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.     

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.     

A referenceless Nyquist ghost correction workflow for echo planar imaging of hyperpolarized [1‐13C]pyruvate and [1‐13C]lactate

Single‐shot echo planar imaging (EPI), which allows an image to be acquired using a single excitation pulse, is used widely for imaging the metabolism of hyperpolarized ¹³C‐labelled metabolites in vivo as the technique is rapid and minimizes the depletion of the hyperpolarized signal. However, EPI suffers from Nyquist ghosting, which normally is corrected for by acquiring a reference scan. In a dynamic acquisition of a series of images, this results in the sacrifice of a time point if the reference scan involves a full readout train with no phase encoding. This time penalty is negligible if an integrated navigator echo is used, but at the cost of a lower signal‐to‐noise ratio (SNR) as a result of prolonged T₂* decay. We describe here a workflow for hyperpolarized ¹³C EPI that requires no reference scan. This involves the selection of a ghost‐containing background from a ¹³C image of a single metabolite at a single time point, the identification of phase correction coefficients that minimize signal in the selected area, and the application of these coefficients to images acquired at all time points and from all metabolites. The workflow was compared in phantom experiments with phase correction using a ¹³C reference scan, and yielded similar results in situations with a regular field of view (FOV), a restricted FOV and where there were multiple signal sources. When compared with alternative phase correction methods, the workflow showed an SNR benefit relative to integrated ¹³C reference echoes (>15%) or better ghost removal relative to a ¹H reference scan. The residual ghosting in a slightly de‐shimmed B₀ field was 1.6% using the proposed workflow and 3.8% using a ¹H reference scan. The workflow was implemented with a series of dynamically acquired hyperpolarized [1‐¹³C]pyruvate and [1‐¹³C]lactate images in vivo, resulting in images with no observable ghosting and which were quantitatively similar to images corrected using a ¹³C reference scan.     

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.     

An indirect method for in vivo T2 mapping of [1‐13C] pyruvate using hyperpolarized 13C CSI

An indirect method for in vivo T₂ mapping of ¹³C–labeled metabolites using T₂ and T₂* information of water protons obtained a priori is proposed. The T₂ values of ¹³C metabolites are inferred using the relationship to T₂′ of coexisting ¹H and the T₂* of ¹³C metabolites, which is measured using routine hyperpolarized ¹³C CSI data. The concept is verified with phantom studies. Simulations were performed to evaluate the extent of T₂ estimation accuracy due to errors in the other measurements. Also, bias in the ¹³C T₂* estimation from the ¹³C CSI data was studied. In vivo experiments were performed from the brains of normal rats and a rat with C6 glioma. Simulation results indicate that the proposed method provides accurate and unbiased ¹³C T₂ values within typical experimental settings. The in vivo studies found that the estimated T₂ of [1‐¹³C] pyruvate using the indirect method was longer in tumor than in normal tissues and gave values similar to previous reports. This method can estimate localized T₂ relaxation times from multiple voxels using conventional hyperpolarized ¹³C CSI and can potentially be used with time resolved fast CSI.     

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.     

Assessing the influence of isoflurane anesthesia on cardiac metabolism using hyperpolarized [1‐13C]pyruvate

Isoflurane is a frequently used anesthetic in small‐animal dissolution dynamic nuclear polarization‐magnetic resonance imaging (DNP‐MRI) studies. Although the literature suggests interactions with mitochondrial metabolism, the influence of the compound on cardiac metabolism has not been assessed systematically to date. In the present study, the impact of low versus high isoflurane concentration was examined in a crossover experiment in healthy rats. The results revealed that cardiac metabolism is modulated by isoflurane concentration, showing increased [1‐¹³C]lactate and reduced [¹³C]bicarbonate production during high isoflurane relative to low isoflurane dose [average differences: +16% [1‐¹³C]lactate/total myocardial carbon, –22% [¹³C]bicarbonate/total myocardial carbon; +51% [1‐¹³C]lactate/[¹³C]bicarbonate]. These findings emphasize that reproducible anesthesia is important when studying cardiac metabolism. As the depth of anesthesia is difficult to control in an experimental animal setting, careful study design is required to exclude confounding factors.     

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.     

