| Multinuclear MRS Studies of Metabolism |
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Wednesday 22 April 2009 |
| Room 315 |
16:00-18:00 |
Moderators: |
Sebastián Cerdán and Matthew Merritt |
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16:00 |
528. |
Sources of Hepatic Glycogen
Synthesis During an Oral Glucose Tolerance Test:
Effect of Transaldolase Exchange on Flux Estimates |
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Teresa Cardoso
Delgado1, Claúdia Silva1,
Isabel Fernandes2, Madalena Caldeira2,
Margarida Bastos3, Carla Baptista3,
Manuela Carvalheiro3, Carlos F.G.C.
Geraldes1, John Griffith Jones1
1NMR 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 |
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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. |
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16:12 |
529. |
A Novel 13C MRS-Based Marker
of Pyruvate Cycling in Perfused Mouse Liver Using
[2-13C] Pyruvate and 13C MRS< |
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Corin O'Dell Miller1,
Haiying Liu1, Leslie Balogh2,
Jin Cao1, Mike R. Tota3,
Reshma Patel3
1Imaging, Merck, Rahway, NJ, USA; 2Laboratory
Animal Research, Merck, Rahway, NJ, USA; 3Metabolic
Disorders, Merck, Rahway, NJ, USA |
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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. |
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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 |
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Sven Gottschalk1,
Tom S. Chan1, Valérie-Ann Raymond1,
Dieter Leibfritz2, Claudia Zwingmann1,2,
Marc Bilodeau1
1Département de sciences biomédicales,
Université de Montréal, Montréal, Québec, Canada;
2Department of Organic Chemistry,
University of Bremen, Bremen, Germany |
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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). |
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16:36 |
531. |
Detection of Altered Hepatic
Gluconeogenesis by Nuclear Magnetic Resonance (NMR)
and Hyperpolarized 13C Pyruvate |
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Matthew E. Merritt1,
Crystal Harrison1, Craig R. Malloy1,
A Dean Sherry1, Shawn C. Burgess1,2
1Advanced Imaging Research, UT Southwestern
Medical Center, Dallas, TX, USA; 2Pharmacology,
UT Southwestern Medical Center, Dallas, TX, USA |
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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. |
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16:48 |
532. |
Real Time Assessment of Krebs
Cycle Metabolism with Hyperpolarized [2-13C]Pyruvate |
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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
1Physiology, Anatomy & Genetics, University of
Oxford, Oxford, Oxfordshire, UK; 2Biochemistry,
University of Cambridge, Cambridge, Cambridgeshire,
UK |
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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. |
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17:00 |
533. |
A 13C Isotopomer
Model for Accurate NMR Quantification of Substrate
Selection and Anaplerosis |
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Wei Li1,
Fang Bian1, Priyanajana Chaudhuri1,
Xin Yu1
1Biomedical Engineering, Case Western Reserve
University, Cleveland, OH, USA |
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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. |
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17:12 |
534. |
Novel Application of Deuterium
MRS: In Vivo Monitoring of Glucose
Consumption |
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Allen Ye1,
Jeffrey L. Duerk1,2, Chris A. Flask1,2,
Gheorghe D. Mateescu3
1Biomedical Engineering, Case Western Reserve
University, Cleveland, OH, USA; 2Radiology,
Case Western Reserve University, Cleveland, OH, USA;
3Chemistry, Case Western Reserve
University, Cleveland, OH, USA |
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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. |
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17:24 |
535. |
Resolution of H3proS
and H3proR Deuterations in [2-13C]glutamate |
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Tiago Brandao
Rodrigues1, Inês Ribeiro Violante1,
Sebastián Cerdán1
1LISMAR, Instituto de Investigaciones
Biomédicas CSIC, Madrid, Spain |
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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. |
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17:36 |
536. |
Hyperpolarized 13C Magnetic Resonance
Detection of Carboxypeptidase G2 Activity |
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Yann Jamin1,
Cristina Gabellieri1, Lynette Smyth1,
Steve Reynolds2, Simon P. Robinson1,
Caroline J. Springer1, Martin O. Leach1,
Geoffrey S. Payne1, Thomas R. Eykyn1
1Institute of Cancer Research and Royal
Marsden NHS trust, Sutton, UK; 2Oxford
Instruments Molecular Biotools Ltd, Abingdon, UK |
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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. |
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17:48 |
537. |
Generation of Hyperpolarized
Substrates by Secondary Labelling with [1,1- 13C]
Acetic Anhydride |
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David M. Wilson1,
Ralph E. Hurd2, Albert P. Chen2,
Mark Van Criekinge1, Kayvan Keshari1,
Sarah J. Nelson1, Daniel B. Vigneron1,
John Kurhanewicz1
1Department of
Radiology, University of California, San Francisco,
San Francisco, CA, USA; 2GE Healthcare |
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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. |
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