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Sample records for isotope labeled coa

  1. Stable isotopes labelled compounds

    International Nuclear Information System (INIS)

    1982-09-01

    The catalogue on stable isotopes labelled compounds offers deuterium, nitrogen-15, and multiply labelled compounds. It includes: (1) conditions of sale and delivery, (2) the application of stable isotopes, (3) technical information, (4) product specifications, and (5) the complete delivery programme

  2. SAIL--stereo-array isotope labeling.

    Science.gov (United States)

    Kainosho, Masatsune; Güntert, Peter

    2009-11-01

    Optimal stereospecific and regiospecific labeling of proteins with stable isotopes enhances the nuclear magnetic resonance (NMR) method for the determination of the three-dimensional protein structures in solution. Stereo-array isotope labeling (SAIL) offers sharpened lines, spectral simplification without loss of information and the ability to rapidly collect and automatically evaluate the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as before. This review gives an overview of stable isotope labeling methods for NMR spectroscopy with proteins and provides an in-depth treatment of the SAIL technology.

  3. Melatonin labeled with hydrogen isotopes

    International Nuclear Information System (INIS)

    Dmitrevskaya, L.I.; Smushkevich, Yu.I.; Kurkovskaya, L.N.; Ponomarenko, N.K.; Suvorov, N.N.

    1989-01-01

    A study has been made of isotope exchange between melatonin and deuterium (D 2 O) or tritium (HTO) oxide under different conditions. The ease of isotope exchange for the indole ring hydrogens of melatonin in an acidic medium decreases over the series H 4 > H 2 H 6 >> H 7 , enabling the authors to process a route for production of melatonin labeled with hydrogen isotopes at positions 4,6, and 2 of the indole ring. A method has been suggested for producing melatonin labeled with hydrogen isotopes at position 2 by desulfurization of 2-(2,4-dinitro-phenylsulfenyl)melatonin at Ni(Re) (D)

  4. Melatonin labelled by hydrogen isotopes

    International Nuclear Information System (INIS)

    Dmitrevskaya, L.I.; Smushkevich, Yu.I.; Kurkovskaya, L.N.; Ponomarenko, N.K.; Suvorov, N.N.

    1988-01-01

    Isotope exchange of melatonin with deuterium (D 2 O) and tritium (HTO) oxides under different conditions is studied. Simplicity of isotope exchange of hydrogens of the indole ring of melatonin in the acidic medium decreases in series H 4 >H 2 >H 6 >>H 7 , that permits to suggest the way of melatonin preparation labelled by hydrogen isotopes in positions 4,6 and 2 of the indole ring. The way of melatonin preparation labelled by hydrogen isotopes in position 2 according to the reaction of desulfation 2-(2,4-dinitrophenylsulphenyl) melatonin at catalyst Ni(Re)(D) is suggested

  5. Isotopically labelled pyrimidines and purines

    International Nuclear Information System (INIS)

    Balaban, A.T.; Bally, I.

    1987-01-01

    Among the three diazines, pyrimidine is by far the most important one because its derivatives uracil, thymine and cytosine are constituents of the ubiquitous deoxynucleic acids (DNA) and ribonucleic acids (RNA). Other derivatives of pyrimidine without condensed rings include barbiturates, alloxan, orotic acid and thiamine or vitamin B 1 . From the polycyclic derivatives of pyrimidine such as pteridine, alloxazine, and purine, the latter, through its derivatives adenine and guanine complete the list of bases which occur in DNA and RNA: in addition, other purine derivatives such as hypoxanthine, xanthine, theobromine, theophylline, caffeine and uric acid are important natural products with biological activity. The paper presents methods for preparing isotopically labeled pyrimidines as well as purine derivatives. For convenience, the authors describe separately carbon-labeled with radioisotopes 11 C (T 1/2 = 20.3 min) and 14 C (T 1/2 = 5736 years) or the stable isotope 13 C (natural abundance 1.1%) and then hydrogen-labeled systems with the radioisotope 3 H ≡ T (T 1/2 = 12.346 years) or with the stable isotope 2 H ≡ D (natural abundance 0.015%). We do not separate stable from radioactive isotopes because the synthetic methods are identical for the same element; however, the introduction of hydrogen isotopes into organic molecules is often performed by reactions such as isotope exchange which cannot take place in the case of carbon isotopes

  6. Synthesis of O-[11C]acetyl CoA, O-[11C]acetyl-L-carnitine, and L-[11C]carnitine labelled in specific positions, applied in PET studies on rhesus monkey

    International Nuclear Information System (INIS)

    Jacobson, Gunilla B.; Watanabe, Yasuyoshi; Valind, Sven; Kuratsune, Hirohiko; Laangstroem, Bengt

    1997-01-01

    The syntheses of L-carnitine, O-acetyl CoA, and O-acetyl-L-carnitine labelled with 11 C at the 1- or 2-position of the acetyl group or the N-methyl position of carnitine, using the enzymes acetyl CoA synthetase and carnitine acetyltransferase, are described. With a total synthesis time of 45 min, O-[1- 11 C]acetyl CoA and O-[2- 11 C]acetyl CoA was obtained in 60-70% decay-corrected radiochemical yield, and O-[1- 11 C]acetyl-L-carnitine and O-[2- 11 C]acetyl-L-carnitine in 70-80% yield, based on [1- 11 C]acetate or [2- 11 C]acetate, respectively. By an N-methylation reaction with [ 11 C]methyl iodide, L-[methyl- 11 C]carnitine was obtained within 30 min, and O-acetyl-L-[methyl- 11 C]carnitine within 40 min, giving a decay-corrected radiochemical yield of 60% and 40-50%, respectively, based on [ 11 C]methyl iodide. Initial data of the kinetics of the different 11 C-labelled L-carnitine and acetyl-L-carnitines in renal cortex of anaesthetized monkey (Macaca mulatta) are presented

  7. Synthesis of O-[{sup 11}C]acetyl CoA, O-[{sup 11}C]acetyl-L-carnitine, and L-[{sup 11}C]carnitine labelled in specific positions, applied in PET studies on rhesus monkey

    Energy Technology Data Exchange (ETDEWEB)

    Jacobson, Gunilla B.; Watanabe, Yasuyoshi; Valind, Sven; Kuratsune, Hirohiko; Laangstroem, Bengt

    1997-07-01

    The syntheses of L-carnitine, O-acetyl CoA, and O-acetyl-L-carnitine labelled with {sup 11}C at the 1- or 2-position of the acetyl group or the N-methyl position of carnitine, using the enzymes acetyl CoA synthetase and carnitine acetyltransferase, are described. With a total synthesis time of 45 min, O-[1-{sup 11}C]acetyl CoA and O-[2-{sup 11}C]acetyl CoA was obtained in 60-70% decay-corrected radiochemical yield, and O-[1-{sup 11}C]acetyl-L-carnitine and O-[2-{sup 11}C]acetyl-L-carnitine in 70-80% yield, based on [1-{sup 11}C]acetate or [2-{sup 11}C]acetate, respectively. By an N-methylation reaction with [{sup 11}C]methyl iodide, L-[methyl-{sup 11}C]carnitine was obtained within 30 min, and O-acetyl-L-[methyl-{sup 11}C]carnitine within 40 min, giving a decay-corrected radiochemical yield of 60% and 40-50%, respectively, based on [{sup 11}C]methyl iodide. Initial data of the kinetics of the different {sup 11}C-labelled L-carnitine and acetyl-L-carnitines in renal cortex of anaesthetized monkey (Macaca mulatta) are presented.

  8. Stable isotope labeling strategy based on coding theory

    Energy Technology Data Exchange (ETDEWEB)

    Kasai, Takuma; Koshiba, Seizo; Yokoyama, Jun; Kigawa, Takanori, E-mail: kigawa@riken.jp [RIKEN Quantitative Biology Center (QBiC), Laboratory for Biomolecular Structure and Dynamics (Japan)

    2015-10-15

    We describe a strategy for stable isotope-aided protein nuclear magnetic resonance (NMR) analysis, called stable isotope encoding. The basic idea of this strategy is that amino-acid selective labeling can be considered as “encoding and decoding” processes, in which the information of amino acid type is encoded by the stable isotope labeling ratio of the corresponding residue and it is decoded by analyzing NMR spectra. According to the idea, the strategy can diminish the required number of labelled samples by increasing information content per sample, enabling discrimination of 19 kinds of non-proline amino acids with only three labeled samples. The idea also enables this strategy to combine with information technologies, such as error detection by check digit, to improve the robustness of analyses with low quality data. Stable isotope encoding will facilitate NMR analyses of proteins under non-ideal conditions, such as those in large complex systems, with low-solubility, and in living cells.

  9. Stable isotope labeling strategy based on coding theory

    International Nuclear Information System (INIS)

    Kasai, Takuma; Koshiba, Seizo; Yokoyama, Jun; Kigawa, Takanori

    2015-01-01

    We describe a strategy for stable isotope-aided protein nuclear magnetic resonance (NMR) analysis, called stable isotope encoding. The basic idea of this strategy is that amino-acid selective labeling can be considered as “encoding and decoding” processes, in which the information of amino acid type is encoded by the stable isotope labeling ratio of the corresponding residue and it is decoded by analyzing NMR spectra. According to the idea, the strategy can diminish the required number of labelled samples by increasing information content per sample, enabling discrimination of 19 kinds of non-proline amino acids with only three labeled samples. The idea also enables this strategy to combine with information technologies, such as error detection by check digit, to improve the robustness of analyses with low quality data. Stable isotope encoding will facilitate NMR analyses of proteins under non-ideal conditions, such as those in large complex systems, with low-solubility, and in living cells

  10. Stereoselective synthesis of stable-isotope-labeled amino acids

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, C.J.; Martinez, R.A.; Silks, L.A. III [Los Alamos National Laboratory, NM (United States); Lodwig, S.N. [Centralia College, WA (United States)

    1994-12-01

    For magnetic resonance and vibrational spectroscopies to reach their full potential, they must be used in combination with sophisticated site-specific stable isotope labeling of biological macromolecules. Labeled amino acids are required for the study of the structure and function of enzymes and proteins. Because there are 20 common amino acids, each with its own distinguishing chemistry, they remain a synthetic challenge. The Oppolzer chiral auxiliary provides a general tool with which to approach the synthesis of labeled amino acids. By using the Oppolzer auxiliary, amino acids can be constructed from several small molecules, which is ideal for stable isotope labeling. In addition to directing the stereochemistry at the {alpha}-carbon, the camphorsultam can be used for stereo-specific isotope labeling at prochiral centers in amino acids. By using the camphorsultam auxiliary we have the potential to synthesize virtually any isotopomer of all of the common amino acids.

  11. Stereoselective synthesis of stable-isotope-labeled amino acids

    International Nuclear Information System (INIS)

    Unkefer, C.J.; Martinez, R.A.; Silks, L.A. III; Lodwig, S.N.

    1994-01-01

    For magnetic resonance and vibrational spectroscopies to reach their full potential, they must be used in combination with sophisticated site-specific stable isotope labeling of biological macromolecules. Labeled amino acids are required for the study of the structure and function of enzymes and proteins. Because there are 20 common amino acids, each with its own distinguishing chemistry, they remain a synthetic challenge. The Oppolzer chiral auxiliary provides a general tool with which to approach the synthesis of labeled amino acids. By using the Oppolzer auxiliary, amino acids can be constructed from several small molecules, which is ideal for stable isotope labeling. In addition to directing the stereochemistry at the α-carbon, the camphorsultam can be used for stereo-specific isotope labeling at prochiral centers in amino acids. By using the camphorsultam auxiliary we have the potential to synthesize virtually any isotopomer of all of the common amino acids

  12. Isotopically labelled vitamin D derivatives and processes for preparing same

    International Nuclear Information System (INIS)

    Deluca, H.R.; Schnoes, H.K.; Napoli, J.L.; Fivizzani, M.A.

    1981-01-01

    This invention relates to 26,27-isotopically labelled vitamin D 3 compounds, including radiolabelled vitamin D 3 compounds of high specific activity, methods for their preparation, and intermediates obtained in their synthesis. The method involves reacting an ester of a 26,27-dinor-vitamin D-25-carboxylic acid with an isotopically labelled methyl Grignard reagent or methyl lithium reagent to obtain a 26,27-isotopically labelled compound in which at least some of the H atoms and/or C atoms are heavy isotopes. (author)

  13. Custom synthesis of isotope-labelled Apis mellifera Pheromone

    International Nuclear Information System (INIS)

    Conanan, Aida P.; Cortes, Nicole Marie A.; Daguno, Cristel Lyn R.; Templonuevo, Jose Angelo A.; Sucgang, Raymond J.

    2012-01-01

    The object of this study is to determine the optimum conditions for the synthesis of the isotope-labelled isopentyl acetate. Isopentyl acetate is widely used as a raw material in industries, in syntheses, and is utilized as a sex attractant (pheromone) by the bee species, Apis mellifera. The isotope labelling of isopentyl acetate will allow tracking of the fate and movement of the isopentyl acetate in the environment, in chemical transformations, and in biological systems. Esterification by alcoholysis of acetic acid was optimized for the preparation of Carbon-14( 14 C)-labelled isopentyl acetate from 14 C-labelled acetic acid and isoamyl alcohol. The different conditions studied were: (1) The effects of acid catalysis and/or reflux on the incorporation and retention of the isotope label on the product. The efficiency of label incorporation and retention was determined through the beta radioactivity of Carbon 14 in each of the synthetic constructs. Determination of the beta radioactivity concentration of 14 C in the isopentyl acetate product was done using low level liquid scintillation spectrometry. Each of the synthetic products was mixed with Ultima Gold scintillation cocktail in a low potassium glass scintillation vial, and analysed in a low-level Wallac 1414 scintillation counter. The application of catalysis without reflux resulted in the highest yield (35%). The same condition also resulted in the highest abundance of carbon isotope label with 2.40 Bequerels per cubic centimetre, Bq/cc (measurement unit for radioactivity). (author)

  14. Protein labelling with stable isotopes: strategies

    International Nuclear Information System (INIS)

    Lirsac, P.N.; Gilles, N.; Jamin, N.; Toma, F.; Gabrielsen, O.; Boulain, J.C.; Menez, A.

    1994-01-01

    A protein labelling technique with stable isotopes has been developed at the CEA: a labelled complete medium has been developed, performing as well as the Luria medium, but differing from it because it contains not only free aminated acids and peptides, but also sugars (96% of D-glucopyrannose) and labelled nucleosides. These precursors are produced from a labelled photosynthetic micro-organisms biomass, obtained with micro-algae having incorporated carbon 13, nitrogen 15 and deuterium during their culture. Labelling costs are reduced. 1 fig., 1 tab., 3 refs

  15. Custom isotope-labelling of apis mellifera pheromone

    International Nuclear Information System (INIS)

    Conanan, Aida P.; Cortes, Nicole Marie A.; Daguno, Cristel Lyn R.; Templonuevo, Jose Angelo A.; Sucgang, Raymond J.

    2012-01-01

    The object of this study is to determine the optimum conditions for the synthesis of isotope-labelled isoamyl acetate. Esterification by alcoholysis of acetic acid was optimized for the preparation of Carbon - 14 ( 14 C)-labelled isopentyl acetate from 14 C-labelled acetic acid and isopentyl alcohol. The optimization procedure defined the effects of catalysis, reflux time, and temperature. The application of catalysis without reflux resulted to the highest yield (40%); the same condition also resulted to the highest abundance of carbon isotope label with 2.40 disintegrations per minute per cubic centimetre, DPM/cc (measurement unit for radioactivity). Determination of the beta radioactivity concentration of 14 C in the isopentyl acetate product was done using low level liquid scintillation spectrometry. Each of the synthetic constructs was mixed with Ultima Gold scintillation cocktail in a glass scintillation vial, and analysed in a low-level Wallac 1414 scintillation counter. Samples were counted for 2 hours in a chamber temperature maintained at 14 degree centegrade. The catalysed reaction without reflux was established to be the most efficient scheme for the radiolabelling. The radiolabelled isoamyl acetate can give way to the synthesis of more complex substances which can be then tracked when they are introduced to a system through the carbon isotope label. (author)

  16. [Progress in stable isotope labeled quantitative proteomics methods].

    Science.gov (United States)

    Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

    2013-06-01

    Quantitative proteomics is an important research field in post-genomics era. There are two strategies for proteome quantification: label-free methods and stable isotope labeling methods which have become the most important strategy for quantitative proteomics at present. In the past few years, a number of quantitative methods have been developed, which support the fast development in biology research. In this work, we discuss the progress in the stable isotope labeling methods for quantitative proteomics including relative and absolute quantitative proteomics, and then give our opinions on the outlook of proteome quantification methods.

  17. Pharmaceuticals labelled with stable isotopes

    International Nuclear Information System (INIS)

    Krumbiegel, P.

    1986-11-01

    The relatively new field of pharmaceuticals labelled with stable isotopes is reviewed. Scientific, juridical, and ethical questions are discussed concerning the application of these pharmaceuticals in human medicine. 13 C, 15 N, and 2 H are the stable isotopes mainly utilized in metabolic function tests. Methodical contributions are given to the application of 2 H, 13 C, and 15 N pharmaceuticals showing new aspects and different states of development in the field under discussion. (author)

  18. Stable isotope dimethyl labelling for quantitative proteomics and beyond

    Science.gov (United States)

    Hsu, Jue-Liang; Chen, Shu-Hui

    2016-01-01

    Stable-isotope reductive dimethylation, a cost-effective, simple, robust, reliable and easy-to- multiplex labelling method, is widely applied to quantitative proteomics using liquid chromatography-mass spectrometry. This review focuses on biological applications of stable-isotope dimethyl labelling for a large-scale comparative analysis of protein expression and post-translational modifications based on its unique properties of the labelling chemistry. Some other applications of the labelling method for sample preparation and mass spectrometry-based protein identification and characterization are also summarized. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644970

  19. 13C metabolic flux analysis: optimal design of isotopic labeling experiments.

    Science.gov (United States)

    Antoniewicz, Maciek R

    2013-12-01

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

  20. Differences among Adult COAs and Adult Non-COAs on Levels of Self-Esteem, Depression, and Anxiety.

    Science.gov (United States)

    Dodd, David T.; Roberts, Richard L.

    1994-01-01

    Examined self-esteem, depression, and anxiety among 60 adult children of alcoholics (COAs) and 143 adult non-COAs. Subjects completed Children of Alcoholics Screening Test, demographic questionnaire, Beck Depression Inventory, State-Trait Anxiety Inventory, and Coopersmith Self-Esteem Inventory. Found no significant differences between COAs and…

  1. Gluconeogenesis from labeled carbon: estimating isotope dilution

    International Nuclear Information System (INIS)

    Kelleher, J.K.

    1986-01-01

    To estimate the rate of gluconeogenesis from steady-state incorporation of labeled 3-carbon precursors into glucose, isotope dilution must be considered so that the rate of labeling of glucose can be quantitatively converted to the rate of gluconeogenesis. An expression for the value of this isotope dilution can be derived using mathematical techniques and a model of the tricarboxylic acid (TCA) cycle. The present investigation employs a more complex model than that used in previous studies. This model includes the following pathways that may affect the correction for isotope dilution: 1) flux of 3-carbon precursor to the oxaloacetate pool via acetyl-CoA and the TCA cycle; 2) flux of 4- or 5-carbon compounds into the TCA cycle; 3) reversible flux between oxaloacetate (OAA) and pyruvate and between OAA and fumarate; 4) incomplete equilibrium between OAA pools; and 5) isotope dilution of 3-carbon tracers between the experimentally measured pool and the precursor for the TCA-cycle OAA pool. Experimental tests are outlined which investigators can use to determine whether these pathways are significant in a specific steady-state system. The study indicated that flux through these five pathways can significantly affect the correction for isotope dilution. To correct for the effects of these pathways an alternative method for calculating isotope dilution is proposed using citrate to relate the specific activities of acetyl-CoA and OAA

  2. Isotope labeling strategies for NMR studies of RNA

    International Nuclear Information System (INIS)

    Lu, Kun; Miyazaki, Yasuyuki; Summers, Michael F.

    2010-01-01

    The known biological functions of RNA have expanded in recent years and now include gene regulation, maintenance of sub-cellular structure, and catalysis, in addition to propagation of genetic information. As for proteins, RNA function is tightly correlated with structure. Unlike proteins, structural information for larger, biologically functional RNAs is relatively limited. NMR signal degeneracy, relaxation problems, and a paucity of long-range 1 H- 1 H dipolar contacts have limited the utility of traditional NMR approaches. Selective isotope labeling, including nucleotide-specific and segmental labeling strategies, may provide the best opportunities for obtaining structural information by NMR. Here we review methods that have been developed for preparing and purifying isotopically labeled RNAs, as well as NMR strategies that have been employed for signal assignment and structure determination.

  3. Isotopically labelled benzodiazepines

    International Nuclear Information System (INIS)

    Liebman, A.A.

    1987-01-01

    This paper reports on the benzodiazepines which are a class of therapeutic agents. Improvements in the analytical methodology in the areas of biochemistry and pharmacology were significant, particularly in the application of chromatographic and spectroscopic techniques. In addition, the discovery and subsequent development of tritium and carbon-14 as an analytical tool in the biological sciences were essentially post-world war II phenomena. Thus, as these new chemical entities were found to be biologically active, they could be prepared in labeled form for metabolic study, biological half-life determination (pharmacokinetics), tissue distribution study, etc. This use of tracer methodology has been liberally applied to the benzodiazepines and also more recently to the study of receptor-ligand interactions, in which tritium, carbon-11 or fluorine-18 isotopes have been used. The history of benzodiazepines as medicinal agents is indeed an interesting one; an integral part of that history is their use in just about every conceivable labeled form

  4. Simple, rapid method for the preparation of isotopically labeled formaldehyde

    Science.gov (United States)

    Hooker, Jacob Matthew [Port Jefferson, NY; Schonberger, Matthias [Mains, DE; Schieferstein, Hanno [Aabergen, DE; Fowler, Joanna S [Bellport, NY

    2011-10-04

    Isotopically labeled formaldehyde (*C.sup..sctn.H.sub.2O) is prepared from labeled methyl iodide (*C.sup..sctn.H.sub.3I) by reaction with an oxygen nucleophile having a pendant leaving group. The mild and efficient reaction conditions result in good yields of *C.sup..sctn.H.sub.2O with little or no *C isotopic dilution. The simple, efficient production of .sup.11CH.sub.2O is described. The use of the .sup.11CH.sub.2O for the formation of positron emission tomography tracer compounds is described. The reaction can be incorporated into automated equipment available to radiochemistry laboratories. The isotopically labeled formaldehyde can be used in a variety of reactions to provide radiotracer compounds for imaging studies as well as for scintillation counting and autoradiography.

  5. Characterization and relative quantification of phospholipids based on methylation and stable isotopic labeling[S

    Science.gov (United States)

    Cai, Tanxi; Shu, Qingbo; Liu, Peibin; Niu, Lili; Guo, Xiaojing; Ding, Xiang; Xue, Peng; Xie, Zhensheng; Wang, Jifeng; Zhu, Nali; Wu, Peng; Niu, Lili; Yang, Fuquan

    2016-01-01

    Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function as second messengers in signal transduction and play a pivotal role in numerous cellular processes, including cell growth, survival, and motility. Here, we present an integrated novel method that combines a fast and robust TMS-diazomethane-based phosphate derivatization and isotopic labeling strategy, which enables simultaneous profiling and relative quantification of PLs from biological samples. Our results showed that phosphate methylation allows fast and sensitive identification of the six major PL classes, including their lysophospholipid counterparts, under positive ionization mode. The isotopic labeling of endogenous PLs was achieved by deuterated diazomethane, which was generated through acid-catalyzed hydrogen/deuterium (H/D) exchange and methanolysis of TMS-diazomethane during the process of phosphate derivatization. The measured H/D ratios of unlabeled and labeled PLs, which were mixed in known proportions, indicated that the isotopic labeling strategy is capable of providing relative quantitation with adequate accuracy, reproducibility, and a coefficient of variation of 9.1%, on average. This novel method offers unique advantages over existing approaches and presents a powerful tool for research of PL metabolism and signaling. PMID:26733148

  6. Isotope effect study of κ-(BEDT-TTF)2Cu(NCS)2: Labeling in the anion

    International Nuclear Information System (INIS)

    Kini, A.M.; Wang, H.H.; Schlueter, J.A.

    1995-01-01

    Since the initial discovery of organic superconductivity in 1979, a large number of organic superconductors have now been synthesized. However, the mechanism of electron-pairing in these novel superconductors has remained largely unresolved. Isotope effect studies constitute an important experimental tool for the investigation of whether or not the electron-pairing mechanism in organic superconductors is phonon-mediated, as in conventional superconductors. Recent isotope effect studies in the authors' laboratory, involving seven different isotopically labeled BEDT-TTF (or ET) derivatives, have demonstrated the following: (1) intramolecular phonon modes involving C double-bond C and Csingle bondS stretching vibrations in the ET donor molecule are not the dominant mediators of electron-pairing, and (2) in κ-(ET) 2 Cu(NCS) 2 , there exist two competing isotope effects--a normal mass effect, i.e., lowering of T c upon isotopic labeling, when the ET molecular mass is increased by concurrent 13 C and 34 S labeling, in addition to an inverse isotope effect upon deuterium labeling in ET. It is of great interest to investigate if there is an isotope effect when the charge-compensating anions, which are also located within the non-conducting layer in the superconducting cation-radical salts, are isotopically labeled. The existence of an isotope effect when the anions are labeled would be indicative of electron-pairing with the mediation of vibrational frequencies associated with the anions. In this paper, the authors present the results of the first isotope effect study in which isotopic labeling in the anion portion of κ-(ET) 2 Cu(NCS) 2 is carried out. The authors find no isotope effect when the carbon and nitrogen atoms of the thiocyanate groups in the anion are replaced with 13 C and 15 N isotopes

  7. Isotopic labelling with carbon-14 and tritium

    International Nuclear Information System (INIS)

    Evans, E.A.

    1980-01-01

    In this paper general methods of isotopic labelling with 14 C and with 3 H are briefly reviewed with special attention to examples of compounds likely to be of wide interest in biological research. (author)

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

    Science.gov (United States)

    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.

  9. NMR studies of isotopically labeled RNA

    Energy Technology Data Exchange (ETDEWEB)

    Pardi, A. [Univ. of Colorado, Boulder, CO (United States)

    1994-12-01

    In summary, the ability to generate NMR quantities of {sup 15}N and {sup 13}C-labeled RNAs has led to the development of heteronuclear multi-dimensional NMR techniques for simplifying the resonance assignment and structure determination of RNAs. These methods for synthesizing isotopically labeled RNAs are only several years old, and thus there are still relatively few applications of heteronuclear multi-dimensional NMR techniques to RNA. However, given the critical role that RNAs play in cellular function, one can expect to see an increasing number of NMR structural studies of biologically active RNAs.

  10. Advances in stable isotope assisted labeling strategies with information science.

    Science.gov (United States)

    Kigawa, Takanori

    2017-08-15

    Stable-isotope (SI) labeling of proteins is an essential technique to investigate their structures, interactions or dynamics by nuclear magnetic resonance (NMR) spectroscopy. The assignment of the main-chain signals, which is the fundamental first step in these analyses, is usually achieved by a sequential assignment method based on triple resonance experiments. Independently of the triple resonance experiment-based sequential assignment, amino acid-selective SI labeling is beneficial for discriminating the amino acid type of each signal; therefore, it is especially useful for the signal assignment of difficult targets. Various combinatorial selective labeling schemes have been developed as more sophisticated labeling strategies. In these strategies, amino acids are represented by combinations of SI labeled samples, rather than simply assigning one amino acid to one SI labeled sample as in the case of conventional amino acid-selective labeling. These strategies have proven to be useful for NMR analyses of difficult proteins, such as those in large complex systems, in living cells, attached or integrated into membranes, or with poor solubility. In this review, recent advances in stable isotope assisted labeling strategies will be discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Synthesis of 14C-labeled and stable isotope-labeled CGS 16617

    International Nuclear Information System (INIS)

    Chaudhuri, N.K.; Markus, B.; Sung Mingsang

    1988-01-01

    The synthesis of a 14 C-labeled and two stable isotope-labeled analogs of CGS 16617 is described. The synthetic method involved the preparation of tetrahydro-3-bromo-1-benzazepin-2-one, labeled with a 14 C or four deuterium atoms, followed by introduction of two side chains at 1- and 3-positions. The labeled bromobenzazepinones were prepared by Beckmann rearrangement of bromo-oximes of α-tetralones, obtained by cyclization of labeled benzenebutanoic acids. The 14 C-labeled acid was prepared by hydrolysis of the nitrile, prepared by reaction of 3-bromopropylbenzene and K 14 CN. The tetradeutero acid was prepared from ethyl phenylpropynoate by catalytic reduction of the triple bond with deuterium gas, followed by reduction of the deuterated ester with lithium aluminium hydride and conversion of the resulting alcohol into the carboxylic acid. The acetic acid side chain was introduced by N-alkylation with ethyl bromoacetate or ethyl bromoacetate-1, 2- 13 C 2 followed by hydrolysis, and the L-lysine side chain, by reaction with L-(-)-3-amino-ε-caprolactam followed by hydrolysis of the caprolactam ring. (author)

  12. Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells

    International Nuclear Information System (INIS)

    Takahashi, Hideo; Shimada, Ichio

    2010-01-01

    The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells.

  13. Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

    Science.gov (United States)

    Kim, Kyoung-Jin; Kim, Yoon-Woo; Kim, Yun-Gon; Park, Hae-Min; Jin, Jang Mi; Hwan Kim, Young; Yang, Yung-Hun; Kyu Lee, Jun; Chung, Junho; Lee, Sun-Gu; Saghatelian, Alan

    2015-01-01

    Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling-based quantitative targeted glycomics (i-QTaG) technique for the comparative and quantitative analysis of total N-glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N-glycans using a model glycoprotein (bovine fetuin). Moreover, the i-QTaG using MALDI-TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of (13) C6 /(12) C6 -2-aminobenzoic acid-labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N-glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N-glycan peaks from i-QTaG method showed a good linearity (R(2) > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2-AA labeled N-glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up-regulation of the Lewis antigen (~82%) in sera from prostate cancer patients. In this proof-of-concept study, we demonstrated that the i-QTaG method, which enables to achieve a reliable comparative quantitation of total N-glycans via MALDI-TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers.

  14. Doubly labeled water method: in vivo oxygen and hydrogen isotope fractionation

    International Nuclear Information System (INIS)

    Schoeller, D.A.; Leitch, C.A.; Brown, C.

    1986-01-01

    The accuracy and precision of the doubly labeled water method for measuring energy expenditure are influenced by isotope fractionation during evaporative water loss and CO 2 excretion. To characterize in vivo isotope fractionation, we collected and isotopically analyzed physiological fluids and gases. Breath and transcutaneous water vapor were isotopically fractionated. The degree of fractionation indicated that the former was fractionated under equilibrium control at 37 0 C, and the latter was kinetically fractionated. Sweat and urine were unfractionated. By use of isotopic balance models, the fraction of water lost via fractionating routes was estimated from the isotopic abundances of body water, local drinking water, and dietary solids. Fractionated water loss averaged 23% (SD = 10%) of water turnover, which agreed with our previous estimates based on metabolic rate, but there was a systematic difference between the results based on O 2 and hydrogen. Corrections for isotopic fractionation of water lost in breath and (nonsweat) transcutaneous loss should be made when using labeled water to measure water turnover or CO 2 production

  15. Preparation of radioactive labelled compounds. Pt. 2. 82Br labelled organic bromine compounds by isotopic exchange

    International Nuclear Information System (INIS)

    Otto, R.

    1988-05-01

    Studies on isotopic exchange between organic bromine compounds and 82 Br labelled dioxane dibromide in the presence of AlCl 3 are described. The results obtained enable to develop a simple and quick preparation method for the labelling with 82 Br [fr

  16. Availability of phosphorus in cow slurry using isotopic labelling technique

    International Nuclear Information System (INIS)

    Pongsakul, P.; Bertelsen, F.; Gissel-Nielsen, G.

    1988-01-01

    A pot experiment was conducted to evaluate the influence of cow slurry on P uptake by corn and to estimate the readily available P in the slurry by using an isotopic labelling techique. Water-soluble P in soil was increased and isotopic equilibrium of available P was attained after labelled slurry was mixed thoroughly throughout the soil. Labelled slurry applied at planting increased the P uptake by corn, whereas the same amount applied one week before harvest did not affect the P uptake. It was estimated that 46-54% of the total P uptake in plants is derived from the slurry. The readily available P (the L-value) in the slurry was at least 26 mg/kg which equals 3.7% of the total P. (author)

  17. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    Science.gov (United States)

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

  18. Mechanistic Insights into Catalytic Ethanol Steam Reforming Using Isotope-Labeled Reactants.

    Science.gov (United States)

    Crowley, Stephen; Castaldi, Marco J

    2016-08-26

    The low-temperature ethanol steam reforming (ESR) reaction mechanism over a supported Rh/Pt catalyst has been investigated using isotope-labeled EtOH and H2 O. Through strategic isotope labeling, all nonhydrogen atoms were distinct from one another, and allowed an unprecedented level of understanding of the dominant reaction pathways. All combinations of isotope- and non-isotope-labeled atoms were detected in the products, thus there are multiple pathways involved in H2 , CO, CO2 , CH4 , C2 H4 , and C2 H6 product formation. Both the recombination of C species on the surface of the catalyst and preservation of the C-C bond within ethanol are responsible for C2 product formation. Ethylene is not detected until conversion drops below 100 % at t=1.25 h. Also, quantitatively, 57 % of the observed ethylene is formed directly through ethanol dehydration. Finally there is clear evidence to show that oxygen in the SiO2 -ZrO2 support constitutes 10 % of the CO formed during the reaction. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Synthesis of 15N isotope labeled alanine

    International Nuclear Information System (INIS)

    Oliveira, Claudineia R. de; Bendassolli, Jose Albertino; Sant'Ana, Carlos Roberto; Tagliassachi, Romulo Barbieri; Maximo, Everaldo; Prestes, Clelber Vieira

    2005-01-01

    The application of light chemical elements and their stable isotopes in biological studies have been increased over the last years. The use of 15 N labeled amino acids is an important tool for elucidation of peptides structures. This paper describe a method for the synthesis of 15 N isotope labeled alanine at lower costs than international ones, as well as the details of the recovery system of the nitrogen residues. In the present work an amination of α-haloacids, with the bromopropionic carboxylic acid and labeled aqua ammonia ( 15 NH 3 aq) was carried out. In order to avoid eventually losses of 15 NH 3 , special cares were adopted, since the production cost is high. Although the acquisition cost of the 13 N (radioactive) labeled compounds is lower, the obtained stable tracer will allow the accomplishment of important studies of the nitrogen cycling in living things, less occupational and environment hazards, and the time limitation problems in field studies. The tests took place in triplicates with NH 3 (aq) being employed. With the establishment of the system for 15 NH 3 recovery, an average of 94 % of the ammonia employed in the synthesis process was recovered. The purity of the amino acid was state determined by TLC (Thin Layer Chromatography) and HPLC (High-Performance Liquid Chromatography) with a fluorescence detector. The Rf and the retention time of the synthesized sample were similar the sigma standard. Finally, regarding the established conditions, it was possible to obtain the alanine with a production cost about 40 % lower than the international price. (author)

  20. Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA▿

    OpenAIRE

    Uchino, Keiichi; Saito, Terumi; Gebauer, Birgit; Jendrossek, Dieter

    2007-01-01

    Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from 14C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in...

  1. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    International Nuclear Information System (INIS)

    Chandra, Subhash

    2004-01-01

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13 C and 15 N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13 C 15 N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39 K, 23 Na and 40 Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors

  2. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, Subhash

    2004-06-15

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes {sup 13}C and {sup 15}N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK{sub 1} kidney cells at mass 28 ({sup 13}C{sup 15}N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of {sup 39}K, {sup 23}Na and {sup 40}Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

  3. A free-air system for long-term stable carbon isotope labeling of adult forest trees

    Science.gov (United States)

    Stable carbon (C) isotopes, in particular employed in labeling experiments, are an ideal tool to broaden our understanding of C dynamics in trees and forest ecosystems. Here, we present a free-air exposure system, named isoFACE, designed for long-term stable C isotope labeling in...

  4. Stable-isotope-labeled carbohydrates and nucleosides: Synthesis and applications in chemistry and biology

    Energy Technology Data Exchange (ETDEWEB)

    Serianni, A.S. [Univ. of Notre Dame, IN (United States)

    1994-12-01

    Carbohydrates play important roles in many key biochemical processes in living cells. For example, they are metabolized to produce energy, mediate cell-cell recognition, and play an indirect role (as constituents of DNA and RNA) in DNA replication, RNA transcription, and protein synthesis. These roles, and others of comparable biochemical significance, have been studied to varying extends with the use of stable isotopically labeled molecules, usually in conjunction with NMR spectroscopy and/or mass spectrometry. For example, carbohydrate metabolism has been monitored in vitro and in vivo with the use of isotopically labeled compounds. Molecular aspects of cell-cell recognition, mediated by cell-surface glycoproteins and glycolipids, have been probed through NMR studies of isotopically labeled oligosaccharides. More recently, the solution behavior of DNA and RNA has been examined through the use of labeled oligonucleotides. In all of these pursuits, the effort and expense to prepare labeled molecules, both of which can be substantial, are more than offset by the wealth of information derived from these studies. This information often cannot be accessed, or can be accessed only with great difficulty, using natural (unlabeled) compounds.

  5. Stable-isotope-labeled carbohydrates and nucleosides: Synthesis and applications in chemistry and biology

    International Nuclear Information System (INIS)

    Serianni, A.S.

    1994-01-01

    Carbohydrates play important roles in many key biochemical processes in living cells. For example, they are metabolized to produce energy, mediate cell-cell recognition, and play an indirect role (as constituents of DNA and RNA) in DNA replication, RNA transcription, and protein synthesis. These roles, and others of comparable biochemical significance, have been studied to varying extends with the use of stable isotopically labeled molecules, usually in conjunction with NMR spectroscopy and/or mass spectrometry. For example, carbohydrate metabolism has been monitored in vitro and in vivo with the use of isotopically labeled compounds. Molecular aspects of cell-cell recognition, mediated by cell-surface glycoproteins and glycolipids, have been probed through NMR studies of isotopically labeled oligosaccharides. More recently, the solution behavior of DNA and RNA has been examined through the use of labeled oligonucleotides. In all of these pursuits, the effort and expense to prepare labeled molecules, both of which can be substantial, are more than offset by the wealth of information derived from these studies. This information often cannot be accessed, or can be accessed only with great difficulty, using natural (unlabeled) compounds

  6. Mutations in COA3 cause isolated complex IV deficiency associated with neuropathy, exercise intolerance, obesity, and short stature.

    Science.gov (United States)

    Ostergaard, Elsebet; Weraarpachai, Woranontee; Ravn, Kirstine; Born, Alfred Peter; Jønson, Lars; Duno, Morten; Wibrand, Flemming; Shoubridge, Eric A; Vissing, John

    2015-03-01

    We investigated a subject with an isolated cytochrome c oxidase (COX) deficiency presenting with an unusual phenotype characterised by neuropathy, exercise intolerance, obesity, and short stature. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis showed an almost complete lack of COX assembly in subject fibroblasts, consistent with the very low enzymatic activity, and pulse-labelling mitochondrial translation experiments showed a specific decrease in synthesis of the COX1 subunit, the core catalytic subunit that nucleates assembly of the holoenzyme. Whole exome sequencing identified compound heterozygous mutations (c.199dupC, c.215A>G) in COA3, a small inner membrane COX assembly factor, resulting in a pronounced decrease in the steady-state levels of COA3 protein. Retroviral expression of a wild-type COA3 cDNA completely rescued the COX assembly and mitochondrial translation defects, confirming the pathogenicity of the mutations, and resulted in increased steady-state levels of COX1 in control cells, demonstrating a role for COA3 in the stabilisation of this subunit. COA3 exists in an early COX assembly complex that contains COX1 and other COX assembly factors including COX14 (C12orf62), another single pass transmembrane protein that also plays a role in coupling COX1 synthesis with holoenzyme assembly. Immunoblot analysis showed that COX14 was undetectable in COA3 subject fibroblasts, and that COA3 was undetectable in fibroblasts from a COX14 subject, demonstrating the interdependence of these two COX assembly factors. The mild clinical course in this patient contrasts with nearly all other cases of severe COX assembly defects that are usually fatal early in life, and underscores the marked tissue-specific involvement in mitochondrial diseases. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  7. A new method for the labelling of proteins with radioactive arsenic isotopes

    Energy Technology Data Exchange (ETDEWEB)

    Jennewein, M. [Institute of Nuclear Chemistry, Johannes Gutenberg University of Mainz, Fritz-Strassmann-Weg 2, 55128 Mainz (Germany); Hermanne, A. [VUB Cyclotron, University of Brussels, Laarbeeklaan 103, 1090 Brussels (Belgium); Mason, R.P. [Department of Radiology, Advanced Radiological Sciences, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas (United States); Thorpe, P.E. [Department of Pharmacology and Simmons and Hamon Cancer Centers, University of Texas Southwestern Medical Center at Dallas, Dallas, TX (United States); Roesch, F. [Institute of Nuclear Chemistry, Johannes Gutenberg University of Mainz, Fritz-Strassmann-Weg 2, 55128 Mainz (Germany)]. E-mail: frank.roesch@uni-mainz.de

    2006-12-20

    Radioarsenic labelled radiopharmaceuticals could be a valuable asset to positron emission tomography. In particular, the long half-lives of {sup 72}As (T{sub 1/2}=26h) and {sup 74}As (T{sub 1/2}=17.8d) allow to investigate slow physiological or metabolical processes, like the enrichment and distribution of monoclonal antibodies (mab) in tumour tissue. In this work, a new method for the labelling of proteins with various radioactive arsenic isotopes was developed. For this purpose, two proteins, namely a chimeric IgG{sub 3} monoclonal antibody, ch3G4, directed against anionic phospholipids, and Rituxan (Rituximab), were labelled as a proof of principle with no-carrier-added radioarsenic isotopes ({sup 74}As and {sup 77}As). The developed labelling chemistry gives high yields (>99.9%), is reliable and could easily be transferred to automated labelling systems in a clinical environment. At least for the mab used in this work, this route of radioarsenic labelling does not affect the immunoreactivity of the product. The arsenic label stays stable for up to 72h at the molecular mass of the monoclonal antibody, which is in particular relevant to follow the pharmacology and pharmacokinetics of the labelled mab for several days.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  9. Kinetic isotope effects in reaction of ferment oxidation of tritium-labelled D-galactosamine

    International Nuclear Information System (INIS)

    Akulov, G.P.; Korsakova, N.A.

    1992-01-01

    Primary, secondary and intramolecular kinetic isotopic effects in reaction of ferment oxidation of D-galactosamine labelled by tritium in position 6, were measured. When comparing values of the effects with previously obtained results for similar reaction D-[6- 3 H]galactose, it was ascertained that the presence of aminogroup in galactopyranosyl mainly affects kinetics of substrate-ferment complex formation stage. The possibility to use kinetic isotope effects for increase in molar activity of D-galactosamine, labelled by tritium in position 6, is shown

  10. Copper absorption from foods labelled intrinsically and extrinsically with Cu-65 stable isotope.

    Science.gov (United States)

    Harvey, L J; Dainty, J R; Beattie, J H; Majsak-Newman, G; Wharf, S G; Reid, M D; Fairweather-Tait, S J

    2005-03-01

    To determine copper absorption from copper containing foods labelled either intrinsically or extrinsically with a highly enriched Cu-65 stable isotope label. A longitudinal cross-over study. The study was conducted at the Institute of Food Research, Human Nutrition Unit, Norwich, UK. Subjects were recruited locally via advertisements placed around the Norwich Research Park. A total of 10 volunteers (nine female, one male) took part in the study, but not all volunteers completed each of the test meals. A highly enriched Cu-65 stable isotope label was administered to volunteers in the form of a reference dose or in breakfast test meals consisting of red wine, soya beans, mushrooms or sunflower seeds. Faecal monitoring and mass spectrometry techniques were used to estimate the relative quantities of copper absorbed from the different test meals. True copper absorption from the reference dose (54%) was similar to extrinsically labelled red wine (49%) and intrinsically labelled sunflower seeds (52%), but significantly higher than extrinsically labelled mushrooms (35%), intrinsically (29%) and extrinsically (15%) labelled soya beans and extrinsically labelled sunflower seed (32%) test meals. The use of Cu-65 extrinsic labels in copper absorption studies requires validation according to the food being examined; intrinsic and extrinsic labelling produced significantly different results for sunflower seeds.

  11. Development of an isotope labeling ultra-high performance liquid chromatography mass spectrometric method for quantification of acylglycines in human urine

    Energy Technology Data Exchange (ETDEWEB)

    Stanislaus, Avalyn; Guo, Kevin [Department of Chemistry, University of Alberta, Edmonton, Alberta (Canada); Li Liang, E-mail: Liang.Li@ualberta.ca [Department of Chemistry, University of Alberta, Edmonton, Alberta (Canada)

    2012-10-31

    Graphical abstract: - Abstract: Acylglycines play a crucial regulatory and detoxification role in the accumulation of the corresponding acyl CoA esters and are an important class of metabolites in the diagnoses of inborn errors of metabolism. Sensitive quantification of a large number of acylglycines not only improves diagnosis but also enables the discovery of potential new biomarkers of diseases. We report an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS) method for quantifying acylglycines in human urine with high sensitivity. This method is based on the use of a newly developed isotope labeling reagent, p-dimethylaminophenacyl (DmPA) bromide, to label acylglycines to improve detection sensitivity. Eighteen acylglycines, namely acetylglycine, propionylglycine, isobutyrylglycine, butyrylglycine, 4-hydroxyphenylacetylglycine, 2-furoylglycine, tiglylglycine, 2-methybutyrylglycine, 3-methylcrotonylglycine, isovalerylglycine, valerylglycine, hexanoylglycine, phenylacetylglycine, phenylpropionylglycine, glutarylglycine, heptanoylglycine, octanoylglycine and suberylglycine, were measured. This method uses calibration standards prepared in surrogate matrix (un-derivatized urine) and stable-isotope labeled analytes as the internal standards. The analysis was carried out in the positive ion detection mode using multiple reaction monitoring (MRM) survey scans. The calibration curves were validated over the range of 1.0-500 nM. The method achieved a lower limit of quantitation (LLOQ) of 1-5 nM for all analytes, as measured by the standard derivations associated with calibration curves and confirmed in surrogate matrix; the signal-to-noise ratio at LLOQ ranged from 12.50 to 156.70. Both accuracy (% RE or relative error) and precision (% CV) were <15%. Matrix effects were minimized using the surrogate matrix. All eighteen analytes were stable in urine for at least 5 h at room temperature, autosampler (4 Degree-Sign C) for 24 h, 7 weeks at -20

  12. Development of versatile isotopic labeling reagents for profiling the amine submetabolome by liquid chromatography–mass spectrometry

    International Nuclear Information System (INIS)

    Zhou, Ruokun; Huan, Tao; Li, Liang

    2015-01-01

    Highlights: • Two new reagents were developed for chemical isotope labeling mass spectrometry (MS). • They could be used to label amine-containing metabolites in a metabolomic sample. • The labeled metabolites could be detected with much improved sensitivity in MS. • One of the reagents could also help generate useful MS/MS spectra for structural analysis. • These reagents should be useful for quantitative metabolomics. - Abstract: Metabolomic profiling involves relative quantification of metabolites in comparative samples and identification of the significant metabolites that differentiate different groups (e.g., diseased vs. controls). Chemical isotope labeling (CIL) liquid chromatography–mass spectrometry (LC–MS) is an enabling technique that can provide improved metabolome coverage and metabolite quantification. However, chemical identification of labeled metabolites can still be a challenge. In this work, a new set of isotopic labeling reagents offering versatile properties to enhance both detection and identification are described. They were prepared by a glycine molecule (or its isotopic counterpart) and an aromatic acid with varying structures through a simple three-step synthesis route. In addition to relatively low costs of synthesizing the reagents, this reaction route allows adjusting reagent property in accordance with the desired application objective. To date, two isotopic reagents, 4-dimethylaminobenzoylamido acetic acid N-hydroxylsuccinimide ester (DBAA-NHS) and 4-methoxybenzoylamido acetic acid N-hydroxylsuccinimide ester (MBAA-NHS), for labeling the amine-containing metabolites (i.e., amine submetabolome) have been synthesized. The labeling conditions and the related LC–MS method have been optimized. We demonstrate that DBAA labeling can increase the metabolite detectability because of the presence of an electrospray ionization (ESI)-active dimethylaminobenzoyl group. On the other hand, MBAA labeled metabolites can be fragmented

  13. Development of versatile isotopic labeling reagents for profiling the amine submetabolome by liquid chromatography–mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Ruokun; Huan, Tao; Li, Liang, E-mail: Liang.Li@ualberta.ca

    2015-06-30

    Highlights: • Two new reagents were developed for chemical isotope labeling mass spectrometry (MS). • They could be used to label amine-containing metabolites in a metabolomic sample. • The labeled metabolites could be detected with much improved sensitivity in MS. • One of the reagents could also help generate useful MS/MS spectra for structural analysis. • These reagents should be useful for quantitative metabolomics. - Abstract: Metabolomic profiling involves relative quantification of metabolites in comparative samples and identification of the significant metabolites that differentiate different groups (e.g., diseased vs. controls). Chemical isotope labeling (CIL) liquid chromatography–mass spectrometry (LC–MS) is an enabling technique that can provide improved metabolome coverage and metabolite quantification. However, chemical identification of labeled metabolites can still be a challenge. In this work, a new set of isotopic labeling reagents offering versatile properties to enhance both detection and identification are described. They were prepared by a glycine molecule (or its isotopic counterpart) and an aromatic acid with varying structures through a simple three-step synthesis route. In addition to relatively low costs of synthesizing the reagents, this reaction route allows adjusting reagent property in accordance with the desired application objective. To date, two isotopic reagents, 4-dimethylaminobenzoylamido acetic acid N-hydroxylsuccinimide ester (DBAA-NHS) and 4-methoxybenzoylamido acetic acid N-hydroxylsuccinimide ester (MBAA-NHS), for labeling the amine-containing metabolites (i.e., amine submetabolome) have been synthesized. The labeling conditions and the related LC–MS method have been optimized. We demonstrate that DBAA labeling can increase the metabolite detectability because of the presence of an electrospray ionization (ESI)-active dimethylaminobenzoyl group. On the other hand, MBAA labeled metabolites can be fragmented

  14. Expeditious syntheses of stable and radioactive isotope-labeled anticonvulsant agent, JNJ-26990990, and its metabolites.

    Science.gov (United States)

    Lin, Ronghui; Weaner, Larry E; Hoerr, David C; Salter, Rhys; Gong, Yong

    2013-01-01

    Syntheses of stable and radioactive isotope-labeled anticonvulsant agent, JNJ-26990990, that is, N-(benzo[b]thien-3-ylmethyl)-sulfamide and its metabolites are described. [(13)C(15)N]Benzo[b]thiophene-3-carbonitrile was first prepared by coupling of 3-bromo-benzo[b]thiophene with [(13)C(15)N]-copper cyanide. The resultant [(13)C(15)N]benzo[b]thiophene-3-carbonitrile was reduced with lithium aluminum deuteride to give [(13)CD2(15)N]benzo[b]thiophen-3-yl-methylamine; which was then coupled with sulfamide to afford [(13)CD2(15)N]-N-(benzo[b]thien-3-ylmethyl)-sulfamide, the stable isotope-labeled compound with four stable isotope atoms. Direct oxidation of [(13)CD2(15)N]-N-(benzo[b]thien-3-ylmethyl)-sulfamide with hydrogen peroxide and peracetic acid gave the stable isotope-labeled sulfoxide and sulfone metabolites. On the other hand, radioactive (14)C-labeled N-(benzo[b]thien-3-ylmethyl)-sulfamide was prepared conveniently by sequential coupling of 3-bromo-benzo[b]thiophene with [(14)C]-copper cyanide, reduction of the carbonitrile to carboxaldehyde, and reductive amination with sulfamide. Copyright © 2013 John Wiley & Sons, Ltd.

  15. Heating Isotopically Labeled Bernal Stacked Graphene: A Raman Spectroscopy Study

    Czech Academy of Sciences Publication Activity Database

    Ek Weis, Johan; da Costa, Sara; Frank, Otakar; Kalbáč, Martin

    2014-01-01

    Roč. 5, č. 3 (2014), s. 549-554 ISSN 1948-7185 R&D Projects: GA MŠk LL1301 Institutional support: RVO:61388955 Keywords : Bernal * graphene * isotopic labeling Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 7.458, year: 2014

  16. Isotopic labelling studies for a gold-catalysed skeletal rearrangement of alkynyl aziridines

    Directory of Open Access Journals (Sweden)

    Neil Spencer

    2011-06-01

    Full Text Available Isotopic labelling studies were performed to probe a proposed 1,2-aryl shift in the gold-catalysed cycloisomerisation of alkynyl aziridines into 2,4-disubstituted pyrroles. Two isotopomers of the expected skeletal rearrangement product were identified using 13C-labelling and led to a revised mechanism featuring two distinct skeletal rearrangements. The mechanistic proposal has been rationalised against the reaction of a range of 13C- and deuterium-labelled substrates.

  17. CoaSim Guile Manual — Using the Guile-based CoaSim Simulator

    DEFF Research Database (Denmark)

    Mailund, T

    2006-01-01

    CoaSim is a tool for simulating the coalescent process with recombination and geneconversion, under either constant population size or exponential population growth. It effectively constructs the ancestral recombination graph for a given number of chromosomes and uses this to simulate samples...

  18. Synthesis of 13C and 2H labelled retinals: spectroscopic investigations on isotopically labelled rhodopsin and bacteriorhodopsin

    International Nuclear Information System (INIS)

    Pardoen, J.A.

    1986-01-01

    In order to develop probes of the structure of chromophores, the author introduces isotopic modifications at specific chromophoric positions as structural probes. To obtain bacteriorhodopsin, rhodopsin and their photoproducts labelled in the chromophore at selected positions, bacterioopsin and opsin were reacted with the appropriate labelled a11-trans and 11-cis retinals. The author describes the synthesis of a11-trans retinal selectively 13 C labelled at different positions. The characterization of these labelled a11-trans retinals by mass spectrometry, 300 MHz 1 H NMR and 75 MHz 13 C NMR spectroscopy is given. The photochemical preparation and isolation of the pure 9-, 11- and 13-cis forms is described in the experimental part. (Auth.)

  19. CoAs: The line of 3 d demarcation

    Science.gov (United States)

    Campbell, Daniel J.; Wang, Limin; Eckberg, Chris; Graf, Dave; Hodovanets, Halyna; Paglione, Johnpierre

    2018-05-01

    Transition metal-pnictide compounds have received attention for their tendency to combine magnetism and unconventional superconductivity. Binary CoAs lies on the border of paramagnetism and the more complex behavior seen in isostructural CrAs, MnP, FeAs, and FeP. Here we report the properties of CoAs single crystals grown with two distinct techniques along with density functional theory calculations of its electronic structure and magnetic ground state. While all indications are that CoAs is paramagnetic, both experiment and theory suggest proximity to a ferromagnetic instability. Quantum oscillations are seen in torque measurements up to 31.5 T and support the calculated paramagnetic Fermiology.

  20. Sensing site-specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides.

    Science.gov (United States)

    Keiderling, Timothy A

    2017-12-01

    Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site-specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini-review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra. © 2017 Wiley Periodicals, Inc.

  1. Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule

    Energy Technology Data Exchange (ETDEWEB)

    Yagi, Hirokazu [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan); Nakamura, Masatoshi [National Institute of Agrobiological Sciences, Genetic Resources Conservation Research Unit, Genetic Resources Center (Japan); Yokoyama, Jun [Taiyo Nippon Sanso Corporation, Tsukuba Laboratories (Japan); Zhang, Ying; Yamaguchi, Takumi [National Institutes of Natural Sciences, Institute for Molecular Science and Okazaki Institute for Integrative Bioscience (Japan); Kondo, Sachiko [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan); Kobayashi, Jun [Yamaguchi University, Department of Biological and Environmental Sciences, Faculty of Agriculture (Japan); Kato, Tatsuya; Park, Enoch Y. [Shizuoka University, Laboratory of Biotechnology, Research Institute of Green Science and Technology (Japan); Nakazawa, Shiori [Nagoya University, Sugashima Marine Biological Laboratory, Graduate School of Science (Japan); Hashii, Noritaka; Kawasaki, Nana [National Institute of Health Sciences, Division of Biological Chemistry and Biologicals (Japan); Kato, Koichi, E-mail: kkato@phar.nagoya-cu.ac.jp [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan)

    2015-06-15

    Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing {sup 15}N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a {sup 15}N-enrichment ratio of approximately 80 %. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.

  2. Existing and emerging technologies for measuring stable isotope labelled retinol in biological samples: isotope dilution analysis of body retinol stores.

    Science.gov (United States)

    Preston, Tom

    2014-01-01

    This paper discusses some of the recent improvements in instrumentation used for stable isotope tracer measurements in the context of measuring retinol stores, in vivo. Tracer costs, together with concerns that larger tracer doses may perturb the parameter under study, demand that ever more sensitive mass spectrometric techniques are developed. GCMS is the most widely used technique. It has high sensitivity in terms of sample amount and uses high resolution GC, yet its ability to detect low isotope ratios is limited by background noise. LCMSMS may become more accessible for tracer studies. Its ability to measure low level stable isotope tracers may prove superior to GCMS, but it is isotope ratio MS (IRMS) that has been designed specifically for low level stable isotope analysis through accurate analysis of tracer:tracee ratios (the tracee being the unlabelled species). Compound-specific isotope analysis, where GC is interfaced to IRMS, is gaining popularity. Here, individual 13C-labelled compounds are separated by GC, combusted to CO2 and transferred on-line for ratiometric analysis by IRMS at the ppm level. However, commercially-available 13C-labelled retinol tracers are 2 - 4 times more expensive than deuterated tracers. For 2H-labelled compounds, GC-pyrolysis-IRMS has now become more generally available as an operating mode on the same IRMS instrument. Here, individual compounds are separated by GC and pyrolysed to H2 at high temperature for analysis by IRMS. It is predicted that GC-pyrolysis-IRMS will facilitate low level tracer procedures to measure body retinol stores, as has been accomplished in the case of fatty acids and amino acids. Sample size requirements for GC-P-IRMS may exceed those of GCMS, but this paper discusses sample preparation procedures and predicts improvements, particularly in the efficiency of sample introduction.

  3. Getting Started with CoaSim — An Introduction to the Simulator CoaSim

    DEFF Research Database (Denmark)

    Mailund, T

    2005-01-01

    CoaSim is a tool for simulating the coalescent process with recombination and geneconversion, under either constant population size or exponential population growth. It effectively constructs the ancestral recombination graph for a given number of chromosomes and uses this to simulate samples...

  4. Isotopically modified compounds

    International Nuclear Information System (INIS)

    Kuruc, J.

    2009-01-01

    In this chapter the nomenclature of isotopically modified compounds in Slovak language is described. This chapter consists of following parts: (1) Isotopically substituted compounds; (2) Specifically isotopically labelled compounds; (3) Selectively isotopically labelled compounds; (4) Non-selectively isotopically labelled compounds; (5) Isotopically deficient compounds.

  5. Performance of isobaric and isotopic labeling in quantitative plant proteomics

    DEFF Research Database (Denmark)

    Nogueira, Fábio C S; Palmisano, Giuseppe; Schwämmle, Veit

    2012-01-01

    , and quantitation. In the present work, we have used LC-MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in...

  6. Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.

    Science.gov (United States)

    Kleifeld, Oded; Doucet, Alain; Prudova, Anna; auf dem Keller, Ulrich; Gioia, Magda; Kizhakkedathu, Jayachandran N; Overall, Christopher M

    2011-09-22

    Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and

  7. Synthetic routes to some isotopically labelled intermediates for diterpenoid biosynthesis

    International Nuclear Information System (INIS)

    Dawson, R.M.; Godfrey, I.M.; Hogg, R.W.; Knox, J.R.

    1989-01-01

    The exo-15-hydrogen of ent-kaurene can be exchanged through a reversible ene reaction in a convenient and efficient procedure which has the potential for giving high specific activity 3 H-labelling. Copalol, the (Z)-double bond stereoisomer, and the allylic alcohol isomers ent-manool and ent-epimanool have been obtained through divergent synthetic pathways involving a 15,16-bisnor ketone intermediate. These pathways have also allowed the four compounds to be obtained with 14 C-labelling. A method, involving a Wittig reaction to form a vinyl bromide intermediate, has been developed for obtaining copalol, as the trityl ether derivative, with stereospecific isotopic labelling of one or the other of the hydrogens of the exocyclic methylene group. 27 refs., figs

  8. Affordable uniform isotope labeling with {sup 2}H, {sup 13}C and {sup 15}N in insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Sitarska, Agnieszka; Skora, Lukasz; Klopp, Julia; Roest, Susan; Fernández, César; Shrestha, Binesh; Gossert, Alvar D., E-mail: alvar.gossert@novartis.com [Novartis Institutes for BioMedical Research (Switzerland)

    2015-06-15

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

  9. Syntheses with isotopically labelled carbon. Methyl iodide, formaldehyde and cyanide

    International Nuclear Information System (INIS)

    Finn, R.D.; Boothe, T.E.; Vora, M.M.; Hildner, J.C.; Emran, A.M.; Kothari, P.J.

