Investigating the origin of the 13 C lactate signal in the anesthetized healthy rat brain in vivo after hyperpolarized [1-13 C]pyruvate injection.

The goal of this study was to investigate the origin of brain lactate (Lac) signal in the healthy anesthetized rat after injection of hyperpolarized (HP) [1-13 C]pyruvate (Pyr). Dynamic two-dimensional spiral chemical shift imaging with flow-sensitizing gradients revealed reduction in both vascular and brain Pyr, while no significant dependence on the level of flow suppression was detected for Lac. These results support the hypothesis that the HP metabolites predominantly reside in different compartments in the brain (i.e., Pyr in the blood and Lac in the parenchyma). Data from high-resolution metabolic imaging of [1-13 C]Pyr further demonstrated that Lac detected in the brain was not from contributions of vascular signal attributable to partial volume effects. Additionally, metabolite distributions and kinetics measured with dynamic imaging after injection of HP [1-13 C]Lac were similar to Pyr data when Pyr was used as the substrate. These data do not support the hypothesis that Lac observed in the brain after Pyr injection was generated in other organs and then transported across the blood-brain barrier (BBB). Together, the presented results provide further evidence that even in healthy anesthetized rats, the transport of HP Pyr across the BBB is sufficiently fast to permit detection of its metabolic conversion to Lac within the brain.

Hyperpolarized NMR study of the impact of pyruvate dehydrogenase kinase inhibition on the pyruvate dehydrogenase and TCA flux in type 2 diabetic rat muscle.

The role of pyruvate dehydrogenase in mediating lipid-induced insulin resistance stands as a central question in the pathogenesis of type 2 diabetes mellitus. Many researchers have invoked the Randle hypothesis to explain the reduced glucose disposal in skeletal muscle by envisioning an elevated acetyl CoA pool arising from increased oxidation of fatty acids. Over the years, in vivo NMR studies have challenged that monolithic view. The advent of the dissolution dynamic nuclear polarization NMR technique and a unique type 2 diabetic rat model provides an opportunity to clarify. Dynamic nuclear polarization enhances dramatically the NMR signal sensitivity and allows the measurement of metabolic kinetics in vivo. Diabetic muscle has much lower pyruvate dehydrogenase activity than control muscle, as evidenced in the conversion of [1-13C]lactate and [2-13C]pyruvate to HCO3- and acetyl carnitine. The pyruvate dehydrogenase kinase inhibitor, dichloroacetate, restores rapidly the diabetic pyruvate dehydrogenase activity to control level. However, diabetic muscle has a much larger dynamic change in pyruvate dehydrogenase flux than control. The dichloroacetate-induced surge in pyruvate dehydrogenase activity produces a differential amount of acetyl carnitine but does not affect the tricarboxylic acid flux. Further studies can now proceed with the dynamic nuclear polarization approach and a unique rat model to interrogate closely the biochemical mechanism interfacing oxidative metabolism with insulin resistance and metabolic inflexibility.

Automated detection of critical findings in multi-parametric brain MRI using a system of 3D neural networks.

With the rapid growth and increasing use of brain MRI, there is an interest in automated image classification to aid human interpretation and improve workflow. We aimed to train a deep convolutional neural network and assess its performance in identifying abnormal brain MRIs and critical intracranial findings including acute infarction, acute hemorrhage and mass effect. A total of 13,215 clinical brain MRI studies were categorized to training (74%), validation (9%), internal testing (8%) and external testing (8%) datasets. Up to eight contrasts were included from each brain MRI and each image volume was reformatted to common resolution to accommodate for differences between scanners. Following reviewing the radiology reports, three neuroradiologists assigned each study to abnormal vs normal, and identified three critical findings including acute infarction, acute hemorrhage, and mass effect. A deep convolutional neural network was constructed by a combination of localization feature extraction (LFE) modules and global classifiers to identify the presence of 4 variables in brain MRIs including abnormal, acute infarction, acute hemorrhage and mass effect. Training, validation and testing sets were randomly defined on a patient basis. Training was performed on 9845 studies using balanced sampling to address class imbalance. Receiver operating characteristic (ROC) analysis was performed. The ROC analysis of our models for 1050 studies within our internal test data showed AUC/sensitivity/specificity of 0.91/83%/86% for normal versus abnormal brain MRI, 0.95/92%/88% for acute infarction, 0.90/89%/81% for acute hemorrhage, and 0.93/93%/85% for mass effect. For 1072 studies within our external test data, it showed AUC/sensitivity/specificity of 0.88/80%/80% for normal versus abnormal brain MRI, 0.97/90%/97% for acute infarction, 0.83/72%/88% for acute hemorrhage, and 0.87/79%/81% for mass effect. Our proposed deep convolutional network can accurately identify abnormal and critical intracranial findings on individual brain MRIs, while addressing the fact that some MR contrasts might not be available in individual studies.

