Total and free Mg 2+ contents in erythrocytes: a simple but still undisclosed cell model

Transcription

1 Magnesium Research 2007; 20 (3): REVIEW ARTICLE Total and free Mg 2+ contents in erythrocytes: a simple but still undisclosed cell model Theodor Günther Charité Universitätsmedizin Berlin, Campus Benjamin Franklin, Institut für Molekularbiologie und Biochemie, Arnimallee 22, Berlin, Germany Correspondence: T. Günther, Waldhüterpfad 63, D14169 Berlin, Germany. Tel.: (+00 49) Abstract. The concentration of intracellular free Mg 2+ ([Mg 2+ ] i ) in erythrocytes, measured by means of 31 P NMR and using a dissociation constant for MgATP of lm, amounted to 0.2 mm [Mg 2+ ] i in the erythrocytes of various species, was not significantly different and was independent of their total Mg 2+ content. The more probable value of [Mg 2+ ] i using the more realistic K D of Mg ATP or the null-point method may amount to 0.4 mm [Mg 2+ ] i in erythrocytes is lower than the [Mg 2+ ] i in nucleated mammalian cell types. The lower [Mg 2+ ] i may be caused by a different regulation of Mg 2+ influx and Mg 2+ efflux by intracellular Mg 2+ in erythroblasts. Free and reversibly bound Mg 2+ represent a Mg 2+ buffer. The main Mg 2+ -binding substances are ATP and 2,3-bisphosphoglycerate (2,3-BPG). Total Mg 2+ content in the erythrocytes of various species is correlated to the concentrations of ATP and 2,3-BPG. The changed Mg 2+ level in erythrocytes during deoxygenation, maturation, cold storage, in Mg 2+ deficiency and in sickle cell anemia was reviewed. Key words: intracellular Mg 2+, erythrocyte doi: /mrh Intracellular Mg 2+ consists of free and bound Mg 2+. Bound Mg 2+ is a heterogeneous fraction containing chelated Mg 2+ with a low dissociation constant and ranges up to interactions of Mg 2+ with negatively charged ligands, which also can be described by a low activity coefficient of free Mg 2+. The level of [Mg 2+ ] i is the result of Mg 2+ influx, Mg 2+ efflux, transfer of Mg 2+ between cytosol and cell organelles as well as intracellular Mg 2+ binding. A general scheme has been shown elsewhere [1]. A simpler model for intracellular Mg 2+ is represented by mammalian erythrocytes. These cells do not contain organelles such as nucleus, ribosomes, mitochondria and endoplasmic reticulum. Transport of Mg 2+ into and out of cell organelles and binding of Mg 2+ within cell organelles are absent. [Mg 2+ ] i in mammalian erythrocytes thus results only from Mg 2+ influx, Mg 2+ efflux and Mg 2+ binding. Mg 2+ efflux operates via Na + /Mg 2+ antiport and as Na + -independent Mg 2+ efflux [2]. The Na + /Mg 2+ antiporter can perform 28 Mg 2+ / 24 Mg 2+ exchange [2]. Net uptake of Mg 2+ in erythrocytes at physiological Na + and Mg 2+ gradients was not detectable [3]. Mg 2+ -binding substances in erythrocytes are nucleotides (ATP), phosphate esters particularly 2,3- BPG, phospholipids of the cell membrane and proteins. Moreover, considering [Mg 2+ ] i in erythrocytes of various mammalian species with different contents of Mg 2+, ATP and 2,3-BPG as well as different Mg 2+ efflux may offer a more comprehensive analysis of the mechanisms that determine the intracellular Mg 2+ level in erythrocytes. Total Mg 2+ content in erythrocytes of various species As shown in table 1 and in more detail elsewhere [4], total Mg 2+ content varies between 0.5 cells in the erythrocytes of cattle up to 4.0 cells in pig erythrocytes. Erythrocytes of various species express very different concentrations of Na +,K +, Mg 2+, ATP and 2,3-BPG as well as different activity of Mg 2+ efflux, indicating that these cells are functioning under very different conditions of electrolyte and energy metabolism. 161

