HDAC inhibition in glioblastoma monitored by hyperpolarized 13C MRSI

Vorinostat is a histone deacetylase (HDAC) inhibitor that inhibits cell proliferation and induces apoptosis in solid tumors, and is in clinical trials for the treatment of glioblastoma (GBM). The goal of this study was to assess whether hyperpolarized ¹³C MRS and magnetic resonance spectroscopic imaging (MRSI) can detect HDAC inhibition in GBM models. First, we confirmed HDAC inhibition in U87 GBM cells and evaluated real‐time dynamic metabolic changes using a bioreactor system with live vorinostat‐treated or control cells. We found a significant 40% decrease in the ¹³C MRS‐detectable ratio of hyperpolarized [1‐¹³C]lactate to hyperpolarized [1‐¹³C]pyruvate, [1‐¹³C]Lac/Pyr, and a 37% decrease in the pseudo‐rate constant, k_PL, for hyperpolarized [1‐¹³C]lactate production, in vorinostat‐treated cells compared with controls. To understand the underlying mechanism for this finding, we assessed the expression and activity of lactate dehydrogenase (LDH) (which catalyzes the pyruvate to lactate conversion), its associated cofactor nicotinamide adenine dinucleotide, the expression of monocarboxylate transporters (MCTs) MCT1 and MCT4 (which shuttle pyruvate and lactate in and out of the cell) and intracellular lactate levels. We found that the most likely explanation for our finding that hyperpolarized lactate is reduced in treated cells is a 30% reduction in intracellular lactate levels that occurs as a result of increased expression of both MCT1 and MCT4 in vorinostat‐treated cells. In vivo ¹³C MRSI studies of orthotopic tumors in mice also showed a significant 52% decrease in hyperpolarized [1‐¹³C]Lac/Pyr when comparing vorinostat‐treated U87 GBM tumors with controls, and, as in the cell studies, this metabolic finding was associated with increased MCT1 and MCT4 expression in HDAC‐inhibited tumors. Thus, the ¹³C MRSI‐detectable decrease in hyperpolarized [1‐¹³C]lactate production could serve as a biomarker of response to HDAC inhibitors.     

Robust hyperpolarized 13C metabolic imaging with selective non‐excitation of pyruvate (SNEP)

In vivo metabolic imaging using hyperpolarized [1‐¹³C]pyruvate provides localized biochemical information and is particularly useful in detecting early disease changes, as well as monitoring disease progression and treatment response. However, a major limitation of hyperpolarized magnetization is its unrecoverable decay, due not only to T₁ relaxation but also to radio‐frequency (RF) excitation. RF excitation schemes used in metabolic imaging must therefore be able to utilize available hyperpolarized magnetization efficiently and robustly for the optimal detection of substrate and metabolite activities. In this work, a novel RF excitation scheme called selective non‐excitation of pyruvate (SNEP) is presented. This excitation scheme involves the use of a spectral selective RF pulse to specifically exclude the excitation of [1‐¹³C]pyruvate, while uniformly exciting the key metabolites of interest (namely [1‐¹³C]lactate and [1‐¹³C]alanine) and [1‐¹³C]pyruvate‐hydrate. By eliminating the loss of hyperpolarized [1‐¹³C]pyruvate magnetization due to RF excitation, the signal from downstream metabolite pools is increased together with enhanced dynamic range. Simulation results, together with phantom measurements and in vivo experiments, demonstrated the improvement in signal‐to‐noise ratio (SNR) and the extension of the lifetime of the [1‐¹³C]lactate and [1‐¹³C]alanine pools when compared with conventional non‐spectral selective (NS) excitation. SNEP has also been shown to perform comparably well with multi‐band (MB) excitation, yet SNEP possesses distinct advantages, including ease of implementation, less stringent demands on gradient performance, increased robustness to frequency drifts and B₀ inhomogeneity as well as easier quantification involving the use of [1‐¹³C]pyruvate‐hydrate as a proxy for the actual [1‐¹³C] pyruvate signal. SNEP is therefore a promising alternative for robust hyperpolarized [1‐¹³C]pyruvate metabolic imaging with high fidelity. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis of 13C and 14C labeling in pyruvate and lactate in tumor and blood of lymphoma‐bearing mice injected with 13C‐ and 14C‐labeled pyruvate

