Mar 22, 2022

Public workspacePyruvate and Urea preparation for dissolution DNP V.1

DOI
dx.doi.org/10.17504/protocols.io.5jyl858m8l2w/v1
  • Dave Korenchan1,
  • Hikari Yoshihara1,
  • Sukumar Subramaniam1,
  • Romelyn DeLos Santos1,
  • Renuka Sriram1,
  • John Kurhanewicz1
  • 1University of California, San Francisco
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DOI: dx.doi.org/10.17504/protocols.io.5jyl858m8l2w/v1
Document Citation: Dave Korenchan, Hikari Yoshihara, Sukumar Subramaniam, Romelyn DeLos Santos, Renuka Sriram, John Kurhanewicz 2022. Pyruvate and Urea preparation for dissolution DNP. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl858m8l2w/v1
License: This is an open access document distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: June 14, 2021
Last Modified: March 22, 2022
Document Integer ID: 50752
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Abstract
This document codifies the probe preparation of pyruvate and urea for dissolution dynamic nuclear polarization. Additionally, this also describes the steps recommended for estimation of T1 and polarization and quality assurance to ensure repeatability and robustness of hyperpolarized 13C probe formulation.
Contents
I) General Remarks
a.Glassing and high compound concentration
b.Stability of compound formulations
II) Trityl Radicals
a.Storage and handling
b.Structures and molecular weights
III) Recipe of [1-13C]pyruvic acid and 13C Urea
IV) Quality assurance (QA) procedure for hyperpolarized (HP) Formulations using Hypersense

I) General Remarks

A. Glassing and high compound concentration
The highest levels of nuclear spin polarization and MR signal are attained when the 13C concentration is as high as possible.The ideal compound formulations therefore are small molecule liquids prepared neat.If needed, the compounds must be formulated with appropriate (co)solvents and at a concentration such that they reliably form an amorphous glass when cooled rapidly.Glassing is critical to achieve high levels of polarization.

B. Stability of compound formulations
The various compounds are stable to various degrees and the radicals are light sensitive.Formulated compounds are best stored frozen and protected from light. Gadolinium(III) chelates, e.g. Dotarem / Gd-DOTA, are relatively unstable at low pH, such as when dissolved in pyruvic acid, and will dissociate to free Gd3+ ions when in liquid state.This can have a significant negative impact on the T1 relaxation rate upon dissolution, which results in the hyperpolarized signal decaying faster.To summarize:
Note:
  • Protect from light exposure whenever possible
  • Do not let compound formulations sit at room temperature for extended periods

II) Trityl Radicals

A. The stabilized trityl radicals used as the source of spin polarization are costly and must be stored and handled properly.They are best stored long term cold, dry and protected from light, e.g. in a jar containing a dessicating agent in the freezer.To prevent the buildup of water condensation, allow them to warm to room temperature before handling.The trityl radicals are typically light, lyophilized solids of various shades of green.To reduce their tendency to cling to surfaces when handling them, it helps to use an antistatic gun to dissipate static electricity on the plasticware and weighing containers.

B. The structures and molecular weights of the radicals:
R = Name Formula Molecular Weight
-CH2CH2OH OX-63 C52H60Na3O18S12 1426.77
-CH2CH2OCH3 “GE Trityl” / AH-111501 C64H84Na3O18S12 1595.09
-CH3 Finland C40H36Na3O18S12 1066.46


III) Recipes
(Note: this is for a large batch; scale down as needed)

A. [1-13C] pyruvic acid
Materials
  1. Amount130 mg GE-Trityl
  2. Amount6.0 g (~Amount4.9 mL ) [1-13C] pyruvic acid (Aldrich: 677175, CIL: CLM-8077) MW: Amount89.05 g /mol ; density 1.281
  3. Amount14.4 µL Dotarem / Concentration500 millimolar (mM) Gd-DOTA

Protocol:
  1. Set sonicator bath to 40°C.
  2. Remove pyruvic acid from freezer and allow to warm to room temperature.
  3. Weigh Amount130 mg GE-Trityl and transfer to Concentration15 millimolar (mM) conical plastic centrifuge tube.
  4. Add Amount6 g pyruvic acid to tube.
  5. Dissolve GE-Trityl with a combination of vortex mixing and bath sonication.
  6. Add Gd-DOTA to get net Concentration1.5 millimolar (mM)
  7. Aliquot Amount200 µL into Amount1 mL screw-top cryovials and store in Temperature-80 °C freezer