    1984-01-01

    Many of the uniquely labelled synthetic precursors currently employed in the design of sophisticated radiolabelled compounds have their origins in the field of hot atom chemistry. Particularly, the development during the past few years of automated, on-line synthetic procedures which combine the nuclear reaction, hot atom and classical chemistry, and rapid purification methods has allowed the incorporation of useful radionuclides into suitable compounds of chemical and biochemical interest. The application of isotopically labelled methyl iodide, formaldehyde, and cyanide anion as synthetic intermediates in research involving human physiology and nuclear medicine, as well as their contributions to other scientific methodology, is reviewed. (author)

  10. Role of Feedback Regulation of Pantothenate Kinase (CoaA) in Control of Coenzyme A Levels in Escherichia coli

    Science.gov (United States)

    Rock, Charles O.; Park, Hee-Won; Jackowski, Suzanne

    2003-01-01

    Pantothenate kinase (CoaA) is a key regulator of coenzyme A (CoA) biosynthesis in Escherichia coli, and its activity is controlled by feedback inhibition by CoA and its thioesters. The importance of feedback inhibition in the control of the intracellular CoA levels was tested by constructing three site-directed mutants of CoaA that were predicted to be feedback resistant based on the crystal structure of the CoaA-CoA binary complex. CoaA[R106A], CoaA[H177Q], and CoaA[F247V] were purified and shown to retain significant catalytic activity and be refractory to inhibition by CoA. CoaA[R106A] retained 50% of the catalytic activity of CoaA, whereas the CoaA[H177Q] and CoaA[F247V] mutants were less active. The importance of feedback control of CoaA to the intracellular CoA levels was assessed by expressing either CoaA or CoaA[R106A] in strain ANS3 [coaA15(Ts) panD2]. Cells expressing CoaA[R106A] had significantly higher levels of phosphorylated pantothenate-derived metabolites and CoA in vivo and excreted significantly more 4′-phosphopantetheine into the medium compared to cells expressing the wild-type protein. These data illustrate the key role of feedback regulation of pantothenate kinase in the control of intracellular CoA levels. PMID:12754240

  11. Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain

    International Nuclear Information System (INIS)

    Miyanoiri, Yohei; Ishida, Yojiro; Takeda, Mitsuhiro; Terauchi, Tsutomu; Inouye, Masayori; Kainosho, Masatsune

    2016-01-01

    We recently developed a practical protocol for preparing proteins bearing stereo-selectively 13 C-methyl labeled leucines and valines, instead of the commonly used 13 C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

  12. Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain

    Energy Technology Data Exchange (ETDEWEB)

    Miyanoiri, Yohei [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Ishida, Yojiro [Rutgers University-Robert Wood Johnson Medical School, Center for Advanced Biotechnology and Medicine (United States); Takeda, Mitsuhiro [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Terauchi, Tsutomu [Tokyo Metropolitan University, Graduate School of Science and Engineering (Japan); Inouye, Masayori [Rutgers University-Robert Wood Johnson Medical School, Center for Advanced Biotechnology and Medicine (United States); Kainosho, Masatsune, E-mail: kainosho@tmu.ac.jp [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

    2016-06-15

    We recently developed a practical protocol for preparing proteins bearing stereo-selectively {sup 13}C-methyl labeled leucines and valines, instead of the commonly used {sup 13}C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

  13. Mass spectrometric studies of stable isotope-labelled carboxylic acid derivatives

    International Nuclear Information System (INIS)

    Andersson, B.Aa.; Dinger, F.; Dinh-Nguyen, N.

    1975-01-01

    Low resolution mass spectra of deuterium and carbon-13 labelled fatty acid pyrrolidides are discussed. The simple fragmentation pattern of pyrrolidides makes them superior to other derivatives, regarding location of isotopes. Deuteriation of ethylenic fatty acid pyrrolidides therefore seems to be an improved method to locate carbon-carbon double bonds by mass spectrometry. (author)

  14. Raman spectroscopy of isotopically labeled two-layer graphene

    Czech Academy of Sciences Publication Activity Database

    Kalbáč, Martin; Kong, J.; Kavan, Ladislav; Dresselhaus, M. S.

    2012-01-01

    Roč. 249, č. 12 (2012), s. 2500-2502 ISSN 0370-1972 R&D Projects: GA AV ČR IAA400400911; GA AV ČR IAA400400804; GA AV ČR KAN200100801; GA MŠk ME09060; GA ČR GAP204/10/1677; GA ČR GBP208/12/G016; GA ČR(CZ) GAP208/12/1062 Institutional support: RVO:61388955 Keywords : electrochemical doping * isotope labeling * graphene Subject RIV: CG - Electrochemistry Impact factor: 1.489, year: 2012

  15. Efficient segmental isotope labeling of multi-domain proteins using Sortase A

    Energy Technology Data Exchange (ETDEWEB)

    Freiburger, Lee, E-mail: lee.freiburger@tum.de; Sonntag, Miriam, E-mail: miriam.sonntag@mytum.de; Hennig, Janosch, E-mail: janosch.hennig@helmholtz-muenchen.de [Helmholtz Zentrum München, Institute of Structural Biology (Germany); Li, Jian, E-mail: lijianzhongbei@163.com [Chinese Academy of Sciences, Tianjin Institute of Industrial Biotechnology (China); Zou, Peijian, E-mail: peijian.zou@helmholtz-muenchen.de; Sattler, Michael, E-mail: sattler@helmholtz-muenchen.de [Helmholtz Zentrum München, Institute of Structural Biology (Germany)

    2015-09-15

    NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to existing protocols are (1) the efficient removal of cleaved peptide fragments by centrifugal filtration and (2) a strategic design of cleavable and non-cleavable affinity tags for purification. Our approach enables routine production of milligram amounts of purified segmentally labeled protein for NMR and other biophysical studies.

  16. Automatic isotope gas analysis of tritium labelled organic materials Pt. 1

    International Nuclear Information System (INIS)

    Gacs, I.; Mlinko, S.

    1978-01-01

    A new automatic procedure developed to convert tritium in HTO hydrogen for subsequent on-line gas counting is described. The water containing tritium is introduced into a column prepared from molecular sieve-5A and heated to 550 deg C. The tritium is transferred by isotopic exchange into hydrogen flowing through the column. The radioactive gas is led into an internal detector for radioactivity measurement. The procedure is free of memory effects, provides quantitative recovery with analytical reproducibility better than 0.5% rel. at a preset number of counts. The experimental and analytical results indicate that isotopic exchange between HTO and hydrogen over a column prepared from alumina or molecular sieve-5A can be successfully applied for the quantitative transfer of tritium from HTO into hydrogen for on-line gas countinq. This provides an analytical procedure for the automatic determination of tritium in water with an analytical reproducibility better than 0.5% rel. The exchange process will also be suitable for rapid tritium transfer from water formed during the decomposition of tritium-labelled organic compounds or biological materials. The application of the procedure in automatic isotope gas analysis of organic materials labelled with tritium will be described in subsequent papers (Parts II and III). (T.G.)

  17. Tests of intestinal absorption using carbon-14-labeled isotopes

    International Nuclear Information System (INIS)

    Fromm, H.; Sarva, R.P.

    1983-01-01

    Beta radiation-emitting isotopes are being used increasingly in diagnostic gastroenterology for the study of absorption. The major reason for the popularity of radioisotopes is that their use is convenient for patient and physician alike. They often obviate naso- or orointestinal intubation and the collection, storage, and analysis of stool. The radioactivity used for the studies of digestive and absorptive processes is small and is not hazardous. In spite of the safety of the radiolabeled compounds, their use is restricted in children and pregnant women. Therefore, for most tests, promising alternative methods that make use of the stable isotope of carbon, /sup 13/C, instead of the radioactive /sup 14/C have been developed. The analysis of stable isotopes requires more sophisticated technology than that of radioactive compounds, however. Only a few centers presently are equipped and staffed to analyze stable isotopes on a routine basis. In contrast, the analysis of radioactive isotopes has become a routine procedure in almost ever major laboratory. The last decade has brought the development of several radioactive absorption tests. The clinically most useful tests relate to the study of bile acid, fat, lactose, and xylose absorption. All of these tests utilize the excretion rate of /sup 14/CO/sub 2/ in breath after ingestion of a /sup 14/C-labeled compound as a measure of the rate of its absorption or malabsorption

  18. Sulfur Isotope Exchange between S-35 Labeled Inorganic Sulfur-Compounds in Anoxic Marine-Sediments

    DEFF Research Database (Denmark)

    FOSSING, H.; THODEANDERSEN, S.; JØRGENSEN, BB

    1992-01-01

    Isotope exchange reactions between S-35-labeled sulfur compounds were studied in anoxic estuarine sediment slurries at 21-degrees-C and pH 7.4-7.7. Two experiments labeled with radioactive elemental sulfur (S-35-degrees) and one labeled with radioactive sulfate ((SO42-)-S-35) were performed as time......% of the total S-35 was recovered in the SIGMA-HS- pool in less than 1.5 h. With no detectable SIGMA-HS- (less than 1-mu-M) in the slurry, 58% of the total S-35 was observed in the pyrite pool within 1.5 h. The FeS pool received up to 31% of all S-35 added. The rapid S-35 incorporation from S-35-degrees...... into SIGMA-HS- and FeS pools was explained by isotope exchange reactions. In contrast, there was evidence that the radioactivity observed in the 'pyrite pool' was caused by adhesion of the added S-35-degrees to the FeS2 grains. In all S-35-degrees-labeled experiments we also observed oxidation...

  19. Hydroponic isotope labeling of entire plants and high-performance mass spectrometry for quantitative plant proteomics.

    Science.gov (United States)

    Bindschedler, Laurence V; Mills, Davinia J S; Cramer, Rainer

    2012-01-01

    Hydroponic isotope labeling of entire plants (HILEP) combines hydroponic plant cultivation and metabolic labeling with stable isotopes using (15)N-containing inorganic salts to label whole and mature plants. Employing (15)N salts as the sole nitrogen source for HILEP leads to the production of healthy-looking plants which contain (15)N proteins labeled to nearly 100%. Therefore, HILEP is suitable for quantitative plant proteomic analysis, where plants are grown in either (14)N- or (15)N-hydroponic media and pooled when the biological samples are collected for relative proteome quantitation. The pooled (14)N-/(15)N-protein extracts can be fractionated in any suitable way and digested with a protease for shotgun proteomics, using typically reverse phase liquid chromatography nanoelectrospray ionization tandem mass spectrometry (RPLC-nESI-MS/MS). Best results were obtained with a hybrid ion trap/FT-MS mass spectrometer, combining high mass accuracy and sensitivity for the MS data acquisition with speed and high-throughput MS/MS data acquisition, increasing the number of proteins identified and quantified and improving protein quantitation. Peak processing and picking from raw MS data files, protein identification, and quantitation were performed in a highly automated way using integrated MS data analysis software with minimum manual intervention, thus easing the analytical workflow. In this methodology paper, we describe how to grow Arabidopsis plants hydroponically for isotope labeling using (15)N salts and how to quantitate the resulting proteomes using a convenient workflow that does not require extensive bioinformatics skills.

  20. Analysis of acyl CoA ester intermediates of the mevalonate pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Seker, Tamay; Møller, Kasper; Nielsen, Jens

    2005-01-01

    The mevalonate pathway plays an important role in providing the cell with a number of essential precursors for the synthesis of biomass constituents. With respect to their chemical structure, the metabolites of this pathway can be divided into two groups: acyl esters [acetoacetyl CoA, acetyl Co......A, hydroxymethylglutaryl (HMG) CoA] and phosphorylated metabolites (isopentenyl pyrophosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate). In this study, we developed a method for the precise analysis of the intracellular concentration of acetoacetyl CoA, acetyl CoA and HMG CoA; and we...... used this method for quantification of these metabolites in Saccharomyces cerevisiae, both during batch growth on glucose and on galactose and in glucose-limited chemostat cultures operated at three different dilution rates. The level of the metabolites changed depending on the growth phase...

  1. Noninvasive imaging of protein metabolic labeling in single human cells using stable isotopes and Raman microscopy

    NARCIS (Netherlands)

    van Manen, H.J.; Lenferink, Aufrid T.M.; Otto, Cornelis

    2008-01-01

    We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C−D

  2. Isotope labeling for NMR studies of macromolecular structure and interactions

    International Nuclear Information System (INIS)

    Wright, P.E.

    1994-01-01

    Implementation of biosynthetic methods for uniform or specific isotope labeling of proteins, coupled with the recent development of powerful heteronuclear multidimensional NMR methods, has led to a dramatic increase in the size and complexity of macromolecular systems that are now amenable to NMR structural analysis. In recent years, a new technology has emerged that combines uniform 13 C, 15 N labeling with heteronuclear multidimensional NMR methods to allow NMR structural studies of systems approaching 25 to 30 kDa in molecular weight. In addition, with the introduction of specific 13 C and 15 N labels into ligands, meaningful NMR studies of complexes of even higher molecular weight have become feasible. These advances usher in a new era in which the earlier, rather stringent molecular weight limitations have been greatly surpassed and NMR can begin to address many central biological problems that involve macromolecular structure, dynamics, and interactions

  3. Isotope labeling for NMR studies of macromolecular structure and interactions

    Energy Technology Data Exchange (ETDEWEB)

    Wright, P.E. [Scripps Research Institute, La Jolla, CA (United States)

    1994-12-01

    Implementation of biosynthetic methods for uniform or specific isotope labeling of proteins, coupled with the recent development of powerful heteronuclear multidimensional NMR methods, has led to a dramatic increase in the size and complexity of macromolecular systems that are now amenable to NMR structural analysis. In recent years, a new technology has emerged that combines uniform {sup 13}C, {sup 15}N labeling with heteronuclear multidimensional NMR methods to allow NMR structural studies of systems approaching 25 to 30 kDa in molecular weight. In addition, with the introduction of specific {sup 13}C and {sup 15}N labels into ligands, meaningful NMR studies of complexes of even higher molecular weight have become feasible. These advances usher in a new era in which the earlier, rather stringent molecular weight limitations have been greatly surpassed and NMR can begin to address many central biological problems that involve macromolecular structure, dynamics, and interactions.

  4. Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

    International Nuclear Information System (INIS)

    Nemazanyy, Ivan; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T.

    2006-01-01

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy β and originally identified CoA synthase, CoASy α. The transcript specific for CoASy β was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy β. In contrast to CoASy α, which shows ubiquitous expression, CoASy β is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation

  5. Stable isotope labeled n-alkanes to assess digesta passage kinetics through the digestive tract of ruminants

    NARCIS (Netherlands)

    Warner, D.; Ferreira, L.M.M.; Breuer, M.J.H.; Dijkstra, J.; Pellikaan, W.F.

    2013-01-01

    We describe the use of carbon stable isotope (13C) labeled n-alkanes as a potential internal tracer to assess passage kinetics of ingested nutrients in ruminants. Plant cuticular n-alkanes originating from intrinsically 13C labeled ryegrass plants were pulse dosed intraruminally in four

  6. Mitochondrial storage form of acetyl CoA carboxylase in fasted and alloxan diabetic rats

    International Nuclear Information System (INIS)

    Roman-Lopez, C.R.; Allred, J.B.

    1986-01-01

    Sodium dodecyl sulfate-denatured biotinyl proteins will bind [ 14 C]methyl avidin which remains bound through polyacrylamide gel electrophoresis. The method has been used to demonstrate the presence of two high molecular weight subunit forms of acetyl CoA carboxylase in rat liver cytoplasm, both of which are precipitated by antibody to purifed rat liver acetyl CoA carboxylase prepared from sheep serum. Rat liver mitochondria contained five distinct biotinyl protein subunits, the two largest of which have been identified as acetyl CoA carboxylase subunits on the basis of precipitation by anti-acetyl CoA carboxylase antibody. The small quantity of acetyl CoA carboxylase associated with rat liver microsomes could be attributed to cytoplasmic contamination. The binding of radioactive avidin is sufficiently tight to use as a measure of the quantity of acetyl CoA carboxylase. The quantity and activity of the cytoplasmic enzyme was reduced in fasted and in alloxan diabetic rats compared to that in fed controls but the quantity of the enzyme associated with isolated mitochondria was not reduced. The results indicate that there is a mitochondrial storage form of acetyl CoA carboxylase

  7. Auto-inducing media for uniform isotope labeling of proteins with 15N, 13C and 2H

    International Nuclear Information System (INIS)

    Guthertz, Nicolas; Klopp, Julia; Winterhalter, Aurélie; Fernández, César; Gossert, Alvar D.

    2015-01-01

    Auto-inducing media for protein expression offer many advantages like robust reproducibility, high yields of soluble protein and much reduced workload. Here, an auto-inducing medium for uniform isotope labelling of proteins with 15 N, 13 C and/or 2 H in E. coli is presented. So far, auto-inducing media have not found widespread application in the NMR field, because of the prohibitively high cost of labeled lactose, which is an essential ingredient of such media. Here, we propose using lactose that is only selectively labeled on the glucose moiety. It can be synthesized from inexpensive and readily available substrates: labeled glucose and unlabeled activated galactose. With this approach, uniformly isotope labeled proteins were expressed in unattended auto-inducing cultures with incorporation of 13 C, 15 N of 96.6 % and 2 H, 15 N of 98.8 %. With the present protocol, the NMR community could profit from the many advantages that auto-inducing media offer

  8. Addressing Raman features of individual layers in isotopically labeled Bernal stacked bilayer graphene

    Czech Academy of Sciences Publication Activity Database

    da Costa, Sara; Ek Weis, Johan; Frank, Otakar; Fridrichová, Michaela; Kalbáč, Martin

    2016-01-01

    Roč. 3, č. 2 (2016), 025022 ISSN 2053-1583 R&D Projects: GA MŠk LL1301 Institutional support: RVO:61388955 Keywords : graphene bilayer * Raman spectroscopy * isotope labeling Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 6.937, year: 2016

  9. Testing isotopic labeling with [¹³C₆]glucose as a method of advanced glycation sites identification.

    Science.gov (United States)

    Kielmas, Martyna; Kijewska, Monika; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2012-12-01

    The Maillard reaction occurring between reducing sugars and reactive amino groups of biomolecules leads to the formation of a heterogeneous mixture of compounds: early, intermediate, and advanced glycation end products (AGEs). These compounds could be markers of certain diseases and of the premature aging process. Detection of Amadori products can be performed by various methods, including MS/MS techniques and affinity chromatography on immobilized boronic acid. However, the diversity of the structures of AGEs makes detection of these compounds more difficult. The aim of this study was to test a new method of AGE identification based on isotope (13)C labeling. The model protein (hen egg lysozyme) was modified with an equimolar mixture of [(12)C(6)]glucose and [(13)C(6)]glucose and then subjected to reduction of the disulfide bridges followed by tryptic hydrolysis. The digest obtained was analyzed by LC-MS. The glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. This method allowed identification of 38 early Maillard reaction products and five different structures of the end glycation products. This isotopic labeling technique combined with LC-MS is a sensitive method for identification of advanced glycation end products even if their chemical structure is unknown. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation

    International Nuclear Information System (INIS)

    Wood, Matthew J.; Komives, Elizabeth A.

    1999-01-01

    Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P.pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P.pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P.pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10-100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N- labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment

  11. Stable isotope labeling – Liquid chromatography/mass spectrometry for quantitative analysis of androgenic and progestagenic steroids

    International Nuclear Information System (INIS)

    Guo, Ning; Liu, Ping; Ding, Jun; Zheng, Shu-Jian; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-01-01

    Steroid hormones play important roles in mammal at very low concentrations and are associated with numerous endocrinology and oncology diseases. Therefore, quantitative analysis of steroid hormones can provide crucial information for uncovering underlying mechanisms of steroid hormones related diseases. In the current study, we developed a sensitive method for the detection of steroid hormones (progesterone, dehydroepiandrosterone, testosterone, pregnenolone, 17-hydroxyprogesterone, androstenedione and 17α-hydroxypregnenolone) in body fluids by stable isotope labeling coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. In this respect, a pair of isotopes labeling reagents, Girard reagent P (GP) and d_5-Girard reagent P (d_5-GP), were synthesized and utilized to label steroid hormones in follicular fluid samples and steroid hormone standards, respectively. The heavy labeled standards were used as internal standards for quantification to minimize quantitation deviation in MS analysis due to the matrix and ion suppression effects. The ionization efficiencies of steroid hormones were greatly improved by 4–504 folds through the introduction of a permanent charged moiety of quaternary ammonium from GP. Using the developed method, we successfully quantified steroid hormones in human follicular fluid. We found that the contents of testosterone and androstenedione exhibited significant increase while the content of pregnenolone had significant decrease in follicular fluid of polycystic ovarian syndrome (PCOS) patients compared with healthy controls, indicating that these steroid hormones with significant change may contribute to the pathogenesis of PCOS. Taken together, the developed stable isotope labeling coupled LC-ESI-MS/MS analysis demonstrated to be a promising method for the sensitive and accurate determination of steroid hormones, which may facilitate the in-depth investigation of steroid hormones related

  12. Stable isotope labeling – Liquid chromatography/mass spectrometry for quantitative analysis of androgenic and progestagenic steroids

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Ning; Liu, Ping; Ding, Jun; Zheng, Shu-Jian; Yuan, Bi-Feng; Feng, Yu-Qi, E-mail: yqfeng@whu.edu.cn

    2016-01-28

    Steroid hormones play important roles in mammal at very low concentrations and are associated with numerous endocrinology and oncology diseases. Therefore, quantitative analysis of steroid hormones can provide crucial information for uncovering underlying mechanisms of steroid hormones related diseases. In the current study, we developed a sensitive method for the detection of steroid hormones (progesterone, dehydroepiandrosterone, testosterone, pregnenolone, 17-hydroxyprogesterone, androstenedione and 17α-hydroxypregnenolone) in body fluids by stable isotope labeling coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. In this respect, a pair of isotopes labeling reagents, Girard reagent P (GP) and d{sub 5}-Girard reagent P (d{sub 5}-GP), were synthesized and utilized to label steroid hormones in follicular fluid samples and steroid hormone standards, respectively. The heavy labeled standards were used as internal standards for quantification to minimize quantitation deviation in MS analysis due to the matrix and ion suppression effects. The ionization efficiencies of steroid hormones were greatly improved by 4–504 folds through the introduction of a permanent charged moiety of quaternary ammonium from GP. Using the developed method, we successfully quantified steroid hormones in human follicular fluid. We found that the contents of testosterone and androstenedione exhibited significant increase while the content of pregnenolone had significant decrease in follicular fluid of polycystic ovarian syndrome (PCOS) patients compared with healthy controls, indicating that these steroid hormones with significant change may contribute to the pathogenesis of PCOS. Taken together, the developed stable isotope labeling coupled LC-ESI-MS/MS analysis demonstrated to be a promising method for the sensitive and accurate determination of steroid hormones, which may facilitate the in-depth investigation of steroid hormones

  13. Investigation into reaction of heterogenous isotopic exchange with gaseoUs tritium in solution for preparation labelled lipid compounds

    International Nuclear Information System (INIS)

    Shevchenko, V.P.; Myasoedov, N.F.

    1983-01-01

    The applicability of the method of heterogeneous catalytic isotopic exchange with gaseous tritium in the solution for the production of labelled lipide preparations is studied. Labelled saturated and unsaturated aliphatic acids, prostaglandins, phospholipides and sphingolipides are prepared

  14. Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture

    DEFF Research Database (Denmark)

    Liberski, A. R.; Al-Noubi, M. N.; Rahman, Z. H.

    2013-01-01

    Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only ra...

  15. IsoMS: automated processing of LC-MS data generated by a chemical isotope labeling metabolomics platform.

    Science.gov (United States)

    Zhou, Ruokun; Tseng, Chiao-Li; Huan, Tao; Li, Liang

    2014-05-20

    A chemical isotope labeling or isotope coded derivatization (ICD) metabolomics platform uses a chemical derivatization method to introduce a mass tag to all of the metabolites having a common functional group (e.g., amine), followed by LC-MS analysis of the labeled metabolites. To apply this platform to metabolomics studies involving quantitative analysis of different groups of samples, automated data processing is required. Herein, we report a data processing method based on the use of a mass spectral feature unique to the chemical labeling approach, i.e., any differential-isotope-labeled metabolites are detected as peak pairs with a fixed mass difference in a mass spectrum. A software tool, IsoMS, has been developed to process the raw data generated from one or multiple LC-MS runs by peak picking, peak pairing, peak-pair filtering, and peak-pair intensity ratio calculation. The same peak pairs detected from multiple samples are then aligned to produce a CSV file that contains the metabolite information and peak ratios relative to a control (e.g., a pooled sample). This file can be readily exported for further data and statistical analysis, which is illustrated in an example of comparing the metabolomes of human urine samples collected before and after drinking coffee. To demonstrate that this method is reliable for data processing, five (13)C2-/(12)C2-dansyl labeled metabolite standards were analyzed by LC-MS. IsoMS was able to detect these metabolites correctly. In addition, in the analysis of a (13)C2-/(12)C2-dansyl labeled human urine, IsoMS detected 2044 peak pairs, and manual inspection of these peak pairs found 90 false peak pairs, representing a false positive rate of 4.4%. IsoMS for Windows running R is freely available for noncommercial use from www.mycompoundid.org/IsoMS.

  16. Site-specific Orientation of an α-helical Peptide Ovispirin-1 from Isotope Labeled SFG Spectroscopy

    Science.gov (United States)

    Ding, Bei; Laaser, Jennifer E.; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T.; Chen, Zhan

    2013-01-01

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single isotope labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138 degrees from the surface normal, and the transition dipole of the isotope labeled C=O group is tilted at 23 degrees from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrated that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution. PMID:24228619

  17. Site-specific orientation of an α-helical peptide ovispirin-1 from isotope-labeled SFG spectroscopy.

    Science.gov (United States)

    Ding, Bei; Laaser, Jennifer E; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T; Chen, Zhan

    2013-11-27

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single-isotope-labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138° from the surface normal, and the transition dipole of the isotope-labeled C═O group is tilted at 23° from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrate that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope-labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution.

  18. Quantification of isotope-labelled and unlabelled folates in plasma, ileostomy and food samples.

    Science.gov (United States)

    Büttner, Barbara E; Öhrvik, Veronica E; Witthöft, Cornelia M; Rychlik, Michael

    2011-01-01

    New stable isotope dilution assays were developed for the simultaneous quantitation of [(13)C(5)]-labelled and unlabelled 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, folic acid along with unlabelled tetrahydrofolic acid and 10-formylfolic acid in clinical samples deriving from human bioavailability studies, i.e. plasma, ileostomy samples, and food. The methods were based on clean-up by strong anion exchange followed by LC-MS/MS detection. Deuterated analogues of the folates were applied as the internal standards in the stable isotope dilution assays. Assay sensitivity was sufficient to detect all relevant folates in the respective samples as their limits of detection were below 0.62 nmol/L in plasma and below 0.73 μg/100 g in food or ileostomy samples. Quantification of the [(13)C(5)]-label in clinical samples offers the possibility to differentiate between folate from endogenous body pools and the administered dose when executing bioavailability trials.

  19. Temperature-induced strain and doping in monolayer and bilayer isotopically labeled graphene

    Czech Academy of Sciences Publication Activity Database

    Verhagen, Timotheus; Drogowska, Karolina; Kalbáč, Martin; Vejpravová, Jana

    2015-01-01

    Roč. 92, č. 12 (2015), "125437-1"-"125437-9" ISSN 1098-0121 R&D Projects: GA ČR GA15-02196S; GA MŠk LL1301 Institutional support: RVO:68378271 ; RVO:61388955 Keywords : isotopically labeled graphene * temperature dependence * Raman spectroscopy * phonons Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 3.736, year: 2014

  20. Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

    International Nuclear Information System (INIS)

    Burrier, R.E.; Manson, C.R.; Brecher, P.

    1987-01-01

    The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. 14 C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell

  1. Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

    Energy Technology Data Exchange (ETDEWEB)

    Burrier, R.E.; Manson, C.R.; Brecher, P.

    1987-05-01

    The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. /sup 14/C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell.

  2. A general procedure for isotopic (deuterium) labelling of non-steroidal antiinflammatory 2-arylpropionic acids

    International Nuclear Information System (INIS)

    Castell, J.V.; Martinez, L.A.; Universidad Politecnica de Valencia; Miranda, M.A.; Tarrega, Pilar

    1994-01-01

    Alkaline treatment of nonsteroidal antiinflammatory 2-arylpropionic acids in deuterium oxide led in all cases to isotopic exchange of the proton located at the α-position of the side chain. Monodeuteration was observed in the case of carprofen, ibuprofen, ketoprofen, fenoprofen, flurbiprofen and naproxen. Additional exchange of one or two protons of the heterocyclic ring occurred in indoprofen, suprofen and tiaprofenic acid. The isotopic labelling survived under the conditions required to perform in vitro photoallergic studies (photolysis in non-deuterated aqueous media). (Author)

  3. A general procedure for isotopic (deuterium) labelling of non-steroidal antiinflammatory 2-arylpropionic acids

    Energy Technology Data Exchange (ETDEWEB)

    Castell, J.V. (Valencia Univ. Hospital (Spain). Centro de Investigacion); Martinez, L.A. (Valencia Univ. Hospital (Spain). Centro de Investigacion Universidad Politecnica de Valencia (Spain). Dept. de Quimica); Miranda, M.A.; Tarrega, Pilar (Universidad Politecnica de Valencia (Spain). Dept. de Quimica)

    1994-01-01

    Alkaline treatment of nonsteroidal antiinflammatory 2-arylpropionic acids in deuterium oxide led in all cases to isotopic exchange of the proton located at the [alpha]-position of the side chain. Monodeuteration was observed in the case of carprofen, ibuprofen, ketoprofen, fenoprofen, flurbiprofen and naproxen. Additional exchange of one or two protons of the heterocyclic ring occurred in indoprofen, suprofen and tiaprofenic acid. The isotopic labelling survived under the conditions required to perform in vitro photoallergic studies (photolysis in non-deuterated aqueous media). (Author).

  4. UNiquant, a Program for Quantitative Proteomics Analysis Using Stable Isotope Labeling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xin; Tolmachev, Aleksey V.; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A.; Smith, Richard D.; Chan, Wing C.; Hinrichs, Steven; Fu, Kai; Ding, Shi-Jian

    2011-03-04

    We present UNiquant, a new software program for analyzing stable isotope labeling (SIL) based quantitative proteomics data. UNiquant surpassed the performance of two other platforms, MaxQuant and Mascot Distiller, using complex proteome mixtures having either known or unknown heavy/light ratios. UNiquant is compatible with a broad spectrum of search engines and SIL methods, providing outstanding peptide pair identification and accurate measurement of the relative peptide/protein abundance.

  5. Sequencing of Isotope-Labeled Small RNA Using Femtosecond Laser Ablation Time-of-Flight Mass Spectrometry

    Science.gov (United States)

    Kurata-Nishimura, Mizuki; Ando, Yoshinari; Kobayashi, Tohru; Matsuo, Yukari; Suzuki, Harukazu; Hayashizaki, Yoshihide; Kawai, Jun

    2010-04-01

    A novel method for the analysis of sequences of small RNAs using nucleotide triphosphates labeled with stable isotopes has been developed using time-of-flight mass spectroscopy combined with femtosecond laser ablation (fsLA-TOF-MS). Small RNAs synthesized with nucleotides enriched in 13C and 15N were efficiently atomized and ionized by single-shot fsLA and the isotope ratios 13C/12C and 15N/14N were evaluated using the TOF-MS method. By comparing the isotope ratios among four different configurations, the number of nucleotide contents of the control RNA sample were successfully reproduced.

  6. COA based robust output feedback UPFC controller design

    Energy Technology Data Exchange (ETDEWEB)

    Shayeghi, H., E-mail: hshayeghi@gmail.co [Technical Engineering Department, University of Mohaghegh Ardabili, Ardabil (Iran, Islamic Republic of); Shayanfar, H.A. [Center of Excellence for Power System Automation and Operation, Electrical Engineering Department, Iran University of Science and Technology, Tehran (Iran, Islamic Republic of); Jalilzadeh, S.; Safari, A. [Technical Engineering Department, Zanjan University, Zanjan (Iran, Islamic Republic of)

    2010-12-15

    In this paper, a novel method for the design of output feedback controller for unified power flow controller (UPFC) using chaotic optimization algorithm (COA) is developed. Chaotic optimization algorithms, which have the features of easy implementation, short execution time and robust mechanisms of escaping from the local optimum, is a promising tool for the engineering applications. The selection of the output feedback gains for the UPFC controllers is converted to an optimization problem with the time domain-based objective function which is solved by a COA based on Lozi map. Since chaotic mapping enjoys certainty, ergodicity and the stochastic property, the proposed chaotic optimization problem introduces chaos mapping using Lozi map chaotic sequences which increases its convergence rate and resulting precision. To ensure the robustness of the proposed stabilizers, the design process takes into account a wide range of operating conditions and system configurations. The effectiveness of the proposed controller for damping low frequency oscillations is tested and demonstrated through non-linear time-domain simulation and some performance indices studies. The results analysis reveals that the designed COA based output feedback UPFC damping controller has an excellent capability in damping power system low frequency oscillations and enhance greatly the dynamic stability of the power systems.

  7. The method for production of high purity carrier free ortophosphoric acid labeled with isotopes Phosphorus-32 and Phosphorus-33

    International Nuclear Information System (INIS)

    Abdukayumov, M.N.; Abdusalyamov, A.N.; Chistyakov, P.G.; Yuldashev, B.S.

    2001-01-01

    Extensive application for various radioactive isotopes was found in an extremity of the 20-Th century in a science and production. Labeled compounds are used with growing effectiveness in a molecular biology, gene engineering, medicine and other areas. Phosphorus-32 and Phosphorus-33 isotopes as a different labeled compounds that are used mainly in molecular biology are produced at the Radiopreparat enterprise of the Institute of Nuclear Physics of Academy of Sciences of Uzbekistan Republic. The quality of labeled preparations is very high. The specifications for above mentioned preparations corresponds to demands most of customers in different countries. P-32 or P-33 labeled orthophosphoric acid has high radiochemical purity (more than 99 %) and specific radioactivity close to theoretical. Orthophosphoric acid prepared by the described above method has radiochemical purity about 95 % and output of the target product 99%

  8. Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation.

    Science.gov (United States)

    Pabst, Martin; Benešová, Iva; Fagerer, Stephan R; Jacobsen, Mathias; Eyer, Klaus; Schmidt, Gregor; Steinhoff, Robert; Krismer, Jasmin; Wahl, Fabian; Preisler, Jan; Zenobi, Renato

    2016-01-04

    We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS.

  9. Effect of elevated total CoA levels on metabolic pathways in cultured hepatocytes

    International Nuclear Information System (INIS)

    Steffen, C.A.; Smith, C.M.

    1987-01-01

    Livers from fasted rats have 30% higher total CoA levels than fed rats. To determine whether this increase of total CoA influences metabolism, the rates of gluconeogenesis, fatty acid oxidation and ketogenesis were measured in hepatocytes with cyanamide (CYM) or pantothenate (PA) deficient medium used to vary total CoA levels independently of hormonal status. Primary cultures of rat hepatocytes were incubated 14 hrs with Bt 2 cAMP, dexamethasone + theophylline in PA deficient medium or with CYM (500 μM) + PA, rinsed and preincubated 0.5 hr to remove the CYM. Hepatocytes treated with CYM had total CoA levels 10-24% higher than PA deficient cells and lower rates of glucose production from lactate + pyruvate (L/P) or from alanine (0.23 +/- 0.05 and 0.089 +/- 0.02 μm/mg protein, respectively in CYM treated cells compared to 0.33 +/- 0.06 and 0.130 +/- 0.006 in PA deficient cells). This decrease was not due to CYM per se, as the direct addition of CYM stimulated glucose production from L/P. CYM treated cells with 15-40% higher total CoA and 30% higher fatty acyl-CoA levels had the same rates of [ 14 C]-palmitate oxidation as PA deficient cells. However, rates of ketogenesis were lower in CYM treated cells (163 +/- 11 nm/mg compared to 217 +/- 14 nm/mg protein). These results suggest that physiological alterations of hepatic total CoA levels are not necessary for fasting rates of gluconeogenesis, fatty acid oxidation and ketogenesis

  10. Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones.

    Science.gov (United States)

    Liokatis, Stamatios

    2017-03-26

    Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

  11. Accurate and sensitive determination of molar fractions of "1"3C-Labeled intracellular metabolites in cell cultures grown in the presence of isotopically-labeled glucose

    International Nuclear Information System (INIS)

    Fernández-Fernández, Mario; Rodríguez-González, Pablo; Hevia Sánchez, David; González-Menéndez, Pedro; Sainz Menéndez, Rosa M.; García Alonso, J. Ignacio

    2017-01-01

    This work describes a methodology based on multiple linear regression and GC-MS for the determination of molar fractions of isotopically-labeled intracellular metabolites in cell cultures. Novel aspects of this work are: i) the calculation of theoretical isotopic distributions of the different isotopologues from an experimentally measured value of % 13C enrichment of the labeled precursor ii) the calculation of the contribution of lack of mass resolution of the mass spectrometer and different fragmentation mechanism such as the loss or gain of hydrogen atoms in the EI source to measure the purity of the selected cluster for each metabolite and iii) the validation of the methodology not only by the analysis of gravimetrically prepared mixtures of isotopologues but also by the comparison of the obtained molar fractions with experimental values obtained by GC-Combustion-IRMS based on "1"3C/"1"2C isotope ratio measurements. The method is able to measure molar fractions for twenty-eight intracellular metabolites derived from glucose metabolism in cell cultures grown in the presence of "1"3C-labeled Glucose. The validation strategies demonstrate a satisfactory accuracy and precision of the proposed procedure. Also, our results show that the minimum value of "1"3C incorporation that can be accurately quantified is significantly influenced by the calculation of the spectral purity of the measured cluster and the number of "1"3C atoms of the labeled precursor. The proposed procedure was able to accurately quantify gravimetrically prepared mixtures of natural and labeled glucose molar fractions of 0.07% and mixtures of natural and labeled glycine at molar fractions down to 0.7%. The method was applied to initial studies of glucose metabolism of different prostate cancer cell lines. - Highlights: • Determination of molar fractions of "1"3C-labeled metabolites in cell cultures. • The method is based on multiple linear regression and GC-MS. • Validation of the method by

  12. Accurate and sensitive determination of molar fractions of {sup 13}C-Labeled intracellular metabolites in cell cultures grown in the presence of isotopically-labeled glucose

    Energy Technology Data Exchange (ETDEWEB)

    Fernández-Fernández, Mario [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo (Spain); Rodríguez-González, Pablo, E-mail: rodriguezpablo@uniovi.es [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo (Spain); Hevia Sánchez, David; González-Menéndez, Pedro; Sainz Menéndez, Rosa M. [University Institute of Oncology (IUOPA), University of Oviedo, Julián Clavería 6, 33006 Oviedo (Spain); García Alonso, J. Ignacio [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo (Spain)

    2017-05-29

    This work describes a methodology based on multiple linear regression and GC-MS for the determination of molar fractions of isotopically-labeled intracellular metabolites in cell cultures. Novel aspects of this work are: i) the calculation of theoretical isotopic distributions of the different isotopologues from an experimentally measured value of % 13C enrichment of the labeled precursor ii) the calculation of the contribution of lack of mass resolution of the mass spectrometer and different fragmentation mechanism such as the loss or gain of hydrogen atoms in the EI source to measure the purity of the selected cluster for each metabolite and iii) the validation of the methodology not only by the analysis of gravimetrically prepared mixtures of isotopologues but also by the comparison of the obtained molar fractions with experimental values obtained by GC-Combustion-IRMS based on {sup 13}C/{sup 12}C isotope ratio measurements. The method is able to measure molar fractions for twenty-eight intracellular metabolites derived from glucose metabolism in cell cultures grown in the presence of {sup 13}C-labeled Glucose. The validation strategies demonstrate a satisfactory accuracy and precision of the proposed procedure. Also, our results show that the minimum value of {sup 13}C incorporation that can be accurately quantified is significantly influenced by the calculation of the spectral purity of the measured cluster and the number of {sup 13}C atoms of the labeled precursor. The proposed procedure was able to accurately quantify gravimetrically prepared mixtures of natural and labeled glucose molar fractions of 0.07% and mixtures of natural and labeled glycine at molar fractions down to 0.7%. The method was applied to initial studies of glucose metabolism of different prostate cancer cell lines. - Highlights: • Determination of molar fractions of {sup 13}C-labeled metabolites in cell cultures. • The method is based on multiple linear regression and GC-MS.

  13. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    Science.gov (United States)

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.

  14. Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*

    Science.gov (United States)

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-01-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms—including humans—are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)1 with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

  15. Auto-inducing media for uniform isotope labeling of proteins with {sup 15}N, {sup 13}C and {sup 2}H

    Energy Technology Data Exchange (ETDEWEB)

    Guthertz, Nicolas [Institute of Cancer Research, Division of Structural Biology (United Kingdom); Klopp, Julia; Winterhalter, Aurélie; Fernández, César; Gossert, Alvar D., E-mail: alvar.gossert@novartis.com [Novartis Institutes for BioMedical Research (Switzerland)

    2015-06-15

    Auto-inducing media for protein expression offer many advantages like robust reproducibility, high yields of soluble protein and much reduced workload. Here, an auto-inducing medium for uniform isotope labelling of proteins with {sup 15}N, {sup 13}C and/or {sup 2}H in E. coli is presented. So far, auto-inducing media have not found widespread application in the NMR field, because of the prohibitively high cost of labeled lactose, which is an essential ingredient of such media. Here, we propose using lactose that is only selectively labeled on the glucose moiety. It can be synthesized from inexpensive and readily available substrates: labeled glucose and unlabeled activated galactose. With this approach, uniformly isotope labeled proteins were expressed in unattended auto-inducing cultures with incorporation of {sup 13}C, {sup 15}N of 96.6 % and {sup 2}H, {sup 15}N of 98.8 %. With the present protocol, the NMR community could profit from the many advantages that auto-inducing media offer.

  16. Quantitative proteomics by amino acid labeling in C. elegans

    DEFF Research Database (Denmark)

    Fredens, Julius; Engholm-Keller, Kasper; Giessing, Anders

    2011-01-01

    We demonstrate labeling of Caenorhabditis elegans with heavy isotope-labeled lysine by feeding them with heavy isotope-labeled Escherichia coli. Using heavy isotope-labeled worms and quantitative proteomics methods, we identified several proteins that are regulated in response to loss or RNAi-med......-mediated knockdown of the nuclear hormone receptor 49 in C. elegans. The combined use of quantitative proteomics and selective gene knockdown is a powerful tool for C. elegans biology.......We demonstrate labeling of Caenorhabditis elegans with heavy isotope-labeled lysine by feeding them with heavy isotope-labeled Escherichia coli. Using heavy isotope-labeled worms and quantitative proteomics methods, we identified several proteins that are regulated in response to loss or RNAi...

  17. Metabolic flux analysis of the phenylpropanoid pathway in wound-healing potato tuber tissue using stable isotope-labeled tracer and LC-MS spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Fumio; Morino, Keiko; Miyashita, Masahiro; Miyagawa, Hisashi [Kyoto Univ. (Japan). Department of Agriculture

    2003-05-01

    The metabolic flux of two phenylpropanoid metabolites, N-p-coumaroyloctopamine (p-CO) and chlorogenic acid (CGA), in the wound-healing potato tuber tissue was quantitatively analyzed by a newly developed method based upon the tracer experiment using stable isotope-labeled compounds and LC-MS. Tuber disks were treated with aqueous solution of L-phenyl-d{sub 5}-alanine, and the change in the ratio of stable isotope-labeled compound to non-labeled (isotope abundance) was monitored for p-CO and CGA in the tissue extract by LC-MS. The time-dependent change in the isotope abundance of each metabolite was fitted to an equation that was derived from the formation and conversion kinetics of each compound. Good correlations were obtained between the observed and calculated isotope abundances for both p-CO and CGA. The rates of p-CO formation and conversion (i.e. fluxes) were 1.15 and 0.96 nmol (g FW){sup -1}h{sup -1}, respectively, and for CGA, the rates 4.63 and 0.42 nmol (g FW){sup -1}h{sup -1}, respectively. This analysis enabled a direct comparison of the biosynthetic activity between these two compounds. (author)

  18. Synthesis of stable isotopically labeled peptides with filter-assisted enzymatic labeling for the diagnosis of hepatitis B virus infection utilizing mass spectrometry-based proteomics strategy

    International Nuclear Information System (INIS)

    Tsai, Hsing-Fen; Hsiao, He-Hsuan

    2017-01-01

    A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic "1"6O/"1"8O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol–1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per μl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease. - Highlights: • Facile synthesis of an inexpensive and highly reproducible stable isotopically labeled peptides. • Complete incorporation of two "1"8O atoms into synthesized peptides with filter-assisted enzymatic labeling. • Targeted analysis with parallel reaction monitoring assay for the disease diagnosis.

  19. Synthesis of stable isotopically labeled peptides with filter-assisted enzymatic labeling for the diagnosis of hepatitis B virus infection utilizing mass spectrometry-based proteomics strategy

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, Hsing-Fen; Hsiao, He-Hsuan, E-mail: hhhsiao@dragon.nchu.edu.tw

    2017-03-01

    A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic {sup 16}O/{sup 18}O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol–1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per μl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease. - Highlights: • Facile synthesis of an inexpensive and highly reproducible stable isotopically labeled peptides. • Complete incorporation of two {sup 18}O atoms into synthesized peptides with filter-assisted enzymatic labeling. • Targeted analysis with parallel reaction monitoring assay for the disease diagnosis.

  20. Evaluation of the impact of matrix effect on quantification of pesticides in foods by gas chromatography-mass spectrometry using isotope-labeled internal standards.

    Science.gov (United States)

    Yarita, Takashi; Aoyagi, Yoshie; Otake, Takamitsu

    2015-05-29

    The impact of the matrix effect in GC-MS quantification of pesticides in food using the corresponding isotope-labeled internal standards was evaluated. A spike-and-recovery study of nine target pesticides was first conducted using paste samples of corn, green soybean, carrot, and pumpkin. The observed analytical values using isotope-labeled internal standards were more accurate for most target pesticides than that obtained using the external calibration method, but were still biased from the spiked concentrations when a matrix-free calibration solution was used for calibration. The respective calibration curves for each target pesticide were also prepared using matrix-free calibration solutions and matrix-matched calibration solutions with blank soybean extract. The intensity ratio of the peaks of most target pesticides to that of the corresponding isotope-labeled internal standards was influenced by the presence of the matrix in the calibration solution; therefore, the observed slope varied. The ratio was also influenced by the type of injection method (splitless or on-column). These results indicated that matrix-matching of the calibration solution is required for very accurate quantification, even if isotope-labeled internal standards were used for calibration. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Synthesis and magnetic properties of superparamagnetic CoAs nanostructures

    Science.gov (United States)

    Desai, P.; Ashokaan, N.; Masud, J.; Pariti, A.; Nath, M.

    2015-03-01

    This article provides a comprehensive guide on the synthesis and characterization of superparamagnetic CoAs nanoparticles and elongated nanostructures with high blocking temperature, (TB), via hot-injection precipitation and solvothermal methods. Cobalt arsenides constitute an important family of magnetically active solids that find a variety of applications ranging from magnetic semiconductors to biomedical imaging. While the higher temperature hot-injection precipitation technique (300 °C) yields pure CoAs nanostructures, the lower temperature solvothermal method (200 °C) yields a mixture of CoAs nanoparticles along with other Co-based impurity phases. The synthesis in all these cases involved usage of triphenylarsine ((C6H5)3As) as the As precursor which reacts with solid Co2(CO)8 by ligand displacement to yield a single source precursor. The surfactant, hexadecylamine (HDA) further assists in controlling the morphology of the nanostructures. HDA also provides a basic medium and molten flux-like conditions for the redox chemistry to occur between Co and As at elevated temperatures. The influence of the length of reaction time was investigated by studying the evolution of product morphology over time. It was observed that while spontaneous nucleation at higher temperature followed by controlled growth led to the predominant formation of short nanorods, with longer reaction time, the nanorods were further converted to nanoparticles. The size of the nanoparticles obtained, was mostly in the range of 10-15 nm. The key finding of this work is exceptionally high coercivity in CoAs nanostructures for the first time. Coercivity observed was as high as 0.1 T (1000 Oe) at 2 K. These kinds of magnetic nanostructures find multiple applications in spintronics, whereas the superparamagnetic nanoparticles are viable for use in magnetic storage, ferrofluids and as contrast enhancing agents in MRI.

  2. Cooperation of CMEA member states in the field of the manufacture and use of stable isotopes and compounds thus labelled

    International Nuclear Information System (INIS)

    Ertel, G.; Ewald, G.

    1977-01-01

    The contribution presents a survey of scientific-technical cooperation of CMEA member states in the field of stable isotopes, it deals with the specialization of stable isotope production and compounds thus labelled, and gives the prospects for further development of this cooperation. (HK) [de

  3. Isotope label-aided mass spectrometry reveals the influence of environmental factors on metabolism in single eggs of fruit fly.

    Directory of Open Access Journals (Sweden)

    Te-Wei Tseng

    Full Text Available 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 ((13C(6-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.

  4. Comparison of a Label-Free Quantitative Proteomic Method Based on Peptide Ion Current Area to the Isotope Coded Affinity Tag Method

    Directory of Open Access Journals (Sweden)

    Young Ah Goo

    2008-01-01

    Full Text Available Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advantage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded affinity tag (ICAT method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90% confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis.

  5. Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

    Science.gov (United States)

    Geary, Bethany; Magee, Kieran; Cash, Phillip; Young, Iain S; Whitfield, Phillip D; Doherty, Mary K

    2016-05-01

    The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Carbon allocation belowground in Pinus pinaster using stable carbon isotope pulse labeling technique

    Science.gov (United States)

    Dannoura, M.; Bosc, A.; Chipeaux, C.; Sartore, M.; Lambrot, C.; Trichet, P.; Bakker, M.; Loustau, D.; Epron, D.

    2010-12-01

    Carbon allocation belowground competes with aboveground growth and biomass production. In the other hand, it contributes to resource acquisition such as nutrient, water and carbon sequestration in soil. Thus, a better characterization of carbon flow from plant to soil and its residence time within each compartment is an important issue for understanding and modeling forest ecosystem carbon budget. 13C pulse labeling of whole crown was conducted at 4 seasons to study the fate of assimilated carbon by photosynthesis into the root on 12 year old Pinus pinaster planted in the INRA domain of Pierroton. Maritime pine is the most widely planted species in South-West Europe. Stem, root and soil CO2 effluxes and their isotope composition were measured continuously by tunable diode laser absorption spectroscopy with a trace gas analyzer (TGA 100A; Campbell Scientific) coupled to flow-through chambers. 13CO2 recovery and peak were observed in respiration of each compartment after labeling. It appeared sequentially from top of stem to bottom, and to coarse root. The maximum velocity of carbon transfer was calculated as the difference in time lag of recovery between two positions on the trunk or on the root. It ranged between 0.08-0.2 m h-1 in stem and between 0.04-0.12 m h-1 in coarse root. This velocity was higher in warmer season, and the difference between time lag of recovery and peak increased after first frost. Photosynthates arrived underground 1.5 to 5 days after labeling, at similar time in soil CO2 effluxes and coarse root respiration. 0.08-1.4 g of carbon was respired per tree during first 20 days following labeling. It presented 0.6 -10% of 13C used for labeling and it is strongly related to seasons. The isotope signal was detected in fine root organs and microbial biomass by periodical core sampling. The peak was observed 6 days after labeling in early summer while it was delayed more than 10 days in autumn and winter with less amount of carbon allocated

  7. Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase.

    Science.gov (United States)

    Luk, Louis Y P; Ruiz-Pernía, J Javier; Adesina, Aduragbemi S; Loveridge, E Joel; Tuñón, Iñaki; Moliner, Vincent; Allemann, Rudolf K

    2015-07-27

    Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes ((2) H, (13) C, (15) N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  8. Regulation of schistosome egg production by HMG CoA reductase

    International Nuclear Information System (INIS)

    VandeWaa, E.A.; Bennett, J.L.

    1986-01-01

    Hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase) catalyzes the conversion of HMG CoA to mevalonate in the synthesis of steroids, isoprenoids and terpenes. Mevinolin, an inhibitor of this enzyme, decreased egg production in Schistosoma mansoni during in vitro incubations. This was associated with a reduction in the incorporation of 14 C-acetate into polyisoprenoids and a reduction in the formation of a lipid-linked oligosaccharide. In vivo, mevinolin in daily doses of 50 mg/kg (p.o., from days 30-48 post-infection) caused no change in gross liver pathology in S. mansoni infected mice. However, when parasites exposed to mevinolin or its vehicle in vivo were cultured in vitro, worms from mevinolin-treated mice produced six times more eggs than control parasites. When infected mice were dosed with 250 mg/kg mevinolin daily (p.o., from days 35-45 post-infection), liver pathology was reduced in comparison to control mice. Thus, during in vivo exposure to a high dose of the drug egg production is decreased, while at a lower dose it appears unaffected until the parasites are cultured in a drug-free in vitro system wherein egg production is stimulated to extraordinarily high levels. It may be that at low doses mevinolin, by inhibiting the enzyme, is blocking the formation of a product (such as an isoprenoid) which normally acts to down-regulate enzyme synthesis, resulting in enzyme induction. Induction of HMG CoA reductase is then expressed as increased egg production when the worms are removed from the drug. These data suggest that HMG CoA reductase plays a role in schistosome egg production

  9. The application of high-resolution IR spectroscopy and isotope labeling for detailed investigation of TiO2/gas interface reactions

    Czech Academy of Sciences Publication Activity Database

    Civiš, Svatopluk; Ferus, Martin; Zukalová, Markéta; Kavan, Ladislav; Zelinger, Zdeněk

    2013-01-01

    Roč. 36, č. 1 (2013), s. 159-162 ISSN 0925-3467 R&D Projects: GA ČR GAP208/10/2302; GA ČR GA13-07724S Institutional support: RVO:61388955 Keywords : isotope exchange * titania * isotope labeling Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.075, year: 2013

  10. Validation of 13CO2 breath analysis as a measurement of demethylation of stable isotope labeled aminopyrine in man

    International Nuclear Information System (INIS)

    Schneider, J.F.; Schoeller, D.A.; Nemchausky, B.; Bayer, J.L.; Klein, P.

    1978-01-01

    Interval sampling of expired breath as a simple, non-invasive assessment of the effect of liver disease upon hepatic microsomal drug metabolism, has been demonstrated with [ 14 C] dimethylaminoantipyrine (aminopyrine). In order to eliminate radiation risk the authors have validated the use of aminopyrine labeled with the stable, non-radioactive isotope 13 C. Simultaneous oral administration of both [ 14 C]- and [ 13 C] aminopyrine to five adult subjects without liver disease as well as five patients with known liver disease, resulted in the excretion of label at nearly identical rates in both individual time collections (r=0.94) as well as cumulative excretion for three hours (r=0.97). An oral dose of 2-mg/kg of [ 13 C) aminopyrine resulted in rates of production of 13 CO 2 significantly greater than baseline variations in 13 CO 2 production in the fasting, resting subject. Measurements of a single peak value at one half hour correlated closely with the determination of cumulative appearance over three hours (r=0.96). A consistent reproducible increase in the peak production of 13 CO 2 was observed when five patients received phenobarbital. Stable isotope labeled aminopyrine may be used to detect the effects of disease and treatment upon hepatic N-demethylation activity in human subjects without incurring any risk from radiation. Furthermore, the availability of another isotopic carbon label should make possible the study of direct drug-drug interaction utilizing CO 2 analysis. (Auth.)

  11. Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR

    Energy Technology Data Exchange (ETDEWEB)

    Eddy, Matthew T. [Massachusetts Institute of Technology, Department of Chemistry (United States); Belenky, Marina [Brandeis University, Department of Chemistry (United States); Sivertsen, Astrid C. [Massachusetts Institute of Technology, Francis Bitter Magnet Laboratory (United States); Griffin, Robert G. [Massachusetts Institute of Technology, Department of Chemistry (United States); Herzfeld, Judith, E-mail: herzfeld@brandeis.edu [Brandeis University, Department of Chemistry (United States)

    2013-10-15

    The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new 'redox' approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-{sup 13}C] acetate does not label {alpha} carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-{sup 13}C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA-CO coupling in the backbone should improve the resolution of NCACX spectra.

  12. Identification of miRNA targets with stable isotope labeling by amino acids in cell culture

    DEFF Research Database (Denmark)

    Vinther, Jeppe; Hedegaard, Mads Marquardt; Gardner, Paul Phillip

    2006-01-01

    miRNAs are small noncoding RNAs that regulate gene expression. We have used stable isotope labeling by amino acids in cell culture (SILAC) to investigate the effect of miRNA-1 on the HeLa cell proteome. Expression of 12 out of 504 investigated proteins was repressed by miRNA-1 transfection...

  13. Modification degrees at specific sites on heparan sulphate: an approach to measure chemical modifications on biological molecules with stable isotope labelling

    Science.gov (United States)

    Wu, Zhengliang L.; Lech, Miroslaw

    2005-01-01

    Chemical modification of biological molecules is a general mechanism for cellular regulation. A quantitative approach has been developed to measure the extent of modification on HS (heparan sulphates). Sulphation on HS by sulphotransferases leads to variable sulphation levels, which allows cells to tune their affinities to various extracellular proteins, including growth factors. With stable isotope labelling and HPLC-coupled MS, modification degrees at various O-sulphation sites could be determined. A bovine kidney HS sample was first saturated in vitro with 34S by an OST (O-sulphotransferase), then digested with nitrous acid and analysed with HPLC-coupled MS. The 34S-labelled oligosaccharides were identified based on their unique isotope clusters. The modification degrees at the sulphotransferase recognition sites were obtained by calculating the intensities of isotopic peaks in the isotope clusters. The modification degrees at 3-OST-1 and 6-OST-1 sites were examined in detail. This approach can also be used to study other types of chemical modifications on biological molecules. PMID:15743272

  14. Preparation of H3-labelled methyl ethers of saturated fatty acids by heterogeneous catalytic isotope exchange in solution with gaseous tritium

    International Nuclear Information System (INIS)

    Shevchenko, V.P.; Myasoedov, N.F.

    1980-01-01

    A simple method of preparing 3 H-labelled methyl ethers of saturated fatty acids in the dioxane solution using the method of isotopic heterogenous catalytic exchange with gaseous tritium, is suggested. 3 H-labelled natural fatty acids (C 12 -C 18 ) are prepared by alkaline hydrolysis [ru

  15. Development of high-performance chemical isotope labeling LC-MS for profiling the human fecal metabolome.

    Science.gov (United States)

    Xu, Wei; Chen, Deying; Wang, Nan; Zhang, Ting; Zhou, Ruokun; Huan, Tao; Lu, Yingfeng; Su, Xiaoling; Xie, Qing; Li, Liang; Li, Lanjuan

    2015-01-20

    Human fecal samples contain endogenous human metabolites, gut microbiota metabolites, and other compounds. Profiling the fecal metabolome can produce metabolic information that may be used not only for disease biomarker discovery, but also for providing an insight about the relationship of the gut microbiome and human health. In this work, we report a chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method for comprehensive and quantitative analysis of the amine- and phenol-containing metabolites in fecal samples. Differential (13)C2/(12)C2-dansyl labeling of the amines and phenols was used to improve LC separation efficiency and MS detection sensitivity. Water, methanol, and acetonitrile were examined as an extraction solvent, and a sequential water-acetonitrile extraction method was found to be optimal. A step-gradient LC-UV setup and a fast LC-MS method were evaluated for measuring the total concentration of dansyl labeled metabolites that could be used for normalizing the sample amounts of individual samples for quantitative metabolomics. Knowing the total concentration was also useful for optimizing the sample injection amount into LC-MS to maximize the number of metabolites detectable while avoiding sample overloading. For the first time, dansylation isotope labeling LC-MS was performed in a simple time-of-flight mass spectrometer, instead of high-end equipment, demonstrating the feasibility of using a low-cost instrument for chemical isotope labeling metabolomics. The developed method was applied for profiling the amine/phenol submetabolome of fecal samples collected from three families. An average of 1785 peak pairs or putative metabolites were found from a 30 min LC-MS run. From 243 LC-MS runs of all the fecal samples, a total of 6200 peak pairs were detected. Among them, 67 could be positively identified based on the mass and retention time match to a dansyl standard library, while 581 and 3197 peak pairs could be putatively

  16. An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts

    International Nuclear Information System (INIS)

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan

    2015-01-01

    Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein 15 N and 13 C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor

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

    DEFF Research Database (Denmark)

    Bornø, Andreas; van Hall, Gerrit

    2014-01-01

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

  18. The Antibiotic CJ-15,801 is an Antimetabolite which Hijacks and then Inhibits CoA Biosynthesis

    Science.gov (United States)

    van der Westhuyzen, Renier; Hammons, Justin C.; Meier, Jordan L.; Dahesh, Samira; Moolman, Wessel J. A.; Pelly, Stephen C.; Nizet, Victor; Burkart, Michael D.; Strauss, Erick

    2012-01-01

    SUMMARY The natural product CJ-15,801 is an inhibitor of Staphylococcus aureus, but not other bacteria. Its close structural resemblance to pantothenic acid, the vitamin precursor of coenzyme A (CoA), and its Michael acceptor moiety suggest that it irreversibly inhibits an enzyme involved in CoA biosynthesis or utilization. However, its mode of action and the basis for its specificity have not been elucidated to date. We demonstrate that CJ-15,801 is transformed by the uniquely selective S. aureus pantothenate kinase, the first CoA biosynthetic enzyme, into a substrate for the next enzyme, phosphopantothenoylcysteine synthetase, which is inhibited through formation of a tight-binding structural mimic of its native reaction intermediate. These findings reveal CJ-15,801 as a vitamin biosynthetic pathway antimetabolite with a mechanism similar to that of the sulfonamide antibiotics, and highlight CoA biosynthesis as a viable antimicrobial drug target. PMID:22633408

  19. Improved segmental isotope labeling of proteins and application to a larger protein

    International Nuclear Information System (INIS)

    Otomo, Takanori; Teruya, Kenta; Uegaki, Koichi; Yamazaki, Toshio; Kyogoku, Yoshimasa

    1999-01-01

    A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591-5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally 13 C/ 15 N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples

  20. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    Energy Technology Data Exchange (ETDEWEB)

    Wubben, T.; Mesecar, A.D. (Purdue); (UIC)

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

  1. Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

    Science.gov (United States)

    Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

    2016-08-01

    The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

  2. The synthesis of tritium, carbon-14 and stable isotope labelled selective estrogen receptor degraders.

    Science.gov (United States)

    Bragg, Ryan A; Bushby, Nick; Ericsson, Cecilia; Kingston, Lee P; Ji, Hailong; Elmore, Charles S

    2016-09-01

    As part of a Medicinal Chemistry program aimed at developing an orally bioavailable selective estrogen receptor degrader, a number of tritium, carbon-14, and stable isotope labelled (E)-3-[4-(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl]prop-2-enoic acids were required. This paper discusses 5 synthetic approaches to this compound class. Copyright © 2016 John Wiley & Sons, Ltd.