Measurements of hyperpolarized ¹³C label exchange between injected [1‐¹³C]pyruvate and the endogenous tumor lactate pool can give an apparent first‐order rate constant for the exchange. The determination of the isotope flux, however, requires an estimate of the labeled pyruvate concentration in the tumor. This was achieved here by measurement of the tumor uptake of [1‐¹⁴C]pyruvate, which showed that <2% of the injected pyruvate reached the tumor site. Multiplication of this estimated labeled pyruvate concentration in the tumor with the apparent first‐order rate constant for hyperpolarized ¹³C label exchange gave an isotope flux that showed good agreement with a flux determined directly by the injection of non‐polarized [3‐¹³C]pyruvate, rapid excision of the tumor after 30 s and measurement of ¹³C‐labeled lactate concentrations in tumor extracts. The distribution of labeled lactate between intra‐ and extracellular compartments and the blood pool was investigated by imaging, by measurement of the labeled lactate concentration in blood and tumor, and by examination of the effects of a gadolinium contrast agent and a lactate transport inhibitor on the intensity of the hyperpolarized [1‐¹³C]lactate signal. These measurements showed that there was significant export of labeled lactate from the tumor, but that labeled lactate in the blood pool produced by the injection of hyperpolarized [1‐¹³C]pyruvate showed only relatively low levels of polarization. This study shows that measurements of hyperpolarized ¹³C label exchange between pyruvate and lactate in a murine tumor model can provide an estimate of the true isotope flux if the concentration of labeled pyruvate that reaches the tumor can be determined.     

Dynamic 2D and 3D mapping of hyperpolarized pyruvate to lactate conversion in vivo with efficient multi‐echo balanced steady‐state free precession at 3 T

The aim of this study was to acquire the transient MRI signal of hyperpolarized tracers and their metabolites efficiently, for which specialized imaging sequences are required. In this work, a multi‐echo balanced steady‐state free precession (me‐bSSFP) sequence with Iterative Decomposition with Echo Asymmetry and Least squares estimation (IDEAL) reconstruction was implemented on a clinical 3 T positron‐emission tomography/MRI system for fast 2D and 3D metabolic imaging. Simulations were conducted to obtain signal‐efficient sequence protocols for the metabolic imaging of hyperpolarized biomolecules. The sequence was applied in vitro and in vivo for probing the enzymatic exchange of hyperpolarized [1–¹³C]pyruvate and [1–¹³C]lactate. Chemical shift resolution was achieved using a least‐square, iterative chemical species separation algorithm in the reconstruction. In vitro, metabolic conversion rate measurements from me‐bSSFP were compared with NMR spectroscopy and free induction decay‐chemical shift imaging (FID‐CSI). In vivo, a rat MAT‐B‐III tumor model was imaged with me‐bSSFP and FID‐CSI. 2D metabolite maps of [1–¹³C]pyruvate and [1–¹³C]lactate acquired with me‐bSSFP showed the same spatial distributions as FID‐CSI. The pyruvate‐lactate conversion kinetics measured with me‐bSSFP and NMR corresponded well. Dynamic 2D metabolite mapping with me‐bSSFP enabled the acquisition of up to 420 time frames (scan time: 180‐350 ms/frame) before the hyperpolarized [1–¹³C]pyruvate was relaxed below noise level. 3D metabolite mapping with a large field of view (180 × 180 × 48 mm³) and high spatial resolution (5.6 × 5.6 × 2 mm³) was conducted with me‐bSSFP in a scan time of 8.2 seconds. It was concluded that Me‐bSSFP improves the spatial and temporal resolution for metabolic imaging of hyperpolarized [1–¹³C]pyruvate and [1–¹³C]lactate compared with either of the FID‐CSI or EPSI methods reported at 3 T, providing new possibilities for clinical and preclinical applications.     