2 T. GÜNTHER Table 1. Total concentrations of Na +,K +,Mg 2+, ATP, 2,3-bisphosphoglycerate (2,3-BPG), concentration of intracellular free Mg 2+ ([Mg 2+ ] i ), activity of the Na + /Mg 2+ antiport and Na + -independent efflux of oxygenated erythrocytes from various species. Species Na + K + Total Mg 2+ 2,3-BPG ATP mm [Mg 2+ ] i mm Na + /Mg 2+ antiport cells Na + -indep. Mg 2+ efflux x60min Pig Rabbit n.d Man a Rat Dog n.d Cat n.d Cattle < Values for more species were given in [4-6]. Values for 2,3-BPG were calculated from [6]. Values for ATP were taken from [5]. ATP content of cat erythrocytes was calculated from [6]. n.d., not determined. a Mean value taken from the values provided by various investigators listed in table 2. [Mg 2+ ] i was measured using 31 P NMR. Mg 2+ efflux via Na + /Mg 2+ antiport and Na + -independent Mg 2+ efflux were measured in Mg 2+ -loaded erythrocytes. The total Mg 2+ content in the erythrocytes of various species correlates to their concentrations of ATP and 2,3-BPG [4-6] (table 1). ATP and 2,3-BPG are the main Mg 2+ -binding substances in erythrocytes, as reflected by their contents and dissociation constants. The dissociation constant of MgATP and its uncertainty were given in [7]. Values of mm [17], 1.67 mm [18], mm [19] and 3 mm [20] have been published for the dissociation constant of Mg-2,3-BPG. [Mg 2+ ] i in erythrocytes of various species Using 31 P MNR, a value of 0.22 mm mm was obtained for [Mg 2+ ] i in oxygenated erythrocytes from various species (tables 1 and 2). In these measurements, dissociation constants for MgATP of 38 lm, 46 lm and 50 lm were used. Using the more realistic value for the Mg ATP dissociation constant of 87 lm (for details see [7]), the more realistic value of [Mg 2+ ] i would be 0.4 mm. An [Mg 2+ ] i of 0.38 mm was obtained with the null-point method [21, 22]. Remarkably, in the erythrocytes of cattle, [Mg 2+ ] i is not significantly lower than in the erythrocytes of other species, although their contents of ATP and 2,3-BPG are extremely low. There are differences in total and free Mg 2+ concentration among humans. In erythrocytes of HLA B35+ subjects, [Mg 2+ ] i amounted to 0.27 mm and total Mg 2+ to 1.93 cells. In erythrocytes of Table 2. Concentration of intracellular free Mg 2+ ([Mg 2+ ] i ) in oxygenated human erythrocytes measured using 31 P NMR. The used MgATP dissociation constants (K D ) and experimental conditions are listed. K D lm ph T C [Mg 2+ ] i mm [8] [9] [10] [11] [12] [13] [14] [15] [16] Ref. 162

3 Mg 2+ LEVEL IN ERYTHROCYTES Table 3. [Mg 2+ ] i (mm) in oxygenated and deoxygenated human erythrocytes measured using various methods. The erythrocytes were completely [14, 26] or partially [8, 21] deoxygenated. Oxygenated Deoxygenated Method Ref P NMR [14] P NMR [8] Null-point [21] Mag-fura-2 [26] Calculation [17] HLA B35- subjects, [Mg 2+ ] i amounted to 0.36 mm and total Mg 2+ to 2.32 cells [23]. The reason for the higher total Mg 2+ and [Mg 2+ ] i may be a lower activity of Na + /Mg 2+ antiport in HLA B35 subjects [24]. Mg 2+ level during maturation of erythrocytes Total Mg 2+ content in reticulocytes is higher than in erythrocytes [25]. [Mg 2+ ] i measured by 31 P NMR (K D ofmgatp=38lm) amounted to 150 lm in reticulocytes and to 250 lm in erythrocytes. The concentration of ATP decreases during maturation. The concentration of 2,3-BPG remained constant or decreased (for lit see [25]). The decrease in total Mg 2+ content can be explained