Name: measuring the spin-lattice relaxation magnetic field dependence of hyperpolarized [1-13c]pyruvate
Uploaded: 2019-09-13T12:30:00
Description: Watch this Scientific Journal Video about Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate at JoVE.com

The authors would like to thank the Ontario Institute for Cancer Research, Imaging Translation Program and the Natural Sciences and Engineering Research Council of Canada for funding this research. We also like to acknowledge useful discussions with Albert Chen, GE Healthcare, Toronto, Canada, Gianni Ferrante, Stelar s.r.l., Italy, and William Mander, Oxford Instruments, UK.

1. Sample Preparation
NOTE: Steps 1.1-1.8 are performed just once
<ol>
	<li>Prepare 1 mL of stock 13C-enriched pyruvic acid solution, widely used for in vivo research1,2,5,6, consisting of 15-mmol/L of triarylmethyl radical dissolved in [1-13C]pyruvic acid (see Table of Materials). Aliquots from this stock solution wi...

<ol>
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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=Multi-compound+polarization+by+DNP+allows+simultaneous+assessment+of+multiple+enzymatic+activities+in+vivo.">Multi-compound polarization by DNP allows simultaneous assessment of multiple enzymatic activities in vivo.</a> Journal of Magnetic Resonance. 205 (1), 141-147 (2010).</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=Production+of+hyperpolarized+[1,4-13C2]malate+from+[1,4-13C2]fumarate+is+a+marker+of+cell+necrosis+and+treatment+response+in+tumors.">Production of hyperpolarized [1,4-13C2]malate from [1,4-13C2]fumarate is a marker of cell necrosis and treatment response in tumors.</a> Proceedings of the National Academy of Science of the United States of America. 106 (47), 19801-19806 (2009).</li><li>Chen, A. P., 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=Feasibility+of+using+hyperpolarized+[1-13C]lactate+as+a+substrate+for+in+vivo+metabolic+13C+MRSI+studies.">Feasibility of using hyperpolarized [1-13C]lactate as a substrate for in vivo metabolic 13C MRSI studies.</a> Magnetic Resonance Imaging. 26 (6), 721-726 (2008).</li><li>Gallagher, F. A., Kettunen, M. I., Day, S. E., Lerche, M., Brindle, K. 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J., 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+lactate,+pyruvate,+and+alanine:+noninvasive+biomarkers+for+prostate+cancer+detection+and+grading.">Hyperpolarized 13C lactate, pyruvate, and alanine: noninvasive biomarkers for prostate cancer detection and grading.</a> Cancer Research. 68 (20), 8607-8615 (2008).</li><li>Chen, A. P., 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+C-13+spectroscopic+imaging+of+the+TRAMP+mouse+at+3T-initial+experience.">Hyperpolarized C-13 spectroscopic imaging of the TRAMP mouse at 3T-initial experience.</a> Magnetic Resonance in Medicine. 58 (6), 1099-1106 (2007).</li><li>Lupo, J. 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=Analysis+of+hyperpolarized+dynamic+13C+lactate+imaging+in+a+transgenic+mouse+model+of+prostate+cancer.">Analysis of hyperpolarized dynamic 13C lactate imaging in a transgenic mouse model of prostate cancer.</a> Magnetic Resonance Imaging. 28 (2), 153-162 (2010).</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=Imaging+of+blood+flow+using+hyperpolarized+[(13)C]urea+in+preclinical+cancer+models.">Imaging of blood flow using hyperpolarized [(13)C]urea in preclinical cancer models.</a> Journal of Magnetic Resonance Imaging. 33 (3), 692-697 (2011).</li><li>Brindle, K. M., Bohndiek, S. E., Gallagher, F. A., Kettunen, M. 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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=Detection+of+tumor+response+to+a+vascular+disrupting+agent+by+hyperpolarized+13C+magnetic+resonance+spectroscopy.">Detection of tumor response to a vascular disrupting agent by hyperpolarized 13C magnetic resonance spectroscopy.</a> Molecular Cancer Therapeutics. 9 (12), 3278-3288 (2010).</li><li>Witney, T. H., 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+treatment+response+in+a+model+of+human+breast+adenocarcinoma+using+hyperpolarised+[1-13C]pyruvate+and+[1,4-13C2]fumarate.">Detecting treatment response in a model of human breast adenocarcinoma using hyperpolarised [1-13C]pyruvate and [1,4-13C2]fumarate.</a> British Journal of Cancer. 103 (9), 1400-1406 (2010).</li><li>Levitt, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Spin dynamics: basics of nuclear magnetic resonance. , (2001).</li><li>Mieville, P., Jannin, S., Bodenhausen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxometry+of+insensitive+nuclei:+optimizing+dissolution+dynamic+nuclear+polarization.">Relaxometry of insensitive nuclei: optimizing dissolution dynamic nuclear polarization.</a> Journal of Magnetic Resonance. 210 (1), 137-140 (2011).</li><li>Redfield, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shuttling+device+for+high-resolution+measurements+of+relaxation+and+related+phenomena+in+solution+at+low+field,+using+a+shared+commercial+500+MHz+NMR+instrument.">Shuttling device for high-resolution measurements of relaxation and related phenomena in solution at low field, using a shared commercial 500 MHz NMR instrument.</a> Magnetic Resonance in Chemistry. 41 (10), 753-768 (2003).</li><li>Grosse, S., Gubaydullin, F., Scheelken, H., Vieth, H. -. M., Yurkovskaya, A. 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The use of DNP to enhance signal acquisition is a technical solution to insufficient magnetic resonance signal available from 13C nuclei at limited concentrations, as those used in animal injections, but presents other experimental challenges. Each relaxation measurement shown in Figure 7 represents a measurement of a uniquely prepared sample because it cannot be repolarized after dissolution for remeasurement. This inevitably leads to experimental variability due to minor differe...

