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Glioblastoma (GBM) is the most aggressive brain tumor type in adults. GBM is heterogeneous, with a compact core lesion surrounded by an invasive tumor front. This front is highly relevant for tumor recurrence but is generally non-detectable using standard imaging techniques. Recent studies demonstrated distinct metabolic profiles of the invasive phenotype in GBM. Magnetic resonance (MR) of hyperpolarized 13C-labeled probes is a rapidly advancing field that provides real-time metabolic information. Here, we applied hyperpolarized 13C-glucose MR to mouse GBM models. Compared to controls, the amount of lactate produced from hyperpolarized glucose was higher in the compact GBM model, consistent with the accepted “Warburg effect”. However, the opposite response was observed in models reflecting the invasive zone, with less lactate produced than in controls, implying a reduction in aerobic glycolysis. These striking differences could be used to map the metabolic heterogeneity in GBM and to visualize the infiltrative front of GBM.

The metabolic shift induced in human CD4+ T lymphocytes by stimulation is characterized by an upregulation of glycolysis, leading to an augmentation in lactate production. This adaptation has already been highlighted with various techniques and reported in several previous studies. We herein propose a method to rapidly and noninvasively detect the associated increase in flux from pyruvate to lactate catalyzed by lactate dehydrogenase using hyperpolarized 13C magnetic resonance, a technique which can be used for in vivo imaging. It was shown that the conversion of hyperpolarized 13C-pyruvate to 13C-lactate during the one-minute measurement increased by a mean factor of 3.6 in T cells stimulated for 5 days as compared to resting T cells. This method can be extended to other metabolic substrates and is therefore a powerful tool to noninvasively analyze T cell metabolism, possibly in vivo.

Hyperpolarized (HP)(13)C magnetic resonance enables non-invasive probing of metabolism in vivo. To date, only (13)C-molecules hyperpolarized with persistent trityl radicals have been injected in humans. We show here that the free radical photo-induced in alpha-ketoglutaric acid (α-KG) can be used to hyperpolarize photo-inactive (13)C-molecules such as [1-(13)C]lactate. α-KG is an endogenous molecule with an exceptionally high radical yield under photo-irradiation, up to 50%, and its breakdown product, succinic acid, is also endogenous. This radical precursor therefore exhibits an excellent safety profile for translation to human studies. The labile nature of the radical means that no filtration is required prior to injection while also offering the opportunity to extend the (13)C relaxation time in frozen HP(13)C-molecules for storage and transport. The potential for in vivo metabolic studies is demonstrated in the rat liver following the injection of a physiological dose of HP [1-(13)C]lactate.

Glioblastoma (GBM) is the most aggressive brain tumor type in adults. GBM is heterogeneous, with a compact core lesion surrounded by an invasive tumor front. This front is highly relevant for tumor recurrence but is generally non-detectable using standard imaging techniques. Recent studies demonstrated distinct metabolic profiles of the invasive phenotype in GBM. Magnetic resonance (MR) of hyperpolarized 13C-labeled probes is a rapidly advancing field that provides real-time metabolic information. Here, we applied hyperpolarized 13C-glucose MR to mouse GBM models. Compared to controls, the amount of lactate produced from hyperpolarized glucose was higher in the compact GBM model, consistent with the accepted “Warburg effect”. However, the opposite response was observed in models reflecting the invasive zone, with less lactate produced than in controls, implying a reduction in aerobic glycolysis. These striking differences could be used to map the metabolic heterogeneity in GBM and to visualize the infiltrative front of GBM.

