Simultaneous determination of intracellular magnesium and pH from the three ³¹P NMR chemical shifts of ATP

The simultaneous determination of intracellular [Mg_T]/[ATP_T] and pH from the three ³¹P NMR chemical shifts of ATP has been demonstrated using two-dimensional calibrations. The resulting pH will more accurately represent that of healthy tissue than by using the standard NMR technique. As a result of other possible complexes of MgATP and uncertainties in intracellular pH, errors in the values of K_D^MgATP(pH), the MgATP NMR shift limits, and thus intracellular magnesium levels are reduced by using this self-consistent analytical method. Direct determination of free magnesium from the (γ-β) shifts of ATP may be more sensitive in the alkaline pH range than with the commonly used (α-β) shifts. In addition, the calibration data sets allow for a graphical representation of the uncertainty in magnesium due to the uncertainty in the measured chemical shifts and pH. Our results indicate that K_D^MgATP is consistent with the literature and favors a value near 50 μM at pH 7.2, but that free magnesium levels will be lower than most prior estimates.