This work was supported in part by the Cancer Prevention and Research Institute of Texas (RP140021-P5), a Julia Jones Matthews Cancer Research Scholar CPRIT research training award (RP140106, CMW), and the National Institutes of Health (P30-CA016672).

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
<ol>
	<li>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.</li>
	<li>Prepare 50 mM NADH solution. Weigh out 17.8 mg of &#946;-nicotinamide adenine dinucleotide (NADH), reduced dipotassium salt and dissolve in 280 &#181;l of the buffer solution that was prepared in step one.</li>
	<li>Prepare 250 U/ml Enzyme solution. Weigh out 78.75 activity units of lactate dehydrogenase (LDH) and dissolve in 315 &#181;l buffer from step one.</li>
	<li>Prepare pyruvic acid mix. Weigh out 21.4 mg Ox063 trityl radical and dissolve in 1.26 g (~1 ml) [1-13C] pyruvic acid.</li>
	<li>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.</li>
	<li>Prepare 1:10 gadoteridol solution (50 mM). Mix 1 &#181;l of gadoteridol in 9 &#181;l of dH2O. Preparation of 8 M [13C] urea. Weigh out 1.465 5 [13C] urea and dissolve in 3 ml dH2O.</li>
</ol>
2. Preparation of Hyperpolarized Pyruvate
<ol>
	<li>In a sample cup for a Dynamic Nuclear Polarization (DNP) system, pipette 0.3 &#181;l of the gadoteridol solution and 13 mg (~10 &#181;l) of the pyruvic acid solution.</li>
	<li>Briefly stir this mixture in the sample cup with the pipette tip.</li>
	<li>Insert Sample into the DNP system.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Initiate the polarization.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
3. Preparation of the Enzyme Phantom
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare high activity enzyme mixture (17.14 mM NADH, 44.57 U/ml LDH). Mix together 240 &#181;l NADH solution, 125 &#181;l LDH solution and 335 &#181;l buffer and keep in a 3 ml syringe that can be attached to the injection line. 
	NOTE: Once combined with the 500 &#181;l 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.</li>
	<li>Prepare low activity enzyme mixture (17.14 mM NADH 26.79 U/ml LDH) Mix together 240 &#181;l NADH solution, 75 &#181;l LDH solution and 385 &#181;l buffer and keep in a separate 3 ml syringe that can be attached to the injection line. 
	NOTE: Once combined with the 500 &#181;l 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.</li>
</ol>
4. Run Any Quality Assurance (QA) and Positioning Scans
<ol>
	<li>Initial positioning.
	<ol>
		<li>Load a new FLASH positioning scan in operation mode [1H] TX/RX Volume mode. Change set up dimensions to 2: Spectrometer Control Tool -&#62; Edit GS -&#62; Setup Dimensions -&#62; 2. Press GSP on the Spectrometer Control and move phantom until centered in the magnet. Press STOP then Press GOP on the Spectrometer Control.</li>
	</ol>
	</li>
	<li>Pilot Scan.
	<ol>
		<li>Load a new TriPiolt positioning scan in operation mode [1H] TX/RX Volume mode. Position Slice: Scan Control -&#62; Slice Tool- &#62; move slices (hold M key click and drag; select slice to move with the slice package slider).</li>
		<li>Wobble 1H Coil: Spectrometer Control Tool - &#62; Acq -&#62; 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.</li>
	</ol>
	</li>
</ol>
5. Radial Echo Planar Spectral Imaging Scan Setup
<ol>
	<li>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).</li>
	<li>Wobble 13C Coil by clicking Spectrometer Control Tool - &#62; Acq -&#62; Wobble. Set the receiver gain to 1,000-2,000 on the Spectrometer.</li>
	<li>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.</li>
</ol>
6. Run Dissolution
<ol>
	<li>When the pyruvate has attained &#62;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.</li>
	<li>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.</li>
	<li>When the DNP system delivers the hyperpolarized pyruvate (~ 2 min after heating starts) withdraw 500 &#181;l 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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