Dynamic metabolic imaging of copolarized [2-13 C]pyruvate and [1,4-13 C2 ]fumarate using 3D-spiral CSI with alternate spectral band excitation.

PURPOSE: Developing a method for simultaneous metabolic imaging of copolarized [2-13 C]pyruvate and [1,4-13 C2 ]fumarate without chemical shift displacement artifacts that also permits different excitation flip angles for substrates and their metabolic products. METHODS: The proposed pulse sequence consists of 2 frequency-selective radiofrequency pulses to alternatingly excite 2 spectral sub-bands each one followed by a fast 3D spiral CSI (3D-spCSI) readout. Spectrally selective radiofrequency pulses were designed to excite differential flip angles on substrates and products in each spectral sub-band. Number of signal averages analysis was used to determine a spectral width suitable to resolve the metabolites of interest in each of the sub-bands. RESULTS: Phantom experiments verified the copolarization strategy and radiofrequency pulse design following differential flip angle used in our method. The signal behavior of the resonances in each sub-band was unaffected by the excitation of the respective alternate frequency band. Dynamic 3D 13 C CSI data demonstrated the ability of the sequence to image metabolites like pyruvate-hydrate, lactate, alanine, fumarate, and malate simultaneously and detect metabolic changes in the liver in a rat model of carbon tetrachloride-induced liver damage. CONCLUSION: The presented method allows the dynamic CSI of a mixture of [2-13 C]pyruvate and [1,4-13 C2 ]fumarate without chemical shift displacement artifacts while also permitting the use of different flip angles for substrate and product signals. The method is potentially useful for combined in vivo imaging of inflammation and cell necrosis.

3D-multi-echo radial imaging of 23 Na (3D-MERINA) for time-efficient multi-parameter tissue compartment mapping.

PURPOSE: This work demonstrates a 3D radial multi-echo acquisition scheme for time-efficient sodium (23 Na) MR-signal acquisition and analysis. Echo reconstructions were used to produce signal-to-noise ratio (SNR)-enhanced 23 Na-images and parameter maps of the biexponential observed transverse relaxation time ( T2*) decay. METHODS: A custom-built sequence for radial multi-echo acquisition was proposed for acquisition of a series of 3D volumetric 23 Na-images. Measurements acquired in a phantom and in vivo human brains were analyzed for SNR enhancement and multi-component T2* estimation. RESULTS: Rapid gradient refocused imaging acquired 38 echoes within a repetition time of 160 ms. Signal averaging of multi-echo time (TE) measurements showed an average brain tissue SNR enhancement of 34% compared to single-TE images across subjects. Phantom and in vivo measurements detected distinguishable signal decay characteristics for fluid and solid media. Mapping results were investigated in phantom and in vivo experiments for sequence timing optimization and signal decay analysis. The T2* mapping results were consistent with previously reported values and facilitated fluid-signal discrimination. CONCLUSION: The proposed method offers an efficient 23 Na-imaging scheme that extends existing 23 Na-MRI sequences by acquiring signal decay information with no increase in time or specific absorption rate. The resultant SNR-enhanced 23 Na-images and estimated T2* signal decay characteristics offer great potential for detailed investigation of tissue compartment characterization and clinical application. Magn Reson Med 79:1950-1961, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

3D printed phantoms mimicking cortical bone for the assessment of ultrashort echo time magnetic resonance imaging.

