Delta alkalinity: a simple method to measure cellular net acid-base fluxes.

Abstract

Acid-base transport across cell plasma membranes is important for cell homeostasis and growth. Current techniques for quantitatively measuring net acid-base fluxes are generally limited by either assumptions concerning properties of intracellular compartments or use of poorly buffered, nonphysiological solutions. We adapted an approach from marine chemistry to quantitate net acid-base changes in standard physiological media that obviates these problems. This method is based on conservation of charge and involves a simple acid titration of the extracellular medium to an end point, the equivalence point, pHe. For standard physiological solutions containing buffers such as bicarbonate and phosphate, pHe exists in the range of pH 4.0-4.7 and is identified as the pH where dpH/dH+ is maximal in an HCl titration. By determining the quantity of H+ required to reach pHe, one can determine precisely total quantity of proton acceptors (alkalinity) present in physiological pH range. Alkalinity (in meq) is a relative measure of the charge capable of interacting with protons. We show that, unlike pH, changes in alkalinity (delta alkalinity) result only from net acid-base changes in medium. Therefore, by monitoring extracellular delta alkalinity associated with cell function, it is possible to quantitate precisely net acid-base fluxes. Moreover, through a second titration procedure, delta alkalinity can be divided into bicarbonate and nonbicarbonate fractions. As an example, we performed the first direct measurement of net Cl(-)-HCO3- exchange in intact human erythrocytes and observed a Cl(-)-HCO3- exchange ratio of 1.01 +/- 0.03. Overall, delta alkalinity measurements are applicable to numerous cell systems, can be performed with solutions containing a mixture of buffers at normal physiological concentrations (e.g., 25 mM HCO3- and 2 mM HPO4(-2), do not require corrections for CO2 diffusion or loss of CO2 from the solution, and avoid assumptions about intracellular or extracellular buffer properties.