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Dissolution Dynamic Nuclear Polarization of Non-Self-Glassing Agents: Spectroscopy and Relaxation of Hyperpolarized [1- C]Acetate. 13

Alessandra Flori, Matteo Liserani, Sean Bowen, Jan Henrik Ardenkjaer-Larsen, and Luca Menichetti J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/jp511972g • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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The Journal of Physical Chemistry

Dissolution Dynamic Nuclear Polarization of Non-SelfGlassing

Agents:

Spectroscopy

and

Relaxation

of

Hyperpolarized [1-13C]Acetate

Alessandra Flori,1,2 Matteo Liserani,3 Sean Bowen,4 Jan Henrik ArdenkjaerLarsen,4,5 and Luca Menichetti*,1,6

1

Fondazione CNR/Regione Toscana G. Monasterio, Pisa, Italy

2

Institute of Life Sciences, Scuola Superiore Sant’Anna, Pisa, Italy

3

Department of Physics, University of Pisa, Pisa, Italy

4

Department of Electrical Engineering, Technical University of Denmark,

KongensLyngby, Denmark 5

GE Healthcare, Broendby, Denmark

6

Institute of Clinical Physiology, National Council of Research, Pisa, Italy

*Correspondingauthor: Dr. Luca Menichetti Institute of ClinicalPhysiology, Via Moruzzi 1, I-56124, Pisa, Italy E-mail: [email protected], Tel.: +39 050 315 2137, Fax: +39 050 315 2166

1 ACS Paragon Plus Environment


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Abstract The intrinsic physicochemical properties of the sample formulation are the key factor for efficient hyperpolarization through dissolution-DNP. In this paper we provide a comprehensive characterization of the DNP process for Na-[113

C]acetate selected as model for non-self-glassing agents: the solid-state

polarization dynamics of different formulations and the effect of the paramagnetic agent (trityl radical) on the pattern of polarization and the relaxation profile were extensively analyzed. We quantified the effects of the glassing agent and Gd3+chelate on DNP performance.The results reported here describe the constraints of the acetate formulation useful for future studies in this field with non-self-glassing enriched molecules.

Keywords: Hyperpolarization; dissolution-DNP; [13C]pyruvate; trityl radical; spin diffusion; 13C.


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Introduction Over the last decade, dissolution dynamic nuclear polarization (dissolutionDNP) has attracted significant interest as a technique for enhancing nuclear magnetic signals,1 and its use in a number of Magnetic Resonance Spectroscopy (MRS) and Chemical Shift Imaging (CSI) applications is increasing.2,3 Empirical evidence, supported by theoretical analysis of the polarization process, has shown that specific physicochemical properties of the formulation are required for efficient dissolution-DNP polarization and application in vivo. Of these characteristics, the liquid-state longitudinal relaxation time (T1) of a compound is a primary factor that needs to be sufficiently long to retain the polarization after dissolution. A long T1 also provides a longer total acquisition time for in vivo MRS studies (>3×T1), and thus, enables the tracking of the metabolic fate of a molecule over a wider biochemical window once injected. Three key factors related to the enhancement of polarization need to be considered when preparing the dissolution-DNP samples: the glassy state of the matrix, and the 13C and radical concentrations in the sample. A homogeneous glassy matrix is crucial for effective hyperpolarization.2,4,5 In particular, an amorphous matrix ensures close proximity between the radicals and enriched molecules, leading to the efficient transfer of polarization from the free electron spins to the

13

C nuclei. Hence, a glassing agent is usually supplied with

non-self-glassing formulations, with dimethyl-sulfoxide (DMSO) and glycerol most commonly used for dissolution-DNP experiments.5-7



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As a starting condition, the

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13

C concentration in the hyperpolarized sample

should be maximized for imaging and spectroscopy applications, because the spectroscopic signal is proportional to the amount of hyperpolarized