Purpose Inflammation is involved in many disease processes. However, accurate imaging tools permitting diagnosis and characterization of inflammation are still missing. As inflamed tissues exhibit a high rate of glycolysis, pyruvate metabolism may offer a unique approach to follow the inflammatory response and disease progression. Therefore, the aim of the study was to follow metabolic changes and recruitment of inflammatory cells after onset of inflammation in arthritic ankles using hyperpolarized 1-13C-pyruvate magnetic resonance spectroscopy (MRS) and 19F magnetic resonance imaging (MRI), respectively. Procedure Experimental rheumatoid arthritis (RA) was induced by intraperitoneal injection of glucose-6-phosphate-isomerase-specific antibodies (GPI) containing serum. To monitor pyruvate metabolism, the transformation of hyperpolarized 1-13C-pyruvate into hyperpolarized 1-13C-lactate was followed using MRS. To track phagocytic immune cell homing, we intravenously injected a perfluorocarbon emulsion 48 h before imaging. The animals were scanned at days 1, 3, or 6 after GPI-serum injection to examine the different stages of arthritic inflammation. Finally, to confirm the pyruvate metabolic activity and the link to inflammatory cell recruitment, we conducted hematoxylin-eosin histopathology and monocarboxylase transporter (MCT-1) immune histochemistry (IHC) of inflamed ankles. Results Hyperpolarized 1-13C-pyruvate MRS revealed a high rate of lactate production immediately at day 1 after GPI-serum transfer, which remained elevated during the progression of the disease, while 19F-MRI exhibited a gradual recruitment of phagocytic immune cells in arthritic ankles, which correlated well with the course of ankle swelling. Histopathology and IHC revealed that MCT-1 was expressed in regions with inflammatory cell recruitment, confirming the metabolic shift identified in arthritic ankles. Conclusions Our study demonstrated the presence of a very early metabolic shift in arthritic joints independent of phagocytic immune cell recruitment. Thus, hyperpolarized 1-13C-pyruvate represents a promising tracer to monitor acute arthritic joint inflammation, even with minor ankle swelling. Furthermore, translated to the clinics, these methods add a detailed characterization of disease status and could substantially support patient stratification and therapy monitoring.

Level of Evidence: 5. Technical Efficacy Stage: 1.

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 similar to 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.

Hyperpolarised 13C MRI (HP-MRI) is a novel imaging technique that allows real-time analysis of metabolic pathways in vivo. 1 The technology to conduct HP-MRI in humans has recently become available and is starting to be clinically applied. As knowledge of molecular biology advances, it is increasingly apparent that cancer cell metabolism is related to disease outcomes, with lactate attracting specific attention. 2 Recent reviews of breast cancer screening programs have raised concerns and increased public awareness of over treatment. The scientific community needs to shift focus from improving cancer detection alone to pursuing novel methods of distinguishing aggressive breast cancers from those which will remain indolent. HP-MRI offers the opportunity to identify aggressive tumour phenotypes and help monitor/predict therapeutic response. Here we report one of the first cases of breast cancer imaged using HP-MRI alongside correlative conventional imaging, including breast MRI.

Objectives To enhance detection of the products of hyperpolarized [2-13C]dihydroxyacetone metabolism for assessment of three metabolic pathways in the liver in vivo. Hyperpolarized [2-13C]DHAc emerged as a promising substrate to follow gluconeogenesis, glycolysis and the glycerol pathways. However, the use of [2-13C]DHAc in vivo has not taken off because (i) the chemical shift range of [2-13C]DHAc and its metabolic products span over 144 ppm, and (ii) 1H decoupling is required to increase spectral resolution and sensitivity. While these issues are trivial for high-field vertical-bore NMR spectrometers, horizontal-bore small-animal MR scanners are seldom equipped for such experiments. Methods Real-time hepatic metabolism of three fed mice was probed by 1H-decoupled 13C-MR following injection of hyperpolarized [2-13C]DHAc. The spectra of [2-13C]DHAc and its metabolic products were acquired in a 7 T small-animal MR scanner using three purpose-designed spectral-spatial radiofrequency pulses that excited a spatial bandwidth of 8 mm with varying spectral bandwidths and central frequencies (chemical shifts). Results The metabolic products detected in vivo include glycerol 3-phosphate, glycerol, phosphoenolpyruvate, lactate, alanine, glyceraldehyde 3-phosphate and glucose 6-phosphate. The metabolite-to-substrate ratios were comparable to those reported previously in perfused liver. Discussion Three metabolic pathways can be probed simultaneously in the mouse liver in vivo, in real time, using hyperpolarized DHAc.

Dynamic nuclear polarization (DNP) provides the opportunity to boost liquid state magnetic resonance (MR) signals from selected nuclear spins by several orders of magnitude. A cryostat running at a temperature of ~ 1 K and a superconducting magnet set to between 3 and 10 T are required to efficiently hyperpolarize nuclear spins. Several DNP polarizers have been implemented for the purpose of hyperpolarized MR and recent systems have been designed to avoid the need for user input of liquid cryogens. We herein present a zero boil‐off DNP polarizer that operates at 1.35 ± 0.01 K and 7 T, and which can polarize two samples in parallel. The samples are cooled by a static helium bath thermally connected to a 1 K closed‐cycle 4He refrigerator. Using a modified version of the commercial fluid path developed for the SPINlab polarizer, we demonstrate that, within a 12‐minute interval, the system can produce two separate hyperpolarized 13C solutions. The 13C liquid‐state polarization of [1‐13C]pyruvate measured 26 seconds after dissolution was 36%, which can be extrapolated to a 55% solid state polarization. The system is well adapted for in vitro and in vivo preclinical hyperpolarized MR experiments and it can be modified to polarize up to four samples in parallel., European Union's Horizon 2020 European Research Council (ERC Consolidator Grant). Grant Number: 682574

