Name: hyperpolarized 13c metabolic magnetic resonance spectroscopy and imaging
Uploaded: 2016-12-30T13:00:00
Description: Watch this Scientific Journal Video about Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging at JoVE.com

E.K. gratefully acknowledges the support of the Graduate School of Bioengineering (GSB) at Technische Universit&#228;t M&#252;nchen. This work was supported by the German Research Foundation (DFG) within the SFB Collaborative Research Center 824, "Imaging for Selection, Monitoring, and Individualization of Cancer Therapies."

1. Sample Stock Solution Preparation
<ol>
	<li>Add gadoterate meglumine (GadM, 0.5 mol/L) to concentrated [1-13C]pyruvic acid to give a final concentration of 1-mmol/L GadM. Add trityl radical tris (8-carboxy-2,2,6,6-tetra-(hydroxyethyl)-benzo-[1,2-4,5]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (OX063) to this mixture to give a final concentration of 15 mmol/L. Vortex until complete dissolution. 
	NOTE: This stock solution preparation is designed for usage with a 3.35-T preclinical DNP hyperpolarizer....

13CMRSI with hyperpolarized probes is a promising method to monitor metabolism in real time in vitro and in vivo. One very important aspect when employing this experimental process is the proper standardization, especially regarding in vitro experiments. First, the preparation of the sample needs to be done properly and consistently to achieve the same concentration of hyperpolarized material in each experiment. This requires a precise weighing of both the sample to be hyperpolarized...

