The phenomenon of separate intra‐ and extracellular resonances of difluorophosphate in 31P and 19F NMR spectra of erythrocytes

Abstract

Trifluoroacetate and trifluoroacetamide, when added to a suspension of human red blood cells, give rise to separate 19F NMR signals from the intra- and extracellular species. This phenomenon has recently been exploited for measuring the membrane potential of erythrocytes. However, the separation of the peaks was incorrectly ascribed to a difference in magnetic susceptibility between the intra- and extracellular environments. Previously, we have reported well-resolved resonances in 31P NMR spectra for the intra- and extracellular populations of some phosphoryl compounds; in these cases, however, the intracellular peak is shifted to low frequency which is the opposite to the situation with the fluorinated compounds. By using difluorophosphate, which rapidly equilibrates across the membrane of human erythrocytes and which has both the phosphoryl and fluorine functional groups, we observed the separate intra- and extracellular resonances. But, the intracellular resonance was shifted to high frequency of the extracellular resonance in the 19F spectra and to low frequency in the 31P spectra. The basis for the phenomenon in both cases is thought to be the reduced hydrogen bonding inside the cells between the solvent water and the phosphoryl oxygen or fluorine atoms.