in a neat CCl 4 solution of 1-Ni possessing the 13-C dbnd 16O free from any interaction. The skeletal C-C/C-N band selectively coupled with the 13-C dbnd O, not with the 3-C dbnd O, when the difference in their peak maxima was less than 20 cm -1.

Synthesizing the Use of Carbon Isotope (14C and 13C) Approaches to Understand Rates and Pathways for Permafrost C Mobilization and Mineralization

NASA Astrophysics Data System (ADS)

Estop-Aragones, C.; Olefeldt, D.; Schuur, E.

2015-12-01

To better understand the permafrost carbon (C) feedback it is important to synthesize our current knowledge, and knowledge gaps, of how permafrost thaw can cause in situ mineralization or downstream mobilization of aged soil organic carbon (SOC) and the rate of this release. This potential loss of old SOC may occur via gaseous flux of CO2 and CH4 exchanged between soil and the atmosphere and via waterborne flux as DOC, POC (and their subsequent decomposition and release to the atmosphere). Carbon isotope (14C and 13C) approaches have been used to estimate both rates and pathways for permafrost C mobilization and mineralization. Radiocarbon (14C) has been used to estimate the contribution of aged C to overall respiration or waterborne C export. We aim to contrast results from radiocarbon studies, in order to assess differences between ecosystems (contrasting wet and dry ecosystems), thaw histories (active layer deepening or thermokarst landforms), greenhouse gas considered (CO2 and CH4) and seasons. We propose to also contrast methodologies used for assessing the contribution of aged C to overall C balance, and include studies using 13C data. Biological fractionation of 13C during both uptake and decomposition has been taken advantage of both in order to aid the interpretation of 14C data and on its own to assess sources and mineralization pathways. For example, 13C data has been used to differentiate between CH4 production pathways, and the relative contribution of anaerobic CO2 production to overall respiration. Overall, carbon isotope research is proving highly valuable for our understanding of permafrost C dynamics following thaw, and there is a current need to synthesize the available literature.

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

PubMed

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

Metabolism and excretion kinetics of {sup 14}C-labeled and non-labeled difloxacin in pigs after oral administration, and antimicrobial activity of manure containing difloxacin and its metabolites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sukul, Premasis; Lamshoeft, Marc; Kusari, Souvik

2009-04-15

Fluoroquinolones are amongst the most important antibiotics used in veterinary medicine. On this account the behavior of difloxacin (DIF) and its metabolites was investigated by administering the {sup 14}C-labeled and non-labeled veterinary drug to fattening pigs. The excretion kinetics were determined after daily collection of manure. Sarafloxacin (SAR) was found to be the major metabolite, three further trace metabolites were also recovered, applying high-resolution (HR) mass spectrometric technique. The identification of DIF and SAR was confirmed by comparison with the spectroscopic and chromatographic data of the authentic references. The identification of the three trace metabolites was performed by HR-MS/MS. Onlymore » 8.1% of the administered radioactivity remained in the pig after 10 days and DIF accounted for 95.9% of the radioactivity excreted. More than 99% of the labeled compounds were detected and identified in the manure. The mean recoveries for all single electrolytes were {>=}94%. Linearity was established over concentration range 10-10,000 {mu}g/kg manure with a correlation coefficient {>=}0.99. By using in vitro antimicrobial activity tests against a group of standard pathogenic control strains, the results showed that the residual antibiotic concentrations in the manure of pigs are high enough to exhibit antibacterial activity.« less

A perspective on tritium versus carbon-14: ensuring optimal label selection in pharmaceutical research and development.

PubMed

Krauser, Joel A

2013-01-01

Tritium ((3) H) and carbon-14 ((14) C) labels applied in pharmaceutical research and development each offer their own distinctive advantages and disadvantages coupled with benefits and risks. The advantages of (3) H have a higher specific activity, shorter half-life that allows more manageable waste remediation, lower material costs, and often more direct synthetic routes. The advantages of (14) C offer certain analytical benefits and less potential for label loss. Although (3) H labels offer several advantages, they might be overlooked as a viable option because of the concerns about its drawbacks. A main drawback often challenged is metabolic liability. These drawbacks, in some cases, might be overstated leading to underutilization of a perfectly viable option. As a consequence, label selection may automatically default to (14) C, which is a more conservative approach. To challenge this '(14) C-by-default' approach, pharmaceutical agents with strategically selected (3) H-labeling positions based on non-labeled metabolism data have been successfully implemented and evaluated for (3) H loss. From in-house results, the long term success of projects clearly would benefit from a thorough, objective, and balanced assessment regarding label selection ((3) H or (14) C). This assessment should be based on available project information and scientific knowledge. Important considerations are project applicability (preclinical and clinical phases), synthetic feasibility, costs, and timelines. Copyright © 2013 John Wiley & Sons, Ltd.

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

PubMed

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

2008-06-01

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

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

PubMed

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

2015-01-01

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

(14)C, delta(13)C and total C content in soils around a Brazilian PWR nuclear power plant.

PubMed

Dias, Cíntia Melazo; Telles, Everaldo C; Santos, Roberto Ventura; Stenström, Kristina; Nícoli, Iêda Gomes; da Silveira Corrêa, Rosangela; Skog, Göran

2009-04-01

Nuclear power plants release (14)C during routine operation mainly as airborne gaseous effluents. Because of the long half-life (5730 years) and biological importance of this radionuclide (it is incorporated in plant tissue by photosynthesis), several countries have monitoring programs in order to quantify and control these emissions. This paper compares the activity of (14)C in soils taken within 1km from a Brazilian nuclear power plant with soils taken within a reference area located 50km away from the reactor site. Analyses of total carbon, delta(13)C and (137)Cs were also performed in order to understand the local soil dynamics. Except for one of the profiles, the isotopic composition of soil organic carbon reflected the actual forest vegetation present in both areas. The (137)Cs data show that the soils from the base of hills are probably allocthonous. The (14)C measurements showed that there is no accumulation due to the operation of the nuclear facility, although excess (14)C was found in the litter taken in the area close to power plant. This indicates that the anthropogenic signal observed in the litter fall has not been transferred yet to the soil. This study is part of an extensive research programme in which other samples including air, vegetation and gaseous effluents (taken in the vent stack of the Brazilian nuclear power reactors Angra I and II) were also analyzed. The present paper aimed to evaluate how (14)C emissions from the nuclear power plant are transferred and stored by soils present in the surroundings of the reactor site. This is the first study concerning anthropogenic (14)C in soils in Brazil.

Characterizing The Microbial Lability And Isotopic (14C, 13C) Signatures Of Marine Organic Matter With A Novel Culture Vessel System

NASA Astrophysics Data System (ADS)

Beaupre, S. R.; Mahmoudi, N.; Pearson, A.

2016-02-01

The rate at which non-living organic matter is respired in the ocean is an unconstrained and important property of the marine carbon cycle. Studies of inherent mineralization rates are complicated by the fact that marine organic matter is a mixture of compounds that vary in reactivity and concentration. While natural radiocarbon ages (14C, half-life = 5730 yr) have served as proxies for lability, they have not been used extensively to characterize that fraction of marine organic matter that is biologically accessible. To address this problem, we developed a novel batch culture system to monitor the time-dependent production rates and isotopic signatures of CO2 released during microbial degradation of natural organic matter. The system simulated a nepheloid layer by maintaining a slurry of decarbonated sediment and minimal media (M9) in a custom 2-liter culture vessel. The natural microbial community was allowed to develop within the sediment, and respired CO2 was continuously sparged from the medium with helium and oxygen, quantified in real time with an infrared gas analyzer, and isolated as a series of contiguous fractions for subsequent isotopic (∆14C, d13C) characterization. Control experiments indicated the accumulation of just 4.5 mg of background carbon per hour of continuous gas flow, which constituted ≤ 10 % of the respired carbon mass in each fraction. Since ∆14C values are conserved during molecular transformations, this low-blank system enables the detection of subtle shifts in the "age" of organic matter respired during the course of a culture experiment. Analyses of sediments from Falmouth, MA revealed both a variable CO2 production rate and an increase in post-bomb ∆14C values during a 10-day incubation. This suggests that the microbial lability of organic matter at this site decreased non-linearly with apparent 14C age, and that the least labile fraction observed was not more than 50 years old. These results underscore the complex

Dynamics of group II chaperonin and prefoldin probed by 13C NMR spectroscopy.

PubMed

Kurimoto, Eiji; Nishi, Yohei; Yamaguchi, Yoshiki; Zako, Tamotsu; Iizuka, Ryo; Ide, Naoki; Yohda, Masafumi; Kato, Koichi

2008-03-01

Group II chaperonin (CPN) cooperates with prefoldin (PFD), which forms a jellyfish-shaped heterohexameric complex with a molecular mass of 87 kDa. PFD captures an unfolded protein with the tentacles and transfers it to the cavity of CPN. Although X-ray crystal structures of CPN and PFD have been reported, no structural information has been so far available for the terminal regions of the PFD tentacles nor for the C-terminal segments of CPNs, which were regarded to be functionally significant in the previous studies. Here we report 13C NMR analyses on archaeal PFD, CPN, and their complex, focusing on those structurally uncharacterized regions. The PFD and CPN complexes selectively labeled with 13C at methionyl carbonyl carbons were separately and jointly subjected to NMR measurements. 13C NMR spectral data demonstrated that the N-terminal segment of the alpha and beta subunits of PFD as well as the C-terminal segments of the CPN hexadecamer retain significant degrees of freedom in internal motion even in the complex with a molecular mass of 1.1 MDa. 2007 Wiley-Liss, Inc.

