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.     

Application of hyperpolarized [1-(13) C]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 © 2012 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

In vivo detection of d‐amino acid oxidase with hyperpolarized d‐[1‐13C]alanine

d ‐amino acid oxidase (DAO) is a peroxisomal enzyme that catalyzes the oxidative deamination of several neutral and basic d ‐amino acids to their corresponding α‐keto acids. In most mammalian species studied, high DAO activity is found in the kidney, liver, brain and polymorphonuclear leukocytes, and its main function is to maintain low circulating d ‐amino acid levels. DAO expression and activity have been associated with acute and chronic kidney diseases and with several pathologies related to N‐methyl‐d ‐aspartate (NMDA) receptor hypo/hyper‐function; however, its precise role is not completely understood. In the present study we show that DAO activity can be detected in vivo in the rat kidney using hyperpolarized d ‐[1‐¹³C]alanine. Following a bolus of hyperpolarized d ‐alanine, accumulation of pyruvate, lactate and bicarbonate was observed only when DAO activity was not inhibited. The measured lactate‐to‐d ‐alanine ratio was comparable to the values measured when the l ‐enantiomer was injected. Metabolites downstream of DAO were not observed when scanning the liver and brain. The conversion of hyperpolarized d ‐[1‐¹³C]alanine to lactate and pyruvate was detected in blood ex vivo, and lactate and bicarbonate were detected on scanning the blood pool in the heart in vivo; however, the bicarbonate‐to‐d ‐alanine ratio was significantly lower compared with the kidney. These results demonstrate that the specific metabolism of the two enantiomers of hyperpolarized [1‐¹³C]alanine in the kidney and in the blood can be distinguished, underscoring the potential of d ‐[1‐¹³C]alanine as a probe of d ‐amino acid metabolism.     

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.     

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.     

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.     

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.     

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.     

Detection of myocardial medium‐chain fatty acid oxidation and tricarboxylic acid cycle activity with hyperpolarized [1–13C]octanoate

Under normal conditions, the heart mainly relies on fatty acid oxidation to meet its energy needs. Changes in myocardial fuel preference are noted in the diseased and failing heart. The magnetic resonance signal enhancement provided by spin hyperpolarization allows the metabolism of substrates labeled with carbon‐13 to be followed in real time in vivo. Although the low water solubility of long‐chain fatty acids abrogates their hyperpolarization by dissolution dynamic nuclear polarization, medium‐chain fatty acids have sufficient solubility to be efficiently polarized and dissolved. In this study, we investigated the applicability of hyperpolarized [1–¹³C]octanoate to measure myocardial medium‐chain fatty acid metabolism in vivo. Scanning rats infused with a bolus of hyperpolarized [1–¹³C]octanoate, the primary metabolite observed in the heart was identified as [1–¹³C]acetylcarnitine. Additionally, [5‐¹³C]glutamate and [5‐¹³C]citrate could be respectively resolved in seven and five of 31 experiments, demonstrating the incorporation of oxidation products of octanoate into the tricarboxylic acid cycle. A variable drop in blood pressure was observed immediately following the bolus injection, and this drop correlated with a decrease in normalized acetylcarnitine signal (acetylcarnitine/octanoate). Increasing the delay before infusion moderated the decrease in blood pressure, which was attributed to the presence of residual gas bubbles in the octanoate solution. No significant difference in normalized acetylcarnitine signal was apparent between fed and 12‐hour fasted rats. Compared with a solution in buffer, the longitudinal relaxation of [1–¹³C]octanoate was accelerated ~3‐fold in blood and by the addition of serum albumin. These results demonstrate the potential of hyperpolarized [1–¹³C]octanoate to probe myocardial medium‐chain fatty acid metabolism as well as some of the limitations that may accompany its use.     

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.     

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.     

