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PURPOSE This study is to investigate time-resolved 13 C MR spectroscopy (MRS) as an alternative to imaging for assessing pyruvate metabolism using hyperpolarized (HP) [1-13 C]pyruvate in the human brain. METHODS Time-resolved 13 C spectra were acquired from four axial brain slices of healthy human participants (n = 4) after a bolus injection of HP [1-13 C]pyruvate. 13 C MRS with low flip-angle excitations and a multichannel 13 C/1 H dual-frequency radiofrequency (RF) coil were exploited for reliable and unperturbed assessment of HP pyruvate metabolism. Slice-wise areas under the curve (AUCs) of 13 C-metabolites were measured and kinetic analysis was performed to estimate the production rates of lactate and HCO3- . Linear regression analysis between brain volumes and HP signals was performed. Region-focused pyruvate metabolism was estimated using coil-wise 13 C reconstruction. Reproducibility of HP pyruvate exams was presented by performing two consecutive injections with a 45-minutes interval. RESULTS [1-13 C]Lactate relative to the total 13 C signal (tC) was 0.21-0.24 in all slices. [13 C] HCO3- /tC was 0.065-0.091. Apparent conversion rate constants from pyruvate to lactate and HCO3- were calculated as 0.014-0.018 s-1 and 0.0043-0.0056 s-1 , respectively. Pyruvate/tC and lactate/tC were in moderate linear relationships with fractional gray matter volume within each slice. White matter presented poor linear regression fit with HP signals, and moderate correlations of the fractional cerebrospinal fluid volume with pyruvate/tC and lactate/tC were measured. Measured HP signals were comparable between two consecutive exams with HP [1-13 C]pyruvate. CONCLUSIONS Dynamic MRS in combination with multichannel RF coils is an affordable and reliable alternative to imaging methods in investigating cerebral metabolism using HP [1-13 C]pyruvate.

The interplay between glycolysis and gluconeogenesis is central to carbohydrate metabolism. Here, we describe novel methods to assess carbohydrate metabolism using [(13)C]-probes derived from glycerate, a molecule whose metabolic fate in mammals remains underexplored. Isotope-based studies were conducted via NMR and mass spectrometry analyses of freeze-clamped liver tissue extracts after [2,3-(13)C(2)]glycerate infusion. The ex vivo investigations were correlated with in vivo measurements using hyperpolarized [1-(13)C]glycerate. Application of [(13)C]glycerate to N-nitrosodiethylamine (DEN)-treated rats provided further assessments of intermediary carbohydrate metabolism in hepatocellular carcinoma. This method afforded direct analyses of control versus DEN tissues, and altered ratios of (13)C metabolic products as well as unique glycolysis intermediates were observed in the DEN liver/tumor. Isotopomer studies showed increased glycerate uptake and altered carbohydrate metabolism in the DEN rats.

The role of pyruvate dehydrogenase in mediating lipid-induced insulin resistance stands as a central question in the pathogenesis of type 2 diabetes mellitus. Many researchers have invoked the Randle hypothesis to explain the reduced glucose disposal in skeletal muscle by envisioning an elevated acetyl CoA pool arising from increased oxidation of fatty acids. Over the years, in vivo NMR studies have challenged that monolithic view. The advent of the dissolution dynamic nuclear polarization NMR technique and a unique type 2 diabetic rat model provides an opportunity to clarify. Dynamic nuclear polarization enhances dramatically the NMR signal sensitivity and allows the measurement of metabolic kinetics in vivo. Diabetic muscle has much lower pyruvate dehydrogenase activity than control muscle, as evidenced in the conversion of [1-(13)C]lactate and [2-(13)C]pyruvate to HCO(3)(−) and acetyl carnitine. The pyruvate dehydrogenase kinase inhibitor, dichloroacetate, restores rapidly the diabetic pyruvate dehydrogenase activity to control level. However, diabetic much has a much larger dynamic change in pyruvate dehydrogenase flux than control. The dichloroacetate induced surge in pyruvate dehydrogenase activity produces a differential amount of acetyl carnitine but does not affect the tricarboxylic acid flux. Further studies can now proceed with the dynamic nuclear polarization approach and a unique rat model to interrogate closely the biochemical mechanism interfacing oxidative metabolism with insulin resistance and metabolic inflexibility.

Transient disruption of the blood-brain barrier (BBB) with focused ultrasound (FUS) is an emerging clinical method to facilitate targeted drug delivery to the brain. The focal noninvasive disruption of the BBB can be applied to promote the local delivery of hyperpolarized substrates. In this study, we investigated the effects of FUS on imaging brain metabolism using two hyperpolarized 13C-labeled substrates in rodents: [1-13C]pyruvate and [1-13C]glycerate. The BBB is a rate-limiting factor for pyruvate delivery to the brain, and glycerate minimally passes through the BBB. First, cerebral imaging with hyperpolarized [1-13C]pyruvate resulted in an increase in total 13C signals (p = 0.05) after disrupting the BBB with FUS. Significantly higher levels of both [1-13C]lactate (lactate/total 13C signals, p = 0.01) and [13C]bicarbonate (p = 0.008) were detected in the FUS-applied brain region as compared to the contralateral FUS-unaffected normal-appearing brain region. The application of FUS without opening the BBB in a separate group of rodents resulted in comparable lactate and bicarbonate productions between the FUS-applied and the contralateral brain regions. Second, 13C imaging with hyperpolarized [1-13C]glycerate after opening the BBB showed increased [1-13C]glycerate delivery to the FUS-applied region (p = 0.04) relative to the contralateral side, and [1-13C]lactate production was consistently detected from the FUS-applied region. Our findings suggest that FUS accelerates the delivery of hyperpolarized molecules across the BBB and provides enhanced sensitivity to detect metabolic products in the brain; therefore, hyperpolarized 13C imaging with FUS may provide new opportunities to study cerebral metabolic pathways as well as various neurological pathologies.