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

NASA Astrophysics Data System (ADS)

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

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

Simultaneous measurement of neuronal and glial metabolism in rat brain in vivo using co-infusion of [1,6- 13C 2]glucose and [1,2- 13C 2]acetate

NASA Astrophysics Data System (ADS)

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

2009-02-01

In this work the feasibility of measuring neuronal-glial metabolism in rat brain in vivo using co-infusion of [1,6- 13C 2]glucose and [1,2- 13C 2]acetate was investigated. Time courses of 13C spectra were measured in vivo while infusing both 13C-labeled substrates simultaneously. Individual 13C isotopomers (singlets and multiplets observed in 13C spectra) were quantified automatically using LCModel. The distinct 13C spectral pattern observed in glutamate and glutamine directly reflected the fact that glucose was metabolized primarily in the neuronal compartment and acetate in the glial compartment. Time courses of concentration of singly and multiply-labeled isotopomers of glutamate and glutamine were obtained with a temporal resolution of 11 min. Although dynamic metabolic modeling of these 13C isotopomer data will require further work and is not reported here, we expect that these new data will allow more precise determination of metabolic rates as is currently possible when using either glucose or acetate as the sole 13C-labeled substrate.

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

NASA Astrophysics Data System (ADS)

Lenhart, Katharina; Keppler, Frank

2017-04-01

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

Dissimilation of [(13)C]methanol by continuous cultures of Bacillus methanolicus MGA3 at 50 degrees C studied by (13)C NMR and isotope-ratio mass spectrometry.

PubMed

Pluschkell, Stefanie B; Flickinger, Michael C

2002-10-01

Using a continuous culture of Bacillus methanolicus MGA3 limited by 100 mM methanol in the feed and growing at a dilution rate D=0.25 h(-1), transients in dissolved methanol were studied to determine the effects of methanol toxicity and the pathway of methanol dissimilation to CO(2). Steady-state cultures were disturbed by pulses of methanol resulting in a rapid change in concentration of 6.4-12.8 mM. B. methanolicus MGA3 responded to a sudden increase in available methanol by a transient decline in the biomass concentration in the reactor. In most cases the culture returned to steady state between 4 and 12 h after pulse addition. However, at a methanol pulse of 12.8 mM, complete biomass washout occurred and the culture did not return to steady state. Integrating the response curves of the dry biomass concentration over a 12 h time period showed that a methanol pulse can cause an average transient decline in the biomass yield of up to 22%. (13)C NMR experiments using labelled methanol indicated that the transient partial or complete biomass washout was probably caused by toxic accumulation of formaldehyde in the culture. These experiments also showed accumulation of formate, indicating that B. methanolicus possesses formaldehyde dehydrogenase and formate dehydrogenase activity resulting in a methanol dissimilation pathway via formate to CO(2). Studies using isotope-ratio mass spectrometry provided further evidence of a methanol dissimilation pathway via formate. B. methanolicus MGA3, growing continuously under methanol limitation, consumed added formate at a rate of approximately 0.85 mmol l(-1) h(-1). Furthermore, significant accumulation of (13)CO(2) in the reactor exhaust gas was measured in response to a pulse addition of [(13)C]formic acid to the bioreactor. This indicates that B. methanolicus dissimilates methanol carbon to CO(2) in order to detoxify formaldehyde by both a linear pathway to formate and a cyclic mechanism as part of the RuMP pathway.

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

PubMed

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.

Novel Imaging Contrast Methods for Hyperpolarized 13 C Magnetic Resonance Imaging

NASA Astrophysics Data System (ADS)

Reed, Galen Durant

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

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

DOEpatents

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

2004-07-20

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

Experimental soil warming and cooling alters the partitioning of recent assimilates: evidence from a (14)C-labelling study at the alpine treeline.

PubMed

Ferrari, A; Hagedorn, F; Niklaus, P A

2016-05-01

Despite concerns about climate change effects on ecosystems functioning, little is known on how plant assimilate partitioning changes with temperature. Particularly, large temperature effects might occur in cold ecosystems where critical processes are at their temperature limit. In this study, we tested temperature effects on carbon (C) assimilate partitioning in a field experiment at the alpine treeline. We warmed and cooled soils of microcosms planted with Pinus mugo or Leucanthemopsis alpina, achieving daily mean soil temperatures (3-10 cm depth) around 5.8, 12.7 and 19.2 °C in cooled, control and warmed soils. We pulse-labelled these systems with (14)CO2 for one photoperiod and traced (14)C over the successive 4 days. Plant net (14)C uptake increased steadily with soil temperature. However, (14)C amounts in fungal hyphae, soil microbial biomass, soil organic matter, and soil respiration showed a non-linear response to temperature. This non-linear pattern was particularly pronounced in P. mugo, with five times higher (14)C activities in cooled compared to control soils, but no difference between warmed and control soil. Autoradiographic analysis of the spatial distribution of (14)C in soils indicated that temperature effects on the vertical label distribution within soils depended on plant species. Our results show that plant growth, in particular root metabolism, is limited by low soil temperature. As a consequence, positive temperature effects on net C uptake may not be paralleled by similar changes in rhizodeposition. This has important implications for predictions of soil C storage, because rhizodeposits and plant biomass vary strongly in their residence times.

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

NASA Astrophysics Data System (ADS)

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

2011-03-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

Detection of C-13O radio emission from C-13-rich carbon stars

NASA Technical Reports Server (NTRS)

Jura, M.; Kahane, C.; Omont, A.

1988-01-01

A high ratio of C-13O radio emission in the J = 1-0 rotational line has been detected from three mass-losing carbon stars which optical data indicate have high C-13/C12 ratios. Since chemical fractionation, isotope-dependent photodissociation and opacity in the rotational and vibrational lines may not raise significantly the C-13O ratio above the actual C-13/C-12 ratio in these circumstellar envelopes, the relative abundance of C-13 in these stars might be even greater by perhaps a factor of two than previously believed. About 15 percent of all luminous carbon stars are C-13-rich, and these stars may play a significant role in the enhancement in the C-13/C12 ratio that has occurred during the past 4.6 billion years since the formation of the sun.

Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi.

PubMed

Steffen, K T; Hofrichter, M; Hatakka, A

2000-12-01

Within a screening program, 27 soil litter-decomposing basidiomycetes were tested for ligninolytic enzyme activities using agar-media containing 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate), a humic acid or Mn2+ ions as indicator substrates. Most active species were found within the family Strophariaceae (Agrocybe praecox, Stropharia coronilla, S. rugosoannulata) and used for mineralisation experiments with a 14C-ring-labelled synthetic lignin (14C-DHP). The fungi mineralised around 25% of the lignin to 14CO2 within 12 weeks of incubation in a straw environment; about 20% of the lignin was converted to water-soluble fragments. Mn-peroxidase was found to be the predominant ligninolytic enzyme of all three fungi in liquid culture and its production was strongly enhanced in the presence of Mn2+ ions. The results of this study demonstrate that certain ubiquitous litter-decomposing basidiomycetes possess ligninolytic activities similar to the wood-decaying white-rot fungi, the most efficient lignin degraders in nature.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jing; Weitz, Eric

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 ontomore » 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.« less

Determination of 15N/14N and 13C/12C in Solid and Aqueous Cyanides

USGS Publications Warehouse

Johnson, C.A.

1996-01-01

The stable isotopic compositions of nitrogen and carbon in cyanide compounds can be determined by combusting aliquots in sealed tubes to form N2 gas and CO2 gas and analyzing the gases by mass spectrometry. Free cyanide (CN-aq + HCNaq) in simple solutions can also be analyzed by first precipitating the cyanide as copper(II) ferrocyanide and then combusting the precipitate. Reproducibility is ??0.5??? or better for both ??15N and ??13C. If empirical corrections are made on the basis of carbon yields, the reproducibility of ??13C can be improved to ??0.2???. The analytical methods described herein are sufficiently accurate and precise to apply stable isotope techniques to problems of cyanide degradation in natural waters and industrial process solutions.

Convergent synthesis of 13N-labelled Peptidic structures using aqueous [13N]NH3.

PubMed

Blower, Julia E; Cousin, Samuel F; Gee, Antony D

2017-01-01

Nitrogen-13 has a 10-min half-life which places time constraints on the complexity of viable synthetic methods for its incorporation into PET imaging agents. In exploring ways to overcome this limitation, we have used the Ugi reaction to develop a rapid one-pot method for radiolabelling peptidic molecules using [ 13 N]NH 3 as a synthetic precursor. Carrier-added [ 13 N]NH 3 (50 μL) was added to a solution of carboxylic acid, aldehyde, and isocyanide in 2,2,2-TFE (200 μL). The mixture was heated in a microwave synthesiser at 120 °C for 10 min. Reactions were analysed by radio-HPLC and radio-LCMS. Isolation of the target 13 N-labelled peptidic Ugi compound was achieved via semi-preparative radio-HPLC as demonstrated for Ugi 1. Radio-HPLC analysis of each reaction confirmed the formation of radioactive products co-eluting with their respective reference standards with radiochemical yields of the crude products ranging from 11% to 23%. Two cyclic γ-lactam structures were also achieved via intra-molecular reactions. Additional radioactive by-products observed in the radio-chromatogram were identified as 13 N-labelled di-imines formed from the reaction of [ 13 N]NH 3 with two isocyanide molecules. The desired 13 N-labelled Ugi product was isolated using semi-preparative HPLC. We have developed a one-pot method that opens up new routes to radiolabel complex, peptidic molecules with 13 N using aqueous [ 13 N]NH 3 as a synthetic precursor.

