Jae Mo Park

Metabolic response of glioma to dichloroacetate measured in vivo by hyperpolarized 13C magnetic resonance spectroscopic imaging

BACKGROUND The metabolic phenotype that derives disproportionate energy via glycolysis in solid tumors, including glioma, leads to elevated lactate labeling in metabolic imaging using hyperpolarized [1-(13)C]pyruvate. Although the pyruvate dehydrogenase (PDH)-mediated flux from pyruvate to acetyl coenzyme A can be indirectly measured through the detection of carbon-13 ((13)C)-labeled bicarbonate, it has proven difficult to visualize (13)C-bicarbonate at high enough levels from injected [1-(13)C]pyruvate for quantitative analysis in brain. The aim of this study is to improve the detection of (13)C-labeled metabolites, in particular bicarbonate, in glioma and normal brain in vivo and to measure the metabolic response to dichloroacetate, which upregulates PDH activity. METHODS An optimized protocol for chemical shift imaging and high concentration of hyperpolarized [1-(13)C]pyruvate were used to improve measurements of lactate and bicarbonate in C6 glioma-transplanted rat brains. Hyperpolarized [1-(13)C]pyruvate was injected before and 45 min after dichloroacetate infusion. Metabolite ratios of lactate to bicarbonate were calculated to provide improved metrics for characterizing tumor metabolism. RESULTS Glioma and normal brain were well differentiated by lactate-to-bicarbonate ratio (P = .002, n = 5) as well as bicarbonate (P = .0002) and lactate (P = .001), and a stronger response to dichloroacetate was observed in glioma than in normal brain. CONCLUSION Our results clearly demonstrate for the first time the feasibility of quantitatively detecting (13)C-bicarbonate in tumor-bearing rat brain in vivo, permitting the measurement of dichloroacetate-modulated changes in PDH flux. The simultaneous detection of lactate and bicarbonate provides a tool for a more comprehensive analysis of glioma metabolism and the assessment of metabolic agents as anti-brain cancer drugs.

Measuring mitochondrial metabolism in rat brain in vivo using MR Spectroscopy of hyperpolarized [2‐13C]pyruvate

Hyperpolarized [1‐13C]pyruvate ([1‐13C]Pyr) has been used to assess metabolism in healthy and diseased states, focusing on the downstream labeling of lactate (Lac), bicarbonate and alanine. Although hyperpolarized [2‐13C]Pyr, which retains the labeled carbon when Pyr is converted to acetyl‐coenzyme A, has been used successfully to assess mitochondrial metabolism in the heart, the application of [2‐13C]Pyr in the study of brain metabolism has been limited to date, with Lac being the only downstream metabolic product reported previously. In this study, single‐time‐point chemical shift imaging data were acquired from rat brain in vivo. [5‐13C]Glutamate, [1‐13C]acetylcarnitine and [1‐13C]citrate were detected in addition to resonances from [2‐13C]Pyr and [2‐13C]Lac. Brain metabolism was further investigated by infusing dichloroacetate, which upregulates Pyr flux to acetyl‐coenzyme A. After dichloroacetate administration, a 40% increase in [5‐13C]glutamate from 0.014 ± 0.004 to 0.020 ± 0.006 (p = 0.02), primarily from brain, and a trend to higher citrate (0.002 ± 0.001 to 0.004 ± 0.002) were detected, whereas [1‐13C]acetylcarnitine was increased in peripheral tissues. This study demonstrates, for the first time, that hyperpolarized [2‐13C]Pyr can be used for the in vivo investigation of mitochondrial function and tricarboxylic acid cycle metabolism in brain. Copyright

Application of hyperpolarized [1-13C]lactate for the in vivo investigation of cardiac metabolism

In addition to cancer imaging, 13C‐MRS of hyperpolarized pyruvate has also demonstrated utility for the investigation of cardiac metabolism and ischemic heart disease. Although no adverse effects have yet been reported for doses commonly used in vivo, high substrate concentrations have lead to supraphysiological pyruvate levels that can affect the underlying metabolism and should be considered when interpreting results. With lactate serving as an important energy source for the heart and physiological lactate levels one to two orders of magnitude higher than for pyruvate, hyperpolarized lactate could potentially be used as an alternative to pyruvate for probing cardiac metabolism. In this study, hyperpolarized [1‐13C]lactate was used to acquire time‐resolved spectra from the healthy rat heart in vivo and to measure dichloroacetate (DCA)‐modulated changes in flux through pyruvate dehydrogenase (PDH). Both primary oxidation of lactate to pyruvate and subsequent conversion of pyruvate to alanine and bicarbonate could reliably be detected. Since DCA stimulates the activity of PDH through inhibition of PDH kinase, a more than 2.5‐fold increase in bicarbonate‐to‐substrate ratio was found after administration of DCA, similar to the effect when using [1‐13C]pyruvate as the substrate. Copyright

