Glucose metabolism via the pentose phosphate pathway, glycolysis and Krebs cycle in an orthotopic mouse model of human brain tumors

It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using orthotopic mouse models of human glioblastoma (GBM) and renal cell carcinoma metastatic to brain, we estimated the activity of the PPP relative to glycolysis by infusing [1,2‐¹³C₂]glucose. The [3‐¹³C]lactate/[2,3‐¹³C₂]lactate ratio was similar for both the GBM and brain metastasis and their respective surrounding brains (GBM, 0.197 ± 0.011 and 0.195 ± 0.033, respectively (p = 1); metastasis: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than the PPP flux in these tumors, and that the PPP flux into the lactate pool is similar in both tumors. Remarkably, ¹³C–¹³C coupling was observed in molecules derived from Krebs cycle intermediates in both tumor types, denoting glucose oxidation. In the renal cell carcinoma, in contrast with GBM, ¹³C multiplets of γ‐aminobutyric acid (GABA) differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. In addition, the orthotopic renal tumor, the patient's primary renal mass and brain metastasis were all strongly immunopositive for the 67‐kDa isoform of glutamate decarboxylase, as were 84% of tumors on a renal cell carcinoma tissue microarray of the same histology, suggesting that GABA synthesis is cell autonomous in at least a subset of renal cell carcinomas. Taken together, these data demonstrate that ¹³C‐labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Hyperpolarized singlet lifetimes of pyruvate in human blood and in the mouse

Hyperpolarized NMR is a promising technique for non‐invasive imaging of tissue metabolism in vivo. However, the pathways that can be studied are limited by the fast T₁ decay of the nuclear spin order. In metabolites containing pairs of coupled nuclear spins‐1/2, the spin order may be maintained by exploiting the non‐magnetic singlet (spin‐0) state of the pair. This may allow preservation of the hyperpolarization in vivo during transport to tissues of interest, such as tumors, or to detect slower metabolic reactions. We show here that in human blood and in a mouse in vivo at millitesla fields the ¹³C singlet lifetime of [1,2‐¹³C₂]pyruvate was significantly longer than the ¹³C T₁, although it was shorter than the T₁ at field strengths of several tesla. We also examine the singlet‐derived NMR spectrum observed for hyperpolarized [1,2‐¹³C₂]lactate, originating from the metabolism of [1,2‐¹³C₂]pyruvate. © 2013 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd.     

Probing carbohydrate metabolism using hyperpolarized 13C‐labeled molecules

Glycolysis is a fundamental metabolic process in all organisms. Anomalies in glucose metabolism are linked to various pathological conditions. In particular, elevated aerobic glycolysis is a characteristic feature of rapidly growing cells. Glycolysis and the closely related pentose phosphate pathway can be monitored in real time by hyperpolarized ¹³C‐labeled metabolic substrates such as ¹³C‐enriched, deuterated D‐glucose derivatives, [2‐¹³C]‐D‐fructose, [2‐¹³C] dihydroxyacetone, [1‐¹³C]‐D‐glycerate, [1‐¹³C]‐D‐glucono‐δ‐lactone and [1‐¹³C] pyruvate in healthy and diseased tissues. Elevated glycolysis in tumors (the Warburg effect) was also successfully imaged using hyperpolarized [U‐¹³C₆, U‐²H₇]‐D‐glucose, while the size of the preexisting lactate pool can be measured by ¹³C MRS and/or MRI with hyperpolarized [1‐¹³C]pyruvate. This review summarizes the application of various hyperpolarized ¹³C‐labeled metabolites to the real‐time monitoring of glycolysis and related metabolic processes in normal and diseased tissues.     

