In Vivo Pyruvate Dehydrogenase Flux Measured by Hyperpolarized Magnetic Resonance Correlates with ex Vivo Pyruvate Dehydrogenase Activity
Michael Samuel Dodd^1,2, Helen J. Atherton¹, Marie A. Schroeder¹, Lisa C. Heather¹, Lowri E. Cochlin¹, Kieran Clarke¹, George K. Radda¹, Damian J. Tyler^1
1Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, Oxfordshire, United Kingdom; ²Department of Cardiovascular Medicine, Oxford University, Oxford, Oxfordshire, United Kingdom

The recent advent of hyperpolarized ¹³C-MRS has opened a new window on in vivo cardiac metabolism. The use of hyperpolarized [1-¹³C]pyruvate has previously been shown to provide an in vivo measure of pyruvate dehydrogenase (PDH) flux, which directly correlates with disease severity. The aim of this work was to compare in vivo measurements of PDH flux with ex vivo measurements of PDH enzymatic activity. Using well established mechanisms for modulating PDH activity, we have shown that in vivo PDH flux, as measured by hyperpolarized ¹³C MRS, significantly correlates with ex vivo  PDH activity, as measured by well established biochemical assay.

Dynamic Interleaved Imaging of Hyperpolarized Metabolites for Lactate Dehydrogenase Kinetics
Kevin Kai-Chi Leung^1,2, Albert Pofu Chen³, Wilfred W. Lam¹, Angus Zoen Lau^1,2, Charles H. Cunningham^1,2
1Imaging Research, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada; ²Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; ³GE Healthcare, Toronto, Ontario, Canada

This abstract describes the use of spectral-spatial RF pulses and rapid flyback echo planar encoding techniques to acquire 13C images of pyruvate and lactate at high spatial and temporal resolution, upon the injection of hyperpolarized [1-13C]pyruvate into in vitro lactate dehydrogenase enzyme mixture and in vivo rat model. The comparable pyruvate-to-lactate conversion time course and fit to a two-pool kinetic model obtained with dynamic imaging and MR spectroscopy demonstrate the feasibility of mapping first order enzymatic conversion rates in heterogeneous tumors and tissue types non-invasively with hyperpolarized 13C MR imaging.

Hyperpolarized ¹³C MR Spectroscopic Imaging of Disease State in a Switchable MYC-Oncogene Model of Liver Cancer
Simon Hu¹, Asha Balakrishnan², Robert Bok¹, Peder E. Larson¹, Sarah J. Nelson¹, John Kurhanewicz¹, Andrei Goga², Daniel B. Vigneron^1
1Dept. of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA, United States; ²Dept. of Medicine, Division of Hematology/Oncology, University of California, San Francisco, San Francisco, CA, United States

Development of hyperpolarized technology utilizing dynamic nuclear polarization has enabled the monitoring of ¹³C metabolites in vivo at very high SNR. In this work, hyperpolarized ¹³C 3D-MRSI was used to measure liver metabolism in mice after expression of the MYC proto-oncogene was switched on and then off in the liver. Mice in various disease stages were studied, and significant differences in hyperpolarized lactate and alanine levels were detected (P < 0.01). In addition, biochemical assays showed increased LDH expression and activity in the MYC-driven tumors.

Hyperpolarized [1-¹³C]pyruvate and [1,4-¹³C]fumarate Magnetic Resonance Spectroscopy Can Detect Response to the Vascular Disrupting Agent, Combretastatin-A4-Phosphate
Sarah E. Bohndiek^1,2, Mikko I. Kettunen^1,2, De-en Hu^1,2, Timothy H. Witney^1,2, Ferdia A. Gallagher^1,2, Kevin M. Brindle^1,2
1Department of Biochemistry, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom; ²Cancer Research UK Cambridge Research Institute, Cambridge, Cambridgeshire, United Kingdom

Hyperpolarization dramatically increases the sensitivity of the 13C magnetic resonance experiment, allowing the uptake and metabolism of hyperpolarized substrates to be followed in vivo. Vascular disrupting agents target the proliferating endothelial cells in tumour vasculature, so rarely cause tumour shrinkage. Our aim was to assess whether hyperpolarized [1-13C]pyruvate and [1,4-13C]fumarate magnetic resonance spectroscopy could detect response to treatment with Combretastatin-A4-Phosphate within 24 hours of treatment and to compare these methods with data obtained by Dynamic Contrast Enhanced MRI (using Gd-DTPA) and Diffusion Weighted Imaging.

Imaging of Elevated Branched Chain Amino Acid Metabolism in Tumors with Hyperpolarized  ¹³C Ketoisocaproate - not available
Magnus Karlsson^1,2, Pernille Rose Jensen^1,2, Rene in 't Zandt^1,3, Georg Hansson¹, Anna Gisselsson^1,3, Jensen Duus⁴, Sebastian Meier⁴, Mathilde Hauge Lerche^1,2
1Imagnia AB, Malmoe, Sweden; ²Albeda Research Aps, Valby, Denmark; ³Eijdo Research AB, Malmoe, Sweden; ⁴Carslberg Research Center, Valby, Denmark

Hyperpolarized ¹³C magnetic resonance (MR) spectroscopy has in many cases the potential to deliver the sensitivity and detailed spectral information to report on the chemical fate of tracer molecules in different tissues. In a preclinical study we here show that á-ketoisocaproic acid (KIC) can be used to assess molecular signatures of tumors using hyperpolarized MR spectroscopy. KIC is metabolized to leucine by the enzyme branched-chain aminotransferase (BCAT), which is a putative marker for metastasis and a target of the proto-oncogene c-myc.

