The pH plays an important role in many diseases, such as cancer and inflammation^{1, 2}. Various attempts have therefore been made to translate a reliable and non-invasive pH imaging technique into the clinic. Recently, magnetic resonance imaging based pH imaging methods using hyperpolarized molecules like bicarbonate³, Goods buffers⁴, and pyridine derivatives⁵ have shown great potential for pH imaging. However, none of these techniques has reached the clinical stage yet.

Here we present the spectroscopic characteristics of a new molecule for in vivo pH imaging, called zymonic acid (ZA). We measured the pH dependent chemical shift of all atoms and synthesized deuterium enriched [1,5-¹³C₂]ZA_d, [2,4-¹³C₂]ZA_d, and [3,6-¹³C₂]ZA_d to determine the spin lattice relaxation time (T₁) of each individual hyperpolarized ¹³C atom. Blood phantom measurements with [1,5-¹³C₂]ZA_d on a pre-clinical 7T MRI system reveal a great potential of ZA for in vivo pH measurements.

¹³C-labelled ZA was synthesized in one step using commercially available ¹³C-labelled pyruvic acid and deuterium enriched solvents⁶. The purification was carried out on a C18 reversed phase HPLC (2% – 20% acetonitrile in water) and the product was freeze dried.

High field NMR spectra were recorded on a 600 MHz Bruker Avance spectrometer. 4 M ZA in DMSO were doped with 15 mM trityl radical (OX063) and 5 mM Dotarem and hyperpolarized in a HyperSense polarizer. The dissolution was performed in 0.1 g/L EDTA and an adequate amount of NaOD in D₂O. T₁ measurements were performed on solutions with a final concentration of 25 mM (50 mM for MRI experiments) on a SpinsolveCarbon benchtop NMR spectrometer (Magritek, B0=1T) using a 10 degree flip angle and a repetition time of 3 s. T₁ decay curves were flip angle corrected and fitted by a mono-exponential decay curve.

Imaging experiments were performed on human blood, the pH adjusted with NaOH or HCl, on a 7T small animal scanner (Agilent/ GE) using chemical shift imaging with FOV=6cm, matrix size=16x16, TR =118ms, FA=3°.

Zymonic acid consists of six carbon atoms and bears a stable enol group with its pKa in the physiological range (pK_a~7.0). Deprotonation of the hydroxyl proton changes the electron density of all neighboring carbon atoms. This leads to pH dependent chemical shift changes up to 3.0 ppm/ pH, which are fast on the NMR time scale (Fig. 1). Carbon atoms number one, two, three, and five exhibit changes in chemical shift values of ∆δ~1.5-3.0 ppm between pH 6.3-7.5. The resonance signals of C₄ and C₆ exhibit smaller chemical shift differences of ∆δ~0.7 ppm and ∆δ~0.5 ppm. Carbon 13 NMR spectra of hyperpolarized [1,5-¹³C₂]ZA at several different pH values reveal the stability of the molecule throughout the dissolution process and its ability to report pH after hyperpolarization (Fig. 2).The T₁ of the carbon atoms one and five is 53±3 s and 115±3 s, respectively (n=3). The T₁ of carbon atoms two, three, four, and six is 30±1 s, 16 s, 51±1 s, and 13 s (n=3 and n=1).

Phantom measurements of [1,5-¹³C₂]ZA_d dissolved in human blood show that ZA is able to spatially resolve different pH values in a bodily fluid (Fig. 3). The pH values calculated by the chemical shift difference of ZA_d correlate well with the values measured with a pH electrode after the imaging experiment.High field NMR measurements demonstrate that zymonic acid bears four highly pH sensitive ¹³C atoms. Furthermore, we have shown that these atoms have a long T₁ and that the molecule is not harmed by the dissolution process. As a first step for going from in vitro towards in vivo pH measurements, blood phantom measurements indicate that [1,5-¹³C₂]ZA is a suitable pH sensor for pre-clinical and potentially also for clinical applications.