Imaging porcine cardiac substrate selection modulations by glucose,insulin and potassium intervention: A hyperpolarized [1‐13C]pyruvate study

Cardiac metabolism has received considerable attention in terms of both diagnostics and prognostics, as well as a novel target for treatment. As human trials involving hyperpolarized magnetic resonance in the heart are imminent, we sought to evaluate the general feasibility of detection of an imposed shift in metabolic substrate utilization during metabolic modulation with glucose–insulin–potassium (GIK) infusion, and thus the limitations associated with this strategy, in a large animal model resembling human physiology. Four [1‐¹³C]pyruvate injections did not alter the blood pressure or ejection fraction over 180 min. Hyperpolarized [1‐¹³C]pyruvate conversion showed a generally high reproducibility, with intraclass correlation coefficients between the baseline measurements at 0 and 30 min as follows: lactate to pyruvate, 0.85; alanine to pyruvate, 1.00; bicarbonate to pyruvate, 0.83. This study demonstrates that hyperpolarized [1‐¹³C]pyruvate imaging is a feasible technique for cardiac studies and shows a generally high reproducibility in fasted large animals. GIK infusion increases the metabolic conversion of pyruvate to its metabolic derivatives lactate, alanine and bicarbonate, but with increased variability.     

Simultaneous imaging of hyperpolarized [1,4‐13C2]fumarate, [1‐13C]pyruvate and 18F–FDG in a rat model of necrosis in a clinical PET/MR scanner

A co‐polarization scheme for [1,4‐¹³C₂]fumarate and [1‐¹³C]pyruvate is presented to simultaneously assess necrosis and metabolism in rats with hyperpolarized ¹³C magnetic resonance (MR). The co‐polarization was performed in a SPINlab polarizer. In addition, the feasibility of simultaneous positron emission tomography (PET) and MR of small animals with a clinical PET/MR scanner is demonstrated. The hyperpolarized metabolic MR and PET was demonstrated in a rat model of necrosis. The polarization and T₁ of the co‐polarized [1,4‐¹³C₂]fumarate and [1‐¹³C]pyruvate substrates were measured in vitro and compared with those obtained when the substrates were polarized individually. A polarization of 36 ± 4% for fumarate and 37 ± 6% for pyruvate was obtained. We found no significant difference in the polarization and T₁ values between the dual and single substrate polarization. Rats weighing about 400 g were injected intramuscularly in one of the hind legs with 200 μL of turpentine to induce necrosis. Two hours later, ¹³C metabolic maps were obtained with a chemical shift imaging sequence (16 × 16) with a resolution of 3.1 × 5.0 × 25.0 mm³. The ¹³C spectroscopic images were acquired in 12 s, followed by an 8‐min ¹⁸F‐2‐fluoro‐2‐deoxy‐d ‐glucose (¹⁸F–FDG) PET acquisition with a resolution of 3.5 mm. [1,4‐¹³C₂]Malate was observed from the tissue injected with turpentine indicating necrosis. Normal [1‐¹³C]pyruvate metabolism and ¹⁸F–FDG uptake were observed from the same tissue. The proposed co‐polarization scheme provides a means to utilize multiple imaging agents simultaneously, and thus to probe various metabolic pathways in a single examination. Moreover, it demonstrates the feasibility of small animal research on a clinical PET/MR scanner for combined PET and hyperpolarized metabolic MR.     

In vivo measurement of aldehyde dehydrogenase‐2 activity in rat liver ethanol model using dynamic MRSI of hyperpolarized [1‐13C]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 ¹³C‐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 ¹³C‐labeled studies. In this work, we show that dynamic MRSI of hyperpolarized [1‐¹³C]pyruvate and its conversion to [1‐¹³C]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 ¹³C‐lactate labeling following the bolus injection of hyperpolarized pyruvate are highly correlated with changes in ALDH2 activity (R² = 0.76). Copyright © 2012 John Wiley & Sons, Ltd.     

Assessing the effect of hypoxia on cardiac metabolism using hyperpolarized 13C magnetic resonance spectroscopy

