DNP System Output Volume Reduction Using Inert Fluids

Abstract

Purpose

To present a method for significantly increasing the concentration of a hyperpolarized compound produced by a commercial DNP polarizer, enabling the polarization process to be more suitable for pre-clinical applications.

Materials and Methods

Using a HyperSense® DNP polarizer, we have investigated the combined use of perfluorocarbon and water to warm and dissolve the hyperpolarized material from the polarization temperature of 1.4 K to produce material at temperatures suitable for injection.

Results

By replacing 75% of the water in the dissolution volume with a chemically and biologically inert liquid that is immiscible with water, the injection volume can be reduced fourfold Rapid separation of the water and perfluorocarbon mixture enables the aqueous layer containing polarized material to be easily and rapidly collected.

Conclusion

The approach provides a significantly increased concentration of compound in a volume for injection that is more appropriate for small animal studies. This is demonstrated for ¹³C labeled pyruvic acid and ¹³C labeled succinate, but may be applied to the majority of nuclei and compounds hyperpolarized by the DNP method.

Introduction

There is increasing interest in using dynamic nuclear polarization (DNP) to enhance the available NMR signal from a variety of compounds and nuclei for use in MR imaging and spectroscopy studies. For a commercial polarizer system (HyperSense®, Oxford Instruments, Oxford UK), the standard injection volume is not optimized for small animal research since a relatively large volume of water (typically around 4 ml) containing EDTA, along with a buffering agent, is used to warm and extract a concentrated smaller volume (typically around 30 μl) of a frozen solution comprised of the polarized compound mixed with radical. Following this dissolution process, the resultant volume may be significantly more dilute than desired, or too large a volume for safe, rapid bolus injection into a small animal. The recommended injection volume and rate are 5 ml/kg and 0.05 ml/s for both mice and rats (1). For a 200 g rat this results in a typical injection of 1 ml taking 20 s. For a 20 g mouse, this becomes 0.1 ml in 2 seconds, which is a short, tight bolus, but it is a very small volume compared to the typical polarizer output. When following the recommended injection procedure, this results in only about 25% of the total polarized solution injected into an adult rat and only 2.5% of the solution for a mouse. The outcome is a significant waste of the hyperpolarized compound and an inordinately large bolus. In both cases this can result in a suboptimal study due to the large and dilute dissolution solution.

Maximizing the signal available from ¹³C labeled compounds is critical for in vivo or in vitro imaging or spectroscopy. Consequently, this work has explored the possibility of reducing the volume of injectable solution produced by the polarizer. To achieve this, we have investigated replacing a fraction of the water used for extraction with a chemically and biologically inert compound that is also immiscible with water and possesses a high relative density for easy and rapid separation. Here we show that the DNP polarizer has the capability to generate smaller volumes of hyperpolarized solution more appropriate for analytical or pre-clinical small animal in vivo ¹³C spectroscopic studies without any physical or methodological modifications to the polarizer or its operation. The generation of smaller volumes for injection provides a more concentrated solution, which enables a potential increase in SNR for both in vitro and in vivo studies. Alternatively, the same approach would enable a smaller amount of polarized compound to be used. In this study, we expand upon a preliminary report (2) and demonstrate that by using a perfluorocarbon (Fluorinert® FC-72 or Fluorinert® FC-3283, 3M, St Paul, MN, USA) to replace a fraction of the water for dissolution of polarized compounds, a volume for injection more appropriate for preclinical studies may be routinely prepared. While pyruvate and succinate were used to demonstrate the approach, this technique is likely applicable to many other compounds that are insoluble in perfluorocarbon. The effects of the approach on the liquid state polarization levels of the output volume were investigated. In addition,, we have investigated the temperature of the prepared solutions and also compared the longitudinal relaxation times of pyruvate and succinate solutions prepared using water only, the water/perfluorocarbon mixture and a deuterated water/perfluorocarbon mixture.

