Probing hepatic metabolism of [2-13C]dihydroxyacetone in vivo with 1H-decoupled hyperpolarized 13C-MR.

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.

Measuring Tumor Glycolytic Flux in Vivo by Using Fast Deuterium MRI.

Background Tumor cells frequently show high rates of aerobic glycolysis, which provides the glycolytic intermediates needed for the increased biosynthetic demands of rapid cell growth and proliferation. Existing clinical methods (fluorodeoxyglucose PET and carbon 13 MRI and spectroscopy) do not allow quantitative images of glycolytic flux. Purpose To evaluate the use of deuterium (hydrogen 2 [2H]) MR spectroscopic imaging for quantitative mapping of tumor glycolytic flux and to assess response to chemotherapy. Materials and Methods A fast three-dimensional 2H MR spectroscopic imaging pulse sequence, with a time resolution of 10 minutes, was used to image glycolytic flux in a murine tumor model after bolus injection of D-[6,6'-2H2]glucose before and 48 hours after treatment with a chemotherapeutic agent. Tumor lactate labeling, expressed as the lactate-to-water and lactate-to-glucose signal ratios, was also assessed in localized 2H MR spectra. Statistical significance was tested with a one-sided paired t test. Results 2H MR spectroscopic imaging showed heterogeneity in glycolytic flux across the tumor and an early decrease in flux following treatment with a chemotherapeutic drug. Spectroscopy measurements on five animals showed a decrease in the lactate-to-water signal ratio, from 0.33 ± 0.10 to 0.089 ± 0.039 (P = .005), and in the lactate-to-glucose ratio, from 0.27 ± 0.12 to 0.12 ± 0.06 (P = .04), following drug treatment. Conclusion Rapidly acquired deuterium (hydrogen 2) MR spectroscopic images can provide quantitative and spatially resolved measurements of glycolytic flux in tumors that can be used to assess treatment response. Published under a CC BY 4.0 license. Online supplemental material is available for this article. See also the editorial by Ouwerkerk in this issue.

Assessing Oxidative Stress in Tumors by Measuring the Rate of Hyperpolarized [1-13C]Dehydroascorbic Acid Reduction Using 13C Magnetic Resonance Spectroscopy.

Rapid cancer cell proliferation promotes the production of reducing equivalents, which counteract the effects of relatively high levels of reactive oxygen species. Reactive oxygen species levels increase in response to chemotherapy and cell death, whereas an increase in antioxidant capacity can confer resistance to chemotherapy and is associated with an aggressive tumor phenotype. The pentose phosphate pathway is a major site of NADPH production in the cell, which is used to maintain the main intracellular antioxidant, glutathione, in its reduced state. Previous studies have shown that the rate of hyperpolarized [1-13C]dehydroascorbic acid (DHA) reduction, which can be measured in vivo using non-invasive 13C magnetic resonance spectroscopic imaging, is increased in tumors and that this is correlated with the levels of reduced glutathione. We show here that the rate of hyperpolarized [1-13C]DHA reduction is increased in tumors that have been oxidatively prestressed by depleting the glutathione pool by buthionine sulfoximine treatment. This increase was associated with a corresponding increase in pentose phosphate pathway flux, assessed using 13C-labeled glucose, and an increase in glutaredoxin activity, which catalyzes the glutathione-dependent reduction of DHA. These results show that the rate of DHA reduction depends not only on the level of reduced glutathione, but also on the rate of NADPH production, contradicting the conclusions of some previous studies. Hyperpolarized [1-13C]DHA can be used, therefore, to assess the capacity of tumor cells to resist oxidative stress in vivo However, DHA administration resulted in transient respiratory arrest and cardiac depression, which may prevent translation to the clinic.

A referenceless Nyquist ghost correction workflow for echo planar imaging of hyperpolarized [1-13 C]pyruvate and [1-13 C]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 13 C-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 13 C EPI that requires no reference scan. This involves the selection of a ghost-containing background from a 13 C 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 13 C 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 13 C reference echoes (>15%) or better ghost removal relative to a 1 H reference scan. The residual ghosting in a slightly de-shimmed B0 field was 1.6% using the proposed workflow and 3.8% using a 1 H reference scan. The workflow was implemented with a series of dynamically acquired hyperpolarized [1-13 C]pyruvate and [1-13 C]lactate images in vivo, resulting in images with no observable ghosting and which were quantitatively similar to images corrected using a 13 C reference scan.

Increased hyperpolarized [1-13 C] lactate production in a model of joint inflammation is not accompanied by tissue acidosis as assessed using hyperpolarized 13 C-labelled bicarbonate.