<ol>
	<li>Merritt, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarized+13C+allows+a+direct+measure+of+flux+through+a+single+enzyme-catalyzed+step+by+NMR.">Hyperpolarized 13C allows a direct measure of flux through a single enzyme-catalyzed step by NMR.</a> Proceedings of the National Academy of Sciences of the United States of America 104. 104, 19773-19777 (2007).</li><li>Rodrigues, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+tumor+glycolysis+using+hyperpolarized+13C-labeled+glucose.">Magnetic resonance imaging of tumor glycolysis using hyperpolarized 13C-labeled glucose.</a> Nature medicine. 20, 93-97 (2014).</li><li>Day, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+tumor+response+to+treatment+using+hyperpolarized+13C+magnetic+resonance+imaging+and+spectroscopy.">Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy.</a> Nature medicine. 13, 1382-1387 (2007).</li><li>Keshari, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarized+13C+dehydroascorbate+as+an+endogenous+redox+sensor+for+in+vivo+metabolic+imaging.">Hyperpolarized 13C dehydroascorbate as an endogenous redox sensor for in vivo metabolic imaging.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 18606-18611 (2011).</li><li>Gallagher, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+pH+in+vivo+using+hyperpolarized+13C-labelled+bicarbonate.">Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate.</a> Nature. 453, 940-943 (2008).</li><li>Larson, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+tumor+hyperpolarized+[1-13C]-pyruvate+dynamics+using+time-resolved+multiband+RF+excitation+echo-planar+MRSI.">Investigation of tumor hyperpolarized [1-13C]-pyruvate dynamics using time-resolved multiband RF excitation echo-planar MRSI.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 63, 582-591 (2010).</li><li>Cunningham, C. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+dynamic+3D+MR+spectroscopic+imaging+with+compressed+sensing+and+multiband+excitation+pulses+for+hyperpolarized+13C+studies.">Fast dynamic 3D MR spectroscopic imaging with compressed sensing and multiband excitation pulses for hyperpolarized 13C studies.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 65, 610-619 (2011).</li><li>Mayer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+subsecond+spiral+chemical+shift+imaging+to+real-time+multislice+metabolic+imaging+of+the+rat+in+vivo+after+injection+of+hyperpolarized+13C1-pyruvate.">Application of subsecond spiral chemical shift imaging to real-time multislice metabolic imaging of the rat in vivo after injection of hyperpolarized 13C1-pyruvate.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 62, 557-564 (2009).</li><li>Walker, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Catalyzing+Phantom+for+Reproducible+Dynamic+Conversion+of+Hyperpolarized+[1-C-13]-Pyruvate.">A Catalyzing Phantom for Reproducible Dynamic Conversion of Hyperpolarized [1-C-13]-Pyruvate.</a> PloS one. 8, e71274 (2013).</li><li>Levin, Y. S., Mayer, D., Yen, Y. F., Hurd, R. E., Spielman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+fast+spiral+chemical+shift+imaging+using+least+squares+reconstruction:+application+for+hyperpolarized+(13)C+metabolic+imaging.">Optimization of fast spiral chemical shift imaging using least squares reconstruction: application for hyperpolarized (13)C metabolic imaging.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 58, 245-252 (2007).</li><li>von Morze, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+multiagent+hyperpolarized+(13)C+perfusion+imaging.">Simultaneous multiagent hyperpolarized (13)C perfusion imaging.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 72, 1599-1609 (2014).</li><li>Sogaard, L. 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Publication of this video-article is supported by Bruker corporation.

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...

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 imaging1-5. During sequence development and verification, dynamic chemical conversion is generally achieved through in vivo or in vitro models6-9 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 needed12-14. This system is ideal for assessment of imaging strategies that aim to provide a quantitative measure of apparent chemical conversion rates.

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

use of a multi-compartment dynamic single enzyme phantom for studies of hyperpolarized magnetic resonance agents

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.

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...

