Simultaneous Determination of Low Free Mg2+ and pH in Human Sickle Cells using 31P NMR Spectroscopy

Abstract

Magnesium is the second most abundant intracellular cation after potassium and is important in the regulation of more than 300 enzymes (1-3) and ion transport across cell membranes (4, 5). Some diseases, such as hypertension, pre-eclampsia, and sickle cell anemia exhibit pathologies linked to low magnesium levels (6 -8). The genetic defect in sickle cell anemia results in the synthesis of an abnormal ␤ hemoglobin (Hb) subunit, which polymerizes upon deoxygenation. This polymerization produces the characteristic sickle deformation in red blood cells and leads to microcirculatory occlusion and consequent tissue ischemia. Sickling depends strongly on the intracellular Hb concentration (9) and is enhanced by dehydration of erythrocytes, a process involving potassium and sodium transport across cell membranes, which is in turn thought to be regulated by cytosolic free magnesium, [Mg 2ϩ ] free , 1 via the KCl cotransporter (KCC1) (5, 10). Thus dehydration of sickle cells, and thereby the process of sickling, may be controlled by changes in the magnesium content (8, 11).To assess its role in sickle cell anemia, an accurate and reproducible method was required for the measurement of [Mg 2ϩ ] free . Although several methods have been developed to determine [Mg 2ϩ ] free in red blood cells, such as null point titration with metallochromic dyes (12), magnesium-sensitive electrodes (13,14), use of divalent cation ionophores (15), and 31 P NMR spectroscopy (16,17), the reported values vary 3-fold, from 0.2 to 0.6 mM (18 -20). Of these methods, the one noninvasive technique that has been used extensively is that of 31 P NMR spectroscopy (16 -19, 21-25). However, there are a number of potential errors associated with using the observed NMR signal to determine [Mg 2ϩ ] free , including uncertainty in the binding constant between ATP and magnesium (26), the effect of intracellular ionic content on this interaction (27), and the effect of metabolite-Hb interactions (23). Gupta and co-workers (2), the first to use this method, addressed the first two problems by determining an apparent MgATP binding constant, K (app)bMgATP , from the difference between the end points of the chemical shifts of the magnesium bound and unbound ␣ and ␤ phosphorus nuclei of ATP, ␦ ␣␤-MgATP and ␦ ␣␤-ATP . This decreased the number of equilibria that had to be considered and overcame the need to rely on adjusted published values of K (app)b , which varied 100-fold (28). However, as with any simplification, problems are associated with this approach, which include the possible difference in ionic strength between solutions and in vivo conditions, the accuracy of the chemical shift end points required to determine K (app)b (29), and the assumption that ␦ ␣-ATP did not vary.Because the chemical shifts of 31 P NMR phosphorus peaks depend on many different factors (30), accurate analysis should take all factors into account, which has become possible with the increased capacity of computation. Previous analyses using phosphorus chemical shifts have be...

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