Response of soil organic carbon mineralization in typical Karst soils following the addition of 14C-labeled rice straw and CaCO3.

PubMed

Hu, Lening; Su, Yirong; He, Xunyang; Wu, Jinshui; Zheng, Hua; Li, Yang; Wang, Aihua

2012-03-30

Organic substrates and calcium are important factors controlling organic matter turnover in Karst soils. To understand their effects on soil organic carbon (SOC) mineralization, an incubation experiment was conducted involving a control treatment (CK), the addition of a (14)C-labeled rice straw (T1), CaCO(3) (T2), and both (14)C-labeled rice straw and CaCO(3) (T3) to two types of Karst soils (terra fusca and rendzina) and a red soil from southwestern China. Cumulative mineralization of the rice straw over 100 days in rendzina (22.96 mg kg(-1)) and terra fusca (23.19 mg kg(-1)) was higher than in the red soil (15.48 mg kg(-1); P < 0.05). Cumulative mineralization of native SOC decreased following addition of (14)C-labeled rice straw in the rendzina and terra fusca but increased in the red soil (negative and positive priming effects on native SOC). The turnover times of (14)C-labeled microbial biomass C (MBC) in the red soil, terra fusca and rendzina were 71 ± 2, 243 ± 20 and 254 ± 45 days, respectively. By adding CaCO(3), the accumulation of SOC was greater in the Karst soils than in the red soil. Although the interactions between rice straw decomposition and priming effects on native SOC are not yet understood, there was considerable variation between Karst and red soils. Soil calcium was a positive factor in maintaining SOC stability. MBC from rice straws was stable in terra fusca and rendzina, whereas it was active in the red soil. The Karst soils (terra fusca and rendzina) used in this study benefited SOC accumulation. Copyright © 2011 Society of Chemical Industry.

Isotope Label-Aided Mass Spectrometry Reveals the Influence of Environmental Factors on Metabolism in Single Eggs of Fruit Fly

PubMed Central

Tseng, Te-Wei; Wu, June-Tai; Chen, Yu-Chie; Urban, Pawel L.

2012-01-01

In order to investigate the influence of light/dark cycle on the biosynthesis of metabolites during oogenesis, here we demonstrate a simple experimental protocol which combines in-vivo isotopic labeling of primary metabolites with mass spectrometric analysis of single eggs of fruit fly (Drosophila melanogaster). First, fruit flies were adapted to light/dark cycle using artificial white light. Second, female flies were incubated with an isotopically labeled sugar (13C6-glucose) for 12 h – either during the circadian day or the circadian night, at light or at dark. Third, eggs were obtained from the incubated female flies, and analyzed individually by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS): this yielded information about the extent of labeling with carbon-13. Since the incorporation of carbon-13 to uridine diphosphate glucose (UDP-glucose) in fruit fly eggs is very fast, the labeling of this metabolite was used as an indicator of the biosynthesis of metabolites flies/eggs during 12-h periods, which correspond to circadian day or circadian night. The results reveal that once the flies adapted to the 12-h-light/12-h-dark cycle, the incorporation of carbon-13 to UDP-glucose present in fruit fly eggs was not markedly altered by an acute perturbation to this cycle. This effect may be due to a relationship between biosynthesis of primary metabolites in developing eggs and an alteration to the intake of the labeled substrate – possibly related to the change of the feeding habit. Overall, the study shows the possibility of using MALDI-MS in conjunction with isotopic labeling of small metazoans to unravel the influence of environmental cues on primary metabolism. PMID:23185587

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ashburn, D.A.; Garcia, K.; Hanners, J.L.

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

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

PubMed

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

2015-10-01

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

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

PubMed Central

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

2011-01-01

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

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

PubMed

Yang, Tae Hoon

2013-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2011-03-01

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

Synthesis of the olanzapine labeled by carbon-14.

PubMed

Saadatjoo, Naghi; Javaheri, Mohsen; Saemian, Nader; Amini, Mohsen

2016-06-30

Olanzapine is one of the most widely used antipsychotic drugs, which acts as an antagonist for multiple neurotransmitter receptor sites. 2-Methyl-4-(4-methyl-1-piperazinyl)-10H-thieno [2,3-b][1,5] benzodiazepine (Olanzapine) labeled with carbon-14 in the four positions has been synthesized as part of a three-step sequence from 2-amino-5-methylthiophene-3-carbonitrile-[carbonitrile-(14) C]. Copyright © 2016 John Wiley & Sons, Ltd.

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.

PubMed

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.

Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone.

PubMed Central

Nelson, P S; Kent, M; Muthini, S

1992-01-01

Novel CE-phosphoramidite (7a-e) and CPG (8a, c, d, e) reagents have been prepared from a unique 2-aminobutyl-1,3-propanediol backbone. The reagents have been used to directly label oligonucleotides with fluorescein, acridine, and biotin via automated DNA synthesis. The versatile 2-aminobutyl-1,3-propanediol backbone allows for labeling at any position (5', internal, and 3') during solid phase oligonucleotide synthesis. Multiple labels can be achieved by repetitive coupling cycles. Furthermore, the 3-carbon atom internucleotide phosphate distance is retained when inserted internally. Using this method, individual oligonucleotides possessing two and three different reporter molecules have been prepared. PMID:1475185

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

PubMed

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

2013-05-01

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

A Radiochemical Biotechnological Approach: Preliminary Study of Lactose Uptake Rate by Kefir Cells, Using 14C-labeled Lactose, in Anaerobic Fermentation

NASA Astrophysics Data System (ADS)

Golfinopoulos, A.; Soupioni, M.; Kanellaki, M.; Koutinas, A. A.

2008-08-01

The effect of initial lactose concentration on lactose uptake rate by kefir free cells, during the lactose fermentation, was studied in this work. For the investigation 14C-labelled lactose was used due to the fact that labeled and unlabeled molecules are fermented in the same way. The results illustrated lactose uptake rates are about up to two fold higher at lower initial ∘Bé densities as compared with higher initial ∘Bé densities.

Imprint of CO2 emission in atmosphere and biosphere on the basis of 14C and 13C measurements

NASA Astrophysics Data System (ADS)

Pazdur, Anna; Gabryś, Alicja; Kuc, Tadeusz; Pawełczyk, Sławomira; Piotrowska, Natalia; Rakowski, Andrzej; Różański, Kazimierz; Sensuła, Barbara

2015-04-01

As is shown in the IPCC (Intergovernmental Panel on Climate Change) report, the observed climate changes are caused, among others, by human activity. Mainly emission of CO2 to the atmosphere coming from the burning of fossil fuels, can have dire consequences for life on Earth and development of humankind. The report uses, among others, data obtained from isotopic measurements in the biosphere. Measurements of 14C and 13C concentration in modern atmospheric carbon dioxide and biosphere allow the determination of the decrease of the concentration of this isotope. Furthermore, the magnitude of emission to the atmosphere of carbon dioxide not containing the isotope 14C can be estimated on this basis. Such emission stems from fossil fuel combustion - petroleum, natural gas and black coal. A sensitive bioindicator of the emission are annual tree rings. The measurements of 14C concentration in tree ring material using AMS allow to see its seasonal changes. Trees, treated as an archive of changes in conjunction with information about the isotopic composition of carbon can be used for monitoring of environment as sensitive bioindicators on local, as well as on the global scale. Regular investigations of isotopic composition of carbon in trees have been carried out in the GADAM Centre for the urban areas of both Poland and worldwide. This method can be applied in the study of the emission of CO2 to the atmosphere and its spatial and temporal distribution connected with the production of energy by power plants based on fossil fuel combustion for the area of southern Poland. Modelling of CO2 emission using both 14C and 13C carbon isotopes measured in pine tree rings on the background of climatic changes will be presented. The national ecological policy in the era of global warming requires the manufacturers of energy to get involved in the development of methods suitable for monitoring the state of the environment. Hence, the interest in the area of monitoring the fossil fuel

Three new HLA-C alleles (HLA-C*14:02:13, HLA-C*15:72 and HLA-C*15:74) in Saudi bone marrow donors.

PubMed

Fakhoury, H A; Jawdat, D; Alaskar, A S; Al Jumah, M; Cereb, N; Hajeer, A H

2015-10-01

Three new HLA-C alleles were identified by sequence-based typing method (SBT) in donors for the Saudi Bone Marrow Donor Registry (SBMDR). HLA-C*14:02:13 differs from HLA-C*14:02:01 by a silent G to A substitution at nucleotide position 400 in exon 2, where lysine at position 66 remains unchanged. HLA-C*15:72 differs from HLA-C*15:22 by a nonsynonymous C to A substitution at nucleotide position 796 in exon 3, resulting in an amino acid change from phenylalanine to leucine at position 116. HLA-C*15:74 differs from HLA-C*15:08 by a nonsynonymous C to T substitution at nucleotide position 914 in exon 3, resulting in an amino acid change from arginine to tryptophan at position 156. © 2015 John Wiley & Sons Ltd.

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

PubMed Central

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

2014-01-01

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

Determination of urea kinetics by isotope dilution with [13C]urea and gas chromatography-isotope ratio mass spectrometry (GC-IRMS) analysis.