13

C that is

injected. However, the radical concentration significantly affects the final polarization enhancement and build-up time constant, with a shorter build-up time constant favored for in vivo studies as it allows repeated hyperpolarizations in the same experimental session.8,9 [1-13C]pyruvate is widely used for metabolic studies with MRS of hyperpolarized

13

C, and it ensures optimal performance in terms of the T1

relaxation time,10-13 glassy properties, and final

13

C concentration of the

formulation.2,4 Nonetheless, the need to explore other metabolic pathways led to the development of other formulations that are based on different substrates,2,4 with [1-13C]acetate arising as an interesting candidate for in vivo dissolution-DNP. Acetate is a short-chain fatty acid (SCFA) and its role as an energy substrate of the skeletal muscle and heart could be exploited to investigate the metabolic pathways complementary to glycolysis.14,15 Moreover, [1-13C]acetate can be used as a working model for the properties of optimized formulations that are alternatives to self-glassing compounds. Matrix crystallization hampers the direct dissolution-DNP of

13

C-labeled

acetic acid.6 Furthermore, the volatility of the organic acid could affect the stability of the medium during preparation. As previously demonstrated,16-19 the hyperpolarization

of

the

corresponding

[1-13C]acetate

salts,

using

tris(hydroxymethyl)aminomethane(Tris) or Na as counter ions, is a more


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convenient approach. Nevertheless, the limited salt solubility makes the preparation of these formulations challenging, since it hampers the formation of a homogeneous glassy matrix. This is even more critical when dealing with large doses (sample volumes >300 µL), as required in pig studies with hyperpolarized 13

C-acetate. In this case, the concentration of [1-13C]acetate to be administered in

vivo is also crucial for providing the required signal-to-noise ratio (SNR).20 Na-[1-13C]acetate has been previously polarized for dissolution-DNP experiments5,21-23 and in vivo studies,24 especially those involving small animals.16,18,19 However, to the best of our knowledge, a comprehensive investigation of the DNP properties of non-self-glassing enriched molecules has not been reported in the literature. Hence, the results reported in this paper describe the requirements and properties of formulations suitable for dissolutionDNP studies, and the development of formulations as alternatives to the commonly used self-glassing compounds, such as [1-13C]- and [2-13C]-pyruvic acid. In this study, we assessed the impact of the

13

C spin density on the DNP spectra,

polarization mechanisms, and performance by changing the

13

C-enrichment of the

samples. We also focused on the quantification of the effect of different trityl radical concentrations on the solid-state DNP dynamics. Finally, we investigated the T1 relaxation through liquid-state NMR measurements.



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Experimental Methods Na-[1-13C]acetate (Cambridge Isotope Laboratories Inc, Andover, MA, USA) was mixed with the trityl radical (OX063, Oxford Instr. Ltd, Abingdon, UK) and a Gd3+-chelate (Dotarem, Guebert, RoissyCdGCedex, France) in a 60:40 w:w ultrapure (mQ, Millipore, Billerica, MA, USA) water/glycerol (Sigma-Aldrich, St. Louis, MO, USA) mixture to obtain final concentration values of [13C-acetate] = 7.3 M, [OX063] = 25 mM, and [Gd3+-chelate] = 1.4 mM. The mixture was sonicated at 60 °C for about 25 min until dissolution. We will refer to this formulation as the standard formulation (number 3 in Table 1) in the following text. Starting from the standard formulation, samples with different 100%, corresponding to a

13

C isotopic enrichments (20%, 50%,

13

C concentration in the mixture of 1.5, 3.6, and 7.3 M,

respectively) were prepared by mixing natural abundance Na-acetate and

13

C-

labeled Na-[1-13C]acetate. Furthermore, we varied the OX063 radical concentration to obtain a final value of 16, 21, and 25 mM, respectively ([13C] and [Gd] were fixed at 7.3 M and 1.4 mM, respectively). All formulations are detailed in Table 1.


