Simultaneous determination of low free Mg2+ and pH in human sickle cells using 31P NMR spectroscopy.
The concentrations of free magnesium, [Mg(2+)](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 [Mg(2+)](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 [Mg(2+)](free), over a pH range of 5.75-8.5 and [Mg(2+)](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 [Mg(2+)](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 [Mg(2+)](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 [Mg(2+)](free) could be attributed to increased [DPG] and therefore greater magnesium binding capacity of sickle cells.
Willcocks
James P
JP
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Mulquiney
Peter J
PJ
Ellory
J Clive
JC
Veech
Richard L
RL
Radda
George K
GK
Clarke
Kieran
K
D019794:2,3-Diphosphoglycerate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000755:Anemia, Sickle Cell / Q000097:blood*; D004912:Erythrocytes / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D013696:Temperature
D006454:Hemoglobins; D019794:2,3-Diphosphoglycerate; D000255:Adenosine Triphosphate; D008274:Magnesium