  3. ISOTOPE METHODS IN HOMOGENEOUS CATALYSIS.

    Energy Technology Data Exchange (ETDEWEB)

    BULLOCK,R.M.; BENDER,B.R.

    2000-12-01

    The use of isotope labels has had a fundamentally important role in the determination of mechanisms of homogeneously catalyzed reactions. Mechanistic data is valuable since it can assist in the design and rational improvement of homogeneous catalysts. There are several ways to use isotopes in mechanistic chemistry. Isotopes can be introduced into controlled experiments and followed where they go or don't go; in this way, Libby, Calvin, Taube and others used isotopes to elucidate mechanistic pathways for very different, yet important chemistries. Another important isotope method is the study of kinetic isotope effects (KIEs) and equilibrium isotope effect (EIEs). Here the mere observation of where a label winds up is no longer enough - what matters is how much slower (or faster) a labeled molecule reacts than the unlabeled material. The most careti studies essentially involve the measurement of isotope fractionation between a reference ground state and the transition state. Thus kinetic isotope effects provide unique data unavailable from other methods, since information about the transition state of a reaction is obtained. Because getting an experimental glimpse of transition states is really tantamount to understanding catalysis, kinetic isotope effects are very powerful.

  4. Studies of phosphorus-containing fertilizer uptake in soils by 32P isotope labelling

    International Nuclear Information System (INIS)

    Fueleky, Gyoergy; Osztoics, Andrasne; Papne Kranitz, Erzsebet

    1983-01-01

    Breeding experiments were carried out with rye-grass (Lolium perenne L.) on two soil types to determine the plant uptake of phosphorus from naturally occuring element and from that added to the soil by superphosphate fertilizers. 32 P isotope labelling and radiometric measuring method were applied. In addition to the determination of phosphorus uptake, the phosphorus contents of the soil from its natural stock and from the fertilizer for both soil types can be determined by this method. (A.L.)

  5. Formation of Hydroxymethyl DNA Adducts in Rats Orally Exposed to Stable Isotope Labeled Methanol

    Science.gov (United States)

    Lu, Kun; Gul, Husamettin; Upton, Patricia B.; Moeller, Benjamin C.; Swenberg, James A.

    2012-01-01

    Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol’s well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [13CD4]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [13CD4]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account. PMID:22157354

  6. Isotope labelling study of CO oxidation-assisted epoxidation of propene. Implications for oxygen activation on Au catalysts.

    Science.gov (United States)

    Jiang, Jian; Oxford, Sean M; Fu, Baosong; Kung, Mayfair C; Kung, Harold H; Ma, Jiantai

    2010-06-07

    (18)O isotope labelling studies of the CO oxidation-assisted epoxidation of propene, catalyzed by a mixture of Au/TiO(2) and TS-1, using a methanol-H(2)O solvent showed the O in the epoxide was exclusively from O(2) and not H(2)O or methanol.

  7. Synthesis of {sup 15}N isotope labeled alanine; Sintese da alanina enriquecida com {sup 15}N

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Claudineia R. de; Bendassolli, Jose Albertino; Sant' Ana, Carlos Roberto; Tagliassachi, Romulo Barbieri; Maximo, Everaldo; Prestes, Clelber Vieira [Centro de Energia Nuclear na Agricultura (CENA), Piracicaba, SP (Brazil). Dept. de Isotopos Estaveis]. E-mail: crolivei@cena.usp.br

    2005-07-01

    The application of light chemical elements and their stable isotopes in biological studies have been increased over the last years. The use of {sup 15}N labeled amino acids is an important tool for elucidation of peptides structures. This paper describe a method for the synthesis of {sup 15}N isotope labeled alanine at lower costs than international ones, as well as the details of the recovery system of the nitrogen residues. In the present work an amination of {alpha}-haloacids, with the bromopropionic carboxylic acid and labeled aqua ammonia ({sup 15}NH{sub 3} aq) was carried out. In order to avoid eventually losses of {sup 15}NH{sub 3}, special cares were adopted, since the production cost is high. Although the acquisition cost of the {sup 13}N (radioactive) labeled compounds is lower, the obtained stable tracer will allow the accomplishment of important studies of the nitrogen cycling in living things, less occupational and environment hazards, and the time limitation problems in field studies. The tests took place in triplicates with NH{sub 3} (aq) being employed. With the establishment of the system for {sup 15}NH{sub 3} recovery, an average of 94 % of the ammonia employed in the synthesis process was recovered. The purity of the amino acid was state determined by TLC (Thin Layer Chromatography) and HPLC (High-Performance Liquid Chromatography) with a fluorescence detector. The Rf and the retention time of the synthesized sample were similar the sigma standard. Finally, regarding the established conditions, it was possible to obtain the alanine with a production cost about 40 % lower than the international price. (author)

  8. Recycling of an amino acid label with prolonged isotope infusion: Implications for kinetic studies

    International Nuclear Information System (INIS)

    Schwenk, W.F.; Tsalikian, E.; Beaufrere, B.; Haymond, M.W.

    1985-01-01

    To investigate whether recycling of a labeled amino acid would occur after 24 h of infusion, two groups of normal volunteers were infused with [ 3 H]leucine and alpha-[ 14 C]-ketoisocaproate for 4 h and [ 2 H 3 ]leucine for either 4 or 24 h (groups I and II, respectively). Entry of [ 2 H 3 ]leucine at steady state into the plasma space was indistinguishable from its infusion rate for group I but 30% higher (P less than 0.001) than this rate for group II, demonstrating significant recycling of label. After discontinuation of the infusions, isotope disappearance from the plasma space was followed for 2 h. The 3 H and 14 C decay data for both groups suggest that plasma leucine and alpha- ketoisocaproate are derived from a single intracellular pool in the postabsorptive state. In group I, the 3 H and 2 H labels decayed identically; whereas, in group II, the decay of [ 2 H 3 ]-leucine and alpha- [ 2 H 3 ]ketoisocaproate was slower (P less than 0.01) than the decay of [ 3 H]leucine and alpha-[ 3 H]ketoisocaproate, confirming re-entry of label after a 24-h infusion. Therefore kinetic values calculated from models assuming no recycling of labeled amino acids are most likely not quantitative and must be interpreted with care when flux does not change or decreases

  9. Synthesis of carbon-14-labeled sodium palmoxirate and its coenzyme A ester

    Energy Technology Data Exchange (ETDEWEB)

    Weaner, L.E.; Hoerr, D.C.

    1986-04-01

    Synthetic procedures for the preparation of carbon-14-labeled sodium palmoxirate (TDGA), labeled either in the carboxyl position or in the tetradecyl hydrocarbon chain, are described. In addition, the synthesis of the coenzyme A ester of TDGA-14C with a specific activity of 51 mCi/mmol is reported. The coenzyme A ester was prepared by formation of the acyl chloride with oxalyl chloride followed by reaction with coenzyme A (CoA) in a borate-buffered tetrahydrofuran solution. Purification methods and analytical and stability data are reported for the compounds.

  10. iMS2Flux – a high–throughput processing tool for stable isotope labeled mass spectrometric data used for metabolic flux analysis

    Directory of Open Access Journals (Sweden)

    Poskar C Hart

    2012-11-01

    Full Text Available Abstract Background Metabolic flux analysis has become an established method in systems biology and functional genomics. The most common approach for determining intracellular metabolic fluxes is to utilize mass spectrometry in combination with stable isotope labeling experiments. However, before the mass spectrometric data can be used it has to be corrected for biases caused by naturally occurring stable isotopes, by the analytical technique(s employed, or by the biological sample itself. Finally the MS data and the labeling information it contains have to be assembled into a data format usable by flux analysis software (of which several dedicated packages exist. Currently the processing of mass spectrometric data is time-consuming and error-prone requiring peak by peak cut-and-paste analysis and manual curation. In order to facilitate high-throughput metabolic flux analysis, the automation of multiple steps in the analytical workflow is necessary. Results Here we describe iMS2Flux, software developed to automate, standardize and connect the data flow between mass spectrometric measurements and flux analysis programs. This tool streamlines the transfer of data from extraction via correction tools to 13C-Flux software by processing MS data from stable isotope labeling experiments. It allows the correction of large and heterogeneous MS datasets for the presence of naturally occurring stable isotopes, initial biomass and several mass spectrometry effects. Before and after data correction, several checks can be performed to ensure accurate data. The corrected data may be returned in a variety of formats including those used by metabolic flux analysis software such as 13CFLUX, OpenFLUX and 13CFLUX2. Conclusion iMS2Flux is a versatile, easy to use tool for the automated processing of mass spectrometric data containing isotope labeling information. It represents the core framework for a standardized workflow and data processing. Due to its flexibility

  11. Determination of symbiotic nitrogen fixation by labelling the soil atmosphere with sup(15)N sub(2) at low isotope enrichment

    International Nuclear Information System (INIS)

    Trivelin, P.C.O.

    1982-01-01

    A direct method to determine the total symbiotic nitrogen fixation during the leguminous plants cycles has been, developed, by labelling the soil atmosphere with sup(15)N sub(2) at low isotope enrichment, of about 1 atom % excess. The soil explored by the root system of leguminous plants was confined by means of a chamber in the field and by sealed pots in greenhouse experiments in order to maintain the soil air labelled with sup(15)N sub(2). The average sup(15)N concentration in the soil atmosphere, necessary to calculate dinitrogen fixation, was obtained by integration of the exponential functions of isotope dilution. Those functions were obtained by periodic sampling and analysis of the N sub(2) in the soil atmosphere. The field experiment with labelled atmosphere was carried out from the 22 sup(nd) to the 31 sup(st) day of the bean crop cycle and 5.5 mg N/plant (24% of total plant N) was derived from fixation. In pot experiments, under greenhouse conditions, integrated determination of fixation was made in Phaseolus beans (from the 19 sup(th) to the 67 sup(th) day from planting) and in soybeans (from the 24 sup(th) to the 70 sup(th) day from planting). The soil atmosphere was labelled with sup(15)N sub(2) in both cases. Average fixation obtained for Phaseolus beans was 80 mg N/plant (65% of total plant N) and for soybeans 265 mg N/plant (71% of total plant N). Evaluation of the basic concept of the isotope dilution method to determine nitrogen fixation in pots experiments, as proposed by Fried and Middelboe (1977) has also been made in the present paper. Simultaneous determinations of fixation in soybeans, using the isotope dilution method of Fried and Middelboe, natural variation of the sup(15)N/ sup(14)N ratios, and total-N differences, indicated the same results for pot experiments, harvested at the end of the plant cycle. (author)

  12. Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol

    Energy Technology Data Exchange (ETDEWEB)

    Torizawa, Takuya; Shimizu, Masato [Crest, Jst (Japan); Taoka, Masato [Tokyo Metropolitan University, Graduate School of Science (Japan); Miyano, Hiroshi [Ajinomoto Co., Inc. Institute of Life Sciences (Japan); Kainosho, Masatsune [Crest, Jst (Japan)], E-mail: kainosho@nmr.chem.metro-u.ac.jp

    2004-11-15

    We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy. These methods are efficient and overcome two critical problems associated with the use of conventional Escherichia coli extract systems. Endogenous amino acids normally present in E. coli S30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein. This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids. The second problem encountered in conventional E. coli cell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra. This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag. Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage. We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis.

  13. Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol

    International Nuclear Information System (INIS)

    Torizawa, Takuya; Shimizu, Masato; Taoka, Masato; Miyano, Hiroshi; Kainosho, Masatsune

    2004-01-01

    We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy. These methods are efficient and overcome two critical problems associated with the use of conventional Escherichia coli extract systems. Endogenous amino acids normally present in E. coli S30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein. This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids. The second problem encountered in conventional E. coli cell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra. This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag. Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage. We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis

  14. Closing the gap between T-cell life span estimates from stable isotope-labeling studies in mice and humans

    NARCIS (Netherlands)

    Westera, Liset; Drylewicz, Julia; den Braber, Ineke; Mugwagwa, Tendai; van der Maas, Iris; Kwast, Lydia; Volman, Thomas; van de Weg-Schrijver, Elise H. R.; Bartha, István; Spierenburg, Gerrit; Gaiser, Koos; Ackermans, Mariëtte T.; Asquith, Becca; de Boer, Rob J.; Tesselaar, Kiki; Borghans, José A. M.

    2013-01-01

    Quantitative knowledge of the turnover of different leukocyte populations is a key to our understanding of immune function in health and disease. Much progress has been made thanks to the introduction of stable isotope labeling, the state-of-the-art technique for in vivo quantification of cellular

  15. Isotopic labeling as a tool to establish intramolecular vibrational coupling: The reaction of 2-propanol on Mo(110)

    International Nuclear Information System (INIS)

    Uvdal, P.; Wiegand, B.C.; Serafin, J.G.; Friend, C.M.

    1992-01-01

    The reactions of 2-propanol on Mo(110) were investigated using temperature programmed reaction, high resolution electron energy loss, and x-ray photoelectron spectroscopies. 2-Propanol forms 2-propoxide upon adsorption at 120 K on Mo(110). The 2-propoxide intermediate deoxygenates via selective γ C--H bond scission to eliminate propene as well as C--O bond hydrogenolysis to form trace amounts of propane. The C--O bond of 2-propoxide is estimated to be nearly perpendicular to the surface. Selective isotopic labeling was used to establish the coupling between the C--O stretch and modes associated with the hydrocarbon framework. The degree of coupling was strongly affected by bonding to the surface, primarily due to weakening of the C--O bond when 2-propoxide is bound to Mo(110). Selective isotopic labeling was, therefore, essential in making vibrational assignments and in identifying key reaction steps. Only a small kinetic isotope effect was observed during reaction of (CD 3 )(CH 3 )CHOH, consistent with a substantial component of C--O bond breaking in the transition state for propene elimination. Coupling of the C--O stretch to motion of the methyl group is also suggested to be important in the transition state for propene elimination

  16. An isotope approach based on C-13 pulse-chase labelling vs. the root trenching method to separate heterotrophic and autotrophic respiration in cultivated peatlands

    Energy Technology Data Exchange (ETDEWEB)

    Biasi, C.; Pitkamaki, A. S.; Tavi, N. M.; Koponen, H. T.; Martikainen, P. J. [Univ.of Eastern Finland, Kuopio (Finland). Dept. of Environmental Science], e-mail: christina.biasi@uef.fi

    2012-11-01

    We tested an isotope method based on C-13 pulse-chase labelling for determining the fractional contribution of soil microbial respiration to overall soil respiration in an organic soil (cutaway peatland, eastern Finland), cultivated with the bioenergy crop, reed canary grass. The plants were exposed to CO{sub 2}-13 for five hours and the label was thereafter determined in CO{sub 2} derived from the soil-root system. A two-pool isotope mixing model was used to separate sources of respiration. The isotopic approach showed that a minimum of 50% of the total CO{sub 2} originated from soil-microbial respiration. Even though the method uses undisturbed soil-plant systems, it has limitations concerning the experimental determination of the true isotopic signal of all components contributing to autotrophic respiration. A trenching experiment which was comparatively conducted resulted in a 71% fractional contribution of soil-microbial respiration. This value was likely overestimated. Further studies are needed to evaluate critically the output from these two partitioning approaches. (orig.)

  17. Stable isotope applications in biomolecular structure and mechanisms. A meeting to bring together producers and users of stable-isotope-labeled compounds to assess current and future needs

    International Nuclear Information System (INIS)

    Trewhella, J.; Cross, T.A.; Unkefer, C.J.

    1994-12-01

    Knowledge of biomolecular structure is a prerequisite for understanding biomolecular function, and stable isotopes play an increasingly important role in structure determination of biological molecules. The first Conference on Stable Isotope Applications in Biomolecular Structure and Mechanisms was held in Santa Fe, New Mexico, March 27--31, 1994. More than 120 participants from 8 countries and 44 institutions reviewed significant developments, discussed the most promising applications for stable isotopes, and addressed future needs and challenges. Participants focused on applications of stable isotopes for studies of the structure and function of proteins, peptides, RNA, and DNA. Recent advances in NMR techniques neutron scattering, EPR, and vibrational spectroscopy were highlighted in addition to the production and synthesis of labeled compounds. This volume includes invited speaker and poster presentations as well as a set of reports from discussion panels that focused on the needs of the scientific community and the potential roles of private industry, the National Stable Isotope Resource, and the National High Magnetic Field Laboratory in serving those needs. This is the leading abstract. Individual papers are processed separately for the database

  18. Stable isotope applications in biomolecular structure and mechanisms. A meeting to bring together producers and users of stable-isotope-labeled compounds to assess current and future needs

    Energy Technology Data Exchange (ETDEWEB)

    Trewhella, J.; Cross, T.A.; Unkefer, C.J. [eds.

    1994-12-01

    Knowledge of biomolecular structure is a prerequisite for understanding biomolecular function, and stable isotopes play an increasingly important role in structure determination of biological molecules. The first Conference on Stable Isotope Applications in Biomolecular Structure and Mechanisms was held in Santa Fe, New Mexico, March 27--31, 1994. More than 120 participants from 8 countries and 44 institutions reviewed significant developments, discussed the most promising applications for stable isotopes, and addressed future needs and challenges. Participants focused on applications of stable isotopes for studies of the structure and function of proteins, peptides, RNA, and DNA. Recent advances in NMR techniques neutron scattering, EPR, and vibrational spectroscopy were highlighted in addition to the production and synthesis of labeled compounds. This volume includes invited speaker and poster presentations as well as a set of reports from discussion panels that focused on the needs of the scientific community and the potential roles of private industry, the National Stable Isotope Resource, and the National High Magnetic Field Laboratory in serving those needs. This is the leading abstract. Individual papers are processed separately for the database.

  19. Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility

    International Nuclear Information System (INIS)

    Kobayashi, Hiroshi; Swapna, G. V. T.; Wu, Kuen-Phon; Afinogenova, Yuliya; Conover, Kenith; Mao, Binchen; Montelione, Gaetano T.; Inouye, Masayori

    2012-01-01

    A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS 2 ) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS 2 -tag is replaced with non-isotope labeled PrS 2 -tag, silencing the NMR signals from PrS 2 -tag in isotope-filtered 1 H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS 2 -tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS 2 (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS 2 -tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS 2 -tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone 1 H, 15 N and 13 C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear 1 H– 15 N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.

  20. Lifetimes of organic photovoltaics: Using TOF-SIMS and 18O2 isotopic labelling to characterise chemical degradation mechanisms

    DEFF Research Database (Denmark)

    Norrman, K.; Krebs, Frederik C

    2006-01-01

    The lifetimes of organic photovoltaic cells based on conjugated polymer materials were studied. The device geometry was glass:ITO:PEDOT:PSS:C-12-PSV:C-60:aluminium. To characterise and elucidate the parts of the degradation mechanisms induced by molecular oxygen, 1802 isotopic labelling was emplo......The lifetimes of organic photovoltaic cells based on conjugated polymer materials were studied. The device geometry was glass:ITO:PEDOT:PSS:C-12-PSV:C-60:aluminium. To characterise and elucidate the parts of the degradation mechanisms induced by molecular oxygen, 1802 isotopic labelling...

  1. A Cost-effective Amino-acid-type Selective Isotope Labeling of Proteins Expressed in Leishmania tarentolae

    Czech Academy of Sciences Publication Activity Database

    Foldynová-Trantírková, Silvie; Matulová, J.; Dötsch, V.; Löhr, F.; Cirstea, I.; Alexandov, K.; Breitling, R.; Lukeš, Julius; Trantírek, Lukáš

    2009-01-01

    Roč. 26, č. 6 (2009), s. 755-761 ISSN 0739-1102 R&D Projects: GA ČR GP204/08/P585; GA AV ČR 1QS600220554; GA AV ČR KAN200100801; GA MŠk 2B06129 Institutional research plan: CEZ:AV0Z60220518 Keywords : NMR * isotope labeling * protein expression * Leishmania * low-level enrichment Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.124, year: 2009

  2. Quantitative imaging of subcellular metabolism with stable isotopes and multi-isotope imaging mass spectrometry

    Science.gov (United States)

    Steinhauser, Matthew L.; Lechene, Claude P.

    2014-01-01

    Multi-isotope imaging mass spectrometry (MIMS) is the quantitative imaging of stable isotope labels in cells with a new type of secondary ion mass spectrometer (NanoSIMS). The power of the methodology is attributable to (i) the immense advantage of using non-toxic stable isotope labels, (ii) high resolution imaging that approaches the resolution of usual transmission electron microscopy and (iii) the precise quantification of label down to 1 part-per-million and spanning several orders of magnitude. Here we review the basic elements of MIMS and describe new applications of MIMS to the quantitative study of metabolic processes including protein and nucleic acid synthesis in model organisms ranging from microbes to humans. PMID:23660233

  3. The labelling of Nanocoll[reg] with [111In] for dual-isotope scanning

    International Nuclear Information System (INIS)

    Mitterhauser, Markus; Wadsak, Wolfgang; Key Mien, Leonhard-; Eidherr, Harald; Roka, Sebastian; Zettinig, Georg; Angelberger, Peter; Viernstein, Helmut; Kletter, Kurt; Dudczak, Robert

    2003-01-01

    Visualization and biopsy of sentinel lymph nodes play an important role in planning and controlling the therapy of breast cancer. Hitherto two methods--scintigraphy or gamma probe detection after injection of [ 99m Tc]-nanocolloids and visual detection after injection of patent blue dye--are used routinely. There are no conclusive publications elucidating such important parameters as injection site, injection method and colloidal parameters. The present work aims to label Nanocoll[reg] with [ 111 In] to provide an alternative method, a simultaneous one-compound dual-isotope application. Methods: [ 111 In]-Indiumchloride was buffered with acetate and transferred to the nanocolloid. The colloid labelling reaction was complete after 30 min and filtrated through 100 nm Nuclepore[reg] filters. Results: Incorporation yield of [ 111 In]-Indium into the nanocolloid was nearly quantitative, the step associated with the major loss of activity was the particle sizing with a mean yield of 55%. Conclusion: The presented method allows for the routine supply of [ 111 In]-nanocolloids. Size-filtered [ 111 In]-Nanocoll[reg] shows the same particle size range as [ 99m Tc]-Nanocoll[reg

  4. An economic approach to efficient isotope labeling in insect cells using homemade {sup 15}N-, {sup 13}C- and {sup 2}H-labeled yeast extracts

    Energy Technology Data Exchange (ETDEWEB)

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan, E-mail: Stephan.Grzesiek@unibas.ch [University of Basel, Focal Area Structural Biology and Biophysics, Biozentrum (Switzerland)

    2015-07-15

    Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein {sup 15}N and {sup 13}C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor.

  5. Development of chemical isotope labeling liquid chromatography mass spectrometry for silkworm hemolymph metabolomics

    International Nuclear Information System (INIS)

    Shen, Weifeng; Han, Wei; Li, Yunong; Meng, Zhiqi; Cai, Leiming; Li, Liang

    2016-01-01

    Silkworm (Bombyx mori) is a very useful target insect for evaluation of endocrine disruptor chemicals (EDCs) due to mature breeding techniques, complete endocrine system and broad basic knowledge on developmental biology. Comparative metabolomics of silkworms with and without EDC exposure offers another dimension of studying EDCs. In this work, we report a workflow on metabolomic profiling of silkworm hemolymph based on high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) and demonstrate its application in studying the metabolic changes associated with the pesticide dichlorodiphenyltrichloroethane (DDT) exposure in silkworm. Hemolymph samples were taken from mature silkworms after growing on diet that contained DDT at four different concentrations (1, 0.1, 0.01, 0.001 ppm) as well as on diet without DDT as controls. They were subjected to differential "1"2C-/"1"3C-dansyl labeling of the amine/phenol submetabolome, LC-UV quantification of the total amount of labeled metabolites for sample normalization, and LC-MS detection and relative quantification of individual metabolites in comparative samples. The total concentration of labeled metabolites did not show any significant change between four DDT-treatment groups and one control group. Multivariate statistical analysis of the metabolome data set showed that there was a distinct metabolomic separation between the five groups. Out of the 2044 detected peak pairs, 338 and 1471 metabolites have been putatively identified against the HMDB database and the EML library, respectively. 65 metabolites were identified by the dansyl library searching based on the accurate mass and retention time. Among the 65 identified metabolites, 33 positive metabolites had changes of greater than 1.20-fold or less than 0.83-fold in one or more groups with p-value of smaller than 0.05. Several useful biomarkers including serine, methionine, tryptophan, asymmetric dimethylarginine, N

  6. Development of chemical isotope labeling liquid chromatography mass spectrometry for silkworm hemolymph metabolomics

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Weifeng [Key Laboratory of Detection for Pesticide Residues, Ministry of Agriculture (China); Sericultural Research Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou (China); Han, Wei; Li, Yunong [Department of Chemistry, University of Alberta, Edmonton, Alberta (Canada); Meng, Zhiqi [Sericultural Research Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou (China); Cai, Leiming, E-mail: cailm@mail.zaas.ac.cn [Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou (China); Li, Liang, E-mail: Liang.Li@ualberta.ca [Department of Chemistry, University of Alberta, Edmonton, Alberta (Canada)

    2016-10-26

    Silkworm (Bombyx mori) is a very useful target insect for evaluation of endocrine disruptor chemicals (EDCs) due to mature breeding techniques, complete endocrine system and broad basic knowledge on developmental biology. Comparative metabolomics of silkworms with and without EDC exposure offers another dimension of studying EDCs. In this work, we report a workflow on metabolomic profiling of silkworm hemolymph based on high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) and demonstrate its application in studying the metabolic changes associated with the pesticide dichlorodiphenyltrichloroethane (DDT) exposure in silkworm. Hemolymph samples were taken from mature silkworms after growing on diet that contained DDT at four different concentrations (1, 0.1, 0.01, 0.001 ppm) as well as on diet without DDT as controls. They were subjected to differential {sup 12}C-/{sup 13}C-dansyl labeling of the amine/phenol submetabolome, LC-UV quantification of the total amount of labeled metabolites for sample normalization, and LC-MS detection and relative quantification of individual metabolites in comparative samples. The total concentration of labeled metabolites did not show any significant change between four DDT-treatment groups and one control group. Multivariate statistical analysis of the metabolome data set showed that there was a distinct metabolomic separation between the five groups. Out of the 2044 detected peak pairs, 338 and 1471 metabolites have been putatively identified against the HMDB database and the EML library, respectively. 65 metabolites were identified by the dansyl library searching based on the accurate mass and retention time. Among the 65 identified metabolites, 33 positive metabolites had changes of greater than 1.20-fold or less than 0.83-fold in one or more groups with p-value of smaller than 0.05. Several useful biomarkers including serine, methionine, tryptophan, asymmetric dimethylarginine, N

  7. Development of uniformly stable isotope labeling system in higher plants for hetero-nuclear NMR experiments in vitro and in vivo

    International Nuclear Information System (INIS)

    Kikuchi, J.

    2005-01-01

    Full text: Novel methods for measurement of living systems are making new breakthroughs in life science. In the era of the metabolome (analysis of all measurable metabolites), a MS-based approach is considered to be the major technology, whereas a NMR-based method is recognized as minor technology due to its low sensitivity. Therefore, my laboratory is currently focusing to develop novel methodologies for an NMR-based metabolomics. This will be achieved by uniform stable isotope labeling of higher plants allowing application of multi-dimensional NMR experiments used in protein structure determination. Using these novel methods, I will analyze the dynamic molecular networks inside tissues. Especially, use of stable isotope labeling methods has enormous advantage for discrimination of incorporated or de novo synthesized compounds. Furthermore, potentiality of in vivo-NMR metabolomics will be discussed in the conference. (author)

  8. Assessing of distribution, mobility and bioavailability of exogenous Pb in agricultural soils using isotopic labeling method coupled with BCR approach

    International Nuclear Information System (INIS)

    Huang, Zhi-Yong; Xie, Hong; Cao, Ying-Lan; Cai, Chao; Zhang, Zhi

    2014-01-01

    Highlights: • Large amounts of exogenous Pb were found to distribute in reducible fractions. • Very few of exogenous Pb were found to distribute in acid-extractable fractions. • More than 60% of exogenous Pb in rhizosphere soils lost after planting. • Isotopic labeling method and SEP enable to explore Pb bioavailability in soil. -- Abstract: The contamination of Pb in agricultural soils is one of the most important ecological problems, which potentially results in serious health risk on human health through food chain. Hence, the fate of exogenous Pb contaminated in agricultural soils is needed to be deeply explored. By spiking soils with the stable enriched isotopes of 206 Pb, the contamination of exogenous Pb 2+ ions in three agricultural soils sampled from the estuary areas of Jiulong River, China was simulated in the present study, and the distribution, mobility and bioavailability of exogenous Pb in the soils were investigated using the isotopic labeling method coupled with a four-stage BCR (European Community Bureau of Reference) sequential extraction procedure. Results showed that about 60–85% of exogenous Pb was found to distribute in reducible fractions, while the exogenous Pb in acid-extractable fractions was less than 1.0%. After planting, the amounts of exogenous Pb presenting in acid-extractable, reducible and oxidizable fractions in rhizospheric soils decreased by 60–66%, in which partial exogenous Pb was assimilated by plants while most of the metal might transfer downward due to daily watering and applying fertilizer. The results show that the isotopic labeling technique coupled with sequential extraction procedures enables us to explore the distribution, mobility and bioavailability of exogenous Pb contaminated in soils, which may be useful for the further soil remediation

  9. Assessing of distribution, mobility and bioavailability of exogenous Pb in agricultural soils using isotopic labeling method coupled with BCR approach

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Zhi-Yong, E-mail: zhyhuang@jmu.edu.cn [College of Bioengineering, Jimei University, Xiamen 361021 (China); Xie, Hong [College of Bioengineering, Jimei University, Xiamen 361021 (China); Shandong Vocational Animal Science and Veterinary College, Weifang 261061 (China); Cao, Ying-Lan [College of Bioengineering, Jimei University, Xiamen 361021 (China); Cai, Chao [Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021 (China); Zhang, Zhi [College of Bioengineering, Jimei University, Xiamen 361021 (China)

    2014-02-15

    Highlights: • Large amounts of exogenous Pb were found to distribute in reducible fractions. • Very few of exogenous Pb were found to distribute in acid-extractable fractions. • More than 60% of exogenous Pb in rhizosphere soils lost after planting. • Isotopic labeling method and SEP enable to explore Pb bioavailability in soil. -- Abstract: The contamination of Pb in agricultural soils is one of the most important ecological problems, which potentially results in serious health risk on human health through food chain. Hence, the fate of exogenous Pb contaminated in agricultural soils is needed to be deeply explored. By spiking soils with the stable enriched isotopes of {sup 206}Pb, the contamination of exogenous Pb{sup 2+} ions in three agricultural soils sampled from the estuary areas of Jiulong River, China was simulated in the present study, and the distribution, mobility and bioavailability of exogenous Pb in the soils were investigated using the isotopic labeling method coupled with a four-stage BCR (European Community Bureau of Reference) sequential extraction procedure. Results showed that about 60–85% of exogenous Pb was found to distribute in reducible fractions, while the exogenous Pb in acid-extractable fractions was less than 1.0%. After planting, the amounts of exogenous Pb presenting in acid-extractable, reducible and oxidizable fractions in rhizospheric soils decreased by 60–66%, in which partial exogenous Pb was assimilated by plants while most of the metal might transfer downward due to daily watering and applying fertilizer. The results show that the isotopic labeling technique coupled with sequential extraction procedures enables us to explore the distribution, mobility and bioavailability of exogenous Pb contaminated in soils, which may be useful for the further soil remediation.

  10. Dansylation isotope labeling liquid chromatography mass spectrometry for parallel profiling of human urinary and fecal submetabolomes

    Energy Technology Data Exchange (ETDEWEB)

    Su, Xiaoling [State Key Laboratory and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003 (China); Wang, Nan [State Key Laboratory and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003 (China); Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 (Canada); Chen, Deying [State Key Laboratory and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003 (China); Li, Yunong [Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 (Canada); Lu, Yingfeng [State Key Laboratory and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003 (China); Huan, Tao [Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 (Canada); Xu, Wei [State Key Laboratory and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003 (China); Li, Liang, E-mail: Liang.Li@ualberta.ca [State Key Laboratory and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003 (China); Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 (Canada); Li, Lanjuan, E-mail: ljli@zju.edu.cn [State Key Laboratory and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003 (China)

    2016-01-15

    Human urine and feces can be non-invasively collected for metabolomics-based disease biomarker discovery research. Because urinary and fecal metabolomes are thought to be different, analysis of both biospecimens may generate a more comprehensive metabolomic profile that can be better related to the health state of an individual. Herein we describe a method of using differential chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) for parallel metabolomic profiling of urine and feces. Dansylation labeling was used to quantify the amine/phenol submetabolome changes among different samples based on {sup 12}C-labeling of individual samples and {sup 13}C-labeling of a pooled urine or pooled feces and subsequent analysis of the {sup 13}C-/{sup 12}C-labeled mixture by LC-MS. The pooled urine and pooled feces are further differentially labeled, mixed and then analyzed by LC-MS in order to relate the metabolite concentrations of the common metabolites found in both biospecimens. This method offers a means of direct comparison of urinary and fecal submetabolomes. We evaluated the analytical performance and demonstrated the utility of this method in the analysis of urine and feces collected daily from three healthy individuals for 7 days. On average, 2534 ± 113 (n = 126) peak pairs or metabolites could be detected from a urine sample, while 2507 ± 77 (n = 63) peak pairs were detected from a fecal sample. In total, 5372 unique peak pairs were detected from all the samples combined; 3089 and 3012 pairs were found in urine and feces, respectively. These results reveal that the urine and fecal metabolomes are very different, thereby justifying the consideration of using both biospecimens to increase the probability of finding specific biomarkers of diseases. Furthermore, the CIL LC-MS method described can be used to perform parallel quantitative analysis of urine and feces, resulting in more complete coverage of the human metabolome

  11. Dansylation isotope labeling liquid chromatography mass spectrometry for parallel profiling of human urinary and fecal submetabolomes

    International Nuclear Information System (INIS)

    Su, Xiaoling; Wang, Nan; Chen, Deying; Li, Yunong; Lu, Yingfeng; Huan, Tao; Xu, Wei; Li, Liang; Li, Lanjuan

    2016-01-01

    Human urine and feces can be non-invasively collected for metabolomics-based disease biomarker discovery research. Because urinary and fecal metabolomes are thought to be different, analysis of both biospecimens may generate a more comprehensive metabolomic profile that can be better related to the health state of an individual. Herein we describe a method of using differential chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) for parallel metabolomic profiling of urine and feces. Dansylation labeling was used to quantify the amine/phenol submetabolome changes among different samples based on "1"2C-labeling of individual samples and "1"3C-labeling of a pooled urine or pooled feces and subsequent analysis of the "1"3C-/"1"2C-labeled mixture by LC-MS. The pooled urine and pooled feces are further differentially labeled, mixed and then analyzed by LC-MS in order to relate the metabolite concentrations of the common metabolites found in both biospecimens. This method offers a means of direct comparison of urinary and fecal submetabolomes. We evaluated the analytical performance and demonstrated the utility of this method in the analysis of urine and feces collected daily from three healthy individuals for 7 days. On average, 2534 ± 113 (n = 126) peak pairs or metabolites could be detected from a urine sample, while 2507 ± 77 (n = 63) peak pairs were detected from a fecal sample. In total, 5372 unique peak pairs were detected from all the samples combined; 3089 and 3012 pairs were found in urine and feces, respectively. These results reveal that the urine and fecal metabolomes are very different, thereby justifying the consideration of using both biospecimens to increase the probability of finding specific biomarkers of diseases. Furthermore, the CIL LC-MS method described can be used to perform parallel quantitative analysis of urine and feces, resulting in more complete coverage of the human metabolome. - Highlights: • A

  12. Synthesizing labeled compounds

    International Nuclear Information System (INIS)

    London, R.E.; Matwiyoff, N.A.; Unkefer, C.J.; Walker, T.E.

    1983-01-01

    A metabolic study is presented of the chemical reactions provided by isotopic labeling and NMR spectroscopy. Synthesis of 13 C-labeled D-glucose, a 6-carbon sugar, involves adding a labeled nitrile group to the 5-carbon sugar D-arabinose by reaction with labeled hydrogen cyanide. The product of this reaction is then reduced and hydrolyzed to a mixture of the labeled sugars. The two sugars are separated by absorption chromotography. The synthesis of 13 C-labeled L-tyrosine, an amino acid, is also presented

  13. In Vivo Isotopic Labeling of Symbiotic Bacteria Involved in Cellulose Degradation and Nitrogen Recycling within the Gut of the Forest Cockchafer (Melolontha hippocastani).

    Science.gov (United States)

    Alonso-Pernas, Pol; Bartram, Stefan; Arias-Cordero, Erika M; Novoselov, Alexey L; Halty-deLeon, Lorena; Shao, Yongqi; Boland, Wilhelm

    2017-01-01

    The guts of insects harbor symbiotic bacterial communities. However, due to their complexity, it is challenging to relate a specific symbiotic phylotype to its corresponding function. In the present study, we focused on the forest cockchafer ( Melolontha hippocastani ), a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining in vivo stable isotope probing (SIP) with 13 C cellulose and 15 N urea as trophic links, with Illumina MiSeq (Illumina-SIP), we unraveled bacterial networks processing recalcitrant dietary components and recycling nitrogenous waste. The bacterial communities behind these processes change between larval and adult stages. In 13 C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In 15 N urea-fed insects, the genera Burkholderia and Parabacteroides were isotopically labeled in larvae and adults, respectively. Additionally, the PICRUSt-predicted metagenome suggested a possible ability to degrade hemicellulose and to produce amino acids of, respectively, 13 C cellulose- and 15 N urea labeled bacteria. The incorporation of 15 N from ingested urea back into the insect body was confirmed, in larvae and adults, by isotope ratio mass spectrometry (IRMS). Besides highlighting key bacterial symbionts of the gut of M. hippocastani , this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.

  14. In Vivo Isotopic Labeling of Symbiotic Bacteria Involved in Cellulose Degradation and Nitrogen Recycling within the Gut of the Forest Cockchafer (Melolontha hippocastani

    Directory of Open Access Journals (Sweden)

    Pol Alonso-Pernas

    2017-10-01

    Full Text Available The guts of insects harbor symbiotic bacterial communities. However, due to their complexity, it is challenging to relate a specific symbiotic phylotype to its corresponding function. In the present study, we focused on the forest cockchafer (Melolontha hippocastani, a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining in vivo stable isotope probing (SIP with 13C cellulose and 15N urea as trophic links, with Illumina MiSeq (Illumina-SIP, we unraveled bacterial networks processing recalcitrant dietary components and recycling nitrogenous waste. The bacterial communities behind these processes change between larval and adult stages. In 13C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In 15N urea-fed insects, the genera Burkholderia and Parabacteroides were isotopically labeled in larvae and adults, respectively. Additionally, the PICRUSt-predicted metagenome suggested a possible ability to degrade hemicellulose and to produce amino acids of, respectively, 13C cellulose- and 15N urea labeled bacteria. The incorporation of 15N from ingested urea back into the insect body was confirmed, in larvae and adults, by isotope ratio mass spectrometry (IRMS. Besides highlighting key bacterial symbionts of the gut of M. hippocastani, this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.

  15. CoCoa: a software tool for estimating the coefficient of coancestry from multilocus genotype data.

    Science.gov (United States)

    Maenhout, Steven; De Baets, Bernard; Haesaert, Geert

    2009-10-15

    Phenotypic data collected in breeding programs and marker-trait association studies are often analyzed by means of linear mixed models. In these models, the covariance between the genetic background effects of all genotypes under study is modeled by means of pairwise coefficients of coancestry. Several marker-based coancestry estimation procedures allow to estimate this covariance matrix, but generally introduce a certain amount of bias when the examined genotypes are part of a breeding program. CoCoa implements the most commonly used marker-based coancestry estimation procedures and as such, allows to select the best fitting covariance structure for the phenotypic data at hand. This better model fit translates into an increased power and improved type I error control in association studies and an improved accuracy in phenotypic prediction studies. The presented software package also provides an implementation of the new Weighted Alikeness in State (WAIS) estimator for use in hybrid breeding programs. Besides several matrix manipulation tools, CoCoa implements two different bending heuristics, in case the inverse of an ill-conditioned coancestry matrix estimate is needed. The software package CoCoa is freely available at http://webs.hogent.be/cocoa. Source code, manual, binaries for 32 and 64-bit Linux systems and an installer for Microsoft Windows are provided. The core components of CoCoa are written in C++, while the graphical user interface is written in Java.

  16. Stable isotope N-phosphoryl amino acids labeling for quantitative profiling of amine-containing metabolites using liquid chromatography mass spectrometry.

    Science.gov (United States)

    Zhang, Shanshan; Shi, Jinwen; Shan, Changkai; Huang, Chengting; Wu, Yile; Ding, Rong; Xue, Yuhua; Liu, Wen; Zhou, Qiang; Zhao, Yufen; Xu, Pengxiang; Gao, Xiang

    2017-07-25

    Stable isotope chemical labeling liquid chromatography-mass spectrometry (LC-MS) is a powerful strategy for comprehensive metabolomics profiling, which can improve metabolites coverage and quantitative information for exploration of metabolic regulation in complex biological systems. In the current work, a novel stable isotope N-phosphoryl amino acids labeling strategy (SIPAL) has been successful developed for quantitative profiling of amine-containing metabolites in urine based on organic phosphorus chemistry. Two isotopic reagents, 16 O 2 - and 18 O 2 -N-diisopropyl phosphoryl l-alanine N-hydroxysuccinimide esters ( 16 O/ 18 O-DIPP-L-Ala-NHS), were firstly synthesized in high yields for labeling the amine-containing metabolites. The performance of SIPAL strategy was tested by analyzing standard samples including 20 l-amino acids, 10 d-amino acids and small peptides by using LC-MS. We observed highly efficient and selective labeling for SIPAL strategy within 15 min in a one-pot derivatization reaction under aqueous reaction conditions. The introduction of a neutral phosphate group at N-terminus can increase the proton affinity and overall hydrophobicity of targeted metabolites, leading to the better ionization efficiency in electrospray ionization processes and chromatographic separations of hydrophilic metabolites on reversed-phase column. Furthermore, the chiral metabolites, such as d-amino acids, could be converted to diastereomers after SIPAL and successfully separated on regular reversed-phase column. The chirality of labeled enantiomers can be determined by using different detection methods such as 31 P NMR, UV, and MS, demonstrating the potential application of SIPAL strategy. In addition, absolute quantification of chiral metabolites in biological samples can be easily achieved by using SIPAL strategy. For this purpose, urine samples collected from a healthy volunteer were analyzed by using LC-ESI-Orbitrap MS. Over 300 pairs of different amine

  17. Labeling pharmaceuticals with radioactive isotopes. Technical progress report, December 1, 1975--November 30, 1976

    International Nuclear Information System (INIS)

    Blau, M.; Bender, M.A.

    1976-01-01

    The purpose of this research is to prepare iodo- and bromo-aliphatic amino acid analogs labeled with γ-emitting isotopes ( 131 I, 123 I and 77 Br) for possible use as pancreas localizing agents. Studies on the halogen exchange reaction (I- for Cl-) for the synthesis of β-iodo-α-aminobutyric acid (a valine analog) have suggested that the iodo compound was formed initially. However, the desired compound cannot be isolated because of its chemical instability. Distribution studies in rats with the crude halogen exchange reaction mixture confirmed this finding. Studies on the addition of hydrogen iodine to allylglycine under various conditions for the synthesis of γ-iodo-α-aminopentanoic acid (a leucine analog) suffered the same obstacle; the chemical instability of the desired iodo compound precludes isolation and characterization. Convinced that the iodo analogs were too unstable for use as practical localizing agents, we turned to the possible use of Br for CH 3 substituted amino acids. The 14 C labeled β-bromo-α-aminobutyric acid methyl ester was synthesized. This methyl ester will be hydrolyzed and the distribution of free amino acid will be studied. Labeled with 77 Br this compound might be useful for pancreas localization

  18. Direct isotope determination of isotopically labelled lipids by field desorption mass spectrometry

    International Nuclear Information System (INIS)

    Lehmann, W.D.; Kessler, M.

    1982-01-01

    Lipids labelled with deuterium or carbon-14 have been investigated by field desorption mass spectrometry for determination of their degree of labelling. This application is demonstrated for free fatty acids, cholesterol, cholesteryl esters, triglycerides, and L-α-phosphatidylcholines. Comparison of the molecular ion groups of the non-labelled and of the labelled compounds enables a fast and reliable determination of the degree of labelling. For multiply labelled compounds the label distribution is also obtained from the molecular ion group. In addition, for cholesteryl esters and for phosphatidylcholines structurally significant fragment ions provide information about the position of the label. Several hundred nanograms of the compound are typically required for a single analysis with a relative standard error of 0.5-2% in the value calculated for atom% hydrogen-2 or for the specific carbon-14 activity. (orig.) [de

  19. Stable isotope labeling, in vivo, of D- and L-tryptophan pools in lemna gibba and the low incorporation of label into indole-3-acetic acid

    International Nuclear Information System (INIS)

    Baldi, B.G.; Maher, B.R.; Slovin, J.P.; Cohen, J.D.

    1991-01-01

    The authors present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [ 15 N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of L-[ 15 N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled L-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into D-tryptophan. D-[ 15 N]trytophan supplied to Lemna at rates of approximately 400 times excess of endogenous D-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of L-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that L-tryptophan is a more direct precursor to IAA than the D isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that L-tryptophan also may not be a primary precursor to IAA in plants

  20. Extrinsic labelling of staple food crops with isotopic iron does not consistently result in full equilibration: Revisiting the methodology

    Science.gov (United States)

    Extrinsic isotopic labeling of food Fe has been used for over 50 years to measure Fe absorption. This method is based on the assumption that complete equilibration occurs between the extrinsic and the intrinsic Fe prior to intestinal absorption. The present study tested this assumption via use of in...

  1. Isotope effects: definitions and consequences for pharmacologic studies

    International Nuclear Information System (INIS)

    Van Langenhove, A.

    1986-01-01

    The use of stable isotope-labeled compounds for pharmacologic studies requires careful consideration of the nature of the stable isotope label (2H, 13C, 15N, 18O) and its position of incorporation in the molecule. When deuterium is used, improper positioning can lead to significant primary isotope effects. Primary isotope effects occur when the breaking of the bond to the heavy isotope is the rate-limiting step in a reaction (or metabolic transformation). A reaction will proceed slower for the molecule with the heavy isotope label because of the mass difference between the light and the heavy isotope. In addition to these primary isotope effects, smaller but nevertheless important secondary isotope effects, physicochemical isotope effects, active hydrogen/deuterium exchange, or isotope effects associated with either the enzyme-catalyzed biotransformation or the mass spectrometric ionization and fragmentation can be operative. In mechanistic studies, isotope effects are used to their advantage; however, in pharmacokinetic studies, the occurrence of isotope effects can lead to grossly misleading biologic and analytic results: the metabolism of the drug will differ when in vivo isotope effects are operative, and isotope effects occurring during the analysis procedure will obscure the true metabolic profile of the drug

  2. Synthesis of isotopically labelled salicylates

    International Nuclear Information System (INIS)

    Hawkins, D.R.; Pryor, R.W.

    1981-01-01

    [ 13 C-carboxyl]Salicylic acid has been prepared by carbonation of 2-benzyloxybromobenzene followed by reductive debenzylation. Deuterium and tritium labelled salicylic acid and 2 H 2 / 13 C-salicylic acid were prepared by reduction of the 3,5-dibromo derivatives using Raney Ni-Al. Deuterium labelled salicylic acid containing up to four deuterium atoms was prepared by catalytic exchange with Raney Ni-Al in 5% NaOD/D 2 O. (author)

  3. From position-specific isotope labeling towards soil fluxomics: a novel toolbox to assess the microbial impact on biogeochemical cycles

    Science.gov (United States)

    Apostel, C.; Dippold, M. A.; Kuzyakov, Y.

    2015-12-01

    Understanding the microbial impact on C and nutrient cycles is one of the most important challenges in terrestrial biogeochemistry. Transformation of low molecular weight organic substances (LMWOS) is a key step in all biogeochemical cycles because 1) all high molecular substances pass the LMWOS pool during their degradation and 2) only LMWOS can be taken up by microorganisms intact. Thus, 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 microbial metabolic network and its control mechanism. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools but studies were nearly exclusively based on uniformly labeled substances. However, such tracers do not allow the differentiation of the intact use of the initial substances from its transformation 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 basic metabolites and quantification of isotope incorporation in CO2 and bulk soil enabled following the basic metabolic pathways of microorganisms. However, the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites like phospholipid fatty acids (PLFA) or amino sugars revealed new insights into the soil fluxome: First, it enables tracing specific anabolic pathways in diverse microbial communities in soils e.g. carbon starvation pathways versus pathways reflecting microbial growth. Second, it allows identification of specific pathways of individual functional microbial groups in soils in situ. Tracing metabolic pathways and understanding their regulating factors are crucial for soil C fluxomics i.e. the unravaling of the complex network of C transformations

  4. Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

    Science.gov (United States)

    Zhou, Shiyue; Tello, Nadia; Harvey, Alex; Boyes, Barry; Orlando, Ron; Mechref, Yehia

    2016-06-01

    Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Isotopic measurements (C,N,O) of detonation soot produced from labeled and unlabeled Composition B-3 indicate source of solid carbon residues

    Science.gov (United States)

    Podlesak, David; Manner, Virginia; Amato, Ronald; Dattelbaum, Dana; Gusavsen, Richard; Huber, Rachel

    2017-06-01

    Detonation of HE is an exothermic process whereby metastable complex molecules are converted to simple stable molecules such as H2 O, N2, CO, CO2, and solid carbon. The solid carbon contains various allotropes such as detonation nanodiamonds, graphite, and amorphous carbon. It is well known that certain HE formulations such as Composition B (60% RDX, 40% TNT) produce greater amounts of solid carbon than other more oxygen-balanced formulations. To develop a greater understanding of how formulation and environment influence solid carbon formation, we synthesized TNT and RDX with 13 C and 15 N at levels slightly above natural abundance levels. Synthesized RDX and TNT were mixed at a ratio of 60:40 to form Composition B and solid carbon residues were collected from detonations of isotopically-labeled as well as un-labelled Composition B. The raw HE and detonation residues were analyzed isotopically for C, N, O isotopic compositions. We will discuss differences between treatments groups as a function of formulation and environment. LA-UR - 17-21266.

  6. Analysis of the differentially expressed low molecular weight peptides in human serum via an N-terminal isotope labeling technique combining nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Leng, Jiapeng; Zhu, Dong; Wu, Duojiao; Zhu, Tongyu; Zhao, Ningwei; Guo, Yinlong

    2012-11-15

    Peptidomics analysis of human serum is challenging due to the low abundance of serum peptides and interference from the complex matrix. This study analyzed the differentially expressed (DE) low molecular weight peptides in human serum integrating a DMPITC-based N-terminal isotope labeling technique with nano-liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (nano-LC/MALDI-MS). The workflow introduced a [d(6)]-4,6-dimethoxypyrimidine-2-isothiocyanate (DMPITC)-labeled mixture of aliquots from test samples as the internal standard. The spiked [d(0)]-DMPITC-labeled samples were separated by nano-LC then spotted on the MALDI target. Both quantitative and qualitative studies for serum peptides were achieved based on the isotope-labeled peaks. The DMPITC labeling technique combined with nano-LC/MALDI-MS not only minimized the errors in peptide quantitation, but also allowed convenient recognition of the labeled peptides due to the 6 Da mass difference. The data showed that the entire research procedure as well as the subsequent data analysis method were effective, reproducible, and sensitive for the analysis of DE serum peptides. This study successfully established a research model for DE serum peptides using DMPITC-based N-terminal isotope labeling and nano-LC/MALDI-MS. Application of the DMPITC-based N-terminal labeling technique is expected to provide a promising tool for the investigation of peptides in vivo, especially for the analysis of DE peptides under different biological conditions. Copyright © 2012 John Wiley & Sons, Ltd.

  7. Isotopic exchange between CO2 and H2O and labelling kinetics of photosynthetic oxygen

    International Nuclear Information System (INIS)

    Gerster, Richard

    1971-01-01

    The reaction of carbon dioxide with water has been studied by measuring the rate of oxygen exchange between C 18 O 2 and H 2 16 O. The mathematical treatment of the kinetics allows to determine with accuracy the diffusion flow between the gas and the liquid phase, in the same way as the CO 2 hydration rate. The velocity constant of this last process, whose value gives the in situ enzymatic activity of carbonic anhydrase, has been established in the case of chloroplast and Euglena suspensions and of aerial leaves. The study of the isotopic exchange between C 18 O 2 and a vegetable submitted to alternations of dark and light has allowed to calculate the isotopic abundance of the metabolized CO 2 whose value has been compared to that of the intracellular water and that of photosynthetic oxygen. In addition, a new method using 13 C 18 O 2 gives the means to measure with accuracy eventual isotopic effects. The labelling kinetics of the oxygen evolved by Euglena suspensions whose water has been enriched with 18 O have been established at different temperatures. (author) [fr

  8. Cross-Course Collaboration in the Undergraduate Chemistry Curriculum: Isotopic Labeling with Sodium Borodeuteride in the Introductory Organic Chemistry Laboratory

    Science.gov (United States)

    Kjonaas, Richard A.; Fitch, Richard W.; Noll, Robert J.

    2017-01-01

    A microscale isotopic labeling experiment is described for the introductory organic chemistry laboratory course wherein half of the students use sodium borohydride (NaBH[subscript 4]) and the other half use sodium borodeuteride (NaBD[subscript 4]) to reduce acetophenone to 1-phenylethanol and then compare spectral data. The cost is reasonable, and…

  9. Isotope exchange reactions of the hydrogen H-5 of selected pyrimidine derivatives and the preparation of tritium-labeled pyrimidines

    Czech Academy of Sciences Publication Activity Database

    Dračínský, Martin; Jansa, Petr; Elbert, Tomáš

    2011-01-01

    Roč. 76, č. 12 (2011), s. 1567-1577 ISSN 0010-0765 R&D Projects: GA AV ČR KJB400550903; GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : isotopic labeling * NMR spectroscopy * nucleobases * pyrimidines Subject RIV: CC - Organic Chemistry Impact factor: 1.283, year: 2011

  10. Protein-based stable isotope probing.

    Science.gov (United States)

    Jehmlich, Nico; Schmidt, Frank; Taubert, Martin; Seifert, Jana; Bastida, Felipe; von Bergen, Martin; Richnow, Hans-Hermann; Vogt, Carsten

    2010-12-01

    We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon ((13)C)- or nitrogen ((15)N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d.

  11. Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

    Science.gov (United States)

    Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

    1999-01-01

    A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

  12. Systematic NMR Analysis of Stable Isotope Labeled Metabolite Mixtures in Plant and Animal Systems: Coarse Grained Views of Metabolic Pathways

    Science.gov (United States)

    Chikayama, Eisuke; Suto, Michitaka; Nishihara, Takashi; Shinozaki, Kazuo; Hirayama, Takashi; Kikuchi, Jun

    2008-01-01

    Background Metabolic phenotyping has become an important ‘bird's-eye-view’ technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of ‘top-down’ chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling and analysis of metabolite mixtures in plant and animal systems. Methodology/Principal Findings The analysis method includes a stable isotope labeling technique for use in living organisms; a systematic method for simultaneously identifying a large number of metabolites by using a newly developed HSQC-based metabolite chemical shift database combined with heteronuclear multidimensional NMR spectroscopy; Principal Components Analysis; and a visualization method using a coarse-grained overview of the metabolic system. The database contains more than 1000 1H and 13C chemical shifts corresponding to 142 metabolites measured under identical physicochemical conditions. Using the stable isotope labeling technique in Arabidopsis T87 cultured cells and Bombyx mori, we systematically detected >450 HSQC peaks in each 13C-HSQC spectrum derived from model plant, Arabidopsis T87 cultured cells and the invertebrate animal model Bombyx mori. Furthermore, for the first time, efficient 13C labeling has allowed reliable signal assignment using analytical separation techniques such as 3D HCCH-COSY spectra in higher organism extracts. Conclusions/Significance Overall physiological changes could be detected and categorized in relation to a critical developmental phase change in B. mori by coarse-grained representations in which the organization of metabolic pathways related to a specific developmental phase was visualized on the basis of constituent changes of 56 identified metabolites. Based on the observed intensities of 13C atoms of

  13. Methyl-coenzyme M reductase from methanogenic archaea: isotope effects on label exchange and ethane formation with the homologous substrate ethyl-coenzyme M.

    Science.gov (United States)

    Scheller, Silvan; Goenrich, Meike; Thauer, Rudolf K; Jaun, Bernhard

    2013-10-09

    Ethyl-coenzyme M (CH3CH2-S-CH2CH2-SO3(-), Et-S-CoM) serves as a homologous substrate for the enzyme methyl-coenzyme M reductase (MCR) resulting in the product ethane instead of methane. The catalytic reaction proceeds via an intermediate that already contains all six C-H bonds of the product. Because product release occurs after a second, rate-limiting step, many cycles of intermediate formation and reconversion to substrate occur before a substantial amount of ethane is released. In deuterated buffer, the intermediate becomes labeled, and C-H activation in the back reaction rapidly leads to labeled Et-S-CoM, which enables intermediate formation to be detected. Here, we present a comprehensive analysis of this pre-equilibrium. (2)H- and (13)C-labeled isotopologues of Et-S-CoM were used as the substrates, and the time course of each isotopologue was followed by NMR spectroscopy. A kinetic simulation including kinetic isotope effects allowed determination of the primary and α- and β-secondary isotope effects for intermediate formation and for the C-H/C-D bond activation in the ethane-containing intermediate. The values obtained are in accordance with those found for the native substrate Me-S-CoM (see preceding publication, Scheller, S.; Goenrich, M.; Thauer, R. K.; Jaun, B. J. Am. Chem. Soc. 2013, 135, DOI: 10.1021/ja406485z) and thus imply the same catalytic mechanism for both substrates. The experiment by Floss and co-workers, demonstrating a net inversion of configuration to chiral ethane with CH3CDT-S-CoM as the substrate, is compatible with the observed rapid isotope exchange if the isotope effects measured here are taken into account.

  14. Cell-free expression and stable isotope labelling strategies for membrane proteins

    International Nuclear Information System (INIS)

    Sobhanifar, Solmaz; Reckel, Sina; Junge, Friederike; Schwarz, Daniel; Kai, Lei; Karbyshev, Mikhail; Loehr, Frank; Bernhard, Frank; Doetsch, Volker

    2010-01-01

    Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems provides a very interesting alternative to cell-based systems, since it circumvents many problems such as toxicity or necessity for the transportation of the synthesized protein to the membrane, and constitutes the only system that allows for direct production of membrane proteins in membrane-mimetic environments which may be suitable for liquid state NMR measurements. The unique advantages of the cell-free expression system, including strong expression yields as well as the direct incorporation of almost any combination of amino acids with very little metabolic scrambling, has allowed for the development of a wide-array of isotope labelling techniques which facilitate structural investigations of proteins whose spectral congestion and broad line-widths may have earlier rendered them beyond the scope of NMR. Here we explore various labelling strategies in conjunction with cell-free developments, with a particular focus on α-helical transmembrane proteins which benefit most from such methods.

  15. Evaluation of a radioisotope labelling technique for measuring bacterial adherence on fabrics

    International Nuclear Information System (INIS)

    Youlo Hsieh; Timm, Debra; Merry, Joanne

    1986-01-01

    A technique utilizing tritiated thymidine labelled bacteria to quantify bacteria on fabrics has been evaluated. Quenching or self-absorption of isotope solution and labelled bacteria suspension by some of the fabrics has been observed. The extents of self-absorption of both isotope and labelled bacteria solutions on various fabrics was found to be dependent upon the fiber contents, i.e. the chemical compositions, of the substrata. This observation confirms that reduction of scintillation efficiency or self-absorption does occur when radio-labelled substances in suspensions were measured with the presence of some fabrics. Cautions should be taken when radio-labelling techniques are applied to detect isotope-labelled micro-organisms or other substances which are in contact with fabrics in the form of solutions. However, when there is no excess and nonattached labelled bacteria in the aqueous surrounding of the fabric, scintillation counting efficiency of the labelled bacteria on all fabrics studied remained constant over a period of 8 h. This indicates that the application of the described isotope labelling procedure is appropriate for quantifying adherent bacteria on fibrous substrate. (author)

  16. Synthesis and pharmacokinetics of thiacoccide labeled with tritium

    International Nuclear Information System (INIS)

    Shestakov, A.D.; Kaminskii, Yu.L.; Nurova, I.M.; Nikitina, V.N.; Zaionts, V.I.; Maksimova, O.V.; Mikhailov, G.A.

    1986-01-01

    To obtain information on the pharmacokinetics of thiacoccide, the authors effect the synthesis of an analog of thiacoccide, viz., the hydrochloride of 2-methyl-N-(2'-n-propyl-4'-aminopyrimid-5'-ylmethyl)pyridinium chloride (I) labeled with tritium. The simplest way to introduce a tritium label into (I) is by isotopic exchange of hydrogen of (I) with H 3 HO. Typical material obtained from isotopic exchange with water labeled with tritium is shown. The dynamics of 3 H distribution in the organism of the chick and the rate of its elimination on single administration of thiacoccide containing isotope in both the methyl group and in the pyridine ring are shown

  17. Labelled compounds. (Pt. B)

    International Nuclear Information System (INIS)

    Buncel, E.; Jones, J.R.