PURPOSE: Human cortical bone has a rapid T2∗ decay, and it can be visualized using ultrashort echo time (UTE) techniques in magnetic resonance imaging (MRI). These sequences operate at the limits of gradient and transmit-receive signal performance. Development of multicompartment anthropomorphic phantoms that can mimic human cortical bone can assist with quality assurance and optimization of UTE sequences. The aims of this study were to (a) characterize the MRI signal properties of a photopolymer resin that can be 3D printed, (b) develop multicompartment phantoms based on the resin, and (c) demonstrate the feasibility of using these phantoms to mimic human anatomy in the assessment of UTE sequences. METHODS: A photopolymer resin (Prismlab China Ltd, Shanghai, China) was imaged on a 3 Tesla MRI system (Siemens Skyra) to characterize its MRI properties with emphasis on T2∗ signal and longevity. Two anthropomorphic phantoms, using the 3D printed resin to simulate skeletal anatomy, were developed and imaged using UTE sequences. A skull phantom was developed and used to assess the feasibility of using the resin to develop a complex model with realistic morphological human characteristics. A tibia model was also developed to assess the suitability of the resin at mimicking a simple multicompartment anatomical model and imaged using a three-dimensional UTE sequence (PETRA). Image quality measurements of signal-to-noise ratio (SNR) and contrast factor were calculated and these were compared to in vivo values. RESULTS: The T2∗ and T1 (mean ± standard deviation) of the photopolymer resin was found to be 411 ± 19 µs and 74.39 ± 13.88 ms, respectively, and demonstrated no statistically significant change during 4 months of monitoring. The resin had a similar T2∗ decay to human cortical bone; however, had lower T1 properties. The bone water concentration of the resin was 59% relative to an external water reference phantom, and this was higher than in vivo values reported for human cortical bone. The multicompartment anthropomorphic head phantom was successfully produced and able to simulate realistic air cavities, bony anatomy, and soft tissue. Image quality assessment in the tibia phantom using the PETRA sequence showed the suitability of the resin to mimic human anatomy with high SNR and contrast making it suitable for tissue segmentation. CONCLUSIONS: A solid resin material, which can be 3D printed, has been found to have similar magnetic resonance signal properties to human cortical bone. Phantoms replicating skeletal anatomy were successfully produced using this resin and demonstrated their use for image quality and segmentation assessment of ultrashort echo time sequences.

Feasibility of free breathing Lung MRI for Radiotherapy using non-Cartesian k-space acquisition schemes.

OBJECTIVE: To test a free-breathing MRI protocol for anatomical and functional assessment during lung cancer radiotherapy by assessing two non-Cartesian acquisition schemes based on T1 weighted 3D gradient recall echo sequence: (i) stack-of stars (StarVIBE) and (ii) spiral (SpiralVIBE) trajectories. METHODS: MR images on five healthy volunteers were acquired on a wide bore 3T scanner (MAGNETOM Skyra, Siemens Healthcare, Erlangen, Germany). Anatomical image quality was assessed on: (1) free breathing (StarVIBE), (2) the standard clinical sequence (volumetric interpolated breath-hold examination, VIBE) acquired in a 20 second (s) compliant breath-hold and (3) 20 s non-compliant breath-hold. For functional assessment, StarVIBE and the current standard breath-hold time-resolved angiography with stochastic trajectories (TWIST) sequence were run as multiphase acquisitions to replicate dynamic contrast enhancement (DCE) in one healthy volunteer. The potential application of the SpiralVIBE sequence for lung parenchymal imaging was assessed on one healthy volunteer. Ten patients with lung cancer were subsequently imaged with the StarVIBE and SpiralVIBE sequences for anatomical and structural assessment. For functional assessment, free-breathing StarVIBE DCE protocol was compared with breath-hold TWIST sequences on four prior lung cancer patients with similar tumour locations. Image quality was evaluated independently and blinded to sequence information by an experienced thoracic radiologist. RESULTS: For anatomical assessment, the compliant breath-hold VIBE sequence was better than free-breathing StarVIBE. However, in the presence of a non-compliant breath-hold, StarVIBE was superior. For functional assessment, StarVIBE outperformed the standard sequence and was shown to provide robust DCE data in the presence of motion. The ultrashort echo of the SpiralVIBE sequence enabled visualisation of lung parenchyma. CONCLUSION: The two non-Cartesian acquisition sequences, StarVIBE and SpiralVIBE, provide a free-breathing imaging protocol of the lung with sufficient image quality to permit anatomical, structural and functional assessment during radiotherapy. Advances in knowledge: Novel application of non-Cartesian MRI sequences for lung cancer imaging for radiotherapy. Illustration of SpiralVIBE UTE sequence as a promising sequence for lung structural imaging during lung radiotherapy.