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13

Formulation

1

13

CEnrichment

Cacetate

(%)

(M)

20%

1.5

50%

2

3.6

100%

3

7.3

100%

4

7.3

100%

5

Table 1.

7.3

OX063 (mM)

25

25

25

21

16

13

C spins/radical concentration

τ(s)

ഥ મ (a.u.)

1207±17

5.6±0.5

n=4

n=4

841±62

5.0±0.8

n=4

n=4

437±18

5.7±0.6

n=4

n=4

528±86

5.9±0.6

n=6

n=6

992±67

6.8±0.4

n=6

n=6

58

144

289

346

453

13

C-enrichment, [1-13C]acetate and OX063 concentrations, and the ratio

of the13C spins to radical concentration are reported for the different hyperpolarized Na-[1-13C]acetate formulations, together with the measured build-up time constants ഥ ) (mean ± SD, n is the number of repetitions (τ) and normalized plateau values (Π for each set of measurements).

The Tris-[1-13C]acetate formulation previously published by Jensen et al.17 was slightly modified for our purposes and used as a reference in the solid-state dissolution-DNP experiments with Na-[1-13C]acetate. For our formulation, the amount of glycerol was increased by up to 10% in weight and the OX063 concentration was increased by 30% compared to the published formulation.



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Tris-[1-13C]acetate was produced by adding 1 g of [1-13C]acetic acid (Cambridge Isotope Laboratories, Inc.) to a 0.54 M Tris base solution (SigmaAldrich)until a pH of 7.4-7.8 was achieved. The resulting mixture was freeze-dried overnight. The DNP sample was formulated by mixing Tris-[1-13C]acetate, OX063 (181 µmol/g solution), and 50 mM Gd3+-chelate in a 65/35 w:w ultrapure (mQ) water/glycerol solution. The mixture was then sonicated at 60 °C for 10 min to cause dissolution. The final concentrations were [13C-acetate] = 4.7 M, [OX063] = 15.5 mM, and [Gd3+-chelate] = 0.93 mM. The [1-13C]pyruvic acid samples were prepared by mixing [1-13C]pyruvic acid (Cambridge Isotope Laboratories Inc.) with trityl radicals (OX063) and the Gd3+-chelate (Dotarem, Guebert) to obtain dissolution-DNP samples with final concentrations of [13C-pyruvic acid] = 14 M, [OX063] = 15 mM, and [Gd3+-chelate] = 1 mM. DNP was performed using a commercial system (HyperSense, Oxford Instruments plc, UK).The samples were polarized at 3.35 T and 1.4 K for about 50 min. The detected build-up curves were fitted withMatlab (The MathWorks Inc., Natick, MA, USA) and the build-up time constants (τ) and normalized plateau ഥ ) (referred to the number of values (Π

13

C spins in the sample), were estimated and

compared for the different formulations. Statistical analysis of the dataset was performed using a two-tailed t-test (p = 0.05 level of significance).


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DNP spectra were measured and compared for the different formulations and the optimum microwave (mw) frequency for DNP was determined. The solid-state

13

C longitudinal relaxation time (T1) of the Na-[13C]acetate

standard formulation was measured as follows: three samples (n = 3) were hyperpolarized for 40 min, the mw was subsequently switched off and the magnetization was detected using a pulse-and-acquire sequence (flip angle = 5°, TR = 300 s). The T1 was then obtained by a mono-exponential fit of the decay curves performed with Matlab, taking into account RF pulse-induced decay.25 The

13

C T1 of Na-[1-13C]acetate was characterized atdifferent temperatures

and concentrations (9.4 T VnmrS Scanner , Agilent Technologies, Santa Clara, CA, USA) with the 50 mM, 200 mM, and 1 M Na-[1-13C]acetate samples over the temperature range from 25 °C to 95 °C. In the experiments, the liquid-state T1 was determined by the monoexponential fitting of the curves obtained using an inversion recovery (IR) sequence. A Shigemi NMR tube was used to prevent convection effects during high temperature measurements.