    1991-01-01

    Since the end of World War II there has been a tremendous increase in the number of compounds that have been synthesized with radioactive or stable isotopes. They have found application in many diverse fields, so much so, that hardly a single area in pure and applied science has not benefited. Not surprisingly it has been reflected in appearance of related publications. The early proceedings of the Symposia on Advances in Trace Methodology were soon followed by various Euratom sponsored meetings in which methods of preparing and storing labelled compounds featured prominently. In due course a resurgence of interest in stable isotopes, brought about by their greater availability (also lower cost) and partly by development of new techniques such as gas chromatography - mass spectrometry (gc-ms), led to the publication of proceedings of several successful conferences. More recently conferences dealing with the synthesis and applications of isotopes and isotopically labelled compounds have been established on a regular basis. In addition to the proceedings of conferences and journal publications individuals left their mark by producing definitive texts, usually on specific nuclides. Only the classic two volume publication of Murray and Williams (Organic syntheses with isotopes, New York 1985), now over 30 years old and out of print, attempted to do justice to several nuclides. With the large amount of work that has been undertaken since then it seems unlikely that an updated edition could be produced. The alternative strategy was to ask scientists currently active to review specific areas and this is the approach adopted in the present series of monographs. In this way it is intended to cover the broad advances that have been made in the synthesis and applications of isotopes and isotopically labelled compounds in the physical and biomedical sciences. (author). refs.; figs.; tabs

  18. Dynamics of N2O production pathways analyzed by 15N18O isotope labeling

    DEFF Research Database (Denmark)

    Jensen, Marlene Mark; Ma, Chun; Lavik, Gaute

    Nitrous oxide production associated with biological nitrogen transformations can contribute substantially to the CO2 footprint of both man-made and natural systems, but the pathways and regulation of N2O production are poorly understood. We developed a 15N/18O dual isotope labelling technique...

  19. On structural identifiability analysis of the cascaded linear dynamic systems in isotopically non-stationary 13C labelling experiments.

    Science.gov (United States)

    Lin, Weilu; Wang, Zejian; Huang, Mingzhi; Zhuang, Yingping; Zhang, Siliang

    2018-06-01

    The isotopically non-stationary 13C labelling experiments, as an emerging experimental technique, can estimate the intracellular fluxes of the cell culture under an isotopic transient period. However, to the best of our knowledge, the issue of the structural identifiability analysis of non-stationary isotope experiments is not well addressed in the literature. In this work, the local structural identifiability analysis for non-stationary cumomer balance equations is conducted based on the Taylor series approach. The numerical rank of the Jacobian matrices of the finite extended time derivatives of the measured fractions with respect to the free parameters is taken as the criterion. It turns out that only one single time point is necessary to achieve the structural identifiability analysis of the cascaded linear dynamic system of non-stationary isotope experiments. The equivalence between the local structural identifiability of the cascaded linear dynamic systems and the local optimum condition of the nonlinear least squares problem is elucidated in the work. Optimal measurements sets can then be determined for the metabolic network. Two simulated metabolic networks are adopted to demonstrate the utility of the proposed method. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Endophilin-A1 BAR domain interaction with arachidonyl CoA.

    Science.gov (United States)

    Petoukhov, Maxim V; Weissenhorn, Winfried; Svergun, Dmitri I

    2014-01-01

    Endophilin-A1 belongs to the family of BAR domain containing proteins that catalyze membrane remodeling processes via sensing, inducing and stabilizing membrane curvature. We show that the BAR domain of endophilin-A1 binds arachidonic acid and molds its coenzyme A (CoA) activated form, arachidonyl-CoA into a defined structure. We studied low resolution structures of endophilin-A1-BAR and its complex with arachidonyl-CoA in solution using synchrotron small-angle X-ray scattering (SAXS). The free endophilin-A1-BAR domain is shown to be dimeric at lower concentrations but builds tetramers and higher order complexes with increasing concentrations. Extensive titration SAXS studies revealed that the BAR domain produces a homogenous complex with the lipid micelles. The structural model of the complexes revealed two arachidonyl-CoA micelles bound to the distal arms of an endophilin-A1-BAR dimer. Intriguingly, the radius of the bound micelles significantly decreases compared to that of the free micelles, and this structural result may provide hints on the potential biological relevance of the endophilin-A1-BAR interaction with arachidonyl CoA.

  1. A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters

    Directory of Open Access Journals (Sweden)

    Dominik M. Peter

    2016-04-01

    Full Text Available Coenzyme A (CoA is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies. In this work we systematically tested five chemo-enzymatic methods for the synthesis of the three most abundant acyl-CoA thioester classes in biology; saturated acyl-CoAs, α,β-unsaturated acyl-CoAs (i.e., enoyl-CoA derivatives, and α-carboxylated acyl-CoAs (i.e., malonyl-CoA derivatives. Additionally we report on the substrate promiscuity of three newly described acyl-CoA dehydrogenases that allow the simple conversion of acyl-CoAs into enoyl-CoAs. With these five methods, we synthesized 26 different CoA-thioesters with a yield of 40% or higher. The CoA esters produced range from short- to long-chain, include branched and α,β-unsaturated representatives as well as other functional groups. Based on our results we provide a general guideline to the optimal synthesis method of a given CoA-thioester in respect to its functional group(s and the commercial availability of the precursor molecule. The proposed synthetic routes can be performed in small scale and do not require special chemical equipment, making them convenient also for biological laboratories.

  2. Simultaneous determination of glucose turnover, alanine turnover, and gluconeogenesis in human using a double stable-isotope-labeled tracer infusion and gas chromatography-mass spectrometry analysis

    International Nuclear Information System (INIS)

    Martineau, A.; Lecavalier, L.; Falardeau, P.; Chiasson, J.L.

    1985-01-01

    We have developed and validated a new method to measure simultaneously glucose turnover, alanine turnover, and gluconeogenesis in human, in steady and non-steady states, using a double stable-isotope-labeled tracer infusion and GC-MS analysis. The method is based on the concomitant infusion and dilution of D-[2,3,4,6,6-2H5]glucose and L-[1,2,3-13C3]alanine. The choice of the tracers was done on the basis of a minimal overlap between the ions of interest and those arising from natural isotopic abundances. Alanine was chosen as the gluconeogenic substrate because it is the major gluconeogenic amino acid extracted by the liver and, with lactate, constitutes the bulk of the gluconeogenic precursors. The method was validated by comparing the results obtained during simultaneous infusion of trace amounts of both stable isotope labeled compounds with the radioactive tracers (D-[3-3H]glucose and L-[1,2,3-14C3]alanine) in a normal and a diabetic subject; the radiolabeled tracers were used as the accepted reference procedure. A slight overestimation of glucose turnover (7.3 versus 6.8 in normal and 10.8 versus 9.2 mumol/kg min in diabetic subject) was noticed when the stable isotope-labeled tracers were used. For the basal turnover rate of alanine, similar values were obtained with both methods (6.2 mumol/kg min). For gluconeogenesis, higher values were observed in the basal state with the stable isotopes (0.42 versus 0.21 mumol/kg min); however, these differences disappeared in the postprandial period after the ingestion of a mixed meal. Despite those minor differences, the overall correlation with the reference method was excellent for glucose turnover (r = 0.87) and gluconeogenesis (r = 0.86)

  3. Synthesis of deuterium-labeled prochlorperazine.

    Science.gov (United States)

    Hawes, E M; Gurnsey, T S; Shetty, H U; Midha, K K

    1983-06-01

    The propylpiperazine side chain of prochlorperazine was labeled with two, four, or six deuterium atoms by lithium aluminum deuteride reduction of the appropriate amide. The isotopic purity of the products after correcting for chlorine isotopes was greater than 95.7%.

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

    Science.gov (United States)

    Dippold, Michaela; Kuzyakov, Yakov

    2015-04-01

    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

  5. Stable isotopes

    International Nuclear Information System (INIS)

    Brazier, J.L.; Guinamant, J.L.

    1995-01-01

    According to the progress which has been realised in the technology of separating and measuring isotopes, the stable isotopes are used as preferable 'labelling elements' for big number of applications. The isotopic composition of natural products shows significant variations as a result of different reasons like the climate, the seasons, or their geographic origins. So, it was proved that the same product has a different isotopic composition of alimentary and agriculture products. It is also important in detecting the pharmacological and medical chemicals. This review article deals with the technology, like chromatography and spectrophotometry, adapted to this aim, and some important applications. 17 refs. 6 figs

  6. Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis

    DEFF Research Database (Denmark)

    Harkness, Linda; Prokhorova, Tatyana A; Kassem, Moustapha

    2012-01-01

    The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass...... spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we...

  7. Isotopic Abundance and Chemical Purity Analysis of Stable Isotope Deuterium Labeled Sudan I

    Directory of Open Access Journals (Sweden)

    CAI Yin-ping;LEI Wen;ZHENG Bo;DU Xiao-ning

    2014-02-01

    Full Text Available It is important that to analysis of the isotopic abundance and chemical purity of Sudan I-D5, which is the internal standard of isotope dilution mass spectrometry. The isotopic abundance of Sudan I-D5 is detected by “mass cluster” classification method and LC-MS. The repeatability and reproducibility experiments were carried out by using different mass spectrometers and different operators. The RSD was less than 0.1%, so the repeatability and reproducibility were satisfactory. The accuracy and precision of the isotopic abundance analysis method was good with the results of F test and t test. The high performance liquid chromatography (HPLC had been used for detecting the chemical purity of Sudan I-D5 as external standard method.

  8. Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein α-subunit

    International Nuclear Information System (INIS)

    Abdulaev, Najmoutin G.; Zhang Cheng; Dinh, Andy; Ngo, Tony; Bryan, Philip N.; Brabazon, Danielle M.; Marino, John P.; Ridge, Kevin D.

    2005-01-01

    Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein α-subunit (G α ) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled G α chimera (∼40 kDa polypeptide) has been tested. The results show that a prodomain fused G α chimera can be expressed to levels approaching 6-8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of G α isolated from natural sources. To assay for the functional integrity of the purified G α chimera at NMR concentrations and probe for changes in the structure and dynamics of G α that result from activation, 15 N-HSQC spectra of the GDP/Mg 2+ bound form of G α obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the 15 N-HSQC spectra reveals a number of changes in chemical shifts of the 1 HN, 15 N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with G α activation

  9. Preparation of radiopharmaceuticals labelled with bromine positron emitting isotopes for the study of dopaminergic receptors of the central nervous system using positron emission tomography

    International Nuclear Information System (INIS)

    Loc'h, C.

    1988-04-01

    The in vivo study of dopaminergic receptors of the central nervous system using positron emission tomography requires the preparation of radiopharmaceuticals labelled with β + emitting isotopes. The chemical and pharmacological properties of these ligands are evaluated. Cyclotron produced 75 and 76 bromine β + emitting isotopes are incorporated into dopaminergic ligands by electrophilic substitution using peracetic acid in a no-carrier added form. Purity, lipophilicity and specific activity are analyzed. Pharmacological criteria (specificity, saturability, displacement, localization) required for ligand-receptor binding studies are evaluated in vitro on striatal membranes and in vivo in the rat. Positron emission tomographic studies show that the study of dopaminergic D2 receptors is possible using 75 and 76 bromine labelled bromospiperone and bromolisuride. These ligands are used in physiological and pharmacological studies of the central nervous system [fr

  10. Classification-based quantitative analysis of stable isotope labeling by amino acids in cell culture (SILAC) data.

    Science.gov (United States)

    Kim, Seongho; Carruthers, Nicholas; Lee, Joohyoung; Chinni, Sreenivasa; Stemmer, Paul

    2016-12-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a practical and powerful approach for quantitative proteomic analysis. A key advantage of SILAC is the ability to simultaneously detect the isotopically labeled peptides in a single instrument run and so guarantee relative quantitation for a large number of peptides without introducing any variation caused by separate experiment. However, there are a few approaches available to assessing protein ratios and none of the existing algorithms pays considerable attention to the proteins having only one peptide hit. We introduce new quantitative approaches to dealing with SILAC protein-level summary using classification-based methodologies, such as Gaussian mixture models with EM algorithms and its Bayesian approach as well as K-means clustering. In addition, a new approach is developed using Gaussian mixture model and a stochastic, metaheuristic global optimization algorithm, particle swarm optimization (PSO), to avoid either a premature convergence or being stuck in a local optimum. Our simulation studies show that the newly developed PSO-based method performs the best among others in terms of F1 score and the proposed methods further demonstrate the ability of detecting potential markers through real SILAC experimental data. No matter how many peptide hits the protein has, the developed approach can be applicable, rescuing many proteins doomed to removal. Furthermore, no additional correction for multiple comparisons is necessary for the developed methods, enabling direct interpretation of the analysis outcomes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes

    International Nuclear Information System (INIS)

    Lo, Andy; Weiner, Joel H.; Li, Liang

    2013-01-01

    Graphical abstract: -- Highlights: •Investigating a strategy of reciprocal isotope labeling of comparative samples. •Filtering out incorrect peptide identification or quantification values. •Analyzing the proteome changes of E. coli cells grown aerobically or anaerobically. •Presenting guidelines for reciprocal labeling experimental design. -- Abstract: Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed

  12. Study of reactions of isotopic exchange of trans-zeatin with tritium

    International Nuclear Information System (INIS)

    Sidorov, G.V.; Myasoedov, N.F.

    2006-01-01

    Reactions of isotopic exchange of trans-zeatin with high-radioactive tritium water, with gaseous tritium in solution and solid-phase catalytic hydrogenation are studied to prepare trans-zeatin and dihydrozeatin labelled with tritium. It is shown that reaction of isotopic exchange of trans-zeatin with gaseous tritium both in solutions and without solvents at 160 Deg C and above leads to practically total hydrogenation of initial compound with formation of dihydrozeatin labelled with tritium. Isotopic exchange with tritium water permits to prepare zeatin labelled with tritium with 67 % yield and specific radioactivity 0.68 PBq/mol. It is determined that in the case of solid-phase isotopic exchange within 150-155 Deg C temperature interval both dihydrozeatin and trans-zeatin labelled with tritium are formed [ru

  13. Failure to label baboon milk intrinsically with iron

    International Nuclear Information System (INIS)

    Figueroa-Colon, R.; Elwell, J.H.; Jackson, E.; Osborne, J.W.; Fomon, S.J.

    1989-01-01

    The widely held belief that 50% of the iron in human milk is absorbed is based on studies that have used an extrinsic radioactive iron tag. To determine the validity of an extrinsic tag, it is necessary to label the milk intrinsically with one isotope and to compare absorption of this isotope with absorption of another isotope added as the extrinsic tag. We chose the baboon as a model and infused 59Fe intravenously. In each of three attempts we failed to label the milk intrinsically

  14. A cost-effective approach to produce 15N-labelled amino acids employing Chlamydomonas reinhardtii CC503.

    Science.gov (United States)

    Nicolás Carcelén, Jesús; Marchante-Gayón, Juan Manuel; González, Pablo Rodríguez; Valledor, Luis; Cañal, María Jesús; Alonso, José Ignacio García

    2017-08-18

    The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15 N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15 NH 4 Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15 N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only 13

  15. Biotin augments acetyl CoA carboxylase 2 gene expression in the hypothalamus, leading to the suppression of food intake in mice.

    Science.gov (United States)

    Sone, Hideyuki; Kamiyama, Shin; Higuchi, Mutsumi; Fujino, Kaho; Kubo, Shizuka; Miyazawa, Masami; Shirato, Saya; Hiroi, Yuka; Shiozawa, Kota

    2016-07-29

    It is known that biotin prevents the development of diabetes by increasing the functions of pancreatic beta-cells and improving insulin sensitivity in the periphery. However, its anti-obesity effects such as anorectic effects remain to be clarified. Acetyl CoA carboxylase (ACC), a biotin-dependent enzyme, has two isoforms (ACC1 and ACC2) and serves to catalyze the reaction of acetyl CoA to malonyl CoA. In the hypothalamus, ACC2 increases the production of malonyl CoA, which acts as a satiety signal. In this study, we investigated whether biotin increases the gene expression of ACC2 in the hypothalamus and suppresses food intake in mice administered excessive biotin. Food intake was significantly decreased by biotin, but plasma regulators of appetite, including glucose, ghrelin, and leptin, were not affected. On the other hand, biotin notably accumulated in the hypothalamus and enhanced ACC2 gene expression there, but it did not change the gene expression of ACC1, malonyl CoA decarboxylase (a malonyl CoA-degrading enzyme), and AMP-activated protein kinase α-2 (an ACC-inhibitory enzyme). These findings strongly suggest that biotin potentiates the suppression of appetite by upregulating ACC2 gene expression in the hypothalamus. This effect of biotin may contribute to the prevention of diabetes by biotin treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. A Breast Cell Atlas: Organelle analysis of the MDA-MB-231 cell line by density-gradient fractionation using isotopic marking and label-free analysis

    Directory of Open Access Journals (Sweden)

    Marianne Sandin

    2015-09-01

    Full Text Available Protein translocation between organelles in the cell is an important process that regulates many cellular functions. However, organelles can rarely be isolated to purity so several methods have been developed to analyse the fractions obtained by density gradient centrifugation. We present an analysis of the distribution of proteins amongst organelles in the human breast cell line, MDA-MB-231 using two approaches: an isotopic labelling and a label-free approach.

  17. Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment

    International Nuclear Information System (INIS)

    Fan Ying; Shi Lichi; Ladizhansky, Vladimir; Brown, Leonid S.

    2011-01-01

    Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly 13 C, 15 N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution 13 C and 15 N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.

  18. Assessing of distribution, mobility and bioavailability of exogenous Pb in agricultural soils using isotopic labeling method coupled with BCR approach.

    Science.gov (United States)

    Huang, Zhi-Yong; Xie, Hong; Cao, Ying-Lan; Cai, Chao; Zhang, Zhi

    2014-02-15

    The contamination of Pb in agricultural soils is one of the most important ecological problems, which potentially results in serious health risk on human health through food chain. Hence, the fate of exogenous Pb contaminated in agricultural soils is needed to be deeply explored. By spiking soils with the stable enriched isotopes of (206)Pb, the contamination of exogenous Pb(2+) ions in three agricultural soils sampled from the estuary areas of Jiulong River, China was simulated in the present study, and the distribution, mobility and bioavailability of exogenous Pb in the soils were investigated using the isotopic labeling method coupled with a four-stage BCR (European Community Bureau of Reference) sequential extraction procedure. Results showed that about 60-85% of exogenous Pb was found to distribute in reducible fractions, while the exogenous Pb in acid-extractable fractions was less than 1.0%. After planting, the amounts of exogenous Pb presenting in acid-extractable, reducible and oxidizable fractions in rhizospheric soils decreased by 60-66%, in which partial exogenous Pb was assimilated by plants while most of the metal might transfer downward due to daily watering and applying fertilizer. The results show that the isotopic labeling technique coupled with sequential extraction procedures enables us to explore the distribution, mobility and bioavailability of exogenous Pb contaminated in soils, which may be useful for the further soil remediation. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    Science.gov (United States)

    Sonawane, Prashant; Vishwakarma, Rishi Kishore; Khan, Bashir M

    2013-07-01

    Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25°C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×10(6) M(-1) s(-1)), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20-50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Modern spectrometric methods for the analysis of labelled compounds

    International Nuclear Information System (INIS)

    Kaspersen, F.M.; Funke, C.W.; Wagenaars, G.N.; Jacobs, P.L.

    1988-01-01

    A proper analysis of chemical compounds should give information about the chemical identity (not only the structure but also enantiomeric form), the chemical purity and chemical composition (e.g. giving information about counter-ions, solvents of crystallization). For labelled compounds information is also needed about isotopic purity (defined as the % of isotope present in the compound), the position/distribution of the isotope in the molecule and degree of labelling/specific activity. In the past ten years the possibilities for spectrometric analyses of labelled compounds have increased enormously and this chapter will give an overview of these methods with the exception of (radio)chromatography that will be dealt with in another chapter. (author)

  1. Shifts in rotifer life history in response to stable isotope enrichment: testing theories of isotope effects on organismal growth

    Science.gov (United States)

    2017-01-01

    In ecology, stable isotope labelling is commonly used for tracing material transfer in trophic interactions, nutrient budgets and biogeochemical processes. The main assumption in this approach is that the enrichment with a heavy isotope has no effect on the organism growth and metabolism. This assumption is, however, challenged by theoretical considerations and experimental studies on kinetic isotope effects in vivo. Here, I demonstrate profound changes in life histories of the rotifer Brachionus plicatilis fed 15N-enriched algae (0.4–5.0 at%); i.e. at the enrichment levels commonly used in ecological studies. These findings support theoretically predicted effects of heavy isotope enrichment on growth, metabolism and ageing in biological systems and underline the importance of accounting for such effects when using stable isotope labelling in experimental studies. PMID:28405367

  2. Reductive methods for isotopic labeling of antibiotics

    International Nuclear Information System (INIS)

    Champney, W.S.

    1989-01-01

    Methods for the reductive methylation of the amino groups of eight different antibiotics using 3 HCOH or H 14 COH are presented. The reductive labeling of an additional seven antibiotics by NaB 3 H 4 is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented

  3. Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein {alpha}-subunit

    Energy Technology Data Exchange (ETDEWEB)

    Abdulaev, Najmoutin G. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States); Zhang Cheng; Dinh, Andy [University of Texas Health Science Center, Center for Membrane Biology, Department of Biochemistry and Molecular Biology (United States); Ngo, Tony; Bryan, Philip N. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States); Brabazon, Danielle M. [Loyola College in Maryland, Department of Chemistry (United States); Marino, John P. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States)], E-mail: marino@carb.nist.gov; Ridge, Kevin D. [University of Texas Health Science Center, Center for Membrane Biology, Department of Biochemistry and Molecular Biology (United States)

    2005-05-15

    Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein {alpha}-subunit (G{sub {alpha}}) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled G{sub {alpha}} chimera ({approx}40 kDa polypeptide) has been tested. The results show that a prodomain fused G{sub {alpha}} chimera can be expressed to levels approaching 6-8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of G{sub {alpha}} isolated from natural sources. To assay for the functional integrity of the purified G{sub {alpha}} chimera at NMR concentrations and probe for changes in the structure and dynamics of G{sub {alpha}} that result from activation, {sup 15}N-HSQC spectra of the GDP/Mg{sup 2+} bound form of G{sub {alpha}} obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the {sup 15}N-HSQC spectra reveals a number of changes in chemical shifts of the {sup 1}HN, {sup 15}N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with G{sub {alpha}} activation.

  4. Temperature-induced evolution of strain and doping in an isotopically labeled two-dimensional graphene-C-70 fullerene peapod

    Czech Academy of Sciences Publication Activity Database

    Verhagen, Timotheus; Valeš, Václav; Kalbáč, Martin; Vejpravová, Jana

    2017-01-01

    Roč. 75, May (2017), s. 140-145 ISSN 0925-9635 R&D Projects: GA ČR(CZ) GA15-01953S; GA MŠk LL1301 Institutional support: RVO:68378271 ; RVO:61388955 Keywords : two-dimensional peapod * Raman spectroscopy * isotope labelling * topography Subject RIV: BM - Solid Matter Physics ; Magnetism; CF - Physical ; Theoretical Chemistry (UFCH-W) OBOR OECD: Condensed matter physics (including formerly solid state physics, supercond.); Physical chemistry (UFCH-W) Impact factor: 2.561, year: 2016

  5. Isotopic labeling affects 1,25-dihydroxyvitamin D metabolism

    International Nuclear Information System (INIS)

    Halloran, B.P.; Bikle, D.D.; Castro, M.E.; Gee, E.

    1989-01-01

    Isotope substitution can change the biochemical properties of vitamin D. To determine the effect of substituting 3H for 1H on the metabolism of 1,25(OH)2D3, we measured the metabolic clearance rate and renal metabolism of unlabeled and 3H-labeled 1,25(OH)2D3. Substitution of 3H for 1H on carbons 26 and 27 [1,25(OH)2[26,27(n)-3H]D3] or on carbons 23 and 24 [1,25(OH)2[23,24(n)-3H]D3] reduced the in vivo metabolic clearance rate of 1,25(OH)2D3 by 36% and 37%, respectively, and reduced the in vitro renal catabolism of 1,25(OH)2D3 by 11% and 54%, respectively. Substitutions of 3H for 1H on carbons 23 and 24 as opposed to carbons 26 and 27 reduced conversion of [3H]1,25(OH)2D3 to [3H]1,24,25(OH)2D3 by 25% and to putative 24-oxo-1,23,25-dihydroxyvitamin D3 by 1600%. These results indicate that substitution of 3H for 1H on carbons 26 and 27 or on carbons 23 and 24 can reduce the metabolic clearance rate and in vitro metabolism of 1,25(OH)2D3 and quantitatively alter the pattern of metabolic products produced

  6. Flexible DAQ card for detector systems utilizing the CoaXPress communication standard

    International Nuclear Information System (INIS)

    Neue, G.; Hejtmánek, M.; Marčišovský, M.; Voleš, P.

    2015-01-01

    This work concerns the design and construction of a flexible FPGA based data acquisition system aimed for particle detectors. The interface card as presented was designed for large area detectors with millions of individual readout channels. Flexibility was achieved by partitioning the design into multiple PCBs, creating a set of modular blocks, allowing the creation of a wide variety of configurations by simply stacking functional PCBs together. This way the user can easily toggle the polarity of the high voltage bias supply or switch the downstream interface from CoaXPress to PCIe or stream directly HDMI. We addressed the issues of data throughput, data buffering, bias voltage generation, trigger timing and fine tuning of the whole readout chain enabling a smooth data transmission. On the current prototype, we have wire-bonded a MediPix2 MXR quad and connected it to a XILINX FPGA. For the downstream interface, we implemented the CoaXPress communication protocol, which enables us to stream data at 3.125 Gbps to a standard PC

  7. NMR studies of two spliced leader RNAs using isotope labeling

    Energy Technology Data Exchange (ETDEWEB)

    Lapham, J.; Crothers, D.M. [Yale Univ., New Haven, CT (United States)

    1994-12-01

    Spliced leader RNAs are a class of RNA molecules (<200 nts) involved in the trans splicing of messenger RNA found in trypanosomes, nematodes, and other lower eukaryotes. The spliced leader RNA from the trypanosome Leptomonas Collosoma exists in two alternate structural forms with similar thermal stabilities. The 54 nucleotides on the 5{prime} end of the SL molecule is structurally independent from the 3{prime} half of the RNA, and displays the two structural forms. Furthermore, the favored of the two structures was shown to contain anomalous nuclease sensitivity and thermal stability features, which suggests that there may be tertiary interactions between the splice site and other nucleotides in the 5{prime} end. Multidimensional NMR studies are underway to elucidate the structural elements present in the SL RNAs that give rise to their physical properties. Two spliced leader sequences have been studied. The first, the 54 nucleotides on the 5{prime} end of the L. Collosoma sequence, was selected because of earlier studies in our laboratory. The second sequence is the 5{prime} end of the trypanosome Crithidia Fasciculata, which was chosen because of its greater sequence homology to other SL sequences. Given the complexity of the NMR spectra for RNA molecules of this size, we have incorporated {sup 15}N/{sup 13}C-labeled nucleotides into the RNA. One of the techniques we have developed to simplify the spectra of these RNA molecules is isotope labeling of specific regions of the RNA. This has been especially helpful in assigning the secondary structure of molecules that may be able to adopt multiple conformations. Using this technique one can examine a part of the molecule without spectral interference from the unlabeled portion. We hope this approach will promote an avenue for studying the structure of larger RNAs in their native surroundings.

  8. The evaluation of new and isotopically labeled isoindoline nitroxides and an azaphenalene nitroxide for EPR oximetry

    Science.gov (United States)

    Khan, Nadeem; Blinco, James P.; Bottle, Steven E.; Hosokawa, Kazuyuki; Swartz, Harold M.; Micallef, Aaron S.

    2011-01-01

    Isoindoline nitroxides are potentially useful probes for viable biological systems, exhibiting low cytotoxicity, moderate rates of biological reduction and favorable Electron Paramagnetic Resonance (EPR) characteristics. We have evaluated the anionic (5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl; CTMIO), cationic (5-(N,N,N-trimethylammonio)-1,1,3,3-tetramethylisoindolin-2-yloxyl iodide, QATMIO) and neutral (1,1,3,3-tetramethylisoindolin-2-yloxyl; TMIO) nitroxides and their isotopically labeled analogues (2H12- and/or 2H12-15N-labeled) as potential EPR oximetry probes. An active ester analogue of CTMIO, designed to localize intracellularly, and the azaphenalene nitroxide 1,1,3,3-tetramethyl-2,3-dihydro-2-azaphenalen-2-yloxyl (TMAO) were also studied. While the EPR spectra of the unlabeled nitroxides exhibit high sensitivity to O2 concentration, deuteration resulted in a loss of superhyperfine features and a subsequent reduction in O2 sensitivity. Labeling the nitroxides with 15N increased the signal intensity and this may be useful in decreasing the detection limits for in vivo measurements. The active ester nitroxide showed approximately 6% intracellular localization and low cytotoxicity. The EPR spectra of TMAO nitroxide indicated an increased rigidity in the nitroxide ring, due to dibenzo-annulation. PMID:21665499

  9. [Solid state isotope hydrogen exchange for deuterium and tritium in human gene-engineered insulin].

    Science.gov (United States)

    Zolotarev, Yu A; Dadayan, A K; Kozik, V S; Gasanov, E V; Nazimov, I V; Ziganshin, R Kh; Vaskovsky, B V; Murashov, A N; Ksenofontov, A L; Haribin, O N; Nikolaev, E N; Myasoedov, N F

    2014-01-01

    The reaction of high temperature solid state catalytic isotope exchange in peptides and proteins under the action of catalyst-activated spillover hydrogen was studied. The reaction of human gene-engineered insulin with deuterium and tritium was conducted at 120-140° C to produce insulin samples containing 2-6 hydrogen isotope atoms. To determine the distribution of the isotope label over tritium-labeled insulin's amino acid residues, oxidation of the S-S bonds of insulin by performic acid was performed and polypeptide chains isolated; then their acid hydrolysis, amino acid analysis and liquid scintillation counts of tritium in the amino acids were conducted. The isotope label was shown to be incorporated in all amino acids of the protein, with the peptide fragment FVNQHLCGSHLVE of the insulin β-chain showing the largest incorporation. About 45% of the total protein isotope label was incorporated in His5 and His10 of this fragment. For the analysis of isotope label distribution in labeled insulin's peptide fragments, the recovery of the S-S bonds by mercaptoethanol, the enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius and HPLC division of the resulting peptides were carried out. Attribution of the peptide fragments formed due to hydrolysis at the Glu-X bond in the β-chain was accomplished by mass spectrometry. Mass spectrometry analysis data of the deuterium-labeled insulin samples' isotopomeric composition showed that the studied solid state isotope exchange reaction equally involved all the protein molecules. Biological studying of tritium-labeled insulin showed its physiological activity to be completely retained.

  10. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Applied to Quantitative Proteomics of Bacillus subtilis

    DEFF Research Database (Denmark)

    Soufi, Boumediene; Kumar, C.; Gnad, F.

    2010-01-01

    We applied stable isotope labeling by amino acids in cell culture (SILAC) to large-scale quantitative proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic...... of the most comprehensive quantitative proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under...

  11. Evaluation of a new component used for isotopic lymphography: colloidal rhenium sulfide sup(99m)Tc labelled

    International Nuclear Information System (INIS)

    Pecking, A.; Le Mercier, N.; Gobin, R.; Bardy, A.; Najean, Y.

    1978-01-01

    We have studied for lymphatic scintigraphy a new radiopharmaceutical, sup(99m)Tc-labelled rhenium sulfocolloid. This preliminary study includes 20 adults patients with lymphomas and lymphoedemas. The principal advantage of this drug is its absence of toxicity and local pain, so that a rapid sub-cutaneous injection without local anesthesia is made possible. Good results have been obtained, as well in morphological studies of para-aortic and mammary lymph nodes as for kinetic studies of lymphatic flow in lymphoedemas. No liver and spleen uptake of radio-isotope was observed after foot injection [fr

  12. Isotopes in day to day life

    Science.gov (United States)

    1984-06-01

    Developments are reported in the use of isotopic labeling and isotope irradiation in agriculture, medical science, hydrology, geochemistry, geophysics, environment pollution detection, and industries. Radioisotope instruments are described as well as techniques for gamma radiography, neutron radiography, and autoradiography. Isotope dating in geology and archaeology is covered. Basic scientific research topics in various areas are listed.

  13. Liquid–liquid extraction combined with differential isotope dimethylaminophenacyl labeling for improved metabolomic profiling of organic acids

    International Nuclear Information System (INIS)

    Peng, Jun; Li, Liang

    2013-01-01

    Graphical abstract: -- Highlights: •An improved method for profiling the carboxylic acid sub-metabolome is reported. •Liquid–liquid extraction was used for separating the organic acids from the amines. • 12 C/ 13 C-p-dimethylaminophenacyl (DmPA) labeling of the organic acids was carried out on the extract. •Detection interference by amines and labeling efficiency reduction by water were reduced. •About 2500 12 C/ 13 C-peak pairs or putative metabolites could be detected from 20 μL of human urine. -- Abstract: A large fraction of the known human metabolome belong to organic acids. However, comprehensive profiling of the organic acid sub-metabolome is a major analytical challenge. In this work, we report an improved method for detecting organic acid metabolites. This method is based on the use of liquid–liquid extraction (LLE) to selectively extract the organic acids, followed by using differential isotope p-dimethylaminophenacyl (DmPA) labeling of the acid metabolites. The 12 C-/ 13 C-labeled samples are analyzed by liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC–FTICR–MS). It is shown that this LLE DmPA labeling method offers superior performance over the method of direct DmPA labeling of biofluids such as human urine. LLE of organic acids reduces the interference of amine-containing metabolites that may also react with DmPA. It can also remove water in a biofluid that can reduce the labeling efficiency. Using human urine as an example, it is demonstrated that about 2500 peak pairs or putative metabolites could be detected in a 30-min gradient LC–MS run, which is about 3 times more than that detected in a sample prepared using direct DmPA labeling. About 95% of the 1000 or so matched metabolites to the Human Metabolome Database (HMDB) are organic acids. It is further shown that this method can be used to handle as small as 10 μL of urine. We believe that this method opens the possibility of generating a

  14. Liquid–liquid extraction combined with differential isotope dimethylaminophenacyl labeling for improved metabolomic profiling of organic acids

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Jun; Li, Liang, E-mail: Liang.Li@ualberta.ca

    2013-11-25

    Graphical abstract: -- Highlights: •An improved method for profiling the carboxylic acid sub-metabolome is reported. •Liquid–liquid extraction was used for separating the organic acids from the amines. •{sup 12}C/{sup 13}C-p-dimethylaminophenacyl (DmPA) labeling of the organic acids was carried out on the extract. •Detection interference by amines and labeling efficiency reduction by water were reduced. •About 2500 {sup 12}C/{sup 13}C-peak pairs or putative metabolites could be detected from 20 μL of human urine. -- Abstract: A large fraction of the known human metabolome belong to organic acids. However, comprehensive profiling of the organic acid sub-metabolome is a major analytical challenge. In this work, we report an improved method for detecting organic acid metabolites. This method is based on the use of liquid–liquid extraction (LLE) to selectively extract the organic acids, followed by using differential isotope p-dimethylaminophenacyl (DmPA) labeling of the acid metabolites. The {sup 12}C-/{sup 13}C-labeled samples are analyzed by liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC–FTICR–MS). It is shown that this LLE DmPA labeling method offers superior performance over the method of direct DmPA labeling of biofluids such as human urine. LLE of organic acids reduces the interference of amine-containing metabolites that may also react with DmPA. It can also remove water in a biofluid that can reduce the labeling efficiency. Using human urine as an example, it is demonstrated that about 2500 peak pairs or putative metabolites could be detected in a 30-min gradient LC–MS run, which is about 3 times more than that detected in a sample prepared using direct DmPA labeling. About 95% of the 1000 or so matched metabolites to the Human Metabolome Database (HMDB) are organic acids. It is further shown that this method can be used to handle as small as 10 μL of urine. We believe that this method opens the

  15. Metabolic Flux Analysis in Isotope Labeling Experiments Using the Adjoint Approach.

    Science.gov (United States)

    Mottelet, Stephane; Gaullier, Gil; Sadaka, Georges

    2017-01-01

    Comprehension of metabolic pathways is considerably enhanced by metabolic flux analysis (MFA-ILE) in isotope labeling experiments. The balance equations are given by hundreds of algebraic (stationary MFA) or ordinary differential equations (nonstationary MFA), and reducing the number of operations is therefore a crucial part of reducing the computation cost. The main bottleneck for deterministic algorithms is the computation of derivatives, particularly for nonstationary MFA. In this article, we explain how the overall identification process may be speeded up by using the adjoint approach to compute the gradient of the residual sum of squares. The proposed approach shows significant improvements in terms of complexity and computation time when it is compared with the usual (direct) approach. Numerical results are obtained for the central metabolic pathways of Escherichia coli and are validated against reference software in the stationary case. The methods and algorithms described in this paper are included in the sysmetab software package distributed under an Open Source license at http://forge.scilab.org/index.php/p/sysmetab/.

  16. Quantitating subcellular metabolism with multi-isotope imaging mass spectrometry

    Science.gov (United States)

    Steinhauser, Matthew L.; Bailey, Andrew; Senyo, Samuel E.; Guillermier, Christelle; Perlstein, Todd S.; Gould, Alex P.; Lee, Richard T.; Lechene, Claude P.

    2011-01-01

    Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter1,2 but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with sub-micron resolution3,4. Here we apply MIMS to diverse organisms, including Drosophila, mice, and humans. We test the “immortal strand hypothesis,” which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labeling mice with 15N-thymidine from gestation through post-natal week 8, we find no 15N label retention by dividing small intestinal crypt cells after 4wk chase. In adult mice administered 15N-thymidine pulse-chase, we find that proliferating crypt cells dilute label consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human hematopoietic system. These studies show that MIMS provides high-resolution quantitation of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research. PMID:22246326

  17. Enantio-specific C(sp3)-H activation catalyzed by ruthenium nanoparticles: application to isotopic labeling of molecules of biological interest

    International Nuclear Information System (INIS)

    Taglang, Celine

    2015-01-01

    Isotopic labeling with deuterium and tritium is extensively used in chemistry, biology and pharmaceutical research. Numerous methods of labeling by isotopic exchange allow high isotopic enrichments but generally require harsh conditions (high temperatures, acidity). As a consequence, a general, regioselective and smooth labeling method that might be applicable to a wide diversity of substrates remains to develop. In the first part of this thesis, we demonstrated that the use of ruthenium nanoparticles, synthesized by Pr. Bruno Chaudret's team (INSA Toulouse), allowed the mild (2 bar of deuterium gas at 55 C), effective and selective H/D exchange reaction of a large variety of nitrogen-containing compounds, such as pyridines, indoles and primary, secondary and tertiary alkyl amines. The usefulness and the efficiency of this novel methodology was demonstrated by the deuteration of eight nitrogen-containing molecules of biological interest without altering their chemical and stereochemical properties. However, the conservation of the original stereochemistry of an activated chiral C-H center remains a major issue. We studied the reactivity of RuNP(at)PVP on different categories of nitrogen-containing substrates (amines, aminoacids and peptides) in water or in organic solvents. Our results showed that C-H activation of chiral carbons C(sp3) took place efficiently, selectively and, in all cases, with total retention of configuration. The wide range of applications of this procedure was demonstrated by the labeling of three chiral amines, fourteen aminoacids, three aromatic amino esters and four peptides. Moreover, our collaboration with Pr. Romuald Poteau's team (INSA Toulouse) led to the identification of two mechanisms by ab initio simulation in agreement with our experimental results: the σ-bond metathesis mechanism and the oxidative addition mechanism. These two mechanisms imply two vicinal ruthenium atoms leading to the formation an original

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

    Science.gov (United States)

    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.

  19. Direct infusion electrospray ionization–ion mobility–mass spectrometry for comparative profiling of fatty acids based on stable isotope labeling

    Energy Technology Data Exchange (ETDEWEB)

    Leng, Jiapeng, E-mail: jpleng@126.com [Department of Chemical Engineering, North University of China, Taiyuan 030051 (China); Guan, Qing [Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center (FUSCC), Shanghai 200032 (China); Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032 (China); Sun, Tuanqi, E-mail: tuanqisun@163.com [Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center (FUSCC), Shanghai 200032 (China); Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032 (China); Wang, Haoyang [National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032 (China); Cui, Jianlan; Liu, Qinghao [Department of Chemical Engineering, North University of China, Taiyuan 030051 (China); Zhang, Zhixu; Zhang, Manyu [Agilent Technologies China Co., Ltd, Shanghai 200080 (China); Guo, Yinlong, E-mail: ylguo@sioc.ac.cn [National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032 (China)

    2015-08-05

    A rapid method for fatty acids (FAs) comparative profiling based on carboxyl-specific stable isotope labeling (SIL) and direct infusion electrospray ionization–ion mobility–mass spectrometry (ESI–IM–MS) is established. The design of the method takes advantage of the three-dimensional characteristics of IM–MS including drift time, m/z and ion intensity, for comparison of d0-/d6-2,4-dimethoxy-6-piperazin-1-yl pyrimidine (DMPP)-labeled FAs. In particular, without chromatographic separation, the method allowed direct FAs profiling in complex samples due to the advantageous priority of DMPP in signal enhancement as well as the extra resolution that IM–MS offered. Additionally, the d0-/d6-DMPP-labeled FAs showed expected features, including very similar drift times, 6 Da mass deviations, specific reporter ions, similar MS responses, and adherence to the drift time rule regarding the influence of carbon chain length and unsaturation on relative drift times. Therefore, the introduction of isotope analogs minimized the matrix effect and variations in quantification and ensured accurate identification of non-targeted FAs by those typical features. Peak intensity ratios between d0-/d6-DMPP-labeled ions were subsequently used in relative quantification for the detected FAs. The established strategy has been applied successfully in the rapid profiling of trace free FAs between normal and cancerous human thyroid tissues. Sixteen free FAs were found with the increased level with a statistically significant difference (p < 0.05) compared to the normal tissue samples. The integrated SIL technique and ESI–IM–MS are expected to serve as an alternative tool for high-throughput analysis of FAs in complex samples. - Highlights: • A novel method based on IM–MS and SIL was developed for FAs comparative profiling. • Without LC separation, the method allowed direct infusion profiling of FAs in complex samples. • Both of the efficiency and accuracy for FAs analyses

  20. Field experimentation in isotope-aided studies

    International Nuclear Information System (INIS)

    Zapata, F.

    1990-01-01

    Isotopic-aided studies involve the application of isotopically labelled fertilizer as tracers for the quantitative and precise determination of the fate of specific nutrient elements in the soil/plant system. The planning of isotopic-aided studies requires a different approach from that followed in the design of normal fertilizer trials because of the cost and supply of isotopically labelled materials, the use of highly specialized equipment and the need for skillful trained staff in the use of isotope techniques both in the field/greenhouse and the laboratory. This report is intended to highlight the main points to be considered while applying those techniques in the field or greenhouse. It has been well established that nuclear techniques are a powerful tool in agricultural research. One should take advantage of the use of such techniques if the following criteria are met: The isotope method is the only way to solve a particular question or to obtain a specific piece of information. There are other methods available for such a purpose but the nuclear method provides a direct and quick means to obtain the needed information resulting in higher economic return

  1. Evaluation of stable isotope labelling strategies for the quantitation of CP4 EPSPS in genetically modified soya

    Energy Technology Data Exchange (ETDEWEB)

    Ocana, Mireia Fernandez [Centre for Chemical and Bioanalytical Sciences, Royal Holloway, University of London, Egham TW20 0EX (United Kingdom)], E-mail: Mireia.FernandezOcana@pfizer.com; Fraser, Paul D. [Centre for Chemical and Bioanalytical Sciences, Royal Holloway, University of London, Egham TW20 0EX (United Kingdom); Patel, Raj K.P.; Halket, John M. [Specialist Bioanalytical Services Ltd., Royal Holloway, University of London, Egham TW20 0EX (United Kingdom); Bramley, Peter M. [Centre for Chemical and Bioanalytical Sciences, Royal Holloway, University of London, Egham TW20 0EX (United Kingdom)

    2009-02-16

    The introduction of genetically modified (GM) crops into the market has raised a general alertness relating to the control and safety of foods. The applicability of protein separation hyphenated to mass spectrometry to identify the bacterial enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein expressed in GM crops has been previously reported [M.F. Ocana, P.D. Fraser, R.K.P. Patel, J.M. Halket, P.M. Bramley, Rapid Commun. Mass Spectrom. 21 (2007) 319.]. Herein, we investigate the suitability of two strategies that employ heavy stable isotopes, i.e. AQUA and iTRAQ, to quantify different levels of CP4 EPSPS in up to four GM preparations. Both quantification strategies showed potential to determine whether the presence of GM material is above the limits established by the European Union. The AQUA quantification procedure involved protein solubilisation/fractionation and subsequent separation using SDS-PAGE. A segment of the gel in which the protein of interest was located was excised, the stable isotope labeled peptide added at a known concentration and proteolytic digestion initiated. Following recovery of the peptides, on-line separation and detection using LC-MS was carried out. A similar approach was used for the iTRAQ workflow with the exception that proteins were digested in solution and generated tryptic peptides were chemically tagged. Both procedures demonstrated the potential for quantitative detection at 0.5% (w/w) GM soya which is a level below the current European Union's threshold for food-labelling. In this context, a comparison between the two procedures is provided within the present study.

  2. Evaluation of stable isotope labelling strategies for the quantitation of CP4 EPSPS in genetically modified soya

    International Nuclear Information System (INIS)

    Ocana, Mireia Fernandez; Fraser, Paul D.; Patel, Raj K.P.; Halket, John M.; Bramley, Peter M.

    2009-01-01

    The introduction of genetically modified (GM) crops into the market has raised a general alertness relating to the control and safety of foods. The applicability of protein separation hyphenated to mass spectrometry to identify the bacterial enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein expressed in GM crops has been previously reported [M.F. Ocana, P.D. Fraser, R.K.P. Patel, J.M. Halket, P.M. Bramley, Rapid Commun. Mass Spectrom. 21 (2007) 319.]. Herein, we investigate the suitability of two strategies that employ heavy stable isotopes, i.e. AQUA and iTRAQ, to quantify different levels of CP4 EPSPS in up to four GM preparations. Both quantification strategies showed potential to determine whether the presence of GM material is above the limits established by the European Union. The AQUA quantification procedure involved protein solubilisation/fractionation and subsequent separation using SDS-PAGE. A segment of the gel in which the protein of interest was located was excised, the stable isotope labeled peptide added at a known concentration and proteolytic digestion initiated. Following recovery of the peptides, on-line separation and detection using LC-MS was carried out. A similar approach was used for the iTRAQ workflow with the exception that proteins were digested in solution and generated tryptic peptides were chemically tagged. Both procedures demonstrated the potential for quantitative detection at 0.5% (w/w) GM soya which is a level below the current European Union's threshold for food-labelling. In this context, a comparison between the two procedures is provided within the present study

  3. Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential assignment

    International Nuclear Information System (INIS)

    Schedlbauer, Andreas; Auer, Renate; Ledolter, Karin; Tollinger, Martin; Kloiber, Karin; Lichtenecker, Roman; Ruedisser, Simon; Hommel, Ulrich; Schmid, Walther; Konrat, Robert; Kontaxis, Georg

    2008-01-01

    Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when 13 C β and 13 C' shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using 13 C α connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs

  4. Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

    DEFF Research Database (Denmark)

    Hurtado-Guerrrero, Ramón; Pena Diaz, Javier; Montalvetti, Andrea

    2002-01-01

    A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower propo...

  5. Secondary isotope effects on alpha-cleavage reactions

    International Nuclear Information System (INIS)

    Ingemann, S.; Hammerum, S.

    1980-01-01

    Kinetic deuterium isotope effects on mass spectral reactions have in several instances been utilized to provide structural information and to answer mechanistic questions. Typically, the influence of the deuterium label on the rate of one of a number of competing reactions has been studied. Secondary isotope effects have usually been assumed to be relatively insignificant in comparison with the observed kinetic effects, even though various workers have shown that secondary isotope effects may indeed exert a considerable influence on the rates of competing simple cleavages. Recent studies have provided quantitative data to show that the mere presence of deuterium atoms up to six bonds away may influence the rate of a simple cleavage reaction. In relation to an investigation of rearrangements accompanying simple cleavage reactions, a semi-quantitative measure was needed of the variation of the secondary isotope effect with the number of bonds between the deuterium label and the point of rupture. The influence has therefore been examined of the presence of remote deuterium atoms on a typical simple cleavage reaction, the α-cleavage of aliphatic amines. As a model compound, N-methyldipentylamine was chosen, systematically labelled with deuterium. (author)

  6. Evaluation of in-vitro cell labelling of mouse epithelia with tritiated thymidine

    International Nuclear Information System (INIS)

    Mackenzie, I.C.; Ettinger, R.L.

    1977-01-01

    Various factors affecting the epithelial labelling index recorded following in-vitro incubation of specimens of mouse skin and palatal mucosa with tritiated thymidine were examined. Isotope concentration, specimen size and the period of exposure of autoradiographs prior to development markedly influenced the labelling index recorded but, following standardization of such factors, a reproducible index could be obtained. Labelling indices comparable to those obtained by the standard in-vivo labelling method could be produced by adjustment of isotope concentration in incubation media. Comparison of labelling indices recorded for tissues labelled in-vitro by a standardized method appeared valid but the absolute values of indices so obtained and their comparison with indices resulting from in-vivo labelling methods were of doubtful significance. (author)

  7. Dual isotope assays

    International Nuclear Information System (INIS)

    Smith, G.F.W.; Stevens, R.A.J.; Jacoby, B.

    1980-01-01

    Dual isotope assays for thyroid function are performed by carrying out a radio-immunoassay for two of thyroxine (T4), tri-iodothyronine (T3), thyroid stimulating hormone (TSH), and thyroxine binding globulin (TBG), by a method wherein a version of one of the thyroid components, preferably T4 or T3 is labelled with Selenium-75 and the version of the other thyroid component is labelled with a different radionuclide, preferably Iodine-125. (author)

  8. Mass spectrometric measurements of norepinephrine synthesis in man from infusion of stable isotope-labelled L-threo-3,4-dihydroxyphenylserine

    International Nuclear Information System (INIS)

    Suzuki, T.; Sakoda, S.; Ueji, M.; Kishimoto, S.

    1985-01-01

    The kinetics of stable isotope-labelled L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS), an immediate precursor of (-)-norepinephrine, was studied to investigate the pharmacologic mechanism of its therapeutic effect on orthostatic hypotension in familial amyloid polyneuropathy (FAP) and on akinesia and freezing in parkinsonism. [ 13 C,D]-L-threo-DOPS was synthesized, and 100 mg of the compound was infused for 2 h into two normal subjects, two FAP patients and two patients with the degenerative diseases of the central nervous system. Labelled and endogenous norepinephrine in urine and plasma was assayed simultaneously by gas chromatography/mass spectrometry. The results indicate that the increase in norepinephrine in biological fluids after administration of L-threo-DOPS is attributable mostly to norepinephrine derived from L-threo-DOPS, not to pre-formed endogenous norepinephrine released by L-threo-DOPS

  9. Synthesis of deuterium-labelled compounds for FOTEK project

    International Nuclear Information System (INIS)

    Joergensen, O.; Egsgaard, H.; Larsen, E.

    1996-01-01

    In the FoTech project there have been utilized labelled compounds of stable isotopes as internal standards. Some of these compounds are commercially available ( 13 C-labelled PCB congeners, 13 C-labelled diethylstilbestrol for determination of anabolic steroids). Others, like D 9 -clenbuterol, D 3 -clenbuterol, D 3 -zeramol and D 3 -dimetridazol have been synthesized. General aspects of deuterium compounds labelling are considered. (EG)

  10. A theory of stable-isotope dilution mass spectrometry

    International Nuclear Information System (INIS)

    Pickup, J.F.; McPherson, C.K.

    1977-01-01

    In order to perform quantitative analysis using stable isotope dilution with mass spectrometry, an equation is derived which describes the relationship between the relative proportions of natural and labelled material and measured isotope ratios

  11. IRMS detection of testosterone manipulated with 13C labeled standards in human urine by removing the labeled 13C

    International Nuclear Information System (INIS)

    Wang, Jingzhu; Yang, Rui; Yang, Wenning; Liu, Xin; Xing, Yanyi; Xu, Youxuan

    2014-01-01

    Highlights: • 13 C labeled testosterone can be used to adjust the isotope ratio of testosterone. • The novel testosterone cannot be detected by the regular IRMS method in doping test. • A method was explored to remove the labeled 13 C. • The established method can be used to detect the manipulated testosterone. - Abstract: Isotope ratio mass spectrometry (IRMS) is applied to confirm testosterone (T) abuse by determining the carbon isotope ratios (δ 13 C value). However, 13 C labeled standards can be used to control the δ 13 C value and produce manipulated T which cannot be detected by the current method. A method was explored to remove the 13 C labeled atom at C-3 from the molecule of androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ 13 C values between Andro and ANAD (Δδ 13 C Andro–ANAD , ‰) would change significantly in case manipulated T is abused. Twenty-one volunteers administered T manipulated with different 13 C labeled standards. The collected urine samples were analyzed with the established method, and the maximum value of Δδ 13 C Andro–ANAD post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ 13 C Andro–ANAD for positive result was suggested as 1.2‰. The developed method could be used to detect T manipulated with 3- 13 C labeled standards

  12. Synthesis of isotopically labeled ketamine

    OpenAIRE

    Stuchlíková, Lucie

    2011-01-01

    In this work were synthesized ketamine isotopomers. Ketamine is used in human medicine and veterinary sectors. It has very broad spectrum of pharmacological effects: anesthetic, analgesic, hallucinogenic, bronchodilator, cardiovascular and antidepressive, which is currently in the research. At first was synthesized precursor of ketamine, N- desmethylketamine which was subsequently labeled the deuterium, tritium and carbon- 14. For the determination of purity and identity mass spectrometry and...

  13. Isotope-labelled folic acid derivatives

    International Nuclear Information System (INIS)

    Lewin, N.; Wong, E.T.

    1976-01-01

    The suggestion deals with the production of folic acid derivatives suitable as indicators or tracers for analyses of serum folates. These folic acid derivatives contain folic acid which is bound by one or both carboxyl groups to the amino nitrogen of compounds such as, e.g., tyramine, glycyl tyrosine, tyrosine, or the methyl ester of tyrosine. The derivative obtained can be substituted by a gamma emitter, e.g. the iodine isotope I 125. The radioactive derivative is used in the method for the competitive protein bonding to determine endogenic folates in the serum. (UWI) [de

  14. Negative ion ESI-MS analysis of natural yellow dye flavonoids--An isotopic labelling study

    Science.gov (United States)

    McNab, Hamish; Ferreira, Ester S. B.; Hulme, Alison N.; Quye, Anita

    2009-07-01

    Flavonoids are amongst the most commonly used natural yellow colourants in paintings, as lakes, and in historical textiles as mordant dyes. In this paper, evidence from isotopically labelled substrates is used to propose negative ion electrospray collision induced decomposition mechanisms of flavones, flavonols and an isoflavone. These mechanisms include a retro-Diels-Alder fragmentation (observed for flavones and flavonols) and an M-122 fragmentation (characteristic of 3',4'-dihydroxyflavonols). In addition, the presence of a m/z 125 fragment ion is shown to be characteristic of 2'-hydroxyflavonols and an ion at m/z 149 is shown to be characteristic of 4'-hydroxyflavones. Applications of these methods are exemplified by the identification of a minor component of Dyer's camomile (Anthemis tinctoria L.) and the identification of the dye source in green threads sampled from an 18th Century Scottish tartan fragment.

  15. Deciphering systemic wound responses of the pumpkin extrafascicular phloem by metabolomics and stable isotope-coded protein labeling.

    Science.gov (United States)

    Gaupels, Frank; Sarioglu, Hakan; Beckmann, Manfred; Hause, Bettina; Spannagl, Manuel; Draper, John; Lindermayr, Christian; Durner, Jörg

    2012-12-01

    In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling.

  16. Isotopic labeling for the understanding of the alteration of limestone used in built cultural heritage

    Science.gov (United States)

    Saheb, Mandana; Chabas, Anne; Mertz, Jean-Didier; Rozenbaum, Olivier; Verney-Carron, Aurélie

    2015-04-01

    This project belongs to a specific work aiming at developing isotopic tools to better understand the alteration of materials used in the built cultural heritage. It is focused on the study of the alteration of limestone used in the facades of historic buildings subject to atmospheric polluted environment. Actually in the elevated parts of the buildings, water as rainfall (runoff or wet deposition) or in vapor form (condensation or dry deposition) is the main agent of alteration. Thus, the rock/water interactions need to be well understood to propose adapted solution to better preserve the buildings. To identify the water transfer within the porous limestone and locate the reaction preferential sites, two isotopic tracers (D and 18O) are used to monitor the alteration solution (D) and locate the zones containing the secondary phases (18O). The Saint-Maximin limestone used in many monuments in the suburbs of Paris (France) as a building and restoration stone has been specifically studied. Pristine materials, stones from monuments (monuments in the Paris area) and samples altered in laboratory constitute the analytical corpus to compare different stages of alteration. In a first step the stones are characterized at different scales to identify the alteration pattern (SEM-EDS, Raman microspectrometry, XRD, rugosimetry) and study the water transfers (X-ray tomography, mercury porosimetry, imbibition kinetics). The samples are then altered in the laboratory by realistic and controlled wet or dry deposition using isotopically labeled solutions to locate the reaction zones by SIMS. The multiscale characterization of the alteration pattern has allowed proposing alteration mechanisms linked to the properties of the stones and their location inside the building. Moreover, the location of the reactive zones inside the materials determined by the isotopic experiments helps examining the role of the evolution of porosity and formation of alteration products within the material

  17. Development of new and improved labelling procedures for introducing isotopic hydrogen and carbon-11 into organic compounds

    International Nuclear Information System (INIS)

    Al-Qahtani, M.H.S.

    1999-10-01

    New and improved methods for introducing radioisotopic hydrogen (tritium) and carbon (positron-emitting short-lived carbon-11, t 1/ 2 = 20.4 min) into organic molecules for application in biological research have been explored. In Chapter 1 the applications of radioactive isotopes in biological and clinical research is surveyed, with particular emphasis on the value of β-emitting tritium and positron-emitting carbon-11. In Chapter 2 we report the use of the non-radioactive hydrogen isotope, deuterium, as a surrogate for tritium in the development of microwave-enhanced labelling procedures, based on catalytic hydrogen transfer to olefins (e.g. styrene, styrene derivatives, cinnamic acid and its derivatives). Hydrogen or deuterium donors (e.g. formate salts) were used alone or in combination with other sources (e.g. D 2 O). The method was found to give fully hydrogenated products using very short microwave irradiation times (∼ 2 min) and was highly reproducible. Importantly, the method is environmentally clean, as when extended to tritiated formates little or no radioactive waste is produced. In Chapter 3 we explored the labelling of CGP 62349 {3-[1-(R)-[3-(4-methoxybenzyl)phosphinyl-2-(S)-hydroxy-propyl- amino]ethyl]benzoic acid}, a γ-aminobutyric acid type B (GABA B ) receptor antagonist, with carbon-11 in order to provide a prospective radioligand for medical imaging with positron emission tomography (PET). Labelling agents, [ 11 C]iodomethane and [ 11 C]methyl triflate, prepared by improved methods, were used in the rapid methylation of desmethyl-CGP 62349. Substantially higher radiochemical yields (78%) of [ 11 C]CGP 62349 were achieved by the new methods compared to that produced in a previously published procedure (9%). In addition, the use of [ 11 C]methyl triflate rather than [ 11 C]iodomethane has the advantage of giving a high radiochemical yield and a lower amount of carrier. In Chapter 4 we report on the use of [ 11 C]carbon monoxide as a labelling

  18. Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism.

    Science.gov (United States)

    Steinhauser, Matthew L; Bailey, Andrew P; Senyo, Samuel E; Guillermier, Christelle; Perlstein, Todd S; Gould, Alex P; Lee, Richard T; Lechene, Claude P

    2012-01-15

    Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter, but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with submicrometre resolution. Here we apply MIMS to diverse organisms, including Drosophila, mice and humans. We test the 'immortal strand hypothesis', which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labelling mice with (15)N-thymidine from gestation until post-natal week 8, we find no (15)N label retention by dividing small intestinal crypt cells after a four-week chase. In adult mice administered (15)N-thymidine pulse-chase, we find that proliferating crypt cells dilute the (15)N label, consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human haematopoietic system. These studies show that MIMS provides high-resolution quantification of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.

  19. Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose.

    Directory of Open Access Journals (Sweden)

    Darren J Creek

    2015-03-01

    Full Text Available Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate.

  20. Simultaneous determination of the intravenous and oral pharmacokinetic parameters of D,L-verapamil using stable isotope-labelled verapamil.

    Science.gov (United States)

    Eichelbaum, M; Somogyi, A; von Unruh, G E; Dengler, H J

    1981-01-01

    Following i.v. administration, the plasma concentration-time curve of verapamil could best be described by either a mono- or biexponential equation. Total plasma clearance (1.26 1/min) approached liver blood flow (1.51/min), so it can be concluded that its clearance is liver blood flow-dependent. Although absorption was almost complete after oral administration, absolute bioavailability (20%) was low, due to extensive hepatic first-pass metabolism. The approach using stable isotope-labelled and unlabelled drug permits simultaneous administration by the intravascular and extravascular routes, thus allowing determination of absolute bioavailability in a single experiment.

  1. Synthesis of carbon-13 and carbon-14 labeled paldimycin tri-sodium salt

    International Nuclear Information System (INIS)

    Hsi, R.S.P.; Witz, D.F.; Visser, J.; Stolle, W.T.; Ditto, C.L.