PubMed

Kloppenburg, W D; Wolthers, B G; Stellaard, F; Elzinga, H; Tepper, T; de Jong, P E; Huisman, R M

1997-07-01

1. Stable urea isotopes can be used to study urea kinetics in humans. The use of stable urea isotopes for studying urea kinetic parameters in humans on a large scale is hampered by the high costs of the labelled material. We devised a urea dilution for measurement of the distribution volume, production rate and clearance of urea in healthy subjects and renal failure patients using the inexpensive single labelled [13C]urea isotope with subsequent analysis by headspace chromatography-isotope ratio MS (GC-IRMS) of the [13C]urea enrichment. 2. The method involves measurement of the molar percentage excess of [13C]urea in plasma samples taken over a 4 h period after an intravenous bolus injection of [13C]urea. During the sample processing procedure, the plasma samples together with calibration samples containing a known molar percentage excess of [13C]urea are acidified with phosphoric acid to remove endogenous CO2, and are subsequently incubated with urease to convert the urea present in the plasma samples into CO2. The 13C enrichment of the generated CO2 is analysed by means of GC-IRMS. This method allows measurement of the molar percentage excess of [13C]urea to an accuracy of 0.02%. 3. Reproducibility studies showed that the sample processing procedure [within-run coefficient of variation (CV) < 2.8% and between-run CV < 8.8%] and the GC-IRMS analysis (within-day CV < 1.3% and between-day CV < 1.3%) could be repeated with good reproducibility. 4. In clinical urea kinetic studies in a healthy subject and in a renal failure patient without residual renal function, reproducible values of the distribution volume, production rate and clearance of urea were determined using minimal amounts of [13C]urea (25-50 mg). 5. Because only low [13C]urea enrichments are needed in this urea dilution method using GC-IRMS analysis, the costs of urea kinetic studies are reduced considerably, especially in patients with renal failure.

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

PubMed Central

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

2012-01-01

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 [2-13C]glucose or [3-13C]glucose. The activity of the PPP was several fold lower than glycolysis in both types of neurons. While metabolism of 13C-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 13C labeling from glucose is incorporated into glutamate proves that both the oxidative and the nonoxidative stages of the PPP are active in neurons. PMID:22714050

A novel design for a dual stable isotope continuous labeling chamber: results on labeling efficiency and C and N allocation in Andropogon gerardii

NASA Astrophysics Data System (ADS)

Soong, J.; Stewart, C.; Reuss, D.; Pinney, C.; Cotrufo, F. M.

2010-12-01

The use of stable isotope enriched plant material can provide an unobstructed method of studying ecosystem nutrient dynamics between plants, soil, and atmosphere. However, the production of uniformly labeled perennial plant material is challenging due to plant physiological constraints and the mechanics of building and operating an isotope labeling system. In this study we present the design of a novel dual 13C and 15N continuous isotope labeling chamber located at Colorado State University. The chamber is equipped with automatic controls for CO2 concentration, temperature, and humidity, and has successfully been used to grow and label the tallgrass perennial Andropogon gerardii in pots from rhizomes. Three different nitrogen fertilization levels were applied to assess how substrate availability may alter growth and overall performance in the system. The efficiency of the 13C and 15N labeling chamber, its design and overall performance, as well as a full C, N, 13C, and 15N budget of the aboveground biomass, belowground biomass, and soil will be presented. Solid samples were analyzed on an EA-IRMS, while air samples from the chamber were analyzed using a precon-GC-IRMS system. The dual stable isotope labeled A. gerardii produced from this chamber will be used in a decomposition experiment to quantify the relative contribution of aboveground litter derived C to soil respiration, dissolved organic carbon, and various soil organic matter pools. Based on the results of our A. gerardii 13C and 15N labeling experiment we believe that this chamber design can be used to successfully produce dual stable isotope labeled plants for a wide variety of terrestrial nutrient flux experiments.

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

PubMed

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

2014-01-01

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

A potent IκB kinase-β inhibitor labeled with carbon-14 and deuterium.

PubMed

Latli, Bachir; Eriksson, Magnus; Hrapchak, Matt; Busacca, Carl A; Senanayake, Chris H

2016-06-30

3-Amino-4-(1,1-difluoro-propyl)-6-(4-methanesulfonyl-piperidin-1-yl)-thieno[2,3-b]pyridine-2-carboxylic acid amide (1) is a potent IκB Kinase-β (IKK-β) inhibitor. The efficient preparations of this compound labeled with carbon-14 and deuterium are described. The carbon-14 synthesis was accomplished in six radiochemical steps in 25% overall yield. The key transformations were the modified Guareschi-Thorpe condensation of 2-cyano-(14) C-acetamide and a keto-ester followed by chlorination to 2,6-dichloropyridine derivative in one pot. The isolated dichloropyridine was then converted in three steps in one pot to [(14) C]-(1). The carbon-14 labeled (1) was isolated with a specific activity of 54.3 mCi/mmol and radiochemical purity of 99.8%. The deuterium labeled (1) was obtained in eight steps and in 57% overall chemical yield using 4-hydroxypiperidine-2,2,3,3,4,5,5,6,6-(2) H9 . The final three steps of this synthesis were run in one pot. Copyright © 2016 John Wiley & Sons, Ltd.

Improved δ(13)C analysis of amino sugars in soil by ion chromatography-oxidation-isotope ratio mass spectrometry.

PubMed

Dippold, Michaela A; Boesel, Stefanie; Gunina, Anna; Kuzyakov, Yakov; Glaser, Bruno

2014-03-30

Amino sugars build up microbial cell walls and are important components of soil organic matter. To evaluate their sources and turnover, δ(13)C analysis of soil-derived amino sugars by liquid chromatography was recently suggested. However, amino sugar δ(13)C determination remains challenging due to (1) a strong matrix effect, (2) CO2 -binding by alkaline eluents, and (3) strongly different chromatographic behavior and concentrations of basic and acidic amino sugars. To overcome these difficulties we established an ion chromatography-oxidation-isotope ratio mass spectrometry method to improve and facilitate soil amino sugar analysis. After acid hydrolysis of soil samples, the extract was purified from salts and other components impeding chromatographic resolution. The amino sugar concentrations and δ(13)C values were determined by coupling an ion chromatograph to an isotope ratio mass spectrometer. The accuracy and precision of quantification and δ(13)C determination were assessed. Internal standards enabled correction for losses during analysis, with a relative standard deviation <6%. The higher magnitude peaks of basic than of acidic amino sugars required an amount-dependent correction of δ(13)C values. This correction improved the accuracy of the determination of δ(13)C values to <1.5‰ and the precision to <0.5‰ for basic and acidic amino sugars in a single run. This method enables parallel quantification and δ(13)C determination of basic and acidic amino sugars in a single chromatogram due to the advantages of coupling an ion chromatograph to the isotope ratio mass spectrometer. Small adjustments of sample amount and injection volume are necessary to optimize precision and accuracy for individual soils. Copyright © 2014 John Wiley & Sons, Ltd.

Metabolism of 14C-labeled doxylamine succinate (Bendectin) in the rhesus monkey (Macaca mulatta).

PubMed

Slikker, W; Holder, C L; Lipe, G W; Korfmacher, W A; Thompson, H C; Bailey, J R

1986-01-01

The time-course of the metabolic fate of [14C]doxylamine was determined after the p.o. administration of 13 mg/kg doxylamine succinate as Bendectin plus [14C]doxylamine succinate to the rhesus monkey. Urine and plasma samples were analyzed by reversed-phase high performance liquid chromatography (HPLC), chemical derivatization, and mass spectrometry. The cumulative 48-hr urinary metabolic profile contained 81% of the administered radiolabeled dose and consisted of at least six radiolabeled peaks. They were peak 1: unknown polar metabolites (8% of dose); peak 2: 2-[1-phenyl-1-(2-pyridinyl)ethoxy] acetic acid, 1-[1-phenyl-1(2-pyridinyl)ethoxy] methanol, and another minor metabolite(s) (31%); peak 3: doxylamine-N-oxide (1%); peak 4a: N,N-didesmethyldoxylamine (17%); peak 4b: doxylamine (4%); and peak 5: N-desmethyldoxylamine (20%). The plasma metabolic profile was the same as the urinary profile except for the absence of doxylamine-N-oxide. The maximum plasma concentrations and elapsed time to attain these concentrations were as follows. Peak 1: 540 ng/mL, 4 hr; peak 2: 1700 ng/mL, 1 hr; peak 4a: 430 ng/mL, 4 hr; peak 4b: 930 ng/mL, 2 hr; and peak 5: 790 ng/mL, 2 hr. These data suggest that in the monkey, doxylamine metabolism follows at least four pathways: a minor pathway to the N-oxide; a minor pathway to unknown polar metabolites; a major pathway to mono- and didesmethyldoxylamine via successive N-demethylation; and a major pathway to side-chain cleavage products (peak 2) via direct side-chain oxidation and/or deamination.

[2,4-13C2]-β-Hydroxybutyrate Metabolism in Human Brain

PubMed Central

Pan, Jullie W.; de Graaf, Robin A.; Petersen, Kitt F.; Shulman, Gerald I.; Hetherington, Hoby P.; Rothman, Douglas L.

