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Jensen P.R.,Technical University of Denmark | Jensen P.R.,Albeda Research | Meier S.,Technical University of Denmark
Analyst | Year: 2016

The influx of exogenous substrates into cellular reaction cascades on the seconds time scale is directly observable by NMR spectroscopy when using nuclear spin polarization enhancement. Conventional NMR assignment spectra for the identification of reaction intermediates are not applicable in these experiments due to the non-equilibrium nature of the nuclear spin polarization enhancement. We show that ambiguities in the intracellular identification of transient reaction intermediates can be resolved by experimental schemes using site-specific isotope labelling, optimised referencing and response to external perturbations. © The Royal Society of Chemistry 2016.

Chiavazza E.,University of Turin | Viale A.,University of Turin | Karlsson M.,Albeda Research | Aime S.,University of Turin
Contrast Media and Molecular Imaging | Year: 2013

The synthesis, NMR properties and preliminary polarization tests on protonated and perdeuterated forms of α-trimethylglutamine (NMe3Gln), α-trimethylglutamate (NMe3Glu) and ε-trimethyllysine (NMe3Lys) are reported. The 15N-permethylated, perdeuterated amino acids display very long 15N-T1 values, ranging between 190 and 330 s, are well polarized by the dynamic nuclear polarization (DNP) procedure, yielding good polarization levels (10%), and appear to be well tolerated by cells and mice. The obtained results make perdeuterated amino acids excellent candidates for innovative DNP 15N-MRI applications such as perfusion or targeting studies. Copyright © 2013 John Wiley & Sons, Ltd. 15N-permethylated, perdeuterated amino acids display very long-T1 values for the (CD3)3 15N resonances thus representing particularly suitable substrates for applications such as perfusion or targeting studies. © 2013 John Wiley & Sons, Ltd.

Jensen P.R.,Technical University of Denmark | Jensen P.R.,Albeda Research | Meier S.,Technical University of Denmark
Chemical Communications | Year: 2016

Organic phosphate metabolites contain functional groups with pKa values near the physiologic pH range, yielding pH-dependent 13C chemical shift changes of adjacent quaternary carbon sites. When formed in defined cellular compartments from exogenous hyperpolarised 13C substrates, metabolites can thus yield localised pH values and correlations of organelle pH and catalytic activity. © The Royal Society of Chemistry 2016.

Meier S.,Carlsberg Laboratory | Solodovnikova N.,Carlsberg Laboratory | Jensen P.R.,Albeda Research | Wendland J.,Carlsberg Laboratory
ChemBioChem | Year: 2012

Detecting the molecular targets of xenobiotic substances in vivo poses a considerable analytical challenge. Here, we describe the use of an NMR-based tracer methodology for the instantaneous in vivo observation of sulfur(IV) action on cellular metabolism. Specifically, we find that glycolytic flux is directed towards sulfite adducts of dihydroxyacetone phosphate and pyruvate as off-pathway intermediates that obstruct glycolytic flux. In particular, the pyruvate-sulfite association hinders the formation of downstream metabolites. The apparent in vivo association constant of pyruvate and sulfite agrees with the apparent inhibition constant of CO2 formation, thus supporting the importance of pyruvate interception in disturbing central metabolism and inhibiting NAD regeneration. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Meier S.,Carlsberg Laboratory | Jensen P.R.,Albeda Research | Duus J.O.,Carlsberg Laboratory
FEBS Letters | Year: 2011

The direct tracking of cellular reactions in vivo has been facilitated with recent technologies that strongly enhance NMR signals in substrates of interest. This methodology can be used to assay intracellular reactions that occur within seconds to few minutes, as the NMR signal enhancement typically fades on this time scale. Here, we show that the enhancement of 13C nuclear spin polarization in deuterated glucose allows to directly follow the flux of glucose signal through rather extended reaction networks of central carbon metabolism in living Escherichia coli. Alterations in central carbon metabolism depending on the growth phase or upon chemical perturbations are visualized with minimal data processing by instantaneous observation of cellular reactions. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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