Hypoxia plays a role in many diseases and can have a wide range of effects on cardiac metabolism depending on the extent of the hypoxic insult. Noninvasive imaging methods could shed valuable light on the metabolic effects of hypoxia on the heart in vivo. Hyperpolarized carbon‐13 magnetic resonance spectroscopy (HP ¹³C MRS) in particular is an exciting technique for imaging metabolism that could provide such information. The aim of our work was, therefore, to establish whether hyperpolarized ¹³C MRS can be used to assess the in vivo heart's metabolism of pyruvate in response to systemic acute and chronic hypoxic exposure. Groups of healthy male Wistar rats were exposed to either acute (30 minutes), 1 week or 3 weeks of hypoxia. In vivo MRS of hyperpolarized [1‐¹³C] pyruvate was carried out along with assessments of physiological parameters and ejection fraction. Hematocrit was elevated after 1 week and 3 weeks of hypoxia. 30 minutes of hypoxia resulted in a significant reduction in pyruvate dehydrogenase (PDH) flux, whereas 1 or 3 weeks of hypoxia resulted in a PDH flux that was not different to normoxic animals. Conversion of hyperpolarized [1‐¹³C] pyruvate into [1‐¹³C] lactate was elevated following acute hypoxia, suggestive of enhanced anaerobic glycolysis. Elevated HP pyruvate to lactate conversion was also seen at the one week timepoint, in concert with an increase in lactate dehydrogenase (LDH) expression. Following three weeks of hypoxic exposure, cardiac metabolism of pyruvate was comparable with that observed in normoxia. We have successfully visualized the effects of systemic hypoxia on cardiac metabolism of pyruvate using hyperpolarized ¹³C MRS, with differences observed following 30 minutes and 1 week of hypoxia. This demonstrates the potential of in vivo hyperpolarized ¹³C MRS data for assessing the cardiometabolic effects of hypoxia in disease.     

Ketone bodies effectively compete with glucose for neuronal acetyl‐CoA generation in rat hippocampal slices

Ketone bodies can be used for cerebral energy generation in situ, when their availability is increased as during fasting or ingestion of a ketogenic diet. However, it is not known how effectively ketone bodies compete with glucose, lactate, and pyruvate for energy generation in the brain parenchyma. Hence, the contributions of exogenous 5.0 mM [1‐¹³C]glucose and 1.0 mM [2‐¹³C]lactate + 0.1 mM pyruvate (combined [2‐¹³C]lactate + [2‐¹³C]pyruvate) to acetyl‐CoA production were measured both without and with 5.0 mM [U‐¹³C]3‐hydroxybutyrate in superfused rat hippocampal slices by ¹³C NMR non‐steady‐state isotopomer analysis of tissue glutamate and GABA. Without [U‐¹³C]3‐hydroxybutyrate, glucose, combined lactate + pyruvate, and unlabeled endogenous sources contributed (mean ± SEM) 70 ± 7%, 10 ± 2%, and 20 ± 8% of acetyl‐CoA, respectively. With [U‐¹³C]3‐hydroxybutyrate, glucose contributions significantly fell from 70 ± 7% to 21 ± 3% (p < 0.0001), combined lactate + pyruvate and endogenous contributions were unchanged, and [U‐¹³C]3‐hydroxybutyrate became the major acetyl‐CoA contributor (68 ± 3%) – about three‐times higher than glucose. A direct analysis of the GABA carbon 2 multiplet revealed that [U‐¹³C]3‐hydroxybutyrate contributed approximately the same acetyl‐CoA fraction as glucose, indicating that it was less avidly oxidized by GABAergic than glutamatergic neurons. The appearance of superfusate lactate derived from glycolysis of [1‐¹³C]glucose did not decrease significantly in the presence of 3‐hydroxybutyrate, hence total glycolytic flux (Krebs cycle inflow + exogenous lactate formation) was attenuated by 3‐hydroxybutyrate. This indicates that, under these conditions, 3‐hydroxybutyrate inhibited glycolytic flux upstream of pyruvate kinase. Copyright © 2015 John Wiley & Sons, Ltd.     

Metabolism of hyperpolarized [1‐13C]pyruvate through alternate pathways in rat liver

