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.
