Simultaneous determination of low free Mg2+ and pH in human sickle cells using 31P NMR spectroscopy

Abstract

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 pHi 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 pHi 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 pHi (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.