The advent of dissolution dynamic nuclear polarization (DNP) led to the emergence of a new kind of magnetic resonance (MR) measurements providing the opportunity to probe metabolism in vivo in real time. It has been shown that, following the injection of hyperpolarized substrates prepared using dissolution DNP, specific metabolic bioprobes that can be used to differentiate between healthy and pathological tissue in preclinical and clinical studies can be readily detected by MR thanks to the tremendous signal enhancement. The present article aims at reviewing the studies of cerebral function and metabolism based on the use of hyperpolarized MR. The constraints and future opportunities that this technology could offer are discussed.

Pyruvate membrane crossing and its lactate dehydrogenase-mediated conversion to lactate in cells featuring different levels of expression of membrane monocarboxylate transporters (MCT4) were probed by dissolution dynamic nuclear polarization-enhanced NMR. Hyperpolarized 13 C-1-labeled pyruvate was transferred to suspensions of rodent tumor cell carcinoma, cell line 39. The pyruvate-to-lactate conversion rate monitored by dissolution dynamic nuclear polarization-NMR in carcinoma cells featuring native MCT4 expression level was lower than the rate observed for cells in which the human MCT4 gene was overexpressed. The enzymatic activity of lactate dehydrogenase was also assessed in buffer solutions, following the real-time pyruvate-to-lactate conversion speeds at different enzyme concentrations. Copyright © 2016 John Wiley & Sons, Ltd.

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

A wide range of organisms host photosynthesizing symbionts. In these animals the metabolic exchange between host and symbionts has prevented in situ host anabolic turnover to be studied without the confounding effect of translocated photosynthates. Using the symbiotic coral Stylophora pistillata as a model organism and [1-13C]-pyruvate and [2,3-13C]-pyruvate in different incubation conditions (light, light + DCMU, and darkness), we employed NanoSIMS isotopic imaging to quantify host anabolism, with and without translocated metabolites from their photosynthesizing dinoflagellate symbionts. Under our experimental conditions, host de novo lipid synthesis accounted for ~40% of the total holobiont lipid reserve, and dinoflagellate recycling of metabolic 13CO2 enhanced host tissue 13C-enrichment by 13–22% in the epidermis, 40–58% in the gastrodermis, and 135–169% in host lipid bodies. Furthermore, we show that host anabolic turnover in different tissue structures differs, in a manner consistent with the localisation, function and cellular composition of these structures., Gibbin et al. use [1–13C]-pyruvate and [2,3–13C]-pyruvate in different incubation conditions (light, light+DCMU, and dark) to discern and quantify coral host anabolism, with and without translocated metabolites from their photosynthesizing symbionts.

Intratumoral genetic heterogeneity and the role of metabolic reprogramming in renal cell carcinoma (RCC) have been extensively documented. However, the distribution of these metabolic changes within the tissue has not been explored. We report on the first-in-human in vivo non-invasive metabolic interrogation of RCC using hyperpolarized carbon-13 ((13)C) magnetic resonance imaging (HP-MRI) and describe the validation of in vivo lactate metabolic heterogeneity against multi-regional ex vivo mass spectrometry. HP-MRI provides an in vivo assessment of metabolism and provides a novel opportunity to safely and non-invasively assess cancer heterogeneity.

The metabolic shift induced in human CD4+ T lymphocytes by stimulation is characterized by an upregulation of glycolysis, leading to an augmentation in lactate production. This adaptation has already been highlighted with various techniques and reported in several previous studies. We herein propose a method to rapidly and noninvasively detect the associated increase in flux from pyruvate to lactate catalyzed by lactate dehydrogenase using hyperpolarized 13C magnetic resonance, a technique which can be used for in vivo imaging. It was shown that the conversion of hyperpolarized 13C-pyruvate to 13C-lactate during the one-minute measurement increased by a mean factor of 3.6 in T cells stimulated for 5 days as compared to resting T cells. This method can be extended to other metabolic substrates and is therefore a powerful tool to noninvasively analyze T cell metabolism, possibly in vivo.

