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 ...

<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. H., Dominguez Viqueira, W., Hurd, R. E., Chen, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+correction+method+for+improved+spatial+correlation+of+hyperpolarized+13C+metabolites+and+anatomy.">Frequency correction method for improved spatial correlation of hyperpolarized 13C metabolites and anatomy.</a> NMR in biomedicine. 27, 212-218 (2014).</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=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. V., Schilling, F., Janich, M. A., Menzel, M. I., Ardenkjaer-Larsen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+apparent+diffusion+coefficients+of+hyperpolarized+(1)(3)C-labeled+metabolites.">In vivo measurement of apparent diffusion coefficients of hyperpolarized (1)(3)C-labeled metabolites.</a> NMR in biomedicine. 27, 561-569 (2014).</li><li>Patrick, P. S., 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=Detection+of+transgene+expression+using+hyperpolarized+13C+urea+and+diffusion-weighted+magnetic+resonance+spectroscopy.">Detection of transgene expression using hyperpolarized 13C urea and diffusion-weighted magnetic resonance spectroscopy.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 73, 1401-1406 (2015).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1045">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">BioSpect 7T</td>
			<td style="width:147px;">Bruker</td>
			<td style="width:195px;">BioSpec 70/30 USR</td>
			<td style="width:445px;">7 Tesla Pre-Clinical MRI Scanner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">HyperSense</td>
			<td style="width:147px;">Oxford Instruments</td>
			<td style="width:195px;">Hypersense DNP Polarizer</td>
			<td>Dynamic Nuclear Polarizer for MRI agents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">1-13C-Pyrvic Acid</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:195px;">677175</td>
			<td>Carbon 13 labled neat pyruvic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Trityl Radical</td>
			<td style="width:147px;">GE Healthcare</td>
			<td style="width:195px;">OX063</td>
			<td>Free radical used in Dynamic Nuclear Polarization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">NaOH</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:195px;">S8045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">EDTA</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:195px;">E6758</td>
			<td style="width:445px;">Ethylenediaminetetraacetic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">LDH</td>
			<td style="width:147px;">Worthingthon</td>
			<td style="width:195px;">LS002755</td>
			<td>Lactate Dehydrogenase from rabbit muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">NADH</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:195px;">N4505</td>
			<td>&#946;-Nicotinamide adenine dinucleotide, reduced dipotassium salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Trizma</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:195px;">T7943</td>
			<td>Trizma&#174;&#160;Pre-set crystals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">NaCl</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:195px;">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 ...

