Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents

Summary

A multi-compartment dynamic phantom is used to simulate some biology of interest for metabolic studies using hyperpolarized magnet resonance agents.

Abstract

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due to fundamental constraints imposed by the hyperpolarized state, exotic imaging and reconstruction techniques are commonly used. A practical system for characterization of dynamic, multi-spectral imaging methods is critically needed. Such a system must reproducibly recapitulate the relevant chemical dynamics of normal and pathological tissues. The most widely utilized substrate to date is hyperpolarized [1-¹³C]-pyruvate for assessment of cancer metabolism. We describe an enzyme-based phantom system that mediates the conversion of pyruvate to lactate. The reaction is initiated by injection of the hyperpolarized agent into multiple chambers within the phantom, each of which contains varying concentrations of reagents that control the reaction rate. Multiple compartments are necessary to ensure that imaging sequences faithfully capture the spatial and metabolic heterogeneity of tissue. This system will aid the development and validation of advanced imaging strategies by providing chemical dynamics that are not available from conventional phantoms, as well as control and reproducibility that is not possible in vivo.

Introduction

The clinical impact of hyperpolarized magnetic resonance imaging (MRI) of ¹³C-labeled compounds is critically dependent on its ability to measure chemical conversion rates through real time magnetic resonance spectroscopy and spectroscopic imaging^1-5. During sequence development and verification, dynamic chemical conversion is generally achieved through in vivo or in vitro models^6-9 that offer limited control and reproducibility. For robust testing and quality assurance, a more controlled system that preserves the chemical conversion endemic to this measurement would be preferred. We outline a method to achieve this conversion in a reproducible manner using a dynamic single enzyme phantom.

Most studies with hyperpolarized ¹³C agents focus on imaging hyperpolarized substrates in a functioning biological environment. This is the obvious choice if the goal is to study biological processes or determine potential for impact on clinical care. However, if characterization of some measurement system or data processing algorithm is desired, biological models have numerous drawbacks such as inherent spatial and temporal variability¹⁰. However, conventional static phantoms lack the chemical conversion that drives the primary clinical interest in MRI of hyperpolarized substrates, and cannot be used to characterize detection of conversion rates or other dynamic parameters¹¹. Using a single enzyme system we can provide controllable and reproducible chemical conversion, enabling rigorous examination of dynamic imaging strategies.

This system is directed to investigators who are developing imaging strategies for hyperpolarized substrates and wish to characterize performance for comparison against alternate approaches. If static measurements are the desired endpoint then static ¹³C-labled metabolite phantoms will suffice¹¹. On the other end if more complex biological characterization is critical to the method (delivery, cellular density, etc.) then actual biological models will be needed^12-14. This system is ideal for assessment of imaging strategies that aim to provide a quantitative measure of apparent chemical conversion rates.

Protocol

NOTE: (Phantom Design) Two 3 ml chambers were machined out of Ultem and fitted with PEEK tubing (1.5875 mm OD and 0.762 mm ID) for injection and exhaust. The Chambers were placed in a 50 ml centrifuge tube filled with water (Figure 1). To avoid signal voids created by bubbles, the chambers and the lines were pre filled with deionized water (dH₂O).

1. Solution Preparation

1. Prepare 1 L buffer solution (81.3 mM Tris pH 7.6, 203.3 mM NaCl). Weigh out 11.38 g Trizma preset crystals pH 7.6 and 11.88 g NaCl and dissolve in 1 L of dH₂O.
2. Prepare 50 mM NADH solution. Weigh out 17.8 mg of β-nicotinamide adenine dinucleotide (NADH), reduced dipotassium salt and dissolve in 280 µl of the buffer solution that was prepared in step one.
3. Prepare 250 U/ml Enzyme solution. Weigh out 78.75 activity units of lactate dehydrogenase (LDH) and dissolve in 315 µl buffer from step one.
4. Prepare pyruvic acid mix. Weigh out 21.4 mg Ox063 trityl radical and dissolve in 1.26 g (~1 ml) [1-¹³C] pyruvic acid.
5. Prepare dissolution media (40 mM Tris pH 7.6, 40 mM NaOH, 0.27 mM EDTA and 50 mM NaCl). Weigh out 5.96 g of Trizma preset crystals pH 7.6, 1.6 g of NaOH, 0.1 g ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) and 2.9 g NaCl and dissolve in 1 L dH₂O.
6. Prepare 1:10 gadoteridol solution (50 mM). Mix 1 µl of gadoteridol in 9 µl of dH₂O. Preparation of 8 M [¹³C] urea. Weigh out 1.465 5 [¹³C] urea and dissolve in 3 ml dH₂O.