Resting skeletal muscle has a preference for the oxidation of lipids compared to carbohydrates and a shift towards carbohydrate oxidation is observed with increasing exercise. Lactate is not only an end product in skeletal muscle but also an important metabolic intermediate for mitochondrial oxidation. Recent advances in hyperpolarized MRS allow the measurement of substrate metabolism in vivo in real time. The aim of this study was to investigate the use of hyperpolarized 13C lactate as a substrate for metabolic studies in skeletal muscle in vivo. Carbohydrate metabolism in healthy rat skeletal muscle at rest was studied in different nutritional states using hyperpolarized [1 13C]lactate a substrate that can be injected at physiological concentrations and leaves other oxidative processes undisturbed. 13C label incorporation from lactate into bicarbonate in fed animals was observed within seconds but was absent after an overnight fast representing inhibition of the metabolic flux through pyruvate dehydrogenase (PDH). A significant decrease in 13C labeling of alanine was observed comparing the fed and fasted group and was attributed to a change in cellular alanine concentration and not a decrease in enzymatic flux through alanine transaminase. We conclude that hyperpolarized [1 13C]lactate can be used to study carbohydrate oxidation in resting skeletal muscle at physiological levels. The herein proposed method allows probing simultaneously both PDH activity and variations in alanine tissue concentration which are associated with metabolic dysfunctions. A simple alteration of the nutritional state demonstrated that the observed pyruvate alanine and bicarbonate signals are indeed sensitive markers to probe metabolic changes in vivo. © 2014 Springer Science+Business Media New York.

Free radicals generated by UV-light irradiation of a frozen solution containing a fraction of pyruvic acid (PA) have demonstrated their dissolution dynamic nuclear polarization (dDNP) potential, providing up to 30 % [1-13C]PA liquid-state polarization. Moreover, their labile nature has proven to pave a way to nuclear polarization storage and transport. Herein, differently from the case of PA, the issue of providing dDNP UV-radical precursors (trimethylpyruvic acid and its methyl-deuterated form) not involved in any metabolic pathway was investigated. The 13C dDNP performance was evaluated for hyperpolarization of [U-13 C6 ,1,2,3,4,5,6,6-d7 ]-d-glucose. The generated UV-radicals proved to be versatile and highly efficient polarizing agents, providing, after dissolution and transfer (10 s), a 13 C liquid-state polarization of up to 32 %.

Various tumors develop addiction to glutamine to support uncontrolled cell proliferation. Here we identify the nuclear receptor liver receptor homolog 1 (LRH-1) as a key regulator in the process of hepatic tumorigenesis through the coordination of a noncanonical glutamine pathway that is reliant on the mitochondrial and cytosolic transaminases glutamate pyruvate transaminase 2 (GPT2) and glutamate oxaloacetate transaminase 1 (GOT1), which fuel anabolic metabolism. In particular, we show that gain and loss of function of hepatic LRH-1 modulate the expression and activity of mitochondrial glutaminase 2 (GLS2), the first and rate-limiting step of this pathway. Acute and chronic deletion of hepatic LRH-1 blunts the deamination of glutamine and reduces glutamine-dependent anaplerosis. The robust reduction in glutaminolysis and the limiting availability of α-ketoglutarate in turn inhibit mTORC1 signaling to eventually block cell growth and proliferation. Collectively, these studies highlight the importance of LRH-1 in coordinating glutamine-induced metabolism and signaling to promote hepatocellular carcinogenesis.

The tremendous polarization enhancement afforded by dissolution dynamic nuclear polarization (DNP) can be taken advantage of to perform preclinical in vivo molecular and metabolic imaging. Following the injection of molecules that are hyperpolarized via dissolution DNP, real-time measurements of their biodistribution and metabolic conversion can be recorded. This technology therefore provides a unique and invaluable tool for probing cellular metabolism in vivo in animal models in a noninvasive manner. It gives the opportunity to follow and evaluate disease progression and treatment response without requiring ex vivo destructive tissue assays. Although its considerable potential has now been widely recognized, hyperpolarized magnetic resonance by dissolution DNP remains a challenging method to implement for routine in vivo preclinical measurements. The aim of this article is to provide an overview of the current state-of-the-art technology for preclinical applications and the challenges that need to be addressed to promote it and allow its wider dissemination in the near future. (C) 2016 Elsevier Inc. All rights reserved.