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-(13)C]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 (13)C-pyruvate imaging studies had focused solely on [1-(13)C]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 (13)C-bicarbonate in cancer tissue, which does provide such information, has proven difficult, however. In this study, we report the reliable measurement of (13)C-bicarbonate production in both the healthy brain and a highly glycolytic experimental glioblastoma model using an optimized (13)C MRS imaging protocol. With the capacity to obtain signal in all tumors, we also confirm for the first time that the ratio of (13)C-lactate to (13)C-bicarbonate provides a more robust metric relative to (13)C-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.

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

PURPOSE: MRS of hyperpolarized [2-(13)C]pyruvate can be used to assess multiple metabolic pathways within mitochondria as the (13)C label is not lost with the conversion of pyruvate to acetyl-CoA. This study presents the first MR spectroscopic imaging of hyperpolarized [2-(13)C]pyruvate in glioma-bearing brain. METHODS: Spiral chemical shift imaging with spectrally undersampling scheme (1042 Hz) and a hard-pulse excitation was exploited to simultaneously image [2-(13)C]pyruvate, [2-(13)C]lactate, and [5-(13)C]glutamate, the metabolites known to be produced in brain after an injection of hyperpolarized [2-(13)C]pyruvate, without chemical shift displacement artifacts. A separate undersampling scheme (890 Hz) was also used to image [1-(13)C]acetyl-carnitine. Healthy and C6 glioma-implanted rat brains were imaged at baseline and after dichloroacetate administration, a drug that modulates pyruvate dehydrogenase kinase activity. RESULTS: The baseline metabolite maps showed higher lactate and lower glutamate in tumor as compared to normal-appearing brain. Dichloroacetate led to an increase in glutamate in both tumor and normal-appearing brain. Dichloroacetate-induced %-decrease of lactate/glutamate was comparable to the lactate/bicarbonate decrease from hyperpolarized [1-(13)C]pyruvate studies. Acetyl-carnitine was observed in the muscle/fat tissue surrounding the brain. CONCLUSION: Robust volumetric imaging with hyperpolarized [2-(13)C]pyruvate and downstream products was performed in glioma-bearing rat brains, demonstrating changes in mitochondrial metabolism with dichloroacetate.

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 (13)C-pyruvate to assess metabolic parameters in a CCl4 model of liver damage in rats. Dynamic 3D (13)C 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 (13)C-pyruvate can be a successful tool to non-invasively assess liver damage in vivo.

Hyperpolarized 13C NMR observation of lactate kinetics in skeletal muscle.

The production of glycolytic end products, such as lactate, usually evokes a cellular shift from aerobic to anaerobic ATP generation and O2 insufficiency. In the classical view, muscle lactate must be exported to the liver for clearance. However, lactate also forms under well-oxygenated conditions, and this has led investigators to postulate lactate shuttling from non-oxidative to oxidative muscle fiber, where it can serve as a precursor. Indeed, the intracellular lactate shuttle and the glycogen shunt hypotheses expand the vision to include a dynamic mobilization and utilization of lactate during a muscle contraction cycle. Testing the tenability of these provocative ideas during a rapid contraction cycle has posed a technical challenge. The present study reports the use of hyperpolarized [1-(13)C]lactate and [2-(13)C]pyruvate in dynamic nuclear polarization (DNP) NMR experiments to measure the rapid pyruvate and lactate kinetics in rat muscle. With a 3 s temporal resolution, (13)C DNP NMR detects both [1-(13)C]lactate and [2-(13)C]pyruvate kinetics in muscle. Infusion of dichloroacetate stimulates pyruvate dehydrogenase activity and shifts the kinetics toward oxidative metabolism. Bicarbonate formation from [1-(13)C]lactate increases sharply and acetyl-l-carnitine, acetoacetate and glutamate levels also rise. Such a quick mobilization of pyruvate and lactate toward oxidative metabolism supports the postulated role of lactate in the glycogen shunt and the intracellular lactate shuttle models. The study thus introduces an innovative DNP approach to measure metabolite transients, which will help delineate the cellular and physiological role of lactate and glycolytic end products.

The feasibility of assessing branched-chain amino acid metabolism in cellular models of prostate cancer with hyperpolarized [1-(13)C]-ketoisocaproate.