    1989-01-01

    Carbon-14 labeled paldimycin trisodium salt was prepared by addition of N-acetyl-L-cysteine to [ 14 C]paulomycin, the radioactive antibiotic produced by fermentation of Streptomyces paulus in the presence of L-methionine labeled with carbon-14 in the S-methyl group. Carbon-13 nuclear magnetic resonance (NMR) spectra of paulomycin produced when the fermentation was carried out in the presence of L-[S-methyl- 13 C]methionine showed that the isotope incorporation had occurred specifically at the methoxy group of ring C, i.e., the 2-deoxy sugar portion of paulomycin. With sustained slow feed of labeled precursors during the optimum antibiotic production period, carbon-14 isotope yields of up to 17.5% with specific activity of up to 11.4 μCi per milligram of paulomycin, and carbon-13 isotope yields of up to 24% with 17-fold isotope enrichment over natural abundance, were achieved. (author)

  2. Selenium-75-labelled foliate compounds

    International Nuclear Information System (INIS)

    1974-01-01

    A saturation method to analyze a foliate is presented; it uses competitive reaction of the compound to be measured and of a radioactive-labelled version of this compound with a reagent specific to this compound present in insufficient quantity to combine with the whole of the compound and its labelled version, separation of the bound compound from its non-bound homologue and measurement of the radioactivity concentration in the bound compound, the non-bound compound or both. The radioactive isotope used in the labelled foliate is selenium 75 [fr

  3. Insulin signaling regulates fatty acid catabolism at the level of CoA activation.

    Directory of Open Access Journals (Sweden)

    Xiaojun Xu

    2012-01-01

    Full Text Available The insulin/IGF signaling pathway is a highly conserved regulator of metabolism in flies and mammals, regulating multiple physiological functions including lipid metabolism. Although insulin signaling is known to regulate the activity of a number of enzymes in metabolic pathways, a comprehensive understanding of how the insulin signaling pathway regulates metabolic pathways is still lacking. Accepted knowledge suggests the key regulated step in triglyceride (TAG catabolism is the release of fatty acids from TAG via the action of lipases. We show here that an additional, important regulated step is the activation of fatty acids for beta-oxidation via Acyl Co-A synthetases (ACS. We identify pudgy as an ACS that is transcriptionally regulated by direct FOXO action in Drosophila. Increasing or reducing pudgy expression in vivo causes a decrease or increase in organismal TAG levels respectively, indicating that pudgy expression levels are important for proper lipid homeostasis. We show that multiple ACSs are also transcriptionally regulated by insulin signaling in mammalian cells. In sum, we identify fatty acid activation onto CoA as an important, regulated step in triglyceride catabolism, and we identify a mechanistic link through which insulin regulates lipid homeostasis.

  4. Combining Solvent Isotope Effects with Substrate Isotope Effects in Mechanistic Studies of Alcohol and Amine Oxidation by Enzymes*

    Science.gov (United States)

    Fitzpatrick, Paul F.

    2014-01-01

    Oxidation of alcohols and amines is catalyzed by multiple families of flavin-and pyridine nucleotide-dependent enzymes. Measurement of solvent isotope effects provides a unique mechanistic probe of the timing of the cleavage of the OH and NH bonds, necessary information for a complete description of the catalytic mechanism. The inherent ambiguities in interpretation of solvent isotope effects can be significantly decreased if isotope effects arising from isotopically labeled substrates are measured in combination with solvent isotope effects. The application of combined solvent and substrate (mainly deuterium) isotope effects to multiple enzymes is described here to illustrate the range of mechanistic insights that such an approach can provide. PMID:25448013

  5. Studies of Human 2,4-Dienoyl CoA Reductase Shed New Light on Peroxisomal β-Oxidation of Unsaturated Fatty Acids

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Tian; Wu, Dong; Ding, Wei; Wang, Jiangyun; Shaw, Neil; Liu, Zhi-Jie [Nankai; (Chinese Aca. Sci.)

    2012-10-15

    Peroxisomes play an essential role in maintaining fatty acid homeostasis. Although mitochondria are also known to participate in the catabolism of fatty acids via β-oxidation, differences exist between the peroxisomal and mitochondrial β-oxidation. Only peroxisomes, but not mitochondrion, can shorten very long chain fatty acids. Here, we describe the crystal structure of a ternary complex of peroxisomal 2,4-dienoyl CoA reductases (pDCR) with hexadienoyl CoA and NADP, as a prototype for comparison with the mitochondrial 2,4-dienoyl CoA reductase (mDCR) to shed light on the differences between the enzymes from the two organelles at the molecular level. Unexpectedly, the structure of pDCR refined to 1.84 Å resolution reveals the absence of the tyrosine-serine pair seen in the active site of mDCR, which together with a lysine and an asparagine have been deemed a hallmark of the SDR family of enzymes. Instead, aspartate hydrogen-bonded to the Cα hydroxyl via a water molecule seems to perturb the water molecule for protonation of the substrate. Our studies provide the first structural evidence for participation of water in the DCR-catalyzed reactions. Biochemical studies and structural analysis suggest that pDCRs can catalyze the shortening of six-carbon-long substrates in vitro. However, the Km values of pDCR for short chain acyl CoAs are at least 6-fold higher than those for substrates with 10 or more aliphatic carbons. Unlike mDCR, hinge movements permit pDCR to process very long chain polyunsaturated fatty acids.

  6. Isotope release cytotoxicity assay applicable to human tumors: the use of 111-indium

    Energy Technology Data Exchange (ETDEWEB)

    Frost, P; Wiltrout, R; Maciorowski, Z; Rose, N R

    1977-01-01

    We have demonstrated that human tumors can be labelled efficiently with the 111indium-oxine chelate. Subsequently, this isotope can be released by cytotoxic lymphoid cells. Both natural and induced cytotoxicity can be demonstrated utilizing this isotope release method. Because of the slow spontaneous release of 111indium and its efficient labelling of human tumor cells, this isotope release assay can be utilized in long-term cytotoxic assays in the study of human tumor immunology.

  7. Laboratory of radioisotopes of IOCB ASCR - recent results of labeling by 3H and 125I

    Czech Academy of Sciences Publication Activity Database

    Elbert, Tomáš; Veselá, Iva

    2009-01-01

    Roč. 52, 7/8 (2009), s. 253-254 ISSN 0362-4803. [15th Workshop of the International Isotope Society - Central European Division: The synthesis and applications of isotopes and isotopically labelled compounds. 12.06.2009-13.06.2008, Bad Soden] Institutional research plan: CEZ:AV0Z40550506 Keywords : tritium * 125-I labeled peptides Subject RIV: CC - Organic Chemistry

  8. Stable isotopes - separation and application

    International Nuclear Information System (INIS)

    Lockhart, I.M.

    1980-01-01

    In this review, methods used for the separation of stable isotopes ( 12 C, 13 C, 14 N, 15 N, 16 O, 17 O, 18 O, 34 S) will be described. The synthesis of labelled compounds, techniques for detection and assay, and areas of application will also be discussed. Particular attention will be paid to the isotopes of carbon, nitrogen, and oxygen; to date, sulphur isotopes have only assumed a minor role. The field of deuterium chemistry is too extensive for adequate treatment; it will therefore be essentially excluded. (author)

  9. Radiation oxidation of polypropylene: A solid-state 13C NMR study using selective isotopic labeling

    International Nuclear Information System (INIS)

    Mowery, Daniel M.; Assink, Roger A.; Derzon, Dora K.; Klamo, Sara B.; Bernstein, Robert; Clough, Roger L.

    2007-01-01

    Polypropylene samples, in which the three different carbon atoms along the chain were selectively labeled with carbon-13, were subjected to radiation under inert and air atmospheres, and to post-irradiation exposure in air at various temperatures. By using solid-state 13 C NMR measurements at room temperature, we have been able to identify and quantify the oxidation products. The isotopic labeling provides insight into chemical reaction mechanisms, since oxidation products can be traced back to their positions of origin on the macromolecule. The major products include peroxides and alcohols, both formed at tertiary carbon sites along the chain. Other products include methyl ketones, acids, esters, peresters, and hemiketals formed from reaction at the tertiary carbon, together with in-chain ketones and esters from reaction at the secondary chain carbon. No evidence is found of products arising from reactions at the methyl side chain. Significant temperature-dependent differences are apparent; for example much higher yields of chain-end methyl ketones, which are the indicator product of chain scission, are generated for both elevated temperature irradiation and for post-irradiation treatment at elevated temperatures. Time-dependent plots of yields of the various oxidation products have been obtained under a wide range of conditions, including the post-irradiation oxidation of a sample at room temperature in air that has been monitored for 2 years. Radiation-oxidation products of polypropylene are contrasted to products measured for 13 C-labeled polyethylene in an earlier investigation: the peroxides formed in irradiated polypropylene are remarkably longer lived, the non-peroxidic products are significantly different, and the overall ratios of oxidation products in polypropylene change relatively little as a function of the extent of oxidation

  10. Synthesis of stereoarray isotope labeled (SAIL) lysine via the "head-to-tail" conversion of SAIL glutamic acid.

    Science.gov (United States)

    Terauchi, Tsutomu; Kamikawai, Tomoe; Vinogradov, Maxim G; Starodubtseva, Eugenia V; Takeda, Mitsuhiro; Kainosho, Masatsune

    2011-01-07

    A stereoarray isotope labeled (SAIL) lysine, (2S,3R,4R,5S,6R)-[3,4,5,6-(2)H(4);1,2,3,4,5,6-(13)C(6);2,6-(15)N(2)]lysine, was synthesized by the "head-to-tail" conversion of SAIL-Glu, (2S,3S,4R)-[3,4-(2)H(2);1,2,3,4,5-(13)C(5);2-(15)N]glutamic acid, with high stereospecificities for all five chiral centers. With the SAIL-Lys in hand, the unambiguous simultaneous stereospecific assignments were able to be established for each of the prochiral protons within the four methylene groups of the Lys side chains in proteins.

  11. Fatty acids labelled in the. omega. -position with iodine isotopes

    Energy Technology Data Exchange (ETDEWEB)

    Mathieu, J.P.; Busquet, G.; Comet, M. (Universite Scientifique et Medicale de Grenoble, 38 - La Tronche (France)); Riche, F.; Vidal, M. (Laboratoire d' Etudes Dynamiques et Structurales de la Selectivite, 38 - Grenoble (France)); Coornaert, S.; Bardy, A. (CEA, Centre de Saclay, 91 - Gif-sur-Yvette (France)); Godart, J. (Institut des Sciences Nucleaires, 38 - Grenoble (France))

    1982-01-01

    The synthesis of saturated acetylenic and olefinic (Z or E) ..omega..-iodinated fatty acids has been carried out and their labelling with iodine-131 or 123 by exchange I/sup -/, *I/sup -/ has been studied. The influence of several parameters -water and fatty acid concentrations, specific activity, labelling solution acidity, iodine carrier presence- on this exchange reaction has been noted, enabling experimental conditions to be defined that produce labelling yields of greater than 95%. These results should lead to widespread clinical use of iodine labelled fatty acids.

  12. Synthesis of isotopically labelled angiotensin II receptor antagonist GR138950X

    International Nuclear Information System (INIS)

    Carr, R.M.; Cable, K.M.; Newman, J.J.; Sutherland, D.R.

    1996-01-01

    Syntheses of [ 13 C] and [ 14 C]-labelled versions of angiotensin II receptor antagonist GR138950X, labelled in the imidazole carboxamide residue, are described. These involved preparation of an iodoimidazole substrate by a novel iododecarboxylation procedure, followed by cyanation with a mixture of carbon-labelled potassium cyanide and copper (l) iodide in DMF at high temperature. The preparation of a mass-labelled (M+5) version of GR138950X is also described. This involved the synthesis of an [ 13 C 3 , 15 N 2 ]-labelled imidazole from a 1,2,3-tricarbonyl compound, [ 13 C 3 ]propionaldehyde and [ 15 N]ammonia. The labelled imidazole was further elaborated into multiply-labelled GR138950X. (Author)

  13. Voltammetry coupled to mass spectrometry in the presence of isotope {sup 18}O labeled water for the prediction of oxidative transformation pathways of activated aromatic ethers: Acebutolol

    Energy Technology Data Exchange (ETDEWEB)

    Bussy, Ugo; Tea, Illa [LUNAM Université de Nantes, CNRS, Chimie et Interdisciplinarité: Synthèse, Analyse et Modélisation (CEISAM), UMR 6230, 2 rue de la Houssinière, BP 92208, F-44322 Nantes cedex 3 (France); Ferchaud-Roucher, Véronique; Krempf, Michel [Université de Nantes, Plateforme Spectrométrie de Masse, CRNH, SFR Santé F. Bonamy, Institut du Thorax, UMR S1087, IRT-UN, BP 70721, 8 Quai Moncousu, 44007 Nantes cedex 1 (France); Silvestre, Virginie; Galland, Nicolas [LUNAM Université de Nantes, CNRS, Chimie et Interdisciplinarité: Synthèse, Analyse et Modélisation (CEISAM), UMR 6230, 2 rue de la Houssinière, BP 92208, F-44322 Nantes cedex 3 (France); Jacquemin, Denis [LUNAM Université de Nantes, CNRS, Chimie et Interdisciplinarité: Synthèse, Analyse et Modélisation (CEISAM), UMR 6230, 2 rue de la Houssinière, BP 92208, F-44322 Nantes cedex 3 (France); Institut Universitaire de France, 103, Boulevard Saint-Michel, 75005 Cedex 5 France (France); Andresen-Bergström, Moa; Jurva, Ulrik [CVGI iMed DMPK, AstraZeneca R and D Mölndal, Mölndal (Sweden); and others

    2013-01-31

    Highlights: ► Voltammetry coupled to mass spectrometry method as a useful tool for on-line predictions of electrochemical transformations. ► Evidence of the O-dealkoxylation reaction pathway of acebutolol in the presence of labeled water. ► New approach for on line EC-MS applications. -- Abstract: The coupling between an electrochemical cell (EC) and a mass spectrometer (MS) is a useful screening tool (EC-MS) to study the oxidative transformation pathways of various electroactive species. For that purpose, we showed that the EC-MS method, carried out in the presence and absence of isotope {sup 18}O labeled water leads not only to a fast identification of oxidation products but also leads to a fast elucidation of the mechanism pathway reaction. We examined herein the case of the electrochemical hydrolysis of activated aromatic ether. Acebutolol (β-blockers) was selected herein as model of activated aromatic ether, and its electrochemical oxidation was examined in both the presence and absence of isotope {sup 18}O labeled water. To elucidate electrochemical hydrolysis pathway reaction: O-dealkylation or O-dealkoxylation, our approach was used to prove its applicability. The electrochemical oxidation mechanism was then elucidated showing an O-dealkoxylation reaction. In addition, density functional theory (DFT) calculations fully support the experimental conclusions.

  14. Isotope labeling-based quantitative proteomics of developing seeds of castor oil seed (Ricinus communis L.)

    DEFF Research Database (Denmark)

    Nogueira, Fábio C S; Palmisano, Giuseppe; Schwämmle, Veit

    2013-01-01

    In this study, we used a mass spectrometry-based quantification approach employing isotopic (ICPL) and isobaric (iTRAQ) labeling to investigate the pattern of protein deposition during castor oil seed (Ricinus communis L.) development, including that of proteins involved in fatty acid metabolism...... give important insights into certain aspects of the biology of castor oil seed development such as carbon flow, anabolism, and catabolism of fatty acid and the pattern of deposition of SSPs, toxins, and allergens such as ricin and 2S albumins. We also found, for the first time, some genes of SSP......, seed-storage proteins (SSPs), toxins, and allergens. Additionally, we have used off-line hydrophilic interaction chromatography (HILIC) as a step of peptide fractionation preceding the reverse-phase nanoLC coupled to a LTQ Orbitrap. We were able to identify a total of 1875 proteins, and from these 1748...

  15. Isotopic evaluations of dynamic and plant uptake of N in soil amended with 15N-labelled sewage sludge

    International Nuclear Information System (INIS)

    Kchaou, R.; Khelil, M. N.; Rejeb, S.; Gharbi, F.; Henchi, B.; Hernandez, T.; Destain, J. P.

    2010-01-01

    Field experiments were conducted to evaluate the use of a novel 15N isotope technique for comparing the dynamics of N derived from sewage sludge applied to sorghum to the dynamics of N derived from the commercial fertilizer, urea. The treatments included a control, sludge applied at three rates (3, 6 and 9 t/ha, or 113, 226 and 338 kg N/ha) and N-urea applied at three rates (150, 250 and 350 kg N/ha). Recovery of 15N -labelled sludge was similar for the different nitrogen rates applied , with a mean value of 27%. However, the recovery of 15N -urea decreased as the rate of N application increased (from 38% to 27%). Approximately 22% and 19% of the 15N from sludge and urea, respectively, remained in the 0-60 cm layer of soil, most of which was present in the 0-20 cm layer. Furthermore, losses of 15N -labelled fertilizer were not affected by the N fertilization source, and the greatest losses, which were measured in response to the highest N application rate, were 59%. (authors)

  16. Deuterium- and tritium-labelled compounds. Applications in the life sciences

    International Nuclear Information System (INIS)

    Atzrodt, Jens; Derdau, Volker; Kerr, William J.; Reid, Marc

    2018-01-01

    Hydrogen isotopes are unique tools for identifying and understanding biological and chemical processes. Hydrogen isotope labelling allows for the traceless and direct incorporation of an additional mass or radioactive tag into an organic molecule with almost no changes in its chemical structure, physical properties, or biological activity. Using deuterium-labelled isotopologues to study the unique mass-spectrometric patterns generated from mixtures of biologically relevant molecules drastically simplifies analysis. Such methods are now providing unprecedented levels of insight in a wide and continuously growing range of applications in the life sciences and beyond. Tritium ( 3 H), in particular, has seen an increase in utilization, especially in pharmaceutical drug discovery. The efforts and costs associated with the synthesis of labelled compounds are more than compensated for by the enhanced molecular sensitivity during analysis and the high reliability of the data obtained. In this review, advances in the application of hydrogen isotopes in the life sciences are described. (copyright 2018 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim)

  17. IRMS detection of testosterone manipulated with {sup 13}C labeled standards in human urine by removing the labeled {sup 13}C

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jingzhu, E-mail: wangjingzhu@chinada.cn [National Anti-Doping Laboratory, China Anti-Doping Agency, Beijing (China); Yang, Rui [Sport Science College, Beijing Sport University Beijing, Beijing (China); Yang, Wenning [School of Pharmacy, Beijing University of Chinese Medicine, Beijing (China); Liu, Xin; Xing, Yanyi; Xu, Youxuan [National Anti-Doping Laboratory, China Anti-Doping Agency, Beijing (China)

    2014-12-10

    Highlights: • {sup 13}C labeled testosterone can be used to adjust the isotope ratio of testosterone. • The novel testosterone cannot be detected by the regular IRMS method in doping test. • A method was explored to remove the labeled {sup 13}C. • The established method can be used to detect the manipulated testosterone. - Abstract: Isotope ratio mass spectrometry (IRMS) is applied to confirm testosterone (T) abuse by determining the carbon isotope ratios (δ{sup 13}C value). However, {sup 13}C labeled standards can be used to control the δ{sup 13}C value and produce manipulated T which cannot be detected by the current method. A method was explored to remove the {sup 13}C labeled atom at C-3 from the molecule of androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ{sup 13}C values between Andro and ANAD (Δδ{sup 13}C{sub Andro–ANAD}, ‰) would change significantly in case manipulated T is abused. Twenty-one volunteers administered T manipulated with different {sup 13}C labeled standards. The collected urine samples were analyzed with the established method, and the maximum value of Δδ{sup 13}C{sub Andro–ANAD} post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ{sup 13}C{sub Andro–ANAD} for positive result was suggested as 1.2‰. The developed method could be used to detect T manipulated with 3-{sup 13}C labeled standards.

  18. ICT: isotope correction toolbox.

    Science.gov (United States)

    Jungreuthmayer, Christian; Neubauer, Stefan; Mairinger, Teresa; Zanghellini, Jürgen; Hann, Stephan

    2016-01-01

    Isotope tracer experiments are an invaluable technique to analyze and study the metabolism of biological systems. However, isotope labeling experiments are often affected by naturally abundant isotopes especially in cases where mass spectrometric methods make use of derivatization. The correction of these additive interferences--in particular for complex isotopic systems--is numerically challenging and still an emerging field of research. When positional information is generated via collision-induced dissociation, even more complex calculations for isotopic interference correction are necessary. So far, no freely available tools can handle tandem mass spectrometry data. We present isotope correction toolbox, a program that corrects tandem mass isotopomer data from tandem mass spectrometry experiments. Isotope correction toolbox is written in the multi-platform programming language Perl and, therefore, can be used on all commonly available computer platforms. Source code and documentation can be freely obtained under the Artistic License or the GNU General Public License from: https://github.com/jungreuc/isotope_correction_toolbox/ {christian.jungreuthmayer@boku.ac.at,juergen.zanghellini@boku.ac.at} Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. Uses of stable isotopes

    International Nuclear Information System (INIS)

    Axente, Damian

    1998-01-01

    The most important fields of stable isotope use with examples are presented. These are: 1. Isotope dilution analysis: trace analysis, measurements of volumes and masses; 2. Stable isotopes as tracers: transport phenomena, environmental studies, agricultural research, authentication of products and objects, archaeometry, studies of reaction mechanisms, structure and function determination of complex biological entities, studies of metabolism, breath test for diagnostic; 3. Isotope equilibrium effects: measurement of equilibrium effects, investigation of equilibrium conditions, mechanism of drug action, study of natural processes, water cycle, temperature measurements; 4. Stable isotope for advanced nuclear reactors: uranium nitride with 15 N as nuclear fuel, 157 Gd for reactor control. In spite of some difficulties of stable isotope use, particularly related to the analytical techniques, which are slow and expensive, the number of papers reporting on this subject is steadily growing as well as the number of scientific meetings organized by International Isotope Section and IAEA, Gordon Conferences, and regional meeting in Germany, France, etc. Stable isotope application development on large scale is determined by improving their production technologies as well as those of labeled compound and the analytical techniques. (author)

  20. Isotopically labeled sulfur compounds and synthetic selenium and tellurium analogues to study sulfur metabolism in marine bacteria

    Directory of Open Access Journals (Sweden)

    Nelson L. Brock

    2013-05-01

    Full Text Available Members of the marine Roseobacter clade can degrade dimethylsulfoniopropionate (DMSP via competing pathways releasing either methanethiol (MeSH or dimethyl sulfide (DMS. Deuterium-labeled [2H6]DMSP and the synthetic DMSP analogue dimethyltelluriopropionate (DMTeP were used in feeding experiments with the Roseobacter clade members Phaeobacter gallaeciensis DSM 17395 and Ruegeria pomeroyi DSS-3, and their volatile metabolites were analyzed by closed-loop stripping and solid-phase microextraction coupled to GC–MS. Feeding experiments with [2H6]DMSP resulted in the incorporation of a deuterium label into MeSH and DMS. Knockout of relevant genes from the known DMSP demethylation pathway to MeSH showed in both species a residual production of [2H3]MeSH, suggesting that a second demethylation pathway is active. The role of DMSP degradation pathways for MeSH and DMS formation was further investigated by using the synthetic analogue DMTeP as a probe in feeding experiments with the wild-type strain and knockout mutants. Feeding of DMTeP to the R. pomeroyi knockout mutant resulted in a diminished, but not abolished production of demethylation pathway products. These results further corroborated the proposed second demethylation activity in R. pomeroyi. Isotopically labeled [2H3]methionine and 34SO42−, synthesized from elemental 34S8, were tested to identify alternative sulfur sources besides DMSP for the MeSH production in P. gallaeciensis. Methionine proved to be a viable sulfur source for the MeSH volatiles, whereas incorporation of labeling from sulfate was not observed. Moreover, the utilization of selenite and selenate salts by marine alphaproteobacteria for the production of methylated selenium volatiles was explored and resulted in the production of numerous methaneselenol-derived volatiles via reduction and methylation. The pathway of selenate/selenite reduction, however, proved to be strictly separated from sulfate reduction.

  1. DNA stable-isotope probing (DNA-SIP).

    Science.gov (United States)

    Dunford, Eric A; Neufeld, Josh D

    2010-08-02

    DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

  2. Labelled compounds for agrochemical residue studies in developing countries

    International Nuclear Information System (INIS)

    1977-01-01

    Potential applications of stable and radioactive isotopic tracers for assessing undesirable contaminants in agriculture, fisheries and food are discussed as related to developing countries. Sources and types of residues are considered, and their local implications; also, the availability of suitably labelled compounds, including possible international cooperation to facilitate more centralized and economic preparation, and the distribution of labelled intermediates and compounds for use by local scientists. The provision of training courses and their syllabus are reviewed. Experience in the Joint FAO/IAEA chemical residue and pollution programme has indicated a need for longer-lived radioisotopically labelled pesticides (insecticides, acaricides, fungicides, herbicides, fumigants, etc.) for studying their behaviour. 15 N-, 13 C- or 2 H-labelled fertilizers and fertilizer additives such as nitrification inhibitors will shortly be needed, for studying the behaviour of fertilizer nitrogen residues, and their regulation and conservation, under conditions prevailing in the developing countries. Compounds labelled with stable isotopes are considered particularly valuable under field conditions. The report reviews the present situation and presents specific recommendations to the Directors General of FAO and IAEA

  3. Synthesis of analogues of purine nucleotides selectively labeled by tritium on the C-8 of the purine ring and evaluation of the stability of tritium label

    Czech Academy of Sciences Publication Activity Database

    Elbert, Tomáš

    2010-01-01

    Roč. 53, č. 3 (2010), s. 156-157 ISSN 0362-4803. [Workshop of the International Isotope Society - Central European Division. The Synthesis and applications of Isotopes and Isotopically Labelled Compounds /16./. 01.10.2009-02.10.2009, Bad Soden] Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleotide analogues * tritium Subject RIV: CC - Organic Chemistry

  4. Pyrolysis of Cigarette Ingredients Labelled with Stable Isotopes

    Directory of Open Access Journals (Sweden)

    Stotesbury S

    2014-12-01

    Full Text Available It is important to know how tobacco additives behave when cigarettes are smoked, whether they transfer intact to the smoke or whether there is any decomposition during smoking. Pyrolysis-GC-MS is a technique that can be focussed upon the effects of combustion from a single material free from interference from the complex mixture of different components present in the smoke. However, because pyrolysis is a model technique, the results need to be validated by comparison with cigarette smoke chemistry. In a previous paper we presented such a method for modelling the smoke chemistry from a burning cigarette using pyrolysis-GC-MS. The transfer and the extent of degradation of anisole, p-anisaldehyde, benzaldehyde, isoamylisovalerate, methyl trans-cinnamate and vanillin within a burning cigarette were estimated using this pyrolysis method. When these data were compared with results from smoke studies from 14C-analogues of the materials, the high levels of transfer predicted by pyrolysis were found to be generally consistent with the smoke chemistry data. However, there were still two outstanding issues. Firstly, there was some ambiguity in the labelled study about whether vanillin actually transferred without degradation or not. Furthermore, the results from the 14C-labelled study showed a greater extent of degradation for p-anisaldehyde than that indicated from the pyrolysis experiments. The purpose of the current study was to present some new information obtained to address these questions by better understanding the effect upon the smoke chemistry from adding vanillin and p-anisaldehyde, and the relationship between the smoke chemistry and the pyrolysis results. Components were identified in the smoke from cigarettes loaded with p-anisaldehyde and vanillin labelled with 18O and 13C. The extent of degradation from each additive was estimated by identifying labelled degradation products in the smoke. Because there was a clear distinction between the

  5. Trends in the use of stable isotopes in biochemistry and pharmacology

    International Nuclear Information System (INIS)

    Matwiyoff, N.A.; Walker, T.E.

    1977-01-01

    Recent trends in the use of the stable isotopes 13 C, 15 N and 18 O in biochemistry and pharmacology are reviewed with emphasis on the studies that have employed nuclear magnetic resonance (nmr) spectroscopy and mass spectrometry as analytical techniques. Pharmacological studies with drugs and other compounds labelled with stable isotopes have developed in parallel with the rapid progress in the enhancement of sensitivity and selectivity of gas chromatography - mass spectrometric analyses, and have been directed largely to an evaluation of pharmako-kinetics and drug metabolic pathways. In these studies, illustrated with selected samples, isotopically labelled compounds have been used to advantage as internal standards for the mass spectrometric analyses and as in vivo tracers for metabolites. In the broader discipline of biochemistry, stable isotopes and isotopically labelled compounds have been used increasingly in conjuction with both nmr spectroscopy and mass spectrometry in tracer and structural studies. The more recent trends in the use of stable isotopes in these biochemical studies are discussed in the context of the improvements in analytical techniques. Specific examples will be drawn from investigations of the biosynthesis of natural products by micro-organisms; the protein, fat and carbohydrate fluxes in humans; and the structure and function of enzymes, membranes and other macro-molecular assemblages. The potential for the future development of stable isotopes in biochemistry and pharmacology are considered briefly, together with some of the problems that must be solved if their considerable potential is to be realized. (author)

  6. Generation of Stoichiometric Ethylene and Isotopic Derivatives and Application in Transition Metal-Catalyzed Vinylation and Enyne Metathesis

    DEFF Research Database (Denmark)

    Min, Geanna; Bjerglund, Klaus Meier; Kramer, Søren

    2013-01-01

    Ethylene is one of the most important building blocks in industry for the production of polymers and commodity chemicals. 13C- and D-isotope-labeled ethylenes are also valuable reagents with applications ranging from polymer-structure determination, reaction-mechanism elucidation to the preparation...... of more complex isotopically labeled compounds. However, these isotopic derivatives are expensive, and are flammable gases, which are difficult to handle. We have developed a method for the controlled generation of ethylene and its isotopic variants including, for the first time, fully isotopically...... labeled ethylene, from simple alkene precursors by using Ru catalysis. Applying a two-chamber reactor allows both the synthesis of ethylene and its immediate consumption in a chemical transformation permitting reactions to be performed with only stoichiometric amounts of this two carbon olefin...

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

    Science.gov (United States)

    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.

  8. Differential isotope dansylation labeling combined with liquid chromatography mass spectrometry for quantification of intact and N-terminal truncated proteins

    International Nuclear Information System (INIS)

    Tang, Yanan; Li, Liang

    2013-01-01

    Graphical abstract: -- Highlights: •LC–MS was developed for quantifying protein mixtures containing both intact and N-terminal truncated proteins. • 12 C 2 -Dansylation of the N-terminal amino acid of proteins was done first, followed by microwave-assisted acid hydrolysis. •The released 12 C 2 -dansyl labeled N-terminal amino acid was quantified using 13 C 2 -dansyl labeled amino acid standards. •The method provided accurate and precise results for quantifying intact and N-terminal truncated proteins within 8 h. -- Abstract: The N-terminal amino acids of proteins are important structure units for maintaining the biological function, localization, and interaction networks of proteins. Under different biological conditions, one or several N-terminal amino acids could be cleaved from an intact protein due to processes, such as proteolysis, resulting in the change of protein properties. Thus, the ability to quantify the N-terminal truncated forms of proteins is of great importance, particularly in the area of development and production of protein-based drugs where the relative quantity of the intact protein and its truncated form needs to be monitored. In this work, we describe a rapid method for absolute quantification of protein mixtures containing intact and N-terminal truncated proteins. This method is based on dansylation labeling of the N-terminal amino acids of proteins, followed by microwave-assisted acid hydrolysis of the proteins into amino acids. It is shown that dansyl labeled amino acids are stable in acidic conditions and can be quantified by liquid chromatography mass spectrometry (LC–MS) with the use of isotope analog standards

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

    NARCIS (Netherlands)

    Kloppenburg, Wybe; Wolthers, BG; Stellaard, F; Elzinga, H; Tepper, T; deJong, PE; Huisman, RM

    1. Stable urea isotopes can be used to study urea kinetics in humans, The use of stable urea isotopes far 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,

  10. Automated NMR structure determination of stereo-array isotope labeled ubiquitin from minimal sets of spectra using the SAIL-FLYA system

    Energy Technology Data Exchange (ETDEWEB)

    Ikeya, Teppei [Goethe University Frankfurt am Main, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance (Germany); Takeda, Mitsuhiro; Yoshida, Hitoshi; Terauchi, Tsutomu; Jee, Jun-Goo; Kainosho, Masatsune [Tokyo Metropolitan University, Graduate School of Science (Japan)], E-mail: kainosho@nmr.chem.metro-u.ac.jp; Guentert, Peter [Goethe University Frankfurt am Main, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance (Germany)], E-mail: guentert@em.uni-frankfurt.de

    2009-08-15

    Stereo-array isotope labeling (SAIL) has been combined with the fully automated NMR structure determination algorithm FLYA to determine the three-dimensional structure of the protein ubiquitin from different sets of input NMR spectra. SAIL provides a complete stereo- and regio-specific pattern of stable isotopes that results in sharper resonance lines and reduced signal overlap, without information loss. Here we show that as a result of the superior quality of the SAIL NMR spectra, reliable, fully automated analyses of the NMR spectra and structure calculations are possible using fewer input spectra than with conventional uniformly {sup 13}C/{sup 15}N-labeled proteins. FLYA calculations with SAIL ubiquitin, using a single three-dimensional 'through-bond' spectrum (and 2D HSQC spectra) in addition to the {sup 13}C-edited and {sup 15}N-edited NOESY spectra for conformational restraints, yielded structures with an accuracy of 0.83-1.15 A for the backbone RMSD to the conventionally determined solution structure of SAIL ubiquitin. NMR structures can thus be determined almost exclusively from the NOESY spectra that yield the conformational restraints, without the need to record many spectra only for determining intermediate, auxiliary data of the chemical shift assignments. The FLYA calculations for this report resulted in 252 ubiquitin structure bundles, obtained with different input data but identical structure calculation and refinement methods. These structures cover the entire range from highly accurate structures to seriously, but not trivially, wrong structures, and thus constitute a valuable database for the substantiation of structure validation methods.

  11. Automated NMR structure determination of stereo-array isotope labeled ubiquitin from minimal sets of spectra using the SAIL-FLYA system

    International Nuclear Information System (INIS)

    Ikeya, Teppei; Takeda, Mitsuhiro; Yoshida, Hitoshi; Terauchi, Tsutomu; Jee, Jun-Goo; Kainosho, Masatsune; Guentert, Peter

    2009-01-01

    Stereo-array isotope labeling (SAIL) has been combined with the fully automated NMR structure determination algorithm FLYA to determine the three-dimensional structure of the protein ubiquitin from different sets of input NMR spectra. SAIL provides a complete stereo- and regio-specific pattern of stable isotopes that results in sharper resonance lines and reduced signal overlap, without information loss. Here we show that as a result of the superior quality of the SAIL NMR spectra, reliable, fully automated analyses of the NMR spectra and structure calculations are possible using fewer input spectra than with conventional uniformly 13 C/ 15 N-labeled proteins. FLYA calculations with SAIL ubiquitin, using a single three-dimensional 'through-bond' spectrum (and 2D HSQC spectra) in addition to the 13 C-edited and 15 N-edited NOESY spectra for conformational restraints, yielded structures with an accuracy of 0.83-1.15 A for the backbone RMSD to the conventionally determined solution structure of SAIL ubiquitin. NMR structures can thus be determined almost exclusively from the NOESY spectra that yield the conformational restraints, without the need to record many spectra only for determining intermediate, auxiliary data of the chemical shift assignments. The FLYA calculations for this report resulted in 252 ubiquitin structure bundles, obtained with different input data but identical structure calculation and refinement methods. These structures cover the entire range from highly accurate structures to seriously, but not trivially, wrong structures, and thus constitute a valuable database for the substantiation of structure validation methods

  12. Automated NMR structure determination of stereo-array isotope labeled ubiquitin from minimal sets of spectra using the SAIL-FLYA system.

    Science.gov (United States)

    Ikeya, Teppei; Takeda, Mitsuhiro; Yoshida, Hitoshi; Terauchi, Tsutomu; Jee, Jun-Goo; Kainosho, Masatsune; Güntert, Peter

    2009-08-01

    Stereo-array isotope labeling (SAIL) has been combined with the fully automated NMR structure determination algorithm FLYA to determine the three-dimensional structure of the protein ubiquitin from different sets of input NMR spectra. SAIL provides a complete stereo- and regio-specific pattern of stable isotopes that results in sharper resonance lines and reduced signal overlap, without information loss. Here we show that as a result of the superior quality of the SAIL NMR spectra, reliable, fully automated analyses of the NMR spectra and structure calculations are possible using fewer input spectra than with conventional uniformly 13C/15N-labeled proteins. FLYA calculations with SAIL ubiquitin, using a single three-dimensional "through-bond" spectrum (and 2D HSQC spectra) in addition to the 13C-edited and 15N-edited NOESY spectra for conformational restraints, yielded structures with an accuracy of 0.83-1.15 A for the backbone RMSD to the conventionally determined solution structure of SAIL ubiquitin. NMR structures can thus be determined almost exclusively from the NOESY spectra that yield the conformational restraints, without the need to record many spectra only for determining intermediate, auxiliary data of the chemical shift assignments. The FLYA calculations for this report resulted in 252 ubiquitin structure bundles, obtained with different input data but identical structure calculation and refinement methods. These structures cover the entire range from highly accurate structures to seriously, but not trivially, wrong structures, and thus constitute a valuable database for the substantiation of structure validation methods.

  13. Molecular analysis of virulent genes (coa and spa) of staphylococcus aureus involved in natural cases of bovine mastitis

    International Nuclear Information System (INIS)

    Khan, A.; Javed, M.T.; Mahmood, F.; Hussain, R.

    2013-01-01

    The present study was undertaken to determine the distribution and genotypic characteristics of Staphylococcus aureus isolates recovered from naturally occurring mastitis in cattle and buffaloes. For this purpose a total of 1445 lactating cattle (653) and buffaloes (792) present at two experimental livestock farms Okara (Bahadarnagar) and Sahiwal (Qadiarabad), in and around district Faisalabad and slaughtered at an abattoir due to low milk yield and were screened for mastitis. California Mastitis Test (CMT) was used to detect sub clinical mastitis. The positive quarter milk samples were collected for culturing of S. aureus isolates. taphylococcus aureus isolates were identified on the basis of growth features, biochemical characteristics, coagulase test and as well as amplification of coagulase (coa) and spa (spa-X) genes specific to its virulence. S. aureus isolates (n=265) were characterized by Polymerase chain reaction to determine the frequency of coagulase (coa) and spa (spa-X) genes. From these isolates the amplification of the coagulase (coa) gene yielded three different PCR products approximately 204bp to 490bp while spa (spa-X) gene produced five different products ranging in size from 190bp to 320bp. PCR revealed that from all the coagulase positive S. aureus isolates 261(98.5%) had spa (spa-X) gene. The results of the present study indicated that S. aureus isolates recovered from bovine mastitis were genetically different within and among the various herds which may provide essential and valuable strategies to control staphylococcal infections in future. (author)

  14. Molecular analysis of virulent genes (coa and spa) of staphylococcus aureus involved in natural cases of bovine mastitis

    Energy Technology Data Exchange (ETDEWEB)

    Khan, A.; Javed, M. T.; Mahmood, F. [University of Agriculture, Faisalabad (Pakistan). Dept. of Pathology; Hussain, R. [The Islamia Univ. of Bahawalpur, Pakistan (Pakistan). Dept. of Veterinary and Animal Sciences

    2013-12-15

    The present study was undertaken to determine the distribution and genotypic characteristics of Staphylococcus aureus isolates recovered from naturally occurring mastitis in cattle and buffaloes. For this purpose a total of 1445 lactating cattle (653) and buffaloes (792) present at two experimental livestock farms Okara (Bahadarnagar) and Sahiwal (Qadiarabad), in and around district Faisalabad and slaughtered at an abattoir due to low milk yield and were screened for mastitis. California Mastitis Test (CMT) was used to detect sub clinical mastitis. The positive quarter milk samples were collected for culturing of S. aureus isolates. taphylococcus aureus isolates were identified on the basis of growth features, biochemical characteristics, coagulase test and as well as amplification of coagulase (coa) and spa (spa-X) genes specific to its virulence. S. aureus isolates (n=265) were characterized by Polymerase chain reaction to determine the frequency of coagulase (coa) and spa (spa-X) genes. From these isolates the amplification of the coagulase (coa) gene yielded three different PCR products approximately 204bp to 490bp while spa (spa-X) gene produced five different products ranging in size from 190bp to 320bp. PCR revealed that from all the coagulase positive S. aureus isolates 261(98.5%) had spa (spa-X) gene. The results of the present study indicated that S. aureus isolates recovered from bovine mastitis were genetically different within and among the various herds which may provide essential and valuable strategies to control staphylococcal infections in future. (author)

  15. An isotope-labeled chemical derivatization method for the quantitation of short-chain fatty acids in human feces by liquid chromatography–tandem mass spectrometry

    International Nuclear Information System (INIS)

    Han, Jun; Lin, Karen; Sequeira, Carita; Borchers, Christoph H.

    2015-01-01

    Highlights: • 3-Nitrophenylhydrazine was used to derivatize short-chain fatty acids (SCFAs) for LC-MS/MS. • 13 C 6 analogues were produced for use as isotope-labeled internal standards. • Isotope-labeled standards compensate for ESI matrix effects in LC-MS/MS. • Femtomolar sensitivities and 93–108% quantitation accuracy were achieved for human fecal SCFAs. - Abstract: Short-chain fatty acids (SCFAs) are produced by anaerobic gut microbiota in the large bowel. Qualitative and quantitative measurements of SCFAs in the intestinal tract and the fecal samples are important to understand the complex interplay between diet, gut microbiota and host metabolism homeostasis. To develop a new LC-MS/MS method for sensitive and reliable analysis of SCFAs in human fecal samples, 3-nitrophenylhydrazine (3NPH) was employed for pre-analytical derivatization to convert ten C 2 –C 6 SCFAs to their 3-nitrophenylhydrazones under a single set of optimized reaction conditions and without the need of reaction quenching. The derivatives showed excellent in-solution chemical stability. They were separated on a reversed-phase C 18 column and quantitated by negative-ion electrospray ionization – multiple-reaction monitoring (MRM)/MS. To achieve accurate quantitation, the stable isotope-labeled versions of the derivatives were synthesized in a single reaction vessel from 13 C 6 -3NPH, and were used as internal standard to compensate for the matrix effects in ESI. Method validation showed on-column limits of detection and quantitation over the range from low to high femtomoles for the ten SCFAs, and the intra-day and inter-day precision for determination of nine of the ten SCFAs in human fecal samples was ≤8.8% (n = 6). The quantitation accuracy ranged from 93.1% to 108.4% (CVs ≤ 4.6%, n = 6). This method was used to determine the SCFA concentrations and compositions in six human fecal samples. One of the six samples, which was collected from a clinically diagnosed type 2

  16. An isotope-labeled chemical derivatization method for the quantitation of short-chain fatty acids in human feces by liquid chromatography–tandem mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Han, Jun; Lin, Karen; Sequeira, Carita [University of Victoria – Genome BC Proteomics Centre, University of Victoria, Vancouver Island Technology Park, 3101–4464 Markham Street, Victoria, BC V8Z 7X8 (Canada); Borchers, Christoph H., E-mail: christoph@proteincentre.com [University of Victoria – Genome BC Proteomics Centre, University of Victoria, Vancouver Island Technology Park, 3101–4464 Markham Street, Victoria, BC V8Z 7X8 (Canada); Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Road, Victoria, BC V8P 5C2 (Canada)

    2015-01-07

    Highlights: • 3-Nitrophenylhydrazine was used to derivatize short-chain fatty acids (SCFAs) for LC-MS/MS. • {sup 13}C{sub 6} analogues were produced for use as isotope-labeled internal standards. • Isotope-labeled standards compensate for ESI matrix effects in LC-MS/MS. • Femtomolar sensitivities and 93–108% quantitation accuracy were achieved for human fecal SCFAs. - Abstract: Short-chain fatty acids (SCFAs) are produced by anaerobic gut microbiota in the large bowel. Qualitative and quantitative measurements of SCFAs in the intestinal tract and the fecal samples are important to understand the complex interplay between diet, gut microbiota and host metabolism homeostasis. To develop a new LC-MS/MS method for sensitive and reliable analysis of SCFAs in human fecal samples, 3-nitrophenylhydrazine (3NPH) was employed for pre-analytical derivatization to convert ten C{sub 2}–C{sub 6} SCFAs to their 3-nitrophenylhydrazones under a single set of optimized reaction conditions and without the need of reaction quenching. The derivatives showed excellent in-solution chemical stability. They were separated on a reversed-phase C{sub 18} column and quantitated by negative-ion electrospray ionization – multiple-reaction monitoring (MRM)/MS. To achieve accurate quantitation, the stable isotope-labeled versions of the derivatives were synthesized in a single reaction vessel from {sup 13}C{sub 6}-3NPH, and were used as internal standard to compensate for the matrix effects in ESI. Method validation showed on-column limits of detection and quantitation over the range from low to high femtomoles for the ten SCFAs, and the intra-day and inter-day precision for determination of nine of the ten SCFAs in human fecal samples was ≤8.8% (n = 6). The quantitation accuracy ranged from 93.1% to 108.4% (CVs ≤ 4.6%, n = 6). This method was used to determine the SCFA concentrations and compositions in six human fecal samples. One of the six samples, which was collected from a

  17. CoaSim: A Flexible Environment for Simulating Genetic Data under Coalescent Models

    DEFF Research Database (Denmark)

    Mailund; Schierup, Mikkel Heide; Pedersen, Christian Nørgaard Storm

    2005-01-01

    get insight into these. Results We have created the CoaSim application as a flexible environment for Monte various types of genetic data under equilibrium and non-equilibrium coalescent variety of applications. Interaction with the tool is through the Guile version scripting language. Scheme scripts......Background Coalescent simulations are playing a large role in interpreting large scale intra- polymorphism surveys and for planning and evaluating association studies. Coalescent of data sets under different models can be compared to the actual data to test different evolutionary factors and thus...

  18. Chromium downregulates the expression of Acetyl CoA Carboxylase 1 gene in lipogenic tissues of domestic goats: a potential strategy for meat quality improvement.

    Science.gov (United States)

    Najafpanah, Mohammad Javad; Sadeghi, Mostafa; Zali, Abolfazl; Moradi-Shahrebabak, Hossein; Mousapour, Hojatollah

    2014-06-15

    Acetyl CoA Carboxylase 1 (ACC1) is a biotin-dependent enzyme that catalyzes the carboxylation of Acetyl CoA to form Malonyl CoA, the key intermediate metabolite in fatty acid synthesis. In this study, the mRNA expression of the ACC1 gene was evaluated in four different tissues (liver, visceral fat, subcutaneous fat, and longissimus muscle) of the domestic goat (Capra hircus) kids feeding on four different levels of trivalent chromium (0, 0.5, 1, and 1.5mg/day) as food supplementation. RT-qPCR technique was used for expression analyses and heat shock protein 90 gene (HSP-90) was considered as reference gene for data normalization. Our results revealed that 1.5mg/day chromium significantly reduced the expression of the ACC1 gene in liver, visceral fat, and subcutaneous fat tissues, but not in longissimus muscles (Pmeat quality in domestic animals. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Compensation of matrix effects in gas chromatography-mass spectrometry analysis of pesticides using a combination of matrix matching and multiple isotopically labeled internal standards.

    Science.gov (United States)

    Tsuchiyama, Tomoyuki; Katsuhara, Miki; Nakajima, Masahiro

    2017-11-17

    In the multi-residue analysis of pesticides using GC-MS, the quantitative results are adversely affected by a phenomenon known as the matrix effect. Although the use of matrix-matched standards is considered to be one of the most practical solutions to this problem, complete removal of the matrix effect is difficult in complex food matrices owing to their inconsistency. As a result, residual matrix effects can introduce analytical errors. To compensate for residual matrix effects, we have developed a novel method that employs multiple isotopically labeled internal standards (ILIS). The matrix effects of ILIS and pesticides were evaluated in spiked matrix extracts of various agricultural commodities, and the obtained data were subjected to simple statistical analysis. Based on the similarities between the patterns of variation in the analytical response, a total of 32 isotopically labeled compounds were assigned to 338 pesticides as internal standards. It was found that by utilizing multiple ILIS, residual matrix effects could be effectively compensated. The developed method exhibited superior quantitative performance compared with the common single-internal-standard method. The proposed method is more feasible for regulatory purposes than that using only predetermined correction factors and is considered to be promising for practical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Deuterium- and tritium-labelled compounds. Applications in the life sciences

    Energy Technology Data Exchange (ETDEWEB)

    Atzrodt, Jens; Derdau, Volker [Isotope Chemistry and Metabolite Synthesis, Integrated Drug Discovery, Medicinal Chemistry, Frankfurt (Germany); Kerr, William J.; Reid, Marc [Department of Pure and Applied Chemistry, WestCHEM, University of Strathclyde, Glasgow (United Kingdom)

    2018-02-12

    Hydrogen isotopes are unique tools for identifying and understanding biological and chemical processes. Hydrogen isotope labelling allows for the traceless and direct incorporation of an additional mass or radioactive tag into an organic molecule with almost no changes in its chemical structure, physical properties, or biological activity. Using deuterium-labelled isotopologues to study the unique mass-spectrometric patterns generated from mixtures of biologically relevant molecules drastically simplifies analysis. Such methods are now providing unprecedented levels of insight in a wide and continuously growing range of applications in the life sciences and beyond. Tritium ({sup 3}H), in particular, has seen an increase in utilization, especially in pharmaceutical drug discovery. The efforts and costs associated with the synthesis of labelled compounds are more than compensated for by the enhanced molecular sensitivity during analysis and the high reliability of the data obtained. In this review, advances in the application of hydrogen isotopes in the life sciences are described. (copyright 2018 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim)

  1. Labelling of endotoxins with Na/sup 51/CrO/sub 4/

    Energy Technology Data Exchange (ETDEWEB)

    Oginski, M; Lipinska-Piotrowska, I [Akademia Medyczna, Lodz (Poland)

    1974-01-01

    The authors modified the method of Braude of labelling of endotoxins with /sup 51/Cr. A higher uptake of the isotope by endotoxin was obtained (98.4%) which has a favourable effect on the accuracy of measurements with labelled endotoxins.

  2. Positional isotope exchange studies on enzyme using NMR spectroscopy

    International Nuclear Information System (INIS)

    Matsunaga, T.O.

    1987-01-01

    The isotopically enriched compounds, 18 O-β,γ-ATP and 18 O bridge-labeled pyrophosphate, synthesized previously in this laboratory, were used to investigate and measure the exchange vs. turnover of substrates and products from their central complexes in four selected enzyme systems. Using hi-field 31 P NMR, we were able to differentiate between 18 O labeled in the bridge vs. the non-bridge positions by virtue of the isotope shift upon the phosphorus nuclei. The bridge to non-bridge scrambling of the label was quantitated and the exchange vs. turnover ratios under a variety of conditions was determined. Using the substrate inhibitor carboxycreatinine, PIX experiments with 18 O-β,γ-ATP and creatine kinase were conducted. It was shown that carboxycreatinine and creatine kinase promoted exchange of the 18 O label as determined by NMR. We have concluded that carboxycreatinine is either a substrate that catalyzes very slow turnover or it catalyzes exchange by a dissociative (SN 1 /sub P/) type of mechanism

  3. Production, purification and detergent exchange of isotopically labeled Bacillussubtilis cytochrome b₅₅₈ (SdhC).

    Science.gov (United States)

    Baureder, Michael; Hederstedt, Lars

    2011-11-01

    Cytochrome b₅₅₈ of the gram-positive bacterium Bacillussubtilis is the membrane anchor subunit of the succinate:quinone oxidoreductase of the citric acid cycle. The cytochrome consists of the SdhC polypeptide (202 residues) and two protoheme IX groups that function in transmembrane electron transfer to menaquinone. The general structure of the cytochrome is known from extensive experimental studies and by comparison to Wolinellasuccinogenes fumarate reductase for which the X-ray crystal structure has been determined. Solution state NMR can potentially be used to identify the quinone binding site(s) and study, e.g. redox-linked, dynamics of cytochrome b₅₅₈. In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome b₅₅₈ produced in Escherichia coli. We have also evaluated several detergents suitable for NMR for their effectiveness in maintaining the cytochrome solubilized and intact for days at room temperature. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    Energy Technology Data Exchange (ETDEWEB)

    Melton, Elaina M. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Center for Cardiovascular Sciences, Albany Medical College, Albany, NY (United States); Cerny, Ronald L. [Department of Chemistry, University of Nebraska, Lincoln, NE (United States); DiRusso, Concetta C. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Black, Paul N., E-mail: pblack2@unl.edu [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States)

    2013-11-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  5. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    International Nuclear Information System (INIS)

    Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

    2013-01-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  6. Fatty acids labelled with iodine 123 or 131 in. omega. position; myocardial evolution

    Energy Technology Data Exchange (ETDEWEB)

    Riche, F.; Vidal, M. (Grenoble-1 Univ., 38 (France)); Mathieu, J.P.; Busquet, G.; Comet, M. (Grenoble-1 Univ., 38 - La Tronche (France)); Coornaert, S.; Bardy, A. (CEA Centre d' Etudes Nucleaires de Saclay, 91 - Gif-sur-Yvette (France). Office des Rayonnements Ionisants); Godart, J. (Grenoble-1 Univ., 38 (France). Inst. des Sciences Nucleaires)

    A simple and rapid method of labelling a number of saturated acetylenic and Z or E ethylenic acids has been developed. The fatty acids are labelled with /sup 123/I- or /sup 131/I- in the ..omega.. position by isotopic exchange labelled NaI in acetone. Myocardial metabolism was studied by injecting the labelled fatty acids into mice.

  7. Isotope products manufacture in Russia and its prospects

    International Nuclear Information System (INIS)

    Malyshev, S.V.; Okhotina, I.A.; Kalelin, E.A.; Krasnov, N.N.; Kuzin, V.V.; Malykh, J.A.; Makarovsky, S.B.

    1997-01-01

    At the present stage of the world economy development, stable and radioactive isotopes,preparations and products on their base are widely used in many fields of the national economy, medicine and scientific researches. The Russian Federation is one of the largest worldwide producers of a variety of nuclide products on the base of more than 350 isotopes, as follows: stable isotopes reactor, cyclotron, fission product radioactive isotopes, ion-radiation sources compounds, labelled with stable and radioactive isotopes, radionuclide short-lived isotope generators, radiopharmaceuticals, radionuclide light and heat sources; luminous paints on base of isotopes. The Russian Ministry for Atomic Energy coordinates activity for development and organization of manufacture and isotope products supply in Russia as well as for export. Within many years of isotope industry development, there have appeared some manufacturing centres in Russia, dealing with a variety of isotope products. The report presents the production potentialities of these centres and also an outlook on isotope production development in Russia in the next years

  8. Isotope separations using chromatographic methods

    International Nuclear Information System (INIS)

    Leseticky, L.

    1985-01-01

    A survey is given of chromatographic separations of compounds only differing in isotope composition. Isotope effects on physical properties which allow chromatographic separation (vapour tension, adsorption heat, partition coefficient) are very small, with the exception of the simplest molecules. Therefore, separation factors only assume the value of several per cent. From this ensues the necessity of using columns which are specially and very carefully prepared and have a separation efficiency of the order of 10 4 theoretical plates. Briefly discussed is liquid chromatography on ion exchangers which with a varied degree of success was used for separating simple inorganic compounds or ions. Ion exchange chromatography of amino acids labelled with tritium, and chromatography of tritium labelled steroids also provided only a certain degree of separation. A detailed analysis is presented of gas chromatography separation of various deuterium and tritium labelled low-molecular compounds, to which a number of studies has been devoted in the literature. Very promising is the method of complexation gas chromatography based on the reversible formation of a complex of the ligand (the compound being separated) and the compound of the (transition) metal as the steady-state phase. (author)

  9. Differential isotope dansylation labeling combined with liquid chromatography mass spectrometry for quantification of intact and N-terminal truncated proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yanan; Li, Liang, E-mail: Liang.Li@ualberta.ca

    2013-08-20

    Graphical abstract: -- Highlights: •LC–MS was developed for quantifying protein mixtures containing both intact and N-terminal truncated proteins. •{sup 12}C{sub 2}-Dansylation of the N-terminal amino acid of proteins was done first, followed by microwave-assisted acid hydrolysis. •The released {sup 12}C{sub 2}-dansyl labeled N-terminal amino acid was quantified using {sup 13}C{sub 2}-dansyl labeled amino acid standards. •The method provided accurate and precise results for quantifying intact and N-terminal truncated proteins within 8 h. -- Abstract: The N-terminal amino acids of proteins are important structure units for maintaining the biological function, localization, and interaction networks of proteins. Under different biological conditions, one or several N-terminal amino acids could be cleaved from an intact protein due to processes, such as proteolysis, resulting in the change of protein properties. Thus, the ability to quantify the N-terminal truncated forms of proteins is of great importance, particularly in the area of development and production of protein-based drugs where the relative quantity of the intact protein and its truncated form needs to be monitored. In this work, we describe a rapid method for absolute quantification of protein mixtures containing intact and N-terminal truncated proteins. This method is based on dansylation labeling of the N-terminal amino acids of proteins, followed by microwave-assisted acid hydrolysis of the proteins into amino acids. It is shown that dansyl labeled amino acids are stable in acidic conditions and can be quantified by liquid chromatography mass spectrometry (LC–MS) with the use of isotope analog standards.

  10. Deciphering Systemic Wound Responses of the Pumpkin Extrafascicular Phloem by Metabolomics and Stable Isotope-Coded Protein Labeling1[C][W

    Science.gov (United States)

    Gaupels, Frank; Sarioglu, Hakan; Beckmann, Manfred; Hause, Bettina; Spannagl, Manuel; Draper, John; Lindermayr, Christian; Durner, Jörg

    2012-01-01

    In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling. PMID:23085839

  11. Correlated optical and isotopic nanoscopy

    Science.gov (United States)

    Saka, Sinem K.; Vogts, Angela; Kröhnert, Katharina; Hillion, François; Rizzoli, Silvio O.; Wessels, Johannes T.

    2014-04-01

    The isotopic composition of different materials can be imaged by secondary ion mass spectrometry. In biology, this method is mainly used to study cellular metabolism and turnover, by pulsing the cells with marker molecules such as amino acids labelled with stable isotopes (15N, 13C). The incorporation of the markers is then imaged with a lateral resolution that can surpass 100 nm. However, secondary ion mass spectrometry cannot identify specific subcellular structures like organelles, and needs to be correlated with a second technique, such as fluorescence imaging. Here, we present a method based on stimulated emission depletion microscopy that provides correlated optical and isotopic nanoscopy (COIN) images. We use this approach to study the protein turnover in different organelles from cultured hippocampal neurons. Correlated optical and isotopic nanoscopy can be applied to a variety of biological samples, and should therefore enable the investigation of the isotopic composition of many organelles and subcellular structures.

  12. Labeling of Pest Insects Using Radioisotopes to Study Dispersal Pattern, Migration and Estimation of Population Density

    International Nuclear Information System (INIS)

    Singgih Sutrisno

    2008-01-01

    To study insects behaviour in their habitat such as dispersal, migration and flight range, insects are needed to be labelled to trace their movement. One of the most promising labeling methodology for internal labeling is the use of radioisotopes. Radioisotopes that have been used for labeling insects are 3 H, 32 P, 14 Ca, 45 K, 35 S, 59 Fe, 60 Co, and 14 C. Insect labeling with isotopes has more advantages as compared to dyes due to isotopes used for labeling is bonded to the tissue such as 3 H, 32 P, 14 Ca, K, 131 I. Several consideration have to be taken to determine isotopes that will be used in line with the time consuming for experiments. This have to be carried out due to the phenomenon that several isotopes are toxic to insects such as 45 Ca, 59 Fe, 86 Rb, 110 Ag, 115 Cd, and 131 J. Precautions have to be fulfilled for insect radiolabeling which are save to insects, environment, easy to apply, materials are available and acceptable to the public. Radioisotope 32 P with a correct dose is very convenience to be used in such experiments due to its relatively short half live, which is only 14.3 days. If it is an stable isotope it can be kept for a long time so the sample analyzed can be conducted convenience for long periods of time. Stable elements such as Rb can be changed to be radioisotopes by bombardment of neutrons in a nuclear reactor or accelerator. Then the element that has been activated can be identified using solid scintillation counter, multichannel analyzer or can be detected using autoradiography. (author)

  13. IRMS detection of testosterone manipulated with 13C labeled standards in human urine by removing the labeled 13C.

    Science.gov (United States)

    Wang, Jingzhu; Yang, Rui; Yang, Wenning; Liu, Xin; Xing, Yanyi; Xu, Youxuan

    2014-12-10

    Isotope ratio mass spectrometry (IRMS) is applied to confirm testosterone (T) abuse by determining the carbon isotope ratios (δ(13)C value). However, (13)C labeled standards can be used to control the δ(13)C value and produce manipulated T which cannot be detected by the current method. A method was explored to remove the (13)C labeled atom at C-3 from the molecule of androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ(13)C values between Andro and ANAD (Δδ(13)CAndro-ANAD, ‰) would change significantly in case manipulated T is abused. Twenty-one volunteers administered T manipulated with different (13)C labeled standards. The collected urine samples were analyzed with the established method, and the maximum value of Δδ(13)CAndro-ANAD post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ(13)CAndro-ANAD for positive result was suggested as 1.2‰. The developed method could be used to detect T manipulated with 3-(13)C labeled standards. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Preparation of 35S-labelled albendazole sulfinyl (ABZO)

    International Nuclear Information System (INIS)

    Zhang Lufang; Wu Yuanfang; Yang Zhiming

    1992-01-01

    Albendazole is an important insecticide and albendazole sulfinyl (ABZO) is the effective component. In order to investigate its absorption, distribution and excretion in vivo, 35 S-labelled tracer was necessary for experiment. 35 S-labelled intermediate was made by isotopic exchange. The radiochemical yield was over 90%. After purification by TLC, the radiochemical purity of the 35 S-ABZO determined by PC and electrophoresis was over 95%

  15. Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria.

    Science.gov (United States)

    Chang, Shih Chieh; Galea, Charles A; Leung, Eleanor W W; Tajhya, Rajeev B; Beeton, Christine; Pennington, Michael W; Norton, Raymond S

    2012-10-01

    The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (T(EM)). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T(EM) cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni²⁺ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of ¹⁵N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a K(d) of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for T(EM) cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of ¹⁵N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Advisory group meeting on stable isotope labelled compounds in biomedical studies

    International Nuclear Information System (INIS)

    Vera Ruiz, H.; Parr, R.M.