Purpose Noninvasive imaging with hyperpolarized (HP) pyruvate can capture in vivo cardiac metabolism. For proper quantification of the metabolites and optimization of imaging parameters, understanding MR characteristics such as T 2 ∗ s of the HP signals is critical. This study is to measure in vivo cardiac T 2 ∗ s of HP [1-13 C]pyruvate and the products in rodents and humans. Methods A dynamic 13 C multi-echo spiral imaging sequence that acquires [13 C]bicarbonate, [1-13 C]lactate, and [1-13 C]pyruvate images in an interleaved manner was implemented for a clinical 3 Tesla system. T 2 ∗ of each metabolite was calculated from the multi-echo images by fitting the signal decay of each region of interest mono-exponentially. The performance of measuring T 2 ∗ using the sequence was first validated using a 13 C phantom and then with rodents following a bolus injection of HP [1-13 C]pyruvate. In humans, T 2 ∗ of each metabolite was calculated for left ventricle, right ventricle, and myocardium. Results Cardiac T 2 ∗ s of HP [1-13 C]pyruvate, [1-13 C]lactate, and [13 C]bicarbonate in rodents were measured as 24.9 ± 5.0, 16.4 ± 4.7, and 16.9 ± 3.4 ms, respectively. In humans, T 2 ∗ of [1-13 C]pyruvate was 108.7 ± 22.6 ms in left ventricle and 129.4 ± 8.9 ms in right ventricle. T 2 ∗ of [1-13 C]lactate was 40.9 ± 8.3, 44.2 ± 5.5, and 43.7 ± 9.0 ms in left ventricle, right ventricle, and myocardium, respectively. T 2 ∗ of [13 C]bicarbonate in myocardium was 64.4 ± 2.5 ms. The measurements were reproducible and consistent over time after the pyruvate injection. Conclusion The proposed metabolite-selective multi-echo spiral imaging sequence reliably measures in vivo cardiac T 2 ∗ s of HP [1-13 C]pyruvate and products.

PURPOSE This study aimed to investigate the role of regional f0 inhomogeneity in spiral hyperpolarized 13 C image quality and to develop measures to alleviate these effects. METHODS Field map correction of hyperpolarized 13 C cardiac imaging using spiral readouts was evaluated in healthy subjects. Spiral readouts with differing duration (26 and 45 ms) but similar resolution were compared with respect to off-resonance performance and image quality. An f0 map-based image correction based on the multifrequency interpolation (MFI) method was implemented and compared to correction using a global frequency shift alone. Estimation of an unknown frequency shift was performed by maximizing a sharpness objective based on the Sobel variance. The apparent full width half at maximum (FWHM) of the myocardial wall on [13 C]bicarbonate was used to estimate blur. RESULTS Mean myocardial wall FWHM measurements were unchanged with the short readout pre-correction (14.1 ± 2.9 mm) and post-MFI correction (14.1 ± 3.4 mm), but significantly decreased in the long waveform (20.6 ± 6.6 mm uncorrected, 17.7 ± 7.0 corrected, P = .007). Bicarbonate signal-to-noise ratio (SNR) of the images acquired with the long waveform were increased by 1.4 ± 0.3 compared to those acquired with the short waveform (predicted 1.32). Improvement of image quality was observed for all metabolites with f0 correction. CONCLUSIONS f0 -map correction reduced blur and recovered signal from dropouts, particularly along the posterior myocardial wall. The low image SNR of [13 C]bicarbonate can be compensated with longer duration readouts but at the expense of increased f0 artifacts, which can be partially corrected for with the proposed methods.

Dynamic nuclear polarization (DNP) coupled with 15N magnetic resonance imaging (MRI) provides an opportunity to image quantitative levels of biologically important metal ions such as Zn2+, Mg2+ or Ca2+ using appropriately designed 15N enriched probes. For example, a Zn-specific probe could prove particularly valuable for imaging the tissue distribution of freely available Zn2+ ions, an important known metal ion biomarker in the pancreas, in prostate cancer, and in several neurodegenerative diseases. In the present study, we prepare the cell-permeable, 15N-enriched, d6-deuterated version of the well-known Zn2+ chelator, tris(2-pyridylmethyl)amine (TPA) and demonstrate that the polarized ligand had favorable T1 and linewidth characteristics for 15N MRI. Examples of how polarized TPA can be used to quantify freely available Zn2+ in homogenized human prostate tissue and intact cells are presented.

Tissue pH is tightly regulated in vivo, being a sensitive physiological biomarker. Advent of dissolution dynamic nuclear polarization (DNP) and its translation to humans stimulated development of p...

PURPOSE: Previous cardiac imaging studies using hyperpolarized (HP) [1-(13)C]pyruvate were acquired at end-diastole (ED). Little is known about the interaction between cardiac cycle and metabolite content in the myocardium. In this study, we compared images of HP pyruvate and products at end-systole (ES) and ED. METHODS: A dual-phase (13)C MRI sequence was implemented to acquire two sequential HP images within a single cardiac cycle at ES and ED during successive R-R intervals in an interleaved manner. Each healthy volunteer (n = 3) received two injections of HP [1-(13)C]pyruvate for the dual-phase imaging on the short-axis (SA) and the vertical long-axis (LA) planes. Spatial distribution of HP (13)C metabolites at each cardiac phase was correlated to multi-phase (1)H MRI to confirm the mechanical changes. Ratios of myocardial HP metabolites were compared between ES and ED. Segmental analysis was performed on the mid-cavity SA plane. RESULTS: Besides mechanical changes, metabolic profiles of the heart detected by HP [1-(13)C]pyruvate differed between ES and ED. The myocardial signal of [(13)C]bicarbonate relative to [1-(13)C]lactate was significantly smaller at ED than the ratio at ES (p < 0.05), particularly in mid-anterior and mid-inferoseptal segments. The distinct metabolic profiles in the myocardium likely reflect the technical aspects of the imaging approach such as the coronary flow in addition to the cyclical changes in metabolism. CONCLUSION: The study demonstrates that metabolic profiles of the heart, measured by HP [1-(13)C]pyruvate, are affected by the cardiac cycle that the data is acquired in.

PURPOSE: This study is to investigate time-resolved (13)C MR spectroscopy as an alternative to imaging for assessing pyruvate metabolism using hyperpolarized [1-(13)C]pyruvate in the human brain. METHODS: Time-resolved (13)C spectra were acquired from four axial brain slices of healthy human participants (n=4) after a bolus injection of hyperpolarized [1-(13)C]pyruvate. (13)C MR spectroscopy with low flip-angle excitations and a multichannel (13)C/(1)H dual-frequency RF coil were exploited for reliable and unperturbed assessment of hyperpolarized pyruvate metabolism. Slice-wise area under the curves (AUCs) of (13)C-metabolites were measured and kinetic analysis was performed to estimate the production rates of lactate and HCO(3)(–). Linear regression analysis between brain volumes and hyperpolarized signals was performed. Region-focused pyruvate metabolism was estimated using coil-wise (13)C reconstruction. Reproducibility of hyperpolarized pyruvate exams was presented by performing two consecutive injections with a 45-min interval. RESULTS: [1-(13)C]Lactate relative to the total (13)C signal (tC) was 0.21–0.24 in all slices. [(13)C]HCO(3)(–)/tC was 0.065–0.091. Apparent conversion rate constants from pyruvate to lactate and HCO(3)(–) were calculated as 0.014–0.018s(−1) and 0.0043–0.0056s(−1), respectively. Pyruvate/tC and lactate/tC were in moderate linear relationships with fractional grey matter volume within each slice. White matter presented poor linear regression fit with hyperpolarized signals, and moderate correlations of the fractional cerebrospinal fluid volume with pyruvate/tC and lactate/tC were measured. Measured hyperpolarized signals were comparable between two consecutive exams with hyperpolarized [1-(13)C]pyruvate. CONCLUSIONS: Dynamic MRS in combination with multichannel RF coils is an affordable and reliable alternative to imaging methods in investigating cerebral metabolism using hyperpolarized [1-(13)C]pyruvate.

