Assessing the pentose phosphate pathway using [2, 3‐13C2]glucose

The pentose phosphate pathway (PPP) is essential for reductive biosynthesis, antioxidant processes and nucleotide production. Common tracers such as [1,2‐¹³C₂]glucose rely on detection of ¹³C in lactate and require assumptions to correct natural ¹³C abundance. Here, we introduce a novel and specific tracer of the PPP, [2,3‐¹³C₂]glucose. ¹³C NMR analysis of the resulting isotopomers is informative because [1,2‐¹³C₂]lactate arises from glycolysis and [2,3‐¹³C₂]lactate arises exclusively through the PPP. A correction for natural abundance is unnecessary. In rats receiving [2,3‐¹³C₂]glucose, the PPP was more active in the fed versus fasted state in the liver and the heart, consistent with increased expression of key enzymes in the PPP. Both the PPP and glycolysis were substantially increased in hepatoma compared with liver. In summary, [2,3‐¹³C₂]glucose and ¹³C NMR simplify assessment of the PPP.     

Simultaneous imaging of hyperpolarized [1,4‐13C2]fumarate, [1‐13C]pyruvate and 18F–FDG in a rat model of necrosis in a clinical PET/MR scanner

A co‐polarization scheme for [1,4‐¹³C₂]fumarate and [1‐¹³C]pyruvate is presented to simultaneously assess necrosis and metabolism in rats with hyperpolarized ¹³C magnetic resonance (MR). The co‐polarization was performed in a SPINlab polarizer. In addition, the feasibility of simultaneous positron emission tomography (PET) and MR of small animals with a clinical PET/MR scanner is demonstrated. The hyperpolarized metabolic MR and PET was demonstrated in a rat model of necrosis. The polarization and T₁ of the co‐polarized [1,4‐¹³C₂]fumarate and [1‐¹³C]pyruvate substrates were measured in vitro and compared with those obtained when the substrates were polarized individually. A polarization of 36 ± 4% for fumarate and 37 ± 6% for pyruvate was obtained. We found no significant difference in the polarization and T₁ values between the dual and single substrate polarization. Rats weighing about 400 g were injected intramuscularly in one of the hind legs with 200 μL of turpentine to induce necrosis. Two hours later, ¹³C metabolic maps were obtained with a chemical shift imaging sequence (16 × 16) with a resolution of 3.1 × 5.0 × 25.0 mm³. The ¹³C spectroscopic images were acquired in 12 s, followed by an 8‐min ¹⁸F‐2‐fluoro‐2‐deoxy‐d ‐glucose (¹⁸F–FDG) PET acquisition with a resolution of 3.5 mm. [1,4‐¹³C₂]Malate was observed from the tissue injected with turpentine indicating necrosis. Normal [1‐¹³C]pyruvate metabolism and ¹⁸F–FDG uptake were observed from the same tissue. The proposed co‐polarization scheme provides a means to utilize multiple imaging agents simultaneously, and thus to probe various metabolic pathways in a single examination. Moreover, it demonstrates the feasibility of small animal research on a clinical PET/MR scanner for combined PET and hyperpolarized metabolic MR.     

Doublet asymmetry for estimating polarization in hyperpolarized 13C–pyruvate studies

Hyperpolarized ¹³C MRS allows in vivo interrogation of key metabolic pathways, with pyruvate (Pyr) the substrate of choice for current clinical studies. Knowledge of the liquid‐state polarization is needed for full quantitation, and asymmetry of the C₂ doublet, arising from 1% naturally abundant [1,2‐¹³C]Pyr in any hyperpolarized [1‐¹³C]Pyr sample, has been suggested as a direct measure of in vivo C₁ polarization via the use of an in vitro calibration curve. Here we show that different polarization levels can yield the same C₂‐doublet asymmetry, thus limiting the utility of this metric for quantitation. Furthermore, although the time evolution of doublet asymmetry is poorly modeled using the expected dominant relaxation mechanisms of carbon‐proton dipolar coupling and chemical shift anisotropy, the inclusion of a C‐C dipolar coupling term can explain the observed initial evolution of the C₂ doublet asymmetry beyond its expected thermal equilibrium value.     