Watch this Scientific Journal Video about Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents at JoVE.com

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

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

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due ...

use-multi-compartment-dynamic-single-enzyme-phantom-for-studies

Research

JoVE Journal

Engineering

6.8K Views.  University of Texas M.D. Anderson Cancer Center. A multi-compartment dynamic phantom is used to simulate some biology of interest for metabolic studies using hyperpolarized magnet resonance agents.

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

Hyperpolarized Xenon for NMR and MRI Applications

Nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) suffer from intrinsic low sensitivity because even strong external magnetic fields of ~10 T generate only a small detectable net-magnetization of the sample at room temperature 1. Hence, most NMR and MRI applications rely on the detection of molecules at relative high concentration (e.g., water for imaging of biological tissue) or require excessive acquisition times. This limits our ability to exploit the very useful molecular specificity of NMR signals for many biochemical and medical applications. However, novel approaches have emerged in the past few years: Manipulation of the detected spin species prior to detection inside the NMR/MRI magnet can dramatically increase the magnetization and therefore allows detection of molecules at much lower concentration 2.
Here, we present a method for polarization of a xenon gas mixture (2-5% Xe, 10% N2, He balance) in a compact setup with a ca. 16000-fold signal enhancement. Modern line-narrowed diode lasers allow efficient polarization 7 and immediate use of gas mixture even if the noble gas is not separated from the other components. The SEOP apparatus is explained and determination of the achieved spin polarization is demonstrated for performance control of the method.
The hyperpolarized gas can be used for void space imaging, including gas flow imaging or diffusion studies at the interfaces with other materials 8,9. Moreover, the Xe NMR signal is extremely sensitive to its molecular environment 6. This enables the option to use it as an NMR/MRI contrast agent when dissolved in aqueous solution with functionalized molecular hosts that temporarily trap the gas 10,11. Direct detection and high-sensitivity indirect detection of such constructs is demonstrated in both spectroscopic and imaging mode.

The production of hyperpolarized xenon by means of spin exchange optical pumping (SEOP) is described. This method yields a ~10000-fold enhancement of the nuclear spin polarization of Xe-129 and has applications in nuclear magnetic resonance spectroscopy and imaging. Examples of gas phase and solution state experiments are given.

Nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) suffer from intrinsic low sensitivity because even strong external magnetic fields of ~10 ...

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.

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.

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

Cancer Research

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

Chemistry

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Bioengineering

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Medicine

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

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

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

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental replicas of a tissue or organ of interest, provide a low cost, flexible solution. However, without access to expensive and specialized equipment, constructing stable phantoms with high fat fractions (e.g., &#62;50% fat fraction levels such as those seen in brown adipose tissue) can be difficult due to the hydrophobic nature of lipids. This work presents a detailed, low cost protocol for creating 5x 100 mL&#160;phantoms with fat fractions of 0%, 25%, 50%, 75%, and 100% using basic lab supplies (hotplate, beakers, etc.) and easily accessible components (distilled water, agar, water-soluble surfactant, sodium benzoate, gadolinium-diethylenetriaminepentacetate (DTPA) contrast agent, peanut oil, and oil-soluble surfactant). The protocol was designed to be flexible; it can be used to create phantoms with different fat fractions and a wide range of volumes. Phantoms created with this technique were evaluated in the feasibility study that compared the fat fraction values from fat-water magnetic resonance imaging to the target values in the constructed phantoms. This study yielded a concordance correlation coefficient of 0.998 (95% confidence interval: 0.972-1.00). In summary, these studies demonstrate the utility of&#160;fat phantoms for validating adipose tissue imaging techniques across a range of clinically relevant tissues and organs.

The purpose of this work is to describe a protocol for creating a practical fat-water phantom that can be customized to produce phantoms with varying fat percentages and volumes.

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental ...