Arthritic conditions are a major source of chronic pain. Furthering our understanding of disease mechanisms creates the opportunity to develop more targeted therapeutics. In rheumatoid arthritis (RA), measurements of pH in human synovial fluid suggest that acidosis occurs, but that this is highly variable between individuals. Here we sought to determine if tissue acidosis occurs in a widely used rodent arthritis model: complete Freund's adjuvant (CFA)-induced inflammation. CFA robustly evoked paw and ankle swelling, concomitant with worsening clinical scores over time. We used magnetic resonance spectroscopic imaging of hyperpolarized [1-13 C]pyruvate metabolism to demonstrate that CFA induces an increase in the lactate-to-pyruvate ratio. This increase is indicative of enhanced glycolysis and an increased lactate concentration, as has been observed in the synovial fluid from RA patients, and which was correlated with acidosis. We also measured the 13 CO2 /H13 CO3- ratio, in animals injected with hyperpolarized H13 CO3- , to estimate extracellular tissue pH and showed that despite the apparent increase in glycolytic activity in CFA-induced inflammation there was no accompanying decrease in extracellular pH. The pH was 7.23 ± 0.06 in control paws and 7.32 ± 0.09 in inflamed paws. These results could explain why mice lacking acid-sensing ion channel subunits 1, 2 and 3 do not display any changes in mechanical or thermal hyperalgesia in CFA-induced inflammation.

Single shot three-dimensional pulse sequence for hyperpolarized 13 C MRI.

PURPOSE: Metabolic imaging with hyperpolarized 13 C-labeled cell substrates is a promising technique for imaging tissue metabolism in vivo. However, the transient nature of the hyperpolarization, and its depletion following excitation, limits the imaging time and the number of excitation pulses that can be used. We describe here a single-shot three-dimensional (3D) imaging sequence and demonstrate its capability to generate 13 C MR images in tumor-bearing mice injected with hyperpolarized [1-13 C]pyruvate. METHODS: The pulse sequence acquires a stack-of-spirals at two spin echoes after a single excitation pulse and encodes the kz-dimension in an interleaved manner to enhance robustness to B0 inhomogeneity. Spectral-spatial pulses are used to acquire dynamic 3D images from selected hyperpolarized 13 C-labeled metabolites. RESULTS: A nominal spatial/temporal resolution of 1.25 × 1.25 × 2.5 mm3 × 2 s was achieved in tumor images of hyperpolarized [1-13 C]pyruvate and [1-13 C]lactate acquired in vivo. Higher resolution in the z-direction, with a different k-space trajectory, was demonstrated in measurements on a thermally polarized [1-13 C]lactate phantom. CONCLUSION: The pulse sequence is capable of imaging hyperpolarized 13 C-labeled substrates at relatively high spatial and temporal resolutions and is robust to moderate system imperfections. Magn Reson Med 77:740-752, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Dual-modality gene reporter for in vivo imaging.

The ability to track cells and their patterns of gene expression in living organisms can increase our understanding of tissue development and disease. Gene reporters for bioluminescence, fluorescence, radionuclide, and magnetic resonance imaging (MRI) have been described but these suffer variously from limited depth penetration, spatial resolution, and sensitivity. We describe here a gene reporter, based on the organic anion transporting protein Oatp1a1, which mediates uptake of a clinically approved, Gd(3+)-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid). Cells expressing the reporter showed readily reversible, intense, and positive contrast (up to 7.8-fold signal enhancement) in T1-weighted magnetic resonance images acquired in vivo. The maximum signal enhancement obtained so far is more than double that produced by MRI gene reporters described previously. Exchanging the Gd(3+) ion for the radionuclide, (111)In, also allowed detection by single-photon emission computed tomography, thus combining the spatial resolution of MRI with the sensitivity of radionuclide imaging.

(13) C magnetic resonance spectroscopy measurements with hyperpolarized [1-(13) C] pyruvate can be used to detect the expression of transgenic pyruvate decarboxylase activity in vivo.

PURPOSE: Dissolution dynamic nuclear polarization can increase the sensitivity of the (13) C magnetic resonance spectroscopy experiment by at least four orders of magnitude and offers a novel approach to the development of MRI gene reporters based on enzymes that metabolize (13) C-labeled tracers. We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC 4.1.1.1), which catalyzes the decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide. METHODS: Pyruvate decarboxylase from Zymomonas mobilis (zmPDC) and a mutant that lacked enzyme activity were expressed using an inducible promoter in human embryonic kidney (HEK293T) cells. Enzyme activity was measured in the cells and in xenografts derived from the cells using (13) C MRS measurements of the conversion of hyperpolarized [1-(13) C] pyruvate to H(13) CO3-. RESULTS: Induction of zmPDC expression in the cells and in the xenografts derived from them resulted in an approximately two-fold increase in the H(13) CO3-/[1-(13) C] pyruvate signal ratio following intravenous injection of hyperpolarized [1-(13) C] pyruvate. CONCLUSION: We have demonstrated the feasibility of using zmPDC as an in vivo reporter gene for use with hyperpolarized (13) C MRS. Magn Reson Med 76:391-401, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging.

Glycosylation is a ubiquitous post-translational modification, present in over 50% of the proteins in the human genome, with important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases, including cancer. We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal MRI probe. Significant N-azidoacetylgalactosamine dependent T1  contrast was observed in vivo two hours after probe administration. Tumor, kidney, and liver showed significant contrast, and several other tissues, including the pancreas, spleen, heart, and intestines, showed a very high contrast (>10-fold). This approach has the potential to enable the rapid and non-invasive magnetic resonance imaging of glycosylated tissues in vivo in preclinical models of disease.

Hyperpolarized [U-(2) H, U-(13) C]Glucose reports on glycolytic and pentose phosphate pathway activity in EL4 tumors and glycolytic activity in yeast cells.

PURPOSE: A resonance at ∼181 ppm in the (13) C spectra of tumors injected with hyperpolarized [U-(2) H, U-(13) C]glucose was assigned to 6-phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3-phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG (13) C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) METHODS: Tumor-bearing mice were injected with (13) C6 glucose and the (13) C-labeled and total 6PG and 3PG concentrations measured. (13) C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-(2) H, U-(13) C]glucose and again (13) C-labeled and total 6PG and 3PG were measured by LC-MS/MS RESULTS: Tumor (13) C-6PG was more abundant than (13) C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG. (13) C MR spectra of wild-type and zwf1Δ yeast cells showed a resonance at ∼181 ppm after labeling with hyperpolarized [U-(2) H, U-(13) C]glucose, however, there was no 6PG in zwf1Δ cells. In the wild-type cells 3PG was approximately four-fold more abundant than 6PG CONCLUSION: The resonance at ∼181 ppm in (13) C MR spectra following injection of hyperpolarized [U-(2) H, U-(13) C]glucose originates predominantly from 6PG in EL4 tumors and 3PG in yeast cells.

Magnetic resonance imaging with hyperpolarized [1,4-(13)C2]fumarate allows detection of early renal acute tubular necrosis.

Acute kidney injury (AKI) is a common and important medical problem, affecting 10% of hospitalized patients, and it is associated with significant morbidity and mortality. The most frequent cause of AKI is acute tubular necrosis (ATN). Current imaging techniques and biomarkers do not allow ATN to be reliably differentiated from important differential diagnoses, such as acute glomerulonephritis (GN). We investigated whether (13)C magnetic resonance spectroscopic imaging (MRSI) might allow the noninvasive diagnosis of ATN. (13)C MRSI of hyperpolarized [1,4-(13)C(2)]fumarate and pyruvate was used in murine models of ATN and acute GN (NZM2410 mice with lupus nephritis). A significant increase in [1,4-(13)C(2)]malate signal was identified in the kidneys of mice with ATN early in the disease course before the onset of severe histological changes. No such increase in renal [1,4-(13)C(2)]malate was observed in mice with acute GN. The kidney [1-(13)C]pyruvate/[1-(13)C]lactate ratio showed substantial variability and was not significantly decreased in animals with ATN or increased in animals with GN. In conclusion, MRSI of hyperpolarized [1,4-(13)C(2)]fumarate allows the detection of early tubular necrosis and its distinction from glomerular inflammation in murine models. This technique may have the potential to identify a window of therapeutic opportunity in which emerging therapies might be applied to patients with ATN, reducing the need for acute dialysis with its attendant morbidity and cost.

Analysis of image heterogeneity using 2D Minkowski functionals detects tumor responses to treatment.

PURPOSE: The acquisition of ever increasing volumes of high resolution magnetic resonance imaging (MRI) data has created an urgent need to develop automated and objective image analysis algorithms that can assist in determining tumor margins, diagnosing tumor stage, and detecting treatment response. METHODS: We have shown previously that Minkowski functionals, which are precise morphological and structural descriptors of image heterogeneity, can be used to enhance the detection, in T1 -weighted images, of a targeted Gd(3+) -chelate-based contrast agent for detecting tumor cell death. We have used Minkowski functionals here to characterize heterogeneity in T2 -weighted images acquired before and after drug treatment, and obtained without contrast agent administration. RESULTS: We show that Minkowski functionals can be used to characterize the changes in image heterogeneity that accompany treatment of tumors with a vascular disrupting agent, combretastatin A4-phosphate, and with a cytotoxic drug, etoposide. CONCLUSIONS: Parameterizing changes in the heterogeneity of T2 -weighted images can be used to detect early responses of tumors to drug treatment, even when there is no change in tumor size. The approach provides a quantitative and therefore objective assessment of treatment response that could be used with other types of MR image and also with other imaging modalities.

Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy.

Measurements of early tumor responses to therapy have been shown, in some cases, to predict treatment outcome. We show in lymphoma-bearing mice injected intravenously with hyperpolarized [1-(13)C]pyruvate that the lactate dehydrogenase-catalyzed flux of (13)C label between the carboxyl groups of pyruvate and lactate in the tumor can be measured using (13)C magnetic resonance spectroscopy and spectroscopic imaging, and that this flux is inhibited within 24 h of chemotherapy. The reduction in the measured flux after drug treatment and the induction of tumor cell death can be explained by loss of the coenzyme NAD(H) and decreases in concentrations of lactate and enzyme in the tumors. The technique could provide a new way to assess tumor responses to treatment in the clinic.

Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate.

As alterations in tissue pH underlie many pathological processes, the capability to image tissue pH in the clinic could offer new ways of detecting disease and response to treatment. Dynamic nuclear polarization is an emerging technique for substantially increasing the sensitivity of magnetic resonance imaging experiments. Here we show that tissue pH can be imaged in vivo from the ratio of the signal intensities of hyperpolarized bicarbonate (H(13)CO(3)(-)) and (13)CO(2) following intravenous injection of hyperpolarized H(13)CO(3)(-). The technique was demonstrated in a mouse tumour model, which showed that the average tumour interstitial pH was significantly lower than the surrounding tissue. Given that bicarbonate is an endogenous molecule that can be infused in relatively high concentrations into patients, we propose that this technique could be used clinically to image pathological processes that are associated with alterations in tissue pH, such as cancer, ischaemia and inflammation.

Spin echo measurements of the extravasation and tumor cell uptake of hyperpolarized [1-(13) C]lactate and [1-(13) C]pyruvate.

PURPOSE: To assess the blood-tissue distribution of hyperpolarized (13) C-labeled molecules in vivo. METHODS: Spin-echo experiments with simultaneous acquisition of the free induction decay (FID) signal following the excitation pulse and the spin-echo signal, were used to monitor hyperpolarized [1-(13) C]lactate, [1-(13) C]pyruvate, and the perfusion marker, [(13) C]HP001, following their intravenous injection into tumor-bearing mice. Apparent T2 relaxation times and diffusion coefficients were also measured. RESULTS: An increasing tumor echo/FID ratio was observed for all three molecules, which could be explained by their extravasation into the tumor interstitial space, where T2 relaxation times were longer and diffusion coefficients smaller. Inhibition of the monocarboxylate transporter, which decreased by 40% the label exchange between pyruvate and lactate, reduced the increase in the echo/FID ratio for pyruvate and lactate, but not for HP001, demonstrating that some of the increase in the echo/FID ratio was due to cell uptake of lactate and pyruvate. The different relaxation and diffusion behavior of the intravascular and extravascular signals affected measurements of the apparent label exchange rate constants. CONCLUSION: Simultaneous collection of both FID and echo signals can provide information on cell uptake thus giving further insight into the kinetics of hyperpolarized (13) C label exchange. Care is needed when comparing exchange rate constants determined in spin-echo-based studies.

Probing lactate dehydrogenase activity in tumors by measuring hydrogen/deuterium exchange in hyperpolarized l-[1-(13)C,U-(2)H]lactate.

(13)C magnetic resonance spectroscopy and spectroscopic imaging measurements of hyperpolarized (13)C label exchange between exogenously administered [1-(13)C]pyruvate and endogenous lactate, catalyzed by lactate dehydrogenase (LDH), has proved to be a powerful approach for probing tissue metabolism in vivo. This experiment has clinical potential, particularly in oncology, where it could be used to assess tumor grade and response to treatment. A limitation of the method is that pyruvate must be administered in vivo at supra-physiological concentrations. This problem can be avoided by using hyperpolarized [1-(13)C]lactate, which can be used at physiological concentrations. However, sensitivity is limited in this case by the relatively small pyruvate pool size, which would result in only low levels of labeled pyruvate being observed even if there was complete label equilibration between the lactate and pyruvate pools. We demonstrate here a more sensitive method in which a doubly labeled lactate species can be used to measure LDH-catalyzed exchange in vivo. In this experiment exchange of the C2 deuterium label between injected hyperpolarized l-[1-(13)C,U-(2)H]lactate and endogenous unlabeled lactate is observed indirectly by monitoring phase modulation of the spin-coupled hyperpolarized (13)C signal in a heteronuclear (1)H/(13)C spin-echo experiment.

Production of hyperpolarized [1,4-13C2]malate from [1,4-13C2]fumarate is a marker of cell necrosis and treatment response in tumors.

Dynamic nuclear polarization of (13)C-labeled cell substrates has been shown to massively increase their sensitivity to detection in NMR experiments. The sensitivity gain is sufficiently large that if these polarized molecules are injected intravenously, their spatial distribution and subsequent conversion into other cell metabolites can be imaged. We have used this method to image the conversion of fumarate to malate in a murine lymphoma tumor in vivo after i.v. injection of hyperpolarized [1,4-(13)C(2)]fumarate. In isolated lymphoma cells, the rate of labeled malate production was unaffected by coadministration of succinate, which competes with fumarate for transport into the cell. There was, however, a correlation with the percentage of cells that had lost plasma membrane integrity, suggesting that the production of labeled malate from fumarate is a sensitive marker of cellular necrosis. Twenty-four hours after treating implanted lymphoma tumors with etoposide, at which point there were significant levels of tumor cell necrosis, there was a 2.4-fold increase in hyperpolarized [1,4-(13)C(2)]malate production compared with the untreated tumors. Therefore, the formation of hyperpolarized (13)C-labeled malate from [1,4-(13)C(2)]fumarate appears to be a sensitive marker of tumor cell death in vivo and could be used to detect the early response of tumors to treatment. Given that fumarate is an endogenous molecule, this technique has the potential to be used clinically.

Hyperpolarized [1-13C]-ascorbic and dehydroascorbic acid: vitamin C as a probe for imaging redox status in vivo.

Dynamic nuclear polarization (DNP) of (13)C-labeled metabolic substrates in vitro and their subsequent intravenous administration allow both the location of the hyperpolarized substrate and the dynamics of its subsequent conversion into other metabolic products to be detected in vivo. We report here the hyperpolarization of [1-(13)C]-ascorbic acid (AA) and [1-(13)C]-dehydroascorbic acid (DHA), the reduced and oxidized forms of vitamin C, respectively, and evaluate their performance as probes of tumor redox state. Solution-state polarization of 10.5 ± 1.3% was achieved for both forms at pH 3.2, whereas at pH 7.0, [1-(13)C]-AA retained polarization of 5.1 ± 0.6% and [1-(13)C]-DHA retained 8.2 ± 1.1%. The spin-lattice relaxation times (T(1)'s) for these labeled nuclei are long at 9.4 T: 15.9 ± 0.7 s for AA and 20.5 ± 0.9 s for DHA. Extracellular oxidation of [1-(13)C]-AA and intracellular reduction of [1-(13)C]-DHA were observed in suspensions of murine lymphoma cells. The spontaneous reaction of DHA with the cellular antioxidant glutathione was monitored in vitro and was approximately 100-fold lower than the rate observed in cell suspensions, indicating enzymatic involvement in the intracellular reduction. [1-(13)C]-DHA reduction was also detected in lymphoma tumors in vivo. In contrast, no detectable oxidation of [1-(13)C]-AA was measured in the same tumors, consistent with the notion that tumors maintain a reduced microenvironment. This study demonstrates that hyperpolarized (13)C-labeled vitamin C could be used as a noninvasive biomarker of redox status in vivo, which has the potential to translate to the clinic.

Detection of tumor glutamate metabolism in vivo using (13)C magnetic resonance spectroscopy and hyperpolarized [1-(13)C]glutamate.

Dynamic nuclear polarization can be used to increase the sensitivity of solution state (13)C magnetic resonance spectroscopy by four orders of magnitude. We show here that [1-(13)C]glutamate can be polarized to 28%, representing a 35,000-fold increase in its sensitivity to detection at 9.4 T and 37°C. The metabolism of hyperpolarized glutamate to α-ketoglutarate, catalyzed by the enzyme alanine transaminase, was detected in vitro in human hepatoma cells (HepG2). Incubation of the cells with sodium pyruvate increased the level of the hyperpolarized label in the α-ketoglutarate pool, with an associated increase in the apparent rate constant describing flux of hyperpolarized (13)C label between glutamate and α-ketoglutarate. The metabolism of hyperpolarized glutamate was observed in vivo following coadministration of pyruvate in a murine lymphoma model. This represents a new method to probe glutamate metabolism and citric acid cycle activity in vivo; as glutamate is an endogenous molecule, it has the potential to be used in the clinic.