2. Preparation of Hyperpolarized Pyruvate

1. In a sample cup for a Dynamic Nuclear Polarization (DNP) system, pipette 0.3 µl of the gadoteridol solution and 13 mg (~10 µl) of the pyruvic acid solution.
2. Briefly stir this mixture in the sample cup with the pipette tip.
3. Insert Sample into the DNP system.
   1. Ensure the door to the DNP system is closed. Begin the sample insertion process by clicking the insert sample button on the DNP system console. On the sample wizard select normal sample and press next.
   2. Keeping the sample cup vertical gently place the insertion rod over the top of the sample cup. When prompted, open the DNP system and insert the cup into the variable temperature insert (VTI) using the insertion rod.
   3. Pull on the plunger at the end of the sample insertion rod to release the sample in the variable temperature index. Remove the sample insertion rod from the system and click the next button on the DNP system console.
4. Initiate the polarization.
   1. Click the start polarization button on the DNP system console. In the RINMR software type .HYPERSENSENMR to launch polarization monitoring software. Set build up configuration to 1 and press enter. Then click solid build up.
   2. Set the location and name of the save file. Select the profile for 13-C in the drop down tab on the DNP system console and click next. Check the box to sample during the buildup, set sample time to 300 sec and click finish.
5. Measure out 3.85 g (~4 ml) of the dissolution media either by volume with a 5 ml syringe or by weight using a scale.

3. Preparation of the Enzyme Phantom

1. Fill a microcentrifuge tube with ~3 ml [¹³C] urea solution and place it in the 50 ml centrifuge tube. Fill the 50 ml centrifuge tube with dH₂O.
2. Pre-fill the two enzyme chambers and the lines with dH₂O by injecting ~3 ml dH₂O into the injection lines of the phantom, taking care to flush any bubbles formed.
3. Place the phantom in the center of the magnet with easy access to the injection lines. Ensure that there is some container to catch the liquid that will vent out to the exhaust line.
4. Prepare high activity enzyme mixture (17.14 mM NADH, 44.57 U/ml LDH). Mix together 240 µl NADH solution, 125 µl LDH solution and 335 µl buffer and keep in a 3 ml syringe that can be attached to the injection line.
   NOTE: Once combined with the 500 µl of 40 mM pyruvate from the DNP system, the final phantom volume will be 1.2 ml with concentrations of 16.7 mM Pyruvate, 10 mM NADH, and 26 U/L LDH in a tris buffered solution with pH ~ 7.5.
5. Prepare low activity enzyme mixture (17.14 mM NADH 26.79 U/ml LDH) Mix together 240 µl NADH solution, 75 µl LDH solution and 385 µl buffer and keep in a separate 3 ml syringe that can be attached to the injection line.
   NOTE: Once combined with the 500 µl of 40 mM pyruvate from the DNP system the final phantom volume will be 1.2 ml with concentrations of 16.7 mM Pyruvate, 10 mM NADH, and 15.625 U/L LDH in a tris buffered solution with pH ~ 7.5.

4. Run Any Quality Assurance (QA) and Positioning Scans

1. Initial positioning.
   1. Load a new FLASH positioning scan in operation mode [¹H] TX/RX Volume mode. Change set up dimensions to 2: Spectrometer Control Tool -> Edit GS -> Setup Dimensions -> 2. Press GSP on the Spectrometer Control and move phantom until centered in the magnet. Press STOP then Press GOP on the Spectrometer Control.
2. Pilot Scan.
   1. Load a new TriPiolt positioning scan in operation mode [¹H] TX/RX Volume mode. Position Slice: Scan Control -> Slice Tool- > move slices (hold M key click and drag; select slice to move with the slice package slider).
   2. Wobble ¹H Coil: Spectrometer Control Tool - > Acq -> Wobble. Tune and match ¹H coil behind the magnet and press STOP. While holding the shift key press the traffic light on the scan control window.