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

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

Robust hyperpolarized 13C metabolic imaging with selective non‐excitation of pyruvate (SNEP)

In vivo metabolic imaging using hyperpolarized [1‐¹³C]pyruvate provides localized biochemical information and is particularly useful in detecting early disease changes, as well as monitoring disease progression and treatment response. However, a major limitation of hyperpolarized magnetization is its unrecoverable decay, due not only to T₁ relaxation but also to radio‐frequency (RF) excitation. RF excitation schemes used in metabolic imaging must therefore be able to utilize available hyperpolarized magnetization efficiently and robustly for the optimal detection of substrate and metabolite activities. In this work, a novel RF excitation scheme called selective non‐excitation of pyruvate (SNEP) is presented. This excitation scheme involves the use of a spectral selective RF pulse to specifically exclude the excitation of [1‐¹³C]pyruvate, while uniformly exciting the key metabolites of interest (namely [1‐¹³C]lactate and [1‐¹³C]alanine) and [1‐¹³C]pyruvate‐hydrate. By eliminating the loss of hyperpolarized [1‐¹³C]pyruvate magnetization due to RF excitation, the signal from downstream metabolite pools is increased together with enhanced dynamic range. Simulation results, together with phantom measurements and in vivo experiments, demonstrated the improvement in signal‐to‐noise ratio (SNR) and the extension of the lifetime of the [1‐¹³C]lactate and [1‐¹³C]alanine pools when compared with conventional non‐spectral selective (NS) excitation. SNEP has also been shown to perform comparably well with multi‐band (MB) excitation, yet SNEP possesses distinct advantages, including ease of implementation, less stringent demands on gradient performance, increased robustness to frequency drifts and B₀ inhomogeneity as well as easier quantification involving the use of [1‐¹³C]pyruvate‐hydrate as a proxy for the actual [1‐¹³C] pyruvate signal. SNEP is therefore a promising alternative for robust hyperpolarized [1‐¹³C]pyruvate metabolic imaging with high fidelity. Copyright © 2015 John Wiley & Sons, Ltd.     

In vivo measurement of apparent diffusion coefficients of hyperpolarized 13C‐labeled metabolites

The combination of hyperpolarized MRS with diffusion weighting (dw) allows for determination of the apparent diffusion coefficient (ADC), which is indicative of the intra‐ or extracellular localization of the metabolite. Here, a slice‐selective pulsed‐gradient spin echo sequence was implemented to acquire a series of dw spectra from rat muscle in vivo to determine the ADCs of multiple metabolites after a single injection of hyperpolarized [1‐¹³C]pyruvate. An optimal control optimized universal‐rotation pulse was used for refocusing to minimize signal loss caused by B₁ imperfections. Non‐dw spectra were acquired interleaved with the dw spectra and these were used to correct for signal decay during the acquisition as a result of T₁ decay, pulse imperfections, flow etc. The data showed that the ADC values for [1‐¹³C]lactate (0.4–0.7 µm²/ms) and [1‐¹³C]alanine (0.4–0.9 µm²/ms) were about a factor of two lower than the ADC of [1‐¹³C]pyruvate (1.1–1.5 µm²/ms). This indicates a more restricted diffusion space for the former two metabolites consistent with lactate and alanine being intracellular. The higher ADC for pyruvate (similar to the proton ADC) reflected that the injected substance was not confined inside the muscle cells but also present extracellular. Copyright © 2014 John Wiley & Sons, Ltd.     

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

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

In vivo investigation of cardiac metabolism in the rat using MRS of hyperpolarized [1‐13C] and [2‐13C]pyruvate

