Intracellular pH in stored erythrocytes. Refinement and further characterisation of the 31P-NMR methylphosphonate procedure

Methylphosphonate in conjunction with ³¹P-NMR spectroscopy was used for the measurement of transmembrane Δ pH in human erythrocytes stored at 4°C for up to 5 weeks in a nutrient medium. Intra- and extracellular pH was determined using calibration curves based on the pH-dependent separation between the NMR resonances of methylphosphonate and orthophosphate (P_i). A comprehensive statistical procedure is presented for the determination of the variance of NMR-based pH estimates. The entry of methylphosphonate into erythrocytes was more rapid at low pH and uptake was fully inhibited by the band 3 reagent, disodium 4,4-diisothiocyano-2,2′-disulphonic acid stilbene. The distribution ratio of methylphosphonate concentration inside and outside the cells was used to calculate the membrane potential; the analysis depends on a consideration of the Donnan equilibrium for an anion with one or two charges. Furthermore, the analysis does not depend on the pH estimates but relies solely on concentration estimates. The chemical shift of methylphosphonate was not subject to the variations associated with specific intracellular binding encountered with many other phosphorus compounds, including P_i. On the other hand, the ionic strength dependence of the chemical shift of methylphosphonate, contrary to earlier reports, is comparable in magnitude (but opposite in sign) to that of P_i.