Presently, the most widely used clinical method for tumor staging, restaging, treatment response monitoring, and recurrence detection of a wide variety of cancers is [18F]-FDG PET.1 However, recently, several novel and alternative approaches have emerged. One of those methods is 13CMRSI. This technique involves the introduction of the 13C-molecule into a biological sample, followed by minimally invasive MRI to assess the metabolism in vitro or in vivo in real time. Nevertheless, the biggest challenge of 13CMRSI, compared to the other methods such as [18F]-FDG PET or computed tomography, is its low signal-to-noise ratio.
The NMR signal is directly proportional to the level of polarization, a ratio of the spin &#189; nuclei population difference in two energy states to the total population (Figure 1A). The polarization is a product of the gyromagnetic ratio (&#947;) of the nuclei and the applied magnetic field strength over the temperature. A typical polarization of 1H nuclei is in the order of 0.001% to 0.005% at 3 T, which gives a relatively poor signal-to-noise ratio. Today&#39;s state-of-the-art MRI has been a successful imaging method only due to the high abundance of 1H in biological samples and the high gyromagnetic ratio of 1H (&#947;1H = 42.576 MHz/T). However, observing other nuclei, such as carbon, is more demanding. The only stable, magnetically active carbon isotope, 13C, makes up only 1.1% of all carbon atoms. In addition, the gyromagnetic ratio of 13C (&#947;13C = 10.705 MHz/T) is four times lower than that of 1H, leading to a lower detection efficiency. In summary, the low 13C abundance and low &#947;13C cause thermal 13C measurements to achieve 0.0176% of the sensitivity of a 1H-NMR measurement in vivo.
Dynamic Nuclear Polarization
A method to overcome the relatively poor sensitivity of 13C measurements is DNP. It was originally described for metals in 1953 by Albert W. Overhauser. In his article, he stated: &#34;It is shown that if the electron spin resonance of the conduction electrons is saturated, the nuclei will be polarized to the same degree they would be if their gyromagnetic ratio were that of the electron spin.&#34;2 Later that year, Carver and Slichter experimentally confirmed Overhauser&#39;s hypothesis3. In 1958, Abragam and Proctor described this effect for electrons in liquids and named it the &#34;solid effect.&#34; At temperatures below 4 K, electron-spin polarization reaches nearly 100% and is more than three orders of magnitude higher than the nuclear-spin polarization (Figure 1B)4. This occurs because the gyromagnetic ratio of the electron (&#947;e = 28024.944 MHz/T) is three orders of magnitude higher than the nuclear gyromagnetic ratios. The weak interactions between electrons and nuclei, such as the Overhauser effect, the solid effect, the cross effect, and the thermal mixing effect, allow the transfer of polarization from electron spins to nuclear spins using microwave irradiation with a frequency close to the corresponding electron paramagnetic resonance (EPR) frequency5,6. DNP theory has been further developed to involve more electrons and thermal mixing. Nevertheless, to date, no unified quantitative theoretical description of DNP has been published7,8.
<img alt="Figure 1" src="/files/ftp_upload/54751/54751fig1.jpg" /> 
Figure 1: Understanding Dynamic Nuclear Polarization and Hyperpolarization. A) A schematic comparison of the spin population in the thermal equilibrium polarization state and the hyperpolarized state. B) The polarization is dependent upon temperature. The polarization of an electron (e-) reaches 100% below 1.4 K. The DNP allows the transfer of the polarization from the e- to the 13C nuclei, which increases their polarization up to 105-fold. <a href="http://cloudflare.jove.com/files/ftp_upload/54751/54751fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To introduce DNP in studies of biological systems using 13C NMR, subsequent rapid sample dissolution had to be developed. 50 years after Overhauser&#39;s hypothesis, Jan H. Ardenkjaer-Larsen et al. solved the technically challenging issue of bringing the hyperpolarized frozen sample into the liquid state with minimal hyperpolarization loss6. Dissolution DNP opened a new field of research called 13CMRSI, providing a new method to investigate and characterize various disease states9,10. As stable carriers of an unpaired electron, a trityl radical tris (8-carboxy-2,2,6,6-tetra-(hydroxyethyl)-benzo-[1,2-4,5]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (OX063) or (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO) is usually used. These are mixed with the desired 13C-labeled molecule and exposed to microwave irradiation with a frequency close to the corresponding EPR frequency. Using this technique, the polarization of 13C nuclei can be increased up to 37%11. This results in a 105-fold polarization enhancement compared to the thermal equilibrium polarization11,12. However, as soon as the microwave irradiation is stopped and/or the 13C-molecule is transferred to the liquid state, the polarization decays with the longitudinal relaxation time (T1) of the 13C nucleus that was polarized. Thus, the invention of fast dissolution techniques or any subsequent technique shortening the time before experimental measurement (i.e., injection) is crucial for biological applications13.
