Magnesium in cellular energetics
Most cellular magnesium is bound, yet it is the concentration of free magnesium, [Mg2+]free, in red blood cells that is vital in the regulation of enzyme activity and ion transport. It is unknown how changes in total blood magnesium affect the [Mg2+]free within red blood cells or in tissue, because the presence of other cations, especially H+ and potassium, K+ , affects the degree to which Mg2+ is bound. Consequently, this Thesis presents a new 31P NMR spectroscopic method to measure [Mg2+]free in blood, which analyses the changes in the phosphorus chemical shifts of ATP and 2,3-DPG using theoretical equations expressing the observed chemical shift as a function of pH, K+ and [Mg2+]free, over the pH range of 5.75 to 8.5 and [Mg2+]free range 0 to 5 mM. The equations were adjusted for the binding of haemoglobin to ATP and DPG, which required knowledge of the intracellular concentrations of ATP, DPG, K+ and Hb. These equations enabled, for the first time, the simultaneous analyses of the chemical shifts of 3P-DPG and β-ATP to measure both intracellular 04- pH and [Mg2+]free in normal and sickle blood. To simulate in vivo 100% oxygenated blood, samples were prepared for analysis by equilibration with a mixture of O2 and CO2, adjusted to give a pCO2 of 40 mmHg and pO2 > 150 mmHg. Under these conditions, normal whole blood had an intracellular pH of 7.20 ± 0.02 and a [Mg2+]free of 0.41 ± 0.03 mM (n = 33). Further work determined blood pH and [Mg2+]free for several clinical conditions including sickle cell anaemia, pre-eclampsia, hypoxia, patients with sub-arachnoid haemorrhage and chronic fatigue syndrome. This Thesis has demonstrated the potential of this new technique to evaluate the importance of [Mg2+]free in the regulation of metabolite concentration and metabolic function, and to elucidate some of the properties of magnesium transport across the erythrocyte cell membrane.