5. Radial Echo Planar Spectral Imaging Scan Setup

1. Load a new radial echo planar spectroscopic imaging (radEPSI) scan in operation mode [¹³C] TX/RX Volume mode. Position Slice: Slice Tool on the Scan control and move slices (hold M key click and drag; select slice to move with the slice package slider).
2. Wobble ¹³C Coil by clicking Spectrometer Control Tool - > Acq -> Wobble. Set the receiver gain to 1,000-2,000 on the Spectrometer.
3. Perform the final system check. Depending on the sequence, observe carbon 13 signal from the urea chamber in a scout protocol.
   NOTE: This ensures that the system is set up properly before beginning the irreversible dissolution process.

6. Run Dissolution

1. When the pyruvate has attained >90% polarization (~ 1 hr), the solutions and phantom are ready, and the scan is configured click the run dissolution button on the DNP system console.
2. When prompted move the dissolution stick into its operating position and inject dissolution media. Close the DNP system and click the finished button on the DNP system console. Move dissolution stick back to resting position when prompted then click finish.
3. When the DNP system delivers the hyperpolarized pyruvate (~ 2 min after heating starts) withdraw 500 µl of the pyruvate solution into each the high and low enzyme concentration solution syringes. Slowly (~10 sec) inject each syringe into an injection line.
   NOTE: Scanning could have been initiated prior to injection or anytime up to 3 min post injection depending on the scan protocol used.

7. Image Processing

NOTE: This phantom was designed for use with many imaging strategies. See Figure 2, as an example of how the rad-EPSI images were processed using Matlab.

1. Load raw data from the fid file. Reshape the data to match the number of projections, read out points, echoes and account for data stored as real and imaginary pairs. Separate out the even and odd echo points.
2. Fourier transform either the even or odd echoes along the echo dimensions. Visually identify the frequency bands for pyruvate and lactate. For simplicity the absolute value of the spectrum was used.
3. Separate each metabolite band and Fourier transform along the frequency encode direction to yield isolated sinograms for each metabolite. Inverse radon transform the separate sinograms to produce image of either lactate or pyruvate.

Results

Slice-selective 2D images were acquired using a snapshot radEPSI sequence. Metabolite images were reconstructed using filtered back projection. The metabolite images were well aligned with proton images, as seen in Figure 2. In this system hyperpolarized lactate signal can only be generated from the enzymatic conversion of hyperpolarized pyruvate. In Figure 4, the bottom chamber, with higher LDH concentration, has a stronger lactate and weaker pyruvate si...

Discussion

Real time imaging of hyperpolarized metabolites has many unique challenges for sequence design, validation, and quality control. The ability to resolve spatiotemporal and spectral heterogeneity offers substantial clinical potential but precludes QA and validation methods associated with conventional MRI. Complex imaging sequences or reconstruction algorithms can have subtle dependencies that render them difficult to characterize or validate outside of the imaging experiment. Biological heterogeneity and other practical c...

Materials

Name	Company	Catalog Number	Comments
BioSpect 7T	Bruker	BioSpec 70/30 USR	7 Tesla Pre-Clinical MRI Scanner
HyperSense	Oxford Instruments	Hypersense DNP Polarizer	Dynamic Nuclear Polarizer for MRI agents
1-13C-Pyrvic Acid	Sigma Aldrich	677175	Carbon 13 labled neat pyruvic acid
Trityl Radical	GE Healthcare	OX063	Free radical used in Dynamic Nuclear Polarization
NaOH	Sigma Aldrich	S8045
EDTA	Sigma Aldrich	E6758	Ethylenediaminetetraacetic acid
LDH	Worthingthon	LS002755	Lactate Dehydrogenase from rabbit muscle
NADH	Sigma Aldrich	N4505	β-Nicotinamide adenine dinucleotide, reduced dipotassium salt
Trizma	Sigma Aldrich	T7943	Trizma Pre-set crystals
NaCl	Sigma Aldrich	S7653