BACKGROUND: Pyruvate dehydrogenase (PDH) and lactate dehydrogenase are essential for adenosine triphosphate production in skeletal muscle. At the onset of exercise, oxidation of glucose and glycogen is quickly enabled by dephosphorylation of PDH. However, direct measurement of PDH flux in exercising human muscle is daunting, and the net effect of covalent modification and other control mechanisms on PDH flux has not been assessed. PURPOSE: To demonstrate the feasibility of assessing PDH activation and changes in pyruvate metabolism in human skeletal muscle after the onset of exercise using carbon 13 ((13)C) MRI with hyperpolarized (HP) [1-(13)C]-pyruvate. MATERIALS AND METHODS: For this prospective study, sedentary adults in good general health (mean age, 42 years ± 18 [standard deviation]; six men) were recruited from August 2019 to September 2020. Subgroups of the participants were injected with HP [1-(13)C]-pyruvate at resting, during plantar flexion exercise, or 5 minutes after exercise during recovery. In parallel, hydrogen 1 arterial spin labeling MRI was performed to estimate muscle tissue perfusion. An unpaired t test was used for comparing (13)C data among the states. RESULTS: At rest, HP [1-(13)C]-lactate and [1-(13)C]-alanine were detected in calf muscle, but [(13)C]-bicarbonate was negligible. During moderate flexion-extension exercise, total HP (13)C signals (tC) increased 2.8-fold because of increased muscle perfusion (P = .005), and HP [1-(13)C]-lactate–to-tC ratio increased 1.7-fold (P = .04). HP [(13)C]-bicarbonate–to-tC ratio increased 8.4-fold (P = .002) and returned to the resting level 5 minutes after exercise, whereas the lactate-to-tC ratio continued to increase to 2.3-fold as compared with resting (P = .008). CONCLUSION: Lactate and bicarbonate production from hyperpolarized (HP) [1–carbon 13 {(13)C}]-pyruvate in skeletal muscle rapidly reflected the onset and the termination of exercise. These results demonstrate the feasibility of imaging skeletal muscle metabolism using HP [1-(13)C]-pyruvate MRI and the sensitivity of in vivo pyruvate metabolism to exercise states. © RSNA, 2021 Online supplemental material is available for this article.

The TCA cycle is a central metabolic pathway for energy production and biosynthesis. A major control point of metabolic flux through the cycle is the decarboxylation of 2-ketoglutarate by the TCA cycle enzyme 2-ketoglutarate dehydrogenase (2-KGDH). In this project, we developed (13)C labeled 2-ketoglutarate derivatives to monitor 2-KGDH activity in vivo. (13)C NMR analysis of liver extracts revealed that uniformly (13)C labeled 2-ketogutarate, in its cell permeable ester form, was rapidly taken up and hydrolyzed in liver and underwent extensive metabolism to produce labeled glutamate, succinate, lactate and other metabolites. Diethyl [1,2-(13)C(2)]-2-ketoglutarate was successfully polarized by dynamic nuclear polarization and within seconds after injection into rats, the probe produced hyperpolarized [(13)C]bicarbonate in the liver reflecting flux through the TCA cycle. These experiments demonstrate that this tracer offers the possibility of directly monitoring flux through 2-KGDH in vivo.

Transient disruption of the blood-brain barrier (BBB) with focused ultrasound (FUS) is an emerging clinical method to facilitate targeted drug delivery to the brain. The focal noninvasive disruption of the BBB can be applied to promote local delivery of hyperpolarized substrates. In this study, we investigated effects of FUS on imaging brain metabolism using two hyperpolarized (13)C-labeled substrates in rodents: [1-(13)C]pyruvate and [1-(13)C]glycerate. The BBB is a rate-limiting factor for pyruvate delivery to the brain and glycerate minimally passes through the BBB. First, cerebral imaging with hyperpolarized [1-(13)C]pyruvate resulted in an increase in total (13)C signals (p = 0.05) after disrupting the BBB with FUS. Significantly higher levels of both [1-(13)C]lactate (lactate/total (13)C signals, p = 0.01) and [(13)C]bicarbonate (p = 0.008) were detected in the FUS-applied brain region as compared to the contralateral FUS-unaffected normal-appearing brain region. Application of FUS without opening the BBB in a separate group of rodents resulted in comparable lactate and bicarbonate productions between the FUS-applied and the contralateral brain regions. Second, (13)C imaging with hyperpolarized [1-(13)C]glycerate after opening the BBB showed increased [1-(13)C]glycerate delivery to the FUS-applied region (p = 0.04) relative to the contralateral side, and [1-(13)C]lactate production was consistently detected from the FUS-applied region. Our findings suggest that FUS accelerates the delivery of hyperpolarized molecules across the BBB and provides enhanced sensitivity to detect metabolic products in the brain; therefore, hyperpolarized (13)C imaging with FUS may provide new opportunities to study cerebral metabolic pathways as well as various neurological pathologies.

Purpose/Objective(s) Radiation therapy is commonly used for treatment of patients with intrathoracic malignancies, such as lung cancer, esophageal cancer, and lymphoma. Radiation-Induced Heart Disease (RIHD) is a major source of morbidity and mortality in patients receiving thoracic radiation. Therefore, there is an acute need for development of non-invasive approaches for detection of RIHD at a stage that offers potential for early intervention and reversibility. Cardiac mitochondrial dysfunction is a hallmark of radiation-induced cardiac injury. During aerobic respiration, pyruvate enters the tricarboxylic acid cycle and is metabolized into bicarbonate in mitochondria. We hypothesized that radiation-induced mitochondrial dysfunction results in decreased conversion of pyruvate to bicarbonate in the mitochondria and increased conversion to lactate in cytosol. We sought to non-invasively assess radiation-induced changes in mitochondrial myocardial metabolism by tracking the fate of hyperpolarized (HP) C-13 pyruvate utilization using Magnetic Resonance Spectroscopy (MRS). Materials/Methods Sprague-Dawley rats (n = 10) underwent baseline echocardiography and HP C-13 pyruvate MRS. Rats in the cardiac radiation group (n = 5) underwent image-guided cardiac radiation with cone-beam CT, to a total dose of 40 Gy in 5 fractions. All rats underwent repeat echocardiography and hyperpolarized C-13 pyruvate MRS one week later. Results For the first time, we have demonstrated feasibility of employing HP C-13 pyruvate MRS for detecting radiation-induced myocardial mitochondrial metabolic changes in a pre-clinical rat model. In the cardiac radiation group, C-13 pyruvate MRS demonstrated a statistically significant decrease in cardiac bicarbonate-to-lactate ratio compared to pre-radiation baseline (P = 0.02, one-tailed paired t-test), suggesting increased metabolism of pyruvate into lactate in the cytoplasm (at the expense of metabolism into bicarbonate in the mitochondria) due to mitochondrial dysfunction. No significant decrease in this ratio was observed in the non-irradiated, age matched controls (P = 0.90). No significant changes in left ventricular ejection fraction or global longitudinal strain were observed in either the cardiac irradiation or control group of rats at this time point. Conclusion Radiation-induced myocardial mitochondrial dysfunction is an early event and can be detected in vivo by hyperpolarized C-13 pyruvate MRS within 1 week after radiation, and prior to onset of echocardiographic changes. Due to its non-invasive nature, this technology has the potential to serve as a platform for building radiation-focused cardio-oncology programs for early detection and mitigation of radiation-induced cardiac injury in hundreds of thousands of patients receiving thoracic radiation annually.