2010-01-01

Summary Infusions of [2,4-13C2]-β-hydroxybutyrate and 1H–13C polarization transfer spectroscopy were used in normal human subjects to detect the entry and metabolism of β-hydroxybutyrate in the brain. During the 2-hour infusion study, 13C label was detectable in the β-hydroxybutyrate resonance positions and in the amino acid pools of glutamate, glutamine, and aspartate. With a plasma concentration of 2.25 ± 0.24 mmol/L (four volunteers), the apparent tissue β-hydroxybutyrate concentration reached 0.18 ± 0.06 mmol/L during the last 20 minutes of the study. The relative fractional enrichment of 13C-4-glutamate labeling was 6.78 ± 1.71%, whereas 13C-4-glutamine was 5.68 ± 1.84%. Steady-state modeling of the 13C label distribution in glutamate and glutamine suggests that, under these conditions, the consumption of the β-hydroxybutyrate is predominantly neuronal, used at a rate of 0.032 ± 0.009 mmol · kg−1 · min−1, and accounts for 6.4 ± 1.6% of total acetyl coenzyme A oxidation. These results are consistent with minimal accumulation of cerebral ketones with rapid utilization, implying blood–brain barrier control of ketone oxidation in the nonfasted adult human brain. PMID:12142574

Twoplex 12/13 C6 aniline stable isotope and linkage-specific sialic acid labeling 2D-LC-MS workflow for quantitative N-glycomics.

PubMed

Albrecht, Simone; Mittermayr, Stefan; Smith, Josh; Martín, Silvia Millán; Doherty, Margaret; Bones, Jonathan

2017-01-01

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run-to-run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC-MS E workflow for the fractionation and relative quantitation of twoplex isotopically labeled N-linked oligosaccharides using neutral 12 C 6 and 13 C 6 aniline (Δmass = 6 Da). Additional linkage-specific derivatization of sialic acids using 4-(4,6-dimethoxy-1,3,5-trizain-2-yl)-4-methylmorpholinium chloride offered simultaneous and advanced in-depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N-glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex 12 C 6 / 13 C 6 aniline 2D LC-MS platform is ideally suited for differential glycomic analysis of structurally complex N-glycan pools due to combination and analysis of samples in a single LC-MS injection and the associated minimization in technical variation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of /sup 125/I-labeled and /sup 14/C-Labeled peptides of the major outer membrane protein of Chlamydia Trachomatis Strain L2/434 separated by high-performance liquid chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Judd, R.C.; Caldwell, H.D.

1985-01-01

The objective of this study was to determine if in-gel chloramine-T radioiodination adequately labels OM proteins to allow for accurate and precise structural comparison of these molecules. Therefore, intrinsically /sup 14/C-amino acid labeled proteins and /sup 125/I-labeled proteins were cleaved with two endopeptidic reagents and the peptide fragments separated by HPLC. A comparison of retention times of the fragments, as determined by differential radiation counting, thus indicated whether /sup 125/Ilabeling identified of all the peptide peaks seen in the /sup 14/Clabeled proteins. Results demonstrated that radioiodination yields complete and accurate information about the primary structure of outer membrane proteins. Inmore » addition, it permits the use of extremely small amounts of protein allowing for method optimization and multiple separations to insure reproducibility.« less

Low-level (submicromole) environmental 14C metrology

NASA Astrophysics Data System (ADS)

Currie, L. A.; Kessler, J. D.; Marolf, J. V.; McNichol, A. P.; Stuart, D. R.; Donoghue, J. C.; Donahue, D. J.; Burr, G. S.; Biddulph, D.

2000-10-01

Accelerator mass spectrometry (AMS) measurements of environmental 14C have been employed during the past decade at the several micromole level (tens of μg carbon), but advanced research in the atmospheric and marine sciences demands still higher (μg) sensitivity, an extreme example being the determination of 14C in elemental or "black" carbon (BC) at levels of 2-10 μg per kg of Greenland snow and ice (Currie et al., 1998). A fundamental limitation for 14C AMS is Poisson counting statistics, which sets in at about 1 μg modern-C. Using the small sample (25 μg) AMS target preparation facility at NOSAMS (Pearson et al., 1998), and the microsample combustion-dilution facility at NIST, we have demonstrated an intrinsic modern-C quantification limit ( mQ) of ca. 0.9 μg, based on a 1-parameter fit to the empirical AMS variance function. (For environmental 14C, the modern carbon quantification limit is defined as that mass ( mQ) corresponding to 10% relative standard deviation (rsd) for the fraction of modern carbon, σ( fM)/ fM.) Stringent control, required for quantitative dilution factors (DL), is achieved with the NIST on-line manometric/mass spectrometry facility that compensates also for unsuspected trace impurities from vigorous chemical processing (e.g., acid digestion). Our current combustion blank is trivial (mean: 0.16 ± 0.02 μg C, n=13) but lognormally distributed (dispersion [σ]: 0.07 ± 0.01 μg). An iterative numerical expression is introduced to assess the quantitative impacts of fossil and modern carbon blank components on mQ; and a new "clean chemistry" BC processing system is described for the minimization of such blanks. For the assay of soot carbon in Greenland snow/ice, the overall processing blank has been reduced from nearly 7 μg total carbon to less than 1 μg, and is undetectable for BC.

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

PubMed

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

2017-01-01

The 13 C turnover of neurotransmitter amino acids (glutamate, GABA and aspartate) were determined from extracts of forebrain nerve terminals and brain homogenate, and fronto-parietal cortex from anesthetized rats undergoing timed infusions of [1,6- 13 C 2 ]glucose or [2- 13 C]acetate. Nerve terminal 13 C fractional labeling of glutamate and aspartate was lower than those in whole cortical tissue at all times measured (up to 120 min), suggesting either the presence of a constant dilution flux from an unlabeled substrate or an unlabeled (effectively non-communicating on the measurement timescale) glutamate pool in the nerve terminals. Half times of 13 C labeling from [1,6- 13 C 2 ]glucose, as estimated by least squares exponential fitting to the time course data, were longer for nerve terminals (Glu C4 , 21.8 min; GABA C2 21.0 min) compared to cortical tissue (Glu C4 , 12.4 min; GABA C2 , 14.5 min), except for Asp C3 , which was similar (26.5 vs. 27.0 min). The slower turnover of glutamate in the nerve terminals (but not GABA) compared to the cortex may reflect selective effects of anesthesia on activity-dependent glucose use, which might be more pronounced in the terminals. The 13 C labeling ratio for glutamate-C4 from [2- 13 C]acetate over that of 13 C-glucose was twice as large in nerve terminals compared to cortex, suggesting that astroglial glutamine under the 13 C glucose infusion was the likely source of much of the nerve terminal dilution. The net replenishment of most of the nerve terminal amino acid pools occurs directly via trafficking of astroglial glutamine.

Global ocean climatology of the 13C Suess effect and preindustrial δ13C

NASA Astrophysics Data System (ADS)

Eide, Marie; Olsen, Are; Ninnemann, Ulysses; Eldevik, Tor; Johannessen, Truls

2017-04-01

We present the first observationally based estimate of the full global ocean 13C Suess effect since preindustrial times. This was constructed by using Olsen and Ninnemann's [2010] back-calculation method to calculate the 13C Suess effect with data from 29 cruises spanning the world ocean. We find a strong 13C Suess effect in the upper 1000 m of all basins, with strongest decrease in the Subtropical Gyres of the Northern Hemisphere, where δ13C has decreased by more than 0.8‰ since the industrial revolution. At greater depths, a significant 13C Suess effect can only be detected in the northern parts of the North Atlantic Ocean. The magnitude of the 13C Suess effect is correlated with the concentration of anthropogenic carbon, but their relationship varying strongly between water masses, reflecting the degree to which source waters are equilibrated with the atmospheric 13C Suess effect before sinking. From the 13C Suess effect estimates, we have estimated the preindustrial δ13C (δ13CPI) along the 29 sections. Further, we developed regional multilinear regression equations, which were applied on the World Ocean Atlas data to construct the δ13CPI climatology, which reveals the natural δ13C distribution in the global ocean. Compared to the modern distribution, the preindustrial δ13C spans a larger range of values, and we find that in some regions in the high northern latitudes, the gradient in modern ocean δ13C is completely reversed compared to the preindustrial. Maximum δ13CPI, of up to 1.8‰, are found in the subtropical gyres of all basins, in the upper and intermediate waters of the North Atlantic, as well as in mode waters with a Southern Ocean origin. Particularly strong gradients occur at intermediate depths, revealing a strong potential for using δ13C as a tracer for changes in water mass geometry at these levels. Further, we identify a much tighter relationship between δ13C and Apparent Oxygen Utilization (AOU) than between δ13C and phosphate that

Hyperpolarized [U-(2) H, U-(13) C]Glucose reports on glycolytic and pentose phosphate pathway activity in EL4 tumors and glycolytic activity in yeast cells.

PubMed

Timm, Kerstin N; Hartl, Johannes; Keller, Markus A; Hu, De-En; Kettunen, Mikko I; Rodrigues, Tiago B; Ralser, Markus; Brindle, Kevin M

2015-12-01

A resonance at ∼181 ppm in the (13) C spectra of tumors injected with hyperpolarized [U-(2) H, U-(13) C]glucose was assigned to 6-phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3-phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG (13) C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) METHODS: Tumor-bearing mice were injected with (13) C6 glucose and the (13) C-labeled and total 6PG and 3PG concentrations measured. (13) C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-(2) H, U-(13) C]glucose and again (13) C-labeled and total 6PG and 3PG were measured by LC-MS/MS RESULTS: Tumor (13) C-6PG was more abundant than (13) C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG. (13) C MR spectra of wild-type and zwf1Δ yeast cells showed a resonance at ∼181 ppm after labeling with hyperpolarized [U-(2) H, U-(13) C]glucose, however, there was no 6PG in zwf1Δ cells. In the wild-type cells 3PG was approximately four-fold more abundant than 6PG CONCLUSION: The resonance at ∼181 ppm in (13) C MR spectra following injection of hyperpolarized [U-(2) H, U-(13) C]glucose originates predominantly from 6PG in EL4 tumors and 3PG in yeast cells. © 2014 Wiley Periodicals, Inc.

