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Chemical Shift Imaging of pH with Hyperpolarized [2-13C,D10] Diethylmalonic Acid

David Korenchan^1,2, Celine Taglang¹, Cornelius von Morze¹, Joseph Blecha¹, Jeremy Gordon¹, Peder Larson^1,2, Henry VanBrocklin¹, John Kurhanewicz^1,2, David Wilson¹, and Robert Flavell¹

¹Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA, United States, ²Bioengineering, University of California, Berkeley, Berkeley, CA, United States

Synopsis

Imaging tumoral pH may aid in characterizing aggressiveness, metastasis, and therapeutic response. The study of hyperpolarized (HP) ¹³C chemical shift-based probes addresses some of the limitations of previously reported ratiometric methods, including HP ¹³C bicarbonate. We report the development of HP [2-¹³C,D₁₀] diethylmalonic acid as a pH imaging agent, which exhibits a significant ¹³C chemical shift difference over the physiologic pH range. We demonstrate that this compound can be copolarized with tert-butanol to accurately measure pH in phantom studies. Furthermore, this HP compound showed high in vivo SNR in a murine model of prostate cancer.

Purpose

To characterize and develop a dicarboxylic acid as a high-resolution HP ¹³C magnetic resonance spectroscopic (MRS) imaging agent for pH.

Methods

All spectroscopic studies were performed at 11.7T and 37°C. All imaging was performed on a 3T clinical MRI scanner. Spectral pH values were compared with standard pH electrode measurements. 2D chemical shift imaging (2D CSI) had a 25kHz spectral bandwidth and 2048 spectral points. Imaging data were processed using SIVIC software¹.

Dicarboxylate screening: 250mM solutions of ten different compounds were prepared with urea (chemical shift standard) at pH 6.5 and 7.4., and ¹³C MRS chemical shifts (Δppm) for each solution were measured.

Diethylmalonic acid characterization: 5mM solutions of [2-¹³C,D₁₀] diethylmalonic acid (DEMA) and [1-¹³C,D₉] tert-butanol (tBuOH, chemical shift standard) were prepared from pH 5.6-8.8. The Δppm between the labeled carbons was measured, plotted versus pH, and fitted to a sigmoidal curve² to obtain an MR titration curve. ¹³C spectra of 5mM DEMA and tBuOH were also acquired in fetal bovine serum at pH 6.6 and 7.4.

DEMA hyperpolarization: ~3.8M DEMA in N,N-dimethylacetamide was prepared with 15mM OX063 trityl radical and 2mM Gd-DOTA and copolarized with tBuOH and OX063 in glycerol. After dissolution and NaOH titration, the HP solution-state T₁ and polarization were determined via dynamic ¹³C MRS (5° hard pulses, flip angle correction, TR = 3s, n = 3). The pH was calculated at ~170s post-dissolution using the ¹³C Δppm and MR titration curve.

pH imaging phantom: HP DEMA and tBuOH were diluted to ~5mM each and titrated in five separate tubes to various pH values. The phantom was imaged with a ¹³C 2D CSI sequence (10x10 matrix, 10° hard pulses, 7.5mm isotropic in-plane resolution). After imaging, dynamic ¹³C MRS was performed for 3T T₁ measurement (10° hard pulses, TR = 3s, n = 2).

Mouse tumor pH imaging: A transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse was injected with 350μL of ~50mM DEMA and tBuOH. A 2D CSI sequence was performed 45s after injection start (6x8 matrix, 3cm axial slab excitation, variable flip angle scheme, centric phase encoding in axial and lateral directions, 5mm isotropic in-plane resolution). Spectra were 4x zero-filled and 10Hz apodized.

Results

Two of ten compounds demonstrated a significant ¹³C Δppm over the physiologic pH range (Figure 1). Of these two, DEMA was chosen for further development based upon preliminary T₁ relaxation measurements (87s versus 38.2s). DEMA had a pK_a near physiologic pH (pK_a,2 = 7.39, 37°C) and a pH-sensitive ¹³C nucleus that shifted 1.66ppm downfield from pH 6.5 to 7.4 (Figure 2). The measured pK_a matched closely with the commonly reported value of 7.20³. The solution-state T₁ and back-calculated polarization measured at 11.7T were 105.6 ± 5.2s and 13.7 ± 0.6%, respectively. At 3T, the measured T₁ value for DEMA was 84.3 ± 1.4s. HP spectral and electrode pH measurements agreed within 0.1 pH unit, both in 11.7T spectroscopy (Figure 3) and 3T phantom imaging (Figure 4). Changes in chemical shift were maintained over the physiologic range in serum (Figure 5a). Imaging in a TRAMP mouse demonstrated excellent signal to noise ratio (Figure 5b). No toxic effects were observed.

Discussion

Unlike ¹³C-bicarbonate, which represents the mainstay of in vivo HP interstitial pH imaging^4-7 and calculates the pH from intensities of two separate resonances, DEMA exhibits a single resonance with a pH-dependent chemical shift. We have recently reported a similar ¹³C approach for measuring pH via chemical shift⁸. Such compounds may circumvent low SNR concerns regarding peak intensity quantification, particularly at pH values far from the pK_a. The pH-sensitive ¹³C nucleus of DEMA is a quaternary carbon, and it exhibits a long T₁ and upfield chemical shift relative to the pH-insensitive ¹³C nucleus of tBuOH⁹. By copolarizing these two compounds, we demonstrated accurate pH measurement in vitro. We obtained high SNR imaging data in a preliminary TRAMP mouse experiment, with some heterogeneity in the Δppm observed between the two HP resonances. While a small shift towards acidic pH was observed in the TRAMP tumor, the acidic shift was not as large as expected based on prior studies^5,6, and ongoing studies are investigating the rationale for this finding and how to correct it.