Materials and Methods

Polarization

To demonstrate the approach, a modification of the dissolution of 1-¹³C labeled pyruvic acid and 1,4-¹³C₂ succinate (Cambridge Isotope Laboratories, Andover, MA, USA) for injection has been investigated using the Oxford Instruments’ DNP polarizer (Oxford Instruments HyperSense®, Tubney Woods, Abingdon, Oxfordshire, UK). All polarization steps up to the dissolution process remain unchanged from that described in the operating manual and a previous report describing the DNP process (3). Briefly, the polarization/dissolution process consists of 5 steps:

1. Place sample cup containing approximately 30 μL of sample into the 3.35 T magnet at the center of the DNP polarizer and irradiate at approximately 94.1 MHz at a temperature of 1.4 K.

2. Place the mixture of 3 ml perfluorocarbon and 1 ml water into the dissolution chamber and heat until the pressure reaches 9.87 atm (10 bar).

3. Trigger extraction of the sample, allowing superheated mixture to warm and dissolve the sample.

4. Direct warmed mixture of perfluorocarbon and sample into a graduated cylinder containing a pH modifying solution.

5. Draw off the less dense polarized aqueous solution from the top of the mixture in the graduated cylinder for in vitro or in vivo experiments, while leaving the denser perfluorocarbon in the graduated cylinder for later reuse if desired.

The dissolution mixture was selected empirically to minimize the injectable aqueous volume while providing sufficient aqueous volume to completely dissolve the polarized sample. Empirical testing consisted of reducing the amount of water from 100% to 0%, while systematically replacing the water with perfluorocarbon.

A pH modifying solution was used to adjust the pH of the hyperpolarized sample to a value close to physiological levels (4). The solution (total volume of 200 μl, containing the base sodium hydroxide (NaOH) and the buffer sodium bicarbonate (NaHCO₃) or Tris (Sigma-Aldrich, St Louis, MO, USA)), was pre-loaded into a graduated cylinder which was used to collect the dissolution mixture prior to injection. The solution was pH balanced outside of the polarizer, however the pH balancing solution can also be placed in the dissolution chamber as well.

The temperature of the resulting aqueous solution was measured using a microprobe thermometer (Mettler-Toledo Inc., Columbus, OH) in order to confirm the thermal suitability of the solution for in vivo administration.

Phantom Experiments: liquid state polarization

Five experiments were performed in order to determine the liquid state polarization for full and reduced volume dissolutions. An Oxford Instruments MQC liquid state polarimeter (Oxford Instruments, Tubney Woods, Abingdon, Oxfordshire, UK) was connected to the polarizer with an automatic trigger line, and the dissolution was performed directly into the NMR tube on the polarimeter. The polarizer was set to trigger the polarimeter 9 s post dissolution to allow for compound transfer and settling times. For the liquid state polarization experiments, only 10 μl of pyruvate was used in order to conserve the pyruvate. Of the five experiments, two had full volume dissolutions of 4 ml of water/EDTA solution. The 3 remaining dissolutions had ¼, ½, and ¾ fractions of FC-3283 to water respectively with a full volume of 4 ml for all dissolutions.

Phantom Experiments: longitudinal relaxation time measurement

Experiments were performed in order to determine the ¹³C longitudinal (T₁) relaxation times when using the more concentrated dissolution solutions. All experiments were performed using a Varian 4.7 T imaging system (Varian Inc, Palo Alto, Ca) and home-built dual tuned ¹H (5 cm diameter) and ¹³C (3.8 cm diameter) surface coil. In order to measure the ¹³C T₁ relaxation times, a pulse-acquire sequence with variable repetition times (between 0.5 s and 2 s) was used to determine the separate effects of RF and T₁ decay of the polarized compound. For reduced volume compounds, the full 1 ml of sample was used, and with standard volume solutions, the full 4 ml of sample was used. The RF and T₁ effects were subsequently fitted independently using the fminsearch function in MATLAB (Mathworks, Natick, MA).