    1985-11-01

    The programme of the meeting was restricted to topics involving applications of stable isotopes of the lighter elements (H, C, N, O). The current status of stable isotope techniques and applications in nutritional and biomedical studies, the applicability of these techniques in developing countries and the IAEA's future programmes on this topic were discussed

  17. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Mitsuhiro [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Ono, Akira M.; Terauchi, Tsutomu [SAIL Technologies Co., Inc. (Japan); Kainosho, Masatsune, E-mail: kainosho@nmr.chem.metro-u.ac.j [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

    2010-01-15

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines ({epsilon}- and {zeta}-SAIL Phe) and tyrosine ({epsilon}-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized {delta}-SAIL Phe and {delta}-SAIL Tyr, which allow us to observe and assign {delta}-{sup 13}C/{sup 1}H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the {delta}-, {epsilon}- or {zeta}-{sup 13}C/{sup 1}H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the {delta}-, {epsilon}-, and {zeta}-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly {sup 13}C, {sup 15}N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of {zeta}-SAIL Phe and {epsilon}-SAIL Tyr would be practically the best choice for protein structural determinations.

  18. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination.

    Science.gov (United States)

    Takeda, Mitsuhiro; Ono, Akira M; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-01-01

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized delta-SAIL Phe and delta-SAIL Tyr, which allow us to observe and assign delta-(13)C/(1)H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the delta-, epsilon- or zeta-(13)C/(1)H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the delta-, epsilon-, and zeta-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly (13)C, (15)N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of zeta-SAIL Phe and epsilon-SAIL Tyr would be practically the best choice for protein structural determinations.

  19. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination

    International Nuclear Information System (INIS)

    Takeda, Mitsuhiro; Ono, Akira M.; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-01-01

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (ε- and ζ-SAIL Phe) and tyrosine (ε-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized δ-SAIL Phe and δ-SAIL Tyr, which allow us to observe and assign δ- 13 C/ 1 H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the δ-, ε- or ζ- 13 C/ 1 H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the δ-, ε-, and ζ-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly 13 C, 15 N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of ζ-SAIL Phe and ε-SAIL Tyr would be practically the best choice for protein structural determinations.

  20. Sulfur and selenium isotope separation by distillation

    International Nuclear Information System (INIS)

    Mills, T. R.; McInteer, B. B.; Montoya, J. G.

    1988-01-01

    Sulfur and selenium isotopes are used for labeled compounds and as precursors for radioisotope production; however, both limited availability and high costs are problems. A new method is needed for large-scale separation of these isotopes. Experimental distillation columns were used to measure isotopic separations for sulfur and selenium compounds. The maximum total isotope separation of 32 S vs. 34 S were 1.127 for H 2 S, 1.048 for COS, 0.838 for SF 4 , and 1.058 for CH 3 SH. Relative volatilities of 32 S vs. 34 S are 1.0006 for COS and 0.9976 for SF 4 . There is a reverse isotope effect for carbon in COS. No isotopic separation was observed for dimethyl selenide. The lower mass selenium isotopes in H 2 Se are more volatile. Distillation is a promising method for separating sulfur isotopes on a production scale. Existing distillation technology produced separated isotopes with an effect similar to that found for sulfur in SF 4 . 8 refs., 2 tabs

  1. Sulfur and selenium isotope separation by distillation

    International Nuclear Information System (INIS)

    Mills, T.R.; McInteer, B.B.; Montoya, J.G.

    1989-01-01

    Sulfur and selenium isotopes are used for labeled compounds and as precursors for radioisotope production; however, both limited availability and high costs are problems. A new method is needed for large-scale separation of theses isotopes. Experimental distillation columns were used to measure isotopic separations for sulfur and selenium compounds. The maximum total isotope separations of 32 S vs. 34 S were 1.127 for H 2 S, 1.048 for COS, 0.838 for SF 4 , and 1.058 for CH 3 SH. Relative volatilities of 32 S and 34 S are 1.0006 for COS and 0.9976 for SF 4 . There is a reverse isotope effect for carbon in COS. No isotopic separation was observed for dimethyl selenide. The lower mass selenium isotopes in H 2 Se are more volatile. Distillation is a promising method for separating sulfur isotopes on a production scale. Existing distillation technology produces separated isotopes with an effect similar to that found for sulfur in SF 4 . (author). 8 refs.; 2 tabs

  2. Calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry assays and its application in supporting microdose absolute bioavailability studies.

    Science.gov (United States)

    Gu, Huidong; Wang, Jian; Aubry, Anne-Françoise; Jiang, Hao; Zeng, Jianing; Easter, John; Wang, Jun-sheng; Dockens, Randy; Bifano, Marc; Burrell, Richard; Arnold, Mark E

    2012-06-05

    A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

  3. Benzylic rearrangement stable isotope labeling for quantitation of guanidino and ureido compounds in thyroid tissues by liquid chromatography-electrospray ionization mass spectrometry

    International Nuclear Information System (INIS)

    Fan, Ruo-Jing; Guan, Qing; Zhang, Fang; Leng, Jia-Peng; Sun, Tuan-Qi; Guo, Yin-Long

    2016-01-01

    Benzylic rearrangement stable isotope labeling (BRSIL) was explored to quantify the guanidino and ureido compounds (GCs and UCs). This method employed a common reagent, benzil, to label the guanidino and ureido groups through nucleophilic attacking then benzylic migrating. The use of BRSIL was investigated in the analysis of five GCs (creatine, L-arginine, homoarginine, 4-guanidinobutyric acid, and methylguanidine) and two UCs (urea and citrulline). The labeling was found simple and specific. The introduction of bi-phenyl group and the generation of nitrogen heterocyclic ring in the benzil-d0/d5 labeled GCs and UCs improved the retention behaviors in liquid chromatography (LC) and increased the sensitivity of electrospray ionization mass spectrometry (ESI MS) detection. The fragment ion pairs of m/z 182/187 and m/z 210/215 from the benzil-d0/d5 tags facilitated the discovery of potential GCs and UCs candidates residing in biological matrices. The use of BRSIL combined with LC-ESI MS was applied for simultaneously quantitation of GCs and UCs in thyroid tissues. It was demonstrated that nine GCs and UCs were detected, six of which were further quantified based on corresponding standards. It was concluded that five GCs and UCs (L-arginine, homoarginine, 4-guanidinobutyric acid, methylguanidine, and citrulline) were statistically significantly different (p < 0.05) between the para-carcinoma and carcinoma thyroid tissue samples. - Highlights: • A common reagent, benzil-d0/d5 was employed to label the GCs and UCs through BRSIL. • The benzil-d0/d5 labeling improved the retention behavior in RPLC and increased the sensitivity by ESI MS detection. • BRSIL coupled with LC-ESI MS was applied to the qualitation and quantitation of GCs and UCs in thyroid tissues.

  4. Benzylic rearrangement stable isotope labeling for quantitation of guanidino and ureido compounds in thyroid tissues by liquid chromatography-electrospray ionization mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Ruo-Jing [State Key Laboratory of Organmetallic Chemistry and National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032 (China); Guan, Qing [Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center, Shanghai, 200032 (China); Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032 (China); Zhang, Fang, E-mail: fzhang@sioc.ac.cn [State Key Laboratory of Organmetallic Chemistry and National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032 (China); Leng, Jia-Peng [State Key Laboratory of Organmetallic Chemistry and National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032 (China); Sun, Tuan-Qi, E-mail: tuanqisun@163.com [Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center, Shanghai, 200032 (China); Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032 (China); Guo, Yin-Long, E-mail: ylguo@sioc.ac.cn [State Key Laboratory of Organmetallic Chemistry and National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032 (China)

    2016-02-18

    Benzylic rearrangement stable isotope labeling (BRSIL) was explored to quantify the guanidino and ureido compounds (GCs and UCs). This method employed a common reagent, benzil, to label the guanidino and ureido groups through nucleophilic attacking then benzylic migrating. The use of BRSIL was investigated in the analysis of five GCs (creatine, L-arginine, homoarginine, 4-guanidinobutyric acid, and methylguanidine) and two UCs (urea and citrulline). The labeling was found simple and specific. The introduction of bi-phenyl group and the generation of nitrogen heterocyclic ring in the benzil-d0/d5 labeled GCs and UCs improved the retention behaviors in liquid chromatography (LC) and increased the sensitivity of electrospray ionization mass spectrometry (ESI MS) detection. The fragment ion pairs of m/z 182/187 and m/z 210/215 from the benzil-d0/d5 tags facilitated the discovery of potential GCs and UCs candidates residing in biological matrices. The use of BRSIL combined with LC-ESI MS was applied for simultaneously quantitation of GCs and UCs in thyroid tissues. It was demonstrated that nine GCs and UCs were detected, six of which were further quantified based on corresponding standards. It was concluded that five GCs and UCs (L-arginine, homoarginine, 4-guanidinobutyric acid, methylguanidine, and citrulline) were statistically significantly different (p < 0.05) between the para-carcinoma and carcinoma thyroid tissue samples. - Highlights: • A common reagent, benzil-d0/d5 was employed to label the GCs and UCs through BRSIL. • The benzil-d0/d5 labeling improved the retention behavior in RPLC and increased the sensitivity by ESI MS detection. • BRSIL coupled with LC-ESI MS was applied to the qualitation and quantitation of GCs and UCs in thyroid tissues.

  5. Isotopic scintigraphy in kidney grafting

    International Nuclear Information System (INIS)

    Renfro, Richard.

    1976-01-01

    Isotopic explorations of kidney transplants were performed on sixty-six patients. Three scintigraphic techniques were used: labelled ferrous ascorbate scintigraphy, sequential 99m technetium DTPA scintigraphy and the 131 I hippuran nephrogram. The aim of this study is to analyse the results obtained under different pathological circumstances affecting the transplant, to discuss the advantages of the techniques and to propose a working procedure. The most reliable and accurate technique is the 131 I hippuran nephrogram combined with sequential 99mTc DTPA, by which renal vascularisation may be judged labelled ferrous ascorbate on the other hand is too insensitive. Although the information supplied is mostly contained in the scintigraphic images, the nephrographic curves and the blood radioactivity decay time and rad V/rad R ratio measurements are very helpful in the early diagnosis and differential diagnosis of complications affecting the transplant. The proper use of isotopic scintigraphy in kidney grafting should provide optimum conditions for better survival of the transplant at minimum risk to the patient [fr

  6. Bacterial production of site specific {sup 13}C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ramaraju, Bhargavi; McFeeters, Hana; Vogler, Bernhard; McFeeters, Robert L., E-mail: robert.mcfeeters@uah.edu [University of Alabama in Huntsville, Department of Chemistry (United States)

    2017-01-15

    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 {sup 13}C 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 {sup 13}C chemical shifts and multiple magnetically equivalent {sup 1}H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with {sup 13}C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated {sup 1}H-{sup 13}C 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.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  8. Overcoming interference with the detection of a stable isotopically labeled microtracer in the evaluation of beclabuvir absolute bioavailability using a concomitant microtracer approach.

    Science.gov (United States)

    Jiang, Hao; Titsch, Craig; Zeng, Jianing; Jones, Barry; Joyce, Philip; Gandhi, Yash; Turley, Wesley; Burrell, Richard; Aubry, Anne F; Arnold, Mark E

    2017-09-05

    The oral absolute bioavailability of beclabuvir in healthy subjects was determined using a microdose (100μg) of the stable isotopically labeled tracer via intravenous (IV) infusion started after oral dosing of beclabuvir (150mg). To simultaneously analyze the concentrations of the IV microtracer ([ 13 C 6 ]beclabuvir) and beclabuvir in plasma samples, a liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) method was initially developed. Surprisingly beclabuvir significantly interfered with the IV microtracer detection when using the selected reaction monitoring (SRM) in the assay. An interfering component from the drug substance was observed using a high resolution mass spectrometer (HRMS). The mass-to-charge (m/z) of the interfering component was -32ppm different from the nominal value for the IV microtracer and thus could not be differentiated in the SRM assay by the unit mass resolution. To overcome this interference, we evaluated two approaches by either monitoring an alternative product ion using the SRM assay or isolating the interfering component using the parallel reaction monitoring (PRM) assay on the HRMS. This case study has demonstrated two practical approaches for overcoming interferences with the detection of stable isotopically labeled IV microtracers in the evaluation of absolute bioavailability, which provides users the flexibility in using either LC-MS/MS or HRMS to mitigate unpredicted interferences in the assay to support microtracer absolute bioavailability studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Gas cleaning with hot char beds studied by stable isotopes

    DEFF Research Database (Denmark)

    Egsgaard, Helge; Ahrenfeldt, Jesper; Ambus, Per

    2014-01-01

    The chemistry taking place in a high temperature char bed used for binding aromatic tar compounds has been studied in detail. 13C labelled tar compounds were used to trace the incorporation into the char bed using isotope ratio mass spectrometry (IRMS) and GC-MS. Furthermore, compounds labelled...

  10. New trends and applications in carboxylation for isotope chemistry.

    Science.gov (United States)

    Bragg, Ryan A; Sardana, Malvika; Artelsmair, Markus; Elmore, Charles S

    2018-05-08

    Carboxylations are an important method for the incorporation of isotopically labeled 14 CO 2 into molecules. This manuscript will review labeled carboxylations since 2010 and will present a perspective on the potential of recent unlabeled methodology for labeled carboxylations. The perspective portion of the manuscript is broken into 3 major sections based on product type, arylcarboxylic acids, benzylcarboxylic acids, and alkyl carboxylic acids, and each of those sections is further subdivided by substrate. © 2018 AstraZeneca. Journal of Labelled Compounds and Radiopharmaceuticals Published by John Wiley & Sons, Ltd.

  11. Measurement of apolipoprotein E and amyloid β clearance rates in the mouse brain using bolus stable isotope labeling

    Science.gov (United States)

    2012-01-01

    Background Abnormal proteostasis due to alterations in protein turnover has been postulated to play a central role in several neurodegenerative diseases. Therefore, the development of techniques to quantify protein turnover in the brain is critical for understanding the pathogenic mechanisms of these diseases. We have developed a bolus stable isotope-labeling kinetics (SILK) technique coupled with multiple reaction monitoring mass spectrometry to measure the clearance of proteins in the mouse brain. Results Cohorts of mice were pulse labeled with 13 C6-leucine and the brains were isolated after pre-determined time points. The extent of label incorporation was measured over time using mass spectrometry to measure the ratio of labeled to unlabeled apolipoprotein E (apoE) and amyloid β (Aβ). The fractional clearance rate (FCR) was then calculated by analyzing the time course of disappearance for the labeled protein species. To validate the technique, apoE clearance was measured in mice that overexpress the low-density lipoprotein receptor (LDLR). The FCR in these mice was 2.7-fold faster than wild-type mice. To demonstrate the potential of this technique for understanding the pathogenesis of neurodegenerative disease, we applied our SILK technique to determine the effect of ATP binding cassette A1 (ABCA1) on both apoE and Aβ clearance. ABCA1 had previously been shown to regulate both the amount of apoE in the brain, along with the extent of Aβ deposition, and represents a potential molecular target for lowering brain amyloid levels in Alzheimer's disease patients. The FCR of apoE was increased by 1.9- and 1.5-fold in mice that either lacked or overexpressed ABCA1, respectively. However, ABCA1 had no effect on the FCR of Aβ, suggesting that ABCA1 does not regulate Aβ metabolism in the brain. Conclusions Our SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain. We expect that

  12. SIMSISH technique does not alter the apparent isotopic composition of bacterial cells.

    Directory of Open Access Journals (Sweden)

    Olivier Chapleur

    Full Text Available In order to identify the function of uncultured microorganisms in their environment, the SIMSISH method, combining in situ hybridization (ISH and nanoscale secondary ion mass spectrometry (nanoSIMS imaging, has been proposed to determine the quantitative uptake of specific labelled substrates by uncultured microbes at the single cell level. This technique requires the hybridization of rRNA targeted halogenated DNA probes on fixed and permeabilized microorganisms. Exogenous atoms are introduced into cells and endogenous atoms removed during the experimental procedures. Consequently differences between the original and the apparent isotopic composition of cells may occur. In the present study, the influence of the experimental procedures of SIMSISH on the isotopic composition of carbon in E. coli cells was evaluated with nanoSIMS and compared to elemental analyser-isotopic ratio mass spectrometer (EA-IRMS measurements. Our results show that fixation and hybridization have a very limited, reproducible and homogeneous influence on the isotopic composition of cells. Thereby, the SIMSISH procedure minimizes the contamination of the sample by exogenous atoms, thus providing a means to detect the phylogenetic identity and to measure precisely the carbon isotopic composition at the single cell level. This technique was successfully applied to a complex sample with double bromine - iodine labelling targeting a large group of bacteria and a specific archaea to evaluate their specific (13C uptake during labelled methanol anaerobic degradation.

  13. SIMSISH Technique Does Not Alter the Apparent Isotopic Composition of Bacterial Cells

    Science.gov (United States)

    Chapleur, Olivier; Wu, Ting-Di; Guerquin-Kern, Jean-Luc; Mazéas, Laurent; Bouchez, Théodore

    2013-01-01

    In order to identify the function of uncultured microorganisms in their environment, the SIMSISH method, combining in situ hybridization (ISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) imaging, has been proposed to determine the quantitative uptake of specific labelled substrates by uncultured microbes at the single cell level. This technique requires the hybridization of rRNA targeted halogenated DNA probes on fixed and permeabilized microorganisms. Exogenous atoms are introduced into cells and endogenous atoms removed during the experimental procedures. Consequently differences between the original and the apparent isotopic composition of cells may occur. In the present study, the influence of the experimental procedures of SIMSISH on the isotopic composition of carbon in E. coli cells was evaluated with nanoSIMS and compared to elemental analyser-isotopic ratio mass spectrometer (EA-IRMS) measurements. Our results show that fixation and hybridization have a very limited, reproducible and homogeneous influence on the isotopic composition of cells. Thereby, the SIMSISH procedure minimizes the contamination of the sample by exogenous atoms, thus providing a means to detect the phylogenetic identity and to measure precisely the carbon isotopic composition at the single cell level. This technique was successfully applied to a complex sample with double bromine – iodine labelling targeting a large group of bacteria and a specific archaea to evaluate their specific 13C uptake during labelled methanol anaerobic degradation. PMID:24204855

  14. Quantitating subcellular metabolism with multi-isotope imaging mass spectrometry

    OpenAIRE

    Steinhauser, Matthew L.; Bailey, Andrew; Senyo, Samuel E.; Guillermier, Christelle; Perlstein, Todd S.; Gould, Alex P.; Lee, Richard T.; Lechene, Claude P.

    2012-01-01

    Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter 1,2 but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with sub-micron resolution 3,4 . Here we apply MIMS to diverse organisms, including Drosophila, mice, and humans. We test the “immortal strand hypothesis,” which predicts that during asymmetric stem cell division ch...

  15. Contribution of stable isotopes to the study of pharmacokinetics of magnesium salts; Apport des isotopes stables a l'etude de la pharmacocinetique de sels de magnesium

    Energy Technology Data Exchange (ETDEWEB)

    Benech, H

    1999-05-28

    The use of stable isotopes as labels is becoming an attractive tool for the study of magnesium behavior in humans. It has been used two stable isotopes of magnesium, {sup 25}Mg and {sup 26}Mg, to measure the absolute bioavailability of a pharmaceutical form of magnesium. (N.C.)

  16. The 2-nd Conference on Isotopic and Molecular Processes. Abstracts

    International Nuclear Information System (INIS)

    Bogdan, Mircea

    2001-01-01

    The proceedings of the 2-nd Conference on Isotopic and Molecular Processes held on September 27 - 29, 2001 in Cluj - Napoca, Romania, contains contributions presented as: 11 plenary lectures, 24 oral presentations and 103 posters in two sections, namely, isotopic processes and molecular processes. The main topics treated in this conference were isotope production, separation and enrichment as well as stable isotope applications. Also, studies on isotope effects in different fields are reported. Besides reports on isotope effects, exchange and separation, new methods of preparation and labelling compounds used particularly in nuclear medicine are presented. Environmental studies by means of stable isotope and radon monitoring are described. Applications of radiation effects and different nuclear methods in medicine are also addressed

  17. The role of isotopes in the development of β-adrenoceptor blocking agents

    International Nuclear Information System (INIS)

    Allen, J.

    1987-01-01

    This chapter is devoted to the applications of labelled β-blocking agents in drug development and to the procedures which have been described in the literature for labelling this class of compounds with radioactive or stable isotopes. 149 refs.; 4 figs

  18. Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris.

    Science.gov (United States)

    Guo, Yu-Qi; Wu, Qing-Ping; Shao, Xiao-Xia; Shen, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

    2015-06-01

    Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.

  19. Effect of Different Carbon Substrates on Nitrate Stable Isotope Fractionation During Microbial Denitrification

    DEFF Research Database (Denmark)

    Wunderlich, Anja; Meckenstock, Rainer; Einsiedl, Florian

    2012-01-01

    -labeled water and 18O-labeled nitrite were added to the microcosm experiments to study the effect of putative backward reactions of nitrite to nitrate on the stable isotope fractionation. We found no evidence for a reverse reaction. Significant variations of the stable isotope enrichment factor ε were observed......In batch experiments, we studied the isotope fractionation in N and O of dissolved nitrate during dentrification. Denitrifying strains Thauera aromatica and “Aromatoleum aromaticum strain EbN1” were grown under strictly anaerobic conditions with acetate, benzoate, and toluene as carbon sources. 18O...... of nitrate transport across the cell wall compared to the kinetics of the intracellular nitrate reduction step of microbial denitrification....

  20. Synthesis of the derivatives of 6-amino-uracil labelled with C-14

    Czech Academy of Sciences Publication Activity Database

    Elbert, Tomáš; Hezký, Petr; Jansa, Petr

    2016-01-01

    Roč. 59, č. 14 (2016), s. 615-618 ISSN 0362-4803. [International Isotope Symposium on the Synthesis and Applications of Isotopes and Isotopically Labelled Compounds /12./. Princeton, 07.06.2015-11.06.2015] R&D Projects: GA MŠk LG13002 Institutional support: RVO:61388963 Keywords : 6-amino-uracil derivatives * specific activity assay using NMR * [C-14]cyanoacetic acid Subject RIV: CC - Organic Chemistry Impact factor: 1.745, year: 2016

  1. Isotope dilution analysis for urinary fentanyl and its main metabolite, norfentanyl, in patients by isotopic fractionation using capillary gas chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Sera, Shoji; Goromaru, Tsuyoshi [Fukuyama Univ., Hiroshima (Japan). Faculty of Pharmacy and Pharmaceutical Sciences; Sameshima, Teruko; Kawasaki, Koichi; Oda, Toshiyuki

    1998-07-01

    Isotope dilution analysis was applied to determine urinary excretion of fentanyl (FT) and its main metabolite, norfentanyl (Nor-FT), by isotopic fractionation using a capillary gas chromatograph equipped with a surface ionization detector (SID). Urinary FT was determined quantitatively in the range of 0.4-40 ng/ml using deuterium labeled FT (FT-{sup 2}H{sub 19}), as an internal standard. We also performed isotope dilution analysis of Nor-FT in urine. N-Alkylation was necessary to sensitively detect Nor-FT with SID. Methyl derivative was selected from 3 kinds of N-alkyl derivatives to increase sensitivity and peak resolution, and to prevent interference with urinary compound. Nor-FT concentration was quantitatively determined in the range of 10-400 ng/ml using deuterium labeled Nor-FT (Nor-FT-{sup 2}H{sub 10}). No endogenous compounds or concomitant drugs interfered with the detection of FT and Nor-FT in the urine of patients. The present method will be useful for pharmacokinetic studies and the evaluation of drug interactions in FT metabolism. (author)

  2. Isotope dilution analysis for urinary fentanyl and its main metabolite, norfentanyl, in patients by isotopic fractionation using capillary gas chromatography

    International Nuclear Information System (INIS)

    Sera, Shoji; Goromaru, Tsuyoshi; Sameshima, Teruko; Kawasaki, Koichi; Oda, Toshiyuki

    1998-01-01

    Isotope dilution analysis was applied to determine urinary excretion of fentanyl (FT) and its main metabolite, norfentanyl (Nor-FT), by isotopic fractionation using a capillary gas chromatograph equipped with a surface ionization detector (SID). Urinary FT was determined quantitatively in the range of 0.4-40 ng/ml using deuterium labeled FT (FT- 2 H 19 ), as an internal standard. We also performed isotope dilution analysis of Nor-FT in urine. N-Alkylation was necessary to sensitively detect Nor-FT with SID. Methyl derivative was selected from 3 kinds of N-alkyl derivatives to increase sensitivity and peak resolution, and to prevent interference with urinary compound. Nor-FT concentration was quantitatively determined in the range of 10-400 ng/ml using deuterium labeled Nor-FT (Nor-FT- 2 H 10 ). No endogenous compounds or concomitant drugs interfered with the detection of FT and Nor-FT in the urine of patients. The present method will be useful for pharmacokinetic studies and the evaluation of drug interactions in FT metabolism. (author)

  3. Contribution of stable isotopes to the study of pharmacokinetics of magnesium salts; Apport des isotopes stables a l'etude de la pharmacocinetique de sels de magnesium

    Energy Technology Data Exchange (ETDEWEB)

    Benech, H

    1999-05-28

    The use of stable isotopes as labels is becoming an attractive tool for the study of magnesium behavior in humans. It has been used two stable isotopes of magnesium, {sup 25}Mg and {sup 26}Mg, to measure the absolute bioavailability of a pharmaceutical form of magnesium. (N.C.)

  4. Proceedings of the Conference on Isotopic and Molecular Processes

    International Nuclear Information System (INIS)

    Pamula, A.

    1999-01-01

    The proceedings of the Conference on Isotopic and Molecular Processes held on September 23 - 25, 1999 in Cluj - Napoca, Romania contains 8 plenary lectures, 12 oral presentations and 34 posters on isotopic processes (Section A) and 12 oral presentations plus 61 posters on molecular processes (Section B). The main topics treated in plenary lectures were isotope production, separation and enrichment as well as stable isotope applications. Also in this section studies on isotope effects in different fields are reported. In the section A, besides reports on isotope effects, exchange and separation, new methods of preparation and labelling compounds used particularly in nuclear medicine are presented. Also environmental studies by means of stable isotope and radon monitoring are described. In the section B several communications are treating the applications of radiation effects and different nuclear methods in medicine

  5. Quantifying the effect of plant growth on litter decomposition using a novel, triple-isotope label approach

    Science.gov (United States)

    Ernakovich, J. G.; Baldock, J.; Carter, T.; Davis, R. A.; Kalbitz, K.; Sanderman, J.; Farrell, M.

    2017-12-01

    Microbial degradation of plant detritus is now accepted as a major stabilizing process of organic matter in soils. Most of our understanding of the dynamics of decomposition come from laboratory litter decay studies in the absence of plants, despite the fact that litter decays in the presence of plants in many native and managed systems. There is growing evidence that living plants significantly impact the degradation and stabilization of litter carbon (C) due to changes in the chemical and physical nature of soils in the rhizosphere. For example, mechanistic studies have observed stimulatory effects of root exudates on litter decomposition, and greenhouse studies have shown that living plants accelerate detrital decay. Despite this, we lack a quantitative understanding of the contribution of living plants to litter decomposition and how interactions of these two sources of C build soil organic matter (SOM). We used a novel triple-isotope approach to determine the effect of living plants on litter decomposition and C cycling. In the first stage of the experiment, we grew a temperate grass commonly used for forage, Poa labillardieri, in a continuously-labelled atmosphere of 14CO2 fertilized with K15NO3, such that the grass biomass was uniformly labelled with 14C and 15N. In the second stage, we constructed litter decomposition mescososms with and without a living plant to test for the effect of a growing plant on litter decomposition. The 14C/15N litter was decomposed in a sandy clay loam while a temperate forage grass, Lolium perenne, grew in an atmosphere of enriched 13CO2. The fate of the litter-14C/15N and plant-13C was traced into soil mineral fractions and dissolved organic matter (DOM) over the course of nine weeks using four destructive harvests of the mesocosms. Our preliminary results suggest that living plants play a major role in the degradation of plant litter, as litter decomposition was greater, both in rate and absolute amount, for soil mesocosms

  6. Solid state NMR of isotope labelled murine fur: a powerful tool to study atomic level keratin structure and treatment effects

    Energy Technology Data Exchange (ETDEWEB)

    Wong, Wai Ching Veronica; Narkevicius, Aurimas; Chow, Wing Ying; Reid, David G.; Rajan, Rakesh [University of Cambridge, Department of Chemistry (United Kingdom); Brooks, Roger A. [University of Cambridge, Department of Trauma and Orthopaedic Surgery, Addenbrooke’s Hospital (United Kingdom); Green, Maggie [University of Cambridge, Central Biomedical Resources, School of Clinical Medicine (United Kingdom); Duer, Melinda J., E-mail: mjd13@cam.ac.uk [University of Cambridge, Department of Chemistry (United Kingdom)

    2016-10-15

    We have prepared mouse fur extensively {sup 13}C,{sup 15}N-labelled in all amino acid types enabling application of 2D solid state NMR techniques which establish covalent and spatial proximities within, and in favorable cases between, residues. {sup 13}C double quantum–single quantum correlation and proton driven spin diffusion techniques are particularly useful for resolving certain amino acid types. Unlike 1D experiments on isotopically normal material, the 2D methods allow the chemical shifts of entire spin systems of numerous residue types to be determined, particularly those with one or more distinctively shifted atoms such as Gly, Ser, Thr, Tyr, Phe, Val, Leu, Ile and Pro. Also the partial resolution of the amide signals into two signal envelopes comprising of α-helical, and β-sheet/random coil components, enables resolution of otherwise overlapped α-carbon signals into two distinct cross peak families corresponding to these respective secondary structural regions. The increase in resolution conferred by extensive labelling offers new opportunities to study the chemical fate and structural environments of specific atom and amino acid types under the influence of commercial processes, and therapeutic or cosmetic treatments.

  7. Validation of the doubly labeled water method using off-axis integrated cavity output spectroscopy and isotope ratio mass spectrometry.

    Science.gov (United States)

    Melanson, Edward L; Swibas, Tracy; Kohrt, Wendy M; Catenacci, Vicki A; Creasy, Seth A; Plasqui, Guy; Wouters, Loek; Speakman, John R; Berman, Elena S F

    2018-02-01

    When the doubly labeled water (DLW) method is used to measure total daily energy expenditure (TDEE), isotope measurements are typically performed using isotope ratio mass spectrometry (IRMS). New technologies, such as off-axis integrated cavity output spectroscopy (OA-ICOS) provide comparable isotopic measurements of standard waters and human urine samples, but the accuracy of carbon dioxide production (V̇co 2 ) determined with OA-ICOS has not been demonstrated. We compared simultaneous measurement V̇co 2 obtained using whole-room indirect calorimetry (IC) with DLW-based measurements from IRMS and OA-ICOS. Seventeen subjects (10 female; 22 to 63 yr) were studied for 7 consecutive days in the IC. Subjects consumed a dose of 0.25 g H 2 18 O (98% APE) and 0.14 g 2 H 2 O (99.8% APE) per kilogram of total body water, and urine samples were obtained on days 1 and 8 to measure average daily V̇co 2 using OA-ICOS and IRMS. V̇co 2 was calculated using both the plateau and intercept methods. There were no differences in V̇co 2 measured by OA-ICOS or IRMS compared with IC when the plateau method was used. When the intercept method was used, V̇co 2 using OA-ICOS did not differ from IC, but V̇co 2 measured using IRMS was significantly lower than IC. Accuracy (~1-5%), precision (~8%), intraclass correlation coefficients ( R = 0.87-90), and root mean squared error (30-40 liters/day) of V̇co 2 measured by OA-ICOS and IRMS were similar. Both OA-ICOS and IRMS produced measurements of V̇co 2 with comparable accuracy and precision compared with IC.

  8. Visualizing Microbial Biogeochemistry: NanoSIMS and Stable Isotope Probing (Invited)

    Science.gov (United States)

    Pett-Ridge, J.; Weber, P. K.

    2009-12-01

    Linking phylogenetic information to function in microbial communities is a key challenge for microbial ecology. Isotope-labeling experiments provide a useful means to investigate the ecophysiology of microbial populations and cells in the environment and allow measurement of nutrient transfers between cell types, symbionts and consortia. The combination of Nano-Secondary Ion Mass Spectrometry (NanoSIMS) analysis, in situ labeling and high resolution microscopy allows isotopic analysis to be linked to phylogeny and morphology and holds great promise for fine-scale studies of microbial systems. In NanoSIMS analysis, samples are sputtered with an energetic primary beam (Cs+, O-) liberating secondary ions that are separated by the mass spectrometer and detected in a suite of electron multipliers. Five isotopic species may be analyzed concurrently with spatial resolution as fine as 50nm. A high sensitivity isotope ratio ‘map’ can then be generated for the analyzed area. NanoSIMS images of 13C, 15N and Mo (a nitrogenase co-factor) localization in diazotrophic cyanobacteria show how cells differentially allocate resources within filaments and allow calculation of nutrient uptake rates on a cell by cell basis. Images of AM fungal hyphae-root and cyanobacteria-rhizobia associations indicate the mobilization and sharing (stealing?) of newly fixed C and N. In a related technique, “El-FISH”, stable isotope labeled biomass is probed with oligonucleotide-elemental labels and then imaged by NanoSIMS. In microbial consortia and cyanobacterial mats, this technique helps link microbial structure and function simultaneously even in systems with unknown and uncultivated microbes. Finally, the combination of re-engineered universal 16S oligonucleotide microarrays with NanoSIMS analyses may allow microbial identity to be linked to functional roles in complex systems such as mats and cellulose degrading hindgut communities. These newly developed methods provide correlated

  9. Contribution of stable isotopes to the study of pharmacokinetics of magnesium salts

    International Nuclear Information System (INIS)

    Benech, H.

    1999-01-01

    The use of stable isotopes as labels is becoming an attractive tool for the study of magnesium behavior in humans. It has been used two stable isotopes of magnesium, 25 Mg and 26 Mg, to measure the absolute bioavailability of a pharmaceutical form of magnesium. (N.C.)

  10. Antibodies and isotopes, a chemical approach to tumour targeting

    International Nuclear Information System (INIS)

    Vaughan, A.T.M.; Yankuba, S.C.S.; Anderson, P.

    1986-01-01

    In this study, scandium-47 and yttrium-90 have been used as representatives of potential cytotoxic labels. Both isotopes have a high yield of energetic beta particles and half-lives of the same order as indium-111. In addition they are both members of Group III and so may be used as a base for chemical comparisons in the future with radiotoxic isotopes from other chemical groups

  11. Sulfur Isotope Exchange between S-35 Labeled Inorganic Sulfur-Compounds in Anoxic Marine-Sediments

    DEFF Research Database (Denmark)

    FOSSING, H.; THODEANDERSEN, S.; JØRGENSEN, BB

    1992-01-01

    of isotope exchange, specific radioactivities of the reduced sulfur pools were poorly defined and could not be used to calculate their rates of formation. Such isotope exchange reactions between the reduced inorganic sulfur compounds will affect the stable isotope distribution and are expected to decrease...

  12. Are isotope pairs in inorganic electrolyte systems comparable with ion pairs

    International Nuclear Information System (INIS)

    Heumann, K.G.; Gindner, F.; Hoffmann, R.; Kloeppel, H.; Schwarz, A.

    1977-03-01

    Intensive studies on the causes of isotope effects in electrolyte studies have been carried out with the final target of making possible chemical pre-enrichment of stable isotopes which are of increasing importance for labelling purposes. The findings are also of general interest for the behaviour of ions in solutions. (orig.) [de

  13. Study of isotopic exchange reactions of azidothymidine with tritium

    International Nuclear Information System (INIS)

    Sidorov, G.V.; Zverkov, Yu.B.; Myasoedov, N.F.

    2003-01-01

    Different reactions of isotopic exchange of azidothymidine (3 - azido-3 - desoxythymidine) with tritium, such as solid- and liquid-phase catalytic isotopic exchange with gaseous tritium and isotopic exchange in solution with tritium water, are investigated. It is determined that catalytic reactions of azidothymidine with gaseous tritium in solution lead to practically full reduction of azido group up to amino group. In reactions of solid-phase catalytic hydrogenation this process takes place too and 3 - azido-3 - desoxythymidine yield is from 20 to 70 %. Molar radioactivity of labelled with tritium azidothymidine prepared in reactions of solid-phase catalytic isotopic exchange with gaseous tritium and so by isotopic exchange in solution with tritium water does not exceed 0.5 Cu/mmol [ru

  14. Isotope effects in pericyclic reactions, ch. 2

    International Nuclear Information System (INIS)

    Dolbier, W.R. Jr.

    1975-01-01

    A distinction is made between primary and secondary isotope effects, the primary ones being based on the relative large vibrational frequencies and ensuing large zeroprint energies for C-H vibrations whereas the secondary effects are observed in systems where the vibrational modes of the isotopically labelled site are perturbed during transformation from reactant to product. Both effects are utilised in the elucidation of mechanism in pericyclic processes. The main processes dealt with in this chapter are cycloadditions of all types and sigmatropic rearrangements

  15. Synthesis of coenzyme A and nicotineamide-adenine dinucleotide labelled with tritium

    International Nuclear Information System (INIS)

    Sidorov, G.V.; Zverkov, Yu.B.; Myasoedov, N.F.

    1999-01-01

    Isotopic exchange in solution with tritium water and with gaseous tritium and solid-phase reaction of isotopic exchange of NAD with tritium were investigated. For synthesis of labelled with tritium coenzyme A solid-phase reaction of isotopic exchange with gaseous tritium was used. It was determined that 98% of tritium was contained in nicotineamide part of molecule of NAD. In the case of coenzyme A studying of intramolecular distribution of tritium demonstrated that 90% of tritium were localized in adenine fragment [ru

  16. Determination of photooxygenation products of rotenone with isotope dilution method

    International Nuclear Information System (INIS)

    Chubachi, Mitsuo; Hamada, Masayuki

    1975-01-01

    When rotenone dissolved in certain solvent was photochemically oxidized, rotenolones, dehydrorotenone and rotenonone were obtained as main products. In order to determine the quantitative yields of these compounds in photooxygenation products, four compounds mentioned above were labeled with carbon-14 and the isotope dilution method by these labeled compounds was applied to the product analysis. (auth.)

  17. Problems and prospects in future applications of stable isotopes in the life sciences and medicine

    International Nuclear Information System (INIS)

    Matwiyoff, N.A.; Unkefer, C.J.; Walker, T.E.

    1982-01-01

    In the last decade, there has been a resurgence of interest in the use of stable isotopes of carbon, oxygen, and nitrogen in the life sciences and medicine fueled by the increased availability of the isotopes and isotopically labeled compounds and of instruments for their detection. Accelerated development of 13 C, 15 N, and 17 18 O can be expected in the future for studies of drug bioavailability, nutrition and body protein economy, viability of organs for transplant, and for non-invasive tests of metabolic diseases and dysfunctions. These accelerated developments depend on continued improvements in nmr and ms instrumentation and in methods for the synthesis of isotopically labeled compounds. The main part of this paper explores the possibilities of biosynthesis for the selective enrichment of natural products, especially amino acids, with 13 C

  18. Isotope methods in hydrology

    International Nuclear Information System (INIS)

    Moser, H.; Rauert, W.

    1980-01-01

    Of the investigation methods used in hydrology, tracer methods hold a special place as they are the only ones which give direct insight into the movement and distribution processes taking place in surface and ground waters. Besides the labelling of water with salts and dyes, as in the past, in recent years the use of isotopes in hydrology, in water research and use, in ground-water protection and in hydraulic engineering has increased. This by no means replaces proven methods of hydrological investigation but tends rather to complement and expand them through inter-disciplinary cooperation. The book offers a general introduction to the application of various isotope methods to specific hydrogeological and hydrological problems. The idea is to place the hydrogeologist and the hydrologist in the position to recognize which isotope method will help him solve his particular problem or indeed, make a solution possible at all. He should also be able to recognize what the prerequisites are and what work and expenditure the use of such methods involves. May the book contribute to promoting cooperation between hydrogeologists, hydrologists, hydraulic engineers and isotope specialists, and thus supplement proven methods of investigation in hydrological research and water utilization and protection wherever the use of isotope methods proves to be of advantage. (orig./HP) [de

  19. Multi-column chromatography and the use of isotopes in the study of steroid metabolism

    International Nuclear Information System (INIS)

    Sayegh, J.F.; Vestergaard, P.

    1978-01-01

    Multi-column liquid chromatography is demonstrated to be a technique well suited for isotope experiments involving administration of labelled cortisol. It has potential for secretion rate determinations, for dynamic studies of cortisol metabolism and for work with stable isotopes. (author)

  20. Isotope production

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Dewi M.

    1995-07-15

    Some 2 0% of patients using radiopharmaceuticals receive injections of materials produced by cyclotrons. There are over 200 cyclotrons worldwide; around 35 are operated by commercial companies solely for the production of radio-pharmaceuticals with another 25 accelerators producing medically useful isotopes. These neutron-deficient isotopes are usually produced by proton bombardment. All commonly used medical isotopes can be generated by 'compact' cyclotrons with energies up to 40 MeV and beam intensities in the range 50 to 400 microamps. Specially designed target systems contain gram-quantities of highly enriched stable isotopes as starting materials. The targets can accommodate the high power densities of the proton beams and are designed for automated remote handling. The complete manufacturing cycle includes large-scale target production, isotope generation by cyclotron beam bombardment, radio-chemical extraction, pharmaceutical dispensing, raw material recovery, and labelling/packaging prior to the rapid delivery of these short-lived products. All these manufacturing steps adhere to the pharmaceutical industry standards of Good Manufacturing Practice (GMP). Unlike research accelerators, commercial cyclotrons are customized 'compact' machines usually supplied by specialist companies such as IBA (Belgium), EBCO (Canada) or Scanditronix (Sweden). The design criteria for these commercial cyclotrons are - small magnet dimensions, power-efficient operation of magnet and radiofrequency systems, high intensity extracted proton beams, well defined beam size and automated computer control. Performance requirements include rapid startup and shutdown, high reliability to support the daily production of short-lived isotopes and low maintenance to minimize the radiation dose to personnel. In 1987 a major step forward in meeting these exacting industrial requirements came when IBA, together with the University of Louvain-La-Neuve in Belgium, developed the Cyclone-30

  1. Parallel β-sheet vibrational couplings revealed by 2D IR spectroscopy of an isotopically labeled macrocycle: quantitative benchmark for the interpretation of amyloid and protein infrared spectra.

    Science.gov (United States)

    Woys, Ann Marie; Almeida, Aaron M; Wang, Lu; Chiu, Chi-Cheng; McGovern, Michael; de Pablo, Juan J; Skinner, James L; Gellman, Samuel H; Zanni, Martin T

    2012-11-21

    Infrared spectroscopy is playing an important role in the elucidation of amyloid fiber formation, but the coupling models that link spectra to structure are not well tested for parallel β-sheets. Using a synthetic macrocycle that enforces a two stranded parallel β-sheet conformation, we measured the lifetimes and frequency for six combinations of doubly (13)C═(18)O labeled amide I modes using 2D IR spectroscopy. The average vibrational lifetime of the isotope labeled residues was 550 fs. The frequencies of the labels ranged from 1585 to 1595 cm(-1), with the largest frequency shift occurring for in-register amino acids. The 2D IR spectra of the coupled isotope labels were calculated from molecular dynamics simulations of a series of macrocycle structures generated from replica exchange dynamics to fully sample the conformational distribution. The models used to simulate the spectra include through-space coupling, through-bond coupling, and local frequency shifts caused by environment electrostatics and hydrogen bonding. The calculated spectra predict the line widths and frequencies nearly quantitatively. Historically, the characteristic features of β-sheet infrared spectra have been attributed to through-space couplings such as transition dipole coupling. We find that frequency shifts of the local carbonyl groups due to nearest neighbor couplings and environmental factors are more important, while the through-space couplings dictate the spectral intensities. As a result, the characteristic absorption spectra empirically used for decades to assign parallel β-sheet secondary structure arises because of a redistribution of oscillator strength, but the through-space couplings do not themselves dramatically alter the frequency distribution of eigenstates much more than already exists in random coil structures. Moreover, solvent exposed residues have amide I bands with >20 cm(-1) line width. Narrower line widths indicate that the amide I backbone is solvent

  2. Stable isotope utilization for research on human nutrition

    International Nuclear Information System (INIS)

    Desjeux, J.F.

    1994-01-01

    In the framework of nutritional molecule metabolism research, this paper presents the various stable isotopes used as labels for biological molecules, the reasons for their application in human nutritional study (mainly because of their non toxicity) and the various analysis methods (isotope ratio mass spectrometry, coupled gaseous chromatography and mass spectrometry, nuclear magnetic resonance). Several application examples in nutrition research are then discussed: metabolic conversion measurement for a molecule into its different metabolites, energetic losses. 23 refs

  3. LC/MS Method for the Determination of Stable Isotope Labeled Promethazine in Human Plasma

    Science.gov (United States)

    Zuwei, Wang; Boyd, Jason; Berens, Kurt L.; Putcha, Lakshmi

    2004-01-01

    Promethazine (PMZ) is taken by astronauts orally (PO), intramuscularly (IM) or rectally (PR) for space motion sickness. LC/MS method was developed with off-line solid phase extraction to measure plasma concentrations of PMZ given as stable isotope-labeled (SIL) formulations by the three different routes of administration simultaneously. Samples (0.5ml) were loaded on to Waters Oasis HLB co-polymer cartridges and eluted with 1.0 mL methanol. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 6 min. Acetonitrile/ ammonium acetate (30 mM) in water (3:2, v/v), pH 5.6 plus or minus 0.1, was used as the mobile phase for separation. Concentrations of PMZ, PMZ-d4 and PMZ-d7 and chlorpromazine (internal standard) were determined using a Micromass ZMD single quadrupole mass spectrometer with Electrospray Ionization (ESI). ESI mass spectra were acquired in positive ion mode with selected ion monitoring of [M+ H]dot plus. The method is rapid, reproducible and the assay specific parameters are listed in a table. A novel, sensitive and specific method for the measurement of PMZ and SIL PMZ in human plasma is reported.

  4. Stable isotopes. Enriched wheat: a new chance for nutrition research

    International Nuclear Information System (INIS)

    Chagvardieff, P.

    1996-01-01

    The Department of Plant Eco-physiology (DEV) from the CEA/Life Sciences Department of Cadarache (France) has artificially produced two kg of carbon 13 labelled wheat for nutrition research. It is the first successful stable isotope labelling of complex nutriments with a 10% enrichment in carbon 13. This wheat has been used for the manufacturing of pastas to follow the assimilation of nutrients by the organism. This short paper gives some details about the experimental procedure of labelled wheat cultivation. (J.S.)

  5. The development of a decision analytic model of changes in mean deviation in people with glaucoma: the COA model.

    Science.gov (United States)

    Kymes, Steven M; Lambert, Dennis L; Lee, Paul P; Musch, David C; Siegfried, Carla J; Kotak, Sameer V; Stwalley, Dustin L; Fain, Joel; Johnson, Chris; Gordon, Mae O

    2012-07-01

    To create and validate a statistical model predicting progression of primary open-angle glaucoma (POAG) assessed by loss of visual field as measured in mean deviation (MD) using 3 landmark studies of glaucoma progression and treatment. A Markov decision analytic model using patient level data described longitudinal MD changes over 7 years. Patient-level data from the Collaborative Initial Glaucoma Treatment Study (n = 607), the Ocular Hypertension Treatment Study (OHTS; n = 148; only those who developed POAG in the first 5 years of OHTS) and Advanced Glaucoma Intervention Study (n = 591), the COA model. We developed a Markov model with transition matrices stratified by current MD, age, race, and intraocular pressure categories and used a microsimulation approach to estimate change in MD over 7 years. Internal validation compared model prediction for 7 years to actual MD for COA participants. External validation used a cohort of glaucoma patients drawn from university clinical practices. Change in visual field as measured in MD in decibels (dB). Regressing the actual MD against the predicted produced an R(2) of 0.68 for the right eye and 0.63 for the left. The model predicted ending MD for right eyes of 65% of participants and for 63% of left eyes within 3 dB of actual results at 7 years. In external validation the model had an R(2) of 0.79 in the right eye and 0.77 in the left at 5 years. The COA model is a validated tool for clinicians, patients, and health policy makers seeking to understand longitudinal changes in MD in people with glaucoma. Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

  6. Theoretical Proof and Empirical Confirmation of a Continuous Labeling Method Using Naturally 13C-Depleted Carbon Dioxide

    Institute of Scientific and Technical Information of China (English)

    Weixin Cheng; Feike A. Dijkstra

    2007-01-01

    Continuous isotope labeling and tracing is often needed to study the transformation, movement, and allocation of carbon in plant-soil systems. However, existing labeling methods have numerous limitations. The present study introduces a new continuous labeling method using naturally 13C-depleted CO2. We theoretically proved that a stable level of 13C-CO2 abundance In a labeling chamber can be maintained by controlling the rate of CO2-free air injection and the rate of ambient airflow with coupling of automatic control of CO2 concentration using a CO2 analyzer. The theoretical results were tested and confirmed in a 54 day experiment in a plant growth chamber. This new continuous labeling method avoids the use of radioactive 14C or expensive 13C-enriched CO2 required by existing methods and therefore eliminates issues of radiation safety or unaffordable isotope cost, as well as creating new opportunities for short- or long-term labeling experiments under a controlled environment.

  7. A guide through the computational analysis of isotope-labeled mass spectrometry-based quantitative proteomics data: an application study

    Directory of Open Access Journals (Sweden)

    Haußmann Ute

    2011-06-01

    Full Text Available Abstract Background Mass spectrometry-based proteomics has reached a stage where it is possible to comprehensively analyze the whole proteome of a cell in one experiment. Here, the employment of stable isotopes has become a standard technique to yield relative abundance values of proteins. In recent times, more and more experiments are conducted that depict not only a static image of the up- or down-regulated proteins at a distinct time point but instead compare developmental stages of an organism or varying experimental conditions. Results Although the scientific questions behind these experiments are of course manifold, there are, nevertheless, two questions that commonly arise: 1 which proteins are differentially regulated regarding the selected experimental conditions, and 2 are there groups of proteins that show similar abundance ratios, indicating that they have a similar turnover? We give advice on how these two questions can be answered and comprehensively compare a variety of commonly applied computational methods and their outcomes. Conclusions This work provides guidance through the jungle of computational methods to analyze mass spectrometry-based isotope-labeled datasets and recommends an effective and easy-to-use evaluation strategy. We demonstrate our approach with three recently published datasets on Bacillus subtilis 12 and Corynebacterium glutamicum 3. Special focus is placed on the application and validation of cluster analysis methods. All applied methods were implemented within the rich internet application QuPE 4. Results can be found at http://qupe.cebitec.uni-bielefeld.de.

  8. Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology.

    Science.gov (United States)

    von Bergen, Martin; Jehmlich, Nico; Taubert, Martin; Vogt, Carsten; Bastida, Felipe; Herbst, Florian-Alexander; Schmidt, Frank; Richnow, Hans-Hermann; Seifert, Jana

    2013-10-01

    The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ((13)C, (15)N, (36)S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.

  9. Isotope ratios as pollutant source and behaviour indicators

    International Nuclear Information System (INIS)

    1975-01-01

    Recent years have witnessed significant advances in isotope techniques for identifying origins and for studying the behaviour of trace contaminants and pollutants of the environment under actual existing environmental conditions. Improvements in the supply of stable isotopes and their labelled compounds, instrumental analysis and information on stable or radioactive isotopic ratios of existing environmental contaminants as a function of origin or behaviour have provided relatively new tools for the environmental scientist. While variations in natural or existing environmental stable and radioactive nuclides could be regarded as 'background noise' in conventional tracer experiments they promised unique information about sources and behaviour to those who listened carefully. (author)

  10. Progress in stable isotope analysis and new possibilities of clinical investigations

    International Nuclear Information System (INIS)

    Roth, E.

    1989-01-01

    The use of stable isotopes in medicine rests on three possibilities offered by labelling: identification of an element, a molecule, or a fragment of a molecule along its biological pathway; quantification of biological pools by isotopic dilution; measurement of metabolization rates, and more generally of clearances. Whenever a corporal function experiences a disregulation reflected either by changes in metabolic activity or modifications of the importance of pools of certain molecules, the possibility exists of making use of isotopes in diagnosis. Examples of practical applications of stable isotopes are given and analytical problems that had to be solved are underlined

  11. Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jordan N.; Tyrrell, Kimberly J.; Hansen, Joshua R.; Thomas, Dennis G.; Murphree, Taylor A.; Shukla, Anil K.; Luders, Teresa; Madden, James M.; Li, Yunying; Wright, Aaron T.; Piehowski, Paul D.

    2017-12-06

    Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins, and quantify heavy:light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ~70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein

  12. Synthesis of {sup 13}C- and {sup 14}C-labeled 1192U90, an ortho-amino benzamide with a preclinical atypical antipsychotic profile

    Energy Technology Data Exchange (ETDEWEB)

    Norman, M.H.; Gabriel, S.D. [Glaxo Wellcome Inc., Research Triangle Park, NC (United States)

    1996-03-01

    Three isotopic forms of potential antipsychotic agent 1192U90 (2-amino-N-(4-(4-(1,2-benzisthiazol-3-yl)-piperazinyl)butyl)benzam ide) were synthesized: one containing {sup 13}C-isotopes and two containing {sup 14}C-isotopes. The compound in which the ortho-amino benzamide ring is completely {sup 13}C-labeled was prepared in a four-step sequence starting from [{sup 13}C{sub 6}]aniline. The {sup 14}C-labeled compounds were prepared by methods analogous to those previously described for the unlabeled material. The key step involved the condensation of 3-(4-(4aminobutyl)-1-piperazinyl)-1,2-benzisothiazole with isatoic anhydride. The first {sup 14}C-labeled compound (3) was prepared from {sup 14}C-labeled 3-(4-(4-aminobutyl)-1-piperazinyl)-1,2-benzisothiazole, while the second compound (4) derived its isotopic label from [{sup 14}C]isatoic anhydride. Compound 3 had a specific activity of 26.55 mCi/mmol, a radiochemical purity of 99.3%, and a radiochemical yield of 3.4%. Compound 4 had a specific activity of 22.67 mCi/mmol and a radiochemical purity of 99.2%. (author).

  13. Biomolecular tracing using long-lived isotopes

    International Nuclear Information System (INIS)

    Vogel, J.S.; Turteltaub, K.W.; Frantz, C.E.; Keating, G.; Felton, J.S.; Southon, J.R.; Roberts, M.R.; Gledhill, B.L.

    1992-01-01

    Accelerator mass spectrometry (AMS) was developed over the past 15 years as an essential tool for detecting long-lived, cosmogenic radio-isotopes in the earth and space sciences. We apply this technology to the measurement of chemical kinetics, primarily in biomedical systems, which had heretofore employed short-lived isotopes and/or long counting times to quantify radio-isotopic labels. AMS provides detection efficiencies of ∼ 1%, 10 3 to 10 6 better than decay-counting. Long-lived isotopes are used and detected with AMS at concentrations which reduce sample size, chemical dose, radiation safety hazards and radiolysis. We measure 3 H, 7,1O Be, 14 C, 26 Al, 36 CI, 41 Ca and 129 I, but most of our current program uses 14 C. Initial experiments involved research on the genotoxicity of mutagens in cooked foods and reversible binding of compounds to antibodies. Through collaborations, we apply AMS detection to research in carcinogenesis, pharmacokinetics of toxins, elemental metabolism, distribution of topical medications and nutrition

  14. Liberdade e coação no direito de Kant

    Directory of Open Access Journals (Sweden)

    Pinheiro, Celso de Moraes

    2007-01-01

    Full Text Available Kant divide a filosofia moral em duas partes: Ética e Teoria da Justiça. Cada uma é compota de diferentes descrições de deveres e direitos. A ética contém deveres e direitos internos, voluntários e não-coercitivos. A teoria da justiça contém deveres e direitos externos e coercitivos. Os dois tipos de deveres e direitos são definidos em sua relação um com o outro. O que distingue os deveres éticos, ou deveres de virtude, dos deveres jurídicos, é que a compulsão externa para o dever de virtude é baseado na livre coerção própria. Assim, a finalidade deste artigo é pesquisar a noção de dever, e a relação entre dever, liberdade e coação

  15. Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography–tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jingshun [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China); Lai, Shiyun [Beingmate Research Institute, Beingmate Baby and Child Food Co., Ltd., Hangzhou 310007 (China); Cai, Zengxuan [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China); Chen, Qi [Beingmate Research Institute, Beingmate Baby and Child Food Co., Ltd., Hangzhou 310007 (China); Huang, Baifen [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China); Ren, Yiping, E-mail: renyiping@263.net [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China)

    2014-06-01

    Highlights: • A UHPLC–MS/MS method for quantification of bovine lactoferrin was developed. • Tryptic fragment LRPVAAEIYGTK was chosen as signature peptide of bovine lactoferrin. • A winged peptide containing isotopically-labeled signature peptide was designed as internal standard. • The method for determining lactoferrin does not discriminate between the different forms of lactoferrin. • Meet the growing demand to quantify bovine lactoferrin in different dairy products. Abstract: A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3 mg/100 g and 1.0 mg/100 g, respectively. Bovine lactoferrin within the concentration range of 10–1000 nmol L⁻¹ showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD < 6.5% and RSD < 7.1%, respectively. Excellent repeatability (RSD < 6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present method

  16. Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography–tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard

    International Nuclear Information System (INIS)

    Zhang, Jingshun; Lai, Shiyun; Cai, Zengxuan; Chen, Qi; Huang, Baifen; Ren, Yiping

    2014-01-01

    Highlights: • A UHPLC–MS/MS method for quantification of bovine lactoferrin was developed. • Tryptic fragment LRPVAAEIYGTK was chosen as signature peptide of bovine lactoferrin. • A winged peptide containing isotopically-labeled signature peptide was designed as internal standard. • The method for determining lactoferrin does not discriminate between the different forms of lactoferrin. • Meet the growing demand to quantify bovine lactoferrin in different dairy products. - Abstract: A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3 mg/100 g and 1.0 mg/100 g, respectively. Bovine lactoferrin within the concentration range of 10–1000 nmol L −1 showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD < 6.5% and RSD < 7.1%, respectively. Excellent repeatability (RSD < 6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present

  17. Validation of a dual-isotope plasma ratio method for measurement of cholesterol absorption in rats

    International Nuclear Information System (INIS)

    Zilversmit, D.B.; Hughes, L.B.

    1974-01-01

    Several methods for measuring cholesterol absorption in the rat have been compared. After administration of an oral dose of labeled cholesterol ( 14 C or 3 H) and an intravenous dose of colloidal labeled cholesterol ( 3 H or 14 C) the ratio of the two labels in plasma or whole blood 48 hr or more after dosing compared closely to the ratio of areas under the respective specific activity-time curves. The area ratio method is independent of a time lag between the appearance of oral and intravenous label in the bloodstream. Both measures of cholesterol absorption agree fairly well with a method based on measuring the unabsorbed dietary cholesterol in a pooled fecal sample. The plasma isotope ratio method gave more reproducible results than the fecal collection method when the measurement was repeated in the same animals 5 days after the first measurement. Cholesterol absorption was overestimated by the use of Tween 20-solubilized labeled cholesterol for the intravenous dose. The plasma disappearance curves of injected labeled colloidal cholesterol and cholesterol-labeled chylomicrons infused intravenously over a 3.5-h period in the same animal coincided within experimental error from the first day until 75 days after injection. The plasma isotope ratio method for cholesterol absorption gave the same results in rats practicing coprophagy as in those in which this practice was prevented. The addition of sulfaguanidine to the diet lowered cholesterol absorption as measured by the plasma isotope ratio to the same degree as that measured by the fecal collection method. (U.S.)

  18. Syntheses of therapeutically active labelled molecules for metabolic and pharmacokinetic studies. Synthesis, preservation and radiochemical purity problems

    International Nuclear Information System (INIS)

    Pichat, L.

    1977-01-01

    Molecules labelled with radioactive isotopes are without question an essential tool for metabolic and pharmacokinetic studies. Carbon 14 is often preferred to tritium since it allows better observation of the fate of carbonated structures. The fact that 14 CO 2 alone is used as basic material is the distinguishing feature of syntheses with isotopic carbon. In many cases the synthesis schemes of labelled drugs diverge considerably or entirely from those normally adopted for the unlabelled product. It is usually necessary to work on micro-quantities in order to maintain high specific activities, which implies the use of special synthesis techniques and of chromatographic separation and purification methods. Radiochemical purity tests are carried out by thin layer, column and gas phase chromatography, purity and identity checks by mass spectrometry and by 13 C and proton RMN. Labelled products are radiolysed by interaction of excited species with the molecules of the compound, a phenomenon much faster with tritiated than with 14 C-labelled molecules. The radiolysis rates may be reduced by molecule dilution. For ethical reasons it is not convenient to use 14 C molecules in human experiments, but molecular labelled with stable isotopes ( 13 C, 15 N, D) can serve instead [fr

  19. Seasonal patterns of carbon allocation to respiratory pools in 60-yr-old deciduous (Fagus sylvatica) and evergreen (Picea abies) trees assessed via whole-tree stable carbon isotope labeling.