Metabolite kinetics in C6 rat glioma model using magnetic resonance spectroscopic imaging of hyperpolarized [1‐13C]pyruvate

In addition to an increased lactate‐to‐pyruvate ratio, altered metabolism of a malignant glioma can be further characterized by its kinetics. Spatially resolved dynamic data of pyruvate and lactate from C6‐implanted female Sprague–Dawley rat brain were acquired using a spiral chemical shift imaging sequence after a bolus injection of a hyperpolarized [1‐13C]pyruvate. Apparent rate constants for the conversion of pyruvate to lactate in three different regions (glioma, normal appearing brain, and vasculature) were estimated based on a two‐site exchange model. The apparent conversion rate constant was 0.018 ± 0.004 s−1 (mean ± standard deviation, n = 6) for glioma, 0.009 ± 0.003 s−1 for normal brain, and 0.005 ± 0.001 s−1 for vasculature, whereas the lactate‐to‐pyruvate ratio, the metabolic marker used to date to identify tumor regions, was 0.36 ± 0.07 (mean ± SD), 0.24 ± 0.07, and 0.12 ± 0.02 for glioma, normal brain, and vasculature, respectively. The data suggest that the apparent conversion rate better differentiate glioma from normal brain (P = 0.001, n = 6) than the lactate‐to‐pyruvate ratio (P = 0.02). Magn Reson Med, 2012.

In vivo investigation of cardiac metabolism in the rat using MRS of hyperpolarized [1-13C] and [2-13C]pyruvate.

Hyperpolarized 13C MRS allows the in vivo assessment of pyruvate dehydrogenase complex (PDC) flux, which converts pyruvate to acetyl‐coenzyme A (acetyl‐CoA). [1‐13C]pyruvate has been used to measure changes in cardiac PDC flux, with demonstrated increase in 13C‐bicarbonate production after dichloroacetate (DCA) administration. With [1‐13C]pyruvate, the 13C label is released as 13CO2/13C‐bicarbonate, and, hence, does not allow us to follow the fate of acetyl‐CoA. Pyruvate labeled in the C2 position has been used to track the 13C label into the TCA (tricarboxylic acid) cycle and measure [5‐13C]glutamate as well as study changes in [1‐13C]acetylcarnitine with DCA and dobutamine. This work investigates changes in the metabolic fate of acetyl‐CoA in response to metabolic interventions of DCA‐induced increased PDC flux in the fed and fasted state, and increased cardiac workload with dobutamine in vivo in rat heart at two different pyruvate doses. DCA led to a modest increase in the 13C labeling of [5‐13C]glutamate, and a considerable increase in [1‐13C]acetylcarnitine and [1,3‐13C]acetoacetate peaks. Dobutamine resulted in an increased labeling of [2‐13C]lactate, [2‐13C]alanine and [5‐13C]glutamate. The change in glutamate with dobutamine was observed using a high pyruvate dose but not with a low dose. The relative changes in the different metabolic products provide information about the relationship between PDC‐mediated oxidation of pyruvate and its subsequent incorporation into the TCA cycle compared with other metabolic pathways. Using a high dose of pyruvate may provide an improved ability to observe changes in glutamate. Copyright

Effects of isoflurane anesthesia on hyperpolarized 13C metabolic measurements in rat brain

Commonly used anesthetic agents such as isoflurane are known to be potent cerebral vasodilators, with reported dose‐dependent increase in cerebral blood flow and cerebral blood volume. Despite the widespread use of isoflurane in hyperpolarized 13C preclinical research studies, a quantitative assessment of its effect on metabolic measurements is limited. This work investigates the effect of isoflurane anesthesia dose on hyperpolarized 13C MR metabolic measurements in rat brain for [1‐13C]pyruvate and 2‐keto[1‐13C]isocaproate.

Dynamic metabolic imaging of hyperpolarized [2-(13) C]pyruvate using spiral chemical shift imaging with alternating spectral band excitation.