Magnetic resonance elastography (MRE) is a powerful technique to assess the mechanical properties of living tissue. However, it suffers from reduced sensitivity in regions with short T2 and T2 * such as in tissue with high concentrations of paramagnetic iron, or in regions surrounding implanted devices. In this work, we exploit the longer T2 * attainable at ultra-low magnetic fields in combination with Overhauser dynamic nuclear polarization (DNP) to enable rapid MRE at 0.0065 T. A 3D balanced steady-state free precession based MRE sequence with undersampling and fractional encoding was implemented on a 0.0065 T MRI scanner. A custom-built RF coil for DNP and a programmable vibration system for elastography were developed. Displacement fields and stiffness maps were reconstructed from data recorded in a polyvinyl alcohol gel phantom loaded with stable nitroxide radicals. A DNP enhancement of 25 was achieved during the MRE sequence, allowing the acquisition of 3D Overhauser-enhanced MRE (OMRE) images with (1.5 × 2.7 × 9) mm(3) resolution over eight temporal steps and 11 slices in 6 minutes. In conclusion, OMRE at ultra-low magnetic field can be used to detect mechanical waves over short acquisition times. This new modality shows promise to broaden the scope of conventional MRE applications, and may extend the utility of low-cost, portable MRI systems to detect elasticity changes in patients with implanted devices or iron overload.

[1-(13)C]pyruvate is the most widely used hyperpolarized metabolic magnetic resonance imaging agent. Using a custom-built 7.0 T polarizer operating at 1.0 K and trityl radical-doped [1-(13)C]pyruvic acid, unextrapolated solution-state (13)C polarization greater than 60% was measured after dissolution and rapid transfer to a spectrometer magnet, demonstrating the signal enhancement attainable using optimized hardware. Slower rates of polarization under these conditions can be largely overcome with higher radical concentrations.

Hyperpolarized 13C Magnetic Resonance Imaging (13C-MRI) provides a highly sensitive tool to probe tissue metabolism in vivo and has recently been translated into clinical studies. We report the cerebral metabolism of intravenously injected hyperpolarized [1–13C]pyruvate in the brain of healthy human volunteers for the first time. Dynamic acquisition of 13C images demonstrated 13C-labeling of both lactate and bicarbonate, catalyzed by cytosolic lactate dehydrogenase and mitochondrial pyruvate dehydrogenase respectively. This demonstrates that both enzymes can be probed in vivo in the presence of an intact blood-brain barrier: the measured apparent exchange rate constant (kPL) for exchange of the hyperpolarized 13C label between [1–13C]pyruvate and the endogenous lactate pool was 0.012 ± 0.006 s−1 and the apparent rate constant (kPB) for the irreversible flux of [1–13C]pyruvate to [13C]bicarbonate was 0.002 ± 0.002 s−1. Imaging also revealed that [1–13C]pyruvate, [1–13C]lactate and [13C]bicarbonate were significantly higher in gray matter compared to white matter. Imaging normal brain metabolism with hyperpolarized [1–13C]pyruvate and subsequent quantification, have important implications for interpreting pathological cerebral metabolism in future studies., Graphical abstract Image 1

To probe the cardiac metabolism of carbohydrates and short chain fatty acids simultaneously in vivo following the injection of a hyperpolarized javax.xml.bind.JAXBElement@47d46327 C-labeled substrate mixture prepared using photo-induced nonpersistent radicals. Droplets of mixed [1- javax.xml.bind.JAXBElement@f125d2a C]pyruvic and [1- javax.xml.bind.JAXBElement@3a61d95c C]butyric acids were frozen into glassy beads in liquid nitrogen. Ethanol addition was investigated as a means to increase the polarization level. The beads were irradiated with ultraviolet light and the radical concentration was measured by ESR spectroscopy. Following dynamic nuclear polarization in a 7T polarizer, the beads were dissolved, and the radical-free hyperpolarized solution was rapidly transferred into an injection pump located inside a 9.4T scanner. The hyperpolarized solution was injected in healthy rats to measure cardiac metabolism in vivo. Ultraviolet irradiation created nonpersistent radicals in a mixture containing javax.xml.bind.JAXBElement@2f4af5f8 C-labeled pyruvic and butyric acids, and enabled the hyperpolarization of both substrates by dynamic nuclear polarization. Ethanol addition increased the radical concentration from 16 to 26 mM. Liquid-state javax.xml.bind.JAXBElement@4b63666d C polarization was 3% inside the pump at the time of injection, and increased to 5% by addition of ethanol to the substrate mixture prior to ultraviolet irradiation. In the rat heart, the in vivo javax.xml.bind.JAXBElement@56058926 C signals from lactate, alanine, bicarbonate, and acetylcarnitine were detected following the metabolism of the injected substrate mixture. Copolarization of two different javax.xml.bind.JAXBElement@629a1183 C-labeled substrates and the detection of their myocardial metabolism in vivo was achieved without using persistent radicals. The absence of radicals in the solution containing the hyperpolarized javax.xml.bind.JAXBElement@1a07cd1d C-substrates may simplify the translation to clinical use, as no radical filtration is required prior to injection.