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Results and Discussion Recent studies that used

13

C-acetate24 and

13

C-butyrate26 as a metabolic

probe of SCFA metabolism found several limitations arising from the low fluxes of conversion, low SNR, and initial low level of polarization. In this regard, optimizing the available formulations to increase the level of polarization could be a key issue for the future development of this technique.

NMR measurements As a first step we performed NMR measurements of the liquid-state T1 of Na [1-13C]acetate/water solutions at variable temperatures and acetate concentrations, in order to characterize the relaxation dynamics of the molecule. In Fig. 1 the T1 value in the range 25->95°C for three concentrations is shown. At lower temperatures (25->65°C) we recorded a T1 increase with temperature for all the concentrations analyzed, which is consistent with a prevalence of mechanisms such as dipolar interaction or chemical shift anisotropy (CSA) on the relaxation profile in this conditions (22, 27-29). In particular, CSA is expected to prevail in the case of carbonyls like Na [1-13C]acetate and it is also mainly effective at higher magnetic fields (4, 27). Conversely, T1 decreased for temperatures in the range 65->95°C likely due to a dominant effect of the spinrotation interaction (30) on the relaxation mechanisms, consistent with increased molecular rotations at higher temperatures (27). The maximum T1 value was found at a temperature of 65 °C. The T1 of Na [1-13C]acetate measured at 35 °C (close to the physiological temperature required for in vivo experiments) was 59.0 ± 0.7 s,


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58.6 ± 0.2 s and 54.9 ± 0.1 s, for the 50 mM, 200 mM and 1 M sample respectively (Fig. 1). These results, obtained for different concentrations and temperatures at 9.4 T, anticipate long T1 values at clinically relevant magnetic field strengths (1.5-3 T), particularly interesting for in vivo studies with hyperpolarized

13

C-MRS. These

findings also confirm the feasibility of using Na [1-13C]acetate as substrate for dissolution-DNP experiments in vivo.

Fig. 1. T1 dependence on temperature measured at 9.4 T, for the 50-mM, 200-mM and 1-M Na [1-13C]acetate samples; the error reported in the Figure is the fit error.



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Solid-State DNP The solid-state hyperpolarization performance of Na-[1-13C]acetate was investigated by evaluating the effect of the different components in the sample formulation, with particular attention paid to the

13

C-enrichment and trityl OX063

concentration. At the same time, the elements resulting in an improved DNP performance of the mixture were identified. In the analysis, we did not estimate the effective polarization enhancement due to the critical measurement of the solid-state thermal polarization because of the extremely long acquisition time. Instead, we selected the normalized plateau ഥ , as a reference parameter for directly comparing the polarization value, Π ഥ was assumed to be proportional to the solid-state polarization. The performance; Π polarization dynamics were studied by means of the build-up time constant, τ. We first estimated the effect of the glassy matrix on the polarization using the Tris-[1-13C]acetate salt. For these experiments, we referred to the formulation previously published by Jensen et al.17 because it had been shown to be effective for in vivo studies.16 In particular, we evaluated the impact of the addition of water and glycerol to the Tris-acetate formulation; glycerol was selected as the glassing agent because of its excellent glassing properties.31 The addition of water and glycerol to the DNP mixture had two major effects; on the one hand, the polarization performance was improved, resulting in an increase of the polarization. On the other hand, the τ constant increased, which was attributed to the spin diffusion component of the polarization process being


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modified.7,32,33 The detailed results of these measurements are available in the Supporting Information (SI). Taking into account the increased polarization obtained for Tris-[113

C]acetate, glycerol was also added to the Na-[1-13C]acetate standard formulation,

leading to a 60:40 water:glycerol (w:w) solvent matrix. The fraction of added glycerol was maintained under the 40% to get the maximum achievable concentration of