by the loss of cell organelles (ribosomes) and by the decrease in ATP. All fractions of Mg 2+ efflux in reticulocytes were the same as in erythrocytes [22]. The increase in [Mg 2+ ] i during maturation of erythrocytes has not as yet been explained. As discussed above, the most probable [Mg 2+ ] i in erythrocytes is 0.4 mm. In any case [Mg 2+ ] i in erythrocytes is lower than in nucleated cell types. This difference has not as yet been explained. Mg 2+ in erythrocytes is buffered by reversible binding of Mg 2+. The low [Mg 2+ ] i in erythrocytes is remarkable because of the rather high Mg 2+ dissociation constant of 2.3-BPG (see above), the main Mg 2+ - binding substance in erythrocytes. The low [Mg 2+ ] i may be established by the ratio of Mg 2+ influx and Mg 2+ efflux during the maturation of erythrocytes. [Mg 2+ ] i and the properties of the Mg 2+ transporters in erythroblasts and their behavior during maturation are not known. It may be assumed that the Na + /Mg 2+ antiporter in erythroblasts has a higher affinity for intracellular Mg 2+ than the Na + /Mg 2+ antiporter in other nucleated cell types. [Mg 2+ ] i during oxygenation-deoxygenation [Mg 2+ ] i in erythrocytes is changed during oxygenation-deoxygenation. The alterations are shown in table 3. During deoxygenation, ph in erythrocytes increases from 7.14 to 7.29 [27]. Total concentrations of ATP and 2,3-BPG were insignificantly [8] or slightly changed by deoxygenation [14]. The increased binding of ATP and 2,3-BPG to hemoglobin during deoxygenation liberates Mg 2+ and increases [Mg 2+ ] i [8]. The values for [Mg 2+ ] i in completely deoxygenated erythrocytes may be overestimated. Under physiological conditions deoxygenation occurs only to about 30% [21]. The deoxygenation-induced increase in [Mg 2+ ] i in these experiments (table 3) is not compensated by Mg 2+ efflux. Thus [Mg 2+ ] i may oscillate during oxygenation-deoxygenation in vivo. Absence of significant Mg 2+ efflux during oxygenationdeoxygenation can also be concluded from the missing Na + and Mg 2+ gradients as driving forces of the Na + /Mg 2+ antiport in the erythrocytes of dog, cat and cattle (table 1). The decreased [Mg 2+ ] i in deoxygenated erythrocytes [14] (table 3) is unexpected. The authors [14] have intensively discussed their controversial results which may have been caused by problems in the calculation of [Mg 2+ ] i using the 31 P NMR method. Difficulties in 31 P NMR due to deoxygenation of erythrocytes [27] may be overcome by measuring [Mg 2+ ] i with other methods also listed in table 3. Experimental alterations of the Mg 2+ level In hypomagnesemic patients, [Mg 2+ ] i in erythrocytes measured with 31 P NMR was reduced from mm to mm.total Mg 2+ was not reported [13]. In Mg 2+ -deficient rats, the total Mg 2+ content of erythrocytes was reduced from 2.95 cells in control rats to 1.73 cells in the Mg 2+ -deficient rats. Simultaneously, [Mg 2+ ] i was reduced from 163

4 T. GÜNTHER 0.38 mm to 0.17 mm [22]. The reduction in total Mg 2+ content can be explained by the reduction in ATP and 2,3-BPG in Mg 2+ -deficient erythrocytes [28]. The activity of Na + /Mg 2+ antiport in Mg 2+ -loaded erythrocytes from Mg 2+ -deficient rats was increased [22], probably by an alteration in the membrane phospholipids and an increase in cell membrane fluidity [29]. The reduction of [Mg 2+ ] i may be explained by the increased activity of the Na + /Mg 2+ antiport. Possibly, a not yet characterized catecholamine-induced effect in Mg 2+ deficiency may be involved in the reduction of [Mg 2+ ] i. Incubation of human