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J. Biol. Chem., Vol. 277, Issue 51, 49911-49920, December 20, 2002

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

James P. Willcocks, Peter J. Mulquiney^§, J. Clive Ellory^¶, Richard L. Veech, George K. Radda, and Kieran Clarke^**

From the Departments of Biochemistry and ^¶ Physiology, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom, and the  National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland 20852

The concentrations of free magnesium, [Mg²⁺]_free, [H⁺], and [ATP] are important in the dehydration of red blood cells from patients with sickle cell anemia, but they are not easily measured. Consequently, we have developed a rapid, noninvasive NMR spectroscopic method using the phosphorus chemical shifts of ATP and 2,3-diphosphoglycerate (DPG) to determine [Mg²⁺]_free and pH_i simultaneously in fully oxygenated whole blood. The method employs theoretical equations expressing the observed chemical shift as a function of pH, K⁺, and [Mg²⁺]_free, over a pH range of 5.75-8.5 and [Mg²⁺]_free range 0-5 mM. The equations were adjusted to allow for the binding of hemoglobin to ATP and DPG, which required knowledge of the intracellular concentrations of ATP, DPG, K⁺, and hemoglobin. Normal oxygenated whole blood (n = 33) had a pH_i of 7.20 ± 0.02, a [Mg²⁺]_free of 0.41 ± 0.03 mM, and [DPG] of 7.69 ± 0.47 mM. Under the same conditions, whole sickle blood (n = 9) had normal [ATP] but significantly lower pH_i (7.10 ± 0.03) and [Mg²⁺]_free (0.32 ± 0.05 mM) than normal red cells, whereas [DPG] (10.8 ± 1.2 mM) was significantly higher. Because total magnesium was normal in sickle cells, the lower [Mg²⁺]_free could be attributed to increased [DPG] and therefore greater magnesium binding capacity of sickle cells.

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