Determination of total concentration of chemically labeled metabolites as a means of metabolome sample normalization and sample loading optimization in mass spectrometry-based metabolomics.

PubMed

Wu, Yiman; Li, Liang

2012-12-18

For mass spectrometry (MS)-based metabolomics, it is important to use the same amount of starting materials from each sample to compare the metabolome changes in two or more comparative samples. Unfortunately, for biological samples, the total amount or concentration of metabolites is difficult to determine. In this work, we report a general approach of determining the total concentration of metabolites based on the use of chemical labeling to attach a UV absorbent to the metabolites to be analyzed, followed by rapid step-gradient liquid chromatography (LC) UV detection of the labeled metabolites. It is shown that quantification of the total labeled analytes in a biological sample facilitates the preparation of an appropriate amount of starting materials for MS analysis as well as the optimization of the sample loading amount to a mass spectrometer for achieving optimal detectability. As an example, dansylation chemistry was used to label the amine- and phenol-containing metabolites in human urine samples. LC-UV quantification of the labeled metabolites could be optimally performed at the detection wavelength of 338 nm. A calibration curve established from the analysis of a mixture of 17 labeled amino acid standards was found to have the same slope as that from the analysis of the labeled urinary metabolites, suggesting that the labeled amino acid standard calibration curve could be used to determine the total concentration of the labeled urinary metabolites. A workflow incorporating this LC-UV metabolite quantification strategy was then developed in which all individual urine samples were first labeled with (12)C-dansylation and the concentration of each sample was determined by LC-UV. The volumes of urine samples taken for producing the pooled urine standard were adjusted to ensure an equal amount of labeled urine metabolites from each sample was used for the pooling. The pooled urine standard was then labeled with (13)C-dansylation. Equal amounts of the (12)C-labeled

Effect of body size and body mass on δ 13 C and δ 15 N in coastal fishes and cephalopods

NASA Astrophysics Data System (ADS)

Vinagre, C.; Máguas, C.; Cabral, H. N.; Costa, M. J.

2011-11-01

Carbon and nitrogen isotopes have been widely used in the investigation of trophic relations, energy pathways, trophic levels and migrations, under the assumption that δ 13C is independent of body size and that variation in δ 15N occurs exclusively due to ontogenetic changes in diet and not body size increase per se. However, several studies have shown that these assumptions are uncertain. Data from food-webs containing an important number of species lack theoretical support on these assumptions because very few species have been tested for δ 13C and δ 15N variation in captivity. However, if sampling comprises a wide range of body sizes from various species, the variation of δ 13C and δ 15N with body size can be investigated. While correlation between body size and δ 13C and δ 15N can be due to ontogenetic diet shifts, stability in such values throughout the size spectrum can be considered an indication that δ 13C and δ 15N in muscle tissues of such species is independent of body size within that size range, and thus the basic assumptions can be applied in the interpretation of such food webs. The present study investigated the variation in muscle δ 13C and δ 15N with body size and body mass of coastal fishes and cephalopods. It was concluded that muscle δ 13C and δ 15N did not vary with body size or mass for all bony fishes with only one exception, the dragonet Callionymus lyra. Muscle δ 13C and δ 15N also did not vary with body size or mass in cartilaginous fishes and cephalopods, meaning that body size/mass per se have no effect on δ 13C or δ 15N, for most species analysed and within the size ranges sampled. The assumption that δ 13C is independent of body size and that variation in δ 15N is not affected by body size increase per se was upheld for most organisms and can be applied to the coastal food web studied taking into account that C. lyra is an exception.

Depletion of eugenol residues from the skin-on fillet tissue of rainbow trout exposed to 14C-labeled eugenol

USGS Publications Warehouse

Meinertz, Jeffery R.; Schreier, Theresa M.; Porcher, Scott T.; Smerud, Justin R.; Gaikowski, Mark P.

2014-01-01

The U.S. is lagging in access to an approved immediate-release sedative, i.e. a compound that can be safely and effectively used to sedate fish and has no withdrawal period. AQUI-S® 20E (10% active ingredient, eugenol) is under investigation as an immediate-release sedative for freshwater finfish. Because of its investigational status, data are needed to characterize the depletion, distribution, and identity of AQUI-S® 20E residues in fillet tissue. Rainbow trout (Oncorhynchus mykiss) were exposed to uniformly ring labeled 14C-eugenol at a nominal concentration of 10 mg/L for 60 min in 18 °C water. Fish (n = 6) were sampled immediately after the exposure (0 min) then at 30, 60, 120, and 240 min. Eugenol concentrations and characterization of 14C residues in the fillet tissue were determined by high pressure liquid chromatography and flow-through liquid scintillation counting techniques. Total 14C-residue burdens in fillet tissue were determined by tissue oxidation and static liquid scintillation counting techniques. Maximum eugenol and 14C-eugenol equivalent residue concentrations in the fillet tissue were measured immediately after the exposure (44.5 and 38.8 μg/g, respectively). Eugenol was the primary 14C-residue (> 90% of all 14C-residues) in extracts from fillet tissue taken from fish sampled immediately after the exposure (0 min) and from fish sampled at 30 and 60 min after the exposure. The depletion of 14C-eugenol residues from the fillet tissue was rapid (t1/2 = 26.25 min) after transferring the exposed fish to fresh flowing water.

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

PubMed Central

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

2015-01-01

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

Two Phase 1, Open‐Label, Mass Balance Studies to Determine the Pharmacokinetics of 14C‐Labeled Isavuconazonium Sulfate in Healthy Male Volunteers

PubMed Central

Kato, Kota; Hale, Christine; Kowalski, Donna; Lademacher, Christopher; Yamazaki, Takao; Akhtar, Shahzad; Desai, Amit

2017-01-01

Abstract Isavuconazonium sulfate is the water‐soluble prodrug of the active triazole isavuconazole. Two phase 1 studies were conducted to identify the metabolic profile and mass balance of isavuconazole and BAL8728 (inactive cleavage product). Seven subjects in study 1 (isavuconazole mass balance) received a single oral dose of [cyano‐14C]isavuconazonium sulfate corresponding to 200 mg isavuconazole. Six subjects in study 2 (BAL8728 mass balance) received a single intravenous dose of [pyridinylmethyl‐14C]isavuconazonium sulfate corresponding to 75 mg BAL8728. Pharmacokinetic parameters of radioactivity in whole blood and plasma and of isavuconazole and BAL8728 in plasma were assessed. Radioactivity ratio of blood/plasma, percentage of dose, and cumulative percentage of radioactive dose recovered in urine and feces for isavuconazole and BAL8728 were assessed. Metabolic profiling was carried out by high‐performance liquid chromatography and mass spectrometry. Mean plasma isavuconazole pharmacokinetic parameters included apparent clearance (2.3 ± 0.7 L/h), apparent volume of distribution (301.8 ± 105.7 L), and terminal elimination half‐life (99.9 ± 44.6 hours). In study 1, isavuconazole‐derived radioactivity was recovered approximately equally in urine and feces (46.1% and 45.5%, respectively). In study 2, BAL8728‐derived radioactivity was predominantly recovered in urine (96.0%). Isavuconazole (study 1) and M4 (cleavage metabolite of BAL8728; study 2) were the predominant circulating components of radioactivity in plasma. PMID:28750160

Determination of chemical purity and isotopic composition of natural and carbon-13-labeled arsenobetaine bromide standards by quantitative(1)H-NMR.

PubMed

Le, Phuong-Mai; Ding, Jianfu; Leek, Donald M; Mester, Zoltan; Robertson, Gilles; Windust, Anthony; Meija, Juris

2016-10-01

In this study, we report the characterization of three arsenobetaine-certified reference materials by quantitative NMR. We have synthesized an arsenobetaine bromide high-purity standard of natural isotopic composition (ABET-1) and two carbon-13-labeled isotopic standards (BBET-1 and CBET-1). Assignments of the chemical purity and isotopic composition are not trivial in the case of arsenobetaine, and in this study we utilized quantitative(1)H-NMR techniques for the determination of the mass fractions (chemical purity). The isotopic purity of all three standards was also assessed by NMR from the carbon-13 satellite signals. The standards are non-hygroscopic, high-purity (ca. 0.99 g/g), and the carbon-13 enrichment for both isotopic standards is x((13)C)≈0.99. These standards are designed for use as primary calibrators for mass spectrometric determination of arsenobetaine in environmental samples.

Thawing permafrost increases old soil and autotrophic respiration in tundra: partitioning ecosystem respiration using δ(13) C and ∆(14) C.

