Calcium and magnesium buffer solutions: the need for standardisation

To measure intracellular Ca2+ concentrations and the binding of Ca2+ and Mg2+ to physiological compounds one must manufacture respectively nmolar and μmolar buffers. To calculate the free ionic concentration the apparent equilibrium constant (Kapp) in the buffer solution has either to be measured or calculated with appropriate temperature, ionic strength and pH. Due to the binding of water, the ligand concentration must also be determined. The principal experimental methods are based on partition methods, macroelectrodes, calcium indicators and pH titration. Only macroelectrodes and pH titration measure both purity and Kapp. The pH method of Moisescu & Pusch (1975) accurately determines ligand purity but for Kapp determinations assumes incorrectly that 2 H+ ions are released for 1 Ca2+ ion bound. It is only an approximation. The Bers (1982) method using Ca2+ macroelectrodes cannot be used with Mg2+ macroelectrodes (Luethi et al. (1997). Unless the Ca2+ macroelectrode is linear, the method overestimates both purity and Kapp and also has a subjective element. The improved iterative method (Oiki et al. 1994) cannot be used with Mg2+ macroelectrodes. The most general method to measure both Kapp and ligand purity (Luethi et al. 1997) is also based on macrolelectrodes, does not depend on the linearity of the macroelectrode and is not subjective but it is iterative, tedious and time consuming. These problems have now been overcome with a MS Excel program (Kay et al. this meeting, C78). An additional problem with such buffer solutions is the precision of the measurement. However, there are no defined limits of allowable coefficient of variation (CV). If ligand purity is known then Kapp can be calculated from internet programs (Maxchelator, BAD and Chelator) or calculated directly from the constants in Martell & Smith (1974). From the various programs only Chelator is similar to the experimentally estimated calcium concentration in Ca2+ buffer solutions. In conclusion, there is no defined method to estimate ligand purity and Kapp, no upper limit to CV and calculated concentrations can vary by a factor of 2 for Ca2+ or between 3 and 4.5 for Mg2+ (Luethi et al. 1997). International standardisation of Ca2+ and Mg2+ buffer solutions is now needed.