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      Dynamic nuclear polarization facilitates monitoring of pyruvate metabolism inTrypanosoma brucei

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      Journal of Biological Chemistry
      American Society for Biochemistry & Molecular Biology (ASBMB)

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          Abstract

          <p class="first" id="d1296305e196">Dynamic nuclear polarization provides sensitivity improvements that make NMR a viable method for following metabolic conversions in real time. There are now many <i>in vivo</i> applications to animal systems and even to diagnosis of human disease. However, application to microbial systems is rare. Here we demonstrate its application to the pathogenic protozoan, <i>Trypanosoma brucei</i>, using hyperpolarized <sup>13</sup>C1 pyruvate as a substrate and compare the parasite metabolism with that of commonly cultured mammalian cell lines, HEK-293 and Hep-G2. Metabolic differences between insect and bloodstream forms of <i>T. brucei</i> were also investigated. Significant differences are noted with respect to lactate, alanine, and CO <sub>2</sub> production. Conversion of pyruvate to CO <sub>2</sub> in the <i>T. brucei</i> bloodstream form provides new support for the presence of an active pyruvate dehydrogenase in this stage. </p>

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          Increase in signal-to-noise ratio of > 10,000 times in liquid-state NMR.

          A method for obtaining strongly polarized nuclear spins in solution has been developed. The method uses low temperature, high magnetic field, and dynamic nuclear polarization (DNP) to strongly polarize nuclear spins in the solid state. The solid sample is subsequently dissolved rapidly in a suitable solvent to create a solution of molecules with hyperpolarized nuclear spins. The polarization is performed in a DNP polarizer, consisting of a super-conducting magnet (3.35 T) and a liquid-helium cooled sample space. The sample is irradiated with microwaves at approximately 94 GHz. Subsequent to polarization, the sample is dissolved by an injection system inside the DNP magnet. The dissolution process effectively preserves the nuclear polarization. The resulting hyperpolarized liquid sample can be transferred to a high-resolution NMR spectrometer, where an enhanced NMR signal can be acquired, or it may be used as an agent for in vivo imaging or spectroscopy. In this article we describe the use of the method on aqueous solutions of [13C]urea. Polarizations of 37% for 13C and 7.8% for 15N, respectively, were obtained after the dissolution. These polarizations correspond to an enhancement of 44,400 for 13C and 23,500 for 15N, respectively, compared with thermal equilibrium at 9.4 T and room temperature. The method can be used generally for signal enhancement and reduction of measurement time in liquid-state NMR and opens up for a variety of in vitro and in vivo applications of DNP-enhanced NMR.
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            Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy.

            Measurements of early tumor responses to therapy have been shown, in some cases, to predict treatment outcome. We show in lymphoma-bearing mice injected intravenously with hyperpolarized [1-(13)C]pyruvate that the lactate dehydrogenase-catalyzed flux of (13)C label between the carboxyl groups of pyruvate and lactate in the tumor can be measured using (13)C magnetic resonance spectroscopy and spectroscopic imaging, and that this flux is inhibited within 24 h of chemotherapy. The reduction in the measured flux after drug treatment and the induction of tumor cell death can be explained by loss of the coenzyme NAD(H) and decreases in concentrations of lactate and enzyme in the tumors. The technique could provide a new way to assess tumor responses to treatment in the clinic.
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              NMR and pattern recognition methods in metabolomics: from data acquisition to biomarker discovery: a review.

              Metabolomics is the discipline where endogenous and exogenous metabolites are assessed, identified and quantified in different biological samples. Metabolites are crucial components of biological system and highly informative about its functional state, due to their closeness to functional endpoints and to the organism's phenotypes. Nuclear Magnetic Resonance (NMR) spectroscopy, next to Mass Spectrometry (MS), is one of the main metabolomics analytical platforms. The technological developments in the field of NMR spectroscopy have enabled the identification and quantitative measurement of the many metabolites in a single sample of biofluids in a non-targeted and non-destructive manner. Combination of NMR spectra of biofluids and pattern recognition methods has driven forward the application of metabolomics in the field of biomarker discovery. The importance of metabolomics in diagnostics, e.g. in identifying biomarkers or defining pathological status, has been growing exponentially as evidenced by the number of published papers. In this review, we describe the developments in data acquisition and multivariate analysis of NMR-based metabolomics data, with particular emphasis on the metabolomics of Cerebrospinal Fluid (CSF) and biomarker discovery in Multiple Sclerosis (MScl). Copyright © 2012 Elsevier B.V. All rights reserved.
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                Author and article information

                Journal
                Journal of Biological Chemistry
                J. Biol. Chem.
                American Society for Biochemistry & Molecular Biology (ASBMB)
                0021-9258
                1083-351X
                November 03 2017
                November 03 2017
                November 03 2017
                September 08 2017
                : 292
                : 44
                : 18161-18168
                Article
                10.1074/jbc.M117.807495
                5672039
                28887303
                25b7f954-3ea8-4e8c-bc57-0a6caae873a1
                © 2017
                History

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