In vivo Experiments

In vivo hyperpolarized 1-¹³C pyruvate metabolism experiments were performed to test tolerance and feasibility of the reduced volume dissolution procedure. Five healthy ICR mice were placed under terminal anesthesia and 4 healthy Sprague-Dawley rats were placed under non-terminal anesthesia using isoflurane in accordance with local IRB guidelines, and their tail veins were cannulated to allow administration of the pyruvate solution while positioned in the scanner. The respiratory rate was monitored throughout the imaging experiment. A dual tuned ¹³C and ¹H surface coil was positioned under the liver of each animal. The volume of the injection was between 0.5 and 0.8 ml of injectable which was produced with the reduced volume procedure. The injections lasted between 10 and 20 s, and followed previously published guidelines (1). The ¹³C spectra were acquired using a global pulse and FID acquire spectroscopy sequence.

In vivo hyperpolarized 1,4-¹³C₂ succinate experiments were performed to verify the use of the perfluorocarbon and water dissolution solution with a second hyperpolarized compound. One healthy ICR mouse and 2 healthy Sprague-Dawley rats were placed under non-terminal anesthesia using isoflurane. All other parameters were identical to the pyruvate experiments discussed above, and all were in accordance with local IRB guidelines.

Results

Polarization

The sample extraction and collection was demonstrated to provide rapid separation between the perfluorcarbon and hyperpolarized solution for all experiments. For all ratios of water and perfluorocarbon, it was observed that once extracted into the graduated cylinder, the mixture rapidly settled into two layers. The separation time is negligible, occurring in less than a second following completion of the polarizer purge process. The dense and immiscible (Table 1) perfluorocarbon provides a colorless lower layer (Figure 1) while the polarized solution in the green-colored upper layer separates completely and can be easily drawn off for injection.

Table 1.

Comparison of the physical properties of water, and FC-72 and FC-3283 perfluorocarbons.

	Average Molecular Weight	Boiling Point (1 atm) (°C)	Density* (kg/m3)	Kinematic Viscosity* (centistokes)	Specific Heat (Jkg−1C−1)	Solubility in water* (ppmw)	Water Solubility* (ppmw)
Water**	18	100	1000	1.00	4181	NA	<5
FC-72***	338	56	1680	0.38	1100	<5	10
FC-3283****	521	128	1820	0.75	1100	<5	7

^*at 25°C

^**CRC Handbook of Chemistry and Physics, 58th Edition (1977–1978), CRC Press Inc.

^***from: 3M Perfluorocarbon FC-72 http://multimedia.3m.com/mws/mediawebserver?66666UuZjcFSLXTtnxTE5XF6EVuQEcuZgVs6EVs6E666666--

^****from: 3M Perfluorocarbon FC-3283 http://multimedia.3m.com/mws/mediawebserver?66666UuZjcFSLXTtnxTEoxF6EVuQEcuZgVs6EVs6E666666--

Figure 1.

Photograph of the pyruvate output seconds after completion of the dissolution process when using 3 ml FC-72 and 1 ml water. The dense FC-72 perfluorocarbon provides a colorless lower layer and the green-colored pyruvate/radical solution forms an easily identifiable upper layer that can be drawn off for injection. Note that approximately 1 ml of FC-72 has been lost in the dissolution, however further evacuation of the polarizer allows for the reclamation of all but 0.3 ml of perfluorocarbon. This is similar to the amount of water lost during water only dissolutions. Note that all evacuated perfluorocarbon may be reused, which is approximately a 90% reclamation rate.

Solutions with higher percentages of perfluorocarbon result in an incomplete clearance of the polarized compound from the cup due to the immiscible nature of the perfluorocarbon. By reducing the percentage of water in the dissolution mixture, it was determined empirically that the mixture should have a minimum of 1 ml water which together with 3 ml of perfluorocarbon ensures a complete flushing of the 30 μl of polarized solution from the sample cup. This provides approximately 1.2 ml of pH adjusted solution suitable for injection. The 1 ml water and 3 ml perfluorocarbon mixture provides full clearance of the polarized compound while delivering a significantly reduced aqueous output volume. When using an initial sample of 30 μl of pyruvate the 1 ml water and 3 ml perfluorocarbon mixture results in a 362 mM solution of pyruvate, compared to a 100 mM solution when using only water for dissolution. Note that this calculation takes into account a base/buffer solution which causes the concentration gain to be slightly less than 4. For succinate, a 30 μl initial sample produces a 22 mM solution when using 4 ml of water, and a 90 mM solution when using 1 ml water and 3 ml perfluorocarbon as the dissolution liquid. It should be noted that because of the immiscible nature of the perfluorocarbon with water, the majority of the perfluorocarbon is easily recovered at the end of the dissolution. The recovered perfluorocarbon can also be reused, thereby mitigating the cost of the perfluorocarbon purchase. With the cost of 1-13C Pyruvate approximately $20 per experiment (30 μl), and the 4 ml of perfluorocarbon costs approximately $0.6 per experiment, which results in a 3% more expensive experiment, it is clear that using perfluorocarbon is a cost effective strategy for injection volume reduction. It should also be noted that when compared to other ¹³C labeled compounds, 1-13C Pyruvate is relatively inexpensive. Some custom compounds will be far more valuable, further underlining the cost effectiveness of the reduced volume technique.