The source of hyperpolarized (HP) [¹³C]bicarbonate in the liver during metabolism of HP [1‐¹³C]pyruvate is uncertain and likely changes with physiology. Multiple processes including decarboxylation through pyruvate dehydrogenase or pyruvate carboxylase followed by subsequent decarboxylation via phosphoenolpyruvate carboxykinase (gluconeogenesis) could play a role. Here we tested which metabolic fate of pyruvate contributed to the appearance of HP [¹³C]bicarbonate during metabolism of HP [1‐¹³C]pyruvate by the liver in rats after 21 h of fasting compared to rats with free access to food. The ¹³C NMR of HP [¹³C]bicarbonate was observed in the liver of fed rats, but not in fasted rats where pyruvate carboxylation and gluconeogenesis was active. To further explore the relative fluxes through pyruvate carboxylase versus pyruvate dehydrogenase in the liver under typical conditions of hyperpolarization studies, separate parallel experiments were performed with rats given non‐hyperpolarized [2,3‐¹³C]pyruvate. ¹³C NMR analysis of glutamate isolated from the liver of rats revealed that flux from injected pyruvate through pyruvate dehydrogenase was dominant under fed conditions whereas flux through pyruvate carboxylase dominated under fasted conditions. The NMR signal of HP [¹³C]bicarbonate does not parallel pyruvate carboxylase activity followed by subsequent decarboxylation reaction leading to glucose production. In the liver of healthy well‐fed rats, the appearance of HP [¹³C]bicarbonate exclusively reflects decarboxylation of HP [1‐¹³C]pyruvate via pyruvate dehydrogenase. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.     

Enhancing the [13C]bicarbonate signal in cardiac hyperpolarized [1‐13C]pyruvate MRS studies by infusion of glucose,insulin and potassium

A change in myocardial metabolism is a known effect of several diseases. MRS with hyperpolarized ¹³C‐labelled pyruvate is a technique capable of detecting changes in myocardial pyruvate metabolism, and has proven to be useful for the evaluation of myocardial ischaemia in vivo. However, during fasting, the myocardial glucose oxidation is low and the fatty acid oxidation (β‐oxidation) is high, which complicates the interpretation of pyruvate metabolism with the technique. The aim of this study was to investigate whether the infusion of glucose, insulin and potassium (GIK) could increase the myocardial glucose oxidation in the citric acid cycle, reflected as an increase in the [¹³C]bicarbonate signal in cardiac hyperpolarized [1‐¹³C]pyruvate MRS measurements in fasted rats. Two groups of rats were infused with two different doses of GIK and investigated by MRS after injection of hyperpolarized [1‐¹³C]pyruvate. No [¹³C]bicarbonate signal could be detected in the fasted state. However, a significant increase in the [¹³C]bicarbonate signal was observed by the infusion of a high dose of GIK. This study demonstrates that a high [¹³C]bicarbonate signal can be achieved by GIK infusion in fasted rats. The increased [¹³C]bicarbonate signal indicates an increased flux of pyruvate through the pyruvate dehydrogenase enzyme complex and an increase in myocardial glucose oxidation through the citric acid cycle. Copyright © 2013 John Wiley & Sons, Ltd.     

Pyruvate‐lactate exchange and glucose uptake in human prostate cancer cell models. A study in xenografts and suspensions by hyperpolarized [1‐13C]pyruvate MRS and [18F]FDG‐PET

Reprogramming of energy metabolism in the development of prostate cancer can be exploited for a better diagnosis and treatment of the disease. The goal of this study was to determine whether differences in glucose and pyruvate metabolism of human prostate cancer cells with dissimilar aggressivenesses can be detected using hyperpolarized [1‐¹³C]pyruvate MRS and [¹⁸F]FDG‐PET imaging, and to evaluate whether these measures correlate. For this purpose, we compared murine xenografts of human prostate cancer LNCaP cells with those of more aggressive PC3 cells. [1‐¹³C]pyruvate was hyperpolarized by dissolution dynamic nuclear polarization (dDNP) and [1‐¹³C]pyruvate to lactate conversion was followed by ¹³C MRS. Subsequently [¹⁸F]FDG uptake was investigated by static and dynamic PET measurements. Standard uptake values (SUVs) for [¹⁸F]FDG were significantly higher for xenografts of PC3 compared with those of LNCaP. However, we did not observe a difference in the average apparent rate constant k_pl of ¹³C label exchange from pyruvate to lactate between the tumor variants. A significant negative correlation was found between SUVs from [¹⁸F]FDG PET measurements and k_pl values for the xenografts of both tumor types. The k_pl rate constant may be influenced by various factors, and studies with a range of prostate cancer cells in suspension suggest that LDH inhibition by pyruvate may be one of these. Our results indicate that glucose and pyruvate metabolism in the prostate cancer cell models differs from that in other tumor models and that [¹⁸F]FDG‐PET can serve as a valuable complementary tool in dDNP studies of aggressive prostate cancer with [1‐¹³C]pyruvate.     