PURPOSE: Noninvasive imaging with hyperpolarized pyruvate can capture in vivo cardiac metabolism. For proper quantification of the metabolites and optimization of imaging parameters, understanding MR characteristics such as T(2)*s of the hyperpolarized signals is critical. This study is to measure in vivo cardiac T(2)*s of hyperpolarized [1-(13)C]pyruvate and the products in rodents and humans. METHODS: A dynamic (13)C multi-echo spiral imaging sequence that acquires [(13)C]bicarbonate, [1-(13)C]lactate, and [1-(13)C]pyruvate images in an interleaved manner was implemented for a clinical 3T system. T(2)* of each metabolite was calculated from the multi-echo images by fitting the signal decay of each region of interest mono-exponentially. The performance of measuring T(2)* using the sequence was validated using a (13)C phantom, then with rodents following a bolus injection of hyperpolarized [1-(13)C]pyruvate. In humans, T(2)* of each metabolite was calculated for left ventricle (LV), right ventricle (RV) and myocardium. RESULTS: Cardiac T(2)*s of hyperpolarized [1-(13)C]pyruvate, [1-(13)C]lactate and [(13)C]bicarbonate in rodents were measured as 24.9 ± 5.0, 16.4 ± 4.7, and 16.9 ± 3.4 ms, respectively. In humans, T(2)* of [1-(13)C]pyruvate was 108.7 ± 22.6 ms in LV and 129.4 ± 8.9 ms in RV. T(2)* of [1-(13)C]lactate was 40.9 ± 8.3, 44.2 ± 5.5, and 43.7 ± 9.0 ms in LV, RV and myocardium, respectively. T(2)* of [(13)C]bicarbonate in myocardium was 64.4 ± 2.5 ms. The measurements were reproducible and consistent over time after the pyruvate injection. CONCLUSIONS: The proposed metabolite-selective multi-echo spiral imaging sequence reliably measures in vivo cardiac T(2)*s of hyperpolarized [1-(13)C]pyruvate and products.

Background Glioblastoma remains incurable despite treatment with surgery, radiation therapy, and cytotoxic chemotherapy, prompting the search for a metabolic pathway unique to glioblastoma cells.13C MR spectroscopic imaging with hyperpolarized pyruvate can demonstrate alterations in pyruvate metabolism in these tumors. Methods Three patients with diagnostic MRI suggestive of a glioblastoma were scanned at 3 T 1–2 days prior to tumor resection using a 13C/1H dual-frequency RF coil and a 13C/1H-integrated MR protocol, which consists of a series of 1H MR sequences (T2 FLAIR, arterial spin labeling and contrast-enhanced [CE] T1) and 13C spectroscopic imaging with hyperpolarized [1-13C]pyruvate. Dynamic spiral chemical shift imaging was used for 13C data acquisition. Surgical navigation was used to correlate the locations of tissue samples submitted for histology with the changes seen on the diagnostic MR scans and the 13C spectroscopic images. Results Each tumor was histologically confirmed to be a WHO grade IV glioblastoma with isocitrate dehydrogenase wild type. Total hyperpolarized 13C signals detected near the tumor mass reflected altered tissue perfusion near the tumor. For each tumor, a hyperintense [1-13C]lactate signal was detected both within CE and T2-FLAIR regions on the 1H diagnostic images (P = .008). [13C]bicarbonate signal was maintained or decreased in the lesion but the observation was not significant (P = .3). Conclusions Prior to surgical resection, 13C MR spectroscopic imaging with hyperpolarized pyruvate reveals increased lactate production in regions of histologically confirmed glioblastoma.

Hyperpolarized (HP) 13C magnetic resonance is an emerging modality that provides a dramatically enhanced signal-to-noise ratio (SNR) over conventional magnetic resonance imaging (MRI). By injecting a biologically active hyperpolarized substrate and then imaging both the injected substrate and its metabolic products, this technology permits, for the first time, real-time in vivo imaging of dynamic metabolic processes in the body. Among the polarizable substrates, observing key aspects of glucose metabolism with HP 13C-labeled pyruvate (Pyr) has generated considerable clinical excitement with an initial focus on cancer and cardiac applications. However, reliably quantifying in vivo HP 13C studies, particularly in terms of biomarkers closely tied to underlying metabolic processes remains a challenge. Among the complicating factors are supraphysiological bolus injections, MRI relaxation effects, underlying enzyme kinetics, and chemical exchange processes. In this chapter, we review these effects and discuss approaches currently used to quantify in vivo data. Quantitation is of particular importance for comparing results across sites and informing on the underlying pathologies.

Despite the textbook pronouncements about muscle lactate (lac) as a glycolytic end-product, which many use to characterize a hypoxia threshold, and as a precursor for recycling in liver, recent studies have begun to challenge these edicts. Indeed, experiment observations have detected lac formation under aerobic conditions and lac shuttling between muscle fibers and within the myocyte as a metabolic precursor. The observations have spawned novel hypothesis to explain aerobic lac formation as part of a dynamic glycogen shunt during a muscle twitch. Since nuclear magnetic resonance (NMR) has detected a large energy fluctuation during each contraction, the cell must dynamically restore glycogen in order to sustain any extended period of contractions. Because of enhanced signal sensitivity to measure kinetics in seconds, dynamic nuclear polarization (DNP) NMR experiments can test key elements of these models. Both the glycogen shunt and intracellular lac shuttle models require rapid mobilization and utilization of lac. If lac cannot mobilize rapidly, then the results invalidate immediately the hypotheses in both models. Can muscle then use and mobilize lac? Indeed, the DNP experiment results show rapid lac utilization, which appears to support the validity of the glycogen shunt and lac shuttle model.