13

C in the sample and, at the same time, to avoid unwanted

precipitation of the acetate salt. The glycerol enabled the dissolution of Na-[113

C]acetate up to a maximum

13

C concentration of 7.3 M (standard formulation,

number 3 in Table 1). Calorimetric measurements were performed for this Na-[1-13C]acetate formulation by means of differential scanning calorimetry (DSC): the absence of contributions from the melting of crystalline fractions when heating the compound above the glass transition temperature (Tg) confirmed the glassy nature of the mixture. Therefore, the different Na-[1-13C]acetate samples were prepared by varying the trityl concentration and for samples with different

13

ഥ were compared C-enrichment: as a first step, τ and Π

13

C-enrichments but the same concentration of the trityl

radical (25 mM, formulations 1-3 in Table 1), Fig. 2. In the Figure the values of τ ഥ have been normalized for the value obtained for the Na-[1-13C]acetate and Π standard formulation-3 (Table 1) to facilitate the comparison among different formulations.



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Fig. 2 panel a) shows that as the

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13

C concentration increases from 1.5 to 7.3 M, a

2.7-fold decrease in τ occurs, which was statistically significant (p < 0.05, n = 4). This behavior could be attributed to the inherent modification of the spin diffusion component of the polarization process,7,21,32,33 because a decrease in the enrichment increases the distance between

13

C-

13

ഥ C atoms. On the other hand, the Π

values are not significantly affected by the different

13

C-enrichments (Fig. 2panel

a)), which shows the same level of polarization can be obtained, but with a slower process. Our findings, especially for the τconstant, agree with results reported by Comment et al.21 for acetate samples in a different solvent matrix (water/ethanol vs. water/glycerol in this study).


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Figure 2. (a) The solid-state build-up time constants, τ, and normalized plateau ഥ , for the hyperpolarized Na-[1-13C]acetate samples with different values, Π

13

C–

enrichments (formulations 1-3, Table 1), which are reported as a function of the 13C concentration. ഥ have been normalized for the value of the Na-[1-13C]acetate The values of τ and Π standard formulation-3 (Table 1).



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Figure 2 (b) The solid-state build-up time constants, τ, and normalized plateau ഥ , measured for the hyperpolarized Na-[1-13C]acetate samples with values, Π different OX063 concentrations (formulations 3-5, Table 1) and Tris-[1-13C]acetate, which are reported as a function of the ratio of

13

C spins to radical concentration.

ഥ have been normalized for the value of the Na-[1-13C]acetate The values of τ and Π standard formulation-3 (Table 1). We conclude that a higher

13

C concentration is beneficial for reducing the

duration of the experiments because it shortens the build-up time constant. Even though we did not record any significant effects of

13

C-enrichment on the level of

polarization, it should be mentioned that maximizing the

13

C concentration is

preferable for increasing the sensitivity of in vivo studies. As reported in the literature,34-37 DNP enhancements can be described as the interplay of different mechanisms, namely the solid effect (SE), cross effect (CE), and thermal mixing (TM). The SE involves interactions between only one electron spin and one nuclear spin, with the two peaks in the DNP spectrum separated by twice the

13

C Larmor frequency. On the other hand, CE involves two

electronic spins and one nuclear spin. Finally, TM involves transitions among all electron spins. According these models, the peaks in the DNP spectrum affected by CE and TM are apparently closer compared to peaks affected by SE.35,38 When more than one mechanism is active, the different interactions can overlap, leading to a broadening of the bandwidth.34,39



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In the DNP spectra recorded for our samples (see Fig. 3), the separation between maxima is about 63 MHz. It is feasible that this polarization profile is not 13

C frequency at 3.35 T is 72 MHz.

exclusively determined by the SE, as twice the

Therefore, we speculate that there is a concurrent effect of all the mechanisms present in the DNP profile. Comparing the DNP spectra (Fig. 3) acquired for Na-acetate formulations with different

13

C-enrichments (numbers 1-3 in Table 1) shows that the bandwidth

of the two peaks increases with increasing

13

C concentration (the spectra were

normalized with the maximum value of the signal to take into account the different 13

C concentrations). This behavior parallels with the shortening of the build-up time

constant (as reported in Table 1): based on the different models applied in the literature to analyze the DNP profiles,34-37,39 an inherent modification of the mechanisms underlying the polarization process cannot be completely excluded. Further experiments are necessary to address this issue; for example, using different compounds in order to estimate the extent of the phenomenon.


