Multinuclear MRS Studies of Metabolism
Wednesday 22 April 2009
Room 315 16:00-18:00


Sebastián Cerdán and Matthew Merritt

16:00 528. Sources of Hepatic Glycogen Synthesis During an Oral Glucose Tolerance Test: Effect of Transaldolase Exchange on Flux Estimates
    Teresa Cardoso Delgado1, Claúdia Silva1, Isabel Fernandes2, Madalena Caldeira2, Margarida Bastos3, Carla Baptista3, Manuela Carvalheiro3, Carlos F.G.C. Geraldes1, John Griffith Jones1
NMR Laboratory, Center for Neurosciences and Cell Biology of Coimbra, Coimbra, Portugal; 22Chemistry Department, Faculty of Sciences and Technology, Coimbra University, Coimbra, Portugal; 3Department of Endocrinology, Diabetes and Metabolism, University Hospital of Coimbra, Coimbra, Portugal
    Sources of hepatic glycogen synthesis during a glucose tolerance test were evaluated in healthy subjects by enrichment of a glucose load with 6.67% [U-13C]glucose and 3.33% [U-2H7]glucose and 2H/13C NMR analysis of plasma glucose and hepatic UDP-glucose enrichments (sampled as urinary menthol glucuronide).The direct pathway contribution, as estimated from the dilution of [U-13C]glucose between plasma glucose and hepatic UDP-glucose, was unexpectedly low (36±5%).With [U-2H7]glucose, direct pathway estimates based on the dilution of position 3 2H-enrichment between plasma glucose and glucuronide were significantly higher (50±6%).These differences reflect the exchange of the carbon 456 moiety of fructose-6-phosphate and glyceraldehyde-3-phosphate catalyzed by transaldolase.
16:12 529. A Novel 13C MRS-Based Marker of Pyruvate Cycling in Perfused Mouse Liver Using [2-13C] Pyruvate and 13C MRS<
    Corin O'Dell Miller1, Haiying Liu1, Leslie Balogh2, Jin Cao1, Mike R. Tota3, Reshma Patel3
Imaging, Merck, Rahway, NJ, USA; 2Laboratory Animal Research, Merck, Rahway, NJ, USA; 3Metabolic Disorders, Merck, Rahway, NJ, USA
    A 13C MRS approach was employed in perfused mouse livers metabolizing substrate levels of [2-13C] pyruvate to develop a novel ex-vivo marker of pyruvate recycling based on the 13C-enrichment in lactate. Inhibition of each of the pyruvate recycling pathways (PEPCK via 3-mercapto-picolinic acid, and Malic enzyme via tartronic acid) reduced this marker in liver extracts while simultaneous inhibition of each pathway further reduced this marker to levels near the LOQ. Real time data from whole perfused livers agreed with data from extracts of freeze-clamped livers indicating that this approach may be translatable to whole tissues and possibly in vivo.
16:24 530.

Liver Tissue Repair in a Mouse Model of Toxicant-Induced Liver Injury Is Associated with Increased Hepatic Energy Metabolism: A Multinuclear Magnetic Resonance Study