Spatial resolution of metabolic imaging with hyperpolarized 13C-labeled substrates is limited owing to the multidimensional nature of spectroscopic imaging and the transient characteristics of dissolution dynamic nuclear polarization. In this study, a patch-based algorithm (PA) is proposed to enhance spatial resolution of hyperpolarized 13C human brain images by exploiting compartmental information from the corresponding high-resolution 1H images. PA was validated in simulation and phantom studies. Effects of signal-to-noise ratio, upsampling factor, segmentation, and slice thickness on reconstructing 13C images were evaluated in simulation. PA was further applied to low-resolution human brain metabolite maps of hyperpolarized [1-13C] pyruvate and [1-13C] lactate with 3 compartment segmentations (gray matter, white matter, and cerebrospinal fluid). The performance of PA was compared with other conventional interpolation methods (sinc, nearest-neighbor, bilinear, and spline interpolations). The simulation and the phantom tests showed that PA improved spatial resolution by up to 8 times and enhanced the image contrast without compromising quantification accuracy or losing the intracompartment signal inhomogeneity, even in the case of low signal-to-noise ratio or inaccurate segmentation. PA also improved spatial resolution and image contrast of human 13C brain images. Dynamic analysis showed consistent performance of the proposed method even with the signal decay along time. In conclusion, PA can enhance low-resolution hyperpolarized 13C images in terms of spatial resolution and contrast by using a priori knowledge from high-resolution 1H magnetic resonance imaging while preserving quantification accuracy and intracompartment signal inhomogeneity.

Spatial resolution of metabolic imaging with hyperpolarized 13C-labeled substrates is limited owing to the multidimensional nature of spectroscopic imaging and the transient characteristics of dissolution dynamic nuclear polarization. In this study, a patch-based algorithm (PA) is proposed to enhance spatial resolution of hyperpolarized 13C human brain images by exploiting compartmental information from the corresponding high-resolution 1H images. PA was validated in simulation and phantom studies. Effects of signal-to-noise ratio, upsampling factor, segmentation, and slice thickness on reconstructing 13C images were evaluated in simulation. PA was further applied to low-resolution human brain metabolite maps of hyperpolarized [1-13C] pyruvate and [1-13C] lactate with 3 compartment segmentations (gray matter, white matter, and cerebrospinal fluid). The performance of PA was compared with other conventional interpolation methods (sinc, nearest-neighbor, bilinear, and spline interpolations). The simulation and the phantom tests showed that PA improved spatial resolution by up to 8 times and enhanced the image contrast without compromising quantification accuracy or losing the intracompartment signal inhomogeneity, even in the case of low signal-to-noise ratio or inaccurate segmentation. PA also improved spatial resolution and image contrast of human 13C brain images. Dynamic analysis showed consistent performance of the proposed method even with the signal decay along time. In conclusion, PA can enhance low-resolution hyperpolarized 13C images in terms of spatial resolution and contrast by using a priori knowledge from high-resolution 1H magnetic resonance imaging while preserving quantification accuracy and intracompartment signal inhomogeneity.

Purpose To evaluate the utility of hyperpolarized [1-13 C]-l-lactate to detect hepatic pyruvate carboxylase activity in vivo under fed and fasted conditions. Methods [1-13 C]-labeled sodium L-lactate was polarized using a dynamic nuclear polarizer. Polarization level and the T1 were measured in vitro in a 3 Telsa MR scanner. Two groups of healthy rats (fasted vs. fed) were prepared for in vivo studies. Each rat was anesthetized and intravenously injected with 60-mM hyperpolarized [1-13 C]-l-lactate, immediately followed by dynamic acquisition of 13 C (carbon-13) MR spectra from the liver at 3 Tesla. The dosage-dependence of the 13 C-products was also investigated by performing another injection of an equal volume of 30-mM hyperpolarized [1-13 C]-l-lactate. Results T1 and liquid polarization level of [1-13 C]-l-lactate were estimated as 67.8 s and 40.0%, respectively. [1-13 C]pyruvate and [1-13 C]alanine, [13 C]bicarbonate ( HCO 3 - ) and [1-13 C]aspartate were produced from hyperpolarized [1-13 C]-l-lactate in rat liver. Smaller HCO 3 - and larger aspartate were measured in the fed group compared to the fasted group. Pyruvate and alanine production were increased in proportion to the lactate concentration, whereas the amount of HCO 3 - and aspartate production was consistent between 30-mM and 60-mM lactate injections. Conclusion This study demonstrates that a unique biomarker of pyruvate carboxylase flux, the appearance of [1-13 C]aspartate from [1-13 C]-l-lactate, is sensitive to nutritional state and may be monitored in vivo at 3 Tesla. Because [13 C] HCO 3 - is largely produced by pyruvate dehydrogenase flux, these results suggest that the ratio of [1-13 C]aspartate and [13 C] HCO 3 - (aspartate/ HCO 3 - ) reflects the saturable pyruvate carboxylase/pyruvate dehydrogenase enzyme activities.

Tissue pH is tightly regulated in vivo, being a sensitive physiological biomarker. Advent of dissolution dynamic nuclear polarization (DNP) and its translation to humans stimulated development of pH-sensitive agents. However, requirements of DNP probes such as biocompatibility, signal sensitivity, and spin-lattice relaxation time (T(1)) complicate in vivo translation of the agents. Here, we developed a (13)C-labeled alanine derivative, [1-(13)C]-L-alanine ethyl ester, as a viable DNP probe whose chemical shift is sensitive to the physiological pH range, and demonstrated the feasibility in phantoms and rat livers in vivo. Alanine ethyl ester readily crosses cell membrane while simultaneously assessing extracellular and intracellular pH in vivo. Following cell transport, [1-(13)C]-L-alanine ethyl ester is instantaneously hydrolyzed to [1-(13)C]-L-alanine, and subsequently metabolized to [1-(13)C]lactate and [(13)C]bicarbonate. The pH-insensitive alanine resonance was used as a reference.