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 © 2013 John Wiley & Sons, Ltd.     

Probing the cardiac malate–aspartate shuttle non‐invasively using hyperpolarized [1,2‐13C2]pyruvate

Previous studies have demonstrated that using hyperpolarized [2‐¹³C]pyruvate as a contrast agent can reveal ¹³C signals from metabolites associated with the tricarboxylic acid (TCA) cycle. However, the metabolites detectable from TCA cycle‐mediated oxidation of [2‐¹³C]pyruvate are the result of several metabolic steps. In the instance of the [5‐¹³C]glutamate signal, the amplitude can be modulated by changes to the rates of pyruvate dehydrogenase (PDH) flux, TCA cycle flux and metabolite pool size. Also key is the malate–aspartate shuttle, which facilitates the transport of cytosolic reducing equivalents into the mitochondria for oxidation via the malate–α‐ketoglutarate transporter, a process coupled to the exchange of cytosolic malate for mitochondrial α‐ketoglutarate. In this study, we investigated the mechanism driving the observed changes to hyperpolarized [2‐¹³C]pyruvate metabolism. Using hyperpolarized [1,2‐¹³C]pyruvate with magnetic resonance spectroscopy (MRS) in the porcine heart with different workloads, it was possible to probe ¹³C–glutamate labeling relative to rates of cytosolic metabolism, PDH flux and TCA cycle turnover in a single experiment non‐invasively. Via the [1‐¹³C]pyruvate label, we observed more than a five‐fold increase in the cytosolic conversion of pyruvate to [1‐¹³C]lactate and [1‐¹³C]alanine with higher workload. ¹³C–Bicarbonate production by PDH was increased by a factor of 2.2. Cardiac cine imaging measured a two‐fold increase in cardiac output, which is known to couple to TCA cycle turnover. Via the [2‐¹³C]pyruvate label, we observed that ¹³C–acetylcarnitine production increased 2.5‐fold in proportion to the ¹³C–bicarbonate signal, whereas the ¹³C–glutamate metabolic flux remained constant on adrenergic activation. Thus, the ¹³C–glutamate signal relative to the amount of ¹³C–labeled acetyl‐coenzyme A (acetyl‐CoA) entering the TCA cycle was decreased by 40%. The data strongly suggest that NADH (reduced form of nicotinamide adenine dinucleotide) shuttling from the cytosol to the mitochondria via the malate–aspartate shuttle is limited on adrenergic activation. Changes in [5‐¹³C]glutamate production from [2‐¹³C]pyruvate may play an important future role in non‐invasive myocardial assessment in patients with cardiovascular diseases, but careful interpretation of the results is required.     

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 © 2013 John Wiley & Sons, Ltd.     

In vivo MRSI of hyperpolarized [1-(13)C]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 © 2010 John Wiley & Sons, Ltd.     

Hyperpolarized 13C‐lactate to 13C‐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 © 2016 John Wiley & Sons, Ltd.     

Real‐time ALT and LDH activities determined in viable precision‐cut mouse liver slices using hyperpolarized [1‐13C]pyruvate—Implications for studies on biopsied liver tissues

Precision‐cut liver slices (PCLS) are widely used in liver research as they provide a liver model with all liver cell types in their natural architecture. The purpose of this study was to demonstrate the use of PCLS for hyperpolarized metabolic investigation in a mouse model, for potential future application in liver biopsy cores. Fresh normal liver was harvested from six mice. 500 μm PCLS were prepared and placed in a 10 mm NMR tube in an NMR spectrometer and perfused continuously. ³¹P spectra were acquired to evaluate the presence of adenosine triphosphate (ATP) and validate viability in all samples. Hyperpolarized [1‐¹³C]pyruvate was flushed into the NMR tube in the spectrometer. Consecutive ¹³C NMR spectra were acquired immediately after the injection using both non‐selective (five injections, two livers) and selective RF excitation (six injections, three livers). The ³¹P spectra showed the characteristic signals of ATP, confirming the viability of the PCLS for more than 2.5 h in the spectrometer. After each of the [1‐¹³C]pyruvate injections, both [1‐¹³C]lactate and [1‐¹³C]alanine signals were detected. Selective RF excitation aimed at both [1‐¹³C]lactate and [1‐¹³C]alanine enabled better visualization and quantification of the metabolic activity. Using this acquisition approach only the newly formed metabolites are observed upon excitation, and their intensities relative to those of hyperpolarized pyruvate enable quantification of metabolite production rates. This rate of lactate and alanine production appeared to be constant throughout the measurement time, with alanine production about 2.3 times higher than lactate. In summary, the viability of PCLS in an NMR spectrometer was demonstrated and hyperpolarized [1‐¹³C]pyruvate metabolism was recorded. This study opens up the possibility of evaluating alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities in human liver biopsies, while preserving the tissue architecture and viability. In healthy, well‐perfused liver slices the ratio of ALT to LDH activity is about 2.3.     

