Modelling intracellular regulation of Ca2+/Mg2+ is important in physiology but the validity of conclusions drawn from such studies depends on the accuracy of measured values for resting [Ca2+]/[Mg2+], their changes and the pK/ values for the intracellular binding of Ca2+/Mg2+. The correctness of these parameters depends on the accuracy of Ca2+/Mg2+ buffers in which the [Ca2+]/[Mg2+ ] can either be calculated or measured. Calculation is usual. There are seven freely available software programs; all give different answers for the [Ca2+]/[Mg2+] (by a factor of around four). This is due to: 1) Calculations for changes in ionic strength are based on single ion activity coefficients, which cannot be experimentally determined; 2) pHa (activity) has to be converted to pHc (concentration); not possible, because of potential changes at the reference electrode. If values in buffers are calculated, the coefficient of variation cannot be determined as a measure of their accuracy, nor can the calculated values be related to SI units because of unverifiable assumptions. Since calculation is not an option, concentrations must be measured. There are two methods for this, both based on the Ligand Optimisation Method (LOM); Method 1, calibration solutions from 0.5 to 4 mmol/l (McGuigan et al, 2017); Method 2, calibration solutions from 1 µmol/l to 2 mmol/l (Allen et al, 1977). Both methods can be used to calibrate Ca2+/Mg2+-electrodes. Only Method 2 can be directly used to calibrate fluorochromes and photochromes (e.g. Aequorin). The Excel program (Kay et al, 2008) to calculate [Ca2+]/[Mg2+] in buffers when using Ca2+/Mg2+-electrodes, has been extended using the statistical program “R” for electrodes and now for fluorochromes and Aequorin. Method 1 is routine and laboratory glassware can be used if adequately washed (Tran et al, 2018). All it requires is a Ca2+/Mg2+-electrode, a 3 mol/l KCl reference electrode and a pH meter in mV mode. Method 2 is exact, but requires special laboratory facilities to reduce Ca2+/Mg2+ contamination to an absolute minimum. Measurement of buffer [Ca2+]/[Mg2+] must be routinely carried out; best done by calibrating Ca2+/Mg2+-electrodes with LOM and software written in “R”. Such calibrated electrode can be used to calibrate fluorochromes and photoproteins. If [Ca2+]/[Mg2+] in buffers are calculated, the parameters used in modelling show the same degree of variability as the software programs. The values for resting [Ca2+], its changes and the pK/ values for intracellular binding of Ca2+/Mg2+ based on calculated values in buffers are not only wrong, but have to be re-determined using buffers with measured [Ca2+]/[Mg2+]. Uncritical acceptance of parameters based on calculated values in Ca2+/Mg2+ buffers means that conclusions reached from such modelling of intracellular regulation of Ca2+/Mg2+ must now be critically re-examined.
Physiology 2019 (Aberdeen, UK) (2019) Proc Physiol Soc 43, PC023
Poster Communications: Ionised concentrations in Ca2+ and Mg2+ buffers must be measured not calculated
J. A. McGuigan1, J. W. Kay2, H. Y. Elder3
1. Institute of Physiology, Berne, Switzerland. 2. School of Mathematics and Statistics, Glasgow, United Kingdom. 3. School of Life Sciences, Glasgow, United Kingdom.
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