Rationale and Objectives
Recent reports suggest that cancer cells may use glutamine, instead of glucose, as an alternative source of metabolic energy. This suggests that hyperpolarized 13 C glutamine may be useful as a magnetic resonance spectroscopy (MRS) imaging agent for detecting changes in glutamine metabolism in cancerous cells or tissues.
Materials and Methods
Synthesis of [5- 13 C-4- 2 H 2 ]-L-glutamine was accomplished through a seven-step synthetic pathway with a 44% overall yield. The introduction of two stable isotopes was performed by a NaB 2 H 4 -mixed anhydride reduction and K 13 CN-nuclophilic substitution, respectively. The desired [5- 13 C-4- 2 H 2 ]-L-glutamine was successfully obtained by a one-pot reaction of deprotection and controlled cyanide hydrolysis. Hyperpolarized [5- 13 C-4- 2 H 2 ]-L-glutamine samples were tested in human glioma cells (myc upregulated glia cells, SF188-Bcl-x L ). MRS signals were obtained with a 9.4 Tesla 89-mm bore nuclear magnetic resonance spectrometer and a direct-detection multi-nuclear probe.
Results
The initial degree of polarization for [5- 13 C-4- 2 H 2 ]-L-glutamine was ∼5% and the initial 13 C signal to noise ratio was ∼100:1. Glutamate was detected within seconds after the injection of hyperpolarized glutamine into the cells. The ratio of glutamate to glutamine was very high, indicating rapid conversion to glutamate. Similar cell uptake studies using [ 3 H]-L-glutamine also demonstrated cell uptakes higher than that of [ 18 F]fluorodeoxyglucose.
Conclusion
We are reporting the first example of using specifically deuterated [5- 13 C-4- 2 H 2 ]-L-glutamine in conjunction with hyperpolarized MRS for studying “glutaminolysis” in proliferating tumor cells.
It is well-known that cancer cells actively consume glucose as their energy source under aerobic conditions leading to the formation of lactate. This is referred to as the “Warburg effect” . This increased aerobic glycolysis in major tumor types is the foundation for using [ 18 F]fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging as a tool in diagnosing cancer . Yet, some malignant tumors show ostensibly negative FDG-PET scans and cannot be reliably detected with FDG-PET. For example, primary tumors of the prostate that have not metastasized are generally not enhanced by FDG . In addition, FDG-PET only provides information about glucose uptake (Glut) and hexokinase levels. Inflammation interferes with interpretation because it will show up on the scan as a false positive. Recently, a series of articles have suggested that the FDG-negative tumors may use a different metabolic pathway, dubbed “glutaminolysis” . The results, at least partially, provide a probable explanation for the observation that FDG-PET sometimes fails to spot tumors in cancer patients. Recent reports have indicated that glutamine utilization (glutaminolysis) can be linked to upregulations of the oncogene, myc . Normally, upregulation of the oncogene, myc, as part of PI3K/Akt/mTOR signal pathway, stimulates glucose uptake and utilization through an aerobic glycolysis pathway leading to the production of lactic acid. However, during starvation or other stressed situations, cancer cells will switch their metabolic energy source from glucose to amino acids, such as glutamine, and other more abundant metabolites .
There is a high concentration of glutamine (0.5–1.0 mM) in the blood circulation and it has the highest concentration among all amino acids. Glutamine is a highly active molecule that can provide a source of nitrogen for various building blocks in the cells and can also be used as a source of energy. Actively proliferating tumors with a significant upregulation of myc gene can use glutamine to produce ATP as well as NADPH and the excess lactate . The major difference between aerobic glycolysis and glutaminolysis is that the latter needs functioning mitochondria and an active tricarboxylic acid (TCA) cycle. Glutamine on entering the cell releases a molecule of ammonia from the amide group and then glutamine transaminase strips off the second molecule of ammonia. Alpha-ketoglutarate is the entrance point to the TCA cycle for this metabolic pathway for energy production. To date, regulation of glutaminolysis has not been examined extensively. In a preliminary study, the oncogene myc was found to transcriptionally regulate glutamine uptake and glutaminase, which converts glutamine to glutamate . It has also been found that cancers with high levels of myc are glutamine addicted and undergo apoptosis when deprived of this amino acid .
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Results and discussion
Chemical Synthesis
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![Scheme 1, Synthesis of desired deuterated C-13 labeled [5- 13 C-4- 2 H 2 ]-L-glutamine, 8 .](https://storage.googleapis.com/dl.dentistrykey.com/clinical/FacileSynthesis513C42H2LGlutamineforHyperpolarizedMRSImagingofCancerCellMetabolism/0_1s20S1076633211002182.jpg)
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Hyperpolarization
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Cell Uptake Study by MR Spectroscopy
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![Figure 1, Time elapsed sequence of magnetic resonance spectroscopy signal after injection of hyperpolarized [5- 13 C-4- 2 H 2 ]-L-glutamine into the test tube containing SF188-Bcl-x L tumor cells. 13 C spectra were acquired with 20° pulses and a 4.5-second interpulse delay. The glutamine (Gln) signal was very high, and there was a rapid conversion of glutamine (Gln) to glutamate (Glu).](https://storage.googleapis.com/dl.dentistrykey.com/clinical/FacileSynthesis513C42H2LGlutamineforHyperpolarizedMRSImagingofCancerCellMetabolism/1_1s20S1076633211002182.jpg)
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Materials and methods
Synthetic Chemistry
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Hyperpolarization
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Cell Uptake Studies by MRS
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Dual-isotope Cell Uptake Study
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