The Concentrations of Free and Bound Magnesium in Rat Tissues

Abstract The levels of total magnesium in rat liver, brain, and kidney have been determined, and the distribution of the magnesium between free and bound forms has been calculated. Total tissue magnesium measured by atomic absorption was found to be 6.92, 8.25, and 10.4 µmoles per g wet weight of brain, kidney cortex, and liver, respectively. The calculation of the intracellular free magnesium concentrations (free Mg2+) in these tissues was approached experimentally in two different ways. In the first method the concentration of the magnesium binding sites and the corresponding stability constants were determined experimentally for the various fractions of tissue and were used together with the total Mg to calculate the free Mg2+. Operationally, the tissue was separated into three fractions: the insoluble binding sites (those sedimented at 40,000 x g for 30 min), the soluble binding sites (large molecules of molecular weight g1000 remaining in the supernatant after centrifugation), and the (small molecules of molecular weight l1000). Using various tissue dilutions and magnesium concentrations, the binding to insoluble and soluble binding sites was studied with centrifugation and equilibrium dialysis techniques. By Scatchard-type plots estimates of the concentrations of these binding sites and the corresponding constants could be made. The concentrations of insoluble binding sites were estimated to be 3.0, 2.0, and 2.8 µmoles per g wet weight of brain, kidney, and liver, respectively, with corresponding binding constants of 4,300, 1,860, and 3,740 kg wet weight of tissue per mole of binding sites. The concentration of soluble binding sites was estimated to be 4.7 and 10.6 µmoles per g wet weight in kidney and liver, respectively, with corresponding binding constants of 727 and 532 kg wet weight of tissue per mole of binding sites. From these values and the concentrations of a number of metabolites measured in freeze-clamped tissue and their binding constants, the free Mg2+ could be calculated from a complex polynomial of the form: see PDF for equation where free Mg2+ = MgB1/(B1 - MgB1)K1 and MgB1 is the concentration of the magnesium complex and K1 the binding constant of the first binding agent; and Bi and Ki are the concentration and binding constant respectively of the ith binding agent. The second method for the calculation of the free Mg2+ involves the use of the equilibrium constant of aconitate hydratase (EC 4.2.1.3). The measured Σcitrate/Σisocitrate (concentrations of total citrate/total ls(+)-isocitrate) in freeze-clamped tissue was compared with the equilibrium ratios in vitro for the aconitate hydratase reactions at various free Mg2+, 38°, ionic strength = 0.25, K+ = 125 mm, and Na+ = 25 mm. Free intracellular Mg2+ was calculated to range between 0.6 and 1.3 µmoles per g wet weight in all tissues studied. The values obtained with the two independent methods showed good agreement. From the similarity of these values with those estimated by other authors in various extracellular fluids, it is implied that there is little or no free Mg2+ gradient between intracellular and extracellular fluids in spite of very different total magnesium concentrations between tissue and extracellular fluid. It is also implied that the free Mg2+ like pH is relatively constant in various tissues and extracellular fluids, the free Mg2+ being buffered by relatively large concentrations of bound magnesium and magnesium-binding sites.