Sonal Josan

In vivo MRSI of hyperpolarized [1-13C]pyruvate metabolism in rat hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than 1 year following diagnosis. Most patients with HCC are diagnosed at an advanced stage, and no efficient marker exists for the prediction of prognosis and/or response(s) to therapy. We have reported previously a high level of [1‐13C]alanine in an orthotopic HCC using single‐voxel hyperpolarized [1‐13C]pyruvate MRS. In the present study, we implemented a three‐dimensional MRSI sequence to investigate this potential hallmark of cellular metabolism in rat livers bearing HCC (n = 7 buffalo rats). In addition, quantitative real‐time polymerase chain reaction was used to determine the mRNA levels of lactate dehydrogenase A, nicotinamide adenine (phosphate) dinucleotide dehydrogenase quinone 1 and alanine transaminase. The enzyme levels were significantly higher in tumor than in normal liver tissues within each rat, and were associated with the in vivo MRSI signal of [1‐13C]alanine and [1‐13C]lactate after a bolus intravenous injection of [1‐13C]pyruvate. Histopathological analysis of these tumors confirmed the successful growth of HCC as a nodule in buffalo rat livers, revealing malignancy and hypervascular architecture. More importantly, the results demonstrated that the metabolic fate of [1‐13C]pyruvate conversion to [1‐13C]alanine significantly superseded that of [1‐13C]pyruvate conversion to [1‐13C]lactate, potentially serving as a marker of HCC tumors. Copyright

Dynamic and high-resolution metabolic imaging of hyperpolarized [1-13C]-pyruvate in the rat brain using a high-performance gradient insert

Fast chemical shift imaging (CSI) techniques are advantageous in metabolic imaging of hyperpolarized compounds due to the limited duration of the signal amplification. At the same time, reducing the acquisition time in hyperpolarized imaging does not necessarily lead to the conventional penalty in signal‐to‐noise ratio that occurs in imaging at thermal equilibrium polarization levels. Here a high‐performance gradient insert was used in combination with undersampled spiral CSI to increase either the imaging speed or the spatial resolution of hyperpolarized 13C metabolic imaging on a clinical 3T MR scanner. Both a single‐shot sequence with a total acquisition time of 125 ms and a three‐shot sequence with a nominal in‐plane resolution of 1.5 mm were implemented. The k‐space trajectories were measured and then used during image reconstruction. The technique was applied to metabolic imaging of the rat brain in vivo after the injection of hyperpolarized [1‐13C]‐pyruvate. Dynamic imaging afforded the measurement of region‐of‐interest‐specific time courses of pyruvate and its metabolic products, while imaging at high spatial resolution was used to better characterize the spatial distribution of the metabolite signals. Magn Reson Med, 2011.

Double half RF pulses for reduced sensitivity to eddy currents in UTE imaging

Ultrashort echo time imaging with half RF pulse excitation is challenging as eddy currents induced by the slice‐select gradient distort the half pulse slice profile. This work presents two pulses with T2‐dependent slice profiles that are less sensitive to eddy currents. The double half pulse improves the slice selectivity for long T2 components, while the inverted double half pulse suppresses the unwanted long T2 signal. Thus, both approaches prevent imperfect cancellation of out‐of‐slice signal from contaminating the desired slice. Experimental results demonstrate substantially improved slice selectivity and R2* quantitation accuracy with these pulses. These pulses are effective in making short T2 imaging and quantitation less sensitive to eddy currents and provide an alternative to time‐consuming gradient characterization. Magn Reson Med, 2009.

Quantification of in vivo metabolic kinetics of hyperpolarized pyruvate in rat kidneys using dynamic 13C MRSI.

With signal‐to‐noise ratio enhancements on the order of 10,000‐fold, hyperpolarized MRSI of metabolically active substrates allows the study of both the injected substrate and downstream metabolic products in vivo. Although hyperpolarized [1‐13C]pyruvate, in particular, has been used to demonstrate metabolic activities in various animal models, robust quantification and metabolic modeling remain important areas of investigation. Enzyme saturation effects are routinely seen with commonly used doses of hyperpolarized [1‐13C]pyruvate; however, most metrics proposed to date, including metabolite ratios, time‐to‐peak of metabolic products and single exchange rate constants, fail to capture these saturation effects. In addition, the widely used small‐flip‐angle excitation approach does not correctly model the inflow of fresh downstream metabolites generated proximal to the target slice, which is often a significant factor in vivo. In this work, we developed an efficient quantification framework employing a spiral‐based dynamic spectroscopic imaging approach. The approach overcomes the aforementioned limitations and demonstrates that the in vivo 13C labeling of lactate and alanine after a bolus injection of [1‐13C]pyruvate is well approximated by saturatable kinetics, which can be mathematically modeled using a Michaelis–Menten‐like formulation, with the resulting estimated apparent maximal reaction velocity Vmax and apparent Michaelis constant KM being unbiased with respect to critical experimental parameters, including the substrate dose, bolus shape and duration. Although the proposed saturatable model has a similar mathematical formulation to the original Michaelis–Menten kinetics, it is conceptually different. In this study, we focus on the 13C labeling of lactate and alanine and do not differentiate the labeling mechanism (net flux or isotopic exchange) or the respective contribution of various factors (organ perfusion rate, substrate transport kinetics, enzyme activities and the size of the unlabeled lactate and alanine pools) to the labeling process. Copyright

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.

Application of double spin echo spiral chemical shift imaging to rapid metabolic mapping of hyperpolarized [1 − 13C]-pyruvate

Undersampled spiral CSI (spCSI) using a free induction decay (FID) acquisition allows sub-second metabolic imaging of hyperpolarized ¹³C. Phase correction of the FID acquisition can be difficult, especially with contributions from aliased out-of-phase peaks. This work extends the spCSI sequence by incorporating double spin echo radiofrequency (RF) pulses to eliminate the need for phase correction and obtain high quality spectra in magnitude mode. The sequence also provides an added benefit of attenuating signal from flowing spins, which can otherwise contaminate signal in the organ of interest. The refocusing pulses can potentially lead to a loss of hyperpolarized magnetization in dynamic imaging due to flow of spins through the fringe field of the RF coil, where the refocusing pulses fail to provide complete refocusing. Care must be taken for dynamic imaging to ensure that the spins remain within the B₁-homogeneous sensitive volume of the RF coil.

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