Metabolism of [U‐13C]glucose in human brain tumors in vivo

Glioblastomas and brain metastases demonstrate avid uptake of 2‐[¹⁸F]fluoro‐2‐deoxyglucose by positron emission tomography and display perturbations of intracellular metabolite pools by ¹H MRS. These observations suggest that metabolic reprogramming contributes to brain tumor growth in vivo. The Warburg effect, excess metabolism of glucose to lactate in the presence of oxygen, is a hallmark of cancer cells in culture. 2‐[¹⁸F]Fluoro‐2‐deoxyglucose‐positive tumors are assumed to metabolize glucose in a similar manner, with high rates of lactate formation relative to mitochondrial glucose oxidation, but few studies have specifically examined the metabolic fates of glucose in vivo. In particular, the capacity of human brain cancers to oxidize glucose in the tricarboxylic acid cycle is unknown. Here, we studied the metabolism of human brain tumors in situ. [U‐¹³ C]Glucose (uniformly labeled glucose, i.e. d ‐glucose labeled with ¹³ C in all six carbons) was infused during surgical resection, and tumor samples were subsequently subjected to ¹³C NMR spectroscopy. The analysis of tumor metabolites revealed lactate production, as expected. We also determined that pyruvate dehydrogenase, turnover of the tricarboxylic acid cycle, anaplerosis and de novo glutamine and glycine synthesis contributed significantly to the ultimate disposition of glucose carbon. Surprisingly, less than 50% of the acetyl‐coenzyme A pool was derived from blood‐borne glucose, suggesting that additional substrates contribute to tumor bioenergetics. This study illustrates a convenient approach that capitalizes on the high information content of ¹³C NMR spectroscopy and enables the analysis of intermediary metabolism in diverse cancers growing in their native microenvironment. Copyright © 2012 John Wiley & Sons, Ltd.     

Cancer metabolism in a snapshot: MRS(I)

The contribution of MRS(I) to the in vivo evaluation of cancer‐metabolism‐derived metrics, mostly since 2016, is reviewed here. Increased carbon consumption by tumour cells, which are highly glycolytic, is now being sampled by ¹³C magnetic resonance spectroscopic imaging (MRSI) following the injection of hyperpolarized [1‐¹³C] pyruvate (Pyr). Hot‐spots of, mostly, increased lactate dehydrogenase activity or flow between Pyr and lactate (Lac) have been seen with cancer progression in prostate (preclinical and in humans), brain and pancreas (both preclinical) tumours. Therapy response is usually signalled by decreased Lac/Pyr ¹³C‐labelled ratio with respect to untreated or non‐responding tumour. For therapeutic agents inducing tumour hypoxia, the ¹³C‐labelled Lac/bicarbonate ratio may be a better metric than the Lac/Pyr ratio. ³¹P MRSI may sample intracellular pH changes from brain tumours (acidification upon antiangiogenic treatment, basification at fast proliferation and relapse). The steady state tumour metabolome pattern is still in use for cancer evaluation. Metrics used for this range from quantification of single oncometabolites (such as 2‐hydroxyglutarate in mutant IDH1 glial brain tumours) to selected metabolite ratios (such as total choline to N‐acetylaspartate (plain ratio or CNI index)) or the whole ¹H MRSI(I) pattern through pattern recognition analysis. These approaches have been applied to address different questions such as tumour subtype definition, following/predicting the response to therapy or defining better resection or radiosurgery limits.     

Effects of fasting on serial measurements of hyperpolarized [1‐13C]pyruvate metabolism in tumors

Imaging of the metabolism of hyperpolarized [1‐¹³C]pyruvate has shown considerable promise in preclinical studies in oncology, particularly for the assessment of early treatment response. The repeatability of measurements of ¹³C label exchange between pyruvate and lactate was determined in a murine lymphoma model in fasted and non‐fasted animals. The fasted state showed lower intra‐individual variability, although the [1‐¹³C]lactate/[1‐¹³C]pyruvate signal ratio was significantly greater in fasted than in non‐fasted mice, which may be explained by the higher tumor lactate concentrations in fasted animals. These results indicate that the fasted state may be preferable for the measurement of ¹³C label exchange between pyruvate and lactate, as it reduces the variability and therefore should make it easier to detect the effects of therapy. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.     