Hyperpolarized ¹³C MRS allows the in vivo assessment of pyruvate dehydrogenase complex (PDC) flux, which converts pyruvate to acetyl‐coenzyme A (acetyl‐CoA). [1‐¹³C]pyruvate has been used to measure changes in cardiac PDC flux, with demonstrated increase in ¹³C‐bicarbonate production after dichloroacetate (DCA) administration. With [1‐¹³C]pyruvate, the ¹³C label is released as ¹³CO₂/¹³C‐bicarbonate, and, hence, does not allow us to follow the fate of acetyl‐CoA. Pyruvate labeled in the C₂ position has been used to track the ¹³C label into the TCA (tricarboxylic acid) cycle and measure [5‐¹³C]glutamate as well as study changes in [1‐¹³C]acetylcarnitine with DCA and dobutamine. This work investigates changes in the metabolic fate of acetyl‐CoA in response to metabolic interventions of DCA‐induced increased PDC flux in the fed and fasted state, and increased cardiac workload with dobutamine in vivo in rat heart at two different pyruvate doses. DCA led to a modest increase in the ¹³C labeling of [5‐¹³C]glutamate, and a considerable increase in [1‐¹³C]acetylcarnitine and [1,3‐¹³C]acetoacetate peaks. Dobutamine resulted in an increased labeling of [2‐¹³C]lactate, [2‐¹³C]alanine and [5‐¹³C]glutamate. The change in glutamate with dobutamine was observed using a high pyruvate dose but not with a low dose. The relative changes in the different metabolic products provide information about the relationship between PDC‐mediated oxidation of pyruvate and its subsequent incorporation into the TCA cycle compared with other metabolic pathways. Using a high dose of pyruvate may provide an improved ability to observe changes in glutamate. Copyright © 2013 John Wiley & Sons, Ltd.     

In vivo T2 relaxation time measurement with echo‐time averaging

The accuracy of metabolite concentrations measured using in vivo proton (¹H) MRS is enhanced following correction for spin–spin (T₂) relaxation effects. In addition, metabolite proton T₂ relaxation times provide unique information regarding cellular environment and molecular mobility. Echo‐time (TE) averaging ¹H MRS involves the collection and averaging of multiple TE steps, which greatly simplifies resulting spectra due to the attenuation of spin‐coupled and macromolecule resonances. Given the simplified spectral appearance and inherent metabolite T₂ relaxation information, the aim of the present proof‐of‐concept study was to develop a novel data processing scheme to estimate metabolite T₂ relaxation times from TE‐averaged ¹H MRS data. Spectral simulations are used to validate the proposed TE‐averaging methods for estimating methyl proton T₂ relaxation times for N‐acetyl aspartate, total creatine, and choline‐containing compounds. The utility of the technique and its reproducibility are demonstrated using data obtained in vivo from the posterior‐occipital cortex of 10 healthy control subjects. Compared with standard methods, distinct advantages of this approach include built‐in macromolecule resonance attenuation, in vivo T₂ estimates closer to reported values when maximum TE ≈ T₂, and the potential for T₂ calculation of metabolite resonances otherwise inseparable in standard ¹H MRS spectra recorded in vivo. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolism of hyperpolarized [1‐13C]pyruvate through alternate pathways in rat liver

The source of hyperpolarized (HP) [¹³C]bicarbonate in the liver during metabolism of HP [1‐¹³C]pyruvate is uncertain and likely changes with physiology. Multiple processes including decarboxylation through pyruvate dehydrogenase or pyruvate carboxylase followed by subsequent decarboxylation via phosphoenolpyruvate carboxykinase (gluconeogenesis) could play a role. Here we tested which metabolic fate of pyruvate contributed to the appearance of HP [¹³C]bicarbonate during metabolism of HP [1‐¹³C]pyruvate by the liver in rats after 21 h of fasting compared to rats with free access to food. The ¹³C NMR of HP [¹³C]bicarbonate was observed in the liver of fed rats, but not in fasted rats where pyruvate carboxylation and gluconeogenesis was active. To further explore the relative fluxes through pyruvate carboxylase versus pyruvate dehydrogenase in the liver under typical conditions of hyperpolarization studies, separate parallel experiments were performed with rats given non‐hyperpolarized [2,3‐¹³C]pyruvate. ¹³C NMR analysis of glutamate isolated from the liver of rats revealed that flux from injected pyruvate through pyruvate dehydrogenase was dominant under fed conditions whereas flux through pyruvate carboxylase dominated under fasted conditions. The NMR signal of HP [¹³C]bicarbonate does not parallel pyruvate carboxylase activity followed by subsequent decarboxylation reaction leading to glucose production. In the liver of healthy well‐fed rats, the appearance of HP [¹³C]bicarbonate exclusively reflects decarboxylation of HP [1‐¹³C]pyruvate via pyruvate dehydrogenase. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.     

