Introduction

The ratio of CO₂ to HCO₃^- has been used as an index of tissue pH using hyperpolarized (HP) ¹³C-MRI (1). Various salts of ¹³C-enriched bicarbonate (H¹³CO₃^-) and CO₂-precurosrs can be polarized using DNP and upon dissolution, the resulting HP-H¹³CO₃^- rapidly equilibrates to form HP-¹³CO₂ (2-4). The short T₁ and low polarization levels of ¹³C-bicarbonate salts remain major challenges for this method. In this study, we report the development of HP ¹³C-enriched mixed anhydrides as esterase-sensitive precursors (Fig. 1) for the HP ¹³CO₂ and H¹³CO₃^- buffer pair for tissue pH measurement. We hypothesized that the ester functional group of mixed anhydrides can be readily hydrolyzed by enzyme esterase, producing HP-H¹³CO₃^-/¹³CO₂ that can be imaged for estimating tissue pH.

Methods

¹³C-enriched ethyl acetyl carbonate (¹³C-EAC) was synthesized from ¹³C-enrihced ethyl chloroformate and acetic acid and isolated as a clear liquid. ¹³C-EAC was polarized neat for 2 h in a HyperSense polarized using BDPA (40 mM) as a radical. For T₁ and signal enhancement measurements, dissolution of HP ¹³C-EAC was done using ethanol ([EAC] = 2 mM) and ¹³C NMR spectra were acquired at 9.4 T. For hydrolysis studies of HP ¹³C-EAC, 2 mL of HP ¹³C-EAC in PBS was mixed with 2 mL of rat plasma or rat liver homogenate ([¹³C-EAC] = 2 mM, 37 °C). Series of ¹³C NMR spectra were acquired with a delay time of 2 s with 10-deg pulses. In vivo pH measurements of rat livers were done in a 4.7T small animal scanner. Healthy Sprague-Dawley rats (250-350 g) were injected with HP ¹³C-EAC via a tail vein catheter (3 mL, 80 mM). ¹³C NMR spectra were acquired with 10-deg pulses every 3 s using a volume coil. pH values were calculated from H¹³CO₃^-/¹³CO₂ intensity ratios.

Results and Discussions

A polarization buildup of ¹³C-EAC is shown in Figure 2A. ¹³C-EAC polarization reached the maximum level in a very short time (~30 min). T₁ of ¹³C-EAC measured at 9.4T was 30 s. The liquid state signal enhancement of this HP probe was ~35,000-fold, corresponding to ~28% polarization. Some degree of HP-¹³C-EAC hydrolysis was observed in PBS as confirmed by the presence of ¹³C-enriched monoacetyl carbonate (¹³C-MAC) at 159.4 ppm (Fig. 3). ¹³CO₂ and H¹³CO₃^- resonances are also visible at 124.5 and 160.0 ppm, respectively. ¹³C NMR spectra of HP ¹³C-EAC in plasma and liver homogenate show strong peaks of ¹³C-MAC, ¹³CO₂, and H¹³CO₃^-. As expected, signal intensities of all ¹³C-EAC hydrolyzed products are much more pronounced in these biological samples. The presence of enzyme esterase caused rapid hydrolysis of HP-¹³C-EAC to HP ¹³C-MAC. The signal-intensity plot of (Fig. 3B) confirms that HP-¹³C-MAC was produced instantly in plasma and liver homogenate. A HP-¹³C spectrum of the liver summed over 21-s period after HP ¹³C-EAC injection is shown in Figure 4A. Our results showed that HP ¹³C-MAC, ¹³CO₂, and H¹³CO₃^- peaks appeared soon after the injection of HP ¹³C-EAC. HP ¹³C-EAC signal was very small compared to its downstream metabolites, indicating that ¹³C-EAC hydrolysis by esterase is very rapid in vivo. Although the HP ¹³C-MAC and HP H¹³CO₃^- peaks in this in vivo result are somewhat overlapped, the two resonances are resolved. Intensities of H¹³CO₃^- and ¹³CO₂ in the liver quantified by Gaussian peak fittings are plotted as a function of time in Figure 4B. A ratio between HP-H¹³CO₃^- and HP-¹³CO₂ signal intensities was calculated over 21 s (7 time points) while HP ¹³CO₂ signal intensity can be reliably measured. From these values, the average HP-H¹³CO₃^--¹³CO₂ signal intensity ratio was 11.9:1, agreeing well with the reported value (5). pH value of the liver was estimated from the calculated intensity ratios (1). The average pH value over this period from 7 values was 7.24 ± 0.08. Interestingly, this value agrees very well with the intracellular pH measured by ³¹P NMR (6, 7).

Conclusions

In conclusion, we have demonstrated that tissue pH can be measured in vivo using HP ¹³C-EAC. Taking advantage of the abundant and fast mammalian enzymes esterase and carbonic anhydrase, HP ¹³CO₂ and H¹³CO₃^- can be rapidly produced in situ and tissue pH can be calculated from HP ¹³C signal ratios of the physiological buffer pair. The long T₁, good chemical stability, and high polarization level of HP ¹³C-EAC are also advantageous for HP ¹³C imaging applications, potentially allowing for pre-injection quality control procedures such as radical removal without a significant signal loss. The results suggest that HP ¹³C-EAC is an attractive pH imaging agent for in vivo assessment of abnormal tissue pH associated with many diseases.

Acknowledgements

We thank the following agencies for financial support: NIH 2P41-EB015908 (CRM), NIH 5R37-HL034557 (ADS), and W81XWH-12-1-0134 (CK).