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the results demonstrated that the metabolic fate of [1‐¹³C]pyruvate conversion to [1‐¹³C]alanine significantly superseded that of [1‐¹³C]pyruvate conversion to [1‐¹³C]lactate, potentially serving as a marker of HCC tumors.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.     

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.     

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.     

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 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.     

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.     

Assessing inflammatory liver injury in an acute CCl4 model using dynamic 3D metabolic imaging of hyperpolarized [1‐13C]pyruvate

To facilitate diagnosis and staging of liver disease, sensitive and non‐invasive methods for the measurement of liver metabolism are needed. This study used hyperpolarized ¹³C‐pyruvate to assess metabolic parameters in a CCl₄ model of liver damage in rats. Dynamic 3D ¹³C chemical shift imaging data from a volume covering kidney and liver were acquired from 8 control and 10 CCl₄‐treated rats. At 12 time points at 5 s temporal resolution, we quantified the signal intensities and established time courses for pyruvate, alanine, and lactate. These measurements were compared with standard liver histology and an alanine transaminase (ALT) enzyme assay using liver tissue from the same animals. All CCl₄‐treated but none of the control animals showed histological liver damage and elevated ALT enzyme levels. In agreement with these results, metabolic imaging revealed an increased alanine/pyruvate ratio in liver of CCl₄‐treated rats, which is indicative of elevated ALT activity. Similarly, lactate/pyruvate ratios were higher in CCl₄‐treated compared with control animals, demonstrating the presence of inflammation. No significant differences in metabolite ratios were observed in kidney or vasculature. Thus this work shows that metabolic imaging using ¹³C‐pyruvate can be a successful tool to non‐invasively assess liver damage in vivo. Copyright © 2015 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.     

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.     

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.     

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.     

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.     

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.     

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.     

Accelerated spectroscopic imaging of hyperpolarized C‐13 pyruvate using SENSE parallel imaging

The ability to accelerate the spatial encoding process during a chemical shift imaging (CSI) scan of hyperpolarized compounds is demonstrated through parallel imaging. A hardware setup designed to simultaneously acquire ¹³C data from multiple receivers is presented here. A system consisting of four preamplifiers, four gain stages, a transmit coil, and a four receive channel rat coil was built for single channel excitation and simultaneous multi‐channel detection of ¹³C signals. The hardware setup was integrated with commercial scanner electronics, allowing the system to function similar to a conventional proton multi‐channel setup, except at a different frequency. The ability to perform parallel imaging is demonstrated in vivo. CSI data from the accelerated scans are reconstructed using a self‐calibrated multi‐spectral parallel imaging algorithm, by using lower resolution coil sensitivity maps obtained from the central region of k‐space. The advantages and disadvantages of parallel imaging in the context of imaging hyperpolarized compounds are discussed. Copyright © 2009 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.     

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.     

A comparison of quantitative methods for clinical imaging with hyperpolarized 13C‐pyruvate

Dissolution dynamic nuclear polarization (DNP) enables the metabolism of hyperpolarized ¹³C‐labelled molecules, such as the conversion of [1‐¹³C]pyruvate to [1‐¹³C]lactate, to be dynamically and non‐invasively imaged in tissue. Imaging of this exchange reaction in animal models has been shown to detect early treatment response and correlate with tumour grade. The first human DNP study has recently been completed, and, for widespread clinical translation, simple and reliable methods are necessary to accurately probe the reaction in patients. However, there is currently no consensus on the most appropriate method to quantify this exchange reaction. In this study, an in vitro system was used to compare several kinetic models, as well as simple model‐free methods. Experiments were performed using a clinical hyperpolarizer, a human 3 T MR system, and spectroscopic imaging sequences. The quantitative methods were compared in vivo by using subcutaneous breast tumours in rats to examine the effect of pyruvate inflow. The two‐way kinetic model was the most accurate method for characterizing the exchange reaction in vitro, and the incorporation of a Heaviside step inflow profile was best able to describe the in vivo data. The lactate time‐to‐peak and the lactate‐to‐pyruvate area under the curve ratio were simple model‐free approaches that accurately represented the full reaction, with the time‐to‐peak method performing indistinguishably from the best kinetic model. Finally, extracting data from a single pixel was a robust and reliable surrogate of the whole region of interest. This work has identified appropriate quantitative methods for future work in the analysis of human hyperpolarized ¹³C data. © 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.     

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.