There are three major requirements that the candidate molecule needs to fulfill for successful 13CMRSI studies. First, the 13C nucleus of interest has to have a sufficiently long T1 (&#62; 10 s). The choice of the 13C-label is crucial. The best candidate nuclei are carbons with no direct contact with 1H-nuclei via a bond. It also needs to be rapidly metabolized within 2 - 3 T1 times, resulting in a downstream metabolic product with a significantly different chemical shift from the original substance. The sample mixture must also form an amorphous glass when in a solid state so that the spatial distribution decreases the distance between the electron and 13C, allowing the transfer of polarization. If the candidate molecule does not form amorphous glass naturally, it needs to be highly soluble in a glassing agent, such as glycerol or dimethyl sulfoxide14. These requirements result in a relatively small number of candidate molecules. However, even after the successful discovery of a suitable molecule, developing a working protocol for hyperpolarization can be technically challenging9,14,15.
In recent years, several substrates have been successfully polarized, such as [1-13C]pyruvate12,16-36, [2-13C]pyruvate37, [1-13C]ethyl pyruvate38, [1-13C]lactate39, [1-13C]fumarate40-43, 13C-bicarbonate36,44,45, [1-13C]sodium acetate43,46-49, 13C-urea6,36,50,51, [5-13C]glutamine15,52,53, [1-13C]glutamate53,54, [1-13C]2-oxoglutarate55, [1-13C]alanine, and others14,56. A particularly interesting and commonly used substrate for hyperpolarization is [1-13C]pyruvate. It is widely used in preclinical studies to investigate the cellular energy-metabolism in various diseases14,17,22. [1-13C]pyruvate meets all the requirements for successful hyperpolarization, including a relatively long T1 and rapid transport across the cell membrane before subsequently being metabolized. Preclinical studies with [1-13C]pyruvate are currently being translated into the clinic57.
Metabolism of Pyruvate
It is well known that there is a direct link between mutations in a cancer cells&#39; DNA and changes in their metabolic pathways. Already in the 1920s, Otto Warburg discovered that there is an increased metabolism of glucose and production of lactate in tumors compared to healthy tissue58-60. Subsequently, various alternations in other metabolic pathways, such as the pentose-phosphate pathway, the tricarboxylic acid cycle, oxidative phosphorylation, and the synthesis of nucleotides and lipids, have been described.
Pyruvate is the final product of glycolysis. In the tumor, it undergoes anaerobic glycolysis catalyzed by LDH61 and reacts with the reduced form of the coenzyme nicotinamide adenine dinucleotide (NADH), resulting in lactate and the oxidized form of the coenzyme (NAD+). Alternatively, pyruvate undergoes a transamination reaction with glutamate to form alanine, catalyzed by alanine transaminase (ALT). Both reactions are readily reversible. Pyruvate also undergoes decarboxylation catalyzed by pyruvate dehydrogenase (PDH) to carbon dioxide and acetyl-CoA, representing an irreversible reaction at this step. Alternations in these reaction rates can be linked to tumor metabolism17,21,22,25,62. The metabolic pathways are summarized in Figure 2.
<img alt="Figure 2" src="/files/ftp_upload/54751/54751fig1.jpg" /> 
Figure 2: Diagram of the major metabolic reaction of pyruvate. Pyruvate/lactate conversion is catalyzed by LDH, and pyruvate/alanine conversion is catalyzed by ALT. Pyruvate is irreversibly converted to acetyl-CoA and CO2 by PDH, and CO2 is in a pH-dependent equilibrium with bicarbonate80. <a href="http://cloudflare.jove.com/files/ftp_upload/54751/54751fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The detection of hyperpolarized [1-13C]pyruvate and its metabolites has been previously demonstrated in the rat heart37,63-65, liver66, muscle, and kidney62,67. One study demonstrated significant differences in the lactate-to-alanine ratio between the normal and fasted rat liver66 and demonstrated a highly elevated and hyperpolarized [1-13C]lactate level in liver cancer68,69. There is evidence that the tumor grade can be identified in a transgenic adenocarcinoma of mouse prostate (TRAMP) using hyperpolarized [1-13C]pyruvate22, with the hyperpolarized lactate levels showing a high correlation with the histological grade of the excised tumors. The alanine catalyzed from pyruvate by ALT has also been suggested as a useful marker in rat hepatocellular carcinoma23.
Measuring the pyruvate-lactate metabolic flux has been used for monitoring ischemia63,65,70 and as a response to treatment with cytotoxic chemotherapy17,40, targeted drugs24,25,41, or radiotherapy26 in animal models. It has also been used for the detection of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 response in glioblastoma and breast cancer mouse models25. Changes in pyruvate metabolism in brain tumors26 and prostate cancer24,71 have also been observed after treatment.
Prostate Carcinoma
Prostate carcinoma is the predominant cancer in elderly men and the second leading cancer related to death in men worldwide72. To date, no reliable, non-invasive methods are available for an early diagnosis and characterization of prostate cancer73,74, emphasizing the urgent need for novel metabolic imaging techniques to enable stringent detection and staging of patients. Prostate carcinoma was used as a model to demonstrate the possibilities of dissolution DNP combined with 13CMRSI in patients57. This work was continued in a first clinical trial employing [1-13C]pyruvate and 13CMRSI for the imaging of prostate cancer, and it has just recently has been completed (NCT01229618).
The motivation behind this work was to illustrate in more detail and for a wider audience the application of the 13CMRSI method in a preclinical setting with cells. Measuring the LDH-catalyzed metabolism of [1-13C]pyruvate to [1-13C]lactate in vitro in the PC3 prostate carcinoma cell line, we demonstrate the possible application of dissolution DNP in in vitro studies and address the crucial steps and challenges during experiments.