Hyperpolarized ketone body metabolism in the rat heart

The aim of this work was to investigate the use of ¹³C‐labelled acetoacetate and β‐hydroxybutyrate as novel hyperpolarized substrates in the study of cardiac metabolism. [1‐¹³C]Acetoacetate was synthesized by catalysed hydrolysis, and both it and [1‐¹³C]β‐hydroxybutyrate were hyperpolarized by dissolution dynamic nuclear polarization (DNP). Their metabolism was studied in isolated, perfused rat hearts. Hyperpolarized [1‐¹³C]acetoacetate metabolism was also studied in the in vivo rat heart in the fed and fasted states. Hyperpolarization of [1‐¹³C]acetoacetate and [1‐¹³C]β‐hydroxybutyrate provided liquid state polarizations of 8 ± 2% and 3 ± 1%, respectively. The hyperpolarized T₁ values for the two substrates were 28 ± 3 s (acetoacetate) and 20 ± 1 s (β‐hydroxybutyrate). Multiple downstream metabolites were observed within the perfused heart, including acetylcarnitine, citrate and glutamate. In the in vivo heart, an increase in acetylcarnitine production from acetoacetate was observed in the fed state, as well as a potential reduction in glutamate. In this work, methods for the generation of hyperpolarized [1‐¹³C]acetoacetate and [1‐¹³C]β‐hydroxybutyrate were investigated, and their metabolism was assessed in both isolated, perfused rat hearts and in the in vivo rat heart. These preliminary investigations show that DNP can be used as an effective in vivo probe of ketone body metabolism in the heart.     

In vivo detection of intermediate metabolic products of [1-(13) C]ethanol in the brain using (13) C MRS

In this study, in vivo ¹³C MRS was used to investigate the labeling of brain metabolites after intravenous administration of [1‐¹³C]ethanol. After [1‐¹³C]ethanol had been administered systemically to rats, ¹³C labels were detected in glutamate, glutamine and aspartate in the carboxylic and amide carbon spectral region. ¹³C‐labeled bicarbonate HCO (161.0 ppm) was also detected. Saturating acetaldehyde C1 at 207.0 ppm was found to have no effect on the ethanol C1 (57.7 ppm) signal intensity after extensive signal averaging, providing direct in vivo evidence that direct metabolism of alcohol by brain tissue is minimal. To compare the labeling of brain metabolites by ethanol with labeling by glucose, in vivo time course data were acquired during intravenous co‐infusion of [1‐¹³C]ethanol and [¹³C₆]‐D ‐glucose. In contrast with labeling by [¹³C₆]‐D ‐glucose, which produced doublets of carboxylic/amide carbons with a J coupling constant of 51 Hz, the simultaneously detected glutamate and glutamine singlets were labeled by [1‐¹³C]ethanol. As ¹³C labels originating from ethanol enter the brain after being converted into [1‐¹³C]acetate in the liver, and the direct metabolism of ethanol by brain tissue is negligible, it is suggested that orally or intragastrically administered ¹³C‐labeled ethanol may be used to study brain metabolism and glutamatergic neurotransmission in investigations involving alcohol administration. In vivo ¹³C MRS of rat brain following intragastric administration of ¹³C‐labeled ethanol is demonstrated. Published in 2011 by John Wiley & Sons, Ltd.     

Probing carbohydrate metabolism using hyperpolarized 13C‐labeled molecules

Glycolysis is a fundamental metabolic process in all organisms. Anomalies in glucose metabolism are linked to various pathological conditions. In particular, elevated aerobic glycolysis is a characteristic feature of rapidly growing cells. Glycolysis and the closely related pentose phosphate pathway can be monitored in real time by hyperpolarized ¹³C‐labeled metabolic substrates such as ¹³C‐enriched, deuterated D‐glucose derivatives, [2‐¹³C]‐D‐fructose, [2‐¹³C] dihydroxyacetone, [1‐¹³C]‐D‐glycerate, [1‐¹³C]‐D‐glucono‐δ‐lactone and [1‐¹³C] pyruvate in healthy and diseased tissues. Elevated glycolysis in tumors (the Warburg effect) was also successfully imaged using hyperpolarized [U‐¹³C₆, U‐²H₇]‐D‐glucose, while the size of the preexisting lactate pool can be measured by ¹³C MRS and/or MRI with hyperpolarized [1‐¹³C]pyruvate. This review summarizes the application of various hyperpolarized ¹³C‐labeled metabolites to the real‐time monitoring of glycolysis and related metabolic processes in normal and diseased tissues.