erythrocytes with isoproterenol, forskolin (activator of adenylate cyclase) or dbcamp induced a decrease in [Mg 2+ ] i measured by 31 P NMR. The decrease in [Mg 2+ ] i was not caused by increased Mg 2+ efflux [16]. This result is in agreement with our results, which have shown that Mg 2+ efflux via the Na + /Mg 2+ antiport in rat erythrocytes was not affected by protein kinase A (PKA) [2]. HA-1004, an inhibitor of PKA, increased [Mg 2+ ] i in Mg 2+ -free medium [16]. These results show that PKA may induce a redistribution of Mg 2+ in erythrocytes. The concentration of 2,3-BPG was not changed (6.15 versus 6.04 mm) whereas the concentration of ATP was reduced by 24%. Therefore the reduction of [Mg 2+ ] i cannot be explained by increased binding of Mg 2+ to ATP and 2,3-BPG. The binding substance was not defined. Cold storage of erythrocytes During cold storage of human erythrocytes at 4 C, the concentration of ATP and 2,3- BPG decreased. Total Mg 2+ content remained constant while [Mg 2+ ] i, measured with 31 P NMR, continuously and reversibly dropped from mm to mm after days, even when the erythrocytes were stored in the presence of 5 mm Mg 2+ [10, 11]. The decrease in [Mg 2+ ] i was not caused by increased Mg 2+ efflux or by an alteration of intracellular ph, K +,Na + or by an increase in inorganic phosphate [11]. The results may indicate increased intracellular Mg 2+ binding during cold storage. The free Mg 2+ that disappeared may have been partly bound to pyrophosphate which increased during cold storage [10]. The cold storage experiments were repeated with [Mg 2+ ] i measured using the null- point method [30]. In these experiments, [Mg 2+ ] i increased from mm to mm after days of cold storage. No satisfying explanation was given for this discrepancy (for a possible explanation see [14]). Total Mg 2+ concentration was higher than the sum of [Mg 2+ ] i, and the calculated concentrations of MgATP and Mg-2,3- BPG. As binding of Mg 2+ to hemoglobin was neglected (the dissociation constants of MgHb and MgHbO 2 amount to 400 mm [8]), an unidentified Mg 2+ -binding substance was suggested. This substance would amount to 1.36 mm (38% of total Mg 2+ ) in fresh and 2.37 mm (67% of total Mg 2+ ) in stored erythrocytes [30] (see also Mg 2+ buffering). Mg 2+ buffering in erythrocytes Free Mg 2+ and reversibly bound Mg 2+ represent an Mg 2+ buffer system consisting of various Mg 2+ binding substances. In these experiments, the concentration of free and total Mg 2+ was measured in ATP-depleted, cell membrane-free hemolysates of Mg 2+ -loaded and non Mg 2+ -loaded erythrocytes which were titrated with MgCl 2. The concentration of free Mg 2+ was measured using an Mg 2+ -sensitive electrode and total Mg 2+ was determined by atomic absorption spectrometry (AAS) [31]. These experiments yielded a total Mg 2+ buffer capacity of 4.85 mm with a K D of 1.1 mm. Due to its concentration and K D, this substance may be 2,3-BPG. In other experiments Mg 2+ buffering in erythrocytes was investigated by increasing intracellular Mg 2+ with A23187 at increasing concentrations of extracellular Mg 2+. The resulting [Mg 2+ ] i and total Mg 2+ were measured using the null-point method and AAS. By this method, three Mg 2+ buffer systems of increasing capacity and decreasing affinity were postulated: 0.15 mm, with a K m of 10-7 M, (K m =K D ), 1.6 mm, with a K m of 0.08 mm (probably ATP), 21-25mM,withaK m of 3.6 mm [32]. In similar extended experiments [33] ATP, 2,3- BPG, PO 4, ph, free and total Mg 2+ in differently Mg 2+ - loaded erythrocytes at three metabolic states (control, ATP and 2,3-BPG depletion) were measured. Binding of Mg 2+,H +,K + and the intraction of H + and Mg 2+ with various Mg 2+ -binding substances (ATP, ADP, 2,3-BPG, PO 4, hemoglobin) were calculated using the dissociation constants provided by various authors. Also, the formation of Mg 2 ATP and Mg 2-2,3- BPG at high [Mg 2+ ] i were considered in the computer models. The calculated bound Mg 2+ at increasing [Mg 2+ ] i was lower for all metabolic states than the measured bound Mg 2+, although the dissociation constants used for MgATP (10 lm),mg-2,3-bpg (0.107 mm) and MgADP (0.304 mm) were much lower than usually reported. (For the K D of MgATP (87 lm) see [2], for 164

5 Mg 2+ LEVEL IN ERYTHROCYTES the K D of Mg-2,3-BPG (up to 3 mm) see above. For the K D of MgADP values of 0.43 mm [8] and 0.59 mm [34] were given). Only when 2:1 Mg 2+ complexes of ATP and 2,3-BPG and binding of Mg 2+ to Hb, using the very low Mg 2+ dissociation constant of 45 mm for MgHb were added, did the measured bound Mg 2+ correspond to the calculated bound Mg 2+. Again the reported K D for MgHb is higher and ranges from mm [8, 33]. Binding of Mg 2+ to negatively charged phospholipids of the inner side of the cell membrane was neglected because of their high intrinsic Mg 2+ dissociation constants [33]. However, the surface-fixed charges produce a charge density-dependent surface potential leading to an enrichment of ions in the aqueous phase at the surface, depending on the valence and concentration of the ions. The enrichment of Mg 2+ at the inner cell membrane, as also reflected by the very low apparent Mg 2+ dissociation constants (10-5 to 10-4 M [1]), cannot be quantified, but may compensate the arbitrarily used low Mg 2+ dissociation constants in the calculated balance of bound Mg 2+. Mg 2+ level in sickle cell anemia red (SS) cells SS cells were fractionated by discontinuous density gradient centrifugation in SS discocytes (1.087 < d < 1.18) and a dense SS fraction (d > 1.118) [35]. In SS discocytes, total Mg 2+ was only slightly changed. [Mg 2+ ] i in oxygenated and deoxygenated SS discocytes was not significantly different from normal erythrocytes. In the dense SS cells, total Mg 2+ content was 22% lower and [Mg 2+ ] i was 58% higher than in normal controls. During deoxygenation, [Mg 2+ ] i in SS discocytes increased similarly by about 40% as in normal erythrocytes, whereas in dense SS cells, [Mg 2+ ] i increased by about 147%. While the concentration of 2,3-BPG in SS discocytes was not reduced, the concentration of 2,3-BPG in dense SS cells was reduced by 50% (however other investigators have found a higher concentration of 2,3-BPG by about 30% in SS erythrocytes [36]). Although the concentration of 2,3-BPG is lower in dense SS cells, the increase in [Mg 2+ ] i due to ATP and 2,3-BPG binding to hemoglobin is much higher than in normal erythrocytes or SS discocytes. The increase in [Mg 2+ ] i in dense erythrocytes may be caused by an increase in Mg 2+ permeability [35]. Its mechanism is not defined. Mg 2+ efflux via the Na + /Mg 2+ antiport in Mg 2+ -loaded SS erythrocytes was four times higher than in normal erythrocytes. Besides V max,other kinetic properties e.g. lower cooperativity of intracellular Mg 2+ and regulation of the Na + /Mg 2+ antiport by protein kinase C was changed in SS erythrocytes [37]. The relationship between these alterations and the primary effect, the mutation in HbS (exchange of glutamate by valine in position 6 of the b-chain), remains to be elucidated. Conclusion Essential results are: [Mg 2+ ] i is not significantly