Determination of Intracellular pH and Compartmentation Using Diffusion-Weighted NMR Spectroscopy with pH-Sensitive Indicators

The intracellular pH (pHi) of a series of cancer cell lines was determined using the pH-sensitive indicators imidazole (Im) or histidine (His) and diffusion-weighted (DW) proton NMR spectroscopy. The DW method allows the observation at high magnetic field gradient values of only the slow-moving (intracellular) components, thus ensuring complete separation between intra- and extracellular components. Using the chemical shift difference (Δδ) between the imidazole ring C2-H and C4(5)-H peaks, we were able to measure the pHi independently of chemical shift standardization. With His, the cell lines gave pHi values of ∼6.5-7.0, whereas with Im, a second, more acidic compartment (pHi = 5.5-5.8) was also observed. An inverse correlation was also found between pHi and the intracellular lactate concentration. This method may be applicable to in vivo pH determinations.