66 research outputs found
Development of Novel 3D NMR-Compatible Bioreactors and Dynamic Nuclear Polarization (DNP) Techniques for the Study of Real-Time Metabolism
It is well known that 13C NMR spectroscopy (as well as other nuclei) has been limited in its ability to characterize metabolic states due to its low sensitivity. This low sensitivity is a result of the low natural abundance of 13C (1.1%) in addition to the low gyromagnetic ratio of carbon. Traditionally, this is compensated by high sample concentrations as well as the introduction of 13C‐enriched substrates. Dynamic nuclear polarization (DNP) results from the coupling of nuclear spins in an amorphous solid, at ~1°K, to unpaired electrons of a stable organic free radical and their subsequent irradiation. Recent advances in the field of low temperature physics, electronic paramagnetic resonance (EPR) and NMR have provided for the adaptation of the DNP technique, to metabolically appreciable systems. NMR‐compatible engineered bioreactor and 3D perfusion systems have been used to study cellular metabolism on the order of hours primarily by 31P and 13C metabolic tracers. Due to the inherent ability to translate in vitro derived biomarkers to clinical in vivo MRI/MRSI, these model systems have been of great interest, though they have been limited by low sensitivity. In this dissertation, methods are described to combine hyperpolarized techniques with an NMR‐compatible bioreactor and observe real time metabolism in a fluidized packed bed design of rat hepatoma cells (JM1). This was extended to a novel hollow fiber bioreactor design, which provided the advantage of immobilization of cells and tissues for imaging. Prostate adenocarcinoma cells (PC3) and primary prostate tissue slices were shown to be viable for greater than 2 days demonstrating the long‐term viability of these culture systems. Real time metabolism via hyperpolarized MR was also observed in the hollow fiber cartridge with the potential for future primary culture experiments. Additionally, new hyperpolarized probes and techniques (secondary and multi‐probe polarization) that allow the study of metabolism, physiology (pH, apoptosis/necrosis, bioenergetics), and ligand‐host binding were developed and tested. In summary, the findings of this dissertation research demonstrate the feasibility of fusing hyperpolarized MR with new NMR‐compatible bioreactors to provide a robust platform for studying biological systems as well as chemical reactions useful in drug design and development
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Editorial commentary for the special issue: technological developments in hyperpolarized 13C imaging-toward a deeper understanding of tumor metabolism in vivo.
Noninvasive in vivo imaging of diabetes-induced renal oxidative stress and response to therapy using hyperpolarized 13C dehydroascorbate magnetic resonance.
Oxidative stress has been proposed to be a unifying cause for diabetic nephropathy and a target for novel therapies. Here we apply a new endogenous reduction-oxidation (redox) sensor, hyperpolarized (HP) (13)C dehydroascorbate (DHA), in conjunction with MRI to noninvasively interrogate the renal redox capacity in a mouse diabetes model. The diabetic mice demonstrate an early decrease in renal redox capacity, as shown by the lower in vivo HP (13)C DHA reduction to the antioxidant vitamin C (VitC), prior to histological evidence of nephropathy. This correlates with lower tissue reduced glutathione (GSH) concentration and higher NADPH oxidase 4 (Nox4) expression, consistent with increased superoxide generation and oxidative stress. ACE inhibition restores the HP (13)C DHA reduction to VitC with concomitant normalization of GSH concentration and Nox4 expression in diabetic mice. HP (13)C DHA enables rapid in vivo assessment of altered redox capacity in diabetic renal injury and after successful treatment
Generating contrast in hyperpolarized 13C MRI using ligand–receptor interactions
We report the imaging of β-cyclodextrin/benzoic acid binding at 14T using hyperpolarized 13C magnetic resonance (MR). Benzoic acid was polarized using a dynamic nuclear polarization (DNP) approach and combined with β-cyclodextrin in aqueous solution. As anticipated, decreases in the spin-lattice relaxation constant (T1) were observed with decreases in the ligand/ receptor ratio. The calculated log K was approximately 1.7, similar to previously reported binding constants. Hyperpolarized [1-13C] benzoic acid was used to interrogate solutions of variable β-cyclodextrin concentration, with the mixtures imaged at 14T using a 3D frequency-selective MR sequence. Differences in β-cyclodextrin concentration were easily visualized. These results suggest that hyperpolarized 13C MR could be used in vivo to determine the presence, and density of receptors for a given ligand-receptor pair
Hyperpolarized 13C Spectroscopic Evaluation of Oxidative Stress in a Rodent Model of Steatohepatitis.
Nonalcoholic fatty liver disease (NAFLD) has become highly prevalent, now considered the most common liver disease in the western world. Approximately one-third of patients with NASH develop non-alchoholic steatohepatitis (NASH), histologically defined by lobular and portal inflammation, and accompanied by marked oxidative stress. Patients with NASH are at increased risk for cirrhosis and hepatocellular carcinoma, and diagnosis currently requires invasive biopsy. In animal models of NASH, particularly the methionine-choline deficient (MCD) model, profound changes are seen in redox enzymes and key intracellular antioxidants. To study antioxidant status in NASH non-invasively, we applied the redox probe hyperpolarized [1-13C] dehydroascorbic acid (HP DHA), which is reduced to Vitamin C (VitC) rapidly in the normal liver. In MCD mice, we observed a significant decrease in HP DHA to VitC conversion that accompanied hepatic fat deposition. When these animals were subsequently placed on a normal diet, resonance ratios reverted to those seen in control mice. These findings suggest that HP DHA, a potentially clinically translatable imaging agent, holds special promise in imaging NASH and other metabolic syndromes, to monitor disease progression and response to targeted therapies
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Imaging glutathione depletion in the rat brain using ascorbate-derived hyperpolarized MR and PET probes.
