Calibration of Mg(2+)-selective macroelectrodes down to 1 mumol l-1 in intracellular and Ca(2+)-containing...

Using Mg(2+)-selective macroelectrodes based on the neutral carriers ETH 7025 and ETH 5506, methods were developed to determine accurately the apparent binding constant (Kapp) and purity, and hence the ionized magnesium concentration ([Mg2+]), in Ca(2+)-free, Mg2+ buffer solutions manufactured with either CDTA (trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid monohydrate) or EDTA. In nominally Ca(2+)-free solutions, calibration of the macroelectrodes was possible down to 1 mumol l-1 in both intracellular (ETH 7025)- and extracellular (ETH 5506)-like physiological solutions. The measured [Mg2+] in the buffer solutions overlapped with the [Mg2+] set by dilution alone, suggesting that the method was reliable. These buffer solutions could then be used to manufacture standard Mg2+ solution containing known [Mg2+] at a set calcium concentration. Ca2+ sensitivity limits the use of ETH 7025 and, for extracellular measurements in Ca(2+)-containing solutions, Mg(2+)-selective macroelectrodes manufactured with ETH 5506 were the electrodes of choice. In 0.5-0.9 mmol l-1 Ca+, measurement of [Mg2+] was possible down to 10 mumol l-1. At [Mg2+] greater than 0.25 mmol l-1 there was no interference from Ca2+ (0.5-1.5 mmol l-1). With CDTA and/or EDTA buffer solutions there was a wide variation between the calculated values for the [Mg2+] and the measured [Mg2+] (the calculated values differed by a factor of up to 4.5 and 3, respectively). At present, measurement of the Kapp and ligand purity in the appropriate solution at the desired pH and temperature would seem to be the best strategy to adopt rather than attempting to calculate the constant. Since no recognized international standard exists for [Mg2+] at the micromolar level, values in the literature for Kd, etc. in this range can only be regarded as approximate.