Ketone bodies effectively compete with glucose for neuronal acetyl‐CoA generation in rat hippocampal slices

Ketone bodies can be used for cerebral energy generation in situ, when their availability is increased as during fasting or ingestion of a ketogenic diet. However, it is not known how effectively ketone bodies compete with glucose, lactate, and pyruvate for energy generation in the brain parenchyma. Hence, the contributions of exogenous 5.0 mM [1‐¹³C]glucose and 1.0 mM [2‐¹³C]lactate + 0.1 mM pyruvate (combined [2‐¹³C]lactate + [2‐¹³C]pyruvate) to acetyl‐CoA production were measured both without and with 5.0 mM [U‐¹³C]3‐hydroxybutyrate in superfused rat hippocampal slices by ¹³C NMR non‐steady‐state isotopomer analysis of tissue glutamate and GABA. Without [U‐¹³C]3‐hydroxybutyrate, glucose, combined lactate + pyruvate, and unlabeled endogenous sources contributed (mean ± SEM) 70 ± 7%, 10 ± 2%, and 20 ± 8% of acetyl‐CoA, respectively. With [U‐¹³C]3‐hydroxybutyrate, glucose contributions significantly fell from 70 ± 7% to 21 ± 3% (p < 0.0001), combined lactate + pyruvate and endogenous contributions were unchanged, and [U‐¹³C]3‐hydroxybutyrate became the major acetyl‐CoA contributor (68 ± 3%) – about three‐times higher than glucose. A direct analysis of the GABA carbon 2 multiplet revealed that [U‐¹³C]3‐hydroxybutyrate contributed approximately the same acetyl‐CoA fraction as glucose, indicating that it was less avidly oxidized by GABAergic than glutamatergic neurons. The appearance of superfusate lactate derived from glycolysis of [1‐¹³C]glucose did not decrease significantly in the presence of 3‐hydroxybutyrate, hence total glycolytic flux (Krebs cycle inflow + exogenous lactate formation) was attenuated by 3‐hydroxybutyrate. This indicates that, under these conditions, 3‐hydroxybutyrate inhibited glycolytic flux upstream of pyruvate kinase. Copyright © 2015 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.     

Hyperpolarized δ‐[1‐13C]gluconolactone as a probe of the pentose phosphate pathway

The pentose phosphate pathway (PPP) is thought to be upregulated in trauma (to produce excess NADPH) and in cancer (to provide ribose for nucleotide biosynthesis), but simple methods for detecting changes in flux through this pathway are not available. MRI of hyperpolarized ¹³C–enriched metabolites offers considerable potential as a rapid, non‐invasive tool for detecting changes in metabolic fluxes. In this study, hyperpolarized δ‐[1‐¹³C]gluconolactone was used as a probe to detect flux through the oxidative portion of the pentose phosphate pathway (PPP_ox) in isolated perfused mouse livers. The appearance of hyperpolarized (HP) H¹³CO₃^? within seconds after exposure of livers to HP‐δ‐[1‐¹³C]gluconolactone demonstrates that this probe rapidly enters hepatocytes, becomes phosphorylated, and enters the PPP_ox pathway to produce HP‐H¹³CO₃^? after three enzyme catalyzed steps (6P–gluconolactonase, 6‐phosphogluconate dehydrogenase, and carbonic anhydrase). Livers perfused with octanoate as their sole energy source show no change in production of H¹³CO₃^? after exposure to low levels of H₂O₂, while livers perfused with glucose and insulin showed a twofold increase in H¹³CO₃^? after exposure to peroxide. This indicates that flux through the PPP_ox is stimulated by H₂O₂ in glucose perfused livers but not in livers perfused with octanoate alone. Subsequent perfusion of livers with non‐polarized [1,2‐¹³C]glucose followed by ¹H NMR analysis of lactate in the perfusate verified that flux through the PPP_ox is indeed low in healthy livers and modestly higher in peroxide damaged livers. We conclude that hyperpolarized δ‐[1‐¹³C]gluconolactone has the potential to serve as a metabolic imaging probe of this important biological pathway.     