    Sven Gottschalk1, Tom S. Chan1, Valérie-Ann Raymond1, Dieter Leibfritz2, Claudia Zwingmann1,2, Marc Bilodeau1
Département de sciences biomédicales, Université de Montréal, Montréal, Québec, Canada; 2Department of Organic Chemistry, University of Bremen, Bremen, Germany
    Due to its ability regenerate, the liver is an ideal model for studying tissue repair mechanisms. Only little is known about the repair-associated changes in cellular metabolic pathways. Energy-intensive repair processes should be reflected in alterations in energy metabolism. An in vivo liver-injury model was used to generate an onset of liver tissue-repair. We assessed the extent of liver-injury and NMR-spectroscopy was used to characterize changes in energy metabolism and metabolites. Our results showed that induction of liver-regeneration was consistent with an up-regulation of the cells overall energy metabolism and a higher demand for TCA-cycle intermediates (eg. for amino-acids synthesis).
16:36 531. Detection of Altered Hepatic Gluconeogenesis by Nuclear Magnetic Resonance (NMR) and Hyperpolarized 13C Pyruvate
    Matthew E. Merritt1, Crystal Harrison1, Craig R. Malloy1, A Dean Sherry1, Shawn C. Burgess1,2
Advanced Imaging Research, UT Southwestern Medical Center, Dallas, TX, USA; 2Pharmacology, UT Southwestern Medical Center, Dallas, TX, USA
    Abnormal hepatic gluconeogenesis is a hallmark feature of obesity, insulin resistance and diabetes, but its measurement requires sophisticated tracer approaches and/or serial MRS measurements over several hours. We used the DNP process to hyperpolarize [1-13C]pyruvate and followed its incorporation into hepatic metabolites over 60 seconds by 13C NMR in isolated perfused mouse liver. In addition to lactate and alanine, several resonances consistent with mitochondrial intermediates of the TCA cycle were observed. The appearance of these metabolites was diminished in livers with impaired gluconeogenesis. The data demonstrate that HP-pyruvate will be useful to observe a number of hepatic pathways, including gluconeogenesis.
16:48 532. Real Time Assessment of Krebs Cycle Metabolism with Hyperpolarized [2-13C]Pyruvate
    Damian John Tyler1, Marie Allen Schroeder1, Helen Jennifer Atherton1, Daniel R. Ball1, Mark Aaron Cole1, Lisa Claire Heather1, Julian L. Griffin2, Kieran Clarke1, George K. Radda1
Physiology, Anatomy & Genetics, University of Oxford, Oxford, Oxfordshire, UK; 2Biochemistry, University of Cambridge, Cambridge, Cambridgeshire, UK
    The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in energetic imbalances characteristic of heart disease. This study presents a novel magnetic resonance spectroscopy technique that enables real-time monitoring of Krebs cycle metabolism in whole hearts. Hyperpolarized [2-13C]pyruvate was infused into isolated perfused hearts, in both healthy and ischaemic states. The conversion of pyruvate to lactate, acetyl-carnitine, citrate and glutamate was observed. Following ischaemia, the appearance of 13C-labelled citrate and glutamate was decreased relative to the healthy state, indicating that hyperpolarized [2-13C]pyruvate may be useful when studying impaired Krebs cycle metabolism in heart disease.
17:00 533. A 13C Isotopomer Model for Accurate NMR Quantification of Substrate Selection and Anaplerosis
    Wei Li1, Fang Bian1, Priyanajana Chaudhuri1, Xin Yu1
Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA
    Pyruvate carboxylation is important in energy metabolism. However, accurate quantification of pyruvate carboxylation with NMR remains to be demonstrated. In this study, a 13C isotopomer model for citrate acid cycle was developed and applied to the quantification of pyruvate carboxylation and the comparison of NMR and GC-MS. Two groups of rat hearts were perfused with buffer containing 5.5 mM glucose and 0.6 mM palmitate with either glucose or palmitate being uniformly labeled with 13C. Pyruvate carboxylation was determined from NMR analysis of experiments. NMR and GCMS were equally good in quantifying substrate utilization, but different in quantifying anaplerosis.
17:12 534. Novel Application of Deuterium MRS: In Vivo Monitoring of Glucose Consumption
    Allen Ye1, Jeffrey L. Duerk1,2, Chris A. Flask1,2, Gheorghe D. Mateescu3
Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA; 2Radiology, Case Western Reserve University, Cleveland, OH, USA; 3Chemistry, Case Western Reserve University, Cleveland, OH, USA
    Multinuclear MRS provides a powerful platform for studying metabolism in many diseases. We have developed a novel application of Deuterium MRS to measure mitochondrial function in vivo. This straightforward method consists of the administration of deuteriated glucose to live organisms (mice, larvae) resulting in the formation of detectable levels of deuteriated metabolic water (D2O/HDO). Preliminary glucose consumption data are reported at 9.4T. A particularly important aspect of this novel approach is the simultaneous determination of glucose and oxygen consumption. Potential translation into the clinic will be useful in the diagnosis and treatment of mitochondrial diseases.
17:24 535. Resolution of H3proS and H3proR Deuterations in [2-13C]glutamate
    Tiago Brandao Rodrigues1, Inês Ribeiro Violante1, Sebastián Cerdán1
LISMAR, Instituto de Investigaciones Biomédicas CSIC, Madrid, Spain
    We provide a protocol to discriminate the 2H substitution of the H3proR or H3proS hydrogens of [2-13C]glutamate. Using 13C MRS at 18.8T, we detected the existence of two different isotopically shifted singlets in the glutamate C2 resonance of brain extracts of deuterated animals as derived from the deuterium substitutions in 3R or 3S, respectively. We show that the H3S depicted a lower value of vicinal isotopic shift (δ 1=-0.058ppm) while the H3R originated the higher-field resonance (δ 1=-0.071ppm), measured relatively to the [2-13C]glutamate C2 resonance.
17:36 536.

Hyperpolarized  13C Magnetic Resonance Detection of Carboxypeptidase G2 Activity

    Yann Jamin1, Cristina Gabellieri1, Lynette Smyth1, Steve Reynolds2, Simon P. Robinson1, Caroline J. Springer1, Martin O. Leach1, Geoffrey S. Payne1, Thomas R. Eykyn1
Institute of Cancer Research and Royal Marsden NHS trust, Sutton, UK; 2Oxford Instruments Molecular Biotools Ltd, Abingdon, UK
    Carboxypeptidase G2 (CPG2) is used in cancer chemotherapy to activate selectively non toxic prodrugs into potent cytoxics in tumors. Employing Dynamic nuclear polarisation (DNP) and natural abundance 13C MRS, we demonstrate the dynamic detection of CPG2 activity in vitro, using the reporter molecule 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu). This strategy could be translated in vivo to monitor CPG2-based therapy.
17:48 537. Generation of Hyperpolarized Substrates by Secondary Labelling with [1,1- 13C] Acetic Anhydride
    David M. Wilson1, Ralph E. Hurd2, Albert P. Chen2, Mark Van Criekinge1, Kayvan Keshari1, Sarah J. Nelson1, Daniel B. Vigneron1, John Kurhanewicz1
Department of Radiology, University of California, San Francisco, San Francisco, CA, USA; 2GE Healthcare
    Hyperpolarized [1,1-13C] acetic anhydride was used to achieve rapid, high SNR analysis of amino acid mixtures and to perform secondary polarization of other biomolecules in solution. This electrophile can be well polarized (6%), has a relatively long T1 relaxation time (33.9s at 11.7T), and preferentially reacts with amine nucleophiles in aqueous solution. This approach was used to reproducibly and near-quantitatively (mean yield – 89.8%) resolve a mixture of amino acids Gly, Ser, Val, Leu, and Ala, with a signal enhancement of up to 1400 fold. The method can also be used to create a hyperpolarized low molecular weight drug like acetylcysteine.