Imaging of Blood Flow Using Hyperpolarized ¹³C-Urea in Preclinical Murine Models
Cornelius von Morze¹, Peder E. Larson¹, Simon Hu¹, Kayvan Keshari¹, David M. Wilson¹, Jan Henrik Ardenkjaer-Larsen², John Kurhanewicz¹, Daniel B. Vigneron^1
1Department of Radiology and Biomedical Imaging, UCSF, San Francisco, CA, United States; ²GE Healthcare, Hillerød, Denmark

We demonstrate regional imaging of blood flow in preclinical murine models with hyperpolarized (DNP) ¹³C-urea. A bSSFP pulse sequence was developed, with progressively increasing flip angles for efficient sampling of the hyperpolarized magnetization. This allowed temporal and volumetric imaging at a spatial resolution of 2.5mm x 2.5mm x 8mm with a time resolution of 6 s. Regional signal dynamics were quantified, and estimates of relative blood flow to the kidneys and the liver were made. Differences were observed in blood flow patterns to normal and cancerous hepatic tissues. The blood flow maps were compared to results of metabolic maps of 1-¹³C-pyruvate.

Detecting Response to Treatment in Human Breast Adenocarcinoma Using a Co-Administration of Hyperpolarized [1-¹³C]pyruvate and [1,4-¹³C₂]fumarate
Timothy H. Witney^1,2, Mikko I. Kettunen^1,2, De-en Hu^1,2, Ferdia A. Gallagher^1,2, Kevin M. Brindle^1,2
1Department of Biochemistry, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom; ²Cancer Research UK Cambridge Research Institute, Cambridge, Cambridgeshire, United Kingdom

In the current study, we used a co-administration of hyperpolarized [1-¹³C]pyruvate and [1,4-¹³C₂]fumarate as a sensitive marker of cell death in a model of human breast adenocarcinoma following treatment with a DNA damaging agent. The results show that a decrease in pyruvate - lactate exchange coincides with the induction of cell death in breast cancer cells both in vitro and in vivo, with an increase in fumarate - malate exchange shown to correlate to the onset of necrosis.

Analysis of Mitochondrial Metabolism in Cancer Cells by Combining Hyperpolarization and Isotopomer Analysis
Crystal E. Harrison^1,2, Ralph J. DeBerardinis^3,4, Ashish K. Jindal¹, Chendong Yang³, A Dean Sherry^1,5, Craig R. Malloy^1,6
1Advanced Imaging Research Center, UT Southwestern, Dallas, TX, United States; ²Physics, UT Dallas, Richardson, TX, United States; ³Pediatrics, UT Southwestern, Dallas, TX, United States; ⁴McDermott Center for Human Growth and Development, UT Southwestern, Dallas, TX, United States; ⁵Chemistry, UT Dallas, Richardson, TX, United States; ⁶Veterans Affairs, NorthTexas Health Care System, Dallas, TX, United States

While most research in cancer metabolism has focused on lactate formation (the Warburg effect), less is known about the mitochondrial pathways utilized during cell growth.  Hyperpolarized [1-13C]-pyruvate provides insight into both the Warburg effect and mitochondrial metabolism, including activity of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC).  To combine the sensitivity of hyperpolarization with the precision of isotopomer analysis, we pre-incubated glioblastoma cells with [3-13C]-pyruvate prior to a short incubation with hyperpolarized [1-13C]-pyruvate.  Using this technique, we observed real-time accumulation of hyperpolarized, 13C-labeled lactate and bicarbonate, and determined that the latter arose from the direct activity of PDH.

Investigating the Metabolic Effects of Heart Failure Progression In Vivo Using Hyperpolarized Magnetic Resonance
Helen Jennifer Atherton¹, Michael S. Dodd¹, Carolyn A. Carr¹, Daniel J. Stuckey¹, Kieran Clarke¹, George K. Radda¹, Damian J. Tyler^1
1Physiology, Anatomy and Genetics, University of Oxford, Oxford, Oxfordshire, United Kingdom

Using hyperpolarized magnetic resonance spectroscopy (MRS), we determined in vivo the temporal metabolic changes associated with heart failure progression post myocardial infarction (MI). Two weeks post MI, PDH flux was equivalent in failing and control hearts. In contrast levels of [1-¹³C]citrate, [1-¹³C]acetyl carnitine and [5-¹³C]glutamate were reduced in infarcted hearts reflecting a perturbation in Krebs cycle metabolism. Reduced [1-¹³C]lactate was also observed post MI indicating decreased glucose uptake and/or glycolysis. This study highlights the importance of assessing metabolism at multiple time points in vivo, and demonstrates the potential of hyperpolarized MRS for investigating the metabolic effects of progressive diseases.

Indirect Detection of Enzymatic Processes by Hyperpolarized NMR: Temporal Information, Enhanced Spectral Resolution and Slow Spin Relaxation
Talia Harris¹, Patrick Giraudeau¹, Lucio Frydman^1
1Chemical Physics, Weizmann Institute of Science, Rehovot, Israel

The outstanding sensitivity arising from ex situ DNP has triggered high expectations concerning the in vivo monitoring of metabolism and disease. So far such gains have materialized for experiments focusing on low-γ nuclei, whose relatively long T₁s enables them to withstand the transfer from the cryogenic hyperpolarizer to the reacting centers of interest. This study demonstrates that, when suitably merged with spatially-encoded methods, also indirectly-detected ¹H NMR spectroscopy can be exploited in time-resolved hyperpolarized analyses. The principles and opportunities opened by this approach are exemplified by Choline’s phosphorylation by Choline Kinase, and by Acetylcholine’s hydrolization by Acetylcholine Esterase.