PubMed

Hicks Pries, Caitlin E; Schuur, Edward A G; Crummer, Kathryn G

2013-02-01

Ecosystem respiration (Reco ) is one of the largest terrestrial carbon (C) fluxes. The effect of climate change on Reco depends on the responses of its autotrophic and heterotrophic components. How autotrophic and heterotrophic respiration sources respond to climate change is especially important in ecosystems underlain by permafrost. Permafrost ecosystems contain vast stores of soil C (1672 Pg) and are located in northern latitudes where climate change is accelerated. Warming will cause a positive feedback to climate change if heterotrophic respiration increases without corresponding increases in primary production. We quantified the response of autotrophic and heterotrophic respiration to permafrost thaw across the 2008 and 2009 growing seasons. We partitioned Reco using Δ(14) C and δ(13) C into four sources-two autotrophic (above - and belowground plant structures) and two heterotrophic (young and old soil). We sampled the Δ(14) C and δ(13) C of sources using incubations and the Δ(14) C and δ(13) C of Reco using field measurements. We then used a Bayesian mixing model to solve for the most likely contributions of each source to Reco . Autotrophic respiration ranged from 40 to 70% of Reco and was greatest at the height of the growing season. Old soil heterotrophic respiration ranged from 6 to 18% of Reco and was greatest where permafrost thaw was deepest. Overall, growing season fluxes of autotrophic and old soil heterotrophic respiration increased as permafrost thaw deepened. Areas with greater thaw also had the greatest primary production. Warming in permafrost ecosystems therefore leads to increased plant and old soil respiration that is initially compensated by increased net primary productivity. However, barring large shifts in plant community composition, future increases in old soil respiration will likely outpace productivity, resulting in a positive feedback to climate change. © 2012 Blackwell Publishing Ltd.

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

NASA Astrophysics Data System (ADS)

Kanamori, Keiko; Ross, Brian D.

2001-12-01

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

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

PubMed

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

2016-10-15

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

21 CFR 1230.13 - Labeling of “poison”.

Code of Federal Regulations, 2013 CFR

2013-04-01

... FEDERAL CAUSTIC POISON ACT Labeling § 1230.13 Labeling of “poison”. The following are styles of...-point size are required on a label in stating the word “poison” they must not be smaller than those...

21 CFR 1230.13 - Labeling of “poison”.

Code of Federal Regulations, 2012 CFR

2012-04-01

... FEDERAL CAUSTIC POISON ACT Labeling § 1230.13 Labeling of “poison”. The following are styles of...-point size are required on a label in stating the word “poison” they must not be smaller than those...

21 CFR 1230.13 - Labeling of “poison”.

Code of Federal Regulations, 2014 CFR

2014-04-01

... FEDERAL CAUSTIC POISON ACT Labeling § 1230.13 Labeling of “poison”. The following are styles of...-point size are required on a label in stating the word “poison” they must not be smaller than those...

21 CFR 1230.13 - Labeling of “poison”.

Code of Federal Regulations, 2011 CFR

2011-04-01

... FEDERAL CAUSTIC POISON ACT Labeling § 1230.13 Labeling of “poison”. The following are styles of...-point size are required on a label in stating the word “poison” they must not be smaller than those...

21 CFR 1230.13 - Labeling of “poison”.

Code of Federal Regulations, 2010 CFR

2010-04-01

... FEDERAL CAUSTIC POISON ACT Labeling § 1230.13 Labeling of “poison”. The following are styles of...-point size are required on a label in stating the word “poison” they must not be smaller than those...

C-14 content of ten meteorites measured by tandem accelerator mass spectrometry

NASA Technical Reports Server (NTRS)

Brown, R. M.; Andrews, H. R.; Ball, G. C.; Burn, N.; Imahori, Y.; Milton, J. C. D.; Fireman, E. L.

1984-01-01

Measurements of C-14 in three North American and seven Antarctic meteorites show in most cases that this cosmogenic isotope, which is tightly bound, was separated from absorbed atmospheric radiocarbon by stepwise heating extractions. The present upper limit to age determination by the accelerator method varies from 50,000 to 70,000 years, depending on the mass and carbon content of the sample. The natural limit caused by cosmic ray production of C-14 in silicate rocks at 2000 m elevation is estimated to be 55,000 + or - 5000 years. An estimation is also made of the 'weathering ages' of the Antarctic meteorites from the specific activity of loosely bound CO2 which is thought to be absorbed from the terrestrial atmosphere. Accelerator measurements are found to agree with previous low level counting measurements, but are more sensitive and precise.

Metabolism of /sup 14/C-labeled doxylamine succinate (Bendectin) in the rhesus monkey (Macaca mulatta)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slikker, W. Jr.; Holder, C.L.; Lipe, G.W.

The time-course of the metabolic fate of (/sup 14/C)doxylamine was determined after the p.o. administration of 13 mg/kg doxylamine succinate as Bendectin plus (/sup 14/C)doxylamine succinate to the rhesus monkey. Urine and plasma samples were analyzed by reversed-phase high performance liquid chromatography (HPLC), chemical derivatization, and mass spectrometry. The cumulative 48-hr urinary metabolic profile contained 81% of the administered radiolabeled dose and consisted of at least six radiolabeled peaks. They were peak 1: unknown polar metabolites (8% of dose); peak 2: 2-(1-phenyl-1-(2-pyridinyl)ethoxy) acetic acid, 1-(1-phenyl-1(2-pyridinyl)ethoxy) methanol, and another minor metabolite(s) (31%); peak 3: doxylamine-N-oxide (1%); peak 4a: N,N-didesmethyldoxylamine (17%); peakmore » 4b: doxylamine (4%); and peak 5: N-desmethyldoxylamine (20%). The plasma metabolic profile was the same as the urinary profile except for the absence of doxylamine-N-oxide. The maximum plasma concentrations and elapsed time to attain these concentrations were as follows. Peak 1: 540 ng/mL, 4 hr; peak 2: 1700 ng/mL, 1 hr; peak 4a: 430 ng/mL, 4 hr; peak 4b: 930 ng/mL, 2 hr; and peak 5: 790 ng/mL, 2 hr. These data suggest that in the monkey, doxylamine metabolism follows at least four pathways: a minor pathway to the N-oxide; a minor pathway to unknown polar metabolites; a major pathway to mono- and didesmethyldoxylamine via successive N-demethylation; and a major pathway to side-chain cleavage products (peak 2) via direct side-chain oxidation and/or deamination.« less

Nicotine, acetanilide and urea multi-level2H-,13C- and15N-abundance reference materials for continuous-flow isotope ratio mass spectrometry

USGS Publications Warehouse

Schimmelmann, A.; Albertino, A.; Sauer, P.E.; Qi, H.; Molinie, R.; Mesnard, F.

2009-01-01

Accurate determinations of stable isotope ratios require a calibration using at least two reference materials with different isotopic compositions to anchor the isotopic scale and compensate for differences in machine slope. Ideally, the S values of these reference materials should bracket the isotopic range of samples with unknown S values. While the practice of analyzing two isotopically distinct reference materials is common for water (VSMOW-SLAP) and carbonates (NBS 19 and L-SVEC), the lack of widely available organic reference materials with distinct isotopic composition has hindered the practice when analyzing organic materials by elemental analysis/isotope ratio mass spectrometry (EA-IRMS). At present only L-glutamic acids USGS40 and USGS41 satisfy these requirements for ??13C and ??13N, with the limitation that L-glutamic acid is not suitable for analysis by gas chromatography (GC). We describe the development and quality testing of (i) four nicotine laboratory reference materials for on-line (i.e. continuous flow) hydrogen reductive gas chromatography-isotope ratio mass-spectrometry (GC-IRMS), (ii) five nicotines for oxidative C, N gas chromatography-combustion-isotope ratio mass-spectrometry (GC-C-IRMS, or GC-IRMS), and (iii) also three acetanilide and three urea reference materials for on-line oxidative EA-IRMS for C and N. Isotopic off-line calibration against international stable isotope measurement standards at Indiana University adhered to the 'principle of identical treatment'. The new reference materials cover the following isotopic ranges: ??2Hnicotine -162 to -45%o, ??13Cnicotine -30.05 to +7.72%, ?? 15Nnicotine -6.03 to +33.62%; ??15N acetanilide +1-18 to +40.57%; ??13Curea -34.13 to +11.71%, ??15Nurea +0.26 to +40.61% (recommended ?? values refer to calibration with NBS 19, L-SVEC, IAEA-N-1, and IAEA-N-2). Nicotines fill a gap as the first organic nitrogen stable isotope reference materials for GC-IRMS that are available with different ??13N

The dynamics of folic acid metabolism in an adult given a small tracer dose of 14C-folic acid.

PubMed

Clifford, A J; Arjomand, A; Dueker, S R; Schneider, P D; Buchholz, B A; Vogel, J S

1998-01-01

Folate is an essential nutrient that is involved in many metabolic pathways, including amino acid interconversions and nucleotide (DNA) synthesis. In genetically susceptible individuals and populations, dysfunction of folate metabolism is associated with severe illness. Despite the importance of folate, major gaps exist in our quantitative understanding of folate metabolism in humans. The gaps exist because folate metabolism is complex, a suitable animal model that mimics human folate metabolism has not been identified, and suitable experimental protocols for in vivo studies in humans are not developed. In general, previous studies of folate metabolism have used large doses of high specific activity tritium and 14C-labeled folates in clinical patients. While stable isotopes such as deuterium and 13C-labeled folate are viewed as ethical alternatives to radiolabeled folates for studying metabolism, the lack of sensitive mass spectrometry methods to quantify them has impeded advancement of the field using this approach. In this chapter, we describe a new approach that uses a major analytical breakthrough, Accelerator Mass Spectrometry (AMS). Because AMS can detect attomole concentrations of 14C, small radioactive dosages (nCi) can be safely administered to humans and traced over long periods of time. The needed dosages are sufficiently small that the total radiation exposure is only a fraction of the natural annual background radiation of Americans, and the generated laboratory waste may legally be classified non-radioactive in many cases. The availability of AMS has permitted the longest (202 d) and most detailed study to date of folate metabolism in a healthy adult human volunteer. Here we demonstrate the feasibility of our approach and illustrate its potential by determining empirical kinetic values of folate metabolism. Our data indicate that the mean sojourn time for folate is in the range of 93 to 120 d. It took > or = 350 d for the absorbed portion of small

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

PubMed

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

2014-04-01

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

Carbon transitions from either Calvin cycle or transitory starch to heteroglycans as revealed by (14) C-labeling experiments using protoplasts from Arabidopsis.

