Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-¹³C]pyruvate

We want to estimate the loss of polarization of a hyperpolarized solution during transport from the hyperpolarizing equipment to the MRI scanner as it traverses through different magnetic fields. Our technique estimates the spin-lattice relaxation time of hyperpolarized solutions at low magnetic fields with high accuracy. This method is not limited to dynamic nuclear polarization and can be used to estimate the spin-lattice relaxation time of other methods of polarization, including parahydrogen-induced polarization.

When performing this technique, make sure that the hyperpolarized sample doesn’t cross or stay at the zero-field region for any period of time, otherwise you may completely lose the polarization. Prepare one milliliter of stock carbon-13 enriched pyruvic acid solution, widely used for in vivo research, consisting of 15 millimoles per liter of triarylmethyl radical dissolved in 1-13C pyruvic acid. On the dynamic nuclear polarizer software interface, click on the Cooldown button to lower the temperature of the variable temperature insert to 1.4 kelvin.

Once the DNP has reached the desired temperature, load 10 microliters of the stock solution in a sample cup. Open the turret doors, and insert the cup into the VTI using an insertion wand specifically designed for this task. Quickly extract the wand, and make sure the cup is released.

Then, close the turret doors, and allow the temperature of the VTI to return to 1.4 kelvin. Meanwhile, prepare the DNP to run a microwave sweep in order to find the optimal radio frequency for the hyperpolarization of the stock solution. At the end of the microwave sweep, recover the sample.

Then, set the system in idle, and record the optimal frequency where the maximum polarization is achieved. This optimal frequency is defined as the polarization frequency that provides the maximum polarization. This frequency will be used for hyperpolarizing all the aliquots obtained from that specific stock solution of pyruvic acid.

Prepare 250 milliliters of stock dissolution medium as described in the text protocol, and add EDTA at a concentration of 100 milligrams per liter to sequester any metal ion contamination. Also, prepare 500 milliliters of stock cleaning solution consisting of 100 milligrams per liter EDTA dissolved in deionized water. Approximately 10 milliliters of this cleaning solution is used after each polarization to clean the dissolution path of the DNP.

Cool the DNP apparatus to 1.4 kelvin in preparation of hyperpolarizing a 1-13C pyruvic acid sample by selecting the Cooldown button in the DNP main window. Weigh out 30 milligrams of the prepared pyruvic acid stock solution in a sample cup. When the desired VTI temperature is achieved, click on Insert Sample, select Normal Sample, and then click on Next.

Following the safety precautions displayed on the screen, insert the cup in the cold DNP apparatus using a long wand specifically designed for this task. Once the cup is inserted, remove the wand and close the DNP doors. On the DNP software interface, click Next and then Finish.

Wait until the temperature has returned to 1.4 kelvin, and then click on the Polarize Sample button. In the new popup window, set the frequency value to that obtained from the microwave sweep. In the same window, set the power to 50 milliwatts and the sampling time to 300 seconds.

Click on Next, check the Enable Build-up Monitoring box, and then click on Finish. Polarize until the build-up of the solid-state magnetization reaches at least 95%of maximum. When the desired polarization is achieved, click on Run Dissolution.

Under Method, select Pyruvic Acid test, and then click on Next. Following the instructions on the screen, open the DNP turret doors. Load the heating and pressurizing chamber at the top of the apparatus with approximately 4.55 milliliters of the dissolution medium.

This produces a concentration of 80 millimoles per liter of pyruvate upon dissolution at a pH of 7.75 and a temperature of 37 degrees Celsius. Position the recovering wand in the right position, and close the turret doors. On the DNP software interface, click on Next and then on Finish.

At that point, the dissolution media will be superheated until the pressure reaches 10 bar. Once the 10-bar pressure is attained, the frozen and hyperpolarized pyruvate is automatically lifted from the liquid helium bath, quickly mixed, and thawed with the superheated dissolution media. The mixture is then ejected through a capillary tubing into a pear-shaped flask.