The dynamics of l-lactate transport across the blood-brain barrier (BBB) and its cerebral metabolism are still subject to debate. We studied lactate uptake and intracellular metabolism in the mouse brain using hyperpolarized 13 C magnetic resonance spectroscopy (MRS). Following the intravenous injection of hyperpolarized [1- 13 C]lactate, we observed that the distribution of the 13 C label between lactate and pyruvate, which has been shown to be representative of their pool size ratio, is different in NMRI and C57BL/6 mice, the latter exhibiting a higher level of cerebral lactate dehydrogenase A ( Ldha) expression. On the basis of this observation, and an additional set of experiments showing that the cerebral conversion of [1- 13 C]lactate to [1- 13 C]pyruvate increases after exposing the brain to ultrasound irradiation that reversibly opens the BBB, we concluded that lactate transport is rate-limited by the BBB, with a 30% increase in lactate uptake after its disruption. It was also deduced from these results that hyperpolarized 13 C MRS can be used to detect a variation in cerebral lactate uptake of

BACKGROUND: The heart relies on continuous energy production and imbalances herein impair cardiac function directly. The tricarboxylic acid (TCA) cycle is the primary means of energy generation in the healthy myocardium, but direct noninvasive quantification of metabolic fluxes is challenging due to the low concentration of most metabolites. Hyperpolarized 13C magnetic resonance spectroscopy (MRS) provides the opportunity to measure cellular metabolism in real time in vivo. The aim of this work was to noninvasively measure myocardial TCA cycle flux (VTCA) in vivo within a single minute. METHODS AND RESULTS: Hyperpolarized [1-13C]acetate was administered at different concentrations in healthy rats. 13C incorporation into [1-13C]acetylcarnitine and the TCA cycle intermediate [5-13C]citrate was dynamically detected in vivo with a time resolution of 3s. Different kinetic models were established and evaluated to determine the metabolic fluxes by simultaneously fitting the evolution of the 13C labeling in acetate, acetylcarnitine, and citrate. VTCA was estimated to be 6.7+/-1.7mumol.g-1.min-1 (dry weight), and was best estimated with a model using only the labeling in citrate and acetylcarnitine, independent of the precursor. The TCA cycle rate was not linear with the citrate-to-acetate metabolite ratio, and could thus not be quantified using a ratiometric approach. The 13C signal evolution of citrate, i.e. citrate formation was independent of the amount of injected acetate, while the 13C signal evolution of acetylcarnitine revealed a dose dependency with the injected acetate. The 13C labeling of citrate did not correlate to that of acetylcarnitine, leading to the hypothesis that acetylcarnitine formation is not an indication of mitochondrial TCA cycle activity in the heart. CONCLUSIONS: Hyperpolarized [1-13C]acetate is a metabolic probe independent of pyruvate dehydrogenase (PDH) activity. It allows the direct estimation of VTCA in vivo, which was shown to be neither dependent on the administered acetate dose nor on the 13C labeling of acetylcarnitine. Dynamic 13C MRS coupled to the injection of hyperpolarized [1-13C]acetate can enable the measurement of metabolic changes during impaired heart function.

Hyperpolarization by dissolution dynamic nuclear polarization (DNP) is a versatile technique to dramatically enhance the nuclear magnetic resonance (NMR) signal intensity of insensitive long-T1 nuclear spins such as (6)Li. The (6)Li longitudinal relaxation of lithium ions in aqueous solutions strongly depends on the concentration of paramagnetic species, even if they are present in minute amounts. We herein demonstrate that blood oxygenation can be readily detected by taking advantage of the (6)Li signal enhancement provided by dissolution DNP, together with the more than 10% decrease in (6)Li longitudinal relaxation as a consequence of the presence of paramagnetic deoxyhemoglobin.

Hyperpolarised (HP) (13)C NMR allows enzymatic activity to be probed in real time in live biological systems. The use of in vitro models gives excellent control of the cellular environment, crucial in the understanding of enzyme kinetics. The increased conversion of pyruvate to lactate in cancer cells has been well studied with HP (13)C NMR. Unfortunately, the equally important metabolic step of lactate transport out of the cell remains undetected, because intracellular and extracellular lactate are measured as a single resonance. Furthermore, typical experiments must be performed using tens of millions of cells, a large amount which can lead to a costly and sometimes highly challenging growing procedure. We present a relatively simple set-up that requires as little as two million cells with the spectral resolution to separate the intracellular and extracellular lactate resonances. The set-up is tested with suspensions of prostate cancer carcinoma cells (PC3) in combination with HP [1-(13)C]pyruvate. We obtained reproducible pyruvate to lactate label fluxes of 1.2 and 1.7 nmol/s per million cells at 2.5 and 5.0 mM pyruvate concentrations. The existence of a 3-Hz chemical shift difference between intracellular and extracellular lactate enabled us to determine the lactate transport rates in PC3. We deduced a lactate export rate of 0.3 s(-1) and observed a decrease in lactate transport on addition of the lactate transport inhibitor α-cyano-4-hydroxycinnamic acid.