In vivo measurement of aldehyde dehydrogenase‐2 activity in rat liver ethanol model using dynamic MRSI of hyperpolarized [1‐13C]pyruvate

To date, measurements of the activity of aldehyde dehydrogenase‐2 (ALDH2), a critical mitochondrial enzyme for the elimination of certain cytotoxic aldehydes in the body and a promising target for drug development, have been largely limited to in vitro methods. Recent advancements in MRS of hyperpolarized ¹³C‐labeled substrates have provided a method to detect and image in vivo metabolic pathways with signal‐to‐noise ratio gains greater than 10 000‐fold over conventional MRS techniques. However aldehydes, because of their toxicity and short T₁ relaxation times, are generally poor targets for such ¹³C‐labeled studies. In this work, we show that dynamic MRSI of hyperpolarized [1‐¹³C]pyruvate and its conversion to [1‐¹³C]lactate can provide an indirect in vivo measurement of ALDH2 activity via the concentration of NADH (nicotinamide adenine dinucleotide, reduced form), a co‐factor common to both the reduction of pyruvate to lactate and the oxidation of acetaldehyde to acetate. Results from a rat liver ethanol model (n = 9) show that changes in ¹³C‐lactate labeling following the bolus injection of hyperpolarized pyruvate are highly correlated with changes in ALDH2 activity (R² = 0.76). Copyright © 2012 John Wiley & Sons, Ltd.     

Lithium compartmentation in brain by 7Li MRS: effect of total lithium concentration

In previous work at 4.7 T, the individual components of biexponential ⁷Li transverse (T₂) spin relaxation in rat brain in vivo were tentatively identified with intra‐ and extracellular Li. The goal in this work was to estimate Li's compartmental distribution as a function of total Li concentration in brain from the biexponential decays. Here a localized, biexponential ⁷Li T₂ MR spin‐relaxation study with isotopically enriched ⁷LiCl is reported in rat brain in vivo at 7 T. Additionally, a simple linear interpolation using the biexponential T₂ values to estimate intracellular Li from individual monoexponential T₂ decays was assessed. Intracellular T₂ was 14.8 ± 4.3 ms and extracellular T₂ was 295 ± 61 ms. The fraction of intracellular brain Li ranged from 37.3 to 64.8% (mean 54.5 ± 6.7%) and did not correlate with total Li concentration. The estimated intracellular Li concentration ranged from 47 to 80% (mean 68.3 ± 8.5%) of the total brain Li concentration and was highly correlated with it. The monoexponential estimates of the intracellular‐Li fractions and derived concentrations averaged about 15% higher than the corresponding biexponential estimates. This work supports the previous conclusion that a large fraction of Li in the brain is within the intracellular compartment. Copyright © 2013 John Wiley & Sons, Ltd.     

In vivo detection of intermediate metabolic products of [1-(13) C]ethanol in the brain using (13) C MRS

In this study, in vivo ¹³C MRS was used to investigate the labeling of brain metabolites after intravenous administration of [1‐¹³C]ethanol. After [1‐¹³C]ethanol had been administered systemically to rats, ¹³C labels were detected in glutamate, glutamine and aspartate in the carboxylic and amide carbon spectral region. ¹³C‐labeled bicarbonate HCO (161.0 ppm) was also detected. Saturating acetaldehyde C1 at 207.0 ppm was found to have no effect on the ethanol C1 (57.7 ppm) signal intensity after extensive signal averaging, providing direct in vivo evidence that direct metabolism of alcohol by brain tissue is minimal. To compare the labeling of brain metabolites by ethanol with labeling by glucose, in vivo time course data were acquired during intravenous co‐infusion of [1‐¹³C]ethanol and [¹³C₆]‐D ‐glucose. In contrast with labeling by [¹³C₆]‐D ‐glucose, which produced doublets of carboxylic/amide carbons with a J coupling constant of 51 Hz, the simultaneously detected glutamate and glutamine singlets were labeled by [1‐¹³C]ethanol. As ¹³C labels originating from ethanol enter the brain after being converted into [1‐¹³C]acetate in the liver, and the direct metabolism of ethanol by brain tissue is negligible, it is suggested that orally or intragastrically administered ¹³C‐labeled ethanol may be used to study brain metabolism and glutamatergic neurotransmission in investigations involving alcohol administration. In vivo ¹³C MRS of rat brain following intragastric administration of ¹³C‐labeled ethanol is demonstrated. Published in 2011 by John Wiley & Sons, Ltd.     