    Science.gov (United States)

    Kuptz, Daniel; Fleischmann, Frank; Matyssek, Rainer; Grams, Thorsten E E

    2011-07-01

    • The CO(2) efflux of adult trees is supplied by recent photosynthates and carbon (C) stores. The extent to which these C pools contribute to growth and maintenance respiration (R(G) and R(M), respectively) remains obscure. • Recent photosynthates of adult beech (Fagus sylvatica) and spruce (Picea abies) trees were labeled by exposing whole-tree canopies to (13) C-depleted CO(2). Label was applied three times during the year (in spring, early summer and late summer) and changes in the stable C isotope composition (δ(13) C) of trunk and coarse-root CO(2) efflux were quantified. • Seasonal patterns in C translocation rate (CTR) and fractional contribution of label to CO(2) efflux (F(Label-Max)) were found. CTR was fastest during early summer. In beech, F(Label-Max) was lowest in spring and peaked in trunks during late summer (0.6 ± 0.1, mean ± SE), whereas no trend was observed in coarse roots. No seasonal dynamics in F(Label-Max) were found in spruce. • During spring, the R(G) of beech trunks was largely supplied by C stores. Recent photosynthates supplied growth in early summer and refilled C stores in late summer. In spruce, CO(2) efflux was constantly supplied by a mixture of stored (c. 75%) and recent (c. 25%) C. The hypothesis that R(G) is exclusively supplied by recent photosynthates was rejected for both species. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  20. Impact of transamination reactions and protein turnover on labeling dynamics in C-13-labeling experiments

    DEFF Research Database (Denmark)

    Grotkjær, Thomas; Åkesson, M.; Christensen, Bjarke

    2004-01-01

    A dynamic model describing carbon atom transitions in the central metabolism of Saccharomyces cerevisiae is used to investigate the influence of transamination reactions and protein turnover on the transient behavior of C-13-labeling chemostat experiments. The simulations performed suggest...... that carbon exchange due to transamination and protein turnover can significantly increase the required time needed for metabolites in the TCA cycle to reach isotopic steady state, which is in agreement with published experimental observations. On the other hand, transamination and protein turnover will speed...... behavior until after three residence times. These observations suggest that greater caution should be used while also pointing to new opportunities in the design and interpretation of C-13-labeling experiments....

  1. Synthesis and Preliminary Biological Evaluations of Fluorescent or 149Promethium Labeled Trastuzumab-Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Jonathan Fitzsimmons

    2015-12-01

    Full Text Available Background: Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody. Methods: The multistep synthesis of a fluorescent or 149Promethium-labeled Trastuzumab-polyethyleneimine (PEI, Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds. Results: Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab was concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with 149Promethium and conjugated to Trastuzumab. The purified 149Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM. Conclusion: The results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.

  2. Labelling of eggs of Ceratitis capitata (Wiedmann) through radioactive sperm ( 32p)

    International Nuclear Information System (INIS)

    Wiendl, F.M.; Pacheco, J.M.

    1975-06-01

    The labelling of Med-fly eggs, using the sperm in the transmission of radioisotope 32 P is described. A hundred hatched couples were used, the males fed on a diet made of 5 g sugar and 1.66g of hydrolized protein. This diet was labelled with a solution of Na 2 HPO 4 , in which the atom of phosphorus was labelled with 32 P isotope. It showed an activity calculated at 343,9 μCi. Statistical treatment of the data indicated that the eggs became labelled and remained labelled until the 5th day after mating, even on eggs laid by female who mated with untreated males

  3. Quantitation of some amino-terminal residues in proteins using 3H-labelled dansyl chloride and 14C labelled amino acids

    International Nuclear Information System (INIS)

    Flengsrud, R.

    1979-01-01

    A method for quantitation of amino-terminal residues in proteins is presented. The method is a modification of a double isotope-labelling technique, using 3 H-labelled dansyl chloride and 14 C-labelled amino acids as internal standards. The method is demonstrated on human fibrinogen, horse myoglobin and on mouse myoloma IgA. A linear relationship between the ratio 3 H/ 14 C in the separated amino-terminal amino acid of the protein and the amount of protein added in the labelling mixture was obtained with standard deviations of +- 7.4%, +-3.4% and +-10.3%, respectively. An application of the method is demonstrated by measuring the increase in amino-terminal glycine in fibrinogen following the proteolytic action of thrombin. The method seems to be useful when 0.1 nmol or more of protein is used. (author)

  4. Some methods for labelling organic compounds by deuterium

    International Nuclear Information System (INIS)

    Moustapha, C.

    1988-01-01

    The rapid growth of knowledge in the fields of biochemistry, physiology, and molecular biology reflects to a considerable degree the utilization of stable isotopes (specially deuterium) in the study of chemical reactions and fragmentation mechanisms in mass spectrometry, as well as in the pharmacological and biological studies. Organic compounds maybe labelled by deuterium through classic organic reactions by using special deuterated solvents and reagents. This article discusses some reactions, with examples on how to prepare labelled compounds with high isotopic purety. These reactions are: exchange reactions in acid and alkaline media (the exchange in the chromatographic column in liquid and gas phases, the exchange in homogenous medium), reduction reactions of functional groups as well as saturation of the double bounds by deuterium using hydrogenation catalystes, electrochemical reactions using KOLBE, and photochemical reactions. This article also deals with spectroscopic properties of deuterium and the methods which are used to identify its compounds such as infrared, nuclear magnetic resonance, and mass spectroscopy. 37 refs., 2 figs

  5. The doubly-labelled water method for measuring energy expenditure. Technical recommendations for use in humans

    International Nuclear Information System (INIS)

    Prentice, A.M.

    1990-01-01

    The doubly-labelled water method using stable isotopes of hydrogen and oxygen, is rapidly becoming established as an important new tool for investigating energy metabolism. It is the first genuinely non-invasive method for measuring energy expenditure in free-living people, providing estimates of habitual expenditure over a time period of 10-20 days. The accuracy and precision of these estimates should be superior to those obtained by traditional factorial methods. The DLW method involves several assumptions about the behaviour of the isotopes, the body water pool and the exchange rates within that pool in the labelled animal. These assumptions are: (1) The volume of the body water pool remains constant throughout the measurement period. (2) The rates of water influx, and water and CO 2 efflux are constant throughout the measurement period. (3) The isotopes label only the H 2 O and CO 2 in the body. (4) The isotopes leave the body only in the form of H 2 O and CO 2 . (5) The concentrations of the isotopes in H 2 O and CO 2 leaving the body are the same as those in body water at that time (i.e. there is no isotopic fractionation). (6) No H 2 O or CO 2 that has left the body re-enters the body. (7) The natural abundance, or ''background'' levels of the isotopes remain constant during the measurement interval. The recommendations presented in this document are based on a variety of criteria including: (a) which procedure among several is theoretically correct in a given application; (b) which procedure is simplest and least prone to methodological errors; and (c) which procedure yields the lowest error in validation studies. Refs, figs and tabs

  6. Synthesis of l-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome.

    Science.gov (United States)

    Ono, Katsuhiko; Jung, Minkyung; Zhang, Tianli; Tsutsuki, Hiroyasu; Sezaki, Hiroshi; Ihara, Hideshi; Wei, Fan-Yan; Tomizawa, Kazuhito; Akaike, Takaaki; Sawa, Tomohiro

    2017-05-01

    Cysteine persulfide is an L-cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L-cysteine derivatives containing isotopic sulfur atoms and application of this method to a reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L-cysteine. We cloned three cysteine synthases-CysE, CysK, and CysM-from the Gram-negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized 34 S-labeled L-cysteine from O-acetyl-L-serine and 34 S-labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L-cysteine at both sulfur ( 34 S) and nitrogen ( 15 N) atoms was also achieved by performing enzyme reactions with 15 N-labeled L-serine, acetyl-CoA, and 34 S-labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L-cysteine derivatives including L-cystine, L-cystine persulfide, S-sulfo-L-cysteine, L-cysteine sulfonate, and L-selenocystine. We also prepared 34 S-labeled N-acetyl-L-cysteine (NAC) by incubating 34 S-labeled L-cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low-molecular-weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using 34 S-labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L-cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology. Copyright © 2017 Elsevier Inc

  7. Radio-isotopic tracers

    International Nuclear Information System (INIS)

    Wolfangel, R.G.

    1976-01-01

    The invention concerns the dispersions that may be used for preparing radio-isotopic tracers, technetium labelled dispersions, processes for preparing these dispersions and their use as tracers. Technetium 99m sulphur colloids are utilized as scintillation tracers to give a picture of the reticulo-endothelial system, particularly the liver and spleen. A dispersion is provided which only requires the addition of a radioactive nuclide to form a radioactively labelled dispersion that can be injected as a tracer. It is formed of a colloid of tin sulphur dispersed in an aqueous buffer solution. Such a reagent has the advantage of being safe and reliable and is easier to use. The colloid can be prepared more quickly since additions of several different reagents are avoided. There is no need to heat up and no sulphuretted hydrogen, which is a toxic gas, is used [fr

  8. Kinetic isotope effect in the thermolysis of methylenecyclobutane

    International Nuclear Information System (INIS)

    Chickos, J.S.

    1979-01-01

    The intramolecular kinetic isotope effect for the thermolysis of equilibrated methylenecyclobutane-d 2 was investigated at 515 0 C as a function of pressure. A high-pressure value of k/sub H/k/sub D/ (ethylene/ethylene-d 2 ) = 0.9 was obtained at 13 cm of N 2 pressure. This value decreased to 0.86 at 70 μm total pressure. No intermolecular kinetic isotope effect was measured for the formation of ethylene from labeled and unlabeled methylenecyclobutane. The pressure and temperature dependence of the intramolecular kinetic isotope effect was used as evidence in establishing the inverse nature of the effect. The isotope effect observed was explained in terms of competing equilibrium and kinetic isotope effects in which the equilibrium isotope effects dominate. It was concluded on the bases of these results that an acyclic intermediate is involved in the fragmentation of methylenecyclobutane to ethylene and allene. The results also support the notion that deuterium prefers to accumulate at the methylene group with the greatest p character in the carbon--hydrogen bond. 1 figure, 4 tables

  9. Isotopic analysis of radioactive waste packages (an inexpensive approach)

    International Nuclear Information System (INIS)

    Padula, D.A.; Richmond, J.S.

    1983-01-01

    A computer printout of the isotopic analysis for all radioactive waste packages containing resins, or other aqueous filter media is now required at the disposal sites at Barnwell, South Carolina, and Beatty, Nevada. Richland, Washington requires an isotopic analysis for all radioactive waste packages. The NRC (Nuclear Regulatory Commission), through 10 CFR 61, will require shippers of radioactive waste to classify and label for disposal all radioactive waste forms. These forms include resins, filters, sludges, and dry active waste (trash). The waste classification is to be based upon 10 CFR 61 (Section 1-7). The isotopes upon which waste classification is to be based are tabulated. 7 references, 8 tables

  10. The introduction of tritium in lactose and saccharose by isotope exchange with gaseous tritium

    International Nuclear Information System (INIS)

    Akulov, G.P.; Snetkova, E.V.; Kaminskij, Yu.L.; Kudelin, B.K.; Efimova, V.L.

    1991-01-01

    Methods for conducting reactions of catalytic protium-tritium isotopic exchange with gaseous tritium were developed in order to synthesize tritium labelled lactose and saccharose. These methods enabled to prepare these labelled disaccharides with high molar activity. The yield was equal to 50-60%, radiochemical purity ∼ 95%

  11. Synthesis of Melamine-d6 and the Feasibility of Deuterium Labeled Compounds as Internal Standard

    Directory of Open Access Journals (Sweden)

    GUO Yang-zhen

    2015-11-01

    Full Text Available S-triazine is an important chemical intermediate. Melamine belongs to s-triazine, which has been widely used as an additive in the food industry. To study the stable isotope labeling method of heterocyclic triazine compounds and its application, one step synthesis of melamine-d6 was achieved with a yield of 30% (calculate in ND4OD, which started from ND4OD by the reaction with cyanuric chloride. According to the exchange mechanism of H/D, the feasibility and the necessary conditions were discussed for applying deuterium labeled compounds in isotope dilution mass spectrometry method.

  12. Scintigraphy with labelled analogues of the somatostatin

    International Nuclear Information System (INIS)

    Duet, M.; Ajzenberg, C.; Warnet, A.; Mundler, O.

    1998-01-01

    The receptors of the somatostatin have been localized in a big number of tumors, whom a great number are neuro-endocrine tumors. However, some tumors that have not this differentiation (breast cancer, lymphomas, cerebral tumors) possess them as well. Analogues of somatostatin, labelled with isotopes having a gamma emission, allow from now their detection in vivo. (N.C.)

  13. DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

    Science.gov (United States)

    Jameson, Eleanor; Taubert, Martin; Coyotzi, Sara; Chen, Yin; Eyice, Özge; Schäfer, Hendrik; Murrell, J Colin; Neufeld, Josh D; Dumont, Marc G

    2017-01-01

    Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13 C, 18 O, or 15 N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labeled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labeled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing of clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labeling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, metagenomes, or metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labeled microorganisms. Analysis of labeled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allow the use of labeled substrates at ecologically relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies

  14. Synthesis of deuterium-labelled compounds for FOTEK project; Syntese af deuterium-maerkede forbindelser til FOeTEK projektet

    Energy Technology Data Exchange (ETDEWEB)

    Joergensen, O.; Egsgaard, H.; Larsen, E. [Forskningscenter Risoe, Roskilde (Denmark)

    1996-06-01

    In the FoTech project there have been utilized labelled compounds of stable isotopes as internal standards. Some of these compounds are commercially available ({sup 13}C-labelled PCB congeners, {sup 13}C-labelled diethylstilbestrol for determination of anabolic steroids). Others, like D{sub 9}-clenbuterol, D{sub 3}-clenbuterol, D{sub 3}-zeramol and D{sub 3}-dimetridazol have been synthesized. General aspects of deuterium compounds labelling are considered. (EG).

  15. Consensus structures of the Mo(v) sites of sulfite-oxidizing enzymes derived from variable frequency pulsed EPR spectroscopy, isotopic labelling and DFT calculations.

    Science.gov (United States)

    Enemark, John H

    2017-10-10

    Sulfite-oxidizing enzymes from eukaryotes and prokaryotes have five-coordinate distorted square-pyramidal coordination about the molybdenum atom. The paramagnetic Mo(v) state is easily generated, and over the years four distinct CW EPR spectra have been identified, depending upon enzyme source and the reaction conditions, namely high and low pH (hpH and lpH), phosphate inhibited (P i ) and sulfite (or blocked). Extensive studies of these paramagnetic forms of sulfite-oxidizing enzymes using variable frequency pulsed electron spin echo (ESE) spectroscopy, isotopic labeling and density functional theory (DFT) calculations have led to the consensus structures that are described here. Errors in some of the previously proposed structures are corrected.

  16. Use of the doubly labeled water technique in humans during heavy sustained exercise

    International Nuclear Information System (INIS)

    Westerterp, K.R.; Saris, W.H.; van Es, M.; ten Hoor, F.

    1986-01-01

    We measured energy expenditure with the doubly labeled water technique during heavy sustained exercise in the Tour de France, a bicycle race lasting more than 3 wk. Four subjects were observed for consecutive intervals of 7, 8, and 7 days. Each interval started with an oral isotope dose to reach an excess isotope level of 200 ppm 18O and 130 ppm 2H. The biological half-lives of the isotopes were between 2.25 and 3.80 days. Energy expenditure was compared with simultaneous measurements of energy intake, and body mass and body composition did not change significantly. The doubly labeled water technique gave higher values for energy expenditure than the food record technique. The discrepancy showed a systematic increment from the first to the third interval, being 12.9 +/- 7.9, 21.4 +/- 9.8, and 35.3 +/- 4.4% of the energy expenditure calculated from dietary intake, respectively. Possible explanations for the discrepancy are discussed. The subjects reached an average daily metabolic rate of 3.4-3.9 or 4.3-5.3 times basal metabolic rate based on the food record technique and the doubly labeled water technique, respectively. Thus, when measured with the same technique, the energetic ceiling for performance in humans is comparable with that of animals like birds

  17. Measurement parameter selection for quantitative isotope dilution gas chromatography/mass spectrometry

    International Nuclear Information System (INIS)

    Colby, B.N.; Rosecrance, A.E.; Colby, M.E.

    1981-01-01

    By use of the two-isotope model of isotope dilution, selection criteria were developed for identifying optimum m/z's for quantitation of compounds by gas chromatography/mass spectrometry. In addition, it was possible to predict the optimum ratio of naturally abundant to labeled compound and to identify appropriate data reduction methods. The validity of these predictions was confirmed by using experimental GC/MS data for several organic compounds

  18. Quality Assurance of technitium-labelled radiopharmaceuticals in the Radiation and Isotopes Centre of Khartoum (RICK)

    International Nuclear Information System (INIS)

    Adlan, A.A.

    2005-09-01

    This descriptive, exploratory study was conducted in the nuclear medicine department at the Radiation and Isotopes Center of Khartoum (RICK) during 2003-2005 the aim of the study was explore and define the dimensions of a problem which was regarded as urgent by the people working in the field of nuclear medicine in Sudan. The problem concerned the quality of technitium-labelled radiopharmaceuticals which are used in more than 90% of the nuclear medicine imaging studies performed in nuclear medicine. Impure 9 ''9''m Tc-labelled radiopharmaceuticals may create problems, and could lead to false diagnosis. These agents must be tested for determination of the levels of radionuclides, radiochemical and chemicals, before administration to patients. They should also be sterile and pyrogen-free. A number of data collection methods, were used by the researcher for adequate exploration of the dimensions of the problem including interviews, questionnaires and close observations to all activities related to the preparation of radiopharmaceuticals in the hot laboratory. Information concerning all the aspects of quality assurance were collected. These aspects were management and organisation of the work, equipment and tools, knowledge and practical experience of the staff members and methods of preparation and administration of the radioactive agents. Data from different sources were then compared with observation results for more validation and finally lead to the following results: All the quality control tests were not normally performed in the department, therefore the levels of impurities in these agents were not exactly determined, moreover these preparations were subject to contamination with microorganisms, due to low level of cleanliness at the work area. The study detected a number of defaults which were likely to be the causes behind these problems. These were, bad management and organisation, in availability of equipment, tools and materials necessary for testing

  19. Analysis of a benthic community food web using isotopically labelled potential food

    International Nuclear Information System (INIS)

    Beviss-Challinor, M.H.; Field, J.G.

    1982-01-01

    A series of experiments was designed to reveal the trophic structure of a benthic community using kelp holdfasts as microcosms within the kelp-bed community. The experimental food comprised zooplankton represented by 3 H 2 O-labelled Artemia sp. eggs and nauplii (200 to 300 μm), detritus derived from 14 C-labelled kelp (60 to 90 μm), and phytoplankton represented by 14 C-labelled Dunaliella primolecta (5 to 10 μm) cultures. Separate experiments of short duration (1 to 2 h) were used to indicate the primary consumers on each type of food, while longer experiments (4, 8 and 16 h) suggested the secondary consumers (coprophagous and carnivorous species). Several species were found to be omnivorous. (orig.)

  20. Future trends in the application of isotopes and radiation

    Energy Technology Data Exchange (ETDEWEB)

    Glubrecht, H [Institut fuer Biophysik, Technische Universitaet Hannover (Germany)

    1977-12-15

    Expected trends for the development of new methods and different applications of isotopes in agriculture, medicine and industrial production are discussed. In agriculture stable-isotope-labelled compounds can also be applied in field studies on the fate and metabolic processes of pesticide residues, as well as in long-lasting experiments on animal nutrition. Radioactive isotopes will continue to play a predominant role in trace element investigations, especially studies on the interaction and synergism of various trace elements in soil, plants and animals. The use of radioactive-labelled substrates and reagents will also become indispensable in enzyme research. Of the many other applications of isotopes in agriculture which might develop further, one item should be mentioned: the importance of non-radioactive labelling in ecology. Two areas of radiation application in agriculture may expect some progress from the use of neutron irradiation instead of gamma rays: plant breeding and the sterile insect technique (SIT). The advantage of sterilizing male insects by neutrons instead of gamma rays has become apparent with tsetse flies, which suffer less physiological harm when exposed to moderate neutron doses and, though sterile, remain fully competitive with wild tsetse fly males. There are also considerable further developments to be expected in the medical field. In vivo radionuclide imaging, if done by scanning, can only inform on local distribution of a radioactive-labelled pharmaceutical. Dynamic studies of function and physiologic processes have to be performed by using gamma-cameras which combine spatial localization with time variation. Short-lived radioisotopes, including cyclotron-produced positron emitters, are becoming more available, and the list of industrially manufactured radiopharmaceuticals is increasing. A most important development is tomography with positron emitters using annihilation gamma radiation. One of the most successful methods in clinical

  1. A new combined method of stable isotope-labeling derivatization-ultrasound-assisted dispersive liquid-liquid microextraction for the determination of neurotransmitters in rat brain microdialysates by ultra high performance liquid chromatography tandem mass spectrometry.

    Science.gov (United States)

    Zheng, Longfang; Zhao, Xian-En; Zhu, Shuyun; Tao, Yanduo; Ji, Wenhua; Geng, Yanling; Wang, Xiao; Chen, Guang; You, Jinmao

    2017-06-01

    In this work, for the first time, a new hyphenated technique of stable isotope-labeling derivatization-ultrasound-assisted dispersive liquid-liquid microextraction has been developed for the simultaneous determination of monoamine neurotransmitters (MANTs) and their biosynthesis precursors and metabolites. The developed method was based on ultra high performance liquid chromatography tandem mass spectrometry detection using multiple-reaction monitoring mode. A pair of mass spectrometry sensitizing reagents, d 0 -10-methyl-acridone-2-sulfonyl chloride and d 3 -10-methyl-acridone-2-sulfonyl chloride, as stable isotope probes was utilized to facilely label neurotransmitters, respectively. The heavy labeled MANTs standards were prepared and used as internal standards for quantification to minimize the matrix effects in mass spectrometry analysis. Low toxic bromobenzene (extractant) and acetonitrile (dispersant) were utilized in microextraction procedure. Under the optimized conditions, good linearity was observed with the limits of detection (S/N>3) and limits of quantification (S/N>10) in the range of 0.002-0.010 and 0.015-0.040nmol/L, respectively. Meanwhile, it also brought acceptable precision (4.2-8.8%, peak area RSDs %) and accuracy (recovery, 96.9-104.1%) results. This method was successfully applied to the simultaneous determination of monoamine neurotransmitters and their biosynthesis precursors and metabolites in rat brain microdialysates of Parkinson's disease and normal rats. This provided a new method for the neurotransmitters related studies in the future. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Applications of stable isotopes of 2H, 13C and 15N to clinical problems

    International Nuclear Information System (INIS)

    Klein, P.D.; Szczepanik, P.A.; Hachey, D.L.

    1974-01-01

    The function of the Argonne Program is to provide synthetic, analytical instrumental capability in a core facility for the clinical investigator who needs to use 2 H, 13 C, or 15 N labelled compounds for metabolic or clinical research on pregnant women, newborn infants, young children, or for mass screening. To carry out such application development, there were six stages which were recurrent steps in every application. Five fundamental strategies should be adopted to establish the use of stable isotopes in clinical work. The instrument required for measurements was a combined gas chromatograph-mass spectrometer, and its use was schematically illustrated. Some of the successful experiences with compounds labelled by stable isotopes, such as deuterium labelled chenodeoxycholic acid, and respective 13 C and 15 N-labelled glycine were described. Deutrium labelled bile acid enabled easy and safe determination of the size of the bile acid pool and the replacement rate, providing clearer diagnoses for cholestatic liver disease and gallstones. 13 C and 15 N labelled compounds were used in clinical studies, of children with genetic disorders of amino acid metabolism, i.e., non ketotic hyperflycinemia, B 12 -responsive methyl malonic acidemia, and Lesch-Nyhan syndrome. 15 N-labelled glycine was also studied in a child with Lesch-Nyhan syndrome. (Mukohata, S.)

  3. Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Skelton, David; Goodyear, Abbey [Florida State University, Department of Chemistry and Biochemistry (United States); Ni, DaQun; Walton, Wendy J.; Rolle, Myron; Hare, Joan T. [Florida State University, Institute of Molecular Biophysics (United States); Logan, Timothy M., E-mail: tlogan@fsu.ed [Florida State University, Department of Chemistry and Biochemistry (United States)

    2010-10-15

    NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-{sup 13}C-glucose and {sup 15}N-glutamate as labeled precursors. This study suggests that uniformly {sup 15}N,{sup 13}C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

  4. Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

    International Nuclear Information System (INIS)

    Skelton, David; Goodyear, Abbey; Ni, DaQun; Walton, Wendy J.; Rolle, Myron; Hare, Joan T.; Logan, Timothy M.

    2010-01-01

    NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U- 13 C-glucose and 15 N-glutamate as labeled precursors. This study suggests that uniformly 15 N, 13 C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

  5. Extrinsic labelling of zinc and calcium in bread

    International Nuclear Information System (INIS)

    Fredlund, Kerstin; Rossander-Hulthen, Lena; Isaksson, Mats; Almgren, Annette; Sandberg, A.-S.

    2002-01-01

    This study investigated the effect of different means of extrinsic administration of 65 Zn and 47 Ca in white wheat flour bread on the measured absorption. Eight healthy subjects were served 80 g of labelled bread as a standardized breakfast after an overnight fast on three occasions. Extrinsic labelling of the meals with 65 Zn and 47 Ca was done in three ways: (a) by adding the isotopes to the bread 16 h before it was served, (b) by adding the isotopes shortly before serving or (c) by adding the isotopes to the water used in dough making. Zinc and calcium chloride corresponding to 3.2 mg (49 μmol) zinc and 275 mg (6.9 mmol) calcium in one portion were added to the dough. Whole-body retention was measured by whole-body counting. The fractional absorption of zinc was (a) 0.243±0.122, (b) 0.217±0.101 and (c) 0.178±0.063 (mean±SD), and the fractional absorption of calcium (expressed as calcium retention on day 7) was (a) 0.351±0.108, (b) 0.357±0.131 and (c) 0.334±0.117 (mean±SD). No significant difference (p>0.05) was seen between the different ways for either zinc nor calcium

  6. Plutonium isotope ratios in polychaete worms

    International Nuclear Information System (INIS)

    Beasley, T.M.; Fowler, S.W.

    1976-01-01

    Reference is made to recent reports that suggest that terrestrial and aquatic organisms may preferentially take up 238 Pu compared with sup(239+240)Pu. It is stated that although kinetic isotope effects are known to occur in biological systems for low mass number elements, such as H, C and N, such effects are generally discounted with higher mass numbers, and differences in the biological 'uptake' of isotopes of high mass number elements, such as those of Pu, are normally attributable to differences in the chemical or physical forms of the isotopes or to different quantities of isotopes available to organisms. This has been applied to explain differential Pu isotope behaviour in animals under controlled laboratory conditions, but it is not certain that it can be applied to explain anomalies of Pu isotope behaviour in organisms contaminated by nuclear test debris or by wastes from nuclear fuel reprocessing plants. Geochemical weathering may also have an effect. Described here are experiments in which it was found that deposit feeding marine worms living in sediments contaminated in different ways with Pu isotopes did not show preferential accumulation of 238 Pu. The worms had been exposed to different chemical and physical forms of the isotopes, including exposure to laboratory-labelled sediment, sediment collected from a former weapons test site, and sediment contaminated by wastes from a nuclear fuel reprocessing plant. The worms were allowed to accumulate Pu for times of 5 to 40 days. Isotope ratios were determined by α-spectrometric techniques. It is considered that the results are important for environmental samples where Pu activity levels are low. (U.K.)

  7. Placentography with technetium-99m labelled RBC - an evaluation

    International Nuclear Information System (INIS)

    Jayachandran, C.A.; Benjamin, C.S.; Balakrishnan; Narasimhan

    1983-01-01

    During 1979-80, placentography was performed in 40 cases of pregnant women with third trimester bleeding. Random labelled RBC with sup(99m)Tc of activity 2-3 mci was used, depending upon the weight of the patient. In 98% of cases, the isotope finding was confirmed. (author)

  8. Capillary absorption spectrometer and process for isotopic analysis of small samples

    Energy Technology Data Exchange (ETDEWEB)

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

    2018-04-24

    A capillary absorption spectrometer and process are described that provide highly sensitive and accurate stable absorption measurements of analytes in a sample gas that may include isotopologues of carbon and oxygen obtained from gas and biological samples. It further provides isotopic images of microbial communities that allow tracking of nutrients at the single cell level. It further targets naturally occurring variations in carbon and oxygen isotopes that avoids need for expensive isotopically labeled mixtures which allows study of samples taken from the field without modification. The process also permits sampling in vivo permitting real-time ambient studies of microbial communities.

  9. Capillary absorption spectrometer and process for isotopic analysis of small samples

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-03-29

    A capillary absorption spectrometer and process are described that provide highly sensitive and accurate stable absorption measurements of analytes in a sample gas that may include isotopologues of carbon and oxygen obtained from gas and biological samples. It further provides isotopic images of microbial communities that allow tracking of nutrients at the single cell level. It further targets naturally occurring variations in carbon and oxygen isotopes that avoids need for expensive isotopically labeled mixtures which allows study of samples taken from the field without modification. The method also permits sampling in vivo permitting real-time ambient studies of microbial communities.

  10. Preparation of pyronaridine labelled with tritium

    International Nuclear Information System (INIS)

    Jiang Shangen; Zhang Liufang; Zheng Dongzhu; Feng Zheng; Wu Zufan

    1987-01-01

    Pyronaridine is a high efficient and low toxic new antimalarial drug. 3 H-pyronaridine was prepared by catalytic isotopic exchange in solution with tritium gas using PdO/BaSO 4 as catalyst. That crude product was purified by extraction. 3 H-NMR spectra of pyronaridine showed that tritium was labelled at the 6-position. Specific activity of 3 H-pyronaridine was 5.5 Ci/mmol and radiochemical purity over 95%

  11. Automatic isotope gas analysis of tritium labelled organic materials Pt. 3

    International Nuclear Information System (INIS)

    Gacs, I.; Mlinko, S.; Payer, K.; Otvos, L.; Banfi, D.; Palagyi, T.

    1978-01-01

    An isotope analytical procedure and an automatic instrument developed for the determination of tritium in organic compounds and biological materials by internal gas counting are described. The sample is burnt in a stream of oxygen and the combustion products including water vapour carrying the tritium are led onto a column of molecular sieve-5A heated to 550 deg C. Tritium is retained temporarily on the column, then transferred into a stream of hydrogen by isotope exchange. After addition of butane, the tritiated hydrogen is led into an internal detector and enclosed there for radioactivity measurement. The procedure, providing quantitative recovery, is completed in five minutes. It is free of memory effect and suitable for the determination of tritium in a wide range of organic compounds and samples of biological origin. (author)

  12. Preparation of ⁶⁸Ga-labelled DOTA-peptides using a manual labelling approach for small-animal PET imaging.

    Science.gov (United States)

    Romero, Eduardo; Martínez, Alfonso; Oteo, Marta; García, Angel; Morcillo, Miguel Angel

    2016-01-01

    (68)Ga-DOTA-peptides are a promising PET radiotracers used in the detection of different tumours types due to their ability for binding specifically receptors overexpressed in these. Furthermore, (68)Ga can be produced by a (68)Ge/(68)Ga generator on site which is a very good alternative to cyclotron-based PET isotopes. Here, we describe a manual labelling approach for the synthesis of (68)Ga-labelled DOTA-peptides based on concentration and purification of the commercial (68)Ga/(68)Ga generator eluate using an anion exchange-cartridge. (68)Ga-DOTA-TATE was used to image a pheochromocytoma xenograft mouse model by a microPET/CT scanner. The method described provides satisfactory results, allowing the subsequent (68)Ga use to label DOTA-peptides. The simplicity of the method along with its implementation reduced cost, makes it useful in preclinical PET studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Validation of doubly labeled water for measuring energy expenditure during parenteral nutrition

    International Nuclear Information System (INIS)

    Schoeller, D.A.; Kushner, R.F.; Jones, P.J.

    1986-01-01

    The doubly labeled water method was compared with intake-balance for measuring energy expenditure in five patients receiving total parenteral nutrition (TPN). Because parenteral solutions were isotopically different from local water, patients had to be placed on TPN at least 10 days before the metabolic period. Approximately 0.1 g 2H2O and 0.25 g H2(18)O per kg total body water were given orally. We collected saliva before, 3 h, and 4 h after the dose for measurement of total body water and urine before, 1 day, and 14 days after the dose for measurement of isotope eliminations. On day 14, total body weight was remeasured and change in body energy stores was calculated, assuming constant hydration. Intake was assessed from weights of TPN fluids plus dietary record for any oral intake. Energy expenditure from doubly labeled water (+/- SD) averaged 3 +/- 6% greater than intake-balance. Doubly labeled water method is a noninvasive, nonrestrictive method for measuring energy expenditure in patients receiving TPN

  14. Application of isotope-labelled compounds in the study of the chemical stability of pesticides

    International Nuclear Information System (INIS)

    Roesseler, M.; Luther, D.; Abendroth, H.C.; Koch, H.

    1980-01-01

    The user of pesticides requires specific biological modes of action from the corresponding commercial products. Impurities and degradation products may cause uncontrollable toxicological reactions. Profound knowledge of the chemical stability of the effective substance in question and its formulations under storage conditions as well as under those of analytical sample preparation and detection is required. Radioisotope labelled effective substances dimethoate and 1-butyl-amino-cyclohexane-phosphonic acid dibutyl ester are used to study storage stability of the pure effective substance and its formulations; effects of selected impurities, such as technical by-products, moisture or water content, binding or carrier materials, organic solvents, chemical stabilizers and other formulation components on storage properties; temperature dependence of storage stability; selection of suitable analytical techniques for quantitative determination of the effective substance without interference effects from any by-product; reduction of the necessary analytical expense; disclosure of sources of error in the application of usual analytical techniques; improvement of possibilities of an immediate and clearer discrimination between types and amounts of compounds in a chemical system consisting of one pesticide and its degradation or reaction products at the beginning and at the end of an experimental or reaction period. Radiochemical analytical techniques, such as radio thin-layer chromatography (also combined with liquid scintillation counting), radio gas chromatography, autoradiography and isotope dilution analysis were used. Results are discussed, especially of experiments on dimethoate and its technical by-products

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

    DEFF Research Database (Denmark)

    Sandberg, Troy E.; Long, Christopher P.; Gonzalez, Jacqueline E.

    2016-01-01

    13C-Metabolic flux analysis (13C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia...

  16. The method of obtaining of sodium orthoiodohippurate labelled with iodine-131

    International Nuclear Information System (INIS)

    Aripov, D.; Abdukayumov, M.; Shukurov, A.Sh.

    1994-01-01

    The method of labelling of sodium orthoiodohippurate was elaborated with the purpose of increasing the preparation quality. Method includes the reaction of isotopic exchange between orthoiodhippur acid and sodium iodide solution labelled with iodine-131 with volume activity 150-200 mCu/mL and pH=6,5-7,0. Reaction occurs at temperature 120-130 C during 1,1-1,3 hours and the compound obtained is dissolved in 1% sodium bicarbonate solution. (author)

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

    Science.gov (United States)

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

    2016-04-01

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

  18. Evaluation of Isotope 32P Method to Mark Culex pipiens (Diptera: Culicidae in a Laboratory

    Directory of Open Access Journals (Sweden)

    Chongxing Zhang

    2016-01-01

    Full Text Available Background: The aim of the current study was to develop a marking technique as an internal marker to mark post blood meal mosquitoes by using stable phosphate isotope 32P and determine the optimal concentration of it.Methods: An isotonic physiological saline solution, containing different concentration of radioactive isotope 32P-labeled disodium phosphate (Na2H32PO4 was injected into rabbits via the jugular vein in the laboratory. Emerged Cx. pipiens were marked after feeding on rabbit. At the same time, the labeled conditions of emerged Cx. pipiens were also measured by placing feces of No. 6 rabbit into containers with mosquito larvae and pupae inside.Results: According to the label condition of Cx. pipiens after taking blood and the effect of different dosage Na2H32PO4 on rabbit health, the optimal concentration of radioactive isotope was determined, that is, 0.1211 mCi/kg. By placing feces of No. 6 rabbit into containers with mosquito larvae and pupae inside, the emerged mosquitoes were also labeled. Therefore, feeding mosquitoes on the animal injected with radioactive Na2H32PO4 was more prac­tical for detecting and tracing mosquitoes.Conclusion: The method was less time-consuming, more sensitive and safer. This marking method will facilitate post-bloodmeal studies of mosquitoes and other blood-sucking insects.

  19. Potential application of labeled antibodies for thrombus detection

    Energy Technology Data Exchange (ETDEWEB)

    Ezekowitz, M.D.; Coller, B.S.; Srivastava, S.C.

    1986-01-01

    Labeling platelets with monoclonal antibodies in whole blood for imaging thrombi is less cumbersome than the established /sup 111/In-oxine method. 7E3, a murine monoclonal antibody directed against glycoprotein IIb and/or IIIa on both human and dog platelets was used to label canine platelets. Thrombi were induced by transcatheter placement of a copper coil followed by electrocoagulation. 7E3 was iodinated with /sup 131/I and labelled with /sup 111/In using 7E3-DTPA conjugate. Whole blood was incubated with 0.5 - 1.0 ..mu..g labeled 7E3/mL blood. In 4/4 dogs, experimental deep vein thrombi were identified using both /sup 131/I-and /sup 111/In-labeled 7E3 within 5-30 min after injection. For both isotopes, 1 h blood clearance was 54 + - 9%. In 1/3 dogs, experimental coronary thrombus could be identified ex vivo at 4 h. Clot to blood ratios ranged between 7 to 13:1. Using the /sup 111/In-oxine method, 0/3 coronary thrombi were seen. Thus, /sup 131/I-and /sup 111/In-labeled 7E3 may be used to readily identify peripheral venous thrombi. For reliable and prompt identification of coronary thrombi, more rapid clearance of the labeled platelets is required.

  20. Evaluation of a Method for Nitrotyrosine Site Identification and Relative Quantitation Using a Stable Isotope-Labeled Nitrated Spike-In Standard and High Resolution Fourier Transform MS and MS/MS Analysis

    Directory of Open Access Journals (Sweden)

    Kent W. Seeley

    2014-04-01

    Full Text Available The overproduction of reactive oxygen and nitrogen species (ROS and RNS can have deleterious effects in the cell, including structural and possible activity-altering modifications to proteins. Peroxynitrite is one such RNS that can result in a specific protein modification, nitration of tyrosine residues to form nitrotyrosine, and to date, the identification of nitrotyrosine sites in proteins continues to be a major analytical challenge. We have developed a method by which 15N-labeled nitrotyrosine groups are generated on peptide or protein standards using stable isotope-labeled peroxynitrite (O15NOO−, and the resulting standard is mixed with representative samples in which nitrotyrosine formation is to be measured by mass spectrometry (MS. Nitropeptide MS/MS spectra are filtered using high mass accuracy Fourier transform MS (FTMS detection of the nitrotyrosine immonium ion. Given that the nitropeptide pair is co-isolated for MS/MS fragmentation, the nitrotyrosine immonium ions (at m/z = 181 or 182 can be used for relative quantitation with negligible isotopic interference at a mass resolution of greater than 50,000 (FWHM, full width at half-maximum. Furthermore, the standard potentially allows for the increased signal of nitrotyrosine-containing peptides, thus facilitating selection for MS/MS in a data-dependent mode of acquisition. We have evaluated the methodology in terms of nitrotyrosine site identification and relative quantitation using nitrated peptide and protein standards.

  1. Applications of stable isotopes

    International Nuclear Information System (INIS)

    Gregg, C.T.

    1977-01-01

    Clinical applications include the galactose breath test which consists of oral administration of 13 C-labeled galactose and measurement of the 13 C content of respired CO 2 as a function of time in patients with cirrhotic livers for diagnosis of liver dysfunction. Another application was the breath test to study glucose metabolism in children. Respired 13 CO 2 from ingested glucose- 13 C was measured for normal and diabetic children. Studies on mice in which 60 percent of the body carbon was replaced with 13 C failed to show significant effects of the isotope. Studies on biochemical applications include nuclear magnetic resonance studies of 13 C-labeled amino acids from Chlorella pyrenoidosa; studies on 15 N nmr spectra of arginine-guanidino- 13 C-2,3-- 15 N 2 as a function of pH; and isolation of fatty acids from algae

  2. Recent progress in fluorine-18 labelled peptide radiopharmaceuticals

    Energy Technology Data Exchange (ETDEWEB)

    Okarvi, S.M. [Cyclotron and Radiopharmaceuticals Department, King Faisal Specialist Hospital and Research Centre, Riyadh (Saudi Arabia)

    2001-07-01

    The application of biologically active peptides labelled with positron-emitting nuclides has emerged as a useful and interesting field in nuclear medicine. Small synthetic receptor-binding peptides are currently the preferred agents over proteins and antibodies for diagnostic imaging of various tumours. Due to the smaller size of peptides, both higher target-to-background ratios and rapid blood clearance can often be achieved with radiolabelled peptides. Hence, short-lived positron emission tomography (PET) isotopes are potential candidates for labelling peptides. Among a number of positron-emitting nuclides, fluorine-18 appears to be the best candidate for labelling bioactive peptides by virtue of its favourable physical and nuclear characteristics. The major disadvantage of labelling peptides with {sup 18}F is the laborious and time-consuming preparation of the {sup 18}F labelling agents. In recent years, various techniques have been developed which allow efficient labelling of peptides with {sup 18}F without affecting their receptor-binding properties. Moreover, the development of a variety of prosthetic groups has facilitated the efficient and site-specific labelling of peptides with {sup 18}F. The {sup 18}F-labelled peptides hold enormous clinical potential owing to their ability to quantitatively detect and characterise a wide variety of human diseases when using PET. Recently, a number of {sup 18}F-labelled bioactive peptides have shown great promise as diagnostic imaging agents. This review presents the recent developments in {sup 18}F-labelled biologically active peptides used in PET. (orig.)

  3. Recent progress in fluorine-18 labelled peptide radiopharmaceuticals

    International Nuclear Information System (INIS)

    Okarvi, S.M.

    2001-01-01

    The application of biologically active peptides labelled with positron-emitting nuclides has emerged as a useful and interesting field in nuclear medicine. Small synthetic receptor-binding peptides are currently the preferred agents over proteins and antibodies for diagnostic imaging of various tumours. Due to the smaller size of peptides, both higher target-to-background ratios and rapid blood clearance can often be achieved with radiolabelled peptides. Hence, short-lived positron emission tomography (PET) isotopes are potential candidates for labelling peptides. Among a number of positron-emitting nuclides, fluorine-18 appears to be the best candidate for labelling bioactive peptides by virtue of its favourable physical and nuclear characteristics. The major disadvantage of labelling peptides with 18 F is the laborious and time-consuming preparation of the 18 F labelling agents. In recent years, various techniques have been developed which allow efficient labelling of peptides with 18 F without affecting their receptor-binding properties. Moreover, the development of a variety of prosthetic groups has facilitated the efficient and site-specific labelling of peptides with 18 F. The 18 F-labelled peptides hold enormous clinical potential owing to their ability to quantitatively detect and characterise a wide variety of human diseases when using PET. Recently, a number of 18 F-labelled bioactive peptides have shown great promise as diagnostic imaging agents. This review presents the recent developments in 18 F-labelled biologically active peptides used in PET. (orig.)

  4. AMP-acetyl CoA synthetase from Leishmania donovani: identification and functional analysis of 'PX4GK' motif.

    Science.gov (United States)

    Soumya, Neelagiri; Kumar, I Sravan; Shivaprasad, S; Gorakh, Landage Nitin; Dinesh, Neeradi; Swamy, Kayala Kambagiri; Singh, Sushma

    2015-04-01

    An adenosine monophosphate forming acetyl CoA synthetase (AceCS) which is the key enzyme involved in the conversion of acetate to acetyl CoA has been identified from Leishmania donovani for the first time. Sequence analysis of L. donovani AceCS (LdAceCS) revealed the presence of a 'PX4GK' motif which is highly conserved throughout organisms with higher sequence identity (96%) to lower sequence identity (38%). A ∼ 77 kDa heterologous protein with C-terminal 6X His-tag was expressed in Escherichia coli. Expression of LdAceCS in promastigotes was confirmed by western blot and RT-PCR analysis. Immunolocalization studies revealed that it is a cytosolic protein. We also report the kinetic characterization of recombinant LdAceCS with acetate, adenosine 5'-triphosphate, coenzyme A and propionate as substrates. Site directed mutagenesis of residues in conserved PX4GK motif of LdAceCS was performed to gain insight into its potential role in substrate binding, catalysis and its role in maintaining structural integrity of the protein. P646A, G651A and K652R exhibited more than 90% loss in activity signifying its indispensible role in the enzyme activity. Substitution of other residues in this motif resulted in altered substrate specificity and catalysis. However, none of them had any role in modulation of the secondary structure of the protein except G651A mutant. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Synthesis of deuterium labelled ibuprofen

    International Nuclear Information System (INIS)

    Cappon, V.J.; Halstead, G.W.; Theis, D.L.

    1986-01-01

    The preparations of [ar- 2 H 4 ]-ibuprofen and [ar, 3,3,3- 2 H 7 ]-ibuprofen are described. The deuterium was incorporated into the aromatic ring of [ar- 2 H 4 ]-ibuprofen which is a metabolically stable position. [ar, 3,3,3- 2 H 7 ]-ibuprofen was synthesized by the same route using [ 2 H 3 ]-CH 3 I instead of CH 3 I for use as a GC/MS internal standard in stable isotope labelled bioavailability studies. (author)

  6. RNAi inhibition of feruloyl CoA 6'-hydroxylase reduces scopoletin biosynthesis and post-harvest physiological deterioration in cassava (Manihot esculenta Crantz) storage roots.

    Science.gov (United States)

    Liu, Shi; Zainuddin, Ima M; Vanderschuren, Herve; Doughty, James; Beeching, John R

    2017-05-01

    Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24-72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava's marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6'-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6'-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6'-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.

  7. Preparation of pyronaridine labelled with tritium

    Energy Technology Data Exchange (ETDEWEB)

    Shangen, Jiang; Liufang, Zhang; Dongzhu, Zheng; Zheng, Feng; Zufan, Wu

    1987-12-01

    Pyronaridine is a high efficient and low toxic new antimalarial drug. /sup 3/H-pyronaridine was prepared by catalytic isotopic exchange in solution with tritium gas using PdO/BaSO/sub 4/ as catalyst. That crude product was purified by extraction. /sup 3/H-NMR spectra of pyronaridine showed that tritium was labelled at the 6-position. Specific activity of /sup 3/H-pyronaridine was 5.5 Ci/mmol and radiochemical purity over 95%.

  8. In Vitro Immunologic Studies with an {sup 131}I-Labelled Component of Complement

    Energy Technology Data Exchange (ETDEWEB)

    Spar, I. L.; Benz, L. L. [Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY (United States)

    1970-02-15

    Most of the in vitro immunological studies using radioactive isotopes have involved labelling of the reacting antigen or antibody. For meaningful results, it is also necessary to use a relatively purified preparation of either the antigen or antibody. This has been difficult to obtain when the antigenic component is ill- defined, as in tissue or tumour immunity. It seemed possible that detection of reactions of this type could be done more easily by using labelled components of haemolytic complement, since some of the factors involved in this 9-11 factor system are known to firmly bind to most antigen-antibody combinations. As an initial step in this study, {sup 125}I- or {sup 131}I-labelled components of complement were used to detect and quantitate known antigen-antibody systems. The initial reacting component of guinea pig complement, C'l, was partially purified and labelled with {sup 125}I or {sup 131}I. It was found that labelled C'l would react with ovalbumin- antiovalbumin (OA) precipitates and would bind to sensitized sheep cells (EA) in proportion to the amount of haemolysin bound to the cells. EDTA elusion of such bound {sup 125}I-C'l yielded a product that would react with EA or OA to a much greater extent than the starting material. In addition, lysis of EA would occur after binding of eluted {sup 125}I-C'l if the remaining complement components were added to the system. In further studies it was found that {sup 131}I-C'l could be used to detect reactions between anti-kidney antisera and hpmogenates of kidney, liver and lung. Extension of this work with isotopically labelled components of complement to a study involving tissue sections after incubation with antisera could lead to defection of tissue-antitissue antibody binding in situ. By utilizing autoradiographic techniques, one can further extend this system to define the site of antibody fixation. A distinct advantage of this approach is that the isotopically labelled reactant, complement, is

  9. Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.

    Science.gov (United States)

    Lueders, Tillmann; Manefield, Mike; Friedrich, Michael W

    2004-01-01

    Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.

  10. Double isotopic method using dansyl chloride for the determination of GABA in rat C6 astrocytoma cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Kohl, R.L.; Quay, W.B.; Perez-Polo, J.R.

    1986-01-01

    Methods are described for the quantitative measurement of GABA in culture. The method can be adapted to any amino acid or dansyl-chloride-reactive species. The sensitivity and selectivity of the procedure result from the double isotopic design in which (/sup 14/C)-labeled internal standard was added to the samples before reaction with (3M)-labeled dansyl chloride. Values obtained by ion-exchange amino acid analysis of cultures agree closely with the values obtained by the double isotopic method. This method is sensitive enough to measure GABA intracellularly and the condition medium.

  11. Inhibitory effect on in vitro LDL oxidation and HMG Co-A reductase activity of the liquid-liquid partitioned fractions of Hericium erinaceus (Bull.) Persoon (lion's mane mushroom).

    Science.gov (United States)

    Rahman, Mohammad Azizur; Abdullah, Noorlidah; Aminudin, Norhaniza

    2014-01-01

    Oxidation of low-density lipoprotein (LDL) has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A) reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM), hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), and aqueous residue (AQ). The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins) for the formation of conjugated diene (CD) at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL) of thiobarbituric acid reactive substances (TBARS) at 1 mg/mL. It also mostly inhibited (59.91%) the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases.

  12. Effects of sample injection amount and time-of-flight mass spectrometric detection dynamic range on metabolome analysis by high-performance chemical isotope labeling LC-MS.

    Science.gov (United States)

    Zhou, Ruokun; Li, Liang

    2015-04-06

    The effect of sample injection amount on metabolome analysis in a chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) platform was investigated. The performance of time-of-flight (TOF) mass spectrometers with and without a high-dynamic-range (HD) detection system was compared in the analysis of (12)C2/(13)C2-dansyl labeled human urine samples. An average of 1635 ± 21 (n = 3) peak pairs or putative metabolites was detected using the HD-TOF-MS, compared to 1429 ± 37 peak pairs from a conventional or non-HD TOF-MS. In both instruments, signal saturation was observed. However, in the HD-TOF-MS, signal saturation was mainly caused by the ionization process, while in the non-HD TOF-MS, it was caused by the detection process. To extend the MS detection range in the non-HD TOF-MS, an automated switching from using (12)C to (13)C-natural abundance peaks for peak ratio calculation when the (12)C peaks are saturated has been implemented in IsoMS, a software tool for processing CIL LC-MS data. This work illustrates that injecting an optimal sample amount is important to maximize the metabolome coverage while avoiding the sample carryover problem often associated with over-injection. A TOF mass spectrometer with an enhanced detection dynamic range can also significantly increase the number of peak pairs detected. In chemical isotope labeling (CIL) LC-MS, relative metabolite quantification is done by measuring the peak ratio of a (13)C2-/(12)C2-labeled peak pair for a given metabolite present in two comparative samples. The dynamic range of peak ratio measurement does not need to be very large, as only subtle changes of metabolite concentrations are encountered in most metabolomic studies where relative metabolome quantification of different groups of samples is performed. However, the absolute concentrations of different metabolites can be very different, requiring a technique to provide a wide detection dynamic range to allow the detection of as

  13. Sub-cellular localisation of a 15N-labelled peptide vector using NanoSIMS imaging

    Science.gov (United States)

    Römer, Winfried; Wu, Ting-Di; Duchambon, Patricia; Amessou, Mohamed; Carrez, Danièle; Johannes, Ludger; Guerquin-Kern, Jean-Luc

    2006-07-01

    Dynamic SIMS imaging is proposed to map sub-cellular distributions of isotopically labelled, exogenous compounds. NanoSIMS imaging allows the characterisation of the intracellular transport pathways of exogenous molecules, including peptide vectors employed in innovative therapies, using stable isotopes as molecular markers to detect the compound of interest. Shiga toxin B-subunit (STxB) was chosen as a representative peptide vector. The recombinant protein ( 15N-STxB) was synthesised in Escherichia coli using 15NH 4Cl as sole nitrogen source resulting in 15N enrichment in the molecule. Using the NanoSIMS 50 ion microprobe (Cameca), different ion species ( 12C 14N -, 12C 15N -, 31P -) originating from the same sputtered micro volume were simultaneously detected. High mass resolving power enabled the discrimination of 12C 15N - from its polyatomic isobars of mass 27. We imaged the membrane binding and internalisation of 15N-STxB in HeLa cells at spatial resolutions of less than 100 nm. Thus, the use of rare stable isotopes like 15N with dynamic SIMS imaging permits sub-cellular detection of isotopically labelled, exogenous molecules and imaging of their transport pathways at high mass and spatial resolution. Application of stable isotopes as markers can replace the large and chemically complex tags used for fluorescence microscopy, without altering the chemical and physical properties of the molecule.

  14. An Overview of Advanced SILAC-Labeling Strategies for Quantitative Proteomics.

    Science.gov (United States)

    Terzi, F; Cambridge, S

    2017-01-01

    Comparative, quantitative mass spectrometry of proteins provides great insight to protein abundance and function, but some molecular characteristics related to protein dynamics are not so easily obtained. Because the metabolic incorporation of stable amino acid isotopes allows the extraction of distinct temporal and spatial aspects of protein dynamics, the SILAC methodology is uniquely suited to be adapted for advanced labeling strategies. New SILAC strategies have emerged that allow deeper foraging into the complexity of cellular proteomes. Here, we review a few advanced SILAC-labeling strategies that have been published during last the years. Among them, different subsaturating-labeling as well as dual-labeling schemes are most prominent for a range of analyses including those of neuronal proteomes, secretion, or cell-cell-induced stimulations. These recent developments suggest that much more information can be gained from proteomic analyses if the labeling strategies are specifically tailored toward the experimental design. © 2017 Elsevier Inc. All rights reserved.

  15. Obtaining of culture of feengus-pathogene of pine root sponge-with double label (phosphorus-32 and sulfur-35)

    Energy Technology Data Exchange (ETDEWEB)

    Kobets, E V

    1979-01-01

    The culture of Fomitopsis annosa (Fr) Karst. is shown to be labelled with phosphorus and sulfur in the period of its development in the nutrient medium, which contains /sup 23/P and /sup 35/S radioactive isotopes. The labelled fungus culture retains the label in the process of its growing in the agar must medium, the correlation of sulfur-35 and phosphorus-32 activities being approximately stable.

  16. Understanding N timing in corn yield and fertilizer N recovery: An insight from an isotopic labeled-N determination

    Science.gov (United States)

    de Almeida, Rodrigo Estevam Munhoz; Pierozan Junior, Clovis; Lago, Bruno Cocco; Trivelin, Paulo Cesar Ocheuze

    2018-01-01

    Early fertilizer nitrogen (N) application on cover crops or their residues during the off-season is a practice adopted in Brazil subtropical conditions under no-tillage corn (Zea mays L.) systems. However, the effect of early N application on yield, plant N content, and N recovery efficiency (NRE) for corn is not yet well documented. Five fertilizer N timings in an oat-corn system were evaluated in two studies utilizing an isotopic-labeled N determination, 15N isotope. The N fertilization timings were: (i) oat tillering, (ii) 15 days before corn planting time, over the oat residues, (iii) at corn planting time, (iv) in-season at the three-leaf growth stage (V3), and (v) in-season split application at V3 and six-leaf (V6) growth stages. Based on the statistical analysis, the N fertilization timings were separated into three groups: 1) N-OATS, designated to N applied at oat; 2) N-PLANT, referred to pre-plant and planting N applications; and 3) N-CORN, designated to in-season corn N applications. Corn yield was not affected by the N fertilization timing. However, the N-CORN N fertilization timings enhanced NRE by 17% and 35% and final N recovery system (plant plus soil) by 16% and 24% all relative to N-OATS and N-PLANT groups, respectively. Overall, N-OATS resulted in the largest N derived from fertilizer (NDFF) amount in the deeper soil layer, in overall a delta of 10 kg N ha-1 relative to the rest of the groups. Notwithstanding corn yield was not affected, early N fertilization under subtropical conditions is not a viable option since NRE was diminished and the non-recovery N increased relative to the in-season N applications. PMID:29462178

  17. Understanding N timing in corn yield and fertilizer N recovery: An insight from an isotopic labeled-N determination.

    Science.gov (United States)

    Maciel de Oliveira, Silas; Almeida, Rodrigo Estevam Munhoz de; Ciampitti, Ignacio A; Pierozan Junior, Clovis; Lago, Bruno Cocco; Trivelin, Paulo Cesar Ocheuze; Favarin, José Laércio

    2018-01-01

    Early fertilizer nitrogen (N) application on cover crops or their residues during the off-season is a practice adopted in Brazil subtropical conditions under no-tillage corn (Zea mays L.) systems. However, the effect of early N application on yield, plant N content, and N recovery efficiency (NRE) for corn is not yet well documented. Five fertilizer N timings in an oat-corn system were evaluated in two studies utilizing an isotopic-labeled N determination, 15N isotope. The N fertilization timings were: (i) oat tillering, (ii) 15 days before corn planting time, over the oat residues, (iii) at corn planting time, (iv) in-season at the three-leaf growth stage (V3), and (v) in-season split application at V3 and six-leaf (V6) growth stages. Based on the statistical analysis, the N fertilization timings were separated into three groups: 1) N-OATS, designated to N applied at oat; 2) N-PLANT, referred to pre-plant and planting N applications; and 3) N-CORN, designated to in-season corn N applications. Corn yield was not affected by the N fertilization timing. However, the N-CORN N fertilization timings enhanced NRE by 17% and 35% and final N recovery system (plant plus soil) by 16% and 24% all relative to N-OATS and N-PLANT groups, respectively. Overall, N-OATS resulted in the largest N derived from fertilizer (NDFF) amount in the deeper soil layer, in overall a delta of 10 kg N ha-1 relative to the rest of the groups. Notwithstanding corn yield was not affected, early N fertilization under subtropical conditions is not a viable option since NRE was diminished and the non-recovery N increased relative to the in-season N applications.

  18. Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences.

    Science.gov (United States)

    Rajesh, Mathur; Wang, Gang; Jones, Roger; Tretyakova, Natalia

    2005-02-15

    The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene

  19. Novel methodology for labelling mesoporous silica nanoparticles using the 18F isotope and their in vivo biodistribution by positron emission tomography

    International Nuclear Information System (INIS)

    Rojas, Santiago; Gispert, Juan Domingo; Menchón, Cristina; Baldoví, Herme G.; Buaki-Sogo, Mireia; Rocha, Milagros; Abad, Sergio; Victor, Victor Manuel; García, Hermenegildo; Herance, José Raúl

    2015-01-01

    Nanoparticles have been proposed for several biomedical applications due to their potential as drug carriers, diagnostic and therapeutic agents. However, only a few of them have been approved for their use in humans. In order to gauge the potential applicability of a specific type of nanoparticle, in vivo biodistribution studies to characterize their pharmacokinetic properties are essential. In this regard, mesoporous silica nanoparticles (30–130 nm) have been functionalized with amino groups in order to react with N-succinimidyl 4-[ 18 F]fluorobenzoate and thus anchor the 18 F positron emission isotope by using a novel and easy labelling strategy. In vivo biodistribution was characterized in mice after intravenous administration of radiolabelled nanoparticles by positron emission tomography. Our results indicated that radiolabelled mesoporous silica nanoparticles were excreted into bile and urine and accumulated mainly in the organs of the reticuloendothelial system and lungs

  20. Analysis of [U-13C6]glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry compared with two other mass spectrometry techniques

    NARCIS (Netherlands)

    Schierbeek, H.; Moerdijk-Poortvliet, T.C.W.; van den Akker, C.H.P.; te Braake, F.W.J.; Boschker, H.T.S.; van Goudoever, J.B.

    2009-01-01

    The use of stable isotope labelled glucose provides insight into glucose metabolism. The 13C-isotopic enrichment of glucose is usually measured by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). However, in both techniques

  1. Study on the meat isotopic composition for origin identification

    Directory of Open Access Journals (Sweden)

    Irina Chernukha

    2018-03-01

    Full Text Available Russian consumer and governmental authorities are equally concerned to know where food products come from. This requires more accurate and specialized methods for the evaluation of geographical location. The following methods are used: chemometrics, histological and histochemical, genomic and proteomic, microbiological, immunochemical and mass spectrometric. Method of stable isotope analysis is becoming increasingly promising nowadays for the identification of meat and meat products' place of origin. The isotope ratios of the four elements - carbon, nitrogen, oxygen and hydrogen, are mainly determined. The method is successfully used to identify a country of origin of wines, juices and water. The aim of the research was to study the stable isotope ratios for pork and beef samples purchased in Moscow supermarkets (Russian Federation. The country of production of meat samples was determined according to specifications and/or labels. The geography of countries of meat samples origin includes Europe, both America continents and Australia. Databases collected by the All-Russian Scientific Research Institute of the Brewing, Non-Alcoholic and Wine Industrywere used for the analysis and interpretation of the results. Values of 13С/12С, δ13С, 18О/16О, δ18О, 2Н/1Н, δ2Н for 30 pork and beef samples from 13 countries were obtained. Differences in stable isotope ratios were found depending on place of origin. The data correlated with the oxygen isotope characteristics for wine, which were in the range from 2.5 to 4.5 ppm. According to the 13С/12С, δ13С results, the assumption was made about a false indication of the region for the beef sample. Despite the fact that beef was labeled as a product of Lithuania, the region of origin was most probably defined as Germany. The studies carried out showed the possibility to identify the region of raw meat origin by the stable isotope ratio.

  2. Isotopic diagnosis of peripheral thrombosis

    International Nuclear Information System (INIS)

    Cornu, Pierre; Scalet, Michel

    1975-01-01

    Radio-isotope diagnosis of peripheral venous thrombosis, using tracer doses of iodine-labelled fibrinogen, provides an important contribution to the solution of the worrying problem of pulmonary embolism due to latent phlebitis. This elegant and precise technique permits early diagnosis of venous thrombosis of the lower limbs at a subclinical stage. It has permitted determination of the frequency, both after surgery and after myocardial infarction, and above all, it provides an objective criterion for assessment of the efficacy of prophylactic measures proposed [fr

  3. Assessment of vitamin A status in rats by isotope dilution: A simplified model

    International Nuclear Information System (INIS)

    Furr, H.C.; Cooper, D.A.; Olson, J.A.