different in the erythrocytes of various species, independent of their total Mg 2+ content. Total Mg 2+ content is correlated to the ATP and 2,3-BPG concentrations. These results provide a basis for concluding what determines the level of Mg 2+ in erythrocytes. The primary mechanism may be the regulation of net Mg 2+ influx and net Mg 2+ efflux by intracellular Mg 2+ during the maturation of erythrocytes. The notyet-identified net Mg 2+ uptake (possibly via TRPM7) may be regulated by inhibition of intracellular Mg 2+, and net Mg 2+ efflux via the Na + /Mg 2+ antiport is allosterically activated by intracellular Mg 2+. The regulatory mechanisms in erythroblasts may express a higher sensitivity for intracellular Mg 2+ than in other nucleated cell types, resulting in the lower [Mg 2+ ] i in mature erythrocytes. Secondarily, Mg 2+ is bound to Mg 2+ -binding substances and is enriched at negatively charged structures as the membranes and proteins. References 1. Günther T. Functional compartmentation of intracellular magnesium. In: Sigel H, Sigel A, eds. Metal ions in biological systems. Vol 26. Marcel Dekker Basel: New York, 1990: Günther T. Mechanisms, regulation and pathologic significance of Mg 2+ efflux from erythrocytes. Magnes Res 2006; 19: Günther T, Vormann J. Removal and reuptake of intracellular magnesium. Magnes Bull 1985; 7: Büttner S, Günther T, Schäfer A, Vormann J. Magnesium metabolism in erythrocytes of various species. Magnes Bull 1998; 20:

6 T. GÜNTHER 5. Miseta A, Bogner P, Berényi E, Kellermayer M, Galambos A, Wheatley DN, Cameron IL. Relationship between cellular ATP, potassium, sodium and magnesium concentrations in mammalian and avian erythrocytes. Biochim Biophys Acta 1993; 1175: Rapoport S, Guest GM. Distribution of acid-soluble phosphorus in the blood cells of various vertebrates. J Biol Chem 1941; 138: Günther T. Concentration. compartmentation and metabolic function of intracellular free Mg 2+. Magnes Res 2006; 19: Gupta RK, Benovic JL, Rose ZB. The determination of the free magnesium level in the human red blood cell by 31 P NMR. J Biol Chem 1978; 253: Resnick LM, Gupta RK, Laragh JH. Intracellular free magnesium in erythrocytes of essential hypertension: Relation to blood pressure and serum divalent cations. Proc Natl Acad Sci USA 1984; 81: Bock JL, Wenz B, Gupta RK. Changes in intracellular Mg adenosine triphosphate and ionized Mg 2+ during blood storage: Detection by 31 P nuclear magnetic resonance spectroscopy. Blood 1985; 65: Bock JL, Wenz B, Gupta RK. Studies on the mechanism of decreased NMR-measured free magnesium in stored erythrocytes. Biochim Biophys Acta 1987; 928: Woods KL, Walmsley D, Heagerty AM, Turner DL, Lian L-Y. 31 P nuclear magnetic resonance measurement of free erythrocyte magnesium concentration in man and its relation to blood pressure. Clin Sci 1988; 74: Ryzen E, Servis KL, DeRusso P, Kershaw A, Stephen T, Rude RK. Determination of intracellular free magnesium by nuclear magnetic resonance in human magnesium deficiency. J Am Coll Nutrit 1989; 8: Petersen A, Kristensen SR, Jacobsen JP, Horder M. 31 P- NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg 2+ in human erythrocytes. Biochim Biophys Acta 1990; 1035: Resnick LM, Altura BT, Gupta RK, Laragh JH, Alderman MH, Altura BM. Intracellular and extracellular magnesium depletion in type 2 (non-insulin- dependent) diabetes mellitus. Diabetologia 1993; 36: Matsuura T, Kanayama Y, Inoue T, Takeda T, Morishima I. camp-induced changes of intracellular free Mg 2+ levels in human erythrocytes. Biochim Biophys Acta 1993; 1220: Mulquiney