The hemodynamic and metabolic effects of spironolactone treatment in acute kidney injury assessed by hyperpolarized MRI

Renal ischemia‐reperfusion injury (IRI) is one of the most common types of acute kidney injury. Spironolactone has shown promising kidney protective effects in renal IRI in rats. We investigated the hemodynamic and metabolic effects of spironolactone (100 mg/kg) administered immediately after 40 min unilateral kidney ischemia in rats. Hyperpolarized MRI using co‐polarized [1‐¹³C]pyruvate and [¹³C,¹⁵N₂]urea as well as ¹H dynamic contrast‐enhanced (DCE) MRI was performed 24 h after induction of ischemia. We found a significant decrease in renal blood flow (RBF) in the ischemic kidney compared with the contralateral one measured using DCE and [¹³C,¹⁵N₂]urea. The RBF measured using [1‐¹³C]pyruvate and [¹³C,¹⁵N₂]urea was significantly altered by spironolactone. The RBFs in the ischemic kidney compared with the contralateral kidney were decreased similarly as measured using both [¹³C,¹⁵N₂]urea and [1‐¹³C]pyruvate in the spironolactone‐treated group. Spironolactone treatment increased the perfusion‐corrected pyruvate metabolism by 54% in both the ischemic and contralateral kidney. Furthermore, we showed a correlation between vascular permeability using a histological Evans blue analysis and the ratio of the volumes of distribution (VoDs), ie VoD‐[¹³C,¹⁵N₂]urea/VoD‐[1‐¹³C]pyruvate. This suggests that [¹³C,¹⁵N₂]urea/[1‐¹³C]pyruvate VoD ratio may be a novel indicator of renal vascular permeability associated with renal damage in rodents.     

T1 nuclear magnetic relaxation dispersion of hyperpolarized sodium and cesium hydrogencarbonate‐13C

In vivo pH mapping in tissue using hyperpolarized hydrogencarbonate‐¹³C has been proposed as a method to study tumor growth and treatment and other pathological conditions related to pH changes. The finite spin–lattice relaxation times (T₁) of hyperpolarized media are a significant limiting factor for in vivo imaging. Relaxation times can be measured at standard magnetic fields (1.5 T, 3.0 T etc.), but no such data are available at low fields, where T₁ values can be significantly shorter. This information is required to determine the potential loss of polarization as the agent is dispensed and transported from the polarizer to the MRI scanner. The purpose of this study is to measure T₁ dispersion from low to clinical magnetic fields (0.4 mT to 3.0 T) of different hyperpolarized hydrogencarbonate formulations previously proposed in the literature for in vivo pH measurements. ¹³C–enriched cesium and sodium hydrogencarbonate preparations were hyperpolarized using dynamic nuclear polarization, and the T₁ values of different samples were measured at different magnetic field strengths using a fast field‐cycling relaxometer and a 3.0 T clinical MRI system. The effects of deuterium oxide as a dissolution medium for sodium hydrogencarbonate were also analyzed. This study finds that the cesium formulation has slightly shorter T₁ values compared with the sodium preparation. However, the higher solubility of cesium hydrogencarbonate‐¹³C means it can be polarized at greater concentration, using less trityl radical than sodium hydrogencarbonate‐¹³C. This study also establishes that the preparation and handling of sodium hydrogencarbonate formulations in relation to cesium hydrogencarbonate is more difficult, due to the higher viscosity and lower achievable concentrations, and that deuterium oxide significantly increases the T₁ of sodium hydrogencarbonate solutions. Finally, this work also investigates the influence of pH on the spin–lattice relaxation of cesium hydrogencarbonate‐¹³C measured over a pH range of 7 to 9 at 0.47 T.     

Real‐time assessment of 13C metabolism reveals an early lactate increase in the brain of rats with acute liver failure

Intracranial hypertension is a severe complication of acute liver failure (ALF) secondary to brain edema. The pathogenesis of cerebral edema in ALF is not clear, but seems to be related to energy metabolism in which lactate may have an important role. The aim of this study was to follow the synthesis of brain lactate using a novel in vivo metabolic technology in a rat model of ALF. Time‐resolved ¹³C MRS of hyperpolarized ¹³C₁‐pyruvate was used to quantitatively follow the in vivo conversion of pyruvate to its substrates in a model of devascularized ALF in rats. Rats with ALF showed a significant increase in the lactate to pyruvate ratio from 36% to 69% during the progression of liver disease relative to rats with portocaval anastomosis. Rats with ALF also showed a significant increase in the alanine to pyruvate ratio from 72% to 95%. These increases were detectable at very early stages (6 h) when animals had no evident disease signs in their behavior (without loss of righting or corneal reflexes). This study shows the dynamic consequences of cerebral in vivo ¹³C metabolism at real time in rats with ALF. The early detection of the de novo synthesis of lactate suggests that brain lactate is involved in the physiopathology of ALF. Hyperpolarization is a potential non‐invasive technique to follow the in vivo metabolism, and both the development and optimization of ¹³C‐labeled substrates can clarify the mechanism involved in ALF. Copyright © 2014 John Wiley & Sons, Ltd.     