Apparent rate constant mapping using hyperpolarized [1–13C]pyruvate

Hyperpolarization of [1‐13C]pyruvate in solution allows real‐time measurement of uptake and metabolism using MR spectroscopic methods. After injection and perfusion, pyruvate is taken up by the cells and enzymatically metabolized into downstream metabolites such as lactate, alanine, and bicarbonate. In this work, we present comprehensive methods for the quantification and interpretation of hyperpolarized 13C metabolite signals. First, a time‐domain spectral fitting method is described for the decomposition of FID signals into their metabolic constituents. For this purpose, the required chemical shift frequencies are automatically estimated using a matching pursuit algorithm. Second, a time‐discretized formulation of the two‐site exchange kinetic model is used to quantify metabolite signal dynamics by two characteristic rate constants in the form of (i) an apparent build‐up rate (quantifying the build‐up of downstream metabolites from the pyruvate substrate) and (ii) an effective decay rate (summarizing signal depletion due to repetitive excitation, T1‐relaxation and backward conversion). The presented spectral and kinetic quantification were experimentally verified in vitro and in vivo using hyperpolarized [1‐13C]pyruvate. Using temporally resolved IDEAL spiral CSI, spatially resolved apparent rate constant maps are also extracted. In comparison to single metabolite images, apparent build‐up rate constant maps provide improved contrast by emphasizing metabolically active tissues (e.g. tumors) and suppression of high perfusion regions with low conversion (e.g. blood vessels). Apparent build‐up rate constant mapping provides a novel quantitative image contrast for the characterization of metabolic activity. Its possible implementation as a quantitative standard will be subject to further studies. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Simultaneous investigation of cardiac pyruvate dehydrogenase flux, Krebs cycle metabolism and pH, using hyperpolarized [1,2-(13)C2]pyruvate in vivo

(13)C MR spectroscopy studies performed on hearts ex vivo and in vivo following perfusion of prepolarized [1-(13)C]pyruvate have shown that changes in pyruvate dehydrogenase (PDH) flux may be monitored non-invasively. However, to allow investigation of Krebs cycle metabolism, the (13)C label must be placed on the C2 position of pyruvate. Thus, the utilization of either C1 or C2 labeled prepolarized pyruvate as a tracer can only afford a partial view of cardiac pyruvate metabolism in health and disease. If the prepolarized pyruvate molecules were labeled at both C1 and C2 positions, then it would be possible to observe the downstream metabolites that were the results of both PDH flux ((13)CO(2) and H(13)CO(3)(-)) and Krebs cycle flux ([5-(13)C]glutamate) with a single dose of the agent. Cardiac pH could also be monitored in the same experiment, but adequate SNR of the (13)CO(2) resonance may be difficult to obtain in vivo. Using an interleaved selective RF pulse acquisition scheme to improve (13)CO(2) detection, the feasibility of using dual-labeled hyperpolarized [1,2-(13)C(2)]pyruvate as a substrate for dynamic cardiac metabolic MRS studies to allow simultaneous investigation of PDH flux, Krebs cycle flux and pH, was demonstrated in vivo.     

T2 relaxation times of 13C metabolites in a rat hepatocellular carcinoma model measured in vivo using 13C‐MRS of hyperpolarized [1‐13C]pyruvate

A single‐voxel Carr‐Purcell‐Meibloom‐Gill sequence was developed to measure localized T₂ relaxation times of ¹³C‐labeled metabolites in vivo for the first time. Following hyperpolarized [1‐¹³C]pyruvate injections, pyruvate and its metabolic products, alanine and lactate, were observed in the liver of five rats with hepatocellular carcinoma and five healthy control rats. The T₂ relaxation times of alanine and lactate were both significantly longer in HCC tumors than in normal livers (p < 0.002). The HCC tumors also showed significantly higher alanine signal relative to the total ¹³C signal than normal livers (p < 0.006). The intra‐ and inter‐subject variations of the alanine T₂ relaxation time were 11% and 13%, respectively. The intra‐ and inter‐subject variations of the lactate T₂ relaxation time were 6% and 7%, respectively. The intra‐subject variability of alanine to total carbon ratio was 16% and the inter‐subject variability 28%. The intra‐subject variability of lactate to total carbon ratio was 14% and the inter‐subject variability 20%. The study results show that the signal level and relaxivity of [1‐¹³C]alanine may be promising biomarkers for HCC tumors. Its diagnostic values in HCC staging and treatment monitoring are yet to be explored. Copyright © 2010 John Wiley & Sons, Ltd.     

Assessing high‐intensity focused ultrasound treatment of prostate cancer with hyperpolarized 13C dual‐agent imaging of metabolism and perfusion