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Figure 3. DNP spectra of Na-[1-13C]acetate formulations with different

13

C-

enrichments, normalized with the maximum value of the signal.

Hence, three formulations were selected where the concentration of the trityl radical ranged from 16 to 25 mM (formulations 3-5 in Table 1). The resulting values ഥ were compared as a function of the ratio of of τ and Π

13

C spins to radical

concentration of the mixture, as shown in Figs. 2 panel b) (n = 6). As can be seen, there is a significant decrease in the build-up time constant (from about 1000 s to about 450 s) with the trityl radical concentration increasing from 16 to 25 mM (p < 0.05); in particular, there is a large decrease from 16 to 21 mM of OX063 (p
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Table of Contents Image


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Fig. 1. T1 dependence on temperature measured at 9.4 T, for the 50-mM, 200-mM and 1-M Na [113C]acetate samples; the error reported in the Figure is the fit error. 1057x793mm (72 x 72 DPI)

ACS Paragon Plus Environment


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Figure 2. (a) The solid-state build-up time constants, τ, and normalized plateau values, Π ̅, for the hyperpolarized Na-[1-13C]acetate samples with different 13C–enrichments (formulations 1-3, Table 1), which are reported as a function of the 13C concentration. 1057x793mm (72 x 72 DPI)


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Figure 2 (b) The solid-state build-up time constants, τ, and normalized plateau values, Π ,̅ measured for the hyperpolarized Na-[1-13C]acetate samples with different OX063 concentrations (formulations 3-5, Table 1) and Tris-[1-13C]acetate, which are reported as a function of the ratio of 13C spins to radical concentration. 1057x793mm (72 x 72 DPI)



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Figure 3. DNP spectra of Na-[1-13C]acetate formulations with different 13C-enrichments, normalized with the maximum value of the signal. 1057x793mm (72 x 72 DPI)


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Figure 4. Comparison of the DNP spectra of Na-[1-13C]acetate and Tris-[1-13C]acetate formulations, normalized with the maximum value of the signal. 1057x793mm (72 x 72 DPI)



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Figure 5. Comparison of representative hyperpolarization build-up curves of the standard formulation of Na[1-13C]acetate (number 3 in Table 1) and [1-13C]pyruvate obtained at 1.4 K and 3.35 T. The curves are divided by the number of 13C spins in the sample. 1057x793mm (72 x 72 DPI)


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Figure 1: (a) The zoom of the initial part of the build-up curves of the Tris [1-13C]acetate is reported for the same sample dehydrated and subsequently added of H2O up to 50% in weight compared to the dehydrated sample; in (b) the τconstant and plateau value (Π ̅) are reported. 93x94mm (299 x 299 DPI)



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Figure2:Normalized plateau value, Π ̅, and build-up time constant,τ, obtained at 3.35 T/1.4 K for Tris [113C]acetate samples without and with addition of 10% w:w glycerol to the formulation. 1057x793mm (72 x 72 DPI)


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1057x793mm (72 x 72 DPI)



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Figure 3: The liquid-state percentage of polarization (%LSP) obtained for Na [1-13C]acetate standard formulation samples (25 mM OX063; 7.3 M 13C-acetate) is shown as a function of the normalized solidstate build-up plateau value(Π ̅): a linear dependence can be found for the reported dataset. 1057x793mm (72 x 72 DPI)


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Table of Contents Image 1410x793mm (72 x 72 DPI)


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