Background: Dissolution dynamic nuclear polarization is a novel method that increases more than 10,000-fold the 13 C signal-to-noise ratio and emerges as a useful tool for detection of molecules much less prevalent than water in the human body. Hyperpolarized [1- 13 C] pyruvate (HP) permits the noninvasive, nonradioactive study of metabolic flux via pyruvate dehydrogenase, a key enzyme in the complex cardiac adaptation to physical activity, energy sources, and in various disease states. The aim of this study is to define an analysis pipeline of multiparametric, non-steady-state, real-time human cardiac magnetic resonance spectroscopic investigations. Methods: HP was infused intravenously into five human subjects. Dynamic spectroscopic data was acquired for [1- 13 C]- pyruvate, 13 C-alanine, [1- 13 C]-lactate, and 13 C-bicarbonate at two consecutive times for each subject. Imaging data were exported into MATLAB. We employed a first-order kinetic model to fit the signal for the hyperpolarized metabolites assuming bidirectional flow between pyruvate and lactate as well as pyruvate and alanine. A gamma variate input function was used to model the initial pyruvate bolus. We also calculated non-parametric values to describe the signal for each metabolite, specifically area-under-curve (AUC) and time-to-peak (TTP). The repeatability analysis was performed using an ANOVA-based method. Results: Rate constants derived from the kinetic model: kPL 0.016 ± 0.008s -1 , kPA 0.012 ± 0.006s -1 , kPB 0.024±0.0095s -1 are of similar order of magnitude to prior studies. The reverse constant for lactate to pyruvate conversion (kLP) is relatively unchanged across the subjects (0.0099±0.0005), and reverse constant for alanine to pyruvate (kAP) conversion is almost negligible (0.0008 ± 0.0012). The model well describes the metabolites signal over time. Within subject repeatability was higher for TTP (lactate: 0.981 ± 0.018) ) than for rate constants (kPL 0.823 ± 0.153) and AUC (lactate 0.697 ± 0.244) with kPB having the lowest repeatability (0.325 ± 0.424) due to a single outlier. Conclusions: We present a real-time pipeline for analysis and modelling of pyruvate metabolism in the human heart and confirm that it is reproducible within the same subject under same conditions.

// Sui Seng Tee 1, 5, * , Jae Mo Park 1, 6, 7, * , Ralph E. Hurd 2 , Kyle R. Brimacombe 3, 4 , Matthew B. Boxer 3 , Tarik F. Massoud 1 , Brian K. Rutt 1 and Daniel M. Spielman 1 1 Department of Radiology, Stanford University, Stanford, CA, USA 2 Applied Sciences Laboratory, GE Healthcare, Menlo Park, CA, USA 3 National Center for Advancing Translational Sciences, NIH, Bethesda, MD, USA 4 NIH Chemical Genomics Center, Bethesda, MD, USA 5 Current/Present address: Memorial Sloan Kettering Cancer Center, New York, NY, USA 6 Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, USA 7 Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX, USA * These authors have contributed equally to this work Correspondence to: Sui Seng Tee, email: tees@mskcc.org Keywords: molecular imaging, metabolic imaging, hyperpolarized MRI, cancer metabolism, PKM2 Received: October 19, 2016 Accepted: July 06, 2017 Published: July 26, 2017 ABSTRACT Cancer metabolism has emerged as an increasingly attractive target for interfering with tumor growth. Small molecule activators of pyruvate kinase isozyme M2 (PKM2) suppress tumor formation but have an unknown effect on established tumors. We demonstrate that TEPP-46, a PKM2 activator, results in increased glucose consumption, providing the rationale for combining PKM2 activators with the toxic glucose analog, 2-deoxy-D-glucose (2-DG). Combination treatment resulted in reduced viability of a range of cell lines in standard cell culture conditions at concentrations of drugs that had no effect when used alone. This effect was replicated in vivo on established subcutaneous tumors. We further demonstrated the ability to detect acute metabolic differences in combination treatment using hyperpolarized magnetic resonance spectroscopy (MRS). Combination treated tumors displayed a higher pyruvate to lactate 13 C-label exchange 2 hr post-treatment. This ability to assess the effect of drugs non-invasively may accelerate the implementation and clinical translation of drugs that target cancer metabolism.

Reactive oxygen species (ROS) are essential cellular metabolites widely implicated in many diseases including cancer, inflammation, and cardiovascular and neurodegenerative disorders. Yet, ROS signaling remains poorly understood, and their measurements are a challenge due to high reactivity and instability. Here, we report the development of 13C-thiourea as a probe to detect and measure H2O2 dynamics with high sensitivity and spatiotemporal resolution using hyperpolarized 13C magnetic resonance spectroscopic imaging. In particular, we show 13C-thiourea to be highly polarizable and to possess a long spin–lattice relaxation time (T1), which enables real-time monitoring of ROS-mediated transformation. We also demonstrate that 13C-thiourea reacts readily with H2O2 to give chemically distinguishable products in vitro and validate their detection in vivo in a mouse liver. This study suggests that 13C-thiourea is a promising agent for noninvasive detection of H2O2 in vivo. More broadly, our findings outline a vi...

Hyperpolarized 13C magnetic resonance spectroscopy (MRS) provides unprecedented opportunities to obtain clinical diagnostic information through in vivo monitoring of metabolic pathways. The continuing advancement of this field relies on the identification of molecular probes that can effectively interrogate pathways critical to disease. In this report, we describe the synthesis, development, and in vivo application of sodium [1-13C]-glycerate ([13C]-Glyc) as a novel probe for evaluating glycolysis using hyperpolarized 13C MRS. This agent was prepared by a concise synthetic route and formulated for dynamic nuclear polarization. [13C]-Glyc displayed a high level of polarization and long spin–lattice relaxation time—both of which are necessary for future clinical investigations. In vivo spectroscopic studies with hyperpolarized [13C]-Glyc in rat liver furnished metabolic products, [13C]-labeled pyruvate and lactate, originating from glycolysis. The levels of production and relative intensities of these metabolites were directly correlated with the induced glycolytic state (fasted versus fed groups). This work establishes hyperpolarized [13C]-Glyc as a novel agent for clinically relevant 13C MRS studies of energy metabolism and further provides opportunities for evaluating intracellular redox states in biochemical investigations.

PURPOSE The intracellular lactate to pyruvate concentration ratio is a commonly used tissue assay biomarker of redox, being proportional to free cytosolic [NADH]/[NAD+ ]. In this study, we assessed the use of hyperpolarized [1-13 C]alanine and the subsequent detection of the intracellular products of [1-13 C]pyruvate and [1-13 C]lactate as a useful substrate for assessing redox levels in the liver in vivo. METHODS Animal experiments were conducted to measure in vivo metabolism at baseline and after ethanol infusion. A solution of 80-mM hyperpolarized [1-13 C]alanine was injected intravenously at baseline (n = 8) and 45 min after ethanol infusion (n = 4), immediately followed by the dynamic acquisition of 13 C MRS spectra. RESULTS In vivo rat liver spectra showed peaks from [1-13 C] alanine and the products of [1-13 C]lactate, [1-13 C]pyruvate, and 13 C-bicarbonate. A significantly increased 13 C-lactate/13 C-pyruvate ratio was observed after ethanol infusion (8.46 ± 0.58 at baseline versus 13.58 ± 0.69 after ethanol infusion; P