While the hyperpolarized pyruvate and dissolution media mixture is ejected, constantly swirl the flask to ensure a homogeneous mixture. When all of the mixture has been ejected, quickly draw 1.1 milliliters of the liquid into a syringe. Transfer the mixture to a pre-warmed, 10-milliliter-diameter NMR tube, and rapidly transport to the field-cycling relaxometer.

Immediately clean the DNP fluid path using clean dissolution medium, followed by ethanol. Then, remove the cup, and blow helium gas through the fluid path to remove any remaining cleaning fluids and purge the path of oxygen. Clean all glassware.

After each measurement, record the pH of the samples from the benchtop spectrometer. Also, record the pH of the samples on the field-cycling relaxometer. Prior to dissolution, the relaxometer flip angle must be calculated, and the relaxometer must be set up and ready for measurement of the hyperpolarized solution.

To perform T-one measurements, make sure the external shim coil is installed and energized. In the instrument software, select the Main Par tab. Then, click on the cell next to the Experiment label, and scroll down in the popup window to select the pulse sequence HPUB/S.

Now, set the acquisition parameters. Set radio frequency attenuation to 25 decibels, maximum T1 to values between three and five seconds, switching time to 0.2 seconds, recycle delay to zero seconds, and relaxation field to the desired relaxation field in megahertz. Then, select the Acquisition Parameters tab, followed by the Basic subtab.

Click on the cell next to the Nucleus label, and scroll down in the popup window to select carbon-13. Then, set system frequency to eight megahertz, sweep width to one megahertz, block size to 652, and filter bandwidth to 50, 000 hertz. Next, select the Configuration subtab.

Set the 90-degree pulse width time to the previously determined value, the receiver inhibit time to 25 microseconds, and the acquisition delay time to 25 microseconds. Select the Pulse subtab, and set the main RF pulse flip angle to five degrees. Then, select the Number of Dimensions subtab, and set the number of blocks to 100.

Wait and get ready to receive the hyperpolarized solution to initiate the data acquisition. Immediately before inserting the sample into the relaxometer, manually start the pulse sequence from the console to avoid inserting the sample into a null magnetic field. For this reason, it is important to ignore the first free induction decay, or FID, during the data analysis.

It is important to start the data acquisition before introducing the sample into the relaxometer to avoid null magnetic fields that will cause a loss in polarization. Once the acquisition is done, save the data by clicking the Save button. Using the analysis software, integrate the magnitude of each FID signal to produce a data series comprised of sample magnetization as a function of time.

An example of a high-resolution, full-range microwave sweep for pyruvic acid is shown. For the presented case, that optimal microwave frequency corresponds to 94.128 gigahertz. Shown here is a typical series of decaying FIDs as the hyperpolarized magnetization is sampled.

The relaxation curve for hyperpolarized 1-13C pyruvate was obtained from the data of the previous figure. Each blue point on the curve represents the area under an FID. The T1 value of 53.9 plus or minus 0.6 seconds was obtained by a non-linear least-squares fit of the signal equation to the decay curve data, which included the effects of the flip angle used for excitation.

The T1 results for all 26 measurements over a range of 0.237 millitesla to 0.705 tesla at 37 degrees Celsius are shown. Each T1 measurement at a given relaxation field is a separate hyperpolarized dissolution from the DNP apparatus. The solid line represents the formula, and the dashed lines represent the 95%confidence bands.

Analysis of the results showed that the relaxation time for the C-1 nucleus is 46.9 seconds at Earth’s magnetic field, compared with 65 seconds at three tesla, which represents a decrease of 28%It is important to install and energize the external shim coil and to start acquisition right before inserting the sample to avoid null magnetic fields and the potential loss of polarization. Following this method, the sample procedure could be used with deuterated dissolution media to extend the spin-lattice relaxation times of the hyperpolarized solution.