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HyperSense DNP Polariser</td>
			<td style="width:375px;">Oxford Instruments</td>
			<td style="width:136px;"></td>
			<td style="width:191px;">3.35 T preclinical DNP hyperpolarizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GE/Agilent MR901</td>
			<td style="width:375px;">GE Healthcare/Agilent Technologies</td>
			<td style="width:136px;"></td>
			<td>7 T preclinical MRI scanner, with small bore designed for experiments onrodent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Spinsolve Carbon</td>
			<td style="width:375px;">Magritek</td>
			<td style="width:136px;"></td>
			<td>1 T NMR spectrometer with permanent magnet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Deuterium Oxide</td>
			<td style="width:375px;">Sigma Aldrich</td>
			<td style="width:136px;">7789-20-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium phosphate dibasic</td>
			<td style="width:375px;">Sigma Aldrich</td>
			<td style="width:136px;">7558-79-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium phosphate monbasic</td>
			<td style="width:375px;">Sigma Aldrich</td>
			<td style="width:136px;">7558-80-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium hydroxide</td>
			<td style="width:375px;">Sigma Aldrich</td>
			<td style="width:136px;">1310-73-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Disodium edetate</td>
			<td style="width:375px;">Sigma Aldrich</td>
			<td style="width:136px;">6381-92-6</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:247px;">Pyruvic acid - 13C1</td>
			<td style="width:375px;">Cambridge Isotopes Laboratories</td>
			<td style="width:136px;">CLM-8077-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Dotarem (0.5 mmol/L)</td>
			<td style="width:375px;">Guerbet</td>
			<td style="width:136px;"></td>
			<td>gadoterate meglumine&#160;&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:247px;">tris (8-carboxy-2,2,6,6-tetra-(hydroxyethyl)-benzo-[1,2&#8211;4,5]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (OX063)</td>
			<td style="width:375px;">GE Healthcare</td>
			<td style="width:136px;"></td>
			<td>trityl radical used as a sourse of free electron</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PC3 cell line</td>
			<td style="width:375px;">ATCC</td>
			<td style="width:136px;">CRL1435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">F-12K medium&#160;</td>
			<td style="width:375px;">ATCC</td>
			<td style="width:136px;">30-2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:375px;">ATCC</td>
			<td style="width:136px;">SCRR-30-2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsine-EDTA Solution, 1X</td>
			<td style="width:375px;">ATCC</td>
			<td style="width:136px;">30-2101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sample plastic cup</td>
			<td style="width:375px;">Oxford Instruments</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Trypan blue</td>
			<td style="width:375px;">Bio-Rad</td>
			<td>145-0013-MSDS</td>
			<td></td>
		</tr>
	</tbody>
</table>