The goal of the study was to establish early hyperpolarized (HP) ¹³C MRI metabolic and perfusion changes that predict effective high‐intensity focused ultrasound (HIFU) ablation and lead to improved adjuvant treatment of partially treated regions. To accomplish this a combined HP dual‐agent (¹³C pyruvate and ¹³C urea) ¹³C MRI/multiparametric ¹H MRI approach was used to measure prostate cancer metabolism and perfusion 3–4 h, 1 d, and 5 d after exposure to ablative and sub‐lethal doses of HIFU within adenocarcinoma of mouse prostate tumors using a focused ultrasound applicator designed for murine studies. Pathologic and immunohistochemical analysis of the ablated tumor demonstrated fragmented, non‐viable cells and vasculature consistent with coagulative necrosis, and a mixture of destroyed tissue and highly proliferative, poorly differentiated tumor cells in tumor tissues exposed to sub‐lethal heat doses in the ablative margin. In ablated regions, the intensity of HP ¹³C lactate or HP ¹³C urea and dynamic contrast‐enhanced (DCE) MRI area under the curve images were reduced to the level of background noise by 3–4 h after treatment with no recovery by the 5 d time point in either case. In the tissues that received sub‐lethal heat dose, there was a significant 60% ± 12.4% drop in HP ¹³C lactate production and a significant 30 ± 13.7% drop in urea perfusion 3–4 h after treatment, followed by recovery to baseline by 5 d after treatment. DCE MRI K^trans showed a similar trend to HP ¹³C urea, demonstrating a complete loss of perfusion with no recovery in the ablated region, while having a 40%–50% decrease 3–4 h after treatment followed by recovery to baseline values by 5 d in the margin region. The utility of the HP ¹³C MR measures of perfusion and metabolism in optimizing focal HIFU, either alone or in combination with adjuvant therapy, deserves further testing in future studies.     

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 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.     

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.     

Detection of lentiviral suicide gene therapy in C6 rat glioma using hyperpolarised [1‐13C]pyruvate

Hyperpolarised [1‐¹³C]pyruvate MRI has shown promise in monitoring therapeutic efficacy in a number of cancers including glioma. In this study, we assessed the pyruvate response to the lentiviral suicide gene therapy of herpes simplex virus‐1 thymidine kinase with the prodrug ganciclovir (HSV‐TK/GCV) in C6 rat glioma and compared it with traditional MR therapy markers. Female Wistar rats were inoculated with 10⁶ C6 glioma cells. Treated animals received intratumoural lentiviral HSV‐TK gene transfers on days 7 and 8 followed by 2‐week GCV therapy starting on day 10. Animals were repeatedly imaged during therapy using volumetric MRI, diffusion and relaxation mapping, as well as metabolic [1‐¹³C]pyruvate MRS imaging. Survival (measured as time before animals reached a humane endpoint and were euthanised) was assessed up to day 30 posttherapy. HSV‐TK/GCV gene therapy lengthened the median survival time from 12 to 25 days. This was accompanied by an apparent tumour growth arrest, but no changes in diffusion or relaxation parameters in treated animals. The metabolic response was more evident in the case‐by‐case analysis than in the group‐level analysis. Treated animals also showed a 37 ± 15% decrease (P < 0.05, n = 5) in lactate‐to‐pyruvate ratio between therapy weeks, whereas a 44 ± 18% increase (P < 0.05, n = 6) was observed in control animals. Hyperpolarised [1‐¹³C]pyruvate MRI can offer complementary metabolic information to traditional MR methods to give a more comprehensive picture of the slowly developing gene therapy response. This may benefit the detection of the successful therapy response in patients.     

Modeling non‐linear kinetics of hyperpolarized [1‐13C] pyruvate in the crystalloid‐perfused rat heart

Hyperpolarized ¹³C MR measurements have the potential to display non‐linear kinetics. We have developed an approach to describe possible non‐first‐order kinetics of hyperpolarized [1‐¹³C] pyruvate employing a system of differential equations that agrees with the principle of conservation of mass of the hyperpolarized signal. Simultaneous fitting to a second‐order model for conversion of [1‐¹³C] pyruvate to bicarbonate, lactate and alanine was well described in the isolated rat heart perfused with Krebs buffer containing glucose as sole energy substrate, or glucose supplemented with pyruvate. Second‐order modeling yielded significantly improved fits of pyruvate–bicarbonate kinetics compared with the more traditionally used first‐order model and suggested time‐dependent decreases in pyruvate–bicarbonate flux. Second‐order modeling gave time‐dependent changes in forward and reverse reaction kinetics of pyruvate–lactate exchange and pyruvate–alanine exchange in both groups of hearts during the infusion of pyruvate; however, the fits were not significantly improved with respect to a traditional first‐order model. The mechanism giving rise to second‐order pyruvate dehydrogenase (PDH) kinetics was explored experimentally using surface fluorescence measurements of nicotinamide adenine dinucleotide reduced form (NADH) performed under the same conditions, demonstrating a significant increase of NADH during pyruvate infusion. This suggests a simultaneous depletion of available mitochondrial NAD⁺ (the cofactor for PDH), consistent with the non‐linear nature of the kinetics. NADH levels returned to baseline following cessation of the pyruvate infusion, suggesting this to be a transient effect. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.     

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.