Assessing the transport rate of hyperpolarized pyruvate and lactate from the intra‐ to the extracellular space

The use of [1‐ ¹³ C]pyruvate hyperpolarized by means of dynamic nuclear polarization provides a direct way to track the metabolic transformations of this metabolite in vivo and in cell cultures. The identification of the intra‐ and extracellular contributions to the ¹³ C NMR resonances is not straightforward. In order to obtain information about the rate of pyruvate and lactate transport through the cellular membrane, we set up a method that relies on the sudden ‘quenching’ of the extracellular metabolites' signal. The paramagnetic Gd–tetraazacyclododecane triacetic acid (Gd‐DO3A) complex was used to dramatically decrease the longitudinal relaxation time constants of the ¹³ C‐carboxylate resonances of both pyruvate and lactate. When Gd‐DO3A was added to an MCF‐7 cellular culture, which had previously received a dose of hyperpolarized [1‐ ¹³ C]pyruvate, the contributions of the extracellular pyruvate and lactate signals were deleted. From the analysis of the decay curves of the ¹³ C‐carboxylate resonances of pyruvate and lactate it was possible to extract information about the exchange rate of the two metabolites across the cellular membrane. In particular, it was found that, in the reported experimental conditions, the lactate transport from the intra‐ to the extracellular space is not much lower than the rate of lactate formation. The method reported herein is non‐destructive and it could be translated to in vivo studies. It opens a route for the use of hyperpolarized pyruvate to assess altered activity of carboxylate transporter proteins that may occur in pathological conditions. Copyright © 2016 John Wiley & Sons, Ltd.     

Real‐time measurement of hyperpolarized lactate production and efflux as a biomarker of tumor aggressiveness in an MR compatible 3D cell culture bioreactor

We have developed a 3D cell/tissue culture bioreactor compatible with hyperpolarized (HP) ¹³C MR and interrogated HP [1‐¹³C]lactate production and efflux in human renal cell carcinoma (RCC) cells. This platform is capable of resolving intracellular and extracellular HP lactate pools, allowing the kinetic measurement of lactate production and efflux in the context of cancer aggressiveness and response to therapy. HP ¹³C MR studies were performed on three immortalized human renal cell lines: HK2, a normal renal proximal tubule cell line from which a majority of RCCs arise, UMRC6, a cell line derived from a localized RCC, and UOK262, an aggressive and metastatic RCC. The intra‐ (Lac_in) and extracellular (Lac_ex) HP lactate signals were robustly resolved in dynamic ¹³C spectra of the cell lines due to a very small but reproducible chemical shift difference (0.031 ± 0.0005 ppm). Following HP [1‐¹³C]pyruvate delivery, the ratio of HP Lac_in/Lac_ex was significantly lower for UOK262 cells compared with both UMRC6 and HK2 cells due to a significant (p < 0.05) increase in the Lac_ex pool size. Lac_in/Lac_ex correlated with the MCT4 mRNA expression of the cell lines, and inhibition of MCT4 transport using DIDS resulted in a significant reduction in the HP Lac_ex pool size. The extension of these studies to living patient‐derived RCC tissue slices using HP [1,2‐¹³C₂]pyruvate demonstrated a similarly split lactate doublet with a high Lac_ex pool fraction; in contrast, only a single NMR resonance is noted for HP [5‐¹³C]glutamate, consistent with intracellular localization. These studies support the importance of lactate efflux as a biomarker of cancer aggressiveness and metastatic potential, and the utility of the MR compatible 3D cell/tissue culture bioreactor to study not only cellular metabolism but also transport. Additionally, this platform offers a sophisticated way to follow therapeutic interventions and screen novel therapies that target lactate export. Copyright © 2015 John Wiley & Sons, Ltd.