Recent advancements in the field of hyperpolarized (13)C magnetic resonance spectroscopy (MRS) have yielded powerful techniques capable of real-time analysis of metabolic pathways. These non-invasive methods have increasingly shown application in impacting disease diagnosis and have further been employed in mechanistic studies of disease onset and progression. Our goals were to investigate branched-chain aminotransferase (BCAT) activity in prostate cancer with a novel molecular probe, hyperpolarized [1-(13)C]-2-ketoisocaproate ([1-(13)C]-KIC), and explore the potential of branched-chain amino acid (BCAA) metabolism to serve as a biomarker. Using traditional spectrophotometric assays, BCAT enzymatic activities were determined in vitro for various sources of prostate cancer (human, transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse and human cell lines). These preliminary studies indicated that low levels of BCAT activity were present in all models of prostate cancer but enzymatic levels are altered significantly in prostate cancer relative to healthy tissue. The MR spectroscopic studies were conducted with two cellular models (PC-3 and DU-145) that exhibited levels of BCAA metabolism comparable to the human disease state. Hyperpolarized [1-(13)C]-KIC was administered to prostate cancer cell lines, and the conversion of [1-(13)C]-KIC to the metabolic product, [1-(13)C]-leucine ([1-(13)C]-Leu), could be monitored via hyperpolarized (13)C MRS.

Hyperpolarized [1,4-(13)C]-diethylsuccinate: a potential DNP substrate for in vivo metabolic imaging.

The tricarboxylic acid (TCA) cycle performs an essential role in the regulation of energy and metabolism, and deficiencies in this pathway are commonly correlated with various diseases. However, the development of non-invasive techniques for the assessment of the cycle in vivo has remained challenging. In this work, the applicability of a novel imaging agent, [1,4-(13)C]-diethylsuccinate, for hyperpolarized (13)C metabolic imaging of the TCA cycle was explored. In vivo spectroscopic studies were conducted in conjunction with in vitro analyses to determine the metabolic fate of the imaging agent. Contrary to previous reports (Zacharias NM et al. J. Am. Chem. Soc. 2012; 134: 934-943), [(13)C]-labeled diethylsuccinate was primarily metabolized to succinate-derived products not originating from TCA cycle metabolism. These results illustrate potential issues of utilizing dialkyl ester analogs of TCA cycle intermediates as molecular probes for hyperpolarized (13)C metabolic imaging.

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

PURPOSE: In contrast to [1-(13) C]pyruvate, hyperpolarized [2-(13) C]pyruvate permits the ability to follow the (13) C 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-(13) C]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. METHODS: This study introduces a sequence for three-dimensional CSI of [2-(13) C]pyruvate using spectrally selective excitation of limited frequency bands containing a subset of metabolites. Dynamic CSI data were acquired alternately from multiple frequency bands in phantoms for sequence testing and in vivo in rat heart. RESULTS: Phantom experiments verified the radiofrequency pulse design and demonstrated that the signal behavior of each group of resonances was unaffected by excitation of the other frequency bands. Dynamic three-dimensional (13) C CSI data demonstrated the sequence capability to image pyruvate, lactate, acetylcarnitine, glutamate, and acetoacetate, enabling the analysis of organ-specific spectra and metabolite time courses. CONCLUSIONS: The presented method allows CSI of widely separated resonances without chemical shift displacement artifact, acquiring multiple frequency bands alternately to obtain dynamic time-course information. This approach enables robust imaging of downstream metabolic products of acetyl-coenzyme A with hyperpolarized [2-(13) C]pyruvate.

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

Hyperpolarized (13)C MRS allows the in vivo assessment of pyruvate dehydrogenase complex (PDC) flux, which converts pyruvate to acetyl-coenzyme A (acetyl-CoA). [1-(13)C]pyruvate has been used to measure changes in cardiac PDC flux, with demonstrated increase in (13)C-bicarbonate production after dichloroacetate (DCA) administration. With [1-(13)C]pyruvate, the (13)C label is released as (13 CO2 /(13)C-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 (13)C label into the TCA (tricarboxylic acid) cycle and measure [5-(13)C]glutamate as well as study changes in [1-(13)C]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 (13)C labeling of [5-(13)C]glutamate, and a considerable increase in [1-(13)C]acetylcarnitine and [1,3-(13)C]acetoacetate peaks. Dobutamine resulted in an increased labeling of [2-(13)C]lactate, [2-(13)C]alanine and [5-(13)C]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.