For pyruvate, the output sample temperature remained in the physiologic range. Replacement of 3 ml of water with 3 ml of FC-72 reduces the dissolution output temperature from 35 °C to 28°C. This reduction in the temperature is still within the acceptable range for animal injection and was well tolerated by the animals studied. FC-3283 was also used, which has a reported boiling point of 128 °C at 1 atm compared to 56 °C at 1 atm for FC-72. By using FC-3283, the dissolution output temperature only decreased from 35 °C to 30 °C, demonstrating that the output temperature may be changed by using perfluorocarbons with different physical properties. The drop in dissolution output temperature when using FC-72 can also be alleviated, to some extent, by using 4 ml of FC-72 rather than 3 ml, which increases the total dissolution volume to 5 ml, thereby achieving a temperature of 31 °C.

Phantom Experiments: liquid state polarization

For the 5 liquid state polarization experiments, all polarizations were determined to be between 15 % and 16 %. The two 4 ml water dissolutions were 15.4 % and 15.6 %, and the ¼, ½, and ¾ fraction water/FC-3283 dissolutions were 15.1 %, 15.3 %, and 15.4 % respectively for a 4 ml total volume. The values reported are for maximum solid state polarization and are scaled by both the input and output volumes. This can be seen in Table 2, which shows the wasted volume for a variety of experimental volumes.

Table 2.

Iso-molar comparison of injection duration and volume for normal and reduced volume solutions.

		Normal Volume				Reduced Volume
Pyruvate Sample Volume	Moles (×10−3)	Total Volume Injection Duration (s)	Volume (mL)	Wasted Injection Volume (mL) (400 g Rat)	Wasted Injection Volume (mL) (40 g Mouse)	Total Volume Injection Duration (s)	Volume (mL)	Wasted Injection Volume (mL) (400 g Rat)	Wasted Injection Volume (mL) (20 g Mouse)
7.5 μL	0.107	20	1	0	0.8	5	0.25	0	0.05
15 μL	0.213	40	2	0	1.8	10	0.5	0	0.3
30 μL	0.426	80	4	2	3.8	20	1	0	0.8
60 μL	0.852	160	8	6	7.8	40	2	0	1.8
120 μL	1.703	320	16	14	15.8	80	4	2	3.8

Note that the total volume injection duration column is the injection time for the whole volume which is produced, not the injected volume. For a 400 g rat, the maximum advisable injection volume is 2 ml, and for the 40 g mouse, it is 0.2 ml. This corresponds with maximum injection durations of 40 s for a rat and 4 s for a mouse. Also note that reduced volume solutions of 120 μL pyruvate sample volume have identical injection characteristics as 30 μL normal volume solutions.

Phantom Experiments: longitudinal relaxation time measurements

In vitro ¹³C-pyruvate T₁ measurements in a 4.7 T magnetic field show that when a 3:1 perfluorcarbon to water ratio is used, the T₁ of the concentrated solution is reduced from approximately 71±8 s (at 0% perfluorcarbon) to approximately 63±2 s (at 75% perfluorocarbon). As illustrated in Figure 2, the in vitro signal of the reduced volume solution (75% perfluorcarbon) and the signal of 100% water are not equivalent, when taking T1 values into account, until after 5 minutes have elapsed, which is the timescale for an entire study and is far longer than the time between dissolution and injection (Figure 2). When using 75% perfluorocarbon (3 ml) and 25% (1 ml) deuterated water, the T₁ was found to be 83±2 s. For ¹³C succinate, T₁ relaxation times were reduced from 31±1.5 s (no perfluorocarbon, 4 ml water) to 25±1.6 s (3ml perfluorocarbon, 1 ml water).

