New method for calculating pHi from accurately measured changes in pHi induced by a weak acid and base.

From the combined Henderson-Hasselbalch relations for a weak acid and a weak base, we derive an expression for steady-state intracellular pH(pHi). The derivation requires only accurate measurements of the pHi changes when the two electrolytes are applied, rather than absolute values. The method is based on the assumption of equilibrium of the neutral forms of the two compounds across the plasma membrane, that is, absence of permeation of the charged forms. It is also assumed that no significant pHi regulation takes place, that there is no significant permeability to intracellular buffers and that intracellular buffering power, over the measured span of pHi, is the same. This precludes the use of CO2/bicarbonate buffered systems. We find that under our experimental conditions steady-state pHi values calculated in this way agree closely with those measured directly with pH-sensitive microelectrodes both in snail neurones and crab muscle. The method would allow the intracellular calibration of other pHi measuring techniques.