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

Hyperpolarized [1-(13) C]pyruvate ([1-(13) C]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-(13) C]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-(13) C]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-(13) C]Glutamate, [1-(13) C]acetylcarnitine and [1-(13) C]citrate were detected in addition to resonances from [2-(13) C]Pyr and [2-(13) C]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-(13) C]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-(13) C]acetylcarnitine was increased in peripheral tissues. This study demonstrates, for the first time, that hyperpolarized [2-(13) C]Pyr can be used for the in vivo investigation of mitochondrial function and tricarboxylic acid cycle metabolism in brain.

Metabolic response of glioma to dichloroacetate measured in vivo by hyperpolarized (13)C 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.

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

PURPOSE: 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 (13)C 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 (13)C MR metabolic measurements in rat brain for [1-(13)C]pyruvate and 2-keto[1-(13)C]isocaproate. METHODS: Dynamic 2D and 3D spiral chemical shift imaging was used to acquire metabolic images of rat brain as well as kidney and liver following bolus injections of hyperpolarized [1-(13)C]pyruvate or 2-keto[1-(13)C]isocaproate. The impact of a "low dose" vs. a "high dose" of isoflurane on cerebral metabolite levels and apparent conversion rates was examined. RESULTS: The cerebral substrate signal levels, and hence the metabolite-to-substrate ratios and apparent conversion rates, were found to depend markedly on isoflurane dose, while signal levels of metabolic products and their ratios, e.g. bicarbonate/lactate, were largely insensitive to isoflurane levels. No obvious dependence on isoflurane was observed in kidney or liver for pyruvate. CONCLUSION: This study highlights the importance of careful attention to the effects of anesthesia on the metabolic measures for hyperpolarized (13)C metabolic imaging in brain.

In vivo measurement of aldehyde dehydrogenase-2 activity in rat liver ethanol model using dynamic MRSI of hyperpolarized [1-(13) C]pyruvate.

To date, measurements of the activity of aldehyde dehydrogenase-2 (ALDH2), a critical mitochondrial enzyme for the elimination of certain cytotoxic aldehydes in the body and a promising target for drug development, have been largely limited to in vitro methods. Recent advancements in MRS of hyperpolarized (13) C-labeled substrates have provided a method to detect and image in vivo metabolic pathways with signal-to-noise ratio gains greater than 10 000-fold over conventional MRS techniques. However aldehydes, because of their toxicity and short T1 relaxation times, are generally poor targets for such (13) C-labeled studies. In this work, we show that dynamic MRSI of hyperpolarized [1-(13) C]pyruvate and its conversion to [1-(13) C]lactate can provide an indirect in vivo measurement of ALDH2 activity via the concentration of NADH (nicotinamide adenine dinucleotide, reduced form), a co-factor common to both the reduction of pyruvate to lactate and the oxidation of acetaldehyde to acetate. Results from a rat liver ethanol model (n = 9) show that changes in (13) C-lactate labeling following the bolus injection of hyperpolarized pyruvate are highly correlated with changes in ALDH2 activity (R(2) = 0.76).

Exchange-linked dissolution agents in dissolution-DNP (13)C metabolic imaging.

PURPOSE: The use of unlabeled exchange-linked dissolution agents in hyperpolarized metabolic imaging was studied to examine pool size limits and saturation relative to the availability of NADH. METHODS: Three-dimensional dynamic metabolic images were obtained, and compared following injection of a bolus of hyperpolarized [1-(13)C]pyruvate, prepared with and without unlabeled sodium lactate in the dissolution buffer. Comparisons were made on the basis of apparent rate constants and [1-(13)C]lactate signal-to-noise ratio. Range finding data were obtained for different bolus compositions. Isotope exchange was also probed in the reverse direction, following injection of a bolus of hyperpolarized [1-(13)C]lactate, with and without unlabeled sodium pyruvate in the dissolution buffer. RESULTS: Liver, kidney, and vascular regions of interest all showed an increase in [1-(13)C]lactate signal with addition of unlabeled sodium lactate in the dissolution buffer. Injection of hyperpolarized [1-(13)C]lactate with unlabeled sodium pyruvate in the dissolution buffer, provided exchange rate constants Klp for kidney and vascular regions of interest. CONCLUSIONS: These results are consistent with a high level of (13)C-exchange, and with labeling rates that are limited by steady-state pool sizes in vivo.