Free magnesium-ion concentration in erythrocytes by31P NMR: the effect of metabolite–haemoglobin interactions

Mulquiney, Peter; Kuchel, Philip W.

doi:10.1002/(sici)1099-1492(199705)10:3<129::aid-nbm459>3.0.co;2-2

Cited by 13 publications

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References 35 publications

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“…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 .…”

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“…However, by 1983 (48), this complexity in the method was lost, perhaps because, according to their equilibrium constants, Hb bound MgATP and ATP equally strongly, which would allow the Hb effect to be ignored (23). Also, this added complexity, combined with the lack of computational power at the time, perhaps made solving the necessary multiple equilibria too difficult to be achieved routinely.…”

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“…Also, this added complexity, combined with the lack of computational power at the time, perhaps made solving the necessary multiple equilibria too difficult to be achieved routinely. A disregard of the effect of Hb has existed ever since, until 1997 when the effect of metabolite-Hb interactions was reevaluated (23).…”

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confidence: 99%

“…This can be achieved, to a first approximation, by assuming that chemical shifts did not change when metabolite species bound to Hb. This is true within a 5% error in oxygenated erythrocytes (2,23). Simultaneous equations were then formulated which included the effect that Hb binding had on the equilibria for ATP and DPG and hence on the overall observed chemical shifts (Equations 10 and 11 and Appendix II).…”

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

Willcocks

Mulquiney

Ellory

et al. 2002

Journal of Biological Chemistry

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

Willcocks

Mulquiney

Ellory

et al. 2002

Journal of Biological Chemistry

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Using the β/α peak-height ratio of ATP in31P NMR spectra to measure free [Mg2+]: theoretical and practical problems

Mulquiney

Kuchel

1999

NMR Biomed.

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A recently described alternative method for determining free [Mg2+] in cells and tissue with 31P NMR spectroscopy, which is based on the peak‐height ratio of the β and β resonances of ATP (hβ/α) [J. Biol. Chem. 271, 21141–21150 (1996)], was found to have numerous problems. These problems include a flaw in the basic multiple equilibrium analysis, the inapplicability of the calibration curve to in vivo studies, and a lack of sensitivity of the measured ratio to changes in free [Mg2+] at ‘clinical’ field strengths. This suggests that the use of this procedure will be very limited. Copyright © 1999 John Wiley & Sons, Ltd.

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The interaction of haemoglobin, magnesium, organic phosphates and band 3 protein in nucleated and anucleated erythrocytes

Luca

Gugliotta

Scuteri

et al. 2004

Cell Biochemistry & Function

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The anion influx was measured in order to study the interaction among organic phosphates, magnesium, haemoglobin and the N-terminal of the cytoplasmic domain of band 3 protein in human, chicken and trout erythrocytes. The rate constant for SO(4)(2-) influx in human and trout erythrocytes increased significantly when it was measured with an increased concentration of intracellular Mg(2+). The SO(4)(2-) influx was also measured in human erythrocyte ghosts in the presence and absence of Mg(2+). The smaller activation provoked by Mg(2+) in ghosts could be caused by the presence of a small quantity of haemoglobin which remained inside. The SO(4)(2-) uptake in chicken erythrocytes in the presence and in absence of Mg(2+) was characterized by very similar rate constants. The results suggest that the small increase in intracellular Mg(2+) in the erythrocytes involves an increase in the formation of Mg(2+)-ATP and Mg(2+)-2,3 BPG complexes reducing the affinity of the organic phosphates for Hb. This new situation may influence the functions of the anion transporter with consequent variations of SO(4)(2-) influx throughout the erythrocyte membrane in human and in trout erythrocytes, whereas in chicken RBCs this function cannot occur and, in fact, no increase in sulphate influx was noticeable. The measurement of Hb/O(2) affinity by the use of alternating fixed and variable concentrations of organic phosphates and Mg(2+), confirms the interactions between these elements and their effect on the mechanism of the affinity. When we measured the sulphate influx in the presence of DIDS we found some differences in the three types of cells.

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Free magnesium-ion concentration in erythrocytes by31P NMR: the effect of metabolite–haemoglobin interactions

Cited by 13 publications

References 35 publications

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

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

Using the β/α peak-height ratio of ATP in31P NMR spectra to measure free [Mg2+]: theoretical and practical problems

The interaction of haemoglobin, magnesium, organic phosphates and band 3 protein in nucleated and anucleated erythrocytes

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