Obtaining High-Quality Hyperpolarized Xenon-129 Magnetic Resonance Images

Troubleshooting and Quality Assurance in Hyperpolarized Xenon Magnetic Resonance Imaging: Tools for High-Quality Image Acquisition

Hyperpolarized (HP) xenon magnetic resonance imaging (129Xe MRI) is a recently federal drug administration (FDA)-approved imaging modality that produces high-resolution images of an inhaled breath of xenon gas for investigation of lung function. However, implementing 129Xe MRI is uniquely challenging as it requires specialized hardware and equipment for hyperpolarization, procurement of xenon imaging coils and coil software, development and compilation of multinuclear MR imaging sequences, and reconstruction/analysis of acquired data. Without proper expertise, these tasks can be daunting, and failure to acquire high-quality images can be&#160;frustrating, and expensive. Here, we present some quality control (QC) protocols, troubleshooting practices, and helpful tools for129Xe MRI sites, which may aid in the acquisition of optimized, high-quality data and accurate results. The discussion will begin with an overview of the process for implementing HP 129Xe MRI, including requirements for a hyperpolarizer lab, the combination of 129Xe MRI coil hardware/software, data acquisition and sequence considerations, data structures, k-space and image properties, and measured signal and noise characteristics. Within each of these necessary steps lies opportunities for errors, challenges, and unfavorable occurrences leading to poor image quality or failed imaging, and this presentation aims to address some of the more commonly encountered issues. In particular, identification and characterization of anomalous noise patterns in acquired data are necessary to avoid image artifacts and low-quality images; examples will be given, and mitigation strategies will be discussed. We aim to make the 129Xe MRI implementation process easier for new sites while providing some guidelines and strategies for real-time troubleshooting.

Author Spotlight: Using Hyperpolarized Xenon-129 MRI to Study Lung Diseases 

Here, we present a protocol for obtaining high-quality hyperpolarized xenon-129 magnetic resonance images, covering hardware, software, data acquisition, sequence selection, data management, k-space utilization, and noise analysis.

Hyperpolarized (HP) xenon magnetic resonance imaging (129Xe MRI) is a recently federal drug administration (FDA)-approved imaging modality that produces ...

Hyperpolarized 129Xe Lung MRI and Spectroscopy in Mechanically Ventilated Mice

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can be exploited to quantify pulmonary physiology, including ventilation, restricted diffusion (alveolar-airspace size), and gas exchange. In humans, it has been used to evaluate disease severity and progression in a variety of pulmonary disorders and is approved for clinical use in the United States and United Kingdom. Beyond its clinical applications, the ability of 129Xe MRI to noninvasively assess pulmonary pathophysiology and provide spatially resolved information is valuable for preclinical research. Among animal models, mice are the most widely used due to the accessibility of genetically modified disease models. Here, 129Xe MRI is promising as a minimally invasive, radiation-free, and sensitive technique to longitudinally monitor lung disease progression and therapy response (e.g., in drug discovery). This technique can extend to preclinical applications by incorporating an MRI-triggered, free-breathing apparatus or mechanical ventilator to deliver gas. Here, we describe the steps and provide checklists to ensure robust data collection and analysis, including creating a thermally polarized xenon gas phantom for quality control, optimizing polarization, animal handling (sedation, intubation, ventilation, and care for mice), and protocols for ventilation, restricted diffusion, and gas exchange data. While preclinical 129Xe MRI can be applied in various animal models (e.g., rats, pigs, sheep), this protocol focuses on mice due to the challenges posed by their small anatomy, which are balanced by their affordability and the availability of many disease models.

Hyperpolarized xenon MRI can quantify regional lung microstructure (air-space dimensions) and physiology (ventilation and gas exchange) in translational research and clinical care. Although challenging, it can provide comparable pulmonary insights in preclinical studies. This protocol describes the infrastructure and procedures needed to perform routine xenon lung MRI in mice.

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can ...

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

Summary

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Chapters in this video

Hyperpolarized Xenon for NMR and MRI Applications

Hyperpolarized ¹³C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

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

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol

Troubleshooting and Quality Assurance in Hyperpolarized Xenon Magnetic Resonance Imaging: Tools for High-Quality Image Acquisition

Hyperpolarized ¹²⁹Xe Lung MRI and Spectroscopy in Mechanically Ventilated Mice