    1990-01-01

    Isotope-dilution analysis of vitamin A status requires giving a known quantity of labeled vitamin A to the subject and measuring the ratio of labeled to unlabeled retinol in the blood after a period for equilibration. To calculate total body stores from the isotopic ratio of plasma retinol, several assumptions must be made. In considering new ways of better calculating liver vitamin A stores from isotope-dilution data, the authors used the data of Green et al. to estimate loss of vitamin A tracer as a function of time and of vitamin A status. This correction markedly improves the correlation between calculated and analyzed liver vitamin A stores and also quantitively explains the hyperbolic relationship between fraction of tracer dose recovered in liver and mass of liver vitamin A stores. Agreement of this model with experimental data suggests that efficiency of absorption and storage of vitamin A is not affected by vitamin A status. This model can be used to estimate both the amount of tracer needed for a given lower limit of detection and an optimum sampling time

  4. Measurement of Endogenous versus Exogenous Formaldehyde-Induced DNA-Protein Crosslinks in Animal Tissues by Stable Isotope Labeling and Ultrasensitive Mass Spectrometry.

    Science.gov (United States)

    Lai, Yongquan; Yu, Rui; Hartwell, Hadley J; Moeller, Benjamin C; Bodnar, Wanda M; Swenberg, James A

    2016-05-01

    DNA-protein crosslinks (DPC) arise from a wide range of endogenous and exogenous chemicals, such as chemotherapeutic drugs and formaldehyde. Importantly, recent identification of aldehydes as endogenous genotoxins in Fanconi anemia has provided new insight into disease causation. Because of their bulky nature, DPCs pose severe threats to genome stability, but previous methods to measure formaldehyde-induced DPCs were incapable of discriminating between endogenous and exogenous sources of chemical. In this study, we developed methods that provide accurate and distinct measurements of both exogenous and endogenous DPCs in a structurally specific manner. We exposed experimental animals to stable isotope-labeled formaldehyde ([(13)CD2]-formaldehyde) by inhalation and performed ultrasensitive mass spectrometry to measure endogenous (unlabeled) and exogenous ((13)CD2-labeled) DPCs. We found that exogenous DPCs readily accumulated in nasal respiratory tissues but were absent in tissues distant to the site of contact. This observation, together with the finding that endogenous formaldehyde-induced DPCs were present in all tissues examined, suggests that endogenous DPCs may be responsible for increased risks of bone marrow toxicity and leukemia. Furthermore, the slow rate of DPC repair provided evidence for the persistence of DPCs. In conclusion, our method for measuring endogenous and exogenous DPCs presents a new perspective for the potential health risks inflicted by endogenous formaldehyde and may inform improved disease prevention and treatment strategies. Cancer Res; 76(9); 2652-61. ©2016 AACR. ©2016 American Association for Cancer Research.

  5. Positional enrichment by proton analysis (PEPA). A one-dimensional "1H-NMR approach for "1"3C stable isotope tracer studies in metabolomics

    International Nuclear Information System (INIS)

    Vinaixa, Maria; Yanes, Oscar; Rodriguez, Miguel A.; Capellades, Jordi; Aivio, Suvi; Stracker, Travis H.; Gomez, Josep; Canyellas, Nicolau

    2017-01-01

    A novel metabolomics approach for NMR-based stable isotope tracer studies called PEPA is presented, and its performance validated using human cancer cells. PEPA detects the position of carbon label in isotopically enriched metabolites and quantifies fractional enrichment by indirect determination of "1"3C-satellite peaks using 1D-"1H-NMR spectra. In comparison with "1"3C-NMR, TOCSY and HSQC, PEPA improves sensitivity, accelerates the elucidation of "1"3C positions in labeled metabolites and the quantification of the percentage of stable isotope enrichment. Altogether, PEPA provides a novel framework for extending the high-throughput of "1H-NMR metabolic profiling to stable isotope tracing in metabolomics, facilitating and complementing the information derived from 2D-NMR experiments and expanding the range of isotopically enriched metabolites detected in cellular extracts. (copyright 2017 The Authors. Published by Wiley-VCH Verlag GmbH and Co. KGaA.)

  6. Bacterial diversity exploration in hydrocarbon polluted soil: metabolic potential and degrader community evolution revealed by isotope labeling

    International Nuclear Information System (INIS)

    Martin, F.

    2011-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds produced by incomplete combustion of organic matter. They are a source of environmental pollution, especially associated to oil product exploitation, and represent a threat for living organisms including human beings because of their toxicity. Many bacteria capable of degrading PAHs have been isolated and studied. However, since less than 5% of soil bacteria can be cultivated in the laboratory, bacterial species able to degrade PAHs in situ have been poorly studied. The first goal of this study was to identify bacteria that degrade PAHs in soil using culture-independent molecular methods. To this end, a strategy known a stable isotope probing has been implemented based on the use of phenanthrene, a three rings PAH, in which the natural isotope of carbon was replaced by 13 C. This molecule has been introduced as a tracer in microcosms containing soil from a constructed wetlands collecting contaminated water from highway runoff. Bacteria having incorporated the 13 C were then identified by 16 S rRNA gene sequence analysis after PCR amplification from labeled genomic DNA extracted from soil. The results show that so far little studied Betaproteobacteria, belonging to the genera Acidovorax, Rhodoferax, Hydrogenophaga and Thiobacillus, as well as Rhodocyclaceae, were the key players in phenanthrene degradation. Predominance of Betaproteobacteries was established thanks to quantitative PCR measurements. A dynamic analysis of bacterial diversity also showed that the community structure of degraders depended on phenanthrene bioavailability. In addition, the phylogenetic diversity of ring-hydroxylating di-oxygenases, enzymes involved in the first step of PAH degradation, has been explored. We detected new sequences, mostly related to di-oxygenases from Sphingomonadales and Burkholderiales. For the first time, we were able to associate a catalytic activity for oxidation of PAHs to partial gene sequences

  7. One-carbon 13C-labeled synthetic intermediates. Comparison and evaluation of preparative methods

    International Nuclear Information System (INIS)

    Ott, D.G.

    1978-01-01

    Frequently the biggest stumbling block to the synthesis of a structurally complex labeled compound is obtaining the required low molecular weight, structurally simple, isotopic intermediates. Selection of a particular scheme from various alternatives depends on the available capabilities and quantity of product desired, as well as on anticipated future requirements and need for related compounds. Many of the newer reagents for organic synthesis can be applied effectively to isotopic preparations with improvements of yields and simplification of procedures compared to established classical methods. New routes developed for higher molecular weight compounds are sometimes not directly adaptable to the one-carbon analogs, either because of isolation difficulties occasioned by physical properties or by chemical reactivities peculiar to their being first members of homologous series. Various routes for preparation of carbon-13 labeled methanol, formaldehyde, and cyanide are compared

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

    Science.gov (United States)

    Guo, Kevin; Bamforth, Fiona; Li, Liang

    2011-02-01

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

  9. Qualitative metabolome analysis of human cerebrospinal fluid by 13C-/12C-isotope dansylation labeling combined with liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry.

    Science.gov (United States)

    Guo, Kevin; Bamforth, Fiona; Li, Liang

    2011-02-01

    Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by (13)C-dansyl and (12)C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner. © American Society for Mass Spectrometry, 2011

  10. On-target labeling of intracellular metabolites combined with chemical mapping of individual hyphae revealing cytoplasmic relocation of isotopologues.

    Science.gov (United States)

    Hu, Jie-Bi; Chen, Yu-Chie; Urban, Pawel L

    2012-06-05

    A microscale analytical platform integrating microbial cell culture, isotopic labeling, along with visual and mass spectrometric imaging with single-cell resolution has been developed and applied in the monitoring of cellular metabolism in fungal mycelium. The method implements open chips with a two-dimensional surface pattern composed of hydrophobic and hydrophilic zones. Two hydrophilic islands are used as medium reservoirs, while the hydrophobic area constitutes the support for the growing aerial hyphae, which do not have direct contact with the medium. The first island, containing (12)C(6)-glucose medium, was initially inoculated with the mycelium (Neurospora crassa), and following the initial incubation period, the hyphae progressed toward the second medium island, containing an isotopically labeled substrate ((13)C(6)-glucose). The (13)C atoms were gradually incorporated into cellular metabolites, which was revealed by MALDI-MS. The fate of the chitin-biosynthesis precursor, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), was monitored by recording mass spectra with characteristic isotopic patterns, which indicated the presence of various (12)C/(13)C isotopologues. The method enabled mapping the (13)C-labeled UDP-GlcNAc in fungal mycelium and recording its redistribution in hyphae, directly on the chip.

  11. Absorption of food cobalamins assessed by the double isotope method in healthy volunteers and in patients with chronic diarrhoea

    International Nuclear Information System (INIS)

    Kittang, B.; Hamborg, B.; Schjoensby, H.

    1985-01-01

    To make a food preparation containing radioactively labelled cobalamins, rabbits were given repeated injections with 57 Co-labelled cyanocobalamin. The liver was removed, homogenized and fried for one min. or boiled for 30 min. Of the radioactivity in the fried homogenate, 41.7% was recovered in the centrifuged supernatant compared with 50.8% in the boiled homogenate. The radioactivity in the supernatants had a molecular size close to that of free 57 Co-labelled cyanocobalamin. 42% of the radioactivity in the whole homogenate had been incorporated into 5-deoxyadenosyl-, 10% into methyl-and 16.5% into hydroxy-cobalamin. To assess the validity of a double-isotope method for measuring the intestinal absorption of doses of 57 Co-labelled liver cobalamins, 51 CrCl 3 was used as a non-absorbable marker. In 14 healthy volunteers the correlation coefficient between the absorption measured by the double-isotope technique and the faecal excretion test was highly significant (r = 0.96, p 57 Co/ 51 Cr ratio in successive stool collections. In 11 patients with chronic diarrhoea there was a significant correlation between the absorption measured by the double-isotope technique and the faecal excretion test (r = 0.92, p 57 / 51 Cr ratio in successive stool collections

  12. Synthesis of deuterium and tritium labelled tyrosine

    International Nuclear Information System (INIS)

    Kanska, M.; Drabarek, S.

    1980-01-01

    A new method of synthesis of tyrosine labelled with deuterium and tritium in the aromatic ring has been developed. Deuterated and tritiated tyrosine was obtained by isotope exchange between tyrosine and deuterated or tritiated water at elevated temperature in hydrochloric acid medium using K 2 PtCl 4 as a catalyst. For synthesis of tritiated tyrosine 1 Ci HTO was used; the specific activity of the product was 5 mCi/mMol. (author)

  13. Validation of single-sample doubly labeled water method

    International Nuclear Information System (INIS)

    Webster, M.D.; Weathers, W.W.

    1989-01-01

    We have experimentally validated a single-sample variant of the doubly labeled water method for measuring metabolic rate and water turnover in a very small passerine bird, the verdin (Auriparus flaviceps). We measured CO 2 production using the Haldane gravimetric technique and compared these values with estimates derived from isotopic data. Doubly labeled water results based on the one-sample calculations differed from Haldane values by less than 0.5% on average (range -8.3 to 11.2%, n = 9). Water flux computed by the single-sample method differed by -1.5% on average from results for the same birds based on the standard, two-sample technique (range -13.7 to 2.0%, n = 9)

  14. A new high-quality set of singly (H-2) and doubly (H-2 and O-18) stable isotope labeled reference waters for biomedical and other isotope-labeled research

    NARCIS (Netherlands)

    Faghihi, V.; Verstappen-Dumoulin, B. M. A. A.; Jansen, H. G.; van Dijk, G.; Aerts-Bijma, A. T.; Kerstel, E. R. T.; Groening, M.; Meijer, H. A. J.

    2015-01-01

    RATIONALE: Research using water with enriched levels of the rare stable isotopes of hydrogen and/or oxygen requires well-characterized enriched reference waters. The International Atomic Energy Agency (IAEA) did have such reference waters available, but these are now exhausted. New reference waters

  15. Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

    Energy Technology Data Exchange (ETDEWEB)

    Monneau, Yoan R. [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States); Ishida, Yojiro [Rutgers University, Center for Advanced Biotechnology and Medicine (United States); Rossi, Paolo; Saio, Tomohide; Tzeng, Shiou-Ru [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States); Inouye, Masayori, E-mail: inouye@cabm.rutgers.edu [Rutgers University, Center for Advanced Biotechnology and Medicine (United States); Kalodimos, Charalampos G., E-mail: ckalodim@umn.edu [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States)

    2016-06-15

    A simple and cost effective method to independently and stereo-specifically incorporate [{sup 1}H,{sup 13}C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [{sup 1}H,{sup 13}C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [{sup 1}H,{sup 13}C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-{sup 13}C,2,3-{sup 2}H{sub 2},{sup 15}N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[{sup 13}CH{sub 3}]{sup γ2} labeled protein with an optimal isotope incorporation using up to 50 % less labeled Thr than the traditional E. coli strain without the need for {sup 2}H-glycine to prevent scrambling.

  16. Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses.

    Directory of Open Access Journals (Sweden)

    Jang Eun Lee

    Full Text Available Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2 regulates fatty acid oxidation (FAO by inhibiting carnitine palmitoyltransferase 1 (CPT1, a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses.

  17. Isotope determination of sulfur by mass spectrometry in soil samples

    Directory of Open Access Journals (Sweden)

    Alexssandra Luiza Rodrigues Molina Rossete

    2012-12-01

    Full Text Available Sulphur plays an essential role in plants and is one of the main nutrients in several metabolic processes. It has four stable isotopes (32S, 33S, 34S, and 36S with a natural abundance of 95.00, 0.76, 4.22, and 0.014 in atom %, respectively. A method for isotopic determination of S by isotope-ratio mass spectrometry (IRMS in soil samples is proposed. The procedure involves the oxidation of organic S to sulphate (S-SO4(2-, which was determined by dry combustion with alkaline oxidizing agents. The total S-SO4(2- concentration was determined by turbidimetry and the results showed that the conversion process was adequate. To produce gaseous SO2 gas, BaSO4 was thermally decomposed in a vacuum system at 900 ºC in the presence of NaPO3. The isotope determination of S (atom % 34S atoms was carried out by isotope ratio mass spectrometry (IRMS. In this work, the labeled material (K2(34SO4 was used to validate the method of isotopic determination of S; the results were precise and accurate, showing the viability of the proposed method.

  18. Diagnostic utility of alpha-methylacyl CoA racemase (P504S) on prostate needle biopsy.

    Science.gov (United States)

    Jiang, Zhong; Woda, Bruce A

    2004-11-01

    Alpha-methylacyl CoA racemase (AMACR), also known as P504S, was identified by the analysis of cDNA library subtraction in conjunction with high throughput microarray screening from prostate tissue and has been proven to be one of the very few biomarkers that can distinguish cancer from benign cells with high sensitivity and specificity for prostate carcinoma. It is a successful example of the translation of molecular findings into clinical practice. This review focuses on the study of AMACR (P504S) expression in small focal prostate cancer and atypical small acinar proliferation (ASAP) on needle biopsies and emphasizes the utility of AMACR (P504S) in routine surgical pathology practice. We also discuss the potential pitfalls and caveats in the interpretation of immunostaining results.

  19. Biocatalysis of a Paclitaxel Analogue: Conversion of Baccatin III to N-Debenzoyl-N-(2-furoyl)paclitaxel and Characterization of an Amino Phenylpropanoyl CoA Transferase.

    Science.gov (United States)

    Thornburg, Chelsea K; Walter, Tyler; Walker, Kevin D

    2017-11-07

    In this study, we demonstrate an enzyme cascade reaction using a benzoate CoA ligase (BadA), a modified nonribosomal peptide synthase (PheAT), a phenylpropanoyltransferase (BAPT), and a benzoyltransferase (NDTNBT) to produce an anticancer paclitaxel analogue and its precursor from the commercially available biosynthetic intermediate baccatin III. BAPT and NDTNBT are acyltransferases on the biosynthetic pathway to the antineoplastic drug paclitaxel in Taxus plants. For this study, we addressed the recalcitrant expression of BAPT by expressing it as a soluble maltose binding protein fusion (MBP-BAPT). Further, the preparative-scale in vitro biocatalysis of phenylisoserinyl CoA using PheAT enabled thorough kinetic analysis of MBP-BAPT, for the first time, with the cosubstrate baccatin III. The turnover rate of MBP-BAPT was calculated for the product N-debenzoylpaclitaxel, a key intermediate to various bioactive paclitaxel analogues. MBP-BAPT also converted, albeit more slowly, 10-deacetylbaccatin III to N-deacyldocetaxel, a precursor of the pharmaceutical docetaxel. With PheAT available to make phenylisoserinyl CoA and kinetic characterization of MBP-BAPT, we used Michaelis-Menten parameters of the four enzymes to adjust catalyst and substrate loads in a 200-μL one-pot reaction. This multienzyme network produced a paclitaxel analogue N-debenzoyl-N-(2-furoyl)paclitaxel (230 ng) that is more cytotoxic than paclitaxel against certain macrophage cell types. Also in this pilot reaction, the versatile N-debenzoylpaclitaxel intermediate was made at an amount 20-fold greater than the N-(2-furoyl) product. This reaction network has great potential for optimization to scale-up production and is attractive in its regioselective O- and N-acylation steps that remove protecting group manipulations used in paclitaxel analogue synthesis.

  20. Positional enrichment by proton analysis (PEPA). A one-dimensional {sup 1}H-NMR approach for {sup 13}C stable isotope tracer studies in metabolomics

    Energy Technology Data Exchange (ETDEWEB)

    Vinaixa, Maria; Yanes, Oscar [Department of Electronic Engineering-Universitat Rovira i Virgili, Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM), Reus (Spain); Rodriguez, Miguel A.; Capellades, Jordi [Universitat Rovira i Virgili, Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM), Reus (Spain); Aivio, Suvi; Stracker, Travis H. [Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (Spain); Gomez, Josep; Canyellas, Nicolau [Department of Electronic Engineering-, Universitat Rovira i Virgili, Tarragona (Spain)

    2017-03-20

    A novel metabolomics approach for NMR-based stable isotope tracer studies called PEPA is presented, and its performance validated using human cancer cells. PEPA detects the position of carbon label in isotopically enriched metabolites and quantifies fractional enrichment by indirect determination of {sup 13}C-satellite peaks using 1D-{sup 1}H-NMR spectra. In comparison with {sup 13}C-NMR, TOCSY and HSQC, PEPA improves sensitivity, accelerates the elucidation of {sup 13}C positions in labeled metabolites and the quantification of the percentage of stable isotope enrichment. Altogether, PEPA provides a novel framework for extending the high-throughput of {sup 1}H-NMR metabolic profiling to stable isotope tracing in metabolomics, facilitating and complementing the information derived from 2D-NMR experiments and expanding the range of isotopically enriched metabolites detected in cellular extracts. (copyright 2017 The Authors. Published by Wiley-VCH Verlag GmbH and Co. KGaA.)

  1. Novel methodology for labelling mesoporous silica nanoparticles using the {sup 18}F isotope and their in vivo biodistribution by positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Rojas, Santiago; Gispert, Juan Domingo; Menchón, Cristina [PRBB, Institut d’Alta Tecnologia PRBB Fundació Privada (IAT) (Spain); Baldoví, Herme G.; Buaki-Sogo, Mireia [Polytechnic University of Valencia, University Institute of Chemical Technology (Spain); Rocha, Milagros [Foundation for the Promotion of Healthcare and Biomedical Research in the Valencian Community (FISABIO) (Spain); Abad, Sergio [PRBB, Institut d’Alta Tecnologia PRBB Fundació Privada (IAT) (Spain); Victor, Victor Manuel [Foundation for the Promotion of Healthcare and Biomedical Research in the Valencian Community (FISABIO) (Spain); García, Hermenegildo, E-mail: hgarcia@qim.upv.es [Polytechnic University of Valencia, University Institute of Chemical Technology (Spain); Herance, José Raúl, E-mail: jrherance@yahoo.es [PRBB, Institut d’Alta Tecnologia PRBB Fundació Privada (IAT) (Spain)

    2015-03-15

    Nanoparticles have been proposed for several biomedical applications due to their potential as drug carriers, diagnostic and therapeutic agents. However, only a few of them have been approved for their use in humans. In order to gauge the potential applicability of a specific type of nanoparticle, in vivo biodistribution studies to characterize their pharmacokinetic properties are essential. In this regard, mesoporous silica nanoparticles (30–130 nm) have been functionalized with amino groups in order to react with N-succinimidyl 4-[{sup 18}F]fluorobenzoate and thus anchor the {sup 18}F positron emission isotope by using a novel and easy labelling strategy. In vivo biodistribution was characterized in mice after intravenous administration of radiolabelled nanoparticles by positron emission tomography. Our results indicated that radiolabelled mesoporous silica nanoparticles were excreted into bile and urine and accumulated mainly in the organs of the reticuloendothelial system and lungs.

  2. Correlation of changes in rate of sterol synthesis with changes in HMG CoA reductase activity in cultured lens epithelial cells

    International Nuclear Information System (INIS)

    Cenedella, R.J.; Hitchener, W.R.

    1986-01-01

    In the present study, the authors correlated changes in HMG CoA reductase activity with changes in relative rates of sterol synthesis measured from either 3 H 2 O or 1- 14 C-acetate for bovine lens epithelial cells cultured in the presence or absence of lipoproteins. Enzyme activity and rates of incorporation of 3 H 2 O or 1- 14 C-acetate into digitonin precipitable sterols were measured in cells on the 4th day of subculture in DMEM containing 9% whole calf serum (WM) or 9% lipoprotein deficient serum (LDM). In three experiments, HMG CoA reductase activity (U/10 6 cells) averaged 2.2 +/- 0.1 times greater for cells grown in LDM than WM. Sterol synthesis averaged 3.0 +/- 0.4 times greater when measured with 3 H 2 O and 4.0 +/- 1.1 times greater when measured with 14 C-acetate. Thus, 3 H 2 O and 14 C-acetate appear to be comparable substrates for estimating changes in relative rates of sterol synthesis by cultured cells. The larger increases in rates of sterol synthesis than in reductase activity in response to decreased cholesterol could reflect stimulation at additional metabolic steps in the cholesterol pathway beyond mevalonic acid

  3. Up-regulation of hepatic Acyl CoA: Diacylglycerol acyltransferase-1 (DGAT-1) expression in nephrotic syndrome.

    Science.gov (United States)

    Vaziri, Nosratola D; Kim, Choong H; Phan, Dennis; Kim, Sara; Liang, Kaihui

    2004-07-01

    Nephrotic syndrome is associated with hypercholesterolemia, hypertriglyceridemia, and marked elevations of plasma low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL). Hypertriglyceridemia in nephrotic syndrome is accompanied by increased hepatic fatty acid synthesis, elevated triglyceride secretion, as well as lipoprotein lipase, VLDL-receptor, and hepatic triglyceride lipase deficiencies, which lead to impaired clearance of triglyceride-rich lipoproteins. Acyl CoA: diacylglycerol acyltransferase (DGAT) is a microsomal enzyme that joins acyl CoA to 1, 2-diacylglycerol to form triglyceride. Two distinct DGATs (DGAT-1 and DGAT2) have recently been identified in the liver and other tissues. The present study tested the hypothesis that the reported increase in hepatic triglyceride secretion in nephrotic syndrome may be caused by up-regulation of DGAT. Male Sprague-Dawley rats were rendered nephrotic by two sequential injections of puromycin aminonucleoside (130 mg/kg on day 1 and 60 mg/kg on day 14) and studied on day 30. Placebo-treated rats served as controls. Hepatic DGAT-1 and DGAT-2 mRNA abundance and enzymatic activity were measured. The nephrotic group exhibited heavy proteinuria, hypoalbuminemia, hypercholesterolemia, hypertriglyceridemia, and marked elevation of VLDL concentration. Hepatic DGAT-1 mRNA, DGAT-1, and total DGAT activity were significantly increased, whereas DGAT-2 mRNA abundance and activity were unchanged in the nephrotic rats compared to the control animals. The functional significance of elevation of DGAT activity was illustrated by the reduction in microsomal free fatty acid concentration in the liver of nephrotic animals. Nephrotic syndrome results in up-regulation of hepatic DGAT-1 expression and activity, which can potentially contribute to the associated hypertriglyceridemia by enhancing triglyceride synthesis. Thus, it appears that both depressed catabolism and increased synthetic capacity contribute to

  4. Site-specific labeling of proteins with NMR-active unnatural amino acids

    International Nuclear Information System (INIS)

    Jones, David H.; Cellitti, Susan E.; Hao Xueshi; Zhang Qiong; Jahnz, Michael; Summerer, Daniel; Schultz, Peter G.; Uno, Tetsuo; Geierstanger, Bernhard H.

    2010-01-01

    A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.

  5. The introduction of tritium label into natural and modified prostaglandins

    International Nuclear Information System (INIS)

    Shevchenko, V.P.; Bezuglov, V.V.; Nagayev, I.Y.; Myasoedov, N.F.

    1989-01-01

    Studies on the role of the nature of both heterogeneous catalysts and the solvent on the reduction selectively of 5,6-double bonds showed that the largest yield could be obtained by using the Lindlar catalyst and ethyl acetate. The use of different isotopes of hydrogen in the protium-deuterium-tritium series markedly decreased the hydrogenation reaction rate, but the selectivity of the process practically remained unaltered. Homogeneous catalysts were also used in the production of natural tritium-labelled prostaglandins and of their fluorine and deoxy analogues. The label was introduced by selective hydrogenation in the presence of (Ph 3 P) 3 RhCl and dioxane as solvent. Different ways have been studied of tritium-label introduction into prostaglandins modified at the carboxyl group. The synthesis of similar preparations was performed either by selective dehalogenation in the presence of heterogeneous catalysts treated with quinoline or triethylamine, or by condensation of prostaglandins at the carboxyl group by tritium-labelled amino acid. (author). 4 refs.; 1 fig

  6. REDOR NMR of stable-isotope-labeled protein binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Schaefer, J. [Washington Univ., St. Louis, MO (United States)

    1994-12-01

    Rotational-echo, double resonance (REDOR) NMR, a new analytical spectroscopic technique for solids spinning at the magic angle, has been developed over the last 5 years. REDOR provides a direct measure of heteronuclear dipolar coupling between isolated pairs of labeled nuclei. In a solid with a {sup 13}C-{sup 15}N labeled pair, for example, the {sup 13}C rotational echoes that form each rotor period following a{sup 1}H-{sup 13}C cross-polarization transfer can be prevented from reaching full intensity by insertion of a {sup 15}N {pi} pulse each half rotor period. The REDOR difference (the difference between a {sup 13}C NMR spectrum obtained under these conditions and one obtained with no {sup 15}N {pi} pulses) has a strong dependence on the {sup 13}C-{sup 15}N dipolar coupling, and hence, the {sup 13}C-{sup 15}N internuclear distance. REDOR is described as double-resonance even though three radio frequencies (typically {sup 1}H, {sup 13}C, and {sup 15}N) are used because the protons are removed from the important evolution part of the experiment by resonant decoupling. The dephasing of magnetization in REDOR arises from a local dipolar {sup 13}C-{sup 15}N field gradient and involves no polarization transfer. REDOR has no dependence on {sup 13}C or {sup 15}N chemical-shift tensors and does not require resolution of a {sup 13}C-{sup 15}N coupling in the chemical-shift dimension.

  7. Inhibitory Effect on In Vitro LDL Oxidation and HMG Co-A Reductase Activity of the Liquid-Liquid Partitioned Fractions of Hericium erinaceus (Bull. Persoon (Lion’s Mane Mushroom

    Directory of Open Access Journals (Sweden)

    Mohammad Azizur Rahman

    2014-01-01

    Full Text Available Oxidation of low-density lipoprotein (LDL has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM, hexane (HEX, dichloromethane (DCM, ethyl acetate (EA, and aqueous residue (AQ. The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins for the formation of conjugated diene (CD at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL of thiobarbituric acid reactive substances (TBARS at 1 mg/mL. It also mostly inhibited (59.91% the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases.

  8. Synthesis of deuterium-labelled viloxazine. [Antidepressant

    Energy Technology Data Exchange (ETDEWEB)

    Mamada, Kumiko; Furuta, Takashi; Kasuya, Yasuji

    1988-06-01

    The synthesis of deuterium-labelled viloxazine with high isotopic purity is described. The synthetic procedures employ alkylation of 2-(benzyloxy)phenol with (/sup 2/H/sub 5/)ethyl iodide for the introduction of deuterium. Catalytic removal of the benzyl group of the deuterated product followed by reaction with epichlorohydrin afforded 1,2-epoxy-3-(2'-pentadeuteroethoxy-phenoxy)propane. Addition of 2-aminoethyl hydrogen sulphate to the epoxide and subsequent ring formation into a morpholine derivative produced the desired (/sup 2/H/sub 5/)viloxazine.

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

    International Nuclear Information System (INIS)

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

    1989-01-01

    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 of 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

  10. Demonstration of de novo synthesis of enzymes by density labelling with stable isotopes

    International Nuclear Information System (INIS)

    Huebner, G.; Hirschberg, K.

    1977-01-01

    The technique of in vivo density labelling of proteins with H 2 18 O and 2 H 2 O has been used to investigate hormonal regulation and developmental expression of enzymes in plant cells. Buoyant density data obtained from isopycnic equilibrium centrifugation demonstrated that the cytokinine-induced nitrate reductase activity and the gibberellic acid-induced phosphatase activity in isolated embryos of Agrostemma githago are activities of enzymes synthesized de novo. The increase in alanine-specific aminopeptidase in germinating A. githago seeds is not due to de novo synthesis but to the release of preformed enzyme. On the basis of this result it is possible to apply the enzyme aminopeptidase as an internal density standard in equilibrium centrifugation. Density labelling experiments on proteins in pea cotyledons have been used to study the change in the activity of acid phosphatase, alanine-specific aminopeptidase, and peroxidase during germination. The activities of these enzymes increase in cotyledons of Pisum sativum. Density labelling by 18 O and 2 H demonstrates de novo synthesis of these three enzymes. The differential time course of enzyme induction shows the advantage of using H 2 18 O as labelling substance in cases when the enzyme was synthesized immediately at the beginning of germination. At this stage of development the amino-acid pool available for synthesis is formed principally by means of hydrolysis of storage proteins. The incorporation of 2 H into the new proteins takes place in a measurable amount at a stage of growth in which the amino acids are also synthesized de novo. The enzyme acid phosphatase of pea cotyledons was chosen to demonstrate the possibility of using the density labelling technique to detect protein turnover. (author)

  11. Isotopic study of the wear of sliding bearings with plastic friction surface

    International Nuclear Information System (INIS)

    Pandur, J.; Varkonyi, A.

    1978-01-01

    A new complex device has been elaborated for the investigation of the duration of bearings in the Institute of Isotopes of the Hungarian Academy of Sciences. The simultaneous determination of wear by an isotopic method the coefficient of friction by means of a Wheatstone bridge and the bearing temperature by means of a thermoresistor is described. Dynamic loading and variable revolution per minute are applied to produce a forced wear of the bearings. The isotopically labelled wear products are removed by oil and the collected sample is measured by a scintillation detector. Wear of a steel axle in plastic housing and plastic coated axle in cast iron housing was determined. (V.N.)

  12. Isotope enrichment by electron spin resonance transitions of the intermediate radical pair

    International Nuclear Information System (INIS)

    Okazaki, M.; Shiga, T.; Sakata, S.; Konaka, R.; Toriyama, K.

    1988-01-01

    Microwave effects on the spin adduct yield were observed in the photoreduction of menadione in micellar solutions with ordinary sodium dodecyl sulfate (SDS), deuterium-labeled SDS, and a mixture of them. A large isotope effect was found in the microwave modulation of the spin adduct yield, which is due to the ESR transitions of the transient radical pair in the reaction. It is demonstrated for the first time that the microwave field can be used to enrich one of the isotopes which coexist in the system

  13. Installation of an isotope separator in Debrecen

    International Nuclear Information System (INIS)

    Gacsi, Z.; Gulyas, J.; Vitez, A.; Csige, L.; Krasznahorkay, A.

    2005-01-01

    Complete text of publication follows. An isotope separator named OSIRIS was decommissioned in Studsvik, Sweden last July. Researchers there offered this equipment to us for dismantling and moving it over to ATOMKI in Debrecen for installation at the cyclotron lab and save and use it in nuclear physics and other sciences where stable and radioactive isotopes are used extensively for fundamental and applied research. Since the separator was used to separate radioactive isotopes, the ion source with its beam extracting, shaping, and transporting accessories, as well as the lining inside the bending magnet, furthermore the beam diagnostic and shaping elements in the 'switchyard' part of the separator had to stay in Studsvik because of the high radioactive contamination. In order to operate this equipment, first we have to design and manufacture these parts together with a new endstation for the collection and handling of the separated isotopes. Parallel with the installation, we also concentrate on different applications of an isotope separator, including separation of stable isotopes for labelling special compounds used in many branches of sciences, medical care, and industry, and on studying single ion implantation possibilities, as well as on the production of special targets for nuclear physics research. First we want to separate stable isotopes, and then, when we overcome all technical pitfalls, we will consider using this equipment to separate radioactive isotopes as well. Our intention is to have this equipment available to anyone at ATOMKI and elsewhere interested in using its capabilities in their own research fields. Consequently, all comments, suggestion, and ideas are welcome now and continuously, since the design and manufacture of parts can then be oriented by taking into account all the suggestions as much as possible. (author)

  14. Site-selective 13C labeling of proteins using erythrose

    International Nuclear Information System (INIS)

    Weininger, Ulrich

    2017-01-01

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

  15. Quantitative Isotope-Dilution High-Resolution-Mass-Spectrometry Analysis of Multiple Intracellular Metabolites in Clostridium autoethanogenum with Uniformly 13C-Labeled Standards Derived from Spirulina.

    Science.gov (United States)

    Schatschneider, Sarah; Abdelrazig, Salah; Safo, Laudina; Henstra, Anne M; Millat, Thomas; Kim, Dong-Hyun; Winzer, Klaus; Minton, Nigel P; Barrett, David A

    2018-04-03

    We have investigated the applicability of commercially available lyophilized spirulina ( Arthrospira platensis), a microorganism uniformly labeled with 13 C, as a readily accessible source of multiple 13 C-labeled metabolites suitable as internal standards for the quantitative determination of intracellular bacterial metabolites. Metabolites of interest were analyzed by hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry. Multiple internal standards obtained from uniformly (U)- 13 C-labeled extracts from spirulina were used to enable isotope-dilution mass spectrometry (IDMS) in the identification and quantification of intracellular metabolites. Extraction of the intracellular metabolites of Clostridium autoethanogenum using 2:1:1 chloroform/methanol/water was found to be the optimal method in comparison with freeze-thaw, homogenization, and sonication methods. The limits of quantification were ≤1 μM with excellent linearity for all of the calibration curves ( R 2 ≥ 0.99) for 74 metabolites. The precision and accuracy were found to be within relative standard deviations (RSDs) of 15% for 49 of the metabolites and within RSDs of 20% for all of the metabolites. The method was applied to study the effects of feeding different levels of carbon monoxide (as a carbon source) on the central metabolism and Wood-Ljungdahl pathway of C. autoethanogenum grown in continuous culture over 35 days. Using LC-IDMS with U- 13 C spirulina allowed the successful quantification of 52 metabolites in the samples, including amino acids, carboxylic acids, sugar phosphates, purines, and pyrimidines. The method provided absolute quantitative data on intracellular metabolites that was suitable for computational modeling to understand and optimize the C. autoethanogenum metabolic pathways active in gas fermentation.

  16. Doubly labelled water assessment of energy expenditure: principle, practice, and promise.

    Science.gov (United States)

    Westerterp, Klaas R

    2017-07-01

    The doubly labelled water method for the assessment of energy expenditure was first published in 1955, application in humans started in 1982, and it has become the gold standard for human energy requirement under daily living conditions. The method involves enriching the body water of a subject with heavy hydrogen ( 2 H) and heavy oxygen ( 18 O), and then determining the difference in washout kinetics between both isotopes, being a function of carbon dioxide production. In practice, subjects get a measured amount of doubly labelled water ( 2 H 2 18 O) to increase background enrichment of body water for 18 O of 2000 ppm with at least 180 ppm and background enrichment of body water for 2 H of 150 ppm with 120 ppm. Subsequently, the difference between the apparent turnover rates of the hydrogen and oxygen of body water is assessed from blood-, saliva-, or urine samples, collected at the start and end of the observation interval of 1-3 weeks. Samples are analyzed for 18 O and 2 H with isotope ratio mass spectrometry. The doubly labelled water method is the indicated method to measure energy expenditure in any environment, especially with regard to activity energy expenditure, without interference with the behavior of the subjects. Applications include the assessment of energy requirement from total energy expenditure, validation of dietary assessment methods and validation of physical activity assessment methods with doubly labelled water measured energy expenditure as reference, and studies on body mass regulation with energy expenditure as a determinant of energy balance.

  17. Validation of the doubly-labeled water (H3H18O) method for measuring water flux and energy metabolism in tenebrionid beetles

    International Nuclear Information System (INIS)

    Cooper, P.D.

    1981-01-01

    Doubly-labeled water (H 3 H 18 O) has been used to determine water flux and energy metabolism in a variety of vertebrates. This study examines the applicability of this technique to arthropods. The theory of the technique depends upon the assumption that doubly-labeled water introduced into the animal's body water equilibrates with water and carbon dioxide by the action of carbonic anhydrase. Tritium ( 3 H) is lost from the animal only with water while oxygen-18 is lost with both water and carbon dioxide. The difference bwtween the rates of loss of the two isotopes is proportional to CO 2 loss rate. Validation of the use of tritiated water for measuring water flux was accomplished by comparing gravimetric measurements of water gain with flux rates determined by loss of tritiated water. At room humidity, an overestimate for influx calculated from labeled water calculations was found, averaging 12 mg H 2 O (g.d) -1 . Comparison of CO 2 loss rate determined isotopically with rates of CO 2 loss determined by standard metabolic rates also yielded overestimates for the isotopic technique, overestimates ranging between 20 and 30%. The relevance of this for studies using labeled water for studying water fluxes and free metabolism of free-ranging arthropods is discussed

  18. Radioiodination via isotope exchange in pivalic acid

    International Nuclear Information System (INIS)

    Weichert, J.P.; Van Dort, M.E.; Groziak, M.P.; Counsell, R.E.

    1986-01-01

    A variety of benzoic and aryl aliphatic mono and polyiodinated acids and esters (sterol, triglyceride) were radioiodinated in 55-99% radiochemical yield by isotope exchange with Na 125 i in a melt of pivalic acid. In general, the reaction was complete in 1 h at 155 0 C with little or no substrate decomposition. High specific activity studies afforded 125 I-labeled iopanoic acid with a specific activity of over 700 Ci/mmol. (author)

  19. Biodistribution of Ru-97-labeled DTPA, DMSA and transferrin

    International Nuclear Information System (INIS)

    Som, P.; Oster, Z.H.; Fairchild, R.G.; Atkins, H.L.; Brill, A.B.; Gil, M.C.; Srivastava, S.C.; Meinken, G.E.; Goldman, A.G.; Richards, P.

    1980-01-01

    Ruthenium-97 is being produced at the Brookhaven Linac Isotope Producer (BLIP). The favorable physical properties of Ru-97 and chemical reactivity of ruthenium offer a potential for using this isotope to label compounds useful for delayed scanning. Diethylenetriamine pentaacetic acid (DTPA), 2,3-Dimercaptosuccinic acid (DMSA), and Transferrin (TF) were labeled with Ru-97-chloride. Ru-97-DTPA and In-111-DTPA, injected intravenously, showed similar organ distribution, kinetics, and more than 80% excretion by 0.5 h. Ru-97-DTPA and In-111-DTPA injected into the cisterna magna of dogs showed similar kinetics in brain, blood, and urinary bladder. The energy deposited by 1 mCi In-111-DTPA is twice that from 1 mCi Ru-97-DTPA. High quality camera images of the CSF space in the dog were obtained with both isotopes. Ru-97-DMSA was prepared with and without the addition of SnCl 2 .2H 2 O. Tin-free DMSA was rapidly excreted via the kidneys, whereas for maximum cortical deposition, the tin-containing preparation was superior. This compound is suitable for delayed imaging of both normal and impaired kidneys. Tissue distribution studies were performed in abscess-bearing rats with Ru-97-transferrin. Although blood levels were higher than with Ga-67-citrate, the abscess had twice as much Ru-97-TF as Ga-67-citrate and the Ru-97 muscle activity was one-third that of Ga-67. Imaging of abscess-bearing rabbits with Ru-97-TF visualized the abscesses as early as 1/2 hr after injection. Since the initial images visualize the abscess so clearly and since the TF portion of the compound binds to the abscess, Tc-99m-TF is being studied for the same purpose. Ru-97-labeled compounds are a promising replacement for In-111 and possibly also for Ga-67 compounds with the advantages of lower radiation dose and high quality image

  20. 15N-labelled glycine synthesis

    International Nuclear Information System (INIS)

    Tavares, Claudineia R.O.; Bendassolli, Jose A.; Sant'Ana Filho, Carlos R.; Prestes, Clelber V.; Coelho, Fernando

    2006-01-01

    This work describes a method for 15 N-isotope-labeled glycine synthesis, as well as details about a recovery line for nitrogen residues. To that effect, amination of α-haloacids was performed, using carboxylic chloroacetic acid and labeled aqueous ammonia ( 15 NH 3 ). Special care was taken to avoid possible 15 NH 3 losses, since its production cost is high. In that respect, although the purchase cost of the 13 N-labeled compound (radioactive) is lower, the stable tracer produced constitutes an important tool for N cycling studies in living organisms, also minimizing labor and environmental hazards, as well as time limitation problems in field studies. The tests were carried out with three replications, and variable 15 NH 3(aq) volumes in the reaction were used (50, 100, and 150 mL), in order to calibrate the best operational condition; glycine masses obtained were 1.7, 2, and 3.2 g, respectively. With the development of a system for 15 NH 3 recovery, it was possible to recover 71, 83, and 87% of the ammonia initially used in the synthesis. With the required adaptations, the same system was used to recover methanol, and 75% of the methanol initially used in the amino acid purification process were recovered. (author)

  1. 99mTc labelled peptides for imaging of peripheral receptors

    International Nuclear Information System (INIS)

    Mikolajczak, R.; Markiewicz, A.; Deptula, C.Z.; Zulczyk, W.; Birnbaum, G.; Zakrezewska, E.; Wozniak, I.

    2001-01-01

    The first trials of 99m Tc labelling by direct method using dithionite as a reducing agent (prepared in the freeze-dried form) gave the yields of around 30%. RC-160 labelling with 125 I by chloramine-T method resulted in 40-80% labelling yield. Our efforts were focused on BFC approach. HYNIC-TOC and HYNIC-RC-160 conjugates obtained in our laboratory were successfully labelled with 99m Tc with the yields over 90%. HPLC and TLC methods were applied for quality control (QC) of the labelled preparation. Methods of in vitro (stability and protein binding) testing of the labelled preparations were adopted to our laboratory conditions. First attempts on dry kit formulation based on HYNIC-TOC conjugates with tricine, tricine/nicotinic acid and EDDA were described. Various amounts of tin (II) (as SnCl 2 ) were added to the kits. Incubation conditions (time, temperature) were investigated. The kits were tested for labelling yield and radiochemical purity. It was shown that the results are at the same level or better than obtained in liquid phase but the procedure of labelling is significantly easier. Kit produced with tricine as co-ligand was labelled with 97% labelling yield after 30 min of incubation at room temperature, which is considered acceptable for diagnostic radiopharmaceutical preparation. Tricine/nicotinic acid kit requires heating to get labelling of around 95%. Similarly EDDA kit gives around 70% labelling after 30 min incubation at 80 deg. C. Further experiments on optimal kit composition and stability are required. Results of DOTA-RC-160 labelling with 90 Y show that this isotope, manufactured by Radioisotope Centre POLATOM, can be successfully used for medical applications. (author)

  2. Intrinsic mineral labeling of edible plants: methods and uses

    International Nuclear Information System (INIS)

    Weaver, C.M.

    1985-01-01

    The fate of minerals can be conveniently studied through intrinsic labeling techniques. The mineral of interest is biologically incorporated into the food in a form that can be distinguished analytically from the natural form of the element. Radiolabels have traditionally been used to study such problems as the uptake of minerals by plants, the gross and subcellular mineral distribution in plant tissues, the form and associations of the deposited mineral, and the bioavailability of minerals to animals and humans. The use of stable (nonradioactive) isotopes as a label offers the potential of safely studying bioavailability of minerals from individual foods in human population groups of all ages using foods processed in normal food handling and processing facilities. 114 references

  3. Automation of dimethylation after guanidination labeling chemistry and its compatibility with common buffers and surfactants for mass spectrometry-based shotgun quantitative proteome analysis

    Energy Technology Data Exchange (ETDEWEB)

    Lo, Andy; Tang, Yanan; Chen, Lu; Li, Liang, E-mail: Liang.Li@ualberta.ca

    2013-07-25

    Graphical abstract: -- Highlights: •Dimethylation after guanidination (2MEGA) uses inexpensive reagents for isotopic labeling of peptides. •2MEGA can be optimized and automated for labeling peptides with high efficiency. •2MEGA is compatible with several commonly used cell lysis and protein solubilization reagents. •The automated 2MEGA labeling method can be used to handle a variety of protein samples for relative proteome quantification. -- Abstract: Isotope labeling liquid chromatography–mass spectrometry (LC–MS) is a major analytical platform for quantitative proteome analysis. Incorporation of isotopes used to distinguish samples plays a critical role in the success of this strategy. In this work, we optimized and automated a chemical derivatization protocol (dimethylation after guanidination, 2MEGA) to increase the labeling reproducibility and reduce human intervention. We also evaluated the reagent compatibility of this protocol to handle biological samples in different types of buffers and surfactants. A commercially available liquid handler was used for reagent dispensation to minimize analyst intervention and at least twenty protein digest samples could be prepared in a single run. Different front-end sample preparation methods for protein solubilization (SDS, urea, Rapigest™, and ProteaseMAX™) and two commercially available cell lysis buffers were evaluated for compatibility with the automated protocol. It was found that better than 94% desired labeling could be obtained in all conditions studied except urea, where the rate was reduced to about 92% due to carbamylation on the peptide amines. This work illustrates the automated 2MEGA labeling process can be used to handle a wide range of protein samples containing various reagents that are often encountered in protein sample preparation for quantitative proteome analysis.

  4. Determination of the energetics of the UDP-glucose pyrophosphorylase reaction by positional isotope exchange inhibition

    International Nuclear Information System (INIS)

    Hester, L.S.; Raushel, F.M.

    1987-01-01

    A method has been developed for obtaining qualitative information about enzyme-catalyzed reactions by measuring the inhibitory effects of added substrates on positional isotope exchange rates. It has been demonstrated for ordered kinetic mechanisms that an increase in the concentration of the second substrate to add to the enzyme will result in a linear increase in the ratio of the chemical and positional isotope exchange rates. The slopes and intercepts from these plots can be used to determine the partitioning ratios of binary and ternary enzyme complexes. The method has been applied to the reaction catalyzed by UDP-glucose pyrophosphorylase. A positional isotope exchange reaction was measured within oxygen-18-labeled UTP as a function of variable glucose 1-phosphate concentration in the forward reaction. In the reverse reaction, a positional isotope exchange reaction was measured within oxygen-18-labeled UDP-glucose as a function of increasing pyrophosphate concentration. The results have been interpreted to indicate that the interconversion of the ternary central complexes is fast relative to product dissociation in either direction. In the forward direction, the release of UDP-glucose is slower than the release of pyrophosphate. The release of glucose 1-phosphate is slower than the release of UTP in the reverse reaction

  5. Isotopes in agriculture

    International Nuclear Information System (INIS)

    1983-01-01

    Part 1: The stable isotope of nitrogen 15N has become widely used as tracer in agriculture, medicine and biology research. The film gives an overview of the sample preparation and analytical procedures followed in the analysis of the nitrogen isotopic composition (14N/15N ratio) by optical emission spectrometry at the Seibersdorf Laboratory. The subsampling of plant material and the several steps of chemical pretreatment such as Kjeldahl digestion, distillation, titration and adjustment of the proper N concentration in the extract are demonstrated. The preparation of the discharge tubes is shown in detail. Final measurement of the 14N/15N ratio is carried out with the NOI-5 and JASCO emission spectrometers. Part 2: This training film deals with the use of 32P-labelled materials in field and greenhouse experimentation in soil-plant relationships studies. All technical aspects, including safe handling and radiation protection procedures to be considered in the layout and harvesting of field experiments are documented in detail. Procedures followed up in the evaluation of P fertilizers such as rock phosphates under greenhouse conditions are described. Several soil injection techniques available for determination of the root activity pattern of trees are shown

  6. A facile method for steroid labeling by heavy isotopes of hydrogen

    Czech Academy of Sciences Publication Activity Database

    Marek, Aleš; Klepetářová, Blanka; Elbert, Tomáš

    2015-01-01

    Roč. 71, č. 29 (2015), s. 4874-4882 ISSN 0040-4020 R&D Projects: GA AV ČR IAA400550801 Institutional support: RVO:61388963 Keywords : brassinosteroids * alpha-hydroxy ketones * reductive dehalogenation * tritium * labeled compounds Subject RIV: CC - Organic Chemistry Impact factor: 2.645, year: 2015

  7. The calibration of the intramolecular nitrogen isotope distribution in nitrous oxide measured by isotope ratio mass spectrometry.

    Science.gov (United States)

    Westley, Marian B; Popp, Brian N; Rust, Terri M

    2007-01-01

    Two alternative approaches for the calibration of the intramolecular nitrogen isotope distribution in nitrous oxide using isotope ratio mass spectrometry have yielded a difference in the 15N site preference (defined as the difference between the delta15N of the central and end position nitrogen in NNO) of tropospheric N2O of almost 30 per thousand. One approach is based on adding small amounts of labeled 15N2O to the N2O reference gas and tracking the subsequent changes in m/z 30, 31, 44, 45 and 46, and this yields a 15N site preference of 46.3 +/- 1.4 per thousand for tropospheric N2O. The other involves the synthesis of N2O by thermal decomposition of isotopically characterized ammonium nitrate and yields a 15N site preference of 18.7 +/- 2.2 per thousand for tropospheric N2O. Both approaches neglect to fully account for isotope effects associated with the formation of NO+ fragment ions from the different isotopic species of N2O in the ion source of a mass spectrometer. These effects vary with conditions in the ion source and make it impossible to reproduce a calibration based on the addition of isotopically enriched N2O on mass spectrometers with different ion source configurations. These effects have a much smaller impact on the comparison of a laboratory reference gas with N2O synthesized from isotopically characterized ammonium nitrate. This second approach was successfully replicated and leads us to advocate the acceptance of the site preference value 18.7 +/- 2.2 per thousand for tropospheric N2O as the provisional community standard until further independent calibrations are developed and validated. We present a technique for evaluating the isotope effects associated with fragment ion formation and revised equations for converting ion signal ratios into isotopomer ratios. Copyright 2007 John Wiley & Sons, Ltd.

  8. N-15-labelled pyrazines of triterpenic acids

    Czech Academy of Sciences Publication Activity Database

    Vlk, M.; Mičolová, P.; Urban, M.; Kvasnica, Miroslav; Šaman, David; Šarek, J.

    2016-01-01

    Roč. 308, č. 2 (2016), s. 733-739 ISSN 0236-5731 R&D Projects: GA ČR GJ15-08202Y; GA MŠk(CZ) LO1304; GA MŠk(CZ) LO1204 Grant - others:GA MŠk(CZ) LK21310; GA TA ČR(CZ) TA03010027; CTU(CZ) SGS15/094/OHK4/1T/14 Institutional support: RVO:61389030 ; RVO:61388963 Keywords : N-15 * Triterpenic acid * Isotopic labelling Subject RIV: CC - Organic Chemistry Impact factor: 1.282, year: 2016

  9. Quantitative Peptidomics with Five-plex Reductive Methylation labels

    Science.gov (United States)

    Tashima, Alexandre K.; Fricker, Lloyd D.

    2018-05-01

    Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of 13C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach. [Figure not available: see fulltext.

  10. Rhenium 188 labelling of peptide conjugates

    International Nuclear Information System (INIS)

    Melendez-Alafort, Laura

    2001-01-01

    Many human tumours express high levels, of somatostatin receptors. In order to make possible a radiotherapeutic treatment of this kind for tumour a series of somatostatin analogues that can tightly chelate beta emitting isotopes have been developed in recent years. The work carried out for this thesis has been aimed towards development of a new therapeutic radiopharmaceutical for treatment of somatostatin receptor positive tumours. The first chapters describe work with technetium-99m to establish the labelling and analytical conditions for a somatostatin analogue, [Tyr 3 ]-octreotide (TOC), as a precursor to undertaking labelling studies with the beta emitter rhenium-188. 6-Hydrazinopyridine-3-carboxylic acid (HYNIC) was conjugated to TOC and labelled with 99m using different coligands. Then the stability, receptor binding and biodistribution of each complex were assessed. 99m Tc-HYNIC-TOC using EDDA as coligand showed the best characteristics, and was superior for tumour imaging in humans than the commercially available 111 In-DTPA-octreotide. The conditions for labelling the HYNIC-TOC conjugate with 188 Re were then optimised using tricine as a co-ligand. A labelling yield of ∼80% was achieved. After purification however, the stability of the complex was low. The use of other coligand systems which had proved useful for 99m Tc labelling was explored, but yields were very poor. Other chelators such as diethylenetriamine pentaacetic acid (DTPA), dimercaptosuccinic acid (DMSA) and mercaptoacetyltriglycine (MAG 3 ) were studied as potential co-ligand agents to label the HYNIC-TOC conjugate with 188 Re but, again low yields of the labelled peptide complexes were achieved. A novel 188 Re-HYNIC complex was prepared in high yields using N-N-disubstituted dithiocarbamates as coligands. However to date, the specific activities achieved with this system are relatively low. The use of the [ 99m Tc(CO) 3 (H 2 O) 3 ] complex to label the HYNIC-TOC conjugate was investigated

  11. Simultaneous tracing of 76Se-selenite and 77Se-selenomethionine by absolute labeling and speciation

    International Nuclear Information System (INIS)

    Suzuki, Kazuo T.; Somekawa, Layla; Kurasaki, Kazuki; Suzuki, Noriyuki

    2006-01-01

    Nutritional selenocompounds are transformed into the assumed common intermediate selenide, which is utilized for the synthesis of selenoenzymes or transformed into methylated metabolites for excretion. Hence, selenocompound metabolites can be traced only with labeled selenium. Here we applied a new tracer method for the metallomics of biometals using simultaneous speciation of each metallome labeled with different homo-elemental isotopes to metabolism and availability of selenium. Rats were depleted of endogenous natural abundance selenium by feeding a single selenium stable isotope ( 82 Se-selenite) and then administered 76 Se-selenite and 77 Se-selenomethionine ( 77 Se-SeMet)simultaneously. Biological samples were subjected to quantification and speciation analysis by HPLC-ICPMS. Metabolites of the labeled 76 Se and 77 Se and interaction with endogenous selenium were traced and examined without interference from the corresponding endogenous natural abundance isotopes. Differences in the distribution and metabolism among organs and between the two nutritional selenocompounds were compared under exactly identical biological and analytical conditions: (1) selenite was distributed more efficiently than SeMet in organs and body fluids except the pancreas. (2) SeMet was taken up by organs in its intact form. (3) Selenium of SeMet origin was distributed selectively in the pancreas and mostly bound to a protein together with intact SeMet. (4) Selenosugars A and B but not trimethylselenonium (TMSe) were detected in the liver. (5) Selenosugar B and TMSe were detected in the kidneys

  12. Use of isotopes in fisheries research

    International Nuclear Information System (INIS)

    Ayyappan, S.

    2001-01-01

    Fisheries is an interdisciplinary science, incorporating not only different aspect of biology like anatomy, physiology, ecology, etc., but also oceanography, biochemistry, biotechnology and such emerging areas in the recent years. Isotopes are being employed for a variety of studies like quantification of aquatic production processes, nutrient cycles and food chain dynamics (primary production, bacterial activity, sediment-water nutrient interactions, nitrification, fertilizer use efficiencies, organic mineralisation, amino acid uptake in algal-bacterial assemblages, hydrological studies including water transport and budgeting, partitioning of trophic pathways), fish nutrition (feed utility and digestibility), fatty acid metabolism, compartmentalization of food spectra in different species of fish and shellfish), fish physiology, genetics and immunology (radioimmunoassay, enzyme assays, evaluation of hormone levels, gene markers -DNA labeling and plasmid incorporation, immuno diagnostics), fish health management and pollution (host-parasite interactions chemical/pesticide uptake and accumulation), etc. Radioisotopes commonly used in ecological studies are beta emitters like 14 C, 3 H, 32 P and 35 S, while a stable isotope like 15 N is used in quantifying nitrogen fixation rates in water and sediment media. Results of some of the studies and potential applications of isotopes in fish research are presented. (author)

  13. Pharmacokinetics of lidocaine and bupivacaine and stable isotope labelled analogues : a study in healthy volunteers

    NARCIS (Netherlands)

    Burm, A.G.D.; de Boer, A G; van Kleef, J.W.; Vermeulen, N P; de Leede, L G; Spierdijk, J; Breimer, D D

    1988-01-01

    The pharmacokinetics of lidocaine and bupivacaine and tri-deuteromethyl-labelled lidocaine and bupivacaine were investigated in healthy volunteers. The deuterium-labelled and the unlabelled form of the drug to be investigated were simultaneously infused in 10 min. Plasma concentrations were

  14. Isotope derived criteria for the measurement of soil and fertilizer micronutrient availability

    International Nuclear Information System (INIS)

    Tiller, K.G.

    1975-01-01

    Field experiments on long-lived gamma-emitting isotopes such as zinc-65 are unlikely to be acceptable because of health hazards, costs, and long-term losses of experimental field sites after completion of the trials. The use of glasshouse experiments for the assessment, by isotopic dilution procedures, of the efficiencies of different fertilizer formulations and their agronomic use is more advantageous. The measurement of nutrient absorbed from the fertilizer need not be restricted to the use of radioactively labelled fertilizers with its attendant technological problems in manufacture, transport, etc. Efficiency of locally available fertilizers, farm and industrial byproducts, could be related to the labelled native soil source of zinc. The dose rate of carrier-free zinc required can be minimized by limiting pot size and restricting fertilizer rates to realistic levels. Radioisotope derived criteria, while clearly valuable in rice micronutrient studies, require complementary field studies involving soil and plant analysis and fertilizer evaluation under conditions of local farm management

  15. Metabolism and pharmacokinetic of cyclo-peptides and peptides. Use of radioelement and stable isotopes

    International Nuclear Information System (INIS)

    Aninat, C.

    2003-10-01

    More and more peptides and proteins are used in therapeutic. Three mainly techniques are used for pharmacokinetic and metabolism studies: immunoassay, radioactively labeled molecules and mass spectrometry. In the first part of this work, we have used uniformly labelled peptides (C-peptide and insulin) with stables ( 13 C, 15 N, and 13 C/ 15 N) or radioactive ( 14 C) isotopes to investigated these kind of studies. These works are based on isotope dilution mass spectrometry assay. In a second time we have investigated the metabolism of a particular cyclo-peptides families composed of two amino acids: the diketo-piperazine. These compounds are found in mammals and in microorganisms. There are not recognized by proteolytic enzymes. We have estimated if the main enzymes implicated in the metabolism of xenobiotics, the P450 cytochrome mono-oxygenases, were able to recognized them

  16. In vivo stability and inertness of various direct labelled and chelate-tagged protein

    International Nuclear Information System (INIS)

    Janoki, A.; Korosi, L.; Klivenyi, G.; Spett, B.

    1987-01-01

    There were looking for methods giving precise information about composition and activity distribution of protein components, both in the initial samples and serum samples after intravenous administration. It was tested the applicability of electroimmunoassay, polyacrilamide gel electrophoresis and high performance liquid chromatography for the assessment of in vivo stability and labelled proteins. The model compound was human serum albumin (HSA) labelled with 99m Tc and 125 I, respectively. Bifunctional chelate labelling was done with desferrioxamine, in this case protein was labelled with 67 Ga. Biodistribution of the labelled compounds and their elimination from the blood were studied in rabbits. Experience with various labelling proteins, especially with Tc-Sn-HSA system indicate that in vivo stability of this compounds are generally low. Following intravenous injection of proteins labelled with metal isotopes, due to dilution and to the presence of considerable amount of compatitive protein in the serum, part of the label is being detached from the carrier protein. Distribution of the detached metal is different from the original distribution of the protein. This problem arises also with radiopharmaceuticals based on monoclonal antibodies. (M.E.L.) [es

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

    Science.gov (United States)

    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.

  18. Early detection of venous thromboses of the lower limb in traumatology using labelled fibrinogen

    International Nuclear Information System (INIS)

    Pasteyer, J.; Lanata, E.; Bonnard, P.; Jean, N.; Kher, A.; Patel, A.

    1975-01-01

    In emergency traumatology, the iodine 125 labelled fibrinogen test is useful in the early detection of phlebitis in patients in whom the routine use of heparin is not possible: fractures of the cervical spine, severe cranial trauma, elderly subjects. It makes possible the institution of appropriate heparin therapy, whithout waiting for clinical signs to become evident. Isotopic venous thromboses occured very frequently and very early and clinical signs, when present (50% of cases) occured 24 to 48 hours after the appearance of isotopic signs [fr

  19. Radioisotope labeling technique for vapor density measurements of volatile inorganic species

    International Nuclear Information System (INIS)

    Peterson, E.J.; Caird, J.A.; Hessler, J.P.; Hoekstra, H.R.; Williams, C.W.

    1979-01-01

    A new method for complexed metal ion vapor density measurement involving labeling the metal ions of interest with a radioactive isotope is described. The isotope chosen in the present work is unstable and leads to emission of a characteristic γ ray. Thus the γ-counting rate was related to the number density of complexed metal ions in the vapor phase. This technique is applicable to the study of any volatile inorganic species, but in the present study has been used to measure vapor densities of complex species in the TbCl 3 -AlCl 3 system by using tracer 160 Tb. 4 figures, 2 tables

  20. Water enriched in the rare stable isotopes : Preparation, measurement and applications

    NARCIS (Netherlands)

    Faghihi, Vahideh

    2016-01-01

    The subject of this thesis is water with increased abundances of the rare stable isotopes 2H and 18O (and to some extent also 17O). Such artificially enriched (or "labelled") waters are often used in biomedicine, for establishing the total amount of body water (and thus body composition) of humans