Hyperpolarized 13C NMR studies of glucose metabolism in living breast cancer cell cultures

The recent development of dissolution dynamic nuclear polarization (DNP) gives NMR the sensitivity to follow metabolic processes in living systems with high temporal resolution. In this article, we apply dissolution DNP to study the metabolism of hyperpolarized U‐¹³C,²H₇‐glucose in living, perfused human breast cancer cells. Spectrally selective pulses were used to maximize the signal of the main product, lactate, whilst preserving the glucose polarization; in this way, both C₁‐lactate and C₃‐lactate could be observed with high temporal resolution. The production of lactate by T47D breast cancer cells can be characterized by Michaelis–Menten‐like kinetics, with K_m = 3.5 ± 1.5 mm and V_max = 34 ± 4 fmol/cell/min. The high sensitivity of this method also allowed us to observe and quantify the glycolytic intermediates dihydroxyacetone phosphate and 3‐phosphoglycerate. Even with the enhanced DNP signal, many other glycolytic intermediates could not be detected directly. Nevertheless, by applying saturation transfer methods, the glycolytic intermediates glucose‐6‐phosphate, fructose‐6‐phosphate, fructose‐1,6‐bisphosphate, glyceraldehyde‐3‐phosphate, phosphoenolpyruvate and pyruvate could be observed indirectly. This method shows great promise for the elucidation of the distinctive metabolism and metabolic control of cancer cells, suggesting multiple ways whereby hyperpolarized U‐¹³C,²H₇‐glucose NMR could aid in the diagnosis and characterization of cancer in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Polymerization of 1,4-butadiene over MgCl2-supported cobalt catalysts combined with trimethylaluminium

MgCl₂-supported Co catalysts with different loading amounts were prepared from the reaction of CoBr₂[P(C₆H₅)₃]₂ or CoBr₂(C₅H₅N)₂ and MgCl₂ in toluene solution. Polymerization of 1,3-butadiene was conducted with them using Al(CH₃)₃ as cocatalyst, which gave polybutadiene composed of 1,2 and cis-1,4 units. The content of 1,2 units could easily be regulated from approximately 0% up to 90% by controlling the loading amount of Co as well as by changing the amount of suitable Lewis base added. The sequence distribution of each unit was determined by means of ¹³C NMR using hydrogenated polybutadiene. Addition of ethylene to the polymerization system caused a marked decrease in the molecular weight of polybutadiene, but ethylene was incorporated neither in the main chain nor at the chain ends. The chain end analysis of very low-molecular-weight polybutadiene by ¹³C NMR suggested that 1,2 and 2,1 insertion reactions predominantly proceed at the initiation and propagation steps, respectively.     

Delineation of substrate selection and anaplerosis in tricarboxylic acid cycle of the heart by 13C NMR spectroscopy and mass spectrometry

¹³C NMR and mass spectrometry (MS) provide complementary information regarding the ¹³C labeling of intermediary metabolites. Currently, these two techniques are rarely used together because of the complexity of modeling the distribution of both positional and mass isotopomers. In this study, we developed a matrix‐based model for the assessment of ¹³C label distribution in the tricarboxylic acid cycle and related metabolites. The model was applied to the analysis of NMR‐ and MS‐measured ¹³C isotopomers for quantification of substrate utilization and anaplerotic fluxes in isolated perfused rat hearts. NMR and MS data were acquired from two groups of rat hearts perfused with substrates in complementary labeling patterns, i.e. the ¹³C‐PAL + GLC group (0.6 mM [¹³C₁₆]palmitate + 5.5 mM glucose) and the PAL + ¹³C‐GLC group (0.6 mM palmitate + 5.5 mM [¹³C₆]glucose). Relative flux parameters were obtained by fitting the model to the NMR data, MS data and their combination, respectively. Our results suggest that, although both NMR and MS can provide accurate quantification of substrate selection in oxidative metabolism, the accuracy of estimation of anaplerotic fluxes relies on the combination of these two experimental methods. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of brain glycogen concentration and turnover through localized 13C NMR of both the C1 and C6 resonances