Conclusion

We report a novel compound for pH measurement via ¹³C MRS, [2-¹³C,D₁₀] diethylmalonic acid (DEMA). The pH is measured via changes in MR chemical shift, potentially circumventing SNR limitations found with HP bicarbonate. The HP imaging pH accuracy, long T₁ values, and absence of observable toxicity make DEMA an excellent candidate for high spatial resolution in vivo pH mapping.

Acknowledgements

The authors wish to thank Dr. Robert Bok and Zihan Zhu for their help with animal handling and injection.

Grants: R01-CA166766; SNMMI-ERF Mitzi and William Blahd, MD, Pilot Grant; RSNA Research Fellow Grant

References

1. Crane, J. C., Olson, M. P. & Nelson, S. J. SIVIC: Open-Source, Standards-Based Software for DICOM MR Spectroscopy Workflows. International Journal of Biomedical Imaging 2013, 1–12 (2013).

2. Gillies, R. J., Liu, Z. & Bhujwalla, Z. ³¹P-MRS measurements of extracellular pH of tumors using 3-aminopropylphosphonate. American Journal of Physiology-Cell Physiology 267, C195–C203 (1994).

3. Nachod, F. C. Determination of organic structures by physical methods. (Academic Press, 1955).

4. Gallagher, F. A. et al. Magnetic resonance imaging of pH in vivo using hyperpolarized ¹³C-labelled bicarbonate. Nature 453, 940–943 (2008).

5. Wilson, D. M. et al. Multi-compound polarization by DNP allows simultaneous assessment of multiple enzymatic activities in vivo. Journal of Magnetic Resonance 205, 141–147 (2010).

6. Korenchan, D. E. et al. Dynamic nuclear polarization of biocompatible 13C-enriched carbonates for in vivo pH imaging. Chemical Communications 52, 3030–3033 (2016).

7. Drachman, N. et al. In vivo pH mapping of injured lungs using hyperpolarized [1- ¹³C]pyruvate. Magnetic Resonance in Medicine (2016). doi:10.1002/mrm.26473

8. Flavell, R. R. et al. Application of Good's buffers to pH imaging using hyperpolarized ¹³C MRI. Chemical Communications 51, 14119–14122 (2015).

9. Grant, A. K., Vinogradov, E., Wang, X., Lenkinski, R. E. & Alsop, D. C. Perfusion imaging with a freely diffusible hyperpolarized contrast agent. Magnetic Resonance in Medicine 66, 746–755 (2011).

Figures

Figure 1: Investigation of dicarboxylates as ¹³C MR pH sensors. The downfield chemical shift migration from pH 6.5 to 7.4 is listed near each labeled ¹³C nucleus. Spectra were acquired at 37°C. Two molecules demonstrating the largest chemical shift difference over this pH range are highlighted in yellow: diethylmalonic acid (top right) and cyclopropyl-1,1-dicarboxylic acid (lower left).

Figure 2: MR pH titration curve for [2-¹³C,D₁₀] diethylmalonic acid (DEMA): (a) Representative ¹³C MR spectrum of 5mM DEMA (upfield) and 5mM [1-¹³C,D₉] tert-butanol (tBuOH, downfield). (b) Plot of chemical shift difference between DEMA and tBuOH vs. pH, along with best-fit equation to the data. This titration curve was used for all subsequent pH calculations from HP spectra.

Figure 3: HP DEMA measurements: (a) Overlaid HP ¹³C spectra of DEMA resonance at two different pH values, demonstrating high SNR and pH-dependent chemical shift. Electrode pH measurements and ppm difference between DEMA and tBuOH listed for each color-coded spectrum. (b) Plot of pH values calculated from HP spectra vs. electrode measurements, demonstrating agreement within 0.1 pH unit.

Figure 4: HP phantom imaging with diethylmalonic acid: (a) T₂-weighted ¹H image. Each tube contains ~5mM copolarized DEMA and tBuOH at varying pH values. Electrode pH measurements are displayed near each tube. (b) Overlaid ¹³C spectra from color-coded voxels highlighting pH-dependent DEMA chemical shift observed via imaging. Spectra are referenced to tBuOH peak. (c) Plot of pH values calculated from spectra in 4b vs. electrode measurements, demonstrating agreement within 0.1 pH unit. The highest and lowest pH values demonstrated chemical shifts that were very close to the minimum and maximum chemical shift differences, respectively, seen in the MR titration curve.

Figure 5: (a) DEMA ¹³C resonance in serum at 5mM and two different pH values, demonstrating preservation of pH-dependent chemical shift. (b) Preliminary HP ¹³C MR spectroscopic imaging of copolarized [2-¹³C,D₁₀] diethylmalonic acid (upfield) and [1-¹³C,D₉] tert-butanol (downfield) in a transgenic murine model of prostate cancer. 2D chemical shift imaging spectra from voxels across normal tissue and tumor regions demonstrate high SNR and a small difference in chemical shift between normal and tumor tissues.

Proc. Intl. Soc. Mag. Reson. Med. 25 (2017)

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