Simultaneous Assessment of Intracellular and Extracellular pH Using Hyperpolarized [1-¹³C]Alanine Ethyl Ester

Tissue pH is tightly regulated in vivo, being a sensitive physiological biomarker. Advent of dissolution dynamic nuclear polarization (DNP) and its translation to humans stimulated development of pH-sensitive agents. However, requirements of DNP probes such as biocompatibility, signal sensitivity, and spin-lattice relaxation time (T₁) complicate in vivo translation of the agents. Here, we developed a ¹³C-labeled alanine derivative, [1-¹³C]-l-alanine ethyl ester, as a viable DNP probe whose chemical shift is sensitive to the physiological pH range, and demonstrated the feasibility in phantoms and rat livers in vivo. Alanine ethyl ester readily crosses cell membrane while simultaneously assessing extracellular and intracellular pH in vivo. Following cell transport, [1-¹³C]-l-alanine ethyl ester is instantaneously hydrolyzed to [1-¹³C]-l-alanine, and subsequently metabolized to [1-¹³C]lactate and [¹³C]bicarbonate. The pH-insensitive alanine resonance was used as a reference.