Carbon‐13 magnetic resonance spectroscopy (MRS) following oral intake of 13C‐labeled glucose is the gold standard for imaging glycogen metabolism in humans. However, the temporal resolution of previous studies has been >13 minutes. Here, we describe a high‐sensitivity 13C MRS method for imaging hepatic glycogen synthesis with a temporal resolution of 1 minute or less. Nuclear magnetic resonance spectra were acquired from the liver of 3 healthy volunteers, using a 13C clamshell radiofrequency transmit and paddle‐shaped array receive coils in a 3 Tesla magnetic resonance imaging system. Following a 15‐minute baseline 13C MRS scan of the liver, [1‐13C]‐glucose was ingested and 13C MRS data were acquired for an additional 1‐3 hours. Dynamic change of the hepatic glycogen synthesis level was analyzed by reconstructing the acquired MRS data with temporal resolutions of 30 seconds to 15 minutes. Plasma levels of 13C‐labeled glucose and lactate were measured using gas chromatography–mass spectrometry. While not detected at baseline 13C MRS, [1‐13C]‐labeled α‐glucose and β‐glucose and glycogen peaks accumulated rapidly, beginning as early as ~2 minutes after oral administration of [1‐13C]‐glucose. The [1‐13C]‐glucose signals peaked at ~5 minutes, whereas [1‐13C]‐glycogen peaked at ~25 minutes after [1‐13C]‐glucose ingestion; both signals declined toward baseline levels over the next 1‐3 hours. Plasma levels of 13C‐glucose and 13C‐lactate rose gradually, and approximately 20% of all plasma glucose and 5% of plasma lactate were 13C‐labeled by 2 hours after ingestion. Conclusion: We observed rapid accumulation of hepatic [1‐13C]‐glycogen following orally administered [1‐13C]‐glucose, using a dynamic 13C MRS method with a temporal resolution of 1 minute or less. Commercially available technology allows high temporal resolution studies of glycogen metabolism in the human liver., In this study, we describe a high‐sensitivity 13C MRS method for imaging hepatic glycogen synthesis with improved temporal resolution. We observed rapid accumulation of hepatic [1‐13C]‐glycogen following orally administered [1‐13C]‐glucose, using a dynamic 13C MRS method with a temporal resolution of 1 minute or less.

To assess whether simultaneous hyperpolarized C-13 magnetic resonance spectroscopy (MRS)/positron emission tomography (PET)/multiparametric magnetic resonance (mpMR) imaging is feasible in an orthotopic canine prostate cancer (PCa) model using a clinical PET/MR system and whether the combined imaging datasets can be fused with transrectal ultrasound (TRUS) in real time for multimodal image fusion-guided targeted biopsy of PCa. Institutional Animal Care and Use Committee approval was obtained for this study. Canine prostate adenocarcinoma (Ace-1) cells were orthotopically injected into the prostate of four dogs. Once tumor engraftment was confirmed by TRUS, simultaneous hyperpolarized C-13 MRS of [1-13C]pyruvate, PET (2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG), [68Ga]NODAGA-SCH1), and mpMR (T2W, DWI) imaging was performed using a clinical PET/MR system. Multimodality imaging data sets were then fused with TRUS and image-guided targeted biopsy was performed. Imaging results were then correlated with histological findings. Successful tumor engraftment was histologically confirmed in three of the four dogs (dogs 2, 3, and 4) and simultaneous C-13 MRS/PET/mpMR was feasible in all three. In dog 2, C-13 MRS showed increased lactate signal in the tumor (lactate/totalC = 0.47) whereas mpMR did not show any signal changes. In dog 3, [18F]FDG-PET (SUVmean = 1.90) and C-13 MRS (lactate/totalC = 0.59) showed elevated metabolic activity in the tumor. In dog 4, [18F]FDG (SUVmean = 2.43), [68Ga]NODAGA-SCH1 (SUVmean = 0.75), and C-13 MRS (Lac/totalC = 0.53) showed elevated uptake in tumor compared to control tissue and multimodal image fusion-guided biopsy of the tumor was successfully performed. Simultaneous C-13 MRS/PET/mpMR imaging and multimodal image fusion-guided biopsy is feasible in a canine PCa model.

Altered branched-chain amino acids (BCAAs) metabolism is a distinctive feature of various cancers and plays an important role in sustaining tumor proliferation and aggressiveness. Despite the therapeutic and diagnostic potentials, the role of BCAA metabolism in cancer and the activities of associated enzymes remain unclear. Due to its pivotal role in BCAA metabolism and rapid cellular transport, hyperpolarized 13C-labeled α-ketoisocaproate (KIC), the α-keto acid corresponding to leucine, can assess both BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase complex (BCKDC) activities via production of [1-13C]leucine or 13CO2 (and thus H13CO3−), respectively. Here, we investigated BCAA metabolism of F98 rat glioma model in vivo using hyperpolarized 13C-KIC. In tumor regions, we observed a decrease in 13C-leucine production from injected hyperpolarized 13C-KIC via BCAT compared to the contralateral normal-appearing brain, and an increase in H13CO3−, a catabolic product of KIC through the mitochondrial BCKDC. A parallel ex vivo13C NMR isotopomer analysis following steady-state infusion of [U-13C]leucine to glioma-bearing rats verified the increased oxidation of leucine in glioma tissue. Both the in vivo hyperpolarized KIC imaging and the leucine infusion study indicate that KIC catabolism is upregulated through BCAT/BCKDC and further oxidized via the citric acid cycle in F98 glioma.

Hyperpolarized [1-(13)C]pyruvate MRS provides a unique imaging opportunity to study the reaction kinetics and enzyme activities of in vivo metabolism because of its favorable imaging characteristics and critical position in the cellular metabolic pathway, where it can either be reduced to lactate (reflecting glycolysis) or converted to acetyl-coenzyme A and bicarbonate (reflecting oxidative phosphorylation). Cancer tissue metabolism is altered in such a way as to result in a relative preponderance of glycolysis relative to oxidative phosphorylation (i.e. Warburg effect). Although there is a strong theoretical basis for presuming that readjustment of the metabolic balance towards normal could alter tumor growth, a robust noninvasive in vivo tool with which to measure the balance between these two metabolic processes has yet to be developed. Until recently, hyperpolarized (13)C-pyruvate imaging studies had focused solely on [1-(13)C]lactate production because of its strong signal. However, without a concomitant measure of pyruvate entry into the mitochondria, the lactate signal provides no information on the balance between the glycolytic and oxidative metabolic pathways. Consistent measurement of (13)C-bicarbonate in cancer tissue, which does provide such information, has proven difficult, however. In this study, we report the reliable measurement of (13)C-bicarbonate production in both the healthy brain and a highly glycolytic experimental glioblastoma model using an optimized (13)C MRS imaging protocol. With the capacity to obtain signal in all tumors, we also confirm for the first time that the ratio of (13)C-lactate to (13)C-bicarbonate provides a more robust metric relative to (13)C-lactate for the assessment of the metabolic effects of anti-angiogenic therapy. Our data suggest a potential application of this ratio as an early biomarker to assess therapeutic effectiveness. Furthermore, although further study is needed, the results suggest that anti-angiogenic treatment results in a rapid normalization in the relative tissue utilization of glycolytic and oxidative phosphorylation by tumor tissue.