Oxidative stress is a critical feature of several common neurologic disorders. The brain is well adapted to neutralize oxidative injury by maintaining a high steady-state concentration of small-molecule intracellular antioxidants including glutathione in astrocytes and ascorbic acid in neurons. Ascorbate-derived imaging probes for hyperpolarized 13C magnetic resonance spectroscopy and positron emission tomography have been used to study redox changes (antioxidant depletion and reactive oxygen species accumulation) in vivo. In this study, we applied these imaging probes to the normal rat brain and a rat model of glutathione depletion. We first studied hyperpolarized [1-13C]dehydroascorbate in the normal rat brain, demonstrating its robust conversion to [1-13C]vitamin C, consistent with rapid transport of the oxidized form across the blood-brain barrier. We next showed that the kinetic rate of this conversion decreased by nearly 50% after glutathione depletion by diethyl maleate treatment. Finally, we showed that dehydroascorbate labeled for positron emission tomography, namely [1-11C]dehydroascorbate, showed no change in brain signal accumulation after diethyl maleate treatment. These results suggest that hyperpolarized [1-13C]dehydroascorbate may be used to non-invasively detect oxidative stress in common disorders of the brain
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Elevated Tumor Lactate and Efflux in High-grade Prostate Cancer demonstrated by Hyperpolarized 13C Magnetic Resonance Spectroscopy of Prostate Tissue Slice Cultures.
Non-invasive assessment of the biological aggressiveness of prostate cancer (PCa) is needed for men with localized disease. Hyperpolarized (HP) 13C magnetic resonance (MR) spectroscopy is a powerful approach to image metabolism, specifically the conversion of HP [1-13C]pyruvate to [1-13C]lactate, catalyzed by lactate dehydrogenase (LDH). Significant increase in tumor lactate was measured in high-grade PCa relative to benign and low-grade cancer, suggesting that HP 13C MR could distinguish low-risk (Gleason score ≤3 + 4) from high-risk (Gleason score ≥4 + 3) PCa. To test this and the ability of HP 13C MR to detect these metabolic changes, we cultured prostate tissues in an MR-compatible bioreactor under continuous perfusion. 31P spectra demonstrated good viability and dynamic HP 13C-pyruvate MR demonstrated that high-grade PCa had significantly increased lactate efflux compared to low-grade PCa and benign prostate tissue. These metabolic differences are attributed to significantly increased LDHA expression and LDH activity, as well as significantly increased monocarboxylate transporter 4 (MCT4) expression in high- versus low- grade PCa. Moreover, lactate efflux, LDH activity, and MCT4 expression were not different between low-grade PCa and benign prostate tissues, indicating that these metabolic alterations are specific for high-grade disease. These distinctive metabolic alterations can be used to differentiate high-grade PCa from low-grade PCa and benign prostate tissues using clinically translatable HP [1-13C]pyruvate MR
Metabolic adaptation supports enhanced macrophage efferocytosis in limited-oxygen environments
Effect of Oxygen Concentration on Viability and Metabolism in a Fluidized-Bed Bioartificial Liver Using 31 P and 13 C NMR Spectroscopy
Many oxygen mass-transfer modeling studies have been performed for various bioartificial liver (BAL) encapsulation types; yet, to our knowledge, there is no experimental study that directly and noninvasively measures viability and metabolism as a function of time and oxygen concentration. We report the effect of oxygen concentration on viability and metabolism in a fluidized-bed NMR-compatible BAL using in vivo 31P and 13C NMR spectroscopy, respectively, by monitoring nucleotide triphosphate (NTP) and 13C-labeled nutrient metabolites, respectively. Fluidized-bed bioreactors eliminate the potential channeling that occurs with packed-bed bioreactors and serve as an ideal experimental model for homogeneous oxygen distribution. Hepatocytes were electrostatically encapsulated in alginate (avg. diameter, 500 μm; 3.5×107 cells/mL) and perfused at 3 mL/min in a 9-cm (inner diameter) cylindrical glass NMR tube. Four oxygen treatments were tested and validated by an in-line oxygen electrode: (1) 95:5 oxygen:carbon dioxide (carbogen), (2) 75:20:5 nitrogen:oxygen:carbon dioxide, (3) 60:35:5 nitrogen:oxygen:carbon dioxide, and (4) 45:50:5 nitrogen:oxygen:carbon dioxide. With 20% oxygen, β-NTP steadily decreased until it was no longer detected at 11 h. The 35%, 50%, and 95% oxygen treatments resulted in steady β-NTP levels throughout the 28-h experimental period. For the 50% and 95% oxygen treatment, a 13C NMR time course (∼5 h) revealed 2-13C-glycine and 2-13C-glucose to be incorporated into [2-13C-glycyl]glutathione (GSH) and 2-13C-lactate, respectively, with 95% having a lower rate of lactate formation. 31P and 13C NMR spectroscopy is a noninvasive method for determining viability and metabolic rates. Modifying tissue-engineered devices to be NMR compatible is a relatively easy and inexpensive process depending on the bioreactor shape
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