We have recently shown that at isotopic steady state ¹³C NMR can provide a direct measurement of glycogen concentration changes, but that the turnover of glycogen was not accessible with this protocol. The aim of the present study was to design, implement and apply a novel dual‐tracer infusion protocol to simultaneously measure glycogen concentration and turnover. After reaching isotopic steady state for glycogen C1 using [1‐¹³C] glucose administration, [1,6‐¹³C₂] glucose was infused such that isotopic steady state was maintained at the C1 position, but the C6 position reflected ¹³C label incorporation. To overcome the large chemical shift displacement error between the C1 and C6 resonances of glycogen, we implemented 2D gradient based localization using the Fourier series window approach, in conjunction with time‐domain analysis of the resulting FIDs using jMRUI. The glycogen concentration of 5.1 ± 1.6 mM measured from the C1 position was in excellent agreement with concomitant biochemical determinations. Glycogen turnover measured from the rate of label incorporation into the C6 position of glycogen in the α‐chloralose anesthetized rat was 0.7 µmol/g/h. Copyright © 2009 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.     

Metabolism of [U‐13C]glucose in human brain tumors in vivo

Glioblastomas and brain metastases demonstrate avid uptake of 2‐[¹⁸F]fluoro‐2‐deoxyglucose by positron emission tomography and display perturbations of intracellular metabolite pools by ¹H MRS. These observations suggest that metabolic reprogramming contributes to brain tumor growth in vivo. The Warburg effect, excess metabolism of glucose to lactate in the presence of oxygen, is a hallmark of cancer cells in culture. 2‐[¹⁸F]Fluoro‐2‐deoxyglucose‐positive tumors are assumed to metabolize glucose in a similar manner, with high rates of lactate formation relative to mitochondrial glucose oxidation, but few studies have specifically examined the metabolic fates of glucose in vivo. In particular, the capacity of human brain cancers to oxidize glucose in the tricarboxylic acid cycle is unknown. Here, we studied the metabolism of human brain tumors in situ. [U‐¹³ C]Glucose (uniformly labeled glucose, i.e. d ‐glucose labeled with ¹³ C in all six carbons) was infused during surgical resection, and tumor samples were subsequently subjected to ¹³C NMR spectroscopy. The analysis of tumor metabolites revealed lactate production, as expected. We also determined that pyruvate dehydrogenase, turnover of the tricarboxylic acid cycle, anaplerosis and de novo glutamine and glycine synthesis contributed significantly to the ultimate disposition of glucose carbon. Surprisingly, less than 50% of the acetyl‐coenzyme A pool was derived from blood‐borne glucose, suggesting that additional substrates contribute to tumor bioenergetics. This study illustrates a convenient approach that capitalizes on the high information content of ¹³C NMR spectroscopy and enables the analysis of intermediary metabolism in diverse cancers growing in their native microenvironment. Copyright © 2012 John Wiley & Sons, Ltd.     

Substrate selection in hearts subjected to ischemia/reperfusion: role of cardioplegic solutions and gender

In conditions of ischemia/reperfusion (I/R), the relative use of all available substrates by the heart has a significant effect on the recovery of the organ. This substrate preference in perfused hearts is influenced by ischemia. We followed the metabolic fate of [U‐¹³C]glucose and [3‐¹³C]lactate in hearts preserved in Celsior (Cs) and histidine buffer solution (HBS) for 4 or 6 h and subsequently perfused with a Krebs–Henseleit solution (KH) containing [U‐¹³C]glucose and [3‐¹³C]lactate. We also assessed gender‐specific metabolic modulation in our I/R experimental conditions. Hearts from male and female Wistar rats (6–8 weeks) were subjected to moderate (0–240 min) or prolonged (240–360 min) cold ischemia whilst immersed in Cs and HBS, and perfused for 30 min with KH containing [U‐¹³C]glucose and [3‐¹³C]lactate. After perfusion, hearts were freeze‐clamped and metabolites were extracted for ¹³C NMR isotopomer analysis. In control conditions, there were no differences with regard to lactate origin in hearts from males and females. After 6 h of preservation in Cs, lactate origin was mostly from [U‐¹³C]glucose in hearts from males and from [3‐¹³C]lactate in hearts from females. During the 6 h of organ preservation in HBS, the lactate pool showed a strong contribution from unenriched sources in male hearts and from [U‐¹³C]glucose in female hearts. The glutamate C2/C4 ratio was stable or increased in hearts from females after I/R, and the alanine index increased in hearts from both males and females. Octanoate was, as predicted, the preferential substrate during perfusion. Glucose and lactate suffer a distinct metabolic fate in our I/R conditions, which is related to the cardioplegic solution used during organ storage, and the gender. Hearts from females appear to be less sensitive to I/R injury, and heart preservation in HBS proved to be effective in enhancing anaplerosis during perfusion, especially in hearts from females. Copyright © 2011 John Wiley & Sons, Ltd.     

