A phosphate-analogue probe of red cell pH using phosphorus-31 nuclear magnetic resonance

In order to find a suitable marker of intraerythrocytic pH in a phosphorus-31 nuclear magnetic resonance (31P NMR) system, analogues of inorganic phosphate were studied. Fluorophosphate (pKa = 4.7) and phosphite (pKa = 6.4) chemical shifts and coupling constants were found to be pH sensitive but at pH ranges too low to be useful for the study of fresh red cells. Methylphosphonate (pKa = 7.6) showed ideal characteristics for a pH probe: its chemical shift was far downfield for red cell phosphates and showed a large pH dependence near its pKa (delta pH/delta delta = 0.46 pH/ppm). Methylphosphonate readily entered red cells [influx 7.8 microM (mL of RBC)-1 h-1] and did not appear to alter glucose consumption or hemoglobin-oxygen affinity in intact cells. NMR spectra were obtained on eight samples of fresh red cells incubated for 30-60 min with methylphosphonate. Chemical shift differences between the extracellular and intracellular methylphosphonate signals were found to be predictive of the transmembrane pH gradient. The extracellular pH was 7.336 +/- 0.031 (range 7.31-7.41), while the intracellular pH was 7.202 +/- 0.034 (range 7.14-7.23), and the transmembrane pH gradient measured 0.129 +/- 0.008 (range 0.11-0.14). Methylphosphonate is a useful probe of pH in the 31P NMR spectroscopic study of red cells.