Magnetic resonance spectroscopic imaging (MRSI) can produce spatial maps of metabolites detected by spectroscopic imaging, but its practical use is often limited by its relatively low sensitivity. This low sensitivity of in vivo magnetic resonance imaging and spectroscopy methods stems from the small degree of nuclear magnetization achievable at body temperatures and reasonable magnetic field strengths. However, this limitation can be overcome by the use of dynamic nuclear polarization (DNP) to greatly enhance the in vitro magnetization of liquid substrates, which are subsequently injected to probe in vivo metabolism using MRSI1,2,3,4. DNP is capable of enhancing the magnetization of most nuclei with non-zero nuclear spin and has been used to increase in vivo MRSI sensitivity of 13C-enriched compounds such as pyruvate5,6, bicarbonate7,8, fumarate9, lactate10, glutamine11, and others by more than four orders of magnitude12. Its applications include imaging of vascular disease13,14,15, organ perfusion13,16,17,18, cancer detection1,19,20,21,22, tumor staging23,24, and quantification of therapeutic response2,6,23,24,25,26.
Slow spin-lattice relaxation is essential for in vivo detection with MRSI. Spin-lattice relaxation times (T1s) on the order of tens of seconds are possible for nuclei with low gyromagnetic ratios within small molecules in solution. Several physical factors influence the transfer of energy between a nuclear spin transition and its environment (lattice) leading to relaxation, including the magnetic field strength, temperature, and molecular conformation27. Dipolar relaxation is reduced in molecules for carbon positions with no protons directly attached, and deuteration of dissolution media can further reduce intermolecular dipolar relaxation. Unfortunately, deuterated solvents have limited abilities to extend in vivo relaxation. Increased relaxation of carbonyls or carboxylic acids (such as pyruvate) can occur at high magnetic field strengths due to chemical shift anisotropy. The presence of paramagnetic impurities from the fluid path during dissolution after polarization can cause rapid relaxation and need to be avoided or eliminated using chelators.
Very little data exist for the relaxation of 13C-containing compounds at low fields, where spin-lattice relaxation could be significantly faster. However, it is important to measure T1 at low fields to understand relaxation during preparation of the agent used for in vivo imaging, since the hyperpolarized contrast agents are usually dispensed from the DNP apparatus near or at the earth&#8217;s field. Additional physical factors such as 13C-enriched substrate concentration, solution pH, buffers and temperature also influence relaxation, and consequently have an effect on the formulation of the agent. All these factors are essential in the determination of key parameters in optimizing the DNP dissolution process, and the calculation of the magnitude of signal loss that occurs in transportation of the sample from the DNP apparatus to the imaging magnet.
Nuclear magnetic resonance dispersion (NMRD) measurements, i.e., T1 measurements, as a function of magnetic field are typically acquired using an NMR spectrometer. To acquire these measurements, a shuttling method could be used where the sample is first shuttled out of the spectrometer to relax at some field determined by its position in the fringe field of the magnet28,29,30 and then rapidly transferred back into the NMR magnet to measure its remaining magnetization. By repeating this process at the same point in the magnetic field but with increasing periods of relaxation, a relaxation curve can be obtained, which can then be analyzed to estimate T1.
We use an alternative technique known as fast field-cycling relaxometry31,32,33 to acquire our NMRD data. We have modified a commercial field-cycling relaxometer (see Table of Materials), for T1 measurements of solutions containing hyperpolarized 13C nuclei. Compared with the shuttle method, field-cycling enables this relaxometer to systematically acquire NMRD data over a smaller range of magnetic fields (0.25 mT to 1 T). This is accomplished by rapidly changing the magnetic field itself, not the sample location in the magnetic field. Therefore, a sample can be magnetized at a high field strength, &#34;relaxed&#34;&#160;at a lower field strength, and then measured by acquisition of a free-induction-decay at a fixed field (and Larmor frequency) to maximize signal. This means that the sample temperature can be controlled during the measurement, and the NMR probe does not need to be tuned at each relaxation field promoting automatic acquisition over the entire magnetic field range.
Focusing our efforts to the effects of dispensing and transporting the hyperpolarized solutions at low magnetic fields, this work presents a detailed methodology to measure the spin-lattice relaxation time of hyperpolarized 13C-pyruvate using fast field-cycling relaxometry for magnetic fields in the range of 0.237 mT to 0.705 T. The main results of using this methodology have been previously presented for [1-13C]pyruvate34 and 13C-enriched sodium and cesium bicarbonate35 where other factors such as radical concentration and dissolution pH have also been studied.