Ring‐Opening Copolymerization of 2‐Aryl‐1‐methylenecyclopropanes with Carbon Monoxide Initiated by Pd–bpy Complexes

[PdCl(Me)(bpy)] and a mixture of the complex with cocatalysts; NaBARF (BARF = [B{C₆H₃(CF₃)₂‐3,5}₄]^?), NaBF₄, AgBARF, AgBF₄, and AgOTf, catalyze the copolymerization of 2‐phenyl‐1‐methylenecyclopropane with carbon monoxide to produce a new polyketone accompanied by ring opening of the monomer. ¹H and ¹³C{¹H} NMR spectra indicate that the polymers have two isomeric repeating units in which the phenyl substituents occupy different positions. The molecular weights of the polyketones formed by the reactions with a [Pd]/[cocatalyst]/[2‐phenyl‐1‐methyleneyclopropane] ratio of 1:3:70 are in the range of M _n = 13 100–86 000. The polymer obtained by the reaction promoted by [PdCl(Me)(bpy)]/MBARF, where M = Ag or Na, shows a narrow molecular weight distribution, M _w/M _n = 1.44 and 1.59, respectively. The catalysis is effective also for the ring‐opening copolymerization of 2‐aryl‐1‐methylenecyclopropanes bearing Me and F substituents on the phenyl ring. Isotope‐labeled experiments revealed the mechanism of the polymerization, which involves a 1,2‐insertion of the monomer into the Pd–acyl bond to produce a cyclopropylmethyl palladium intermediate, and subsequent β‐alkyl elimination to give the Pd–alkyl complex.

     

PRESS timings for resolving 13C4‐glutamate 1H signal at 9.4 T: Demonstration in rat with uniformly labelled 13C‐glucose

MRS of ¹³C₄‐labelled glutamate (¹³C₄‐Glu) during an infusion of a carbon‐13 (¹³C)‐labelled substrate, such as uniformly labelled glucose ([U‐¹³C₆]‐Glc), provides a measure of Glc metabolism. The presented work provides a single‐shot indirect ¹³C detection technique to quantify the approximately 2.51 ppm ¹³C₄‐Glu satellite proton (¹H) peak at 9.4 T. The methodology is an optimized point‐resolved spectroscopy (PRESS) sequence that minimizes signal contamination from the strongly coupled protons of N‐acetylaspartate (NAA), which resonate at approximately 2.49 ppm. J‐coupling evolution of protons was characterized numerically and verified experimentally. A (TE₁, TE₂) combination of (20 ms, 106 ms) was found to be suitable for minimizing NAA signal in the 2.51 ppm ¹H ¹³C₄‐Glu spectral region, while retaining the ¹³C₄‐Glu ¹H satellite peak. The efficacy of the technique was verified on phantom solutions and on two rat brains in vivo during an infusion of [U‐¹³C₆]‐Glc. LCModel was employed for analysis of the in vivo spectra to quantify the 2.51 ppm ¹H ¹³C₄‐Glu signal to obtain Glu C4 fractional enrichment time courses during the infusions. Cramér‐Rao lower bounds of about 8% were obtained for the 2.51 ppm ¹³C₄‐Glu ¹H satellite peak with the optimal TE combination.     

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.     

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.