hyperpolarized 13c metabolic magnetic resonance spectroscopy and imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

The results of the &#8220;microwave sweep&#8221; are illustrated in Figure 3. It shows that the optimal microwave frequency for the [1-13C]pyruvate sample is at 94.156&#160;GHz for the local 3.35-T hyperpolarizer. All following hyperpolarization experiment (n = 14) were performed using this microwave frequency with a power of 100&#160;mW. The microwave irradiation was applied for 60 to 80 min, leading to a solid-state hyperpolarization higher than 90%. The results are presented in Figu...

Watch this Scientific Journal Video about Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging at JoVE.com

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

The overall goal of this procedure is to hyperpolarize carbon 13-labelled molecules using dynamic nuclear polarization, and to measure their enyzmatic conversion using carbon 13 magnetic resonance, retroscopic imaging, in vitro. This method increases the resonant signal of carbon by 10, 000 to 100, 000 fold, allowing the layered compounds to be distinguished from their metabolic products, and to be localized in the body by imaging. The main advantage of this method, compared to say, the VR techniques, is that it provides a view of the metabolism of naturally occurring substances in living organisms in real time.Also this method can provide insight into the metabolism of cancer and as it is in vitro. It can also be applied to animal models and human patients. The application of this technique exceeds tumor localization.It can be used to find and distinguish metabolic tumor sub-types, and to monitor treatment alongside state-of-the-art techniques. To begin the procedure, first prepare a one millimolar solution of gadoterate meglumine, and concentrated one-carbon 13 pyruvate. Add trityl radical OX063 to the mixture to achieve a 15 millimolar final concentration, and dissolve it completely.In the DNP Polarizer software, click Cool Down'to decrease the temperature of the variable temperature insert, or VTI, below 1.4 kelvin. Once the VTI has cooled, place about eight microliters of the carbon 13-labelled sample solution in a plastic sample cup. Attach the cup to the sample insertion rod.In the software, click Insert Sample'Select Normal Sample'and then Next'This raises the sample holder and opens the inlet valves to the VTI, allowing sample insertion. At the prompt, use the insertion rod to push the sample cup into the VTI. Detach the sample cup and carefully withdraw the insertion rod.Confirm that the sample was detached from the rod and was not pushed out of the VTI during the closing procedure. Click Next'to close the valves and lower the sample cup into the liquid helium, and then click Finish'To find an optimal polarization frequency, first start the RINMR software. In the configuration screen, click Select Sample'and Do Micro Sweep'Create the file where the data will be stored.Switch back to the DNP Polarizer software. Select the Calibrate'tab, set the microwave sweep parameters, and then start the microwave sweep process. Once the microwave sweep is finished, note the frequency with a maximal measured signal amplitude.Return to the RINMR software, and select Solid Build Up'Then, in the DNP Polarizer software, click Polarization'Enter the optimal microwave frequency and the power. Click Next'Enable Polarization Buildup Monitoring'and click Finish'Polarize the sample for a sufficient length of time to achieve over a 95%polarization. During polarization, connect a collection flask to the hyperpolarizer sample exhaust tube, and place it near the spectrometer.Set up a one tesla NMR spectrometer with the 13 carbon scan series of 110 degree RF pulses at three second intervals. During pyruvate polarization, begin cell culture preparation. Remove the cell medium from cell culture flasks.Wash the cells with about 10 milliliters of phosphate-buffered saline. Add five milliliters of trypsin to the culture flask. Incubate the culture for three to five minutes at 37 degrees celsius, and then deactivate the trypsin with about five milliliters of F12K medium containing 10%fetal calf serum by volume.Then, transfer the volume of cell suspension containing the desired number of cells to a plastic centrifuge tube. Centrifuge the cell sample at 1200 G for five minutes. Discard the supernatant and re-suspend the pallet in sufficient F12K medium with 10%FCS to achieve a final volume of 800 microliters.Transfer the suspension to a 1.5 milliliter microcentrifuge tube, and place the tube in a plastic container filled with warm water. To begin data collection via carbon 13 magnetic resonance spectroscopy, once the cell sample is prepared and the pyruvate polarization is greater than 95 percent, click Run Dissolution'in the DNP Polarizer software. Load the meted volume of dissolution agent via the top valve of the dissolution chamber.Move the dissolution stick into the active position. Confirm by clicking Next'Once the cell sample is ready