<table border="0" cellpadding="0" cellspacing="0" style="width:825px;" width="824">
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	</colgroup>
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:263px;">[1-13C]Pyruvic Acid</td>
			<td style="width:244px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:79px;">677175</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">10mm NMR Tube</td>
			<td style="width:244px;">Norell, Inc., Morganton NC, USA</td>
			<td style="width:79px;">1001-8</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">De-ionized water</td>
			<td style="width:244px;"></td>
			<td style="width:79px;"></td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td style="width:244px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:79px;">E5134</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="294">
			<td height="294" style="height:294px;width:263px;">HyperSense Dynamic Nuclear Polarizer</td>
			<td style="width:244px;">Oxford Instruments, Abingdon, UK</td>
			<td style="width:79px;"></td>
			<td style="width:240px;">Includes the following: &#34;DNP-NMR Polarizer&#34; software used to control and monitor the whole DNP polarizer; &#34;RINMR&#34; used to monitor the solid state polarization levels; &#34;HyperTerminal&#34; used to communicate the DNP software with the RINMR software that monitors the solid state polarization level. Also includes the MQC bench top spectrometer to monitor the liquid state polarization in conjunction with it own RINMR software</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:263px;">MATLAB R2017b</td>
			<td>MathWorks, Natick, MA</td>
			<td style="width:79px;"></td>
			<td style="width:240px;">Include scripts for non-linear fitting of magnetization decay over time and T1 NMRD analysis of hyperpolarized pyruvic acid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">OX063 Triarylmethyl radical</td>
			<td style="width:244px;">Oxford Instruments, Abingdon, UK</td>
			<td style="width:79px;"></td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">pH meter - SympHony</td>
			<td style="width:244px;">VWR International, Mississauga, ON., Canada</td>
			<td style="width:79px;">SB70P</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">ProHance</td>
			<td style="width:244px;">Bracco Diagnostics Inc.</td>
			<td style="width:79px;"></td>
			<td style="width:240px;">Gadoteridol, Gd-HP-DO3A</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Pure Ethanol (100% pure)</td>
			<td style="width:244px;">Commercial Alcohols, Toronto, ON, Canada</td>
			<td style="width:79px;">P016EAAN</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Shim Coil</td>
			<td style="width:244px;"></td>
			<td style="width:79px;"></td>
			<td style="width:240px;">Developed in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Sodium Chloride</td>
			<td style="width:244px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:79px;">S7653</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Sodium Hydroxide</td>
			<td style="width:244px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:79px;">S8045&#160;</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:263px;">SpinMaster FFC2000 1T C/DC</td>
			<td style="width:244px;">Stelar s.r.l., Mede (PV) Italy</td>
			<td style="width:79px;"></td>
			<td style="width:240px;">Includes the software &#34;AcqNMR&#34; that is used to set experimental parameters, monitor the tuning and matching of the RF coil, loading different pulse sequences, calibrate flip angle, data acquisition and curve fitting, among other functions. Also includes a depth gauge, some weights and a depth stopper.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Trizma Pre-Set Crystals (pH 7.6)</td>
			<td style="width:244px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:79px;">T7943</td>
			<td style="width:240px;"></td>
		</tr>
	</tbody>
</table>

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.

Figure 2 presents an example of a high-resolution full-range microwave sweep for pyruvic acid. For the presented case, that optimal microwave frequency corresponds to 94.128 GHz, highlighted in the figure insert. Our DNP system can normally work in the range of 93.750 GHz to 94.241 GHz with step size of 1 MHz, polarization time of up to 600 s, and power of up to 100 mW. A full range of frequencies is investigated only for novel substrates. However, based on previous experience with 13</...

Watch this Scientific Journal Video about Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate at JoVE.com

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.

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 ...

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