Hyperpolarized δ‐[1‐13C]gluconolactone as a probe of the pentose phosphate pathway

The pentose phosphate pathway (PPP) is thought to be upregulated in trauma (to produce excess NADPH) and in cancer (to provide ribose for nucleotide biosynthesis), but simple methods for detecting changes in flux through this pathway are not available. MRI of hyperpolarized ¹³C–enriched metabolites offers considerable potential as a rapid, non‐invasive tool for detecting changes in metabolic fluxes. In this study, hyperpolarized δ‐[1‐¹³C]gluconolactone was used as a probe to detect flux through the oxidative portion of the pentose phosphate pathway (PPP_ox) in isolated perfused mouse livers. The appearance of hyperpolarized (HP) H¹³CO₃^? within seconds after exposure of livers to HP‐δ‐[1‐¹³C]gluconolactone demonstrates that this probe rapidly enters hepatocytes, becomes phosphorylated, and enters the PPP_ox pathway to produce HP‐H¹³CO₃^? after three enzyme catalyzed steps (6P–gluconolactonase, 6‐phosphogluconate dehydrogenase, and carbonic anhydrase). Livers perfused with octanoate as their sole energy source show no change in production of H¹³CO₃^? after exposure to low levels of H₂O₂, while livers perfused with glucose and insulin showed a twofold increase in H¹³CO₃^? after exposure to peroxide. This indicates that flux through the PPP_ox is stimulated by H₂O₂ in glucose perfused livers but not in livers perfused with octanoate alone. Subsequent perfusion of livers with non‐polarized [1,2‐¹³C]glucose followed by ¹H NMR analysis of lactate in the perfusate verified that flux through the PPP_ox is indeed low in healthy livers and modestly higher in peroxide damaged livers. We conclude that hyperpolarized δ‐[1‐¹³C]gluconolactone has the potential to serve as a metabolic imaging probe of this important biological pathway.     

Detection of lung transplant rejection in a rat model using hyperpolarized [1‐13C] pyruvate‐based metabolic imaging

The current standard for noninvasive imaging of acute rejection consists of X‐ray/CT, which derive their contrast from changes in ventilation, inflammation and edema, as well as remodeling during rejection. We propose the use of hyperpolarized [1‐¹³C] pyruvate MRI—which provides real‐time metabolic assessment of tissue—as an early biomarker for tissue rejection. In this preliminary study, we used μCT‐derived parameters and HP ¹³C MR‐derived biomarkers to predict rejection in an orthotopic left lung transplant model in both allogeneic and syngeneic rats. On day 3, the normalized lung density—a parameter that accounts for both lung volume (mL) and density (HU)—was ?0.335 (CI: ‐0.598, ?0.073) and ? 0.473 (CI: ‐0.726, ?0.220) for the allograft and isograft, respectively (not significant, 0.40). The lactate‐to‐pyruvate ratios—derived from the HP ¹³C MRI—for the allograft and isograft were 0.200 (CI: 0.161, 0.240) and 0.114 (CI: 0.074, 0.153), respectively (significant, 0.020). Both techniques showed tissue rejection on day 7. A separate sub‐study revealed CD8+ cells as the primary source of the lactate‐to‐pyruvate signal. Our study suggests that hyperpolarized (HP) [1‐¹³C] pyruvate MRI is a promising early biomarker for tissue rejection that provides metabolic assessment in real time based on changes in cellularity and metabolism of lung tissue and the infiltrating inflammatory cells, and may be able to predict tissue rejection earlier than X‐ray/CT.