Most cellular magnesium is bound, yet it is the concentration of free magnesium, [Mg<sup>2+</sup>]<sub>free</sub>, 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 [Mg<sup>2+</sup>]<sub>free</sub> within red blood cells or in tissue, because the presence of other cations, especially H<sup>+</sup> and potassium, K<sup>+</sup> , affects the degree to which Mg<sup>2+</sup> is bound. Consequently, this Thesis presents a new <sup>31</sup>P NMR spectroscopic method to measure [Mg<sup>2+</sup>]<sub>free</sub> 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<sup>+</sup> and [Mg<sup>2+</sup>]<sub>free</sub>, over the pH range of 5.75 to 8.5 and [Mg<sup>2+</sup>]<sub>free</sub> 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<sup>+</sup> 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 [Mg<sup>2+</sup>]<sub>free</sub> in normal and sickle blood. To simulate in vivo 100% oxygenated blood, samples were prepared for analysis by equilibration with a mixture of O<sub>2</sub> and CO<sub>2</sub>, adjusted to give a pCO<sub>2</sub> of 40 mmHg and pO<sub>2</sub> > 150 mmHg. Under these conditions, normal whole blood had an intracellular pH of 7.20 ± 0.02 and a [Mg<sup>2+</sup>]<sub>free</sub> of 0.41 ± 0.03 mM (n = 33). Further work determined blood pH and [Mg<sup>2+</sup>]<sub>free</sub> 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 [Mg<sup>2+</sup>]<sub>free</sub> in the regulation of metabolite concentration and metabolic function, and to elucidate some of the properties of magnesium transport across the erythrocyte cell membrane.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:249500 |
| Date | January 2002 |
| Creators | Willcocks, James Peter |
| Contributors | Clarke, Kieran |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:f4f5de76-8c72-4b42-8bd4-eb151485d47e |
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