for the experiment, click Finish'in the DNP Polarizer software to initialize the dissolution process. Wait until the dissolution process is finished.Add 200 microliters of the hyperpolarized pyruvate solution to 800 microliters of the cell sample. Mix well with a pipette, and then place about 600 microliters of the mixture in a five millimeter NMR tube. Load the NMR tube into the spectrometer and click Run'to start the measurement.Then, move the dissolution stick with the attached sample cup to the cleaning position and click Finish'in the DNP Polarizer software. To begin carbon 13 magnetic resonance imaging, after starting the hyperpolarization process, transfer 800 microliters of re-suspended cell sample with a cell number of at least 10 to the eighth into a three milliliter syringe. Connect the syringe to a long catheter and place the syringe on an MRI scanning bed.Make sure that the catheter is long enough to reach outside of the MRI scanner. Place a calibration phantom, containing ten molar carbon 13 urea with 10%gadoterate meglumine next to the syringe. Place a carbon 13-tuned RF receiver coil on the syringe.Insert the scanning bed into the scanner so that the object of interest is in the isocenter of the MRI scanner. Connect the connection flask to the hyperpolarizer sample exhaust tube and place it close to the end of the catheter. Run a standard three plane localization sequence and adjust the syringe position if necessary.Then, set up and run a proton T2-weighted anatomical sequence covering the syringe localization. From the resulting anatomical images, select five continuous slices of the region of interest, including the carbon 13 phantom. Set up a carbon 13 spectroscopic calibration acquisition, and select the 2D block siegert calibration sequence from the pulse sequence library.Download the pulse sequence to the scanner, and run a spectroscopic pre-scan. In the spectrum magnitude plot, set the carbon 13 calibration phantom peak to the center of the scanner frequency. Set the receiver gains to maximum and click Scan'to run the calibration sequence.Record the reported transmit gain and centric frequency. Then, set up a carbon 13 chemical shift imaging acquisition for the chosen anatomical slices. Choose the 2D echo-planar spectroscopic imaging sequence and download the pulse sequence to the scanner.Run the spectroscopic pre-scan, and adjust the centric frequency and transmit gain based on the calibration values to finish setting up the MRI spectrometer. Once the pyruvate reaches 95%polarization, click Run Dissolution'in the DNP Polarizer software and load the dissolution agent. Move the dissolution stick into the active position.Click Next'and then Finish'When the dissolved hyperpolarized sample reaches the collection flask, draw about 1 milliliter of the hyperpolarized solution into a syringe, and inject it into the catheter so that 200 microliters reaches the cell sample. Click Scan'to start the acquisition of the experimental data. Once data collection is finished, move the dissolution stick with the attached sample cup to the cleaning position, and click Finish'in the DNP NMR software.The metabolic exchange rate of pyruvate to lactate was studied in prostate carcinoma PC3 cells by monitoring the carbon 13 NMR spectroscopy signal intensities of hyperpolarized lactate and pyruvate with respect to frequency and time. The lactate kinetic values were evaluated by a simple ratio model and by a two-site exchange differential model, and normalized to the number of cells. In both models, a trend of decreasing lactate production with an increasing number of cells was observed.This is attributed to higher cell concentrations and greater viscosity of the sample. Samples with cell counts below two times ten to to the seven show a very small lactate signal to noise ratio and therefore large standard deviation values indicating that samples should contain at least that number of cells to obtain informative data. Spatial localization was achieved with MRI with a good spectral resolution allowing carbon 13 images to be acquired for specific frequency regions.The spatial distribution was determined from the raw data. The carbon 13 image could also be co-registered with the previously acquired proton image. Once mastered and properly performed this experiment can be done in less than 60 minutes.After watching this video, you should have a good understanding of how to perform a hyperpolarization of a desired molecule using the dynamic nuclear polarization technique and how to do simple NMR spectroscopy and cell imaging experiments. While attempting this procedure, it's important to be very fast. After the hyperpolarized sample leaves the polarizer, remember that you lose all your gained NMR signal in less than two minutes.In conclusion, dissolution DNP can be used for a variety of applications. This includes studying diseases such as diabetes, or looking at metabolic changes of various types of cancer, but also measuring tissue pH.

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