PJ, Kuchel PW. Model of the ph-dependence of the concentrations of complexes involving metabolites, haemoglobin and magnesium ions in the human erythrocyte. Eur J Biochem 1997; 245: Gerber G, Berger H, Jänig G-R, Rapoport SM. Interaction of haemoglobin with ions. Quantitative discription of the state of magnesium, adenosine 5 - triphosphate, 2,3-bisphosphoglycerate, and human haemoglobin under simulated intracellular conditions. EurJBiochem 1973; 38: Bunn HF, Ransil BJ, Chao A. The interaction between erythrocyte organic phosphates, magnesium ion, and hemoglobin. J Biol Chem 1971; 246: Dietsch P, Siegmund P. Equilibrium constants and thermodynamic data of the reaction of 2,3-diphosphoglycerate with earthalkaline metal ions. Z Naturforsch 1972; 27b: Flatman PW. The effect of buffer composition and deoxygenation on the concentration of ionized magnesium inside human red blood cells. J Physiol 1980; 300: Günther T, Vormann J, Höllriegl V. Concentration of intracellular free Mg 2+ and Mg 2+ efflux from magnesium-deficient erythrocytes. Magnes Bull 1990; 12: Santarromana M, Delepierre M, Féray J-C, Franck G, Garay R, Henrotte JG. Correlation between total and free magnesium levels in human red blood cells. Influence of HLA antigens. Magnes Res 1989; 2: Féray J-C, Franck G, Garay R, Henrotte J-G. Interindividual differences in red cell Mg 2+ contents are related to the activity of a Na + :Mg 2+ exchanger. Possible relationship with HLA-associated genetic factors. Magnes Res 1989; 2: Jelicks LA, Weaver J, Pollack S, Gupta RK. NMR studies of intracellular free calcium, free magnesium and sodium in the guinea pig reticulocyte and mature red cell. Biochim Biophys Acta 1989; 1012: Barbul A, Zipser Y, Nachles A, Korenstein R. Deoxygenation and elevation of intracellular magnesium induce tyrosine phosphorylation of band 3 in human erythrocytes. FEBS Lett 1999; 455: Labotka RJ. Measurement of intracellular ph and deoxyhemoglobin concentration in deoxygenated erythrocytes by phosphorus-31 nuclear magnetic resonance. Biochemistry 1984; 23: Oken MM, Lichtman MA, Miller DR, Leblond P. Spherocytic hemolytic disease during magnesium deprivation in the rat. Blood 1971; 38: Tongyai S, Rayssiguier Y, Motta C, Gueux E, Maurois P, Heaton FW. Mechanism of increased erythrocyte membrane fluidity during magnesium deficiency in weanling rats. Am J Physiol 1989; 257: C270-C Bock JL, Yusuf Y. Further studies on alterations in magnesium binding during cold storage of erythrocytes. Biochim Biophys Acta 1988; 941: Günther T, Vormann J, McGuigan JAS. Buffering and activity coefficient of intracellular free magnesium concentration in human erythrocytes. Biochem Molec Biol Internat 1995; 37: Flatman PW, Lew VL. Magnesium buffering in intact human red blood cells measured using the ionophore A J Physiol 1980; 305: Raftos JE, Lew VL, Flatman PW. Refinement and evaluation of a model of Mg 2+ buffering in human red cells. Eur J Biochem 1999; 263:

7 Mg 2+ LEVEL IN ERYTHROCYTES 34. Sillén LG, Martell AE. Stability Constants of Metal-ion Complexes. London: The Chemical Society Burlington House, Ortiz OE, Lew VL, Bookchin RM. Deoxygenation permeabilizes sickle cell anaemia red cells to magnesium and reverses its gradient in the dense cells. J Physiol 1990; 427: Lam Y-F, Lin A-L, Ho C. A phosphorus-31 nuclear magnetic resonance investigation of intracellular environment in human normal and sickle cell blood. Blood 1979; 54: Rivera A, Ferreira A, Bertoni D, Romero JR, Brugnara C. Abnormal regulation of Mg 2+ transport via Na/Mg exchanger in sickle erythrocytes. Blood 2005; 105: