In vivo measurement of normal rat intracellular pyruvate and lactate levels after injection of hyperpolarized [1-(13)C]alanine

Hyperpolarized technology utilizing dynamic nuclear polarization has enabled rapid and high-sensitivity measurements of ¹³C metabolism in vivo. The most commonly used in vivo agent for hyperpolarized ¹³C metabolic imaging thus far has been [1-¹³C]pyruvate. In preclinical studies, not only is its uptake detected, but also its intracellular enzymatic conversion to metabolic products including [1-¹³C]lactate and [1-¹³C]alanine. However, the ratio of ¹³C-lactate/¹³C-pyruvate measured in this data does not accurately reflect cellular values since much of the [1-¹³C]pyruvate is extracellular depending on timing, vascular properties, and extracellular space and monocarboxylate transporter activity. In order to measure the relative levels of intracellular pyruvate and lactate, in this project we hyperpolarized [1-¹³C]alanine and monitored the in vivo conversion to [1-¹³C]pyruvate and then the subsequent conversion to [1-¹³C]lactate. The intracellular lactate-to-pyruvate ratio of normal rat tissue measured with hyperpolarized [1-¹³C]alanine was 4.89±0.61 (mean±S.E.) as opposed to a ratio of 0.41±0.03 when hyperpolarized [1-¹³C]pyruvate was injected.     

Feasibility of using hyperpolarized [1-13C]lactate as a substrate for in vivo metabolic 13C MRSI studies

The development of dynamic nuclear polarization in solution has enabled in vivo 13C MR studies at high signal-to-noise ratio following injection of prepolarized 13C substrates. While prior studies have demonstrated the ability to observe metabolism following injection of hyperpolarized 13C pyruvate, the goal of this study was to develop and test a new hyperpolarized agent for investigating in vivo metabolism, [1-13C]lactate. A preparation for prepolarized 13C lactate and the requisite dissolution media were developed to investigate the feasibility for in vivo 13C MRS/MRSI studies following injection of this hyperpolarized agent. This study demonstrated, for the first time, not only the ability to detect hyperpolarized [1-13C]lactate in vivo but also the metabolic products 13C pyruvate, 13C alanine and 13C bicarbonate following injection in normal rats. The use of 13C lactate as a substrate provided the opportunity to study the conversion of lactate to pyruvate in vivo and to detect the secondary conversions to alanine and bicarbonate through pyruvate. This study also demonstrated the potential value of this hyperpolarized agent to investigate in vivo lactate uptake and metabolism in preclinical animal models.     

Frequency-specific SSFP for hyperpolarized C metabolic imaging at 14.1 T

Metabolic imaging of hyperpolarized [1-¹³C] pyruvate co-polarized with [¹³C]urea by dynamic nuclear polarization with rapid dissolution is a promising new method for assessing tumor metabolism and perfusion simultaneously in vivo. Novel pulse sequences are required to enable dynamic imaging of multiple ¹³C spectral lines with high spatiotemporal resolution. The goal of this study was to investigate a new frequency-specific approach for rapid metabolic imaging of multiple ¹³C resonances using the spectral selectivity of steady-state free precession pulse (SSFP) trains. Methods developed in simulations were implemented in a dynamic frequency-cycled balanced SSFP pulse sequence on a 14.1-T animal magnetic resonance imaging scanner. This acquisition was tested in thermal and hyperpolarized phantom imaging studies and in a transgenic mouse with prostate cancer.     

Investigating tumor perfusion and metabolism using multiple hyperpolarized (13)C compounds: HP001, pyruvate and urea

The metabolically inactive hyperpolarized agents HP001 (bis-1,1-(hydroxymethyl)-[1-(13)C]cyclopropane-d(8)) and urea enable a new type of perfusion magnetic resonance imaging based on a direct signal source that is background-free. The addition of perfusion information to metabolic information obtained by spectroscopic imaging of hyperpolarized [1-(13)C]pyruvate would be of great value in exploring the relationship between perfusion and metabolism in cancer. In preclinical normal murine and cancer model studies, we performed both dynamic multislice imaging of the specialized hyperpolarized perfusion compound HP001 (T(1)=95 s ex vivo, 32 s in vivo at 3 T) using a pulse sequence with balanced steady-state free precession and ramped flip angle over time for efficient utilization of the hyperpolarized magnetization and three-dimensional echo-planar spectroscopic imaging of urea copolarized with [1-(13)C]pyruvate, with compressed sensing for resolution enhancement. For the dynamic data, peak signal maps and blood flow maps derived from perfusion modeling were generated. The spatial heterogeneity of perfusion was increased 2.9-fold in tumor tissues (P=.05), and slower washout was observed in the dynamic data. The results of separate dynamic HP001 imaging and copolarized pyruvate/urea imaging were compared. A strong and significant correlation (R=0.73, P=.02) detected between the urea and HP001 data confirmed the value of copolarizing urea with pyruvate for simultaneous assessment of perfusion and metabolism.     

Development of high resolution 3D hyperpolarized carbon-13 MR molecular imaging techniques

The goal of this project was to develop and apply techniques for T₂ mapping and 3D high resolution (1.5 mm isotropic; 0.003 cm³) ¹³C imaging of hyperpolarized (HP) probes [1-¹³C]lactate, [1-¹³C]pyruvate, [2-¹³C]pyruvate, and [¹³C,¹⁵N₂]urea in vivo. A specialized 2D bSSFP sequence was implemented on a clinical 3T scanner and used to obtain the first high resolution T₂ maps of these different hyperpolarized compounds in both rats and tumor-bearing mice. These maps were first used to optimize timings for highest SNR for single time-point 3D bSSFP acquisitions with a 1.5 mm isotropic spatial resolution of normal rats. This 3D acquisition approach was extended to serial dynamic imaging with 2-fold compressed sensing acceleration without changing spatial resolution. The T₂ mapping experiments yielded measurements of T₂ values of > 1 s for all compounds within rat kidneys/vasculature and TRAMP tumors, except for [2-¹³C]pyruvate which was ~ 730 ms and ~ 320 ms, respectively. The high resolution 3D imaging enabled visualization the biodistribution of [1-¹³C]lactate, [1-¹³C]pyruvate, and [2-¹³C]pyruvate within different kidney compartments as well as in the vasculature. While the mouse anatomy is smaller, the resolution was also sufficient to image the distribution of all compounds within kidney, vasculature, and tumor. The development of the specialized 3D sequence with compressed sensing provided improved structural and functional assessments at a high (0.003 cm³) spatial and 2 s temporal resolution in vivo utilizing HP ¹³C substrates by exploiting their long T₂ values. This 1.5 mm isotropic resolution is comparable to ¹H imaging and application of this approach could be extended to future studies of uptake, metabolism, and perfusion in cancer and other disease models and may ultimately be of value for clinical imaging.     

Perfusion and diffusion sensitive C stimulated-echo MRSI for metabolic imaging of cancer

Metabolic imaging with hyperpolarized [1-¹³C]-pyruvate can rapidly probe tissue metabolic profiles in vivo and has been shown to provide cancer imaging biomarkers for tumor detection, progression, and response to therapy. This technique uses a bolus injection followed by imaging within 1–2 minutes. The observed metabolites include vascular components and their generation is also influenced by cellular transport. These factors complicate image interpretation, especially since [1-¹³C]lactate, a metabolic product that is a biomarker of cancer, is also produced by red blood cells. It would be valuable to understand the distribution of metabolites between the vasculature, interstitial space, and intracellular compartments. The purpose of this study was to better understand this compartmentalization by using a perfusion and diffusion-sensitive stimulated-echo acquisition mode (STEAM) MRSI acquisition method tailored to hyperpolarized substrates. Our results in mouse models showed that among metabolites, the injected substrate ¹³C-pyruvate had the largest vascular fraction overall while ¹³C-alanine had the smallest vascular fraction. We observed a larger vascular fraction of pyruvate and lactate in the kidneys and liver when compared to back muscle and prostate tumor tissue. Our data suggests that ¹³C-lactate in prostate tumor tissue voxels was the most abundant labeled metabolite intracellularly. This was shown in STEAM images that highlighted abnormal cancer cell metabolism and suppressed vascular ¹³C metabolite signals.     

Multi-channel metabolic imaging, with SENSE reconstruction, of hyperpolarized [1-(13)C] pyruvate in a live rat at 3.0 tesla on a clinical MR scanner

We report metabolic images of (13)C, following injection of a bolus of hyperpolarized [1-(13)C] pyruvate in a live rat. The data were acquired on a clinical scanner, using custom coils for volume transmission and array reception. Proton blocking of all carbon resonators enabled proton anatomic imaging with the system body coil, to allow for registration of anatomic and metabolic images, for which good correlation was achieved, with some anatomic features (kidney and heart) clearly visible in a carbon image, without reference to the corresponding proton image. Parallel imaging with sensitivity encoding was used to increase the spatial resolution in the SI direction of the rat. The signal to noise ratio in was in some instances unexpectedly high in the parallel images; variability of the polarization among different trials, plus partial volume effects, are noted as a possible cause of this.     

Cancer recurrence monitoring using hyperpolarized [1-13C]pyruvate metabolic imaging in murine breast cancer model

The purpose of this work was to study the anatomic and metabolic changes that occur with tumor progression, regression and recurrence in a switchable MYC-driven murine breast cancer model. Serial ¹H MRI and hyperpolarized [1-¹³C]pyruvate metabolic imaging were used to investigate the changes in tumor volume and glycolytic metabolism over time during the multistage tumorigenesis. We show that acute de-induction of MYC expression in established tumors results in rapid tumor regression and significantly reduced glycolytic metabolism as measured by pyruvate-to-lactate conversion. Moreover, cancer recurrences occurring at the tumor sites independently of MYC expression were observed to accompany markedly increased lactate production.     

Analysis of hyperpolarized dynamic C lactate imaging in a transgenic mouse model of prostate cancer

This study investigated the application of an acquisition that selectively excites the [1-¹³C]lactate resonance and allows dynamic tracking of the conversion of ¹³C-lactate from hyperpolarized ¹³C-pyruvate at a high spatial resolution. In order to characterize metabolic processes occurring in a mouse model of prostate cancer, 20 sequential 3D images of ¹³C-lactate were acquired 5 s apart using a pulse sequence that incorporated a spectral–spatial excitation pulse and a flyback echo-planar readout to track the time course of newly converted ¹³C-lactate after injection of prepolarized ¹³C-pyruvate. The maximum lactate signal (MLS), full-width half-maximum (FWHM), time to the peak ¹³C-lactate signal (TTP) and area under the dynamic curve were calculated from the dynamic images of 10 TRAMP mice and two wild-type controls. The regional variation in ¹³C-lactate associated with the injected pyruvate was demonstrated by the peak of the ¹³C-lactate signal occurring earlier in the kidney than in the tumor region. The intensity of the dynamic ¹³C-lactate curves also varied spatially within the tumor, illustrating the heterogeneity in metabolism that was most prominent in more advanced stages of disease development. The MLS was significantly higher in TRAMP mice that had advanced disease.     

Measuring the link between cardiac mechanical function and metabolism during hyperpolarized 13C-pyruvate magnetic resonance experiments

PurposeThe goal of this study was to develop a methodology to investigate the relationship between contractile function and hyperpolarized (HP) [1-¹³C]pyruvate metabolism in a small animal model. To achieve sufficient signal from HP ¹³C compounds, HP ¹³C MRS/MRSI has required relatively large infusion volumes relative to the total blood volume in small animal models, which may affect cardiac function.MethodsEight female Sprague Dawley rats were imaged on a 4.7T scanner with a dual tuned ¹H/¹³C volume coil. ECG and respiratory gated k-t spiral MRSI and an IDEAL based reconstruction to determine [1-¹³C]pyruvate metabolism in the myocardium. This was coupled with ¹H cine MRI to determine ventricular volumes and mechanical function pre- and post-infusion of [1-¹³C]pyruvate. For comparison to the [1-¹³C]pyruvate experiments, three female Sprague Dawley rats were imaged with ¹H cine MRI to determine myocardial function pre- and post-saline infusion.ResultsWe demonstrated significant changes in cardiac contractile function between pre- and post-infusion of [1-¹³C]pyruvate. Specifically, there was an increase in end-diastolic volume (EDV), stroke volume (SV), and ejection fraction (EF). Additionally, the ventricular vascular coupling ratio (VVCR) showed an improvement after [1-¹³C]pyruvate infusion, indicating increased systolic performance due to an increased arterial load. There was a moderate to strong relationship between the downstream metabolic conversion of pyruvate to bicarbonate and a strong relationship between the conversion of pyruvate to lactate and the cardiac mechanical function response.ConclusionThe infusion of [1-¹³C]pyruvate resulted in demonstrable increases in contractile function which was related to pyruvate conversion to bicarbonate and lactate. The combined effects of the infusion volume and inotropic effects of pyruvate metabolism likely explains the augmentation in myocardial mechanical function seen in these experiments. Given the relationship between pyruvate metabolism and contractile function observed in this study, this methodological approach may be utilized to better understand cardiac metabolic and functional remodeling in heart disease.     

Design of spectral-spatial outer volume suppression RF pulses for tissue specific metabolic characterization with hyperpolarized C pyruvate

[1-¹³C] pyruvate pre-polarized via DNP has been used in animal models to probe changes in metabolic enzyme activities in vivo. To more accurately assess the metabolic state and its change from disease progression or therapy in a specific region or tissue in vivo, it may be desirable to separate the downstream ¹³C metabolite signals resulting from the metabolic activity within the tissue of interest and those brought into the tissue by perfusion. In this study, a spectral-spatial saturation pulse that selectively saturates the signal from the metabolic products [1-¹³C] lactate and [1-¹³C] alanine was designed and implemented as outer volume suppression for localized MRSI acquisition. Preliminary in vivo results showed that the suppression pulse did not prevent the pre-polarized pyruvate from being delivered throughout the animal while it saturated the metabolites within the targeted saturation region.     

Rapid sequential injections of hyperpolarized [1-C]pyruvate in vivo using a sub-kelvin,multi-sample DNP polarizer

The development of hyperpolarized technology utilizing dynamic nuclear polarization (DNP) has enabled the rapid measurement of ¹³C metabolism in vivo with very high SNR. However, with traditional DNP equipment, consecutive injections of a hyperpolarized compound in an animal have been subject to a practical minimum time between injections governed by the polarization build-up time, which is on the order of an hour for [1-¹³C]pyruvate. This has precluded the monitoring of metabolic changes occurring on a faster time scale. In this study, we demonstrated the ability to acquire in vivo dynamic magnetic resonance spectroscopy (MRS) and 3D magnetic resonance spectroscopic imaging (MRSI) data in normal rats with a 5 min interval between injections of hyperpolarized [1-¹³C]pyruvate using a prototype, sub-Kelvin dynamic nuclear polarizer with the capability to simultaneously polarize up to 4 samples and dissolve them in rapid succession. There were minimal perturbations in the hyperpolarized spectra as a result of the multiple injections, suggesting that such an approach would not confound the investigation of metabolism occurring on this time scale. As an initial demonstration of the application of this technology and approach for monitoring rapid changes in metabolism as a result of a physiological intervention, we investigated the pharmacodynamics of the anti-cancer agent dichloroacetate (DCA), collecting hyperpolarized data before administration of DCA, 1 min after administration, and 6 min after administration. Dramatic increases in ¹³C-bicarbonate were detected just 1 min (as well as 6 min) after DCA administration.     

Multiband excitation pulses for hyperpolarized 13C dynamic chemical-shift imaging

Hyperpolarized 13C offers high signal-to-noise ratios for imaging metabolic activity in vivo, but care must be taken when designing pulse sequences because the magnetization cannot be recovered once it has decayed. It has a short lifetime, on the order of minutes, and gets used up by each RF excitation. In this paper, we present a new dynamic chemical-shift imaging method that uses specialized RF pulses designed to maintain most of the hyperpolarized substrate while providing adequate SNR for the metabolic products. These are multiband, variable flip angle, spectral-spatial RF pulses that use spectral selectivity to minimally excite the injected prepolarized 13C-pyruvate substrate. The metabolic products of lactate and alanine are excited with a larger flip angle to increase SNR. This excitation was followed by an RF amplitude insensitive double spin-echo and an echo-planar flyback spectral-spatial readout gradient. In vivo results in rats and mice are presented showing improvements over constant flip angle RF pulses. The metabolic products are observable for a longer window because the low pyruvate flip angle preserves magnetization, allowing for improved observation of spatially varying metabolic reactions.     

Fast volumetric spatial-spectral MR imaging of hyperpolarized C-labeled compounds using multiple echo 3D bSSFP

Purpose

The goal of this work was to develop a fast 3D chemical shift imaging technique for the noninvasive measurement of hyperpolarized ¹³C-labeled substrates and metabolic products at low concentration.

Materials and Methods

Multiple echo 3D balanced steady state magnetic resonance imaging (ME-3DbSSFP) was performed in vitro on a syringe containing hyperpolarized [1,3,3-2H3; 1-¹³C]2-hydroxyethylpropionate (HEP) adjacent to a ¹³C-enriched acetate phantom, and in vivo on a rat before and after intravenous injection of hyperpolarized HEP at 1.5 T. Chemical shift images of the hyperpolarized HEP were derived from the multiple echo data by Fourier transformation along the echoes on a voxel by voxel basis for each slice of the 3D data set.

Results

ME-3DbSSFP imaging was able to provide chemical shift images of hyperpolarized HEP in vitro, and in a rat with isotropic 7-mm spatial resolution, 93 Hz spectral resolution and 16-s temporal resolution for a period greater than 45 s.

Conclusion

Multiple echo 3D bSSFP imaging can provide chemical shift images of hyperpolarized ¹³C-labeled compounds in vivo with relatively high spatial resolution and moderate spectral resolution. The increased signal-to-noise ratio of this 3D technique will enable the detection of hyperpolarized ¹³C-labeled metabolites at lower concentrations as compared to a 2D technique.     

The feasibility of assessing branched-chain amino acid metabolism in cellular models of prostate cancer with hyperpolarized [1-C]-ketoisocaproate

Recent advancements in the field of hyperpolarized ¹³C magnetic resonance spectroscopy (MRS) have yielded powerful techniques capable of real-time analysis of metabolic pathways. These non-invasive methods have increasingly shown application in impacting disease diagnosis and have further been employed in mechanistic studies of disease onset and progression. Our goals were to investigate branched-chain aminotransferase (BCAT) activity in prostate cancer with a novel molecular probe, hyperpolarized [1-¹³C]-2-ketoisocaproate ([1-¹³C]-KIC), and explore the potential of branched-chain amino acid (BCAA) metabolism to serve as a biomarker. Using traditional spectrophotometric assays, BCAT enzymatic activities were determined in vitro for various sources of prostate cancer (human, transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse and human cell lines). These preliminary studies indicated that low levels of BCAT activity were present in all models of prostate cancer but enzymatic levels are altered significantly in prostate cancer relative to healthy tissue. The MR spectroscopic studies were conducted with two cellular models (PC-3 and DU-145) that exhibited levels of BCAA metabolism comparable to the human disease state. Hyperpolarized [1-¹³C]-KIC was administered to prostate cancer cell lines, and the conversion of [1-¹³C]-KIC to the metabolic product, [1-¹³C]-leucine ([1-¹³C]-Leu), could be monitored via hyperpolarized ¹³C MRS.     

Use of hyperpolarized [1-C]pyruvate and [2-C]pyruvate to probe the effects of the anticancer agent dichloroacetate on mitochondrial metabolism in vivo in the normal rat

Development of hyperpolarized technology utilizing dynamic nuclear polarization has enabled the measurement of ¹³C metabolism in vivo at very high signal-to-noise ratio (SNR). In vivo mitochondrial metabolism can, in principle, be monitored with pyruvate, which is catalyzed to acetyl-CoA via pyruvate dehydrogenase (PDH). The purpose of this work was to determine whether the compound sodium dichloroacetate (DCA) could aid the study of mitochondrial metabolism with hyperpolarized pyruvate. DCA stimulates PDH by inhibiting its inhibitor, pyruvate dehydrogenase kinase. In this work, hyperpolarized [1-¹³C]pyruvate and [2-¹³C]pyruvate were used to probe mitochondrial metabolism in normal rats. Increased conversion to bicarbonate (+ 181±69%, P=.025) was measured when [1-¹³C]pyruvate was injected after DCA administration, and increased glutamate (+ 74±23%, P=.004), acetoacetate (+ 504±281%, P=.009) and acetylcarnitine (+ 377±157%, P=.003) were detected when [2-¹³C]pyruvate was used.     

Transmit-Only/Receive-Only Radiofrequency System for Hyperpolarized 13C MRS Cardiac Metabolism Studies in Pigs

Hyperpolarized ¹³C magnetic resonance spectroscopy in pig models enables metabolic activity mapping, providing a powerful tool for the study of the heart physiology, but requires the development of dedicated radiofrequency coils, capable of providing large field of view with high signal-to-noise ratio (SNR) data. This work describes the simulations and the tests of a transmit-only (TX) volume coil/receive-only (RX) surface coil both designed for hyperpolarized studies of pig heart with a clinical 3T scanner. The coil characterization is performed by developing an SNR model for coil performance in terms of coil resistance, sample-induced resistance and magnetic field pattern. In particular, coil resistances were calculated from Ohm’s law, while magnetic field patterns and sample-induced resistances were calculated using a numerical finite-difference time-domain algorithm. Experimental phantom chemical shift image, showed good agreement with the theoretical SNR-vs-depth profiles and highlighted the advantage of the novel configuration over the single transmit–receive coils throughout the volume of interest for cardiac imaging in pig. Finally, the TX-birdcage/RX-circular configuration was tested by acquiring metabolic maps with hyperpolarized [1-13C] pyruvate injected i.v. in a pig. The results of the phantom and pig experiments show the ability of the coil configuration to image well the metabolites distribution.     

Double spin-echo sequence for rapid spectroscopic imaging of hyperpolarized 13C

Dynamic nuclear polarization of metabolically active compounds labeled with (13)C has been introduced as a means for imaging metabolic processes in vivo. To differentiate between the injected compound and the various metabolic products, an imaging technique capable of separating the different chemical-shift species must be used. In this paper, the design and testing of a pulse sequence for rapid magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized (13)C is presented. The pulse sequence consists of a small-tip excitation followed by a double spin echo using adiabatic refocusing pulses and a "flyback" echo-planar readout gradient. Key elements of the sequence are insensitivity to calibration of the transmit gain, the formation of a spin echo giving high-quality spectral information, and a small effective tip angle that preserves the magnetization for a sufficient duration. Experiments in vivo showed three-dimensional coverage with excellent spectral quality and SNR.     

Hyperpolarized 13C MRS Cardiac Metabolism Studies in Pigs: Comparison Between Surface and Volume Radiofrequency Coils

Cardiac metabolism assessment with hyperpolarized ¹³C magnetic resonance spectroscopy in pig models requires the design of dedicated coils capable of providing large field of view with high signal-to-noise ratio (SNR) data. This work presents a comparison between a commercial ¹³C quadrature birdcage coil and a homebuilt ¹³C circular coil both designed for hyperpolarized studies of pig heart with a clinical 3T scanner. In particular, the simulation of the two coils is described by developing an SNR model for coil performance prediction and comparison. While coil resistances were calculated from Ohm’s law, the magnetic field patterns and sample-induced resistances were calculated using a numerical finite-difference time-domain algorithm. After the numerical simulation of both coils, the results are presented as SNR-versus-depth profiles using experimental SNR extracted from the [1-¹³C]acetate phantom chemical shift image and with a comparison of metabolic maps acquired by hyperpolarized [1-¹³C]pyruvate injected in a pig. The accuracy of the developed SNR models was demonstrated by good agreement between the theoretical and experimental coil SNR-versus-depth profiles.     

Towards hyperpolarized (13)C-succinate imaging of brain cancer

We describe a novel (13)C enriched precursor molecule, sodium 1-(13)C acetylenedicarboxylate, which after hydrogenation by PASADENA (Parahydrogen and Synthesis Allows Dramatically Enhanced Nuclear Alignment) under controlled experimental conditions, becomes hyperpolarized (13)C sodium succinate. Fast in vivo 3D FIESTA MR imaging demonstrated that, following carotid arterial injection, the hyperpolarized (13)C-succinate appeared in the head and cerebral circulation of normal and tumor-bearing rats. At this time, no in vivo hyperpolarized signal has been localized to normal brain or brain tumor. On the other hand, ex vivo samples of brain harvested from rats bearing a 9L brain tumor, 1 h or more following in vivo carotid injection of hyperpolarized (13)C sodium succinate, contained significant concentrations of the injected substrate, (13)C sodium succinate, together with (13)C maleate and succinate metabolites 1-(13)C-glutamate, 5-(13)C-glutamate, 1-(13)C-glutamine and 5-(13)C-glutamine. The (13)C substrates and products were below the limits of NMR detection in ex vivo samples of normal brain consistent with an intact blood-brain barrier. These ex vivo results indicate that hyperpolarized (13)C sodium succinate may become a useful tool for rapid in vivo identification of brain tumors, providing novel biomarkers in (13)C MR spectral-spatial images.