Figure 2.

Signal per unit volume for the H₂O, FC-72/H₂O mixture, and FC-72/D₂O mixture for 1-¹³C Pyruvate polarized solutions. Note that the signal per unit volume for the more concentrated solution remains higher than that of the H₂O only solution for more than 5 minutes after dissolution. The T₁ times used for the H₂O, FC-72/H₂O, and FC-72/D₂O solutions were 63, 71, and 83 s respectively.

In vivo Experiments

For all 9 animals administered pyruvate, the higher concentration of the compound was tolerated well and vital signs were stable with a slight increase in respiratory rate after injection. To assess the tolerance of the animals to the reduced volume solution, the animals were observed for 15 minutes following recovery from anesthesia. All animals exhibited normal behavior and activity, and all continued to exhibit normal behavior and activity 24 hours after injection. Figure 3 shows a time series of an in vivo spectrum from a typical animal experiment which demonstrates the efficacy of the 3:1 perfluorocarbon to water mixture despite an increase in the osmolarity of the injected solution. Normal hepatic metabolism was observed as reported previously (5).

Figure 3.

In vivo 4x volume reduction ¹³C spectra was acquired from the liver of a rat following IV injection of a 362 mM 1-¹³C Pyruvate solution produced using a four-fold reduction in dissolution output using FC-3283. Spectral localization was achieved by positioning the animal prone over a 3.8 cm diameter ¹³C surface coil. The spectra shown were obtained in 1 s using pulse and acquire (~5 degree flip angle) however for visualization only every 5^th spectrum is shown. The plot, representing the first 100 s of the study, reveals a typical in vivo decay rate. The peaks from left to right are lactate, pyruvate hydrate, alanine and pyruvate together with signal from an ex vivo urea reference sample.

For all 3 animals injected with succinate, the increased concentration of the compound was tolerated well. No hepatic metabolism was observed. Other studies have also yet to report observable hyperpolarized succinate metabolism in normal tissues (6).

Discussion

In conclusion, the reduced dissolution volume consisting of perfluorocarbon and water has proven to be a reliable method for increasing the concentration of the injected polarized material, thus decreasing the injection time and creating a more compact injection bolus. This work shows that the technique is robust, well tolerated, and applicable to the majority of compounds and nuclei for in vivo or in vitro applications. While this paper focuses on two specific perfluorocarbons to demonstrate feasibility, other perfluorocarbons or immiscible liquids could potentially be used to prepare solutions containing a wide variety of polarizable nuclei and compounds, pyruvate and succinate being two such examples.

For pyruvate, it is believed that the specific heat (1100 Jkg⁻¹C⁻¹) of FC-72 combined with its low boiling point (56 °C at 1 atm, Table 1) results in the perfluorocarbon boiling during dissolution, thereby reducing the temperature of the aqueous output solution. The higher and more desirable dissolution temperature of 30 °C achieved with FC-3283 is likely due to its higher boiling point given that its heat capacity is the same as FC-72 (1.1 kJ/kgC). Consequently, it is likely that the approach may be refined further using suitable compounds other than FC-72 and FC-3283 after considering their physical properties in the context of the desired application.

The increased concentration of sample in the resultant solution is accompanied by a decrease in the ¹³C T₁ relaxation time. However, the reduced relaxation time effect is more than offset by the increased concentration. Moreover, the relatively modest reduction in relaxation time is only relevant during the time interval immediately prior to injection because both high and low concentration solutions will rapidly approach the same in vivo relaxation time as they mix with the blood volume.

This study shows that without physical modification of the DNP polarizer, the standard operating procedure may, with minimal modification, be used to routinely generate volumes of hyperpolarized labeled compound with a concentration tailored to the desired study.