In addition to the dedicated spin exchange optical pumping (SEOP) technique, an alternative method to produce hyperpolarized xenon gas using dynamic nuclear polarization (DNP) followed by a sublimation procedure was proposed. The challenge associated with sublimation DNP is to obtain solid-state samples with the required homogeneity following the incorporation of xenon into a glass-forming solvent. We show that the maximum achievable 129Xe polarization strongly depends on the xenon concentration dissolved in the solvent and the properties of the solvent itself. Using a 5-T home-built polarizer, the 129Xe spins in natural abundance xenon were polarized up to 30% in about 90 min at 1.15±0.05 K. The gas was subsequently extracted to perform 129Xe MRI in a distant 9.4-T rodent scanner. We demonstrate that by careful optimization of the sample preparation, sublimation DNP can be used to hyperpolarize 129Xe at a polarization level and quantities that are sufficiently large for biomedical applications.

The heart relies on continuous energy production and imbalances herein impair cardiac function directly. The tricarboxylic acid (TCA) cycle is the primary means of energy generation in the healthy myocardium, but direct noninvasive quantification of metabolic fluxes is challenging due to the low concentration of most metabolites. Hyperpolarized (13)C magnetic resonance spectroscopy (MRS) provides the opportunity to measure cellular metabolism in real time in vivo. The aim of this work was to noninvasively measure myocardial TCA cycle flux (VTCA) in vivo within a single minute.Hyperpolarized [1-(13)C]acetate was administered at different concentrations in healthy rats. (13)C incorporation into [1-(13)C]acetylcarnitine and the TCA cycle intermediate [5-(13)C]citrate was dynamically detected in vivo with a time resolution of 3s. Different kinetic models were established and evaluated to determine the metabolic fluxes by simultaneously fitting the evolution of the (13)C labeling in acetate, acetylcarnitine, and citrate. VTCA was estimated to be 6.7±1.7 μmol·g(-1)·min(-1) (dry weight), and was best estimated with a model using only the labeling in citrate and acetylcarnitine, independent of the precursor. The TCA cycle rate was not linear with the citrate-to-acetate metabolite ratio, and could thus not be quantified using a ratiometric approach. The (13)C signal evolution of citrate, i.e. citrate formation was independent of the amount of injected acetate, while the (13)C signal evolution of acetylcarnitine revealed a dose dependency with the injected acetate. The (13)C labeling of citrate did not correlate to that of acetylcarnitine, leading to the hypothesis that acetylcarnitine formation is not an indication of mitochondrial TCA cycle activity in the heart.Hyperpolarized [1-(13)C]acetate is a metabolic probe independent of pyruvate dehydrogenase (PDH) activity. It allows the direct estimation of VTCA in vivo, which was shown to be neither dependent on the administered acetate dose nor on the (13)C labeling of acetylcarnitine. Dynamic (13)C MRS coupled to the injection of hyperpolarized [1-(13)C]acetate can enable the measurement of metabolic changes during impaired heart function.

Hyperpolarization via dissolution dynamic nuclear polarization (DNP) is a versatile method to dramatically enhance the liquid-state NMR signal of X-nuclei and can be used for performing metabolic and molecular imaging. It was recently demonstrated that instead of incorporating persistent radicals as source of unpaired electron spins, required for DNP, non-persistent radicals can be photo-induced in frozen beads of neat pyruvic acid (PA), the most common substrate for metabolic imaging. In the present work, it is shown that the same radicals can be created in frozen solutions containing a fraction of PA in addition to 13C- or 6Li-labeled salts, or 129Xe nuclei. The use of these non-persistent radicals prevents the loss of a substantial part of the polarization during the transfer of hyperpolarized solutions into iron-shielded high-field MRI scanners. It is also demonstrated that UV-irradiated d4-PA yields non-persistent radicals exhibiting similarities with the most efficient and widely used persistent trityl radicals.