Purpose MRS of hyperpolarized [2-13C]pyruvate can be used to assess multiple metabolic pathways within mitochondria as the 13C label is not lost with the conversion of pyruvate to acetyl-CoA. This study presents the first MR spectroscopic imaging of hyperpolarized [2-13C]pyruvate in glioma-bearing brain. Methods Spiral chemical shift imaging with spectrally undersampling scheme (1042 Hz) and a hard-pulse excitation was exploited to simultaneously image [2-13C]pyruvate, [2-13C]lactate, and [5-13C]glutamate, the metabolites known to be produced in brain after an injection of hyperpolarized [2-13C]pyruvate, without chemical shift displacement artifacts. A separate undersampling scheme (890 Hz) was also used to image [1-13C]acetyl-carnitine. Healthy and C6 glioma-implanted rat brains were imaged at baseline and after dichloroacetate administration, a drug that modulates pyruvate dehydrogenase kinase activity. Results The baseline metabolite maps showed higher lactate and lower glutamate in tumor as compared to normal-appearing brain. Dichloroacetate led to an increase in glutamate in both tumor and normal-appearing brain. Dichloroacetate-induced %-decrease of lactate/glutamate was comparable to the lactate/bicarbonate decrease from hyperpolarized [1-13C]pyruvate studies. Acetyl-carnitine was observed in the muscle/fat tissue surrounding the brain. Conclusion Robust volumetric imaging with hyperpolarized [2-13C]pyruvate and downstream products was performed in glioma-bearing rat brains, demonstrating changes in mitochondrial metabolism with dichloroacetate. Magn Reson Med, 2015. © 2015 Wiley Periodicals, Inc.

Summary Traumatic brain injury (TBI) involves complex secondary injury processes following the primary injury. The secondary injury is often associated with rapid metabolic shifts and impaired brain function immediately after the initial tissue damage. Magnetic resonance spectroscopic imaging (MRSI) coupled with hyperpolarization of 13C-labeled substrates provides a unique opportunity to map the metabolic changes in the brain after traumatic injury in real-time without invasive procedures. In this report, we investigated two patients with acute mild TBI (Glasgow coma scale 15) but no anatomical brain injury or hemorrhage. Patients were imaged with hyperpolarized [1-13C]pyruvate MRSI 1 or 6 days after head trauma. Both patients showed significantly reduced bicarbonate (HCO3–) production, and one showed hyperintense lactate production at the injured sites. This study reports the feasibility of imaging altered metabolism using hyperpolarized pyruvate in patients with TBI, demonstrating the translatability and sensitivity of the technology to cerebral metabolic changes after mild TBI.

To facilitate diagnosis and staging of liver disease, sensitive and non-invasive methods for the measurement of liver metabolism are needed. This study used hyperpolarized (13)C-pyruvate to assess metabolic parameters in a CCl4 model of liver damage in rats. Dynamic 3D (13)C chemical shift imaging data from a volume covering kidney and liver were acquired from 8 control and 10 CCl4-treated rats. At 12 time points at 5 s temporal resolution, we quantified the signal intensities and established time courses for pyruvate, alanine, and lactate. These measurements were compared with standard liver histology and an alanine transaminase (ALT) enzyme assay using liver tissue from the same animals. All CCl4-treated but none of the control animals showed histological liver damage and elevated ALT enzyme levels. In agreement with these results, metabolic imaging revealed an increased alanine/pyruvate ratio in liver of CCl4-treated rats, which is indicative of elevated ALT activity. Similarly, lactate/pyruvate ratios were higher in CCl4-treated compared with control animals, demonstrating the presence of inflammation. No significant differences in metabolite ratios were observed in kidney or vasculature. Thus this work shows that metabolic imaging using (13)C-pyruvate can be a successful tool to non-invasively assess liver damage in vivo.

Cancer cells reprogram their metabolism to maintain viability via genetic mutations and epigenetic alterations, expressing overall dynamic heterogeneity. The complex relaxation mechanisms of nuclear spins provide unique and convertible tissue contrasts, making magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) pertinent imaging tools in both clinics and research. In this review, we summarized MR methods that visualize tumor characteristics and its metabolic phenotypes on an anatomical, microvascular, microstructural, microenvironmental, and metabolomics scale. The review will progress from the utilities of basic spin-relaxation contrasts in cancer imaging to more advanced imaging methods that measure tumor-distinctive parameters such as perfusion, water diffusion, magnetic susceptibility, oxygenation, acidosis, redox state, and cell death. Analytical methods to assess tumor heterogeneity are also reviewed in brief. Although the clinical utility of tumor heterogeneity from imaging is debatable, the quantification of tumor heterogeneity using functional and metabolic MR images with development of robust analytical methods and improved MR methods may offer more critical roles of tumor heterogeneity data in clinics. MRI/MRS can also provide insightful information on pharmacometabolomics, biomarker discovery, disease diagnosis and prognosis, and treatment response. With these future directions in mind, we anticipate the widespread utilization of these MR-based techniques in studying in vivo cancer biology to better address significant clinical needs.

The tricarboxylic acid (TCA) cycle performs an essential role in the regulation of energy and metabolism, and deficiencies in this pathway are commonly correlated with various diseases. However, the development of non-invasive techniques for the assessment of the cycle in vivo has remained challenging. In this work, the applicability of a novel imaging agent, [1,4-13C]-diethylsuccinate, for hyperpolarized 13C metabolic imaging of the TCA cycle was explored. In vivo spectroscopic studies were conducted in conjunction with in vitro analyses to determine the metabolic fate of the imaging agent. Contrary to previous reports (Zacharias NM et al. J. Am. Chem. Soc. 2012; 134: 934–943), [13C]-labeled diethylsuccinate was primarily metabolized to succinate-derived products not originating from TCA cycle metabolism. These results illustrate potential issues of utilizing dialkyl ester analogs of TCA cycle intermediates as molecular probes for hyperpolarized 13C metabolic imaging. Copyright © 2014 John Wiley & Sons, Ltd.

Hyperpolarized (13)C MRS allows the in vivo assessment of pyruvate dehydrogenase complex (PDC) flux, which converts pyruvate to acetyl-coenzyme A (acetyl-CoA). [1-(13)C]pyruvate has been used to measure changes in cardiac PDC flux, with demonstrated increase in (13)C-bicarbonate production after dichloroacetate (DCA) administration. With [1-(13)C]pyruvate, the (13)C label is released as (13 CO2 /(13)C-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 (13)C label into the TCA (tricarboxylic acid) cycle and measure [5-(13)C]glutamate as well as study changes in [1-(13)C]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 (13)C labeling of [5-(13)C]glutamate, and a considerable increase in [1-(13)C]acetylcarnitine and [1,3-(13)C]acetoacetate peaks. Dobutamine resulted in an increased labeling of [2-(13)C]lactate, [2-(13)C]alanine and [5-(13)C]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.