The overall goal of this procedure is to measure the conversion of hyperpolarized pyruvate to lactate by magnetic resonance imaging or MRI in a controlled phantom environment. This method can help answer key questions in the hyperpolarized MRI field such as the ability of a system to detect chemical conversion of pyruvate by magnetic resonance. The main advantage of this technique is that chemical conversion of pyruvate proceeds similarly to metabolism in vivo but is more controllable and repeatable than in living systems.The implications of this technique extend towards diagnosis of cancer because the elevated conversion of pyruvate to lactate common in most cancers is simulated by the phantom environment. Though this method can provide insight into cancer it can also be applied to other metabolic imaging such as cardiac metabolism. Generally individuals new to this method may struggle because the time constraints inherent to hyperpolarized media are short.Visual demonstration of this method is critical as dissolution and ejection steps are difficult to learn because they have to happen rapidly and must be performed precisely. In a sample cup for a dynamic nuclear polarization or DNP system pipette 0.3 micro-liters of the gadoteridol solution and 13 milligrams of the pyruvic acid solution. Briefly stir this mixture in the sample cup with a pipette tip.Begin the sample insertion process by clicking the insert sample button on the DNP system console. On the sample wizard select normal sample and click next. 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 using the insertion rod. Pull on the plunger at the end of the sample insertion rod to release the sample in the variable temperature insert. Remove the sample insertion rod from the system and click the next button on the DNP system console.Then initiate the polarization by clicking the polarise sample button on the DNP system console. In the RINMR software type HYPERSENSENMR to polarization monitoring software. Set build up configuration to one and press enter.Then click solid build up. After setting the location and name of the save file select the profile for carbon 13 in the drop down tab on the DNP system console. Click next.Check the box to enable sampling during the build up. Set sample time to 300 seconds and click finish up. Finally measure out 3.85 grams of the dissolution media either by volume with a five milliliter syringe or by weight using a scale.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. Prepare the high activity enzyme mixture by mixing 240 micro-liters of NADH solution 125 micro-liters of LDH solution and 335 micro-liters of buffer.Keep the solution in a three milliliter syringe that can be attached to the injection line. Then prepare the low activity enzyme mixture by mixing together 240 micro-liters of NADH solution 75 micro-liters of LDH solution and 385 micro-liters of buffer. Keep this mixture in a separate three milliliter syringe that can be attached to the injection line.To perform initial positioning load a new localizer scan. Wobble the proton coil by selecting Acq/Reco. Display and open the adjustment platform.On the adjustment panel select Wobble Adjust and click ppen. Set the sweep width to ten megahertz and click set up. After a moment the protons coils tuning and matching should appear in the acquisition window.Wobble the carbon coil by changing the coil element to 13C or element two and setting the sweep width to five megahertz. After a moment the carbons coils tuning and matching should appear in the acquisition window and if tuned properly, hit stop. To return to scan control press apply then back and finally press continue to begin the scan.It is critical that the scan in this step is fully set up before beginning dissolution. After dissolution has begun it should not be stopped and their will be little time to adjust sequence parameters before the hyperpolarized pyruvate is delivered. Load a new radio echo planar spectroscopic imaging scan.Set the slice thickness to 30 millimeters so as to cover the whole reaction chamber. Set the operation mode to carbon 13 by selecting the system tab and changing the operation mode to 13C transmit receive. Once the pyruvate has attained greater than 90%polarization the solutions and phantom are ready and the scan is configured.Click the run dissolution button on the DNP system console. When prompted move the dissolution stick into its operating position and inject the dissolution media. Close the DNP system and click the finished button on the DNP system console.It is important that injection of pyruvate and enzyme is done smoothly. This will ensure the enzyme mixture is properly mixed and delivered to the phantom chamber before the chemical conversion has completed. When the DNP system delivers the hyperpolarized pyruvate aspirate 500 micro-liters of the pyruvate solution into each of the high and low enzyme concentration solution syringes.Slowly inject each syringe into an injection line. Scanning can be initiated prior to injection or immediately after injection depending on the scan protocol used. After the dissolution is complete move the dissolution stick back to the resting position when prompted and then click finish.Representative results of a radio echo planar spectral imaging sequence are shown here. The pyruvate image shows the strong pyruvate signal in both chambers. The lactate image shows a weaker lactate signal but is still localized to the chambers.The signal ratio of hyperpolarized lactate and pyruvate can be used to estimate the enzyme activity in each chamber. The signal ratios in each chamber match the enzyme activity present. Once mastered, this technique can be done in one hour.While attempting this procedure it is important to remember to have the phantom mixture and imaging sequence ready before beginning the dissolution process. Following this procedure other hyperpolarized agents and enzymes can be used in order to answer additional questions like how well a sequence can image other chemical reactions. This technique paves the way for researchers in the field of hyperpolarized MRI to explore sequence performance using reproduceable phantoms.After watching this video you should have a good understanding of how to prepare an enzyme mixture to facilitate the conversion of hyperpolarized pyruvate to lactate and to polarize carbon 13 enriched pyruvate for imaging. Don't forget that working with strong magnetic fields can be extremely hazardous and precautions such as controlled access to the scan room should always be taken.

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6.8K vistas.  University of Texas M.D. Anderson Cancer Center. The overall goal of this procedure is to measure the conversion of hyperpolarized pyruvate to lactate by magnetic resonance imaging or MRI in a controlled phantom environment. This method can help answer key questions in the hyperpolarized MRI field such as the ability of a system to detect chemical conversion of pyruvate by magnetic resonance. The main advantage of this technique is that chemical conversion of pyruvate proceeds similarly to metabolism in vivo but is more controllable and rep...