Ionized concentrations in Ca2+ and Mg2+ buffers must be measured, not calculated

Modelling intracellular regulation of Ca2+/Mg2+ is now an established part of physiology. However, the conclusions drawn from such studies depend on accurate knowledge of intracellular [Ca2+], ∆[Ca2+] and the pK′ values for the intracellular binding of Ca2+/Mg2+. Calculation of [Ca2+]/[Mg2+] in buffers is normal. The eight freely available programs all give different values for the [Ca2+]/[Mg2+] in the buffer solutions, varying by up to a factor of 4.3. As a result, concentrations must be measured. There are two methods to do this, both based on the ligand optimization method (LOM): (1) calibration solutions from 0.5 to 4 mmol l−1; and (2) calibration solutions from 0.1 µmol l−1 to 2 mmol l−1. Both methods can be used to calibrate Ca2+/Mg2+ electrodes. Only Method 2 can be used directly to calibrate fluorochromes and aequorin. Software in the statistical program R to calculate the [Ca2+]/[Mg2+] in buffers is provided for both methods. The LOM has now been generalized for use with electrodes, fluorochromes and aequorin, making it the ideal method to determine the pK′ values for intracellular binding of Ca2+/Mg2+. The [Ca2+]/[Mg2+] in buffers must be measured routinely, which is best done by calibrating electrodes with the LOM and software written in R. If [Ca2+]/[Mg2+] in buffers are calculated, the parameters used in modelling show the same degree of variability as the software programs. Uncritical acceptance of such parameters means that conclusions reached from such studies are relative, not absolute, and must now be re‐examined