PubMed

Malinova, Irina; Steup, Martin; Fettke, Joerg

2013-09-01

Plants metabolize transitory starch by precisely coordinated plastidial and cytosolic processes. The latter appear to include the action of water-soluble heteroglycans (SHGin ) whose monosaccharide pattern is similar to that of apoplastic glycans (SHGex ) but, unlike SHGex , SHGin strongly interacts with glucosyl transferases. In this study, we analyzed starch metabolism using mesophyll protoplasts from wild-type plants and two knock-out mutants [deficient in the cytosolic transglucosidase, disproportionating isoenzyme 2 (DPE2) or the plastidial phosphoglucomutase (PGM1)] from Arabidopsis thaliana. Protoplasts prelabeled by photosynthetic (14) CO2 fixation were transferred to an unlabeled medium and were darkened or illuminated. Carbon transitions from the Calvin cycle or from starch to both SHGin and SHGex were analyzed. In illuminated protoplasts, starch turn-over was undetectable but darkened protoplasts continuously degraded starch. During illumination, neither the total (14) C content nor the labeling patterns of the sugar residues of SHGin were significantly altered but both the total amount and the labeling of the constituents of SHGex increased with time. In darkened protoplasts, the (14) C-content of most of the sugar residues of SHGin transiently and strongly increased and then declined. This effect was not observed in any SHGex constituent. In darkened DPE2-deficient protoplasts, none of the SHGin constituents exhibited an essential transient increase in labeling. In contrast, some residues of SHGin from the PGM1 mutant exhibited a transient increase in label but this effect significantly differed from that of the wild type. Two conclusions are reached: first, SHGin and SHGex exert different metabolic functions and second, SHGin is directly involved in starch degradation. © 2013 Scandinavian Plant Physiology Society.

Linking of Microorganisms to Phenanthrene Metabolism in Soil by Analysis of 13C-Labeled Cell Lipids

PubMed Central

Johnsen, Anders R.; Winding, Anne; Karlson, Ulrich; Roslev, Peter

2002-01-01

Phenanthrene-metabolizing soil microbial communities were characterized by examining mineralization of [14C]phenanthrene, by most-probable-number (MPN) counting, by 16S-23S spacer DNA analysis of the numerically dominant, culturable phenanthrene-degrading isolates, and by examining incorporation of [13C]phenanthrene-derived carbon into sterols and polar lipid fatty acids (PLFAs). An unpolluted agricultural soil, a roadside soil diffusely polluted with polycyclic aromatic hydrocarbons (PAHs), and two highly PAH-polluted soils from industrial sites were analyzed. Microbial phenanthrene degraders were not detected by MPN counting in the agricultural soil and the roadside soil. In the industrial soils, phenanthrene degraders constituted 0.04 and 3.6% of the total number of CFU. 16S-23S spacer DNA analysis followed by partial 16S DNA sequencing of representative isolates from one of the industrial soils showed that one-half of the isolates belonged to the genus Sphingomonas and the other half were closely related to an unclassified beta-proteobacterium. The 13C-PLFA profiles of the two industrial soils were relatively similar and resembled the profiles of phenanthrene-degrading Sphingomonas reference strains and unclassified beta-proteobacterium isolates but did not match the profiles of Pseudomonas, Mycobacterium, or Nocardia reference strains. The 13C-PLFA profiles of phenanthrene degraders in the agricultural soil and the roadside soil were different from each other and different from the profiles of the highly polluted industrial soils. Only in the roadside soil were 10me/12me18:0 PLFAs enriched in 13C, suggesting that actinomycetes metabolized phenanthrene in this soil. The 13C-PLFA profiles of the unpolluted agricultural soil did not resemble the profiles of any of the reference strains. In all of the soils investigated, no excess 13C was recovered in the 18:2ω6,9 PLFA, suggesting that fungi did not contribute significantly to assimilation of [13C

Assay for vitamin B12 absorption and method of making labeled vitamin B12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, Peter J; Dueker, Stephen; Miller, Joshua

2012-06-19

The invention provides methods for labeling vitamin B12 with .sup.14C, .sup.13C, tritium, and deuterium. When radioisotopes are used, the invention provides for methods of labeling B12 with high specific activity. The invention also provides labeled vitamin B12 compositions made in accordance with the invention.

Carbon transfer dynamics from bomb- 14C and δ 13C time series of a laminated stalagmite from SW France - modelling and comparison with other stalagmite records

NASA Astrophysics Data System (ADS)

Genty, Dominique; Massault, Marc

1999-05-01

Twenty-two AMS 14C measurements have been made on a modern stalagmite from SW France in order to reconstruct the 14C activity history of the calcite deposit. Annual growth laminae provides a chronology up to 1919 A.D. Results show that the stalagmite 14C activity time series is sensitive to modern atmosphere 14C activity changes such as those produced by the nuclear weapon tests. The comparison between the two 14C time series shows that the stalagmite time series is damped: its amplitude variation between pre-bomb and post-bomb values is 75% less and the time delay between the two time series peaks is 16 years ±3. A model is developed using atmosphere 14C and 13C data, fractionation processes and three soil organic matter components whose mean turnover rates are different. The linear correlation coefficient between modeled and measured activities is 0.99. These results, combined with two other stalagmite 14C time series already published and compared with local vegetation and climate, demonstrate that most of the carbon transfer dynamics are controlled in the soil by soil organic matter degradation rates. Where vegetation produces debris whose degradation is slow, the fraction of old carbon injected in the system increases, the observed 14C time series is much more damped and lag time longer than that observed under grassland sites. The same mixing model applied on the 13C shows a good agreement ( R2 = 0.78) between modeled and measured stalagmite δ 13C and demonstrates that the Suess Effect due to fossil fuel combustion in the atmosphere is recorded in the stalagmite but with a damped effect due to SOM degradation rate. The different sources of dead carbon in the seepage water are calculated and discussed.

Metabolic characterization of cultured mammalian cells by mass balance analysis, tracer labeling experiments and computer-aided simulations.

PubMed

Okahashi, Nobuyuki; Kohno, Susumu; Kitajima, Shunsuke; Matsuda, Fumio; Takahashi, Chiaki; Shimizu, Hiroshi

2015-12-01

Studying metabolic directions and flow rates in cultured mammalian cells can provide key information for understanding metabolic function in the fields of cancer research, drug discovery, stem cell biology, and antibody production. In this work, metabolic engineering methodologies including medium component analysis, (13)C-labeling experiments, and computer-aided simulation analysis were applied to characterize the metabolic phenotype of soft tissue sarcoma cells derived from p53-null mice. Cells were cultured in medium containing [1-(13)C] glutamine to assess the level of reductive glutamine metabolism via the reverse reaction of isocitrate dehydrogenase (IDH). The specific uptake and production rates of glucose, organic acids, and the 20 amino acids were determined by time-course analysis of cultured media. Gas chromatography-mass spectrometry analysis of the (13)C-labeling of citrate, succinate, fumarate, malate, and aspartate confirmed an isotopically steady state of the cultured cells. After removing the effect of naturally occurring isotopes, the direction of the IDH reaction was determined by computer-aided analysis. The results validated that metabolic engineering methodologies are applicable to soft tissue sarcoma cells derived from p53-null mice, and also demonstrated that reductive glutamine metabolism is active in p53-null soft tissue sarcoma cells under normoxia. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Analysis of concentration and (13)C enrichment of D-galactose in human plasma.

PubMed

Schadewaldt, P; Hammen, H W; Loganathan, K; Bodner-Leidecker, A; Wendel, U

2000-05-01

A stable-isotope dilution method for the sensitive determination of D-galactose in human plasma was established. D-[(13)C]Galactose was added to plasma, and the concentration was measured after D-glucose was removed from the plasma by treatment with D-glucose oxidase and the sample was purified by ion-exchange chromatography. For gas chromatographic-mass spectrometric analysis, aldononitrile pentaacetate derivatives were prepared. Monitoring of the [MH-60](+) ion intensities at m/z 328, 329, and 334 in the positive chemical ionization mode allowed the assessment of 1-(12)C-, 1-(13)C-, and U-(13)C(6)-labeled D-galactose, respectively. The D-galactose concentration was quantified on the basis of the (13)C-labeled internal standard. The method was linear (range examined, 0.1-5 micromol/L) and of good repeatability in the low and high concentration ranges (within- and between-run CVs <15%). The limit of quantification for plasma D-galactose was <0.02 micromol/L. Measurements in plasma of postabsorptive subjects yielded D-galactose concentrations (mean +/- SD) of 0.12 +/- 0.03 (n = 16), 0.11 +/- 0.04 (n = 15), 1.44 +/- 0.54 (n = 10), and 0.17 +/- 0.07 (n = 5) micromol/L in healthy adults, diabetic patients, patients with classical galactosemia, and obligate heterozygous parents thereof, respectively. These data were considerably lower (3- to 18-fold) than the values of a conventional enzymatic assay. The procedure was also applied successfully in a stable-isotope turnover study to evaluate endogenous D-galactose formation. The present findings establish that detection of D-galactose from endogenous sources is feasible in human plasma and show that erroneously high results may be obtained by enzymatic methods.