In contrast to [1‐13C]pyruvate, hyperpolarized [2‐13C]pyruvate permits the ability to follow the 13C label beyond flux through pyruvate dehydrogenase complex and investigate the incorporation of acetyl‐coenzyme A into different metabolic pathways. However, chemical shift imaging (CSI) with [2‐13C]pyruvate is challenging owing to the large spectral dispersion of the resonances, which also leads to severe chemical shift displacement artifacts for slice‐selective acquisitions.

Volumetric spiral chemical shift imaging of hyperpolarized [2-13c]pyruvate in a rat c6 glioma model

MRS of hyperpolarized [2‐13C]pyruvate can be used to assess multiple metabolic pathways within mitochondria as the 13C label is not lost with the conversion of pyruvate to acetyl‐CoA. This study presents the first MR spectroscopic imaging of hyperpolarized [2‐13C]pyruvate in glioma‐bearing brain.

Hyperpolarized (13)C-lactate to (13)C-bicarbonate ratio as a biomarker for monitoring the acute response of anti-vascular endothelial growth factor (anti-VEGF) treatment.

Hyperpolarized [1‐13C]pyruvate MRS provides a unique imaging opportunity to study the reaction kinetics and enzyme activities of in vivo metabolism because of its favorable imaging characteristics and critical position in the cellular metabolic pathway, where it can either be reduced to lactate (reflecting glycolysis) or converted to acetyl‐coenzyme A and bicarbonate (reflecting oxidative phosphorylation). Cancer tissue metabolism is altered in such a way as to result in a relative preponderance of glycolysis relative to oxidative phosphorylation (i.e. Warburg effect). Although there is a strong theoretical basis for presuming that readjustment of the metabolic balance towards normal could alter tumor growth, a robust noninvasive in vivo tool with which to measure the balance between these two metabolic processes has yet to be developed. Until recently, hyperpolarized 13C‐pyruvate imaging studies had focused solely on [1‐13C]lactate production because of its strong signal. However, without a concomitant measure of pyruvate entry into the mitochondria, the lactate signal provides no information on the balance between the glycolytic and oxidative metabolic pathways. Consistent measurement of 13C‐bicarbonate in cancer tissue, which does provide such information, has proven difficult, however. In this study, we report the reliable measurement of 13C‐bicarbonate production in both the healthy brain and a highly glycolytic experimental glioblastoma model using an optimized 13C MRS imaging protocol. With the capacity to obtain signal in all tumors, we also confirm for the first time that the ratio of 13C‐lactate to 13C‐bicarbonate provides a more robust metric relative to 13C‐lactate for the assessment of the metabolic effects of anti‐angiogenic therapy. Our data suggest a potential application of this ratio as an early biomarker to assess therapeutic effectiveness. Furthermore, although further study is needed, the results suggest that anti‐angiogenic treatment results in a rapid normalization in the relative tissue utilization of glycolytic and oxidative phosphorylation by tumor tissue. Copyright

Assessing inflammatory liver injury in an acute CCl4 model using dynamic 3D metabolic imaging of hyperpolarized [1-(13)C]pyruvate.

To facilitate diagnosis and staging of liver disease, sensitive and non‐invasive methods for the measurement of liver metabolism are needed. This study used hyperpolarized 13C‐pyruvate to assess metabolic parameters in a CCl4 model of liver damage in rats. Dynamic 3D 13C chemical shift imaging data from a volume covering kidney and liver were acquired from 8 control and 10 CCl4‐treated rats. At 12 time points at 5 s temporal resolution, we quantified the signal intensities and established time courses for pyruvate, alanine, and lactate. These measurements were compared with standard liver histology and an alanine transaminase (ALT) enzyme assay using liver tissue from the same animals. All CCl4‐treated but none of the control animals showed histological liver damage and elevated ALT enzyme levels. In agreement with these results, metabolic imaging revealed an increased alanine/pyruvate ratio in liver of CCl4‐treated rats, which is indicative of elevated ALT activity. Similarly, lactate/pyruvate ratios were higher in CCl4‐treated compared with control animals, demonstrating the presence of inflammation. No significant differences in metabolite ratios were observed in kidney or vasculature. Thus this work shows that metabolic imaging using 13C‐pyruvate can be a successful tool to non‐invasively assess liver damage in vivo. Copyright