B. 13C Urea
Materials
  1. Amount216 mg OX-63
  2. Amount3100 mg 13C Urea (Aldrich: 603430) MW: Amount61.05 g /mol
  3. Amount7150 mg Glyerol (Aldrich 56-81-5), MW: 92.09
  4. Amount3.2 µL of Concentration500 millimolar (mM) Gd-DOTA

Protocol:
  1. Set sonicator to Temperature60 °C
  2. Static gun zap Amount15 mL conical tube
  3. Mix Amount3100 mg urea with Amount7150 mg glycerol in the conical tube. Dissolve by vortexing and sonication.
  4. Static gun zap weigh paper and Amount15 mL conical tube
  5. Weigh Amount216 mg OX-63 into the Amount15 mL conical tube
  6. Cut tip off a pl000 pipette tip
  7. Pipette the urea+glycerol mix into the radical tube
  8. Shake, vortex, sonicate (Duration00:00:30 ), and centrifuge to dissolve
  9. Add Amount3.2 µL Gd-Dota (Concentration500 millimolar (mM) ).
  10. Aliquot Amount450 µL intoAmount1 mL screwtop cryovials and store in Temperature-80 °C freezer

IV) QA Procedure for HP Formulations using Hypersense

A)Microwave sweep, polarization buildup:
  1. Load 50-­100 µL of HP formulation (pyruvate and urea separately) into HyperSense cup, load into polarizer
  2. Perform microwave sweep to obtain polarization frequency
a. Start with these parameters : 300 min buildup time,100 MHz sweepwidth, 25 mW power
b. Recommended to do a coarse frequency sweep (5 MHz resolution) to capture full lobe of sweep curve, then do a fine frequency sweep (2-­‐3 MHz resolution) to identify maximum amplitude
c. If polarizer contains background signal, perform a sweep using same parameters prior to loading sample
d. Perform routine sweeps every 2-‐3 months
3. Start polarization at optimal microwave frequency, monitor buildup using RINMR program
4. Note the following parameters, compare with historic values:
a. Microwave frequency
b.Buildup curve maximum amplitude (solid state build-up)
c. Buildup curve time constant (Typical time-constant in HyperSense 957 seconds)


B) HP dynamic 13C­ NMR spectroscopy to measure % polarization, T1:
  1. Prepare NMR spectrometer for HP polarization and T1 measurement:
a. Insert sample with similar ionic strength, tune coils (1H,13C) and shim
b. Set up arrayed 13C sequence, verify that it runs properly. Ensure the following parameters are set properly:
i. Spectral width to cover the signal of interest (Pyruvate at 171 ppm and Urea at 163.5 ppm)
ii. Transmit + receive frequency
iii. Pulsewidth + power for a low flip angle pulse (5 - 15°)
iv. Acquire multiple transients to capture the entire relaxation of the HP signal
2. Prepare empty NMR tube with cap for measurement
3. Prepare polarizer for dissolution, inject dissolution buffer, dissolute. Note the time of start of dissolution
4. Quickly inject HP dissolution into NMR tube, insert into spectrometer, start sequence
5. Use timer to record transfer time from polarizer to spectrometer
6. Save data once acquisition finishes

C) Thermal 13C NMR spectroscopy to measure %polarization, concentration:
  1. Setup NMR spectrometer using the following parameters
a. TR = 5 * T1 (Pyruvate is ~ 48 s and Urea is ~44 s @ 11.7 T or measured in HP dynamic spectra section IV.D.1)
b. Alternatively, add 1/100 volume of Concentration500 millimolar (mM) Gd-DOTA to reduce required TR
c. Tip angle = 90°
d. averages = 8 - 32 (depending on final agent concentration in dissolution)
2. Start acquisition, save once finished

D) Calculation of T1 and % polarization
1. ForT1: fit HP dynamic peak integrals to exponential decay
Tip angle correction: multiply each nth transient by sec(α)n-1, where α is the tip angle
2. For % polarization (PHP): use the peak integrals in (a) the 1st HP spectrum (𝑆HP); and (b) the thermal spectrum (𝑆T), using the following equation:

a.

is the polarization at the NMR spectrometer field strength
b. Depending on the NMR spectrometer, 𝑆T may need to be normalized by the number of averages
c. The “back-­‐calculated” % polarization to the time of dissolution is often reported, assuming constant T1 loss over the transfer time (ttransfer). This is calculated as

For each batch of HP probe preparation, the % polarization and T1 are logged (along with polarizer characteristics to enable QA. Typically the % polarization achieved with pyruvate is ~ 14-20% .