Given their high sensitivity and ability to limit the field of view (FOV), surface coils are often used in magnetic resonance spectroscopy (MRS) and imaging (MRI). A major downside of surface coils is their inherent radiofrequency (RF) B1 heterogeneity across the FOV, decreasing with increasing distance from the coil and giving rise to image distortions due to non-uniform spatial responses. A robust way to compensate for B1 inhomogeneities is to employ adiabatic inversion pulses, yet these are not well adapted to all imaging sequences - including to single-shot approaches like echo planar imaging (EPI). Hybrid spatiotemporal encoding (SPEN) sequences relying on frequency-swept pulses provide another ultrafast MRI alternative, that could help solve this problem thanks to their built-in heterogeneous spatial manipulations. This study explores how this intrinsic SPEN-based spatial discrimination, could be used to compensate for the B1 inhomogeneities inherent to surface coils. Experiments carried out in both phantoms and in vivo rat brains demonstrate that, by suitably modulating the amplitude of a SPEN chirp pulse that progressively excites the spins in a direction normal to the coil, it is possible to compensate for the RF transmit inhomogeneities and thus improve sensitivity and image fidelity.

The mammalian brain relies primarily on glucose as a fuel to meet its high metabolic demand. Among the various techniques used to study cerebral metabolism, 13C magnetic resonance spectroscopy (MRS) allows following the fate of 13C-enriched substrates through metabolic pathways. We herein demonstrate that it is possible to measure cerebral glucose metabolism in vivo with sub-second time resolution using hyperpolarized 13C MRS. In particular, the dynamic 13C-labeling of pyruvate and lactate formed from 13C-glucose was observed in real time. An ad-hoc synthesis to produce [2,3,4,6,6-2H5, 3,4-13C2]-D-glucose was developed to improve the 13C signal-to-noise ratio as compared to experiments performed following [U-2H7, U-13C]-D-glucose injections. The main advantage of only labeling C3 and C4 positions is the absence of 13C-13C coupling in all downstream metabolic products after glucose is split into 3-carbon intermediates by aldolase. This unique method allows direct detection of glycolysis in vivo in the healthy brain in a noninvasive manner.

The mammalian brain relies primarily on glucose as a fuel to meet its high metabolic demand. Among the various techniques used to study cerebral metabolism, 13C magnetic resonance spectroscopy (MRS) allows following the fate of 13C-enriched substrates through metabolic pathways. We herein demonstrate that it is possible to measure cerebral glucose metabolism in vivo with sub-second time resolution using hyperpolarized 13C MRS. In particular, the dynamic 13C-labeling of pyruvate and lactate formed from 13C-glucose was observed in real time. An ad-hoc synthesis to produce [2,3,4,6,6-2H5, 3,4-13C2]-D-glucose was developed to improve the 13C signal-to-noise ratio as compared to experiments performed following [U-2H7, U-13C]-D-glucose injections. The main advantage of only labeling C3 and C4 positions is the absence of 13C-13C coupling in all downstream metabolic products after glucose is split into 3-carbon intermediates by aldolase. This unique method allows direct detection of glycolysis in vivo in the healthy brain in a noninvasive manner.

Hyperpolarization via dynamic nuclear polarization (DNP) is pivotal for boosting magnetic resonance imaging (MRI) sensitivity and dissolution DNP can be used to perform in vivo real-time 13C MRI. The type of applications is however limited by the relatively fast decay time of the hyperpolarized spin state together with the constraint of having to polarize the 13C spins in a dedicated apparatus nearby but separated from the MRI magnet. We herein demonstrate that by polarizing 13C with photo-induced radicals, which can be subsequently annihilated using a thermalization process that maintains the sample temperature below its melting point, hyperpolarized 13C-substrates can be extracted from the DNP apparatus in the solid form, while maintaining the enhanced 13C polarization. The melting procedure necessary to transform the frozen solid into an injectable solution containing the hyperpolarized 13C-substrates can therefore be performed ex situ, up to several hours after extraction and storage of the polarized solid.

Hyperpolarized magnetic resonance allows for noninvasive measurements of biochemical reactions in vivo. Although this technique provides a unique tool for assaying enzymatic activities in intact organs, the scope of its application is still elusive for the wider scientific community. The purpose of this review is to provide key principles and parameters to guide the researcher interested in adopting this technology to address a biochemical, biomedical, or medical issue. It is presented in the form of a compendium containing the underlying essential physical concepts as well as suggestions to help assess the potential of the technique within the framework of specific research environments. Explicit examples are used to illustrate the power as well as the limitations of hyperpolarized magnetic resonance.